[1S04e] 14. Chemical Biotechnology - Part 3

5:15 PM - 5:30 PM JST(8:15 AM - 8:30 AM UTC)

[1Chemi-17] Modulation of Hydrophobicity and Negative Charge of Molecular Block with High Tumor Microenvironment-sensitive Cytotoxicity by Regulating Endocytic Cell Uptake

○Kazuki Moroishi^1,2, Marie Kawahara³, Masahiko Nakamoto¹, Satoshi Fujita², Ryohei Katayama³, Michiya Matsusaki^1,2 (1. The University of Osaka (Japan), 2. Advanced Photonics and Biosensing Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology (Japan), 3. The Cancer Chemotherapy Center, Japanese Foundation for Cancer Research (Japan))

Keywords:

pH sensitivity,cancer cell,ursodeoxycholic acid,Molecular block

[Purpose]
Drug delivery systems (DDS) have been widely studied to achieve the selective accumulation of anticancer drugs using sub-100 nm nanomaterials via the enhanced permeability and retention effect. However, the trade-off exists between achieving sufficient penetration into tumor tissue with small-sized particles and increasing retention with large-sized ones. To overcome this problem, we have developed a biocompatible poly(vinyl alcohol) (PVA) functionalized with ursodeoxycholic acid (UDCA), named molecular block (MB). MB selectively forms hydrophobic self-aggregates and subsequently induces cell death in tumor microenvironment (pH 6.5), providing both tumor stroma permeability and retention at the tumor site (K. Moroishi et al., Biomacromolecules 2023). In this study, we focused on engineering polymer aggregation and investigated their aggregation properties in detail to further improve tumor microenvironment-selective cytotoxicity.
[Method]
MBs were synthesized by conjugating UDCA to PVA at grafting degree of 3% (PVA-U3), 15% (PVA-U15), and 25% (PVA-U25). Pancreatic cancer (MIAPaCa-2) cells were treated with each PVA-U at pH 7.4 and pH 6.5 for 24 hours and their cell viability was measured by WST-8 assay to calculate half maximal inhibitory concentration (IC₅₀) values of each PVA-U. MIAPaCa-2 cells stained with Cell Tracker Deep red were treated with 10 µg mL^-1 of PVA-U labeled with rhodamine B isothiocyanate (RBITC) and incubated for 24 hours while being observed via time-lapse imaging using a confocal microscope. The mice bearing tumor derived from MIAPaCa-2 cells were administrated 144 µM of UDCA and 100 µg mL^-1 PVA and PVA-U15 five days a week, and tumor volume was assessed over a period of three weeks.
[Results]
The IC₅₀ values for PVA-U15 and PVA-U25 were lower at pH 6.5 than at pH 7.4, indicating cytotoxicity in response to a weakly acidic environment. In contrast, PVA-U3 exhibited no cytotoxicity at either pH due to its low grafting degree of UDCA to PVA. Notably, PVA-U15 showed a greater difference in IC₅₀ values between pH 7.4 and pH 6.5 compared to PVA-U25, indicating higher tumor microenvironment selectivity. To further understanding, RBITC-labeled PVA-U15 and PVA-U25 were added to MIAPaCa-2 cells under pH 7.4 and pH 6.5 conditions and observed by confocal microscopy to evaluate its cell adsorption property. PVA-U15 showed pH-sensitive aggregation and cell adsorption whereas PVA-U25 was taken up by cells via an endocytosis pathway independent of pH, suggesting that grafting degree of UDCA contributes to its cell adsorption behavior.
[Consideration]
PVA-U15 was not adsorbed onto the cell membrane at pH 7.4 due to charge repulsion by its high negative charge, while it aggregated due to increased hydrophobicity at pH 6.5 and induced cell death. In contrast, PVA-U25 showed cellular uptake at both pHs due to its intrinsically high hydrophobicity.
[Conclusion]
We demonstrated that the tumor microenvironment-sensitive cytotoxicity of PVA-U could be modulated by controlling the grafting degree of UDCA to PVA. To achieve these optimal tumor microenvironment-selectivity, it is also important to regulate hydrophobicity and negative charge, modulating cell adsorption behavior.