CN113499454A - 一种超声纳米诊疗剂及其制备方法和应用 - Google Patents
一种超声纳米诊疗剂及其制备方法和应用 Download PDFInfo
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Abstract
一种超声纳米诊疗剂,包括一个外壳体,外壳体由二硬脂酰磷脂酰胆碱及端羟基聚乳酸乙醇酸共聚物杂合而成,在外壳体的表面镶嵌有紫杉醇,外壳体内具有一个内核,内核中包裹有全氟正戊烷和基因药物miR‑221抑制剂组成的水溶液混合物,外壳体的粒径为500~600纳米。本发明提供了上述超声纳米诊疗剂的制备方法。本发明还提供了上述超声纳米诊疗剂在制备用于治疗癌症的药物中的用途。本发明在超声波的刺激下,内核中的PFP被刺激后产生气体,气体的出现使超声的谐波信号增强。同时,大量气体使纳米外壳变薄,外加之外部较低功率的超声波,使外壳被破坏,包埋在外壳中的紫杉醇和内核中的miR‑221抑制剂被释放,从而杀死肿瘤。
Description
技术领域:
本发明属于生物医药领域,涉及一种纳米诊疗剂,具体来说是一种超声纳米诊疗剂及其制备方法和应用。
背景技术:
三阴性乳腺癌(TNBC)具有高度侵袭性和转移潜力。传统化疗是当前主要治疗手段,但受副作用的影响,预后情况仍不尽如人意。如何选择合适治疗方案以减少副作用、提高诊疗效果已然成为临床难点。近年来的纳米技术发展为解决部分问题带来了一线希望,具体优势体现在以下几个方面:
1.纳米颗粒的外壳能嵌入或包裹药物,以提高功效、并减少全身毒性。
2.能同时携带不同药物,实现共同给药。
3.外壳上可以增加靶向修饰,以提高特异性靶区递送药物效率。
4.通过内外部刺激因素,实现持续或刺激触发的药物释放。
目前纳米技术的发展主要体现在治疗效率的提升上。已有少数NP经过FDA许可,用于治疗肿瘤、内分泌疾病等。然而,此类NP无法了解肿瘤的空间结构。为此,我们联合了超声技术。
临床使用的微米级超声造影剂是一种提高对比度的显像介质,常用于进行肿瘤血池显像和评估肿瘤发生发展。纳米级超声造影剂(NP)能透过肿瘤血管内皮间隙进入微血管,达到满意的显像效果。常用NP外壳多由脂质、高分子材料或介孔硅材料组成,可封装放射性物质、响应刺激源的物质或药物分子。内核多选择惰性气体和全氟类气体。但含气NP性状较稳定,给予较大外源刺激才会被破坏。出于安全性问题,目前学者多以内源刺激来进行释药,这一做法导致NP外壳所需响应刺激源的物质需要增加、载药率减低,最终使靶区药物浓度不足的后果。
目前主流的纳米材料不易受外部刺激所激活,故所使用的释药机制主要为内源性刺激,如PH的改变等。外部刺激需要较大功率的刺激才可以释药,这直接影响了生物安全性,亦间接降低了NP载药率。此外,响应内源性刺激的释药机制存在不稳定性及不可控性,这对精准治疗提供了难度及不确定性。再者,为使超声能提供肿瘤内部结构,纳米内核中包裹了显像的气体,这就导致了药物无法被包裹于内核中、而被包埋于纳米外壳的原因。因此,同时提高载药率及成像能力是当前纳米材料需解决的关键。但这两个因素主要与纳米外壳的材料有很大的关联。常用的NP外壳为脂质、高分子材料,但前者封装药物的效率较低,而后者存在体内成像能力差等缺陷。为优化载药率,本发明中的纳米材料表面无靶向物质修饰,因此缺乏靶向性,需结合其他方式增加靶向性。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种超声纳米诊疗剂及其制备方法和应用,所述的这种超声纳米诊疗剂及其制备方法和应用要解决现有技术中的纳米诊疗剂载药率、成像能力、靶向性效果不佳的技术问题。
本发明提供了一种超声纳米诊疗剂,包括一个外壳体,所述的外壳体由二硬脂酰磷脂酰胆碱及端羟基聚乳酸乙醇酸共聚物杂合而成,在所述的外壳体的表面镶嵌有紫杉醇,所述的外壳体内具有一个内核,所述的内核中包裹有全氟正戊烷和基因药物miR-221抑制剂组成的水溶液混合物,所述的外壳体的粒径为500~600纳米。
进一步的,所述的端羟基聚乳酸乙醇酸共聚物、二硬脂酰磷脂酰胆碱和紫杉醇的质量比为50mg:3~5mg:3~5mg。
进一步的,所述的miR-221抑制剂水溶液是将miR-221抑制剂溶解在由焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水中,在所述的miR-221抑制剂水溶液中,所述的miR-221抑制剂的浓度为0.5nmol/mL。
进一步的,miR-221抑制剂水溶液与全氟正戊烷的体积比为1:1。
进一步的,所述纳米诊疗剂中,所述紫杉醇的含量为7~8wt.%。
进一步的,所述纳米诊疗剂在去离子水和磷酸缓冲盐溶液中的水动力学粒径为700~800nm。
本发明还提供了上述超声纳米诊疗剂的制备方法,包括如下步骤:
1)先制备miR-221抑制剂水溶液,所述的miR-221抑制剂水溶液是将miR-221抑制剂溶解在由焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水中,所述的miR-221抑制剂的浓度为0.5nmol/mL;
2)按照物料比称取PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)、PTX(紫杉醇)、miR-221抑制剂水溶液、全氟正戊烷;PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)、PTX(紫杉醇)、miR-221抑制剂、全氟正戊烷的物料比为50mg:3~5mg:3~5mg:3~7nmol:0.05~0.2ml;
3)将PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)和PTX(紫杉醇)完全溶解在一个装有二氯甲烷的容器中;
4)在另外一个容器中加入miR-221抑制剂水溶液和全氟正戊烷,在冰浴中超声处理;
5)将前两步获得的液体混合后进行冰浴超声,获得乳液;
6)随后在乳液中添加0.03~0.05g/ml聚乙烯醇水溶液,聚乙烯醇水溶液和二氯甲烷的体积比为10:0.5~2,再均质,将均质后的乳化液转入到已装有超纯水的离心管中,再均质;
7)冰浴搅拌混合物6-8小时,使二氯甲烷完全蒸发;
8)用去离子水洗涤所得混合物并离心,获得纳米诊疗剂,低温保存备用。
本发明还提供了上述超声纳米诊疗剂在制备用于治疗癌症的药物中的用途。
本发明所制备的纳米诊疗剂具有成像效果好、疗效显著等优点,可用于人体或其他哺乳动物癌症实现诊疗一体化的模式。
anti-miR-221为公知技术,该物质为一种小RNA,序列为:
mG*mA*mA*mA*mC*mC*mC*mA*mG*mC*mA*mG*mA*mC*mA*mA*mU*mG*mU*mA*mG*mC*mU。m代表甲基化修饰。
在文献(Li S,Li Q,LüJ,et al.Targeted Inhibition ofmiR-221/222PromotesCell Sensitivity to Cisplatin in Triple-Negative Breast Cancer MDA-MB-231Cells.Front Genet.2020Jan 14;10:1278.doi:10.3389/fgene.2019.01278.PMID:32010177;PMCID:PMC6971202.)有记载。该物质在肿瘤细胞中对miR-221/222的靶向抑制作用可促进顺铂诱导的细胞凋亡,并提高体外细胞对顺铂的敏感性。并且,在小鼠体内提高了肿瘤细胞对于顺铂敏感性。因此,该物质可以在体内体外与化疗药物联用,抑制乳腺癌细胞生长,达到抑癌目的。因此,在本发明中,将其与化疗药PTX联用,达到相似目的。
本发明引入外部刺激源超声进行释药,纳米级超声造影剂液态全氟正戊烷(PFP,沸点29℃)作为刺激源的响应器。PFP易受外部刺激如温度、超声等而发生液气相变,液态转变为气态的PFP时,产生的气体增强显像效果。同时气态PFP使纳米颗粒体积变大,加之外部较低功率的超声波刺激,外壳材料易发生破碎,使外壳及内核中的药物可控性地释药,达到有效将肿瘤细胞杀死的效果。
本发明由于使用了脂质和高分子材料杂合而成的杂合壳,适量的脂质可以提高外壳的弹性,使成像能力得到提高;而聚合物又使NP具备一定抗压能力,能使球体具有一定抗压性。
本发明的纳米诊疗剂在超声波的刺激下,内核中的PFP被刺激后产生气体,气体的出现使超声的谐波信号增强。同时,大量气体使纳米外壳变薄,外加之外部较低功率的超声波,使外壳易被破坏,包埋在外壳中的紫杉醇和内核中的miR-221抑制剂被释放,从而杀死肿瘤。这一体系实现了超声的诊疗一体化模式。
经典的纳米材料多以主动靶向或被动靶向方式到达靶区,然此类靶向的递送效率低下。研究证实,活细胞载药纳米粒,并运送到肿瘤的可行性。活细胞给药系统能极大保留了纳米载体的载药量、亦能产生更强的谐波信号。纳米载体也可以保护活细胞免受miR-221抑制剂和PTX的毒性。
所述纳米诊疗剂本身无靶向性,需要与活细胞载体共孵育后,所获得的复合体可具备天然靶向性。
本发明和已有技术相比,其技术效果是显著的。本发明扩展了纳米材料的释药机制,设计引入易响应超声刺激而发生液气相变的纳米级显像剂,利用超声的机械效应,达到在肿瘤部位能进行显像及治疗的诊疗一体化模式,实现精准治疗的目的。此外,为进一步优化了NP的载药率及成像能力,我们选择了合适的外壳材料,并将所得纳米材料与活细胞共孵育,借由活细胞作为给药载体以增加靶向性。
本发明同时还具有以下的优点:
1)本发明制备的纳米诊疗剂载药率高,具有实时成像、可控释药,能有效提高肿瘤的治疗效果。
2)本发明制备的纳米诊疗剂表面无靶向物质修饰,故而本身不具备靶向性,需要与活细胞载体一起共孵育后获得复合体,根据不同活细胞的特性,以复合体形式达到靶区。
3)本发明制备的纳米诊疗剂具有相容性好,稳定性高的特点。
4)本发明工艺简易,产品易制备。本发明制备的纳米诊疗剂拥有实时超声成像、可控释药及治疗等特点,可快速实现肿瘤的诊疗一体化。同时实现不同药物协同治疗,对推进精准治疗有重要意义。但本发明本身不具备靶向性,需要借助活细胞作为递药载体。
5)本发明将活细胞载体、超声技术、化疗、基因治疗与纳米材料相结合,具有天然靶向性、实时成像、可控释药、超声成像联合治疗的特性,可用于人体或其他哺乳动物癌症的诊断和高效治疗。
附图说明:
图1是实施例1中诊疗剂的透射电镜照片。
图2是实施例1中诊疗剂在磷酸缓冲盐溶液PBS和细胞培养基中的粒径。
图3是实施例1中诊疗剂在在不同功率的超声波刺激5s后的的光学显微镜照片。
图4是实施例1中最高浓度诊疗剂的超声成像效果。
图5是实施例1中不同浓度诊疗剂的超声成像效果及回声时间强度曲线分析。
图6是实施例1中诊疗剂在不同功率、不同时间的超声波刺激下的超声成像效果。
图7是实施例1中诊疗剂与活细胞载体共孵育后获得复合体的内通效率。
图8是实施例1中诊疗剂与活细胞载体共孵育后获得复合体的共聚焦照片。
图9是实施例1中在2.2w/cm2超声照射下,荷瘤Balb/C裸鼠经尾静脉注射定量超声显像照片。
图10是实施例1中经不同治疗后裸鼠的肿瘤体积随时间的变化关系。
具体实施方式:
下述实施方法仅用于说明本发明,而非限制本发明。
实施例1
采用改进的双乳化法制备了携带PTX和anti-miR-221的脂质/PLGA-PFP纳米粒。
首先,将50mg PLGA、3mg DSPC和3mg PTX完全溶解在1ml二氯甲烷中。
其次,在冰浴中超声处理0.1ml RNA水溶液(含5nmol anti-miR-221(anti-miR-221由南京金斯瑞生物科技有限公司根据上述的序列制备))和0.1ml PFP(全氟正戊烷)80s(20%功率;美国超音速材料公司)。
第三,将前两步获得的液体混合,再在冰浴中通过120秒超声,采用脉冲模式(20%功率;超音速材料公司),在该模式下,关闭超声波3秒并打开1秒,以防止热量积聚,获得乳液。
随后,添加10mL PVA(聚乙烯醇)水溶液(4%w/v),然后再均质2min(IKA T10basic均质机,5档,大概20000-25000转/分)。
上述乳化液转入到已装有10ml的超纯水的50ml离心管中,用IKA T10 basic均质机(上海坚融实业有限公司,中国上海),旋转2min。
第四,用磁力搅拌器冰浴上搅拌混合物6-8小时,使二氯甲烷完全蒸发。最后,用去离子水洗涤所得混合物并离心(8000转/分,5min)三次以获得PTX/anti-miR-221PLGA/Lipid PFP纳米粒。使用透射电镜观察(图1),可证明已经得到单纯的纳米颗粒。将其置于磷酸缓冲盐溶液PBS和细胞培养基中可以了解到该纳米颗粒可以在体外以及体内等渗溶液中稳定存在(图2),即可进行后续实验。将纳米材料在不同功率的超声波刺激5s后的光学显微镜照片(图3),以证明粒子具有很好的稳定性和分散性,可以长期保存。
以类似的方法制备了PLGA/Lipid-PFP(即空NP)、PTX PLGA/Lipid PFP(即PNP)和anti-miR-221PLGA/Lipid PFP纳米粒(即ANP)。所有纳米颗粒保存在4℃的冰箱中。
具体的,所述的miR-221抑制剂水溶液是将miR-221抑制剂溶解在由焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水中。
实施例2
材料自身的超声成像性能:将实施例1的纳米诊疗剂分别配置成浓度为5ug/mL、50ug/mL、500ug/mL、5mg/mL、50mg/mL的溶液,分别置于注入由质量百分比浓度为2%琼脂糖凝胶制成的仿体中的锥形空室中,在超声造影模式下采集纳米诊疗剂在仿体中的声像图(图4),分析注射前后的时间依赖性回声强度曲线(图5)。从图5可以看出浓度升高可以看出,随着浓度增多,超声图像呈变亮趋势。说明超声性能与药物浓度相关,在实验浓度范围内与药物浓度显示良好的线性关系,显示出良好的超声造影性能,表明在超声造影成像方面具有良好的应用前景。
实施例3
材料在不同超声功率及时间作用下的成像性能:选择浓度为5mg/mL的实施例1的纳米诊疗剂,分别置于几个质量百分比浓度为2%琼脂糖凝胶制成的仿体中的锥形空室中,在不同超声功率(0.8W/cm2、1.0W/cm2、1.2W/cm2、1.4W/cm2、1.6W/cm2)及时间(20s、40s、60s)作用下,记录超声显像实验(图6),证明了纳米材料时间依赖性超声成像效果。
实施例4
材料在被特定细胞吞噬后可在体内具有靶向肿瘤的能力,此为该材料的另一种应用方式。
将实施例1的纳米材料与巨噬细胞共同孵育不同时间(3小时、6小时、12小时、24小时)后,活细胞可将吞噬材料,并可以在体内定向移动。
图7证明了共同孵育不同时间的活细胞吞噬效率。图8为活细胞吞噬后的荧光照片。也可以证明没有爆破的情况下,纳米材料内部的药物已经不会直接释放,没有生物毒性。
实施例5
将6周大雌性裸鼠,乳腺部位注射106个肿瘤细胞,标准饲养四周后,再取荷瘤裸鼠(肿瘤直径为0.6-1.0cm),通过尾静脉注射实施例1的纳米定量材料。然后用2.2W/cm2超声照射肿瘤部位,通过超声造影记录肿瘤超声图像。得到图图9证明,该纳米材料可以在小鼠体内按预期进入肿瘤细胞,并可以被超声爆破。
实施例6
将不同内容物的纳米诊疗剂分散于PBS溶液中,取100uL尾静脉注射到荷231乳腺癌肿瘤模型Balb/C裸鼠内。通过超声功率为2.5W/cm2照射2分钟。如图10,实验结果表明,注射纳米诊疗剂的荷瘤鼠肿瘤体积的增长受到明显抑制。而同等条件下,仅注射PBS水组的荷瘤鼠肿瘤增长明显。值得注意的是,本发明纳米诊疗剂比单纯药物组更加抑制肿瘤生长。这一实验结果表明超声协同治疗的引入能够显著提高药物的疗效。
序列表
<110> 上海市东方医院(同济大学附属东方医院)
<120> 一种超声纳米诊疗剂及其制备方法和应用
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Claims (10)
1.一种超声纳米诊疗剂,其特征在于:包括一个外壳体,所述的外壳体由二硬脂酰磷脂酰胆碱及端羟基聚乳酸乙醇酸共聚物杂合而成,在所述的外壳体的表面镶嵌有紫杉醇,所述的外壳体内具有一个内核,所述的内核中含有由全氟正戊烷和特异性靶向基因药物miR-221抑制剂水溶液组成的混合溶液,所述的外壳体的粒径为500~1000纳米。
2.如权利要求1所述的一种超声纳米诊疗剂,其特征在于:所述的端羟基聚乳酸乙醇酸共聚物、二硬脂酰磷脂酰胆碱和紫杉醇的质量比为50mg∶3~5mg∶3~5mg。
3.如权利要求1所述的一种超声纳米诊疗剂,其特征在于:所述的miR-221抑制剂水溶液是将miR-221抑制剂溶解在由焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水中,所述的miR-221抑制剂的浓度为0.5nmol/mL。
4.如权利要求1所述的一种超声纳米诊疗剂,其特征在于:所述的miR-221抑制剂水溶液与全氟正戊烷的体积比为1∶1。
5.如权利要求1所述的一种超声纳米诊疗剂,其特征在于:所述纳米诊疗剂中,所述紫杉醇的含量为7~8wt%。
6.如权利要求1所述的一种超声纳米诊疗剂,其特征在于:所述纳米诊疗剂在去离子水和磷酸缓冲盐溶液中的水动力学粒径为700~800nm。
7.权利要求1所述的超声纳米诊疗剂的制备方法,其特征在于包括如下步骤:
1)先制备miR-221抑制剂水溶液,所述的miR-221抑制剂水溶液是将miR-221抑制剂溶解在由焦碳酸二乙酯处理过并经高温高压灭菌的MiliQ纯水中,所述的miR-221抑制剂的浓度为0.5nmol/mL;
2)按照物料比称取PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)、PTX(紫杉醇)、miR-221抑制剂水溶液和全氟正戊烷。PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)、PTX(紫杉醇)、miR-221抑制剂、全氟正戊烷的物料比为50mg∶3~5mg∶3~5mg∶3~7nmol∶0.05~0.2ml;
3)将PLGA(聚乳酸-羟基乙酸共聚物)、DSPC(二硬脂酰磷脂酰胆碱)和PTX(紫杉醇)完全溶解在一个装有二氯甲烷的容器中;
4)在另外一个容器中加入miR-221抑制剂水溶液和全氟正戊烷,在冰浴中超声处理;
5)将前两步获得的液体混合后进行冰浴超声,获得乳液;
6)随后在乳液中添加0.03~0.05g/ml聚乙烯醇水溶液,聚乙烯醇水溶液和二氯甲烷的体积比为10∶0.5~2,再均质,将均质后的乳化液转入到已装有超纯水的离心管中,再均质;
7)冰浴搅拌混合物6~8小时,使二氯甲烷完全蒸发;
8)用去离子水洗涤所得混合物并离心,获得纳米诊疗剂,低温保存备用。
8.权利要求1所述的超声纳米诊疗剂在制备用于治疗癌症的药物中的用途。
9.一种靶向纳米诊疗剂,其特征在于:由权利要求1所述的超声纳米诊疗剂与细胞载体复合而成。
10.权利要求9所述的靶向纳米诊疗剂在制备用于治疗癌症的药物中的用途。
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