CN113941010B - 一种协同no气体治疗并增强声动力治疗效果的纳米粒及其制备方法与应用 - Google Patents
一种协同no气体治疗并增强声动力治疗效果的纳米粒及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种协同NO气体治疗并增强声动力治疗的纳米粒及其制备方法与应用。该纳米粒是以适配体修饰的介孔二氧化硅为载体,再装载L‑精氨酸和血卟啉,通过自组装制备而得。该纳米粒经适配体修饰后,能够靶向识别表皮生长受体突变的肿瘤细胞;血卟啉作为一种声敏剂,经超声后产生活性氧,产生细胞毒性,可发挥其声动力治疗效果;L‑精氨酸作为NO供体,经细胞代谢后可产生NO气体,和活性氧进一步反应生成毒性更高的亚硝酸盐ONOO‑,从而选择性地杀死肺癌细胞。该纳米粒,利用适配体修饰的载体作为递送系统,联合NO气体治疗与声动力治疗,靶向识别肿瘤细胞并最大程度抑制肿瘤细胞增殖。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种协同NO气体治疗并增强声动力治疗效果的纳米粒及其制备方法与应用。
背景技术
肺癌是全世界最常见的癌症之一,严重危害着人类的公共健康。肺癌从组织学的角度分为非小细胞肺癌(NSCLC)和小细胞肺癌(SCLC),其中85%的肺癌属于NSCLC。NSCLC与SCLC 相比,范围更广,病因也更加复杂,且NSCLC被发现时大多已处于晚期。放疗、化疗等全身疗法广泛用于肺癌治疗,然而,因放疗、化疗存在肿瘤选择性差且易引起高毒副作用等缺点,刺激了其他治疗手段的发展。
肿瘤细胞中的过表达受体为正常和恶性组织的选择性提供了理想的靶点,这些癌症生物标志物也是癌症细胞筛选、检测、诊断、预后、预测、癌症发展和疾病复发监测所必需的。表皮生长因子受体(EGFR)在正常细胞中正常表达,而在肿瘤细胞中该受体发生基因突变,使得其功能活化 ,进而过表达。核酸适配体(Apt)是由一段DNA或RNA构成的单链寡核苷酸,能够与靶标进行特异性结合,具有选择性高,亲和力强,易合成、稳定好等优点。采用具有靶向 EGFR 突变型肺癌细胞的适配体修饰纳米载体进行药物靶向输送,提高药物对靶细胞的选择性,为提高药物疗效提供了新的思路。
声动力治疗(SDT)是以超声波为主的新型治疗手段,SDT由光动力治疗(PDT)发展而来,相比于PDT,超声介导的SDT具有非侵害性、深组织穿透力、高精度等独特的优点,超声能够到达更深层次的器官,如胰腺、肝脏和肿瘤。实际SDT作用的原理较为复杂,不像PDT引起光动力效应是单一的由光辐射激活光敏剂通过能量及电子转移等过程诱导产生活性氧(ROS),SDT还可能包括了空化气穴效应、热效应及直接诱导细胞凋亡等机制。血卟啉(HP)是目前广泛使用的第一代声敏剂,它在超声下展现出不俗的抗肿瘤效果。
一氧化氮(NO)是首次发现的气体信号分子,它在生理学上也被称为血管内皮舒张因子(EDRF)。作为一种内源性的气体,NO的半衰期非常短,约3~5 s,它在体内由一氧化氮合成酶(NOS)合成。NO在体内参与着许多的生理功能的调节,包括血流调节,抗病反应,神经传递和增强免疫反应等。NO对肿瘤起着双重作用,高水平的NO对肿瘤有杀伤的效果,低水平的NO则在某种程度上有利于肿瘤发生发展。因此提高NO的浓度水平是一种潜在的肿瘤治疗手段。L-精氨酸,作为NO的供体,能够和肿瘤微环境过剩的H2O2等发生氧化还原反应,自身的残基被氧化进而生产NO。在胞内,NO能够和ROS进一步发生反应生成细胞毒性更强的活性氮(如过亚硝酸盐ONOO-)进而增强声动力治疗效果。
介孔二氧化硅纳米粒(MSN)是一种相当独特的介孔纳米材料,早已被用作纳米药物递送系统,具有粒径小、孔径可调、表面积大、生物相容性良好等优点。肿瘤治疗时的副作用始终是不可忽视的,而MSN的毒性低,生物降解性优良,一定程度上降低了药物输送载体的副作用。明确的介孔结构和独特的中空结构,高表面积,高孔体积和可调整的孔径,确保了MSN巨大的负载能力。MSN表面的氨基具有较高的反应活性,通过表面修饰靶向分子如Apt能够提高该纳米材料的靶向药物递送能力。
基于以上研究背景,本发明以靶向表皮生长因子突变的核酸适配体(Apt)修饰的MSN作为靶向药物递送的载体(AM),在AM内部负载L-精氨酸(L-Arg)和血卟啉(HP),制得了一种靶向共载药纳米粒AMLH。该纳米粒能够靶向识别表皮生长因子受体突变的NSCLC细胞,在胞内,L-Arg作为NO供体,能够产生NO,Hp在超声作用下产生活性氧(ROS), ROS能够和NO进一步反应生成细胞毒性更高的ONOO-,既协同NO气体治疗,同时能够增强声动力治疗,最终呈现出协同治疗的效果,用于非小细胞肺癌治疗。
发明内容
本发明的目的在于提供一种协同NO气体治疗并增强声动力治疗效果的纳米粒及其制备方法与应用,该纳米粒利用适配体修饰的MSN作为载体靶向递送HP和L-Arg至EGFR突变的NSCLC细胞,超声激活声敏剂HP产生ROS,诱导L-Arg在肿瘤细胞内产生NO,协同声动力治疗和气体治疗,ROS和NO进一步反应生成细胞毒性更强的ONOO-,增强SDT,最大程度抑制肿瘤细胞增殖。
为达到上述目的,本发明采取了如下技术方案:
一种协同NO气体治疗并增强声动力治疗效果的纳米粒AMLH,所述的纳米粒是由介孔二氧化硅纳米粒、L-精氨酸、血卟啉以及羧基修饰的核酸适配体自组装形成,其中,L-精氨酸的包封率为30-90%,血卟啉的包封率为30-90%
上述一种纳米粒AMLH的制备方法,其是以适配体修饰的介孔二氧化硅纳米粒为载体,再自组装包载L-精氨酸和血卟啉,从而构建得到一种协同NO气体治疗并增强声动力治疗的纳米粒AMLH。
上述一种纳米粒AMLH的制备方法, 具体包括以下步骤:
(1)MSN-OH的制备:将十六烷基三甲基氯化铵和三乙醇胺混合并溶于水中,于80-105 ℃下搅拌反应1-2 h;向反应液中滴加四乙氧基硅烷,继续搅拌反应1 h;待反应液冷却至室温,将其离心,移去上清,洗涤沉淀;将洗涤后的沉淀加入至氯化钠-甲醇混合液中,在室温下搅拌反应4h;将反应液离心,移除上清,洗涤沉淀;将洗涤后的沉淀冷冻干燥,即得到MSN-OH;
(2)MSN-NH2的制备:将3-氨丙基三乙氧基硅烷和MSN-OH混合并溶于无水乙醇中,在50-80 ℃下搅拌反应2-8 h;将反应液离心,移除上清,洗涤沉淀;将洗涤后的沉淀冷冻干燥,即得到MSN-NH2;
(3)AM的制备:将1-乙基-3-(3-二甲氨基)碳二亚胺和N-羟基琥珀酰亚胺混合并溶于水中;向反应液中加入羧基修饰的核酸适配体E-APt,室温下搅拌反应3 h;向反应液中加入MSN-NH2,室温下搅拌反应24h,使得核酸适配体E-APt与MSN-NH2共价连接;将反应液离心,移除上清,洗涤并重悬沉淀,即得到AM溶液;
(4)AMLH的制备:将AM溶液离心,移除上清,将沉淀溶于氢氧化钠溶液中;在磁力搅拌下向反应液中加入L-精氨酸和血卟啉,室温下搅拌反应3-7天;将反应液离心,移除上清,将所得沉淀冷冻干燥,即得到纳米粒AMLH。
其中,步骤(1)中,所述十六烷基三甲基氯化铵:三乙醇胺:四乙氧基硅烷的投料比为0.2-40:1:10-4-10-2 w/w/v;所述氯化钠-甲醇混合液中氯化钠:甲醇的比例为0.1-10:100 w/v。
步骤(2)中,所述MSN-OH:3-氨丙基三乙氧基硅烷的比例为10000-500000:20-500w/v。
步骤(3)中,所述核酸适配体E-APt的连接量为0.01-0.50 nmol/mg。
步骤(4)中,所述AM在氢氧化钠溶液中的浓度为1-10 mg/mL;所述氢氧化钠溶液的pH为8-11,浓度为10-6-10-3 mol/L;所述血卟啉的终浓度为10-1000 μg/mL;所述L-精氨酸的终浓度为10-1000 μg/mL。
上述一种纳米粒AMLH在制备用于治疗表皮生长因子突变非小细胞肺癌的药物中的应用。
本发明的显著优点在于:
本发明以共载声敏剂和一氧化氮供体的方式制得的二氧化硅纳米粒,其表面用适配体修饰,提供对肿瘤细胞表面特异性蛋白的识别能力,协同NO气体治疗并显著增强声动力治疗,选择性地抑制表皮生长因子突变的非小细胞肺癌增殖。
附图说明
图1为本发明制备的MSN-OH、MSN-NH2的粒径分布和电势图。其中,1A为粒径分布图,1B为电势图。
图2为本发明制备的AM的表征实验。其中,2A为琼脂糖凝胶电泳成像,2B为傅里叶变换红外扫描图谱。
图3为本发明制备的各纳米粒复合物的HP紫外图谱。
图4为HP和L-Arg的标准曲线。其中,4A为HP在10-3 mol/L氢氧化钠溶液中标准曲线,4B为L-Arg和茚三酮反应标准曲线。
图5为本发明制备的AM的靶向识别实验。
图6为本发明制备的各纳米粒的细胞摄取实验。
图7为本发明制备的各纳米粒对PC9细胞的体外协同治疗实验。
图8为本发明制备的各纳米粒的胞内活性氧检测实验。
图9为本发明制备的各纳米粒的胞内的NO检测实验。
图10为本发明制备的各纳米粒的胞内的ONOO-检测实验。
具体实施方式
下面结合具体实施例,对本发明进行进一步说明,有助于本领域的普通技术人员进一步理解本发明,但不以任何形式限制本发明。
以下实施例中所用到的DMEM培养基中含有体积分数为10%的胎牛血清和体积分数为1%的青霉链霉素。
以下实施例中所用到的1640培养基中含有体积分数为10%的胎牛血清和体积分数为1%的青霉链霉素。
实施例1
分别称取2.0 g十六烷基三甲基氯化铵(CTAC)和0.1 g三乙醇胺(TEA)置于同一50mL的圆底烧瓶中,混合,向其中加入20 mL超纯水,将烧瓶置于95℃油浴下搅拌反应1 h。反应结束后,利用1 mL注射器向反应液中逐滴滴加1.5 mL的四乙氧基硅烷(TEOS),然后在95℃油浴下继续搅拌反应1 h。随后,待反应液冷却至室温,将其在12000 rpm的条件下离心30min,移去上清后,将离心后的沉淀用无水乙醇洗涤三次。将洗涤后的沉淀加入到20 mL氯化钠-甲醇混合液(1 g NaCl溶于100 mL甲醇)中,室温搅拌反应4 h,随后将反应液在10000rpm的条件下离心10 min,移除上清,将离心后的沉淀依次用无水乙醇和超纯水各洗涤3次,再将洗涤后的沉淀冷冻干燥,得到MSN-OH。
实施例2
称取100 mg MSN-OH置入50 mL圆底烧瓶,量取200 μL 3-氨丙基三乙氧基硅烷(APTES)加入到同一烧瓶中,随后向其中加入20 mL无水乙醇,将烧瓶置于60℃油浴下磁力搅拌反应4 h,随后将反应液在10000 rpm的条件下离心10 min,移去上清,将离心后的沉淀依次用无水乙醇和超纯水各洗涤3次,再将洗涤后的沉淀冷冻干燥,得到MSN-NH2(MSN)。
实施例3
分别称取10 mg 1-乙基-3-(3-二甲氨基)碳二亚胺(EDC)和8 mgN-羟基琥珀酰亚胺(NHS)加入到同一5 mL的圆底烧瓶中,混合,向其中加入2 mL超纯水混匀15 min后,向其中加入0.2 nmol羧基修饰的靶向EGFR的核酸适配体E-APt(购自生工生物工程(上海)股份有限公司,其具体序列参见文献Zhu F, Xu L, Li X, Li Z, Wang J, Chen H, et al.Co-delivery of gefitinib and hematoporphyrin by aptamer-modified fluorinateddendrimer for hypoxia alleviation and enhanced synergistic chemo-photodynamictherapy of NSCLC. Eur J Pharm Sci 2021;167:106004.),在室温下搅拌反应3 h以活化羧基。随后向反应液中加入10 mg MSN-NH2,室温下搅拌反应24 h,使得核酸适配体E-APt与MSN-NH2共价连接。随后将反应液在12000 rpm的条件下离心10 min,移去上清,将离心后的沉淀用超纯水洗涤3次,将沉淀重悬于1 mL超纯水中,得到10 mg/mL的AM溶液。AM溶液冻干后所得的固体产物即为AM。
实施例4
用二甲基亚砜(DMSO)配制10 mg/mL的血卟啉(HP)溶液,用超纯水配制10 mg/mL的L-精氨酸(L-Arg)溶液。
取1 mL 10 mg/mL的AM溶液在15000 rpm的条件下离心10 min,移去上清,向离心后的沉淀中加入1 mL氢氧化钠溶液(pH=9,10-4 mol/L),并将反应液转移至5 mL圆底烧瓶中,在磁力搅拌下向反应液中逐滴加入10μL 10 mg/mL的Hp溶液和10 μL10 mg/mL的L-Arg溶液,室温下搅拌反应3天,随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,移除上清(将该上清编号为①),将所得沉淀冷冻干燥,即得到纳米粒AMLH。
取1 mL 10 mg/mL的AM溶液在15000 rpm的条件下离心10 min,移去上清,向离心后的沉淀中加入1 mL氢氧化钠溶液(pH=9,10-4 mol/L),并将反应液转移至5 mL圆底烧瓶中,在磁力搅拌下向反应液中逐滴加入10 μL10 mg/mL的L-Arg溶液,室温下搅拌反应3天,随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,移除上清(将该上清编号为②),将所得沉淀冷冻干燥,即得到纳米粒AML。
取1 mL 10 mg/mL的AM溶液在15000 rpm的条件下离心10 min,移去上清,向离心后的沉淀中加入1 mL氢氧化钠溶液(pH=9,10-4 mol/L),并将反应液转移至5 mL圆底烧瓶中,在磁力搅拌下向反应液中逐滴加入10μL 10 mg/mL的HP溶液,室温下搅拌反应3天,随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,移除上清(将该上清编号为③),将所得沉淀冷冻干燥,即得到纳米粒AMH。
取MSN 10 mg分散在1 mL氢氧化钠溶液 (pH=9,10-4 mol/L)中,磁力搅拌下向反应液中逐滴加入10μL 10 mg/mL的HP溶液和10 μL10 mg/mL的L-Arg溶液,室温下搅拌反应3天。随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,将所得沉淀冷冻干燥,即得到纳米粒MLH。
取MSN 10 mg分散在1 mL氢氧化钠溶液 (pH=9,10-4 mol/L)中,磁力搅拌下向反应液中逐滴加入10 μL10 mg/mL的L-Arg溶液,室温下搅拌反应3天。随后将反应液在12000rpm的条件下离心10 min,以去除未包载的游离药物,将所得沉淀冷冻干燥,即得到纳米粒ML。
取MSN 10 mg分散在1 mL氢氧化钠溶液 (pH=9,10-4 mol/L)中,磁力搅拌下向反应液中逐滴加入10μL 10 mg/mL的HP溶液,室温下搅拌反应3天。随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,将所得沉淀冷冻干燥,即得到纳米粒MH。
实施例5
将MSN-OH和MSN-NH2分别分散在超纯水中,使二者终浓度均为200 μg/mL,用动态光散射粒径仪分别测定MSN-OH和MSN-NH2的水合粒径与Zeta电位。结果如图1所示,MSN-OH粒径为181.2±4.0 nm,分散系数为0.381±0.05,电势为-26.1±2.4 mV,MSN-NH2粒径为182.8±12.0 nm,分散系数为0.499±0.06,电势为21.0±2.5 mV;电势由MSN-OH负电转变为MSN-NH2正电性,表明MSN-NH2即MSN成功合成。
实施例6
称取0.4 g琼脂糖(生工生物,上海)至20 mL 0.5×TBE中,微波炉加热溶解,向其中加入2 μL GelRed(Genstar,中国)混匀,然后将溶液倒至8孔模具中,插入8孔梳,待溶液冷却至室温制得琼脂糖凝胶。取各样品上样,80V恒定电压下于0.5×TBE电泳缓冲溶液中电泳50 min,用荧光成像仪(Bio-Rad,USA)对琼脂糖凝胶成像。将AM溶液冻干,取所得固体用傅里叶变换红外光谱仪(尼高力仪器公司,美国)测定红外吸收。琼脂糖凝胶电泳成像结果如图2A所示,泳道2游离的适配体在电泳的作用下迁移至凝胶下端,泳道3有微弱适配体的条带,而MSN-OH和MSN无适配体干扰信号,且当适配体与MSN结合后,整体分子量增大,迁移路径变短,表明E-Apt成功和MSN连接。AM红外吸收图谱结果如图2B所示,AM在1705 cm-1和1568 cm-1处有红外吸收,分别归属于适配体羰基的伸缩振动和胞嘧啶碳氮双键的特定吸收,而MSN-OH和MSN无此吸收,进一步佐证适配体E-Apt和MSN的成功连接。
实施例7
将Hp、AM、AML、AMH和AMLH分别溶解于氢氧化钠溶液(pH=9,10-4 mol/L)中,用紫外可见分光光度计(Quawell,USA)扫描各样品紫外吸收图谱。结果如图3所示,AMH和AMLH有Hp的特定吸收峰,表明Hp被成功包载。
实施例8
测定实施例4所制备的各纳米粒的HP和L-Arg的包封率。
对于HP:用氢氧化钠溶液配制不同浓度梯度(1,2,5,10,20,25,50 μg/mL)的HP标准溶液,用酶标仪测定其在374 nm处的校正曲线,标准曲线如图4A所示。分别将上清①、②、③稀释20倍,用酶标仪测得其在374 nm处的吸光度值,根据校正曲线计算各上清中HP含量(μg)。包封率计算公式为:包封率=(加入量-上清量*20)/加入量*100%。其中,所述加入量为10 μg。
对于L-Arg:配制pH=4.5的醋酸缓冲溶液(醋酸钠0.9 g,冰醋酸490 μL,超纯水50mL);用无水乙醇配制浓度为5 mg/mL茚三酮溶液;用超纯水配制不同浓度梯度的(1,2,5,10,20,50,100,500,1000 μg/mL)L-Arg标准溶液。分别取不同浓度梯度的L-Arg标准溶液50μL,向其中加入100 μL pH4.5的醋酸缓冲溶液和200 μL茚三酮溶液,混合,置于105℃油浴锅中加热反应8 min;待反应液冷却至室温后取80 μL加至96孔板中,用酶标仪测定其在570nm处的吸光度值,绘制吸光度-浓度校正曲线,结果如图4B所示。然后用酶标仪测得上清①、②、③在570 nm处的吸光度值,根据校正曲线计算上清L-Arg量(μg)。包封率计算公式为包封率=(加入量-上清量)/加入量*100%。其中,所述加入量为10 μg。
各纳米粒中HP和L-Arg的包封率如表1所示。
表1 各纳米粒包封率
实施例9
用二甲基亚砜(DMSO)配制10 mg/mL的血卟啉(HP)溶液,用超纯水配制10 mg/mL的L-精氨酸(L-Arg)溶液。
取10 mg AM ,将其分散在1 mL氢氧化钠溶液(pH=9,10-4 mol/L)中,在磁力搅拌下向其中逐滴加入10 mg/mL的HP溶液和10 mg/mL的L-Arg溶液各1 μL,室温下搅拌反应3天。随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,将所得沉淀冷冻干燥,即得到AMLH(ii)。依据实施例8中包封率测定方法,测得本实施例所制备的AMLH(ii)中Hp的包封率为90.53±0.39(%),L-Arg的包封率为87.32±0.76(%)。
实施例10
用二甲基亚砜(DMSO)配制10 mg/mL的血卟啉(HP)溶液,用超纯水配制10 mg/mL的L-精氨酸(L-Arg)溶液。
取1 mg AM ,将其分散在1 mL氢氧化钠溶液(pH=9,10-4 mol/L)中,在磁力搅拌下向其中逐滴加入10 mg/mL的HP溶液和10 mg/mL的L-Arg各10 μL,室温下搅拌反应3天。随后将反应液在12000 rpm的条件下离心10 min,以去除未包载的游离药物,将所得沉淀冷冻干燥,即得到AMLH(iii)。依据实施例8中包封率检测方法,测得本实施例所制备的AMLH(iii)中Hp的包封率为29.87±0.52(%),L-Arg的包封率为30.54±0.43(%)。
实施例11
采用DMEM培养基培养Helf细胞,采用1640培养基培养PC9细胞。
取细胞密度80%的PC9细胞,经0.25%的胰蛋白酶(含EDTA)消化后分别铺到12孔板中,使得每孔细胞数量为1×105个,放入培养箱(37℃,5% CO2)过夜孵育,随后吸弃旧培养基,然后进行Apt+AM处理或AM处理。对于Apt+AM组细胞:预先向每孔中加入500 μL 0.2 μM的适配体E-Apt在37℃下孵育1 h,吸弃适配体E-Apt溶液后,再用500 μL 50 μg/mL的AM溶液(FAM标记)在37℃下孵育细胞4 h。对于AM组细胞:直接用500 μL 50 μg/mL的AM溶液(FAM标记)在37℃下孵育细胞4 h。孵育结束后,用0.01M pH7.4的PBS缓冲液洗涤细胞两次,用200 μL 0.25%的胰蛋白酶(不含EDTA)进行消化后,加入200 μL 1640培养基终止消化,然后将各孔细胞悬液分别全部转移至离心管中,在1900 rpm的条件下离心5 min,弃上清,用500μL 0.01M pH7.4的PBS缓冲液重悬细胞沉淀,待测流式。
取细胞密度80%的Helf细胞,经0.25%胰蛋白酶(含EDTA)消化后分别铺到12孔板中,使得每孔细胞数量为1×105个,放入培养箱(37℃,5% CO2)过夜孵育,随后吸弃旧培养基,然后进行Apt+AM处理或AM处理。对于Apt+AM组细胞:预先向每孔中加入500 μL 0.2 μM的适配体E-Apt在37℃下孵育1 h,吸弃适配体E-Apt溶液后,加入500 μL 50 μg/mL的AM溶液(FAM标记)在37℃下孵育4 h。对于AM组细胞:直接用500 μL 50 μg/mL的AM溶液在37℃下孵育4 h。孵育结束后,用0.01M pH7.4的PBS缓冲液洗涤细胞两次,用200 μL 0.25%的胰蛋白酶(不含EDTA)进行消化后,加入200 μL 1640培养基终止消化,然后将各孔细胞悬液分别全部转移至离心管中,在1900 rpm的条件下离心5 min,弃上清,用500 μL 0.01M pH7.4的PBS缓冲液重悬细胞沉淀,待测流式。
结果如图5所示:PC9细胞对AM摄取(图5B)明显多于Helf细胞(图5A);当PC9细胞预先用适配体处理后,对AM摄取减弱,这是由于适配体先结合部分EGFR受体,导致AM靶向结合减少。结果表明,AM作为载体能够靶向识别EGFR突变的细胞,可以作为靶向药物递送的载体。
实施例12
采用1640培养基培养PC9细胞。
取生长状态良好的PC9细胞,用0.25%胰酶(含EDTA)胰蛋白酶消化后按每孔1×105个铺至12孔板,放入5% CO2,37℃培养箱贴壁培养过夜,各孔分别加入上述制得的不同的纳米粒:MLH、MH、AMH、AMLH,并使得各纳米粒中所包载的HP或L-Arg在培养基中的含量均为5 μg。将孔板放至5% CO2,37℃培养箱中孵育4 h,弃去旧培养基,用预冷的0.01M pH7.4的PBS缓冲液洗涤细胞3次,用200 μL 0.25%的胰蛋白酶(不含EDTA)进行消化后,加入200 μL1640培养基终止消化,然后将各孔细胞悬液分别全部转移至离心管中,在1900 rpm的条件下离心5 min,弃上清,用500 μL 0.01M pH7.4的PBS缓冲液重悬细胞沉淀,待测流式。摄取结果如图6,PC9细胞对AMH和AMLH摄取量分别比MH和MLH高,结果表明,E-Apt修饰的MSN具有靶向识别能力,可以靶向递送包载物至EGFR敏感突变的NSCLC细胞。
实施例13
采用1640培养基培养PC9细胞。
取生长状态良好的PC9细胞,按每孔10,000个的细胞密度接种至96孔板中,置于5%CO2,37℃培养箱中孵育24 h,弃去旧培养基。向1640培养基中分别加入上述制得的不同的纳米粒:MLH、AML、AMH、AMLH,并使得各纳米粒中所包载的HP或L-Arg在培养基中的含量均为5 μg。将含有纳米粒的1640培养基避光分别加入到PC9细胞对应的孔中,37℃孵育24 h,弃去旧培养基。对于超声组:将细胞接种至6孔板中,在含纳米粒的1640培养基中37℃孵育4 h后超声(0.1 W/cm2)1 min,继续孵育20 h后,弃去旧培养基,随后采用四甲基偶氮唑盐微量酶反应比色法(MTT法)测定各纳米粒对PC9细胞的抑制作用,计算细胞存活率。未超声组除不进行超声外,其他操作与超声组相同。协同治疗效果如图7所示,AMLH比MLH细胞毒性更强,这是因为Apt的靶向性,PC9细胞对AMLH的摄取比MLH高; AMLH比AMH细胞毒性更强,证明L-Arg协同HP产生更强的抗肿瘤治疗的能力;AML、AMH、MLH及AMLH各组细胞施加超声后细胞活性均低于未超声组,AMLH联合声动力治疗,肿瘤细胞致死量最高,表明AMLH给药后施加超声,HP被激活,能够发挥声动力治疗作用,同时,协同NO气体治疗,显著抑制肿瘤细胞增殖。
实施例14
采用1640培养基培养PC9细胞。
取生长状态良好的PC9细胞,按每孔1×105个铺至12孔板,放入5% CO2,37℃培养箱贴壁培养过夜,然后每孔分别加入上述制得的不同的纳米粒:MLH、AML、AMH、AMLH,并使得各纳米粒中所包载的HP或L-Arg在培养基中的含量均为5 μg。将孔板放至5% CO2,37℃培养箱中孵育24 h。对于超声组:细胞给药24 h后,在0.1W/cm2的超声强度下超声1 min,随后弃去旧培养基,用预冷的PBS缓冲液(0.01M,pH7.4)洗涤细胞3次,随后利用活性氧检测试剂盒(购自北京鼎国昌盛生物技术有限公司)测定细胞内活性氧的水平。未超声组除不进行超声外,其他操作与超声组相同。胞内ROS产生的结果如图8所示,AMLH超声组比AMLH未超声组产生明显ROS;在相同的超声条件下,AML组能产生少量ROS;AMLH组比AMH组产生更多的ROS,这是因为L-Arg可以促进细胞摄取(图6),因此共包载L-Arg和HP比单载HP可以产生更多的ROS,证明AMLH在超声条件下可以发挥显著的声动力治疗效果。
实施例15
采用1640培养基培养PC9细胞。
取生长状态良好的PC9细胞,按每孔1×105个铺至12孔板,放入5% CO2,37℃培养箱贴壁培养过夜,然后每孔分别加入上述制得的不同的纳米粒:MLH、AML、AMH、AMLH,并使得各纳米粒中所包载的HP或L-Arg在培养基中的含量均为5 μg。将孔板放至5% CO2,37℃培养箱中孵育24 h。对于超声组:细胞给药24 h后,在0.1W/cm2的超声强度下超声1 min,随后弃去旧培养基,用预冷的PBS缓冲液(0.01M,pH7.4)洗细胞3次,随后将500 μL 5 μM的DAF-FM DA探针(购自碧云天生物技术有限公司)与细胞于37 ℃下共孵育30 min,再次用预冷的PBS缓冲液(0.01M,pH7.4)洗涤细胞3次,然后用200 μL 0.25%的胰蛋白酶(不含EDTA)进行消化后,加入200 μL 1640培养基终止消化,然后将各孔细胞悬液分别全部转移至离心管中,在1900 rpm的条件下离心5 min,弃上清,用500 μL 0.01M pH7.4的PBS缓冲液重悬细胞沉淀,待测流式。未超声组除不进行超声外,其他操作与超声组相同。胞内NO产生的结果如图9所示:AMLH组在超声前后NO产量无显著差异;在相同超声条件下, AMH组仅有微弱的NO产生,而AML组有显著的NO产生。结果表明,L-Arg能够作为供体在胞内产生NO。
实施例16
采用1640培养基培养PC9细胞。
取生长状态良好的PC9细胞,按每孔1×105个铺至12孔板中,放入5% CO2,37℃培养箱孵育过夜,然后每孔分别加入上述制得的不同的纳米粒:MLH、AML、AMH、AMLH,并使得各纳米粒中所包载的HP或L-Arg在培养基中的含量均为5 μg。将孔板放入5% CO2,37℃培养箱中孵育24 h。对于超声组:细胞给药24 h后,在0.1W/cm2的超声强度下超声1 min,随后弃去旧培养基,用预冷的PBS缓冲液(0.01M,pH7.4)洗涤细胞3次,随后用500 μL 5 μM的二氢罗丹明123(购自大连美仑生物技术有限公司)于37 ℃孵育细胞30 min,再用预冷的PBS缓冲液(0.01M,pH7.4)洗涤细胞3次,然后用200 μL 0.25%的胰蛋白酶(不含EDTA)进行消化后,加入200 μL 1640培养基终止消化,然后将各孔细胞悬液全部转移至离心管中,在1900 rpm的条件下离心5 min,弃上清,用500 μL 0.01M pH7.4的PBS缓冲液重悬细胞沉淀,待测流式。未超声组除不进行超声外,其他操作与超声组相同。胞内ONOO-产生的结果如图10所示:AMLH超声组能够比未超声组产生明显的亚硝酸盐;在相同超声条件下,AMLH双载L-Arg和HP后,亚硝酸盐含量明显多于AML组和AMH组。结果表明,L-Arg和HP存在协同作用,在超声下ROS和NO进一步反应,显著产生细胞毒性更强的亚硝酸盐,既协同NO气体治疗同时增强声动力治疗,显著抑制肿瘤细胞增殖。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (7)
1.一种协同NO气体治疗并增强声动力治疗效果的纳米粒AMLH的制备方法,其特征在于:其是以适配体修饰的介孔二氧化硅纳米粒为载体,再自组装包载L-精氨酸和血卟啉,从而构建得到一种协同NO气体治疗并增强声动力治疗的纳米粒AMLH,其中,L-精氨酸的包封率为30~90%,血卟啉的包封率为30~90%;所述制备方法包括以下步骤:
1)MSN-OH的制备:将十六烷基三甲基氯化铵和三乙醇胺混合并溶于水中,于80~105℃下搅拌反应1~2 h;向反应液中滴加四乙氧基硅烷,继续搅拌反应1 h;待反应液冷却至室温,将其离心,移去上清,洗涤沉淀;将洗涤后的沉淀加入至氯化钠-甲醇混合液中,在室温下搅拌反应4 h;将反应液离心,移除上清,洗涤沉淀;将洗涤后的沉淀冷冻干燥,即得到MSN-OH;
2)MSN-NH2的制备:将3-氨丙基三乙氧基硅烷和MSN-OH混合并溶于无水乙醇中,在50~80℃下搅拌反应2~8 h;将反应液离心,移除上清,洗涤沉淀;将洗涤后的沉淀冷冻干燥,即得到MSN-NH2;
3)AM的制备:将1-乙基-3-(3-二甲氨基)碳二亚胺和N-羟基琥珀酰亚胺混合并溶于水中;向反应液中加入羧基修饰的核酸适配体E-APt,室温下搅拌反应3 h;向反应液中加入MSN-NH2,室温下搅拌反应24 h,使得核酸适配体E-APt与MSN-NH2共价连接;将反应液离心,移除上清,洗涤并重悬沉淀,即得到AM溶液;
4)AMLH的制备:将AM溶液离心,移除上清,将沉淀溶于氢氧化钠溶液中;在磁力搅拌下向反应液中加入L-精氨酸和血卟啉,室温下搅拌反应3~7天;将反应液离心,移除上清,将所得沉淀冷冻干燥,即得到纳米粒AMLH。
2.根据权利要求1所述的制备方法,其特征在于:步骤(1)中,所述十六烷基三甲基氯化铵:三乙醇胺:四乙氧基硅烷的投料比为0.2~40:1:10-4 ~10-2 w/w/v;所述氯化钠-甲醇混合液中氯化钠:甲醇的比例为0.1~10:100 w/v。
3.根据权利要求1所述的制备方法,其特征在于:步骤(2)中,所述MSN-OH:3-氨丙基三乙氧基硅烷的比例为10000~500000:20~500 w/v。
4.根据权利要求1所述的制备方法,其特征在于:步骤(3)中,所述核酸适配体E-APt的连接量为0.01~0.50 nmol/mg。
5.根据权利要求1所述的的制备方法,其特征在于:步骤(4)中,所述AM在氢氧化钠溶液中的浓度为1~10 mg/mL;所述氢氧化钠溶液的pH为8~11,浓度为10-6~10-3 mol/L;所述血卟啉的终浓度为10~1000 μg/mL;所述L-精氨酸的终浓度为10~1000 μg/mL。
6.一种如权利要求1所述的制备方法制得的纳米粒AMLH。
7.一种如权利要求6所述的纳米粒AMLH在制备用于治疗表皮生长因子突变非小细胞肺癌的药物中的应用。
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