A comparative study of cationic liposomes for gene delivery

In the field of gene delivery, non-viral vectors have become more attractive carriers for nucleic acids since they can overcome the significant drawbacks of viral systems such as safety, immunogenicity, and oncogenicity. Among non-viral vectors, cationic liposome-mediated gene delivery gives promising results for gene therapy approaches. This study aimed to develop cationic liposomes and examine the effectiveness in terms of gene delivery. For this purpose, cholesterol, lecithin, and cationic lipid containing liposomes have been developed by film hydration method. Two different cationic lipids, DDAB and EQ, and their different mole ratios were investigated. Characterization studies showed that obtained liposomes have appropriate physiochemical characteristics (similar to 100 nm, homogenous in size and positive zeta potential) for gene delivery. Gel retardation assay revealed that they have DNA binding and protection ability against nucleases. According to the cytotoxicity evaluation performed on L929 cell line, EQ containing liposomes shows significantly less toxicity comparing DDAB containing liposomes (p<0.05). Furthermore, in vitro transfection study revealed that increasing the EQ mole ratio has increased transfection ability. The stability results showed that the optimal liposome which contains EQ in a higher mole ratio is stable during 90 days at 4 degrees C. Based on these findings, we propose that the developed optimal liposome system in this study could be considered as a suitable nucleic acid delivery vehicle base for gene therapy.TUBITAK (The Scientific and Technological Research Council of Turkey) [218S840]; Izmir Katip Celebi University Research Fund [2020-ODL-ECZF-0001, 108S083]This study has been financially supported by a grant from TUBITAK (The Scientific and Technological Research Council of Turkey) (project no: 218S840) and Izmir Katip Celebi University Research Fund (project no: 2020-ODL-ECZF-0001). We appreciate the project 108S083 (TUBITAK) in which the Malvern Zetasizer Instrument was purchased

Investigation of the potential therapeutic effect of cationic lipoplex mediated fibroblast growth factor-2 encoding plasmid DNA delivery on wound healing

Background Developing an alternative and efficient therapy for wound healing has been an important research topic for pharmaceutical sciences. A straightforward but effective system for delivering fibroblast growth factor-2 (FGF-2) encoding plasmid DNA (pFGF-2) for wound healing therapy was aimed to develop in this study. Methods In order to provide the delivery of pFGF-2, a delivery vector, namely, cationic lipid nanopArticle (cLN) was developed by the melt-emulsification process, complexed with pFGF-2 to form a lipoplex system and further characterized. The pFGF-2 binding and protecting ability of lipoplexes were evaluated. The cytotoxicity and transfection efficiency of the lipoplexes, FGF-2 expression levels, and in vitro wound healing ability have been investigated on the L929 fibroblast cell line. Results The obtained lipoplex system has a pArticle size of 88.53 nm with a low PDI (0.185), and zeta potential values of 27.8 mV with a spherical shape. The ability of cLNs to bind pFGF-2 and protect against nucleases was demonstrated by gel retardation assay. Furthermore, the developed FGF-2 carrying lipoplexes system showed significant transfection and FGF-2 expression ability comparing naked plasmid. Finally, scratch assay revealed that the developed system is able to promote in vitro cell proliferation/migration in 48 h. Conclusion Promising results have been achieved with the use of lipoplexes carrying pFGF-2, and this approach could be considered as a potentially applicable concept for the future gene-based wound healing therapies.This project was supported by Izmir Katip Celebi University, Scientific Research Projects Coordinatorship (Project Number: 2019-ONAP-ECZF-0002).Izmir Katip Celebi University, Scientific Research Projects Coordinatorship [2019-ONAP-ECZF-0002

Gen taşınımı için katı witepsol nanopartiküllerinin geliştirilmesi ve değerlendirilmesi

Objectives: Gene therapy approaches have become increasingly attractive in the medical, pharmaceutical, and biotechnological industries due to their applicability in the treatment of diseases with no effective conventional therapy. Non-viral delivery using cationic solid lipid nanoparticles (cSLNs) can be useful to introduce large nucleic acids to target cells. A careful selection of components and their amounts is critical to obtain a successful delivery system. In this study, solid Witepsol nanoparticles were formulated, characterized, and evaluated in vitro for gene delivery purposes. Materials and Methods: Solid Witepsol nanoparticles were formulated through the microemulsion dilution technique using two grades of Witepsol and three surfactants, namely Cremephor RH40, Kolliphor HS15, and Peceol. Dimethyldioctadecylammonium bromide was incorporated into the system as a cationic lipid. Twelve combinations of these ingredients were formulated. The obtained nanoparticles were then evaluated for particle size, zeta potential, DNA binding and protection ability, cytotoxicity, and transfection ability. Results: Particle sizes of the prepared cationic cSLNs were between 13.43 +/- 0.06 and 68.80 +/- 0.78 nm. Their zeta potential, which is important for DNA binding efficiency, was determined at >+40 mV. Gel retardation assays revealed that the obtained cSLNs can form a compact complex with plasmid DNA (pDNA) encoding green fluorescent protein and that this complex can protect pDNA from DNase I-mediated degradation. Cytotoxicity evaluation of nanoparticles was performed on the L929 cell line. In vitro transfection data revealed that solid Witepsol nanoparticles could effectively transfect fibroblasts. Conclusion: Our findings indicate that solid Witepsol nanoparticles prepared using the microemulsion dilution technique are promising non-viral delivery systems for gene therapy.Scientific and Technological Research Council of Turkey (TUBITAK) [218S840]; Izmir Katip Celebi University Research Fund [2020-ODL-ECZF-0001]This study has been financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant code 218S840 and Izmir Katip Celebi University Research Fund under grant code 2020-ODL-ECZF-0001. This paper is dedicated to the memory of our co-author dear Selen Isar, who sadly passed away on Wednesday, December 9th, 2020

Recruitment of solid lipid nanopArticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing

Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials ; methods: Solid lipid nanopArticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.Scientific and Technological Research Council of Turkey [TUBITAK-SBAG-218S682]This study was funded by the Scientific and Technological Research Council of Turkey (grant no. TUBITAK-SBAG-218S682). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Development and evaluation of antisense shRNA-encoding plasmid loaded solid lipid nanoparticles against 5-alpha reductase activity

WOS: 000428980600030In this study, we aimed to develop and evaluate an effective system for nucleic acid delivery. For this purpose, shRNA-encoding plasmid against 5-alpha reductase (p5 alpha-Red) activity was selected as a model. In order to achieve the delivery of p5 alpha-Red and to obtain lower cytotoxicity, higher transfection and silencing efficiency, we developed solid lipid nanoparticles (SLNs) as a delivery vector by the melt-emulsification process. As a first step, microemulsions were prepared by using Compritol ATO 888 as internal oil phase; Tween 80 as a surfactant (S); ethanol as co-surfactant (CoS) and ultra-pure water as the continuous water phase. Then, obtained o/w microemulsion was dispersed in cold ultra-pure distilled water (0-4 degrees C) to form SLNs. The formulated nanoparticles were electrostatically bound to p5 alpha-Red to form SLN: p5 alpha-Red vectors. The SLN: p5 alpha-Red vectors have particle sizes of 62.65 nm, and zeta potential values of 12.9 mV. DNase I protection analysis showed that developed formulation is able to protect the p5 alpha-Red from degradation. The permeation study revealed that SLN: p5 alpha-Red vectors are able to pass the cellulose membrane and 80.2% of the SLN: p5 alpha-Red vectors were dialyzed after 6 h. According to cytotoxicity test results, no significant cytotoxicity was observed on DU-145 cells in the concentration range of 0.3-0.6 mu g/well. Furthermore, in vitro gene silencing experiment demonstrated that SLN: p5 alpha-Red vector effectively reduced 5 alpha-Red enzyme level 48 h after administration in DU-145 cell line. Considering the role of 5-alpha reductase in related diseases such as benign prostatic hyperplasia, androgenic alopecia and prostate cancer, the developed SLN: p5 alpha-Red vector system may have promises in future therapy.Ege University Research Fund [BAP] [14-ECZ-030]This work was supported by Ege University Research Fund [BAP, 14-ECZ-030, 2016]

Enhanced Cellular Uptake and Gene Silencing Activity of Survivin-siRNA via Ultrasound-Mediated Nanobubbles in Lung Cancer Cells

WOS: 000556247900001PubMed: 32761250Purpose Paclitaxel is a first-line drug for the therapy of lung cancer, however, drug resistance is a serious limiting factor, related to overexpression of anti-apoptotic proteins like survivin. To overcome this phenomenon, developing novel ultrasound responsive nanobubbles - nanosized drug delivery system- for the delivery of paclitaxel and siRNA in order to silence survivin expression in the presence of ultrasound was aimed. Methods Paclitaxel-carrying nanobubble formulation was obtained by modifying the multistep method. Then, the complex formation of the nanobubbles - paclitaxel formulation with survivin-siRNA, was examined in terms of particle size, polydispersity index, zeta potential, and morphology. Furthermore, siRNA binding and protecting ability, cytotoxicity, cellular uptake, gene silencing, and induction of apoptosis studies were investigated in terms of lung cancer cells. Results Developed nanobubbles have particle sizes of 218.9-369.6 nm, zeta potentials of 27-34 mV, were able to protect siRNA from degradation and delivered siRNA into the lung cancer cells. Survivin expression was significantly lower compared with the control groups and enhanced apoptosis was induced by the co-delivery of survivin-siRNA and paclitaxel. Furthermore, significantly higher effects were obtained in the presence of ultrasound induction. Conclusion the ultrasound responsive nanobubble system carrying paclitaxel and survivin-siRNA is a promising and effective approach against lung cancer cells.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [116S213]This study has been financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant code 116S213. the authors would like to thank Ege University, Faculty of Pharmacy, Department of Pharmaceutical Technology (for DLS measurement), Ege University, Pharmaceutical Sciences Research Center (for Varioskan Multiplate Reader), Izmir Katip Celebi University, Central Research Labs (for SEM imaging) and Middle East Technical University, Central Laboratory (for TEM imaging)

Development and in vitro evaluation of positive-charged solid lipid nanoparticles as nucleic acid delivery system in glioblastoma treatment

WOS: 000438786000019In this study, we aimed to develop a novel positive charged nucleic acid delivery system for the treatment of glioblastoma. For this purpose, Epidermal Growth Factor Receptor (EGFR) which plays a prominent role in glioblastoma was selected as a target. Cationic solid lipid nanoparticles (cSLN) were developed by microemulsion dilution method using cetyl palmitate as matrix lipid, Cremephor RH40 and Peceol as surfactants, and ethanol as cosurfactant. Characterization studies showed that obtained nanoparticles are positively charged and has an appropriate particle size for nucleic acid delivery (<20 nm). Gel retardation assay revealed that cSLNs have complexation ability with siRNA EGFR (cSLN:siRNAEGFR) and this complex is able to protect siRNA from serum- mediated degradation up to 6 h. The cytotoxicity evaluation of nanoparticles performed on U87 Human Glioblastoma Cell Line. Furthermore, in vitro delivery of siRNA-EGFR via cSLN inhibited EGFR expression significantly at 50nM siRNA dose compared to free siRNA-EGFR at the same dose on U87 cell line (p<0.05). Based on these findings, we propose that the developed cSLN system may has a potential as a siRNA delivery system for glioblastoma.Scientific Human Resources Development Program (OYP) of the Turkish Higher Education Council (YOK); TUBITAK-SBAGTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108S083]This work has been supported by Scientific Human Resources Development Program (OYP) of the Turkish Higher Education Council (YOK). We appreciate the project 108S083 (TUBITAK-SBAG) which the Malvern Zetasizer Instrument was purchased. Dr. Bilge Debelec Butuner from the Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University is greatly appreciated for kindly providing the U87 cells

Development and characterization of nanobubbles containing paclitaxel and survivin inhibitor YM155 against lung cancer

Kotmakci, Mustafa/0000-0002-5593-8608WOS: 000472733600015PubMed: 31129344Lung cancer remains 23% of cancer-related death worldwide, ranking on first place for men and second place for women. Almost each cancer type has a great deal in common, overexpression of the apoptosis inhibitor survivin. Chemotherapy with anticancer drugs is leading to side effects. Drug targeting by the use of nanobubbles is a useful strategy to reduce side effects. Nanobubbles in cancer are one of the most investigated carriers in the last years. the size of nanobubbles (1-500 nm) is bigger than the pore size of healthy tissues, but smaller than the pores of cancer tissues. Thus, it is not possible for the drug to leave the blood stream and enter the tissue, but it can enter the cancer tissue through the pores, where it can accumulate. Therefore, the probability of undesired side effects decreases. For that reason, the development of nanobubbles containing paclitaxel and survivin inhibitor sepantronium bromide (YM155) were carried out. Characterization studies in terms of particle size, size distribution, zeta potential and morphology, and investigation of their effects on lung cancer cells were performed. To the best of our knowledge, there is no information in the literature about combining paclitaxel and YM155 loaded nanobubbles with ultrasound exposure.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [116S213]This study has been financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant code 116S213