[Preparation of Lip-TTO-PHMB and evaluation of its in vitro antibacterial activity and biocompatibility].

Abstract

Objective: To prepare a lecithin liposome-tea tree essential oil-polyhexamethylene biguanide hydrochloride complex (Lip-TTO-PHMB) and to investigate its in vitro antibacterial activity and biocompatibility. Methods: This study was an experimental research including group design, factorial design, and repeated measurement design. Lip-TTO-PHMB was prepared by the thin-film hydration method. Through single-factor experiments combined with the Box-Behnken response surface methodology, the optimal preparation condition for Lip-TTO-PHMB was determined as follows: mass ratio of egg yolk lecithin to cholesterol of 3.77:1.00, volume fraction of tea tree essential oil of 0.64%, and volume fraction of polysorbate 80 of 0.13%. The model-predicted encapsulation efficiency was 51.035%. Lip-TTO-PHMB was prepared under the optimal preparation condition and its encapsulation efficiency was measured by ultracentrifugation ultraviolet method. Mouse fibroblasts L929 were divided into lecithin liposome-polyhexamethylene biguanide hydrochloride (Lip-PHMB) group treated with Lip-PHMB, Lip-TTO-PHMB group treated with Lip-TTO-PHMB, and polyhexamethylene biguanide hydrochloride (PHMB) group treated with PHMB, with the final mass concentrations of PHMB adjusted to 62.5, 31.3, and 15.6 μg/mL, and the cell viability was detected by the thiazolyl blue assay after 24 h of culture. Another batch of L929 cells were divided into the same groups as before and treated with the corresponding materials with the final mass concentration of PHMB at 15.0 μg/mL, and the cell viability was detected by live/dead cell staining after 24 h of culture. The sample size for the above experiments was 4. Human immortalized keratinocytes (HaCaTs) were divided into control group cultured with serum-free MEM medium, PHMB group treated with PHMB, and Lip-TTO-PHMB group treated with Lip-TTO-PHMB, with the final mass concentration of PHMB at 15.0 μg/mL. The scratch test was performed to detect the cell migration rates at 24 and 48 h after scratch (with the sample size of 3). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PHMB in three materials against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739 were determined by the broth microdilution twofold dilution method, with the sample size of 5. Results: The measured encapsulation efficiency of Lip-TTO-PHMB was (52.35±0.24)%, which was close to the model-predicted value. The results detected by thiazolyl blue assay showed that after 24 h of culture, at PHMB final mass concentrations of 62.5, 31.3, and 15.6 μg/mL, the viability of L929 cells in Lip-PHMB group and Lip-TTO-PHMB group was significantly higher than that in PHMB group (P<0.05), and the viability of L929 cells in Lip-PHMB group was significantly higher than that in Lip-TTO-PHMB group (P<0.05). The results detected by live/dead cell staining showed that after 24 h of culture, the viability of L929 cells in PHMB group was significantly lower than that in Lip-PHMB group and Lip-TTO-PHMB group (with P values both <0.05). At 24 and 48 h after scratch, the migration rates of HaCaT cells in Lip-TTO-PHMB group were significantly higher than those in PHMB group (P<0.05); at 48 h after scratch, the migration rate of HaCaT cells in control group was significantly higher than that in PHMB group (P<0.05). The MICs of Lip-PHMB, Lip-TTO-PHMB, and PHMB against Staphylococcus aureus were 31.3, 15.6, and 15.6 μg/mL, respectively, and those against Escherichia coli were 31.3, 15.6, and 7.8 μg/mL, respectively. The MBCs of Lip-PHMB, Lip-TTO-PHMB, and PHMB against Staphylococcus aureus were 20, 10, and 15 μg/mL, respectively, and those against Escherichia coli were 30, 20, and 20 μg/mL, respectively. Conclusions: The Lip-TTO-PHMB is prepared successfully, and the material possesses excellent antibacterial activity while retaining favorable biocompatibility.