Nanoencapsulation of Cyanidin-3-O-glucoside Enhances Protection

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Nanoencapsulation of Cyanidin-3-O-glucoside Enhances Protection Against UVB-induced Epidermal Damage through Regulation of p53-mediated Apoptosis in Mice Zhaohan Liu, Yunfeng Hu, Xia Li, Zhouxiong Mei, Shi Wu, Yong He, Xinwei Jiang, Jianxia Sun, Jianbo Xiao, Liehua Deng, and Weibin Bai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01002 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Journal of Agricultural and Food Chemistry

Nanoencapsulation of Cyanidin-3-O-glucoside Enhances Protection Against UVB-induced Epidermal Damage through Regulation of p53-mediated Apoptosis in Mice Zhaohan Liu†^, Yunfeng Hu†^, Xia Li‡^, Zhouxiong Mei§, Shi Wu†, Yong He†, Xinwei Jiang‡, Jianxia Sun§, Jianbo Xiao$, Liehua Deng*, †, Weibin Bai*, ‡

†

Department of Dermatology, The First Affiliated Hospital, Jinan University, Guangzhou, China

‡

Department of Food Science and Engineering, Institute of Food Safety and Nutrition, Guangdong

Engineering Technology Center of Food Safety Molecular Rapid Detection, Jinan University, Guangzhou, China §

Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology,

Guangzhou, China $

Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese

Medicine, University of Macau, Taipa, Macau, China

*

Correspondence: E-mail: [email protected]; Fax: +86-20-8522630; Tel.: +86-20-8522630

(Weibin Bai); Fax: +86-20-38688115; Tel.: +86-20-38688115; E-mail:[email protected] (Liehua Deng).

^

These authors have contributed equally to this work.

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ABSTRACT: Excess ultraviolet (UV) radiation causes numerous forms of skin damage. The aim

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of the present study was to assess and compare the photoprotective effects of cyanidin-3-O-glucoside

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(C3G) alone and encapsulated in chitosan nanoparticles (Nano-C3G) in a UVB-induced acute

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photodamage mouse model. Nano-C3G was developed from chitosan and sodium tripolyphosphate

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(TPP) by ionic gelation. The particle size, zeta potential, entrapment efficiency, drug loading, and in

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vitro release in 6 days were determined. Kunming (KM) mice were treated with Nano-C3G (125, 250,

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500 µM) or C3G (500 µM) after part of the dorsal skin area was dehaired, and then exposed to 2 J/cm2

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of UVB. The nanocapsules were successfully produced and had a uniform and complete spherical

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shape without agglomeration. The size, zeta potential, entrapment efficiency, and drug loading of

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Nano-C3G was 288 nm, +30 mV, 44.90%, and 4.30%, respectively. C3G in the nanocapsules was

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released quite rapidly, and the release rate slowed down at higher pH. The animal experiment

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demonstrated that Nano-C3G could effectively reduce the UVB-induced lipid peroxidation,

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malondialdehyde, and 8-hydroxy-2’-deoxyguanosine contents; downregulate p53, Bcl-2 associated X

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(Bax), and caspase-3 and -9 expression; and balance the B-cell lymphoma-2/leukemia-2 ratio.

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Moreover, Nano-C3G (125, 250, 500 µM) improved the visual appearance, skin moisture, histologic

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appearance, and apoptotic index (based on TUNEL staining) under UVB exposure. In conclusion,

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these results suggest that Nano-C3G can reduce UVB-induced epidermal damage through the

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p53-mediated apoptosis signaling pathway. Moreover, Nano-C3G was more efficient than C3G at the

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same concentration (500 µM).

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KEYWORDS: Ultraviolet B (UVB), Cyanidin-3-O-glucoside (C3G), Nanoparticles, Mitochondrial apoptosis, Skin photodamage

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INTRODUCTION

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Excessive sunlight exposure, especially ultraviolet (UV) radiation, causes short-term and

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long-term deleterious effects such as sunburn, photoaging, and sun-induced skin cancers1-2. The UV

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spectrum (200–400 nm) is divided into three bands: UVA, UVB (280–320 nm), and UVC. As absorbed

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by the ozone layer, virtually no UVC can reach the surface of Earth. Compared to UVA, almost every

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solar erythema is caused by UVB, as it is up to 1000 times more erythemogenic than UVA3. Our

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research team, including dermatologists, mainly focuses on UVB-generated skin damage at the

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following three levels: the macroscopic level as erythema, microscopic level as edema and

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morphology of sunburn cells, and molecular level as the apoptotic cascade (visible histologically as

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sunburn cells) 4.

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The human skin has its own defense system against photodamage through endogenous

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anti-oxidative enzymes as well as antioxidants, including glutathione, coenzymes, and vitamins.

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However, this capability can be overwhelmed by an extreme photosensitized reaction. To compensate

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for this imbalance, supplementation of additional photochemoprotective agents has been considered,

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especially natural products derived from fruits and seeds, some of which have already been developed

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into cosmetic or medical products on the commercial market5–8. Phenolic compounds (including

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flavonoids) 9, polysaccharides10, polypeptides11, and vitamins12, have been reported to inhibit

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UVB-induced photodamage. In addition, previous studies have shown that anthocyanins, a group of

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flavonoids, have remarkable anti-oxidant, anti-inflammatory, and anti-tumor effects13–16. In particular,

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the anthocyanin cyanidin-3-O-glucoside (C3G) was shown to inhibit UVB-induced apoptosis in

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human HaCaT keratinocytes17-18.

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However, it is unknown whether C3G can protect against UVB-induced epidermal damage in vivo.

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In addition, C3G easily degrades, which has limited its clinical application. Regarding the new tide of

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the development of cosmetic or pharmaceutical dosage forms, polymeric nanoparticles now play a

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vital role in the promotion of therapeutic and calleidic systems, owing to their ability for controlling

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drug release and the improvement of the stability of drugs. Therefore, we hypothesized that

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encapsulating C3G in polymeric nanoparticles might improve the therapeutic effects in vivo19–22. ACS Paragon Plus Environment

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To test this possibility, we assessed the photochemoprotective effects of C3G nanoparticles

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(Nano-C3G) in an acute UVB photodamage mouse model, and evaluated whether encapsulation in

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chitosan (CS) would improve the protective effects. In particular, we investigated the effects of

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Nano-C3G and C3G on oxidative stress, biomarkers of DNA damage, and regulatory pathways for

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apoptosis, including caspases and members of the B-cell lymphoma/leukemia-2 (Bcl-2) family.

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CHEMICALS & METHODS

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Chemicals. C3G (purity > 98%) was purchased from Biosynth AS (Sandnes, Norway).

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Nano-C3G was manufactured using the methods described in detail below22 (average size: 288 nm;

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zeta potential: 30 mV; encapsulation efficiency: 44.9%). Antibodies for the western blot assay (p53,

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caspase-3, and caspase-9) were purchased from Affinity Biologicals (ON, Canada). Commercial kits

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used to detect the lipid peroxidation (LPO) and malondialdehyde (MDA) levels were purchased from

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Jiancheng Institute of Biotechnology (Nanjing, China). The contents of 8-hydroxy-2’-deoxyguanosine

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(8-OHdG), Bcl-2, and Bcl-2-associated X protein (Bax) were measured by enzyme-linked

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immunosorbent assay (ELISA; CUSABIO, Inc. Wuhan, Hubei, China). Other chemicals and reagents

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were of analytical grade.

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Preparation of Nano-C3G. According to the pre-experimental optimization conditions,

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Nano-C3G was synthesized by ionic gelation. A certain amount of CS was weighed and dissolved in 1%

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(v/v) glacial acetic acid to reach a final concentration of 1 mg/mL. C3G was added to the solution at a

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concentration of 0.1 mg/mL. This solution was stirred for 1 h in the dark for sufficient dissolution and

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then the pH was adjusted to 4.7 with 6 mol/L sodium hydroxide solution. Nano-C3G was obtained

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upon the addition of sodium tripolyphosphate (TPP) aqueous solution (1 mg/mL, CS:TPP = 5:1, w:w)

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to a CS and C3G aqueous solution at a rate of 1 mL/min under magnetic stirring at room temperature23.

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The blank nanoparticles (CS-TPP) were prepared as described above without the addition of C3G.

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Transmission Electron Microscopy (TEM). The morphology of Nano-C3G was observed by

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TEM. An appropriate amount of Nano-C3G was dropped onto the copper mesh of the carbon support

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film and the excess water was blotted with filter paper. Nano-C3G was stained with 1%

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phosphotungstic acid solution and then dried to prepare for TEM. ACS Paragon Plus Environment

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Fourier-transform Infrared (FTIR) Spectroscopy. FTIR spectra of freeze-dried Nano-C3G and

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CS-TPP were taken with potassium bromide pellets by an FTIR spectrometer at a scanning range from

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400 cm-1 to 4000 cm-1.

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Size and Zeta Potential Measurement. The average size and surface zeta potential of

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Nano-C3G in the solution were measured by Malvern Zetasizer Nano ZS. One milliliter of the

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Nano-C3G solution was placed in a measuring dish and sonicated for 10 min, and then tested at 25°C.

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Determination of the Entrapment Efficiency and Drug Loading of Nano-C3G. The

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Nano-C3G solution was centrifuged at 12,000 rpm for 15 min, and then washed three times with

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ultrapure water. The supernatant was collected to measure the C3G content by the pH difference

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method. The centrifuge tubes were freeze-dried together with the precipitate to calculate the weight of

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the precipitate. The entrapment efficiency and drug loading of Nano-C3G were calculated as follows:

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Entrapment efficiency = (Wo − Wc)/Wo

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Drug loading = (Wo − Wc)/Wm

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where Wm, Wo, and Wc is the mass of Nano-C3G, added C3G, and free C3G, respectively.

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In Vitro Release. Ten milligrams of freeze-dried Nano-C3G was treated with 10 mL

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phosphate-buffered saline (PBS) at pH 5.3 (Rong Meiti), pH 6.8 (tumor), pH 5.3+ lysozyme (1

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mg/mL), and pH 7.4 (cytosolic), respectively, and shaken at 37°C. A 350-µL sample was respectively

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removed in 6 days and centrifuged (10,000 rpm, 10 min), and then 300 µL of the sample was

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transferred to a 96-well plate at 100 µL per well. The precipitate was mixed with 300 µL of fresh PBS

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and then transferred to the original PBS buffer simulation system. The 96-well plates were stored at

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−20°C and the absorbance was detected at 520 nm.

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Animal Model. All animals were fed and treated according to the Guidance of Animal Care and

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Use Committee of Jinan University. Kunming (KM) mice (18–20 g, female) were purchased from

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Guangdong Medical Lab Animal Center, and maintained in a specific pathogen-free environment (23 ±

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2°C, 55 ± 10% humidity, 12-h light/dark cycle, with a standard laboratory diet and water). Prior to the

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start of the experiment, the mice were acclimatized to the laboratory conditions for at least 1 week.

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UVB Irradiation and Mouse Treatment. The mice were randomly divided into seven groups ACS Paragon Plus Environment

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(10 mice per group) after acclimatization. Initially, part of the dorsal skin area (2 × 3 cm2) of the mice

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was dehaired using a baby shaver for subsequent UVB stimulation. The mice were exposed to UVB

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irradiation using ULTRA-VITALUX UVB lamps (OSRAM Company, Germany), except for the

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normal control group (NC group). We used an electronic controller to regulate the UV dosage. To

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establish the acute UVB photodamage model, the mice were exposed to 2 J/cm2 of UVB, which is four

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times the minimal erythemal dose. The solutions of C3G and Nano-C3G were dissolved in PBS at pH

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6.8. After UVB irradiation, the focal part of the dorsal surface of each mouse was treated with 0.3 mL

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of the following formulations: the solvent control group (SC group) was treated with non-loaded

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nanoparticles (Nano); groups 1, 2, and 3 were treated with 125 µM, 250 µM, and 500 µM Nano-C3G,

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respectively; and group 4 was treated with 500 µM C3G. Non-irradiated mice (NC group) and

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untreated irradiated mice (UC group) served as controls. Twenty-four hours later, all mice were

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anesthetized and euthanized for further experiments.

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Skin Moisture and Elasticity Test. Twenty-four hours later after the irradiation, the mice were

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photographed and measured under anesthesia by 1.5% pentobarbital intraperitoneal injection. The

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moisture and elasticity measurements were performed on the dehaired skin area (above the sacral

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vertebrae) of each mouse with the Soft Plus 5.5 device (Callegari S.P.A., Italy). After these

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measurements, all mice were euthanized and dorsal skin samples (1.5 × 2.5 cm2, from the same region)

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were collected for further analyses.

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Histology. The skin tissue was excised and fixed in 10% paraformaldehyde before being

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embedded in paraffin blocks, and then cut into slide sections (4–6 µm each). Hematoxylin and eosin

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(H&E) staining was employed to examine the cell morphology24–26.

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Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) Staining. For

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immunohistochemical microscopy, some of the slides prepared as described above were rehydrated in

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citrate buffer (10 mM, pH 6) for antigen retrieval. The sunburn cells (epidermis cell apoptosis) were

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then detected with a TUNEL staining kit (KeyGEN, Nanjing, China). The positive cells were counted

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under a microscope (Olympus). The apoptotic index was defined as the ratio of TUNEL-positive

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epidermis cells to all epidermis cells, and expressed as a percentage. ACS Paragon Plus Environment

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Determination of LPO and MDA Contents. Tissue samples (400 mg) were harvested and

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homogenized (10,000 rpm, 20 s) in 9 volumes of 0.9% sodium chloride aqueous solution (4°C) to

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obtain the 10% tissue homogenate. The total homogenate was used to measure the LPO and MDA

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contents according to the manufacturer protocols.

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Determination of 8-OHdG, Bcl-2, and Bax Contents. Other tissue samples (400 mg) were

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homogenized (10,000 rpm, 20 s) in 9 volumes of cold PBS (4°C) to obtain the 10% skin tissue

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homogenate. Before collecting the total supernatant, the homogenate was centrifuged (3000 ×g, 20 min,

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4°C) to remove the undissolved pellet. Secreted 8-OHdG, Bcl-2, and Bax levels were estimated using

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ELISA kits, and the protein content was determined according to the manufacturer instructions.

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Western Blot Analysis. Immunoblot analysis was carried out to examine changes in p53,

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caspase-3, and caspase-9 expression in the tissue samples. The results were normalized to the level of

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GAPDH expression. Following protein estimation, the samples were separated by sodium dodecyl

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sulfate-polyacrylamide gel electrophoresis and the separated molecules were blotted onto a

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polyvinylidene fluoride membrane as described previously27.

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Statistical Analysis. Data are expressed as mean ± standard error of the mean. Analysis of

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variance and the Student-Newman-Keuls post-hoc test were used to determine differences among

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different groups. A value of p < 0.05 was considered to be statistically significant. All analyses were

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performed using GraphPad Prism 6.0.

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RESULTS

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Characterization of Nano-C3G (Figure 1). FTIR spectroscopy of Nano-C3G and CS-TPP was

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performed to characterize the chemical structure of the nanoparticles. FTIR spectra of Nano-C3G and

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Nano are shown in Figure 1B. A band at 3400 cm-1 corresponded to the hydrogen bond association

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peaks of the -NH and -OH group stretching vibration in CS. The absorption peaks overlapped and

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broadened. A band at 2980 cm-1 was attributed to the C=O group. The band at 1600 cm-1 corresponded

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to the -NH group stretching vibration peak. The bands at 1385 cm-1 and 1370 cm-1 corresponded to the

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vibration absorption peaks of the C-C skeleton of tertiary carbon. A band at 1098 cm-1 was attributed to

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the C-O-C absorption peak, and a band at 899 cm-1 corresponded to the vibration absorption peak of ACS Paragon Plus Environment

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the pyranyl ring. The peak of the band at 1644 cm-1 disappeared, and two small sharp peaks of bands at

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1630 cm-1 appeared, which could be observed clearly in Nano-C3G, and the bending vibration peak of

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-NH2 shifted from the band at 1602 cm-1 to the band at 1543 cm-1, which showed that the amino groups

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of CS were cross-linked with TPP by electrostatic interaction.

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TEM observation of Nano-C3G showed that the nanoparticles have a uniform and complete

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spherical shape without agglomeration (Figure 1C). The size of Nano-C3G observed by TEM in the

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dry state was smaller than the average particle size measured by the laser particle size analyzer

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according to the hydrodynamic radius of Nano-C3G in aqueous solution. The size of the nanoparticles

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followed a Gaussian distribution; the mean size of Nano-C3G was 288 nm (Figure 1D) and the zeta

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potential was +30 mV (Figure 1E). These nanoparticles are also known as nano-gel particles, and their

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particle size represents the most basic property.

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The dosage of C3G and CS was 1:10, and the entrapment efficiency of Nano-C3G was 44.90%

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and drug loading was 4.30%. The entrapment efficiency of drugs obtained by the nano-embedding

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method was reported to be about 70%28. The entrapment efficiency and drug loading differ depending

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on the embedding material, dosage, and cross-linking method. The increase of CS concentration

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increased the diameter of Nano-C3G, and the increase in the dosage ratio reduced drug loading,

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resulting in a waste of drugs. Therefore, the dosage of 1:10 C3G:CS, and the concentration of 1 mg/mL

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CS were deemed to be suitable for the preparation of Nano-C3G.

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Figure 1F shows that in a lower pH environment, the C3G was released quite rapidly and the

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release rate slowed down at higher pH. The cumulative release was significantly higher than that at

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physiological pH. This indicated that the in vitro release of C3G from Nano-C3G was sensitive to pH.

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In general, there was a sudden release detected initially, followed by a slow and steady release, with a

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maximum cumulative release rate of 75% due to the release of C3G attached to the surface of

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Nano-C3G. Thus, Nano-C3G had a good sustained release effect.

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Nano-C3G and C3G Inhibit UVB-induced Cutaneous Erythema, Edema, and Formation of

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Sunburn Cells. UVB contributes to the development of cutaneous erythema, edema, and disorders of

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the dermal blood vessels6. Therefore, we assessed the effects of Nano-C3G and C3G on UVB-induced ACS Paragon Plus Environment

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erythema. After UVB exposure, apparent large, macroscopic erythema was observed in the SC and UC

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groups. The dorsal skin of these mice appeared to be red and swollen, as if they had been ironed or

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burned without vesiculation. By contrast, the skin of the groups treated with Nano-C3G and C3G

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(groups 1, 2, 3, and 4) was visually better, with less macroscopic erythema and a more similar

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appearance to that of the NC group (Figure 2A). H&E staining of skin tissue slides revealed that UVB

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irradiation to the skin also led to microscopic changes such as the formation of sunburn cells, with

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mixed interstitial fluid in the dermis (considered as edema). H&E staining of skin tissue slides was

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independently examined by different dermatopathologists. Overall, the slides of the NC group showed

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the characteristics of the normal epidermis, with nearly no sunburn cells or edema, whereas those of

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the SC and UC groups showed many sunburn cells and different degrees of edema. As expected, the

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macroscopic and microscopic conversions induced by UVB were inhibited by the treatments of

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Nano-C3G and C3G, since the slides of groups 1, 2, 3, and 4 showed fewer sunburn cells and lighter

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edema (Figure 2B).

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Nano-C3G and C3G Improve Skin Moisture. As non-invasive tests, we assessed the effects of

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Nano-C3G and C3G on skin moisture and elasticity in the UVB-irradiated mice before the skin biopsy.

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The data demonstrated that the Nano-C3G or C3G treatment (group 1, 2, 3, or 4) could improve the

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increase of dorsal moisture caused by UVB irradiation (UC group), and the effect of Nano-C3G (group

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3) was stronger than that of C3G (group 4) under the same concentration (Figure 2C).

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Nano-C3G and C3G Protect Epidermis Cells from UVB-mediated Apoptosis. Sunburn cells

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in the epidermis represent apoptotic keratinocytes and can frequently be observed in normal skin

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subjected to UVB. TUNEL staining is a method that is commonly used for detecting DNA

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fragmentation resulting from apoptotic signaling cascades. TUNEL staining clearly demonstrated that

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topical treatment of Nano-C3G and C3G (groups 1, 2, 3, and 4) resulted in marked inhibition in the

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number of TUNEL-positive cells, and the effect of Nano-C3G (group 3) was again stronger than that

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of C3G (group 4) under the same concentration (Figure 2D).

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Nano-C3G and C3G Improve UVB-induced Oxidative Stress. UVB irradiation also leads to

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oxidative stress by generating excessive reactive oxygen species (ROS) production and oxidative DNA ACS Paragon Plus Environment

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damage. LPO induced by ROS is a major manifestation of oxidative stress5. To assess the effects of

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Nano-C3G and C3G on UVB-exposed skin, we measured the concentrations of LPO and MDA (a

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major by-product of LPO). The results showed that UVB irradiation induced a significant increase in

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the formation of LPO and MDA in KM mouse skin (UC group) when compared to the control group

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(NC group). However, there was no significant change when the mice were treated with Nano only

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(SC group). Moreover, the treatment of Nano-C3G or C3G (groups 1, 2, 3, and 4) could effectively

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inhibit the UVB-induced increase of epidermal LPO and MDA formation (Figure 3A, B).

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Nano-C3G and C3G Inhibit UVB-induced DNA Damage. After UVB exposure, 8-OHdG is

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formed in the epidermal DNA, which is considered to be an important biomarker of DNA damage.

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Therefore, ELISA was used to measure the change in 8-OHdG levels and assess the effects of

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Nano-C3G and C3G on UVB-induced DNA damage. Topical treatment of Nano-C3G or C3G (group 1,

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2, 3, or 4) resulted in marked inhibition of 8-OHdG, and the effect of Nano-C3G (group 3) was

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stronger than that of C3G (group 4) under the same concentration (Figure 3C).

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Nano-C3G and C3G Reduce UVB-induced p53 Expression. The p53 protein is also known as

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the "guardian of the genome", since p53 responds to cytotoxic stress, including UVB exposure, to

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protect the integrity of DNA28. Western blot analysis revealed that the protein expression of p53 was

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significantly decreased in the Nano-C3G groups (groups 1, 2, and 3) and C3G group (group 4)

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compared to that of the control group (SC group), and the effect of Nano-C3G (group 3) was stronger

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than that of C3G (group 4) under the same concentration (Figure 3D).

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Nano-C3G and C3G Balance UVB-induced Bcl-2 Family Disorder. The mitochondrial

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membranes contain proteins that either activate (Bax) or inhibit (Bcl-2) apoptosis. p53 protein

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activates the transcription of Bax and inhibits the transcription of Bcl-2, with a net result favoring

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apoptosis. Overexpression of the survival factor Bcl-2 blocks apoptosis and thus protects the cells

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against radiation. ELISA revealed that the level of Bcl-2 was markedly increased in the Nano-C3G

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groups and C3G group (groups 1, 2, 3, and 4), while the Bax level was decreased compared to that of

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the control group (SC group), and the effect of Nano-C3G (group 3) was stronger than that of C3G

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(group 4) under the same concentration (Figure 3E, F). ACS Paragon Plus Environment

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Nano-C3G and C3G Inhibit UVB-induced Activation of Caspase Family Proteins. The

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caspase family plays a key role in the mitochondrial apoptosis cascade, especially caspase-3, after

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caspase-9 pulls the trigger. The results of western blot analysis indicated that overdose UVB irradiation

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to the mouse skin (UC group) resulted in significant disorder of the caspase family, including increases

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in the levels of caspase-3 and caspase-9. In addition, Nano-C3G or C3G (group 1, 2, 3, or 4) could

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markedly reduce this UVB-mediated caspase-3 and caspase-9 increase as compared to the Nano-alone

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group (SC group). The effect of Nano-C3G (group 3) for caspase-3 was stronger than that of C3G

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(group 4) under the same concentration (Figure 3G, H).

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DISCUSSION

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Nano-C3G could effectively protect against UVB-induced apoptosis, and the effects were

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stronger than those of C3G alone at the same concentrations. Apoptosis is a regulated cellular suicide

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mechanism characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA

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fragmentation29-30. Thus, apoptosis, or programmed cell death, is a major control mechanism by which

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cells die if DNA damage is not repaired31-32. DNA damage caused by UVB appears to result from the

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direct absorption of destructive UVB radiation by nucleotides33. At the same time, the radiation can

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also damage DNA indirectly. After absorption of photons, energy can be transferred either to DNA

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(non-photodynamic reaction) or to molecular oxygen, resulting in increased oxidative stress, which can

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then damage the DNA in turn (photodynamic reaction). Following DNA damage, p53 is activated to

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induce transcription of the Bcl-2 family of proteins, including the anti-apoptotic protein Bcl-2 and the

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pro-apoptotic protein Bax34. Caspases, a family of cysteine proteases, are the central regulators of

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apoptosis35. Once activated, the initiator caspases (including caspase-9, playing a key role in the

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mitochondrial-controlled apoptosis signaling pathway) cleave and activate downstream effector

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caspases (including caspase-3), which in turn execute apoptosis cascades.36–42

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Natural antioxidants are among the most promising photochemoprotective agents43-44. Indeed,

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botanicals such as C3G are increasingly being used in cosmetic or medical products as well as in skin

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care to protect against the adverse effects of UVB radiation45-46.

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The skin is composed of the epidermis, dermis, and subcutaneous tissue. Most cell components ACS Paragon Plus Environment

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are concentrated in the epidermis, the outermost layer of the skin. Keratinocytes are the major cell type

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in the epidermis, accounting for approximately 90% of the total cell population47, which was

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confirmed with our microscopic observations (Figure 2B). According to the results of slide

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observations (Figure 2B, D), we believe that Nano-C3G and C3G exert their actions on keratinocytes.

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We previously demonstrated that C3G inhibits UVB-induced apoptosis in human HaCaT

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keratinocytes in vitro. In particular, we found that C3G decreased the intracellular ROS level and DNA

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damage, suppressed COX-2 expression and inflammation, inhibited apoptosis by affecting the p53

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level or the MAPK and Akt signaling pathways, and decreased the levels of downstream factors

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ATM/ATR, p38, ERK, and JNK17-18. Furthermore, in the present study, we demonstrated that

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Nano-C3G increased its photodamage resistance ability against UVB in vivo through the regulation of

275

p53-mediated apoptosis.

276

Nano-C3G was successfully synthesized, and characterized by FTIR and TEM, respectively. We

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produced nanoparticles with an average size of 288 nm. It is generally considered that a diameter range

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of 1–1000 nm could be considered the nanoparticle level; however, the size range often reported in

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previous studies is 100–500 nm48.

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Nano-C3G and C3G were applied to the dorsal skin of mice after UVB exposure for assessing the

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therapeutic or repairing effects, as they were purple products. The results showed that Nano-C3G could

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improve the visual appearance and skin moisture of the model mice, as well as the histologic

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appearance and apoptotic index based on TUNEL staining. Further, Nano-C3G effectively reduced the

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LPO, MDA, and 8-OHdG contents induced by UVB; downregulated the p53 expression level;

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balanced members of the Bcl-2 family; and downregulated the caspase family in the acute UVB

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photodamage mouse model.

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Overall, these results suggest that Nano-C3G could protect against UVB-induced apoptosis

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through resistance to type Ⅰ and type Ⅰ photosensitized reactions through the regulation of

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p53-mediated apoptosis in mice, and the effects of Nano-C3G are stronger than those of C3G under the

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same concentration (500 µM). The fact that the superior effect of Nano-C3G could only be observed at

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the high dose (500 µM) might be due to the fact that the UVB-induced damage was very severe, and ACS Paragon Plus Environment

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thus only the high dose was sufficient to achieve an obvious improvement effect. We will further

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investigate this issue with more in-depth quantification in future studies.

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The mechanism of action of Nano-C3G contributed to its efficiency in skin protection. There are

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two potential reasons to explain the greater efficiency of Nano-C3G than the un-embedded C3G. First,

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as shown by our in vitro study of Nano-C3G stability, C3G could be released from nanoparticles under

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every pH condition tested, and the released C3G could be efficient to treat the damaged skin tissue.

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Second, nanoparticles could directly permeate through the skin lipid membrane and then release the

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drug in a sustained manner49. In addition, nanoparticles increased the permeation of drug into the skin8,

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50–52

. However, the specific mechanism requires further investigation.

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The mitochondrial control of the apoptosis signaling pathway acts in a network, and not as

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isolated branches. Therefore, a limitation of this study is that we did not detect the potential “blockers”

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of this pathway. Therefore, further research is needed to find out precisely how Nano-C3G affects the

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p53-mediated apoptosis signaling pathway, especially in vitro, and to quantify the advantage of

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nano-encapsulation techniques of C3G.

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Despite its preliminary nature, this study clearly indicated that Nano-C3G could protect the skin

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of mice against UVB-induced epidermal damage through the regulation of p53 apoptosis, and that

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these protective effects are stronger than those of C3G under the same concentration. Conclusively, our

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results provide a basis for the powerful photochemopreventive effect of Nano-C3G, and suggest that it

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may be a useful agent against UVB-induced damage for the skin toward potential development as a

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skin care product.

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AUTHOR INFORMATION

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Corresponding Author

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* Weibin Bai. E-mail: [email protected], Fax: +86-20-8522630, Tel.: +86-20-852263

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* Liehua Deng. Email: [email protected], Fax: +86-20-38688115, Tel.: +86-20-38688115

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Funding

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This work is supported by the Science and Technology Program of Guangzhou (No.

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201704020050). The authors also thank the National Science Foundation of China (NSFC) (No. ACS Paragon Plus Environment

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319 320 321 322 323 324 325 326 327 328

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31471588 and No. 31771983).

CONFLICT OF INTEREST The authors declare no competing financial interest.

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FIGURE CAPTIONS

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Figure 1 Characterization of Nano-C3G. (A) Nano-C3G. (B) FTIR spectra of Nano-C3G and

460

CS-TPP. (C) TEM of Nano-C3G. (D) Size of Nano-C3G. (E) Zeta potential of Nano-C3G. (F)

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Nano-C3G release in buffers of different pH (n = 3).

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Figure 2 Nano-C3G increases the protective effects of C3G against UVB-induced epidermal

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damage in mice (n = 10). (A) Visual appearance. (B) H&E staining of the skin. (C) Skin moisture and

464

elasticity. (D) TUNEL staining and the apoptosis index. #Group: NC (negative control group), UC

465

(UVB control group), SC (solvent control group), G1 (Nano-C3G low-dose group), G2 (Nano-C3G

466

middle-dose group), G3 (Nano-C3G high-dose group), G4 (C3G high-dose group). *p < 0.05, **p

Nanoencapsulation of Cyanidin-3-O-glucoside Enhances Protection

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