Oxidized Bacterial Cellulose Membrane Improves Photothermal Therapy For Inhibiting Postoperative HNSCC Residue & Case Report

Part one The preparation of implantable versatile oxidized bacterial cellulose membrane(TB/αPD-1@AuNCs/OBC composites)Objective:Studies have indicated that surgical resection is considered the primary option for treating head and neck squamous cell carcinoma(HNSCC),assisted by radiotherapy and chemotherapy in the clinic.However,more than half of patients with advanced HNSCC after surgery will develop local recurrence and outbreak lifethreatening.Conventional chemotherapy and radiotherapy as adjuvant treatments to avoid the recurrence of cancer after surgery are limited by systemic toxicity,drug resistance.Thus,Local drug delivery for postoperative cancer treatment have attracted much attention,combining clinical needs of clinician.In this part,we designed and constructed a multifunctional oxidized bacterial cellulose membrane(OBC)based therapeutic system for local photothermal therapy(TB/αPD-1@AuNCs/OBC),which could reduce the risk of residual tumor recurrence.Methods:The AuNCs were prepared using the galvanic replacement method.And it was characterized by transmission electron microscope(TEM),field-emission scanning electron microscope(FESEM),dynamic light scattering(DLS).The OBC was prepared by TEMPO-mediated oxidation.And it was characterized by FESEM,X-ray diffraction(XRD),Fourier-transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS).After absorption of αPD-1@AuNCs,the formed αPD1@AuNCs/OBC composites were then added with thrombin(TB)he obtained TBloaded αPD-1@AuNCs/OBC(TB/αPD-1@AuNCs/OBC)composites were stored at 4℃ for further experiments.TB/αPD-1@AuNCs/OBC was characterized by XRD,FESEM,FTIR,XPS and thermogravimetric analysis(TGA).H&E staining of BC and OBC in the tissue were observed the degradability of OBC after implantation into the surgical sites of mice for 14 days.Results:The characterization results of materials confirmed that the successful incorporation of AuNCs and TB into the OBC.The OBC-based nanocomposites have inherited the advantages of BC,such as high porosity,high crystallinity and high drug loadings.The OBC-based nanocomposites have remarkable degradability.Conclusions:TB/αPD-1@AuNCs/OBC as a locally therapy drug delivery system has excellent drug loading capacity and degradability.Part two Photothermal performance of TB/αPD-1@AuNCs/OBC composites and photothermal therapy ability in vitro by αPD-1@AuNCsObjective:Photothermal therapy is a non-invasive and highly spatiotemporal treatment that kills tumor cells using high heat energy from near-infrared(NIR)light-irradiated photothermal agents.On the basis of the first section,we have test photothermal performance of TB/αPD-1@AuNCs/OBC.Meanwhile,the potential mechanisms of SCC7 mice HNSCC cells were studied in vitro.Methods:The photothermal conversion performance of AuNCs was evaluated under NIR laser irradiation by an infrared thermal camera.The antibacterial activity of different treatments was determined against Gram-negative Escherichia coli by bacterial colonies.The behavior of αPD-1@AuNCs entering tumor cells was determined by flow cytometry.The cell viability was evaluated using CCK-8 assays.Meanwhile,fluorescence staining technique was used in cytotoxicity and HMGB1 detection.Western blot analysis was used to detect the protein expression levels of pyroptosis-related proteins in tumor cells after αPD-1@AuNCs treatment.The LDH release was performed using the LDH cytotoxicity assay.Meanwhile,the ATP was detected using an ATP assay kit.Results:TB/αPD-1@AuNC s/OB C have remarkable photothermal conversion performance and antibacterial activity.αPD-1@AuNCs under NIR laser irradiation could induce to undergo GSDMD-mediated pyroptosis by increasing reactive oxygen species(ROS),releasing damage-associated molecular patterns(DAMPs)and proinflammatory cytokines,such as HMGB1,ATP and LDH.Conclusions:TB/αPD-1@AuNC s/OBC as a locally photothermal therapy drug delivery system has excellent photothermal conversion performance.TB/αPD-1@AuNCs/OBC could induce pyroptosis of tumor cells and have antibacterial activity by photothermal therapy.Part three Study of improving antitumor immunity by implantable versatile oxidized bacterial cellulose membrane(TB/αPD-1@AuNCs/OBC composites)Objective:Recently,immunotherapy has emerged a treatment for HNSCC.However,the majority of patients remain unresponsive to the current immunotherapies that are based on immune checkpoint inhibitors.Improving the response rate to immune checkpoint blockades of HNSCC has already become present research focus.In this part,we applied TB/αPD-1@AuNCs/OBC to the HNSCC postoperative mice model to explore the possibility of inhibiting residual tumor recurrence and the effect of improving the systemic immune capacity.Methods:The TB/αPD-1@AuNC s/OBC was applied to the HNSCC postoperative mice model for treatment.The information such as tumor sizes and mice weights were measured.To investigate immune cell infiltration in the lymph nodes and spleen,monodispersed cells were detected using flow cytometry.Finally,the immunohistochemistry(IHC)was used to detect expression of immune related molecules in mice tumor tissue.Meanwhile,TB/αPD-1@AuNCs/OBC was applied to the HNSCC postoperative survival mice model to detect the life cycle of mice after receiving different treatments.The survival of mice was correlated with in vivo bioluminescence imaging in the corresponding period.The systemic biosafety of TB/αPD-1@AuNC s/OBC was detected by the haematological indicators of the routine blood test and the blood biochemical test.The major organs of the heart,liver,spleen,lung and kidney were stained by H&E staining to detect biological safety.Results:TB/αPD-1@AuNCs/OBC was remarkably inhibiting residual tumor recurrence an prolonging the survival of mice.Flow cytometry analysis indicated that the TB/αPD-1@AuNCs/OBC+L successfully promoted the DCs maturation,exhibited a significant increase in CD8+ T cells and reduced accumulation of myeloid-derived suppressor cells(MDSCs).Mice haematological indicators(WBC,RBC,MCH,PLT,ALT,AST,BUN and CRE)were within the normal range,which shown excellent biosafety of TB/αPD-1@AuNCs/OBC.H&E staining of the heart,liver,spleen,lung,kidney from tumor-bearing mice after various treatments demonstrated that there was no significant difference between TB/αPD-1@AuNCs/OBC group and PBS group.Conclusions:The TB/αPD-1@AuNCs/OBC developed herein enhanced systemic and tumor microenvironment antitumor immunity by increasing T-cell infiltration in the tumor site and immune organs,which could be effective in preventing tumor recurrence following surgery,prolonging survival,and could have excellent biosafety.Thus TB/αPD-1@AuNCs/OBC has multimodal interdisciplinary effects and potential clinical applications.