pH-Sensitive Polymeric Vesicles from Coassembly of Amphiphilic

Jul 30, 2012 - Herein we report a coassembly method toward the preparation of pH-sensitive polymeric vesicular aggregates, using comb-shaped ...
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pH-Sensitive Polymeric Vesicles from Coassembly of Amphiphilic Cholate Grafted Poly(L‑lysine) and Acid-Cleavable Polymer−Drug Conjugate Lijun Zhu, Lingling Zhao, Xiaozhong Qu,* and Zhenzhong Yang* State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China

ABSTRACT: Herein we report a coassembly method toward the preparation of pH-sensitive polymeric vesicular aggregates, using comb-shaped amphiphilic polymers, i.e., cholate grafted poly(L-lysine) (PLL-CA), with an amphiphilic poly(ethylene glycol)−doxorubicin conjugate (PEG-DOX). Because the drug conjugate includes a low-pH labile bond, i.e., benzoic imine, the permeability of the coassembled polymeric vesicles can be tuned by changing either the PLL-CA/PEG-DOX weight ratio or the environmental pH from 7.4 to 6.5. Furthermore, at lower pH values such as 5.0, the vesicles destabilize. The pH sensitivity leads to enhanced uptake of the vesicles by cancer cells (MCF-7) under a condition close to the extracellular environment of solid tumor (pH = 6.5) and subsequent efficient endosome escape after the endocytosis.

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INTRODUCTION

vesicle formation for the amphiphilic polymers by increasing the hydrophobic interaction of the system.27 Meanwhile, the functionalization of polymeric vesicles with “stealthy” and/or stimuli responsive feature is an important step to fabricate biofunctional materials, for their applications such as in biomedical field.13,28 So far, polymeric vesicles with pH, temperature, redox, and irradiation sensitive shells have been reported;29−36 for example, those with a tailored pH sensitivity between 5.0 and 7.4, corresponding to endosomal pH and physiological pH, are promising for intracellular drug delivery.37 However, challenge remains for developing systems that enable to respond tinier pH changes within biologically relevant conditions, e.g., 6.5−7.4, which corresponds to the pH variation caused by the enhancement of anaerobic glycolysis in solid tumor tissues,38,39 because it will be especially interesting if the polymeric vesicles are sensitive to the extracellular stimuli and hence become tumor site-specific. In the present work, we studied on a novel way for engineering polymeric vesicles based on comb-shaped amphiphilic polymers, using cholate grafted poly(L-lysine) (PLL-CA) as an example, by coassembling with an amphiphilic polymer−drug conjugate, i.e., poly(ethylene glycol)−doxorubicin (PEG-DOX). Coassembly of polymers has been proven as an efficient way for creating multifunctional systems.40−45

The construction of vesicular structured polymeric aggregates has attracted considerable attention because it helps the fundamental understanding of self-assembly rationale of amphiphilic polymers and the exploration of new ways for constructing biomimic structures.1−8 According to the works of Israelachvili,9,10 the increase of relative size of the hydrophobic moiety in amphiphiles results in structural transformation of their self-assemblies in aqueous solution from spherical micelles to rodlike micelles and eventually to bilayered vesicles. In polymer science, this has been experimentally evidenced from block copolymers,11,12 including AB type diblock9,13−15 and ABA or ABC type triblock copolymers with defined chain length.16−18 Comparably, the reports on vesicle aggregation from other types of amphiphilic polymers, e.g., graft copolymers or comb-shaped polymers (hydrophilic backbone with hydrophobic pendants), are rear.19−21 Nevertheless, previous investigations have shown that with proper substitution levels water-soluble polymers bearing hydrophobic pendant groups, e.g., amphiphilic poly(L-lysine) (PLL) and polyethylenimine (PEI) derivatives, can form various aggregates rather than micelles, such as vesicular and disk-shaped assemblies.22−26 However, among those regimes, the window for forming nonmicellar structures, i.e., the range of hydrophilic to hydrophobic segmental ratio, is relatively narrow.22,25,26 Therefore, the addition of hydrophobic molecules, e.g., cholesterol, has been applied to improve the tendency of © 2012 American Chemical Society

Received: April 18, 2012 Revised: June 14, 2012 Published: July 30, 2012 11988

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slowly evaporated with a rotary evaporator under vacuum to form a thin film and further dried for 3−6 h to ensure a complete removal of residual organic solvent. Subsequently, phosphate buffer (PB, 10 mM, pH 7.4) was added into the flask to get dispersion at a PLL-CA concentration of 0.5 mg/mL. The dispersion was sonicated in a bath sonicator for 1 h at 30 °C and then passed through a 0.80 μm nylon filter and further stirred for 12 h at room temperature. The dispersions of PLL-CA/PEG-DOX coassemblies were kept at 4 °C prior to further characterizations. Characterizations. 1H NMR analysis was performed on polymer solutions using a Bruker Advance 400 MHz NMR spectrometer (Bruker Instrument, Switzerland). Elemental analysis was carried out using a Flash EA 1112 NC Analyzer (Thermo Electron Corp., Milan, Italy) to determine the C, H, and N content in the polymers. The critical aggregation concentration (CAC) of PEG-DOX and PLL-CA in aqueous media was determined by fluorometry using pyrene as a probe. Aliquots of pyrene stock solution (1.0 × 10−5 M) in acetone were pipetted into 10 mL glass vials, and the solvent was evaporated under a nitrogen gas stream. The polymer solutions with a series of concentrations were then added into the vials. Final concentration of pyrene was 1.0 × 10−6 M. The solutions were allowed to equilibrate for 24 h at 37 °C. Pyrene emission fluorescence at 372 and 383 nm (I372, I383) were monitored using a fluorescence spectrophotometer (Cary Eclipse, Varian) with an excitation wavelength of 335 nm. CAC was estimated as the first inflection point on the plot of the intensity ratio of I372/I383 against the polymer concentration. Transmission electron microscopy (TEM) was carried out on a JEM-1011 microscope at an operating voltage of 100 kV. The sample dispersions were dropped onto carbon-coated copper grids, followed by staining using 0.5% phosphotungstic acid solution (pH = 7.4), and then air-dried. The size distribution and zeta potential of the selfassemblies were performed in aqueous dispersion using a Zetasizer (Nano Series, Malvern Instruments, UK) at 25 °C and repeated for three times. For investigating the pH sensitivity of the samples, a small amount of 1 M HCl and 1 M NaOH were used to adjust the pH of the dispersions. Calcein Leakage. A dry film of PLL-CA/PEG-DOX mixture, prepared as above-described, was hydrated using phosphate buffer (10 mM, pH 7.4) containing 50 μg/mL calcein dye. Free calcein was removed by dialysis (MWCO 3.5 kDa, 4 changes within 12 h), and pH of the resultant solution was adjusted to desired value by adding HCl (1 M) and then diluted for 3 times in volume with buffer solution at the same pH. A reference solution was also prepared by diluting with buffer containing 0.1% (w/v) Triton X-100 in order to disrupt the structure of polymeric vesicles. The fluorescent intensity of calcein in those solutions was then monitored using a fluorescence spectrophotometer at 37 °C with an excitation and an emission wavelength of 488 and 517 nm, respectively. The percent leakage was defined as leakage (%) = 100 × (Rt − R0)/(1 − R0), where R0 and Rt are ratio of the initial and intermediate fluorescence intensity of the sample solution to the intensity of the reference solution.13,51 In-Vitro FITC-Dextran Release. FITC-dextran (MW = 4 kDa) was loaded into PLL-CA/PEG-DOX vesicles by hydrating the polymer film with FITC-dextran containing phosphate buffer (10 mM, pH 7.4). Free dextran was removed by dialysis, and the resultant solution was freeze-dried. The dextran encapsulation efficiency and loading content were determined by fluorometry on the redispersed FITC-dextranloaded vesicle dispersion in phosphate buffer supplemented with 2% (w/v) Triton X-100. Release of FITC-dextran from the polymeric vesicles was investigated using dialysis method (MWCO 14 kDa) at 37 °C with 2 mL of FITC-dextran-loaded vesicle dispersion against 20 mL of PB (10 mM, pH 7.4 or 6.5) or citrate-phosphate buffer (10 mM, pH 5.0). At desired time intervals, 2 mL of release media was taken out and replenished with an equal volume of fresh media. The amount of released dextran was determined by fluorescence measurements (excitation 488 nm, emission 515 nm). The release experiments were conducted in triplicate. Cellular Uptake. MCF-7 cells (European Collection of Cell Cultures, UK) were cultured using Dulbecco’s Modified Eagle

However, few gains have been reported on the morphological control of aggregates by this strategy, especially aiming to the production of vesicular nanostructures. Herein, we can not only show the vesicle formation from mixing of two micelle-forming polymers, i.e., PLL-CA and PEG-DOX, but also demonstrate that the size and wall thickness of the vesicles are changed by varying the PLL-CA/PEG-DOX weight ratio. Doxorubicin (DOX) is a clinical-used antitumor drug; thus, the use of PEGylated DOX conjugate as one of the building blocks of the coassemblies will favor the delivery of the drug together with other therapeutics, since polymeric vesicles are an ideal carrier for both hydrophobic and hydrophilic encapsulates.7 Besides, we used an acid labile covalent linkage, i.e., benzoic imine bond, for the PEG-DOX conjugation. The characteristics of this linkage have been investigated in our previous works, which shows desired stability at physiological pH (7.4) and lability at pH 6.8 and more acidic conditions.46−49 Therefore, the stability, permeability, and cell uptake of the coassembled PLL-CA/PEG-DOX vesicles are responsive within the range of physiological pH, extracellular pH of tumor, and endosome/ lysosome pH, as described below.

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EXPERIMENTAL SECTION

Materials. Poly(L-lysine) hydrobromide (MW = 15−30 kDa), cholic acid (CA), 4-(dimethylamino)pyridine (DMAP), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), calcein, FITC-dextran (MW = 4 kDa, FITC:glucose = 1:250 mol/mol), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Benzaldehydeterminated methoxy poly(ethylene glycol) (PEG-CHO, Mn of PEG: 2 kDa) was synthesized according to ref 46. 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Roche (Basel, Switzerland). LysoTracker Green DND-26 was purchased from Invitrogen Co. (Eugene, OR). Doxorubicin hydrochloride (DOX) was supplied by Huafeng Lianbo Technology Co. (Beijing, China). Solvents and other compounds were obtained from Beijing Chemical Reagents Co., China. Synthesis of PEG-DOX. Conjugation of DOX to PEG-CHO was accomplished through Schiff’s reaction between the amino group of DOX and the benzaldehyde end group of PEG-CHO. PEG-CHO (140 mg) and an excess amount of DOX (80 mg) were dissolved in 50 mL of methanol. The solution was protected from light and stirred at room temperature for 24 h. The resultant dark orange solution was concentrated to ca. 3 mL and applied to an open-column filled with Sephadex LH-20 gel (GE Healthcare, Sweden) and eluted using methanol to separate PEG-DOX from the unbound residual DOX. The eluate was evaporated to gain red color product (145 mg, yield 83%). The product was evaluated using HPLC to confirm absence of free DOX and 1H NMR to check the chemical structure. Synthesis of Cholate Grafted Poly(L-lysine) (PLL-CA). Cholate grafted poly(L-lysine) (PLL-CA) was synthesized via a two-step procedure.46 CA was first activated by coupling with N-hydroxysuccinimide in the presence of DCC. Then a desired amount of the cholic acid succinimide ester (0.30−2.0 mmol) in 10 mL of DMSO was added dropwise under stirring to 10 mL of anhydrous DMSO containing poly(L-lysine) hydrobromide (1 g, 4.8 mmol of lysine segment) and TEA (1.8 mL). The reaction mixture was stirred for 24 h at room temperature, dialyzed (MWCO 14 kDa) against 75% v/v ethanol for 3 days (2 L with four changes per day), and subsequently against distilled water for 2 days (2 L with four changes per day). The dialysate was freeze-dried to gain fiber-like PLL-CA in 80−90% yield (calculated with respect to the starting polymer weight). The CA substitution level was determined by elemental analysis, and the chemical composition was examined using 1H NMR.26,50 Preparation of PLL-CA/PEG-DOX Coassemblies. The mixture of PLL-CA and PEG-DOX with varied weight ratio was dissolved in a 25 mL round-bottomed flask containing methanol. The solution was 11989

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Figure 1. 1H NMR spectra of CA grafted PLL (PLL-CA25) (a) and PEG-DOX (b) in DMSO-d6. Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 mg/mL) and plated on microscope slides in a culture dish at 5 × 104 cells/well. After 24 h, the culture medium was replaced with the medium dispersion of FITC-dextran loaded PLL-CA/PEG-DOX vesicles at the desired pH (7.4 or 6.5). After incubation at 37 °C and 5% CO2 for predetermined times, the medium was removed, and the cells on microscope plates were washed three times with PBS. The cells were then fixed with 4% formaldehyde for 20 min and further washed with PBS for 3 times. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min followed by an other wash with PBS for 3 times. In a separate experiment, in order to observe the endosome/lysosome compartments formed in the cells, empty PLL-CA/PEG-DOX vesicles and pH-insensitive vesicles formed by PLL-CA and NaBH4 reduced PEGDOX were incubated with the cells and then with LysoTracker Green DND-26 (75 nM) for 15 min at the end of incubation. The cells were then washed three times with ice-cold PBS and stored at −4 °C. Fluorescence images of cells were observed using a FV 1000-IX81 confocal laser scanning microscope (CLSM, Olympus, Japan), with an

excitation wavelength of 488 nm and emission wavelength of 560−620 nm for DOX and 500−530 nm for FITC and LysoTracker.

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RESULTS AND DISCUSSION Synthesis of PLL-CA and PEG-DOX. The synthesis of PLL-CA has been previously reported. The pendant grafting degree of PLL-CA was determined by elemental analysis and the structure was confirmed by 1H NMR (Figure 1a).26,50 As listed in Table 1, CA grafting level was adjusted within 7−38 mol %, calculated as mole of CA molecules per 100 lysine segments, by changing the PLL/CA feed ratio. PEG-DOX was prepared via Schiff’s reaction between benzaldehyde-terminated PEG and DOX hydrochloride in methanol. The molecular structure of the PEG-DOX was examined using 1H NMR in DMSO-d6 (Figure 1b). The appearance of proton peak at 8.4 ppm indicates the formation of imine bond, and the absence of the aldehyde proton peak (10 ppm) proves that free PEGCHO is not present in the purified PEG-DOX.48 Meanwhile, 11990

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aggregates are observed by TEM as shown in Figure 3, and the size is around 56 nm measured by DLS, much larger than the micelles formed from low molecular weight surfactants, and thus implies a multicore structure of the aggregates. The PLLCAs also self-assemble into micelles in PB buffer whenever the polymer has a CA grafting level within 7−25%, as indicated by the mean hydrodynamic diameter of ∼10 nm detected by DLS (Table 1). Besides, some larger aggregates are also observed in the TEM images (Figure 3). In contrast, vesicular structure is formed by the PLL-CA38 (TEM, Figure 3) with a mean size of 158 nm (DLS). This structural evolution, i.e., from micelle to vesicle with the increase of grafting pendants, is consistent with previous observations on cetylated or palmitoylated linear PEI and PLL, which is governed by the hydrophobic−hydrophilic balance of the graft copolymers.22,23,26 It is also noticed that the CAC of the micelle-forming PLL-CAs decreases with the increase of CA grafting level (Table 1). However, for the PLLCA38, which forms the vesicular structure, the CAC abruptly increases due to less entropic gain from the aggregation.25 Further increasing the hydrophobicity of the PLL-CA such as to a CA grafting of 50% will result in the formation of solid nanoparticles.26 The coassembly of micelle-forming PLL-CAs (PLL-CA7, PLL-CA15, and PLL-CA25) with PEG-DOX leads to the formation of vesicular aggregates, whereas for the vesicleforming PLL-CA38, solid particles are generated at a PLL-CA/ PEG-DOX ratio of 1:2 w/w, as revealed by TEM (Figure 3). The influence of PLL-CA/PEG-DOX ratio on the structural transition of PLL-CA25 was further investigated. TEM observation shows vesicle dominated morphology at PLLCA25/PEG-DOX weight ratio of 4:1, 1:1, and 1:2. However, the wall of the vesicles becomes thicker with the increase of PEG-DOX content. And finally condensed nanoparticles are formed at a PLL-CA25/PEG-DOX weight ratio of 1:4 (Figure 3). The size variation of the coassemblies upon the change of PLL-CA25/PEG-DOX ratio is plotted in Figure 4a. At low PEG-DOX contents, i.e., PLL-CA25/PEG-DOX = 4:1 and 1:1 (w/w), multiple populations of aggregates are described by the DLS diagrams whereas only one population is viewed at higher PEG-DOX contents, i.e., PLL-CA25/PEG-DOX = 1:2 and 1:4 (w/w), irrespective of the morphological change of these two samples from vesicle to solid particle as shown in the TEM images (Figure 3). A similar phenomenon is noticed from the size diagram of PEG-DOX/PLL-CA mixtures with different PLL-CA, in which the PLL-CA7/PEG-DOX displays two peaks

Table 1. Synthesis and Characterization of PLL-CA and PEG-DOX

PEG-DOX PLL-CA7 PLL-CA15 PLL-CA25 PLL-CA38

CA grafting level (mol %)a

CAC (mg/mL)b

7 15 25 38

0.16 0.084 0.034 0.01 0.25

size (nm)c 55.5 12.0 9.1 9.3 157.7

± ± ± ± ±

2.6 1.7 0.7 0.1 0.3

morphology micelled micelled,e micelled,e micelled,e vesicled

a

Determined by elemental analysis. bDetermined by I372/I383 ratio (I1/ I3) of pyrene fluorescent emission as a function of polymer concentration. cMean value measured using DLS. dObserved using TEM. eAccording to the size data.

the stability and acid labile ability of the PEG-DOX was also investigated using 1H NMR in deuterated water. No aldehyde proton is observed at pH 7.4, whereas the cleavage of benzoic imine bond at weak acidic pHs, e.g. 6.5 and 5.0, can be identified via the recovery of aldehyde proton peak at different intensity (Figure 2).47,52,53

Figure 2. Enlarged 1H NMR spectra of PEG-DOX dispersed in deuterated water (D2O) at different pHs. The samples were equilibrated for 24 h before the scans.

Self-Assembly and Coassembly of PLL-CA and PEGDOX. Both PEG-DOX and PLL-CA self-assemble in PB buffer (pH = 7.4) due to their amphiphilic nature. CAC of PEG-DOX is 0.16 mg/mL, determined using fluorescence spectrophotometer with pyrene as the probe (Table 1). Spherical

Figure 3. TEM images of PEG-DOX aggregates, PLL-CA aggregates, and the coassemblies of PLL-CA and PEG-DOX in PB buffer (pH = 7.4). 11991

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Figure 5. Zeta potential of the aggregates of PLL-CA25, PEG-DOX, and PLL-CA25/PEG-DOX coassemblies (1:2 w/w) as a function of pH.

PLL-CA micelles. In contrast, we can hypothesize that the PEG-DOX molecules would feed only part of the PLL-CA micelles to form vesicles while the other micelles are fasting. However, at high PEG-DOX/PLL-CA ratios, the PEG-DOX molecules will be incorporated by the PLL-CA/PEG-DOX vesicles since no extra peak, corresponding to the PEG-DOX micelles, is observed in the DLS diagrams. Instead, as revealed in Figure 3, the wall of the vesicles becomes thicker due to the fill of PEG-DOX. It should be pointed out that the micelle-tovesicle transition process may need further investigation. For example, the third population in aggregates of PLL-CA25/ PEG-DOX at the weight ratio of 1:1 has not been clarified (Figure 4a). Besides, the presented data are not sufficient to identify whether the vesicles contain a bilayer or multilayer structure especially for those with thicker walls. Permeability of the PLL-CA/PEG-DOX Vesicles. The permeability of the PLL-CA/PEG-DOX vesicles is found influenced by two variables, i.e., the wall thickness and environmental pH, which is demonstrated by the calcein leakage and the release behavior of FITC labeled dextran. The carrier capacity of the vesicles is first confirmed using CLSM on FITC-dextran loaded PLL-CA25/PEG-DOX dispersion (1:2 w/w). As shown in Figure 6, ring-shaped DOX fluorescence is observed as an indication of vesicle structure of the coassemblies. And the green fluorescence is mainly located inside the cavity which proves the encapsulation of FITCdextran is in the inner aqueous phase of the vesicles. Calcein leakage is frequently used to investigate the permeability of liposomes as well as polymeric vesicles.51 Figure 7a plots the leakage of calcein dye against time from the PLL-CA25/PEG-DOX vesicles at pH 7.4 with varied PLL-CA/ PEG-DOX ratio. As expected, the permeability of the vesicles becomes weaker with the increase of PEG-DOX content due to the thickening of the shell. However, the permeability of the PLL-CA/PEG-DOX vesicles with the change of solution pH is novel (Figures 7b and 8). As shown in Figure 7b, the release rate of calcein becomes slower at pH 6.5 rather than that at 7.4, while a burst release is observed at lower pH, i.e., 5.0. The results indicate that permeability of the PLL-CA25/PEG-DOX (1:2 w/w) has a two-stage response to the pH changes within the narrow interval. In the first pH drop from 7.4 to 6.5, TEM images can prove that the vesicular structure of the PLL-CA25/PEG-DOX is well preserved under the mild acidic condition (Figure 9a, inset); besides, DLS also reveals no obvious change of particle size within a period of 4 h (Figure 9b). However, the zeta

Figure 4. DLS diagrams of PLL-CA/PEG-DOX coassemblies at different PLL-CA25 to PEG-DOX weight ratios (a) and with various PLL-CAs at a fixed PLL-CA/PEG-DOX ratio of 1:2 (w/w) (b). The concentration of PLL-CA in the dispersions was 0.5 mg/mL, and the dispersions were equilibrated for 4 h before the measurements.

(Figure 4b). By comparing the aggregation size of the parent polymers (Table 1), the peaks at ∼10 and >200 nm in the DLS diagrams in Figure 4 are characteristic to the size of PLL-CA micelle and the PLL-CA/PEG-DOX coassemblies, respectively. It is considered that the PLL-CA micelles are more stable than the PEG-DOX, since the critical aggregation concentration of the former is lower (Table 1). However, for micelles formed from a comb-shaped polymer, especially containing a polyelectrolyte backbone, the pendants that form hydrophobic microdomains are more loosely packed than that formed by traditional block copolymers and thus leave larger voids at the interface. Therefore, being a linear amphiphile, the PEG-DOX molecules could easily submerge into the PLL-CA micelles through the voids to reduce the interface free energy of the system rather than to form less stable PEG-DOX micelles. This mechanism is different from the induced vesicle formation via increasing the hydrophobic interaction of amphiphilic polymers by the incorporation of hydrophobic or counterionic hydrophilic molecules54−56 because in this work, the amphiphilic nature of the PEG-DOX is determinative during the micelle-tovesicle transformation. The mixed aggregates exhibit a neutral zeta potential at pH 7.4, as demonstrated in Figure 5, which proves that the PEG moieties are distributed on the exterior surface of the polymeric vesicles. Besides, the DLS data imply that the formation of PLL-CA/PEG-DOX vesicles may be thermodynamically driven. The presence of PLL-CA micelles with the coassembled vesicles at lower PEG-DOX fractions or with PLL-CA containing less hydrophobic pendants indicates that the PEG-DOX molecules are not equally taken up by the 11992

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Figure 8. Cumulative release of FITC-dextran from the PLL-CA25/ PEG-DOX (1:2 w/w) vesicles at different pHs.

affected. While the detached PEG moiety diffuses into the aqueous phase, the DOX molecules leave in the vesicle membrane and enhance the hydrophobic interaction, which led to a more compact packing of molecules in the shell. However, the release of FITC-dextran is less influenced by the pH change from 7.4 to 6.5 (Figure 8). This is attributed to larger size of the macromolecule which hampers its penetration across the vesicle membrane, implying a filtration effect of the vesicle to encapsulants. By further decreasing the solution pH to 5.0, the polymeric vesicles destabilize due to the complete hydrolysis of the PEGDOX (Figure 2) which disrupts the hydrophilic−hydrophobic balance in the coassemblies.47 TEM observation reveals discontinuous and fractured morphologies from the PLLCA25/PEG-DOX solution at pH of 5.5 and 4.0, respectively. Meanwhile, the dispersions become unstable at these pH values as the DLS appears two peaks and shifts with time in a 4 h period (Figure 9). The dissociation of the vesicular structure causes the burst release of calcein dye as well as the FITCdextran at pH 5.0 (Figures 7b and 8). The residue PLL-CA will aggregate into micelles and solubilize part of DOX molecules in case the polymer concentration is above the CAC, which can explain the multiple peaks seen in the DLS (pH 5.5 and 4.0) in Figure 9. Cell Uptake and Intracellular Delivery. The cell uptake of FITC-dextran loaded PLL-CA25/PEG-DOX (1:2 w/w) vesicles was investigated by CLSM on MCF-7 cell line at two representative pH values, i.e., 7.4 and 6.5. As shown in Figure 10a, after incubation at pH 7.4 for 15 min, only weak fluorescence can be observed on the cell membranes. However, strong fluorescence of both the carrier (DOX moieties) and the encapsulated molecules (FITC-dextran) is visualized in the cytosol of the cells when incubated at pH 6.5. With a prolonged incubation, e.g., 1 h, the fluorescence distribution and intensity in the MCF-7 cells still show a significant dependence on the pH of the media (Figure 10b). In comparison with a cytoplasm distribution at physiological pH (7.4), major DOX and FITC fluorescence is already in the nucleus region of the cells in the acidic media (pH 6.5). Considering that the vesicular structure is not damaged in the PLL-CA25/PEG-DOX coassemblies at pH 6.5, the favorable cell uptake of the PLL-CA25/PEG-DOX at this pH value is attributed to the change of surface property;47 that is, the vesicile surface becomes positively charged due to the detachment of PEG, which results in stronger electrostatic interaction of the vesicles with the cell membrane and thus accelerates the endocytosis.57 It is known

Figure 6. CLSM observation of FITC-dextran (green) loaded PLLCA25/PEG-DOX (1:2 w/w) vesicles (red). Inset is the image of empty PLL-CA25/PEG-DOX vesicles.

Figure 7. Dependence of calcein leakage from the PLL-CA25/PEGDOX vesicles on (a) PLL-CA/PEG-DOX ratio and (b) solution pH. Concentration of PLL-CA was fixed at 0.5 mg/mL for all tests. For testing the pH dependence (b), the PLL-CA/PEG-DOX weight ratio was fixed at 1:2 (w/w).

potential of the vesicles shows a remarkable increase with the decrease of pH (Figure 5). The finding suggests the detachment of PEG chains from the vesicle surface at pH 6.5. Nevertheless, the cleavage of PEG-DOX in this pH condition should be in partial since the vesicle architecture was not 11993

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Figure 9. DLS diagrams of the PLL-CA25/PEG-DOX (1:2 w/w) vesicles in PB buffer at different pHs for 1 h (a) and 4 h (b). Inset: TEM images of PLL-CA25/PEG-DOX (1:2 w/w) dispersed in buffer at the corresponding pHs for 1 h.

membrane disruptive.46 To evidence this, LysoTracker Green was used to stain the acidic compartments in the MCF-7 cells after incubated with the PLL-CA25/PEG-DOX vesicles at physiological pH, by comparing with pH-insensitive vesicles. The pH-insensitive vesicle was synthesized by a similar coassembly procedure of PLL-CA25 with the PEGylated DOX conjugate in which the imine bond was reduced using NaBH4. Figure 11 shows that a large fraction of the vesicles,

Figure 10. CLSM observation on MCF-7 cells incubated with the FITC-dextran loaded PLL-CA25/PEG-DOX (1:2 w/w) vesicles at pH 7.4 and 6.5 for 15 min (a) and 1 h (b), respectively.

that a pH value of 6.5 corresponds to the extracellular environment of solid tumors. Therefore, it is reasonable to expect that the PLL-CA/PEG-DOX vesicles will show better specificity in tumor tissues. The PLL-CA/PEG-DOX vesicles can also gain a faster endosome/lysosome escape after the cell uptake. The pH value in the endosome (pH = 4.5−5.5) is sufficient to disassociate the vesicles and hence generate PLL-CA residues which are very

Figure 11. Assessment of endosome/lysosome escape using CLSM for PLL-CA25/PEG-DOX (1:2 w/w) vesicles by comparing with pHinsensitive PLL-CA25/reduced PEG-DOX vesicles. The endosome/ lysosome compartments were stained with LysoTracker Green DND26. The images are merged from vesicles (red fluorescence from DOX) and LysoTracker (green fluorescence) channels. Scale bar is 10 μm. 11994

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despite their pH sensitivity, is localized in acidic compartments of the cells after incubation for 30 min, as revealed by the appearance of numbers of yellow dots (merged colors of DOX and LysoTracker) in the images. However, the DOX fluorescence becomes dominant in the cells no longer than 90 min of incubation with the pH-sensitive vesicles, indicating an inhibited lysosome trafficking of DOX molecules, whereas with the pH-insensitive vesicles, the LysoTracker fluorescence (yellow and green dots) is still clearly visible. Therefore, by the inclusion of the benzoic imine bond in the PEG-DOX, the coassembled vesicles demonstrate multifunctionality during the internalization process, i.e., tumor pH enhanced cell uptake and fast endosome escape.

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CONCLUSIONS Polymeric vesicles were prepared by coassembly of combshaped amphiphilic polymer, i.e., PLL-CA, with an amphiphilic polymer−drug conjugate, i.e., PEG-DOX. At low PEG-DOX content, PLL-CA/PEG-DOX vesicles generated coexistent with PLL-CA micelles. With the increase of PEG-DOX amount, all PLL-CA micelles transferred into coassembled vesicles, and the shell of the vesicles became thicker with further increase of PEG-DOX fraction while individual PEG-DOX micelles would unlikely form. The results indicate a favorable incorporation of the drug conjugate with the PLL-CA due to the thermodynamic stability of their coassemblies. Furthermore, the inclusion of low-pH labile bond in the PEG-DOX results in pH responsive permeability and triggered dissociation of the PLL-CA/PEGDOX vesicles following an environmental pH dropping from 7.4 to 5.0. In-vitro tests showed that the polymeric vesicles could be more efficiently taken up by cells via endocytosis at an acidic pH close to the extracellular condition of solid tumor, and subsequently a fast endosome/lysosomes escape was achieved. Therefore, PLL-CA/PEG-DOX vesicles should be promising for antitumor drug and gene delivery.

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AUTHOR INFORMATION

Corresponding Author

*Tel +86-10-82619206; fax +86-10-62559373; e-mail quxz@ iccas.ac.cn (X.Q.), [email protected] (Z.Y.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (20720102041, 50733004) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-H19).

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