Background/Objectives: An inhibitor of small Rho GTPase Cdc42, CASIN, has been shown to reduce cancer cell proliferation, migration, and invasion, yet it has several limitations, including rapid drug elimination and low bioavailability, which prevents its systemic administration. In this study, we designed and characterized a nanoparticle-based delivery system for CASIN encapsulated within poly(lactide-co-glycolide)-block-poly(ethylene glycol)-carboxylic acid endcap nanoparticles (PLGA-PEG-COOH NPs) for targeted inhibition of Cdc42 activity in colon cancer. Methods: We applied DLS, TEM, and UV–vis spectroscopy methods to characterize the size, polydispersity index, zeta potential, encapsulation efficiency, loading capacity, and in vitro drug release of the synthesized nanoparticles. The CCK-8 cell viability test was used to study colorectal cancer cell growth in vitro. Results: We showed that CASIN-PLGA-PEG-COOH NPs were smooth, spherical, and had a particle size of 86 ± 1 nm, with an encapsulation efficiency of 66 ± 5% and a drug-loading capacity of 5 ± 1%. CASIN was gradually released from NPs, reaching its peak after 24 h, and could effectively inhibit the proliferation of HT-29 (IC50 = 19.55 µM), SW620 (IC50 = 9.33 µM), and HCT116 (IC50 = 10.45 µM) cells in concentrations ranging between 0.025–0.375 mg/mL. CASIN-PLGA-PEG-COOH NPs demonstrated low hemolytic activity with a hemolytic ratio of less than 1% for all tested concentrations. Conclusion: CASIN-PLGA-PEG-COOH NPs have high encapsulation efficiency, sustained drug release, good hemocompatibility, and antitumor activity in vitro. Our results suggest that PLGA-PEG-COOH nanoparticles loaded with CASIN show potential as a targeted treatment for colorectal cancer and could be recommended for further in vivo evaluation.

Background: CASIN, a selective Cdc42 inhibitor, shows strong antitumor potential in colorectal cancer (CRC) but its clinical translation is limited by rapid clearance and low bioavailability. To overcome these challenges, we developed poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG-COOH) nanoparticles for efficient encapsulation and delivery of CASIN. Materials and methods: CASIN-loaded PLGA-PEG-COOH nanoparticles were prepared via one-step nanoprecipitation. The resulting particles were characterized for morphology, size, zeta potential, drug loading, encapsulation efficiency, and stability. Blood compatibility was assessed by hemolysis assays, while in vitro antitumor activity was evaluated in CRC cell lines (HT-29, SW620, HCT116) using CCK-8 assays. Results: The CASIN-PLGA-PEG nanoparticles were spherical and uniform, with an average size of 86 ± 1 nm. Encapsulation efficiency reached 66 ± 5% with a drug loading of 5 ± 1%. The formulation provided sustained drug release over 24 hours. Hemolysis assays demonstrated excellent biocompatibility (<1% hemolysis across tested doses). In vitro studies showed potent antiproliferative activity, with IC₅₀ values of 19.55 µM (HT-29), 9.33 µM (SW620), and 10.45 µM (HCT116). Conclusion: Encapsulation of CASIN into PLGA-PEG-COOH nanoparticles improved drug stability, release profile, and safety, while maintaining strong antitumor efficacy in vitro. This nanoplatform represents a promising strategy for targeted Cdc42 inhibition in CRC and warrants further investigation in in vivo models. Acknowledgement: This research has been funded by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP26100973) and Nazarbayev University, Collaborative Research Project (CRP) Grant No. 211123CRP1611. Key words: Colorectal cancer, Cdc42 inhibition, PLGA-PEG nanoparticles, Drug delivery.

Tumor hypoxia is a hallmark of solid tumors that promotes aggressiveness and resistance to therapy. Within hypoxic tumors, hypoxia-inducible factor (HIF)-dependent transcriptional programs drive metabolic reprogramming, lactate accumulation, extracellular acidosis, angiogenesis, immune suppression, and invasive phenotypes. Translation of these adaptations into increased tumor cell motility and invasion requires coordinated remodeling of the cytoskeleton and cell polarity machinery. Cdc42, a Rho-family small GTPase regulating actin dynamics, cell polarity, and migration, may therefore link hypoxic signaling to invasive tumor behavior. Here, we review current evidence on the regulation and function of Cdc42 in hypoxic solid tumors and its therapeutic implications. Available studies indicate that hypoxia can induce rapid and potentially reversible Cdc42 activation in selected cancer models. In some experimental systems, interference with Cdc42-associated signaling attenuates hypoxia-induced HIF-1α and VEGF responses, suggesting a role in pro-angiogenic adaptation. However, effects of hypoxia on Cdc42 expression, localization, and activation vary by tumor type, oxygen concentration, exposure duration, and microenvironmental context. Cdc42 therefore remains an emerging rather than validated therapeutic target. Future studies should employ physiologically relevant in vivo models of chronic and cycling hypoxia, directly measure Cdc42-GTP, and more accurately distinguish tumor-cell-intrinsic effects from those occurring in stromal components of the tumor microenvironment.