Multilevel Response Hydrogel Loaded With Gambogic Acid In The Treatment Of Osteosarcoma

Objective:Osteosarcoma（OS）is a bone tumor with high mortality and poor prognosis.Currently,in addition to surgical treatment,chemotherapy is commonly used in clinical treatment,but traditional chemotherapy drugs lack tumor targeting and are easy to induce drug resistance.Besides,the specificity of bone tissue makes it difficult for drugs to reach the lesions,resulting in poor treatment and serious drug side effects.As a star molecule in natural active ingredients,Gambogic acid（GA）has great potential in the treatment of osteosarcoma,but its poor water solubility and short half-life limit its clinical translation.Therefore,aiming at the clinical problems of osteosarcoma treatment,this thesis explores a formulation process of gambogic acid liposome（GL）and a process scale-up method.Subsequently,a multilevel response gambogic acid hydrogel formulation for topical administration was synthesized.By studying anti-osteosarcoma effect and mechanism,it is expected to provide more options for clinical treatment of osteosarcoma.Methods:Firstly,PEG-ylated gambogic acid（GM）was synthesized through an esterification reaction.Subsequently,GL was obtained by ethanol injection method,after optimization,the stability of GL obtained by the process was investigated.Secondly,the acute toxicity of GL was investigated with Balb/c mice.Moreover,enzymatic and p H responsive hydrogel was synthesized by fibrinogen,thrombin and dicalcium phosphate nanowires.Then,morphology and structure characterization of the prepared hydrogel after loading GL.Finally,the anti-tumor effects and mechanisms of gambogic acid in osteosarcoma were explored at the molecular and cellular levels.Results:Firstly,GM was successfully synthesized,which was further prepared to obtain GL.Subsequently,a scale-up experiment of the formulation process was carried out to successfully prepare a 500 m L scale GL sample with uniform and stable quality.Next,through the acute toxicity test,it was found that the median lethal dose(LD₅₀)of GL（65.250 mg/kg）was significantly higher than that of the free drug GA（47.211 mg/kg）.In addition,physiological and biochemical indicators also showed that GL was less toxic than free GA.On this basis,we prepared GL-loaded multi-level response hydrogels and showed that the drugs were successfully loaded into the backbone of the hydrogels through morphological characterization.Moreover,in vitro release experiments found that the drug release in the hydrogel is acid responsive.In addition,we found that gambogic acid can induce exogenous calcium into osteosarcoma cells,causing intracellular calcium overload,thereby promoting osteosarcoma cell apoptosis,and inhibiting tumor migration and proliferation.Finally,we show at the molecular protein level that the combined treatment of gambogic acid and exogenous calcium in osteosarcoma cells can enhance anti-tumor effect.Conclusion:In this thesis,500 m L of homogeneous and stable GL was successfully prepared,which significantly improved the safety of gambogic acid and laid a foundation for the clinical translational research of gambogic acid.In addition,we also prepared enzyme-responsive and acid-responsive,biodegradable slow-release hydrogels with three-dimensional network structure,which can be loaded with GL for local delivery of osteosarcoma.It is expected to overcome the defects of short half-life and poor selectivity of gambogic acid.Finally,through in vitro cell studies,we found that gambogic acid can promote the entry of calcium into osteosarcoma cells,thereby causing intracellular calcium overload and inducing apoptosis through endoplasmic reticulum stress,mitochondrial damage and other pathways.