CN111956807A - 马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究 - Google Patents
马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究 Download PDFInfo
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Abstract
本发明公开了一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,发明中涉及马齿苋多糖(POP)作为主链,高碘酸钠(NaIO4),阿仑膦酸钠(ALN),通过选择性氧化、席夫碱反应制得马齿苋多糖‑阿仑膦酸钠偶联物,最后马齿苋多糖‑阿仑膦酸钠(POP‑ALN)偶联物和顺铂的配合物共价键合制得最终产物(POP‑ALN‑CIS‑Pt),通过MTT法测定细胞半致死数(IC50),细胞免疫荧光染色法测定其免疫作用,本发明制备的POP‑ALN‑CIS‑Pt偶联物合成条件温和,制备过程容易重复,能抑制宫颈癌细胞的生长,对其还有免疫调节作用,在肿瘤协同治疗中具有广阔的应用前景。
Description
技术领域
本发明属于抗癌药物制备技术领域,涉及一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究。
背景技术
近年来癌症依然是危害人类生命和健康最严重的疾病之一,且恶性肿瘤的发病死亡率均呈持续上升态势,化学药物疗法是目前肿瘤治疗的主要手段之一,与其它的治疗方案相比,化疗是一种全身性的治疗方案且能够最大程度地杀死肿瘤细胞,因此,化疗在抗肿瘤治疗中占有非常重要的比重,常规的化疗药物具有明显的毒副作用,所以新的治疗方案被不断发现和改进,比如,化学治疗和免疫治疗相结合是利用机体自身的免疫系统增强药物的抗肿瘤效果,还可以降低毒副作用,铂类的化疗药物毒副作用比较严重,限制其临床应用,因此,以多糖为载体合成低毒性的多糖复合物受到关注。
多糖作为生物大分子,具有稳定、低毒、良好的生物相容性和生物降解性和可以修饰的化学基团等优点,作为载体材料进行功能化修饰,制备复合的多糖药物,马齿苋是一年生的植物,拥有悠久的入药与食用历史,属于传统中药,能清热解毒,可用作退烧药、杀菌剂等,马齿苋多糖(purslane polysaccharide,POP)是从马齿苋中提取的主要有效成分,属于活性多糖中植物多糖的一种,具有多种生物活性,如增强免疫力、抗氧化、抗癌等生物活性,研究发现,两种不同分子量的马齿苋多糖都具有抗肿瘤活性和增强免疫力的作用,所以,协同治疗药物用于癌症治疗更受到青睐,因此,构造新的含铂金属药物,提高抗肿瘤治疗效果,以马齿苋多糖为主链,通过功能化修饰共价键和制成多糖复合药物,在联合治疗肿瘤方面有着广阔的应用前景。
发明内容
为解决上述问题,本发明将马齿苋多糖、阿仑膦酸钠和顺铂结合,设计制备了一种马齿苋多糖与顺铂复合物并研究抗肿瘤活性和免疫活性。
马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于该方法包括步骤如下:
步骤1,按照马齿苋多糖与高碘酸钠的质量比为1:(1~1.5)各自溶解在水溶液中,在反应温度为25~50℃及磁力搅拌器下搅拌25~50h的情况下,溶液的颜色逐渐由红棕色变为浅黄色,反应充分后,透析得到含醛基的马齿苋多糖;
步骤2,测定马齿苋多糖中醛基的含量,按照醛基与阿仑膦酸钠的物质的量比为1:1(0.5g:0.623g)分别溶于HAc/NaAc(pH=4~6)缓冲溶液中,反应温度为25~50℃,搅拌48h,透析得到POP-ALN偶联物,然后冷冻真空干燥;
步骤3,将180mg的马齿苋多糖-阿仑膦酸钠偶联物和45mg的顺铂配合物混合,在温度范围为25~40℃,搅拌反应48h,透析冷冻干燥得POP-ALN-CIS-Pt偶联物,制备过程以式一所示:
式一
POP-ALN-CIS-Pt偶联物抗肿瘤活性和免疫活性研究方法如下:
将人正常肝细胞(LO-2)和人宫颈癌细胞(HeLa)分别加入含100μL的1640培养液和DMEM培养液,在37℃恒温培养箱中孵育贴壁生长24h,各自加入100μL不同浓度梯度的样品溶液,48h后加入MTT(20μL,5mg/mL),放入恒温培养箱继续培养4h,待细胞沉淀为紫色,除去上清液,用200µL二甲基亚砜溶液溶解紫色沉淀,摇床摇匀后测定吸光度,计算细胞IC50值。
将含HeLa的DMEM培养液加入20mm共聚焦皿中,在37℃恒温培养箱中孵育贴壁生长,当细胞密度为50-60%时,加入各样品溶液(IC50值作为各样品的最终浓度),连续培养24h,然后,弃掉上清液,固定细胞后,依次在两种抗体中孵育,用冷的PBS冲洗,用DAPI标记细胞核后,在蔡司共焦显微镜(LMS710,Germany)上采集图像,使用ZentM178软件操作处理图像。
步骤1中,马齿苋多糖和高碘酸钠的质量比为1:(1~1.5),优选1:1.2,马齿苋多糖和高碘酸钠混合后反应温度30~50℃,搅拌反应时间48h以上,优选反应温度40℃。
步骤2中,所配制的HAc/NaAc缓冲溶液的pH在4~6,优选pH为5,含醛基的马齿苋多糖HAc/NaAc缓冲溶液与阿仑膦酸钠的HAc/NaAc缓冲溶液,反应温度在40~50℃,优选反应温度50℃。
步骤3中,制备POP-ALN-CIS-Pt偶联物的反应温度25~40℃,优选反应温度25℃,细胞毒性实验中加药的时间为12~48h,优选时间为48h,抗肿瘤药物能够通过协同免疫作用增强抑制宫颈癌细胞生长。
由于采用上述技术方案,本发明所具有的优点和积极效果是:马齿苋多糖与顺铂复合物的协同抗肿瘤活性研究采用马齿苋多糖为主链,与阿仑膦酸钠和顺铂的水化分子以共价键的形式结合在一起,不需要温度很高,可以减少能量的消耗,准备方法简单,本发明的新型POP-ALN-CIS-Pt偶合物具有抑制宫颈癌细胞的生长作用,同时也具有免疫调节能力,为多糖作为药物载体材料提供了新的思路。
附图说明
下面结合附图和实施例对本发明进一步说明,本发明有如下8幅附图:
图1为本发明中POP-ALN-CIS-Pt偶联物的红外光谱图之一,
图2为本发明中POP-ALN-CIS-Pt偶联物的红外光谱图之二,
图3为本发明中POP-ALN-CIS-Pt偶联物的核磁共振谱图,
图4为本发明中POP-ALN-CIS-Pt偶联物的X射线衍射谱图,
图5为本发明中POP-ALN-CIS-Pt偶联物的扫描电子显微镜图,放大5000倍的表面形貌之一,
图6为本发明中POP-ALN-CIS-Pt偶联物的扫描电子显微镜图,放大5000倍的表面形貌之二,
图7为本发明中POP-ALN-CIS-Pt偶联物的细胞增殖图,
图8为本发明中POP-ALN-CIS-Pt偶联物的细胞免疫染色图。
具体实施方式
为了易于了解本实验的技术方案,给出下列实施例进一步描述,但是本发明的实施方式不限于此。
实施例1 POP-ALN-CIS-Pt偶联物的制备
将含1.5g的高碘酸钠的水溶液逐滴加入1.5g纯化后的马齿苋多糖水溶液中,在30℃条件下搅拌反应48h,用透析袋(MW=3500Da)透析,冷冻干燥得到含醛基的马齿苋多糖;将0.5g的含醛基的马齿苋多糖和0.623g的ALN分别溶于pH=4.0的HAc/NaAc缓冲溶液中,在30℃条件下搅拌反应48h,透析冷冻干燥得到POP-ALN偶联物;180mg的POP-ALN偶联物的水溶液和45mg的顺铂配合物溶液发生交联,氮气保护下维持25℃,搅拌反应24h,透析冷冻干燥得到POP-ALN-CIS-Pt偶联物样品。
实施例2 POP-ALN-CIS-Pt偶联物的制备
将含1.5g的高碘酸钠的水溶液逐滴加入1.8g纯化后的马齿苋多糖水溶液中,在40℃条件下搅拌反应48h,用透析袋(MW=3500Da)透析,冷冻干燥得到含醛基的马齿苋多糖;将0.5g的含醛基的马齿苋多糖和0.623g的ALN分别溶于pH=5.0的HAc/NaAc缓冲溶液中,在30℃条件下搅拌反应48h,透析冷冻干燥得到POP-ALN偶联物;180mg的POP-ALN偶联物的水溶液和45mg的顺铂配合物溶液发生交联,氮气保护下维持37℃,搅拌反应24h,透析冷冻干燥得到POP-ALN-CIS-Pt偶联物样品。
实施例3 POP-ALN-CIS-Pt偶联物的制备
将含1.5g的高碘酸钠的水溶液逐滴加入2.25g纯化后的马齿苋多糖水溶液中,在50℃条件下搅拌反应48h,用透析袋(MW=3500Da)透析,冷冻干燥得到含醛基的马齿苋多糖;将0.5g的含醛基的马齿苋多糖和0.623g的ALN分别溶于pH=5.0的HAc/NaAc缓冲溶液中,在30℃条件下搅拌反应48h,透析冷冻干燥得到POP-ALN偶联物;180mg的POP-ALN偶联物的水溶液和45mg的顺铂配合物溶液发生交联,氮气保护下维持25℃,搅拌反应24h,透析冷冻干燥得到POP-ALN-CIS-Pt偶联物样品。
实施例4 POP-ALN-CIS-Pt偶联物的制备
将含1.5g的高碘酸钠的水溶液逐滴加入1.8g纯化后的马齿苋多糖水溶液中,在40℃条件下搅拌反应48h,用透析袋(MW=3500Da)透析,冷冻干燥得到含醛基的马齿苋多糖;将0.5g的含醛基的马齿苋多糖和0.623g的ALN分别溶于pH=6.0的HAc/NaAc缓冲溶液中,在50℃条件下搅拌反应48h,透析冷冻干燥得到POP-ALN偶联物;180mg的POP-ALN偶联物的水溶液和45mg的顺铂配合物溶液发生交联,氮气保护下维持37℃,搅拌反应24h,透析冷冻干燥得到POP-ALN-CIS-Pt偶联物样品。
实施例5 POP-ALN-CIS-Pt偶联物的制备
将含1.5g的高碘酸钠的水溶液逐滴加入1.8g纯化后的马齿苋多糖水溶液中,在40℃条件下搅拌反应48h,用透析袋(MW=3500Da)透析,冷冻干燥得到含醛基的马齿苋多糖;将0.5g的含醛基的马齿苋多糖和0.623g的ALN分别溶于pH=5.0的HAc/NaAc缓冲溶液中,在50℃条件下搅拌反应48h,透析冷冻干燥得到POP-ALN偶联物;180mg的POP-ALN偶联物的水溶液和45mg的顺铂配合物溶液发生交联,氮气保护下维持25℃,搅拌反应24h,透析冷冻干燥得到POP-ALN-CIS-Pt偶联物样品。
实施例6 POP-ALN-CIS-Pt偶联物的红外光谱测试
本发明制备的POP-ALN-CIS-Pt偶联物的红外光谱测试结果,如图2所示,分别取适量样品放入玛瑙研钵中,加入相对质量比的KBr粉末混合,研磨成细粉并使用压片机压片,放入测试样品的模具中,用红外光谱仪(Bruker Vertex 80)对已压片的样品进行扫描,扫描的波数范围是4000~500cm-1,
从图1中看出位于1725cm-1处出现了醛基峰,由此可以确定马齿苋多糖成功被高碘酸钠氧化,磷酸化马齿苋多糖出现位于1152cm-1、924cm-1分别是P=O和P-O-P的特征吸收,可以看出马齿苋多糖被阿仑磷酸钠成功修饰。
实施例7 POP-ALN-CIS-Pt偶联物的核磁共振谱测试
分别取适量样品溶解于D2O,转移至核磁管中,用核磁共振光谱仪(Bruker AVANCE IIIHD,500MHz,Germany)在常温下进行测试磷谱,
如图2所示,在PPS-ALN偶联物的31PNMR谱中,ALN的P-C-P信号峰出现在18.02ppm证实了PPS-ALN的成功合成;信号约为39.10ppm,属于铂双膦酸盐中的两个磷原子的信号峰,因为磷酸盐氧原子与金属铂离子协同的作用,它们在磁场上是等价的,并转移到较低的磁场中。
实施例8 本发明POP-ALN-CIS-Pt偶联物的X射线衍射谱图
取适量样品,用研钵研碎,转移至测试的载玻片上,放入测试样品的仪器,采用粉末X射线衍射仪(Malvern Panalytical X'Pert3)对样品进行了分析,设置仪器扫描范围为5~80°(2θ)。
如图3所示,POP在2θ≈19.72°处只有较宽的一个衍射峰,表明PPS是具有典型非晶体材料性质的粉末衍射图样,先前的研究表明,具有非晶态性质的多糖更稳定,POP-ALN与POP-ALN-CIS-Pt偶合物的衍射图谱没有明显的峰,呈弥散状态,表明改性POP的非晶相增多,结果表明,改性后的多糖分子不仅发生了化学结构的变化,而且结构的交联导致了非晶态。
实施例9 本发明POP-ALN-CIS-Pt偶联物的表面形貌测试
取适量样品放在导电胶带上,镀金,采用扫描电子显微镜(Nova Nano SEM 450)对样品的微观形貌观察。
如图4所示,图中是POP和POP-ALN-CIS-Pt偶联物放大5000×的表面形貌,本发明使用的POP表面结构是片状结构,POP-ALN-CIS-Pt偶联物表面结构是不规则的碎片,还有部分是圆状的颗粒。
实施例10 本发明POP-ALN-CIS-Pt偶联物的细胞增殖图,
将人正常肝细胞(LO-2)和人宫颈癌细胞(HeLa)分别加入含100μL的1640培养基和DMEM培养基在96孔板中,在37℃恒温培养箱中孵育贴壁生长24h,每孔分别加入浓度梯度在0~1000ug/mL的样品溶液,处理48h后加入MTT(20μL,5mg/mL),放入恒温培养箱继续培养4h,待细胞沉淀为紫色,除去上清液,用200µL二甲基亚砜溶液溶解紫色沉淀后测定吸光度,立即用酶标仪测定490nm(OD490)的吸光度。
经SPSS软件处理数据,计算得出细胞最大半致死数IC50,通过绘制图形,能够进行详细的比较。
如图5所示,POP对2株细胞几乎无毒性,而POP-ALN、POP-ALN-CIS-Pt的细胞毒性由IC50的值依次下降来证实,说明修饰后的产物有一定的细胞毒性,而且,ALN对HeLa有明显的影响。
实施例11 本发明POP-ALN-CIS-Pt偶联物的细胞免疫染色图
免疫荧光法检测钙网蛋白在HeLa细胞中的分布,将含HeLa的DMEM培养基加入20mm共聚焦皿中,在37℃恒温培养箱中孵育贴壁生长,当细胞密度为50-60%时,加入各样品溶液(IC50值作为各样品的最终浓度),连续培养24h。
然后,从20mm激光共聚焦培养皿中取出培养液,用PBS清洗,并用4%聚甲醛覆盖20min,在PBS中清洗三次,每次5min,用0.1%TritonX-100渗透10min,在PBS中清洗三次,每次5min。
最后,5%牛血清白蛋白阻断1h,孵育1/500抗钙网蛋白抗体,在第一次抗孵育后,样品在PBS中冲洗三次5min,随后,携带荧光标记物的山羊抗兔IgG抗体(AlexaFluor®488)培养1h,并应用DAPI标记细胞核,在蔡司共焦显微镜(LMS710,Germany)上采集图像,使用ZentM178软件操作处理图像,
如图6所示,用免疫荧光法研究钙网蛋白在HeLa细胞中的表达,DAPI染色(蓝色荧光)辅助定位细胞核,对照组HeLa细胞的抗体标记CRT(绿色荧光)的表达,POP、POP-ALN和POP-ALN-CIS-Pt偶联物处理HeLa细胞后,其CRT表达量依次增加,因此,POP-ALN-CIS-Pt偶联物在调节肿瘤细胞免疫活性方面具有一定的作用。
Claims (8)
1.一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于该方法包括步骤如下:
步骤1,按照马齿苋多糖与高碘酸钠的质量比为1:(1~1.5)各自溶解在水溶液中,在反应温度为25~50℃及磁力搅拌器下搅拌25~50h的情况下,溶液的颜色逐渐由红棕色变为浅黄色,反应充分后,透析得到含醛基的马齿苋多糖;
步骤2,测定马齿苋多糖中醛基的含量,按照醛基与阿仑膦酸钠的物质的量比为1:1(0.5g:0.623g)分别溶于HAc/NaAc(pH=4~6)缓冲溶液中,反应温度为25~50℃,搅拌48h,透析得到POP-ALN偶联物,然后冷冻真空干燥;
步骤3,将180mg的马齿苋多糖-阿仑膦酸钠偶联物和45mg的顺铂配合物混合,在温度范围为25~40℃,搅拌反应48h,透析冷冻干燥得POP-ALN-CIS-Pt偶联物,制备过程以式一所示:
式一
POP-ALN-CIS-Pt偶联物抗肿瘤活性和免疫活性研究方法如下:
将人正常肝细胞(LO-2)和人宫颈癌细胞(HeLa)分别加入含100μL的1640培养液和DMEM培养液,在37℃恒温培养箱中孵育贴壁生长24h,各自加入100μL不同浓度梯度的样品溶液,48h后加入MTT(20μL,5mg/mL),放入恒温培养箱继续培养4h,待细胞沉淀为紫色,除去上清液,用200µL二甲基亚砜溶液溶解紫色沉淀,摇床摇匀后测定吸光度,计算细胞IC50值;
将含HeLa的DMEM培养液加入20mm共聚焦皿中,在37℃恒温培养箱中孵育贴壁生长,当细胞密度为50-60%时,加入各样品溶液(IC50值作为各样品的最终浓度),连续培养24h,然后,弃掉上清液,固定细胞后,依次在两种抗体中孵育,用冷的PBS冲洗,用DAPI标记细胞核后,在蔡司共焦显微镜(LMS710,Germany)上采集图像,使用ZentM178软件操作处理图像。
2.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:步骤1中,马齿苋多糖和高碘酸钠的质量比为1:(1~1.5)。
3.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:步骤1中,马齿苋多糖和高碘酸钠混合后反应温度为25~50℃。
4.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于,步骤2中,所配制的HAc/NaAc缓冲溶液的pH在4~6。
5.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:步骤2中,含醛基的马齿苋多糖HAc/NaAc缓冲溶液与阿仑膦酸钠的HAc/NaAc缓冲溶液,反应温度在30~50℃。
6.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:步骤3中,制备POP-ALN-CIS-Pt偶联物的反应温度25~40℃。
7.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:细胞毒性实验中加药的时间为12~48h。
8.根据权利要求1所述的一种马齿苋多糖与顺铂复合物的制备及其协同抗肿瘤活性研究,其特征在于:所加入的多糖复合药物能够对人宫颈癌细胞产生免疫作用。
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CN115920076B (zh) * | 2022-11-07 | 2024-05-31 | 盐城师范学院 | 一种马齿苋多糖-去甲基斑蝥素-甲氨蝶呤偶联物及其制备方法和应用 |
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