Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles

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.

Figure 1

Aspect and calibration of the prototyped “IgHk calibrator beads”. (A) Calibration line of mouse IgG coated µ-QIFIkit calibrator beads on CyTOFLEX S (Beckman coulter, Villepinte France) for the calibration of human IgG kappa light chains coated beads (IgHk). (B) Quantification of Human IgHk on all prototyped IgHk calibrated beads using µ-QIFIkit as reference. IgHk beads have been first saturated with unlabeled L1C1 Mab, washed and fluorescently stained with PE-labeled anti-mouse IgG sencondary reagent in parallel with the µ-QIFIkit beads used as reference. (C) Superposition of histograms from the prototyped “IgHk calibrator beads” as seen on the PE channel of CytoFLEX S under yellow laser excitation. A limited series of 5 beads regularly distributed over the measuring range were chosen from the larger series of all (n = 13) calibrated beads to be later used in direct IF experiments with L1C1-PE as “IgHk calibrator beads”. (D) Calibration line of the prototyped “IgHk calibrator beads” generated for this study.

Figure 2

FCM analysis of non-fluorescent immunoliposomes and instrument background noise (A), DiD+ immunoliposomes (B), DiD+ Trastuzumab uncoated liposomes (C) and DiD+ Trastuzumab coated immunoliposomes (D). Inset figures present FSC-SSC plots showing the distribution of background noise events in blue dots (insets A and B) and specific detection of DID stained liposomes with red dots (inset B).

Figure 3

Overlay plot in the revelation Antibody PE fluorescence channel. Color lines: IgGH calibrator beads coated with 0 (green), 18 (pink), 175 (violet), 830 (blue), 2850 (orange) and 7450 (red) IgGH antibodies respectively. Black line represents PE distribution of trastuzumab coated DiD+ immunoliposomes.

Figure 4

Number of coated trastuzumab on three immunoliposome batches over time^a. ^aValues are mean ± SEM of three or more experiments.

Figure 5

Monitoring of cell population (%) in early (A) and late (B) apoptosis when treated with three batches of free drugs, liposomes and immunoliposomes for 12 and 72 hours^a. ^aValues are mean of three or more experiments.

Figure 6

Monitoring of dtomato fluorescent MDA-MB-453 when seeded in 4000 cell spheroids and treated at day 3 and day 10^a. ^aExperiment was in triplicate.

Figure 7

Cell viability (%) of MDA-MB-453 spheroids 14 days after seeding^a. ^aValues are mean of three or more experiments.