Abstract
Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles.
Highlights
Development of nanoparticles (NP) for targeted delivery and controlled drug release may improve the therapeutic index of drugs, especially that of anticancer agents
As described in detail in the Supplemental data section, the 10 μm QIFIkit calibrator, was scaled-down to generate a series of prototype 1 μm-sized “μ-QIFIkit” calibrator beads covering an approximate range of ~20 to ~20,000 mouse IgG/ bead
Based on the expression of two kappa light chains per IgG, we considered that immunoliposomes may theoretically bind 2 molecules of PE anti-Human IgG reagent, resulting in dividing by 2 each result
Summary
Development of nanoparticles (NP) for targeted delivery and controlled drug release may improve the therapeutic index of drugs, especially that of anticancer agents. This technique has already demonstrated its high interest in the characterization of both biological and synthetic particles[11,12] Such FCM-based quantitative analysis of immuno-staining should be applied on an absolute rather than only relative basis through ad hoc calibration[13]. This allows reproducible measurements at various time points providing more meaningful results expressed as molecules/cell rather than in arbitrary units (a.u.) of fluorescence[13,14,15,16,17,18]. The aims of this work were first to develop an original QFCM assay to measure the number of antibodies coated on the surface of submicron-sized particles and second to evaluate the potential impact of antibodies coating level on immunoliposomes cytotoxic effect
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