Preparation And Pharmacodynamics Study Of Enzyme-responsive Drug Delivery System Based On Heparanase-1

Melanoma is an extremely malignant,highly metastatic tumor that has seriously threatened human life and health.Although researchers have developed a variety of treatments for melanoma,clinically,the poor prognosis of melanoma is still frustrating,and the tumor recurrence rate and patient survival rate after surgery or chemotherapy are very low.In order to solve this dilemma,based on a three-dimensional anti-cancer strategy,this project used a combination of chemotherapy and immunotherapy to treat melanoma,which can greatly reduce the drug resistance of melanoma and improve the cure rate of cancer patients.In this process,based on the characteristics of melanoma highly expressing heparanase-1,enzyme-responsive smart nanoparticles(PTX/Alloferon-1 NP)were prepared to achieve the co-loading of the chemotherapy drug paclitaxel(PTX)and the immunotherapy drug alloferon-1,and the anti-tumor effect of PTX/Alloferon-1 NP was far superior to other groups.This proved that this project has completed the efficient combination application of chemotherapy and immunotherapy,which has an important guiding significance for the application of immunochemotherapy in melanoma and its clinical transformation.The main contents of this project include:preparation,characterization and in vitro properties study of PTX/Alloferon-1 NP(preparation and prescription screening of related nanoparticles,physicochemical property characterization,fluorescence colocalization study,serum stability study,enzyme degradation study and in vitro release studies);in vitro cytology studies of related nanoparticles(B16F10 cell culture,cell uptake experiments,cytotoxicity experiments,apoptosis experiments and cell cycle experiments);in vivo distribution studies and pharmacodynamic studies of related nanoparticles(establishment of tumor model and experimental grouping,in vivo imaging and study of drug distribution in isolated organs,study of antitumor activity in mice,detection of TUNEL apoptosis and Ki-67 proliferation in mice tumor tissue);in vivo immune mechanism research and safety evaluation(immunofluorescence staining to determine the mechanism of alloferon-1 in vivo,HE staining studies of major organs and hematology studies).The main conclusions of this project include:the key mass ratios in PTX/Alloferon-1 NP included PTX/DOTAP=1:12,protamine/heparin=13.82:80,(heparin/protamine)/DOTAP=4.85:1.TEM photos showed that PTX/Alloferon-1 NP was round and uniformly distributed.The average particle size measured by dynamic light scattering was 106.1±1.113nm(0.147±0.005),and the ?-potential was-45.1 ±0.455mV,which contributed to improve its stability during low temperature storage,and avoided hemolytic effect during blood circulation,enhanced the safety and stability of nanoparticles in blood circulation.Enzymatic degradation experiments and in vitro release studies have shown that PTX/Alloferon-1 NP can rapidly realize the process of charge turnover and particle size reduction under the combined action of enzyme degradation and ion diffusion,which was conducive to the penetration of nanoparticles into melanoma core to achieve better efficacy.The in vitro cytology study of PTX/Alloferon-1 NP proved that nanoparticles were more likely to induce endocytosis.Respectively,the IC50 of free PTX group,free combo group,PTX NP group and PTX/alloferon-1 NP group were 57.22?g/mL,52.49?g/mL,14.42?g/mL and 13.16?g/mL,which proved that PTX/Alloferon-1 NP is more likely to cause toxicity to B16-F10 cells,and the subsequent apoptosis experiments and cell cycle experiments have verified it again and explored mechanism.In vivo distribution studies and pharmacodynamic studies have proved that PTX/Alloferon-1 NP is significantly enriched in the tumor site,and the in vivo anti-tumor of each formulation group was evaluated by the tumor inhibition rate experiment.The results showed that the PTX/alloferon-1 NP group had the best tumor-inhibiting effect,which greatly prolonged the survival time of mice,and it also did not affect the normal growth of mice.Subsequently,TUNEL fluorescence staining and Ki67 histochemical staining of tumor tissue sections also explained the mechanism of experimental results of tumor inhibition rate from the molecule level.The in vivo immune mechanism study of PTX/Alloferon-1 NP proved that alloferon-1 cannot directly kill tumor cells,but can activate NK cells to reverse suppression of tumor cells to other immune cells.The results of in vivo safety evaluation experiments showed that free PTX can cause severe damage to the liver,spleen,lungs and kidneys of mice,while PTX/Alloferon-1 NP has no organ toxicity.The above results showed that the PTX/Alloferon-1 NP prepared in this project had a very specific response to melanoma that highly expresses heparanase-1,and the PTX and alloferon-1 encapsulated in it can play a very important role in melanoma.The good inhibitory effect proved the efficient combined application of immunotherapy and chemotherapy,which provided a theoretical basis and experimental basis for the clinical transformation of this nanoparticle.