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Engineering of a Nanosized Biocatalyst for Combined Tumor Starvation and Low-Temperature Photothermal Therapy Jun Zhou, Menghuan Li, Yanhua Hou, Zhong Luo, Qiufang Chen, Hexu Cao, Runlan Huo, Chencheng Xue, Linawati Sutrisno, Lan Hao, Yang Cao, Haitao Ran, Lu Lu, Ke Li, and Kaiyong Cai ACS Nano, Just Accepted Manuscript • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Engineering

of

a

Nanosized

Biocatalyst

for

Combined Tumor Starvation and Low-Temperature Photothermal Therapy Jun Zhoua, Menghuan Lia, Yanhua Houc, Zhong Luoa,b*, Qiufang Chenb, Hexu Caoa, Runlan Huoa, Chencheng Xuea, Linawati Sutrisnob, Lan Haod, Yang Caod, Haitao Rand, Lu Lub, Ke Lib, Kaiyong Caib* a

School of Life Science, Chongqing University, Chongqing 400044, P. R. China.

b

Key Laboratory of Biorheological Science and Technology, Ministry of Education, Chongqing

University, Chongqing 400044, P. R. China c

Chongqing Engineering Research Center of Pharmaceutical Sciences. Chongqing Medical

and Pharmaceutical College, Chongqing 401331, China d

Laboratory of Ultrasound Molecular Imaging, Second Affiliated Hospital of Chongqing

Medical University, Chongqing 400010,China Corresponding authors: Zhong Luo / [email protected] Kaiyong Cai / [email protected]

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ABSTRACT: Tumor hypoxia is one of the major challenges for the treatment of tumor, as it may negatively affect the efficacy of various anticancer modalities. In this study, a tumortargeted redox-responsive composite biocatalyst is designed and fabricated, which may combine tumor starvation therapy and low-temperature photothermal therapy for the treatment of oxygendeprived tumors. The nanosystem was prepared by loading porous hollow Prussian Blue nanoparticles (PHPBNs) with glucose oxidase (GOx) and then coating their surface with hyaluronic acid (HA) via redox-cleavable linkage, therefore allowing the nanocarrier to bind specifically with CD44-overexpressing tumor cells while also exerting control over the cargo release profile. The nanocarrier are designed to enhance the efficacy of the hypoxia-suppressed GOx-mediated starvation therapy by catalyzing the decomposition of intratumoral hydroperoxide into oxygen with PHPBNs, and the enhanced glucose-depletion by the two complementary biocatalysts may consequently suppress the expression of heat shock proteins (HSPs) after photothermal treatment to reduce their resistance to the PHPBN-mediated low-temperature photothermal therapies. KEYWORDS. starvation therapy, low-temperature photothermal therapy, thermoresistance, anaerobic glycolysis, porous hollow Prussian Blue nanoparticles

Starvation therapy is an emerging treatment paradigm for the clinical management of tumors and have drawn great interest in recent years.1-3 It’s well-known that the rapidly proliferating tumor cells require a large amount of energy (ATP) to sustain their biological activity, which was predominantly produced through anaerobic glycolysis (Warburg effect). Therefore, it’s widely


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accepted that the growth of tumors is highly dependent on glucose supply.4 In most of the previous reports, tumor starvation was usually induced by cutting off the blood supply to the tumor tissues, and moderate success has been achieved in slowing down the tumor growth rate.5-7 More recently, scientists have discovered that tumor starvation may be used conjunctionally with other anticancer modalities such as chemotherapy and radiotherapy to improve their therapeutic efficacy, which may further expand the application range of tumor starvation therapy and facilitate the development of combinational anticancer therapies with greater clinical relevance.810

Photothermal therapy (PTT) is a non-chemotherapeutic intervention option against many types

of tumors, which utilizes near-infrared (NIR) light-absorbing materials to remove tumor tissues through thermal ablation.11-17 In comparison with traditional tumor chemotherapy of smallmolecule drugs, NIR-induced PTT features low toxicity and high therapy specificity due to the controllable NIR irradiation setups such as exposure time, positioning of laser source and power output.18, 19 Nevertheless, high temperature photothermal treatment may confer collateral damage to healthy cells and tissues nearby by the inevitable heat diffusion. Moreover, a large localized concentration of photothermal converters is usually required to achieve the high photothermal temperature, which may result in increasing health risks due to overdose of the nanoagents.12, 2023

Therefore, scientists have sought to use low hyperthermia to induce the death of tumor cells,

but its actual performance was compromised by the upregulated expression of intracellular heat shock proteins after NIR treatment.24-26 Heat shock proteins (HSPs) were a molecular chaperone capable of repairing the heat-denatured proteins,27, 28 and their expression would increase rapidly upon exposure to hyperthermia, which would result in enhanced tumor thermoresistance. As the expression of HSPs is intrinsically associated with ATP level, inhibiting the intratumoral ATP


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production (tumor starvation) may help to overcome the HSPs-dependent tumor resistance and improve the efficacy of low-hyperthermia photothermal therapy.29

In this study, the tumor starvation is achieved via employing glucose oxidase (GOx) as a glucose consumer in the tumor region. GOx is a natural-occurring oxido-reductase which is capable of converting glucose into gluconic acid with oxygen as the oxidant (1), and has been vigorously studied for tumor starvation therapy.2, 30, 31 Nevertheless, due to the complexity of tumor microenvironment, the clinical translation of GOx-based therapeutics has been impeded by a series of practical issues. Specifically, the oxygen concentration in most solid tumors are significantly lower than that in normal tissues, which would severely limit the catalytic efficiency of GOx.32, 33 Moreover, GOx is highly susceptible to degradation after exposure to the biological environment. To address these issues, we have developed a multifunctional tumortargeted redox-responsive nanoplatform to starve the tumor while sensitizing the cancer cells to low-hyperthermia photothermal therapy. Porous hollow Prussian Blue nanoparticles (PHPBNs) was used as the carrier substrate of GOx, which is a biologically-friendly photothermal agent with high absorption coefficient in the NIR region.34-36 Moreover, it also possesses catalase-like activity that could be used to catalyze the decomposition of the intratumoral H2O2 for rapid reoxygenation (2), which may help to circumvent the tumor hypoxia-related issues.37, 38 In this system, GOx was loaded into the central cavity of PHPBNs, and the surface of the GOx-loaded nanoparticles was subsequently conjugated with hyaluronic acid (HA) using disulfide bond. HA is an extensively explored targeting moiety that could bind specifically to the CD44 receptor overexpressed on the surface of HepG2 cell and enhance the tumor-specific accumulation of the


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nanocarrier.39, 40 After successful endocytosis, the disulfide bond could be readily cleaved by the glutathione (GSH) within tumor cells for efficient GOx release, which may rapidly deplete the intratumoral glucose due to the oxygen replenishment from the PHPBN-catalyzed H2O2 decomposition. Upon NIR irritation, the PHPBNs could absorb the photons in the incident light and convert them into thermal energy, resulting in a mild temperature increase in the tumor region. Meanwhile, the hyperthermia-induced upregulation of HSPs in the affected tumor cells would be inhibited by the restricted ATP supply, therefore attenuating the photothermal-induced treatment resistance thereof. The in vitro and in vivo results suggest that by using our multifunctional nanoplatform, the GOx-mediated tumor starvation may be combined with lowhyperthermia photothermal therapy for enhanced antitumor efficacy. RESULTS AND DISCUSSION Preparation and Characterization of the PEGylated HA-Functionalized GOx-Loaded PHPBNs. As the first step for the synthesis of the PEGylated HA-functionalized GOx-loaded PHPBNs (PHPBNs-S-S-HA-PEG@GOx), solid Prussian Blue nanoparticles (PBNs) were synthesized with polyvinylpyrrolidone (PVP) and K3[Fe(CN)6] at 80 °C and then etched by hydrochloric acid under the protection of PVP.41 As shown by the transmission electron microscopy (TEM) and the scanning electron microscopy (SEM) images in Figure 1(a-c, a1-c1), the as-prepared PHPBNs have an average diameter of 130 nm, which is consistent with the result of the dynamic light scattering (DLS) (Figure S1). The thickness of the shell in the PHPBNs was around 28 nm and the diameter of the central cavity was about 74 nm. The results of XRD (Figure S2) revealed the PHPBNs retained the crystalline feature of the PBNs. According to the N2 absorption-desorption isotherms (Figure S3 and Table S1), the BET surface area of the nanoparticles increased dramatically from 302.56 m2/g to 922.14 m2/g after the etching process,


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and the pore size was revealed to be around 10.12 nm using BJH method. The characterization results above evidently confirmed the porosity and hollow feature of the as-prepared Prussian Blue nanostructures, which is vital for the efficient loading of glucose oxidase (GOx). In order to optimize the release profile of GOx, PHPBNs were subsequently modified with the dual-purpose hyaluronic acid-containing ligand, which could effectively block the pore openings while simultaneously conferring targeting specificity against HepG2 cells overexpressed with CD44. The preparation process of PHPBNs-S-S-HA-PEG@GOx was illustrated in Scheme 1. Briefly, the PVP on the surface of the PHPBNs were replaced by poly(acrylic acid) (PAA, MW = 2k) via carboxyl coordination. Then, excess amount of cystamine dihydrochloride (Cys) was introduced for coupling with the PAA on the nanoparticle surface by the amidation reaction between amines and carboxylic acids. Afterwards, GOx was loaded into the cavity of the cystamine dihydrochloride (Cys) modified PHPBNs, followed by the coating of hyaluronic acid (HA, MW = 5–150k) via the amidation reaction between amino and carboxyl groups as well, and the resultant intermediate was denoted as PHPBN-S-S-HA. Finally, amine terminated PEG (mPEG-NH2, MW = 5k) was grafted to the outer shell of the PHPBN-S-S-HA to prolong their blood circulation time, and the product was denoted as PHPBNs-S-S-HA-PEG. As shown by the long term DLS monitoring (Figure S4), after 4 days of incubation in biomimicking buffer solution, the average hydrodynamic diameter of PHPBNs-S-S-HA-PEG only changed minimally and the PDI value was also smaller than 0.25, which demonstrated the good stability of PHPBNs-S-S-HA-PEG@GOx in physiological environment. TGA tests were also carried out which revealed the chemical composition (w/w) of the final product. According to Figure S5, PHPBNs-S-S-HA-PEG@GOx comprised 4.5 % PAA, 1.6 % Cys, 6 % HA, 3 % mPEG-NH2, and had a loading capacity of 4.7 % for GOx. The modification process was also monitored stepwise


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by the Fourier transform infrared spectroscopy (FTIR) and zeta potential analysis (Figure S6 and Figure S7). As revealed in Figure S7, it was found that the zeta potential of PHPBNs-COOH changed from 10.1 mV to -22.3 mV due to the carboxyl groups in PAA when the free PHPBNs were coated with the PAA. The zeta potential of PHPBNs-S-S-NH2 increased to 7.8 mV after the addition of Cys, indicating the successful conjugation of Cys onto PHPBNs-COOH. Furthermore, after the conjugation of HA to PHPBNs-COOH, the zeta potential of the PHPBNsS-S-HA decreased to -37.1 mV, which was due to the carboxyl-containing HA on the surface of nanoparticles. After the PEGylation, the zeta potential has increased slightly to -21.2 mV as a result of the reaction between amines in the mPEG-NH2 and carboxyl groups in HA. Consistently, according to the dynamic light scattering (DLS) results (Figure S1), the average hydrodynamic diameter of PHPBNs-S-S-HA-PEG@GOx was around 150 nm, while that of PHPBNs was only about 130 nm, which suggested the successful surface modification of PHPBNs. Additionally, UV-VIS spectrophotometer was applied to investigated the GOx loading in the PHPBNs-S-S-HA-PEG@GOx (Figure S8). The GOx loading capacity (GLC) was calculated using the equation: GLC (%) = (mass of GOx in PHPBNs-S-S-HA-PEG@GOx/mass of PHPBNs-S-S-HA-PEG@GOx)×100%, while the GOx encapsulation efficiency (GEE) was calculated by the equation GEE (%) = (mass of GOx in PHPBNs-S-S-HA-PEG@GOx/mass of total GOx)×100%. Therefore, the GOx loading capacity was determined to be around 4.7% with an encapsulation efficiency at approximately 18%. As shown in the Figure 1d, PHPBNs-S-S-HA-PEG possess high absorption coefficient in the range from 500 nm to 900 nm and the absorption peak was centered at 780 nm. To evaluate the photothermal capability of PHPBNs-S-S-HA-PEG@GOx, a series of PHPBN-S-S-HAPEG@GOx solutions with different concentrations at 0.05, 0.1, 0.2 and 0.5 mg/mL were irritated


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by NIR laser (808 nm, 1 W/cm2). According to the temperature curves (Figure 1e), it was found that the temperature increasing rate was dependent on the concentration of PHPBNs-S-S-HAPEG@GOx and the temperature eventually reached beyond 60°C after 5 min of irradiation when the concentration was 0.5 mg/mL, while the temperature of the pure water group only increased minimally (nearly 5°C). Additionally, it was observed in Figure S9 that photothermal conversion efficiency of PHPBNs-S-S-HA-PEG still remained at a high level even after four cycles of NIR laser irradiation (808 nm laser at a power density of 1 W/cm2, each cycle lasted 3 min). On the other hand, the absorption band of the PHPBNs-S-S-HA-PEG solution from 400 nm to 1000 nm showed no apparent change even after irradiation for 60 min (Figure 1d). The results above demonstrated the photothermal stability of PHPBNs-S-S-HA-PEG, which is favorable for photothermal therapy. Redox-Responsive GOx Release. In this work, the release behavior of GOx from PHPBNs-SS-HA-PEG@GOx under GSH has also been systematically investigated. After the loading of GOx, PHPBN-S-S-HA-PEG@GOx solutions were exposed to Tris buffer containing 0 mM, 1 mM and 10 mM GSH for 24 hours, respectively. The amount of released GOx at different incubation periods was determined using a UV-VIS spectrophotometer (Figure 2f) by BCA protein assay. When no GSH was present, only around 8% of the total GOx was released from PHPBNs-S-S-HA-PEG@GOx within 24 hours, suggesting the excellent GOx retention capability of PHPBNs-S-S-HA-PEG@GOx. In comparison, the amount of GOx releasd from PHPBNs-S-S-HA-PEG@GOx increased to 41% and 80% after 24 hours under GSH concentrations of 1 mM and 10 mM, respectively. The above observations indicated that the GOx release is initiated by the GSH-mediated removal of hyaluronic acid by cleaving disulfide linkage, and its release rate was positively dependent on the GSH concentration.


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Biocatalysis-Enabled Glucose Consumption and Reoxygenation In Vitro. For the investigation of the GOx-mediated glucose decomposition, the H2O2 concentration and pH value were measured after the reaction of GOx and glucose. As shown in Figure S10, the amount of H2O2 and gluconic acid (reflected with pH) increased gradually with the gradient glucose concentration upon the released GOx from PHPBNs-S-S-HA-PEG@GOx, which indicated that the glucose could be effectively catalyzed into gluconic acid by the GOx released from PHPBNsS-S-HA-PEG@GOx. Moreover, according to the time plot in Figure S11, the concentrations of H2O2 and gluconic acid increased rapidly as the reaction continued and reached the maximum after 1 hour under air exposure, while only negligible H2O2 and gluconic acid were detected when pumped with N2. All results revealed that GOx released from the nanocarrier still possesses intact activity and the GOx-catalyzed glucose conversion is oxygen-dependent. To further evaluate the catalase-like activity of PHPBNs-S-S-HA-PEG against H2O2 and the glucose consuming ability of GOx, the concentration of reactive oxygen species (ROS) in HepG2 cells was measured using DCFH-DA as the indicator, which has no intrinsic fluorescence but can emit green light after being oxidized by intracellular ROS.42, 43 Specifically, HepG2 cell samples were treated individually with PBS, PHPBNs-S-S-HA-PEG, GOx and PHPBNs-S-S-HA-PEG@GOx. As revealed in the Figure 2(a-e, g) and Figure S12, the cellular DCF fluorescence intensity of the GOx group has increased around 4-fold to the PBS group, suggesting that GOx could effectively catalyze the glucose into the H2O2. However, after the treatment with PHPBNs-S-S-HA-PEG, the fluorescence intensity has decreased significantly. It was attributed to the catalase-like activity of PHPBNs-S-S-HA-PEG that consumed the intracellular H2O2. In comparison with the GOx group, similar decreasing trend was observed for the fluorescence intensity in the PHPBNsS-S-HA-PEG@GOx group, which consistently supported the H2O2 decomposition capacity of


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PHPBNs-S-S-HA-PEG. The trends observed after the DCF staining were further validated by quantitative flow cytometric analysis, which was summarized in Figure 2g and Figure S12. Based on these preliminary results, we further examined the oxygen concentration in vitro under seven conditions including H2O2, PHPBNs-S-S-HA-PEG, PHPBNs-S-S-HA-PEG + H2O2, glucose, GOx, glucose + GOx and glucose + GOx + PHPBNs-S-S-HA-PEG (Figure 2h and 2i). It could be found that oxygen concentration in the PHPBNs-S-S-HA-PEG + H2O2 group increased rapidly within the 10 min, while no change was detected in the PHPBNs-S-S-HA-PEG and H2O2 solution, indicating that H2O2 has been decomposed to oxygen by PHPBNs. Additionally, the oxygen concentration in the glucose + GOx group decreased dramatically from 5.1 mg/L to 0.04 mg/L after the 10 min incubation, which immediately suggests that the solubilized oxygen has been consumed by the GOx-mediated glucose oxidization. After the addition of PHPBNs-S-S-HA-PEG, the decreasing rate of oxygen concentration in solution has become lower, which was theoretically consistent with previously reported catalase-like activity of PHPBNs. PHPBN-S-S-HA-PEG@GOx Induced ATP and HSP Suppression In Vitro. To investigate the effects of the PHPBNs-S-S-HA-PEG@GOx nanocomposite on the ATP generation in malignant cells and normal cells, the ATP level in HepG2 and HL-7702 cells was quantified after different treatments. According to Figure 3a, the ATP generation in HepG2 cells was significantly reduced after treatment with PHPBNs-S-S-HA-PEG@GOx, which confirmed that the glucose depletion by GOx could effectively reduce the ATP level in malignant cells. In comparison, the ATP level showed no substantial decrease in the HL-7702 after same treatment. These results demonstrated that the PHPBNs-S-S-HA-PEG@GOx nanocomposite could suppress the ATP generation in malignant cells while imposing negligible impact on that in the


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normal cells, which was in accordance with the results shown in Figure S13. Furthermore, the effect of the GOx and PHPBNs-S-S-HA-PEG@GOx system on the ATP generation in HepG2 cells under hypoxic condition was also explored. The results (shown in Figure 3b) indicated the ATP level in the GOx + 1% O2 group showed no apparent change after treatment for 24 hours, while a significant decrease was found in the PHPBNs-S-S-HA-PEG@GOx + 1% O2 group after 24 hours. These results demonstrated that hypoxia could impede the glucose depletion capacity of GOx, while PHPBNs-S-S-HA-PEG could efficiently reoxygenate the hypoxic tumor cells by decomposing H2O2. Based on these preliminary results, we further examined the level of HSPs family (HSP90, HSP70) in HepG2 cells with different treatments via western blotting (Figure 3c and Figure S14) to evaluate the impact of ATP suppression on the expression of HSPs. After incubation at 40°C or treatment with PHPBNs-S-S-HA-PEG + NIR, greater amount of HSP70 and HSP90 were produced compared with the control group, which confirmed that the increase in ambient temperature would stimulate the expression of HSPs. Specifically, the HSP70 level has increased to 1.3 and 1.29 fold in the mild hyperthermia (40°C) and PHPBNs-S-S-HA-PEG + NIR group, respectively, while that of HSP90 in the two groups was 1.5 and 1.49 fold, respectively. Furthermore, in the PHPBNs-S-S-HA-PEG@GOx group, the relative HSP70 and HSP90 levels were reduced dramatically to 0.41 and 0.18 compared to the PBS control, which demonstrated that starving HepG2 cells by depleting glucose could effectively lower the HSP expression within cells. Similar decreasing trend has also been observed for the expression of HSP90 and HSP70 in the two groups treated by PHPBNs-S-S-HA-PEG@GOx + hyperthermia 40°C and PHPBNs-S-S-HA-PEG@GOx + NIR.


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Endocytic Efficiency of the Nanocarrier and the Efficacy of the Starvation Sensitized Low-Temperature Photothermal Therapy In Vitro. The cell endocytosis and intracellular distribution of PHPBNs-PEG and PHPBNs-S-S-HA-PEG by HepG2 cells were characterized by CLSM (Figure S15), in which both nanospecies were fluorescently marked with FITC. The HepG2 cells were incubated with FITC-labeled PHPBNs-PEG and PHPBNs-S-S-HA-PEG for 12 hours under 37°C, and the CLSM observations demonstrated that both PHPBNs-PEG and PHPBNs-S-S-HA-PEG have good biocompatibility, indicated by the intact cell nuclei. Furthermore, it was also shown in Figure S15e and S15f that the amount of PHPBN-S-S-HAPEG uptake by HepG2 cells was visibly higher in comparison with PHPBNs-PEG and would decrease significantly if the HepG2 cells were pre-incubated with free HA for 4 hours to saturate the CD44 receptor, suggesting the targeting efficiency of HA on PHPBNs-S-S-HA-PEG against CD44 overexpressed tumor cells. However, in healthy cells (HL-7702) where the expression level of CD44 was normal, the amount of nanoparticle uptake by HL-7702 in all three groups remained at a similar level to the PHPBNs-PEG group in the HepG2 cells with no statistically significant difference. Based on these findings, we further quantitatively evaluated the phagocytosis of nanoparticles using ICP assay by using Fe element as an indicator, and the results illustrated that the amount of iron in HepG2 cells for the PHPBNs-S-S-HA-PEG group was approximately 2 times higher than the PHPBNs-PEG and PHPBNs-S-S-HA-PEG + free HA group in HepG2 cells and all groups in HL-7702 cells (Figure S15f). The increasing intracellular iron level consistently supported the targeting capacity of HA to CD44 positive cells. Subsequently, the tumor starvation capability and PTT-sensitizing effect of PHPBNs-S-S-HAPEG@GOx were investigated on HepG2 cells by treating them with PBS, PHPBNs-S-S-HAPEG, PHPBNs-S-S-HA-PEG@GOx, PHPBNs-PEG + NIR, PHPBNs-S-S-HA-PEG + NIR and


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PHPBNs-S-S-HA-PEG@GOx + NIR, respectively. The biosafety, potency of sole tumor starvation or photothermal therapy and HA-mediated targeting efficiency of various nanoparticles samples were firstly revealed in Figure S16 and Figure S17, and the results were consistent with the previous characterizations. It was further observed that the PHPBNs-S-SHA-PEG@GOx + NIR group showed the lowest cell viability in all the six groups, which was about 21%. The superior antitumor efficacy of PHPBNs-S-S-HA-PEG@GOx under NIR illumination was evidently due to the combination of the GOx-mediated tumor starvation, PHPBN-mediated reoxygenation, low-temperature photothermal therapy and HA-dependent tumor targeting. Furthermore, the cell killing effect after different treatment was investigated with live/dead cell fluorescence staining and flow cytometry and the results were in accordance with the MTT results (Figure 3(d-e), Figure S18 and Table S2). To further investigate the glucose-mediated starvation effect in vitro, the MTT cell viability assay was carried out after treatment with different concentrations of glucose. Consistently, as revealed in Figure S13, we have found that the cell viability of malignant cells (HepG2) dropped significantly along with the decreasing glucose concentration. In comparison, the impact of varying the glucose concentration for normal cells (HL-7702) was found to be modest at most, which was attributed to the aerobic respiration of normal cells even when the glucose concentration remained at a low level. These observations demonstrated that the glucose-mediated starvation had only negligible side effects to normal tissues. Efficacy of the Biocatalysis-Enabled Tumor Starvation In Vivo. To investigate the efficacy of PHPBN-S-S-HA-PEG@GOx-mediated starvation therapy on tumor tissue, the intratumoral oxygen saturation (sO2) was investigated on tumor-bearing mouse models by mapping the oxygenation state of hemoglobin via photoacoustic (PA) imaging. It’s well known that the light


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absorption peak of hemoglobin would transit from 750 nm to 850 nm when it changes from the deoxygenated state to the oxygenated state, resulting in excellent PA contrast.44 Specifically, HepG2 tumor-bearing mice were divided into four groups of PBS, PHPBNs-S-S-HA-PEG, GOx and PHPBNs-S-S-HA-PEG@GOx groups. All samples were administered through intratumoral injection. The photoacoustic oxygenation map was recorded at selected time points. As revealed by Figure 4(a-d, a1-d1) and Figure 4e, the blood oxygenation level dramatically dropped after the injection of PHPBNs-S-S-HA-PEG@GOx as the average intratumoral blood oxygen saturation (average sO2) decreased from ~25% before injection to ~10% at 2 hours after injection. It’s worth noting that the tumor oxygenation levels (average sO2) in the PHPBNs-S-SHA-PEG group was higher than the PBS group (Figure 4e) while the H2O2 concentration of tumor tissues dropped significantly (Figure 4f), which was attributed to the catalase-like H2O2decomposing effect of PHPBNs-S-S-HA-PEG. Similar trends have also been observed in oxygen partial pressure (Figure 4g). Compared with the PBS group, the tumor H2O2 concentration in GOx group increased to about 5 µmol/g due to the GOx-catalyzed conversion of glucose. However, the tumor H2O2 concentration in the PHPBNs-S-S-HA-PEG@GOx group was still maintained at 2.7 µmol/g similar to the PBS group, as the PHPBN substrate could break down H2O2 into oxygen. These results collectively verified the superior glucose-depleting ability of PHPBNs-S-S-HA-PEG@GOx in tumor regions. Furthermore, the tumor ATP level was measured after various treatments to investigate their tumor starvation efficacy in vivo. As shown in the Figure 4h, the tumor ATP level in the GOx group has been significantly reduced compared to the PBS group, and the ATP level in the PHPBNs-S-S-HA-PEG@GOx group was even lower. The results demonstrated that GOx could impede the ATP production in the tumor tissues by catalyzing the oxygen dependent conversion of glucose into gluconic acid that couldn’t no longer


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be utilized to produce energy (ATP), and the PHPBNs-S-S-HA-PEG could decompose the intratumoral H2O2 to reoxygenate the hypoxic tumor and enhance the starvation effect thereof. Efficacy of the Combined Starvation and Low-Temperature Photothermal Treatment In Vivo. Before evaluating the overall anticancer performance of the nanosystem on live animal models, we firstly studied its plasma stability and distribution in various organs after systemic administration using ICP analysis, which is a well-established technique for the accurate determination of nanoparticle concentration in living organisms.45 It was found in Figure S19 that the circulation half-life of PHPBNs-S-S-HA-PEG@GOx was as long as 5.26 ± 0.53 hours, and the observed serum stability was due to the combination of size effect and PEGylation. Moreover, the concentration of circulating nanoparticles decreased to almost 0 after 24 hours post-injection while the nanoparticle deposition in the tumor tissues simultaneously reached the maximum as indicated by the changes in the nanoparticle accumulation in different organs and tissues (Figure S20). Therefore, the laser illumination was implemented 24 h after the intravenous injection to maximize the therapeutic effect. To evaluate the photothermal efficacy of PHPBNs-S-S-HA-PEG@GOx on HepG2 tumor-bearing mice, the changes in tumor temperature against time was recorded using an IR thermal camera. As illustrated in Figure 5, the tumor temperature in PHPBNs-S-S-HA-PEG@GOx + NIR and PHPBNs-S-S-HA-PEG + NIR groups increased rapidly with the NIR laser radiation time. The maximum temperature reached about 45 °C after 5 min of NIR laser radiation, which was within the temperature range for lowtemperature photothermal therapy. Moreover, the maximum tumor temperature in the PHPBNsPEG + NIR group was around 40 °C and lower than the above two groups. It was possibly due to the targeting effect of HA, which facilitated the accumulation of the nanoparticles in the tumor


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tissues. In comparison, the PBS + NIR group showed only a minimal tumor temperature increase (less than 4 °C), which confirmed the low biological invasiveness of NIR laser at 808 nm. Based on these results, we further investigated the therapeutic efficacy of these nanocomposites in vivo on HepG2 tumor-bearing mice. As revealed in Figure 5e and 5g, for the sample group that received only PHPBNs-S-S-HA-PEG treatment, the tumor volume showed a rapid and uncontrolled growth pattern similar to the PBS/PBS+NIR groups, which demonstrated the biocompatibility and biosafety of the PHPBN-based nanocarriers. However, after the incorporation of GOx (the PHPBNs-S-S-HA-PEG@GOx group), moderate growth inhibition has been observed in the HepG2 tumors, which was evidently associated with the tumor starvation effect of GOx. In comparison, the potency of PHPBN-mediated photothermal therapy is significantly higher than tumor starvation therapy under the current experimental setting, which was indicated by the strong tumor inhibition effect observed in the three groups treated by PHPBNs-PEG+NIR, PHPBNs-S-S-HA-PEG+NIR and PHPBNs-S-S-HA-PEG@GOx + NIR. Nevertheless, the tumor ablation was more efficient in the PHPBNs-S-S-HA-PEG + NIR group than PHPBNs-PEG + NIR, and the better photothermal efficacy could be explained by the HAfacilitated tumor accumulation and uptake. The best anticancer efficacy was observed in the PHPBNs-S-S-HA-PEG@GOx + NIR group, in which the average tumor volume has decreased by 32.5% at the end of the incubation period. The observed trends in tumor volume were another direct proof for the therapeutic efficacy of the as-developed biocatalytic nanocomposites. Extending from the analysis on the tumor volume changes, the extracted tumors were further sliced into sections for the immunofluorescence staining of HSP 90 and HSP 70 to investigate the effectiveness of inhibition of those resistance-related proteins after PHPBN-S-S-HAPEG@GOx-mediated tumor starvation therapy. As shown in Figure 6(a1-g1, a2-g2, h, i), the


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green (HSP 90) and red (HSP 70) fluorescence in the PHPBNs-PEG + NIR group was 1.34 and 1.42 fold to the PBS group, and a comparable increase was also observed for the PHPBNs-S-SHA-PEG + NIR group at 1.28 and 1.37 fold, respectively. These results indicated that photothermal therapy could enhance the expression of HSP 90 and HSP 70 in the tumor tissue. On the other hand, the expression levels of HSP 90 and HSP 70 in the PHPBNs-S-S-HAPEG@GOx and PHPBNs-S-S-HA-PEG@GOx + NIR group were significantly lower than the PBS group, which was attributed to the tumor starvation effect of GOx in PHPBNs-S-S-HAPEG@GOx. The observations above was in agreement with the trends of tumor volume and consistently support our previous hypothesis that the as-developed multifunctional nanosystem could be utilized to reverse the tumor thermoresistance for more effective low temperature PTT. Additionally, the tumor sections were also stained with hematoxylin and eosin (H&E) to reveal the histological details after various treatments and evaluate their pro-apoptotic efficiency (Figure 6(a-g)). Particularly, the tumor cell apoptosis was most severe in the PHPBNs-S-S-HAPEG@GOx + NIR group, indicated by the large areas of shrunken cells and dissolved nuclei (arrows). Overall, the extent of cancer cell apoptosis in each group was consistent with the measured tumor volumes/weights (Figure 5g and Figure 6j). In addition to the investigations on the therapeutic efficacy of the nanosystem, we also studied their in vivo safety using multiple complementary techniques. As shown in Figure S21, the final weight of mice in all seven groups showed no significant differences, which again confirmed the excellent biocompatibility of the PHPBN-based nanosystem. Furthermore, we have employed immunochemistry staining to investigate the systemic toxicity of the nanoplatform, in which major organs from the tumor-bearing mice in all above groups were harvested on the twenty-first days and stained with H&E for histological analysis. As shown in Figure S22, no significant


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histological damages were observed in major organs, which demonstrated the low toxicity of the nanoparticles while also being consistent with the trends of nanoparticle deposition in different organ systems revealed in Figure S20. Specifically, a decreasing trend was found in the amount of nanoparticles accumulated in major organs when the incubation period was extended beyond 24h, especially the liver and spleen, which suggested that the nanoparticles could be excreted from the host and therefore reduce the tissue damage caused by nanoparticle accumulation. These results collectively demonstrated the biocompatibility and biosafety of PHPBNs-S-S-HAPEG@GOx in vivo. The survival time of each HepG2 tumor-bearing nude mice in all sample groups was also recorded to evaluate the biological impact and therapeutic effect of the nanosystem in vivo. Median survival time of HepG2 tumor-bearing nude mice in PBS, PBS + NIR, PHPBNs-S-SHA-PEG, PHPBNs-S-S-HA-PEG@GOx, PHPBNs-PEG + NIR, PHPBNs-S-S-HA-PEG + NIR and PHPBNs-S-S-HA-PEG@GOx + NIR groups was 35, 34, 33, 41, 48.5, 53 and 59 days, respectively (Figure 6k). The PBS, PBS + NIR and PHPBNs-S-S-HA-PEG groups were found with the shortest survival time, which was attributed to the rapid progression of HepG2 tumor without any therapeutic intervention. The small increase in the survival time of the PHPBNs-SS-HA- PEG@GOx group was due to the moderate tumor starvation effect of GOx. It was also found that the survival time in the PHPBNs-PEG + NIR group was longer than PHPBNs-S-SHA-PEG@GOx group, indicating the potent antitumor efficacy of the PHPBN-mediated photothermal therapy. It should also be noted the average survival time for the PHPBNs-S-SHA-PEG + NIR group was even longer than their non-targeted counterparts, which was a direct proof of the importance of the incorporation of tumor-targeting HA units. Nevertheless, PHPBNs-S-S-HA- PEG@GOx + NIR group displayed the highest survival time compared with


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all other groups, which was the collective result of the tumor starvation and sensitized photothermal therapy, again demonstrating the superior antitumor efficacy of PHPBNs-S-S-HAPEG@GOx when illuminated with NIR laser. CONCLUSION In this study, we successfully developed a tumor-targeted multifunctional nanoplatform for combined tumor starvation and low temperature PPT. The nanocarrier system was developed around the NIR-absorbing hollow porous Prussian Blue nanoparticles, which was loaded with GOx and capped with HA moieties via disulfide linkages. The incorporation of the HA units could enhance the accumulation and uptake of the nanocarrier by the CD44 overexpressing tumors and afterwards the PHPBN-protected GOx could be released by the high level of intratumoral GSH, both of which could enhance the delivery of the degradation-susceptible GOx. The released GOx could deplete the glucose in the tumor tissues by consuming oxygen and the PHPBNs were capable of breaking down intratumoral H2O2 into oxygen to amplify the tumor starvation effect in hypoxic tumors. The GOx-mediated tumor starvation could not only suppress the growth of tumors directly, but also block the hyperthermia-induced expression of HSPs to enhance their susceptibility to the PHPBN-mediated low-temperature photothermal treatment. The improved therapeutic efficacy from the combination of tumor starvation therapy and lowtemperature photothermal therapy in this study may be indicative for the creation of combinational therapeutic systems with clinical significance. MATERIALS AND METHODS Preparation of Solid PBNs. The protocol for solid PBN synthesis was adapted from a previous report.41 Under stirring condition, K3[Fe(CN)6]·3H2O (131.7 mg) and PVP (3 g) were dissolved into HCl solution (0.01 M, 40 mL). After stirring for 30 min, the mixture solution


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became transparent and then heated at 80°C for 20 hours with a muffle furnace. The reaction solution was cooled down when the heating was completed and the synthetic products were finally collected by centrifugation and extensively washed with deionized water and ethanol for several times, which was denoted as PBN. Preparation of PHPBNs. The synthesis procedure for PHPBNs was adapted from a previous report.41 PBNs (20 mg) and PVP (100 mg) were dissolved into HCl solution (1.0 M, 20 mL) under stirring. After stirring for 2.5 hours, the mixture solution was added into a Teflon vessel inside a stainless autoclave and heated at 140°C for 4 hours in a muffle furnace. After heating, the reaction solution was cooled down and the synthetic products were finally collected by centrifugation and extensively washed with deionized water and ethanol several times, which was denoted as PHPBNs. Preparation of PHPBNs-PEG, PHPBNs-S-S-HA-PEG and PHPBNs-S-S-HA-PEG@GOx. PHPBNs-PEG and PHPBNs-S-S-HA-PEG were prepared using a reported protocol with some changes.46 To synthesize the PHPBNs-PEG, 10 mg (5 mL) of PHPBNs was added into 10 mL of PAA solution (3 mg/mL) dropwise under stirring. After 3 hours of stirring, the mixture solution was filtered with filters (100 kDa MWCO, Millipore) to remove the unreacted PAA and the product was denoted as PHPBNs-COOH. Furthermore, the above PHPBN-COOH solution was added into 20 mL of mixture solution containing 2 mg of EDC and 2 mg of NHS. After stirring for 4 h, 10 mg of mPEG-NH2 was added into the above solution and dispersed by ultrasonication. After reacting for 48 hours, the mixture solution was filtered with filters (100 kDa MWCO, Millipore) to remove the unreacted mPEG-NH2. The product was denoted as PHPBNs-PEG.


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To prepare the PHPBNs-S-S-HA-PEG, the above PHPBNs-COOH solution was added again into 20 mL mixture solution of EDC (2 mg) and NHS (2 mg). After stirring for 4 hours, 20 mL of Cys (0.5 mg/mL) solution was added into the mixture. After reacting for 48 hours, the mixture solution was filtered with filters (100 kDa MWCO, Millipore) to remove the unreacted Cys, EDC and NHS to obtain PHPBNs-S-S-NH2. Subsequently, the PHPBN-S-S-NH2 solution was added into the 10 mL aqueous solution containing 2 mg of EDC and 2 mg of NHS and further stirred for 4 hours. Then, 15 mg of HA was added into the mixture solution and further reacted for 48 hours, and PHPBN-S-S-HA was extracted by centrifugation. At last, EDC and NHS (2 mg each) was added into the above PHPBN-S-S-HA-containing mixture solution. After stirring for 4 hours, 10 mg of mPEG-NH2 was added into the above solution upon ultrasonication and then stirred for 48 hours. The PHPBN-S-S-HA-PEG was extracted and washed by repetitive centrifuging. To obtain PHPBNs-S-S-HA-PEG@GOx, PHPBN-S-S-NH2 was dispersed into 10 mL of mixture solution containing 2 mg of EDC and 2 mg of NHS and stirred for 4 hours. The unreacted EDC and NHS were removed by centrifugation. Then, 15 mg of HA and 50 mg of GOx were added into the mixture solution to further react for 48 hours and PHPBN-S-SHA@GOx was obtained by centrifugation. EDC (2 mg) and NHS (2 mg) were subsequently added into the above mixture solution. After stirring for 4 hours, 10 mg of mPEG-NH2 was added into the above solution with ultrasonication and then stirred for 48 hours. The PHPBNs-SS-HA-PEG@GOx were extracted by centrifugation. Preparation of FITC Labeled PHPBNs-PEG and PHPBNs-S-S-HA-PEG. To prepare the FITC-labelled sample series, 20 mg of PHPBNs-S-S-NH2 were dispersed in 20 mL of deionized water and then 5 mg of FITC was added into the mixture solution. The reaction would continue


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for 24 hours under stirring in the dark. Afterwards, the PHPBNs-FITC were extracted by centrifuging (12000 rpm, 10 min) for several times and then further purified through dialysis. At last, EDC and NHS (each for 4 mg) were added into the mPEG-COOH (20 mg) solution. After stirring for 4 hours, the PHPBNs-FITC was added into the above solution under ultrasonication and then stirred for 48 hours. The PHPBNs-PEG-FITC was washed and centrifuged for several times. For the preparation of the PHPBNs-S-S-HA-PEG-FITC, 20 mg of the PHPBNs-S-S-NH2 were dispersed in 20 mL of deionized water and then 5 mg of FITC was added into the mixture solution. The reaction continued for 24 hours under the stirring condition in the dark. Afterwards, PHPBNs-S-S-FITC were extracted by centrifuging (12000 rpm, 10 min) for several times and then further purified through dialysis. Furthermore, the obtained PHPBNs-S-S-FITC solution was added into 10 mL mixture solution containing 4 mg of EDC and 4 mg of NHS and stirred for 4 hours. Then, 30 mg of HA was added into the mixture solution to further react for 48 hours and PHPBN-S-S-HA-FITC was collected by centrifugation. At last, 4 mg of EDC and NHS each was added into the above PHPBNs-S-S-HA-FITC mixture solution. After stirring for 4 hours, 20 mg of mPEG-NH2 was added into the above solution upon ultrasonication and then stirred for 48 hours. The PHPBNs-S-S-HA-PEG-FITC were collected by centrifuging for several times. Redox-Responsive Glucose Oxidase Release from PHPBNs-S-S-HA-PEG@GOx. The redox-responsive release behavior of GOx from PHPBNs-S-S-HA-PEG@GOx was investigated under GSH condition. The GOx level was determined by BCA protein assay. Firstly, PHPBNsS-S-HA-PEG@GOx was added into Tris buffer (pH 7.4) containing different amounts of GSH


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(0, 1 mM and 10 mM) at 37 °C under continuous stirring. Then, at different time points, 80 µL of the incubation medium was taken out for measurement and replenished with fresh ones. In Vitro Oxygen Concentration Assessment. The oxygen concentration in different solution of H2O2, PHPBNs-S-S-HA-PEG, PHPBNs-S-S-HA-PEG + H2O2, Glucose, GOx, Glucose + GOx and Glucose + GOx + PHPBNs-S-S-HA-PEG was determined every 60 seconds by a portable dissolved oxygen meter through the oxygen electrode probe (IMP-211, Inter Medical co., Itd., Japan). The concentration of H2O2, PHPBNs-S-S-HA-PEG, Glucose and GOx was 10 mM, 115.1 µg/mL, 1 mg/mL and 4.7 µg/mL, respectively. Cell Culture. Human hepatocellular liver carcinoma cell (HepG2) line was purchased from American Type Culture Collection (ATCC). The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL) and penicillin (100 U/mL) at 37°C under 5% CO2 atmosphere. The cell culture medium was replaced every day. Intracellular ROS Assay. For the analysis of intracellular ROS content, HepG2 cells were seeded into the 35 mm × 12 mm CLSM cell culture dish (NEST Biotechnology Co., Ltd, China). After the cell confluence reached about 70%, the original culture medium was replaced by fresh ones containing PBS (control), PHPBNs-S-S-HA-PEG (115.1 µg/mL), GOx (4.7 µg/mL) and PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL) and further incubated for 1 hour, noting that the net concentration of PHPBN substrate in all sample groups have been maintained at 100 µg/mL. After that, the HepG2 cells were rinsed with serum-free medium and stained with DCFH-DA under 37 °C for 20 min and then analyzed using a confocal microscope (LSM 510 Metanlo, Zeiss Co., Germany). Quantitative determination of the ROS content in the cell samples was further carried out as a complement to the characterizations above. The cells were collected by


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digestion after same treatment as above and then were incubated with DCFH-DA for 20 min. The DCF fluorescence intensity of the cells was eventually measured with flow cytometry. Characterization of Endocytosis and Intracellular Distribution of Nanoparticles within HepG2 cells and HL-7702 Cells. To monitor the intracellular distribution of nanoparticles, HepG2 cells and HL-7702 cells were cultured with FITC, PHPBNs-PEG-FITC, PHPBNs-S-SHA-PEG-FITC and PHPBNs-S-S-HA-PEG-FITC + free HA for 12 hours, respectively. The equivalent FITC concentration was kept at 5 µg/mL among different groups. After culturing for 12 hours at 37°C, typan blue (200 µg/mL) was added into the culture medium to quench the cytoplasmic fluorescent molecules. Then, cells were fixed by 2% glutaraldehyde at 4°C for 40 min and permeabilized by 0.2% Triton X-100 at 4°C for 2 min. After that, these cells were stained using 5 U/mL of rhodamine-phalloidin at 4°C for 24 hours and then with 10 µg/mL of Hoechst 33258 at room temperature for 10 min. Finally, the above cells were mounted using 90% glycerinum. The green fluorescence of internalized nanoparticles and the blue cell nuclei were imaged using CLSM. The intracellular distribution of nanoparticles was further investigated by ICP, the treated cells in each sample were collected by digestion and rinsed with PBS by centrifugation. Then each sample was added with lysis buffer (1% SDS, 1% Triton X100, 40 mM Tris acetate, 0.5 mL) for cell disintegration. NaOH (2 mL, 50%) was also added to decompose the PHPBNs. After 5 min of centrifuging at 1000 rpm, the Fe concentration in cells was measured by ICP on iCAP 6300 Duo. Assessment on Intracellular ATP Level. The ATP levels in various cell samples were detected for the evaluation of the intracellular ATP production after different treatments. Firstly, HepG2 cells or HL-7702 cells were seeded into a 6-well plate with an initial cell density of 2 × 104 cells/cm2. After the cell confluence reached about 70%, the cells were incubated with


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PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL) for 12 and 24 hours, respectively. For the hypoxia groups, the cells were incubated with GOx and PHPBNs-S-S-HA-PEG@GOx under 1% O2 condition for 12 and 24 hours. Subsequently, the cells were collected and counted to ensure that each sample comprises same amount of cells. Ultimately, the intracellular ATP content was measured following the user manual of ATP assay kit. The ATP level is measured by monitoring the fluorescence produced by firefly luciferase, which could catalyze luciferin to generate fluorescence in an ATP-consuming manner. The fluorescence intensity is proportional to the ATP concentration when firefly luciferase and luciferin were excessive, therefore allowing the quantification of ATP concentration with high sensitivity. Intracellular Western Blotting Characterization of HSP70 and HSP90. For the analysis of the expression level of HSP70 and HSP90, HepG2 cells were seeded into a 6-well plate with an initial cell density of 2 × 104 cells/cm2. After the cell confluence reached about 70%, the cells were treated with normal medium (blank control), hyperthermia at 40°C, PHPBNs-S-S-HA-PEG with NIR, PHPBNs-S-S-HA-PEG@GOx, PHPBNs-S-S-HA-PEG@GOx + hyperthermia at 40°C and PHPBNs-S-S-HA-PEG@GOx with NIR. Then Laemmli Sample Buffer (Bio-Rad) was used to lyse these cells and the total protein amount was quantified with a BCA protein assay Kit (Beyotime). The protein was loaded onto 12% SDS-containing polyacrylamide (SDS-PAGE) gel and the relevant proteins were extracted and transferred to a polyvinylidenedi-fluoride (PVDF) (Immobilon P, Millipore) membrane. After blocking with 5% skim milk, HSP70/HSP90 primary antibodies and secondary horseradish peroxidase-conjugated antibodies were utilized to probe the HSP70 and HSP90 levels, which were imaged with chemiluminescence and quantified by Molecular Imager Versa Doc MP 4000 System (Bio-Rad).


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In Vitro Cytotoxicity Assay. To investigate the in vitro toxicity of PHPBNs-S-S-HAPEG@GOx combined with NIR treatment to HepG2 cells, the original culture medium was removed and added with fresh ones containing PBS (control), PHPBNs-S-S-HA-PEG (115.1 µg/mL), PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL), PHPBNs-PEG (107.5 µg/mL), PHPBNsS-S-HA-PEG (115.1 µg/mL) and PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL), in which the net concentration of PHPBN substrate in all sample groups has been maintained at 100 µg/mL. The samples were further incubated for 24 hours. After that, PHPBN-PEG, PHPBN-S-S-HA-PEG and PHPBN-S-S-HA-PEG@GOx groups were irradiated using an 808 nm laser with a power density of 1 W/cm2 for 5 min. At last, the cell viability was determined using MTT Kit (Beyotime, C0009). Cell Live/Dead Staining Assessment. For the analysis of cell survival and death, HepG2 cells were seeded into the 35 mm × 12 mm CLSM cell culture dish (NEST Biotechnology Co., Ltd, China). After the cell confluence reached about 70%, the original culture medium was replaced by fresh one containing PBS (control), PHPBNs-S-S-HA-PEG (115.1 µg/mL), PHPBNs-S-SHA-PEG@GOx (119.8 µg/mL), PHPBNs-PEG (107.5 µg/mL), PHPBNs-S-S-HA-PEG (115.1 µg/mL) and PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL), in which the net concentration of PHPBN substrate in all sample groups has been maintained at 100 µg/mL. The samples were further incubated for 24 hours. After that, PHPBNs-PEG, PHPBNs-S-S-HA-PEG and PHPBNsS-S-HA-PEG@GOx groups were irradiated using an 808 nm laser under the 1 W/cm2 condition for 5 min. Then, these treated cells were stained with Calcein AM/Propidium Iodide (PI) for 15 min and analyzed using a confocal microscope (LSM 510 Metanlo, Zeiss Co., Germany). Flow Cytometry Characterization of Cell Apoptosis. To determine the cell apoptosis ratio (%) after treatment with PHPBNs-S-S-HA-PEG@GOx + NIR system, HepG2 cells were seeded


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into a 6-well plate at an initial cell density of 2 × 104 cells/cm2. After the cell confluence reached about 70%, the original culture medium was replaced by fresh ones containing PBS (control), PHPBNs-S-S-HA-PEG (115.1 µg/mL), PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL), PHPBNsPEG (107.5 µg/mL), PHPBNs-S-S-HA-PEG (115.1 µg/mL) and PHPBNs-S-S-HA-PEG@GOx (119.8 µg/mL), in which the net concentration of PHPBN substrate in all sample groups has been maintained at 100 µg/mL. The samples were further incubated for 24 hours. After that, PHPBNsPEG, PHPBNs-S-S-HA-PEG and PHPBNs-S-S-HA-PEG@GOx groups were irradiated using an 808 nm laser at a power density of 1 W/cm2 for 5 min. Then, these treated cells were collected by centrifugation at 1500 rpm and stained with the Annexin V/PI cell assay kit (Invitrogen) following the instructions. At last, the BD FACS Calibur Flow Cytometer was applied to determine the cell apoptosis in each sample. Animal Tumor Model. The nude mice (average weight 18.5 ± 0.8 g) were provided by Daping Hospital of The Third Military Medical University (Chongqing, China) for in vivo experiments. All animal experiments were conducted under the Animal Management Rules of the Ministry of Health of the People's Republic of China (Document No. 55, 2001) and the protocols of the Care and Use of Laboratory Animals of the Third Military Medical University. HepG2 cells (0.2 mL, 1.5×107 cells mL-1 in PBS) were injected into subcutaneous tissue in the back of the nude mice to develop the tumor model. Assessment on the Tumor Hemoglobin Oxygen Saturation, H2O2 Level and ATP Level. The intratumoral hemoglobin oxygen saturation (sO2) was monitored with a preclinical photoacoustic (PA) imaging instrument (VevoLAZR, VisualSonic Inc., Toronto). The PA/US images of tumors were obtained by ‘Oxyhemo’ mode. The PA signal was recorded at the wavelength of 750 and 850 nm. The hemoglobin oxygen saturation (sO2) was calculated with a


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rate of 1 Hz based on the received PA signal. The HepG2 tumor-bearing nude mice were anesthetized with 0.25% pentobarbital sodium. The hemoglobin oxygen saturation (sO2) images of PA/US were obtained immediately before and 2 hours after intratumoral injection of PBS (50 µL), PHPBNs-S-S-HA-PEG (50 µL, 0.92 mg/mL), GOx (50 µL, 0.0376 mg/mL) and PHPBNsS-S-HA-PEG@GOx (50 µL, 0.958 mg/mL) to monitor the intratumoral sO2, in which the net concentration of PHPBN substrate in all sample groups was maintained at 0.8 mg/mL. After the PA imaging, the nude mice were sacrificed and the tumors were extracted. The intratumoral H2O2 and ATP content were detected via H2O2 and ATP assay kit, respectively. Plasma Concentration Analysis of PHPBNs-S-S-HA-PEG@GOx by ICP. Six HepG2 bearing nude mice were prepared and then 50 µL of PHPBNs-S-S-HA-PEG@GOx (4.79 mg/mL) was injected through tail vein. At various time points, 50 µL of blood was obtained by the orbital sinus. Then, 0.5 mL of lysis buffer (1% SDS, 1% Triton X-100, 40 mM Tris acetate) were added to lyse the blood and 0.5 mL of NaOH (50%) was also added to resolve PHPBNs into Fe3+. The Fe content of each blood sample was determined by ICP assay with iCAP 6300 Duo after centrifugation. Time-Dependent Biodistribution Analysis of PHPBNs-S-S-HA-PEG@GOx by ICP. HepG2 bearing nude mice (six mice per group) were prepared and then 50 µL of PHPBNs-S-SHA-PEG@GOx (4.79 mg/mL) was injected into each mouse through tail vein. These nude mice were euthanized at various time points of 1h, 4h, 8h, 12h, 24h, 72h after injection and the primary organs (heart, liver, spleen, lungs, kidneys) and tumors were obtained. These tissues were lysed by 0.5 mL of lysis buffer (1% SDS, 1% Triton X-100, 40 mM Tris acetate) after trituration and then the internal PHPBNs were resolved into Fe3+ with 0.5 mL of NaOH (50%).


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The Fe content of each tissue sample was determined by ICP assay with iCAP 6300 Duo after centrifugation. In Vivo Photothermal Tumor Therapy. In this experiment, when the tumor size reached about 300 mm3, 42 HepG2 tumor-bearing nude mice were randomly divided into seven groups (each group comprises six mice). The treatment setup was: Group 1: PBS; Group 2: PBS + NIR; Group 3: PHPBNs-S-S-HA-PEG; Group 4: PHPBNs-S-S-HA-PEG@GOx; Group 5: PHPBNsPEG + NIR; Group 6: PHPBNs-S-S-HA-PEG + NIR; Group 7: PHPBNs-S-S-HA-PEG@GOx + NIR. 50 µL of PBS, PHPBNs-PEG (4.3 mg/mL), PHPBNs-S-S-HA-PEG (4.6 mg/mL) or PHPBNs-S-S-HA-PEG@GOx (4.79 mg/mL) was intravenously injected into each nude mice of different groups, respectively and the equivalent concentration of net PHPBNs was 4 mg/mL. After injection, the HepG2 tumor-bearing nude mice in the Group 2, Group 5, Group 6 and Group 7 were irritated by the 808-nm NIR laser (Hi-Tech Opto-electronics Co., Ltd. Beijing, China) at 1 W/cm2 for 5 min. During the illumination process, the temperature variation of tumor in different groups was recorded by an IR thermal camera (FLIR-Systems Corporation, P20, USA). All these tumor-bearing nude mice were treated every other day. The tumor volume and body weight were recorded and compared. The length and width of the tumors were measured with a digital caliper and the corresponding tumor volumes were calculated using the following formula: volume = width2× length/2. 21 days post treatment, the tumor and main organs of nude mice in different groups were collected and fixed by 10% formalin for two days under 4 oC. Subsequently, these fixed tissues were embedded with paraffin and then sectioned for further hematoxylin and eosin (H&E) staining. At last, these stained sections were imaged by an optical microscope. The relevant tumors were also frozen-sliced and immobilized into glass slides for further HSP90 and HSP 70 staining.


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Animal Survival. HepG2 tumor models were first established to study the nude mice survival after different treatments, when the tumor grew up to about 300 mm3, 42 HepG2 tumor-bearing nude mice were randomly divided into seven groups (six mice per group), and the setup was: Group 1: PBS; Group 2: PBS + NIR; Group 3: PHPBNs-S-S-HA-PEG; Group 4: PHPBNs-S-SHA-PEG@GOx; Group 5: PHPBNs-PEG + NIR; Group 6: PHPBNs-S-S-HA-PEG + NIR; Group 7: PHPBNs-S-S-HA-PEG@GOx + NIR. 50 µL of PBS, PHPBNs-PEG (4.3 mg/mL), PHPBNs-S-S-HA-PEG (4.6 mg/mL) or PHPBNs-S-S-HA-PEG@GOx (4.79 mg/mL) was intravenously injected into each nude mice of different groups, respectively and the equivalent concentration of net PHPBNs was 4 mg/mL. After injection, the HepG2 tumor-bearing nude mice in the Group 2, Group 5, Group 6 and Group 7 were radiated by the 808 nm NIR laser (HiTech Opto-electronics Co., Ltd. Beijing, China) with 1 W/cm2 for 5 min. All of these tumorbearing nude mice were treated every other day for the initial 21 days and the whole observation time is 60 days. During the whole observation period, the survival time of each nude mouse was recorded for subsequent analysis. Statistical Analysis. The statistical analysis was implemented with OriginPro software (version 9.0) via one-way analysis of variance (ANOVA) and Student’ s t-test and the confidence levels were set at 95% and 99%, and the results were shown as Mean ± SD.

ASSOCIATED CONTENT Supporting Information. Supporting Information is available from the ACS Publications Website or from the authors. The authors declare no competing financial interest. AUTHOR INFORMATION


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Funding Sources This work was financially supported by Natural Science Foundation of China (51602034 and 51603024), National Key R&D Program of China (2016YFC1100300), China Postdoctoral Scien ce Foundation (2017M612919) and Fundamental Research Funds for the Central Universities (10611CDJXZ238826). ACKNOWLEDGMENT The authors sincerely thank the Department of General Surgery in Third Military Medical University for providing the murine tumor models. REFERENCES 1.

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Scheme 1. Schematic presentation of the multifunctional porous hollow Prussian Blue nanoparticles (PHPBNs) for combined tumor starvation and enhanced low-temperature photothermal therapy against hypoxic tumors. (a) Synthesis scheme of PHPBNs-S-S-HAPEG@GOx. (b) Illustration of GOx induced starvation for enhanced low-temperature


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photothermal therapy in hypoxic tumor microenvironment. The hypoxia-induced photothermal resistance was circumvented via the PHPBN-mediated tumor reoxygenation.


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Figure 1. Representative SEM and TEM images of Prussian Blue nanoparticles (PBNs, a, a1), porous hollow Prussian Blue nanoparticles (PHPBNs, b, b1) and PHPBNs-S-S-HA-PEG@GOx (c, c1). SEM scale bar: 200 nm and TEM scale bar: 100 nm. (d) UV-vis-NIR absorption spectra of ICG and PHPBNs-S-S-HA-PEG solutions before and after exposure to an 808-nm laser at the power density of 1 W/cm2. (e) Photothermal temperature curves of PHPBNs-S-S-HAPEG@GOx dispersed in water at various concentrations of 0, 0.05, 0.1, 0.2 and 0.5 mg/mL. The power density of the 808-nm was 1 W/cm2 and the treated lasted 5 min.


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Figure 2. Representative CLSM images of HepG2 cells: (a) blank control, (b) samples stained by DCFH-DA after treatment of PBS, (c) PHPBNs-S-S-HA-PEG, (d) GOx, (e) PHPBNs-S-SHA-PEG@GOx. Green: positive staining of ROS. (f) Redox-responsive release profiles of GOx from PHPBNs-S-S-HA-PEG@GOx. (g) Flow cytometry analysis of intracellular DCF fluorescence intensity. (h) The changes in oxygen concentration for PHPBNs-S-S-HA-PEG + H2O2, PHPBNs-S-S-HA-PEG and H2O2 groups. (i) The oxygen concentration plotted against time in different solutions of glucose, GOx, glucose + GOx and glucose + GOx + PHPBNs-S-SHA-PEG. (n = 6, **p < 0.01)


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Figure 3. (a) ATP level in HepG2 cells and HL7702 cells treated with PHPBNs-S-S-HAPEG@GOx for 12 and 24 hours, respectively. The untreated cells were used as the control. (b) ATP level in HepG2 cells after treatment with GOx or PHPBNs-S-S-HA-PEG@GOx under


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hypoxic condition (1% O2) for 12 and 24 hours, respectively. The untreated cells were used as control. (c) Representative western blotting of HSP 90 and HSP 70 expression under different conditions: control group (I), incubation at 40°C (II), PHPBNs-S-S-HA-PEG with NIR (III), PHPBNs-S- S-HA-PEG@GOx (IV), PHPBNs-S-S-HA-PEG@GOx and incubation at 40°C (V), PHPBNs-S-S-HA-PEG@GOx with NIR (VI). Representative CLSM live/dead cell images of HepG2 cells stained by Calcein-AM/PI, the samples are blank control; PHPBNs-S-S-HA-PEG, PHPBNs-S-S-HA-PEG@GOx, PHPBNs-PEG + NIR, PHPBNs-S-S-HA-PEG + NIR and PHPBNs-S-S-HA-PEG@GOx + NIR groups, respectively. Scale bar: 100 µm. (n = 6, **p < 0.01)


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Figure 4. Representative PA images of HepG2 tumors regarding the oxyhemoglobin saturation in tumor tissue before and 2 hours after different treatment: PBS (a, a1), PHPBNs-S-S-HA-PEG (b, b1), GOx (c, c1), PHPBNs-S-S-HA-PEG@GOx (d, d1). (e) Quantitative analysis of the oxyhemoglobin saturation of HepG2 tumor treated with PBS, PHPBNs-S-S-HA-PEG, GOx, PHPBNs-S-S-HA-PEG@GOx. (f) H2O2 concentration in HepG2 tumor tissue after treatment with PBS, PHPBNs-S-S- HA-PEG, GOx and PHPBNs-S-S-HA-PEG@GOx. (g) Partial oxygen pressure (pO2) of HepG2 tumor in different sample groups: PBS, PHPBNs-S-S- HA-PEG, GOx


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and PHPBNs-S-S-HA-PEG@GOx. (h) ATP level in HepG2 tumor tissue after treatment with PBS, PHPBNs-S-S-HA-PEG, GOx, PHPBNs-S-S-HA-PEG@GOx. (n = 6, **p < 0.01)


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Figure 5. (a) IR thermographic images of HepG2 tumor-bearing mice after the exposure to an 808 nm laser at a power density of 1 W/cm2 in different groups including PBS + NIR (a-a5), PHPBNs-PEG + NIR (b-b5), PHPBNs-S-S-HA-PEG + NIR (c-c5), PHPBNs-S-S-HAPEG@GOx + NIR (c-c5), taken at different time points. (e) Representative photos of HepG2 tumor tissues obtained after treatment with PBS (I), PBS + NIR (II), PHPBNs-S-S-HA-PEG (III), PHPBNs-S-S-HA-PEG@GOx (IV), PHPBNs-PEG + NIR (V), PHPBNs-S-S-HA-PEG + NIR (VI), PHPBNs-S-S- HA-PEG@GOx + NIR (VII) for 21 days, respectively. (f) The in vivo tumor temperature changes plotted against time for PBS + NIR, PHPBNs-PEG + NIR, PHPBNsS-S-HA-PEG + NIR and PHPBNs-S-S-HA-PEG@GOx + NIR groups, the readings were recorded with an IR thermal camera. (g) The changes in HepG2 tumor volumes monitored every other day. (n = 6, **p < 0.01)


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Figure 6. Representative photos of HepG2 tumor tissue sections extracted at the 21st day after treatment by PBS (a), PBS + NIR (b), PHPBNs-S-S-HA-PEG (c), PHPBNs-S-S-HA-PEG@GOx (d), PHPBNs-PEG + NIR (e), PHPBNs-S-S-HA-PEG + NIR (f), PHPBNs-S-S-HA-PEG@GOx + NIR (g) for histological apoptosis observation stained by hematoxylin and eosin (H&E). Black


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arrows indicate the shrunken cells and dissolved nuclei. The scale bar is 20 µm. Representative CLSM immunofluorescence images of HepG2 tumor slices stained with anti-HSP 90 and antiHSP 70 antibody after treatment with PBS (a1-a3), PBS + NIR (b1-b3), PHPBNs-S-S-HA-PEG (c1-c3), PHPBNs-S-S-HA-PEG@GOx (d1-d3), PHPBNs-PEG + NIR (e1-e3), PHPBNs-S-SHA-PEG + NIR (f1-f3) and PHPBNs-S-S-HA-PEG@GOx + NIR (g1-g3) for 3 weeks. Blue: cell nucleus. Green: HSP 90 expressing region. Red : HSP 70 protein expressing region. The scale bar is 30 µm. Quantitative fluorescence analysis of the HSP90 (h) and HSP 70 (i) expression. (j) The weights of tumors after various treatment. (k) Survival rate of HepG2 tumor-bearing mice in different treatment groups for 60 days, respectively. (n = 6, **p < 0.01)


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