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Bimonjugate Chem. 1993,4, 296-299
TECHNICAL NOTES Synthesis of an End-Group Functionalized Polyethylene Glycol-Lipid Conjugate for Preparation of Polymer-Grafted Liposomes Samuel Zalipsky Liposome Technology, Inc., 1050 Hamilton Court, Menlo Park, California 94025. Received March 9, 1993
Synthesis of a distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) conjugate, bearing a hydrazide group at the unattached end of the polymer chain, was achieved using a new heterobifundional polymeric reagent. The heterobifunctional PEG derivative carrying on one end a reactive succinimidyl carbonate (SC) group and a t the other terminal a tert-butyloxycarbonyl (Boc) protected hydrazide group was prepared by an efficient four step process from readily available PEG-2000. The SC-end group of the polymer reacted readily with the amino group of DSPE forming a stable urethane attachment between lipid and PEG. Acidolytic removal of the Boc group yielded a hydrazide-PEGlipid conjugate suitable for preparation of polymer-grafted liposomes. Taking advantage of the well-documentedchemical versatility of hydrazide groups, various biologically relevant ligands can be linked to this type of functionalized liposomes.
INTRODUCTION Lack of toxicity, excellent solubility, and superb biocompatibility made polyethylene glycol (PEG') one of the most popular modifiers of biologicals (1). Examples of PEG conjugates range from low molecular weight drugs prepared to alter pharmacokinetics, biodistribution, or toxicity (2, 3) to surfaces of biomaterials used to reduce protein adsorption and thrombogenicity (4). PEG has been used extensively to alter in vivo properties of biological macromolecules, particularly proteins (5-7). More recently, liposomes carrying PEG chains on their exterior were discovered to have extended half-lifes in bloodstream (up to t1p = 48 h in humans). With this, one of the most important limitations of liposomal drug delivery was overcome (8-12). Such liposomeswere named Stealth2 liposomes for their ability to avoid uptake by reticuloendothelial system. In the scientific literature they have been referred to as sterically stabilized liposomes on the basis of a proposed mechanism where protein adsorption is inhibited by a steric barrier created by the polymeric "brush" (for a comprehensive review see ref 9). Now that long-circulating liposomes are within our reach, the next frontier is to use them as carriers for various ligands. One can envisage use of such conjugates for targeting and immunomodulation. Ligands of potential interest can be directly linked to polar groups of conventional lipids coincorporated into liposomes with PEG-PE. Not surprisingly, this approach has a drawback in that PEG chains interfere with the interaction between a ligand and its target (12). The polymer would also be expected to interfere with chemical reactions between reactive groups on the surface of a liposome and a potential ligand. ~~
~
'Abbreviations used: PEG, polyethylene glycol; mPEG, methoxy-PEG; PMA, polymethacrylic acid; PE, phosphatidylethanolamine;DSPE, distearoyl-PE;Boc, tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide;DSC, disuccinimidylcarbonate; SC, succinimidyl carbonate. Wealth is a registered trademark of Liposome Technology, Inc.
Thus, for numerous applications it seems attractive to link potential ligands to the far end of PEG chains. In light of the fact that all the PEGlipids used to date for preparation of liposomes have been derived from commercially available methoxy-PEG (mPEG) and thus carry unreactive methoxy end groups, it is essential to develop a new approach toward functionalized PEG-lipid conjugates. In this paper I am presenting an efficient method for synthesis of a new type of functionalized PEGlipid conjugate, namely DSPE-PEGhydrazide (l),suitable for preparation of PEGliposomes with reactive hydrazide groups positioned on the exterior. RESULTS AND DISCUSSION DSPE was previously shown to be the starting material of choice for preparation of modified lipids suitable for incorporation into liposomes. For example, urethanelinked mPEGDSPE incorporated into liposomes performed well by improving pharmacokinetics and biodistribution (9).The functionalized conjugate sought as the target of this study in its simplest form is an analog of mPEGDSPE in which the inert methoxy group is replaced by hydrazide. It is apparent that efficient preparation of such a material is dependent on the availability of a heterobifunctional derivative of PEG having a reactive acyl group on one terminal and a hydrazide group on the other. The hydrazide group has to be in a protected form to prevent undesirable reaction between the two terminal groups of the polymer. Succinimidyl carbonate (SC) derivatives of PEG were recently introduced for modification and cross-linking of proteins (13,141 and preparation of high molecular weight polymers (15,161.Both applications were successful due to very efficient urethane formation reactions of SC groups with amino-containing molecules. Preliminary experiments showed that SC-PEG reacted within minutes with DSPE, producing a product identical to the mPEGDSPE obtained by the previously published procedure (10).Given this result, the SC group was chosen as the active acyl
7Q43-~8Q2/93/29Q~O296$Q4.QQlQ 0 1993 American Chemical Society
Technical Notes Scheme I
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Scheme I1
H
1. 2 I triethylamine
2. 4 M HCI in dioxane
C17H33
O-l
1
group for the heterobifunctional PEG. The choice of the hydrazido-protecting group (tert-butyloxycarbonyl, Boc) was governed by the stability of the mPEGDSPE under acydolytic deprotection conditions. Scheme I illustrates the sequence of reactions used to obtain Boc-protected hydrazido PEG having a reactive SC group at the opposite terminal (2). First, the w-hydroxy acid derivative of PEG (3) was prepared using a modification of the previously published procedure (17). Ethyl isocyanatoacetate was employed for partial introduction of carboxyl groups onto PEG of molecular weight 2000 forming a mixture of dicarboxylated PEG, w-hydroxy acid derivative, and unreacted starting polymer (18). The w-hydroxy acid derivative (3) purified by ion exchange chromatography was coupled with tert-butyl carbazate in the presence of dicyclohexylcarbodiimide,thus introducing a protected hydrazide group on one end of the polymer (derivative 4). Activation of the hydroxy end group with disuccinimidyl carbonate in the presence of pyridine was performed by a modification of the published procedure for Sepharose activation (19). This deviation from the original protocol, requiring treatment of the polymer with phosgene as the first step (13,14), was necessary due to the instability of both the Boc and hydrazide groups under these conditions. However,incorporation of the SC groups under the new set of conditions proceeded quantitatively, as was confirmed by NMR and titration of the active acyl groups. A slight excess of 2 reacted with chloroform-suspended DSPE in the presence of triethylamine (Scheme 11). The lipid derivative was quickly solubilizedduring this reaction. Taking advantage of ita very low critical micelle concentration, the product was purified by dialysis using a 300 000 molecular weight cutoff (MWCO) membrane and then lyophilized. The structure of the Boc-protected conjugate was confirmed by NMR,and it wa8 shown to be pure by TLC analysis. Deprotection with 4 M HC1 in dioxane produced the target DSPE-PEGhydrazide (l),as was ascertained by comparison of ita NMR spectra with those
of the Boc-protected starting material, urethane-linked mPEGDSPE, and mPEG-oxycarbonyl-glycine hydrazide. The last material was obtained by hydrazinolysis of the corresponding ethyl ester (20). Treatment of 1in borate buffer solution with trinitrobenzene sulfonate produced the red-maroon color (A, = 500 nm) characteristic to hydrazides. This color reaction failed when 1 was preincubated with an excess of valeraldehyde or 4-carboxybenzaldehyde. These observations are consistent with the known hydrazide-aldehyde reactivity leading to hydrazone formation and indicate that the conjugate 1is functionally reactive. The conjugate 1 can be formulated with lecithin and cholesterol into a liposomalpreparation containing reactive hydrazide groups. Preliminary experiments demonstrated that such PEGhydrazide liposomes showed pharmacokinetic profiles similar to the more conventional m P E G DSPE counterparts. The chemicalversatility of hydrazide group, ita use as a starting material for introduction of other functional groups, and ita ability to participate in numerous types of conjugation schemes is well-documented in literature (21). Particularly useful is ita reactivity toward various glycoproteins, e.g. immunoglobulins (22). Studies of hydrazide-containing PEGliposomal conjugates are currently underway and will be reported in the future. This paper presents a facile and an efficient procedure for a new heterobifunctional PEG derivative (2), employing readily available and inexpensive PEG-diol as a starting material. Unlike alternative procedures for synthesis of PEGS with two different end groups (see for example, Yokoyama et al. (23) and references cited therein), this method does not involve polymerization of toxic and explosive ethylene oxide. The scope of potential applications of the heterobifunctional polymer 2 goes beyond the synthesis of the lipid conjugate shown in Scheme 11. For example, it is suitable for attachment of ligands to surfaces, linking cofactors to enzymes, and preparation of various other macromolecular conjugates. One can take advantage of the presence of 1 equiv of glycine in the reagent for characterization of such conjugates by amino acid analysis of their hydrolysates (20). EXPERIMENTAL PROCEDURES
Preparation of 3. PEG-diol of molecular weight 2000 (42 g, 42 mequiv of OH) was dissolved in toluene (200 mL), azeotropically dried, and then treated with ethyl isocyanatoacetate (2.3 mL, 21 mmol) and triethylamine
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(1.5 mL, 10 mmol). After overnight reaction at 25 OC the solution was evaporated to dryness. The residue was dissolved in 0.2 M NaOH (100 mL), a trace of toluene was evaporated, and pH 1 2 was maintained with periodic dropwise additions of 4 M NaOH. When the pH stopped dropping, the solution was acidified to pH 3.0 and the product extracted with methylene chloride (100 mL X 2). TLC on silica gel G (iPrOH/HzO/concentrated ammonia 10:2:1) gave the typical chromatogram of partially carboxylated PEG (17,18)consisting of unreacted PEG (Rf = 0.67) and monocarboxylated (Rj= 0.55) and dicarboxylated (Rf= 0.47) derivatives of the polymer. This solution was dried (MgSOd), filtered, and evaporated to dryness. The remaining PEG mixture was dissolved in water (50 mL). One third of this solution (30mL, =14 g of derivatized PEG) was loaded onto DEAE-Sephadex A-25 (115 mL of gel in borate form). After the underivatized PEG was washed off the column with water (confirmed by a negative PMA test a gradient of ammonium bicarbonate (220 mM at increments of 1-2 mM every 200 mL) was applied, and 50-mL fractions were collected. It was determined by the PMA test and TLC that fractions 1-25 contained only PEG monoacid. These fractions were pooled together, concentrated to -70 mL, acidified to pH 2, and extracted with methylene chloride (50 mL X 2). The CHzClz solution was dried (MgSOd), concentrated, and poured into cold stirring ether. The precipitated product was dried in uucuo. Yield: 7 g. Titration of carboxyl groups gave 4.6.10""equiv/g (97% of theoretical value). Preparation of 4. The w-hydroxy acid derivative of PEG (3,5 g, 2.38 mmol) and tert-butyl carbazate (0.91 g, 6.9 mmol) were dissolved in CHzClz-ethyl acetate (l:l,7 mL). The solution was cooled on ice and treated with DCC (0.6 g, 2.9 mmol) predissolved in the same solvent mixture. After 30 min the ice bath was removed and the reaction was allowed to proceed for an additional 3 h. The reaction mixture was filtered from dicyclohexylurea and evaporated. The product was recovered and purified by two precipitations from ethyl acetate-ether (1:l)and dried in uucuo over PzO5. Yield: 5.2 g, 98%. TLC of the product gave one spot (Rf= 0.68) different from the starting material (Rf = 0.55). H NMR (CDCl3): 6 1.46 (s, tBu, 9H), 3.64 (s, PEG, 178H), 3.93 (br d, J = 4.5 Hz, HNCHZCO, 2H), 4.24 (t, J 4.5 Hz, CHzOCONH, 2H) ppm. 13C NMR (CDCl3): 6 28.1 (tBu),43.4 (CHzof Gly), 61.6 (CHzOH), 64.3 (CHzOCONH), 69.3 (CHzCHzOCONH), 70.5 (PEG), 72.4 (CHzCHzOH), 81.0 (CMes), 155.1 (C=O of Boc), 156.4 (C-0 of urethane), 168.7 (C=O of Gly hydrazide) ppm. Preparation of 2. The w-hydroxy Boc-hydrazide derivative of PEG (4, 5 g, 2.26 mmol) was dissolved in pyridine (1.1mL), CHzClz (5 mL), and CH3CN (2 mL) and treated with DSC (1.4 g, 5.5 mmol) at 25 "C overnight. The solution was filtered and gradually added to cold ethyl ether (100 mL). The precipitated product was dissolved in warm ethyl acetate (45 mL), chilled, and mixed with an equal volume of ethyl ether. The precipitate was collected by filtration and dried in uucuo over P205. Yield: 4.8 g 90%. SC groups content 4.15.1V mequiv/g (98% of theoretical value) was determined by titration (13). H NMR (CDCl3): 6 1.46 (e, tBu, 9H), 2.83 (s, succinimide), 3.64 (8, PEG, 178H), 3.79 (t, J = 4.7 Hz, CHzCHzOCOz, 2H), 3.93 (br d, J = 4.5 Hz, CHZof Gly, 2H), 4.24 (t, J = 4.5 Hz, CHzOCONH, 2H), 4.46 (t,J = 4.7 Hz, CHzOCOz, 2H) ppm. 13C NMR: 6 25.5 (succinimide), 28.2 ((CH&C), 43.3 (NHCHzCO), 64.3 (CHzOCONH), 68.3 (CHzOCOz), 69.4 (CHzCHzOCOz), 70.6 (PEG), 80.9 ((CH3)3C), 151.6
(In),
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Zalipsky
(OCOz), 155.3 (C=O of Boc), 156.7 (C=O of urethane), 168.6 (C=O of succinimide), 169.1 (C=O of Gly) ppm. Preparation of 1. Boc-hydrazide-PEGSC (2,693 mg, 0.29 mmol) was dissolved in chloroform (4 mL) and treated with solid DSPE (200 mg, 0.27 mmol) and triethylamine (0.1mL, 0.72 mmol). The suspension was vigorouslymixed and maintained at 45 "C for a10 min. During this time a clear solution was formed. TLC (chloroform-methanolwater 9018:2) showed complete disappearance of ninhydrin-positive DSPE (Rf = 0.22) and formation of a new product (Rf= 0.55, IZ vapor visualized) in addition to a weak spot due to the unreacted PEG reagent (Rf= 0.73). Acetic acid (42 mL, 0.73 mmol) was added to the reaction mixture and it was evaporated to dryness. The solid residue was slowly dissolved in water (5 mL) and the trace of remaining chloroform evaporated. The solution (pH = 6) was transferred into Spectrapor CE dialysis tubing (MWCO 300,000)and dialyzed against saline solution (=50 mM) at 4 OC until the dialysis solution became negative to the PMA test for PEG (17) (3 X 1000 mL, 8-16 h per period). The conjugate solution was further dialysed against deionized water, filtered through a 0.2-pm sterile filter, and lyophilized, producing white solid Boc-hydrazide-PEGDSPE (520 mg, 65%). H NMR (CDCl3): 6 0.88 (t, J = 6.8 Hz, CH3, 6H), 1.26 (s,CHz, 56H), 1.46 ( 8 , tBu, 9H), 1.58(br, t, J = 7.3 Hz, 4H), 2.28 (2 overlapping t, J = 7.3 Hz, CH2C=O, 4H), 3.36 (br, m, OCHzCHzNH, 2H), 3.64 ( 8 , PEG, =180H), 3.94 (br m, CHZof Gly and CHZCH~O-P, 4H), 4.17 (dd,J = 7.0,12 Hz, glycerol CHzOP, 2H), 4.19-4.25 (br m, CHZ-0of both urethanes, 4H), 4.39 (dd, J = 3.2, 12 Hz, glycerol CHzO-C=O, 2 H), 5.20 (m, CH glycerol, IH) ppm. I3C NMR (CDCL): 6 14.1 (CHs), 22.7 (CHZCH~), 25.0 (CHzCHzC=O), 28.3 (CH3 of tBu), 29.7 (polyCHz), 31.9 (CHZCHZCH~), 34.2 and 34.4 (two CHzC=O), 42.4 (HNCHzC=O), 43.4 (HNCHzCHz), 62.8 (CHzOC=O), 63.4 and 63.5 (CH20-P), 64.3 and 64.4 (CHzOCONH),69.5 (CHOC=O), 70.6 (PEG), 81.0 (Cq of tBu), 155.4 (C=O of Boc), 156.6 (urethane C=O), 169.2 (C=O of Gly), 173.0 and 173.4 (C=O of 2 esters) ppm. Deprotection was carried out in 4 M HC1 in dioxane for 60 min. The product was recovered as a white solid after removal of the volatiles and thorough drying in uucuo. Yield: 98 % . Disappearance of the tert-butyl peak at 1.46 ppm of the H-NMR spectra and 28.3,81.1, and 155.4 ppm peaks from the 13C-NMRspectra confirmed the complete removal of the Boc group. The remainder of the NMR spectra of 1 were essentially the same as those of the protected conjugate. ACKNOWLEDGMENT
I wish to thank Dr. Virginia W. Miner of Acorn NMR for her skillful acquisition of NMR spectra, Dr. Aleksander L. Klibanov of the University of Pittsburgh for his helpful suggestionon the purification of PEG-lipids; and Liposome Technology, Inc., for support and for publication of this work. LITERATURE CITED (1) Topchieva, I. N.(1990)Synthesis of biologically active polyethylene glycol derivatives. A review. Polymer Sci. USSR 32,833-851. (2) Ouchi, T., Yuyama, H., and Vogl, 0. (1987)Synthesis of 5Fluorouracil-terminatedmonomethoxypoly(ethy1ene glycol)s, their hydrolysis behavior, and their antitumor activities. J. Macromol. Sci.-Chem., A24, 1011-1032. (3) Zalipsky, S.,Gilon, C., and Zilkha, A. (1983)Attachment of drugs to polyethylene glycols. Eur. Polym. J. 19,1177-1183. (4)Merrill, E. W. (1992)Poly(ethy1ene oxide) and blood contact: A chronicle of one laboratory. Poly(Ethy1ene Glycol)
Technical Notes Chemistry: Biotechnical and Biomedical Applications (J.M. Harris, Eds.) pp 199-220, Plenum Press, New York. (5) Veronese, F. M., Caliceti, P., Schiavon, O., and Sartore, L. (1992) Preparation and properties of monomethoxypoly(ethylene glycol)-modified enzymes for therapeutic applications. Poly(Ethy1ene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Eds.) pp 127-137, Plenum Press, New York. (6) Zalipsky, S., and Lee, C. (1992)Use of functionalized polyethylene glycols for modification of polypeptides. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications (J.M. Harris, Eds.) pp 347-370,Plenum Press, New York. (7) Dreborg, S., and Akerblom, E. B. (1990)Immunotherapy with monomethoxypolyethylene glycol modified allergens. Crit. Rev. Ther. Drug Carrier Syst. 6,315-365. ( 8 ) Papahadjopoulos,D., Allen,T. M., Gabizon,A.,Mayhew, E., Matthay, K., Huang, S. K., Lee, K.-D., Woodle, M. C., Lasic, D. D., Redemann, C., and Martin, F. J. (1991) Sterically stabilized liposomes: Improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U.S.A. 88,11460-11464. (9) Woodle, M. C., and Lasic, D. D. (1992)Sterically stabilized liposomes. Biochim. Biophys. Acta 1113,171-199. (10) Woodle, M. C., Matthay, K. K., Newman, M. S., Hidayat, J. E., Collins, L. R., Redemann, C., Martin, F. J., and Paphadjopoulos, D. (1992)Versatility in lipid compositions showing prolonged circulation with sterically stabilized liposomes. Biochim. Biophys. Acta 1105,193-200. (11) Senior, J., Delgado, C., Fisher, D., Tilcock, C., and Gregoriadis, G. (1991)Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: Studies with poly(ethy1ene glycol)-coated vesicles. Biochim. Biophys. Acta 1062,77-82. (12) Klibanov, A. L., Maruyama, K., Beckerleg,A. M., Torchilin, V. P., and Huang, L. (1991)Activity of amphiphathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target. Biochim. Biophys. Acta 1062,142-148. (13) Zalipsky, S.,Seltzer, R., and Nho, K. (1991)Succinimidyl carbonates of polyethylene glycol: Useful reactive polymers
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for preparation of protein conjugates. Polymeric Drugs and Drug Delivery Systems (R. L. Dunn, and R. M. Ottenbrite, Eds.) pp 91-100, American Chemical Society, Washington, DC. (14) Zalipsky, S.,Seltzer, R., and Menon-Rudolph, S. (1992) Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins. Biotechnol. Appl. Biochem. 15,100-114. (15) Nathan, A., Zalipsky, S., Erthel, S. I., Agathos, S. N., Yarmush, M. L., and Kohn, J. (1993)Copolymers of lysine and polyethylene glycol: A new family of functionalized drug carriers. Bioconjugate Chem. 4,54-62. (16) Nathan, A., Bolikal, D., Vyavahare, N., Zalipsky, S., and Kohn, J. (1992)Hydrogels based on water-soluble poly(ether urethane) derived from L-lysine and poly(ethy1ene glycol). Macromolecules 25,4476-4484. (17)Zalipsky, S.,and Barany, G. (1990)Facile synthesis of a-hydroxy-w-carboxymethylpolyethyleneoxide. J. Bioact. Compatible Polym. 5,227-231. (18) Zalipsky, S.,and Barany, G. (1986)Preparation of polyethylene glycol derivatives with two different groups at the termini. Polym. Prepr. Am. Chem. SOC.Diu. Polym. Chem. 27(1), 1-2. (19) Wilchek, M., and Miron, T. (1985)Activation of sepharose with N,N’-disuccinimidyl carbonate. Appl. Biochem. Biotechnol. 11, 191-193. (20) Zalipsky, S.,Albericio, F., Slomczynska, U., and Barany, G. (1987)A convenient general method for synthesis of N e - or Nu-dithiasuccinoyl (Dts) amino acids and dipeptides: Application of polyethylene glycol as a carrier for functional purification. Int. J. Pept. Protein Res. 30,740-783. (21) Inman, J. K. (1974)Covalent linkage of functional groups, ligands and proteins to polyacrylamide beads. Methods Enzymol. 34,30-58. (22) Wilchek, M., and Bayer, E. A. (1987)Labeling glycoconjugates with hydrazide reagents. Methods Enzymol. 138,429442. (23) Yokoyama, M., Okano,T., Sakurai,Y., Kikuchi,A., Ohsako, N., Nagasaki, Y., and Kataoka, K. (1992)Synthesis of poly(ethylene oxide) with heterobifunctional reactive groups at its terminals by an anionic initiator. Bioconjugate Chem. 3,275276.
