Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The absence of CpG in plasmid DNA-chitosan polyplexes enhances transfection efficiencies and reduces inflammatory responses in murine lungs.

ABSTRACT: Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200-400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(-)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(-)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(-)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.

SUBMITTER: Wongrakpanich A

PROVIDER: S-EPMC3993893 | biostudies-other | 2014 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Plasmid CpG depletion improves degree and duration of tumor gene expression after intravenous administration of polyplexes.

Project description:PURPOSE: Tumor gene expression after the intravenous (i.v.) administration of current polymer-based gene delivery systems is generally low and short-lived. Immune stimulatory CpG dinucleotides, present within the plasmid DNA of the polyplexes are likely to contribute to this. The effect of CpG replacement on the levels of transgene expression was studied, after the i.v. administration of polyethylenimine (PEI) polyplexes. METHODS: Tumor transfection and immune stimulation of PEI polyplexes containing plasmid DNA encoding for luciferase and rich in CpG motifs was monitored and compared to polyplexes containing the same gene but devoid of CpG motifs. Lipoplexes based on 1,2-dioleyl-3-trimethylammonium-propane/dioleoylphosphatidylethanolamine liposomes were included as a control. RESULTS: The replacement of CpGrich DNA by CpGfree DNA did neither affect the physical properties of the DNA complexes nor did it affect their in vitro transfection activity or cytotoxicity. The immune stimulation (interleukin-12) after i.v. administration of the PEI DNA complexes was low and unaffected by the presence of CpG motifs. The absence of CpG motifs within the different DNA complexes improved the degree and the duration of organ and tumor gene expression. CONCLUSION: The depletion of CpG dinucleotides within the plasmid DNA of polyplexes enhances the degree and duration of in vivo transgene expression.

| S-EPMC2440937 | biostudies-other

Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells.

Project description:Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca2+) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg2+ or Na+ did not show similar enhancement. Interestingly, addition of Ca2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.

| S-EPMC8216448 | biostudies-literature

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

Project description: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.

| S-EPMC8293032 | biostudies-literature

Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes.

Project description:The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.

| S-EPMC2951560 | biostudies-other

Increased tumor distribution and expression of histidine-rich plasmid polyplexes.

Project description:BackgroundSelecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays.MethodsAfter solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo.ResultsBy contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex.ConclusionsAlthough mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer.

| S-EPMC4242722 | biostudies-literature

Starch-Chitosan Polyplexes: A Versatile Carrier System for Anti-Infectives and Gene Delivery.

Project description:Despite the enormous potential of nanomedicine, the search for materials from renewable resources that balance bio-medical requirements and engineering aspects is still challenging. This study proposes an easy method to make nanoparticles composed of oxidized starch and chitosan, both isolated from natural biopolymers. The careful adjustment of C/N ratio, polymer concentration and molecular weight allowed for tuning of particle characteristics. The system's carrier capability was assessed both for anti-infectives and for nucleic acid. Higher starch content polyplexes were found to be suitable for high encapsulation efficiency of cationic anti-infectives and preserving their bactericidal function. A cationic carrier was obtained by coating the anionic polyplex with chitosan. Coating allowed for a minimal amount of cationic polymer to be employed and facilitated plasmid DNA loading both within the particle core and on the surface. Transfection studies showed encouraging result, approximately 5% of A549 cells with reporter gene expression. In summary, starch-chitosan complexes are suitable carriers with promising perspectives for pharmaceutical use.

| S-EPMC6415184 | biostudies-literature

Multiple cytosolic DNA sensors bind plasmid DNA after transfection.

Project description:Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) bound plasmid DNA in the cytosol within 15 minutes of transfection and at consistent levels for 4 h. Interferon activated gene 204 protein (p204) and DEAH box helicase 9 (DHX9) also bound pDNA, peaking 15 and 30 min respectively. Plasmid DNA was not detectably bound by DEAD box helicase 60 (DDX60) protein, despite a similar level of mRNA upregulation to DAI/ZBP1, or by cyclic GMP-AMP synthase (cGAS), despite its presence in the cell cytosol. Taken together, these results indicate several DNA sensors may participate and cooperate in the complex process of cytosolic DNA sensing.

| S-EPMC6821305 | biostudies-literature

Eradication of Intracellular Salmonella Typhimurium by Polyplexes of Acid-Transforming Chitosan and Fragment DNA.

Project description:Antibiotics are highly successful against microbial infections. However, current challenges include rising antibiotic resistance rates and limited efficacy against intracellular pathogens. A novel form of a nanomaterial-based antimicrobial agent is investigated for efficient treatment of an intracellular Salmonella enterica sv Typhimurium infection. A known antimicrobial polysaccharide, chitosan, is engineered to be readily soluble under neutral aqueous conditions for systemic administration. The modified biologic, named acid-transforming chitosan (ATC), transforms into an insoluble, antimicrobial compound in the mildly acidic intracellular compartment. In cell culture experiments, ATC is confirmed to have antimicrobial activity against intracellular S. Typhimurium in a concentration- and pH-dependent manner, without affecting the host cells, RAW264.7 macrophages. For improved cellular uptake and pharmacokinetic/pharmacodynamic properties, ATC is further complexed with fragment DNA (fDNA), to form nano-sized spherical polyplexes. The resulting ATC/fDNA polyplexes efficiently eradicated S. Typhimurium from RAW264.7 macrophages. ATC/fDNA polyplexes may bind with microbial wall and membrane components. Consistent with this expectation, transposon insertion sequencing of a complex random mutant S. Typhimurium library incubated with ATC does not reveal specific genomic target regions of the antimicrobial. This study demonstrates the utility of a molecularly engineered nanomaterial as an efficient and safe antimicrobial agent, particularly against an intracellular pathogen.

| S-EPMC8295212 | biostudies-literature

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes.

Project description:The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

| S-EPMC7602608 | biostudies-literature

Near-infrared light-sensitive liposomes for enhanced plasmid DNA transfection.

Project description:Near-infrared (NIR) light-responsive liposomes are attractive carriers for targeted and controlled drug delivery to the superficial organ or tissue (e.g., skin). This work describes the development of NIR-responsive liposomes by incorporating gold nanostars within liposomes composed of Phospholipon 90 g and cholesterol. Following cellular delivery, photothermal effect around the gold nanostar upon NIR stimulation induces microcavitation and liposome phase transition which consequently triggers the release of encapsulated molecules. Taking GFP plasmid as an example, we demonstrate enhanced gene transfection into fibroblasts following NIR treatment.

| S-EPMC5689532 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data