Evaluating the Biological Activities of Oleanolic Acid and Apigenin in a 2-in-1 Nanoparticulate System: A Proof-of-concept Study

Background: Apigenin (APG) and Oleanolic Acid (OA) exhibit diverse biological activities; however, their pharmaceutical applications are limited by poor solubility, low bioavailability, and restricted permeability. This study developed a hybrid nanoparticulate system consisting of apigenin nanoparticles loaded into oleanolic acid micelles, named 2-in-1-NPs, to enhance their therapeutic potential. It was designed as a pilot study to identify the most promising biological activity of the 2-in-1-NPs. Methods: APG nanoparticles were prepared using the liquid anti-solvent method, while OA micelles were formulated via thin-film hydration. The hybrid system was characterized in terms of particle size, zeta potential, polydispersity index, encapsulation efficiency, and an in vitro drug release study. results: 3. Results and Discussion 3.1. Optimization of 2in1-NPS The formulations were optimized by using a 3-factor, 2-level Box–Behnken statistical design. A 3D response surface plot was used to visually assess the effect of independent variables on the dependent variables. 3.1.1. Effect of formulation variables on particle size Particle size of the prepared formulations (F1–F17) varied from 150 nm to 310 nm, as represented in (Table 1). Particle size is a key parameter in evaluating drug delivery systems—smaller sizes (typically below 200 nm) can enhance absorption, stability, and penetration into target tissues. The data shows variability among formulations, which could be due to differences in composition or preparation method. Table 1. The composition of different formulations, consisting of apigenin-loaded nanoparticles and oleanolic acid-loaded micelles, resulted final formulation 2in1-NPs, and their effects on particle size and % entrapment efficiency were evaluated. Selected independent and dependent variables were used to optimize the 2in1-NPs using the Box-Behnken design (* center point). Factors Responses Std Run A:Drug (mg) B:Plox-407 (g) C:TPGS (mg) Particle size (nm) Entrapment efficiency (%) 2 F1 6 67 23 215 65 3 F2 4 69 23 205 73 5 F3 4 68 22 280 60 9 F4 5 67 22 230 70 7 F5 4 68 24 310 58 1 F6 4 67 23 261 68 6 F7 6 68 22 301 62 4 F8 6 69 23 255 63 14 F9* 5 68 23 153 83 13 F10* 5 68 23 150 84 15 F11* 5 68 23 155 82 10 F12 5 69 22 210 75 8 F13 6 68 24 206 71 11 F14 5 67 24 257 76 17 F15* 5 68 23 156 82 12 F16 5 69 24 291 59 16 F17* 5 68 23 151 65 The formulation (F10), prepared using plox-407 (68mg), TPGS (23 mg), and OA (5 mg), showed the smallest particle size while the formulation (F5), prepared with plox-407 (68 mg), TPGS (24 mg), and OA (4 mg) showed the largest particle size. The influence of independent variables on particle size is illustrated in the 3D response plot (Fig. 2). Increasing the concentration of plox-407 and decreasing the concentration of OA increased in particle size, likely due to the corresponding increase in the solution's viscosity. TPGS rapidly coated the drug particles, forming a dense network to stabilize the formulation, leading to an increase in particle size as the TPGS concentration rises. This finding aligns with previously published research [45]. Fig. (2). 3D response surface plots (a-c) and contour plots (aˊ-cˊ) showing the effect of independent variables A (Drug), B (ploxamer-407), and C (TPGS) on particle size of apigenin-loaded nanoparticles (APG-NP) and oleanolic acid-loaded micelles (OA-MM) resulted in combined formulation 2in1-NPs. 3.1.2. Effect of formulation variables on entrapment efficiency The Results: The optimized hybrid 2-in-1 nanoparticle formulation exhibited a mean particle size of 150.4 ± 0.055 nm with sustained drug release over 72 hours, and high encapsulation efficiencies of 83 ± 0.577% for OA-MM and 92 ± 0.431% for APG-NP. While APG and OA alone showed limited antimicrobial activity against Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Bacillus cereus, the hybrid nanoparticle system significantly enhanced anti-inflammatory activity, demonstrated by a marked reduction in IL-6 levels (p < 0.0001) compared with free or individually loaded drugs, and showed increased cytotoxicity against HepG-2 liver cancer cells with an IC₅₀ of 16.988 μg/mL, highlighting its potential for improved therapeutic efficacy. discussion: 3. Results and Discussion 3.1. Optimization of 2in1-NPS The formulations were optimized by using a 3-factor, 2-level Box–Behnken statistical design. A 3D response surface plot was used to visually assess the effect of independent variables on the dependent variables. 3.1.1. Effect of formulation variables on particle size Particle size of the prepared formulations (F1–F17) varied from 150 nm to 310 nm, as represented in (Table 1). Particle size is a key parameter in evaluating drug delivery systems—smaller sizes (typically below 200 nm) can enhance absorption, stability, and penetration into target tissues. The data shows variability among formulations, which could be due to differences in composition or preparation method. Table 1. The composition of different formulations, consisting of apigenin-loaded nanoparticles and oleanolic acid-loaded micelles, resulted final formulation 2in1-NPs, and their effects on particle size and % entrapment efficiency were evaluated. Selected independent and dependent variables were used to optimize the 2in1-NPs using the Box-Behnken design (* center point). Factors Responses Std Run A:Drug (mg) B:Plox-407 (g) C:TPGS (mg) Particle size (nm) Entrapment efficiency (%) 2 F1 6 67 23 215 65 3 F2 4 69 23 205 73 5 F3 4 68 22 280 60 9 F4 5 67 22 230 70 7 F5 4 68 24 310 58 1 F6 4 67 23 261 68 6 F7 6 68 22 301 62 4 F8 6 69 23 255 63 14 F9* 5 68 23 153 83 13 F10* 5 68 23 150 84 15 F11* 5 68 23 155 82 10 F12 5 69 22 210 75 8 F13 6 68 24 206 71 11 F14 5 67 24 257 76 17 F15* 5 68 23 156 82 12 F16 5 69 24 291 59 16 F17* 5 68 23 151 65 The formulation (F10), prepared using plox-407 (68mg), TPGS (23 mg), and OA (5 mg), showed the smallest particle size while the formulation (F5), prepared with plox-407 (68 mg), TPGS (24 mg), and OA (4 mg) showed the largest particle size. The influence of independent variables on particle size is illustrated in the 3D response plot (Fig. 2). Increasing the concentration of plox-407 and decreasing the concentration of OA increased in particle size, likely due to the corresponding increase in the solution's viscosity. TPGS rapidly coated the drug particles, forming a dense network to stabilize the formulation, leading to an increase in particle size as the TPGS concentration rises. This finding aligns with previously published research [45]. Fig. (2). 3D response surface plots (a-c) and contour plots (aˊ-cˊ) showing the effect of independent variables A (Drug), B (ploxamer-407), and C (TPGS) on particle size of apigenin-loaded nanoparticles (APG-NP) and oleanolic acid-loaded micelles (OA-MM) resulted in combined formulation 2in1-NPs. 3.1.2. Effect of formulation variables on entrapment efficiency The Discussion: The results demonstrate that the developed system successfully transports the drugs through the cell membrane and exhibits synergistic or additive biological activities, including antiinflammatory and anticancer effects. conclusion: 4. Conclusion The 2in1-NPs loaded with APG and OA were successfully formulated. Both APG-NP and OA-MM exhibited very small particle sizes and high encapsulation efficiency. The objective of the present study was to develop a hybrid delivery system combining APG-NP and OA-MM as 2in1-NPs to enhance the bioavailability of the drugs and reduce the required dosage. The results demonstrated that the 2in1-NPs were more effective than the individual drugs in addressing various health conditions. They exhibited negligible antimicrobial activity against gram-negative bacteria (Escherichia coli, Salmonella typhi) and gram-positive bacteria (Staphylococcus aureus, Bacillus cereus). However, the 2in1-NPs showed significant anti-inflammatory effects by reducing the levels of the pro-inflammatory cytokine IL-6 in carrageenan-induced rat paw oedema. In comparison to the individual drugs, the 2in1-NPs reduced cell viability more effectively and exhibited a lower IC50 in HepG-2 cells, indicating enhanced anti-cancer activity. This improvement was attributed to the simultaneous uptake of both drugs by the HepG-2 cells. The sustained release of the drugs was achieved through their encapsulation within the inner core of the nanoparticles. In conclusion, the hybrid 2in1-NP system showed strong potential for therapeutic applications in the treatment of various diseases. Future studies will focus on in vivo evaluations and further optimization to improve the clinical efficacy of this hybrid system. Conclusion: The hybrid 2-in-1-NPs system presents a promising platform for the treatment of various diseases, offering enhanced bioactivity and therapeutic efficacy.