Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Biodegradable poly(ethylene glycol)-peptide hydrogels with well-defined structure and properties for cell delivery.

In this study, biodegradable PEG-peptide hydrogels have been synthesized using Click chemistry. A series of Arg-Gly-Asp (RGD) containing peptides were prepared via a solid phase synthesis approach, which were further functionalized with azide to yield peptide azide or peptide diazide. A tetra-hydroxy terminated 4-arm PEG was functionalized with acetylene and was reacted with peptide azide/diazide and/or PEG diazide to produce hydrogels via a copper mediated 1,3-cycloaddition (Click chemistry) generating a triazole linkage as the networking forming reaction. The gelation time ranged from 2 to 30 min, depending on temperature, catalyst and precursor concentration, as well as peptide structure. The resulting hydrogels were characterized by swelling, viscoelastic properties and morphology as well as their ability for cell attachment and proliferation. Hydrogels cross-linked by peptide diazide yielded higher storage modulus (G') with shorter spacers between azide groups. As expected, the swelling degree decreased while the G' increased with increasing the concentration of the precursors as a result of increased cross-linking density. Primary human dermal fibroblasts were used as model cells to explore the possibility of using the RGD peptide hydrogels for cell-based wound healing. The attachment and proliferation of the cells on the hydrogels were evaluated. The RGD peptide hydrogels synthesized with a peptide concentration of 2.7-5.4mm achieved significantly improved cell attachment and greater cell proliferation rate when compared to the hydrogels without RGD peptides. These hydrogels may provide a platform technology to deliver cells for tissue repair.

The self-assembly of biodegradable cationic polymer micelles as vectors for gene transfection.

Cationic micelles self-assembled from a biodegradable amphiphilic copolymer, poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-co-CES)) have recently been reported for efficient gene delivery and co-delivery of drug and nucleic acid. In this study, poly(ethylene glycol) (PEG) of various molecular weights (Mn=550, 1100 and 2000) was conjugated to P(MDS-co-CES) having different cholesterol grafting degrees to improve the stability of micelle/DNA complexes in the blood for systemic in vivo gene delivery. DNA binding ability, gene transfection efficiency and cytotoxicity of P(MDS-co-CES), PMDS, PEGylated PMDS and PEGylated P(MDS-co-CES) micelles were studied and compared. As with P(MDS-co-CES), PEG-P(MDS-co-CES) polymers could also self-assemble into stable micelles of small size. However, PMDS and PEG-PMDS without cholesterol could not form stable micelles but formed large particles. PEGylation of polymers significantly decreased their gene transfection efficiency in HEK293, HepG2, HeLa, MDA-MB-231 and 4T1 cells. However, increasing N/P ratio promoted gene transfection. An increased cholesterol grafting degree led to greater gene expression level possibly because of the more stable core-shell structure of the micelles. PEG550-P(MDS-co-CES) micelles induced high gene transfection level, comparable to that provided by P(MDS-co-CES) micelles. PEGylated polymers were much less cytotoxic than P(MDS-co-CES). PEGylated P(MDS-co-CES) micelles may provide a promising non-viral vector for systemic in vivo gene delivery.

PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion.

We have developed a new block copolymer gene carrier that comprises of a polyethylene glycol segment and a degradable cationic polyphosphoramidate (PPA) segment. This PEG-b-PPA copolymer carrier formed micelles upon condensation with plasmid DNA in aqueous solution. PEG-b-PPA/DNA micelles exhibited uniform and reduced particle size ranging from 80 to 100 nm and lowered surface charge, compared with complexes of DNA with the corresponding cationic PPA carrier. PEG-b-PPA/DNA micelles maintained similar transfection efficiency as PPA/DNA complexes, which was comparable to that of PEI/DNA complexes in HepG2 cells, but yielded about 16-fold lower transgene expression in primary rat hepatocytes than PPA/DNA complexes. Following bile duct infusion in Wistar rats, PEG-b-PPA/DNA micelles mediated 4-fold higher and more uniform gene expression in the liver than PPA/DNA complexes. Liver function tests and histopathological examination indicated that PEG-b-PPA/DNA micelles showed low toxicity and good biocompatibility in the liver. This study demonstrated the potential of PEG-b-PPA/DNA micelles as an efficient carrier for liver-targeted gene delivery.