Tumor cells, especially metastatic and cancer stem cells, in comparison to somatic cells, are characterized by overexpression of tumor-specific marker CD44, which have a high affinity to hyaluronic acid fragments [1]. Therefore we decided to use hyaluronic acid fragments as targeting moiety grafted to fluorescent nanodiamond particles [2] for monitoring of malignant process.
We conjugated the hyaluronic acid fragments (8-15 kDa) with amino terminated nanodiamond particles by reductive amination using sodium cyanoborohydride NaBH3CN as a reducing agent. Amino terminated nanodiamonds were prepared from nanodiamonds having sp2 hybridisation on the surface by arylation of in situ generated diazonium salt of (4-(2-aminoethyl)aniline. The resulting conjugate (hyaluronate-grafted nanodiamond) was isolated from unreacted hyaluronic acid fragments by centrifugation and thorough washing.
Further we tested how is the hyaluronic acid attached to the surface of the nanodiamonds. The addition of citric acid to the conjugate would result in the exchange of the hyaluronate for the citrate anions in case the hyaluronate is attached by physisorbtion onto the surface of the nanodiamond. The acidic environment would also cleave the Schiff base, in case the reductive amination is not successful. If the hyaluronate is covalently attached on the nanodiamond particles, the addition of citric acid (followed with rinsing) would not substantially affect the zeta potential of the conjugate. We measured the zeta potential of the amino terminated nanodiamonds, which showed a positive value. After the reductive amination the zeta potential abruptly changed to negative values. The fact, that the zeta potential remained strongly negative after the washing of the hyaluronate-grafted nanodiamond surface by citric acid proves the covalent attachment of hyaluronic acid to the nanodiamond particles.
Acknowledgements:
This work was partly-financed by Technology Agency of the Czech Republic, TA ČR alfa, No. TA02010760 and TA01011165, and FP7 DINAMO No. 245122, Ministry of Education and Sport Czech Rep. No.: 7E11071 and CZ.1.07/2.3.00/20.0306 (OPVK project); and Academy of Sciences of the Czech Republic No.: RVO 61388963 and RVO 61389005.
References:
[1]. Platt, M., Szoka Jr., F.C. Mol Pharm, 2008, 5, 474-486.
[2]. Petrakova, V. et al. Adv. Functional Mat., 2012, 22, 4, 812-819.
