Non-viral Gene Delivery Systems Research Articles

Peptide-based gene delivery vectors.

Gene therapy as a strategy for disease treatment requires safe and efficient gene delivery systems that encapsulate nucleic acids and deliver them to effective sites in the cell. Due to the insecurity of viral vectors, non-viral vectors have gained great attention recently. Additional advantages of non-viral vectors include their safety, flexibility in packaging nucleic acids, and ease of production. To construct an ideal gene carrier, peptides can be incorporated into non-viral gene delivery systems as functional motifs to overcome the current barriers in gene delivery. In this review, we summarize recent developments in peptide-based gene delivery vector research.

Tuning optimum transfection of gemini surfactant-phospholipid-DNA nanoparticles by validated theoretical modeling.

Gemini nanoparticles (NPs) are a family of non-viral gene delivery systems with potential for applications in non-invasive gene therapy. Translation of these non-viral gene delivery systems requires improvement of transfection efficiency (TE) through fine-tuning of their physicochemical properties such as electric charge and exact ratios of their components. Since high-throughput experimental screening of minute differences in NP compositions is not routinely feasible, we have developed a coarse-grained model to quantitatively study the energetics of the formation of gene delivery complexes with cationic gemini surfactants (G) (m-s-m type) and helper lipids (H) (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DOPE/1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)), in order to use it as a tool to predict effective compositions. The model is based on the polymorphic structural conformational flip of NPs and incorporates the electrostatic, entropic and elastic energies, to predict the formation energy and stability of different polymorphic structures as a function of the electric charge of cationic surfactants and concentration of constituent helper lipids. Our results show that these two factors are intertwined in determining the behavior of gene delivery vectors. Specifically, we show that increasing H/G lowers free energy per DNA base pair and increases the stability of the complex. At pH 7, low H/G and charge ratio (ρ±), where the lamellar structure is favored, the formation free energy per DNA base pair is between 0 and -14kBT. At higher values of H/G (2-3) and ρ±, where HII and cubic structures are formed, the formation free energy drops down to values ≈-50kBT, indicating the stable existence of these polymorphic structures in the NPs. At pH 5, the structural transformation of NPs in the endosomes to the lamellar/HII structure with free energy values of about -40kBT is beneficial for endosomal escape, and correlates with increased transfection efficiency. The theoretical model is supported by transfection data in A7 astrocytes with a panel of 16-3-16 gemini NPs, which validates the mathematical model and supports the hypothesis that the NP polymorphic phase transition increases transfection efficiency.

Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosystems.

Gene therapy holds great promise for various medical and biomedical applications. Nonviral gene delivery systems formed by cationic polymer and nucleic acids (e.g., polyplexes) have been extensively investigated for targeted gene therapy; however, their in vitro and in vivo stability is affected by both their intrinsic properties such as chemical compositions (e.g., polymer molecular weight and structure, and N/P ratio) and a number of environmental factors (e.g., shear stress during circulation in the bloodstream, interaction with the serum proteins, and physiological ionic strength). In this review, we surveyed the effects of a number of important intrinsic and environmental factors on the stability of polymeric gene delivery systems, and discussed various strategies to enhance the stability of polymeric gene delivery systems, thereby enabling efficient gene delivery into target cells. Future opportunities and challenges of polymeric nucleic acid delivery nanosystems were also briefly discussed.

A hierarchical assembly strategy to engineer dextran-enveloped polyurethane nanopolyplexes for robust ovarian cancer gene therapy

A hierarchical assembly strategy is herein investigated to generate bio-responsive, dextran-enveloped, bioreducible polyurethane nanopolyplexes for nonviral gene therapy against ovarian tumor. Initially, a group of poly(urethane amine)s were designed and characterized for in vitro gene transfection. The polyurethane containing 1,4-bis(3-aminopropyl)piperazine residue (PUBAP) could induce the best in vitro transfection efficacy against SKOV-3 or A2780 ovarian cancer cells. Next, dextran-enveloped PUBAP polyplexes (e-polyplexes) were constructed by a hierarchical assembly procedure involving gene neutralization with PUBAP and subsequent gene condensation with a cationic dextran (SSDP800). Such dextran comprised dextran (15 kDa) as the main chain and multiple disulfide-linked branched polyethylenimine (BPEI) oligomers as the side grafts. Additionally, folate-dextran-enveloped PUBAP polyplexes (FA-e-polyplexes) were fabricated by folate-modified SSDP800. These nanoscale-enveloped polyplexes elicited an improved colloidal stability against salt ions and negatively charged heparin, efficient endosomal escaping, and bioreduction-triggered intracellular gene release. In vitro transfection against SKOV-3 cells illustrated that FA-e-polyplexes exerted higher transfection efficiency in the serum than e-polyplexes and 25 kDa BPEI-polyplexes. In vivo, FA-e-polyplexes yielded higher transgene expression level than e-polyplexes in an SKOV-3 tumor-bearing nude mouse model. In the tumor gene therapy with a small hairpin RNA silencing vascular endothelial growth factor, FA-e-polyplexes afforded higher tumor growth inhibition than polyplexes of folate-PEGylated PUBAP and 25 kDa linear polyethylenimine as positive controls. Importantly, such gene therapy had minor toxic effects on the health of the mouse. This work highlights a practical hierarchical assembly method to construct innovative enveloped polyurethane nanopolyplexes enabling robust ovarian cancer gene therapy. Statement of SignificanceIt is indispensable to rationally update binary cationic polyplexes into ternary polyplexes for vigorous tumor gene therapy. In this work, we have confirmed that a hierarchical assembly strategy, by using initial gene neutralization and subsequent gene condensation, is facile and effective to promote cationic polyurethane polyplexes into ternary folate-dextran-enveloped polyurethane polyplexes with a relatively high gene-loading capacity. The enveloped polyplex system enables more efficient gene transfection than the PEGylated polyplex counterpart in ovarian cancer in vitro and in vivo, thereby affording robust ovarian cancer gene therapy. The development of innovative enveloped polyplexes may be a new direction for a non-viral gene delivery system.

Diethylaminoethyl- chitosan as an efficient carrier for siRNA delivery: Improving the condensation process and the nanoparticles properties

Chitosan has been indicated as a promising carrier for the preparation of small interfering RNA (siRNA) delivery systems due to its remarkable properties. However, its weak interactions with siRNA molecules makes the condensation of siRNA molecules into nanoparticles difficult. In this work, a non-viral gene delivery system based on diethylaminoethyl chitosan (DEAE-CH) derivatives of varied Mw (25–230 kDa) having a low degree of substitution of 15% was investigated. The presence of secondary and tertiary amino groups strengthened the interaction of siRNA and DEAE-CH derivatives of higher Mw (130 kDa to 230 kDa) and provided the preparation of spherical nanoparticles at low charge ratios (N/P 2 to 3) with low polydispersities (0.15 to 0.2) in physiological ionic strength. Nanoparticles prepared with all derivatives exhibited remarkable silencing efficiencies (80% to 90%) on different cell lines (HeLa, MG-63, OV-3) by adjusting the charge ratios. A selected PEG-folic acid labeled derivative (FA-PEG-DEAE15-CH230) was synthesized and its nanoparticles completely inhibited the mRNA expression level of TNF-α in RAW 264.7 macrophages. The study demonstrates that the insertion of DEAE groups provides improved physical properties to chitosan-siRNA nanoparticles and holds potential for in vivo applications.

Retinal Multipotent Stem-Cell Derived “MiEye” Spheroid 3D Culture Model for Preclinical Screening of Non-viral Gene Delivery Systems

Non-viral retinal gene therapy is a promising therapeutic approach towards the management of retinal degenerative diseases especially glaucoma. Current methods of in vitro preclinical screening of candidate nanoparticle systems in monolayer cell cultures are not reliable in predicting in vivo performance. In this paper, we describe the development of a multipotent stem-cell derived three-dimensional “mini-retina” culture model (MiEye) that aims to simulate an in vivo clinical model for more reliable gene delivery system screening. Through the utilization of multiplex gene expression profiling, we have shown that retinal stem cells can be differentiated in 3D culture to generate retinal neurospheres comprising of multiple retinal cell types. The 3D cell culture model combined with confocal microscopy imaging and fluorescence profiling techniques is a powerful tool as a retinal gene and drug delivery screening model.

Reverse Multidrug Resistance in Human HepG2/ADR by Anti-miR-21 Combined with Hyperthermia Mediated by Functionalized Gold Nanocages.

Multidrug resistance (MDR) remains a formidable challenge to effective clinical cancer therapy. Herein, a nonviral gene delivery system HA/anti-miR-21/PPAuNCs to overcome MDR was reported. This system could condense the microRNA-21 inhibitor (anti-miR-21) into hyaluronic acid-conjugated and polyethylenimine-modified PEGylated gold nanocages (AuNCs) and had good stability. In vitro studies demonstrated that HA/anti-miR-21/PPAuNCs could enhance intracellular DOX accumulation in DOX-resistant HCC cells (HepG2/ADR cells) and increase the sensitivity to DOX of HepG2/ADR cells through upregulating PTEN protein expression mediated by anti-miR-21 and downregulating P-gp protein expression mediated by the hyperthermia of HA/PPAuNCs upon mild near-infrared irradiation. Furthermore, the therapeutic effects had been enhanced due to the combination of chemotherapy, gene therapy, and photothermal therapy. Besides, HA/anti-miR-21/PPAuNCs have a good biocompatibility. These findings can provide new insights and strategies for the treatment of cancers with MDR.

PEGylation of polypropylenimine dendrimers: effects on cytotoxicity, DNA condensation, gene delivery and expression in cancer cells

Diaminobutyric polypropylenimine (DAB) dendrimers have been shown to be highly efficient non-viral gene delivery systems for cancer therapy. However, their cytotoxicity currently limits their applications. To overcome this issue, PEGylation of DAB dendrimer, using various PEG molecular weights and dendrimer generations, has been attempted to decrease the cytotoxicity and enhance the DNA condensation, size and zeta potential, cellular uptake and transfection efficacy of these dendriplexes. Among all the PEGylated dendrimers synthesized, generation 3- and generation 4-DAB conjugated to low molecular weight PEG (2 kDa) at a dendrimer: DNA ratio of 20:1 and 10:1 resulted in an increase in gene expression on almost all tested cancer cells lines (by up to 3.2-fold compared to unmodified dendrimer in A431 cells). The highest level of β-galactosidase gene expression (10.07 × 10−3 ± 0.09 × 10−3 U/mL) was obtained following treatment of B16F10-Luc cells with G4-dendrimer PEGylated with PEG2K at a dendrimer: DNA ratio of 20:1. These delivery systems significantly decreased cytotoxicity on B16F10-Luc cells, by more than 3.4-fold compared to unmodified dendrimer. PEGylated generations 3- and 4-DAB dendrimers are therefore promising gene delivery systems for cancer therapy, combining low cytotoxicity and high transfection efficacy.

Preparation and characterization of non-viral gene delivery systems with pEGFP-C1 Plasmid DNA

In recent years, non-viral delivery systems for plasmid DNA have become particularly important. They can overcome the disadvantages of viral systems such as insertional mutagenesis and unpredicted immunogenicity. Some additional advantages of non-viral gene delivery systems are; good stability, low cost, targetability, delivery of a high amount of genetic materials. The aim of the study was to develop novel non-viral nanosystems suitable for gene delivery. Two formulations were developed for this purpose: water-in-oil microemulsion (ME) and solid lipid nanoparticles (SLN). The microemulsion was composed of Peceol, Tween 80, Plurol oleique, ethanol and water. The SLN was consisting of Precirol, Esterquat-1 (EQ1), Tween 80, Lecithin, ethanol and water. Characterization studies were carried out by measuring particle size, zeta potential, viscosity and pH. TEM imaging was performed on SLN formulations. Protection against DNase I degradation was examined. Cytotoxicity and transfection efficacy of selected formulations were tested on L929 mouse fibroblast cells. Particle sizes of complexes were below 100 nm and with high positive zeta potential. TEM images revealed that SLNs are spherical. The SLN:DNA complexes have low toxicity and good transfection ability. All results showed that the developed SLN formulations can be considered as suitable non-viral gene delivery systems.

Oral Nonviral Gene Delivery for Chronic Protein Replacement Therapy.

Efficient nonviral oral gene delivery offers an attractive modality for chronic protein replacement therapy. Herein, the oral delivery of insulin gene is reported by a nonviral vector comprising a copolymer with a high degree of substitution of branched polyethylenimine on chitosan (CS‐g‐bPEI). Protecting the plasmid from gastric acidic degradation and facilitating transport across the gut epithelium, the CS‐g‐bPEI/insulin plasmid DNA nanoparticles (NPs) can achieve systemic transgene expression for days. A single dose of orally administered NPs (600 µg plasmid insulin (pINS)) to diabetic mice can protect the animals from hyperglycemia for more than 10 d. Three repeated administrations spaced over a 10 d interval produce similar glucose‐lowering results with no hepatotoxicity detected. Positron‐emission‐tomography and computed‐tomography images also confirm the glucose utilization by muscle cells. While this work suggests the feasibility of basal therapy for diabetes mellitus, its significance lies in the demonstration of a nonviral oral gene delivery system that can impact chronic protein replacement therapy and DNA vaccination.

Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery

AbstractThe successful clinical translation of nonviral gene delivery systems has yet to be achieved owing to the biological and technical obstacles to preparing a safe, potent, and cost‐effective vector. Hyperbranched polymers, compared with other polymers, have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification, which makes them more feasible for scale‐up and manufacturing. Here, we compare hyperbranched poly(amino acids) synthesised by copolymerising histidine and lysine, with hyperbranched polylysine prepared using the well‐known “ultrafacile” thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water‐soluble hyperbranched polylysine‐co‐histidine of three different molar ratios with molecular masses varying from 13 to 30 kDa. Spectroscopic, rheological, and thermal analyses indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 cells indicated that all the new hyperbranched polymers were well tolerated, but, surprisingly, the copolymers containing histidine were not more effective in transfecting a luciferase gene than were hyperbranched polylysines synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and we obtain structure–function information relating histidine content with polymer stiffness, pKa, and ability to form stable polyplexes with DNA. The results are of significance to nanomedicine design as they indicate that addition of histidine as a co‐monomer in the synthetic route to hyperbranched polymers not only changes the buffering capacity of the polymer but has significant effects on the overall structure, architecture, and gene delivery efficacy.

Nonviral Delivery Systems for Cancer Gene Therapy: Strategies and Challenges.

Gene therapy has been receiving widespread attention due to its unique advantage in regulating the expression of specific target genes. In the field of cancer gene therapy, modulation of gene expression has been shown to decrease oncogenic factors in cancer cells or increase immune responses against cancer. Due to the macromolecular size and highly negative physicochemical features of plasmid DNA, efficient delivery systems are an essential ingredient for successful gene therapy. To date, a variety of nanostructures and materials have been studied as nonviral gene delivery systems. In this review, we will cover nonviral delivery strategies for cancer gene therapy, with a focus on target cancer genes and delivery materials. Moreover, we will address current challenges and perspectives for nonviral delivery-based cancer gene therapeutics.

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas.

Polyelectrolyte layer assembly of bacterial nanocellulose whiskers with plasmid DNA as biocompatible non-viral gene delivery system

The nanocrystalline fraction of the natural biopolymer bacterial nanocellulose (BNC) was isolated by controlled degradation resulting in anionic surface modifications. The potential of those nanowhiskers for safe and efficient gene delivery was tested using electrostatic layer assemblies with different poly(ethylene imine)s (2.5 and 25 kDa) and plasmid DNA. For different isolation techniques (sulfuric acid hydrolysis and oxidation) the influence of varying preparation conditions such as concentration of the degrading agent, reaction time and temperature on the physicochemical characteristics of the nanowhiskers like morphology, size, and surface charge could be demonstrated. All nanowhiskers were found to be highly cyto- and hemo-compatible in vitro in short-term (2–8 h) or long-term (24–72 h) studies as well as ex ovo in a shell-less hen’s egg model over 8 h. The use of a polyelectrolyte layer technique based on electrostatic interactions with different poly(ethylene imine)s and plasmid DNA enabled the formation of small sized (ca. 200 nm), DNA stabilizing nanowhiskers and facilitated the transfection of CHO-K1 cells with high biocompatibility. In conclusion, by the use of BNC as sustainable raw material, in combination with optimized and controllable preparation conditions and surface modifications, BNC nanowhiskers modified by a polyelectrolyte layer approach could be used as renewable, “green” drug delivery system for bioactive substances such as nucleic acids.

Rational Design of Polypeptide‐Based Block Copolymer for Nonviral Gene Delivery

AbstractThe present work describes the development, characterization, and in vitro evaluation of novel poly(L‐lysine)‐based polyplexes as nonviral gene delivery systems. Initially, a well‐defined hybrid block copolymer comprising poly(ethylene glycol) methacrylate) (POEGMA) and poly(L‐lysine) (PLL) blocks was successfully synthesized and characterized. The hybrid copolymer shows high ability to condense DNA into stable polyplexes in aqueous media with sizes of approx. 100 nm. The nanoplexes were evaluated for cellular toxicity in A549 alveolar and HepG2 (hepatocarcinoma) cell lines. The nanoparticles cell internalization and transfection ability were assessed in HepG2 cells. The initial experiments showed that DNA was successfully transfected into the nucleus of human liver cancer cells and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. These results revealed that the newly synthesized POEGMA‐b‐PLL diblock copolymer might be very attractive candidate as a nonviral gene delivery vector.

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6–14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R6) and nona-arginine (R9)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6–14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.

Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization.

The objective of this study is to design a graphene‐based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR‐7b overexpression plasmid. GO‐PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR‐7b after GO‐PEI‐miR‐7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell‐specific transmembrane protein. However, osteoclastogenesis occurs without cell–cell fusion and preosteoclast (POC) is preserved. Through preservation of POC, GO‐PEI‐miR‐7b transfection promotes mesenchymal stem cell osteogenesis and endothelial progenitor cells angiogenesis in the coculture system. Platelet‐derived growth factor‐BB secreted by POC is increased by GO‐PEI‐miR‐7b both in vitro and in vivo. In treating osteoporotic ovariectomized mice, GO‐PEI‐miR‐7b significantly enhances bone mineral density, bone volume as well as bone vascularization through increasing CD31hiEmcnhi cell number. This study provides a cell–cell fusion targeted miRNA transfection drug delivery strategy in treating bone disorders with excessive osteoclastic bone resorption.

Effective in vivo gene delivery with reduced toxicity, achieved by charge and fatty acid -modified cell penetrating peptide

Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.

Polyethyleneimine-capped silver nanoclusters for microRNA oligonucleotide delivery and bacterial inhibition.

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this paper, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were prepared for the purpose of microRNA (miRNA) delivery. The resultant PEI-AgNCs were characterized by a photoluminescence assay and transmission electron microscopy. A cytotoxicity assay showed that PEI-AgNCs exhibit relatively low cytotoxicity. Interestingly, PEI-AgNCs were confirmed to transfect miRNA mimics more effectively than PEI in HepG2 and 293A cells. In this regard, hsa-miR-21 or hsa-miR-221 mimics (miR-21/221m) were transported into HepG2 cells by using PEI-AgNCs. The miR-21/221 expression was determined post-transfection by quantitative real-time polymerase chain reaction. Compared with the negative control, PEI-AgNCs/miR-21/221m groups exhibited higher miR-21/221 levels. In addition, AgNCs endow PEI with stronger antibacterial activity, and this advantage provided PEI-AgNCs the potential to prevent bacterial contamination during the transfection process. Furthermore, we showed that PEI-AgNCs are viable nanomaterials for plain imaging of the cells by laser scanning confocal microscopy, indicating great potential as an ideal fluorescent probe to track the transfection behavior. These results demonstrated that PEI-AgNCs are promising and novel nonviral vectors for gene delivery.

Direct reprogramming of mouse fibroblasts into neural cells via Porphyra yezoensis polysaccharide based high efficient gene co-delivery

BackgroundThe cell source for transplantation therapy is always a prerequisite question to be solved in clinical applications. Neural cells are considered non-regenerable, which highly restrict their application in the treatment for nerve injury. Therefore, neural trans-differentiation based on gene transfection provides a new solution to this issue. Compared to viral strategy, non-viral gene delivery systems are considered as a more promising way to achieve this aim. This study centers on a novel application of Porphyra yezoensis polysaccharide as a non-viral gene carrier for the neural trans-differentiation of mouse fibroblasts.ResultsEthanediamine modified P. yezoensis polysaccharide (Ed-PYP) served as a gene carrier and a group of plasmids that encode Ascl1, Brn4, and Tcf3 (pABT) self-assembled into nanoparticles. Results demonstrated that Ed-PYP–pABT nanoparticles at Ed-PYP: pABT weight ratio of 40:1 was the optimal candidate for gene delivery. ELISA assay revealed the highest expression levels of NGF, BDNF and SHH at 14 days after last transfection. Immunofluorescence and western blot assays also showed robust expression of neural markers including Nestin, GFAP, β-3tubulin, NF200, GAP43 and MAP2, in induced 3T6 cells at this time point.ConclusionOverall, these findings indicated that the P. yezoensis polysaccharide-based non-viral gene co-delivery system is a promising strategy for the generation of neural cells, which might facilitate the developments in the recovery of neural injuries.