Self-assembly Of Inorganic Nanoparticles Mediated By Bombvx Mori Silk Protein For Tumor Therapy

Inorganic nanoparticles(NPs)have been widely studied in cancer therapy due to its special physical and chemical properties.Self-assembly is a simple regulation method,which can change the shape and arrangement of NPs and promote its application.Based on the good biocompatibility and stable structure,in this study,Bombyx mori(B.mori)silk protein(silk fibroin and silk sericin)were used as biotemplates to regulate the self-assembly of NPs.In this dissertation,silica nanoparticles(SiO2)and gold nanoparticles(AuNPs)were chosen as study objects.We explored the optimum condition for regulating the self-assembly of silica and gold nanoparitcles by silk protein,and made an elucidation about the regulation mechanism.After that,the assembled silica/silk protein and AuNPs/Silk protein nanocomposites were applied on the chemotherapy and photothermal therapy of tumor,respectively.The therapy efficiency was also evaluated.The results indicated the self-assembled nanoparticles can effectively inhibit tumor growth.Details are given in the following parts.(1)Nucleation and assembly of silica regulated by silk proteinWe first adopted silk fiber(SF fiber and SF nanofiber)as templates to regulate the self-assembly of silica.The results indicated silk fiber can regulate the silica into microtubes.The silica microtubes prepared using SF fiber as template had large pore size but unstable structure.And silica microtubes prepared using SF nanofiber as template was more stable with pore diameter at 500 nm and smooth wall.Therfore,silk fibroin(SF)and silk sericin(SS)were used as soft templates to regulate the nucleation and assembly of silica.The results indicated SF having a concentration from 0.1 mg/mL to 10 mg/mL can mediate the assembly of silica into nanospheres(termed as Si/SF)while SS with concentration varied from 0.01 mg/mL to 1 mg/mL can mediate the assembly of silica into nanofibers(termed as Si/SS).Moreover,the size and morphology of assembled Si/SF nanospheres and Si/SS nanofibers can both be adjusted by changing the concentration of APTES and TEOS.During the formation of silica nanocomposites,the secondary structure and surface potential also changed,which indicated silk protein mediated the nucleation and self-assembly of silica by hydrogen bond and electrostatic attraction.(2)Anti-cancer drug carriers based on silica/silk protein nanocomposites for tumor therapy.The assembled Si/SF nanospheres and Si/SS nanofibers were used as drug carriers for loading anti-tumor drugs,DOX and PTX.The release of drug was also studied.The results indicated both Si/SF nanospheres and Si/SS nanofibers have high drug loading efficiency,the loading efficiency of DOX can reach to 30%and for PTX also near 10%.The results of drug release indicated DOX can sustained release from silica nanocomposites and was sensitive to pH.Then Si/SF nanospheres and Si/SS nanofibers were cultured with HeLa cells for 12 and 24 h,the uptake and cytotoxicity of nanopaticles were studied.The results indicated Si/SF nanospheres can enter HeLa cells while Si/SS nanofibers can only adhere on the surface of HeLa cells.The Si/SF nanospheres and Si/SS nanofibers are both biocompatible with IC50 on HeLa cells overpassed 1 mg/mL.Then,DOX-loaded silica nanocomposites were cultured with HeLa cells for 1,2,3 and 5 d,respectively,the cell viability of HeLa cells was evaluated.The results indicated DOX-loaded Si/SF nanospheres and Si/SS nanofibers can easy to reach tumor cells,inhibiting the growth of tumor cells faster.So,compared to free DOX,DOX-loaded silica nanocomposites can kill tumor cells more effectively.(3)Self-assembly of AuNPs mediated by SFIn this study,positeve charged AuNPs were synthesized by sol-gel method with modification by PEI.Silk fibroin was used to mediate the assembly of AuNPs.The results indicated SF having a concentration from 25 ?gg/mL to 200 ?g/mL can mediate the AuNPs to form fibrous array(termed as AuNPs/SF).The morphology of assembly of AuNPs can be adjusted by changing the concentration of SF,time and the molecule weight of PEI.The change of surface potential and secondary structure of SF indicated it mediated the assembly of AuNPs by electrostatic attraction.The secondary structure of SF changed from alpha helix to beta sheet during the process.(4)Photothermal reagent based on AuNPs/SF nanofibers for photothermal therapy of tumor.The absorption spectrum of AuNPs/SF was firstly analysied.The result indicated the absorption peak of AuNPs shifted to red through self-assembly.AuNPs/SF nanofibers had higher light absorption in the near-infrared region.The thermal imaging indicated AuNPs/SF nanofibers had higher photothermal conversion efficiency than monodispersed AuNPs.Then,the photothermal therapy on tumor of AuNPs/SF was studied by using breast cancer cells,MCF-7 and Bcap-37,and nude mice bearing tumor as models.The in vitro results indicated,AuNPs/SF nanofibers with concentration at 100 ?g/mL can kill nearly all tumor cells under irradiation using 808 nm laser for 6 min.But AuNPs with same concentration can only kill half of tumor cells.In vivo results indicated in situ injection of AuNPs/SF can efficiently inhibit the growth of tumor.The tumor can hardly be observed posttreatment 2 weeks.But,the tumor in PBS and AuNPs groups continued to grow.Therefore,AuNPs/SF nanofibers can kill tumor cells more efficiently and inhibit the growth of tumor tissue.This study succeed to mediate the self-assembly of silica and gold nanoparticles with silk protein as templats.The loading efficiency of drug and inhibition ability to tumor cells of silica nanoparticles were enhanced by self-assembly,on the other hand,the optical propertie of Au nanoparticles was improved,increasing the photothermal conversion efficiency and broadening the application of AuNPs in photothermal therapy.So,our study provided a new idea that using Bombyx silk protein to mediate the assembly of inorganic nanoparticles,as to improve the properties of nanoparticles and increase the biological application.At last,our study broaden the application range of silk proein biomaterials.