The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes

Abstract

This study evaluated the antimicrobial activity of lauric acid (LA) and its liposomal derivatives against Propionibacterium acnes (P. acnes), the bacterium that promotes inflammatory acne. First, the antimicrobial study of three free fatty acids (lauric acid, palmitic acid and oleic acid) demonstrated that LA gives the strongest bactericidal activity against P. acnes. However, a setback of using LA as a potential treatment for inflammatory acne is its poor water solubility. Then the LA was incorporated into a liposome formulation to aid its delivery to P. acnes. It was demonstrated that the antimicrobial activity of LA was not only well maintained in its liposomal derivatives but also enhanced at low LA concentration. In addition, the antimicrobial activity of LA-loaded liposomes (LipoLA) mainly depended on the LA loading concentration per single liposomes. Further study found that the LipoLA could fuse with the membranes of P. acnes and release the carried LA directly into the bacterial membranes, thereby killing the bacteria effectively. Since LA is a natural compound that is the main acid in coconut oil and also resides in human breast milk and liposomes have been successfully and widely applied as a drug delivery vehicle in the clinic, the LipoLA developed in this work holds great potential of becoming an innate, safe and effective therapeutic medication for acne vulgaris and other P. acnes associated diseases.

Figure 1

Antimicrobial effects of free fatty acids on P. acnes. P. acnes (1×10⁷ CFU/mL) was incubated with 0∓100 μg/mL of lauric acid (LA), palmitic acid (PA), or oleic acid (OA) in 5% DMSO in PBS for 5 hours under anaerobic condition. After incubation, P. acnes suspension was diluted 1:10 –1:10⁶ with PBS, and 5 μL of the dilutions were spotted on a Brucella Broth agar plate supplemented with 5% defibrinated sheep blood, vitamin K and hemin. After liquid in the P. acnes suspension was absorbed into the agar, the plate was incubated under anaerobic condition for 3 days before quantifying CFU of P. acnes. Data represents mean ± SD of three individual experiments.

Figure 2

Characterization of lauric acid-loaded liposomes (LipoLA). LA at various concentrations ranging from 0~200 μg/mL was mixed with other lipid components to prepare LipoLA. The size (diameter, nm) and surface zeta potential (mV) of the LipoLA were determined by dynamic light scattering. Data represents mean ± SD of three individual experiments.

Figure 3

Quantification of LA loading in LipoLA by HPLC. (A) UV absorption intensity of derivatized LA at the concentrations of a-f: 50, 100, 200, 500, 1000, and 1500 μg/mL. Inset: the corresponding linear calibration standard curve. (B) The loading of LA in the LipoLA formulations with an initial LA input of 0, 25, 50, 100, and 200 μg/mL was 0, 12, 33, 80, and 102 μg/mL, respectively. Data represents mean ± SD of three individual experiments.

Figure 4

Antimicrobial activity of LipoLA against P. acnes. Two sets of LipoLA were incubated with P. acnes (1×107 CFU/mL), respectively, for 5 hours under anaerobic condition to test their antimicrobial activity. (A) LipoLA with a LA loading concentration of 0, 12, 33, 80, and 102 μg/mL, respectively. In this set, the LipoLA concentration of each sample was constant while the LA concentration per liposome was different. The results showed that 102 μg/mL LipoLA completely killed P. acnes. (B) LipoLA with a LA loading concentration of 25.5, 51, and 102 μg/mL, respectively. The 25.5 μg/mL LipoLA and the 51 μg/mL LipoLA were obtained by diluting the 102 μg/mL LipoLA solution with PBS fourfold and twofold, respectively. In this set, the LipoLA concentration of each sample was different while the LA concentration per liposome was constant. The results showed that both 51 μg/mL LipoLA and 102 μg/mL LipoLA completely killed bacteria. Incubation with PBS buffer and empty liposome solution (without LA) served as negative controls. Data represents mean ± SD of three individual experiments. UD: undetectable.

Figure 5

FRET measurements of the fusion between LipoLA and P. acnes. A florescent donor (C₆NBD) and a fluorescent acceptor (DMPE-RhB) were simultaneously incorporated into the LipoLA (51 μg/mL LA) with a proper molar ratio that the acceptor completely quenched the fluorescence emission from the donor. The FRET-pair labeled LipoLA was incubated with P. acnes at a concentration of a-d: 8×107, 1.2×10⁸, 1.6×10⁸, and 3.2×10⁸ CFU/mL) for 10 minutes. After removing the excess LipoLA, all samples were excited at 470 nm. A rise in emission intensity of C₆NBD (donor) at 520 nm was observed with the increasing bacterial concentration, indicating the occurrence of fusion between LipoLA and bacteria which caused the spatial separation of C₆NBD and DMPE-RhB.