Serum-Resistant Ternary DNA Polyplexes for Suicide Gene Therapy of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a prevalent benign tumor in women that frequently gives rise to a multitude of reproductive complications. The use of suicide gene therapy has been proposed as a highly promising method for treating UL. To achieve successful gene therapy, it is essential to develop carriers that can efficiently transport nucleic acids into targeted cells and tissues. The instability of polyplexes in blood and other biological fluids is a crucial factor to consider when using non-viral carriers. In this study, we present serum-resistant and cRGD-modified DNA complexes for targeted delivery genes to UL cells. Ternary polyplexes were formed by incorporating cystine-cross-linked polyglutamic acid modified with histidine residues. We employed two techniques in the production of cross-linked polyanionic coating: matrix polymerization and oxidative polycondensation. In this study, we investigated the physicochemical properties of ternary DNA complexes, including the size and zeta-potential of the nanoparticles. Additionally, we evaluated cellular uptake, toxicity levels, transfection efficiency and specificity in vitro. The study involved introducing the HSV-TK gene into primary UL cells as a form of suicide gene therapy modeling. We have effectively employed ternary peptide-based complexes for gene delivery into the UL organtypic model. By implementing in situ suicide gene therapy, the increase in apoptosis genes expression was detected, providing conclusive evidence of apoptosis occurring in the transfected UL tissues. The results of the study strongly suggest that the developed ternary polyplexes show potential as a valuable tool in the implementation of suicide gene therapy for UL.

Peptide-Based Nanoparticles for αvß3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells.

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 αvß3 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.

Development of iRGD-Modified Peptide Carriers for Suicide Gene Therapy of Uterine Leiomyoma.

Uterine leiomyoma (UL) is one of the most common benign tumors in women that often leads to many reproductive complications. Suicide genetherapy was suggested as a promising approach for UL treatment. In the present study, we describe iRGD ligand-conjugated cysteine-rich peptide carrier RGD1-R6 for targeted DNA delivery to αvß3 integrin-expressing primary UL cells. The physico-chemical properties, cytotoxicity, transfection efficiency and specificity of DNA/RGD1-R6 polyplexes were investigated. TheHSV-1thymidine kinase encoding plasmid delivery to PANC-1pancreatic carcinoma cells and primary UL cells resulted in significant suicide gene therapy effects. Subsequent ganciclovir treatment decreased cells proliferative activity, induced of apoptosis and promoted cells death.The obtained results allow us to concludethatthe developed RGD1-R6 carrier can be considered a promising candidate for suicide gene therapy of uterine leiomyoma.

Characterization of iRGD-Ligand Modified Arginine-Histidine-Rich Peptides for Nucleic Acid Therapeutics Delivery to αvß3 Integrin-Expressing Cancer Cells.

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 αvß3 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 αvß3 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.

Synergistic Anti-Angiogenic Effects Using Peptide-Based Combinatorial Delivery of siRNAs Targeting VEGFA, VEGFR1, and Endoglin Genes.

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-ß-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.

Macrophages are a source of IL-17 in the human placenta.

PROBLEM: To determine if placental macrophages (Hofbauer cells) can synthesize and secrete cytokines of the IL-17 family throughout pregnancy and to reveal the patterns of cytokine expression in early and late gestation. METHODS OF STUDY: Macrophages were isolated from the first-trimester and term placental villous tissues from normal pregnancies. Basal and stimulated intracellular production of IL-17A, IL-17E, IL-17F as well as IL-17A secretion was quantified by flow cytometry and cytometric bead array, respectively. The expression of IL-17 and IL-23 receptors was determined on the surface of the placental macrophages by flow cytometry after antibody staining. RESULTS: In early and late gestation, a substantial proportion of the placental macrophages synthesized IL-17A and IL-17F, but not IL-17E, as determined by intracellular staining of the cytokines. Neither the intracellular production nor the secretion of IL-17 was significantly affected by LPS stimulation and spontaneous labour. The level of secretion decreased slightly but significantly at term. The IL-23 receptor was absent on the surface of cells, whereas variable expression of the IL-17 receptor was observed. CONCLUSION: Placental macrophages constitutively produced IL-17 at different gestational ages and represent thus a source of this cytokine in the human placenta. Patterns of the cytokine and receptor expression suggest that this cell population may participate in non-immune processes and contribute to the regulation of placental development and function.

IL-11 expression in human term placental macrophages.

PROBLEM: IL-11 is a cytokine with pleiotropic activities, including gestational effects. Whereas IL-11 production by maternal reproductive tissues is extensively studied, there is poor information about IL-11 sources in the fetal counterpart of the maternal-fetal unit. METHOD OF STUDY: We investigated the expression of IL-11 in the purified human term placenta macrophages, using flow cytometry and immunoenzyme assay. RESULTS: Intracellular IL-11 was detected in a substantial proportion of cultured CD68(+) cells (median 38%). IL-11 secretion by the placental macrophages was observed after 23-hr cultivation (median 4.3 pg/10(5) cells). Stimulation with lipopolysaccharide did not significantly change both intracellular expression and secretion of the cytokine. CONCLUSION: Demonstrated IL-11 expression by placental macrophages suggests that non-trophoblast cells contribute to IL-11 production at the maternal-fetal interface and thus account for the reproductive effects of the cytokine.

Characterization of cytokine production by human term placenta macrophages in vitro.

PROBLEM: Macrophages are apparently the only immune cells within placenta villi, yet functions of these cells remain obscure. It has been postulated that placental macrophages accomplish regulatory roles at the fetal-maternal interface by means of wide variety of secreted cytokines. We attempt to analyze the patterns of cytokine production in an isolated population of placental macrophages. METHOD OF STUDY: Macrophages were obtained from term placentas in the absence of spontaneous labor. The basal and lipopolysaccharide (LPS)-stimulated levels of intracellular cytokines were detected by flow cytometry. The basal cytokine secretion was determined by BD Cytometry Bead Array (BD Biosciences, San Diego, CA, USA). RESULTS: Intracellular IL-1alpha, IL-1beta, IL-6, and TNFalpha were detected in 31, 27, 4, and 3% CD68+ cells, respectively. Stimulation with LPS increased the proportions of cytokine-producing CD68+ cells to 48, 50, 28, and 49%, respectively. Under basal conditions, levels of released TNFalpha and IL-6, respectively, were 20- and 25-fold higher when compared with IL-1b while IL-10 was secreted in small but detectable amounts. When a secretory activity was estimated for cytokine-producing cells, the secretion rate for TNFalpha and IL-6 overwhelmingly surpassed that for IL-1beta (TNFalpha:IL-6:IL-1beta ratio was 192:145:1). CONCLUSION: These results suggest functional heterogeneity of the placental macrophage population and contribute to the elucidation of regulatory roles of these cells in gestation.