Lochmann Dirk, Weyermann Jörg, Georgens Christiane, Prassl Ruth, Zimmer Andreas
Institute for Pharmaceutical Technology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Eur J Pharm Biopharm. 2005 Apr;59(3):419-29. doi: 10.1016/j.ejpb.2004.04.001.
In this paper, a ternary system of albumin-protamine-oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). 'Initial complexes' of HSA and protamine sulphate with a mean hydrodynamic diameter (dh) of about 10-14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a dh in a range of 230-320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 microg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7-10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to -60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.
在本文中，介绍了Vogel等人最近研发的白蛋白-鱼精蛋白-寡核苷酸纳米颗粒（AlPrO-NP）三元体系[V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert，寡核苷酸-鱼精蛋白-白蛋白纳米颗粒：制备、物理性质及细胞内加工过程，《控制释放杂志》（即将发表）]，其可作为反义寡核苷酸的潜在药物递送系统。先前对二元鱼精蛋白-寡核苷酸纳米颗粒的研究显示出两个主要缺点：（i）在有盐存在的情况下，颗粒在几分钟内发生聚集；（ii）鱼精蛋白与寡核苷酸，尤其是硫代磷酸酯之间的细胞内解离率较低。为克服这些问题，引入了作为无毒、可生物降解大分子的人血清白蛋白（HSA）作为保护胶体。通过小角X射线散射（SAXS）、荧光相关光谱（FCS）、光子相关光谱（PCS）测量以及扫描电子显微镜（SEM）对AlPrO-NP的组装过程进行了研究。发现HSA与硫酸鱼精蛋白的“初始复合物”的平均流体动力学直径（dh）约为10 - 14 nm。加入寡核苷酸（未修饰的、硫代磷酸酯DNA和小干扰RNA）后，纳米颗粒（NP）在水中以及等渗介质中组装，对于大多数制剂，其dh在230 - 320 nm范围内。通过高效液相色谱和荧光光谱法研究了颗粒的化学组成。以1:2的质量比（寡核苷酸/鱼精蛋白）将全部寡核苷酸（30μg）包封于颗粒中。约7 - 10%（w/w）的HSA与颗粒结合。颗粒的表面电荷根据鱼精蛋白浓度和离子条件在约 +12至 -60 mV范围内变化。根据FCS数据计算了各组分、初始复合物以及两种模型NP制剂的大小和分子量。这些数据证实了PCS、SEM和SAXS测量结果。
