Injectable Hydrogel As Drugs Carrier Releases Artesunate And Alendronate Locally To Inhibit Breast Cancer Bone Metastatic Osteolysis

Advanced breast cancers easily metastasize to bone and this happens to about 70%of patients with advanced breast cancers.Bone metastasis can lead to osteolysis and bone destruction,resulting in bone pain,pathological fracture,hypercalcemia,spinal cord compression and other skeletal-related events which have serious impact on patients’ lives.The treatment of osteolysis caused by breast cancer bone metastasis bases on systemic chemotherapy.And local treatment can improve the symptoms of bone metastasis.Bisphosphonates and denosumab are the routine therapy for osteolysis accompanied by nonnegligible side effects such as mandibular osteonecrosis and hypocalcemia.Some traditional Chinese herb extractions were studied for their ability to inhibit the activation of osteoclast induced by cancer bone metastasis and obtained optimistic results.However,these extractions’ low bioavailability,poor solubility,short half-life and toxicity to liver and kidney limit their application such as artesunate used in this research.Some studies showed that artemisinin and it’s derivants which were usually used to cure malaria in clinic could inhibit osteoclasts activation caused by receptor activator of nuclear factor-KB ligand(RANKL)and not influence the function of osteoblasts.Similarly,the preliminary experiment indicated that ALN and ATS could synergistically inhibit osteoclasts formation induced by RANKL.Delightedly,nanotechnology and nanomaterials provide new strategies to build superior drug delivery systems that make drugs more efficient and secure to the body.These drug delivery systems,like nanoparticles,can reduce the influence from outside environment and protect drug’s activity.In the field of bone tissue engineering and regenerative medicine,many implantable biomaterials have been designed with good biocompatibility and osteoconductive ability to promote the bone tissue regeneration such as polymer scaffolds,calcium phosphorus composition and hydrogel.Actually,these implantable materials also possess good potentiality of loading and releasing drugs which can be explored to treat osteolysis.Inspired by these novel implantable materials and nanotechnology,this research resorted to BSA NPs and liposomes to encapsulate ALN and ATS respectively.And injectable collagen hydrogel was utilized to carry these two kinds of NPs to fabricate a local drug delivery system of ALN and ATS(COL/ATS/ALN)to suppress the osteolysis.The result showed that COL/ATS/ALN could release ATS and ALN sustainedly to curb the activation of osteoclasts and breast cancer cells’ proliferation but not to inhibit the function of osteoblasts.Vivo bone cancer pain model showed COL/ATS/ALN was able to alleviate hyperpathia.This thesis will demonstrate the work in following two parts:Part 1:The fabrication and character of injectable hydrogel loading ATS and ALNDesolvation method was used to fabricate BSA NPs encapsulating ALN(ALN-BSA).Ats liposomes(ATS-Lipo)were made through ethanol injection method.The morphology of the NPs were characterized by TEM.The particle size and dispersion index were tested through Zetasizer Nano analyzer.UV spectrophotometer was applied to analyze the encapsulation rate.The results showed that these two kinds of NPs were fabricated successfully with the size of about 200nm and encapsulation efficiency of 57.46%and 46.18%for ALN-BSA and ATS-Lipo respectively.Injectable hydrogel was composed of bovine type I collagen which was crosslinked by genipin.The hydrogel was thermosensitive that it kept liquid at 4℃ for a certain time and solidified rapidly at 37℃.The solidifying time could be regulated through the concentration of genipin.In the research,the collagen hydrogel with 20%concentration of genipin was utilized as the carrier of ALN-BSA and ATS-Lipo for the local release of ALN and ATS at injection site.Before the hydrogel solidified,two kinds of NPs were blended in it.After that,the hydrogel was placed in 37℃ to solidify(10-15min).FE-SEM,ATR-FTIR and Universal mechanical testing machine were used to test hydrogel’s physical and chemical property.The results showed that the hydrogel with 3D polyporous structure served as a good carrier of ALN-BSA and ATS-Lipo which also optimized the mechanical property of hydrogel(elasticity modulus 58.48KPa,crushing stress 0.0198MPa).The degradation property and swell ratio(SR)of hydrogel were also tested.The enzymolysis ratio and hydrolysis ratio of COL/ATS/ALN in 6 days were 87.74%±0.49%and 42.86%±0.16%respectively and the SR was 12.47±0.69.UV spectrophotometer was used to analyze the drug release of hydrogel.The results showed that COL/ATS/ALN hydrogel could release drugs in a more sustained way that the cumulative release of ALN and ATS were 72.29%and 81.7%respectively.Part 2:In vitro and in vivo researches of the hydrogel inhibiting osteolysisHuman breast cancer cells MDA-MB-231 and mouse macrophage RAW264.7 were cocultured with the help of transwell cell culture chamber with 0.4 um pore so as to build vitro co-culture model.Then,the extract liquid of hydrogel was added in the co-culture system.CCK8 method,TRAP activity test,immunofluorescent staining,RTq-PCR and western blot methods were taken to analyze the proliferation,differentiation and activation of osteoclasts.The release of ATS and ALN from COL/ATS/ALN could inhibit the proliferation,differentiation and relevant gene expression of osteoclasts.The extract liquid of hydrogel was added in the culture medium of MC3T3-E1 cells.CCK8 method,ALP activity test and Alizarin red S staining were taken to test the proliferation and function of osteoblasts.The ALP activity increased in COL/ATS/ALN group.The extract liquid was added in the culture medium of MDA-MB-231 cells.CCK8 method and Live-dead cell fluorescent staining were taken to test the proliferation of breast cancer cells.The results indicated that the ALN and ATS released sustainedly from the injectable hydrogel COL/ATS/ALN were not only able to suppress osteoclasts’ formation and activation and breast cancer cells’ proliferation but also promote the function of osteoblasts partly.Walker 256 cells were injected into the marrow of female SD rats’ left tibia to establish bone metastatic pain model.Prepared injectable hydrogel was injected into the left tibia marrow of model rats.Before and after the injection,the paw withdrawal threshold(PWT)of static mechanical stimulation of rats’left legs was tested to valuate the ability of COL/ATS/ALN to relieve hyperpathia.Imageological analysis was taken to analyze the bone destruction of metastatic tibia.And it was showed that COL/ATS/ALN hydrogel could alleviate the hyperpathia and osteolysis caused by breast cancer bone metastasis.The results in this part demonstrated that injectable hydrogel COL/ATS/ALN releasing ATS and ALN sustainedly had great potential of inhibiting osteolysis and alleviating the bone pain caused by breast cancer bone metastasis.