The application of STEP-technology® for particle and protein dispersion detection studies in biopharmaceutical research.

Particle detection and analysis techniques are essential in biopharmaceutical industries to evaluate the quality of various parenteral formulations regarding product safety, product quality and to meet the regulations set by the authority agencies. Several particle analysis systems are available on the market, but for the operator, it is quite challenging to identify the suitable method to analyze the sample. At the same time these techniques are the basis to gain a better understanding in biophysical processes, e.g. protein interaction and aggregation processes. The STEP-Technology® (Space and Time resolved Extinction Profiles), as used in the analytical photocentrifuge LUMiSizer®, has been shown to be an effective and promising technique to investigate particle suspensions and emulsions in various fields. In this study, we evaluated the potentials and limitations of this technique for biopharmaceutical model samples. For a first experimental approach, we measured silica and polystyrene (PS) particle standard suspensions with given particle density and refractive index (RI). The concluding evaluation was performed using a variety of relevant data sets to demonstrate the significant influences of the particle density for the final particle size distribution (PSD). The most challenging property required for successful detection, turbidity, was stated and limits have been set based on the depicted absorbance value at 320 nm (A320 values). Furthermore, we produced chemically cross-linked protein particle suspensions to model physically "stable" protein aggregates. These results of LUMiSizer® analysis have been compared to the orthogonal methods of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) and micro-flow imaging (MFI). Sedimentation velocity distributions showed similar tendencies, but the PSDs and absolute size values could not be obtained. In conclusion, we could demonstrate some applications as well as limitations of this technique for biopharmaceutical samples. In comparison to orthogonal methods this technique is a great complementary approach if particle data e.g. density or refractive index can be determined.

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.