Combinatorial Synthesis of Peptoid Arrays via Laser-Based Stacking of Multiple Polymer Nanolayers.

Here, the combinatorial synthesis of molecule arrays via a laser-assisted process is reported. Laser-transferred polymer nanolayers with embedded monomers, activators, or bases can be reliably stacked on top of each other, spot-by-spot, to synthesize molecule arrays. These various chemicals in the nanometer-thin layers are mixed by heat or solvent vapor, inducing coupling reactions. As an example, peptoid arrays with a density of 10 000 spots per cm2 with the sub-monomer or monomer method are generated. Moreover, successful reactions spot-by-spot are verified by laser-transferring MALDI-matrix (Matrix-assisted laser desorption/ionization) followed by MALDI mass spectrometry imaging.

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS).

Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2 . To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

Phenolic excipients of insulin formulations induce cell death, pro-inflammatory signaling and MCP-1 release.

Skin reactions at the infusion site are a common side effect of continuous subcutaneous insulin infusion therapy. We hypothesized that local skin complications are caused by components of commercial insulin formulations that contain phenol or m-cresol as excipients. The toxic potential of insulin solutions and the mechanisms leading to skin reactions were explored in cultured cells. The toxicity of insulin formulations (Apidra, Humalog, NovoRapid, Insuman), excipient-free insulin, phenol and m-cresol was investigated in L929 cells, human adipocytes and monocytic THP-1 cells. The cells were incubated with the test compounds dose- and time-dependently. Cell viability, kinase signaling pathways, monocyte activation and the release of pro-inflammatory cytokines were analyzed. Insulin formulations were cytotoxic in all cell-types and the pure excipients phenol and m-cresol were toxic to the same extent. P38 and JNK signaling pathways were activated by phenolic compounds, whereas AKT phosphorylation was attenuated. THP-1 cells incubated with sub-toxic levels of the test compounds showed increased expression of the activation markers CD54, CD11b and CD14 and secreted the chemokine MCP-1 indicating a pro-inflammatory response. Insulin solutions displayed cytotoxic and pro-inflammatory potential caused by phenol or m-cresol. We speculate that during insulin pump therapy phenol and m-cresol might induce cell death and inflammatory reactions at the infusion site in vivo. Inflammation is perpetuated by release of MCP-1 by activated monocytic cells leading to enhanced recruitment of inflammatory cells. To minimize acute skin complications caused by phenol/m-cresol accumulation, a frequent change of infusion sets and rotation of the infusion site is recommended.