Polyphenols as Functional Replacements for Cholesterol in Liposome Formulations: A 21-Day Stability Study

Introduction

Cholesterol is commonly incorporated into liposome formulations to enhance bilayer stability, regulate membrane fluidity, and reduce membrane permeability. However, identifying alternative membrane stabilizers has gained interest, particularly naturally derived amphiphilic molecules. Polyphenols represent a promising class of compounds due to their hydrophobic aromatic structures and ability to interact with lipid bilayers in ways similar to cholesterol. These interactions may influence membrane packing, rigidity, and overall vesicle stability. The objective of this study was to evaluate polyphenols as potential cholesterol substitutes in liposome formulations. Specifically, liposomes containing the polyphenol compound-1 (PC-1) were compared with conventional cholesterol-containing liposomes to assess short-term physical stability during refrigerated storage.

Methods

Liposomes were prepared using three formulations: DSPC: Cholesterol (10:4 w/w), DSPC: PC-1 (10:4 w/w), and DSPC: PC-1 (10:2 w/w). Vesicles were produced using a microfluidic mixing technique to ensure controlled and uniform nanoparticle formation. The prepared liposome suspensions were stored at 4 °C and evaluated for physical stability over 21 days. Samples were analyzed on days 0, 1, 7, 14, and 21. Particle size, polydispersity index (PDI), and zeta potential were measured using dynamic light scattering (DLS) to monitor changes in vesicle size distribution and surface charge. In addition, samples are currently being analyzed using differential scanning calorimetry (DSC) to evaluate lipid bilayer phase behavior and potential effects of polyphenols on membrane thermotropic properties; these results are not included in the present analysis.

Results

All formulations produced nanosized liposomes that remained relatively stable over the 21-day storage period. The cholesterol formulation (DSPC: Chol, 10:4 w/w) showed minimal changes in physicochemical properties, with particle size increasing slightly from 61.12 nm (±2.76) on day 0 to 62.46 nm (±2.02) on day 21. The PDI remained low (0.05–0.06), indicating a narrow size distribution, while zeta potential changed modestly from −12.22 mV to −8.61 mV. The polyphenol-containing formulation DSPC: PC-1 (10:4 w/w) showed a particle size change from 57.94 nm (±1.30) to 58.56 nm (±3.54), with PDI remaining similar (0.18–0.17). Zeta potential decreased from −37.43 mV to −25.60 mV during storage. The DSPC: PC-1 (10:2 w/w) formulation exhibited a modest increase in size from 50.60 nm (±5.20) to 55.01 nm (±9.10), while PDI remained relatively consistent (0.184–0.191). Zeta potential decreased from −37.18 mV to −26.88 mV, maintaining a strongly negative surface charge throughout the study.

Discussion and Conclusion

PC-1–containing liposomes exhibited physical stability comparable to conventional cholesterol-based liposomes during 21-day refrigerated storage. Particle sizes remained within the nanoscale range with only minor increases, indicating maintained vesicle integrity. Although the cholesterol formulation showed lower PDI and narrower size distribution, PC-1 formulations remained within acceptable stability limits. PC-1 liposomes also displayed more negative zeta potentials than cholesterol liposomes, suggesting stronger electrostatic repulsion and improved colloidal stability. Despite a moderate decrease during storage, the zeta potential remained sufficiently negative. Overall, these findings suggest that PC-1 may serve as a promising alternative to cholesterol in DSPC-based liposome formulations, warranting further evaluation with additional polyphenols and DSC analysis.