The Radiation Protection Of WSC-DTPA Nanoparticles

Objective: To prepare WSC-DTPA polymers and their nanoparticles, andinvestigate their capability of penetrating into cellular and radiation protection.Methods: N-acylation reaction was used to prepare WSC-DTPA polymers, and ioncrossing-linking method was used to prepare their nanoparticles, which structures andmorphological properties were characterized. The FITC-WSC-DTPA nanoparticlesprobe was synthesized by the reaction between the amino-group on the WSC-DTPAnanoparticles and isothiocyanate group of FITC. The capability of FITC-WSC-DTPAnanoparticles probe penetrating through cellular membrane was observed by Live cellstation （LCS）. MTT assay was performed to evaluate their effects on the viability of thecells after γ-rays irradiation. Flow cytometry （FCM） with propidium iodide （PI） stainingwas used to measure the effects of WSC-DTPA nanoparticles on cell cycle phases whichwere exposed to6Gy⁶⁰Co_γirradiation. The apoptosis rates, intracellular Ca²⁺ level ofBRL cells, mitochondrion membrane potential （MMP） and reactive oxygen species（ROS） were detected by AnnexinⅤ-FITC/PI double staining, Fluo-3AM, JC-1andH₂DCFA commercial kits respectively. Surviving fraction （SF） of BRL cells exposed toWSC-DTPA nanoparticles combined with ionizing irradiation were analyzed by colonyformation assay.Results: The WSC-DTPA polymers were successfully synthesized, which wereidentified with FT-IR and¹H-NMR spectrum, and their content free amino groups were92.7%,74.3%,14.1%,1.59%respectively, which were analyzed by TNBS reagent. Themean diameters of WSC nanoparticles,92.7%,74.3%,14.1%and1.59%WSC-DTPAnanoparticles were about7.31,8.42,8.34and14.53nm respectively, and their PDI werebelow0.03. Detailed imaging analysis of the particle morphology showed that the WSC-DTPA nanoparticles were spherical with uniform size. LCS in this studydemonstrated that WSC nanoparticles and WSC-DTPA nanoparticles could penetrateinto cellular within2hours, while WSC and WSC-DTPA polymers could not passthrough the membrane. The results of cell viability indicated that the WSC-DTPAnanoparticles had no cytotoxity in the range from1.56μg/mL to50.0μg/mL. Comparedwith_γ-rays alone group,1.59%WSC-DTPA nanoparticles and92.7%WSC-DTPAnanoparticles with the concentration less than6.25μg/mL groups had no efficacy inradiation protection to BRL cells from damage induced by6Gy⁶⁰Co_γ-rays, whileWSC, WSC nanoparticles and92.7%WSC-DTPA nanoparticles with the concentrationmore than6.25μg/mL had good efficacy in evidence of higher survival rate （p＜0.05）.Compared with_γ-rays irradiation group, the proportions of SubG1of BRL cells afterexposure to6Gy⁶⁰Co_γ irradiation significantly decreased （p＜0.05） in WSC, WSCnanoparticles and92.7%WSC-DTPA nanoparticles with the concentration more than6.25μg/mL groups. And the proportions of SubG1did not changed in1.59%WSC-DTPA nanoparticles and92.7%WSC-DTPA nanoparticles with the concentrationless than6.25μg/mL groups. All results showed that apoptosis rate, intracellular Ca²⁺level and ROS of BRL cells in WSC, WSC nanoparticles and92.7%WSC-DTPAnanoparticles with the concentration more than6.25μg/mL groups significantlydecreased （p<0.05）, while MMP in these groups statistically decreased （p<0.05）. Andthese in1.59%WSC-DTPA nanoparticles and92.7%WSC-DTPA nanoparticles withthe concentration less than6.25μg/mL groups did not change. An excellent correlationwas found between the apoptosis rates and intracellular Ca2+level, the linear R-Squarein the range from0.9⁶⁰3to0.9940. The results of colony formation assay proved thatthe PF of1.59%WSC-DTPA nanoparticles and92.7%WSC-DTPA nanoparticles withthe concentration less than6.25μg/mL groups were less than1, while WSC, WSCnanoparticles and92.7%WSC-DTPA nanoparticles with the concentration more than6.25μg/mL groups were more than1respectively. The experimental protocols used inthe present study were to examine all of these inter-related toxic events in BRL cells.There was a good correlation between the change of all detecting results which made a protective effect clear derived from pre-exposure to WSC, WSC nanoparticles and92.7%WSC-DTPA nanoparticles with the concentration more than6.25μg/mL before6Gy⁶⁰Co_γ irradiation, while1.59%WSC-DTPA nanoparticles and92.7%WSC-DTPAnanoparticles with the concentration less than6.25μg/mL had no radiation protectiveeffects.Conclusion: The content free amino groups of92.7%,74.3%and1.59%WSC-DTPA nanoparticles have been developed successfully, and they can penetrateinto cellular within2hours by Live Cell Station. In vitro studies indicated that92.7%WSC-DTPA nanoparticles with the concentration less than6.25μg/mL have radiationprotective effects on BRL cells after6Gy⁶⁰Co_γ irradiation, which may be beneficial tothe further investigation.