CN117582546A - 一种可注射水凝胶及其制备方法与应用 - Google Patents
一种可注射水凝胶及其制备方法与应用 Download PDFInfo
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- CN117582546A CN117582546A CN202311571337.3A CN202311571337A CN117582546A CN 117582546 A CN117582546 A CN 117582546A CN 202311571337 A CN202311571337 A CN 202311571337A CN 117582546 A CN117582546 A CN 117582546A
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- hyaluronic acid
- solution
- dopamine
- oxidized
- injectable hydrogel
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Abstract
本发明提供了一种可注射水凝胶及其制备方法与应用,属于生物医用材料开发技术领域,所述可注射水凝胶的制备方法包括:将氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸分别溶解于缓冲液中,获得氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液,将氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液混合,静置,获得可注射水凝胶。本发明通过以天然多糖为基础,利用一些特殊的结构制得了一种安全性高、生物相容性好、可实现心脏部位给药且能持续发挥支撑效果的可注射水凝胶。
Description
技术领域
本发明属于生物医用材料开发技术领域,尤其涉及一种可注射水凝胶及其制备方法与应用。
背景技术
心肌细胞凋亡是多种心脏急慢性损伤的直接后果之一,包括但不限于缺血(如心肌梗死)、药物损伤(如阿霉素心肌病)、疾病损伤(如急慢性心肌炎),进而导致心肌细胞外基质(ECM)重塑或是肥大,形成瘢痕。如不加以限制,则最终会导致心力衰竭。目前临床治疗通过利尿、扩血管或降低心脏负荷来延缓心衰的过程。然而,现有的治疗手段存在不能改变心衰的恶化进程、药物生物利用度低及大多具有全身给药副作用等问题。
随着生物材料的发展,一些具备组织功能恢复的生物材料也逐步被用于损伤的心脏组织的治疗。而利用水凝胶为损伤的心脏提供结构支撑、软化瘢痕、保护心肌细胞免受氧化应激以及炎症的损伤等策略已经被证实对心脏的结构恢复有益。
局部注射治疗是一种实用性强且高效的给药方式,相比于传统的心脏手术治疗,可注射水凝胶可以利用局部注射进行微创治疗,减少手术带来的风险和局部软组织的继发性破坏。而实现可注射效果的方式多种多样,如,热敏材料、酶响应成胶材料、调控成胶时间、注射后自愈合等方式。其中,热敏材料主要是以N-异丙基丙烯酰胺为基础材料,亚甲基双丙烯酰胺为交联结构,通过碳碳双键聚合实现材料的构建;N-异丙基丙烯酰胺和亚甲基双丙烯酰胺均可以实现注射后37℃下形成水凝胶;但该类合成材料存在体内代谢的问题,极大程度上限制了该类材料的临床转化。酶响应成胶材料大多来源于生物提取,例如猪来源的脱细胞的细胞外基质。该材料可以在注射后、细胞外环境中多种酶的作用下重新形成水凝胶状态的细胞外基质。但该材料存在原料获取困难且受到伦理限制的问题。
同时,可注射水凝胶需要具备能够在体内驻留并黏附于器官的性能。该性能通常可以利用N-羟基马来酰亚胺对水凝胶进行改性或使用聚多巴胺实现。然而,N-羟基马来酰亚胺通常与含有硫醚官能基的化合物反应,因此应用范围受到了限制,同时N-羟基马来酰亚胺的反应通常是可逆的,改性可能会不稳定。且聚多巴胺的制备及保存过程中的不稳定性限制了聚多巴胺的应用。
发明内容
有鉴于此,本发明的目的在于提供一种可注射水凝胶及其制备方法与应用,该可注射水凝胶不仅能够实现心脏部位的可注射给药,黏附于心脏并持续发挥支撑效果,还具有一定的自由基清除能力和抗氧化应激等的作用。
为实现上述目的,本发明提供了以下技术方案:
本发明提供了一种可注射水凝胶的制备方法,包括如下步骤:
将氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸分别溶解于缓冲液中,获得氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液,将氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液混合,静置,获得可注射水凝胶。
优选的,所述氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸的质量比为1~5:2~10:1~5,所述缓冲液为pH 7.2~7.5的PBS缓冲液,所述静置的时间为2~3min。
优选的,所述氧化透明质酸的制备方法包括如下步骤:
将高碘酸钠溶液滴加至透明质酸溶液中,透析、冻干,获得氧化透明质酸。
进一步优选的,所述透明质酸溶液的浓度为0.010~0.015g/ml,所述高碘酸钠溶液的浓度为0.10~0.15g/ml,所述滴加后静置4.5~5.5h,所述静置的温度为23~27℃,所述透析的时间为70~75h,所述冻干的温度为-70~-80℃,所述冻干的真空度为15~25MPa。
优选的,所述多巴胺改性透明质酸的制备方法包括如下步骤:
将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解于二甲基亚砜溶液中,获得催化溶液;将透明质酸溶液与催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,调节pH,透析、冻干获得多巴胺改性透明质酸。
进一步优选的,所述透明质酸溶液的浓度为0.005~0.015g/ml,所述调节pH后将混合溶液静置45~50h,所述静置的温度为23~27℃。
本发明提供了一种所述制备方法获得的可注射水凝胶。
本发明还提供了所述可注射水凝胶在制备治疗心脏急慢性损伤的药物中的应用。
优选的,所述心脏急慢性损伤包括心肌梗死、阿霉素心肌病、急慢性心肌炎、心力衰竭。
与现有技术相比,本发明具有如下有益效果:
本发明制备可注射水凝胶的原理为:在高碘酸钠的氧化下,能够使透明质酸的邻二羟基断裂形成醛基;盐酸多巴胺可在1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐及N-羟基琥珀酰亚胺的催化下,与透明质酸结构中的羧基形成酰胺;同时,氧化透明质酸断裂形成的醛基与羧甲基壳聚糖中的氨基在弱碱性环境下发生席夫碱反应,形成碳氮双键;进而羧甲基壳聚糖链与透明质酸链交联形成网状结构并最终形成水凝胶;此外,多巴胺的儿茶酚结构与透明质酸糖链、羧甲基壳聚糖糖链可形成氢键进一步加强水凝胶的交联作用,且儿茶酚结构也为水凝胶提供了一定的组织黏附效果。
本发明通过以天然的多糖为基础,利用一些特殊的结构,例如席夫碱、苯硼酸酯、离子键等等来实现对成胶时间的控制和实现自愈的效果,并能规避现有技术中存在的问题。本发明制得的基于天然多糖的生物材料不仅能够模拟天然的细胞外基质,而且通过改性可以实现多种功能。
本发明制得的可注射水凝胶能够实现心脏部位的可注射给药,黏附于心脏并持续发挥支撑效果。该水凝胶不仅能够持续发挥抗氧化应激的效果以实现心肌细胞的保护,还可以考虑作为药物递送和释放的平台。同时,由于该水凝胶的原料均来自于天然多糖,生物相容性良好。也可以作为干细胞递送的良好基础平台。
同时,本发明制得的可注射水凝胶具有如下特点:可拉伸、可注射、可组织黏附;可在活性氧环境中(100μM)实现响应降解:具有一定的自由基清除(DPPH检测)能力;能够抗细胞氧化应激;且生物相容性极好。且相比现有的技术,本发明提供的方法制备简单,使用的材料天然,安全性较好。
附图说明
图1为试验例1测得的结果;
图2为可注射水凝胶成胶示意图;
图3为添加多巴胺改性透明质酸前、后测得的可注射水凝胶的储能模量(G′)与损耗模量(G″)随转动频率变化曲线(其中,从上到下依次为添加多巴胺改性透明质酸前测得的变化曲线和添加多巴胺改性透明质酸后测得的变化曲线);
图4为添加多巴胺改性透明质酸前、后测得的可注射水凝胶储能模量(G′)与损耗模量(G″)随剪切应变变化曲线(其中,从上到下依次为添加多巴胺改性透明质酸前测得的变化曲线和添加多巴胺改性透明质酸后测得的变化曲线);
图5为水凝胶成胶时间的实验结果;
图6为水凝胶可注射和黏附效果,其中,从左至右依次为可注射性效果图、可注射性效果图、黏附效果图;
图7为水凝胶在100μM的H2O2溶液和PBS缓冲溶液中的降解图;
图8为水凝胶对DPPH自由基的清除效果图;
图9为水凝胶对H9C2细胞系的抗氧化应激结果,其中,Control代表“正常培养基”的处理组,H2O2代表“模拟ROS环境”的处理组,H2O2+Hydrogel代表“ROS环境下水凝胶搭载细胞”的处理组;
图10为水凝胶与RAW 264.7细胞系及H9C2细胞系的生物相容性结果。
具体实施方式
本发明提供了一种可注射水凝胶的制备方法,包括如下步骤:
将氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸分别溶解于缓冲液中,获得氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液,将氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液混合,静置,获得可注射水凝胶。
在本发明中,所述氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸的质量比优选为1~5:2~10:1~5,进一步优选为2~4:4~8:2~4,更进一步优选为3:6:3;所述缓冲液优选为pH 7.2~7.5的PBS缓冲液,进一步优选为pH7.3~7.4;所述溶解时使用的是相同体积的缓冲液;所述静置的时间优选为2~3min,进一步优选为2.5min。
在本发明中,所述氧化透明质酸的制备方法优选的包括如下步骤:
将高碘酸钠溶液滴加至透明质酸溶液中,透析、冻干,获得氧化透明质酸。
在本发明中,所述高碘酸钠溶液和透明质酸溶液的制备方法为:将透明质酸和高碘酸钠分别溶解于缓冲液中获得透明质酸溶液和高碘酸钠溶液。所述缓冲液优选为pH7.2~7.5的PBS缓冲液,进一步优选为7.3~7.4;所述透明质酸溶液的浓度优选为0.010~0.015g/ml,进一步优选为0.012~0.014g/ml,更进一步优选为0.013g/ml;所述高碘酸钠溶液的浓度优选为0.10~0.15g/ml,进一步优选为0.12~0.14g/ml,更进一步优选为0.13g/ml;所述滴加后优选的静置4.5~5.5h,进一步优选为4.8~5.2h,更进一步优选为5.0h;所述静置的温度优选为23~27℃,进一步优选为24~26℃,更进一步优选为25℃;所述透析的时间优选为70~75h,进一步优选为72~74h,更进一步优选为73h;所述冻干的温度优选为-70~-80℃,进一步优选为-72~-78℃,更进一步优选为-74~-76℃;所述冻干的真空度优选为15~25MPa,进一步优选为18~23MPa,更进一步优选为20~22MPa;所述透明质酸的平均分子量优选为1.5×106。
在本发明中,所述多巴胺改性透明质酸的制备方法优选的包括如下步骤:
将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解于二甲基亚砜溶液中,获得催化溶液;将透明质酸溶液与催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,调节pH,透析、冻干获得多巴胺改性透明质酸。
在本发明中,所述透明质酸溶液的浓度优选为0.005~0.015g/ml,进一步优选为0.008~0.012g/ml,更进一步优选为0.010g/ml;所述调节pH后优选的将混合溶液静置45~50h,进一步优选为46~48h,更进一步优选为47h;所述静置的温度优选为23~27℃,进一步优选为24~26℃,更进一步优选为25℃。
本发明提供了一种所述制备方法获得的可注射水凝胶。
本发明还提供了所述可注射水凝胶在制备治疗心脏急慢性损伤的药物中的应用。
在本发明中,所述心脏急慢性损伤优选的包括心肌梗死、阿霉素心肌病、急慢性心肌炎、心力衰竭。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
一种可注射水凝胶的制备方法,包括如下步骤:
将2g、平均分子量为1.5×106的透明质酸溶解于190ml、pH7.4的PBS缓冲液中,获得透明质酸溶液;将1.4g、1.2eq的高碘酸钠溶解于10ml、pH7.4的PBS缓冲液中获得浓度为0.14g/ml的高碘酸钠溶液,将获得的高碘酸钠溶液全部滴加至透明质酸溶液中,于25℃静置5h,Mw为3500透析72h、于-80℃、20MPa冻干,获得氧化透明质酸(AHA)。
将1g、平均分子量为6×105的透明质酸溶解至100ml去离子水中,获得浓度为0.010g/ml的透明质酸溶液;将0.569g盐酸多巴胺溶解至50ml二甲基亚砜中,获得盐酸多巴胺溶液;将690mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和414mg N-羟基琥珀酰亚胺溶解于100ml二甲基亚砜溶液中,获得催化溶液;将100ml、浓度为0.010g/ml的透明质酸溶液与100ml催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,使用1M盐酸调节pH=5,于25℃静置48h,透析、冻干获得多巴胺改性透明质酸(HA-Dop)。
取125mg上述获得的氧化透明质酸、250mg羧甲基壳聚糖和125mg上述获得的多巴胺改性透明质酸分别溶解于pH7.4的PBS缓冲液中,直至获得10ml的氧化透明质酸溶液、10ml的羧甲基壳聚糖溶液和10ml的多巴胺改性透明质酸溶液,将获得的溶液等体积混合,静置2min,获得可注射水凝胶。
实施例2
一种可注射水凝胶的制备方法,包括如下步骤:
将1.9g、平均分子量为1.5×106的透明质酸溶解于190ml、pH7.2的PBS缓冲液中,获得透明质酸溶液;将1g、1.2eq的高碘酸钠溶解于10ml、pH7.2的PBS缓冲液中获得浓度为0.10g/ml的高碘酸钠溶液,将获得的高碘酸钠溶液全部滴加至透明质酸溶液中,于23℃静置5.5h,Mw为3400透析75h、于-70℃、25MPa冻干,获得氧化透明质酸(AHA)。
将0.5g、平均分子量为4×105的透明质酸溶解至100ml去离子水中,获得浓度为0.005g/ml的透明质酸溶液;将0.569g盐酸多巴胺溶解至50ml二甲基亚砜中,获得盐酸多巴胺溶液;将680mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和424mg N-羟基琥珀酰亚胺溶解于100ml二甲基亚砜溶液中,获得催化溶液;将100ml、浓度为0.005g/ml的透明质酸溶液与100ml催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,使用1M盐酸调节pH=5,于23℃静置50h,透析、冻干获得多巴胺改性透明质酸(HA-Dop)。
取120mg上述获得的氧化透明质酸、240mg羧甲基壳聚糖和120mg上述获得的多巴胺改性透明质酸分别溶解于pH7.2的PBS缓冲液中,直至获得10ml的氧化透明质酸溶液、10ml的羧甲基壳聚糖溶液和10ml的多巴胺改性透明质酸溶液,将获得的溶液等体积混合,静置2~3min,获得可注射水凝胶。
实施例3
一种可注射水凝胶的制备方法,包括如下步骤:
将2.85g、平均分子量为1.5×106的透明质酸溶解于190ml、pH7.5的PBS缓冲液中,获得透明质酸溶液;将1.5g、1.2eq的高碘酸钠溶解于10ml、pH7.2~7.5的PBS缓冲液中获得浓度为0.15g/ml的高碘酸钠溶液,将获得的高碘酸钠溶液全部滴加至透明质酸溶液中,于27℃静置4.5h,Mw为3600透析75h、于-75℃、15MPa冻干,获得氧化透明质酸(AHA)。
将1.5g、平均分子量为8×105的透明质酸溶解至100ml去离子水中,获得浓度为0.015g/ml的透明质酸溶液;将0.569盐酸多巴胺溶解至50ml二甲基亚砜中,获得盐酸多巴胺溶液;将700mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和404mg N-羟基琥珀酰亚胺溶解于100ml二甲基亚砜溶液中,获得催化溶液;将100ml、浓度为0.015g/ml的透明质酸溶液与100ml催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,使用1M盐酸调节pH=5,于27℃静置45h,透析、冻干获得多巴胺改性透明质酸(HA-Dop)。
取130mg上述获得的氧化透明质酸、260mg羧甲基壳聚糖和173mg上述获得的多巴胺改性透明质酸分别溶解于pH7.5的PBS缓冲液中,直至获得10ml的氧化透明质酸溶液、10ml的羧甲基壳聚糖溶液和10ml的多巴胺改性透明质酸溶液,将获得的溶液等体积混合,静置2~3min,获得可注射水凝胶。
试验例1
根据实施例1的方法制备可注射水凝胶。不同的是:设定添加1%、3%、5%和7%的羧甲基壳聚糖(CMCs)以及添加1.5%、2%、2.5%、3%和3.5%的氧化透明质酸(AHA)制备不同羧甲基壳聚糖及氧化透明质酸含量的可注射水凝胶。研究不同羧甲基壳聚糖含量对可注射水凝胶的影响。结果如图1所示。(图中,A、B、C、D依次为添加1%、3%、5%和7%的羧甲基壳聚糖制得的可注射水凝胶的形态图)。
由图1可知,既能满足可注射要求,又具有一定的凝胶强度的处理组是添加2.5%氧化透明质酸和5%的羧甲基壳聚糖制得的可注射水凝胶。
试验例2
根据实施例1的方法制备可注射水凝胶,其中,在进行等体积混合时,先将氧化透明质酸溶液与羧甲基壳聚糖溶液混合,再加入多巴胺改性透明质酸溶液。使用HAAKE MARSII旋转流变仪,使用平板夹具,测试温度设置为25℃,设置零间隙为1.5mm,将圆形水凝胶样品静置在流变仪上,选择震荡模式-时间扫描进行测试。
采用旋转流变仪检测可注射水凝胶添加多巴胺改性透明质酸前、后,可注射水凝胶的储能模量(G′)与损耗模量(G″)随转动频率变化的情况和随剪切应变变化的情况。结果如图3和图4所示。
结果显示,水凝胶在震荡、蠕动的情况下仍然能保持凝胶状态,能够满足在心脏部位注射的需求。
试验例3
根据实施例2的方法制备可注射水凝胶,并在将获得的溶液等体积混合后记录水凝胶的成胶时间。结果如图5所示。
由图5可知,水凝胶的成胶时间在110s-130s之间,能够满足注射后成胶的需求。
试验例4
使用罗丹明b(购自Adamas公司)对实施例2制得的可注射水凝胶进行染色,使用25G注射针头分别将染色后的可注射水凝胶注入干燥平板中及PBS缓冲液(pH=7.4)中,观察可注射水凝胶的成胶形态。
同时,将染色后的可注射水凝胶贴附在猪皮表面,观察其黏附情况。结果如图6所示。
由图6可知,本发明制得的可注射水凝胶可以通过25G注射针头注射后成胶,在PBS(pH=7.4)中能够保持完整的成胶形态。且水凝胶能够黏附在组织表面不会掉落,能够满足注射后的贴附效果。
试验例5
将实施例3制得的可注射水凝胶分别置于PBS(pH=7.4)缓冲溶液中(Control组)、含有200μmol/mL过氧化氢的PBS溶液(pH=7.4)中(H2O2处理组),观察记录(1-12d)水凝胶在不用环境下的降解情况。结果如图7所示。
由图7可知,水凝胶在无过氧化氢的环境中缓慢降解,在添加过氧化氢的环境中(即模拟损伤后高ROS环境),水凝胶的降解加快,有利于水凝胶包载的药物加速释放。
试验例6
按照实施例3的方法制备可注射水凝胶,并在等体积混合后、成胶前将混合液分别稀释至25μg/mL、50μg/mL、100μg/mL、200μg/mL。根据《DPPH法测定典型植物油的抗氧化活性》(纪璐璐,张丽,张思访等.DPPH法测定典型植物油的抗氧化活性[J].广东化工,2022,49(22):220-222.)中记载的DPPH(1,1-二苯基-2-三硝基苯肼,购自上海麦克林生化科技股份有限公司)法检测不同浓度的可注射水凝胶的自由基清除能力。具体的:
取7.89mg二苯代苦味酰基自由基(DPPH)溶解到200mL 80%(v/v)甲醇溶液中,随后使用80%甲醇溶液将储备液稀释至再527nm下的吸光值为0.7,备用。取不同浓度的可注射水凝胶样品加入到2.95mL DPPH稀释液中,25℃孵育1h,使用酶标仪检测527nm下的吸光值并计算自由基清除比例。结果如图8所示。
由图8可知,可注射水凝胶在稀释后仍有良好的自由基清除能力,并且随着浓度的增加自由基清除能力增加,有利于在注射后改善损伤后的高氧化应激环境。
随后,将H9C2细胞(购自北纳生物BNCC)以1×106个/孔接种在6孔板中,加入200μmol/mL过氧化氢完全培养基,模拟高ROS环境培养24h,PBS(pH=7.4)清洗细胞3次,每次100μL,每次1min;使用100μL 10ug/mL DCFH-DA(2',7'-二氯荧光素二乙酸酯,购自上海麦克林生化科技股份有限公司,其中,以pH=7.4的PBS溶液作为溶剂配置10ug/mL DCFH-DA)对细胞中的ROS(DCFH-DA溶液与清洗后的细胞共同孵育15min,37℃)进行染色,通过FITC荧光通道检测。
由图9可知,ROS环境中的细胞内有明显的ROS累积,而与可注射水凝胶稀释液共同培育的组与对照组的绿色荧光几乎无差异,表明可注射水凝胶有清除微环境中的ROS、减少细胞内ROS累积的效果。
试验例7
将800mg实施例3制得的可注射水凝胶浸没在4ml完全培养基(DMEM高糖培养基+10%胎牛血清+1%青霉素链霉素,均购自武汉普诺赛生命科技有限公司)中培养24h,并稀释制备200μg/mL,100μg/mL,50μg/mL,25μg/mL,10μg/mL的水凝胶浸出液。
将RAW 264.7细胞(购自BNCC)以及H9C2细胞系(购自BNCC)均以1×106个/孔接种至6孔板中,加入不同浓度的水凝胶浸出液,与细胞共同培育48h,PBS(pH=7.4)清洗细胞3次,每次100μL,每次1min,使用钙黄绿素(AM)/碘化丙啶(PI)(购自碧云天生物技术)配置10μg/mL的PBS(pH=7.4,AM与PI均为10μg/mL)混合溶液进行活/死细胞染色,染色时间为20min。结果如图10所示。
由图10可知,不同浓度下的RAW 264.7、H9C2细胞系并没有出现明显的细胞死亡,表明该可注射水凝胶的生物相容性极好,能够满足体内注射的需要。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种可注射水凝胶的制备方法,其特征在于,包括如下步骤:
将氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸分别溶解于缓冲液中,获得氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液,将氧化透明质酸溶液、羧甲基壳聚糖溶液和多巴胺改性透明质酸溶液混合,静置,获得可注射水凝胶。
2.根据权利要求1所述的制备方法,其特征在于,所述氧化透明质酸、羧甲基壳聚糖和多巴胺改性透明质酸的质量比为1~5:2~10:1~5,所述缓冲液为pH 7.2~7.5的PBS缓冲液,所述静置的时间为2~3min。
3.根据权利要求1所述的制备方法,其特征在于,所述氧化透明质酸的制备方法包括如下步骤:
将高碘酸钠溶液滴加至透明质酸溶液中,透析、冻干,获得氧化透明质酸。
4.根据权利要求3所述的制备方法,其特征在于,所述透明质酸溶液的浓度为0.010~0.015g/ml,所述高碘酸钠溶液的浓度为0.10~0.15g/ml,所述滴加后静置4.5~5.5h,所述静置的温度为23~27℃,所述透析的时间为70~75h,所述冻干的温度为-70~-80℃,所述冻干的真空度为15~25MPa。
5.根据权利要求1所述的制备方法,其特征在于,所述多巴胺改性透明质酸的制备方法包括如下步骤:
将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解于二甲基亚砜溶液中,获得催化溶液;将透明质酸溶液与催化溶液混合,获得混合溶液;将盐酸多巴胺溶液滴入混合溶液中,调节pH,透析、冻干获得多巴胺改性透明质酸。
6.根据权利要求5所述的制备方法,其特征在于,所述透明质酸溶液的浓度为0.005~0.015g/ml,所述调节pH后将混合溶液静置45~50h,所述静置的温度为23~27℃。
7.如权利要求1~6任一项所述的制备方法获得的可注射水凝胶。
8.如权利要求7所述的可注射水凝胶在制备治疗心脏急慢性损伤的药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述心脏急慢性损伤包括心肌梗死、阿霉素心肌病、急慢性心肌炎、心力衰竭。
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