Journot Céline M A, Nicolle Laura, Lavanchy Yann, Gerber-Lemaire Sandrine
Group for Functionalized Biomaterials, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, EPFL SB ISIC SCI-SB-SG, Station 6, CH-1015 Lausanne, Switzerland.
Molecular and Hybrid Materials Characterization Center, Ecole Polytechnique Fédérale de Lausanne, EPFL STI MHMC MHMC-GE, Station 12, CH-1015 Lausanne, Switzerland.
Polymers (Basel). 2020 Jun 2;12(6):1274. doi: 10.3390/polym12061274.
In the field of gene therapy, chitosan (CS) gained interest for its promise as a non-viral DNA vector. However, commercial sources of CS lack precise characterization and do not generally reach sufficient solubility in aqueous media for in vitro and in vivo evaluation. As low molecular weight CS showed improved solubility, we investigated the process of CS depolymerization by acidic hydrolysis, using either long time heating at 80 °C or short time microwave-enhanced heating. The resulting depolymerized chitosan (dCS) were analyzed by gel permeation chromatography (GPC) and H nuclear magnetic resonance (NMR) to determine their average molecular weight (Mn, Mp and Mw), polydispersity index (PD) and degree of deacetylation (DD). We emphasized the production of water-soluble CS (solubility > 5 mg/mL), obtained in reproducible yield and characteristics, and suitable for downstream functionalization. Optimal microwave-assisted conditions provided dCS with a molecular weight (MW) = 12.6 ± 0.6 kDa, PD = 1.41 ± 0.05 and DD = 85%. While almost never discussed in the literature, we observed the partial post-production aggregation of dCS when exposed to phase changes (from liquid to solid). Repeated cycles of freezing/thawing allowed the selection of dCS fractions which were exempt of crystalline particles formation upon solubilization from frozen samples.
在基因治疗领域，壳聚糖（CS）因其作为非病毒DNA载体的潜力而受到关注。然而，商业来源的壳聚糖缺乏精确的表征，并且在水性介质中通常达不到足够的溶解度以用于体外和体内评估。由于低分子量壳聚糖显示出改善的溶解度，我们研究了通过酸性水解使壳聚糖解聚的过程，采用在80℃长时间加热或短时间微波增强加热的方法。通过凝胶渗透色谱法（GPC）和氢核磁共振（NMR）对所得的解聚壳聚糖（dCS）进行分析，以确定其平均分子量（Mn、Mp和Mw）、多分散指数（PD）和脱乙酰度（DD）。我们着重于生产水溶性壳聚糖（溶解度>5mg/mL），其产率和特性具有可重复性，并且适用于下游功能化。最佳的微波辅助条件下得到的dCS分子量（MW）=12.6±0.6kDa，PD=1.41±0.05，DD=85%。虽然文献中几乎从未讨论过，但我们观察到dCS在经历相变（从液体到固体）时会出现部分产后聚集现象。反复的冻融循环使得能够选择出在从冷冻样品溶解时不会形成结晶颗粒的dCS级分。
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