Hydrophobicity Reversable and Redox-Sensitive

Guillemard , V.; Saragovi , H. U. Novel approaches for targeted cancer therapy Curr. Cancer Drug Targets 2004, 4 (4) 313– 26. [Crossref], [PubMed], ...
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Hydrophilicity/Hydrophobicity Reversable and Redox-Sensitive Nanogels for Anticancer Drug Delivery Hao Yang,§,† Qin Wang,§,‡ Wei Chen,† Yanbing Zhao,† Tuying Yong,† Lu Gan,*,† Huibi Xu,† and Xiangliang Yang† †

National Engineering Research Center for Nanomedicine, College of Life Science and Technology and ‡School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China S Supporting Information *

ABSTRACT: Long circulation in the blood, efficient cellular internalization, and intracellular drug release in the tumor cells are major challenges in the development of ideal anticancer drug delivery systems. In this paper, hydrophilicity/hydrophobicity reversable and redox-sensitive poly(oligo(ethylene glycol) methacrylates-ss-acrylic acid) (P(OEGMAs-ss-AA)) nanogels were constructed as drug carriers for cancer therapy. The nanogels underwent a pH-dependent hydrophilic/hydrophobic change. The nanogels were hydrophilic under physiological conditions (pH 7.4, 37 °C), resulting in fewer opsonization of proteins and less phagocytosis by macrophage RAW264.7 cells, while they were hydrophobic in the tumor tissues (pH 6.5, 37 °C), resulting in strong internalization by Bel7402 cells. The doxorubicin (DOX) release from DOXloaded nanogels was increased in intracellular reductive and lysosome acidic environments. DOX-loaded nanogels exhibited higher cellular proliferation inhibition to GSH-OEt-pretreated Bel7402 cells at pH 6.5 than to unpretreated cells at pH 7.4. Further studies showed that the loaded DOX and nanogels were internalized into the cells together via both lipid raft/ caveolae- and clathrin-mediated endocytic pathways. After internalization, the DOX-loaded nanogels were transported via the specific route in endo/lysosomal system. The loaded DOX was released from the nanogels with the introduction of intracellular GSH and entered the nucleus. This study indicated that the hydrophilicity/hydrophobicity reversable and redoxsensitive nanogels might be used as potential carriers for anticancer drugs, which provided a foundation for designing an effective drug delivery system for cancer therapy. KEYWORDS: nanogels, hydrophilicity/hydrophobicity reversal, stimuli-responsiveness, drug delivery, intracellular tracking responsiveness to external stimuli such as temperature,5 pH,6 redox,7 and enzyme.8 Several major challenges existed in the development of anticancer drug delivery systems including long circulation in the blood, efficient cellular internalization, and intracellular drug release in the tumor cells. 9 PEGylation, namely conjugating polyethylene glycol (PEG) onto the surface of nanoparticles, was the most commonly used strategy to avoid recognition and clearance of the nanoparticles by reticuloendo-

1. INTRODUCTION Chemotherapy plays an important role in the treatment of cancer. However, systemic toxicity and insufficient drug concentration in cancer cells might be major problems leading to chemotherapy failure.1 Nanotechnology-based therapeutics have exhibited the great advantages compared with free drugs including enhanced drug accumulation in tumors via the enhanced permeability and retention (EPR) effect, better efficacy, and safety.2−4 Among various nanocarriers, nanogels with hydrophilic three-dimensional polymer networks have attracted growing attention as drug delivery carriers due to their unique advantages such as high loading capacity, good stability, large surface area for multivalent bioconjugation, and smart © 2015 American Chemical Society

Received: Revised: Accepted: Published: 1636

January 22, 2015 March 20, 2015 April 1, 2015 April 1, 2015 DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Molecular Pharmaceutics thelial system (RES), which increases the circulation half-life of nanoparticles.10,11 However, PEGylation, which provides a hydrated barrier to prevent the phagocytosis by RES, has been reported to have an adverse effect on the internalization of nanoparticles into tumor cells.12 Several strategies have been developed to achieve a longer plasma half-life as well as better cellular uptake to improve the therapeutic effects of anticancer drugs. For example, removing PEG from PEGylated nanoparticles in response to tumor extracellular microenvironment stimuli, that is, pH,13 proteases,14 and redox,15 has been reported to be an option. Recently, Yuan et al. developed surface charge switchable nanoparticles based on zwitterionic polymer for enhance drug delivery to tumor.16 The negatively charged nanoparticles that were resistant to nonspecific protein absorption switched to being positively charged to promote internalization by tumor cells in response to tumor extracellular pH. Thermoresponsive materials exhibited a phase transition between hydrophilic and hydrophobic properties at the lower critical solution temperature (LCST), and the LCST of thermoresponsive materials could be easily adjusted by many factors including the pH value of medium.17 Copolymers of oligo(ethylene glycol) methacrylates (OEGMAs) have been shown to have the similar biocompatibility as PEG and a controllable LCST behavior with a more uniform profile of heating and cooling cycles than poly(N-isopropylacrilamide), a well-known thermoresponsive polymer.18,19 Therefore, polymers based on OEGMAs have been developed as novel excellent biocompatible thermoresponsive materials.20,21 Here, we reported hydrophilicity/hydrophobicity reversable and redox-sensitive nanogels based on OEGMAs and acrylic acid (AA), which were cross-linked by N,N′-bis(acryloyl)cystamine (BAC) (the prepared nanogels were denoted as P(OEGMAsss-AA) nanogels in this paper). The nanogels were hydrophilic in bloodstream (pH 7.4) for protection from the clearance by RES, but hydrophobic in the tumor tissues (pH 6.5) for easy cellular uptake. When the disulfide bond-cross-linked nanogels nanogels were internalized via endo/lysosome compartments into tumor cells, they were disintegrated by the high intracellular GSH concentration in cancer cells, which led to the rapid drug release to enter the nucleus (Figure 1).

Figure 1. Schematic illustration of the hydrophlicity/hydrophobicity reversable and redox-sensitive P(OEGMAs-ss-AA) nanogels for the drug delivery in cancer.

Academy of Sciences (Shanghai, China). Bel7402 cells were cultured in RPMI 1640 medium, and RAW264.7 cells were cultured in Dulbecco’s modified eagle medium (DMEM) medium at 37 °C in 5% CO2 in a humidified atmosphere. All media contained 10% fetal bovine serum (FBS) (Gibco BRL/ Life Technologies, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. 2.3. Preparation of P(OEGMAs-ss-AA) Nanogels. P(OEGMAs-ss-AA) nanogels cross-linked by BAC were prepared by emulsion polymerization as described elsewhere.22 Briefly, comonomers MEO2MA (645 μL, 3.5 mmol), OEGMA (465 μL, 1 mmol) and AA (35 μL, 0.5 mmol), surfactant SDS (0.06 g), and cross-linker BAC (65 mg, 0.25 mmol) were dissolved in 100 mL of water in a three-necked round-bottom flask. The resulting solution was purged with nitrogen for at least 30 min to remove oxygen and then heated up to 95 °C. Potassium persulfate (KPS, 0.03 g) was then added to initiate the free radical polymerization. The reaction was maintained for 5 h under the nitrogen atmosphere at 95 °C with stirring. The resulting reaction mixture was dialyzed in a dialysis bag (the cutoff molecular weight was 14 000) against ultrapure water for 2 weeks to remove unreacted monomers and other low molecular weight impurities. 2.4. Preparation of DOX-Loaded P(OEGMAs-ss-AA) Nanogels. To form free DOX solution, DOX·HCl (1.0 mg/ mL) was dissolved in chloroform (CHCl3) with an excess of triethylamine (TEA) (DOX·HCl/TEA molar ratio was 1:5). Then DOX-loaded P(OEGMAs-ss-AA) nanogels were prepared as described.21 Briefly, 0.6 mg of DOX solution was added to 10 mg of aqueous dispersions of P(OEGMAs-ss-AA) nanogels in a total volume of 4 mL. The solution was stirred at room temperature for 2 h, then for overnight at 40 °C to evaporate

2. MATERIALS AND METHODS 2.1. Materials and Plasmids. Di(ethylene glycol) methyl ether methacrylate (MEO2MA, Mw 188.22), OEGMA (average Mn 475), BAC, sodium dodecyl sulfate (SDS), 3-(4,5-dimethyl2-thiazolyl)-2,5-Diphenyltetrazolium bromide (MTT), glutathione reduced ethyl ester (GSH-OEt), methyl-β-cyclodextrin (MβCD), chlorpromazine, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were purchased from Sigma-Aldrich (St Louis, MO, USA). LysoTracker Green and 4′6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) (purity >98.0%) was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, China). Plasmids expressing enhanced green fluorescent protein (EGFP)-Rab5a and EGFP-Rab7 were kindly provided by Prof. Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). All other chemicals used were of analytical grade and commercially available. 2.2. Cell Culture. The human hepatocellular carcinoma cell line Bel7402 and the murine macrophage cell line RAW264.7 were purchased from Type Culture Collection of the Chinese 1637

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Molecular Pharmaceutics

sample was withdrawn and replenished with an equal volume of fresh medium. The amount of DOX release was quantified by fluorescence measurement with the excitation wavelength at 480 nm using a DOX calibration curve. 2.9. Protein Absorption of P(OEGMAs-ss-AA) Nanogels. Samples of 10 mg/mL P(OEGMAs-ss-AA) nanogels were suspended in PBS in the presence or absence of 10% FBS at different pH values. After incubation at 37 °C for 4 h, the mixtures were centrifuged at 12 000 rpm to precipitate the nanogels. The pellets were washed with PBS twice, and the concentration of protein absorbed on nanogels was measured using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. 2.10. Phagocytosis of P(OEGMAs-ss-AA) Nanogels by Macrophage RAW264.7 Cells. 5-aminofluorescein-conjugated P(OEGMAs-ss-AA) nanogels were first constructed through the classical reaction between amino and carboxylic groups under the activation of EDC and NHS.25 Samples of 0.5 mg/mL 5-aminofluorescein-conjugated nanogels were pretreated in DMEM medium with or without 10% FBS for 1 h at different pH values. Then RAW264.7 cells were incubated in DMEM medium containing 5-aminofluorescein-conjugated nanogels in the presence or absence of 10% FBS for 2 h at different pH values, respectively. The cells were washed with PBS and harvested for flow cytometric analysis (FC500, Beckman Coulter, Fullerton, CA, USA). 2.11. Cellular Accumulation of DOX in Bel7402 Cells. Bel7402 cells were seeded at a density of 2 × 105 cells/well in 12-well plates overnight. The cells were pretreated with serumfree RPMI 1640 medium in the presence or absence of 10 mM GSH-OEt for 2 h.26 After being washed with PBS twice, the cells were incubated in serum-free medium containing 10 μg/ mL free DOX or DOX-loaded nanogels at different pH values for 4 h. The cells were washed with PBS and harvested for flow cytometric analysis. 2.12. In Vitro Cytotoxicity of DOX-Loaded P(OEGMAsss-AA) Nanogels. The in vitro cytotoxicity of DOX-loaded P(OEGMAs-ss-AA) nanogels was evaluated by MTT assay.27 Briefly, Bel7402 cells were seeded in 96-well plates at 8 × 103 cells/well and incubated overnight. Cells were preincubated with fresh medium in the presence or absence of 10 mM GSHOEt for 2 h. After being washed with PBS twice, the cells were incubated in serum-free medium containing different concentrations of free DOX or DOX-loaded nanogels at different pH values. After treatment for 48 h, the cells were washed with PBS, and then 20 μL of 5 mg/mL MTT solution was added to the cells in each well. Plates were incubated for an additional 4 h at 37 °C. The medium containing MTT was removed, and 150 μL DMSO was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 490 nm using a Labsystems iEMS microplate reader (Helsinki, Finland). 2.13. Endocytic Pathway of P(OEGMAs-ss-AA) Nanogels. Bel7402 cells were preincubated in serum-free RPMI 1640 medium containing 10 mM MβCD (30 min), 10 μg/mL chlorpromazine (30 min), 10 μg/mL cytochalasin D (30 min), and 50 μM EIPA (1 h), respectively.28 The medium was then changed to fresh serum-free medium containing the inhibitors plus 0.5 mg/mL 5-aminofluorescein-conjugated nanogels and further incubated for 1 h at 37 °C. The cells were washed with PBS, fixed with 4% paraformaldehyde solution, and imaged by Andor Revolution spinnig disk confocal microscope. For

CHCl3. The DOX-loaded nanogels were purified in an ultrafilter tube (Millipore; cutoff Mw 10 000) by centrifugation (4000 rpm/20 min). The DOX concentration in the supernatant was measured by UV−vis absorption spectroscopy at 480 nm. The drug loading capacity and encapsulation efficiency of P(OEGMAs-ss-AA) nanogels were determined according to the following equations.23 Drug loading capacity (%) = (amount of drug in nanogels)/(amount of drug-loaded nanogels) × 100. Encapsulation efficiency (%) = (amount of drug in nanogels)/(total amount of feeding drug) × 100. The entire procedure was performed in the dark. 2.5. Characterization of P(OEGMAs-ss-AA) Nanogels. The hydrodynamic diameters and zeta potentials of the blank and DOX-loaded P(OEGMAs-ss-AA) nanogels were measured by dynamic light scattering (DLS; Zetasizer Nano-ZS 90, Malvern Instruments Ltd., Worcestershire, UK) equipped with a He−Ne laser (λ = 633 nm) with the scattering angle of 90°.22 All samples were diluted with water to 0.5 mg/mL and maintained for 3 min at the designed temperature range from 20−50 °C before testing. The morphologies of the blank and DOX-loaded nanogels were observed by a transmission electron microscope (TEM; Tecnai G2 20, FEI Corp., The Netherlands).22 Briefly, a drop of nanogel aqueous dispersion (0.1 mg/mL) was spread onto a 400-mesh carbon-coated copper grid, followed by staining with phosphomolybdic acid and drying in air overnight. The accelerating voltage was 200 kV. 2.6. Hydrophilic/Hydrophobic Properties of P(OEGMAs-ss-AA) Nanogels. The hydrophobic fluorescence probe pyrene (Py) was used to measure the temperature dependence of hydrophilic/hydrophobic properties of P(OEGMAs-ss-AA) nanogels.22 Briefly, 2.5 mg/mL of P(OEGMAs-ss-AA) nanogels was dispersed in phosphatebuffered saline (PBS) at two pH values (pH 7.4 and 6.5), and then Py stock solution (dissolved in acetone) was dropped into the aqueous dispersions of nanogels to obtain a final Py concentration of 1.2 μM. Acetone was subsequently removed by evaporation at room temperature for 2 h. The fluorescence spectra of the dispersions were recorded at an excitation wavelength of 334 nm at various temperatures on an F-4500 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). The LCST of P(OEGMAs-ss-AA) nanogels at different pH values was calculated according to the ratios of the third emission peak to the first one (I3/I1) of Py at different temperatures. 2.7. GSH-Responsive DOX Release from DOX-Loaded P(OEGMAs-ss-AA) Nanogels. DOX-loaded P(OEGMAs-ssAA) nanogels at the final DOX concentration of 10 μg/mL were incubated with different concentrations of GSH in aqueous solutions at 37 °C.23 The fluorescence intensity of DOX was recorded with the excitation wavelength at 480 nm and emission wavelength at the interval of 500−700 nm on an F-4500 fluorescence spectrophotometer. 2.8. In Vitro Drug Release from DOX-Loaded P(OEGMAs-ss-AA) Nanogels. The in vitro pH- and GSHresponsive release behaviors of DOX from DOX-loaded P(OEGMAs-ss-AA) nanogels were monitored by dialysis method.24 Briefly, 1 mg/mL of DOX-loaded P(OEGMAs-ssAA) nanogels was placed in a dialysis bag (the cutoff molecular weight was 14 000) and submerged fully into 25 mL of PBS in the presence of different concentrations of GSH at different pH values. The release experiments were carried out at 37 °C with shaking at 100 rpm. At the desired time intervals, 1 mL of the 1638

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Figure 2. Characterization of the blank P(OEGMAs-ss-AA) nanogels and DOX-loaded P(OEGMAs-ss-AA) nanogels. (A) Synthesis of P(OEGMAsss-AA) nanogels. (B) The hydrodynamic sizes of the blank P(OEGMAs-ss-AA) nanogels and DOX-loaded P(OEGMAs-ss-AA) nanogels. (C) TEM images of the blank P(OEGMAs-ss-AA) nanogels and DOX-loaded P(OEGMAs-ss-AA) nanogels.

cells were washed with PBS twice and then incubated with RPMI 1640 medium containing 5 μg/mL DAPI for 20 min or 1 μM LysoTracker Green for 15 min. After they were fixed with 4% paraformaldehyde, the cells were observed under an Andor Revolution spinning disk confocal microscope. To further explore the specific route in endo/lysosome compartments, Bel7402 cells were transfected with plasmids expressing different Rab proteins fused with EGFP by electroporation.30 After 24 h transfection, the cells were treated with DOX-loaded P(OEGMAs-ss-AA) nanogels at the final DOX concentration of 10 μg/mL for 5 min, washed with PBS, and then incubated with RPMI 1640 medium for different time courses. The cells were washed with PBS, fixed with 4% paraformaldehyde, and observed under an Andor Revolution spinning disk confocal microscope. 2.16. Statistical Analysis. Experiments were carried out with three or four replicates. Statistical analyses were performed by Student’s t test. Values with P < 0.05 are considered significant.

quantitative analysis of the intracellular concentration of 5aminofluorescein-conjugated nanogels after treatment with the inhibitors, the cells were harvested for flow cytometric analysis. 2.14. Intracellular Process of DOX-Loaded P(OEGMAsss-AA) Nanogels. DOX was loaded into 5-aminofluoresceinconjugated nanogels as stated above. Bel7402 cells were treated with DOX-loaded 5-aminofluorescein-conjugated nanogels at the final DOX concentration of 10 μg/mL for different time courses. The cells were washed with PBS twice and then incubated with medium containing 5 μg/mL DAPI for 20 min. After they were fixed with 4% paraformaldehyde, the cells were observed under an Andor Revolution spinning disk confocal microscope.29 2.15. Subcellular Distribution of DOX-Loaded P(OEGMAs-ss-AA) Nanogels. For assessment of the colocalization of DOX-loaded P(OEGMAs-ss-AA) nanogels with nucleus or lysosomes, Bel7402 cells were treated with DOXloaded P(OEGMAs-ss-AA) nanogels at the final DOX concentration of 10 μg/mL for different time courses. The 1639

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Molecular Pharmaceutics

Figure 3. pH-responsive hydrophilic/hydrophobic properties of P(OEGMAs-ss-AA) nanogels. (A) The temperature-responsive curves of the sizes of P(OEGMAs-ss-AA) nanogels in PBS at pH 7.4, 6.5, and 4.5, respectively. (B) The temperature-responsive curves of the fluorescence intensities of Py in P(OEGMAs-ss-AA) nanogels at pH 7.4 and 6.5. Data as mean values ± SD (n = 3).

Figure 4. Redox/pH-responsive release of DOX from P(OEGMAs-ss-AA) nanogels. (A) The DOX fluorescence spectra of DOX-loaded P(OEGMAs-ss-AA) nanogels after treatment with different concentrations of GSH for 24 h. (B) The DOX fluorescence spectra of DOX-loaded P(OEGMAs-ss-AA) nanogels after treatment with 10 mM GSH for different time courses. (C) The in vitro release profile of DOX from DOX-loaded P(OEGMAs-ss-AA) nanogels in PBS containing different concentrations of GSH at different pH values at 37 °C. Data as mean values ± SD (n = 3).

3. RESULTS AND DISCUSSION

elaborately to adjust the LCST of the obtained nanogels close to 37 °C with pH-responsiveness. BAC containing disulfide bond was employed as cross-linker to afford nanogels GSH responsiveness. DOX, as an anticancer drug model, was then incorporated into nanogels by hydrophobic interaction. The

3.1. Preparation and Characterization of P(OEGMAsss-AA) Nanogels. P(OEGMAs-ss-AA) nanogels were synthesized by emulsion polymerization (Figure 2A). The molar ratio of comonomers of MEO2MA, OEGMA, and AA was chosen 1640

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Figure 5. pH-responsive cellular uptake and GSH-responsive intracellular release of P(OEGMAs-ss-AA) nanogels in RAW264.7 cells and Bel7402 cells. (A) The absorbed FBS in P(OEGMAs-ss-AA) nanogels at pH 7.4 and 6.5. (B) Percentages of the internalized fluorescence intensity of P(OEGMAs-ss-AA) nanogels when RAW264.7 cells were treated with 0.5 mg/mL 5-aminofluorescein-conjugated P(OEGMAsss-AA) nanogels in the presence or absence of 10% FBS for 2 h at pH 7.4 and 6.5. Results from one representative experiment are shown on the left. Quantitative results are shown on the right. (C) Percentages of the internalized fluorescence intensity of DOX-loaded P(OEGMAsss-AA) nanogels when Bel7402 cells were treated with DOX-loaded P(OEGMAs-ss-AA) nanogels at the DOX concentration of 10 μg/mL for 2 h at pH 7.4 and 6.5. Results from one representative experiment are shown on the left. Quantitative results are shown on the right. (D) Percentages of the internalized fluorescence intensity of DOX-loaded P(OEGMAs-ss-AA) nanogels when Bel7402 cells pretreated with or without 10 mM GSH-OEt for 2 h were incubated with DOX-loaded P(OEGMAs-ss-AA) nanogels at the DOX concentration of 10 μg/mL for 2 h at pH 7.4 and 6.5. Results from one representative experiment are shown on the left. Quantitative results are shown on the right. Data as mean values ± SD (n = 3). ∗, P < 0.05.

Figure 6. In vitro cytotoxicity of DOX-loaded P(OEGMAs-ss-AA) nanogels in Bel7402 cells. (A) Cell viability in Bel7402 cells treated with DOX-loaded P(OEGMAs-ss-AA) nanogels or free DOX for 48 h at pH 7.4 and 6.5. Data as mean values ± SD (n = 3). ∗, P < 0.05 compared with DOX-loaded nanogels-treated group at the corresponding DOX concentration at pH 7.4. (B) Cell viability in Bel7402 cells pretreated with or without 10 mM GSH-OEt for 2 h and then followed by treatment with DOX-loaded P(OEGMAs-ss-AA) nanogels or free DOX for 48 h. ∗, P < 0.05 compared with DOX-loaded nanogels-treated group at the corresponding DOX concentration.

LCST, in which its properties (such as hydrophility/hydrophobility, refractive index, viscosity, etc.) sharply change. The LCST could be adjusted by many factors including composition of polymers and properties of surrounding media. To confirm the temperature- and pH-sensitive properties of P(OEGMAsss-AA) nanogels, the sizes of the nanogels at different temperatures and pH values were determined by DLS. As shown in Figure 3, panel A, the size of P(OEGMAs-ss-AA) nanogels considerably decreased as temperature increased at pH 4.5, 6.5, or 7.4 (mimicking the pH in lysosomes, tumor extracellular microenvironment, blood and normal tissues, respectively), which confirmed the thermoresponsiveness of the prepared P(OEGMAs-ss-AA) nanogels. In addition, the sizes of the nanogels varied with pH in the medium at the same temperature. Especially, the sizes of the nanogels at pH 4.5 were much smaller than that at pH 6.5 and 7.4, which might be due to the presence of carboxyl group in the nanogels derived from the comonomer AA. As pH in the medium decreased, the ionization degree of carboxyl groups was decreased, and the interaction between the nanogels and water was reduced

encapsulation efficiency and the drug loading capacity of the nanogels were about 80% and 4%, respectively. As shown in Figure 2, panel B, the mean hydrodynamic diameters of the blank and DOX-loaded P(OEGMAs-ss-AA) nanogels were about 150 and 180 nm, respectively. The zeta potentials of the blank and DOX-loaded P(OEGMAs-ss-AA) nanogels were around −25 and −17.4 mV, respectively, which provided the electrostatic repulsion force to increase the stability of nanogels. TEM imaging showed that the blank and DOX-loaded P(OEGMAs-ss-AA) nanogels were nearly monodisperse and spherical (Figure 2C). 3.2. pH-Responsive Hydrophilic/Hydrophobic Properties of P(OEGMAs-ss-AA) Nanogels. Thermoresponsive copolymers of OEGMAs undergo a coil-granule transition at 1641

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Figure 7. Confocal microscopy images of the intracellular tracking of DOX-loaded P(OEGMAs-ss-AA) nanogels after Bel7402 cells were treated with DOX-loaded 5-aminofluorescein-conjugated P(OEGMAs-ss-AA) nanogels at the DOX concentration of 10 μg/mL for different time courses and then labeled with 5 μg/mL DAPI. The scale bar is 25 μm.

GSH concentration in human plasma) compared to control group, which indicated the stability of the DOX-loaded nanogels in blood circulation. However, the fluorescence intensity of DOX-loaded P(OEGMAs-ss-AA) nanogels dramatically increased after treatment with GSH at the concentration above 10 mM (corresponding to GSH concentration in tumor cells). Furthermore, the fluorescence intensity of DOX increased in a time-dependent manner when DOX-loaded P(OEGMAs-ss-AA) nanogels were treated with 10 mM GSH (Figure 4B). These data indicated a GSH-triggered DOX release from P(OEGMAs-ss-AA) nanogels. To further confirm the GSH-responsive release of DOX-loaded P(OEGMAs-ssAA) nanogels, the in vitro release behavior of DOX-loaded P(OEGMAs-ss-AA) nanogels was investigated in PBS in the presence or absence of GSH by dialysis method. As shown in Figure 4, panel C, the cumulative release of DOX from P(OEGMAs-ss-AA) nanogels was about 18% in the absence of GSH at pH 7.4 in 100 h, which indicated a low drug release from the nanogels under physiological blood circulation. The DOX release behavior of DOX-loaded P(OEGMAs-ss-AA) nanogels did not change in response to 2 μM GSH. However, the cumulative release of DOX increased to about 60% in the presence of 10 mM GSH under a physiological pH 7.4. The effects of pH on the DOX release from P(OEGMAs-ssAA) nanogels were further determined in PBS in the presence or absence of GSH at different pH values by dialysis method. As shown in Figure 4, panel C, the DOX release rate from P(OEGMAs-ss-AA) nanogels at pH 6.5 was similar to that at pH 7.4 in the presence of the same concentration of GSH. Nevertheless, the DOX release from P(OEGMAs-ss-AA) nanogels at pH 4.5 was faster than that under the condition of physiological pH 7.4. For example, the cumulative release

accordingly, which resulted in the decline in the hydrophilicity and size of the nanogels. The LCSTs calculated from the size curves (Figure 3A) were consistent with the LCSTs calculated from the hydrophilic/ hydrophobic curves (Figure 3B). The LCST of P(OEGMAs-ssAA) nanogels was about 42 °C at pH 7.4, higher than the physiological temperature (37 °C), while the LCST decreased to about 36 °C at pH 6.5, lower than the physiological temperature. The results indicated that P(OEGMAs-ss-AA) nanogels were hydrophilic and swollen in blood and normal tissues due to its higher LCSTs than 37 °C, which might make nanogels avoid the detection and clearance by RES. However, P(OEGMAs-ss-AA) nanogels turned to hydrophobic and shrunken in the tumor tissues due to its lower LCST than 37 °C, which might make nanogels be easily internalized by tumor cells. 3.3. Redox/pH-Responsive Release of DOX from P(OEGMAs-ss-AA) Nanogels. It is well-known that the disulfide linkages are stable under normal physiological conditions but cleaved to free thiols under reductive condition. The reduction of the disulfide linkages of nanogels cross-linked by BAC would result in the destabilization of the nanogels and a rapid release of loaded cargoes.7 It was reported that because of the self-quenching effect of DOX in nanoparticles, DOX fluorescence was enhanced when DOX was released from nanoparticles.31 To explore the redox-responsive effects of P(OEGMAs-ss-AA) nanogels, the fluorescence intensity of DOX-loaded nanogels was determined after treatment with different concentrations of GSH for different time courses. As shown in Figure 4, panel A, little alteration in DOX fluorescence intensity was observed when P(OEGMAs-ss-AA) nanogels were incubated with 2 μM GSH (corresponding to 1642

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Molecular Pharmaceutics

Figure 8. Endocytic pathway of P(OEGMAs-ss-AA) nanogels in Bel7402 cells. (A) Confocal microscopy images of Bel7402 cells preincubated with serum-free medium containing 10 mM MβCD, 10 μg/mL chlorpromazine, 10 μg/mL cytochalasin D, and 50 μM EIPA followed by coincubation with 0.5 mg/mL 5-aminofluorescein-conjugated P(OEGMAs-ss-AA) nanogels for 1 h, respectively. The scale bar is 25 μm. (B) Percentages of the internalized fluorescence intensity in Bel7402 cells treated with the specific endocytic inhibitors as above followed by coincubation with 0.5 mg/mL 5-aminofluorescein-conjugated P(OEGMAs-ss-AA) nanogels for 1 h by flow cytometry. Data as mean values ± SD (n = 3). ∗, P < 0.05 compared with control group.

Figure 9. Confocal microscopy images of the intracellular localization of DOX-loaded P(OEGMAs-ss-AA) nanogels after Bel7402 cells were treated with DOX-loaded nanogels at the DOX concentration of 10 μg/mL for different time courses and then labeled with (A) 1 μM LysoTracker Green for 15 min or (B) 5 μg/mL DAPI for 20 min. The scale bar is 5 μm.

rate of DOX from P(OEGMAs-ss-AA) nanogels in the absence of GSH at pH 4.5 reached about 35% in 100 h, higher than that

at pH 7.4, which suggests that the drug release from P(OEGMAs-ss-AA) nanogels might be accelerated in endo1643

DOI: 10.1021/acs.molpharmaceut.5b00068 Mol. Pharmaceutics 2015, 12, 1636−1647

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Figure 10. Confocal microscopy images of the intracellular localization of DOX-loaded P(OEGMAs-ss-AA) nanogels after Bel7402 cells transfected with (A) GFP-Rab5a or (B) GFP-Rab7 were treated with DOX-loaded nanogels at the DOX concentration of 10 μg/mL for 5 min, washed with PBS, and then incubated with serum-free RPMI 1640 medium for different time courses, respectively. The scale bar is 5 μm. The inserts were the amplification of the designated areas (the scale bar is 25 μm).

Bel7402 cells at pH 6.5 and 7.4 by flow cytometry. As shown in Figure 5, panel C, the fluorescence intensity of DOX was significantly higher in cells incubated with DOX-loaded P(OEGMAs-ss-AA) nanogels at pH 6.5 than that at pH 7.4. In contrast, no difference was observed when the cells were incubated with free DOX at pH 6.5 and 7.4. These results demonstrated that the response of DOX-loaded P(OEGMAsss-AA) nanogels to the pH in the tumor microenvironment could enhance the cellular uptake by tumor cells. 3.5. Redox-Responsive Intracellular Release of P(OEGMAs-ss-AA) Nanogels in Bel7402 Cells. To verify the feasibility of P(OEGMAs-ss-AA) nanogels for intracellular drug release in cancer therapy, the cellular uptake and intracellular release behaviors of DOX-loaded nanogels were determined by flow cytometry in Bel7402 cells pretreated with or without 10 mM GSH-OEt, which can obviously enhance the intracellular GSH concentration. As shown in Figure 5, panel D, pretreatment with GSH-OEt significantly increased the fluorescence intensity of the internalized DOX-loaded nanogels, which was consistent with the in vitro data that the DOX fluorescence intensity increased when DOX was released from nanogels in response to GSH (Figure 3C), suggesting that the enhanced fluorescence intensity in the GSH-OEt-pretreated Bel7402 cells might be due to the intracellular release of DOX caused by the reduction responsive degradation. Furthermore, the fluorescence intensity of DOX-loaded nanogels was improved in Bel7402 cells incubated at pH 6.5 compared with that at pH 7.4, which further confirmed the higher

somal/lysosomal compartments. One reason might be that the solubility of DOX was elevated at pH 4.5, which accelerated the drug release. Another might be that the shrinkage of the nanogels at pH 4.5 (Figure 3A) could press the cargoes out of the nanogels and increase the drug release. 3.4. pH-Responsive Cellular Uptake of P(OEGMAs-ssAA) Nanogels by RAW264.7 Cells and Bel7402 Cells. To confirm whether the hydrophilic/hydrophobic property of P(OEGMAs-ss-AA) nanogels regulated by pH could affect their clearance by RES, the interaction of P(OEGMAs-ss-AA) nanogels with FBS at pH 6.5 and 7.4 was first evaluated. As shown in Figure 5, panel A, P(OEGMAs-ss-AA) nanogels showed stronger protein absorption at pH 6.5 than that at pH 7.4. Furthermore, more P(OEGMAs-ss-AA) nanogels were phagocytosed by RAW264.7 macrophage cells in the presence of 10% FBS at pH 6.5 than that at pH 7.4; however, no significant difference in the phagocytosis of P(OEGMAs-ss-AA) nanogels was found when the cells were incubated in serumfree medium at pH 6.5 and 7.4 (Figure 5B). Here we noticed that the size of the nanogels did not change significantly in PBS containing different concentrations of FBS (Supplementary Figure 1, Supporting Information). These data implied the potential of P(OEGMAs-ss-AA) nanogels for prolonging their circulation time in blood. To further confirm whether the hydrophilic/hydrophobic property of P(OEGMAs-ss-AA) nanogels regulated by pH could affect the internalization by tumor cells, the in vitro cellular uptake of DOX-loaded nanogels was determined in 1644

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involved in the internalization of P(OEGMAs-ss-AA) nanogels in Bel7402 cells. 3.9. Intracellular Localization of DOX-Loaded P(OEGMAs-ss-AA) Nanogels. To further investigate the intracellular trafficking of DOX-loaded P(OEGMAs-ss-AA) nanogels, Bel7402 cells were treated with DOX-loaded nanogels for different time courses, and confocal microscopy was used to demonstrate the colocalization of DOX-loaded nanogels with cell compartments. As shown in Figure 9, panel A, the DOX fluorescence in Bel7402 cells almost totally colocalized with LysoTracker Green-labeled lysosomes after 1 h of treatment. However, the colocalization of DOX and lysosomes gradually decreased as incubation time increased, and nearly no colocalization was observed after 8 h of treatment. On the other hand, the DOX was found in the cytoplasm after 1 h of treatment, began to enter into the nucleus after 3 h of treatment, and majorly accumulated in the nucleus after 8 h of treatment (Figure 9B). These data suggested that after endocytosis, DOX-loaded P(OEGMAs-ssAA) nanogels were first delivered to lysosomes and then translocated from the lysosomes to release DOX into nucleus, which might induce cytotoxicity against Bel7402 cells. To confirm whether DOX-loaded P(OEGMAs-ss-AA) nanogels were internalized via endo/lysosome compartments, Bel7402 cells were transfected with plasmids for expression of fluorescent proteins targeted to Rab proteins and then exposed to a 5 min pulse of DOX-loaded P(OEGMAs-ss-AA) nanogels followed by a chase in nanogels-free and serum-free media for different time courses. It was known that small GTPases Rab family not only functions as components of the intracellular trafficking machinery, but also provides specific identity to the endosomal compartments.32−34 Rab5a mediates the endocytosis and early endosome fusion,35,36 while Rab7 is involved in the maturation of late endosomes and the subsequent fusion with lysosomes.37 As shown in Figure 10, panel A, the colocalization of DOX and Rab5a appeared in Bel7402 cells after a 5−40 min chase and then declined after a 60 min chase. On the other hand, the colocalization of DOX and Rab7 increased in Bel7402 cells as the chase time increased. DOX almost completely colocalized with Rab7 after a 60 min chase (Figure 10B). These data indicated that after endocytosis, DOX-loaded P(OEGMAs-ss-AA) nanogels were internalized via the specific route in endo/lysosome compartments. The previous finding that the faster DOX release from DOX-loaded nanogels at pH 4.5 (Figure 4) provided the possibility that the location of DOX-loaded nanogels at endo/lysosome compartments might result in the faster release of DOX, which further contributed to the strong cytotoxicity of DOX-loaded nanogels.

internalization efficiency of P(OEGMAs-ss-AA) nanogels at pH 6.5. 3.6. In Vitro Cytotoxicity of DOX-Loaded P(OEGMAsss-AA) Nanogels. To evaluate the potential application of P(OEGMAs-ss-AA) nanogels as a drug carrier for cancer therapy, the cytotoxicity of DOX-loaded nanogels against Bel7402 cells was determined by MTT assay. As shown in Figure 6, panel A, free DOX and DOX-loaded nanogels showed dose-dependent cytotoxicity to Bel7402 cells at any conditions. The cytotoxic effects of DOX-loaded nanogels to Bel7402 cells at pH 6.5 were significantly higher than that at pH 7.4. However, no significant difference in cytotoxicity of free DOX at pH 6.5 and 7.4 was observed. The fact that free DOX possessed higher cytotoxicity than DOX-loaded nanogels at the same concentration of DOX might be that free DOX can be quickly transported into the cells and enter nucleus by passive diffusion. Furthermore, Bel7402 cells pretreated with GSH-OEt exhibited higher proliferation inhibition efficiency to DOXloaded nanogels (Figure 6B). No cytotoxicity was found when Bel7402 cells were treated with the blank nanogels (data not shown). These data revealed that the enhanced cellular uptake of P(OEGMAs-ss-AA) nanogels at pH 6.5 and the faster DOX release from DOX-loaded nanogels triggered by higher intracellular GSH concentration might result in the strong cytotoxicity of DOX-loaded nanogels. 3.7. Intracellular Tracking of DOX-Loaded P(OEGMAsss-AA) Nanogels. The understanding of cellular uptake and intracellular tracking is necessary for clarifying the biological activity of nanomaterials. To investigate the intracellular tracking of DOX-loaded P(OEGMAs-ss-AA) nanogels, Bel7402 cells were treated with DOX-loaded nanogels labeled with 5-aminofluorecein for different time courses and the fluorescence of DOX/5-aminofluorecein was observed by confocal microscope. As shown in Figure 7, the fluorescence of DOX and 5-aminofluorescein totally merged when the cells were treated for 0.5 and 2 h, which suggests that DOX and the nanogels entered the cells together. However, DOX was found to colocalize with DAPI (labeling the nucleus) when the cells were incubated for 8 h. These data indicated that DOX was released from the nanogels and diffused into the nucleus once DOX-loaded P(OEGMAs-ss-AA) nanogels entered the cells. 3.8. Endocytic Pathway of P(OEGMAs-ss-AA) Nanogels. Given that DOX entered into cells with P(OEGMAs-ssAA) nanogels together, the endocytic pathway of P(OEGMAsss-AA) nanogels was investigated using several specific endocytic inhibitors.28 As shown in Figure 8, panel A, MβCD, a cholesterol-depleting agent to disrupt several lipid raft/caveolae-mediated endocytic pathways and chlorpromazine, an inhibitor to probe clathrin-mediated endocytosis, significantly decreased the internalization of 5-aminofluoreceinlabeled P(OEGMAs-ss-AA) nanogels in Bel7402 cells. Cytochalasin D, a potent inhibitor of actin polymerization to inhibit both caveolin- and clathrin-mediated pathways as well as macropinocytosis, also significantly reduced the cellular uptake of 5-aminofluorecein-labeled P(OEGMAs-ss-AA) nanogels. However, EIPA, an inhibitor of epithelial sodium channels (ENaC) as well as Na+/H+ antiporters (NHE) to inhibit macropinocytosis did not lower the cellular uptake of 5aminofluorecein-labeled P(OEGMAs-ss-AA) nanogels. The qualitative results from confocal microscopy studies were consistent with the quantitative results obtained from flow cytometry (Figure 8B). These data suggested that both lipid raft/caveolae- and clathrin-mediated endocytic pathways were

4. CONCLUSIONS Hydrophilicity/hydrophobicity reversible and redox-sensitive P(OEGMAs-ss-AA) nanogels with the size of about 150 nm were constructed by emulsion polymerization. The nanogels underwent a pH-dependent hydrophilic/hydrophobic change. The nanogels were hydrophilic under physiological conditions, which might make nanogels avoid the clearance by RES. The nanogels became hydrophobic at pH 6.5 in the tumor microenvironment, which might make nanogels be easily internalized by tumor cells. The loaded DOX and nanogels were internalized into the cells together, and both lipid raft/ caveolae- and clathrin-mediated endocytic pathways were involved in the cellular uptake of the nanogels in Bel7402 cells. Furthermore, DOX-loaded P(OEGMAs-ss-AA) nanogels 1645

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were internalized via the specific route in endo/lysosome compartments. Once internalized, DOX was released from DOX-loaded P(OEGMAs-ss-AA) nanogels in response to intracellular GSH and entered nucleus to exert cytotoxicity. The hydrophilicity/hydrophobicity reversible and redoxsensitive P(OEGMAs-ss-AA) nanogels might be a promising carrier for the effective intracellular delivery of anticancer drugs.

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ASSOCIATED CONTENT

S Supporting Information *

The size of P(OEGMAs-ss-AA) nanogels in PBS containing different concentrations of FBS. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 27 87792147. Fax: +86 27 8779 2234. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Marino Zerial for the plasmids. We also thank the Analytical and Testing Center of Huazhong University of Science and Technology for related analysis. This work was supported by National Basic Research Program of China (973 Programs, 2012CB932500, and 2015CB931800) and the National Natural Science Foundation of China (81473171, 81372400, and 51103051).

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