Preliminary Study On The Functionalization, Cytotoxicity Of Single-Walled Carbon Nanotubes And Their Applications As Gene Carrier For Pain Therapy

Objective:To prepare functionalized single-walled carbon nanotubes suspension; To evaluate the cytotoxicity of single-walled carbon nanotubes; To investigate the gene silencing effect of intrathecally injecting functionlized single-walled nanotubes/NR2B-siRNA complex on inflammatory pain of murine.Methods:(1) Disperse single-walled carbon nanotubes (SWNTs) with surfactant cetyltrimethyl ammonium bromide (CTAB) and ultrasound dispersing method. Get mixture of functionalized single-walled carbon nanotube and NR2B-siRNA with a series of mass ratio, keep still and then centrifuge at high speed, test the combination of single-walled carbon nanotube with NR2B-siRNA by conducting agarose gel electrophoresis and then take photograph with gel imaging systems.(2) Cultivate Hela cell and evaluate the cytotoxicity of SWNTs, SWNT-COOH, CTAB and SWNT-COOH/CTAB by cell viability (MTT) and cell apoptosis experiments(Hochest33342fluorescent dye).(3) Forty male C57BL/6mice, weighing about18-22g, were randomly divided into four groups(n=ten each):blank control group (group C), the negative control group (group N), the experimental group (group A) and the positive control group(group P). Then seven days latter, the formalin inflammatory pain mice models were established and spontaneous pain behavioral changes were observed one hour postoperation, then lumbar segments of spinal cords were taken in each group to detect the expression of NR2BmRNA.Results:(1) Surfactant cetyltrimethyl ammonium bromide (CTAB) modified single-walled carbon nanotubes can form stable suspension of functionalized single-walled carbon nanotubes (f-SWNTs) in deionized water; f-SWNTs can well combine with siRNA and the mass ratio between (f-SWNT) and NR2B-siRNA may reach saturation between1:1.5and1:2.(2) SWNTs contain more kinds of impurities compared with acid-purified SWNT(SWNT-COOH). SWNTs were more toxic than SWNT-COOH. Concentration analysis indicated that cytotoxicity of the surfactant conjugated to SWNT-COOH was more related to the toxicity of the surfactant molecules cetyltrimethyl ammonium bromide(CTAB), the cytotoxicity of CTAB and SWNT-COOH/CTAB were acceptable at low concentrations (0.5-50μg/ml).(3) The nociceptive response in the acute phase were indistinguishable among all groups (P>0.05), the nociceptive response in the tonic phase significantly decreased in the experimental group (group A) and the positive control group(group P) compared to other groups(group C and group N)(P<0.02); the expression of NR2BmRNA in the experimental group (group A) and the positive control group(group P) were lower than that in other three control group (P<0.05).Conclusion:(1) Single-walled carbon nanotubes could well dispersed by surfactant cetyltrimethyl ammonium bromide (CTAB) to form functionalized single-walled carbon nanotubes suspension.(2) Compared with SWNTs, acid-purified SWNT(SWNT-COOH) could greatly decrease cytotoxicity, Concentration analysis of SWNT-COOH/CTAB indicated that the cytotoxicity of SWNT-COOH/CTAB were acceptable at low concentrations (0.5-50μg/ml).(3) CTAB-SWNT/NR2B-siRNA complex could effectively inhibit the expression of NR2B in spinal cord and significantly reduce the formalin inflammatory pain in the tonic phase.