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Encapsulation of Plasmid DNA in Stabilized Plasmid – Lipid Particles

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Encapsulation of Plasmid DNA in Stabilized Plasmid – Lipid Particles Jotma/ o/ Drug Twgerois. ?Mw), Vol. 7. No. 6, pp. 423-437 (('I ?OM OPA (Overseas Publishers Association) N.V. Reprints ovailable directly rrom the publisher Published by license under Photocopying permitted by license only the Hamood Academic Publishers imprint, part or The Gordon and Bmch Publishing Group. Printed in Malaysia. Encapsulation of Plasmid DNA in Stabilized Plasmid- Lipid Particles Composed of Different Cationic Lipid Concentration for Optimal Transfection Activity E.G. SARAVOLAC".+, 0. LUDKOVSKI", R. SKIRROW", M. OSSANLOU", Y.P. ZHANG", C. GIESBRECHT", J. THOMPSON', S. THOMAS", H. STARK', P.R. CULLIS".b and P. SCHERRER".b.* 'INEX Plitrr~ii~celclicrrlsiceir~ic~rlsCorp., 100-8900 Glerilyon Purkwuy, Burnuby B.C. VSJ 5J8, Canuth; bDepurtment of Bioclietnistry trritl Moleciilur Biologj. U~iiversityof British Columbia. Vuncouver, B.C. V6T 123. Cunudu; CImritrrtefor Molekulurbiologie und Tirmorforschruig. Emil-Muniikopf~Strrrsse2. 35037 Murhrrrg. Germany (Received 16 5111s1999: Revised II October 1999: In Jnul forni I8 October 1999) In previous work (Wheeler ef al. (1 999) Gene Therupy 6, 27 1-28 1) we have shown that plasmid DNA can be entrapped in "stabilized plasmid-lipid particles" (SPLP) using low levels (5- 10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanola- mine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG- CerCzo). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1 : 1 lipid ratio. The objective of this study was to construct SPLPs with increased For personal use only. cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this techniquecan be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 f6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for 91 vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an Journal of Drug Targeting Downloaded from informahealthcare.com by University British Columbia on 08/16/12 intraperitoneal B 16 tumor model and intraperitoneal administration of SPLP showed * Corresponding author. Department of Biochemistry and Molecular Biology, Liposome Research Unit, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C. V6T 123, Canada. E-mail: [email protected]. Current address: Johnson and Johnson Research Ry. Ltd., G.P.O.3331, Sydney, 2001 NSW,Australia. 423 424 E.G. SARAVOLAC et al. maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4°C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity. Keywords: Non-viral gene delivery, Cationic lipid, Cancer gene therapy, Plasmid encapsulation, Liposomes INTRODUCTION (Fraley et al., 1979; 1980; Lurquin, 1979; Wang and Huang, 1987). Plasmid delivery has primarily relied upon two The focus in our lab has been on the development approaches; virus based gene delivery and non-viral of small (approximately 100 nm diameter), well- gene delivery. Recombinant viral delivery systems defined lipid-based plasmid carrier systems, where while specific are rapidly cleared from the circula- the plasmid is fully encapsulated within 'a lipid tion limiting delivery to first pass organs such as the envelope. Previous studies have shown that plasmid liver (Huard et al., 1995; Worgall et al., 1997). Non- DNA can be encapsulated with high efficiency viral delivery systems have included liposomes, (> 50%) by a detergent dialysis procedure using cationic lipid-DNA complexes or lipoplexes, cat- low cationic lipid concentration (Wheeler et a/., ionic polymers and cation coated nanoparticles 1999). The encapsulation efficiency was a sensitive (reviews see Ledley, 1995; Felgner, 1997; Lasic, function of the cationic lipid concentration used. 1997; Zabner, 1997; Sorgi and Huang, 1997; Chonn The particles formed, of approximately 70 nm diam- and Cullis, 1998; Maurer et af., 1999; Pollard et af., eter, contain one plasmid per particle and are 1998). The highly charged lipoplexes and polymers stabilized with a polyethylene glycol (PEG) coating. are quite efficient in gene delivery to cells in vitro The plasmid in these stabilized plasmid-lipid (Gao and Huang, 1991; Felgner et al., 1994; Zabner particles (SPLP) is fully protected from serum nu- et al., 1995; Hofland et al., 1996). However, these cleases and the particles are stable during circula- For personal use only. charged and often large systems are generally tion in the blood stream in vivo (Monck et al., 1999). cleared rapidly following i.v. injection limiting the It was shown that the transfection potency of SPLP potential transfection sites to first-pass organs, such is dependent on the type of ceramide used as anchor as lung liver and spleen (Thierry et al., 1995; Hong for the PEG polymer. The higher transfection et al., 1997; Hofland et al., 1997; Templeton et al., potency observed with ceramide groups containing 1997; Huang and Li, 1997; Liu et al., 1997). The shorter acyl groups was attributed to the faster DNA is not completely sequestered by these carrier dissociation rate of PEG-Cer with shorter acyl systems, and it is susceptible to degradation by groups, thereby destabilizing the particle and im- nucleases (Wheeler et al., 1999). High concentra- proving cell interaction and uptake into target cells. tions of cationic lipids are known to cause toxicity The inhibitory effect of PEG coating for association in vivo and in vitro (Harrison et al., 1995, Li and and fusion are well known (Holland et al., 1996a,b). Huang, 1997). Furthermore, these complexes often Initial studies have shown that the transfection Journal of Drug Targeting Downloaded from informahealthcare.com by University British Columbia on 08/16/12 exhibit limited stability, have a tendency for activity of SPLP could be increased significantly aggregation and exhibit transfection potencies that by increasing the cationic lipid content in the SPLP can be sample and time dependent. Methods for (Zhang et al., 1999). The formulation method passive encapsulation of plasmid into liposomes are described resulted in efficient encapsulation of highly inefficient and result in low DNA/lipid ratios DNA into well-defined SPLP containing up to OPTIMAL CATIONIC LIPID CONCENTRATION IN SPLP 425 25 mol% cationic lipid. Citrate was used as counter- was synthesized as described in Webb et al. (1998) ion to efficiently shield the positive charge on the and generously provided by Dr. Zhao Wang (INEX lipid intermediate structures, such as cylindrical Pharmaceuticals Corp.). Dioleylphosphatidyl- micelles and lamellar sheets formed during dialysis ethanolamine (DOPE) was obtained from North- to levels compatible with good entrapment. Citrate ern Lipids (Vancouver, BC, Canada). Plasmids concentrations below the optimal range resulted in p BS KS+ (2.964kb), pINEX TKO2 (4.262kb) aggregate formation while at concentrations above pGFPemd-c[R] (4.356 kb), pINEX TKO5 the optimal range, interaction of the plasmid with (5.046 kb), pINEX LO18 (pCMVLuc; 5.650 kb), lipid structures was inhibited and little or no encap- pINEX DO01 (5.963 kb), pINEX PLOl (8.151), sulation occurred. However this protocol was not pINEX PO05 (1 1.125 kb), pINEX BOO1 (15.600kb) successful for cationic lipid concentrations higher were supplied by the Core Support Group (INEX than 25-30 mol%. Pharmaceuticals). A-phage DNA (48.502 kb) was The focus of this work was to develop a formula- supplied by New England Biolabs (Mississauga, tion protocol that permits efficient encapsulation of Ont., Canada). Monobasic potassium phosphate plasmid in SPLP containing 6-50 mol% cationic (ACS), bovine serum albumin, octyl-P-D-gluco- lipid and to determine the optimal cationic lipid pyranoside (OGP), Triton X-lOOTM and DEAE- concentration in SPLP for transfection in vitro and Sepharose CL-6B were obtained from Sigma (St. it7 viiw. Formation of SPLP with equimolar con- Louis,
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