Martin C. Lok

Poly(amido amine)s as gene delivery vectors: effects of quaternary nicotinamide moieties in the side chains

To evaluate the effect of quaternary nicotinamide pendant groups on gene delivery properties, a series of poly(amido amine) (co)polymers were synthesized by Michael addition polymerization of N, N′‐cystaminebisacrylamide with variable ratios of 1‐(4‐aminobutyl)‐3‐carbamoylpyridinium (Nic‐BuNH2), and tert‐butyl‐4‐aminobutyl carbamate (BocNH‐BuNH2), yielding poly(amido amine)s (NicX‐NHBoc) with X=0, 10, 30, and 50 % of quaternary nicotinamide groups in the polymer side chains. Deprotection of the pendant Boc‐NH groups afforded an analogous series of polymers (NicX‐NH2) with higher charge density (due to the presence of protonated primary amino groups in the side chains) and subsequent acetylation yielded a series of polymers (NicX‐NHAc) of lower hydrophobicity than the Boc‐protected polymers. The polymers with the Boc‐protected or the acetylated amino groups showed high buffer capacity in the range pH 5.1–7.4, which is a property that can contribute to endosomal escape of polyplexes. The presence of quaternary nicotinamide groups has distinct beneficial effects on the gene vector properties of these polymers. The polymers containing ≥30 % of quaternary nicotinamide groups in their side chains condense DNA into small, nanosized particles (≤200 nm) with positive surface charge (≥+15 mV). Fluorescence experiments using ethidium bromide as a competitor showed that the quaternary nicotinamide groups intercalate with DNA, contributing to a more intimate polymer–DNA binding and shielding. Polyplexes of nicotinamide‐functionalized poly(amido amine)s NicX‐NHBoc and NicX‐NHAc, formed at 12/1 polymerDNA mass ratio, efficiently transfect COS‐7 cells with efficacies up to four times higher than that of PEI (Exgen 500), and with essentially absence of cytotoxicity. NicX‐NH2 polymers, possessing protonated primary amino groups in their side chains, have a higher cytotoxicity profile under these conditions, but at lower 3/1 polymer‐DNA mass ratio also these polymers are capable of efficient transfection, while retaining full cell viability.

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.

Analysis of High Throughput Protein Expression in Escherichia coli

The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.