Polyplex gene delivery modulated by redox potential gradients

Polyplexes sensitive to redox potential gradients represent a promising class of vectors for delivery of nucleic acids. This review focuses on the recent advances in the development of these vectors. The biological rationale for the design of redox-sensitive polyplexes is discussed together with the basic synthetic approaches for introducing reducible disulfide bonds into the structure of the polyplexes. The biological properties of the redox-sensitive polyplexes of plasmid DNA, mRNA, antisense oligonucleotides and siRNA are reviewed with emphasis on in vitro cellular delivery, cytotoxicity and in vivo activity. Overall, redox-sensitive polyplexes represent a promising platform for further development as vectors for delivery of a wide variety of therapeutic nucleic acids.     

Polycation gene delivery systems: escape from endosomes to cytosol

Clinical success of gene therapy based on oligonucleotides (ODNs), ribozymes, RNA and DNA will be greatly dependent on the availability of effective delivery systems. Polycations have gained increasing attention as a non-viral gene delivery vector in the past decades. Significant progress has been made in understanding complex formation between polycations and nucleic acids, entry of the complex into the cells and subsequent entry into the nucleus. Sophisticated molecular architectures of cationic polymers have made the vectors more stable and less susceptible to binding by enzymes or proteins. Incorporation of specific ligands to polycations has resulted in more cell-specific uptake by receptor-mediated mechanisms. However, there are still other barriers limiting the transfection efficiency of polycation gene delivery systems. There is a consensus that polycation-DNA complexes (polyplexes) enter cells via the endocytotic pathway. It is not clearly understood, however, how the polyplexes escape (if they do) from endosomes, how DNA is released from the polyplexes or how the released DNA is expressed. The primary focus of this article is to review various polycation gene delivery systems, which are designed to translocate DNA from endosomes into cytosol. Many polycation gene delivery systems have tried to mimic the mechanisms that viruses use for the endosomal escape. Polycation gene delivery systems are usually coupled with synthetic amphipathic peptides mimicking viral fusogenic peptides, histidine-based gene delivery systems for pH-responsive endosomal escape, polycations with intrinsic endosomolytic activity by the proton sponge mechanism and polyanions to mimic the anionic amphiphilic peptides.     

Cationic liposome systems in gene therapy

Cationic liposomes are well known to assist the delivery of genes and other nucleic acids to cells in vitro. This has held out the tantalizing possibility that cationic liposome systems could play a major role as vectors (ie, gene delivery vehicles) for the delivery of therapeutic genes and/or other nucleic acids into diseased cells and organs of patients, in order to treat disease by gene therapy. How realistic is this? Certainly, a number of cationic liposome systems have been used to mediate the delivery of nucleic acids in vivo with gene therapy applications in mind. However, this approach is still fraught with technical problems and further progress in basic research will be necessary if cationic liposomes are to be a gene therapy vector of the future.     

Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery

The successful delivery of therapeutic genes to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. Nonviral gene delivery is currently a subject of increasing attention because of its relative safety and simplicity of use; however, its use is still far from being ideal because of its comparatively low efficiency. Most of the currently available nonviral gene vectors rely on two main components, cationic lipids and cationic polymers, and a variety of functional devices can be added to further optimize the systems. The design of these functional devices depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fate is usually linked to the entry mechanism. Therefore, the successful design of a nonviral gene delivery system requires a deep understanding of gene/carrier interactions as well as the mechanisms involved in the interaction of the systems with the target cells. In this article, we review the different uptake pathways that are involved in nonviral gene delivery from a gene delivery point of view. In addition, available knowledge concerning cellular entry and the intracellular trafficking of cationic lipid-DNA complexes (lipoplexes) and cationic polymer-DNA complexes (polyplexes) is summarized.     

Polyplex gene delivery modulated by redox potential gradients

Polyplexes sensitive to redox potential gradients represent a promising class of vectors for delivery of nucleic acids. This review focuses on the recent advances in the development of these vectors. The biological rationale for the design of redox-sensitive polyplexes is discussed together with the basic synthetic approaches for introducing reducible disulfide bonds into the structure of the polyplexes. The biological properties of the redox-sensitive polyplexes of plasmid DNA, mRNA, antisense oligonucleotides and siRNA are reviewed with emphasis on in vitro cellular delivery, cytotoxicity and in vivo activity. Overall, redox-sensitive polyplexes represent a promising platform for further development as vectors for delivery of a wide variety of therapeutic nucleic acids.     

Bioresponsive small molecule polyamines as noncytotoxic alternative to polyethylenimine

Nonviral gene therapy continues to require novel synthetic vectors to deliver therapeutic nucleic acids effectively and safely. The majority of synthetic nonviral vectors employed in clinical trials to date have been cationic liposomes; however, cationic polymers are attracting increasing attention. One of the few cationic polymers to enter clinical trials has been polyethylenimine (PEI); however, doubts remain over its cytotoxicity, and in addition it displays lower levels of transfection than viral systems. Herein, we report on the development of a series of small molecule analogues of PEI that are bioresponsive to the presence of pDNA, forming poly(disulfide)s that are capable of efficacious transfection with no associated toxicity. The most effective small molecule developed, a cyclic disulfide based upon a spermine backbone, is shown to form very well-defined polyplexes (100-200 nm in diameter) that mediate murine lung transfection in vivo to within an order of magnitude of in vivo jetPEI, and at the same time display a much improved cytotoxicity profile.     

Cationic lipids for gene delivery in vitro and in vivo

Certain disease states can be corrected by using nucleic acids as therapeutic agents. To achieve this, nucleic acids must be delivered into the affected cells efficiently. At the core of a successful gene therapy protocol is the design of the nucleic acid carrier. Cationic lipids, as one of the gene delivery systems, have a wide potential in delivering nucleic acids both in vivo and in vitro. They are synthetic in origin and, hence, can be produced in required quantities and are biologically safe. Significant inputs from synthetic chemists in the recent past have resulted in the exploration of cationic lipids with very interesting functionalities. Transfection efficiencies of cationic lipids are comparable to viral-mediated transfection in vitro. However, viral-based methods for gene delivery in vivo are comparatively more efficient. Current understanding of lipid-mediated transfection is partially due to incomplete characterisation of the lipoplex, poor understanding of cell biology of transfection and cell type variations in transfection efficiencies. The published patents and research demonstrates the need for incorporation of the biological information in the design of the gene delivery formulations. In this review, the cell biological aspects critical for lipid-mediated transfection are emphasised. The parameters that influence the colloidal stability of the lipoplexes, cell biological processes relevant to gene delivery, such as cell association/uptake, cytoplasmic stability of the DNA and nuclear import, are discussed. The main focus of this review is patents published in the last 5 years.     

Recent development of nonviral gene delivery systems with virus-like structures and mechanisms

The concept of gene therapy includes not only the addition of normal genes to genetically deficient cells, but also the use of transgenes encoding several peptides that function to enhance the capacity of normal cells or to regulate cell differentiation. The application of gene therapy has been widely considered for various diseases, as well as for the field of tissue engineering. To overcome the problems with viral vectors, a broad range of nonviral systems for gene delivery have been developed, including systems composed of cationic lipids (lipoplexes) and cationic polymers (polyplexes). However, most of these systems are still much less efficient than viral vectors, especially for in vivo gene delivery. Paradoxically, to achieve a maximum transgene expression in the targeted cells, there is no question that natural viruses are the most effective nanocarriers. In this article, we highlight the approaches currently being taken to improve nonviral gene delivery systems so that they better replicate the typical structures and mechanisms of viruses, such as DNA (RNA) condensation in the core, surrounding structures with targeting molecules for specific receptors, as well as the toxic and immunogenic problems which should be avoided, with the ultimate goal of bringing these systems into a clinical setting.     

Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers

DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3–4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery.This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major importance for turning gene therapy into a successful therapeutic method. Nonviral gene delivery relies mainly on the complexes formed from cationic liposomes (or cationic polymers) and DNA, i.e., lipoplexes (or polyplexes). Many lipoplex formulations have been studied, but in vivo activity is generally low compared to that of viral systems. This review gives a concise overview of studies on the application of cationic liposomes in vivo in animal models of diseases and in clinical studies. The transfection efficiency, the pharmacokinetic and pharmacodynamic properties of the lipid-DNA complexes, and potentially relevant applications for cationic liposomes are discussed. Furthermore, the toxicity of, and the induction of an inflammatory response in association with the administration of lipoplexes are described. Increasing understanding of lipoplex behavior and gene transfer capacities in vivo offers new possibilities to enhance their efficiency and paves the path to more extensive clinical applications in the future.     

非病毒载体在肿瘤基因治疗领域的研究进展

随着肿瘤基因治疗领域的研究进展，临床应用逐渐增多。载体是癌症基因治疗的主要难题。当前广泛使用的病毒载体存在的安全问题越来越受到人们的重视，已经有多种非病毒载体用于肿瘤基因治疔，如：裸DNA直接注射、阳离子脂质、阳离子聚合物。研究非病毒载体的目标是：它能像靶向的合成病毒载体那样对肿瘤组织表现出高度特异性；具有很高的转染效率；潜在的安全性问题能够被控制。     

Localized,non-viral delivery of nucleic acids: Opportunities,challenges and current strategies

Localized delivery of drugs is an emerging field both with regards to drug delivery during disease as well as in tissue engineering. Despite significant achievements made in the last decades, the efficient delivery of proteins and peptides remains challenging, especially in cases requiring long-term release of proteins after application. The localized delivery of nucleic acids (NA) represents an interesting alternative due to higher physicochemical stability of NA, increased efficiency by harnessing cells as bioreactors for the production of required proteins and improved versatility with regards to expression of specific proteins through plasmid DNA or repression of gene products through siRNA. However, unlike most proteins and peptides, NA must be delivered to the cytoplasm or nucleus to be efficacious, resulting in significant delivery challenges. We herein describe frequently used non-viral vectors for the delivery of NA including polyplexes, lipoplexes and lipopolyplexes and summarize recent developments in the field of nucleic acid delivery systems for local application based on hydrogels, solid scaffolds and physical delivery methods. The challenges associated with the different approaches are identified and options to address these challenges are discussed.     

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 polyethylenimine-derived nanoparticles for in vivo gene delivery

Introduction: Branched and linear polyethylenimines (PEIs) are cationic polymers that have been used to deliver nucleic acids both in vitro and in vivo. Owing to the high cationic charge, the branched polymers exhibit high transfection efficiency, and particularly PEI of molecular weight 25 kDa is considered as a gold standard in gene delivery. These polymers have been extensively studied and modified with different ligands so as to achieve the targeted delivery.

Areas covered: The application of PEI in vivo promises to take the polymer-based vector to the next level wherein it can undergo clinical trials and subsequently could be used for delivery of therapeutics in humans. This review focuses on the various recent developments that have been made in the field of PEI-based delivery vectors for delivery of therapeutics in vivo.

Expert opinion: The efficacy of PEI-based delivery vectors in vivo is significantly high and animal studies demonstrate that such systems have a potential in humans. However, we feel that though PEI is a promising vector, further studies involving PEI in animal models are needed so as to get a detailed toxicity profile of these vectors. Also, it is imperative that the vector reaches the specific organ causing little or no undesirable effects to other organs.     

Polyplexes based on cationic polymers with strong nucleic acid binding properties

Cationic polymers have been studied for nucleic acid delivery both in vitro and in vivo. However, many polymer-based formulations suffer from lack of stability in biologic fluids due to interactions with anionic biomacromolecules such as proteins and polysaccharides. Likely, the stronger the electrostatic interactions between a cationic polymer and nucleic acids, the higher the stability of the polyplexes in biologic fluids will be. To get evidence for this hypothesis, quaternized poly[3,5-bis(dimethylaminomethylene)-p-hydroxyl styrene] (QNPHOS) with two permanently charged cationic sites per monomer unit as well as its block copolymer with PEG were synthesized and compared with the standard transfectant pDMAEMA, in terms of nucleic acid binding strength, gene silencing and transfection activities of the complexes which these polymers form with siRNA and plasmid DNA, respectively. It was shown that siRNA complexes based on QNPHOS and QNPHOS-PEG dissociate in the presence of a fourfold higher heparin concentration than necessary to destabilize pDMAEMA complexes. Under the same conditions, complexes of DNA and QNPHOS or QNPHOS-PEG did not show any dissociation, in contrast to pDMAEMA polyplexes. The DNA polyplexes based on QNPHOS or QNPHOS-PEG did not show transfection activity, which might be ascribed to their high physicochemical stability. On the other hand, siRNA complexes based on QNPHOS and QNPHOS-PEG showed a low cytotoxicity and an improved siRNA delivery and high gene silencing activity, even higher than those based on pDMAEMA. This might be due to the excellent binding characteristics of QNPHOS and QNPHOS-PEG to siRNA which in turn is ascribed to the presence of two permanently charged cationic groups per monomer unit. Based on the results of this study, it is concluded that formation of strong siRNA complexes with polymers containing double charges per monomer is advantageous.     

Influence of Physicochemical Properties on Pharmacokinetics of Non-viral Vectors for Gene Delivery

The influence of physicochemical properties on the in vivo pharmacokinetics of gene delivery vectors after systemic administration is reviewed based on our studies. We have been studying the development of DNA delivery systems, such as plasmid DNA complexed with cationic polymers (polyplexes) and cationic liposomes (lipoplexes). Even if target-recognizable ligand is incorporated into the system, the overall physicochemical properties, notably size and charge, are predominant factors influencing in vivo disposition characteristics of the vector. Based on this consideration, liver cell-specific carrier systems via receptor-mediated endocytosis were successfully developed by optimizing physicochemical characteristics. In conclusion, rational design of gene delivery vectors requires an understanding of their pharmacokinetics in relation to the physicochemical properties. Optimization of the physicochemical properties is important for successful in vivo gene delivery by non-viral vectors.     

Antisense strategies and non-viral gene therapy for cancer

Effective gene therapy for cancer remains an elusive goal, even after more than a decade of intensive research. There has been, however, tremendous progress in the development of increasingly sophisticated non-viral (or synthetic) delivery vectors for local and systemic administration of nucleic acids. Recent clinical data has also indicated the feasibility of using antisense oligonucleotides to inhibit inappropriately expressed or mutated genes in human cancers. The purpose of this review is to provide an update of the patent literature on the development of non-viral approaches for cancer gene therapy. In particular, patents on lipoplexes and polyplexes for delivery of therapeutic genes and antisense oligonucleotides are reviewed. The diverse range of antisense strategies being developed and recent clinical data are also highlighted.     

Influence of physicochemical properties on pharmacokinetics of non-viral vectors for gene delivery

The influence of physicochemical properties on the in vivo pharmacokinetics of gene delivery vectors after systemic administration is reviewed based on our studies. We have been studying the development of DNA delivery systems, such as plasmid DNA complexed with cationic polymers (polyplexes) and cationic liposomes (lipoplexes). Even if target-recognizable ligand is incorporated into the system, the overall physicochemical properties, notably size and charge, are predominant factors influencing in vivo disposition characteristics of the vector. Based on this consideration, liver cell-specific carrier systems via receptor-mediated endocytosis were successfully developed by optimizing physicochemical characteristics. In conclusion, rational design of gene delivery vectors requires an understanding of their pharmacokinetics in relation to the physicochemical properties. Optimization of the physicochemical properties is important for successful in vivo gene delivery by non-viral vectors.     

Cell-penetrating peptides for the delivery of nucleic acids

Introduction: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo.

Areas covered: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs.

Expert opinion: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.