CN112494711A - 高粘附性双组分自交联消化道粘膜保护胶及其应用 - Google Patents
高粘附性双组分自交联消化道粘膜保护胶及其应用 Download PDFInfo
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- oxidized
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Abstract
本发明提供高粘附性双组分自交联消化道损伤粘膜保护胶及其应用，高粘附性双组分自交联消化道损伤粘膜保护胶包括含醛基高分子生物材料和含氨基高分子生物材料。两组分通过双腔注射器混合后通过内窥镜注射到消化道损伤粘膜表面，两组分成分中的醛基和氨基交联形成胶体膜，含醛基高分子生物材料与创面暴露的蛋白质氨基结合牢固粘附创面上，避免肠道消化液对消化道损伤粘膜的刺激作用，促进创面的愈合。动物试验表明本发明能明显促进损伤性粘膜的修复，因此对食道、胃、肠道粘膜损伤以及皮肤创面具有促进愈合和保护作用。
Description
技术领域
本发明涉及生物医药技术领域，特别是涉及一种适用于内窥镜喷涂到消化道损伤粘膜表面的高粘附性双组分自交联消化道粘膜保护胶及其应用。
背景技术
消化道粘膜损伤从食道、胃、十二指肠到直肠都会发生，一些损伤是有疾病引起的，有些是手术造成的。内窥镜下胃黏膜切除手术和胃黏膜剥离术是早期胃癌和胃良性肿瘤切除的微创手术，不仅可达到根治早期胃癌的目的，且具有创伤小、对患者生存质量影响小等优点，已逐渐取代部分传统外科手术。食道和结肠的早期癌变和良性肿瘤切除的微创手术也取代部分的传统外科手术。但是，目前这种手术后造成的粘膜创面仅做止血处理，尚无良好的创面保护措施。裸露的粘膜创面在消化液的作用下经历溃疡活动期、愈合期和瘢痕期，并且创面的愈合时间也较长。临床研究资料显示病人经内窥镜下胃黏膜切除手术和胃黏膜剥离术6周的愈合率为69％左右。因此，有必要提供一种可以直接通过内镜应用到损伤消化道黏膜表面的产品。
虽然早在1994年就有人试图采用黄芪白芨胶(中药中提取的天然多糖成分)和α氰基丙烯酸酯类粘合剂在胃镜下覆盖胃溃疡创面，但是由于这些材料的不足限制了这类技术的推广应用。国外在2017年有研究人员采用处理的人新鲜羊膜同种异体材料在动物狗的胃损伤模型进行了研究，证明采用新鲜羊膜缝合覆盖到胃损伤粘膜上观察21天发现没有覆盖的创面没有观察到内皮化，而采用新鲜羊膜覆盖的创面大部分被新生的内皮细胞覆盖，证明胃损伤粘膜覆盖生物材料可以非常显著的促进损伤粘膜的重建和内皮化。随着修复材料的发展，目前较具有代表性的是Takimoto是等报道的在猪胃、十二指肠等部位用PGA膜片对创面进行覆盖，应用纤维蛋白胶联合PGA膜片，或用止血夹联合PGA膜片，可避免膜片滑脱。覆盖PGA膜片具有可避免消化液与创面接触，保持创面清洁，尤其是胃部大创面。修复材料PGA本身可引导细胞爬行，促进创面愈合。使用PGA联合生物蛋白胶处理ESD术后创面，可降低ESD术后十二指肠发生穿孔的风险，在食管、十二指肠等部位巨大创面应用PGA膜片可有效预防术后消化道因瘢痕而引起的狭窄。Spiliopoulos等报道使用马心包膜作为兔胃壁全层穿孔的补片，该实验用腹腔镜进行，在术后3d、1周、2周测胃压并行病理学和免疫组织化学检查，结果显示创面愈合较快，修补具有很强的抗压能力，病理学和免疫组织化学检查显示炎性细胞、肉芽组织及生长因子均多或高于对照组。新型修复材料虽然疗效获得了肯定，大多数材料是切成条状的，通过活组织检查孔道送达创面部位，尤其对于较大创面而言，完整的材料铺设对创面具有较好的保护作用。但这些材料难以固定在创面限制了其在临床的使用。国内在2016年柴宁莉等人选取上消化道早癌接受ESD治疗的26例患者为研究对象，以创面是否覆盖聚羟基乙酸(PGA)膜随机分为覆膜组和对照组。分析两组间术后并发症的发生情况、创面愈合程度、肝肾功能及血常规的改变，对比不同的固定方式对PGA膜与创面的贴合情况。结果对照组和覆膜组术后均无并发症发生。覆膜组术后1个月创面愈合度明显优于对照组(P<0.05)。国内外的研究表明，消化道粘膜损伤表面覆盖生物医用材料可以明显促进创面的愈合。目前国内外均无类似产品在临床应用，国外也只是处于临床前动物试验研究阶段。
海藻酸盐作为优良的药物制剂基质，医药上多用作亲水性乳化剂、凝胶剂和增稠剂。中国专利文件CN101933894B公开了一种胃粘膜保护胶，包括成胶物质，酸碱调节剂、交联剂和粘合剂；所述成胶物质，交联剂和粘合剂重量比为1∶0.01-0.3∶0.1-0.5，酸碱调节剂的用量为调pH在6.5-8.5。该专利仅利用物理学原理保护胃黏膜，使用的交联剂为酸溶解性钙，这种钙在中性溶液中不溶解呈颗粒状态，适合口服使用，不适合通过胃镜注射到创面使用，还有就是这种产品具有流动性，胃酸分泌液需要时间，因此这种产品注射到损伤表面会快速流走不能粘附到损伤粘膜表面，这种产品和胃损伤粘膜通过物理性结合较弱极易脱落，不能牢固粘附损伤粘膜表面，不能充分发挥损伤胃粘膜的保护作用。因此解决流动性和创面牢固结合问题，才能真正解决胃损伤粘膜的保护问题。另外这种产品的交联需要酸性环境，在非酸性环境不能发生交联，只适用于胃粘膜损伤，不能用于食管和肠道部位。
发明内容
针对上述技术领域中的不足，本发明提供一种全新的保护消化道损伤粘膜保护胶产品及其应用。
具体的，本发明提供一种高粘附性双组分自交联消化道损伤粘膜保护胶包括两种组分，分别为含醛基高分子生物材料和含氨基高分子生物材料。
所述含醛基高分子生物材料为氧化海藻酸钠、氧化透明质酸钠、氧化葡聚糖、双醛纤维素及衍生物、氧化壳聚糖及衍生物、双醛淀粉、双醛聚乙二醇、双醛硫酸软骨素、双醛普鲁兰多糖、氧化果胶、氧化魔芋葡甘聚糖、双醛聚异麦芽糖、氧化黄原胶、氧化β-环糊精、氧化卡拉胶、氧化结冷胶的一种或多种。
双醛纤维素及衍生物是氧化羟乙基纤维素、含双醛基的氧化羟丙基纤维素、含双醛基的氧化羧甲基纤维素钠盐或钾盐中的一种或多种。
氧化壳聚糖及衍生物是含双醛基的氧化壳聚糖、含双醛基的氧化羟乙基壳聚糖、含双醛基的氧化羟丙基壳聚糖、含双醛基的氧化羧甲基壳聚糖钠盐或钾盐、含双醛基的氧化羟乙基甲壳素、含双醛基的氧化羟丙基甲壳素、含双醛基的氧化羧甲基甲壳素钠盐或钾盐中一种或多种。
所述的含氨基高分子生物材料为胶原蛋白、明胶、氨基化明胶、羧甲基壳聚糖、羧乙基壳聚糖、羟丙基壳聚糖、胺化壳聚糖、胺化羧甲基壳聚糖、胺化羧乙基壳聚糖、胺化羟丙基壳聚糖、多聚赖氨酸、多聚赖氨酸-聚乙烯醇-多聚赖氨酸共聚物、聚谷氨酸、聚乙烯亚胺、白蛋白、纤维蛋白原中的一种或多种。
本发明一个实施例中，含有氨基的壳聚糖和氧化海藻酸钠的交联反应如下：
所述的胶原蛋白包括动物来源和重组类人源胶原蛋白。
所述的含有醛基的高分子生物材料溶液的浓度为2％-15％。溶解液为磷酸盐溶液或硼酸溶液中的一种或多种。
所述含有氨基的高分子生物材料溶液的浓度为2％-20％。溶解液为纯水、生理盐水、或磷酸盐溶液中的一种或多种。
其中，含醛基高分子生物材料组分还可以包括离子敏感生物材料、触变剂溶液。
所述的离子敏感生物材料为海藻酸钠、低甲氧基果胶、酰胺化低甲氧基果胶和去乙酰结冷胶中的一种或多种，用量含醛基高分子生物材料组分总重量的0.0％-1.5％。
所述的触变剂为硅酸镁锂，用量为含醛基高分子生物材料组分总重量的1％-5％；
含氨基高分子生物材料组分还可以包括促进细胞迁移的生物材料、触变剂、含钙化合物溶液。
所述的含钙化合物为氯化钙、乳酸钙、葡萄糖酸钙中的一种或多种。
此外，含有氨基的高分子生物材料组分还包括促进细胞增殖迁移的生物材料。所述的促进细胞迁移生物材料为甘草多糖、β-葡聚糖、党参多糖、索拉胶、杏鲍菇葡聚糖、燕麦葡聚糖、灰树花多糖、谷物葡聚糖、酵母葡聚糖、灵芝多糖、胶原肽、肝细胞生长因子、表皮细胞生长因子、胰岛素样生长因子1、成纤维细胞生长因子、血小板来源生长因子、富组蛋白1、角质细胞生长因子、抗菌肽中的一种或多种。用量为0.001％-0.5％(W/V)。
本发明的高粘附性双组分自交联消化道损伤粘膜保护胶还可以含有治疗溃疡、肿瘤的有效药物量的药物化合物或组合物。
本发明还提供一种高粘附性双组分自交联消化道损伤粘膜保护胶在治疗消化性溃疡、应激性溃疡、溃疡性结肠炎、消化道出血、胃黏膜切除和剥离术、食道粘膜剥离术和肠道粘膜剥离术后的创面中的应用。
本发明的高粘附性双组分自交联消化道损伤粘膜保护胶也可以用于皮肤急性创面和慢性溃疡创面的治疗中的应用。
本发明的高粘附性双组分自交联消化道损伤粘膜保护胶产品为液体状态，包括含醛基高分子生物材料溶液组分和含氨基高分子生物材料溶液组分，通过双腔注射器可以非常方便地经过内窥镜喷洒到损伤粘膜表面，并且两组分混合后含醛基高分子生物材料的醛基和含氨基高分子生物材料的氨基通过席夫碱反应交联形成固体凝胶膜，同时含醛基高分子生物材料的醛基和创面蛋白的氨基通过席夫碱反应结合牢固粘附创面，避免消化道内消化液对损伤创面的刺激和消化作用，保护损伤粘膜创面，促进损伤粘膜的愈合。另外双组分中含醛基高分子生物材料组分中含有少量海藻酸钠，含氨基高分子生物材料组分中含有少量钙，因此当双组分混合注射到表面会实现轻度交联避免流动。而且双组分混合含醛基高分子生物材料和氨基高分子生物材料通过共价键进一步交联增强保护膜的强度，醛基高分子生物材料还可以和创面蛋白的氨基通过席夫碱反应结合牢固粘附创面，这是本发明的关键技术点，解决了粘膜保护胶的流动性和创面牢固结合问题。另外本发明产品的交联不需要酸性环境，在消化道从食管到直肠都可以形成胶体，应用的范围更广。本发明采用的氧化或醛基化海藻酸钠等多糖具有和含氨基的高分子物质通过化学键交联的特性，而没有氧化或醛基化海藻酸钠等多糖没有这种特性。本发明充分利用氧化或醛基化海藻酸钠等多糖能和含氨基的高分子物质交联实现消化道损伤粘膜的保护胶形成。
高粘附性双组分自交联消化道损伤粘膜保护胶不需要缝合，可以牢固的和创面的蛋白结合，不易从创面脱落，高粘附性双组分自交联消化道损伤粘膜保护胶属于具有国际原创的医疗器械创新产品。
具体实施方式
下面结合实施例对本发明作进一步的详细说明。
实施例1
组分1：氧化海藻酸钠10克溶解在预先混悬1.5克硅酸镁锂的100ml0.02％硼酸溶液中，溶解完全呈透明液体，每瓶10ml包装。高温蒸汽灭菌。
组分2：2克硅酸镁锂溶解在100ml纯水中溶解成透明液体，然后加入明胶10克、β-葡聚糖0.2克、氯化钙0.2克溶解完全，每瓶10ml包装。高温蒸汽灭菌。
实施例2
组分1：双醛淀粉5克，海藻酸钠0.2克溶解在预先混悬1.5克硅酸镁锂的100ml0.02％硼酸溶液中，溶解完全呈透明液体，每瓶10ml包装。高温蒸汽灭菌。组分2：2克硅酸镁锂溶解在100ml纯水中溶解成透明液体，然后加入羧甲基壳聚糖3克、β-葡聚糖0.2克、氯化钙0.1克溶解完全，每瓶10ml包装。高温蒸汽灭菌。
实施例3
组分1：氧化透明质酸钠10克，海藻酸钠0.5克溶解在预先混悬1.5克硅酸镁锂的100ml0.01％硼酸溶液中，溶解完全呈透明液体，每瓶10ml包装。高温蒸汽灭菌。
组分2：1.5克硅酸镁锂溶解在100ml纯水中溶解成透明液体，然后加入羟丙基壳聚糖3克、杏鲍菇葡聚糖0.2克、氯化钙0.2克溶解完全，每瓶10ml包装。高温蒸汽灭菌。
实施例4
组分1：双醛淀粉5克，海藻酸钠0.2克溶解在预先混悬1.5克硅酸镁锂的100ml0.02％硼酸溶液中，溶解完全呈透明液体，每瓶10ml包装。高温蒸汽灭菌。组分2：1.5克硅酸镁锂溶解在100ml纯水中溶解成透明液体，然后加入多聚赖氨酸5克、β-葡聚糖0.2克、氯化钙0.3克溶解完全，每瓶10ml包装。高温蒸汽灭菌。
实施例5
组分1：双醛纤维素6克，海藻酸钠0.5克溶解在预先混悬1.5克硅酸镁锂的100ml0.02％硼酸溶液中，溶解完全呈透明液体，每瓶10ml包装。高温蒸汽灭菌。组分2：1.5克硅酸镁锂溶解在100ml纯水中溶解成透明液体，然后加入聚谷氨酸5克、灰树花多糖0.2克、氯化钙0.3克溶解完全，每瓶10ml包装。高温蒸汽灭菌。
实验例效果评价
以上液体直接用于试验。
1测试例体外胶体膜形成试验
分别取上述制品的组分1和组分2各1ml混匀后涂布在预温到37℃新鲜的猪皮内侧面上，然后放置在37℃恒温箱中30分钟，观察胶体膜形成的情况。结果是实施例1到实施例5均形成胶体膜。
2测试例安全性研究试验
将实施例1-5进行以下生物学试验
1)口腔粘膜刺激：将制品的组分1和组分2各2.5ml混匀后倒入直径9cm的无菌培养皿中，然后放置在37℃恒温箱中30分钟使其成膜，然后按照3cm2/ml比例加入生理盐水，在37℃浸提72小时制备试验液。将样品的实验液制成直径不大于5mm棉球浸透置于3只金黄地鼠一侧颊囊中。接触时间每次最少5min，每天一次，共4次，末次接触后24h肉眼观察颊囊,无痛处死地鼠,取颊囊有代表性部位的组织样品放入4％甲醛溶液中固定后并制作组织切片后进行组织学评价。结果刺激指数均为0，被试样品无口腔粘膜刺激性。
2)细胞毒性：将制品的组分1和组分2各2.5ml混匀后倒入直径9cm的无菌培养皿中，然后放置在37℃恒温箱中30分钟使其成膜，然后按照3cm2/ml比例加入培养基，在37℃浸提24小时制备试验液。然后按GB/T16886.5中规定的细胞毒性试验采用MTT法测定，细胞增值率均在75％-90％级范围内。
3)致敏试验：将制品的组分1和组分2各2.5ml混匀后倒入直径9cm的无菌培养皿中，然后放置在37℃恒温箱中30分钟使其成膜，然后按照3cm2/ml比例加入生理盐水，在37℃浸提72小时制备试验液。然后按GB/T16886.10中规定的方法进行皮肤致敏试验，均未观察到致敏作用。
3测试例组织粘附力的测定(组织留存量法)：
将实施例1-5分别进行以下试验
取SD大鼠,禁食24h用戊巴比妥钠溶液(40mg/kg)经腹腔注射麻醉,解剖取出胃置生理盐水(37℃)中切取胃,用生理盐水将胃内壁清洗干净，清洗的胃在2小时内使用。剪取一定面积的胃组织(2cm×2cm),固定于聚乙烯薄膜上，将制品的组分1和组分2各等量混匀后0.5ml均匀涂布于预温到37℃的胃黏膜创面上，喷涂固定液。胃组织在相对湿度为92.5％的恒湿密闭容器中放置20分钟，将经上述处理的胃组织固定于冲洗斜槽上,将斜槽的角度调至60度，调节蠕动泵流速为20ml/min,将胃组织用0.1mol/L盐酸冲洗5mins,冲洗液收集于一已知重量的烧杯中,在70℃烘干,称重,组织粘附力用粘附百分率表示。
计算方法如下:
胃组织粘附百分率(Bg/％)Bg/％＝{[M-(G-g-m)]/M}x100％
式中M为黏膜保护胶的重量(0.5ml同条件下烘干)；g为空烧杯重；G为烧杯与烘干后残渣总重；m为同体积冲洗液所含固体物质的量(空白对照)。B值越大表示粘附力越大。
结果显示：实施例1：95％，实施2：96％，实施例3：97％，实施例4：95％，实施例5：95％。试验表明实施例1-5的黏膜保护胶对组织的粘附力较强，不易脱落。
4测试例动物试验
动物分两组，体重3kg左右。实验组6只，对照组6只。
手术前24小时禁食，不禁水。
麻醉方法：推荐家兔用质量浓度30g/L戊巴比妥钠静脉注射1.0ml/kg。
家兔仰面固定手术台上，腹部去毛。按外科常规手术要求以2％碘酊和75％乙醇溶液消毒试验区域。
在上腹部逐层切开皮肤、肌肉层和腹膜，如有出血结扎止血。暴露胃部，在胃大弯处切开胃，用生理盐水清洗胃部，在胃体内的胃大弯侧面的黏膜下，1：10000肾上腺素生理盐水进行黏膜下注射，注射1ml生理盐水形成胃黏膜突出，然后用环套器切除直径1cm的黏膜形成创面，喷洒凝血酶(1ml内含凝血酶50U)并压迫止血完全，测量创面的直径。对照组不做任何处理，实验组涂布组分1和组分2各等量混匀后0.5ml保护胶，然后缝合胃部。再逐层缝合腹壁。放饲养笼中，禁食一天。
结果观察：与手术后1周安乐死动物，沿原来的切口切开胃壁，观察创面愈合情况，测量创面的直径，结果发现实验组手术后1周溃疡的实验组5只家兔愈合，0只未愈合，愈合率100％；对照组2只愈合，4只未愈合，愈合率33％。以上结果可以看出,按实施例1能明显促进的胃黏膜损伤性溃疡愈合，对胃溃疡有明显的治疗作用。
本文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，但并不会偏离本发明的精神。
Claims (10)
1.高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，高粘附性双组分自交联消化道损伤粘膜保护胶包括两种组分，含醛基高分子生物材料和含氨基高分子生物材料。
2.根据权利要求1所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，所述含有醛基的高分子生物材料为氧化海藻酸钠、氧化透明质酸钠、氧化葡聚糖、双醛纤维素及衍生物、氧化壳聚糖及衍生物、双醛淀粉、双醛聚乙二醇、双醛硫酸软骨素、双醛普鲁兰多糖、氧化果胶、氧化魔芋葡甘聚糖、双醛聚异麦芽糖、氧化黄原胶、氧化β-环糊精、氧化卡拉胶、氧化结冷胶的一种或多种。
3.根据权利要求2所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，双醛纤维素及衍生物是氧化羟乙基纤维素、含双醛基的氧化羟丙基纤维素、含双醛基的氧化羧甲基纤维素钠盐或钾盐；氧化壳聚糖及衍生物是含双醛基的氧化壳聚糖、含双醛基的氧化羟乙基壳聚糖、含双醛基的氧化羟丙基壳聚糖、含双醛基的氧化羧甲基壳聚糖钠盐或钾盐、含双醛基的氧化羟乙基甲壳素、含双醛基的氧化羟丙基甲壳素、含双醛基的氧化羧甲基甲壳素钠盐或钾盐中的一种或多种。
4.根据权利要求1所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，所述的含有氨基的高分子生物材料为胶原蛋白、明胶、氨基化明胶、羧甲基壳聚糖、羧乙基壳聚糖、羟丙基壳聚糖、胺化壳聚糖、胺化羧甲基壳聚糖、胺化羧乙基壳聚糖、胺化羟丙基壳聚糖、多聚赖氨酸、多聚赖氨酸-聚乙烯醇-多聚赖氨酸共聚物、聚谷氨酸、聚乙烯亚胺、白蛋白、纤维蛋白原中的一种或多种。
5.根据权利要求1所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，含醛基高分子生物材料组分还可以包括离子敏感生物材料、触变剂溶液，所述的离子敏感生物材料为海藻酸钠、低甲氧基果胶、酰胺化低甲氧基果胶和去乙酰结冷胶中的一种或多种。
6.根据权利要求1所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，含氨基高分子生物材料组分还可以包括促进细胞迁移的生物材料、触变剂、含钙化合物溶液，所述的含钙化合物为氯化钙、乳酸钙、葡萄糖酸钙中的一种或多种。
7.根据权利要求6所述的高粘附性双组分自交联消化道损伤粘膜保护胶。其特征在于，所述的促进细胞迁移生物材料为甘草多糖、β-葡聚糖、党参多糖、索拉胶、杏鲍菇葡聚糖、燕麦葡聚糖、灰树花多糖、谷物葡聚糖、酵母葡聚糖、灵芝多糖、胶原肽、肝细胞生长因子、表皮细胞生长因子、胰岛素样生长因子1、成纤维细胞生长因子、血小板来源生长因子、富组蛋白1、角质细胞生长因子、抗菌肽中的一种或多种。
8.根据权利要求1-7任意一项所述的高粘附性双组分自交联消化道损伤粘膜保护胶，其特征在于，还包括治疗溃疡、肿瘤的有效药物量的药物化合物或组合物。
9.如权利要求1-7任意一项所述的高粘附性双组分自交联消化道损伤粘膜保护胶在制备治疗消化性溃疡、应激性溃疡、溃疡性结肠炎、消化道出血、胃黏膜切除和剥离术、食道粘膜剥离术和肠道粘膜剥离术后的创面药物中的应用。
10.如权利要求1-7任意一项所述的高粘附性双组分自交联消化道损伤粘膜保护胶在制备皮肤急性创面和慢性溃疡创面的治疗药物中的应用。
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