SEPTEMBER/OCTOBER 1999 Volume 10, Number 5 © Copyright 1999 by the American Chemical Society
COMMUNICATIONS New Detachable Poly(ethylene glycol) Conjugates: Cysteine-Cleavable Lipopolymers Regenerating Natural Phospholipid, Diacyl Phosphatidylethanolamine Samuel Zalipsky,* Masoud Qazen,† John A. Walker II,‡ Nasreen Mullah, Yolanda P. Quinn, and Shi Kun Huang Alza Corporation, 1050 Hamilton Court, Menlo Park, California 94025. Received March 16, 1999
A new strategy for the reversible attachment of methoxypoly(ethylene glycol) (mPEG) to an aminocontaining substrate is described. The strategy is based on formation of a benzyl carbamate linkage substituted with a disulfide in the para or ortho position. While being stable under nonreducing conditions, the dithiobenzyl (DTB) urethane linkage is susceptible to cleavage by mild thiolysis with cysteine resulting in release of the parent amino component of the conjugate in its original form. The method is exemplified by preparation of mPEG-DTB-alcohol, its activation and attachment to distearoylphosphatidylethanolamine (DSPE). The resulting lipopolymer incorporates into liposomes, which are capable of losing their polymer coating under conditions approximating those existing in vivo. Implications for drug delivery are briefly discussed.
Lipopolymers, polymer-lipid conjugates, have proven to be very useful in micellar and liposomal drug delivery systems (1). In particular, the use of poly(ethylene glycol)-lipid [usually distearoylphosphatidylethanolamine (DSPE)]1 conjugates (2) has become routine to convey * To whom correspondence should be addresses. Phone: (650) 564-4651. Fax: (650) 617-3080. E-mail:
[email protected]. † Current address: CellGate, Inc., 552 Del Rey Ave., Sunnyvale, CA 94086. ‡ Current address: Protogene Laboratories, 1454 Page Mill Road, Palo Alto, CA 94304. 1 Abbreviations: PEG, poly(ethylene glycol); mPEG, methoxyPEG; DTB, dithiobenzyl; DSPE, distearoylphosphatidylethanolamine; DOPE, dioleoylphosphatidylethanolamine; PHPC, partially hydrogenated phosphatidylcholine; MALDI-TOFMS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry.
favorable pharmacokinetic and in vivo distribution characteristics to various injectable particles. The presence of grafted PEG chains on the surface of particulates results in reduced nonspecific interactions with various biological cells and macromolecules. This is caused by the exclusion effect of the flexible, very mobile, well water solvated PEG chains. However, for a variety of applications interaction with target cells is desirable. These might include, for example, intracellular delivery of a liposomal drug payload, delivery of oligonucleotides, or delivery of genes. In these instances, it might be desirable to be able to facilitate a process of removal of the surfacegrafted polymer chains. We recently demonstrated some of the useful properties of cleavable PEG-lipids in their liposomal formulations (3). The first generation of detachable PEG-bearing liposomes contained aliphatic di-
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Figure 1. p-Dithiobenzyl urethane-linked mPEG-DSPE conjugate and its thiolytic cleavage. The o-DTB lipopolymer undergoes similar decomposition reaction. mPEG ) CH3O(CH2CH2O)45-.
sulfide-linked conjugate with 3,3′-dithiodipropionate as the linking moiety. Consequently, cleavage of the mPEG grafts from these liposomes required a relatively potent thiolytic agent, 1,4-dithiothreitol, which is unacceptable for the in vivo use. Furthermore, a modified phospholipid, 3-mercaptopropionyl-DSPE product, was generated as a result of the disulfide cleavage (3). In this communication, we report the preparation and some of the properties of a new mPEG-DSPE conjugate, containing o- or p-ditiobenzyl carbamate (DTB-urethane) linkage moiety (Figure 1). In contrast to the earlier version of the disulfide-linked conjugate, these new lipopolymers are cleavable with cysteine, and as a result of this reaction, phosphatidylethanolamine lipid is regenerated in its natural, unmodified form. In essence in this application, the mPEG moiety is acting as a macromolecular protecting group, which is removable under mild thiolytic conditions achievable in vivo. To the best of our knowledge this is the first demonstration of this type of reversible PEGylation strategy applied to any substrate. Thiolytic decomposition of benzyl urethane-linked drugs containing aromatic disulfides in the para or ortho positions has been utilized by Senter et al. as a basis for an elegant prodrug strategy (4). We decided to adapt the dithiobenzyl urethane as a linkage between the lipid, DSPE, and mPEG components of the cleavable lipopolymer. The main attraction of this linking strategy, as shown in Figure 1, lies in its ability to regenerate the amino component of the carbamate upon reaction with thiols. Moreover, cleavage of mixed aliphatic-aromatic disulfides is usually easier than of their symmetrical aliphatic counterparts. We anticipated that this would allow the use of a mild, nontoxic thiol such as cysteine as the cleaving reagent. The syntheses of the polymer-lipid conjugates are schematically depicted in Figure 2. First, we prepared a novel mPEG-2000 derivative with a methoxycarbonyldithioalkyl end group. This was accomplished by reacting 2-(methoxycarbonyldithio)ethaneamine with mPEG-chloroformate. The former compound was obtained through the reaction of 2-aminoethanethiol hydrochloride with an equivalent amount of methoxycarbonylsulfenyl chloride2 according to the previously published procedures (6, 7). The polymeric chloroformate was easily prepared by phosgenation of an anhydrous mPEG-OH solution (8). The methoxycarbonyldithio functionality, positioned in this instance at the end of mPEG, is known to be useful
Figure 2. Schematic depiction of the synthesis of mPEG-DTBDSPE.
for the formation of mixed disulfides (6, 9, 10). Furthermore, it is sufficiently active to react with even weakly nucleophilic aromatic thiols (7). Both para and ortho isomers of mercaptobenzyl alcohol (11) coupled cleanly with the polymeric acyl disulfide, yielding the mPEGbearing dithiobenzyl alcohol end group. Active carbonate introduction proceeded as with the underivatized mPEG2 To avoid complication due to various impurities potentially present in commercially available, aged methoxycarbonylsulfenyl chloride in some experiments, we used instead freshly prepared ethoxycarbonylsulfenyl chloride (5) with no bearing on the outcome of the syntheses.
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Figure 3. Cysteine-mediated cleavage of mPEG-DTB-DSPE conjugates (33 µM) in either micellar or liposomal (PHPC/cholesterol/ lipopolymer at 95:5:3 mole ratio, ∼120 nm diameter, closed symbols) solutions in PBS, pH 7.2, containing 5 mM EDTA at 37 °C with or without cysteine (150 µM). Disappearance of the conjugates was monitored by HPLC (Phenomenex C8 Prodigy, 4.6 × 50 mm column, detection at 277 nm, mobile phase methanol/water 95:5 with 0.1% trifluoroacetic acid at 1 mL/min). Incubation in buffer only of ortho conjugate (*) and para conjugate (+). Incubations in cysteine-PBS of para conjugate (0), ortho conjugate (O); or liposomes containing para conjugate (9), ortho conjugate (b).
OH. We prepared both succinimidyl and p-nitrophenyl carbonates. However, most of the conjugation reactions with DSPE were performed with the latter derivatives, due to the ease of their preparation, shelf life stability, and avoidance of the potential side reactions associated with the succinimidyl carbonate chemistry (12). Both oand p-DTB urethane linked lipopolymers were purified by silica gel chromatography (methanol gradient 0 to 10% in chloroform, ∼70% isolated yield) and characterized by NMR3 and MALDI-TOFMS. The latter technique confirmed the average molecular masses of the conjugates, 3127 and 3139 Da for para and ortho isomers, respectively (theoretical molecular mass ≈ 3100 Da). As was previously observed for various PEG conjugates (13, 14), the spectra consisted of bell-shaped distributions of signals equally spaced at 44 Da, corresponding to the ethylene oxide repeating units. Consistent with the known tendency of MALDI-MS to, at least partially, cleave disulfides (15), separate distributions corresponding to mPEG-thiol, average molecular mass 2240 Da, were also observed. The stabilities of both DTB-linked lipopolymers in buffered aqueous solution (pH 7.2) in the presence and absence of cysteine was monitored by HPLC. The results are illustrated in Figure 3. In these experiments, the o-DTB conjugate exhibited a slightly faster rate of decomposition than its para counterpart (T1/2 ≈ 12 min vs ∼18 min, at [Cys] ) 150 µM). Both ortho and para conjugates were slightly more resistant to the thiolytic cleavage when incorporated into liposomes rather than in micellar solutions. In the absence of thiols, both conjugates appeared to be stable. Examination of com3 1H NMR for para conjugate: (d -DMSO, 360 MHz) δ 0.86 6 (t, CH3, 6 H), 1.22 (s, CH2 of lipid, 56H), 1.57 (m, CH2CH2CO2, 4H), 2.50 (2xt, CH2CO2, 4H), 2.82 (t, CH2S, 2H), 3.32 (s, OCH3, 3H), 3.51 (m, PEG, ∼180 H), 4.07 (t, PEG-CH2OCONH, 2H), 4.11 and 4.28 (2 x dd CH2CH of glycero, 2H), 4.98 (s, benzylCH2, 2H), 5.09 (m, CHCH2 of lipid), 7.35 and 7.53 (2 x d, aromatic, 4H) ppm. The ortho conjugate differed only in benzyl and aromatic signals at 5.11 (s, CH2, 2H), and 7.31 (d, 1H), 7.39 (m, 2H) 7.75 (d, 1H) ppm.
pleted thiolysis reactions of both para and ortho conjugates by TLC (silica gel G, chloroform/methanol/water 90:18:2) revealed the presence of DSPE as the sole lipid component (16) and another spot corresponding to a thiolbearing, lipid-free mPEG derivative. Due to the very large polar headgroup of PEG-lipid conjugates, they allow liposomal bilayer accommodation of lipids that normally do not form vesicles, e.g., DOPE, a hexagonal phase forming lipid. If a cleavable lipopolymer is used to facilitate the formation of this type of liposome, then chemical detachment of the grafted polymer chains would trigger decomposition of the vesicles and conversion of the lipids into the hexagonal phase. This, as was previously demonstrated (3), can be used for instant release of the liposomal contents and liposomal fusion. The content release characteristic of such liposomes, or the rate of the dequenching of the liberated fluorophores, provides for a convenient quantitative evaluation of the cleavable PEG-bearing liposomes. As illustrated in Figure 4, both dithiobenzyl-linked conjugates described here performed well in this assay, resulting in a cysteine-triggered release of encapsulated dyes. From these experiments, it is also apparent that the ortho conjugate was more susceptible to the thiolytic cleavage. For example, 300 µM cysteine liberates within 20 min most of the contents of DOPE liposomes stabilized by the o-DTB lipopolymer. Under the same conditions, only a very small fraction of the liposomes is decomposed when p-DTB-linked conjugate is used. After incubation for 20 min at 150 µM cysteine, half of the content release occurs by 20 min for o-DTB formulation, while only approximately 10% of the content release occurs in p-DTB liposomes even at the doubled cysteine concentration. Since we observed in Figure 3 that both ortho and para conjugates have half-lifetimes of less than 20 min, it is apparent that more than half of the original 3 mol % of PEG-lipids have to be cleaved in order to see a burst in the content release. Small, yet clearly not negligible, decomposition of the mPEG-DTB-DSPE/DOPE liposomes took place at 15 µM cysteine, which is the average plasma concentration in both humans and rodents (17), in the
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DSPE conjugates are of practical value. It is clear that DTB-linked PEG-lipids are cleavable with cysteine under mild conditions attainable in vivo. We are currently studying liposomes containing these cleavable lipopolymers in various biological systems. The results of these experiments will be reported in a due course. We anticipate that the new mPEG-DTB reagents and the described conjugation strategy will be of general applicability to a variety of amino-containing substrates including, peptides, proteins, aminoglycosides, and suitably functionalized biomaterials. LITERATURE CITED
Figure 4. Fluorescent dequenching accompanying release of entrapped fluorophores (p-xylene-bis-pyridinium bromide, trisodium 8-hydroxypyrenetrisulfonate, 30 mM each) from liposomes (DOPE/mPEG-DTB-DSPE at 100: 3 molar ratio, ∼100 nm diameter) incubated in HEPES buffer, pH 7.2, at 37 °C in the presence of cysteine (concentrations indicated for each curve). [Cys] ) 15 µM ([), 150 µM (1), 300 µM (2), 1 mM (9), 1.5 mM (b). The assays and calculations were performed according to the previously published procedures (3). Control experiments in the absence of reducing agents or with noncleavable urethane-linked mPEG-DSPE containing liposomes produced no content release.
time frame of these experiments (60 min). This suggests that DTB-linked lipopolymers should have sufficiently long lifetimes in plasma to allow the PEG-grafted vesicles to distribute systemically in vivo or to accumulate in a specific site either passively or through ligand-mediated targeting. Furthermore, these results suggest that a prolonged exposure to the natural plasma cysteine concentration (∼15 µM) might be sufficient to decompose most of these lipopolymers. When faster cleavage is required, local or short-term increases in cysteine concentration can potentially be achieved by its intravenous or intraarterial administration. The only example of the potential usefulness of in vivo reversible PEGylation was described by Garman and Kalindjian (18), who prepared acid-labile PEG-plasminogen activator by reacting mPEG-maleic anhydride reagent with amino groups of the protein. In this example, however, the conjugate was susceptible to cleavage not only under acidic conditions but even close to neutral pH and had to be stored frozen to minimize premature loss of the PEG attachments. Herein, we presented a new type of reversible PEGylation chemistry exemplified by preparation of mPEG-o/p-DTB-DSTE conjugates, which are rather stable in neutral pH in the absence of reducing agents. The linkages employed, p- or o-disulfide of a benzyl urethane, when subjected to mild thiolytic conditions can regenerate the original amine component, in this case DSPE, in its unmodified form. This is a very desirable outcome, since unnatural, modified lipids generated as a result of alternative cleavage strategies (see, for example, ref 3) are more likely to have unpredictable, potentially negative in vivo effects. Since all the reactions shown in Figure 2 are clean high yielding transformations, the syntheses of mPEG-DTB reagents and their
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