CN117338900A - 促进糖尿病伤口愈合的外用凝胶制剂及其制备方法和应用 - Google Patents
促进糖尿病伤口愈合的外用凝胶制剂及其制备方法和应用 Download PDFInfo
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- CN117338900A CN117338900A CN202311317079.6A CN202311317079A CN117338900A CN 117338900 A CN117338900 A CN 117338900A CN 202311317079 A CN202311317079 A CN 202311317079A CN 117338900 A CN117338900 A CN 117338900A
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- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; Compounds thereof
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Abstract
本发明公开了一种促进糖尿病伤口愈合的外用凝胶制剂及其制备方法和应用,属于生物医药技术领域,所述外用凝胶制剂包括苯硼酸酯键水凝胶和树状活性多肽,其利用树状分子的多价效应增强了铜肽GHK‑Cu的药理活性,通过树状结构形成的一种空间位阻障碍,阻碍了蛋白酶进入活性位点并提高其酶稳定性;同时利用硼酸基团与PVA的羟基形成动态硼酸酯键来制备水凝胶,使得制得的水凝胶具有良好的ROS响应特性。本发明的树状铜肽水凝胶能够响应糖尿病伤口处高水平的过氧化氢并将其清除,同时释放树状铜肽,从而发挥抗炎抗氧化、促血管生成、促成纤维细胞的增殖和迁移作用,这种水凝胶能够顺应伤口愈合的过程,大幅提高糖尿病伤口的愈合效果。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种促进糖尿病伤口愈合的外用凝胶制剂及其制备方法和应用。
背景技术
糖尿病伤口是一种典型的慢性伤口,它的难愈合不但给患者造成严重的疾病负担,还会诱发心理健康问题。正常的伤口愈合通常会经历四个阶段:止血、炎症、增殖和重塑,糖尿病伤口的愈合在不同阶段均可出现停滞。伤口微环境中持续的高水平活性氧(ROS)、炎症细胞及炎症因子过多、新生血管不足、细胞增殖能力减弱是导致糖尿病伤口难愈合的关键因素。目前针对糖尿病伤口常见治疗的策略中敷料治疗是最常用的治疗手段,具有成本低、安全性高、与伤口贴合较好、隔绝细菌等优点。水凝胶敷料与其他敷料相比,因其具有生物相容性好、含水量高等的特点,成为最有前景的伤口敷料之一。但水凝胶通常仅具有单一的生物功能,仅针对糖尿病伤口难愈合诸多因素中的单一因素,因此治疗效果差。
GHK(甘氨酰-L-组氨酸-L-赖氨酸)是从血浆中分离出的一种天然多肽分子,与Cu2+络合后形成铜肽(GHK-Cu),具有抗炎、抗氧化、招募愈合细胞的作用,同时能够活化生长因子并促进血管生成和胶原蛋白的合成,以及促进细胞的增殖、分化、迁移,广泛用于伤口愈合和组织修复。然而,GHK-Cu容易被多种蛋白酶水解,导致其疗效低下甚至失效,因此需要提高其稳定性。
发明内容
本发明的目的在于提供一种促进糖尿病伤口愈合的外用凝胶制剂及其制备方法,通过响应糖尿病伤口处高水平的过氧化氢(H2O2)并将其清除,同时释放具有高的药理活性和酶稳定性的树状活性多肽分子,从而达到促进糖尿病伤口愈合的目的。
本发明的目的是通过以下技术方案来实现:
一种促进糖尿病伤口愈合的外用凝胶制剂,包括苯硼酸酯键水凝胶和树状铜肽G2(GHK-Cu),所述树状铜肽由树状活性多肽G2(GHK)与铜离子络合后得到;所述树状活性多肽G2(GHK)包括赖氨酸母体结构与活性多肽分支单元,赖氨酸母体与分支单元通过酰胺键连接,所述分支单元为赖氨酸和/或组氨酸和/或甘氨酸通过酰胺键连接的活性多肽,所述树状活性多肽G2(GHK)具有如下结构:
其中,R2选自-CnH2n+1或-CnH2n,n=0-18的整数;R1为:
进一步的,所述树状活性多肽的氨基酸数量为2-11;优选的氨基酸数量为3-7。
进一步的,所述树状活性多肽为如下的结构式:
另一方面,本发明提供上述外用凝胶制剂的制备方法,包括如下步骤:
步骤S1,树状活性多肽G2(GHK)的制备:
步骤S11:将(S)-2-氨基-3-(1-三苯甲基-1H-咪唑-4-基)丙酸甲酯盐酸盐(H-His(Trt)-OMe·HCl)、N-Boc-甘氨酸(Boc-Gly-OH)溶解于第一有机溶剂中,加入缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到二肽GH-OMe,脱甲酯后得到GH-COOH;
步骤S12:将GH-COOH溶解于第二有机溶剂中,加入(S)-2-氨基-6-((叔丁氧羰基)氨基)己酸甲酯盐酸盐(H-Lys(Boc)-OMe·HCl)和缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到三肽GHK-OMe,脱甲酯后得到GHK-COOH;
步骤S13:将GHK-COOH溶解于第三有机溶剂中,加入L-赖氨酸二甲酯二盐酸盐(H-Lys-OMe·2HCl)和缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到G2(GHK-Boc-Trt-OMe);
步骤S14:将G2(GHK-Boc-Trt-OMe)溶解于第四有机溶剂中,加入三氟乙酸,氮气保护,反应8-20小时;用冰乙醚洗涤,旋干得到G2(GHK)-OMe;
步骤S15:将G2(GHK)-OMe溶解于第五有机溶剂中,脱甲酯得到树状活性多肽G2(GHK)。
步骤S2,树状铜肽G2(GHK-Cu)的制备:将树状活性多肽G2(GHK)与相同摩尔比的硫酸铜(CuSO4)溶液等体积混合,得到树状铜肽G2(GHK-Cu)。
步骤S3交联剂PBA的制备,所述交联剂PBA具有如下结构:
其中,n=2-18的整数;
步骤S4,将凝胶基质溶胀于水中,将上述制备得到的树状铜肽溶于水中得到树状铜肽水溶液,与经过溶胀的凝胶基质涡旋混合均匀;
步骤S5,将步骤S3制备得到的交联剂PBA溶于水,得到PBA水溶液,加入步骤S4中的混合溶液中,快速搅拌均匀得到凝胶制剂。
进一步的,步骤S11-S15中所述缩合剂为四甲基脲六氟磷酸盐(HATU)、1-羟基-7-偶氮苯并三氮唑(HOAT)、1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐(PYBOP)、1-羟基苯并三唑(HOBT)中的一种或两种;其中缚酸剂为N,N-二异丙基乙胺(DIPEA)或三乙胺(TEA)。
进一步的,所述第一有机溶剂、第二有机溶剂和第三有机溶剂均为N,N-二甲基甲酰胺(DMF);所述第四有机溶剂为二氯甲烷;所述第五有机溶剂为甲醇。
进一步的,所述交联剂PBA为如下结构式:
其中,步骤S3制备交联剂PBA的具体内容包括:
步骤S31:将4-羧基苯硼酸、乙二胺二盐酸盐溶解于第六有机溶剂中,加入缩合剂,氮气保护,反应15-30小时;其中缩合剂为HATU、HOAT,缚酸剂为DIPEA;所述第六有机溶剂为DMF;
步骤S32:减压蒸发除去反应溶剂,残余物中加入去离子水;
步骤S33:往滤饼中加入第七有机溶剂,超声使其成为混悬液,抽滤,取滤饼;重复操作多次直至滤饼变成乳白色,真空干燥得到交联剂PBA;所述第七有机溶剂为乙酸乙酯。
进一步的,步骤S4中所述凝胶基质为PVA-1788和PVA-0588中的一种或两种;所述凝胶基质的浓度为5-20%(w:w)。
进一步的,步骤S4中制备得到的凝胶制剂中凝胶基质、树状铜肽和交联剂PBA的体积比为1:0.5-0.8:0.2-0.5。
本发明还提供了所述的水凝胶在制备通过局部涂抹施以治疗皮肤伤口中的药物中的应用。
与现有技术相比,本发明的有益效果是:
1、本发明所述的外用凝胶制剂以GHK-Cu为分支单元制备成树状铜肽,通过树状分子的多价效应增强了天然三肽分子GHK-Cu的药理活性,同时利用树状结构形成的空间位阻障碍,提高其酶稳定性,使其具有更强的促伤口愈合能力,大幅提高其应用前景。
2、本发明所制备得到的促进糖尿病伤口愈合的外用凝胶制剂使用含苯硼酸酯键的ROS响应性水凝胶作为药物载体,能够清除糖尿病伤口处的大量的H2O2,并释放树铜肽起到协同抗氧化作用,进一步促进伤口愈合。
3、本发明所制备得到的外用凝胶制剂具有优异的细胞相容性,药理活性高,具有重要的理论价值和应用前景。
附图说明
图1为本发明实施例制备得到的树状活性多肽G2(GHK)的1H-NMR谱图;
图2为本发明实施例制备得到的ROS响应凝胶交联剂PBA的1H-NMR谱图;
图3为本发明实施例中制备的树状铜肽G2(GHK-Cu)的紫外光谱图;
图4为本发明实施例中制备的树状铜肽G2(GHK-Cu)的电子顺磁共振波谱(EPR)图;
图5为本发明实施例中制备的树状铜肽G2(GHK-Cu)酶稳定性结果分析图;
图6为本发明实施例制备的树状铜肽水凝胶Gel/G2(GHK-Cu)的扫描电镜(SEM)结果分析图;
图7为本发明实施例制备的树状铜肽水凝胶Gel/G2(GHK-Cu)的体外释放结果分析图;
图8为本发明实施例制备的树状铜肽水凝胶Gel/G2(GHK-Cu)的细胞毒性实验结果分析图;
图9为本发明实施例制备的树状铜肽水凝胶Gel/G2(GHK-Cu)的过氧化氢清除结果分析图;
图10为本发明实施例制备的树状铜肽水凝胶Gel/G2(GHK-Cu)的细胞划痕结果分析图;
图11为本发明实施例中进行糖尿病小鼠伤口治疗的结果分析图;
图12为本发明实施例中进行糖尿病小鼠伤口皮肤组织免疫组化染色结果示意图。
具体实施方式
为了相关技术领域人员更好的理解本发明专利的内容,下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的内容不限于下述的实例。
下述各实施例中,(S)-2-氨基-3-(1-三苯甲基-1H-咪唑-4-基)丙酸甲酯盐酸盐(H-His(Trt)-OMe·HCl)、(S)-2-氨基-6-((叔丁氧羰基)氨基)己酸甲酯盐酸盐(H-Lys(Boc)-OMe·HCl)、L-赖氨酸甲酯二盐酸盐(H-Lys-OMe·2HCl)、1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐(PYBOP)、1-羟基苯并三唑(HOBT)、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)、N-Boc-甘氨酸(Boc-Gly-OH)4-羧基苯硼酸、市售GHK-Cu均购自上海毕得医药生物科技股份有限公司。
N,N-二异丙基乙胺(DIPEA)、乙二胺二盐酸盐、1-羟基-7-偶氮苯并三氮唑(HOAT)均购自上海阿拉丁股份有限公司。
三氟乙酸(TFA)购自上海麦克林生化科技股份有限公司。
无水N,N-二甲基甲酰胺(DMF)购自上海泰坦科技股份有限公司。
本发明通过将树状铜肽装载到ROS响应性水凝胶中来构建一种树状铜肽水凝胶,其利用树状分子的多价效应增强GHK-Cu的药理活性,通过树状结构形成的一种空间位阻障碍,阻碍了蛋白酶进入活性位点并提高其酶稳定性;同时利用硼酸基团与PVA的羟基形成动态硼酸酯键来制备水凝胶,使得制得的水凝胶具有良好的ROS响应特性,本发明的树状铜肽水凝胶能够响应糖尿病伤口处高水平的过氧化氢(H2O2)并将其清除,同时释放树状铜肽,从而发挥抗炎抗氧化、促血管生成、促成纤维细胞的增殖和迁移作用,这种水凝胶能够顺应伤口愈合的过程,大幅提高糖尿病伤口的愈合效果。
实施例1:树状铜肽水凝胶制备方法
1)树状活性多肽G2(GHK)的制备
步骤1:精密称取H-His(Trt)-OMe·HCl 5.00g、Boc-Gly-OH 2.15g、HATU 8.50g、HOAT 3.00g置于三颈圆底烧瓶中,用橡胶塞密封。在氮气保护氛围下,抽真空,缓慢加入超干DMF 80mL使反应物充分溶解。在冰水浴条件下搅拌10min后,分批次缓慢加入DIPEA7.75mL,移除冰浴,室温下反应24h。反应结束后,减压蒸发除去反应溶剂,残余物用乙酸乙酯300mL溶解。依次用5%的碳酸氢钠溶液、pH=3的盐酸溶液、饱和NaCl溶液反复洗涤三次。用过量无水硫酸钠干燥1h。过滤除去干燥剂,减压浓缩滤液制得粗品。采用柱层析法对固体粗产物进行纯化(二氯甲烷:甲醇=60:1,V:V),得到GH-OMe纯产物。
步骤2:精密称取GH-OMe 6g于单口烧瓶中,在冰水浴条件下缓慢加入氢氧化钠甲醇溶液(1mol/L)50mL,快速搅拌直至反应物完全溶解,撤去冰浴,室温反应6h。取微量反应体系液体进行TLC监测,反应结束后,用pH=2的柠檬酸溶液调节反应体系pH 2~3,在室温下减压蒸发除去甲醇,残余物用乙酸乙酯300mL稀释,用饱和NaCl溶液洗涤乙酸乙酯层,收集合并有机相并用过量无水硫酸钠干燥1h。过滤除去干燥剂,减压浓缩滤液制得粗品,真空干燥过夜得到GH-COOH固体纯产物。
步骤3:精密称取GH-COOH 5.4g、H-Lys(Boc)-OMe·HCl 3.46g、HATU 7.42g、HOAT2.64g于250mL三颈圆底烧瓶中,用橡胶塞密封。在氮气保护氛围下,抽真空,缓慢加入超干DMF 80mL使反应物充分溶解。在冰水浴条件下搅拌10min后,分批次缓慢加入DIPEA6.66mL,移除冰浴,室温下反应24h。反应结束后,减压蒸发除去反应溶剂,残余物用乙酸乙酯300mL溶解。依次用5%的碳酸氢钠溶液、pH=3的盐酸溶液、饱和NaCl溶液反复洗涤三次。用过量无水硫酸钠干燥1h。过滤除去干燥剂,减压浓缩滤液制得粗品。采用柱层析法对固体粗产物进行纯化(二氯甲烷:甲醇=40:1,V:V),得到GHK-OMe纯产物。
步骤4:精密称取GHK-OMe 5g于单口烧瓶中,在冰水浴条件下缓慢加入氢氧化钠甲醇溶液(1mol/L)50mL,快速搅拌直至反应物完全溶解,撤去冰浴,室温反应6h。取微量反应体系液体进行TLC监测,反应结束后,用pH=2的柠檬酸溶液调节反应体系pH 2~3,在室温下减压蒸发除去甲醇,残余物用乙酸乙酯300mL稀释,用饱和NaCl溶液洗涤乙酸乙酯层,收集合并有机相并用过量无水硫酸钠干燥1h。过滤除去干燥剂,减压浓缩滤液制得粗品,真空干燥过夜得到GHK-COOH固体纯产物。
步骤5:精密称取GHK-COOH 1.94g、H-Lys-OMe·2HCl 0.24g、PYBOP 1.62g、HOBT0.42g于三颈圆底烧瓶中,用橡胶塞密封。在氮气保护氛围下,抽真空,缓慢加入超干DMF40mL使反应物充分溶解。在冰水浴条件下搅拌10min后,分批次缓慢加入DIPEA1.08mL,移除冰浴,使该反应体系在室温下反应24h。反应结束后,减压蒸发除去反应溶剂,残余物用乙酸乙酯200mL溶解。依次用5%的碳酸氢钠溶液、pH=3的盐酸溶液、饱和NaCl溶液反复洗涤3次,用过量无水硫酸钠干燥1h。过滤除去干燥剂,减压浓缩滤液制得粗品。将采用柱层析法对固体粗产物进行纯化(二氯甲烷:甲醇=20:1,V:V)得到G2(GHK-Boc-Trt-OMe)纯产物。
步骤6:精密称取G2(GHK-Boc-Trt-OMe)1g于三颈圆底烧瓶中,用橡胶塞密封。在氮气保护氛围下,抽真空,在冰浴条件下缓慢加入二氯甲烷2.6mL使反应物充分溶解。快速搅拌10min后,分批次缓慢加入三氟乙酸2.6mL,移除冰浴,室温下反应6h。取微量反应体系液体进行TLC监测,反应结束后,减压蒸发除去反应溶剂,残余物用二氯甲烷50mL溶解,再次减压蒸发除去反应溶剂,所得粘稠液体用冰乙醚10mL匀速打浆直至出现固体沉淀物,离心、收集沉淀物,重复操作以上步骤3次直至出现白色沉淀。白色沉淀真空干燥过夜即得G2(GHK-OMe)固体纯产物。
步骤7:精密称取G2(GHK-OMe)0.80g于单口烧瓶中,在冰水浴条件下缓慢加入氢氧化钠甲醇溶液(1mol/L)50mL,快速搅拌直至反应物完全溶解,撤去冰浴,室温反应6h。取微量反应体系液体进行TLC监测,反应结束后,用pH=2的柠檬酸溶液调节反应体系pH 5~6,在室温下减压蒸发除去甲醇,向残余物中依次加入乙酸乙酯100mL、去离子水200mL,超声使其成为混悬液,离心,弃去上清液,重复操作以上步骤3次。收集并合并水相,室温减压浓缩水相至10mL,以去离子水为透析介质透析72h,透析完成后,冷冻干燥72h,即得树状活性多肽G2(GHK)。
树状铜肽1H-NMR谱见图1。1H NMR(400MHz,D2O)δ7.78(s,2H),7.01(s,2H),4.71–4.60(m,2H),4.44–4.03(m,3H),3.80–3.74(m,4H),3.32–2.88(m,10H),1.85–1.11(m,18H)。
2树状铜肽G2(GHK-Cu)的制备
量取上述制备得到的0.024mol/mLG2(GHK)溶液1mL于西林瓶中,在搅拌条件下缓慢滴加0.024mol/mLCuSO4溶液1mL,室温搅拌过夜待用。
3)交联剂PBA的制备
精密称取4-羧基苯硼酸3.65g、乙二胺二盐酸盐1.33g、HATU 11.40g、HOAT 4.08g于三颈圆底烧瓶中,用橡胶塞密封。在氮气保护氛围下,抽真空,缓慢加入超干DMF 40mL使反应物充分溶解。在冰水浴条件下搅拌10min后,分批次缓慢加入DIPEA10.45mL,移除冰浴,室温下反应24h。反应结束后,减压蒸发除去反应溶剂,残余物加入去离子水200mL,超声使其成为混悬液,抽滤,取滤饼,再次加入乙酸乙酯200mL,超声使其成为混悬液,抽滤,取滤饼,重复操作以上步骤3次直至滤饼变成乳白色。收集滤饼,真空干燥过夜即得交联剂PBA。
交联剂PBA的1H-NMR谱见图2。1H NMR(300MHz,DMSO)δ8.63(d,J=5.2Hz,2H),8.09(d,J=72.6Hz,4H),7.83(q,J=8.2Hz,8H),3.44(d,J=4.5Hz,4H)。
4)树状铜肽水凝胶Gel/G2(GHK-Cu)的制备
取15% PVA-1788(w/w)溶液1mL于5mL离心管中,加入G2(GHK-Cu)溶液650μL,用涡旋仪充分震荡,混合均匀后,加入交联剂5mg/mL PBA溶液350μL,快速搅拌10s制得树状铜肽水凝胶Gel/G2(GHK-Cu)。
对比例铜肽水凝胶Gel/(GHK-Cu)的制备
取15% PVA-1788(w/w)溶液1mL于5mL离心管中,加GHK-Cu溶液650μL,用涡旋仪充分震荡,混合均匀后,加入交联剂5mg/mL PBA溶液350μL,快速搅拌10s制得Gel/G2(GHK-Cu)。
实施例2树状铜肽G2(GHK-Cu)的紫外光谱分析
分别取0.012mmol/mL G2(GHK)溶液、0.012mmol/mL G2(GHK-Cu)溶液、市售的0.012mmol/mL GHK-Cu溶液、0.012mmol/mL CuSO4溶液各500μL于紫外石英比色皿中,校准背景后进行200~800nm波长范围内的紫外光谱扫描。
如图3为上述各溶液的紫外光谱结果分析图,由图可知,树状铜肽G2(GHK-Cu)与市售GHK-Cu相同在600nm处有最大吸收峰,表明G2(GHK)与Cu2+成功配位。
实施例3:树状铜肽G2(GHK-Cu)的EPR光谱分析
使用EPR观察化合物G2(GHK)和G2(GHK-Cu)中铜离子的金属价态。用毛细管分别吸取0.012mmol/mL G2(GHK)溶液、0.012mmol/mL G2(GHK-Cu)溶液样品,将一端封口后放入导管中并在低温下进行EPR扫描。
由图4可知在3426mT处有吸收峰,证明G2(GHK)与Cu2+成功配位。
实施例4:树状铜肽G2(GHK-Cu)酶稳定性分析
分别取1mM的G2(GHK-Cu)和GHK-Cu溶液1mL于5mL离心管中。分别加入蛋白酶K溶液(酶活力为10U/mL)100μL,在37℃条件下孵育0、0.5、1、2、4h。孵育结束后,每管加入甲醇400μL来终止反应。每管样品在4℃、12000rpm条件下离心10min,离心结束后,收集上清液,采用高效液相色谱法来定量检测样品对蛋白酶K的抗降解能力。
由图5可知,当实验进行第1小时,GHK-Cu被迅速降解,溶液中剩余大约58%的底物,而G2(GHK-Cu)仅被降解了8%,剩余92%的底物;在第4小时,GHK-Cu降解达到最大值,溶液中剩余大约50%的底物,而G2(GHK-Cu)剩余大约87%。以上结果表明树状铜肽具有更高的抗蛋白酶水解能力。实施例5树状铜肽水凝胶Gel/G2(GHK-Cu)的扫描电镜(SEM)分析
取实施例1制备的树状铜肽水凝胶Gel/G2(GHK-Cu)于离心管中,适当铺平,将离心管浸入液氮中使水凝胶快速冻结;将冷冻的水凝胶趁冷放入冻干机,冻干过夜;冻干凝胶继续放入37℃真空干燥箱中,干燥24h以便完全除去水分;干燥后的凝胶取自然断面,喷金后在加速电压5.0kV下拍摄微观形态。
由图6可知,SEM显示清晰可见的孔洞,总体呈现三维网络状结构,证明水凝胶制备成功。
实施例6树状铜肽水凝胶Gel/G2(GHK-Cu)的体外释放实验
取装载有20mg G2(GHK-Cu)的凝胶1mL于3.5KDa的透析袋中,将透析袋两端扎紧后放入50mL离心管中,加入释放介质12mL(PBS缓冲液或1mM的H2O2溶液),平行三组实验;将离心管置于37℃的恒温空气摇床中,80rpm振摇,分别于0、2、4、6、8、12、24、36、48、60、72h取出释放介质1mL,每次取样后补加等温等体积的释放介质;取得的样品用紫外分光光度计测量600nm处的吸光度值,并按下述公式计算药物的累积释放量和累积释放比。其中,Qn是药物的累积释放百分比,V是离心管中的释放介质体积(12mL),Vi是在不同时间点采样的释放介质体积(1mL),Cn是不同时间点离心管中的浓度,Ci为第i(n-1)个时间点离心管中G2(GHK-Cu)的浓度,A为G2(GHK-Cu)的初始药量。
由图7可知,在PBS缓冲液中,凝胶在24h内释放迅速,累积释放率达到48%,在72h内达65%。在H2O2溶液中,凝胶在24h内释放迅速,累积释放率达到70%,在72h内达90%。在H2O2溶液中累积释放率高于PBS缓冲液,说明该凝胶具有良好的H2O2响应性。
实施例7树状铜肽水凝胶Gel/G2(GHK-Cu)的细胞毒性实验
将密度为1×105个/mL的NIH/3T3细胞(获得自药用辅料及仿创药物研发中心)悬液接种于96孔板内,每孔接种100μL,置于37℃、5%CO2、饱和湿度的细胞恒温培养箱中孵育24h使细胞贴壁。称取实施例1中制备的树状铜肽水凝胶1g于15mL离心管中,加入PBS缓冲液(pH=7.4)10mL,待凝胶变成接近溶胶状态时,即得凝胶提取液。用DMEM高糖基础培养基稀释至树状铜肽的终浓度分别为200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL)作为实验组给药,每孔给药100μL(n=4),加入DMEM高糖基础培养基100μL作为空白对照组,孵育24小时。用PBS洗涤3次,加入1mg/mL的MTT溶液孵育4小时。加入150μLDMSO 37℃振摇15min,用酶标仪在490nm波长下测定各孔的吸光度并按下述公式计算细胞的相对存活率。其中A2为实验组吸光度值,A1为空白对照组吸光度值,A0为调零孔吸光度值。
细胞毒性试验结果见图8。树状铜肽水凝胶在浓度为3.125μg/mL~200μg/mL区间时没有显著性毒性,表明具有良好的生物相容性。此外,在6.25μg/mL~12.5μg/mL浓度范围内,树状铜肽水凝胶的相对细胞活力为153.90%,显著高于空白对照组,说明在较低浓度下具有促进细胞增殖的作用。
实施例8树状铜肽水凝胶Gel/G2(GHK-Cu)的清除过氧化氢实验
将密度为1×105个/mL的NIH/3T3细胞悬液接种于六孔板内,每孔接种2mL,置于37℃、5%CO2、饱和湿度的细胞恒温培养箱中孵育12h。待细胞铺满培养皿底部70%时,吸掉旧培养基,用无菌PBS缓冲液轻轻冲洗细胞表面三次,加入含树状铜肽溶液、凝胶提取液的500μM H2O2溶液2mL作为实验组,加入1mM H2O2溶液2mL作为阴性对照组(NC),加入DMEM基础培养基2mL为空白对照组(BC),放回培养箱继续培养1h。孵育完成后,弃掉培养液,用无菌PBS缓冲液轻轻冲洗细胞表面三次,确保完全除去残留的H2O2。每孔加入DCFH-DA探针(10μM)1mL,放回培养箱继续培养20min。孵育完成后,用无菌PBS缓冲液轻轻冲洗细胞表面三次,立即使用倒置荧光显微镜观察实验结果。
DCF的荧光结果如图9所示,凝胶载体组、树状铜肽组、树状铜肽水凝胶组均有明显荧光强度的降低,树状铜肽水凝胶组荧光强度最低,说明树状铜肽与ROS响应水凝胶具有协同清除H2O2能力。
实施例9树状铜肽水凝胶Gel/G2(GHK-Cu)的细胞划痕实验
将密度为4×105个/mL的L929细胞悬液接种于六孔板内,每孔接种2mL,置于37℃、5%CO2、饱和湿度的细胞恒温培养箱中孵育24h。待贴壁细胞长满6孔板底部95%时,用无菌200μL枪头垂直于细胞表面,由孔一端划向另一端,吸掉旧培养液,再用pH=7.4的无菌PBS缓冲液轻轻冲洗细胞表面三次来去除划下的细胞。加入不同浓度6.25μg/mL的G2(GHK-Cu)和GHK-Cu溶液2mL加入到6孔板中作为实验组,加入DMEM基础培养基2mL作为空白对照组(BC)。使用倒置荧光显微镜观察并拍照记录0h、8h、18h和30h时的细胞迁移情况,用Image J对划痕面积进行测定计算伤口愈合率。实验结果如图10,G2(GHK-Cu)的细胞迁移率是GHK-Cu的1.6倍。
实施例10糖尿病小鼠伤口治疗
雄性C57BL/6J小鼠(18~22g)(斯贝福(苏州)生物技术有限公司)饲养于12h的光照/黑暗周期、高糖高脂饮食且自由饮水条件下。实验动物体重大于30g时开始实验,具体操作步骤如下:(1)禁食12h,尾静脉取血测空腹血糖并记录体重;(2)取新鲜配制预冷的STZ溶液,按照小鼠50mg/kg体重剂量通过腹腔注射方式给药,注射后给与充足的食物和水;(3)连续注射5天后,尾静脉取血,用血糖仪测定小鼠空腹血糖并记录体重。当连续两天空腹血糖值高于16.65mmol/L时,判定为糖尿病模型,若血糖值低于16.65mmol/L时,以小鼠20mg/kg体重剂量通过腹腔注射追加给药直至糖尿病模型成功建立。
取9只普通模型小鼠并标记为空白对照组(BC),取27只糖尿病模型小鼠,并随机分成3组:阴性对照组(NC)、铜肽凝胶组(Gel/GHK-Cu)、树状铜肽凝胶组(Gel/G2(GHK-Cu))。剃除小鼠背部毛发并正常饲养两天,以0.1mL/10g体重剂量通过腹腔注射5%三氯水合氯醛溶液,待小鼠麻醉后,用75%乙醇对小鼠背部剃除毛发区域消毒,用6mm直径打孔器在背部打孔并用镊子挑除皮下筋膜,给与充足的食物和水源并保持勤换垫料。
各组分别于第0天、第3天、第7天、第10天给药,其中BC组和NC组在伤口处给予无菌PBS溶液,其余各组给予相应制剂治疗。拍照记录伤口面积,用Image J软件对伤口图片进行量化处理。实验结果如图11,树状铜肽水凝胶具有最佳的愈合效果。
实施例11小鼠糖尿病伤口治疗皮肤组织免疫组化染色实验
基于实施例10,第7天处死小鼠,进行小鼠皮肤炎性因子TNF-α、血管内皮生长因子VEGF的免疫组化染色。
实验结果如图12,糖尿病小鼠在通过树状铜肽水凝胶治疗下,相比较其他组别,小鼠TNF-α因子下调明显,VEGF上调明显。
测试结果表明,本发明实施例所得到的外用凝胶制剂具有良好的清除ROS特性,且树状铜肽与ROS响应性水凝胶具有协同抗氧化作用。体内初步药效学实验显示,所制备的树状铜肽水凝胶可明显促进糖尿病伤口愈合。
以上所述仅为本发明的较佳实施例,并不以上述实施方式为限,但凡本领域普通技术人员根据本发明所揭示内容所作的等效修饰、等同替换和改进等,皆应纳入权利要求书中记载的保护范围。
Claims (10)
1.一种促进糖尿病伤口愈合的外用凝胶制剂,其特征在于,包括苯硼酸酯键水凝胶和树状铜肽G2(GHK-Cu),所述树状铜肽由树状活性多肽G2(GHK)与铜离子络合后得到;所述树状活性多肽G2(GHK)包括赖氨酸母体结构与活性多肽分支单元,赖氨酸母体与分支单元通过酰胺键连接,所述分支单元为赖氨酸和/或组氨酸和/或甘氨酸通过酰胺键连接的活性多肽,所述树状活性多肽G2(GHK)具有如下结构:
其中,R2选自-CnH2n+1或-CnH2n,n=0-18的整数;R1为:
2.根据权利要求1所述的一种促进糖尿病伤口愈合的外用凝胶制剂,其特征在于,所述树状活性多肽的氨基酸数量为3-7。
3.根据权利要求1所述的一种促进糖尿病伤口愈合的外用凝胶制剂,其特征在于,所述树状活性多肽为如下的结构式:
4.一种权利要求1-3任一项所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,包括如下步骤:
步骤S1,树状活性多肽G2(GHK)的制备:
步骤S11:将(S)-2-氨基-3-(1-三苯甲基-1H-咪唑-4-基)丙酸甲酯盐酸盐(H-His(Trt)-OMe·HCl)、N-Boc-甘氨酸(Boc-Gly-OH)溶解于第一有机溶剂中,加入缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到二肽GH-OMe,脱甲酯后得到GH-COOH;
步骤S12:将GH-COOH溶解于第二有机溶剂中,加入(S)-2-氨基-6-((叔丁氧羰基)氨基)己酸甲酯盐酸盐(H-Lys(Boc)-OMe·HCl)和缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到三肽GHK-OMe,脱甲酯后得到GHK-COOH;
步骤S13:将GHK-COOH溶解于第三有机溶剂中,加入L-赖氨酸二甲酯二盐酸盐(H-Lys-OMe·2HCl)和缩合剂,氮气保护,反应15-30小时;反应液洗涤、除水后纯化,得到G2(GHK-Boc-Trt-OMe);
步骤S14:将G2(GHK-Boc-Trt-OMe)溶解于第四有机溶剂中,加入三氟乙酸,氮气保护,反应8-20小时;用冰乙醚洗涤,旋干得到G2(GHK)-OMe;
步骤S15:将G2(GHK)-OMe溶解于第五有机溶剂中,脱甲酯得到树状活性多肽G2(GHK);
步骤S2,树状铜肽G2(GHK-Cu)的制备:将树状活性多肽G2(GHK)与相同摩尔比的硫酸铜(CuSO4)溶液等体积混合,得到树状铜肽G2(GHK-Cu);
步骤S3,交联剂PBA的制备,所述交联剂PBA具有如下结构:
其中,n=2-18的整数;
步骤S4,将凝胶基质溶胀于水中,将上述步骤S2制备得到的树状铜肽溶于水中得到树状铜肽水溶液,与经过溶胀的凝胶基质涡旋混合均匀得到混合溶液;
步骤S5,将步骤S3制备得到的交联剂PBA溶于水,得到PBA水溶液,加入步骤S4中的混合溶液中,快速搅拌均匀得到凝胶制剂。
5.根据权利要求4所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,步骤S11-S15中所述缩合剂为四甲基脲六氟磷酸盐、1-羟基-7-偶氮苯并三氮唑、1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐、1-羟基苯并三唑中的一种或两种,其中缚酸剂为N,N-二异丙基乙胺或三乙胺。
6.根据权利要求4所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,所述第一有机溶剂、第二有机溶剂和第三有机溶剂均为N,N-二甲基甲酰胺(DMF);所述第四有机溶剂为二氯甲烷;所述第五有机溶剂为甲醇。
7.根据权利要求4所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,所述交联剂PBA为如下结构式:
其制备方法包括:
步骤S31:将4-羧基苯硼酸、乙二胺二盐酸盐溶解于N,N-二甲基甲酰胺中,加入缩合剂,氮气保护,反应15-30小时;所述缩合剂为四甲基脲六氟磷酸盐、1-羟基-7-偶氮苯并三氮唑,缚酸剂为N,N-二异丙基乙胺;
步骤S32:减压蒸发除去反应溶剂,残余物中加入去离子水;
步骤S33:往滤饼中加入乙酸乙酯,超声使其成为混悬液,抽滤,取滤饼;重复操作多次直至滤饼变成乳白色,真空干燥得到交联剂PBA。
8.根据权利要求4所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,步骤S4中所述凝胶基质为PVA-1788和PVA-0588中的一种或两种;所述凝胶基质的浓度为5-20%(w:w)。
9.根据权利要求8所述的促进糖尿病伤口愈合的外用凝胶制剂的制备方法,其特征在于,步骤S5中制备得到的凝胶制剂中凝胶基质、树状铜肽和交联剂PBA的体积比为1:0.5-0.8:0.2-0.5。
10.权利要求1-3任一项所述的水凝胶或权利要求4-9任一项所述的制备方法所得到的水凝胶在制备通过局部涂抹以治疗皮肤伤口的药物中的应用。
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