CN105963278B - 一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法 - Google Patents
一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法 Download PDFInfo
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Abstract
本发明涉及一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法。本发明使用采用巯基壳聚糖、羧甲基壳聚糖、三聚磷酸钠、盐酸阿霉素、过氧化氢为原料,将巯基壳聚糖溶液与盐酸阿霉素溶液混合得混合液一,将羧甲基壳聚糖溶液与三聚磷酸钠溶液混合得混合液二,搅拌下,将混合液一滴入混合液二中,控制羧甲基壳聚糖与巯基壳聚糖的用量,调节溶液酸碱度至pH6‑8,超声混匀,完成离子交联和聚合物凝聚,而后向体系中滴加过氧化氢完成氧化交联,室温搅拌反应分离产物,干燥得到具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒,可逃逸肿瘤细胞内体/溶酶体,可基于肿瘤细胞独特微环境,自发调控实现胞质释放,提高阿霉素的递药效率和药效,在医药、医用材料等许多方面具有良好的研究和开发应用背景。
Description
技术领域
本发明涉及一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法。
背景技术
纳米药物可实现对化疗药物的靶向输送和缓慢释放因而成为研究的热点。针对药物的体内靶向递送是自发应对体内环境和肿瘤微环境调控药物释放的特点,药物载体需要具备与其相适应的功能特性。肿瘤细胞具有独特的环境特性(如pH,还原特性,温度,酶等),环境敏感载体通过对单一或多重环境特性响应,自发控制药物在正确的时空释放。一方面,内体/溶酶体的内环境呈弱酸性(pH 4.5-6.5),基于原酸酯、腙或乙缩醛等基团的pH敏感载体,可以在内体/溶酶体的酸性环境中解体释药。但如果药物在内体/溶酶体滞留,酸性环境及酶的存在会导致药物结构破坏而失活,需要药物及时从内体/溶酶体中逃逸。O型羧甲基壳聚糖(CMCS),在保留壳聚糖氨基基团的前提下,分子链上还同时具有羧基基团,使其成为一种两性电解质。当pH<4.8时,CMCS分子骨架上的氨基质子化,pH在4.8-6.0时,部分的羧甲基基团去质子化,CMCS达到其等电点,当pH>6时,CMCS的氨基和羧基去质子化。因此CMCS具有pH响应性,在内体/溶酶体的酸性环境中,去质子化的羧甲基基团可吸附质子,引发内体/溶酶体渗透溶胀而解体,实现纳米粒逃逸。另一方面,肿瘤细胞质中谷胱甘肽(GSH)的含量明显高于正常组织,使肿瘤细胞质呈高还原性。基于二硫键交联的还原敏感载体,如巯基化壳聚糖,可在细胞质中解体,使药物在胞质释放。pH/氧化还原双响应载药纳米粒可结合肿瘤组织独特的微环境,自发调控药物在正确的时空释放。
发明内容
本发明的目的在于提供一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法,采用巯基壳聚糖、羧甲基壳聚糖、三聚磷酸钠、盐酸阿霉素、过氧化氢为原料,通过离子交联、氧化交联、聚合物凝聚法,制备的pH/氧化还原双响应的载阿霉素壳聚糖纳米粒可逃逸肿瘤细胞内体/溶酶体,自发调控实现胞质释放,提高阿霉素的递药效率和药效。
本发明具体实施方式包括以下步骤。
(1) 将巯基壳聚糖溶于稀酸溶液,其中稀酸浓度为1 mM,巯基壳聚糖浓度为0.1%(w/v),向该溶液中加入浓度为1 mg/mL的盐酸阿霉素溶液,室温搅拌30 min,控制盐酸阿霉素用量,得混合液一。
(2) 将羧甲基壳聚糖溶于去离子水,羧甲基壳聚糖浓度为0.1%(w/v),向该溶液中加入浓度为0.2%(w/v)三聚磷酸钠溶液,室温搅拌30 min,控制羧甲基壳聚糖和三聚磷酸钠的用量,得混合液二。
(3)在搅拌下,将混合液一滴入混合液二中,其添加量为羧甲基壳聚糖与巯基壳聚糖的质量比是1.5-2:1,三聚磷酸钠和巯基壳聚糖质量比是0.1-0.5:1,滴加速度为30滴/分钟,调节溶液酸碱度至pH6-8,室温搅拌反应1 h,超声混匀,超声功率200 W,时间5 min,开2s,停1 s。
(4)将浓度为30%的过氧化氢滴入步骤三的体系中,过氧化氢与巯基壳聚糖游离巯基的摩尔比为1:1,室温搅拌反应2 h,15000转/分钟离心30 min分离产物,干燥得到固体产品,即具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒。
本发明中的所用巯基壳聚糖是壳聚糖与巯基化合物通过酰胺键、酯键或脒基等形成的偶联物,巯基壳聚糖分子量为150 kDa,取代度范围为大于150 μmol/g;羧甲基壳聚糖的分子量范围是150-1500 kDa,脱乙酰度大于90%;所指稀酸溶液可以是浓度为1 mM盐酸或者是浓度为1 mM醋酸;所指的羧甲基壳聚糖可以是O-羧甲基壳聚糖,也可以是N,O-羧甲基壳聚糖,羧甲基壳聚糖取代度范围是大于80%;所用将产物进行粒径和Zeta电位分析,结果表明,所制备pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的粒径分布较窄,平均粒径在150-250 nm,Zeta电位为-31- -27 mv,盐酸阿霉素的包封率为54.9-70.7%。
本发明具有操作方便,制备技术工艺简便等优点。所形成的包载盐酸阿霉素的巯基壳聚糖/羧甲基壳聚糖纳米控释微粒呈球形。本发明的重要意义在于形成的纳米粒对氧化还原及pH具有双响应性,赋予载体对肿瘤细胞递药的时序特性相适应的材料特性,可基于肿瘤细胞独特微环境,逃逸内体/溶酶体对药物的破坏,实现胞质内有效释放,提高药物的输送效率。在医药、医用材料等许多方面具有良好的研究和开发应用背景。
以下结合附表、图表和实施例对本发明做出进一步说明。
附图说明
图1、pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的透射电镜照片。
图2、pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒对氧化还原响应性释药曲线。
图3、pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒对pH响应性释药曲线。
具体实施方式
下面通过实施例对本发明做出进一步具体说明,但本发明不局限于这些实施例。
实施例1
称取2 mg巯基壳聚糖,溶于2 mL 1 mM的盐酸溶液中,在搅拌条件下滴入0.3 mL 1mg/ml盐酸阿霉素溶液,得混合液一,取3 mg分子量为150 kDa的羧甲基壳聚糖溶于3 mL去离子水中,在搅拌条件下滴入0.1 mL浓度为2%(w/v)的三聚磷酸钠溶液得混合液二,将混合液一逐滴加入到混合液二中,滴加速度为30滴/分钟,用盐酸调节酸碱度至pH 7.5,室温下搅拌反应1 h,超声混匀,超声功率200 W,时间5 min,开2 s,停1 s,在搅拌条件下滴加0.05mL 30%的过氧化氢,室温下搅拌反应2 h,15000转/分钟离心30 min,去除上清,冷冻干燥即得具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒。
实施例2
称取2 mg巯基壳聚糖,溶于2 mL 1 mM的盐酸溶液中,在搅拌条件下滴入0.3 mL 1mg/ml盐酸阿霉素溶液,得混合液一,取4 mg分子量为400 kDa的羧甲基壳聚糖溶于4 mL去离子水中,在搅拌条件下滴入0.2 mL浓度为2%(w/v)的三聚磷酸钠溶液得混合液二,将混合液一逐滴加入到混合液二中,滴加速度为30滴/分钟,用盐酸调节酸碱度至pH 8.0,室温下搅拌反应1 h,超声混匀,超声功率200 W,时间5 min,开2 s,停1 s,在搅拌条件下滴加0.05mL 30%的过氧化氢,室温下搅拌反应2 h,15000转/分钟离心30 min,去除上清,冷冻干燥即得具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒。
实施例3
称取2 mg巯基壳聚糖,溶于2 mL 1 mM的盐酸溶液中,在搅拌条件下滴入0.3 mL 1mg/ml盐酸阿霉素溶液,得混合液一,取3 mg分子量为1500 kDa的羧甲基壳聚糖溶于3 mL去离子水中,在搅拌条件下滴入0.5 mL浓度为2%(w/v)的三聚磷酸钠溶液得混合液二,将混合液一逐滴加入到混合液二中,滴加速度为30滴/分钟,用盐酸调节酸碱度至pH 6.0,室温下搅拌反应1 h,超声混匀,超声功率200 W,时间5 min,开2 s,停1 s,在搅拌条件下滴加0.05mL 30%的过氧化氢,室温下搅拌反应2 h,15000转/分钟离心30 min,去除上清,冷冻干燥即得具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒。
Claims (2)
1.一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法,其特征在于0.1%(w/v)的巯基壳聚糖溶液与1 mg/mL的盐酸阿霉素溶液混合得混合液一,将0.2%(w/v)的三聚磷酸钠溶液与0.1%(w/v)的羧甲基壳聚糖溶液混合得混合液二,在搅拌下,将混合液一滴入混合液二中,其中,羧甲基壳聚糖与巯基壳聚糖的质量比是1.5-2:1,三聚磷酸钠和巯基壳聚糖质量比是0.1-0.5:1,调节溶液酸碱度至pH6-8,超声混匀,向上述体系中滴加入30%的过氧化氢,过氧化氢与巯基壳聚糖游离巯基的摩尔比为1:1,室温下搅拌,得到稳定的包载盐酸阿霉素的纳米分散体系,分离干燥,即得到所述产品。
2.根据权利1所述的一种具有pH/氧化还原双响应的阿霉素控释壳聚糖纳米粒的制备方法,其特征在于巯基壳聚糖是壳聚糖与巯基化合物通过酰胺键、酯键或脒基形成的偶联物。
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