Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz-b

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Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz‑b‑PLA Micelles with Varied Hydrophilic/Hydrophobic Ratios and Intracellular Trafficking Routes and Fate of the Copolymer Dishi Wang,† Yanxia Zhou,† Xinru Li,† Xiaoyou Qu,† Yunqiang Deng,† Ziqi Wang,† Chuyu He,† Yang Zou,† Yiguang Jin,*,‡ and Yan Liu*,† †

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China ‡ Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China S Supporting Information *

ABSTRACT: pH-responsive polymeric micelles have shown promise for the targeted and intracellular delivery of antitumor agents. The present study aimed to elucidate the possible mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles in detail, further unravel the effect of hydrophilic/hydrophobic ratio of the micelles on their cellular internalization, and examine the intracellular trafficking routes and fate of PEOz-b-PLA after internalization of the micelles. The results of variations in the size and Zeta potential of PEOz-b-PLA micelles and cross-sectional area of PEOz-b-PLA molecules with pH values suggested that electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain at pH lower than its pKa was responsible for pH-sensitivity of PEOz-b-PLA micelles. Furthermore, the studies on internalization of PEOz-bPLA micelles by MCF-7 cells revealed that the uptake of PEOz-b-PLA micelles was strongly influenced by their structural features, and showed that PEOz-b-PLA micelles with hydrophilic/hydrophobic ratio of 1.7−2.0 exhibited optimal cellular uptake. No evident alteration in cellular uptake of PEOz-b-PLA micelles was detected by flow cytometry upon the existence of EIPA and chlorpromazine. However, the intracellular uptake of the micelles in the presence of MβCD and genistein was effectively inhibited. Hence, the internalization of such micelles by MCF-7 cells appeared to proceed mainly through caveolae/lipid raft-mediated endocytosis without being influenced by their hydrophilic/hydrophobic ratio. Confocal micrographs revealed that late endosomes, mitochondria and endoplasmic reticulum were all involved in the intracellular trafficking of PEOz-b-PLA copolymers following their internalization via endocytosis, and then part of them was excreted from tumor cells to extracellular medium. These findings provided valuable information for developing desired PEOz-b-PLA micelles to improve their therapeutic efficacy and reducing the potential safety risks associated with their intracellular accumulation. KEYWORDS: PEOz-b-PLA, hydrophilic/hydrophobic ratio, pH-responsive polymeric micelles, pH-sensitivity mechanism, intracellular trafficking, endocytosis mechanism

1. INTRODUCTION Conventional chemotherapy has been proved to be only partially successful in treating and prolonging the lives of patients due to serious systemic toxicities of many anticancer drugs.1 Further, only small fraction of the drugs can be delivered to the cancer site and take effect quickly in tumor cells.2 To ameliorate this challenge, different types of pHresponsive nanosized anticancer drug carriers have been developed due to the intrinsic difference between various solid tumors and surrounding normal tissues in acidity, and a pH gradient from 6.5−7.2 in extracellular medium to 5.5−6.5 in endosomes in their cell entry pathway and from 5.5−6.5 in endosomes to 4.5−5.0 in lysosomes in their trafficking pathway inside cells. Among them, pH-responsive polymeric micelles have been by far extensively noticed and studied, and presented © 2017 American Chemical Society

indeed their great potential in more effective delivery as well as rapid drug release inside cells for antitumor drugs. For example, mPEG-b-(PPLG-g-MSA) micelles,3 mPEG-b-(PLA-co-PAE) micelles,4 mPEG-b-P(Asp) micelles,5 mPEG-b-PDEAEMAPCL micelles,6 DSPE-PEG/PEG-b-PHis mixed micelles,7 PEG-b-P(Asp-hyd-DOX) micelles8 and PEG-b-P(TMBPECco-AC) micelles.9 What is common to these micelles is that their pH-responsive area locates in the core of the micelles and that their physicochemical properties in drug delivery such as size and size distribution, structural stability, drug solubilization, drug release, and their interaction with target cells as well as Received: December 20, 2016 Accepted: February 10, 2017 Published: February 10, 2017 6916

DOI: 10.1021/acsami.6b16376 ACS Appl. Mater. Interfaces 2017, 9, 6916−6930

Research Article

ACS Applied Materials & Interfaces

2. MATERIALS AND METHODS

organ disposition and treatment efficacy have been mainly focused on in the past few years. Generally, well-designed pH-responsive polymeric micelles should have high loading efficiency and drug-incorporation stability in circulation to efficiently deliver drugs to the target, and respond to endo/lysosomal acidic pH stimulus to rapid release the encapsulated drugs inside tumor cells. In addition, it is also important for pH-responsive polymeric micelles to be internalized in a greater amount by tumor cells. There are considerable evidence that block copolymer structure in micelles influences the extent and mechanism of micellar internalization by cancer cells.10−13 Consequently, it is essential to understand the effect of chemical manipulations on the extent of micellar uptake by target cells in the design and development of pH-responsive polymeric micelles. On the other hand, the safety of developed nanocarriers should not be overlooked. To ensure that micelles forming materials are safe, it is essential to clarify whether the block copolymers are accumulated inside the cell or undergo extracellular efflux. In recent years, we developed pH-sensitive polymeric micelles based on amphiphilic copolymers poly(2-ethyl-2oxazoline)-b-poly((D,L-lactide)) (denoted as PEOz-b-PLA) with pH-responsive hydrophilic blocks for delivery of anticancer drugs.14−17 PEOz-b-PLA micelles were demonstrated to exhibit desired performance featured by nanoscaled size to benefit their EPR effect, high drug encapsulation efficiency, favorable pH-sensitivity to promote endosome escape and quick drug release, promising tumor targeting behavior, leading to enhanced antitumor efficacy with negligible systemic toxicity. Consequently, PEOz-b-PLA copolymers are promising encapsulants in drug delivery domains. However, many factors remain unknown, and more researches need to be done. For example, the detailed mechanism of pH-sensitivity of PEOz-bPLA micelles is confused. No systematic study on the effect of the structural features of PEOz-b-PLA copolymers on the cellular uptake of their micelles has yet been reported and the pathway of their cell entry is currently lacking. Moreover, little is known on the intracellular trafficking routes and fate of PEOz-b-PLA copolymers after internalization of PEOz-b-PLA micelles. To address these issues in the present study, a series of PEOz-b-PLA copolymers with various hydrophilic/hydrophobic ratios were synthesized and characterized, and their micelles were constructed and characterized. The possible mechanism of pH-sensitivity of PEOz-b-PLA micelles was investigated in detail based on variations of the size and Zeta potential of PEOz-b-PLA micelles and cross-sectional area of PEOz-b-PLA molecules with pH values. Furthermore, the effect of PEOz-b-PLA chemical composition on cellular internalization of the micelles was elucidated by use of a panel of PEOz-b-PLA analogues differing in hydrophilic/hydrophobic ratio. In addition, the potential mechanism involved in internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios by MCF-7 cells was disclosed. Finally, the trafficking routes of PEOz-b-PLA, from intracellular uptake to extracellular efflux, was evaluated. Mechanistic understanding of these issues may give new insights into more rational design of PEOz-b-PLA copolymers with optimal chemical manipulation to fabricate desired pH-responsive micelles.

2.1. Materials. Paclitaxel (PTX) was purchased from Guilin Huiang Biopharmaceutical Co. Ltd. (Guilin, China). D,L-Lactide obtained from Daigang Biological Technology Co. Ltd. (Jinan, China) was purified by recrystallization with the mixture of benzene and ethyl acetate (6:4, v/v).18 Stannous octoate was the product of Aladdin reagent company (Shanghai, China). 2-ethyl-2-oxazoline supplied by Sigma-Aldrich (St Louis, MO) was dried by vacuum distillation over calcium hydride. Thionyl chloride (SOCl2) was purchased from Ouhechem Technology Co., Ltd. (Beijing, China). 7-N,N-diethylamino-coumarin-3-carboxylic acid (DEC-3-COOH) was purchased from Bide Pharmatech Ltd. (Shanghai, China). Coumarin-6 (C6), sulforhodamine B sodium salt (SRB), chlorpromazine, 5-(N-ethyl-Nisopropyl)-amiloride (EIPA), methyl-β-cyclodextrin (MβCD), and genistein were obtained from Sigma-Aldrich (St Louis, MO). DiO and DiI were supplied by J&K Chemical Ltd. (Shanghai, China). CellLight Late Endosomes-RFP, LysoTracker Red DND-99, ER-Tracker Red, Bodipy TR Ceramide complexed to BSA and MitoTracker Deep Red FM were products of Life Technologies (Brooklyn, NY). RPMI-1640 cell culture medium, fetal bovine serum (FBS), trypsin-EDTA, penicillin-streptomycin solution, 6-well and 96-well tissue culture plates, and 25 and 75 cm2 plastic culture flasks were supplied by M&C Gene Technology (Beijing, China). Glycerin jelly and 4% paraformaldehyde solution were obtained from Biodee Biotechnology Co., Ltd. (Beijing, China). All other reagents and chemicals were of analytical grade or better. 2.2. Synthesis and Characterization of PEOz-b-PLA Copolymers. PEOz-b-PLA diblock copolymers were synthesized via a twostep reaction procedure as previously reported.14 A series of monohydroxyl poly(2-ethyl-2-oxazoline) (PEOz−OH) polymers with different chain lengths were first synthesized by controlling the mass ratio of 2-ethyl-2-oxazoline (EOz) to methyl p-toluenesulfonate (MeOTs) through the cationic ring-opening polymerization. Then the resulting PEOz−OH, used as an initiator, was polymerized with D,LLA to prepare PEOz-b-PLA copolymers with varied PLA chain lengths by controlling the feed ratio of D,L-LA to PEOz−OH using stannous octoate as the catalyst through ring-opening polymerization. The obtained PEOz−OH and PEOz-b-PLA were confirmed by the 1H NMR spectrum. Gel permeation chromatography was used to determine molecular weight and the molecular weight distribution of the synthesized PEOz−OH. PEOz-b-PLA diblock copolymers with different PEOz and PLA chain lengths were referred to as PEOzx-b-PLAy, wherein x and y represented the averaged molecular weight of PEOz and PLA in kD, respectively. 2.3. Synthesis and Characterization of DEC-Labeled PEOz-bPLA. 2.3.1. Synthesis of DEC-Labeled PEOz-b-PLA. To track the intracellular transport pathway of PEOz-b-PLA, the copolymer was labeled with DEC-3-COOH. In brief, DMF (50 μL) as the catalyst was added to the solution of DEC-3-COOH (50 mg) in thionyl chloride (4 mL), and then the mixture was stirred at 80 °C under reflux for 6 h. After removing all the solvents by rotary evaporation, the residue and 500 mg of PEOz6.7-b-PLA4 were dissolved in 5 mL of CH2Cl2, and then 50 μL of trimethylamine (TEA) was added as the acid-binding agent. After stirring the reaction mixture in an ice bath in the dark for 12 h, the solvent was then removed by rotary evaporation. The solution of the crude product in methanol was dialyzed using a dialysis bag (MWCO 3500, Millipore Co. Ltd.) first against DMSO for 7 days to remove unconjugated DEC-3-COOH, and then against deionized water for 24 h to remove any organic solvent, followed by lyophilization for storage. DEC-labeled PEOz-b-PLA (denoted as PEOz-b-PLA-DEC) was confirmed by thin layer chromatography (TLC) and 1H NMR spectrum. 2.3.2. Spectral Measurement and Fluorescence Stability. The excitation and emission spectra of PEOz-b-PLA-DEC copolymers in methanol at 1.0 mg/mL were recorded on a Shimadzu fluorescence spectrophotometer (RF5301, Japan). Then, to explore whether the fluorescence stability of PEOz-b-PLA-DEC is affected by the physiological and acidic environment, PEOz-b-PLA-DEC micelles were redispersed in fresh serum-free cell culture medium with different 6917


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ACS Applied Materials & Interfaces pH at 37 °C and the fluorescent intensity of each micelle solution at concentration of 2.0 mg/mL for PEOz-b-PLA-DEC was determined as a function of time (0−10 h) at 398 nm of maximum excitation wavelength. 2.4. Determination of pKa of PEOz-b-PLA. Acid dissociation constant (pKa) of PEOz6.7-b-PLA4 was determined by acid−base titration with sodium hydroxide as previously reported.14 2.5. Measurement of Surface Pressure-Molecular Area (π-A) Isotherms of PEOz-b-PLA. The surface pressure-molecular area (πA) isotherms of amphiphilic copolymer PEOz-b-PLA molecules were measured on the surface of phosphate buffered saline (PBS, 10 mM) solution with different pH values (5.0, 6.5, and 7.4) at 25 °C using a Minitrough film balance (KSV Instruments, Finland) equipped with dual barriers and a Pt Wilhelmy plate-sensing device according to the published reports.19,20 The Teflon trough has an area of 24 300 mm2 and a width of 75 mm. Prior to each test, the cleanness of the subphase surface was verified by compressing the surface area to a minimum and monitoring the change in surface pressure. In brief, 100 μL of PEOz-bPLA solution (0.2 mg/mL) in chloroform was carefully spread on the air/PBS interface using a Hamilton microsyringe. The PEOz-b-PLA layer on the subphase surface was symmetrically compressed by two mobile barriers at a constant speed (10 mm/min) after the complete evaporation of chloroform (about 20 min). The π-A isotherms were recorded in real time with a Wilhelmy plate. In the solid phase, an extrapolation of the linear portion of the π-A isotherm to the surface pressure of zero to obtain the average area per molecule which corresponds to the actual cross-sectional area of the molecule.21 2.6. Preparation of Drug-Loaded Polymeric Micelles. PTXloaded PEOz-b-PLA polymeric micelles were prepared by the dialysis method. Briefly, PEOz-b-PLA (50 mg) and PTX (5 mg) (10:1, w/w) were dissolved in 20 mL of methanol, then the mixture was dialyzed against 1 L of deionized water using a dialysis bag (MWCO 3500, Millipore Co. Ltd.), which was refreshed every 3 h in the course over 24 h, followed by filtration through a Sephadex G-25 column to remove nonencapsulated PTX and lyophilization for storage. C6-, DiI/DiO-loaded PEOz-b-PLA polymeric micelles were prepared as mentioned above except that the C6/polymer ratio was 1:1000 (w/w) for C6-loaded polymeric micelles, the DiO/polymer and DiI/polymer ratio was 1:500 (w/w) for (DiI/DiO)-loaded micelles (denoted as FRET micelles), respectively. 2.7. Physicochemical Characterization of Polymeric Micelles. The particle size and size distribution (polydispersity index, PDI), and Zeta potential of PEOz-b-PLA micelles were analyzed by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, UK) with a scattering angle of 90° at 25 °C after diluting the micelle solution to an appropriate volume with deionized water. Each measurement was repeated three times and an average value was reported. The morphology of PEOz-b-PLA micelles was visualized by transmission electron microscope (TEM, JEM-1230, JEOL, Japan). Before examination, the sample was stained with a drop of 1 wt % phosphotungstic acid solution, and then placed on a copper grid with carbon film followed by removal of the excess fluid with filter paper, and dried for 48 h. The loading content (LC) of the micelles was determined as previously reported.15,22 In vitro release of PTX from the micelles was conducted using a dialysis-bag diffusion technique with little modification.15 Briefly, 2 mL of PTX-loaded micelle solution was introduced into a dialysis bag (MWCO 3500), and then the sealed bag was immersed into 25 mL of PBS (10 mM, pH 5.0, 6.5, and 7.4) with 0.2% Tween 80, which was kept at 37 °C with continuously shaking at 100 rpm. At predetermined time intervals, 1 mL of the release medium was taken out and immediately replaced with 1 mL of fresh medium. The content of released PTX in the medium was analyzed with a Shimadzu series HPLC system (Shimadzu LC-10AT, Kyoto, Japan) equipped with a UV detector (Shimadzu SPD-10A) and reversed phase column (ODS C18, 5 μm, 4.6 × 250 mm, Dikma, China). The mobile phase consisted of water (containing 0.28% SDS and 0.14% phosphoric acid): acetonitrile =50:50 (v/v), and was pumped at a flow rate of 1.0 mL/min. The detection wavelength was 227 nm. The column

temperature was set to 25 °C. In the assessment of drug release behavior, the accumulative release amount of PTX was calculated, and the cumulative release percentages of PTX from micelles by the amount of loaded drug were plotted against time. 2.8. Cell Culture. MCF-7 cells were obtained from Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% Penicillin-Streptomycin in 5% CO2 humidified atmosphere at 37 °C. 2.9. Micelle Integrity Analysis after Being Internalized by Cells. The integrity of the micelle structure was evaluated through the release of core-loaded hydrophobic fluorescent probes from micelles by using the Förster resonance energy transfer (FRET) method after they were internalized by cells.15 In brief, MCF-7 cells were seeded on a 35 mm glass bottom culture dish coated with poly-L-lysine (SigmaAldrich, St. Louis, MO) at a density of 3.0 × 105 cells per dish and were allowed to culture at 37 °C under 5% CO2 for adherence prior to study. After their confluency was about 80%, the culture medium was removed and refreshed with the medium containing FRET micelles (the final concentration of both DiO and DiI was 50 ng/mL, respectively). After 1 h of incubation, the cells were rinsed three times with cooled PBS and fixed with 5% paraformaldehyde for 15 min at 37 °C, followed by rinsing thrice with PBS, and finally sealed with glycerin jelly for visualizing FRET images using confocal laser scanning microscope (CLSM, TCS, SP5, Leica, Germany). FRET images were acquired by detecting sensitized emission at 555−655 nm for DiI (excitation at 549 nm) with the excitation at 488 nm for DiO (emission at 500−530 nm). 2.10. Cytotoxicity Assessment of Endocytosis Inhibitors. Prior to the investigation toward endocytosis pathway of PEOz-b-PLA micelles, the cytotoxicity of different endocytosis inhibitors was evaluated using the SRB assay.23 Briefly, MCF-7 cells were seeded in 96-well plates at a density of 1.0 × 104 cells per well and incubated in a 5% CO2 humidified atmosphere at 37 °C for 24 h. Then the culture medium was removed, 200 μL of fresh serum-free medium containing various endocytosis inhibitors with serial concentration was added to each well and cultured for a further 4.5 h. The medium was then removed, cells were rinsed with cold PBS for three times followed by fixing with 200 μL of 10% trichloroacetic acid (TCA) for 1 h at 4 °C. After TCA was removed, cells were rinsed five times with deionized water, dried in the air and stained with 100 μL of 4% (w/v) SRB in 1% (v/v) acetate solution at room temperature for 30 min followed by rinsing five times with 1% acetic acid and drying at 37 °C. The absorbance of each well was determined at 540 nm using a microplate reader (BIO-RAD model 680, Shanghai, China) after the bound dye in each well was dissolved in 200 μL of 10 mM Tris. The fresh serum-free RPMI-1640 was used as negative control. The cytotoxicity of tested samples expressed by the relative cell viability was the ratio of the absorbance of the tested groups to that of the negative control (100%). 2.11. Cellular Uptake Analysis of Micelles by Flow Cytometry. MCF-7 cells were seeded in 6-well plates at a density of approximately 3 × 105 cells per well and incubated for 24 h at 37 °C under 5% CO2. The culture medium was then removed, and the cells were washed three times with PBS, followed by incubation with C6loaded or DEC-labeled micelles in serum-free medium at 37 °C. The final concentration of C6 was 100 ng/mL, and DEC-labeled polymer was 2 mg/mL. MCF-7 cells incubated with serum-free medium were used as control. After 4 h incubation, the medium was removed, and the cells were rinsed three times with cold PBS, trypsinized and harvested with 0.4 mL of 0.25% (w/v) trypsin-0.1% (w/v) EDTA solution, collected by centrifugation followed by washing thrice with PBS, and then resuspended in 1 mL of precold PBS followed by filtration through a nylon mesh. Finally, the intracellular fluorescence intensity was determined by a FACScan flow cytometer (FACScan, Becton Dickinson, San Jose, CA) at 488 nm excitation wavelength and 560 nm emission wavelength for C6, and 405 nm excitation wavelength and 455 nm emission wavelength for DEC, respectively. For each sample, 10 000 events were collected and the data were analyzed in triplicate. 6918


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ACS Applied Materials & Interfaces 2.12. Endocytosis Pathway of Micelles. To investigate whether the cellular uptake of PEOz-b-PLA micelles is energy-dependent, the cells were preincubated at 4 °C for 0.5 h and then treated with C6loaded micelles with a final C6 concentration of 100 ng/mL for another 4 h at 4 °C. The fluorescence intensity of intracellular C6 was collected by flow cytometry as described in Section 2.11. To explore the endocytosis pathway of micelles, MCF-7 cells were preincubated at 37 °C in culture medium with each endocytosis inhibitor at a safe and effective concentration. Following the preincubation for 0.5 h, the medium was removed, and C6-loaded PEOz-b-PLA micelles in serum-free medium with endocytosis inhibitors was added and further incubated for another 4 h. The fluorescence intensity of intracellular C6 was collected by flow cytometry as described in Section 2.11. 2.13. Intracellular Trafficking Tracked by Confocal Microscope. To investigate the intracellular trafficking process of micelles, the colocalization of PEOz-b-PLA micelles with the intracellular compartments was observed visually with a confocal microscope.15 The intracellular compartments were labeled by organelle-specific fluorescent probes in accordance with the manufacturer’s instructions. Specifically, for endosomes labeled with CellLight Late EndosomesRFP and lysosomes labeled with Lyso-Tracker Red DND-99, the cells were incubated with fresh culture medium with CellLight Late Endosomes-RFP (5 μL) at 37 °C for 30 h and with Lyso-Tracker Red DND-99 (200 nM) at 37 °C for 30 min before treated with DEClabeled micelles in culture medium, respectively. For endoplasmic reticulum (ER) marked by ER-Tracker Red, Golgi apparatus stained with Bodipy-TR C5-ceramide complexed to BSA, and mitochondria labeled with Mito-Tracker Deep RED, the cells were incubated with fresh culture medium with ER-Tracker Red (4 μM) at 37 °C for 30 min, with Bodipy-TR C5-ceramide complexed to BSA (10 μM) at 4 °C for 30 min and then at 37 °C for another 30 min and with MitoTracker Deep RED (200 nM) at 37 °C for 30 min after treated with DEC-labeled micelles in culture medium, respectively. Briefly, MCF-7 cells (3.0 × 105) were seeded on 35 mm glass-bottom dishes coated with poly-L-lysine in medium containing 10% FBS and 100 U/mL penicillin/streptomycin. After 24 h incubation at 37 °C under 5% CO2, the culture medium was removed, and the cells were washed thrice with HBSS and then exposed to DEC-labeled micelles in culture medium with a final polymer concentration of 2 mg/mL. At a predetermined time after addition of the micelles, the cells were washed thrice with HBSS and kept in HBSS for imaging with CLSM (TCS SP8, Leica, Germany). 2.14. Determination of Intracellular Level of the Polymers. MCF-7 cells (3.0 × 105) were seeded in 6-well plates and cultured in RPMI-1640 cell culture medium containing 10% FBS and 100 U/mL penicillin/streptomycin. The culture medium was removed after incubation for 24 h, the cells were rinsed with serum-free medium three times followed by addition of the micelle solution with a final DEC-labeled polymers concentration of 2 mg/mL. Following 3 h incubation, the medium was removed, and the cells were washed three times with serum-free medium. After an additional 1, 2, 4, and 6 h incubation in complete medium, respectively, the medium was removed, and the cells were rinsed thrice with cold PBS, trypsinized and harvested with 0.4 mL of 0.25% (w/v) trypsin-0.1% (w/v) EDTA solution, collected by centrifugation followed by washing thrice with PBS, and then resuspended in 1 mL of precold PBS followed by filtration through a nylon mesh. Finally, the intracellular fluorescence intensity of PEOz-b-PLA-DEC was determined by a FACScan flow cytometer (FACScan, Becton Dickinson, San Jose, CA) at 405 nm excitation wavelength and 455 nm emission wavelength. 2.15. Statistical Analysis. All results were presented as mean ± SD unless particularly outlined. The statistical significance of differences among more than two groups was determined by a oneway ANOVA. A p-value of 0.05 or less was considered to be statistically significant.

3. RESULTS 3.1. Synthesis and Characterization of PEOz-b-PLA Copolymers. A series of monohydroxyl poly(2-ethyl-2-oxazoline) (PEOz−OH) with different chain lengths were first synthesized and confirmed by 1H NMR spectra in CDCl3. The peaks at 3.04 and 3.38 ppm (Supporting Information (SI) Figure S1A) were attributed to the protons from the terminal methyl and methylene groups in the backbone of the PEOz− OH, respectively. The peaks at 1.05 and 2.33 ppm were assigned to the protons of the methyl and methylene groups in the side chains, respectively. The number-averaged molecular weight (Mn) and its distribution of all synthesized PEOz−OH polymers determined by GPC were presented in Table S1 (SI). The results suggested that the obtained PEOz−OH had wellcontrolled Mn with narrow distribution. Next, PEOz-b-PLA block copolymers with varied PEOz and PLA chain lengths were synthesized by controlling the mass ratio of PEOz−OH to D,L-LA through ring-opening polymerization. The characteristic peaks of PEOz (1.05, 2.33, and 3.38 ppm) and PLA (1.49 ppm, 5.10 ppm) were identified (SI Figure S1 B), which were in good agreement with our previous reports.14,15 As the number-averaged molecular weight of PEOz−OH was measured by GPC, the molecular weight of PLA listed in Table 1 was determined by calculating the peak area of the protons in characteristic groups in PEOz and PLA extracted from the 1H NMR spectra. Table 1. Characterization of PEOz-b-PLA Copolymers

a

Mn (kD) of PEOz

Mn (kD) of PLA

abbreviation of copolymers

6.0 6.0 6.0 6.0 6.0 6.0 2.6 3.3 4.5 5.6 6.7 8.9

1.1a 2.2 3.9 8.5 10.0 13.7 3.9 3.7 4.5 4.5 3.7 4.4

PEOz6-b-PLA1.1 PEOz6-b-PLA2.2 PEOz6-b-PLA3.9 PEOz6-b-PLA8.5 PEOz6-b-PLA10 PEOz6-b-PLA13.7 PEOz2.6-b-PLA4 PEOz3.3-b-PLA4 PEOz4.5-b-PLA4 PEOz5.6-b-PLA4 PEOz6.7-b-PLA4 PEOz8.9-b-PLA4

determined by 1H NMR.

Further, the pKa of the synthesized PEOz6.7-b-PLA4 was determined to be 6.5, obtained from acid−base titration profiles (SI Figure S2). 3.2. Synthesis and Characterization of DEC-labeled PEOz-b-PLA. In order to track the intracellular transport pathway of PEOz-b-PLA, DEC-3-COOH was conjugated to the end of the hydrophobic segment of PEOz6.7-b-PLA4. DEC-3COOH was first activated by treatment with thionyl chloride (SI Figure S3), which was supported by TLC of the product DEC-3-COCl compared with DEC-3-COOH (Figure 1C1). Afterward, PEOz6.7-b-PLA4-DEC was synthesized by conjugation of DEC-3-COCl to the terminal hydroxyl group of PEOz6.7-b-PLA4. The successful esterification was verified by 1 H NMR spectra (Figure 1A) and TLC (Figure 1C2). In addition to the characteristic peaks of PEOz6.7-b-PLA4, the triple peak of 1.20, 1.22, and 1.24 ppm, which was assigned to the methyl protons of DEC-3-COOH (Figure 1B), was also observed in Figure 1A, implying that the conjugate had been 6919


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Figure 1. 1H NMR spectra of PEOz-b-PLA-DEC (A) and 7-N,N-diethylamino-coumarin-3-carboxylic acid (DEC-3-COOH) (B) in CDCl3. (C) The schematic diagrams of thin layer chromatograms of DEC-3-COOH (DECA), DEC-3-COCl (DOCl), DEC-3-COOCH3 (DCH), PEOz-b-PLA (PP), and PEOz6.7-b-PLA4-DEC (PPD). PEOz6.7-b-PLA4 was developed to be faint yellow spots by iodine fumigation; PEOz6.7-b-PLA4-DEC, DEC-3COOH, DEC-3-COCl, and DEC-3-COOCH3 showed light blue fluorescence under 365 nm UV lamp.

Figure 2. (A) Fluorescence excitation spectrum (red) and emission spectrum (black) of PEOz6.7-b-PLA4-DEC at a concentration of 1.0 mg/mL in methanol. (B) Morphological characteristics of the representative PEOz4.5-b-PLA4 micelles observed by TEM.

Table 2. Physicochemical Characteristics of Various Drug-loaded Micelles (n = 3) encapsulated drug C6

PTX

PEOzx-b-PLAy PEOz6-b-PLA1.1 PEOz6-b-PLA2.2 PEOz6-b-PLA3.9 PEOz6-b-PLA8.5 PEOz6-b-PLA10 PEOz6-b-PLA13.7 PEOz2.6-b-PLA4 PEOz3.3-b-PLA4 PEOz4.5-b-PLA4 PEOz5.6-b-PLA4 PEOz6.7-b-PLA4 PEOz8.9-b-PLA4 PEOz6-b-PLA4

diameter (nm) 28.82 34.89 60.48 71.93 83.27 128.1 44.81 48.74 58.22 58.23 63.12 66.94 38.20

± ± ± ± ± ± ± ± ± ± ± ± ±

1.34 3.98 6.41 4.12 3.12 1.54 2.78 5.31 4.13 3.11 2.64 3.41 0.73

PDI 0.29 0.70 0.15 0.21 0.21 0.23 0.11 0.17 0.13 0.05 0.14 0.14 0.21

± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.15 0.03 0.01 0.04 0.05 0.01 0.02 0.05 0.02 0.03 0.05 0.03

LC (wt %) 0.18 0.18 0.19 0.20 0.19 0.19 0.19 0.19 0.19 0.19 0.19 0.19 8.66

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.01 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.01 0.10

fluorescence spot, being similar to that of DEC-3-COOH, of PEOz6.7-b-PLA4-DEC was found at the same site of PEOz6.7-

successfully synthesized. Furthermore, TLC suggested the formation of PEOz6.7-b-PLA4-DEC, in which the nattier blue 6920


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Figure 3. (A) In vitro release profiles of PTX from PEOz6-b-PLA4 micelles in PBS with 0.2% Tween 80 at 37 °C (n = 3). (B) Variations of the size for PEOz6-b-PLA4 micelles as a function of time in PBS with different pH values at 37 °C (n = 3). (C) Zeta potential of the blank PEOz6-b-PLA4 micelles as a function of pH of medium. All micelles were stabilized at each pH for 10 min prior to measuring Zeta potential (n = 3). (D) Surface pressure-molecular area isotherms from the PEOz6-b-PLA4 monolayer on PBS with different pH values. The compression speed was kept at 10 mm/min.

b-PLA4, whereas this phenomenon was not observed for PEOz6.7-b-PLA4 (Figure 1C2). Further, the synthesized PEOz6.7-b-PLA4-DEC exhibited stable excitation−emission spectra (Figure 2A) with 456 nm emission wavelength and 398 nm excitation wavelength. Thus, it is suitable for imaging in the subsequent studies. 3.3. Characterization of Polymeric Micelles. To investigate the effect of the structure of PEOz-b-PLA on the internalization of its micelles, a series of C6-loaded PEOz-bPLA micelles were prepared by the dialysis method and characterized by mean size and size distribution (polydispersity index, PDI) and loading content. As listed in Table 2, all PEOzb-PLA copolymers with different PEOz and PLA chain lengths could self-assemble into polymeric micelles with nanoscaled size ranging from 28 to 128 nm in diameter and narrow distribution. Notably, the mean size of micelles became larger with the increase of PEOz and PLA chain length, and the influence of PLA chain length on the micelle size was found to be stronger than that of PEOz chain length, which might be attributed to the difficulty to form compact micelles for PEOzb-PLA copolymers with longer PLA chain length. Further, all C6-loaded PEOz-b-PLA micelles exhibited comparable loading content of about 0.2%. In contrast, PTX-loaded PEOz-b-PLA micelles had smaller diameter and higher loading content, which might be assigned to stronger interaction of PTX than that of C6 with the inner core of PEOz-b-PLA micelles and lower rigidity of PTX than that of C6. A typical representation of the morphological characteristics for the micelles examined by TEM was presented in Figure 2B. It was clearly seen that the micelles were spherical or spheroid in shape, and the size was slightly appreciable decreased compared with the result of DLS measurements, which might be attributed to the shrinkage of the micelles on the grid surface during the drying process of TEM sample and the swelling or

stretching of the shell-forming polymer chain in the hydrated state of the micelles for DLS measurement. 3.4. Variations in Physicochemical Properties of PEOz-b-PLA Micelles and Their Components with pH. 3.4.1. In Vitro pH-Dependent Release of PEOz-b-PLA Micelles. The in vitro drug release behavior of PTX-loaded PEOz6-bPLA4 micelles was determined by a dialysis method under physiological conditions (pH 7.4) that mimics the blood environment and in the tumor acidic microenvironment (pH 6.5) and endo/lysosome mimetic acidic circumstance (pH 5.0). As anticipated, PTX release from the micelles was pHdependent and markedly accelerated with decreasing pH value (Figure 3A). More precisely, at pH 7.4, PEOz6-b-PLA4 micelles showed slower release during the whole experiment period, and about 67% of PTX was released within 48 h, however, PTX release was enhanced at pH 6.5 and pH 5.0, and the cumulative release was almost 75% and 90% after 48 h. Specifically, within the first 12 h, the release of PTX was suppressed at pH 7.4, and the PTX release curve reached a plateau with accumulative release of about 61%. Comparatively, at pH 6.5, about 65% of PTX was leaked at 12 h, and the release was sustained thereafter. PTX release was considerably accelerated at pH 5.0, and the release of PTX burst to approximately 77% at 12 h, and the release was sustained thereafter. These results suggested that PEOz-b-PLA micelles could distinguish endo/lysosomal pH and tumor extracellular pH from physiological pH by accelerating drug release. This release behavior of triggering the rapid release of hydrophobic anticancer drugs in the tumor microenvironment and endo/ lysosomal acidic environment for PEOz-b-PLA micelles, which was consistent with our previous studies,14,15 was highly advantageous for targeted cancer therapy owing to the fact that the premature leakage of drug encapsulated in micelles might be significantly suppressed during circulation in the 6921


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Figure 4. Flow cytometry analysis results of the cellular uptake for various C6-loaded PEOz-b-PLA micelles with varying PLA chain lengths (A) and with varying PEOz chain lengths (B) by MCF-7 cells (n = 3). *p < 0.05, **p < 0.01 compared with PEOz6-b-PLA3.9 micelles and PEOz6.7-b-PLA4 micelles, respectively. (C) Effects of hydrophilic/hydrophobic ratio (MPEOz/MPLA) of PEOz-b-PLA on the cellular uptake of C6-loaded PEOz-b-PLA micelles by MCF-7 cells.

bloodstream and the drug might be quickly released inside cells once the micelles have been internalized via endocytosis, thereby providing a sufficient amount of free drug to rapid and effectively kill the cancer cells. 3.4.2. Effect of pH on the Size and Zeta Potential of PEOzb-PLA Micelles. To examine the pH-dependence of the size and Zeta potential of PEOz-b-PLA micelles, the blank micelles with 1.0 mg/mL of the copolymer were first exposed to PBS (10 mM) with different pH values (5.0, 6.5 and 7.4) at 37 °C for 30 min, and then size and Zeta potential of the micelles were recorded as a function of time (0−4 h). As shown in Figure 3B, no obvious change in size of the micelles was observed within 4 h at the same pH value. On the other hand, the micelles did not show markedly variation in size with decreasing pH value. The pH-responsive feature of the micelle surface was evaluated by measuring Zeta potential. Figure 3C illustrated the variation of Zeta potential of the blank micelles with pH of medium. Remarkably, however, PEOz6-b-PLA4 micelles exhibited a slightly negative charge with −2.33 mV of Zeta potential under physiological conditions (pH 7.4), which could be attributed to the adsorption of anions (Cl− or OH−) from the dispersed medium. As pH declined from 7.4 to 5.0, the surface charge of the micelles was switched from negative to positive, and Zeta potential increased from −2.33 mV at pH 7.4 to 1.39 mV at pH 6.5 and 4.07 mV at pH 5.0, suggesting that

the charge reversion of the micelles is due to the ionization of amide groups of PEOz located in the outer shell of the micelles, leading to partial charge neutralization. These results revealed that positive charge density of PEOz-b-PLA micelles surface increased with decreasing pH from 6.5 to 5.0 as the pH value in the environment was lower than the pKa of PEOz-b-PLA. 3.4.3. Effect of pH on the Cross-Sectional Area of PEOz-bPLA. It has been well-known that determination of the surface pressure-molecular area isotherm gives an insight into the monolayer film behavior of the amphiphilic substance. Further, the monolayer film behavior changes with the amphiphilic substance and depends largely on the molecular structure and interaction between the amphiphilic molecules.24 Accordingly, to obtain the information about pH-dependence of the interaction between PEOz-b-PLA molecules, PEOz6-b-PLA4 solution in chloroform was spread on the air/PBS interface by using a Langmuir minitrough to form a monolayer anchored by the PLA blocks, and the π-A isotherms for the monolayer was determined.19,20 Figure 3D displayed a series of π-A isotherms of PEOz6-b-PLA4 Langmuir monolayers at different pH values obtained at a compression rate of 10 mm/min at a subphase temperature of 25 °C. Noticeable surface area dependence was found in surface pressure, clearly indicating that PEOz6-b-PLA4 formed an insoluble monolayer on the subphase surface, and there was no exchange of the copolymer between the 6922


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Figure 5. FRET analysis of (DiO/DiI)-loaded PEOz6-b-PLA4 micelles after they were incubated with MCF-7 cells for 1 h. The Ex/Em of DiO and DiI lines was DiO/DiO, DiI/DiI (488/501 nm, 549/563 nm). The Ex/Em of FRET line was DiO/DiI (488/563 nm).

monolayer and the subphase.25 Even more importantly, the average molecular area of PEOz6-b-PLA4 in the monolayer increased gradually as the pH of the subphase declined from 7.4 to 5.0. Specifically, the average molecular area of PEOz6-bPLA4 at pH 7.4, 6.5, and 5.0 was about 380 Å2, 430 Å2 and 550 Å2, respectively, and the average molecular area of PEOz6-bPLA4 at pH 6.5 was slightly larger than that at pH 7.4, implying that there might exist the electrostatic repulsion between PEOz blocks. 3.5. Effect of Chemical Composition of PEOz-b-PLA on Cellular Uptake of the Micelles. Effects of PEOz and PLA chain lengths on the internalization of PEOz-b-PLA micelles by MCF-7 cells were estimated by flow cytometry. Figure 4 presented the cellular uptake of encapsulated C6 for incubating MCF-7 cells with C6-loaded PEOz-b-PLA analogues micelles for 4 h. Among the tested PEOz6-b-PLAy analogues micelles with an identical PEOz chain length and varied PLA chain length (Figure 4A), maximum uptake was observed at a PLA chain length of about 3.9k. Although there was no statistically significant difference in cellular uptake among PEOz6-bPLA1.1, PEOz6-b-PLA2.2 and PEOz6-b-PLA3.9 micelles (p > 0.05), the cellular uptake of PEOz6-b-PLA3.9 micelles was obviously more than that of PEOz6-b-PLA1.1 and PEOz6-bPLA2.2 micelles, respectively. The cellular uptake was still noteworthy, but not as pronounced for PEOz6-b-PLA8.5, PEOz6-b-PLA10 and PEOz6-b-PLA13.7 micelles compared with PEOz6-b-PLA3.9 micelles (p < 0.01). Furthermore, the internalization of PEOz-b-PLA micelles with similar PLA chain lengths and varied PEOz chain length of 2.6k to 8.9k was also assessed (Figure 4B). After 4 h incubation of PEOz-b-PLA micelles with MCF-7 cells, an increase in PEOz chain length from 2.6k to 6.7k, the cellular uptake of PEOzx-bPLA4 micelles was increased by almost 1.01-, 0.97-, 1.22and1.34-fold, respectively. Further increase in the chain length of PEOz block from 6.7k to 8.9k, the micellar uptake was reduced by 1.10-fold. Thus, PEOzx-b-PLA4 copolymers with a PEOz chain length of about 6.7k exhibited the maximum micellar uptake. Based on these results, it could be concluded that the structural composition of PEOz-b-PLA had a remarkable effect on cellular uptake of the micelles. Notably, as illustrated in Figure 4, the effect of PEOz (the outer shell forming block) chain length (Figure 4A) has greater impact on the internalization of PEOz-b-PLA micelles by MCF-7 cells compared with PLA (the inner core forming block) chain length (Figure 4B), which was in agreement with previous research toward PEG-b-PCL micelles.13 Consequently, the chemical composition of PEOz-b-PLA contributed to micellar uptake to a certain extent.

Moreover, variations in the length of both hydrophilic PEOz chain and lipophilic PLA chain result in PEOz-b-PLA copolymer analogues with different molecular weight and distinct hydrophilic−hydrophobic properties. Therefore, to gain a better view of the relation between cellular uptake and micelle composition, the ratio of the molecular weight of PEOz chain (MPEOz) to that of PLA chain (MPLA), defined as hydrophilic/hydrophobic ratio, which was herein used to represent the general hydrophilic/hydrophobic property of PEOz-b-PLA, was plotted against intracellular fluorescence intensity of C6 after 4 h incubation of C6-loaded PEOz-b-PLA micelles with different composition with MCF-7 cells. As shown in Figure 4C, an interesting phenomenon was observed. The effect of structural composition of PEOz-b-PLA on cellular uptake of the micelles was PEOz and PLA chain length dependent, and the relationship between MPEOz/MPLA and micellar uptake exhibited a bell-shaped curve for PEOz-b-PLA analogues micelles with identical PEOz chain length and similar PLA chain length, respectively. Specifically, for PEOz-b-PLA analogues micelles with identical PEOz chain length, MPEOz/ MPLA was around 2.0 at the maximum cellular uptake. Meanwhile, PEOz-b-PLA analogues micelles with similar PLA chain length showed a similar phenomenon, cellular uptake of the micelles peaked when MPEOz/MPLA was around 1.7. These suggested the existence of more potent copolymers with MPEOz/MPLA of 1.7 to 2.0, at which PEOz-b-PLA micelles were apt to be internalized by MCF-7 cells. In conclusion, the composition or hydrophilic/hydrophobic ratio of the block copolymers appeared to play an important role in cellular uptake of their micelles. 3.6. Micelles Integrity Analysis after Their Entry into Cells. FRET method was used to evaluate the structure integrity of PEOz-b-PLA micelles after they were taken up by tumor cells.26 DiO and DiI were therefore physically coencapsulated into the inner core of PEOz-b-PLA micelles (denoted as FRET micelles) with an average diameter of 70.50 ± 4.13 nm and PDI of 0.17 ± 0.02 measured by dynamic light scattering due to the fact that the fluorescence excitation spectrum of DiO is partially overlapped with the fluorescence emission spectrum of DiI,27 which is necessary for FRET pair. As known, FRET occurs as DiI and DiO are in the close proximity to each other (10 nm),28 resulting in weaker emission at 563 nm and stronger emission at 501 nm.26 SI Figure S4 presented the fluorescence spectra of FRET micelles dispersed in deionized water and acetone with the excitation at 488 nm, respectively. As expected, the emission intensity of DiO at 501 nm was 6923


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Figure 6. (A) Cytotoxicity of various endocytosis inhibitors at different concentration against MCF-7 cells after incubation for 30 min (n = 5). (B) Effects of endocytosis inhibitors on the cellular uptake of C6-loaded PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratio by MCF-7 cells (n = 3). * p < 0.05, ** p < 0.01 compared with the control group.

markedly lower than that of DiI at 563 nm in deionized water, which implied the energy transfer from donor DiO to acceptor DiI. Thus, FRET micelles exhibited a strong FRET effect between two interacting partners with a FRET ratio IR/(IR +IG) of 0.61, where IR and IG are fluorescence intensity of DiI at 563 nm and that of DiO at 501 nm, respectively, while FRET ratio was 0.27 in acetone. These indicated that the FRET micelles were constructed successfully and could be thus used to detect whether the core-loaded molecules are inside micelles. After FRET micelles were incubated with MCF-7 cells for 1 h at 37 °C, a strong FRET phenomenon between two interacting partners was also observed inside the cells and colocalization of DiO with DiI was obvious without discrete green or red light spots (Figure 5), suggesting that the FRET pair, DiO and DiI, was still closely encapsulated inside the core of the micelles after being internalized by cells. Consequently, it could be concluded that PEOz6-b-PLA4 micelles were taken up by MCF-7 cells in the form of intact micelles, which was consistent with the published report.15 3.7. Internalization Pathway of PEOz-b-PLA Micelles. To ensure the safety and effectiveness of endocytosis inhibitors used in the present study, the appropriate concentration was first determined according to their cytotoxicity to MCF-7 cells evaluated by using the SRB assay in a wide concentration range

used in the published reports. Based on the results of cytotoxicity presented in Figure 6A, the final concentration of 50 μM, 25 μg/mL, 1 mM and 25 μM was selected for 5-(Nethyl-N-isopropyl)-amiloride (EIPA), genistein, methyl-β-cyclodextrin (MβCD) and chlorpromazine, respectively, in the subsequent studies. First, in order to investigate if the cellular uptake of PEOz-bPLA micelles was an active process, the cellular uptake was carried out at 4 °C by treating MCF-7 cells with C6-loaded PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratio. As shown in Figure 6B, on reducing the incubation temperature, the internalization of all tested micelles was markedly lowered to 31.4−64.9% compared with the control (p < 0.01) (at 37 °C), thus, strongly suggesting that all PEOz-bPLA micelles were taken up by an energy-dependent active process. On this basis, we posited that endocytosis was a prominent pathway for internalization of PEOz-b-PLA micelles. Second, to more clearly delineate the specific endocytotic pathways involved in the cellular internalization of PEOz-b-PLA micelles, MCF-7 cells were pretreated with biochemical inhibitors of clathrin-mediated endocytosis (CME), cholesterol-dependent process, caveolae-mediated endocytosis (CvME), and macropinocytosis for 30 min and then the cellular uptake of the micelles was analyzed by flow cytometry. Figure 6B 6924


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ACS Applied Materials & Interfaces depicted the effect of various inhibitors on the uptake of polymeric micelles into the cells. Upon pretreatment of MCF-7 cells with chlorpromazine, which leads to the accumulation of clathrin in late endosomes, thereby blocking coated pit endocytosis, no significant change in the cellular uptake for all tested groups was found in comparison to the control group (p > 0.05) irrespective of the hydrophilic/hydrophobic ratio of PEOz-b-PLA, indicating that none of the tested PEOz-b-PLA micelles appeared to enter cells via clathrin-mediated endocytosis. To explore whether lipid raft is involved in the internalization of PEOz-b-PLA micelles, the plasma membrane cholesterol was extracted by treatment with MβCD, which is a known inhibitor of lipid raft, selectively depleting cholesterol without incorporation into the plasma membrane.29,30 The results showed pretreatment of MCF-7 cells with MβCD caused 3- to 5-fold decrease in cellular uptake for the tested micelles (p < 0.01) (Figure 6B), implying that the internalization of PEOz-b-PLA micelles was cholesterol-dependent process. The order in the extent of inhibition on cellular uptake from highest to lowest was PEOz8.9-b-PLA4 > PEOz6-b-PLA8.5 > PEOz2.6-b-PLA4 > PEOz6-b-PLA4 > PEOz6-b-PLA1.1. Moreover, the effect of genistein, a tyrosine kinase inhibitor which is used to inhibit caveolae-mediated endocytosis,31 on the cellular uptake of PEOz-b-PLA micelles was further evaluated. Obviously, the cellular uptake of all tested PEOz-bPLA micelles treated with genistein was remarkably decreased compared with untreated cells (Figure 6B). The greatest inhibition (55%) was observed for PEOz6-b-PLA4 micelles (p < 0.01), while the cellular uptake was inhibited by about 25% for the other four micelles (p < 0.01). These results thus suggested that the uptake of PEOz-b-PLA micelles by MCF-7 cells appeared to predominantly involve in caveolae-mediated endocytosis. EIPA blocks the sodium-proton exchange and alters the concentrations of sodium ions, which is involved micropinocytosis.32 The internalization of the PEOz6-b-PLA4 and PEOz8.9-b-PLA4 micelles was slightly increased (just only 111% and 108% of its control, respectively) in the presence of EIPA (p < 0.05) (Figure 6B). However, there was no significant effect of EIPA on cell uptake of the other three micelles (p > 0.05). These indicated that macropinocytosis mechanism played little role in cell uptake of the tested PEOz-b-PLA micelles, which was consistent with the reports that particles smaller than 150 nm may not generally involve in such endocytosis pathway.33 3.8. Tracing Intracellular Trafficking of PEOz-b-PLA Copolymer. To elucidate intracellular trafficking of PEOz-bPLA copolymer following their micelles uptake, the localization of DEC labeled PEOz6.7-b-PLA4 (PEOz6.7-b-PLA4-DEC) copolymer with different organelles was examined using confocal microscopy. The organelles, including late endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus and mitochondria were stained using specific organelle trackers (red fluorescence). As shown in Figure 7, colocalization of the internalized PEOz6.7-b-PLA4-DEC with late endosomes, mitochondria and endoplasmic reticulum was observed, whereas lysosomes and Golgi apparatus were not involved in the intracellular trafficking of PEOz6.7-b-PLA4-DEC. 3.9. Extracellular Efflux of PEOz-b-PLA Inside Cells to Medium. Given that fluorescence stability of PEOz6.7-bPLA4-DEC copolymer under physiological and acidic environment may have effect on the experimental accuracy of efflux

Figure 7. Confocal images of colocalization of PEOz6.7-b-PLA4-DEC with different organelles after incubation of PEOz6.7-b-PLA4-DEC micelles with MCF-7 cells for 0.5 or 3 h. Green, PEOz6.7-b-PLA4DEC; red, specific organelle probes; yellow, colocalization of green and red signals. Late endosome, lysosome, mitochondria, endoplasmic reticulum (ER) and Golgi apparatus were labeled with CellLightLate Endosomes-RFP, Lyso-Tracker RED, Mito-Tracker RED, ER-Tracker RED and BODIPY-ceramide, respectively.

study, the fluorescence stability of PEOz6.7-b-PLA4-DEC copolymer in serum-free culture medium with different pH at 37 °C was therefore evaluated. The fluorescence intensity of PEOz6.7-b-PLA4-DEC was presented in Figure 8A as a function of time (0−10 h). As anticipated, no obvious change in the fluorescence intensity of PEOz-b-PLA-DEC copolymer at different pH was observed, suggesting that PEOz6.7-b-PLA4DEC exhibited favorable fluorescence stability in serum-free culture medium with different pH within the whole period of 10 h. Thus, the possibility of fluorescence quenching in cells for PEOz6.7-b-PLA4-DEC could be ruled out, and PEOz6.7-bPLA4-DEC could be used in subsequent studies. To investigate the fate of PEOz-b-PLA copolymers after their trafficking to ER, time-dependent change of intracellular amount of PEOz6.7-b-PLA4-DEC copolymers was determined by flow cytometry. As shown in Figure 8B, the intracellular amount of PEOz6.7-b-PLA4-DEC copolymers decreased rapidly within the first 2 h, and then this change tended to be slowly throughout the period of 2−4 h. Afterward, the intracellular amount of PEOz6.7-b-PLA4-DEC was nearly leveled off. These results indicated the possible exocytosis of PEOz6.7-b-PLA4-DEC. 6925


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Figure 8. (A) Variation of fluorescent intensity at excitation wavelength of 488 nm for PEOz6.7-b-PLA4-DEC copolymer in serum-free culture medium with different pH at 37 °C as a function of time (n = 3). The concentration of PEOz6.7-b-PLA4-DEC was 2.0 mg/mL. (B) Time-dependent change in intracellular amount of PEOz6.7-b-PLA4-DEC polymers (n = 3).

Scheme 1. Schematic Illustration of Ionization of PEOz Chains at pH Lower Than Its pKa

Scheme 2. (A) Schematic Illustrations of the Formation and pH-Dependent Evolution of PEOz-b-PLA Micelles, (B) Schematic Illustration of Cellular Uptake and Intracellular Trafficking of PEOz-b-PLA Copolymer in Tumor Cellsa

a

“Cargo” mainly refers to nutrients, antigens, growth factors and receptors, etc.

4. DISCUSSION

distribution could greatly affect the performance of drugs loaded in nanoparticles. Therefore, unraveling of the composition-cellular internalization relationship of PEOz-bPLA and the mechanism for cellular internalization of PEOz-bPLA micelles are of significance. Moreover, detailed information about the clearance of nanoparticles-formed polymers from target cells after they have released encapsulated drugs is important from the perspective of safety. This study addressed in great detail such a need for elucidating the mechanism of pH-sensitivity and cellular internalization of PEOz-b-PLA

pH-responsive polymeric micelles based on synthetic polymers have attracted much attention in the targeted delivery of antitumor drugs. A promising example of such polymers is PEOz-b-PLA block copolymers, which have been evaluated as drug delivery agents in recent years.14,15,34,35 Unfortunately, the mechanism of pH-sensitivity for PEOz-b-PLA micelles remains under debate. Further, in the field of drug delivery, the extent and pathway of cell entry of nanoparticles and their intracellular 6926


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PLA micelles by target cells exhibited a strong dependence on the chain length of the shell (Figure 4B) and core (Figure 4A) forming blocks of PEOz-b-PLA micelles, which was consistent with structurally diverse block copolymer PEG-b-PCL micelles.13 The present finding further suggested that PEOzb-PLA micelles with hydrophilic/hydrophobic ratio MPEOz/ MPLA between 1.7 and 2.0 were apt to be internalized by MCF7 cells (Figure 4C). This phenomenon might be assigned to the fact that the hydrophilic/hydrophobic ratio of PEOz-b-PLA influenced the interaction of PEOz-b-PLA micelles with cell membranes consisting of lipid bilayers when they were internalized by cells.40,41 As has been demonstrated earlier, PEOz-b-PLA micelles entered into cells by caveolae/lipid raftmediated endocytosis in which caveolae are small, flask-shaped, hydrophobic plasma membrane microdomains that are rich in cholesterol and glycosphingolipids.42 Therefore, it is possible that polymeric micelles with appropriate hydrophilic/hydrophobic ratio might exhibit stronger interaction with caveolae/ lipid raft, thereby resulting in higher cellular uptake. Altogether, a delicate balance between hydrophilic and lipophilic chains for PEOz-b-PLA molecules might be useful for developing PEOzb-PLA micelles to provide the best cellular uptake. This observation pointed to the importance of the chemical composition for core/shell of micelles in defining their cellular uptake. On the other hand, the effect of subtle distinction in endocytosis pathway of PEOz-b-PLA micelles on their cellular uptake could be ignored owing to the fact that the hydrophilic/ hydrophobic ratio had hardly effect on endocytosis pathway of PEOz-b-PLA micelles (Figure 6B). The study on the mechanisms of uptake pathways is important to the rational design of nanosized drug carriers. As known, endocytosis has been established as the main cell uptake mechanism of nanoparticles in most cells.43 Clathrinmediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, clathrin- and caveolae-independent endocytosis have characterized as morphologically distinct endocytosis pathways.44 Previous documents reported that different nanoparticles employ different endocytosis mechanisms to gain cellular entry.45 For example, uptake of poly(ethylene glycol)-bpoly(aspartate)-DOX conjugate micelles by HeLa cells proceeded mainly through clathrin-mediated and caveolaemediated endocytosis.46 P85 micelles were reported to be internalized exclusively through clathrin-mediated endocytosis.47 Polyion complex micelles composed of both polyethylene glycol-b-poly(L-lysine) and DNA were found to enter human bronchial epithelial cells via a caveolae-mediated pathway.48 However, the mechanism involved in the internalization of PEOz-b-PLA micelles by tumor cells is still unclear. On the other hand, as the amphiphilicity of copolymers is the essential molecular requirement for their self-assembly to micelles, copolymers with different hydrophilic/hydrophobic ratio of the building blocks exhibit different characteristics.40 Thus, it is possible for the interaction of polymeric micelles with tumor cell membrane consisting of lipid bilayers to influence their cellular uptake pathway. In current study, therefore, PEOz-bPLA micelles were first validated to be internalized by MCF-7 cells in the form of integrity. Then, our mechanistic studies for the first time provided important convincing evidence for their uptake mechanism by MCF-7 cells based on the results of effects of biochemical inhibitors of selected endocytosis pathways on the internalization of PEOz-b-PLA micelles with various hydrophilic/hydrophobic ratios. Measurement of the fluorescent intensity of C6 inside treated cells allowed

micelles and exploring intracellular trafficking trace and fate of PEOz-b-PLA polymers. Previous studies showed that PEOz-b-PLA micelles exhibited remarkable pH-dependent drug release behavior,14,15,35 which was further confirmed by the results presented in Figure 3A. Wang et al. documented that the above phenomenon was assigned to the deformation of the core−shell structure promoted by the aggregation of PEOz chains owing to the interaction of inter- and intrahydrogen bonding between protonated nitrogen and carbonyl groups by examining the effect of pH value on the size and Zeta potential of PEOz-bPLA micelles.35 However, we disagreed with Wang et al. about the underlying mechanism of pH-sensitivity of the micelles due to the fact that the tertiary amide groups along PEOz chains are very little basic and cannot be protonated especially close to neutral pH. A possible explanation for the pH-sensitivity of PEOz-b-PLA micelles might be attributed to the ionization of the tertiary amide groups along PEOz chain at pH lower than its pKa (Scheme 1). Thus, the positive charges loaded on nitrogen atoms may lead to electrostatic repulsion between PEOz chains in outer shell of PEOz-b-PLA micelles, thereby inducing loosening of the micelle structure. We therefore set out to examine this hypothesis by first evaluating the effect of variations of pH value on the size and Zeta potential of PEOzb-PLA micelles. With decreasing pH value, the micelles did not show markedly variation in size (Figure 3B), however, Zeta potential of the micelles increased (Figure 3C) and positive charge density of PEOz-b-PLA micelles surface increased, suggesting no micelle aggregation and no disruption of the micellar structure, and the tertiary amide groups along PEOz chains were positively charged at pH lower than its pKa. Then Langmuir film balance was used to measure the molecular area of PEOz-b-PLA at different pH to gain additional understanding of electrostatic interaction between PEOz chains. As expected, the mean molecular area of PEOz-b-PLA increased gradually as the pH of the subphase declined from 7.4 to 5.0 (Figure 3D). This provided further evidence supporting the hypothesis that there existed stronger electrostatic repulsion between PEOz chains located in the outer shell of PEOz-b-PLA micelles at pH lower than its pKa possibly due to the charged PEOz chains. Based on these results, it could be concluded that the increased electrostatic repulsion between PEOz chains with decreasing pH value induced the loose outer shell of the micelles, thereby resulting in rapid drug release of PEOz-b-PLA micelles at acidic pH. A schematic diagram depicting the formation and pH-dependent evolution of micelles was shown in Scheme 2A. It was reported that the presence of hydrophilic shell could not only improve the stability of nanoparticles, but also inhibit the adsorption of opsonins to the surface of nanoparticles. On the other hand, excessive hydration of the shell on the surface of nanoparticles, however, could trigger steric repulsive forces and hinder the cellular uptake of nanoparticles.36 The higher the chain length and surface density of the hydrophilic block on the outer shell of nanoparticles were, the more difficult nanoparticles were internalized by cells.37−39 Accordingly, understanding of the effect of chemical manipulations on the uptake of micelles by target cells is very important for design and development of polymeric micelles to effectively deliver drugs to subcellular targets. The effect of chain length of the shell and core forming blocks on the uptake of C6 loaded in PEOz-b-PLA micelles by MCF-7 cells was investigated in the present study. As shown in Figure 4, cellular uptake of PEOz-b6927


Research Article


ER-to-Golgi transfer was not involved in intracellular transport pathway of PEOz-b-PLA-DEC copolymers as previous reports on doxorubicin-bound PEG-b-poly(aspartic acid) block copolymers.46,61 Interestingly, the present findings seemed to provide the evidence supporting the latter viewpoint mentioned above about intracellular trafficking pathway. Moreover, gradual decrease of the amount of intracellular PEOz6.7-b-PLA4-DEC polymers (Figure 8B) indicated that part of PEOz6.7-b-PLA4DEC localized in ER was possibly transported to plasma membrane and then cleared by efflux. Taken together, based on the results of this study, we inferred that PEOz-b-PLA polymers were internalized into cells mainly by caveolae/lipid raftmediated endocytosis, then transported to endosomes, followed by escape from late endosomes, then distributed to the ER and mitochondria (Scheme 2B). Meanwhile, another intracellular trafficking pathway might also exist simultaneously: PEOz-b-PLA polymers internalized through caveolae/lipid raftmediated endocytosis transported directly to the ER and mitochondria (Scheme 2B). Of course, this need to be further identified. Finally, part of the polymers was excreted from tumor cells to extracellular medium. Details about molecular mechanisms involved in the intracellular trafficking and extracellular efflux of PEOz-b-PLA block copolymers will be elucidated in our subsequent studies to improve the drug delivery efficiency and reduce the safety risk of PEOz-b-PLA micelles.

quantification of cellular uptake of PEOz-b-PLA micelles in various treatment groups. Reduced uptake of PEOz-b-PLA micelles after their incubation with MCF-7 cells at 4 °C was an indication for the involvement of energy-dependent mechanism (Figure 6B). This was in accordance with previous documents on the uptake of various polymeric micelles.13,49 Furthermore, pretreatment of cells with chlorpormazine and EIPA had no effect on the micellar internalization, however, the intracellular uptake of the micelles in the presence of MβCD and genistein (Figure 6B), respectively, was effectively inhibited. Based on these results, we inferred that caveolae/lipid raft-mediated endocytosis was a major uptake mechanism for PEOz-b-PLA micelles by MCF-7 cells even though subtle differences were observed among the tested five micelles. Thus, it was concluded that PEOz-b-PLA micelles might bypass degradative endo/ lysosomal trafficking, which was further validated by intuitive visualization of intracellular colocalization of PEOz-b-PLA-DEC with organelles (Figure 7), and the hydrophilic/hydrophobic ratio ranging from 0.68 to 5.45 of PEOz-b-PLA micelles appeared not to influence their internalization mechanism (Figure 6B). The present findings provided important convincing evidence for such micelles to be suitable for the intracellular delivery of lysosomal degradable drugs.50−52 Notably, none of the specific inhibitors led to complete inhibition (no more than 80% in Figure 6B) of cellular uptake, most likely indicating the role of clathrin- and caveolaeindependent pathways for internalization. As known, there are many factors that are involved in the selection of uptake pathways of nanoparticles, such as particle size, surface charge, shape, cell type, and even culture condition.13,40,49 This was further confirmed by the present findings that uptake of PEOz6-b-PLA4 micelles was strongly caveolae-dependent among the tested micelles. The reason behind this diversity is not clear and requires further investigations. As known, the intracellular trafficking of nanoparticles and polymers is dependent on their endocytosis pathway,42 surface properties and composition.53 In general, endocytosis of nanoparticles and polymers via caveolae is considered a nonacidic and nondigestive route of uptake, implying that the cargo does not suffer a drop in pH and most of cargo can be directly transported to endoplasmic reticulum and/or Golgi apparatus, thus avoiding the endosome-lysosome pathway,54 just like cholesterol being evidenced for a direct pathway from endosomes to the ER.55 In contrast, as reported by different researchers, caveosomes sometimes join the classical endocytic pathway, meaning that fusion with lysosomes cannot be avoided56,57 as the case of Pluronic P85.58 In addition, previous documents mainly focused on intracellular trafficking of drugloaded polymeric micelles after being internalized by tumor cells,59,60 but little is known about the intracellular trafficking and fate of the micelles forming polymers or drug-released polymeric micelles. Our study presented a more complete understanding of intracellular trafficking of PEOz-b-PLA copolymers through monitoring intracellular colocalization of DEC-labeled PEOz6.7-b-PLA4 with organelles by confocal microscopy. Confocal micrographs revealed that late endosomes, mitochondria and endoplasmic reticulum were all involved in the intracellular trafficking of PEOz6.7-b-PLA4DEC, and PEOz6.7-b-PLA4-DEC was not transported to lysosomes and Golgi apparatus (Figure 7). This was in agreement with our previous study demonstrating that pHresponsive PEOz-b-PLA micelles could rapidly escape from endosomes, thereby bypassing lysosomes.15 On the other hand,

5. CONCLUSIONS In this study, a series of PEOz-b-PLA micelles with various hydrophilic/hydrophobic ratios were prepared and characterized, and their favorable pH-dependent drug release performance was further confirmed. The pH-sensitivity of PEOz-b-PLA micelles was evidenced to result from the electrostatic repulsion between PEOz chains at pH lower than pKa of PEOz-b-PLA. The hydrophilic/hydrophobic ratio of PEOz-b-PLA micelles appeared to influence the extent of micellar internalization, and PEOz-b-PLA micelles with hydrophilic/hydrophobic ratio of 1.7−2.0 exhibited optimal cellular uptake. Mechanistic studies demonstrated that PEOz-b-PLA micelles were internalized by MCF-7 cells mainly through caveolae/lipid raft-mediated endocytosis without being influenced by the hydrophilic/ hydrophobic ratio. In addition, the intracellular trafficking routes and fate of PEOz-b-PLA copolymers were identified. In summary, the findings of this study might be of general significance for developing favorable PEOz-b-PLA micelles with higher therapeutic efficacy and lower safety risks.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b16376. 1 H NMR spectra of PEOz−OH and PEOz-b-PLA in CDCl3, the molecular weight and its distribution of PEOz−OH, acid/base titration profiles of PEOz6.7-bPLA4, synthesis routes of DEC-labeled PEOz-b-PLA and emission spectra of DiO/DiI-loaded PEOz6-bPLA4 micelles (FRET micelles) dispersed in distilled water and acetone (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Y.L.) Phone: +86 10 82801508; e-mail: [email protected]. 6928


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ACS Applied Materials & Interfaces ORCID

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Yiguang Jin: 0000-0002-3528-1397 Yan Liu: 0000-0001-5531-2376 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (No.81673366) and the National Key Science Research Program of China (973 Program, 2015CB932100).

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Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz-b

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