Cationic lipids, lipoplexes and intracellular delivery of genes.

As a consequence of several setbacks encountered by viral technology in achieving efficient and safe gene therapy in clinical trials, non-viral gene delivery vectors are considered to date as a valuable alternative and to hold promise for future therapeutic applications. Nevertheless, the transfection efficiency mediated by these non-viral gene delivery vectors has to be improved, especially in vivo, to benefit fully from their advantages. Cationic lipid/nucleic acid complexes or lipoplexes have been the subject of intensive investigations in recent years to understand the parameters governing the efficiency of transfection. Specifically, the comprehension of such mechanisms, from the formation of the complexes to their intracellular delivery, will lead to the design of better adapted non-viral vectors for gene therapy applications. Here, we will discuss some recent developments in the field on the structure/function relationship of cationic lipids in the mechanism of transfection, and where appropriate, we will make a comparison with mechanisms of viral and polyplex-mediated gene delivery. Cationic lipids are often used in combination with helper lipids such as DOPE or cholesterol. The effect of DOPE on lipoplex assembly and the relevance of the structural properties of the lipoplexes in destabilizing endosomal membranes and mediating endosomal escape of DNA will be discussed.     

Enhancement of Microbubble Mediated Gene Delivery by Simultaneous Exposure to Ultrasonic and Magnetic Fields

It has been shown in previous studies that gene delivery can be enhanced by a variety of minimally-invasive techniques including: (1) exposure of cells to ultrasound in the presence of DNA and gas microbubbles and (2) exposure of cells to a magnetic field in the presence of DNA conjugated to magnetic nanoparticles. The aim of this work was to investigate whether it was possible to combine the advantages of both these techniques. It was found that transfection of Chinese hamster ovary cells by naked plasmid DNA was enhanced by combined exposure of the cells to ultrasound (10 s at 1 kHz pulse repetition frequency with 40 cycle 1 MHz sinusoidal pulses, 1 MPa peak to peak pressure) and a magnetic field (provided by five square cross-section N52 grade NdFeB magnets 25 × 10 × 10 mm with transversal magnetisation Br = 1.50 T arranged in a Halbach array), in the presence of one of two different microbubble/nanoparticle preparations. The first preparation consisted of phospholipid coated microbubbles mixed with micelles containing magnetic nanoparticles. The second consisted of microbubbles which were themselves magnetically active. These preparations were found to be more effective than either magnetic micelles or phospholipid coated microbubbles alone by a factor of 2.8 (total flux ∼4 versus 1.4 × 10⁶ photon/s) and the results were found to be statistically significant (p < 0.01). Two mechanisms are proposed to explain these observations: firstly, that the magnetic field facilitates close proximity between the cells and the microbubbles and hence increases the likelihood of transfection; second, that there is sensitisation of the cells, as a result of exposure to the magnetic field in the presence of the micelles, which increases their ability to be transfected upon exposure to ultrasound. Further work is in progress to determine which of these mechanisms is the most significant and the potential for other therapeutic applications. (E-mail: e_stride@meng.ucl.ac.uk)     

Cationic liposomes as non-viral carriers of gene medicines: resolved issues, open questions, and future promises

The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "transfection vectors." The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling and preparation techniques; (c) ability to inject large lipid:DNA complexes; and (d) low immunogenic response. The present review highlights the major achievements in the area of designing efficacious cationic transfection lipids, some of the more recent advances in the field of cationic liposomes-mediated gene transfer and targeted gene delivery, some unresolved issues and challenges in liposomal gene delivery, and future promises of cationic liposomes as gene-carriers in non-viral gene therapy.     

Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo

Low efficiencies of nonviral gene vectors, the receptor-dependent host tropism of adenoviral or low titers of retroviral vectors limit their utility in gene therapy. To overcome these deficiencies, we associated gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. This potentiated the efficacy of any vector up to several hundred-fold, allowed reduction of the duration of gene delivery to minutes, extended the host tropism of adenoviral vectors to nonpermissive cells and compensated for low retroviral titer. More importantly, the high transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection in the gastrointestinal tract and in blood vessels. Magnetofection provides a novel tool for high throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo.     

Functionalized gold nanostructures: promising gene delivery vehicles in cancer treatment

Surface-modified gold nanoparticles are recognized as promising gene delivery vehicles in the treatment of cancer owing to their excellent biocompatibility with biomolecules (like DNA or RNA) and their unique optical and structural properties. In this context, this review article focuses on the diverse transfection abilities of the gene to the targeted cell on the basis of different shapes and sizes of gold nanoparticles in order to promote its effective expression for cancer treatment. In addition, recent trends in gold nanoparticle mediated gene silencing, gene delivery, detection and combinatory therapies are highlighted considering their cytotoxic effects on healthy human cells.

Various functions of gold nanoparticles in conjugation with nucleic acids.     

Efficient transfection of MG‐63 osteoblasts using magnetic nanoparticles and oscillating magnetic fields

Aims: To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG‐63) and investigate the effects of a novel non‐viral oscillating nanomagnetic gene transfection system (magnefect‐nano?) in enhancing transfection efficiency (TE). Methods: MG‐63 cells were transfected using MNPs coupled with a GFP‐carrying plasmid. The magnefect‐nano system was evaluated for transfection efficiency and potential associated effects on cell viability. Results: MG‐63 cells were efficiently transfected using MNPs and the magnefect‐nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. Conclusion: Non‐viral transfection using MNPs and the magnefect‐nano system can be used to transfect MG‐63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Theoretical and experimental characterisation of magnetic microbubbles

In addition to improving image contrast, microbubbles have shown great potential in molecular imaging and drug/gene delivery. Previous work by the authors showed that considerable improvements in gene transfection efficiency were obtained using microbubbles loaded with magnetic nanoparticles under simultaneous exposure to ultrasound and magnetic fields. The aim of this study was to characterise the effect of nanoparticles on the dynamic and acoustic response of the microbubbles. High-speed video microscopy indicated that the amplitude of oscillation was very similar for magnetic and nonmagnetic microbubbles of the same size for the same ultrasound exposure (0.5 MHz, 100 kPa, 12-cycle pulse) and that this was minimally affected by an imposed magnetic field. The linear scattering to attenuation ratio (STAR) was also similar for suspensions of both bubble types although the nonlinear STAR was ~50% lower for the magnetic microbubbles. Both the video and acoustic data were supported by the results from theoretical modelling.     

Poly(ethylenimine) and its role in gene delivery

Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.     

Transfection of primary chondrocytes using chitosan-pEGFP nanoparticles.

Chitosan-pEGFP nanoparticles were synthesized through the complex coacervation of the cationic polymer with pEGFP, in order to examine the potential of chitosan as a non-viral gene delivery vector to transfer exogenous gene into primary chondrocytes for the treatment of joint diseases. The nanoparticles were prepared at an N/P ratio of 3.8 and showed a spherical or irregular shape. The mean particle size and zeta potential of the nanoparticles freshly prepared with chitosan of different molecular weight were in the range of 100-300 nm and varied from +1 to +23 mV, respectively. Both the particle size and the zeta potential altered in DMEM of different pH. The transfection of primary chondrocytes was performed in different conditions by varying pH of transfection medium, molecular weight of chitosan and different plasmid dosage. Analysis of FACS demonstrated that the transfection efficiency could reach a much high level and the percentage of positive cells could exceed 50% in certain condition. These results suggest that chitosan-DNA nanoparticles have favorable characteristics for non-viral gene delivery to primary chondrocytes, and have the potential to deliver therapeutic genes directly into joint.     

Human serum albumin-polyethylenimine nanoparticles for gene delivery.

Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration.     

Protein kinase A inhibition modulates the intracellular routing of gene delivery vehicles in HeLa cells, leading to productive transfection

Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin- and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the degradative clathrin-mediated pathway towards lysosomes, and promoting that along the caveolae-mediated pathway. Here, we demonstrate the effective internalization of branched polyethylenimine polymers (BPEI), complexed with nucleic acids, by HeLa cells along both pathways. However, transfection efficiency or nuclear ODN delivery primarily occurs via the caveolae-mediated pathway, along which delivery into lysosomes is avoided. Interestingly, inhibition of intracellular protein kinase A (PKA) activity modulates the intracellular trafficking of both poly- and lipoplexes along the clathrin-mediated pathway by impeding trafficking into the late endosomal/lysosomal compartments, thus avoiding degradation. In case of BPEI polyplexes this promotes their transfection efficiency by 2-3 fold. Evidence excludes early endosomes as a major site for BPEI-mediated release/delivery. Rather, we identify a novel compartment, tentatively characterized as a transferrin⁻/rab9⁻/LAMP1⁻ compartment, to which cargo within the clathrin-mediated pathway of endocytosis is rerouted upon inhibition of PKA, and which may act as an alternative and effective site of cargo release in gene delivery. Our findings offer new opportunities for improving gene delivery by non-viral based nanoparticles.     

Non-viral gene delivery for p53

Abnormality in the tumor suppressor gene p53 is one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 function is seen as a particularly promising approach for cancer gene therapy. In recent years, considerable research effort has centered upon developing and improving non-viral delivery systems as alternatives to viral vectors for gene delivery. These methods include the use of lipoplexes and polyplexes, and even delivery of naked DNA. Optimally effective cancer gene therapy requires treatment of metastatic as well as local disease, and to achieve this end, systemic delivery systems for therapeutic genes will be required. This review will discuss some of the recent advances in ways to improve targeting, transfection efficiency and stability for systemic, non-viral p53 gene therapy.     

Intracellular tracking of protamine/antisense oligonucleotide nanoparticles and their inhibitory effect on HIV-1 transactivation.

Membrane transport of antisense oligonucleotides (ODN) is an inefficient process which requires special carriers for their intracellular delivery. We have developed a delivery system for AS-ODN and their phosphorothioate analogues (AS-PTO) directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA for efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges in the range of -18 to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides. A double labeling technique with fluorescently tagged protamine and AS-ODN was used to follow the intracellular fate of the nanoparticles. Protamine/AS-ODN nanoparticles showed release of the antisense compound leading to specific inhibition of tat mediated HIV-1 transactivation. In contrast, protamine/AS-PTO complexes were stable over 72 h, and failed to release AS-PTO. These results demonstrate that protamine/AS-ODN nanoparticles are useful for future therapeutical application to inhibit viral gene expression.     

Less is more: Investigating the influence of cellular nanoparticle load on transfection outcomes in neural cells

Genetic engineering of cell transplant populations offers potential for delivery of neurotherapeutic factors to modify the regenerative microenvironment of the injured spinal cord. The use of magnetic nanoparticle (MNP)‐based vectors has reduced the traditional reliance on viral methods and their associated obstacles in terms of scale up and safety. Studies utilizing magnetic assistive platforms for MNP‐mediated gene delivery have found transfection efficiency in astrocytes (a major transplant and homeostatic neural cell type) to be both frequency‐ and amplitude‐dependent. It is widely assumed that increased intracellular particle load will enhance transfection efficiency in a cell population. Therefore, we tested repeat delivery of MNP:plasmid complexes in conjunction with oscillating magnetic field parameters—a process termed “magneto‐multifection”—in astrocytes of primary origin in an attempt to enhance transfection levels. We show (a) levels of transfection using magneto‐multifection equal that seen with viral methods; (b) reporter protein expression using two reporter plasmids shows a diverse profile of single/dual transfected cells with implications for delivery of a “cocktail” of neurotherapeutic proteins; and (c) contrary to expectation, an inverse relationship exists between particle load and reporter protein expression.     

Recent advances on biocompatible and biodegradable nanoparticles as gene carriers

ABSTRACT

Introduction: Gene therapy mainly depends on the use of appropriate delivery vehicles with no induction of immune responses and toxicity. The limitations of viral gene carriers such as induction of immunogenicity, random integration in the genome of the host, limitations in the size, has led to a movement toward non-viral systems with much safer properties. Biodegradable and biocompatible polymeric nanocarriers due to several unique properties such as excellent biocompatibility, prolonged gene circulation time, prevented gene degradation, passive targeting by using the enhanced permeability and retention (EPR) effect, and possibility of modulating polymers structure to obtain desirable therapeutic efficacy, are among the most promising systems for gene delivery. However, biodegradable gene delivery systems have some limitations such as inadequate stability and slow release of therapeutics which have to be overcome. Thus, a variety of advanced functional biodegradable delivery systems with more efficient gene delivery activity has recently been introduced.

Areas covered: This review summarizes different aspects of biodegradable and biocompatible nano carriers including formulation, mechanism of intracellular uptake, various potential applications of biodegradable nanoparticles and finally recent studies on the therapeutic efficacy of these nanoparticles in sustained delivery of genes.

Expert opinion: Biocompatible and biodegradable polymers will play a necessary and important role in developing new and safe carriers for oligonucleotide delivery. More working and the development of optimized polymers will reveal more their efficacy in the treatment of patients via helping in better gene therapy.     

Self-assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery.

A self-assembled nanoparticle was prepared using a hydrophobically modified glycol chitosan for gene delivery. A primary amine of glycol chitosan was modified with 5beta-cholanic acid to prepare a hydrophobically modified glycol chitosan (HGC). The modified chitosan spontaneously formed DNA nanoparticles by a hydrophobic interaction between HGC and hydrophobized DNA. As the HGC content increased, the encapsulation efficiencies of DNA increased while the size of HGC nanoparticles decreased. Upon increasing HGC contents, HGC nanoparticle became less cytotoxic. The increased HGC contents also facilitated endocytic uptakes of HGC nanoparticles by COS-1 cells, which were confirmed by a confocal microscopy. The HGC nanoparticles showed increasing in vitro transfection efficiencies in the presence serum. In vivo results also showed that the HGC nanoparticles had superior transfection efficiencies to naked DNA and a commercialized transfection agent. The HGC nanoparticles composed of hydrophobized DNA and hydrophobically modified glycol chitosan played a significant role in enhancing transfection efficiencies in vitro as well as in vivo.     

Polyphosphoramidate gene carriers: effect of charge group on gene transfer efficiency

Cationic polymeric carriers have been widely used for gene delivery. However, the structure-function relationship, especially the effect of charge groups of cationic polymeric carriers on the transfection activity, is poorly understood. To examine this important parameter, a series of cationic polymers, polyphosphoramidates (PPAs) with an identical backbone, same side chain spacer, similar molecular weights but different charge groups containing primary to quaternary amino groups (PPA-EA, PPA-MEA, PPA-DMA and PPA-TMA, Figure 1) were synthesized. The DNA-binding affinity of these four PPAs increased in the order of PPA-EAPPA-MEA>PPA-DMA>PPA-TMA. Particle size and zeta potential of four different types of PPA/DNA nanoparticles did not show significant correlation with PPA structure. These PPAs did not show significant buffering capacity within pH 5-7, even though transfection mediated by PPA-EA was the only one that seemed to be limited by endolysomal escape. Endocytosis of DNA mediated by PPAs was also similar (17-22%) for all four PPAs. However, the transfection efficiency of these PPAs varied significantly. In vitro transfection efficiency of PPAs decreased in the order of PPA-EA>PPA-MEA>PPA-DMA approximately PPA-TMA. Nanoparticles with PPA-EA containing primary amino groups gave the highest transfection efficiency in cell lines at the charge ratios from 6/1 to 20/1 (+/-). Matching the trend of transfection efficiency observed in vitro, PPA-EA mediated the highest transgene expression, comparable to that of polyethylenimine, in the spinal cord following intrathecal injection of the nanoparticles. These results establish that PPA gene carriers with primary amino group side chains are more potent than those with secondary, tertiary or quaternary amino groups in vitro and in the intrathecal gene delivery model.     

Delivery of immunostimulatory RNA oligonucleotides by gelatin nanoparticles triggers an efficient antitumoral response

RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-α. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8+ T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic.     

Calcium phosphate nanoparticles: second-generation nonviral vectors in gene therapy

Adverse effects of viral vectors, instability of naked DNA, cytotoxicity and low transfection of cationic lipids, cationic polymers and other synthetic vectors are currently severe limitations in gene therapy. In addition to targeting a specific cell type, an ideal nonviral vector must manifest an efficient endosomal escape, render sufficient protection of DNA in the cytosol and help provide an easy passage of cytosolic DNA to the nucleus. Virus-like size calcium phosphate nanoparticles have been found to overcome many of these limitations in delivering genes to the nucleus of specific cells. This review has focused on some applications of DNA-loaded calcium phosphate nanoparticles as nonviral vectors in gene delivery, and their potential use in gene therapy, as well as highlighting the mechanistic studies to probe the reason for high transfection efficiency of the vector. It has been demonstrated that calcium ions play an important role in endosomal escape, cytosolic stability and enhanced nuclear uptake of DNA through nuclear pore complexes. The special role of exogenous calcium ions to overcome obstacles in practical realization of this field suggests that calcium phosphate nanoparticles are not 'me too' synthetic vectors and can be designated as second-generation nonviral vectors for gene therapy.