Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy
Introduction
In the post-genome era, the analysis of disease-related genes has rapidly advanced, and the medical application of the information obtained from gene analysis is being put into practice. In particular, the development of effective method to transfer the gene of interest selectively and efficiently into the targeted cells is essential for the gene therapy of refractory diseases, in-vivo functional analysis of genes and establishment of animal models for diseases. However, a suitable carrier for selective and efficient gene transfer to the targeted cells is still being developed. Although various types of viral and non-viral carriers have been developed for gene transfer, they are limited to use by viral-associated pathogenesis and low transfection efficiency, respectively. For the cell-selective gene transfer, many investigators have focused on ligand-modified non-viral carriers such as liposomes [1], [2], [3], [4], emulsions [5], micelles [6] and polymers [7], because of their high productivity and low toxicity. On the other hand, since the gene transfection efficiency by non-viral carriers is poor, it is difficult to obtain the effective therapeutic effects by gene therapy using non-viral carriers. Moreover, in the gene transfection using conventional ligand-modified non-viral carriers, since the carriers need to be taken up into cells via endocytosis following by interaction with targeted molecules on the cell membrane, the number of candidates which are suitable as ligands for targeted gene delivery is limited.
Some researchers have attempted to develop the transfection method using external stimulation, such as electrical energy [8], physical pressure [9] and water pressure [10], to enhance the gene transfection efficiency. Among these, gene transfection method using US exposure and microbubbles enclosing US imaging gas, called “sonoporation method”, have been focused as effective drug/gene delivery systems [11], [12], [13], [14]. In the sonoporation method, microbubbles are degraded by US exposure with optimized intensity, then cavitation energy is generated by the destruction of microbubbles. Consequently, the transient pores are created on the cell membrane, and large amount of nucleic acids are directly introduced into the cytoplasm through the created pores [13], [15], [16]. However, the in-vivo gene transfection efficiency by conventional sonoporation method administering the nucleic acids and microbubbles separately is low because of the rapid degradation of nucleic acids in the body [17], the large particle size of conventional microbubbles [15] and the different pharmacokinetic profiles of the nucleic acids and microbubbles. Moreover, to transfer the gene into the targeted cells selectively by sonoporation method in vivo, the control of in-vivo distribution of nucleic acids and microbubbles, which are separately administered, is necessary.
In our previous report [16], we have demonstrated the effective transfection by combination-use method using our mannosylated lipoplexes composed of Man-C4-chol: DOPE [1], and conventional Bubble liposomes (BLs) [12] with US exposure. However, this combination-use method is complicated because of the necessity for multiple injections of mannosylated lipoplexes and BLs; therefore, it is difficult to apply for medical treatments using multiple transfections. In addition, the difference of in-vivo distribution characteristics between mannosylated lipoplexes and BLs might be decreased its transfection efficacy. Therefore, it is essential to develop the US-responsive and cell-selective gene carriers constructed with ligand-modified gene carriers and microbubbles.
Taking these into considerations, we examined the gene transfection system for effective DNA vaccine therapy using physical stimulation and ligand-modification. First, we developed US-responsive and mannose-modified gene carriers, Man-PEG2000 bubble lipoplexes (Fig. 1), by enclosing perfluoropropane gas into mannose-conjugated PEG2000-DSPE-modified cationic liposomes (DSTAP: DSPC: Man-PEG2000-DSPE (Fig. 1))/pDNA complexes. Then, we evaluated the enhanced and cell-selective gene expression in the APCs by intravenous administration of Man-PEG2000 bubble lipoplexes and external US exposure in mice. Finally, we examined high anti-tumor effects by applying this method to DNA vaccine therapy using OVA-expressing pDNA.
Section snippets
Mice and cell lines
Female ICR mice (4∼5 weeks old) and C57BL/6 mice (6∼8 weeks old) were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). All animal experiments were carried out in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the US National Institutes of Health and the guideline for animal experiments of Kyoto University. CD8-OVA1.3 cells, T cell hybridomas with specificity for OVA 257∼264-kb, were kindly provided
In-vitro gene transfection properties by Man-PEG2000 lipoplexes
Polyethylene-glycol (PEG) modification of particles is necessary to enclose US imaging gas stably and to prepare the small-sized microbubbles for in-vivo administration [12]. Firstly, we developed mannose-conjugated PEG2000-modified lipids (Man-PEG2000-DSPE (Fig. 1)) to prepare the APC-targeted small-sized microbubbles and determined the in-vitro and in-vivo transfection characteristics of mannose-conjugated PEG2000-modified lipoplexes (Man-PEG2000 lipoplexes) containing Man-PEG2000 lipids. The
Discussion
To obtain high therapeutic effects by DNA vaccination using tumor-specific antigen-coding gene, it is essential to transfer the gene selectively and efficiently into the APCs, such as macrophages and dendritic cells [31], [32]. However, it is difficult to transfer the gene into the APCs selectively because of the number of APCs is limited in the organ [33]. Since the APCs are expressed a large number of mannose receptors [28], [29], we and other groups have developed mannose-modified non-viral
Conclusion
In this study, we developed the gene transfection method using Man-PEG2000 bubble lipoplexes and US exposure. This transfection method enabled APC-selective and efficient gene expression, and moreover, effective anti-tumor effects was obtained by applying this method to DNA vaccine therapy against cancer. This method could be widely used in a variety of targeted cell-selective and efficient gene transfection methods by substituting mannose with various ligands reported previously [2], [3], [4],
Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health and Labour Sciences Research Grants for Research on Noninvasive and Minimally Invasive Medical Devices from the Ministry of Health, Labour and Welfare of Japan, and by the Programs for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and by the Japan Society for the
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