TARGETED DRUG DELTVERY BASED ON IU)HESION DOMAINS OF IMMUNOGLOBULIN SUPERFAMILY: Po AS A MODEL

A Thesis Submitted to the College of Graduate Studies and Research in Partial FuIfillment of the Requirements

for the Degree of Doctor of PhiIosophy in the College of Pharmacy and Nutrition University of Saskatchewan Saskatoon

BY Mahmoud Reza Jaafari Spring 1999

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College of Graduate Studies and Research SIJMMARY OF DISSERTATION

Submitted in partial fùlfïllment

of the requirements for the DEGREE OF DOCTOR OF PHTTIOSOPHY

by Mahmoud Reza Jaafari

Division of Phannaceufical Sciences

College of Phmacy and Nutrition

University of Saskatchewan

Spring 1999

Dr. L. Babiuk 1IX$Sai/W#&#gnwDean's Designate, Chair CoIlege of Graduate Studies and Research Dr. M. Foldvari Supe~sor,College of Phannacy and Nutrition Dr. S.J. Whiting Chair of Advisory Cornmittee, College of Pharmacy and Nutrition Dr. J.R Dimmock College of Phmacy and Nutrition Dr. GA. Zeilo College of Pharmacy and Nutrition Dr. J.S. Lee Department of Biochernistry

Extemd Examiner:

Dr. Nigel C. Phillips Faculte de Pharmacie Universite de Montreai C.P.6128, Succursale Centre-ville Montreal, PQ H3C 357 Targeted Drug Delivery Based on Adhesion Domains of Immunoglobulin Superfarnily: Po Protein as a Mode1 The objectives of this study were (i) to investigate the feasibility of targeting Liposomes reconstituted with Po protein (an immunoglobulin Dg] superfamily ce11 adhesion molecule fkom peripheral ) to melanoma cells with high level of intercellular adhesion rnolecule-1 (ICAM-1) expression, (ii) to analyze selected sequences of Po protein or leukocyte function associated antigen-1 (LFA-1, the counter receptor of ICA.-1) to hdpeptides with adhesive activity for ICAM-1 and (iii) to investigate whether these peptides could be used as ligands for targebiig liposomes toward ICAM-1 expressing cells. Liposome uptake by the cells was quantitated using radioactive . The presence of intact Po protein in liposome bilayer Po liposomes) increased the extent of binding of liposomes to human M2 1 (7.80 fold) and A-375 (4.62 fold) melanoma cells compared to control liposomes of the sarne composition but no protein, whereas with MeM 50-10 melanoma cells no significant increase was found (1.70 fold). The extent of binding of Po liposomes to the melanoma ceiis correlated with the level of ICAM-1 expression (2 = 0.9996). M21 and A-375 cells express ICAM-1 (percentage of stained cells, PSC, was 95% and 85%, respectively), whereas MeM 50-10 cells do not (5% PSC). The indirect flow cytornetry experiments using biotinyIated Po protein showed that Poprotein itself in solution also binds to M21 cells but does not bind to MeM 50-10 celIs. To identiQ peptides with adhesive activity for ICAM-1 three synthetic Po peptides (Po- peptide-l, YTDNGTF, extracellular Ig-like domain; Po-peptide-2, VALLVAV, transmembrane region; Po-peptide-3, KAhlEKK, basic intracellular dornain) and one peptide f?om LFA-1 (a 12 amino acids long fkom '1' domain) were selected. Po-peptide-1 and Po-peptide -3 decreased the binding of Po liposomes to M21 cells by 30 and 40%, respectively, while Po-peptide2 had no effect. Binding of phorbol myristate acetate-activated Jurkat T cells to interferon-y (IFN-y)- stirnulated keratinocytes WC), which is mediated by the interaction of LFA- l/ICAM- 1, decreased by 31% in the presence of LFA-1-peptide and Po-peptide4 and by 21% in the presence of Po-peptide-3, while Po-peptide-;! and RGD-peptide (as control) had no effect. Po-peptide-1, when Iinked to the liposome surface, increased the extent of binding of liposomes to M21 (6.36 fold) and A-375 (1.85 fold) compared to control liposomes of the same lipid composition but without peptide, whereas with MeM 50-10 (1.02 fold) cells no increase was found. Due to the high content of Lys, Po-peptide-3 nonspecifically increased binding of liposomes to ail the three melanoma ceIl lines. LFA-1- and RGD-peptides had no effect in the binding of liposomes to M21 cells. The extent of binding of Po-peptide-1-liposomes to hurnan melanoma cell lines correlated with the level of ICAM-1 expression (2= 0.868). Po-peptide-1 aiso increased the binding of liposomes to both native (2.96 fold) and IFN-y- stimdated KCs (6.19 fold) compared to controI Liposomes, whereas with LFA-1 -denved peptide and RGD-peptide no increase was found. Po-peptide3 non-specificaIly mediated binding of liposomes to both native and IFN-y-stimulated KCs. Since increase in the binding of Po-peptide- 1-liposomes to KCs in the presence of IFN-y was 2.1 times of native KCs, it was concluded that part of increase in the binding might be due to the ICAM-1 expression by iFN-y-stimulated KCs. Po protein and Po-peptide-1 that mediate specific targeting of liposomes to ICAM-1 expressing cells may be usehl for the development of hgdelivery systems for the treatment of malignant melanomas and idammatory skin disorders which have high level of ICAM-I expression. BIOGRAPHICAL

Born in Bojnourd, Iran

Doctor of Pharmacy, Mashhad University of Medical Sciences

Scholarship award for higher education b y the Ministry of Heaith and Medical Education of Iran, 1993-1996.

The Parke-Davis Centennial Phannacy Research Award, 1996-1997 and 1997-1998.

PUBLICATIONS

Research Articles (1) Foldvari, M., Jaafari, M*RyMezei, C., and Mezei, M. 1998. Targeting of liposomes through immunoglobulin superfamily domains: Po protein as a model. Dnrg Delivev 5: 1-13. (2) Jaafari, M.R and Foldvari, M. 1998. Po protein mediated targeting of liposomes to melanoma cells with high level of ICAM-1 expression. Int. J. Cancer (submited). To my wife, Anahita, and rny son, Arash,

for their patience and support PERMISSION TO USE

in presenting this thesis in partial fulfillment of the requirements for a postgraduate degree fiom the University of Saskatchewan, 1 agree that the Libraries of this University may make it freely available for inspection. I lürther agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor who supervised my thesis work or, in their absence, by the Dean of the College in which my thesis work kvas done. Tt is understood that any copying or publication or use of this thesis or parts of thereof for financial gain shall not be allowed without rny written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis.

Requests for permission to copy or to make any other use of matenal in this thesis in whole or in part should be addressed to: Dean of the College of Phaimacy and Nutrition University of Saskatchewan Saskatoon, Saskatchewan S7N SC9 The objectives of this study were (i) to investigate the fcasibility of targeting liposomes reconstituted with Po protein (an Immunoglobulin [IgJ superfamily ceil adhesion molecule fkom peripheral nerve myelin) to melanoma cells with high level of intercellular adhesion molecuIe- 1 (IChM- 1) expression, (ii) to anaIyze seiected sequences of Po pro tein or leukocyte function associated antigen- 1 (LFA- 1, the counrer receptor of ICAii-1) to find peptides with adhesive activity for ICXM-1 and (iii) to investigate whether these peptides could be used a. ligands for targeting liposomes toward ICAM-1 expressing cells. Liposome uptake by the cells was quantitated using radioactive lipids. The presence of intact Po protein in liposome bilayer (Po liposomes) increased the extent of binding of Liposomes to human M2 1 (7.80 fold) and A475 (4.62 fold) melanoma cells compared to control liposomes of the sarne lipid composition but no protein, whereas with MeM 50-10 melanoma cells no significant increase was found (1.70 fold). The binding of Po liposomes to M21 cells was inhibited by anti-chick Po Fab. A controi transrnernbrane , A, had no effect on the binding of liposome to M21 cells. The extent of binding of Po liposomes to the melanoma cells correiated with the level of ICAM-1 expression (r2 = 0.9996). MZl and A-375 cells express 1CA.M-i (percentage of stained ceils, PSC, was 95% and S5%, respectively), whereas lMeM 50-10 ceIls do not (5% PSC). Po protein also increased binding of liposomes to Po protein expressing CHO-X2 cells (4.36 fold, as a positive control) compared to control liposomes. The indirect flow cytometry expenmenrs using biotinylated Po protein showed that Po protein itself in solution also binds to M21 cells but does not bind to MeM 50-10 cells. Preincubation of M21 ceIls with anti-ICAM-1 monoclonal antibody decreased the binding of biotinylated Po to M21 celis by 35%. To identify peptides with adhesive activity for ICAM-1 three synthetic Po peptides (Po-peptide-1, YTDNGTF, extracellular Ig-like dornain; Po-peptide-2, VALLVAV, transmernbrane region; Po-peptide-3, KAAAEKK, basic intracellular domain) and one peptide from LFA-1 (a 12 amino acids long from '1' domain) were selected. Po-peptide- 1 and Pa-peptide -3 decreased the binding of Po liposomes to hl2 1 cells by 30 and 40%, respectively, while Po-peptide-2 had no effect. Blnding of phorbol rnyristate acetate-activated Jurkat T cells to interferon-y (EN-y)-stimulated keratinocytes (KC), which is mediated b y the interaction of LFA- 1/ICAM- 1, decreased by 3 1% in the presence of LFA-1-peptide and Po-peptide4 and by 21 % in the presence of Po-peptide-3, whiie Po-peptide-') and RGD-peptide (as control) had no effect. The effect of these peptides in the inhibition of LFA-1IICA.M-1 interaction might be through adhesion to ICAM-1. Peptides were covalently linked to the liposomes containing N-glutaryL phosphatidylethanolamine in the presence of 1-ethyl-3-(3-dimethylaminopropy1)- carbodiimide and N-hydroxysulfosuccinimide. The yield of linking, measured by capillary electrophoresis, was 85% on average for the different peptides. Po-peptide-1, when linked to the liposome surface, increased the extenc of binding of liposomes to MZ1 (6.36 fold) and A-375 (1.85 fold) compared to control liposomes of the same lipid composition but without peptide, whereas with MeM 50-10 (7.02 fold) cells no increase was found. Due to the high content of positively charged amino acids, Po-peptide-3 nonspecifically increased binding of liposomes to al1 the three melanoma ce11 lines. LFA-1- and RGD-peptides had no effect in the binding of liposomes to M21 cells. The extent of binding of Po-peptide-1-liposomes to human melanoma ce11 lines corre1ated with the levei of ICAM-1 expression (r' = 0.868). Pa-peptide-1 also increased the binding of liposomes to both native (2.96 fold) and EN-y-stimulated KCs (6.19 fold) compared to control liposomes, whereas with LFA- 1-derived peptide and RGD-peptide no increase was found. Po-peptide-) non- specifically mediated binding of liposomes to both native and IFN-y-stimufated KCs. Since increase in the binding of Po-peptide-1-liposomes to KCs in the presence cf IFN-y was 2.1 times of native KCs, it was concluded that part of increase in the binding might be due to the ICAM-1 expression by EN-y-stimulated KCs.

Po protein and Po-peptide-1 that mediate specific targeting of liposomes to ICAM-I expressing cells may be useful for the development of dmg delivery systems for the treatment of malignant melanomas and inflammatory skin disorders which have high level of ICAM-1 expression. I would like to express my sincere gatitude and appreciation to my supervisor, Dr. Marianna Foldvari, for her cnticisms, guidance and consistent encouragement throughout the course of this research work. Her advice and assistance in the preparation of this thesis is thankfully appreciated. 1 would like to gratehlly thank my advisory committee member, Dr. J.R. Dimmock, Dr. J.S. Lee, Dr. S.J. Whiting, Dr. G.A. Zello and Dr. E.M. Hawes (the previous chair of advisory committee) for their comments, guidance and supports. 1 would like to thank Veterinary Infectious Diseases Organization for providing tissue culture facility and flow cytornetry unit. I would like to thank PharmaDerm Laboratories Ltd for providing their research facilities. 1 am grateful to Drs. D. Godson and P. Gnebel for their discussions with regards to the flow cytometry results. I thank Dr. M. Baca-strada for helping me in ce11 culture techniques. I thank Dr. S. Attah-poku for his hitful discussion with regards to the linking of peptides to liposomes.

I express rny appreciation to Dr. T. Ghose for providing the human M21 melanoma ce11 line, Dr. R.S. Kerbel for the human MeM 50-10 melanoma ce11 line and Dr. M.T. Filbin for the CHO-= ce11 line. 1 thank Mr. T. Beskorwane for skillful technical assistance with flow cytornetry and Mr. Y. Yano for his assistance with the transmission electron microscope unit. I thank Ms. Corinne Braun for her assistance in cornputer problems. Financial support in the form of scholarship award for higher education from the Ministry of Health and Medical Education of Iran is gratefully acknowledged. Finally, 1 would like to thank al1 my family rnembers especially, rny wife, Anahita and son, Arash, for without their understanding, sacrifice and help this endeavor would have not been possible. TABLE OF CONTENTS Page

Permission to Use ...... 1 .. Abstract ...... *.-11

Acknowledgements ...... IV Table of Contents ...... v ... List of Tables ...... List of Figures ......

.* List of Abbreviations ...... XVIL Chapter One: RATIONALE AND OBJECTIVES ...... 1 1.1 Rationale ...... 1 1.2 Objectives ...... 4

Chapter Two: BACKGROUND INFORiMATION ...... 6 2.1 Drug targeting ...... 6 2.1 -1 Principles of dnig targeting ...... 6 2.1 -2 Modes of drug targeting ...... 7 2.1 -3 Obstacles to dnig targeting ...... 8 2.1.4 Macrornolecules in drus targeting ...... 1 1 2.1.4.1 in dmg targeting ...... 1 1 2.1.4.1.1 Low molecular weight antineoplastic agents ...... 12 2.1.4.1 .2 Immunotoxins ...... 12 2 14 13 Radionuclides ...... L 3 2.1.4.1.4 ...... 14 2.1.4.2 ...... 15 2.1.4.3 Lectins ...... 17 2.1.4.4 Hormones ...... 17 Cells in dmg targeting ...... 18 2.1 3.1 Erythrocytes ...... 19 2.1 .j -2 Leukocytes ...... 20 Synthetic carriers in drug tarsetins ...... 20 2.1.6.1 Liposomes ...... 20 2.1.6.1.1 Basic aspects ...... 20 2.1 .6.1.2 Liposome-cell interactions ...... 25 2.1.6.1.3 Targeting of liposomes ...... 28 2.1.6.1.4 Linking of to liposomes ...... 35 2.2 Adhesion molecules 36 Immunoglobulin superfamily ...... 36 2.2.1.1 Poprotein ...... 39 2.2.1.1.1 The structure of Po protein ...... 40 3.3.1.1.2 The fùnction of Pa protein ...... 42 22-12 IlntercelluIar adhesion rnolecule- 1 ...... 45

2.2.2.1 Leukocyte function associated antigen-l ...... 4s

Selectins ...... - ...... 51

Chapter Three: MATEMALS AND METHODS ...... 53 3.1 Purification of Po protein ...... 53 3.1.1 Purification of Po protein by preparative polyacrylamide gel

- 9 electrophoresis ...... -23 3.12 Purification of Po protein by reversed-phase high performance .. Irquid chromatography ...... 54 3.1.3 Analytical polyacrylarnide gel electrophoretic anaIysis of protein - - sarnples ...... 33 3 -2 Preparation of liposomes ...... 56 3.2.1 Reconstitution of Poprotein into lipid bilayer ...... 56 3 .2.2 Preparation of liposomes containing N-Glut-PE ...... 57 3.2.3 Linking of peptides to liposomes containing N-Glut-PE ...... 57 3.2.4 Negative staining of liposomes ...... -58 Maintenance of cell culture systems ...... 58 3.3.1 Hurnan M2 1 melanoma celis ...... 35 3 -3-2 Human A-375 melanoma celk ...... 59 3 .3.3 Human MelM 50- 10 rnelanoma cells ...... 59 3 -3-4 CHO-X2 Cells ...... 59 3 .3.5 Jurkat T cells ...... 60 3.3 -6 HUT 78 T cells ...... 60 3 -4 Adult normal human epidermal keratinocyte in ce11 culture ...... 60 3.4.1 Isolation and growth of keratinocytes ...... 60 3 .4.2 Frozen storage of keratinocytes ...... 63 3.4.3 Phase contrast microscopy of keratinocytes ...... 63 3.4.4 Transmission electron rnicroscopy of keratinocytes ...... 63 3 -5 Liposome-ce11 interaction assays ...... 64 3.5. L Quantitative detemination of the cellular uptake of Po liposomes in human MZ 1, A-375, MeM 50-10 melanorna cells and CHO-X2 ce 1Ls ...... 64 3.5.1.1 Quantitative determination of the cellular uptake of Po liposomes in human M21 cells in the presence of anti- chick Po Fab ...... 65 3.5.1.2 Quantitative determination of the cellular uptake of liposomes in human M21 cells in the presence or absence of Po-peptides ...... 63 3.5.2 Quantitative determination of the cellular uptake of peptide- liposomes in hurnan M2 1, A-375 and MeM 50-10 melanoma

cells *...*...... *...... +...... *...... 65 3.5.3 Quantitative determination of the cellular uptake of peptide- liposomes by human keratinocytes ...... 66 6 Biotifiylation of Po protein and peptides ...... 67 3.6.1 Biotinylation of Po protein ...... 67 3.6.2 Biotinylation of peptides ...... 67 3.6.2.1 Biotinylation of Po-peptides ...... 67 3.6.2.2 Biotinylation of LFA-1-derived and RGD-peptides ...... 65 3.7 Flow cytornetry ...... , ...... 69 3 .7.1 Direct irnmunofiuorescence flow cytornetry ...... 69 3.7.1.1 Quantitative determination of the differential expression of ICAM-l on hurnan LM& A-375, and MeM 50-10 ceils ...... 69 3.7.1.2 Determination of the expression of IChV-1 on human epidermal keratinocytes in the presence and absence of EN-./...... 70 3 -7.3 Indirect irnmuno fluorescence flow cytornetry using biotinylated- Po protein and -peptides and streptavidin-PE ...... 70 3.7.2.1 Binding of biotinylated Po protein to human MZ 1, MeM 50-10 melanoma cells and CHO-X2 celis ...... 70 3 -7.2.2 Binding of biotinylated Pa-peptides and LFA- 1-derïved peptide to human M2 1 melanoma cells ...... 71 3 -7.2.3 Binding of biotinylated RGD-peptide to HUT 78 T Cells ...... 71

3.8 Cell- assay ...... ,...... 71 3.8.1 Binding of Jurkat T cells to human keratinocytes ...... 71 3.8.2 Binding of Jurkat T cells to human M21, A-375, and MeM 30-10 cells ...... 72 3.9 Statistical analysis of the results ...... 73

3.9.1 . Unpaired t-test ...... 73 3.9.2 One-way ANOVA ...... 73

Chapter Four: RESULTS ...... 75 4.1 Study of association of Po protein reconstituted liposomes with cells ...... 73 Purification of Po protein ...... 75 Charactenzation of Po-liposomes ...... 76 Interaction of Po- and Glycophorin A-liposomes with human M2 1 melanoma cells and the inhibition of interaction with anti-chick Po antibody...... 80 4.1.4 Charactenzing the binding domains of Po protein reconstituted into liposomes by Po-peptides cornpetition studies ...... 82 4.1.5 Charactenzing of human melanoma ce11 lines for the expression of ICM-L ...... 83 4.1.6 Po protein mediated binding of Po liposomes to human MX, A- 375, MeM 50-10 melanoma cells and CHO-= cells ...... 86 4.2 Study of association of biotinylated Po protein with cells using indirect flow cvometry ...... 91

* . 4.2.1 B totinylation of Po protein ...... ,.,...,...... 93 4.2.2 Association of biotinylated Po protein with human M21 and MeM 50- 10 melanoma cells and CHO-X2 cells ...... 93 4-23 Inhibition of association of biotinylated Po protein to hurnan M2 1 melanoma cells by preincubation with anti-ICAM-1 ...... 96 4.3 Study of the effect of peptides on the interaction of anti-ICAM-1 with ICAM- 1 on M2 I cells ...... 96 4.3.1 Effect of Pa-peptides on the interaction of ICAM- 1 and anti- ICAM- 1 ...... 98 4.3.2 Effect of LFA-derived peptide on the interaction of ICAM-1 and anti-ICAM- 1 ...... 103 4.4 Study of association of biotinylated peptides with cells using indirect flow cytornetry ...... 103 4.4.1 Biotinylation of peptides ...... 101 4.4.1 .1 Biotinylation of Po-peptide-2 ...... 101 4.4.1.2 Biotinylation of Po-peptide- 1...... 106 4.4.1.3 Biotinylation of Po-peptide3...... 109 4.4.1.4 Biotinylation of LFA- 1 denved peptide ...... 109 3-4-13 Biotinylation of RGD-peptide ...... 110 4.4.2 Association of biotinylated Po-peptides with human MZ 1 melanoma celk ...... 120 4.4.3 Association of biotinylated LFA- 1-derived peptide with hurnan M3 1 melanoma cells ...... 114 4.4.4 Association of biotinylated RGD-peptide with KLJT 78 T celis ...... 115 4.5 Study of the inhibition of adherence of PMA-activated Jurkat T celis to EN-ystimulated human keratinocytes by peptides ...... 116 4.5.1 Isolation and growth of adult normal human epidermal keratinocytes ...... 117 4.5.1 .I Growth characteristics of keratinocytes ...... 118 4.5.1.2 Ultrastructural characterization of keratinocytes by transmission electron microscopy ...... 120 4 1.3 Induction of expression of ICAiM-1 in keratinocytes by interferon-y (IFN-y) ...... ~...... 121 4-52 HUT 75 T cells binding to humm keratinocytes ...... 126 4.5.3 Jurkat T cells binding to human keratinocytes ...... 130 4.5.4 Inhibition of adherence of PMA-activated Jurkat T cells to IFN-y- stimulated human keratinocytes by LFA- 1-derived, RGD-, and Po-peptides ...... 133->? 4.6 Linking of peptides to the liposomes containing N-Glut-PE ...... 139 4.6.1 Linking of LFA- 1-derived peptide ...... ,...... 130 4.6.2 Linking of RGD-peptide ...... 131 4.6.3 Linking of Po-peptide4 ...... 131 4.6.4 L inking of Po-peptide-3 ...... 147 4.6.5 Electron rnicroscopic characterization of peptide-liposomes and control liposomes by negative staining ...... 149 4.7 Study of association of peptide-linked liposomes with melanoma cell lines ...... 149 4.7.1 Peptide rnediated adhesion of liposomes to human M2 1 melanoma cells ...... 150 4.7.2 Po-peptide-1- and Po-peptide-3-linked mediated adhesion of liposomes to human M21. A.375. and MeM 50-10 melanoma cells ...... 151 4-73 Concentration-dependent adhesion of Po-peptide- 1-1inked iiposornes to human MX melanoma cells ...... 155 4.8 Study of the association of peptide-linked liposomes to human keratinocytes ...... 157 4.5.1 Peptide mediated adhesion of liposomes to human keratinocytes in the presence and absence of EN-y ...... 157 4-82 Concentration-dependent adhesion of Po-peptide-1-linked liposomes to hurnan keratinocytes in the presence and absence of EN-y ...... 162

Chapter Five: DISCUSSION AND PERSPECTIVES ...... 165 5 .1 Discussion ...... 165 j2 Perspectives ...... 181

References ...... 152 LIST OF TABLES

Table 2.1 Chernical structure of phospholipids

4.1 The effect of Po-peptides on the interaction of ICAM- 1 on human M2 1 melanoma celIs and FITC-conjugated anti-human CD54 db(0.25 PL). pgso 99

4.2 The effect of DMSO on the interaction of ICAM-1 on human M21 melanorna cells and FITC-conjugated anti-human CD54 mAb (0.25 PL). ps/so 1O0 4.3 Determining the exclusion of the effect of DMSO on the interaction of ICALM-1 on human M21 melanoma ceils and FITC-conjusated anti- human CD54 mAb (0.25 p=/50 PL)

4.4 The effect of LFA-f derived peptide (peptide-4) on the interaction of ICAM-I on human M21 melanoma cells and FITC-conjugated anti- human CD54 mAb (0.25 p3/50 PL) r 04 4.5 Binding of biotinylated RGD-peptide (peptide-5) to HUT 78 T cells and human M21 melanorna cells: indirect imrnunofluorescence flow analysis streptavidin-PE (0.5 pg50 cytornetric using PL). Il6

4.6 Adhesion of calcein-labeled HUT 78 T cells to keratinocyte

monolayers for 60 min at 37OC 137

4.7 Adhesion of calcein-labeled HUT 78 T cells (2 x 10~/well)to

keratinocyte rnonolayers for 30 min at room temperature 130

4.8 Statistical analysis of the effect of LFA- l -denved- and RGD-peptides on the adhesion of PMA-activated-Jurkat T ceils/lFN-y-stimulated-

keratinocytes by ANOVA and Tukey test 135

xii 4.9 Inhibition of adherence of PMA-activated Jurkat T ceIls to EN-y-

stimulated keratinocytes by Po-peptides dissolved in different solvents 137

4.10 Summary of results invoIving peptides in the detemuning the probable

adhesive peptides for targeting to ICAM-1 L 40

4.1 1 Linking of LFA- 1-derived-peptide (peptide-4), RGD-peptide (peptide- s), Po-peptide-1 and Po-peptide-3 to liposome containing N-Glut-PE

143

4.12 Association of LFA- 1-derived-liposomes (containing 4.0 mR/f covalently linked peptides) and control liposomes with human iM2 1

melanoma cells for 1, 2,4, and 8 hour at 37OC 151 4.13 The supernatant analysis cf association of Po-peptide- I -, Po-peptide-3- , peptide-+, peptide-5-liposomes (containing 4.0 mM covalently linked peptides) and control liposomes to human keratinocytes in the presence and absence of IFN-y (500 IU / rnL for 36 houn) for 1 hour

4.14 The supernatant analysis of association of Po-peptide- 1-liposomes (containing 4.0, 2.0, and 1.0 ml covalently iinked peptides) and control liposomes to human keratinocytes in the presence and absence of IFN-y (500 IU / mL for 36 houn) for 1 hour at 37°C 164 LIST OF FIGURES Figure Page

2.1 A schernatic drawing of the structure of liposomes --33

2.2 ~Mechanismsof interactions of liposomes with cells 26

2.3 Selected members of immunojlobulin superfmily 37

2.4 The peripheral nerve rnyelin 43 2.5 Schematic representation of leukocyte fiction-associated antijen-l 49 3.1 Flow diagram of procedures for establishment of normal human epidermal keratinocytes fiom breast skin

4.1 Purification of Po protein 4.2 Transmission electron micrographs of Po-liposomes and contro2 liposomes by negative staining using phosphotungstic acid

4.3 SDS-PAGE analysis of the Po-liposomes 4.4 Association of Po-, control-, and GLycophorin A-liposomes with human lM3 1 rnelanoma celis

4.5 Effect of preincubation with anti-chick Po-antibody Fab on the binding of Po-liposomes and control-liposomes to hurnan M2 1 melanorna celis

4.6 Association of Po-liposomes and control-liposomes with human

M2 1 meIanoma cells in the presence of Po-peptides 85

4.7 Flow cytometric analysis of human M21, A-375 and MeM 50-10

melanoma cells for ICAM- 1 expression 87

4.5 Association of Po- and controI-liposomes with human M21, A-375,

MeM 50-10 mehoma ceils and CHO42 ceIIs 89

4.9 Correlation between ICAM-1 expression on human MeM 50-1 0, A- 375, and M2 1 melanoma cell Iines and binding of Po-liposomes 90

xiv Biotin-strep tavidin indirect imrnuno fluorescence staining of ce11 surfaces

SDS-PAGE analysis of the biotinylated Po-protein Indirect immuno fluorescence flow cytometric analysis of human Ml1 and MeM 50-10 melanoma celiç and CHO-= celk for binding of biotinylated Po protein

hdirect irnmunofluorescence flow cytornetric analysis of hurnan M21 melanoma cells for binding of biotinylated Po protein in the presence of anti-human CD54 rnAb

B iotinylat ion of Po-peptide-2 Biotinylation of Po-peptide-1 Biotinylation of Po-peptide-3 B iotinylation of LFA- 1-denved peptide (peptide-4) Biotinylation of RGD-peptide (peptide-5). Phase contrat light micrographs of human epidermal keratinocytes Transmission electron rnicrographs of cultured epiderrnal keratinocytes

Induction of expression of ICAM-1 on human epidennal keratinocytes by EN-y Determination of the sensitivity of the cell-ce11 adhesion assay using calcein AM Adherence of calcein-labelied Jurkat T cells to keratinocyte monolayers for 30 min at room temperature

Inhibition of adherence of PMA-activated Jurkat T cells to 1FN-y- stimulated keratinocytes by LFA- 1-derived and RGD-pep tides

Inhibition of adherence of PMA-activated Jurkat T cells to IFN-y- stirnulated keratinocytes by Po-peptide- 1 and -3 EDC/sulfo-NHS rnediated coupling of peptides to liposomes containing N-GIut-PE 142

Linking of LFA- 1-derived peptide (peptide-4) to liposomes containin%N-Glut-PE 144

Linking of RGD-peptide (peptide-5) to liposomes containing N- GIut-PE 145

Linking of Po-peptide- 1 to liposomes containin; N-Glut-PE 146

Linking of Pa-peptide3 to liposomes containing N-Glut-PE 143 Association of Po-peptide-l-, Po-peptide-3-, peptide-4, peptide-5- liposomes and control-liposomes with human M2 1 cells 150 Association of Po-peptide-1-, and Po-peptide-3-liposomes and control-liposomes with hurnan MZ 1, A-375, and MeLM 50- 10 melanoma cells

Correlation between ICAM- 1 expression on human MeM 50- 10, A- 375, and MX melanoma ce11 lines and bindin~of Po-peptide-l- liposomes

Concentratio n-dependent binding of Po-peptide- I -liposomes and control-liposomes with human MZl melanoma cells for 1 hour at

Association of Po-peptide-1 -, Po-peptide-3-, peptide-4, peptide-5- liposomes and control-liposomes with human keratinocytes in the presence and absence of IFN-y 155

Phase contrat light micrographs of hurnan epidermal keratinocytes in the absence and presence of IFN-y 160

Concentration dependent binding of Po-peptide- 1-liposomes and control liposomes with human keratinocytes in the presence and absence of IFN-y 163

xvi ADEPT Antibody directed prodrug therapy ara- A Adenine-9-P-D-arabino furaneside

AUC Ares under cuwe BBS Sodium borate, Na2B~07.10H20 (50 mu); NaCl(0.1 M); pH 3.0 C AiM Calcein acetoxymethylester CE Capillary electrophoresis CEA Carcino-embryonic antigen CH Congenital hypomyelination CHO Chinese hamster ovary Cho1 Cho lester0 1 CMT Charco t-Mane-Tooth CNS Central nervous system cm Compartment of uncoupling of receptor and ligand DMF Dimethylformamide DMEM Dulbecco's modified Eagie's medium DMSO Dimethylsulfoxide DPPC Dipalmitoy Iphosphatidylcho Iine DRV Dehydration-rehydration vesicle DSS Dejerine-sottas syndrome EDC 1-Ethyl-3-(3-dimethylarninopropyl)carbodiimide EDTA Ethylene diamine tetrâacetic acid EGF Epidermal growth factor EIV Ether injection vesicle Fab Fragment of antigen bindin;

xvii FACS Fluorescein zctivated ce11 sorter FCS Fetal calf serum FITC FLuorescein isothiocyanate FPV French press vesicle

ICALiI- 1 Intercellular adhesion molecule- 1 ICAM-2 Intercellular adhesion rnolecule-2 TCAM-3 Intercellular adhesion molecule-3 ICAM-4 Intercellular adhesion molecule-4 IgSF Immunoglobulin superfamily IP Intraperiod line ru International unit JC Jurkat T Cells KC Keratinocytes K-SFM Keratinocyte semm fiee growth medium 13 Immunoglobulin IgSF Immunoglobulin superfami Iy m-y inter fer on-y IL- 1 Interleukin- 1 Da Kilo dalton LAD-I Leukocyte adhesion molecules deficiency type I L-CAM Liver LFA-1 Leukocyte function associated antigen- 1 LW Large unilarnellar vesicle mAb Monoclonal antibody nid Major dense line MesBS 2-[N-Morpholino]ethanesulfonic acid (5 mM); M); pH 5.5 rn1CA.M-I Membrane-bound ICAM- 1

xviii MLV Multilarneilar vesicle MHC Major histcompatibilty complex MWCO Molecular weight cut off LMW~ MolecuIar weight NA Not applicable N-CAM Neural ce11 adhesion molecuIe N-Glut-PE N-glutaryl p hosp hatidylethano lamine NHS N-hydroxysuccinimide NHS-biotin N-hydroxysuccinimide-biotin hrS Not significant PAGE Polyacrylamide gel electrophoresis

PBS-BSA-Az PBS (0.0 1 M)-bovine serum albumin (1%)-sodium azide (0.0 1%) PDGFR Platelet-denved growth factor receptor PE Phosphatidyiethanolamine PEG Polyethylene glyco 1 p-lgR Po ly-immunoglo bulin recep tor PMA Phorbol myrktate acetate PNS Perip heral nervous system PSA Penicillin-streptornycin-amphotericin-B RGD kg-Gly-ASP RER Rough endoplasmic reticulum RES Reticuloendothelial systern RP-HPLC Reversed-phase high performance liquid chromatography REV Reverse-phase evaporation vesicle SICAM-I Soluble ICAiM- 1 SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel eiectrophoresis

xix SPDP N-succinimidyl 3-(2-pyridyldirhio) propionate Sulfo N-hydroxysuccinirnide Phycoerythnn SUV Small unilamellar vesicle TcR T-cell receptor Transmission electron microscopy Transit ion temperature

Turnor necrosis factor-a

Ultraviolet CHAPTER ONE

RATIONALE ,AND OBJECTIVES

1.1 Rationaie Liposomes are microscopie vesicles compnsing phospholipid bilayers which enclose aqueous spaces. Drugs, both water and lipid soluble, can be included into the liposomes to increase the efficacy and specificity of drugs. It is also possible to target liposomes to specific cells or tissues in the body by various rneans. The usual strategy is to attach a ligand, which is specific for a determinant on the appropriate cell, to the surEace of liposomes. There are many biomolecules such as antibodies (Weinstein et al., 1978; Ghose et al., 1988; Moradpour et al., 1995; Huwyler et al., 1996; Kirpotin et al., 1997), lectins (Juliano and Starnp, 1976; Yarnazaki et al., 1992; Chen et al., 1996), antigens (Leserman et al., 1979) and others (h/ia.rgaIit et al., 1992; Schreier et al., 1993; Sarti et al., 1996), which have been used in this way. The ce11 adhesion molecules of immunoglobulin superfamily (IgSF), which mediate highly specific interactions in the cell-ce11 adhesiodrecognition process, can be used as targets or targeting ligands for dmgs or dmg delivery systems. Among the ceIl adhesion molecules of IgSF, Po protein, which has one of the simplest structure, is a suitable mode1 for studying liposome targeting. Po protein, the major glycoprotein of peripheral nerve myelin, is a mernber of the IgSF (Uyemura et al., 1987; Lemke et al., 1988). It has a single transmembrane region, a small basic intracellular domain, and a large extracellular domain with a single V-like immunogIobulin homology unit which contains an asparagine-linked oligosaccharide. In the peripheral nervous system (PNS) Po protein is responsible for the compact nature of myelin through homophilic interaction of Po extracellular dornains on adjacent concentric bilayers of the Schwann cells (the rnyelinating ce11 of the PNS) and by electrostatic interaction of the intracellular domain, rich in basic amino acids, with acid lipids in the opposing membranes (Filbin and Tennekoon, 1992; Ding and Brunden, 1994). Po protein, like other IgSF members, fünctions as a homophilic adhesion molecule, by interacting via its Ig-Iike extracellular domain holding the membranes of myelin together at the intraperiod line (Filbin et al., 1990; D'Urso et al., 1990; Schneider-Schaulise et al., 1990; Doyle et al., 1995). Using transfected Chinese hamster ovary (CHO) ceiIs expressing hl1 [ength Po protein and cell aggregation experiments, it has been shown that Po protein cm confer a capacity for adhesion on ce11 surfaces (Filbin et al., 1990). For investigatinj whether Po protein cm be used as a tarseting agent for liposome-ceIl fusion, Foldvan et al. (1990) reconstituted highly purified avian Po protein into artificial lipid vesicles (liposomes) of known and well-defined lipid composition. The prepared Po containing liposomes (Po - or proteoliposomes) had a simple lipid composition of dipalmitoylphosphatidylcholine: cholesterol (10: 1 molar ratio) and yielded stable, unilamellar liposomes of 80 prn averaje diameter that encapsulated water soluble dmgs efficiently into the intravesicular cornpartment. By cell-liposome interaction açsays it was dernonstrated that Po protein reconstituted into these liposomes was mediating the binding and uptake of these lipid vesicles into human M21 melanoma cells (Foldvari et al., 1991). Po liposomes had an interaction rate with M21 cells three tirnes higher than control vesicles of the sarne lipid composition but without protein after incubation at 37 OC or 4 OC. The presence of Po protein on the surface of melanoma cells was demonstrated by irnmunofluorescence after incubation with fluorescent Po liposomes (J?oldvari et al., 1991). Binding to the celi surface and endocytosis of Po liposomes was suggested from the sensitivity of ce11 associated liposomes to trypsin, metabolic inhibitors and low temperature (Foldvari et al., 1991). The Po protein mediated binding of liposomes to M21 cells may be due to the hydrophobie character of Po glycoprotein which may bind the ce11 membrane or more specifically rnay be due to receptor mediated endocytosis through heterophilic interaction of Po glycoprotein with some cell surface proteins of M21 cells. Most of the melanoma cells express ICAM-1 (Scheibenbogen et al., 1993; and Aitomonte et al., 1993), also an IgSF cell adhesion molecule. ICAM-I has three dornains: intracellular, transmembrane, and extracellular. In the extracelIular portion, it has five immunojlobulin like domains; thus, there is a possibility of heterophilic interaction between Po glycoprotein and these irnrnunoglobulin-like dornains. ICAIM-1 serves as a ligand for leukocyte function associated antigen-1 (LFA-l), a cell surface glycoprotein which belongs to the P2 farniIy of integins (Sprïnger, 1990; Pardi et al., 1992). LFA-I is expressed on al1 leukocytes and by binding to ICM-1 mediates adhesion of leukocytes to target cells in the inflammatory and immune responses (Marlin and Springer, L 987; Staunton et al., 1988). NormaI epidermal keratinocytes do not express ICAM-1, but under infiammatory conditions such as allergic contact eczerna, psoriasis, and urticaria, ICAM-1 is expressed on keratinocytes (Wantzin et al., 1988; Griffiths et al., 1989; Vejlsgaard et al., 1989). Expression of ICAM-1 on cultured epidermal keratinocytes cm also be induced with certain inflammatory mediators such as interferon-y (IFN-y) and turnor necrosis factor-a (TNF-a)(Gatto et al., 1994; Griffiths et al., 1989). It has also been shown (Altomonte et al., 1993) that rnost human metastatic melanoma lesions (fÏom skin or lymph node of patients with rnetastatic melanoma) express high levels of ICAM-I . Meanwhile, normal human melanocytes, similarly to epidermal keratinocytes, do not express ICAh4-1, excep: in the presence of inflammatory cytokines (Krutrnann, 1992). There is a direct relationship between ICAM-1 expression and disease progression. Therefore, ICAM-1 could be a good candidate as a target for dmgs or drug delivery systems in the inflammatory skin diseases or skin/Iyrnph node melanomas. If the increase of interaction rate of liposomes with MI1 melanoma celIs is due to the heterophilic interaction of Po protein with ICAM-1, it may possible to use Po protein as a mode1 ligand to target ICAM-I expressing cells. Using synthetic short peptides as a targeting ligands for liposomes may have some advantages over whole protein molecules, since they might be less immunojenic, their handling and prese~ationis easier and allow longer circulation of liposomes in the blood Stream. Also, bulk production of short synthetic peptides should be easier than proteins as targeting ligands. Therefore, the principal objective of this project was to analyze selected sequences of Po protein or LFA-1 as models to find peptides with adhesive activity for ICAM-I and investigate whether these peptides could be used as ligands for targeting liposomes toward ICAM-1 expressing cells.

1.2 Objectives

The overall objective of this study was to derennine the feasibility of targeting liposomes through ce11 adhesion molecules, using Po protein as a model. The specific objectives of the snidy were as follows:

1) Evaluate the specificity of heterophilic interaction of Po Iiposomes with human M2 1 melanoma celIs by:

a) Determining the cellular (human M2 1 melanoma cells) uptakc of Po liposomes in the presence of anti-chick Po antibody Fab.

b) Determining the cellular (human M2 1 melanoma cells) uptake of liposomes which are reconstituted with a non-adhesive transmembrane protein, Glycophorin A, instead of Po protein. 2) IdentiS possible short adhesive peptide-sequences in the Po protein by conducting cornpetition studies with synthetic Po-peptides based on the chicken Po sequence.

3) Evaluate the invoivement of [CAM-l/Po protein interaction in the Po protein mediated binding of liposomes to hurnan MZ 1 melanoma cells by:

a) Determining the relative expression of ICAM-1 on human iMZ1 melanoma celk and on other selected human melanoma ce11 lines by flow cytometry.

b) Deteminhg the cellular uptake of Po liposomes by different human melanoma ceIl Iines to evaluate whether the Ievei of ICAM-1 expression on melanoma cells correlates with the extent of binding of Po liposomes to the cells. 4) Evaluate the heterophiiic and homophilic interaction of Po protein in solution by deteminin; the adhesive activiry of biotinylated Po protein with different melanoma ce11 lines and CHO-= cells using indirect £low cytornetry.

j) Identify peptides which have possible adhesive activity for ICAM-1 by:

a> Evaluating the inhibitory effects of peptides on the interaction of ICAM-L and anti-TCAM-1 by flow cytometry in order to find out the possible adhesive peptides for targeting to IChiI- 1

b) Evaluating the adhesive activity of peptides by determining the binding capacity of biotinylated peptides to human MX melanoma cells, which have a hi& level of IChV-1 expression, using indirect Row cytometry. Evaluating the inhibitory effects of peptides on the interaction aLherence of PM-activated Jurkat T cells to IFN-y-stimulated human keratinocytes which is rnediated by the interaction of LFA-l/ICAM-1 in order to find out the possible adhesive peptides for targeting to ICAM- 1.

6) Develop liposomal systems with covalently linked peptides.

7) Determine the cellular uptake of peptide-linked liposomes in different hurnan melanornz ce11 lines and evaluate whether the level of ICAM-1 expression on melanoma ceIls correlates with the extent of binding of peptide-linked liposomes to the cells. 8) Determine the cellular uptake of peptide-linked liposomes in human keratinocytes in the presence and absence of EN-y. CH-APTER TWO

LITERATURE RJZVIEW

2.1 Drug targeting 2.1.1 Principles of drug targeting Drug tarseting is the systemic or local administration of a dry-carrier conjugate that delivers the active dm; selectively to a specific tissue or ce11 population within the body (Gregoriadis, 1977 and 1981; Poznansky and Juliano, 1984; Friend and Pangbum, 1987; Rihova, 1997; Langer, 1998). The specific delivery of dmgs to their site of acrion should increase their therapeutic activity while minimizing the adverse effects. Behveen the site of administration of a drug and its site of action, there are molecules, cells, membranes and organs that dru; rnust traverse to be effective. In many instances, a drug has limited or no access to its site of action or is rapidly metabolized or excreted. In other instances, the dnig distributes freely throughout the body; however, it not only acts on the desired target sites but also causes undesirable effects on non-target sites. These difficulties may be circumvented by the use of targeted dnig delivery systems that will not only protect the non-target sites from the drug, but also the dm3 from the environment. At the same tirne these carriers will direct the dmgs where they are needed. In an ideal drug-carrier conjugate, it is speculated that the dmg carrier unit will preserve its inte,gïty, avoid interception by molecules and normal cells, penetrate ce11 membranes, and selectively recogize and associate with the targets. Furthemore, the carier rnust be non-toxic, nonimmunogenic and ideally biodegradable. There have been many carrier systems, such as macrornolecules (antibodies, glycoproteins, lectins), homones, carbohydrates, cellular (erythrocyte, other cells) and synthetic carriers (liposomes) that have been used for drug targeting purposes. 2.1.3 Modes of drug targeting Targeted drug delivery systems can be classified as passive, physical, or active targeting according to the rnethods used for locaiization of drus (Poste and Kirsh, 1983). In passive targeting, the natural distribution pattern of the carrier in the body determines the selectivity of the targeting. The disposition pattern of the caniers in the body mainly depends on their particle size and shape, surface charactenstics (hydrophobicitylhydrophilicity) and surface charge. In this respect, for instance, it has been shown that the conventional liposomes are taken up by the cells of the reticuloendothelial system @ES) (Juliano, 1982). Encapsulating antirnonial dmgs to liposomes to treat leishmaniasis, a parasitic infection which involves macrophages, increased the therapeutic index because the drug containing liposomes accumulate preferentially in the ceIls which are the site of parasitic infection (Alvins, 1982). Due to this charactenstic of liposomes, Liposomal amphotericin B has been developed and clinically used for the treatrnent of visceral leishmaniasis (di Martino et al., 1997; Coukell and Brogden, 1998). In another study, liposomes encapsulatin; imunornodulators have been used to stimulate the macrophages to increase their tumoricidal activity (Poste et al., 1982). The accumulation of the drug-carrier complex in the RES cells are not aIways desirable, because this sequestration by RES ceI1s not only decreases the effrcacy of the systern for tqetin; to other ceIls, but can also damage the RES cells which are critical for the immune systems, particularly if the dmg is highly cytotoxic. In physical targeting, the environment is manipulated to direct the drug-carrier systems to a specific place in the body or to cause seiective reIease of its content in the target place. This is usually accomplished by an extemal mechanisms such as induced local hyperthermia or magnetic field. For instance, liposomes composed of iipids, which have their transition temperature slightly above body temperature, destabilized and released the encapsulated rnethotrexate upon temperature rise on the tumor (Weinstein et al., 1979). In another study doxorubicin was delivered to solid tumors using thermosensitive liposomes and local hyperthermia in mice (Unezaki et al., 1991). To prolong the circulation time of these liposomes in the blood saeam, thsy also incorporated polyethylene glycol (PEG) in their formulation. The concentration of doxorubicin in the tumor afier injection of these liposomes containing doxorubicin with local hyperthermia was si,pificantly greater than fkee doxonibicin and this combination increased the survival of the animals. In another study the therrnosensitive liposomes were efficiently targeted to the mouse liver cells with the aid of the extemal magnetic field and local hyperthermia (Viroonchatapan et al., 1996).

In active targeting, the natural disposition pattern of hgor dm;-camer is modified by attachrnent of cell-specific ligands to target it to the specific organ, tissue, or ce11 population. Ligands are here defined as biomolecules that have specific affinity for a determinant on the appropriate cells. Ligands, which have been investigated for active site-specific drus delivery, include antibodies (Blakey, 1997; Pietersz and Krauer, 1994; Panchapnula and Dey, 1997), hormones (Liu et al., 1988), peptides (Slepushkin et al., 1996; Banejee et al., 1998), glycoproteins (Fiume et al.. 19S2), lectins (Yarnazaki et al., 1997) and carbohydrates (Wadhwa and Rice, 1995). Active dnig targeting using targeting ligands to ce11 surface receptors on specific cells is a more promising approach and it should decrease adverse side effects resulting fiom the interaction of drugs with normal cells and increase the pharmacological activity of the dru; by having a greater amount of dmg in target cells compared to the normal cells.

3.1.3 Obstacles to drug targeting The aim ofdrug tarsetins is to deliver drugs to the specific site of action through a carrier. In order for a dnig-carrier unit to successfûlly reach to the site of target ceIls it must pass a nurnber of barriers (Poznansky and luliano, 1984). When a drug-carrier unit is administered systemically, there is an endothelial barrier between the lumen of the vasculature and extravascular cornpartment. Vascular endotheliurn morphologically is similar to any simple epithelium, however it has higher permeability for water and small water soluble molecules. Furthemore, the vascular endothelium, unlike the epithelium, has also relatively hi$ pemeability for large water soluble macromolecules ransing Born albumin (68 kDa) to lipoproteins (3,000 ma). It is not well understood how solutes are transported fiom the plasma to tissue fluid. In al1 capillaries (except liver, spleen and bone marrow), the capillary endothelial is supported by a basement membrane or basal lamina. Basal lamina consists of fibrillar molecules such as laminin, fibronectin, collagen (type IV) and . The basement membrane has the ability to ultrafilter macrornolecules. Furthemore, some components of basal tamina, such as fibronectin, have the ability ro specifically bind to other macromolecules. Therefore, the penetration of a drug-carrier complex through the basal lamina depends not only on its size and charge, but also on the specific binding characteristics of the macrornolecular camer.

The other obstacle to drug targeting is the celIs of RES. The RES cells (eg. Kupffer cells, macrophages of the spleen, blood phagocytes and monocytes, tissue and alveolar macrophages) are phagocytic and take up foreign particIes and macromolecules. In order for a drug-carrier complex to successfuliy reach its site of action, it has to escape from the cells of the RES. The cells of RES rapidly take up particulate drug carriers such as liposomes and microspheres. By manipulating the chernical and physical charactenstics of the liposomes, it is possible to avoid t~ sorne degree, the phagocytic action of the RES cells (Poste, 1983). The cells of the RES can also sequester macromolecular carriers such as antibodies. The sequestration of dmg- carrier complex by the RES will result in a decrease in the amount of available drug- canier complex to reach its site of action, and sometirnes more importantly this may damage the host defense if the drug-carrier complex is harmful to the cells of the RES. Another obstacle to dmg targeting is the cellular barrier. AAer escaping from the cells of RES and passing through the endothelial and basal lamina barriers, a dmg- carrier cornplex must reach its specific site of action. If the site of action of the dmg is on the ce11 surface, then the camer by bnnging the dnig close to the ce11 surface has achieved its job. However, if the site of action of the dmg is intracellular, then the dmg- carrier cornplex has to go inside the cells. Cells cm intemalize particles and macromolecules at least three different ways: phagocytosis, pinocytosis and receptor- mediated endocytosis. Phagocytosis is mainly used by specialized cells (e.g. macrophages 2nd neutrophils) to take up large particles (100 nm or greater), cells and microorganisms (Silverstein et al., 1977). The foreig particies are first engulfed into membrane-bound vesicles and internalized by the cells. Then, the vesicles are fused with lysosomes. Some nonspecialized cells like fibroblast can also phagocytose large particles adsorbed to their surface (Okada et al., 198 1). In pinocytosis, the ce11 membrane invaginates and pinches off inside of the cells forming intracellular vesicles of about 25 nm in diameter containing extracellular fluid. These vesicles finally fuse and deliver their content to the lysosomes (Silverstein et al., 1977). Unlike the phagocytosis which is usually observed in specialized cells, pinocytosis is common to al1 cells in which the cells takes in liquid or liquid containin; solutes (e.g. drug-carrier conjugate) 6om the extracellular space. The receptor-mediated endocytosis is the best characterized way of internalization of rnacromolecules (Pastan and Willingham, 1983; Shephered, 1 989). This mechanism is used for internalization of many proteins, hormones, growth factors, viruses and toxins. In receptor-mediated endocytosis, after specific binding of the ligand to the receptor, the ce11 membrane invaginates into the ce11 to fom a coated vesicle and internalizes into the cell. Inside the cell, the vesicle is converted to an endosorne. The endosome then fuses with a vesicle called CURL (cornpartment of uncouplin; of receptor and ligand). CURL has an acidic pH of about 5 and induces the separation of ligand fiom its receptor. The separated receptors concentrate in the membrane of the CURL and are recycled to the ce11 membrane. However, the ligand accumulates in the vesicular portion of the CURL and Fuses with a lysosome where the ligand is degraded by the Iysosomal enzymes. If the lisand is conjujated to a dnig or a carrier containing drug (e.g. liposomes and microspheres), the dmjcould be released in rhe lysosome. In addition to the ban-iers mentioned above for drug targeting, there is a problem of possible irnmunogenicity induced by macromolecule carriers. The antibodies developed could elirninate the carrier-hg cornplex. ThereFore, the macromolecule cher-drug complex should not be irnmunogenic. 3.1.4 iMacromolecules in drug targeting 1.1.4.1 Antibodies in drug targeting Due to their hijh specificity, antibodies, especially monoclonal antibodies (mAbs) and their Fab fragments, have been used as carriers for drug tarjeting, particularly in cancer chemotherapy (Blakey, 1992; Pietersz and Krauer, 1994; Panchagnula and Dey, 1997). Ln cancer chemotherapy, the cytotoxic dmgs nonselectiveIy kiIl the tumor cells as well as normal proriferative cells (e-g. cells of bone marrow and ~astrointestinaltract). Monoclonal antibodies raised against specific antigens on turnors, cm be conjugated to cytotoxic dmgs to specifically deliver the dmgs to the cancer cells and hence reduce damage to normal cells. Low molecular weight antineoplastic agents, protein toxins (immunotoxins), radionuclides and enzymes which are able to convert the inactive prodmgs to cytotoxic dnqs have been conjugated to mAbs for selective deIivery to turnor cells.

One of the main problems associated with the use of mAb in cancer chemotherapy is the antigenic heterogeneity of the tumor cells. The antigens produced by cancer cells are constantly changing and therefore decreasing the specificity of antibody-cytotoxic cornplex prepared for an earlier form of cancer cells (Pimm et al., 1988; Hinman et al., 1993; Pietersz and Krauer, 1994). The other problem is the turnor accessibility of the antibody-cytotoxic conjugate which is not always that sigificant in

3aeneral, due to the poor vascularization of the tumors. An additional problern related to the tumor accessibility, is the shedding of the turnor antigens into the systemic circulation. The rnAb specific for these antigens wouId react in the blood Stream, thus decreasing the site specificity and access of the antibody-cytotoxic complex to the tumor. Some of the tumor specific mAbs do not have complete tumor selectivity and are cross-reactive with some ce11 surface antigens of normal cells, therefore, have decreased site specificity. Beside these problems, in the developrnent of antibody-cytotoxic conjugate for targeting purposes, the process of conjugation must not affect the specificity of the antibody or decease the binding ability of the antibody, also the toxicity of the cytotoxic asent should remain intact. Furthemore, the antibody- cytotoxic ajent should remain stable in the systernic circulation before reachin; the target site (Upeslacis and Hinman; 1988). 2.1.4.1.1 Low rnolecuIar weight antineoplastic agents Several low molecular weight antineoplastic asents including methoîrexate (Shih et al., 1988; Pimm et al., 1988; Ballantyne et al., 1985), melphalan (Srnyth et al., 1957), cisplatin (Schechter et al., 1987), 5-fluorouracil (Hunvitz et al., 1985), doxonibicin (Trail et al., 1993) and vinca alkaloids (Smith, 1985) have been conjugated to tumor specific mAbs in order to selectively deliver the dmgs to the tumor cells. The main problern with this method of antibody based dmg tarjeting is the low amount of drug delivered to the target site, because only a limited nurnber of drug can be conjugated to the antibody. Therefore, different conjugation procedures have been devised to deliver the maximum number of dmgs per molecule of antibody under conditions that both antigen bindinj activity and the cytotoxic activity of the dmg are lefi intact. These procedures include: a> Covalent linking of the dnig to amino acids or carbohydrate residues of the antibody. b) Linking of the drug to the antibody through spacer molecules which are cleavable by lysosomal enzymes. c> Linking of the dmg to a high molecular weight carrier like albumin, poly-L- Lysine or dextran which is then attached to the antibody. mong the different procedures, the high molecular weight carriers result in the greatest nurnber of drug per antibody molecule. However, due to their very large size, tumor accessibility is still a problem. In spite of al1 these difficulties, the antibody-drug conjugate has shown higher anticancer effects in animal mode1 systems compared to Free dmgs (Starling et al,, 1989; Blakey, 1992 and Pietersz and Krauer, 19%).

2.1.4.1.2 Irnmunotoxins In this group, the antibody is conjugated to a potent toxin of bactenal or plant origin (Blakey et al., 1988; Frankel, 1958; Ghetie et al, 1997). The main advantage of these toxins over usual antineoplastic agents is their 1000-10000 fold higher potency. Diphtheria toxin and Pseudomonas endotoxin A from the bacterial toxin and ricin and abrin fiom plants are the most widely used toxins. A11 of these toxins have at Ieast nvo regions of catalytic and bindins domains. The bindins domains of these toxins mediate the bindins of the toxins to the surface of rnost hurnan cells. Afier binding to the ceIl surface, the toxin is intemalized and in the cytosol catalytically inactivates the prorein synthesis. Only a small nurnber of toxin molecules need to get access to the ce11 cytosol to kill the cells, since these molecules act catalytically. If the whole protein of toxin is used for conjugation to the antibody, the conjugate will non-specifically bind to the cells (both normal and cancerous cells) and cause systemic toxicity. Therefore, the conjugation of the antibody is carrïed out only with the catalytic domain of these toxins (Youle et al., 1988). Then the antibody provides specific binding capacity and the catalytic domain contributes to the cytolytic effects (Byers et al., 1989). The main problems with the use of irnmunotoxins are the immunogenicity of the conjugate, instability and reduced tumor accessibility due to their large size. Attempts have been made to decrease the size of irnmunotoxins through using univalent Fab fragments which have had prornising effects in some cancers (Chaudhary et al., 1990). The immuno jenicity problem with imrnunotoxins is greater, since both antibody and toxin are immunogenic. In clinical trials the antibody response to the irnmunotoxin has been seen even 10-17 days afier the initiation of immunotoxin injection (Byers et al., 1989). Gelber and Vitetta (1998) utilized the irnrnunosuppresssive agents ro prevent the immunogenicity of the immunotoxins. They showed that the administration of a ricin A chain-containing immunotoxin to the mice in the presence of immunosuppresssives (a combination of hCTLA4Ig and anti-CD40L) prevented the anti-immunotoxin response and the effect of immunotoxin in tumor killing in the presence of immunosuppresssive was 1.5 fold greater than the irnmunotoxin alone.

2.1 Al.3 Radionuclides Antibodies conjugated to radionuclides have also been used for the treatment of cancers (Ballau et al., 1979; Vogol, 1987; Scholm, 1986; Zhu et al., 1997). Arnong the different radionuclides, iodine 131 and yttrium 90 which emit high and intermediate energy beta particles, respectively, and half lives of 2.7 and 8 days, respectively, are suitable for targeting to cancer cells. The beta particles have path lengths and cm pass many cell diameters. Therefore when antibodies are conjugated to these radionuclides, they do not need to get inside the cells to kill them. This is the main advanrage of radionuclides over anti-neoplastic agents and imrnunotoxins. Furthermore, the radionuclides might be able to overcome the problem of antigenic heterogenicity and tumor accessibility, since the radionuclide-antibody conjujate cannot only kill the target ce11 but also the sumunding cells. Therefore this property of the radionuclide would mediate the destruction of adjacent turnor cells which either do not have the target tumor specific antigen or are in areas of poor accessibility (e.g. poor vascularization). On the other hand, since these beta particles can span over many cells diameter, as long as the radionuclide-antibody complex is in the systernic circulation, they can cause damage on normal cells and this limit the dose that can be used. Meredith et al (1995) showed that administration of increasing doses of munne 17-1A IgG2a antibody, which reacts ajainst hurnan gastrointestinal cancers, conjugated to "'1 in patients with metastatic colorectal cancer had no significant acute or late side adverse effects. They concluded that high doses of lZ51 conjugated to 17-lA IgGîa antibody could be administered in patients with metastatic colorectal cancer without sigificant toxicity.

2.1 Al.4 Enzymes In this category of antibody-based dmg targeting, an enzyme which cm convert a non-toxic prodrug to a porent cytotoxic drug, is covalently linked to an antibody which is specific for a tumor antigen. Afier localization of the enzyme-antibody conjugate at the tumor site and complete clearance of the conjugate from the systemic circulation, the prodrug is administered. The prodrug is converted to a cytotoxic agent at the tumor site, therefore, minimizing the toxicity in the normal cells. This method of antibody based targeting is called antibody directed enzyme prodrug therapy (ADEPT) (Bagshawe et al., 1988; Senter et al., 1989; Kerr et al., 1990; Melton and Shenvood, 1996). ADEPT has many advantages over other antibody based targeting strategies. In ADEPT, the enzyme-antibody conjujate is not necessary to be intemalized to the tumor cells, since the low molecular weight cytotoxic agents can be produced from produgs at the tumor site (on the surface of targeted cell), then they cm easily diffuse into tumor cells. In ADEPT, it is also possible to use a tumor specific antibody that cannot be intemalized. The ADEPT, like radionuclide, can also overcome the problems of antigenic heterogenity and tumor accessibility, since cytotoxic agents produced outside the tumor cells cm diffuse to the adjacent -or cells which either do not posses the tqet tumor specific antigen or are in the areas of poor accessibility. However, unlike the radionuclide, the antibody-enzyme complex and prodrug are relatively non-toxic, and they should not cause darnage when they are in the systemic circulation- The other advantage of ADEPT is that a single enzyme can convert a large number of prodrugs to the cytotoxic agent, therefore, the potency of the drug should not be a problem. .LDEPT will result in toxicity in normal cells, if: a) A residual amount of enzyme-antibody conjugate remains in the systemic circulation or normal tissue when the prodrug is administered. b) The body itself has enzymes that can convert the prodrug to the cytotoxic agent. c) The tumor specific antibody has cross reactivity with the ce11 surface antisens of normal cells and delivers the enzyme to the normal tissue. d) The produced cytotoxic ajent at the tumor site gers back to the systernic circuIation. Care should be taken to minimize these possibilities to decrease the toxicity in normal cells in the development of an ADEPT system. hother approach in ADEPT is to conjujate enzymes to the surface of immunoliposomes (liposomes conjugated to antibodies) (Vingerhoeds et al., 1996). The advantage of this method is that the enzyme density is higher on the surface of liposomes. Utilizing this technique, Vingerhoeds et al. (1996) showed the cornplete conversion of daunorubicin-gLucuronide to daunorubicin.

Glycoproteins are proteins that are covalently associated with carbohydrates. There are two types of attachment of carbohydrates to proteins: N-linked and O-linked. In N-linked glycosylation the carbohydrate is linked to the amide nitrogen of an Asn in the sequence Asn-X-Ser or Asn-X-Thr. X is any arnino acid residue except Pro or Asp. In O-linked glycosylation the carbohydrate is linked to the carboxyl grooup of either Ser or Thr. In general, glycoproteins do not exhibit site selectivity. However, when the sialic acid (N-acetyl-neuraminic acid) residues are rcmoved frorn the temiinal sugar branches, the resulted are then recognized and sequestered by certain liver cells, cells of RES, and fibroblasts; dependin; on the sujar type exposed (Ashwell and Morell, 1973; Neufeld and Ashwell, 1980). According to this principle, modified glycoproteins have been used to target drugs to certain liver cells, cells of RES and fibroblasts. The receptor responsible for the recognition of asialo,alycoprotein by hepatocytes has been characterized (Ashwelt and Harford, 1982). This receptor recognizes both galactose and N-acetyl-galactosamine. Other characterized recognition systems for asialoglycoproteins include: L-fucose by mammalian macrophages, N- acetyl-glucosamine or mannose by mammalian RES cells and mannose-6-phosphate by hurnan fibroblasts (Neufeld and Ashwell, 1980). After binding of to its receptor on ce11 surfaces, the glycoprotein is intemalized into the cells by receptor- mediated endocytosis and ends up in the lysosome. Hepatocytes are among the best studied cells for tarseting through asialoglycoproteins, because they are targets of various infective elernents, especially those causing hepatitis, and they have a receptor for recognizing asialoglycoprotein. For targeting to hepatocytes, gaIactosyI-terminated fetuin (asialofetuin) has been developed aid coupled to adenine-9-P-D-arabinofuranoside(ara-A), a drug used for the treatment of chronic hepatitis B (Fiume et al., 1982). Ara-A has serious adverse side effects, gastrointestinal and neurological disorders and depression of bone marrow, in high doses which limit its use. The asialofetuin-ara-A conjugate selectiveIy delivered the ara- A to the mouse hepatocytes. Since the asialofetuin-ara-A conjugate showed immunogenicity, the ara-A was conjugated to lactosarninated human serum albumin (Fiume et al., 1983), which has also been shown to bind to hepatocytes. They showed that the quantity of ara-A required for antiviral activity was about ten times smaller when the drug was conjugated to lactosaminated serum alburnin than when it was used as a fiee drus. 2.1.4.3 Lectins Lectins are proteins that specifically bind to carbohydrate residues. Lectins have been evaluated as drug targeting ligands (Juliano and Stamp 1976; Yamazaki et al., 1993; Chen et aI,, 1996). The main disadvantages of the lectins as ligands in site specific drug delivery are the lack of their selectivity and their adverse effects. Sugar residues are distributed a11 over the body which cm interact with and reduce the selectivity of the lectins. The other drawback of lectins is their adverse effects. Lectins are irnmunosuppressive and at doses as low as 10 can cause a severe long lasting inflammatory response (Friend and Pangbum, 1987). Another possibility for using lectins in targeted drug delivery systems is to target the lectins in the body since it has been shown that the mmalian cell membranes contain lectins which are targetable with carbohydrate residues (Neufeld and Ashwell, 1985). In this approach, a dmg was conjugated to a specific oligosaccharide structure which has specificity for certain lectins in the body (Wadhwa and Rice, 1995). Upon binding of the drug-oligosaccharide conjugate to lectins on the ce11 surface, the conjugate is endocytosed. The main advantage of this type of dmg targeting over others (e-g. antibodies) is the small size of dmg-conjugate. The molecular masses of oligosaccharides could typically range fkom 500-5,000 Da, which is much smaller than antibodies (about 150,000 Da). Because of the relatively small size of dmg- olipsaccharide conjugates, the obstacles of dmj targeting such as capillary endothelium, basai lamina, and cells of RES should be less. This is one of the most promising ways of tarseting. For instance, when allopunnol riboside was linked to the rnannosylated poly-L-lysine carrier molecule, it was 50 times more effective rhan the fiee drus in the treatrnent of leishmaniasis in a rnunne mode1 (Nerge et al., 1992).

2.1.4.4 Hormones Hormones can also be used as targeting ligands since they have specific receptors on their target cells and exert their effects by binding them. Hormones are intemalized by receptor rnediated endocytosis (Varga, 1985). The advantages of using hormones compared to other targeting ligands are: a) Easy to obtain in pure form, b) Lack of imrnunogenicity,

C) Not phagocytosed by the cells of ES. However, hormones, as targeting ligands, have not been as successful in vivo as they have been in virro. For instance, melatonin, a 13-amino acid peptide which binds to melanocytes and stimulates the production of rnelanin, has a specific receptor on melanoma cells. Therefore, to target melanoma ceils for cancer chemotherapy, melatonin was conjugated to daunomycin (Varsa et al., 1977). This snidy showed that the conjugate enters the ce11 lysosome and the &ee dmg is released by hydrolysis. Even though the conjugate was about 3 times more effective than the eee dmg in melanoma cells in vitro, in the murine mode1 the conjugate was only marginaily effective. Several reasons, including the presence of melatonin receptors on other cells (in addition to normal melanocytes and melanoma cells), cornpetition with endogenous ligands for the receptor on the target and the short half-life of the conjujate, were cited to explain the lack of success of the conjugate in vivo (Varga and Asato, 1983). In another interesting study, a conjugate of hormone and antibody, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes (Liu et al., 1958). The hormone (an analogue of melatonin) was conjugated to an antibody to the CD3 cornponent of the T-cell receptor. The hormone which has a specific receptor on melanoma cells was used as a targetin; 1i;and. The antibody to the CD3 component of the T ce11 can activate the cytotoxic T lymphocytes to destroy the target cells. This study showed that the conjugate of hormone-antibody mediates significant specific lysis of human melanoma cells, whereas the antibody by itself showed oniy little specific lysing of the celis.

2.1.5 Ceils in drug targeting In cellular drug tarsetins, actual cells such as erythrocytes and leukocytes are used as a vehicle for drug delivery (Poznansky and Juliano, 1984; Friend and Pangbum, 1987). Lack of imrnunogenicity, especially if the patient's own cells are used, and biodegradability are the advantages of cells as targeted drus delivery vehicles. The main disadvantage of the cells as drug carriers is their inability to deliver their content to the cells by fusion.

2.1.5.1. Erythrocytes Red blood cells have been extensively evaluated as a drug targeting camer (Krantz, 1997; de Flora et al., 1993). The most widely used technique for incorporation of drugs into erythrocytes is to subject them to hypotonic lysis (Ihler et al., 1973). However, during this process, the erythrocytes lose some of their hemoglobin and membrane proteins which decrease their Iife tirne in the blood Stream. Since then attempts have been made to avoid the disadvantage of hypotonic lysis by loading the erythrocytes under isotonic condition (Billah et al., 1977) and external electric field (Scheurich, et al., 1980; Mangaf and Kauer, 1991). The external electrïc field produces pores at 4 OC in the ce11 membranes of erythrocytes that allows equilibration with the added dmg. Upon raising the temperature to 37 OC, the pores are sealed and the drug is captured in the erythrocytes.

Since erythrocytes do not migrate out of the blood stream, their use as dmj delivery camers is limited to the systemic circulation. Active targeting of erythrocytes has been achieved by noncovalent binding of antibody (specific for a determinant on the target cell) to erythrocytes through (or streptavidin)-biotin system (Sarnokhin et al., 19S3; Muzikahtov and Murciano, 1996). In this system, erythrocytes and the antibody were biotinylated separately. The biotinylated erythrocytes were first incubated with avidin (or streptavidin) and then the biotinylated antibody. Erythrocytes have been also used as a dmg delivery system for targeting to the cells of the RES since, it has been shown that alteration of the erythrocyte ce11 surface by hypotonic lysis can lead to their rapid removal from the blood stream by the celis of the RES (Tyrrell and Ryrnan, 1976). Erythrocytes have been also used for the sustained delivery of drugs and enzymes in the systemic circulation. For instance, it has been shown that desferrioxamine (an iron-chelating agent) when loaded to erythrocytes, is more effective than the free drus in patients suffering Eom high titration of iron in their blood (thalascemic patients) (Green et al., 198 1).

2.1.5.2 Leukocytes Among the leukocytes, neutrophils are fiequently used as dnig delivery camers, because of their availability in Iarge numbers in the blood and easy isoIation wirh high purity. These leukocytes have the advantage that they can Ieave the blood Stream unlike erythrocytes. Since neutrophils tend to accumulate in large numbers in the areas of acute inflammation, they have been used as a drug delivery vehicle for diagostic and therapeutic agents in inflamrnatory conditions (Gziney and McDougall, 1954; Smith, 1994). Among different dnigs, the antimicrobial agents have been evaluated more for their increased effects for the treatment of infectious disease through loading to the neutrophiles (Frank et al., 1997; Paul et al., 1997). Frank et al. (1 992) demonstrated that neutrop hils loaded with cipro floxacin migrate toward chemoattractant agent on agar and inhibit the growth of Streptococcus pyogenes. The empty neutrophils migrated toward chemoattractant agent on agar but did not show significant inhibition of bacterial growth. Paul et al. (1997) demonstrated that neutrophils loaded with azithrornycin could migrate and deliver the antibiotic to Chlamydia trachomatis-infected polarized human endornetrial epithelial cells. The delivered antibiotic had a LethaI effect on the Chlamydia trachomatis and reduced the viability of infectious progeny. Whcn the polarized human endornetrial epithelial cells infected with Chlamydia trachomatis were exposed to emp ty neutrop hi 1s. thz Chlamydia trachomatis was remained alive.

3.1.6 Synthetic carriers in drug targeting 2.1.6.1 Liposomes

2.1.6.1.1 Basic aspects Liposomes are rnicroscopic vesicles consistin; of phospholipid bilayers which enclose aqueous compartments (Juliano, 1981; Gregoriadis, 198 1). They are usually ciassified on the basis of their size and the number of bilayers: multiIarnel1ar vesicles (MLV, 0.5-20 pm), srnail unilameliar vesicles (SUV, 25-100 nrn), and large unilarnellar vesicles (LW, 100-500 nm). SUV and LWhave only one single lipid bilayer and one enclosed aqueous compartment, but MLVs have more than one lipid bilayer and aqueous compartrnents (Figure 2.1). Liposomes are sometimes classified based on the rnethod of preparation: reverse-phase evaporation vesicles, REVs; French press vesicles, FPVs; ether injection vesicles, EIVs; dehydration-rehydration vesicles, DRVs (Weiner et al., 1989).

Mer liposomes were discovered by Bangham et al. (1965), they were used in biological sciences for many purposes, especially as models of biological membranes. Liposomes are aIso used as dmg delivery vehicles to increase the efficacy and specificity of drugs. Liposomes, because of their amptiiphilic nature, can serve as dm; camers for water soluble as well as lipid soluble drujs. Lipid soluble dmgs can be introduced into the bitayer, and water soluble dmgs can be encapsulated into the aqueous compartment of liposomes (Figure 2.1). The location of the drug in the liposomes depends on the partition coefficient of the drug between the lipid bilayer and aqueous phase (Margalit et al., 199 1). Liposomes have several characteristics which contnbute to their use as a vehicle for dru; delivery. Liposomes can protect the encapsulated dmg from metabolic degradation, increase the half-life of the drug, and reduce the systemic toxicity of the dmgs. They cm be used as sustained release vehicles, and also it is possible to target them to selected tissues or cells. Furthemore, they are biodegradable and biocompatible due to the nature of their composition. The main constituent of liposomes is phospholipids. PhosphoIipids are also the major lipid components of biological membranes. Phosphotipids consist of glycero-3- phosphate esterified to fatty acids at positions C 1 and C2 of glycerol and through a phosphoryl group to a group, X (Table 1.1). Phospholipids are arnphiphilic molecules since they have hydrophobic fatty acid 'tails' and polar phosphoryl-X heads. Phospholipids when hydrated adapt a bilayer structure because of their amphipathic structure. In the presence of water, the bilayer structure is the lowest energy macrornolecular organization (thermodynamically the most stable), in which the polar head groups orient toward the aqueous phase and the hydrophobic fatty acid tails are MLY

cholesterol

@ lipid soluble drugs

>-:.=:. 2.. * ...> -.*: 32: water soluble drugs

Figure 2.1 A schematic drawing of the structure of liposomes (Redrawn with permission from Foldvari, 1996). Table 2.1 Chernical structure of phosphclipids.

Narne of X-OH Formula of -X Name of phospholipid

Water -H Phosphatidic acid

E thanolamine -CH~CH~MI~+ Phosphatidyiethanolamine

Serine -CH~CH(NH~+)COO- Phosphatidyisenne

Gly cero 1 -CH2CH(OH)CH20H Phosp hatidylglycero 1

N-Glut-ethanolamine -CH2CHzNKCO(CH2)3COO- N-Glut-PE

sequestered fkom water. For preparing liposomes, phospholipids wiîh different head groups and fatty acid chah length are used. Cholesterol is often included in the liposome formulation. Cholesterol, because of its ngid steroid ring system which interferes with the motion of fatty acid tails, stabilizes the lipid bilayer and decreases the leakage of encapsulated drug. There are several methods available for the preparation of liposomes which have been reviewed in detail (New, 1992a; Woodle and Papahadjopoulos, 1989). The most commonly used method for the preparation of MLVs is the solvent evaporation method (Bangham et al., 1974). In this method, lipids are dissolved in organic solvent(s) in a round bottom flask. The soIvent(s) are removed by rotary evaporation under vacuum resulting in deposition of a thin lipid film on the walls. Then the lipid film is hydrated with an aqueous phase at a temperature higher dian the phase transition temperature (Tm). Upon an increase in temperature, phospholipids change fiom an ordered gel-like state to a liquid crystalline state. The temperature at which phospholipids changes f?om gel state to Iiquid crystalline is called the phase transition temperature. In the liquid crystalline state, both the lipid molecules as a whole and their hydrophobie fatty acid tails are highly mobile and capable of reorientation to forrn bilayer structures. Tm depends on the nature of fatty acid tails and polar head group. A decrease in the length of fatty acid tail and also unsaturation reduces the Tm. Difference in the polar head group has also effect on the Tm, for instance, the Tm of analogous O E phosphatidylcholine is 20°C less than analogous of phosp hatidylethanol- amines. The Tm of dipalmitoytphosphatidylcholine (DPPC) is 31°C,therefore, DPPC liposomes are stable in room temperature (Tyrrell et al., 1976). The reproducibility of the MLVs formed depends on the thickness of the lipid film, time and method of hydration (Weiner et al., 1989). SWs are usually prepared from MLVs by sonication (Saunders et al., 1962; Barenholz et al., 1977) or using French press (Hamilton et al., 1980). The required tirne for sonication depends on the type of sonication. A few minutes might be enough using a probe-type sonicator; however by bath-type sonicator a longer time, sometimes up to 2 hours is needed. Of course, bath type sonicators have some advantages: they produce less heat, the process cm be carried out in a closed container under nitrogen to prevent oxidation of phospholipids, and also the possibility of contamination by metals which may result from probe-type sonicator is not a problern @eamer and Uster, 1983). French press method involves high pressure extrusion of MLVs through a small orifice. The size of SWs depends on the applied pressure and the number of extrusions. Higher pressures result in liposomes with smaller sizes. The other procedure for the preparation of SUVs is the rapid injection of a phospholipid solution in ethanol into the aqueous phase and subsequent removal of the ethanol from the formed liposomes (Batzri and Kom, 1973). SWs can also be prepared by solubilizing the lipids in an aqueous solution that contains detersent. This technique is gentle and particularly good for the reconstitution of proteins into the liposome bilayer (Kagawa and Racjer, 1971; Helenius et al., 1977; Eidelman et al., 1984; Foldvmi et ai., 1990). The reverse-phase evaporation technique is usually used for the preparation of LUVs (Szoka and Paphadjopoulos, 1978). In REV the lipids are solubilized in orsanic solvent(s), mixed with aqueous phase and sonicated to produce an emulsion. Then, the organic solvent(s) are removed under the vacuum.

2.1.6.1.2 Liposome-ce11 interactions The general purpose of liposomal dnig targeting is to achieve specific interaction of the liposomes with a specific cells or tissue and then intemalization of the drus or the liposome containinj dmg by the cells. Whether a drug will reach its intracellular site of action is mainly dependent on the mechanism of intemalization, and also the physicochemical characteristics of the drug. There are four principal ways by which liposomes can interact with a celt: stable adsorption, fusion with the plasma membrane, endocytosis and lipid exchange behveen the liposome and ceIl membrane (Figure 2.2) (Pagano and Weinstein, 1978; Weinstein and Leseman, 1981; Foldvari et ai., 1992). Liposomes can adsorb to the surface of the cells without intemalization. In this situation, the intemalization of dmg depends on its release from liposome followed by its absorption through the ce11 membrane. In this system, the liposomes are only concentrating the dmg at the surface of the target cells. This approach has the disadvantage that the released dmg might diffuse quickly back to the systemic circulation and decrease the specificity of the dmg delivery systern (Blumenthal et al., 1992). In fusion, the liposome bilayer merses with the ce11 membrane and the liposome content is released into the . If the liposome is a multiiamellar one, the outmost bilayer of the liposome fuses with the ce11 membrane and the rest of the liposome and the encapsulated dmg are released into the cytoplasm. The liposome in the cytoplasm will possibly fuse with a lysosome. If the liposome is a unilamellar one, the only liposome bilayer rnerjes with the ce11 membrane and the encapsulated dmg is expelled into the cytoplasm. In the context of fusion, there is the possibility of dmg delivery to the cytoplasm by just brief touching of the liposome to the ceil membrane, without complete merging of liposome bilayer with the ce11 membrane, which is called "kissing" fusion. In the past there has been reports for the fusion of liposomes with Iipid exchange

rupture of vesicle .:> . -

pinocytosis nonspecific coated pit adsorptive pinocytosis

Figure 2.2 Mechanisms of interactions of liposomes with cells [Reproduced with permission from Foldvari et al., 19921. Tlipylipid transferred fiom liposomes to cells; Sb, liposomes bound to the ce11 surface; Shi, intact liposomes bound to the ce11 surface; DL, dmg to lipid ratio in liposomes; DLo, dmg to lipid ratio in liposomes originally; DI, drug intemalized by fusion or juxtapositional transfer; DL>dmg intemalized in fiee form by leakage of liposomes; M, mitochondnon; N, nucleus; Ly, lysosome; Gy Golgi apparatus. Any combination of the above interactions cm occur simultaneously during incubation of cells with liposomes. cells (Paphadjopoulos et al., 1974; Weinstein et al., 1978), however, it became clear that in these situations probably other mechanisms are involved in the reported delivsry of the drus into the cytoplasm (Szoka et al, 1980; Straubinger et al, 1983). There have been reports about designing fusogenic liposomes (Straubinger et al., 1985; Nayar and Schroits, 1985), however, almost al1 of them relied on acidic pH to tngger the Fusion process. Sufficiently acidic pH is not available extracellularly, and only endocytic vacuoles provide the acidic pH. Therefore, the liposome-encapsulated drug has first to be endocytosed to start a hsogenic process. Endocytosis is believed to be the most common mechanism by which liposomes are intemalized by cells. Liposomes can bind to the ceII surface by non-specific and specific interactions. Both interactions may result in endocytosis. In the specific interaction, usually a component of liposomes (e-g. mAb, glycoprotein, oligosacchande and etc.) is recognized by a ce11 surface receptor. The interaction tnggers receptor- mediated endocytosis via the clathrh coated pit pathway. The internalized liposome ends up in the lysosomal system through the endosome compartment where they may fuse with endocytic vacuoles. In the lysosome, the liposome phospholipids are desaded by the lysosomal acidic hydrolases and the encapsulated dru; will be released. if the dmg is resistant to the lysosomal enzymes and small enough, it may escape the lysosome and reach its site of action. Non-specific bindins of liposomes to the ce11 membranes cm lead to adsorptive pinocytosis. These adsorbed liposomes are then taken in by invagination of the ce11 membrane and produce intracellular vesicles. These vesicIes fuse and deliver their contents to the lysosome. Pinocytosis can also be mediated by specific interaction of a macromolecule reconstituted or linked to a liposome bilayer and a ce11 surface marker. This is a kind of receptor-mediated endocytosis and is called "piggy-back" endocytosis. Lipid exchange involves the exchange of lipid between the liposome bilayer and the cells. In this process the liposome content does not reach to the ce11 cytoplasm (Kew et al., 199%). 2.1.6.1.3 Targeting of liposomes Site specific drus delivery by liposomes can be achieved by manipularing the physical characteristics of liposomes, by linking a ligand to the surface of Liposomes and by reconstituting a ligand into the liposome bilayer. Liposomes have demonstrated considerable potential as a camer for the specific delivery of dnigs. However, one of the main drawbacks is that most liposomes afier intravenous injection are rapidly cleared from the blood by the phagocytic cells ot' RES. Tt has been found that altering the liposome surface by adding hydrophilic substituents, such as polyethylene glycol derivatives (PEG-liposomes), reduces RES uptake and prolongs the circulation of the liposomes in the blood (Yuda et al., 1996; Bedu Addo et al., 1996). PEGs are used in the liposome formulations as a cholesterol and phospholipid derivatives, or are directly linked to glycerol. PEG derivatives in any form prolong the circulation tirne of the liposomes, which depends on the MWt of PEG: 4800 > 2600 > 1700 > 800 (Yuda et al., 1996). The mechanism by which PEG- derivatives prolong the circulation time of liposomes is not hlly understood; however, Lasic et al. (1991) suggested that the highly hydrated groups of PEG on the surface of liposomes stencaily prevent the eiectrostatic and hydrophobic interactions of liposomes with macrophages. Due to the improved pharmacokinetic characteristics, PEG- liposomes have considerable potential as drus carriers. For instance, doxorubicin encapsulated in PEG-liposomes have significantly longer circulatinj half-life and a Lower volume of distribution compared to conventional (without PEG derivatives) liposomal doxonibicin and fiee doxorubicin (Gabizon and Martin, 1997). PEG- liposomes encapsulated with doxorubicin produced higher intratumoral drus concentration and better therapeutic responses in breast, prostate, pancreatic and ovarian cancer cornpared to conventional liposornal doxorubicin and fiee dmg. The adverse effects of doxorubicin were also fewer with PEG-liposomes. The better effect of PEG-liposomal doxorubicin is attnbuted to the nature of tumor vasculature. Tumor vasculature, due to the higher permeability of their endothelium and structural abnormalities associated with tumor angiogenesis, are more permeable than normal vasculature. This allows higher extravasation of the liposomes to the tumor (Gabizon and Martin, 1997). in physical manipulation, for instance, liposomes composed of lipids which have their transition temperature slightly above body temperature would be destabilized and release the encapsulated dru; upon temperature rise at a selected area of body. Chelvi and Ralhan (1997) showed that encapsulation of melp halan in thennosensitive liposomes potentiates its anticancer activity in murine rnelanoma wirh the aid of local hyperthermia. Thermosensitive liposomes containing chemotherapeutic agents (cisplatin) and local hyperthermia (41°C) has also been used for the hgdelivery to the brain tumor (Kakinuma et al., 1996). This treatment increased the concentration of cisplatin on the tumor compared to free drug and thermosensitive liposome containing cisplatin but without hyperthermia. Thermosensitive liposomes containing PEG- derivatives have been prepared to avoid RES uptake (Iga et al., 1991). Hyperthermia mediated targeted dmg delivery to the tumor using PEG-thermosensitive liposomes should be more useful than the conventional therrnosensitive liposomes, since they are circulating longer in the blood (Unezaki et al., 1994). The pH-sensitive liposomes are also an another example of physical manipulation of the liposomes for the purpose of dmg targeting. Since pH in the vicinity of some tumors is slightly acidic, it is possible to make liposomes that destabike at iow pH and release their contents faster (Yatvin et al., 1980). Slepushkin et al. (1997) have shown that pH sensitive liposomes containing PEG derivatives could deliver their content intracellularly and have a longer circulation time i,i vivo. Superoxide dismutase, which is an antioxidant, has been delivered to the pulmonary epithelium through pH sensitive liposomes to reduce lung damage due to the treatment with oxygen supplemenations (Briscoe et al., 1995). Liposomes have recently been investigated as a vehicle for the delivery of genes or antisense oligonucleotides to mammalian cells (Li et al., 1996). Positively charged liposomes bind to the negatively charged DNA, providing transfection for many cell types. Positively charged liposomes have also been used as a vehicle for DNA vaccination (Gregoriadis et al., 1997). When naked plasmid DNA is injected intrarnusculariy, following the uptake of DNA by the muscle celIs, the antigen is expressed on the ce11 surface and released to the extracellular regions. The antigen can then lead to both humoral and cell-mediated immunity. The disadvantages of the injection of naked DNA is that most of the DNA cannot be taken up by the cells and the DNA is attacked by nucleases present in the extracellular fluid. Therefore, a high dose of DNA is required. Using positively char~edliposomes as a delivery system for DNA could circumvent these disadvantages. Administration of DNA in a liposome formulation facilitates its uptake and protects the DNA from the effect of nucleases. Positively charsed lipids are used for the preparation of the positively charged liposomes. It has been shown that positively charged lipids are toxic for the phagocytic cells (Filion and Phillips, 1997a). The positively charged lipids inhibit the synthesis of pro-inflammatory mediators, nihic oxide and turnor necrosis factor-a, in the activated macrophages. It has been also shown that the positively charged lipids have an anti- inflammatory effect that is mediated by the inhibition of C (Filion and Phillips, 1997b). The positively charged lipids should therefore be used carefully as a carrier for DNA delivery, since they are toxic and posses anti-inflarnmatory activity. Liposomes has been shown to function as an efficient immunoadjuvant in inducing immune responses to proteins (Phillips et al., 1996; Baca-Estrada et al., 1997; Nakanishi et al., 1997). Phillips et al. (1996) showed that antibody responses to the protein and peptide antigens encapsulated in Liposomes depends on the phospholipid composition of the liposomes. They also showed that for inducing immune response liposomal incorporation of the antigens is necessary. By encapsulating the protein antigen and cytokines in liposomes Baca-Estrada et al. (1997) influenced the development of the immune response. They showed that by utilizing cytokines along with antigen it is possible to get immune response with different antigen-specific Th subsets. Nakanishi et al (1997) compared the immunoadjuvant activity of three different kinds of liposomes encapsulated with soluble proteins. They showed that the positively chaqed liposomes are taken up by macrophages more efficiently than the negatively charged and neutral liposomes. The immune responses to positively charged liposomes containing soluble chicken egg albumin were also more potent than the negatively charged and neutral liposomes. They concluded that the presence of positive charges in the liposome surface enhances the immunoadjuvancy of the liposomes. Liposomes have been targeted to a specific organ, tissue or ce11 population by attachent of ligands such as antibodies, peptides and carbohydrates. The ligands are specific for a determinant on the appropnate cells. These liposomes are usually internaiized into the cells by receptor-mediated endocytosis. However, the intemalization is not always guaranteed. In this situation, the liposomes remain on ce11 surface untiI destroyed and gradualIy release their contents. htibodies and their Fab Ea3gnents due to their high specificity have been intensely investigated as a ligand for targeting liposomes (immunoliposomes) (Park et al., 1997). The conventional immunoliposomes containing anticancer dmjs were unsuccessful in the treatment of cancer, since they were taken up rapidly by the cells of RES. Further there was a limited availability of specific tumor targeting antibodies. By the development of stencally stabilized liposomes using PEG-derivatives and finding more specific tumor targeting antibodies, the above problems began to be solved in the fom of irnmurioIiposornes with higher therapeutic index. An irnrnunoliposorne containing adriamycin showed greater anti-turnor activity cornpared to free dmg against pancreatic cancer due to enhanced tumor accessibility (Akaishi et al., 1995). The intratumor concentration of the adriamycin from immunoliposomes was signi ficantly higher then the liposomal (without antibody) adriamycin and free drug. Moradpour et al. (1997) conjugated mAb AF20 to liposomes ta specifically target the liposomes to human hepatocellular carcinoma cells. The mAb AF20 specifically binds to a surface glycoprotein which is highly expressed on hurnan hepatocel1ula.r carcinoma cell lines and other hurnan cancer ce11 lines. The interaction of AFZO-immunoliposomes with different cancer ceII lines was 5-200 times greater than liposomes conjugated with an irrelevant antibody and liposome alone. Phillips and Tsoukas (1990) specifically targeted the CD~+cells in human blood using an anti-Leu3A (CD4) conjugated- liposome. They prepared hvo types of iiposomes, a conventional and a stencally stabilized liposome preparation. The conventional liposomes were rapidly cleared up from the mice circulation after intravenous injection; however, stencally stabilized liposomes remained in the circulation longer than 5 hours. Conventional liposomes were binding to monocytes but no t to lymphocyte. The stencally stabi Iized liposomes did not interact with either monocytes or lymphocytes; however, they were binding to both lymphocytes and monocytes after conjugation with anti-Leu3A antibody. Phi llips and Tsoukas (1990) concluded that the stencally stabilized liposomes conjugated to anti-Leu3A antibody rnight be suitable for targetinj of anti-viral drugs in HIV-infzcted patients. Fab fia3gnents of' antibodies could be more usehl as a targeting ligand for liposome, since they have the same specificity of their parent antibody and due to their smaller sizes they rnight reach to their site of action easier. Mmyama et al. (1997) showed that immunoliposomes containing PEG-derivatives conjugated to the Fab fragment of antibody evaded RES uptake and had an increased circulation tirne. These immunoliposomes had an enhanced intratumor concentration in the solid tumor compared to the irnmunoliposomes conjugated to intact antibody. Sterically stabiked liposomes containing PEG-derivative was conjugated to the Fab fragment of a mAb (anti-HEW) raised asainst the extracellular dornain of glycoprotein p 1 8sHERZin order to target the liposomes to human breast cancer cells (Kirpotin et al., 1997). Glycoprotein 185HEu, which is a member of epidermal growth factor receptor family, is encoded by HER2heu protooncogene. This protein, which is a receptor tyrosine kinase, is highly expressed on a variety of cancers, such as breast, lung, and ovarian carcinoma. This rnAb specifically mediated the binding of liposomes to the hurnan breast cancer cells. The pharmacokinetics of anti-HERZ immuno liposomes was determined afier intravenous injection in rats (Park et al., 1997). They showed that the half life of immunoliposomes (empty or loaded with doxorubicin) is 10 hours, while the half life of the free drug was only few minutes and the antibody itself was 1-2 hours. The anti-HERî imrnunoliposomes were Iocalized in nude mice bearing subcutaneous HE= tumor xenografts. For this purpose, colloidal jold-loaded anti-HER2 immunoliposomes were injected intravenously to the mice. After 24 hours the mice were sacrificed and the tumors were excised. The presence of imrnunoliposomes was then shown in the cytoplasm of individual tumor cells. The antitumor efficacy of doxorubicin-Ioaded anti-HER2 imrnunoliposomes were also determined. For this purpose the liposome preparations and free drug were injected intravenously to the nude mice bezing turnor xenograft and the tumor growth suppression was measured. The antitumor activity of these immunoliposomes was significantly greater than free dmg or PEG-liposomes without the antibody (Park et al., 1997). Immunoliposomes have also been used for the targeting of drugs for the treatment of diseases other than cancer. For exarnple, liposomes conjugated with antibodies that recognize E- (an endothelial-specific ce11 surface molecule) have been tarseted to vascular endothelia1 cells for specific dmg delivery to cardiovascular system (Sprag; et al., 1997). Liposomes have been also reconstitutec! with proteins, mostly trammembrane, for drus delivery purposes (Foldvari et al., 1991). In this regard, viral glycoproteins have been reconstituted into liposomes and evaluated for their ability to mediate fusion for drug delivery purposes (Loyter and Volsky, 1982). Adhesive protein derived synthetic short peptides have been also used as a ligand for targeting liposomes. The advantages of utilizing synthetic peptides instead of whole proteins as tarseting ligands for liposomes is that they might be less irnmunogenic, their production is easier and allow 1on;er circulation of the liposomes in the blood Stream, and because of smaller sizes they might also diffuse into the tissues easier. Slepushkin et al. (1996) showed that arnong different CD4-derived peptides, a syEk!ir peptide fiorn the complementarity determining region 2 (CDR-2)-like domair., when linked to the liposome surface, can mediate specific binding of liposomes to the human imrnunodeficiency virus type-1 @IV-1) -infected cells. It has already been shown when the recombinant transmembrane CD4 molecules are reconstituted into the liposome bilayer, or recombinant soluble CD3 molecules are covalently linked to the liposome surface, specific binding of liposomes to HIV-1-infected cells could be achieved (Flasher et al., 1994). The peptides were selected fiom the CDR-2 regions of CD4, since this part of CD4 is considered a possible site of interaction with HIV envelope protein gp120 (Jarneson et al., 1988 and Brand et al., 1995). Peptides were also selected fkom the CDR-3 region of CD4, based on their antiviral activity (Slepushkin et al., 1996). However, peptide derived fiom CDR-3 did not mediate the binding of liposomes to the HIV-1-infected cells. The extent of binding of CDR-2 peptide-liposomes to the HIV- l -infected cells was comparable with soluble recombinant CD4-coupled liposomes (Slepushkin et al., 1996). They then concluded that CDR-2 derived peptide might be a suitable ligand for targeting liposomes containing anti-HN dmgs to HIV-1-infected cells. Banerjee et al. (1998) used a chernotactic peptide (N-formyl-Met-Leu-Phe) to target liposomes to macrophages. They showed that the uptake of peptide-linked liposomes is fast and concentration dependent. The anti-leishrnaniasis activity of primaquine loaded peptide-liposomes \vas sigificantly greater than the primaquine Ioaded Iiposomes or the free dmg. They then concluded that this peptide-linked liposome might be a suitable delivery system for the targeting of dmgs to macrophages. In another study, liposomes conjugated with macrophage activator tetrapeptide (tufisin) have been used as a carrier for rifampin in the treatment of tuberculosis in mice (Aganval et al., 1994). They showed that the antitubercular activity of rifampin loaded peptide-liposome was sigificantly greater than the nfampin loaded liposomes (without the peptide) or the free dnig. Gyongyossy- Issa et al. (1998) linked the RGD-containing peptides to the liposome surface through disulfide linkage. The resulting lipopeptides (liposomes linked to the peptides) were homogeneous and the RGD-containing peptides had a functional orientation on the surface of liposomes mediating biochemical interaction with their receptor, the glycoprotein 1%-IIIa. The phage-displayed peptides have been used to show the potential of peptides for targeting purposes into selected tissues. With this respect, the phase mimics the targeting of Iiposomes. The advantage of phage over Liposome is that the phage is easily detectable in tissues by utilizing immunohistochemistry or by counting the infectious phage particles. Pasqualini et al. (1997) targeted phage displayinj an RGD-containing peptide to the turnor in the mice. The vasculanire of the tumon undergoes continuous angiogenesis. The angiogenic endothelium expresses ce11 surface molecules that are specific for tumors. One of these molecules is avB3 integrin (Conforti et al., 1992; Brooks et al., 1994). Many , especially the av integins, bind to the RGD sequence. Pasqualini et al. (1997) therefore evaluated the possibility of targeting an RGD containing peptide with a high affinity for aV integrins to tumor in the mice. This RGD-containing peptide (CDCRGDCFC) has a cyclic conformation with hvo disulfide bonds and it has been shown that it is very specific for aV integrins (Koivunen et al., 1995). Pasqualini et al. (1997) intravenously injected the phage-dispiaying RGD- containing peptide and control phage to tumor-bearing mice and determined the amount of phage in the tumor and normal tissue. They showed that the aV-directed RGD-phage concentrated specifically in turnor. They recovered sigificantly more aV-directed RGD-phage than control phage korn tumors. They conchded that the UV-directed RGD containing peptide might be suitable ligand for tarjeting to tumon.

2.1.6.1.4 Linking of proteins to liposomes Proteins are covalently linked to the surface of liposomes through reactive functional groups on the head groups of phospholipids. The amines, sulfhydryls, aldehydes and carboxylates on the protein molecules are usually targeted for linking to the Liposomes. There are many ways to link the proteins to the surface of Liposomes and some of them are discussed briefly.

A) Linking throtigh the NHS ester of palmitic acid. The N-hydroxysuccinimide ester denvatives of palmitic acid react with amine soups on the protein molecule, producing stable amide bond linkages. Then the complex of lipid- protein is used for the preparation of liposomes (Huang et al., 1980). B) Littking throrgh carbodiinzide cotipling to phospliaridyletliartolantine (PE) containirig [iposornes. The carboxylate groups of proteins are fint activated with EDC, then the activated ester intermediate can react with amine group of PE in the liposome surface to fom amide linkages (Martin et al., 1990).

C) Lirzh-ing throrrgh glzifaraldehyde coupling to PE. Liposomes containing FE are coupled to glutaraldehyde, which is a homobifunctional cross-linker. The carboxylate jroup of glutaraldehyde is activated using EDC and NHS, then the activated ester intemediate can react with amine goups of proteins to form amide linkages (Bogdanov et al., 1988). D) Linkingthroz~ghperiodnteo.ridaiio~zofl~ydroxylic-contairzir~giipidfollorvedby redzxtive amfiration. Hydrosylic-containing Iipid cornponents, such as phospha- tidylglycerol, in the liposome bilayer is oxidized with sodium periodate to produce reactive aldehyde groups on the liposome surface. In the presence of a reducing agent such as cyanoborohydnde, the labile Schiff base linkages which are formed between the amine goups of proteins and aldehydes, are reduced to form stable secondary amine bonds (Heath et aI., 198 1). E) Linking throzrgh SPDP cotqding to PE. The PE residues in the surface of liposome bilayer are coupled to N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), which is a heterobifunctional cross-linker. The produced pyridyl- disulfide derivatives of the liposome surface react with sulfhydryls of proteins to form disulfide Iinkazes (Carlsson et al., 1978).

F) Linking rhrozrgh biotirtylated PE. Liposomes containing biotinylated PE are first incubated with avidin and then biotinyiated proteins. Avidin, which has four identical subunits, acts as a bridge and connects the proteins and liposome to each another (Plant et al., 1989).

2.2 Adhesion rnoIecuIes Cells are adhered to other cells and also to the extracellular rnatrix through adhesion molecules. The processes of adhesion are needed for cells to interact and to migrate to their place of destination. They have roles in rnany biological processes, like embryonic development, irnmunology, and hematopoiesis. In general, four groups of cell adhesion molecules have been descnbed: imrnunoglobuIin superfamily, integins, , and (Obrink, 199 1; Dianzani and Malavasi, 1995).

2.2.1 Irnmunoglobulin superfamily The IgSF molecules consist of a diverse collection of glycoproteins that share a

common structural ' homology unit. The soluble and ce11 surface molecules of the immune systern and also molecules that are participating in the process of cell-ce11 recognition and embryonic development are the members of IgSF (Figure 2.3). This group of molecules has become known as the IgSF because immunogIobuIins were the first members of this farnily to be sequenced (Williams, 1985; Hunkapitler and Hood, 1986; Buck, 1992). Most of the IgSF molecules are ceIl surface proteins and have an extracellular, a hydrophobic transmembrane, and an intracellular region, however, some of them are secreted proteins or attached to the ce11 surface via a glycophospholipid anchor. The criteria for inclusion of molecules in the IgSF is the presence of an immunoglobulin CO4

CD2 CD37 Cû7 CO 8 CO28 THY-1 Blast-1 (CTLA- 4) aP

Figure 2.3 Selected members of imrnunoglobulin superfamily Feproduced with permission fiom Williams and Barclay, 19881. IgM, immunoglobulin M; V, Ig variable-like domain; C, Ig constant-like domain; H, primordial (C2) Ig-like domain; TcR, T-ce11 receptor; MHC, major histcompatibilty cornplex; FcRy, Fc receptor y; ICAM-1, intercellualr adhesion molecule- 1; Po, Po protein; p-IgR, poly-Ig receptor; N-CAM, neural ce11 adhesion molecule-1; PDGFR, platelet- derived growth factor receptor; CEA, carcino-embryonic antigen. homolojy unit or domain in the extracellular region (Williams, 1987). h individual IQSF molecule rnay have one or more imrnunoglobulin homology domains. In general, each homology dornain has about 70-1 10 amino acids organized into approximately 7-9 B-strands. The f3-strands have 5-10 amino acids and are placed in an antiparallel rnanner and making nvo O-sheets. Benveen the sheets, there is a hydrophobic interaction due to the hydrophobic side chains of amino acids. These are facing one another in the interior of the molecule and thereby exposing the hydrophilic side chains to the external environment. in addition, there is a conserved disulfide bond in the hornology domain of IgSF which further stabilizes the interaction behveen the sheets (Williams and Barclay, 198s). The homology domains of IgSF are classified as V- or C-like on the basis of the closest homology to the V- or C-domain of the imrnunoglobulin molecule. The V and C homology units differ in that the V domain contains 9 B-strands, whereas the C domain contains only 7 B-strands. Because of these two extra B-strands, the V-domain has an extra loop in the middle of the domain. There is a third type of Ig homology domain that is called C2 or H domain. C2 or H domain is sirniIar in Iength to C dornain, however, it has sequence typical of both V and C domains. In al1 interactions in which IgSF are participating the homolojy domain represents specific determinants for recognition reaction on the faces of 8-sheets or at the bends between the fi-strands. Most of the time the determinant is protein in nature, but it is possible that it may be a carbohydrate structure which is a dominant feature in most of the IgSF molecules (Williams and Barclay, 1988). Conserved patterns of sequence are seen arnong the IgSF homology domains, especially behveen D-strands. Sorne of these conserved sequences are common to V and C domains while others are specific to either the V or C domain type. The transmembrane sequences and cytoplasrnic domains of IgSF molecule show a great diversity. IgSF molecules usually have one hydrophobic transmembrane sequence. The cytoplasrnic domains of IgSF (may Vary from 3-543 amino acids) are mostly completely unrelated without any apparent homology. Their roles are usually unknown (Williams and Barclay, 1988). In general, the IgSF molecules have adhesion or binding functions that trigger a subsequent event at the ce11 surface. The interactions of IgSF receptors with counter receptors, which can be other IgSF molecule or other receptor families such as the integins and selectins, have been classified generally as either homophilic (protein A binds protein A) or heterophilic (protein A binds protein B) (Anderson et al., 1988). This interaction offen occus between molecules on an opposed membrane surface, with the exception of antibodies. Several members of IgSF have more than one irnrnunoglobulin homoiogy domain and it has been shown that some of these molecules have more than one counter receptor which interact with different domains of molecules. These multiple homology domains rnay be due to the creation of multiple binding sites. Each of these dornains may be considered as a unit binding site which, depending on its particular sequences, may interact with other domains or various family rnembers (Diamond et al., 199 1).

2.2.1.1 Po protein Myelin is a rnultilamellar membrane formed in both the CNS (central nervous system) and the PNS as an extension of the myelinating cell's plasma membrane which then wraps around axons, insulates them, and accounts functionally for fast nerve conduction (Kirschner and Blaurock, 1992). This axonal insulation is crucial to the correct and efficient functioning of the nervous system. The most abundant protein of PNS myelin is a glycoprotein termed Po (Roomi et al,. 1978). This protein is restricted to PNS myelin in mammals, but is also found in the CNS in some fish (Saavedra et al., 1989). It is beIieved that Po protein functions as an adhesion molecule and is responsible for the compact nature of the peripheral myelin. It has been shown by homology search that Po protein is a member of the irnmunoglobulin superfarnily (Uyemura et al., 1987; Lemke et al., 1988), and perhaps the closest relative of the ancestral gene for this whole family of molecules which are all believed to be involved in adhesion /recognition processes (Williams and Barclay, 1988). 2.2.1.1.1 The structure of Poprotein The prirnary structure of Po protein has been detennined in rat (Lemke and .&xel, 1985), chicken (Barbu, 1990), human (Hayasaka et al., I991), uid shark (Saavedra et al., 1989) indirectly by the nucleotide sequencing of their cDNAs, and in bovine directly by protein chernical methods (Sakamoto et al., 1987). These studies have revealed that the Po protein is a primitive rnember of the irnmunoglobulin superfarnily and has been highly conserved during evolution. The polypeptide chah of Po has 220 amino acid residues which make up three different regions: an extracellular arnino- terminal domain with a single V-like Ig hornology domain (residue 1 to 121), a single transmembrane sequence (125 to 150), and an intracellular carboxyl-terminal domain (151 to 220). The analysis of cDNA genes which encode Po has revealed the presence of a signal peptide with 29 amino acids residue at the begimin; of the protein. Al1 the proteins which are destined for insertion in the ce11 membrane (transmembrane proteins) are synthesized with ribosomes associated with the endoplasmic reticulum (rough endoplasmic reticulum, RER). Al1 these proteins are synthesized with a leadins 13-36 residue signal peptide that helps the protein molecule pass the RER membrane. The signal peptide is separated fkom the main Po protein after entering into the rough endoplasmic reticulum. The hydropathy profiles of Po proteins show that the region of residues 125 to 150 is highly hydrophobic (9 Leu or Ile, 8 Val, 4 Gly, 3 Ala; 2 Tyr and 1 Ser, according to the chicken Po sequence, Barbu, 1990) suggesting a transmembrane sqgnent (Sakamoto et al., 1987). The carboxyl-terminal domain which contains nurnerous basic arnino acids (23 out of 70 arnino acids, based on chicken Po sequence, Barbu, 1990) forrns a basic intraceltular domain. Po protein has a molecular weight of 28-30 kDa, about 6% of which is carbohydrate (Roomi et al., 1978) attached via a single N-linkase at Asp 93 (Lernke and Axel, 1985; Sakamoto et al., 1987). Sorne of the sugar residues of this N-Iinked glycoside are sulfated (Matthieu et al., 1975; Poduslo, 1990; Lemieux et al., 1995). Heterogeneity in the glycosylation pattern of Po has been reported. This includes the presence or absence of fucose, sialic acid, galactose, and also CO-existenceof complex and high-mannose type Po glycoproteins (Poduslo, 1984). Po protein is also palmitoylated (Agrawal et al., 1983; Lemieux et al., 1995), and phosphorylated (Singh and Spritz, 1976; Brunden and Poduslo, 1987; Suniki et al., 1990; Hilmi et al., 1993; Lemieux et al-, 1995; Iyer et al., 1996). The palmitoylation site of Po glycoprotein has been deterrnined in rat (Bizzozero et al., 1994). This is the Cys 153 which is located at the junction of the putative transmembrane and cytoplasmic domain. This Cys has been conserved in other species such as human, bovine, mice, and chicken. It is suggested that this site of esterification may allow interaction bettveen the fatty acids and the adjacent membrane lipids, thus perhaps holding the domains of Po in a functionally active conformation (Sakamoto et al., 1986). The cytoplasmic domain of Po protein is phosphorylated (Suzuki et al., 1990). Serine residues at amino acids 181, 204, and 214 have been shown to undergo phosphorylation and dephosp horylation in mature myelin. It is suggested this could be associated with the temporal changes of myelin structure (Brunden and Poduslo, 1987) and this process rnay be a result of Po protein's involvement in signal transduction (Fiibin and Tennekoon, 1993).

The secondary structure of Po protein waç first predicted, based on the method of Chou and Fasman (1978), fiom bovine prirnaiy amino acid sequence (Uyemura et al., 1987). According to this rnethod Po protein has seven helical regions, eleven B- strand region and eight turns. The extracellular dornain is 0-strand dominant, while the intracellular dornain is mostly helical. Later, the secondary structure of extracellular domain of Poprotein was predicted using a computerized rnolecular modeling prograrn (Wells et al., 1993). According to this molecular modeling prediction, there are 10 13- strands in the extracellular domain. The structure of the B-strands is similar to that of immunoglobulin V domain consisting of hvo O-sheets with antiparallel O-strands. This secondary structure is further stabilized by a conserved disulfide bond between Cys 21 and Cys 98. By site directed mutagenesis on the Cys 21 of Po protein, it has been shown that formation of a disulfide bond in the irnrnunoglobulin-like domain of Po protein is essential for its adhesion (Zhang and Filbin, 1994). The secondary structure of the whole Po protein has not been directly detemined using X-ray crystallography since it is a transmembrane protein and it is not possible to crystallize it. However, Shapiro et al. (1996) has made and crystallized just the extracelluIar part of the Po protein. The X- ray cryçtallography showed that the secondary structure of extracellular domain of Po protein is similar to V domain of immunoglobulins. There are ten P strands that make two p sheets. The X-ray crystallogaphy studies confirmed the prediction of the rnolecular rnodeling program.

2.2.1.1.2 The function of Po protein In the peripheral nervous system Schwann cells produce the myelin membranes. Approximately 70% of the dry weight of the myelin sheath is composed of lipid with the rernaining 30% being protein. When Schwann cells wrap around the axon, the cytoplasmic sides of its membranes as well as the extracellular sides corne into close contact. The close apposition of the extracellular side of membrane is defined as the minor dense line (intraperiod iine); the attachent of the cytoplasmic sides of membrane by the exclusion of the cytoplasm of the Schwann ce11 is defined as the major dense line (Figure U)(Kirschner and Blaurock, 1992). The proteins of rnyelin, especially Po protein, are believed to play critical roles in the formation and maintenance of the highly organized, multilayered rnyelin sheath. Po protein may constitute more than 80% of the protein in the mature myelin membranes (Doyle and Coiman, 1993). The importance of the presence of Po protein in the peripheral nerve myelin has been shown in mice with knocked out Po gene (Gieses et al., 1992; Martini et al., 1995a and 1995b). The peripheral nerve of these mice had extensive myelin degeneration and there were only remnants of loose membranes that poorly ivrapped around some axons. Point mutations in the Po protein gene have been associated with Charcot-Marie-Tooth (CMT), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH) (PateI and Lupski, 1994; Wamer et al., 1996) which are hereditary demyeiinating disorders. One of the proposed functions for Po protein is the involvement of its extracellular domain in adhesion and thus in holding together the intrapenod line of myelin. Studies utilizing cells in culture that express transfected Po cDNA constructs, have demonstrated (Filbin et al., 1990; and D7Urso et al., 1990) that Po protein is capable of behaving like a homophilic adhesion molecule by its extracellular domain. The extracellular domain of Po protein interacts with Po protein (extracellular domain) Major dense Line Minor dense line (intraperiod line) (period lk) Axolemma ,a / i ./ Axon /

Schwann ce11 cyropl~m

Node of Ranvier-. -.

Schwann ceIl

Figure 2.4 The penpheral nerve myelin peproduced with permission fiom Cedric, 1984). A: Longlitudinal section of a mature myelinated nerve. B: Cross section of a mature myelinated nerve (A = kuon, ip = intraperiod line, md = major dense line). in the opposinj Schwann cells and keepins the cells in close and uniforni contact with each other. According to recent results of X-ray crystalIography of the estracelluIar domain of Po protein, a new model for the organization of Po extracellular domain and its homophilic interactions has been suggested (Shapiro et al., 1996; Choe, 1996; Baringa, 1996). Accordin; to this model, the extracellular dornains of Po protein exist on the ce11 surface of Schwann cells as tetrarners. Each tetramer interacts with four other tetramers protruding kom the opposing Schwann ce11 membrane, linking the membrane tightly tosether. They have also suggested that the extracellular domain of Po protein directly mediates adhesion through a direct interaction of tryptophan in the extracellular domain with the opposing ce11 membrane. Both the homophilic interactions of tetramers of Po protein and direct tryptophan-membrane interactions contribute to the maintaining of a constant intemembrane spacing. Glycosylation of Po protein in the extracellular domain has an important role in the homophilic adhesion. It was dernonstrated that Po protein in the homophilic pair must be glycosylated for adhesion to take place (Grif5th et al., 1992; Filbin and Temekoon, 1993). Furthermore, the adhesive properties of Po protein depend not only on the existence of N-Iink glycosylation, but also on the type of oligosacchande residue. It was shown that expression of the cornplex form of Po glycoprotein greatly increases the adhesiveness of the cells, whereas expression of the high-mannose form of Po glycoprotein does not (Filbin and Temekoon, 199 1). In order to identify regions of Po protein which are functional in the Po-Po binding mechanism, Yazaki et al. (1992) prepared synthetic oligopeptides from different region of Po protein and perfonned cornpetition analysis of the aggregation of Po protein using C6 glioma cells transfected with PocDNA. Yazaki et al. (1 992) showed that only one peptide from the extracellular region (residue 90-96, YSDNGTF) ivhich includes the glycoside attachment site, significantly inhibits ce11 aggregation by about 50%. Yazaki et al. (1992) also showed a from the same area (residue 91- 95) with N-glycoside, obtained fkorn bovine Po, inhibits ce11 aggregation by 85%. In another similar study usin; transfected CHO cells expressing Po protein, it has been shown that the same peptide from the extracellular domain (residue 91-95, SDNGT) can block ceil aggregation (Zhang et al., 1996). The cytoplasmic dornain of Po protein contains many basic amino acids, and it has been show that it interacts with phospholipids in the opposing membrane (Ding and Brunden, 1994). Accordingly, Ding and Bnuiden (1994) suggested that the intracellular domain of Po protein may also have adhesive propenies, and then by interacting with the opposing membrane it is responsible for the formation and maintenance of the myelin major dense line. The probability of homophilic interaction behveen intracellular domain of Po protein is low, because of the repulsive interaction among the positively charsed amino acids. Furthemore, the presence of the intracellular domain of Po protein is required for the homophilic interactions of its extracellular domain (Wons and Filbin, 1994), since the CHO cells transfected with only the extracellular domain had little aggregation cornpared to the CHO cells that were transfected with whole Pa protein.

2.2.1.2 IntercelIular adhesion molecule-1 ICAM-1 (CD54) is a transmembrane glycoprotein with an extracellular resion containing five Ig-Iike domains (Staunton et al., 1988). The prirnary structure of ICAM-l protein has been detemined in murine (Siu et al., 1989), rat (Kita et al., 1992), canine (Manning et al., 1995), and human (Staunton et al., 1988) by nucleotide sequencing of their cDNA. There is limited homology (5565%) between the sequences of different species. The polypeptide chain of human ICAiM-1 has 505 amino acid residues which make up three different regions: the extracellular region (453 residues) containing five Ig-like domain; a single hydrophobie transmembrane domain (24 residues), and a positively charged cytoplasmic domain (28 residues). The polypeptide molecular rnass of ECAM-1 is 60 kDa. There are eight N-linked glycosyiation sites in ICAM-1 that are differentially glycosylated depending on ce11 type, resulting in a glycoprotein with a molecular mass between 80-1 14 kDa. The secondary structure of the Ig-domains is similar to the H (C2) Ig-like domain; each Ig domain consists of 90- 100 arnino acids that make two P-sheets with antiparallel P-strands.

ICAM-1 binds ligands LFA-1 (CD1 laCD18, a P2 integrin), (Marlin and Springer, l987), Mac- 1 (CD 1 1bKD18, a P2 integrin), Diarnond et al., 1990). CD43 (sialophorin), (Rosenstein et al., 1991), and serves as a receptor for soluble fibrinogen (Languino, 1993), hyaluronan (an extracellular matrïx component), (McCourt, 1994), rhinovinises (Staunton et al., 1989a), and Plasmodizirn fakipnnrm infected erythroc>.tes (Berendt, et al., 1989). LFA-1 is expressed on al1 leukocytes. The first amino terminal Ig-like dornain of ICAM-1 is the binding site for LFA-1 (Staunton et al., 1990). Mac-1 is also expressed on leukocytes but only a subpopulation of Mac-1 molecules rnediate effective interaction with ICAM- 1 @iarnond and Sprinjer, 1993). The third Ig-like domain of ICAM-1 is the bindin~site for Mac-1 (Diarnond et al., 199 1). LFA-1 and Mac-1 can probably bind to ICAM-1 at the same time (Staunton et al., 1985). The usual integrin ligands contain Arg-Gly-Asp (RGD) sequence that mediates bindinj of integrin to the ligand. ICAM-1 does not contain RGD sequence and its binding to inte,g-in is not mediated by this sequence (Marlin and Springer, L987; Staunton et al., 1990). CD43 is expressed on leukocytes and platelets, however, its contribution in ICAiM-1 mediated adhesion has not been charactenzed. The binding site of fibrinogen on [CAM-1 is Iocated in the first Ig-like domain, but it is distinct from the LFA-1- binding site (Languino et al., 1993). The binding site of rhinoviruses on ICAM-1 partially overlaps with the binding site for LFA-I (Staunton et al, 1990; Giranda, et al., 1990). Plasnzodirrnz fnlcipam infected erythrocytes bind to the first Ig-like domain of ICAM-1 but this is also distinct £Yom the site of binding of LFA-1 (Ockenhouse et al., 1997). The binding site of LFA- I on ICAM-1 is hijhly conserved arnong species. This may indicate that LFA-I is the most important ligand of ICAM-1 (Manning et al., 1995; Staunton et al., 1988; Ockenhouse et al., 1992). Functionally, [CAM- 1 has an important role in imrnunological responses. Mice deficient in ICAM-1 develop normally, however, their immune and inflarnmatory responses are impaired (Sligh et al., 1993). These mice have increased blood neutrophil counts; however, activation and migration of leukocytes to sites of inflammation were diminished. These mice were îlso tesistant to a lethal septic shock induced by bacterial lipopolysaccharide, indicating a pathological role of ICAM-1 in septic shock (XLIet al., 1991). It is generally believed that the interaction of ICAM-1 with LFA-1 mediates adhesion of leukocytes with the target cells in the inflammatory and immune responses (Springer, 1990). Increasing expression of ICAM-1 has been reported in various cancers, especially malignant melanomas (Alromonte et al., 1993). Interaction of LFA- 1IICAi.i.- 1 on tumor cells recruits leukocytes and also mediates T ce11 mediated killing of the cells (Webb et al., 1990; Pandolfi et al., 1991). There is a direct relationship benveen the ievel of ICAM-1 expression and tumor progression (metastasis) (Kageshita et al., 1993). This is in contrast with the function of ICPuM-1 that mediates the killinp of tumor cells. The possible reason for this contrast is the SICAV-1. It has been shown (Giaraui et al., 1992) that melanoma cells shed ICM-1 in vitro. Indeed, ICAiil-1 is present in the senun of patients with malignant melanornas and its concentration in the semm is increased with disease progression (Kageshita et al., 1993). The SICAM-1 may compete with the interaction of LFA-1 with rn1CA.M-I and prevent attack of cytotoxic T cells to melanoma cells. In rnaligant cells induction of ICAM-1 on tumor cells would be therapeutically more usehl, because it will enhance T ce11 rnediated tumor killing. The other therapeutic options could be the specific dnig delivery to ICAii-1 expressing cancer cells (for example in metastatic melanomas). In this respect, an anti- ICAM-1 immunotoxin (a mAb to ICM-I conjugated to ricin A-chah) has been show to be cytotoxic to human myeloma ce11 lines (Huang, 1993). In an alternative approach, mAbs to ICAM-1 have been conjugated to liposomes for targeting purposes (Bloemen et al., 1995).

2.2.2 Integrins Inte,gins are a large farnily of cell surface receptors that rnediate interaction with other cells, extracellular matnx components, and also soluble ligands (Hynes, 1992). They have important roles in embryogenesis, tumor jrowth and metastasis, imrnunology, platelet aggregation and wound healing. Structurall y, they are heterodimers consisting of an a-chain (120-180 kDa) and a P-chah (90-1 10 ma). Both the a and p chains are transmembrane glycoproteins with a short intracellular domain (about 40-60 amino acids residues). The a and P chains are associated to each other noncovalently. There are at lest 16 kinds of a-chains and 8 P-chains. Each P-chain can interact with multiple a-chains, forming integrins that bind different ligands. They are subclassified based on their P-chahs: pl integins, which bind to extracellular matrix cornponents such as fibronectin, laminin, and collagen; P? integins, which are present on leukocytes, mediate cell-ceIl interactions; and p3 inteagins, which bind soluble Iisands like Eibrinogen. Ligand binding sites have been identified in both u and the p chains (Ilogg et al., 1994). The binding of integrins is divalent cation dependent. They may need activation in order to bind their ligands. The cytoplasrnic dornain of both u and P chains can interact with the cytoskeIeton. The presence of an intracellular domain affects the adhesiveness of integins (Hayashi, et aI., 1990; Hibbs et al., 199 1).

LXZ.1 Leu kocyte function associated antigen -1

LFA-1 (CD 1laKD18) is a ce11 surface glycoprotein which belongs to the P3 family of integrins (Springer, 1990; Pardi et al., 1992). It contains noncovalently associated a (CD 1 1a) and p (CD 18) chains (Figure 2.5). The human u chain cDNA has been cloned (Law et al., 1987; Larson, et al., 1989). The a chain is composed of 1145 amino acids with a polypeptide molecular mass of 126 kDa. There are 12 N-linked glycosylation sites in a chain that cm result in a molecular mass of 170-1 85 kDa. The a chain contains a large extracelluIar region, one transmernbrane sequence (23 amino acids, mainly hydrophobic), and a relatively short cytoplasrnic region (56 amino acids). The extracellular region of the a chain contains seven homologous dornains (1-VII). Domains V-VI1 contain characteristic divalent cation binding sites (EF-hand-like) similar to ca2*-binding proteins such as calmodulin (Arnaout, 1990a). Behveen domains II and III of the cc chain there is an "inserted" domain or "1" dornain of approximately 200 amino acids. The "1" domain is homologous to the "A" domains in von Willebrandt factor, a large (up to approximately 10' kDa) rnultimeric ptasma glycoprotein that mediates adhesion of platelets to collagen and other subendothelial matrices (Sadler, 1991). It has been shown that the "I" domain is important in ligand binding (Landis et al., 1993, 1994; Randi and Hogg, 1994).

The humm LFA-1 P chain (CD18)is composed of 747 amino acids (Law et ai., 1987) with a polypeptide molecular mass of 82.6 kDa. It contains 6 N-linked gIycosy1ation sites that result in a molecular masses of 95- 105 kDa. The LFA- 1 P chain Conserved Cys-nch reg ion reg ion

CelI membrane

B I subunit a subunit

Figure 2.5 Schematic representation of leukocyte fimction-associated antigen-1 [Reproduced with permission fiom Hogg et al., 19941. (a) Schematic representation of LFA-1 a and P subunits, @) Hypothetical mode1 of LFA-1 structure, (+) indicahg the putative divalent cation binding sites, filled boxes indicating ligand binding sites in a subunit and open boxes indicating ligand buidhg sites in P subunit. has three regions: an extracellular region, a single transmembrane region (23 amino acids, mainly hydrophobic) and an intracellular region. The intracellular domain of the p chain is smatl (46 arnino acids) and contains a Tyr and several Ser and Thr residues that serve as phosphorylation sites. Upon activation, the cytoplasmic dornain of the P chain is phosphorylated (Arnaout, 1990b). It has been shown that the intracellular domain of the p chah is important in the regulation of adhesion of LFA-1 to IC.2,bI-1 (Hibbs et al., 199 1). The extracellular region contains a Cys-nch dornain near the cd1 membrane which is highly conserved in a11 P chains. The Cys-rich dornain is compnsed of four repeated units of approximately 40 amino acids. In the N-terminal side of the Cys-rich domain there is another conserved region of 250 amino acids. This rejion contains the RGD-binding site (Hogg et al., 1994). LFA-1 is expressed on al1 leukocytes and by binding to ligand ICAiM-1 provides an adhesion force behveen leukocytes and their target cells (Marlin and Springer, 1987; Staunton et al., 1988). The LFA-l/ICAiM- 1 mediated adhesion is important in a variety of leukocyte functions including T and B ce11 proliferation, T ce11 mediated cytotoxicity and interaction of leukocytes with other tissues including the endothelium (Dustin and Springer, 1991). The adhesiveness of LFA-1 for ICAM- 1 is transiently increased upon T ce11 activation. In addition to ICAM-1, LFA-1 has been shown to bind ICAM-2 (CD102, Staunton et al., 1989b, de Fougerolles et al., 199L), ICAM-3 (CDSO, de Fougerolles and Spnnger, 1992; de FougerolIes et al., 1993), and ICAM-4 (Bailly et al., 1994). Al1 of these fCAMs are glycoproteins and belong to the IgSF molecules. ICAM-2 and ICAM-4 contain hvo Ig-like domains in their extracellular region, however, ICAM-3 has five Ig-like domains. The intensity of expression of ICAM- on resting endothelial cells is greater than ICAM-1, therefore, it may have a major function in the binding of leukocytes to resting endothelial cells (Gahmberg et ai., 1997). ICAM-3 is highly expressed on resting leukocytes and it has an important role in the onset of immune responses (de Fougerolles et al., 1993). [CAM-3, unlike ICAM-I and ICAM-2, is not expressed on endothelial cells (Gahmberg et al., 1997). ICAM-4 is only expressed on red blood cells, and its function is unknown (Gahmberg et al., 1997). Mutations in die /32 chah of inte,grhs may result in a lack of association with u chains. In this situation, the unassociated u and p chains cannot be intracellularly transported to the ce11 surface and results in a deficiency of P? integrin. This is a hereditary disease and is called leukocyte adhesion molecules deficiency type I (LAD- 1) (Arnaout et al., 1990b). These patients are imrnunodeficient and display recurrent Me-threatening bactenal infections and poor wound healing. LAD-1 due to P3 integin deficiency usually results in death in infancy. The ligand binding sites in LFA-L have been identified in bot11 the u and P chains (Hojg et al., 1994). In the a chain, domains V-VI contain an ICAQI-1 binding site (Stanley et al., 1994). It has been shown that the "1" dornain in the a chain has also a binding site for ICAM- 1 and ICAM-3 (Landis et al., 1993 and 1994; Randi and Hogg, 1994). Removal of this domain from LFA-I causes a substantial decrease in the interaction of LFA-I/IChM-1.Randi and Hogg (1994) showed that an isolated recombinant form of the "Y' domain can directly bind to soluble ICIZIM-1 and can also block LFA- 1 dependent adhesion of T cells to ICAM- 1. The third ligand-binding site of LFA-1 is on the P chain. The conserved region of the P chain contains the RGD- binding site (Hogg et al., 1994).

2.2.3 Selectins Selectins are carbohydrate binding proteins that mediate calcium dependent heterotypic interactions between blood cells and endothelial cells during lymphocyte homing and leukocyte adhesion (Lasky, 1992). They also have an important role in the recruitrnent of leukocytes into sites of inflammation. There are three kinds of selectins: L (1eukocyte)-selectin, P (platelet)-selectin, and E (endothe1ium)-selectin. Structurally, they are transmembrane proteins and consist of the following domains starting Frorn the aminoterminal: aminoteminal calcium-dependent lectin domain which mediates specific interactions with carbohydrate ligands on the opposing ce11 membrane; epidermai growth factor (EGF)-like domain; varying number of short consensus repeat which is cysteine-rich and is homologous to the complementary regulatory protein; transmembrane domain; and short cytoplasmic domain. Both lectin and the EGF domain are needed for cell adhesion; however, only the lectin domain is directly involved in binding to carbohydrate ligands. Shon consensus repeats contribute to the flexibility of the molecule and increase the binding afgi-ity. The cytoplasmic domain of selectins is usually required for adhesion and by binding to regulates the function.

2.3.4 Cadherins Cadherins are a farnily of calcium dependent adhesion molecules that mediate homotypic interactions (Takaichi, 1991). They are expressed by al1 cells that form solid tumors and have important roles in tissue rnorphogenesis and differentiation and tumor metastasis. Four subcIasses have been described: E (epithe1ium)-cadherins, P @lacental)-cadherins, N (neural)-cadherins, and L-CAM (liver ce11 adhesion molecule). Cadherins are transmembrane glycoproteins. The extracellular domain consists of 3- j repeats of about t 10 amino acid residues, which are not hornologous to the Ig-domains. The first N-terminal repeat participates directly in the homotypic interaction and the repeats of 1-3 have a putative calcium-binding site. The cytoplasrnic dornain is shon and highly conserved. It has potential phosphorylation sites and interacts with the cytoskeleton. The presence of the cytoplasmic domain is important for binding. CHAPTER THREE

3.1 Purification of Poprotein Po protein was purified from partially purified Po protein fraction III (FIII), by preparative polyacrylamide gel electrophoresis (PAGE) or reverse-phase hijh performance liquid chrornatography (RP-HPLC). Po protein FI11 fraction was previously extracted and partially puriFIed kom the myelin, which was isolated and purified from sciatic nemes of 7-1 1 day old White Leghorn chicks, by the solvent extraction and colurnn chromatographie procedures( Foldvari et al., 1990).

3.1.1 furification of Po protein by preparative polyacryIamide gel electrophoresis The purification of Po protein from partiaIly purified Po protein FIII fraction using preparative PAGE was canied out as descnbed by Foldvari et al., 1990. The FI11 Fraction of Po protein, 4 mg, was dissolved in 1600 pL 0.01 EVI phosphate bufFer (pH 7.2) containing 1% SDS and 2% 2-rnercaptoethanol, then 400 pL 50% glycerol was added. In order to visualize the protein bands after electrophoresis, 0.25 mg FI11 fraction was dissolved in 100 pL of the above buffer and derivatized with fluorescarnine as follows. To the sample, borate buffer 40 pL (0.2 M, pH 9.0), distilled water 60 PL,and fluorescarnine 100 pL (5 mg/mL in DMSO) were added and kept in the dark for 30 minutes at room temperaiure. Then bromophenol-blue ciye 133 PL (25 mL of brornophenol-blue 0.5% in distilled water rnixed with 75 mL of glycerol) and phosphate buffer 567 pL (0.01 M, pH 7.2) were added. The preparative PAGE was camed out in a Protean II Xi (Bio-Rad Laboratones Ltd. Mississuga, ON) apparatus. The gel consisted of a 10% (wh) acrylamide containing Nnning gel and a 3% (w/v) acrylamide containing stacking gel. In the stacking gel 40 mm wide wells were prepared using a 3- well comb, 0.3 cm thickness. One of the wells \vas loaded with 1 rnL denvatized sample, which also contained the trackins dye, the other bvo weIIs ivere loaded with FI11 fraction (2 mg/mL/well). The electrophoresis buffer was 25 mM Tris, 192 n-&f

3ulycine, 0.1% SDS, pH 8.3. Electrophoresis was initially camed out at 50 rn.A constant current per slab gel. When the bromophenol-blue tracking dye in the derivatized protein sample reached the running gel the current was changed to 79 mA per slab gel and electrophoresis was carried out for 4-5 hrs. After electrophoresis, the Po protein band was located using an ultraviolet lamp. The position of the Po protein in the other two wells was determined based on the position of the derivatized Po protein. The located Po protein bands were cut out of the polyacrylarnide gel. The gel slices containing the Po protein were rninced using a ,olass rod and extracted with 3 mL 0.1% Triton X-100 solution in warer by stimng for 3-4 hrs. The protein solution was then separated from gel by filtering through a Millipore filter unit (0.22 pm) The extraction was repeated two more times using 0.1% Triton X-100 in water, 2 mL each tirne. The protein soIutions were pooled and the concentration was determined according to Lowry et al. (195 1).

3.1.2 Purification of Po protein by reversed-phase high performance liquid chromatography The purification of Po protein from partially purifieci Po protein FI11 fraction using RP-HPLC was camed out as described by Brunden et al. (1987). The FI11 fraction of Po protein, 1.5 mg, was dissolved in a minimum (2 mL) volume of acetic acid/water (111). This sohbilized protein was used for RP-HPLC employing a Beckman System Gold V8 10 data systern/controller equipped with Beckman System Gold programmable solvent module 126 and a Beckman System GoId diode array detector module 168. The column used for separation was a Vydac proteidpeptide reverse phase column 214TP, protein C4. The mobile phase consisted of acetic acidlwater (111 v1v) (solution A), with a gradient of acetic acid/2-propanol (111 v/v) (solution B) as follows: 0-2 min 100% A; 2-4 min 0-10% B; 4-29 min 10-35% i3; 29-35.5 min 35-100% B; 35-50 min 200% B. Flow rate was 0.3 dhinand the eluting components were monitored at 280 m. The Po protein fiactions collected fiom RE'-HPLC were freezed-dried under vacuum at - 50 OC and dissolved in 0.1% Triton X-100 solution, or were concentrated under nitrogen in a 40°C water bath using a Turbo Vap LV evaporator (Zyrnark) and then dialyzed against 0.1% Tnton X-IO0 solution four times, each time 12 hrs, using a Spectrapore dialyzin~membrane with a molecular weight cut off 6000-8000. The fractions were then analyzed by analytical sodium dodecyl sulfate polyacrylamide gel electropnoresis (SDS-PAGE).

3.1.3 ~naiyticalpolyacrylamide gel electropboretic analysis of protein samples The analytical PAGE was carried out in a Mini-Protean II (Bio-Rad Laboratories Ltd. Mississuga, ON) apparatus. The gel consisted of a 10% (wh) acs.lamide containing running gel and a 3% (4v) acrylamide containing stacking gel. The gel thickness was 1 mm. The electrophoresis buffer was 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3. Electrophoresis was camed out at 150 v constant voltage for 45 min. After the electrophoresis the gels were stained for protein with Coomassie- blue R-250 (Pierce Chernical Comp., Rockford, IL) or silver. Coomassie-blue staining procedure: AAer the electrophoresis the sel was transferred to 25% isopropanol/lO% acetic acid/65% water for fixation, hvo times, 30 minutes each tirne. Then, the gel was stained with 0.05% Coomassie-blue in 25% isopropanol and 10% acetic acid for 10 minutes. The stain was removed and the gel was nnçed a few times with distilled water. Then the gel was destained with a destaining solution consisting 10% acetic acid and 10% isopropanol in water. The destaining process was repeated several times until the protein bands were revealed. Silver staining procedure: Afier the electrophoresis the gel was transferred to j0% methanol/lo% acetic acid for fixation ovemight. Then the gel was placed into distilled water for at ieast 30 min with three changes of water. The gel was stained in solution C for 15 min, which was prepared by titrating solution B (a mixture of 21 mL 0.36% NaOH and 1.4 mL concentrated NHJOH solution) with solution A (0.8 g silver nitrate in 4.0 mL of distilled water) dropwise with constant vigorous stimng allowinj the brown precipitate to clear and diIuted to 100 mL. After rinsing the gel nvice with distilled water and soaking for 2 min with gentle shaking, it was placed into the developer solution (ImL of 1% citnc acid and 0.1 mL of 38% formaldehyde mixed and diluted to 200 rnL with distilled water) until the bands appeared (usually 5-10 min). Finally the gel was washed with distilled water and transferred to 5% acetic acid to stop the staining process.

3.2 Preparation of liposomes

3.2.1 Reconstitution of Po protein into lipid bilayer Unilamellar liposomes containing Po protein @roteoliposomes) were prepared by the detergent solubilization method (Foldvari et al., 1990). The lipid phase consistin; of L-a-dipalrnitoylphosphatidylcholine (DPPC, Sigma Chemical Company, St. Louis, MO), ~-rneth~l-~~]DPPC (1 rnCi/rnL, TRK 673, Amersham Canada Ltd., Oakville, ON) or [N-methyl-'"CI DPPC (0.05 mCi/mL, Du Pont Canada Ltd., Mississauga, ON) and cholester01 (chol) (10:l mot= ratio of total DPPC and chol) was dissolved in chloroform:methanol; 2: 1, v/v in a round-bottom flask. The solvent was removed by rotary evaporation (Buchii RE 11IRotavapor, Buchii Laboratoriums, AG FlawiVSheiz, Switzerland) resulting in the deposition of a thin lipid film on the walls. This lipid film was then freeze-dned (Labconco freeze-dryer apparatus, Kansas City, MO) overnight to ensure total removal of the solvent. The lipid film was solubilized in the aqueous phase which contained Tnton X-100 (1% dv), the appropriate arnount of Po protein (400 pg / mL of liposome, the initial Lipid:protein ratio was 20: 1, w/w), NaCL solution (90 mg / 10 mL, 100 pL for 1 mL liposome) and water. Then, the detergent solution of lipids and protein (detergent and protein in 150 mM NaCI solution) was sonicated for 15 minutes in a Branson bath sonicator to compIete the soktbilization. To remove the detergent, the detergent:lipid:protein mixture was treated (three times, first overnight then 2h each time) with 300 mg/mL of moist SM-2 Bio-Beads (spherical, macroreticular divinyl benzene-styrene copolymers; 20-50 mesh; capacity 70 mg Tnton X- 1001100 mg beads, from Bio-Rad Laboratories, Richnond, CA). After removal of the detergent, the liposome preparation was separated from the Bio-Beads using a syringe and centrifuged at 100,000 x g for Ih and the pellet was resuspended in 0.9% NaCl solution. To prepare the control liposomes, the sarne procedure as described for proteoliposomes was followed escept the Po protein was ornitted. To prepare the Glycophorin A-liposomes. the sarne proced~irewas also used except Po protein was replaced by Glycophorin r\ (SiDrna, G-9511, fiorn human blood), and the initial 1ipid:protein ratio was 40: 1, w/w.

3.2.2 Preparation of liposomes containing N-Glut-PE Uniiamellar liposomes containing DPPC, N-glutaryl-phosphatidylethanolamine (N-%lut-PE, Avanti Polar Lipids, Inc, Alabaster, AL), and cholesterol (9:3:1, molar ratio) were prepared by the detersent solubilization method (Foldvari et al., 1990). The lipid phase consisting of DPPC, p-methyl-3~]DPPC (for quantitative detemination of cellular uptake), N-glut-PE and cholesterol was dissolved chloroform:methanol; 2: 1, V/Vin a round-bottom flask. The solvent was removed by rotary evaporation resulting in the deposition of a thin lipid film on the walls. This lipid film was then freeze-dned ovemight to ensure total removal of the solvent. The lipid film was solubilized in the aqueous phase which contained Tnton X-100 (1% v/v) in MesBS (5 mM 2-[N- MorpholinoJethanesulfonic acid, Sigma M5287; 0.15 M NaCl; pH 5.5). Then, the detergent solution of lipids was sonicated for 15 minutes in a Branson bath sonicator to complete the solubilization. To remove the detergent, the detergent: Iipid mixture was treated (three tirnes, first overnight then 2h each time) with 300 mgmL of moist SM-? B io-Beads. After removal of the detergent, the liposome preparation was separated from the Bio-Beads and centrifûged at 100,000 x g for Ih and the pellet was resuspended in MesB S.

3.2.3 Lin king of peptides to liposomes containing N-Glu t-PE The linking of peptides to liposomes containin; N-GIut-PE was camed out as described by Bogdanov et al., 1988. For the covalent attachent of the peptides, 200 pL of liposomes (containing 1.44 pmole N-glut-PE) in MesBS was activated by 150 pL of 0.25 M EDC, 1-ethyl-3-(3-dimethy1aminopropyl)carbodiimide (Sigma E-7750). in water and 150 pL of N-hydroxysulfosuccinimide (sulfo-NHS, Pierce No. 245 10. Rockford, IL) for 10 min at room temperature with mixing. The activated liposomes were incubated with BBS solution (50 mM sodium borate, NarB~Oi.10H20, Si,oma S- 9640; 0.1 M NaCl; pH 8.0) containing 0.96 prnole peptides for 2.5 hours at room temperature with mixing. The liposomes were then separated from the unbound

peptides by centrifugation at 100,000 x g for lh and washed three times with BBS (200 PL each time), and resuspended in 200 pL 0.9% NaCl solution. For control liposomes, liposomes containin; N-glut-PE were treated similarly but without the peptides. The amount of linked peptide to the liposomes was determined by capillary electrophoresis (CE) analysis of supernatants after washes with BBS. The CE employing a Beckman System Gold V8 10 data systern/controller equipped with a Beckman P/ACE System 5500 and a Beckman P/ACE diode array detector or ultra violet (W) detector. The column used for separation was a Bechan uncoated capillary tubing, 57 cm (sometimes 37 and 27 cm) length and 75 pm diameter. The buffer was sodium borate pH 8.75 (0.6% sodium borate.10 Hz0 and 0.4% boric acid). The samples were injected by pressure injection method for 5 seconds and the components were separated under 20 (sometimes 10) KV voltage at 23°C. The eluting components were monitored at 214 m.

3.2.4 Negative staining of liposomes Po liposomes, peptide-liposomes and control liposomes (10 PL) were rnixed with 10 pl of 1% sodium phosphotungstate pH 7 for 10-15 seconds. Then they were pipetted ont0 carbon and formvar coated copper grids. ARer the excess stain was removed by filter paper, the samples were viewed and photographed with a CiM 10 Philips transmission electron microscope at an accelerating voltage of 80 KV.

3.3 Maintenance of ce11 culture systerns 3.3.1 Human M21 Melanoma cells Human M21 melanoma cells (provided by Dr T. Ghose, Dalhousie University) were grown in RPMI-1640 culture medium (88%) supplemented with 10% heat inactivated fetal calf serurn (FCS), 1% v/v antibiotics (antibiotic antimycotic solution with 10,000 nits penicillin, 10 mg streptomycin and 25 p; amphotericin B per mL in

0.9% sodium chloride; 100 x, SIGMA A9909) and 1% v/v L-glutamine (200 mbl, 100 x, GIBCO 25030-081) in a humidified incubator at 37"C, 5% COy95% air atmosphere. For detaching the cells Versenz solution (ethylenediarnine tetraacetic acid disodium salt 0.2 g, KCl 0.2 g, NaCl 8.0 g, NaHe04 1.15 g, glucose 0.2 3, per LOO0 mL distilled water) was used.

3.3.2 Human A-375 melanoma cells Human A-375 (ATCC CRL 1619, Rockville, IMD) melanoma cells were gown in Dulbecco's Modified Eagle Medium (DMEM, 88%) supplemented with 10% heat inactivated FCS, 1% v/v antibiotics (SIGMA A9909) and L-glutamine (GIBCO BRL 25030-081) in a humidified incubator at 37OC, 5% C02/95% air atrnosphere. For detaching the cells Versene solution was used.

3.3.3 Human MeM 50-10 melanoma cells Human MeM 50-10 melanoma cells (provided by Dr R.S. Kerbel, Sumybrook Health Science Centre, Toronto, ON) were grown in Dulbecco's Modified EajIe Medium (DMEM, 88%) supplemented with 10% heat inactivated FCS and 1% v/v antibiotics (SIGMA A9909) and 1% v/v L-glutamine (GIBCO BRL 25030-081) in a humidified incubator at 37OC, 5% C02/95% air atmosphere. For detaching the cells Versene soIution was used.

3.3.4 CHO-X2 celis Chinese hamster ovary cells expressing full length of wild type rat Po protein (provided by Dr. M.T. Filbin, Hunter College of the City University of New York, New York, NY) (CHO-XZ cells) were grown in DMEM 87% supplemented with 10% dialyzed FCS, 1% v/v antibiotics (SIGMA A9909), 1% vlv L-glutamine (GIBCO BRL 25030-OH), 1% PGT (L-proline 200 mg, glycine 37.5 mg, and thymidine 3.65 mg in 50 mL of DMEM), and 0.5 pM of methotrexate in a humidified incubator at 37"C, 5% COd95% air atmosphere. For detaching the cells 0.0025% trypsin in Versene solution for 2-3 min in room temperature \vas used.

3.3.5 Jurkat T ceI1s Jurkat T cells (ATCC TB 152, acute T cell leukemia, human) were grou-n in RPMI-1640 culture medium (88%) supplemented with 10% heat inactivated fetal calf serurn (FCS), 1% v/v antibiotics (SIGMA A9909) and 1% v/v L-glutamine in (GIBCO 25030-081) a humidified incubator at 37"C, 5% C02/95% air atmosphere. These cells grow in suspension.

3.3.6 HUT 78 T ceils HUT 78 T cells (ATCC TE3 161, cutaneous T ce11 lymphoma, human) were grown in WMI- 1640 culture medium (88%) supplemented with 10% heat inactivated fetal caIf semm (FCS), 1% v/v antibiotics (SIGMA A9909) and 1% v/v L-glutamine (GIBCO 25030-081) in a humidified incubator at 37OC, 5% CO7/95%- air atmosphere. These celk gow in suspension.

3.4 Adult normal human epidermal keratinocyte in ce11 culture

3.4.1 IsoIation and growth of keratinocytes Epidemal keratinocytes were isolated from the human brevt or abdominal skin after cosrnetic surgery (skins were provided by the Royal University Hospital, University of Saskatchewan, Saskatoon, SK) (Figure 3.1). Immediately after operation, the skin biopsies were placed in Hank's balanced solution containing penicillin / streptomycin / arnphotericin-B (SIGMA A9909) (Hank's-PSA) and stored at 4OC. The skin biopsies were used to initiate cultures within 48 h. Al1 steps descnbed below were performed under sterile conditions. The subcutaneous connective tissue of the skins was trimmed fiom the dermal surface under Hank's-PSA. Keeping the skin submerged in Hank's-PSA, they were cut into pieces of approximately 5-mm squares. For separating the epidermis fiom the demis five different procedures were used as follows: Digest on

rn Separate Epidermis from Derrnis

Apply Trypsin-EDTA on the

1 Release Individual Cells 1 I by Pipetting I J

1 Pool Supernatant I

Centrifuge,

Figure 3.1 Flow diagram of procedures for establishment of normal human epidemal keratinocytes ftom breast skin. hcubating the tissue pieces with 625 U/mL collagenase type III (GIBCO, Cat. No. 17102-021, hom Clostridium histolyticum) in defined semm fiee keratinocyte groowth medium (K-SFM, GIBCO, Cat. No. 17005-015) Tor minimum 2 hours at 37°C.

Incubating the tissue pieces with a 625 U/mL collagenase type III (GIBCO, Cat. No. 17102-02 1, from Clostridium histolyticum) in K-SFM, over night at 4°C. Incubating the tissue pieces in a solution of 0.3% trypsin (GIBCO, Cat. No. 25095-019), 0.1% EDTA in a calcium and magnesium fiee Hank's solution (CLW-Hank's)for 3 hours at 37°C. Incubatins the tissue pieces on the top of filter papes moistened with a solution of 1% trypsin (GIBCO, Cat- No. 25095-019), 0.025% EDTA in CMF-Hank's for 3 hours at 37OC. Incubating the tissue pieces on the top of filter papers moistened with a solution of 1% trypsin (SIGMA, T8642, Type XIII, fiom bovine pancreas), 0.025% EDTA in a CMF-Hank's for 3 hours at 37OC. Among the different methods, only treatment with trypsin (SIGMA, T8642, Type XIII, from bovine pancreas) gave good results for separating the epidermis fiom demis. The separated epidermis was placed in Hank's solution at room temperature. Hank's solution was aspirated off, and replaced with a solution of 0.025% trypsin and 0.01% EDTA in CMF-Hank's solution. To release individual cells, the epidemal fiagments were pipetted up and down for 10 min. The ce11 suspension was withdrawn fiom the remaininj tissue f?aaments, the trypsin was neutralized by fetal bovine semm

(FBS) and centrifuged at 200 x g for 8 min. The semm solution was aspirated from the ce11 pellet, and resuspended in a low calcium (0.1 mM CaClz), K-SFM medium. Cells were counted with a hemocytometer. Viability ofcells was tested by trypan blue. Cells were inoculated into plastic tissue culture flasks containing K-SFM medium, which were gassed with 5% CO2 at 37°C for 30 minutes, at a density of 25,000 cells/cm2 and incubated in an atrnosphere of 95% air, 5% C02, at 37OC. After 3 days the medium was gently removed and replaced with fkesh K-SFM. Six days afier inoculation, the medium was removed, the cultures were washed twice with Hank's solution, and fiesh medium was added. This procedure was repeated every 3 days. It took 15 days for the culture to be confluent. For the subsequent passages, the cells were detached by usinp 0.025% trypsin, 0.0 1% EDTA (5 min, 37°C)-

3.4.2 Frozen storage of keratinocytes Upon reaching confluency, the flasks of hurnan epidermal keratinocytes were washed twice with Hank's solution. For detaching the cells, 1 to 2 mL of 0.025% trypsin, 0.01 % EDTA in CMF-Hank's were added to the flasks and incubated for 5-10 minutes at 37°C. The cells were then detached by washing with PBS and the trypsin was neutralized by 10% solution of FBS in RPlMI-1640. The cells were centrifuged at

200 x g for 8 minutes. The cells were resuspended in K-SFM medium at a ce11

concentration of 4 x 106 cells / rnL and kept on ice for 10 minutes. An equal voiume of cold (4°C) K-SFM medium containing 20% dimethyl sulfoxide (DMSO) and 20% FBS waç slowly (dropwise) added to the cell suspension. The cells were aliquoted into cryovials (2 x 106 cells / mL / vial) and kept ovemight at -20 OC, and nt -70 OC another ovemight. The vials were then ~ansferredto a liquid nitrogen tank.

3.4.3 Phase contrast microscopy of keratinocytes The keratinocyte cultures were and at different growth stages usin; an Olympus phase contrast microscope.

3.4.4 Transmission electron rnicroscopy of keratinocytes Secondary cultures of human epidermal keratinocytes were gown in serum free keratinocytes growth medium in a humidified incubator at 37"C, 5% C02/95% air atmosphere. After 90% confluence, the cells were collected by detaching with 0.015% trypsin, 0.01% EDTA (5 min, 37OC). The trypsin was neutralized by 10% FBS in WMI- 1640 medium and washed twice with warmed RPMI-1640 medium-without semm in order to remove the proteins of serum. Then the cells were fixed for 2 hrs at 4'C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. The cells were washed three tirnes (10 min each tirnes, 4°C) in the sarne buffer, then postfixed in 2% osmium tetroaide in distilled water for 1 hour at 4°C. The cells were washed twice (10 min each times, 4°C) in the water, and stainzd with 2% uranyl acetate in water for 2 hrs at CC. Again, the cells were washed twice and dehydrared as follows: 50% acetone, roorn temperarure, 5 min; 75% acetone, room temperature, 5 min; 90% acetone, room temperature, 5 min; 100% acetone, room temperature, three times, 5 min. Then, the cells were embedded in low-viscosity epoxy resin [ERL 4206 (vinyl cyciohexene dioxide) 5.0 g; DER 736 (apolyglycol diepoxide 3 -03; NSA (nonenyl succinic anhydride) 13 .Os; DMAE (2- dimethylaminoethanol) 0.7gl as follows: acetonekesin 1: 1 I hour; acetonekesin 1:2 1hour; resin 5 hrs; resin over-night; resin and leave to polymerize for 21h at 70°C. Thin sections of about 70-100 nrn (silver-gold sections) were cut on a Sorvall Porter-Blum MT-2 Ultramicrotome with glass knives (LKB Knikmaker Type 7801 B). Sections were picked up on 300 rn mesh copper gnds and stained with 2% aqueous solution uranyl acetate for 30 min and ~e~nold'slead citrate for 5-10 min. Then the cells were viewed and photographed with a CM 10 Philips transmission electron microscope at an accelerating voltage of 80 KV. The negatives were developed on Ilford Multigrade Paper with Ilford Multigrade filter.

3.5 Liposome-ce11 interaction assays 3.5.1 Quantitative determination of the cellular uptake of Po liposomes in humao MZI, A-375, MeM 50-10 melanoma celIs and CHO-X2 cells

The cellular uptake of liposomes was determined as descnbed by Foldvari et al. (199 1). Before the incubation with liposomes, the cells were collected when, after subcultivation, they reached a confluent rnonolayer. Cells were washed with 0.0 1 M PBS, and the viability of cells was tested by trypan blue dye. Cells were counted using a

Coulter counter and/or hemocytometer and a ce11 suspension of 5 x 1o6 cellsl2 mL medium with 10% serum was prepared and plated into each weli of six-well plates (35mm diameter). The cells were allowed to attach to the bottom of the dish for 12 h.

Liposome preparations (proteoliposomes and control liposomes) containing 3~-~~~~ lipids were added at a maximum volume of 10-15 pL to the wells. The final lipid concentration of the incubation medium was 54.5 pM which was constant in al1 experiments. Cells were incubated ai 37°C for 1 hou. After incubation, the medium (with the unbound liposomes) was completely removed, the cells were covered immediately with EDTA solution, then collected into conical tubes, centrifuged at 300 x

3D for 3 min and washed with 10 mL 0.01 M PBS three times. The ce11 pellet was dissolved in the scintillation liquid and transferred into vials for the determination of radioactivity. The amount of liposomal lipid associated with cells was determined by liquid scintillation counting using a Beckrnan LS 3801 liquid scintillation counter. Ready Value (Beckman) scintillation liquid was used for al1 sarnples.

3.5.1.1. Quantitative determination of the cellular uptake of Po liposomes in human M21 cells in the presence of anti-chick Po Fab

Quantitative determination of the cellular uptake of liposomes was camed out as explained in the previous section (3.5.1), except 30 min before adding the liposomes, the cells were incubated with anti-chick Po Fab (fragment of antigen binding was prepared fkom anti-chick Po antibody) at a concentration of 90 pg / 5 x 106 cells.

3.5.1.2 Quantitative determination of the cellular uptake of liposomes in human M21 cells in the presence or absence of Po-peptides Three peptides were synthesized (Alberta Peptide Institute, Edmonton, m) representing the extracellular (Po-peptide-1: residues 90-96: Tyr-Thr-Asp-Asn-Gly-Thr- Phe), the membrane spanning (Po-peptide-2: residues 140- 146: Val-Ala-Leu-Leu-Val- Ala-Val) and intraceildar domain (Po-peptide-3: residues 201-307: Lys-Ala-Ala-Ala- Glu-Lys-Lys). Quantitative detemination of the cellular uptake of liposomes was carried out as explained in section 3.5.1, except 30 min before adding the liposomes, the cells were incubated with peptides at a concentration of 90 pg/5 x 106cells.

3.5.2 Quantitative determination of the cellular uptake of peptide-liposomes in human M21, A-375 and MeM 50-10 rnelanoma cells Quantitative determination of cellular uptake of peptide-liposomes in human M21, A-375, MeM j0-10 melanoma celis was carried out as explained in section 3.5-1 with the following changes. Liposome preparations (peptide-liposomes and control liposomeç) containing ~H-DPPClipids were added at a maximum volume of 10-15 uL

to the 6-well plates containhg 5 x 1o6 celld:! mL medium. The final total lipid concentration of the incubation medium wz 78.7 pM lipid which was constant in al1 experiments. C&j were incubated at 37OC for 1 hour. Afier incubation, the medium (with the unbound liposomes) was completely removed. The cells washed inside the 6- well plate wiih 2 mL 0.01 M PBS three times to remove the unbound liposomes (the peptide-linked liposomes and control liposomes conta in in^ N-glut-PE due to their

surface charges were precipitatinp with centrifugation at 300 x g for 3 min, and hence the radioactive readings for both control liposomes and peptide-liposomes were higher than usual, therefore, it was decided to wash the cells in wells with PBS to remove the unbound liposomes before collecting them with EDTA solution). Then cells were

covered with EDTA solution, collected into conical tubes, centrifuged at 300 x g for 3 min and washed one more time with 10 mL 0.01 M PBS. The cell pellet was dissolved in the scintillation liquid and aansferred into vials for the determination of radioactivity. The amount of liposomal lipid associated with cells was detemined by liquid scintillation counting using a Bechan LS 3801 liquid scintillation counter.

3.5.3 Quantitative determination of the cellular uptake of peptide-liposomes by human keratinocytes Quantitative determination of the cellular uptake of peptide-liposomes into human keratinocytes was carried out as explained in the previous section (33.1) with the following changes. The human keratinocytes were seeded and grown in triplicate in

Bat-bottom 6-well plates at 4 x 10' cells per well. The confluent monolayers were used for the cell-liposome adhesion assay. In some expenments, keratinocyte monolayers were incubated with IFN-y 500 IU/mL for 36 houn. On the day of liposome-ce11 adhesion assay, the supernatant of human keratinocytes was removed, the monolayers were washed once with K-SFM 2 mL per well. K-SFM (1 mL per well) was then added to each well and the keratinocyte monolayers were incubated with liposome preparations (peptide-liposomes and control liposomes) containing 3~ - DPPC lipids (at a maximum volume of LO-15 pL per well) at 37°C for 1 hou. The final total lipid concentration of the incubation medium was 78.7 pM per well which was constant in al1 expenments. After incubation, the medium (with the unbound liposomes) was completely rernoved, the cells were washed with 2 mL 0.01 M PBS three times in the wells. The cells were covered immediately with trypsin-EDTA solution (0.25%, Gibco 2478) for 2 min at 37"C, then coliected into conical tubes containing RPMI-1610 with

10% FCS to neutralize the trypsin, centrifuged at 300 x g for 3 min. Then the amount of liposomai lipid associated with cells was determined by Iiquid scintillation countin; in both ce11 pellet and the supematmt.

3.6 Biotinylation of Po protein and peptides 3.6.1 Biotinyiation of Poprotein

Po-protein (HPLC-purified) (40 pg/344.83 PL of 0.1% Triton X-100) was incubated with I mg of N-hydroxysuccinimide-biotin (NHS-biotin, SIGMA, H 1759) (dissolved in 50 pL of DMSO) in the presence of 0.59 mg triethylamine for 5 hrs at room temperature under nitrogen with very gentle stimng. The biotinylation was monitored by analytical SDS-page. The biotinylated Po-protein was purified by dialysis procedure. The final reaction mixture was put in dialysis tubinj (MWCO 6000-8000) and dialyzed against 750 mL of 0.1% Triton X-100 solution, four times, at 4°C (12 hrs for each dialysis step). Then the protein concentration was determined by the Lowy protein assay method (1951) and/or densitometry using a Bio-Rad Mode1 GS-670 lmaging Densitometer.

3.6.2 Biotinylation of peptides 3.6.2.1 Biotinylation of Po-peptides The peptides were dissolved in the minimum volume of dimethylfomamide (DMF) (Po-peptide-1 and -2) or dimethylsulfoxide (DMSO) (Po-peptide-3). NHS-biotin at a 3-fold moIar excess over peptides was added. Then, a 2-fold moIar excess of tnethylamine over NHS-biotin was added and the mixture left for 24 hours at room temperature under nitrogen with gentle stirring. The progress of the reaction was rnonitored by capillary electrophoresis (CE) emp loyinp a Beckrnan System Gold V8 10 data systern/controlleq equipped with a Beckman WACE System 5500 and a Beckman P/ACE diode array deiector. The column used for separation was a Bechan uncoated capiliary tubing, 57 cm length and 75 pm diarneter. The buffer was sodium borate pH 8.75 (0.6% sodium borate.10 Hz0 and 0.4% boric acid). The samples were injected by pressure injection method for 5 seconds and the components were separated under 20 KV voltage at 23 OC. The eluting components were monitored at 214 m. The biotinylated peptides were purified by dialysis using SpectralPor non-sterile cellulose ester dialysis membrane, MWCO 500. Prior to dialysis, due to the incompatibility of the cellular membrane dialysis tubing with DMF and DMSO, the final reaction mixtures were freezed-dried under vacuum at -50 OC. Then, the dned powder was dissolved in toluene/ methanol (91) and dialyzed against 750 mL of 50 mM sodium acetate buffer pH 4.5, 750 mL sodium borate buffer pH 8, and three changes of water (12 hrs for each dialysis step, at 4°C). AAer completion of the dialysis procedure, the organic solvents and aqueous phase inside the dialysis bag were collected and the organic solvents removed by rotary evaporation and the water was removed by freeze- drying. The dry powder of biotinylated peptides were weighed and analyzed by CE.

3.6.2.2 Biotinylation of LFA-1-derived and RGD-peptides The LFA-1-denved peptide (a 12 amino acid long from the "I" dornain of LFA- 1, Alberta Peptide Institute, Edmonton, AB) and Arg-Gly-Asp peptide (BACHEM Bioscience Inc., Kin; of Prussia, PA) were dissolved in a minimum volume of NaHCO, buffer (0.1 M, pH 8.3). N-hydroxysuccinirnide-biotin (NHS-biotin, SIGMA, H 1759), 3- fold rnolar excess over peptides was dissolved in dimethylsulfoxide (DMSO) and added to the peptide solutions. Then, a 2-fold rnolar excess of triethyiarnine over NHS- biotin was added and the mixture Ieft for 24 hours at room temperature under nitrogen with gentle stirring. The progress of reaction was monitored by CE as explained in the previous section (3.6.2.1). The biotinylated peptides were purified by dialysis using SpectraPor non-steri le ceilulose ester dialysis membrane, MWCO 500. Ptior to dialysis, the ha1 reaction mixtures were Eeezed-dned under vacuum at -50°C, the dried powder was dissolved in water and purifÏed by dialysis as descnbed in section 3.6.2.1.

3.7 Flow cytometry \ 3.73 Direct irnmunofluorescence flow cytometry 3.7.1.1 Quantitative determination of the differential expression of ICAM-1 on human 1-21, A-375, and iMeM 50-10 cells The percent of stained cells and rnean fluorescence intensity (MFI) of ICAiiYf-1 (CD54)expression were determined on human M2 1, A-375, and MeM 50- 1O melanoma cells by direct immunofiuorescence flow cytornetry as descnbed by Maio et al., 1990. The cells were collected when, afier subcultivation, they reached a confluent monolayer. Cells were washed with 0.01 M PBS, and the viability of the cells was tested by trypan blue exclusion. Cells were counted using a Coulter counter andor hernocytometer and a ce11 suspension of 1 x 10' cells/mL in PBS (0.0 1 M)-bovine serum albumin (1%)-sodium azide (0.01%) PBS-BSA-A21 was prepared, and preincubated for 30 min at 4°C with occasional gentle shaking. Then, 50 pL of the ce11 suspension was incubated with 50 pL fluorescein isothiocyanate conjugated mouse monoclonal antibody to human CD54 (FITC-conjugated anti-human CD54 mAb; Cedarlane, product code MHCD5400) (1/40 dilution in [PBS-BSA-A21 from 0.2 rng/mL stock solution), and also separately with 50 PL mouse FITC-conjugated anti- bovine CD8 monoclonal antibody (1/250 dilution in [PBS-BSA-AZ]) as a control, for 30 min on 4°C. Cells were washed three times with [PBS-BSA-AZ] and fixed with 200

pL of 2% formaldehyde in PBS. Then the cells (1 x 10" volume gate) were analyzed with a Becton Dickinson FACScan flow cytometer. In the direct imrnunofluorescence flow cytornetry expenments in the presence of peptides, the peptides were incubated with cells for 30 min on 4°C before incubation with FITC-conjugated anti-human CD54 mAb. 3.7.1.2 Determination of the expression of ICM-1 on hurnan epidermal keratinocytes in the presence and absence of IFNy

The human keratinocytes (5 x 105 cells/well) were seeded into 6-well plates and

3qown. The keratinocyte monolayers when reached to confluency washed with PBS and incubated with various concentration of EN-y (0-1000 IU/rnL, Actimrnune IFN-y, 1 m=/rnL, activity: 20-40 million IU I mL) in K-SFM for 24-36 hrs. The keratinocytes were then detached using 0.022% trypsin, 0.01% EDTA in ClMF-Hank's. The trypsin was neutralized using 10% solution of FCS in RPMI-1640. The cells were centnfuged at 200 x g for 8 minutes. Cells were washed with 0.01 M PBS, and viability of cells was tested by trypan blue dye. Cells were counted using a Couiter counter andlor hernocytometer, and a ce11 suspension of 1 x 107 cells/ml in [PBS-BSA-A21 was prepared, stained with anti-human CD54 mAb and analyzed by a FACScan flow cytorneter as described previously (section 3.7.1.1).

3.7.2 Indirect immunofluorescence floiv cytometry using biotinylated-Po protein and -peptides and streptavidin-PE

3.7.2.1 Binding of biotinylated-Po protein to human MX, MeM 50-10 melanoma ceIk and CHO-X2 cells The binding capability of biotinylated-Po-protein to hurnan M21, MeM 50- 10 melanoma cells and CHO-X2 cells was investigated usin; indirect immunofluorescence flow cytometry as described by Maio et al. (1990). The cells were collected when, after subcultivation, they reached a confluent monolayer. Cells were washed with 0.01 M PBS, and the viability of ceIls was tested by trypaii blue exclusion. Cells were counted using a Coulter counter andlor hemocytometer and a ceIl suspension of 1 x 10' cells/mL in [PBS-BSA-AZ] was prepared, and preincubated for 30 min at 4°C with occasional gentle shaking. Then 50 yL oFcel1 suspension was incubated with 20 gL of biotinylated Po-protein (5-700 nM) for 30 min at 4'C. Cells were washed three times with [PBS- BSA-AZ], resuspended in 50 pL of [PBS-BSA-AZ] and incubated with 50 pL of R- p hycoerythrin conjugated streptavidin (strep tavidin-P E, Jackson ImmunoResearch Laboratories, code number 016-1 10-084) (1/100 dilution in [PBS-BSA-AZ] from 0.5 mg/mL stock solution) for 30 min at 4'C. Cells were washed three times with PBS-

BSA-AZ] and fixed with 200 PL of 2% fonnaldehyde in PBS. Then the cells (1 x 10" volume sate) were analyzed using a FACScan flow cytometer.

3.7.1.2 Binding of biotinylated Po-peptides and LFA-1-derived peptide to human M3I melanoma cells The binding capability of biotinylated-Po-peptides and -LFA-1-derived-peptide (5-200 FM) to human M21 melanoma cells was investigated using indirect immuno- fluorescence flow cytometry as described previously (section 3 .7.2.l).

3.7.2.3 Binding of biotinylated RGD-peptide to HUT 78 T celIs

The binding capability of biotinylated-Ars-Gly-Asp-peptide (25-500 uM) to =TT 78 T cells was investigated using indirect immunofluorescence flow cytometry as described previously (section 3 -7-2.1).

3.8 Cell-ce11 adhesion assay 3.8.1 Binding of Jurkat T celIs to human keratinocytes The cell-ce11 adhesion assay was carried out as described by Braut-Boucher et al. (1995) with some modifications. The human keratinocytes were seeded in triplicate in flat-bottom 96-well microtiter plates at 4 x 10' cells per well and groum. The confluent rnonolayers were used for the cell-ce11 adhesion assay. In some experirnents, keratinocytes were incubated with IFN-y 500 IU/rnL for 36 hours. On the day of the adhesion assay, the Jurkat T cells were washed three times with PBS, and the viability of the cells was tested by trypan blue and counted. The T-cells were then rnixed in equal volume with Calcein-AM (CAM, Molecular Probes, Eugene, OR) in PBS containing 1% bovine serum aIbumin (final concentration of CAM 20 PM) and incubated for 20 min at 37OC. The T cells were washed three times with cold WMI-1640 containing 10% fetal calf serum (RPMI-FCS), resuspended in RPMI-FCS, activated with phorbol myristate acetate (PMA, Sigma ) 1 nghL for 30 min at room temperature (dark place) and added to the washed (with K-SFM) EN-y- or non-EN-y-treated keratinocyes plates at concentration of 2 x 10' cells per well. After 30 min incubation at room temperature (dark place), nonadherent T cells were removed by repeated rvashes (20 times) with WMI-FCS using a multiple channel pipettor. The remaining cells were lysed using 2% Triton X-100 in RPM-1640, kept at least bvo hours in 4°C to complete the extraction process and to allow the fluorescent probe to equilibrate, then the flu~rescencewas determined at 485-538 nrn using a Labsystems Fluorskan fluororneter. In the peptide inhibition expenrnents, the peptides were dissolved in RPMI-FCS and added to the welk at vanous concentrations 10 min before the addition of CAM-loaded T cells. The % adherence of Jurkat T cells adhering to the keratlnocytes was calculated as the ratio of the fluorescence intensity after binding (washing) and the fluorescence intensity without washing (total input) x 100.

3.8.2 Binding of Jurkat T cells to human MX, A-375, and MeM 50-10 melanoma cells The cetl-ce11 adhesion assays were carried out as described before (section 3.5.1) with minor changes. The human M21, A-375, and MeM 50-10 cells were seeded in triplicate in collagen coated (Type 1, SiPa, 100 pg/rnL of 0.001 M acetic acid, 1 hour at 37"C, then washed three times with PBS) flat-bottom 96 well plate at 7.5 x 10' cells per well the day before experiment. The cells were seeded on collagen-coated wells to increase the binding strength of the cells to the wells; othenvise, the cells would corne off during the washing of the well. The next day the Jurkat T cells were loaded with calcein-AM, activated with PMA (0.5 ng/mL), and added to the different melanoma ce11 plates at 2 x 10' cells per well. After 30 min incubation at room temperature, the non- adherent Jurkat T cells were removed by washes (7 times) with room temperature RPMI-FCS. The remaining ceHs were lysed and the fluorescence was detemined. 3.9 Statistical analysis

3.9.1 Unpaired t-test Statistical evaIuations of the differences bebveen results of various treamients in each expenment were carried out using unpaired t-test. The unpaired t-test was used for most of the experirnents except inhibition of T cell/keratinocyte binding.

t = (XB- XA)I ./(s2$n,) + (s2~n~), XB - XI\is the difference of means between group B and goup A, S is the standard deviation, n is the number of replicates in each of group A or B, If t > q,, .,then Ho: PB = pA is rejected, cr is the significance level,

v is the degree of keedom (DF) = (n, + n,) - 2. Since in the most of the involved expenments there were more than two treatment groups in each expenment, Bonferroni correction was performed on the unpaired t-test results (Hassard, 1991). When in an experiment there are more than two treatrnent groups, the cornparison involves the testing of al1 pairs of treatment means by unpaired t-test. This way could provide the differences among the treatment groups; however, repeated testing in this way may increase the nsk of obtaining false significant difference by accident which is called type 1 error. To overcome this problem, Bonfenoni correction, which is a more sever significance level as die basic critical value for the unpaired t-test, is used. If we consider that the results are significant at p values less than or equal to 0.05, according to the Bonferroni correction the significance level would be: 0.0Yc where c is the nurnber of cornparison to be made for each experiment.

3.9.2 One-way ANOVA To evaluate the effect of peptides in the inhibition of T cell/keratinocyte binding, the various peptide treatment groups were cornpared using one-way ANOVA and in case of sipnificant F values multiple cornparison Tukey test was used to compare the means of different treatment groups. q=(XB- XA)/SE, XB - XA is the difference of means between goup B and goup A,

SE = ds2/2(I/ns + ~/n,), s2is the error mean square from the analysis of variance, n is the number of replicates in exh of goup A or B, If q > q,, .,k then Ho: pB = FA is rejected, a is the significance IeveI,

v is the error DF for the analysis of variance, k is the total number of means being tested. Results with p c 0.05 were considered to be statistically significant. CHAPTER FOUR

RESULTS

4.1 Study of association of Po protein reconstituted liposomes with cells

Highly purified Po protein was obtained fiom partially purified Po protein fiaction ID: by preparative PAGE or RP-HPLC. The FUI fraction partially purified Pa protein has been already produced fiom the myelin of chicks by the solvent extraction and the Sepharose CL-6B column chromatography by Foldvari et al. (1990). They have shown that the FI11 fraction contains about 80% Po protein (as monomer and oligomer) and about 20 % basic proteins of 21 kDa molecular rnass that are the highest contaminating components of the fraction. After the electrophoresis the Po protein band was cut fiom the gel and extracted with 0.1% Triton X-100. Po protein concentration was detemined by the Lowry method (Lowry et al., 1951). The purity of Po protein solution in 0.1% Triton X-100 was confirmed by analytical SDS-PAGE using Coomassie blue staining. The Po protein migrated as a single band on polyacrylamide gei with a molecular rnass of 30 ma. The identity of the Po protein band on SDS-PAGE has aIready been confirmed with Western blotting using anti-Po protein antibody (Foldvari et al., 1990). The partially purified FI11 fraction of Po protein was also further purified by RP- HPLC using a proteidpeptide C4 column according to Brunden et al., 1987. The HPLC chromatogram of FI11 fraction is shown in Figure 4.1A. To investigate the presence of protein in each peak, fractions of each peak were collected separately, fkeeze-dried and analyzed with analytical SDS-PAGE using Coomassie blue or silver staining. SDS- PAGE analysis of the peak eluting at 36.1 min (fractions collected 35-38 min) was identified as the Po protein pr th a molecular mass of 30 kDa (Figure 4. IB, line 3 and 4). In this peak beside the monomer of Po protein, there are proteins migrating with rnolecular masses around 60 and 90 kDa. It has already been shown that these nvo bands on SDS-PAGE can be identified as Po protein by Western blottinj using anri-Po protein antibody poldvari et al., 1990). The 90 and 60 kDa protein bands are presumed to be a trimer and dirner of Po protein, respectively. It has also been shown that purified Po protein has a tendency to form oligomers upon SDS-PAGE analysis (Mezei and Verpoorte, 1981). The Poprotein standard is shown in lane 2 (Figure 4. LA). The SDS-PAGE analysis of the peak eluting at 1.5 min (&actions collected 1-3 min) revealed a single protein band with a molecular mass of 21 kDa, the (MBP), which is the major contaminant of the FI11 fraction (Figure 4.18, line 5). The additional smaller peaks (30 min and 40 min) were not protein related.

4.1.2 Characterization of Po-liposomes The unilarnellar Po-liposomes and control liposomes were prepared by a Bio- Bead batch method as described by Foldvari et al. (1 990). Figure 4.2 shows the electron micrographs of Po-liposomes and control liposomes. The pictures were taken ten days after preparation of the liposomes (stored at 4OC. The Bio-Bead batch method gave liposomes of a unifom size distribution (80-90 nrn average apparent diameter). The SDS-PAGE analysis of the Po-liposome pellet after centrifugation revealed nearly 100% Po protein incorporation into the liposome bilayer (Figure 4.3, line 4 and 5). The arnount of Po protein in the supernatant of centrifuged Po-liposomes was negligible and was not visible on the gel by Coomassie blue staining (Figure 4.3, lane 6). I O lil,,ll, II,, ri1 ( ILI, 1 o .ao 10.00 20.00 30.00 40.00 50.00 52.41 Tirne (min) Figure 4.1 Purification of Po protein. A) RP-HPLC chromatogram of partially purified Po protein Fm &action. Partially purified Po protein (1.5 mg) was dissolved in 2 mL of acetic acid/water (1/1), injected ont0 C4-RP proteidpeptide column, and the eluting components were monitored at 280 m. The mobile phase consisted of acetic acidhater (M) (solution A), with a gradient of acetic acid.12-propanol (111) (solution B) as follows: 0-2 min 100% A; 2-4 min 0-10% B; 4-29 min 10-35% B; 29-35.5 min 35-100% B; 35-50 min 100% B, fiow rate was 0.3 mllrnin. B) SDS-PAGE analysis of RP-HPLC isolated Po protein (10% running gel, 3% staclàng gel, Coomassie blue staining). 1) Pharmacia standard (fiom top 94,67,43, 30, 20.2 Da). 2) Po protein standard. 3) Fractions collected 35-38 min (10 PL). 4) Fractions collected 35-38 mùi (5 PL). 5) Fractions collected 1-3 min. Figure 4.2 Transmission electron rnicrographs of Po-liposomes and control liposomes by negative staining using phosphotungstic acid. Po-Iiposomes (A, 38,000 x) and control liposomes (E!, 41,000 x) prepared by the Bio-Bead batch method with DPPC:cholesterol (10:l molar ration), stained with 1% phosphotungstate and viewed in an electron n~icroscope. Figure 4.3 SDS-PAGE analysis of the Po-liposomes (10% running gel, 3% stacking gel, Coomassie blue staining). 1) Pharmacia standard (fiom top 94,67,43,30,20.2, 14.4 kDa). 2) Po protein standard. 3) HPLC-purified Po protein. 4) Po-liposome pellet &er centrifugation and resuspension (5 PL). 5) Po-liposome pellet after centrifugation and resuspension (1 0 PL). 6) Supernatant of Po-liposomes (20 PL). 4.1.3 Interaction of Po- and Glycophorin A-liposomes with human hl31 melanoma cells and the inhibition of interaction with anti-chick Po antibody The ceIlular uptake of Po protein and Glycophorin-A reconstituted liposomes was deterrnined in human M21 melanorna celIs to determine the effect of Po protein and Glycophorin-A (as a control transmembrane protein) in the binding of liposomes to M21 cells. Glycophorin-A, like Po protein, has three domains: extracellular, transmernbrane, and intracellular; but it does not have any known adhesion properties. The expenrnent was camied out with Po (SDS-PAGE purified)-, Glycophorin A-, and control-liposomes at 37OC for 1, 4, and 8 hours. Incorporation of Glycophorin A into liposomes was confirmed by PAGE. The analysis of liposome uptake indicated that Po-liposomes associated with M21 cells 3-4 times more efficiently than control liposomes of the sarne lipid composition but devoid of Po protein at 37°C @ c 0.001, Po-liposomes cczpared to control liposome in three different time) (Figure 4.4). The association of Po-liposomes and control liposomes with M21 cells was time dependent and increased with tirne. The resufts of this expenment confirmed the previous information in the Iiterature with regards to the ability of Po protein in mediating heterophilic interaction with M21 cells (Foldvari et al., 199 1). The degree of cell-associated Glycophorin A-liposomes was similar to the binding rate of control-liposomes (p > 0.2, Glycophorin A-liposomes compared to control liposome in three different time), where it was 3-4 times lower than Pa- liposomes @ < 0.001, Po-liposomes compared to Glycophorin A-liposome in three different tirne) (Figure 4.4). Therefore, it was concluded that incorporation of Glycopho~n-Ainto the liposomal bilayer did not change the cellular uptake of liposomes indicating that specific adhesive properties are required for binding. To determine the molecular nature and specificity of the heterophilic interaction of Po protein with M21 melanoma cells the effect of anti-chick Po antibody Fab (fragment of antigen binding was prepared from anti-chick Po antibody) on Pa-liposome binding with M21 melanorna cells was investigated. Po- or control-liposomes were incubated with M2 1 cells at a concentration of 80 pg phosphoIipid/j x 106 cells in the Time (hours)

Figure 4.4 Association of Po-, control-, and Glycophonn A-liposomes with human M2 1 melanoma cells. Monolayers of M21 cells (5 x 106/well) were incubated with Po liposomes, Glycophorin A-liposomes and control liposomes composed of [14~]-~~~~:chol(10:l molar ratio) prepared by the Bio-Bead batch method at a lipid concentration of 54.5 plM for 1,4 and 8 hours at 37OC. The free proteins were separated fkom liposomes by ultracentrifugation. After incubation, the cells were separated Eom unbound liposomes by washing with PBS. The arnount of liposomes associated with the cells was determined from the uptake of radioactivity. Each bar represents the mean f SD of 3 separate experhents. The standard deviation for control liposomes at 1 hour was 0.004. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences @ c 0.001) between Po-liposomes compared to control- and glycophonn A-liposomes at al1 time points examined. There were no significant differences @ > 0.2) between control-liposomes and glycophorin A-liposomes at al1 time points examined. presence or absence of anti-chick Po antibody Fab (90 pg 1 5 x 106 cells) at 37°C for 1 hour. The xmount of cell-associated Po-liposomes was about three times of control liposomes (same lipid composition but no Po protein, p < 0.001 Po-liposomes cornpared to control liposome) (Figure 4.5). However, the arnount of cell-associated Po-liposomes in the presence of anti-chick Po antibody Fab was si,qïficantly decreased (p c 0.001) to sirnilar levels of control liposomes @ > 0.30). Meanwhile, the binding of control liposomes in the presence of anti-chick Po antibody Fab was unaffected @ > 0.30). In summary, the binding of Po-liposomes to the cells was inhibited by anti-chick Po antibody indicating that Po protein plays a specific role in the binding of liposomes to M21 cells.

4.1.4 Characterizing the binding domains of Po protein reconstituted into liposomes by Po-peptide competition studies

The purpose of this part of the study was to further charactenze the binding domains of Po protein reconstituted into liposomes by conducting competition studies with synthetic Po peptides based on the chicken Po sequence. Three peptides were synthesized representing the extracellula. (Po-peptide-1: residues 90-96: Tyr-Thr-Asp- Asn-Gly-Thr-Phe), the membrane spanning (Po-peptide-2: residues 140-146: Val-Ala- Leu-Leu-Val-Ala-Val) and the intracellular domain (Po -peptide-3 : residues 20 1-207: Lys-Aia-Ala-Ala-Glu-Lys-Lys).The choice of Po-peptides fiom di fferent regions of Po protein was based on the function of each region in the Po protein. Po-peptide2 and Po- peptide-3 were selected as a control for Po-peptide-l. Po-peptide2 contains mainly hydrophobic amino acids and represents the hydrophobic trammembrane region of Po protein. The Po-peptide-3 contains the positively charged amino acid Lys and represents the basic intracellular domain of Po protein. Po-peptide-1 was selected from the Ig-like extracellular region of Po that contains the glycoside attachment site (Asn 93). Figure 4.5 Effect of preincubation with anti-chick Po-antibody Fab on the binding of Po-liposomes and control-liposomes to human M2 1 melanoma cells. Monolayers of M21 cells (5 x 106/well) were incubated with Po liposomes and control liposomes composed of [14~]~~~~:chol(1 0: 1 molar ratio) in the presence (n=5) or absence (n=3) of anti-chick Po antibody Fab (90 pg / 5 x 106cells) for 1 hour at 37°C as described in the legend of Figure 4.4. Each bar represents the mean + SD of 3 separate experiments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were ~i~gnificantdifferences @ < 0.001) between Po-liposomes uptake compared to control-liposomes in the presence and absence of anti-chick Po Fab, and Po-liposomes in the presence of anti-chick Po Fab. There were no significant differences (p > 0.3) arnong conbol-liposomes, Po-liposomes Fab and control-liposomes Fab. Po (SDS-PAGE purified)- or control-liposomes were incubated with M21 cells at a concentration 80 pg phospholipid / 5 x 106 iLi71 cells in the presence or absence of Po peptides (90 pg 1 5 x 106 cells) at 37°C for 1 hour. The results indicated that the association of Po-liposomes with M21 cells decreased by 30% in the presence of Po- peptide-l @ < 0.008) and by 40% in the presence of Po -peptide-3 @ < 0.001), while the effect of Po-peptide-2 was no significant @ > 0.04) (Figure 4.6A). The presence of Po- peptide-l and Po-peptide-2 increased the association of control-liposomes with M21 cells by 240% @ c 0.001) and 290% @ c 0.001), respectively (Figure 4.6B). Po- peptide-3 had no effect on control liposome interaction with cells (p > 0.008). According to this liposome-cell adhesion assay, it was concluded that both extracellular and intracellular Po-peptides (1 and 3) showed cornpetition and may be involved in the heterophilic interaction of Po protein with M21 cells. The results may also be indicative of the adhesion of Po-peptide-1 and -3 to the M21 cells. Therefore, Po-peptide-1 and Po-peptide-3 could be candidates as ligands for liposomes for targeting to M21 melanorna cells. The effect of Po-peptide-1 and -2 on control liposomes binding may be indicative of adhesion of these peptides to the liposome surface and mediatins cellular uptake of liposomes.

4.1.5 Characterization of human melanoma ce11 lines for the expression of ICAM-1 The purpose of this part of the study was to determine whether human M21 melanoma cells express ICAM- 1 and compare quantitatively the expression of ICA%- 1 on M21 cells with melanoma ce11 lines of A-375 and MeM 50-10. Previous studies indicated a relativety high level of TCAM-1 expression for human A-375 rnelanoma cells (Scheibenbogen et al., 1993; and Altomonte et al., 1993), and relatively low levels for human MeM 50-10 melanoma cells (Altomonte et al., 1993). The expression of ICAM-I on the melanoma cells was quantitated by flow cytometry using FITC- conjugated anti-human CD54 mAb (0.25 pg / 50 PL PBS-BSA-AZ). The results I 1 I 1 a O 20 40 60 80 1O0 Percent binding compared to Po-liposomes

Percent binding compared to control liposomes

Figure 4.6 Association of Po-liposomes (A) and control-liposomes (B) with hurnan M21 melanoma cells in the presence of Po-peptides. Monolayers of M21 cells (5 x 10~/well)were incubated with Po liposomes (A) and control-liposomes (B) composed of [L4~]~~~~:chol (10:l molar ratio) in the presence or absence of Po peptides (90 pg 1 5 x 106 cells) for 1 hour at 37OC as described in the legend of Figure 4.4. Each bar represents the mean k SD of 3 separate expenments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. In Figure 4.6A there were significant differences between Po-liposomes and Po liposomes Po-peptide-1 @ < 0.008), Po-liposomes and Po liposomes Po-peptide-3 @ < 0.001); there \vas no sigificant difference behveen Po- liposomes and Po liposomes Po-peptide2 (p > 0.04). In Figure 4.6B there was sigificant difference @ < 0.001) between control-liposomes and control-liposomes in the presence of Po-peptide-1 and Po-peptide-2; there was no sigificant difference bebveen Po-liposomes and Po liposomes Po- peptide-3 (p > 0.05). indicated that human M2L rnelanoma cells express high Ievel of ICAM-1 and that there are significant differences in the expression of ICAM-1 arnon,o the melanoma ce11 lines @ < 0.001, cornparison of PSC and MF1 arnonj the three different melanoma ce11 lines, Figure 4.7). The percentage of stained cells with anti-human CD54 mAb was about 5% for MeM 50-10 cells, 95% for M21 celis and 85% A-375 cells. Analysis of the mean fluorescence intensity (MFI) values showed that M21 cells have a higher level of ICAM-1 expression compared to A-375 and MeM 50-10 ce11 lines (1 54% and 326% respectively) .

4.1.6 Po protein mediated binding of liposomes to human M21, A-375,

MeM 50-10 melanoma celis and CHO-X2 cells

It is hypothesized that the increased binding of Po-liposomes to M21 cells may be due to the heterophilic interaction of Po protein with ceIl adhesion proteins such as ICAM- 1 (CD54) on the M2 1 ce11 surface. The purpose of this part of the study was to evaluate the possibility of interaction of Po protein with ICAM-1 by determining the extent of binding of Po-liposomes to human MY, A-375 and MeM 50-10 rnelanoma ce11 lines and to evaluate whether the level of ICAM-1 expression on the different hurnan melanoma ce11 lines correlates with the extent of binding of Po-liposomes to the cells. The other airn was ta detemine the extent of bindins of Po-liposomes to CHO-X2 cells that are expressing Po protein as a positive control (in this case the interaction of Po-liposomes with CHO-X2 cells would be hornophilic). Therefore, the unilamellar Po- liposomes (using HPLC-purîfied Po protein) and control liposomes were prepared by the Bio-Bead batch rnethod and incubated with M21, A-375, MeM 50-10 and CHO-Xî cells at a concentration of 80 pg phospholipid per 5 x 106 cells for 1 hour at 37'C. t ; 1 Percent of stained cells 1 -1 Mean fluorescence intensity ! ' I I

Figure 4.7 Flow cytometric analysis of human M21, A-375 and MeM 50-10 melanoma cells for ICAM- 1 expression. Melanoma cells (5 x 105 / 50 pL) were resuspended in [PBS-BSA-AZ] and incubated with FTTC-conjugated anti-hurnan CD54 mAb. Then cells (1 x 104 volume gated) were analyzed by flow cytometry. Each bar represents the mean + SD of 3 separate expenments. Statistical analysis was camed out by unpaired t test with Bonferroni correction. There were significant differences among the melanoma ce11 lines for expression of ICAM-1 with respect to PSC @ c 0.001) and also MF1 @ c 0.001). The results of the liposome ce11 binding assay indicated that the presence of intact Po protein in the liposome bilayer hiwy increases the extent of bindins of liposomes with M21 (7.80 fold) and A475 (4.62 fold) melanoma cells cornpared to control liposomes of same lipid composition but no Po protein, whereas with MeM 50- 10 melanoma cells no si,gificant increase was found (1.70 fold) (Figure 4.8). The magnitude of binding of control liposomes to the three different melanoma ce11 lines was similar and the differences were nonsipifkant (p > 0.05). However, there were significant differences (p < 0-001) in the magnitude of binding of Po-liposomes between the three different melanoma ce11 lines. The level of binding of PQ-liposomes to the M21 cells was higher than A-375 and MeM 50-10 cells (138% and 510%, respectively), and also the binding ievel of Po-liposomes was higher for A-375 cells compared to the MeM 50-10 cells (368%). The extent of binding of PO-liposomeswith different melanoma cells correlated with the level of ICAM-1 expression on their ce11 surface and it was more pronounced with melanoma cells that express ICAM- 1 (Figure 3.9, 2 = 0.9996). The results of the binding assay also indicated that Po-liposomes associated with CHO-X2 (4.36 fold) more efficiently than control liposomes (p < 0.001, Figure 4.8). The results also indicated that the magnitude of binding of Po-liposomes and control liposomes among different ceIl lines is the highest for CHO-X2 cells. The increased binding of Po-liposomes to CHO-XZ cells could be indicative of homophilic interaction of Po protein. The Po protein on the surface of CHO-M cells interacts with the Po protein on the surface of liposomes resulting in increased extent of binding. The CHO- X2 ce11 line was used as a positive control for the bindin~assay. According to these results, even though the Po-liposomes had the highest extent of binding with CHO-XZ cells, the magnitude of binding for CHO-X2 cells was only marginally higher than MZ1 cells (compare 2.66 to with 2.09, Figure 4.8). In sumrnary, Po protein reconstituted into liposomes mediates heterophilic interactions in ceII culture and increases the extent of binding of liposomes to M2 I and A-375 cells but not MeM 50-10 ceIIs, the level of binding for M2 Z cells was higher than A-375 cells. The extent of binding of Po liposomes with human melanoma ce11 lines correlated with the level of ICAM-1 expression on their ce11 surface and it was more Po-liposomes 1-1 1-1 - Control liposomes

M2 1 A-375 MeM 50-1 O CHO-X2 Human melanoma ce11 line

Figure 4.8 Association of Po- and control-liposomes with hurnan MX, A-375, MeM 50-10 melanoma cells and CHO-X2 cells. Monolayers of cells (5 x 106/well)were incubated with Po liposomes and control liposomes composed of [3~~~~~:chcl(10: 1 rnolar ratio) prepared by the Bio-Bead batch method at a lipid concentration of 54.5 pM for 1 hour at 37°C. The Po liposomes contained 0.27 nM protein/pM phospholipid; the fkee protein was separated from liposomes by ultracentrifugation. After incubation, the cells were separated from unbound liposomes by washing with PBS. The arnount of liposomes associated with the cells was deterrnined fkom the uptake of radioactivity. Each bar represents the mean + SD of 3 separate expenments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences (p < 0.001) in Po-liposome uptake in the different melanoma ce11 lines. There were no significant differences @ > 0.05) in the control liposome uptake in the different melanoma ce11 lines. There were significant differences (p < 0.001) between Po-liposomes and control-liposomes uptakes in al1 the ce11 lines except in MeM 50-10 cells @ > 0.01). 20 25 30 35 Mean fluorescence intensity

Figure 4.9 Correlation between ICAM-1 expression on human MeM 50- 10, A-3 75, and M21 melanoma ce11 Iines and binding of Po-liposomes. The extent of binding of Po liposomes to rnelanoma ceIl lines was plotted against the intensity of ICAM-1 expression for each ce11 line, then linear regression was carried out. R~ = 0.9996. pronounced with melanoma cells that express ICAM- 1. Therefore, it is speculated that the increase in the extent of binding of Po liposomes to hurnan Ml1 and A-375 melanoma cells may partly be due to the interaction of Po protein with ICkM-1.

4.2 Study of association of biotinylated Po protein with cells using indirect flow cytometry

In this part of study the binding ability of the Po protein itself in solution with different cells was evaluated. To be able to visualize and quantitate the binding of Po protein to the cells a biotin-streptavidin indirect immunofluorescence flow cytometry was used. In the biotin-streptavidin system, cells were first incubated with biotin- conjugated Po protein and then subsequently incubated with streptavidin-conjugated ro FITC or PE (Figure 4.10). The streptavidin (or avidin)-biotin bond is one of the strongest known biological interactions behveen a ligand and a protein (dissociation constant, K = IO-'' M) (Green, 1963). Biotin is a small hydrophobie molecule (MWt 244.31 Da) that exists in ail living cells and functions as a coenzyme of carboxylases (Knappe, 1970). Both avidin (66-68 kDa) and streptavidin (60 kDa) are tetramenc protein and each of them binds four molecules of biotin. Because of the rapid interaction of avidin and streptavidin with biotin and the stability of the formed complex, this system has been used in many bioanalytical procedures such as irnmunoassays and localization of antigens in cells and tissues (Wilcheck and Bayer, 1988). Avidin, because of its positively charged amino acids and oligosaccharide moiety, which consists of mostly mannose and glucosamine, can interact with nejative charges on ce11 surfaces or nonspecifically with Iectin receptors and this may cause backgound problems in histochemical and cytochemical applications (Bmch and White, 1982). Streptavidin, which is a near-neutral protein, causes less background problems than avidin (Hiller et al., 1987). -3 Wash

lncubate Wash

Analyze by FACS

Y Ceii surface maiecuie

Figure 4.10 Biotin-streptavidin indirect immunofluorescence staining of ce11 surfaces. 4.2.1 Biotinylation of Pa protein The Po protein was biotinylated by incubation with NHS-biotin in the presence of trïethylamine for 5 hrs at room temperame and purified by dialysis. The SDS-PAGE analysis of biotinylated Po protein in 0.1% Triton X-100 solution revealed a protein with a molecular mass of 34.1 kDa (Fiawe 4.11, Iine 3 and 4). The standard Po protein solution and HPLC-purîfied Po protein had a protein band with an apparent rnolecular mass of 30 kDa (Figure 4.1 1, line 1 and 2, respectively).

4.3.2 Association of biotinylated Po protein with human M21 and MeM 50-10 melanoma cells and CHO-X2 ceils The results of indirect flow cytometry indicated that the biotinylated Po protein binds to M21 cells in a concentration dependent manner (10 nM - 700 fi) and produced about 95% bindin; (Figure 4.12A), whereas with MeM 50-10 melanoma cells no binding was found (Figure 4.12B). The perceil; of stained cells for M21 cells with 200 mM of biotinylated Po protein was about 95%, however, for MeM 50-10 cells it was only 5%. Also, the MF1 for M21 cells staining increased by increasing the biotinylated Po protein concentration. MF1 of MeM 50-10 ceils did not change in the Pa protein concentration range tested. MeM 50-10 cells have a higher autofluorescence (3 1.67) than the other ce11 lines in this study. The flow cytometry results also indicated that the biotinylated Po protein binds to CHO-X2 cells in a concentration dependent manner (Figure 4.12C). The binding began with 10 nM concentration and reached to more than 95% by 160 nM. The percent and intensity of binding of biotinylated Po protein to CHO-X2 cells was comparable with M21 cells at al1 concentrations. Control experirnents with sirnilar volumes of 0.1% Triton X-100 used for biotinylated Po protein bindin; (to keep Po protein solubilized) showed no effect on PSC and MF1 values. Since biotinylated Po protein binds to the M21 cells but does not bind to MeM 50-10 cetls it was concluded that the interaction of Po protein with M21 cells may be mediated by ICAM-1, because M21 cells express ICAM-I but the MeM 50-10 cells do not. Also, it was concluded that the heterophilic interaction of biotinylated Po protein Figure 4.11 SDS-PAGE analysis of the biotinylated Po-protein (10% nuining gel, 3% stacking gel, Coomassie blue staining). 1) Po protein standard. 2) HPLC-purified Po protein. 3) Biotinylated Po protein (5 PL). 4) Biotinylated Po protein (1 0 PL). 5) Pharmacia standard (fkom top 94, 67,43,30, kDa). Percent O F stained cells 1-1 1-1 M ean fluorescence intensity

Biotinylated Po protein concentration (nM)

Figure 4.12 Indirect irnmunofluorescence flow cytometric analysis of human M21 and MeM 50-10 rnelanoma cells and CHO-X2 cells for binding of biotinylated Po protein. M21 (A), MeM 50-10 (B) and CHO-X2 (C) cells (5 x 10' / 50 PL) were resuspended in [PBS-BSA-AZ] and sequentially incubated with biotinylated Po protein and streptavidin-FE (0.5 pg / pL). Then cells (1 x lo4 volume eated) were analyzed by Dow cytornetry. with M21 cells is more likely specific. If it was non-specific, there should not be a difference in the interaction of Po protein with M21 cells and h4eM 50-10 cells.

42.3 Inhibition of association of biotinylated Po protein to human M2l melanoma cells by preincubation with anti-ICAM-1 To further charactenze the involvement of ICAM-1 in the binding of Po protein to M21 cells, the indirect flo~cytometry using biotinylated Po protein was camed out on M21 cells in the presence of anti-human CD54 ab.For this purpose, M21 cells were first incubated with FITC-conjusated anti-hurnan CD54 (0.50 pg 1 75 krL, the sarne range of concentration which had been used for direct flow cytornetry) for 15 min at 4°C then incubated with biotinylated Po protein 100 nM for 30 min at 4°C. Cells were waslied and incubated with streptavidin-PE (0.5 pg / PL) for 30 min at 4OC and analyzed with a FACScan flow cytometer. The results indicated that the association of biotinylated Po protein to M21 ceils decreased significantly (p e 0-05, cornparison of PSC and MF1 in the absence and presence of anti-CD54 mAb) in the presence of anti- human ICAM- 1 (Fijure 4.13). The binding of biotinylated Po protein to M2 1 cells were comparable to the previous expenments, however, preincubation of M21 cells with anti- human ICAM-1 rnAb decreased the percent of stained cells by 32% and the mean fluorescent intensity by 37%. It should be noted that even though the antibody used in this study was a monoclonal antibody and its binding site on ICAM-1 could be different fkom Po protein, the mAb decreased the binding of Po protein. Accordingly, it was concluded that ad-human ICAM-1 partially interferes with the binding of biotinylated Po protein to the M21 cells and then the binding of Po protein to rM21 cells may be mediated by interaction with ICAM-1.

4.3 Study of the effect of peptides on the interaction of anti-ICAM-1 with ICAM-1 on M31 ceils In order to find an adhesive peptide for targeting toward ICAM-1, immunofluorescence flow cytometry experiments were camed out on human M2 1 cells M21 cells 1 1-1 1-1 M2I cells + anti-CD54 /

Percent of stained Mean fluorescence cells (PSC) intensity (MFI)

Figure 4.13 Indirect irnrnunofluorescence flow cytometric analysis of human iMZ 1 melanoma cells for binding of biotinylated Po protein in the presence of anti-human CD54 mAb. Human M21 melanoma cells (5 x lo5 / 50 pL) were resuspended in PBS-BSA-AZ], first incubated with anti-human CD54 mAb (0.50 pg i 75 yL) and then sequentially with biotinylated Po protein (100 LM) and streptavidin-PE (0.5 pg/50 PL). Cells (1 x 104 volume gated) were then analyzed by flow cytornetry. Each bar represents the mean + SD of 3 separate experiments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There was a significant difference (p c 0.05) in the PSC values. The difference in the MF1 values was only significant at the significance level of p < 0.1 (0.05 -= p c 0.1). using FITC-labeled anti-ICAM-1 in the presence of peptides. The aim of this part of the study was to investigate the possible interaction of peptides with ICAM-1. If thsre is interaction between peptides with ICAM-1 on the surface of M21 cells, there wilI be a reduction in the MF1 of cells due to the interaction of FITC-labeled anti-ICAM-1 with ECAM-1 on the surface of M21 cells. The evaluated peptides consisted of Pa-petides and an LFA- 1 àerived peptide. LFA-1 is the counter receptor of ICM-1. Tt has been shown that the "I" domain of LFA-1 a chain contains a ligand binding site for 1C.ELbI-I (tandis et aI., 1993 and 1994; Randi and Hogg, 1994). Removal of the "1" dornain from LFA- 1 causes a substantial decrease in the interaction between LFA- 1nCAM-1. Randi and Hogg (1994) showed that an isolated recombinant fom of the "1" domain can directly bind to soluble ICAM-1 and cm also block LFA-1 dependent adhesion of T cells to ICAM-1. The LFA-1 denved peptide (peptide-4) is a synthetic 12 amino acid long peptide selected hmthe "1" domain of LFA-1.

4.3.1 Effect of Po-peptides on the interaction of ICAM-1 and anti-ICAM-1

In this experiment, the cells were resuspended in PBS-BSA-AZ (5 x 10' cells /

50 PL), preincubated with Po-peptides (2.5, 5, and 12.5 pg / 5 x 105 cells) at 4OC for 30 min, and incubated with FITC-conjugated anti-human CD54 mAb (0.25 pg / 50 PL PBS-BSA-AZ). As a negative control, the FITC conjugated anti-bovine CD8 mAb was used. The results indicated that the MF1 values for the ICAM-I stainins decreased by 65.67% in the presence of Po-peptide-3, by 59.43% in the presence of Po-peptide-2, and by 17.75% in the presence of Po-peptide-1, at 12.5 pg / 5 x 10' cells Po-peptides concentration (Table 4.1). The results of this experiment seemed good at first, except that these results were not in agreement with the Po-peptides competition studies of M21 and Po- liposomes interactions (Figure 4.6). In the competition studies, the association of Po- liposomes decreased by Po-peptide-l and -3, but the Pa-peptide-:! had no effect. In this experiment, to improve the solubility of Po-peptides, DMSO had been used as a CO- solvent. It was speculated that DMSO may have some effect on the results since the amounts of DMSO used for Po-peptide-1, -2, and -3 were 8.66, 18.38 and 21.61 PL, respectively, and the degree of inhibition appeared to be correlating with the increasing concetration of DMSO. It was therefore decided to repeat the flow cytometry experirnent to determine whether DLMSOhas any effect in the interaction of ICA,?-1 on M3 1 cells with FITC-conjugated anti-human CD54 mAb.

TabIe 4.1 The effect of Po-peptides on the interaction of ICA??-1 on human hl21 melanoma cells and FITC-conjugated anti-human CD54 rnAb (0.25

Treatment condition FITC-conjugated MF1 * SD, n=3 % decrease of II mAb MFI value* /I

None (n = 1) anti-bovine CD8 7.98 None 1 anti-human CD54 1 51.78 * 2.26 Po-peptide- l,2.5 pg anti-human CD54 50.85 * 2.26

1 Po-peptide-1, 5 pg 1 anti-human CD54 1 46.90 & 3.60

1 Po-peptide-2, 2.5 pg 1 anti-human CD54 1 40.10 & 0.91 1 Po-peptide-& 5 p; 1 anti-human CD54 1 37.74*3.57 1 Po-peptide-2, 12.5 pg 1 anti-human CD54 1 21.00 * 0.98 1 Po-peptide-3, 2.5 pg ( anti-human CD54 ( 4 1.52 * 0.83 1 Po-peptide-3, 5 pg 1 anti-human CD54 1 36.05 * 0.53 1 Po-peptide-3, 12.5 pg 1 anti-human CD54 1 17.77 * 1.92

* % decrease of MF1 value for ICAM-1 expression in the presence of Pa-peptides.

The cells were preincubated with DMSO (8.66, 18.38, and 21.64 pL) for 30 min at 4'C, and then incubated with FITC-conjugated anti-human CD54 mAb (0.25 pg 150 yL PBS-BSA-AZ). The results indicated that DLMSOinterferes with the interaction of TCAM-I on M2l cek and anti-ICAM-1 mAb and decreases the MF1 value for the 1CALM-Z staining (Table 4.2). This effect of DMSO was dose-dependent and increased with increased DMSO concentration.

Table 4.2 The effect of DMSO on the interaction of ICAbl-1 on human A421 melanoma cells and FITC-conjugated anti-human CD54 rnAb (0.25 ps/so PL).

~one 1 anti-bovine CD8 1 8.86 1 -

.. - None 1 anti-human CD54 1 63.26 -

-- DMSO, 8.66 pL 1 anti-hurnan ~D54 1 38.2 / 39.61 DMSO, 18.38 pL 1 anti-hurnan CD54 1 22.72 1 64.08 ( DMSO, 2 1.64 p~ 1 anti-hurnan CD% 1 16.52 1 73.88 (

--

* % decrease of MF1 value for ICAM- 1 expression in the presence of DMSO.

In addition to the false positive results caused by DMSO, these espenrnents were limited by the poor soiubility of Po-peptides. The other organic solvents such as DMF, propylene glycol (PG) and polyethylene glycol (PEG) 400, in which Po-peptides are soluble, were toxic to the cells. Therefore flow cytornetry experiments were carried out with the following modifications to avoid the effect of DMSO:

A) Washing the cells three times with PBS-BSA-AZ after incubating the cells with Po-peptides in DMSO. B) Carrying out the ffow cytorneîq experiment as usual but excluding the dead cells from the ce11 population by usinj propidium iodide in the flow cytometry protocol.

A) Washin~the celIs : In this flow cytometry experiment to exclude the effect of DMSO in the interaction of ICAM-1 on the surface of M21 cells with FITC-conjugated anti-human CD54 rnAb, the cells were incubated with Po-peptides in DLMSO for 30 min at 4°C. Then the cells were washed three tirnes with PBS-BSA-AZ and incubated for an another 30 min with FITC-conjugated anti-human CD54 db. The idea for this experiment was that if Po-peptides have adhesive activity for ICAILI-1 on the surface of M21 cells, after washing they will remain bound but the DMSO will be washed away. The results indicated that even afler washing the cells, the DMSO still has its effect and somehow prevents the interaction of ICAM-1 with anti-ICAM-1 (Table 4.3). Perhaps incubating the cells with DMSO affects the conformation of ICAM-1 molecules such that they cannot interact completely with anti-ICAM-1. The effect of Po-peptides was similar to their DMSO control. BI Excludin ~idiumiodide in the flow cvtometrv experiment: In these experiments the dead cells were excluded from ce11 population by using propidium iodide (PI). The idea for this experiment was that the dead cells, resulting from incubation with DMSO, could not interact properly with anti-ICAM-1 and then decreasin; the MFI. The protocol was the same as before, but instead of PBS-BSA-AZ just PBS was used and after incubation the cells with FITC- conjugated anti-human CD54 mAb and washing, the cells were not fixed with formaldehyde. However, just afler finishing the experiment, 2 pg/rnL of PI were added to the cells and analyzed in FACScan. By this procedure, the dead cells cm be excluded from the analysis by setting gates that exclude al1 PI-positive cells. The results indicated that even with the separation of dead cells, DMSO still affects the interaction of ICAM-1 with anti-ICAM-1, and the MF1 is decreased (Table 4.3). Also, the percent of dead cells increases with the increasing of arnount of DMSO. In surnmary, in spite of various efforts to exclude the effect of DMSO, the binding of Po-peptides to ICAM-1 on M21 cells could not be demonstrated. Interpreting the present data (Table 4.2 and 4.3), it seems that the Po-peptides at the Table 4.3 Determining the exclusion of the effect of DIMSO on the interaction of ICFLIM-1 on human M21 melanoma cells and FITC-conjugated anti- human CD54 mAb (0.25 pJ5O uL).

Treatment condition FITC-conjugated Mean fluorescence 1 Percent of dead mAb intensity ceils

- - A) Effect of was& the cells -1 None - 7.5 1 -

None anti-bovine CD8 7.78 - None anti-human CD54 43.95 - Po-peptide- l,25 pg anti-human CD54 37.52 - I 1 Po-peptide-2, 25 pg 1 anti-human CD54 I 32.38 -

1 DMSO, 25 CIL** ( anti-human CD54 I I - I I B) Excluding the dead cells using propidium iodide I None - 7.86 5.42

None anti-human CD54 47.5 5.23 DMSO,8.66 pL anti-human CD54 33.8 1 13.34 DMSO, 18.88 pL anti-human CD54 34.39

DMSO, 2 1.64 pL anti-human CD54 23.78 43.34

iF Control DMSO for Po-peptide-1. ** Control DMSO for Po-peptide-2 and 3.

mentioned concentrations have no effect on the interaction of ICAM-1 and anti-IChi- 1 and the effect was due to DMSO. Therefore, these results may indicate that Po- peptides do not interact with ICILM-1. However, it is also possible that Po-peptides and anti-human CD54 mAb interact with different epitopes on the ICAM-1 mo1ecuIe.

1.3.2 Effect of LFA-1-derived peptide on the interaction of ICAiM-I and anti- ICAM-1

Direct immunofluorescence flow cytometry experiment was camed out with human M21 melanoma celk using FITC-conjugated anti-hurnan CD54 mAb in the presence of different concentrations of LFA-1 derived peptide (peptide-4, iMWt 1353.5 Da) to evaluate the effect of the peptide on the interaction of ICAM-1 and anti-ICAM- 1. Peptide-4, which is fiom the "I" domain of LFA-1, was expected to bind to IChiS4-1 on M21 cells and therefore compete with the interaction of anti-ICAM-1 mAb and ICAM-1 and decrease the fluorescence intensity. In this expenment, the cells were resuspended in PBS-BSA-AZ (5 x lo5 cells I 50 PL), preincubated with peptide-3

(0.125, 0.25, 0.5, 1, 2, 5, 10, 20 pg 1 5 x 105 cells) at 4OC for 30 min, and incubated with FITC- conjusated anti-human CD54 mAb (0.25 pg / 50 pL PBS-BSA-AZ). As a negative control, the FITC conjugated anti-bovine CD8 mAb was used. The results indicated that preincubation of M21 cells with peptide4 had no effect on the interaction of anti-ICAM-1 mAb with ICAM-1 on M21 cells (Table 4.4). The percent of stained cells and MFI were similar in the presence and absence of peptide-4. According to these results it was concluded that LFA- 1 denved peptide does not interact with ICAiM- 1 or LFA-1 denved peptide and anti-ICAM-1 mAb bind to different epitopes on ICAii- 1.

4.4 Study of association of biotinylated peptides with cells using indirect flow cytornetry

In this part of the study the ce11 binding ability of peptides was evaluated by indirect imrnunofluorescence flow cytometry. For this purpose, the peptides were biotinylated and afier the binding the cells were stained with streptavidin-PE for analysis. 1Olt

Table 4.4 The effect of LFA-1 derived peptide (peptide+ on die interaction of ICAM-1 on human M21 melanoma cells and FITC-conjugated anti- human CD54 rnAb (0.25 pJ50 PL).

fluorescence II II intensity

I None anti-bovine CD8 I None 1 anti-human CD54

-- -- r Peptide-4, 0.25 pg 1 anti-human CD 54 1 Peptide-4, 0.5 pg 1 anti-human CD54 Peptide-4, 1 pg anti-human CD54

Peptide-4, 2 pg anti-human CD54 1 Peptide-4, 5 pg 1 anti-human CD54 1 Peptide-4, 10 pg 1 anti-human CD54

- -- / Peptide-4,20 pg 1 anti-human CD54

4.4.1 Biotinylation of peptides Peptides were biotinylated with NHS-biotin in the presence of triethylamine. The biotinylation process of the peptides was monitored using CE (Appendix 1).

3.4.1.1 Biotinylation of Po-peptide-2 Due to its hydrophobic nature, the Po-peptide-2 was dissolved in chloroform/rnethanol (2/1 v/v), incubated with 3-fold rnolar excess of NHS-biotin and 2-fold molar excess of trÏethylamine over MIS-biotin ovemi~htat room temperature. To monitor the reaction, TLC was first used (developing system chlorofom /methmol hater 69231v/v; or n-butanol 1 glacial acetic acidl water 100/10/30 dv) and staining with ninhydrin or iodine vapor did not give acceptable results due to sensitivity or separation problerns. At this point capillary electrophoresis became available for the precise monitoring of the reaction. The biotinylated Po-peptide-2 was purified by diaiysis using a cellulose ester membrane (MWt cut off 500). Since the chloroforrn was incompatible with the dialysis membrane, the solvents of the reaction mixture were removed by rotary evaporation prior to dialysis. The dry reaction mixture had to be dissolved in a solvent(s) that was compatible with the cellulose ester membrane. The reaction mixture was soluble in methanol, but the methanol by itself was not suitable for the membrane. Methanollwater (20:80 v/v) was tried, however this did not result in the complete removal of Mis-biotin (as shown by TLC). The other solvents that were compatible tvith the membrane and could dissolve both peptide and NHS-biotin were hexans and toluene. First methanolhexane (20:80 vlv) tvas used, however, a11 of the hexane evaporated and after dialysis the TLC showed that the purification was not successful. Then a solution of methanol in toluene (20:80 vlv) that has lower volatility than hexane was used. The purification with this solvent systern was successhl as confirmed by TLC and later by CE. The CE electropherograrn of biotinylated Po-peptide2 is shown in Figure 4.14A. To analyze the reaction mixture in CE, the product after purification was dissolved in methanollwater (20:80 vlv). The CE electropherograrn of the control Po- peptide-2 (100 p=/120 pL of methanol/water 20% v/v) indicated that the Po-peptide-? elutes at 3.42 min, and its purity is 63% (Figure 4.14B). In addition to Po-peptide-2, there were some impurities that eluted at 4.26, 5.32, 5.59 and 6.21 min. The peak at 3.03 min is the rnethanol as sho~mby the contro1 CE of methanollwater (20:80 dv). According to the CE electropherogram of the purified product, there was some unreacted Po-peptide2 (peak 3.43 min) that constitutes 30% of the total product (Figure 4.14A). The peak at 2.94 min was methanol, and the peaks of 4.32 min and 6.07 min should be the biotinylated peptides, because in comparing with the electropherogram of control Po-peptide-2, these were new. Also, according to this electropherogram (Figure 4. MA) there was not any NHS-biotin in the final product. The control NHS-biotin was eluted at 3.91 min (as a solution in methanoVwater, 20:80 v/v, Figure 4.lrlC). According to the CE results, the purïty of NHS-biotin is 79%, the other main peak (6.57 min which comprises 12%) could be the hydrolyzed fom of the NHS-biotin, since the NHS-biotin hydrolyzes rapidly in aqueous solutions. Since the percent of umeacted Po-peptide-2 was relatively high (30%) afier purification, it was decided to repeat the biotinylation of Po-peptide-? to improve the reaction. The biotinylation reaction was carried out in dirnethylformarnide instead of chloroform/methanol because of its greater polarity in cornparison with chloroform, and the duration of reaction was increased to 24 hrs. The results cf biotinylation (Figure 4. MD) showed a signi ficant improvement and the percent of unreacted Po-peptide-2 was @eak 3.33 min) reduced to 15%. Reactions with longer time were tried ro improve the reaction, however it was not successfil. Then it was decided to use the biotinylated peptide product with 85% punty for flow cytometry experiment.

4.4.1.2 Biotinylation of Po-peptide-1 The biotinylation of Po-peptide-1 and the purification of biotinylated Po-peptide- 1 was can-ied out as explained in materials and methods. The CE electropherogam of control Po-peptide-1 (100 pd100 pL of methanoUwater, 20:80 v/v) showed that Po- peptide-l eluted at 4.37 min and its purity was 83% (Figure 4.15A). The peak at 2.94 min is rnethanol and the rest are impurities. The CE electropherogram of biotinylated Po-peptide-1 atter purification and dissolving in rnethanol/water (20:80 v/v) showed that there was no unreacted Po-peptide-1 which would elute at 4.37 min (Figure 4.1 SB). Therefore, it was concluded that the biotinylation of Po-peptide-1 was complete. The peak at 2.96 min was methanol and the peak at 4.08 min is the biotinylated Po-peptide- 1, and there are also other minor peaks that could be the biotinylated form of the Po- peptide-l impurities. Figure 4.14 B iotinylation off0-peptide-2. The Po-peptide-2 was biotinylated as explained in materials and methods and the progress of reaction was monitored by CE. A) The CE electropherograrn of biotinylated Po-peptide-2 (carried out in chloroform / rnethano 1) dissolved in methanolhater (20230 vlv). B) The CE electropherograrn of control Po-peptide-? dissolved in methanoVwater (20230 v/v). C) The CE electropherogram of NHS-biotin (control) dissolved in methanoVwater (2030 v/v). D) The CE electropherogram of biotinylated Po-peptide-2 (carried in DMF) dissolved in methanovwater (20:80 v/v). Figure 4.15 Biotinylation of Po-peptide-1. A) The CE electropherogram of control Po-peptide-1 dissolved in methanovwater (20:80 v/v). B) The CE electropherogram of biotinylated Po-peptide-1 dissolved in methanoYwater (2090 v/v). 3.4.1.3 Biotinylation of Po-peptide3 The Po-peptide-3 did not dissolve well in Dm. Therefore, for biotinyiation of Pa-peptide-3, DMSO was used and the biotinyiation reaction was camed out as explained in materials and methods. The CE electropherogram of contro l Po-peptide4 (100 pg/100 uL of methmolhater 20% v/v) indicated that Po-peptide-3 eluted at 2-52 min and its purity was 96 % (Figure 4.16A). The peak at 2.93 min is rnethanol and the peak at 3.27 min could be impurity (4 %). The CE electropherogram of biotinylated Po- peptide-3 after purification and dissolving in methanoVwater (20% v/v) showed that there is no unreacted Po-peptide-3 that would elute in 2.52 min (Figure 4.16B). Therefore, it was concluded that al1 Po-peptide3 was biotinylated, and the biotinylated Po-peptide-) was rnost likely the 3.66 min peak. The peak at 2.95 min is methanol and the peak at 4.10 min is the biotinylated form of the Po-peptide-3 impurity.

4.4.1.3 Biotinylation of LFA-1 derived peptide The LFA-1 derived peptide (peptide-4) was dissolved in NaHC03 buffer (0.1 MypH 8.3) and incubated with 3-fold molar excess of NHS-biotin dissolved in DMSO and 2-fold molar excess of tneihylamine over NHS-biotin for 24 hours at roorn temperature. To puri& the biotinylated peptide, the reaction mixture (freeze-dried to remove DMSO) was dissolved in water and dialyzed as explained in methods.

Figure 4.17A shows the CE electropherogm of control peptide-4 (1 mg 1 mL in water) which elutes at 4.08 min and its purity is 823%. The CE electropherogram of biotinylated peptide-4 aRer freeze-drying and dissolving in water showed two peaks at 3.900 and 4.017 min, where the first one was most Likely the biotinylated peptide-4 and the second one was peptide-4 (the peaks at 2.647 min, 4.770 min, and 5.200 min are DMSO, NHS-biotin and the hydrolyzed form of NHS-biotin, respectively) (Figure 4.17B). To confirm the position of biotinylated peptide-4, the sample was spiked with 0.5 pL of the peptide-4 solution in water (10 pJpL) (Figure 4.17C). According to this analysis the second peak was peptide-4 and the first peak was the biotinylated peptide- 4. The ratio of biotinylated peptide4 to peptide-4 was 75% (by analyzing the area under curve of the two peaks in Figure 4.17B). hcreased incubation tirne of more than 24 hours resulted in no further increase of biotinylated peptide-4 yield.

4.4.1.5 Biotinylation of RGD peptide The RGD peptide (peptide-5) was dissolved in NaHC03 buffer (0.1 M, pH 8.3) and incubated with 3-fold molar excess of NHS-biotin dissolved in DMSO and 2-fo[d molar excess of triethylamine over NKS-biotin for 24 hows at room temperature. To puri& the biotinylated peptide, the reaction mixture (freeze-dried to remove DMSO) was dissolved in water and dialyzed as explained in methods. Figure 4.1SA shows the CE electropherogram of control peptide-5 (1 mg I rnL in water) which elute at 1.90 min and its punty is 97 %. The CE electropherograrn of biotinylated peptide-5 afier freeze- drying and dissolving in water showed two peaks at 1.743 and 1.8 10 min, where the first one was most likely the biotinylated peptide-5 and the second one was peptide3 (the peaks at 2.367 min, and 2.533 min are MIS-biotin and the hydrolyzed form of MIS-biotin, respectively) (Figure 4.18B). To confirm the position of biotinylated peptide-5, the sarnple was spiked with 5 PL of the peptide4 solution in water (0.25 pg/pL) (Figure 4.18C). The CE electropherogrm showed that the second peak is peptide-5 and the first peak is the biotinylated peptide-5. According to the CE analysis the percent of biotinylated peptide-5 was 87.62% (Figure 4.18B).

4.4.2 Association of biotinylated Prrpetides with human M21 melanoma cells The results of staining of M21 cells with biotinylated Po-peptides (20, 50, 100, and 200 PM) were negative and none of the Po-peptides had adhesive activity for M3 1 cells. The intensity of staining was the same for control M21 cells and the cells in the presence ofdifferent concentrations of biotinylated Po-peptides (results not shown). The MF1 and PSC values were about 6 and 3, respectively, and did not change simificantly. To exclude the possibility that the lack of binding was not due to suboptimal conditions, the flow cytometry experirnent was re~eatedwith the following changes: 1) To improve the solubility of biotinylated Po-peptides, they were dissolved in a 0.1% Tri ton X- 100 soIution in water.

h E Omo C 0-

0031 0-

0-

0- A I

t

0- I 4 0-

Figure 4.17 Biotinylation of LFA-l -derived peptide (peptide-4). A) The CE electropherogram of control peptide-4, 1 pg/pL in water. B) The CE electropherogram of peptide-4 biotinylation reaction afier fieeze-drying anddissolving in water. C) The CE electrop lerogram of biotinylated peptide-4 solution in water (5 pL) spiked with peptide-4 solution in water (10 pg/pL, 0.5 PL). Figure 4.1 8 Biotinylation of RGD-peptide (peptide-5). A) The CE electropherogram of control peptide-5, 1 p&L in water. B) The CE electropherograrn of peptide-5 biotinylation reaction afier freeze-drying and dissolving in water. B) The CE electropherograrn of biotinylated peptide-5 solution in water (5 PL) spiked with peptide-5 solution in water (0.25 &PL, W)* To determine the interference effect of bovine serum albumin (BSA) in th2 flow cytornetry experiment (BSA were used as a 1% solution in PBS as a blocking agent to resuspend and wash the cells), the flow cytometry experiment was atso carried out in just PBS.

The results again showed that the biotinylated Po-peptides do not bind to MZ1 cells. There were no differences in the results of staining in the presence of PBS and PBS-BSA. Also. dissolving the peptides in the 0.1% Triton X-100 solution did not improve the binding ability of the Po-peptides. In surnmary, the biotinylated Po-peptides up to 200 pM had no adhesive activity for M2l cells. This may indicate either the Po-peptides do not have binding affinity for W 1 cells or the biotinylation affects the conformation of the Po-peptides such that they cannot bind to M21 cells.

4.4.3 Association of biotinylated LFA-1-derived peptide (peptide-4) with human M2 L melanoma cells

The results indicated that biotinylated peptide-4 (1, 2.5, 5, 10, 25, 50, 100, and 200 FM) does not have adhesive activity for M21 cells. The percent of staining and the intensity of staining were the same for control M2 1 cells and the cells in the presence of different concentrations of biotinylated peptide-4. To exciude the possibility that the Iack of binding is not due to suboptimal conditions, the flow cytometry experiment was repeated with the following changes:

1) To determine the interference effect of bovine semm albumin in the flow cytornetry experiment, the flow cytornetry experiment was repeated just using PBS instead of [PBS-BSA-AZ].

1) Since LFA-1 requires divalent cations in order to interact (Marlin and Springer, 1987; Dransfield and Hogg, 1989; Hynes, 1992), the flow cytornetry experiments were carried out in the presence of ImM MgC12. Overall the flow cytometry results showed that the biotinylated peptide4 up to 200 pM does not bind to M21 cells. There were no differences in the results of staining in the presence of PBS and PBS-BSA. Also, the presence of MgCl2 (used as a L mL1 solution in [PBS-BSA-AZ]) did not improve the binding ability of the peptide-4. The fiow cytometry results may indicate that either the LFA-1-denved peptide does not have binding affinity for Ad21 cells or the biotinylation affects the conformation of the peptide that it can not bind to Ml1 cells.

4.4.4 Association of biotinylated RGD-peptide (peptide-5) with HUT 78 T cells

It is well lsnotrin that LFA-1, and integrins in general, bind to Iigands containing the RGD (Arg-Gly-Asp) sequence (Ruoslahti and Pierschbacher, 1986 and 1987). For this reason the RGD peptide (peptide-5) was selected for biotinylation and indirect flow cytometry. To carry out the indirect flow cytometry expenment, a ce11 Iine which expresses LFA-1 was needed. Al1 leukocytes express LFA-1, however, LFA-1 to be able to react should be activated by e.g. phorbol esters. Among the different ce11 lines, it has been shown that HUT 78 T cells are a continuously activated T cells and does not need activation with phorbol esters (Nickoloff et al., 1992). Therefore, HUT 78 T tells were selected to detennine the adhesive activity of the biotinylated RGD-peptide. Therefore, Hm78 cells and M21 celis (as control) were first incubated with biotinylated peptide-5 (28, 66, 208, 323 and 484 FM), washed and then incubated with streptavidin-PE, and analyzed by flow cytometry. The results indicated that the biotinylated peptide-5 has adhesive activity for HUT 78 T cells but not for M21 ceils (Table 4.5). The binding of biotinylated peptide-5 to HUT 78 T cells gradually increased with increasing concentration of peptide and reached about 30% at 484 FM. In the case of M21 cells, biotinylated peptide-5 even at the highest concentration showed no adhesive activity. The binding of biotinylated peptide4 to HUT 78 T cells indicates that LFA-1 binds to RGD sequence as shown by others (Ruoslahti and Pienchbacher, 1986 and 1987). This expenment also indicates that indirect flow cytometry expenment using biotinylated peptides is a useful method for evaluating the adhesive activity of a peptide provided that the biotinylation does not affect the conformation of the peptide or its solubility. It should also be noted that in this TabIe 3.5 Bindinj of biotinylated RGD-peptide (peptide-5) to HUT 78 T cells and human M21 melanoma cells: indirect irnrnunofluorescence flow cytometric analysis using streptavidin-PE (0.5 pJ5O .ILL). rt))Treatment condition I None (control), 1 0.46 3.16 1 1.20 3.49 1

* Percent of stained cells. # Mean fluorescent intensity.

experiment the biotinylated peptide-5 was adhesive only at a relatively hijh concentration. In the previous flow cytometry experiments, using the biotinylated Po- peptide- l, -2, and -3 and also peptide4 the maximum concentration was 200 FM. Due to solubility restrictions, it was not possible to cary out the experiment with a concentration of more than 200 PM, especially with Po-peptides.

4.5 Study of inhibition of the adherence of PMA-activated Jurkat T cells to IFN-y-stimulated human keratinocytes by peptides In order to identiw the probable adhesive peptides from Po protein or LFA-1 for tarjeting toward ICAM-1, peptide cornpetition studies were camed out to inhibit the adherence PMA-activated Jurkat T cells to IFN-y-stirnulated human keratinocytes. It has been shown that the PMA-activated Jurkat T cells bind to IFN-y stimulated human keratinocytes and this binding is mediated by the interaction of adhesion molecules LFA-1 (T cells) and ICAV-2 (keratinocytes) (Nickoloff et al., 1988 and 1992; Stoof et al., 1992; Bruynzeel et al., 1995). Among the different T ce11 lines, it has been sho~m that HUT 78 T cells also bind to the noncytokine stimulated keratinocytes using an adhesion pathway other than LFA-1IICAiM-1 (Nickoloff et al., 1992). For this reason the Jurkat T ceII line was selected for our experiments since it interacts only with cytokine stimuIated keratinocytes (Nickoloff et al., 1992). However, for the inirial development of the cell-ce11 adhesion assay HUT 78 T cells were used, since the Jurkar T ce11 line was not available yet. In the initial cell-ce11 adhesion assay, neutral red dye was tried. Live cells can take up the neutrai red and result in a dark color which is measurable at 540 nm using a spectrophotometer. In general, the results using neutral red dye had large variation and the sensitivity of the method was limited. Therefore, instead a highly sensitive fiuorimetric method using calcein AM (Braut-Boucher et al., 1995; Fargeas et al., 1995) tvas used. Caicein AM (acetoxymethylester of calcein) is a nonfluorescent cornpound which is taken up by cells as the ester. Live cells hydrolyze cakein AM to the intensely fluorescent calcein by intracellular estrases. Unlike the other fluorescent probes where staininj of the cells depends on the intracellular pH, the staining of the cells by calcein is pH-independent (Kolber et ai., 1988). Extensive studies have shown that calcein has a high ievel of intracellular retention (Wang et al., 1993). Moreover, it has been found that calcein AM has no effect on the adhesion properties of calcein-labeled lymphocytes (Braut-Boucher et al., 1995), and no deleterious effects on ce11 function and is suitable for in vivo studies and it is actually a ce11 viability marker (Weston and Parish, 1992). Also the sensitivity of the method using calcein AM is comparable to adhesion assays using radiolabelled compounds (Braut-Boucher et al., 1995).

4.5.1 Isolation and growth of adult normal human epidermal keratinocytes In this part of the study a human epidermal keratinocyte ce11 line was estab lished. The keratinocytes were iso lated fkom human breast or abdominal skin a Aer cosmetic surgery. We developed a primary culture of epidemal keratinocytes and induced expression of ICAM-1 with IFN-y in order to use them in the cell-ce11 adhesion assay and as a mode1 for idammatory conditions and dmg targeting studies.

3.5.1.1 Growth c haracteristics of keratinocytes The morphological appearance of human epidermal keratinocytes observed under phase contrast light microscopy during different stages is shown in Figure 4.194~- E. The l-day-old isolated keratinocytes consisted of srnail translucent round cells and Iarger angulated flat cells (Figure 4.19A). The cells aîtached to the plastic tissue culture flask within 3 days. Most of the small round cells were attached firmly to the flask as opposed to the angulated flat cells that were attached loosely. Figure 4.19B shows a 3- day-old culture. This picture was taken after the first replacement of medium without rinsing the culture. There was no difference in the appearance of the cells between the 3- and 1-day-old culture. Figure 4.19C shows a 8-day-old culture. This was taken after the second medium change and washing hvice with Hank's solution. There was a sigificant difference in the appearance of the cells at this stage compared to earlier stages. The culture consisted of only the small round cells, and most of them were proliferating and appeared to be in a growing stage. The angulated fIat cells were completely washed away by rinsing the culture at this stage. These flat cells were most likely connective tissue fibroblasts that loosely attach to the tissue culture flask and were washed away by rinsing. The K-SFM medium with 0.1 mM ~a"is a defined medium for selectively growing epidermal keratinocytes and suppressing the growth of fibroblasts (it has been shown by Boyce et al., 1983 that extracellular 0.1 mM ca2+ concentration can control the differentiation of hurnan epidermal keratinocytes). Figure 4.19D shows a 12-day-old culture. At this stage the cultures were about 70% confluent, and almost al1 of the cells were in a proliferative stage. Figure 4.19E shows a 16-day- old culture that is fully confluent. After this stage the cells did not grow. With respect to the growth of human epidermal keratinocytes three stages were obsenred. A lag phase of 5-days during which the cells were attached to the tissue gu re 4.19 Phase contrast light micrographs of human epidermal keratinocytes. The human keratinocyte cultures were photographed in different stages of growth using a phase contrast microscope. A, 1-day-old culture; B, 3- day-old culture; 8-day-old culture; D, 12-day-old culture; E, 16-day-old culture (mapification x 200). culture flask and did not gow. A multiplication phase of about 10-days when the cells were dividing until the culture reached a confluent monolayer, followed by a plateau phase where the cells stopped growing. For subdividing or freezing the cells, a culture with approximately 70-80% confluency was used, othenvise the viability of the cells was decreased.

4.5.1.2 Ultrastructural characterization of keratinocytes by transmission electron microscopy The aim of this part of study was to characterize the ultrastructure of the isolated epidermal keratinocytes, to demonstrate the selective culturing of keratinocytes and to dernonstrate the absence of other contaminant cells such as melanocytes, Langerhans cells, Merkel cells or fibroblasts.

The epidermis consists of a mixed population of ce11 types. Arnong the keratinocytes that constitute the majonty of the population, there are nonkeratinizing cells that migrate into the epidermis. The main immigrant cells are rnelanocytes and Langerhans cells. Ultrastructurally, the main features of keratinocytes are bundles of keratin filaments and melanosomes in their cytoplasm that surround the nucIeus. Melanocytes have numerous melanosomes but they do not have keratin filaments. The characteristic feature of Langerhans cells is the presence of tennis racket-shaped Birebeck granules and the lack of keratin filaments in the cytoplasm. Merkel cells are also found in the epidermis. They are charactenzed by electron dense granules. During the isolation of epidermal keratinocytes, usually fibroblasts are contarninating the culture but later they are selectively removed using special K-SFM medium and rinsing. The cytoplasm of fibroblasts is full of organelles especially abundant in rough endoplasmic reticulurn and mitochondna and they lack keratin filaments (Montagana et al., 1992). The electron rnicropphs of human epidermal keratinocytes, which were fixed with glutaraldehyde and osmium tetroxide, are shown in Figures 4.20A-C. A total of 190 cells from different sections were examined under the microscope. Al1 examined ceils had keratin filaments, 26 of them had meIanosomes. Other mentioned organelles that may be specific for Langerhans cells, Merkel cells, and fibroblasts could nor be seen. Based on the EM evatuation, the isolated cells were accepted as a pure culture without contaminating melanocytes or other cells.

4.5.1.3 Induction of expression of ICAM-1 in keratinocytes by in terferon-y We induced expression of ICAM-1 in cultured epidermal keratinocytes with different concentrations of EN-y in order to fmd the optimum concentration of IFN-y for later studies. The human epidermal keratinocytes (5 x 10' celIs/weIl) were seeded into 6-well plates and groum. The confluent monolayers of keratinocytes were incubated with or without the addition of various concentrations of IFN-y (0, 10, 25, 50, 100, 250, 500, 1000 IU/ml medium) for 24 hrs. Then the celIs were detached, counted and stained wiîh FITC-anti human CD54 mAb and analyzed by a FACScan flow cytorneter.

The results indicated that IFN-y upregulates the expression of ICAM-1 (Figure 4.21). The percent of stained cells reached near 100% with 100 IU EN-y, and also the MF1 reached almost a plateau under the same conditions. Accordinj to these results, IFN-y with a concentration of 100-500 W/mL would be suitable for induction of ICAM- 1 expression in human keratinocytes. Figure 4.20 Transmission electron micrographs of cultured epidermal keratinocytes. Cultureci human keratinocytes were detached, fixed in 2.5% glutaraldehyde and 2% osmium tetroxide, stained with 2% uranyl acetate and Renold's lead citrate as described in materials and methods, then the cells viewed and photographed under transmission electron microscope (F = keratin filaments, M = melanosomes, N = nucleus, A: 15000 x, B: 13000 x, C: 23000 x). Figure 4.20 continued Figure 4.20 continued / 1-1 Facent of stained cells (PSC)

Figure 4.21 Induction of expression of ICAM- 1 on hurnan epidermal keratinocytes by IFN-y. The confluent monolayers of keratinocytes were incubated with or without the addition of EN-y. Then the cells were detached, resuspended in [PBS-BSA-AZ] (5 x 10' / 50 PL) and incubated with FITC-conjugated anti-human CD54 rnAb (0.25 pg / 50 PL). Then the cells (1 x 104 volume gated) were analyzed by flow cytometry. 4.5.2 HUT 78 T cells binding to hurnan keratinocytes The cell-cell adhesion assay was carried out to evaluate the binding characteristics of HUT 78 T cells to hurnan keratinocytes and also to determine the optimal conditions for the assay. The cell-ce11 adhesion assay was camed out as explained in Materials and Methods, however, initially the following parameters were different:

A) The incubation tirne of T cells with keratinocyte rnonolayers was 60 min at 37°C incubator.

B) The nonadherent T cells were removed fkom keratinocyte monolayers by washing with ice-cold RPMI-FCS, 3 tirnes. In this experîment the following controls were also included:

1) Biank, consisting of non-labeled T cells deposited on keratinocyte monolayer.

2) RPMI- 1640 media to measure its fluorescence. 3) Supernatant of the cells after incubating with CAM, after the third washing.

4) Release of CAM fiom T cell-labeled, by incubation of 200,000 T cells (CAW loaded) for 60 min at 37OC then centrifugation at 800 rpm for 10 min and measuring the fluorescence of the supernatant, The results of the cell-ce11 adhesion assay indicated that HUT 75 T cells bind to

both stimulated and .non-stimulated keratinocytes (Table 4.6). The percent O f adherence for IFN-y-stimulated keratinocytes was higher than the non-stirnulated keratinocytes. However, the extent of binding in both situations was very high, even higher than the data shown in the literature (% adherence of about 20, Nickoloff et al., 1992). The fluorescent unit values of the blank, non-labeled T cells deposited on keratinocyte monolayer, was quite low (1.35 k 0.14). The control value of RPMI media was also small (0.81 t 0.08). The fluorescent unit of supematant of the HUT 78 T cells after the third washing was 1.50 + 0.13 which is also srnall compared to those values obtained in the binding assay (74.40). The fluorescence unit for release of calcein aAer incubation of 100,000 calcein-loaded T cells for 60 min at 37OC was 5.90 k 0.96. This amount accounts only for 7.97% of the total input.

Table 4.6 Adhesion of calcein-labeled HUT 78 T cells to keratinocyte monolayers for 60 min at 37OC.

------No of HUT 75 Fluorescence Fluorescence Percent of units T cells adherence \vas hed*, per well

r(r Fluorescence intensity after removing the nonadherent T cells by washlng. ff Fluorescence intensity without washing (total input).

To determine the sensitivity of the cell-ce11 adhesion assay, the fluorescence emissions of calcein-labeled HUT 78 T cells (1-20 x 10' cells / well) was also detemined in this experiment. The fluorescence intensity emitted by 1000 cells was statistically different (p < 0.005) fiom that emitted by the blank (Figure 4.22). There Blank 1O00 2000 4000 8000 20000 Number of HUT 78 T cells

Figure 4.22 Determination of the sensitivity of the ceIl-ce11 adhesion assay using calcein AM. CAM-labeled lymphocytes (1,000-20,000 cells / well) were seeded in 96-well plates. After 30 min incubation the cells were lysed using Triton X-100 and the arnount of fluorescence intensity in each well was quantified using a fluororneter at excitation 485 nm/ emission 530 nrn as described in Materials and Methods. Each bar represents the mean ISD of 3 separate experiments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences @ < 0.005) arnong the fluorescence intensities of different numbers of T celIs and also compared to blank. was also significant difference (p c 0.004) in the fluorescence intensity emitted by the different nurnber of cells examined in the experiment. Since the percent adherence of the HUT 78 T cells to keratinocytes was higher than expected (Table 4.6), the cell-cell adhesion assay was repeated with the following changes :

1) The incubation condition was changed from 60 min at 37°C to 30 min at room temperature.

2) Afier 30 min incubation at room temperature, the wells were washed with ice- cold RPMI-FCS, 7 times instead of 3 times. The percent of adherence of HüT 78 T cells to keratinocytes (stimulated with 500 IU of IFN-y) was reduced to 52.41 + 5.00. Even though this time the percent of adherence was less than the previous experiment, it was still higher than previous data in the literature. Then, the ceII-ce11 adhesion experiment was repeated with two more changes. First, washing the wells afier 30 min incubation at room temperature was c&ed out with room temperature WMI-FCS instead of ice-cold RPMI-FCS, and second the wells were washed 20 times instead of 7 times. The results obtained by this protocol were comparable with previous data in the literature (Table 4.7). The results indicated that the HUT 78 T cells bind to both stimulated and non-stimulated keratinocytes, however, the extent of binding was geater with the stimulated keratinocytes. Incubation of keratinocytes with both IFN-y and PMA increased the binding of rrtlT 78 T cells dose dependently. The highest percent of adherence was achieved with the combination of IFN-y and PMA. The extensive washing procedure was needed in this protocol to remove the nonadherent and Ioosely attached T cells; othenvise, the percent of binding would have been high and there would not have been any differences between various treatments of cells (as seen in Table 4.6). On the other hand, the extensive washing procedure to remove nonadherent cells in cell-ce11 adhesion assay has been used by others, too. Nickoloff et al. (1992) washed the keratinocyte monolayers 20-30 times to remove the nonadherent HUT 78 T cells or Jurkat T cells from the monolayer, It is known that both EN-y and PLMA upregulate the expression of ICAM-1 in human keratinocytes (Nickoloff et al., 1992). The rnolecular basis of the adhesion HLiT 78 T ceiis to the IFN--f- andor PM- stirnulated keratinocytes is attributed to the interaction of LFA-I (HUT 78 78 cclls) and CAM- 1 (keratinocytes). In summary, the conditions for the cell-ce11 adhesion assay were optirnized. The optimum results were achieved with incubation time of 30 min at room temperature, and washing the wells with room temperature WMI-FCS 20 times. According to the cell-ce11 adhesion results, HUT 78 T cells binds to the IFN--f- andor PMA-stimulated keratinocytes and it is more pronounced in the presence of both.

Table 4.7 Adhesion of calcein-labeled HUT 78 T cells (2 x 10~/well) to keratinocyte monolayers for 30 min at room temperature. 1 Y"" Fluorescence unit ,,i_ "::E [ NO&# (unwashed, total) ' 91.10 + 4.76 -

PMA 5 nghL 36 hrs 17.75 + 1-80 19.48 + 1.98 IFN-y 250 IU/mL 3 6 hrs 21 -65 + 0.59 23.77 It: 0.65 PMA 5 ngmL 36 hrs

if Fluorescence intensity without washing (total input). * Fluorescence intensity aRer removing the nonadherent T cells by washing.

4.5.3 Jurkat T cells binding to human keratinocytes

CeII-ce11 adhesion assay was carried out to establish conditions for the PMA- activated Jurkat T cells to IFN-y stirnulated human keratinocytes binding protocol. The cell-cell adhesion assay was carried out as explained for HUT 78 T cells and human keratinocytes. The Jurkat T cells, after loading with calcein AV and washing 3 times, were activated with different concentrations of Pm4 for 30 min at room temperature and added to the washed (with serum free keratinocyte medium) IFW-/- and/or PMA-

treated keratinocyte plates at concentration of 2 x 105 cells per well. Afier 30 min incubation at room temperature, non-adherent Jurkat T cells were rernoved by repeated washing and after lyzing the cells the fluorescence intensities were measured. In this experiment the fo llowing contro 1s were also included:

1) Blank, consisting of non-labeled T cells deposited on the keratinocyte mono layer.

2) Release of calcein AM fiom hrkat T cell-labeled, by incubation of 200,000 T cells (calcein loaded) for 30 min at room temperature then centrifugation at S00 rpm for 10 min and rneasuring the fluorescence of the supernatant. The results indicated that binding of native- and PMA-activated Jurkat T cells to unstimulated keratinocytes was minimal, but T cells activated with PMA showed a strong adhesion to IM-y-stimulated keratinocytes @ 0.001) (Figure 4.23). Different concentrations of PMA to activate Jurkat T cells had little effect on their adherence to the EN-y-stimulated keratinocytes. The combined keratinocyte treatrnent with IFN--y and PMA synergistically increased Jurkat T celis binding @ < 0.05, compared with just [FN-y-stimuiated keratinocytes). PMA treatment of keratinocytes also mediated binding of PMA-activated Jurkat T cells (p c 0.00 1, compareci with unstimulated keratinocytes), however, it was less than the IFN-y-stimulated keratinocytes @ c 0.003). The blank wells consisting of non-labelled T cells deposited on the keratinocyte monolayers had little background fluorescence (1.14 k 0.04). The spontaneous release of calcein from labeled Jurkat T cells by incubation of 200,000 T cells (calcein loaded) for 30 min at room temperature was 2.80 + 0.48 which is less than 5% of the total fluorescence. The ability of IFN-y to induce the binding of PMA-activated Jurkat T cells to the keratinocytes, suggests involvement of ICAM-1. To confirrn the expression of IC~~-1 in human keratinocytes in the presence ofIFh*-y, the cells were detached from 96 well KC PMA 5 ng/rnL JC PMA 5 ng/mL

1 KC IFN-y 500 U/mL * and PMA 5 ngi mL, JC PMA 5 ngImL

KC IFN-y 500 U/mL 1 JC PMA 5 ng/mL

KC IFN-y 500 U/mL JC PMA 0.5 ng/mL

KC IFN-y 500 UImL 1 1 JC PMA 0.1 ng/mL 1 !

% Adherence

Figure 4.23 Adherence of calcein-Iabelled Jurkat T cells to keratinocyte monolayers for 30 min at roorn temperature. Keratinocyte (KC) rnonolayers were incubated with EN-y or PMA for 36 hrs and washed once with K-SFM media before the adhesion assay. Jurkat T cells (JC) were labelled with CAM, activated with PMA for 30 min at room temperature pnor to the adhesion assay. The T-cells were added (2 x 105/well)to keratinocyte monolayers without washing for 30 min at room temperature. The non-adherent T cells were removed by repeated washes and the percent of adherence was determined as described in Materials and Methods. Each bar represents the mean + SD of 3 separate experiments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There was no significant difference @ > 0.1) between the binding of native and Pm-activated T cells to native keratinocytes. There were significant differences (p < 0.001) in the binding of PMA-activated T cells (al1 concentrations) to FN-y stimulated keratinocytes compared to the binding of native and PMA-activated T celk to native keratinocytes. plates and analyzed by direct flow cytometry. The FACS analysis revealed that I~-;I upreylates the expression of ICAM- 1 in human epidermal keratinocytes. The treatmenr of keratinocytes with EN-y 500 IU for 36 hrs increased the percent of stained cells fiom 8.00 to 94.6 and the mean fluorescence intensity fiom 7.97 to 112.55. In brie6 according to the cell-ce11 adhesion assay, the PMA-activated Jurkat T cells bind to the EN-y- andor PM-stirnulated keratinocytes and it is more pronounced in the presence of both (Figure 4.23). This binding is believed to be mediated by the adhesion moIecules LFA-1 (T cells) / ICAM-1 (keratinocytes).

4.5.4 Inhibition of adherence of PMA-activated Jurkat T celIs to IFX-y- stimulated human keratinocytes by LFA-1-derived-, RGD-, and Po-peptides

To determine whether LFA-1-denved- and RGD-peptides can interfere with the binding of PMA-activated Jurkat T ceils to EN-y-stimulated keratinocytes peptide cornpetition studies were cax-ried out. The peptides were added to the wells at various concentrations 10 minutes before the addition of the calcein-loaded Jurkat T cells. In this expenment the effect of mouse monoclonal antibody to human CD54 (ICAM-1) on the adhesion of PMA-activated Jurkat T cells to EN-y-stimulated keratinocytes was also investigated. For this purpose, the human keratinocyte cells were incubated with anti-CD54 mAb (1 pd100 pL/well) 10 minutes before the addition of calcein-loaded T

The results indicated that the percent adherence of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes decreased sisificantly to 69.40, 78 -75, and 83.45 in the presence of 1, 0.1, 0.0; mM of LFA-1 derived peptide, respectively Figure 4.21). The effect of RGD-peptide was smaller (statistically nonsignificant), a decrease to 85.97, 91.40, and 96.71 in the presence of 1, 0.1 and 0.01 mM of the peptide was measured. The anti-CD54 mAb apparently did not have any effect on the binding of PMA-activated Jurkat T celIs to EN-y-stimulated keratinocytes.

One-way ANOVA was camed out to detemine whether there are significant differences in the inhibition study using the LFA-1 denved and RGD peptide. The one- RGD-pep tide 0.01 mM RGD-pep tide 0.1 rnM RGD-peptide 1 rnM LFA- 1-peptide 0.01 rnM LFA- 1-peptide 0.1 mM LFA- 1-peptide 1 rd4 Anti-CD54 rnAb 1 pg / 100 & None

% Adherence

Figure 4.24 Inhibition of adherence of PMA-activated Jurkat T cells to IFN-y- stimuIated keratinocytes by LFA- 1-denved and RGD-peptides. Keratinocyte rnonolayers were incubated with EN-y (500 IU/rnL) for 36 hrs and washed once with K-SFM media before the adhesion assay. CAM-loaded Jurkat T cells (JC) were activated with PMA (1 ng/mL) for 30 min prior to the adhesion assay. The T-cells were added (2 x l~'/well) to keratinocyte monolayers without washing for 30 min at room temperature. The peptides and anti-CD54 mAb were added to the weIls 10 min before adding the T cells. The non-adherent T cells were removed by repeated washes and the percent of adherence was detemined as desdbed in Materials and Methods. The adhesion in the presence of peptides and rnAb is expressed as a percent relative to the adhesion of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes which considered as 100%. Each bar represents the mean c SD of 3 separate experiments, Statistical analysis was carried out by one- way ANOVA with multiple cornparison Tukey test to compare the means of different treatment groups. The result of statistical analysis is presented in Table 4.8. way PSLTOVA test showed a siagificant difference @ < 0.001) for the resutcs. Then a multiple cornparison Tukey test was used to compare the means of different treatment g-roups. The multiple cornparison test indicated that the effect of the LFA-1 derived peptide in the reduction of adhesion is significant at al1 concentrations (Table 4.8). However, the effect of the RGD-peptide in the reduction of adhesion was not significant at any concentration tested-

To compare the effect of the LFA-1-derived- and RGD-peptide at different concentrations, hvo-way ANOVA was carried out. This analysis revealed that there are significant differences between the effect of the bvo peptides in the reduction of adhesion (p < 0.001)-

Table 4.8 Statistical analysis of the effect of LFA- 1-derived- and RGD-peptides on the adhesion of PMA-activated-Jurkat T cells/IFN-y-stimdated- keratinocytes by ANOVA and Tukey test.

Cornparison Significance level

-4

With regards to the Po-peptides, due to the solubility problerns, especially Po- peptide-2, various solvents (DMSO, PG, PEG 400) were tried to dissolve the Po- peptides for conducting the competition study. The cell-ce11 adhesion assay was carried out as explained in the Materials and Methods. The results showed strong inhibition of adherence of PLMA-activatedJurkat T cells to IFN-y-stimulated keratinocytes by Po-peptides solutions in DMSO (Table 4.9A). However, the DMSO itself also showed bindins inhibition. The effect of Pa-peptide solutions in DMSO appeared to be greater than the DMSO alone. To avoid the interference by DMSO, it was decided to repeat the experiment using propylene glycol as solvent for Po-peptides. Among the peptides, Po-peptide-7 had good solubility in propylene glycol, Po-peptide4 was relatively soluble, and Po-peptide- 3 was alrnost insoluble. However, after rnixing the Po-peptides solution with WMI- FCS, the Po-peptide-l and -3 ais0 resulted in a clear solution. The result of this experiment was similar to the DMSO one (Table 4.9B). To exclude solvent effects in the assay, the solubilization of Po-peptides in RPMI-FCS was attempted. Po-peptide4 and Po-peptide-3 were soluble at 1 mihl concentration. However, Po-peptide-2 was not soluble even at very low concentrations. The cell-ce11 adhesion assay was carried out only in the presence of Po-peptide-1 and Po- peptide-3 that were dissolved in WMI-FCS at l rnM concentration. The results indicated that the Po-peptide-1 significantly @ < 0.001) interferes with the LFA- 1flCAM-1 mediated adhesion of Jurkat T cells to keratinocyte (Figure 4.35). The percent of adherence of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes decreased by 3 1% in the presence of Po-peptide-1. The effect of Po-peptide3 was smaller and a decrease of 21% was found @ c 0.01). The effect of Po-peptide1 was slightly higher than Po-peptide-3 (0.05 < p < 0.1). In an another experiment, Po-peptide-2 (1 rnM) was tried one more time as a solution in polyethylene glycol (PEG) 400. The cell-ce11 adhesion assay indicated that the effect of Po-peptide-2 (1mM) dissolved in PEG 400 was similar to control PEG 400 (Table 4.9C). Therefore, it was concluded that the Po-peptide-2 does not compete with PMA-activated Jurkat T cells and IFN-y-stimulated keratinocytes binding.

In summary, it was found that the LFA-1-denved peptide and Po-peptide-1 inhibit PMA-activatecl-Jurkat T cellslIFN-y-stimulated-keratinocytes adhesion which is mediated by the interaction of ICAM-ILFA-1. The magnitude of the effect of the LFA- 1-denved peptide and Po-peptide-1 in the inhibition LFA- 1AC AM- 1 interaction was Table 4.9 Inhibition of adherence of PM-activated Jurkat T cells ro EN-y- stimulated keratinocytes by Po-peptides dissolved in different solvents.

Peptide treatrnent Fluorescence units % adhesion" II II A) DMSO None (unwashed, total input) 39.60 t 1-00 - I None* (washed) 20-05 k 0.90 50.63 + 2.27 Po-peptide-1 1rnM in DMSO 6.53 t 1.14 16.46 t 3-58 Po-peptide-2 1mM in DMSO 0.20 3- 0.02 0.51 I0.05

Po-peptide4 1rnM in DMSO 7.92 t_ 1.5 1 20.00 r: 3.5 1 DMSO control 9.82 C 1.68 24.80 ir 4.24 ~one'(washed, no IFN-y) 6.86 t 0.96 17.32 4 2-43

1 B) Propylene Glycol None (unwashed, total input) 1 77.63 + 7.07 - None* (washed) 63-15 k 2.09 80.06 t 2.69

PG control 36.36 + 3.82 46.84 t 4.92 I ~one'(washed, no EN-y) 34.64 k 6.57 43.62 + 8.46 1 I C) PEG 400 None (unwashed, total input) 1 99.57 k 5.05 - I None* (washed) 40.38 k 5.43 40.55 + 5.45 IY Po-peptide-2 ImM in PEG 400 1 3.20 t 0.29 3.21 + 0.29 I 1 PEG 400 control 4.63 k 1.O9 4.65 + 1.09 ~one'(washed, no IFN-y) 21.10 k 4.92 21.19 -t- 4.94

Binding of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes. Binding of PMA-activated Jurkat T cells to keratinocytes. Ratio of fluorescence intensity aRer binding (washing) and fluorescence intensity without washing (total input) x 100. Figure 4.25 Inhibition of adherence of PMA-activated Jurkat T cells to IFN-y- stimulated keratinocytes by Po-peptide4 and -3. The PMA (hg/ mL 30 min)-activated Jurkat T cells/IFN-y (500 IU / mL 36 hours)-stimulated keratinocytes adhesion assay in the presence of Po- peptide-l and -3 was carried out as described in the legend of Figure 4.24. Each bar represents the mean + SD of 3 separate experiments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences between none and Po- peptide-l @ < 0.001), and none and Po-peptide-3 @ < 0.01). There was no significant difference between Po-peptide4 and Po-peptid-3 (0.1 < p c 0.05). comparable. The Po-peptide-3 also had an effect but it was lower than the LFX-1- denved peptide and Po-peptide-1. Po-peptide-? and RGD-peptide had no effect- The cell-ce11 adhesion assay also indicates that the available epitope of anti-CD54 m4b is not interfenns in the interaction of ICAX-ILFA-1. The effect of LFA-1-derived peptide and Po-peptide-1 in the inhibition of LFA-LIICAM-1 interaction might be through adhesion to ICAM-1; therefore, these peptides may be useful as ligands for targeting to IChM- 1.

4.6 Linking of peptides to the liposomes containing N-@ut-PE

Table 4.10 shows the surnmary of various experiments that have been carried out to determine probable adhesive peptides for targetins to ICAM- 1. The Po-peptide-1 and Po-peptide-3 competed with the association of Po-liposomes to huma? MZ1 cells and decreased the binding by about 30 and 40 percent, respectively. The LFA-1-derived peptide and Po-peptide-1 and Po-peptide-3 partially inhibited the LFA-1IICAiM-1 rnediated adhesion of PMA-activated Jurkat T CelIs to EN-y-stimulated keratinocytes. We concluded that the effect of these peptides rnight be through adhesion to ICM-1 hence these peptides may be useful as ligands for targeting to ICAM-1. Therefore, the purpose of this part of the study was to covalently link the LFA-1-denved peptide and Po-peptide-1 and Po-peptide-3 to liposomes containing N-Glut-PE, to detennine their direct adhesive activity for melanoma cells and human keratinocytes.

The linking of peptides to the liposomes containing N-glut-PE was carrïed out by two-stage immobilization procedure as described by Bogdanov et al., 1988 (Figure 4.26). In this procedure, first the carboxylic group on the surface of liposomes is activated using EDC for a short period of time (10 min) at pH 5.5 to form an O- acylisourea intermediate. Then at the second stage, the peptide solution is added with sirnultaneous change of pH to 8; the O-acylisourea intermediate reacts with amines resulting in the formation of a peptide bond. Using EDC alone at the first stage has sorne drawbacks resulting in a decrease in the yield of coupling. The O-acylisourea intermediate hydrolyses rapidly and results to the starting reaction components (EDC and N-Glut-PE). Also, the O-acylisourea isomerizes and converts to the non-active N- acylurea. However, the presence of sulfo-NHS at the first stage of the reaction forms the active N-hydroxysuIfosuccinimide esters that are more resistant ro hydrolysis than the O-acylisourea intermediate and also they are not isomerizable. Therefore, adding sulfo- NHS to EDC mediated coupling of peptides would result in a higher coupling yield.

Table 4.10 Summary of results involving peptides in detemining the probable adhesive peptides for targeting to ICAM- 1.

Experirnent LFA-1 -derived peptide

- -- . mPo-liposome mediated 30% 40% NA* 1 adhesion to M21 cells inhibition inhibition inhibition ICAM-1 and anti No effect No effect No effect No effect ICAM-1 mAb interaction

1 ~dhesionof the Negative Negative Negative Neg ative biotinylated peptides to M2 1 cells

31 % No effect 21 % 3 1 % inhibition mediated adhesion of inhibition of inhibition of of adherence at PMA-activated Jurkat T adherence at adherence at 1 mM cells to IFN-y-stimulated 1mM ImM keratinocytes

* Not applicable.

4.6.1 Lin king of LFA-1-derived peptide To monitor the linking of the peptide to the liposomes and determine the yield of linking, CE was used by an indirect method of analyzing the liposome supernatant for fiee (unlinked) peptide. Figures 4.27A and 4.27B shows the CE electropherogram of the supernatant of peptide-4-liposomes and control liposomes after the fint centrifugation (first washing), respectively. Peptide-4-liposomes have a peak at 5.42 min, representing the unlinked peptide Baction. Control peptide4 (1 pg /PL in water) elutes at 5.14 min (Figure 4.27C). The other peaks in the supematant of both peptide4 and control Iiposomes at 2.4, 3.6 and 11.3 min were identified as EDC, an urea derivative (which is released kom EDC during reaction), and sulfo-NHS, respectively. The supematants of both control liposomes and peptide-4-liposomes after the second and third washes showed no peak at 5.42 min. The percent of unlinked peptide in the supematant of peptide-4-liposomes was quantitated by comparing it to a peptide standard (Table 4.1 1). This analysis revealed that there was 16.2% unlinked peptide-4 in the liposome supematant. Therefore, the iiposome-linking efficiency of peptide-4 is considered to be 83 -8%.

4.6.2 Linking of RGD-peptide Figures 4.28A and 4.28B shows the CE electropherograrn of the supematant of peptide-5-liposomes and control liposomes after the first centrifugation (first washing), respectively. These nvo CE electropherogams were very similar, indicatins virtually cornplete linking of peptide3 to the liposomes. The control peptide4 (2.2 pg /PL in water) elutes at 4.300 min (Figure 4.28C). The percent of unlinked peptide in the supematant of peptide-5-liposomes was quantitated by comparing it to a peptide standard (Table 4.1 1). This analysis revealed that there was 10.0% unlinked peptide4 in the liposome supematant. Therefore, the liposome-linking efficiency of peptide4 is considered to be 90.0%.

4.6.3 Linking of Po-peptide4 Figures 4.29A and 4.29B shows the CE electropherogram of the supematant of Po-peptide-1-liposomes and control liposomes aRer the first centrifugation (first washing), respectively. Po-peptide-1-liposomes have a peak at 4.963 min representinj the unlinked peptide fraction. Control Po-petide-l (1 pg /PL in water) elutes at 5.037 O=C I OH Liposomes confaining N-Glut-PE groups

O II C-O

+ HN - peptide 2

2.5 hrs

I N- I Peptide

Figure 4.26 EDC/sulfo-NKS mediated coupling of peptides to liposomes containing N-Glut-PE.

Figure 4-27 Linking of LFA-1-derived peptide (peptide-4) to liposomes containing N-Glut-PE. A) The CE electropherogram of peptide-4-liposomes supernatant. B) The CE electropherogram of control-liposomes supernatant. C) The CE electropherogram of peptide4 standard, 1 &pL in water. looro

Figure 4.28 Linking of RGD-peptide (peptideJ) to liposomes containing N-Glut-PE. A) The CE electrophero gram of pep tide-5-liposomes supematant. B) The CE electropherogram of control-liposomes supematant. C) The CE electropherogram of peptide-5 standard, 2.25 pg/pL in water. Figure 4.29 Linking of Po-peptide4 to liposomes contaking N-Glut-PE. C) ~heCE electrophero gram of Po-peptide- l -liposomes sup ernatant. D) The CE electropherogram of control-liposomes supernatant. D) The CE electropherograrn of Po-peptide-1 standard, 1 pg/pL in water. min (Figure 4.29C). The percent of unlinked peptide in the supernatant of Po-peptide-1- liposomes was quantitated by cornparin; to a peptide standard (Table 4.1 1). This analysis revealed that there was 19.3% unlinked Po-peptide-1 in the liposome supematant. Therefore, the liposome-linking efficiency of Po-peptide-1 is considered to be 80.7%.

4.6.4 Linking of Po-peptide3 Figures 4.30A and 4.29B (since Po-peptide-1 and Po-peptide4 were Iinked to the liposomes at the sarne tirne, the CE electropherogarn of the supernatant of control liposomes is the same for both peptides) shows the CE electropherogam of the supernatant of Po-peptide-3-liposomes and control liposomes after the first centrifugation (tirst washing). respectively. These bvo CE electropherograms were very similar, indicatin; virtually complete linking of Po-peptide4 to the liposomes. The control Po-peptide3 (1 pg /PL in water) elutes at 3.467 min (Figure 4.30B). In the supematant of Po-peptide-3-liposomes there was a peak (3.56 min peak in Figure 4.3OA) which appeared related to the control Po-peptide-3 peak (3.467 min peak in Figure 4.30B). On the other hand, the control liposome supernatant had also the same peak (3.577 min peak in Figure 4.29B). To determine whether Po-peptide-3 peak in the supematant of Po-peptide-3-liposomes elutes at the same time of 3.577 min peak in the supematant of control liposome, 8 PL of Po-peptide3 liposome supernatant was spiked with 2 pL of Po-peptide-3 (1 pg/ PL) in water. The CE electropherograrn (Figure 4.3OC) revealed that the Po-peptide-3 eluted at 3.56 min which corresponds to the peak of 3.577 min in the supematant of the control liposomes. The percent of unlinked peptide in the supematant of Po-peptide-3-liposomes was quantitated by cornparhg it to a peptide standard (Table 4.11) (to obtain the AUC of Po-peptide-3 in the supernatant of Po- peptide-3-liposome, the difference of AUC of peak 3.56 min in the supematant of Po- peptide-3-liposome and pe& 3.577 min in the supernatant of contrcil liposome was used; average of three CE analysis for each one was considered). This analysis revealed that there was 12.3% unlinked Po-peptide-3 in the liposome supematant. Therefore, the liposome-linking efficiency of Po-peptide-3 is considered to be 87.7%. Figure 4.30 Linking of Po-peptide-3 to liposomes containhg N-Glut-PE. A) The CE electropherogram of Po-peptide-3 -liposomes supernatant. B) The CE electropherogram of Po-peptide-3 standard, 1 pg/pL in water. C) The CE electropherogram of Po-peptide-3-liposome supernatant (8 PL) spiked with 2 pL of Po-peptide-3 (1 pg/pL in water). 4.6.5 EIectron rnicroscopic characterization of peptide-liposomes and control Iiposomes by negative staining Electron microscopic examinations of Po-peptide-1 -, Po-peptide-3-, peptide-+, peptide-5, and control-liposome preparations revealed that the liposomes after linking of the peptides rernained intact with a uniform size distribution (45-70 nm averaze diameter).

4.7 Study of association of peptide-linked liposomes with melanoma cet1 lines

4.7.1 Peptide rnediated adhesion of liposomes to human M21 melanoma cells The liposome-ce11 binding assays using LFA- 1-derived-peptide-liposome (peptide-4-liposome), Po-peptide-1-liposome, Po-peptide-3-liposomes, RGD-peptide- liposomes (peptide-5-liposomes) and control liposomes were carried out to determine their adhesive activity for hurnan M21 meIanoma cells. Therefore, the peptide- liposomes and control liposomes were incubated with M21 cells at a concentration of 78.7 pM total lipid / 5 million cells for 1 hour at 37°C. Considering the linking efficiency achieved with the peptides with an average of 85.6 % + 4.1, the average concentration of peptides on the surface of liposomes was calculated to be 4.0 mM t 0.20. The results of the binding assay indicated that Po-peptide4 and Po-peptide-3, linked to the surface of liposomes, increased the extent of binding of liposomes to M21 cells (6.4 and 6.1 fold, respectively) compared to control liposomes of the same lipid composition but without peptides (Figure 4-31), whereas with peptide-4 (LFA-I- derived) and peptide4 (RGD-peptide) no increase was found. The magnitude of binding of peptide-4- and peptide-5-liposomes was similar to control liposomes and the differences were nonsipi ficant @ > 0.7). However, there were signi ficant differences @ c 0.001) in the magnitude of binding of Po-peptide-1- and Po-peptide-3-liposomes to M2 1 cells compared to the contrd liposomes, and peptide-4- and peptide-5-liposomes. The level of binding of Po-peptide-1-liposomes was somewhat higher than the Po- peptide-3-liposomes but the difierence statistically was not significant (p > 0.2). Percent binding compared to the control liposome

Figure 4.3 1 Association of Po-peptide-1-, Po-peptide-3-, LFA- l -derived- (peptide-4), RGD-peptide- (peptide-5) and control-liposomes with human M21 cells. Monolayers of iM2 1 cells (5 x 1o6/we11) were incubated with peptide- liposomes and control liposomes composed of [3~]~~~~:~-~lut- PE:chol (10:3:1 rnolar ratio) prepared by the Bio-Bead batch method at a lipid concentration of 78.7 PM and for 1 hour at 37°C- The peptide- liposomes contained 4.0 mM of peptides; the fiee peptides were separated fiom liposomes by ultracentrifugation. After incubation, the cells were separated fkom unbound liposomes by washing with PBS. The amount of liposomes associated with the cells was detemined from the uptake of radioactivity. Each bar represents the mean ISD of 3 separate experiments. Statistical analysis was camed out by unpaired t test with Bonferroni correction. There were significant differences (p < 0.001) in the Po-peptide-1- and -3-liposome uptake cornpared to control- , peptide-4- and peptide-5-liposomes. There were no significanr differences (p > 0.2) among the control-, peptide-4- and peptide-5- liposome uptake and also between Po-peptide-1- and -3-liposomes. Previously it was demonstrated that LFA- 1-derived pep tide (pep tide-?) partially inhibited PMA-activated-Jurkat T celIs/IFN-y-stimulated-keratinocytes adhesion which is mediated by the interaction of ICkM-l/LFA-1. It was speculated List peptide-4 may have adhesive activity for M21 cells that are expressing ICkM-I. However, the liposornes linked with peptide-4 showed no adhesive activity for M21 cells. Therefore, it was decided to repeat the peptide-4-liposomes adhesion assay and incubate the cells with liposomes longer (1, 2, 4, 8 hours). The analysis of liposome uptake indicated that at 37°C the association of liposomes (peptide-4-liposomes and control liposomes) with M21 cells is time dependent, and increases with time (Table 4.12). However, there was no difference in the extent of binding of control-liposomes compared to the peptide-4- liposomes at any time examined.

Table 4.12 Association of LFA-1-derived-liposomes (containing 4.0 mM covalently linked peptides) and control liposomes with human MZI melanoma cells for l,3,4, and 8 hours at 37°C.

Time Control4iposomes Pep tide-4-liposome (hours) % of total added liposomal lipid % of total added liposomal lipid

('H-DPPC) associated with 5 x lo6 (-'H-DPPC) associated with 5 x lo6 celIs sf: SD, n=3 cells + SD, n=3

1

-7

4

8

4.7.2 Pa-peptide-I- and Po-peptide-3-linked mediated adhesion of liposomes to human M21, A-375 and MeM 50-10 melanoma cells

Po-peptide-1-, Po-peptide-3- and control-liposomes were incubated with M2 1, A- 375, and MeM SO-10 cells at a concentration of 78.7 pM total lipid / 5 million cells for 1 hour at 37OC. The results of this Liposome-ce11 binding assay indicated that the linking of Po-peptide-1 to the liposome surface increased the extent of binding of liposomes to MX (6.36 fold) cells and rnoderately with A-375 cells (1.85 folds) compared to the control liposomes of the same lipid composition but no Po-peptide-1, whereas with MeM 50-10 cells no increase was found (Figure 4.32). The linking of Po-peptide-3 to the liposome surfaces highly increased the extent of binding of liposomes to al1 three human rnelanoma ceII lines of M21, A-375, and MeM 50-10 cells (6.05, 4.98, and 2.67 folds respectively) compared to the control liposomes of the same Lipid composition but no Po-peptide-3. The magnitude of binding of Po-peptide-1-liposomes compared to the control liposomes were significant @ c 0.001) for M21 cells and A-375 cells but it was not significant @ > 0.2) for MeM 50-10 cells. The extent of binding of Po-peptide-1- liposomes to A42 1 cells was significantly (p <0.001) higher than A-375 and MeM 50-10 cells (292% and 380%, respectively), and also the binding extent of Po-peptide-1- liposomes was higher (p = 0.0105) for A-375 cells compared to the MeM 50-10 cells (130%). The extent of binding Po-peptide-1-liposomes to different melanorna cells correlated with the level of ICAM-1 expression on their ce11 surface and it was more pronounced in rnelanoma cells that express ICAM-1 (Figure 4.33, r' = 0.868).

With regards to Po-peptide-3-liposomes, there was a significant differences @ < 0.001) between the extent of binding of the three melanorna ce11 lines compared to the control liposomes, however, there was no difference O, > 0.2) in the magnitude of binding of Po-peptide-3-liposomes between A-375 and M21 cells. The extent of binding of Po-peptide-3-liposomes to M21 and A-375 cells was slightly higher (p 0.005) than the MeM 50-10 cells (139% and 154% respectively). The higher extent of binding of Po-peptide-3-liposomes with al1 three melanoma cell lines indicated that this peptide nonspecifically (electrostatically) binds to ail three ce11 lines which is probably due to the three positively charged lysines in the peptide sequence. Comparing the magnitude of binding of Po-peptide-1-liposomes and Po-peptide- 3 liposomes, there was no significant difference (p > 0-2) between the binding capacity of Po-peptide-1-liposomes and Po-peptide-3-liposomes to M21 cells. However, the Po-peptide-[ -liposome 1 - Po-peptide-3-liposome 1 - Control liposome 1

M21 A-375 MeM 50-1 0

Figure 4.32 Association of Po-peptide-1-, and Po-peptide-3-liposomes and control- liposomes with human M21, A-375, and MeM 50-10 rnelanoma cells. Melanoma cells were incubated with Po-peptide-1-, and Po-peptide-3- liposomes (containing 4.0 rnM linked peptides) and control liposomes for 1 hour at 37'C as described in the legend of Figure 4.3 1. Each bar represents the mean k SD of 3 separate experirnents. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences 0, < 0.00 1) between Po-peptide-1-liposomes and control-liposome uptake in M21 and A-375 cells but not in MeM 50-10 cells @ > 0.3)- There was a significant difference (p < 0.001) between Po-peptide-3-liposomes and control-liposome uptake by al1 the three melanoma ce11 lines. There was a significant difference (p c 0.001) between Po-peptide-1-liposomes uptake by M2 1 cells and A-3 75 or MeM 50-10 cells, and also between A-375 and MeM 50- 10 cells (p = 0.0105). There was no significant difference @ > 0.2) between Po- peptide-3-liposomes uptake by M21 and A-375 cells, but there was a significant difference (p c 0.005) between Po-peptide-3-liposome uptake by M21 and A-375 cells compared to MeM 50-10 cells. There was no significant difference @ > 0.2) between Po-peptide-1-liposomes and Po- peptide-3-liposomes uptake by M21 cells but it was significant (p < 0.001) with A-375 and MeM 50-10 cells. MeM 50- IO/

IO 15 20 25 30 35 40 45 Mean fluorescence intensity

Figure 4.33 Correlation between ICAM-1 expression on human MeM 50-10, A-375, and M2 1 melanoma cell lines and binding of Po-peptide-1 -liposomes. The extent of binding of the Po-peptide-1-liposomes to melanoma ceIl lines was plotted against the intensity of ICAM-1 expression for each ce11 line, then linear regression was carried out. R' = 0.868. magnitude of binding of Po-peptide-3-liposomes to A475 and MeM 50-10 cells was sigificantly higher than @ < 0.00 1) the Po-peptide- 1-liposomes (307% and 263%, respectively). This again indicates that Po-peptide-3 rnediates nonspecific bindinj to the ce11 line. The extent and the mamitude of binding of control Iiposornes to the different melanoma cells were overall very similar- In summary, the binding assay indicated that Po-peptide4 when linked to the liposome surface, mediates liposome adhesion to M21 cells and A-375 cells but not to MeM 50-10 cells, and the maagitude of adhesion for M21 cells was higher than A-375 cells. According to these results, it was first concluded that the adhesive properties of Po-peptide-1 is specific, othenvise, there should not be differences in the adhesion properties of Po-peptide-1 for different melanoma ce11 lines. Secondly, it was also concluded that the adhesion of Po-peptide-1 with human melanoma cells correlated with the level of ICAM-1 expression on their ce11 surface and it was more pronounced in melanoma cells that express ICAM-1. Therefore, the increased extent of bindins of liposomes to the cells may be mediated through the interaction of Po-peptide-1 and ICAM-1. The binding assay also indicated that the Po-peptide3 mediates binding to human melanoma ce11 Iines nonspecifically.

4.7.3 Concentration-dependent adhesion of Po-peptide-1-Linked-Iiposomes to human M21 meIanorna ceIis The purpose of this part of the study was to determine whether the binding of Po-peptide- L is concentration-dependent. Therefore, the Po-peptide- 1-liposomes containing three different concentrations of Po-peptide-1 (4, 2, and 1 mM linked on their surface) and control liposomes were incubated with MSI cells at a concentration of78.7 pM total lipid / 5 million cells for 1 hour at 37°C. The liposome-ce11 adhesion assay indicated that Po-peptide-1 when linked to the liposomes dose dependently increases the extent of binding of liposomes to human M21 cells (Figure 4.34). The extent of binding of Po-peptide-1-liposomes to M2 1 cells was significant (p c 0.00 1) with al1 the three concentrations of Po-peptide-1 compared to the control liposomes. According to the Po-peptide- l -liposome 4mM

Percent binding compared to the control liposome

Figure 4.34 Concentration-dependent binding of Po-peptide- 1-liposomes and contro l- liposomes with human M21 melanoma cells for 1 hour at 37°C. Melanoma cells were incubated with Po-peptide-1-liposomes (containing 4.0, 2.0, 1.0 mM linked peptides) and control liposomes for 1 hour at 37OC as described in the Iegend of Figure 4.3 1. Each bar represents the mean + SD of 3 separate expenments. Statistical analysis was carried out by unpaired t test with Bonferroni correction. There were significant differences @ < 0.00 1) behveen the Po-peptide-1 -liposome uptake with al1 the three concentrations compared to control liposomes. There were significant differences in the Po-peptide-1-liposomes uptake between 1 mM and 4 rnM @ <0.001), 1 mM and 2 rnM (p < 0.021, and 2 mM and 4 rnM (p < 0.05). results, it was concluded that the linkins of Po-peptide-1 to the liposome surface concentration-dependently increases the extent of binding of liposomes to M3 1 cells.

4.8 Study of the association of peptide-linked Liposomes to human keratinocytes 1.8.1 Peptide mediated adhesion of liposomes to human keratinocytes in the presence and absence of IFN-y Liposome-human keratinocyte adhesion assays using LFA- 1-denved-peptide- liposome (peptide-4-liposome), Po-peptide- 1-liposome, Po-peptide-3 -liposome, RGD- peptide-liposomes (peptide-5-liposomes, as a control) and control liposomes were carried out to determine their adhesive activity for human keratinocytes in the presence and absence of IFN-y. Therefore, peptide-liposomes and control liposomes were incubated with keratinocyte monolayers in the presence and absence of IM-y (500 IU I mL for 36 hours) at a concentration of 78.7 pM total Iipid / keratinocyte monolayer for 1 hour at 37°C. The peptide-linked liposomes contained 4.0 mM of the li~kedpeptides. The results of liposome-keratinocyte binding assay indicated that the Po-peptide- 1 and Po-peptide-3 increases the extent of binding of liposomes to human keratinocytes in the presence of IFN-y by 6.2 and 3.2 fold respectively, compared to the control Liposomes of the sarne lip id composition but without peptides, whereas with pep tide-4 and peptide-5 no increase was found (Figure 4.35). The magnitude of binding of peptide-4- and peptide-5-liposomes was similar to the control liposomes and the differences were nonsignificant @ > 0.2). However, there were significant differences @ c 0.001) in the magnitude of binding of Po-peptide-1- and Po-peptide-3-liposomes to human keratinocytes in the presence of IFN-y compared to the control liposomes, and also compared to peptide-4- and peptide-5-liposomes. The level of binding of Po- peptide-l-liposomes to human keratinocytes in the presence of IFN-y was significantly higher (p c 0.00 1) than Po-peptide-3-liposomes (192%). The liposome-keratinocyte binding assay also indicated that Po-peptide- 1- and Po-peptide-3- liposomes have a higher extent of binding @ < 0.001) with keratinocytes (2.96 and 2.44 folds, respectively) in the absence of EN-y, compared to the control keratinocyte keratinocyte + IFN-y

Figure 4.35 Association of Po-peptide-1 -, Po-peptide-3-, peptide-4 peptide-5- liposomes and control-liposomes with human keratinocytes in the presence and absence of IFN-y. Human keratinocytes confluent monolayers were incubated with peptide- liposomes and control liposomes cornposed of [3~]~~~~:~-~lut- PE:chol(10:3:1 molar ratio) prepared by the Bio-Bead batch method at a lipid concentration of 78.7 pM and for 1 hour at 37OC in the presence and absence oCIFN-y (500 IU / rnL for 36 hours). The peptide-liposomes contained 4.0 rnM of peptides; the Gee peptides were separated from liposomes by ultracentrifugation. After incubation, the cells were separated fiom unbound liposomes by washing with PBS. The cells were collected using Trypsin-EDTA as explained in Matenals and Methods. The amount of liposomes associated with the cells was detemined frorn the uptake of radioactivity. Each bar represents the mean + SD of 3 separate experiments. Statistical analysis was carrïed out by unpaired t test with Bonferroni correction. There were significant differences @ < 0.00 1) in the Po-peptide-1 - and Po-peptide-3-Liposome uptake compared to peptide-4-, peptide-5- and control-liposome uptake but there were no significant differences @ > 0.2) among the peptide-4-, peptide-5- and control-liposome uptake in either native or [FN-y stimulated keratinocytes. liposomes (Figure 4.35). However, there were again no differences @ > 0.2) in the extent of binding of peptide-4- and 5-liposomes wirh the native keratinocytes compared to control liposomes. The extent of binding of Po-peptide-1-liposomes to human keratinocytes was slightly hiJher than the Po-peptide-3-liposomes (12 1 %). The binding assay also revealed that there were si,onificant differences (p < 0.01) in the extent of binding of liposornes to the keratinocytes in the presence and absence of IFN-y. In general, the extent of binding of al1 liposome prepvations to native keratinocytes was greater than to EN-y-stirnulated keratinocytes. The magnitude of binding of control-, peptide-4-, peptide+, and Po-peptide-3-liposomes to native keratinocytes was 325%, 328%, 326% and 244%, respectively; compared to keratinocytes in the presence of EN-y. However, the extent of binding of Po-peptide4- liposome to native keratinocytes was only 155%, cornpared to keratinocytes in the presence of IFN-y. Cornparhg the binding ability of Po-peptide-1-liposomes to human keratinocytes in the presence and absence of EN-y, the binding assay showed that Po-peptide-1 increases the extent of binding in both cases cornpared to the controI liposome. However, the increase in the extent of binding for human keratinocytes in the presence of EN-y (619%) was 2.1 times that of native keratinocytes (296%). This difference rnight be due to the expression of ICAM-1 in human keratinocytes in the presence of EN-y The Po-peptide-1 interacts with ICAM-1 on the surface of keratinocytes and mediates binding. The increased binding of Po-peptide-3 to the keratinocytes (in the presence and absence of IFN-y) was nonspecific, sirnilar to that shown with melanoma ce11 lines. The extent of binding of this peptide to native- and IFN-y-stimulated-keratinocyte was 244% and 322%, respectively, compared to control liposomes. As has been discussed before, this basic peptide could bind electrostatically to ce11 membranes. The extent of binding of liposomes to native keratinocytes was generally higher than the EN-y-stimulated keratinocytes. Figures 4.36A and B show the phase contrast rnicrographs of native and IFN-y-stimulated keratinocytes. Accordin; to these Figure 4.36 Phase contrast light micrographs of human epidennal keratinocytes in the absence and presence of IFN-y. The human keratinocyte confluent monolayers were incubated with IFN- y, 500 IU / / for 36 hours. Then keratinocyte cultures were photographed using a phase contrast microscope. (A), in the absence of IFN-y; (B), in the presence of EN-y (magnification x 215). rnicroscopic evaluations them was a difference in the morpholog of the cells in the presence and absence of LN-/, which could be a reason for the difference in the binding magnitude. In the liposome-keratinocyte adhesion assay, after incubating the cells lv-ith liposome preparations, a trypsin-EDTA solution was used to detach the cells. Since the tiypsin could digest the peptides and hence detach the liposomes from the surface of keratinocytes, the amount of radioactivity was also detemined in the supematant of lhe detached cells afier collecting into 15 rnL tubes and centriQing to separate the cells from the supematant. Table 4.13 shows the results of analysis of the supematant of liposome-keratinocyte binding assay. The results were very similar to the analysis of the cells itself. The amount of released Po-peptide-1- and Po-peptide-3-liposomes from the surface of the cells was 2.0 and 1.48 times compared to the control liposome, respectively, with keratinocytes in the presence of EN-y; this was 1.75 and 1.27 times, respectively, for native keratinocytes. There was no difference in the extent of release of peptide-4- and peptide-5-liposomes compared to the control liposomes in both native and IFN-y-stimulated keratinocytes. The results also showed that the control liposomes also have considerable liposome release. However, the control liposomes did not have any linked peptide. Therefore, al1 of these released liposomes from the ce11 surface were not related to only trypsin and the trypsin-EDTA solution most likely rdeased the loosely-attached liposomes frorn the surface of the cells. Accordingly, the results that are presented in Figure 4.35 (analysis of the cells) show the real extent of binding of liposomes to the cells. In summary, it was found that Po-peptide-1, when linked to the liposome surface, mediates adhesion of liposomes to native and IFN-y-stirnulated keratinocytes. Since increase in the interaction rate for human keratinocytes in the presence of IFN- y (619%) was 2.1 times of native keratinocyte (296%), it was concluded that part of the increase in the interaction rate might be due to the ICAM-1 expression by IFN-y- stimulated keratinocytes. The binding assay also indicated that the Po-peptide-3 mediates binding to both native and IFN-y-stimulated keratinocytes, nonspecifically. Table 4.1 3 The supernatant analysis of association of Po-peptide- 1-, Po-peptide4 -, peptide+, peptide-5-liposomes (containin; 4.0 mM covalently linked peptides) and control liposomes to human keratinocytes in the presencs and absence of EN-y (500 TU / mL for 36 hours) for 1 hou at 37OC.

- - - . - - - - Liposome preparation Human keratinocyte Human keratinocyte + EN-y % of total added liposomal % of total added liposomal lipid ('H-DPPC) associated lipid ('H-DPPC) associated

/ weIl f: SD, n=3 / well+ SD, n=3

Control liposome

4.8.2 Concentration-dependent adhesion of Pa-peptide-l-Iinked-liposomes to human keratinocytes in the presence and absence of IFN-y

Po-peptide- 1-liposomes containing three different concentrations of Po-peptide-1 (4, 2, and 1 mM linked on their surface) and control liposomes were incubated with keratinocytes in the presence and absence of IFN-y (500 TU / mL 36 houn) at a concentration of 78.7 pM total / keratinocyte monolayer for 1 hour at 37T. The liposome-ce11 adhesion assay indicated that Po-peptide4 when linked to the liposomes dose dependently increases the extent of binding of liposomes to native and IFN-y- stimulated keratinocytes (Figure 4.37). The extent of binding of Po-peptide- 1-liposomes to both native and IFN-y-stimulated was significant @ < 0.01) with al1 the three concentration of Pa-peptide-l compared to control liposomes. However, the increase in the extent of binding for human keratinocytes in the presence of IFN-y Control liposome

Human Human keratinocyte keratinocyte + IFN-y

Figure 4.37 Concentration dependent binding of Po-peptide-l -liposomes and contro 1 liposomes with human keratinocytes in the presence and absence of IFN-y. Human keratinocytes monolayers incubated with peptide- 1-liposomes (containing 4.0, 2.0, and 1.0 mM linked peptide) and control liposomes in the presence and absence of IFN-y (500 IU / mL for 36 hours) for 1 hour at 37°C as described in the Zegend of Figure 4.35. Each bar represents the mean t SD of 3 separate experiments. Statisticzl analysis was canied out by unpaired t test with Bonferroni correction. There were significant differences (p c 0.001) in the Po-peptide-1 -liposome uptake with al1 the three concentrations compared to control liposomes in both native and IFN-y stimulated keratinocytes. (615 %, 497%, and 369%, respectively) was about 2 times that of the native keratinocyte (267%, 264%, and ZOO%, respectively). In this experiment the supernatant was also analyzed after the trypsin-EDTA treatrnent as explained in the previous section. The analysis of supematant also revealed that the arnount of Po-peptide-1-liposomes released fiom the surface of native keratinocyte was concentration-dependent (Table 4.14).

Table 4.14 The supematant ana!ysis of the association of Po-peptide-1-liposomes (containing 4.0, 2.0, and 1.0 rnM covalently linked peptides) and control liposomes to hurnan keratinocytes in the presence and absence of IFN-y (500 IU / mL for 36 hours) for 1 hour at 37OC.

- - -- Liposome preparat ion Human keratinocyte Human keratinocyte + iFhr-y Po-peptide-1 % of total added liposornal % of total added liposomal concentration lipid OH-DPPC) associated lipid ('H-DPPC) associated

/ well Itr SD, n=3 / well k SD, n=3

Control liposome CHAPTER FIVE

DISCUSSION AND PERSPECTIVES

5.1 Discussion Liposome targeted dmg delivery to selected tissues or cells is one of the promisin; ways of improving controlled or selective treatment of various diseases. In the present study the major glycoprotein of peripheral nerve myelin (Po protein), a ce11 adhesion molecule from IgSF. was selected as a rnodel for targeted dmg delivery study. It has already been shown that when Po protein reconstituted into liposome bilayer mediates heterophilic interaction and increases the interaction rate of liposomes with human MZ 1 melanoma cells Foldvari et al., 199 1). They also showed that liposome encapsulation highly increases the association of model dmgs (methotrexate and inulin) with M2I cells. In this study the heterophilic interaction of Po protein was further charactenzed and we showed that the extent of interaction correlates with the expression of ICILLI-1 on the ceIl surfaces. Therefore, it was concluded that Poprotein could be used as a targeting ligand for liposomes for specific drus delivery to ICAM-1 expressing melanoma cells. Compared to whole proteins, synthetic short peptides might be Iess immunogenic and their bulk production, handling and preservation are easier. Therefore, an adhesive short Po protein- derived peptide which is effectively capable of mediating binding and attachment of liposomes to the ICAM-I expressing cells could be a more feasible way for liposome targeted dmg delivery. Therefore, we analyzed selected sequences of Po protein to identiQ an adhesive peptide that can be used as a ligand for targeting. In this regard a peptide based on the LFA- 1 (the counter receptor of ICAM-1) structure was also evaluated. Various peptide cornpetition studies showed that some of the peptides selected in this study might have adhesiveness for f CAM- 1. kVhen these peptides were linked to the !iposome surface, one peptide (Po-peptide-1) specifically increased the extent of binding of liposomes to ICAM-1 expressing cells. Po-peptide4 targeted liposome systems might be useful in inflammatory disorders of skin and human lymph node/skin melanoma, which have high expression of ICAM-1 (Griffiths et al., 1989 and Altomonte et al., 1993). In tissue culture models, we showed that Po-peptide-1 when it is Iinked to the liposome surfàce, increases the extent of binding of liposomes to IC.4iM-1 expressing melanoma ceIl lines and LM-7- stimulated keratinocytes. Highly purified Po protein was obtained fiom partially purified Po protein FI11 fraction initially by preparative SDS-PAGE and later by reversed-phase HPLC usins a proteidpeptide C4 column. Compared to preparative SDS-PAGE, RP-HPLC is a more rapid and gentler purification procedure as it does not involve detergent. The SDS-PAGE has the disadvantage that not only SDS denatures the protein, but also 2-rnercaptoethanol may break the disulfide bond of the extracellular Ig-like domain of P, protein. The integity of the disulfide bond of Po protein is necessary for its homophilic interaction (Zhang and Filbin, 1994). Therefore, Po protein was also purified by a published RF'-HPLC procedure (Brunden et al., 1987).

Brunden et al. (1987) have show that the solubilization and chromatography in acetic acid/tpropanol solution do not affect the pnmary structure and N-iinked glycosylation of Po protein. They have determined the polypeptide sequence of the RP- HPLC purified Poprotein that agrees with the sequence predicted from cDNA of the Po protein gene. They have also determined the amount of siaiic acid in the RP-HPLC purified Po protein. The molar ratio of sialic acid to Po glycoprotein was approximately one, in agreement with the expected amount of sialic acid. These results confirmed the integrity of the prirnary structure of Po glycoprotein; however, the effects of solubilization and chromatography steps on the conformation of the protein are unhom. On the other hand. it has been demonstrated that many proteins after RP-HPLC treatment regain their biological activity (Luiken et al., 1984). The presence of intact Po protein (PAGE-purified) in the tiposome bilayer increased the extent of binding of liposomes to human MX mehoma cells 3-4 times in cornparison with control liposomes of the sarne lipid composition but without Po protein. These results confirmed the previous results with regards to the ability of Po protein for rnediating heterophilic interactions (Foldvari et al., 1991). A control protein, Glycophorin A (a transrnembrane glycoprotein), when reconstinited in liposomes had no effect on the binding of liposomes with M2 1 cells. The extent of binding of Glycophonn A-Iiposomes to k12 1 cells was simiIar to control-liposomes and it was significantly lower than Po liposomes. The increased binding of Po-liposomes to M21 cells but not control-liposomes was inhibited by anti-chick Po Fab, indicating that Po protein plays a specific role in the binding of liposomes to MZ 1 cells. To charactenze the binding domains of Po protein, it was reconstituted into liposomes and cornpetition studies were carried out in the presence of synthetic Po-peptides based on the chicken Po sequence. Cell-liposome assay indicated that the association of Po- liposomes with M2 l cells decreased by 30% in the presence of Po-peptide-l and b y 10% in the presence of Po-peptide-3, while Po-peptide2 had no effect. The cell uptake of control liposomes in the presence of Po-peptide-1 and Po-peptide-2 increased by 240% and 290%, respectively. Po-peptide-3 had no effect on control liposome interaction with cells. Both extracellular and intracellular Po-peptides (1 and 3) showed cornpetition and may be involved in the heterophilic interaction of Po protein with M21 cells. The results also indicated that Po-peptide-1 and Po-peptide3 mi& have adhesive activity for M2 1 cells and could be candidates as ligands for targeting liposomes to M21 cells. The increasing effect of Po-peptide-1 and 2 on control liposomes binding rnay be indicative of adhesion of these peptides to the liposome surface and mediating cellular uptake of liposomes.

To conduct the Po-liposome binding expenment in the presence of Po-peptides, due to solubility problems, the peptides were dissolved in a solution containing water and DMSO: Po-peptide-1 and Po-peptide-), 90 pg / 20 pL (&O: DMSO 1 :1 v/v); Po-peptide-2, 90 pg / 20 pL (H,O: DMSO 1:2 v/v). In flow cytornetry experiments we showed that DMSO concentration-dependently kills the cells and decreases the interaction of anti- 1Ch.M-1 mAb with ICAM-I on M31 cells (Table 4.3). One might think that DMSO has affected the results of peptide competition study in the same way and decreased the interaction of Po-liposomes with M2I cells. However, this could be explained with the difference in the concentration of DMSO in these hvo different experiments. In the flow cytornetry expenments the total volume was 100 pL per experirnent. Therefore, the concentration of DMSO in 80w cytornetry experiment, for instance with 8.66 pL which results in 13.34% death in ce11 population, would be 8.66% (v/v). However, in the peptide competition studies the total volume was 2020 pL per well and the concentration of DMSO would be 0.5% (v/v) for Po-peptide4 and Po-peptide-3, and 0.7% (v/v) for Po-peptide-2. Considering DMSO concentration-dependently kills the cells and decreases the interaction of anti-ICPuM-1 rnAb tvith ICAM-1 on MX cells, the effect of O.5-O.7% DMSO, which is at least 10 times less than the concentration of DMSO in flow cytometry experiment, on the Po-liposomes binding experiment in the presence of Po-peptides should be insignificant. Here we showed that Po-peptide4 (YTDNGTF, corresponding to residues 90-96 of chicken Po protein) of the extracellular domain of Po protein is important for its heterophilic interaction with M21 melanoma cells. Zhang et al. (1996) have been show that the sarne segment of the Po extracellular domain is important for the Po-Po homophilic interaction. They have shown a Po-peptide (SDNGT, corresponding to residues 9 1-95 of rat Po protein) and also antibodies to this Po peptide inhibit the adhesion of Po expressing CHO ceils. The difference between Po-peptide-1 and this peptide is in the residue 9 1. Po- peptide-l has Thr but this peptide has Ser. However, these two amino acids are similar to each other and both of them have a hydroxyl group in their side chains. Zhang et al. (1 996) have also shown when Asp 92 and Gly 94 are rnutated to Glu and Ala, Po protein loses its homophilic adhesive activity. Therefore, this part of the Po extracellular doniain is important for its adhesion.

Po protein is a member of IgSF molecules Gemke et al., 1985), and it is well knowm that interaction behveen Ig-like domain molecules on different proteins is important in the immune system and cell-ce11 adhesion/recognition processes within the IgSF (Buck, 1992). There are examples for both homophilic and heterophihc interaction in the IgSF. It has been show that neural cell adhesion molecule (N-CAM) functions via homophilic interactions of its Ig-domain in cell-ce11 adhesion in nerve tissues (Rutishauser et al., 1982). On thz other hand, for instance, poly Ig receptor binds to immunoglobulins through heterophilic interaction of its Ig-domain and transports them through epitheliaI cells (Mostov et al., 1984). This may imply that Po protein, which has only one Ig-domain, cannot only interact with self molecules and mediate hornophilic interactions, but also interacts with other Ig-domain containhg IsSF molecules on ceIl membranes. One of the main ce11 surface protein candidate of melanoma cells for the heterophilic interaction of P, protein is ICAV-1, an IgSF ce11 adhesion molecule with five Ig-like domains in the extracellular regon. The flow cytometry expenment showed that more than 95% of MX melanoma cells express ICAM-1. The expression of ICAM-1 on M21 cells was then quantitatively compared with other human melanoma ce11 lines of A475 (positive control cells) and MeM 50-10 (negative control cells). The percentage of stained ceils for ICXM- 1 expression was about 5% for MeM 50-10 cells, but for M21 cells and A-375 cells it was 95 and 85%, respectively. The intensity of ICAM-1 expression for M21 cells was higher than A-3 75 cells (154%). To determine the possibility of interaction of Po protein with ICAM-1, the extent of binding of Po-liposomes to human MX, A-375 and MeM 50-10 melanoma cell lines was determined and it was correlated with the level of ICAM-1 expression on human melanoma ce11 lines. Po protein (RP-HPLC purified) reconstituted into liposomes mediated heterophilic interactions and highly increased the extent of binding of liposomes with ICAM- 1 expressing melanoma cells oEM2 1 and A-375 (7.80 and 4.62 fo id, respectively) compared to control liposomes cells whereas with the MeM 50-10 cells no significant increase was found. The extent of binding of Po liposomes to M2 l cells was higher than A- 375 cells (138%). The extent of binding of Po liposomes with the hurnan melanoma cell lines correIated with the level of ICAM-1 expression on their ceil surface (9 = 0.9996) and it was more pronounced with melanoma cells that express ICAM-1. Therefore, it was conciuded that the increased binding of Po liposomes &Ohuman M21 and A-375 melanoma cells might partly be due to the interaction of Poprotein with ICAM-1. According to our hypothesis, the extracellular Ig-domain of Po protein interacts with the Ig-domains of ICAM-1 on melanoma cells and mediates heterophilic interactions. In this regard, one aspect of Po protein mediated liposome binding is the confirmation that Po protein is incorporated into the liposome bilayer in a transrnernbrane orientation with the bindin; 1;-domain facing outward. Previous experiments have shown that the incorporation of P, protein into liposomes is either in a transmembrane (50%) or in a hairpin confijuration (50%) (Foldvan et al., 1990). Tne latter arrangement will have both the extracellular and intracellular domains of Po protein exposed on the surface of the liposomes. Therefore, the possibility exists that the basic intracellular regions can also contribute to binding through nonspecific (electrostatic) interactions with cells. Experiments using biotinylated P, protein, however, can exclude this possibility. We showed that biotinylated P, protein binds to iM21 cells but does not bind to MeM 50-10 cells. Furthemore, during biotinylation the basic amino acids are neutralized and Po protein should have shown decreased binding if electrostatic binding was a mechanism. Po protein (based on chicken Posequence) has 23 positively charged amino acids out of the 70 amino acids in the intracellular domain, (Barbu, 1990). SDS-PAGE showed a 4.1 kDa increase in the molecular mass of Po protein after biotinylation, corresponding to 18 molecules of biotin (MWt 227). In total there are 35 positively charged amino acids (Lys, Arg, His) in the molecule of chicken Po protein that can theoretically bind to biotin, 23 of them are intracellular (Barbu et al., 1990). The most likely biotinylation sites are lysines; it is therefors anticipated that 11 of the 22 positively charged amino acids in the intracellular domain that are Lys are neutralized in the process. There is a receptor for biotin on ce11 membranes (Vesely et al., 1987), which mediates the transfer of biotin from the outside of the cells to the intracellular region. Therefore, the biotin receptor could theoretically mediate the binding of biotinylated proteins to ce11 surfaces. To evaluate the effect of biotinylation on the biological activity and binding ability of the proteins, Storm et al. (1996) biotinylated the Clq, CI inhibitor, activated C ls, alpha 1-antitrypsin, ovalbumin, transfemn and soybean trypsin inhibitor. The biotinylated proteins retained their bioiogical activity and antigenicity. ;\mong the biotinylated protein, Clq, Cl inhibitor and activated Cls showed si_gïficant binding to monocytic U937 cells, promyelocytic HL-60 cells, monocytes and granulocytes. Hotvever, the sarne proteins (without biotinylation) did not show binding affinity for the cells when tested by other means. To evaluate the involvement of biotin recepton in the nonspecific binding of these biotinylated proteins, they carried out the experiment in the presence of an excess of fiee d-biotin. However, the free d-biotin did not decrease the binding of biotinylated proteins to the cells. Stom et al. (1996) then showed by hydrophobic interaction chromatography that the hydrophobicity of the biotinylated proteins are si,gnïficantly geater than the unlabelled proteins. They then susgested that the nonspecific interaction of some biotinylated proteins could be due to the increased hydrophobicity. With respect to biotinylated Po protein, the binding up to 200 riiM was most likely specific, since we showed that the biotinylated Poprotein binds to M2 1 and CHO-X2 cells but does not bind to MeM 50-10 cells. If the binding of biotinylated Po protein to the cells was unspecific and it was due to the hydrophobic character of biotinylated Po protein, there should not have been a difference in the binding capacity of the biotinylated Po protein in different ce11 lines.

To further characterize the involvement of ICM-1 in the binding of P, protein to M2 1 cells, indirect flow cytometry using biotinylated Po protein was camed out on M21 cells in the presence of anti-human CD54 mAb. Preincubation of MX cells with anti- ICAM-1 interfered with the binding of biotinylated Po protein to the M21 cells and decreased the binding by about 35%. This was a Merevidence that binding of Poprotein to M21 cells may be partially mediated by the interaction with ICAM- 1. The monoclonal antibody used in this study rnay recognize different epitopes on ICAM-1 than Po protein; complete inhibition of binding was not therefore expected.

Pfexpressing CHO-X2 cells were used as a positive control for the Po-liposome binding assay and the indirect flow cytometry experiment with biotinylated Poprotein. In both the Po-liposome and Po protein binding experiments, the extent of binding to CHO-X2 cells was fairly similar to M21 melanoma cells. According to these results it was concluded that the ability of Po protein in rnediating heterophilic interaction with IChrM- 1 expressing melanoma cells is comparable to homophilic interaction. The results of experiments with CHO-= also confirm the previous information in the literature with regards to homophilic interaction of Po protein (Filbin et al., 1990; D'Un0 et al., 1990; Schneider-Schaulies et al., 1990).

The ceil-liposome adhesior. and flow cytometry experiments showed that there is a possibility for specific dnig delivery to ICAV- 1 expressing cells through Po protein ivhich is incorporated into the liposome bilayer.

ICAM-1, which is the counter receptor of LFA-1, is widely expressed on both nonhaematopoietic (e-8- vascular endothelial cells, thymic epithelial cells, fibroblasts) and haematopoietic (e.g leukocytes, macrophages, dendritic ceIls in tonsils, Peyer's patches) cells (Dustin et al., 1986). The intensity of ICAM-I expression on restinj perïpheral blood leukocytes is low but it is increased on activated T and B lymphocytes. The intensity of ICAM-1 expression on nonhaematopoietic cells is also low, however cytokines such as IFN-y, interleukin-1 (IL- 1) and -or necrosis factor-u (TNF-a),upregulate the expression of ICAM-1 on these cells.

Normal hurnan melanocytes (Knitmann, 1992) and benign melanoma lesions do nor express ICAM-1, whereas malipant melanoma lesions do (Johnson et al., 1989; Natali et al., 1990 and 1997). Arnong the malimant rnelanoma lesions, the expression of ICAW-1 in metastatic melanoma is higher than the primary melanomas (Natali et al., 1990; Kageshita et al., 1993; Altomonte et al., 1993). The extent of expression of ICAiM-1 on prirnary melanoma lesions correlates with their thickness and prognosis (Natali et al., 1990 and 1997). Expression of ICAM-1 on malignant tumor cells mediates the T-ce11 mediated killing of melanoma cells (Pandolfi et al., 1991). Expression of ICAM-1 on malignant melanoma lesions, but not on benign melanoma lesions, provides an opportunity for specific drug delivery. Since we have shown that Poprotein reconstituted into the liposome bilayer increases the extent of binding of liposomes to melanoma cells, which have a high level of ICAM-1 expression, Po protein might be a suitable ligand to target the liposomes to malignant rnelanorna Iesions. The route of administration of liposomes in vivo rnay also have si,anificance in the success of therapy. Systemically administered Po liposomes may bind to soluble ICiGbI-1 in the circulation that is present due to shedding from malisgant melanomas (Kageshita et al., 1993). It is believed that the presence of soluble ICAM-1 in the systemic circulation prevents cytotoxic T cells from attacking malignant melanoma cells (.4ltomonte et al., 2993). Interaction of Po liposomes with ICAM-1 in the circulation rnay reduce the melanoma cell-targeted fraction of Po-liposomes and decrease the efficiency of trearment. Other alternatives may be intralesional or topical treatments where regional delivery to melanoma lesions could be more specific and longer lasting. Application of the adhesion based targeting concept to another melanoma adhesion protein, the integrin u,B,, which is also involved in tumorgenicity, could offer another interesting option for specific drug delivery for the treatment of malignant melanorna lesions. Simildy to ICAM-1, the intensity of a$, expression on malignant melanorna lesions is significantly higher than benign melanoma lesions, and the extcnt of uJ, expression on primary lesions depends on the thichess of the lesions (Natali et al., 1997). However, unlike ICAM- 1, the expression of aJ, on melanorna cells is not upregulated by cytokines such as IFN-y, IL-l and Th?=-a,and a&, has a restricted distribution in normal cells (Natali et al., 1997). In addition, there is no evidence of the shedding of cc$, integin into the systemic circulation.

In order to find an adhesive peptide fiom Po protein to target ICAM-1, three heptapeptides were selected fiorn different regions of Po protein based on the function of each region. Po-peptide-3 represents the basic intracellular domain of Po protein and consists of mainly positively charged amino acids of Lys. Po-peptide-2 represents the hydrop hobic transmembrane region and contains mainly of the hydropho bic amino acids of Val and Leu. Po-peptide-1 represents the extracellular domain of Po protein which has been shown to be involved in hornophilic Po-Po interactions (Filbin et al., 1990; D'Urso et al., 1990). Po-peptide-1 was chosen fiom the region of Po-extracellulardomain that contains the only glycoside attaching site (Asn 93). Although it was proposed ihat both Ig-domain and oligosaccharide moiety of Po extracellular region function as adhesion sites (Lemke et al., 1988; Kucherer et al., 1987), Yazaki et al. (1992) has also shovm that a peptide from the extracellular region of Po protein, which contains the glycoside attaching site (devoid of glycoside rnoiety), inhibit Po-Pomediated ce11 asgregation by 50%. Therefore, in this study Po-peptide-1 represents the Ig-domain of chicken Po protein (residues 90-96) and was used without the carbohydrate residue. It should also be noted that in this sequence hvo amino acids (Asp 92 and Gly 94) are conserved in a large number of V-like domains of IgSF molecules (Williams and Barclay, 1988). Furthemore, according to the crystal structure of the Po extracellular dornain (Shapiro et al., 1996), the amino acids YTDN fiom the Po-peptide-1 sequence (YTDNGTF) are exposed at the surface of Po molecules (E-F loop) and in a position to interact with opposing molecules. To find an adhesive peptide for targeting to ICAM-1, in addition to Po protein, LFA- 1 which binds to ICAIM- 1, was also considered. A peptide, LFA- 1-denved peptide. was chosen from the "1" domain of LFA-1 that contains a lisand binding site for 1C.ki.I-1 and cm separately bind to soluble ICAM-1 molecules.

To identiS adhesive peptides for targeting to ICAM-1, various peptide competition studies were carxied out. The fint experiment was the inhibition of interaction of Po liposomes with M21 cells using Po-peptides. According to this experiment, Po-peptide-1 and Po-peptide-3 showed competition, therefore, it was concluded these two peptides might have adhesive activity for M2 1 cells.

The second peptide competition study was carried out to determine the effects of Po-peptides and LFA- 1-denved peptide on the interaction of anti-ICPUM- 1 with ICAM- 1 on M2 1 cells usin; flow cytometry. The experiments, which involved the Po-peptides,were limited with the solubility. To increase the solubility of the Po-peptides DMSO \vas used as a cosolvent. The DMSO, however, interfered with the interaction of anti-ICAM-1 rnAb with ICM-I on M21 cells. In spite of various efforts to exclude the effect oIDMSO, the results of competition studies with Po-peptides were stili inconclusive. On the other hand there was no difference between the effect oFDMSO alone or peptide solutions in DiLlSO on the interaction of anti-ICAM-l mAb with ECAM-1 on M2 1 cells and the effect was only due to DMSO. It was therefore concluded that the Po-peptides have no effect on the interaction of anti-ICAM-1 mAb (used in this study) and ICXM-1 on 3421 cells. With respect to LFA-1-derived peptide, which did not have a solubility problem, the peptide had no effect on the interaction of anti-ICM-1 mAb ~vithICAU-1 on M71 cells. According to these results it was concluded that Po-peptides and LFA-1 derived peptide do not interact with ICAM-1 or these peptides and anti-ICAM-1 mAb bind to different epitopes on IChV- 1. The other possibility for this experiment could have been using po1yclonaI anti-ICAM-1 antibody or testing several different anti-ICAM-1 mAbs. To detemine the direct adhesive activity of peptides for M21 cells, binding of biotinylated Po-peptides and LFA-1-denved-peptide to M21 cells were evaluated using indirect flow cytometry. The peptides were biotinylated using NHS-biotin. The yield of biotinylation of the peptides was determined by CE. The results indicated that the biotinylated peptides have no adhesive activity for M21 cells. The flow cytometry experirnents, which involved biotinylated Po-peptides, were a;ain limited by a solubility problem. Also the solubility of the peptides was fùrther decreased by the biotinylation. To improve the solubility of biotinylated Po-peptides, they were dissolved in 0.1% Triton X- 100 solution in water. However, with using 0.1% triton X-100 as a solvent no binding was observed. It was therefore concluded that either peptides do not have binding affinity for M21 cells or the biotinylation affects the conformation of the peptides such that they cannot bind to M21 cells.

The third peptide competition study was carried out to determine the effect of peptides on the binding of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes which is mediated by the interaction of LFA-1 with ICAM-1. The keratinocytes were isolated fiom human breast or abdominal skin after cosmetic surgery. The isolated keratinocytes were characterized by msrnission electron microscopy. The PMA-activated Jurkat T cells had a strong adhesion (45.32%) to IM-y-stimulated keratinocpes. The percentage of adhesion of PMA-activated Jurkat T cells to IFN-y-stimulated keratinocytes decreased by 3 1% in the presence of 1 mM of LFA-1-derived peptide and the effect of RGD-peptide as control was insignificant. The cell-ce11 adhesion assays in the presence of Po-peptides were again limited by solubility. Different solvents such as DMSO, PG, and PEG 400 were tested to dissolve the Po-peptides; however, al1 of them interfered with the cell-ce11 adhesion assay. During the experiment with PG, Po-peptide-2 was soluble in PG; however, Po-peptide4 was relatively soluble and Po-peptide4 was alrnost insoluble. However, it was found out that the Po-peptide-1 and Po-peptide4 are soluble in a combination of PG and RPMI-FCS. We then tried RPMI-FCS as a solvent for Po-peptides. Po-peptide-1 and Po-peptide3 were soluble in RPMI-FCS, whereas Po-peptide-2 was not. Using Po-peptide-1 and Po-peptide3 solution in RPMI-FCS we showed that the percentage of adhesion of PU-activated Jurkat T cells to IFN--j-stimulated keratinocytes decreased by 3 1% in the presence of 1 mM of Po-peptide-1 and 21% in the presence of Po-peptide-3. Using different solvents, we showed that Po-peptide2 had no effect on the interaction of PMA-activated Jurkat T cells to IFN-y-stimuiated keratinocytes. The effect of LFA- 1- derived peptide, Po-peptide-1 and Po-peptide-3 on the inhibition of LFA- 1tKkW-1 interaction might be through adhesion to ICAM-1; therefore, these peptides may be candidates as ligands for tarsetins to ICAM-1 expressing cells. In this study, to find peptides with high adhesive activity for ICA%-1 we analyzed selected sequences of Po protein or LFA-1 according to their function in the protein. Available literature data with rezards to the binding epitopes of these proteins was also examined. The other possible way for finding high affinity peptides could have been the phage display peptide Library technique (DeLeo et al., 1995; Pierce et al., 1996; Nevalainen et al., 1997; Dore et al., 1998). In this technique the filamentous bacteriophage that display a peptide library on their surfaces are utilized. A peptide library contains most of the possible peptide sequences that the combination of amino acids could produce. For example, a phage display hexapeptide library contains over 200 million hexapeptide sequences. In this technique, peptide sequences with high affinity binding propeaies for a protein, antibody and specific domains are selected by the sequential analysis of the phase display peptide library for binding to the proteins by procedures such as biopanning and enzyme-linked immunosorbent assay. Nevalainen et al. (1 997) described the isolation of two 10-amino acid peptides usinj phage display peptide library that specifically bind to calmodulin and inhibit calmodulin-dependent kinases I and II. DeLeo et al. (1995) identified the potential sites of interaction of p47-phox and flavocytochrorne b using peptide phase disptay library. Dore et al. (1 998) using phage display heptapeptide library identified the epitope of hvo mAbs that react with syndecan-1 core protein. As cm be seen fiom these illustrations, the peptide phage display iibrary could be a suitable technique for the discovery of high affinity binding peptides for targeting purposes.

According to the different peptide competition studies, Po-peptide-1, Po-peptide-3 and LFA-1-derived peptide showed competition and it was concluded that these peptides might be candidates for ICAM-1 tarsetins. To investigate whether these peptides can mediate specific interactions of liposomes with ICAM-1 expressing cells, the peptides were covalently Iinked to the liposomes and liposome-ce11 adhesion assays were camed out. Liposomes containing N-Glut-PE were activated with EDC and S-NHS in pH 5.5, then linked to the peptides at pH 8.0. The yield of linking, which was measured by CE, was relatively good, about 85% on average, for the different peptides.

The adhesive activity of peptide-linked-liposomes was evaluated in two different tissue culture models of melanoma ce11 lines and human epidermal keratinocytes in the presence of EN-y. These hvo models represent rnalignant melanomas and inflamrnatory disorders of the skin, in which cells have high levels of ICAM-1 expression.

The liposome-ce11 adhesion assay indicated that the linking of Po-peptide-1 and Po- peptide-3 to the liposomes increased the extent of binding of liposomes to human M21 melanoma cells (6.4 and 6.1 fold, respectively) compared to the control liposomes of the same lipid composition but devoid of peptides; however, the LFA-1-denved and the RGD- peptide (as control) does not. The binding of Po-peptide-1-liposomes to M21 cells was dose-dependent, and increased with increasing amounts of liposome-linked peptide. The liposome-ce11 adhesion assay on different melanoma cells indicated Po-peptide-1, when linked to the liposome surface, highly increases the extent of binding of liposomes to MX cells (6.36 fold) and moderately with A-375 cells (1.85 fold), whereas with MeM 50-10 cells (1.02) no increase was found. Po-peptide-3 nonspecifically increased the extent of binding of liposomes to al1 the three melanoma ce11 lines narnely M21, A-375, and MeM 50-10 cells, which is most likely due to its high content of the positively chaged amino acid Lys (three Lys out of seven amino acid). According to these results it was concluded that Po-peptide-1 mediates specific interaction of liposomes with cells. Furthemore, the adhesion of Po-peptide-1 with human melanoma ceil lines correlated with the leveI of ICAM-1 expression (? = 0.868) and it was more pronounced in melanoma cells that express ICAM- 1. Therefore, the increased interaction rate of Po-peptide-l -liposomes with IC&M-1 expressin; cells may be mediated throua the interaction of Po-peptide4 and ICAM- 1. According to these results it was concluded that there is a possibility of targeting of melanoma cells, which have a high leveI of ICAM- 1 expression, through Po-peptide-I which is linked to the liposome surface. As discussed before, normal rnelanocytes and beni~nmelanoma Iesions do not express ICAM-1, however, malignant melanoma lesions express ICAM- 1. Therefore, Po-peptide- 1 might be a suitable ligand to target liposomes ro the melanoma cells in the malignant melanoma Iesions. Since synthetic short peptides compared to the whole protein are less immunogenic, their production is easier and allow longer circulation of the liposomes in the blood strem, they have been recently tried as a targeting ligand for liposomes. Flasher et al., (1991) showed when the recombinant transrnembrane CD4 molecules are reconstituted into the liposome bilayer, or recombinant soluble CD4 molecules are covalently linked to the liposome surface, specific binding of liposomes to human imrnunodeficiency vims type-1 (HIV-1)-infected cells could be achieved. They later showed that arnong the different CD& derived peptides, a synthetic peptide from the complementarity determining region 2 (CDR-2)-like domain (14 amino acids long), when linked to the liposome surface, can mediate specific binding of [iposomes to the HN-1-infected cells (Slepushkin et al., 1996). The extent of binding of CDR-3 peptide-liposomes to the HIV-1-infected cells has been comparable with soluble recombinant CD4-coupled liposomes.

The CDR-2-derived peptide was linked to the liposome surface by two different methods. Linking via the SH-group of the peptide to the preformed liposomes containin: N-(4-(p-maleimidophenyl)butyryl)phospha~ine(MBP-PE), has not been successful(27% effkiency of linking). However, linking through the amino-terminal group of the peptide to the preformed liposomes containing a carboxyl group (cholesteryl hemisuccinate) by a hvo-stage irnmobilization procedure usin= EDC and S-NHS has resulted in 100% efficiency of linking. Only the latter linking method, which is the method used in this study for Po-peptide-1 linking, mediated specific binding of liposomes to HIV- Z -infected cells. Even though the short synthetic peptides by themselves might not induce an immune response, when they are used with adjuvants they cm become imrnunogenic (Amon, 1991). Indeed one of the ways to design vaccines is to utilize appropriate synthetic peptides derived fkom an antigen. For this purpose, the selected peptide is chernically cross- linked to a carrier rnolecule such as keyhole Iimpet hemocyanin or bovine serurn albumin and is utilized for irnrnunization. It has been show that liposomes could function as an efficient immunoadjuvant in inducing immune responses to proteins (Baca-Estrada et al., 1997; Bei et al., 1998). Liposomes have been also effective as an adjuvant to induce immunogenic responses for the encapsulated peptides (Phillips et al., 1996; Samuel et al., 1998). Alving et al. (1995) usinj cornputer-generated molecular modeling analysis of small peptides suggested that small synthetic peptides when linked to the liposome surface could serve as efficient antigenic epitopes. In our study we linked the peptides to the liposome surfaces as a targeting ligand for specific dm; delivery purposes. There is a possibility that liposomes may function as an immunoadjuvant in inducin~immune responses to the peptides. This problem should be therefore addressed in the future studies.

Peptide-linked liposome-ce11 adhesion assays in human keratinocytes in the presence and absence of IFN-y indicated that P,peptide-1 and Po-peptide-3, when linked to the liposome surface, increased the extent of binding of liposomes to both native (2.96 and 2.44 folds, respectively) and LFN-y-stimulated keratinocytes (6.2 and 3.2 folds, respectively) compared to the control liposomes of the same lipid composition but without peptides, whereas with LFA- 1-denved peptide and RGD-peptide (as contro 1) no increase was found. Human keratinocytes express ICAM-1 in the presence of IFN-y. Since the increase in the extent of binding to hurnan keratinocytes in the presence of EN-y (G19%) was 2.1 times that of native keratinocytes (296%), it was concluded that part of the increase in the binding mijht be due to the ICAM-1 expression by IFN-y-stimulated keratinocytes. The Po-peptideJ mediated bindins of liposomes to both native and EN-y-stimulated keratinocytes nonspecifically- The binding of Po-peptide-1-liposomes to both native and EN-y-stimulated keratinocytes was concentration-dependent, and increased with increasing amounts of liposome-linked peptide. The results demonstrated the feasibility of using Po-peptide-1 to target liposomes to keratinocytes when they are expressing ICAM-1. Keratinocytes of normal skin do not express ICAM-1. However, in inflamrnatory conditions such as allergic contact eczema, psoriasis, pemphigoid, exanthema and urticaria, epidermal keratinocytes express [C.&U- 1 (Wantzin et al., 1958; Griffiths et al., 1989; Vejlsgard et al., 1989; de Boer et al., 1991). The intensity of ICAM-I expression is increased by increasing seventy of inflammation in the &in. Therefore, there is a possibility of using Po-peptide-1 to target liposomes to the keratinocytes in the inflammatory skin disorders. For this purpose a topical liposome formulation coiild be a feasible approach, since the site of action would be more accessible.

It has been shown that herpes sirnplex virus (HSV)-infected keratinocyte cultures express ICAM-1 (Bennion et al., 1995). Due to the adhesive affinity of Po-peptide-1 for ICAM-1, there is also the possibility of using this peptide for topical targeting of liposomes to HSV-infected keratinocytes for the specific delivery of ad-HSV drugs such as acyclovir and idoxuridine in the treatment of senital and cutaneous HSV infections. The presence of ICAM-1 expression has also been demonstrated on keratinocytes, stroma1 keratocytes, and endothelial cells after keratopl~tyhm patients wiîh corneal infections due to HSV (Elner et al., 1992). Therefore, topical Po-peptide4 liposomes containinj anti-HSV drugs may also increase the therapeutic efficacy of these dnigs in the treatrnent of eye HSV infection.

In surnmary, we have show that Po-peptide-1 when linked to the liposome surface is capable of rnediating the specific bindin; of liposomes to the melanoma cells with a high level of ICAM-1 expression and keratinocytes in the presence of IFN-y. Therefore, it mipht be feasible to use Po-peptide-1 to target liposomes to cancerous melanocytes in rnalignant melanomas and keratinocytes in inflammatory and HSV-infected skin disorders. 5.3 Perspectives

We showed that LFA-1-derived peptide and Po-peptide-l inhibit LFA-1flC.LM- 1 interaction. Increased expression of IChM-1 has been associated with various disorders such as allergic asthma (Weger et al., 1990), autoimmune diseases (Wuthrich et al., 1990), atherosclerosis (Poston et al., 1992), septic shock (Xu et al., 1994, inflarnrnatory dermatoses (Singer et al., 1989), neurological disorders (Akiyama et al., 1993), and organ transplantation (Hau; et al., 1993). Al1 of these disorders are characterized by localized or systernic inflammation. In these disorders, in teraction of IC&M- ILFA- 1 mediates recruitment of inflarnrnatory elements to the site of infiammation. Various approaches have been tried to inhibit the ICAM-l/LFA-1 mediated adhesion of leukocytes to cure these disorders, e.g. mAbs to ICAM-I or LFA-l (Haug et al., 1993; Fischer, !991); a soluble fom of ICAM-1 (SICAM-1) that will compete with the membrane-bound ICAiM-1 (rn1CA.M-1) (O Roep et al., 1994); blocking the expression of ICAM-1 using antisense 01i;onucleotides (Bennett, 1994); and ICAM-1 derived synthetic peptides (Fecondo et al., 199 1). The LFA- 1-denved peptide and Po-peptide-1, which also inhibit the LFA-1ACAM- I interaction, could also be evaluated in the treatment of these disorders. The present investigation also indicated that Po-peptide4 mediates specific interactions with ICAM- 1 expressing cells. With regards to this ability of Po-peptide-1, the following could be the basis of further investigation:

1) Further characterization of specificity of interaction of Po-peptide-1 and [CAM- 1.

2) By considerin; Po-peptide4 as a mother-peptide, producing peptides with higher adhesive potency and more specificity for targeting toward ICAM-1 by changinj the arnino acid residues of Po-peptide-1.

3) Evaluating the feasibility of targeting Pôpeptide- 1-liposomes to cancerous cells in malignant melanomas.

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