Engineering liposomes as detection reagents for CD4+ T-cells

Abstract

Liposomes are often used for targeted and controlled delivery of sensitive pharmaceutical compounds. They may be synthesized encapsulating drugs within their aqueous cores and their surfaces may be functionalized with antibodies and polyethylene glycol (PEG) for site-specific delivery and increased circulation time, respectively. Here we investigated the use of liposomes for cell detection in clinical diagnostics, relying on a specific biorecognition event rather than non-specific fusion with cell membranes. Streptavidin-conjugated sulforhodamine B-encapsulating liposomes were used to provide fluorescence signal amplification in a microtiter plate-based immunoassay, relying on sandwich complex formation of CD4+ T-cells between anti-CD3 or anti-CD45 antibodies and biotinylated anti-CD4+ detection antibodies. Liposomes were engineered to avoid fusion with T-cells, non-specific binding and self-aggregation, as well as promoting specific binding, encapsulation efficiency and stability. Increasing the mol% coverage of the carboxylated lipid N-glutaryl-DPPE increased the streptavidin coupling and dye encapsulation efficiency. However, increasing amounts of this lipid also increased non-specific binding to T-cells and monocytes. Thus, PEG–lipid conjugates were included in the formulation with varied coverage (0.5–3.0 mol% of total lipid) and PEG chain length (MW 350–5000). Increases in both parameters increased the liposomes' zeta potential and decreased non-specific binding, presumably due to a shielding effect on the negative charge. The optimal formulation was able to detect as few as 30 cells per μL from negatively isolated cell preparations from human blood samples. The assay responded with excellent reproducibility and discrimination between at risk and normal CD4+ cell counts over the CD4+ cell concentration range expected, which makes this method amenable to monitoring cell concentrations in those with other immunosuppressive diseases or currently receiving anti-retroviral therapy. The successful engineering of liposomes as diagnostic tools for immune cell detection expands their use to whole cell-based studies for cancer research and clinical diagnostics.