(M1530-03-20) Overcoming Intracellular Delivery Challenges of Small- and Large-Molecule Through First-in-Class Macropinocytosis Activators

Purpose: Improving the intracellular delivery and bioavailability of anticancer therapeutics is crucial to treatment success, particularly in multidrug resistant cancers (MDRs). The challenge is even greater when it comes to highly aggressive cancers like colorectal cancer (CRC) and triple-negative breast cancer (TNBC), both of which have limited treatment options and frequently exhibit MDR, which limits therapeutic Bioavailability, and the intracellular delivery of many cancer therapeutics remains a major barrier to treatment efficacy. It is especially challenging when dealing with highly aggressive cancers such as colorectal cancer (CRC) and triple negative breast cancer (TNBC) that show limited treatment options and frequently exhibit multidrug resistance (MDR). Macropinocytosis, used by some cancer cells to acquire essential nutrients or under stress, presents a unique route to enhance drug uptake. Hereby, we designed and developed, first-in-class, small-molecule macropinocytosis activators to enhance the intracellular delivery of both small- and large-molecules in CRC models, with the goal of overcoming delivery limitations and improving therapeutic efficacy. Understanding the selectivity, trafficking, and delivery mechanism of these compounds will allow development of adjuvants to overcome MDR cancers.

Methods: We designed and synthesized a series of compounds (known as BAPT series), of which BAPT-78 and BAPT-54 were identified as potent lead macropinocytosis modulator along with well-known agent, MOMIPP in the CRC cell lines (SW620 and HCT116). Uptake of macropinocytosis tracers—green fluorescent-tagged dextran and albumin—was quantified via fluorescence microscopy. Co-localization with late endosomal and lysosomal markers was used to determine endolysosomal trafficking. Functional delivery of large molecules (anti-β-tubulin antibodies, saporin [10.9–350 nM], and siRNA [50 nM]) and low molecular weight (MW) anticancer drugs (doxorubicin and paclitaxel) was assessed using viability assays and fluorescent signal intensity. Additionally, as a proof of concept we used TNBC cell lines SUM159 and paclitaxel resistance PAC200 for drug delivery validation. Cytotoxic synergy was quantified through combination index and IC₅₀ shift analyses using synergyfinder.

Results: BAPT-78 and BAPT-54 significantly increased uptake of fluorescently labeled dextran and albumin in SW620 and HCT116 cells, comparable to MOMIPP. BAPT-78-induced vacuoles displayed co-localization with acidic endolysosomal markers, confirming trafficking through the macropinocytic pathway. Despite robust internalization, delivery of saporin and siRNA was sub-par, likely due to degradation in endolysosomes. In contrast, treatment with BAPT-78, BAPT-54, or MOMIPP markedly enhanced the intracellular accumulation and cytotoxic activity of paclitaxel and doxorubicin across CRC and TNBC models, including paclitaxel drug-resistant PAC200 TNBC cells. Notably, co-treatment with BAPT analogs and doxorubicin or paclitaxel reduced IC₅₀ values for both traditional anticancer agents, indicating enhanced delivery and efficacy.

Conclusion: These findings demonstrate that novel macropinocytosis activators can selectively increase uptake and functional delivery of small-molecule chemotherapeutics, while large molecules were significantly uptaked but trapped in endolysosomes and may need further optimization to avoid endolysosome degradation. Co-administration of BAPT-78 or BAPT-54 with paclitaxel or doxorubicin significantly improves cytotoxic outcomes in CRC and TNBC models, including drug-resistant variants, suggesting a promising strategy for therapeutic enhancement via macropinocytosis modulation.

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Tiwari AK, Karthikeyan C, Amawi HA, Erhardt PW, Trivedi P, inventors; University of Toledo, assignee. Materials and methods useful to induce cancer cell death via methuosis or autophagy or a combination thereof. United States patent application US 18/621,480. 2024 Jul 25.
Neupane R, Malla S, Karthikeyan C, Asbhy Jr CR, Boddu SH, Babu RJ, Tiwari AK. Endocytic highways: Navigating macropinocytosis and other endocytic routes for precision drug delivery. International Journal of Pharmaceutics. 2025 Feb 15:125356.

Acknowledgements: This work was partly supported by Winthrop P. Rockefeller Cancer Institute funds (CC103501/FD208) and Arkansas Bioscience Institute Funds (GR020025/FD224) to A.K.T.


Figure 1. A schematic cartoon shows combination of macropinocytosis inducers with large/small therapeutic molecules.


Figure 2. The uptake study of fluorescently labelled dextran and albumin in HCT116 and SW620 cells with aid of the BAPT analogues (BAPT 31, BAPT 54 and BAPT 78). The uptake of (A) dextran and (B) albumin was significantly enhanced by 24 hours incubation of BAPT 31, BAPT 54 and BAPT 78 with the cells compared to the control. (n=3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).


Figure 3. Combination study of BAPT 78 with doxorubicin (DOX) in SW620 and HCT116 cells. (A) The combination of BAPT 78 with doxorubicin in SW620 cells showed synergistic cytotoxicity with an overall synergy score of 8.16. (B) The combination of BAPT 78 with doxorubicin in HCT116 cell showed synergistic cytotoxicity with overall synergy score of 7.53. (n=6, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)