Increased Transfection Efficiency Research Articles

Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency.

We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.

Synergistic effect of cationic lipids with different polarheads, central core structures and hydrophobic tails on gene transfection efficiency.

Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. The primary approach to improving transfection efficiency is the design and synthesis of novel cationic lipids. Alternatively, using the synergistic effect of different cationic mixtures can provide another approach to increasing transfection efficiency. This paper describes the synergistic effect of lipids with different polarheads, central core structures and hydrophobic tails. The enhancement of cellular transfection into HEK293 cells was observed by combining two lipids having aminoglycerol and di(hydroxylethyl)amino core structures at a 1 : 1 weight ratio. Additionally, the liposome formation of these lipids with the helper lipid, 1,2-dioleoyl-propyl-3-phosphatidylethanolamine (DOPE), at the weight ratio of 1 : 1 can provide higher transfection efficiency into HEK293, MCF-7 and HeLa cells than Lipofectamine™ 2000. Our finding indicated that cationic liposomes comprised of a mixture of lipids with different polarheads, central core structures and hydrophobic tails should be very promising in liposome-mediated gene delivery in vitro and in vivo.

MRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Polyelectrolyte Multilayer Coating of 3D Scaffolds Enhances Tissue Growth and Gene Delivery: Non‐Invasive and Label‐Free Assessment

Layer-by-layer (LbL) deposition is a versatile technique which is beginning to be be explored for inductive tissue engineering applications. Here, it is demonstrated that a polyelectrolyte multilayer film system composed of glycol-chitosan (Glyc-CHI) and hyaluronic acid (HA) can be used to coat 3D micro-fabricated polymeric tissue engineering scaffolds. In order to overcome many of the limitations associated with conventional techniques for assessing cell growth and viability within 3D scaffolds, two novel, real-time, label-free techniques are introduced: impedance monitoring and optical coherence phase microscopy. Using these methods, it is shown that LbL-coated scaffolds support in vitro cell growth and viability for a period of at least two weeks at levels higher than uncoated controls. These polyelectrolyte multilayer coatings are then further adapted for non-viral gene delivery applications via incorporation of DNA carrier lipoplexes. Scaffold-based delivery of the enhanced green fluorescent protein (EGFP) marker gene from these coatings is successfully demonstrated in vitro, achieving a two-fold increase in transfection efficiency compared with control scaffolds. These results show the great potential of Glyc-CHI/HA polyelectrolyte multilayer films for a variety of gene delivery and inductive tissue engineering applications.

Ultrasound microbubbles combined with the NFκB binding motif increase transfection efficiency by enhancing the cytoplasmic and nuclear import of plasmid DNA

Inefficient gene delivery poses a challenge for non‑viral gene therapy. Cytoplasmic and nuclear membrane barriers are responsible for the inefficiency as they restrict the import of exogenous genes. The present study aimed to improve the transfection efficiency using a novel gene delivery system, which consisted of two components: ultrasound microbubbles and the nuclear factor κB (NFκB) binding motif. Ultrasound-targeted microbubble destruction (UTMD) was used to enhance the cytoplasmic import of plasmids and the NFκB binding motif was added to promote the nuclear intake of the plasmid from the cytoplasm. In the present study, human umbilical vein endothelial cells were transfected using UTMD with two different Cy3-labeled plasmids, phSDF-1α and phSDF‑1α‑NFκB. phSDF-1α-NFκB was constructed by inserting a specific DNA targeting sequence (five optimal repeats of the binding motif for the inducible transcription factor NFκB) into phSDF‑1α. The nuclear import and gene expression efficiency of phSDF-1α-NFκB were compared with those of phSDF-1α to investigate the effect of the NFκB binding motif on transfection. The results showed that UTMD significantly increased the cytoplasmic intake of pDNA and maintained high cell viability. The nuclear import and gene expression of phSDF-1α‑NFκB‑transfected cells were significantly higher than those transfected with phSDF-1α. Compared with the NFκB‑free plasmids, the quantity of NFκB plasmids in the nucleus increased 6.5-fold and the expression of SDF-1α was 4.4-fold greater. These results suggest that UTMD combined with NFκB binding motif significantly improve transfection efficiency by enhancing the cytoplasmic and nuclear import of exogenous plasmid DNA.

Improvement of delivery of molecules into corneal endothelium using nanoparticles activated by femtosecond laser pulses

AbstractPurpose NanoFemtoTransfection (NFT) is an innovative and promising technique of gene transfer (Nature nanotechnology 2011). It consists in permeabilizing cell membrane with nanoparticles (NP) of black carbon activated by Ti‐Sapphire femtosecond laser (FsL) in order to safely introduce molecule of interest. Initially described for cell suspension, we adapted during last year NFT to human corneal endothelial cells (EC) Aim: to further optimize NFT of EC in vitroMethods The model of NFT of calcein (a small molecule) in an EC line (B4G12, DMSZ, Germany) was used to optimize exposition times, fluence and Ti‐Saphir FsL beam movements in order to determine the best efficiency/toxicity ratio. Calcein transfer was observed with fluorescent microscopy (IX81, Olympus) and quantified by flow cytometry (FACSCalibur, BD, CA). Viability of EC was assessed by propidium iodide staining. Staining of actine with phalloidin and immunostaining of ZO‐1 was performed to study cell alteration caused by NFTResults With a static laser beam increasing FsL fluence and exposure time increased transfection efficiency but also EC mortality. The use of mobile FsL beam (with a XY micrometric stage, cheaper than a scanner), permitted an irradiation of the entire area of EC culture resulting in a noticeably increased efficiency that reached 38% of transfected EC and a limited toxicity (3%). Calcein positive EC presented the same phalloidin and ZO‐1 patterns as control ECConclusion Optimized NFT parameters are currently being tested in our laboratory to transfect plasmids. Grants: Fondation pour la Recherche Médicale, Fondation des Aveugles de France

Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery

Functionalized carbon nanotubes (CNTs) have been recently emerged as important class of vectors for delivery of DNA and other biomolecules into various cells. In this study, single-walled carbon nanotubes (SWNTs) were functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to polyethyleneimines (PEIs). PEIs of three molecular weights (25, 10 and 1.8kDa) were used. CNTs were functionalized with the PEI series either through phospholipid moiety (via a polyethyleneglycol linker) or through directly-attached long (18 carbons) or intermediate (10 carbons) hydrophobic alkyl moieties. All PEI-functionalized CNTs exhibited good stability and dispersibility in biological media. Visualizing of functionalized CNTs and lack of aggregation were confirmed by atomic force microscopy. The PEI derivatives bound to CNTs retained the ability to fully condense plasmid DNA at low N/P ratios and substantial buffering capacity in the endosomal pH range. PEI-functionalized CNTs exhibited increased transfection efficiency compared to underivatized PEIs up to 19-fold increase being observed in the functionalized CNT with the smallest PEI tested, the smallest hydrophobic attachment moiety tested and no linker. Also PEI-functionalized CNTs were effective gene delivery vectors in vivo following tail vein injection in mice with the largest expression occurring with the vector PEI-functionalized through a polyethyleneglycol linker.

Stable transformation of pleomorphic bloodstream form Trypanosoma brucei

African trypanosomes differentiate between various developmental stages both in mammalian hosts and their tsetse vector to adapt to and survive in the different environments they encounter. In the bloodstream, trypanosomes naturally exist as either proliferative slender-forms or non-proliferative stumpy-forms, the latter being responsible for both prolonged infection and transmission. However, most trypanosome studies are carried out on laboratory-adapted monomorphic cell lines, incapable of differentiating to stumpy-forms or completing the life cycle through the tsetse fly. Partly, this has been due to the inefficiency of transfection of pleomorphic strains which have retained the ability to generate stumpy-forms. Recently, Amaxa Nucleofector® technology was shown to increase transfection efficiency for monomorphic bloodstream forms. Using this technology we have optimised a similar method for pleomorphic bloodstream form transfection, generating transfection efficiencies of 10−7–10−6. This permits routine genetic manipulation of pleomorphic lines, which have the most biological relevance for trypanosomes in the field.

Use of an anti‐apoptotic CHO cell line for transient gene expression

Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti-apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro-apoptotic factors, Bax and Bak, in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO-K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ∼3- to 4-fold higher antibody titers than CHO-K1 cells. As evidence of their anti-apoptotic properties post-transfection, DKO cells maintained higher viability and had reduced levels of active caspase-3 compared to CHO-K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post-transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae- and clathrin-mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection-induced apoptosis and not differences in endocytosis mechanism.

Targeted gene delivery with noncovalent electrostatic conjugates of sgc‐8c aptamer and polyethylenimine

Several strategies have been shown to improve the transfection efficiency of polyethylenimine (PEI) as a nonviral gene delivery vector. In the present study, a nucleic acid aptamer specific for protein tyrosine kinase 7 (PTK7) surface marker, sgc-8c, was conjugated electrostatically to pre-formed 10-kDa PEI/plasmid DNA polyplexes, and the ability of the conjugate to transfer genetic material was evaluated in MOLT-4 human acute lymphoblastic leukemia T-cells, which express PTK7 on their surface. Polyplexes (plasmid DNA-vector conjugates), prepared using PEI-sgc-8c conjugate and pCMVLuc as a reporter gene, were characterized in terms of particle size, surface charge and the extent of DNA condensation. Polyplexes were also evaluated for cytotoxicity using the MTS colorimetric assay, as well as for transfection efficiency in MOLT-4 cells, and compared with the results obtained in U266 cells, which lack cell surface PTK7. Relative to pDNA/PEI, the size of pDNA/PEI/sgc-8c aptamer polyplexes increased with decreasing zeta potential. In MOLT-4 cells, pDNA/PEI/sgc-8c aptamer polyplexes exhibited an almost six- to eight-fold increase in transfection efficiency compared to that of pDNA/PEI polyplex, indicating that conjugation of sgc-8c aptamer to pre-formed 10-kDa PEI/plasmid DNA polyplexes achieved effective targeting without covalent attachment, whereas receptor-mediated conducted transfection was confirmed by performing a competitive transfection experiment and a cellular uptake study. The results of the present study provide an example of the usefulness of a nucleic acid aptamer in the form of noncovalent, electrostatic conjugates as an approach for enhancing the transfection efficiency of a polycation vector such as PEI without significant induced cytotoxicity.

Influence of Histidine Incorporation on Buffer Capacity and Gene Transfection Efficiency of HPMA-co-oligolysine Brush Polymers

One of the major intracellular barriers to nonviral gene delivery is efficient endosomal escape. The incorporation of histidine residues into polymeric constructs has been found to increase endosomal escape via the proton sponge effect. Statistical and diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), oligolysine, and oligohistidine were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization and tested for in vitro transfection efficiency, buffering ability, and polyplex uptake mechanism via the use of chemical endocytic inhibitors. Interestingly, histidine-containing statistical and diblock polymers exhibited increased buffer capacity in different endosomal pH ranges. Statistical copolymers transfected better than block copolymers that contained similar amounts of histidine. In addition, only the polymer containing the highest incorporation of oligohistidine residues led to increases in transfection efficiency over the HPMA-oligolysine base polymer. Thus, for these polymer architectures, high histidine incorporation may be required for efficient endosomal escape. Furthermore, inhibitor studies indicate that nonacidified caveolae-mediated endocytosis may be the primary route of transfection for these copolymers, suggesting that alternative approaches for increasing endosomal escape may be beneficial for enhancing transfection efficiency with these HPMA-oligolysine copolymers.

161. Co-Delivery of siRNA and pDNA for Increased Transfection Efficiency

161. Co-Delivery of siRNA and pDNA for Increased Transfection Efficiency

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine™. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.

Evaluating Gene Delivery Using N-triethyl Chitosan Nanoparticles to AsPC-1 Cells and Predicting the Relation between Transfection, Cytotoxicity and Charge Ratio of Nanoparticles Via Mathematical Models

N-triethyl chitosan (TEC) has recently been proven to be an efficient polymer for improving transport of hydrophilic drugs. Thus, in order to evaluate its gene delivery efficiency, TEC is assembled with Enhanced Green fluorescence protein plasmid DNA (pEGFP) at different polymer to plasmid (N/P) ratios to form TEC/pEGFP nanoparticles. Photon correlation spectroscopy is used to evaluate nanoparticles size and zeta potential. The transfection efficiency and cytotoxicity in gene delivery to AsPC-1 cells (human pancreatic cancer cells) are evaluated via fluorescence microscopy, flow cytometry and MTT assay. Also parameters such as cancer cells’ mean fluorescence intensity (MFI) and cell size changes after transfection are assayed. The results indicate that the incorporation of pEGFP in TEC can form charged nanoparticles at N/P ratios from 10 to 40. Increasing the N/P ratio increases transfection efficiency, MFI, cell size and cytotoxicity. At N/P ratio of 10, transfection increased up to 11.12 fold with the least cytotoxicity. Considering that mathematical models can be used to understand and predict consequences associated with nanomedicine, the relation between TEC /pDNA nanoparticles charge ratio, cell transfection and toxicity was evaluated for the first time with interpolation polynomial method. Keywords: N-triethyl chitosan, nanoparticles, AsPC-1 cells, gene delivery, transfection, cytotoxicity, mathematical models.

Novel Nanosomes for Gene Delivery to Plasmodium falciparum-infected Red Blood Cells

Malaria threatens millions of people annually and is a burden to human health and economic development. Unfortunately in terms of disease control, no effective vaccines are available and the efficacy of treatment is limited by drug resistance. Genetic manipulation in Plasmodium falciparum is hampered due to the absence of robust methods for genetic analyses. Electroporation-based transfection methods have allowed the study of gene function in P. falciparum, with low efficiency. A lipid nanoparticle was developed that allowed nuclear targeting of pDNA with increased efficiency in reporter assay, compared to traditional electroporation method. This method has for the first time, facilitated transfection using both circular and linear DNA in P. falciparum thereby serving as an alternative to electroporation with an increase in transfection efficiency. Availability of a robust method for functional genomic studies in these organisms may be a catalyst for discovery of novel targets for developing drugs and vaccines.

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after −1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after −1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at −1 V compared with the same at ±2–3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.

Comparison of Ectopic Gene Expression Methods in Rat Neural Stem Cells

Neural stem cells (NSCs) have the ability to proliferate and differentiate into various types of cells that compose the nervous system. To study functions of genes in stem cell biology, genes or siRNAs need to be transfected. However, it is difficult to transfect ectopic genes into NSCs. Thus to identify the suitable method to achieve high transfection efficiency, we compared lipid transfection, electroporation, nucleofection and retroviral transduction. Among the methods that we tested, we found that nucleofection and retroviral transduction showed significantly increased transfection efficiency. In addition, with retroviral transduction of Ngn2 that is known to induce neurogenesis in various types of cells, we observed facilitated final cell division in rat NSCs. These data suggest that nucleofection and retroviral transduction provide high efficiency of gene delivery system to study functions of genes in rat NSCs.

Low molecular weight oligochitosans for non-viral retinal gene therapy

Ultrapure oligochitosans have recently been evaluated as a promising tool for corneal gene therapy; however, there are no reports regarding the potential use of this polymer in other ocular tissues. We have prepared and characterized at pH 7.1 oligochitosan/pCMS-EGFP polyplexes to evaluate the transfection efficiency in rat retinas after subretinal and intravitreal administration. Polyplexes were characterized in terms of shape, size, surface charge, DNA condensation, and transfection efficiency in HEK-293 and ARPE-19 culture cells. Polyplexes were positively charged, around 10mV, and size oscillated between 256.5±56 and 67.3±0.44nm, depending on the nitrogenous/phosphate ratio. Polyplexes efficiently protected the plasmid against enzymatic digestion. A drastic increase in transfection efficiency was observed when pH slightly decreased from 7.4 to 7.1 in both HEK-293 (from 19.1% to 51.5%) and ARPE-19 (from 2.0% to 36.5%) cells (data normalized to Lipofectamine™ 2000). In rat retinas, subretinal administrations transfected cells mainly in the RPE layer, whereas intravitreal injections transfected cells in the inner nuclear and plexiform layers of the retina and mainly in the ganglion cell layer. This study establishes the base for future treatments of genetic retinal disorders with low molecular weight oligochitosan polyplexes.

The Use of Nanoparticles for Gene Therapy in the Nervous System

Nanoparticles represent an alternative to viral vectors for genetic material transfer to the nervous system. However, to increase transfection efficiency in the central nervous system and to decrease toxicity, the design of nanoparticles needs to be improved to enhance blood-brain barrier crossing and endosomal escape. This paper reviews the strategies used to solve these difficulties and covers the use of various nanoparticles including natural inorganic particles, natural polymers, cationic lipids, polyethylenimine derivatives, dendrimers, and carbon-based nanoparticles. The effectiveness, both in vivo and in vitro, of each method to deliver genetic material to neural tissue is discussed.

The effects of coating pDNA/chitosan complexes with chondroitin sulfate on physicochemical characteristics and cell transfection

pDNA/chitosan complexes have been investigated as promising non-viral vectors for gene delivery. However, an increase in transfection efficiency and enhancement of physicochemical stability are required for their practical use. In this study, chondroitin sulfate (CS) was employed as a coating agent to increase the stability and transfection efficiency of a pDNA/chitosan complex. The pDNA/chitosan/CS ternary complexes formed with six kinds of CSs having different limiting viscosities (0.2–1.6) and sulfation degrees (5.0–7.0%) showed considerable differences in particle size, surface charge, and morphology. Among them, CS having a medium limiting viscosity (0.5–0.6) and a high sulfation degree (6.9%) showed significant enhancements in cell transfection efficiency. Analyses of cellular uptake and intracellular trafficking revealed that increased cellular uptake via macropinocytosis, together with reduced entry into lysosomes, may explain the promotion of transfection efficiency of ternary complexes.