Biomedical Engineering Reference
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T m increased from 49 to 59 uC. Further increase of the PS content did not
increase T m . These results confirm that the PS backbone modification induces
a more stable complex than the PO backbone, while only 20 mol%
modification should be enough to give the same increment in T m as the
100% one. Our results demonstrate that the complex with the phosphor-
othioate dA 40 was thermally more stable than phosphodiester dA 40 , and the
stability increased to the same level as the fully phosphorothioated one by only
replacing 20-30 mol% of the oxygen of the phosphodiester moiety.
d n 4 y 3 n g | 0
8.3
Application of the Complex to ODN Delivery
8.3.1
Uptake of the Complex by Macrophages
We used RT-PCR to analyze the expression of dectin-1 mRNA in
thioglycolate-elicited macrophages (thio-Mw; Figure 8.4A). 45 The expression
doubled after 24 h and stayed at this level to 48 h (data not shown), which is
consistent with the drastic increase at the protein level for the 24 h pre-cultured
cells measured with a flow cytometer (Figure 8.4B). For Figure 8.4A, the
expression at 0 h is due to elicitation by thioglycolate in vivo, and the further
increment can be ascribed to additional stimulation generated by cellular
adhesion to the culture plate. 46 The expression of CD11b, a macrophage-
specific antigen, was unaffected by 24 h culture (data not shown), indicating
that the macrophage's essential nature was unaltered during the pre-culturing.
We examined the uptake of ODN/SPG by 3-, 24-, and 48-h thio-Mw
(Figure 8.4C). Even some naked ODN was taken up, regardless of the dectin-1
expression level. This result can be interpreted in the knowledge that
phosphorothioate ODN can enter cells via an unidentified pathway. 47 The
uptake of ODN/SPG increased from 3-h to 24-h thio-Mw. This increment can
be ascribed to dectin-1-mediated uptake, because dectin-1 expression at 24 and
48 h was more enhanced than at 3 h, as mentioned above. After 24 h culture
we treated thio-Mws with TAMRA-labeled ODN or its complex with FITC-
labeled SPG, and examined the distributions of these two markers by
fluorescence microscopy (Figure 8.4D). The naked ODN gave no fluorescence,
while the complex gave strong fluorescence with both FITC and TAMRA,
appearing as small dots scattered and sprinkled over the cells. This distribution
suggests that they are localized inside vesicles, most likely endocytosis vesicles.
Superimposition of these two images showed that ODN and SPG were co-
localized in the cells. These results demonstrate that SPG can act a pilot
molecule to transport the bound ODN to APCs by means of dectin-1.
8.3.2 IL-12 Secretion Due to Administration of CpG-ODN/SPG
Complexes
Peritoneal macrophages express TLR9 to induce CpG DNA-mediated IL-12. 49
Accordingly, the complexes with various compositions were applied to the
 
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