Hyperbranched PEGmethacrylate linear pDMAEMA block copolymer as an efficient non-viral gene delivery vector

A unique hyperbranched polymeric system with a linear poly-2-dimethylaminoethyl methacrylate (pDMAEMA) block and a hyperbranched polyethylene glycol methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EGDMA) block was designed and synthesized via deactivation enhanced atom transfer radical polymerisation (DE-ATRP) for efficient gene delivery. Using this unique structure, with a linear pDMAEMA block, which efficiently binds to plasmid DNA (pDNA) and hyperbranched polyethylene glycol (PEG) based block as a protective shell, we were able to maintain high transfection levels without sacrificing cellular viability even at high doses. The transfection capability and cytotoxicity of the polymers over a range of pDNA concentration were analysed and the results were compared to commercially available transfection vectors such as polyethylene imine (branched PEI, 25 kDa), partially degraded poly(amido amine)dendrimer (dPAMAM; commercial name: SuperFect(?)) in fibroblasts and adipose tissue derived stem cells (ADSCs).     

Nonviral approach for targeted nucleic acid delivery

Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.     

Novel polyallylamine-dextran sulfate-DNA nanoplexes: highly efficient non-viral vector for gene delivery

Polyallylamine (PAA), a less investigated polycationic polymer along with polyanionic dextran sulfate (DS) has been complexed with DNA into self assembling PAA–DS–DNA complexes with zinc as stabilizing agent. The complexes prepared were characterized by dynamic light scattering and atomic force microscopy and found to have average hydrodynamic diameter of 150 nm with polydispersity below 0.2, i.e. nanoplexes with narrow size distribution. As expected, the zeta potential values increased with the increase in the PAA in the nanoplexes. Further, PAA–DS–DNA nanoplexes were investigated for in vitro transfecting efficacy on HEK 293 cells. The nanoplexes were found to have better gene transfer efficacy compared to the DNA–PAA complex and commercially available transfecting agent lipofectin. The cytotoxicity was also decreased considerably as revealed by MTT colorimetric assay. The nanoplexes prepared with PAA to DS ratio of 2 were found to be most efficient transfecting agent.     

Novel biotinylated chitosan-graft-polyethyleneimine copolymer as a targeted non-viral vector for anti-EGF receptor siRNA delivery in cancer cells

The major impediments to develop an efficient non-viral siRNA-mediated gene silencing method, as a therapeutic approach, are the low cellular uptake and intracellular delivery and release of non-viral vectors. To overcome these problems, designing a proper vector with high transfection efficiency is obviously under scrutiny of various studies. The present study, evaluate a novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer as an appropriate non-viral vector for targeted delivery of siRNA to cancer cells. The composition of the synthesized Bio-Chi-g-PEI copolymer was thoroughly characterized using ¹H NMR and FTIR spectroscopy, besides the hydroxyazobenzene-2-carboxylic acid (HABA) assay. In vitro cytotoxicity assay of the Bio-Chi-g-PEI copolymers was performed by MTT assay. Cytotoxicity evaluations indicated that the new copolymer was markedly less toxic than PEI 25KD. Physicochemical properties of the Bio-Chi-g-PEI/siRNA complexes such as complex stability, size, zeta potential, and their morphology at various weight ratios, investigated by appropriate methods, revealed the suitability of the complexes for the transfection. The efficient cellular internalization of the complexes for HeLa and OVCAR-3 cells in culture media was confirmed by intracellular tracking of the prepared complexes using confocal laser scanning microscopy and Cy3-labeled anti-epidermal growth factor receptor siRNA. Finally, evaluation of the transfection efficiency and gene silencing by flow cytometry and real-time polymerase chain reaction highlighted the significantly higher efficiency of transfection and silencing for biotinylated copolymer compared with the PEI 25KD and non-biotinylated copolymer.     

Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery

Calcium phosphate nanoparticles present a unique class of non-viral vectors, which can serve as efficient and alternative DNA carriers for targeted delivery of genes. In this study we report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 80 nm in diameter. The DNA encapsulated inside the nanoparticle is protected from the external DNase environment and could be used safely to transfer the encapsulated DNA under in vitro and in vivo conditions. Moreover, the surface of these nanoparticles could be suitably modified by adsorbing a highly adhesive polymer like polyacrylic acid followed by conjugating the carboxylic groups of the polymer with a ligand such as p-amino-1-thio-beta-galactopyranoside using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a coupling agent. We have demonstrated in our studies that these surface modified calcium phosphate nanoparticles can be used in vivo to target genes specifically to the liver.     

pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery

Nanoparticles of calcium phosphate encapsulating plasmid DNA (pDNA) of size 100-120 nm in diameter were prepared. XRD studies of these nanoparticles showed them to be crystalline in nature having hydroxyapatite structure. The maximum loading of pDNA and its release from nanoparticles were studied using gel electrophoresis. The time dependent size measurement of these particles demonstrated that these particles show strong aggregational behaviour in aqueous dispersion. Calcium phosphate nanoparticles were found to be dissolved even in low acidic buffer (pH 5.0) releasing the pDNA, which suggested that DNA release from these particles in the endosomal compartment was possible. In vitro transfection efficiency of these calcium phosphate nanoparticles was found to be higher than that of the commercial transfecting reagent Polyfect.     

Polyethylenimine as a promising vector for targeted siRNA delivery

Recent discovery of RNA interference (RNAi) technology for gene therapy has triggered explosive research efforts towards development of small interfering RNA (siRNA) as therapeutic modality for gene silencing. Owing to its large molecular weight (~13 kDa), polyanionic nature (~40 negative phosphate groups) and rapid enzymatic degradation, delivery of siRNA remains an unresolved issue. Hence, there arises a need of an appropriate delivery vector to overcome the intrinsic, poor intracellular uptake and limited in vitro and in vivo stability. Amongst the various non-viral delivery vectors, the application of polymeric vectors such as polyethylenimine (PEI) or its derivatives has attracted much attention due to its high transfection efficiency and ease of manipulation. PEI has been extensively investigated for DNA delivery, only recently this polymer has been employed for siRNA delivery. This review will focus on studies done on PEI to deliver siRNA, with emphasis on the targeted, self-assembled polymeric nanoparticles with promising potential to evolve as therapeutic tool in gene therapy.     

核苷酸适体在靶向给药系统中的应用进展

适体,即体外合成筛选得到的能特异性地与靶分子结合的一段寡核苷酸序列。由于其独特的性质,在靶向给药系统中有着广阔的应用前景,目前已成为靶向给药研究领域的热点。综述了适体在靶向给药系统中的优势及其应用进展,并对其应用前景和面临的问题进行分析。     

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ∼2.0–7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.     

New vector innovation for drug delivery: development of fusigenic non-viral particles

Efficient and minimally invasive drug delivery systems have been developed to treat intractable human diseases. One approach has been the development of chimeric vector systems combining at least two different vector systems. Based on this concept, chimeric drug delivery systems that combine viral and non-viral features have been developed. Fusigenic non-viral particles have been constructed by conferring viral fusion proteins onto non-viral vectors. HVJ (hemagglutinating virus of Japan; Sendai virus)-liposomes were constructed by the combination of DNA-loaded liposomes with a fusigenic envelope derived from HVJ (hemagglutinating virus of Japan, Sendai virus). Reconstituted HVJ-liposomes were also developed by the insertion of isolated fusion proteins of HVJ into DNA-loaded liposomes. Recently, the technology has been developed to incorporate macromolecules directly into inactivated HVJ particles without liposomes. The resulting HVJ envelope vector introduced plasmid DNA, efficiently and rapidly into both cultured cells in vitro and organs in vivo. Furthermore, proteins, synthetic oligonucleotides and drugs have also been effectively introduced into cells using the HVJ envelope vector. The HVJ envelope vector will be a promising tool for both ex vivo and in vivo gene therapy experiments.     

Folic acid functionalized nanoparticles as pharmaceutical carriers in drug delivery systems

Conventional chemotherapeutic approaches in cancer therapy such as surgery, chemotherapy, and radiotherapy have several disadvantages due to their nontargeted distributions in the whole body. On the other hand, nanoparticles (NPs) based therapies are remarkably progressing to solve several limitations of conventional drug delivery systems (DDSs) including nonspecific biodistribution and targeting, poor water solubility, weak bioavailability and biodegradability, low pharmacokinetic properties, and so forth. The enhanced permeability and retention effect escape from P-glycoprotein trap in cancer cells as a passive targeting mechanism, and active targeting strategies are also other most important advantages of NPs in cancer diagnosis and therapy. Folic acid (FA) is one of the biologic molecules which has been targeted overexpressed-folic acid receptor (FR) on the surface of cancer cells. Therefore, conjugation of FA to NPs most easily enhances the FR-mediated targeting delivery of therapeutic agents. Here, the recent works in FA which have been decorated NPs-based DDSs are discussed and cancer therapy potency of these NPs in clinical trials are presented.     

Reducible HPMA-co-oligolysine copolymers for nucleic acid delivery

Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-l-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol] propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity were attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.     

Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines

Introduction: Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have been licensed for human use. More recently, mRNA-based vaccine alternatives have emerged and might offer certain advantages over their DNA-based counterparts.

Areas covered: This review describes the two main categories of mRNA vaccines: conventional non-amplifying and self-amplifying mRNA. It summarizes the initial clinical proof-of-concept studies and outlines the preclinical testing of the next wave of innovations for the technology. Finally, this review highlights the versatile functionality of the mRNA molecule and introduces opportunities for future improvements in vaccine design.

Expert opinion: The prospects for mRNA vaccines are very promising. Like other types of nucleic acid vaccines, mRNA vaccines have the potential to combine the positive attributes of live attenuated vaccines while obviating many potential safety limitations. Although data from initial clinical trials appear encouraging, mRNA vaccines are far from a commercial product. These initial approaches have spurred innovations in vector design, non-viral delivery, large-scale production and purification of mRNA to quickly move the technology forward. Some improvements have already been tested in preclinical models for both prophylactic and therapeutic vaccine targets and have demonstrated their ability to elicit potent and broad immune responses, including functional antibodies, type 1 T helper cells-type T cell responses and cytotoxic T cells. Though the initial barriers for this nucleic acid vaccine approach seem to be overcome, in our opinion, the future and continued success of this approach lies in a more extensive evaluation of the many non-viral delivery systems described in the literature and gaining a better understanding of the mechanism of action to allow rational design of next generation technologies.     

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery

In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN–DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN–DNA complexes. Further, SLN–DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.     

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery

In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN-DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN-DNA complexes. Further, SLN-DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.     

Directed evolution for drug and nucleic acid delivery

Directed evolution is a term used to describe a variety of related techniques to rapidly evolve peptides and proteins into new forms that exhibit improved properties for specific applications. In this process, molecular biology techniques allow the creation of up to billions of mutants in a single experiment, which are then subjected to high-throughput screening to identify those with enhanced activity. Applications of directed evolution to drug and gene delivery have been recently described, including those that improve the effectiveness of therapeutic enzymes, targeting peptides and antibodies, and the effectiveness or tropism of viral vectors for use in gene therapy. This review first introduces fundamental concepts of directed evolution, and then discusses emerging applications in the field of drug and gene delivery.