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pH-Sensitive Nano-Crystals of Carbonate Apatite Regulate Delivery and Release Kinetics of DNA for Efficient Expression in Mammalian Cells

E.H. Chowdhury and T. Akaike
Tokyo Institute of Technology, JP

Keywords:
carbonate apatite, nano-particle, pH-sensitive, DNA delivery, DNA release, acidic vesicles

Abstract:
The completion of whole human genome sequencing encourages the development of a powerful gene delivery technology for elucidating structure, regulation and function of genes and proteins in addition to the emerging biomedical applications, such as industry-based productions of therapeutic proteins and ‘gene therapy’ (1). Due to some major limitations of viral-mediated delivery, non-viral synthetic systems have become increasingly desirable. However, synthetic systems are notably inefficient compared to the viral ones in gene delivery and expression. Here we report on the development of the simplest, but highly efficient gene delivery device based on generated nano-apatite crystals. The synthesized apatite was characterized as carbonate apatite, as proven by the IR spectrum, with the diameter in the nanometer range, as indicated by scanning electron microscopy (Fig. 1). The nano-particles have high affinity to DNA but fast dissolution kinetics in acidic vesicles, following efficient endocytosis, for effective release of DNA (not showing here) and thus result in 5 to 100-fold higher transgene expression than the existing ones (Figure 2). Fluoride (F-) or strontium (Sr2+) which is known to decrease the solubility of carbonate apatite (2), dramatically reduced the transfection efficiency (Figure 3-a), suggesting that DNA release through particle dissolution, is a crucial factor in gene delivery pathway. Additionally, flexibility in modulating crystal dissolution kinetics enabled to control intracellular DNA release and an intermediate rate of DNA release enhanced survival of DNA (not showing here) and subsequent expression (Figure 3-b). Thus, considering the efficacy, safety and simplicity, this newly developed technology is highly promising for basic research laboratories, industries and clinical settings (3). References: 1. Luo, D. & Saltzman, W. M. Nature Biotechnology 18, 33-37 (2000). 2. Driessens, F. C. M. Nature 243, 420-421 (1973). 3. 4. Toyoda, K. et al. Gene Therapy 7, 1284-1291 (2000).

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