CN110652597B - 壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用 - Google Patents
壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用 Download PDFInfo
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Abstract
本发明涉及壳聚糖纳米级超声造影剂联合Dickkopf‑2基因在制备治疗前列腺癌药物中的应用。本发明首次发现了Dickkopf‑2基因与前列腺癌的关系,在前列腺癌患者组织中Dickkopf‑2基因的表达明显降低,以壳聚糖纳米级超声造影剂为传递载体,将Dickkopf‑2基因定向输送到肿瘤组织中,通过Dickkopf‑2基因的表达抑制肿瘤细胞的增殖。本发明制备的壳聚糖纳米级超声造影剂以壳聚糖为壳膜材料,安全无毒、生物相容性好、成本较低,粒径在纳米级范围,能顺利通过肿瘤组织血管壁间隙,将目的基因传递至肿瘤组织,并且可以联合超声靶向破坏微泡技术显著提高基因转染效率。
Description
技术领域
本发明涉及壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用,属于生物医药技术领域。
背景技术
随着基因治疗关键技术的不断突破,多种疾病的临床治疗将有更多的新选择。许多疾病具有明确的病变部位,尤其是肿瘤。因而,寻找到一种能够有效地将治疗基因递送到病变部位的方法非常重要。在过去的几十年里,病毒载体由于其高效的基因传递效率而在基因治疗领域占据主导地位。然而,其安全问题限制了应用。病毒载体的这一局限性加快了寻找替代非病毒传递载体的步伐。在非病毒载体中,超声造影剂是一种很有前途的基因传递系统,已成功地用于基因的体内外传递。壳聚糖是一种具有良好生物相容性及生物可降解性的天然多糖,其分子链中存在大量氨基且带正电荷,已成功应用于核酸等生物大分子的研究,是一种理想的超声造影剂壳膜材料。
超声造影剂不仅能显著增强超声显像,还可以携载目的基因,实现并增强基因的转染和表达。同时,超声靶向微泡破坏(Ultrasound-targeted microbubble destruction,UTMD)技术可以实现时空可控的基因传递,使治疗效果最大化。因而,超声与超声造影剂相结合,为基因的时空调控传递提供了一个很有前景的平台。
前列腺癌(prostate cancer,PCa)是来源于前列腺上皮的恶性肿瘤,是中老年男性高发的恶性肿瘤。早期前列腺癌患者手术治疗预后良好,中晚期多采用去势治疗为主的内分泌综合治疗,多数患者会逐渐转变为去势抵抗性前列腺癌(castration-resistantprostate cancer,CRPC),死亡率高,治疗效果差。最近的数据表明,Wnt信号通路在调控肿瘤的发生和发展中起着关键作用。Wnt通路受多种分泌拮抗剂家族调控,包括可溶性卷曲相关受体和dickkopfs(DKK)等。
发明内容
针对现有技术的不足,本发明提供了壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用,以壳聚糖纳米级超声造影剂为非病毒载体,携载Dickkopf-2基因,用于制备治疗前列腺癌的药物。
术语说明:
室温:具有本技术领域公知的含义,一般是指25±2℃。
本发明的技术方案如下:
壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用,所述应用是将壳聚糖纳米级超声造影剂与Dickkopf-2基因连接制备得到壳聚糖纳米级超声造影剂/Dickkopf-2基因复合物,并将所述复合物应用于治疗前列腺癌药物的制备。
根据本发明优选的,所述壳聚糖纳米级超声造影剂是由全氟己烷内核和壳聚糖外壳构成,所述壳聚糖和全氟己烷的质量体积比为(25~35):1,单位是g/L。
根据本发明优选的,所述壳聚糖纳米级超声造影剂是按照以下步骤制备得到的:
(1)配制体积分数为1%乙酸溶液,将壳聚糖加入到所述乙酸溶液中,溶解后制备得到壳聚糖溶液;
(2)将全氟己烷、吐温20和卵磷脂分散于去离子水中制成悬液,均质化,得到乳化产物Ⅰ;
(3)将步骤(1)制得的壳聚糖溶液逐滴加入到步骤(2)制得的乳化产物Ⅰ中,均质化,得到乳化产物Ⅱ;
(4)将步骤(3)制得的乳化产物Ⅱ低速离心,取上层液体,即得壳聚糖纳米级超声造影剂。
进一步优选的,步骤(1)所述壳聚糖溶液中壳聚糖的浓度为1~3g/L;最优选的,步骤(1)中所述壳聚糖溶液中壳聚糖的浓度为2g/L,其中,壳聚糖的分子量为160kDa,购自sigma,产品编号MFCD00161512。
进一步优选的,步骤(2)中所述全氟己烷和吐温20的体积比为(24~26):1;所述卵磷脂在悬液中的浓度为1~3g/L;所述卵磷脂和吐温20的质量体积比为(0.5~1):1,单位是g/mL,其中全氟己烷纯度≥98%,购自macklin,产品编号355-42-0;卵磷脂纯度≥99%,购自sigma,产品编号232-715-0;吐温20纯度≥96%,购自solarbio,产品编号82LL0422。
最优选的,步骤(2)中所述全氟己烷和吐温20的体积比为25:1,所述卵磷脂在悬液中的浓度为1.5g/L;所述卵磷脂和吐温20的质量体积比为0.7:1,单位是g/mL。
进一步优选的,步骤(2)中所述均质的速度为18000~20000rpm,时间为1~3min;最优选的,步骤(2)中所述均质的速度为19000rpm,时间为1min。
进一步优选的,步骤(3)中所述均质的速度为13000~15000rpm,时间为1~3min;最优选的,步骤(3)中所述均质的速度为14000rpm,时间为2min。
进一步优选的,步骤(4)中所述低速离心的速度为500~800rpm,时间为1~3min;最优选的,步骤(4)中所述低速离心的速度为500rpm,时间为3min。
根据本发明优选的,所述壳聚糖纳米级超声造影剂的粒径为387.85±36.87nm,Zeta电位为+44.98mV。
本发明制备的壳聚糖纳米级超声造影剂置于4℃保存。
根据本发明优选的,所述壳聚糖纳米级超声造影剂与Dickkopf-2基因连接是将壳聚糖纳米级超声造影剂与Dickkopf-2基因室温下孵育30~50min。
根据本发明优选的,所述治疗前列腺癌药物中含有有效剂量的壳聚糖纳米级超声造影剂/Dickkopf-2基因复合物。
本发明的技术特点:
Wnt信号通路异常或不恰当的活化与肿瘤干细胞的形成以及肿瘤的发生、发展密切相关。Dickkopf蛋白家族是Wnt信号通路的细胞外拮抗剂,在脊椎动物中存在4个成员,分别是Dickkopf-1、Dickkopf-2、Dickkopf-3、Dickkopf-4(Dkk-1、Dkk-2、Dkk-3、Dkk-4)。本发明的前期研究意外发现,在前列腺癌患者组织中Dkk-2的表达降低。
体积分数为1%的乙酸溶液有助于壳聚糖的溶解,壳聚糖溶解后有利于孵育产物的制备,将全氟己烷、吐温20和卵磷脂按比例混合采用微乳液法,得到以壳聚糖为外壳、以全氟己烷为内核的纳米级超声造影剂,经离心后,粒径较大的超声造影剂分布在下层,粒径相对较小的超声造影剂分布在上层,取上层液,得到粒径较小且均匀的纳米级超声造影剂。
本发明制备的壳聚糖纳米级超声造影剂带有正电荷,Dickkopf-2基因带有负电荷,壳聚糖纳米级超声造影剂和Dickkopf-2基因通过静电吸引相互连接,得到壳聚糖纳米级超声造影剂/Dickkopf-2基因复合物。
有益效果:
1、本发明首次发现了Dickkopf-2基因与前列腺癌的关系,在前列腺癌患者组织中Dickkopf-2蛋白的表达明显降低,以壳聚糖纳米级超声造影剂为传递载体,将Dickkopf-2基因定向输送到肿瘤组织中,通过Dickkopf-2基因的表达抑制肿瘤细胞的增殖。
2、纳米级超声造影剂作为基因载体,可以携载目的基因选择性地到达肿瘤组织,可实现肿瘤的精准化、可视化治疗,并可以有效克服传统肿瘤药物治疗过程中,靶向性差、全身毒副作用大、耐药等缺点。本发明制备的壳聚糖纳米级超声造影剂以壳聚糖为壳膜材料,安全无毒、生物相容性好、成本较低,粒径为387.85±36.87nm(聚合物分散指数PDI:0.145),在纳米级范围,能顺利通过肿瘤组织血管壁间隙,将目的基因传递至肿瘤组织。
3、本发明利用微乳液法制备壳聚糖纳米级超声造影剂,制备工艺简便,可以广泛应用。
4、本发明制备的壳聚糖纳米级超声造影剂Zeta电位为+44.98mV,可以通过静电作用吸引Dickkopf-2基因形成纳米复合物,操作简单。
5、本发明制备的壳聚糖纳米级超声造影剂可同时联合超声靶向破坏微泡(UTMD)技术显著提高基因转染效率,有利于目的基因的靶向传递。
附图说明
图1是前列腺癌患者和前列腺增生患者的前列腺组织中Dickkopf-2蛋白的相对表达情况,图中,A为前列腺癌患者和前列腺增生患者的前列腺组织中Dickkopf-2蛋白的免疫组化法检测结果,B为前列腺癌患者和前列腺增生患者的前列腺组织中Dickkopf-2蛋白的相对含量柱状图,纵坐标为蛋白相对含量,BPH为前列腺增生患者,PCa为前列腺癌患者;
图2是实施例1制备的壳聚糖纳米级超声造影剂(CND)的光学显微镜照片;
图3是实施例1制备的壳聚糖纳米级超声造影剂(CND)的透射电镜照片;
图4是实施例1制备的壳聚糖纳米级超声造影剂(CND)的扫描电镜照片;
图5是壳聚糖纳米级超声造影剂与Dickkopf-2基因复合物的琼脂糖凝胶电泳图;
图6是体外自制超声增强显像装置;图中,1为超声探头,2为壳聚糖纳米级超声造影剂,3为夹子,4为滴头;
图7是实施例1制备的壳聚糖纳米级超声造影剂(CND)的体外超声图像;
图8是不同条件下前列腺癌细胞的细胞存活率曲线;
图9是壳聚糖纳米级超声造影剂与Dickkopf-2基因复合物转染后前列腺癌细胞荧光强度柱状图;图中,纵坐标为荧光强度;
图10是壳聚糖纳米级超声造影剂与Dickkopf-2基因复合物转染后前列腺癌细胞的增殖情况柱状图;图中,纵坐标为新增殖细胞占总细胞数量的百分比。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步说明,但是本发明的保护范围并不仅限于此。
实施例中涉及的原料及试剂,若无特殊说明,均为普通市售产品。
实施例中pDNA是将Dickkopf-2基因(GenBank:NM_014421.3)插入到质粒载体中构建得到的重组质粒载体,购自上海博尚生物技术有限公司。
在本发明的前期研究中,选取2017年1月至2018年12月在山东大学齐鲁医院泌尿外科住院患者共40例(前列腺癌患者20例,前列腺增生患者20例)。每个病例的前列腺组织病理诊断均由两名病理学专家确认,且所有患者均未接受术前辅助治疗。
对所得前列腺组织标本应用免疫组化法检测Dickkopf-2蛋白的相对含量,检测结果如图1所示,前列腺增生患者的前列腺组织中Dickkopf-2蛋白的相对含量远高于前列腺癌患者的前列腺组织,应用统计学分析发现前列腺癌患者的前列腺组织中Dickkopf-2蛋白的相对含量与前列腺增生患者的前列腺组织中Dickkopf-2蛋白的相对含量之间差异显著(p<0.01),所有前列腺癌患者的前列腺组织中Dickkopf-2基因的表达均明显降低。
实施例1:壳聚糖纳米级超声造影剂的制备
壳聚糖纳米级超声造影剂的制备,步骤如下:
(1)将50μL乙酸加入到5mL去离子水中,上下颠倒混匀,配制体积分数为1%的乙酸溶液,将壳聚糖加入到乙酸溶液中,壳聚糖溶解后制备得到壳聚糖溶液,所述壳聚糖溶液中壳聚糖的浓度为2g/L;
(2)将0.15mL全氟己烷、0.006mL吐温20和0.004g卵磷脂分散于2.7mL去离子水中制成悬液,将所述悬液在FJ2000-S高速分散均质机中均质化1min,转速为19000r/min,得到乳化产物Ⅰ;
(3)将步骤(1)制备的壳聚糖溶液逐滴加入步骤(2)制得的乳化产物Ⅰ中,高速分散均质机中均质化2min,转速为14000r/min,得到乳化产物Ⅱ;其中,壳聚糖和全氟己烷的质量体积比为30:1,单位是g/L;
(4)将步骤(3)制得的乳化产物Ⅱ500rpm离心3min,取上层液体,即得壳聚糖纳米级超声造影剂(CND)。
所述壳聚糖纳米级超声造影剂置于4℃冰箱内备用。
实施例2:壳聚糖纳米级超声造影剂(CND)的表征
1、采用光学显微镜、透射电镜及扫描电镜进行表面形貌和构成观察
用生理盐水将实施例1制备的CND进行稀释后观察,CND的光学显微镜照片如图2所示,1000×光学显微镜下CND呈圆形,粒径均一,分散均匀无聚集;CND的透射电镜照片如图3所示,透射电镜下CND呈圆形,表面光滑透亮;CND的扫描电镜照片如图4所示,扫描电镜下CND呈现规则球形。
2、粒径与电位
用纯净水将实施例1制备的CND稀释后,用动态光散射法测定其粒径并使用激光粒径仪检测Zeta电位,结果显示CND的粒径为387.85±36.87nm,聚合物分散指数PDI:0.145,Zeta电位为+44.98mV。
实施例3:壳聚糖纳米级超声造影剂(CND)的基因结合及保护能力评价
将实施例1制备的CND与质粒DNA(pDNA)按不同质量比室温下混合孵育30分钟制备CND/pDNA复合物,其中pDNA与CND的质量比分别为1:1,1:5,1:10,1:20,pDNA质量均为1μg。
检测上述CND/pDNA复合物的粒径及Zeta电位,结果如表1所示。
表1.不同质量比的CND/pDNA复合物的粒径及Zeta电位
制备得到的CND/pDNA复合物中,pDNA添加比例越大,CND/pDNA复合物的粒径越大,说明pDNA的添加量越多,CND表面静电结合的pDNA越多;另外,随着pDNA添加量的增加,CND/pDNA复合物表面电位逐渐下降,主要是因为CND带正电荷,pDNA带负电荷,两者通过静电吸引相互结合,两者的电荷相互中和,导致了CND/pDNA复合物表面电位的降低。
将上述CND/pDNA复合物加入1%的琼脂糖凝胶点样孔中,电泳槽中加入适量1×TAE缓冲液进行凝胶电泳分析(100V,30min),然后用紫外透照仪观察凝胶;另外,将上述CND/pDNA复合物与0.5U DNase I在37℃孵育30min,按照上述步骤进行凝胶电泳分析,结果如图5所示,未与DNase I孵育反应的CND/pDNA复合物均没有pDNA目的条带,pDNA被完全阻滞在凝胶孔中;而与DNase I孵育反应后,pDNA泳道中没有条带,说明pDNA被DNase I完全酶解,CND/pDNA复合物的凝胶孔中仍显示有pDNA的存在,说明CND可在一定程度上保护pDNA免受DNase I的降解,表明CND可以作为一种较好的基因保护载体。
实施例4:壳聚糖纳米级超声造影剂(CND)的体外超声造影实验
利用5mL滴管观察实施例1制备的CND的增强显像能力,操作装置如图6所示,在滴头4中加满壳聚糖纳米级超声造影剂2,并用夹子3夹闭,置于37℃水浴中,采用临床诊断超声仪(LOGIQ E9;GE,USA)的超声探头1检测超声显影能力,主要参数为:频率9.0MHz;机械指数:0.5;焦距:3.0cm;动态范围:60dB,二维与超声造影模式同步观察,参数设置保持不变,体外超声造影结果如图7所示,与滴头中为水的对照组相比,37℃水浴下,该壳聚糖纳米级超声造影剂有较强的超声显影能力。
实施例5:壳聚糖纳米级超声造影剂(CND)的生物安全性检测
将LNCaP细胞系(购自ATCC公司)接种于96孔板中,细胞密度为1.0×104个/孔,37℃、5%CO2孵箱中培养过夜后,将培养基更换为含有实施例1制备的壳聚糖纳米级超声造影剂的新鲜10%FBS 1640培养基,壳聚糖纳米级超声造影剂在培养基中的浓度分别为0、0.2、0.4、0.6mg/mL,然后给予超声辐照,根据超声辐照处理方式的不同,可将细胞分为四组,其处理方式如表2所示。给予超声辐照后,37℃、5%CO2孵育箱中培养48h,然后用PBS洗涤细胞,加入含有10μL CCK-8试剂的新鲜10%FBS 1640培养基,继续孵育1h,用酶标仪在480nm波长处检测其光吸收值,用CCK-8法测定CND的细胞毒性。
表2.不同的超声辐照及不同壳聚糖载体浓度处理方式
检测结果如图8所示,当壳聚糖纳米级超声造影剂的浓度≤0.4mg/mL,辐照强度为0.5W/cm2时,细胞的存活率均在80%以上,说明壳聚糖纳米级超声造影剂对LNCaP细胞没有明显的细胞毒性;当将辐照强度提高至1.0W/cm2时,壳聚糖纳米级超声造影剂的浓度≤0.2mg/mL,细胞的存活率均在80%以上,说明壳聚糖纳米级超声造影剂对LNCaP细胞亦没有明显的细胞毒性,以上结果表明在一定的超声强度范围之内,该壳聚糖纳米级超声造影剂细胞毒性小,生物安全性好。
实施例6:CND和Dickkopf-2基因复合物(CND/pDNA复合物)转染前列腺癌细胞(LNCaP)体外实验
将LNCaP细胞接种于6孔板中,在37℃、5%CO2孵箱中培养过夜。将绿色荧光蛋白基因插入到质粒DNA(pDNA)中,将含绿色荧光蛋白的质粒DNA(pDNA)与实施例1制备的CND(按照质量比1:20)室温下孵育30分钟,得到CND/pDNA复合物。将LNCaP细胞分为四组,分别为pDNA组、pDNA超声组、CND/pDNA组、CND/pDNA超声组,用1mL无血清培养基稀释CND/pDNA复合物或pDNA后加入上述6孔板中,在37℃、5%CO2孵箱中培养60分钟。随后pDNA超声组和CND/pDNA超声组进行超声辐照处理(1MHz,1W/cm2,占空比50%)1分钟。超声辐照后,用正常培养基替代无血清培养基。37℃、5%CO2孵箱中培养48小时后,用荧光显微镜定性评价体外转染效率,并通过ImageJ软件进行分析。结果如图9所示,CND/pDNA组、CND/pDNA超声组的荧光强度远高于pDNA组和pDNA超声组,CND具有一定的Dickkopf-2基因转染能力,而携载Dickkopf-2基因的CND在超声辐照的作用下基因转染效率更高。
实施例7:CND和Dickkopf-2基因复合物(CND/pDNA复合物)转染LNCaP细胞后对细胞增殖的影响
将LNCaP细胞接种于96孔板中,在37℃、5%CO2孵箱中培养过夜。将质粒DNA(pDNA)与实施例1制备的CND(按照质量比1:20)室温下孵育30分钟,得到CND/pDNA复合物。将LNCaP细胞分为四组,分别为pDNA组、pDNA超声组、CND/pDNA组、CND/pDNA超声组,用1mL无血清培养基稀释CND/pDNA复合物或pDNA后加入上述96孔板中,在37℃、5%CO2孵箱中培养60分钟。随后pDNA超声组和CND/pDNA超声组进行超声辐照处理(1MHz,1W/cm2,占空比50%)3分钟。超声辐照后,用正常培养基替代无血清培养基。37℃、5%CO2孵箱中培养48小时后,用EdU细胞增殖检测试剂盒观察各组细胞增殖情况,结果如图10所示,CND/pDNA复合物转染LNCaP细胞后,细胞增殖受到抑制,而且CND/pDNA复合物在超声辐照的作用下抑制效率更高,说明壳聚糖纳米级超声造影剂可联合超声靶向破坏微泡(UTMD)技术显著提高基因转染效率,并显著抑制癌细胞的增殖。
Claims (9)
1.壳聚糖纳米级超声造影剂联合Dickkopf-2基因在制备治疗前列腺癌药物中的应用,其特征在于,所述应用是将壳聚糖纳米级超声造影剂与Dickkopf-2基因连接制备得到壳聚糖纳米级超声造影剂/Dickkopf-2基因复合物,并将复合物应用于治疗前列腺癌药物的制备;
其中,所述壳聚糖纳米级超声造影剂是由全氟己烷内核和壳聚糖外壳构成,所述壳聚糖和全氟己烷的质量体积比为(25~35):1,单位是g/L;
所述壳聚糖纳米级超声造影剂是按照以下步骤制备得到的:
(1)配制体积分数为1%乙酸溶液,将壳聚糖加入到乙酸溶液中,溶解后制备得到壳聚糖溶液;
所述壳聚糖溶液中壳聚糖的浓度为1~3g/L;
(2)将全氟己烷、吐温20和卵磷脂分散于去离子水中制成悬液,均质化,得到乳化产物Ⅰ;
所述卵磷脂在悬液中的浓度为1~3g/L;所述卵磷脂和吐温20的质量体积比为(0.5~1):1,单位是g/mL;
(3)将步骤(1)制得的壳聚糖溶液逐滴加入到步骤(2)制得的乳化产物Ⅰ中,均质化,得到乳化产物Ⅱ;
(4)将步骤(3)制得的乳化产物Ⅱ低速离心,取上层液体,即得壳聚糖纳米级超声造影剂。
2.如权利要求1所述的应用,其特征在于,步骤(1)所述壳聚糖的分子量为160kDa。
3.如权利要求1所述的应用,其特征在于,步骤(2)中所述全氟己烷和吐温20的体积比为(24~26):1;其中,全氟己烷纯度≥98%,卵磷脂纯度≥99%,吐温20纯度≥96%。
4.如权利要求1所述的应用,其特征在于,步骤(2)中所述均质的速度为18000~20000rpm,时间为1~3min。
5.如权利要求1所述的应用,其特征在于,步骤(3)中所述均质的速度为13000~15000rpm,时间为1~3min。
6.如权利要求1所述的应用,其特征在于,步骤(4)中所述低速离心的速度为500~800rpm,时间为1~3min。
7.如权利要求1所述的应用,其特征在于,所述壳聚糖纳米级超声造影剂的粒径为387.85±36.87nm,Zeta电位为+44.98mV。
8.如权利要求1所述的应用,其特征在于,所述壳聚糖纳米级超声造影剂与Dickkopf-2基因连接是将壳聚糖纳米级超声造影剂与Dickkopf-2基因室温下孵育30~50min。
9.如权利要求1所述的应用,其特征在于,所述治疗前列腺癌药物中含有有效剂量的壳聚糖纳米级超声造影剂/Dickkopf-2基因复合物。
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