Transient fluid flow improves photoimmunoconjugate delivery and photoimmunotherapy efficacy

Abstract

Circulating drugs in the peritoneal cavity is an effective strategy for advanced ovarian cancer treatment. Photoimmunotherapy, an emerging modality with potential for the treatment of ovarian cancer, involves near-infrared light activation of antibody-photosensitizer conjugates (photoimmunoconjugates) to generate cytotoxic reactive oxygen species. Here, a microfluidic cell culture model is used to study how fluid flow-induced shear stress affects photoimmunoconjugate delivery to ovarian cancer cells. Photoimmunoconjugates are composed of the antibody, cetuximab, conjugated to the photosensitizer, and benzoporphyrin derivative. Longitudinal tracking of photoimmunoconjugate treatment under flow conditions reveals enhancements in subcellular photosensitizer accumulation. Compared to static conditions, fluid flow-induced shear stress at 0.5 and 1 dyn/cm² doubled the cellular delivery of photoimmunoconjugates. Fluid flow-mediated treatment with three different photosensitizer formulations (benzoporphyrin derivative, photoimmunoconjugates, and photoimmunoconjugate-coated liposomes) led to enhanced phototoxicity compared to static conditions. This study confirms the fundamental role of fluid flow-induced shear stress in the anti-cancer effects of photoimmunotherapy.

Keywords: Biotechnology; Cancer; Drug delivery system; Fluidics; Nanotechnology.

Graphical abstract

Figure 1

Transient fluid shear stress-induced effects on PIC purity, cell viability, and EGFR expression (A–C) Comparison of PIC purity following incubation under static or flow conditions. Cell viability (D), morphology (E and F), and EGFR expression (G and H) were evaluated following drug-free treatment under static or 1 dyn/cm² conditions for 30 min. Statistical analysis was performed using one-way ANOVA (C) and t tests (D and H). Error bars represent the standard error of the mean. ∗p ≤ 0.05; ns: nonsignificant.

Figure 2

Longitudinal monitoring of subcellular photosensitizer and protein under transient fluid shear stress Confluent monolayers of OVCAR8 cells were treated with 1 μM PIC under static or FSS (5 dyn/cm²) conditions for 10, 30, 60, 120, 180, 240, and 300 min. At each time point, cells were fractionated using Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Inc.). Longitudinal data for total BPD and protein are plotted for the plasma membrane (A), organelle compartment (B), nucleus (C), and cytosol (D). BPD association profiles are shown for the groups treated under static and FSS conditions with a dotted blue line and a solid blue line, respectively. Statistical analysis was performed using t tests at each time point. Error bars represent the standard error of the mean. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ns: nonsignificant.

Figure 3

Comparison of total BPD, total protein, and BPD/protein at varying fluid shear stresses OVCAR8 cell monolayers plated μ-Slide I^0.6 Luer chips were treated with 1 μM PIC for 30 min under static or FSS (0.5, 1, 5 dyn/cm²) conditions. Total BPD and protein were quantified by UV-Vis spectrophotometry and BCA, respectively. Averaged data (A) and individual datapoints (B) are shown, as well as the normalized BPD/protein (C). Statistical analysis was performed using a one-way ANOVA, and FSS data were compared to static controls using post hoc Dunnet test. Error bars represent the standard error of the mean. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ns: nonsignificant.

Figure 4

Fluid shear stress-dependent changes in subcellular BPD and protein compartmentalization Monolayers of OVCAR8 cells were treated with 1 μM PIC under static or FSS (0.5, 1, 5 dyn/cm²) conditions and then fractionated for subcellular analysis of BPD, protein, and BPD/protein. FSS-dependent changes in BPD and protein (A–D). Individual datapoints for BPD versus protein (E–H). Normalized BPD/protein in each compartment (I–L). Statistical analysis was performed using a one-way ANOVA, and FSS data were compared to static controls using post hoc Dunnet test. Error bars represent the standard error of the mean. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns: nonsignificant.

Figure 5

Percent distribution of BPD and protein among subcellular compartments Total BPD (A) and total protein (B) values were summed across all compartments; then values in each compartment were divided by the sum to determine percentage. Circle diameter is equal to the fold change in total BPD or protein compared to static condition. Fold change is listed inside each circle. Compartment percentages are listed outside each segment.

Figure 6

Cytotoxicity and uptake of three photosensitizer formulations following transient flow-mediated treatment Confluent monolayers of OVCAR8 cells were treated with 1 μM PIC (A), 1 μM PIC-Nal (B), or 1 μM free BPD (C) for 30 min under static or FSS (1 dyn/cm²) conditions. Next, medium was exchanged and chips were irradiated with a 690 nm laser at 10, 20, 40, or 80 J/cm² (100 mW/cm²). After 4 h, cell viability was analyzed by NRU. For uptake studies, cells were incubated under static or FSS conditions with PIC (D), PIC-Nal (E), or BPD (F) and then immediately washed, collected, and analyzed as described in STAR Methods. Statistical analysis was performed using unpaired t tests. Error bars represent the standard error of the mean. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 ns: nonsignificant.