Zuzana Kadlecova

DNA delivery with hyperbranched polylysine: A comparative study with linear and dendritic polylysine

PEI and polylysine are among the most investigated synthetic polymeric carriers for DNA delivery. Apart from their practical use, these 2 classes of polymers are also of interest from a fundamental point of view as they both can be prepared in different architectures (linear and branched/dendritic) and in a wide range of molecular weights, which is attractive to establish basic structure-activity relationships. This manuscript reports the results of an extensive study on the influence of molecular weight and architecture of a library of polylysine variants that includes linear, dendritic and hyperbranched polylysine. Hyperbranched polylysine is a new polylysine-based carrier that is structurally related to dendritic polylysine but possesses a randomly branched structure. Hyperbranched polylysine is attractive as it can be prepared in a one-step process on a large scale. The performance of these 3 classes of polylysine analogs was evaluated by assessing eGFP and IgG production in transient gene expression experiments with CHO DG44 cells, which revealed that protein production generally increased with increasing molecular weight and that at comparable molecular weight, the hyperbranched analogs were superior as compared to the dendritic and linear polylysines. To understand the differences between the gene delivery properties of the hyperbranched polylysine analogs on the one hand and the dendritic and linear polylysines on the other hand, the uptake and trafficking of the corresponding polyplexes were investigated. These experiments allowed us to identify (i) polyplex-external cell membrane binding, (ii) free, unbound polylysine coexisting with polyplexes as well as (iii) polymer buffer capacity as three possible factors that may contribute to the superior transfection properties of the hyperbranched polylysines as compared to their linear and dendritic analogs. Altogether, the results of this study indicate that hyperbranched polylysine is an interesting, alternative synthetic gene carrier. Hyperbranched polylysine can be produced at low costs and in large quantities, is partially biodegradable, which may help to prevent cumulative cytotoxicity, and possesses transfection properties that can approach those of PEI.

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.

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools.

Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3-4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins.

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.

Assessment of transferrin recycling by Triplet Lifetime Imaging in living cells

An optical method is presented that allows the measurement of the triplet lifetime of a fluorescent molecule. This is a characteristic specific to each fluorophore. Based on differences in triplet lifetimes of two fluorescent species (autofluorescence versus label), this novel approach measures relative quantities of a transmembrane receptor and associated fluorescently labeled ligand during its recycling in living cells. Similarly to fluorescence-lifetime based methods, our approach is almost insensitive to photobleaching. A simple theory for unmixing two known triplet lifetimes is presented along with validation of the method by measurements of transferrin recycling in a model system based on chinese hamster ovarian cells (CHO). Transferrin is the delivery carrier for Fe3+ to the cell.

Quantification of Polyethylenimine in Transient Gene Expression: On the Way to GMP Compliance

Transient gene expression (TGE) allows production of virtually any recombinant protein (r-protein) in mammalian cells. Its flexibility, speed, scalability, and cost-effectiveness have been widely demonstrated. However, good manufacturing practices (GMP) have not been established for the production of r-proteins by TGE. In this study, a method was developed for the detection and quantification of polyethylenimine (PEI), the DNA delivery agent in TGE. Currently, there are no established methods to track this polymer during r-protein production and purification. Linear 25 kDa PEI was labelled with fluorescein, and the modified PEI was characterized by NMR and UV/VIS spectroscopy. The optimal conditions for an accurate measurement of PEI by fluorescence were defined, and the limit of detection and quantification were determined. Importantly, the labeling of PEI did not alter its capacity to form polyplexes with plasmid DNA and to efficiently transfect HEK-293 cells in suspension. The assay we developed is expected to be an essential tool for the establishment of GMP protocols for the production of r-proteins by TGE.

Assessing the Cellular Uptake Pathway for Poly-Lysine Analogues using Triplet Lifetime Imaging

Research on synthetic delivery vectors is of major interest for cell imaging and manipulation, as they allow an efficient transfer of nucleic acids, therapeutic proteins or small drugs into the cells. We have developed a library of L-lysine analogues that allow for highly efficient gene delivery with low cytotoxicity. However little is known on the exact mechanism of uptake and the final intracellular destination of the synthetic carriers. Therefore we have developed a novel optical technique based on a modulated excitation allowing for intracellular imaging of the triplet-lifetime and -yield of fluorophores attached to the delivery vector. Both these parameters are highly dependant on the intracellular environment thus provide insight into the subcellular localization of the labelled carrier. The method combines high temporal and spatial resolution and is compatible with a multiplicity of fluorophores. We performed series of model experiments to compare the triplet lifetime and triplet yield behaviour during the natural uptake mechanism to a series of controlled conditions. The latter include microinjection of fluorescently labelled carriers directly into the cytoplasm and cell nucleus as well as in vitro measurements under conditions mimicking physiological, acidic, or DNA rich environments. To validate our technique the results from the triplet imaging were compared with two complementary methods: carrier localization by subcellular fractionation and confocal laser scanning microscopy. --- Reference --- Geissbuehler et al. Triplet imaging of Oxygen consumption during the contraction of a single smooth muscle cell (A7r5). Biophysical Journal (2010) vol. 98 (2) pp. 339-349