Enhanced Transfection Efficiency Research Articles

Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Microfluidic Mechanoporation: Current Progress and Applications in Stem Cells.

Intracellular delivery, the process of transporting substances into cells, is crucial for various applications, such as drug delivery, gene therapy, cell imaging, and regenerative medicine. Among the different approaches of intracellular delivery, mechanoporation stands out by utilizing mechanical forces to create temporary pores on cell membranes, enabling the entry of substances into cells. This method is promising due to its minimal contamination and is especially vital for stem cells intended for clinical therapy. In this review, we explore various mechanoporation technologies, including microinjection, micro-nano needle arrays, cell squeezing through physical confinement, and cell squeezing using hydrodynamic forces. Additionally, we highlight recent research efforts utilizing mechanoporation for stem cell studies. Furthermore, we discuss the integration of mechanoporation techniques into microfluidic platforms for high-throughput intracellular delivery with enhanced transfection efficiency. This advancement holds potential in addressing the challenge of low transfection efficiency, benefiting both basic research and clinical applications of stem cells. Ultimately, the combination of microfluidics and mechanoporation presents new opportunities for creating comprehensive systems for stem cell processing.

Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.

Leveraging the extensive surface area of the lungs for gene therapy, the inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Our results demonstrated the superiority of the MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of the nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful lung-specific mRNA transfection without observable signs of toxicity. This MAP may represent an advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells.

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines' culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

Abstract 7249: Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy

Abstract Cancer poses a global health challenge, driving the quest for innovative therapies. Messenger ribonucleic acid (mRNA) lipid nanoparticles (LNPs) are a promising strategy in cancer immunotherapy. However, a major obstacle lies in efficiently transfecting immune cells like macrophages and dendritic cells (DCs) with LNPs. This study seeks to improve cancer immunotherapy by optimizing mRNA-LNP formulations. Therefore, we systematically evaluated three components of LNPs, namely ionizable lipids, phospholipids, and sterols, and fine-tuned the components and their molar ratios. A comprehensive screening process was conducted to determine the efficiency of LNPs, involving in vitro experiments on macrophages and DCs, as well as in vivo assessments. Among six ionizable lipids assessed in different cell lines (RAW264.7 and DC2.4), SM-102 significantly enhanced the transfection efficiency in macrophages and DCs. Subsequently, we evaluated these LNPs in an in vivo setting. We quantified the bioluminescence signal after administering LNPs loaded with luciferase mRNA. The results demonstrated a significant increase in luciferase expression when using SM-102 and ALC-0315 lipids. To validate this finding, we conducted a T cell response study, which revealed an increased percentage of CD8+ T cells with SM-102-based LNPs compared to other formulations. Having identified SM-102 as a potent ionizable lipid, we proceeded to evaluate three phospholipids in vitro. The results indicated a significant enhancement in transfection efficiency with DOPE lipids compared to DSPC. Consequently, we selected DOPE as the phospholipid for subsequent screening. Lastly, we explored different types of sterols with varying molar ratios in macrophages and DC cells. Our findings unveiled a notable improvement in mRNA delivery, particularly in DCs, when the molar ratio of β-sitosterol was increased to 19.5%, while concurrently decreasing the molar ratio of cholesterol to 19% within the same formulation. It is noteworthy that the addition of β-sitosterol alone led to reduced transfection efficiency in DCs but resulted in a significant enhancement in macrophages. Lastly, we evaluated the top four formulations in vivo, and the results demonstrated a significant increase when β-sitosterol was added to the formulation with cholesterol, at molar ratios of 19.5% and 19%, respectively. Overall, altering lipid composition and their molar ratios profoundly impact mRNA-LNP transfection. The optimized formulations, comprising SM-102, DOPE, and cholesterol with or without β-sitosterol, outperformed the alternatives. These refined formulations hold promise for clinical applications, potentially enhancing passive therapeutic agent delivery into macrophages and DCs, offering promising prospects for advancing cancer immunotherapy. Citation Format: Yasir Alshehry, Matthew Fernandez, Raneem Aldaqqa, Asma Al-Terawi, Douglas Sweet, Sandro da Rocha. Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7249.

Abstract B018: Enhanced transfection efficiency with folic acid conjugated synthetic polymer nanoparticles for high grade serous ovarian cancer

Abstract Objective High grade serous ovarian cancer (HGSOC) represents the predominant subtype of epithelial ovarian cancer. Developing targeted therapies for HGSOC presents challenges due to its distinctive characteristic of copy number variation rather than recurrent somatic mutation. In our recent study, we proposed a novel approach for treating ovarian cancer by combining a gene editing system and nanotechnology, utilizing a cationic synthetic biopolymer, poly (γ-propargyl L-glutamine) (PPLQ). While our findings demonstrated promising results, we identified the presence of a negatively charged polymer layer, required for systematic delivery in vivo, significantly reduced both transfection efficiency and therapeutic efficacy. Therefore, an innovative approach to enhance transfection efficiency is desired to achieve improved treatment outcomes. Folate receptor alpha (FRα) is highly overexpressed in various epithelial cancers, including breast, lung, and ovarian cancer. Previous studies have revealed that FRα exhibits high tumor specificity, with epithelial ovarian cancers demonstrating 10 to 100-fold higher FRα expression levels compared to kidney, lung, and breast epithelial cells. Therefore, FRα is an attractive candidate for targeted therapy for ovarian cancer. Method In this study, we conjugated folic acid (FA), which has high affinity for FRα, to PPLQ to enhance gene delivery efficiency. Polyplexes were synthesized using FA conjugated PPLQ (FA-PPLQ) with pGL3 luciferase reporter vector. The particle size and the zeta potential were analyzed by Zetasizer, and transfection efficiency was evaluated by a luminescence assay. Result Compared to the positively charged PPLQ polyplexes, +55.5 mV, the FA-PPLQ polyplexes showed highly negative surface charges of -43.8 mV. The particle size was slightly increased from 35.07 ± 0.5422 for the PPLQ polyplexes to 48.63 ± 0.4239 for the FA-PPLQ polyplexes. The luminescence assay using a pGL3 luciferase reporter vector demonstrated a 74.4% improvement in transfection efficiency with FA conjugation compared to the negatively charged polymer layer. Conclusion Through the conjugation of folic acid to a cationic biopolymer, we successfully achieved enhanced transfection efficiency compared to a negatively charged polymer layer. Notably, the FA-PPLQ polyplexes exhibited a negative surface charge, which is advantageous for in vivo delivery. The results suggest that folic acid conjugation on the surface of polyplexes has a great promise as an effective approach for targeted gene therapy in HGSOC. Citation Format: Chaebin Lee, Kevin Elias. Enhanced transfection efficiency with folic acid conjugated synthetic polymer nanoparticles for high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B018.

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.

Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

Interferon gene therapy with nadofaragene firadenovec for bladder cancer: from bench to approval.

Bladder cancer is a prevalent malignancy with limited therapeutic options, particularly for patients who are unresponsive to Bacillus Calmette-Guérin (BCG). The approval of interferon-α (IFNα) gene therapy with nadofaragene firadenovec (Adstiladrin®), the first gene therapy for genitourinary malignancies, has provided a promising alternative. This article reviews the research and milestones that led to the development and approval of nadofaragene firadenovec. Bladder cancer is well-suited for gene therapy due to direct access to the bladder and the availability of urine and tissue samples for monitoring. Early challenges included effective gene transfer across the urothelium, which was overcome initially by modulating the expression of coxsackie/adenovirus receptor (CAR) and, ultimately, by disrupting the urothelial barrier with Syn3. Nadofaragene firadenovec is a modified adenoviral vector carrying the IFNα gene. Clinical trials have shown promising results, with high response rates and manageable adverse events. Ongoing research focuses on improving patient selection, identifying biomarkers for response prediction, exploring alternative vectors for enhanced transfection efficiency, and developing combination strategies targeting resistance mechanisms. The approval of nadofaragene firadenovec marks a significant milestone in the field of gene therapy for bladder cancer, and future developments hold promise for further enhancing its efficacy and impact.

Effective mRNA Delivery by Condensation with Cationic Nanogels Incorporated into Liposomes.

The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an ∼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.

Fluorescent Magnetic Mesoporous Nanoprobes for Biotechnological Enhancement Procedures in Gene Therapy

In recent years, nanotechnology has deployed a new set of theragnostic tools, including magnetic resonance contrast agents, nano-delivery systems and magnetic hyperthermia treatments in cancer therapy, exploiting not only the small size of nanoparticles, but also relevant nanoscale properties such as superparamagnetism. Specifically, magnetic nanostructures can be remotely manipulated by external magnetic fields, incrementing their possibilities not only for theragnosis, but also for biotech procedures. Genetic engineering processes involve a set of steps like extracting cells from complex environments, their selection and subsequent cultivation or modification by transfection and can benefit from the use of bioconjugated magnetic nanoparticles. Magnetofection of cells with genes or biological material uploaded on superparamagnetic nanoparticles attracted by a magnetic field greatly increases the efficiency, specificity and speed of the biotechnological procedure in gene transfer systems. This article presents a preliminary investigation into the enhanced transfection efficiency of fluorescent magnetic mesoporous silica nanostructures functionalized with mCherry plasmid, which were used to transfect HeLa cells in just 15 min via magnetic transfection. This method was compared to passive transfection (4 h) and conventional gene transfer using the commercial K2 Transfection System (16 h). The results demonstrated that the fluorescent magnetic mesoporous silica nanostructures were similarly effective to the commercial kit, without the need for reagents that increase costs in clinical therapy. Furthermore, viability assays conducted with HeLa cells showed negligible toxicity at concentrations of up to 50 μg/mL.

GSH-Activatable Aggregation-Induced Emission Cationic Lipid for Efficient Gene Delivery.

The key to gene therapy is the design of biocompatible and efficient delivery systems. In this work, a glutathione (GSH)-activated aggregation-induced-emission (AIE) cationic amphiphilic lipid, termed QM-SS-KK, was prepared for nonviral gene delivery. QM-SS-KK was composed of a hydrophilic biocompatible lysine tripeptide headgroup, a GSH-triggered disulfide linkage, and a hydrophobic AIE fluorophore QM-OH (QM: quinoline-malononitrile) tail. The peptide moiety could not only efficiently compact DNA but also well modulate the dispersion properties of QM-SS-KK, leading to the fluorescence-off state before GSH treatment. The cleavage of disulfide in QM-SS-KK by GSH generated AIE signals in situ with a tracking ability. The liposomes consisted of QM-SS-KK, and 1,2-dioleoylphosphatidylethanolamine (DOPE) (QM-SS-KK/DOPE) delivered plasmid DNAs (pDNAs) into cells with high efficiency. In particular, QM-SS-KK/DOPE had an enhanced transfection efficiency (TE) in the presence of 10% serum, which was two times higher than that of the commercial transfection agent PEI25K. These results highlighted the great potential of peptide and QM-based fluorescence AIE lipids for gene delivery applications.

Magnetofection of miR-21 promoted by electromagnetic field and iron oxide nanoparticles via the p38 MAPK pathway contributes to osteogenesis and angiogenesis for intervertebral fusion

BackgroundMagnetofection-mediated gene delivery shows great therapeutic potential through the regulation of the direction and degree of differentiation. Lumbar degenerative disc disease (DDD) is a serious global orthopaedic problem. However, even though intervertebral fusion is the gold standard for the treatment of DDD, its therapeutic effect is unsatisfactory. Here, we described a novel magnetofection system for delivering therapeutic miRNAs to promote osteogenesis and angiogenesis in patients with lumbar DDD.ResultsCo-stimulation with electromagnetic field (EMF) and iron oxide nanoparticles (IONPs) enhanced magnetofection efficiency significantly. Moreover, in vitro, magnetofection of miR-21 into bone marrow mesenchymal stem cells (BMSCs) and human umbilical endothelial cells (HUVECs) influenced their cellular behaviour and promoted osteogenesis and angiogenesis. Then, gene-edited seed cells were planted onto polycaprolactone (PCL) and hydroxyapatite (HA) scaffolds (PCL/HA scaffolds) and evolved into the ideal tissue-engineered bone to promote intervertebral fusion. Finally, our results showed that EMF and polyethyleneimine (PEI)@IONPs were enhancing transfection efficiency by activating the p38 MAPK pathway.ConclusionOur findings illustrate that a magnetofection system for delivering miR-21 into BMSCs and HUVECs promoted osteogenesis and angiogenesis in vitro and in vivo and that magnetofection transfection efficiency improved significantly under the co-stimulation of EMF and IONPs. Moreover, it relied on the activation of p38 MAPK pathway. This magnetofection system could be a promising therapeutic approach for various orthopaedic diseases.Graphical

Methods to visualise zinc transporter proteins of the SLC39A family in cells.

Zinc is essential to many important biological processes and the SLC39A family of zinc transporters which help to control zinc levels in cells, are increasingly being implicated in disease states. In order to determine their exact roles in these processes, reliable methods for their successful production are now required. Unfortunately, extensive post-translational modification and temporally specific activation makes visualisation of ZIP family transporters difficult. As such, modifications of common molecular cell biology techniques are necessary to maximise success when studying these proteins. These include zinc stimulation and nocodazole synchronisation to enhance the activation of ZIP7 and ZIP6/ZIP10, respectively. Maximal ZIP6/ZIP10 retention can also be achieved through careful handling of loosely adhered mitotic fractions when harvesting cell cultures for lysis. Transfection can also be used to enhance ZIP visualisation, however consideration of transfection periods and inclusion of sodium butyrate are recommended to enhance transfection efficiency. When probing for ZIP family transporters, we recommend that epitope choice considers post-translational cleavage and phosphorylated protein isoforms. Finally, where expression of a particular ZIP transporter is manipulated, researchers should consider parallel evaluation of related ZIP transporter expression, to account for transporter compensation.

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.

Cationic RGD peptidomimetic nanoconjugates as effective tumor targeting gene delivery vectors with antimicrobial potential

Gene delivery combined with systemic targeting approach has shown promising potential in cancer gene therapy. Peptides are ideal functional motif for constructing biocompatible non-viral gene delivery vehicles. RGD peptides, in particular, are known to recognize the integrin αVβ3, which is expressed specifically on angiogenic blood vessels and, therefore, is considered vital for anti-angiogenesis strategies and cancer treatment. In recent times, several RGD peptide-based non-viral gene delivery vectors have been utilized for targeted gene delivery, however, lack in proteolytic stability. In the current study, we have investigated a series of non-naturally modified RGD peptide mimic (MOH) nanoconjugates with low molecular weight branched polyethylenimine (bPEI 1.8 kDa). The projected peptide mimic, Fmoc-FFARKA (MOH), has already been demonstrated to have high binding efficiency for αVβ3 integrins and enhanced cell adhesive ability with high stability compared to the natural RGD counterpart. The polymer-peptide, PEI-MOH (PMOH), nanoconjugate vectors have been designed to enhance the tumor targeting ability, therapeutic proficiency, transfection efficiency and proteolytic stability. The synthesized nanoconjugates showed the ability to protect the bound DNA with low cytotoxicity and their pDNA complexes displayed enhanced transfection efficiency. Furthermore, a competitive study confirmed their selective behavior towards liver cancer cells, HepG2. Lastly, PMOH nanoconjugates also exerted significant antimicrobial effects against drug-resistant pathogens. Altogether, the data suggest that nanosized non-naturally modified RGD peptide mimic-based gene vectors hold great potential as efficient biomaterials for targeted gene delivery and antimicrobial applications.

Poly(Beta-Amino Ester)s as High-Yield Transfection Reagents for Recombinant Protein Production.

PurposeTransient transfection is an essential tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and the resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of a class of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection in comparison to linear 25 kDa PEI, a commonly used transfection reagent.MethodsTransfection efficiency and protein production were assessed in human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions using PBAE-based nanoparticles in comparison to linear 25 kDa PEI. Production of both a cytosolic reporter and secreted antibodies was investigated.ResultsIn both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and enhanced production of a cytosolic reporter compared to linear 25 kDa PEI. This result extended to secreted proteins, as a model PBAE increased the production of 3 different secreted antibodies compared to linear 25 kDa PEI at culture scales ranging from 20 to 2000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticle formulation led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types.ConclusionThese results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.

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.