No abstract

Here we present a “smart” xenon-129 NMR biosensor that undergoes a peptide conformational change and labels cancer cells at acidic pH. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide that is alpha-helical at pH 5.5 and disordered at pH 7.5. The 129Xe NMR chemical shift at rt was strongly pH-dependent (Δδ = 3.4 ppm): δ = 64.2 ppm at pH 7.5 vs. δ = 67.6 ppm at pH 5.5 where Trp(peptide)-cryptophane interactions were evidenced by Trp fluorescence quenching. Using Hyper-CEST NMR, we probed peptido-cryptophane detection limits at low-picomolar (10−11 M) concentration, which compares favourably to other NMR pH sensors at 10−2−10−3 M. Finally, in biosensor-HeLa cell solutions, peptide-cell membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-129Xe NMR chemical shift relative to pH 7.5 studies. This highlights new uses for 129Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for pH-dependent cell labeling in cancer diagnosis and treatment.

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized 129Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized 129Xe NMR experiments.

Malaria is a deadly infectious disease caused by parasites of the Plasmodium spp. that takes an estimated 435,000 lives each year, primarily among young African children. For most children, malaria is a febrile illness that resolves with time, but in ∼1% of cases, for reasons we do not understand, malaria becomes severe and life threatening. Cerebral malaria (CM) is the most common form of severe malaria, accounting for the vast majority of childhood deaths from malaria despite highly effective antiparasite chemotherapy. Thus, CM is one of the most prevalent lethal brain diseases, and one for which we have no effective therapy. CM is, in part, an immune-mediated disease, and to fully understand CM, it is essential to appreciate the complex relationship between the malarial parasite and the human immune system. In this study, we provide a primer on malaria for immunologists and, in this context, review progress identifying targets for therapeutic intervention.