Liposomes Encapsulating Morin: Investigation of Physicochemical Properties, Dermal Absorption Improvement and Anti-Aging Activity in PM-Induced Keratinocytes

Abstract

Recently, a global market for anti-aging skin care using botanicals has been noticeably developing. Morin, 3,5,7,2',4'-pentahydroxyflavone, is a polyphenol with many pharmacological properties including antioxidant, anti-inflammation and photoprotection. However, poor aqueous solubility of morin restricts its application in pharmaceuticals. The present study aimed to encapsulate morin into liposomal vesicles to improve its water solubility and skin penetration, and further investigated its ROS inhibition and anti-aging activity in HaCaT keratinocytes induced by particulate matters (PMs). Our data presented that morin was a strong DPPH^• radical scavenger. Morin displayed a remarkable ROS inhibitory ability and protected keratinocytes against PMs by downregulating matrix metalloproteinase-1 (MMP-1) expression via suppressing p-ERK and p-p38 in the MAPK pathway. Moreover, water solubility of liposomal morin (LM) prepared by the thin film hydration method was significantly better than free form of morin due to particle size reduction of LM. Our results also demonstrated that deformable liposomal vesicles were achieved for increasing dermal absorption. Additionally, LM (morin:lecinolws-50:tween-80:PF-68, 1:2.5:2.5:5) was able to effectively reduce generation of ROS, inactivate p-ERK, p-p38 and MMP-1 in HaCaT cells exposed to PM. In conclusion, our findings suggested that LM would be a bright candidate for various topical anti-aging and anti-pollution products.

Keywords: PM; liposomes; morin; skin anti-aging; skin penetration; water solubility.

Figure 1

DPPH^• radical scavenging activity of morin in water and in ethanol. Data were from triplicate experiments and presented as mean ± SD. * indicates a statistically significant difference between morin in water and morin in ethanol, p < 0.05.

Figure 2

Cell viability of HaCaT keratinocytes treated with morin. Data were from triplicate experiments and presented as mean ± SD. * indicates a statistically significant difference with control, p < 0.05.

Figure 3

Effect of morin on PM-induced ROS generation in HaCaT keratinocytes. +, − mean with or without exposing to PM or morin, respectively. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and morin) and PM group (only treating PM), respectively, p < 0.05.

Figure 4

p-ERK and p-p38 expression. Cells were pre-incubated with morin for 3 h and then treated with PM for 2 h. +, − mean with or without exposing to PM or morin, respectively. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and morin) and PM group (only treating PM), respectively, p < 0.05.

Figure 5

MMP-1 protein expression. Cells were pre-incubated with morin for 3 h and then treated with PM for 6 h. +, − mean with or without exposing to PM or morin, respectively. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and morin) and PM group (only treating PM), p < 0.05.

Figure 6

TEM images of liposomal morin with different ratios of morin:lecithin:tween-80, (a) (F1) 1:20:20, (b) (F2) 1:40:20, (c) (F3) 1:80:20, (d) (F4) 1:1:20, (e) (F5) 1:5:20, (f) (F6) 1:10:20, (g) (F7) 1:2.5:2.5. Images of samples are magnified by ×30.0k. Scale bar is 500 nm.

Figure 7

FTIR spectra of morin, liposomal morin formulation 1 (LMF1), 7 (LMF7); blank liposome formulation 1 (BLF1), 7 (BLF7).

Figure 8

Skin penetration of liposomal morin formulation 1 (1:20:20:5) (LFM1) and 7 (1:2.5:2.5:5) (LMF7) in water using the FDC method and pork skin: (a) stratum corneum, (b) viable epidermis (epidermis without stratum corneum), (c) dermis and (d) total dermal absorption. There was no morin found in the receptor fluid. Data were presented as mean ± SD and from triplicate experiments. * indicates a statistically significant difference between LMF1 and LMF7, p < 0.05.

Figure 9

DPPH^• radical scavenging activity of morin in water compared to liposomal morin formulation 7 (LMF7). Data were presented as mean ± SD and from triplicate experiments. * indicates a statistically significant difference between morin and LMF7 in water, p < 0.05.

Figure 10

Cell viability of HaCaT keratinocytes treated with liposomal morin formulation 7. Data were presented as mean ± SD and from triplicate experiments. * indicates a statistically significant difference with control group, p < 0.05.

Figure 11

Effect of liposomal morin formulation 7 on PM-induced ROS generation in HaCaT keratinocytes. +, − mean with or without exposing to PM or LMF7. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and LMF7) and PM group (only treating LMF7), respectively, p < 0.05.

Figure 12

p-ERK and p-p38 protein expression. Cells were pre-incubated with blank liposome or liposomal morin formulation 7 for 3 h and then treated with PM for 2 h. +, − mean with or without exposing to PM or LMF7, respectively. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and LMF7) and PM group (only treating LMF7), respectively, p < 0.05.

Figure 13

MMP-1 protein expression. Cells were pre-incubated with blank liposome or liposomal morin formulation 7 for 3 h and then treated with PM for 6 h. +, − mean with or without exposing to PM or LMF7, respectively. Data were presented as mean ± SD and from triplicate experiments. *, # indicates a statistically significant difference with control group (without treating PM and LMF7) and PM group (only treating LMF7), respectively, p < 0.05.