Preclinical Evaluation of Angiogenesis Inhibition in Breast Cancer by htsFLT۰۱/MiRGD Nano complex

Gene therapy involves introducing therapeutic genes into cancer cells and tissues to induce cell death or slow tumor growth. Biological nanomaterials based on peptides have been considered for gene delivery due to their structural and chemical diversity, biocompatibility, specific targeting, drug-loading capacity, low toxicity, immunogenicity, and flexibility. MiRGD was specially designed from functional motifs of DNA condensing; it overcomes intracellular and extracellular barriers. We aimed to inhibit pathological angiogenesis by recruiting htsFLT۰۱/MiRGD in in vitro and in vivo breast cancer models. Materials and methods:The htsFLT۰۱ gene was previously designed and constructed. MiRGD peptide was expressed and purified by Ni-NTA affinity chromatography in E. coli expression strain C۴۱ (DE۳). The peptide's capacity to attach to htsFLT۰۱ was tested through an experimental procedure known as gel retardation. Cell viability and toxicity of nano complex were evaluated using MTT assays in MCF۷ cell culture. After transfection by htsFLT۰۱/MiRGD nano complex at N/P = ۱۴, conditioned medium and cell lysate were collected, and htsFLT۰۱ protein expression was checked. Also, expression analysis of the key genes of apoptosis, inflammation, angiogenesis, metabolic pathways, and migration pathways that were extracted from previous studies was performed. In order to complete the in vivo drug tests, first, ۴-week-old female BALB/C mice weighing ۱۵-۲۰ g were given cancer using ۴T۱ cells, and then the htsFLT۰۱/MiRGD Nano complex was injected locally into cancer mice. Histological analyses, including hematoxylin-eosin staining and immunofluorescence of tumors, were performed. Results: The MiRGD nano complex was successfully produced and purified. The gel retardation assay revealed that the peptide-based carrier MiRGD interacts with the plasmid carrying the htsFLT۰۱ gene at an N/P ratio of ۴ and beyond.These nano complexs effectively delivered its contents to MCF۷ breast cancer cells using the iRGD targeting moiety. Protein analysis revealed intracellular production and extracellular secretion of htsFLT۰۱ protein.Also, the results show the very low toxicity of the htsFLT۰۱/MiRGD nano complex compared to the htsFLT۰۱/PEI complex.Examining the expression of VEGF, MMP-۲, MMP-۹, MMP-۱۴, and HIF-۱ genes in angiogenesis and migration pathways showed that the htsFLT۰۱/MiRGD nano complex neutralizes the function of VEGF.On the other hand,a positive effect on regulating the expression of the vital genes IL-۱,COX-۲,NF-kB and TNF-,IL-۶,IL-۸,IL-۱۷,Bcl-۲,BAX,SLC۱۶A۸,and APOE in apoptosis, inflammation, and metabolic pathways.Animal studies showed decreased tumor size after injection of the second dose of the htsFLT۰۱/MiRGD complex (N/P = ۱۴)compared to the control groups.The results of H&E staining in ۴ groups showed decrease in angiogenesis and cell division and increase in apoptosis and necrosis of cancer cells in the treated mouse model that received the htsFLT۰۱/MiRGD nano complex. Immunofluorescence results showed decreased expression of VEGF protein followed by a decrease in the expression of VEGFR.The reduction of CD۳۱ protein as an angiogenesis marker confirmed the other results; it showed the efficiency of htsFLT۰۱/MiRGD in reducing angiogenesis and metastasis in a mouse model of breast cancer. Conclusion: The nanodrug complex htsFLT۰۱/MiRGD introduced a new anti-angiogenic therapy with promising improvements. It may be used as a new medical approach to manage cancer effectively and develop as an antitumor agent for breast cancer treatment.

کلیدواژه ها:

Gene Therapy ، htsFLT۰۱ ، MiRGD ، Anti-angiogenic ، Breast cancer