Effects Of Cell-penetrating Peptide RDP Modified Gold Nanoclusters And Gold Nanoparticles On The Imaging And Axonal Outgrowth Of Neural Cells

Neurodegenerative disease is one of major diseases that are harmful to human life and health. According to previous study, the common feature of this kind of disease is that there is no change for the cell body of lesioned neuron, but the protrusions of these neural cells were significantly shorter than the normal neural cells. Axonal degeneration is one of common pathological features in many neurodegenerative diseases, and the main cause for lesioned central nervous system to lose their basic function. Therefore, the design of drugs with the function of diagnosis and treatment to the lesioned nerve cells will be important for implications of the neurodegenerative diseases and the treatment of theses dieases.Non-invasive imaging of neural cells of central nervous system (CNS) is a promising strategy for theranostics, however, it still remains challenge due to some drawbacks such as low signal-to-noise ratio resulting from scattering of fluorescence signals by thick brain tissues and low strict single-cell resolution. The principal barriers to detect the neural cells in vivo are tissue specificity of fluorescence-based imaging and the cell penetration ability to blood-brain barrier (BBB).To address these problems, coupling of specific BBB-penetrating carrier and stable fluorescence payload to achieve live and long-term imaging of neural cells, may provide new insights that will help elucidate the essential structural and functional information in CNS disorders and then harness the relative diseases.Our group recently developed a brain-targeting peptide RDP, a long peptide consisted of 39 amino acids, which is derived from 330-357 amino acid sequence of rabies virus glycoprotein, one of the key candidates mediates the virus into host cells. RDP has been suggested to have the capability of targeted-delivery several proteins and DNA into mouse brain, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), plasmids containing Lac Z or BDNF gene. Also, repeated administration RDP and its conjugates do not produce obvious toxicity or immunogenicity in the animals. Therefore, we assumed that RDP could be used as a safely targeting ligand in vivo after modification of AuNCs surface.Size less than 10 nm of gold nanomaterials, providing a variety of platforms for the interaction of nanoparticles with biomolecules, have been used in various fields either for diagnostic purpose, including imaging, turn on-and-off near infrared fluorescent bioimaging of cells, biological label, and electrochemical genosensor for DNA, or for cancer therapy as a multifunctional nanomedicine with capability of in vivo noninvasive near-infrared fluorescence. Low toxicity and ultrafine size of gold nanomaterials have captivated the interest of researchers indicating a potential candidate for passage through BBB. Especially, gold nanoclusters (AuNCs), as one of small size gold nanomaterials with the high quantum yields, allowed to be used for molecular location researches both in vitro and in vivo. However, the application of AuNCs is limited due to the lack of cell specificity and a widespread distribution in vivo, so the surface of AuNCs should be modified to improve the cell selectivity in case of bioimaging application.In this study, RDP is attached to AuNCs surface for long-term, nontoxic imaging and neural cell targeting in living cells and in mice. The novel peptide-conjugated AuNCs (RDP-AuNCs) were characterized by using fourier-transform infrared (FTIR) spectra, dynamic light scattering (DLS), Zeta potential analysis, gel electrophoresis, as well as UV-visible spectra. Localization of RDP-AuNCs in neural cells was performed by TEM, and mechanism of cellular attachment and entry into neural cell of the conjugates were indicated as receptor-mediated endocytosis through clathrin-coated pits. Both non-invasive imaging analysis and in vivo animal tests suggested that the RDP-AuNCs is an effective and specific bioimaging agent for neural cells.Another reseach section involved in the RDP modified AuNPs. According to the data of flow cytometric analysis, the cell cycle arrest at the G2/M phase of SH-SY5Y and U87 were declined, after the cells were treated with 6MP-AuNPs-RDP. The mophorlogy of SH-SY5Y cells treated with 6MP-AuNPs-RDP were changed when observed under the microscope and the length of axonal increased significantly. The HE staining of tissue section showed that the 6MP-AuNPs-RDP cannot induce the toxicity wnen mice were treated with short period, however, for the treatment with long period, there will be liver injury.