Voltan Rebecca, Castaldello Arianna, Brocca-Cofano Egidio, Altavilla Giuseppe, Caputo Antonella, Laus Michele, Sparnacci Katia, Ensoli Barbara, Spaccasassi Silvia, Ballestri Marco, Tondelli Luisa
Department of Histology, Microbiology and Medical Biotechnology, Section of Microbiology, University of Padova, Via A. Gabelli 63, 35122, Padova, Italy.
Pharm Res. 2007 Oct;24(10):1870-82. doi: 10.1007/s11095-007-9310-8. Epub 2007 May 3.
This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates.
Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model.
The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice.
This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.
本研究旨在开发新型核壳聚甲基丙烯酸甲酯（PMMA）纳米颗粒作为候选蛋白疫苗的递送系统。
基于乳液聚合的创新合成方法制备了由PMMA核和源自Eudragit L100/55的壳组成的阴离子纳米颗粒。通过无细胞研究对形成的纳米颗粒的尺寸、表面电荷以及可逆结合两种基本模型蛋白（溶菌酶、胰蛋白酶）和一种疫苗相关抗原（HIV-1 Tat）的能力进行了表征。在细胞培养系统中研究了它们的体外毒性以及保存HIV-1 Tat蛋白生物活性的能力。最后，在小鼠模型中研究了它们的安全性和免疫原性。
纳米颗粒表面光滑、呈球形且尺寸分布均匀，平均直径为220 nm。壳的特征是在生理pH下带负电荷的共价结合羧基，能够主要通过特异性静电相互作用可逆地吸附大量（高达20% w/w）的碱性蛋白（溶菌酶、胰蛋白酶和HIV-1 Tat）。纳米颗粒稳定，对细胞无毒，保护HIV-1 Tat蛋白免受氧化，从而保留其生物活性并延长其保质期，并在细胞内有效地递送和释放它。体内实验表明，它们在小鼠中耐受性良好，并能引发针对递送抗原的强烈免疫反应。
本研究表明，这些新型纳米颗粒为蛋白质表面吸附提供了一个通用平台，是一种有前景的递送系统，特别是当疫苗效力需要维持生物活性构象时。
