Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

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.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology.

Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

BACKGROUND: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. RESULTS: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. CONCLUSIONS: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.

Preparation of Chitosan Nanoparticles through a Readily Solvent-Exchange Process for Efficient and Enhanced Gene Delivery.

In view of the excellent prospects of gene therapy and the potential safety and immunogenicity issues challenged by viral vectors, it is of great significance to develop a nonviral vector with low toxicity and low cost. In this work, we report a chitosan nanoparticle (CSNP) to be used as a gene vector prepared through a facile solvent-exchange strategy. Chitosan is first dissolved in ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIM Ac), and then, the solvent is exchanged with water/phosphate-buffered saline (PBS) to remove ionic liquid, forming a final CSNP dispersion after ultrasonication. The prepared CSNP shows a positive surface charge and can condense green fluorescent protein-encoding plasmid (pGFP) at weight ratios (CSNP/pGFP) of 5/1 or higher. Dynamic light scattering size and ζ-potential characterization and gel retardation results confirm the formation of CSNP/pGFP complexes. Compared with plain pGFP, efficient cellular internalization and significantly enhanced green fluorescent protein (GFP) expression are observed by using CSNP as a plasmid vector. Benefitting from the intrinsic biocompatibility, low cost, low immunogenicity, and abundant sources of chitosan, as well as the facile preparation and the efficient gene transfection capacity of CSNP, it is believed that this CSNP could be used as a nonviral gene vector with great clinical translational potentials.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Poly(ß-aminoester) Physicochemical Properties Govern the Delivery of siRNA from Electrostatically Assembled Coatings.

Localized short interfering RNA (siRNA) therapy has the potential to drive high-specificity molecular-level treatment of a variety of disease states. Unfortunately, effective siRNA therapy suffers from several barriers to its intracellular delivery. Thus, drug delivery systems that package and control the release of therapeutic siRNAs are necessary to overcome these obstacles to clinical translation. Layer-by-layer (LbL) electrostatic assembly of thin film coatings containing siRNA and protonatable, hydrolyzable poly(ß-aminoester) (PBAE) polymers is one such drug delivery strategy. However, the impact of PBAE physicochemical properties on the transfection efficacy of siRNA released from LbL thin film coatings has not been systematically characterized. In this study, we investigate the siRNA transfection efficacy of four structurally similar PBAEs in vitro. We demonstrate that small changes in structure yield large changes in physicochemical properties, such as hydrophobicity, pKa, and amine chemical structure, driving differences in the interactions between PBAEs and siRNA in polyplexes and in LbL thin film coatings for wound dressings. In our polymer set, Poly3 forms the most stable interactions with siRNA (Keff,w/w = 0.298) to slow release kinetics and enhance transfection of reporter cells in both colloidal and thin film coating approaches. This is due to its unique physiochemical properties: high hydrophobicity (clog P = 7.86), effective pKa closest to endosomal pH (pKa = 6.21), and high cooperativity in buffering (nhill = 7.2). These properties bestow Poly3 with enhanced endosomal buffering and escape properties. Taken together, this work elucidates the connections between small changes in polymer structure, emergent properties, and polyelectrolyte theory to better understand PBAE transfection efficacy.

Development and characterization of a first-in-class adjustable-dose gene therapy system.

INTRODUCTION: Despite significant potential, gene therapy has been relegated to the treatment of rare diseases, due in part to an inability to adjust dosage following initial administration. Other significant constraints include cost, specificity, antigenicity, and systemic toxicity of current generation technologies. To overcome these challenges, we developed a first-in-class adjustable-dose gene therapy system, with optimized biocompatibility, localization, durability, and cost. METHODS: A lipid nanoparticle (LNP) delivery system was developed and characterized by dynamic light scattering for size, zeta potential, and polydispersity. Cytocompatibility and transfection efficiency were optimized in vitro using primary human adipocytes and preadipocytes. Durability, immunogenicity, and adjustment of expression were evaluated in C57BL/6 and B6 albino mice using in vivo bioluminescence imaging. Biodistribution was assessed by qPCR and immunohistochemistry; therapeutic protein expression was quantified by ELISA. RESULTS: Following LNP optimization, in vitro transfection efficiency of primary human adipocytes reached 81.3 % ± 8.3 % without compromising cytocompatibility. Critical physico-chemical properties of the system (size, zeta potential, polydispersity) remained stable over a broad range of genetic cassette sizes (1,871-6,203 bp). Durable expression was observed in vivo over 6 months, localizing to subcutaneous adipose tissues at the injection site with no detectable transgene in the liver, heart, spleen, or kidney. Gene expression was adjustable using several physical and pharmacological approaches, including cryolipolysis, focused ultrasound, and pharmacologically inducible apoptosis. The ability of transfected adipocytes to express therapeutic transgenes ranging from peptides to antibodies, at potentially clinically relevant levels, was confirmed in vitro and in vivo. CONCLUSION: We report the development of a novel, low-cost therapeutic platform, designed to enable the replacement of subcutaneously administered protein treatments with a single-injection, adjustable-dose gene therapy.

Thermally Robust Solvent-Free Liquid Polyplexes for Heat-Shock Protection and Long-Term Room Temperature Storage of Therapeutic Nucleic Acids.

Nucleic acid therapeutics have attracted recent attention as promising preventative solutions for a broad range of diseases. Nonviral delivery vectors, such as cationic polymers, improve the cellular uptake of nucleic acids without suffering the drawbacks of viral delivery vectors. However, these delivery systems are faced with a major challenge for worldwide deployment, as their poor thermal stability elicits the need for cold chain transportation. Here, we demonstrate a biomaterial strategy to drastically improve the thermal stability of DNA polyplexes. Importantly, we demonstrate long-term room temperature storage with a transfection efficiency maintained for at least 9 months. Additionally, extreme heat shock studies show retained luciferase expression after heat treatment at 70 °C. We therefore provide a proof of concept for a platform biotechnology that could provide long-term room temperature storage for temperature-sensitive nucleic acid therapeutics, eliminating the need for the cold chain, which in turn would reduce the cost of distributing life-saving therapeutics worldwide.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery.

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility. In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a âˆ¼ 3.3 - 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100%), whereas polymer E100 demonstrated dose-dependent behaviour (20-90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42% at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Synthesis and Validation of TRIFAPYs as a Family of Transfection Agents for Therapeutic Oligonucleotides.

Transfection agents play a crucial role in facilitating the uptake of nucleic acids into eukaryotic cells offering potential therapeutic solutions for genetic disorders. However, progress in this field needs the development of improved systems that guarantee efficient transfection. Here, we describe the synthesis of a set of chemical delivery agents (TRIFAPYs) containing alkyl chains of different lengths based on the 1,3,5-tris[(4-alkyloxy-1pyridinio)methyl]benzene tribromide structure. Their delivery properties for therapeutic oligonucleotides were evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs) as a silencing tool. The binding of liposomes to PPRHs was evaluated by retardation assays in agarose gels. The complexes had a size of 125 nm as determined by DLS, forming well-defined concentrical vesicles as visualized by Cryo-TEM. The prostate cancer cell line PC-3 was used to study the internalization of the nanoparticles by fluorescence microscopy and flow cytometry. The mechanism of entrance involved in the cellular uptake was mainly by clathrin-mediated endocytosis. Cytotoxicity analyses determined the intrinsic toxicity caused by each TRIFAPY and the effect on cell viability upon transfection of a specific PPRH (HpsPr-C) directed against the antiapoptotic target survivin. TRIFAPYs C12-C18 were selected to expand these studies in the breast cancer cell line SKBR-3 opening the usage of TRIFAPYs for both sexes and, in the hCMEC/D3 cell line, as a model for the blood-brain barrier. The mRNA levels of survivin decreased, while apoptosis levels increased upon the transfection of HpsPr-C with these TRIFAPYs in PC-3 cells. Therefore, TRIFAPYs can be considered novel lipid-based vehicles for the delivery of therapeutic oligonucleotides.

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Recent Progress in Nucleic Acid Pulmonary Delivery toward Overcoming Physiological Barriers and Improving Transfection Efficiency.

Pulmonary delivery of therapeutic agents has been considered the desirable administration route for local lung disease treatment. As the latest generation of therapeutic agents, nucleic acid has been gradually developed as gene therapy for local diseases such as asthma, chronic obstructive pulmonary diseases, and lung fibrosis. The features of nucleic acid, specific physiological structure, and pathophysiological barriers of the respiratory tract have strongly affected the delivery efficiency and pulmonary bioavailability of nucleic acid, directly related to the treatment outcomes. The development of pharmaceutics and material science provides the potential for highly effective pulmonary medicine delivery. In this review, the key factors and barriers are first introduced that affect the pulmonary delivery and bioavailability of nucleic acids. The advanced inhaled materials for nucleic acid delivery are further summarized. The recent progress of platform designs for improving the pulmonary delivery efficiency of nucleic acids and their therapeutic outcomes have been systematically analyzed, with the application and the perspectives of advanced vectors for pulmonary gene delivery.

Peptide-based non-viral gene delivery: A comprehensive review of the advances and challenges.

Gene therapy is the most effective treatment option for diseases, but its effectiveness is affected by the choice and design of gene carriers. The genes themselves have to pass through multiple barriers in order to enter the cell and therefore require additional vectors to carry them inside the cell. In gene therapy, peptides have unique properties and potential as gene carriers, which can effectively deliver genes into specific cells or tissues, protect genes from degradation, improve gene transfection efficiency, and enhance gene targeting and biological responsiveness. This paper reviews the research progress of peptides and their derivatives in the field of gene delivery recently, describes the obstacles encountered by foreign materials to enter the interior of the cell, and introduces the following classes of functional peptides that can carry materials into the interior of the cell, and assist in transmembrane translocation of carriers, thus breaking through endosomal traps to enable successful entry of genetic materials into the nucleus of the cell. The paper also discusses the combined application of peptide vectors with other vectors to enhance its transfection ability, explores current challenges encountered by peptide vectors, and looks forward to future developments in the field.

PEI, a new transfection method, augments the inhibitory effect of RBM5 on prostate cancer.

PEI is a cationic polymer, serving as a non-viral transfection carrier grounded in nanotechnology that enhances transfection efficiency via the proton sponge effect. RBM5 is an RNA-binding protein that can inhibit tumor development. This study involved the transfection of RBM5 in prostate cancer cells with PEI, Lipo2000, and their combination. Transwell and wound healing assays were used to observe invasion and migration of prostate cancer cells and flow cytometry was used to observe the apoptosis. Detect the expression of invasion and migration-related protein MMP9 through western blotting experiment. An activity detection kit was used to detect the activity of apoptotic protein caspase-3. We found that there was no significant difference in transfection efficiency when PEI and Lipo2000 were used alone but it significantly improved when they are combined. RBM5 reduced invasion, migration, and proliferation of prostate cancer and enhanced apoptosis. MMP9 expression was reduced, and the activity of caspase-3 was increased. PEI transfection could improve the inhibition of RBM5 on tumors more than Lipo2000. The inhibitory effect is more obvious when the two are used together. RBM5 transfected with PEI can amplify its inhibitory effect on prostate cancer, and this effect is more evident when combined with Lipo2000.

Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes.

Promoting gene transfection by ROS responsive silicon nanowire arrays.

The development of a fast and safe reactive oxygen species (ROS)-responsive vector is generally limited by the intracellular unstable ROS concentration, and a relatively long time is still needed for the complete intracellular release of drugs or genes induced by ROS. In this work, a gene transfection platform based on ROS-responsive silicon nanowire arrays (SN) is developed, to promote the gene transfection efficiency for several cell lines. Briefly, the surface of the ROS generating system, gold nanoparticle modified SN (SN-Au), is grafted with poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA), an oxidation-responsive charge-reversal cationic polymer. Plasmid DNA (pDNA) bound on the surface through electrostatic interactions was directly delivered into the cells by the time the nanowires penetrate the cells. SN-Au can generate ROS under light treatment, which has an influence on the surface charge change of B-PDEAEA grafted on gold nanoparticles, realizing effective pDNA release in the cytosol for transfection. Nearly 80% of DNA released from the surface of the platform after treated with 1 mM ROS for 10 min. The transfection efficiency of the platform for several cell types was significantly enhanced after a short period of light exposure (3.2-fold for HeLa cells, 7.6-fold for L929 cells, 2.3-fold for BMSC cells and 6.2-fold for mESC cells). The platform also has good biocompatibility. Overall, our results suggest that ROS-responsive SN is a novel, efficient and safe platform for drug and gene transfection.