Anti-Tumor
Potential of htsFLT01/MiRGD Nano Complex: An In Vitro and In Vivo Study
Mohadeseh Khoshandam1 , Zahra-Soheila Soheili1 *, Saman Hosseinkhani2 , Shahram Samiee3 , Hamid Latifi-Navid1 , Hamid Ahmadieh4 , Hossein Soltaninejad5
- Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
- Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Department of stem cells technology and Tissue Regeneration, Faculty of Interdisciplinary Science and Technologies, Tarbiat Modares University, Tehran, Iran
Abstract: The anti-angiogenic gene htsFLT01 is a novel therapeutic gene designed to inhibit the growth of blood vessels in neovascularized pathologic angiogenesis situations like CNV, retinoblastoma and other solid tumors. The htsFLT01 gene produces a protein called sFLT01, which acts as a decoy receptor for VEGF. The MiRGD complex, comprised of histone H1 motifs for DNA compression and neutralizing its negative charge, the Gp41 motif from HIV for crossing the endosome membrane, and the NLS motif of the SV40 virus for nuclear membrane crossing, was utilized.
Methods: The htsFLT01 gene had been previously designed and constructed. The MiRGD peptide was expressed and purified using Ni-NTA affinity chromatography in the E. coli expression strain C41 (DE3). Cell viability and toxicity of the nanoparticles were determined through MTT assays in MCF7 cell cultures. Following transfection with the htsFLT01/MiRGD Nano complex at N/P = 14, conditioned medium and cell lysate were collected, and the expression of htsFLT01 protein was examined. Additionally, an expression analysis of key genes involved in the apoptosis pathway, obtained from previous studies, was conducted. To proceed with in vivo drug testing, 4-week-old female BALB/C mice were inoculated with cancer cells (4T1), following by the htsFLT01/MiRGD Nano complex locally injection into the cancerous mice. Histological analyses, including hematoxylin-eosin staining and immunofluorescence studies of tumors, were conducted.
Results: The MiRGD nanocarrier was successfully produced and purified. It effectively delivered its contents to MCF7 breast cancer cells using the iRGD targeting moiety. Protein analysis revealed intracellular production and extracellular secretion of htsFLT01 protein. Also, the results showed the very low toxicity of the htsFLT01/MiRGD complex compared to the htsFLT01/PEI complex. The function of the htsFLT01/MiRGD complex culminated to an increase in the expression of FADD, Cas8, and p53 genes in the apoptosis pathway in vitro. The histological results revealed decrease in angiogenesis and cell division, suppressed expression of VEGF, VEGFR, and CD31 protein, and increased apoptosis and necrosis of cancer cells in the treated mouse model
Conclusion: The htsFLT01/MiRGD nanocomplex as a novel anti-angiogenic therapy represented promising inhibition of tumor growth. It may serve as a novel medical approach for solid tumor management specially to control metastasis and invasion.
