Article pubs.acs.org/Biomac
Reduction-Responsive Cholesterol-Based Block Copolymer Vesicles for Drug Delivery Lin Jia,†,‡,§ Di Cui,†,‡,§ Jérôme Bignon,∥ Aurelie Di Cicco,†,‡,§,⊥ Joanna Wdzieczak-Bakala,∥ Jianmiao Liu,*,∥,# and Min-Hui Li*,†,‡,§ †
Institut Curie, Centre de Recherche, 75248 Paris, France CNRS, UMR168, Physico-Chimie Curie, 75248 Paris, France § UPMC Université Paris VI, 75005 Paris, France ⊥ Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, 75248 Paris, France ∥ Institut de Chimie des Substances Naturelles, CNRS UPR2301, 91191 Gif sur Yvette, France ‡
S Supporting Information *
ABSTRACT: We developed a new robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SSPAChol. The stability and robustness were achieved by the smectic physical cross-linking of cholesterol-containing liquid crystal polymer PAChol in the hydrophobic layer. The reductionsensitivity was introduced by the disulfide bridge (-S−S-) that links the hydrophilic PEG block and the hydrophobic PAChol block. We used a versatile synthetic strategy based on atom transfer radical polymerization (ATRP) to synthesize the reduction-responsive amphiphilic block copolymers. The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced in organic solution. The partial destruction of PEG-SS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by glutathione (GSH) was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentrations (≥35 mM in PBS or artificially enhanced in macrophage cells by GSH-OEt pretreatment) and long incubation time (in the order of hours) were, however, necessary to get significant calcein release. These polymersomes could be used as drug carriers with very long circulation profiles and slow release kinetics.
1. INTRODUCTION The in vivo delivery of therapeutic drugs such as anticancer drugs and gene therapeutic nucleic acid substances (e.g., pDNA, siRNA, etc.) to their molecular targets (targeted drug delivery) remains a major challenge. Biomimetic drug carriers based on nanobiotechnology are being conceived with the main objectives of protecting the active molecules against degradation and against interaction with extracellular biomacromolecules, reducing drug toxicity to healthy cells and controlling drug release in time and space.1 Polymer vesicles (also referred to as polymersomes) have attracted special attention due to their vesicular structures, cell- and virus-mimicking dimensions, and functions.2,3 They are particularly interesting for intracellular delivery of hydrophilic substances such as proteins and nucleic acids.4−6 Targeting properties can be endowed to the polymersomes by the grafting of biologically relevant receptorspecific ligands.7,8 The controlled release can be achieved by the structural engineering of amphiphilic copolymers (in particular, their hydrophobic parts) in order to render polymersomes responsive to different chemical or physical stimuli (pH, oxidation, reduction, enzyme, temperature, light, magnetic fields, ...).9−12 Under the action of these stimuli, chemical or physical structure of copolymers can be altered. © XXXX American Chemical Society
This can in turn induce the destabilization or the destruction of polymersomes and the release of encapsulated drugs. Among these stimuli-responsive systems, polymersomes that release drugs in response to intracellular signals, such as acidic pH and reductive environment, are of great interest, because many therapeutic drugs (e.g., protein drugs, pDNA and siRNA) have to be delivered and released inside the cells such as in the cytoplasm or cell nucleus, in order to exert therapeutic effects.13−16 In the present work, we will focus on the reduction-responsive polymersomes liable to release active molecules inside the cells. The cytosol contains 2 to 3 orders higher level of a reducing agent, glutathione tripeptide (L-γ-glutamyl-L-cysteinyl-glycine, GSH), than the extracellular fluids (2−10 mM in cytosol vs 2− 20 μM in extracellular fluids).17−19 The endosomal compartment is also redox-active where the redox potential is modulated by a specific reducing enzyme γ-interferon-inducible lysosomal thiol reductase (GILT) in the copresence of another reducing agent, cysteine.20 Moreover, the redox-active lysosome Received: March 6, 2014 Revised: May 5, 2014
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dx.doi.org/10.1021/bm5003569 | Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Article
Scheme 1. Synthetic Route of ATRP Macroinitiator PEG-SS-Br
contains also low-mass iron species in a reduced state (Fe2+).21 Recently, considerable progress has been made in the design of reduction-responsive nanocarriers for triggered intracellular drug release. The basic idea is to introduce the disulfide bond -S−S- (SS), that is cleavable in the reductive environment, into the molecular structures of the nanocarriers (see ref 22 and refs therein). However, there is only a very limited number of works dealing with reduction-responsive polymersomes. Hubbell’s group23 reported the first example of such polymersomes based on poly(ethylene glycol)-SS-poly(propylene sulfide) (PEG17SS-PPS33). The amphiphilic block copolymer PEG17-SS-PPS33 was obtained by synthesizing first monothiolated-PEG and PPS (PEG-S− and PPS-S−), respectively, and then coupling them by oxidation. Polymersomes formed from this block copolymer were demonstrated to break down in the presence of intracellular concentrations of cysteine. In cellular experiments with macrophages, uptake, disruption, and release were observed within 10 min of exposure to cells, well within the time frame of early endosome and endolysosomal processing. Kim and co-workers reported a reduction-sensitive and biocompatible vesicle (SSCB[6]VC) from an amphiphilic cucurbit[6]uril (CB[6]) derivative containing disulfide bonds between hexaethylene glycol units and the CB[6] core.24 The vesicles were stable in the presence of 3 μM GSH or 15 μM cysteine. However, complete disruption of vesicles occurred in 12 h in response to 5 mM GSH. In general, the polymer self-assembled structures are more stable than those of small molecule self-assemblies (e.g., polymersomes vs liposomes), because critical aggregation concentration (CAC) of polymers is much lower than that of small molecules. Nevertheless, the polymer nanostructures including polymersomes still suffer from the problem of limited stability upon extensive dilution during intravenous administration, which leads to premature release of drugs.25 This is because the individual intermolecular interactions that stabilize the polymer nanostructures, like hydrophobic interactions, van der Waals forces, and hydrogen bonding, are relatively weak, and the extensive dilution leading their concentration to be
lower than CAC can destabilize the polymer nanostructures. For example, major problems encountered in time-controlled delivery of polypeptides using polymer matrices are the fast initial release (in particular, in the first 24 h: “burst release”) and, consequently, the overall bioavailability of the peptide drugs.26 Robust polymersomes were reported by introducing chemical cross-linking in their membrane.27−31 In the following two examples, polymersomes that are robust upon dilution, but sensitive to reduction condition, were constructed by introducing disulfide cross-linking, a reversible chemical crosslinking, in their hydrophobic cores. Mastrobattista and coworkers reported that peptide vesicles could be stabilized by introducing two or three cysteine units into the hydrophobic domain of amphiphilic oligopeptide SA2 (Ac-Ala-Cys-Val-CysLeu-(Leu/Cys)-Leu-Trp-Glu-Glu-COOH), which allow the formation of intermolecular disulfide bridges. Upon disulfide formation, the cross-linked vesicles remained stable under dilution conditions that disrupted the non-cross-linked peptide vesicles.32,33 Zhong and co-workers reported the preparation of disulfide-cross-linked polymersomes from water-soluble poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-b-PAA-b-PNIPAM) triblock copolymers by using the temperature-responsive property of PNIPAM. PEO-b-PAAb-PNIPAM solution in water was heated to above the LCST of PNIPAM block (e.g., 37 °C) to render the triblock copolymer amphiphilic. The polymersomes formed in situ were then crosslinked at the hydrophilic−hydrophobic interface (PAA parts) using cystamine via carbodiimide chemistry.34 The cross-linked polymersomes, while showing remarkable stability against dilution, organic solvent, high salt conditions, and change of temperature in water, were otherwise completely dissociated in 1.5 h in 10 mM DTT media at pH 7.4 at room temperature. Taking account of both responsiveness and stability of polymersomes discussed above, we developed in this work a new robust reduction-sensitive polymersome based on the amphiphilic block copolymers PEG-SS-PAChol (see Scheme 2). The stability and robustness were achieved via a physical cross-linking instead of a chemical one in the hydrophobic B
dx.doi.org/10.1021/bm5003569 | Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Article
Scheme 2. Synthetic Route of Reduction-Sensitive Block Copolymers PEG-SS-PBA and PEG-SS-PAChol
netriamine (PMDETA, 99%), n-butyl acrylate (n-BA, 99%), 2,2′dithiodipyridine (99%), dithiothreitol (DTT, 99%), calcein (mixed isomer), glutathione reduced (L-γ-glutamyl-L-cysteinyl-glycine, GSH, ≥98%), and glutathione reduced ethyl ester (GSH-OEt, >90%) were purchased from Aldrich. 2-Mercaptoethanol (99%) was purchased from Acros. 2-Bromo-2-methylpropionic acid (98%) was purchased from Fluka. The monomer n-BA was filtered through a short column of neutral Al2O3 to remove the inhibitor before use. Dichloromethane (CH2Cl2) (Carlo Erba), 1,4-dioxane (Carlo Erba), and PMDETA were distilled under argon. Tetrahydrofuran (THF; Carlo Erba) was distilled from sodium-benzophenone ketyl under argon. Other chemical reagents were used as purchased without further purification. All water-sensitive reactions were conducted in oven-dried glassware, under a dry argon atmosphere. All flash chromatography was performed using silicone gel (Macherey-Nagel MN Kieselgel 60). Synthesis. Synthesis of Monomer. The LC monomer, cholesteryl acryloyoxy ethyl carbonate (AChol), was synthesized from cholesteryl chloroformate and 2-hydroxyethyl acrylate as described previously.36 Synthesis of ATRP Macroinitiator (PEG-SS-Br) with Disulfide Bond. The ATRP macroinitiator with disulfide bond (PEG-SS-Br) was synthesized as shown in Scheme 1. Hydroxyethyl-mercaptopyridine (1).42 A solution containing 2,2′dithiopyridine (10 g, 0.045 mol), 0.66 mL of glacial acetic acid, and 100 mL of methanol was prepared in a 250 mL round-bottom flask with stir bar. 2-Mercaptoethanol (1.65 g, 0.022 mol) was dissolved in 10 mL of methanol and added dropwise to the 2,2′-dithiopyridine solution. After 4 h of reaction at room temperature, the crude product was concentrated under vacuum using rotation evaporator and purified by flash chromatography with a solvent mixture ethyl acetate/hexane (1:2) as the eluent. The pure compound is transparent oil. Yield: 70%. 1 H NMR (300 MHz, CDCl3, ppm): δ 8.50 (d, 1H, −CHN−, pyridine), 7.57 (t, 1H, −CH, pyridine), 7.40 (d, 1H, −CH, pyridine), 7.14 (m, 1H, −CH, pyridine), 3.79 (m, 2H, −CH2−OH), 2.93 (m, 2H, −S−CH2−). 2-(2-pyridinyldithio)ethyl 2-Bromo-2-methylpropionate (2).43 Hydroxyethyl-mercaptopyridine (1; 0.66 g, 3.5 mmol), 1,3-dicyclohexylcarbodiimide (DCC; 1.1 g, 4.2 mmol), and 4-dimethylaminopyridine (DMAP; 0.062 g, 0.42 mmol) were dissolved in 20 mL of anhydrous dichloromethane. 2-Bromo-2-methylpropionic acid (0.71 g, 4.2 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered, the solvent evaporated under vacuum, and the oily residue purified by column chromatography (ethyl acetate/hexanes, 1:2) to give the compound 2. Yield: 70%. 1H NMR (300 MHz, CDCl3; see Figure SI-1): δ 8.48 (m, 1H, −CH−N, pyridine), 7.68 (m, 2H, −CHCH−, pyridine), 7.09 (m,
PAChol layer. The reduction-sensitivity was introduced by the disulfide bridge (-S−S-) that links the hydrophobic block PEG and hydrophobic block PAChol. Our group has studied, for several years, liquid crystal (LC) polymersomes made of block copolymers in which the hydrophobic block is a LC polymer.35 These LC polymersomes are very stable because of the physical cross-linking of LC nature in the membrane and can be preserved well at room temperature for several years without further morphological modification.36−40 The system made of PEG-b-PAChol36−40 with hydrophobic block bearing cholesterol pendent groups is of special interest for possible biorelated applications, because of the biocompatibility of cholesterol moieties. PAChol is a smectic liquid crystal (LC) polyacrylate (PA) composed of cholesterol monomers (Chol).37 This thermotropic smectic phase (with smecticisotropic transition temperature around 150 °C) in lyotropic bilayer plays the role of physical cross-linking.41 Here PEG-SSPAChol is a homologue of PEG-b-PAChol, but with cleavable disulfide linkage between PEG and PAChol blocks. We described a versatile method based on atom transfer radical polymerization (ATRP) to synthesize the reductionsensitive amphiphilic block copolymers. The copolymers were obtained by polymerizing the LC acrylate monomers using disulfide-containing PEG as ATRP macroinitiator (see Schemes 1 and 2). The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced when copolymers were solubilized in organic solution. The partial destruction of PEGSS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by GSH was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentration (e.g., ≥35 mM in PBS) and long incubation time (in the order of hours) were necessary to get significant calcein release, showing the high stability of PEG-SS-PAChol polymersomes.
2. EXPERIMENTAL SECTION Materials. α-Methoxy-ω-hydroxy poly(ethylene glycol) (MeOPEG45-OH, Mn = 2000 Da), 3-mercaptopropionic acid (⩾99%, Hafnium(IV) chloride tetrahydrofuran complex (HfCl4·2THF, 98%), 1,3-dicyclohexylcarbodiimide (DCC, 99%), 4-dimethylaminopyridine (DMAP, >99%), CuIBr (98%), N,N′,N′,N″,N″-pentamethyldiethyleC
dx.doi.org/10.1021/bm5003569 | Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Article
1H, −CH, pyridine), 4.42 (t, 2H, −CH2−OCO−), 3.08 (t, 2H, −S−CH2−), 1.94 (s, 6H,−CH3). MeO-PEG45-SH (3). The thiol-functionalized poly(ethylene glycol), MeO-PEG45-SH, was synthesized from α-methoxy-ω-hydroxy poly(ethylene glycol) (MeO-PEG45-OH, Mn = 2000) and 3-mercaptopropionic acid (MPA), as reported in the literature.44 In a typical experiment, MeO-PEG45-OH (10 g, 5 mmol) and MPA (2.65 g, 25 mmol) were stirred at 50 °C under argon until a solution was formed, and then toluene (20 mL) along with HfCl4·2THF (116 mg, 0.25 mmol) were added. The mixture was refluxed at 130 °C under argon for 24 h. Toluene was removed under reduced pressure. The MeOPEG45-SH was solubilized CH2Cl2 and reprecipitated into diethyl ether trice. Yield: 95%. 1H NMR (300 MHz, CDCl3; see Figure SI-2): δ 4.27 (t, 2H, −CH2−OCO−), 3.63 (m, 180H, −O−CH2−CH2− O−), 3.37 (s, 3H, −O−CH3), 2.76 (t, 2H, −CH2−SH), 2.68 (t, 2H, −O−CO−CH2−). ATRP Macroinitiator with Disulfide Bond PEG-SS-Br. 2-(2Pyridinyldithio)ethyl 2-bromo-2-methylpropionate (2; 30.6 mg, 0.09 mmol) and acetic acid (16.78 mg, 0.28 mmol) were dissolved in 1 mL of dry CH2Cl2. The solution was then added dropwise into 3 mL CH2Cl2 solution containing MeO-PEG45-SH (3; 100 mg, 0.045 mmol) during a period of 1 h. The mixture was allowed to stir for other 24 h at room temperature. The reaction mixture was concentrated by rotary evaporation, redissolved in 1 mL of CH2Cl2 and then precipitated from 50 mL diethyl ether. The precipitation process was repeated twice to remove the compound 2 in excess. The final ATRP macroinitiator MeO-PEG45-SS-Br is a white solid (97 mg, yield: 90%). 1 H NMR (300 MHz, CDCl3; Figure 1): δ 4.43 (t, 2H, −S−CH2−
by dissolution of the final mixture in chloroform and precipitation in methanol trice. The polymer was dried under vacuum for 48 h (150 mg, yield: 75%). 1H NMR (300 MHz, CDCl3): δ 0.67−2.42 (m, −CH3, −CH(CH3)−, −CH−, −CH2−), 2.76 (t, 2H, −O−CO− CH2−), 2.90 (t, 4H, −CH2−S−S−CH2−), 3.37 (s, 3H, −O−CH3), 3.65 (s, 180H, −O−CH2−CH2−O−), 4.30 (m, (4n+2)H, −OCO− CH2−CH2−OCOO−, −S−CH2−CH2−O−CO−), 4.45 (m, nH, −CHO−), 5.38 (m, nH, −CCH−CH2−). (n is the degree of polymerization of the LC block; see Figure 2 for 1H NMR spectra of PEG-SS-PBA and PEG-SS-PAChol.) The degrees of polymerization of the hydrophobic block (DPPBA or DPPAChol) were calculated according to eq 1 for PEG-SS-PBA, or eq 2 for PEG-SS-PAChol. See Table 1 for detailed information.
DPBA = 1.5 × I4.03/I3.37
(1)
DPLCblock = 3 × I5.38/I3.37
(2)
In eq 1, I4.03 is the integration value of the methylene proton signal (c) in the butyl acrylate units at 4.03 ppm and I3.37 the integration value of the methoxy proton signal (a) in the PEG end group at 3.37 ppm. In eq 2, I5.38 is the integration value of the vinylic proton signal (g) in the cholesteryl group at 5.38 ppm and I3.37 the integration value of the methoxy proton signal (a) in the PEG end group at 3.37 ppm. The copolymer PEG45-b-PAChol10 without the disulfide bridge, which was used as a control copolymer, was synthesized according to previously published procedure.36 See Table 1 for its molecular characterization. Polymer Characterization. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra were recorded using a Bruker HW300 MHz spectrometer. CDCl3 was used as solvent and tetramethylsilane (TMS) as the internal chemical shift reference. Size Exclusion Chromatography (SEC). Molecular weights of the copolymers were characterized by SEC at 40 °C using Waters Styragel HR 5E columns (×2), a Waters 4110 differential refractometer and a Waters 486 UV detector. THF was used as the eluent at 1 mL/min. Molecular weight distributions (Mw/Mn) of the diblock copolymers were evaluated by SEC calibrated with polystyrene (PS) standards (Polymer Laboratory PS calibration kit).45 Polymer Degradation Analyses. The analyses of disulfide bond cleavage in block copolymers were made by recording the size exclusion chromatograms before and after the addition of dithiothreitol (DTT) as the reducing agent. Typically, 10 mg of disulfide-containing copolymers were dissolved in 1 mL THF, then 1.54 mg of DTT (10 equiv mole of disulfide bond) was added into the polymer solution. After 1 h, 24 or 48 h of stirring, a 100 μL of the solution was directly injected to the SEC system for analysis. Polymersome Formation and Characterization. Polymersome Formation. The polymersomes were prepared following a classical nanoprecipitation procedure as reported previously.37 Typically, the copolymer was first dissolved in 1 mL of dioxane (at 0.5, 0.2, or 0.1 wt %) and 1 mL of deionized milli-Q water was injected slowly to the organic solution under mild shaking (2−3 μL of water per minute to 1 mL of polymer solution). The whole process of nanoprecipitation was carried out at 25 °C. The turbid mixtures were then dialyzed against water for 3 days to remove the dioxane using a Spectra/Por regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 3500. The final volume of the polymersome suspension was then adjusted to 2 mL. For the dye-encapsulated polymersomes, deionized water was replaced by PBS (10 mM phosphate buffer saline, pH 7.4) during the preparation. Polymersome Degradation under Reductive Condition. A total of 0.5 mL of DTT aqueous solution (2 mM) was added, under slight hand shaking, into 0.4 mL of polymersome suspension in water (prepared with 0.5 wt % of initial polymer concentration in dioxane). DTT was again of 10 equiv mole of disulfide bond. Then the mixture was kept still without any stirring for 24 h. The morphological changes of polymersomes were finally analyzed by cryo-TEM. Transmission Electron Microscopy (TEM). The morphological analysis of polymersomes was performed by TEM on samples stained by uranyl acetate (2 wt %) or by cryo-TEM on sample deposited onto
Figure 1. 1H NMR (300 MHz) spectrum of ATRP macroinitiator PEG-SS-Br (Mn,PEG = 2000) in CDCl3. CH2−OCO−), 4.27 (t, 2H, −O−CH2−CH2−OCO−), 3.65(m, 180H, −O−CH2−CH2−O−), 3.38 (s, 3H, −O−CH3), 2.96 (t, 4H, −CH2− S−S−CH2−), 2.79 (t, 2H, −O−CO−CH2−), 1.94 (s, 6H, −CH3). As all polymers and copolymers in this work contained methoxy terminated PEG45 (Mn = 2000 Da), the polymer names will be simplified as PEG-SS-Br for ATRP macroinitiator, and PEG-SS-PBA and PEG-SS-PAChol for block copolymers in the following. Synthesis of Diblock Copolymer with Disulfide Bond between the Two Blocks by ATRP Polymerization. In this work, we synthesized two kinds of copolymers, PEG-SS-PBA and PEG-SS-PAChol, from the ATRP PEG-macroinitiator (PEG-SS-Br) containing disulfide bond by typical ATRP polymerization. Taking PEG-SS-PAChol, for example, PEG-SS-Br (50 mg, 0.02 mmol), CuIBr (7.5 mg, 0.05 mmol), cholesteryl acryloyoxy ethyl carbonate (AChol, LC monomer; 150 mg, 1.72 mmol), anhydrous toluene (0.5 mL), and PMDETA (10.5 μL, 0.05 mmol), were added into a Schlenk-type flask. The flask was degassed and exchanged with argon via three freeze−thaw cycles. The reaction mixture was heated at 80 °C for 18 h. The mixture was then diluted with chloroform and passed through a short basic aluminum oxide column to remove Cu salts. The resulting solution was concentrated by rotary evaporation. The copolymer was recovered D
dx.doi.org/10.1021/bm5003569 | Biomacromolecules XXXX, XXX, XXX−XXX
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Article
Figure 2. 1H NMR (300 MHz) spectra of diblock copolymers: (a) PEG-SS-PBA and (b) PEG-SS-PAChol in CDCl3. pixels CCD camera. Calibration was performed with a 2D crystal of purple membrane leading to 0.385 nm/pixel at 45000 magnifications. Nanoparticle Tracking Analysis (NTA). The sizes and size distributions of polymersomes were measured by NTA, using a NanoSight LM14 (NanoSight, United Kingdom) equipped with a 532 nm laser. The sample at right dilutions (in the order of 108−109 particles per mL) was loaded in the sample chamber with a sterile syringe. The NTA 2.2 analytical software was used for capturing and analyzing the data. The samples were measured for 60 s with manual shutter and gain adjustments. All measurements were performed in triplicate at 25 °C. Dye Encapsulation and Reduction-Responsive Release. Encapsulation of Dyes in Polymersomes. The hydrophilic calcein was first dissolved in PBS (v0 = 1 mL, c0‑cal = 0.02 M). This solution was added slowly to the dioxane solution of copolymers (1 mL, 0.1 wt %, cw‑polym = 1 g·L−1) to form the vesicles loaded with calcein. The free dyes and dioxane were removed by 4 days’ dialysis (MWCO 3500) against PBS (changing the buffer twice per day), until the intensity of fluorescence of PBS outside the dialysis tube to be very low and stable (1 μm). When we decreased the initial polymer concentration from 0.5 wt % and 0.2 wt % to 0.1 wt %, the situation was improved. Figure 5b shows the vesicle morphologies of PEG45SS-PAChol16 become quite homogeneous and the average diameter is
Reduction-Responsive Cholesterol-Based Block Copolymer Vesicles for Drug Delivery Lin Jia,†,‡,§ Di Cui,†,‡,§ Jérôme Bignon,∥ Aurelie Di Cicco,†,‡,§,⊥ Joanna Wdzieczak-Bakala,∥ Jianmiao Liu,*,∥,# and Min-Hui Li*,†,‡,§ †
Institut Curie, Centre de Recherche, 75248 Paris, France CNRS, UMR168, Physico-Chimie Curie, 75248 Paris, France § UPMC Université Paris VI, 75005 Paris, France ⊥ Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, 75248 Paris, France ∥ Institut de Chimie des Substances Naturelles, CNRS UPR2301, 91191 Gif sur Yvette, France ‡
S Supporting Information *
ABSTRACT: We developed a new robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SSPAChol. The stability and robustness were achieved by the smectic physical cross-linking of cholesterol-containing liquid crystal polymer PAChol in the hydrophobic layer. The reductionsensitivity was introduced by the disulfide bridge (-S−S-) that links the hydrophilic PEG block and the hydrophobic PAChol block. We used a versatile synthetic strategy based on atom transfer radical polymerization (ATRP) to synthesize the reduction-responsive amphiphilic block copolymers. The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced in organic solution. The partial destruction of PEG-SS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by glutathione (GSH) was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentrations (≥35 mM in PBS or artificially enhanced in macrophage cells by GSH-OEt pretreatment) and long incubation time (in the order of hours) were, however, necessary to get significant calcein release. These polymersomes could be used as drug carriers with very long circulation profiles and slow release kinetics.
1. INTRODUCTION The in vivo delivery of therapeutic drugs such as anticancer drugs and gene therapeutic nucleic acid substances (e.g., pDNA, siRNA, etc.) to their molecular targets (targeted drug delivery) remains a major challenge. Biomimetic drug carriers based on nanobiotechnology are being conceived with the main objectives of protecting the active molecules against degradation and against interaction with extracellular biomacromolecules, reducing drug toxicity to healthy cells and controlling drug release in time and space.1 Polymer vesicles (also referred to as polymersomes) have attracted special attention due to their vesicular structures, cell- and virus-mimicking dimensions, and functions.2,3 They are particularly interesting for intracellular delivery of hydrophilic substances such as proteins and nucleic acids.4−6 Targeting properties can be endowed to the polymersomes by the grafting of biologically relevant receptorspecific ligands.7,8 The controlled release can be achieved by the structural engineering of amphiphilic copolymers (in particular, their hydrophobic parts) in order to render polymersomes responsive to different chemical or physical stimuli (pH, oxidation, reduction, enzyme, temperature, light, magnetic fields, ...).9−12 Under the action of these stimuli, chemical or physical structure of copolymers can be altered. © XXXX American Chemical Society
This can in turn induce the destabilization or the destruction of polymersomes and the release of encapsulated drugs. Among these stimuli-responsive systems, polymersomes that release drugs in response to intracellular signals, such as acidic pH and reductive environment, are of great interest, because many therapeutic drugs (e.g., protein drugs, pDNA and siRNA) have to be delivered and released inside the cells such as in the cytoplasm or cell nucleus, in order to exert therapeutic effects.13−16 In the present work, we will focus on the reduction-responsive polymersomes liable to release active molecules inside the cells. The cytosol contains 2 to 3 orders higher level of a reducing agent, glutathione tripeptide (L-γ-glutamyl-L-cysteinyl-glycine, GSH), than the extracellular fluids (2−10 mM in cytosol vs 2− 20 μM in extracellular fluids).17−19 The endosomal compartment is also redox-active where the redox potential is modulated by a specific reducing enzyme γ-interferon-inducible lysosomal thiol reductase (GILT) in the copresence of another reducing agent, cysteine.20 Moreover, the redox-active lysosome Received: March 6, 2014 Revised: May 5, 2014
A
dx.doi.org/10.1021/bm5003569 | Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Article
Scheme 1. Synthetic Route of ATRP Macroinitiator PEG-SS-Br
contains also low-mass iron species in a reduced state (Fe2+).21 Recently, considerable progress has been made in the design of reduction-responsive nanocarriers for triggered intracellular drug release. The basic idea is to introduce the disulfide bond -S−S- (SS), that is cleavable in the reductive environment, into the molecular structures of the nanocarriers (see ref 22 and refs therein). However, there is only a very limited number of works dealing with reduction-responsive polymersomes. Hubbell’s group23 reported the first example of such polymersomes based on poly(ethylene glycol)-SS-poly(propylene sulfide) (PEG17SS-PPS33). The amphiphilic block copolymer PEG17-SS-PPS33 was obtained by synthesizing first monothiolated-PEG and PPS (PEG-S− and PPS-S−), respectively, and then coupling them by oxidation. Polymersomes formed from this block copolymer were demonstrated to break down in the presence of intracellular concentrations of cysteine. In cellular experiments with macrophages, uptake, disruption, and release were observed within 10 min of exposure to cells, well within the time frame of early endosome and endolysosomal processing. Kim and co-workers reported a reduction-sensitive and biocompatible vesicle (SSCB[6]VC) from an amphiphilic cucurbit[6]uril (CB[6]) derivative containing disulfide bonds between hexaethylene glycol units and the CB[6] core.24 The vesicles were stable in the presence of 3 μM GSH or 15 μM cysteine. However, complete disruption of vesicles occurred in 12 h in response to 5 mM GSH. In general, the polymer self-assembled structures are more stable than those of small molecule self-assemblies (e.g., polymersomes vs liposomes), because critical aggregation concentration (CAC) of polymers is much lower than that of small molecules. Nevertheless, the polymer nanostructures including polymersomes still suffer from the problem of limited stability upon extensive dilution during intravenous administration, which leads to premature release of drugs.25 This is because the individual intermolecular interactions that stabilize the polymer nanostructures, like hydrophobic interactions, van der Waals forces, and hydrogen bonding, are relatively weak, and the extensive dilution leading their concentration to be
lower than CAC can destabilize the polymer nanostructures. For example, major problems encountered in time-controlled delivery of polypeptides using polymer matrices are the fast initial release (in particular, in the first 24 h: “burst release”) and, consequently, the overall bioavailability of the peptide drugs.26 Robust polymersomes were reported by introducing chemical cross-linking in their membrane.27−31 In the following two examples, polymersomes that are robust upon dilution, but sensitive to reduction condition, were constructed by introducing disulfide cross-linking, a reversible chemical crosslinking, in their hydrophobic cores. Mastrobattista and coworkers reported that peptide vesicles could be stabilized by introducing two or three cysteine units into the hydrophobic domain of amphiphilic oligopeptide SA2 (Ac-Ala-Cys-Val-CysLeu-(Leu/Cys)-Leu-Trp-Glu-Glu-COOH), which allow the formation of intermolecular disulfide bridges. Upon disulfide formation, the cross-linked vesicles remained stable under dilution conditions that disrupted the non-cross-linked peptide vesicles.32,33 Zhong and co-workers reported the preparation of disulfide-cross-linked polymersomes from water-soluble poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-b-PAA-b-PNIPAM) triblock copolymers by using the temperature-responsive property of PNIPAM. PEO-b-PAAb-PNIPAM solution in water was heated to above the LCST of PNIPAM block (e.g., 37 °C) to render the triblock copolymer amphiphilic. The polymersomes formed in situ were then crosslinked at the hydrophilic−hydrophobic interface (PAA parts) using cystamine via carbodiimide chemistry.34 The cross-linked polymersomes, while showing remarkable stability against dilution, organic solvent, high salt conditions, and change of temperature in water, were otherwise completely dissociated in 1.5 h in 10 mM DTT media at pH 7.4 at room temperature. Taking account of both responsiveness and stability of polymersomes discussed above, we developed in this work a new robust reduction-sensitive polymersome based on the amphiphilic block copolymers PEG-SS-PAChol (see Scheme 2). The stability and robustness were achieved via a physical cross-linking instead of a chemical one in the hydrophobic B
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Scheme 2. Synthetic Route of Reduction-Sensitive Block Copolymers PEG-SS-PBA and PEG-SS-PAChol
netriamine (PMDETA, 99%), n-butyl acrylate (n-BA, 99%), 2,2′dithiodipyridine (99%), dithiothreitol (DTT, 99%), calcein (mixed isomer), glutathione reduced (L-γ-glutamyl-L-cysteinyl-glycine, GSH, ≥98%), and glutathione reduced ethyl ester (GSH-OEt, >90%) were purchased from Aldrich. 2-Mercaptoethanol (99%) was purchased from Acros. 2-Bromo-2-methylpropionic acid (98%) was purchased from Fluka. The monomer n-BA was filtered through a short column of neutral Al2O3 to remove the inhibitor before use. Dichloromethane (CH2Cl2) (Carlo Erba), 1,4-dioxane (Carlo Erba), and PMDETA were distilled under argon. Tetrahydrofuran (THF; Carlo Erba) was distilled from sodium-benzophenone ketyl under argon. Other chemical reagents were used as purchased without further purification. All water-sensitive reactions were conducted in oven-dried glassware, under a dry argon atmosphere. All flash chromatography was performed using silicone gel (Macherey-Nagel MN Kieselgel 60). Synthesis. Synthesis of Monomer. The LC monomer, cholesteryl acryloyoxy ethyl carbonate (AChol), was synthesized from cholesteryl chloroformate and 2-hydroxyethyl acrylate as described previously.36 Synthesis of ATRP Macroinitiator (PEG-SS-Br) with Disulfide Bond. The ATRP macroinitiator with disulfide bond (PEG-SS-Br) was synthesized as shown in Scheme 1. Hydroxyethyl-mercaptopyridine (1).42 A solution containing 2,2′dithiopyridine (10 g, 0.045 mol), 0.66 mL of glacial acetic acid, and 100 mL of methanol was prepared in a 250 mL round-bottom flask with stir bar. 2-Mercaptoethanol (1.65 g, 0.022 mol) was dissolved in 10 mL of methanol and added dropwise to the 2,2′-dithiopyridine solution. After 4 h of reaction at room temperature, the crude product was concentrated under vacuum using rotation evaporator and purified by flash chromatography with a solvent mixture ethyl acetate/hexane (1:2) as the eluent. The pure compound is transparent oil. Yield: 70%. 1 H NMR (300 MHz, CDCl3, ppm): δ 8.50 (d, 1H, −CHN−, pyridine), 7.57 (t, 1H, −CH, pyridine), 7.40 (d, 1H, −CH, pyridine), 7.14 (m, 1H, −CH, pyridine), 3.79 (m, 2H, −CH2−OH), 2.93 (m, 2H, −S−CH2−). 2-(2-pyridinyldithio)ethyl 2-Bromo-2-methylpropionate (2).43 Hydroxyethyl-mercaptopyridine (1; 0.66 g, 3.5 mmol), 1,3-dicyclohexylcarbodiimide (DCC; 1.1 g, 4.2 mmol), and 4-dimethylaminopyridine (DMAP; 0.062 g, 0.42 mmol) were dissolved in 20 mL of anhydrous dichloromethane. 2-Bromo-2-methylpropionic acid (0.71 g, 4.2 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered, the solvent evaporated under vacuum, and the oily residue purified by column chromatography (ethyl acetate/hexanes, 1:2) to give the compound 2. Yield: 70%. 1H NMR (300 MHz, CDCl3; see Figure SI-1): δ 8.48 (m, 1H, −CH−N, pyridine), 7.68 (m, 2H, −CHCH−, pyridine), 7.09 (m,
PAChol layer. The reduction-sensitivity was introduced by the disulfide bridge (-S−S-) that links the hydrophobic block PEG and hydrophobic block PAChol. Our group has studied, for several years, liquid crystal (LC) polymersomes made of block copolymers in which the hydrophobic block is a LC polymer.35 These LC polymersomes are very stable because of the physical cross-linking of LC nature in the membrane and can be preserved well at room temperature for several years without further morphological modification.36−40 The system made of PEG-b-PAChol36−40 with hydrophobic block bearing cholesterol pendent groups is of special interest for possible biorelated applications, because of the biocompatibility of cholesterol moieties. PAChol is a smectic liquid crystal (LC) polyacrylate (PA) composed of cholesterol monomers (Chol).37 This thermotropic smectic phase (with smecticisotropic transition temperature around 150 °C) in lyotropic bilayer plays the role of physical cross-linking.41 Here PEG-SSPAChol is a homologue of PEG-b-PAChol, but with cleavable disulfide linkage between PEG and PAChol blocks. We described a versatile method based on atom transfer radical polymerization (ATRP) to synthesize the reductionsensitive amphiphilic block copolymers. The copolymers were obtained by polymerizing the LC acrylate monomers using disulfide-containing PEG as ATRP macroinitiator (see Schemes 1 and 2). The reductive cleavage of the disulfide bridge in the block copolymers was first evidenced when copolymers were solubilized in organic solution. The partial destruction of PEGSS-PAChol polymersomes in the presence of a reducing agent was then demonstrated by cryo-electron microscopy. Finally, the calcein release from PEG-SS-PAChol polymersomes triggered by GSH was observed both in PBS suspension and in vitro inside the macrophage cells. High GSH concentration (e.g., ≥35 mM in PBS) and long incubation time (in the order of hours) were necessary to get significant calcein release, showing the high stability of PEG-SS-PAChol polymersomes.
2. EXPERIMENTAL SECTION Materials. α-Methoxy-ω-hydroxy poly(ethylene glycol) (MeOPEG45-OH, Mn = 2000 Da), 3-mercaptopropionic acid (⩾99%, Hafnium(IV) chloride tetrahydrofuran complex (HfCl4·2THF, 98%), 1,3-dicyclohexylcarbodiimide (DCC, 99%), 4-dimethylaminopyridine (DMAP, >99%), CuIBr (98%), N,N′,N′,N″,N″-pentamethyldiethyleC
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1H, −CH, pyridine), 4.42 (t, 2H, −CH2−OCO−), 3.08 (t, 2H, −S−CH2−), 1.94 (s, 6H,−CH3). MeO-PEG45-SH (3). The thiol-functionalized poly(ethylene glycol), MeO-PEG45-SH, was synthesized from α-methoxy-ω-hydroxy poly(ethylene glycol) (MeO-PEG45-OH, Mn = 2000) and 3-mercaptopropionic acid (MPA), as reported in the literature.44 In a typical experiment, MeO-PEG45-OH (10 g, 5 mmol) and MPA (2.65 g, 25 mmol) were stirred at 50 °C under argon until a solution was formed, and then toluene (20 mL) along with HfCl4·2THF (116 mg, 0.25 mmol) were added. The mixture was refluxed at 130 °C under argon for 24 h. Toluene was removed under reduced pressure. The MeOPEG45-SH was solubilized CH2Cl2 and reprecipitated into diethyl ether trice. Yield: 95%. 1H NMR (300 MHz, CDCl3; see Figure SI-2): δ 4.27 (t, 2H, −CH2−OCO−), 3.63 (m, 180H, −O−CH2−CH2− O−), 3.37 (s, 3H, −O−CH3), 2.76 (t, 2H, −CH2−SH), 2.68 (t, 2H, −O−CO−CH2−). ATRP Macroinitiator with Disulfide Bond PEG-SS-Br. 2-(2Pyridinyldithio)ethyl 2-bromo-2-methylpropionate (2; 30.6 mg, 0.09 mmol) and acetic acid (16.78 mg, 0.28 mmol) were dissolved in 1 mL of dry CH2Cl2. The solution was then added dropwise into 3 mL CH2Cl2 solution containing MeO-PEG45-SH (3; 100 mg, 0.045 mmol) during a period of 1 h. The mixture was allowed to stir for other 24 h at room temperature. The reaction mixture was concentrated by rotary evaporation, redissolved in 1 mL of CH2Cl2 and then precipitated from 50 mL diethyl ether. The precipitation process was repeated twice to remove the compound 2 in excess. The final ATRP macroinitiator MeO-PEG45-SS-Br is a white solid (97 mg, yield: 90%). 1 H NMR (300 MHz, CDCl3; Figure 1): δ 4.43 (t, 2H, −S−CH2−
by dissolution of the final mixture in chloroform and precipitation in methanol trice. The polymer was dried under vacuum for 48 h (150 mg, yield: 75%). 1H NMR (300 MHz, CDCl3): δ 0.67−2.42 (m, −CH3, −CH(CH3)−, −CH−, −CH2−), 2.76 (t, 2H, −O−CO− CH2−), 2.90 (t, 4H, −CH2−S−S−CH2−), 3.37 (s, 3H, −O−CH3), 3.65 (s, 180H, −O−CH2−CH2−O−), 4.30 (m, (4n+2)H, −OCO− CH2−CH2−OCOO−, −S−CH2−CH2−O−CO−), 4.45 (m, nH, −CHO−), 5.38 (m, nH, −CCH−CH2−). (n is the degree of polymerization of the LC block; see Figure 2 for 1H NMR spectra of PEG-SS-PBA and PEG-SS-PAChol.) The degrees of polymerization of the hydrophobic block (DPPBA or DPPAChol) were calculated according to eq 1 for PEG-SS-PBA, or eq 2 for PEG-SS-PAChol. See Table 1 for detailed information.
DPBA = 1.5 × I4.03/I3.37
(1)
DPLCblock = 3 × I5.38/I3.37
(2)
In eq 1, I4.03 is the integration value of the methylene proton signal (c) in the butyl acrylate units at 4.03 ppm and I3.37 the integration value of the methoxy proton signal (a) in the PEG end group at 3.37 ppm. In eq 2, I5.38 is the integration value of the vinylic proton signal (g) in the cholesteryl group at 5.38 ppm and I3.37 the integration value of the methoxy proton signal (a) in the PEG end group at 3.37 ppm. The copolymer PEG45-b-PAChol10 without the disulfide bridge, which was used as a control copolymer, was synthesized according to previously published procedure.36 See Table 1 for its molecular characterization. Polymer Characterization. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra were recorded using a Bruker HW300 MHz spectrometer. CDCl3 was used as solvent and tetramethylsilane (TMS) as the internal chemical shift reference. Size Exclusion Chromatography (SEC). Molecular weights of the copolymers were characterized by SEC at 40 °C using Waters Styragel HR 5E columns (×2), a Waters 4110 differential refractometer and a Waters 486 UV detector. THF was used as the eluent at 1 mL/min. Molecular weight distributions (Mw/Mn) of the diblock copolymers were evaluated by SEC calibrated with polystyrene (PS) standards (Polymer Laboratory PS calibration kit).45 Polymer Degradation Analyses. The analyses of disulfide bond cleavage in block copolymers were made by recording the size exclusion chromatograms before and after the addition of dithiothreitol (DTT) as the reducing agent. Typically, 10 mg of disulfide-containing copolymers were dissolved in 1 mL THF, then 1.54 mg of DTT (10 equiv mole of disulfide bond) was added into the polymer solution. After 1 h, 24 or 48 h of stirring, a 100 μL of the solution was directly injected to the SEC system for analysis. Polymersome Formation and Characterization. Polymersome Formation. The polymersomes were prepared following a classical nanoprecipitation procedure as reported previously.37 Typically, the copolymer was first dissolved in 1 mL of dioxane (at 0.5, 0.2, or 0.1 wt %) and 1 mL of deionized milli-Q water was injected slowly to the organic solution under mild shaking (2−3 μL of water per minute to 1 mL of polymer solution). The whole process of nanoprecipitation was carried out at 25 °C. The turbid mixtures were then dialyzed against water for 3 days to remove the dioxane using a Spectra/Por regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 3500. The final volume of the polymersome suspension was then adjusted to 2 mL. For the dye-encapsulated polymersomes, deionized water was replaced by PBS (10 mM phosphate buffer saline, pH 7.4) during the preparation. Polymersome Degradation under Reductive Condition. A total of 0.5 mL of DTT aqueous solution (2 mM) was added, under slight hand shaking, into 0.4 mL of polymersome suspension in water (prepared with 0.5 wt % of initial polymer concentration in dioxane). DTT was again of 10 equiv mole of disulfide bond. Then the mixture was kept still without any stirring for 24 h. The morphological changes of polymersomes were finally analyzed by cryo-TEM. Transmission Electron Microscopy (TEM). The morphological analysis of polymersomes was performed by TEM on samples stained by uranyl acetate (2 wt %) or by cryo-TEM on sample deposited onto
Figure 1. 1H NMR (300 MHz) spectrum of ATRP macroinitiator PEG-SS-Br (Mn,PEG = 2000) in CDCl3. CH2−OCO−), 4.27 (t, 2H, −O−CH2−CH2−OCO−), 3.65(m, 180H, −O−CH2−CH2−O−), 3.38 (s, 3H, −O−CH3), 2.96 (t, 4H, −CH2− S−S−CH2−), 2.79 (t, 2H, −O−CO−CH2−), 1.94 (s, 6H, −CH3). As all polymers and copolymers in this work contained methoxy terminated PEG45 (Mn = 2000 Da), the polymer names will be simplified as PEG-SS-Br for ATRP macroinitiator, and PEG-SS-PBA and PEG-SS-PAChol for block copolymers in the following. Synthesis of Diblock Copolymer with Disulfide Bond between the Two Blocks by ATRP Polymerization. In this work, we synthesized two kinds of copolymers, PEG-SS-PBA and PEG-SS-PAChol, from the ATRP PEG-macroinitiator (PEG-SS-Br) containing disulfide bond by typical ATRP polymerization. Taking PEG-SS-PAChol, for example, PEG-SS-Br (50 mg, 0.02 mmol), CuIBr (7.5 mg, 0.05 mmol), cholesteryl acryloyoxy ethyl carbonate (AChol, LC monomer; 150 mg, 1.72 mmol), anhydrous toluene (0.5 mL), and PMDETA (10.5 μL, 0.05 mmol), were added into a Schlenk-type flask. The flask was degassed and exchanged with argon via three freeze−thaw cycles. The reaction mixture was heated at 80 °C for 18 h. The mixture was then diluted with chloroform and passed through a short basic aluminum oxide column to remove Cu salts. The resulting solution was concentrated by rotary evaporation. The copolymer was recovered D
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Figure 2. 1H NMR (300 MHz) spectra of diblock copolymers: (a) PEG-SS-PBA and (b) PEG-SS-PAChol in CDCl3. pixels CCD camera. Calibration was performed with a 2D crystal of purple membrane leading to 0.385 nm/pixel at 45000 magnifications. Nanoparticle Tracking Analysis (NTA). The sizes and size distributions of polymersomes were measured by NTA, using a NanoSight LM14 (NanoSight, United Kingdom) equipped with a 532 nm laser. The sample at right dilutions (in the order of 108−109 particles per mL) was loaded in the sample chamber with a sterile syringe. The NTA 2.2 analytical software was used for capturing and analyzing the data. The samples were measured for 60 s with manual shutter and gain adjustments. All measurements were performed in triplicate at 25 °C. Dye Encapsulation and Reduction-Responsive Release. Encapsulation of Dyes in Polymersomes. The hydrophilic calcein was first dissolved in PBS (v0 = 1 mL, c0‑cal = 0.02 M). This solution was added slowly to the dioxane solution of copolymers (1 mL, 0.1 wt %, cw‑polym = 1 g·L−1) to form the vesicles loaded with calcein. The free dyes and dioxane were removed by 4 days’ dialysis (MWCO 3500) against PBS (changing the buffer twice per day), until the intensity of fluorescence of PBS outside the dialysis tube to be very low and stable (1 μm). When we decreased the initial polymer concentration from 0.5 wt % and 0.2 wt % to 0.1 wt %, the situation was improved. Figure 5b shows the vesicle morphologies of PEG45SS-PAChol16 become quite homogeneous and the average diameter is
