Purpose: Macropinocytosis is a non-selective, receptor-independent endocytic pathway which is shared by all vertebrate cells and is characterized by membrane ruffling which is caused by actin filament polymerization1. Clathrin mediated endocytosis has been found to be the most effective route for delivering large drug molecules and nano-formulations into cells2. However, the feasibility of macropinocytosis route in drug delivery is yet to be explored3. Our goal is to determine the potential uses of novel macropinocytosis inducers developed in our lab and those that are commercially available in the delivery of therapeutic molecules (large/small) into the cell via macropinocytosis pathway for intracellular targeting. Methods: Retrospective analysis of macropinocytosis inducers (BAPT and MOMIPP) in the induction of methuosis in cancer cells revealed that these compounds enhance the uptake of large and small molecules into cells for intracellular targeting. In the breast cancer cells, SUM159, and its paclitaxel resistant variant (PAC200), paclitaxel was combined with macropinocytosis inducers (BAPT and MOMIPP) to increase the uptake of paclitaxel via macropinocytosis route. The cytotoxicity assay (SRB) was used to determine the enhanced uptake of paclitaxel in the cells. Furthermore, macromolecule delivery with the aid of macropinocytosis inducers was investigated using fluorescently labelled dextran (150 kDa) and fluorescently tagged antibody (anti-β-tubulin). The uptake of dextran and antibody was visualized using fluorescent microscopy. Results: A synergistic effect of combination of macropinocytosis inducers and paclitaxel in SUM159 and resistance variant PAC200 was observed in the uptake of small drugs (paclitaxel) in SRB assays. In addition, we found that co-incubating macromolecules/antibodies with macropinocytosis inducers may facilitate the uptake of antibodies/macromolecules into cells. As shown by higher mean fluorescence compared to the control, macropinocytosis inducing molecules (MOMIPP and BAPT) enhanced uptake of dextran and anti-β-tubulin antibodies in cancer cells. Conclusion: Inducers of macropinocytosis can be used to enhance intracellular delivery of small and large molecules, expanding the possibilities for drug delivery. Using macropinocytosis inducers, we are determining the uptake of formulations (nanotubes), plant-based toxins, and siRNAs. Optimization of micropinocytosis inducing molecules holds greater promise in delivery of biologics (antibodies, siRNAs, nucleic acids) and a wide range of therapeutic molecules. References: 1. Kay, R. R., Macropinocytosis: biology and mechanisms. Cells & Development 2021, 203713. 2. Ju, Y.; Guo, H.; Edman, M.; Hamm-Alvarez, S. F., Application of advances in endocytosis and membrane trafficking to drug delivery. Advanced drug delivery reviews 2020, 157, 118-141. 3. Liu, H.; Qian, F., Exploiting macropinocytosis for drug delivery into KRAS mutant cancer. Theranostics 2022, 12 (3), 1321.
Acknowledgements:The authors declare no conflict of interest.
Figure1: Synergy plots based on viability of SUM159 cells incubated with MOMIPP combined with Paclitaxel over 72 h following sulforhodamine B (SRB) assay. MOMIPP enhanced the uptake of the paclitaxel drug via macropinocytosis into the SUM159 cells inducing better cytotoxicity with average synergy score of 7.8 (Two independent experiments, each performed in triplicate.)
Figure 2: Synergy plots based on viability of paclitaxel resistant variant of SUM159 cells (PAC200) incubated with BAPT54 combined with Paclitaxel over 72h following sulforhodamine B (SRB) assay. BAPT54 enhanced the uptake of the paclitaxel drug via macropinocytosis into the PAC200 cells inducing better cytotoxicity with overall synergy score of 13.49. (Two independent experiments, each performed in triplicate.)
Figure 3: Uptake study of β-tubulin antibody modulated by macropinocytosis inducers (MOMIPP, BAPT) in cancer cells. A: - HCT116 cells incubated with macropinocytosis inducer, MOMIPP, and fluorescent labelled β-tubulin antibody over 6 h, compared to control (without MOMIPP). B: - SW620 cells incubated with macropinocytosis inducer, BAPT54, and fluorescent labelled β-tubulin antibody over 6 h, compared to control (without BAPT54). Higher green fluorescence in cells incubated with macropinocytosis inducers compared to control indicates the uptake of antibody is facilitated by macropinocytosis inducers via macropinocytotic routes.
