CN110507831B - 一种高度生物相容性纳米级超声造影剂及其制备方法与应用 - Google Patents
一种高度生物相容性纳米级超声造影剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种高度生物相容性纳米级超声造影剂及其制备方法与应用。本发明中的纳米级超声造影剂是以全氟己烷为核心,以壳聚糖、棕榈酸、卵磷脂为壳膜材料,全氟己烷作为核心可显著提高造影剂的产出效率,并使超声造影剂的结构更加稳定。所述的高度生物相容性纳米级超声造影剂的粒径为300~900nm,平均粒径为519.6±72.66nm,在纳米级范围,能穿过肿瘤组织血管壁间隙,对于肿瘤的治疗具有靶向性和高效性;该造影剂具有较强的体内增强显像能力,较高的药物包封率及载药量,生物安全性高。
Description
技术领域
本发明涉及一种高度生物相容性纳米级超声造影剂及其制备方法与应用,属于超声分子影像学技术领域。
背景技术
随着超声分子成像技术和生物纳米技术的迅猛0发展,纳米级超声造影剂发展迅速,越来越多的关于载药纳米系统的研究已经证明了它们在癌症治疗中的潜在重要性。纳米给药系统的优点在于能够将集中剂量的药物,以体内肿瘤组织为目标,输送到特定区域。纳米给药系统不仅增加了药物对肿瘤的局部作用,而且减少了化疗药物在血液循环过程中进入其他组织的副作用。然而,大多数纳米药物缓释系统在临床实际应用中仍面临一些问题,例如,如何在纳米药物缓释系统聚集并粘附于肿瘤细胞后增加向细胞内的释放,无法实时观察和控制治疗过程。因此,在治疗过程中不可能对治疗方案进行进一步的评价和改进。超声引导下药物传递系统可以通过超声靶向微泡破坏增强细胞质膜可逆通透性,使药物有效进入肿瘤细胞。这种方法可以同时通过体外超声成像实时精准的观察治疗过程。
超声引导药物传递系统正成为靶向治疗研究的热点之一。为了更容易穿透新生血管内皮细胞间隙,超声造影剂的粒径由微米逐渐减小到纳米。随着纳米级超声造影剂的研究和临床应用的不断深入,其安全性引起了人们的广泛关注,注射用造影剂在临床应用前必须保证生物安全性。在所有用于纳米级超声造影剂的材料中,蛋白质材料可能引起过敏反应。许多固定剂或表面活性剂如戊二醛和吐温对人体有害,而高分子材料对人体安全构成潜在风险。因此,迫切需要制备安全性高、疗效确切的纳米级超声造影剂。壳聚糖是自然界中常见的一种天然多糖。大量的研究表明,壳聚糖具有良好的生物相容性和生物降解性,并具有抗菌和抗肿瘤的特性。中国专利文献CN106139174A(申请号201610693711.0)提供了一种包裹液态氟碳的基于壳聚糖衍生物纳米级超声造影剂的制备方法,是通过酰化反应对羧甲基壳聚糖进行改性,合成具有两亲性的正己酰羧甲基壳聚糖,在此基础上加入液态氟碳,采用超声乳化方法制得由液态氟碳内核和壳聚糖衍生物外壳构成的纳米级超声造影剂。该发明制备得到的纳米级超声造影剂带负电荷,合成的正己酰羧甲基壳聚糖使用了壳聚糖的活跃氨基,不利于造影剂携载药物,并且在制备过程中使用的材料正己酸酐和甲醇都是有毒物质,若无法完全除去则其生物安全性无法保障。中国专利文献CN109260480A(申请号201811219930.0)公开了一种携载阿霉素的壳聚糖纳米级超声造影剂及其制备方法与应用,该纳米级超声造影剂是以壳聚糖为壳膜,阿霉素(DOX)和全氟丙烷气体包裹在壳聚糖壳膜内部,具有较强的增强显像能力,该发明制备的纳米级超声造影剂的形态为纳米液泡,全氟丙烷纳米液泡产出率低,稳定性较差。
发明内容
针对现有技术的不足,本发明提供了一种高度生物相容性纳米级超声造影剂及其制备方法与应用,本发明中的纳米级超声造影剂是以全氟己烷为核心,以壳聚糖、棕榈酸、卵磷脂为壳膜材料,全氟己烷作为核心可显著提高造影剂的产出效率,并使超声造影剂的结构更加稳定。该造影剂具有较强的体内增强显像能力,较高的药物包封率及载药量,生物安全性高。
本发明还提供了上述纳米级超声造影剂在负载抗肿瘤药物和体内治疗肿瘤中的应用。
术语说明:
室温:具有本领域技术人员公知的含义,一般是指25±2℃。
本发明的技术方案如下:
一种高度生物相容性纳米级超声造影剂,所述纳米级超声造影剂以壳聚糖、棕榈酸、卵磷脂为壳膜,壳膜内部包裹有全氟己烷,所述纳米级超声造影剂的粒径为300~900nm。
根据本发明优选的,所述棕榈酸和卵磷脂的质量比为1:4,所述卵磷脂和壳聚糖的质量比为(1~2):4。
上述高度生物相容性纳米级超声造影剂的制备方法,步骤如下:
(1)将棕榈酸加热溶解于超纯水中得棕榈酸溶液,将棕榈酸溶液与卵磷脂混合于超纯水中,再加入溶解于超纯水的壳聚糖溶液,室温下涡旋混匀,得混合溶液;
(2)向步骤(1)制得的混合溶液中加入全氟己烷,室温下涡旋混匀,乳化(1~3)min,得乳化产物;
(3)将步骤(2)制得的乳化产物稀释后超滤离心,即得高生物相容性纳米级超声造影剂。
根据本发明优选的,步骤(1)中所述棕榈酸和卵磷脂的质量比为1:4,所述混合溶液中棕榈酸的浓度为0.1~0.2g/L,其中棕榈酸纯度≥99%,购自sigma,产品编号200-312-9;卵磷脂购自sigma,产品编号232-715-0。
根据本发明优选的,步骤(1)中所述卵磷脂和壳聚糖的质量比为(1~2):4,其中壳聚糖的分子量为100~300kD,购自sigma,产品编号MFCD00161512。
根据本发明优选的,步骤(2)中所述全氟己烷在混合溶液中的体积百分比为0.2~0.4%。
根据本发明优选的,步骤(3)中所述稀释的倍数为5~10倍。
根据本发明优选的,步骤(3)中所述超滤离心使用的超滤离心管的截留分子量为30KD。
根据本发明优选的,步骤(3)中所述超滤离心为500-1000rpm离心1-5min。
上述高度生物相容性纳米级超声造影剂在携载阿霉素制备抗肿瘤药物中的应用。
上述未作详细说明的实验步骤均按照本技术领域常规操作进行。
本发明的技术特点:
本发明以壳聚糖、棕榈酸和卵磷脂为壳膜材料,以全氟己烷为内核得到乳化产物,超滤离心后得到粒径较小且均匀的纳米级超声造影剂。壳聚糖、棕榈酸和卵磷脂均具有良好的生物相容性和生物降解性,是良好的用于制备超声造影剂的材料,另外,棕榈酸和卵磷脂都带有负电荷,与带正电的壳聚糖通过正负电荷吸引形成纳米滴的外壳。
同时卵磷脂也是表面活性剂(既有疏水基团也有亲水基团),容易形成外部亲水内部疏水(内核全氟己烷不溶于水)的纳米液滴结构。
有益效果:
1、本发明制备的高度生物相容性纳米级超声造影剂,是以壳聚糖、棕榈酸、卵磷脂为壳膜,全氟己烷包裹在壳膜内部的纳米液滴,所述超声造影剂的粒径为300~900nm,平均粒径为519.6±72.66nm,在纳米级范围,能穿过肿瘤组织血管壁间隙,对于肿瘤的治疗具有靶向性和高效性。
2、本发明制备的高度生物相容性纳米级超声造影剂以全氟己烷液体为核心,全氟己烷可以显著提高造影剂的产出效率,出泡率高,并使超声造影剂的结构更加稳定,25℃放置6小时后,仍没有明显的形态上的变化。
3.本发明制备的纳米级超声造影剂体内生物相容性高、生物安全性高,80mg/kg小鼠尾静脉注射无明显毒性。
4、本发明制备的高度生物相容性纳米级超声造影剂具有较强的体内增强显像能力,而且可以在较长的时间内维持显影,还具有较高的药物包封率及载药量。
5、本发明采用超声辐照的方式利用本发明制备的高度生物相容性纳米级超声造影剂对肿瘤进行治疗,一方面可以增强纳米级超声造影剂显影,另一方面可以借助超声辐照产生的声孔效应促进携载药物或小分子更多的进入肿瘤细胞内,实现诊疗一体化。
6、本发明制备的高度生物相容性纳米级超声造影剂携载药物进入体内联合超声治疗能显著提高药物的提取,有利于肿瘤疾病的治疗。
附图说明
图1是实施例1制备得到的BCNDs光学显微镜照片;图中,A为未经处理的BCNDs光学显微镜照片;B为在25℃放置6小时后的BCNDs光学显微镜照片;
图2是实施例1制备得到的BCNDs粒径及电位曲线图;图中,A为BCNDs的粒径大小分布图,B为BCNDs的电位分布图;
图3是小鼠脏器的HE染色照片;图中,A-1是对照组小鼠心脏的HE染色照片,A-2是对照组小鼠肝脏的HE染色照片,A-3是对照组小鼠脾脏的HE染色照片,A-4是对照组小鼠肺的HE染色照片,A-5是对照组小鼠肾脏的HE染色照片;B-1是实验组小鼠心脏的HE染色照片,B-2是实验组小鼠肝脏的HE染色照片,B-3是实验组小鼠脾脏的HE染色照片,B-4是实验组小鼠肺的HE染色照片,B-5是实验组小鼠肾脏的HE染色照片;
图4是不同的阿霉素浓度制备得到的DOX-BCNDs的包封率及载药量曲线图;图中,A为DOX-BCNDs的载药量曲线图,横坐标为阿霉素浓度,纵坐标为载药量;B为DOX-BCNDs的包封率曲线图,横坐标为阿霉素浓度,纵坐标为包封率;
图5是DOX-BCNDs体外和体内的超声显影图像;图中,A-1为体外灰阶显像图像,A-2为体外增强显像图像;B-1为体内灰阶显像图像,B-2为体内增强显像图像;
图6是荷瘤小鼠及组织器官的荧光成像结果;图中,A为DOX-BCNDs组和DOX组小鼠在不同时间的活体成像结果;B为小鼠组织器官的荧光成像结果,图中组织器官从左到右依次为肿瘤组织、心脏、脾脏、肺、肝脏、肾脏;
图7是不同处理组小鼠肿瘤体积变化图;图中,A为小鼠解剖后肿瘤体积图片,B为小鼠肿瘤体积变化柱状图;
图8是不同处理组小鼠血液中血肌酐(CREA)、尿素氮(BUN)、乳酸脱氢酶(LDH)和肌酸磷酸激酶(CK)含量水平柱状图;图中横坐标从左到右依次为正常对照组、对照组、DOX组、DOX超声组、DOX-BCNDs超声组、双倍剂量DOX-BCNDs超声组;
图9是不同处理组小鼠肿瘤组织石蜡切片HE染色的显微镜照片;
图10是不同处理组小鼠肿瘤组织细胞凋亡情况的荧光显微镜照片;
图11是不同处理组小鼠肿瘤组织细胞增殖情况的荧光显微镜照片;
图12是不同处理组小鼠肿瘤组织细胞增殖情况柱状图,图中,横坐标从左到右依次为对照组、DOX组、DOX超声组、DOX-BCNDs超声组、双倍剂量DOX-BCNDs超声组,横坐标为ki67平均光密度。
具体实施方式
下面结合实施例对本发明做进一步的说明,但本发明的保护范围并不仅限于此。
棕榈酸购自sigma,产品编号200-312-9;
卵磷脂购自sigma,为Epikuron200,产品编号232-715-0;
壳聚糖购自sigma,分子量为100~300kD,产品编号MFCD00161512;
阿霉素购自sigma,产品编号246-818-3。
本实施例涉及药品及试剂如无特殊说明,均为普通市售产品。
实施例1:高生物相容性纳米级超声造影剂的制备
一种高生物相容性纳米级超声造影剂的制备方法,步骤如下:
(1)将棕榈酸加热溶解于超纯水中得棕榈酸溶液,将棕榈酸溶液与卵磷脂混合于超纯水中,再加入溶解于超纯水的壳聚糖溶液,室温下涡旋混匀,得混合溶液,所述混合溶液中,棕榈酸的浓度为0.1g/L,卵磷脂的浓度为0.4g/L,壳聚糖的浓度为1.6g/L;
(2)向步骤(1)制得的混合溶液中加入全氟己烷,全氟己烷在混合溶液中的体积百分比为0.2~0.4%,室温下涡旋混匀,采用细胞破壁仪乳化1min,得乳化产物;
(3)将步骤(2)制得的乳化产物稀释8倍后1000rpm超滤离心1min,超滤离心管的截留分子量为30KD,即得高生物相容性纳米级超声造影剂(BCNDs)。
取上述制备得到的BCNDs,稀释后滴加到载玻片上,通过光学显微镜观察造影剂的表观形貌,结果如图1所示,A为未经处理的BCNDs光学显微镜照片,B为在25℃放置6小时后的BCNDs光学显微镜照片,1000×光学显微镜下可见造影剂均呈球形,粒径均一,分散均匀无聚集;将BCNDs在25℃放置6小时后,造影剂仍呈球形,分散均匀无聚集,说明本发明制备的BCNDs稳定性强。
取上述制备的纳米级超声造影剂,稀释后,使用纳米激光粒度及Zeta电位分析仪检测超声造影剂的粒径及电位,结果如图2所示,造影剂的粒径为300~900nm,平均粒径为519.6±72.66nm,PI:0.219,表面电位为59.1±24.1mV。
实施例2:BCNDs的体内安全性评价
取实施例1制备的BCNDs,实验组小鼠以总剂量80mg/kg、给药体积0.5mL从小鼠尾静脉注射。对照组小鼠尾静脉注射相同剂量的生理盐水。所有实验动物在实验前禁食12小时。
观察各组小鼠的体重变化,并在尾静脉注射后第14天进行血液生化检测,对心脏、肝脏、脾脏、肺和肾脏进行HE染色,染色结果如图3所示,结果表明实验组小鼠和对照组小鼠的体重和各项血液生化检测指标无明显差异,HE染色结果表明实验组小鼠和对照组小鼠的心脏、肝脏、脾脏、肺和肾脏的病理切片也无明显差异,说明本发明制备的BCNDs对小鼠无毒害作用,体内生物安全性高。
实施例3:BCNDs的药物包封率及载药量检测
取实施例1制备的BCNDs加入适量的阿霉素,阿霉素浓度分别为2.0、2.5、3.0、3.5mg/mL,室温共孵育20min,室温下1000rpm超滤离心1min,超滤离心管的截留分子量为30KD,即得携载阿霉素的BCNDs(DOX-BCNDs),采用酶标仪检测下层液中底层液体在480nm处的吸光度,根据DOX溶液浓度标准曲线计算底层液体中游离的DOX质量,再根据以下公式计算DOX-BCNDs的药物包封率(EE%)和载药量(LD%):
EE%=(载有的DOX质量/加入的初始DOX质量)×100%,
LD%=(载有的DOX质量/加入的纳米级超声造影剂的质量)×100%,
其中,载有的DOX质量=加入的初始DOX质量-游离的DOX质量,
加入的纳米级超声造影剂的质量为加入的初始原料的总质量,包括壳聚糖、阿霉素、棕榈酸、全氟己烷和卵磷脂。
不同阿霉素浓度制备的DOX-BCNDs的包封率及载药量如图4所示,随着阿霉素浓度的升高,DOX-BCNDs的包封率逐渐升高并达到稳定,当阿霉素浓度为3mg/mL时,DOX-BCNDs的载药量最大,大约为9.5%;当阿霉素浓度为2.0-3.0mg/mL时,DOX-BCNDs的包封率最高,为70-80%,所以当阿霉素浓度为3mg/mL时,DOX-BCNDs的载药量及包封率均达到较佳水平,以下实施例涉及到的DOX-BCNDs均在此条件下制备。
实施例4:BCNDs的体内和体外超声显影能力检测
取DOX-BCNDs,PBS溶液稀释后放入显影袋中,进行体外成像实验,将耦合剂涂在GE超声仪M9L探头上后,放置在显影袋侧进行超声成像,设定超声成像仪的力学指标(MI)为0.10,成像深度为4.5cm。
在体内成像实验中,取一只荷瘤小鼠,在肿瘤的突出部位及周围区域进行局部脱毛,将0.1mLDOX-BCNDs混悬液注入肿瘤内,然后立即进行超声成像。实验使用GE超声装置小器官探头,超声参数设置在3cm深度,力学指标为MI0.22。
用超声仪工作站存储图像资料,图像如图5所示,在灰阶显像和增强显像模式下,均能呈现出清晰的图像,表明该造影剂有较强的超声显影能力,并能在体内和体外都获得超声增强显像。
实施例5:DOX-BCNDs体内荧光成像
将20只荷瘤BALB/C小鼠随机分为DOX-BCNDs组和DOX组,每组10只,在所有小鼠肿瘤组织和肿瘤周边组织脱毛,每个肿瘤注射0.1mLDOX-BCNDs或相同剂量的DOX。DOX-BCNDs组注射后立即对肿瘤区域进行超声照射1分钟(输出功率密度2W/cm2)。
所有小鼠异氟醚麻醉,进入ivis动态小动物成像系统。分别在注射后0h、2h、4h和12h进行激发荧光和拍照,所有程序都是在暗室中进行,结果如图6A所示,DOX-BCNDs组肿瘤局部荧光强度随时间衰减更缓慢。
12小时后,处死所有小鼠,将心脏、肝脏、脾脏、肺、肾脏、肿瘤组织都进行解剖,并通过荧光成像进行了组间比较,结果如图6B所示,并检测小鼠肿瘤组织中的DOX荧光强度,荧光强度越强表明DOX浓度越高,结果如表1所示,从以上结果可以看出,两组小鼠中肿瘤组织的荧光强度均高于其他组织;DOX组小鼠肿瘤组织的平均荧光强度为3.42×108,DOX-BCNDs组小鼠肿瘤组织的平均荧光强度为5.33×108,相对于DOX组小鼠,DOX-BCNDs组小鼠肿瘤组织中的荧光强度更高,而且DOX-BCNDs组小鼠肝脏、肾脏组织中的DOX更少,说明DOX-BCNDs具有一定的肿瘤组织靶向性,可以减少对体内肝、肾组织的损害。
表1.DOX-BCNDs组和DOX组小鼠肿瘤中DOX的浓度
实施例6:体内肿瘤抑制实验
将35只balb/c小鼠随机分为5组,分别为对照组、DOX组、DOX超声组、DOX-BCNDs超声组和双倍剂量DOX-BCNDs超声组。将H22肿瘤细胞植入小鼠体内后的第7天,对小鼠进行局部治疗。DOX-BCNDs超声组和双倍剂量DOX-BCNDs超声组小鼠分别在肿瘤部位注射0.1mL和0.2mL的DOX-BCNDs。DOX组和DOX超声组小鼠分别注射等量的DOX。对照组肿瘤部位注射0.1mL生理盐水。DOX超声组、DOX-BCNDs超声组和双倍剂量DOX-BCNDs超声组在注射后立即照射肿瘤区1分钟(输出功率密度2w/cm2)。注射分别在第7、9和11天各进行一次。
1、小鼠体重与肿瘤体积变化
在实验开始和治疗结束时给小鼠称重;计算各组小鼠体重变化。
治疗前后测定荷瘤小鼠的肿瘤体积,计算各组小鼠肿瘤体积变化,并计算肿瘤生长抑制率;
其中V和V0是当前和初始肿瘤体积(V0是治疗开始时的肿瘤体积);
其中Vc为对照组肿瘤体积,Vt为治疗组肿瘤体积;
小鼠体重的变化如表2所示,小鼠肿瘤体积的变化如表2和图7所示。
表2.各组小鼠体重和瘤体体积的变化
注:◆与对照组相比P<0.05,*与DOX组相比P<0.05,▼与DOX超声组相比P<0.05,▲与DOX-BCNDs超声组相比P<0.05。
结果表明,各组小鼠体重变化差异较大,DOX组、DOX超声组和DOX-BCNDs超声组小鼠体重均增加,其中,DOX-BCNDs超声组小鼠增重最为显著,对照组和双倍剂量DOX-BCNDs超声组小鼠体重均下降,说明DOX治疗可在一定程度上改善肿瘤小鼠的生存状态,但对于DOX-BCNDs来说,适当的剂量也是很重要的。
从肿瘤体积的变化来看,DOX组和DOX超声组肿瘤体积的增加比对照组慢。DOX-BCNDs超声组和双倍剂量DOX-BCNDs超声组治疗后肿瘤体积减小;其中DOX-BCNDs超声组的肿瘤缩小比双倍剂量DOX-BCNDs超声组的肿瘤缩小更明显。这些结果表明,DOX-BCNDs可促进DOX在肿瘤治疗中的作用,但是DOX-BCNDs的注射剂量要适宜。
DOX组和DOX超声组的肿瘤抑制率分别为8.35%和15.52%;DOX-BCNDs超声组的肿瘤抑制率为39.50%,超过DOX超声组的两倍;这一结果也表明DOX-BCNDs联合超声能显著提高肿瘤抑制率,促进DOX的治疗作用。
2、小鼠解剖与血液采集
实验第13天,取各组小鼠眼球血进行血液生化和血常规检查,评价不良反应。使用自动细胞计数器(ABX-micros-60细胞计数器Horiba,Inc.)进行血液学分析。
样本的血液学参数如下:白细胞数、淋巴、单核细胞和粒细胞比率、红细胞计数和血红蛋白。将采集的部分血液倒入肝素化试管中,分析血肌酐(CREA)、尿素氮(BUN)、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总蛋白(TP)、白蛋白(ALB)、乳酸脱氢酶(LDH)和肌酸磷酸激酶(CK)的水平,其中,血肌酐(CREA)、尿素氮(BUN)、乳酸脱氢酶(LDH)和肌酸磷酸激酶(CK)的水平在各组之间的差异较大,其水平差异如图8所示。
结果表明,DOX-BCNDs组小鼠的血液中CREA、LDH、CK水平低于DOX组和DOX超声组,肾脏损害时,血肌酐(CREA)因无法通过肾脏排泄而升高;LDH、CK是心肌组织释放的酶,心肌损害时大量释放会导致血液中LDH、CK升高;说明了BCNDs可以减轻肾脏及心肌组织损伤,对机体有保护作用。
3、HE染色、凋亡试验和免疫组化
处死所有小鼠,解剖肿瘤组织。对切除的肿瘤进行组织病理学分析。石蜡包埋的肿瘤切片厚度为5μm,置于玻璃显微镜载玻片上,苏木精和伊红(HE)染色进行显微镜观察(ti50;nikon公司)。根据试剂盒说明书进行细胞凋亡和免疫组化检测,荧光显微镜观察细胞凋亡;用image pro软件读取ki67图片,进行组间比较。
各组小鼠肿瘤组织切片的HE染色结果如图9所示,结果显示对照组肿瘤细胞核无定形、不典型,细胞大小不同,胞浆嗜酸性,核小体增多,排列不规则;DOX组的部分肿瘤细胞出现空泡、变性和坏死;DOX超声组的肿瘤细胞空泡化、变性,坏死、破碎细胞略有增多;DOX-BCNDs超声组和双DOX-BCNDs超声组肿瘤细胞大量变性坏死,细胞碎裂明显。
DOX治疗肿瘤的主要途径是诱导肿瘤细胞凋亡,各组小鼠肿瘤组织细胞凋亡情况如图10所示,荧光越强,凋亡细胞越多,从图中可以看出,DOX组和DOX超声组的肿瘤细胞凋亡数量均大于对照组;DOX-BCNDs超声组和双倍剂量DOX-BCNDs超声组的凋亡细胞数增加更为显著;说明DOX-BCNDs促进了DOX对肿瘤细胞的凋亡作用。
利用单克隆抗体ki67采用免疫组化法检测肿瘤细胞增殖。使用image pro plus专业图像分析系统(media controlneticsinc,bethesda,maryland,usa)测量面积和平均光密度,ki67标记阳性率越高,肿瘤细胞增殖越快。ki67免疫荧光染色结果如图11所示,测量得到的ki67平均光密度如图12所示,结果表明,与对照组相比,DOX组和DOX超声组的ki67阳性细胞数明显减少(p<0.05);DOX-BCNDs超声组ki67阳性细胞数下降更为显著(p<0.05),说明DOX-BCNDs的应用可显著抑制肿瘤细胞的增殖。
综上,相对于DOX组和DOX超声组,DOX-BCNDs联合超声治疗更能够抑制肿瘤细胞的增殖,促进肿瘤细胞的凋亡。
Claims (7)
1.一种高度生物相容性纳米级超声造影剂,其特征在于,所述纳米级超声造影剂以壳聚糖、棕榈酸、卵磷脂为壳膜,壳膜内部包裹有全氟己烷,所述纳米级超声造影剂的粒径为300~900nm;所述棕榈酸和卵磷脂的质量比为1:4,所述卵磷脂和壳聚糖的质量比为1:4;
所述的高度生物相容性纳米级超声造影剂按照如下步骤制备得到:
(1)将棕榈酸加热溶解于超纯水中得棕榈酸溶液,将棕榈酸溶液与卵磷脂混合于超纯水中,再加入溶解于超纯水的壳聚糖溶液,室温下涡旋混匀,得混合溶液;所述混合溶液中棕榈酸的浓度为0.1g/L;
(2)向步骤(1)制得的混合溶液中加入全氟己烷,室温下涡旋混匀,乳化(1~3)min,得乳化产物;所述全氟己烷在混合溶液中的体积百分比为0.2~0.4%;
(3)将步骤(2)制得的乳化产物稀释后超滤离心,即得高生物相容性纳米级超声造影剂。
2.如权利要求1所述的高度生物相容性纳米级超声造影剂,其特征在于,步骤(1)中所述棕榈酸纯度≥99%。
3.如权利要求1所述的高度生物相容性纳米级超声造影剂,其特征在于,步骤(1)中所述壳聚糖的分子量为100~300kD。
4.如权利要求1所述的高度生物相容性纳米级超声造影剂,其特征在于,步骤(3)中所述稀释的倍数为5~10倍。
5.如权利要求1所述的高度生物相容性纳米级超声造影剂,其特征在于,步骤(3)中所述超滤离心使用的超滤离心管的截留分子量为30KD。
6.如权利要求1所述的高度生物相容性纳米级超声造影剂,其特征在于,步骤(3)中所述超滤离心为500-1000rpm离心1-5min。
7.权利要求1所述的高度生物相容性纳米级超声造影剂在携载阿霉素制备抗肿瘤药物中的应用。
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