Article pubs.acs.org/Biomac

Poly(L‑histidine) Based Triblock Copolymers: pH Induced Reassembly of Copolymer Micelles and Mechanism Underlying Endolysosomal Escape for Intracellular Delivery Xiaojun Zhang,†,§ Dawei Chen,†,§ Shuang Ba,† Jia Zhu,† Jie Zhang,† Wei Hong,‡ Xiuli Zhao,† Haiyang Hu,† and Mingxi Qiao*,† †

School of Pharmacy, Shenyang Pharmaceutical University, P.O. Box 42, Wenhua Road 103, Shenyang, Liaoning Province 110016, P.R. China ‡ School of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning Province 110866, P.R. China S Supporting Information *

ABSTRACT: Various poly(L-histidine) based amphiphilic copolymers have been developed for intracellular drug delivery due to the pH responsive properties and the escape from endolysosomal pathway. However, the pH induced reassembly of copolymer micelles and the assumed endolysosome membrane rupture during the copolymer facilitated endolysosomal escape have never been elucidated. To address these issues, a series of poly(ethylene glycol)-poly(D,L-lactide)poly(L-histidine) (mPEG-PLA-PHis) with different degrees of polymerization of PLA and PHis block were synthesized. The self-assembly and reassembly behaviors of the copolymers were characterized using transmission electron microscopy (TEM), 1H NMR, fluorescence probe technique, and dynamic light scattering (DLS). The copolymers self-assembled into micelles with PLA and unprotonated PHis blocks as hydrophobic core and PEG as hydrophilic shell at neutral pH. The changes in TEM images, 1H NMR spectrum of PHis peak, pyrene fluorescene spectrum, and particle size as well as size distribution over the pH range from pH 8.5 to 4.5 suggest that the copolymer micelles reassembled into micelles with PLA as hydrophobic core and protonated PHis and PEG as hydrophilic shell under acidic environment. The pH induced reassembly triggered the incoporated doxorubicin (DOX) release, as indicated by the in vitro accelerated drug release and enhanced cytotoxicity. The integrity of endolysosome membrane during the copolymer facilitated DOX endolysosomal escape was observed by confocal laser scan microscopy (CLSM) and further evaluated by hemolysis test and calculation of the critical size of endolysosomal membrane. The results indicate that the endolysosomal membrane remained intact during the copolymer facilitated endolysosomal escape of DOX. It is more reasonable to ascribe the PHis based copolymer facilitation endolysosomal escape to the “proton sponge” hypothesis without rupturing the endolysosomal membrane.

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The first pH sensitive PHis based copolymer micelles that destabilized at tumor pH was developed by Bae’s group using PHis(5K)-PEG(2K) diblock copolymer.9 However, the copolymer micelles showed poor stability at physiological pH (pH 7.4), as characterized by the triggering pH slightly higher than the acidic tumor interstitial pH (pH 6.5−7.21). In order to tailor the triggering pH of the PHis based copolymer micelles to the more acidic extracellular pH of the tumor, two different approaches have been used to increase the stability of the micelles at physiological pH. One approach is to manipulate the pKa value of the PHis based copolymers by hydrophobic modification. For example, Kim et al. have developed a pH sensitive copolymer of poly(L-histidine-co-L-phenylalanine)-

INTRODUCTION A number of pH-sensitive copolymer micelles taking advantage of the intrinsic pH difference between solid tumors and normal tissues have been developed to efficiently deliver cytotoxic drugs to solid tumors in the past decade.1−6 Among of them, poly(L-histidine) (PHis) based copolymers have attracted great attention in the field of intracellular drug delivery because of their biodegradability, low toxicity, and, more importantly, appropriate pH responsive properties as well as escape from the endolysosomal pathway.7,8 The combination of pH triggered release and endolysosomal escape properties has been recognized to be an efficient approach to deliver a payload to the cytosol, enhancing the effectiveness of the payload by avoiding the lysosomal sequestration and degradation.9,10 Noteworthy, PHis based micelles with endosomal or lysosomal pH triggered release have been demonstrated to overcome multidrug resistance (MDR) of various tumors.11,12 © 2014 American Chemical Society

Received: July 22, 2014 Revised: September 30, 2014 Published: October 12, 2014 4032

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has never been demonstrated to date. Furthermore, a recent study suggests that nanomaterial associated lysosomal membrane destabilization (LMD) is mainly responsible for the nanomaterial-induced toxicity.24 Considering the biocompability of PHis based copolymers25 and lack of evidence for membrane rupture, it is arguable if the PHis based copolymers are capable of inducing the rupture of endolysosomal membrane during the endolysosomal escape. The objectives of the present study are to address the unknown issues about the PHis based copolymers: (1) Can they display pH responsive reassembly of micelles other than pH responsive micellar aggregation or dissociation? (2) Is membrane rupture involved in the endolysosomal escape of a drug facilitated by PHis based copolymers besides the “proton sponge” effect? In this study, a series of mPEG-PLA-PHis copolymers have been synthesized. The self-assembly and pH dependent reassembly behaviors of the copolymer micelles were characterized by transmission electron microscopy (TEM), 1 H NMR, fluorescene probe technique and dynamic light scattering (DLS). The endolysosomal membrane rupture during the endolysosomal escape pathway of the copolymer was studied by evaluating the integrity of the membrane and membrane-lytic property of PHis homopolymer.

PEG by copolymerization of L-phenylalanine, a hydrophobic amino acid, with L-histidine.13 The pKa value of the diblock copolymer could be manipulated by adjusting the ratio of Lhistidine/L-phenylalanine groups in the poly(amino acid) block. It was observed that the diblock copolymers self-assembled into stable micelles at pH 7.4 and started to dissociate below pH 6.7.13 Lee and Youn designed a pH sensitive benzyl (Be) conjugated PHis copolymer (polyBe-His-b-PEG14). The benzyl group was used to adjust the pKa of the copolymer for tuning drug release behavior of the copolymer micelles with pH. The copolymer micelles showed accelerated drug release over the pH range of 7.4−5.5, depending on the content of the benzyl group in the PHis block. The poly(D,L-lactic acid) (PLA) modified PEG-PHis copolymers (PLA-PEG-PHis7 and PEGPHis-PLA15) have also been synthesized to increase the stability of the micelles at physiological pH. The other approach is to construct mixed micelles with another amphiphilic polymer.16 For example, a mixed micellar delivery system composed of PHis(5K)-PEG(2K) and PLLA(3K)-PEG(2K) was fabricated. The pH triggered drug release of the mixed micellar system could be manipulated within a certain pH range by variation of the ratio of the two copolymers. Increasing the fraction of PLLA-PEG in the mixed micelles would increase the stability of the micelles against pH droping and lower the triggering pH for the micelles disassembly. This could be attributed to the stabilizing effect of the micellar core arising from the hydrophobic PLLA blocks.17,18 Another mixed micellar system consisted of poly(His-co-Phe)(5K−5.5K)-PEG(2K) and PLLA(3K)-PEG(2K) was also developed for targeting endosomal pH.19 The micelles showed minimal drug release above pH 6.0, but an accelerated drug release below pH 6.0, indicating the destabilization of the micelles at early endosomal pH. The pH sensitive mixed micelles have also been integrated with active targeting function in order to further enhance the cytosolic drug delivery. For example, the mixed micelle system composed of PHis-PEG and folate conjugated PLLA-PEG showed high cytotoxicity against MDR cells after folate mediated endocytosis.20,21 Later, the mixed micelles constructed from DSPE-PEG(2K), DSPE-PEG(3.4K)-2C5, and PHis-PEG(2K) were developed.22 To sum up, various PHis based copolymer micelles have been developed to achieve intracellular drug delivery due to the attractive pH responsive and endolysosomal escape properties. Regarding the mechanisms underlying pH responsive properties of PHis based copolymer micelles, they have been proposed to be either micellar dissociation or aggregation caused by the pH dependent protonation of the imidazole rings in the PHis block.7,9,10,18,23 Another more favorable pH responsive mechanism, that is, reassembly of PHis based copolymer micelles, has not been reported to date. As compared to the pH induced micellar dissociation or aggregation, pH responsive reassembly ensures the micellar structure after pH stimulus which might offer a promise for further subcelluar drug delivery. Regarding the endolysosomal escape, the mechanism underlying the PHis based copolymers facilitated endolysosomal escape has been attributed to the “proton sponge” effect and rupture of the endosomal membrane.7,9,13 Since PHis becomes a strong polycation at acidic pH, it seems reasonable to assume that the positively charged PHis is capable of binding with the negatively charged phospholipids. However, the rupture of the endolysosomal membrane by the previously developed PHis based copolymers

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EXPERIMENTAL SECTION

Materials. Nα-CBZ-Nim-DNP-L-histidine was purchased from GL Biochem (Shanghai, China). D,L-Lactide was purchased from GLACO (Beijing, China). Poly(ethylene glycol) methyl ether (mPEG, MW: 2000 g/mol) and PAMAM G4.0 solution were purchased from SigmaAldrich (St. Louis, MO). Isopropylamine was purchased from Sinopharm Chemical Reagent Co (Shanghai, China). N,N′-Carbonyldiimidazole (CDI) was purchased from J&K Ltd. (Beijing, China). Doxorubicin (DOX) was purchased from Beijing HuaFeng United Technology Co. Ltd. (Beijing, China). Hoechst 33258 were purchased from Sigma (St. Louis, MO). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) and Lysotracker Green were purchased from Beyotime Biotechnology Co, Ltd. (Nantong, China). Acridine Orange (AO) was purchased from DingGuo Biotechnology Co., Ltd. (Beijing, China). Calcein was purchased from Heng Xing Chemical Preparation Co., Ltd. (Tianjin, China). Purified deionized water was prepared via the Milli-Q plus system (Millipore Co., Billerica, MA). All the other reagents and chemicals were of analytical or chromatographic grade and were purchased from Concord Technology (Tianjin, China). The human breast cancer cell line MCF-7 was purchased from American Type Cell Culture (ATCC, Manassas, VA). Culture plates and dishes were purchased from Corning Inc. (New York, NY). MCF7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin sulfate. All cells were cultured at 37 °C in a humidifier with 5% CO2 atmosphere. Male Sprague−Dawley rats (200 ± 20) g were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University and housed at 22 ± 2 °C with access to food and water ad libitum. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Shenyang Pharmaceutical University. Synthesis and Characterization of mPEG-PLA-PHis Copolymer. The mPEG-PLA-PHis copolymers were synthesized according to our previous research.12 The details of the synthesis and characterization of the intermediates are shown in the Supporting Information. Acid−Base Titration. The buffering capacity of mPEG-PLA-PHis was measured by an acid−base titration method as previously reported,26 In brief, mPEG-PLA-PHis copolymer (30 mg) was dissolved in 0.01 M NaOH solution (10 mL) to obtain the copolymer 4033

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solution of 3 mg/mL. The copolymer solution was first titrated to pH 11 with 1 M NaOH, and then titrated with 0.01 M HCl given in various volume increments. The titration curve was plotted with pH as a function of HCl volume. The buffering capacity is defined as the percentage of unsaturated nitrogen in imidazole groups becoming protonated from pH 7.4 to 4.0, which can be calculated as follows:27,28 buffering capacity =

ΔH+copolymer − ΔH+NaCl n

carbon-coated copper grid and then negatively stained with 2% phosphotungstic acid aqueous solution, followed by air-drying at room temperature. In Vitro Drug Release Experiments. The in vitro DOX release from the copolymer micelles was studied using a dialysis bag (MWCO = 12 000 Da) under sink conditions. Briefly, 2 mL of DOX-loaded micelles was sealed in dialysis bags and incubated in conical flasks containing 50 mL of PBS buffer with different pH values (pH 7.4, pH 6.8, pH 6.0, pH 5.0, ionic strength = 0.1 M). The conical flasks were put in a shaking bed (Incubator shaker KYC 100B, Shanghai FUMA TEST EQUIPMENT Co. Ltd.) with a shaking rate of 100 rpm at 37 °C. At predetermined time intervals, 0.5 mL of the outside medium of the dialysis bag was withdrawn and replaced with fresh buffer solution. The concentration of DOX was determined by using a fluorescence detector with excitation wavelength at 480 nm and emission wavelength at 550 nm.31 The in vitro release experiments were carried out in triplicate at each pH value. The in vitro DOX release profiles were plotted with cumulative drug release as a function of time. Cytotoxicity Test. The in vitro cytotoxicity of DOX-loaded micelles was assessed by a standard MTT assay.17,32 The MCF-7 cells were seeded in 96-well plates at a density of 7000 cells per well and incubated overnight. The DMEM medium with serum was then replaced with fresh medium containing an indicated concentration of the tested formulations (free DOX solution or DOX-loaded micelles solution at pH 7.4, pH 6.5 and pH 6.0). Control wells were treated with equivalent volume of fresh medium. After incubation for 48 h, 20 μL of MTT solution (5 mg/mL in PBS buffer) was added to the cultured supernatants. The plates were further incubated for 4 h at 37 °C, allowing the viable cells to reduce the yellow MTT into purple formazan crystals. At last, the medium was removed completely and 150 μL of DMSO was added to each well to dissolve purple formazan crystals. The absorbance was measured at 570 nm using a multifunctional microplate reader (Tecan, Austria). The relative cell viability (%) was calculated using the following equation.32

× 100%

wherein ΔH+copolymer and ΔH+NaCl are the moles of H+ required to adjust the pH of copolymer solution and 0.1 M NaCl from pH 7.4 to 4.0, respectively, and n is the total moles of unsaturated nitrogen in imidazole groups of the copolymer. Measurement of Fluorescent Spectroscopy. A stock solution of pyrene (6.0 × 10−6 M) was prepared in acetone and dropped into a brown volumetric flask. The solution was then distilled at 60 °C to remove acetone followed by mixing with the solution of copolymer micelles with concentration ranging from 0.5 to 100 μg/mL in PBS 7.4. The final pyrene concentration was 6 × 10−7 M. All the solutions were stored in the dark for 24 h to reach solubilization equilibrium prior to measurements. Fluorescence excitation spectra were measured using fluorescence spectroscopy (Shimadzu RF-5301PC). Excitation and emission slit widths were fixed at 2.5 and 5.0 nm, respectively. The peak intensities at 334 and 336 nm from the excitation spectra were monitored with the emission wavelength set at 397 nm. The critical micelle concentration (CMC) is defined as the intersection of two straight lines drawn through the points of the flat and drastically increasing region from the concentration−intensity ratio (I336/I334) plots. In order to quantify the polarity change of the micellar core as a function of pH, the intensity ratios (I336/I334; higher ratio means a less polar environment) of pyrene at different pH values (pH 8.5−4.5) were measured.7 Preparation of DOX Loaded Copolymer Micelles. DOX·HCl (20 mg) was dissolved in 3 mL of methanol/water (2:1) in the presence of triethyl amine (TEA, 1.5 times molar quantity of DOX) and stirred overnight at room temperature to form DOX base solution. The methanol/water and extra TEA were evaporated under rotary vacuum, and the residue was filtrated to remove residual TEA·HCl and dried to obtain the DOX base.29 DOX-loaded micelles were prepared by thin-film rehydration method.30 To prepare DOX-loaded micelles, DOX and mPEG-PLAPHis copolymers were codissolved in 10 mL of acetonitrile. The solvent was removed by rotary evaporation at 60 °C to obtain a thin film. Residual acetonitrile remaining in the film was further evaporated overnight at room temperature under vacuum. The resultant thin film was hydrated with 10 mL of PBS 7.4 solution for 30 min to obtain a micellar solution. The micellar solution was filtered through a 0.45 μm film to remove the unincorporated DOX aggregates. The blank copolymer micelles were prepared as described above without adding DOX. The drug loading content (DL, %) and encapsulation efficiency (EE, %) were calculated by using the following equations, respectively. DL (%) =

weight of drug in the micelles × 100 weight of drug incorporated micelles

EE (%) =

weight of drug in the micelles × 100 weight of the feeding drug

cell viability =

A sample − A blank Acontrol − A blank

× 100%

wherein Acontrol and Asample are the absorbance at 570 nm in the absence and in the presence of sample treatment, respectively, and Ablank is the absorbance at 570 nm without cells. Confocal Laser Scanning Microscopy (CLSM). MCF-7 cells were seeded on a cover-slide system at a density of 4 × 105 cells/well in a humidifier with 5% CO2 atmosphere for 24 h at 37 °C followed by incubation with the test formulations. After incubation for predetermined time intervals, the cells were stained with 100 nM Lyso-Tracker Green (30 min) and washed three times with cold PBS, and the cells were fixed with 4% paraformaldehyde for 30 min. The cells were stained with 10 μg/mL Hoechst 33258 for 15 min. The microscopic images were captured using a confocal laser scan microscope (Olympus FV1000-IX81, Japan). Endolysosomal Membrane Integrity. Lysosomal membrane integrity was assessed using Acridine Orange (AO).24 MCF-7 cells were grown on a cover-slide system at the density of 4 × 105 cells/well in a humidifier with 5% CO2 atmosphere for 24 h at 37 °C. The cells were exposed to blank copolymer micelles for 4 h, and then rinsed three times with PBS followed by staining with AO (5 μg/mL) in PBS for 10 min at 37 °C. The cell images were captured via CLSM. Calcein Uptake and Endosomal Stability. Calcein, a membrane-impermeable fluorophore, was used as a tracer to monitor the endosomal stability following micelle uptake.33 MCF-7 cells were plated (4 × 105 cells/well) in 6-well plates on round cover glasses and incubated overnight at 37 °C under 5% CO2. Then, calcein (1 mg/ mL) was added to the cells with (test sample) or without (control) copolymer micelles in DMEM medium with serum. After 1 h incubation at 37 °C, the cells were washed three times with PBS and incubated in complete medium for 3 h to allow for intracellular trafficking. The cells were then washed with PBS and fixed with 4%

Particle Size and Distribution. DLS was used to measure the particle size and size distribution of the copolymer micelles (n = 3). All the measurements were carried out on a Zetasizer Nano ZS instrument (Malvern, U.K.) at 25 °C after equilibration for 5 min. The micellar solutions were filtered through a 0.45 μm disposable membrane filter prior to measure. Transmission Electron Microscopy Observations. The morphology of the copolymer micelles was observed using a JEOL JEM-2100 electron microscope (Jeol Ltd., Tokyo, Japan) with an accelerating voltage of 100 kV. Each sample was first placed on a 4034

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Figure 1. Typical 1H NMR spectrum of mPEG45-PLA18-PHis12 copolymer in DMSO-d6 with a drop of TFA.

Figure 2. Typical GPC spectra of mPEG45-PLA18-PHis12, mPEG45-PLA18, and mPEG45 polymers.

Table 1. Characterization Results of mPEG-PLA-PHis Copolymers

a

copolymers

Mw (Da)a

Mn (Da)a

Mw/Mna

Mn (Da)b

buffering capacity (pH7.4−4.0) (%)

CMC (μg/mL)

mPEG45-PLA18-PHis12 mPEG45-PLA13-PHis12 mPEG45-PLA12-PHis12 mPEG45-PLA18-PHis6 mPEG45-PLA18-PHis3 mPEG45-PLA18-PHis0

3882 3783 3194 3822 3474 3232

3512 3518 2971 3530 3213 2971

1.105 1.076 1.075 1.083 1.081 1.088

4940 4580 4508 4118 3707 3296

26.76 28.45 27.52 18.30 10.30

4.26 20.0 23.5 7.60 12.7 11.5

Determined from GPC analysis. bCalculated from 1H NMR spectrum. endosomal membrane.34 Erythrocytes were isolated from rat blood, which was obtained from anesthetized animals by cardiac puncture and drawn into tubes containing EDTA. The blood was centrifuged for 10 min at 1000 rpm and washed several times with saline at pH 7.4 until

(V/V) formaldehyde in PBS (pH 7.4) for 15 min at room temperature. The cell images were captured via CLSM. Hemolysis Studies. The pH-dependent membrane-lytic activity of PHis homopolymer was assessed using erythrocytes as a model of the 4035

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the supernatant was clear and colorless. Finally, 2% erythrocyte suspension was obtained by adding an adequate amount of saline to the erythrocyte tube. The hemolysis assay was performed following the procedure.35 Erythrocyte suspension (100 μL) was seeded into a 96well plate. The sample solution (100 μL) was added to each well of the erythrocyte suspensions in PBS buffer of pH 7.4, pH 6.5, or pH 5.7, and incubated for 1 h at 37 °C. Two controls were prepared by resuspending erythrocyte suspension either in buffer alone (negative control) or in distilled water (positive control). The absorbance of the supernatant from each sample was measured at 540 nm using a microplate reader to determine the released hemoglobin.34 The percentage of hemolysis were determined by comparison the absorbance of samples with that of the positive control samples. Statistical Analysis. All experiments were performed at least three times. Quantitative data are presented as the mean ± SD. Statistical comparisons were determined by analysis of variance (ANOVA) among ≥3 groups or Student’s t test between 2 groups. P-Values < 0.05 and P-values < 0.01 were considered statistically significant.

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RESULTS AND DISCUSSION H NMR and GPC Characterization of the Copolymers. The typical 1H NMR spectrum of the mPEG-PLA-PHis 1

Figure 4. Emission spectra of pyrene as a function of copolymer (mPEG45-PLA18-PHis12) concentration (A); fluorescence intensity ratio of I336/I334 ratio from emission spectra vs log concentration of the mPEG-PLA-PHis copolymers at pH 7.4 (B).

showed pKa values around pH 6.8, which were similar to previous research values.9 As compared to the controls (NaCl and mPEG-PLA copolymer), the mPEG-PLA-PHis copolymers showed obvious buffering capacity in the pH range from 7.4 to 4.0, indicating that PHis block in the copolymers was accounted for in the buffering capacity. The calculated buffering capacity values of the copolymers are shown in Table 1. As the degree of polymerization (DP) of PHis block increased from 3 to 12, the buffering capacity increased from 10.30% to 26.76%. The higher buffering capacity was attributed to the longer PHis block, producing more imidazole rings for binding protons. CMC Values of the Copolymers. The CMC values of the copolymers were measured by the change of fluorescence spectrum of pyrene due to its selective partition into hydrophobic micellar core in the copolymer solution.37 As copolymer concentration increased, the fluorescence intensity increased and the peak shifted from 334 to 336 nm in the excitation spectra of pyrene (Figure 4A). The intensity ratio (I336/I334) of the pyrene as a function of copolymer concentration is shown in Figure 4B. The remarkable increase in the intensity ratio of the pyrene indicates the copolymer unimers self-assembled into micelles.32 The CMC values of the copolymers estimated from the plots are shown in Table 1. As expected, the CMC value of the copolymer decreased with an increase in the DP of either PLA block or PHis block due to the longer hydrophobic block producing stronger associative hydrophobic force. In addition, the copolymer displayed a slightly increase in the CMC value when the pH decreased from 7.4 to 5.0 (data not shown). This was attributed to the

Figure 3. Acid−base titration profiles of mPEG-PLA-PHis copolymers with NaCl and mPEG-PLA copolymer as controls.

copolymer is shown in Figure 1. All the chemical shifts were expressed in parts per million (δ) relative to the tetramethylsilane (TMS) signal. In order to properly display the characteristic peaks of poly(L-histidine) block, the final product was performed with a drop of TFA addition into the DMSO-d6 according to the previous article.36 1H NMR spectrum (DMSO-d6) of the mPEG-PLA-PHis copolymer showed the characteristic peaks of poly(L-histidine) at δf 1.28 ppm (−C(CH3)2−),δg 4.98 ppm (−CH−NH−), δi 8.97 ppm (N− CHN of imidazole ring), and δj 7.49 ppm (N−CHC of imidazole ring) and the characteristic peaks of mPEG-PLA at δc 5.14 ppm (−COCH(CH3)O−), δa 3.51 ppm (−OCH2CH2O−), δb 3.25 ppm (−OCH3), δd 1.45 ppm (−COCH(CH3)O−), and δe 4.28 ppm (−COOCH2CH2O−). The typical GPC chromatogram of the triblock copolymer is shown in Figure 2. The copolymer showed unimodal distribution with a polydispersity less than 1.2. The characterization results of different copolymers are shown in Table 1. pKa and Buffering Capacity of the Copolymers. The pKa and buffering capacity of the copolymers were studied by acid−base titration. The titration curves of the mPEG-PLAPHis copolymers are presented in Figure 3. The copolymers 4036

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Figure 5. TEM images and size distribution of blank copolymer (mPEG45-PLA18-PHis12) micelles under different pH values.

Figure 6. 1H NMR spectra of mPEG45-PLA18-PHis12 copolymer micelles under different pH values (pH 8.0 ∼ pH 5.0).

were replaced by the smaller micelles with more uniform distribution when the pH decreased further down to 4.5. The TEM images at acidic pH revealed no signs of aggregation of copolymer micelles, indicating a different pH responsive mechanism from previously developed PHis based copolymers.7,9,23 The 1H NMR spectra of the copolymer under different pH values are shown in Figure 6. The −CCH3− group of PHis block displayed two separate peaks at pH 8.0 owing to different chemical environment caused by the local shielding of electrondonating group of imidazole, leading to one of the −CCH3− shift to lower field at neutral pH. The two peaks merged into one broad peak when the pH decreased to 5.0, indicating the protonation of imidazole rings in the PHis block, which reduced the electron-donating of imidazole group, resulting in the similar chemical environment of the two −CCH3− groups

protonation of PHis block of the copolymer at acidic pH making it more hydrophilic compared to that at pH 7.4 pH Induced Reassembly of the mPEG-PLA-PHis Copolymer Micelles. The pH responsive mechanism of the mPEG-PLA-PHis copolymers was studied by observing the changes in TEM images, 1H NMR spectra, micropolarity of the micellar inner core, particle size, and size distribution under different pH values. To first characterize the morphology and core−shell structure of the copolymer micelles, the copolymer solutions under different pH values were measured by TEM and 1H NMR, respectively. As shown in Figure 5, the copolymer solution showed spherical shaped and smooth surfaced micelles under both neutral pH and acidic pH. Some big micelles with diameter around 200 nm could be observed when the pH decreased to 5.5. However, these big micelles disappeared and 4037

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core could be a sign of reassembly of the copolymers into the micelles with hydrophobic core composed of PLA block and hydrophilic shell composed of PEG and protonated PHis block. The average particle size and polydispersity index (PDI) of the copolymer micelles at different pH values are shown in Figure 8. The average particle size of the micelles increased remarkably when the pH decreased from 8.5 to 4.5. The PDI of the micelles increased with pH decrease, and reached the maximum value at pH 5.5. It could be noted that the copolymer micelles displayed bimodal size distribution at pH 6.5 and pH 5.5. Moreover, the amount of big particles increased from 28.4% to 59.2% when the pH decreased from 6.5 to 5.5. However, the PDI of the micelles decreased sharply when the pH was further down to 4.5. The PDI variation was different from the previous studies, in which the copolymer micelles showed continuous PDI increase due to the micellar aggregation or dissociation.7,9 To understand the PDI variation of the copolymer micelles, the degree of ionization of PHis blocks was calculated using the formula of pH = pKa + log([N]/[NH+]), which was derived from the Henderson−Hasselbalch equation: pH = pKa + log([A−]/[HA]). The degree of ionization of PHis block was 2.62% (pH 8.5), 21.1% (pH 7.4), 68.0% (pH 6.5), 95.5% (pH 5.5), and 99.5% (pH 4.5). The degree of ionization as a function of pH indicates the hydrophobic to hydrophilic transition of the PHis block. The PHis block was considered to be more hydrophobic when the degree of ionization was relatively low at pH between 8.5 and 7.4. The pH induced hydrophobic to hydrophilic transition probably occurred at pH below 6.5 when the ionization degree was above 50%. The bimodal size distribution of the copolymer micelles at pH 6.5 and pH 5.5 was probably attributed to the ionized PHis blocks becoming immiscible with hydrophobic PLA blocks. The PHis block became completely hydrophilic when the pH dropped to 4.5, due to the degree of ionization being close to 100%. Considering the decreased PDI of the micelles and the hydrophilic PHis block at pH 4.5, it was reasonable to assume that the copolymers reassembled into micelles with hydrophobic PLA core and hydrophilic PHis-PEG shell due to the polarity conversion of the PHis block. Based on the above results, an assumed pH sensitive mechanism of the copolymer micelles was proposed (Figure 9). The mPEG-PLA-PHis copolymers self-assembled into micelles with an inner core of hydrophobic PHis and PLA blocks and an outer shell of hydrophilic PEG blocks at the pH above or equal to pKa + 2. The hydrophobic interaction arising from PLA and deprotonated PHis blocks was the associative force for maintaining the inner core of micelles. As the pH started to decrease, the progressive protonation of imidazole groups in the PHis block caused a hydrophobic to hydrophilic transition of PHis block as indicated by the degree of ionization at different pHs. The ionization of PHis blocks resulted in a progressive increase of the electrostatic repulsive force and the immiscibility of PHis blocks with PLA blocks. The inner core of the micelles had a sensitive electrostatic repulsive force/ hydrophobic force balance between the PHis blocks and PLA blocks. The hydrophobic force of the PLA block was the dominant force that maintained the inner core of the micelles when the ionization degree of PHis blocks was low. As the pH continued to decrease, the increasing degree of ionization of PHis blocks caused a hydrophobic to hydrophilic transition of these blocks. The electrostatic repulsive force and hydrophobicity of PHis blocks probably drove these blocks to

Figure 7. Fluorescence intensity ratio of I336/I334 as a function of pH (mean ± SD, n = 3) in mPEG45-PLA18-PHis12 copolymer (**P < 0.01 indicates significant difference; “−” means no significant difference).

Figure 8. Influence of pH on the particle size and size distribution of blank mPEG45-PLA18-PHis12 copolymer micelles (mean ± SD, n = 3).

in PHis. Compared to 1H NMR spectrum of the copolymer in DMSO, the disappear of PLA characteristic peaks (δ1.45 ppm (−COCH(CH3)O−) and δ 5.14 ppm (−COCH(CH3)O−)) in D2O under both neutral and acidic pH, indicates that the copolymer self-assembled into core−shell structure micelles under different pH conditions. The micropolarity change of the micellar inner core as a function of pH was investigated with pyrene as a probe. The fluorescence intensity ratios (I336/I334) of pyrene under different pH values are shown in Figure 7. The I336/I334 ratio showed insignificant change over the pH from 8.5 to 7.4, but a drastic decrease was observed when the pH dropped from 7.4 to 7.0 and further down to 6.5. However, the I336/I334 ratio drastically increased and leveled off when the pH decreased from 6.5 to 6.0 and further below 6.0, respectively. The fluorescence change of pyrene induced by pH dropping indicates a first increase and then a decrease in the polarity of the micellar core. The initial polarity increase was obviously attributed to the progressive protonation of PHis block of the copolymer with pH decrease, inducing the destabilization of the micellar core.9 The afterward polarity decrease of the micellar 4038

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Figure 9. Schematic illustration of self-assembly and reassembly of the mPEG-PLA-PHis copolymers under different pH values.

Figure 10. CLSM images of MCF-7 cells incubated with mPEG45-PLA18-PHis12/DOX copolymer micelles, mPEG45-PLA18/DOX copolymer micelles, and free DOX solution for 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h at 37 °C (DOX concentration: 2 μg/mL). (Red, DOX; blue, Hoechst 33258; green, LysoTracker Green DND-26.)

loaded copolymer micelles were prepared using film evaporation method. Cellular uptake of the DOX loaded micelles by MCF-7 cells was evaluated using CLSM with free DOX and mPEG-PLA copolymer micelles as controls. The CLSM photos of the DOX loaded copolymer micelles incubated with MCF-7

translocate to the outer shell of the micelles, resulting in the reassembly behavior. Endolysosomal Escape Pathway of the mPEG-PLAPHis Copolymers. To elucidate the endosomal escape facilitated by the mPEG-PLA-PHis copolymers, the DOX 4039

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Figure 11. CLSM images of MCF-7 cells incubated with different mPEG-PLA-PHis/DOX copolymer micelles at 37 °C (red: DOX).

Figure 12. CLSM images of MCF-7 cells showing the integrity of lysosomal membrane visulized by AO staining. (a, negative control, incubation with AO alone; b, incubation with 100 μg/mL of mPEG45-PLA18-PHis12 copolymer micelles; c, incubation with 500 μg/mL of mPEG45-PLA18PHis12 copolymer micelles; d, positive control: incubation with 100 μg/mL PAMAM dendrimer G 4.0 solution).

Figure 13. CLSM images of MCF-7 cells showing the subcellular distribution of calcein fluorescence. The cells were treated with 1 mg/mL calcein (a. negative control: incubation with calcein alone; b.incubation with 100 μg/mL of mPEG45-PLA18-PHis12 copolymer micelles; c. incubation with 500 μg/mL of mPEG45-PLA18-PHis12 copolymer micelles; d. positive control: incubation with 100 μg/mL PAMAM dendrimer G 4.0 solution).

accumulation of the micelles in the endosome. A strong purple fluorescence (the overlap of DOX with nuclei staining Hoechst) was found after incubation for 4 h, indicating the escape of the DOX from endosome into nuclei. As compared to the PHis based copolymer micelles which displayed obvious DOX endosomal escape, the mPEG-PLA micelles showed much weaker purple fluorescence after incubation for 4 h,

cells for different time intervals are presented in Figure 10. The cell nuclei and endosome were labeled with Hoechst 33258 (blue) and LysoTracker DND-26 (green), respectively. The DOX loaded PHis based copolymer micelles were quickly uptaken by MCF-7 cells and encapsulated into the primary endosome after 15 min. With the increase of incubation time, the enhancement of orange fluorescence (the overlap of LysoTracker with DOX) in cells suggests the continuous 4040

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PHis copolymer with higher buffering capacity induced more Cl− ions and water molecules influx into the endolysosome, creating higher osmotic pressure which facilitated the endolysosomal escape behavior.43 Endolysosomal Membrane Integrity during the Endolysosomal Escape Pathway of mPEG-PLA-PHis Copolymer. To investigate if the endolysosomal membrane rupture played a role in endolysosomal escape facilitated by the mPEG-PLA-PHis copolymers, the integrity of the endolysosomal membrane was assessed using the AO relocation assay24 and calcein uptake study.33 In this study, high concentration (100 μg/mL) of PAMAM dendrimer G 4.0 solution was used as positive control, which has been shown to be capable of rupturing the endolysosomal membrane.44 AO is a metachromatic fluorophore and a lysosomotropic base which diffuses into cells and accumulates in the endolysosome by proton trapping. The accumulation of AO in the endolysosome produces a change in the fluorescence of the probe (from cytosolic green to lysosomal red). Therefore, the rupture of the endolysosomal membrane can be indicated by a shift of redorange fluorescence to a yellow-green fluorescence, and an increase in the overall fluorescent intensity.24,45 As shown in Figure 12, the MCF-7 cells treated with AO alone (negative control) revealed red-orange granules in the endolysosomes and a diffuse green fluorescence in the cytosol. The MCF-7 cells treated with PAMAM solution (positive control) caused noticeable endolysosomal membrane damage as evidenced by the significant yellow-green fluorescence due to the overlay of enhance green fluorescence and reduced red fluorescence. The MCF-7 cells incubated with copolymer micelles showed observable red-orange granules but no signs of fluorescent color shift or fluorescent intensity increase, indicating that the endolysosomal membrane was intact. The noticeable decrease in the red-orange granules was probably attributed to the diffuse of AO from endolysosome to cytosol via the enhanced osmotic pressure caused by the “proton sponge” effect. Calcein is a membrane-impermeable fluorophore with green fluorescence, which was used as a tracer to monitor the endosomal stability after particle uptake. The rupture of the endolysosomal membrane can be indicated by a remarkable broad distribution of green fluorescence in cytosol.33,45 As shown in Figure 13, the MCF-7 cells treated with calcein alone (negative control) showed punctuate distribution of green fluorescence in endolysosomes, which was in agreement with previous studies.45 The MCF-7 cells treated with PAMAM solution (positive control) revealed diffusion of green fluorescence in the cytosol, indicating the destabilization of the endolysosomal membrane and releasing calcein to the cell cytosol. The MCF-7 cells treated with copolymer micelles revealed similar punctuation distribution of green fluorescence with negative control cells, but a marginally broader cytosolic distribution of the green fluorescence indicating the endolysosomal membrane remained intact during the endolysosomal escape of the calcein. Therefore, the CLSM results demonstrated that the rupture of endolysosomal membrane was not involved in the endolysosomal escape pathway of mPEG-PLAPHis copolymer micelles. The Presumption of Endolysosomal Membrane Integrity during the Endolysosomal Escape Pathway of Other PHis Based Copolymers. To further shed some lights on the role of membrane rupture in the endolysosomal escape facilitated by previously synthesized PHis based copolymers (PHis(5K)-PEG(2K),9 PHis(5K)-PEG(2K)-PLA-

Table 2. Calculated critical diameters of lysosome at pH4.0 for PHis based copolymers calcd critical diameters of lysosome at pH 4.0 copolymer concn (mg/mL)

PHis(5K)-PEG(2K)9

PHis(5K)-PEG(2K)-PLA(3K)7

0.05 0.5 5

119 μm 11.9 μm 1.19 μm

199 μm 19.9 μm 1.99 μm

Figure 14. pH-sensitive membrane-lytic activity of poly(L-histidine) (5000 Da) after 1 h of incubation with rat erythrocytes at pH 7.4, pH 6.5, and pH 5.7 (mean ± SD, n = 3).

indicating that little DOX was capable of escaping from the endosome. To investigate the effect of the copolymer’s buffering capacity on the endolysosomal escape, different micelles incorporating DOX were observed with CLSM. As shown in Figure 11, the endolysosomal escape facilitated by the micelles was indicated by the DOX red fluorescence distribution in the cell nuclei. For the same incubation time, it could be noted that the copolymer with higher buffering capacity (mPEG45-PLA18-PHis12) displayed stronger DOX fluorescence, indicating that the buffering capacity of the copolymer was a promoting factor for endolysosomal escape. Mechanism Underlying the Endolysosomal Escape Pathway of PHis Based Copolymers. The endolysosomal escape mechanism of PHis based micelles has been ascribed to the “proton sponge” effect38,39 and the rupture of endolysosomal membrane by the previous studies.7,9 The “proton sponge” effect is usually induced by a cationic polymer with a high buffering capacity, leading to its distribution in the cytosol and the spillage of endolysosomal contents.39−41 Once a cationic copolymer based micelles endocytoses into the acidic endolysosome, the protonation of the copolymer results in the binding of protons that are supplied by the VATPase (proton pump). This process keeps the pump functioning and leads to the influx of one Cl− ion and one water molecule per proton.42 The influx of Cl− ions and water molecules into the endolysosome caused an increase in osmotic pressure and tension on the endolysosomal membrane, subsequently leading to the endolysosome swelling and possible rupture of the membrane, depending on the characteristics of the copolymers. Therefore, the mPEG-PLA4041

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Figure 15. Schematic illustration of endolysosomal escape of a drug facilitated by PHis based copolymers via “proton sponge” effect after cellular internalization. (The micelle ends up in the endolysosome after endocytosis; the PHis block becomes protonated in the acidic environment and keeps the proton pump pumping protons and influx of Cl− ions and water molecules; the influx of Cl− ions and water molecules causes the osmotic pressure increase and swelling of endolysosome, inducing the release of incoporated drug into cytosol.)

Table 3. Characterization of DOX-Loaded Copolymer Micelles (mean ± SD, n = 3) copolymers mPEG45-PLA18-PHis12 mPEG45-PLA13-PHis12 mPEG45-PLA12-PHis12 mPEG45-PLA18-PHis6 mPEG45-PLA18-PHis3 mPEG45-PLA18

particle size (nm) 28.03 34.96 67.74 32.60 41.84 28.30

± ± ± ± ± ±

3.65 4.92 8.85 5.66 5.94 4.36

PDI 0.215 0.284 0.303 0.258 0.206 0.133

± ± ± ± ± ±

0.010 0.012 0.015 0.017 0.016 0.011

DL (%) 8.91 8.64 8.69 9.00 8.62 10.0

± ± ± ± ± ±

0.11 0.12 0.15 0.13 0.15 0.17

EE (%) 86.4 71.1 80.1 84.0 80.2 80.5

± ± ± ± ± ±

1.30 1.02 1.13 1.25 1.16 1.14

burst was calculated to be a reasonable value (∼1.2 μm). Even though the approximations are oversimplified, higher concentrations of copolymer than 5 mg/mL are necessary to cause a rupture of lysosomal membrane because many factors are not considered in the calculation. For example, the lysosomal membrane is likely to be more stable than accounted for in the Young−Laplace equation as it contains ∼27 mol % cholesterol,47 so the critical tension is expected to be larger than 10 mJ/m2. Therefore, it is not likely that the PHis based copolymers can rupture the lysosomal membrane under the reasonable concentration range. The membrane-lytic property of poly(L-histidine) (Mn = 5000 Da) was further evaluated with erythrocytes as a model for the endosomal membrane.34 The poly(L-histidine) with the same molecular weight (5000 Da) with the poly(L-histidine)

(3K)7), the critical diameter for the lysosomes before burst was calculated with respect to the nitrogen molarity of PHis block.43 (For a detailed description of these calculations, see Supporting Information “Osmotic pressure and critical size of endo/ lysosomes”.) According to the Young−Laplace equation, the critical diameters of lysosomes were calculated with a critical membrane tension of 10 mJ/m2.46 As shown in Table 2, the calculated critical diameters of lysosome before burst were above 10 μm when incubated with PHis based copolymers at concentration below 0.5 mg/mL, which were beyond the reasonable value that lysosomes could reach. This indicates that these PHis based copolymers were probably not able to rupturethe lysosomal membrane at a concentration below 0.5 mg/mL. When the copolymer concentration increased to a high value (5 mg/mL), the critical diameter of lysosome before 4042

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Figure 16. In vitro DOX release profiles from copolymer micelles under different pH values (pH 7.4, pH 6.8, pH 6.0, and pH 5.0).

previous developed PHis based copolymers (PHis(5K)-PEG(2K) and PHis(5K)-PEG(2K)-PLA(3K)) would have lower membrane-lytic property than poly(L-histidine) due to the PEG shielding and self-assembly behavior in the aqueous medium, preventing the PHis block from interaction with erythrocyte membrane. As shown in Figure 14, poly(L-histidine) induced no membrane-lysis at any pH conditions within the concentration up to 1 mg/mL. The membrane-lytic activity of the PHis homopolymer could be only found at acidic pH (pH 6.5 and pH 5.7) when the concentration increased to 10 mg/mL due to the protonation of imidazole groups, producing positive charge capable of binding to the erythrocyte membrane. Considering the critical diameter for endolysosome bursting and the low membrane-lytic property of the poly(Lhistidine) (even lower for PHis(5K)-PEG(2K) and PHis(5K)PEG(2K)-PLA(3K)), the endolysosomal escape pathway of them was probably attributed to the increased osmotic pressure arising from “proton sponge” effect rather than the membrane rupture. To sum up, we proposed that the endolysosomal escape of a drug facilitated by PHis based copolymers could be attributed to the “proton sponge” effect (Figure 15). The micelles are uptaken via endocytosis and trapped in endolysosomes. The PHis blocks in the copolymers become protonated and keep the proton pump functioning, leading to the influx of Cl− ions and water molecules. This causes an increase of osmotic pressure in endolysosomes and swelling of endolysosomes, resulting in the release of incorporated drug to the cytosol with the membrane remains intact.

Figure 17. In vitro cytotoxicity of mPEG45-PLA18-PHis12/DOX copolymer micelles and DOX solution against MCF-7 cells at pH 7.4 and pH 6.5 (mean ± SD, n = 6). The incubation time was 48 h. All data are presented as mean ± SD (**P < 0.01, significantly different from the micelles at neutral pH (pH 7.4); −P > 0.05, not significantly different from the DOX solution at pH 7.4).

block of the previous developed PHis based copolymers was chosen as the substitute for them. This was because the 4043

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In Vitro pH Dependent Drug Release and Cytotoxicity. To investigate pH-responsive drug release profiles of the copolymer micelles in vitro, the DOX loaded copolymer micelles were prepared using film evaporation method. The physical characterizations of the DOX loaded mPEG-PLA-PHis micelles were summarized in Table 3. DOX release profiles from the copolymer micelles under different pH values (pH 7.4, pH 6.8, pH 6.0 and pH 5.0) are shown in Figure 16. The copolymer micelles showed clear pH dependent DOX release behavior as characterized by the faster DOX release as the pH decreased from 7.4 to 5.0. The mPEG-PLA-PHis copolymer micelles showed quite similar burst release of DOX (20%) within 1 h, but remarkable pH accelerated DOX release afterward. As compared to the PHis based micelles, mPEG-PLA micelles showed similar and incomplete DOX release within 24 h under different pH values (∼55%). The slightly difference in DOX release was induced by the solubility variation of free DOX in different medium. The effect of the DP of PLA block on the triggering pH of the copolymer micelles could also be observed from Figure 16. The trigging pH is defined as the pH at which the accelerate drug release occurs. The triggering pH decreased from 6.8 to 6.0 with increasing the degree of PLA block from 12 to 18. As the DP of PLA block increased, the copolymer became more hydrophobic. Accordingly, the copolymer micelles required stronger electrostatic repulsive force to break the micellar core, inducing the burst release of incorporated drug. Therefore, the trigging pH shifted to a lower value to produce more protonated imidazole rings. This indicates that the triggering pH of the copolymer micelles could be manipulated by adjusting molecular composition of the copolymer. The in vitro cytotoxicity of DOX-loaded micelles at three different pH values characteristic22 of normal tissue (pH 7.4), tumor interstitium (pH 6.5), and early endosomal compartment (pH < 6.0) were also evaluated using the MTT assays.22 (The cell viability data at pH 6.0 are not shown in Figure 17 due to the acidic pH having a negative effect on the growth of the MCF-7 cells.) The free DOX showed similar MCF-7 cell cytotoxicity at both 10 and 25 μg/mL due to the MCF-7 cells exhibiting concentration independent responsiveness to the free DOX in this region (Figure S.1-7, Supporting Information). The DOX loaded micelles showed slightly lower cytotoxicity than DOX solution at neutral pH (pH 7.4) due to the sustained release characteristics of the DOX-loaded micelles and the different cell uptake ways of free DOX molecules and the micelles. However, at pH 6.5, the cytotoxicity of the micelles was significantly increased compared to that at neutral pH due to the accelerated DOX release in the acidic environment.

previously proposed endolysosomal membrane rupture during the endolysosomal escape pathway of the PHis based copolymers.

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis and characterization of mPEG-PLA-PHis and the calculation of osmotic pressure and critical size of endo/ lysosome. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-24-23986308. Fax: +86-24-23986306. E-mail: [email protected]. Author Contributions §

X.Z. and D.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from the National Natural Science Foundation of China (81273448, 81202483 and 81102400) as well as the program for Liaoning Excellent Talents in university (LR 2014029), Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.

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ABBREVIATIONS mPEG-PLA-PHis: Poly(ethylene glycol)-poly(D,L-lactide)poly(L-histidine) TEM: Transmission electron microscopy DLS: Dynamic light scattering DOX: Doxorubicin CLSM: Confocal laser scan microscopy MTT: 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide AO: Acridine Orange D.P: Degree of polymerization REFERENCES

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CONCLUSIONS The PHis based copolymers of mPEG-PLA-PHis with various DP of PLA and PHis were synthesized. A unique pH induced micellar core reassembly as characterized by the transition of PHis-PLA block based core to PLA block based core under acidic environment has been found to be the pH responsive mechanism for the mPEG-PLA-PHis copolymers. The endolysosomal escape mechanism of the copolymer micelles was attributed to the “proton sponge” hypothesis without the rupture of endolysosomal membrane due to the evidence of the membrane integrity. The critical diameter calculation of the endolysosomal membrane and the low membrane-lytic property of PHis homopolymer raised doubts about the 4044

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