CN117598984B - 伏立康唑水凝胶的制备方法及在滴眼液制备中的应用 - Google Patents
伏立康唑水凝胶的制备方法及在滴眼液制备中的应用 Download PDFInfo
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Abstract
本发明属于眼部外用药物技术领域，针对现有伏立康唑滴眼液溶解性和渗透性差，眼表停留时间短的问题，本发明提供伏立康唑水凝胶的制备方法及在滴眼液制备中的应用。将2,3‑二氨基丙酸盐酸和氢氧化钠加入到去离子水中，搅拌至完全溶解制得DAPA溶液，将所述DAPA溶液滴入均苯三甲醛乙醇溶液中，室温反应，离心，沉淀用乙醇洗涤，真空冷冻干燥，得到聚醛低聚物；将聚醛低聚物与AHA反应合成含伏立康唑的水凝胶。伏立康唑水凝胶在眼部外用药物制备中的应用，特别是在机械性损伤或治疗真菌性角膜炎的药物制备中的应用。本发明提高了VCZ的水溶性、渗透性及眼表停留时间，克服了伏立康唑应用于临床时遇到的问题。
Description
技术领域
本发明属于眼部外用药物技术领域，尤其是一种伏立康唑水凝胶的制备方法及在滴眼液制备中的应用。
背景技术
真菌性角膜炎是一种严重的眼部真菌感染，是感染性角膜失明的主要原因之一。目前，局部滴眼液如纳他霉素、伏立康唑(VCZ)、两性霉素B等抗真菌药物是真菌性角膜炎的主要治疗用药。然而，眼组织独特的生理解剖结构限制了这些药物的停留时间和渗透性，导致生物利用度较低。频繁给药虽然是维持有效药物浓度所必需的，但会导致各种副作用并促进抗真菌耐药性。以伏立康唑为例，伏立康唑眼表滴眼药物吸收量少且吸收不规则，需反复给药。伏立康唑的水溶性较低，目前通常使用环糊精作为增溶剂来增加伏立康唑的溶解性，但仍存在伏立康唑水溶液无法长期稳定贮存的问题。另外伏立康唑滴眼液的渗透压偏低，刺激性较大，常用氯化钠、甘油、丙二醇、甘露醇等渗透压调节剂来调节。专利文献CN113509436A中公开了一种滴眼液的制备方法，其采用磺丁基-β-环糊精为增溶剂，并且以HCl、NaOH为pH调节剂，以NaCl为渗透压调节剂，还加入了粘稠剂、抑菌剂。专利文献CN116570558A公开了一种伏立康唑眼用纳米缓释组合物，包含如下组分：伏立康唑和/或其可药用盐、增溶剂(所述增溶剂为羟丙基-β-环糊精、磺丁基-β-环糊精或其组合)、羟丙甲纤维素，利用羟丙甲纤维素水溶液具有一定的粘度，来延长药物在眼部的停留时间，从而达到缓释效果，减少用药次数。控制包埋物的平均粒径小于20nm，D90＜100nm，有一定的粘度，可以适当粘附在眼表并抵挡泪眼屏障，同时由于粒度小可以透过角膜屏障，在眼表处缓慢的释放出来，最终对真菌表现出持续抑制的作用。应用既有的环糊精进行增溶，虽然增加了药物黏度，但第1周内点眼仍然需要每天点12次。同时应用伏立康唑浓度为1％，并未对于渗透性增加进行改善。
纳米酶复合水凝胶是一种生物复合材料，由纳米酶(一种具有酶类活性的纳米材料)和水凝胶(一种高度水合的聚合物网络)组成。在眼科应用中，这种复合物以其优良的生物相容性和可调性展现出巨大潜力。其生物相容性可以避免对眼睛组织产生刺激或伤害，而可调性则允许根据特定眼病的需求提供定制化解决方案。
专利文献CN115300459A提供一种纳米酶复合水凝胶滴眼液的制备方法，先利用六水氯化钴与氨水的配位反应以及硝酸银与单宁酸的氧化还原反应合成一种单宁酸配合的银钴复合纳米粒子(TCN)；以水为媒介，将TCN和丙烯酸酯修饰明胶混合并超声至均匀，然后通过紫外光照射一步制备出纳米酶复合水凝胶材料(TCNH)；进而与过氧化氢溶液混合均匀即得TCNH滴眼液。材料并没有可以增溶伏立康唑的功能，因此该方案无法负载伏立康唑。
专利文献CN108434093 A公开了一种伏立康唑眼内药物控释的可注射水凝胶的制备方法，通过制备水溶性线型聚环糊精，然后将伏立康唑装载于水溶性线型聚环糊精的分子笼中，解决了伏立康唑微溶于水，不能直接装载到交联结构中的问题；可将本发明制备的伏立康唑眼内药物控释的可注射水凝胶在进行中央部玻璃体切除手术过程中同期注射入玻璃体基底部，原位凝胶化。但是该方案属于注射剂，只能应用玻璃体腔，水凝胶成胶为团块状固体且黏附性弱，用于眼表时会产生明显的异物感以及摩擦，且不利于药物在眼表的均匀分布，不适合作为滴眼液用于眼表。
发明内容
针对现有伏立康唑滴眼液溶解性和渗透性差，眼表停留时间短的上述问题，本发明提供伏立康唑水凝胶的制备方法及在滴眼液制备中的应用。本发明通过合成聚醛低聚物(PAO)，以PAO和氨基功能化透明质酸(AHA)为原料，采用希夫碱缩合反应合成含伏立康唑(VCZ)的水凝胶，提高了VCZ的水溶性、渗透性及眼表停留时间，克服了伏立康唑应用于临床时遇到的问题。
本发明是通过以下技术方案实现的：
本发明一方面提供一种伏立康唑水凝胶的制备方法，包括以下步骤：
将2,3-二氨基丙酸盐酸盐(DAPA)和氢氧化钠加入到去离子水中，搅拌至完全溶解制得DAPA溶液，将所述DAPA溶液滴入盛有均苯三甲醛(TFB)乙醇溶液的容器中，室温反应，离心，沉淀用乙醇洗涤，真空冷冻干燥，得到聚醛低聚物(PAO)；
将PAO与VCZ加入PBS缓冲液中，于55-65℃超声分散，使VCZ完全溶解，得到第一分散液；将氨基功能化透明质酸(AHA)加入另外的PBS缓冲液中，得到第二分散液，将上述两种分散液混合，于室温反应，制得负载伏立康唑的纳米酶水凝胶复合材料(NHC)。
氢氧化钠增加溶解性并提供反应的碱性环境。进一步的，每50.0mL去离子水中加入579.6-708.4mg DAPA和780.0-820.0mg氢氧化钠溶解，制得DAPA溶液。
进一步的，将DAPA溶液滴入315-385.0mL浓度为1.43mg/mL的TFB乙醇溶液中。
进一步的，分离PAO沉淀的离心速度为4000 -6000rpm，优选5000rpm。
进一步的，将盐酸乙二胺，N-羟基琥珀酰亚胺(NHS)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和4-二甲氨基吡啶(DMAP)依次加入透明质酸(HA)溶液中，并调整pH至4.8-5.2，于25-28℃搅拌1-2天，然后调整pH至6.8-7.2，去除未反应成分或副产物，冻干，得到白色的AHA；
进一步的，所述HA溶液的浓度为5.0mg/mL，每90.0-110mLHA溶液中加入4.4-5.4g盐酸乙二胺、6.4-7.8g NHS、10.7-13.1g EDC和4.5-5.5mg DMAP。
进一步的，所述AHA的制备过程中采用透析的方法去除未反应成分或副产物，截留分子量为8000-14000Da。
本发明还提供含有纳米酶的伏立康唑水凝胶的制备方法。所述纳米酶由可溶性铜盐与多酚化合物进行金属-酚配位制备，将氨基功能化透明质酸和所述纳米酶加入PBS缓冲液中得到第二分散液。
进一步的，所述可溶性铜盐为硫酸铜或者氯化铜；所述多酚类化合物为原花青素(PC)、大豆异黄酮、儿茶素、槲皮素、花色苷、姜黄素、白藜芦醇中的至少一种。
进一步的，将CuSO4·5H2O、PC和去离子水混合，并搅拌形成溶液，调节pH至7.2-7.6，加热至50-55℃，反应3h,离心，沉淀用去离子水洗涤，于60-65℃干燥，制得CuPC纳米酶，如需保存，保存温度为2-4℃。
进一步的，每100.0mL去离子水溶解1600.0-1900.0mg CuSO4·5H2O和50.0 -58.0mg PC。
进一步的，分离CuPC纳米酶沉淀的离心速度为5000 -7000rpm，优选6000rpm。
进一步的，所述步骤(4)第一分散液，每100.0mL PBS缓冲液中加入500.0mgPAO和250.0mgVCZ；所述第二分散液，每100.0mL PBS缓冲液中加入1500.0mgAHA和4.00mg纳米酶(或不添加CuPC)，两种分散液按照1：1的体积比混合。
本发明另一方面提供所述伏立康唑水凝胶在眼部外用药物制备中的应用，特别是在机械性损伤或治疗真菌性角膜炎的药物制备中的应用。
进一步的，所述真菌性角膜炎的致病菌为镰刀菌、曲霉菌、念珠菌属、青霉菌属或酵母菌等常见真菌。
本发明具有以下有益效果：
(1)本发明制备了促进伏立康唑溶解的PAO，解决了伏立康唑溶解性差的问题。
(2)本发明的药物渗透性好，明显增加了黏性和眼表停留时间，应用的伏立康唑的浓度为仅为0.25％(现有技术的1/4)可以达到较好的效果，同时滴眼液只要每天点2次，减少了用药频率。
(3)本发明制备的NHC为具有触变性能的阴离子水凝胶，可以在眼睑剪切时变稀，易于药物重新分布和减少摩擦，同时具有水凝胶的携带负电具有静电排斥抗真菌的作用。
(4)本发明制备的NHC在HCECs和HCFs中均能够清除ROS。
附图说明
图1为实施例3制备的纳米酶颗粒的表征图。
图2为实施例3制备的NHC以及其前体的FTIR图。
图3为实施例3制备的NHC的SEM图。
图4为实施例3制备的NHC的触变性能表征图。
图5为实施例3制备的NHC的Zeta电位图。
图6为实施例3制备的NHC的多酶模拟活性评价图。
图7为测试例7房水中伏立康唑的药物浓度-时间曲线。
图8为动物实验1NHC修复角膜上皮损伤的照片。
图9为动物实验2NHC治疗真菌性角膜炎的照片。
图10为实施例1制备的NHC的抑菌环。
具体实施方式
下面结合具体实施例及附图对本发明做进一步详细说明。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
一、材料制备
实施例1
(1)制备氨基功能化透明质酸
将盐酸乙二胺(4.4g)，NHS(6.4g)，EDC(10.7g)和4.5mg DMAP依次加入HA溶液(5.0mg/mL,100.0mL)中。将pH调至5.0，于25℃搅拌1天。然后进一步调整pH至6.8，用8000-14000Da截止透析管透析2天，去除未反应成分或副产物。将透析管中的混合物冻干48小时，形成白色的AHA。
(2)制备聚醛低聚物
将DAPA(579.6mg)和氢氧化钠(780.0mg)加入到去离子水(50.0mL)中，以1000rpm搅拌30min至完全溶解。然后将DAPA溶液滴入315.0mL浓度1.43mg/mL TFB乙醇溶液中。室温反应1周，5000rpm离心10min。沉淀用乙醇洗涤3次，真空冷冻干燥48h。
(3)采用希夫碱缩合反应制备NHC
将PAO(500.0mg)与VCZ(250.0mg)混合于PBS(100.0mL)中，55℃超声作用30min，确保VCZ完全溶解。将AHA(1500.0mg)混合于PBS(100.0mL)中，振荡使CuPC分散。将上述溶液按1:1的体积比混合，室温反应5min，制得NHC。
实施例2
(1)制备氨基功能化透明质酸
将盐酸乙二胺(4.9g)，NHS(7.1g)，EDC(11.9g)和5.0mg DMAP依次加入HA溶液(5.0mg/mL,100.0mL)中。将pH调至5.0，于25℃搅拌1天。然后进一步调整pH至7.0，用8000-14000Da截止透析管透析2天，去除未反应成分或副产物。将透析管中的混合物冻干48小时，形成白色的AHA。
(2)制备聚醛低聚物
将DAPA(644.0mg)和氢氧化钠(800.0mg)加入到去离子水(50.0mL)中，以1000rpm搅拌30min至完全溶解。然后将DAPA溶液滴入350.0mL浓度1.43mg/mL TFB乙醇溶液中。室温反应1周，5000rpm离心10min。沉淀用乙醇洗涤3次，真空冷冻干燥48h。
(3)NHC的合成采用希夫碱缩合反应制备NHC
将PAO(500.0mg)与VCZ(250.0mg)混合于PBS(100.0mL)中，65℃超声作用30min，确保VCZ完全溶解。将AHA(1500.0mg)和CuPC(4.00mg)混合于PBS(100.0mL)中，振荡使CuPC分散。将上述溶液按1:1的体积比混合，室温反应5min，制得NHC。
实施例3
(1)金属-酚配位制备CuPC纳米酶
在圆底烧瓶中加入CuSO4·5H2O(1750.0mg)、PC(54.0mg)和去离子水(100.0mL)。将混合物以600rpm搅拌5分钟，形成均匀的溶液。用1.0M NaOH溶液调节pH至7.4，然后加热至50℃并保持3h,以6000rpm离心10min，沉淀用去离子水冲洗3次，于60℃干燥过夜，并在4℃条件下保存以备后续使用。
(2)制备氨基功能化透明质酸，与实施例2相同。
(3)制备聚醛低聚物，与实施例2相同。
(4)用希夫碱缩合反应制备NHC
将PAO(500.0mg)与VCZ(250.0mg)混合于PBS(100.0mL)中，65℃超声作用30min，确保VCZ完全溶解。将AHA(1500.0mg)和CuPC(4.00mg)混合于PBS(100.0mL)中，振荡使CuPC分散。将上述溶液按1:1的体积比混合，室温反应5min，制得NHC。
实施例4
采用氯化铜(941.0mg)代替CuSO4·5H2O制备CuPC纳米酶，其它步骤与实施例3相同。
实施例5
采用单宁酸(127.0mg)代替原花青素制备CuPC纳米酶，其它步骤与实施例3相同。
二、材料的性能
评价物理性质、多酶模拟活性、体外/体内释放量、体外/体内生物相容性和体外抗真菌活性。
测试例1
将实施例3得到的CuPC进行物理特性表征。图1中(A)TEM图像显示CuPC的棒状形态，其长度为数十至数百纳米，(B)XRD图显示了CuPC的晶体结构。CuPC的XRD衍射峰分别位于13.8、16.5、22.8、28.0、30.6、33.4、35.6、41.3、52.7处，与铜单宁酸配位纳米片的衍射峰相对应。
测试例2
将实施例3制备的水凝胶的制备前体AHA、PAO以及NHC进行傅里叶变换红外光谱仪，如图2所示。
测试例3
将实施例3得到的NHC冻干后通过扫描电子显微镜进行观察测试，测试结果如图3所示，NHC具有大孔径，这有利于保持高含水量、透气性以及营养物质的交换。
测试例4
将实施例3得到的NHC进行流变性能测试，观察其触变功能，如图4所示，NHC具有剪切变稀、触变的性能。
测试例5
将实施例3得到的NHC进电位测试，观察到阴离子水凝胶携带负电荷，如图5所示。
测试例6
将实施例3得到的NHC进性清除ROS的测试，观察到纳米复合水凝胶具有过氧化物酶活性、过氧化氢酶活性、超氧化物歧化酶活性，如图6所示，其中(A)不同组成反应体系的吸收光谱；(B)pH、(C)CuPC浓度和(D)反应温度对CuPC过氧化物酶模拟活性的影响；(E)光学照片显示CuPC与H2O2相互作用产生氧气。(F)CuPC和NHC对H2O2和O2·-的去除能力；(G)DCFH-DA染色图显示NHC在HCECs和HCFs中均能够清除ROS。
测试例7
将实施例3得到的NHC应用于新西兰大白兔后在不同时间抽取房水测伏立康唑的浓度，观察到纳米酶复合水凝胶可以提高药物的渗透性，如图7所示。
测试例8
文献报道临床用1％伏立康唑给予新西兰大白兔1次点眼后的药物峰值浓度为3.56±1μg/mL，如图7所示给予NHC 1次点眼后药物的峰值浓度为10.31±0.27μg/mL，为临床1％伏立康唑效果的2倍以上。
三、动物实验
动物实验1
将C57BL/6雌性小鼠通过腹腔注射0.06％戊巴比妥钠麻醉。用2.5mm环形钻在角膜中央的角膜上皮上做标记，刮除角膜上皮制备角膜上皮损伤动物模型。
将实施例1制备的NHC点在小鼠的角膜上，每天2次，在不同的时间观察拍照角膜上皮修复的情况，对照组给予生理盐水每天2次点眼，实验组给予NHC每天2次点眼。如图8所示，实验组36h时角膜上皮损伤得到明显修复，48h时角膜上皮损伤基本修复；对照组修复速度较实验组明显慢，48h时还有部分角膜上皮损伤区域未修复。表明NHC对角膜上皮机械损具有明显的修复作用。
动物实验2
将C57BL/6雌性小鼠通过腹腔注射0.06％戊巴比妥钠麻醉。用2.5mm环形钻在角膜中央的角膜上皮上做标记，刮除角膜上皮。取一张滤纸，用茄病镰刀菌真菌孢子悬浮液(107CFU/mL)浸泡在角膜中央，并用7-0缝线缝合眼睑，制备真菌性角膜炎动物模型。
将实施例1制备的NHC点在小鼠的角膜上，每天2次，在不同的时间观察拍照真菌性角膜炎的治疗情况，对照组以生理盐水代替NHC。如图9所示第3天，真菌性角膜炎的症状明显减轻，第7天真菌性角膜炎的症状消失，表明NHC对真菌性角膜炎具有明显的治疗效果。
以上所述的实施例仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.一种伏立康唑水凝胶的制备方法，其特征在于，包括以下步骤：
将2,3-二氨基丙酸盐酸盐和氢氧化钠加入到去离子水中，搅拌至完全溶解制得DAPA溶液，将所述DAPA溶液滴入均苯三甲醛乙醇溶液中，室温反应，离心，沉淀用乙醇洗涤，真空冷冻干燥，得到聚醛低聚物；
将盐酸乙二胺， N-羟基琥珀酰亚胺、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和4-二甲氨基吡啶依次加入透明质酸溶液中，并调整pH至4.8-5.2，于25-28℃搅拌1-2天，然后调整pH至6.8-7.2，去除未反应成分或副产物，冻干，得到白色的氨基功能化透明质酸；
将聚醛低聚物与伏立康唑加入PBS缓冲液中，于50-65℃超声分散，使伏立康唑完全溶解，得到第一分散液；将氨基功能化透明质酸加入另外的PBS缓冲液中，得到第二分散液，将上述两种分散液混合，于室温反应，制得伏立康唑水凝胶。
2.根据权利要求1所述的制备方法，其特征在于，每50.0 mL去离子水中加入579.6-708.4 mg 2,3-二氨基丙酸盐酸盐和780.0-820.0 mg氢氧化钠进行溶解，制得DAPA溶液，将所述DAPA溶液滴入315-385.0 mL浓度为1.43 mg/mL的均苯三甲醛乙醇中。
3.根据权利要求1所述的制备方法，其特征在于，所述第二分散液中还含有纳米酶，所述纳米酶由可溶性铜盐与多酚化合物进行金属-酚配位制备，将氨基功能化透明质酸和所述纳米酶加入PBS缓冲液中得到第二分散液；所述多酚类化合物为原花青素。
4.根据权利要求3所述的制备方法，其特征在于，所述可溶性铜盐为硫酸铜或者氯化铜。
5.根据权利要求4所述的制备方法，其特征在于，将CuSO4·5H2O、原花青素和去离子水混合，并搅拌形成溶液，调节pH至7.2-7.6，加热至50-55℃反应，离心，沉淀用去离子水洗涤，于60-65°C干燥，保存温度为2-4°C，制得CuPC纳米酶。
6.根据权利要求3-5任一项所述的制备方法，其特征在于，所述第一分散液，每100.0mLPBS缓冲液中加入500.0 mg聚醛低聚物和250.0 mg伏立康唑；所述第二分散液，每100.0mLPBS缓冲液中加入1500.0 mg氨基功能化透明质酸和4.00 mg纳米酶，两种分散液按照1：1的体积比混合。
7.权利要求1-6任一项所述的伏立康唑水凝胶在眼部外用药物制备中的应用。
8.根据权利要求7所述的应用，其特征在于，在机械性损伤或真菌性角膜炎的药物制备中的应用。
9.根据权利要求8所述的应用，其特征在于，所述真菌性角膜炎的致病菌为镰刀菌、曲霉菌、念珠菌属、青霉菌属或酵母菌。
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