Cationic Gene Carriers Research Articles

Enhancing the in vivo stability of polycation gene carriers by using PEGylated hyaluronic acid as a shielding system

Abstract To increase the in vivo stability of cationic gene carriers and avoid the adverse effects of their positive charge, we synthesized a new shielding material by conjugating low molecular weight polyethylene glycol (PEG) to a hyaluronic acid (HA) core. The HA-PEG conjugate assembled with the positively charged complex, forming a protective layer through electrostatic interactions. DNA/polyetherimide/HA-PEG (DNA/PEI/HA-PEG) nanoparticles had higher stability than both DNA/polyethyleneimine (DNA/PEI) and DNA/PEI/HA complexes. Furthermore, DNA/PEI/HA-PEG nanoparticles also showed a diminished nonspecific response toward serum proteins in vivo. The in vivo transfection efficiency was also enhanced by the low cytotoxicity and the improved stability; therefore, this material might be promising for use in gene delivery applications.

PAMAM-Functionalized Cellulose Nanocrystals with Needle-Like Morphology for Effective Cancer Treatment.

Gene therapy is used to correct or compensate for diseases caused by gene defects and abnormalities. Improving the transfection efficiency and reducing the toxicity of gene carriers are the keys to gene therapy. Similar to a typical cationic gene carrier—polyethylenimine (PEI, 25 kDa)—the polyamidoamine (PAMAM) dendrimer also has a large number of amino groups. These amino groups can be complexed with nucleic acids after protonation under physiological conditions. However, the concentrated positive charge can cause undesirable cytotoxicity. Cellulose nanocrystals (CNCs) have good biocompatibility and unique needle-like morphology, and have been proven to be efficiently taken up by cells. In this article, three-dimensional spherical PMAMA dendrimers are conjugated onto the surface of CNCs to obtain a kind of needle-like cationic carrier (CNC-PAMAM). PAMAM dendrimers act as anchors to bind the plasmid DNAs (pDNA) to the surface of the CNC. The prepared CNC-based carrier showed high transfection efficiency and low toxicity. The CNC-PAMAM can effectively deliver the suicide gene to the tumor site, enabling the suicide gene/prodrug system (cytosine deaminase/5-fluorocytosine (CD/5-FC)) to play an effective anti-tumor role in vivo. This research demonstrates that the functionalization of CNCs with PAMAM dendrimers is an effective method for developing novel gene delivery systems.

Bio-inspired gene carriers with low cytotoxicity constructed via the assembly of dextran nanogels and nano-coacervates.

Aim: To achieve safe and biocompatible gene carriers. Materials & methods: A core/shell-structured hierarchical carrier with an internal peptide/gene coacervate 'core' and a dextran nanogel 'shell' on the surface has been designed. Results: The dextran nanogels shield coacervate (DNSC) can effectively condense genes and release them in reducing environments. The dextran nanogel-based 'shell' can effectively shield the positive charge of the peptide/gene coacervate 'core', thus reducing the side effects of cationic gene carriers. In contrast with the common nonviral gene carriers that had high cytotoxicities, the DNSC showed a high transfection efficiency while maintaining a low cytotoxicity. Conclusion: The DNSC provides an effective environmentally responsive gene carrier with potential applications in the fields of gene therapy and gene carrier development.

Ligand-installed anti-VEGF genomic nanocarriers for effective gene therapy of primary and metastatic tumors

The systemic dosage regimen exhibited low therapeutic efficacy and incurred severe adverse effect, thus, the development of tumor-targeted therapeutics is crucial important for tumor precision therapy. Herein, the active targeted modulation of tumor microenvironments was schemed by developing hyaluronic acid-installed genomic nanocarriers (HA-NPs) for effectively ablation of both primary and metastatic tumors through anti-vascular endothelial growth factor (anti-VEGF) approach. The anti-VEGF genomic payloads were strategically packaged into the well-defined synthetic nanocarriers by layer-by-layer preparation strategy, exhibiting high colloidal stability and much lower cell viability than the cationic gene carriers. Besides, the HA-NPs could specifically and efficiently internalize with cancer cells for efficient intracellular gene delivery, leading to high gene transfection efficacy. Moreover, it further demonstrated efficient extravasation, high accumulation and deep penetration in tumors, which markedly facilitated tumor-targeted expression of anti-VEGF genomic payloads for inhabitation of neo-vasculature, consecutively contributing to potent ablation of solid tumors. In addition, the ligand-installed nanocarriers facilitated systemic treatment of melanoma lung metastasis by the expressed anti-VEGF proteins, which were extensively spread along blood circulation and metastatic niches to diminish the formation of neovasculature for tumorigenesis. Therefore, the proposed anti-VEGF genomic nanocarriers could shed intriguing implication in effectively treatment of primary tumors and metastasis.

Bioreducible Polyspermine-Based Gene Carriers for Efficient siRNA Delivery: Effects of PEG Conjugation on Gene Silencing Efficiency

A polyspermine-derived bioreducible polymer was synthesized as efficient siRNA carriers. Spermine was polymerized using disulfide-bearing cross-linkers, resulting in poly(disulfide spermine) (PDS). PDS was also conjugated with poly(ethylene glycol) (PEG) to tailor siRNA condensation efficiency of the polymer. PEG-conjugated PDS (PEG-PDS) was able to condense siRNA into nano-sized polyplexes at high N/P ratios. siRNA condensation efficiency of PDS was reduced by PEGylation due to its weakened interaction with siRNA caused by its decreased cationic charge density. An ethidium bromide exclusion assay demonstrated that siRNA was more efficiently released from the less stable PEG-PDS polyplexes compared to stable PDS polyplexes. In addition, bioreducible siRNA/PEG-PDS polyplexes were efficiently disassembled in the reductive conditions, leading to facilitated siRNA release into the cytoplasm. The lowered siRNA condensation but efficient cytoplasmic release of siRNA by PEG-PDS induced higher gene silencing efficiency in comparison with PDS. Moreover, PEG-PDS exhibited lower cytotoxicity compared to PDS and non-biodegradable branched polyethylenimine. These results suggest that siRNA delivery efficiency of cationic gene carriers can be improved by tailoring their siRNA complexation through PEG conjugation. This study demonstrates that bioreducible PEG-PDS is a promising candidate for safe and efficient siRNA delivery.

Multifunctional hetero-nanostructures of hydroxyl-rich polycation wrapped cellulose-gold hybrids for combined cancer therapy

The development of new hetero-nanostructures for multifunctional applications in cancer therapy has attracted widespread attention. In this work, we put forward a facile approach to synthesize multifunctional hetero-nanostructures of cellulose nanocrystal (CNC)-gold nanoparticle hybrids wrapped with low-toxic hydroxyl-rich polycations to integrate versatile functions for effective cancer therapy. Biocompatible CNCs with the superior rod-like morphology for high cellular uptake were employed as substrates to flexibly load spherical gold nanoparticles (Au NPs) or gold nanorods (Au NRs) through gold-thiolate bonds, producing hetero-layered nanohybrids of CNC-Au NPs or CNC-Au NRs. Profound hydroxyl-rich cationic gene carrier, CD-PGEA (comprising β-cyclodextrin cores and ethanolamine-functionalized poly(glycidyl methacrylate) arms), was then assembled onto the surface of CNC-Au nanohybrids through host-guest interaction and gold-thiolate bonds, where PEG was employed as the intermediate and spacer. The resultant CNC-Au-PGEA hetero-nanostructures exhibited excellent performances as gene carriers. Furthermore, CNC-Au NR-PGEA comprising Au NRs demonstrated favorable optical absorption properties and were validated for photoacoustic imaging and combined photothermal/gene therapy with considerable antitumor effects. The present work provided a flexible strategy for the construction of new multifunctional hetero-nanostructures with high antitumor efficacy.

Targeted delivery of in situ PCR-amplified Sleeping Beauty transposon genes to cancer cells with lipid-based nanoparticle-like protocells.

A Sleeping Beauty (SB) transposon system is made of a transposon plasmid (containing gene encoding a desired functional or therapeutic protein) and a transposase plasmid (encoding an enzyme capable of cutting and pasting the gene into the host cell genome). It is a kind of natural, nonviral gene delivery vehicle, which can achieve efficient genomic insertion, providing long-term transgenic expression. However, before the SB transposon system could play a role in promoting gene expression, it has to be delivered efficiently first across cell membrane and then into cell nuclei. Towards this end, we used a nanoparticle-like lipid-based protocell, a closed bilayer of the neutral lipids with the DNA encapsulated inside, to deliver the SB transposon system to cancer cells. The SB transposon system was amplified in situ inside the protocells by a polymerase chain reaction (PCR) process, realizing more efficient loading and delivery of the target gene. To reach a high transfection efficiency, we introduced two targeting moieties, folic acid (FA) as a cancer cell-targeting motif and Dexamethasone (DEX) as a nuclear localization signaling molecule, into the protocells. As a result, the FA enabled the modified targeting protocells to deliver the DNA into the cancer cells with an increased efficiency and the DEX promoted the DNA to translocate to cell nuclei, eventually leading to the increased chromosome insertion efficiency of the SB transposon. Invivo study strongly suggested that the transfection efficiency of FA-modified protocells in the tumor tissue was much higher than that in other tissues, which was consistent with the invitro results. Our studies implied that with the targeting ligand modification, the protocells could be utilized as an efficient targeting gene carrier. Since the protocells were made of neutral lipids without cationic charges, the cytotoxicity of protocells was significantly lower than that of traditional cationic gene carriers such as cationic liposomes and polyethylenimine, enabling the protocells to be employed in a wider dosage range in gene therapy. Our work shows that the protocells are a promising gene carrier for future clinical applications.

Co-self-assembly of cationic microparticles to deliver pEGFP-ZNF580 for promoting the transfection and migration of endothelial cells.

The gene transfection efficiency of polyethylenimine (PEI) varies with its molecular weight. Usually, high molecular weight of PEI means high gene transfection, as well as high cytotoxicity in gene delivery in vivo. In order to enhance the transfection efficiency and reduce the cytotoxicity of PEI-based gene carriers, a novel cationic gene carrier was developed by co-self-assembly of cationic copolymers. First, a star-shaped copolymer poly(3(S)-methyl-morpholine-2,5-dione-co-lactide) (P(MMD-co-LA)) was synthesized using D-sorbitol as an initiator, and the cationic copolymer (P(MMD-co-LA)-g-PEI) was obtained after grafting low-molecular weight PEI. Then, by co-self-assembly of this cationic copolymer and a diblock copolymer methoxy-poly(ethylene glycol) (mPEG)-b-P(MMD-co-LA), microparticles (MPs) were formed. The core of MPs consisted of a biodegradable block of P(MMD-co-LA), and the shell was formed by mPEG and PEI blocks. Finally, after condensation of pEGFP-ZNF580 by these MPs, the plasmids were protected from enzymatic hydrolysis effectively. The result indicated that pEGFP-ZNF580-loaded MP complexes were suitable for cellular uptake and gene transfection. When the mass ratio of mPEG-b-P(MMD-co-LA) to P(MMD-co-LA)-g-PEI reached 3/1, the cytotoxicity of the complexes was very low at low concentration (20 μg mL−1). Additionally, pEGFP-ZNF580 could be transported into endothelial cells (ECs) effectively via the complexes of MPs/pEGFP-ZNF580. Wound-healing assay showed that the transfected ECs recovered in 24 h. Cationic MPs designed in the present study could be used as an applicable gene carrier for the endothelialization of artificial blood vessels.

Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers

Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with 1H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.

Facile synthesis of wormlike quantum dots-encapsulated nanoparticles and their controlled surface functionalization for effective bioapplications

Semiconductor quantum dots (QDs) are considered as ideal fluorescent probes owing to their intrinsic optical properties. It has been demonstrated that the size and shape of nanoparticles significantly influence their behaviors in biological systems. In particular, one-dimensional (1D) nanoparticles with larger aspect ratios are desirable for cellular uptake. Here, we explore a facile and green method to prepare novel 1D wormlike QDs@SiO2 nanoparticles with controlled aspect ratios, wherein multiple QDs are arranged in the centerline of the nanoparticles. Then, an excellent cationic gene carrier, ethanolamine-functionalized poly(glycidyl methacrylate) (denoted by BUCT-PGEA), was in-situ produced via atom transfer radical polymerization on the surface of the QDs@SiO2 nanoparticles to achieve stable surfaces (QDs@SiO2-PGEA) for effective bioapplications. We found that the wormlike QDs@SiO2-PGEA nanoparticles demonstrated much higher gene transfection performance than ordinary spherical counterparts. In addition, the wormlike nanoparticles with larger aspect ratio performed better than those with smaller ratio. Furthermore, the gene delivery processes including cell entry and plasmid DNA (pDNA) escape and transport were also tracked in real time by the QDs@SiO2-PGEA/pDNA complexes. This work realized the integration of efficient gene delivery and real-time imaging within one controlled 1D nanostructure. These constructs will likely provide useful information regarding the interaction of nanoparticles with biological systems.

Bis-quaternary gemini surfactants as components of nonviral gene delivery systems: A comprehensive study from physicochemical properties to membrane interactions

Gemini surfactants have been successfully used as components of gene delivery systems. In the present work, a family of gemini surfactants, represented by the general structure [CmH2m+1(CH3)2N+(CH2)sN+(CH3)2CmH2m+1]2Br−, or simply m–s–m, was used to prepare cationic gene carriers, aiming at their application in transfection studies. An extensive characterization of the gemini surfactant-based complexes, produced with and without the helper lipids cholesterol and DOPE, was carried out in order to correlate their physico-chemical properties with transfection efficiency. The most efficient complexes were those containing helper lipids, which, combining amphiphiles with propensity to form structures with different intrinsic curvatures, displayed a morphologically labile architecture, putatively implicated in the efficient DNA release upon complex interaction with membranes. While complexes lacking helper lipids were translocated directly across the lipid bilayer, complexes containing helper lipids were taken up by cells also by macropinocytosis. This study contributes to shed light on the relationship between important physico-chemical properties of surfactant-based DNA vectors and their efficiency to promote gene transfer, which may represent a step forward to the rational design of gene delivery systems.

DNA amplification in neutral liposomes for safe and efficient gene delivery.

In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed in vivo gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application.

Comprehensive comparison of two new biodegradable gene carriers

Safety and high transfection efficiency are the prerequisites for an ideal gene vector. Polyethylenimine (PEI), especially PEI 25k (25 kDa), is a well-known cationic gene carrier with high transfection efficiency. However, the high toxicity, depended on its molecular weight, has limited its use as a potential gene carrier. In our research, for the purpose of reducing the toxicity and increasing the transfection efficiency and further to inspect where the degradation of these biodegradable polymers take place would be more beneficial, in cytoplasm or in endocytic vesicles, two kinds of degradable polymers were synthesized. One is an acid-liable PEI derivate (PEI-GA) which was cross-linked by PEI 2k with glutadialdehyde (GA) through imine linkages and the other one (PEI-TEG) was cross-linked PEI 2k with modified triethyleneglycol (TEG) through biscarbamate linkages and can be degraded at neutral environment. By the use of a series of assay methods both in vitro and in vivo, the results showed that PEI-TEG was found to be biodegradable at neutral environment and exhibit high transfection ability with low toxicity, which indicated its potential as a candidate carrier for gene therapy.

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C14H29(CH3)2N+(CH2)2N+(CH3)2C14H29]2Br− and herein designated 14–2–14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14–2–14, 14–2–14:Chol (1:1 molar ratio) and 14–2–14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/− charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14–2–14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/− charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

155. Intracellular Fragmentation of Cationic Gene Carriers Leads to the Enhanced Transgene Expression

155. Intracellular Fragmentation of Cationic Gene Carriers Leads to the Enhanced Transgene Expression

Simultaneous non-invasive analysis of DNA condensation and stability by two-step QD-FRET

Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously.

Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus.

The highly viscous secretions lining the upper airways and bronchi of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy of the lung. In this report we examined the influence of CF mucus components (albumin, DNA, mucin and phospholipids) on the gene transfection activity of cationic DOTAP-based lipoplexes and pegylated GL67-based lipoplexes which previously have been used in CF clinical studies. Upon exposure of the cationic DOTAP:DOPE lipoplexes to either albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these pegylated GL67 lipoplexes. These results suggest that charge shielding of cationic gene carriers with pEG may favor their physicochemical stability in CF mucus and thereby aid in preserving their transfection activity.