pDNA Binding Research Articles

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

A catch-and-release nano-based gene delivery system.

The design of nanomaterial-based nucleic acid formulations is one of the biggest endeavours in the search for clinically applicable gene delivery systems. Biopolymers represent a promising subclass of gene carriers due to their physicochemical properties, biodegradability and biocompatibility. By modifying melanin-like polydopamine nanoparticles with poly-L-arginine and poly-L-histidine blends, we obtained a novel catch-and-release gene delivery system for efficient trafficking of pDNA to human cells. A synergistic interplay of nanoparticle-bound poly-L-arginine and poly-L-histidine was observed and evaluated for pDNA binding affinity, cell viability, gene release and transfection. Although the functionalisation with poly-L-arginine was crucial for pDNA binding, the resulting nanocarriers failed to release pDNA intracellularly, resulting in limited protein expression. However, optimal pDNA release was achieved through the co-formulation with poly-L-histidine, essential for pDNA release. This effect enabled the design of gene delivery systems, which were comparable to Lipofectamine in terms of transfection efficacy and the catch-and-release surface modification strategy can be translated to other nanocarriers and surfaces.

Tailoring head-tail mesoporous silica nanoparticles for enhanced gene transfection.

Plasmid DNA (pDNA) delivery has attracted extensive research interest due to its great potential in gene therapy. The design of efficient nano-vectors to promote cellular delivery and transfection of gene molecules is the key to success. Compared to conventional nanocarriers with spherical geometry, asymmetric nanoparticles have been well documented showing enhanced cellular uptake and drug delivery capability. However, the impact of asymmetric nanostructures on pDNA binding and following intracellular delivery performance has been less reported. Herein, asymmetric head-tail mesoporous silica nanoparticles (HTMSNs) with tailored tail lengths were synthesized and employed as nano-vectors for pDNA delivery. The nanostructures of HTMSNs were carefully characterized by electron tomography. The pDNA binding, cellular uptake and gene transfection capabilities of engineered asymmetric nanoparticles were compared with symmetric dendritic mesoporous silica nanoparticles (DMSNs). The results showed that the asymmetric morphology of nanoparticles promoted pDNA binding and cell internalization, where HTMSNs-66 with a specific tail length of 66 nm achieved the highest transfection efficiency. This study reveals the impact of asymmetric nanostructure on DNA interaction, and provides guidance in future designs of non-viral nano-vectors for efficient gene delivery.

Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery.

Supramolecular polymers formed through host–guest complexation have inspired many interesting developments of functional materials for biological and biomedical applications. Here, we report a novel design of a non-viral gene delivery system composed of a cationic star polymer forming supramolecular complexes with the surface oleyl groups of superparamagnetic iron oxide nanoparticles (SPIONs), for magnetically enhanced delivery of DNA into mammalian cells. The cationic star polymer was synthesized by grafting multiple oligoethylenimine (OEI) chains onto an α-cyclodextrin (α-CD) core. The SPIONs were synthesized from iron(III) acetylacetonate and stabilized by hydrophobic oleic acid and oleylamine in hexane, which were characterized in terms of their size, structure, morphology, and magnetic properties. The synthesized magnetic particles were found to be superparamagnetic, making them a suitable ferrofluid for biological applications. In order to change the hydrophobic surface of the SPIONs to a hydrophilic surface with functionalities for plasmid DNA (pDNA) binding and gene delivery, a non-traditional but simple supramolecular surface modification process was used. The α-CD-OEI cationic star polymer was dissolved in water and then mixed with the SPIONs stabilized in hexane. The SPIONs were “pulled” into the water phase through the formation of supramolecular host–guest inclusion complexes between the α-CD unit and the oleyl surface of the SPIONs, while the surface of the SPIONs was changed to OEI cationic polymers. The α-CD-OEI-SPION complex could effectively bind and condense pDNA to form α-CD-OEI-SPION/pDNA polyplex nanoparticles at the size of ca. 200 nm suitable for delivery of genes into cells through endocytosis. The cytotoxicity of the α-CD-OEI-SPION complex was also found to be lower than high-molecular-weight polyethylenimine, which was widely studied previously as a standard non-viral gene vector. When gene transfection was carried out in the presence of an external magnetic field, the α-CD-OEI-SPION/pDNA polyplex nanoparticles greatly increased the gene transfection efficiency by nearly tenfold. Therefore, the study has demonstrated a facile two-in-one method to make the SPIONs water-soluble as well as functionalized for enhanced magnetofection.

Mineralized polyplexes for gene delivery: Improvement of transfection efficiency as a consequence of calcium incubation and not mineralization

Gene therapy is an emerging field in which nucleic acids are used to control protein expression. The necessity of delivering nucleic acids to specific cell types and intracellular sites demands the use of highly specialized gene carriers. As a carrier modification technique, mineralization has been successfully used to modify viral and non-viral carriers, providing new properties that ultimately aim to increase the transfection efficiency. However, for the specific case of polyplexes used in gene therapy, recent literature shows that interaction with calcium, a fundamental step of mineralization, might be effective to increase transfection efficiency, leaving an ambiguity about of the role of mineralization for this type of gene carriers. To answer this question and to reveal the properties responsible for increasing transfection efficiency, we mineralized poly(aspartic acid) coated polyplexes at various CaCl2 and Na3PO4 concentrations, and evaluated the resultant carriers for physicochemical and morphological characteristics, as well as transfection and delivery efficiency with MC3T3-E1 mouse osteoblastic cells. We found that both mineralization and calcium incubation positively affected the transfection efficiency and uptake of polyplexes in MC3T3-E1 cells. However, this effect originated from the properties achieved by polyplexes after the calcium incubation step that are maintained after mineralization, including particle size increase, improved pDNA binding, and adjustment of zeta potential. Considering that mineralization can be a longer process than calcium incubation, we find that calcium incubation might be sufficient and preferred if improved transfection efficiency in vitro is the only effect desired.

PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

Preparation of a water soluble aminated β-1,3-D-glucan for gene carrier: The in vitro study of the anti-inflammatory activity and transfection efficiency.

β-1,3-D-glucan has been reported to have a series of bioactivities including antitumor, antimicrobial, anti-inflammatory and antioxidative effects; however, its insolubility in neutral aqueous solution significantly restricts the potential application in biological and medicine fields. Herein, a water-soluble aminated β-1,3-D-glucan (AG) was synthesized and the anti-inflammatory effect, cytotoxicity and plasmid DNA (pDNA) binding capacity of AG, serum stability, the particle sizes and zeta potentials of AG/pDNA nanocomposites, and the transfection efficiency and mechanism of action were studied. AG shows no obvious cytotoxicity within the range of working concentration (1-64 μg/ml) and it exerts potent anti-inflammatory effect independent on Dectin-1 and TLR2. AG/pDNA nanocomposites prepared by electrostatic interaction possess an appropriate particle size ranged from 192.8 to 118.4nm and zeta potentials ranged from 20.880 to 27.16 mV with the N/P ratios from 5 to 100. AG/pDNA nanocomposites at the N/P ratios of 10 and 20 were able to show superior transfection efficiencies in RAW 264.7 cells as a result of their suitable particle size, zeta potential, anti-inflammatory effect, and the specific interaction with pattern recognition receptors (Dectin-1 and TLR2). These results indicate that AG is a potential candidate for DNA delivery system due to its potent anti-inflammatory effect and high transfection efficiency.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Enzyme-responsive polysaccharide supramolecular nanoassembly for enhanced DNA encapsulation and controlled release

An enzyme-responsive polysaccharide supramolecular targeted nanoassembly was successfully constructed by the host-guest complexation of positively charged mono-(6-(tetraethylenepentamine)-6-deoxy)-β-cyclodextrin (TEPA-CD) with adamantane-grafted hyaluronic acid (HA-ADA). Possessing a series of positively charged polyamine chains, the obtained polysaccharide nanoassembly could serve as a biocompatible plasmid DNA (pDNA) container. More interestingly, the pDNA could be released from the nanoassembly through the enzymatic degradation of HA skeleton, which realized the controlled pDNA binding and release. Besides, the polysaccharide nanoassembly exhibited lower cytotoxicity than the commercial transfection reagents 25 kDa bPEI (PEI25k), accompanied by similar gene delivery effect. We believe that this work might present a convenient method for targeted, controlled gene delivery.

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.

In the present study, three biodegradable block copolymers composed of a poly(ethylene glycol) block and a copolypeptide block with varying compositions of cationic L-lysine (L-Lys) and hydrophobic benzyl-L-glutamate (Bzl-L-Glu) were designed for gene delivery applications. The polypeptides were synthesized by ring opening polymerization (ROP) and after orthogonal deprotection of Boc-L-Lys side chains, the polymer exhibited an amphiphilic character. To bind or encapsulate plasmid DNA (pDNA), different formulations were investigated: a nanoprecipitation and an emulsion technique using various organic solvents as well as an aqueous pH-controlled formulation method. The complex and nanoparticle (NP) formations were monitored by dynamic light scattering (DLS), and pDNA interaction was shown by gel electrophoresis and subsequent controlled release with heparin. The polypeptides were further tested for their cytotoxicity as well as biodegradability. The complexes and NPs presenting the most promising size distributions and pDNA binding ability were subsequently evaluated for their transfection efficiency in HEK293T cells. The highest transfection efficiencies were obtained with an aqueous formulation of the polypeptide containing the highest L-Lys content and lowest proportion of hydrophobic, helical structures (P1*), which is therefore a promising candidate for efficient gene delivery by biodegradable gene delivery vectors.

Lactobionic acid-chitosan functionalised gold-coated poly(lactide-co-glycolide) nanoparticles for hepatocyte targeted gene delivery

Nanoscale non-viral gene delivery vectors are attractive systems for the efficient and safe delivery of therapeutic genes. This study explored the potential of surface tailored poly(lactide-co-glycolide) (PLGA) systems for targeted gene delivery to human hepatocellular carcinoma (HepG2) cells overexpressing the asialoglycoprotein receptor (ASGPR). Chitosan (CS) functionalised gold (G) PLGA nanoparticles (NPs) were synthesised with and without the targeting ligand, lactobionic acid (LA), and bound to pCMV-Luc-DNA (pDNA) to form nanocomplexes. NPs were physicochemically characterised, and pDNA binding and protection by the NPs were investigated using mobility shift, dye displacement and nuclease digestion assays. MTT and luciferase assays served to evaluate cytotoxicity and transgene expression in vitro. Nanocomplexes of 20–40 nm in size effectively protected the DNA from nuclease digestion and exhibited low cytotoxicity (>90% cell viability). Targeted transgene expression was significantly greater than the untargeted expression, confirming ASPGR uptake. Our results highlight the potential of these hybrid CS-PLGA based nano-platforms for targeted gene therapy, warranting further development, especially for liver-directed gene therapy.

An innovative genosensor for the monitoring of Leishmania spp sequence using binding of pDNA to cDNA based on Cit-AgNPs

Leishmaniasis considered as the most crucial epidemic-prone diseases according to the World Health Organization. Early diagnoses and therapy of Leishmania infection is a great challenge since, it has no symptom and is resistance to drugs. Therefore, there is an urgent need for sensitive and precise detection of this pathogen. In this study, a new method was developed for optical biosensing of Leishmania spp sequence based on hybridization of Citrate capped Ag nanoparticles bonded to specific single stranded DNA probe of Leishmania spp. Aggregation of the Citrate capped Ag nanoparticles in the existence or lack of a cDNA sequence of Leishmania, cause eye catching and considerable significant alter in the UV–vis. The obtained low limit of quantification (LLOQ) of was achieved as 1ZM. Based on experimental results in optimum conditions, quick bioanalysis of Leishmania spp sequence was performed (2 min). So, this probe can be used for the clinical diagnosis of this pathogen and infection disease.

Effects of Decomplexation Rates on Ternary Gene Complex Transfection with α-Poly(l-Lysine) or ε-Poly(l-Lysine) as a Decomplexation Controller in An Easy-To-Transfect Cell or A Hard-To-Transfect Cell.

The tight binding of pDNA with a cationic polymer is the crucial requirement that prevents DNA degradation from undesired DNase attack to safely deliver the pDNA to its target site. However, cationic polymer-mediated strong gene holding limits pDNA dissociation from the gene complex, resulting in a reduction in transfection efficiency. In this study, to control the decomplexation rate of pDNA from the gene complex in a hard-to-transfect cell or an easy-to-transfect cell, either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL) was incorporated into branched polyethylenimine (bPEI)-based nanocomplexes (NCs). Compared to bPEI/pDNA NCs, the addition of APL or EPL formed smaller bPEI-APL/pDNA NCs with similar zeta potentials or larger bPEI-EPL/pDNA NCs with reduced zeta potentials, respectively, due to the different characteristics of the primary amines in the two poly(l-lysine)s (PLs). Interestingly, although both bPEI-APL/pDNA NCs and bPEI-EPL/pDNA NCs showed similar pDNA compactness to bPEI/pDNA NCs, the addition of APL or EPL resulted in slower or faster pDNA release, respectively, from the bPEI-PL/pDNA NCs than from the bPEI/pDNA NCs. bPEI-EPL/pDNA NCs with a decomplexation enhancer (i.e., EPL) improved the transfection efficiency (TE) in both a hard-to-transfect HepG2 cell and an easy-to-transfect HEK293 cell. However, although a decomplexation inhibitor (i.e., APL) reduced the TE of bPEI-APL/pDNA NCs in both cells, the degree of reduction in the TE could be compensated by PL-mediated enhanced nuclear delivery, particularly in HepG2 cells but not HEK293 cells, because both PLs facilitate nuclear localization of the gene complex per its cellular uptake. In conclusion, a decomplexation rate controller could be a potential factor to establish a high TE and design clinically available gene complex systems.

A promising approach to develop nanostructured lipid carriers from solid lipid nanoparticles: preparation, characterization, cytotoxicity and nucleic acid binding ability

We aimed to develop nanostructured lipid carriers (NLCs) displaying similar characteristics – particle size, polydispersity index, and zeta potential – with the model solid lipid nanoparticles (SLNs) for better comparability. By considering the hydrophilic-lipophilic balance values of solid and liquid lipids, five out of six NLCs and eight out of eight cationic NLCs (cNLCs) were successfully prepared with similar characteristics to their precursor SLN and cationic SLNs (cSLNs), respectively. Among cationic formulations, two cSLNs containing different surfactant/co-surfactant concentrations (4% and 8% S/CoS; w/w) and their cNLC versions prepared with Labrafac lipophile WL 1349 (LWL) or Labrafac PG were selected to compare cytotoxicity, stability, and nucleic acid binding ability. All formulations are well-tolerated by L-929 cells, cSLNs being least toxic. The formulations containing 4% S/CoS had higher stability after 24-months. All nanoparticles formed complexes with pDNA (Binding ability: cNLCs > cSLNs). cSLN and LWL-cNLC containing 4% S/CoS showed the highest pDNA binding capacity in each group, and their spherical/oval shape was revealed by electron microscopy. However, they did not form complexes with siRNA. The developed approach has the potential to simplify the production of (c)NLCs having similar physicochemical properties with the optimum (c)SLN and may provide better insight for (c)SLN vs. (c)NLC comparison studies.

Binding of pDNA with cDNA using hybridization strategy towards monitoring of Haemophilus influenza genome in human plasma samples

Haemophilus Influenza leads to respiratory infections such as sinusitis, acute otitis media, pneumonia and bronchitis. In addition, it causes invasive infections such as cellulite, septic arthritis, and meningitis. Therefore, quick and sensitive detection of H. influenza is of great importance in medical microbiology. In this study, a novel DNA-based bioassay was developed to the monitoring of Haemophilus influenza genome in human plasma samples using binding of pDNA with cDNA. DNA hybridization strategy was used to investigation of DNAs binding. For this purpose, silver nanoparticle doped graphene quantum dots inks functionalized by D-penicillamine (Ag NPs-DPA-GQDs) were synthesized and deposited on the surface of glass carbon electrode (GCE). Also, gold nanoparticles functionalized with cysteamine (CysA-AuNPs) were deposited on the surface of the Ag-DPA-GQDs modified GCE. Afterward, thiolated DNA probe was immobilized on the surface of the modified electrode. DNA hybridization was monitored using square wave voltammetry (SWV) technique. Engineered genosensor indicated good performance with high specificity and sensitivity for detection of Haemophilus influenza genome. Under optimal conditions, linear range and low limit of quantitation (LLOQ) were obtained as target concentrations ranging from 1 pM–1 ZM and 1 ZM, respectively. The designed biosensor also showed high capability of discriminating one-base, two-base and three-base mismatched sequences. Also, the prepared genosensor could be easily regenerated and reused to evaluate hybridization process.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

Folic acid directly modified low molecular weight of polyethyleneimine for targeted pDNA delivery

Gene therapy for cancer treatment has been an attractive option in the last 2-3 decades; however, the development of efficient gene delivery systems is still underway. Low molecular weight of polyethyleneimines (LMW PEIs), especially 1.8K PEI, have received much attention due to their low toxicity. Hydrophobic modification is a versatile method to address the poor transfection efficiency of LMW PEIs. In this study, a series of mole ratios of folic acid were grafted to 1.8K PEI for hydrophobic modification and targeting purposes. All PEI-FAX conjugates were characterized by UV absorption, 1H nuclear magnetic resonance spectroscopy (1HNMR), gel electrophoresis, dynamic light scattering (DLS), zeta potential, luminometry, flow cytometry and confocal microscopy. As expected, all PEI-FAX conjugates maintained the low toxicity of 1.8K PEI. With the optimal FA substitution, PEI-FA0.65 conjugate achieved the highest transfection efficiency on several cell lines, nearly 100-fold higher than that of the 1.8K PEI, which could be attributed to the better pDNA binding ability and favourable pH buffering capacity. Furthermore, folate receptor-mediated specific cell uptake of the PEI-FA0.65/pDNA polyplex also played an important role in its high transfection efficiency. With low cytotoxicity and high transfection efficiency, PEI-FA0.65 holds potential for in vivo study.

Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production.

The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.

Controlling complexation/decomplexation and sizes of polymer-based electrostatic pDNA polyplexes is one of the key factors in effective transfection

The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers.

Improving Magnetofection of Magnetic Polyethylenimine Nanoparticles into MG-63 Osteoblasts Using a Novel Uniform Magnetic Field

This study aimed to improve the magnetofection of MG-63 osteoblasts by integrating the use of a novel uniform magnetic field with low molecular weight polyethylenimine modified superparamagnetic iron oxide nanoparticles (PEI-SPIO-NPs). The excellent characteristics of PEI-SPIO-NPs such as size, zeta potential, the pDNA binding and protective ability were determined to be suitable for gene delivery. The novel uniform magnetic field enabled polyethylenimine-modified superparamagnetic iron oxide nanoparticles/pDNA complexes (PEI-SPIO-NPs/pDNA complexes) to rapidly and uniformly distribute on the surface of MG-63 cells, averting local transfection and decreasing disruption of the membrane caused by the centralization of positively charged PEI-SPIO-NPs, thereby increasing the effective coverage of magnetic gene carriers during transfection, and improving magnetofection efficiency. This innovative uniform magnetic field can be used to determine the optimal amount between PEI-SPIO-NPs and pDNA, as well as screen for the optimal formulation design of magnetic gene carrier under the homogenous conditions. Most importantly, the novel uniform magnetic field facilitates the transfection of PEI-SPIO-NPs/pDNA into osteoblasts, thereby providing a novel approach for the targeted delivery of therapeutic genes to osteosarcoma tissues as well as a reference for the treatment of other tumors.