Redox Dual-Responsive Nanogels for On-Demand

Mar 10, 2016 - (I) Intravenous administration of DOX@nanogels; (II) accumulation and penetration of .... were incubated with various amount of GSH for...
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Smart pH/Redox Dual-Responsive Nanogels for On-Demand Intracellular Anticancer Drug Release Hao Yang,†,‡ Qin Wang,†,§ Shan Huang,†,‡ Ai Xiao,§ Fuying Li,‡ Lu Gan,*,‡ and Xiangliang Yang*,‡ ‡

National Engineering Research Center for Nanomedicine, Key Laboratory of Molecular Biophysics of the Ministry of Education, 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: Efficient accumulation and intracellular drug release in cancer cells remain a crucial challenge in developing ideal anticancer drug delivery systems. Here, poly(N-isopropylacrylamide)-ss-acrylic acid (P(NIPAM-ss-AA)) nanogels based on NIPAM and AA cross-linked by N,N’-bis(acryloyl)cystamine (BAC) were constructed by precipitation polymerization. The nanogels exhibited pH/redox dual responsive doxorubicin (DOX) release behavior in vitro and in tumor cells, in which DOX release from nanogels was accelerated in lysosomal pH (pH 4.5) and cytosolic reduction (10 mM GSH) conditions. Moreover, intracellular tracking of DOX-loaded nanogels confirmed that after the nanogels and the loaded DOX entered the cells simultaneously mainly via lipid raft/caveolae-mediated endocytosis, DOX-loaded nanogels were transported to lysosomes and then the loaded DOX was released to nucleus triggered by lysosomal pH and cytoplasmic high GSH. MTT analysis showed that DOX-loaded nanogels could efficiently inhibit the proliferation of HepG2 cells. In vivo animal studies demonstrated that DOX-loaded nanogels were accumulated and penetrated in tumor tissues more efficiently than free DOX. Meanwhile, DOX-loaded nanogels exhibited stronger tumor inhibition activity and fewer side effects. This study indicated that pH/redox dual-responsive nanogels might present a prospective platform for intracellular drug controlled release in cancer therapy. KEYWORDS: pH/redox dual responsiveness, nanogels, controlled drug release, tumor accumulation and penetration, cancer therapy

1. INTRODUCTION Nano drug delivery systems (NDDS) have been considered as one of the most prospective platforms for cancer chemotherapy because of their distinctive physicochemical and biological characterization, including increased drug accumulation in solid tumor by the enhanced permeability and retention (EPR) effect and decreased side effects.1−3 To date, various nanocarriers like liposomes, polymeric micelles, and nanogels have been constructed to deliver anticancer drugs.4−6 However, insufficient drug release from the nanoparticles in tumor cells may prevent the drug from reaching the targeting sites (e.g., nucleus for DOX), which hindered their application in chemotherapy. The desirable anticancer drug delivery system should hold the drug being transported stably in the bloodstream and release the drug in tumor cells.7 To achieve the above requirements, intelligent drug carriers have recently gained great attention because of their controlled on-demand release of the entrapped drugs in response to intracellular microenvironmental stimuli, including temperature,8 pH,9 redox,10 and enzymy,11 which resulted in strong anticancer activity with fewer side effects. Among them, multistimuli responsive vehicles, especially pH/redox dual responsive nanocarriers have received additional consideration for drug delivery.12−14 It is widely known that the pH value in tumor tissues is lower than that of normal tissues. In addition, © XXXX American Chemical Society

the pH values in late endosomes and lysosomes, where the drug delivery vehicles are usually located via endocytosis, are significantly less than extracellular pH in tumor tissues.15 On the other hand, the intracllular concentration of glutathione (GSH) is about 2−3 orders more than that in extracellular plasma.16 The low GSH concentration offers the stability of disulfide bond-cross-linked nanocarriers in plasma, whereas the high intracellular GSH concentration in cells triggers their disintegration, resulting in the quick drug release. Because of the dramatic difference in the pH and GSH concentration inside and outside cells, pH/redox dual responsive nanocarriers might provide the significant potential for the delivery and intracellular release of anticancer drugs. Nanogels are recognized as hydrophilic three-dimensional polymer networks with the hydrodynamic diameter in submicron range. Owing to their distinctive characterization such as good stability, high drug loading capacity, large surface area allowing covalent conjugation and intelligent responsiveness to environmental stimuli,17 nanogels have shown as the potential drug delivery carriers for various low molecular chemotherapeutics, peptides, RNAs and DNAs.18,19 Poly(NReceived: February 5, 2016 Accepted: March 10, 2016

A

DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of P(NIPAM-ss-AA) nanogels for Anticancer Drug Deliverya

a (A) Construction of P(NIPAM-ss-AA) nanogels. (B) Characterization of P(NIPAM-ss-AA) nanogels in response to intracellular microenvironment. The nanogels were shrunken at pH 4.5 and degraded in response to high GSH, which triggered intracellular drug release. (C) Site-directed DOX release delivered by P(NIPAM-ss-AA) nanogels for cancer therapy. (I) Intravenous administration of DOX@nanogels; (II) accumulation and penetration of DOX@nanogels in tumor tissues; (III) endocytosis of DOX@nanogels by tumor cells; (IV) translocation to lysosome compartment, where DOX was partially squeezed out from the nanogels; (V) DOX release from the nanogels in response to high cytosolic GSH; (VI) DOX delivery to nucleus to exert cytotoxicity against cancer cells.

2. EXPERIMENTAL SECTION

isopropylacrylamide) (PNIPAM)-based nanogels showed typical thermosensitive property which helped to be prepared easily in aqueous medium via precipitation polymerization at an elevated temperature higher than its volume phase transition temperature (VPTT).20 The thermosensitive property could also endow the nanogels with hydrophilicity and stability during circulation by copolymerization of NIPAM with other hydrophilic monomer, such as acrylic acid (AA), to render the VPTT of nanogels higher than the body temperature.21 Here, pH/redox dual responsive nanogels (named as P(NIPAM-ss-AA) nanogels) based on NIPAM and AA crosslinked by a reducible cross-linker containing disulfide bond N,N’-bis(acryloyl)cystamine (BAC), have been prepared for on-demand intracellular anticancer drug release (Scheme 1A). The nanogels efficiently enhanced the accumulation of DOX in tumor tissues via EPR effect and allowed DOX to penetrate deeply in tumor tissues. DOX-loaded nanogels (shorted as DOX@nanogles) were internalized into lysosomes in tumor cells, where the nanogels were shrunken in response to lysosomal pH, resulting in partial DOX release. DOX@ nanogels then entered into cytoplasma where nanogels were disintegrated and DOX was completely released triggered by high intracellular GSH. The released DOX was finally translocated to nucleus to exert cytotoxicity against cancer cells (Scheme 1B, C).

2.1. Materials. Doxorubicin hydrochloride (DOX·HCl, with the purity of above 98.0%) was obtained from Beijing Huafeng United Technology CO., Ltd. (Beijing, China). NIPAM with the purity above 98.0% was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and recrystallized from n-hexane. BAC, sodium dodecyl sulfate (SDS), glutathione reduced ethyl ester (GSH-OEt), 3-(4,5-dimethyl-2thiazolyl)-2,5-Diphenyltetrazolium bromide (MTT), methyl-β- cyclodextrin (MβCD), 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and chlorpromazine (CPZ) were obtained from Sigma-Aldrich (St Louis, MO, USA). 4’6-diamidino-2-phenylindole (DAPI) and LysoTracker Green were bought from Beyotime Institute of Biotechnology (Shanghai, China). The other reagents used in this study were commercially available with analytical grade. 2.2. Cell Culture. The human hepatoma HepG2 cell line and mouse hepatoma H22 cell line were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco BRL/Life Technologies, Grand Island, NY, USA), 100 μg/mL streptomycin and 100 U/mL penicillin at 37 °C in 5% CO2 atmosphere. 2.3. Construction of P(NIPAM-ss-AA) Nanogels. P(NIPAM-ssAA) nanogels were constructed through precipitation polymerization as described.22,23 Briefly, comonomers of NIPAM and AA were fed with the molor ratio of 9:1 into ultrapure water with mass percent concentration of ∼1.1%. Cross-linker BAC was added with the amount of 0.4 mol % to all the monomers. SDS was used as a surfactant with the concentration of 0.04%. The above mixture was deoxidized with high-purity nitrogen and then heated to 70 °C. Potassium persulfate (KPS, 0.06 wt %) was added to initiate the polymerization reaction under N2 at 70 °C for at least 4.5 h. The resulting system was purified B

DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DAPI (5 μg/mL, 20 min), respectively. The cells were fixed with 4% paraformaldehyde and visualized using confocal microscope.22 2.10. Cellular Accumulation of DOX@Nanogels. HepG2 cells were preincubated in DMEM medium with or without 10 mM GSHOEt for 2 h.26 After rinsing with PBS, the cells were treated with free DOX or DOX@nanogels (10 μg/mL DOX concentration) for 4 h. The cells were collected for flow cytometric analysis. 2.11. In Vitro Cytotoxicity of DOX@Nanogels. The in vitro cytotoxicity of DOX@nanogels was determined by MTT assay.27 In brief, HepG2 cells were preincubated in fresh medium with or without 10 mM GSH-OEt for 2 h. After rinsing with PBS, the cells were treated with different concentrations of free DOX or DOX@nanogels. At the designed time points, the cells were washed with PBS and then incubated in medium containing 20 μL MTT solution (5 mg/mL) at 37 °C for another 4 h. The medium containing MTT was discarded and 150 μL DMSO was added to dissolve the formazan crystals. The absorbance at 490 nm was determined by a Labsystems iEMS microplate reader (Helsinki, Finland). 2.12. Biodistribution of Drug@Nanogels. Tumor-bearing mice were prepared by subcutaneously inoculating H22 cells (2 × 106) in the back of male BALB/c mice (around 25 g). When tumors reached 300 mm3 in volume, the mice were divided randomly into four groups. DiR (a near-infrared fluorescent dye) was encapsulated into the nanogels using the same method with the preparation of DOX@ nanogels. The mice were injected intravenously with free DOX or DOX@nanogels (5 mg/kg DOX dose), or free DiR or DiR@nanogels (30 μg/kg DiR dose), respectively. Whole-body fluorescence images were acquired at the desired time intervals after intravenous injection of free DiR or DiR@nanogels by a Caliper IVIS Lumina II in vivo imaging system (λex 740 nm, λem 780−820 nm, PerkinElmer, Waltham, MA, USA). At 24 h after injection, the mice were sacrificed by cervical dislocation and the major organs including heart, liver, spleen, lung, kidney and tumor were resected and imaged to evaluate the ex vivo biodistribution of DOX@nanogels or [email protected] 2.13. In Vivo Tumor Penetration of DOX@Nanogels. When tumors reached 300 mm3 in volume, the tumor-bearing mice were intravenously administered with free DOX or DOX@nanogels (5 mg/ kg DOX dose), respectively. At 24 h postinjection, the mice were sacrificed and tumor tissues were collected, rinsed with PBS followed by cryotomy. The frozen tumor sections were stained by FITC-CD31 antibody (Abcam, Cambridge, UK) for 30 min at 37 °C and then rinsed with PBS. The tumor sections were observed by confocal microscope.29 2.14. In Vivo Antitumor Efficacy of DOX@Nanogels. When tumor reached 300 mm3 in volume, the tumor-bearing mice were intravenously administered with free DOX or DOX@nanogels (5 mg/ kg DOX dose) at day 1, 4, 7. The tumor sizes were measured every other day. At day 10, the mice were sacrificed, and the hearts were excised, fixed and sliced for hematoxylin and eosin (H&E) staining. 2.15. Statistical Analysis. Experiments were performed with at least three replicates. Statistical analyses were carried out by Student’s t test. A p-value of less than 0.05 was considered significant.

by dialyzing in a dialysis bag (molecular weight cutoff 14 000) against ultrapure water for more than 10 days to eliminate unreacted monomers and other low molecules, and then lyophilized to obtain the xerogels for further use in a yield of above 90%. 2.4. Preparation of DOX@Nanogels. DOX@nanogels were prepared as described with minor modification.22,24 Briefly, DOX·HCl aqueous solution with the concentration of 1.0 mg/mL was mixed with excess triethylamine (the molar ratio of DOX·HCl to triethylamine was 1:3) in chloroform for 30 min to obtain hydrophobic DOX. Then DOX solution was dropped into the nanogels aqueous solution (2.5 mg/mL) and mixed at room temperature for 2 h, followed by stirring at 40 °C overnight to volatilize chloroform. The obtained DOX@ nanogels were purified in an ultrafilter tube (molecular weight cutoff 10000) by centrifugation at 4000 rmp for 20 min. Drug loading capacity (DLC) and encapsulation efficiency (EE) of the nanogels were defined by the following equations: 2 2 DLC(%) = (W encapsulated DOX in nanogels /W DOX@nanogels ) × 100; EE(%) = (Wencapsulated DOX in nanogels/Wtotal administered DOX) × 100. DOX concentration in the supernatant was determined at 480 nm on a UV− vis spectroscopy. The whole procedure was carried out away from light. 2.5. Characterization of P(NIPAM-ss-AA) Nanogels. The zeta potentials and hydrodynamic diameters of blank nanogels and DOX@ nanogels were determined by dynamic light scattering (DLS, ZetaSizer ZS90, Malvern, Worcestershire, UK). The morphologies of blank nanogels and DOX@nanogels were visualized using transmission electron microscopy (TEM, Tecnai G2−20, FEI, Netherlands). The VPTT of the nanogels was measured from hydrodynamic diametertemperature curves.25 Briefly, 0.25 mg/mL nanogels were dissolved in PBS at pH 7.4 or pH 4.5. The hydrodynamic diameters of the nanogels at various temperatures and various pH values were measured by DLS. The diameter−temperature curves were fitted with Boltzmann function. The fitted ones were then differentiated, and the temperature at the point of inflection was defined as the VPTT. 2.6. In Vitro Drug Release. The GSH-sensitive DOX release from the nanogels was determined by incubating DOX@nanogels (10 μg/ mL DOX concentration) in PBS containing various amount of GSH at 37 °C at various time intervals.22 DOX fluorescence spectrum was measured with λex of 480 nm and λem of 500−700 nm using fluorescence spectrophotometer (F-4500, Hitachi Ltd., Tokyo, Japan). The in vitro pH/GSH-responsive DOX release profiles from the nanogels were further determined by dialysis method.13 Briefly, 1 mg/ mL DOX@nanogels were put in a dialysis bag (molecular weight cutoff 14 000) and immersed in 25 mL PBS containing various amount of GSH at various pH values with shaking at 100 rpm at 37 °C. At given time points, 1 mL of the release medium was removed and replaced with the same volume of fresh medium. The amount of DOX released was determined with λex of 480 nm by fluorescence spectrophotometer. 2.7. Endocytosis of P(NIPAM-ss-AA) Nanogels. Fluoresceinlabeled nanogels were prepared through the condensation reaction between amino group in 5-aminofluorescein and carboxylic groups in nanogels with the catalysis of NHS and EDC.23 HepG2 cells were pretreated with 10 mM MβCD (30 min), 50 μM EIPA (1 h) or 10 μg/ mL CPZ (30 min), respectively. The cells were then incubated in fresh medium containing the inhibitors and 50 μg/mL 5-aminofluoresceinlabeled nanogels for another 2 h. The cells were collected for flow cytometric analysis (FC500, Beckman Coulter, Fullerton, CA, USA). 2.8. Intracellular Process of DOX@Nanogels. HepG2 cells were incubated with 5-aminofluorescein-conjugated DOX@nanogels (10 μg/mL DOX concentration). At the designed time points, the cells were washed with PBS and then labeled with 5 μg/mL DAPI at 37 °C for 20 min. The cells were fixed with 4% paraformaldehyde and visualized by FV 1000 confocal microscope (Olympus, Tokyo, Japan).22 2.9. Subcellular Localization of DOX@Nanogels. For determination of the overlay of DOX@nanogels with lysosomes or nucleus, we incubated HepG2 cells with DOX@nanogels (10 μg/mL DOX concentration). At the designed time points, the cells were washed with PBS and then labeled with LysoTracker Green (1 μM, 30 min) or

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of P(NIPAM-ssAA) Nanogels. P(NIPAM-ss-AA) nanogels were easily constructed in aqueous medium by precipitation polymerization at an elevated temperature above its VPTT.20 Here, AA as a comonomer was used to adjust the VPTT of the nanogels and endow the nanogels with pH-responsiveness. Disulfide bond-containing BAC was used as a cross-linker to impart the nanogels redox-responsiveness, in which the nanogels might be disintegrated in response to high intracellular GSH concentration while keeping stable in blood circulation. The hydrophobic DOX organic solution, as an anticancer drug model, was then mixed with the aqueous solution of nanogels, forming unstable polymer emulsion. With the evaporation of the organic solvents, DOX was forced into the hydrophobic C

DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Characterization of the blank nanogels and DOX@nanogels. (A) Hydrodynamic size of the blank nanogels and DOX@nanogels at pH 7.4. (B) Size of the nanogels at various pH values at 37 °C. (C) TEM images of the blank nanogels and DOX@nanogels. (D) Diameter−temperature curves of the nanogels at pH 7.4 or pH 4.5. Data as mean values ± SD (n = 3).

Figure 2. In vitro pH/redox dual responsive DOX release from P(NIPAM-ss-AA) nanogels. (A) Fluorescence spectra of DOX when DOX@ nanogels were treated with various amount of GSH for 24 h. (B) Fluorescence spectra of DOX when DOX@nanogels were treated with 10 mM GSH at different time intervals. (C) In vitro DOX release behavior from the nanogels in the presence of various amount of GSH at various pH values at 37 °C. Data as mean values ± SD (n = 3).

The size of the nanogels decreased significantly at pH 4.5 compared with that at pH 7.4 or pH 6.5, which could be attributed to the inherent pH-sensitivity of the nanogels originated from the comonomer AA. Namely, the carboxyl groups in the nanogels were partially protonized, resulting in the increment of the interaction between the chains in the nanogels via hydrogen bond and the subsequent decrease in the size at pH 4.5, which was lower than the pKa of poly(acrylic acid).30 In contrast, the interaction in the nanogels tended to decrease due to the electrostatic replusive force between the

microdomains of nanogels to construct the DOX@nanogels. The DLC and EE of the nanogels were about 5.9 and 97.8%, respectively. The mean sizes of the blank nanogels and DOX@nanogels were around 245 and 260 nm at pH 7.4 measured by DLS, respectively (Figure 1A). The zeta potentials of the blank nanogles and DOX@nanogels were −24.6 mV and −26.7 mV, respectively, which increased the stability of the nanogels by offering the electrostatic repulsive force. Furthermore, the size of the nanogels was measured at various pH values (Figure 1B). D

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Figure 3. Redox-sensitive intracellular DOX release from P(NIPAM-ss-AA) nanogels in HepG2 cells. (A) Internalized DOX fluorescence intensity when HepG2 cells preincubated in medium in the presence or absence of 10 mM GSH-OEt for 2 h were treated with different concentrations of DOX@nanogels for 4 h by flow cytometry. (B) Quantitative analysis of DOX fluorescence intensity in the above experiments. Data as mean values ± SD (n = 3). *P < 0.01.

Figure 4. Endocytosis of P(NIPAM-ss-AA) nanogels in HepG2 cells. (A) The internalized fluorescence intensity by flow cytometry when HepG2 cells pretreated with 10 mM MβCD, 10 μg/mL CPZ or 50 μM EIPA were coincubated with 50 μg/mL 5-aminofluorescein-conjugated nanogels for 2 h, respectively. (B) Quantitative analysis of 5-aminofluorescein fluorescence intensity in the above experiments. Data as mean value ± SD (n = 3). *P < 0.05, **P < 0.01 compared with control group.

deprotonized carboxyl groups at an elevated pH (7.4 or 6.5). The blank nanogels and DOX@nanogels were nearly monodisperse with spherical morphology revealed by TEM imaging (Figure 1C). The VPTTs of the nanogels at pH 7.4 and pH 4.5 determined by DLS method were 45.6 and 28.8 °C, respectively. (Figure 1D). These data suggested that the nanogels were hydrophilic during circulation, which might render the nanogels with long circulation capacity to preferentially accumulate at tumor site by EPR effect. Additionally, the nanogels shifted to shrunken and hydrophobic in lysosomes (pH 4.5), which might be beneficial in controlling drug release. 3.2. pH/Redox-Responsive Drug Release from DOX@ Nanogels in Vitro. It has been reported that DOX fluorescence was self-quenched when encapsulated in nanoparticles, while it was increased when DOX was released from nanoparticles.31 To investigate the redox-sensitive DOX release from the nanogels, we measured DOX fluorescence when DOX@nanogels were incubated with various amount of GSH for different time courses. No change in fluorescence intensity of DOX@nanogels was found when treated with 2 μM GSH (equivalent to GSH level in human plasma) compared with control group, suggesting that DOX@nanogels were stable in the bloodstream. However, the fluorescence intensity of DOX@nanogels dramatically increased in response to 10 mM GSH (equivalent to GSH level in tumor cells) (Figure 2A). Moreover, the fluorescence intensity enhanced with time in the presence of 10 mM GSH (Figure 2B). The results

demonstrated that DOX@nanogels showed a GSH-responsive drug release behavior. To explore whether P(NIPAM-ss-AA) nanogels exhibited pH/redox dual-responsive intracellular drug release profile, DOX release from the nanogels was further investigated in PBS containing various concentrations of GSH at various pH values by dialysis method (Figure 2C). As expected, 10 mM GSH, but not 2 μM GSH significantly enhanced the cumulative DOX release from the nanogels at the same pH value. In the mean time, DOX release from the nanogels at pH 4.5 was dramatically enhanced compared to that at pH 7.4 and pH 6.5 in the presence or absence of the same GSH concentration, indicating that DOX release from the nanogels might be expedited in lysosomes. One possibility might be due to the decrease in the size of the nanogels at pH 4.5 (Figure 1B), which resulted in the ejection of the cargoes from the nanogels and then the enhanced drug release. In addition, DOX solubility was increased at pH 4.5, which might speed up the drug release.22 DOX release from the nanogels reached as high as 96% in PBS containing 10 mM GSH at pH 4.5, revealing the almost complete DOX release from the nanogels in the intracellular milieu. 3.3. Redox-Sensitive Intracellular DOX Release from P(NIPAM-ss-AA) Nanogels. To explore the possibility of redox-responsive intracellular drug release from the nanogels in cancer therapy, we determined the effects of GSH-OEt, which can increase the intracellular GSH concentration, on the intracellular accumulation of DOX@nanogels and drug release E

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Figure 5. Intracellular distribution of DOX@nanogels in HepG2 cells. (A) Confocal microscopic images of the intracellular trafficking of DOX@ nanogels in HepG2 cells incubated with 5-aminofluorescein-conjugated DOX@nanogels (10 μg/mL DOX concentration) for different time courses. DAPI was used for nucleus labeling. (B) Confocal microscopic images of the intracellular distribution of DOX@nanogels in HepG2 cells incubated with DOX@nanogels (10 μg/mL DOX concentration) for different time courses. LysoTracker Green was used to label lysosomes (left) and DAPI was used to label nucleus (right). The scale bar is 10 μm.

behavior by flow cytometry. As illustrated in Figure 3A, B, the intracellular accumulation of DOX@nanogels increased in a dose-dependent manner. In the meanwhile, pretreatment with GSH-OEt could significantly upregulate DOX fluorescence intensity in DOX@nanogels-treated HepG2 cells. In contrast, GSH-OEt pretreament did not change the intracellular DOX fluorescence in DOX-treated cells (Figure S1). In view of the increased DOX fluorescence when it was released from nanogels triggered by GSH (Figure 2A), the enhanced DOX fluorescence intensity in DOX@nanogels-treated HepG2 cells might result from the intracellular DOX release, which was attributed to the reduction-triggered degradation. 3.4. Endocytosis of P(NIPAM-ss-AA) Nanogels. Elucidating internalization and intracellular trafficking was important to understand the biological activity of nanomaterials. Several specific endocytic inhibitors were used to investigate the endocytosis of P(NIPAM-ss-AA) nanogels.32 As illustrated in Figure 4A and B, MβCD, an inhibitor of lipid raft/caveolaemediated endocytosis, resulted in a decrease of 75% in the internalization of 5-aminofluorecein-labeled nanogels in HepG2 cells. Meanwhile, CPZ, an inhibitor of clathrin-mediated endocytosis, and EIPA, an inhibitor of micropinocytosis, resulted in the decrease of about 10% in the internalization of the nanogels. These data revealed that besides clathrinmediated endocytosis and macropinocytosis, lipid raft/ caveolae-mediated endocytosis played an important role in

the cellular uptake of P(NIPAM-ss-AA) nanogels in HepG2 cells. 3.5. Intracellular Trafficking of DOX@Nanogels. To clarify the intracellular trafficking of DOX@nanogels, we first incubated HepG2 cells with 5-aminofluorecein-conjugated DOX@nanogels for different time courses. Confocal microscopy data showed that the fluorescence of 5-aminofluorescein and DOX overlaid in cells treated with DOX@nanogels for 1 and 3 h, indicating that the nanogels and DOX went into the cells together. Meanwhile, DOX colocalized with nucleus (labeled with DAPI) when the cells were treated with DOX@ nanogels for 8 h (Figure 5A). The results revealed that after DOX@nanogels entered the cells, DOX was released and translocated to the nucleus. To further demonstrate the intracellular distribution of DOX@nanogels, we incubated HepG2 cells with DOX@ nanogels for different time courses, and the overlay of DOX with cellular compartments was investigated by confocal microscopy. As illustrated in Figure 5B, DOX almost thoroughly colocalized with lysosomes (labeled with LysoTracker Green) after 1 h treatment. Nevertheless, a gradual decrease in the overlay of DOX and lysosomes was observed as the incubation time went on. In the meanwhile, DOX was found to be distributed in the cytoplasm after 1 h incubation, whereas it was mostly located in the nucleus after 8 h treatment. The results confirmed that after internalization, F

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Figure 6. Biodistribution and penetration of P(NIPAM-ss-AA) nanogels in H22-bearing mice. (A) In vivo biodistribution of DiR at different time points after intravenous administration of free DiR or DiR@nanogels (30 μg/kg DiR concentration) in H22-bearing mice by fluorescence imaging. The red circles indicated the location of the tumors. (B) Ex vivo fluorescence imaging of DiR at 24 h after intravenous injection of free DiR or DiR@ nanogels (30 μg/kg DiR concentration) in H22-bearing mice. (C) Ex vivo fluorescence imaging of DOX at 24 h after intravenous injection of free DOX or DOX@nanogels (5 mg/kg DOX concentration) in H22-bearing mice. (D) The tumor penetration of DOX at 24 h after intravenous injection of free DOX or DOX@nanogels (5 mg/kg DOX concentration) in H22-bearing mice. FITC-CD31 was used to label tumor blood vessels. The scale bar is 50 μm.

venously into H22-bearing mice and DiR fluorescence was first investigated by whole-animal fluorescence imaging. As shown in Figure 6A, the distribution of DiR fluorescence increased in tumors with time in free DiR- and DiR@nanogels-treated group. However, DiR@nanogels exhibited stronger DiR fluorescence in tumor than free DiR. To clearly evaluate the tissue distribution of DiR@nanogels, the major organs, including heart, liver, spleen, lung, kidney and tumor, were excised and imaged (Figure 6B). In accord with the wholeanimal imaging results, DiR@nanogels showed higher accumulation in tumor than free DiR. The biodistribution of DOX@ nanogels in H22-bearing mice further confirmed the increased accumulation in tumor compared with that of free DOX (Figure 6C). These data consistently indicated that P(NIPAMss-AA) nanogels loaded with cargoes exhibited good tumor targeting capacity, possibly because of their enhanced EPR effect.28 The poor penetration of anticancer drugs into tumor tissues reduced the overall therapeutic effects owing to the abnormal tumor vasculature structure, high interstitial pressure and dense extracellular matrix in tumors, which was considered as one of the main mechanisms of drug resistance in solid malignan-

DOX@nanogels were first transported to lysosomes and then released DOX into nucleus to exert cytotoxicity. In view of the faster DOX release behavior from the nanogels at pH 4.5 (Figure 2C), the localization of DOX@nanogels at lysosomes might accelerate DOX release in HepG2 cells. DOX was further released in cytoplasma where nanogels were disintegrated in the presence of high intracellular GSH concentration and then delivered into the nucleus. Here, P(NIPAM-ss-AA) nanogels might possess the ability to escape from lysosomes and then release DOX trigged by high intracellular GSH. The reason for the nanogels’ lysosomal escape might be that the nanogels turned to hydrophobic due to its VPTT below the body temperature at lysosomal pH 4.5 (Figure 1D), which resulted in the fusion of lysosome membrane with the nanogels and then membrane destabilization. The fact that the hydrophilicity/ hydrophobicity of nonviral gene carriers affected lysosomal escape capacity had been reported.33,34 3.6. In Vivo Biodistribution and Tumor Penetration of DOX@Nanogels. The in vivo biodistribution of anticancer drugs directly affects their therapeutic effects and possible side reactions. To demonstrate the biodistribution behavior of P(NIPAM-ss-AA) nanogels, we injected DiR@nanogels intraG

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Figure 7. In vitro cytotoxicity of DOX@nanogels in HepG2 cells. (A) Cell viability of HepG2 cells incubated with free DOX or DOX@nanogels for 48 h by MTT assay. (B) Cell viability of HepG2 cells preincubated with or without 10 mM GSH-OEt for 2 h, following treatment with free DOX or DOX@nanogels (5 μg/mL DOX concentration) for 24 h. Data as mean values ± SD (n = 3). *P < 0.05.

Figure 8. In vivo antitumor activity of DOX@nanogels in H22-bearing mice. (A) Tumor growth inhibition profiles in H22-bearing mice after intravenous administration of free DOX or DOX@nanogels (5 mg/kg DOX dose) at day 1, 4, and 7. (B) The body weight of H22-bearing mice throughout tumor growth inhibition experiment. (C) H&E staining of the heart slices in H22-bearing mice at the end of experiment. Black arrow represents neutrophil accumulation. The scale bar is 25 μm. Data as mean values ± SD (n = 6).

cies.35,36 Therefore, enhancing the penetration of anticancer drugs into tumor tissues presents a crucial challenge in cancer therapy. To explore whether P(NIPAM-ss-AA) nanogels might result in enhanced tumor penetration, we injected H22-bearing mice intravenously with free DOX or DOX@nanogels, and the distance between DOX and tumor blood vessels recognized by FITC-conjugated CD31 was determined by confocal microscopy (Figure 6D). Free DOX was mostly localized in blood vessels. However, DOX@nanogels were distributed more widely outside the tumor vessels than free DOX. One possible explanation for the enhanced tumor penetration of DOX@ nanogels was that the nanogels were preferentially accumulated in tumor tissues owing to their long circulation and EPR effect. In the mean time, recent papers showed that some nanoparticles loaded with anticancer drugs could induce apoptosis in cancer cells, be exocytosed from cancer cells and then infect neighboring cells like a virus, which resulted in deep tumor penetration.37,38 Whether P(NIPAM-ss-AA) nanogels exhibited

similar phenomenon to penetrate deeper inside tumor remained to be elucidated. 3.7. In Vitro Cytotoxicity of DOX@Nanogels. To assess the potentials of P(NIPAM-ss-AA) nanogels used as drug carriers in cancer chemotherapy, we evaluated the cell cytotoxicity of DOX@nanogels by MTT assay (Figure 7A). DOX@nanogels and free DOX exhibited dose-dependent cytotoxicity to HepG2 cells under all circumstances. In the meanwhile, DOX@nanogels showed similar cytotoxic effects to HepG2 cells compared with free DOX, which might be due to the successful DOX release from P(NIPAM-ss-AA) nanogels in response to lysosomal pH- and redox-responsive intracellular microenvironment (Figure 2C). Furthermore, pretreatment with GSH-OEt significantly enhanced DOX@nanogels-induced cytotoxicity to HepG2 cells. In contrast, pretreatment with GSH-OEt did not affect the cytotoxicity of free DOX (Figure 7B). These data further confirmed that intracellular DOX release from the nanogels responding to intracellular GSH contributed to the inhibition of cell proliferation. No evident H

DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

of China (81372400, 81473171 and 51103051). We also thank the Analytical and Testing Center of Huazhong University of Science and Technology for related analysis.

cytotoxicity was observed in HepG2 cells treated with the blank nanogels (Figure S2), indicating the low cytotoxicity and biocompatibility of P(NIPAM-ss-AA) nanogels. 3.8. Antitumor Activity of DOX@Nanogels. In view of the efficient accumulation and penetration of DOX@nanogels into tumor tissues and the intracellular DOX release, the antitumor activity of DOX@nanogels was evaluated using H22bearing mice (Figure 8A). DOX@nanogels exhibited a stronger suppression of tumor growth than free DOX. In the meanwhile, the body weight change, as an indirect index of general toxicity, was detected during the whole tumor growth inhibition experiment (Figure 8B). Free DOX-treated mice showed significant weight loss, whereas the body weight of mice treated with DOX@nanogels was similar to that of control group. Cardiotoxicity, a major side effect of DOX-based chemotherapy, was further investigated by H&E staining (Figure 8C). Obvious neutrophil accumulation was observed in the heart slices of free DOX-treated group. However, DOXinduced cardiotoxicity was significantly inhibited in group treated with DOX@nanogels. These results demonstrated that DOX delivered by P(NIPAM-ss-AA) nanogels exhibited stronger anticancer activity but less adverse effects.

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4. CONCLUSIONS The pH/redox dual-responsive P(NIPAM-ss-AA) nanogels for on-demand intracellular anticancer drug release were constructed by precipitation polymerization. After internalization into the cells mainly via lipid raft/caveolae-mediated endocytic pathway, DOX@nanogels were delivered to lysosomes, in which DOX was efficiently released from the nanogels at lysosomal pH. DOX was further released in response to high cytoplasmic GSH concentration and then transported into nucleus to induce cytotoxicity. Moreover, P(NIPAM-ss-AA) nanogels enhanced the accumulation and penetration of DOX into tumor tissues. DOX@nanogels could effectively inhibited tumor growth and reduced the cardiotoxicity of DOX in H22bearing mice. This study demonstrated that pH/redox dualresponsive P(NIPAM-ss-AA) nanogels might be used as potential anticancer drug carriers.

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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.6b01602. Effects of GSH-OEt on the intracellular DOX concentration; biocompatibility of P(NIPAM-ss-AA) nanogels (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 27 87792147. *E-mail: [email protected]. Tel: +86-27-87793539. Author Contributions †

H.Y., Q.W., and S.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding support from National Basic Research Program of China (973 Programs, 2012CB932500 and 2015CB931802) and the National Natural Science Foundation I

DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b01602 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX