Comparative study on the in vitro cytotoxicity of linear, dendritic, and hyperbranched polylysine analogues.

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC(50) values for the LPL analogues were ∼5-250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.

Hyperbranched polylysine: a versatile, biodegradable transfection agent for the production of recombinant proteins by transient gene expression and the transfection of primary cells.

The feasibility of a new transfection agent, HBPL, for the production of recombinant IgG antibody via TGE as well as for the transfection of primary cells is studied. Under the conditions investigated, transfection of CHO-DG44 cells using HBPL results in IgG yields that are comparable to those obtained with PEI. In experiments with CHO-K1 cells and MEFs the use of HPBL allows to achieve transfection efficiencies comparable to or better than those obtained with PEI or Fugene®. HBPL-mediated transfection does not require complex pre-formation, works well in serum-containing media and is biodegradable, which may prevent cumulative cytotoxicity and facilitates downstream processing.

Microfabricated solenoids and Helmholtz coils for NMR spectroscopy of mammalian cells.

NMR-microprobes based on solenoids and Helmholtz coils have been microfabricated and NMR-spectra of mammalian cells have successfully been taken. The microfabrication technology developed for these probes consists of three electroplated copper levels for low resistance coils and three SU-8 layers for the integration of microchannels. This technology allows fabricating solenoids, Helmholtz and planar coils on the same wafer. The coils have inner diameters in the range of 160 to 400 microm and detection volumes of 5 to 22 nL. The solenoid and Helmholtz coils show improved RF-field characteristics compared to a planar coil fabricated with the same process. The fabricated solenoid has a particularly low resistance of only 0.46 Omega at 300 MHz. Moreover, it is very sensitive and has a very uniform RF-field, but shows large line width. The Helmholtz coils are slightly less sensitive, but display a far narrower line width, and are therefore a good compromise. With a Helmholtz coil, a SNR of 620 has been measured after one scan on 9 nL pure water. An NMR-microprobe based on a Helmholtz coil has also been used to take spectra of CHO cells that have been concentrated in the sensitive region of the coil with a mechanical filter integrated into the channel.

NMR spectroscopy and perfusion of mammalian cells using surface microprobes.

NMR spectra of mammalian cells are taken using surface microprobes that are based on microfabricated planar coils. The surface microprobe resembles a miniaturized Petri dish commonly used in biological research. The diameter of the planar coils is 1 mm. Chinese Hamster Ovaries are immobilized in a uniform layer on the microprobe surface or patterned by an ink-jet printer in the centre of the microcoil, where the rf-field of the planar microcoil is most uniform. The acquired NMR spectra show the prevalent metabolites found in mammalian cells. The volumes of the detected samples range from 25 nL to 1 nL (or 50,000 to 1800 cells). With an extended set-up that provides fluid inlets and outlets to the microprobe, the cells can be perfused within the NMR-magnet while constantly taking NMR spectra. Perfusion of the cells opens the way to increased cell viability for long acquisitions or to analysis of the cells' response to environmental change.