Angiogenesis is a process of new blood vessel formation, which plays a significant role in carcinogenesis and the development of diseases associated with pathological neovascularization. An important role in the regulation of angiogenesis belongs to several key pathways such as VEGF-pathways, TGF-β-pathways, and some others. Introduction of small interfering RNA (siRNA) against genes of pro-angogenic factors is a promising strategy for the therapeutic suppression of angiogenesis. These siRNA molecules need to be specifically delivered into endothelial cells, and non-viral carriers modified with cellular receptor ligands can be proposed as perspective delivery systems for anti-angiogenic therapy purposes. Here we used modular peptide carrier L1, containing a ligand for the CXCR4 receptor, for the delivery of siRNAs targeting expression of VEGFA, VEGFR1 and endoglin genes. Transfection properties of siRNA/L1 polyplexes were studied in CXCR4-positive breast cancer cells MDA-MB-231 and endothelial cells EA.Hy926. We have demonstrated the efficient down-regulation of endothelial cells migration and proliferation by anti-VEGFA, anti-VEGFR1, and anti-endoglin siRNA-induced silencing. It was found that the efficiency of anti-angiogenic treatment can be synergistically improved via the combinatorial delivery of anti-VEGFA and anti-VEGFR1 siRNAs. Thus, this approach can be useful for the development of therapeutic angiogenesis inhibition.

Efficient and specific delivery of nucleic acid (NA) therapeutics to tumor cells is extremely important for cancer gene therapy. Various therapeutic strategies include delivery of DNA-therapeutics such as immunostimulatory or suicide genes and delivery of siRNA-therapeutics able to silence expression of cancer-related genes. Peptides are a promising class of non-viral vehicles which are biodegradable and can efficiently condense, protect and specifically deliver NA to the cells. Here we designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells. Combination of iRGD-modified and unmodified arginine–histidine-rich peptides during NA complexation resulted in carriers with different ligand contents. The NA-binding and protecting properties in vitro transfection efficiency and cytotoxicity of the DNA- and siRNA-polyplexes were studied and the most efficient carrier RGD1 was determined. The ability of the peptides to mediate specific intracellular uptake was confirmed inhuman cervical carcinoma (HeLa), human kidney (293T) and human pancreatic (PANC-1) cell lines with different αvβ3 integrins surface expression. By means of RGD1 carrier, efficient delivery of the herpes simplex virus (HSV-1) thymidine kinase gene to PANC-1 cells was demonstrated. Subsequent ganciclovir treatment led to a reduction of PANC-1 cells’ viability by up to 54%. Efficient RNAi-mediated down-regulation of GFP and VEGFA gene expression was achieved in MDA-MB-231-GFP+ breast cancer and EA.hy926 endothelial cells, respectively, by means of RGD1/siRNA polyplexes. Here we demonstrated that the peptide carrier RGD1 can be considered as promising candidate for development of NA therapeutics delivery systems useful in cancer gene therapy.

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvβ3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.

Endometriosis (EM) is a prevalent gynecological disease characterized by the abnormal growth of tissue similar to the endometrium outside of the uterus. This condition is accompanied by the development of new blood vessels in endometriotic lesions. While surgical intervention is effective in removing endometriotic lesions, some patients require multiple surgeries. Therefore, finding non-surgical treatments for EM is of great interest. One of the promising approaches is anti-angiogenic therapy using siRNA-therapeutics to target the expression of the VEGFA gene. Peptide-based polymers have shown promise as siRNA delivery systems due to their biocompatibility and ease of modification. We conducted a study to evaluate the effectiveness of the R6p-cRGD peptide carrier as a non-viral vehicle for delivering siRNA to endothelial cells in vitro and endometrial implants in vivo. We investigated the physicochemical properties of the siRNA-complexes, assessed cellular toxicity, and examined the efficiency of GFP and VEGFA genes silencing. Furthermore, we tested the anti-angiogenic effects of these complexes in cellular and animal models. The transfection with siRNA complexes led to a significant increase in VEGFA gene knockdown efficiency and a decrease in the migration of endothelial cells. For the animal model, we induced endometriosis in rats by transplanting endometrial tissue subcutaneously. We evaluated the efficiency of anti-angiogenic therapy for EM in vivo using anti-VEGF siRNA/R6p-RGD complexes. During this assessment, we measured the volume of the implants, analyzed VEGFA gene expression, and conducted CD34 immunohistochemical staining. The results showed a significant decrease in the growth of endometriotic implants and in VEGFA gene expression. Overall, our findings demonstrate the potential of the R6p-cRGD peptide carrier as a delivery system for anti-angiogenic therapy of EM.

Anti-angiogenic RNAi-based therapy can be considered as a possible strategy for the treatment of endometriosis (EM), which is the most common gynecological disease. Targeted delivery of siRNA therapeutics is a prerequisite for successful treatment without adverse effects. Here we evaluated the RGD1-R6 peptide carrier as a non-viral vehicle for targeted siRNA delivery to endothelial cells in vitro and endometrial implants in vivo. The physicochemical properties of the siRNA complexes, cellular toxicity, and GFP and VEGFA gene silencing efficiency were studied, and anti-angiogenic effects were proved in cellular and animal models. The modification of siRNA complexes with iRGD ligand resulted in a two-fold increase in gene knockdown efficiency and three-fold decrease in endothelial cells’ migration in vitro. Modeling of EM in rats with the autotransplantation of endometrial tissue subcutaneously was carried out. Efficiency of anti-angiogenic EM therapy in vivo by anti-VEGF siRNA/RGD1-R6 complexes was evaluated by the implants’ volume measurement, CD34 immunohistochemical staining, and VEGFA gene expression analysis. We observed a two-fold decrease in endometriotic implants growth and a two-fold decrease in VEGFA gene expression in comparison with saline-treated implants. RNAi-mediated therapeutic effects were comparable with Dienogest treatment efficiency in a rat EM model. Taken together, these findings demonstrate the advantages of RGD1-R6 peptide carrier as a delivery system for RNAi-based therapy of EM.

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.