新型MR对比剂长循环超顺磁氧化铁脂质体纳米微粒的体内外实验研究

The in vitro and in vivo experimental study of long-circulating superparamagnetic iron oxideliposome nanoparticles as novel MR contrast agent

目的评价自制新型MR对比剂：长循环超顺磁性氧化铁（superparamagnetic ironoxide，SPIO）脂质体纳米微粒的药代动力学。方法以加入SPIO为对照组，加入长循环SPIO脂质体纳米微粒为实验组。（1）巨噬细胞的体外吞噬实验：将巨噬细胞株RAW264．7经复苏、培养，细胞达到80％～90％融合后，以每孔2．5×10’个细胞接种于培养板，对照组和实验组细胞与不同浓度药物孵化后，分别测定细胞中Fe浓度和Fe染色情况，并用析因设计的方差分析对结果进行统计学处理。（2）小鼠体内药物分布实验：C57BL／6J雄性小鼠6只，每组2只，设为空白对照组、对照组、实验组，经尾静脉分别注射生理盐水、SPIO以及长循环SPIO脂质体后牺牲动物，经病理检查观察2组药物在体内的分布情况。（3）荷肺癌裸鼠MR成像实验：建立20只BALB／c裸鼠移植型肺癌模型，分为对照组和实验组，每组10只，分别经尾静脉注入SPIO和长循环SPIO脂质体纳米微粒后行MR扫描，测量增强前后2组实验动物各时间点肝脏和肿瘤ROI的信号强度，绘制信噪比（SNR）时间动态变化曲线，采用协方差分析比较2组动物肝脏及肿瘤增强12h后SNR差异有无统计学意义。（4）细胞毒性实验（噻唑蓝，MTT法）：检查2种药物对人肝细胞株HL-7702细胞的毒性作用，用析因设计的方差分析对结果进行统计学处理。结果（1）巨噬细胞的体外实验：析因分析结果表明，对照组细胞内Fe含量明显高于实验组，差异有统计学意义（F=296839．9，P=0．000）。普鲁士蓝染色显示对照组巨噬细胞染色较深，实验组巨噬细胞染色较浅。（2）小鼠体内药物分布实验：对照组肝、脾、肺、肾组织内蓝染颗粒多于实验组。（3）荷肺癌裸鼠MR成像实验：平扫对照组肝脏SNR为31．47±0．56，实验组为30．89±1．41；增强扫描12h后对照组为17．00±0．96，实验组为22．29±0．73。平扫对照组肿瘤SNR为58．41-4-0．61，实验组为58．44±1．08；增强扫描12h后对照组为58．50±0．63，实验组为52．47±1．18。经协方差分析表明增强12h后对照组肝脏SNR显著低于实验组（F=167．022，P=0．000）；而实验组肿瘤SNR显著低于对照组（F=266．106，P=0．000）。（4）细胞毒性实验：随着培养液中Fe浓度的增加，HL-7702细胞生存率均有下降趋势，但析因分析结果表明，实验组药物的细胞毒性较对照组低（F=2256．204，P=0．000）。结论长循环SPIO脂质体纳米微粒在体内外均有较好的抗巨噬系统吞噬功能，在体内实现了长循环，对非网状内皮系统的肿瘤也具有良好的T2负性被动靶向强化效果；同时还降低了SPIO对细胞的毒性作用。

Objective To evaluate pharmacodynamics of prepared long-circulating superparamagnetic iron oxide （SPIO） liposomes. Methods Control and experimental groups wereestablished after adding SPIO or long-circulating SPIO liposomes as agents. （ 1 ） Macrophages experiment in vitro ： the RAW 264. 7 macrophage cell strains were recovered, cultured and seeded in the culture plate at a density of 2. 5 × 105 cells/well until they reached 80%-90% confluence. The intracellular Fe uptake of control and experimental group cells were quantified by Ferrozine assay after incubation with different concentrations of drugs. Factorial design analysis of variance was used as statistics method. Prussian blue staining method was used to detect staining of experimental cells. （2） Drug biodistribntion in mice： C57BL/6J（ n-6） were classified into blank control group （n = 2 ）, control group （n = 2 ） and experimental group（n = 2）. Saline, SPIO and long circulating SPIO were injected via the tail vein in the blank control group, control group and experimental group respectively. Then distribution of drugs in the body was observed by pathological examination. （3） MR imaging of tumor-bearing nude mice： 20 BALB/c nude mice bearing lung cancer models were established and classified into control group and experimental group. After administration of drugs, all animals underwent MR scanning. Signal intensities of livers and tumors were measured, SNR-time dynamic curves were drew. Covariance analysis was used to compare post-enhanced SNR at the 12th hour. （4）Cytotoxicity studies （MTF） ： cytotoxicity of both drugs on human liver cell line HL-7702 was studied, and statistically analyzed using factorial design analysis of variance. Results （ 1 ） Macrophages experiment in vitro： The nanoparticle uptake by macrophage cells evaluated by ferrozine assay showed the uptake of blank SPIO was higher than long-circulating SPIO liposomes. Compared with the blank control group, there was strong blue staining in the macrophages with Prussian staining in the control group and little blue staining in the experimental group. （2） Drug biodistribution in mice： for blue stained ceils composed of iron particles, the amounts in the liver, spleen, lung, kidney of the control group were more than those in the experimental group. （3） MR imaging of tumor-bearing nude mice ： the non-enhanced SNR of livers and tumors in the control group and experimental group were 31.47 ± 0. 56, 30. 89 ± 1.41, 58.41 ± O. 61,58.44 ±1.08, respectively. After injecting of contrast agents, SNR of livers and tumors in the control group and experimental group were 17.00 ± 0. 96, 22. 29 ± 0. 73, 58.50 ± 0. 63, 52.47 ± 1.18, respectively. The covariance analysis showed that SNR of the livers in the control group after 12 hours was significantly lower than the experimental group （ F = 167. 022, P = 0. 000 ） ; while the SNR of the tumors in the experimental group was significantly lower than the control group （ F = 266. 106, P = 0. 000）. （4） Cytotoxicity of nanoparticles by MTF method： the viability of HL-7702 cells tend to decrease with the increase of Fe concentration. Cytotoxicity in the long-circulating SPIO liposomes was lower than the SPIO （ F = 2256. 204,P = 0. 0013）. Conclusions Long-circulating SPIO liposomes we prepared reveal suitable sizes, even distribution, and good anti-macrophage ability in vitro and in vivo. They have long circulation characteristic and T2 negative enhancement effect in the transplanted lung cancers, while they still maintain low cytotoxicity.