Long-Lasting Postoperative Analgesia With Local Anesthetic-Loaded Hydrogels Prevent Tumor Recurrence Via Enhancing CD8~+T Cell Infiltration

Background:Postoperative pain(POP)may promote tumor recurrence and reduce the quality of life of cancer patients.However,in clinical practice,postoperative pain management is relatively independent from oncology treatment.Opioids are commonly used for postoperative pain relief,but they have numerous dose-related side effects.Analgesic regimens based on local anesthetics have attracted much attention because of their effectiveness and low price.However,the short duration of analgesia and the side effects of using local anesthetics at high concentrations have limited their application.Hydrogel is a hydrophilic polymeric three-dimensional network structure gel in which local anesthetics are loaded,which can effectively circumvent the disadvantages of local anesthetics.In addition,more and more studies have found that local anesthetics can directly kill tumor cells and inhibit tumor recurrence after surgery.However,it is not clear whether local anesthetics can exert immunomodulatory effects,such as regulating major histocompatibility complex class 1(MHC-I),which in turn promotes tumor killing by immune cells.Therefore,in this study,we synthesized a local anesthetic-loaded hydrogel and tested its effectiveness in reducing the toxicity of high doses of local anesthetics and prolonging postoperative pain;in addition,the above hydrogel was modified to test its effectiveness in inhibiting tumor recurrence and revealing the immunomodulatory function of local anesthetics.Methods:The local anesthetic ropivacaine was loaded into Pluronic F127(PF)temperature-sensitive hydrogel to synthesize PFR hydrogel.Mice were randomly divided into three groups,namely,control group,free ropivacaine(Rop)group and PFR group,after plantar inoculation with 4T1 tumors.Mice underwent plantar tumor resection under isoflurane anesthesia and butorphanol-based analgesia,in which mice in the Rop and PFR groups were locally injected with 30μL of Rop and PFR hydrogel(in which the concentration of Rop was 1%in both)at the postoperative incision,respectively;the incidence of convulsions in mice was counted.The paw withdrawal latency of thermal stimulation was measured before and 3,10,16 and 24 hours after surgery.The number of p-p38-positive cells in the dorsal horn of the spinal cord of L3～L5 segment was detected using immunofluorescence method by taking the spinal cord of mice from L3～L5 segment 3 days after surgery.In addition,MHC-I was used as an entry point to explore the immunomodulatory ability and anti-tumor effect of the local anesthetic-loaded hydrogel.First,the effect of PFR on MHC-I was tested in vitro.Given that impaired autophagic flow can upregulate cellular MHC-I,cellular autophagic flow was examined.In vitro 4T1 cells were cultured and divided into PF and PFR groups.After being treated with PF and PFR for 8 hours,respectively,lysosomes were stained using Lyso-Tracker and lysosome size was counted;P62 and LC3II protein levels were measured using protein immunoblotting(WB)to assess autophagic flow.Further,the expression level of MHC-I was detected using flow cytometry and immunofluorescence.To further enhance the immunotherapeutic effect of PFR on tumors,a hydrogel PFRM loaded with both ropivacaine and imiquimod was synthesized by incorporating imiquimod into PFR.Mice were subjected to in situ mastectomy,after which they were randomly divided into four groups,namely,control,PFR,PFM and PFRM groups,150μL of PBS,PFR,PFM and PFRM(The concentration of Rop was1%and the concentration of imiquimod was 0.03%)were injected in situ for analgesia immediately after surgery,on day 2 and day 4,respectively,and the tumor size and weight of the mice were measured continuously for the next 20 days,and the percentage of tumor recurrence was calculated.In addition,3 days after the last analgesia,draining lymph nodes were taken from mice,and the percentage of CD80~+CD86~+DC was measured using flow cytometry;20 days after tumor resection,tumor tissues were collected by executing mice,and tumor MHC-I was assessed using immunofluorescence,the ratio of CD8~+T cells,MDSC and Treg cells and the levels of cytokines TNF-αand IFN-γwere measured using flow cytometry.Results:PFR has good temperature-sensitive properties and appears gelatinous at 37℃,which facilitates the in situ retention of the drug;PFR can slowly release ropivacaine for more than 24 h.The injection of 1%free ropivacaine solution at the surgical site triggered convulsions in 16.67%of mice,but no mice showed convulsions after PFR administration;PFR administration significantly increased the threshold of thermal-stimulated paw withdrawal latency in mice for more than 16h after surgery.Moreover,PFR administration significantly reduced the number of p-p38-positive cells in the dorsal horn of the spinal cord of mice after surgery.In addition,compared with the control group,PFR significantly increased the volume of lysosomes in 4T1 cells,upregulated p62 and LC3II protein levels,and increased cellular MHC-I expression,suggesting that PFR has a good frontal immunomodulatory effect.The designed and synthesized PFRM group theoretically had stronger tumor recurrence inhibitory effects,and the results showed that hydrogels loaded with only ropivacaine(PFR)or imiquimod(PFM)could mildly inhibit tumor recurrence and reduce the volume of recurrent tumors.Further inflammation of the mechanism revealed that PFM and PFRM significantly increased the proportion of CD80~+CD86~+DCs in draining lymph nodes and the proportion of CD8~+T cells in tumors,suggesting that they could promote the maturation of DCs and sensitize tumor-specific CD8~+T cells;PFR and PFRM significantly upregulated the proportion of tumor MHC-I and CD8~+T cells,suggesting that they could directly promote recognition of tumor cells by CD8~+T cells.The experimental results also showed that PFRM maximized CD8~+T cell infiltration in tumor tissues,significantly suppressed the proportion of immunosuppressive cells such as MDSCs and Tregs,and increased the levels of cytokines such as TNF-αand IFN-γ.Conclusions:Ropivacaine-loaded Pluronic F127 temperature-sensitive hydrogel reduced the biotoxicity of high concentrations of ropivacaine and prolonged postoperative analgesia;hydrogels co-loaded with ropivacaine and imiquimod significantly inhibited postoperative tumor recurrence and recurrent tumor volume in mice by the mechanism that imiquimod sensitized tumor-specific CD8~+T cells,whereas ropivacaine upregulated tumor cell MHC-I and promoted tumor cell recognition and killing by CD8~+T cells.