RESUMEN
Interleukin-4 (IL-4) is an immune-modulating therapeutic with growing potential for the treatment of inflammatory diseases. Current challenges of IL-4 therapy include a low serum half-life and pleiotropic activity, suggesting effective targeting of IL-4. To develop an interleukin-4 bioconjugate with rapid targeting to inflammatory disease sites, we report the chemical synthesis, bioconjugation, and in vitro characterization of a murine interleukin-4 (mIL-4) conjugate decorated with a fibroblast activation protein inhibitor (FAPI). The FAPI targeting moiety features 2,2',2â³,2â´-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to allow future biodistribution and imaging studies of the FAPI-mIL-4 bioconjugate. We demonstrated site-specific coupling of mIL-4 and FAPI-DOTA deploying chemo-enzyme and enzyme chemistries with a high purity exceeding 95%. The FAPI-DOTA modified mIL-4 was bioactive with polarization of murine macrophages into the M2 state while maintaining specific binding to FAP on fibroblast cells. Together, these results point to future in vivo use of the FAPI-mIL-4 bioconjugate to assess biodistribution and biological effects in animal models of inflammatory joint disease.
RESUMEN
Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.
