Development and in vitro evaluation of ursolic acid-loaded poly(lactic- co-glycolic acid) nanoparticles in cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA), an epithelial biliary tract malignancy, is a significant health concern in the Greater Mekong Subregion, particularly in northeastern Thailand. Prior to the development of advanced stages, CCA is typically asymptomatic, thereby limiting treatment options and chemotherapeutic effectiveness. Ursolic acid (UA), a triterpenoid derived from plants, was previously discovered to inhibit CCA cell growth through induction of apoptosis. Nevertheless, the therapeutic effectiveness of UA is limited by its poor solubility in water and low bioavailability; therefore, dimethyl sulfoxide (DMSO) is utilized as a solvent to treat UA with CCA cells. Enhancing cellular uptake and reducing toxicity, the utilization of polymeric nanoparticles (NPs) proves beneficial. In this study, UA-loaded PLGA nanoparticles (UA-PLGA NPs) were synthesized using nanoprecipitation and characterized through in silico formation analysis, average particle size, surface functional groups and ζ-potential measurements, electron microscopic imaging, drug loading efficiency and drug release studies, stability, hemo- and biocompatibility, cytotoxicity and cellular uptake assays. Molecular dynamics simulations validated the loading of UA into PLGA via hydrogen bonding. The synthesized UA-PLGA NPs had a spherical shape with an average size of 240 nm, a negative ζ-potential, good stability, great hemo- and bio-compatibility and an encapsulation efficiency of 98%. The NPs exhibited a characteristic of a simple diffusion-controlled Fickian process, as predicted by the Peppas-Sahlin drug release kinetic model. UA-PLGA NPs exhibited cytotoxic effects on KKU-213A and KKU-055 CCA cells even when dispersed in media without organic solvent, i.e., DMSO, highlighting the ability of PLGA NPs to overcome the poor water solubility of UA. Rhodamine 6G (R6G) was loaded into PLGA NPs using the same approach as UA-PLGA NPs, demonstrating effective delivery of the dye into CCA cells. These findings suggest that UA-PLGA NPs showed promise as a potential phytochemical delivery system for CCA treatment.

Scheme 1. Schematic representation of UA-PLGA NPs preparation via nanoprecipitation method.

Fig. 1. (A) Hydrodynamic size distribution; (B) TEM image of UA-PLGA NPs (insets: size distribution histogram and zoomed image); (C) SEM image of UA-PLGA NPs; FTIR spectra of (D) PLGA, (E) UA and (F) UA-PLGA NPs. Scale bar: 500 nm.

Fig. 2. Self-assembly of UA-PLGA NPs. (A) Snapshot of UA-PLGA NPs, which was observed at t = 200 ns; (B) and (C) are H-bonds formed between PLGA and UA during self-assembly. Red, cyan, white represent oxygen, carbon, and hydrogen atoms, respectively; (D) the number of H-bonds as a function of time during complex formation; (E) the number of H-bonds as a function of time between water molecules and UA, PLGA, and acetone molecules as a function of simulation times; (F) SASA as a function of simulation time during complex formation.

Fig. 3. In vitro release studies and kinetic release models for UA-PLGA NPs. (A) In vitro release studies of UA at pH 7.4, 37 °C for 360 h, highlighting the burst release pattern over the 24 h; (B) fitting curve from various kinetic drug release models using the DDsolver program, a Microsoft Excel add-in software package.

Fig. 4. (A) Cytotoxic effects of UA-PLGA NPs and free UA on KKU-213A and KKU-055 CCA cells. The UA concentration (μg mL⁻¹) was calculated by converting the UA content to μg mL⁻¹ for comparison with free UA. (B) The cellular uptake of RG6-PLGA NPs was examined in KKU-213A and KKU-055 CCA cell lines using fluorescence microscopy. Cell nuclei were stained with DAPI and visualized by blue fluorescence, whereas R6G in NPs was visualized by red fluorescence. Scale bar: 50 μm.

Fig. 5. Hemolysis assay of UA-PLGA NPs and blank PLGA NPs on sheep red blood cells. The photographs (A) and percentages of hemolysis (B) upon incubation of red blood cells with various concentrations of UA-PLGA NPs (0.125, 0.25, 0.5, 1, 2 and 4 mg mL⁻¹) and blank PLGA NPs (0.25, 1, and 4 mg mL⁻¹). Triton X-100 was used as a positive control and PBS was used as a negative control; (C) microscopic images showing the morphology of red blood cells upon incubation with negative control, 4 mg mL⁻¹ UA-PLGA NPs, 4 mg mL⁻¹ blank PLGA NPs and positive control. Data represent  ± SD (n  =  2). Scale bar: 25 μm.