新型肿瘤靶向的叶酸—羧甲基壳聚糖—超顺磁氧化铁纳米粒的合成及评价
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摘要
恶性肿瘤已经成为威胁人类健康的最严重疾病之一,每年死于恶性肿瘤的患者高达数百万,造成劳动力的巨大损失和社会资源的惊人消耗,同时给患者及其家属带来巨大的经济和精神压力。因此恶性肿瘤已经成为全人类共同关注的重大课题。目前超顺磁氧化铁纳米粒的研究从最初侧重于MRI造影发展到目前的靶向给药、治疗和造影等。具有肿瘤靶向超顺磁氧化铁纳米粒由于其在MRI诊断、热疗及靶向给药中潜能,是目前靶向制剂研究中很热门的课题,本研究课题用三步法合成具有肿瘤靶向的超顺磁氧化铁纳米粒,并体内、体外考察其靶向性、细胞毒性和抗巨噬细胞吞噬作用等。
目的
1.制备肿瘤靶向的叶酸-羧甲基壳聚糖-超顺磁氧化铁纳米粒(Folic acid-o-carboxymethyl chitosans superparamagnetic oxide iron nanoparticles (FA-OCMCS-SPIO-NPs)并对处方工艺进行优化。
2.对中间产物(超顺磁氧化铁纳米粒和羧甲基壳聚糖超顺磁氧化铁纳米粒)及终产物(叶酸-羧甲基壳聚糖超顺磁氧化铁纳米粒)分别进行表征。
3.体外评价FA-OCMCS-SPIO-NPs的细胞毒性、逃避吞噬细胞吞噬作用及叶酸受体肿瘤靶向作用。
4.体内初步评价FA-OCMCS-SPIO-NPs的肿瘤靶向性及组织中分布情况。
方法
1三步法合成FA-OCMCS-SPIO-NPs
即先用共沉淀法合成超顺磁氧化铁纳米粒(superparamagnetic oxide iron nanoparticles SPIO-NPs),依次用羧甲基壳聚糖和叶酸对SPIO-NPs表面进行共价修饰制备FA-OCMCS-SPIO-NPs.
1.1共沉淀法合成超顺磁氧化铁纳米粒
考察体系的PH值、温度、沉淀剂的种类和用量对SPIO-NPs粒径和组成的影响。用X-Ray衍射仪、激光散射粒度分析仪、透射电子显微镜、傅里叶红外光谱仪、超导电子干涉等对产物进行表征。
1.2OCMCS对SPIO-NPs表面进行共价修饰
合成以SPIO-NPs为核,OCMCS为“壳”的OCMCS-SPIO-NPs。为了对合成工艺进行优化,先单因素考察SPIO与OCMCS的比例对纳米粒粒径的影响,确定最佳的SPIO与OCMCS比例,然后,以纳米粒的粒径作为因变量,羧甲基壳聚糖的分子量(A)、浓度(B)、超声时间(C)及超声细胞粉碎仪的功率(D作为自变量,采用正交设计(L824)进行处方优化和工艺筛选。用透射电镜、激光散射粒度分析仪、电位测定仪、X-Ray衍射仪、傅立叶红外和超导电子干涉(superconducting quantum interference measurement device、SQUID)等对OCMCS-SPIO-NPs进行表征。MRI扫描测定弛豫率、邻二氮菲法测定铁含量。
1.3利用OCMCS-SPIO-NPs的氨基基团与叶酸中羧基基团发生酰胺反应合成FA-OCMCS-SPIO-NPs,采用傅里叶红外扫描及X-Ray粉末衍射考察不同嫁接工艺对合成的FA-OCMCS-SPIO-NPs的红外图谱及Fe304晶型的影响,确定FA修饰OCMCS-SPIO-NPs的最佳工艺,按方法1.2对FA-OCMCS-SPIO-NPs进行表征；将FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs和SPIO-NPs溶液4℃留样,观察溶液的形状,初步考察他们的溶液稳定性。
2共沉淀法合成葡聚糖超顺磁氧化铁纳米粒(dextran-SPIO-NPs)并按方法1.2对合成的dextran-SPIO-NPs进行表征。
3体外评价细胞毒性、叶酸受体肿瘤靶向性及抗吞噬细胞吞噬作用。
3.1细胞毒性
LO2细胞(正常肝细胞),叶酸受体高表达的KB细胞(人口腔上皮癌细胞)和叶酸受体不表达的A549细胞(肺小细胞腺癌细胞)常规传代培养后,以5×103/孔接种于96孔板上,培养24h后,分别与不同浓度的FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs、dextran-SPIO-NPs和未包被的SPIO-NPs孵化24h,MTT法测定细胞毒性。
3.2肿瘤靶向性
3.2.1菲洛嗪法定量分析细胞内铁含量
A549细胞常规培养于RPMI1640全培溶液中、Hela细胞(人宫颈癌细胞)和KB细胞常规传代培养于FFRPMI1640全培溶液中,以2.5×105/孔接种于24孔板,培养24h,PBS液洗涤三遍后,与不同浓度的FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs孵化24后,菲洛嗪法测定细胞内铁含量。
3.2.2普鲁士蓝染色
A549细胞、Hela细胞和KB细胞常规传代培养后,以5×105/孔接种于6孔板上,孵化24h,分别加入0.4mgFe/ml的FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs2ml24h后,普鲁士蓝染色,高倍镜下观察。
3.3巨噬细胞摄取实验
3.3.1菲洛嗪法细胞内铁含量
常规培养RAW264.7(小鼠单核巨噬细胞白血病细胞),以2.5×105/孔接种于24孔板,培养24h,PBS液洗三遍后,加入一系列不同浓度的FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs、dextran-SPIO-NPs和未包被的SPIO-NPs,孵化24h后,PBS彻底清洗三遍,用0.5ml浓度为0.1M盐酸分散,菲洛嗪法测定细胞内铁含量。
3.3.2普鲁士蓝染色
常规培养RAW264.7(小鼠单核巨噬细胞白血病细胞),以5×105/孔接种于6孔板上,孵化24h后,分别加入0.4mgFe/ml的上述纳米粒2ml与RAW264.7细胞孵化24h后,进行普鲁士蓝染色,倒置显微镜下观察。
4.体内初步考察FA-OCMCS-SPIO-NPs的肿瘤靶向性及体内分布情况
4.1体内肿瘤靶向性考察
4.1.1实验动物：健康,SPF级裸鼠20只、鼠龄4-6周、雌雄各半,采用随机法将其分成两组：KB肿瘤细胞组(n=10)和A549肿瘤细胞组(n=10)。
4.1.2细胞培养和肿瘤模型建立
A549用细胞RPMI-1640全培常规培养、KB细胞用FFPRMI-1640全培常规传代培养,0.25%胰酶-EDTA消化,PBS液洗3遍后,无血清的培养基稀释制成每毫升含1.0×107/ml的细胞悬液,7号针头注射0.2 ml于裸鼠颈背部皮下。两周后,待瘤体最大直径≥1.0 cm时,选取已成功建立肿瘤模型的小鼠(16只,每组8只)进行动物实验。
4.1.3 MRI扫描
动物分组：将上述已成功荷瘤裸鼠(16只,每组8只)进一步随机分成四组：Ⅰ组,A549. OCMCS-SPIO-NPs(n=4);Ⅱ组,A549. FA-OCMCS-SPIO-NPs(n=4)；Ⅲ组,KB细胞、OCMCS-SPIO-NPs(n=4)；Ⅳ组,KB细胞、FA-OCMCS-SPIO-NPs组(n=4)。
4.1.3.1 MRI扫描：
采用西门子(Siemens Magnetom Vision Plus)1.5T,头部线圈,冠状面扫描,平扫后分别从尾静脉注射OCMCS-SPIO-NPs(5.54 mg Fe/ml)或FA-OCMCS-SPIO-NPs(5.62 mg Fe/ml)混悬液,剂量为0.25ml/只,给药3h后,MRI增强扫描。
4.1.3.2图像分析
T2WI图像测量平扫及增强3h后四组实验动物肿瘤感兴趣区(Region ofinterest, ROI)的信号强度(signal intense, SI)。测量时ROI放置在局部信号相对均匀,无明显伪影的区域,选取同一部位,测量三次,取其平均值并计算肿瘤信号强度下降百分比(percentage of SI loss, PSIL), PSIL=|(SIenhanced-SIunenhanced)|/SIunenhanced×100%,其中SIunenhanced、SIenhanced分别代表平扫、增强后感兴趣区的信号强度。
4.1.4组织病理学检查
将MRI扫描后的荷瘤小鼠脱臼处死,取其肿瘤组织于4%福尔马林溶液中固定24h,脱水、透明、包埋、切片分别进行H&E和普鲁士蓝染色,比较上述四组荷瘤小鼠肿瘤组织中SPIO的聚集情况说明FA-OCMCS-SPIO-NP的肿瘤靶向性。
4.2初步评价FA-OCMCS-SPIO-NPs的体内分布
SPF级昆明小鼠20只(购自南方医科大学实验动物中心),随机分成四组,每组各5只,Ⅰ组,OCMCS-SPIO-NPs;Ⅱ组,FA-OCMCS-SPIO-NPs;Ⅲ组,dextran-SPIO-NPs;Ⅳ组,空白。分别从尾静脉注射OCMCS-SPIO-NPs(5.44 mg Fe /ml)、FA-OCMCS-SPIO-NPs(5.62 mg Fe/ml)、dextran-SPIO-NPs(5.76mgFe/ml)和生理盐水各0.25m112h后,将小鼠脱臼处死,取其心、肝、脾、肺和肾于4%的福尔马林溶液中固定、透明、石蜡包埋、脱蜡至水行普鲁士蓝染色,高倍镜下观察。比较四组小鼠上述各组织中普鲁士蓝染色情况,说明SPIO-NPs在组织内的分布情况及体内抗吞噬作用。
5统计分析：应用SPSS13.0软件包,采用正交实验的析因分析和配对t检验(Paired-Samples T Test)进行统计学处理,P<0.05统计学有显著性差异。
结果
1共沉淀法合成SPIO-NPs的单因素考察结果显示,铁盐溶液中pH值、反应温度及沉淀剂种类是影响SPIO-NPs合成的重要因素,制备超顺磁氧化铁纳米粒的最佳工艺是：铁溶液体系中Fe3+：Fe2+摩尔比为2：1,初始pH值为3.0,反应温度为30℃,沉淀剂为25%的氨水溶液、体积为48ml,消化温度为80℃。
2正交实验析因及极差分析结果显示羧甲基壳聚糖的分子量、超声强度、羧甲基壳聚糖溶液的浓度对OCMCS-SPIO-NPs的粒径影响显著(FA=15.867、PA=0.028；FB=12.243、PB=0.040；FD=20.257、PD=0.02),而超声时间对纳米粒的粒径影响不显著(Fc=2.173,Pc=0.241),考虑到节约时间,最佳工艺处方是A1B1C1D2即羧甲基壳聚糖的浓度为2%、分子量为1-2万、超声时间为30分钟、强度为600W。
3傅里叶红外及X-Ray衍射分析确定叶酸嫁接的最佳方案为：叶酸活性酯和OCMCS的比例为4：1,反应温度为50℃,在充氮、无氧、无水的二甲基亚砜溶液中反应。
4 SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的表征,结果如下：
4.1透射电镜观察四种纳米粒均呈类圆形或椭圆形,大小均匀,表面平滑完整,SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的平均粒径分别为：12.5±3.0nm、13.7±3.6nm,15.4±4.5nm和17.5±4.8nm。
4.2激光粒度分析仪测定SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs及dextran-SPIO-NPs的平均粒径和多分散系数分别为201.6nm和0.234、38.2nm和0.119、41.4和0.132、125.4nm和0.143。
4.3磁性测定结果表明SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs均具有超顺磁性,SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs饱和磁矩分别为：98.0、71.4和69.6 emu/gFe
4.4驰豫率测定结果：OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的驰豫率分别为0.1685×106mol/s、0.1512×106mol/s和0.139×106mol/s,均高于0.062×106mol/s的最低标准,并且表明随着体系中SPIO的浓度增加,横向驰豫时间T2相应缩短。
4.5 Zeta电位测定结果：FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs和dextran-SPIO-NPs分别为-21.36±1.15mV、-27.88±0.73mV和-18.1±1.01mV。
4.6优化处方工艺后制备的OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs溶液中铁含量分别为5.44mgFe/ml、5.62mgFe/ml和5.76mgFe/ml; SPIO-NPs、OCMCS-SPIO-NPs和FA-OCMCS-SPIO-NPs干燥粉末内铁含量分别为663.89mg/g、615.61mg/g和601.40mg/g。
4.7 X-Ray衍射法结果表明我们制备的SPIO-NPs、OCMCS-SPIO-NPs和FA-OCMCS-SPIO-NPs粉末的晶型与标准Fe304粉末晶型一致,而且在该纳米粒的整个制备过程中,SPIO的晶型均未发生明显的改变。傅里叶红外结果说明OCMCS和FA是通过共价结合对SPIO进行修饰的,结果就是我们成功地合成FA-OCMCS-SPIO-NPs。
5纳米粒的体外评价
5.1体外细胞毒性结果
FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs、dextran-SPIO-NPs和SPIO-NPs对L02、A549和KB细胞的细胞毒性实验结果表明随着培养液中SPIO的浓度增加,细胞活力均有下降的趋势(F=366.490,P=0.000),SPIO经过羧甲基壳聚糖和叶酸表面修饰后,对上述三种细胞的细胞毒性均显著下降(F=2256.204,P=0.000),多重比较显示FA-OCMCS-SPIO-NPs与未经叶酸修饰修饰的CMCS-SPIO对KB细胞的细胞毒性(P=0.000),但是FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs对A549细胞的细胞毒性相差不显著(P=0.843)而且OCMCS-SPIO-NPs与dextran-SPIO-NPs的细胞毒性无显著性差异(P=0.122)；FA-OCMCS-SPIO-NPs与dextran-SPIO-NPs的细胞毒性无显著性差异(P=0.190)；
5.2体外叶酸受体肿瘤靶向性评价
菲洛嗪法测定细胞内的铁含量,统计分析表明对于叶酸受体高度表达的KB细或叶酸受体适度表达的Hela细胞,FA-OCMCS-SPIO-NPs组细胞内铁含量明显高于OCMCS-SPIO-NPs组(FKB=656.558,PKB=0.000; FHela=642.423, PHela=0.000)。FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs分别与KB细胞、Hela细胞和A549细胞孵化24h普鲁士蓝染色结果显示：在KB细胞FA-OCMCS-SPIO-NPs组的颜色明显深于OCMCS-SPIO-NPs组；Hela细胞,FA-OCMCS-SPIO-NPs组略深于OCMCS-SPIO-NPs组；A549细胞,FA-OCMCS-SPIO-NPs组和OCMCS-SPIO-NPs组普鲁士蓝染色后颜色变化不大,细胞内染色颜色很浅。
5.3体外巨噬细胞摄取
菲洛嗪法测定细胞内铁含量FA-OCMCS-SPIO-NPs组、OCMCS-SPIO-NPs组、dextran-SPIO-NPs组与未包被SPIO-NPs组RAW264.7细胞内铁含量差异显著((F=2302.289,P=0.000),结合普鲁士蓝结果显示RAW264.7与包被后SPIO-NPs孵化24h后细胞内铁含量远远低于未包被的SPIO-NPs组,而且FA-OCMCS-SPIO-NPs组与dextran-SPIO-NPs组RAW264.7细胞内铁含量,差异显著(P=0.007)。普鲁士蓝染色颜色由浅到深依次是空白,FA-OCMCS-SPIO-NPs,OCMCS-SPIO-NPs和uncoated-SPIO-NPs组。
6体内肿瘤靶向性及逃避网状内皮系统吞噬作用
6.1体内肿瘤靶向性评价
6.1.1 MRI扫描说明体内靶向性
MRI扫描结果表明荷A549细胞肿瘤小鼠尾静脉注射OCMCS-SPIO-NPs或FA-OCMCS-SPIO-NPs3h后,肿瘤部位T2加权信号与平扫信号无显著性差异。荷KB细胞肿瘤小鼠尾静脉注射OCMCS-SPIO-NPs3h后,肿瘤部位T2加权信号与平扫信号无明显下降,而注射FA-OCMCS-SPIO-NPs3h后,T2加权信号比平扫信号降低了27.23%,具有显著性差异(t=15.411,P=0.001)。
6.1.2病理组织学检查
HE染色和普鲁士蓝染色结果表明,只有尾静脉注射FA-OCMCS-SPIO-NPs的荷KB瘤小鼠肿瘤组织中有SPIO-NPs富集,普鲁士蓝染色阳性；而其他肿瘤组织中,均无SPIO-NPs富集,普鲁士蓝染色呈阴性。
6.2体内分布的初步评价
普鲁士蓝染色结果表明FA-OCMCS-SPIO-NPs组小鼠的心、肝、脾、肾、肺组织经普鲁士蓝染色后,所有组织普鲁士蓝染色为阴性,而在dextran-SPIO-NPs组,富含大量Kuffer细胞的肝、脾组织的细胞核和细胞质普鲁士蓝染呈阳性,在心、肺和肾普鲁士蓝染色阴性,OCMCS-SPIO-NPs组小鼠肝、脾有少量组织染成蓝色,浅于dextran-SPIO-NPs组。
结论
1我们成功地合成了具有“典型核壳”结构,内核粒径约为10nm,亲水性好,强超顺磁性,流体粒径<50nm的FA-OCMCS-SPIO-NPs。
2体外细胞毒性结果表明利用羧甲基壳聚糖和叶酸对SPIO-NPs进行共价修饰能明显降低SPIO-NPs的细胞毒性。FA-OCMCS-SPIO-NPs组对叶酸受体高表达的KB细胞明显高于OCMCS-SPIO-NPs组,对于叶酸受体不表达的肿瘤细胞A549和LO2, FA-OCMCS-SPIO-NP和OCMCS-SPIO-NPs的细胞毒性无显著性差异。
3体外靶向性结果表明所合成的FA-OCMCS-SPIO-NP具有很强的叶酸受体靶向性,细胞表面叶酸受体表达越高,细胞内铁含量越高,但是OCMCS-SPIO-NPs无叶酸受体肿瘤靶向性。
4体外抗吞噬结果表明OCMCS的修饰可以明显降低RAW264.7细胞对超顺磁氧化铁纳米粒的摄取,而叶酸的进一步修饰对其吞噬作用影响很小。
5体内实验表明FA-OCMCS-SPIO-NPs具有高的叶酸受体肿瘤靶向性和逃避网状内皮系统吞噬的能力。
本研究的创新点
生物相容性好、低毒、无免疫原性和价格低廉的羧甲基壳聚糖纳米粒为桥梁,将超顺磁氧化铁纳米粒与肿瘤靶向的配体--叶酸有机结合,制备具有肿瘤靶向和长循环作用的FA-OCMCS-SPIO-NPs。
Cancer has been one of the most serious threats to human health. up to several million of patients die from cancer, each year. It is a big burden not only for the patients and their families but also the whole society.Therefore, malignant tumors has become a major issue of concern to all mankind. The study on Superparamagnetic iron oxide nanoparticles were focused on MRI imaging from the initial to the current targeted drug delivery, treatment and imaging, etc.. superparamagnetic iron oxide nanoparticles with tumor targeting has been attracted the attention of many researcher because of their application in MRI diagnosis, hyperthermia and the potential for targeted drug delivery. In our study we synthesized a novel tumor targeting superparamagnetic iron oxide nanoparticles (SPIO-NPs) with three-step and we evaluate their tumor targeting and ability to evade the capture by macrophage in vivo and in vitro.
1. To synthesize folic acid-o-carboxymethyl chitosan-superparamagnetic iron oxide nanoparticles for tumor targetings and optimize their prescription process.
2. To characterize the property of the intermediate products (superparamagnetic iron oxide nanoparticles and o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles) as well as the end products (folic acid-o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles).
3. To evaluate the tumor targeting, cytotoxicity and the ability to t of evade phagocytes phagocytosis of FA-OCMCS-SPIO-NPs in vitro.
4. To preliminary evaluate the tumor targeting in vivo.
Methods
1. The FA-OCMCS-SPIO-NPs was synthesized by " three-step":At first, superparamagnetic oxide iron nanoparticles was synthesized by coprecipitation., then, the o-carboxymethyl chitosan and folic acid was covalently modified on the surface of SPIO-NPs in turn to prepare FA-OCMCS-SPIO-NPs.
1.1 Superparamagnetic iron oxide nanoparticles were synthesized by the coprecipitation of ferric and ferrous salts in anaerobic conditions. We studied the effect of initial pH and temperature of iron salt solutions, the type of precipitation agent (NH3·H2O) and the amount of NH3·H2O on particles size and composition of SPIO-NPs during co-precipitation by one factor design. the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property.
1.2 o-Carboxymethyl chitosans superparamagnetic oxide iron nanoparticles (OCMCS-SPIO-NPs) composed of SPIO-NPs as "core" and OCMCS as "shell" were prepared by covalently modifying SPIO-NPs surface with OCMCS. In order to optimize the synthesis process, Firstly, we explore the effect of mass ratio between the OCMCS and SPIO nanoparticles on the particle size to determine the optimal ratio of SPIO and OCMCS. Then, On the base of initial preparative conditions, The optimum conditions were investigated by Lg (24) orthogonal design (carboxymethyl chitosan molecular weight, concentration of OCMCS in solution, ultrasonic time four factors and power of ultrasonic cell crushing instrument, two levels) through the index of particle size of nanoparticles as the dependent variable.the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property. T2 relaxation rate was calculated according to MRI scanning. The iron content in dry SPIO-NPs and OCMCS-SPIO-NPs suspension was determined by o-Phenanthroline Method.
1.3 The exposed amino group in OCMCS-SPIO-NPs was reacted with carboxyl groups in folic acid to synthesize FA-OCMCS-SPIO-NPs., Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis and the influence of the grafting process of folic acid on the crystal structure of Fe3O4 to determine the best process to modify the OCMCS-SPIO-NPs with FA. The FA-OCMCS-SPIO-NPs were characterized according to the methods 1.2 for characterization,then The FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and SPIO-NPs suspension was stored in 4℃to observe the property of the suspension, preliminary study of their solution stability was studied.
2. Dextran superparamagnetic iron oxide nanoparticles (dextran-SPIO-NPs) was synthesized by Co-precipitation of ferric, ferrous salts and dextran solution in anaerobic conditions. The synthetic dextran-SPIO-NPs were characterized according to the Method 1.2 too.
3. Evaluation the cytotoxicity, folate receptor tumor targeting and the ability to evade the capture by macrophage cells in vitro.
3.1 Cytotoxicity
LO2 cells (normal liver cells), KB cells (human oral epithelial cells, a folate receptor over-expressing cell line) and A549 cells (small cell lung adenocarcinoma cells, folate receptor deficient cell line),routinely culture in full media. Then,the cells were seeded in 96 wells and continued incubating with full media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs and non-coated SPIO-NPs with different concentrations respectively. with after 24h incubation, the viability of the KB, A549 and LO2 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
3.2 Tumor Targeting
3.2.1 Quantification of cellular uptake by ferrozine assay:
A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 24-well plates, after 24h, the cells were washed three times with PBS, Then, the full media containing different concentrations of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs were added, after 24h incubation, the cells were washed thoroughly three times with PBS and dispersed in 0.5ml of 0.1M hydrochloric acid,the intracellular iron content was determined by ferrozine Assay
3.2.2 Prussian blue staining:A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full media containing FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs (iron concentration was 0.4mgFe/ml)were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope..
3.3 Capture by macrophage cells in vitro
3.3.1 Quantification of cellular uptake by ferrozine assay: RAW 264.7 cell were routinely cultured in full DMEM culture medium. cells were seeded in 24-well culture plates with approximately 2.5×105 cells/well. After incubation at 37℃for 24 h, the cells were washed three time with PBS. 1ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs(iron concentration ranging from 50μg/ml to 800μg/ml) was added to RAW264.7 cell respectively. the intracellular iron content was determined by ferrozine Assay.
3.3.2 Prussian blue staining:RAW 264.7 cell were routinely cultured in full DMEM culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope.
4 Preliminary studies of FA-OCMCS-SPIO-NPs tumor targeting and distribution in vivo
4.1 Tumor targeting in vivo
4.1.1 Experimental Animals:SPF- nude mice 20 were bought animal center of Southern Medical University, age 4-6 weeks, male and female in half, randomly divided into two groups: KB tumor cell group (n= 10), and A549 tumor cell group (n= 10).
4.1.2 Cell culture and tumor animal model develop:A549 were routinely cultured in RPMI-1640 full culture medium, KB were routinely cultured in FFPRMI-1640 full culture medium, then were detached by 0.25% trypsin-EDTA solution and washed three time by PBS s, followed by diluting in serum-free medium with 1.0×107 cells/ml,0.2 ml of cell suspension and were injected subcutaneously in the neck of nude mice. Two weeks later, when tumor diameter≥1.0 cm, the mouse with successful tumor model (16,8 for each group) were selected for animal experiments to study tumor targeting.
4.1.3.The study of FA-OCMCS-SPIO-NPs tumor targeting imaging
4.1.3.1.Animals
In this experiment, the 16 mice with successful tumor model (8 for both A549 and KB tumor group) were subdivided into four group at random. namely groupⅠ:A549, OCMCS-SPIO-NPs (n= 4);Ⅱgroup:A549, FA-OCMCS-SPIO-NPs (n= 4);ⅢGroup:KB cells, OCMCS-SPIO-NPs; IV Group:KB cells, FA-OCMCS-SPIO-NPs group (n= 4). (5.54 mg Fe/ml) or OCMCS-SPIO-NPs (5.62 mg Fe/ml) suspension, a dose of 0.25ml/nude mouse was intravenously injected.
4.1.3.2 MR imaging
All MR imaging was performed before and after FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs suspension were injected 3h at field strength of 1.5 Tesla MR scanner
4.1.3.3 MR imaging analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on tumor were measured on MR images
4.1.4. Histopathology: After the completion of MR imaging,tumors were collected Mice were deeply anesthetized with an intraperitoneal injection of chloral hydrate (400 mg/kg). The tumor tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with hematoxylin-eosin (H&E) or Prussian blue stain to identify the iron in the histological sections of the tumor. The images obtained on a OLYMPUS DMR inverted microscope
4.2 Preliminary evaluation the distribution of OCMCS-SPIO-NPs. FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in vivo. SPF level obtained 20 Kunming mice were bought from the Southern Medical University were divided into four groups, n= 5:I group-OCMCS-SPIO-NPs group; II group- FA-OCMCS-SPIO-NPs group;Ⅲgroup -- dextran-SPIO-NPs (5.76mgFe/ml) group, IV group is the control group. The mice in all the four groups were injected with OCMCS-SPIO-NPs (5.44 mg Fe/ml), FA-OCMCS-SPIO-NPs (5.62 mg Fe/ml), dextran-SPIO-NPs (5.76mgFe/ml) or saline 0.25ml respectively, 12h later, the mice were killed, the hearts, livers, spleens, lungs and kidneys of all the mice were collected and the these tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with Prussian blue stainning to identify the iron in the histological sections of,while the HE staining in blank group were used to histologically identify the tissue. The images obtained on a OLYMPUS DMR inverted microscope
5. Statistical analysis: SPSS 13.0 was used as analyzing software. Orthogonal factorial experiment analysis, factorial analysis and Paired-Samples T Test was applied for statistical analysis, P<0.05 considered significant statistical difference.
Results
1.Based on the initial preparative conditions of SPIO-NPs by one factor design,Physicochemical factors such as the PH, temperature in iron salt solution and the ripening temperature for have important effect on the size and size distribution as well as composition of SPIO-NPs.The optimal process-formulation was as follows:molar ratio of Fe+:Fe2+is 2:1, the initial pH and temperature of iron salt solutions were pH 3.0 and 30℃respectively, the precipitant agent was ammonia solution, the volume of 25% ammonia solution was 48ml, the ripening temperature is 80℃.
2 Orthogonal experimental design results shows:The molecular weight of o-carboxymethyl chitosan, concentration of OCMCS and the power of ultrasonic have significant influence on the hydrophilic size of OCMCS-SPIO-NPs, while the ultrasonic time have no significant on the hydrophilic of OCMCS-SPIO-NPs. The optimal formulation-process was A1B1C1D2:namely,carboxymethyl chitosan concentration was 2%, MW of OCMCS was 1-2 million, ultrasonic time was 30minutes, the power of ultrasonic was 600W.
3 Fourier transform infrared spectroscopy and X-Ray diffraction indicated that the best process to modify the OCMCS-SPIO-NPs with FA was as follows. The mass ratio of folic acid active ester and OCMCS the 4:1 ratio,the reaction temperature was 50℃under the conditions of anhydrous dimethyl sulfoxide solution with the nitrogen protecting from oxygen.
4 Characterizations of resulting SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs
4.1TEM showed the resulting SPIO-NPs, OCMCS-SPIO-NPs,FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs are almost spherical or ellipsoidal. The size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs were present as x±s, whose particle size were 12.5±3.0nm,13.7±3.6nm,15.4±4.5nm and 17.5±4.8nm, respectively.
Hydrophilic size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs The average intense size was 201nm and the polydispersity was 0.234 for SPIO-NPs The average intense size was 38.2 nm and the polydispersity was 0.132 for OCMCS-SPIO-NPs, The average intense size was 41.4nm and the polydispersity for FA-OCMCS-SPIO-NPs; The average intense size was 125 nm and the polydispersity was 0.119 for dextran-SPIO-NPs.
4.3.Magneitc properties The results of magnetic properties of the samples which were study by VSM indicated that the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs were superparamagnetic, The saturation magnetization values for the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS SPIO-NPs were:98.0,71.4 and 69.6 emu/gFe.
4.4.T2 rate of the resulting SPIO-NPs:
OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in the T2 rate was 0.1685×106mol/s,0.1512×106mol/s and 0.139×106mol/s, which were higher than 0.062×106mol/s minimum standards.The transverse relaxation time (T2) shorten with the iron concentration increase in SPIO-NPs suspension.
4.5. Zeta potential of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs were-21.36±1.15mV,-27.88±0.73m and-18.1±1.01lmV respectively. 4.6. The iron content in resulting SPIO-NPs was as follows:OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs solution iron content were 5.44 mg Fe /ml,5.62mgFe/ml and 5.76mgFe/ml respectively; iron content in SPIO-NPs, OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs dry powder was 663.89mg/g, 615.61mg/g, and 601.40mg/g respectively.
4.7. X-Ray diffraction results showed that the powder crystals of resulting SPIO-NPs、OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs, were consistent with that of standards Fe3O4, more important, the entire preparation process of nanoparticles have no obvious impact on those powder crystal. FTIR results confirmed that OCMCS and the FA were conjugated on the surface of SPIO-NPs by covalent.
5 Evaluation of resulting SPIO-NPs In vitro
5.1 In vitro cytotoxicity
The cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO- NPs and SPIO-NPs against LO2, A549 and KB cells showed the cytoto xicity increase with the increase of iron concentration in culture media. The modification with OCMCS and FA could significantly decrease the cytotoxicity against all of the three cell. The cytotoxicity of FA-OCMC-SPIO-NPs against KB cell (FR over-expressed) was much higher than OCMCS-SPIO-NPs and dextran-SPIO-NPs(P < 0.05). While there is no significant difference among the cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs against LO2 and A549(FR negative) (P> 0.05).
5.2 Evaluation folate receptor tumor targeting in vitro
Ferroine assay showed that the intracellular iron content of both KB cells and Hela cells in FA-OCMCS -SPIO-NPs group was significantly higher than that in OCMCS-SPIO-NPs (PHela= 0.000<0.05; PKB= 0.000<0.05), while,the intracellular iron content in A549 cells, there was no significant difference between FA-OCMCS-SPIO-NPs group and OCMCS-SPIO-NPs group(P= 0.106> 0.05).
Prussian blue staining results showed both KB cell and Hela cell cultured with FA-OCMCS-SPIO-NPs were have dark prussian blue staining positive, which was much darker than those cells cultured in OCMCS-SPIO-NPs. While, A549 cultured in either FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs, both of them were almost prussian blue staining negative in intracellular. 5.3 Macrophage uptake in vitro
Intracellular iron content in RAW264.7 determined by Ferroine assay cells and Prussian blue staining showed that the iron content in RAW264.7 cultured in media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs was much lower than that cultured in media containing SPIO-NPs group, moreover, the intracellular iron content of RAW264.7 culture in media containing the FA-OCMCS-SPIO-NPs were significantly lower than the that cultured in media containing dextran-SPIO-NPs.
6. Evaluations in vivo
6.1. Tumor targeting in vivo
6.1.1 MRI scan
T2-weighted spin echo images of the tumor-bearing mice showed that the average intensity of the entire KB cell subcutaneous tumors decreases about 27.23% between preinjection and postinjection images injected with FA-OCMCS-SPIO-NPs (p=0.01<0.05). However, the signal intensity had no significant difference between preinjection and postinjection images of subcutaneous tumors for other groups(p>0.05).
6.1.2 Histopathology
HE and Prussian blue staining of tumor tissue slice showed only the bearing KB cell tumor mice were intravenously injected with FA-OCMCS-SPIO-NPs can be seen scatter SPIO-NPs aggregation and Prussian blue staining positive.while in other group there were no SPIO-NPs aggregation in HE staining or Prussian blue positive
6.2 Histological evaluation of the distribution of nanoparticles in vivo
Prussian blue staining results suggested that the mice that were intravenously injected with FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs, there were no stainable iron was observed in the heart, liver, spleen, kidney and lung tissue. While the mice that were intravenously injected with dextran-SPIO-NPs, the stainable iron was observed in KCs in hepatic sinusoid, in splenic cord of the perimeter section of follicle and in splenic red pulp. however there were no stainable iron in the other tissues
Conclusions
1. we have successfully synthesized the FA-OCMCS-SPIO-NPs with a typical "core-shell" structure, those nanoparticles has good hydrophilicity and strong superparamagnetic with a hydrophilic size e<50nm.
2. In vitro cytotoxicity results showed that covalently modify the SPIO-NPs with OCMC and folic acid could significantly reduce the cytotoxicity of SPIO-NPs. The cytotoxicity of FA-OCMCS-SPIO-NPs on cells (FR positive) were higher than that of OCMCS-SPIO-NPs, There was no significant difference on the cytotoxicity of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs against LO2(normal cell line) and A549(FR negative)
3. Tumor targeting results showed the synthesized FA-OCMCS-SPIO-NP had a strong targeting folate receptor, the higher folate receptor expressed on the surface, the higher the intracellular iron content
4. Uptake of all the synthesized SPIO-NPs by RAW264.7 in vitro indicated that the OCMCS surface modification can significantly reduce uptake of superparamagnetic iron oxide nanoparticles by the RAW264.7 cells, while the further modification had no influence on the uptake by RAW264.7 cells.
5.In vivo evaluation results showed that FA-OCMCS-SPIO-NPs had a high folate receptor tumor targeting and could avoid capture by reticuloendothelial system
The innovation of the paper
In this article, the OCMCS was used as a conjugate to associate the SPIO core and tumor target ligand (folic acid) to develop a novel active tumor-targeting SPIO-NPs system-FA-OCMCS-SPIO-NPs.
目的
1.制备肿瘤靶向的叶酸-羧甲基壳聚糖-超顺磁氧化铁纳米粒(Folic acid-o-carboxymethyl chitosans superparamagnetic oxide iron nanoparticles (FA-OCMCS-SPIO-NPs)并对处方工艺进行优化。
2.对中间产物(超顺磁氧化铁纳米粒和羧甲基壳聚糖超顺磁氧化铁纳米粒)及终产物(叶酸-羧甲基壳聚糖超顺磁氧化铁纳米粒)分别进行表征。
3.体外评价FA-OCMCS-SPIO-NPs的细胞毒性、逃避吞噬细胞吞噬作用及叶酸受体肿瘤靶向作用。
4.体内初步评价FA-OCMCS-SPIO-NPs的肿瘤靶向性及组织中分布情况。
方法
1三步法合成FA-OCMCS-SPIO-NPs
即先用共沉淀法合成超顺磁氧化铁纳米粒(superparamagnetic oxide iron nanoparticles SPIO-NPs),依次用羧甲基壳聚糖和叶酸对SPIO-NPs表面进行共价修饰制备FA-OCMCS-SPIO-NPs.
1.1共沉淀法合成超顺磁氧化铁纳米粒
考察体系的PH值、温度、沉淀剂的种类和用量对SPIO-NPs粒径和组成的影响。用X-Ray衍射仪、激光散射粒度分析仪、透射电子显微镜、傅里叶红外光谱仪、超导电子干涉等对产物进行表征。
1.2OCMCS对SPIO-NPs表面进行共价修饰
合成以SPIO-NPs为核,OCMCS为“壳”的OCMCS-SPIO-NPs。为了对合成工艺进行优化,先单因素考察SPIO与OCMCS的比例对纳米粒粒径的影响,确定最佳的SPIO与OCMCS比例,然后,以纳米粒的粒径作为因变量,羧甲基壳聚糖的分子量(A)、浓度(B)、超声时间(C)及超声细胞粉碎仪的功率(D作为自变量,采用正交设计(L824)进行处方优化和工艺筛选。用透射电镜、激光散射粒度分析仪、电位测定仪、X-Ray衍射仪、傅立叶红外和超导电子干涉(superconducting quantum interference measurement device、SQUID)等对OCMCS-SPIO-NPs进行表征。MRI扫描测定弛豫率、邻二氮菲法测定铁含量。
1.3利用OCMCS-SPIO-NPs的氨基基团与叶酸中羧基基团发生酰胺反应合成FA-OCMCS-SPIO-NPs,采用傅里叶红外扫描及X-Ray粉末衍射考察不同嫁接工艺对合成的FA-OCMCS-SPIO-NPs的红外图谱及Fe304晶型的影响,确定FA修饰OCMCS-SPIO-NPs的最佳工艺,按方法1.2对FA-OCMCS-SPIO-NPs进行表征；将FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs和SPIO-NPs溶液4℃留样,观察溶液的形状,初步考察他们的溶液稳定性。
2共沉淀法合成葡聚糖超顺磁氧化铁纳米粒(dextran-SPIO-NPs)并按方法1.2对合成的dextran-SPIO-NPs进行表征。
3体外评价细胞毒性、叶酸受体肿瘤靶向性及抗吞噬细胞吞噬作用。
3.1细胞毒性
LO2细胞(正常肝细胞),叶酸受体高表达的KB细胞(人口腔上皮癌细胞)和叶酸受体不表达的A549细胞(肺小细胞腺癌细胞)常规传代培养后,以5×103/孔接种于96孔板上,培养24h后,分别与不同浓度的FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs、dextran-SPIO-NPs和未包被的SPIO-NPs孵化24h,MTT法测定细胞毒性。
3.2肿瘤靶向性
3.2.1菲洛嗪法定量分析细胞内铁含量
A549细胞常规培养于RPMI1640全培溶液中、Hela细胞(人宫颈癌细胞)和KB细胞常规传代培养于FFRPMI1640全培溶液中,以2.5×105/孔接种于24孔板,培养24h,PBS液洗涤三遍后,与不同浓度的FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs孵化24后,菲洛嗪法测定细胞内铁含量。
3.2.2普鲁士蓝染色
A549细胞、Hela细胞和KB细胞常规传代培养后,以5×105/孔接种于6孔板上,孵化24h,分别加入0.4mgFe/ml的FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs2ml24h后,普鲁士蓝染色,高倍镜下观察。
3.3巨噬细胞摄取实验
3.3.1菲洛嗪法细胞内铁含量
常规培养RAW264.7(小鼠单核巨噬细胞白血病细胞),以2.5×105/孔接种于24孔板,培养24h,PBS液洗三遍后,加入一系列不同浓度的FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs、dextran-SPIO-NPs和未包被的SPIO-NPs,孵化24h后,PBS彻底清洗三遍,用0.5ml浓度为0.1M盐酸分散,菲洛嗪法测定细胞内铁含量。
3.3.2普鲁士蓝染色
常规培养RAW264.7(小鼠单核巨噬细胞白血病细胞),以5×105/孔接种于6孔板上,孵化24h后,分别加入0.4mgFe/ml的上述纳米粒2ml与RAW264.7细胞孵化24h后,进行普鲁士蓝染色,倒置显微镜下观察。
4.体内初步考察FA-OCMCS-SPIO-NPs的肿瘤靶向性及体内分布情况
4.1体内肿瘤靶向性考察
4.1.1实验动物：健康,SPF级裸鼠20只、鼠龄4-6周、雌雄各半,采用随机法将其分成两组：KB肿瘤细胞组(n=10)和A549肿瘤细胞组(n=10)。
4.1.2细胞培养和肿瘤模型建立
A549用细胞RPMI-1640全培常规培养、KB细胞用FFPRMI-1640全培常规传代培养,0.25%胰酶-EDTA消化,PBS液洗3遍后,无血清的培养基稀释制成每毫升含1.0×107/ml的细胞悬液,7号针头注射0.2 ml于裸鼠颈背部皮下。两周后,待瘤体最大直径≥1.0 cm时,选取已成功建立肿瘤模型的小鼠(16只,每组8只)进行动物实验。
4.1.3 MRI扫描
动物分组：将上述已成功荷瘤裸鼠(16只,每组8只)进一步随机分成四组：Ⅰ组,A549. OCMCS-SPIO-NPs(n=4);Ⅱ组,A549. FA-OCMCS-SPIO-NPs(n=4)；Ⅲ组,KB细胞、OCMCS-SPIO-NPs(n=4)；Ⅳ组,KB细胞、FA-OCMCS-SPIO-NPs组(n=4)。
4.1.3.1 MRI扫描：
采用西门子(Siemens Magnetom Vision Plus)1.5T,头部线圈,冠状面扫描,平扫后分别从尾静脉注射OCMCS-SPIO-NPs(5.54 mg Fe/ml)或FA-OCMCS-SPIO-NPs(5.62 mg Fe/ml)混悬液,剂量为0.25ml/只,给药3h后,MRI增强扫描。
4.1.3.2图像分析
T2WI图像测量平扫及增强3h后四组实验动物肿瘤感兴趣区(Region ofinterest, ROI)的信号强度(signal intense, SI)。测量时ROI放置在局部信号相对均匀,无明显伪影的区域,选取同一部位,测量三次,取其平均值并计算肿瘤信号强度下降百分比(percentage of SI loss, PSIL), PSIL=|(SIenhanced-SIunenhanced)|/SIunenhanced×100%,其中SIunenhanced、SIenhanced分别代表平扫、增强后感兴趣区的信号强度。
4.1.4组织病理学检查
将MRI扫描后的荷瘤小鼠脱臼处死,取其肿瘤组织于4%福尔马林溶液中固定24h,脱水、透明、包埋、切片分别进行H&E和普鲁士蓝染色,比较上述四组荷瘤小鼠肿瘤组织中SPIO的聚集情况说明FA-OCMCS-SPIO-NP的肿瘤靶向性。
4.2初步评价FA-OCMCS-SPIO-NPs的体内分布
SPF级昆明小鼠20只(购自南方医科大学实验动物中心),随机分成四组,每组各5只,Ⅰ组,OCMCS-SPIO-NPs;Ⅱ组,FA-OCMCS-SPIO-NPs;Ⅲ组,dextran-SPIO-NPs;Ⅳ组,空白。分别从尾静脉注射OCMCS-SPIO-NPs(5.44 mg Fe /ml)、FA-OCMCS-SPIO-NPs(5.62 mg Fe/ml)、dextran-SPIO-NPs(5.76mgFe/ml)和生理盐水各0.25m112h后,将小鼠脱臼处死,取其心、肝、脾、肺和肾于4%的福尔马林溶液中固定、透明、石蜡包埋、脱蜡至水行普鲁士蓝染色,高倍镜下观察。比较四组小鼠上述各组织中普鲁士蓝染色情况,说明SPIO-NPs在组织内的分布情况及体内抗吞噬作用。
5统计分析：应用SPSS13.0软件包,采用正交实验的析因分析和配对t检验(Paired-Samples T Test)进行统计学处理,P<0.05统计学有显著性差异。
结果
1共沉淀法合成SPIO-NPs的单因素考察结果显示,铁盐溶液中pH值、反应温度及沉淀剂种类是影响SPIO-NPs合成的重要因素,制备超顺磁氧化铁纳米粒的最佳工艺是：铁溶液体系中Fe3+：Fe2+摩尔比为2：1,初始pH值为3.0,反应温度为30℃,沉淀剂为25%的氨水溶液、体积为48ml,消化温度为80℃。
2正交实验析因及极差分析结果显示羧甲基壳聚糖的分子量、超声强度、羧甲基壳聚糖溶液的浓度对OCMCS-SPIO-NPs的粒径影响显著(FA=15.867、PA=0.028；FB=12.243、PB=0.040；FD=20.257、PD=0.02),而超声时间对纳米粒的粒径影响不显著(Fc=2.173,Pc=0.241),考虑到节约时间,最佳工艺处方是A1B1C1D2即羧甲基壳聚糖的浓度为2%、分子量为1-2万、超声时间为30分钟、强度为600W。
3傅里叶红外及X-Ray衍射分析确定叶酸嫁接的最佳方案为：叶酸活性酯和OCMCS的比例为4：1,反应温度为50℃,在充氮、无氧、无水的二甲基亚砜溶液中反应。
4 SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的表征,结果如下：
4.1透射电镜观察四种纳米粒均呈类圆形或椭圆形,大小均匀,表面平滑完整,SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的平均粒径分别为：12.5±3.0nm、13.7±3.6nm,15.4±4.5nm和17.5±4.8nm。
4.2激光粒度分析仪测定SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs及dextran-SPIO-NPs的平均粒径和多分散系数分别为201.6nm和0.234、38.2nm和0.119、41.4和0.132、125.4nm和0.143。
4.3磁性测定结果表明SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs均具有超顺磁性,SPIO-NPs、OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs饱和磁矩分别为：98.0、71.4和69.6 emu/gFe
4.4驰豫率测定结果：OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs的驰豫率分别为0.1685×106mol/s、0.1512×106mol/s和0.139×106mol/s,均高于0.062×106mol/s的最低标准,并且表明随着体系中SPIO的浓度增加,横向驰豫时间T2相应缩短。
4.5 Zeta电位测定结果：FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs和dextran-SPIO-NPs分别为-21.36±1.15mV、-27.88±0.73mV和-18.1±1.01mV。
4.6优化处方工艺后制备的OCMCS-SPIO-NPs、FA-OCMCS-SPIO-NPs和dextran-SPIO-NPs溶液中铁含量分别为5.44mgFe/ml、5.62mgFe/ml和5.76mgFe/ml; SPIO-NPs、OCMCS-SPIO-NPs和FA-OCMCS-SPIO-NPs干燥粉末内铁含量分别为663.89mg/g、615.61mg/g和601.40mg/g。
4.7 X-Ray衍射法结果表明我们制备的SPIO-NPs、OCMCS-SPIO-NPs和FA-OCMCS-SPIO-NPs粉末的晶型与标准Fe304粉末晶型一致,而且在该纳米粒的整个制备过程中,SPIO的晶型均未发生明显的改变。傅里叶红外结果说明OCMCS和FA是通过共价结合对SPIO进行修饰的,结果就是我们成功地合成FA-OCMCS-SPIO-NPs。
5纳米粒的体外评价
5.1体外细胞毒性结果
FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs、dextran-SPIO-NPs和SPIO-NPs对L02、A549和KB细胞的细胞毒性实验结果表明随着培养液中SPIO的浓度增加,细胞活力均有下降的趋势(F=366.490,P=0.000),SPIO经过羧甲基壳聚糖和叶酸表面修饰后,对上述三种细胞的细胞毒性均显著下降(F=2256.204,P=0.000),多重比较显示FA-OCMCS-SPIO-NPs与未经叶酸修饰修饰的CMCS-SPIO对KB细胞的细胞毒性(P=0.000),但是FA-OCMCS-SPIO-NPs和OCMCS-SPIO-NPs对A549细胞的细胞毒性相差不显著(P=0.843)而且OCMCS-SPIO-NPs与dextran-SPIO-NPs的细胞毒性无显著性差异(P=0.122)；FA-OCMCS-SPIO-NPs与dextran-SPIO-NPs的细胞毒性无显著性差异(P=0.190)；
5.2体外叶酸受体肿瘤靶向性评价
菲洛嗪法测定细胞内的铁含量,统计分析表明对于叶酸受体高度表达的KB细或叶酸受体适度表达的Hela细胞,FA-OCMCS-SPIO-NPs组细胞内铁含量明显高于OCMCS-SPIO-NPs组(FKB=656.558,PKB=0.000; FHela=642.423, PHela=0.000)。FA-OCMCS-SPIO-NPs、OCMCS-SPIO-NPs分别与KB细胞、Hela细胞和A549细胞孵化24h普鲁士蓝染色结果显示：在KB细胞FA-OCMCS-SPIO-NPs组的颜色明显深于OCMCS-SPIO-NPs组；Hela细胞,FA-OCMCS-SPIO-NPs组略深于OCMCS-SPIO-NPs组；A549细胞,FA-OCMCS-SPIO-NPs组和OCMCS-SPIO-NPs组普鲁士蓝染色后颜色变化不大,细胞内染色颜色很浅。
5.3体外巨噬细胞摄取
菲洛嗪法测定细胞内铁含量FA-OCMCS-SPIO-NPs组、OCMCS-SPIO-NPs组、dextran-SPIO-NPs组与未包被SPIO-NPs组RAW264.7细胞内铁含量差异显著((F=2302.289,P=0.000),结合普鲁士蓝结果显示RAW264.7与包被后SPIO-NPs孵化24h后细胞内铁含量远远低于未包被的SPIO-NPs组,而且FA-OCMCS-SPIO-NPs组与dextran-SPIO-NPs组RAW264.7细胞内铁含量,差异显著(P=0.007)。普鲁士蓝染色颜色由浅到深依次是空白,FA-OCMCS-SPIO-NPs,OCMCS-SPIO-NPs和uncoated-SPIO-NPs组。
6体内肿瘤靶向性及逃避网状内皮系统吞噬作用
6.1体内肿瘤靶向性评价
6.1.1 MRI扫描说明体内靶向性
MRI扫描结果表明荷A549细胞肿瘤小鼠尾静脉注射OCMCS-SPIO-NPs或FA-OCMCS-SPIO-NPs3h后,肿瘤部位T2加权信号与平扫信号无显著性差异。荷KB细胞肿瘤小鼠尾静脉注射OCMCS-SPIO-NPs3h后,肿瘤部位T2加权信号与平扫信号无明显下降,而注射FA-OCMCS-SPIO-NPs3h后,T2加权信号比平扫信号降低了27.23%,具有显著性差异(t=15.411,P=0.001)。
6.1.2病理组织学检查
HE染色和普鲁士蓝染色结果表明,只有尾静脉注射FA-OCMCS-SPIO-NPs的荷KB瘤小鼠肿瘤组织中有SPIO-NPs富集,普鲁士蓝染色阳性；而其他肿瘤组织中,均无SPIO-NPs富集,普鲁士蓝染色呈阴性。
6.2体内分布的初步评价
普鲁士蓝染色结果表明FA-OCMCS-SPIO-NPs组小鼠的心、肝、脾、肾、肺组织经普鲁士蓝染色后,所有组织普鲁士蓝染色为阴性,而在dextran-SPIO-NPs组,富含大量Kuffer细胞的肝、脾组织的细胞核和细胞质普鲁士蓝染呈阳性,在心、肺和肾普鲁士蓝染色阴性,OCMCS-SPIO-NPs组小鼠肝、脾有少量组织染成蓝色,浅于dextran-SPIO-NPs组。
结论
1我们成功地合成了具有“典型核壳”结构,内核粒径约为10nm,亲水性好,强超顺磁性,流体粒径<50nm的FA-OCMCS-SPIO-NPs。
2体外细胞毒性结果表明利用羧甲基壳聚糖和叶酸对SPIO-NPs进行共价修饰能明显降低SPIO-NPs的细胞毒性。FA-OCMCS-SPIO-NPs组对叶酸受体高表达的KB细胞明显高于OCMCS-SPIO-NPs组,对于叶酸受体不表达的肿瘤细胞A549和LO2, FA-OCMCS-SPIO-NP和OCMCS-SPIO-NPs的细胞毒性无显著性差异。
3体外靶向性结果表明所合成的FA-OCMCS-SPIO-NP具有很强的叶酸受体靶向性,细胞表面叶酸受体表达越高,细胞内铁含量越高,但是OCMCS-SPIO-NPs无叶酸受体肿瘤靶向性。
4体外抗吞噬结果表明OCMCS的修饰可以明显降低RAW264.7细胞对超顺磁氧化铁纳米粒的摄取,而叶酸的进一步修饰对其吞噬作用影响很小。
5体内实验表明FA-OCMCS-SPIO-NPs具有高的叶酸受体肿瘤靶向性和逃避网状内皮系统吞噬的能力。
本研究的创新点
生物相容性好、低毒、无免疫原性和价格低廉的羧甲基壳聚糖纳米粒为桥梁,将超顺磁氧化铁纳米粒与肿瘤靶向的配体--叶酸有机结合,制备具有肿瘤靶向和长循环作用的FA-OCMCS-SPIO-NPs。
Cancer has been one of the most serious threats to human health. up to several million of patients die from cancer, each year. It is a big burden not only for the patients and their families but also the whole society.Therefore, malignant tumors has become a major issue of concern to all mankind. The study on Superparamagnetic iron oxide nanoparticles were focused on MRI imaging from the initial to the current targeted drug delivery, treatment and imaging, etc.. superparamagnetic iron oxide nanoparticles with tumor targeting has been attracted the attention of many researcher because of their application in MRI diagnosis, hyperthermia and the potential for targeted drug delivery. In our study we synthesized a novel tumor targeting superparamagnetic iron oxide nanoparticles (SPIO-NPs) with three-step and we evaluate their tumor targeting and ability to evade the capture by macrophage in vivo and in vitro.
1. To synthesize folic acid-o-carboxymethyl chitosan-superparamagnetic iron oxide nanoparticles for tumor targetings and optimize their prescription process.
2. To characterize the property of the intermediate products (superparamagnetic iron oxide nanoparticles and o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles) as well as the end products (folic acid-o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles).
3. To evaluate the tumor targeting, cytotoxicity and the ability to t of evade phagocytes phagocytosis of FA-OCMCS-SPIO-NPs in vitro.
4. To preliminary evaluate the tumor targeting in vivo.
Methods
1. The FA-OCMCS-SPIO-NPs was synthesized by " three-step":At first, superparamagnetic oxide iron nanoparticles was synthesized by coprecipitation., then, the o-carboxymethyl chitosan and folic acid was covalently modified on the surface of SPIO-NPs in turn to prepare FA-OCMCS-SPIO-NPs.
1.1 Superparamagnetic iron oxide nanoparticles were synthesized by the coprecipitation of ferric and ferrous salts in anaerobic conditions. We studied the effect of initial pH and temperature of iron salt solutions, the type of precipitation agent (NH3·H2O) and the amount of NH3·H2O on particles size and composition of SPIO-NPs during co-precipitation by one factor design. the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property.
1.2 o-Carboxymethyl chitosans superparamagnetic oxide iron nanoparticles (OCMCS-SPIO-NPs) composed of SPIO-NPs as "core" and OCMCS as "shell" were prepared by covalently modifying SPIO-NPs surface with OCMCS. In order to optimize the synthesis process, Firstly, we explore the effect of mass ratio between the OCMCS and SPIO nanoparticles on the particle size to determine the optimal ratio of SPIO and OCMCS. Then, On the base of initial preparative conditions, The optimum conditions were investigated by Lg (24) orthogonal design (carboxymethyl chitosan molecular weight, concentration of OCMCS in solution, ultrasonic time four factors and power of ultrasonic cell crushing instrument, two levels) through the index of particle size of nanoparticles as the dependent variable.the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property. T2 relaxation rate was calculated according to MRI scanning. The iron content in dry SPIO-NPs and OCMCS-SPIO-NPs suspension was determined by o-Phenanthroline Method.
1.3 The exposed amino group in OCMCS-SPIO-NPs was reacted with carboxyl groups in folic acid to synthesize FA-OCMCS-SPIO-NPs., Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis and the influence of the grafting process of folic acid on the crystal structure of Fe3O4 to determine the best process to modify the OCMCS-SPIO-NPs with FA. The FA-OCMCS-SPIO-NPs were characterized according to the methods 1.2 for characterization,then The FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and SPIO-NPs suspension was stored in 4℃to observe the property of the suspension, preliminary study of their solution stability was studied.
2. Dextran superparamagnetic iron oxide nanoparticles (dextran-SPIO-NPs) was synthesized by Co-precipitation of ferric, ferrous salts and dextran solution in anaerobic conditions. The synthetic dextran-SPIO-NPs were characterized according to the Method 1.2 too.
3. Evaluation the cytotoxicity, folate receptor tumor targeting and the ability to evade the capture by macrophage cells in vitro.
3.1 Cytotoxicity
LO2 cells (normal liver cells), KB cells (human oral epithelial cells, a folate receptor over-expressing cell line) and A549 cells (small cell lung adenocarcinoma cells, folate receptor deficient cell line),routinely culture in full media. Then,the cells were seeded in 96 wells and continued incubating with full media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs and non-coated SPIO-NPs with different concentrations respectively. with after 24h incubation, the viability of the KB, A549 and LO2 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
3.2 Tumor Targeting
3.2.1 Quantification of cellular uptake by ferrozine assay:
A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 24-well plates, after 24h, the cells were washed three times with PBS, Then, the full media containing different concentrations of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs were added, after 24h incubation, the cells were washed thoroughly three times with PBS and dispersed in 0.5ml of 0.1M hydrochloric acid,the intracellular iron content was determined by ferrozine Assay
3.2.2 Prussian blue staining:A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full media containing FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs (iron concentration was 0.4mgFe/ml)were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope..
3.3 Capture by macrophage cells in vitro
3.3.1 Quantification of cellular uptake by ferrozine assay: RAW 264.7 cell were routinely cultured in full DMEM culture medium. cells were seeded in 24-well culture plates with approximately 2.5×105 cells/well. After incubation at 37℃for 24 h, the cells were washed three time with PBS. 1ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs(iron concentration ranging from 50μg/ml to 800μg/ml) was added to RAW264.7 cell respectively. the intracellular iron content was determined by ferrozine Assay.
3.3.2 Prussian blue staining:RAW 264.7 cell were routinely cultured in full DMEM culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope.
4 Preliminary studies of FA-OCMCS-SPIO-NPs tumor targeting and distribution in vivo
4.1 Tumor targeting in vivo
4.1.1 Experimental Animals:SPF- nude mice 20 were bought animal center of Southern Medical University, age 4-6 weeks, male and female in half, randomly divided into two groups: KB tumor cell group (n= 10), and A549 tumor cell group (n= 10).
4.1.2 Cell culture and tumor animal model develop:A549 were routinely cultured in RPMI-1640 full culture medium, KB were routinely cultured in FFPRMI-1640 full culture medium, then were detached by 0.25% trypsin-EDTA solution and washed three time by PBS s, followed by diluting in serum-free medium with 1.0×107 cells/ml,0.2 ml of cell suspension and were injected subcutaneously in the neck of nude mice. Two weeks later, when tumor diameter≥1.0 cm, the mouse with successful tumor model (16,8 for each group) were selected for animal experiments to study tumor targeting.
4.1.3.The study of FA-OCMCS-SPIO-NPs tumor targeting imaging
4.1.3.1.Animals
In this experiment, the 16 mice with successful tumor model (8 for both A549 and KB tumor group) were subdivided into four group at random. namely groupⅠ:A549, OCMCS-SPIO-NPs (n= 4);Ⅱgroup:A549, FA-OCMCS-SPIO-NPs (n= 4);ⅢGroup:KB cells, OCMCS-SPIO-NPs; IV Group:KB cells, FA-OCMCS-SPIO-NPs group (n= 4). (5.54 mg Fe/ml) or OCMCS-SPIO-NPs (5.62 mg Fe/ml) suspension, a dose of 0.25ml/nude mouse was intravenously injected.
4.1.3.2 MR imaging
All MR imaging was performed before and after FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs suspension were injected 3h at field strength of 1.5 Tesla MR scanner
4.1.3.3 MR imaging analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on tumor were measured on MR images
4.1.4. Histopathology: After the completion of MR imaging,tumors were collected Mice were deeply anesthetized with an intraperitoneal injection of chloral hydrate (400 mg/kg). The tumor tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with hematoxylin-eosin (H&E) or Prussian blue stain to identify the iron in the histological sections of the tumor. The images obtained on a OLYMPUS DMR inverted microscope
4.2 Preliminary evaluation the distribution of OCMCS-SPIO-NPs. FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in vivo. SPF level obtained 20 Kunming mice were bought from the Southern Medical University were divided into four groups, n= 5:I group-OCMCS-SPIO-NPs group; II group- FA-OCMCS-SPIO-NPs group;Ⅲgroup -- dextran-SPIO-NPs (5.76mgFe/ml) group, IV group is the control group. The mice in all the four groups were injected with OCMCS-SPIO-NPs (5.44 mg Fe/ml), FA-OCMCS-SPIO-NPs (5.62 mg Fe/ml), dextran-SPIO-NPs (5.76mgFe/ml) or saline 0.25ml respectively, 12h later, the mice were killed, the hearts, livers, spleens, lungs and kidneys of all the mice were collected and the these tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with Prussian blue stainning to identify the iron in the histological sections of,while the HE staining in blank group were used to histologically identify the tissue. The images obtained on a OLYMPUS DMR inverted microscope
5. Statistical analysis: SPSS 13.0 was used as analyzing software. Orthogonal factorial experiment analysis, factorial analysis and Paired-Samples T Test was applied for statistical analysis, P<0.05 considered significant statistical difference.
Results
1.Based on the initial preparative conditions of SPIO-NPs by one factor design,Physicochemical factors such as the PH, temperature in iron salt solution and the ripening temperature for have important effect on the size and size distribution as well as composition of SPIO-NPs.The optimal process-formulation was as follows:molar ratio of Fe+:Fe2+is 2:1, the initial pH and temperature of iron salt solutions were pH 3.0 and 30℃respectively, the precipitant agent was ammonia solution, the volume of 25% ammonia solution was 48ml, the ripening temperature is 80℃.
2 Orthogonal experimental design results shows:The molecular weight of o-carboxymethyl chitosan, concentration of OCMCS and the power of ultrasonic have significant influence on the hydrophilic size of OCMCS-SPIO-NPs, while the ultrasonic time have no significant on the hydrophilic of OCMCS-SPIO-NPs. The optimal formulation-process was A1B1C1D2:namely,carboxymethyl chitosan concentration was 2%, MW of OCMCS was 1-2 million, ultrasonic time was 30minutes, the power of ultrasonic was 600W.
3 Fourier transform infrared spectroscopy and X-Ray diffraction indicated that the best process to modify the OCMCS-SPIO-NPs with FA was as follows. The mass ratio of folic acid active ester and OCMCS the 4:1 ratio,the reaction temperature was 50℃under the conditions of anhydrous dimethyl sulfoxide solution with the nitrogen protecting from oxygen.
4 Characterizations of resulting SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs
4.1TEM showed the resulting SPIO-NPs, OCMCS-SPIO-NPs,FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs are almost spherical or ellipsoidal. The size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs were present as x±s, whose particle size were 12.5±3.0nm,13.7±3.6nm,15.4±4.5nm and 17.5±4.8nm, respectively.
Hydrophilic size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs The average intense size was 201nm and the polydispersity was 0.234 for SPIO-NPs The average intense size was 38.2 nm and the polydispersity was 0.132 for OCMCS-SPIO-NPs, The average intense size was 41.4nm and the polydispersity for FA-OCMCS-SPIO-NPs; The average intense size was 125 nm and the polydispersity was 0.119 for dextran-SPIO-NPs.
4.3.Magneitc properties The results of magnetic properties of the samples which were study by VSM indicated that the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs were superparamagnetic, The saturation magnetization values for the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS SPIO-NPs were:98.0,71.4 and 69.6 emu/gFe.
4.4.T2 rate of the resulting SPIO-NPs:
OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in the T2 rate was 0.1685×106mol/s,0.1512×106mol/s and 0.139×106mol/s, which were higher than 0.062×106mol/s minimum standards.The transverse relaxation time (T2) shorten with the iron concentration increase in SPIO-NPs suspension.
4.5. Zeta potential of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs were-21.36±1.15mV,-27.88±0.73m and-18.1±1.01lmV respectively. 4.6. The iron content in resulting SPIO-NPs was as follows:OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs solution iron content were 5.44 mg Fe /ml,5.62mgFe/ml and 5.76mgFe/ml respectively; iron content in SPIO-NPs, OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs dry powder was 663.89mg/g, 615.61mg/g, and 601.40mg/g respectively.
4.7. X-Ray diffraction results showed that the powder crystals of resulting SPIO-NPs、OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs, were consistent with that of standards Fe3O4, more important, the entire preparation process of nanoparticles have no obvious impact on those powder crystal. FTIR results confirmed that OCMCS and the FA were conjugated on the surface of SPIO-NPs by covalent.
5 Evaluation of resulting SPIO-NPs In vitro
5.1 In vitro cytotoxicity
The cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO- NPs and SPIO-NPs against LO2, A549 and KB cells showed the cytoto xicity increase with the increase of iron concentration in culture media. The modification with OCMCS and FA could significantly decrease the cytotoxicity against all of the three cell. The cytotoxicity of FA-OCMC-SPIO-NPs against KB cell (FR over-expressed) was much higher than OCMCS-SPIO-NPs and dextran-SPIO-NPs(P < 0.05). While there is no significant difference among the cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs against LO2 and A549(FR negative) (P> 0.05).
5.2 Evaluation folate receptor tumor targeting in vitro
Ferroine assay showed that the intracellular iron content of both KB cells and Hela cells in FA-OCMCS -SPIO-NPs group was significantly higher than that in OCMCS-SPIO-NPs (PHela= 0.000<0.05; PKB= 0.000<0.05), while,the intracellular iron content in A549 cells, there was no significant difference between FA-OCMCS-SPIO-NPs group and OCMCS-SPIO-NPs group(P= 0.106> 0.05).
Prussian blue staining results showed both KB cell and Hela cell cultured with FA-OCMCS-SPIO-NPs were have dark prussian blue staining positive, which was much darker than those cells cultured in OCMCS-SPIO-NPs. While, A549 cultured in either FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs, both of them were almost prussian blue staining negative in intracellular. 5.3 Macrophage uptake in vitro
Intracellular iron content in RAW264.7 determined by Ferroine assay cells and Prussian blue staining showed that the iron content in RAW264.7 cultured in media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs was much lower than that cultured in media containing SPIO-NPs group, moreover, the intracellular iron content of RAW264.7 culture in media containing the FA-OCMCS-SPIO-NPs were significantly lower than the that cultured in media containing dextran-SPIO-NPs.
6. Evaluations in vivo
6.1. Tumor targeting in vivo
6.1.1 MRI scan
T2-weighted spin echo images of the tumor-bearing mice showed that the average intensity of the entire KB cell subcutaneous tumors decreases about 27.23% between preinjection and postinjection images injected with FA-OCMCS-SPIO-NPs (p=0.01<0.05). However, the signal intensity had no significant difference between preinjection and postinjection images of subcutaneous tumors for other groups(p>0.05).
6.1.2 Histopathology
HE and Prussian blue staining of tumor tissue slice showed only the bearing KB cell tumor mice were intravenously injected with FA-OCMCS-SPIO-NPs can be seen scatter SPIO-NPs aggregation and Prussian blue staining positive.while in other group there were no SPIO-NPs aggregation in HE staining or Prussian blue positive
6.2 Histological evaluation of the distribution of nanoparticles in vivo
Prussian blue staining results suggested that the mice that were intravenously injected with FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs, there were no stainable iron was observed in the heart, liver, spleen, kidney and lung tissue. While the mice that were intravenously injected with dextran-SPIO-NPs, the stainable iron was observed in KCs in hepatic sinusoid, in splenic cord of the perimeter section of follicle and in splenic red pulp. however there were no stainable iron in the other tissues
Conclusions
1. we have successfully synthesized the FA-OCMCS-SPIO-NPs with a typical "core-shell" structure, those nanoparticles has good hydrophilicity and strong superparamagnetic with a hydrophilic size e<50nm.
2. In vitro cytotoxicity results showed that covalently modify the SPIO-NPs with OCMC and folic acid could significantly reduce the cytotoxicity of SPIO-NPs. The cytotoxicity of FA-OCMCS-SPIO-NPs on cells (FR positive) were higher than that of OCMCS-SPIO-NPs, There was no significant difference on the cytotoxicity of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs against LO2(normal cell line) and A549(FR negative)
3. Tumor targeting results showed the synthesized FA-OCMCS-SPIO-NP had a strong targeting folate receptor, the higher folate receptor expressed on the surface, the higher the intracellular iron content
4. Uptake of all the synthesized SPIO-NPs by RAW264.7 in vitro indicated that the OCMCS surface modification can significantly reduce uptake of superparamagnetic iron oxide nanoparticles by the RAW264.7 cells, while the further modification had no influence on the uptake by RAW264.7 cells.
5.In vivo evaluation results showed that FA-OCMCS-SPIO-NPs had a high folate receptor tumor targeting and could avoid capture by reticuloendothelial system
The innovation of the paper
In this article, the OCMCS was used as a conjugate to associate the SPIO core and tumor target ligand (folic acid) to develop a novel active tumor-targeting SPIO-NPs system-FA-OCMCS-SPIO-NPs.
引文
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