Nanodiamond as Hemoglobin based Artificial Blood Substitutes
Y.- C. Lin,1 C.-Y. Lee1, L.-W. Tsai1, E. Perevedentseva,1, 3 K.-J. Huang2, Y.- S. Ye,1
C.- L. Cheng1,*
1
Department of Physics, National Dong Hwa University, Hualien, Taiwan
2
Department of Life Science, National Dong Hwa University, Hualien, Taiwan
3
P. N. Lebedev Physics Institute, Russian Academy of science, 119991 Moscow, Russia
clcheng@mail.ndhu.edu.tw
The vital function and the sacred nature of blood have received numerous studies since
the beginning of modern science. With the increasing needs in blood supplies, the
development of blood substitutes remains an urgent but challenging task. Nanodiamond (ND)
has shown great potential in bio and medical applications owing to its physical-chemical
properties and biocompatibility. Various surface functionalization methods have been
developed. These render the possibility of grafting biomolecules, includes human blood
proteins, on ND surface. The blood proteins adsorbed on ND form stable complex suitable for
medical applications. We have earlier demonstrated ND does not alter the oxygen delivery
properties of red blood cells. This may provide a possible scheme for blood substitutes.
We first address the interaction of nanodiamonds (5 and 100 nm) with human red blood
cells (RBC) in vitro. Measurements have been facilitated using Raman spectroscopy, laser
scanning fluorescence spectroscopy and laser diffractometry (ektacytometery). Data on cell
viability and hemolytic analysis are also presented. Results indicate that the nanodiamonds in
studied condition do not cause hemolysis and the cell viability is not affected. Importantly, the
oxygenation/deoxygenation process was not found altered when nanodiamond interacted with
the RBC. Next, we study the immune responses of 100 nm nanodiamond interacts with the
tissue-resident murine macrophages, Raw 264.7. The cytotoxicity of ND for macrophages
was analysed using MTT assay. The mechanisms of ND engulfment by macrophage were
analyzed via alternatively blocking different possible pathways. ND was found non-cytotoxic
to macrophages and could be engulfed by macrophages through the clathrin-mediated
endocytosis pathway. Upon internalization, ND was entrapped in lysosome; does not induce
macrophages to produce the pro-inflammatory cytokines nor activate those correspondent
transcriptional factors.
With these positive results, we propose to use nanodiamond as haemoglobin based
artificial blood substitute. Blood proteins are adsorbed on ND surface to form ND-Protein
complexes. We study the interaction of ND with blood proteins, such as hemoglobin (Hb) and
blood plasma proteins, human serum albumin (HSA) and -globulin. The surface protein
loading was analyzed using UV-Visible spectroscopy. FTIR spectroscopy, -potential
measurements and dynamic light scattering were applied to access the structural changes of
the proteins. The FTIR absorbance spectra revealed strong adsorption of proteins on ND
surface, but only non-significant transformations of the proteins conformation has been
observed. The oxygenation state of Hb in ND-Hb complex was examined using UV-Visible
and Raman spectroscopy, and compared with human red blood cell on the behaviour of the
oxygen delivery ability of the artificial blood. The results of -potential measurements point
out the binding force between these proteins and NDs is not only electrostatic but also other
mechanisms can be involved, such as hydrophilic and hydrophobic interactions between NDs
and proteins. The study shows at the interaction with blood components, ND does not affect
significantly the structure and function of proteins. The ability of ND to load blood proteins
may have great potential for the development of artificial blood substitutes.