Cellular Transfection Research Articles

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.

High-Throughput Methods for Screening Polymeric Transfection Reagents

The clinical success of gene therapy requires the development of a safe and efficient delivery system for DNA. Cationic polymers are nonviral vectors that can associate electrostatically with plasmid DNA to form nanocomplexes. In some cases, this is sufficient for cellular uptake and transfection, although the precise mechanisms by which polymers facilitate gene delivery remain unclear. A robust and reliable method to screen for efficacy is essential for the development of effective polycationic transfection reagents. Numerous parameters must be controlled and optimized, such as polymer structure, polymer-DNA-binding conditions (mixing method, pH, ionic strength, incubation time, concentrations, ratios), transfection media (type, serum content), DNA dose and incubation time with the cells, cell specificity, and assay conditions. In this protocol, we describe a high-throughput method for assessing polymer-mediated transfection. The technique uses 96-well plates, which allows many transfection parameters to be varied and optimized in parallel. Hundreds of polymers can be tested in quadruplicate in a single day and the technique can easily be automated to efficiently and reproducibly test large material libraries. One limitation is that many plate types, solutions, and equipment must be stocked and sterilized. Moreover, because all polymers are processed simultaneously in very small volumes, it is difficult to validate each step for each polymer to ensure solution uniformity and adequate polymer-DNA complexation. Despite these drawbacks, this high-throughput screening method has already been used successfully in the development of efficient polycation vectors.

Enhanced hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations

Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid, into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, LNPs containing OA delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, LNPs containing OA showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. LNPs containing OA is a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases.

Optimization of Tet1 ligand density in HPMA-co-oligolysine copolymers for targeted neuronal gene delivery

Targeted gene delivery vectors can enhance cellular specificity and transfection efficiency. We demonstrated previously that conjugation of Tet1, a peptide that binds to the GT1b ganglioside, to polyethylenimine results in preferential transfection of neural progenitor cells in vivo. In this work, we investigate the effect of Tet1 ligand density on gene delivery to neuron-like, differentiated PC-12 cells. A series of statistical, cationic peptide-based polymers containing various amounts (1–5 mol%) of Tet1 were synthesized via one-pot reversible addition-fragmentation chain transfer (RAFT) polymerization by copolymerization of Tet1 and oligo-l-lysine macromonomers with N-(2-hydroxypropyl)methacrylamide (HPMA). When complexed with plasmid DNA, the resulting panel of Tet1-functionalized polymers formed particles with similar particle size as particles formed with untargeted HPMA–oligolysine copolymers. The highest cellular uptake in neuron-like differentiated PC-12 cells was observed using polymers with intermediate Tet1 peptide incorporation. Compared to untargeted polymers, polymers with optimal incorporation of Tet1 increased gene delivery to neuron-like PC-12 cells by over an order of magnitude but had no effect compared to control polymers in transfecting NIH/3T3 control cells.

An approach to transgene expression in liver endothelial cells using a liposome-based gene vector coated with hyaluronic acid

Dysfunctional sinusoidal liver endothelial cells (LECs) are associated with liver diseases, such as liver fibrosis, cirrhosis, and portal hypertension. Because of this, gene therapy targeted to LECs would be a useful and productive strategy for directly treating these diseases at the level of genes. Here, we report on the development of a transgene vector that specifically targets LECs. The vector is a liposome-based gene vector coated with hyaluronic acid (HA). HA is a natural ligand for LECs and confers desirable properties on particles, rendering them biodegradable, biocompatible, and nonimmunogenic. In this study, we constructed HA-modified carriers, and evaluated cellular uptake and transfection activity using cultured LECs from KSN nude mice (KSN–LECs). Cellular uptake analyses showed that KSN–LECs recognized the HA-modified carriers more effectively than skin endothelial cells. The transfection assay indicated that the efficient gene expression in KSN–LECs, using the HA-modified carriers, required an adequate lipid composition and a functional device to control intracellular trafficking. This finding contributes to our overall knowledge of transgene expression targeted to LECs.

Enhanced Cellular Transfection by Ternary Non-Viral Gene Vectors Coupled with Adeno-Associated Virus-Derived Peptides

The establishment of efficient and safe gene delivery systems is crucial for biomedical applications. To address this objective, novel, ternary hybrid gene vectors are designed with viral capsid peptides in non-viral gene carriers. The viral peptide, TQVGQKT, is coupled with a membrane active peptide, LK15. Which acts as a linker to tag peptide with plasmid DNA. Additionally, polyethylenimine (PEI) is employed to condense the complexes further, thereby forming ternary DNA/TQVGQKT-LK15/PEI complexes. The ternary complexes result in rapid internalization leading to significantly enhanced cellular transfection. The new moiety, TQVGQKT, as well as enhanced cellular transfection, will certainly provide crucial insights for the design of novel non-viral gene carriers with efficient and safe properties.

Brain-targeting delivery for RNAi neuroprotection against cerebral ischemia reperfusion injury

Nanoparticles (NPs) with modification of brain-targeting molecules have been extensively exploited for therapeutic gene delivery across the blood–brain barrier (BBB). As one of the effective RNA interference (RNAi) approaches, short hairpin RNA (shRNA) has been proved to be promising in the field of gene therapy. Apoptosis signal-regulating kinase 1 (Ask1) has been reported to be an important target for gene therapy against cerebral ischemia reperfusion injury. In this study, dendrigraft poly-l-lysine (DGL) was decorated by dermorphin (a μ-opiate receptor agonist) through PEG for efficient brain-targeting, then complexed with anti-Ask1 shRNA plasmid DNA, yielding the DGL-PEG-dermorphin/shRNA NPs. The DGL-PEG-dermorphin/shRNA NPs were characterized and estimated the brain-targeting ability. In vitro, increased cellular uptake and transfection efficiency were explored; in vivo, preferable accumulation and gene transfection in brain were showed in images. The DGL-PEG-dermorphin/shRNA NPs also revealed high efficiency of neuroprotection. As a result of RNAi, corresponding mRNA was distinctly degraded, expression of Ask1 protein was obviously suppressed, apoptotic cell death was apparently decreased and cerebral infarct area was significantly reduced. Above all, DGL-PEG-dermorphin/shRNA NPs were proved to be efficient and safe for brain-targeting RNAi neuroprotection against cerebral ischemia reperfusion injury.

Combinatorial Synthesis and High-Throughput Screening of Alkyl Amines for Nonviral Gene Delivery

Efficient delivery of plasmid DNA and siRNA into cells is essential for biological and biomedical research. Although significant efforts have been made to develop efficient nonviral vectors, such as cationic lipids and polymers, most of the vectors require multistep synthesis, which complicates both fast structural optimizations and combinatorial synthesis of such vectors. Here, we present a facile, single-step method based on an alkylation of amines, allowing for the fast parallel synthesis of libraries of cationic lipid-like molecules (lipidoids). We exploited the method to synthesize 200 lipidoids, which were screened for their transfection efficiency in HEK293T cells. The screen resulted in about 2% of new lipidoids capable of efficient cell transfection similar or higher than the efficiency of Lipofectamine 2000. In addition, we observed an enhancement of cellular transfection by combining single- with double-chain lipidoids, which was attributed to the different roles of the single- and double-tailed lipids in the mixed liposomes.

Integration of Adeno-Associated Virus-Derived Peptides into Nonviral Vectors to Synergistically Enhance Cellular Transfection

This study describes a simple, versatile approach for developing a nonviral gene carrier by adopting the highly efficient gene delivery properties of the adeno-associated virus (AAV). Specific viral peptides (r3.45_hepBD) extracted from AAV r3.45, which directly evolved to improve gene delivery capabilities in many cell types, were conjugated onto branched polyethylenimine (PEI) to form hybrid gene carriers. AAV r3.45 carries a sequence insertion (LATQVGQKTA; r3.45) within the heparin-binding domain (LQRGNRQA; hepBD), which ultimately comprises a novel sequence (LQRGNLATQVGQKTARQA; r3.45_hepBD) on the capsid. This sequence is hypothesized to be a crucial cue to enhance gene delivery efficiency. Consequently, the intimate interactions of the conjugated r3.45_hepBD with the glycosaminoglycans, including chondroitin sulfate, resulted in significantly enhanced cellular transfection of DNA/PEI-r3.45_hepBD complexes. The successful establishment of a nonviral system that is built with novel peptides will provide a powerful means for developing a substantial number of gene therapy applications.

Enhanced Transfection Efficiency and Reduced Cytotoxicity of Novel Lipid–Polymer Hybrid Nanoplexes

The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency) a system was hypothesized in which DOTAP/phosphatidyl choline (PC) lipoplexes were electrostatically coated with anionic poly(acrylic acid) (PAA) and cationic polyethylenimine (PEI) alternatively to finally shape a robust structure PEI-PAA-DOTAP/PC-lipoplexes (nanoplexes). The nanoplexes were found to have size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine.

Supramolecular pseudo-block gene carriers based on bioreducible star polycations

A series of supramolecular pseudo-block polycations (CD-SS-pDM/Ad-pPEGs) were realized by assembling bioreducible β-cyclodextrin-cored star poly (2-dimethyl amino)ethyl methacrylate with different molecular weight and an adamantine-ended linear poly(poly(ethylene glycol)ethyl ether methacrylate) (pPEGEEMA) via the host–guest interaction. The pseudo-block CD-SS-pDM/Ad-pPEG carriers were investigated in terms of DNA binding capability, cytotoxicity, gene transfection in HepG2 and COS7 cell lines, and in vivo anti-tumor activity. The pseudo-block carriers exhibited undiminished pDNA-condensing abilities compared with the starting star carriers. Meanwhile, the pseudo-block carriers displayed lower cytotoxicity and higher gene transfection efficiencies at various N/P ratios. These results are consistent with the favorable properties of pPEGEEMA as expected. Furthermore, cellular internalization results and in vivo anti-tumor activity analysis demonstrated that assembled pPEGEEMA could enhance the stability of pseudo-block carriers, thus improving their cellular internalization and gene transfection efficiency. The present study demonstrated that supramolecular pseudo-block polycations via the host–guest interaction is an effective means to produce new gene carriers.

Efficient Cell Transfection with Melamine-Based Gemini Surfactants

Gemini surfactants consisting of two melamine scaffolds connected by a n-hexyl linker and functionalized with a 1-propylammonium polar head and a lipophilic chain having variable carbon length (from C8 to C16) were synthesized. These were then used successfully for the transfection of A549, U87 MG, and Bristol 8 cell lines with maxGFP expressing plasmid. The transfection protocol was optimized appropriately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions, the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required), improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA transfection.

The effect of molar mass and degree of hydroxyethylation on the controlled shielding and deshielding of hydroxyethyl starch-coated polyplexes

PEGylation is currently the gold-standard in shielding cationic DNA-polyplexes against non-specific interaction with blood components. However, it reduces cellular uptake and transfection, in what is known as the “PEG-dilemma”. In an approach to solve this problem we developed hydroxyethyl starch (HES)-shielded polyplexes which get deshielded under the action of alpha amylase (AA). In this study, the effect of molar mass and degree of hydroxyethylation on the shielding and deshielding of the polyplexes as well as their in vivo performance were investigated. For this purpose, a battery of HES–polyethylenimine (PEI) conjugates was synthesized, and their rate and extent of biodegradation were investigated using asymmetric flow-field flow fractionation (AF4) and quartz-crystal microbalance with dissipation (QCM-D). Additionally, the transfection efficiency of the polyplexes was tested in Neuro2A cells and tumor-bearing mice. AF4 and QCM results show a rapid degradation for HES with lower degrees of hydroxyethylation. Meanwhile, in vitro transfection experiments showed a better shielding for higher HES molar masses, as well as deshielding with a significant boost in transfection upon addition of AA. Finally, in vivo experiments showed that the biodegradable HES markedly reduced the non-specific lung transcription of the polyplexes, but maintained gene expression in the tumor, contrary to the non-degradable HES and PEG controls, which reduced both tumor and lung expression. This study shows that by controlling the molecular characteristics of HES it is possible to engineer the shielding and deshielding properties of the polyplexes for more efficient gene delivery.

Cellular uptake and transfection activity of DNA complexes based on poly(ethylene glycol)-poly(l-glutamine) copolymer with PAMAM G2.

Poly(ethylene glycol)-poly(l-glutamine) (PEG-PLGA) copolymer EA-G2 (or EA-G1) was prepared by aminolysis of poly(ethylene glycol)-poly(l-benzyl glutamate) (PEG-PBLG) using PAMAM G2 (or G1). The chemical structure of PEG-PLGA was confirmed by FT-IR, 1H-NMR, DSC and GPC. The performances of the EA-G2 (or EA-G1) were assayed by enzyme degradation, MTT method and agarose gel electrophoresis. The particle size, zeta potential and morphology of EA-G2 (or EA-G1)/pDNA complexes were inspected by DLS and AFM. The cellular uptake mechanism was evaluated by endocytic inhibiting test, cell uptake test and observation of CLSM. The transfection activity was measured by flow cytometry. The EA-G2 (or EA-G1) exhibited good biodegradability, low cytotoxicity and great ability to combine with pDNA. The EA-G2 (or EA-G1) complexes exhibited particle sizes in the range 120-180 nm and zeta potentials in the range 20-40 mV, which were suitable for cell uptake. The cellular uptake of the EA-G2 complexes occurred mainly through clathrin-dependent and caveolin-mediated endocytosis, and at 6 h in 10% FBS and in serum-free media, the percentages of complex uptake reached 89.0% or 72.7%, respectively. EA-G2 complexes could efficiently mediate pEGFP-Cl into the cell nuclei. EA-G2 complexes displayed enhancing transfection efficiency and better serum tolerance. The results suggest that the EA-G2 has potential to be used as a biodegradable, efficient and serum-resistant gene vector.

Synthesis of NBD-Labeled DOTAP Analog to Track Intracellular Delivery of Liposome

A DOTAP analog labeled by NBD on the head group (DTNBD) was designed and synthesized to label DOTAP liposome. The structure was confirmed by (1)H NMR and FAB-MS, and the fluorescence of the newly synthesized DT-NBD was observed by fluorescent microscopy. The transfection efficiency of DOTAP liposome containing DT-NBD was comparable to commonly used NBD PE in COS7 and MCF7 cells. Furthermore, the level of cellular uptake and fluorescent intensity of fluorescent liposome containing DT-NBD was higher than NBD PE. Therefore, the novel NBD-labeled DOTAP analog seems to be effectively used for investigation of the cellular interaction and transfection mechanism of DOTAP liposome.

Design and Development of Degradable Polyethylenimines for Delivery of DNA and Small Interfering RNA: An Updated Review

Polyethylenimine (PEI), considered as the most potent and promising alternative carrier to viral vectors, has been studied as the “state of the art” among various polymers for nonviral gene delivery applications for many years. Although PEI-based carrier minimizes the bottlenecks associated with viral vectors such as unwanted immunogenicity and production problems, the toxic side effects of PEI prevent its rapid advancements due to nondegradable nature. In this regard, various degradable cross-linking and/or grafting agents have been linked to synthesize degradable PEIs in order to minimize the toxicity and improve the efficacy of PEI-mediated gene carriers. This paper describes an update on various cross-linkers and grafting agents in the design and development of degradable PEI derivatives and their potential applications for effective delivery of DNAin vitroandin vivo. The molecular weight (MW) of PEI and the structural relationship to its cellular toxicity and transfection ability were also discussed. Finally, the potential applications of various degradable PEIs for small interfering RNA (siRNA)-mediated gene silencing were also covered.

Bioreducible poly(amido amine)s with different branching degrees as gene delivery vectors

Based on the knowledge that cationic polymers with different topographical structures behave differently in gene transfection process, herein, we synthesized three biodegradable poly(amido amine)s (PAAs) with the same repeating units and molecular weights except for degree of branching: linear PAA (LPAA), low-branched PAA (LBPAA), and high-branched PAA (HBPAA). We found that LBPAA could more effectively compact pDNA into positively charged nanoparticles than both HBPAA and LPAA. LBPAA polyplexes had the highest transfection efficiency among the three PAA polyplexes, and the difference in transfection efficiency is mainly attributed to the endocytosis rate. The cytotoxicity of PAAs was negligible at the transfection doses, probably due to the degradable disulfide bonds. Therefore, we could use branching as a parameter to simply tune a polymer's cellular uptake behavior and transfection efficiency.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer–pDNA transfection

BackgroundInteraction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. MethodsThe physicochemical properties of the PAMAM–pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. ResultsPhysicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. ConclusionPresented data shows that the rate-limiting step of PAMAM–pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.

Surface modified poly(β amino ester)-containing nanoparticles for plasmid DNA delivery

The use of biodegradable polymers provides a potentially safe and effective alternative to viral and liposomal vectors for the delivery of plasmid DNA to cells for gene therapy applications. In this work we describe the formulation of a novel nanoparticle (NP) system containing a blend of poly(lactic-co-glycolic acid) and a representative poly(beta-amino) ester (PLGA and PBAE respectively) for use as gene delivery vehicles. Particles of different weight/weight (wt/wt) ratios of the two polymers were characterized for size, morphology, plasmid DNA (pDNA) loading and surface charge. NPs containing PBAE were more effective at cellular internalization and transfection (COS-7 and CFBE41o−) than NPs lacking the PBAE polymer. However, along with these delivery benefits, PBAE exhibited cytotoxic effects that presented an engineering challenge. Surface coating of these blended particles with the cell-penetrating peptides (CPPs) mTAT, bPrPp and MPG via a PEGylated phospholipid linker (DSPE-PEG2000) resulted in NPs that reduced surface charge and cellular toxicity to levels comparable with NPs formulated with only PLGA. Additionally, these coated nanoparticles showed an improvement in pDNA loading, intracellular uptake and transfection efficiency, when compared to NPs lacking the surface coating. Although all particles with a CPP coating outperformed unmodified NPs, respectively, bPrPp and MPG coating resulted in 3 and 4.5× more pDNA loading than unmodified particles and approximately an order of magnitude improvement on transfection efficiency in CFBE41o− cells. These results demonstrate that surface-modified PBAE containing NPs are a highly effective and minimally toxic platform for pDNA delivery.

Antibody-Linked Spherical Nucleic Acids for Cellular Targeting

Spherical nucleic acid (SNA) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through noncovalent functionalization with a complementary antibody-DNA conjugate. As a proof-of-concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.