Table_1_Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.DOCX

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 μg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

纳米颗粒（NPs）在药物递送领域扮演着举足轻重的角色。尽管近年来涉及NPs的研究数量持续增长，但对其对免疫系统可能产生的副作用往往被忽视或被省略。在基于纳米药物递送系统的聚合物中，壳聚糖（Chit）是研究最为广泛的聚合物之一。在科学文献中，尽管壳聚糖的物化性质（如分子量（MW）或脱乙酰化度（DDA））、内毒素污染以及恰当的测试控制措施鲜有报道，但这些因素能够显著影响免疫毒性测试结果。本研究旨在探讨不同DDA和MW的壳聚糖聚合物生产的NPs的免疫毒性，并将其与聚合物本身进行比较。基于壳聚糖与三聚磷酸钠（TPP）的离子凝胶化，制备了壳聚糖NPs，包括壳聚糖80% NPs（80% DDA）和壳聚糖93% NPs（93% DDA）。总体而言，在体外使用人外周血单核细胞（PBMCs）或RAW 264.7细胞系进行测试时，我们发现壳聚糖NPs引起的细胞活力降低程度大于相应的壳聚糖聚合物。此外，与壳聚糖93% NPs相比，壳聚糖80% NPs对PBMCs更具细胞毒性，并在RAW 264.7细胞系中增加了活性氧（ROS）的产生（超过156 μg/mL），同时干扰了凝血的内在途径（在1 mg/mL时）。另一方面，只有壳聚糖93% NPs在2 mg/mL时诱导了血小板聚集。尽管壳聚糖NPs和聚合物在RAW 264.7细胞中并未刺激一氧化氮（NO）的产生，但它们在所有测试浓度下均降低了脂多糖（LPS）诱导的NO产生。壳聚糖NPs和聚合物均未引起溶血，也未诱导PBMCs分泌TNF-α和IL-6细胞因子。根据所获得的结果，我们得出结论，壳聚糖聚合物的DDA和壳聚糖NPs的大小会影响体外免疫毒性测试结果。由于NPs的细胞毒性比相应的聚合物更强，因此在从聚合物外推到NPs的趋势以及在具有不同DDA的壳聚糖制备的递送系统之间的比较时，应谨慎行事。