Improved Transfection Efficiency Research Articles

Immune response of channel catfish (Ictalurus punctatus) against Ichthyophthirius multifiliis post vaccination using DNA vaccines encoding immobilization antigens

The channel catfish (Ictalurus punctatus) immune response against Ichthyophthirius multifiliis (Ich) after vaccination using plasmid DNA vaccines pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, encoding Ich immobilization antigen genes was studied. Parasite infection level, serum anti-Ich antibodies level, fish mortality after theront challenge, and immune-related gene expression were measured. After in vitro transfection of walking catfish gill cells (G1b) with both pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, antigens IAG52A and IAG52B were detected. During the vaccination trial, 76-fold increase in the Iag52b gene expression was observed in the vaccinated fish group h4 post vaccination. Administration of DNA vaccines by IM injection induced significant gene up-regulation in the head kidney, including immunoglobulin M (IgM), cluster of differentiation 4 (CD4), major histocompatibility I (MHC I), and T cell receptor α (TcR-α) from h4 to d5 post immunization. Fish vaccinated with DNA vaccines or theronts showed increased gene expression of the cytokine interferon (IFN-γ), complement component 3 (C3), and toll-like receptor-1 (TLR-1). Anti-Ich antibodies were detected in fish received pcDNA3.1-IAg52a, pcDNA3.1-IAg52b and the combination of both vaccines d10 post vaccination. Fish vaccinated with pcDNA3.1-IAg52b showed mild parasite infection level, partial survival (20%) and longer mean day to death (MDD) after theront challenge. By contrast, a heavy parasite load, 0% survival and short MDD were observed in the sham vaccinated control fish that received pcDNA3.1 (plasmid without genes encoding Ich immobilization antigen). Further research is needed to improve DNA vaccines for Ich that can induce strong protective immunity in fish. Suggested studies include improved transfection efficiency, use of appropriate adjuvants and including additional parasite antigen genes in the plasmid.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.

Polyrotaxane Nanocarriers Can Deliver CRISPR/Cas9 Plasmid to Dystrophic Muscle Cells to Successfully Edit the DMD Gene

AbstractGene editing with clustered regularly interspaced short palindromic repeats and CRISPR‐associated protein 9 (CRISPR/Cas9) has shown promise in models of Duchenne muscular dystrophy (DMD); however, nonviral strategies to deliver CRISPR to muscle have not been widely explored or optimized. Most studies have relied on viral vectors, which are likely limited to single dosing due to their immunogenicity, thus reducing their therapeutic potential. Therefore, there is a need to develop nonviral approaches that allow for delivery and repeat dosing of CRISPR/Cas9 therapies to skeletal muscle. Here, biocompatible multi‐arm polyrotaxane (PRX) nanocarriers, are iteratively optimized for packaging large plasmid DNA for delivery to muscle cells. The PRXs are optimized by addition of a disulfide‐responsive linker that enhances plasmid release. Furthermore, conjugation of peptides leads to quicker uptake and improved transfection efficiency in humanized dystrophic muscle cells in vitro. Finally, in vitro delivery of PRXs complexed with a CRISPR/Cas9 platform demonstrates effective deletion of DMD exons 45–55, a therapeutic strategy with potential to restore the reading frame for half of DMD patients. This work represents the first PRX platform that is optimized and designed for delivery of large plasmid DNA, such as CRISPR/Cas9, to dystrophic muscle cells.

PH-Switchable Coordinative Micelles for Enhancing Cellular Transfection of Biocompatible Polycations

Inefficient transfection of biocompatible low-molecular-weight (LMW) polycations, such as 1.8k polyethylenimine (PEI), is a major challenge for successful nucleic acid delivery. Current strategies to improve transfection efficiency are bottlenecked by maintaining the balance between efficient gene encapsulation and on-demand cargo release. Here, we developed a new class of Zn(II)-coordinated micelles, which showed tight small interfering RNA (siRNA) binding and pH-switchable release. The dipicolylamine-modified PEI 1.8k (PD) and dopamine-conjugated cholesterol (Chol-Dopa) assemble into coordinative micelles (Zn-PD/Chol-Dopa) via the coordination of 2,2'-dipicolylamine (DPA) and Dopa through Zn(II) as a bridge. The high phosphate-binding affinity of Zn-DPA enhanced the siRNA packaging and the interaction between cholesterol and cell membranes enhanced cellular uptake. Moreover, the coordination effect weakened in the acidic environment of lyso/endosome, triggering the disassembly of micelles and siRNA release. These properties of the micelles resulted in strong siRNA transfection efficiencies in various cell lines. Our strategy of constructing coordinative micelles improves the transfection efficiency of LMW PEI and holds tremendous potential to develop the endogenous responsive gene delivery systems.

A microparticle approach for non-viral gene delivery within 3D human mesenchymal stromal cell aggregates

Three-dimensional (3D) multicellular aggregates, in comparison to two-dimensional monolayer culture, can provide tissue culture models that better recapitulate the abundant cell-cell and cell-matrix interactions found in vivo. In addition, aggregates are potentially useful building blocks for tissue engineering. However, control over the interior aggregate microenvironment is challenging due to inherent barriers for diffusion of biological mediators (e.g. growth factors) throughout the multicellular aggregates. Previous studies have shown that incorporation of biomaterials into multicellular aggregates can support cell survival and control differentiation of stem cell aggregates by delivering morphogens from within the 3D construct. In this study, we developed a highly efficient microparticle-based gene delivery approach to uniformly transfect human mesenchymal stromal cells (hMSC) within multicellular aggregates and cell sheets. We hypothesized that release of plasmid DNA (pDNA) lipoplexes from mineral-coated microparticles (MCMs) within 3D hMSC constructs would improve transfection in comparison to standard free pDNA lipoplex delivery in the media. Our approach increased transfection efficiency 5-fold over delivery of free pDNA lipoplexes in the media and resulted in homogenous distribution of transfected cells throughout the 3D constructs. Additionally, we found that MCMs improved hMSC transfection by specifically increasing macropinocytosis-mediated uptake of pDNA. Finally, we showed up to a three-fold increase of bone morphogenetic protein-2 (BMP-2) expression and enhanced calcium deposition within 3D hMSC constructs following MCM-mediated delivery of a BMP-2 encoding plasmid and culture in osteogenic medium. The technology described here provides a valuable tool for achieving efficient and homogenous transfection of 3D cell constructs and is therefore of particular value in tissue engineering and regenerative medicine applications. Statement of SignificanceThis original research describes a materials-based approach, whereby use of mineral-coated microparticles improves the efficiency of non-viral gene delivery in three-dimensional human mesenchymal stromal cell constructs. Specifically, it demonstrates the use of mineral-coated microparticles to enable highly efficient transfection of human mesenchymal stromal cells in large, 3D culture formats. The manuscript also identifies specific endocytosis pathways that interact with the mineral coating to afford the improved transfection efficiency, as well as demonstrates the utility of this approach toward improving differentiation of large cell constructs. We feel that this manuscript will impact the current understanding and near-term development of materials for non-viral gene delivery in broad tissue engineering and biofabrication applications, and therefore be of interest to a diverse biomaterials audience.

Interleukin-1 receptor antagonist protein (IL-1Ra) and miR-140 overexpression via pNNS-conjugated chitosan-mediated gene transfer enhances the repair of full-thickness cartilage defects in a rabbit model.

ObjectivesPreviously, we reported the improved transfection efficiency of a plasmid DNA-chitosan (pDNA-CS) complex using a phosphorylatable nuclear localization signal-linked nucleic kinase substrate short peptide (pNNS) conjugated to chitosan (pNNS-CS). This study investigated the effects of pNNS-CS-mediated miR-140 and interleukin-1 receptor antagonist protein (IL-1Ra) gene transfection both in rabbit chondrocytes and a cartilage defect model.MethodsThe pBudCE4.1-miR-140, pBudCE4.1-IL-1Ra, and negative control pBudCE4.1 plasmids were constructed and combined with pNNS-CS to form pDNA/pNNS-CS complexes. These complexes were transfected into chondrocytes or injected into the knee joint cavity.ResultsHigh IL-1Ra and miR-140 expression levels were detected both in vitro and in vivo. In vitro, compared with the pBudCE4.1 group, the transgenic group presented with significantly increased chondrocyte proliferation and glycosaminoglycan (GAG) synthesis, as well as increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and TIMP metallopeptidase inhibitor 1 (TIMP-1) levels. Nitric oxide (NO) synthesis was reduced, as were a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 (ADAMTS-5) and matrix metalloproteinase (MMP)-13 levels. In vivo, the exogenous genes reduced the synovial fluid GAG and NO concentrations and the ADAMTS-5 and MMP-13 levels in cartilage. In contrast, COL2A1, ACAN, and TIMP-1 levels were increased, and the cartilage Mankin score was decreased in the transgenic group compared with the pBudCE4.1 group. Double gene combination produced greater efficacies than each single gene, both in vitro and in vivo.ConclusionThis study suggests that pNNS-CS is a good candidate for treating cartilage defects via gene therapy, and that IL-1Ra in combination with miR-140 produces promising biological effects on cartilage defects.Cite this article: R. Zhao, S. Wang, L. Jia, Q. Li, J. Qiao, X. Peng. Interleukin-1 receptor antagonist protein (IL-1Ra) and miR-140 overexpression via pNNS-conjugated chitosan-mediated gene transfer enhances the repair of full-thickness cartilage defects in a rabbit model. Bone Joint Res 2019;8:165–178. DOI: 10.1302/2046-3758.83.BJR-2018-0222.R1.

SPIONs’ Enhancer Effect on Cell Transfection: An Unexpected Advantage for an Improved Gene Delivery System

The clinical implantation of gene therapy is hindered by the limitations of current gene delivery vectors, namely, safety issues regarding viral vectors and low transfection efficacy regarding nonviral vectors. Thus, the design of safe and efficient gene carriers is a key point for the success of such therapies. In addition, when employing genetically modified cells for further applications, the selection of successfully modified cells becomes crucial. To address these issues, we have developed multicomponent nanoparticles composed of poly(s-amino ester) (pBAE) polymers, plasmid DNA, and superparamagnetic iron oxide nanoparticles (SPIONs). Whereas pBAEs were initially employed as safe and biocompatible carriers with improved transfection efficiency, as compared to commercial vectors, SPIONs were used because of their magnetic character that enables cell selection. Surprisingly, the results presented here revealed an unexpected enhancer effect of SPIONs on the transfection efficiency of pBAE/pDNA polyplexe...

Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

BackgroundWe developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells.ResultsmTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C.ConclusionsThese findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Photothermal-assisted surface-mediated gene delivery for enhancing transfection efficiency.

The development of gene therapy puts forward the requirements for efficient delivery of genetic information into diverse cells. However, in some cases of transfection, especially those for transfecting some primary cells and for delivering large size plasmid DNA (pDNA), the existing conventional transfection methods show poor efficiency. How to further improve transfection efficiency in these hard-to-achieve issues remains a crucial challenge. Here, we report a photothermal-assisted surface-mediated gene delivery based on a polydopamine-polyethylenimine (PDA-PEI) surface. The PDA-PEI surface was prepared through PEI-accelerated dopamine polymerization, which showed efficiency in the immobilization of PEI/pDNA polyplexes and remarkable photothermal properties. Upon IR irradiation, we observed improved transfection efficiencies of two important hard-to-achieve transfection issues, namely the transfection of primary endothelial cells, which are kinds of typical hard-to-transfect cells, and the transfection of cells with large-size pDNA. We demonstrate that the increases of transfection efficiency were due to the hyperthermia-induced pDNA release, the local cell membrane disturbance, and the polyplex internalization. This work highlights the importance of local immobilization and release of pDNA to gene deliveries, showing great potential applications in medical devices in the field of gene therapy.

Multiply clustered gold-based nanoparticles complexed with exogenous pDNA achieve prolonged gene expression in stem cells.

Development of a stable and prolonged gene delivery system is a key goal in the gene therapy field. To this end, we designed and fabricated a gene delivery system based on multiply-clustered gold particles that could achieve prolonged gene delivery in stem cells, leading to improved induction of differentiation.Methods: Inorganic gold nanoparticles (AuNPs) underwent three rounds of complexation with catechol-functionalized polyethyleneimine (CPEI) and plasmid DNAs (pDNAs), in that order, with addition of heparin (HP) between rounds, yielding multiply-clustered gold-based nanoparticles (mCGNPs). Via metal-catechol group interactions, the AuNP surface was easily coordinated with positively charged CPEIs, which in turn allowed binding of pDNAs.Results: Negatively charged HP was encapsulated with the positive charge of CPEIs via electrostatic interactions, making the NPs more compact. Repeating the complexation process yielded mCGNPs with improved transfection efficiency in human mesenchymal stem cells (hMSCs); moreover, these particles exhibited lower cytotoxicity and longer expression of pDNAs than conventional NPs. This design was applied to induction of chondrogenesis in hMSCs using pDNA harboring SOX9, an important chondrogenic transcription factor. Prolonged expression of SOX9 induced by mCGNPs triggered expression of chondrocyte extracellular matrix (ECM) protein after 14 days, leading to more efficient chondrogenic differentiation in vitro and in vivo.

Protein corona of magnetic PEI/siRNA complex under the influence of a magnetic field improves transfection efficiencyviacomplement and coagulation cascades

Magnetic fields enhance the silencing efficiencyviathe alteration of protein corona adsorbed on magnetic PEI/siRNA complex.

Amphoteric poly(amido amine)s with adjustable balance between transfection efficiency and cytotoxicity for gene delivery.

In order to balance transfection efficiency and cytotoxicity as well as screen the optimal polymers for gene delivery, a series of amphoteric copolymers (poly(CBA-AGM/GABA)s) composed of different ratios between agmatine (AGM) and γ-aminobutyric acid (GABA) monomers were synthesized. The AGM containing positively charged guanidinium groups was used to improve transfection efficiency, while the GABA containing negatively charged carboxyl groups was used to decrease cytotoxicity. It is hypothesized that the amphoteric poly(CBA-AGM/GABA)s synthesized at the optimal ratio of both components would well balance transfection efficiency and cytotoxicity. By comparing these polymers' essential features in gene delivery, the ideal ratio between AGM and GABA was optimized. AGM80, which contained 80% AGM and 20% GABA, showed favorable properties for gene delivery, including moderate DNA condensation capacity, high cellular uptake, strong nuclear localization ability, high transfection efficiency, and low cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.

Polymeric nanoengineered HBsAg DNA vaccine designed in combination with β‑glucan

Antigen-specific immune responses following DNA vaccination are hard to achieve, owing to the difficulty to mediate efficient gene delivery. This study proposed the use of PDMAEMA:PβAE/DNA polyplexes (Pol) as the vehicle of a pDNA vaccine encoding the hepatitis B surface antigen (HBsAg), with these Pol designed in combination with a soluble (Glu) or a particulate (GPs) form of β‑glucan. β‑Glucans are recognized adjuvants that activate immune cells, a good strategy to improve transfection efficiency and vaccine efficacy. Results showed that Pol produced at a 19:1 polymer:DNA (+/−) charge ratio were positively charged (+41 mV), had a mean size of 180 nm and presented high stability under different storage conditions. These polyplexes resulted in enhanced transfection activity than the positive control, showing even higher luciferase gene expression in the presence of GPs (COS-7 and RAW 264.7 cell lines). Additionally, no alterations in hemolysis and plasma coagulation time of human blood were found in the non-cytotoxic working range. Mice vaccination studies (pCMV-S), resulted in a seroconversion rate of 40%, regardless of the additional β‑glucan adjuvants. This work showed the potential of this nanosystem together with GPs to enhance in vitro transfection capacity and to be further studied as a DNA vaccination platform.

Senile Octodon degus Reproductive Strategy: Morphometric and Histological Description of the Penis

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

PH-Responsive Charge-Conversional Poly(ethylene imine)-Poly(l-lysine)-Poly(l-glutamic acid) with Self-Assembly and Endosome Buffering Ability for Gene Delivery Systems.

Poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly(ethylene imine) 1.8 kDa (PEI1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK5E9 polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK5E9 or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. In conclusion, PK5E9 polymer with self-assembly and endosome buffering ability was found to possess potentials for efficient gene delivery systems in acidic conditions via charge conversion, which may be applied for tumor microenvironment-targeting.

MicroRNAs delivery into human cells grown on 3D-printed PLA scaffolds coated with a novel fluorescent PAMAM dendrimer for biomedical applications

Many advanced synthetic, natural, degradable or non-degradable materials have been employed to create scaffolds for cell culture for biomedical or tissue engineering applications. One of the most versatile material is poly-lactide (PLA), commonly used as 3D printing filament. Manufacturing of multifunctional scaffolds with improved cell growth proliferation and able to deliver oligonucleotides represents an innovative strategy for controlled and localized gene modulation that hold great promise and could increase the number of applications in biomedicine. Here we report for the first time the synthesis of a novel Rhodamine derivative of a poly-amidoamine dendrimer (G = 5) able to transfect cells and to be monitored by confocal microscopy that we also employed to coat a 3D-printed PLA scaffold. The coating do not modify the oligonucleotide binding ability, toxicity or transfection properties of the scaffold that is able to increase cell proliferation and deliver miRNA mimics (i.e., pre-mir-503) into human cells. Although further experiments are required to optimize the dendrimer/miRNA ratio and improve transfection efficiency, we demonstrated the effectiveness of this promising and innovative 3D-printed transfection system to transfer miRNAs into human cells for future biomedical applications.

Effective and safe in vivo gene delivery based on polyglutamic acid complexes with heterocyclic amine modified-polyethylenimine

Polyethylenimine (PEI) has been extensively used for non-viral gene delivery. Increasing the molecular weight of PEI often improves transfection efficiency, but enhances cytotoxicity and non-specific interaction with plasma proteins, limiting its use in clinical applications. In this study, poly-l-glutamic acid (L-PGA) as an anionic polymer, was introduced to piperazine-modified PEI to improve its in vivo properties. The physicochemical properties, cytotoxicity, in vitro and in vivo tranfection efficiency of these carriers were evaluated. Conjugation of 50% of primary amines of PEI 25 kDa with piperazine in the presence of PGA1% (PEI25Pip50%/PGA1%) could significantly increase transfection efficiency even in the presence of serum compared to PEI 25 kDa. Increasing the PGA content led to lower cytotoxicity of DNA/PEI25Pip50%/PGA1% triplexes. Systemic administration of triplexes in Balb/c mice resulted in significant enhancement of luciferase gene expression in brain, spleen, and liver compared to PEI 25 kDa. In a 30-day survival study, no significant changes were observed in mice body weights in DNA/PEI25Pip50%/PGA1% group. Moreover, this group exhibited a survival rate of 100% compared to 0% in mice receiving PEI 25 kDa. This novel PEI25Pip50%/PGA1% carrier could be used to overcome the serum inhibitory effects on gene expression in vivo, providing a promising gene delivery system for tissue-specific targeting.

In silico and in vitro study of magnetic niosomes for gene delivery: The effect of ergosterol and cholesterol

A low transfection efficiency and failure to deliver therapeutic genes to target organs limit the use of vesicular systems in gene therapy. In this study, magnetic niosomes were used to improve transfection efficiency and overcome limitations. In this light, Tween 60 and Span 60 molecules were employed as the bilayer component and ergosterol and/or cholesterol as membrane-stabilizing agents. We studied the structural and dynamical properties of cholesterol-containing niosomes (ST60/Chol) and ergosterol-containing vesicles (ST60/Ergo) using the molecular dynamics (MD) simulation technique. In in vitro experiments, the protamine-condensed DNA along with magnetic nanoparticles were prepared and incorporated into the niosome to form magnetic niosome-entrapped protamine-condensed DNA (M-NPD). The MD simulation comparison of two bilayers showed that the ST60/Ergo vesicles have better properties for gene delivery. Our in vitro results confirmed the in silico results and revealed that Ergo-niosomes have smaller size, better polydispersity, and slower release of plasmid than Chol-niosome. Moreover, M-NPD-Ergo showed higher cellular uptake and gene expresssion in HEK-293T cell line compared to M-NPD-Chol vesicles.

Hyaluronic acid-modified cationic niosomes for ocular gene delivery: improving transfection efficiency in retinal pigment epithelium.

Recent years, gene therapy to treat retinal diseases has been paid much attention. The key to successful therapy is utilizing smart delivery system to achieve efficient gene delivery and transfection. In this study, hyaluronic acid (HA) modified cationic niosomes (HA-C-niosomes) have been designed in order to achieve retinal pigment epithelium (RPE) cells targeted gene delivery and efficient gene transfection. Cationic niosomes composed of tween 80/squalene/1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) were prepared by the ethanol injection method. After that, HA-DOPE was further added into cationic niosomes to form HA-C-niosomes. Cellular uptake and transfection have been investigated in ARPE-19 cells. In vivo pEGFP transfection efficiency was evaluated in rats. Twenty percentage HA-C-niosomes were about 180 nm, with -30 mV, and showing spherical shape in TEM. 2 times higher transfection efficiency was found in the group of HA-C-niosomes with 20% HA modification. No toxicity was found in niosome preparations. In vivo evaluation in Sprague Dawley (SD) rats revealed that HA-C-niosomes could specifically target to the retina layer. In the group of pEGFP-loaded HA-C-niosomes, 6-6.5 times higher gene transfection has been achieved, compared with naked pEGFP. Hyaluronic acid-C-niosomes might provide a promising gene delivery system for successful retinal gene therapy.