Clickable Albumin Nanoparticles for Pretargeted Drug Delivery toward PD-L1 Overexpressing Tumors in Combination Immunotherapy.

We present a simple methodology to design a pretargeted drug delivery system, based on clickable anti-programmed death ligand 1 (anti-PD-L1) antibodies (Abs) and clickable bovine serum albumin (BSA) nanoparticles (NPs). Pretargeted drug delivery is based on the decoupling of a targeting moiety and a drug-delivering vector which can then react in vivo after separate injections. This may be key to achieve active targeting of drug-delivering NPs toward cancerous tissue. In pretargeted approaches, drug-delivering NPs were observed to accumulate in a higher amount in the targeted tissue due to shielding-related enhanced blood circulation and size-related enhanced tissue penetration. In this work, BSA NPs were produced using the solvent precipitation methodology that renders colloidally stable NPs, which were subsequently functionalized with a clickable moiety based on chlorosydnone (Cl-Syd). Those reactive groups are able to specifically react with dibenzocyclooctyne (DBCO) groups in a click-type fashion, reaching second-order reaction rate constants as high as 1.9 M-1·s-1, which makes this reaction highly suitable for in vivo applications. The presence of reactive Cl-Syd was demonstrated by reacting the functionalized NPs with a DBCO-modified sulfo-cyanine-5 dye. With this reaction, it was possible to infer the number of reactive moieties per NPs. Finally, and with the aim of demonstrating the suitability of this system to be used in pretargeted strategies, functionalized fluorescent NPs were used to label H358 cells with a clickable anti-PD-L1 Ab, applying the reaction between Cl-Syd and DBCO as corresponding clickable groups. The results of these experiments demonstrate the bio-orthogonality of the system to perform the reaction in vitro, in a period as short as 15 min.

Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells.

High ambient glucose exerts its injurious effects on renal cells through nonenzymatic and enzymatic pathways, including altered signal transduction and upregulation of the transforming growth factor-beta (TGF-beta) system. Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade, is activated in mesangial cells cultured in high glucose and in glomeruli of diabetic rats. However, the biologic consequences of ERK activation in the kidney have not been investigated. To clarify the role of ERK activation, mouse mesangial cells were exposed to normal (5.5 mM) or high (25 mM) glucose with or without addition of PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), an upstream kinase activator of ERK. Cells that were exposed to high glucose exhibited significant increases in ERK activity, TGF-beta1 expression (total protein, mRNA levels, and promoter activity), [(3)H]-proline uptake, and alpha1(I) collagen and fibronectin mRNA levels. Treatment with PD98059 (up to 25 microM) significantly inhibited these parameters. In contrast, 25 microM PD98059 had no significant effect on any of the parameters measured in cells that were exposed to normal glucose. Overexpression of MAPK phosphatase CL 100 prevented TGF-beta1 promoter activation by high glucose, confirming the involvement of the MEK-ERK pathway in response to high glucose. The conclusion is that activation of ERK in mesangial cells is responsible for high-glucose-induced stimulation of TGF-beta1 and contributes to the increased extracellular matrix expression.