Atomic Force Microscopy Investigation of an Algorithm for Calculation of Targeted Liposomal Binding Force Based on Affinity Measurements

Background/Objectives: Ligand-target binding force is a valuable quality control parameter for evaluation of targeted nanoparticulate formulations. Atomic force microscopy (AFM) is the method of choice for measuring this parameter, but AFM instruments are expensive and not available to researchers outside of universities, large medical centers, and technology hubs. We have developed a computational method for determining binding force from enzyme-linked immunosorbent assay (ELISA) affinity measurements, which may also be obtained by surface plasmon resonance (SPR) and isothermal titrating calorimetry (ITC) measurements. We have tested a series of tissue plasminogen activator (tPA)-loaded echogenic liposome (TELIP) preparations, in which the tPA fibrin-binding sites are exposed, with AFM to confirm the accuracy and to validate the computational method. Methods: Plastic culture dishes (35 mm) were coated with monoclonal anti-tPA antibodies (5 µg/mL) overnight at 4 °C. After blocking of unoccupied sites with 1% bovine serum albumin (BSA), dishes were incubated with 1.5 mL of TELIP (1 mg lipid/mL 0.02 M phosphate-buffered saline, pH 7.4) for 2 h at 37 °C. After washing with PBS, dishes were scanned with fibrin-coated AFM probes. Binding force was expressed as pN/molecule. Results: The computed binding force of seven TELIP batches ranged from 407 to 441 pN/molecule. AFM values compared favorably with the computed values, but varied over a wider range. AFM measurements were made on four TELIP batches. Conclusions: The computational algorithm has been validated and can be used as a translational tool.