Study On Heparin-Modified Graphene Oxide Transdermal Delivery System And The Tumor Deep Transportation,Tumor Microenvironment Regulation And Immune Activation

Enhancing skin penetration and facilitating tumor infiltration in melanoma treatment through transdermal administration remains an intractable challenge.This study presented a Heparin(Hep)-functionalized graphene oxide(GO)for the transdermal delivery of docetaxel(DTX)and 1-methyl-D-tryptophan(1 MT).Briefly,Hep was grafted onto GO through an amido bond to construct Hep-functionalized-GO(GH),followed by loading of DTX and 1MT throughπ-π conjugation(Scheme 1).DTX-and 1MT-loaded GH(D-1/GH)was entrapped in a carbomer 941 gel for topical application on the surface of the tumor skin.D-1/GH penetrated the skin through the appendage pathway as well as-the intercellular route,which was enables through Hep-mediated stratum corneum(SC)hydration.The EPR-like effect at the junction of the tumor and skin,which was characterized by a relatively leaky tumor structure and enhanced retention owing to the affinity between Hep and proteins,permitted D-1/GH to infiltrate tumors.Thereafter,D-1/GH was trafficked to deeper tumors through the blood and lymphatic vessels.D-1/GH is wrapped into the lymphatic vessels through lymphatic vessel endothelial receptor-1(LYVE-1),forming special structures termed lymphatic transmigratory cups.This is the first report of a Hep-functionalized carrier permeating into tumors through the lymphatic transmigratory cup route by means of topical administration.Owing to satisfactory tumor infiltration,the D-1/GH-mediated synergistic therapy showed strong antitumor performance.In addition to the direct killing effect on tumors,the released damage-associated molecular pattern(DMAPs)induced by ICD initiated antigen presentation and subsequent antitumor immunity.D-1/GH remodeled the TME,including downregulated Tregs,polarized M1 macrophages,relieved hypoxia,eliminated extracellular matrix(ECM),and reduced interstitial pressure(IP),which enabled the infiltration of immune cells.Local administration of D-1/GH has also been proven to inhibit abscopal tumors and lung metastases.Importantly,D-1/GH distribution in the major organs was limited,minimizing the side effects relevant to systemic injection.The main research contents and results are as follows:Chapter 1.Preparation and characterization of D-1/GHThe successful synthesis of Hep-ADH was confirmed by hydrogen nuclear magnetic resonance spectroscopy(1H-NMR).GO was synthesized by the modified Hummers’ method.XRD and UV-Vis pattern verified the successful synthesis of GO.Monolayer-dispersed GO was prepared by probe ultrasonic method,and the optimal ultrasonic time was 1 h by particle size screening.Next,GO-ADH-Hep(GH)nanosheets were constructed by reacting the carboxyl groups of GO with the amino groups in Hep-ADH.Fourier-transform infrared spectroscopy(FT-IR)and elemental analysis demonstrated that Hep was successfully modified to GO.The modification amount of Hep was calculated to be 21.6%by thermogravimetric analysis(TGA).The morphology and thickness of GO and GH characterized by atomic force microscopy(AFM)presented sheet structures with thicknesses of approximately 1 nm and 1.5 nm,respectively.The results of hydration particle size showed that the particle size distribution of GH was uniform,and its average particle size was 188.4 nm.DTX and 1MT were loaded onto GH through π-π conjugation.When the mass ratio of DTX to GH was 2:5,it obtained the best drug loading(DL)and encapsulation efficiency(EE).The DL of DTX and 1MT were 25.13%and 8.94%,respectively.Carbomer 941 was selected to prepare a gel for entrapping D-1/GH to enhance skin retention.D-1/GH had a uniform particle size distribution,with an average hydrodynamic size of about 160 nm,a PDI of 0.26,and a zeta potential of-26 mV.The particle size of D-1/GH remained stable for 72 h,indicating that D-1/GH has good stability.D-1/GH had good photothermal conversion ability and high photothermal stability.In vitro release results showed that D-1/GH had a sustained release trend and an NIR light-triggered release profile.Chapter 2.In vitro antitumor evaluation of D-1/GHCoumarin 6(C6)/GH was prepared by using C6 instead of drugs to fluorescently label GH.Cellular uptake study suggested that Hep modification could enhance the cellular uptake of C6/GH by improving the dispersion of GO in cell culture medium and keeping a smaller size of C6/GH.C6/GH is prone to internalization into tumor cells more than normal cells.The results of the cytotoxicity assay showed that GH had good biological safety and light-triggered cytotoxicity.The cytotoxicity of D-1/GH+L treatment indicated that PTT can augment the cytotoxicity of chemotherapy.Staining of live and dead cells implied that chemotherapy coupled with PTT elicited the strongest cell death effect.After D-1/GH+L treatment,mitochondrial membrane potential substantially decreased,suggesting the increased mitochondrial impairment.D-1/GH+L treatment had the strongest ability to induce apoptosis.EdU staining and colony formation assay showed that D-1/GH+L could inhibit the proliferation of B16F10 cells.After treatment with D-1/GH+L,cells exhibited robust microtubule aggregation,which hindered cell division.Cell cycle experiments demonstrated that the cell cycle was arrested in the G2/M phase.The scratch experiments confirmed that D-1/GH+L treatment distinctly restrained cell migration.Stress fibers and lamellipodia were hardly observed in cells treated with D-1/GH+L.Disrupted actin smoothened the cell morphology,which limited the motility capacity.D-1/GH was shown to inhibit the IDO pathway and downregulate kynurenine(Kyn)concentrations.D-l/GH triggered ICD under NIR irradiation,including the calreticulin(CRT)exposure,high mobility group box 1(HMGB1)release and efflux of heat shock protein 70(HSP70),which contributed to the activation of the immune system.Chapter 3.Ex vivo and in vivo transdermal studies of D-1/GHThe ex vivo skin penetration of D-1/GH gel demonstrated that D-l/GH notably enhanced the transdermal penetration of the drug and photothermal promoted the percutaneous penetration of drugs.The results of distribution in ex vivo skin indicated that C6/GH could enter the skin through the appendages and the intercellular pathway.The results of HE staining of the skin showed that C6/GH can interfere with the arrangement of the stratum corneum to promote percutaneous penetration.The results of skin scanning electron microscope(SEM)suggested that D-l/GH and D-1/GH+L overcame the skin barrier by disturbing the ordered structure of the skin surface.The results of transmission electron microscope(TEM)showed that D-1/GH could enter the skin through the intercellular and adnexal pathways.Skin differential scanning calorimetry(DSC)and FT-IR analysis indicated D-l/GH enhanced skin permeability by interfering with skin structure.In vivo skin and tumor penetration experiments confirmed that C6/GH can penetrate SC through the adnexal pathway.The EPR-like effect between skin and tumor tissue enabled C6/GH to infiltrate and accumulate inside the tumor,and C6/GH can be transported to deeper tumor tissue through blood and lymphatic vessels.Chapter 4,In vivo antitumor evaluation of D-l/GHThe results of in vivo distribution revealed that transdermal administration of D-1/GH offered a feasible means to avoid its distribution in normal tissues,thus lessening unwanted systemic adverse effects.The results of in vivo photothermal performance indicated that D1/GH had excellent in vivo photothermal effect.Tumor hypoxia relief study suggested that the D-1/GH-mediated photothermal effect effectively alleviated tumor hypoxia by dilating blood vessels,increasing blood flow rate,and increasing oxygen supply.Anti-primary tumor studies demonstrated that D-1/GH-mediated synergistic therapy had significant antitumour effects and a good biosafety profile,remodeling the tumour microenvironment(TME),promoting vascular normalisation,down-regulating hypoxia-inducible factor 1α(HIF-1α),cyclooxygenase-2(COX-2)and the immune checkpoint CD47 and programmed death ligand 1(PD-L1),and inducing ICD effects in vivo.Antitumour immune studies have shown that D-1/GH+L treatment significantly promoted dendritic cell(DC)maturation,increased infiltration of helper T cells(Ths)and cytotoxic T cells(CTLs)in tumour tissue,enhanced the proportion of Ths and CTLs in the spleen,downregulated the proportion of Treg,reversed the phenotype of tumourassociated macrophages(TAMs)in tumour tissue and promoted pro-inflammatory cytokine secretion.Anti-abscopal tumor study showed that D-1/GH+L treatment can inhibit the growth of distal tumors and activate the systemic anti-tumor immune response.Anti-metastatic studies confirmed the D-1/GH+L had superior anti-metastatic efficacy.