scholarly journals Effect of Double Substitution in Cationic Chitosan Derivatives on DNA Transfection Efficiency

Polymers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1057 ◽  
Author(s):  
Veronika D. Badazhkova ◽  
Sergei V. Raik ◽  
Dmitry S. Polyakov ◽  
Daria N. Poshina ◽  
Yury A. Skorik
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Substituent Effects ◽  
Maximum Efficiency ◽  
Viral Gene ◽  
Cationic Polymers ◽  
Dna Encoding ◽  
Single Carrier ◽  
Mass Ratios ◽  
Pyridoxal Hydrochloride

Recently, much effort has been expended on the development of non-viral gene delivery systems based on polyplexes of nucleic acids with various cationic polymers. Natural polysaccharide derivatives are promising carriers due to their low toxicity. In this work, chitosan was chemically modified by a reaction with 4-formyl-n,n,n-trimethylanilinium iodide and pyridoxal hydrochloride and subsequent reduction of the imine bond with NaBH4. This reaction yielded three novel derivatives, n-[4-(n’,n’,n’-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS), and n-[4-(n’,n’,n’’-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS). Their structures and degrees of substitution were established by 1H NMR spectroscopy as DS1 = 0.22 for TMAB-CS, DS2 = 0.28 for Pyr-CS, and DS1 = 0.21, DS2 = 0.22 for PyrTMAB-CS. Dynamic light scattering measurements revealed that the new polymers formed stable polyplexes with plasmid DNA encoding the green fluorescent protein (pEGFP-N3) and that the particles had the smallest size (110–165 nm) when the polymer:DNA mass ratio was higher than 5:1. Transfection experiments carried out in the HEK293 cell line using the polymer:DNA polyplexes demonstrated that Pyr-CS was a rather poor transfection agent at polymer:DNA mass ratios less than 10:1, but it was still more effective than the TMAB-CS and PyrTMAB-CS derivatives that contained a quaternary ammonium group. By contrast, TMAB-CS and PyrTMAB-CS were substantially more effective than Pyr-CS at higher polymer:DNA mass ratios and showed a maximum efficiency at 200:1 (50%–70% transfected cells). Overall, the results show the possibility of combining substituent effects in a single carrier, thereby increasing its efficacy.

scholarly journals Biodegradable Gene Carriers Containing Rigid Aromatic Linkage with Enhanced DNA Binding and Cell Uptake

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1080 ◽  
Author(s):  
Ju-Hui Zhang ◽  
Hui-Zhen Yang ◽  
Ji Zhang ◽  
Yan-Hong Liu ◽  
Xi He ◽  
...  
Keyword(s):  
Dna Binding ◽  
Laser Scanning ◽  
Transfection Efficiency ◽  
Laser Scanning Microscopy ◽  
Cell Uptake ◽  
Confocal Laser ◽  
Viral Gene ◽  
Cationic Polymers ◽  
Aromatic Rings ◽  
Gene Carriers

The linking and modification of low molecular weight cationic polymers (oligomers) has become an attracted strategy to construct non-viral gene carriers with good transfection efficiency and much reduced cytotoxicity. In this study, PEI 600 Da was linked by biodegradable bridges containing rigid aromatic rings. The introduction of aromatic rings enhanced the DNA-binding ability of the target polymers and also improved the stability of the formed polymer/DNA complexes. The biodegradable property and resulted DNA release were verified by enzyme stimulated gel electrophoresis experiment. These materials have lower molecular weights compared to PEI 25 kDa, but exhibited higher transfection efficiency, especially in the presence of serum. Flow cytometry and confocal laser scanning microscopy results indicate that the polymers with aromatic rings could induce higher cellular uptake. This strategy for the construction of non-viral gene vectors may be applied as an efficient and promising method for gene delivery.

Effects of lyoprotectants on long-term stability and transfection efficacy of lyophilized poly(lactide-co-glycolide)-graft-polyethylenimine/plasmid DNA polyplexes

Nanomedicine ◽  
2021 ◽  
Author(s):  
Joshua Woo ◽  
Jeoung Soo Lee
Keyword(s):  
Plasmid Dna ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Dna Encoding ◽  
Rat Glioma ◽  
Term Stability ◽  
Long Term Stability ◽  
Glioma C6 ◽  
Transfection Efficacy

Aim: We investigated the effect of lyoprotectants on the long-term stability and transfection efficiency of lyophilized (Lyo.) polyplexes prepared from poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) and plasmid DNA encoding green fluorescent protein (pGFP). Materials & methods: Lyo. PgP/pGFP polyplexes prepared with/without lyoprotectants were stored at -20°C over 6 months. Polyplex stability was analyzed by gel electrophoresis and heparin competition assay. Transfection efficiency and cytotoxicity were evaluated in rat glioma (C6) cells in medium containing 10% serum. Results: Lyo. PgP/pGFP polyplexes prepared with 5% sucrose as a lyoprotectant remained stable up to 6 months and retained transfection efficiency up to 4 months. Conclusion: Lyo. PgP-based polyplexes retain bioactivity during extended storage, potentially enabling transport to remote regions and less stable settings, increasing access to life-changing gene therapy.

scholarly journals Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Jinghua Duan ◽  
Yangde Zhang ◽  
Wei Chen ◽  
Chengrong Shen ◽  
Mingmei Liao ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Hepg2 Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Electrophoretic Analysis ◽  
Dna Encoding ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Low Cytotoxicity ◽  
Green Fluorescent

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA) and chitosan to prepare PBCA nanoparticles (NPs) by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2) cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

scholarly journals Strategies for simultaneous and successive delivery of RNA

2020 ◽  
Vol 98 (12) ◽  
pp. 1767-1779
Author(s):  
Hanieh Moradian ◽  
Andreas Lendlein ◽  
Manfred Gossen
Keyword(s):  
Messenger Rna ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Viral Gene ◽  
Cell Level ◽  
Integrated Method ◽  
Viral Gene Delivery ◽  
Selection Of

AbstractAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

Quaternized Chitosans as Gene Delivery Carriers: Effect of Degree of Quaternization

2014 ◽  
Vol 1060 ◽  
pp. 17-20
Author(s):  
Lalita Leksantikul ◽  
Warayuth Sajomsang ◽  
Theerasak Rojanarata ◽  
Tanasait Ngawhirunpat ◽  
Praneet Opanasopit
Keyword(s):  
Gene Delivery ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Weight Ratio ◽  
Dna Encoding ◽  
Factors Affecting ◽  
Cervical Carcinoma Cell Line ◽  
Cell Line Hela

The objective of this study was to investigate the transfection efficiency of quaternized chitosan or trimethylated chitosans (TMCs) using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human cervical carcinoma cell line (HeLa cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ) and polymer/DNA weight ratio were evaluated. The results revealed that the complexes of TMC30 with DNA had the highest transfection efficiency and safety followed by the complexes of TMC60 and TMC94, respectively. Increasing the DQ of chitosan not only improve the efficiency of gene delivery, but also increase cytotoxicity. In conclusion, TMC30 showed elevated potential as a gene carrier by efficient DNA condensation and mediated the highest level of gene transfection with negligible cytotoxicity in HeLa cells.

scholarly journals A facile and scalable in production non-viral gene engineered mesenchymal stem cells for effective suppression of temozolomide-resistant (TMZR) glioblastoma growth

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Geraldine Xue En Tu ◽  
Yoon Khei Ho ◽  
Zhi Xu Ng ◽  
Ke Jia Teo ◽  
Tseng Tsai Yeo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Transfection Efficiency ◽  
Viral Gene ◽  
Antitumor Effects ◽  
Differentiation Potential ◽  
Highly Efficient

Abstract Background Mesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP). Methods MSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice. Results At > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated. Conclusions Collectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

scholarly journals Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

2004 ◽  
Vol 72 (12) ◽  
pp. 7096-7106 ◽  
Author(s):  
James E. Galen ◽  
Licheng Zhao ◽  
Magaly Chinchilla ◽  
Jin Yuan Wang ◽  
Marcela F. Pasetti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Expression System ◽  
Dna Encoding ◽  
Protein Fusion ◽  
Vector Vaccine ◽  
Interleukin 5 ◽  
Export System ◽  
Domain 4

ABSTRACT Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain

2010 ◽  
Vol 2010 (7) ◽  
pp. pdb.prot5457-pdb.prot5457
Author(s):  
C. Schultz ◽  
A. B. Neef ◽  
T. W. Gadella ◽  
J. Goedhart
Keyword(s):  
Fluorescent Protein ◽  
Lipid Binding ◽  
Binding Domain ◽  
Dna Encoding

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

Flexible cyclic siloxane core enhances the transfection efficiency of polyethylenimine-based non-viral gene vectors

2015 ◽  
Vol 3 (42) ◽  
pp. 8250-8267 ◽  
Author(s):  
Cristina M. Uritu ◽  
Manuela Calin ◽  
Stelian S. Maier ◽  
Corneliu Cojocaru ◽  
Alina Nicolescu ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Viral Gene ◽  
Cyclic Siloxane ◽  
Gene Vectors

cD4H–AGE–PEI conjugates, with a favorable balance between hydrophilic and hydrophobic moieties, are promising carriers for gene delivery.

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