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.     

Target Specific Optimization of Cationic Lipid-Based Systems for Pulmonary Gene Therapy

Purpose. Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. Methods. The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. Results. Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. Conclusions. Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.     

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.     

Cationic nanoparticles for cancer therapy

Importance of the field: The lack of selective delivery of therapeutic molecules to cancer cells remains a problem in cancer therapy. As a result of this non-selectivity, cytotoxic agents are delivered to both healthy and cancerous cells, resulting in severe side effects for the patient, eventually causing termination of therapy or ineffective therapy resulting in progression or recurrence of the disease. In this context, cationic polymers with net positive surface charge emerge as a promising option owing to their very strong cellular interaction properties and good cellular uptake.

Areas covered in this review: In this review, the structure, characteristics and preparation techniques for cationic nanoparticulate drug delivery systems are discussed in the light of cytotoxicity associated with cationic polymers and strong complement activation properties of cationic carrier systems on injection. In vivo behavior and biodistribution of cationic nanoparticles are also reviewed for a better understanding of biological interaction of cationic nanoparticles.

What the reader will gain: This review will give an insight to the properties of cationic polymers, including their advantages and drawbacks and drug/gene delivery systems based on cationic polymers intended for cancer therapy.

Take home message: Cationic polymer-based nanoparticles emerge as a promising group of nanosize carrier systems to the tumor cell level with a wide range of modification and application possibilities.     

Polyethylene Glycol-Conjugated Copolymers for Plasmid DNA Delivery

No Heading Polymeric gene delivery systems have been developed as an alternative for viral gene delivery systems to overcome the problems in the use of viral gene carriers. Polymeric carriers have many advantages as gene carriers such as low cytotoxicity, low immunogenicity, moderate transfection efficiency, no size-limit, low cost, and reproducibility. In the efforts to develop safe and efficient polymeric gene carriers, polyethylene glycol (PEG) has widely been used because of its excellent characteristics. PEG-conjugated copolymers have advantages for gene delivery: 1) The PEG-conjugated copolymers show low cytotoxicity to cells in vitro and in vivo, 2) PEG increases water-solubility of the polymer/DNA complex, 3) PEG reduces the interaction of the polymer/DNA complex with serum proteins and increases circulation time of the complex, 4) PEG can be used as a spacer between a targeting ligand and a cationic polymer. A targeting ligand at the end of a PEG chain is not disturbed by the interaction of a cationic polymer with plasmid DNA, and the PEG spacer increases the accessibility of the ligand to its receptor. In this review, PEG copolymers as gene carriers are introduced, and their characteristics are discussed.     

Self-Assembled and Nanostructured siRNA Delivery Systems

A wide range of organic and inorganic materials have been used in the development of nano-scale self-assembling gene delivery systems to improve the therapeutic efficacy of nucleic acid drugs. Small interfering RNA (siRNA) has recently been recognized as a promising and potent nucleic acid medicine for the treatment of incurable genetic disorders including cancer; however, siRNA-based therapeutics suffer from the same delivery problems as conventional nucleic acid drugs such as plasmid DNA and antisense oligonucleotides. Many of the delivery strategies developed for nucleic acid drugs have been applied to siRNA therapeutics, but they have not produced satisfactory in vivo gene silencing efficiencies to warrant clinical trials. This review discusses recent progress in the development of self-assembled and nanostructured delivery systems for efficient siRNA-induced gene silencing and their potential application in clinical settings.     

Electrohydrodynamic Comminution: A Novel Technique for the Aerosolisation of Plasmid DNA

Purpose Naked plasmid DNA (pDNA) is a potential gene transfer agent for lung gene therapies but cannot be aerosolised without degradation using conventional nebulisation devices. This study investigated the viability of an alternative nebulisation technique, electrohydrodynamic (EHD) comminution for the aerosol delivery of naked DNA in vivo.Methods Naked pDNA was aerosolised using jet and ultrasonic nebulisers, and by EHD comminution. Degradation associated with the aerosolisation process was investigated using gel electrophoresis and by transfection studies in cell culture. Optimised formulations for EHD aerosolisation of pDNA were developed and in vivo deposition and reporter gene expression were investigated in mice.Results Unlike conventional nebulisation devices, EHD comminution of plasmids up to 15 kb in size resulted in no detectable pDNA degradation. EHD formulations containing up to 1 mg/ml pDNA were developed and shown to produce monodisperse aerosols suitable for targeted lung delivery in humans. Aerosolisation studies in vivo demonstrated detectable levels of pDNA deposition and measurable luciferase reporter gene expression in the lungs of exposed mice.Conclusions This study demonstrates for the first time that respirable aerosols of naked pDNA can be generated without plasmid degradation and that EHD comminution is an appropriate technique for the aerosolisation of delicate gene transfer agents.     

Gene Expression Profiles in Mouse Lung Tissue after Administration of Two Cationic Polymers Used for Nonviral Gene Delivery

Purpose This study compared gene expression profiles in mouse lungs after administration of the cationic polymers polyethyleneimine (PEI) or chitosan alone or formulated with a luciferase reporter plasmid (PEI–pLuc, chitosan–pLuc). Methods The polymers and formulations were administered intratracheally to Balb/c mice at doses judged to be nontoxic according to intracellular dehydrogenase activity and tissue morphology. RNA was isolated from the lungs 24 or 72 h after administration, and a dedicated stress and toxicology cDNA array was used to monitor the in vivo response to the gene delivery system in the lung tissue. Results The gene expression profiles differed between the PEI and chitosan groups with regard to both the total number and the type of expressed genes. Chitosan–pLuc upregulated genes that protect the cell from oxidative stress and inflammation, such as heme oxygenase-1 and catalase, whereas PEI–pLuc upregulated genes involved in inflammatory processes, such as the cyclooxygenases 1 and 2, indicating possible involvement in the development of adverse reactions. However, both polymers activated genes involved in reaction to stress, such as DNA damage repair. Furthermore, in the PEI group, chaperone genes and members of the p38 mitogen-activated protein kinase pathway were also upregulated, suggesting a possible explanation for the better performance of PEI in gene delivery systems. Conclusions The results indicate that gene expression profiling is a useful and sensitive tool for the evaluation of tissue responses after administration of polymers or gene delivery systems. The results also suggest a possible explanation for the differences in gene delivery performance between the two polymers in gene delivery systems.     

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer

Purpose. This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol. Method. An acid labile cationic lipid, O-(2R-1,2-di-O-(1`Z, 9`Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls. Results. The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25°C. Addition of BCAT to plasmid DNA in 40%ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FACS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20%higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 g plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs. Conclusions. Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection.     

Cationic Polymer Based Gene Delivery Systems

Gene transfer to humans requires carriers for the plasmid DNA which canefficiently and safely carrythe gene into the nucleus of the desired cells. A series of chemically differentcationic polymers arecurrently being investigated for these purposes. Although many cationic polymersindeed condense DNAspontaneously, which is a requirement for gene transfer in most types of cells,the physicochemical andbiopharmaceutical behavior of the current generation of polyplexes severelylimits an efficient genetransfer in vitro and especially in vivo. This papersummarizes recent physicochemical and biologicalinformation on polyplexes and aims to provide new insights with respect to thistype of gene deliverysystem. Firstly, the chemical structure of frequently studied cationic polymersis represented. Secondly,the parameters influencing condensation of DNA by cationic polymers aredescribed. Thirdly, the surfaceproperties, solubility, aggregration behavior, degradation and dissociation ofpolyplexes are considered.The review ends by describing the in vitro and in vivo genetransfection behavior of polyplexes.     

Sphingosine-Based Liposome as DNA Vector for Intramuscular Gene Delivery

Purpose. The aim of this study was to develop a labile sphingosine-based liposome for intramuscular gene delivery. Methods. Sphingosine-based liposomes were formulated in a range of solutions with phosphatidylcholine, then were associated to DNA. The physico-chemical characteristics of the sphingosine/EPC liposomes and sphingosine/EPC/DNA lipoplexes were determined. DNA stability within sphingosine-based liposomes was evaluated in the presence of a nuclease and mouse serum. In vivo gene transfer was studied by intramuscular injection with and without the electrotransfer technique. Results. By increasing the charge ratios, colloidally stable sphingosine/DNA particles with a 170 nm average diameter and a positive potential were obtained. Ethidium bromide was still able to insert into plasmid DNA within the lipoplexes, even though plasmid DNA was demonstrated to be complexed to the lipid by gel electrophoresis. Additionally, DNA was shown to be accessible to DNase I, but significantly resistant to serum enzymatic digestion. Upon intramuscular injection, lipoplexes induced an inhibition of gene expression as compared with naked DNA. Conclusions. The cationic sphingosine/EPC/DNA complexes form weakly compacted structure, potentially labile in vivo, which might be useful for in vivo gene transfer.     

Lipid-based Nanoparticles for Nucleic Acid Delivery

Abstract Lipid-based colloidal particles have been extensively studied as systemic gene delivery carriers. The topic that we would like to emphasize is the formulation/assembly of lipid-based nanoparticles (NP) with diameter under 100 nm for delivering nucleic acid in vivo. NP are different from cationic lipid–nucleic acid complexes (lipoplexes) and are vesicles composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The diameter of the NP is an important attribute to enable NP to overcome the various in vivo barriers for systemic gene delivery such as: the blood components, reticuloendothelial system (RES) uptake, tumor access, extracellular matrix components, and intracellular barriers. The major formulation factors that impact the diameter and encapsulation efficiency of DNA-containing NP include the lipid composition, nucleic acid to lipid ratio and formulation method. The particle assembly step is a critical one to make NP suitable for in vivo gene delivery. NP are often prepared using a dialysis method either from an aqueous-detergent or aqueous-organic solvent mixture. The resulting particles have diameters about 100 nm and nucleic acid encapsulation ratios are >80%. Additional components can then be added to the particle after it is formed. This ordered assembly strategy enables one to optimize the particle physico-chemical attributes to devise a biocompatible particle with increased gene transfer efficacy in vivo. The components included in the sequentially assembled NP include: poly(ethylene glycol) (PEG)-shielding to improve the particle pharmacokinetic behavior, a targeting ligand to facilitate the particle–cell recognition and in some case a bioresponsive lipid or pH-triggered polymer to enhance nucleic acid release and intracellular trafficking. A number of groups have observed that a PEG-shielded NP is a robust and modestly effective system for systemic gene or small interfering RNA (siRNA) delivery.     

Gelatin-based delivery systems for cancer gene therapy

Gelatin is a natural, biocompatible, nontoxic, edible, and inexpensive macromolecule. These properties result in its wide application in pharmaceutical, medical, and cosmetic products. Recently, it has been used for the delivery of such gene therapeutic entities as plasmid DNA. This review discusses the in vivo and in vitro studies using gelatin for delivery of therapeutic genes to cancerous cells. Recent studies show that present cancer gene therapy using gelatin is lacking in both efficiency and specificity in comparison with viral vectors, whereas complexes of therapeutic DNA with modified gelatin possibly offer a safe and efficient strategy for systemic administration of therapeutic genes to solid tumors compared to injection of naked plasmid DNA. The future of these promising approaches lies in the development of better techniques for preparing gelatin–gene complexes with the aim of a gelatin-based cancer gene therapy with comparable efficiency to viral vectors but with the added advantage of biosafety for patients.     

Gene delivery using cationic liposomes

The development of cationic liposomes for gene delivery has been ongoing for almost 20 years; however, despite extensive efforts to develop a successful therapeutic agent, there has been limited progress towards generating an effective pharmaceutical product. Since the introduction of N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride, an immense number of different cationic lipids have been synthesised and used to formulate cationic liposome–DNA complexes. Structural modification of the cationic lipids and the addition of components within the delivery system that can facilitate the fusion, cellular uptake and targeting of liposome–DNA complexes have all been used in a bid to enhance their transfection efficiency. Unfortunately, the overall impact of these improvements is still nominal, with the vast majority of clinical trials (～ 85%) continuing to rely on more potent viral delivery of DNA despite their associated toxicity issues. Key characteristics of the most effective cationic liposomes for the delivery of plasmid DNA (from a consensus of the literature) is identified here and the problems of converting these attributes into an effective pharmaceutical product are outlined.     

Degradable polyethylenimines as DNA and small interfering RNA carriers

Gene therapy is a powerful approach in the treatment of a wide range of both inherited and acquired diseases. Nonviral delivery systems have been proposed as safer alternatives to viral vectors because they avoid the inherent immunogenicity and production problems that are seen when viral systems are used. Many cationic polymers, including high-molecular-weight polyethylenimine (PEI) have been widely studied as gene-delivery carriers, both, in vitro and in vivo. However, interest has recently developed in degradable polymeric systems. The advantage of degradable polymer is its low in-vivo cytotoxicity, which is a result of its easy elimination from the cells and body. Degradable polymer also enhances transfection of DNA or small interfering RNA (siRNA) for efficient gene expression or silencing, respectively. This review paper summarizes and discusses the recent advances with degradable PEIs, such as cross-linked and grafted PEIs for DNA and siRNA delivery.     

Ultrasonic Nebulization of Cationic Lipid-Based Gene Delivery Systems for Airway Administration

Purpose. This study relates to the development of gene therapies for the treatment of lung diseases. It describes for the first time the use of ultrasonic nebulization for administration of plasmid/lipid complexes to the lungs to transfect lung epithelial cells. Methods. Plasmid complexed to cationic liposomes at a specific stoichiometric ratio was nebulized using an ultrasonic nebulizer. We assessed: (i) the stability of plasmid and plasmid/lipid complexes to ultrasonic nebulization, (ii) the in vitro activity of plasmid in previously nebulized plasmid/lipid complex, (iii) the in vivo transgene expression in lungs following intratracheal instillation of nebulized plasmid/lipid formulations compared to un-nebulized complexes, (iv) the emitted dose from an ultrasonic nebulizer using plasmid/lipid complexes of different size, and (v) the transgene expression in lungs following oral inhalation of aerosolized plasmid/lipid complex generated using an ultrasonic nebulizer. Results. Integrity of plasmid formulated with cationic lipids, and colloidal stability of the plasmid/lipid complex were maintained during nebulization. In contrast, plasmid alone formulated in 10% lactose was fragmented during nebulization. The efficiency of transfection of the complex before and after nebulization was comparable. Nebulization produced respirable aerosol particles. Oral exposure of rodents for 10 minutes to aerosol produced from the ultrasonic nebulizer resulted in transgene expression in lungs in vivo. Conclusions. The performance characteristics of the ultrasonic nebulizer with our optimized plasmid/lipid formulations suggests that this device can potentially be used for administering gene medicines to the airways in clinical settings for the treatment of respiratory disorders.     

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.     

Using Salivary Glands as a Tissue Target for Gene Therapeutics

Gene transfer offers a potential way to correct local and systemic protein deficiency disorders by using genes as drugs, so called gene therapeutics. Salivary glands present an interesting target site for gene therapeutic applications. Herein, we review proofs of concept achieved for salivary glands with in vivo animal models. In that context we discuss problems (general and salivary tissue-specific) that limit immediate clinical use for this application of gene transfer. Ongoing efforts, however, suggest that salivary glands may be suitable as gene therapeutic target sites for drug delivery in the near future.     

Gelucire-stabilized nanoparticles as a potential DNA delivery system

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs?>?DDAB-NPs?>?CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.     

The role of cell cycle in the efficiency and activity of cancer nanomedicines

Introduction: With a wealth of knowledge on the effect of nanoparticle properties, including size, shape, charge and composition, on intracellular delivery, little has been reported on the effect of the cell cycle on the intracellular delivery and activity of nanomedicines including non-viral gene delivery systems. The aim of this review is to shed a light on this topic.

Areas covered: It is now evident that nanoparticle cell uptake varies with the cell cycle phase. This review addresses this variation by dissecting the effect of cell population heterogeneity on the intracellular delivery and activity of nanomedicines with a special focus on non-viral gene delivery and combination therapy modalities that utilize cell cycle inhibitors as co-targets for therapy. In addition, the importance of three-dimensional (3D) culture systems in the drug delivery field within the context of the cell cycle will be addressed.

Expert opinion: The understanding of the cell cycle machinery has improved dramatically over the last few decades. Developing combination therapy modalities that target the cell cycle to achieve better cancer patient outcome should now be the focus. Furthermore, more effort should be placed on developing a reliable, consistent, high throughput 3D cell culture system since these systems more closely resemble the cell cycle status of in vivo tumors. A switch from 2D to 3D culture systems, to more accurately predict the in vivo efficacy of nanoparticle drug delivery systems, is desirable.