Article pubs.acs.org/Langmuir
Labile Incorporation of Cholesterol-Terminated Poly(acrylic acid) for the Facile Surface-Modification of Lipid Vesicles Sang-Min Lee* Department of Chemistry, The Catholic University of Korea, Bucheon, Gyeonggi-do 14662, Korea ABSTRACT: An amphiphilic cholesterol-terminated poly[acrylic acid] (CholPAA) that can be self-aggregated into nanoscale micelles in aqueous media has been prepared via nitroxide-mediated radical polymerization for the facile postformation modification of lipid vesicles. By varying the amount of CholPAA addition, the incorporation of Chol-PAA on the liposome templates was verified with zeta potential while the dynamic light scattering measurements revealed the polymer length of Chol-PAA dictated the hydrodynamic diameter of the resulting polymer-grafted vesicles (PGVs). The membrane incorporation process of Chol-PAA was monitored through the fluorescence resonance energy transfer (FRET) study, which showed the relatively labile incorporation property of Chol-PAA, compared to the cholesterol-free PAA and the native cholesterol. Additionally, the postmodification of liposomes with such labile Chol-PAA exhibited a negligible leakage of calcein payloads, which can be attributed to the partial modification of the external membrane. These results indicated that our Chol-PAA can be exploited for the facile construction of functional polymer-decorated liposomal delivery systems.
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INTRODUCTION Over the past decades, liposomes have attracted considerable attention for versatile delivery system by encapsulating toxic agents for reduced side effects and enhancing the aqueous solubility of hydrophobic drugs for improved pharmacokinetic profiles.1 However, given that the lipid vesicles consist of highly dynamic lipid molecules assembled by weak van der Waals interaction, the conventional liposomes often suffer from the low stability against membrane fusion and rupture, eventually leading to the recurrent drug leakage during the plasma circulation.2,3 Hence, to prevent the in vivo destruction of liposome, which can be triggered by opsonization process,4 steric protection by hydrophilic polymers such as polyethylene glycol (PEG) has been employed to increase the circulation half-life.5 As such, a facile surface-modification strategy has been required for the liposomal systems to enhance the colloidal stability and chemical functionality for targeting ligands and stimuli-sensitive triggers for drug release. In liposome-based delivery system, the membrane-modification strategy has been successfully accomplished by the postparticle-formation inclusion of hydrophilic macromolecules covalently linked to a long-chain phospholipid such as distearoylphosphatidyl-ethanolamine (DSPE) as a hydrophobic anchor.6,7 However, when such phospholipid-based anchors were attached to a large biological ligand and incorporated to the membrane of liposomes for targeted delivery the resulting lipid-attached ligands often suffer from the inefficient grafting yields due to the increased aqueous solubility. Although the grafting efficiency of the lipid-attached macromolecules can be improved by coincubation with liposome templates at elevated temperature, where the membrane exhibits enhanced fluidity, © XXXX American Chemical Society
undesirable drug leakage has been often observed due to the increased permeability of lipid membrane.7−9 Additionally, the narrow range of chemical condition for the compatible inclusion of “detergent-like” amphiphilic macromolecules often leads to the rupture of membrane via vesicle-to-micelle transition.9,10 Previously, we demonstrated a facile construction of hybrid vesicles, containing liposomal interior surrounded by a crosslinked polymer shell, which exhibited both enhanced physical stability and chemical functionality over the conventional liposomes.11 In the fabrication of such surface-modified liposomal nanocarriers that we named polymer-caged nanobins (PCNs), cholesterol has been employed as an alternative hydrophobic anchor to immobilize the hydrophilic polymers such as poly[acrylic acid] (PAA) on the liposome membrane. To this end, cholesterol-terminated poly[acrylic acid] (CholPAA) was synthesized through the cholesterol-attached alkoxyamine initiator for nitroxide-mediated radical polymerization and then subjected to a postformation modification process of liposomes, which can provide a chemical handle for the attachment of targeting ligands and drug-releasing devices by in situ cross-linking on the surface of liposome.12 As an intrinsic component of mammalian cell membrane, cholesterol has been known to regulate the physicochemical property of lipid bilayer.13,14 The inclusion of cholesterol can increase both the lipid fluidity and the packing density of bilayer by spanning the interstitial space between the lipid Received: February 28, 2017 Revised: April 27, 2017
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DOI: 10.1021/acs.langmuir.7b00670 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. (A) Schematic illustration of Chol-PAA micelle formation in aqueous media by direct hydration method. (B) The 1H NMR spectra of cholesterol-terminated poly[tert-butyl acrylate] (Chol-PtBA) in CDCl3 (top) and the cholesterol-terminated poly[acrylic acid] (Chol-PAA) in D2O (bottom). The characteristic proton resonance peaks from cholesterol moiety (0.7−1.2 ppm in dotted box at the top spectrum) were extensively suppressed in bottom spectrum, indicating the hydrophobic aggregation of cholesterol end groups in aqueous phase. *The proton peak at 1.5 ppm in the bottom spectrum is due to the residual tert-butyl group after acidolysis. (C) Hydrodynamic diameter of Chol-PAA micelles measured by DLS in aqueous solutions (pH 7). (D,E) TEM images of Chol-PAA 2.5 kDa (D) and 5.6 kDa (E) dispersed in aqueous solutions. Samples were negatively stained with 4% aqueous uranyl acetate for TEM analyses.
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RESULTS AND DISCUSSION Micellar Structure of Self-Associated Chol-PAA Amphiphiles. We have previously employed a cholesterolattached alkoxyamine radical initiator for the synthesis of a cholesterol-terminated poly[tert-butyl acrylate] (Chol-PtBA)11 via nitroxide-mediated radical polymerization. A bulk polymerization was carried out with excess amount of tert-butyl acrylate monomers as reported previously.19 After 6 and 9 h of polymerization, two representative sets of narrowly dispersed polymers, Chol-PtBA 3.8 kDa (DP ≈ 30, PDI = 1.09) and Chol-PtBA 9.0 kDa (DP ≈ 70, PDI = 1.11), have been obtained, respectively. Subsequent deprotection of tertbutyloxycarbonyl groups by simple acidolysis with trifluoroacetic acid (TFA) provided Chol-PAA 2.5 kDa and Chol-PAA 5.6 kDa, each of which was then subjected to micelle formation via direct hydration method as previously reported with cholesterol-end-capped polymers (Figure 1A).20,21 During the dissolution process of Chol-PAA, the solventdependent structural change was monitored by 1H NMR spectroscopy (Figure 1B). For the Chol-PtBA precursor in organic solvent such as CDCl3, distinct proton resonance signals were clearly observed from both the terminal cholesterol end groups (dotted box) and the polymer backbone (peaks a and b in 1.6−2.4 ppm). In stark contrast, the proton resonance peaks from the cholesterols were significantly suppressed for Chol-PAA dispersed in D2O, indicating that the hydrophobic cholesterol moieties were segregated from the aqueous phase20 while the hydrophilic PAA segments were still hydrated in D2O. Hence, we surmised that Chol-PAA chains can aggregate into a micelle-like structure, containing the hydrophobic cholesterol end groups inside the micellar core stabilized by the surrounding hydrophilic PAA chains in aqueous solution.
molecules, eventually reducing the membrane permeability against the transmembrane diffusion of small molecules. In this sense, when the cholesterol is employed as a membrane anchor for postformation modification, cholesterol can be well suited to occupy the void space between the lipid molecules in the outer leaflet, which can minimize the lateral expansion of membrane during the incorporation process. Additionally, upon the insertion of Chol-PAA, the rapid flip-flop rate of native cholesterol in the liposomal membrane allows for the quick equilibration of transbilayer cholesterol distribution,13 keeping the integrity of bilayer membrane. As such, cholesterol can provide several unique functionalities as a hydrophobic anchor for membrane incorporation and thus has been employed in versatile systems.15−18 Herein, we report the grafting behavior of the amphiphilic Chol-PAA chains immobilized as colloidal micelles to the preformed liposome templates during the incorporation process. The colloidal structure of Chol-PAA micelles and the successful formation of polymer-grafted vesicles (PGVs) were extensively verified by the multiple colloidal characterization methods including dynamic light scattering, zeta potential, and transmission electron microscope as well as the atomic force microscope measurements. The fluorescence resonance energy transfer (FRET) study further demonstrated the labile property of Chol-PAA chains for the incorporation, which is highly favorable for the postformation modification of liposomes with rigid membrane. Additionally, the incorporation study with selfquenched dye-containing liposomes shows the negligible cargoleakage induced by the Chol-PAA addition, which is a critical prerequisite for the surface modification of drug-containing lipid vesicles as a robust delivery system. B
DOI: 10.1021/acs.langmuir.7b00670 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 2. (A) Schematic illustration of theoretical calculation on the amount of end-grafted Chol-PAA required for the interchain cross-linking. (B) TEM images of polymer-grafted vesicles (PGVs) modified with Chol-PAA 2.5k. The toroidal morphology is commonly observed for lipid vesicles due to the evaporation of aqueous cores during the TEM observation. (C) ζ-potential change of PGVs after mixing with various amount of CholPAA 2.5k amphiphiles. Also shown is the ζ of native Chol-PAA micelle. (D) Hydrodynamic diameter (DH) change of vesicles after the incorporation of Chol-PAA amphiphiles. Addition of Chol-PAA 2.5k to bare liposomes (BLs, mean DH = 87 ± 11 nm) produced PGVs with increased mean DH of 101 ± 13 nm. Addition of Chol-PAA 5.6k exhibited further increase in mean DH of PGVs to 124 ± 16 nm.
measurements, demonstrating the formation of micellar aggregates of Chol-PAA amphiphiles in aqueous solution. Modification of Liposomal Membrane with Chol-PAA. For the postformation modification of native liposomes, amphiphilic polymers can incorporate to the external leaflet of liposome membrane while the structural integrity of vesicles should be maintained without significant rupture.6 In previous X-ray diffraction estimation with a lamellar phase lipid bilayer,25 15−20 mol % symmetrical incorporation of lipid-conjugated PEG2k was obtained (compared to the total lipid amounts) on both sides of bilayer membrane, above which the increased lateral hindrance of the grafted polymer chains induces vesicleto-micelle transition, resulting in the rupture of vesicles. Additionally, ∼9 mol % incorporation of dipalmitoylphosphatidylethanolamine-conjugated polyethylene glycol 5000 (DPPE-PEG5k) has been observed for the postmodification of the external layer of liposome membrane regardless of the lipid tail lengths.26 In the PCN fabrication, the interchain distances (D) between the membrane-grafted CholPAA should be sufficiently close each other for interchain crosslinking (D ≤ 2RH, Figure 2A).12 On the basis of the known hydrodynamic radius (RH) of PAA (approximately 1.18 nm for PAA 1.66k)24 and the number of lipid molecules in a single vesicle (∼50 000 lipids for 80 nm vesicle),27 apparently more than 9.8 mol % of Chol-PAA is required for complete coverage of a single vesicle. Hence, 10 mol % of Chol-PAA 2.5k was employed for the maximum amount of membrane modifiers. For the initial verification of surface modification, the mixtures of bare liposomes (BLs, containing DPPC/DOPG/ Cholesterol of 56.4/3.6/40 mol %) and various amounts of Chol-PAA suspension were prepared by the 24 h incubation and the subsequent change of zeta (ζ) potential was observed at pH 7 to compare the apparent degree of incorporation
Also supporting the micelle formation of Chol-PAA in aqueous media is the results observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). When DLS measurements were carried out with Chol-PAA 2.5 kDa and Chol-PAA 5.6 kDa, distinct light scattering signals from both samples appeared at the concentration above about 5−10 μM, which is a concentration range of critical micelle concentration (cmc) commonly observed for the cholesterol-attached amphiphilic polymers in aqueous media.21,22 The resulting aggregates exhibited mean hydrodynamic diameters (DH) of 15 ± 3 and 27 ± 5 nm for Chol-PAA 2.5 and 5.6 kDa, respectively (Figure 1C). These values are quite comparable to the size of micelles composed of distearoyl-phosphatidylethanolamine-conjugated polyethylene glycol 2000 (DSPE-PEG2k), which showed ∼11−16 nm.23 Compared to the previously reported hydrodynamic radius of PAA homopolymers (RH = 3.7 nm for PAA with 18.1 kDa at pH 7, 1 M NaCl),24 apparently large DH was observed for the Chol-PAA micelles. We attributed the relatively large size of the Chol-PAA micelles to the fully extended conformation of PAA chains through the repulsive forces between the anionic acrylate residues at low salt environment (
