| BackgroundMelanoma is a malignant skin cancer with a mortality rate of 79%of skin cancers.Around 50%of melanomas exhibit mutations in the murine sarcoma virus oncogene homolog B1(Braf)gene,which promotes the proliferation and metastasis of melanoma.Currently,available therapies for melanoma include surgical treatment,immunotherapy,radiotherapy,and chemotherapy.However,their clinical application is limited by recurrent metastasis,high dose toxicity,poor drug tissue distribution,drug resistance,and low tumor targeting.Therefore,there is a need to explore novel treatments for melanoma.In recent years,combination therapies have achieved better therapeutic results in melanoma.Photothermal therapy(PTT),small interfering ribonucleic acid(siRNA)-based gene therapy(GT)and calcium ion interference therapy(CIT)have shown good application prospects.However,the poor tumor homing ability of photothermal converters under conventional delivery modalities limits the therapeutic efficacy of PTT.Injectable hydrogels have been widely used in the medical field to prolong the retention time of drugs at the disease site.Therefore,injectable hydrogels can be combined with photothermal conversion agents to overcome the limitations of PTT.In addition,siRNA is easily degraded by ribonucleases in vivo and has a low cellular uptake rate.In contrast,the three-dimensional network structure of injectable hydrogels can wrap,protect and deliver drugs(e.g.,siRNA,proteins and cells)and achieve controlled release of drugs.Therefore,injectable hydrogels are combined with siRNA to avoid the limitations of GT.Hydrogels are prepared by physical cross-linking and chemical cross-linking,and hydrogels formed by physical cross-linking have better biosafety and environmental responsiveness.While metal(such as Ca2+,Mg2+ and Fe2+)ligand crosslinking is widely used in physical crosslinking,among which,Ca2+can also exert the anti-tumor effect of CIT by disrupting Ca2+homeostasis in tumor cells,triggering calcium overload and promoting apoptosis of tumor cells.Therefore,an injectable hydrogel drug delivery system formed by Ca2+ligand cross-linking can be combined with a photothermal conversion agent and an anti-Braf gene siRNA(siBraf)for the treatment of melanoma.Building on the aforementioned research,this thesis explores the use of two hydrogels with pH/NIR responsiveness in treating melanoma by combining CIT,PTT and GT.The hydrogels used were calcium carbonate/polydopamine-sodium alginate hydrogels(CaCO3/PDA-SA hydrogels)and polydopamine@calcium phosphate/siBraf-sodium alginate hydrogels(PDA@CaP/siBraf-SA hydrogels).The aim is to investigate the synergistic therapeutic effects of CIT,GT and PTT using the novel hydrogel formulations.The findings of this thesis are hoped to provide new insights and methods for effectively treating melanoma.Purpose(1)Calcium-based nanoparticles can decompose and release Ca2+under NIR irradiation and weakly acidic environment,thereby inducing the formation of hydrogels from sodium alginate.The effects of NIR,pH and the concentration of calcium-based nanoparticles on the formation of hydrogels were investigated in detail.(2)To evaluate the anti-melanoma effect of hydrogels and initially explore the mechanism to clarify the therapeutic effect of injectable hydrogels-based CIT in concert with PTT on melanoma.(3)siBraf was introduced on the basis of the study of injectable hydrogel delivery system in combination with CIT and PTT for melanoma research.To investigate the inhibitory effect of multifunctional hydrogels on the growth of highly metastatic melanoma in vitro and in vivo,and further confirmed the advantages of combination therapy based on the injectable hydrogels delivery system in the treatment of highly metastatic melanoma.MethodsCalcium-based nanoparticles(calcium-based/siBraf nanoparticles)were prepared by gas diffusion method,and the obtained nanoparticles were dispersed in sodium alginate solution.The formation of hydrogels was induced by adjusting the pH to 6.5.The morphology,composition and structure of the nanoparticles were analyzed by transmission electron microscopy(TEM),X-ray diffraction spectroscopy(XRD),high-resolution transmission electron microscopy(HR-TEM)and X-ray photoelectron spectroscopy(XPS).Meanwhile,the network structure and rheological properties of the hydrogels were also studied by scanning electron microscopy(SEM)and rheometer.The photothermal conversion properties of the obtained hydrogels and their protection against siBraf were investigated.Subsequently,the anti-melanoma(highly metastatic melanoma)effect of the hydrogels and their mechanism of action were investigated,mainly including cytotoxicity test,cell scratching assay,cell invasiveness assay,western blotting test,Braf gene expression level determination,mitochondrial membrane potential determination and intracellular calcium ion level determination.Finally,the therapeutic effects of hydrogels on melanoma(highly metastatic melanoma)were evaluated comprehensively by establishing a tumor-bearing mouse model and a lung metastasis model,and its safety was evaluated in detail.ResultThe prepared nanoparticles were spherical in shape,and both hydrogels also showed a typical three-dimensional network structure with injectable features such as shear thinning and self-healing.The results of photothermal conversion experiments demonstrated that the prepared hydrogels had good photothermal conversion effects.The results of polyacrylamide gel electrophoresis indicated that the hydrogels could effectively protect siBraf from degradation by ribonuclease.In addition,both hydrogels exhibited pH/NIR responsive release behavior.In the weakly acidic environment of the tumor,the hydrogels released large quantities of siBraf and Ca2+,which was further accelerated by exposure to NIR.While inhibiting the expression of Braf gene,it triggers calcium overload and disrupts the mitochondrial Ca2+ buffering capacity,leading to mitochondrial dysfunction,inhibiting ATP synthesis,and promoting apoptosis in tumor cell.Moreover,PDA in the hydrogels(acting as a photothermal conversion agent)under NIR irradiation to increase the temperature of tumor cells,ultimately killing tumor cells.In vitro experimental results showed that both hydrogels combined with NIR irradiation had significant anti-melanoma effects,and the addition of siBraf significantly inhibited the metastasis and proliferation of highly metastatic melanoma.The results of animal experiments further confirmed this conclusion,and both hydrogels have high biosafety. |
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