cIBR effectively targets nanoparticles to LFA-1 on acute lymphoblastic T cells

Leukocyte function associated antigen-1 (LFA-1) is a primary cell adhesion molecule of leukocytes required for mediating cellular transmigration into sites of inflammation via the vascular endothelium. A cyclic peptide, cIBR, possesses high affinity for LFA-1 and conjugation to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles can specifically target and deliver the encapsulated agents to T cells expressing LFA-1. The kinetics of targeted nanoparticle uptake by acute lymphoblastic leukemia T cells was investigated by flow cytometry and microscopy and compared to untargeted nanoparticles. The specificity of targeted nanoparticles binding to the LFA-1 integrin was demonstrated by competitive inhibition using free cIBR peptide or using the I domain of LFA-1 to inhibit the binding of targeted nanoparticles. The uptake of targeted nanoparticles was concentration and energy dependent. The cIBR-conjugated nanoparticles did not appear to localize with lysosomes whereas untargeted nanoparticles were detected in lysosomes in 6 hrs and steadily accumulated in lysosomes for 24 hrs. Finally, T-cell adhesion to epithelial cells was inhibited by cIBR-nanoparticles. Thus, nanoparticles displaying the cIBR ligand may offer a useful targeted drug delivery system as an alternative treatment of inflammatory diseases involving transmigration of leukocytes

Rapid Identification of Fluorochrome Modification Sites in Proteins by LC ESI-Q-TOF Mass Spectrometry

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5′-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain