Enhanced gene delivery in tumor cells using chemical carriers and mechanical loadings (original) (raw)
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Non-viral transfection methods optimized for gene delivery to a lung cancer cell line
Avicenna journal of medical biotechnology, 2013
Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (su...
Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems
Biotechnology Letters, 2018
Objective In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. Results The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p \ 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (* 83.94 and * 77.01% for HEK-293T), and cancer cells (* 71.69 and * 67.19% for TC-1, and * 73.86 and * 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by ?36 GFP reached * 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (* 63.66%) compared to TurboFect (* 32.95%); however, the efficiency level of hPP10 was only * 17.51 and * 16.36% in TC-1 and A549 cells. Conclusions Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.
BMC Cancer, 2009
Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors.
Polymer-based non-viral gene delivery as a concept for the treatment of cancer
Pharmacological Reports, 2009
Gene therapy has become a promising technique for the treatment of cancer. Nevertheless, the success of gene therapy depends on the effectiveness of the vector. The challenge of a gene carrier is to deliver exogenous DNA from the site of administration into the nucleus of the appropriate target cell. Polymer-based vectors are biologically safe, have low production costs and are efficient tools for gene therapy. Although non-degradable polyplexes exhibit high gene expression levels, their application potential is limited due to their inability to be effectively eliminated, which results in cytotoxicity. The development of biodegradable polymers has allowed for high levels of transfection without cytotoxicity. For site-specific targeting of polyplexes, further modifications, such as incorporation of ligands, can be performed. Most expectations have been addressed to polyplexes architecture according it dynamic response with the microenvironment.
Cancer Gene Therapy, 2002
In this study, we measured transfection efficiency in vitro and in vivo using the following nonviral approaches of gene delivery: injection of plasmid DNA, electroporation -assisted, liposome -enhanced, and integrin -targeted gene delivery, as well as the combination of these methods. Four histologically different tumor models were transfected with a plasmid encoding the green fluorescent protein ( GFP ) ( B16 mouse melanoma, P22 rat carcinosarcoma, SaF mouse sarcoma, and T24 human bladder carcinoma ) using adherent cells, dense cell suspensions, and solid tumors. Emphasis was placed on different electroporation conditions to optimise the duration and amplitude of the electric pulses, as well as on different DNA concentrations for effective gene delivery. In addition, transfection efficiency was correlated with cell density of the tumors. The major in vivo findings were: ( a ) electroporationassisted gene delivery with plasmid DNA, employing long electric pulses with low amplitude, yielded significantly better GFP expression than short electric pulses with high amplitude; ( b ) electroporation combined with liposome -DNA complexes yielded the highest percentage of transfected tumor area in B16F1 tumor ( 6% ); ( c ) transfection efficiency of electroporation -assisted plasmid DNA delivery was dependent on tumor type; ( d ) integrin -targeted vector, alone or combined with electroporation, was largely ineffective. In conclusion, our results demonstrate that some nonviral methods of gene delivery are feasible and efficient in transfecting solid tumors. Therefore, this makes nonviral methods attractive for further development.
Polycation-based DNA complexes for tumor-targeted gene delivery in vivo
The Journal of Gene Medicine, 1999
Background Ef®cient and target-speci®c in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological¯uids and extracellular matrix, and binding to a broad variety of non-target cell types. Methods Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin-polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. Results Gene transfer after intratumoral application was 10±100 fold more ef®cient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. Conclusion For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection ef®cacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.
Gene Therapy, 2007
Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.
Enhancement of transfection by physical concentration of DNA at the cell surface
Nature Biotechnology, 2000
Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or
Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology
Gene Therapy, 2001
The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' handheld system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the -galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-␣) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3-5 l plasmid DNA (1 g DNA/l in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with