Construction Of Functionalized Silk Fibroin Nanoparticle Drug Delivery System And Its Anti-breast Cancer Effect

Cancer is a major disease affecting human health.There were approximately 19.3million new cases of cancer and 10 million cancer deaths worldwide in 2020.Among them,the total number of cancer deaths in China was 3 million,accounting for 30%of the cancer deaths worldwide,ranking first in the world.In addition,cancer ranked first among all causes of death in China.Among the many cancer types,breast cancer has become the first cancer type in the world,accounting for 11.8%of the incidence.The American Cancer Society’s latest cancer statistics in 2023 show that breast cancer is the most common cancer among women,accounting for 31%of all new cases of cancer in women.Breast cancer accounts for 15%of all cancer deaths in women.To make matters worse,the incidence of breast cancer in women continues to increase at a rate of about 0.5%per year.At present,chemotherapy is still the classic clinical choice in the treatment of breast cancer.However,repeated treatment of tumor cells with chemotherapy drugs can produce multi-drug resistance,which may lead to tumor recurrence and remote metastasis,which is one of the main causes of tumor chemotherapy failure and cancer-related death.In addition,breast cancer metastasis is also the main cause of death in breast cancer patients,and the survival time of breast cancer patients will be greatly reduced once metastasis occurs.In view of the difficult problems of multi-drug resistance and easy metastasis in the treatment of breast cancer,two kinds of functional silk fibroin nanoparticles were synthesized by means of desolvation method and layer-by-layer self-assmbly method.Firstly,folate-modified composite silk fibroin nanoparticles with the ability to target tumor cells was constructed to deliver chemotherapy drug and hypoxia-inducing factor inhibitor simultaneously.In addition,based on the copper sulfide nanonuclear structure,PD-L1siRNA was loaded using layer-by-layer self-assembly method.And the silk fibroin protein shell was wrapped to obtain functional silk fibroin nanoparticles with four mechanisms of copper death,photothermal effect,chemical kinetic therapy and immune activation,treating metastatic breast cancer synergisticially.In this paper,the synthesis methods,physicochemical properties,in vivo and in vitro therapeutic effects of these two functional silk fibroin nanoparticles and their molecular mechanisms against breast cancer were discussed in detail.Specifically,it includes the following two aspects:（1）PX478-loaded silk fibroin nanoparticles reverse multidrug resistance by inhibiting the hypoxia-inducible factorMultiple responsive silk fibroin nanoparticles,FA-PX478-DOX-SFNs（F-P-D-S）targeted by folic acid（FA）were prepared based on the desolvent method by co-loading the hypoxic-inducing-factor（HIF）inhibitor PX478 and the chemotherapy drug doxorubin hydrochloride（DOX）.It could actively target tumor cells,release HIF inhibitor PX478 in response to tumor microenvironment,inhibit HIF and its related downstream drug-resistant related target genes,and overcome the drug resistance of breast tumor cells.It was noteworthy that the prepared F-P-D-S could accelerate drug release in a simulated tumor microenvironment（low p H,high ROS and high GSH）.Compared with PX478-DOX-SFNs（P-D-S）without targeted modification,FA-functionalized F-P-D-S significantly promoted the uptake of nanoparticles by drug-resistant cells.In addition,after F-P-D-S entered drug-resistant breast cancer cells,lysosome escape could be achieved rapidly,so that DOX could enter the nucleus quickly and play a killing role on drug-resistant cells.Cytotoxicity test showed that the IC₅₀ of DOX against MCF-7/ADR cells was only 1.0μg/m L in F-P-D-S,which is about 26 times lower than that of free DOX（25.6μg/m L）.Real-time fluorescence quantitative PCR results showed that F-P-D-S significantly down-regulated the expression levels of HIF-1αand its downstream target genes MDR1,VEGF,GLUT-1.In particular,the MDR1 gene encodes P-glycoprotein（P-gp）,which is located in the cell membrane to pump drugs into the cell outside the cell,and is a major mechanism for the development of multidrug resistance in tumors.Furthermore,F-P-D-S was not easy to produce hemolytic toxicity,and the blood routine,liver and kidney function indexes and histological analysis of major organs after F-P-D-S injection in individuals indicated that F-P-D-S had good biosafety.（2）Cuproptosis-based silk fibroin nanoplatform loaded PD-L1 siRNA combining photothermal and chemodynamic therapy against metastatic breast cancerCuproptosis is a newly identified form of copper-dependent cell death that differs from other known cell death pathways.This new discovery points to a new direction for exploring the application of copper-based nanomaterials in cancer therapy.Here,a multifunctional copper-based silk fibroin nanoparticle,Cu S-PEI-siRNA-SFNs,was synthesized by using layer-by-layer self-assembling method on the basis of the nanpcore structure of copper sulfide nanoparticles（Cu S）,loaded with the siRNA of immune escape related gene PD-L1 by electrostatic adsorption,and finally wrapped with the shell of silk fibroin.Firstly,the photothermal conversion efficiency of Cu S-PEI-siRNA-SFNs was as high as 52.1%after irradiation by 808 nm near-infrared irradiation,and it had good photothermal heating profiles and photothermal stability.In addition,Cu S-PEI-siRNA-SFNs could produce a large number of reactive oxygen species to kill breast cancer cells through chemical kinetic effects after entering 4T1breast cancer cells.Cu S-PEI-siRNA-SFNs could also significantly reduce the expression of PD-L1 gene both in vivo and in vitro,thus reducing the occurrence of immune escape.Western blot assay showed that Cu S-PEI-siRNA-SFNs could induce the aggregation of lipoylated protein,especially dihydrolipoamide S-acetyltransferase（DLAT）.And Cu S-PEI-siRNA-SFNs could reduce the expression of ferridoxin 1（FDX1）gene,the upstream regulator of lipoacylation of protein in mitochondria,triggering the cuproptosis effect to kill breast cancer cells.The experimental results in mice showed that Cu S-PEI-siRNA-SFNs could accumulate in tumor sites of 4T1tumor-bearing mice with high efficiency and long retention,significantly inhibit subcutaneous tumor of breast cancer,and significantly reduce the number of lung metastatic nodules.Hemolysis test,routine blood parameters,liver and kidney function indexes,main organ sections and the trend of body weight of mice all indicated that Cu S-PEI-siRNA-SFNs had good biosafety.The results of immunohistochemical staining and immunofluorescence staining of tumor tissue showed that Cu S-PEI-siRNA-SFNs could significantly increase the distribution of CD4⁺/CD8⁺T cells in tumor tissues.The silk fibroin shell protected the PD-L1 siRNA in Cu S-PEI-siRNA-SFNs and had a sustained release effect on Cu ions in nanocore.Cu S-PEI-siRNA-SFNs inhibited breast cancer and its lung metastasis through the synergistic effect of cuproptosis,photothermal therapy,chemodynamic therapy and immune activation.In summary,this paper prepared PX478/DOX composite silk fibroin nanoparticles capable of achieving multiple responsive drug release in the tumor microenvironment,and functional silk fibroin nanoparticles integrating the synergistic effects of cuproptosis,photothermal therapy,chemodynamic therapy,and immune activation.They were used to solve the two major problems of multi-drug resistance and easy metastasis in the treatment of breast cancer.These two kinds of functional silk fibroin nanoparticles with good biosafety have achieved significant efficacy in the corresponding in vitro cell experiments and in vivo tumor treatment experiments in mice.These two silk fibroin nanoparticles have broad application prospects in breast cancer drug development and clinical treatment.In addition,the two nanodrug carriers synthesized in this study are based on silk fibroin protein extracted from natural cocoons,and the silk fibroin nanodrug delivery carriers have the advantages of simple preparation,easy chemical modification,multiple responsive drug release,long-acting sustained drug,and good biocompatibility,which play an important role in the therapeutic effect.Therefore,this study also provides a new idea and theoretical basis for the high-value utilization and transformation of silk protein.