Bioconjugate
Chemistry NOWMBEWDECEMBER 1994 Volume 5, Number 6 0 Copyright 1994 by the American Chemical Society
LETTERS New Amphipatic Polymer-Lipid Conjugates Forming Long-Circulating Reticuloendothelial System-Evading Liposomes Martin C. Woodle, Charles M. Engbers, and Samuel Zalipsky* Liposome Technology, Inc., 960 Hamilton Court, Menlo Park, California 94025. Received August 8, 1994@
Lipid-conjugates of two amphipatic polymers, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl2-oxazoline) (PEOZ) (degree of polymerization = 50) were synthesized by linking glutarate esters of the polymers to distearoylphosphatidylethanolamine (DSPE) or alternatively by termination of the polymerization process with DSPE. Surface-modified liposomes (90 f 5 nm) prepared from either conjugate (5 mol % of total lipid) were injected into rats and followed by blood level and tissue distribution measurements. Both polymers PEOZ and PMOZ were found to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as polyethylene glycol (PEG), the best known material for this purpose. This is the first demonstration of protection from rapid recognition and clearance conveyed by alternative polymers, which is equal to the effect of PEG.
Suppression or evasion of biological recognition mechanisms is of primary importance for successful use of implant devices and macromolecular or particulate drug delivery systems. Specifically, in the case of liposomal drug delivery, until recently, most applications were hampered by very short blood lifetimes of conventional lipid vesicles, their preferential accumulation in organs of the reticuloendothelial system (RES'), mainly liver and spleen, and dose-dependent pharmacokinetics. In the last few years several liposomal surface modifiers were
* To whom correspondence should be addressed. Tel. (415) 323-9011;FLU(415)617-3080. Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: RES, reticuloendothelial system; PEG, poly(ethylene glycol); POZ, poly(oxazo1ine); PEOZ, poly(ethy1oxazoline); PMOZ, poly(methyloxazo1ine); DSPE, distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; EPG, egg phosphatidylglycerol; TsO, p-toluenesulfonate; DP, degree of polymerization; DCC, dicyclohexylcarbodiimide; HOSu, N-hydroxysuccinimide. @
introduced, which addressed these problems to some extent. Among the various lipid derivatives used to prepare surface modified vesicles were dicarboxylic acidlipid adducts, synthetic polymer conjugates, and natural (e.g., GM1) and synthetic glycolipids (for recent reviews see refs 1 and 2). Best RES-avoiding vesicles were obtained when poly(ethy1ene glycol) (PEG)-lipids (3) were incorporated into liposomes (4-12). Such liposomes exhibit dose-independent pharmacokinetics accompanied with remarkable persistence in vivo (tllz 2 48 h in humans). While several manuscripts attempted to explain this phenomenon (9-12), heretofore no other liposomal surface modifiers were identified that are a s efficient as PEG in producing these beneficial properties. We report here for the first time dramatic RES-evasion and prolonged circulation effects brought about by the incorporation of lipid conjugates of two amphiphatic polyoxazolines into liposomes. Furthermore, the effect produced by both polymers described in this paper, poly-
1043-1802/94/2905-0493$04.50/00 1994 American Chemical Society
494 Bioconjugate Chem., Vol. 5, No. 6, 1994
Woodle et al.
Scheme 1. Preparation of Poly(oxazo1ine)Conjugates of DSPE"
TsO-CHI
R q N > '0,
0
n-1 moles
H20
I
*
Solubilities of the polymers (DP x 45-50) were tested qualitatively. Key: +, soluble; -, insoluble; f,soluble only upon warming, yet remain in solution a t 25 "C. Taken from ref 30. DSPE I TEA
I
1. DCC I HOSu 2. DSPE I TEA
Key: R = CH3 for PMOZ; R = CzHs for PEOZ; DP = n 50.
Table 1. Comparative Solubility of PEG, PEOZ, and PMOZ" solvent polarity index* PEG PEOZ PMOZ petroleum ether 0.1 2.7 benzene ethyl ether 2.8 3.9 n-butanova-propanol 4.0 tetrahydrofuran 4.1 chloroform 4.4 ethyl acetate 5.8 acetonitrile 6.4 dimethylformamide 10.2 water
*
(ethyloxazoline) (PEOZ) and poly(methyloxazo1ine) (PMOZ), are quantitatively comparable to the effect of PEG. The synthesis of POZ-lipid conjugates is shown in Scheme 1. The polymers were prepared by methyl p-toluenesulfonate-initiated reactions of 2-methyl- and 2-ethyl-2-oxazolines following the previously published procedures (13-1 7). Since oxazoline polymerization is known to proceed via a cationic ring-opening mechanism yielding living polymers (for a review see ref 131,we used initiator -monomer ratios of 1 5 0 , aiming for the same degree of polymerization as PEG of molecular weight 2000 (DP x 46). The latter polymer was previously identified a s optimal for preparation of long-circulating liposomes (5). Hydroxyl-terminated POZs of low dispersity (MwlMn x 1.1-1.4) were obtained after aqueous workup, followed by dialysis and lyophilization. The molecular weights of the polymers used in conjugation with lipid were 4000 for PMOZ and 5000 for PEOZ, as determined by GPC using PEG molecular weight standards (18). Pyridine-catalyzed reaction of PEOZ with glutaric anhydride in refluxing benzene was used to introduce the carboxylic acid end-group onto the polymer. Since PMOZ is insoluble under these conditions (Table I), acetonitrile was used as a solvent for preparation of PMOZ-glutarate under otherwise same conditions. The glutarate derivatives of POZs2 were converted into reactive succinimidyl esters and then coupled with distearoylphosphatidylethanolamine(DSPE) in chloroform in the presence of triethylamine (TEA). To assure complete conversion of DSPE into the conjugates the Glutarates of POZs obtained by terminating 2-oxazoline polymerization with glutaric acid were described by Miyamoto et al. (29).
reactive polymers were used in slight excess to DSPE3. Taking advantage of very low critical micelle concentration of distearoyl lipids, the conjugates were purified by removing excess of free polymer and other reactants by dialysis through 300 000 MWCO membrane (7, 19). An alternative approach to POZ-DSPE conjugates involved direct termination of living oxazolinium-terminated polymers obtained in chloroform (14-16) with DSPE in the presence of TEA. This unoptimized procedure, while considerably simpler, was accompanied by very low recoveries of conjugates (yields 5 5%). However, the purified conjugates (characterized by NMR and TLCI3 obtained by both methods produced equivalent results in animal experiments with liposomal formulations described below. Liposomes containing POZ-DSPE conjugates (or for comparison PEG-DSPE), egg phosphatidylcholine (EPC), and cholesterol were prepared as described previously (5, 6),labeled with 67Ga,and injected intravenously into rats (20). Each liposomal preparation contained 5 mol % of the corresponding polymer-lipid conjugate. Figure 1 illustrates the persistence of the 67Ga-labeledliposomes in the bloodstream. The behavior of a conventional liposomal preparation containing (for the total charge equivalence) egg phosphotidylglycerol (EPG) instead of polymer-lipid conjugate is also shown. The performance of both POZ-grafted liposomes (tl,z 2 15 h) was similar to PEG-DSPE-containing vesicles. A striking similarity was also observed in biodistribution data (Figure 2). Low uptake by the liver and spleen was observed for the polymer-grafted liposomes with the largest portion of the dose remaining in the bloodstream after 24 h, in sharp contrast to the control preparation of conventional liposomes. Of the three polymer grafted vesicles compared here, PMOZ-containing liposomes produced slightly better results (Figures 1 and 2). However, the relative superiority of one polymer over another could be formulation dependent; e.g., each of the polymers might have a different optimal molecular weight. Therefore, a definite statement on this point has to be delayed until our ongoing studies fully examine the interrelationship of all the relevant formulation variables. The ability of PEG to elicit its beneficial effect when grafted onto the surface of liposomes has been explained by its chains' high mobility associated with conformaThe conjugation reactions were followed by TLC on silica gel G (for PMOZ derivatives: CHC13/EtOW30% aqueous ammonia 3:7:1; for PEOZ CHCl&IeOWwater 80:18:2; and 130: 70:8 for derivatives of both POZs). Being only slightly soluble in chloroform, DSPE completely dissolved in the process of the polymer coupling reaction. Characteristic signals of both polymer and lipid components were clearly identifiable on H-NMR (CDC13) spectra of the purified POZ-DSPE conjugates.
Bioconjugate Chem., Vol. 5, No. 6,1994 495
Letters 100
10
1
l ! 0.0
. , 4.0
. , 8.0
.
.
, 12.0
, 16.0
.
, 20.0
.
,I 24.0
Time after injection (h)
Figure 1. Blood lifetimes of 67Ga-labeledliposomes (90 f 5 nm) prepared (5, 20) from the EPC, cholesterol, and DSPE conjugate of either PEG, PMOZ, or PEOZ, or as a control EPG, in a molar ratio of 1.85:1:0.15. Four Sprague-Dawrey rats were injected with each liposomal preparation via the tail vein. Samples obtained by retro-orbital bleeding at various time points were used to determine radioactivity in the blood.
chemical versatility (13) have been considerably less studied than PEG, and only a few biomedical applications of these polymers were heretofore contemplated (26-29). However, the above-mentioned properties thought to be responsible for the “PEG-effect” could be reasonably attributable to the POZs as well. For example, similar to PEG, they have carbon-carbon-heteroatom repeating units, with tendency to be engaged in hydrogen bonds, which explains similarity in solubility properties of PEG and the two PEOZs (see Table 1). The PEOZ and PMOZ conjugates described in this paper are the first polymers to be able to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as PEG. Since previous efforts to identify liposomal surface modifiers other than PEG to exert measurable protection from biological recognition had only very limited success (1,2, 4), our findings not only have important practical implications, but they should also help to advance the understanding of the properties that these amphiphatic polymers exert on the surfaces modified by them. ACKNOWLEDGMENT
The authors are grateful to Ms. J a n Oaks and Ms. Bhagya Puntambekar for their enthusiastic and skillful technical assistance and Liposome Technology, Inc. , for support and release for publication of this work.
100
PEG PMOZ 0 PEOZ ?A EPG
LITERATURE CITED
80
60
40
Blood
Liver
Spleen
L+S/B
Figure 2. Selected tissue distribution at 24 h. The atlLnals treated as described in legend for Figure 1were sacrificed after 24 h and tissues removed for determination of radioactive content. Ratio of summed liver and spleen to blood levels (L S/B) is a commonly used measure for RES uptake (5, 6 , 8).
+
tional flexibility and water-binding ability-all of which contribute to the so-called steric stabilization effect, which results in the well-known propensity of PEG to exclude proteins, other macromolecules, and particulates from its surroundings (9-12). Taking advantage of these properties, PEG grafting has been widely used in other systems as a method for reduction of various undesirable consequences of biological recognition manifested by immunogenicity and antigenicity in the case of proteins (21-23), and thrombogenicity, cell adherence, and protein adsorption in the case of artificial biomaterials (24, 25).
Polyoxazolines, despite their low toxicity and superb
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