Non-viral Gene Transfection Research Articles

Enhanced non-viral gene delivery via calcium phosphate/DNA co-precipitates with low-voltage pulse electroporation in NK-92 cells for immunocellular therapy.

Achieving high cell transfection efficiency is essential for various cell types in numerous disease applications. However, the efficient introduction of genes into natural killer (NK) cells remains a challenge. In this study, we proposed a design strategy for delivering exogenous genes into the NK cell line, NK-92, using a modified non-viral gene transfection method. Calcium phosphate/DNA nanoparticles (pDNA-CaP NPs) were prepared using co-precipitation methods and combined with low-voltage pulse electroporation to facilitate NK-92 transfection. The results demonstrated that the developed pDNA-CaP NPs exhibited a uniform diameter of approximately 393.9 nm, a DNA entrapment efficiency of 65.8%, and a loading capacity of 15.9%. Furthermore, at three days post-transfection, both the transfection efficiency and cell viability of NK-92 were significantly improved compared to standalone plasmid DNA (pDNA) electroporation or solely relying on the endocytosis pathway of pDNA-CaP NPs. This study provides valuable insights into a novel approach that combines calcium phosphate nanoparticles with low-voltage electroporation for gene delivery into NK-92 cells, offering potential advancements in cell therapy.

Gene Therapy for Regenerative Medicine.

The development of biological methods over the past decade has stimulated great interest in the possibility to regenerate human tissues. Advances in stem cell research, gene therapy, and tissue engineering have accelerated the technology in tissue and organ regeneration. However, despite significant progress in this area, there are still several technical issues that must be addressed, especially in the clinical use of gene therapy. The aims of gene therapy include utilising cells to produce a suitable protein, silencing over-producing proteins, and genetically modifying and repairing cell functions that may affect disease conditions. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, non-viral gene transfection agents are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Gene therapy based on viral vectors may induce pathogenicity and immunogenicity. Therefore, significant efforts are being invested in non-viral vectors to enhance their efficiency to a level comparable to the viral vector. Non-viral technologies consist of plasmid-based expression systems containing a gene encoding, a therapeutic protein, and synthetic gene delivery systems. One possible approach to enhance non-viral vector ability or to be an alternative to viral vectors would be to use tissue engineering technology for regenerative medicine therapy. This review provides a critical view of gene therapy with a major focus on the development of regenerative medicine technologies to control the in vivo location and function of administered genes.

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.

Lignin-Based Nonviral Gene Carriers Functionalized by Poly[2-(Dimethylamino)ethyl Methacrylate]: Effect of Grafting Degree and Cationic Chain Length on Transfection Efficiency.

Lignin is a natural renewable biomass resource with great potential for applications, while its development into high value-added molecules or materials is rare. The development of biomass lignin as potential nonviral gene delivery carriers was initiated by our group through the “grafting-from” approach. Firstly, the lignin was modified into macroinitiator using 2-bromoisobutyryl bromide. Then cationic polymer chains of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) were grown from the lignin backbone using atom transfer radical polymerization (ATRP) to yield lignin-PDMAEMA graft copolymers (LPs) with branched structure. To gain a deep understanding of the relationship between the nonviral gene transfection efficiency of such copolymers and their structural and compositional factors, herein eight lignin-based macroinitiators with different modification degrees (MDs, from 3.0 to 100%) were synthesized. Initiated by them, a series of 20 LPs were synthesized with varied structural factors such as grafting degree (GD, which is equal to MD, determining the cationic chain number per lignin macromolecule), cationic chain length (represented by number of repeating DMAEMA units per grafted arm or degree of polymerization, DP) as well as the content of N element (N%) which is due to the grafted PDMAEMA chains and proportional to molecular weight of the LPs. The in vitro gene transfection capability of these graft copolymers was evaluated by luciferase assay in HeLa, COS7 and MDA-MB-231cell lines. Generally, the copolymers LP-12 (N% = 7.28, MD = 36.7%, DP = 13.6) and LP-14 (N% = 6.05, MD = 44.4%, DP = 5.5) showed good gene transfection capabilities in the cell lines tested. Overall, the performance of LP-12 was the best among all the LPs in the three cell lines at the N/P ratios from 10 to 30, which was usually several times higher than PEI standard. However, in MDA-MB-231 at N/P ratio of 30, LP-14 showed the best gene transfection performance among all the LPs. Its gene transfection efficiency was ca. 11 times higher than PEI standard at this N/P ratio. This work demonstrated that, although the content of N element (N%) which is due to the grafted PDMAEMA chains primarily determines the gene transfection efficiency of the LPs, it is not the only factor in explaining the performance of such copolymers with the branched structure. Structural factors of these copolymers such as grafting degree and cationic chain length could have a profound effect on the copolymer performance on gene transfection efficiency. Through carefully adjusting these factors, the gene transfection efficiency of the LPs could be modulated and optimized for different cell lines, which could make this new type of biomass-based biomaterial an attractive choice for various gene delivery applications.

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...

Synthesis and Evaluation of Cytocompatible Alkyne-Containing Poly(β-amino ester)-Based Hydrogels Functionalized via Click Reaction.

Although poly(β-amino esters) (PAEs) have been widely applied in nonviral gene transfection, drug delivery systems, and regenerative medicine, the multifunctional modification of PAEs and bio-orthogonal strategies of PAE-based hydrogel functionalization is still a challenge. Herein, a strategy of poly(β-amino ester)-based hydrogel functionalization was developed via bio-orthogonal reactions in this study. Acrylate-terminated poly(β-amino esters) containing alkyne groups were synthesized by Michael addition reaction. Alkyne groups on poly(β-amino esters) could conjugate bioactive molecules with azide of K(N3)RGD via copper-catalyzed azide-alkyne cycloaddition, and terminal acrylate groups could in situ polymerize to prepare a hydrogel. A biomimetic peptide K(N3)RGD functionalized hydrogel was prepared by polymerization of acrylate-terminated poly(β-amino esters) containing conjugated peptide and polyethylene glycol diacrylate (PEGDA). The storage modulus and mechanical properties exhibited an increased trend with the increased concentration; nevertheless, swelling ratio and surface wetting properties demonstrated a decreased tendency by increased concentrations. Cell proliferation and live/dead staining showed that Schwann cells plated on the hydrogel with an elastic modulus of 25.39 KPa are more suitable for proliferation and function exertion of Schwann cells compared with that of 42.11 and 57.86 KPa, and KRGD-conjugated hydrogel could increase the elongation of Schwann cells relative to nonconjugated hydrogels. This azide-alkyne strategy may be a promising candidate for hydrogel functionalization in tissue engineering and other biomedical applications.

Optimization of electroporation and other non-viral gene delivery strategies for T cells.

CAR-T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+ B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR-T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time-consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non-viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non-viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.

MRNA as a Tool for Gene Transfection in 3D Cell Culture for Future Regenerative Therapy.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

Prostate cancer cell-specific BikDDA delivery by targeted polymersomes

In this paper, we report the polymersome-mediated intracellular delivery of pro-apoptotic BikDDA gene using two different peptide–copolymer covalent conjugation strategies specific for prostate cancer targeting. The BikDDA gene was used as a therapeutic agent on prostate cancer cells. The transfection efficiency of BikDDA-loaded poly[oligo(ethyleneglycol) methacrylate]-co-poly[2-(diisopropylamino) ethyl methacrylate] (P(OEG10MA)20-PDPA100) polymersomes revealed that they could serve as a suitable non-viral gene transfection tool. The targeted delivery of BikDDA into prostate cancer cells (LNCaP) using polymersomes was successfully carried out by conjugating the PSMA-targeting moiety (peptide 563) to P(OEG10MA)20-PDPA100 copolymer using either succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a bifunctional linker between the thiol-bearing targeting peptide and amino-bearing P(OEG10MA)20-PDPA100 copolymer or attaching a maleimide-modified targeting peptide onto a thiol-terminated P(OEG10MA)20-PDPA100 copolymer. The pH-responsive and biocompatible polymersomes, conjugated with peptide 563, exhibited an enhanced cellular uptake by LNCaP cells in comparison to the healthy prostate epithelial cell line PNT1A, thus indicating the cell-specific delivery. The increased Bik mRNA expression and cell death in these LNCaP cells indicates high effectiveness of the targeting polymersomes. According to these results, we believe more efficient gene delivery systems via specifically targeted pH-sensitive polymersomes can be a promising approach and promote the development of novel therapies against prostate cancer.

Preparation and In Vivo Expression of CS-PEI/pCGRP Complex for Promoting Fracture Healing

Background/Objective. CGRP is a calcitonin gene-related peptide that is capable of promoting bone development and bone regeneration. Chitosan is a nontoxic and degradable biomaterial. However, the gene transfection efficiency of chitosan is low, whereas PEI (polyethyleneimine) has higher capability of transfection efficiency. In this paper, PEI was covalently linked to chitosan, and the rat CGRP plasmid was encapsulated in a CS-PEI complex to construct CS-PEI/pCGRP nanoparticles. The characterization and biological effects of CS-PEI/pCGRP nanoparticles were investigated in vivo. Methods. CS-PEI/pCGRP nanoparticles were prepared by a complex coacervation method. The PEI distribution degree on chitosan was measured with a dialysis method and 1H-NMR analysis. The particle size and zeta potential of CS-PEI/pCGRP nanoparticles were detected by dynamic light scattering. The binding of CS-PEI to pCGRP was detected by gel retardation assay. The transfection effect was evaluated by RT-qPCR. A rat femoral fracture model was established and treated with PBS, pCGRP, CS-PEI, and CS-PEI/pCGRP to detect the expression of CGRP and downstream genes in early healing of fractures by RT-qPCR, western blot, and immunohistochemistry (IHC). Results. The particle size and zeta potential of CS-PEI/pCGRP nanoparticles were stable when the mass ratio of CS-PEI and pCGRP was higher than 5 : 1, the ratio which could also effectively protect pCGRP from DNase I degradation. CS-PEI/pCGRP could obviously increase CGRP expression in rat bone marrow stromal cells. In vivo fracture healing experiments demonstrated that CGRP could be delivered to the body via the CS-PEI and expressed in situ after a 3-week treatment. Moreover, CS-PEI/pCGRP significantly enhanced the mRNA and protein levels of downstream RUNX2 and ALP. Conclusion. CS-PEI/pCGRP nanoparticles were an effective nonviral gene transfection system that could upregulate CGRP expression in vivo and accelerate the expression of key biomarkers for early healing of fractures.

Clay-Based Biohybrid Materials for Biomedical and Pharmaceutical Applications

ABSTRACTClays have traditionally been linked to health care, being used for centuries in the fight against infections and diseases. Similarly, biohybrids produced by combinations of clays and biological species through ‘bottom-up’ approaches have been evaluated over the past decade for biomedical and pharmaceutical uses. These biohybrids show interesting features such as biocompatibility and biodegradability which make them suitable for healthcare applications. The aim of the present communication was to review recent research contributions describing progress and the role of biohybrid materials based on clays in biomedicine and pharmacy disciplines. Emphasis will be on the authors’ own experience of this topic, particularly on aspects related to controlled drug-delivery systems, adjuvants of vaccines, and vectors for non-viral gene transfection. Bionanocomposites offer several advantages for use in the design of new and efficient pharmacological formulations for cutaneous and oral administration. In these systems, the drug is typically entrapped in the clay and protected by a biopolymer matrix, and both components contribute to a gradual release of the drug. Clay-based hybrids have also shown their efficacy in vaccines as they can act as nanocarriers of viral particles, due to the biomimetic interface created on the clay surface after adsorption of suitable biomolecules such as phospholipids, while the clay acts as an adjuvant to increase the efficacy of the vaccine. Finally, a new application of clays as non-viral vectors for controlled gene delivery is attracting increasing interest in the treatment of diverse diseases; clays such as sepiolite have demonstrated their ability to act as nanocarriers of nucleic acids and facilitate their transfection in mammalian cells.

Lipids for Nucleic Acid Delivery: Cationic or Neutral Lipoplexes, Synthesis, and Particle Formation.

Lipidic vesicles have been extensively studied for their capacity to condensate and deliver nucleic acids to the cells. Many different amphiphilic lipidic structures have been proposed each of them bringing some advances in nonviral gene transfection. The ionic or neutral nature of the lipids induces tremendous differences in the behavior of the corresponding liposomes, from the complexation of nucleic acid to the delivery to the cell. An efficient delivery in vitro or in vivo also depends closely on the structure of the lipids and very often, efficient liposomes in vitro have been found useless for in vivo administration.We describe in this chapter the chemical synthesis of two different lipids, one cationic and the other essentially neutral, and the formulation to obtain liposomes and DNA-liposome complexes. The different ways and tricks for the formulation of the two different structures are especially highlighted.

Photochemical Internalization Enhanced Nonviral Suicide Gene Therapy.

Nonviral gene transfection overcomes some of the disadvantages of viral vectors, such as undesired immune responses, safety concerns, issues relating to bulk production, payload capacity, and quality control, but generally have low transfection efficiency. Here we describe the effects of a modified form of photodynamic therapy (PDT), i.e., photochemical internalization (PCI) to: (1) greatly increase nonviral cytosine deaminase gene (CD) transfection into tumor cells, significantly increasing the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and (2) enhance the toxic efficacy of the locally produced 5-FU to induce cell death on both transfected and non-transfected bystander cells.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model

Gene therapy for bone tissue engineering has been widely developed. Recently, non-viral DNA-based gene therapy has been reported to be a safer and more efficient method of delivering DNA into target cells. We used a non-viral gene transfection reagent to delivery bone morphogenetic protein-2 (BMP-2) gene into bone marrow stromal cells (BMSCs). Primary BMSCs were isolated from rat femurs and transfected with BMP-2 plasmids. The transfection rate was analyzed using flow cytometry. The concentration of BMP-2 protein was quantified using an enzyme-linked immunosorbent assay. Levels of osteopontin and osteocalcin were measured to evaluate osteogenic differentiation. In vivo, we designed a critical-size calvarial defect rat model to study new bone regeneration, using Matrigel as a scaffold to carry BMP-2-transfected bone marrow stromal cells into the defect site. New bone formation was assessed by micro-computed tomography, X-ray, immunohistochemical staining and histomophometry. The transfection rate after 72 h was 31.5%. The BMP-2 protein level as well as osteopontin and osteocalcin expressions were higher in the experimental group (transfected with BMP-2) than the control group (transfected with green fluorescent protein, GFP). The in vivo study suggested that bone healing occurred 12 weeks after scaffold implantation. In addition, BMP-2-transfected bone marrow stromal cells provided better osteogenic differentiation than primary bone marrow stromal cells. Our findings suggest that non-viral gene therapy may be useful in bone tissue engineering. SignificanceThe study has clinical implications for the wider use of BMP-2-transfected BMSCs for cell-based transplantation therapy in bone regeneration.

Surface mediated non-viral gene transfection on titanium substrates using polymer electrolyte and nanostructured silicate substituted calcium phosphate pDNA (NanoSiCaPs) composites

Exploiting gene delivery from the surfaces of bio-functionalized implants is a unique strategy to facilitate tissue regeneration and integration. However, it has been challenging, due to the difficulty for incorporation and delivey of sufficient payloads of plasmid DNA (pDNA) to cells from the implant surface allowing efficient gene expression. Herein, we describe a novel and simple coating approach using nanostructured silicate substituted calcium phosphate pDNA complexes termed as NanoSiCaPs, that preferentially adsorb onto titanium (Ti) substrates coated with poly (diallyldimethylammonium chloride) (PDADMAC). The Ti-polyelectrolyte NanoSiCaPs assemblies aptly called PNA, deliver pDNA to MC3T3-E1 cells (pre-osteoblast cell line), induce protein production, without eliciting any cytotoxicity, while simultaneously encouraging cell attachment on the substrate.

Lysine-based amino-functionalized lipids for gene transfection: 3D phase behaviour and transfection performance

Based on previous work, the influence of the chain composition on the physical-chemical properties of five new transfection lipids (TH10, TT10, OH10, OT10 and OO10) containing the same lysine-based head group has been investigated in aqueous dispersions. For this purpose, the chain composition has been gradually varied from saturated tetradecyl (T, C14:0) and hexadecyl (H, C16:0) chains to longer but unsaturated oleyl (O, C18:1) chains with double bonds in the cis configuration. In this work, the lipid dispersions have been investigated in the absence and presence of the helper lipid DOPE and calf thymus DNA by small-angle and wide-angle X-ray scattering (SAXS/WAXS) supplemented by differential scanning calorimetry (DSC), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and Fourier-transform Raman spectroscopy (FTRS). Lamellar and inverted hexagonal mesophases have been observed in single-component systems. In the binary mixtures, the aggregation behaviour changes with an increasing amount of DOPE from lamellar to cubic. The lipid mixtures with DNA show a panoply of mesophases. Interestingly, TT10 and OT10 form cubic lipoplexes, whereas OO10 complexes the DNA sandwich-like between lipid bilayers in a lamellar lipoplex. Surprisingly, the latter is the most effective lipoplex.

PH‐Triggered Pinpointed Cascading Charge‐Conversion and Redox‐Controlled Gene Release Design: Modularized Fabrication for Nonviral Gene Transfection

Although pH and reduction responses are widely applied on gene and drug delivery system, the undefined molecule and disconnected response to corresponding transfection barriers still hamper their further application. Here, a multistage‐responsive lipopeptides polycation‐DNA nanoparticles (namely KR‐DC) as gene vector is designed, consisting of three functional modules. It provides the following outstanding “smart” characteristics: i) facile manufacture and ease to adjust ingredients for different conditions, ii) negatively charged surface to remain stable and increase biocompatibility in physiological environment, iii) pH‐triggered cascading charge‐conversion corresponding to tumor extracellular pH and endo/lysosomal pH, iv) the first stage of charge reversal for uptake enhancement at tumor site, v) the second stage of charge conversion for rapid endosomal escape, vi) the third stage of redox degradation aiming at DNA controlled release and nuclear entry, vii) cell‐penetrating peptides mimicking arginine‐rich periphery targeting to membrane penetration capacity improvement, and viii) lipid forming hydrophobic cavity for potential fat‐soluble drug encapsulation. Finally, KR‐DC nanoparticles achieve significantly enhanced in vitro transfection efficiency by almost four orders of magnitude in manual tumor environment with reduced side effects and satisfying gene expression in Hela xenograft tumor model in vivo.

Injectable Supramolecular Hydrogels as Delivery Agents of Bcl-2 Conversion Gene for the Effective Shrinkage of Therapeutic Resistance Tumors.

Injectable hydrogels to deliver therapeutic genes in a minimally invasive manner and to achieve long term sustained release at tumor sites to minimize side effects are attractive for cancer therapy and precision medicine, but its rational design remains a challenge. In this report, an injectable supramolecular hydrogel system is designed based on the polypesudorotaxane formation between α-cyclodextrin (α-CD) and cationic methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene imine) (MPEG-PCL-PEI) copolymer, with the ability to form polyplexes with anionic plasmid DNA for effective sustained gene delivery. To be mentioned, the MPEG-PCL-PEI copolymers show similar pDNA binding ability, better gene transfection efficiency, lower cytotoxicity than nonviral gene transfection gold standard PEI (25 kDa), due to the formation of micelles and more stable polyplexes. More importantly, this MPEG-PCL-PEI/α-CD/pDNA supramolecular hydrogel shows a sustained release of pDNA in form of polyplex for up to 7 d. By taking these advantages, this supramolecular hydrogel system is applied as an injectable carrier for sustained Bcl-2 conversion gene release, in an in vivo rodent model of therapeutic resistant hepatocarcinoma with high expression of antiapoptotic Bcl-2 protein. This work represents the first time that injectable MPEG-PCL-PEI/α-CD supramolecular hydrogels possess good controllable release effect of Bcl-2 conversion genes in the form of polyplex to effectively inhibit in vivo tumor growth and this "enemy to friend" strategy will benefit various applications, including on-demand gene delivery and personalized medicine.

Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector.

Nanoparticle-based non-viral gene delivery vectors have been extensively used in regenerative medicine, however their intrinsic effects on mesenchymal stem cell (MSC) differentiation remain poorly understood. This paper demonstrates that different classes of commonly used non-viral vectors are not inert and they have a strong effect on cell morphology, stress fiber formation and gene transcription in MSCs, which in turn modulates their capacity to differentiate towards osteogenic, adipogenic and chondrogenic lineages. These results also point to the need for careful and tissue-specific selection of nanoparticle-based delivery vectors to prevent undesired phenotypic changes and off-target effects when delivering therapeutic genes to damaged or diseased tissues.