Tissue adhesive indocyanine green‐locking granular gel‐mediated photothermal therapy combined with checkpoint inhibitor for preventing postsurgical recurrence and metastasis of colorectal cancer

Abstract Developing effective therapy to inhibit postoperative recurrence and metastasis of colorectal cancer (CRC) is challenging and significant to reduce mortality and morbidity. Here, a granular hydrogel, assembled from gelatin microgels by dialdehyde starch and interpenetrated with in situ polymerized poly(sulfobetaine methacrylate‐co‐N‐isopropylacrylamide) (P(SBMA‐co‐NIPAM)), is prepared to load and lock Food and Drug Administration (FDA)‐approved indocyanine green (ICG) with definite photothermal function and biosafety for photothermal therapy (PTT) combining with checkpoint inhibitor. The presence of P(SBMA‐co‐NIPAM) endows granular hydrogel with high retention to water‐soluble ICG, preventing easy diffusion and rapid scavenging of ICG. The ICG‐locking granular hydrogel can be spread and adhered onto the surgery site at wet state in vivo, exerting a persistent and stable PTT effect. Combined with αPD‐L1 treatment, ICG‐locking granular hydrogel‐mediated PTT can eradicate postsurgery residual and metastatic tumors, and prevent long‐term tumor recurrence. Further mechanistic studies indicate that combination treatment effectively promotes dendritic cells maturation in lymph nodes, enhances the number and infiltration of CD8+ T and CD4+ T cells in tumor tissue, and improves memory T cell number in spleen, thus activating the antitumor immune response. Overall, ICG‐locking gel‐mediated PTT is expected to exhibit broad clinical applications in postoperative treatment of cancers, like CRC.


Translational Impact Statement
This work demonstrates the application of ICG, the clinical photothermal agent, in postoperative treatment of colorectal cancer.The designed granular hydrogel not only prevents the diffusion of hydrophilic ICG to guarantee the PTT effect but also adheres stably to the whole wound area at body temperature.Therefore, ICG-locking gel-mediated PTT combined with a PD-L1 treatment is expected to exhibit broad clinical applications in the postoperative treatment of cancers like CRC.

| INTRODUCTION
Surgery resection is the first-line treatment for colorectal cancer (CRC), which is the most common malignant tumor of the digestive system worldwide. 1Unfortunately, 20%-30% of CRC patients are in the advanced stage, which cannot be easily rooted out through surgery.In addition, postoperative metastasis and recurrence severely affect patient survival. 2Currently, systemic radiotherapy and chemotherapy are the common intervention managements for preventing local tumor recurrence and distance metastasis after surgery.However, both radiotherapy and chemotherapy usually cause serious side effects and destroy immune systems, which limit their efficacy in prolonging patients' overall survival. 3Notably, surgery produces a transient immunosuppressive tumor microenvironment associated with wound healing, which leads to tumor cell proliferation, migration, invasion, and even immune escape. 4Therefore, it is urgent to develop new technologies to deal with postsurgery tumor recurrence and metastasis in CRC.
Cancer immunotherapy represented by immune checkpoint blockade (ICB) has held new promise for CRC treatment. 5For example, the anti-programmed cell death receptor-1 antibody (anti-PD-1 Ab) has been approved for high microsatellite instability (MSI-H) or mismatch repair deficiency (d MMR) in advanced CRC. 6 However, only approximately 5% of CRC patients have the characteristics of MSI-H/d MMR.
Most CRC patients have a low response rate to ICB, mainly because tumors lack immunogenicity and immunosuppressive microenvironment. 7[11] Photothermal therapy is a noninvasive means of cancer therapy, during which tumor tissues are ablated by the thermal effect of nearinfrared (NIR) light-absorbing materials that can convert photon energy to heat to kill tumor cells. 12Compared to other traditional treatments, PTT has already showed plenty of merits such as noninvasiveness, better controllability, and low incidence of complication. 13Furthermore, when combined with immunotherapy, PTT not only removes tumor tissues by thermal ablation but also induces tumor-specific antigen release so as to activate the body's immune response, leading to inhibiting recurrence and metastasis. 14,15Thus, a material-based platform for local cancer treatment is a promising strategy, which could focus PTT on the surgical site, allow one to break local immune tolerance, and stimulate the systemic antitumor immune response, thus inhibiting the tumor recurrence/metastasis and avoiding system side effects.

Various materials with photothermal function have been reported
and applied in PTT treatment. 16Given that most reported photothermal materials have not been verified in a clinic, U.S. Food and Drug Administration (FDA) approved indocyanine green (ICG), which possesses outstanding photothermal conversion efficiency upon NIR irradiation, as well as definite biocompatibility and biosafety, has received more expectations of clinical transformation. 17However, for local cancer treatment that focuses PTT on the surgical site, photothermal materials have to be easily applied to the surgical site and firmly fixed to the surgical site to ensure that they can play a photothermal function during the follow-up treatment.While ICG is amphiphilic, the application of ICG alone as the photothermal reagent in PTT on the surgical site is almost impossible due to its water-soluble nature and high diffusivity and rapid scavenging in vivo. 18Thus, there is a need to develop an ICG carrier, which can not only improve ICG retention and limit ICG diffusion but also realize convenient and accurate delivery of ICG onto the residual tumor site after the operation.0][21] However, it is very difficult to prevent amphiphilic ICG release from hydrogels that usually with an amphiphilic nature.In addition, in the humoral environment of most organs, hydrogel carriers lacking tissue adhesion performance will fall off from the surgical site, further limiting the curative effect. 22us, it is of high clinical value and also of great challenge to develop an injectable hydrogel with high ICG retention and tissue adhesive ability in the humoral environment to perform guaranteed PTT on the surgical site.Herein, according to the molecular structure of ICG with negatively charged hydrophilic side and hydrophobic side, 23,24 we designed an ICG-locking injectable hydrogel based on gelatin microspheres/dialdehyde starch granular hydrogel compounded with poly(sulfobetaine methacrylate-co-N-isopropylacrylamide) (P(SBMA-co-NIPAM)).The P(SBMA-co-NIPAM) that also possesses a negatively charged hydrophilic side and a hydrophobic side at body temperature was expected to effectively prevent ICG from releasing and diffusion.At the same time, the granular hydrogel was expected to be spread easily to the surgery site and adhere tightly to the surgery site for CRC postsurgical relapse and metastasis prevention.We hypothesized that such hydrogel could realize ICG application on the tumor site thus amplifying PTT efficiency.The combination of αPD-L1 was expected to eliminate residual tumor cells and prevent tumor recurrence and metastasis by promoting both innate and adaptive antitumor immune responses (Figure 1).and penicillin-streptomycin were obtained from Gibco BRL (Carlsbad, CA, USA).RPMI 1640 and phosphate-buffered saline (PBS) were obtained from Hyclone (Thermo Fisher Scientific, Waltham, MA, USA).Cell Counting Kit-8 (CCK-8) and Annexin V-FITC apoptosis detection kit were purchased from Dojindo (Kumamoto, Japan).In situ cell death detection kit, TMR red (TUNEL) was acquired from Roche (Roche Diagnostics GmbH, Germany).HMGB1 of human and mouse were purchased from Arigo Biolaboratories (Hsinchu City, Taiwan, ROC).ATP assay kit was acquired from Beyotime (Cat.S0026).

| Preparation of gelatin microspheres
Gelatin (0.2 g) and NHS (0.117 g) were dissolved in 2 mL of deionized water at 50 C. EDC•HCl (0.228 g) was dissolved in 1 mL of deionized water.Span (2 mL) was mixed into petroleum ether (30 mL)   and emulsified under 24,000 r/min for 30 s.The above gelatin/NHS solution and EDC•HCl solution were mixed evenly and quickly added into the emulsified petroleum ether (water oil ratio = 1:10) for further emulsification for 2 min, which was transferred into the round bottom flask containing 120 mL petroleum ether, and stirred at the stirring speed of 650 r/s.After 9 h, microspheres were obtained with three times ethanol washing and 24 h of vacuum drying.
Microspheres in dry and wet states were observed by scanning electron microscope (SEM) (SU-1500, HITACHI) and stereomicroscope (S9EDI, Leica), respectively.Size statistics were achieved during stereomicroscope observation via its dimension measurement software.

| Preparation of granular hydrogel
Dialdehyde starch was dispersed in deionized water at 70 C (concentration of 10 wt%), followed by being mixed with gelatin microspheres for centrifugation at 2000 rpm for 10 min.A granular hydrogel (GEL-DAS) was obtained.A solution containing NIPAM, SBMA, and the photoinitiator LAP (total solid concentration of 10 wt%) was added into vacuum-dried granular hydrogel (GEL-DAS), which was then irradiated under ultraviolet light for in situ radical polymerization for 30 min and then dialyzed in deionized water to obtain the composite granular hydrogel (GEL-DAS-PSN).At the same time, a solution only contained SBMA and the photoinitiator LAP (total solid concentration of 10 wt%) was added into vacuum-dried granular hydrogel (GEL-DAS) and for polymerization under the same protocol to obtain the composite granular hydrogel (GEL-DAS-PS) as the control group.A solution only contained NIPAM and the photoinitiator LAP (total solid concentration of 10 wt%) was added into vacuumdried granular hydrogel (GEL-DAS) and for polymerization under the same protocol to obtain the composite granular hydrogel (GEL-DAS-PN) as another control group.
For the rheological properties test, the granular hydrogels, GEL-DAS and GEL-DAS-PSN were transferred to the fixture of the rheometer with a 12 mm flat plate clamp.The modulus-frequency relationship was tested under the frequency scanning range of 0.01-100 Hz and the strain of 0.5%.Strain scanning measurements were carried out at a constant frequency of 1 Hz and a strain range from 0.1% to 1000%.The shear-thinning behavior was studied by measuring the viscosity change with the shear rate increased from 0.001 to 100 s À1 continuously. 25r the swelling test, the vacuum-dried granular hydrogels were weighted and immersed in deionized water at 25 C and weighed at 5, 10, 30, 60, 180, 300, 600, and 1800 s.The swelling ratio was calculated according to the following formula: SR = (W x -W 0 )/W 0 , where W x was the wet weight of the hydrogels at different time points, and W 0 was the dry weight of the hydrogels. 25

| ICG retention in granular hydrogels
ICG solution was added into the granular hydrogels, GEL-DAS, GEL-DAS-PSN, GEL-DAS-PN, and GEL-DAS-PS, and immersed in 0.01 mol/L PBS solution at 37 C for 24 h.Then, PBS solutions were collected into a cuvette, and evaluated on a Spectrophotometer (SpectraMax M2, Molecular Devices) at the wavelength of 660 nm.
To determine the amount of ICG released from hydrogels, ICG adsorbed in hydrogels were immersed in PBS.At 1 day and 5 days, PBS was collected to measure the concentration of the released ICG via the UV-Vis absorption spectra to determine ICG release. 23

| Tissue adhesive force of granular hydrogels
The lap shear tests were employed to measure the tissue adhesion capability of granular hydrogels.The gelatin solution was spread onto glass slides and dried at 37 C. Granular hydrogels, GEL-DAS and GEL-DAS-PSN were injected into the overlapped area between two glass slides at room temperature and compressed for 1 min.DXLL-10000 tensile machine (D&G Measure Instruments, China) was used to carry out stretching at a shear velocity of 1.3 mm/min until two glass slides were wholly separated. 26

| In vitro cytotoxicity assays
HCT116 and CT26 were seeded into 96-well plates separately at a density of 1 Â 10 4 cells per well and maintained with 100 μL RPMI 1640 medium.After 24 h, the culture media were replaced with 100 μL of fresh medium containing different concentrations of ICG and ICG@Hydrogel (CT26:0, 0.625, 1.25, 2.5, 5, 10, and 20 ug/mL; HCT116: 0, 0.625, 1.25, 2.5, 5, and 10 ug/mL).After 3 h, both of the above colorectal cells were washed by PBS and irradiated with or without 808 nm laser (1 W/cm 2 , MDL-F-808, Changchun New Industries Optoelectronics Technology Co., Ltd.) for 5 min, then changed into complete medium and incubated for another 24 h.The relative cell viability of the different groups was evaluated by the CCK-8 assay (Dojindo). 27e cells were stained with PI (dead cells, red) and Calcein-AM (live cells, green), respectively.The fluorescence images were obtained with an inverted fluorescence microscope (Leica). 277 | Flow cytometric cytotoxicity assay CT26 and HCT116 (5 Â 10 4 cells/well) were seeded in 24-well plates with 500 μL of culture medium overnight.Subsequently, the ICG or ICG@Hydrogel was added to the culture media containing different concentrations (CT26:0, 2.5, 5, 10, and 20 ug/mL; HCT116: 0, 1.25, 2.5, 5, and 10 ug/mL) for 3 h.After they were washed by PBS, the cells were exposed to 808 nm laser (1 W/cm2) for 5 min, then change into a complete medium.After incubation for 24 h, the cell suspension with a density of 1 Â 10 6 /mL was diluted by binding buffer. Ten, 5 μL annexin V-FITC and 5 μL PI solution were added and incubated for 15 min in the dark, followed by an analysis with a flow cytometer (Beckman).27

| In vitro photothermal characteristics
In vitro photothermal traits of the ICG@Hydrogel were characterized by monitoring the temperature change of ICG@Hydrogel aqueous solution under 808 nm laser (1 W/cm 2 ) irradiation for 5 min.Besides, ICG@Hydrogel was just prepared by dissolving powder-like hydrogel into 5 mg/ mL ICG aqueous solution.Laser irradiation-induced temperature elevation was recorded with an infrared imaging device (Fotric 111#L28). 129 | Immunogenic cell death (ICD) HCT116 and CT26 cells (5 Â 10 4 cells/well) were seeded into 24-well plates separately and maintained with 500 μL culture medium.After 24 h, the culture media were replaced with 500 μL of fresh medium containing different samples (PBS+L, ICG@Hydrogel, free ICG+L, ICG@Hydrogel+L).After 3 h later, cells were washed by PBS and irradiated with 808 nm laser (1 W/cm 2 ) for 5 min, then change into a complete medium and incubated for another 24 h.The supernatants were collected to determine for Human or Mouse HMGB1 ELISA Kit, and cells were collected for ATP detection, according to the manufacturer's instructions.11 For the CRT exposure assay, the cells were seeded in 35-mm confocal dishes at a density of 2 Â 10 5 cells/well for 22 h treated as described for the HMGB1 study.After incubation for 24 h, cells were fixed with 4% polyoxymethylene for 5 min, washed with PBS for 5 min Â 3, permeabilized with 0.1% Triton X-100 for 5 min, and washed with PBS for 5 min Â 3. Ater being blocked with 5% fetal bovine serum in PBS for 1 h, followed by staining with Alexa Fluor 488-CRT antibody (ab196158) for 4 C overnight, then washed with PBS three times and DAPI for 5 min and washed with PBS for 5 min Â 3. Finally, the samples were observed with the Confocal Laser Scanning Microscope (CLSM 510; Carl Zeiss, Inc.).11

| In vivo evaluation of hydrogel degradation and biocompatibility
All of the animal care, housing, and study procedures for mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Shanghai Putuo Hospital Ethics Committee (No. DWEC-A-202203005).The degradation of granular hydrogel in vivo was imaged by IVIS Spectrum In Vivo Imaging System (PerkinElmer IVIS Lumina III).BALB/c mice (6-8 weeks old) were subcutaneously coated with ICG@Hydrogel (ICG: 5 mg/mL) on the right flank during the surgery and then sutured.The mice were anesthetized with 2.5% pentobarbital sodium and imaged by IVIS to record the degradation of the granular hydrogel (ICG:Ex = 780 nm, Em = 831 nm)until the hydrogels disappeared.
Afterward, the wound was routinely coated with hydrogel for the hydrogel groups.Meantime, tumor tissue of the same size was inoculated on the left side to establish a bilateral mouse model, in order to test whether to eliminate the distant tumor.During the surgery, the mice were anesthetized with 2.5% pentobarbital sodium and sutured with sterile suture.For the laser groups, the entire wound region was irradiated at 0, 2, and 4 days (808 nm, 1 w/cm 2 ) for 5 min after the operation.And for the αPD-L1 groups, the mice were injected intravenously at 1, 3, and 5 days.Thermal imaging was captured using an infrared thermal imaging camera simultaneously.Tumor volume on both sides and weight of mice were measured every 2 days after the operation.Tumor volume was calculated using the following formula: tumor volume = 0.5 Â length Â width 2 .
For studies on the effect of PTT, when the tumor reached around 2500 mm 3 , all the mice were sacrificed.The lymph nodes, spleens, and tumor were harvested at the same time to detect immune cells.
Simultaneously, the main organs including heart, liver, spleen, lung, and kidney were also collected for histology analysis.For studies on the tumor recurrence model, mice were randomly divided into four treatment groups (n = 6): (1) Ctrl, (2) αPD-L1, (3) ICG@Hydrogel (ICG: 5 mg/mL)+L, (4) ICG@Hydrogel (ICG: 5 mg/mL) +L+αPD-L1.The operation and treatment scheme were the same as the bilateral mouse model.CT26 cells were inoculated subcutaneously on the opposite side of the mouse on the 30th day after the operation, the mice were observed for 60 days after the operation, during which the tumor volume was recorded every 2 days, and the survival rate was also counted.

| In vivo bioluminescence imaging
At 7, 14, 21, and 28 days after the operation, the tumor-bearing mice were anesthetized with 2.5% pentobarbital sodium and injected with d-luciferin (150 mg/kg), imaged by IVIS to monitor tumor and lung metastasis. 28

| In vivo photothermal characteristics(thermal image)
To examine the photothermal effect of the ICG@Hydrogel in vivo, the ICG concentration was 5 mg/kg, which was injected subcutaneously into the right blank of BALB/c mice.Then, the injection points were irradiated with 808 nm (1 W/cm 2 ) laser for 5 min, and the temperature change was detected by an infrared imaging device. 12

| In vivo toxicity assays (organ HE and skin HE)
The hydrogel was applied to the right blank skin of mice.The mice were sacrificed on days 7, 14, 21, 28, and 36 postsurgery, and in situ hydrogel and surrounding skin tissue sections were retrieved from the surgical site.
The major organs, including the heart, liver, spleen, lungs, kidneys, and tumors were also collected after the experiment finished, all these organizations were dissected, fixed in 10% formalin, embedded in paraffin, and sliced at 4 μm thickness.Then, slides were stained with hematoxylin and eosin (H&E) following a standard protocol and observed by a microscope (Leica). 11

| Cell flow cytometry analysis
Tumors, lymph nodes, and spleens were harvested, and the tissues were cut into small pieces with scissors and gently filtered through a 70 μm cell strainer.Red blood cells were removed with Red Blood Cell Lysis Buffer.Single-cell suspensions were washed and resuspended with PBS.The cells were incubated with Fc-Block (BioLegend) for 5 min on ice to avoid nonspecific binding.Subsequently, cells were stained with fluorophore-conjugated antibodies in the dark for 30 min on ice. 11r analysis of T cells in tumors and spleens, cell suspensions were stained with CD3-PE (BioLegend), CD4-FITC (BD Pharmingen), and CD8a-APC (BD Pharmingen).

| Immunohistochemistry assays
Tumors in different groups were collected, fixed in 10% formalin, and paraffin-embedded tissue sections were performed.After blocking and permeabilization, tumor tissue sections were stained with Ki67 (ab15580) antibody, rinsed in PBS three times, and then incubated with secondary goat anti-rabbit immunoglobulin G (IgG) at room temperature for 30 min.The slides were incubated with DAB (Abcam) for visualization after rinsing with PBS.Subsequently, the slides were washed in tap water, stained with hematoxylin for 15 s, rinsed in tap water again, and seal the slices. 11

| Immunofluorescence and TUNEL assays
For immunofluorescence staining, tumor sections were collected and permeated for 20 min, incubated with primary CD8 (ab217344) antibodies overnight at 4 C, followed by the secondary donkey anti-rabbit IgG at room temperature for 1 h.DAPI was employed for nucleus staining for 5 min and sealing the slices.In the TUNEL assay, the slides were stained with the TUNEL technique on the in situ cell death detection kit according to the manufacturer's instructions.The fluorescent images were acquired on the CLSM. 11

| Statistical analysis
All data are presented as mean ± standard deviation (SD).Statistical analysis was performed using GraphPad Prism software version 8.01.To compare the difference between groups, the one-way ANOVA test was utilized.T. Significant differences between the groups are expressed in the figures as *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.

| RESULTS
Using tissue adhesive hydrogel with photothermal function to cover surgical site for inhibiting postoperative recurrence and metastasis of CRC is a method with great potential to reduce mortality and morbidity.This study designed a granular hydrogel for carrying and trapping ICG with a photothermal function.The granular hydrogel was assembled from gelatin microgels by dialdehyde starch and interpenetrated with in situ co-polymerized poly(sulfobetaine methacrylate-co-N-isopropylacrylamide) (P(SBMA-co-NIPAM)).The main functions of the granular gel were tissue adhesion, ICG-locking, and injectability.
Hydrogel-carrying ICG was to perform definite photothermal function and biosafety for photothermal therapy (PTT) combined with a checkpoint inhibitor.

| Preparation and injectability of granular hydrogel
In this study, gelatin microspheres were prepared by amidation cross-linking combined with the emulsion method.There are abundant carboxyl and amino groups in gelatin molecules, which can be cross-linked by amidation between these carboxyl and amino groups with the presence of EDC and NHS in the aqueous phase.Thus, cross-linked gelatin microspheres by amide bonds were prepared in a water-in-oil emulsion system.According to the SEM image in Figure 2a, dry regular spherical particles were obtained.In water, the microspheres could adsorb water and swell with volume increase, forming microgels with a diameter range of 1-16 μm (Figure 2b,c).
Gelatin microspheres were assembled by dialdehyde starch.Dialdehyde starch contains a large number of aldehyde groups, which can form Schiff base interaction with gelatin. 30The dialdehyde starch suspension heated at 70 C and mixed with stacked gelatin microspheres could immediately form a bulk hydrogel as shown in Figure 2d.This hydrogel was named GEL-DAS granular hydrogel.Subsequently, SBMA and NIPAM monomers in GEL-DAS granular hydrogel copolymerized in situ to form poly (sulfobetaine methacrylate-co-N-isopropylacrylamide) (P(SBMA-co-NIPAM)) that penetrating through GEL-DAS granular hydrogel.The in situ co-polymerized P(SBMA-co-NIPAM) inside GEL-DAS granular hydrogel formed an interpenetrating network with gelatin microspheres, further physically cross-linking the microspheres, forming composite granular hydrogel, GEL-DAS-PSN (Figure 2e).Of note, P(SBMA-co-NIPAM) was not the key to the formation of gelatin microspheres and GEL-DAS-PSN.The introduction of P(SBMA-co-NIPAM) did not significantly change the size of gelatin microspheres (Figure S1).Its functions, including ICG-locking and adhesion strength promotion, were illustrated in the following evaluations.The temperature sensitivity of P(SBMA-co-NIPAM) was adjusted by changing the proportion of the two monomers (Table 1).
The introduction of SBMA significantly raised the lower critical dissolution temperature (LCST) of the copolymer.The LCST of P3 was the closest to body temperature (37 C).At the same time, due to the lower LCST of P1 and P2, the requirements for injection ambient temperature of granular hydrogels that are made from P1 and P2 might be more stringent.Thus, P3 was used in the related studies.As shown in Figure 2f,g, both GEL-DAS and GEL-DAS-PSN granular hydrogel were composed of assembled microspheres.However, the microstructure of the two granular hydrogels was different.Compared with GEL-DAS, GEL-DAS-PSN granular hydrogel showed a denser assembly of gelatin microspheres.There were multi-networks in GEL-DAS-PSN granular hydrogel, including a chemical cross-linking network of gelatin microspheres, Schiff Base interaction between dialdehyde starch and gelatin microspheres, and interpenetrating network among P(SBMA-co-NIPAM), as well as hydrophobic aggregation of PNIPAM at body temperature.For one thing, as shown in Figure 2h, the storage modulus (G 0 ) was greater than the loss modulus (G 00 ) under the scanning range of 0.01 to 100 Hz (25 C, strain 0.1%), illustrating that both GEL-DAS and GEL-DAS-PSN granular hydrogel exhibited elasticity of solid hydrogel.In addition, due to the addition of P(SBMA-co-NIPAM), the G 0 of GEL-DAS-PSN granular hydrogel was significantly higher than that of GEL-DAS granular hydrogel (Figure 2i).
For another, the reversible Schiff Base interaction also realized the injectability of granular hydrogels at a temperature lower than 37 C.As shown in Figure 3a, when the strain was small, G 0 was higher than G 00 .When the strain increased, G 0 began to decline, while G 00 began to increase to intersect with G 0 .The intersection point indicated the yield strain of hydrogel.When the yield strain was exceeded, G 00 was greater than G 0 , indicating that the granular hydrogels showed viscous behavior.Of note, the yield strain of GEL-DAS-PSN granular hydrogel was higher than that of GEL-DAS granular hydrogel.In addition, the viscosity of hydrogel showed a straight downward as the shear rate increased from 0.001 to 100 s À 1 .The granular hydrogels changed from the solid state to the "liquid state" with low viscosity, revealing the flow ability of the granular hydrogel during injection (Figure 3b).Besides, Figure 3c showed that the granular gels maintained their moduli after cyclic low-high strain action.
Under low strain, G 0 was greater than the G 00 .When the strain increased to 500%, the hydrogel was damaged, G 0 decreased quickly and was lower than G 00 .When the strain returned to the initial 1%, G 0 immediately recovered without significant decrease, indicating the self-healing property of the granular gels.The introduction of P(SBMA-co-NIPAM) did not show a significant effect on gel recovery.
Due to the shear-thinning, the solid granular hydrogel could evenly flow from nozzle and recovered to the solid state immediately after extrusion, forming a regular filament that extended vertically without breaking at room temperature (Figure 3d).The filament showed even and regular morphology without extravasation.After extrusion, as shown in Figure 3e, the filament was bent and stacked with no collapse on the surface glass surface.According to SEM observation (Figure 3f), the filament was composed of densely assembled microspheres.While GEL-DAS-PSN granular hydrogel could not be extruded when the temperature was higher than 37 C because of the gelation of P(SBMA-co-NIPAM).This was one of the reasons why P3 was chosen in the following studies.

| ICG retention in granular hydrogel
The GEL-DAS-PSN granular hydrogel was then employed as a carrier to combine with ICG for preparing ICG granular hydrogel.As shown in Figure 4a, the swelling behavior showed that both GEL-DAS and GEL-DAS-PSN exhibited quick adsorption of water after being immersed in PBS at room temperature.The swelling ratio quickly reached over 600 wt% within 60 s.Besides, the equilibrium swelling degree of GEL-DAS-PSN granular hydrogel was higher than that of GEL-DAS granular hydrogel.At the same time, due to the presence of P(SBMA-co-NIPAM), the equilibrium swelling degree of GEL-DAS-PSN granular hydrogel at 37 C was reduced.Thus, one of the unique features of the granular hydrogel was that it could quickly adsorb aqueous solution, such as ICG solution.As shown in Figure 4b, ing the effect of charge interaction on ICG-locking.Thus, the synergistic effect of charge interaction and hydrophobic association realized the tight bond of ICG to the GEL-DAS-PSN, so as to guarantee the continuous photothermal effect during the treatment period.The long-term in vitro (Figure 4h) and in vivo (Figure S3) ICG release from GEL-DAS hydrogel and GEL-DAS-PSN hydrogel was also monitored.
As shown in Figure 4h, Only 20% of ICG in total was released from GEL-DAS-PSN hydrogel, leaving nearly 80% of ICG locked in hydrogel, indicating that ICG release from GEL-DAS-PSN hydrogel was significantly limited when compared to that from GEL-DAS hydrogel, ensuring the application of photothermal performance in the treatment scheme.Besides, the in vivo ICG release from GEL-DAS hydrogel was relatively faster in 3 weeks when compared to that from GEL-DAS-PSN hydrogel, indicating the tight bond of ICG to the GEL-DAS-PSN (Figure S3).These results were consistent with our in vitro study (Figure 4h).
Thus, the photothermal conversion efficiency activity of ICG granular gel was evaluated by measuring the temperature changes under 808 nm radiation.As expected, the temperature increased rapidly along with time within 5 min then remained at 68 C (Figure 4I).
Moreover, during in vivo treatment, there were three times of irradiation within 7 days.Thus, ICG granular gel immersed in PBS in vitro for 5 and 7 days was also subjected to photothermal conversion evaluation.As shown in Figure 4i, the ICG granular gel kept its photothermal conversion efficiency without significant change, indicating that ICG granular gel possessed guaranteed photothermal conversion property for in vivo treatment.The GEL-DAS-PSN granular hydrogel carrying and retaining ICG was thus defined as ICGlocking gel.

| Tissue adhesion performance of ICGlocking gel
Due to the presence of dialdehyde starch, the granular gel exhibited significant tissue adhesive ability.As shown in Figure 5a, the GEL-DAS-PSN granular hydrogel could adhere to different matrices, including plastic, metal, rubber, and glass, which could bear a certain load in the air at room temperature.During in vivo application, dialdehyde starch could provide abundant hydrogen bonding and Schiff Base bonding with tissues to realize tissue attachment. 32,33As shown in Figure 5b, the granular hydrogel containing ICG could be extruded and spread onto pig skinlike creams.Of note, the ICG-locking gel adhered tightly to the pig skin even when being rinsed with warm water (Figure 5c).
Then, the ICG-locking gel was injected into glasses covered with gelatin to evaluate its tissue adhesive ability at body temperature.As shown in Figure 5d, two glasses coated with gelatin were adhered with ICG-locking gel.The lap shear tests illustrated that GEL-DAS-PSN showed larger displacement and higher adhesive strength at 37 C.According to Figure S2, granular hydrogel, GEL-DAS without P(SBMA-co-NIPAM) copolymer also showed high adhesive strength, while gelatin microspheres with P(SBMA-co-NIPAM) copolymer without dialdehyde starch showed significantly lower adhesive strength, indicating the important contribution of dialdehyde starch to hydrogel adhesion.Of note, in water, the adhesive strength of GEL-DAS significantly reduced, while in water at 37 C, the adhesive strength of GEL-DAS-PSN did not reduce, and was higher than that at room temperature.This might be related to the thermosensitivity of the P(SBMAco-NIPAM) copolymer.At 37 C, the P(SBMA-co-NIPAM) gelled to further enhance the granular hydrogel, so as to improve the adhesive force.At the same time, the P(SBMA-co-NIPAM) showed hydrophobic characteristics to prevent excessive water molecules from destroying the adhesion between gel and tissue.
Thus, gelatin and dialdehyde starch contributed the most to construction of granular hydrogel and its tissue adhesion ability, while the introduction of P(SBMA-co-NIPAM) copolymer not only realized the ICG retention in hydrogel but also ensured sufficient adhesion of the hydrogel in a wet environment.

| In vivo biodegradation of granular hydrogel
The biodegradation and biocompatibility properties of implanted hydrogels are critical for clinical applications. 30At first, the degradation of GEL-DAS-PSN hydrogel was estimated in vivo.The hydrogels were placed through extrusion subcutaneously into the flank of Balb/c mouse.The nodules that formed right after implantation were collected and photographed at different time points.As shown in Figure 6a, the hydrogels gradually degraded and disappeared completely in 5 weeks.At the same time, The degradation of ICGlocking gel was monitored by fluorescence in vivo imaging system (IVIS) imaging, and it was noticed that total degradation was achieved at 5 weeks, which was consistent with the above result (Figure 6b).
The addition of ICG had no influence on the degradation of the hydrogel.It was vital that the surrounding tissue at the implantation site returned to normal.In addition, no skin damage and toxicity were observed during the test period by hematoxylin and eosin (H&E) (Figure 6c).The in vitro cytotoxicity assay was further determined to confirm the biosafety of hydrogels.No obvious toxicity was observed both in CT26 and HCT116 cells (Figure 7a, Figure S4).These results indicated that the granular gel had good biocompatibility in vivo and in vitro.

| In vitro-induced ICD
The CCK-8 assay was used to investigate the cytotoxicity in HCT116 and CT26 cells treated with different concentrations of ICG-locking gel with 808 nm laser irradiation.The viability of the cells decreased with increasing concentration of ICG-locking gel under light exposure concentration (Figure 7b, Figure S5).The IC50 value was calculated to be 6.68 ± 0.89 μg/mL and 6.80 ± 1.19 μg/mL for the HCT116 cells and 9.80 ± 2.67 μg/mL and 6.26 ± 1.61 μg/mL for the CT26 cells, respectively.
In addition, the cell viability of HCT116 and CT26 cells treated with ICG-locking gel with 808 nm laser irradiation was also performed by calcein-AM/PI costaining (Figure 7c, Figure S6).The results were in good agreement with the CCK-8 assay.Subsequently, in order to eval- ICD is known to recruit antigen-presenting cells, dendritic cells (DCs), and promote them to engulf tumor antigen, then activate their maturation. 31ICD is characterized by the release of DAMPs, including CRT, HMGB1, and ATP. 34Given that PTT could induce ICD in tumor cells, we further explored whether ICG-locking gel-mediated PTT could exert an effect on CRT, ATP, and HMGB1.As indicated in upon ICG-locking gel treatment by ELISA assay (Figure 7g, h, Figure S9).To further investigate whether ICG-locking gel-mediated PTT-induced DCs maturation, a transwell system was employed.The upper chamber containing CT26 cells was pretreated with PBS+laser, ICG@Hydrogel, ICG+laser, or ICG@Hydrogel+laser, and the lower chamber was seeded with DCs harvested from BALB/c mice for 24 h.We then used flow cytometry to detect the expression of CD11c, CD80, and CD86, which represents mature DCs.As shown in Figure S10, the ICG+laser group showed a higher level of DCs maturation (72.23%) than the PBS+laser treatment (56.28%) and the ICG@Hydrogel (60.46%) treatment.And the ICG@Hydrogel plus a laser group led to the highest level of DCs maturation (81.19%).Our findings suggested that ICG-locking gel-mediated PTT could effectively induce ICD in CRC cells, which might hold excellent potential in antitumor immunotherapy.

| Inhibition of postsurgery tumor local recurrence and distant metastasis in vivo
Immune checkpoint inhibitors have been approved for the treatment of metastatic MSI-H/d MMR CRC. 35The antitumor effect of the ICGlocking gel-mediated PTT combined with αPD-L1 on CRC was further investigated in a bilateral tumor model.In order to simulate the clinical situation that some tumors could not be removed completely by surgery, especially for advanced-stage cancers, we performed the surgery results, no noticeable damage was observed in the major organs of mice (Figure S11B).These results confirmed that ICG-locking gel had no obvious systemic toxicity.
To further elucidate the underlying mechanisms of the antitumor efficacy, we analyzed the immune cell in the main immune organs by flow cytometry.Both innate and adaptive immune responses are indispensable for antitumor immunotherapy. 36As shown in Figure 9a, the number of NK cells in spleen was significantly increased in the ICG@Hydrogel+L+αPD-L1 group, suggesting that innate immunity was activated by PTT.In addition, we further investigated dendritic cells in lymph nodes, which play key roles in activating adaptive antitumor response.Our data indicated that ICG@Hydrogel+L(2.19-fold) and ICG@Hydrogel+L+αPD-L1(3.33-fold) groups effectively promoted DCs mature compared with other groups (Figure 9b).Apart from that, an increased population of CD3 + CD8 + T and CD3 + CD4 + T cells of tumor and spleen tissues were detected, especially in mice treated with ICG@Hydrogel+L+αPD-L1 among all the groups, indicating that ICG-locking gel implantation combined with PD-L1 triggers T cell significantly mediated adaptive antitumor immune response (Figure 9c,d, Figure S12).
Owing to the immunosuppressive post-resection microenvironment, the activity of antitumor leukocytes, such as DCs, NK cells, and cytotoxic T lymphocytes, might be suppressed, leading to tumor recurrence and metastasis. 37MDSC has the inhibitory function of immune cells, especially T cells.MDSCs could inhibit T cell proliferation and function by down-regulating the expression of IL-2, INF-γ, and granzyme B. 38 MDSC also reduces the number of CD8 + T cells and inhibits its cytotoxicity on tumor cells. 39,40As shown in Figure 9e, the number of MDSC notably dropped after treatment with ICG@Hy-drogel+L (75%) and ICG@Hydrogel+L+αPD-L1(80%) groups, which suggested that ICG-locking gel with αPD-L1 could reprogram a postsurgery immunosuppressive TME into an immunostimulatory one.
These data together confirmed that our designed ICG-locking gelmediated PTT combined with αPD-L1 produced a practical paradigm for preventing tumor recurrence and diminishing distance metastasis after surgery.

| Antilung metastatic tumors and long-term immune memory effect
Tumor metastasis as well as recurrence after surgery are the main factors for poor survival rate in CRC.To further verify the antimetastatic tumor activity of ICG-locking gel that combined with αPD-L1, the lung metastatic tumor model was established.The primary tumors were inoculated on day À7, and lung metastasis was established by injecting CT26 cells via tail vein on day 7 (Figure 10a).According to the luciferase intensity of mice, the ICG@hydrogel+L+αPD-L1 group showed less lung metastatic tumor lesions as compared with that in other groups, which was further confirmed by H&E staining of lung tissues (Figure 10b-d).
To examine the long-term immune memory effects, we established recurrent CT26 tumor models.To the recurrence of the tumor after the indicated treatment, the tumors were incompletely eradicated by ICGlocking gel and irradiation treatment or surgery (with a residue of ≈1% of the original volume) after the treatment for 3 weeks (Figure 10e).The mice receiving ICG-locking gel and irradiation treatment were better protected from local tumor recurrence (with a recurrence rate of 40%) (Figure 10f), and displayed slower growth of the tumor (Figure 10g), compared with untreated and αPD-L1 groups.Notably, mice in the ICG@hydrogel+L+αPD-L1 group had no tumor recurrence and exhibited the highest survival rate, as compared with other groups.The survival time of mice in the combination group was also significantly prolonged when compared to other groups (Figure 10h).The effector memory T cells (CD3+CD8+CD44+CD62LÀ) (Tem) in the spleens of the mice were examined by flow cytometry.ICG-locking gel and irradiation and αPD-L1 treatment generated much more Tem cells than the other groups (Figure 10i, j, Figure S13), suggesting that the combination treatment evoked an immune memory response.Although photothermal therapy combined with checkpoint inhibitor has been reported to show outstanding curative effect, various photothermal materials have not been verified in the clinic.Thus, the present chose ICG as the photothermal component, which has been approved by U.S. Food and Drug Administration (FDA) for clinical application, possessing outstanding photothermal conversion efficiency upon NIR irradiation, as well as definite biocompatibility and biosafety. 17 Besides, the PTT guarantee, convenient application onto surgical site was also one of the characteristics of the present ICG-locking granular gel.Previous studies including ours showed that granular hydrogel, a kind of bulk hydrogel formed by densely assembled microparticles, can exhibit properties of thixotropy under shear force, thus performing injectability through the design of particle interaction.The flowing granular gel can recover back to solid gel immediately after injection. 43,44Thus, the granular hydrogel is a "solid-to-solid" injectable system.Its thixotropy makes it act like the commonly used creams in dermatology that can be easily applied to the whole tissue area via spread without further extra external stimuli for gelation. 45mbined photothermal and immunotherapy demonstrates great

For
the tumor model of lung metastasis, the mice were intravenously infused with 2 Â 10 6 CT26 (Luc) tumor cells to generate a local tumor, the cells were washed with PBS before use and dissolved in 200 uL PBS.At 21 days after injection, imaged by IVIS to monitor local tumor and lung metastasis.

F I G U R E 3
Figure4c, the volume shrinkage was not found significantly in the present granular hydrogel.This might be because the granular hydrogel was constructed by densely assembled gelatin microgels, which mainly determined the volume of the hydrogel.Although the interpenetrating PNIPAM-based copolymers collapsed at 37 C, their collapse was not

5
Tissue adhesive ability of ICG granular gel.(a) General observation of granular gel adhered on different matrixes, including plastic, metal, rubber, and glass in the air at room temperature.(b) and (c) General observation of ICG-locking gel spread and attached to pig skin.(d) Lap shear tests and displacement-adhesive strength curves.n = 3, *p < 0.05.
uate the cell apoptosis caused by ICG-locking gel under the laser, the flow cytometry analysis was examined by Annexin V-FITC/PI apoptosis detection kit.As shown in Figure 7d, e and Figure S7, ICG-locking gel with 808 nm laser irradiation could effectively induce dosedependent cell apoptosis.Taken together, ICG laser irradiation possessed superior antitumor PTT activity.

Figure
Figure 7f and Figure S8, ICG-locking gel with irradiation-induced excellent CRT exposure according to the immunofluorescence in CT26 cells.Meanwhile, the extracellular HMGB1 concentration increased while the intracellular ATP level decreased significantly

F I G U R E 7
The antitumor effects of ICG-locking gel and evaluations of ICD in vitro.Cell viability of CT26 cells after treatment with free ICG solution and ICG-locking gel (a) without or (b) with NIR irradiation (808 nm, 1 W/cm 2 ) for 24 h determined by CCK-8 assay.(c) Calcein-AM/PI double staining of CT26 cells treated with different concentrations of ICG-locking gel with NIR irradiation (808 nm, 1 W/cm 2 ) (bar scale: 100 μm).(d) Apoptosis analysis of CT26 cells resulting from combined treatment with different concentrations of ICG-locking gel and NIR irradiation (808 nm, 1 W/cm 2 ) for 24 h.(e) Quantification for the apoptosis rate of CT26 cells.(f) CLSM images of CRT exposure on the surface of CT26 cells after different treatments with ICG-locking gel and NIR irradiation (808 nm, 1 W/cm 2 ) for 24 h (bar scale: 20 μm).(g, h) Extracellular HMGB1 and intracellular ATP levels of CT26 cells after different treatments as indicated.Data are presented as the mean ± SD (n = 3) for (e, g, h).(PBS +L = PBS and irradiation treatment; ICG@Hydrogel = ICG-locking gel treatment; Free ICG+L = ICG and irradiation treatment; ICG@Hydrogel +L = ICG-locking gel and irradiation treatment).(*p < 0.05, **p < 0.05, and ***p < 0.001).

8
photothermal conversion property in vivo.We verified the effect of ICG-locking gel-mediated PTT combined with αPD-L1 on recurrence after colon cancer resection.As shown in the bioluminescent IVIS imaging and tumor growth curves, the ICG@Hydrogel+L+αPD-L1 group (ICG-locking gel and irradiation treatment combined with αPD-L1) most effectively prevented local tumor recurrence and distance metastasis, while Hydrogel group (granular gel without ICG treatment), ICG@Hydrogel group (ICGlocking gel treatment), and αPD-L1 group (αPD-L1 treatment) showed a limited effect in inhibiting the tumor growth on both sides (Figure8c).Owing to the PTT-elicited antitumor immune responses enhanced by ICG-locking gel, the treatment in the ICG@Hydrogel+L group (ICG-locking gel and irradiation treatment) resulted in the elimination of four out of six local tumors on the right side, and obviously delayed growth of distant tumors on the left sides.Importantly, the efficacy was further amplified by αPD-L1, as indicated by the elimination of five out of six local tumors on the right side, and the elimination of all distant tumors on the left side.Meanwhile, we counted the survival rate within 60 days of mice in different groups (Figure8d, e), ICG@Hydrogel+L+αPD-L1 group showed a remarkable extension of survival time (more than 60 days) when compared to other groups (Figure8f).The tumor cell proliferation of tumor sections was evaluated by Immunohistochemical Ki67 staining.As indicated in Figure8g, the combination of ICG-locking gel and αPD-L1 exhibited much lower Ki67 expression, which indicated markedly decreased tumor cell proliferation after treatment.In TUNEL-stained tumor sections, the highest apoptotic rate was also detected in the combination group.In addition, there was no visible body weight loss among different groups, which was shown in FigureS11A.According to H&E staining
However, ICG alone cannot be fixed on the surgical site for its water-soluble feature and rapid scavenging in vivo.In order to ensure that ICG can play an adequate role in photothermal therapy after surgery, this study designed multi-levels of guarantees.For one level, a tissue adhesive function was designed into hydrogel to ensure the fixing of the ICG hydrogel.Dialdehyde starch could provide abundant hydrogen bonding and Schiff Base bonding with tissue to realize tissue attachment.In addition, thermosensitive P(SBMA-co-NIPAM) at 37 C showed hydrophobic characteristics to prevent excessive water molecules from destroying the adhesion between gel and tissue.The P(SBMA-co-NIPAM) gelled to further enhance the granular hydrogel itself, so as to improve the adhesive force and hydrogel stability.For the other level, hydrogel adhered on the surgical site should prevent water-soluble ICG from releasing.This was attributed to the presence of P(SBMA-co-NIPAM), which could form electrostatic attractive interactions and hydrophobic interactions with ICG at 37 C, significantly improved the retention of ICG in granular hydrogel and limited the diffusion of ICG.Besides, the degradation was as long as 5 weeks, revealing that the ICG granular gel was stable enough for the PTT course.Such multiple guarantees made the ICG stable and firmly fixed at the surgical site to play the role of PTT.
potential in the treatment of cancer postsurgical relapse and metastasis.Our strategy combined photothermal tumor ablation via ICG-locking hydrogel together with anti-PD-L1 checkpoint blockade therapy to achieve superior antitumor efficacy in CRC.Generally speaking, the combination therapy with ideal inhibition effect on the growth of primary tumor, and triggered an abscopal effect controlling distant tumor growth, as well as the immune memory protection to prevent tumor relapse may be explained by three mechanisms.First, PTT of ICG-locking gel kills local tumor cells by inducing apoptosis and necrosis, which could also generate tumor-associated antigens to elicit specific immune responses.Second, our gel-mediated PTT could effectively induce ICD in CRC cells by stimulating CRT exposure and releasing HMGB1 and ATP, as well as promote in vitro DCs maturation, thus promoting antitumor cell immunity.Third, our strategy could reprogram the postsurgery immunosuppressive TME by promoting DCs maturation, increasing the percentage of NK cells, enhancing the infiltration of effector T cells, and decreasing the number of MDSC in mice's main immune organs, leading to maximized antitumour immunity of anti-PD-L1 for synergistic cancer immunotherapy.Distinguished from previously reported hydrogel-based immunotherapy strategies, our hydrogel platform locked FDA-approved ICG with definite photothermal function and biosafety for PTT combined with anti-PD-L1, which is more suitable for clinical translation and application.The limitations of this hydrogel-based photothermal therapy for CRC are probably as follows: (1) it is hard to keep the hydrogel immobilized at the surgical site for a long time result from the unique position of the colon, which is the fundamental challenge for most hydrogel-based photothermal therapy in CRC; (2) the hydrogel is likely to interfere with postoperative healing; (3) because of the poor tissue penetration of NIR light in vivo, it is possibly difficult for PTT to achieve great antitumor effect.Therefore, it is urgent to establish hydrogels that not only have great biocompatibility but also could adhere tightly to colon tissue.It is worth mentioning that, endoscopebased clinical devices with imbedded laser optical fibers could be used to treat tumors located deeply inside the body to overcome the limitation of the penetration depth of light in tissues with NIR lasers.5 | CONCLUSION In summary, we demonstrated a combined photothermal immunotherapy strategy by spreading ICG-locking gel to tumor resection cavity for preventing CRC postsurgical relapse and metastasis.With the presence of P(SBMA-co-NIPAM), ICG was highly retained in hydrogel.With the photothermal action of ICG-locking gel on the postsurgical site, the combination treatment not only elicited systemic antitumor immunity but also reprogramed an impressive microenvironment, preventing local tumor growth, distance metastasis, and long-term recurrence.Considering the biosafety, biodegradability, and wellperformed tissue adhesive ability, the ICG-locking gel is not only suitable for postoperative treatment of CRC but also for other human cancers that can be resected.AUTHOR CONTRIBUTIONS Yuan Zeting: Data curation (equal); investigation (equal); methodology (equal); project administration (equal); supervision (equal); validation (equal); writingoriginal draft (equal); funding acquisition (equal).Ma Shuli: Investigation (equal); methodology (equal); validation (equal); writingoriginal draft (equal).Li Yue: Methodology (equal); validation (equal); writingreview and editing (equal).Fang Haowei: Methodology (equal); resources (equal).Shang Jing: Methodology (equal); validation (equal).Zhan Yueping: Formal analysis (equal); software (equal).Wang Jie: Formal analysis (equal); resources (equal).Chen Teng: Conceptualization (equal); resources (equal).Deng Wanli: Conceptualization (equal); funding acquisition (equal); resources (equal); validation (equal).Kunxi Zhang: Conceptualization (equal); funding acquisition (equal); project administration (equal); resources (equal); writingreview and editing (equal).Yin Peihao: Conceptualization (equal); funding acquisition (equal); project administration (equal); resources (equal); writingreview and editing (equal).ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants No. 81973700, 51973108, 81973625), The Science and Technology Innovation Project of Shanghai Putuo District Health Commission (Grants No. ptkwws202004), Clinical Specialized Discipline of Health System of Putuo District in Shanghai (Grants No. 2021tszk01), Shanghai Key Medical Specialty Construction Project (Grants No. ZK2019B18), Clinical Specialized Disease Construction Project of Shanghai Putuo District Municipal Health Commission (Grants No. 2019tszb01), The National Key Research and Development Program of China (Grants No. 2019YFC1316000).We also thank the funding support from the Fifth Batch of "Xinglin Scholar" of Shanghai University of Traditional Chinese Medicine, and the Reserve Outstanding Chinese Medicine Talent of Shanghai University of Traditional Chinese Medicine.
ZK2019B18; The National Key Research and Development Program of China, Grant/Award Number: 2019YFC1316000; The Reserve Outstanding Chinese Medicine Talent of Shanghai University of Traditional Chinese Medicine; The Science and Technology Innovation Project of Shanghai Putuo District Health Commission, Grant/Award Number: ptkwws202004 locking gel-mediated PTT is expected to exhibit broad clinical applications in postoperative treatment of cancers, like CRC.
Synthesis of copolymer.