Redox-Sensitive Nanogels for Near-Infrared

Jun 30, 2017 - Thermosensitive poly(N-isopropylacrymide) (pNIPAM)-based nanogels with chemically cross-linked three-dimensional polymer networks have ...
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Zwitterionic Temperature/Redox-Sensitive Nanogels for NearInfrared Light-Triggered Synergistic Thermo-Chemotherapy Fuying Li,†,§ Hao Yang,†,§ Nana Bie,† Qingbo Xu,† Tuying Yong,† Qin Wang,*,‡ Lu Gan,*,† and Xiangliang Yang† †

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

ABSTRACT: Ideal anticancer nano drug delivery systems (NDDSs) need to overcome a series of physiological barriers including blood circulation, tumor accumulation, tumor penetration, internalization by cancer cells, lysosomal escape, and on-demand intracellular drug release following systemic administration. However, it remains a big challenge to construct NDDSs that can overcome all the barriers at the same time. Here, we develop zwitterionic temperature/redoxsensitive nanogels loaded with near-infrared (NIR) dye Indocyanine green (ICG) and anticancer drug doxorubicin (I/D@NG). I/D@NG exhibits enhanced photothermal effects, and NIR irradiation markedly decreases its diameter. NIR irradiation at tumor sites significantly enhances tumor accumulation, tumor penetration, and cellular uptake of I/D@NG with prolonged blood circulation time. Furthermore, I/D@ NG can effectively escape from lysosomes by singlet oxygen-induced lysosomal disruption, and DOX is then sufficiently released from the nanogels to the nucleus in response to high intracellular GSH and photothermal effects. This nanoplatform for thermochemotherapy not only efficiently exerts synergistic cytotoxicity but also overcomes all the physiological barriers of therapeutic agent, thereby providing a substantial in vivo anticancer effect. The multiple functions of I/D@NG provide new insights into designing nanoplatforms for synergistic cancer therapy. KEYWORDS: nanogels, temperature/redox responsiveness, tumor accumulation and penetration, photothermal effect, synergistic therapy vascular permeability,12−14 thereby promoting the delivery of medications to tumor. For instance, local hyperthermia can lead to tumor-site-specific accumulation and penetration of systemically administrated thermal-responsive NDDSs.12,15 Thus, hyperthermia may potentially function as an effective strategy to improve the in vivo process of NDDSs to achieve the synergistic thermo-chemotherapy. Recently, near-infrared (NIR) light-induced photothermal therapy (PTT) as a minimally invasive means can achieve efficient hyperthermia with a considerably deep tissue penetration capacity by irradiating photoabsorbing agents, including cyanine dyes and some photothermal nanoparticles such as gold nanoparticles, carbon nanomaterials, platinated nanoparticles, and so on.16−22 Indocyanine green (ICG), a NIR dye that has been approved by Food and Drug Administration (FDA) in the United States, is regarded as a perfect photoabsorbing agent for PTT because of its excellent biocompatibility and multifunctions, such as

1. INTRODUCTION Nanotechnology-based drug-delivery systems (NDDSs) exhibit promising therapeutic efficacy and reduced side effects in cancer therapy owing to an enhanced permeability and retention (EPR) effect.1−3 Although a number of NDDSs, including PEGylated liposomal doxorubicin (Doxil) and albumin-bound paclitaxel (Abraxane) have been clinically approved, they only offer modest survival benefits.4 NDDSs must overcome a series of physiological barriers from the intravenous injection site to the targeting site, including blood circulation, tumor accumulation, tumor penetration, internalization by cancer cells, lysosomal escape, and on-demand intracellular drug release.5 Although some smart NDDNs responding to pH,6,7 redox,8,9 enzyme,10 and temperature11 have been developed to overcome these barriers, they often encounter difficulty in circumventing all the barriers at the same time and achieve limited therapeutic efficiency. Hyperthermia is one of the therapy methods used for cancer treatment. It not only kills cancer cells directly and enhances the sensitivity of cancer cells to chemotherapy but also increases blood flow to the heated area and augments tumor © 2017 American Chemical Society

Received: June 6, 2017 Accepted: June 30, 2017 Published: June 30, 2017 23564

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of I/D@NG for Synergistic Thermo-Chemotherapy: (A) Characterization of I/D@NG; (B) NIR Light-Induced In Vivo Transport Processing of I/D@NG

NIR fluorescence (NIRF) imaging and photodynamic therapy (PDT) to actualize cancer theranostics and comprehensive treatment.23 However, the limited in vivo stability and photostability as well as lack of tumor selectivity restrict its clinical application. Although some nanoparticles including micelles, vesicles, and liposomes have been used to effectively deliver ICG with enhanced stability and targeting,24−26 these nanocarriers usually involve complicated synthesis, unstable structure, and lack of responsiveness. For example, micelles and vesicles are formed via physical self-assembly of amphiphilic molecules, which need complicated multistep synthesis and undergo potential disassembly at an elevated temperature because the amphiphilic property might vary with the temperature.27,28 Liposomes can undergo a gel-to-liquid phase transition above their phase transition temperature.29,30 The nanostructure disintegration might result in an uncontrolled release of coencapsulated ICG and chemotherapeutic agent before entering into cancer cells, compromising their synergistic anticancer effects. Therefore, it is highly required to construct feasible nanoparticles combining enhanced photothermal and chemotherapeutic effects to achieve synergistic anticancer activity. Thermosensitive poly(N-isopropylacrymide) (pNIPAM)based nanogels with chemically cross-linked three-dimensional polymer networks have been widely used as drug delivery systems owing to their biocompatibility, easy preparation, excellent stability, and intelligence.31,32 The nanogels just shrink in size while keeping the integral network structure above their volume-phase-transition temperature (VPTT),31 which might contribute to deep tumor penetration and cellular uptake.33,34 In addition, betaine-based zwitterionic polymers with antifouling and long circulation properties have been used to replace PEGylated polymers as NDDSs.35 Here, we reported zwitterionic temperature/redox-sensitive nanogels based on NIPAM, zwitterionic sulfobetaine methacrylate (SBMA) and hydrophilic methylallyl amine (MAA) as comonomers and disulfide bond-containing N,N′-bis(acryloyl) cystamine (BAC) as a cross-linker. The obtained nanogels encapsulating ICG and anticancer drug doxorubicin (DOX) (I/D@NG) not only synergized thermo-chemotherapy but also overcame a series of

physiological barriers of chemotherapeutic agent following systemic administration, resulting in excellent antitumor effect (Scheme 1). The nanogels ameliorated the photothermal effect of ICG, and their diameter significantly decreased upon NIR irradiation. I/D@NG exhibited prolonged circulation time in blood. NIR irradiation markedly improved its tumor accumulation, tumor penetration, and cellular uptake by cancer cells. Furthermore, I/D@NG escaped from lysosomes through singlet oxygen-induced lysosomal disruption after internalization and DOX was then efficiently released on-demand to nucleus in response to intracellular high GSH and NIR lightinduced photothermal effects, resulting in enhanced synergism of thermo-chemotherapy.

2. EXPERIMENTAL SECTION 2.1. Materials. NIPAM with the purity >98.0% was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). MAA, SBMA, BAC, sodium dodecyl sulfate (SDS), ICG, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and L-ascorbic acid (Vc) were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). Doxorubicin hydrochloride (DOX·HCl) with the purity >98.0% was obtained from Beijing Huafeng United Technology CO., Ltd. (Beijing, China). Dulbecco’s modified eagle medium (DMEM), RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL/Life Technologies (Grand Island, NY, U.S.A.). 4′6-Diamidino-2phenylindole (DAPI) and LysoTracker Green were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Dihydroethidium (DHE) was purchased from Wusheng Company (Shanghai, China). 2.2. Cells and Animals. HepG2 cells and H22 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HepG2 cells were maintained in DMEM media, and H22 cells were maintained in RPMI 1640 media supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/ mL penicillin at 37 °C in a 5% CO2 incubator. BALB/c mice (male, 18−20 g) and Sprague-dawley (SD) rats (male, 250−300 g) were obtained from the Center for Disease Control and Prevention in Hubei Province (Wuhan, China). H22-bearing mice were prepared by subcutaneously implanting 2 × 106 H22 cells into the right hind leg of BALB/c mice. All animal experiments were performed according to the guidelines authorized by the Institutional Animal Care and Use 23565

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

Research Article

ACS Applied Materials & Interfaces

2.8. Intracellular Singlet Oxygen Generation. The generation of intracellular singlet oxygen was detected by fluorescence microscopy using DHE as the indicator.38 Briefly, HepG2 cells were incubated with I/D@NG containing different concentrations of ICG for 6 h. After the cells were rinsed by PBS, 0.2 mL of DHE solution (5 μM in PBS) was added to the cells and incubated for 30 min at 37 °C, followed with or without 3 min photoirradiation (808 nm, 1.5 W/ cm2). The cells were rinsed by PBS and then detected using Olympus 1X71 fluorescent microscope (Japan). 2.9. NIR Light-Induced Lysosomal Disruption and Intracellular Drug Release. HepG2 cells were pretreated with or without 2 mM Vc for 2 h, followed by incubation with I/D@NG at ICG concentration of 20 μg/mL for 30 min. The cells were rinsed by PBS, then subjected to or not to photoirradiation (808 nm, 1.5 W/cm2) for 1 min. After rinsing by PBS, lysosomes were subsequently dyed with 50 nM LysoTracker Green for 30 min, and nuclei were dyed with 2.5 μg/mL DAPI for 15 min at 37 °C, respectively. The cells were rinsed by PBS and detected under an Andor Revolution spinning disk confocal microscope (Andor Technology, Germany). 2.10. In Vitro Cytotoxicity. HepG2 cells were treated with free I/ D solution, I@NG, D@NG, or I/D@NG at different concentrations of ICG (the added DOX concentration was correspondingly calculated according to the drug loading capacity of nanogels) for 24 h, followed with or without photoirradiation (808 nm, 1.5 W/cm2) for 3 min. The cells were incubated for another 4 h, and then the cell viability was determined by MTT assay.38 2.11. Pharmacokinetic Study. SD rats were randomly grouped and intravenously administrated with free I/D solution or I/D@NG at the ICG dose of 20 mg/kg and DOX dose of 5 mg/kg, respectively. Then 250 μL of blood was drawn and centrifuged to harvest plasma at the indicated time points. Subsequently, 150 μL of plasma was taken out and incubated with 150 μL of 100 mM GSH for 2 h. DOX was extracted by deproteinating plasma with 600 μL methanol and then centrifuged at 10 000 rpm for 10 min. DOX fluorescence in the supernatants was measured at λex 480 nm and λem 565 nm by a fluorescence spectrophotometer, and DOX content was counted according to a DOX standard curve following the same procedure. 2.12. In Vivo NIR Light-Induced Fluorescence Imaging. H22bearing mice with the tumor size of about 100 mm3 were intravenously administrated with free I/D solution or I/D@NG at the ICG dose of 10 mg/kg, followed with or without photoirradiation (808 nm, 1.5 W/ cm2) for 15 min at the tumor sites. The NIRF images of mice at the tumor sites were obtained at the predetermined time using a Caliper IVIS Lumina II in vivo imaging system (PerkinElmer, Waltham, MA, U.S.A.) at λex 745 nm. 2.13. NIR Light-Induced Tumor Accumulation. H22-bearing mice with the tumor size about 100 mm3 were intravenously administrated with free I/D solution or I/D@NG at the ICG dose of 10 mg/kg, followed with or without photoirradiation (808 nm, 1.5 W/cm2) for 15 min at the tumor sites. After 24 h administration, the mice were executed, and tumor tissues were harvested and washed by PBS. The tumor tissues were first imaged using Caliper IVIS Lumina II in vivo imaging system to obtain the ex vivo biodistribution of ICG. Subsequently, the tumor tissues were homogenized in 0.2 mL of tissue lysis buffer, and DOX in tumor lysates was extracted with 200 mM GSH for 2 h and methanol for 30 min. DOX content in tumor tissues was measured at λex 480 nm and λem 565 nm by fluorescence spectrometer using a DOX calibration curve made in accordance with the same procedure as above. 2.14. NIR Light-Induced Tumor Penetration. H22-bearing mice with the tumor size about 100 mm3 were intravenously administrated with free I/D solution or I/D@NG at the ICG dose of 10 mg/kg, followed with or without photoirradiation (808 nm, 1.5 W/cm2) for 15 min at the tumor sites. After 24 h administration, the mice were executed and the tumor tissues were harvested, washed by PBS and then frozen sectioned. The cryosectioned tumors were dyed by fluorescein isothiocyanate isomer I (FITC)-conjugated CD31 antibody at 37 °C for 30 min, rinsed by PBS and then detected by confocal microscope. DOX fluorescence distribution from blood

Committee at Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). 2.3. Construction of Nanogels. The nanogels were prepared by a simple precipitation polymerization method as described elsewhere.36,37 Briefly, the calculated amount of NIPAM, SBMA, MAA, and BAC with the corresponding feeding molar ratio of 85.7:4.8:9.5:0.02 were added into a three-necked round-bottom flask and dissolved with water under magnetic stirring at the monomer concentration of about 1.2 wt %. 0.02 wt % of SDS was used as a surfactant. The reactive system was bubbled with nitrogen gas for more than 30 min and then heated to 75 °C, followed by adding potassium persulfate (KPS, 0.05 wt %) to trigger the polymerization reaction. After reacting for 4.5 h at 75 °C in nitrogen environment, the resulting system was cooled to room temperature and then dialyzed in water for 14 days (the dialysis bag’s cutoff molecular weight was 14 000), followed by lyophilization for the further use. 2.4. Preparation and Characterization of I/D@NG. I/D@NG was prepared as described.26 First, 0.5 mg DOX·HCl dissolved in chloroform was blended with triethylamine (TEA) at the DOX·HCl/ TEA molar ratio of 1:5 for 30 min. DOX solution was then added to 10 mg of the nanogel aqueous dispersions in a total volume of 4 mL, blended by sonication for 20 min and evaporated using rotary evaporator at 40 °C for 1 h. Subsequently, 2 mg of ICG dissolved in 1 mL of methanol was added to DOX-loaded nanogels (D@NG), blended and evaporated using the same procedure. I/D@NG was purified in an ultrafilter tube (Millipore, cutoff molecular weight 10 000) by centrifugation at 4000 rpm for 20 min. The nanogels only encapsulating ICG (I/NG) were prepared according to the same operation process as that of I/D@NG, except not adding DOX. The concentration of ICG and DOX in the outer centrifuge tube was measured by UV−vis spectroscopy at 780 and 480 nm, respectively. The hydrodynamic diameter of I/D@NG was measured by dynamic light scattering (DLS; Zetasizer Nano-ZS 90, Malvern Instruments Ltd., Worcestershire, UK). The morphology of I/D@NG was detected by transmission electron microscopy (TEM; Tecnai G2 20, FEI Corp., The Netherlands). 2.5. In Vitro Photothermal Effect. Free ICG and DOX (I/D) solution, I@NG or I/D@NG at the final ICG concentration of 50 μg/ mL or I/D@NG containing different concentrations of ICG were stored in transparent plastic vials. The samples were then irradiated by an 808 nm NIR laser (1.5 W/cm2) for different time courses. Simultaneously, the temperature of the solutions was monitored by an FLIR E50 Infrared (IR) camera (FLIR Systems Inc., U.S.A.). H2O was used as a negative control in this experiment. 2.6. GSH and NIR Light-Responsive DOX Release. I/D@NG at the final DOX concentration of 10 μg/mL was incubated in PBS containing different concentrations of GSH at 37 °C. The samples were or were not then subjected to NIR irradiation (808 nm, 1.0 W/ cm2) for 5 min at the indicated time points (0 and 8 h). After 24 h incubation, DOX fluorescence intensity at 510−650 nm (λex = 480 nm) was determined by an F-4500 fluorescence spectrophotometer. Dialysis bag diffusion method was further used to determine the cumulative DOX release profile from I/D@NG. Briefly, 1 mL of I/D@ NG solution containing 30 μg of DOX was put into a dialysis bag with a molecular weight cutoff of 14 kDa, followed by immersion into 29 mL of PBS containing different concentrations of GSH (pH 7.4). The dialysis was performed in a shaking incubator at 37 °C. The samples were or were not then subjected to NIR irradiation (808 nm, 1.5 W/ cm2) for 1 min at the indicated time points (2, 4, 6, and 8 h). One mL of sample was taken out, and then 1 mL of fresh medium was compensated at the desired time intervals. The amount of released DOX at λex 480 nm and λem 565 nm was measured by a fluorescence spectrophotometer using a DOX standard curve. 2.7. Cellular Uptake. HepG2 cells were treated with free I/D solution or I/D@NG at ICG concentration of 20 μg/mL and DOX concentration of 5 μg/mL, followed with or without photoirradiation (808 nm, 1.5 W/cm2) for 3 min. After 4 h incubation, the cells were rinsed by PBS, harvested, and intracellular DOX fluorescence was then analyzed by flow cytometry (FC500, Beckman Coulter, Fullerton, CA, U.S.A.). 23566

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

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Figure 1. Characterization of I/D@NG. (A) Hydrodynamic size of I/D@NG by DLS at pH 7.4 and 25 °C. (B) Morphology of I/D@NG by TEM. (C) Hydrodynamic size of I/D@NG after incubation in PBS (pH 7.4) at different temperatures. vessels to tumor tissues on the designated line was analyzed by software image J 1.45S. 2.15. In Vivo Photothermal Effect. H22-bearing mice with the tumor size about 100 mm3 were intravenously administrated with free I/D solution or I/D@NG at the ICG dose of 10 mg/kg, followed with or without photoirradiation (808 nm, 1.5 W/cm2) for 15 min at the tumor sites. At 24 h postinjection, the tumors were then irradiated (808 nm, 1.5 W/cm2) for different time courses, and the temperature of the tumor tissues was simultaneously monitored by an IR camera. 2.16. In Vivo Anticancer Efficacy. H22-bearing mice with the tumor size about 100 mm3 were intravenously administrated with various formulations including saline, free I/D solution, D@NG, I@ NG, or I/D@NG at the ICG dose of 10 mg/kg and DOX dose of 2.5 mg/kg on day 1, 3, and 5, respectively. Following the first injection, the mice were or were not subjected to photoirradiation (808 nm, 1.5 W/cm2) at the tumor sites for 15 min. On day 3, 5, 7 and 9, the mice were or were not subjected to photoirradiation (808 nm, 1.5 W/cm2) at the tumor sites for 5 min. The tumor size of each mouse was measured every day. On day 11, the mice were executed, and the tumors and major organs (including heart, liver, spleen, lung, and kidney) were harvested. The tissues were fixed by 4% paraformaldehyde and sliced for hematoxylin-eosin (H&E) staining. The serum samples were harvested for biochemical analysis. 2.17. Statistical Analysis. All data were presented as mean value ± SD. Student’s t-test was used to evaluate the statistical significance where P < 0.05 was regarded as significant difference.

of zwitterionic SBMA might contribute to endow them with prolonged blood circulation. 3.2. In Vitro Photothermal Effects and GSH/NIR LightInduced DOX Release. The NIR light-triggered photothermal effects of I/D@NG were examined by monitoring the temperature of the aqueous solutions following 808 nm irradiation (1.5 W/cm2). Upon NIR irradiation for different time courses, the temperature of I@NG and I/D@NG appreciably increased compared with that of free I/D solution (Figure 2A,B), which might be largely due to the enhanced stability of ICG in nanogels. Temperature increase of I/D@NG under NIR irradiation showed a similar profile with that of I@ NG, suggesting that DOX loading did not affect the

3. RESULTS AND DISCUSSION 3.1. Characterization. The zwitterionic temperature/ redox-responsive nanogels were constructed by using NIPAM, SBMA, and MAA as comonomers and disulfide bond-containing BAC as a cross-linker by one-step precipitation polymerization. The VPTT of the nanogels was adjusted to over the physiological temperature 37 °C by modulating the molar ratio of NIPAM, SBMA, and MAA. DOX and ICG were encapsulated into the nanogels by the solvent evaporation method. The drug loading capacity of the nanogels was 4.5% for DOX and 4.75% for ICG. The hydrodynamic size of I/D@ NG at pH 7.4 and 25 °C was about 110 nm by DLS analysis (Figure 1A). TEM showed that I/D@NG was almost uniform with spherical morphology (Figure 1B). To determine the thermosensitive property of nanogels, their hydrodynamic sizes at different temperatures at pH 7.4 were determined (Figure 1C). The nanogels exhibited a temperature-dependent size decrease. The VPTT of the nanogels was further calculated to be 40.6 °C by Boltzmann fitting and corresponding differential, suggesting that the nanogels maintained swollen state (hydrophilic) in blood (37 °C, pH 7.4). The hydrophilic state of the nanogels in combination with the antiprotein adsorption nature

Figure 2. In vitro photothermal effects and GSH/NIR light dualstimuli responsive DOX release profiles. (A) Temperature images of different formulations at different time points under NIR irradiation (808 nm, 1.5 W/cm2) recorded by an IR camera. (B) Highest temperature of different formulations at different time points under NIR irradiation. (C) Hydrodynamic size of I/D@NG at different time points under NIR irradiation (808 nm, 1.5 W/cm2). (D) DOX release profiles from I/D@NG in PBS containing different concentration of GSH with or without NIR irradiation (808 nm, 1.5W/cm2). The arrows indicate NIR irradiation at different time points. Data as mean value ± SD (n = 3). 23567

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

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Figure 3. Pharmacokinetics and in vivo tumor accumulation of I/D@NG. (A) Plasma DOX concentrations at different time points in SD rats intravenously injected with free I/D solution or I/D@NG at the DOX dose of 5 mg/kg, respectively. (B) In vivo ICG fluorescence images of H22 tumor-bearing mice at 24, 48, 72, and 96 h after intravenous injection of free I/D solution or I/D@NG at the ICG dose of 10 mg/kg followed with or without 15 min NIR irradiation (808 nm, 1.5 W/cm2), respectively. The black rounds denoted the position of the tumors. (C) Ex vivo imaging of ICG in tumor tissues of H22-bearing mice at 24 h after intravenous injection of free I/D solution or I/D@NG at the ICG dose of 10 mg/kg followed with or without 15 min NIR irradiation. (D,E) ICG fluorescence intensity (D) and DOX content (E) in tumor tissues of H22-bearing mice at 24 h postinjection with free I/D solution or I/D@NG at the ICG dose of 10 mg/kg followed with or without 15 min NIR irradiation. Data as mean value ± SD (n = 3). *P < 0.05, **P < 0.01.

photothermal effect of I@NG. Furthermore, I/D@NG exhibited a concentration-dependent photothermal effect (Figure S1) and easily reached the temperature above 42 °C at relatively low concentrations, which can potentially induce photothermal destruction of cancer cells. Correspondingly, the hydrodynamic size of I/D@NG dramatically decreased ranging from 117 to 66 nm under 808 nm irradiation for 8 min (Figure 2C). To confirm whether the nanogels could release DOX in response to photothermal effect of ICG and intracellular redox environment, I/D@NG was incubated in PBS containing different concentrations of GSH for 24 h with or without 808 nm irradiation, and then DOX fluorescence intensity was measured (Figure S2). DOX fluorescence slightly increased upon NIR irradiation in the presence or absence of 2 μM GSH (comparable to GSH content in human plasma), which might result from NIR irradiation-induced reduction in the size of nanogels, resulting in the ejection of DOX from the nanogels

and subsequent increased DOX release. However, NIR irradiation dramatically increased DOX fluorescence when I/ D@NG was incubated with 10 mM GSH (comparable to GSH content in tumor cells). Furthermore, DOX release profiles from I/D@NG with or without 808 nm irradiation were evaluated using dialysis in PBS with different concentrations of GSH at 37 °C (Figure 2D). As expected, the cumulative DOX release from I/D@NG was about 18% and 47% responding to 2 μM and 10 mM GSH for 10 h, suggesting that I/D@NG was stable during blood circulation and intracellular high GSH concentration benefited drug release. Moreover, DOX release rate can be increased up to 84% by cotreatment with 10 mM GSH and NIR irradiation, confirming a synergistically GSH and NIR light dual-stimuli responsive DOX release. 3.3. Pharmacokinetics and NIR Light-Induced Tumor Accumulation of I/D@NG. Achieving long circulation of nanoparticles would contribute to their accumulation in tumor tissues by EPR effect.39 The pharmacokinetic behavior of free 23568

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

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Figure 4. Colocalization of I/D@NG or free I/D (red) with CD31 antibody-labeled endothelial cells (green) in tumor sections of H22-bearing mice at 24 h postinjection at the ICG dose of 10 mg/kg followed with or without NIR irradiation (808 nm, 1.5 W/cm2) for 15 min. Distribution profile of DOX from blood vessels to tumor tissues on the specified white line was shown on the right. Scale bar is 200 μm.

Figure 5. NIR light-induced cellular uptake and lysosomal escape of I/D@NG. (A) Amounts of internalized DOX by HepG2 cells treated with free I/D solution or I/D@NG containing 5 μg/mL DOX for 4 h with or without 3 min NIR irradiation (808 nm, 1.5 W/cm2), respectively. (B) DHE staining of HepG2 cells treated with I/D@NG containing different ICG concentrations with or without 3 min NIR irradiation (808 nm, 1.5 W/cm2) by fluorescent microscope. Scale bar is 100 μm. (C) Intracellular delivery of DOX in HepG2 cells after treatment with I/D@NG in the presence or absence of 2 mM Vc with or without NIR irradiation (808 nm, 1.5 W/cm2) by confocal microscopy. Lysosomes were dyed with LysoTracker Green and nuclei were dyed with DAPI. Scale bar is 2.5 μm. (D) Cell viability of HepG2 cells incubated with free I/D solution, D@NG, I@NG or I/D@ NG at different concentrations of ICG for 24 h with or without 3 min NIR irradiation (808 nm, 1.5 W/cm2), respectively. Data as mean value ± SD (n = 3). **P < 0.01.

I/D solution and I/D@NG was first assessed in SD rats (Figure 3A). It revealed that the area under the curve (AUC) and Cmax of I/D@NG was 3.4- and 3.8-fold higher than those of free I/D solution, respectively. In addition, the nanogels extended the elimination half-life of DOX from 2.5 to 8.0 h. These results indicated that I/D@NG exhibited prolonged blood circulation compared with free I/D solution. The biodistribution of chemotherapeutic drugs has a direct effect on their treatment outcomes and unwanted side effects.40 To determine their in vivo thermoresponsive character, NIR

light-induced tumor accumulation of I/D@NG was assessed in H22-bearing mice. H22-bearing mice were intravenously administrated with free I/D solution or I/D@NG and then the tumor tissues were irradiated with or without 808 nm for 15 min. Fluorescence signals of ICG by whole-animal fluorescence imaging showed that ICG accumulation significantly enhanced in tumors of both free I/D solution- and I/D@NG-treated mice after NIR irradiation, and more I/D@NG accumulated in tumor than free I/D solution regardless of NIR irradiation (Figure 3B). The enhanced tumor accumulation of ICG in I/ 23569

DOI: 10.1021/acsami.7b08047 ACS Appl. Mater. Interfaces 2017, 9, 23564−23573

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oxidation into 2-hydroxyehtidium by singlet oxygen.24,38 Weak red fluorescence was observed in I/D@NG-treated HepG2 cells containing 0.1 μg/mL ICG after NIR irradiation, whereas no fluorescence was detected in the cells without NIR irradiation (Figure 5B). The red fluorescence intensity enhanced in cells treated with I/D@NG containing higher concentrations of ICG. However, stronger fluorescence was detected in I/D@NG-treated group upon NIR irradiation than that without NIR irradiation (Figure 5B, S3), demonstrating that intracellular singlet oxygen was effectively generated in a concentration-dependent manner in I/D@NG-treated cells upon NIR irradiation. The intracellular singlet oxygen was reported to effectively disrupt lysosomes which contributed to lysosomal escape of nanoparticles.24 To demonstrate that I/D@NG was efficiently escaped from lysosomes upon NIR light-triggered singlet oxygen and then DOX was sufficiently released into nucleus to exert cytotoxic effects, HepG2 cells were treated with I/D@ NG in the presence or absence of antioxidant Vc for 30 min, washed with PBS, and then irradiated with 808 nm laser for 1 min. The colocalization of DOX with lysosomes or nucleus was detected by confocal microscopy (Figure 5C). DOX was majorly distributed in lysosomes when HepG2 cells were treated with I/D@NG without NIR irradiation. However, stronger intracellular DOX fluorescence was detected and more DOX was localized in nucleus after NIR irradiation, suggesting that DOX was efficiently released and then translocated into nucleus in view that DOX fluorescence intensity increased when released from I/D@NG (Figure 2D). In the meanwhile, less LysoTracker Green-labeled fluorescence intensity and smaller size of lysosomes were detected after NIR irradiation, which might be due to singlet oxygen-induced lysosomal disruption upon NIR irradiation. However, pretreatment with Vc reversed NIR light-induced increase in intracellular DOX fluorescence and DOX nucleus translocation, further confirming that NIR light-triggered generation of singlet oxygen resulted in lysosomal disruption and lysosomal escape of I/D@ NG, followed by releasing DOX to nucleus in response to intracellular high GSH concentration and NIR light-induced photothermal effect. To investigate the synergistic antitumor activity of I/D@NG from photothermal effect of ICG and chemotherapy of DOX, HepG2 cells were treated with different formulations for 24 h, followed with or without 808 nm irradiation for 3 min, and then the cytotoxicity was determined by MTT assay (Figure 5D). No cytotoxicity of I@NG and similar cytotoxicity of D@ NG and I/D@NG were found in HepG2 cells in the absence of NIR irradiation, revealing that ICG without irradiation functioned as a nontoxic material. Free I/D solution, I@NG, and I/D@NG upon photoirradiation exhibited significantly greater cytotoxicity than those without photoirradiation, respectively. Importantly, I/D@NG displayed the strongest cytotoxicity against HepG2 cells in response to NIR irradiation, indicating that I/D@NG with NIR irradiation could generate effective synergistic effects of thermo-chemotherapy, possibly owing to its multiple effects including NIR light-induced photothermal effect, enhanced cellular uptake and sufficient intracellular DOX release. 3.6. In Vivo Photothermal Effects. Considering NIR light-induced enhanced accumulation of I/D@NG in tumor tissues, their in vivo photothermal effects were evaluated. H22bearing mice were intravenously administrated with PBS, free I/D solution, I@NG, or I/D@NG, followed by 808 nm

D@NG-treated mice without irradiation might be due to EPR effect of the nanogels, and NIR light-induced photothermal effect of ICG further increased their tumor accumulation. To directly evaluate the thermoresponsive accumulation of I/D@ NG in tumors, the ex vivo ICG fluorescence and DOX concentration were determined in tumors of H22-bearing mice at 24 h after intravenous administration followed by NIR irradiation for 15 min (Figure 3C−E). Consistently, both ICG fluorescence and DOX concentration in tumors of the irradiated mice were significantly increased than those of unirradiated mice, confirming enhanced accumulation of I/D@ NG after NIR irradiation. The increased accumulation of ICG and DOX at tumor sites might contribute to achieve excellent photothermal therapy and chemotherapy, respectively. 3.4. NIR Light-Induced Deep Tumor Penetration. Because of the heterogeneous vascular network, dense extracellular matrix, and elevated interstitial fluid pressure in tumor tissues, nanoparticles are mostly localized in the proximity of tumor vasculatures, which decreases the chance of carrying chemotherapeutic drugs to cancer cells.41−43 Increasing the tumor penetration ability of nanoparticles was critical for improving therapeutic efficacy. To assess their in vivo thermoresponsive tumor penetration capacity, H22bearing mice were intravenously administrated with free I/D solution or I/D@NG, and then the tumor tissues were irradiated with or without 808 nm for 15 min. At 24 h postinjection, the fluorescent microscopic images of H22 tumor sections were evaluated (Figure 4). Consistent with the tumor accumulation data, the tumors of mice injected with I/D@NG exhibited stronger DOX fluorescence intensity than those with free I/D in the presence or absence of NIR irradiation, further confirming NIR light-triggered increased tumor accumulation of I/D@NG. In the meanwhile, DOX fluorescence from I/D@ NG-treated group upon NIR irradiation was scattered further in distance from FITC-labeled CD31, a blood vasculature endothelial cell marker, while DOX fluorescence from free I/ D solution- and I/D@NG-treated group was mostly restricted in close proximity to blood vasculatures, revealing NIR lightinduced superior tumor penetration capacity of I/D@NG. One reason might be that NIR light-triggered hyperthermia increased tumor vascular permeability and destroyed the compact tumor extracellular matrix to promote tumor penetration of I/D@NG. Another might be that NIR lightinduced hyperthermia resulted in the shrinkage of nanogels, which contributed to their deep tumor penetration. 3.5. NIR Light-Induced Cellular Uptake, Intracellular Drug Release, and In Vitro Cytotoxicity. Efficient cellular uptake plays a key prerequisite for selective cytotoxicity.44 To demonstrate whether NIR light-induced hyperthermia affected the internalization, the cellular uptake of free I/D solution or I/ D@NG in HepG2 cells following with or without NIR irradiation was determined. Figure 5A showed that NIR irradiation did not markedly affect the cellular uptake of DOX in cells treated with free I/D solution. However, NIR irradiation significantly enhanced the cellular uptake of DOX in I/D@NG-treated cells, indicating that photoirradiation can contribute to the internalization of I/D@NG, which might be attributed to NIR light-induced reduction in the size of nanogels. To validate the formation of intracellular singlet oxygen in I/ D@NG-treated HepG2 cells upon NIR irradiation, DHE was used as a probe to determine the production of singlet oxygen since DHE can generate red fluorescence in the cell nuclei after 23570

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Figure 6. In vivo photothermal effects of I/D@NG. (A) NIR light-triggered temperature images of tumor region recorded by an IR camera at 24 h after H22-bearing mice were administrated with PBS, free I/D solution, I@NG or I/D@NG at the ICG dose of 10 mg/kg, followed by NIR irradiation (808 nm, 1.5 W/cm2) for 5 min at the tumor sites. (B) Temperature curves of the four NIR-irradiated groups. Data as mean value ± SD (n = 3).

irradiation for 15 min at tumor sites. At 24 h postinjection, the temperature of tumor tissues irradiated by NIR for different time courses was determined by an IR camera (Figure 6A, B). The irradiated I/D@NG group exhibited a similar temperature increase with irradiated I@NG group, and their highest temperature of the tumor tissues arrived at more than 50 °C, which is high enough to kill cancer cells. In contrast, the highest temperature of irradiated free I/D group only showed a slight increase compared with that of PBS group, which might be due to their not enough tumor accumulation. 3.7. In Vivo Synergistic Thermo-Chemotherapeutic Activity. In view of NIR light-triggered elevated temperature for photothermal therapy and enhanced DOX accumulation, penetration, cellular uptake, and intracellular release for chemotherapy in tumors, the anticancer activity of I/D@NG was further evaluated. As shown in Figure 7A, I/D@NG exhibited more significant inhibition in tumor growth with a tumor inhibition rate of 37.5% as compared to free I/D solution with 22.9% in the absence of NIR irradiation, which might be due to the enhanced tumor accumulation of I/D@ NG via EPR effects. D@NG and I/D@NG without NIR irradiation showed a similar inhibition in tumor growth, confirming that ICG displayed no therapeutic effect in the absence of NIR irradiation. Even though I@NG achieved enhanced anticancer activity upon NIR irradiation with a tumor inhibition rate of 56.1%, the tumors suffering from only photothermal therapy still showed the slow tumor growth. However, I/D@NG exhibited an almost tumor eradication upon NIR irradiation (half tumor ablation in 4 mice), demonstrating a synergistic thermo-chemotherapeutic activity of I/D@NG. The tumor weight excised at the end of treatment also showed the same trend (Figure 7B,C). Furthermore, histological analysis by H&E staining showed severe tumor necrosis with worst structural destruction in tumors of mice administrated with I/D@NG upon NIR irradiation. To test the biosafety of I/D@NG, the histopathological examinations of the major organs were carried out by H&E staining (Figure S4). All treatment groups did not exhibit obvious pathological changes in heart, liver, spleen, lung, and kidney. Furthermore, serological analysis showed that all these treatment did not markedly affect the activity of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) (Figure

Figure 7. In vivo antitumor efficacy of I/D@NG. (A) Tumor growth inhibition curves of H22-bearing mice injected with various formulations at the dose of 10 mg/kg ICG and 2.5 mg/kg DOX on day 1, 3, and 5, followed with or without 15 min NIR irradiation (808 nm, 1.5 W/cm2) at the first injection and 5 min NIR irradiation on day 3, 5, 7, and 9 at the tumor sites. (B) Tumor weight after treatment. (C) Photos of tumors after treatment. (D) Histological observation of tumor tissues after treatment by H&E staining. Scale bar is 100 μm. Data as mean value ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

S5), confirming that I/D@NG did not cause heart, liver, and kidney dysfunction. These results demonstrated that I/D@NG 23571

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(2) Ji, T. Y.; Zhao, Y.; Ding, Y. P.; Nie, G. J. Using Functional Nanomaterials to Target and Regulate the Tumor Microenvironment: Diagnostic and Therapeutic Applications. Adv. Mater. 2013, 25, 3508− 3525. (3) Peer, D.; Karp, J. M.; Hong, S.; Margalit, R.; Farokhzad, O. C.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (4) Venditto, V. J.; Szoka, F. C., Jr. Cancer Nanomedicines: So Many Papers and So Few Drugs. Adv. Drug Delivery Rev. 2013, 65, 80−88. (5) Chauhan, V. P.; Jain, R. K. Strategies for Advancing Cancer Nanomedicine. Nat. Mater. 2013, 12, 958−962. (6) Li, H. J.; Du, J. Z.; Liu, J.; Du, X. J.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. Smart Superstructures with Ultrahigh pHSensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. ACS Nano 2016, 10, 6753−6761. (7) Li, H. J.; Du, J. Z.; Du, X. J.; Xu, C. F.; Sun, C. Y.; Wang, H. X.; Cao, Z. T.; Yang, X. Z.; Zhu, Y. H.; Nie, S.; Wang, J. StimuliResponsive Clustered Nanoparticles for Improved Tumor Penetration and Therapeutic Efficacy. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 4164−4169. (8) Hu, Y. W.; Du, Y. Z.; Liu, N.; Liu, X.; Meng, T. T.; Cheng, B. L.; He, J. B.; You, J.; Yuan, H.; Hu, F. Q. Selective Redox-Responsive Drug Release in Tumor Cells Mediated by Chitosan Based GlycolipidLike Nanocarrier. J. Controlled Release 2015, 206, 91−100. (9) Li, Y.; Xiao, K.; Zhu, W.; Deng, W.; Lam, K. S. StimuliResponsive Cross-Linked Micelles for on-Demand Drug Delivery against Cancers. Adv. Drug Delivery Rev. 2014, 66, 58−73. (10) Thornton, P. D.; Mart, R. J.; Ulijn, R. V. Enzyme-Responsive Polymer Hydrogel Particles for Controlled Release. Adv. Mater. 2007, 19, 1252−1256. (11) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991−1003. (12) Zhang, Z.; Wang, J.; Nie, X.; Wen, T.; Ji, Y.; Wu, X.; Zhao, Y.; Chen, C. Near Infrared Laser-Induced Targeted Cancer Therapy Using Thermoresponsive Polymer Encapsulated Gold Nanorods. J. Am. Chem. Soc. 2014, 136, 7317−7326. (13) Li, L.; ten Hagen, T. L.; Schipper, D.; Wijnberg, T. M.; van Rhoon, G. C.; Eggermont, A. M.; Lindner, L. H.; Koning, G. A. Triggered Content Release from Optimized Stealth Thermosensitive Liposomes Using Mild Hyperthermia. J. Controlled Release 2010, 143, 274−279. (14) Wust, P.; Hildebrandt, B.; Sreenivasa, G.; Rau, B.; Gellermann, J.; Riess, H.; Felix, R.; Schlag, P. M. Hyperthermia in Combined Treatment of Cancer. Lancet Oncol. 2002, 3, 487−497. (15) Deng, Z.; Xiao, Y.; Pan, M.; Li, F.; Duan, W. L.; Meng, L.; Liu, X.; Yan, F.; Zheng, H. R. Hyperthermia-Triggered Drug Delivery from iRGD-Modified Temperature-Sensitive Liposomes Enhances the AntiTumor Efficacy Using High Intensity Focused Ultrasound. J. Controlled Release 2016, 243, 333−341. (16) Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Au Nanoparticles Target Cancer. Nano Today 2007, 2, 18−29. (17) Albert, K.; Hsu, H. Y. Carbon-Based Materials for PhotoTriggered Theranostic Applications. Molecules 2016, 21, 1585−1614. (18) Zhou, B.; Li, Y.; Niu, G.; Lan, M.; Jia, Q.; Liang, Q. NearInfrared Organic Dye-Based Nanoagent for the Photothermal Therapy of Cancer. ACS Appl. Mater. Interfaces 2016, 8, 29899−29905. (19) Guo, Z.; Zou, Y.; He, H.; Rao, J.; Ji, S.; Cui, X.; Ke, H.; Deng, Y.; Yang, H.; Chen, C.; Zhao, Y.; Chen, H. Bifunctional Platinated Nanoparticles for Photoinduced Tumor Ablation. Adv. Mater. 2016, 28, 10155−10164. (20) Zhou, L.; Yang, T.; Wang, J.; Wang, Q.; Lv, X.; Ke, H.; Guo, Z.; Shen, J.; Wang, Y.; Xing, C.; Chen, H. Size-Tunable Gd2O3@Albumin Nanoparticles Conjugating Chlorin e6 for Magnetic Resonance Imaging-Guided Photo-Induced Therapy. Theranostics 2017, 7, 764− 774. (21) Yang, T.; Tang, Y.; Liu, L.; Lv, X.; Wang, Q.; Ke, H.; Deng, Y.; Yang, H.; Yang, X.; Liu, G.; Zhao, Y.; Chen, H. Size-Dependent Ag2S Nanodots for Second Near-Infrared Fluorescence/Photoacoustics

displayed excellent antitumor activity upon NIR irradiation with fewer side effects.

4. CONCLUSIONS We have successfully developed zwitterionic temperature/ redox-sensitive nanogels to codeliver ICG and DOX. I/D@ NG with enhanced photothermal effect exhibited in vitro synergistic photothermal and chemotherapeutic cytotoxicity. More importantly, NIR light-induced hyperthermia significantly improved the in vivo process of I/D@NG after systemic administration. I/D@NG had prolonged circulation time in blood. Its tumor accumulation, tumor penetration, and cellular uptake were significantly improved under NIR irradiation. After internalization into cancer cells, I/D@NG escaped from lysosomes through singlet oxygen-induced lysosomal disruption and DOX was then sufficiently released on-demand to nucleus responding to intracellular high GSH and NIR light-induced photothermal effects. This NDDS not only efficiently exerted synergism of thermo-chemotherapy, but also its photothermal effect contributed to overcome a series of in vivo physiological barriers of chemotherapeutic agent, resulting in excellent antitumor effect. This finding provided a versatile synergistic strategy to obtain the desired therapeutic effects in cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08047. In vitro photothermal effect of I/D@NG; fluorescence spectra analysis of DOX after I/D@NG was incubated with different concentrations of GSH with or without 808 nm irradiation; histological observation of tissues after treatment by H&E staining; analysis of the activity of CK, ALT, AST, and BUN after treatment (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*L.G. Tel: +86 27 87792147. E-mail: [email protected]. *Q.W. Tel: +86-27-87792147. E-mail: [email protected]. cn. ORCID

Lu Gan: 0000-0002-8785-867X Author Contributions §

F.L. and H.Y. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (2015CB931802 and 2012CB932500) and National Natural Science Foundation of China (81672937, 81473171, 81372400, 51473057, and 31371423). We thank the Analytical and Testing Center of Huazhong University of Science and Technology for related analysis.

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