| Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), extracted from the root of Plumbaginaceae plants, has been used in traditional medicine for more than 2500 years. It possesses antiatherosclerotic, antiparasitic, antimicrobial , anti-hepatic fibrosis and antioxidant properties. It has also been shown to exert anticancer and antiproliferative activities in animal models as well as in cells in culture.Lots of research work was done and the anti-tumor effect of plumbagin has been preliminarily explored in our group. However, application of plumbagin to clinical therapy of tumor is blocked because of its poor water solubility and high toxicity.Single walled Carbon nanotubes (SWNTs) are kinds of tube-like nanomaterials formed by curling up a graphite sheet into a cylinder. Protein, DNA, small interfering RNA (siRNA), and drug could be delivered into tumor cells by water- soluble SWNTs as carriers. Objective:1. To prepare water-soluble PLB-PEG-SWNTs conjugate and improve the solubility of PLB by coupling PLB and PEG modified SWNTs.2. To investigate the anti-tumor effect of PLB-PEG-SWNTs in vitro.Methods:1. After oxidation and cutting of SWNTs raw materials by using concentrated strong acid, the obtained short functionalized SWNTs is modified by PEG, and subsequently covalently linked to PLB molecules.2. The composition and structure of PLB-PEG-SWNTs were characterized by Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). And the loading of PLB in PLB-PEG-SWNTs conjugate was determined by using Ultraviolet and visible spectrophotometry (UV-Vis).3. The cytotoxicity of PLB-PEG-SWNTs on Bel-7402, CNE1, HepG-2, and MCF-7 cell lines were detected by MTT assay in vitro.4. The Ultrastructural changes of Bel-7402 cells after PLB-PEG-SWNTs treatment were examined by transmission electron microscope (TEM). The effects of the conjugate on apoptosis and cell cycle of Bel-7402 cells were investigated by the methods of acridine orange (AO) fluorescent staining and Annexin V-FITC/PI double staining FCM assay.ResuLts:1. The as-prepared PLB-PEG-SWNTs conjugate exhibits high water solubility.2. TEM characterization indicated that the length distribution of the oxidized and cut SWNTs is approx. 300 nm, and FT-IR characterization showed that PLB is linked to PEG modified SWNTs by covalent connection.3. The conjugate showed obvious inhibiting effect on Bel-7402, CNE1, HepG-2, and MCF-7 cell lines. And it decreased the survival rate of these tumor cells in a concentration-dependent manner. The IC50 value for Bel-7402 was 80μg/mL, which contained 1.12μg/mL PLB and was lower than the IC50 value of dissociative PLB (7μg/mL).4. PLB-PEG-SWNTs conjugate (50, 150, 450μg/mL) could arrest cell cycle of Bel-7402 at G2/M phase, and after treated with the conjugate for 24h, the related apoptosis ratios were 2.20±0.03%, 3.26±0.67% and 24.33±1.70%, respectively, while the one of the control group was 0.383±0.097%. The results of acridine orange (AO) fluorescent staining showed morphological changes associated with the character of apoptosis of Bel-7402. Condensation of chromatin at margins of nuclei, disintegration of nucleolus and vacuoles in cytoplasm were observed in PLB-PEG-SWNTs group by TEM.Conclusion:1. Water-soluble PLB-PEG-SWNTs conjugate has been synthesized by connecting PLB to PEG modified SWNTs.2. PLB-PEG-SWNTs conjugate displays significant anti-tumor effect in vitro.3. The conjugate can arrest the cell cycle at G2/M phase and induce the apoptosis of Bel-7402 tumor cells.
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