Department of Drug Sciences, University of Pavia, V.le Taramelli, 12, 27100 Pavia, Italy.
Int J Pharm. 2013 Aug 16;452(1-2):390-401. doi: 10.1016/j.ijpharm.2013.05.037. Epub 2013 May 23.
The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19). At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (-2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 μg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4°C, 25°C and 37°C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37°C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2h. At 4°C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA19 loaded nanoparticles cytotoxicity (up to 10.652 mg PLGA/ml) by MTT assay and the study of cellular up-take assessed on human fibroblasts confirmed the feasibility to formulate a dosage form useful for vaccination against S. aureus-mediated infections.
特别是，开发了聚乳酸-共-羟基乙酸（PLGA）纳米粒，以便有效负载和增强亚单位模型疫苗，即纯化的重组胶原结合细菌黏附片段（CNA19）。首先，通过皮下给药评估游离 CNA19 的实际免疫原性，以将其作为亚单位抗原模型。其次，为了开发负载 CNA19 的 PLGA 纳米粒，首先进行了初步研究，即通过 w/o/w 双乳液法在温和条件下利用超声循环制备形态良好的纳米粒，然后优化了冷冻干燥条件和不同的 CNA19 加载方法（包封、吸附在空白或负载 CNA19 的纳米粒上）。制剂方法的建立（收率约为 83%）可获得具有球形、窄粒径分布（187.41 ± 51.2nm）、轻微负 ζ 电位（-2.91 ± 0.64mV）的 CNA19 负载纳米粒，并获得令人满意的蛋白包封效率（75.91 ± 4.22%）和载药量（8.59 ± 0.33μg CNA19/纳米粒 mg）。然后，通过以下方式对负载 CNA19 的 PLGA 纳米粒进行了表征：（i）在不同温度（4°C、25°C 和 37°C）下进行体外释放试验，以测试抗原完整性（SDS-PAGE）和活性（ELISA）；（ii）在 21 天的时间内进行了尺寸和表面电荷方面的体外稳定性研究。在 37°C 下，发现抗原几乎以持续释放的形式，以活性形式释放近 240h，在前 2h 内出现约 20%的爆发式释放。在 4°C 下进行的稳定性试验和活性测定可确定有助于维持 CNA19 活性和易于 NP 重新悬浮的储存条件，同时保持物理特性完整。此外，通过 MTT 测定法评估负载 CNA19 的纳米粒的细胞毒性（高达 10.652mg PLGA/ml），并在人成纤维细胞上评估细胞摄取研究，证实了可用于配制针对金黄色葡萄球菌感染的疫苗剂型的可行性。
