TmDOTA-tetraglycinate encapsulated liposomes as pH-sensitive LipoCEST agents

Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)₄ ⁻) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)₄ ⁻ complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)₄ ⁻ at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼10⁴ compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors.     

Multisite Evaluation of Point of Care CD4 Testing in Papua New Guinea

Laboratory-based CD4 monitoring of HIV patients presents challenges in resource limited settings (RLS) including frequent machine breakdown, poor engineering support and limited cold chain and specimen transport logistics. This study assessed the performance of two CD4 tests designed for use in RLS; the Dynal assay and the Alere PIMA test (PIMA). Accuracy of Dynal and PIMA using venous blood was assessed in a centralised laboratory by comparison to BD FACSCount (BD FACS). Dynal had a mean bias of −50.35 cells/µl (r² = 0.973, p<0.0001, n = 101) and PIMA −22.43 cells/µl (r² = 0.964, p<0.0001, n = 139) compared to BD FACS. Similar results were observed for PIMA operated by clinicians in one urban (n = 117) and two rural clinics (n = 98). Using internal control beads, PIMA precision was 10.34% CV (low bead mean 214.24 cells/µl) and 8.29% (high bead mean 920.73 cells/µl) and similar %CV results were observed external quality assurance (EQA) and replicate patient samples. Dynal did not perform using EQA and no internal controls are supplied by the manufacturer, however duplicate testing of samples resulted in r² = 0.961, p<0.0001, mean bias = −1.44 cells/µl. Using the cut-off of 350 cells/µl compared to BD FACS, PIMA had a sensitivity of 88.85% and specificity of 98.71% and Dynal 88.61% and 100%. A total of 0.44% (2/452) of patient samples were misclassified as “no treat” and 7.30% (33/452) “treat” using PIMA whereas with Dynal 8.91% (9/101) as “treat” and 0% as “no treat”. In our setting PIMA was found to be accurate, precise and user-friendly in both laboratory and clinic settings. Dynal performed well in initial centralized laboratory evaluation, however lacks requisite quality control measures, and was technically more difficult to use, making it less suitable for use at lower tiered laboratories.