Non-viral Transfection Vectors Research Articles

Introduction of tenomodulin by gene transfection vectors for rat bone tissue regeneration.

Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique invitro and invivo was investigated in this study. Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.

Cationic lipid membranes condense double-strand DNA through ion bridging.

Cationic lipid (CL)-DNA complexes have attracted much attention due to their potential application in somatic gene therapy, such as nonviral transfection vectors. The rational design of efficient synthetic vectors requires a systematic understanding of the underlying mechanisms governing CL-DNA assembly. Experimental studies show that DNA double strands form assemblies in divalent cations when confined between CL lipid systems. The physical mechanism of membrane-induced condensation is not well understood. Herein, we present a computational study of a simplified vector system, quasi-random genomic DNA chains intercalated into DOPC/DOTAP membrane bilayers. Using all-atom molecular dynamics simulations and free energy calculations, we observe attraction between dsDNA pairs when the ionic strength of the solution and mole fraction of DOPC mimic experimental conditions. Strikingly, the same salt conditions favoring condensation in CL lead to repulsion without the lipid. We investigated the role of lipids and confined cations to understand the differences in DNA-DNA interactions. Simulations suggest that the head groups of the lipids in contact with the DNA backbone reduce the DNA charges. The cationic DOTAP and zwitterionic DOPC head groups form direct contact with the DNA backbone. In addition, the DOPC phosphate head groups are glued to the DNA backbone by magnesium cations. The already reduced opposite surface of DNA double strands are brought together with dynamically bridging magnesium cations confined between the two helices. Our study offers physical insight into the mechanism of CL-DNA assembly and may help to facilitate the development of liposome transfer vectors for gene delivery.

Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle.

RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues. Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers. Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine® 3.0, a lipid nonviral transfection vector, for local and sustained release. PEGylation of fibrin allowed for controlled release of small interfering RNA (siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo. A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days. Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth. Thus, the increased efficacy observed here for the combination of a lipid nanoparticle, the only type of nonviral vector approved for the clinic, with fibrin, might allow for more rapid translation of injectable hydrogel-based RNA interference.

Novel Non-Viral Vectors Based on Pluronic® F68PEI with Application in Oncology Field.

Copolymers composed of low-molecular-weight polyethylenimine (PEI) and amphiphilic Pluronics® are safe and efficient non-viral vectors for pDNA transfection. A variety of Pluronic® properties provides a base for tailoring transfection efficacy in combination with the unique biological activity of this polymer group. In this study, we describe the preparation of new copolymers based on hydrophilic Pluronic® F68 and PEI (F68PEI). F68PEI polyplexes obtained by doping with free F68 (1:2 and 1:5 w/w) allowed for fine-tuning of physicochemical properties and transfection activity, demonstrating improved in vitro transfection of the human bone osteosarcoma epithelial (U2OS) and oral squamous cell carcinoma (SCC-9) cells when compared to the parent formulation, F68PEI. Although all tested systems condensed pDNA at varying polymer/DNA charge ratios (N/P, 5/1−100/1), the addition of free F68 (1:5 w/w) resulted in the formation of smaller polyplexes (<200 nm). Analysis of polyplex properties by transmission electron microscopy and dynamic light scattering revealed varied polyplex morphology. Transfection potential was also found to be cell-dependent and significantly higher in SCC-9 cells compared to the control bPEI25k cells, as especially evident at higher N/P ratios (>25). The observed selectivity towards transfection of SSC-9 cells might represent a base for further optimization of a cell-specific transfection vehicle.

A new nano hyperbranched β-pinene polymer: Controlled synthesis and nonviral gene delivery

Recently, an extensive research effort has been directed toward the improvement of nonviral transfection vectors, such as polymeric materials. The macromolecular structure of polymers has a substantial effect on their transfection efficacy. In this context, the modern advances in polymer production techniques, such as the deactivation-enhanced radical atom transfer polymerization (DE-ATRP), have been fundamental for the synthesis of controlled architecture nanomaterials. In this study, hyperbranched poly(β-pinene)-PDMAEMA-PEGDMA nanometric copolymers were synthesised at high conversion via DE-ATRP using different concentrations of β-pinene for gene delivery applications. The structural characterization and the biological performance of the materials were investigated. The copolymers’ molar mass (10,434–16,438 mol l-1), dispersity, and conversion (90–95%) varied significantly with β-pinene proportion on the polymerizations. The polymer-gene complexes generated (280–110 nm) presented excellent solution stability due to the β-pinene segment on the copolymers. Gene delivery and transfection were highly dependent on the copolymer composition. The copolymers containing the highest β-pinene proportions exhibited the best results with high transfection effectivity. In conclusion, the incorporation of β-pinene in DMAEMA-PEGMA copolymer formulations is a renewable option to improve the materials’ branching ratio, polyplex stability, and gene delivery performance without causing cytotoxic effects.

Mesenchymal Stem Cells Engineered by Nonviral Vectors: A Powerful Tool in Cancer Gene Therapy.

Due to their “tumor homing” and “immune privilege” characteristics, the use of mesenchymal stem cells (MSCs) has been proposed as a novel tool against cancer. MSCs are genetically engineered in vitro and then utilized to deliver tumoricidal agents, including prodrugs and bioactive molecules, to tumors. The genetic modification of MSCs can be achieved by various vectors, and in most cases viral vectors are used; however, viruses may be associated with carcinogenesis and immunogenicity, restricting their clinical translational potential. As such, nonviral vectors have emerged as a potential solution to address these limitations and have gradually attracted increasing attention. In this review, we briefly revisit the current knowledge about MSC-based cancer gene therapy. Then, we summarize the advantages and challenges of nonviral vectors for MSC transfection. Finally, we discuss recent advances in the development of new nonviral vectors, which have provided promising strategies to overcome obstacles in the gene modulation of MSCs.

Unprecedented Self-Assembly in Dilute Aqueous Solution of Polyethyleneimine: Formation of Fibrillar Network

Polyethyleneimine (PEI), a cationic polyelectrolyte, finds great utility as a nonviral gene transfection vector. The mechanism of gene delivery process across the cell membrane through PEI-based po...

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations.

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human β-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

Carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for siRNA cell delivery

Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard fish embryo test (FET) on Danio rerio in vivo model, with LD50 = 6.26 µM after 48 h of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

Differentially Branched Ester Amine Quadpolymers with Amphiphilic and pH-Sensitive Properties for Efficient Plasmid DNA Delivery.

Development of highly effective nonviral gene delivery vectors for transfection of diverse cell populations remains a challenge despite utilization of both rational and combinatorial driven approaches to nanoparticle engineering. In this work, multifunctional polyesters are synthesized with well-defined branching structures via A2 + B2/B3 + C1 Michael addition reactions from small molecule acrylate and amine monomers and then end-capped with amine-containing small molecules to assess the influence of polymer branching structure on transfection. These Branched poly(Ester Amine) Quadpolymers (BEAQs) are highly effective for delivery of plasmid DNA to retinal pigment epithelial cells and demonstrate multiple improvements over previously reported leading linear poly(beta-amino ester)s, particularly for volume-limited applications where improved efficiency is required. BEAQs with moderate degrees of branching are demonstrated to be optimal for delivery under high serum conditions and low nanoparticle doses further relevant for therapeutic gene delivery applications. Defined structural properties of each polymer in the series, including tertiary amine content, correlated with cellular transfection efficacy and viability. Trends that can be applied to the rational design of future generations of biodegradable polymers are elucidated.

Non-viral transfection vectors: are hybrid materials the way forward?

Transfection is a process by which oligonucleotides (DNA or RNA) are delivered into living cells. This allows the synthesis of target proteins as well as their inhibition (gene silencing). However, oligonucleotides cannot cross the plasma membrane by themselves; therefore, efficient carriers are needed for successful gene delivery. Recombinant viruses are among the earliest described vectors. Unfortunately, they have severe drawbacks such as toxicity and immunogenicity. In this regard, the development of non-viral transfection vectors has attracted increasing interests, and has become an important field of research. In the first part of this review we start with a tutorial introduction into the biological backgrounds of gene transfection followed by the classical non-viral vectors (cationic organic carriers and inorganic nanoparticles). In the second part we highlight selected recent reports, which demonstrate that hybrid vectors that combine key features of classical carriers are a remarkable strategy to address the current challenges in gene delivery.

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.

Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery.

Herein, we present the first evaluation of cationic dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) as nonviral vectors for transfection of short interfering RNA (siRNA) in cell cultures. The study encompassed dendrimers of generation one to four (G1–G4), modified to bear 6–48 amino end-groups, where the G2–G4 proved to be capable of siRNA complexation and protection against RNase-mediated degradation. The dendrimers were nontoxic to astrocytes, glioma (C6), and glioblastoma (U87), while G3 and G4 exhibited concentration dependent toxicity towards primary neurons. The G2 showed no toxicity to primary neurons at any of the tested concentrations. Fluorescence microscopy experiments suggested that the dendrimers are highly efficient at endo-lysosomal escape since fluorescently labeled dendrimers were localized specifically in mitochondria, and diffuse cytosolic distribution of fluorescent siRNA complexed by dendrimers was observed. This is a desired feature for intracellular drug delivery, since the endocytic pathway otherwise transfers the drugs into lysosomes where they can be degraded without reaching their intended target. siRNA-transfection was successful in C6 and U87 cell lines using the G3 and G4 dendrimers followed by a decrease of approximately 20% of target protein p42-MAPK expression.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Efficient gene delivery to primary human retinal pigment epithelial cells: The innate and acquired properties of vectors

The purpose of this study is designing non-viral gene delivery vectors for transfection of the primary human retinal pigment epithelial cells (RPE). In the design process of gene delivery vectors, considering physicochemical properties of vectors alone does not seem to be enough since they interact with constituents of the surrounding environment and hence gain new characteristics. Moreover, due to these interactions, their cargo can be released untimely or undergo degradation before reaching to the target cells. Further, the characteristics of cells itself can also influence the transfection efficacy. For example, the non-dividing property of RPE cells can impede the transfection efficiency which in most studies was ignored by using immortal cell lines. In this study, vectors with different characteristics differing in mixing orders of pDNA, PEI polymer, and PLGA/PEI or PLGA nanoparticles were prepared and characterized. Then, their characteristics and efficacy in gene delivery to RPE cells in the presence of vitreous or fetal bovine serum (FBS) were evaluated. All formulations showed no cytotoxicity and were able to protect pDNA from premature release and degradation in extracellular media. Also, the adsorption of vitreous or serum proteins onto the surface of vectors changed their properties and hence cellular uptake and transfection efficacy.

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.

Engineering biodegradable micelles of polyethylenimine-based amphiphilic block copolymers for efficient DNA and siRNA delivery

Polycationic micelles have shown advantageous properties as nucleic acid delivery vectors both in vitro and in vivo. In contrast to polycationic micelles reported so far, we designed particles integrating a sufficient nucleic acid condensation capability by polycationic polyethylenimine (PEI) segments as well as only a mild cytotoxic behavior. The micelles composed of a hydrophobic oligoester core with glycolide units resulting in fast degradation after cellular internalization in combination with PEG moieties acting as shielding agents. By grafting branched 25kDa polyethylenimine (PEI25) and poly(ethylene glycol) (PEG) on poly[(ε-caprolactone)-co-glycolide] (CG), amphiphilic PEI-CG-PEI and PEG-CG block copolymers were used to form a series of micelles via self-assembly of PEI-CG-PEI or co-assembly of both copolymers for DNA and siRNA delivery. This modular system enabled a systematic investigation of different parameters and their synergetic effects as different functions were introduced. The polyplex formation and serum stability, cytotoxicity, and transfection activity could be tailored by changing the CG chain length in PEI-based copolymer, incorporating PEG-CG, and varying the N/P ratio. All micelle-based polyplex compositions showed high DNA transfection activity according to reporter gene-expression and an exceptionally high knockdown in siRNA delivery experiments. Remarkably, the GFP expression of >99% cells was successfully knocked down by micelle-mediated siRNA interference, resulting in a decrease of two orders of magnitude in fluorescence intensity. Incorporation of PEG-CG in the micelles reduced the PEI-related cytotoxicity, and markedly enhanced the serum stability of both DNA and siRNA polyplexes. Compared with homo-PEI25, these micelles showed several advantages including the lower toxicity, higher siRNA transfection efficiency and higher polyplex stability in the presence of serum. This study therefore provides an effective approach to tune the structure, property and function of polycationic micelles for efficient DNA and siRNA delivery, which could contribute to the design and development of novel non-viral transfection vectors with superb functionality.

Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

PurposePrenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.MethodsAP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.ResultsAll the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation—considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.ConclusionObtained results indicate that APs have a potential as non-viral vectors for cell transfection.

Graphene Nanoribbon-Based Platform for Highly Efficacious Nuclear Gene Delivery.

Current efforts in the design and development of nonviral vectors for gene delivery and transfection have focused on the development of versatile agents that can load short or large sized genetic material, and are efficacious without eliciting toxicity in dividing and nondividing cells. Herein, we have investigated oxidized graphene nanoribbons (O-GNRs) as nonviral vectors for gene therapy and report in vitro studies that detail their cytotoxicity, intracellular and nuclear uptake, and gene delivery and transfection efficiencies. Our results indicate that, without additional functionalization with positively charged groups or other nonviral vectors, O-GNRs could load large amounts of small-sized single-stranded or large-sized double stranded genetic materials. O-GNRs at potential therapeutic doses (20-60 μg/mL) elicited lower cytotoxicity compared to widely used commercial nonviral gene delivery vectors (Polyethylenimine and Fugene 6). The O-GNR-plasmid DNA complexes showed uptake into vesicular structures of dividing Henrietta Lacks (HeLa) and nondividing Human umbilical vein endothelial cells (HUVEC), release into the cell's cytoplasm and entry into the nucleus. In these cells, O-GNRs loaded with enhanced green fluorescence protein (EGFP) plasmid or siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed a concentration- and time- dependent increase in gene delivery and gene transfection efficiencies up to 96-98%. The results suggest that O-GNRs are promising candidates as versatile and efficient nonviral vectors of small- or large-sized genetic material in primary and secondary cell types for gene therapy.

Hydroxyapatite nanoparticles modified by branched polyethylenimine are effective non-viral vectors for siRNA transfection of hepatoma cells in vitro.

Small interfering RNA (siRNA) technology is a powerful tool in biomedical research and holds great potential for RNA interference-based therapies for HIV, hepatitis and cancer. However, the absence of a safe and efficient method for the delivery of siRNA has become a bottleneck for their development. Nanocrystallized hydroxyapatite (nHAP) appears to be an optimal candidate non-viral gene vector for several reasons, including its good biocompatibility and ease of production, however, nHAP microemulsions cannot remain monodispersed for long periods of time. Due to their high surface energy, nHAP particles gradually aggregate into large ones that are difficult for the cell to take up. To overcome this we modified nHAP with polyethylenimine (PEI) to generate a compound (MnHAP) with a tight size-distribution of <200 nm. The positive surface potential of MnHAP inhibited particle aggregation and thus made it easier to conjugate more siRNA. The transfection efficiency of MnHAP/fluorescent FAM-labeled siRNA complex was tested using flow cytometry, and the transfected cells were observed using fluorescence microscopy. The cytotoxicity of MnHAP/siRNA complexes to the human liver cancer cell line BEL-7402 was assessed in vitro by a formazan dye assay. Our results show that the in vitro transfection efficiency of MnHAP/siRNA was equivalent to that of the commercially available transfection agent Lipofectamine® 2000, but with decreased cytotoxicity. The MnHAP nanoparticles were also able to deliver siRNA for silencing of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in BEL-7402 cells, which supports that MnHAP might be a promising non-viral vector for biomedical research and gene delivery.