[CANCER RESEARCH 47, 735-739, February 1, 1987] Cytotoxicity of Diphtheria Toxin A Fragment to Toxin-resistant Murine Cells Delivered by pH-sensitive Immunoliposomes1

David Collins and Leaf Huang2

Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840

ABSTRACT have been shown to be resistant to DT, though they bind and internalize DT normally (8,11) and possess a DT-sensitive EF- pH-sensitive immunoliposomes composed of dioleoylphosphatidyl- 2 (12). Thus, DT resistance in mouse cells seems to result from ethanolamine and oleic acid (8:2 molar ratio) mediated the delivery of a block in the translocation of DTA from the endosóme into the cytotoxic fragment A of diphtheria toxin to the cytoplasm of target the cytoplasm. Since pH-sensitive immunoliposomes are able L-929 cells. Free fragment A, fragment A encapsulated in antibody-free liposomes, or fragment A encapsulated in pH-insensitive immunolipo to deliver their encapsulated contents to the cytoplasm of target somes was not effective in the inhibition of the cellular protein synthesis. cells, they should be able to overcome the DTA translocation pH-sensitive immunoliposomes containing diphtheria fragment A were block in DT-resistant mouse cells if delivery is only dependent not toxic to nontarget diphtheria-resistant A31 cells or to nontarget on liposome-endosome fusion. We have chosen the mouse cell diphtheria-sensitive Vero cells. Pretreatment of target L-929 cells with line L-929 to test this hypothesis. Our results, supporting this the weak bases NI I4i'l or chloroquine, agents which raise the endosóme/ model, show that pH-sensitive immunoliposomes are able to lysosome pH, blocked the cytotoxic effect of the pH-sensitive immuno release active DTA from an acidic cellular compartment follow liposomes containing fragment A. Excess free antibody or excess empty ing receptor-mediated endocytosis of the immunoliposomes by pH-sensitive immunoliposomes also blocked the cytotoxic effect. Since target cells. Furthermore, we have demonstrated the potential it is known that fragment A alone cannot cross lipid membranes, the of the liposome system as a target-specific cytotoxic reagent for results of this study indicate that pi I-sensitive immunoliposomes are able to release the toxin into the cytoplasm, probably by fusing with the cancer chemotherapy. endosómemembrane following a receptor-mediated endocytosis of the immunoliposome. MATERIALS AND METHODS

Materials. DOPE and DOPC were purchased from Avanti Polar INTRODUCTION Lipids, Inc. Chloroquine, NH4('l. and oleic acid were purchased from Sigma Chemical Co. Diphtheria toxin was purchased from Connaught Liposomes composed of DOPE3 and acylated amino acids Laboratories (Ontario, Canada). [3H]Leucine and [3H]NAD were pur (1), fatty acids such as arachidonic acid4 (1, 2), OA (3, 4), chased from New England Nuclear. NAD* (free acid) was purchased palmitic acid (4), or cholesteryl hemisuccinate (5) are stable at from Calbiochem. Ricin toxin was isolated from castor beans by the neutral pH but become unstable and fusion-active at the weakly method of Nicholson and Blaustein (13). acidic pH of 5.0-6.5 (1-5). These pH-sensitive liposomes can Antibody Preparation. Ami // 7A*antibody from the murine hybrid be coated with fatty acid-derivatized antibody to enhance the orna cell line 11-4.1 was purified, labeled with I2SI,and derivati/ed with cytoplasmic delivery of encapsulated fluorescent dye (6), anti- the ,V hydroxysuceinimide ester of palmitic acid, as described by Huang, tumor drugs (7), or nucleic acids5 to target cells. Cytoplasmic et ai. (14). Preparation of Diphtheria Fragment A. Fragment A was prepared delivery is thought to be achieved through receptor-mediated from intact diphtheria toxin by the method of Gill and Dinius (9). DTA endocytosis of the immunoliposomes. The immunoliposomes was then labeled with ' " 1by the chloramine T method. then encounter the acidic pH of the endosóme and are thought Liposome Preparation. Dehydration-rehydration vesicles were pre to mediate cytoplasmic delivery after fusion with the endosóme pared by a modification of the method reported by Gregoriadis and membrane (6, 7). Kirby (15). Sonicated unilamellar vesicles composed of DOPE:OA (8:2 To test this hypothesis we have used DTA as a marker for molar ratio) or DOPC were prepared in 1/10 x PBS. The pH of the cytoplasmic delivery. DT is secreted by Corynebacterium diph- preparation was maintained at 8.0-8.5 with l N NaOH since the pH theriae as a single polypeptide chain with a molecular weight was found to decrease during sonication. The sonicated vesicles were then added to an equal volume of DTA (0.2 mg/ml) in 1/10 x PBS. of 67,000 (8). The toxin can be cleaved by trypsin and reduced Fatty acid-derivatized antibody in 1/10 x PBS, 0.15% deoxycholate by thiol compounds to yield two fragments which differ in was added to the mixture to obtain a final lipid antibody molar ratio of activity (8-10). DT fragment B is involved in binding of the 4x10'. The mixture was then dialysed for 24 h against three changes toxin to the cell surface and mediating the translocation of the of 1/10 x PBS (pH 8.0). The mixture was frozen as a thin shell by active moiety, DTA, into the cytoplasm (10). DTA inhibits swirling in a dry ice-ethanol bath and lyophilized overnight. The freeze- protein synthesis in eukaryotes by catalyzing the transfer of dried sample was rehydrated with distilled H2O at one-tenth of the ADP-ribose from NAD to eukaryotic EF-2 (8). Mouse cell lines original vesicle volume, vortexed vigorously, and allowed to stand for 2-3 h, vortexing occasionally. PBS was added to bring the final lipid Received 8/27/86; revised 10/24/86; accepted 10/27/86. concentration to 16.5 mM. The liposomes were then extruded through The costs of publication of this article were defrayed in part by the payment a 0.1-/im polycarbonate filter for better size uniformity (16). Unencap- of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sulated DTA was separated from liposomes by passage over a Sephadex ' This investigation was funded by NIH Grant ÇA245S3. G-200 column. Liposomes without antibody were prepared identically, 2 Research Career Development Awardee (CA 00718). To whom requests for except no palmitoyl antibody was added. Empty (DTA-free) liposomes reprints should be addressed, at University of Tennessee, Department of Bio chemistry, M407 Walters Life Science Building, Knoxville, IN 37996-0840. and immunoliposomes were also prepared. 3The abbreviations used are: DOPE, dioleoylphosphatidylethanolamine; Acid-induced Liposome Leakage. Liposome leakage in response to DOPC, dioleoylphosphatidylcholine; OA, oleic acid; DT, diphtheria toxin; DTA, pH was measured by using the self-quenching dye, calcein (17), as an diphtheria toxin fragment A; PBS, phosphate-buffered saline (137 min NaCl, 2.7 aqueous space marker. DOPE:OA and DOPC immunoliposomes were HIMKCI, 5.5 min KH2PO4, 1.12 mM NajHPO«);1/10 x PBS, one-tenth concen tration of phosphate-buffered saline; EF-2, elongation factor 2. prepared as described above to encapsulate 50 imi calcein. Unencap- 4 S. Kanda and I.. Huang, unpublished data. sulated calcein was removed from liposomes by passage over a Sepha- 9C. Y. Wang and L. Huang, unpublished data. rose 4B column. pH-dependent leakage of calcein from liposomes was 735 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. TARGETED DELIVERY BY pH-SENSITIVE IMMUNOLIPOSOMES assayed spectrofluorometrically as described previously (1). ADP-ribosylation Assay. A crude extract of EF-2 was obtained from untoasted wheat germ by the method of Clemens (18). Aliquots of the extract were stored at —70°Cuntiluse. ADP-ribosylation of wheat germ EF-2 (19) was carried out in 400 ti\ reaction mixtures containing 20 /¿g EF-2 (2.8 mg/ml) in 0.25 M Tris-HCl (pH 8.2), 0.04 M dithiothreitol, and 0.1 IHMEDTA. 16 »toxins were added. After 3 h at 37°C,the Gregoriadis and Kirby reported encapsulation efficiencies for cells were washed and fresh medium with 10% fetal calf serum was dehydration-rehydration vesicles of between 26 and 72% of added. After 18 h, the medium was replaced with fresh medium con available solute (15). Using the modified dehydration-rehydra taining pHjleucine (10 /iCi/ml) and incubation was continued for an tion vesicles method, we consistently encapsulated between 10 additional 6 h. The cells were then washed and the proteins were precipitated by adding 200 ti\ of ice-cold 5% trichloroacetic acid to and 20% of the available DTA (Table 1). DTA encapsulation each well. After 10 min, the supernatant was removed and an additional was generally lower for DOPE:OA liposomes than for DOPC 200 n\ of 5% trichloroacetic acid per well was added and incubated an liposomes. Immunoliposomes also encapsulated less DTA than additional 10 min. The supernatant was removed again and the precip liposomes without palmitoyl antibody. Antibody incorporation itate was dissolved with 0.1 M NaOH (100 pi/well) and processed for into the liposome membranes ranged 32-57% in different ex scintillation counting. Untreated control cells incorporated between periments and was generally lower for DOPE:OA immunoli 7,000 to 20,000 cpin in different experiments. All experiments were posomes than for DOPC immunoliposomes (Table 1). The done in quadruplicate. Cytotoxicity Blocking. Underivatized ami // _'A' antibody or empty enzymatic activities of free and encapsulated DTA were also immunoliposomes were incubated with the L-929 cells for 1 h at 37°C assayed. When the immunoliposomes were lysed with Triton- 100 to release all the encapsulated DTA, approximately 72- prior to addition of pH-sensitive immunoliposomes containing DTA. The free antibody concentration was a 10-fold excess of the antibody 77% of the activity was detected as compared with that of the concentration in the immunoliposomes. Empty pH-sensitive immuno free DTA. This result indicates that the encapsulation proce liposomes were added to a S-fold excess of the lipid concentration of dure only slightly reduced the activity of DTA. Comparing the the DTA-containing pH-sensitive immunoliposomes. Following incu activities assayed in the presence and absence of Triton X-100, bation with the immunoliposomes containing DTA, the cells were DTA which was encapsulated in DOPE:OA immunoliposomes washed, labeled with [3H]leucine, and treated as described above. had 86% latent activity while DTA encapsulated in DOPC immunoliposomes had 83% latent activity. It is concluded that RESULTS the majority of the DTA was encapsulated inside the immuno liposomes without appreciable loss of activity. Addition of Liposomes composed of DOPE:OA (8:2 molar ratio) have Triton had no significant effect on the ADP-ribosylating activity been shown to become fusion competent at the weakly acidic of free DTA (Table 1). pH of 5-6.5 (1-7) while DOPC liposomes are stable and do The toxicity of the liposome preparations to target L-929 not fuse in this pH range (1, 3). When DOPE:OA (8:2) lipo cells is shown in Fig. 2. pH-insensitive DOPC liposomes con somes are coated with fatty acid-derivatized antibody against taining DTA did not inhibit protein synthesis in the concentra the mouse major histocompatibility antigen H-2Kk, cytoplasmic tion range used. Toxicity of the DOPC liposomes could not be delivery of encapsulated materials to target cells is enhanced enhanced by incorporation of antibody into the liposome mem (6, 7). The target cells used are mouse L-929 cells of the k brane. It has been shown previously that DOPC liposomes do haplotype and express the H-2K* antigen. A31 cells and Vero not release their encapsulated contents into the cytoplasm, since cells were chosen as nontarget cells since neither expresses they are delivered to the lysosome and degraded (20). DTA- H-2K11. Furthermore, Vero cells are DT-sensitive while both containing pH-sensitive DOPE:OA liposomes without antibody L-929 and A31 cells are resistant to the toxin (8). were also nontoxic. This result is probably due to a lack of Calcein is a fluorescent dye which is self quenching at high sufficient binding and internalization of the liposomes. concentrations but upon dilution the fluorescence increases DOPE:OA immunoliposomes containing DTA showed a (17). Therefore, calcein is a convenient aqueous marker for marked increase in toxicity over free DTA. The IC50 for the liposome leakage. Fig. 1 shows the pH-dependent leakage of DOPE:OA immunoliposomes containing DTA was approxi mately 2.5 x 10~5mg/ml. Unencapsulated DTA or nicked DT calcein from DOPE:OA or DOPC immunoliposomes. Consist ent with earlier data from our laboratory (1), DOPE:OA im had no effect on protein synthesis while the cells were fully munoliposomes release calcein in response to a decrease in pH. sensitive to ricin toxin. Empty DOPErOA immunoliposomes The pH required for 50% release of contents is approximately were nontoxic to cells. 6.35 for the DOPE:OA immunoliposomes. DOPC immunoli Fig. 3 shows the toxicity of the various liposome preparations posomes are not acid-sensitive since no significant leakage of on nontarget A31 cells. Neither free DTA nor nicked DT were aqueous contents occurs even at pH 4.0 (Fig. 1). able to inhibit protein synthesis. None of the liposome or 736

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Table 1 Parameters of liposome preparations of available of available palmitoyl DTA encap antibody DTA:antibody:lipid sulated9.6 incorporated39 molarratio8x %'0.141 Latency, 10-«:4x10-3:16.51 DOPE:OA:DTA: ±3.012.0 ±7 ±0.018ND" 16.0ND0.132±0.008 86 ± antibody x 10-3: :16.5 DOPE:OA:DTA ±4.9 1.4X10-3:16.51.7 10-3:3.9x DOPC:DTA: 17.1±8.120.5 50 ±7Activity*Final 0.022±0.016ND0.192 ±16.8ND0.1±0.010 83 antibody x IO"3: :16.5—Triton0.020 DOPODTA ±6.7% DTA (free)% ±0.016-HTriton* 82 ±0.022 * ADP-ribosyltransferase activity is defined as pmol ADP-ribose incorporated into trichloroacetic acid insoluble material per »igDTA. * Liposomes were lysed with 0.1 % Triton N I(10prior to addition to reaction mixture. ' [activity (-Triton)/activity (+Triton)] x 100%. rfND, not done.

IO- 10" 10* 10"° IO4 10 10 10 10 10 Toxin Concentration (mg/ml) Toxin Concentration (mg/ml) Fig. 2. Toxicity of free or encapsulated toxins toward target cells were incu Fig. 3. Toxicity of free or encapsulated toxins toward nontarget cells. A31 bated with DOPE:OA (8:2) immunoliposomes (A), DOPC immunoliposomes (:0. cells were incubated with DOPE:OA (8:2) immunoliposomes (A), DOPC immu DOPE:OA (8:2) nontargeted liposomes (A), and DOPC nontargeted liposomes noliposomes (a), DOPE:OA (8:2) nontargeted liposomes (A), and DOPC nontar geted liposomes (•)allcontaining DTA [DTA (•),nicked DT (x), and ricin toxin (•)allcontaining DTA. DTA (•)nickedDT (x) and ricin toxin CJ) were incubated (B)]. Cytotoxicity was assayed as the inhibition of (3H]leucine incorporation. with the cells as were empty DOPE:OA (8:2) immunoliposomes (O). Cytotoxicity was assayed as the inhibition of [3H]leucine incorporation. munoliposome-mediated translocation of DTA into the cyto immunoliposome preparations were toxic to the cells. Ricin plasm. toxin was used as a positive control to show that the assay We also investigated the dependence of our delivery system works for this cell line. on specific cell-surface binding of immunoliposomes. L-929 All the liposome preparations used in this study were non- cells were preincubated for l h in the presence of an excess of toxic to DT-sensitive Vero cells (Fig. 4). Unencapsulated DTA free anti-//-2A'A antibody prior to immunoliposome addition. was also nontoxic to the cells. A dose-dependent toxicity was As seen in Fig. 5, such cells were protected from the toxic effect observed with Vero cells when incubated with nicked DT or of encapsulated DTA. Pretreatment of cells with empty ricin toxin. DOPE:OA immunoliposomes prior to addition of pH-sensitive To see if endosome/lysosome acidification was required for immunoliposomes containing DTA also effectively blocked pH-sensitive (DOPE:OA) immunoliposome-mediated cytotox- DTA delivery. icity to L-929 cells, we repeated the cytotoxicity experiments in the presence of either NH4C1 or chloroquine. These drugs DISCUSSION are weak bases which raise the internal pH of acidic organelles in the cell (21). Cells which were pretreated with NH4C1 or It has been shown that DT-resistant cells, such as L-929 cells, chloroquine prior to immunoliposome addition were protected are not defective in the binding and internalization of DT (10), from intoxification by immunoliposome-encapsulated DTA rather there is a block in the translocation of DTA from the (Fig. 5). Neither NH4C1 nor chloroquine alone had an effect on endosóme into the cytoplasm. As shown in Fig. 2, pH-sensitive protein synthesis (data not shown). Therefore, endosome/lyso immunoliposomes were able to overcome this translocation some acidification appears to be required for pH-sensitive im block and release active DTA into the cell cytoplasm, and thus 737

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120 immunoliposomes toward either DT-resistant (Fig. 3) or DT- sensitive nontarget cells (Fig. 4). Furthermore, the cytotoxic effect of immunoliposomes on target L-929 cells could be 100 blocked by either empty immunoliposomes or free antibody (Fig. 5). Weak bases such as NH4C1 and chloroquine also blocked the immunoliposome-mediated toxicity (Fig. 5). These drugs are known to inhibit a variety of cellular events which require acidification of the endosóme, such as the release of the Semliki Forest virus genome (22) and the translocation of DTA in DT-sensitive cells (23, 24). Thus, endosome/lysosome acid ification is required for cytoplasmic delivery by pH-sensitive immunoliposomes. As can be seen in Fig. 2, the maximum inhibition of protein synthesis by pH-sensitive immunoliposomes containing DTA was approximately 60% at the highest concentration used. Cytotoxicity could not be enhanced by increasing the time of exposure to immunoliposomes or increasing the incubation period after immunoliposome removal (data not shown). The lack of cytotoxicity was not due to heterogeneity in cell-surface antigen expression as measured by immunofluorescence (data not shown) nor to the existence of "immunoliposome-resistant" variants in the population (data not shown). It seems therefore 10 10"° 10" 10" 10 that the heterogeneity responsible for the lack of killing resides Toxin Concentration (mg/ml) in the liposomes rather than in the cell population. One possi Fig. 4. Toxicity of free or encapsulated toxins to nontarget cells. Vero cells bility which cannot be ruled out at the present time is that the were incubated with DOPE:OA (8:2) immunoliposomes ¡(A),DOIT immunoli posomes (a), DOPE:OA nontargeted liposomes (A), and DOPC nontargeted distribution of DTA in the immunoliposome population is liposomes (•)allcontaining DTA [DTA (•),nickedDT (x), and nein toxin (H)]. heterogeneous. The numbers given in Table 1 for liposome- Cytotoxicity was assayed as the inhibition of [3H]leucine incorporation. associated DTA concentrations refer only to averages over the entire liposome population. A heterogeneous distribution of 15 . DTA might result in certain cells receiving loaded immunoli posomes and others receiving empty ones. The empty immu noliposomes would therefore act as competitive blockers of the immunoliposome effect (Fig. 5). This might also explain the observation that the cytotoxicity of encapsulated DTA is less than expected theoretically (12). Nevertheless, it is clear that pH-sensitive immunoliposomes are able to mediate the cyto plasmic delivery of encapsulated DTA. 10 _ The observation that the pH-sensitive immunoliposomes are able to bypass the translocation block suggests that the site of DTA release is at the endosóme. This notion is consistent with previous work in our laboratory in which pH-sensitive immu noliposomes were able to mediate the cytoplasmic delivery of 1-iS-D-arabinofuranosylcystosine (7). l-/3-D-Arabinofuranosyl- cytosine is lysosome-sensitive in that exposure of the drug to lysosomal enzymes leads to its degradation and inactivation (2). It has been extensively shown that pH-sensitive immunoli posomes become fusion-active when exposed to an acidic envi ronment (pH 5-6.5) (1-5). This is the range of the endosóme pH (21). We therefore suggest that pH-sensitive immunolipo somes, like Semliki forest virus (22), influenza virus (25), and vesicular stomatitis virus (26), release their contents into the cytoplasm from the endosomes and that the release step is DOPElOA DOPE:OA OOPE:OA DOPE:OA DOPE:OA (DTA) (DTA) (DTA) (DTA) (DTA) probably a result of fusion of the liposome with the endosóme

Free Ab DOPEiOA membrane in response to the acidification of the organelle. Fig. 5. Blocking of encapsulated DTA toxicity. L-929 cells were pretreated Another possible mechanism of release is that pH-sensitive with excess free antibody, excess empty DOPE:OA immunoliposomes, chloro- immunoliposomes become leaky at the endosomal pH (27) and quine, or NH¿C1priorto addition of pH-sensitive immunoliposomes containing DTA (DOPE:OA:DTA). [3H]Leucine incorporation into protein was then as release their contents in the interior of the endosóme. Free sayed. DTA then passes into the cytoplasm. However, since DTA has been shown to be unable to cross lipid membranes in the absence the pH-sensitive lipid composition was required for cytoplasm it- of the B fragment of DT (9, 21, 28), it is unlikely that this delivery, Antibody on the pH-sensitive liposome membrane was mechanism of delivery would result in the toxicity observed in required since nontargeted pH-sensitive liposomes had no effect our system (Fig. 2). Since it has been shown that DOPE:OA on protein synthesis (Fig. 2). The target specificity of the system liposomes undergo a bilayer-to-hexagonal phase transition at is indicated by the lack of cytotoxicity of the pH-sensitive low pH (27), it is possible that pH-sensitive immunoliposomes 738

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. TARGETED DELIVERY BY pH-SENSITIVE IMMUNOLIPOSOMES induce endosóme rupture, thereby releasing DTA into the cy 13. Nicholson, G., and Blaustein, J. The interaction of Ricinus communi* agglu- tinin with normal and tumor cell surfaces. Biochim. Biophys. Acta, 266: toplasm. While the precise mechanism of delivery cannot be 543-547, 1972. determined at the present time, it is clear that pH-sensitive 14. Huang, A., Tsao, Y. S., Kennel, S. J., and Huang, L. Characterization of immunoliposomes provide an effective vehicle for targeted, antibody covalently coupled to liposomes. Biochim. Biophys. Acta, 7/6:140- 150, 1982. cytoplasmic delivery. To our knowledge, this is the first report 15. KIrin, C., and Gregoriadis, G. Dehydration-rehydration vesicles: a simple in which liposomal delivery of DTA has been achieved using method for high yield drug entrapment in liposomes. Biotechnology, 1:979- 984, 1984. pure lipid vesicles. In earlier reports, liposome carriers for DTA 16. Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., and Papahadjopoulos, D. were either ineffective (22) or effective only when viral fusion Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta, 557:9-23, 1979. proteins were included in the liposome membrane (12,30). The 17. Allen, T. M., and Cleland, L. G. Serum-induced leakage of liposome contents. system at present has potential as a targeted carrier for use in Biochim. Biophys. Acta, 597:418-426, 1980. cancer chemotherapy as well as the delivery of macromolecules 18. Clemens, M. J. Translation of eukaryotic messenger RNA in cell-free ex tracts. In: B. D. Hames, and S. J. Higgins (eds.), Transcription and Trans such as enzymes, nucleic acids, and immunomodulators to lation: A Practical Approach, pp. 231-270, Oxford, England: 1RL Press, targeted cell populations. 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739 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1987 American Association for Cancer Research. Cytotoxicity of Diphtheria Toxin A Fragment to Toxin-resistant Murine Cells Delivered by pH-sensitive Immunoliposomes

David Collins and Leaf Huang

Cancer Res 1987;47:735-739.

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