Establishment And Evaluation Of Silk Fibroin Based Drug Loaded Nanoparticle And Hydrogel Systems For Anticancer Therapy

Purpose:Drug carriers for anti-cancer agents are effective to enhance antitumor efficacy.Aimed to establish safe,efficient and biocompatible drug carriers,silk fibroin was chosen as the structural material in this dissertation.This dissertation focused on:1.developing paclitaxel loaded silk fibroin without toxic chemicals and evaluating its antitumor efficacy in vitro and in vivo;2.based on intra-tumoural heterogeneity and cancer stem cells theories,developing salinomycin and paclitaxel silk fibroin nanoparticles incorporated hydrogel and evaluating its antitumor efficacy.Methods:Paclitaxel loaded silk fibroin nanoparticles(Ptx-SF-NPs)were fabricated by self-assembling of silk fibroin in aqueous solution under room temperature.Nanoparticle characterization and silk fibroin conformation studies were carried out by transmission electron microscopy,dynamic light scattering,Fourier transform infrared spectroscopy and X-ray diffraction.The cellular uptake studies were investigated on human gastric cell line BGC-823 and SGC-7901 by fluorescein loaded silk fibroin nanoparticles.In vitro cytotoxicity studies and apoptosis studies were also carried on those cell lines.Antitumor efficacy was evaluated on a BGC-823 xenograft model by monitoring tumor volume,tumor weight and pathological study.Salinomycin loaded silk fibroin nanoparticles(Sal-SF-NPs)were fabricated by self-assembling of silk fibroin in aqueous solution under room temperature.Nanoparticle characterization and silk fibroin conformation studies were carried out by transmission electron microscopy,dynamic light scattering,Fourier transform infrared spectroscopy and X-ray diffraction.Salinomycin and paclitaxel silk fibroin nanoparticles incorporated hydrogels(Sal-Ptx-NP-Gel)were prepared by dispersing Sal-SF-NPs and Ptx-SF-NPs in ultrasound processed silk fibroin solutions.The properties of injectability,swelling,biodegradation,drug loading homogeneity and drug release pattern were studied in vitro.In vivo degradability,biocompatibility,toxicity,antitumor and cancer stem cell inhibition on a murine hepatic carcinoma H22 subcutaneous tumor model were also investigated.Results:PTX-SF-NPs with a diameter of 130 nm were formed in an aqueous solution at room temperature by self-assembling of SF protein,which demonstrated mainly silk I conformation in the NPs.In cellular uptake experiments,coumarin-6 loaded SF NPs were taken up efficiently by two human gastric cancer cell lines BGC-823 and SGC-7901.In vitro cytotoxicity studies demonstrated that PTX kept its pharmacological activity when incorporating into PTX-SF-NPs,while SF showed no cytotoxicity to cells.The in vivo antitumor effects of PTX-SF-NPs were evaluated on gastric cancer nude mice exnograft model.We found that locoregional delivery of PTX-SF-NPs demonstrated superior antitumor efficacy by delaying tumor growth and reducing tumor weights compared with systemic administration.Furthermore,the organs of mice in NP reated groups didn't show obvious toxicity,indicating the in vivo safety of SF NPs.Sal-SF-NPs with a diameter of 120nm were fabricated under mild and non-toxic condition.The drug loaded SF-NPs were dispersed in the ultrasound processed SF solution before gelation.The resulting SF hydrogel(Sal-Ptx-NP-Gel)retained its injectable property,and demonstrate homogenous drug distribution compared to the non-NP incorporated hydrogel.Sal and Ptx were released from the hydrogel in a sustained manner.The in vivo degradation study in ICR mice proved the slow degradation of hydrogel for longer than 28 days after subcutaneous injection with low immunogenicity.The toxicity study showed that mice all survived after subcutaneous injection of the Sal-Ptx-NP-Gel containing Sal above the lethal dose of pristine Sal.After the above safety evaluation,anti-tumor efficacy studies were conducted on the mouse liver cancer cell H22 subcutaneous tumor model.Sal-Ptx-NP-Gel showed significantly better inhibition of tumor growth compared to single drug loaded hydrogel and systemic dual drug administration.Sal-Ptx-NP-Gel also significantly reduced CD44+CD133+tumor cells and demonstrated tand he least tumor formation in the in vivo tumor seeding experiment,indicating the superior inhibition of cancer stem cells.Conclusions:Ptx-SF-NPs enhanced antitumor efficacy by systemic and locoregional administration;Sal-Ptx-NP-Gel designed based on intra-tumoural heterogeneity and cancer stem cells theories demonstrated superior local tumor control.Thus,silk fibroin nanoparticles and hydrogels are promising drug carriers for anti-cancer agents,and they a potential future clinical cancer treatment regimen.