CN112353950A - 一种siRNA纳米递送系统的制备方法及其在前列腺癌中的应用 - Google Patents
一种siRNA纳米递送系统的制备方法及其在前列腺癌中的应用 Download PDFInfo
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Abstract
本发明公开了一种新型siRNA纳米递送系统的制备及其在前列腺癌中的应用。制备方法是将靶向skp2的siRNA与天然黄酮类药物槲皮素通过氢键,π‑π作用,疏水作用组装形成纳米粒子。本发明可将槲皮素和siRNA同时携带进入前列腺肿瘤细胞,二者协同促进skp2蛋白下调,抑制前列腺癌细胞增殖迁移。本发明通过分子水平及体外活性评价,证明了该系统优于同时输送各单一组分,能够显著提高其抗癌活性,具有明确的协同治疗效果。本发明抗肿瘤效果明确,所用材料均有抗肿瘤效果,且具有良好生物相容性、可生物降解性、无毒性。制备工艺简单,易于操作。
Description
技术领域
本发明属于纳米药物领域,具体地涉及以槲皮素为载体递送siRNA的制备方法及其在前列腺癌中的应用。
背景技术
siRNA可以选择性下调靶基因,在多种疾病治疗中具有广泛的应用前景。然而,由于siRNA不易递送,降低了其治疗效果。因此,在基于siRNA治疗的临床转化中,一个好的递送系统起到了重要的作用。目前,阳离子聚合物及脂质体是siRNA递送主要的载体。然而,这些载体由于带正电荷,易产生潜在毒性,限制了其在临床上的应用。除此之外,阳离子纳米复合物会吸附血浆蛋白,导致其易被网状内皮系统清除。因此,急需开发一种新型的siRNA递送系统。
槲皮素是一种黄酮醇类化合物,具有抗氧化,抗癌的作用。其可以通过促进肿瘤细胞凋亡和细胞周期停滞而抑制肿瘤细胞增殖。有研究表明,药物之间可以通过亲疏水作用和氢键作用进行自组装形成纳米颗粒,协同发挥抗癌效果。受此启发,本发明设计利用槲皮素(Quercetin,Que)与siRNA 通过π-π作用、亲疏水作用和氢键作用自组装形成纳米药物,递送siRNA。该siRNA纳米粒子中的槲皮素不仅可作为载体,也具有抗癌活性,与siRNA共同发挥双重抗肿瘤效果。该发明以药物作为载体进行制备siRNA递送系统,实现“ 零载体”概念。
发明内容
本发明的目的是提供一种新型siRNA纳米递送系统的制备及其在前列腺癌中的应用。本发明新型siRNA纳米粒子是利用靶向skp2的siRNA与具有抗肿瘤效果的天然黄酮类药物槲皮素通过氢键,π-π作用,疏水作用,组装形成纳米粒子。本发明可将槲皮素和siRNA同时携带进入前列腺肿瘤细胞,二者协同促进skp2蛋白下调,抑制前列腺癌细胞增殖迁移。siRNA序列:sense5'-3':GGAGUGACAAAGACUUUGU,antisense5'-3':ACAAAGUCUUUGUCACUCC。
为实现上述目的,本发明采用如下技术方案:
一种新型siRNA纳米粒子的制备方法,所述新型siRNA纳米粒子的制备方法包括以下步骤:
1)在无菌操作台中将 5 nmol siRNA 干粉溶于25 ul DMSO配成 200 uM,0.0035 g槲皮素粉末(Quercetin,Que)在1158.12 ul DMSO 中充分溶解,配制成10 mM(浓度);
2) 取步骤1)槲皮素母液1 ul与10 ul siRNA 溶液充分混匀,轻微震荡 10 s,再加入89 ul DEPC 水充分混匀;
3)将步骤2)所得溶液 置于金属浴中 95℃中反应 5 min,缓慢降至室温;
4) 将步骤3)所得溶液用透析管(100kDa)透析24h,换4次DEPC水,除去未反应的槲皮素与siRNA,即得槲皮素与siRNA自组装纳米粒子,置于 4℃ 保存备用。
本发明的有益效果在于:本发明设计利用槲皮素(Quercetin,Que)与siRNA 通过π-π作用、亲疏水作用和氢键作用自组装形成纳米药物,递送siRNA。该siRNA纳米粒子中的槲皮素不仅可作为载体,也具有抗癌活性,与siRNA共同发挥双重抗肿瘤效果。该发明以药物作为载体进行制备siRNA递送系统,实现“ 零载体”概念。
附图说明
图1为本发明新型siRNA纳米递送系统的透射电镜图。
图2为前列腺癌细胞对本发明siRNA纳米递送系统或游离siRNA的摄取图。
图3为槲皮素(Que)或同时单独加槲皮素与游离siRNA(siSkp2+Que)或对照siRNA纳米递送系统(siNC/Que NPs)或本发明skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞的skp2靶蛋白的抑制效果。
图4为同时单独加槲皮素与游离siRNA(siSkp2+Que)或对照siRNA纳米递送系统(siNC/Que NPs)或skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞生长的抑制效果。
图5为本发明同时单独加槲皮素与游离siRNA(siSkp2+Que)或对照siRNA纳米递送系统(siNC/Que NPs)或skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞迁移的抑制效果。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1 新型siRNA纳米粒子的制备:
在无菌操作台中将 5 nmol siRNA 干粉溶于25 ul DMSO配成 200 uM,0.0035g槲皮素粉末(Quercetin,Que)在1158.12 ul DMSO 中充分溶解,配制成10mM (浓度);槲皮素母液1ul与10 ul siRNA 溶液充分混匀,轻微震荡 10 s,再加入89 ul DEPC 水充分混匀;将所得溶液置于金属浴中 95℃中反应 5 min,缓慢降至室温;用透析管(100kDa)透析24h,换4次DEPC水,除去未反应的槲皮素与siRNA,即得槲皮素与siRNA自组装纳米粒子,置于 4℃ 保存备用。
图1为所得新型siRNA纳米递送系统的TEM图。图中可以看出其为单分散、平均粒径77.4 nm的球形纳米粒子。
实施例2 前列腺细胞对本发明siRNA纳米递送系统与游离siRNA的摄取能力考察
1. 制备Cy5-siSkp2和Cy5-siSkp2/Que NPs:
为确定siRNA纳米递送系统是否可以有效促进siRNA被细胞摄取,设计siRNA的antisense链5’端修饰Cy5荧光分子,从上海吉玛制药技术有限公司订购。用标记有Cy5荧光分子的siRNA(Cy5-siSkp2)按实施例1合成新型siRNA纳米粒子(Cy5-siSkp2/Que NPs)。
2. Cy5-siSkp2和Cy5-siSkp2/Que NPs的细胞摄取实验:
将培养的前列腺癌细胞用 0.05%胰酶消化,然后重悬在含有终浓度10%FBS, 1%双抗的1640 完全培养基中并计数;将均匀分散好的细胞接种在共聚焦皿中, 3 ×104 个细胞/皿;在含 5% CO2 的培养箱中孵育 24 h,然后将 Cy5-siRNA/Que NPs 或游离Cy5-siRNA分散在 500 μL 培养基溶液并加到相应的共聚焦皿中,轻柔混合均匀后继续在培养箱中孵育 4 h;吸出培养基,用无菌 PBS 清洗 3 次;加入 Hoechst 33342(10 μg/mL)染液,用于细胞核的染色; 15 min 后吸出染液,并用无菌 PBS 轻柔洗 3 次,使用激光共聚焦显微镜系统采集荧光图像。
图2 为前列腺癌细胞H33342对本发明siRNA纳米递送系统或游离siRNA的摄取图:结果显示与游离的 Cy5-siSkp 2 相比,细胞浆中 Cy5-siSkp2/Que NPs 的荧光更强, 说明siSkp2 与 Que NPs 形成纳米递送系统后更易被细胞摄取, 有利于 siSkp2 发挥基因沉默的作用。
实施例3 本发明新型siRNA纳米递送系统对前列腺肿瘤细胞的skp2靶蛋白的抑制效果考察。
1.游离siRNA与槲皮素混合组(siSkp2+Que),对照siRNA纳米递送系统组(siNC/Que NPs)siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)的制备:
为比较siRNA纳米递送系统是否可以有效递送siRNA,提高siRNA的治疗效果。设计游离siRNA与槲皮素混合组(siSkp2+Que),对照siRNA纳米递送系统组(siNC/Que NPs)作为对照。siSkp2+Que:游离的siRNA与培养液混合加样,再加入槲皮素。siNC/Que NPs:从上海吉玛制药技术有限公司订购作为随机对照的无作用的siRNA,命名为siNC,序列为:sense(5'-3'):UUCUCCGAACGUGUCACGUTT,antisense(5'-3'):ACGUGACACGUUCGGAGA ATT。按实施例1合成对照siRNA纳米粒子(siNC/Que NPs)。从上海吉玛制药技术有限公司订购skp2沉默性siRNA。siRNA序列:sense(5'-3'):GGAGUGACAAAGACUUUGU,antisense(5'-3'):ACAAAGUCUUUGUCACUCC。按实施例1所述方法制备siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)
2.蛋白质印迹实验
1)将细胞接种至 6孔板中培养,细胞贴壁后加入不同浓度槲皮素,以及siSkp2+Que,siNC/Que NPs,siSkp2/Que NPs。另外,设置一组空白对照组(control组)即不加任何药物处理,其他操作相同的组别。药物与细胞孵育30h后,用细胞刮刀收取细胞;按照 100:1 的比例加入 RIPA(裂解液) :PMSF(蛋白酶抑制剂);用BCA 试剂盒测定所提取蛋白的浓度。
2)蛋白变性: 取提好的蛋白上清于新的 1.5 mL 离心管中,加入 5× loadingbuffer混匀(二者体积比为 4:1);上述离心管夹好防爆夹后,置于 100℃沸水煮 10 min,接着冷却至室温,放于-20℃保存备用;
3)制胶:先制分离胶,加入 200 μL 异丙醇封顶,凝胶洗净,再制浓缩胶;上样:按Marker 4 μL,Tubulin 5 μg,目的蛋白skp2 25 μg 的上样量上样,先在 90 V 电压下电泳至溴酚蓝染料从浓缩胶进入分离胶后,将电压调至120 V 继续电泳;
4)转膜: PVDF 膜先在甲醇中浸泡 30 s,再放入电转液中, 100 V 转膜 1 h(具体时间根据蛋白分子量大小调整);
5)封闭:取 PVDF 膜,蛋白面朝上, 放入 TBST 中洗 5 min,封闭 1 h,再放入 TBST洗 10 min 倒掉,重复一次;
6)抗体孵育:将封闭好的 PVDF 膜放入稀释后的一抗(skp2(D3G5)XP Rabbit mAb)中,封口室温孵育 1h 后再放于 4℃孵育过夜,后用 TBST 洗 3 次, 每次 5 min;加入二抗(Anti-rabbit lgG,HRP-linked Antibody),室温孵育 1 h,后用 TBST 洗 3 次, 每次 8min;
7)显影:将膜放入化学发光成像仪中,利用 ECL 发光液进行显影并拍照。
图3A 结果显示, 与空白对照组(control组)相比,槲皮素(Que) 可以下调前列腺癌细胞中 Skp2 的蛋白表达量,但是需要达到比较高的浓度。图3B结果显示, 在前列腺癌细胞中,siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)能够提高 siRNA 沉默靶基因 Skp2 在蛋白水平表达的能力。
实施例4 本发明新型siRNA纳米递送系统对前列腺癌细胞增殖及迁移能力考察
1.游离siRNA与槲皮素混合组(siSkp2+Que),对照siRNA纳米递送系统组(siNC/QueNPs)siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)的制备
按实施例3所述方法制备游离siRNA与槲皮素混合组(siSkp2+Que),对照siRNA纳米递送系统组(siNC/Que NPs),siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)。
2. 采用实时无标记细胞分析仪记录细胞生长曲线,每2天加一次药,监测4天。考察同时单独加槲皮素与游离siRNA(siSkp2+Que)或对照siRNA纳米递送系统(siNC/QueNPs)或skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞生长的抑制效果,并与空白对照组(control组)进行对比。空白对照组即不加任何药物处理,其他操作相同的组别。
如图4结果所示,与空白对照组(control组)相比,skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞生长有明显的抑制作用,且抑制效果显著强于单独加槲皮素与游离siRNA组(siSkp2+Que)以及对照siRNA纳米递送系统组(siNC/Que NPs)。
3.采用transwell技术检测游离siRNA与槲皮素混合组(siSkp2+Que),对照siRNA纳米递送系统组(siNC/Que NPs)和siSkp2 沉默性siRNA纳米递送系统(siSkp2/Que NPs)对细胞迁移能力的影响:
将培养的前列腺癌细胞用 0.05%胰酶胰酶消化,然后重悬在含有10%胎牛血清, 1%双抗的 1640 完全培养基中并计数;将均匀分散好的细胞以8 ×104 个细胞/孔接种于transwell小室中,下室加入500 µL含有 10%胎牛血清,1%双抗的1640完全培养基,在含 5%CO2 的培养箱中孵育;待细胞贴壁后,将上室换成含有siSkp2+Que、siNC/Que NPs或siSkp2/Que NPs的无血清培养基,轻柔混合均匀后继续在培养箱中孵育 24 h;另外,设置一组空白对照组(control组)即不加任何药物处理,其他操作相同的组别。弃上下室培养液,用湿润的棉签轻柔擦拭上室内表面未迁移的细胞,用PBS洗后吸干液体;每个小室加1mL 4%多聚甲醛固定15 min,弃固定液,倒置风干;用PBS洗,再用500 µL 0.1%结晶紫染色15min,弃染色液;再用PBS洗,用棉签擦拭多余液体,晾干;显微镜观察拍照,随机选取5个细胞均匀穿膜的视野(200 X)。
由图5结果显示,与空白对照组(control组)相比,skp2沉默性siRNA纳米递送系统(siSkp2/Que NPs)对前列腺肿瘤细胞迁移能力的抑制作用显著,且明显强于单独加槲皮素与游离siRNA组(siSkp2+Que)以及对照siRNA纳米递送系统组(siNC/Que NPs)。
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。
SEQUENCE LISTING
<110> 闽江学院
<120> 一种siRNA纳米递送系统的制备方法及其在前列腺癌中的应用
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Claims (5)
1.一种siRNA纳米粒子,其特征在于,包含槲皮素和skp2沉默性siRNA;siRNA序列:sense5'-3':GGAGUGACAAAGACUUUGU,antisense5'-3':ACAAAGUCUUUGUCACUCC。
2.根据权利要求1所述的siRNA纳米粒子,其特征在于,skp2沉默性siRNA最终浓度为10μM-40μM,槲皮素浓度为50μM -200μM。
3.根据权利要求1所述的siRNA纳米粒子,其特征在于,skp2沉默性siRNA最终浓度为优选20μM;槲皮素浓度为100μM。
4.一种如权利要求1所述siRNA纳米粒子的制备方法,其特征在于,所述siRNA属纳米粒子的制备方法包括以下步骤:
1)在无菌操作台中将siRNA 干粉溶于DMSO中制备得到siRNA 溶液,槲皮素粉末在DMSO中充分溶解,得到槲皮素母液;
2) 取步骤1)槲皮素母液与siRNA 溶液充分混匀,轻微震荡 10 s,再加入到DEPC 水中,充分混匀;
3)将步骤2)所得溶液置于金属浴中 95℃中反应 5 min,缓慢降至室温;
4) 将步骤3)所得溶液用100kDa透析管透析24h,换4次水,除去未反应的槲皮素与siRNA,即得槲皮素与siRNA自组装纳米粒子,置于 4℃ 保存备用。
5.如权利要求1所述的新型siRNA纳米粒子在制备抗前列腺癌药物中的应用。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112933242A (zh) * | 2021-04-08 | 2021-06-11 | 曜迪生物技术徐州有限公司 | 儿茶酚与胶原三肽自组装纳米复合物及制备和应用 |
CN114224869A (zh) * | 2021-12-21 | 2022-03-25 | 湖南省人民医院(湖南师范大学附属第一医院) | 一种高效递送siRNA的载药纳米粒及其制备方法与应用 |
CN115721713A (zh) * | 2022-09-14 | 2023-03-03 | 福州大学 | 一种癌细胞膜包裹的siRNA-维替泊芬自组装纳米药物及其制备方法和应用 |
CN116549440A (zh) * | 2023-06-30 | 2023-08-08 | 福州大学 | 小分子化合物棘壳孢素A在抑制Skp2蛋白及抗前列腺癌中的应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060264394A1 (en) * | 2003-04-15 | 2006-11-23 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Livin-specific sirnas for the treatment of therapy-resistant tumors |
CN109847062A (zh) * | 2019-04-09 | 2019-06-07 | 中山大学 | 一种槲皮素金属纳米药物及其制备方法和应用 |
CN111481665A (zh) * | 2020-02-24 | 2020-08-04 | 中国药科大学 | 一种具有荧光分子开关特性的无载体纳米粒及其制备方法与应用 |
-
2020
- 2020-12-11 CN CN202011447637.7A patent/CN112353950B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060264394A1 (en) * | 2003-04-15 | 2006-11-23 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Livin-specific sirnas for the treatment of therapy-resistant tumors |
CN109847062A (zh) * | 2019-04-09 | 2019-06-07 | 中山大学 | 一种槲皮素金属纳米药物及其制备方法和应用 |
CN111481665A (zh) * | 2020-02-24 | 2020-08-04 | 中国药科大学 | 一种具有荧光分子开关特性的无载体纳米粒及其制备方法与应用 |
Non-Patent Citations (3)
Title |
---|
JINGNA SU ET AL.: "Curcumin inhibits cell growth and invasion and induces apoptosis through down-regulation of Skp2 in pancreatic cancer cells", 《AM J CANCER RES》 * |
LIU CHIN-CHIH: "quercetin and EGCG inhibit the growth of human prostate carcinoma cells by down-regulation of skp2 protein", 《台湾大学机构典藏》 * |
刘萍: "纳米银和黄酮相互作用研究及分析应用", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112933242A (zh) * | 2021-04-08 | 2021-06-11 | 曜迪生物技术徐州有限公司 | 儿茶酚与胶原三肽自组装纳米复合物及制备和应用 |
CN112933242B (zh) * | 2021-04-08 | 2024-02-02 | 杭州睿导基因科技有限公司 | 儿茶酚与胶原三肽自组装纳米复合物及制备和应用 |
CN114224869A (zh) * | 2021-12-21 | 2022-03-25 | 湖南省人民医院(湖南师范大学附属第一医院) | 一种高效递送siRNA的载药纳米粒及其制备方法与应用 |
CN115721713A (zh) * | 2022-09-14 | 2023-03-03 | 福州大学 | 一种癌细胞膜包裹的siRNA-维替泊芬自组装纳米药物及其制备方法和应用 |
CN116549440A (zh) * | 2023-06-30 | 2023-08-08 | 福州大学 | 小分子化合物棘壳孢素A在抑制Skp2蛋白及抗前列腺癌中的应用 |
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