Bactericidal Effect of Lauric Acid-Loaded PCL-PEG-PCL Nano-Sized Micelles on Skin Commensal Propionibacterium acnes

Acne is the over growth of the commensal bacteria Propionibacterium acnes (P. acnes) on human skin. Lauric acid (LA) has been investigated as an effective candidate to suppress the activity of P. acnes. Although LA is nearly insoluble in water, dimethyl sulfoxide (DMSO) has been reported to effectively solubilize LA. However, the toxicity of DMSO can limit the use of LA on the skin. In this study, LA-loaded poly(ɛ-caprolactone)-poly(ethylene glycol)-poly(ɛ-caprolactone) micelles (PCL-PEG-PCL) were developed to improve the bactericidal effect of free LA on P. acnes. The block copolymers mPEG-PCL and PCL-PEG-PCL with different molecular weights were synthesized and characterized using 1H Nuclear Magnetic Resonance spectroscopy (1H NMR), Fourier-transform infrared spectroscopy (FT-IR), Gel Permeation Chromatography (GPC), and Differential Scanning Calorimetry (DSC). In the presence of LA, mPEG-PCL diblock copolymers did not self-assemble into nano-sized micelles. On the contrary, the average particle sizes of the PCL-PEG-PCL micelles ranged from 50–198 nm for blank micelles and 27–89 nm for LA-loaded micelles. The drug loading content increased as the molecular weight of PCL-PEG-PCL polymer increased. Additionally, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of free LA were 20 and 80 μg/mL, respectively. The MICs and MBCs of the micelles decreased to 10 and 40 μg/mL, respectively. This study demonstrated that the LA-loaded micelles are a potential treatment for acne.

S3 of S12 (b) (c) Figure S2. DSC thermograms of triblock copolymers: (a) PC20E40C20; (b) PC50E40C50 and (c) PC100E40C100. The upper half of the thermogram represents the second cooling curve and the other displayed the second heating curve.

Preparation and Characterization of Diblock PEG-PCL Copolymer
In this section, mPEG-PCL diblock copolymers were synthesized by ring-opening polymerization reaction using Sn(Oct)2 as a catalyst and mPEG (5000 Da) as an initiator. Theoretical molecular weights of PCL segment were designed to be 7500, 10000, 15000 Da corresponding to PC75, PC100 and PC150, respectively. The reaction was carried out at 130 °C for 5 h. Moreover, the ratios of hydrophilic segment: hydrophobic segment were 1:0.75, 1:1, 1:2, respectively. The physicochemical properties of copolymers were characterized by 1 H NMR, FTIR and DSC, respectively. Lastly, the micelles were prepared and characterized by particle size analyzer. These results were used to compare to that of triblock copolymers PCL-PEG-PCL.

Critical Micelle Concentration of the Micelles
Regarding to the pyrene 1:3 ratio method, CMCs of copolymer were determined based on the relationship of I1/I3 intensity ratio of pyrene included in micelles and concentrations. As shown in Figure S7, the CMC was determined at the center point of the sigmoid. It was found that the CMCs of PC75, PC100, PC150 (shown in Table S1) were 16.4 × 10 −3 , 8.91 × 10 −3 and 4.47 × 10 −3 wt %, respectively. Apparently the CMCs reduced from 16.4 × 10 −3 to 4.47 × 10 −3 (wt %) when the molecular weight (chain length) of the hydrophobic PCL segment increased from 2500 Da to 10,000 Da. The values of polymeric micelles mainly depend on the hydrophobic segment of copolymers. The comparison of CMC of diblock and triblock copolymer is described in details in Section 3.2.1 of the main text.

1 H NMR and FT-IR Characterization of Molecular Structure of Diblock Copolymers
mPEG-PCL diblock copolymers were synthesized using the ring-opening polymerization of ɛ-CL in the presence of mPEG. The hydroxyl end group initiated the ring opening of ɛ-CL. The Chemical structure of mPEG-PCL diblock copolymers was determined by 1 H NMR in CDCl3. The presence of CH2 group in PCL was observed around 1.3 ppm, 1.6 ppm, 2.3 ppm and 4 ppm as shown in Figure S4. The methoxy protons (OCH3 group) of mPEG was observed at 3.4 ppm and the peak at 3.64 ppm was assigned as the methylene protons (CH2 group) of mPEG. Table S1 summarized the characteristics of synthesized diblock copolymers. Functional groups of mPEG-PCL diblock copolymers were characterized by FT-IR spectrophotometer. As one can see in Figure S5, all spectra show typical peaks of C-H stretching in PCL segment at 2890.7-2946.7 cm −1 . In addition, typical peaks of C=O groups in PCL segment appeared at 1722.8-1731.7 cm −1 . Lastly, a specific peak at 1110.8-1180.2 cm −1 indicated C-O-C stretching in PEG segment. Moreover, when increasing molecular weight of PCL segment in the diblock copolymer, the intensity of C=O of PCL became stronger.

DSC Thermograms of Diblock Copolymers
As observed in Figure S6, the components of hydrophobic segment affected the melting point of copolymers. Particularly, the melting point decreased from 56.65 to 54.83 °C, when increasing molecular weight of PCL segment from 2500 to 10,000 Da. Figure S6. DSC Thermograms of PC75 (a); PC100 (b) and PC150 (c) diblock copolymers; the upper half side of the diagram represents for the second cooling curve and the other displays the second heating curve. Figure S7. The properties of the micelles: (a) the suspension solution of LA-loaded micelles; (b) the particle morphology of LA-loaded micelle (PC75LA) observed under the upright microscopy, the scale bar is 10 μm; (c) the particle size of blank micelle and (d) the particle size of LA-loaded micelle determined by DLS (PDI < 0.3). Please refer to the section 3.2.2 for the results and discussion for this figure.