The 25-kDa linear polyethylenimine exerts specific antiviral activity against pseudorabies virus through interferencing its adsorption via electrostatic interaction.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which is responsible for enormous economic losses to the global pig industry. Although vaccination has been used to prevent PRV infection, the effectiveness of vaccines has been greatly diminished with the emergence of PRV variants. Therefore, there is an urgent need to develop anti-PRV drugs. Polyethylenimine (PEI) is a cationic polymer and has a wide range of antibacterial and antiviral activities. This study found that a low dose of 1 µg/mL of the 25-kDa linear PEI had significantly specific anti-PRV activity, which became more intense with increasing concentrations. Mechanistic studies revealed that the viral adsorption stage was the major target of PEI without affecting viral entry, replication stages, and direct inactivation effects. Subsequently, we found that cationic polymers PEI and Polybrene interfered with the interaction between viral proteins and cell surface receptors through electrostatic interaction to exert the antiviral function. In conclusion, cationic polymers such as PEI can be a category of options for defense against PRV. Understanding the anti-PRV mechanism also deepens host-virus interactions and reveals new drug targets for anti-PRV.IMPORTANCEPolyethylenimine (PEI) is a cationic polymer that plays an essential role in the host immune response against microbial infections. However, the specific mechanisms of PEI in interfering with pseudorabies virus (PRV) infection remain unclear. Here, we found that 25-kDa linear PEI exerted mechanisms of antiviral activity and the target of its antiviral activity was mainly in the viral adsorption stage. Correspondingly, the study demonstrated that PEI interfered with the virus adsorption stage by electrostatic adsorption. In addition, we found that cationic polymers are a promising novel agent for controlling PRV, and its antiviral mechanism may provide a strategy for the development of antiviral drugs.

Evaluation of the Effect of Polyethylenimine on Boron Adsorption by Soil Minerals.

The removal of hazardous ions from water is crucial for safeguarding both the environment and human health. Soil minerals, integral components of soil, play a vital role as adsorbents for various contaminants, including heavy metal ions, organic dyes, and detergents. This study investigates the interaction between boron ions and soil minerals (gibbsite, kaolinite, and montmorillonite) in the presence of polyethylenimine (PEI). The minerals underwent characterization based on specific surface area, particle size distribution, zeta potential, and the presence of functional groups. The influence of PEI addition on the stability of the soil mineral suspension was evaluated by turbidimetry. Mineral-boron and mineral-boron-PEI interactions were explored under varying conditions, including pH, initial boron concentration, and mineral quantity, with all adsorption experiments conducted over 24 hours. Using the Langmuir isotherm, the maximum adsorption capacity of the studied minerals was determined for boron both without and in the presence of PEI. For gibbsite, kaolinite and montmorillonite, it was 30.63, 24.55 and 26.62 mg g-1, respectively, while in the presence of PEI, it increased to 33.11, 26.61 and 45.47 mg g-1, respectively. The addition of PEI enhanced boron adsorption from aqueous solutions, increasing the removal efficiency from 65 % to about 80 %.

An optimized polymeric delivery system for piggyBac transposition.

The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.

Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells: when less is more.

Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. KEY POINTS: • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields.

Contribution of Adsorption and Hematocrit Levels to Ganciclovir Clearance in an in Vitro Continuous Hemodiafiltration Model.

Estimation of the continuous hemodiafiltration (CHDF) clearance (CLCHDF) of ganciclovir (GCV) is crucial for achieving efficient treatment outcomes. Here, we aimed to clarify the contribution of diafiltration, adsorption, and hematocrit level to the CLCHDF of GCV in an in vitro CHDF model using three membranes: polyacrylonitrile and sodium methallyl sulfonate copolymer coated with polyethylenimine (AN69ST); polymethylmethacrylate (PMMA); and polysulfone (PS). In vitro CHDF was performed with effluent flow rates (Qe) of 800, 1500, and 3000 mL/h. The initial GCV concentration was 10 µg/mL while that of human serum albumin (HSA) was 0 or 5 g/dL. The CLCHDF, diafiltration rates, and adsorption rates were calculated. The whole blood-to-plasma ratio (R) of GCV for a hematocrit of 0.1 to 0.5 was determined using blood samples with 0.5 to 100 µg/mL of GCV. The in vitro CHDF experiment using AN69ST, PMMA, and PS membranes showed that the total CLCHDF values were almost the same as the Qe and not influenced by the HSA concentration. The diafiltration rate exceeded 88.1 ± 2.8% while the adsorption rate was lower than 9.4 ± 9.4% in all conditions. The R value was 1.89 ± 0.11 and was similar at all hematocrit levels and GCV concentrations. In conclusion, diafiltration mainly contributes to the CLCHDF of GCV, rather than adsorption. Hematocrit levels might not affect the relationship between the plasma and blood CLCHDF of GCV, and the CLCHDF of GCV can be estimated from the Qe and R, at least in vitro.

One-step enrichment and stepwise elution of glycoproteins and phosphoproteins by hydrophilic Ti4+-immobilized dendrimer poly(glycidyl methacrylate) microparticles functionalized with polyethylenimine and phytic acid.

Glycosylation and phosphorylation rank as paramount post-translational modifications, and their analysis heavily relies on enrichment techniques. In this work, a facile approach was developed for the one-step simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. The core of this approach was the application of the novel titanium (IV) ion immobilized poly(glycidyl methacrylate) microparticles functionalized with dendrimer polyethylenimine and phytic acid. The microparticles possessed dual enrichment capabilities due to their abundant titanium ions and hydroxyl groups on the surface. They demonstrate rapid adsorption equilibrium (within 30 min) and exceptional adsorption capacity for ß-casein (1107.7 mg/g) and horseradish peroxidase (438.6 mg/g), surpassing that of bovine serum albumin (91.7 mg/g). Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to validate the enrichment capability. Experimental results across various biological samples, including standard protein mixtures, non-fat milk, and human serum, demonstrated the remarkable ability of these microparticles to enrich low-abundance glycoproteins and phosphoproteins from biological samples.

Robust and Stable Atomically Precise Metal Nanoclusters Mediated Solar Water Splitting.

Atomically precise metal nanoclusters (NCs) represent an emerging sector of light-harvesting antennas by virtue of peculiar atomic stacking fashion, quantum confinement effect, and molecular-like discrete energy band structure. Nevertheless, precise control of charge carriers over metal NCs has yet to be achieved by the short carrier lifetime and intrinsic instability of metal NCs, which renders the complexity of metal NCs-based photosystems with photoredox mechanisms remaining elusive. Herein, fine tuning of charge migration over metal NCs is demonstrated by constructing directional charge transfer channels in multilayered heterostructure enabled by a facile layer-by-layer (LbL) assembly approach, wherein oppositely charged branched poly-ethylenimine (BPEI) and glutathione (GSH)-capped gold NCs [Aux NCs, Au25 (GSH)18 NCs] are alternately deposited on the metal oxide (MOs: TiO2 , WO3 , Fe2 O3 ) substrates. TheAux (Au25 ) NCs layer serves as light-harvesting antennas for engendering charge carriers, andBPEI interim layer uniformly intercalated at the interface of Aux NCs layer constitutes the tandem hole transport channel for motivating the charge transfer cascade, resulting in the considerably enhanced photoelectrochemical water oxidation performances. Besides, poor photo-stability of Aux NCs is surmounted by stimulating the hole transfer kinetics process.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

The fluorescence and colorimetric dual-readout probe for clinical rapid detection of mycophenolic acid by the poly(ethylenimine)/silica-coated CdTe quantum dots.

It is particularly meaningful to therapeutic drug monitoring (TDM) of mycophenolic acid (MPA) for transplant patients to maximize the drug efficacy and minimize the adverse effect. In this study, a novel fluorescence and colorimetric dual-readout probe was put forward to fast and reliable detect MPA. The blue fluorescence of MPA was largely enhanced in the presence of poly (ethylenimine) (PEI), while the red fluorescence of CdTe@SiO2 (silica-coated CdTe quantum dots) provided a reliable reference signal. Hence, combining PEI70,000 and CdTe@SiO2, a fluorescence and colorimetric dual-readout probe could be constructed. For fluorescence measurement of MPA, the linearity was obtained in the MPA concentration range of 0.5-50 µg/mL, with a limit of detection (LOD) of 33 ng/mL. For the visual detection, the fluorescent colorimetric card was established in the MPA concentration from 0.5 to 50 µg/mL corresponding to the fluorescence color from red to violet and then to blue, which could be used for semi-quantification. Furthermore, in the light of the ColorCollect APP by the smartphone, the ratio of blue and red brightness values was linear with the MPA concentration from 1 to 50 µg/mL; thus, quantification of MPA could be realized by APP with the LOD of 83 ng/mL. The developed method was successfully applied to the analysis of MPA in the plasma samples of three patients after oral administration of mycophenolate mofetil, which was the prodrug of MPA. The result was comparable to those obtained by the clinically widely-used enzyme multiplied immunoassay technique. The developed probe was fast, cost-effective and operational convenience, and possessed high potential for TDM of MPA.

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4.

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 µg mL-1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.

25KDa branched polyethylenimine increases interferon-γ production in natural killer cells via improving translation efficiency.

BACKGROUND: Ex vivo cultivation is a promising strategy for increasing the number of NK cells and enhancing their antitumor activity prior to clinical application. Recent studies show that stimulation with 25KDa branched polyethylenimine (25KbPEI) generates NK cells with enhanced antitumor activity. To better understand how 25KbPEI primes NK cells, we explored the mechanism underlying increase in production of IFN-γ. METHODS: Chemical priming was performed on NK-92MI cells by incubating them with 5 µg/ml of 25KbPEI. The production of IFN-γ was evaluated by RT-qPCR, ELISA, and Flow cytometry. By evaluating the effect of pharmacological inhibition of ERK/mTOR-eIF4E signaling pathways on IFN-γ translation, the function of these signaling pathways in IFN-γ translation was examined. To comprehend the level of 25KbPEI activity on immune-related components in NK cells, RNA sequencing and proteomics analyses were conducted. RESULTS: 25KbPEI enhances the production of IFN-γ by NK cells without transcriptional activation. Activation of ERK and mTOR signaling pathways was found to be associated with 25KbPEI-mediated calcium influx in NK cells. The activation of ERK/mTOR signaling was linked to the phosphorylation of 4E-BP1, which resulted in the activation of translation initiation complex and subsequent IFN-γ translation. Analysis of RNA sequencing and proteomics data revealed that the activity of 25KbPEI to improve translation efficiency in NK cells could be extended to additional immune-related molecules. CONCLUSIONS: This study provides substantial insight into the process by which 25KbPEI primes NK cells. Our data demonstrated that the 25KbPEI mediated activation of ERK/mTOR signaling and subsequent stimulation of eIF4E is the primary mechanism by which the chemical stimulates translation of IFN-γ in NK cells. Video abstract.

Liquid nitric oxide donor for adjuvant therapy of acute ischemic stroke via nasal administration.

OBJECTIVES: The aim of this study was to synthesize and characterize a novel NO donor, PEI-PO-NONOate, using propylene oxide and to investigate its biosafety and therapeutic efficacy via nasal administration in vitro and vivo. EXPERIMENTAL PROCEDURES: The PEI-PO-NONOate was synthesized based on polyethylenimine (PEI) with different molecular weights and characterized using Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and ultraviolet (UV) spectroscopy. Cytotoxicity assays were performed on mouse fibroblast cells L929 and human nasal mucosa epithelial cells (HNEpC), and a rat middle cerebral artery occlusion (MCAO) model was established to evaluate the therapeutic efficacy of PEI-PO-NONOate via nasal administration. RESULTS: The PEI-PO-NONOate was found to be stable under dark, dry, and airproof conditions, and its release was accelerated in an aqueous phase or acidic environment, while it was slowed down in a polyethylene glycol (PEG) mixture system. The NO donor released approximately 0.4, 0.5, and 0.6 µmol of gaseous NO from 1.0 mg of the polymer based on PEI600, PEI1800, and PEI10K, respectively. Cytotoxicity assays showed that the PEI-PO-NONOates had a cryoprotective effect as compared with PEI and PEI-PO. Furthermore, nasal administration of PEI-PO-NONOates resulted in a significant reduction in overall necrotic ratio as compared with the control group (16.4% versus 24.6%, p < 0.05). CONCLUSION: The findings of this study suggest that PEI-PO-NONOates may have potential as an adjuvant therapy for acute ischemic stroke when administered via the nasal route.

Polyethylenimine linked with chitosan improves astaxanthin production in Haematococcus pluvialis.

Astaxanthin is receiving increasing interest as an antioxidant and high value-added secondary metabolite. Haematococcus pluvialis is the main source for astaxanthin production, and many studies are being conducted to increase the production of astaxanthin. In this study, we linked polyethylenimine (PEI) with chitosan to maintain astaxanthin-inducing ability while securing the recyclability of the inducer. Astaxanthin accumulation in H. pluvialis was induced to 86.4 pg cell-1 with the PEI-chitosan fiber (PCF) treatment prepared by cross-linking of 10 µM PEI and low molecular weight (MW) chitosan via epichlorohydrin. PEI concentration affected the astaxanthin accumulation, whereas the MW of chitosan did not. In addition, the PCF treatment in H. pluvialis increased the reactive oxygen species (ROS) content in cells, thereby upregulating the transcription of enzymes involved in astaxanthin biosynthesis. PCF can be reused multiple times with the maintenance of over 90% of the astaxanthin production efficiency. This study offers a reusable PCF stimulation strategy for enhancing natural astaxanthin content, and PCF treatment will easily increase the production scale or reduce production costs by using recyclability that is not available in current methods. KEY POINTS: • Polyethylenimine-chitosan fiber (PCF) was applied to Haematococcus pluvialis • PCF promotes astaxanthin accumulation by enhancing oxidative stress in H. pluvialis • PCF can be reused multiple times with maintaining over 90% production efficiency.

High-Performance Flexible Polymer Memristor Based on Stable Filamentary Switching.

Polymer-based atomic switch memristors via the formation/dissolution of atomic-scale conductive filaments are considered as the leading candidate for next-generation nonvolatile memory. However, the instability of conductive filaments of incomplete bridge makes their switching performances unsatisfied. In this work, we report a flexible polymeric memristor using polyethylenimine incorporated with silver salt. The memristor device exhibited superior performances at room temperature with a favorable endurance, high ON/OFF ratio, good retention, and low operating voltage. These satisfactory performances are attributed to the pre-existing Ag ions in the polymer, guiding the formation of a robust Ag filament. In addition, the device shows stable bipolar switching behavior in bending conditions or after hundreds of bending cycles. In our work, we provide a simple and efficient method to construct robust filament-based memristors for flexible electronics.

Transcriptional Targeting of Dendritic Cells Using an Optimized Human Fascin1 Gene Promoter.

Deeper knowledge about the role of the tumor microenvironment (TME) in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. Whereas some approaches focus on the expression of tumoricidal genes within the TME, DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs) in secondary lymphoid organs, which in turn induce anti-tumor T cell responses. Besides effective delivery systems and the requirement of appropriate adjuvants, DNA vaccines themselves need to be optimized regarding efficacy and selectivity. In this work, the concept of DC-focused transcriptional targeting was tested by applying a plasmid encoding for the luciferase reporter gene under the control of a derivative of the human fascin1 gene promoter (pFscnLuc), comprising the proximal core promoter fused to the normally more distantly located DC enhancer region. DC-focused activity of this reporter construct was confirmed in cell culture in comparison to a standard reporter vector encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer (pCMVLuc). Both plasmids were also compared upon intravenous administration in mice. The organ- and cell type-specific expression profile of pFscnLuc versus pCMVLuc demonstrated favorable activity especially in the spleen as a central immune organ and within the spleen in DCs.

Cellulose Modified with Polyethylenimine (PEI) Using Microwave Methodology for Adsorption of Chromium from Aqueous Solutions.

A large amount of agricultural waste was used to prepare cellulose (Cel) and then the surface was modified with PEI (Cel-PEI) using the microwave method. To be used as a metal adsorbent, the adsorption of Cr (VI) from an aqueous solution by Cel-PEI was measured using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) techniques. The parameters of Cr (VI) adsorption in solution by the Cel-PEI adsorbent were as follows: the pH of the solution was 3, the concentration of the chromium solution was 100 mg/L, and the adsorption time was 180 min at 30 °C using 0.01 g of adsorbent. Cel-PEI had a Cr (VI) adsorption capacity of 106.60 mg/g, while the unadjusted Cel was 23.40 mg/g and the material recovery showed a decrease in efficiency of 22.19% and 54.27% in the second and third cycles, respectively. The absorption isotherm of chromium adsorption was also observed. The Cel-PEI material conformed to the Langmuir model with an R2 value of 0.9997. The kinetics of chromium adsorption showed that under pseudo-second-order analysis, with R2 values of 0.9909 and 0.9958 for Cel and Cel-PEI materials, respectively. The G° and H° values of the adsorption process were negative, indicating that the adsorption is spontaneous and that the adsorption process is exothermic. The efficient preparation adsorbent materials for Cr (VI) was achieved using a short microwave method that is low-cost and environmentally friendly for use in the treatment of Cr-contaminated wastewater.

Linoleic-acid-substituted polyethylenimine to silence heat shock protein 90B1 (HSP90B1) to inhibit migration of breast cancer cells.

INTRODUCTION: Breast cancer continues to be one of the leading causes of death in women, and the lack of treatment options for distant metastasis warrants the need to identify and develop more effective approaches. The aim of this study was to identify and validate targets that are associated with the survival and migration of the breast cancer cells in vitro through RNA interference (RNAi) approach. METHODS: Linoleic-acid-modified polyethylenimine (PEI) polymer was used to screen a short interfering RNA (siRNA) library against numerous cell adhesion and cytoskeleton genes in MDA-MB-231 triple-negative breast cell line, and the functional outcome of silencing was determined by growth and migration inhibition with further target validation studies. RESULTS: Heat shock protein 90B1 (HSP90B1) was identified as a crucial gene that is known to be involved in various breast cancer machineries, including uncontrolled proliferation and brain metastasis. The success of this approach was also due to the use of hyaluronic acid (HA) additive in lipopolymer complexes that showed a profound impact in reducing the cell viability (~50%), migration (~40%), and mRNA transcript levels (~80%) with a physiologically relevant siRNA concentration of 60 nM. The use of Dicer-substrate siRNA proved to be beneficial in target silencing, and a combinational treatment of integrin-ß1 (ITGB1) and HSP90B1 was effective in reducing the migration of the MDA-MB-231 and MDA-MB-436 breast cancer cells. CONCLUSION: This study demonstrates the potential to identify and silence targets using a lipid-modified PEI/siRNA system and highlights the importance of HSP90B1 in the growth and migration of breast cancer cells.

Gene transfection of primary mouse hepatocytes in 384-well plates.

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.

A Polyplex in a Shell: The Effect of Poly(aspartic acid)-Mediated Calcium Carbonate Mineralization on Polyplexes Properties and Transfection Efficiency.

Mineralization by exposure of organic templates to supersaturated solutions is used by many living organisms to generate specialized materials to perform structural or protective functions. Similarly, it was suggested that improved robustness acquired through mineralization under natural conditions could be an important factor for virus survival outside of a host for better transfection of cells. Here, inspired by this fact, we developed a nonviral tricomponent polyplex system for gene delivery capable of undergoing mineralization. First, we fabricated anionic polyplexes carrying pDNA by self-assembly with a lipid-modified cationic polymer and coating by poly(aspartic acid). Then, we submitted the polyplexes to a two-step mineralization reaction to precipitate CaCO3 under various supersaturations. We carried out detailed morphological studies of the mineralized polyplexes and identified which parameters of the fabrication process were influential on transfection efficiency. We found that mineralization with CaCO3 is efficient in promoting transfection efficiency as long as a certain Ca2+/CO32- lower limit ratio is respected. However, calcium incubation can also be used to achieve similar effects at higher concentrations depending on polyplex composition, probably due to the formation of physical cross-links by calcium binding to poly(aspartic acid). We proposed that the improved robustness and transfection efficiency provided by means of mineralization can be used to expand the possible applications of polyplexes in gene therapy.

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 αVß3, 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 αVß3 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.