2.2 Biological activity By in vitro cellular uptake studies, we confirmed 5-BPA ligands on the micelles promote the intracellular delivery in cancer cells by specifically interacting with the SA, as confirmed by blocking the uptake after sialidase treatment. This enhanced SA targeting and intracellular delivery of 5-BPA-DACHPt/m promoted their activity against cancer cells and eliminated the CSC-fraction as determined by flow cytometry after CD44v staining. Motivated by the improved in vitro activity of 5-BPA-DACHPt/m, we proceeded to evaluate the in vivo performance of the micelles. By evaluating the tumor accumulation of the drugs at different time points, we found that 5-BPA-DACHPt/m enhanced the accumulation in head and neck (HSC-2) tumors in vivo (FFiigguurree 22AA). Moreover, the 5-BPA effectively suppressed the growth rate of orthotopic HSC-2 tumors (FFiigguurree 22BB). This antitumor activity enhancement did not come at a cost of treatment side effect, such as weight loss (FFiigguurree 22CC). The enhanced efficacy of 5-BPA-DACHPt/m translated into significant improvement in the survival of mice, with a median-survival of 80 days (FFiigguurree 22DD). Besides the increased accumulation of 5-BPA-DACHPt/m in tumors, the enhanced activity of these micelles against HSC2 tumors was due their inhibitory activity of the CSC-fraction as determined by staining the CD44v cells after drug treatment (FFiigguurree 22EE). 3. Future development Our findings indicate the potential for developing boronic acid-based targeting strategies that can promote the recognition of SA-containing epitopes and particular cell populations inside tumors. Besides improving t delivery of drugs, boronic acid ligands may also engage with sialylated epitopes in immune cells and cancer cells in tumors, which are involved in intratumoral immune signals. In fact, we have recently observed that 5-BPA modified polymers can promote the T cell proliferation at intratumoral pH and the interaction with cancer cells. Thus, we envision tumor-targeted boronic acid-installed systems stimulating antitumor immune cells and/or blocking immunosuppressive cancer cells by controlling the interaction of with SA. 4. References 1. Pinho, S. S. et al. Nat. Rev. Cancer 1155, 540 (2015). 2. Hanahan, D. & Weinberg, R. A. Cell 114444, 646 (2011) 3. Munkley, J. & Elliott, D. J. Oncotarget 77, (2016). 4. Noto, Z. et al. Oral Oncol. 4499, 787 (2013) 5. Büll, C. et al. Cancer Res. 7788, 3574 (2018). 6. Rillahan, C. D. et al. Nat. Chem. Biol. 88, 661 (2012). 7. Mi, P., Cabral, H. et al. Adv. Mater. 1902604 (2019). 8. Matsumoto, A., Cabral, H. et al. Chem. Sci. 88, 6165 (2017). 5. Contact Horacio Cabral, PhD Department of Bioengineering, Graduate School of Engineering, The University of Tokyo. 7-3-1- Hongo Bunkyo-ku. Tokyo. 113-8656 horacio@bmw.t.u-tokyo.ac.jp FFiigguurree 22.. AA.. Accumulation of drugs in HSC2 tumors after intravenous injection. n = 4, *p < 0.05, **p <0.01. BB. Antitumor activity against orthotopic HSC2 tumors after treatment with oxaliplatin (8 mg/kg), DACHPt/m, and PBA- or 5-BPA-DACHPt/m (2 mg/kg on DACHPt basis) injected on days 0, 2, 4 and 15 (arrows). Data are expressed as averages ± S.D., n = 8, ***p <0.001. CC. Body weight of mice during treatment. DD. Animal survival during treatment. n = 8, *p < 0.005. EE. Distribution of CD44v9 CSCs (red) and differentiated cancer cells (involucrin; green). 0123456780510152025303540455055% injected dose/ g of HSC2 tissueTime (h)OxaliplatinDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/mD***0102030405060708005101520253035Tumor volume (mm3)Time (day)ControlDACHPt/mPBA-DACHPt/mE******PBS0510152005101Body weight (g)TimControlDACHPt/mPBA-DACHPt/5-BPA-DACHPOxaliplatinFPBS5-BPA-DACHPt/m/DACHPt/m/ColocalizationPBA-DACHPt/m/DACHPt/m/ColocaA0 1 3 6 0 1 3 6 0.111010005101520253035404550% injected dose/ ml plasmaTime (h)OxaliplatinDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/mC0123456780510152025303540455055% injected dose/ g of HSC2 tissueTime (h)OxaliplatinDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/mD***0102030405060708005101520253035Tumor volume (mm3)Time (day)ControlDACHPt/mPBA-DACHPt/mE******PBS0510152005101520253035Body weight (g)Time (day)ControlDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/mOxaliplatinFPBS0204060801000102030405060708090100% survivalTime (day)OxaliplatinControlDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/mGPBS**B5-BPA-DACHPt/m/DACHPt/m/ColocalizationTime (h) PBA-DACHPt/m/DACHPt/m/ColocalizationTime (h) BDCAACD44v+ (CSCs)/SASA/Involucrin(differentiated cancer cells)00.10.20.30.40.5CSC/SADC/SAColocalization ratioC**BCD44v+ (CSCs)/Involucrin(Differentiated cancer cells)PBSDACHPt/mOxaliplatinPBA-DACHPt/m5-BPA-DACHPt/mMergedDifferentiated cancer cellsCSCD020406080100120ControlOxaliplatinDACHPt/mPBA-DACHPt/m5-BPA-DACHPt/m% areaE***PBSInvolucrin/SAE−49−
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