摘要
发掘大型海藻多糖的新功能,应用于生物医学领域,是提高其附加值的重要途径,也是获得新型生物医学材料的希望之所在。大型海藻多糖,特别是琼脂糖主要缺点是降解慢,因此需要预降解成分子量小、甚至低温可溶解的琼脂糖,以便用于诸如药物载体、组织再生修复用支架等领域;另外琼脂糖没有细胞或蛋白受体,不与它们发生作用,因此需要与其它降解快、可与细胞或蛋白受体发生特殊相互作用、有生物活性的组分(如透明质酸)共混或接枝共聚改性,得到具有生物活性的大型海藻多糖基材料。由于透明质酸能与细胞表面的CD44膜蛋白特异结合,并与蛋白或多肽产生离子相互作用而物理附载它们,因此琼脂糖接枝透明质酸共聚物有望成为一种新型的多肽载体,广泛应用于生物医学领域。
过氧化氢将琼脂糖氧化降解成小分子,以环氧氯丙烷活化琼脂糖,得到琼脂糖环氧基含量超过1.42 mmol/g。加入分子量为5.7 kDa的透明质酸反应,得到琼脂糖接枝透明质酸共聚物。红外光谱及元素分析结果表明透明质酸成功与琼脂糖进行了接枝反应,接枝量受透明质酸加入比例调控。埋植在小鼠皮下降解,结果证实该聚合物(接枝HA量为37.7%)在28天内完全降解,而琼脂糖仅降解23.65±3.33%。生物学初步评价结果表明该材料具有良好的细胞相容性,无热原反应,无全身急性毒性反应,溶血率小于5%,是一种生物安全的可降解功能材料。
以胰岛素为酸性多肽模型药物,在不同pH环境下测试琼脂糖接枝透明质酸共聚物对胰岛素的控制释放性能。建立Caco-2细胞模型,考察琼脂糖接枝透明质酸共聚物是否具有特异促进胰岛素吸收的作用。研究结果表明,琼脂糖接枝透明质酸共聚物对胰岛素的载药量达40%,包封率可达80%,并有一定的缓释作用,初步显示琼脂糖凝胶效应对包封率的积极作用。但受pH值的影响较大,在pH 6.8缓释时间为3 h。Caco-2细胞模型实验表明琼脂糖接枝透明质酸共聚物有特异促进胰岛素吸收的作用,表明HA的特异生物学功能得到维持。
以碱性成纤维细胞生长因子(bFGF)为碱性多肽模型药物,将琼脂糖接枝透明质酸共聚物与bFGF复合后制备成海绵状皮肤敷料,该敷料具有20～22倍的吸水率。体外释放实验结果表明琼脂糖接枝透明质酸海绵中bFGF呈缓慢持续释放特性,14 d释放总量为41.42±5.07%。在小鼠体内血清中探测到bFGF的含量随时间而衰减,进一步证明缓释海绵不断释放bFGF的有效性。MTT实验结果表明琼脂糖接枝透明质酸共聚物具有良好的细胞相容性,同时也证实琼脂糖接枝透明质酸海绵中的bFGF仍保持其生物学活性。建立小鼠全层皮肤创伤模型,研究琼脂糖接枝透明质酸共聚物复合bFGF促进创伤愈合的效果。实验结果表明琼脂糖接枝透明质酸共聚物复合5μg/mL bFGF海绵在伤口愈合的效果理想,皮肤中大部分组织得到再生。。
因此,琼脂糖接枝透明质酸共聚物有望成为一种新型的多肽药物载体,应用于生物医药以及组织再生修复等领域。
Agarose(abbr Ag) is a naturally polysaccharide derived from red-purple seaweed.To find its new features and broaden its application,the key work is to tailor agarose to the need in biomedical fields.The major drawbacks of agarose are that it is slowly degraded and has no specific binding motif for protein or cell.So it is necessary to lower molecular weight of agarose for easier solution at lower temperature or quicker degradation,and composite with some quickly degradable polymer for promoting its degradation rate,or with some bioactive agents(such as hyaluronan,abbr HA) to provide advantages in specific interaction with protein, peptide or cell.Hyaluronan not only can bind to cell surface CD44 receptor,but also can be used as a component to induce the ionic interaction with peptide for efficient loading and sustained release.In addition,peptides can keep their activation when complexing with hydrophilic polymer by a physical loading process.Agarose grafting hyaluronan copolymer might be a promising carrier for protein,peptides or a regenerative tissue scaffold..
Agarose was degraded by hydrogen peroxide to lower molecular weight,then activated with epichlorohydrin,and had epoxy group content of over 1.42 mmol/g, then grafted with 5.7 kDa hyaluronan to obtain agarose grafting hyaluronan copolymer(Ag-g-HA).Results of IR and EA experiments proved that HA was succeedly grafted into agarose chain and the grafting ratio was changed with the adding amount of HA.The result of the implanted experiments Showed that Ag-g-HA copolymer with HA content 37.7%totally degraded in 28d,but the agarose degraded with H_2O_2 for 8h only lost 23.65±3.33%under mice subdermis.Results of preliminary biocompatibility evoluations disclosed that the copolymer had no pyrogen reaction and no irritant toxicity reaction,haemolysis rate being less than 5%.It is concluded that Ag-g-HA copolymer is a degradable biomaterial with good biocompatibility.
As an acidic peptide model,insulin was capsuled into Ag-g-HA copolymer microparticles by an ionic interaction process,and its release behavior in the copolymer was tested in different pH environments.Using Caco-2 cell monolayer model,we also evaluated if the copolymer enhanced insulin transport effects or not in vitro.The copolymer could load 40%weight percentage of insulin and capsule 80% of insulin added into solution.It might disclose that the gel effect of agarose chain in the copolymer does faver to increasing capsulation rate of the copolymer to insulin. Results suggested that the stability of the insulin loaded Ag-g-HA complexes in distinct pH environments significantly affected their insulin release profiles and these microparticles had sustained-release effect in the environment of pH 6.8 with a complete release within 3 h.The result of Caco-2 cell model experiment showed that Ag-g-HA could specificly enhance insulin transportation.It means that HA maintains its biological effect in the copolymer.
As a basic peptide model,basic fibroblast growth factor(bFGF) was loaded into Ag-g-HA sponge for accelerating wound skin healing with an ionic interaction process.Water uptake ratio of the sponge was 20 to 22 times.Results of in vitro measurements showed that the release of bFGF in the sponge was sustained over 14 days and the cumulative release percent was 41.42±5.07%.Also bFGF could be effectively detected in mice plasma which proved again that the bFGF loading sponge could sustainedly release bFGF.MTT test result revealed that the sponge had good biocompatibility and bFGF in it remained active to accelerate cell proliferation. Animal experiments disclosed that the Ag-g-HA sponge loading 5μg/mL bFGF was excellent one for accelerating wound skin healing,and most f tissues in skin are regenerated.
In conculsion,the degradable Ag-g-HA copolymer is a safety biomaterial,and is convienent to load peptides.It has the potential to be a novel peptide carder and be widely applied in biomedical fields.
引文
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