Pluda Stefano, Pavan Mauro, Galesso Devis, Guarise Cristian
Fidia Farmaceutici S.p.A., via Ponte della Fabbrica 3/A, 35031 Abano Terme, PD, Italy.
Fidia Farmaceutici S.p.A., via Ponte della Fabbrica 3/A, 35031 Abano Terme, PD, Italy.
Carbohydr Res. 2016 Oct 4;433:47-53. doi: 10.1016/j.carres.2016.07.013. Epub 2016 Jul 9.
Hyaluronic Acid (HA) is a non-sulphated glycosaminoglycan that, despite its high molecular weight, is soluble in water and is not resistant to enzymatic degradation, the latter of which hinders its wider application as a biomedical material. Auto-crosslinked polymer (ACP) gels of HA are fully biocompatible hydrogels that exhibit improved viscoelastic properties and prolonged in vivo residence times compared to the native polymer. Crosslinking is achieved through a base-catalysed reaction consisting of the activation of HA carboxyl groups by 2-chloro-1-methylpyridinium iodide (CMPI) and subsequent nucleophilic acyl substitution by the hydroxyl groups of HA in organic solvent. In this study, a number of ACP hydrogels have been obtained via reactions using varying ratios of CMPI to HA. The crosslinking reaction was monitored by rheological measurements in organic solvents during CMPI addition to the reaction mixture. The ACP intermediates, powders and hydrogels were characterized, helping to elucidate the crosslinking process. A two-step mechanism was proposed to explain the observed trends in viscosity and particle size. Syntheses were carried out by varying the reaction temperature, respectively at 0 °C, 25 °C and 45 °C in N-Methyl-2-Pyrrolidone (NMP), as well as the solvent respectively in NMP, DMSO and DMF at 25 °C. Interestingly, varying these parameters did not substantially affect the degree of crosslinking but likely did influence the intra/inter-molecular crosslinking ratio and, therefore, the viscoelastic properties. A wide range of crosslinking densities was confirmed through ESEM analysis. Finally, a comparative hyaluronidase degradation assay revealed that the ACPs exhibited a higher resistance toward enzymatic cleavage at low elastic modulus compared to other more chemically resistant, crosslinked HAs. These observations demonstrated the importance of crosslinking density of matrix structures on substrate availability.
透明质酸（HA）是一种非硫酸化糖胺聚糖，尽管其分子量很高，但可溶于水且不耐酶降解，后者阻碍了其作为生物医学材料的更广泛应用。HA的自交联聚合物（ACP）凝胶是完全生物相容的水凝胶，与天然聚合物相比，具有改善的粘弹性和延长的体内停留时间。交联是通过碱催化反应实现的，该反应包括用2-氯-1-甲基碘化吡啶（CMPI）活化HA羧基，随后在有机溶剂中由HA的羟基进行亲核酰基取代。在本研究中，通过使用不同比例的CMPI与HA进行反应获得了多种ACP水凝胶。在向反应混合物中添加CMPI期间，通过在有机溶剂中的流变学测量监测交联反应。对ACP中间体、粉末和水凝胶进行了表征，有助于阐明交联过程。提出了一个两步机制来解释观察到的粘度和粒径趋势。分别在0°C、25°C和45°C下于N-甲基-2-吡咯烷酮（NMP）中改变反应温度进行合成，以及在25°C下分别在NMP、二甲基亚砜（DMSO）和N，N-二甲基甲酰胺（DMF）中改变溶剂进行合成。有趣的是，改变这些参数并没有实质性地影响交联程度，但可能确实影响了分子内/分子间交联比率，因此影响了粘弹性。通过环境扫描电子显微镜（ESEM）分析证实了广泛的交联密度。最后，一项比较性的透明质酸酶降解试验表明，与其他化学抗性更强的交联HA相比，ACP在低弹性模量下对酶切表现出更高的抗性。这些观察结果证明了基质结构的交联密度对底物可用性的重要性。
