CuS Nanodot-Loaded Thermosensitive Hydrogel for Anticancer

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CuS nanodots loaded thermosensitive hydrogel for anti-cancer photothermal therapy Jijun Fu, Jian-ye Zhang, Songpei Li, Lingmin Zhang, Zhongxiao Lin, Lu Liang, Aiping Qin, and Xiyong Yu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00624 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Molecular Pharmaceutics

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CuS nanodots loaded thermosensitive hydrogel for anti-cancer photothermal therapy Ji-Jun Fu 1 2, Jian-Ye Zhang 1 2, Song-Pei Li 1, Ling-Min Zhang 1, Zhong-Xiao Lin 1, Lu Liang 1, Ai-Ping Qin 1 #, Xi-Yong Yu 1 2 # 1 Key Laboratory of Molecular Target & Clinical Pharmacology, School of Pharmaceutical Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China, 511436 2 The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China, 510700 # Address correspondence to [email protected], [email protected] Abstract: Purpose: To establish an injectable hydrogel encapsulating the copper sulfide (CuS) nanodots for photothermal therapy against cancer. Methods: The CuS nanodots were prepared by one-pot synthesis and, the thermosensitive Pluronic F127 was used as the hydrogel matrix. The CuS nanodots and the hydrogel were characterized by morphous, particle size, serum stability, photothermal performance upon repeated 808 nm laser irradiation as well as the rheology features. The effects of the CuS nanodots and the hydrogel were evaluated qualitatively and quantitatively in 4T1 mouse breast cancer cells. The retention, photothermal efficacy, therapeutic effects and systemic toxicity of the hydrogel were assessed in tumor bearing mouse model. Results: The CuS nanodots with a diameter of about 8 nm exhibited satisfying serum stability, photo-heat conversion ability and repeated laser exposure stability. The hydrogel encapsulation did not negatively influence the above features of the photothermal agent. The nanodots loaded hydrogel shows a phase transition at body temperature and, as a result, a long retention in vivo. The photothermal agent embedded hydrogel played a promising photothermal therapeutic effects in tumor bearing mouse model with low systemic toxicity after peritumoral administration. Keywords: CuS; nanodots; hydrogel; thermosensitive; injectable; in-situ; photothermal

Introduction In the past few years, the world has witnessed many new methods in treating cancers [1-10], such as monoclonal antibodies, gene therapy [4, 10], photodynamic therapy (PDT), sonodynamic therapy, photothermal therapy (PTT) [8]. However, cancer therapy is still a big challenge, with the mean survival length of cancer patients only several years. As a traditional dosage form, hydrogel has been extensively investigated in many research fields, especially in regeneration medicine. For example, thermosensitive carbon nanotube-functionalized hydrogel was proposed to support long-term survival of cardiomyocytes and promote its alignment and proliferation [11]. Hydrogel made of hyaluronic acid and gelatin was used to encapsulate stem cell-derived exosomes, and the hydrogel could be used as a novel cell-free scaffold material for articular cartilage regeneration [12]. Injection of microporous annealing particle hydrogels in the stroke cavity was found to reduce gliosis and inflammation, as well as promote neural progenitor cell migration to the lesion [13]. CaSO4 and FGF-18 delivered by chitinpoly(D,L-lactic acid-co-glycolic acid) (PLGA) hydrogel was found to be able to enhance bone regeneration [14]. Application of the hydrogels in treating 1

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cancers is also a newly emerging research field. Lin et al prepared the paclitaxel hydrogels with three different dispersing states of the compound, molecules, nanocrystals and microcrystals. The hydrogel with paclitaxel nanocrystals exhibited the most outstanding long-term stability, retention in tumor tissue and anti-cancer performance [15, 16]. Nowadays, there has been an increasingly widespread interest in PTT. The photothermal agents (PTA) are able to convert laser energy, especially the near infrared light (NIR) which could penetrate deep into tissues, into heat energy. Up to now, PTT is often employed to treat cancers. Sun et al [17] proposed core-shell TiO2 nanoparticles as an excellent PTA. Jiu et al [18] reported an ultrasmall iron-gallic acid nanoparticles as photoacoustic imaging (PAI) guided PTA. MoO3-x nanostructures could also be used as PAI guided photothermal agent [19, 20]. Based on the fact of the elevated negative charges on cancer cell membrane compared to healthy cells, a positively charged superparamagnetic Fe3O4 nanoparticles was proposed to achieve tumor targeting and destruction upon NIR irradiation [21]. Zhan et al [22] fabricated special liposomes encapsulating tetrodotoxin and dexmedetomidine. The liposomes were decorated by gold nanorods (GNR) which could initiate the release of the anesthetics to achieve repeatable and on-demand nerve block upon NIR irradiation for short duration and low irridance. The authors further discovered that encapsulation of the photosensitizer 1,4,8,11,15,18,22,25-octabutoxyphthalocyaninatopalladium(II) (PdPC) in liposomes enhanced the on-demand release of the anesthetics upon NIR irradiation [23]. Normally, the PTA are intravenous administered to play the therapeutic effects. Despite great advances in PTT, the toxicity of the intravenous administered PTA remains a big concern as most of the PTA are made of heavy metal elements, such as gold and bismuth, that are difficult to tackle with. In addition, despite the enhanced penetration and retention effects (EPR) and other targeting methods, the nano-carriers finally distributing in tumor tissues account for only a small proportion of the dose administered, as a result, the unsatisfactory distribution retards superior therapeutic effects and increases systemic toxicity. In this study, an injectable Pluronic F127 hydrogel embedding copper sulfide (CuS) nanodots for peritumoural administration is proposed (Fig. 1). Pluronic F127 is widely used in constructing thermosensitive hydrogel and, is often employed in drug delivery systems for i.v. administration [24-26]. As the hydrogel is locally administered and has a long retention time, it is believed that this system will exhibit better photothermal therapeutic effects and reduced systemic toxicity. As far as we know, PTA loaded hydrogel for cancer therapy was rarely reported.

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Fig. 1 Schematic illustration

Materials and methods Preparation and characterization of CuS nanodots

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21 mg of CuCl2•2H2O and 60 mg of polyvinylpyrrolidone (M.W. 24000) were added into 5 ml of pure water, the mixture was stirred to get clear solution.

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Then, 30 mg of Na2S•9H2O was added with stirring. Then, the solution was

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heated to 90 °C for 15 min with stirring to get dark green nanodispersions. After dialysis in pure water, CuS nanodots was obtained. Appearance of the nanodot was pictured by transmission electron microscopy (300 kV) and, particle size was measured by particle sizing system (Malvern). To investigate stability of the CuS nanodots, serum was added to CuS nanodispersions for a final concentration of 10%, and CuS nanodots according

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to CuS was 0.5 mg/ml. The sample was placed in water bath at 37 °C for 6 d, visible absorption curves of the sample as well as particle size of the nanodots before and after storage were measured. To evaluate the photothermal performance of CuS nanodots, the nanodispersions with different concentrations (0, 20, 50, 100, 200, 500 µg/ml) were exposed to 808 nm laser at 1 W/cm2. Temperatures of the samples at different time points were recorded by infrared imaging camera (Fotric). To further characterize the CuS nanodots, the sample with 0.5 mg/ml CuS was exposed to NIR irradiation for five cycles. After each irradiation for 5 min at 1 W/cm2, the sample was cooled to room temperature naturally, and then exposed to 808 nm laser again. Temperatures of the sample were monitored. Particle size and visible absorption curves of the sample before and after 5-cycle irradiation were measured. Preparation and characterization of CuS nanodots embedded Pluronic F127 hydrogel Pluronic F127 granules were simply added to CuS nanodispersions with stirring in ice bath to get CuS nanodots loaded hydrogel. The concentration of Pluronic F127 was 0.2 g/ml and, the concentration of CuS nanodots was 0.1 mg/ml. Hydrogel loaded with CuS nanodots was frozen in liquid nitrogen overnight, then the frozen hydrogel was lyophilized. Morphous of the final 3



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sample was viewed by scanning electron microscopy (SEM, 3.0 kV). Blank Pluronic F127 hydrogel was used as a control. To investigate the photothermal performance of CuS nanodots loaded hydrogel, the hydrogel was exposed to 808 nm laser at 1 W/cm2. Temperatures of the hydrogel at different time points were monitored. Blank hydrogel was used as a control. To further characterize the CuS nanodots loaded hydrogel, the rheology features of the samples were measured. The frequency was 1 Hz and, the

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heating rate was 1 °C /min. The blank hydrogel was used as a control. To study the stability of CuS nanodots loaded hydrogel, the sample was

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placed at 37 °C for days. The hydrogel sample before and after storage was diluted by water for 10 times, and then measured by particle sizing system to reflect the size of the CuS nanodots. Moreover, the heating curves upon NIR irradiation (808 nm, 1 W/cm2) were gathered before and after different storage time. The similarity between two curves was evaluated by f2 factor according to the following equation and, f2 > 50 means highly similar.

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Rt and Tt mean temperature of the sample before and after storage upon NIR irradiation at t minute. Assessment of in vitro photothermal therapeutic effects

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4T1 mouse breast cancer cells (1ⅹ104) were seeded in 96-plate and

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incubated overnight (37 °C, 5% CO2). Then, CuS nanodispersion was added

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to make final concentration of the nanodots 0, 2, 5, 10, 20 µg/ml, respectively. The cells were incubated for another 24 h. Then, the cells were exposed to 808 nm laser (1 W/cm2) for 1 min and, the cells free from NIR treatment were used as control. After incubation for another 4 h, the viability of the cells was measured by MTT method.

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4T1 mouse breast cancer cells (5ⅹ104) were seeded in 24-plate and

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incubated overnight (37 °C, 5% CO2). Then, CuS nanodispersion was added to make final concentration of the nanodots 20 µg/ml. Then, the cells were exposed to 808 nm laser (1 W/cm2) for 3 min and, the cells treated by PBS and the cells free from NIR treatment were used as controls. After incubation for another 4 h, propidium iodide (final concentration 30 µg/ml) was added and incubated for 30 min to stain the dead cells. Then, the culture media was discarded and the cells were washed by PBS for several times. 4% paraformaldehyde solution was used to fix the cells. After washing by PBS for several times, Hoechst 33342 solution was used to stain the nucleus of all the cells. At last, the cells were photographed by fluorescence microscope to view the photothermal therapeutic effects of the nanodots.

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4T1 mouse breast cancer cells (5ⅹ104) were seeded in 24-plate in 1 ml of

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culture media. 200 µl of the CuS nanodots embedded hydrogel or blank

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hydrogel was added to the upper transwell (8 µm). The cells were incubated at

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37 °C for 24 h. Then, the hydrogels encapsulated transwells were exposed to 808 nm laser (1 W/cm2) for 3 min and, the cells treated by blank hydrogel and the cells free from NIR treatment were used as controls. Finally, the cells were treated and pictured as the above section. Retention of the hydrogels around tumor tissue The study was approved by Ethics Committee of Guangzhou Medical University. The near infrared fluorescent dye DiR labeled hydrogel was prepared by

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adding DiR ethanol solution (100 µg/ml) to the blank hydrogel with stirring in

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ice bath to make the final concentration of 2 µg/ml. The control DiR solution

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was prepared by adding DiR ethanol solution (100 µg/ml) to PBS with stirring

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to make the same final concentration of 2 µg/ml before use At day 0, Balb/c mouse (female, 6-8 week old) was subcutaneously administered 1 million of 4T1 cancer cells to establish armpit tumor bearing

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mouse model. At day 10, 500 µl of DiR labeled hydrogel was peritumoral injected. To ensure homogenous distribution of the hydrogel surrounding the

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tumor tissue, the needle was inserted at five different points and, 100 µl of the

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hydrogel was injected at each point. Mice received 500 µl of the DiR solution were used as control. Then at different time intervals, the DiR signals were captured at 740/820 nm to image the retention of the hydrogels around tumor tissue (Berthold). In vivo anti-cancer performance At day 0, Balb/c mouse (female, 6-8 week old) was subcutaneously administered 1 million of 4T1 cancer cells to establish armpit tumor bearing mouse model. At day 7, the volume of the tumor reached approximately 200

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mm3 according to the following equation: volume=lengthⅹ(width)2/2. Then, 500

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µl of the CuS nanodots loaded hydrogel was administered by peritumoral

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injection. Mice received 500 µl of the blank hydrogel were used as control. To ensure homogenous distribution of the hydrogel surrounding the tumor tissue,

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the needle was inserted at five different points and, 100 µl of the hydrogel was injected at each point. After 2 h, the tumors were exposed to 808 nm laser at 1 W/cm2 for 5 min after the mice were anesthetized. Temperatures of the tumor tissues were recorded by infrared imaging camera. NIR treatment was carried out every day from then on. At day 15, the mice were sacrificed and, the tumors of the test and control group were photographed and weighed. Then, the tumor, heart, liver, spleen, lung and kidney samples were made into paraffin sections and H&E stained. Body weight of the mice was measured everyday. Statistical analysis All values were expressed as mean ± standard deviation (SD). All comparisons were performed by the two-tailed Student’s t test. A p-value less than 0.05 was supposed to be statistically significant and, p-value less than 5



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0.01 was considered to be highly significant.

Results and discussions Characteristics of the CuS nanodots The CuS nanoplatform was prepared by a simple one-pot synthesis. PVP was used as the stabilizer of the nanodispersions due to its favourable hydrophilicity. The final product appeared to be a dark green solution and, the concentration of the nanoplatform could be simply adjusted by dilution through adding pure water.

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Fig. 2 In vitro characterization of the CuS nanodots. A) TEM image of the CuS nanodots; B) Particle sizes of the CuS nanodots before (red) and after 6-day incubation in serum at 37 °C (green); C) The visible absorption curves of CuS nanodots before and after 7



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incubation in serum for 3 and 6 days at 37 °C; D) and E) The thermographs and corresponding heating curves of the CuS nanodots at varied concentrations upon 808 nm laser exposure (1 W/cm2); F) The heating curves of the CuS nanodots upon repeated 808 nm irradiation (1 W/cm2); G) Particle sizes of the CuS nanodots before (red) and after (green) 5-cycle 808 nm irradiation (1 W/cm2); H) The visible absorption profiles of the CuS nanodots before and after 5-cycle 808 nm irradiation (1 W/cm2).

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nanodots exposed to 10% blood serum for 3 and 6 days at 37 °C was compared to that of the original sample to assess the stability of the PTA. The sample after serum exposure still exhibited an increasing visible absorption curve, which was highly comparable to the original one (Fig. 2C). This indicated the outstanding stability of the CuS nanodots in body fluids and, promising photothermal performance in vivo. The stability of the CuS nanodots in the body fluids was also evaluated in the term of particle size, Fig. 2B shows a slight decrease in diameter from 11 nm to 10 nm after serum exposure for 6

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Fig. 2A shows that CuS existed as irregular nanodots in the solution, with a diameter of approximately 8 nm. Particle size of the nanodots was further validated by particle sizing system (Fig. 2B), which shows a single peak around 11 nm. The appearance and size of the CuS nanodots were in accordance with other reports [27]. The NIR is considered to be biologically compatible due to its deep tissue penetration and negligible influence on tissues. PTAs are able to convert the light to heat to play anti-cancer effects by generating intolerable high temperature microenvironment for malignant cells. The light-heat conversion ability of the PTAs can be indicated by visible absorption curve. The visible absorption curve in Fig. 2C saw a steady increase from 600 nm to 1000 nm, implying the encouraging NIR absorption capacity of the CuS nanodots. The stability of PTA in the body fluids is critical for its performance in vivo. To assess the stability of the PTA, the visible absorption curve of the CuS

days at 37 °C, and the data further supported the above results. Fig. 2D and Fig. 2E show the thermo-photographs and heating curves of the CuS nanodots with a serial concentrations of 20, 50, 100, 200, 500 µg/ml upon 808 nm laser exposure. The CuS nanodots exhibited dramatic increase in temperature upon NIR irradiation at any concentration. Moreover, the temperature rises along with the increase of concentration, with the final

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temperature of the five concentrations reaching 35.5, 41.8, 47.0, 55.8, 60.6 °C, respectively. Meanwhile, the NIR exposure had little influence on the pure water control, with the control showing negligible temperature changes. The above results indicated the favorable photothermal effects of the CuS nanodots. Photothermal stability is one of the most important index to assess PTA. Indocyanine green (ICG), a typical PTA belonging to the cypates, shows severe light-bleach and reduced photothermal efficacy after repeated NIR exposure [28, 29]. Despite its excellent photo-heat conversion ability, the photothermal instability hampered the practical use of ICG as PTA. As the CuS nanodots loaded hydrogel would be locally administered, it was hoped that the PTA in the formed hydrogel would be able to exert long-term therapeutic effects. Fig. 2F shows the heating curve of the CuS nanodots upon repeated

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NIR irradiation. The nanodispersion could be re-heated to about 56 °C after 5

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min laser exposure once the sample was naturally cooled to room temperature. In other words, the heating curve of each cycle was identical to the other ones,

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with the highest temperature of each cycle nearly 56 °C, and the heating curve showed negligible changes after repeated laser exposure. These data indicated that the CuS nanodots displayed satisfying photothermal stability. To further validate this feature, the particle size and visible absorption curve of the nanodots before and after 5-cycle NIR irradiation were measured. Fig. 2G shows that the particle size of the sample post-NIR treatment was about 10 nm, slightly smaller than the diameter of 11 nm before NIR treatment. Fig. 2H shows that the two visible absorption curves of the nanodots were obviously identical, with minor variations. Both graphs further support the encouraging photothermal stability of the CuS nanodots. Based on these results, it is estimated that the PTA would maintain the therapeutic effects throughout the whole retention period in vivo and would not be destroyed by body fluids or repeated laser exposure. Features of the CuS nanodots loaded Pluronic F127 hydrogel In order to reduce the systemic toxicity of intravenously administered PTA and overcom the possible problem of unsatisfying photothermal therapeutic effects due to the limited enrichment of the PTA in tumor tissues, thermosensitive in-situ hydrogel was employed to encapsulate the CuS nanodots, which would be peritumorally injected. It was believed that the injectable hydrogel would solidify locally and, form semisolid hydrogel surrounding the tumor tissue and, might exhibit a long retention time. Meanwhile, the local administration would decrease systemic toxicity and increase therapeutic outcomes. In this section, the in vitro characteristics associated with the above aims would be discussed in detail.

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Fig. 3 In vitro characterization of the CuS hydrogel. A) SEM images of the blank Pluronic F127 hydrogel and the CuS nanodots loaded hydrogel; B) and C) The thermographs and corresponding heating curves of the CuS hydrogel and blank hydrogel upon 808 nm irradiation (1 W/cm2); D) Pictures of the blank hydrogel and CuS hydrogel at 4 and 37 °C; E) The complex viscosity and loss modulus curves of the blank hydrogel and CuS 10


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hydrogel; F) Particle size of the CuS nanodots encapsulated in the hydrogel after different storage time at 37 °C; G) The heating profiles of the CuS hydrogel upon 808 nm laser

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exposure (1 W/cm2) after different storage time at 37 °C.

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The CuS nanodots embedded hydrogel was prepared by a simple one-pot method of dissolving Pluronic F127 in the nanodispersions directly. Fig. 3A shows the morphous of the blank hydrogel and the PTA encapsulated hydrogel. Both hydrogels had a highly macroporous network structure, with the pores

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about 50-500 µm in diameter. In detail, the blank hydrogel shows more but smaller pores than the CuS nanodots embedded hydrogel. With 10000-fold magnification, both the blank hydrogel and the CuS nanodots embedded hydrogel displayed a smooth surface. Although the CuS nanodots were supposed to be evenly dispersed in the hydrogel matrix, the nanodots were too small to be detected by SEM. Ideally, the CuS nanodots embedded hydrogel would exhibit pronouncing photothermal performance. Fig. 3B and Fig. 3C show the temperature changes of the PTA encapsulated hydrogel and the blank control hydrogel upon 808 nm laser exposure. The temperature of CuS hydrogel increased

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dramatically to 54.9 °C upon NIR irradiation. The heating curve of the CuS hydrogel shown in Fig. 3C was comparable to that of the nanodispersions illustrated in Fig. 2E. Moreover, the laser exposure had little influence on the blank hydrogel, with negligible temperature changes throughout the whole period. These data indicate that the encapsulation in hydrogel did not negatively influence the photothermal performance of the CuS nanodots and, the nanodots loaded hydrogel was supposed to display excellent photo-heat conversion transition capacity in vivo. To elevate the compliance of the patients, non-invasive treatment is highly required nowadays. Injectable hydrogel is one of the most widely used non-invasive administration route. Pluronic F127 is a commercially available thermosensitive hydrogel constructing material. Fig. 3D shows that both the blank hydrogel and the CuS hydrogel are liquid at 4°C and, the two hydrogels changed into semisolid at body temperature. In other words, the formed in-situ

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hydrogel maintained its fluidity at low temperature (< 20 °C) and, the fluidity assigned the injectable feature of the hydrogel. At the same time, the in-situ hydrogel solidify at body temperature and, the semisolid state would promise a long retention time in vivo. To explain the phase transition of the hydrogels in detail, the rheology characteristics of the hydrogels were studied. Fig. 3E shows the rheological characteristics of the blank hydrogel and the CuS hydrogel. Both the complex viscosity curves exhibited a sharp increase in the complex viscosity in a narrow temperature range. In detail, the complex viscosity profile of the blank hydrogel showed a phase transition temperature

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of 30 °C, while the encapsulation of the CuS nanodots resulted in a decrease

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in phase transition temperature to 26 °C. The phase transition temperatures of both hydrogels indicated by the loss modulus curves were in accordance with the above numbers. Normally, loading water-soluble substances will increase the phase transition temperature of the hydrogel, while embedding water-insoluble agents will decrease the phase transition temperature. Despite

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the water-soluble PVP in the nanodots, the CuS core was water-insoluble and hydrophobic. As a result, there was a reduction in the phase transition temperature of the hydrogel after the CuS nanodots encapsulation. Anyhow, these data indicated the CuS hydrogel would undergo a phase transition process after being locally administered, assigning the hydrogel a long retention time in vivo. One of the advantage of the injectable hydrogel in treating cancers is the sustained therapeutic efficacy. To achieve this, the PTA in the hydrogels must be stable for a considerable period. In this research, the particle size and the heating curve of the CuS nanodots after being encapsulated were measured. Fig. 3F shows that the nanodots in the hydrogel maintained a nearly constant diameter around 7.5 nm, comparable to the original particle size before being loaded. The result indicated that the CuS nanodots remained stable in the hydrogel in the term of particle size. Fig. 3G shows the heating profiles of the

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CuS hydrogel before and after storage at 37 °C for several days. It can be seen that all the heating curves were nearly identical, with the temperature

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reaching about 55 ° C at the end of laser exposure. It seems that the encapsulation and storage at body temperature had little influence on the photothermal performance of the CuS nanodots. Moreover, as all the heating profiles were obtained by recording the thermograph of the same hydrogel sample upon NIR irradiation at different time intervals, it can be concluded that the CuS nanodots remained stable in photo-heat transition ability after repeated 808 nm laser exposure. Statistically, the similarity factor f2 between the initial heating curve and that of the 4th, 6th, 8th, 11th, 13th day were 88.3, 82.3, 88.1, 78.2 and 83.4, respectively. All the similarity factor f2 is over 50, which represented that all the heating profiles were highly comparable. In a word, both results supported that encapsulation in the hydrogels and storage at body temperature would not negatively influence the CuS nanodots and, implied that the PTA would perform sustained therapeutic effects in vivo. In vitro anti-cancer photothermal therapy In this section, the photothermal therapeutic effects of the CuS nanodots and the PTA loaded hydrogel against the 4T1 mouse breast cancer cells were evaluated.

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Fig. 4 The anti-cancer effects of the CuS nanodots and the hydrogel against 4T1 mouse breast cancer cells. A) The fluorescence images of the 4T1 cells after being treated by the CuS nanodots differently, the red color represents the dead cells stained by propidium iodide and the blue color represents the nucleus of all the cells, the white line is the boundary of the 808 nm laser; B) The survival rates of the 4T1 cells measured by MTT test after being treated by the CuS nanodots differently, ** represents statistically high significance (p < 0.01); C) The fluorescence images of the lower 4T1 cells after being treated by the blank hydrogel and CuS hydrogel in the upper transwell differently, the red color represents the dead cells stained by propidium iodide and the blue color represents the nucleus of all the cells.

Fig. 4A illustrates the fluorescence images of the 4T1 cells, with all the cells blue stained and the dead cells red stained. As anticipated, the PBS treatment alone resulted in few cell death, with negligible number of the cells red stained. Moreover, the combination of PBS treatment and 808 nm laser exposure also led to insignificant cell death, with only a few cells red stained. The results indicated that the NIR light did not make detrimental effects on the cell survival and, the NIR light could be regarded as bio-safe to some extents. Furthermore, the CuS nanodots at the concentration of 20 µg/ml did not exert obvious toxicity to cells, as implied by the comparable effects on cell death compared to the PBS control group. The result clued the encouraging biocompatibility of the CuS nanodots. Based on the observation, it was believed that the PTA would produce tolerable side effects to healthy tissues without NIR irradiation. As anticipated, the combination of CuS nanodots treatment and 808 nm laser exposure resulted in significant cell deaths, with almost all the cells under irradiation failed to survive. Furthermore, the overlay image shows a clear dividing line separating the alive and dead cells. Actually, the dividing line was the boundary of the laser light, merely the cells covered by the laser light were sentenced to death. The existence of the dividing line pointed out that the photothermal therapy could be carried out accurately and 13



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precisely. To analyze the toxicity and therapeutic effects of the CuS nanodots quantitatively, MTT test was carried out. Fig. 4B shows the 4T1 cancer cell survival rate after being differently treated. When the cells were free from the laser exposure and treated by the CuS nanodots for 24 h only, the viability of the cells showed a decreasing trend along with the PTA concentration increase, with the survival rate declining to 91.9%, 87.8% and 71.6% at the CuS

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nanodots concentration of 5, 10, 20 µg/ml, respectively. Once the cells treated by the CuS nanodots at the same concentrations were exposed to laser light, the viability of the cells further declined to 70.6%, 44.4% and 23.5%. The NIR exposure made statistically significant difference to the survival rate of the CuS nanodots treated cells when the PTA concentration reached 5, 10 and 20

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µg/ml. Above results suggested that the CuS nanodots treatment plus the NIR irradiation would kill cancer cells and, the malignant cells could be totally wiped out at a high PTA concentration. As a result, it can be anticipated that the enrichment of the CuS nanodots surrounding the tumor tissues by local administration of the PTA encapsulated hydrogel would bring in promising therapeutic effects. In this research, transwell was employed to examine the therapeutic effects of the CuS hydrogel against the 4T1 cancer cells. The transwell membrane separated the upper PTA embedded hydrogel and the lower cells. Fig. 4C shows the fluorescence images of the cells, with all the cells blue stained and the dead cells red stained. It can be seen that the blank hydrogel alone did not induce cell death, indicating the outstanding compatibility of the Pluronic F127 hydrogel matrix. Moreover, the blank hydrogel plus the laser exposure did not produce cell death either. As discussed in the above section, the NIR irradiation did not make any changes to the hydrogel. The absence of cell death during the combination of blank hydrogel and the laser exposure further proved this point. Fig. 4C also shows that the CuS hydrogel alone was not harmful to the cells, with negligible number of cells red stained. It seems that, despite the same incubation time, the cells treated by the PTA loaded hydrogel exhibited much higher survival rate than the cells treated by the same amount of the nanodots directly, as indicated in Fig. 4B. The difference implied that the encapsulation in the hydrogel reduced the undesirable adverse effects of the CuS nanodots. It was supposed that the hydrogel matrix relieved the unwanted toxicity of the chemicals by “prisoning” them and, played the therapeutic effects through photo-heat conversion only. The capacity of relieving the direct toxicity caused by the metal chemicals renders the hydrogel an ideal candidate to decrease the systemic toxicity of the PTA. Fig. 4C also shows that the cancer cells were almost completely wiped out when the cells were exposed to the CuS hydrogel together with the NIR irradiation. Obviously, the temperature rise caused by the CuS hydrogel under the laser exposure killed the malignant cells. It is worth noting that, despite the same irradiation time and strength as well as the PTA dose, the CuS hydrogel produced much bigger cell death area than the free nanodots, as indicated in Fig. 4A. In other words, concentrated PTA played more promising photothermal therapy than the evenly dispersed PTA of the same dose. That is to say, the hydrogel not only reduced the direct chemical toxicity of the CuS nanodots, but also enhanced the photothermal therapeutic effects. The results further indicated 14


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the superiority of the hydrogel in achieving more pronouncing photothermal therapy. Local retention of the CuS nanodots loaded hydrogel In order to reduce the systemic toxicity and enhance the photothermal therapeutic effects, the CuS hydrogel was peritumorally administered. It was supposed that the thermosensitive hydrogel would change into semisolid hydrogel locally, and then the encapsulated CuS nanodots would have a long retention surrounding the tumor tissues and, as a result, play sustained therapeutic effects after one non-invasive administration. The prerequisite of the sustained therapeutic effects is the long retention in vivo. To image the retention of the hydrogel in vivo, near-infrared imaging technique was employed. The near-infrared fluorescence dye DiR labeled hydrogel was used, with the DiR solution used as a control group. Fig. 5A shows the fluorescence images of the breast tumor bearing mice after being peritumorally treated by DiR hydrogel or free DiR solution. The free DiR treated mice showed a rapid signal decrease, with weak fluorescence signal at day 5 and day 10 and almost undetectable fluorescence signal at day 15. The fast fluorescence signal decrease meant the rapid loss of the agent from the tumor site. At the same time, the DiR hydrogel treated mice saw a nearly constant fluorescence signal, with the signal strength at day 10 comparable to the original signal strength at day 0 and only a slight signal decrease at day 15. The stable fluorescence strength indicated little loss of the agent from the tumor site. In other words, the in-situ hydrogel guaranteed a promising retention of the treating agents surrounding the tumor site and, as a result, provided the basis of sustained therapeutic effects.

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Fig. 5 In vivo performances of the CuS hydrogel in tumor bearing mouse model. A) The retention of the DiR labeled hydrogel and free DiR solution after peritumoral administration (Ex/Em: 740/820 nm); B) and C) The thermographs and the corresponding heating curves of the tumor sites after peritumoral administration of the blank hydrogel or CuS hydrogel plus 808 nm irradiation (1 W/cm2); D) Pictures of the tumor tissues after photothermal therapy (1 W/cm2, 5 min) in eight sequential days, the red circle represents visibly undetectable tumor sample; E) Statistic graph of the tumor weights, * represents statistical significance (p < 0.05); F) Body weight curves of the tumor bearing mice throughout the whole experiment period; G) Hematoxylin & Eosin staining of the tumor tissues; H) Hematoxylin & Eosin staining of the heart, liver, spleen, lung and kidney of the CuS hydrogel treated mouse.

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temperature reaching 45.9 °C at the end of irradiation. The temperature increase was probably caused by the direct tissue heating effect of the NIR. Meantime, the CuS hydrogel treated mouse displayed a sharp temperature increase at the tumor site upon the laser exposure, with the temperature

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In vivo anti-cancer performance In this section, the anti-cancer performance of the CuS nanodots embedded hydrogel was evaluated in a breast cancer bearing mouse model, with the blank hydrogel as a control. Fig. 5B shows the thermographs of the CuS hydrogel or blank hydrogel peritumorally treated tumor bearing mice upon the 808 nm laser irradiation. Fig. 5C illustrated the temperatures of the tumor tissue at varied time points. It can be seen that the blank hydrogel treated mouse exhibited a moderate temperature increase at the tumor site upon the NIR exposure, with the

approaching 90 °C at the end of NIR irradiation. The results implied the outstanding photo-heat transition ability of the PTA loaded hydrogel in vivo. The satisfying photo-heat transition capacity was supposed to provide a promising photothermal therapeutic effect against cancer. After the NIR irradiation in eight sequential days, the tumor tissues of the test and control groups were collected, as shown in Fig. 5D. Despite the same laser strength and lasting time, the tumor samples of the blank hydrogel treated mice were obviously bigger than that of the CuS hydrogel treated mice. Specially, one mouse peritumorally treated by the CuS hydrogel plus the NIR irradiation was totally cured, with the tumor tissue visibly undetectable at the end of the experiment. The tumor weights between the test and control group were statistically different (p < 0.05), with the mean tumor weight 0.35 g and 0.76 g respectively, as illustrated in Fig. 5E. The above results indicated the excellent photothermal therapeutic effects of the CuS nanodots encapsulated hydrogel against cancer. To evaluate the systemic toxicity of the treatments, the body weights of the mice were measured. Fig. 5F shows that the mice of both groups had a nearly constant body weight, with a small initial fluctuation probably due to the inadaptation to anesthesia in the first few days. In order to further evaluate the systemic toxicity of the CuS hydrogel, main organs of the mice after the repeated NIR treatment were sliced and H & E stained. Heart, liver, spleen, lung and kidney all displayed normal morphology, as shown in Fig. 5H. The relatively stable body weight and normal morphology of the main organs indicated that the non-invasive administration of the thermosensitive CuS hydrogel plus the laser irradiation induced negligible systemic toxicity. As shown in Fig. 5G, it is worth noting that, comparable to the control group, the necrosis was absent in the tumor tissue of the test group. It was 17



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estimated that the heat generated could not penetrate into the core of the tumor tissue to induce cell death, as a result, the necrosis was undetectable in the remaining tumor tissue. The phenomenon clued that the CuS hydrogel played the therapeutic effects through “burning” the cancer cells layer by layer. In summary, it is clear that the injectable and temperature-responsive in-situ CuS hydrogel is a promising PTA for cancer treatment with low systemic toxicity.

Conclusions In this research, an injectable and thermosensitive hydrogel encapsulating CuS nanoparticles for photothermal therapy against cancer was reported. The CuS particles show nanodots structure with a diameter of about 8 nm. The nanodots evenly distributed in the hydrogel matrix and, maintained its particle size unchanged. The PTA loaded hydrogel shows excellent photo-heat conversion ability and outstanding repeated irradiation stability. The hydrogel not only exhibited thermo-responsive in-situ gel-forming capacity at body temperature, but also reduced the chemical toxicity of the CuS by “prisoning” the nanodots in the matrix. Moreover, the concentrated PTA in the hydrogel exhibited more promising therapeutic effects against cancer cells than the nanodots solution. At last, the CuS nanodots embedded hydrogel displayed pronouncing tumor curing effects after peritumoral administration due to the enhanced retention in vivo and elevated local temperature surrounding the tumor tissue upon the NIR exposure. Meantime, the preparation shows insignificant systemic toxicity.

Acknowledgements The research was supported by the Science and Technology Programs of Guangzhou (Grant No. 201604010087), National Natural Science Foundation (Grant No. 81700382), Guangdong Natural Science Foundation (Grant No. 2015A030310428) and, the funding from Guangzhou Medical University (Grant No. B185004204).

Disclosure The authors report no conflicts of interest in this work.

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CuS Nanodot-Loaded Thermosensitive Hydrogel for Anticancer

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