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Near-Infrared-Triggered in situ Gelation System for Repeatedly Enhanced Photothermal-Brachytherapy with a Single Dose Zhouqi Meng, Yu Chao, Xuanfang Zhou, Chao Liang, Jingjing Liu, Rui Zhang, Liang Cheng, Kai Yang, Wei Pan, Meifang Zhu, and Zhuang Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04544 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Near-Infrared-Triggered

in

situ

Gelation

System

for

Repeatedly

Enhanced

Photothermal-Brachytherapy with a Single Dose Zhouqi Meng†, Yu Chao†, Xuanfang Zhou†, Chao Liang†, Jingjing Liu†, Rui Zhang†, Liang Cheng†, Kai Yang‡, Wei Pan#, Meifang Zhu§, Zhuang Liu†,* †

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu, 215123, China. ‡ State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection & School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, Jiangsu 215123, China. § State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. # College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China. Email: [email protected] ABSTRACT Brachytherapy by placing therapeutic radioactive materials into or nearby tumors has been widely used in the clinic for cancer treatment. The efficacy of brachytherapy, however, may often be limited by the radiation resistance for tumor cells located in the hypoxic region of a solid tumor, as well as the non-optimal distribution of radioactivity inside the tumor. Herein, a hybrid hydrogel system is developed by using 131I-labeled copper sulfide (CuS/131I) nanoparticles as the photothermal-/radiotherapeutic agent, poly(ethylene glycol) double acrylates (PEGDA) as the polymeric matrix, and 2, 2’-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (AIPH) as the thermal initiator, to realize light-induced in situ gelation in the tumor for the combined photothermal-brachytherapy. After local injection, CuS/131I nanoparticles under irradiation by the 915-nm near-infrared (NIR) laser would produce heat to 1 ACS Paragon Plus Environment

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mildly raise the tumor temperature and initiate the polymerization of PEGDA by activating the AIPH thermal initiator, effectively fixing CuS/131I by in situ gelation within the tumor for long-term. By repeated NIR irradiation of tumors, the tumor hypoxia could be relieved for a much longer term, resulting in a significant synergistic photothermal-brachytherapeutic effect to

eliminate

tumors.

This

work

presents an

efficient type

of

NIR-responsive

nanoparticle-encapsulated hydrogel system, inspiring the design of the form of brachytherapy.

KEYWORDS: in situ gelation, nanoparticles, NIR photothermal treatment, brachytherapy, tumor hypoxia relief.

Brachytherapy is a form of radiation therapy by placing radioactive sources within or in the direct vicinity of the tumor, thus enabling the localized destruction of tumors.1-4 During brachytherapy, oxygen radicals are often generated to attack biomolecules such as DNA and then kill tumor cells.2, 5, 6 Therefore, similar to that in external-beam radiation therapy, oxygen also plays a critical role in enhancing the brachytherapy-induced cancer cell killing.7 However, owing to the hypoxic nature inside the tumor microenvironment, tumor cells with insufficient oxygen are known to be much more resistant to ionizing radiation than normal cells, leading to the resistance or failure of brachytherapy to treat certain types of cancers.8-10 Moreover, the local implantation of radioactive sources (e.g.

125

I beads) in some clinical cases may not be

able to offer homogenous dose distribution to the entire tumor, due to the large tumor sizes and sometimes the movement of the radioactive source in certain body organs (e.g. for

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treatment of intestinal cancers), leading to the possible steep dose gradient within tumors and ineffective tumor treatment.11, 12 Hydrogels have attracted tremendous attention on account of their ability to encapsulate bioactive substances by simply mixing them in precursor liquids, which are then transformed into cross-linked hydrogel networks at the targeted lesion (e.g. the tumor) to allow long-term local retention of therapeutic agents with sustained drug release.13-18 We thus hypothesize that radioisotope-encapsulated hydrogels that can undergo sol-to-gel transformation within the tumor may be an attractive approach for easy-to-operate brachytherapy. There have been different ways to trigger in situ formation of hydrogels within the body after injection of liquid solution, such as light,19-22 redox,23-25 and specific enzymes.26,

27

Among those

strategies, the light-induced gelation method by using photo-initiator to trigger polymerization is attractive as it can be controlled with great temporal and spatial precision.28-30 Unfortunately, most of photoinitiator are excited by short-wavelength light sources with limited tissue penetration depth.31-33 To realize an efficient form of brachytherapy, it would thus be interesting the develop near-infrared (NIR) light activatable hydrogel systems, which are able to homogenously restrain therapeutic radioisotopes in the tumor for long-term in a highly

controllable

manner,

and

hopefully

in

the

meanwhile

could

overcome

hypoxia-associated radiation resistance for solid tumors.34 To realize above aims for improving the brachytherapy, herein, we have designed a NIR-triggered in situ hybrid hydrogel system consisting 131I-labeled copper sulfide (CuS/131I) nanoparticles (NPs) as the photothermal-/radiotherapeutic agents, poly(ethylene glycol) double acrylates (PEGDA) as a polymeric matrix, and the 2, 2’-azobis[2-(2-imidazolin-2-yl) 3 ACS Paragon Plus Environment

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propane] dihydrochloride (AIPH) as the thermal initiator. Notably, PEGDA has been found to be a biodegradable polymer based on previous reports,35,

36

although its exact in vivo

degradation behaviors have not been studied in the current work. After local injection of the precursor solution into tumors, photoacoustic imaging is employed to track the intra-tumor diffusion of CuS/131I NPs. At the best timing post injection (p.i.) with whole-tumor homogenous distribution of photoacoustic signals, the tumors on mice were exposed to a 915-nm NIR laser, which could induce photothermal heating of CuS/131I NPs to mildly raise the tumor temperature and initiate the polymerization of PEGDA by activating the AIPH thermal initiator (Figure 1). With in situ gelation, CuS/131I NPs are then fixed within the tumor for long-term without significant leakage into normal organs. Meanwhile, the photothermal heating could result in effective relief of tumor hypoxia by promoting tumor blood flow. Interestingly, repeated NIR irradiation of tumors after a single dose injection of the hybrid solution could lead to tumor hypoxia relief over a much longer term, offering an excellent

synergistic

treatment

response

to

eliminate

tumors

with

such

photothermal-brachytherapy, by “one injection, multiple treatments”.

RESULTS AND DISCUSSION In our work, hydrophilic CuS-PEGDA NPs were firstly prepared in the aqueous solution by the in situ co-precipitation method as described earlier, simply through mixing copper chloride (CuCl2) and sodium sulfide (Na2S) in the PEGDA solution upon stirring at 80 °C.37, 38

As observed under transmission electron microscope (TEM) (Figure 2a-b), multiple CuS

NPs with the individual diameter of 5-10 nm were encapsulated by PEGDA to form clusters 4 ACS Paragon Plus Environment

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with the size of 50-70 nm in the formed CuS-PEGDA sample. This should be attributed to the fact that the PEGDA could simultaneously serve as the nanoreactor, ligands and the polymeric monomers.37, 39-41 Moreover, the high-resolution TEM (HRTEM) image (Figure 2c) indicated the clear lattice fringes of CuS, suggesting the single crystal structure of CuS NPs synthesized by this method. The inter-planar d spacing was determined to be 2.81 Å, agreeing with the (103) lattice fringes of hexagonal structured CuS crystal.37 The as-made CuS-PEGDA NPs could be well dispersed in water with green color when the monomer content exceeded 15% (w/v) (Supporting Figure S1a, inset), showing no obvious agglomeration after being stored for several weeks (Figure 2d, Supporting Figure S1a and S2, inset). As observed by the UV-Vis-NIR absorbance spectra (Supporting Figure S1), the obtained CuS-PEGDA aqueous dispersions showed enhancing optical absorption as the wavelength increases from 550 to 950 nm, similar to the absorption spectrum of citrate-stabilized CuS nanoparticles (CuS-Cit NPs) prepared by the conventional aqueous phase method in the presence of citrate (Figure 2d).42 Due to the strong absorption of CuS-PEGDA in the NIR region, photoacoustic (PA) imaging of CuS-PEGDA dispersions was carried out (Figure 2e). Visibly, the PA signal intensity went up linearly as the increase of Cu concentrations (Cu concentration: 0.8-4 mM) for the CuS-PEGDA samples. Subsequently, the photothermal conversion performance of CuS-PEGDA NPs was evaluated by monitoring the temperature changes of CuS-PEGDA samples with different concentrations (Cu concentration: 32-160 ppm) under the irradiation of a 915-nm laser at the power intensity of 2.0 W cm−2 (Figure 2f). Apparent concentration-dependent photothermal heating effect was

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observed for CuS-PEGDA NPs, which in the meanwhile exhibited great photothermal stability after five cycles of NIR laser irradiation (Supporting Figure S3). To realize the controllable in situ gelation, an efficient and biocompatible polymerization condition should be developed. Here, we selected the precursor solution consisting of CuS-PEGDA (20% w/v) and AIPH (0.2% w/v) for remotely controlled polymerization triggered by the NIR laser. To investigate the gelation behavior of this system, the rheological analysis for the precursor solution with different temperatures was performed (Figure 2g). When the precursor solution was kept at the body temperature of 37 °C, the storage modulus (G’) showed no significant change, suggesting that the polymerization reaction would not occur at this temperature. In contrast, when the sample was placed at 43 °C, the sample modulus showed a rapid increase after 13 min and reached the equilibrium later, indicating the gelation of this sample occurred effectively at 43 °C, a temperature that is above the body temperature but still tolerable for biological tissues.29, 43 Furthermore, as vividly illustrated by the photographs shown in Figure 2h, effective gelation of CuS-PEGDA could be observed by heating the sample to 43 °C under the NIR laser in the presence of AIPH. Photothermal heating of the CuS-PEGDA with AIPH sample to 37 °C, or the CuS-PEGDA sample without AIPH to 43 °C, failed to induce gelation. Moreover, we confirmed that CuS nanoparticles and gelation system would not be notable destroyed or changed after repeated NIR laser irradiation (Supporting Figure S4 and S5). Furthermore, the polymerization of CuS-PEGDA could also be proved by the FTIR spectra (Supporting Figure S6). Our results collectively suggest that NIR photothermal heating of CuS to the temperature only 6 °C above the body

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temperature would be sufficient for AIPH activation to induce polymerization of PEGDA and the subsequent gelation of CuS-PEGDA. In our previous work, we have demonstrated that CuS NPs could be labeled by the radionuclide

131

I by doping into the nano-crystals during their growth.44 Considering the

strong binding between Cu2+ and I- ions, herein we developed an even simpler method for iodide labeling of CuS NPs by simply mixing CuS-PEGDA with sodium iodide (NaI) to form the CuS/I-PEGDA. X-ray photoelectron spectroscopy (XPS) confirmed the existence of iodine in our prepared CuS/I-PEGDA NPs (Supporting Figure S7). Moreover, the energy dispersive X-ray spectroscopy (EDS) element mapping of nanoparticles (Supporting Figure S8) demonstrated that Cu, S and I, the three elements were distributed uniformly inside our obtained CuS/I-PEGDA NPs. This phenomenon could be attributed to the binding of I- ions at the defect sites on the surface of CuS NPs.45 Moreover, the formed CuS/I-PEGDA hydrogel showed similar absorption spectrum to that of CuS-PEGDA aqueous dispersion (Supporting Figure S9). Following, we labeled CuS-PEGDA NPs using radionuclide

131

I in the form of

Na131I. The radiolabeling yield reached to 41.5 ± 0.2 % after mixing CuS-PEGDA NPs with Na131I at 37 °C under constant stirring for an hour. At higher labeling temperature of 43 °C or 60 °C, as much as 46.5±0.8% or 57.0 ± 0.6 % of added

131

I was labeled onto CuS/I-PEGDA

NPs, respectively (Figure 2i).46, 47 Notably, only a minimal amount of 131I was detached from the obtained CuS/131I-PEGDA NPs after incubation in mouse plasma at 37 °C for 7 days (Figure 2j), illustrating that the excellent radiolabeling stability of CuS/131I-PEGDA. Next, we carried out in vitro studies with radiolabeled nanoparticles. 4T1 murine breast cancer cells were cultured with various concentrations of CuS-PEGDA or CuS/131I-PEGDA 7 ACS Paragon Plus Environment

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for 24 h. No significant cytotoxicity was found for CuS-PEGDA NPs in our tested concentration range (Supporting Figure S10), while radiolabeled CuS/131I-PEGDA showed obvious dose-dependent cancer cell killing ability, demonstrating the possibility of using those nanoparticles for brachytherapy. The in vitro combined photothermal-brachytherapy with CuS/131I-PEGDA was then evaluated. As evidenced by both CCK-8 cell viability assay and micrographs of calcein AM/propidium iodide co-stained cells, the combined photothermal-brachytherapy (CuS/131I-PEGDA with laser treatment) showed much stronger cancer cell destruction efficacy compared to brachytherapy alone (CuS/131I-PEGDA without laser) or photothermal therapy alone (CuS-PEGDA with laser) (Supporting Figure S11), achieving an obvious synergistic effect in such combination therapy. Nanoparticles after local administration into the tumor would undergo gradual dispersion by diffusion in the interstitial fluid.48 To achieve the optimal brachytherapy, it is hoped that radiolabeled nanoparticles could be firstly dispersed throughout the whole tumor and then fixed (e.g. by gelation) to prevent their further leaking out to normal tissues. In that sense, the light-triggered gelation should occur at the best timing (Figure 3a). Therefore, in our work, by ultizing the strong NIR absorbance of CuS, we firstly employed photoacoustic (PA) imaging to track the time-dependent intra-tumoral distribution of CuS NPs post local injection. The CuS-PEGDA/AIPH solution (CuS-PEGDA 20% w/v, AIPH 0.2% w/v) was injected into 4T1 tumors on Balb/c mice. As revealed by PA imaging data (Figure 3b, c), the PA signals from CuS showed almost homogenous dispersion throughout the entire tumor at 10 min p.i.. At later time points, however, the absolute PA signals in the tumor showed slight decrease,

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indicating the possible leakage of CuS NPs into surrounding tissues nearby the tumor (e.g. by lymphatic drainage). As the best timing with whole-tumor homogenous distribution of CuS-PEGDA NPs (10 min p.i.), we then evaluated the possibility of using the in vivo photothermal heating of CuS to initiate the polymerization of PEGDA and trigger the subsequent in situ gelation. Balb/c mice bearing subcutaneous 4T1 tumors were intratumorally (i.t.) injected with the precursor solution containing CuS-PEGDA (20% w/v) and AIPH (0.2% w/v). At 10 min p.i., those tumors were exposed to the 915-nm laser with the power density of 1.0 W cm-2 for 12 min. An infrared (IR) thermal camera was employed to monitor the temperature evolution (Figure 3d, e). As expected, under the irradiation of the laser, tumors injected with CuS-PEGDA/AIPH could be heated with the temperature maintaining at 43 °C, while those injected with PBS showed little temperature increase by less than 3 °C (Figure 3d, e). Next, we ought to investigate whether photothermal heating of tumors by the external NIR laser would be able to trigger gelation of the CuS-PEGDA/AIPH system within the tumor. Unlike free nanoparticles that may diffuse into surround tissues, we hypothesize that nanoparticles within the hydrogel would be fixed within the formed gel without much leakage to the surrounding environment. Therefore, in vivo gamma imaging was employed to track the whole body distribution of CuS/131I-PEGDA NPs (Figure 4a, b). Mice bearing 4T1 tumors were i.t. injected with CuS/131I-PEGDA/AIPH and exposed under the NIR laser for photothermal heating at 43 oC (CuS/131I-PEGDA/AIPH, NIR+), and then imaged by a small animal gamma imaging system. For comparison, tumor-bearing mice injected with the free 131

I, CuS/131I, CuS/131I-PEGDA NPs (no AIPH) with/without laser irradiation and 9 ACS Paragon Plus Environment

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CuS/131I-PEGDA/AIPH in the absence of NIR laser irradiation were also investigated under the other identical conditions. It could be clearly found that when CuS/131I NPs or CuS/131I-PEGDA NPs were injected into the tumor, those nanoparticles would be gradually migrated into other organs with little tumor retention at 96 h p.i., regardless of NIR laser irradiation. The tumor retention of CuS/131I-PEGDA/AIPH in the absence of NIR laser exposure was also found to be rather low at later time points (e.g. at 96 h p.i.). In marked contrast, for tumors injected with CuS/131I-PEGDA/AIPH and exposed to the NIR laser, much higher retention of radioactivity was found in the tumor, with minimal leaking out into other parts of the mouse body. Obviously, predominate tumor retention of radioactivity was found even at 192 h p.i. for this group. The quantitative ex vivo biodistribution study was further carried out at different post i.t. injection of different formulations (Figure 4c, d and Supporting Figure S12). It was found that for the CuS/131I-PEGDA/AIPH (NIR+) group, 82.8% of injected radioactivity retained within the tumor at the 24 h p.i. (combined with the radioactive disintegration), with little radioactivities observed in other organs. Even at 96 h and 192 h p.i., the majorities of radioactivity, 79.1 % and 62.8 % of the injection dose (corrected by the decay half-life), respectively, were found to be still retained within the tumor (Supporting Figure S12). In contrast, much lower tumor retention and significant distribution of radioactivities into other organs were observed for other control groups without in situ gelation to fix those radiolabeled nanoparticles (Figure 4d). Free

131

I, on the other hand, would be quickly

excreted without much retention in the tumor and all other organs at 24 h p.i. Therefore, our results confirmed that the NIR laser triggered in situ gelation of the CuS/131I-PEGDA/AIPH 10 ACS Paragon Plus Environment

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system could effectively withhold CuS/131I nanoparticles within the tumor for long term, favorable for more efficient brachytherapy of tumors. With

long-term

tumor

retention,

such

CuS/131I-PEGDA/AIPH

system

after

NIR-triggered in situ gelation within the tumor may be repeatedly heated up under NIR laser irradiation with a single injection of nanoparticles. CuS NPs or CuS-PEGDA/AIPH were injected into the 4T1 tumors on mice, which were then exposed to the 915-nm laser with the power density of 1.0 W cm-2 at 10 min, 48 h and 96 h p.i. (20 min for the 1st, 10 min for the 2nd, and 3rd round of irradiation), as illustrated as Figure 5a. The IR thermal camera was used to monitor the temperature evaluation of tumors, under every round irradiation at 10 min, 48 h and 96 h p.i. (Figure 5b, c). Meanwhile, tumors injected with PBS were also irradiated and imaged under the other identical conditions for comparison. After the 1st round of laser irradiation conducted at 10 min p.i., similar tumor temperature increases to 42-43 °C were observed for tumors injected with two formulations of CuS (Figure 5c). Owing to the gradual diffusion of CuS NPs out from tumors, much weaker photothermal heating effects were observed for tumors injected with CuS NPs at later rounds of NIR laser irradiation (48 and 96 h p.i.). In contrast, consistently strong photothermal heating could be achieved for tumors injected with CuS-PEGDA/AIPH at later rounds of NIR irradiation even at 96 h p.i., on account of the effective long-term retention of CuS in this system after NIR-triggered in situ gelation in the first round. For the brachytherapy, it is known that the tumor damaging effect of ionizing radiation would be much more effective under well-oxygenated conditions compared to that for hypoxic tumors.49, 50 Previous reports have demonstrated that mild local hyperthermia could 11 ACS Paragon Plus Environment

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efficiently relieve tumor hypoxia by promoting intra-tumoral blood flow.7, 51 To investigate such effect, 4T1 tumors on mice were i.t. injected with CuS-PEGDA/AIPH and irradiated by the NIR laser (1.0 W cm-2 for 20 min) at 10 min p.i. Then the tumors were collected at 0 h, 1 h, 4 h, 8 h and 24 h post laser irradiation for immunofluorescence staining using pimonidazole-based hypoxyprobe to examine the tumor hypoxia status (Supporting Figure S13). Due to the local hyperthermia effect to promote intra-tumor blood flow, the tumor hypoxia signals were obviously weakened within the first few hours after NIR-triggered photothermal heating. However, the hypoxia status within the irradiated tumor would be gradually recovered later on, and reached to its initial status at 24 h post laser irradiation. Therefore, we hypothesize that repeated hypothermia heating of tumors may be necessary for improving brachytherapy via tumor hypoxia relief. Inspired by the abovementioned findings, in our experiments, 4T1 tumors on mice were i.t. injected with CuS-PEGDA/AIPH and irradiated by the NIR laser at 10 min, 48 h, and 96 h p.i., and collected after the laser irradiation (1st round at 2 h p.i.) for immunofluorescence staining using pimonidazole-based hypoxyprobe to examine the tumor hypoxia status (Figure 5d, Supporting Figure S14). Tumors with one a single round of NIR photothermal heating at 10 min p.i. were collected at the same time points for comparison. As expected, tumors treated with CuS-PEGDA/AIPH showed greatly weakened hypoxic signals after each round of NIR laser irradiation (Figure 5d, e). In addition, we found that the hypoxia signals were greatly weakened particularly for regions nearby the blood vessels identified by anti-CD31 staining (red), for tumors post NIR photothermal heating, indicating that the mild hyperthermia effect could improve tumor oxygenation by enhancing blood flow into the 12 ACS Paragon Plus Environment

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tumor. However, for tumors with only one round of NIR laser irradiation, their hypoxic status would be recovered at later time points. Therefore, repeated photothermal heating would lead to cycled tumor hypoxia relief, which may be preferred for enhancing brachytherapy. Based on the excellent performance of PEGDA hydrogel for long-term tumor retention of radiolabeled CuS/131I, and the possibility of using NIR laser to trigger repeated photothermal heating and tumor hypoxia relief in this system, the CuS/131I-PEGDA/AIPH system may be employed for photothermal-brachytherapy by “one injection, multiple treatment”. To further verify this conception, BLAB/c mice bearing 4T1 tumors were i.t. injected with 25 µL of the CuS/131I NPs or CuS/131I-PEGDA/AIPH solution, with either single NIR laser irradiation (irradiation for a single time at 10 min p.i.) or multiple laser irradiation (irradiation 3 times at the 10 min, 2 d, and 4 d p.i.) (Figure 5a). For systematic comparison,

tumors

were

i.t.

injected

with

the

non-radioactive

CuS

NPs

or

CuS-PEGDA/AIPH for multiple NIR irradiations under the otherwise identical conditions. For the mild photothermal treatment alone by either CuS NPs or CuS-PEGDA/AIPH, although the tumors showed partially delayed growth after multiple rounds of NIR irradiation, they grew up rapidly later on (Figure 6a), indicating that the mild PTT treatment alone at this temperature would not be able to effectively ablate tumors. Correspondingly, mice from these groups had short life spans of only 20-24 days after treatments (Figure 6b). In the meanwhile, tumors on mice after the combined mild photothermal-brachytherapy by CuS/131I NPs or CuS/131I-PEGDA/AIPH with a single round of NIR laser treatment, as well as after treatment by CuS/131I-PEGDA/AIPH with multiple rounds of NIR laser irradiation, although showed delayed growth, also could not be effectively suppressed at later time points. Apparently, it 13 ACS Paragon Plus Environment

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was found that for mice with tumors injected by CuS/131I-PEGDA/AIPH and exposed to three rounds of NIR laser irradiation, their tumors rapidly shrunk and finally disappeared in ~10 days (Figure 6a). Additionally, mice in this group (CuS/131I-PEGDA/AIPH, multiple NIR+) survived for over 60 days without a single death, while those in all other control groups all died in ~26 days post various treatments (Figure 6b). Such a significant therapeutic effect achieved with CuS/131I-PEGDA/AIPH (multiple NIR+) should be attributed not only to long-term tumor retention of radioactivity after NIR-triggered in situ gelation, but also to the multiple rounds of NIR-triggered tumor hypoxia relief to further enhance the efficacy of radioisotope 131I to destruct tumor cells. Histological examination was then carried out for tumors collected at the 7th day post various treatments (Figure 6c). Micrographs of hematoxylin/eosin (H&E) stained tumor slices revealed the highest degree of tumor cell damages for tumors after the combined photothermal-brachytherapy treated with CuS/131I-PEGDA/AIPH and multiple rounds of NIR irradiations. Consistent to the tumor growth data, partially tumor damages were found in the other control treatment groups. Furthermore, as a result of the long-term tumor retention of radioactivity by in situ gelation with CuS/131I-PEGDA hydrogel (Figure 4a-d), no appreciable systemic toxic effect to the treated animals was noticed, as revealed by the mouse body weight data and histology analysis of major organs collected 10 days after various treatments (Supporting Figure S15, S16).

CONCLUSION

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In summary, we have developed an efficient type of NIR-activated hybrid hydrogel system to enable photothermal-brachytherapy of tumors. In this system CuS NPs synthesized with PEGDA as the stabilizer could be labeled with

131

I upon simple mixing with high

stability. With AIPH added as the thermo-initiator, the CuS/131I-PEGDA/AIPH solution upon NIR-induced photothermal heating could be rapidly transformed into a gel owing to the polymerization

of

PEGDA

triggered

by thermally

activated

AIPH.

After

such

CuS/131I-PEGDA/AIPH solution was injected into the tumor, we could use a 915-nm NIR laser to irradiate the tumor at the best timing with whole-tumor homogenous distribution of nanoparticles. Thereafter, the NIR-triggered gelation of PEGDA could allow long-term local retention of CuS/131I within the tumor so as to avoid the leakage of radioactivity into normal organs. Meanwhile, the long-term tumor retention of CuS/131I-PEGDA would enable repeated NIR-triggered photothermal heating of tumors by means of ‘one injection, multiple treatments’, which resulted in cycled tumor hypoxia relief to further enhance tumor killing, achieving

therapeutic

effects

far

better

than

that

observed

with

conventional

nanoparticle-based treatment (e.g. with CuS/131I). Notably, although single treatment of PTT-brachytherapy with a high temperature can improve the PTT performance, PTT at higher temperatures in certain cases may not be practical considering the limited light penetration depth and unwanted heating damages to surrounding normal tissues. Therefore, such a NIR responsive hybrid hydrogel system would allow remotely controlled in vivo gelation with great temporal and spatial precision, promising not only for local brachytherapy with enhanced efficacy and reduced side effects, but also for controlled release of other types of therapeutics to potentially facilitate disease treatment and tissue engineering. 15 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Materials. Poly (ethylene glycol) methyl ether acrylate (PEGDA, Mn=700), copper (II) chloride dihydrate (CuCl2·2H2O, >99%), sodium sulfide nonahydrate (Na2S·9H2O), sodium iodide (NaI) and 2,2’-azobis [2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) were purchased from Sigma-Aldrich Chemical Co., Ltd.. All reagents (analytical grade) were used as received without further purification. Ultrapure water was obtained from a Milli-Q system (Health Force Bio-meditech Holdings Ltd.). Synthesis of CuS/131I-PEGDA. Firstly, CuS-PEGDA NPs were prepared by a literature procedure.41, 44 Briefly, CuCl2·2H2O (0.04 mmol) and PEGDA (20% w/v) were dispersed into an aqueous solution under constant stirring, and then 0.04 mmol of Na2S·9H2O was added into the above dispersion under magnetic stirring to form a homogeneous solution. The reaction mixture was heated to 80 °C for 20 min and then cooled to room temperature naturally. Finally, the obtained CuS-PEGDA NPs solution was stored at 4 °C for further polymerization. For radiolabeling, Na131I was mixed with CuS-PEGDA NPs under the magnetic stirring for 1 h at different temperatures. After purification by centrifuge filtration (MWCO=10KDa filters) to remove free

131

I, the radioactivity of CuS/131I-PEGDA was detected by the gamma

counter. Meanwhile, CuS/I-PEGDA NPs were also prepared by replacing the Na131I with the cold NaI (0.03 mmol) under other identical condition. After that, 0.2% AIPH (w/v) was added into the CuS/131I-PEGDA solution to form the precursor solution.

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Synthesis of PEGDA based hydrogels. The precursor solution of CuS-PEGDA/AIPH or CuS/I-PEGDA/AIPH (CuS-PEGDA or CuS/I-PEGDA 20% w/v, AIPH 0.2% w/v) was exposed to the 915-nm laser irradiation for 20 min (1.0 W cm−2, maintaining at 43 °C). Characterization. The size and morphology of nanoparticles were characterized by using the TEM (JEM-2100F, JEOL) and a high-resolution scanning transmission electron microscopy (STEM). UV-Vis-NIR absorption spectra were determined by PerkinElmer UV-Vis spectrophotometer. The contents of copper ions in those samples was determined by using inductively-coupled plasma atomic-emission spectroscopy (ICP-AES, Thermo). The photoacoustic signals were obtained by PA imaging system (FujiFilm VisualSonics Inc.) utilizing the PA mode (915-nm). The rheological properties of the solutions or gels were measured using a rotational rheometer (Haake Rheo Stress 6000, Germany) (PP20 H, gap set at 0.5 mm, frequency=1 Hz) at 37 °C and 43 °C. NIR photothermal heating. The solution of CuS-PEGDA NPs with various concentrations in water were irradiated using a 915-nm semiconductor laser device (Xi’an Tours Radium Hirsh Laser Technology Co., Ltd., China) for 5 min (2.0 W cm-2). The temperature was monitored by the photothermal therapy-monitoring system FLIR-A300 (FLIR Systems Inc., USA). The radio-stability examination. 50 µL of K131I was mixed with 450 µL of CuS/131I-PEGDA NPs (20% w/v) at 37 °C for 1 h. After removal of free

131

I by centrifuge

filtration (MWCO=10KDa filters), the remained radioactivity was measured by gamma counting. Cell Experiments 17 ACS Paragon Plus Environment

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The 4T1 murine breast cancer cell were grown in RMPI 1640 culture medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator under 5% CO2, at 37 °C. Cytotoxicity of CuS-PEGDA and CuS/131I-PEGDA NPs were tested in vitro by the CCK-8 cytotoxicity assay (Dojindo) following the standard procedure. Six parallel wells were done for each group. In vitro photothermal-brachytherapy. To directly demonstrate the therapeutic efficacy, the adherent 4T1 cells (96-well plates) were incubated with 100 µL 1640 culture medium containing Free 131I (10 µCi mL-1), CuS-PEGDA (3 µg mL-1), or CuS/131I-PEGDA (3 µg mL-1 of CuS-PEGDA corresponding to 10 µCi mL-1 of

131

I) NPs, and the medium only with 1640

culture medium as control. Then, samples containing the CuS-PEGDA or CuS/131I-PEGDA were also irradiated by 915-nm laser (1.0 W cm-2, 43 °C) for 5 min. The CCK-8 cell viability assay was then conducted. More than three parallel wells were done for each group. Meanwhile, the cell after different treatments were washed with PBS, and co-stained with calcein AM/propidium iodide (PI) for 15 min to analyze the live cells (green) and dead cells (red), according to the manufacturer’s instructions. Then cells were imaged by a confocal fluorescence microscope (Leica TCS-SP5II, Germany). Three replicates were done for each treatment group. Animal Experiments Animal’s experiments were carried out according to the Guidelines for institutional committee for animal use and care regulations. BALB/c nude mice (6 weeks old, 15-20 g, male) were obtained from Soochow University Laboratory Animal Center and used under appropriate protocols. 4T1 cells (2×106/mouse) were injected subcutaneously into the 18 ACS Paragon Plus Environment

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backside of each mouse to prepare tumor-bearing mice. The tumor can be used for experiments, when the surface diameter achieved to 5-6 mm. Multimodal imaging PA imaging. The 4T1 tumor-bearing mice were i.t. injected with 25 µL CuS-PEGDA/AIPH (CuS-PEGDA 20%, AIPH 0.2%, w/v, the content of CuS with 0.08 mmol mL-1) solution. Then the tumor region was imaged with an excitation wavelength at 915-nm at different time intervals p.i. (Visualsonic Vevo 2100 LAZER). IR thermal imaging. Tumor-bearing mice were injected with PBS, CuS NPs 9.5 mg kg-1), CuS-PEGDA NPs (20% w/v, the same content of CuS with CuS group) and CuS-PEGDA/AIPH (CuS-PEGDA 20%, AIPH 0.2%, w/v, the same content of CuS with CuS group) groups (n=3) were exposed to 915-nm laser with 1.0 W cm-2 for 20 min or 10 min (maintaining at 43 °C) at 10 min, 48 h, 96 h p.i.. During this process, the tumor temperature was monitored by the IR thermal camera. Gamma imaging. Mice bearing 4T1 tumors were injected with free mouse), CuS/131I (9.5 mg kg-1,

131

131

I (50 µCi per

I dose = 50 µCi per mouse), CuS/131I-PEGDA/AIPH

(CuS/131I-PEGDA 20% w/v, AIPH 0.2%, 131I dose = 50 µCi per mouse). Tumors from the CuS-PEGDA/131I/AIPH group were irradiated by the 915-nm laser for 20 min for gelation at 10 min p.i.. Then mice were imaged by an in vivo animal imaging system (Kodak, FX Pro) at different time points to record the distribution of radioactivity signals. In vivo cancer therapy For in vivo cancer therapy, the mice were randomly distributed into several groups for intratumoral injection of various agents: PBS, CuS/131I (0.08 mmol mL-1, 19 ACS Paragon Plus Environment

131

I dose = 50 µCi

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per mouse), CuS NPs (9.5 mg kg-1), CuS-PEGDA/AIPH (CuS-PEGDA 20%, AIPH 0.2%, w/v, the same content of CuS with CuS group), CuS/131I-PEGDA/AIPH (CuS/131I-PEGDA 20% w/v, AIPH 0.2%, 131I dose = 50 µCi per mouse). The injection volume was 25 µL. To prevent the possible leakage of the administered agent, the solution was injected into the tumor from the distal end into the intact tumor using an insulin needle. After the injection, we kept the needle inside the tumor for 1 min before it was pulled out from the tumor, to avoid possible leakage. After 10 min p.i., tumors were exposed to 915-nm laser at 1.0 W cm-2 maintaining 43 °C for 20 min as the 1st round irradiation. At 48 h and 96 h p.i., tumor from CuS NPs (NIR+) (M), CuS/131I (NIR+) (M), CuS-PEGDA/AIPH (NIR+) (M) and CuS-PEGDA/131I/AIPH

(NIR+) (M) groups were repeated irradiated by 915-nm laser at 1.0

W cm-2 maintaining 43 °C for 10 min, as the 2nd and 3rd round irradiation. The tumor size of each mouse were monitored every 2 days. The volumes of tumors were calculated by the formula: width2×length×π/6. After 7 days treatment, tumor tissue from groups were collected for further hematoxylin and eosin (H&E) staining. Immunofluorescence staining. The tumors were intratumorally injected with CuS-PEGDA/AIPH (25 µL, 20% w/v of CuS-PEGDA and 0.2% AIPH) and then received a single or multiple irradiation of 915-nm laser (1.0 W cm-2) for 20 min at 10 min, 48 h and 96 h p.i.. Tumors were surgically excised at 0, 1.5 h, 4 h, 8 h, 24 h post laser irradiation to make frozen sections. The pimonidazole hydrochloride (60 mg/kg) (Hypoxyprobe-1 plus kit, Hypoxyprobe Inc) were injected for 1.5 h before surgically excised the tumor. The hypoxia staining was conducted by using the pimonidazole staining method with Hypoxyprobe-1 plus

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kit (Hypoxyprobe Inc) following the standard protocol.49, 52. The images were captured with a confocal microscopy. Histology Analysis. Tumors and organs were harvested right after mice were sacrificed for H&E staining and histological examination. Statistical Analysis. The data of the experiments were presented as mean ± standard deviation (SD). Multiple comparisons among groups were determined using one-way ANOVA analysis followed by Tukey’s post-test; *p