Increased Transfection Efficiency Research Articles

Cationic Carbosilane Dendrimers for Apmnkq2 Aptamer Transfection in Breast Cancer: An Alternative to Traditional Transfectants.

Transfection efficiency is a critical parameter in gene therapy and molecular biology, representing the success rate at which nucleic acids are introduced and expressed in target cells. The combination of aptamers with nanotechnology-based delivery systems has demonstrated remarkable improvements in the transfection efficiency of therapeutic agents and holds significant potential for advancing gene therapy and the development of targeted treatments for various diseases, including cancer. In this work, cationic carbosilane dendritic systems are presented as an alternative to commercial transfection agents, demonstrating an increase in transfection efficiency when used for the internalization of apMNKQ2, an aptamer selected against a target in cancer. Their potential therapeutic use has been evaluated in breast cancer cell lines, MDA-MB-468 and MDA-MB-231, studying the cytotoxicity of the nanoconjugate, the internalization process, and its effect on cellular migration processes.

Biodegradable silica nanoparticles for efficient linear DNA gene delivery

Targeting, safety, scalability, and storage stability of vectors are still challenges in the field of nucleic acid delivery for gene therapy. Silica-based nanoparticles have been widely studied as gene carriers, exhibiting key features such as biocompatibility, simplistic synthesis, and enabling easy surface modifications for targeting. However, the ability of the formulation to incorporate DNA is limited, which restricts the number of DNA molecules that can be incorporated into the particle, thereby reducing gene expression. Here we use polymerase chain reaction (PCR)-generated linear DNA molecules to augment the coding sequences of gene-carrying nanoparticles, thereby maximizing nucleic acid loading and minimizing the size of these nanocarriers. This approach results in a remarkable 16-fold increase in protein expression six days post-transfection in cells transfected with particles carrying the linear DNA compared with particles bearing circular plasmid DNA. The study also showed that the use of linear DNA entrapped in DNA@SiO2 resulted in a much more efficient level of gene expression compared to standard transfection reagents. The system developed in this study features simplicity, scalability, and increased transfection efficiency and gene expression over existing approaches, enabled by improved embedment capabilities for linear DNA, compared to conventional methods such as lipids or polymers, which generally show greater transfection efficiency with plasmid DNA. Therefore, this novel methodology can find applications not only in gene therapy but also in research settings for high-throughput gene expression screenings.

An efficient low cost means of biophysical gene transfection in primary cells

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1–10 μl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.

Increased Transfection Efficiency Boosts AAV Titers for Gene Therapies by 2–4X

Increased Transfection Efficiency Boosts AAV Titers for Gene Therapies by 2–4X

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

Efficient and scalable gene delivery method with easily generated cationic carbon dots

Gene delivery is a complex process with several challenges when attempting to incorporate genetic material efficiently and safely into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal escape to ensure that the genetic material can exert its effect but also minimizing the toxicity of the delivery system, which is vital for safe gene delivery. Of importance, if gene delivery systems are intended for biomedical applications or clinical use, they must be scalable and easy and affordable to manufacture to meet the demand. Here, we show an efficient gene delivery method using a combination of carbon dots coated by PEI through electrostatic binding to easily generate cationic carbon dots. We show a biofunctional approach to generate optimal cationic carbon dots (CCDs) that can be scaled up to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency four times over the standard of PEI polyplexes. Generated CCDs enabled the challenging transfection protocol to produce retroviral vectors via cell cotransfection of three different plasmids into packing cells, showing not only high efficiency but also functionality of the gene delivery, tested as the capacity to produce infective retroviral particles.

Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

Helical polyamines.

Polymer microstructures rely on tacticity, yet exploration in polyamines has focused predominantly on atactic polymers. We introduce a method to synthesize a diverse library of ortho and para-cyanobenzenesulfonyl-activated-methyl aziridines using R, S, and racemic alaninol. Living anionic ring-opening polymerization of racemic sulfonyl aziridines yields soluble polymers, while enantiomerically-pure sulfonyl aziridines follow a dispersion polymerization with complete monomer conversion giving access to stereoblock copolymers. Removal of activation groups is achieved using dodecanethiol and tert-butylimino-tri(pyrrolidino)phosphorane to obtain isotactic or atactic linear polypropylene imines (LPPIs). High-purity L-PPIs are obtained in salt and neutral forms with high yields. Stereoblock copolymers of poly-R-block-S-polysulfonamides and respective polypropylene imine stereoblocks are synthesized, revealing helical structures in water influenced by the monomer type and sequence in CD spectroscopy. Molecular dynamics simulations confirm the helical nature of isotactic LPPIs in water. Bulk characterization demonstrates the first crystalline isotactic polyamines via spherulite growth in polarized light, atomic force microscopy and XRD analyses. In cell-transfection studies, the synthesized isotactic LPPIs exhibit lower toxicity and transfection efficiency than commercial hyperbranched polyethylene imine, with longer chains showing increased transfection efficiency. These isotactic polymers open avenues for complex macromolecular architectures with optically active polyamines akin to poly(amino acid)s but lacking hydrolytically cleavable amide links.

Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.

PEI functionalized cell membrane for tumor targeted and glutathione responsive gene delivery

Polyethylenimine (PEI) is a broadly exploited cationic polymer due to its remarkable gene-loading capacity. However, the high cytotoxicity caused by its high surface charge density has been reported in many cell lines, limiting its application significantly. In this study, two different molecular weights of PEI (PEI10k and PEI25k) were crosslinked with red blood cell membranes (RBCm) via disulfide bonds to form PEI derivatives (RMPs) with lower charge density. Furthermore, the targeting molecule folic acid (FA) molecules were further grafted onto the polymers to obtain FA-modified PEI-RBCm copolymers (FA-RMP25k) with tumor cell targeting and glutathione response. In vitro experiments showed that the FA-RMP25k/DNA complex had satisfactory uptake efficiency in both HeLa and 293T cells, and did not cause significant cytotoxicity. Furthermore, the uptake and transfection efficiency of the FA-RMP25k/DNA complex was significantly higher than that of the PEI25k/DNA complex, indicating that FA grafting can increase transfection efficiency by 15 %. These results suggest that FA-RMP25k may be a promising non-viral gene vector with potential applications in gene therapy.

Polyphosphate coated nanoparticles: Enzyme-activated charge-reversal gene delivery systems

AimThe current study aimed to develop enzyme-activated charge-reversal lipid nanoparticles (LNPs) as novel gene delivery systems. MethodsPalmitic acid was covalently bound to protamine being utilised as transfection promoter to anchor it on the surfaces of LNPs. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was ion paired with various cationic counter ions to achieve high encapsulation in LNPs. Protamine-decorated LNPs were prepared by solvent injection method followed by coating with sodium tripolyphosphate (TPP) to generate a bio-inert anionic outer surface. Resulting LNPs were characterised regarding size, polydispersity, zeta potential and encapsulation efficiency. Enzyme-triggered charge-reversal of LNPs was investigated using isolated alkaline phosphatase (ALP) monitoring changes in zeta potential as well as monophosphate release. Furthermore, monophosphate release, cell viability and transfection efficiency were evaluated on a human alveolar epithelial (A549) cell line. ResultsProtamine-decorated and TPP-coated (Prot-pDNA/DcChol-TPP) LNPs displayed a mean size of 298.8 ± 17.4 nm and a zeta potential of −13.70 ± 0.61 mV. High pDNA encapsulation was achieved with hydrophobic ion pairs of pDNA with 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DcChol). Zeta potential of Prot-pDNA/DcChol-TPP LNPs reversed to positive values with a total Δ26.8 mV shift upon incubation with ALP. Conformably, a notable amount of monophosphate was released upon incubation of Prot-pDNA/DcChol-TPP LNPs with isolated as well as cell-associated ALP. A549 cells well tolerated LNPs displaying more than 95 % viability. Compared with naked pDNA, unmodified LNPs and control LNPs, Prot-pDNA/DcChol-TPP LNPs showed a significantly increased transfection efficiency. ConclusionProt-pDNA/DcChol-TPP LNPs can be regarded as promising gene delivery systems.

A novel use of a needle-free injection system for improved nucleic acid delivery and expression in vivo.

Transfection, a nonviral method of nucleic acid delivery, often exhibits poor efficiency in vivo. The needle-based in vivo delivery of transfection reagents can be invasive. Here, we report a noninvasive protocol for in vivo gene delivery via the needle-free MED-JET H4 MULTIJET (MJH4M) device using both "home-made" glucose-based and commercial transfection reagents. The objective of this study was to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. We observed a 15-fold increase in transfection efficiency using the needle-free MJH4M device when compared to the needle-based delivery method. The highest transfection efficiency was achieved using a 5% glucose solution as the delivery vehicle.

Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.

CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.

Poly(β-amino ester)s-Based Delivery Systems for Targeted Transdermal Vaccination.

Nucleic acid vaccines have become a transformative technology to fight emerging infectious diseases and cancer. Delivery of such via the transdermal route could boost their efficacy given the complex immune cell reservoir present in the skin that is capable of engendering robust immune responses. We have generated a novel library of vectors derived from poly(β-amino ester)s (PBAEs) including oligopeptide-termini and a natural ligand, mannose, for targeted transfection of antigen presenting cells (APCs) such as Langerhans cells and macrophages in the dermal milieu. Our results reaffirmed terminal decoration of PBAEs with oligopeptide chains as a powerful tool to induce cell-specific transfection, identifying an outstanding candidate with a ten-fold increased transfection efficiency over commercial controls in vitro. The inclusion of mannose in the PBAE backbone rendered an additive effect and increased transfection levels, achieving superior gene expression in human monocyte-derived dendritic cells and other accessory antigen presenting cells. Moreover, top performing candidates were capable of mediating surface gene transfer when deposited as polyelectrolyte films onto transdermal devices such as microneedles, offering alternatives to conventional hypodermic administration. We predict that the use of highly efficient delivery vectors derived from PBAEs could advance clinical translation of nucleic acid vaccination over protein- and peptide-based strategies.

Mesoporous Organosilica Nanoparticles with Tetrasulphide Bond to Enhance Plasmid DNA Delivery

Cellular delivery of plasmid DNA (pDNA) specifically into dendritic cells (DCs) has provoked wide attention in various applications. However, delivery tools that achieve effective pDNA transfection in DCs are rare. Herein, we report that tetrasulphide bridged mesoporous organosilica nanoparticles (MONs) have enhanced pDNA transfection performance in DC cell lines compared to conventional mesoporous silica nanoparticles (MSNs). The mechanism of enhanced pDNA delivery efficacy is attributed to the glutathione (GSH) depletion capability of MONs. Reduction of initially high GSH levels in DCs further increases the mammalian target of rapamycin complex 1 (mTORc1) pathway activation, enhancing translation and protein expression. The mechanism was further validated by showing that the increased transfection efficiency was apparent in high GSH cell lines but not in low GSH ones. Our findings may provide a new design principle of nano delivery systems where the pDNA delivery to DCs is important.

Engineering high post-electroporation viabilities and transfection efficiencies for elongated cells on suspended nanofiber networks

The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.

Hydrophilic Surface Modification of Cationic Unimolecular Bottlebrush Vectors Moderate pDNA and RNP Bottleplex Stability and Delivery Efficacy.

A cationic unimolecular bottlebrush polymer with chemically modified end-groups was synthesized to understand the impact of hydrophilicity on colloidal stability, nucleic acid delivery performance, and toxicity. The bottlebrush polymer template was synthesized using grafting-through techniques and was therefore composed of a polynorbornene backbone with poly(2-(dimethylamino)ethyl methacrylate) side chains with dodecyl trithiocarbonate end-groups. Postpolymerization modification was performed to fully remove the end-groups or install hydroxy and methoxy poly(ethylene glycol) functional groups on the bottlebrush exterior. The bottlebrush family was preformulated with biological payloads of pDNA and CRISPR-Cas9 RNP in both water and PBS to understand binding, aggregation kinetics, cytotoxicity, and delivery efficacy. Increasing end-group hydrophilicity and preformulation of bottleplexes in PBS increased colloidal stability and cellular viability; however, this did not always result in increased transfection efficiency. The bottlebrush family exemplifies how formulation conditions, polymer loading, and end-group functionality of bottlebrushes can be tuned to balance expression with cytotoxicity ratios and result in enhanced overall performance.

TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation.

DNA transfection is an essential technique in the life sciences. Non-viral transfection reagents are widely used for transfection in basic science. However, low transfection efficiency is a problem in some cell types. This low efficiency can be primarily attributed to the intracellular degradation of transfected DNA by p62-dependent selective autophagy, specifically by p62 phosphorylated at the S403 residue (p62-S403-P). To achieve efficient DNA transfection, we focused on a phosphorylation process that generates p62-S403-P and investigated whether inhibition of this process affects transfection efficiency. One of the kinases that phosphorylate p62 is TBK1. The TBK1 gene depletion in murine embryonic fibroblast cells by genome editing caused a significant reduction or loss of p62-S405-P (equivalent to human S403-P) and enhanced transfection efficiency, suggesting that TBK1 is a major kinase that phosphorylates p62 at S403. Therefore, TBK1 is a viable target for drug treatment to increase transfection efficiency. Transfection efficiency was enhanced when cells were treated with one of the following TBK1 inhibitors BX795, MRT67307, or amlexanox. This effect was synergistically improved when the two inhibitors were used in combination. Our results indicate that TBK1 inhibitors enhanced transfection efficiency by suppressing p62 phosphorylation.

Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model.

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Anhydride chemistry-based hexanoylation of polyethylenimine increases transfection efficiency and expression of tagged DNA for therapeutic proteins in cultured cells.

Transfection of nucleic acid molecules into mammalian cells can be facilitated using viral vectors, electroporation, or biocompatible cationic materials. However, safety issues and the requirement of specialized equipment limits the use of viral vectors and physical methods of transfection like electroporation and microinjection, respectively. Biocompatible cationic lipids and polymers like branched-polyethyleneimine (bPEI) have a wide transfection range and are user-friendly in most applications. However, bPEI exhibits low transfection efficiency in most cell types. In the present work, we have crosslinked the hexanoyl group to bPEI using anhydride chemistry to enhance its efficiency as a transfection reagent. The efficient association of hexanoyl group to bPEI was assessed using Fourier-transform infrared spectroscopy and other physicochemical methods. Hexanoyl-modified bPEI (FA6-bPEI) was found to exhibit significantly enhanced transfection efficiency in both cell lines and cultured primary cells, as compared to native bPEI and the commercially available transfection reagent, Lipofectamine 3000. Furthermore, our invitro studies indicated that FA6-bPEI can be used for robust transfection for increased production of therapeutic proteins in a cell culture-based system. These results suggested that hexanoyl-modified bPEI can serve as an efficient transfection reagent for studies on hard-to-transfect cells and for enhanced production of therapeutic proteins in vitro.