Inhibition of melanoma using a nanoceria-based prolonged oxygen-generating phototherapy hydrogel

Tumor hypoxic environment is an inevitable obstacle for photodynamic therapy (PDT) of melanoma. Herein, a multifunctional oxygen-generating hydrogel loaded with hyaluronic acid-chlorin e6 modified nanoceria and calcium peroxide (Gel-HCeC-CaO2) was developed for the phototherapy of melanoma. The thermo-sensitive hydrogel could act as a sustained drug delivery system to accumulate photosensitizers (chlorin e6, Ce6) around the tumor, followed by cellular uptake mediated by nanocarrier and hyaluronic acid (HA) targeting. The moderate sustained oxygen generation in the hydrogel was produced by the reaction of calcium peroxide (CaO2) with infiltrated H2O in the presence of catalase mimetic nanoceria. The developed Gel-HCeC-CaO2 could efficiently alleviate the hypoxia microenvironment of tumors as indicated by the expression of hypoxia-inducible factor -1α (HIF-1α), meeting the “once injection, repeat irradiation” strategy and enhanced PDT efficacy. The prolonged oxygen-generating phototherapy hydrogel system provided a new strategy for tumor hypoxia alleviation and PDT.


Introduction
The global morbidity and mortality of melanoma are increased dramatically, which is still one of the severe threats to public health (1)(2)(3). However, traditional tumor treatment often confronts obstacles due to visible toxic side effects and drug resistance (4). Recently, synergetic photodynamic therapy (PDT) has been introduced to overcome melanoma and has shown great potential in the field of tumor therapy (5,6). As a noninvasive technology, PDT addresses spectacular advantages such as high localized tissue damage and minimal side effects (7). In the PDT process, photosensitizers such as chlorin e6 (Ce6) accumulated in tumor tissues are usually activated and energized by a specified light, resulting in reactive oxygen species (ROS) generation and singlet oxygen ( 1 O 2 ) in the presence of biological substrates and oxygen (8). Therefore, oxygen and photosensitizer contents in tumor tissues are restrictive factors for PDT reaction and cytotoxicity (9). However, the therapeutic efficacy of PDT is often unsatisfactory because of the hypoxic microenvironment in most malignant tumors and rapid drug metabolism-induced short-term effects (10)(11)(12). It is of great significance to alleviate hypoxia and prolong photosensitizer supply in the tumor environment for enhancing PDT efficacy.
Cerium nanoparticles (CeNPs) are a remarkably versatile rare earth nanomaterial with excellent catalytic activities (37, 38). It has been reported to mimic multiple types of enzymes due to the electron shuttle between their mixed oxidation states (Ce 3+ /Ce 4+ ) and has emerged as a fascinating material in biological fields (39). Recently, we demonstrated that chemically cytotoxic and oxygen-carrying CeNPs could act as a nanocarrier to deliver photosensitizers into tumor cells, resulting in enhanced PDT efficacy (40,41). In this study, to obtain sufficient oxygen generation for PDT, the CeNPs nanocarrier was introduced into the system and used as an ideal renewable catalase-like enzyme to catalyze the reaction of CaO 2 and H 2 O. Herein, we designed a prolonged oxygen-generating phototherapy hydrogel system (Gel-HCeC-CaO 2 ) to improve the therapeutic efficacy of PDT in treating melanoma.

Synthesis of materials
The naked cerium oxide nanoparticles (MCeNPs), cerium oxide nanoparticles-alendronate (CeNPs-AL), hyaluronic acid-chlorin e6 modified nanoceria (HCeC), and Gel-HCeC-CaO 2 were prepared as described in the literature (17,42 After 15 min, 0.8 ml sodium hydroxide (NaOH) (1M) was added to the mixture and stirred for 30 min. Then, 400 ml Ce6 (10 mM) was quickly added and stirred for 5.5 h. After that, the dialysis bag (Thermo, 10 kDa) was used to dialyze the acquired HCeC for 24 h in dark and rinsed with water repeatedly in a sleeve tube (Millipore, 30 kD). The acquired HCeC were dispersed in ddH 2 O (HCe not contained Ce6).

Characterization of HCe and HCeC
The zeta potential of the HCe and HCeC were determined using dynamic light scattering(Invitrogen, America). The hydration of nanoparticles was determined by dynamic light scattering (DLS) analysis. The HCe and HCeC were characterized by HR-TEM (FEI, America) at an accelerating voltage of 200 kV. The fluorescence spectra of Ce6, HCe, and HCe6 were determined by Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc, France) at 405nm excitation light. The absorption spectra of Ce6, HCe, and HCeC solution were determined by a UV spectrophotometer (KAIAO, China). Moreover, the HCe and HCeC were cultured with ddH 2 OˎPBS ˎDMEMˎDMEM+ 10% FBS for 24 h to observe the stability. The photosensitivity of HCeC was detected by singlet oxygen sensor green reagent (SOSG). And we mixed the HCeC with 50 mM H 2 O 2 to show the CAT-like activity of the HCeC.
2.4 Sol-Gel transition behavior of the Gel-HCeC-CaO 2 system 1 mL Gel and Gel-HCeC-CaO 2 solution were added into a 10 mL bottle, then put the bottle into a 37°C water bath at a different time until the solution transformed to gel, recording the time (43). The test Abbreviations: PDT, photodynamic therapy; Gel-HCeC-CaO2,oxygengenerating hydrogel loaded with hyaluronic acid-chlorin e6 modified nanoceria and calcium peroxide; Ce6, chlorin e6; HA, hyaluronic acid; CaO2, calcium peroxide; HIF-1a, hypoxiainducible factor -1a; ROS, reactive oxygen species; CeNPs, cerium nanoparticles; AOT, docusate sodium; NaOH, sodium hydroxide; DMEM, dulbecco's modified Eagle's medium; FBS, fetal bovine serum. tube inverting method was used to determine the sol-gel transition behavior of the hydrogel (44), 1 mL Gel and Gel-HCeC-CaO 2 solution was placed in a 10 mL bottle at -20°C, 4°C, 25°C, and 37°C to observe the solution transformed to gel. 1 mL Gel-HCeC-CaO 2 solution was placed in a 10 mL bottle and incubated at 37°C for 5 min to gel. 1 mL PBS (pH = 7.4, 37°C) and CaCl 2 solution were gently added into the bottle as a release medium. The bottles were shaken at the speed of 50 rpm in a thermostatic shaker (HENGZI, China) at 37°C while the weight of the gel was measured every 20 min.
2.5 Oxygen generation of the Gel-HCeC-CaO 2 system N 2 was injected into 30 mL PBS for 30 min to produce the deoxygenated PBS. The deoxygenated PBS was divided into two groups, and the PH was adjusted by concentrated hydrochloric acid to 5.4 and 7.2, respectively. 5 mL Gel-HCeC-CaO 2 solution were put into a 50 mL centrifuge tube and incubated at 37°C for 5 min to gel. Then, 30 mL deoxygenated PBS (PH=7.2/5.4, 37°C) was added into Gel-HCeC-CaO 2 solution. Afterward, we added 10 mL cooking oil isolate deoxygenated PBS and air. We used the Dissolved Oxygen Meters (Mettler, Switzerland) to detect oxygen generation.

Cell experiment and design
We obtained the murine melanoma cells (B16F10) lines from the Chinese Academy of Sciences cell bank. Cells were cultured in RPMI 1640 culture medium containing 10% FBS and 1% penicillinstreptomycin in a controlled environment (37°C, 95% air, 5% CO 2 ).

Animal experiment and design
Animal model: All BALB/c-nu mice animal procedures were approved by SPF Biotechnology Co., Ltd in our experiments. The melanoma cancer model was generated by subcutaneous injection of B16F10 cell (5×10 5 ) suspended in PBS into the flank region of the right back of BALB/c mice and allowed them to grow into solid tumors.
Biodistribution of Gel-HCeC-CaO 2 in B16F10 tumor-bearing mice: Mice were injected with 100mL Ce6, HCeC, and Gel-HCeC-CaO 2 after the tumor volume reached about~65 mm 3 . We used an in vivo imaging system (Perkin Elmer, America) to observe the mice at 0,4, and 20 h after injection. And the major organs such as the liver, spleen, kidney, heart, lung, and tumor were excised for further imaging analysis.
Histological analysis and anti-metastatic activity of mice: The tumors of all mice were collected and subjected to the H&E staining or TUNEL assays after the mice were sacrificed. We used a light microscope (Olympus, Japan) to observe the histological change. At the same time, the lungs of mice were also excised and photographed (47).

Quantitative polymerase chain reaction
The oligonucleotide primers were designed by Sangon Biotech (Shanghai, China) and listed in Table 1. The total RNA was extracted by TRIzol reagent and quantified by NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA). The PrimeScript RT Master Mix and SYBR Green PCR Master Mix were used for performing the qPCR (48).

Statistical analysis
The data were expressed as mean values ± SEM, including at least three biological replicates. The Student's t-test and one-way analysis of variance (ANOVA) were utilized to determine the statistical significance of differences among groups. Statistical values are indicated according to the following scale: ⁎p < 0.05, ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001. All statistical analyses were performed by SPSS 19.0 software.

Characterization of HCe and HCeC
The MCeNPs was synthesized by the microemulsion method (49). As shown in Supplementary Figure 1, the solution was milky white in the process of magnetic stirrers, which stratified overnight. The upper popcorn liquid was a toluene organic phase containing MCeNPs and the underlying dark yellow liquid was an aqueous phase containing CeNPS-Al. Due to the lack of effective surface protection, MCNPs was agglomerated in solution and cleared by the endothelial reticular system (ERS) in the body. Therefore, HCe and HCeC were synthesized by probing the interaction between CeNPs and HA to improve the stability of MCeNPs. To measure the size distribution of HCe and HCeC, we performed the DLS. As shown in Figure 1A, the average diameter of HCeC was approximately 25.60 nm while HCe was about 19.86 nm, because the Ce6 molecule increased the molecular diameter of HCe. And the zeta potential of the HCeC was approximately -6mV and the HCe was -5.6mV in Figure 1B, owing to the presence of the carboxyl group in Ce6. To examine the structure of the HCe and HCeC, the detailed morphology was characterized by HR-TEM. Due to the protective effect of HA (50), we found the size of HCe and HCeC are about 2-3nm and 3-4nm, showing good dispersion in water in Figure 1C. The Ce 3+ /Ce 4+ ratio is crucial for the enzyme-mimetic activity, the X-ray photoelectron spectroscopy (XPS) analysis of HCe and HCeC showed high Ce 3+ /Ce 4 + ratio in Figures 1D, E and Supplementary Figures 2A-C. Besides, the results of Fourier transform infrared spectra (FT-IR) showed that HA was conjugated to nanoceria successfully in Supplementary Figure 2D.
To verify that Ce6 was linked to HCe, the fluorescence spectra of Ce6, HCe, and HCeC were determined by a fluorescence spectrometer at 405 nm excitation light in Figure 1F, and the characteristic fluorescence peaks of Ce6 were between 640-690nm. Owing to the electron transfer between HCe and Ce6, HCeC showed significant fluorescence quenching compared with Ce6. The successful preparation of HCeC was validated by UV-vis absorption spectroscopy in Figure 1G. The characteristic absorption peak of Ce6 was at 404nm, with the Q wave between 500-700nm. The absorption peak of HCeC was similar to Ce6 besides a little red shift, which indicated that Ce6 had been successfully covalently linked to HCe. To explore the stability of nanomaterial, HCe and HCeC were mixed in a series of physiological solutions (water, PBS, DMEM, DMEM+10% FBS) for 24 h. As shown in Supplementary Figure 2E, we found there were no obvious agglomeration and precipitation of HCe and HCeC. These results indicated that HA modification could improve the stability of CeNPs effectively as literature report (51,52). Besides, we found that the mass ratio of Ce to Ce6 of the HCeC is about 1:0.33 Supplementary Figures 2F, G and the HA modification could not affect the photosensitivity of HCeC in Figure 1H. The HCeC showed high CAT-like activity in Figures 1I, J. Therefore, we demonstrated that HA had been successfully modified on the surface of nano-sized cerium oxide and covalently bound to Ce6, which provided a foundation for further biological application.

Characterization of the Gel-HCeC-CaO 2 system
Hydrogel was a kind of polymer material that had been widely studied at present (53), which could be converted between liquid and solid state with a change of temperature. To show the sol-gel transition behavior of the hydrogel by the test tube inverting method (44), the Gel and Gel-HCeC-CaO 2 were liquid at -20°Cˎ 4°C ˎ25°Cand 37°C. As shown in Figure 2A, the liquid transformed into gel at -20°C and 37°C and the sol state at 4°C and 25°C. With the extension of the bath time at 37°C, the hydrosol transformed into the gel phase gradually, and the rheological temperature was affected by the addition of CaO 2 and HCeC. The gelation time of Gel-HCeC-CaO 2 was approximately 9 s with the Gel needing 12 s and more time in Figure 2B. To observe the corrosion behavior of the Gel, the release experiment in vitro was performed, which showed the gel is almost completely dissolved at approximately 6 h in Figure 2C, following zero-order kinetics. Besides, the UV-vis absorption spectroscopy was used to investigate the photophysical properties of HCeC, Gel, Gel-CaO 2 , and Gel-HCeC-CaO 2 in Figure 2D, and the absorption peak of Gel-HCeC-CaO 2 was similar to HCeC, indicating that HCeC was mixed with the gel. Furthermore, owing to the catalase activity of HCeC, the O 2 generation of Gel-HCeC-CaO 2 in vitro was detected. As shown in Figure 2E, the generation rate of oxygen from Gel-HCeC-CaO 2 (PH=7.2) was fast compared with other groups, indicating that the catalase activity of HCeC was influenced by the PH value. Owing to the catalase activity of the Gel-HCeC-CaO 2 being affected by PH (54), the catalase activity of the Gel-HCeC-CaO 2 was the highest under neutral conditions while reducing the catalase activity under acidic conditions. Therefore, the Gel-HCeC-CaO 2 could release oxygen slowly to relieve tumor hypoxia in the acidic tumor microenvironment.

In vitro PDT of HCeC and Gel-HCeC-CaO 2 system
To detect the phototoxicity of Gel-HCeC-CaO 2 , cell viability was measured by CCK-8. There was almost no significant phototoxic effect observed in B16F10 cells treated with HCeC with or without irradiation in Figure 3A. However, the B16F10 cells treated with Gel-HCeC-CaO 2 (Laser) had a significant effect on B16F10 cells compared with Gel-HCeC-CaO 2 (Dark) in Figure 3B. Because the Gel-HCeC-CaO 2 (Laser) could release oxygen to improve the PDT efficiency, and produce a large number of 1 O 2 and ROS to kill tumor cells. Moreover, the H 2 O 2 (300 mM) could almost kill 50% of B16F10 cells owing to the toxicity in Supplementary Figure 3A, but the H 2 O 2 (100 mM) had almost no effect, confirming that the toxicity of Gel-HCeC-CaO 2 was not caused by H 2 O 2 .
To test the migration of B16F10 cells treated with Gel-HCeC-CaO 2 (Laser), we performed the scratch test. As shown in Figure 3C, the migration ability of B16F10 cells in the Gel-HCeC-CaO 2 (Laser) group was significantly lower compared with other groups. Therefore, the Gel-HCeC-CaO 2 (Laser) could enhance PDT efficiency and kill B16F10 cells significantly.

In vivo biodistribution of Gel-HCeC-CaO 2 system
To show the biodistribution of Gel-HCeC-CaO 2 in vivo, the fluorescence of Ce6, HCeC, and Gel-HCeC-CaO 2 in mice was detected at 0 h,4 h, and 20 h post-injection. As shown in Figure 3D, only a small amount of fluorescence appeared at the tumor site in Ce6 and HCeC group whereas predominantly observed in the Gel-HCeC-CaO 2 group at 20 h, suggesting that the good retention of the Gel-HCeC-CaO 2 in the mice. The biodistribution of the nanomaterials in mice was quantitatively analyzed in Figure 3E. Fluorescence imaging of some organs like lungs, liver, and kidneys showed that free Ce6 and HCeC were rapidly cleared over time up to 20 h, and the Gel-HCeC-CaO 2 had the good retention of the tumor to improve the PDT efficiency.

In vivo photodynamic effect of Gel-HCeC-CaO 2 system
The athymic nude mouse xenograft B16F10 model was generated for photodynamic therapy of melanoma. To examine the PDT efficiency of different materials to reduce tumor progression in the athymic nude mouse xenograft B16F10 model, 100mL PBS, Gel-CaO 2 , HCeC, Gel-HCeC-CaO 2 were intravenously injected into the mice at 1 week after engraftment, and the NIR irradiation (660 nm, 200 mW/ cm 2 , 5 min) was administered to the tumor site at 4 h and 20 h postinjection in the laser groups in Supplementary Figures 3B, C. The tumors of mice in each group were photographed, the Gel-HCeC-CaO 2 group could significantly inhibit the B16F10 tumor progression in Figures 4A, B. As shown in Figures 4C, D, the HCeC and Gel-HCeC-CaO 2 groups showed significant tumor regression with a reduction of the tumor volume by ∼42%, indicating the chemotherapy toxicity of the HCeC. The laser groups which irradiated one time could delay B16F10 tumor progression by∼69%, indicating that HCeC had obvious phototherapy toxicity. The Gel-HCeC-CaO 2 (Laser*2)had high tumor growth inhibition than HCeC(Laser*2), showing that Gel-HCeC-CaO 2 could satisfy repeat PDT therapy.
The tunnel assay confirmed that the Gel-HCeC-CaO 2 (Laser) group induce apoptosis of B16F10 cells in vivo in Figure 4E. Furthermore, hematoxylin and eosin (H&E) staining revealed large areas of necrosis, inflammatory cell infiltration and broken blood vessels of tumor tissue in Gel-HCeC-CaO 2 (Laser*2) group in Figure 4F. Hypoxia-inducible factor 1 a (HIF-1 a) was an important transcriptional regulator, which was crucial for tumor progression (55). The Gel-HCeC-CaO 2 system could significantly inhibit the expression of HIF-1 a compared with the HCeC in Figure 4G, indicating that Gel-HCeC-CaO 2 could alleviate the hypoxia of the tumor microenvironment. Besides, there were no metastatic tumor nodules observed in the lungs of the mice in Supplementary Figure 3D. To show the systemic toxicity of materials, we observed the body weight and histological change of mice. As shown in Figure 4H, the treatment could not change the body weight of mice. Therefore, the Gel-HCeC-CaO 2 (Laser) could reduce tumor progression in the athymic nude mouse xenograft B16F10 model significantly.

Discussion
Notably, the inadequate amount of oxygen generation and shortterm effects of photosensitizers induced by drug metabolism and clearance in the tumor environment cannot ameliorate the aggravated stuff supply mediated by PDT consumption (10)(11)(12), failing efficient and repeated PDT. Therefore, an efficient long-term oxygen supply and drug delivery system should be explored for PDT efficacy improvement and tumor inhibition. Herein, a prolonged oxygengenerating phototherapy hydrogel system (Gel-HCeC-CaO 2 ) was designed to alleviate tumor hypoxia, prolong drug supply, enhance PDT efficacy, and overcome melanoma. As shown in Figure 5, PSs loaded biocompatible CeNPs nanocarrier (HCeC) was synthesized by an HA-mediated self-assembly method under an alkaline environment, followed by incorporation into a thermo-sensitive hydrogel prepared by simple mixing Pluronic ® F127 and F68, the FDA-approved polymers (42). Sufficient oxygen generation was obtained by introducing an O 2 -evolving agent CaO 2 into the A B D E C FIGURE 3 In vitro photothermal of HCeC and biodistribution of Gel-HCeC- (E) Fluorescence imaging of some organs (Top: liver, spleen, kidney; Bottom: heart, lung, tumor). * p < 0.05; * * p < 0.01; * * * p < 0.001.
hydrogel system, which could react with H 2 O to form H 2 O 2 and followed produce O 2 catalyzed by catalase-like CeNPs. The prolonged oxygen supply was achieved by the prepared hydrogel. The prepared hydrogel could limit the infiltration of H 2 O into the system and moderate the hydrolysis rate of CaO 2 , resulting in sustained hypoxia alleviation in tumor tissues. Besides, the prepared hydrogel could act as a sustained delivery system and be easily injected around the tumor, resulting in prolonged drug accumulation. The accumulated FIGURE 5 Synthesis of the Gel-HCeC-CaO 2 system. The Gel-HCeC-CaO 2 system could alleviate tumor hypoxia microenvironment, prolong drug supply, enhance PDT efficacy, and overcome melanoma. HCeC entered into tumor cells by endocytosis mediated by CD44, a targeted receptor of HA overexpressed on most of the malignant tumors, resulting in a promoted PSs cellular uptake. Synthetically, a "once injection, repeat irradiation" strategy was achieved through the developed hydrogel system to enhance the PDT efficiency and overcome melanoma.

Conclusion
In this study, we prepared a Gel-HCeC-CaO 2 system which showed a significant phototoxic effect of B16F10 tumor by NIR irradiation. The Gel-HCeC-CaO 2 system showed the following advantages: (i) utilized the catalase activity of HCeC to produce O 2 ; (ii) good retention of the HCeC in the tumor; (iii) no severe systemic toxicity; (iv) alleviate tumor hypoxia environment, meeting the repeated photodynamic therapy strategy and effectively inhibiting tumor metastasis. Therefore, we believe the Gel-HCeC-CaO 2 system will provide a new strategy for the treatment of melanoma, which greatly alleviates the tumor hypoxia microenvironment to improve PDT efficiency.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by Centre for Diseases Prevention and Control of Eastern Theater.

Author contributions
LZ and XL had contributed equally to this work. All authors approved the final manuscript. HL, LZ, and XL designed the study and instructed all experiments. LZ and XL carried out the data analysis and drafted the manuscript. SR, YC, and ZC assisted in performing the experiments. YM, YQ, and HL provided many suggestions on the articles and obtained funding. All authors contributed to the article and approved the submitted version.