Preparation of Hybrid Nanoparticle Systems as Nucleic Acid Carriers and Investigation of Their Effectiveness on Cancer Cells
Project no. 30842 · Status: Kapanmış (Yayın bekleniyor) · 2023
Summary
<p>Nanocarrier systems play an important role in gene therapy by enabling selective delivery of genetic material to target cells. Lipid-based and polymer-based carriers are used to enhance the efficiency of genetic material in cells. Among lipid-based carriers, anionic liposomes, pH-sensitive liposomes, and cationic liposomes are commonly used. Among polymer-based carriers, cationic polymers and lipids electrostatically bind the genetic material and facilitate its cellular uptake. These carrier systems protect the therapeutic payload from enzymatic degradation, prevent them from being recognized by the immune system, and reduce their side effects.</p><p>This research aims to enhance transfection efficiency and reduce transfection-related toxicity by creating new nanocarrier hybrid systems. Lipid nanoparticle (LNP) formulations prepared by incorporating lipooligomers providing endosomal escape will be used to increase the efficiency of gene delivery. In the proposed study, pDNA loading will be carried out, and the optimal formulation of new hybrid carrier systems will be determined. The developed formulation's transfection efficiency in specific cell lines will be evaluated. The LNP:lipooligomer:pDNA hybrid systems will be prepared by adding lipooligomers in varying proportions to LNPs composed of DOPC, cholesterol, and DOTAP compounds, and complexing them with pDNA.</p><p>It was planned to examine the prepared complexes using agarose gel electrophoresis and to measure particle size and surface charge by DLS thechnique. To examine the stability of formulations under physiological conditions, it was aimed to test the integrity of complexes in the presence of serum and anionic agents. In cell culture studies, cell viability assay and transfection efficiency were measured. Cells treated with fluorescently labeled complexes were identified by flow cytometry to determine the level of cellular uptake. Furthermore, intracellular localization of the designed complexes were examined under a fluorescence microscope. In conclusion, the primary aim of this study was to enhance the uptake of genetic materials into cells and improve transfection efficiency by developing a new complex formulation.</p><p>For LNP:pDNA complexes, the particle size was measured between 99.13 nm and 153.3 nm at N/P ratios of 6 and 12, respectively, the PDI value ranged from 0.26 to 0.57, and the zeta potential was measured between +31.5 and +44.86 mV. In the hybrid complexes formed with the addition of lipooligomers, the particle size ranged from 93 to 110 nm, the PDI value ranged from 0.27 to 0.38, and the zeta potential ranged from +47.5 to +51.9 mV. It was determined that hybrid complexes were more resistant to serum degradation and maintained their integrity compared to LNP complexes. While LNP complexes could hold pDNA in complex form only at low heparin concentrations, hybrid complexes exhibited higher stability. Hybrid complexes showed higher <em>in vitro</em> transfection efficiency compared to LNPs. In cell uptake and association analyses, hybrid complexes were observed to be more efficient. Cytotoxicity analyses showed that LNP complexes exhibited a maximum of 10% cytotoxicity, while hybrid complexes exhibited a maximum of 7.1% cytotoxicity. Hybrid complexes showed less toxicity compared to complexes formed by lipooligomers alone.</p><p>According to the obtained results, it was observed that hybrid complexes formed more compact, homogeneous, and stable structures. pDNA complexes prepared by combining lipooligomers with LNPs showed high efficiency and low toxicity, making them successful candidates for future applications.</p><p>This study could serve as a reference for future cancer treatments and provide an economical treatment alternative.</p>