DataSheet1_High-Molecular-Weight Fractions of Spruce and Eucalyptus Lignin as a Perspective Nanoparticle-Based Platform for a Therapy Delivery in Liver Cancer.docx

The natural polymer, lignin, possesses unique biodegradable and biocompatible properties, making it highly attractive for the generation of nanoparticles for targeted cancer therapy. In this study, we investigated spruce and eucalyptus lignin nanoparticles (designated as S-and E-LNPs, respectively). Both LNP types were generated from high-molecular-weight (Mw) kraft lignin obtained as insoluble residues after a five-step solvent fractionation approach, which included ethyl acetate, ethanol, methanol, and acetone. The resulting S-and E-LNPs ranged in size from 16 to 60 nm with uniform spherical shape regardless of the type of lignin. The preparation of LNPs from an acetone-insoluble lignin fraction is attractive because of the use of high-Mw lignin that is otherwise not suitable for most polymeric applications, its potential scalability, and the consistent size of the LNPs, which was independent of increased lignin concentrations. Due to the potential of LNPs to serve as delivery platforms in liver cancer treatment, we tested, for the first time, the efficacy of newly generated E-LNPs and S-LNPs in two types of primary liver cancer, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), in vitro. Both S-LNPs and E-LNPs inhibited the proliferation of HCC cells in a dose-dependent manner and did not affect CCA cell line growth. The inhibitory effect toward HCC was more pronounced in the E-LNP-treated group and was comparable to the standard therapy, sorafenib. Also, E-LNPs induced late apoptosis and necroptosis while inhibiting the HCC cell line. This study demonstrated that an elevated number of carbohydrates on the surface of the LNPs, as shown by NMR, seem to play an important role in mediating the interaction between LNPs and eukaryotic cells. The latter effect was most pronounced in E-LNPs. The novel S- and E-LNPs generated in this work are promising materials for biomedicine with advantageous properties such as small particle size and tailored surface functionality, making them an attractive and potentially biodegradable delivery tool for combination therapy in liver cancer, which still has to be verified in vivo using HCC and CCA models.

天然高分子木质素（lignin）具备独特的可生物降解性与生物相容性，使其在制备靶向癌症治疗用纳米颗粒的领域极具应用潜力。本研究针对云杉与桉木木质素纳米颗粒（分别记为S-LNPs与E-LNPs）展开了系统探究。两种木质素纳米颗粒均以经五步溶剂分级法（包含乙酸乙酯、乙醇、甲醇与丙酮）提取得到的高分子量（Mw）牛皮木质素不溶残渣为原料制备而成。所得S-LNPs与E-LNPs的粒径范围为16至60纳米，且无论木质素来源类型如何，均呈现均匀的球形形貌。以丙酮不溶级分木质素制备LNPs的方案颇具吸引力，这是因为该方法所用的高分子量木质素原本大多无法满足多数聚合物应用的要求，同时该制备路径具备规模化应用潜力，且所得LNPs的粒径均一稳定，不受木质素浓度提升的影响。鉴于LNPs作为肝癌递送载体的应用潜力，本研究首次在体外实验中评估了新型制备的E-LNPs与S-LNPs在两种原发性肝癌——肝细胞癌（hepatocellular carcinoma, HCC）与胆管癌（cholangiocarcinoma, CCA）——中的治疗效果。S-LNPs与E-LNPs均以剂量依赖方式抑制HCC细胞的增殖，且不会对CCA细胞系的生长产生影响。其中，E-LNPs处理组对HCC细胞的抑制效果更为显著，其抑制水平可与临床标准疗法索拉非尼（sorafenib）相媲美。此外，E-LNPs在抑制HCC细胞系的同时，还可诱导晚期凋亡与坏死性凋亡。本研究通过核磁共振（NMR）检测发现，LNPs表面富集的碳水化合物可能在介导LNPs与真核细胞的相互作用中发挥了关键作用，且该效应在E-LNPs中表现得更为突出。本研究制备的新型S-LNPs与E-LNPs是颇具前景的生物医药材料，具备粒径小巧、表面功能可定制等优势，使其成为一种极具吸引力且可生物降解的肝癌联合治疗递送工具，不过其实际应用效果仍需借助HCC与CCA模型在体内开展进一步验证。