The success of gene therapy is largely dependent on the development of a delivery system that can selectively and efficiently deliver a gene to target cells with minimal toxicity. Although viruses are efficient in introducing genes to cells, the safety concerns regarding the use of virus in humans make nonviral DDS an attractive alternative. Compared with viral vectors, nonviral DDS for DNA are particularly suitable with respect to simplicity of use, ease of large scale production, and lack of specific immune response, So far, several methods are developed as nonviral DDS : naked DNA injection, gene gun, electroporation, complex formation with cationic liposome or cationic polymer, However, there are some drawbacks with each of these nonviral DDS, including their lower efficiency than viral vectors in gene transfer and their transient gene expressions, For an efficient gene transfer in vivo, nonviral DDS should overcome the delivery barriers existing in the processes of its biodistribution, cellular uptake and intracellular routing. The physicochemical properties of a DNA/DDS complex will affect its passage through capillaries, extravasation, capture by the mononuclear phagocytes, and uptake by target cells. Interaction with blood components would alter these properties of the DNA complex, which results in the altered biodistribution. Since DNA complex is mainly entrapped in the endosome/lysosame after its cellular uptake, it should be released into the cytoplasm and delivered to the nucleus. To escape the endosome, fusogenic lipids or peptides are involved in some nonviral DDS. A DDS having a buffering capacity to the pH drop in the late endosome is also effective in releasing DNA into cytoplasm by destabilizing the endosome. A successful gene therapy could be achieved by overcoming these delivery barriers by developing a well-designed nonviral DDS for DNA. Recent topics on the immune responses against CpG motifs in bacterial DNA and the correction of mutations in genes by chimeric RNA/DNA oligonucleotides are also reviewed.

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Cationic liposomes have been considered as a potential non-viral vector for gene delivery, which possess a low immunogenicity unlike viral vectors. The gene transfer effcieney of cationic liposomes is lower than that of viral vectors but the advance of recent study revealed to enhance the gene expression levels of cationic liposomes. The most problem of cationic liposomes seem to be the lack of organ or cell selectivity because the lung shows highest gene expression organ after intravenous injection. Applying a cell-specific targeting technology to the liposomes would improve in vivo gene delivery and reduce unexpected side effects. Liver parencymal cells and liver non-parenchymal cells exclusively express large numbers of high affinity asialoglycoprotein and mannose receptors, respectively. Receptor-mediated gene delivery systems seems to introduce foreign DNA into specific cell types in vivo. However, we have confirmed that not only the nature of the ligands grafted to carriers but also the overall physicochemical properties of the complexes need to be optimized for the cell-selective targeting of plasmid DNA. In this article, we attempt to evaluate the the gene delivery system based on the physicochemical properties of plasmid DNA and plasmid DNA/cationic liposomes or glycosylated cationic liposomes.

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Dendritic cells(DCs) are the most potent antigen presenting cells and DCs play a crucial role in the generation of antitumor immune responses. Using recently developed methods, it is now possible to culture large numbers of DCs from the adherent fraction of peripheral blood mononuclear cells and CD 34+ hematological stem cells. Antigen loading to DC has been demonstrated using a variety of strategies including direct delivery of exogenous peptide fragments or tumor extracts, or by endogenous antigen expression and processing within DCs after delivery of antigen-encoding DNA molecule. DCs efficiently take up soluble and cell-derived protein antigens, but some studies have recently demonstrated that DCs expressing vector encoded tumor associated antigens (TAA) can induce protective and therapeutic immunity in murine cancer models. The transduction of DCs with a TAA-encoding transgene offers several potential advantages over peptide-pulsing. First, expression of an entire TAA gene would enable them to efficiently present multiple epitopes associated with different MHC class I and II molecules. In this way, gene-modified DCs would likely stimulate multiple T-cell clones, each one reactive to a different epitope from the same tumor antigen. And there is no need to carefully identify and synthesize different peptides to be used with every possible MHC subtype. Second, TAA expression within DCs provide the cell with a renewable supply of antigen for presentation, and DCs can present antigen for long time (at least 6 days). Various methods of gene transfer to DCs were examined, including recombinant adenovirus vector, retroviral vectors, DNA/liposome complex and electroporation. Retroviruses have been used to transduce human DCs in vitro. Human DCs progenitors (CD 34+cell) were shown to differentiate into normal DCs after transduction of retroviruses. But the transduction rates were lower than the adenoviral systems. Adenovirus rarely integrates into host genome. It also transfects a large number of terminally differentiated human DCs. For immunotherapy, DCs expressing adenovirus vector encoded TAA are the most attractive candidate. Although some important questions still need to be answered, this approach to immunotherapy of cancer may be promising.

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Though number of fundamental and clinical studies of gene therapy offered its exciting potential in treating human diseases, several issues such as non-specific immune reaction owing to viral vectors were pointed out. Therefore, increasing demand will be placed on the development of non-viral vectors. The most commonly examined synthetic carriers are based on cationic polymer/DNA complexes or cationic lipid/DNA complexes. However, the insufficient efficacy and toxicity of these delivery systems limited their applications to therapeutic uses. For improving potentiality of the non-viral vector, considerable efforts are recently paid in designing tailor-made biomaterials. Such materials may allow genes and genetic materials i) to be delivered to specific tissues and cells, ii) to be translocated to particular compartment, i.e. cytosol or nucleus, in the cells, and iii) to exhibit enhanced functionality at the site of action. Unsatisfactory collidal stability and non-specific interaction of the complex with proteins and cells resulted in massive uptake of the complex by non-target tissues including reticuloendotherial systems (RES) and interfered with cell-specific delivery. Modification of cationic polymers or lipids with water-soluble polymers, such as poly(ethylene glycol) (PEG) and polysaccharides, has been employed to overcome these shortcoming. Such soluble carriers are further conjugated with cell-specific ligands to increase cell uptake or provide cell-specificity. To facilitate cytosolic delivery of DNA, various peptides or polymers that perturb plasma or endosomal membrane are utilized. Nuclear localizing signal which helps translocation of macromolecules from cytosol to nucleus through nuclear pores was also conjugated to DNA or carrier systems to control final destination. Insufficient binding affinity of oligonucleotides (ODN) with target molecules would be another reason why ODN medicine on the basis of antisense and antigene strategy can not take effects as it expected. Efforts to increase hybridization opportunity of ODN medicine with its target molecules using tailor-made biomaterials are also described in this review.

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Since the first gene therapy started in USA in 1990, more than 300 clinical trials have been performed. The bases of gene therapy are to transfer a therapeutic gene into target cells by using a gene transfer system (vector) and to treat the disease with physiological activities of gene products for the treatment. The vector used in these processes consists of a gene transfer system and a gene used for the treatment of diseases. This system is associated with the relation of a delivery system and a drug in the Drug Delivery System(DDS) that has hitherto been studied. In fact, many findings obtained in the research and development on DDS have been applied to the recent studies on vectors. At the beginning, gene therapy was expected as “a dream therapy” to give rise to therapeutic effects on any kind of diseases. Nevertheless, only very limited cases in gene therapy gave results indicating overt efficacy in clinical practices, although such studies have been made actively until now. It should be noted that one of the characteristics in protocols of studies in which positive results are obtained is a topical application of vectors. These facts suggest that active targeting at the aimed sites can not be accomplished by systemic administrations of most vectors developed until now, and topical applications have been selected as the most effective procedure to ensure an expression of the gene for treatment at the target sites. It is considered that the key for success in the gene therapy in the future is a further improvement of vectors.

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Relationship between permeation of ethanol and leaching of hemoglobin from capillary blood, as a possible index of skin irritancy, was examined in hairless rats. Ethanol solutions were applied in vivo on the abdominal skin for 5h and then content of Fe, instead of hemoglobin, in the application site was measured. Permeation of ethanol through the skin was examined in vitro in the corresponding condition. The index, i. e. Fe content in the skin, shows correlation with cumulative amount of ethanol permeated or maximum flux of ethanol through the skin, while that wasn't correlated with concentration and content of ethanol in the applied solution. Since a slow and continuous permeation of ethanol for 8h didn't damage the skin, the maximum flux is more important parameter for the effect of ethanol on the skin irritation than the cumulative amount of ethanol permeated. Relationship between permeation of ethanol and morphine hydrochloride (MPH) was also examined. The cumulative amount of MPH was correlated with the maximum flux of ethanol. A formulation containing ethanol should be designed carefully so as to attain effective skin permeation of drug while avoiding the occurrence of skin irritation.

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The effects of ultrasound on the skin bioconversion of estradiol esters were investigated in the hairless mouse skin pretreated by ultrasound in vitro. The time-course of the drugs was well described by the first order reaction kinetics with or without ultrasound pretreatment. The ultrasound markedly reduced the rate constants of skin metabolism of β-estradiol diacetate (ED) as well as β-estradiol mono-acetate (EA).

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Diltiazem(DZ) is widely used for the treatment of chronic anginapectoris, hypertension and arrythmia. In order to increase therapeutic efficiency, skin permeation of DZ was investigated employing 2-chamber diffusion cells in which Wistar rat abdominal skin was mounted. DZ suspended solution was used as donor solution. The receiver cell was filled with water. Ethanol(EtOH), isopropanol(IpOH), propyleneglycol(PG) were used as solvent in donor solution. The flux of DZ was increased by the addition of 30% EtOH or 30% IpOH to the donor solution. Further increase of solvent concentration from 30% to 50% led to a slight increase of the flux. To improve the skin permeation of DZ, menthol and O-ethylmenthol (MET) were selected as absorption enhancers. The flux of DZ markedly increased by the addition of these enhancers. Irrespective of kind of enhancers, the concentration of ethanol accumulated in the skin was increased by co-administration of enhancers. The concentration of menthol in the skin increased significantly with an increase of menthol in the donor phase, although the concentration of MET in the skin was very low. Therefore, the solvent dragging in the skin is an important factor to exhibit enhancement action. A little amount of MET can induce the accumulation of ethanol in the skin, however, much greater amount of menthol was required to obtain a similar efficiency. The microviscosity of the stratum corneum lipid was determined by using an electron spin resonance(ESR) spectrometer. In use of menthol, the microviscosity of stratum corneum lipid decreased gradually as an increase of menthol amount in the donor phase. On the other hand, a little amount of MET was sufficient to exhibit such decreasing efficiency. These findings suggest that menthol and MET act directly to the skin to fluidize dense barrier structure of stratum corneum under the co-existence of ethanol.

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