Biomed Res Int. 2014;2014:821607.

D-glucosamine conjugation accelerates the labeling efficiency of quantum dots in osteoblastic cells.

Igawa K1, Xie MF2, Ohba H3, Yamada S1, Hayashi Y1.

1Department of Cariology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan.

2Biotechnology Division of Care Four Company Ltd., Kokura-Kitaku, Kitakyushu 802-0071, Japan.

3Measurement Solution Research Center, National Institute of Advanced Industrial Science and Technology, Tosu, Saga 841-0052, Japan.

 

 

Abstract

Quantum dots (QDs) are useful imaging tools in the medical and biological fields due to their optical properties, such as a high fluorescence intensity, remarkable resistance to photobleaching, broad absorption spectra, and narrow emission spectra. This is the first study to investigate the uptake of carboxylated QDs conjugated with D-glucosamine (core size: approximately 3 nm, final modified size: 20-30 nm) into cultured osteoblastic cells. The QDs attached to the cell surface and were transported into the cytoplasm within approximately three hours of culture, whose process was clearly demonstrated using specific fluorescent staining of the cell membrane. Although the intranuclear distribution was not observed, a dramatic decrease in the transfer of quantum dots into the cytoplasm was recognized after approximately seven days of culture. Other interesting phenomena include the escape of the quantum dots from lysosomes in the cytoplasm, as confirmed by the merging of both QD fluorescence and specific fluorescent staining of lysosomes in the cytoplasm. These findings suggest that D-glucosamine conjugation enhances proton absorption in acid organelles and promotes the lysosomal escape of QDs.

PMID: 24818156

 

Supplement:

D-glucosamine accelerates cell proliferation and differentiation in vitro at a super-low concentration and the ideal tissue regeneration in dental pulp wounds occurs where there is a minimal initial inflammatory reaction (1). Then, D-glucosamine acts as a biocompatibly stable medicament with the initial stage of wound healing compared to the application of chitosan polymer. This study is the first report from our laboratory about the application of D-glucosamine for biomedical fields. After this study, our research interest has directed toward the possibility of cell membrane stability by D-glucosamine. More recently, our group reported that bradykinin and 5-hydroxytryptamine-induced nociceptive responses were significantly suppressed by the direct application of GlcN (2, 3). Then, GlcN may, thus, exhibit an anti-nociceptive effect by binding to these sodium channels, resulting in a longer open time, which produces the hyperpolarization of nerves in relation to the cell membrane stability. These studies were the first electrophysiological investigations for nerve membrane stability by using D-glucosamine.

To investigate further membrane stabilizing activity of D-glucosamine, we chose two unique methods which consisted of QDs (4) and electroporation (5) applications into cultured osteoblastic cells. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells) (Fig. 1). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

We believe that cell membrane stability by D-glucosamine application may be related to the basic and fundamental mechanism for creating human health-care, probably through the antioxidative and immunostimulating properties. D-glucosamine can function as a biological response modifier and can boost natural protective responses to both cells and tissues.

 

References

  1. Matsunaga T, Yanagiguchi K, Yamada S, Ohara N, Ikeda T, Hayashi Y 2006 Chitosan monomer promotes tissue regeneration on dental pulp wounds, Journal of Biomedical Materials Research A 76(4):711-720
  2. Kaida K, Yamashita H, Toda K, Hayashi Y 2013 Effects of glucosamine on the tooth pulpal nociceptive responses in the rat, Journal of Dental Sciences 8(1):68-73
  3. Kaida K, Yamashita H, Toda K, Hayashi Y 2014 Suppressive Effects of D-glucosamine on the 5-HT sensitive nociceptive units in the rat tooth pulpal nerve, BioMed Research International vol. 2014, Article ID 187989, 6 pages
  4. Igawa K, Xie M-F, Ohba H, Yamada S, Hayashi Y 2014 D-glucosamine conjugation accelerates the labeling efficiency of quantum dots in osteoblastic cells, BioMed Research International vol. 2014, Article ID 821607, 5 pages
  5. Igawa K, Ohara N, Kawakubo A, Sugimoto K, Yanagiguchi K, Ikeda T, Yamada S, Hayashi Y 2014 D-glucosamine promotes transfection efficiency during electroporation, BioMed Research International vol. 2014, Article ID 485867, 4 page *The first two authors contributed equally to this work.

 

fig yhayashi

Figure 1  A: GFP-positive cells after electroporation with 0.005% GlcN-containing buffer. B: Phase contrast image of area A. Scale bar = 20 mm. (Igawa, K. et al., BioMed Research International 2014: Article ID 485867. 2014. With permission)

 

Contact:

Yoshihiko Hayashi, DDS, PhD

Professor and Chair

Department of Cariology

Nagasaki University Graduate School of Biomedical Sciences

Nagasaki852-8588, JAPAN

E-mail: hayashi@nagasaki-u.ac.jp

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