Preparation Of Polyleucine Based Thermosensitive Hydrogel And Its Application Of In Situ Therapy For Rectal Cancer

Objective:Worldwide,rectal cancer is one of the most common types of tumors in digestive system.Traditionally,radical surgery,perioperative chemotherapy,and radiotherapy are preferred for treatment of rectal cancer,which have been widely utilized in the clinical treatment of rectal cancer.However,these treatment styles were not suitable and efficient enough for therapy of advanced rectal cancer.Checkpoint blockade immunotherapy,as hot spots in the treatment of rectal cancer in recent years,has been already acknowledged widely.Besides,up-regulated expression of programmed death ligand-1(PD-L1)is noticed after the management of molecular targeted therapy in some studies.This immune accommodation of tumor may hamper the molecular targeted agents-based antitumor efficiency.Therefore,checkpoint blockade immunotherapy that targets upregulation of PD-L1 can synergistically suppress tumor progress together with molecular targeted therapy theoretically.However,the synergistic utilization of checkpoint blockade immunotherapy and molecular targeted therapy may probably cause systematic side-effects.In view of that,we prepared an injectable polyleucine-based thermosensitive hydrogel drug delivery system,in which regorafenib(REG)for molecular targeted therapy,and a small molecule compound of BMS202 for PD-L1 inhibition are loaded.This antitumor agents-loaded drug delivery system is administered for in situ treatment of rectal cancer which,at the same time,can reduce the incidence of systematic side-effects,providing a theoretical and research fundament for clinical treatment of rectal cancer.Methods:Part 1: The L-leucine(L-Leu)based thermosensitive hydrogel(mPEG45-PLeu)was manufactured by the ring-opening polymerization(ROP)of L-Leu N-carboxyanhydrides(L-Leu NCA)in presence of the amino-terminated poly(ethylene glycol)(mPEG45-NH2).To confirm the successful synthesis of mPEG45-PLeu and calculate the degrees of polymerization(DPs),proton nuclear magnetic resonance(1H NMR)and Fourier-transform infrared(FT-IR)spectra were performed.The microscopic morphology of the mPEG45-PLeu was detected via scanning electron microscopy(SEM).The phase diagram,the rheological analyses,and in vitro degradation properties were further investigated to test the physical and chemical properties of mPEG45-PLeu,as a result of which,the sol-gel transition mechanism and the proper concentration of gel for in vivo anticancer assays were explicit.Part 2: CT26-Luc cell line stably expressing luciferase(CT26-Luc) and BALB/c mice were employed to establish an orthotopic rectal tumor model.Compared with the common subcutaneous tumor model,the orthotopic rectal tumor model in this study accurately simulated the tumor microenvironment(TME)when CT26-Luc tumor formed and progressed in the body,which made the in vivo antitumor evaluation of REG and BMS202 more convincing.Through precise submucosal injection,CT26-Luc cells were submucosally injected into the posterior wall of rectum.And tumor formation was monitored through bioluminescent detection using BRUKER Xtreme II.Part 3: After the successful synthesis of mPEG45-PLeu and the establishment of the orthotopic rectal tumor model,the REG and BMS202 carrier(mPEG45-PLeu(REG/BMS202))was administered for in situ antitumor treatment of tumor-bearing mice.After in situ treatment of rectal tumor,tumor burden at the given time points were monitored through bioluminescent detection using BRUKER Xtreme II to evaluate and quantitatively analyze the progress of tumors in different groups.Flow cytometry assays were performed to analyze the differences of CD8+ T cells in tumor tissues,tumor draining lymph nodes(TDLN),and spleens between different groups.Western bloting analysis was performed to further investigate expression of PD-L1 and p-VEGFR2 in tumor tissues.In addition,enzyme-linked immunosorbent assay(ELISA)was utilized to analyze the cytokines(IFN-?,TNF-?)in tumor tissues of mice after treatment.The immunohistochemical staining of each tumor specimen was performed to analyze the expression of casepase-3 and Ki-67.Finally,the systematic toxicity assessment was analyzed through monitoring the body weight of mice and performing histopathological analyses using HE staining of the organ specimens in each group.Results:Part 1: Characteristic peaks of amide bond were noticed at 1548 cm-1and 1653 cm-1 from FT-IR spectra of the copolymer,indicating that mPEG45-PLeu was successfully synthesized by polymerization.When the feeding mass ratio of mPEG45 / L-Leu NCA in the polymerization is 2.0 g/ 2.78 g,the 1H NMR spectra of the obtained copolymer showed that the DPs of leucine units in the copolymer was 17(mPEG45-PLeu17).The phase diagram of mPEG45-PLeu17 indicated that,at the concentration of3% and 4%,PBS solution of mPEG45-PLeu17 showed a process of sol-gel-sol phase transition where the copolymer solution displayed a sol phase under condition of body temperature.As a consequence of that,the copolymer solutions would not promote gelation after in vivo injection at the concentration of 3% and 4%.On the other hand,at the concentration of 5%,mPEG45-PLeu17 solution exhibited a solution-gel phase transition at low temperature and no longer dissolved as the temperature increased,which was suitable as a drug delivery system for in situ treatment.In the rheological analysis,the values of G' and G" intersected at low temperature,indicating a sol-gel phase transition behavior,which was consistent with data of the phase diagram.Microscopic morphology of mPEG45-PLeu17 displayed a porous structure from SEM detection.Based on in vitro degradation assays,we concluded that mPEG45-PLeu17 could be gradually degraded under the action of chymotrypsin and elastase which afforded to simulate the milieu interne of organisms.It is suitable for in-situ injection of drug-loaded copolymers and the antitumor drugs are gradually released.Consequently,mPEG45-PLeu17 was proper as a drug delivery system for in situ treatment of rectal tumor.Part 2: The orthotopic rectal tumor model was established via injecting CT26-Luc cells into the submucosa of the posterior rectal wall of BALB/c mice.And the rectal tumor formation could be monitored through bioluminescent detection using BRUKER Xtreme II.Part 3: After in situ treatment of mPEG45-PLeu17(REG/BMS202),the growth of rectal tumors in mice was significantly inhibited,and the tumor suppression rate(TSR)reached 75.76%,which was significantly higher than other groups.In flow cytometry analysis,the changes of immune cells in tumor tissues,adjacent lymph nodes and spleens were detected.The results showed that after treatment of BMS202,the proportion of CD8+ T cells in tumor infiltrating lymphocytes(TILs)increased significantly.However,the proportion of CD8+ T cells in tumor-draining lymph nodes(TDLN)and spleen cells did not change significantly.Results of western bloting showed that the utilization of BMS202 can inhibit the expression of PD-L1 in tumor tissues.At the same time,we found that the expression level of PD-L1 in the mPEG45-PLeu17(REG/BMS202)group was higher compared with the mPEG45-PLeu17(BMS202)group,indicating that REG treatment caused up-regulated expression of PD-L1.Outcome of ELISA showed that the expression of cytokines IFN-? and TNF-? in the treatment group containing BMS202 were significantly increased,indicating that BMS202 mediated the body to induce effective antitumor immune responses.The results of immunohistochemistry showed that the expression of Ki67 was decreased and the expression of casepase-3 was increased in mouse tumor tissues treated with REG,and the use of BMS202 had a synergistic therapeutic effect.In the results of systemic toxicity assessment,the body weights of the experimental animals in each group did not fluctuate significantly.At the same time,after 21 days of treatment in each group,the HE staining results of the main organs(heart,liver,spleen,lung,and kidney)sections did not show any significant change of damage or obvious toxicity,this indicated that the antitumor treatment in this study was relatively safe and would not cause treatment related side-effects.Conclusion:1.The thermosensitive hydrogel mPEG45-PLeu17 was developed for in situ treatment of mice rectal cancer with favourable properties as a drug carrier.2.Treatment of REG induced upregulated expression of PD-L1 in rectal tumor tissues of mice,which was more efficient in the synergetic treatment with BMS202.3.BMS202 regulated immune microenvironment status of tumor,improved ratio of CD8+ T cells in TILs,and boosted secretion of IFN-?and TNF-?in TME.4.In situ therapy of rectal cancer using mPEG45-PLeu17(REG/BMS202)showed good antitumor effect with no obvious systematic side-effects.