CN115414337A - 一种基于血红蛋白的携氧增敏纳米药物的制备方法及应用 - Google Patents
一种基于血红蛋白的携氧增敏纳米药物的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种基于血红蛋白的携氧增敏纳米药物的制备方法及应用。本发明首先分别将熊果酸、索拉非尼、吲哚菁绿溶于良性溶剂中,分别得到溶液A、溶液B和溶液C,将溶液A、溶液B和溶液C混合得到溶液D,再将适量溶液D滴入到二次蒸馏水中,随后超声处理,再用氮吹仪吹干良性溶剂,得到纳米药物‑1水溶液,最后将红细胞悬液和纳米药物‑1水溶液按照一定比例混合,过膜,即得到所述基于血红蛋白的携氧增敏纳米药物。本发明利用红细胞中血红蛋白的携氧‑释氧功能有效改善肿瘤乏氧环境,实现多重增敏作用,制备方法绿色、简单,结合血红蛋白携氧功能有效减小传统治疗手段的抗性,提高抗肿瘤作用,具有广阔的临床应用前景。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及一种基于血红蛋白的携氧增敏纳米药物的制备方法及应用。
背景技术
2020年世界卫生组织国际癌症研究机构(IARC)的调查结果显示,2020年癌症新发病例1929万,死亡病例996万例,严重危害着人类的健康。传统的肿瘤治疗方法包括手术切除、化疗、放疗、光疗等,这些方法虽然能够一定程度上治疗肿瘤,但是依旧存在易复发、毒副作用强、易耐受等不足。例如手术切除后易复发,放疗副作用强,长时间化疗容易产生耐药性等。因此,研究者们一直致力于探索新的、毒副作用小的治疗方法,或者采用辅助手段达到增效的目的,从而提高治疗效果。
乏氧是肿瘤微环境的一大病理生理特征,与肿瘤的恶性进展、血管新生、肿瘤细胞的侵入、转移和化疗耐药和代谢变化等密切相关。肿瘤细胞在增殖过程不断浸润周围组织和细胞,形成肿瘤微环境,即成为适应肿瘤细胞生长的“土壤”。肿瘤细胞在迅速增殖过程虽然具有形成微血管网的能力,但是由于肿瘤细胞的增殖速度快,消耗大量氧气,超过了微血管网的供氧量,从而使肿瘤部位的含氧量正常组织,处于低氧状态。报道称,正常组织的氧分压约为30 mmHg,而肿瘤组织低至0 mmHg,而且距离越远氧分压越低。乏氧环境下,肿瘤细胞周期发生改变,大多停留在G1/S期,影响细胞内DNA复制转录、翻译等过程,降低靶向DNA化疗药物的疗效,如阿霉素。另一方面,还会诱导低氧诱导因子的活化和P-糖蛋白的表达,对化疗药物疗效产生严格抗性作用。除此之外,对于光动力疗法和声动力疗法等依赖于氧气产生活性氧达到肿瘤细胞杀伤作用,乏氧环境严重限制了其疗效。
为了改善肿瘤乏氧环境,研究者提出利用氧载体输送氧或者原位产氧等策略。中国专利(公开号CN112263679A,实质审查中)采用一步超声法杂交人血清白蛋白载氧血红蛋白,并实现对疏水声敏剂锰卟啉的包裹,将外源氧分子和锰卟啉靶向递送到肿瘤内部,有效改善肿瘤缺氧微环境同时提高声动力疗效。另一项专利(授权号CN105497894B已授权)发明了一种血红蛋白-光敏剂试剂(光敏剂包括吲哚菁绿、Ce6、血卟啉衍生物中的一种或多种),通过生物分子计算分析发现血红蛋白中氧气结合点与光敏剂距离近,氧气能够充分转化为活性氧,从而达到杀伤作用。虽然上述专利能够利用血红蛋白改善乏氧肿瘤微环境,提高癌症治疗效果,但是受限于血红蛋白易氧化的问题,存在潜在的肾毒性、载氧能力低、疗效降低的问题。
本专利制备一种基于血红蛋白的载氧增敏纳米药物,血红蛋白来源于哺乳动物红细胞,无须提纯血红蛋白,将红细胞和纳米粒利用纳米挤出技术得到最终纳米药物,不仅能够利用红细胞膜实现免疫清除逃逸,还能利用红细胞自身酶保护血红蛋白维持其载氧能力,实现肿瘤微环境的乏氧改善,减少光疗和化疗中的抗性作用,从而实现较强的抗肿瘤作用。
发明内容
本发明的目的在于提供一种基于血红蛋白的携氧增敏纳米药物的制备方法及应用,利用血红蛋白作为氧载体,进一步提高纳米药物的稳定性,保持其药理活性的稳定,以实现抗肿瘤作用。
为实现上述目的,本发明采用如下技术方案:
本发明提供了一种基于血红蛋白的携氧增敏纳米药物的制备方法,所述方法具体包括如下步骤:
(1)将熊果酸溶于良性溶剂中得到溶液A,将索拉非尼溶于良性溶剂中得到溶液B,将吲哚菁绿溶于良性溶剂中得到溶液C;
(2)将溶液A、溶液B和溶液C按一定体积比混合均匀得到溶液D,将适量溶液D适量缓慢滴入到二次蒸馏水中,超声处理,再用氮吹仪吹干良性溶剂,得到纳米药物-1的水溶液;
(3)完全麻醉哺乳动物后心脏取血,获得全血,将全血离心,弃去上层清液,将得到的暗红色沉淀用阿氏液重悬,即得到红细胞悬液;
(4)将红细胞悬液和纳米药物-1的水溶液按照一定体积比混合,用0.22μM水相微孔滤膜挤出多次过膜,即得到所述基于血红蛋白的携氧增敏纳米药物的水溶液。
进一步的,上述制备方法的步骤(1)中,所述溶液A、溶液B和溶液C中的熊果酸、索拉非尼和吲哚菁绿浓度范围均为0.5-16 mg/ml,所述良性溶剂为甲醇、乙醇、二氯甲烷、乙酸乙酯中的一种或多种。
进一步的,上述制备方法的步骤(2)中,所述溶液A:溶液B:溶液C的体积比为4:4:1~4:4:5 ;所述溶液D:二次蒸馏水的体积比为1:10~1:40;所述超声时间为2-60 min,超声处理温度为25℃,超声功率为250W,超声频率为40KHz。
进一步的,上述制备方法的步骤(3)中,所述离心转速为500-5000 rpm,时间为3-20 min;所述红细胞悬液的浓度范围为106-109 cells/mL。
进一步的,上述制备方法的步骤(4)中,所述红细胞悬液:纳米药物-1水溶液的体积比为1:10-1:200,所述过膜次数为5-20次。
本发明还提供了一种利用上述制备方法制得的基于血红蛋白的携氧增敏纳米药物。
其中,所述携氧增敏纳米药物的粒径为100-200 nm。
本发明还提供了上述一种基于血红蛋白的携氧增敏纳米药物在制备抗肿瘤药物中的应用。
本发明的显著优点在于:
1、本发明所制得的纳米药物利用分子间作用力自组装形成,制备方法简单,可有效提高水不溶性药物的水溶性,以实现更佳的抗肿瘤效果。
2、本发明所制得的一种基于血红蛋白的携氧增敏纳米药物,由于无载体,可有效解决传统载体的引入所引起的机体代谢和安全性问题,为后续临床新药研发提供了新思路。
3、本发明所制得的一种基于血红蛋白的携氧增敏纳米药物,能够利用血红蛋白载氧功能,实现肿瘤乏氧环境的改善,实现光动力治疗和化疗增敏作用;与此同时,红细胞膜能够帮助纳米实现免疫清除逃逸,实现体内长循环的显著特点。
4、本发明所制得的一种基于血红蛋白的携氧增敏纳米药物联合低毒性天然产物熊果酸和分子靶向药索拉非尼,能够有效抑制肿瘤细胞迁移、增殖和黏附,达到协同增敏作用;另外联合吲哚菁绿实现光/化疗联用,大大增强治疗效果。
5、本发明所制得的一种基于血红蛋白的携氧增敏纳米药物尺寸为100-200 nm,能够利用EPR效应实现肿瘤部位的药物富集,提高治疗效果,减少毒副作用。
附图说明
图1为纳米药物-2的粒径分布图。
图2为纳米药物-1和纳米药物-2的紫外吸收图。
图3为纳米药物-1和纳米药物-2的溶氧曲线。
图4为纳米药物-2光照不同时间后的活性氧检测。
图5为HepG2细胞的毒性实验结果。A,不同药物对HepG2细胞存活率的影响;B,纳米药物-1和纳米药物-2在常氧和低氧环境下对HepG2细胞存活率的影响。
图6为HepG2细胞对不同药物的摄取情况。
具体实施方式
根据下述实施例,可以更好地理解本发明,下面结合具体实施方式对本发明所述的技术方案作进一步的说明,但是本发明不仅限于此。
以下实施例中所用到的阿氏液的配制方法为:取葡萄糖2.05g,氯化钠0.42g,柠檬酸0.055g,柠檬酸钠0.80g,加蒸馏水至100ml;将pH调至6.1,过滤后分装;121℃高压灭菌30min,封口放4℃冰箱备用。
实施例1
本实施例提供了一种基于血红蛋白的携氧增敏纳米药物的制备方法,所述方法具体包括如下步骤:
(1)将熊果酸溶于甲醇中,得到溶液A,其中,熊果酸的浓度为4 mg/ml;将索拉非尼溶于甲醇中,得到溶液B,其中,索拉非尼的浓度为4 mg/ml;将吲哚菁绿溶于甲醇中,得到溶液C,其中,吲哚菁绿的浓度为10 mg/m;
(2)将步骤(1)得到的溶液A、溶液B和溶液C按照4:4:1的体积比进行混合,得到溶液D;在涡旋状态下,将90 μL溶液D缓慢滴加至1 mL二次蒸馏水中,超声20 min(超声处理温度为25℃ ,超声频率为40KHz ,超声功率为250W ),得到纳米药物-1的水溶液;
(3)完全麻醉哺乳动物后心脏取血,获得全血,将全血在2000rpm的条件下离心5min,弃去上层清液,将得到的暗红色沉淀用阿氏液重悬,并调节其浓度为106-109 cells/mL,即为红细胞悬液;
(4)取20μL步骤(3)得到的红细胞悬液与1mL步骤(2)制得的纳米药物-1的水溶液混合均匀,用 0.22μM水相微孔滤膜挤出过膜7次,即得到所述基于血红蛋白的携氧增敏纳米药物(纳米药物-2)的水溶液。
本实施例制得的基于血红蛋白的携氧增敏纳米药物(纳米药物-2)的粒径如图1所示。
实施例2
分别将熊果酸水溶液、索拉非尼水溶液、吲哚菁绿水溶液、实施例1制得的的纳米药物-1水溶液和纳米药物-2水溶液利用紫外可见分光光度计进行进行紫外吸收光谱分析,发现纳米药物-1水溶液和纳米药物-2水溶液相较于熊果酸水溶液、索拉非尼水溶液和吲哚菁绿水溶液峰形出现偏移(图2),证明纳米药物组装成功。
实施例3
将实施例1制得的纳米药物-1水溶液和纳米药物-2水溶液分别取1mL,分别加入到3mL脱氧PBS缓冲液(10mM,pH=7.4)中,利用便携式溶氧仪检测30分钟内体系中氧含量的变化,发现在低氧环境中纳米药物-2释氧能力高于纳米药物-1(图3),说明了本发明制得的纳米药物-2具有携氧-释氧功能。
实施例4
分别取500μL实施例1制得的纳米药物-1水溶液和纳米药物-2水溶液,分别向二者中加入5 μL 9 mM的1,3-二苯基异苯并呋喃(DPBF),再分别808nm激光器,功率为0.5W/cm2,分别光照0、2、4、6、8、10分钟,检测体系在425nm波长处的吸光度值,发现随着光照时间的延长,纳米药物-1和纳米药物-2在425nm波长处的吸光度值均呈现下降趋势,且由于纳米药物-2中血红蛋白增效吲哚菁绿光动力的原因,纳米药物-2吸光度值下降趋势相较于纳米药物-1更显著(图4)。
实施例5
采用MTT法检测纳米药物-1和纳米药物-2对肝癌细胞的毒性影响作用。将HepG2细胞培养至对数生长期,经0.25%含EDTA胰酶消化后获得细胞沉淀,用新鲜的MEM培养基稀释,接种至96孔板,每孔加入100 μL细胞悬液,使其细胞密度为5000-10000 cells/孔,37℃过夜培养(注:96孔板外周一圈按每孔100 μL加入生理盐水作为空白组)。待细胞完全贴壁后,弃去旧培养液,每孔加入100 μL含不同药物MEM培养基(熊果酸组: ;索拉非尼组: ;双药纳米组: ;双药混合组: ),对照组每孔加入100 μL不含药MEM培养基,37℃孵育24 h。24h后,弃去旧培养液,每孔加入100 μL, 0.5 mg/ml的MTT溶液, 继续培养4h后,弃去MTT溶液,每孔加入100 μL DMSO溶解甲瓒。置于摇床上振荡10min后, 酶标仪读取每孔490nm吸光度值(OD)。用下述公式细胞存活率:细胞存活率(%)=(OD实验组-OD空白)/( OD对照组-OD空白)*100%(注:低氧环境采用液体石蜡液封实现相对低氧环境)。结果显示,熊果酸和索拉非尼具有协同作用效果(图5A);纳米药物-1和纳米药物-2在常氧状态没有显示出差距,但是纳米药物-1在低氧状态显示出一定的耐药性,杀伤作用有所减弱,而纳米药物-2在低氧状态下显示出更强的杀伤作用(图5B),说明血红蛋白的携氧作用能够增强纳米药物-2在低氧环境下的杀伤力。
实施例6
将HepG2细胞培养至对数生长期,经0.25%含EDTA胰酶消化后用新鲜的MEM培养基稀释获得细胞悬液,按照细胞密度2x105 cells/孔接种于6孔板,过夜培养,待细胞贴壁,弃去旧培养液,实验分为每孔加入1.5 mL含不同药物MEM培养基,对照组每孔加入1.5 mL不含药MEM培养基,共孵育2h后,生理盐水清洗细胞两遍,再加入0.5 mL 0.25%不含EDTA胰酶放回培养箱消化20 min,加入0.5 mL新鲜的MEM培养基终止消化,轻轻吹打收集细胞,在1500rpm的条件下离心5 min,保留细胞沉淀,用生理盐水清洗细胞两遍,再生理盐水重悬进行流式细胞仪检测。结果显示,HepG2细胞对纳米药物-2表现出最强的细胞摄取能力(图6)。
Claims (8)
1.一种基于血红蛋白的携氧增敏纳米药物的制备方法,其特征在于:包括以下步骤:
a)将熊果酸溶于良性溶剂中得到溶液A,将索拉非尼溶于良性溶剂中得到溶液B,将吲哚菁绿溶于良性溶剂中得到溶液C,;
b)将溶液A、溶液B和溶液C按一定体积比混合均匀得到溶液D,取适量溶液D缓慢滴入到二次蒸馏水中,超声处理,用氮吹仪吹干良性溶剂,即得到纳米药物-1的水溶液;
c)完全麻醉大鼠后心脏取血,获得全血,将全血离心,弃去上层清液,将得到的暗红色沉淀用阿氏液重悬,即得到红细胞悬液;
d)将红细胞悬液和纳米药物-1的水溶液按照一定体积比混合,用水相微孔滤膜挤出多次过膜,即得到所述基于血红蛋白的携氧增敏纳米药物的水溶液。
2.根据权利要求1所述的基于血红蛋白的携氧增敏纳米药物的制备方法,其特征在于,步骤a)中,所述溶液A中熊果酸的浓度范围为0.5-16 mg/ml,所述溶液B中索拉非尼的浓度范围为0.5-16 mg/ml,所述溶液C中吲哚菁绿的浓度范围为0.5-16 mg/ml,所述良性溶剂为甲醇、乙醇、二氯甲烷、乙酸乙酯中的任意一种。
3.根据权利要求1所述的基于血红蛋白的携氧增敏纳米药物的制备方法,其特征在于,步骤b)中,所述溶液A:溶液B:溶液C的体积比为4:4:1-4:4:5 ;所述溶液D:二次蒸馏水的体积比为1:10-1:40 ;所述超声处理时间为2-60 min,超声处理温度为25℃ ,超声处理频率为40KHz ,超声处理功率为250W。
4.根据权利要求1所述的基于血红蛋白的携氧增敏纳米药物的制备方法,其特征在于,步骤c)中,所述离心条件为500-5000 rpm、3-20 min;所述红细胞悬液的浓度范围为106-109 cells/mL。
5.根据权利要求1所述的基于血红蛋白的携氧增敏纳米药物的制备方法,其特征在于,步骤d)中,所述红细胞悬液:纳米药物-1水溶液的体积比为1:10-1:200,所述过膜次数为5-20次。
6.一种利用权利要求1所述的制备方法制得的纳米药物。
7.根据权利要求6所述的纳米药物,其特征在于,所述纳米药物的粒径为100-200 nm。
8.如权利要求6所述的纳米药物在制备抗肿瘤药物中的应用。
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