Large-scaled composite hydrogels (several
centimeters) have been prepared for use in biomedical applications such as cartilage and bone. However, few preparation methods of nanocomposites of PVA and Hap have been reported. In this study, the nano-, microparticles of PVA, HAp and DNA were obtained by using high hydrostatic pressure technology. It is thought that this is achieved by the pressure-induced Inhibitors,research,lifescience,medical quick formation of PVA particles that could incorporate secondary and third substrates, such as DNA and HAp, without phase separation [15, 31]. 3.2. Cytotoxicity Test Figure 4 shows the result of the cytotoxicity test of PVA/DNA and PVA/HAp/DNA complexes. Inhibitors,research,lifescience,medical The high viability of COS-7 cells incubated with them is shown, irrespective of the concentration of PVA and HAp. PVA and HAp are biocompatible materials [32, 33]. The PVA/DNA complex is nontoxic
because of the composite formation of PVA and DNA via hydrogen bonding interaction [16]. HAps were encapsulated in PVA/HAp/DNA complexes. Consequently, it is considered Inhibitors,research,lifescience,medical that the nontoxicity of PVA/HAp/DNA complexes was achieved by these combinations. Figure 4 Viability of COS-7 cells incubated with (white) PVA/DNA complexes and (black) PVA/HAp/DNA complexes for 24h. DNA conc.: 0.0025w/v%. Each value represents the mean ± SD (n = 3). 3.3. Cellular Uptake of PVA/HAp/DNA Nanoparticles In order to investigate cellular Inhibitors,research,lifescience,medical uptake of the HAp/DNA complex, PVA/DNA, and PVA/HAp/DNA nanoparticles, rhodamine-labeled plasmid DNA was used. Figure 4 shows DMXAA in vivo fluorescent microscopic images of COS-7 cells incubated with complexes of PVA, Hap,
and rhodamine-labeled DNA for one and 24h. After 1h incubation, fluorescent spots were poorly observed for DNA and PVA/DNA nanoparticles (Figures 5(a) and 5(c)), whereas Inhibitors,research,lifescience,medical a lot of bright red fluorescent spots on many cells were shown in the case of HAp/DNA and PVA/HAp/DNA complexes (Figures 5(b) and 5(d)), indicating the effective absorption of them onto cells because of their higher specific gravity. However, strong aggregation of HAp/DNA complexes was observed due to the fact that the nature of HAp however particles tends to result in an aggregation in the aqueous medium [34]. For PVA/HAp/DNA nanoparticles, PVA bearing HAp could attenuate the aggregation property of HAp. After 24h incubation, the aggregation of the HAp/DNA composite was still observed (Figure 5(f)). The internalization of PVA/HAp/DNA nanoparticles into cells was exhibited. Also, the subcellular distribution of DNA was observed in some cells (Figure 5(h)) similar to that of PVA/DNA nanoparticles (Figure 5(g)). This strongly suggests that HAp in PVA/HAp/DNA nanoparticles could be dissolved during the intracellular process, probably due to the endocytosis pathway.
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