Non-viral Vectors For Gene Transfection Research Articles

Non-viral vector based gene transfection with human induced pluripotent stem cells derived cardiomyocytes

Non-viral transfection of mammalian cardiomyocytes (CMs) is challenging. The current study aims to characterize and determine the non-viral vector based gene transfection efficiency with human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs differentiated from PCBC hiPSCs were used as a cell model to be transfected with plasmids carrying green fluorescence protein (pGFP) using polyethylenimine (PEI), including Transporter 5 Transfection Reagent (TR5) and PEI25, and liposome, including lipofectamine-2000 (Lipo2K), lipofectamine-3000 (Lipo3K), and Lipofectamine STEM (LipoSTEM). The gene transfection efficiency and cell viability were quantified by flow cytometry. We found that the highest gene transfection efficiency in hiPSC-CMs on day 14 of contraction can be achieved by LipoSTEM which was about 32.5 ± 6.7%. However, it also cuased poor cell viability (60.1 ± 4.5%). Furthermore, a prolonged culture of (transfection on day 23 of contraction) hiPSC-CMs not only improved gene transfection (54.5 ± 8.9%), but also enhanced cell viability (74 ± 4.9%) by LipoSTEM. Based on this optimized gene transfection condition, the highest gene transfection efficiency was 55.6 ± 7.8% or 34.1 ± 4%, respectively, for P1C1 or DP3 hiPSC line that was derived from healthy donor (P1C1) or patient with diabetes (DP3). The cell viability was 80.8 ± 5.2% or 92.9 ± 2.24%, respectively, for P1C1 or DP3. LipoSTEM is a better non-viral vector for gene transfection of hiPSC-CMs. The highest pGFP gene transfection efficiency can reach >50% for normal hiPSC-CMs or >30% for diabetic hiPSC-CMs.

A PEG-b-poly(disulfide-l-lysine) based redox-responsive cationic polymer for efficient gene transfection.

Gene therapy is concerned with the transfer of complement genes to functionally defective cells in a safe and directed manner for the treatment of the most challenging diseases. But safety issues and low transfection efficiency of the gene vectors are the major challenges, which need to be overcome. Recently, redox-responsive bioreducible polymers containing disulfide linkages have been considered as efficient gene vectors, owing to the selective degradation of the disulfide bond in the reducing environment of the cells. This enables spatiotemporal release of pDNA with no or minimum toxicity. Herein, we reported a bioreducible poly(ethyleneglycol)-b-poly(disulfide-l-lysine) cationic polymer (denoted as PEG-SSL) via a Michael addition reaction of poly(ethyleneglycol)tetraacrylate PEG(Ac)4 and the terminal amine group of poly(disulfide-l-lysine). PEG-SSL efficiently condensed the plasmid ZNF580 gene (pZNF580) forming nano-sized polyplexes (155 ± 4 to 285 ± 3 nm) with zeta potentials of 1.9 ± 0.1 to 26.7 ± 0.4 mV. PEG-SSL successfully retarded pZNF580 at a small polymer/pDNA weight ratio of 10/1 and higher. When exposed to a reducing environment of 5 mM DTT, it rapidly released genes even at higher weight ratios of the PEG-SSL polymer in the PEG-SSL/pDNA complexes. The PEG-SSL/pZNF580 complexes exhibited good stability when exposed to DNase I and efficiently protected pDNA from degradation. In vitro transfection and cytotoxicity were investigated in EA.hy926 cells. The results showed that PEG-SSL successfully delivered pZNF580 into the cells with less cytotoxicity compared to PEI25kDa. The flow cytometry and confocal scanning laser microscopy results indicated that PEG-SSL polyplexes exhibited good cellular uptake and nuclear co-localization rates. All these results implied that PEG-SSL had the potential as a non-viral vector for gene transfection.

Dynamic constitutional chemistry towards efficient nonviral vectors

Dynamic constitutional chemistry has been used to design nonviral vectors for gene transfection. Their design has been thought in order to fulfill ab initio the main requirements for gene therapy. As building blocks were used hyperbranched PEI as hydrophilic part and benzentrialdehyde and a diamine linear siloxane as hydrophobic part, connected through reversible imine linkages. The obtaining of the envisaged structures has been confirmed by NMR and FTIR spectroscopy. The dynamic synthesized amphiphiles proved to be able to self-assemble in nano-sized spherical entities as was demonstrated by TEM and DLS, characterized by a narrow dimensional polydispersity. Agarose gel electrophoresis proved the ability of the synthesized compounds to bind DNA, while TEM revealed the spherical morphology of the formed polyplexes.As a proof of the concept, the nonviral vectors promoted an efficient transfection on HeLa cells, demonstrating that dynamic constitutional chemistry can be an important tool in the development of this domain.

Applications of lipid nanoparticles in gene therapy

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been recognized, among the large number of non-viral vectors for gene transfection, as an effective and safety alternative to potentially treat both genetic and not genetic diseases. A key feature is the possibility to be designed to overcome the numerous challenges for successful gene delivery. Lipid nanoparticles (LNs) are able to overcome the main biological barriers for cell transfection, including degradation by nucleases, cell internalization intracellular trafficking, and selectively targeting to a specific cell type. Additionally, they present important advantages: from a safety point of view LNs are prepared with well tolerated components, and from a technological point of view, they can be easily produced at large-scale, can be subjected to sterilization and lyophilization, and have shown good storage stability. This review focuses on the potential of SLNs and NLCs for gene therapy, including the main advances in their application for the treatment of ocular diseases, infectious diseases, lysosomal storage disorders and cancer, and current research for their future clinical application.

A knot polymer mediated non-viral gene transfection for skin cells.

A knot polymer, poly[bis(2-acryloyl)oxyethyl disulphide-co-2-(dimethylamino) ethyl methacrylate] (DSP), was synthesized, optimized and evaluated as a non-viral vector for gene transfection for skin cells, keratinocytes. With recessive dystrophic epidermolysis bullosa keratinocytes (RDEBK-TA4), the DSP exhibited high transfection efficacy with both Gaussia luciferase marker DNA and the full length COL7A1 transcript encoding the therapeutic type VII collagen protein (C7). The effective restoration of C7 in C7 null-RDEB skin cells indicates that DSP is promising for non-viral gene therapy of recessive dystrophic epidermolysis bullosa (RDEB).

Solid Lipid Nanoparticles as Non-Viral Vectors for Gene Transfection in a Cell Model of Fabry Disease.

Here, we demonstrate the ability of solid lipid nanoparticle-based non-viral vectors to increase the α-galactosidase A levels of the IMFE1 cell line, an in vitro model for target cells in Fabry disease. For this purpose, vectors containing the pR-M10-αGal A plasmid, which encodes the α-galactosidase A enzyme, were prepared; the in vitro transfection efficacy was studied in IMFE1 cells, and the results were confirmed by RT-PCR. The cellular uptake of the vectors, intracellular disposition of the plasmid, and probable endocytosis pathways of the nanoparticles were also analyzed. The vectors used for the studies carried protamine (P-DNA-SLN), dextran and protamine (D-P-DNA-SLN), or hyaluronic acid of two different molecular weights and protamine (HA150-P-DNA-SLN or HA500-P-DNA-SLN). The new formulations, which presented a particle size in the range of nanometers (from 218 nm to 348 nm) and a positive superficial charge, were able to increase α-galactosidase A activity up to 4-fold in comparison to non treated IMFE1 cells. The most efficient vectors were those that included HA, and no differences due to changes in the molecular weight of HA were detected. The observed lack of colocalization with each of the four different Nile Red-labeled vectors and transferrin or cholera toxin appears to indicate that clathrin- and caveolae-independent pathways may be involved in their cellular uptake. Additionally, colocalization with LysoTracker indicated that the formulations were exposed to lysosomal activity, which may be responsible for the release of the plasmid from the vector. In conclusion, we reveal the potential of SLN-based vectors to efficiently transfect an immortalized Fabry patient cell line.

In vitro and in vivo gene transfection using biodegradable and low cytotoxic nanomicelles based on dendritic block copolymers.

Dendritic block copolymers with different chain length of poly(ε-caprolactone) (PCL) and poly(2-(dimethyl-amino)ethylmethacrylate) (PDMAEMA) were prepared and investigated as non-viral vectors for gene transfection in vitro and in vivo. Gel retardation analysis showed that all these dendritic copolymers could completely retard the mobility of DNA at and above an N/P ratio of 2.5/1. Dynamic light scattering analysis revealed that, at and above an N/P ratio of 5/1, these dendritic copolymers strongly condensed DNA into nanoscale polyplexes with average sizes ranging from about 50-160 nm and positive surface charges ranging from +13.4 to +30.3 mV. In vitro transfection tests using the plasmid encoding green fluorescence protein as gene reporter indicated that dendritic PCL-b-PDMAEMA copolymers were capable of transfecting MCF-7 and SKOV-3 cells, yielding comparable or higher transfection efficiencies in the absence or presence of serum when compared to 25 kDa branched polyethylenimine (bPEI) or 50 kDa PDMAEMA homopolymer as positive controls. Moreover, by intravenous injection of the polyplexes of these dendritic copolymers into SKOV-3 tumor-bearing nude mice, detectable transgene expression was observed in the tumor and other organs. These dendritic copolymers exhibited low in vitro cytotoxicity against MCF-7 and SKOV-3 cells with >80% viable cells at the tested polymer concentration below 25 μg mL-1 and also caused no death of the mice in vivo at a dose of 0.75 mg kg-1. The results manifested that dendritic PCL-b-PDMAEMA copolymers have high potential as safe and efficient gene delivery vectors.

Analytical model study of complexation of dendrimer as an ion penetrable sphere with DNA

Poly (amidoamine) (PAMAM) dendrimers, being protonated under physiological conditions, have great potential as nonviral vectors for gene transfection. Many experimental and simulation studies have been performed to study the effect of dendrimer size, charge, and salt concentration on the structure and transfection efficiency of condensed DNA aggregates. In this study a theoretical model describing a linear polyelectrolyte (LPE) and ion-penetrable spheres has been developed and applied to investigate the interaction between linearized DNA and positively charged dendrimer of different generations. Throughout the study, we emphasized on the effect of the medium’s environments on the complexation of LPE chain with one dendrimer. It is found that the wrapping degree of the chain around the dendrimer increases by increasing dendrimer’s charge (decreasing pH), Bjerum length, salt concentration, and decreases by increasing the rigidity of the chain. Also, the effect of 1:1 salt concentration on complexation of DNA plasmids with one dendrimer of different generations has been investigated, and found that the optimal wrapping length of the LPE chain around the dendrimer depends on dendrimer generation.

Enhanced gene transfection efficiency of PDMAEMA by incorporating hydrophobic hyperbranched polymer cores: effect of degree of branching

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a well-known cationic polymer candidate for non-viral vectors for gene transfection. However, such an application has been greatly limited due to the cytotoxicity of the polymers. Herein, PDMAEMAs are grafted from hydrophobic hyperbranched PEHO cores (PEHO means poly(3-ethyl-3-(hydroxymethyl)-oxetane)), and the obtained hyperbranched multiarm copolymers of PEHO-g-PDMAEMAs show higher transfection efficiency than that of branched polyethylenimine (PEI) and PDMAEMA homopolymers, due to the improved cytotoxicity, DNA compaction, buffering ability and cellular uptake. In addition, to disclose the structure–property relationship, a series of PEHO-g-PDMAEMAs with different topological architectures are synthesized by changing the degrees of branching (DBs) of the PEHO cores and the lengths of the PDMAEMA arms. The ability of these vectors in DNA compaction, buffering ability, cytotoxicity and gene transfection efficiency is also investigated. It has been found the gene transfection efficiency of the vectors is dependent on the DB of the PEHO cores, but almost independent of the PDMAEMA arms in the experimental range.

PEGylated PEI-based biodegradable polymers as non-viral gene vectors

Novel functional biodegradable gene vectors, poly(L-succinimide)-g-polyethylenimines-g-poly(ethylene glycol) (PSI-g-PEI-g-PEGs) were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG, M(w)=750 Da) to PEI segments (M(w)=800 Da) of PSI-g-PEI. The physicochemical properties of PSI-g-PEI-g-PEGs, including buffering capability, pDNA binding ability, cytotoxicity, zeta potential and the particle size of polymer/pDNA complexes, were explored. The influence of PEGylation was discussed based on a comparative study of PSI-g-PEI-g-PEGs, PSI-g-PEI and PEI25k (M(w)=25 kDa). SEM images revealed that PSI-g-PEI-g-PEG/pDNA particles have a regular shape with the diameter ranging from 70 to 170 nm. PEGylation could suppress the aggregation occurrence between complexes, resulting in a reduction of the polymer/pDNA complex size. PSI-g-PEI-g-PEGs exhibited remarkably lower cytotoxicity compared to PSI-g-PEI and PEI25k. In 293T and HeLa cells, the obtained PSI-g-PEI-g-PEGs showed very high transfection efficiency compared to PEI25k. Fluorescent confocal microscopy demonstrated that PSI-g-PEI-g-PEGs could effectively transport pGL-3 plasmids into the nuclei of HeLa cells. Taking into account the continued high transfection efficacy and decreased toxicity after PEG modification, PSI-g-PEI-g-PEGs show great potential as the non-viral vectors for gene transfection.

A Novel Cationic Lipophosphoramide with Diunsaturated Lipid Chains: Synthesis, Physicochemical Properties, and Transfection Activities

Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.

Convenient Preparation of Biodegradable PEI‐Containing Polymers as Non‐Viral Vectors for Gene Transfection

Poly(L-succinimide)-graft-polyethylenimines (PSPs) were prepared as non-viral vectors for gene transfection. Branched polyethylenimine (Mw= 800, PEI800) was grafted to poly(L-succinimide) (PSI) in a one-step reaction with no catalyst. Gel retardation assay showed that the mobility of PSP/pDNA complexes was completely retarded at the low N/P ratio of 0.42. In vitro transfection experiments showed that, at N/P ratio of 0.84, PSPs can reach the highest transfection level with ten-fold enhancement in 293T cells and five-fold enhancement in HeLa cells as compared with PEI25k (Mw= 25,000). Fluorescent confocal microscopy showed that pGL-3 plasmids condensed by PSPs could be effectively transported into the nuclei of HeLa cells. Significantly reduced cytotoxicity of polymers was observed towards 293T and HeLa cells, with the 50% inhibition concentration of PSPs being almost four times higher than that of PEI25k.

Polycationic graft copolymers of poly(N-vinylpyrrolidone) as non-viral vectors for gene transfection

AbstractNovel graft copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with N-vinylpyrrolidone (NVP) were designed and synthesized by the free radical copolymerization of DMAEMA with precursor polymers of vinyl-functionalized poly(N-vinylpyrrolidone) (PVP). The ability of the PVP- grafted copolymers to bind and condense DNA was confirmed by ethidium bromide displacement assay, agarose gel electrophoresis and transmission electron microscopy. The presence of PVP in the copolymers had a favorable effect on the biophysical properties of polymer/DNA complexes. Colloidal stable complexes obtained from the copolymer systems, were shown to be separate, uniformly spherical nanoparticles by transmission electron microscopy. The approximate diameter of the complexes was 150–200 nm, as determined by dynamic light scattering studies. These results confirm an important role played by the PVP grafts in producing compact stable DNA complexes. The ζ-potential measurements revealed that the incorporation of the PVP grafts reduced the positive surface charge of polymer/DNA complexes. The cytotoxicity of the copolymers decreased with an increasing fraction of PVP. Furthermore, in vitro transfection experiments with these copolymers showed improved ability of transfection in cell culture, demonstrating an important role for PVP grafts in enhancement of the transfection efficiency.

Characterization of Biosurfactant-Containing Liposomes and Their Efficiency for Gene Transfection

Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection.

Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome

Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3β-[ N-( N′ ,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy.

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

Chitosan and its derivatives--a promising non-viral vector for gene transfection.

The ability of a vector to ferry gene into targeted cells is the premise of improving gene-transfer efficiency. Chitosan, a naturally occurring cationic polysaccharide, has been shown to excel in transcellular transport. This attribute has been fully reflected in chitosan-mediated gene transfection systems. The objective of this review is to summarize the recent encouraging advances in unveiling the mechanism of cell entry and application of chitosan and its derivatives as novel non-viral vectors. It is our belief that researchers will uncover more truth about chitosan-based vector and realize the long-term goal of gene transfection--produce the desired clinical effect.