CN114404429B - 一种纳米银修饰的单宁酸-铁网络的载药纳米复合物及其制备方法和逆转肿瘤耐药应用 - Google Patents
一种纳米银修饰的单宁酸-铁网络的载药纳米复合物及其制备方法和逆转肿瘤耐药应用 Download PDFInfo
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Abstract
本发明涉及纳米药物呈递***以及造影剂技术领域,具体涉及一种纳米银修饰的单宁酸‑铁网络的载药纳米复合物及其制备方法和逆转肿瘤耐药应用。本方案的载药纳米复合物,包括连接有细胞穿膜肽的阿霉素形成的内核,在内核外包裹有单宁酸和铁离子形成的网络结构层,在所述网络结构层外修饰有纳米银颗粒。本方案可以解决肿瘤耐药性造成的肿瘤化疗失败的技术问题,可下调耐药细胞内过表达的P‑gp并增加细胞内化疗药物的积累,且可将抗肿瘤药物有效递送到其最终目的地细胞核,且是一种具有成像潜力的双模态(光声/磁共振)的增强造影剂。本方案可以应用于耐药性肿瘤的治疗、可视化监测和引导治疗,具有广阔的应用前景。
Description
技术领域
本发明涉及纳米药物呈递***以及造影剂技术领域,具体涉及一种纳米银修饰的单宁酸-铁网络的载药纳米复合物及其制备方法和逆转肿瘤耐药应用。
背景技术
肿瘤的发生是一个长期、多阶段、多基因改变以及积累的过程,具有基因控制和多因素调节的复杂性。国内外研究表明:肿瘤多重耐药性(MDR)是导致肿瘤化疗失败的主要原因。肿瘤的耐药性涉及细胞内药物的浓度降低、药物靶分子的改变、代谢解毒、DNA损伤修复功能失衡等多种机制。尽管通过科学家们采取各种基于纳米技术的策略来逆转MDR,如通过肿瘤靶向增强药物积累,防止药物靶向途径失活以及联合光热治疗、光动力治疗等,但单一的逆转耐药策略仍不能完全恢复肿瘤细胞对化疗药的敏感性,MDR仍在严重限制着化学治疗的疗效。阿霉素(DOX)是最具代表性的经典化疗药物之一。因为DOX杀伤肿瘤细胞的机制是通过与DNA螺旋或相关酶的相互作用诱导细胞死亡,因此其疗效高度依赖于核通路。然而,纳米载体在释放DOX到达其作用靶点之后,会遭到内涵体捕获和溶酶体降解。此外,由于MDR细胞膜上P糖蛋白(P-gp)的过度表达,DOX到达细胞质后可能会被迅速泵出细胞外,进一步降低了细胞内药物浓度。因此,亟需开发一种高效的纳米复合材料来克服MDR,用以下调耐药细胞内过表达的P-gp并增加细胞内DOX的积累,且可将抗肿瘤药物有效递送到其最终目的地细胞核。
发明内容
本发明意在提供一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,以解决肿瘤多药耐药造成的肿瘤化疗失败的技术问题。
为达到上述目的,本发明采用如下技术方案:
一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其包括连接有细胞穿膜肽的阿霉素形成的内核,在内核外包裹有单宁酸和铁离子形成的网络结构层,在所述网络结构层外修饰有纳米银颗粒。
本方案的原理及优点是:本技术方案利用单宁酸和铁离子形成的网络结构层(TA-Fe)包裹连接有细胞穿膜肽的阿霉素形成的内核(PDOX),再在该网络结构层外修饰纳米银颗粒,不但实现了多种药效成分的装载,还使得药物之间的协同作用变为可能,极大地降低了肿瘤对化疗药物的耐药性。本方案的载药纳米复合物是一种诊疗型纳米复合材料,可同时实现核靶向和内质网应激来增强肿瘤化疗,逆转肿瘤多药耐药。
更具体地,阿霉素是一种抗肿瘤抗生素,可抑制RNA和DNA的合成,抗瘤谱较广,对多种肿瘤均有作用,属周期非特异性药物,对各种生长周期的肿瘤细胞都有杀灭作用。但是,阿霉素本身缺少靶向性,为了使得足够量阿霉素进入细胞核发挥其抑制作用,需要增加用药量,从而增加了肿瘤细胞产生耐药性的可能性,以及增加了药物副作用。本技术方案将细胞穿膜肽连接在阿霉素上,增加了阿霉素的核聚集效果,提升了其治疗效果。为了降低肿瘤对阿霉素的耐药性,本技术方案将连接有细胞穿膜肽的阿霉素形成的纳米颗粒的表面进行金属网络多酚工程化处理,单宁酸和铁离子形成的网络结构层使得纳米复合材料具备ATP响应性分解和ATP消耗特性,提高了生物相容性,同时减少了ATP依赖的药物外泵,增加了肿瘤细胞的药物敏感性,提升了阿霉素的治疗效果。不仅如此,单宁酸和铁离子形成的网络结构层还可以显著下调肿瘤细胞中的P糖蛋白(P-gp)的水平。P糖蛋白是一种分子量170KD的跨膜糖蛋白,它具有能量依赖性“药泵”功能。P糖蛋白既能与药物结合,又能与ATP结合,ATP供能,使细胞内药物泵出细胞外,减低了细胞内的药物浓度使细胞产生耐药性。单宁酸和铁离子形成的网络结构层包裹在阿霉素外,可以通过消耗ATP和下调P糖蛋白的双重方式,降低肿瘤细胞耐药性。本方案将纳米银颗粒引入纳米***中,可诱发细胞内强烈的内质网应激,触发细胞凋亡信号。除此之外,纳米银颗粒附着在单宁酸和铁离子形成的网络结构层上,还可以通过协同增效来实现P糖蛋白的表达的进一步抑制,使耐药细胞恢复对化疗的敏感。因此纳米银颗粒也可以产生双重效应,分别诱导了肿瘤细胞凋亡和增加肿瘤细胞的药物敏感性。此外,纳米复合物具有良好的近红外吸收和顺磁特性,是一种具有成像潜力的双模态(光声/磁共振)的增强造影剂,可一次性实现可视化监测和引导治疗。
综上所述,方案的纳米银修饰的单宁酸-铁网络的载药纳米复合物,可以通过多种机制克服MDR,还可以下调耐药细胞内过表达的P-gp并增加细胞内化疗药物的积累,且可将抗肿瘤药物有效递送到其最终目的地细胞核。
进一步,一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其原料包括连接有细胞穿膜肽的阿霉素、单宁酸和水合三氯化铁;单宁酸和水合三氯化铁的质量之和与连接有细胞穿膜肽的阿霉素中的阿霉素的质量的比值为5:1。在本技术方案中,将质量投放比控制在1:5可以获得较为理想的包封率以及载药量。阿霉素用量过高对载药量的提升帮助不大,造成药物浪费;单宁酸和水合三氯化铁的用量过大会导致阿霉素载药量过小,并且会增加纳米粒的粒径,不利用纳米粒给药以及其通过实体瘤的高通透性和滞留效应进入肿瘤细胞,实验数据详见实施例1。
进一步,连接有细胞穿膜肽的阿霉素的结构式如式(1)所示;
其中,CB5005代表细胞穿膜肽;所述细胞穿膜肽的氨基酸序列如SEQ ID NO.1所示。
本技术方案将一种具有细胞膜穿透和核定位序列的新肽偶联到阿霉素上,改善了耐阿霉素乳腺癌细胞的吞噬和核内积累,合成一种具有强大抗癌活性的纳米***。
进一步,所述纳米银颗粒的粒径为10nm。上述粒径的纳米银颗粒可以发挥较为理想的刺激内质网应激和下调P糖蛋白的效果,并且也可以通过静电吸附作用负载到单宁酸和铁离子形成的网络结构层上。
进一步,一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其粒径为185.9nm,电位为-33.6mv。上述尺寸的纳米粒可通过实体瘤的高通透性和滞留效应进入肿瘤细胞,发挥抗肿瘤作用。
本方案还提供了一种纳米银修饰的单宁酸-铁网络的载药纳米复合物的制备方法包括以下依次进行的步骤:
S1:将连接有细胞穿膜肽的阿霉素溶于二甲基亚砜中,获得DOX/DMSO溶液,然后将DOX/DMSO溶液加入水并声震处理,获得内核悬液;
S2:在内核悬液中依次加入单宁酸溶液和氯化铁溶液,经过声震和涡旋之后,将pH值调整至6.4-7.4,获得含TA的纳米粒悬液;
S3:将纳米银溶液分散于含TA的纳米粒悬液中,获得Ag-TF@PDOX。
本方案通过马来酰亚胺法将细胞穿膜肽和阿霉素偶联,使阿霉素具备了细胞膜穿透和核靶向的能力。然后利用溶剂置换的方法将单宁酸/铁网络包裹于在水溶液中形成纳米颗粒的阿霉素的周围,再利用单宁酸/铁网络中大量存在的羟基和静电吸附作用,将纳米银颗粒修饰在单宁酸-铁网络外形成纳米复合物,使纳米复合物具备诱导细胞内内质网应激的能力。通过细胞膜穿透、细胞核靶向、内质网应激这三大策略,提高化疗药阿霉素的抗癌能力,恢复耐阿霉素乳腺癌肿瘤细胞对化疗的敏感性,逆转肿瘤多药耐药。
进一步,在S1中,连接有细胞穿膜肽的阿霉素、二甲基亚砜和水的用量比为4.4mg:200μL:8mL;声震处理的时间为15s,功率为60W。利用阿霉素的疏水性,通过声震处理的方法,形成本技术方案的纳米粒的内核。
进一步,在S2中,单宁酸溶液和氯化铁溶液的体积比为1:1,单宁酸溶液和氯化铁溶液中的溶质的浓度分别为40mg/mL和10mg/mL;在S3中,纳米银溶液中纳米银颗粒的浓度为0.1mg/mL,纳米银溶液与含TA的纳米粒悬液的体积比为40μL:1mL。利用溶剂置换的方法将单宁酸/铁网络包裹于在水溶液中形成纳米颗粒的阿霉素的周围,上述用量的单宁酸和氯化铁可形成网络,并将内核充分包裹,确保减小肿瘤细胞对阿霉素抗性的产生。上述纳米银颗粒的用量,可实现其在网络结构上的充分负载,发挥促进肿瘤细胞凋亡和增加肿瘤细胞的药物敏感性的效果。
本方案还提供了一种纳米银修饰的单宁酸-铁网络的载药纳米复合物在制备逆转肿瘤耐药性药物中的应用。本方案的纳米复合物具有肿瘤细胞核靶向性,可以促进细胞凋亡以及降低肿瘤细胞的耐药性,可以作为逆转肿瘤耐药性药物进行临床应用。载药纳米复合物逆转耐药性的研究实验数据详见实验例2,体内外治疗效果研究详见实验例4和5。
本方案还提供了一种纳米银修饰的单宁酸-铁网络的载药纳米复合物在制备造影剂中的应用。本方案的纳米复合物具有良好的近红外吸收和顺磁特性,是一种具有成像潜力的双模态(光声/磁共振)的增强造影剂,可一次性实现可视化监测和引导治疗,实验数据详见实验例3。
附图说明
图1为本发明实施例1的Ag-TF@PDOX的合成过程示意图。
图2为本发明实施例1的Ag-TF@PDOX的扫描电镜图。
图3为本发明实施例1的Ag-TF@PDOX的粒径分布图。
图4为本发明实施例1和实施例2的Ag-TF@PDOX和TF@PDOX的电位分布图。
图5为本发明实施例1和实施例2的Ag-TF@PDOX、TF@PDOX、AgNPs、TA-Fe3+、PDOX的紫外-可见光光谱图。
图6为本发明实施例1的Ag-TF@PDOX的14天稳定性测试实验结果。
图7为本发明实施例1的Ag-TF@PDOX在ATP环境中释药曲线。
图8为本发明实验例1的细胞吞噬药物情况的共聚焦荧光显微镜图片。
图9为本发明实验例1的药物核定位研究结果。
图10为本发明实验例1的细胞吞噬药物情况的流式细胞仪检测结果。
图11为本发明实验例2的不同药物处理之后内质网应激相关的蛋白的表达情况。
图12为本发明实验例2的Ag-TF@PDOX处理之后生物透射电镜照片。
图13为本发明实验例2的不同药物处理之后P-gp蛋白表达情况(流式细胞仪检测以及WB检测)。
图14为本发明实验例3的Ag-TF@PDOX的体外光声成像结果。
图15为本发明实验例3的Ag-TF@PDOX的体内光声成像结果。
图16为本发明实验例3的Ag-TF@PDOX的体外磁共振成像结果。
图17为本发明实验例3的Ag-TF@PDOX的体内磁共振成像结果。
图18为本发明实验例4的Ag-TF@PDOX的CCK8实验结果(阿霉素的浓度为50μg/ml)。
图19为本发明实验例4的Ag-TF@PDOX的细胞凋亡情况的流式细胞仪检测结果(阿霉素的浓度为50μg/ml)。
图20为本发明实验例4的Ag-TF@PDOX的细胞活死染色结果(阿霉素的浓度为50μg/ml)。
图21为本发明实验例4的Ag-TF@PDOX的CCK8实验结果(纳米银的浓度为0、0.25、0.5和1.0μg/ml)。
图22为本发明实验例5的不同处理下荷瘤小鼠的照片。
图23为本发明实验例5的不同处理下荷瘤小鼠的相对肿瘤体积变化曲线。
具体实施方式
下面结合实施例对本发明做进一步详细的说明,但本发明的实施方式不限于此。若未特别指明,下述实施例以及实验例所用的技术手段为本领域技术人员所熟知的常规手段,且所用的材料、试剂等,均可从商业途径得到。
实施例1:载药纳米复合物的制备
将4.4mg与细胞穿膜肽偶连后的DOX(PDOX)溶于200μL DMSO溶液中。然后将溶解完全的DOX/DMSO溶液200μL注入8mL去离子水中,并连续声震15s(60w)。将80μL的单宁酸(40mg/mL)和80μL水合三氯化铁(10mg/mL)水溶液依次加入到上述溶液中,并在加液过程中连续声震(60w),然后涡旋25s,再使用1μM氢氧化钠溶液中和溶液的酸碱度,获得的混悬液的pH需要控制在6.4-7.4,在本实施例中具体控制为7.0。接下来,将40μL的纳米银溶液(0.1mg/mL,粒径10nm)加入1mL的上述溶液中并搅拌过夜(约8h),离心25分钟后(13000rpm)使用去离子水重悬,最终得到纳米复合物(Ag-TF@PDOX)。本方案的载药纳米复合物的制备过程参见图1。
在本技术方案中,细胞穿膜肽的序列为:NH2-KLKLALALALAVQRKRQKLMP-COOH(SEQID NO.1),然后在其羧基端加入了一个半胱氨酸(C),形成NH2-KLKLALALALAVQRKRQKLMPC-COOH(SEQ ID NO.2)。利用半胱氨酸的巯基通过现有技术的常规的马来酰亚胺法使得该穿膜肽可以与DOX形成共价连接。细胞穿膜肽偶连后的DOX(PDOX)委托生物技术公司合成,通过马来亚酰胺法将实现蛋白在药物或者载体上的连接,是现有技术的常规手段。PDOX的分子式参见式(1),其中CB5005的氨基酸序列为SEQ ID NO.1。CB5005经改造后形成如SEQ IDNO.2所示的序列,半胱氨酸(C)提供了与马来酰亚胺基团连接的巯基,序列如SEQ ID NO.2的蛋白通过马来酰亚胺基团与阿霉素的氨基连接。
制备得到的纳米复合物呈球形,扫描电镜显示因为纳米银颗粒吸附后表面较粗糙(图2)。马尔文粒径电位仪测得纳米复合物粒径约185.9nm(图3),电位约-33.6mv(图4)。在装载阿霉素后,纳米复合物中属于阿霉素480nm处特征峰红移至495nm处。有紫外-可见光光谱及高效液相色谱法测得(图5),纳米复合物中阿霉素的包封率约79.2%,载药量约35.2%。包封率计算公式如下:包封率(%w/w)=(纳米复合物中阿霉素/阿霉素加入量)×100%。;载药量计算公式如下:载药量(%w/w)=(纳米复合物中阿霉素/纳米复合物总质量)×100%14天内,该纳米复合物的粒径无明显改变(图6),说明纳米复合物的稳定性较好。释药实验显示,将该纳米复合物放置在含5mM的ATP溶液48小时后,可释放约80.4%的阿霉素(图7)。
在本技术方案中,使用氢氧化钠溶液中和酸碱度的操作非常重要,发明人尝试将混悬液的pH调整至6.4和7.4。在上述两种pH值的条件下,后续按照前文所描述的方法加入纳米银,通过搅拌混合,均可以获得分散度良好的纳米复合物的悬浊液。发明人同时也尝试了其他的pH值对于实验效果的影响,具体地采用了经氢氧化钠调节后的混悬液的pH值为9.4和5.5。后续按照前文所描述的方法加入纳米银,发现在后续的搅拌过程中生成大量的絮状沉淀,纳米复合物非常容易聚集,在液相中的分散度差,导致纳米复合物的制作失败。所以,在上述工艺过程中,将pH值维持在6.4-7.4,对纳米银修饰的单宁酸-铁网络的载药纳米复合物的成功制备非常关键。
对PDOX和单宁酸体之间的比例关系进行了研究,结果如表1所示。在表1中,质量投放比是指药效成分DOX(即:PDOX的质量减去细胞穿膜肽和MAL基团的质量)与单宁酸+水合三氯化铁的质量比。表1中的“2”为前文中描述的最佳实施方式,PDOX的质量为4.4mg,换算成DOX为800μg,单宁酸+水合三氯化铁的质量为4mg,所以质量投放比为1:5(800μg:4mg)。由表1的数据可知,将质量投放比控制在1:5可以获得较为理想的包封率以及载药量。
表1:PDOX和单宁酸体之间的比例关系对PDOX的载药量、包封率和粒径的影响
实施例2
本实施例制备了不含有纳米银颗粒的载药纳米复合物,具体制备方法为:
将4.4mg与细胞穿膜肽偶连后的DOX(PDOX)溶于200μlDMSO溶液中。然后将溶解完全的DOX/DMSO溶液200μl注入8ml去离子水中,并连续声震15秒(60w)。将80μl的单宁酸(40mg/ml)和80μl水合三氯化铁(10mg/ml)水溶液依次加入到上述溶液并不断的声震和涡旋后,使用1μM氢氧化钠溶液中和溶液的酸碱度,离心25分钟后(13000rpm)使用去离子水重悬,最终得到纳米复合物(TF@PDOX)。其电位图参见图4,紫外-可见光光谱参见图5。
将4.4mg与细胞穿膜肽偶连后的DOX(PDOX)溶于200μlDMSO溶液中。然后将溶解完全的DOX/DMSO溶液200μl注入8ml去离子水中,并连续声震15秒(60w),获得PDOX纳米粒,其紫外-可见光光谱图参见图5。单宁酸和六水合三氯化铁的粉末溶于去离子水后搅拌获得TA-Fe3+,其紫外-可见光光谱图参见图5。
实验例1:载药纳米复合物的细胞膜穿透和靶向能力
将普通的阿霉素(DOX),经细胞穿膜肽偶连后的阿霉素(PDOX)和包载了实施例2的纳米复合物(TF@PDOX)与耐阿霉素型乳腺癌细胞(MCF-7/ADR)共孵育0.5、1、3、6小时。其中,阿霉素浓度为20μg/ml,细胞密度为1.5×104个细胞/每共聚焦皿。经共聚焦荧光显微镜检测,经细胞穿膜肽偶连后的阿霉素和实施例2的纳米复合物能够大量被吞噬入细胞质和细胞核内,而未经偶连的阿霉素则大部分停留在细胞质中,无法进入细胞核。同时流式细胞仪通过定量检测细胞内的荧光强度,也证明了这一结论。这说明经细胞穿膜肽功能化后的阿霉素具备了细胞膜穿透和细胞核靶向的能力。细胞吞噬药物情况的共聚焦荧光显微镜图片参见图8(蓝色荧光表示细胞核,红色荧光表示阿霉素或者经细胞穿膜肽偶连后的阿霉素),药物核定位研究结果参见图9(从左向右依次为DOX组、PDOX组和TF@PDOX组,蓝色荧光表示细胞核,红色荧光表示阿霉素或者经细胞穿膜肽偶连后的阿霉素),细胞吞噬药物情况的流式细胞仪检测结果参见图10。
实验例2:载药纳米复合物逆转耐药性的研究
在本实验例中,分析了在纳米***中加入纳米银颗粒后(实施例1制备的Ag-TF@PDOX),纳米复合物诱导肿瘤细胞内质网应激的能力。实验方案为:将PBS,TF@PDOX和Ag-TF@PDOX(DOX含量为50μg/ml)分别加入细胞密度为1×106的培养瓶中孵育6h,然后使用westernbolt检测相关蛋白表达。通过western bolt检测发现,相比与未加入纳米银颗粒的纳米复合物以及对照组,在加入纳米银颗粒后,细胞内与内质网应激相关的蛋白表达均明显提高,特别是GRP78、ATF4和Caspase-12等于内质网应激相关的蛋白的表达量大大上调(图11)。生物透射电镜也显示,经纳米复合物(Ag-TF@PDOX)共孵育后,细胞内的内质网明显肿胀(图12,下图为上图的放大图,箭头表示内质网发生明显肿胀的位置),这说明该纳米复合物启动的细胞内内质网应激的信号通路传递。我们采用了不同的试剂检测了其对细胞内P-gp表达的影响。经流式细胞仪和western bolt检测证实,含有纳米银颗粒的纳米复合物能够最大程度下调P-gp,这将有利于恢复细胞对化疗药物的敏感性(图13,左为流式细胞仪检测结果,右为WB检测结果)。在图13中,使用流式软件定量测量PDOX组流式荧光值,其荧光值略高于对照组和DOX组,与WB结果一致。在单宁酸铁的作用下,pDOX对P-gp的上调作用被大大抑制,并且,如果在纳米粒中进一步加入纳米银颗粒,对P-gp的表达的抑制作用会进一步增强。其中,P-gp(P糖蛋白)是一种分子量170KD的跨膜糖蛋白,它具有能量依赖性“药泵”功能。P-gp既能与药物结合,又能与ATP结合,ATP供能,使细胞内药物泵出细胞外,减低了细胞内的药物浓度使细胞产生耐药性。
实验例3:成像效果研究
由于纳米复合物内含有金属多酚网络(单宁酸-铁),使该纳米粒具有近红外区吸收及顺磁性的特性,因此该纳米复合物具备了成为光声成像和磁共振成像的造影剂的潜力。体外实验证明,光声信号(图14)与磁共振(图16)弛豫率与纳米复合物的浓度具有良好的线性关系。同时体内实验也证实,该纳米***能够实现生物活体的光声成像(图15)与磁共振成像(图17)。
实验例4:体外细胞毒性研究
于体内外检测了该纳米复合物对MCF-7/ADR细胞的细胞毒性,将MCF-7/ADR细胞的细胞密度为5×103个细胞/孔,加入待测药物,分别为:空包对照、未连接穿膜肽的阿霉素(DOX)、连接穿膜肽的阿霉素(pDOX)、纳米粒TF@PDOX、纳米粒Ag-TF@PDOX,共培养6h、12h和24h,再进行CCK-8细胞活性检测。其中,除了空白对照之外,其他所有实验组中保持阿霉素的浓度为50μg/ml。经CCK8检测(图18),凋亡细胞检测(图19)及细胞活死双染检测(图20,绿色荧光表示活细胞,红色荧光表示死细胞)我们发现,含有细胞穿膜肽偶连阿霉素以及纳米银颗粒的纳米复合物具备了最强的细胞毒性。这意味着同时具备细胞膜穿透,核靶向以及诱导内质网应激能力的纳米复合物能够最有效的回复耐药细胞对化疗药的敏感性,可以逆转细胞耐药性。
除此之外,对MCF-7/ADR进行了不同形式的纳米银的细胞毒性实验,实验分组包括单独使用纳米银、纳米银联合纳米银抑制剂(TUDCA)、Ag-TF@PDOX、Ag-TF@PDOX联合TUDCA。药物处理时间均为24h,并且将银粒子的浓度分别控制为0、0.25、0.5和1.0μg/ml。实验结果参见图21,可见在纳米银的浓度为1.0μg/ml时,TUDCA可以几乎完全解除纳米银对细胞的抑制作用,由66.7%上升至91.7%,变化量为25.0%。然而,将纳米银装载到纳米复合物上,形成Ag-TF@PDOX之后,使用TUDCA相对于不使用TUDCA,细胞抑制率由21.0%上升至50.3%,变化量为29.3%。TUDCA为一种高效且特异的纳米银抑制剂,上述变化量显示了纳米银独立的对于待测细胞的毒性作用。当纳米银加载到纳米复合物上之后,纳米银的细胞毒性得到了进一步加强,这说明在本方案中,纳米复合物对于纳米银的毒性作用具有协同增效的效应。
实验例5:体内细胞毒性研究
建立MCF-7/ADR荷瘤小鼠模型,并对各种载药纳米复合物肿瘤的抑制能力进行了研究。给药以及实验分组的具体情况为:对照组,DOX组,PDOX组,TF@PDOX组和Ag-TF@PDOX组,每次给药浓度均统一换算为阿霉素的给药浓度,具体浓度为每只小鼠体重注射1.5mg/kgDOX。荷瘤小鼠在不同的药物处理下的照片详见图22,荷瘤小鼠肿瘤生长曲线见图23。体内实验也证明了本方案的纳米复合物对荷瘤小鼠的肿瘤生长抑制最明显。
以上所述的仅是本发明的实施例,方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出,对于本领域的技术人员来说,在不脱离本发明技术方案的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
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Claims (9)
2.根据权利要求1所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其特征在于:其原料包括连接有细胞穿膜肽的阿霉素、单宁酸和水合三氯化铁;单宁酸和水合三氯化铁的质量之和与连接有细胞穿膜肽的阿霉素中的阿霉素的质量的比值为5:1。
3.根据权利要求2所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其特征在于:所述纳米银颗粒的粒径为10nm。
4.根据权利要求3所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物,其特征在于:其粒径为185.9nm,电位为-33.6mv。
5.根据权利要求4所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物的制备方法,其特征在于:包括以下依次进行的步骤:
S1:将连接有细胞穿膜肽的阿霉素溶于二甲基亚砜中,获得DOX/DMSO溶液,然后将DOX/DMSO溶液加入水并声振处理,获得内核悬液;
S2:在内核悬液中依次加入单宁酸溶液和氯化铁溶液,经过声振和涡旋之后,将pH值调整至6.4-7.4,获得含单宁酸TA的纳米粒悬液;
S3:将纳米银溶液分散于含TA的纳米粒悬液中,获得纳米银修饰的单宁酸-铁网络的载药纳米复合物Ag-TF@PDOX。
6.根据权利要求5所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物的制备方法,其特征在于:在S1中,连接有细胞穿膜肽的阿霉素、二甲基亚砜和水的用量比为4.4mg:200μL:8mL;声振处理的时间为15s,功率为60W。
7.根据权利要求5所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物的制备方法,其特征在于:在S2中,单宁酸溶液和氯化铁溶液的体积比为1:1,单宁酸溶液和氯化铁溶液中的溶质的浓度分别为40mg/mL和10mg/mL;在S3中,纳米银溶液中纳米银颗粒的浓度为0.1mg/mL,纳米银溶液与含TA的纳米粒悬液的体积比为40μL:1mL。
8.根据权利要求1-4中任一项所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物在制备逆转肿瘤耐药性药物中的应用。
9.根据权利要求1-4中任一项所述的一种纳米银修饰的单宁酸-铁网络的载药纳米复合物在制备造影剂中的应用。
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