Self-porating polymersomes of PEG–PLA and PEG–PCL: hydrolysis-triggered controlled release vesicles
Introduction
Lipid vesicles or liposomes have been widely investigated as encapsulators of hydrophilic drugs and proteins for several decades. Many if not all conventional liposome systems have proven to be both inherently leaky [1] and short-lived in the circulation [2]. Systems based on chemically active monomers, such as phospholipase sensitive [3], [4] or ph/light destabilized [5], [6], [7], [8] lipids, and polyethyleneglycol (PEG)–lipids [9], [10], [11], [12], [13] have been introduced as a means to control drug release. Chemically reactive polyethylene glycol PEG–lipids can play dual roles as liposome stabilizers that also, upon exposure to an environmental stimulus, effectively destabilize the carrier membrane via thiolytic [9], [10] or hydrolytic [11], [12], [13] cleavage of their PEG–lipid bonds. As stabilizers, a small percentage (5–10%) of PEG–lipid was found, some time ago, to also delay liposome clearance [14]. In other words, PEG imparts stealthiness. Both ideas—controlled release and stealth—are extended here into purely synthetic polymer vesicle systems, which clearly offer broad control over vesicle properties.
The ‘polymersomes’ here are composed of block copolymers of both PEG and a hydrolytically susceptible polyester of either polylactic acid (PLA) or polycaprolactone (PCL). Both PLA [15], [16], [17], [18] and PCL [19], [20] have been widely studied as readily hydrolysable polyesters. PEG–PLA or PEG–PCL block copolymers have both been described before [21], [22], [23], [24], [25], [26], and very recent illustrations of PEG–PLA vesicles [27], [28], [29] highlight the need for detailed characterization of release and degradability. Vesicle formulations of PEG–PLA or PEG–PCL with or without inert PEG–PBD (polybutadiene)—a well-documented vesicle former in water [30]—are shown here to provide programmed control over release kinetics. The dense 100% PEG corona of the PEG–PBD vesicles has recently been shown to deter membrane opsonization, and extend in vivo circulation times significantly beyond stealth liposomes [31]. While broader compatibility of PBD has been explored by others [32], [33], the in vitro focus here is on the general principle of blending degradable and inert copolymers.
The elusiveness of making PEG–PLA vesicles is largely attributable to limited copolymer designs in relation to narrow requirements for a suitable lamellar phase. Extensive theoretical [34], [35] as well as general experimental studies of block copolymer amphiphiles have established that aggregate morphology, in dilution, is principally determined by molecular geometry. Kinetic traps are many (e.g. entanglements, crystallization, or glassiness at high molecular weight, MW), but when solvated selectively, a delicate but now relatively well-understood balance of hydrophilic/hydrophobic segments emerges (Fig. 1A) [27], [36]. This balance allows design of PEG-block based copolymers that—in the absence of degradation—form membranes in preference to other structures. Whereas diblock copolymers with small hydrophilic PEG fractions of fEO<20% and large MW hydrophobic blocks exhibit a strong propensity for sequestering their immobile hydrophobic blocks into solid-like particles (for PEG–PLA [21], [26], [37]), an increased fEO ∼20–42% generally shifts the assembly towards more fluid-like vesicles [27], [28], [30], [38], [39], [40], [41], [42], [43] or other “loose” micellar architectures [44], [45], [46]. For fEO>42%, however, one generally finds both worm micelles (up to ∼50% fEO) [36], [47], [48] and, as noted by others, spherical micelles (for PEG–PLA [45], [46], [49], and PEG–PCL [50]). Lastly, although kinetic traps to equilibrium may deepen with MW, the equilibrium boundaries enumerated above between predominant microphases are only weakly dependent on MW. Recent work indeed shows that the aforementioned fEO's shift to lower values for diblocks only by about 5–6% per addition of 100 EO monomers [36].
The vesicle/micelle transitions outlined above would seem to provide a clear starting point for the design of novel copolymer carriers. While similar mechanisms have been exploited in otherwise conventional liposomal systems [8], [51], [52], [53]. The kinetic aspects of phase transitions are not easily predicted but are of paramount importance when using ‘active’ chains such as the hydrolytically degradable PEG–PLA for release mechanisms. Considerable data in the literature indicate that degradation of PLA nanoparticles occurs on the order of weeks [15], [44]. For the vesicles here, tunable, controlled release that ranges from hours to many days is demonstrated through copolymer blending within the membrane as well as polyester selection and chain architecture (i.e. fEO).
Section snippets
Copolymers and chemicals
The diblocks listed in Table 1, except for OB18 and OL1, were purchased from Polymer Source (Dorval, Quebec, Canada). Note that EO denotes ethylene oxide, and that polyethylene oxide is structurally the same as PEG. Tetramethylrhodamine-5-carbonylazide (TMRCA) was obtained from Molecular Probes (Eugene, OR). Dialysis tubing and dram vials were purchased from Spectrum Laboratories (Rancho Dominguez, CA) and Fisher Scientific (Suwanee, GA), respectively. l-Lactide, mono-methoxy polyethylene
PEG–PLA vesicles and blends
Both PLA and PCL are generally considered hydrophobic provided they are of sufficiently high molecular weight [27]. The spontaneous aggregation and assembly of OL1 copolymer (Table 1: EO43–LA44) into lamellar or bilayer morphology—i.e. a vesicle—in dilute solution is verified by direct cryo-TEM imaging (Fig. 1B). The hydrophobic core of the membrane provides the contrast and has a measured width d≈10.4±1.4 nm.
The miscibility of OL1 block copolymer in a vesicle membrane with OB18 (Table 1: EO80
Copolymer integration into membranes
When hydrated initially, the PEG–polyester copolymers and blends self-assemble into stable bilayer architectures (e.g. Fig. 1B). The core thickness of the PLA membrane is similar to a previously studied PEG–PBD vesicle [39], namely EO50-BD55 (with d≈10.6±1 nm). This OL1 result fits the general scaling found for PBD cores of d∼N0.5. While this may seem surprising because of PLA's high oxygen content, it is to be noted that such high oxygen contents in hydrophobic blocks are of no limitation to
Conclusions
The kinetics of hydrolytically triggered destabilization of polymersomes composed or blended with degradable PEG–PLA or PEG–PCL and the inert PEG–PBD (OB18) have been elucidated by sucrose and fluorophore leakage assays for giant vesicles as well as DLS of nanovesicles. Labeling of the PLA block demonstrates the participation of the polyester chain in stable membrane integration. Subsequent polyester hydrolysis in the core of the membrane transforms these bilayer-forming chains into active,
Acknowledgements
We thank Frank Bates (University of Minnesota), Shastri Prasad (University of Pennsylvania), and Aswin N. Venkat (University of Wisconsin-Madison) for a careful reading of this manuscript as well as the Bates group's for PEG–PLA copolymer and cryo-TEM images. Dr. George Furst (Chemistry, University of Pennsylvania) is acknowledged for the NMR, and Dr. Puay Phuan (Chemistry, University of Pennsylvania) for the use of the rotavap instrument. Funding was provided by an NIH R21, Penn's NSF-MRSEC
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