1,721,000 research outputs found
In vitro characterization and transfection of IL-2 gene complexes
No full textBackground: Interleukin-2 used in the treatment of malignant tumors has an anti-tumor efficacy. In this study, we have studied in vitro characterization and transfection efficiency of a plasmid encoding hIL-2, pCXWN-hIL-2, complexed to chitosan, polyethylenimine or DOTAP with varying ratios. Methods: Plasmid DNA was amplified in Escherichia coli DH5 alpha and isolated by alkali lysis method. The pDNA/chitosan, pDNA/PEI or pDNA/DOTAP complexes were analyzed by agarose gel electrophoresis for complex formation and by ESEM image analysis system for the morphology and DNA/medium relationship of complexes. DNase stability, the particle size and zeta potential values of complexes were determined. Transfection efficiencies of resulting complexes in two different cell lines were assayed by ELISA method. Results: Conclusively, a transfection activity was observed in both cell lines (HeLa and Swiss3T3) with the order of pDNA/DOTAP > pDNA/PEI > pDNA/chitosan complexes. We have observed that the transfection efficiency was higher in HeLa cell line compared to Swiss3T3 cell line. Conclusion: The physicochemical studies like stability, particle size and zeta potential, showed a relationship between the properties of a complex and its transfection efficiency. (c) 2006 Elsevier B.V. All rights reserved
The Effects to GM-CSF Expression and Fibroblast Proliferation of pGM-CSF Containing Chitosan/PVP Hydrogels
No full textThe aim of this study was to develop pGM-CSF containing chitosan/PVP hydrogel system for gene delivery. The hydrogels in semi-interpenetrating polymer network (semi-IPN) structure were synthesized by glutaraldehyde crosslinking between 2% chitosan and 2%, % 4, % 6 PVP. The viscosity, swelling properties and surface morphology of hydrogels were examined. These hydrogels showed highly porous structure. The porosity was increased with enhanced PVP concentration. Viscosity decreased with increasing PVP concentration of the hydrogels. The swelling percentage increased with increasing PVP concentration. In order to examine the effect of cell attachment and proliferation of hydrogels, NIH-3T3 fibroblast cell was used. The cells were able to attach on the top surface of hydrogels. However, the attachment of cells were slow at days 1 and 3. At days 7 and 14, cells looked healthy and normal and proliferated around and top of the hydrogels. With the ELISA study, the highest GM-CSF expression was obtained with pGM-CSF containing 2% chitosan+4% PVP hydrogels. MTT assay showed that chitosan/PVP hydrogels did not induce significant cytotoxic effect. As a result, these hydrogels could be served candidate for local gene delivery in tissue regeneration
Preparation of superoxide dismutase loaded chitosan microspheres: Characterization and release studies
No full textSuperoxide dismutase (SOD) is the most potent antioxidant enzyme. In this study, SOD was encapsulated in chitosan microspheres to obtain suitable sustained protein delivery. Protein-loaded chitosan microspheres with various formulations were prepared based on complex coacervation process. Due to the inherent characteristic of SOD, high encapsulation efficiency could not be obtained with simple preparation method. The pH of chitosan solution is 3.0; when the chitosan microspheres were prepared with this solution, encapsulation was low. Therefore, several strategies have been tested to increase the encapsulation efficiency and good results have been obtained. 70-80% protein encapsulation efficiency was obtained. The addition of PEG to the protein solution enhanced the encapsulation efficiency also. Mean sizes of microspheres were between 1.38 and 1.94 mu m. Factors affecting the release behaviour of SOD from microspheres have been studied. They included pH values of chitosan solution (the pH of chitosan solution is 3.0), addition of PEG to the protein solution and the use of adsorption technique, In general, biphasic release profiles were obtained with these formulations. The protein activity changed between 70 and 100% during the release. In general, the protein activity remained in acceptable limits. The SOD encapsulated chitosan microspheres can be prepared by changing the pH or addition of PEG, allowing the safe incorporation of protein for controlled release. (c) 2006 Elsevier B.V. All rights reserved
The design of biodegradable ofloxacin-based core-shell microspheres: Influence of the formulation parameters on in vitro characterization
No full textOfloxacin (OFL), second-generation fluoroquinolone, is a broad-spectrum antibiotic which is active against both Gram-positive and Gram-negative bacteria. However, OFL has a short biological half life (8-9 h) and poor stability in serum and needs frequently repeated doses during the treatment. The objective of this study was to fabricate the fucospheres and chitosan microspheres containing a poorly soluble drug, OFL, and to compare the formulation parameters influencing the in vitro properties of microparticles such as size, zeta potential, encapsulation efficiency and drug release characteristics. Particle size of fucospheres and chitosan microspheres has been found to be 0.61-1.48 mu m and 1.05-2.08 mu m, respectively. The zeta potentials have changed between 5.6 mV and 28.0 mV for fucospheres; 22.3 mV and 42.4 mV for chitosan microspheres. The fucospheres have had higher drug encapsulation efficiency than those of chitosan microspheres. The particle size, surface charge, encapsulation efficiency and in vitro drug release from both fucospheres and chitosan microspheres have been affected by type and concentration of the polymers used. The release mechanism from most of the microsphere formulations has been fitted to Higuchi kinetic model. It can be concluded that OFL-encapsulated fucospheres can be a potential delivery system for antibiotics.</
Plasmid DNA-loaded chitosan/TPP nanoparticles for topical gene delivery
No full textTopical application of plasmid DNA represents an attractive route of gene delivery. Although chitosan (CS) has been widely investigated as a gene-carrier, there is very limited information about the skin application of CS-based systems for DNA. This study evaluated pDNA-loaded chitosan nanoparticles (CS-NPs) for skin gene delivery. NPs were prepared by inducing the gelation of CS upon interaction with sodium tripolyphosphate. pSV-beta-Gal was used as a reporter gene. The size, surface charge, and the other in vitro characteristics of CS-NPs were examined. Primary human dermal fibroblast cells (HDF) and mouse fibroblast NIH 3T3 cell lines (ATCC CCL-92) were used for in vitro transfection studies. In in vivo study, CS-NPs were applied to the skin of baby and adult Sprague Dawley rats by spreading on the shaved area of the back of animals. During a week animals were sacrificed and skin biopsies were taken for beta-Gal expression. beta-galactosidase enzyme activity was determined spectrophotometrically at 420 nm. The distribution of beta-galactosidase expressing cells within the skin tissue was observed by X-gal histochemical method. beta-galactosidase was continuously expressed at the nanoparticle-treated skin during the 7 days. High and continuous beta-Gal expressions were obtained with CS-NPs, although it was low in the first day. When a comparison was made between the data of baby and adult rats, markedly high transfection were measured in the skin samples of the baby rats. NPs protected pDNA against the enzyme and serum attacks. In conclusion, CS-NPs showed in vivo transfection potential in rats for skin gene delivery
Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes
No full textPURPOSE: Studies on DNA complexes with cationic polymers are prompted by the search for nonviral DNA carriers for gene therapy. Among them, poly(L-Lysine) (PLL) has been extensively studied. On the other hand, these systems deliver DNA as a bolus without long-term release. The aims of this study were to encapsulate plasmid DNA:poly(L-lysine) (pDNA:PLL) complexes into chitosan microspheres as an alternative to the PLL based gene delivery and investigate its in vitro release and transfection characteristics as well as plasmid DNA integrity and stability against serum and DNase I challenge. METHODS: pUC18 plasmid DNA that encoded beta-galactosidase was used as a model. The microspheres were prepared by complex coacervation method and the release and in vitro transfection properties were investigated. pDNA:PLL complexes were prepared at two different mass ratios. In vitro release studies were performed at 37 +/- 0.5 degrees C and drug release was monitored both spectrophotometrically and fluorometrically. Structural integrity of the pDNA:PLL complexes were determined by Southern blotting analysis. Protective effect of encapsulation of pDNA:PLL complexes against DNase I and serum treatment were also studied. In vitro transfection studies were performed by using 3T3 cell line. RESULTS: According to our in vitro release data, the mass ratio of pDNA:PLL significantly affected the release of pDNA:PLL complexes from chitosan microspheres, and the structure of the plasmid DNA did not change during the experiments. pDNA:PLL-loaded chitosan microspheres indicated high stability against fetal bovine serum and DNase I treatment for a week. In vitro transfection data showed that pDNA:PLL-loaded chitosan microspheres could be effectively transfected 3T3 cells in vitro. CONCLUSION: As a conclusion, pDNA:PLL complexes could be encapsulated into chitosan microspheres with maintaining their structural and functional integrity and this system may be a good alternative for polycation based gene carriers
The effects of chitosan/miR-200c nanoplexes on different stages of cancers in breast cancer cell lines
No full textDysregulation of miR-200c in breast cancer has been associated with migration, epithelial mesenchymal transition (EMT), angiogenesis and metastasis of the tumor cells. Therefore, the modulation of miR-200c offers a promising therapeutic approach in breast cancer. However, the major obstacles in the usage of miRNAs in therapy are their low stability, rapid clearance, and poor cellular uptake. The development of efficient and safe delivery systems is important in effective therapy with miRNA. The purpose of this study was to investigate the therapeutic role of chitosan/miR-200c nanoplexes in angiogenesis, EMT, invasion, and apoptosis in breast cancer cell lines. We found that miR-200c levels were downregulated in various breast cancer cell lines by qRT-PCR After transfection with chitosan/miRNA nanoplexes in the appropriate size (294 nm) and zeta potential (12.3 mV), levels of miR-200c increased and reached the endogenous miR-200c levels in the MCF-7, MDA-MB-231, and MDA-MB-435 cells. While the chitosan/miR-200c nanoplexes decreased angiogenesis, invasion, EMT, and metastasis in the cells, the apoptosis levels increased by 3.1, 1.3, and 3 fold in the MCF-7, MDA-MB-231, MDA-MB-435 cell lines, respectively. Consequently, chitosan is a suitable carrier for miR-200c and formed stable nanoplexes with miR-200c. The effect of the chitosan/miRNA nanoplexes on tumor angiogenesis, EMT, invasion, metastasis, and apoptosis, changed depending on the cell-types. Therefore, during the treatment with the chitosan based miR-200c nanoplexes in breast cancers, the type of tumor cells must be considered. (C) 2016 Elsevier B.V. All rights reserved
Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-beta-Containing Chitosan Nanoplexes
No full textMesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-beta. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-beta, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-beta gene silencing efficiencies of PDGF-B and PDGFR-beta targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-beta-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease
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