CN116077684A - 一种组装纳米颗粒PM-HMSN/Arg及制备方法及应用 - Google Patents
一种组装纳米颗粒PM-HMSN/Arg及制备方法及应用 Download PDFInfo
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Abstract
本发明属于生物材料技术领域,具体涉及一种组装纳米颗粒PM‑HMSN/Arg及制备方法及应用。本发明以空心硅酸盐纳米粒子(HSN)、Mn元素、L‑Arg和血小板膜(PM)为原料,利用元素原位生长法、物理静电吸附法和薄膜挤出法制备出包裹血小板膜的含L‑Arg的空心硅酸锰纳米颗粒PM‑HMSN/Arg。本发明所制备的PM‑HMSN/Arg纳米颗粒入血后通过天然血小板膜表面蛋白介导的结合作用主动积聚到肿瘤部位。在超声的激活下,孔中产生大量ROS并催化L‑Arg转化为NO。NO分子以扩散的形式进入到肿瘤组织中,在抑制肿瘤生长的同时作为超声造影剂,产生有效的超声影像。而纳米颗粒PM‑HMSN/Arg中的Mn元素则可用于T1‑MRI核磁共振成像效果的增强。
Description
技术领域
本发明属于纳米材料的制备技术领域,具体涉及一种组装纳米颗粒PM-HMSN/Arg及制备方法及在制备用于肿瘤治疗药物与超声/核磁造影剂中的应用。
背景技术
当前肿瘤治疗面临着诊疗分离、影像学模式单一和治疗效果不佳等诸多问题,这些问题使得治疗频率和毒副作用增加,严重降低了患者的依从性。目前通常利用微/纳米载体来整合诊断剂和治疗剂,以期发挥其各自的功能。但往往存在制备过程复杂、非特异性肿瘤递送、诊断和治疗失调等问题,限制了其临床转化与应用。为了克服这一困境,迫切需要开发一种能同时进行多模态诊断和肿瘤治疗的诊疗系统。
研究发现,以适当的浓度使用一些具有生理意义的气体如氧气(O2)、一氧化氮(NO)、一氧化碳(CO)、硫化氢(H2S)、二氧化硫(SO2)和氢气(H2)时,可以逆转癌细胞中的Warburg效应,在对正常细胞无不利影响的同时,抑制癌细胞的增殖。其中,NO在心血管稳态、对感染的免疫反应等一系列生理过程中发挥重要的调节作用。据报道,NO具有剂量相关的功效,即在高浓度下通过“抗Warburg效应”、线粒体和DNA的亚硝化作用、活性氧(ROS)的产生来抑制肿瘤癌细胞的增殖;在低浓度下则作为一种有效的p-糖蛋白调节剂来克服肿瘤的多药耐药。同时NO还可以促进肿瘤新血管生成、激活MMP-2活性和减少细胞外基质(ECM)、减轻结缔组织压迫和实体瘤缺氧、增强抗肿瘤效应。因此,NO可作为一种有效的肿瘤杀伤剂。并且NO气体的超低分子量使得它们通过生物膜扩散到肿瘤间质时无需任何主动运输机制。不仅如此,NO气泡还可以用作潜在的超声造影剂(UCA)。
但NO在癌症治疗中的存在以下缺陷:首先,常规NO释放系统的肿瘤靶向递送仅依赖于通透性改变和滞留(EPR)效应或主动靶向,其机制单一、疗效低下,常常造成脱靶毒性,如全身性低血压、急性肾损伤或有生命威胁的器官出血。其次,由于拉普拉斯压力、氧代谢、组织内超声能暴露、血流撞击等原因,NO气泡产生后会迅速破裂,使得NO气泡难以在体内稳定存在。再者是体内NO的生成和释放难以操控,如出现过早的NO泄漏。因为气体疗法的疗效和生物安全性均取决于疾病病灶中气体的浓度和滞留时长,所以采用NO进行肿瘤治疗时,开发一种能稳定保护NO气泡的肿瘤靶向递送系统至关重要。
发明内容
为解决目前缺乏靶向富集至肿瘤的可缓慢释放NO的载体的技术问题。
本发明的第一个目的是提供一种组装纳米颗粒PM-HMSN/Arg,其中,组装纳米颗粒PM-HMSN/Arg为包被血小板膜的HMSN/Arg颗粒,所述HMSN/Arg颗粒由硅酸锰空心颗粒在L-Arg溶液中静电吸附L-Arg后制得。
进一步的,所述硅酸锰空心颗粒是以中空介孔二氧化硅纳米颗粒作为模板,将中空介孔二氧化硅纳米颗粒与可溶性锰盐、可溶性铵盐在碱性条件下进行水热反应或在常温水溶液中反应,得到硅酸锰空心颗粒。
进一步的,所述硅酸锰空心颗粒的DLS和PDI分别为193nm和0.224,zeta电位为-18.4mV,所述HMSN/Arg颗粒zeta电位为10.3mV,所述组装纳米颗粒PM-HMSN/Arg的粒径较HMSN颗粒增加了18~20nm,zeta电位为-32.5mV。
空心颗粒组装纳米颗粒PM-HMSN/Arg具有低密度、高表面积与体积比、低热膨胀系数和折射率等特点,使其在生物成像、药物/基因储存、控释等广泛的潜在应用中具有极大的吸引力。空心颗粒的空心内部可以充满气体以充当超声造影剂。并且空心硅酸锰颗粒(HMSN)可作为超声触发活性氧(ROS)生产的平台。而ROS可以与L-Arginine(Arg)反应生成NO。因此,本发明将HMSN和L-Arg构建在一起,利用超声来触发NO的释放。
血小板作为一种内源性血液细胞,其最显著的功能是保持血管系统的完整性,防止出血。除了止血作用外,血小板膜表面表达的膜蛋白(如CD62p和整合素α6)具有一定的肿瘤靶向性。血小板膜仿生涂层已被证实是一种有效靶向肿瘤组织的策略。提取的血小板膜含有血小板所特有的所有蛋白组,包裹在化学合成的载体纳米颗粒表面后,其膜表面的CD62p和整合素α6能使纳米粒子对肿瘤组织具有主动靶向的亲和力。因此,本发明设计了一种具有血小板膜仿生涂层的中空NO纳米搭载平台,超声激活后可用于制备靶向肿瘤气体治疗的药物和超声/核磁造影剂。
本发明的第二个目的是提供所述的组装纳米颗粒PM-HMSN/Arg的制备方法,包括以下步骤:
S01.采用气溶胶辅助自组装法,制备中空介孔二氧化硅纳米颗粒;
S02.以中空介孔二氧化硅纳米颗粒作为模板,将中空介孔二氧化硅纳米颗粒与可溶性锰盐、可溶性铵盐在碱性条件下进行水热反应或在常温水溶液中反应,得到硅酸锰空心颗粒;
S03.将所述HMSN颗粒加入L-Arg溶液中反应,固液分离,将得到的固体洗涤,得到所述HMSN/Arg颗粒;
S04.采用薄膜挤出法制备血小板膜包被的HMSN/Arg颗粒,即组装纳米颗粒PM-HMSN/Arg。
进一步的,步骤S02具体包括:取可溶性锰盐、可溶性铵盐和氨水溶于水配置成透明溶液A;取步骤S01制得的中空介孔二氧化硅纳米颗粒分散于水,得到悬浮溶液B;然后将透明溶液A和悬浮溶液B混合后,固液分离,将得到的固体洗涤,得到沉积Mn2+后形成的硅酸锰空心颗粒;所述的可溶性锰盐、可溶性铵盐、氨水和中空介孔二氧化硅纳米颗粒按0.2mmol:6mmol:1mL:200mg配比。
进一步的,步骤S04包括:先提取血小板,然后将血小板重悬于磷酸盐缓冲溶液,反复超声处理至血小板悬浮液透明,再将HMSN/Arg颗粒滴加到血小板悬浮液中混合均匀,最后从滤膜中挤出,固液分离得到组装纳米颗粒PM-HMSN/Arg。
本发明基于空心硅酸盐纳米粒子(HSN),通过依次包覆Mn元素、L-Arg和血小板膜(PM)来构建血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg。首先用气溶胶辅助自组装法制备出中空的介孔二氧化硅纳米颗粒(HSN)。其次利用Mn元素原位生长方法制备Mn元素沉积的空心硅酸锰纳米颗粒(HMSN)。再通过HMSN负电性的介孔表面和正电性的L-Arg之间的静电吸附作用,制备出负载L-Arg的HMSN(HMSN/Arg)。最后采用薄膜挤出法将血小板膜(PM)包被在HMSN/Arg表面,成功制备PM-HMSN/Arg。
本发明最后目的是提供组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤治疗药物与超声/核磁造影剂中的应用。
所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤治疗药物中的应用,其特征在于,所述组装纳米颗粒PM-HMSN/Arg能够特异性富集至肿瘤细胞,并在超声辐照下释放NO。
PM-HMSN/Arg进入血液后,PM-HMSN/Arg通过天然血小板膜表面蛋白的介导,主动积聚到肿瘤组织中。在超声的激活下,孔中首先产生大量ROS,产生的ROS可催化L-Arg转化为NO。NO分子以扩散的形式进入到肿瘤组织中,能有效抑制肿瘤生长。
进一步的,所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤超声造影剂中的应用,其特征在于,所述组装纳米颗粒PM-HMSN/Arg能够特异性富集至肿瘤细胞,并在超声辐照下释放NO并填充在HMSN颗粒的空心腔内,组装纳米颗粒PM-HMSN/Arg将肿瘤细胞与正常组织区分开。
超声时产生的NO气体分子治疗肿瘤的同时,能作为超声造影剂,产生有效的超声影像。
进一步的,所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤核磁造影剂中的应用。
纳米颗粒PM-HMSN/Arg中的Mn元素则可用于T1-MRI核磁共振成像效果的增强。
与现有技术相比,本发明的优势和有益效果主要体现在:
一、当进入血液时,PM-HMSN/Arg可以通过天然血小板膜表面蛋白的介导,主动积聚在肿瘤组织中。在超声的激活下,超声机械能向化学能的转化导致孔中产生大量ROS,进一步催化L-Arg转化为NO。NO分子以扩散的形式进入到肿瘤组织中,可有效抑制肿瘤生长。
二、PM-HMSN/Arg在超声的激活下所产生的NO可以扩张肿瘤血管,导致更多PM-HMSN/Arg纳米颗粒聚集于此。
三、PM-HMSN/Arg在超声的激活下所产生的NO气泡可以稳定地保存在空心硅酸锰颗粒的空心核心内,用来维持超声的成像,并结合Mn介导的T1-MRI核磁共振进行双模态诊断。
附图说明
图1为实施例2中HSN、HMSN、HMSN/Arg、PM-HMSN/Arg的纳米粒度电位仪结果;
图2为实施例2中HMSN/Arg、PM-HMSN/Arg的透射电镜图像;
图3为实施例2中PM-HMSN/Arg的SDS-PAGE与Western blot蛋白质印迹分析结果图像;
图4为实施例3中PM-HMSN/Arg的溶血测定结果图像;
图5为实施例3中甲基噻唑基四唑(MTT)法测PM-HMSN对HUVEC的细胞毒性时细胞生存率;
图6为实施例4中PM-HMSN/Arg治疗种瘤裸鼠时的体重变化曲线;
图7为实施例5中PM-HMSN/Arg体外NO释放曲线;
图8为实施例8中PM-HMSN/Arg介导的体外超声图像;
图9为实施例9中PM-HMSN/Arg介导的体内超声图像;
图10为实施例10中PM-HMSN/Arg在体内MRI磁共振成像图像。
具体实施方式
下面结合具体实施例对本发明的技术方案进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg,由以下方法制备得到:
首先采用气溶胶辅助自组装法,制备中空介孔二氧化硅纳米颗粒(HSN)。其次利用Mn元素原位生长方法制备Mn元素沉积的空心硅酸锰纳米颗粒(HMSN)。再通过HMSN负电性的介孔表面和正电性的L-Arg之间的静电吸附作用,制备负载L-Arg的HMSN,即HMSN/Arg。最后采用薄膜挤出法制备血小板膜(PM)包被的HMSN/Arg,即PM-HMSN/Arg。
1.1采用气溶胶辅助自组装法,快速大量制备中空介孔二氧化硅纳米颗粒(HSN)。
(1)取3g十六烷基三甲基溴化铵(CTAB)、6g硫酸铵和50g蒸馏水配制水溶液,室温搅拌至溶解,用稀HCl调节至pH≤2,作为水相。
(2)按30g乙醇,10g四乙氧基硅烷(TEOS)取样混合,室温搅拌至溶解,作为乙醇相。
(3)水相和乙醇相分别搅拌0.5h后,二者互相混合,继续搅拌0.5h后,室温静置进行Ostward熟化6h。
(4)将混合物分批加入高压气流雾化器(空气为载气,气压3bar)。然后,被雾化为纳米尺寸的气溶胶小液滴通入高温管式炉(400~500℃)中,进行蒸发诱导二氧化硅-表面活性剂液晶间的自组装。
(5)在高温管式炉出口端滤膜上保持80℃,收集产物。
(6)产物在550℃空气中煅烧6h,得到终产物HSN。
该部分制备方法参考《Aerosol fabrication of hollow mesoporoussilicananoparticles and encapsulation of L-methionine as a candidate drugcargo》文章中的制备手法。
1.2采用了基于HSN的Mn元素原位生长方法制备Mn元素沉积的HSN,即空心硅酸锰纳米颗粒(HMSN)。
(1)将所得200mg HSN纳米颗粒分散在20mL蒸馏水中。
(2)将1mL NH3·H2O(40wt%)添加到10mL含有0.2mmol MnCl2和6mmol NH4Cl的蒸馏水中。
(3)将步骤(2)的混合物倒入HSN纳米颗粒悬浮液中,在室温下搅拌24小时。
(4)取纳米混悬液,10000rpm离心20min,弃去上清,取底部沉淀的HMSN纳米颗粒,分别用水和乙醇交替洗涤3次。
(5)最后,取纳米粒水中混悬液,冷冻干燥,得到终产物HMSN。
该掺杂Mn离子的制备方法参考ZL201711134136.1中的制备手法。
1.3采用物理静电吸附法,通过HMSN负电性的介孔表面和正电性的L-Arg之间的静电吸附作用,制备负载L-Arg的HMSN,即HMSN/Arg。
(1)将1g L-Arg在室温搅拌下溶解于20mL去离子水中。
(2)加入100mg HMSN并搅拌24h以充分吸附L-Arg分子。
(3)反应结束后,以10000rpm离心20min,收集沉淀,用水清洗3次,冷冻干燥,得到HMSN/Arg。
该部分制备方法参考《Ultrasound-Triggered Nitric Oxide Release PlatformBased on Energy Transformation for Targeted Inhibition of Pancreatic Tumor》文章中的制备手法。
1.4采用薄膜挤出法制备血小板膜(PM)包被的HMSN/Arg,即PM-HMSN/Arg。
(1)血小板的提取:取健康雄性BALB/c小鼠全血约10mL,新鲜血样经过肝素抗凝处理后,分别以100g和200g常温离心20min,去除红细胞和白细胞。然后,将含有1mM EDTA和2μM前列腺素E1的PBS添加到上清液中以抑制血小板活化。以800g常温离心20分钟,获得血小板沉淀,并将其重新分散在1mL PBS中,然后加入蛋白酶抑制剂和1mM EDTA。之后,将收获的血小板进行三个重复循环的冻融。最后,以4000g常温离心10分钟,去除上清,加入1mL纯水吹匀沉淀,重复2次。超声分散样品5min,冻冷干燥。
(2)将提取出的PM重悬在磷酸盐缓冲溶液中,浓度为4mg/mL,并反复超声处理,直到溶液变得清澈透明。
(3)将HMSN/Arg中空纳米粒子滴加到PM悬浮液中,浓度为5mg/mL。通过涡旋和超声处理反复分散混合物。
(4)使用微型挤出机(Avanti)配合800nm和200nm滤膜,分别对上述混合物进行物理挤出。最终,通过以10000rpm离心20分钟收集PM-HMSN/Arg纳米颗粒。
该部分制备方法参考《Platelet-Membrane-Coated Nanoparticles EnableVascular Disrupting Agent Combining Anti-Angiogenic Drug for Improved TumorVessel Impairment》文章中的制备手法。
实施例2:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg的表征
2.1HSN、HMSN、HMSN/Arg和PM-HMSN/Arg的流体动力学直径和Zeta电位检测方法:配制HSN、HMSN、HMSN/Arg和PM-HMSN/Arg的纯水分散样品,稀释至适宜浓度。然后,通过纳米粒度电位仪Malvern Zeta-sizer Nano ZS90(Malvern Instruments Ltd.,UK)测量样品的流体动力学直径和Zeta电位。
如图1所示:HSN的粒径分布(DLS)和分布系数(PDI)分别为193.3nm和0.224,zeta电位为-25.5mV,符合介孔硅空心球的均匀单分散和负电性表面特性。此外,相比于HSN,样品HSMN和HMSN/Arg的流体动力学直径没有发生明显变化,说明Mn元素和L-Arg等小分子的负载对于纳米颗粒的尺寸影响较小。然而,可以看到HMSN/Arg相对于HMSN,发生了从-18.4mV到10.3mV的明显zeta电位反转,说明正电性的L-Arg通过静电吸附作用成功负载到HMSN纳米颗粒中。PM-HMSN/Arg的粒径增加了大约20nm,zeta电位恢复到-32.5mV,这表明带负电的PM已成功包被在颗粒表面上。
2.2HMSN/Arg和PM-HMSN/Arg的形貌和结构方法:在120KeV下传导的透射电子显微镜(TEM,JEOL,JEM2000EX,日本)研究HMSN/Arg和PM-HMSN/Arg的形貌和结构。
如图2所示:TEM图像显示HMSN/Arg和PM-HMSN/Arg具有均匀的尺寸分布,明显的壳核结构和内部空心腔。与HMSN/Arg相比,PM-HMSN/Arg在表面显示出明显的约18nm厚的膜涂层。
2.3PM-HMSN/Arg的表征方法:采用TF-G20高分辨率TEM(Thermo FisherScientific,USA)对PM-HMSN/Arg形貌进行表征,并分析了Si、O、Mn、N和P的元素分布。
结果:元素分布mapping显示PM-HMSN/Arg含有Si、O、Mn、N和P元素,证明Mn、L-Arg和血小板膜磷脂分别成功地逐层包被在HSN表面。
2.4PM-HMSN/Arg的蛋白质组成表达方法:取血小板、PM和PM-HMSN/Arg,10000g室温离心20min,沉淀用RIPA裂解液在冰上孵育30min,以裂解血小板膜。再次10000g室温离心20min,取包含膜蛋白的上清通过BCA试剂盒测定蛋白浓度后,将剩余上清液加入SDSloading buffer,煮蛋白10min后,将样品置于-20℃保存。接下来,进行SDS-PAGE和蛋白质印迹实验,测定其广谱和特异性蛋白质组成的表达。
2.4.1SDS-PAGE
(1)上样:取提取的各组样品,以40μg蛋白样品/孔上样。
(2)电泳:将电泳槽置于电泳仪中,加入新鲜电极缓冲液,80V,电泳120min。
(3)染色:电泳结束后,取凝胶置于考马斯亮蓝染色液中,染色2h。
(4)脱色:配制脱色液A、B、C:甲醇分别为300mL、200mL、100mL,乙酸浓度分别为100mL、100mL、50mL,分别加入去离子水定容至1L。将凝胶置于脱色液A、B、C中,分别脱色0.5h、1h、2h。
(5)成像:取凝胶,PBS冲洗后,置于Bio-Rad ChemiDoc Touch成像系统进行拍照。
2.4.2Western blot蛋白质印迹分析
(1)-(2)同其上
(3)进行转膜,将蛋白质从凝胶转移到PVDF膜中。
(4)用5% BSA封闭1小时。
(5)用TBST(含0.02% Tween 20的Tris缓冲液)洗涤两次后,将PVDF膜浸于血小板膜蛋白标记物抗CD41一抗(ab134131)(1:2000稀释)和抗CD62p一抗(ab255822)(1:2000稀释)4℃孵育12小时。
(6)用TBST溶液洗膜3次后,浸泡于HRP标记的二抗(山羊抗兔,1:5000稀释)中孵育2h。
(7)使用Bio-Rad ChemiDoc Touch成像系统(Bio-Rad,美国)对印迹图像进行可视化。
如图3所示:SDS-PAGE考马斯亮蓝染色法的结果表明PM-HMSN/Arg表面包被的血小板膜蛋白组分与天然血小板膜十分相似,表明经过膜挤出法制备的PM-HMSN/Arg几乎能将血小板膜蛋白组分完全保留。此外,血小板膜蛋白CD41和CD62p在PM-HMSN/Arg表面的存在通过Western blot蛋白质印迹分析得到证实,它们是血小板膜的特异性膜蛋白。
实施例3:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg的体外安全性检测
3.1溶血测定
(1)将从新鲜的含肝素钠的小鼠血液中分离出来的红细胞(RBC),于4℃ 2000rpm离心5分钟,用PBS洗涤3次,直至上清液澄清。
(2)将去离子水和生理盐水,及不同浓度(0~5mg/mL)的PM-HMSN/Arg添加到2%(v/v)红细胞悬浮液中,并在37℃下孵育2小时。
(3)将这些孵育溶液以2000rpm离心5分钟,使上清液与红细胞分离。
(4)使用酶标仪(Multiskan MK3,Thermo Fisher Scientific,USA)在570nm处检测上清液的吸光度。
计算溶血率:溶血率(%)=[(ODtest-ODneg)/(ODpos-ODneg)]×100
其中ODtest、ODneg和ODpos为样品的OD570值,生理盐水和去离子水分别作为阴性和阳性对照。
(5)使用数码相机拍摄孵育溶液上清液的图像。
(6)使用显微镜(Leica DMI 4000D,德国)捕获红细胞的形态图像。
如图4所示:PM-HMSN/Arg在0~5mg/mL的宽浓度范围内与红细胞孵育几乎未观察到明显的溶血现象,定量测定结果显示各浓度纳米粒组的溶血率均小于5%,表明PM-HMSN/Arg具有良好的血液相容性。
3.2甲基噻唑基四唑(MTT)试验
(1)人脐静脉内皮细胞(HUVEC)细胞以5000细胞/孔的密度种于96孔板中培养。
(2)待肿瘤细胞贴壁后,加入系列梯度浓度的PM-HMSN溶液进行孵育,每组设置5个重复孔,同时设置空白对照组。将96孔板置于37℃,5%CO2培养箱培养。
(3)培育时间到达后,弃去上清,每孔加入空培稀释的MTT试剂(MTT:空培=1:9)100μL,置于培养箱中孵育4h。
(4)孵育时间达到后除去每孔中的MTT试剂,每孔加入DMSO溶液100μL,避光孵育10min后使用酶标仪在490nm波长处进行吸光度测量。
如图5所示:对照细胞的存活率设为100%。用1mg/mL的PM-HMSN孵育HUVEC细胞后,暴露12小时后,不到10%的细胞死亡。当孵育时间延长至24h时,细胞活力仍保持在85%以上。这些结果表明PM-HMSN可被认为具有低细胞毒性,使其非常适合体内生物应用。
实施例4:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg的体内的安全性监测
4.1抗肿瘤治疗期间每3天记录一次小鼠的体重
常规培养BxPC-3细胞(人原位胰腺腺癌细胞),在对数生长期时用胰蛋白酶消化细胞,并用预冷PBS7.4重悬,调整细胞浓度为8×107cells/mL。采用1mL注射器吸取100μL细胞接种于裸鼠右前肢皮下部位,停针1min后缓缓旋转撤出针头以防止细胞渗漏出来。种瘤后按照操作规程继续正常饲养,并每天观察肿瘤生长情况。待肿瘤体积达100mm3时,将BxPC-3荷瘤裸鼠随机分为6组,每组6只,并作为治疗的第0天开始给药。各给药组分别为:生理盐水组、单纯超声(US alone)组、HMSN/Arg组、PM-HMSN/Arg组、HMSN/Arg联合超声(HMSN/Arg+US)组、PM-HMSN/Arg联合超声(PM-HMSN/Arg+US)组,其中L-Arg的给药剂量为10mg/kg,不含L-Arg的纳米粒组称取与含L-Arg纳米粒等量的载体进行尾静脉注射。同时,联合治疗方案中超声辐照条件为:1.0MHz-1.0W-20%,超声15s,间歇30s,每次超声辐照处理进行20个循环,每天进行12次超声辐照处理。每2天治疗一次,共处理7次,最后一次治疗后2天,处死BxPC-3荷瘤裸鼠,结束实验。在BxPC-3荷瘤裸鼠14天治疗期间内,每2天测定一次各组小鼠的体重,绘制体重变化曲线,如图6。
4.2抗肿瘤治疗结束后采集血样进行血常规检查和血液生化分析在BxPC-3荷瘤裸鼠14天治疗结束后,分别从生理盐水组、单纯超声(US alone)组、HMSN/Arg组、PM-HMSN/Arg组、HMSN/Arg联合超声(HMSN/Arg+US)组、PM-HMSN/Arg联合超声(PM-HMSN/Arg+US)组,随机选择3只小鼠,心脏采集全血,转移至血常规专用抗凝管中,多次轻柔颠倒以保证血液与管壁的抗凝剂充分混匀。然后,通过全自动血球计数仪测定红细胞(RBC)、白细胞(WBC)、血小板(PLT)、血红蛋白(HGB)等血常规参数。
同时,将每组剩余3只荷瘤鼠的全血样本置于冰上,待其自然凝结后于4℃下4000rpm离心15min,取上层淡黄色血清部分转移至新的EP管中,通过全自动生化分析仪测定丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、肌酐(CREA)和尿素氮(BUN)的水平。
4.3抗肿瘤治疗结束后取出主要器官,例如心脏、肝脏、脾脏、肺和肾脏进行HE染色
BxPC-3荷瘤小鼠取血后,每组随机选取3只依次进行生理盐水和4%多聚甲醛的心脏灌流,然后取出心、肝、脾、肺、肾等主要脏器,浸泡于4%多聚甲醛中固定48h,制备石蜡切片并进行H&E染色。最后,将切片置于光学显微镜下观察组织形态并随机选择视野拍照。
结果:通过小鼠抗肿瘤治疗过程中的体重变化、治疗结束后的血常规检查和生化分析,以及主要器官的HE染色,考察了小鼠经过多个周期PM-HMSN/Arg处理的生物安全性。在各治疗组中,没有明显的体重减轻或主要器官的组织学损伤。生理盐水处理小鼠的各项血液学指标和肝肾功能指标与其他任何治疗组均无显著差异,表明PM-HMSN/Arg长期治疗对血液、肝肾功能等没有产生明显的毒性作用。
实施例5:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg体外NO释放实验
方法:使用Griess分析试剂盒研究了纳米颗粒的体外NO释放曲线,重点对纳米颗粒的超声触发响应生成NO的性质进行了考察。简而言之,将PM-HMSN,PM-HSN/Arg和PM-HMSN/Arg分别以20mg L-Arg的相当量,添加到50ml PBS中。然后,进行超声照射,条件为:1.0MHz,1.5W cm-2,5min,每个脉冲的持续时间为15s,两个相邻脉冲之间的间隔为30s。在US触发后,在预定时间点(0分钟、5分钟、15分钟、30分钟、1小时、2小时、4小时、6小时),收集1ml溶液,10000rpm离心5分钟。然后,通过Griess测定试剂盒将试管中的上清液用于NO释放测定。
如图7所示:不含L-Arg的纳米颗粒,无论有无超声辐照,均无无明显的NO释放。相比而言,含L-Arg的纳米颗粒,即PM-HSN/Arg和PM-HMSN/Arg,在超声辐照下均有明显的NO释放,说明L-Arg作为底物生成NO的必要性,同时,也观察到PM-HMSN/Arg明显的依赖超声触发的NO释放特点。此外,PM-HMSN/Arg相对于PM-HSN/Arg的超声触发NO的释放速度和程度也更高,说明Mn元素的加入有助于提高纳米颗粒的NO产生效率,这可能是通过Mn元素增强介孔硅的电孔分离效应进而产生更多活性氧实现的。
实施例6:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg细胞间NO释放实验
方法:将BxPC-3细胞以每孔2×105个细胞的密度接种在24孔玻璃底的细胞培养板上。待细胞稳定生长24小时后,避光条件下将BxPC-3细胞与1μM DAF-FM DA工作液共孵育20min,进行NO探针的细胞原位装载。然后,弃去探针,PBS清洗BxPC-3细胞多次后,细胞分别与PBS、HMSN/Arg或PM-HMSN/Arg纳米粒悬液(L-Arg:1mg/mL)共孵育。然后,进行超声照射。超声条件为:1.0MHz,1.5W cm-2,5分钟,每个脉冲的持续时间为15s,两个相邻脉冲之间的间隔为30s。处理30min后,弃去培养基,PBS清洗细胞,4%多聚甲醛固定细胞。然后,Hoechst33342标记细胞核,置于激光共聚焦显微镜下观察拍照,并用Image J软件对细胞的DAF-FM DA荧光强度半定量以评价纳米颗粒释放到细胞内的NO水平。
结果:PBS对照组和单独超声处理组,细胞内均未观察到明显的NO分布,说明肿瘤细胞正常状态下的低NO生成水平。HMSN/Arg或PM-HMSN/Arg轻微的提高了胞内NO的水平,这可能是纳米颗粒被细胞摄取后,被胞内的少量活性氧催化生成了低水平的NO。在经过超声辐照后,HMSN/Arg或PM-HMSN/Arg在胞内释放了大量NO,这与其体外NO释放的趋势相一致。同时,PM-HMSN/Arg联合超声处理相比于其他各组,产生了最显著的胞内NO释放水平,这可能与PM介导的肿瘤细胞靶向摄取效率提高有关。
实施例7:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg肿瘤内的NO释放实验
方法:在BxPC-3皮下肿瘤模型中,用荧光探针DAF-FM DA评估了PM-HMSN/Arg在肿瘤内的NO释放水平。将肿瘤体积约为100mm3的荷瘤小鼠随机分为6组(n=3)。第1组:生理盐水,(尾静脉注射,100μL);第2组:超声照射;第3组:HMSN/Arg,(尾静脉注射,L-Arg:10mg/kg,100μL);第4组:PM-HMSN/Arg,(尾静脉注射,L-Arg:10mg/kg,100μL);第5组:HMSN/Arg,(尾静脉注射,L-Arg:10mg/kg,100μL)+超声照射;第6组:PM-HMSN/Arg,(尾静脉注射,L-Arg:10mg/kg,100μL)+超声照射。其中,超声照射指的是在便携式聚焦超声治疗仪(Chattanooga,美国)上用超声波照射肿瘤组织,每个周期的参数为1.0MHz-1.0W-20%,持续15s,相邻2次超声之间间隔30s,每个周期进行20次超声照射。每天进行12个周期的照射。在超声治疗的最后一个周期结束时,小鼠瘤内注射DAF-FM DA(2.5mg/kg)。1小时后,收集肿瘤进行冷冻切片,并用Hoechst 33258染色标记细胞核以进行CLSM观察。
结果:生理盐水对照组和单独超声辐照处理的肿瘤组织内只观察到极少量的代表NO的绿色荧光。注射HMSN/Arg和PM-HMSN/Arg后,小鼠肿瘤内NO水平有轻微的升高,这是由于纳米颗粒负载的L-Arg与肿瘤微环境中的部分活性氧反应生成了少量NO。在给予超声辐照后,纳米颗粒在肿瘤内释放了大量NO。PM-HMSN/Arg联合超声辐照显示了最高的瘤内NO生成水平,说明纳米颗粒在血小板膜的主动肿瘤靶向作用下,通过高效肿瘤富集实现了更强的肿瘤部位NO生成和释放。
实施例8:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg在体外超声成像
方法:在配备3.0MHz凸阵列探头的SonoScape扫描仪上,通过对比增强超声(CEUS)模型研究了PM-HMSN/Arg的超声成像能力,机械指数(MI)为0.16。首先,分别将用脱气生理盐水稀释的PM-HMSN,PM-HSN/Arg,PM-HMSN/Arg悬浮液(载体浓度1mg/mL)填充到离心管中,进行超声扫描仪探针引导的扫描。所有测量均在室温下进行。
如图8所示:当该纳米材料不含L-Arg时,即PM-HMSN,在最初5min内可以观察到短暂的超声信号,这可能是来源于纳米颗粒空腔里最初包含的空气内核。然而,PM-HMSN的超声成像只能持续约5min,之后几乎没有观察到明显的超声信号,这可能是由于超声辐射和表面张力介导的内部空气气泡的快速破坏,从而造成很短暂的超声成像“有效时间窗”。然而,当该纳米材料含L-Arg时,PM-HSN/Arg和PM-HMSN/Arg显示超声成像信号强度显着增加,并且在2小时后给予超声辐照仍能观察到显著的超声信号。这可能是因为超声依托纳米颗粒平台将超声波机械能转化为化学能,在颗粒中触发了大量的ROS快速生成,进而将负载的L-Arg分子催化成NO,而这些NO气泡可以持续生成并填充在纳米颗粒的空心腔内作为超声造影剂。在这种情况下,每次暴露于超声辐照时,空心纳米颗粒可以被NO气泡快速“填充”,进行超声造影。另外,包含Mn元素的PM-HMSN/Arg相较于不含Mn元素PM-HSN/Arg,在2h内的超声反射信号明显更强,这是由于Mn元素通过增强的电孔分离效应,能够辅助介孔硅空心球产生更多的活性氧,从而生成更多的NO气泡用于超声造影。
实施例9:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg在体内超声成像
方法:将肿瘤体积约为100mm3的BxPC-3荷瘤小鼠分别尾静脉注射PM-HMSN、HMSN/Arg和PM-HMSN/Arg(L-Arg:10mg/kg)。然后,在不同时间点(2min、0.5h、1h、2h)通过彩色多普勒超声检查仪采集肿瘤部位的超声图像。
如图9所示:生理盐水对照组在注射后2小时内,在肿瘤部位未观察到明显的超声造影信号,这可能与胰腺癌低血供和肿瘤组织缺乏造影剂灌注有关。尾静脉注射不含L-Arg的PM-HMSN纳米颗粒,在最初2min内可以观察到短暂的超声造影,这可能与纳米颗粒空腔里最初包含的空气内核有关。然而,接下来的30min~2h,肿瘤部位几乎没有观察到明显的超声信号,这可能是由于PM-HMSN包含的空气内核在体内快速消耗殆尽,并被代谢清除。相比而言,含L-Arg的HMSN/Arg和PM-HMSN/Arg纳米颗粒在肿瘤部位均能产生持续且清晰的超声造影信号,证明超声触发NO气泡生成和填充在纳米颗粒的空心腔对于超声造影的增强作用。同时,PM-HMSN/Arg相比于HMSN/Arg具有更强的造影信号,这与二者的肿瘤靶向递送效率一致,说明PM介导的肿瘤主动靶向粘附通过提高纳米颗粒的肿瘤蓄积实现更好的超声造影效果。
实施例10:血小板膜包被的搭载Mn元素和L-Arg的空心硅酸盐纳米粒子PM-HMSN/Arg在体内MRI磁共振成像
方法:在室温(TE=50ms,TR=3000ms)下,通过Bruker Biospect 7.0T/20厘米磁共振系统测量PM-HMSN/Arg的体内T1-weighted MRI信号。首先,将肿瘤体积约为100mm3的BxPC-3荷瘤小鼠分别尾静脉注射生理盐水、HMSN/Arg、PM-HSN/Arg、PM-HMSN/Arg(L-Arg:10mg/kg)。然后,通过便携式聚焦超声治疗仪(Chattanooga,美国)用超声波照射肿瘤组织,每个周期的参数为1.0MHz-1.0W-20%,持续15s,相邻2次超声之间间隔30s,每个周期进行20次超声照射。每天进行12个周期的照射。同时,在纳米颗粒注射后的不同时间点(2h、12h)采集肿瘤部位的T1-MRI图像。另外,取适宜数量的荷瘤裸鼠不施加超声辐照,其他纳米颗粒注射和T1-MRI图像采集同法操作,作为对照组。
如图10所示:生理盐水组和不含Mn元素的PM-HSN/Arg,2h和12h的肿瘤部位显示出一片灰暗,表明其T1-MRI信号均很弱,这可能是由于缺乏Mn元素等磁共振对比增强剂。相较而言,其他各组含Mn元素的肿瘤组织,均呈现出一定“亮度”的增强,证明Mn元素的T1-MRI造影剂效果。其中,PM-HMSN/Arg联合超声,显示出最强的肿瘤T1-MRI信号,且各组的磁共振造影效果与体内递送趋势较为一致,说明PM-HMSN/Arg可以通过增强的肿瘤靶向蓄积,实现更好的T1-MRI造影效果。
Claims (9)
1.一种组装纳米颗粒PM-HMSN/Arg,其特征在于,组装纳米颗粒PM-HMSN/Arg为包被血小板膜的HMSN/Arg颗粒,所述HMSN/Arg颗粒由硅酸锰空心颗粒在L-Arg溶液中静电吸附L-Arg后制得。
2.根据权利要求1所述的组装纳米颗粒PM-HMSN/Arg,其特征在于,所述硅酸锰空心颗粒是以中空介孔二氧化硅纳米颗粒作为模板,将中空介孔二氧化硅纳米颗粒与可溶性锰盐、可溶性铵盐在碱性条件下进行水热反应或在常温水溶液中反应,得到硅酸锰空心颗粒。
3.根据权利要求1所述的组装纳米颗粒PM-HMSN/Arg,其特征在于,所述硅酸锰空心颗粒的DLS和PDI分别为193nm和0.224,zeta电位为-18.4mV,所述HMSN/Arg颗粒zeta电位为10.3mV,所述组装纳米颗粒PM-HMSN/Arg的粒径较硅酸锰空心颗粒增加了18~20nm,zeta电位为-32.5mV。
4.如权利要求1~3任一项所述的组装纳米颗粒PM-HMSN/Arg的制备方法,包括以下步骤:
S01.采用气溶胶辅助自组装法,制备中空介孔二氧化硅纳米颗粒;
S02.以中空介孔二氧化硅纳米颗粒作为模板,将中空介孔二氧化硅纳米颗粒与可溶性锰盐、可溶性铵盐在碱性条件下进行水热反应或在常温水溶液中反应,得到硅酸锰空心颗粒;
S03.将所述HMSN颗粒加入L-Arg溶液中反应,固液分离,将得到的固体洗涤,得到所述HMSN/Arg颗粒;
S04.采用薄膜挤出法制备血小板膜包被的HMSN/Arg颗粒,即组装纳米颗粒PM-HMSN/Arg。
5.根据权利要求4所述的制备方法,其特征在于,步骤S02具体包括:取可溶性锰盐、可溶性铵盐和氨水溶于水配置成透明溶液A;取步骤S01制得的中空介孔二氧化硅纳米颗粒分散于水,得到悬浮溶液B;然后将透明溶液A和悬浮溶液B混合后,固液分离,将得到的固体洗涤,得到沉积Mn2+后形成的硅酸锰空心颗粒;所述的可溶性锰盐、可溶性铵盐、氨水和中空介孔二氧化硅纳米颗粒按0.2mmol:6mmol:1mL:200mg配比。
6.根据权利要求5所述的制备方法,其特征在于,步骤S04包括:先提取血小板,然后将血小板重悬于磷酸盐缓冲溶液,反复超声处理至血小板悬浮液透明,再将HMSN/Arg颗粒滴加到血小板悬浮液中混合均匀,最后从滤膜中挤出,固液分离得到组装纳米颗粒PM-HMSN/Arg。
7.如权利要求1~3任一项所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤治疗药物中的应用,其特征在于,所述组装纳米颗粒PM-HMSN/Arg能够特异性富集至肿瘤细胞,并在超声辐照下释放NO。
8.如权利要求1~3任一项所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤超声造影剂中的应用,其特征在于,所述组装纳米颗粒PM-HMSN/Arg能够特异性富集至肿瘤细胞,并在超声辐照下释放NO并填充在HMSN颗粒的空心腔内,组装纳米颗粒PM-HMSN/Arg将肿瘤细胞与正常组织区分开。
9.如权利要求1~3任一项所述的组装纳米颗粒PM-HMSN/Arg在制备用于肿瘤核磁造影剂中的应用。
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