Current Drug Delivery - Volume 3, Issue 1, 2006

The knowledge of the genome and proteome will result in enormous opportunities in the identification of new therapeutic molecules which will ultimately have a major impact on human health. The new types of pharmaceuticals will be DNA and RNA pieces, small peptides, large proteins and recombinant or subunit vaccines. The science and technology of the delivery and targeting of the 'products' of the genome and proteome are the next crucial links in the development of new approaches for the treatment and prevention of diseases. Developing non-invasive delivery approaches for macromolecules in general is a major challenge. This is conspicuously reflected in the articles in this issue dealing with DNA delivery methods. To replace direct injection using needles or reimplanting ex vivo transfected keratinocytes or dermal fibroblasts, novel delivery methods for oligo- and polynucleotides may include less invasive procedures such as microprojectile techniques, electroporation and topically applied formulations. There is also effort to develop non-viral delivery systems in order to avoid the many disadvantages of viral vectors. This issue of Current Drug Delivery contains a collection of articles focusing on non-invasive delivery of DNA for therapeutic and vaccine purposes. The reviews by von drunen Littel-van den Hurk, Foldvari et al., Cui et al. and Choi and Maibach in this issue provide extensive coverage of the various administration methods of DNA into the body, the differences between the requirements for therapeutic and vaccine DNA and the effect of formulation design on DNA delivery. There are specific examples for dermal, oral, pulmonary and electrically-assisted delivery technologies (Lisziewicz et al., Wang et al., Densmore, Cemazar et al. and Foldvari et al.). There are reports on several novel technologies for non-invasive DNA delivery: silicon microneedles, DNAnanoparticles, biphasic vesicles (Biphasix(™)) and an invasin-based gene targeting system. Birchall et al. demonstrated the ability of microfabricated silicon microneedle arrays to create micron-sized channels through the stratum corneum of excised human skin and the resulting ability of the conduits to facilitate localized delivery of charged macromolecules and plasmid DNA. Lisziewicz et al. have developed a non-viral delivery system where the plasmid DNA, encoding appropriate retroviral genes, is encapsulated within pathogenlike nanoparticles. Topical application of these nanoparticles on abraded skin resulted in suppressed viral replication and increased survival time in HIV-infected macaques. Foldvari et al. demonstrated high level of plasmid DNA delivery after topical application on intact human skin using biphasic vesicles and evaluated the quantitative aspects of DNA delivery. Wang et al. described a Yersinia protein, invasin, that binds to a subset of b1 integrin receptors located on the apical membrane of intestinal M-cells, as potential delivery/targeting agents. By coupling invasin to a micro/nanoparticle carrier, the natural transport mechanism can be utilized for the oral delivery of therapeutic genes and gene-based vaccines. This issue intends to give the readers an excellent overview on issues associated with non-invasive delivery of DNA and some recent developments on potential technologies. It is also intended to reflect on the necessity of further significant efforts into delivery system development for macromolecular therapeutics. Even though the pharmaceutics and the engineering of delivery technologies does not seem (to some) as dazzling as the sequencing of the genome or the compiling of the proteome, it is probably the most crucial task necessary for turning genes and proteins into therapeutic products for humans and animals.

To develop a successful protein therapeutic, effective DNA delivery technologies are required that induce high and sustained levels of protein production in appropriate targets sites, whereas robust and long-lasting immune responses need to be induced by a DNA-based vaccine. Vectors for gene therapy and DNA vaccines must be resistant to degradation and attack by the immune system, have a satisfactory safety profile, and be able to express the therapeutic protein for the desired period of time. Effective non-viral vectors, which can express the proteins of interest at high levels, are available. However, since most of the DNA delivered in vivo is degraded before it can enter the nucleus, proper formulation and delivery are critical to the development of effective gene-based therapeutics and vaccines. These systems must be safe for human and veterinary clinical applications and yet ensure that the DNA survives the extra- and intracellular environment and is capable of entering the appropriate cellular compartments. In this review various potential and proven non-invasive chemical, mechanical, physical and biological DNA delivery systems for therapeutic and vaccine applications are discussed. A few of these approaches have been evaluated and proven to be promising in target species. Others, which promise to be less invasive, have only just started to be explored.

Cutaneous gene therapy and DNA vaccination are potential applications of plasmid delivery methods where a gene for an antigen or a therapeutic protein is inserted in the plasmid and applied to the skin. However, the delivery of the DNA plasmid is a major challenge due to the unusual physicochemical properties of the DNA, the tissue and cellular barriers and expression difficulties. Even though the skin is the most accessible organ of the body and it is an ideal target for gene therapy, the delivery of plasmid DNA across the skin is very difficult due to the specific barrier function of the stratum corneum and the inconsistent transfection rate of keratinocytes and other epidermal cells. To date there is no gene delivery system that was shown to be optimal for cutaneous gene therapy. In order to develop an efficient non-viral delivery vehicle we need to design a system that provides the combined properties of effective DNA condensation, cutaneous permeation, cellular transfection and sufficiently sustained expression. This paper reviews the formulation approaches and delivery methods for DNA through the skin in the context of the barriers both at the tissue and cellular levels for both vaccine and gene therapy applications.

Skin has evolved to protect not only by acting as a physical barrier, but also by its role in our powerful immune system. As a frontline of the host's defense against pathogens, skin is well equipped for immune surveillance. For example, compared to many other tissues, the epidermis of the skin contains a high population of Langerhans cells, which are very potent immature dendritic cells. Thus, targeting antigens to the skin epidermis should be able to efficiently induce strong immune responses. However, the forbidden barrier posed by the stratum corneum layer of the epidermis prevents effective entrance of antigens into the epidermis. Nevertheless, non-invasive immunization onto the skin has proven in the last several years to be a viable immunization modality. DNA vaccine is a vaccine made of bacterial plasmid DNA encoding an antigen of interest. Upon uptake of the plasmid, host express and process the encoding antigen, and then mount immune responses against it. DNA vaccine is advantageous over many other types of vaccines. The feasibility of noninvasive immunization onto the skin with DNA vaccine has been confirmed. Although the potency of the immune response has proven to be weak, many skin stratum corneum disrupting chemical and physical approaches and DNA vaccine carriers/adjuvants that significantly enhance the resulting immune response have been reported. In addition, research on elucidating the mechanism of immune induction from non-invasively, topically applied DNA vaccine has also been carried out. With further improvement and optimization, non-invasive immunization onto the skin with DNA vaccine should be able to elicit reliable and efficacious immune response to a variety of antigens.

Topical DNA vaccines have been shown to elicit both broad humoral and cellular immune response in vivo. The skin is an attractive site for the delivery DNA antigens for DNA vaccination. However, due to skin's barrier properties, the penetration of DNA and the applications of topical vaccination are limited. To improve permeability of stratum corneum and the potency of topical DNA vaccines, efficient delivery systems are needed. Topical vaccination has been achieved using topical application of naked DNA with or without tape stripping and DNA/lipid based complex such as liposomes, niosomes, Transfersomes®, or microemulsion. All methods resulted in significant enhancement in humoral and cellular immune response over naked DNA alone. To develop more cost-effective and needle free vaccines, skin targeted immunizations are required. This overview focuses on the comparison of the potency of topical DNA vaccine between naked DNA and DNA-lipid based complex.

The mucosal surface of the gastrointestinal (GI) tract is the first line of defense against foreign pathogens and toxins ingested orally. The content of the GI tract is constantly being sampled by the immune system through specialized epithelial cells known as M-cells, which are present in the Peyer's patches of the gut, providing a thin covering over lymphoid tissue. In this way, once a harmful entity is found an immune response can be activated to eliminate the threat. Many bacterial pathogens, such as Yersinia, Listeria, Salmonella, and Shigella, have evolved ways of exploiting M-cells to gain entrance to the body. The Yersinia species is of particular interest since its extracellular protein invasin provides one of the most direct and efficient manners of host cell invasion. Invasin binds to a subset of β1 integrin receptors located on the apical membrane of intestinal M-cells, thereby facilitating the bacteria's entry into the cells and the lymphatic system underneath. This mechanism is highly specific and effective, making the invasin protein a very attractive modality for use in the oral delivery of molecules that include therapeutic genes and gene-based vaccines. This article provides a brief overview of the molecular structure and properties of the Yersinia invasin as related to the protein's ability to facilitate binding and entry into M-cells. Also discussed are several innovative approaches that demonstrate the use of invasin as an effective targeting agent for biological and synthetic gene carrier systems, and the future prospect of developing invasinbased oral gene delivery formulations.

One of the most noninvasive approaches to drug delivery is via inhalation. The delivery of genes via aerosol holds promise for the treatment of a broad spectrum of pulmonary disorders and offers numerous advantages over more invasive modes of delivery. Delivery of genes expressing secretory therapeutic proteins or peptides may even have application to a number of nonpulmonary diseases. After the cloning of the cystic fibrosis gene, there was great interest in the delivery of genes directly to the lung surfaces via inhalation and most early efforts focused on the use of nonviral vectors, particularly cationic lipids. Early on, nebulization shear forces, inefficient penetration of mucous barriers and inhibitory effects of surfactant and other lung specific features generally resulted in a lack of therapeutic effect. But in recent years, a number of other nonviral and even viral vectors have been delivered successfully in this manner. Polyethyleneimine (PEI)-based formulations have proven stable during nebulization and result in transfection of a very large proportion of epithelial cells throughout the airways (though the level of transgene expression per cell may be relatively low), as well as significant, though lower levels of transfection throughout the lung parenchyma. Most importantly, therapeutic responses have been obtained in several animal lung tumor models when PEI-based complexes of p53 and IL-12 genes were delivered by aerosol. This approach may also prove useful as a means of localized genetic immunization. In addition, inhalation delivery of some formulations seems to be associated with surprisingly low toxicity and has resulted in little or no immunostimulatory response to the unmethylated CpG sequences in bacterially-produced plasmid DNA, which has presented a challenge to repeated gene therapy via many other modes of delivery.

The stratum corneum (SC) represents a significant barrier to the delivery of gene therapy formulations. In order to realise the potential of therapeutic cutaneous gene transfer, delivery strategies are required to overcome this exclusion effect. This study investigates the ability of microfabricated silicon microneedle arrays to create micron-sized channels through the SC of ex vivo human skin and the resulting ability of the conduits to facilitate localised delivery of charged macromolecules and plasmid DNA (pDNA). Microscopic studies of microneedle-treated human epidermal membrane revealed the presence of microconduits (10-20μm diameter). The delivery of a macromolecule, β-galactosidase, and of a 'non-viral gene vector mimicking' charged fluorescent nanoparticle to the viable epidermis of microneedle-treated tissue was demonstrated using light and fluorescent microscopy. Track etched permeation profiles, generated using 'Franz-type' diffusion cell methodology and a model synthetic membrane showed that >50% of a colloidal particle suspension permeated through membrane pores in ∼2 hours. On the basis of these results, it is probable that microneedle treatment of the skin surface would facilitate the cutaneous delivery of lipid:polycation:pDNA (LPD) gene vectors, and other related vectors, to the viable epidermis. Preliminary gene expression studies confirmed that naked pDNA can be expressed in excised human skin following microneedle disruption of the SC barrier. The presence of a limited number of microchannels, positive for gene expression, indicates that further studies to optimise the microneedle device morphology, its method of application and the pDNA formulation are warranted to facilitate more reproducible cutaneous gene delivery.

Electrically-assisted gene delivery is a non-viral gene delivery technique, using application of square wave electric pulses to facilitate uptake of plasmid DNA into the cells. Feasibility and effectiveness of this method in vivo was already demonstrated, elaborating on pulse parameters and plasmid construction. However, there were no studies performed on sequencing and timing of plasmid DNA injection into the tumors and application of electric pulses. For this purpose we measured luciferase expression in two tumor models (LPB fibrosarcoma, B16F1 melanoma) after electricallyassisted gene delivery at varying time intervals between the pCMV-Luc plasmid injection and electroporation. Expression of luciferase was determined by measurement of its activity using luminometer. The results demonstrated that pCMV-Luc plasmid has to be injected before the application of electric pulses, since no measurable expression was detected in the tumors when pCMV-Luc plasmid was injected after electroporation of tumors. In both tumor models the highest transfection efficiency was obtained when pCMV-Luc plasmid was injected not less than 5 minutes but also not more than 30 minutes before the application of electric pulses. The results also demonstrated variability in the transfection efficiency depending on the tumor model. High expression was obtained in B16F1 tumor model (∼5500 pg luc/mg tumor) and lower in LPB fibrosarcoma (∼200 pg luc/mg tumor). In conclusion, our results demonstrate that regardless of the susceptibility of the tumors to electrically-assisted gene delivery, the best timing for pCMV-Luc plasmid is between 30 to 5 minutes prior to the application of electric pulses to the tumors.

DermaVir employs a topical, non-invasive method for vaccine delivery to dendritic cells. The vaccine product contains plasmid DNA as the active ingredient, encoding authentically expressed retroviral genes with appropriate safety modifications. The non-viral delivery system packages the DNA within pathogen-like nanoparticles and studies indicate that vaccine antigens are taken up by epidermal Langerhans cells, the precursors of dendritic cells. DermaVir loaded dendritic cells reach the draining lymph node target but not the bloodstream nor indiscriminately other organ systems. Safety data from DermaVir immunized infected macaques indicate improved survival, absence of apparent toxicities other than transient erythema and lack of recombination between the vaccine DNA and the infectious viral DNA integrated in the host genome. DermaVir represents a potential new approach for the treatment of HIV infection to be utilized either in conjunction with antiretroviral therapy or during structured treatment interruption.

Topical gene delivery to the skin shows great potential for painless, non-invasive administration of novel vaccines and therapeutic agents. The challenge is to develop a pharmaceutically acceptable system that can deliver suitable amounts of plasmid DNA to produce the desired level of response. The purpose of this study was to quantitatively assess DNA delivery by a novel lipid-based biphasic delivery system into the viable layers of excised human skin. Biphasic lipid vesicle formulations, incorporating plasmid DNA were evaluated in vitro in flow-through diffusion cells. Fifty mg DNA formulation containing 10 μg DNA was applied to full-thickness human breast skin for 24 hours. Residual formulation was removed and the skin was washed with PBS, then tape-stripped, followed by DNase treatment to remove surface bound DNA. Skin samples were homogenised and digested overnight with Proteinase K. The resulting supernatant was used as a template for quantitative PCR. Three formulations yielded a significant degree of dermal absorption compared to the controls. Formulation 26-3-2-DNA indicated that approximately 1x10 9 copies of plasmid were absorbed per cm2 skin. Other formulations resulted in 5 x 10 6 copies/cm2 skin (17C3-1-DNA) and 5 x 10 8 copies/cm2 skin (26-3-1- DNA). Biphasic vesicles delivered significant quantities of plasmid DNA into the 'viable' layers of human skin in vitro. The successful delivery of this large (∼ 4, 400 kDa) charged molecule through intact stratum corneum represents a major advance in transdermal macromolecule delivery.

Stratum corneum (SC) is comprised of lipids, protein and low molecular weight water-soluble components. Changes in these skin micro constituents can be understood by instrumental methods like differential scanning calorimetry (DSC) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The former provides information about changes in thermotropic behavior of SC lipids and proteins, whereas the latter provides data about alterations at molecular and conformational level. Most of the DSC thermograms of intact mammalian SC show two reversible and two irreversible transitions in the temperature range of 25-125%C. The reversible endotherms are ascribed to lipid melting transitions, whereas the irreversible endotherms are ascribed to protein denaturation. Similarly, the FTIR spectral bands of SC occurring between 2920-2850 cm-1 and between 1650-1550 cm -1 have been suggested to arise from lipid and protein molecular vibrations, respectively. Treatment of skin with solvents or permeation enhancers alters the composition of lipids or their molecular arrangement in the skin microenvironment, which leads to changes in permeability of drug molecules. Furthermore, inhibition of lipid synthesis in epidermis with concomitant decrease in enthalpy of lipid endothermic transitions and reduction in height and area of asymmetric and symmetric C-H stretching peaks have been found to be directly correlated with enhanced permeation of drugs. In addition, method of skin preparation, type of skin, types of enhancer etc. also influence both the nature and intensity of responses recorded in spectrographs and thermograms. Therefore, the modification in spectrographs and thermograms of skin samples treated with various enhancers, vehicles etc. are expected to provide better insight into their mechanism of action on the skin. This review article shall critically evaluate the thermotropic and infrared spectroscopic data of SC/epidermis after various treatments.

Several groups have shown that vaccine antigens can be encapsulated within polymeric microparticles and can serve as potent antigen delivery systems. We have recently shown that an alternative approach involving charged polylactide co-glycolide (PLG) microparticles with surface adsorbed antigen(s) can also be used to deliver antigen into antigen presenting cell (APC). We have described the preparation of cationic and anionic PLG microparticles which have been used to adsorb a variety of agents, which include plasmid DNA, recombinant proteins and adjuvant active oligonucleotides. These PLG microparticles were prepared using a w/o/w solvent evaporation process in the presence of the anionic surfactants, including DSS (dioctyl sodium sulfosuccinate) or cationic surfactants, including CTAB (hexadecyl trimethyl ammonium bromide). Antigen binding to the charged PLG microparticles was influenced by several factors including electrostatic and hydrophobic interactions. These microparticle based formulations resulted in the induction of significantly enhanced immune responses in comparison to alum. The surface adsorbed microparticle formulation offers an alternative and novel way of delivering antigens in a vaccine formulation.

Although oral drug therapy for tuberculosis exists and is widely followed, its major drawbacks are lack of patient compliance and development of adverse effects like hepatotoxicity on long term use. Absence of new therapeutic agents and the above mentioned demerits have led to search for alternative methods for delivery of antitubercular agents. Colloidal drug carriers, a popularly utilized delivery system has been deeply explored for the cause. The article discusses the advances in the management of tuberculosis by the use of particulate and vesicular drug carriers by parenteral, inhalational and oral routes. Use of this delivery strategy has led to massive reduction in the dosage resulting in toxicity alleviation. As a number of studies have already been undertaken in experimental models, it will be a promising tool in the prevention of relapse and successful treatment of tuberculosis in patients.