Article pubs.acs.org/Biomac
Cite This: Biomacromolecules 2019, 20, 2649−2656
Cancer-Selective Bioreductive Chemotherapy Mediated by Dual Hypoxia-Responsive Nanomedicine upon Photodynamic TherapyInduced Hypoxia Aggravation Rongying Zhu,†,∥ Hua He,‡,∥ Yong Liu,*,§ Desheng Cao,‡ Jin Yan,‡ Shanzhou Duan,† Yongbing Chen,*,† and Lichen Yin*,‡ †
Department of Thoracic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science & Technology, Soochow University, Suzhou 215123, China § Department of Biomedical Engineering, University of Groningen and University Medical Center Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands
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‡
S Supporting Information *
ABSTRACT: Stimuli-responsive drug delivery has rendered promising utilities in cancer treatment. Nevertheless, cancer selectivity as well as sensitivity still remains critical challenges that would undermine the therapeutic efficacy of chemodrugs and cause undesired systemic toxicity. Herein, a dual hypoxiaresponsive drug delivery system was developed to enable photodynamic therapy (PDT)-induced drug release and drug activation intermediated via PDT-induced hypoxia. Particularly, tumor-targeting and hypoxia-dissociable nanoparticles (NPs) were self-assembled from the amphiphilic polyethylenimine−alkyl nitroimidazole [PEI−ANI, (PA)] and hyaluronic acid-chlorin e6 (HA-Ce6) to encapsulate bioreductive chemodrug, tirapazamine (TPZ). After systemic administration, the obtained PA/HA-Ce6@TPZ NPs enabled effective tumor accumulation due to HA-mediated cancer targeting. Upon receptor-mediated endocytosis, light irradiation (660 nm, 10 mW/cm2) produced high levels of reactive oxygen species to mediate PDT and generated a severe local hypoxic environment to dissociate the NPs and selectively release TPZ, as a consequence of hypoxia-triggered hydrophobic-to-hydrophilic transformation of ANI. In the meantime, TPZ was activated under hypoxia, finally contributing to a synergistic anticancer treatment between PDT and hypoxiastrengthened bioreductive chemotherapy. This study, therefore, demonstrates a suitable strategy for cancer-selective drug delivery as well as programmed combination therapy.
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INTRODUCTION Chemotherapy is a widely adopted anticancer modality in the clinical setting, owing to the wide application and accessibility of chemo-drugs.1,2 However, inefficient drug delivery severely impedes the efficacy of chemotherapy, which leads to serious adverse effect and limited clinical outcomes. Nanomedicine is an attractive approach to overcome obstacles related with chemodrugs because it can improve the drug solubility, prolong the circulation time in the blood, enhance tumor accumulation, and enable controlled release.3−5 Recently, various types of stimuli-responsive nanovehicles have been constructed to mediate “on demand” drug release by responding to a specific external or internal stimulus. Late-stage solid tumors are often characterized with hypoxia, mainly because of the overwhelming oxygen consumption of tumor cells and the low oxygen transport capability of the abnormal vasculature in the tumors.6−8 Thus, hypoxia-responsive delivery systems can serve to mediate selective and instantaneous chemodrug release in tumors to © 2019 American Chemical Society
enhance the anticancer potency while reduce the systemic toxicity. However, for most stimuli-responsive carriers, the nonspecific drug leakage still occurs during blood circulation or upon distribution into normal tissues, leading to undesired side toxicity and even drug resistance.9 In comparison to nanocarriers, stimuli-activatable prodrugs serve as an alternative strategy to cope with undesirable properties of chemodrugs, which can completely eliminate the toxicity to normal tissue/cells,10,11 while can be selectively released or activated in response to tumor microenvironment factors, such as GSH,12−15 acid pH,16−18 reactive oxygen species (ROS),19−21 and hypoxia.22−24 Tirapazamine (TPZ) is a clinically used hypoxia-activatable bioreductive prodrug, which can be converted from a nontoxic molecule into a toxic drug via single-electron Received: March 28, 2019 Revised: May 16, 2019 Published: May 24, 2019 2649
DOI: 10.1021/acs.biomac.9b00428 Biomacromolecules 2019, 20, 2649−2656
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Biomacromolecules reduction in the hypoxic environment.25,26 It, thus, imparts selective anticancer efficacy against hypoxic tumors while induced minimal toxicity to noncancerous tissues. To enable anticancer treatment in a more precise and intensive approach, it is ideal that the TPZ be delivered using hypoxia-responsive nanosystems, which could allow simultaneous drug release and drug activation in a dual hypoxia-responsive manner after targeting to and accumulating in tumors. A critical issue related to hypoxia-responsive delivery systems or prodrugs is that the hypoxia level in many tumors is heterogeneous and insufficient, especially in oxygen-rich tumor cells nearby blood vasculatures. Thus, it cannot efficiently provoke the drug liberation from hypoxia-responsive nanovehicles or the activation of hypoxia-responsive chemodrugs.27−30 Photodynamic therapy (PDT), an anticancer mechanism mediated by photosensitizer (PS) that generates high levels of toxic ROS under light irradiation, has attracted wide attention for anticancer treatment.31−34 Specifically, PDT is less invasive and more tumor selective than surgery or chemotherapy, thus ensuring selective eradication of tumor cells with minimal damage to normal cells.35,36 Alternatively, intracellular oxygen can be converted into singlet oxygen (1O2) during PDT, which results in acute hypoxia in tumor cells.37,38 We thus reason that PDT can serve to exacerbate hypoxia in tumor cells to either facilitate drug release from hypoxia-dissociable nanocarriers or activate the bioreductive drug,39 thus realizing cancerselective chemotherapy in synergism with PDT to potentiate the anticancer efficacy. Keeping these understandings in mind, in the current study, we designed tumor-targeting, hypoxia-dissociable nanoparticles (NPs) for the codelivery of PS and a hypoxia-activatable bioreductive chemodrug TPZ, attempting to enable highly precise control over bioreductive chemotherapy via dual hypoxiaresponsiveness toward cancer-selective TPZ release and activation. To this end, alkylated 2-nitroimidazole (ANI)-modified polyethylenimine [PEI-ANI (PA)] and chlorin e6 (Ce6)-grafted hyaluronic acid (HA-Ce6) were synthesized and allowed to self-assemble into NPs to encapsulate TPZ, thus affording the PA/HA-Ce6@TPZ NPs (Scheme 1). We hypothesize that the PA/HA-Ce6@TPZ NPs could enhance the serum stability and prolong the circulation time of the NPs due to the presence of HA molecules that formed a negatively charged and hydrophilic surface. After tumor accumulation and cancer cell internalization, light irradiation of Ce6 could generate high levels of ROS, which would result in cell apoptosis and a local hypoxic environment. With the assistance of NADPH, the hydrophobic ANI could be reduced to the hydrophilic alkyl-2-aminoimidazole (AAI) under hypoxia,40,41 thus resulting in dissociation of NPs to release TPZ (Scheme 1). In the meantime, the aggravated hypoxic condition activated TPZ to generate toxic oxidizing radical species, thus allowing selective toxicity to hypoxic cancer cells with negligible effect on normal cells.
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Scheme 1. Illustration of Dual Hypoxia-Responsive PA/ HA-Ce6@TPZ NPs toward PDT-Strengthened Bioreductive Therapya
a
Tumor-targeting and hypoxia-dissociable NPs were self-assembled from PA and HA-Ce6 via electrostatic interaction to encapsulate TPZ. After i.v. injection, the NPs accumulated in the tumors through the EPR effect and HA-Ce6-mediated active targeting. Light irradiation (660 nm, 10 mW/cm2) of the tumor site generated high levels of ROS to kill cancer cells and a severe local hypoxic environment to induce NP dissociation and TPZ release, as a result of hypoxia-triggered transformation of the hydrophobic ANI to the hydrophilic alkyl AAI. In the meantime, TPZ was activated under hypoxia, finally provoking synergistic anticancer treatment between PDT and hypoxiastrengthened bioreductive chemotherapy.
Female BALB/c mice (6−8 weeks) were purchased from Shanghai Slaccas Experimental Animal Co. Ltd. (Shanghai, China) and housed in an SPF room. All animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Soochow University. Synthesis of PA and HA-Ce6. PA was synthesized as follows: firstl, alkyl-2-nitroimidazole (0.20 g, 1 equiv), potassium carbonate (0.49 g, 2 equiv), and ethyl 6-bromohexanoate (0.41 g, 1.05 equiv) were dissolved in dimethylformamide (DMF) (10 mL) and stirred at 60 °C for 6 days. The mixture was extracted by ethyl acetate (70 mL × 3), followed by washing with water and drying over anhydrous Na2SO4. Following filtering and concentration via rotary evaporation, compound 1 was collected as yellow oil (0.40 g, yield 68%). Subsequently, compound 1 (0.4 g, 1 equiv) was stirred in NaOH solution (0.16 M, 100 mL) at room temperature (RT) overnight, followed by adjustment of pH to 2 with HCl solution (6 M). After extraction with ethyl acetate (70 mL × 3), the organic phase was combined and dried over anhydrous Na2SO4. After filtering and concentrating via rotary
EXPERIMENTAL SECTION
Materials, Cells, and Animals. Branched polyethylenimine (PEI, MW = 1.8 kDa) was provided by J&K (Beijing, China). TPZ was bought from Adooq (Nanjing, China). HA (MW = 35 kDa) was obtained from Shandong Freda Biopharm (Jinan, China). Other chemicals were purchased from Energy Chemical or Sinopharm Chemical (Shanghai, China). 4T1 (mouse mammary carcinoma) and L929 (mouse fibroblast) cells were purchased from ATCC (Rockville, MD) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco, NY) containing 10% fetal bovine serum (FBS). 2650
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N-hydroxysuccinimide (NHS, 96 mg, 30 equiv) were added into a DMF solution (1 mL) of compound 2 (63 mg, 10 equiv) and stirred at RT for 1.5 h. PEI (50 mg, 1 equiv) dissolved in distilled water (1 mL) was then slowly added into the above solution, stirred overnight at RT, and dialyzed against distilled water (MWCO = 1 kDa) for 2 days. After lyophilization, the final product PA was obtained as a pale yellow solid (0.96 g, yield 85%). HA-Ce6 was obtained via the coupling reaction between HA (100 mg, 1 equiv) and Ce6 (33 mg, 20 equiv) as catalyzed by EDC·HCl and NHS in formamide in the dark.42 The Ce6 amount in HA-Ce6 was 24 μg/mg, as determined by UV−vis absorbance at 405 nm. Preparation and Characterization of NPs. PA, TPZ, and HA-Ce6 were dissolved in DMF (2 mg/mL), DMSO (2 mg/mL), and distilled water (10 mg/mL), respectively. Then, PA (200 μL), TPZ (40 μL), and HA-Ce6 (20 μL) were mixed and added into 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (5 mM, pH 6.8, 800 μL) in small droplets. The mixture was stirred at RT for 3 h and dialyzed against HEPES buffer for 6 h (MWCO = 3 kDa), thus obtaining the PA/HA-Ce6@TPZ NPs. The same method was used to formulate the PA/HA-Ce6 NPs (without encapsulating TPZ) and PA/HA@TPZ NPs (without Ce6 conjugation). The size of the NPs was monitored by dynamic laser scanning (DLS), and the morphology was observed by transmission electron microscopy (TEM). To evaluate the serum stability, NPs were incubated with 10% FBS, and the particle size was monitored at various time points. TPZ-loaded NPs were mixed with DMF (30 fold) and shaked at 200 rpm overnight. The TPZ concentration was measured by spectrofluorimetry (λex = 470 nm, λem = 580 nm) to calculate the drug loading content (DLC) and drug loading efficiency (DLE).42 Hypoxia-Triggered NP Dissociation. PA/HA NPs were incubated with 100 μM NADPH for 24 h under normoxia or hypoxia (filled with N2), and the UV−vis absorption spectra were recorded. Additionally, the PA/HA-Ce6@TPZ NPs (with 100 μM NADPH)
Figure 1. Light irradiation triggered dissociation of NPs and TPZ release in vitro. (A) Size of PA/HA-Ce6@TPZ NPs in HEPES buffer containing 100 μM NADPH for 24 h. To create the hypoxic condition, NPs were irradiated (660 nm, 10 mW/cm2) for 30 min. (B) TEM images of PA/HA-Ce6@TPZ NPs before and after light irradiation as described in (A). Bar represents 200 nm. (C) UV−vis spectra of PA/ HA NPs in HEPES buffer containing 100 μM NADPH under hypoxic or normoxic conditions for 24 h. (D) Release profiles of TPZ from PA/ HA-Ce6@TPZ NPs in HEPES buffer containing 100 μM NADPH with or without light irradiation (660 nm, 10 mW/cm2, 30 min). evaporation, the product compound 2 was obtained as yellow solid (0.26 g, yield 65%). Finally, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 150 mg, 30 equiv) and
Figure 2. In vitro cell uptake of NPs and light-triggered intracellular TPZ release. Uptake levels of PA/HA-Ce6@TPZ NPs in 4T1 cells (A) and L929 cells (B) following 4 h incubation as evaluated by flow cytometry. Alternatively, cells were pretreated with free HA (10 mg/mL) for 4 h to block the CD44 on cell membranes. (C) CLSM images of 4T1 cells following incubation of PA/HA-Ce6@TPZ NPs at 37 °C for 4 h and light irradiation (660 nm, 10 mW/cm2) for 30 min. Cell nuclei were stained with DAPI. Bar represents 10 μm. 2651
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Biomacromolecules were irradiated (660 nm, 10 mW/cm2) for 30 min, incubated for 24 h, and subjected to DLS and TEM characterization. Hypoxia-Triggered TPZ Release. PA/HA-Ce6@TPZ NPs containing 100 μM NADPH were placed in a dialysis tube (MWCO = 12 kDa) and irradiated (660 nm, 10 mW/cm2, 30 min). It was then immersed in the same medium (50 mL), and shaked at 200 rpm. At different time points, the release medium (1 mL) was collected, and the cumulative TPZ release amount was measured by UV−vis. Cellular Uptake and Intracellular Distribution. 4T1 and L929 cells were cultured on 12-well plates to reach 70% confluence. PA/ HA-Ce6@TPZ NPs (0.2 μg TPZ/mL) were added and incubated with cells at 37 °C for 4 h. After washing with PBS three times, cells were collected and subjected to follow cytometry analysis. Alternatively, cells were pretreated with HA (10 mg/mL) for 4 h to block the membrane-bound CD44 before incubation with PA/HA-Ce6@TPZ NPs. To evaluate the intracellular TPZ release from PA/HA-Ce6@TPZ NPs, 4T1 cells on 15 mm glass bottom cell culture dish (20% confluence) were incubated with PA/HA-Ce6@TPZ NPs (0.2 μg TPZ/mL) for 24 h. After removal of the NPs, cells were irradiated (660 nm, 2 mW/cm2) for 30 min and cultured for another 4 h. After washing with PBS and staining with DAPI (5 μg/mL), cells were observed by confocal laser scanning microscopy (CLSM). In Vitro Anti-Cancer Efficiency. 4T1 cells on 96-well plates (60% confluence) were incubated with free TPZ, PA/HA@TPZ NPs, PA/HA-Ce6 NPs, and PA/HA-Ce6@TPZ NPs for 24 h before irradiation (660 nm, 2 mW/cm2, 30 min). After incubation for another 48 h, the cell viability was monitored using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. PA/HA-Ce6@TPZ NPs were then used for the following assays. To demonstrate the cancer-targeting effect mediated by HA, cells were preincubated with HA (10 mg/mL, 4 h) before the same treatment as described above. To substantiate the anticancer effect of PDT, cells were preincubated with vitamin C (VC, a ROS scavenger, 200 μM) for 12 h followed by the same treatment as described above in the presence of VC. To probe the effect of hypoxia toward the anticancer potency, cells were preincubated with CoCl2 (a hypoxia inducer, 100 μM) for 12 h, incubated with PA/HA@TPZ NPs and CoCl2 for 48 h, and subjected to viability measurement. In Vivo Photoacoustic Imaging. Mice bearing 4T1 xenograft tumors (∼150 mm3) were obtained as described in the Supporting Information. PA/HA-Ce6@TPZ NPs (TPZ, 2 mg/kg; Ce6, 0.65 mg/kg) or PBS (control) were intratumorally injected. At 1 h postinjection, the tumor site was irradiated (660 nm, 10 mW/cm2) for 30 min, and at 4 h post irradiation, mice were subjected to photoacoustic imaging to detect the tumoral oxygen levels. In Vivo Anticancer Efficacy. PBS, free TPZ, PA/HA@TPZ NPs, PA/HA-Ce6 NPs, and PA/HA-Ce6@TPZ NPs were i.v. injected to 4T1 tumor-bearing mice (tumor volume of ∼50 mm3) on day 1, 4, and 7 (TPZ, 2 mg/kg; Ce6, 0.65 mg/kg). At 6 h post each injection of PA/HA-Ce6 NPs or PA/HA-Ce6@TPZ NPs, the tumor sites were irradiated (660 nm, 10 mW/cm2) for 30 min. The body weight and tumor volume were measured every 2 days. The tumor volume (V, mm3) was calculated as length (L) × width (W) × width (W) × 0.5. Relative tumor volume was calculated as V/V0, wherein V0 is the tumor volume on day 0. Mice were considered dead when the tumor volume exceeded 1000 mm3 or the mice died. On day 12, one mouse from each group was euthanized, and the tumor tissues were harvested and subjected to hematoxylin and eosin (H&E) staining and TUNEL staining as previously described.42 The major organs were also collected and subjected to histological examination after H&E staining. Survival of the remaining animals was recorded for up to 42 days.
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Figure 3. In vitro antitumor efficacy of NPs against 4T1 cells. (A) Cytotoxicity of free TPZ, PA/HA@TPZ NPs, PA/HA-Ce6 NPs, and PA/HA-Ce6@TPZ NPs after 72 h incubation (n = 3). Cells treated with PA/HA-Ce6 NPs and PA/HA-Ce6@TPZ NPs for 24 h were irradiated (660 nm, 2 mW/cm2) for 30 min, and further incubated for 48 h before the MTT assay. (B) Cytotoxicity of PA/HA-Ce6@TPZ NPs toward 4T1 cells pretreated with CoCl2 (100 μM, to induce hypoxia) for 12 h and incubated with NPs for 72 h (n = 3). (C) Cytotoxicity of PA/HA-Ce6@TPZ NPs toward 4T1 cells pretreated with VC (200 μM, to scavenge ROS) for 12 h, treated with NPs for 24 h, irradiated (660 nm, 2 mW/cm2) for 30 min, and further incubated for 48 h (n = 3).
afford compound 2, and their chemical structures were characterized by NMR (Figures S1 and S2).42 Compound 2 was finally covalently grafted to branched PEI, and the average degree of ANI conjugation to PEI was measured around 8, as confirmed by the integral ratio of NMR peaks (Figure S3). Preparation, Characterization, and Hypoxia-Responsiveness of NPs. To prepare NPs, the aqueous solution of HA-Ce6 was mixed with dimethyl sulfoxide (DMSO) solution of PA. The resulting mixture was added into HEPES buffer to form the NPs because of the amphiphilic nature of HA-Ce6 and electrostatic attraction between the oppositely charged HA-Ce6 and PA. The hydrophobic moieties inside of the formed NPs offer the capability to encapsulate TPZ via hydrophobic interaction. After encapsulation, the DLC and DLE for TPZ were determined at 5.2 and 16.5%, respectively. Besides, the resulting NPs possess a DLC of 1.7% and a DLE of 7.3% for the conjugated Ce6. DLS measurement showed that the size of PA/HA-Ce6@TPZ NPs was around 183 nm (Figure 1A), and the spherical morphology was noted in TEM images (Figure 1B). Notably, the PA/HA-Ce6@TPZ NPs were stable in the presence of 10% FBS or normal saline (Figures S4 and S5), as evidenced by the minimal alteration of particle size within 3 d. Such desired salt/serum stability was closely related to the presence of HA-Ce6
RESULTS AND DISCUSSION
Synthesis and Characterization of PA. To synthesize PA, compound 1 was first synthesized via nucleophilic substitution between ANI and ethyl 6-bromohexanoate. Thereafter, compound 1 was subjected to alkaline hydrolysis to 2652
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Figure 4. Pharmacokinetics and biodistribution of NPs. (A) Pharmacokinetics of free DiR or PA/HA-Ce6@DiR NPs following i.v. injection (n = 3). (B) In vivo fluorescence imaging of 4T1 tumor-bearing mice at 1, 6, 12, and 24 h post i.v. injection of free DiR or PA/HA-Ce6@DiR NPs (1.25 mg DiR/kg). Circles refer to the tumor regions. (C) Ex vivo fluorescence imaging of excised tumors and major organs at 6 h post i.v. injection (1: heart; 2: liver; 3: spleen; 4: lung; 5: kidney; and 6: tumor). (D) Biodistribution levels of DiR in tumors and major organs at 6 h post i.v. injection (n = 3).
disassembly of the NPs and liberation of the encapsulated TPZ. Only 20.3% of the loaded TPZ was liberated from the NPs under normoxic conditions within 48 h, while the TPZ release was greatly accelerated upon light irradiation (660 nm, 10 mW/cm2, 30 min) that created hypoxic conditions, yielding an accumulative release of 71.3% within 48 h (Figure 1D). Cellular Internalization and Intracellular Drug Release. As indicated by flow cytometry analysis, 4T1 cells showed remarkable internalization of the NPs, which however, was dramatically reduced when 4T1 cells were pretreated with HA (Figure 2A). HA receptors (CD44) are overexpressed on tumor cell membranes, and therefore, the HA-Ce6 shells of the NPs could target the tumor cells and enhance the internalization.45 Pretreatment with HA could occupy the CD44 on cancer cell membranes and thus reduce the cellular uptake of NPs. In contrast, murine fibroblast L929 cells display low expression levels of CD44, and accordingly, the cell uptake level was negligibly affected by the pretreatment with free HA (Figure 2B). Subsequently, the uptake of NPs and lighttriggered intracellular TPZ release were monitored by CLSM. After internalization, the colocalization ratio between red fluorescence (TPZ) and green fluorescence (Ce6) was 85.4% without light irradiation, while it was greatly decreased to 20.3% upon light irradiation (Figure 2C). Such fluorescence separation thus demonstrated the intracellular TPZ release as a result of hypoxia-induced dissociation of NPs. In Vitro Anti-Cancer Efficacy. 4T1 cells treated with the PA/HA-Ce6 NPs with a concentration up to 500 μg/mL could maintain 90% of their viability, illustrating desired cytocompatibility of the nanocarriers (Figure S6). Comparatively, the PA/HA-Ce6@TPZ NPs (+L) exhibited remarkable anticancer efficacy against 4T1 cells which was notably superior to that of PA/HA-Ce6 NPs (+L), PA/HA@TPZ NPs, and free TPZ. The IC50 of TPZ (0.21 μg/mL) or Ce6 (0.07 μg/mL) for PA/HA-Ce6@TPZ NPs was significantly lower than those of the PA/HA@TPZ NPs (TPZ, >4 μg/mL) or PA/HA-Ce6 (Ce6, 1.38 μg/mL) (Figure 3A). Upon light irradiation, the
Figure 5. Photodynamic therapy-aggravated tumoral hypoxia in vivo as determined by photoacoustic imaging. (A) Representative photoacoustic images of 4T1 xenograft tumors to determine the tumoral oxygenation status by measuring the ratios of oxygenated hemoglobin (λ = 850 nm) and deoxygenated hemoglobin (λ = 750 nm). PA/ HA-Ce6@TPZ NPs were intratumorally injected (TPZ, 2 mg/kg; Ce6, 0.65 mg/kg), and tumors were irradiated (660 nm, 10 mW/cm2, 30 min) at 1 h postinjection and imaged at 4 h post irradiation. (B) Quantification of the oxyhemoglobin saturation in the tumors before and after treatment (n = 3).
that shielded the positive surface charges. Under hypoxic conditions induced by light irradiation (660 nm, 10 mW/cm2) for 30 min, the size of PA/HA-Ce6@TPZ NPs dramatically increased to ∼900 nm (Figure 1A,B). One proposed mechanism of NP swelling is a reductive conversion of hydrophobic ANI in PA into hydrophilic AAI via six electron shift with the assistance of NAPDH, involving intermediates containing nitroso (−NO) and hydroxylamino (−NHOH).42 Another mechanism could be that ROS triggers the dissociation of ANI moieties and thus the transition from hydrophobicity to hydrophilicity, which ultimately causes disassembly of NPs.43 In support of such assumption, UV−vis spectroscopy was adopted to investigate the transformation of the hydrophobic ANI moiety in PEI to hydrophilic AAI under hypoxia. As shown in Figure 1C, the absorption peak at 330 nm under normoxic conditions was replaced by the absorption peak at 272 nm under hypoxic conditions, indicating the conversion of ANI to AAI. This observation was consistent with the previous work.44 Such hydrophobicity to hydrophilicity transition would finally cause the 2653
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Figure 6. Light-activated synergistic anticancer efficacy of PA/HA-Ce6@TPZ NPs in 4T1 xenograft tumor-bearing mice. (A) Protocol of the efficacy study. (B) Tumor volume progression of mice receiving i.v. injections of PBS, free TPZ, PA/HA@TPZ NPs, PA/HA-Ce6 NPs, and PA/ HA-Ce6@TPZ NPs on day 1, 4, and 7 (TPZ, 2 mg/kg; Ce6, 0.65 mg/kg) (n = 8). Mice were irradiated (660 nm, 10 mW/cm2) at the tumor site for 30 min at 6 h post injection of PA/HA-Ce6 NPs and PA/HA-Ce6@TPZ NPs. (C) Photographs of excised tumors on day 12. (D) Survival rates of mice within the observation period of 42 d (n = 7).
Photoacoustic imaging is commonly used to monitor the in vivo hypoxia induced by PDT. As shown in Figure 5A, the PA signal of the oxygenated hemoglobin was extremely low in tumors treated with PA/HA-Ce6@TPZ NPs followed by irradiation. Comparatively, the PA signal was negligibly affected by injection of the PA/HA-Ce6@TPZ NPs followed by no light irradiation. Quantitatively, the tumoral oxyhemoglobin saturation was markedly reduced by ∼65% after injection of PA/HA-Ce6 NPs and light irradiation (Figure 5B). Such discrepancy, thus, demonstrated the aggravation of hypoxia in tumor tissues by Ce6mediated PDT, which would potentially facilitate the TPZ release and activation. In Vivo Antitumor Efficacy. The anticancer efficacy of the PA/HA-Ce6@TPZ NPs against 4T1 xenograft tumors was assessed following the protocol in Figure 6A. As shown in Figures 6B,C and S9, PA/HA@TPZ NPs and free TPZ provoked poor antitumor efficacy, mainly due to the inadequate tumoral hypoxic level to efficiently activate TPZ. PA/HA-Ce6 NPs (w/L) that allowed light-mediated PDT also partially inhibited the tumor growth. In comparison, remarkable inhibition of tumor progression was noted for PA/HA-Ce6@TPZ NPs (w/L), which was ascribed to the cooperation between PDT and PDT-activated TPZ. In consistency with the tumor growth, mice administered with PA/HA-Ce6@TPZ NPs (w/L) showed the highest survival rate (Figure 6D), along with the highest cancer cell remission and apoptosis as revealed by H&E staining and TUNEL staining of tumor tissues, respectively (Figure 7A−C). These findings therefore substantiated the potent and cooperative anticancer effect between PDT and TPZ-mediated chemotherapy, as a consequence of PDT-induced hypoxia that triggered TPZ release and activation. Biocompatibility. In the above in vivo efficacy study, administration of PA/HA-Ce6@TPZ NPs did not induce significant body weight loss (Figure S10), and histological examination of H&E-stained organ slices also showed negligible toxicity (Figure S11), suggesting desired biocompatibility of the NPs. Hematological and biochemical examinations in normal mice also revealed unappreciable abnormality after i.v. injection of PA/HA-Ce6@TPZ NPs (Figure S12), indicating minimal acute toxicity.
PA/HA-Ce6@TPZ NPs could not only produce extensive ROS under light irradiation (Figure S7) but also induce intracellular hypoxia due to the oxygen consumption (Figure S8). To probe the activation of TPZ under hypoxia, 4T1 cells were treated with CoCl2, a commonly used hypoxia inducer,42 before incubation with the PA/HA-Ce6@TPZ NPs. As shown in Figure 3B, the IC50 of TPZ under hypoxic conditions was ∼0.31 μg/mL, six-fold lower than that of TPZ under normoxic conditions (2 μg/mL). This finding, thus, suggested the hypoxiaenhanced TPZ release and activation. To further substantiate the ROS-mediated cancer cell death, cells were treated with PA/HA-Ce6@TPZ NPs and light irradiation along with a ROS scavenger, VC.12 The anticancer efficiency of PA/HA-Ce6@ TPZ NPs was greatly decreased by VC, provoking an increase of the IC50 value for the loaded TPZ from 0.22 μg/mL (without VC) to 1.0 μg/mL (with VC) (Figure 3C). Taken together, these results demonstrated the excellent anticancer efficacy of the PA/HA-Ce6@TPZ NPs under hypoxia conditions and the combination of TPZ-mediated bioreductive chemotherapy and Ce6-mediated PDT. Pharmacokinetics and Biodistribution. After systemic injection to normal mice, the half-life (t1/2) of PA/HA-Ce6@ DiR NPs and free DiR was determined to be 5.52 and 0.75 h, respectively (Figure 4A), suggesting that the HA-decorated NPs could appreciably prolong the blood circulation time, mainly due to the negative surface charges to prevent serum protein adsorption and the opsonization process. Subsequently, the biodistribution of PA/HA-Ce6@DiR NPs and free DiR was explored in 4T1 tumor-bearing mice. Live animal imaging revealed a notably stronger DiR fluorescence intensity in the tumor site of mice treated with NPs than those administered with free DiR, and the fluorescence intensity peaked at 6 h post i.v. injection (Figure 4B). Ex vivo imaging of harvested tissues at this time point further revealed that the fluorescence intensity in tumors treated with NPs was ∼10.4 fold higher than that in free DiR-treated tumors (Figure 4C,D). These results, therefore, collectively evidenced the desired tumor accumulation of the NPs as a consequence of the EPR effect-mediated passive targeting and HA-mediated active targeting. PDT-Aggravated Hypoxia in Vivo. PDT involves the consumption of tissue oxygen and leads to the local hypoxia. 2654
DOI: 10.1021/acs.biomac.9b00428 Biomacromolecules 2019, 20, 2649−2656
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Figure 7. Histological examination of tumor tissues. Representative H&E (A) and TUNEL (B) staining images of tumor sections harvested from mice on day 12 in the efficacy study. Scale bar represents 100 μm. (C) Apoptosis ratio of tumor cells calculated from TUNEL staining images using the ImageJ software (n = 3, 8 random microscope fields for each tumor slice).
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CONCLUSIONS In conclusion, we report the development of a dual hypoxiaresponsive drug delivery system which demonstrates cooperative anticancer effect between PDT and PDT-activated bioreductive chemotherapy intermediated via PDT-induced hypoxia. Hypoxia-dissociable NPs co-encapsulating Ce6 and hypoxiaactivatable chemodrug TPZ were designed to mediate cancertargeted delivery and hypoxia-induced TPZ release as well as activation upon light-induced hypoxia aggravation in tumor cells. Such a dual-responsive approach renders more precise control over the efficacy and toxicity of TPZ, yielding greatly potentiated anticancer potency while diminishing systemic toxicity. To the best of our knowledge, the current study renders the first example of combining hypoxia-dissociable vehicles with a hypoxia-activatable chemodrug, which could enable anticancer treatment in a more precise and intensive approach. With dual control over drug release and drug activity, this strategy enables highly selectively drug delivery in tumors to maximize the therapeutic efficacy and minimize the side toxicity.
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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/acs.biomac.9b00428. 1 H NMR spectra, DLS measurement, cytotoxicity of NPs, intracellular ROS generation, hypoxia levels under light irradiation, and biocompatibility of NPs (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Phone: 86-512-67784776 (Y.C.). *E-mail:
[email protected]. Phone: 31-06-39396957 (Y.L.). *E-mail:
[email protected]. Phone: 86-512-65882039 (L.Y.). ORCID
Yong Liu: 0000-0003-1738-7857 Lichen Yin: 0000-0002-4573-0555 Author Contributions ∥
R.Z. and H.H. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51873142, 51722305, and 51573123), the Ministry of Science and Technology of China (2016YFA0201200), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 111 project, and the Youth Pre-Research Found Project (SDFEYQN1606). 2655
DOI: 10.1021/acs.biomac.9b00428 Biomacromolecules 2019, 20, 2649−2656
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