Preparation, Characterization and ex vivo Skin Permeability Evaluation of Type I Collagen-Loaded Liposomes
I 型胶原负载脂质体的制备、表征和体内外皮肤渗透性评估

Mingyuan Li' , Meng Li', Xinyi Li', Wanhui Shao', Xiujuan Pei $^{2}$ , Ruyue Dong $^{'}$ , Hongmeng Ren $^{'}$ , Lin Jia $^{'}$ , Shiqin Li', Wenlin Ma', Yi Zeng', Yun Liu' $^{'}$ , Hua Sun $^{'}$ , Peng Yu'
Minyuan Li' , Meng Li', Xinyi Li', Wanhui Shao', Xiujuan Pei $^{2}$ , Ruyue Dong $^{'}$ , Hongmeng Ren $^{'}$ , Lin Jia $^{'}$ , Shiqin Li', Wenlin Ma', Yi Zeng', Yun Liu' $^{'}$ , Hua Sun $^{'}$ , Peng Yu''College of Biotechnology/Tianjin Enterprise Key Laboratory for Application Research of Hyaluronic Acid, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China; $^{2}$ Tianjin Shiji Kangtai Biomedical Engineering Co.,Ltd, Tianjin, 300462, People's Republic of China
天津科技大学生物技术学院/透明质酸应用研究天津市企业重点实验室，天津，300457； $^{2}$ 天津世纪康泰生物医学工程有限公司，天津，300462Correspondence: Hua Sun; Peng Yu, Email sunhua@tust.edu.cn; yupeng@tust.edu.cn
通讯：Hua Sun; Peng Yu，电子邮件：sunhua@tust.edu.cn; yupeng@tust.edu.cn

Abstract 摘要

Purpose: In the present study, we prepared collagen liposomes with the addition of polyol, which is expected to not only increase the solubility of collagen but also improve skin penetration. Methods: Collagen liposomes were prepared by the film dispersion method, and their characteristics, integrity and biosafety were evaluated by Fourier transform infrared spectroscopy (FTIR), UV-VIS spectroscopy, polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and transmission electron microscope (TEM). The transdermal absorption of collagen and collagen liposomes were tested by an ex vivo horizontal Valia-Chien diffusion cell system. Results: We first demonstrated that collagen extracted from bovine Achilles tendon was type I collagen. The results of DLS measurement and TEM observation showed that the collagen liposomes were spherical in shape with average diameter ( $75.34 \pm 0.93 nm$ ) and maintained high stability at low temperature $(4^{\circ} C)$ for at least 42 days without toxicity. The encapsulation rate of collagen liposomes was $57.80 \pm 0.51 %$ , and SDS-PAGE analysis showed that collagen was intact in liposomes. Finally, permeability studies indicated that the collagen-loaded liposomes more easily penetrated the skin compared to collagen itself. Conclusion: This study proposed a new method to improve the bioavailability and permeability of bovine type I collagen, which improves the applicability of collagen in biomedicine, cosmeceuticals and pharmaceutical industries.
目的：在本研究中，我们制备了添加多元醇的胶原蛋白脂质体，这不仅有望增加胶原蛋白的溶解度，还能改善皮肤渗透性。制备方法采用薄膜分散法制备胶原蛋白脂质体，并通过傅立叶变换红外光谱（FTIR）、紫外可见光谱（UV-VIS）、聚丙烯酰胺凝胶电泳（SDS-PAGE）、动态光散射（DLS）和透射电子显微镜（TEM）对其特性、完整性和生物安全性进行了评价。通过体外水平 Valia-Chien 扩散细胞系统测试了胶原蛋白和胶原蛋白脂质体的透皮吸收。结果：我们首先证明了从牛跟腱中提取的胶原蛋白是 I 型胶原蛋白。DLS 测量和 TEM 观察结果表明，胶原蛋白脂质体呈球形，平均直径（ $75.34 \pm 0.93 nm$ ），在低温 $(4^{\circ} C)$ 条件下至少能保持 42 天的高稳定性，且无毒性。胶原蛋白脂质体的包封率为 $57.80 \pm 0.51 %$ ，SDS-PAGE 分析表明脂质体中的胶原蛋白完好无损。最后，渗透性研究表明，与胶原蛋白本身相比，负载胶原蛋白的脂质体更容易穿透皮肤。结论本研究提出了一种提高牛 I 型胶原蛋白生物利用度和渗透性的新方法，从而提高了胶原蛋白在生物医学、药妆和制药行业的适用性。

Keywords: collagen, liposome, physicochemical properties of collagen, skin penetration, drug delivery
关键词：胶原蛋白；脂质体；胶原蛋白的理化特性；皮肤渗透；药物输送

Introduction 导言

Collagen is the major protein in human and animal connective tissues, accounting for approximately

25 %

of the total vertebrate protein,

^{1}

and it is essential for the mechanical protection of tissues and organs as well as the physiological regulation of the cellular environment.

^{2}

To date, among the 29 different types of collagen that have been identified,

^{3}

type I collagen is the most abundant and typical one, and it mainly plays a structural role.

^{4}

Type I collagen has a relative molecular mass of approximately 300 kDa , and it is mainly found in skin, lung, bone and connective tissue.

^{5}

Type I collagen is a three-stranded helical structural protein formed by two

α_{1}

peptide chains and one

α_{2}

peptide chain intertwined, and each peptide consists of

(Gly - XY)_{n}

with X and Y representing mainly proline and hydroxyproline, respectively.

^{6}

Due to its many properties, such as low immunity, good biocompatibility and biodegradability,

^{7 - 11}

type I collagen has a wide range of applications in the bioindustry, such as tissue regeneration,

^{12, 13}

pharmaceuticals,

^{14}

food

^{15}

and cosmetics.

^{16}

Although the cost of preparing collagen remains high, the market demand for collagen and its derivatives has been growing at a high rate in recent years.

^{17}

胶原蛋白是人类和动物结缔组织中的主要蛋白质，约占脊椎动物蛋白质总量的

25 %

^{1}

，它对组织和器官的机械保护以及细胞环境的生理调节至关重要。

^{2}

迄今为止，在已发现的 29 种不同类型的胶原蛋白中，

^{3}

Ⅰ型胶原蛋白是最丰富、最典型的一种，它主要起结构作用。

^{4}

I 型胶原蛋白的相对分子质量约为 300 kDa，主要存在于皮肤、肺、骨骼和结缔组织中。

^{5}

Ⅰ型胶原蛋白是一种三股螺旋结构蛋白，由两条

α_{1}

肽链和一条

α_{2}

肽链交织而成，每条肽链由

(Gly - XY)_{n}

组成，其中X和Y分别主要代表脯氨酸和羟脯氨酸。

^{6}

由于 I 型胶原蛋白具有免疫力低、生物相容性好和可生物降解等多种特性，因此在生物产业中有着广泛的应用，如组织再生、

^{12, 13}

制药、

^{14}

食品

^{15}

和化妆品等。

^{16}

虽然制备胶原蛋白的成本仍然很高，但近年来市场对胶原蛋白及其衍生物的需求一直在高速增长。

^{17}

The increasing market demand has led to more aggressive attempts to extract and isolate type I collagen from different animal species. Currently, the best source of type I collagen is mammals

^{18}

due to its high sequence homology with human collagen,

^{19}

and bovine and porcine collagen dominate in terms of quality and quantity.

^{20, 21}

In some
市场需求的不断增长促使人们更加积极地尝试从不同动物物种中提取和分离 I 型胶原蛋白。目前，I型胶原蛋白的最佳来源是哺乳动物

^{18}

，因为它与人类胶原蛋白的序列同源性很高，

^{19}

而牛和猪的胶原蛋白在质量和数量上都占主导地位。

^{20, 21}

在某些情况下

Graphical Abstract 图表摘要

countries, however, the use of pork-derived collagen has been banned,

^{22}

resulting in bovine collagen as the main raw material for pharmaceuticals and cosmetics. Despite the many advantages of type I collagen, its low bioavailability and low absorption efficiency still limit its use, suggesting that encapsulation may be an effective solution in this regard.
然而，许多国家已禁止使用猪肉提取的胶原蛋白，

^{22}

因此牛胶原蛋白成为药品和化妆品的主要原料。尽管 I 型胶原蛋白有许多优点，但其生物利用率低和吸收效率低的特点仍然限制了它的使用，这表明封装可能是这方面的一个有效解决方案。

Using liposomes to encapsulate collagen improves its bioavailability and absorption efficiency.

^{23}

Liposomes are closed vesicles (usually

50 - 500 nm

in diameter) consisting of a phospholipid bilayer

^{24}

with a biofilm-like structure,

^{25}

and they have been widely used to deliver various components, such as DNA,

^{26, 27}

vaccines

^{28, 29}

and drugs

^{30, 31}

into the body, with contributions in the pharmaceutical and cosmetic fields.

^{32}

The use of liposomes loaded with fish-derived peptides improves antioxidant properties and skin penetration characteristics.

^{33 - 35}

Thus, the liposome encapsulation technique may improve the abovementioned problems. To be applied in the cosmetics industry, in addition to natural lecithin, a certain amount of permeability enhancers can also be added in the preparation of liposomes to enhance skin permeability, allowing the effective ingredients to play a better role, and propylene glycol is the most commonly used permeability enhancer.

^{36}

使用脂质体包裹胶原蛋白可提高其生物利用度和吸收效率。

^{23}

脂质体是一种封闭的囊（直径通常为

50 - 500 nm

），由具有生物膜状结构的磷脂双分子层

^{24}

组成，

^{25}

脂质体已被广泛用于向体内输送各种成分，如DNA、

^{26, 27}

疫苗

^{28, 29}

和药物

^{30, 31}

，在制药和化妆品领域做出了贡献。

^{32}

使用含有鱼源性肽的脂质体可提高抗氧化性能和皮肤渗透特性。

^{33 - 35}

因此，脂质体封装技术可以改善上述问题。应用于化妆品行业，除了天然卵磷脂外，还可以在脂质体的制备过程中加入一定量的渗透增强剂，增强皮肤的渗透性，让有效成分发挥更好的作用，丙二醇是最常用的渗透增强剂。

^{36}

In the present study, we prepared collagen liposomes with the addition of polyol, which not only increases the solubility of collagen but also improves skin penetration to some extent. Although liposomes have been used as carriers to improve the absorption of protein, nanoliposomes encapsulating bovine-derived type I collagen have not been developed. Therefore, we used bovine-derived type I collagen to prepare collagen nanoliposomes with high molecular mass, resulting in better skin penetration ability compared to conventional bovine collagen.
在本研究中，我们制备了添加多元醇的胶原蛋白脂质体，这不仅增加了胶原蛋白的溶解度，还在一定程度上改善了皮肤渗透性。虽然脂质体已被用作改善蛋白质吸收的载体，但包裹牛源性 I 型胶原蛋白的纳米脂质体尚未开发出来。因此，我们使用牛源性 I 型胶原蛋白制备了高分子质量的胶原蛋白纳米脂质体，与传统的牛胶原蛋白相比，其皮肤渗透能力更强。

Materials and Methods 材料与方法

Materials 材料

The collagen lyophilized sponge was kindly provided by Tianjin Shiji Kangtai Biomedical Engineering Company, Ltd; BV2 cell line, fetal bovine serum and MEM medium were purchased from Wuhan Procell Life Science & Technology Company; Egg yolk lecithin PL-100M (Injection Grade, Phosphatidylcholine

\geq 98 %

), purchased from AVT (Shanghai) Pharmaceutical Technology Company; 1,2-propanediol,

99 %

, AR, purchased from Shanghai Macklin Biochemical Technology Co.; Fluorescein Isothiocyanate (FITC), 97%, Biotech Grade, BR, purchased from Shanghai Macklin Biochemical Technology Co.; Coumarin-6, 98%, HPLC grade, purchased from Shanghai Aladdin Biochemical Technology Co. All other chemicals were of analytical grade and were purchased from the Shanghai Aladdin Biochemical Technology Company. Double distilled water, obtained from a water purification system (Nano-pure Infinity, Barnstead International. Dubuque. IA), was used to prepare all solutions.
胶原蛋白冻干海绵由天津世纪康泰生物医学工程有限公司提供；BV2 细胞系、胎牛血清和 MEM 培养基购自武汉普罗凯尔生命科技有限公司；蛋黄卵磷脂 PL-100M（注射级，磷脂酰胆碱

\geq 98 %

），购自 AVT（上海）医药科技有限公司；1,2-丙二醇，

99 %

，AR，购自上海麦林生化科技有限公司；荧光素异硫磷，购自上海麦林生化科技有限公司。异硫氰酸荧光素（FITC），97%，生物技术级，BR，购自上海麦林生化科技有限公司；香豆素-6，98%，HPLC 级，购自上海阿拉丁生化科技有限公司；呋喃妥因（FITC），97%，生物技术级，BR，购自上海麦林生化科技有限公司；香豆素-6，98%，HPLC 级，购自上海阿拉丁生化科技有限公司。其他化学品均为分析级，购自上海阿拉丁生化科技有限公司。所有溶液的配制均使用双蒸馏水，该水来自水纯化系统（Nano-pure Infinity, Barnstead International.

Physical-Chemical Properties of Collagen Measurement of Collagen Solubility
胶原蛋白的物理化学特性测量胶原蛋白的溶解度

The solubility of collagen can be determined according to Lawal

^{37}

with slight modifications. 50 mg lyophilized collagen sponge was dissolved in 20 mL ultrapure water, and the pH value of collagen solution was adjusted with

1.0 mol / L HCl

1.0 mol / L NaOH, pH = 2.0 - 12.0

, at 1.0 unit intervals. The above treated collagen solution was swirled at

25^{\circ} C

with a vortex oscillator until it was fully mixed, centrifuged at 8000 rpm for 10 min , and the supernatant was filtered. The amount of soluble collagen and total collagen in the supernatant were determined using the BCA kit, and the protein standard curve was obtained using bovine serum albumin (BSA). The protein concentration at each pH was determined by comparing it with the highest protein content calculated using the following formula.
胶原蛋白的溶解度可根据 Lawal

^{37}

略加修改后测定。将 50 毫克冻干海绵胶原蛋白溶解在 20 毫升超纯水中，用

1.0 mol / L HCl

或

1.0 mol / L NaOH, pH = 2.0 - 12.0

调节胶原蛋白溶液的 pH 值，每隔 1.0 个单位。将上述处理过的胶原蛋白溶液用漩涡振荡器以

25^{\circ} C

的转速旋转，直至完全混合，然后在 8000 rpm 转速下离心 10 分钟，过滤上清液。用 BCA 试剂盒测定上清液中可溶性胶原蛋白和总胶原蛋白的含量，并用牛血清白蛋白（BSA）绘制蛋白质标准曲线。将每种 pH 值下的蛋白质浓度与用下式计算的最高蛋白质含量进行比较，从而确定蛋白质浓度。

Relative solubility (%) = \frac{Collagen content in supernatant (mg)}{Highest collagen content in sample (mg)} \times 100

UV-Vis Spectroscopy of Collagen
胶原蛋白的紫外可见光谱分析

In brief, 10 mg of lyophilized collagen sponge was dissolved in 10 mL of 0.5 M acetic acid (

1 mg / mL

), and the UV-Vis spectra from 190 to 400 nm absorption light were recorded with a Shimadzu UV-2550 PC type UV-Vis spectrophotometer (Shimadzu, Japan).
将 10 毫克冻干海绵胶原蛋白溶于 10 毫升 0.5 M 乙酸（

1 mg / mL

）中，用岛津 UV-2550 PC 型紫外可见分光光度计（日本岛津公司）记录 190 至 400 纳米吸收光的紫外可见光谱。

Fourier Transform Infrared Spectroscopy of Collagen
胶原蛋白的傅立叶变换红外光谱分析

The infrared spectra of collagen were measured in the range of

4000 \sim 400 {cm}^{- 1}

using Fourier transform infrared spectrometer (Thermo Fisher IS50, USA) according to the method of Hamdan.

^{38}

根据 Hamdan 的方法，使用傅立叶变换红外光谱仪（Thermo Fisher IS50，美国）测量了胶原蛋白在

4000 \sim 400 {cm}^{- 1}

范围内的红外光谱。

^{38}

Preparation of Collagen Liposomes
胶原蛋白脂质体的制备

Preparation of Collagen Liposome
胶原蛋白脂质体的制备

Collagen-loaded liposomes were prepared by a combination of film dispersion method and ultrasonication. 240 mg egg yolk lecithin dissolved in anhydrous ethanol was formed into homogeneous lipid films by a rotary evaporator under vacuum at

55^{\circ} C

, and dried in a vacuum drying oven

(- 0.1 MPa)

for 1 h . Then the lipid films were hydrated by 8 mL collagen solution (

5 %

1.2-propylene glycol,

v / v

) at

35^{\circ} C

. The newly formed multilayer lipid capsules were ultrasonically crushed (Power

5 %

, mode

φ 6

, on 2 s , off 2 s ) and squeezed through a 220 nm Millipore Express

^{®}

PES membrane filter unit to form small liposomes (The concentration of egg yolk lecithin is

30 mg / mL

) of uniform size. Blank liposomes were prepared in the same way as a control.
胶原蛋白负载脂质体的制备结合了薄膜分散法和超声波法。将 240 毫克蛋黄卵磷脂溶于无水乙醇，用旋转蒸发仪在

55^{\circ} C

真空条件下制成均匀的脂质膜，并在真空干燥箱

(- 0.1 MPa)

中干燥 1 小时。然后在

35^{\circ} C

下用 8 mL 胶原溶液（

5 %

1.2-丙二醇，

v / v

）对脂膜进行水合。将新形成的多层脂质胶囊用超声波粉碎（功率

5 %

，模式

φ 6

，开 2 秒，关 2 秒），并通过 220 nm Millipore Express

^{®}

PES 膜过滤装置挤压，形成大小均匀的小脂质体（蛋黄卵磷脂的浓度为

30 mg / mL

）。空白脂质体的制备方法与对照组相同。

Preparation of Fluorescein-Labeled Collagen Liposomes
荧光素标记胶原脂质体的制备

Fluorescein-labeled liposomes loaded with collagen were prepared by a combination of thin-film dispersion and ultrasonication. 240 mg egg yolk lecithin with coumarin-6 dissolved in anhydrous ethanol was formed into homogeneous lipid films by a rotary evaporator under vacuum at

55^{\circ} C

, and dried in a vacuum drying oven

(- 0.1 MPa)

for 1 h . The lipid films were then hydrated by 8 mL collagen solution (

5 % 1.2

-propylene glycol,

v / v

) at

35^{\circ} C

. The newly formed
采用薄膜分散和超声相结合的方法制备了负载胶原蛋白的荧光素标记脂质体。将 240 毫克含有香豆素-6 的蛋黄卵磷脂溶于无水乙醇，用旋转蒸发仪在

55^{\circ} C

真空条件下制成均匀的脂膜，并在真空干燥箱

(- 0.1 MPa)

中干燥 1 小时。然后在

35^{\circ} C

下用 8 mL 胶原溶液（

5 % 1.2

- 丙二醇，

v / v

）对脂膜进行水合。新形成的
multilayer lipid capsules were crushed by ultrasonication (Power

5 %

, mode

φ 6

, on 2 s , off 2 s ) and squeezed through a 220 nm Millipore Express® PES membrane filter unit to form small liposomes (The concentration of egg yolk lecithin is

30 mg / mL

) of uniform size.
用超声波（功率

5 %

，模式

φ 6

，开 2 秒，关 2 秒）粉碎多层脂质胶囊，并通过 220 nm 的 Millipore Express® PES 膜过滤装置挤压，形成大小均匀的小脂质体（蛋黄卵磷脂的浓度为

30 mg / mL

）。

Characterization of Liposomes
脂质体的表征

Size, Size Distribution and $ζ$ -Potential Measurements
尺寸、尺寸分布和 $ζ$ 电位测量

The average droplet size, size distribution and

ζ

-potential of liposomes were measured by a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Size distributions were expressed as polydispersity index (PDI) and depicted graphically. Each sample was analyzed at least three times and the mean value was calculated.
用 Malvern Zetasizer Nano ZS90（英国马尔文仪器公司）测量脂质体的平均液滴大小、大小分布和

ζ

电位。粒度分布以多分散指数（PDI）表示，并以图形表示。每个样品至少分析三次，并计算平均值。

Determination of Encapsulation Rate
确定封装率

Unencapsulated (free) collagen was separated from liposomes by a D-TUBE

^{TM}

Dialyzer Maxi with a molecular weight up to 300 kDa according to manufacturer’s instructions. Briefly, 3 mL of sample was placed in the D-TUBE

^{TM}

dialyzer and dialyzed in 300 mL of deionized water for 12 h to remove unencapsulated collagen retained in the continuous phase. Pure liposomes were then disrupted by Triton X-100 (

5 %

, w/v) to release encapsulated collagen. The collagen concentration was determined using a BCA kit. For this purpose,

10 μ L

bovine serum albumin samples with different concentrations were added to

250 μ L

working solution, mixed, incubated at

37^{\circ} C

for 30 min , and then the absorbance was measured at 562 nm using a microplate reader (Biotek Synergy H1, USA). The encapsulation efficiency was calculated using bovine serum albumin (BSA) to obtain a protein standard curve. The encapsulation efficiency was then calculated as a percentage according to the following equation:
根据制造商的说明，用 D-TUBE

^{TM}

透析器 Maxi 从脂质体中分离出未包封（游离）的胶原蛋白，分子量最高可达 300 kDa。简单地说，将 3 mL 样品放入 D-TUBE

^{TM}

透析器中，在 300 mL 去离子水中透析 12 小时，以去除连续相中保留的未包封胶原蛋白。然后用 Triton X-100 (

5 %

, w/v)破坏纯脂质体，以释放包封的胶原蛋白。使用 BCA 试剂盒测定胶原蛋白浓度。为此，在

250 μ L

工作液中加入不同浓度的

10 μ L

牛血清白蛋白样品，混合，在

37^{\circ} C

下孵育 30 分钟，然后使用微孔板阅读器（Biotek Synergy H1，美国）在 562 纳米处测量吸光度。使用牛血清白蛋白（BSA）计算封装效率，得到蛋白质标准曲线。然后根据以下公式计算封装效率的百分比：

Encapsulation efficiency (%) = \frac{Encapsulated Collagen concentration (mg)}{Total collagen concentration (mg)} \times 100

Morphological Analysis 形态分析

The morphology of liposomes was evaluated using transmission electron microscopy (TEM, Talos G2 200x, Thermo Fisher, USA) operating at 200 kV . For TEM sample preparation, approximately

10 μ L

of liposomes was added dropwise on a carbon-coated grid for 30 s, and the excess solution was aspirated using filter paper. The grids were stained with

2 %

phosphotungstic acid and dried overnight at room temperature.
使用透射电子显微镜（TEM，Talos G2 200x，Thermo Fisher，USA）在 200 kV 下对脂质体的形态进行评估。制备 TEM 样品时，在碳涂层网格上滴加大约

10 μ L

的脂质体 30 秒，然后用滤纸吸去多余的溶液。用

2 %

磷钨酸对网格进行染色，并在室温下干燥过夜。

Physical Stability Studies
物理稳定性研究

The stability of collagen liposomes was compared under various storage conditions by assessing different variations of the mean particle size. They were stored at

25^{\circ} C

and

4^{\circ} C

for 56 days, and aliquots of the samples were collected at intervals and analyzed for stability. Each batch was analyzed in triplicate.
通过评估平均粒径的不同变化，比较了胶原蛋白脂质体在不同储存条件下的稳定性。它们在

25^{\circ} C

和

4^{\circ} C

条件下储存了56天，每隔一段时间收集等分样品并分析其稳定性。每批样品一式三份进行分析。

SDS-PAGE

SDS-PAGE was performed according to a previously reported method.

^{39}

Briefly, collagen was dissolved in ultrapure water (

1 mg / mL

), and collagen liposomes were dissociated by Triton X-100 (

5 %

, w/v). Then,

40 μ L

of sample was mixed with

10 μ L

of SDS-PAGE protein loading buffer (bromophenol blue was added as an indicator). Samples were then heated to

100^{\circ} C

for 10 min , centrifuged at 9000 rpm for 10 min and electrophoresed using

8 %

SDS-PAGE gels,

1 \times

Running Buffer and Novex X Cell II devices. The protein bands were visualized by coomassie brilliant blue R250 staining, and the molecular mass was identified using Benchmark pre-stained protein ladders.
SDS-PAGE 按照之前报道的方法进行。

^{39}

简单地说，将胶原蛋白溶解在超纯水（

1 mg / mL

）中，用 Triton X-100 （

5 %

，w/v）溶解胶原蛋白脂质体。然后，将

40 μ L

样品与

10 μ L

SDS-PAGE 蛋白上样缓冲液（添加溴酚蓝作为指示剂）混合。然后将样品加热至

100^{\circ} C

10 分钟，在 9000 rpm 转速下离心 10 分钟，使用

8 %

SDS-PAGE 凝胶、

1 \times

运行缓冲液和 Novex X Cell II 装置进行电泳。蛋白质条带通过库马西亮蓝 R250 染色显现，分子质量通过 Benchmark 预染色蛋白质梯形图鉴定。

Cell Viability Detection (MTT)
细胞活力检测（MTT）

To assess the toxicity of collagen-loaded liposomes, we performed a cell viability test using the BV2 microglial cell line. Cells (

2 \times 10^{5}

cells/well) were seeded into 96 -well culture plates and cultured in MEM medium supplemented with

10 %

fetal bovine serum (FBS) for 24 h . The collagen-loaded liposomes were then mixed with the medium at different concentrations, and cells were cultured for 24 h before performing cell viability tests. In the 3-(4,5-dimethylthiazol-2-yl)2,5 -diphenyltetrazolium bromide (MTT) assay,

20 μ L

MTT solution was added to

100 μ L

medium containing collagen liposome. After an additional 4 h incubation,

100 μ L

of acidified isopropanol was added to dissolve the insoluble
为了评估胶原脂质体的毒性，我们使用 BV2 小神经胶质细胞系进行了细胞活力测试。将细胞（

2 \times 10^{5}

细胞/孔）播种到96孔培养板中，在添加

10 %

胎牛血清（FBS）的MEM培养基中培养24小时。然后将不同浓度的胶原负载脂质体与培养基混合，培养细胞 24 小时后进行细胞活力测试。在 3-(4,5-二甲基噻唑-2-基)2,5-二苯基溴化四氮唑（MTT）试验中，

20 μ L

将 MTT 溶液加入到含有胶原脂质体的

100 μ L

培养基中。再孵育 4 小时后，加入

100 μ L

酸化异丙醇溶解不溶性物质。
methanogenic crystals in the live cells. The absorbance was measured at 570 nm and 630 nm using a microplate reader. At the same time, the cell viability of blank liposomes was also measured according to the above method as a comparison. The effect of liposomes on cytotoxicity was assessed by cell viability, which was calculated using the following formula:
活细胞中的产甲烷晶体。用微孔板阅读器在 570 纳米和 630 纳米处测量吸光度。同时，根据上述方法还测量了空白脂质体的细胞活力作为对比。脂质体对细胞毒性的影响通过细胞存活率来评估，细胞存活率用下式计算：

Cell viability (%) = \frac{O D_{experiment} - O D_{blank} [mean]}{O D_{M E M} - O D_{blank} [mean]} \times 100

Ex vivo Skin Permeability Assessment of Collagen and Collagen Liposomes
胶原蛋白和胶原蛋白脂质体的体外皮肤渗透性评估

The fresh tonguefish skin was provided by the College of Marine and Environmental Sciences at Tianjin University of Science and Technology. The BALB/c mouse skin and nude mouse skin were provided by Professor Yuou Teng in the College of Bioengineering at Tianjin University of Science and Technology. The BALB/c mouse (

6 \sim 8

weeks/20~25 g) and nude mice (

6 \sim 8

weeks

/ 18 \sim 22 g

) were purchased from Vital River Laboratories [Beijing, China; permit number SCXK (jing), 2017-0005], and were well housed and served as the control group in Professor Yuou Teng’s study without any drug or experimental treatment. After her experiment, the animals in the control group also needed to be sacrificed. In this study, the skin tissue was taken from animal carcasses provided by several professors. All procedures were carried in accordance with the Guidelines for Care and Use of Laboratory Animals of Tianjin University of Science and Technology and approved by the Animal Ethics Committee of Tianjin University of Science and Technology (Tianjin, China; protocol code: 2021.2.42.1, approval date: 5 March 2021).
新鲜舌鱼皮肤由天津科技大学海洋与环境科学学院提供。BALB/c 小鼠皮肤和裸鼠皮肤由天津科技大学生物工程学院滕玉鸥教授提供。BALB/c小鼠（

6 \sim 8

周/20~25 g）和裸鼠（

6 \sim 8

周

/ 18 \sim 22 g

）购自生命河实验室[中国北京；许可证号SCXK（京），2017-0005]，饲养良好，作为滕玉鸥教授研究中的对照组，未进行任何药物和实验处理。在她的实验结束后，对照组的动物也需要牺牲。在这项研究中，皮肤组织取自几位教授提供的动物尸体。所有实验过程均按照《天津科技大学实验动物饲养与使用指导原则》进行，并经天津科技大学动物伦理委员会批准（天津，中国；方案代码：2021.2.42.1，批准日期：2021年3月5日）。

Skin Preparation 皮肤准备

The Skin of Tonguefishes
舌鱼的皮肤

The fresh tonguefish skin scraped off the scales carefully, and the skin on the black side was taken, the subcutaneous tissue and fat were removed, rinsed with saline (The procedure for separating the skin of a tonguefish is shown in Figure S1). According to the requirements of the experiment,

2 cm \times 2 cm

skin in vitro was cut, soaked in saline, refrigerated, and used within 24 h . The integrity of the skin was checked by magnifier before the transdermal experiment and hydrated by soaking in saline for 1 h .
将新鲜舌鱼皮仔细刮去鳞片，取黑色一侧的皮，去除皮下组织和脂肪，用生理盐水冲洗干净（舌鱼皮的分离过程如图 S1 所示）。根据实验要求，

2 cm \times 2 cm

体外皮肤被切开，浸泡在生理盐水中，冷藏并在 24 小时内使用。透皮实验前用放大镜检查皮肤的完整性，并在生理盐水中浸泡 1 小时以补充水分。

Nude Mouse Skin 裸鼠皮肤

The isolated back skin of nude mice was taken, remove the nude mouse hair, removing subcutaneous tissue and fat, rinsing with saline. According to the experimental requirements,

2 cm \times 2 cm

skin in vitro was cut, soaking in saline, refrigerating, and using within 24 h . Before the transdermal experiment, the skin integrity was checked with a magnifier and hydrated by soaking in saline for 1 h .
取裸鼠离体背部皮肤，去掉裸鼠毛发，去除皮下组织和脂肪，用生理盐水冲洗。根据实验要求，将

2 cm \times 2 cm

体外皮肤剪开，浸泡在生理盐水中，冷藏，24 小时内使用。透皮实验前，用放大镜检查皮肤的完整性，并在生理盐水中浸泡 1 小时，补充水分。

BALB/c Mouse Skin BALB/c 小鼠皮肤

The isolated back skin of BALB/c mice was taken, remove the BALB/c mouse hair, half of the mouse skin was treated with hair removal cream to remove the remaining hair to simulate normal skin, and the other half was immersed in

8 %

sodium sulfide solution to remove the remaining hair for 15 min to simulate damaged skin. Removing subcutaneous tissue and fat, rinsing with saline, according to the experimental requirements,

2 cm \times 2 cm

skin in vitro was cut, soaking in saline, refrigerating, and using within 24 hours. Before the transdermal experiment, the skin integrity was checked with a magnifier and hydrated by soaking in saline for 1 h .
取BALB/c小鼠离体背部皮肤，去除BALB/c小鼠毛发，一半小鼠皮肤用脱毛膏处理，去除剩余毛发，模拟正常皮肤，另一半小鼠皮肤浸泡在

8 %

硫化钠溶液中15min，去除剩余毛发，模拟受损皮肤。去除皮下组织和脂肪，用生理盐水冲洗，根据实验要求，将

2 cm \times 2 cm

体外皮肤剪开，浸泡在生理盐水中，冷藏，24小时内使用。透皮实验前，用放大镜检查皮肤的完整性，并在生理盐水中浸泡 1 小时，补充水分。

Cumulative Penetration $(Q_{n})$ , Steady-State Permeation Rate $(J_{ss})$ and Absorption Rate (W%) of Collagen
胶原蛋白的累积渗透率 $(Q_{n})$ 、稳态渗透率 $(J_{ss})$ 和吸收率 (W%)

For this experiment, the TK-6H1 Valia-Chien double-chamber transdermal diffusion tester was used. The isolated cryopreserved skin was fixed in the combined part of the diffusion cell with the cuticle of the skin facing the supply cell. The pool was filled with collagen solution and collagen liposomes at a concentration of

1 mg / mL

. The receiving pool was filled with receiving solution (saline). The diffusion cell water bath jacket was connected to a peristaltic pump and a thermostatically heated magnetic stirrer to maintain constant stirring, and the water bath temperature was set to

32.0 \pm {0.5}^{\circ} C

and

36.0 \pm {0.5}^{\circ} C

200 r / \min .^{40}

Samples were taken at

0.5, 1, 2, 4, 6, 8, 10

and 24 h time points, and 1 mL of
本实验使用了 TK-6H1 Valia-Chien 双室透皮扩散测试仪。分离的低温保存皮肤被固定在扩散池的组合部分，皮肤的角质层朝向供应池。池中注入浓度为

1 mg / mL

的胶原蛋白溶液和胶原蛋白脂质体。接收池中注入接收溶液（生理盐水）。扩散池水浴夹套与蠕动泵和恒温加热磁力搅拌器相连，以保持恒定搅拌，水浴温度设定为

32.0 \pm {0.5}^{\circ} C

和

36.0 \pm {0.5}^{\circ} C

时，

200 r / \min .^{40}

和

0.5, 1, 2, 4, 6, 8, 10

和 24 小时时间点取样，并取 1 mL

the receiving medium was removed each time and then replenished with saline of equal temperature to the original volumetric amount. The total collagen concentration of the samples was determined by a BCA kit, and the

Q_{n}, J_{ss}

and absorption

W %

of collagen were calculated using the following respective equations:
每次移除接收培养基，然后补充与原体积等温的生理盐水。样品的总胶原蛋白浓度由 BCA 试剂盒测定，胶原蛋白的

Q_{n}, J_{ss}

和吸收

W %

分别用以下公式计算：

\begin{matrix} Q_{n} = \frac{C_{n} \times V + \sum_{i = 1}^{n - 1} C_{i} \times V_{i}}{A} \\ J_{s s} = \frac{d Q}{A d t} = P C_{n} \\ W = \frac{Q_{n} \times A}{S} \times 100 % \end{matrix}

In the above equation,

Q_{n}

is the cumulative transdermal volume per unit area

(μ g \cdot {cm}^{- 2}), C_{n}

is the concentration of collagen at the nth sampling

(μ g / mL), V

is the volume of the receiving cell ( 5.0 mL ),

C_{i}

is the concentration of collagen at the ith sampling

(μ g / mL), V_{i}

is the sampling volume

(1 mL)

, A is the effective contact area

(1.13 {cm}^{2})

J_{ss}

is the steady-state transmission rate

(μ g \cdot {cm}^{- 2} \cdot h^{- 1})

, W is the cumulative permeation percentage (%), and S is the total amount of collagen (

μ g

).
在上式中，

Q_{n}

是单位面积的累积透皮量

(μ g \cdot {cm}^{- 2}), C_{n}

是第 n 次取样时的胶原蛋白浓度

(μ g / mL), V

是接收池的体积（5.0 mL ），

C_{i}

是第 i 次取样时的胶原蛋白浓度

(μ g / mL), V_{i}

是取样体积

(1 mL)

，A 是有效接触面积

(1.13 {cm}^{2})

，

J_{ss}

是稳态透皮率

(μ g \cdot {cm}^{- 2} \cdot h^{- 1})

，W 是累积渗透百分比 (%)，S 是胶原蛋白总量 (

μ g

)。

Using

Q_{n}

as the vertical coordinate and sampling time t as the horizontal coordinate, the ex vivo transdermal permeation curves of collagen and collagen liposomes were plotted, and the slope of the straight line was the steadystate transdermal rate

(J_{sS})

.
以

Q_{n}

为纵坐标，取样时间t为横坐标，绘制胶原蛋白和胶原蛋白脂质体的体内透皮渗透曲线，直线斜率即为稳态透皮率

(J_{sS})

。

Skin Retention $(Q_{s})$ and Retention Rate $(A_{s})$ of Collagen
皮肤保留 $(Q_{s})$ 和胶原蛋白保留率 $(A_{s})$

After the ex vivo transdermal experiment, the isolated skin was removed, and the surface of the skin was washed repeatedly with saline until the protein on the skin surface was cleaned. Then the drug administration part was placed in a 10 mL centrifuge tube, cut into pieces with ophthalmic scissors, 4 mL of ultrapure water was added, sonicated it in the water bath for 30 min , left it stand for 10 min . The supernatant was taken and the BCA kit was used to determine the collagen concentration, calculation of collagen skin retention

(Q_{s})

and retention rates

(A_{s})

.
体外透皮实验结束后，取出离体皮肤，用生理盐水反复清洗皮肤表面，直至皮肤表面的蛋白质被清洗干净。然后将给药部分置于 10 mL 离心管中，用眼科剪刀剪成碎片，加入 4 mL 超纯水，在水浴中超声 30 分钟，静置 10 分钟。取上清液，用 BCA 试剂盒测定胶原蛋白浓度，计算胶原蛋白皮肤保留率

(Q_{s})

和保留率

(A_{s})

。

\begin{matrix} Q_{s} = \frac{V_{0} \times C}{A} \\ A_{s} = \frac{Q_{s} \times A}{m_{s}} \times 100 % \end{matrix}

In the above equation,

Q_{s}

is the skin retention volume

(μ g \cdot {cm}^{- 2}), V_{0}

is the ultra-pure water volume ( 4.0 mL ), C is the measured collagen concentration (

mg / mL

), A is the effective contact area

(1.13 {cm}^{2}), A_{s}

is the skin retention rate (%), and

m_{s}

is the mouse skin weight (

μ g

).
在上式中，

Q_{s}

是皮肤保留体积

(μ g \cdot {cm}^{- 2}), V_{0}

是超纯水体积（4.0 mL），C是测量的胶原蛋白浓度（

mg / mL

），A是有效接触面积

(1.13 {cm}^{2}), A_{s}

是皮肤保留率（%），

m_{s}

是小鼠皮肤重量（

μ g

）。

Microscopic Observation of Skin Permeation Phenomenon
皮肤渗透现象的显微镜观察

Fluorescein-Labeled Collagen
荧光素标记的胶原蛋白

Fluorescein isothiocyanate (FITC) was added to 20 mL of

1 mg / mL

collagen carbonate buffer (

0.1 M, pH 9.0

), stirred at room temperature and crosslinked at 450 rpm for 18 h in the dark. The labeled collagen solution was centrifuged at 3000 rpm for 15 min , washed five times with carbonate buffer and stored at

4^{\circ} C

in the dark.
将异硫氰酸荧光素（FITC）加入 20 mL 碳酸胶原缓冲液（

0.1 M, pH 9.0

）中，在室温下搅拌，并在黑暗中以 450 rpm 的转速交联 18 h。标记的胶原蛋白溶液在 3000 rpm 转速下离心 15 分钟，用碳酸盐缓冲液洗涤 5 次，然后在黑暗中储存在

4^{\circ} C

中。

Fluorescence Microscope Observation of Skin Permeation
荧光显微镜观察皮肤渗透情况

For this experiment, the TK-6H1 type Valia-Chien double chamber transdermal diffusion tester was used. The normal skin of an isolated cryopreserved BALB/c mouse was fixed in the combined part of the diffusion cell with the cuticle of the skin facing the supply pool, and the pool was filled with fluorescein-labeled collagen liposomes and fluoresceinlabeled collagen solution at a concentration of

1 mg / mL

. The receiving pool was filled with the receiving solution (saline). The diffusion cell outer water bath was connected to a peristaltic pump and a thermostatically heated magnetic
本实验采用 TK-6H1 型 Valia-Chien 双室透皮扩散试验箱。将离体低温保存的 BALB/c 小鼠的正常皮肤固定在扩散池的组合部分，皮肤角质层朝向供池，池中注入浓度为

1 mg / mL

的荧光素标记胶原脂质体和荧光素标记胶原溶液。接收池中注入接收液（生理盐水）。扩散池外水槽连接着一个蠕动泵和一个恒温加热的磁力泵。
stirrer to maintain constant stirring at a water bath temperature of

32.0 \pm {0.5}^{\circ} C

with a speed of

200 r / \min

for 18 h . At the end of the experiment, the skin was treated and cut into slices for observation under a fluorescence microscope (Olympus BX51, Japan).
在水浴温度为

32.0 \pm {0.5}^{\circ} C

、转速为

200 r / \min

的条件下，用搅拌器持续搅拌 18 小时。实验结束后，处理皮肤并切片，在荧光显微镜（Olympus BX51，日本）下观察。

Statistical Analysis 统计分析

One-way analysis of variance (ANOVA) was performed in Statistics Analysis System 9.2 (SAS Institute, NC, USA) statistical software version, and the means were analyzed by Duncan’s multiple polar difference test (

p < 0.05

). Correlation between independent variables and measurements were calculated as Pearson correlation coefficients. All measurements were performed at least three treated samples and reported as mean

\pm

standard deviation.
在统计分析系统 9.2（SAS Institute，NC，USA）统计软件版中进行单因素方差分析（ANOVA），并用邓肯多重极差检验（

p < 0.05

）对均值进行分析。自变量与测量值之间的相关性以皮尔逊相关系数计算。所有测量至少进行了三个处理样本，并以平均值

\pm

标准偏差进行报告。

Results and Discussion 结果与讨论

Physical-Chemical Properties of Collagen
胶原蛋白的物理化学特性

Measurement of Collagen Solubility
胶原蛋白溶解度测量

Figure 1a shows the collagen solubility at different pH values. The solubility curves at different pH values showed higher collagen solubility in the acid pH range of 2 to 5 . Collagen showed maximum solubility at pH 5 and minimum solubility in the pH range of 7 to 9 , especially at

pH = 8

, which is the isoelectric point of collagen. However, collagen solubility slightly increased on the alkaline side of the isoelectric point (pI). The degree of protein solubility is the result of the interaction between electrostatic and hydrophobic protein molecules, and the high electrostatic repulsion of protein
图 1a 显示了胶原蛋白在不同 pH 值下的溶解度。不同 pH 值下的溶解度曲线显示，在 2 至 5 的酸性 pH 值范围内，胶原蛋白的溶解度较高。胶原蛋白在 pH 值为 5 时溶解度最大，在 pH 值为 7 到 9 时溶解度最小，尤其是在

pH = 8

时，这是胶原蛋白的等电点。不过，在等电点（pI）的碱性侧，胶原蛋白的溶解度略有增加。蛋白质的溶解度是静电分子和疏水蛋白质分子相互作用的结果，而蛋白质的高静电斥力则会影响蛋白质的溶解度。

Figure I Physicochemical properties of collagen. (a) Solubility of collagen at different pH ; (b) UV-VIS spectra of collagen; © infrared spectrum of collagen.
图 I 胶原的理化性质。(a) 胶原在不同 pH 值下的溶解度；(b) 胶原的紫外-可见光谱；© 胶原的红外光谱。
molecules increases the solubility of the protein. Due to the increase in the interaction of proteins and water, repulsion occurs, and the solubility in ( pI ) high or low (negative or positive net charge) pH value increases because the attraction and trend under the pI (zero net charge) reduce molecular aggregation.

^{41}

Thus, protein solubility as a function of pH provides an S-shaped curve and solubility minimum corresponding to pI .
分子会增加蛋白质的溶解度。由于蛋白质与水的相互作用增加，发生排斥，在（pI）高或低（净电荷为负或正）pH 值下的溶解度增加，因为在 pI（净电荷为零）下的吸引力和趋势减少了分子聚集。

^{41}

因此，蛋白质的溶解度与 pH 值的函数关系呈 S 型曲线，溶解度最小值与 pI 对应。

UV-Vis Spectroscopy of Collagen
胶原蛋白的紫外可见光谱分析

Collagen has

COOH, {CONH}_{2}

and

C = O

chromogenic groups, resulting in strong UV absorption properties. The UV absorption spectrum is the result of the summation of various UV chromophores of protein molecules and is an important marker for determining the type of collagen. Hydroxyproline (Hyp) and proline (Pro), which have a phenyl ring structure in type I collagen, cause the maximum UV absorption peaks to be mainly concentrated at

220 - 230 nm

due to the

n \to π

leap of the carbonyl group

C = O

. The UV spectrum of collagen is shown in Figure 1 b , and its strongest absorption peak occurred at 225 nm , which is similar to the collagen of bullfrog ( 236 nm ), Japanese schistosome ( 233 nm ) and spotted forktail (232 nm)…

^{42}

Because collagen contains few amino acids with aromatic rings, such as phenylalanine (Phe) and tyrosine (Tyr), there was no significant absorption peak near 280 nm , which indicated that the protein was type I collagen.

^{43}

胶原蛋白具有

COOH, {CONH}_{2}

和

C = O

发色基团，因此具有很强的紫外线吸收特性。紫外线吸收光谱是蛋白质分子的各种紫外线发色团相加的结果，是确定胶原蛋白类型的重要标志。Ⅰ型胶原蛋白中具有苯环结构的羟脯氨酸（Hyp）和脯氨酸（Pro），由于羰基

n \to π

跃迁

C = O

，导致最大紫外线吸收峰主要集中在

220 - 230 nm

处。胶原蛋白的紫外光谱如图1 b所示，其最强吸收峰出现在225 nm处，与牛蛙胶原蛋白（236 nm）、日本血吸虫胶原蛋白（233 nm）和斑点叉尾胶原蛋白（232 nm）相似......

^{42}

由于胶原蛋白中含有较少的苯丙氨酸（Phe）和酪氨酸（Tyr）等芳香环氨基酸，在280 nm附近没有明显的吸收峰，表明该蛋白质为I型胶原蛋白。

^{43}

FTIR Analysis of Collagen
胶原蛋白的傅立叶变换红外分析

The main characteristic absorption peaks of the collagen FTIR spectrum are shown in Figure 1c, which mainly include amide A, B, I, II, III belt,

^{44}

consistent with literature reports. In general, the N-H free-stretching vibration occurred in the range of 3400 to

3440 {cm}^{- 1}

. However, when the

N - H

group combines with the hydrogen bond on the peptide chain, the position of amide A shifts to a lower frequency, approximately

3300 {cm}^{-} 1 .^{45}

Due to the

N - H

free-stretching vibrations, the position of the amide A band of collagen appeared at

3408.57 {cm}^{- 1}

. The amide B band of collagen appeared at

2924.52 {cm}^{- 1}

and is associated with asymmetric stretching vibrations of

= {CH}_{2}

and

{NH}^{3} +,^{46}

and the amide I band of collagen was at approximately

1647.88 {cm}^{- 1}

. The amide I band is mainly associated with

C = O

stretching vibrations of the related peptide moiety, generally in the range of

1600 - 1700 {cm}^{-} 1 .^{47}

The amide II band of collagen had a wave number of

1575.08 {cm}^{- 1}

and is associated with CN -stretching vibrations and NH -bending vibrations (in the range of 1550 to

1600 {cm}^{- 1}) .^{48}

Amide III is generally in the range of

1200 - 1300 {cm}^{- 1}

, representing the combined peak of NH deformation and NH stretching vibrations as well as the pendulum vibration absorption peaks of the

{CH}_{2}

group on the proline side chain and the glycine backbone. Furthermore, it involves the triple helix structure of collagen

^{49, 50}

where the amide III absorption peak of collagen is located at

1240.00 {cm}^{- 1}

, confirming the existence of the collagen helix structure. In conclusion, the triple helix structure of collagen was not damaged, and it belongs to type I collagen.
胶原蛋白傅立叶变换红外光谱的主要特征吸收峰如图 1c 所示，主要包括酰胺 A、B、I、II、III 带，

^{44}

与文献报道一致。一般情况下，N-H 自由伸缩振动发生在 3400 到

3440 {cm}^{- 1}

的范围内，但当

N - H

基团与肽链上的氢键结合时，酰胺 A 的位置移动到较低的频率，约为

3300 {cm}^{-} 1 .^{45}

由于

N - H

自由伸缩振动，胶原蛋白酰胺 A 带的位置出现在

3408.57 {cm}^{- 1}

处。胶原蛋白的酰胺 B 带出现在

2924.52 {cm}^{- 1}

处，与

= {CH}_{2}

和

{NH}^{3} +,^{46}

的不对称伸缩振动有关，胶原蛋白的酰胺 I 带大约在

1647.88 {cm}^{- 1}

处。酰胺 I 波段主要与相关肽分子的

C = O

伸缩振动有关，一般在

1600 - 1700 {cm}^{-} 1 .^{47}

范围内。胶原蛋白的酰胺 II 波段波数为

1575.08 {cm}^{- 1}

，与 CN 伸缩振动和 NH 弯曲振动有关（在 1550 到

1600 {cm}^{- 1}) .^{48}

范围内）、代表 NH 变形振动和 NH 拉伸振动的组合峰，以及脯氨酸侧链和甘氨酸骨架上的

{CH}_{2}

基团的钟摆振动吸收峰。此外，它还涉及胶原蛋白的三重螺旋结构

^{49, 50}

，其中胶原蛋白的酰胺 III 吸收峰位于

1240.00 {cm}^{- 1}

处，证实了胶原蛋白螺旋结构的存在。总之，胶原蛋白的三重螺旋结构没有受到破坏，它属于 I 型胶原蛋白。

Characterization of Liposomes
脂质体的表征

Particle Size, Size Distribution and $ζ$ -Potential Measurements
粒度、粒度分布和 $ζ$ 电位测量

The blank liposomes and collagen liposome solutions prepared by a combination of the film dispersion method and ultrasonication showed a blue opalescence with a distinct Tyndall effect,

^{51}

indicating the unique nanoscale characteristics (Figure 2b). The average sizes of the prepared blank liposomes and collagen liposomes were

73.88 \pm 0.68 nm

and 75.34

\pm 0.93 nm

, respectively (Figure 2a and c) with

ζ

-potentials of

- 15.63 \pm 1.25 mV

and

- 11.21 \pm 0.83 mV

, respectively, and polydispersity indexes (PDI) of

0.287 \pm 0.043

and

0.238 \pm 0.012

, respectively. This is similar to Liu et al

^{52}

particle size potential results for ferritin liposomes. The relatively small mean particle size and polydispersity index of collagen liposomes, with a surface charge of medium anionic

(- 11.21 \pm 0.83 mV)

, suggest that the liposome manufacturing method used in this study is feasible in the production of nanocarriers. Both blank liposomes and collagen liposomes showed good PDI (PDI

< 0.3

), which indicated that the liposomes had reasonable size homogeneity.
结合薄膜分散法和超声波法制备的空白脂质体和胶原蛋白脂质体溶液呈现蓝色乳光，具有明显的廷德尔效应，

^{51}

表明其具有独特的纳米级特征（图 2b）。制备的空白脂质体和胶原蛋白脂质体的平均尺寸分别为

73.88 \pm 0.68 nm

和75.34

\pm 0.93 nm

（图2a和c），

ζ

电位分别为

- 15.63 \pm 1.25 mV

和

- 11.21 \pm 0.83 mV

，多分散指数（PDI）分别为

0.287 \pm 0.043

和

0.238 \pm 0.012

。这与 Liu 等人对铁蛋白脂质体的

^{52}

粒径电位结果相似。胶原蛋白脂质体的平均粒径和多分散指数相对较小，表面电荷为中等阴离子

(- 11.21 \pm 0.83 mV)

，这表明本研究中使用的脂质体制造方法在纳米载体的生产中是可行的。空白脂质体和胶原脂质体都显示出良好的 PDI（PDI

< 0.3

），这表明脂质体具有合理的大小均一性。

Measurement of Encapsulation Rate
封装速率的测量

In the case of applying liposomes as nanocarriers, the effectiveness of encapsulation is the ultimate issue. A protein standard curve with

R^{2} = 0.9998

using BSA as a standard (Figure 2d) was constructed and used to calculate the encapsulation rate based on the collagen content. The encapsulation rate of collagen liposomes was

57.80 \pm 0.51 %

, which was similar to the encapsulation rate of BSA liposomes determined by Liu et al.

^{53}

在应用脂质体作为纳米载体的情况下，封装的有效性是最终问题。以 BSA 为标准，构建了

R^{2} = 0.9998

的蛋白质标准曲线（图 2d），并根据胶原蛋白的含量计算封装率。胶原蛋白脂质体的包封率为

57.80 \pm 0.51 %

，与 Liu 等人测定的 BSA 脂质体的包封率

^{53}

相似。

Figure 2 Characterization of liposomes. (a) Appearance of collagen liposomes; (b) size distribution of blank liposomes; © size distribution of collagen liposomes; (d) protein standard curve with bovine serum albumin (BSA) as standard.
图 2 脂质体的表征。(a) 胶原脂质体的外观；(b) 空白脂质体的大小分布；© 胶原脂质体的大小分布；(d) 以牛血清白蛋白（BSA）为标准的蛋白质标准曲线。

Morphological Analysis 形态分析

TEM imaging is widely used to study the morphological characteristics of nanoparticles as well as their size characteristics. TEM images of blank liposomes and collagen liposomes are shown in Figure 3. TEM observations indicated the formation of nearly circular spherical nanovesicles with smooth outer surfaces (Figure 3) but diameters smaller than the measurements obtained from DLS, which may be attributed to the different degrees of vesicle shrinkage during TEM sample preparation (drying process).

^{54}

The particle size of collagen liposomes was significantly larger than that of blank liposomes as detected by TEM images, and bilayer vesicles were detected in collagen liposomes (Figure 3d), which was also similar to the results of Imam et al.

^{55}

Danaei et al

^{56}

reported that bilayer vesicles have higher stability and controlled release of active compounds compared to other types of vesicles, such as monolayer and multilayer vesicles. Thus, these findings provide a theoretical possibility for the future use of collagen liposomes as carriers of skin nutritional bioactive proteins.
TEM 成像被广泛用于研究纳米粒子的形态特征及其尺寸特征。空白脂质体和胶原脂质体的 TEM 图像如图 3 所示。TEM 观察结果表明，形成的纳米微粒近似圆形球体，外表面光滑（图 3），但直径小于 DLS 的测量值，这可能是由于 TEM 样品制备过程（干燥过程）中囊泡收缩程度不同造成的。

^{54}

通过 TEM 图像检测，胶原蛋白脂质体的粒径明显大于空白脂质体的粒径，并且在胶原蛋白脂质体中检测到了双层囊泡（图 3d），这也与 Imam 等人的研究结果相似。

^{55}

Danaei 等人

^{56}

报道，与单层和多层囊泡等其他类型的囊泡相比，双层囊泡具有更高的稳定性和活性化合物的可控释放性。因此，这些研究结果为今后将胶原蛋白脂质体用作皮肤营养生物活性蛋白的载体提供了理论上的可能性。

Physical Stability Studies
物理稳定性研究

Figure 4 depicts the size distribution of collagen liposomes during storage at

25^{\circ} C

and

4^{\circ} C

for 56 days. With the extension of storage time, the size of collagen liposomes showed an increasing trend (

P < 0.05

). On day 0 , the average size of the collagen liposomes was approximately 72 nm , and the solution was clear and transparent. On day 14 , the size of the collagen liposomes stored at

25^{\circ} C

significantly increased, and some white precipitate was found at the bottom of the bottle, which may be related to the instability caused by the accumulation of the liposomes during storage. The size of collagen liposomes stored at

4^{\circ} C

did not increase significantly until day 56 , indicating that low temperature may prolong the storage time of collagen liposomes.
图 4 描述了胶原蛋白脂质体在

25^{\circ} C

和

4^{\circ} C

储存 56 天期间的大小分布。随着储存时间的延长，胶原蛋白脂质体的大小呈上升趋势（

P < 0.05

）。第 0 天，胶原蛋白脂质体的平均大小约为 72 nm，溶液清澈透明。第 14 天，储存在

25^{\circ} C

条件下的胶原蛋白脂质体的大小明显增大，瓶底出现了一些白色沉淀，这可能与储存过程中脂质体堆积造成的不稳定有关。在

4^{\circ} C

条件下储存的胶原蛋白脂质体的体积直到第56天才明显增大，说明低温可以延长胶原蛋白脂质体的储存时间。

Figure 3 Transmission electron microscopy (TEM) images of blank liposomes (

a

and

b

) and collagen liposomes (c and d).
图 3 空白脂质体（

a

和

b

）和胶原蛋白脂质体（c 和 d）的透射电子显微镜（TEM）图像。

SDS-PAGE

The structural integrity of collagen liposomes was determined by polyacrylamide gel electrophoresis. Figure 5 shows an image of the SDS-PAGE gel containing collagen and collagen liposomes. Collagen has

α_{1}

and

α_{2}

chains with a molecular weight of approximately 130,000 , while the

β

chain has a molecular weight greater than 200,000 , which is consistent with the characteristics of type I collagen. Figure 5 shows that no structural or conformational instability of the protein was observed during the preparation and that the protein was released from the liposomes. Regardless of whether the membrane was broken or not, the blank liposomes had no band, while the collagen-coated liposomes had a shallow band when the membrane was not broken because all of the collagen was not covered. There was no significant difference between the bands of collagen liposomes with broken membrane and those of collagen solution, and there were no other obvious bands, indicating that the collagen covered by liposomes was intact. Moreover, the band depth of collagen liposomes with broken membrane was shallower than that of collagen solution, which was consistent with the above results in Measurement of Encapsulation Rate.
通过聚丙烯酰胺凝胶电泳测定胶原脂质体的结构完整性。图 5 显示了含有胶原蛋白和胶原蛋白脂质体的 SDS-PAGE 凝胶图像。胶原蛋白的

α_{1}

和

α_{2}

链的分子量约为 130,000 ，而

β

链的分子量大于 200,000 ，这符合 I 型胶原蛋白的特征。图 5 显示，在制备过程中没有观察到蛋白质结构或构象的不稳定性，蛋白质从脂质体中释放出来。无论膜是否破损，空白脂质体都没有条带，而包覆胶原蛋白的脂质体在膜未破损时有一条较浅的条带，因为胶原蛋白没有被全部覆盖。破膜胶原蛋白脂质体的条带与胶原蛋白溶液的条带无明显差异，也没有其他明显的条带，表明脂质体覆盖的胶原蛋白完好无损。此外，破膜胶原蛋白脂质体的条带深度比胶原蛋白溶液浅，这与上述 "包封率测定 "中的结果一致。

Figure 4 Size distribution of collagen liposomes stored at

25^{\circ} C

and

4^{\circ} C

for 56 days.
图 4 在

25^{\circ} C

和

4^{\circ} C

下储存 56 天的胶原蛋白脂质体的粒度分布。

Figure 5 SDS-PAGE electrophoresis of collagen and its liposomes (M-protein Marker; 1-Blank liposomes with unbroken membranes; 2-membrane-breaking blank liposomes; 3- ultrapure water + Triton X-I00; 4, 5-membrane-breaking collagen liposomes; 6-collagen liposomes without broken membranes; 7-collagen solution).
图 5 胶原及其脂质体的 SDS-PAGE 电泳图（M-蛋白标记；1-未破膜空白脂质体；2-破膜空白脂质体；3-超纯水 + Triton X-I00；4、5-破膜胶原脂质体；6-未破膜胶原脂质体；7-胶原溶液）。

Cell Viability Detection (MTT)
细胞活力检测（MTT）

To test the biological safety of collagen liposomes, we used BV2 cells for the cell viability test. Figure 6 shows the effect of collagen liposomes on the BV2 cell survival rate, demonstrating that different concentrations of collagen liposomes resulted in BV2 cell survival rates greater than

95 %

with no significant differences among the cell survival rates. These results indicated that the prepared collagen liposomes are biologically safe, and the experimental results provided a certain reference for the application of collagen liposomes in cosmetics and pharmaceutical fields as a natural and safe raw material.

^{57}

为了测试胶原蛋白脂质体的生物安全性，我们使用 BV2 细胞进行了细胞存活率测试。图 6 显示了胶原蛋白脂质体对 BV2 细胞存活率的影响，表明不同浓度的胶原蛋白脂质体使 BV2 细胞存活率均大于

95 %

，且细胞存活率之间无显著差异。这些结果表明，制备的胶原蛋白脂质体在生物学上是安全的，实验结果为胶原蛋白脂质体作为一种天然、安全的原料在化妆品和医药领域的应用提供了一定的参考。

^{57}

Figure 6 Effect of collagen liposomes on the viability of BV2 cells.
图 6 胶原脂质体对 BV2 细胞活力的影响。

Permeability of Collagen and Collagen Liposomes
胶原蛋白和胶原蛋白脂质体的渗透性

Q_{n}

J_{s s}

and

W %

of Collagen
胶原蛋白的

Q_{n}

、

J_{s s}

和

W %

Ex vivo skin permeation studies

^{58}

were performed to assess the permeation effect of collagen was encapsulated in liposome, and the isolated BALB/c mouse skin (divided into normal skin group and injury skin group) was utilized for the initial evaluation of collagen delivery because it has similar skin barrier properties to human and can simulate human skin ex vivo percutaneous research.

^{59}

In addition, the isolated skin of tonguefishes and nude mouse were also selected for preliminary evaluation of collagen delivery, and the results of the assessment were compared with the BALB/c mouse. In addition, the variability of skin penetration results was compared between different animals.

^{58}

为了评估脂质体包裹胶原蛋白的渗透效果，进行了体外皮肤渗透研究，并利用离体BALB/c小鼠皮肤（分为正常皮肤组和损伤皮肤组）进行胶原蛋白递送的初步评估，因为它具有与人类相似的皮肤屏障特性，可以模拟人类皮肤进行体外经皮研究。

^{59}

此外，还选择了舌鱼和裸鼠的离体皮肤进行胶原蛋白输送的初步评估，并将评估结果与 BALB/c 小鼠进行比较。此外，还比较了不同动物皮肤渗透结果的差异性。

In this study,

32^{\circ} C

was set to simulate the temperature of human skin in the normal and stable external environment, and

36^{\circ} C

was set to simulate the temperature of human skin when the external environment temperature rise (for example, after taking a bath).

^{60, 61}

Figure 7 shows the cumulative penetration of collagen and collagen liposomes through the skin of different animals within 24 h (expressed per

{cm}^{2}

). At the same temperature

(32^{\circ} C)

, the cumulative transmission volume of collagen liposomes in the BALB/c mouse normal skin group, the BALB/c mouse injury skin group and the tonguefishes skin group were 1.3, 1.5 and 1.2 times more than the collagen solution, respectively. The cumulative transmission volume of collagen liposomes in nude mouse skin was 1.6 times more than the collagen solution. With the increase of temperature, the cumulative transmittance also proportionally increased. Figure 8 shows the skin absorption curves of collagen and collagen liposomes. At the same temperature

(32^{\circ} C)

, the skin absorption of collagen liposomes was higher than the collagen solution, and the results of multiple relation of absorbance and cumulative permeability of different animal skins were similar. As the temperature increased, the skin absorption rate proportionally increased. The results of ex vivo percutaneous permeation were fitted by the drug release kinetic equation (Table 1), and the results showed that the first-order kinetics equation of the collagen solution group and collagen liposomes group had a higher fitting degree. At the same temperature

(32^{\circ} C)

, the steady-state transmission rate of
在本研究中，

32^{\circ} C

设定为模拟正常稳定的外部环境下人体皮肤的温度，

36^{\circ} C

设定为模拟外部环境温度升高时（例如洗澡后）人体皮肤的温度。

^{60, 61}

图 7 显示了胶原蛋白和胶原蛋白脂质体在 24 小时内通过不同动物皮肤的累积渗透率（以

{cm}^{2}

表示）。在相同温度

(32^{\circ} C)

下，胶原蛋白脂质体在 BALB/c 小鼠正常皮肤组、BALB/c 小鼠损伤皮肤组和舌鱼皮肤组的累积透过量分别是胶原蛋白溶液的 1.3 倍、1.5 倍和 1.2 倍。胶原蛋白脂质体在裸鼠皮肤中的累积透射量是胶原蛋白溶液的 1.6 倍。随着温度的升高，累积透射率也成比例增加。图 8 显示了胶原蛋白和胶原蛋白脂质体的皮肤吸收曲线。在相同温度

(32^{\circ} C)

下，胶原蛋白脂质体的皮肤吸收率高于胶原蛋白溶液，不同动物皮肤的吸收率与累积透过率的倍数关系结果相似。随着温度的升高，皮肤吸收率也成比例增加。用药物释放动力学方程（表 1）对体外经皮渗透的结果进行拟合，结果表明胶原蛋白溶液组和胶原蛋白脂质体组的一阶动力学方程拟合度较高。在相同温度

(32^{\circ} C)

下，胶原蛋白溶液组和胶原蛋白脂质体组的稳态透过率分别为

Figure 7 Cumulative transmission curves of collagen and its liposomes at different temperature; (a) tonguefishes skin; (b) nude mouse skin; © normal skin of BALB/c mice; (d) injury skin of BALB/c mouse.
图 7 不同温度下胶原蛋白及其脂质体的累积透射率曲线；(a) 舌鱼皮肤；(b) 裸鼠皮肤；© BALB/c 小鼠正常皮肤；(d) BALB/c 小鼠损伤皮肤。
collagen liposomes was higher than the collagen solution. The steady state transmission rate increased proportionally with the increase of temperature. Comparison of the permeability results of the BALB/c mouse normal skin group to the BALB/c mouse injury skin group demonstrated that skin barrier damage under the condition, the permeability of collagen and its liposomes correspondingly increased and that the permeability of collagen liposomes significantly increased. These results demonstrated that liposomes loaded with collagen are more easily to penetrate the skin than collagen itself at the same temperature. Similarly, Lv et al

^{62}

conducted similar permeability experiments on vitamin C liposomes and obtained the same results. In addition, rising temperature also promoted the permeation of collagen and its liposomes to a certain extent.
胶原蛋白脂质体的透射率高于胶原蛋白溶液。随着温度的升高，稳态透射率成比例增加。比较 BALB/c 小鼠正常皮肤组和 BALB/c 小鼠损伤皮肤组的渗透性结果表明，在皮肤屏障损伤的条件下，胶原蛋白及其脂质体的渗透性相应增加，胶原蛋白脂质体的渗透性显著增加。这些结果表明，在相同温度下，负载胶原蛋白的脂质体比胶原蛋白本身更容易穿透皮肤。同样，Lv 等人

^{62}

对维生素 C 脂质体进行了类似的渗透性实验，也得到了相同的结果。此外，温度升高也在一定程度上促进了胶原蛋白及其脂质体的渗透。

Skin Retention $(Q_{s})$ and Retention Rate $(A_{s})$ of Collagen
皮肤保留 $(Q_{s})$ 和胶原蛋白保留率 $(A_{s})$

The effect of collagen and collagen liposomes on the skin retention and retention rate of skin different animals after ex vivo infiltration was investigated (Table 2). At the same temperature

(32^{\circ} C)

, the skin accumulation volume and accumulation rate of collagen liposomes were significantly higher than the collagen solution. The skin accumulation volume and accumulation rate of collagen liposomes in BALB/ c normal mouse skin group, BALB/c injury mouse skin liposome group, tonguefishes skin group and nude mouse skin group were approximately

1.7, 1.8, 2.6

and 1.3 times more than the collagen solution, respectively, which indicated that more collagen were retained in the skin when encapsulated by liposomes. With the increase of temperature, the accumulation volume and accumulation rate of the skin slightly decreased because of the increase of temperature, the pores in the skin will open. Thereby accelerating the loss of the water and active ingredients,

^{63}

such as collagen. These results indicated that collagen
研究了胶原蛋白和胶原蛋白脂质体对不同动物体内外浸润后皮肤蓄积量和蓄积率的影响（表 2）。在相同温度

(32^{\circ} C)

下，胶原蛋白脂质体的皮肤蓄积量和蓄积率明显高于胶原蛋白溶液。BALB/ c正常小鼠皮肤组、BALB/c损伤小鼠皮肤脂质体组、舌鱼皮肤组和裸鼠皮肤组的皮肤蓄积量和蓄积率分别约为胶原蛋白溶液的

1.7, 1.8, 2.6

和1.3倍，这表明脂质体包裹后皮肤中保留了更多的胶原蛋白。随着温度的升高，皮肤的蓄积量和蓄积率略有下降，因为温度升高，皮肤中的毛孔会张开。从而加速了水分和活性成分

^{63}

（如胶原蛋白）的流失。这些结果表明，胶原蛋白

Figure 8 Skin absorption curves of collagen and its liposomes at different temperature; (a) tonguefishes skin; (b) nude mouse skin; © normal skin of BALB/c mice; (d) injury skin of BALB/c mouse.
图 8 不同温度下胶原蛋白及其脂质体的皮肤吸收曲线；(a) 舌鱼皮肤；(b) 裸鼠皮肤；© BALB/c 小鼠正常皮肤；(d) BALB/c 小鼠损伤皮肤。
liposomes with a small particle size not only increase the content of collagen in the skin and improve the concentration of collagen in the skin tissue but also increase the retention of collagen in the skin to delay the release of collagen.
粒径小的脂质体不仅能增加皮肤中胶原蛋白的含量，提高皮肤组织中胶原蛋白的浓度，还能增加皮肤中胶原蛋白的存留，延缓胶原蛋白的释放。

Table I Kinetic Parameters of Cumulative Release of Collagen and Its Liposomes
表 I 胶原及其脂质体累积释放的动力学参数

Group 组别		$Q_{t}$	$R^{2}$	$J_{s s} / [μ g / ({c m}^{2} \cdot h)]$
Tonguefishes skin 舌鱼皮肤	Solution $(32^{\circ} C)$ 解决方案 $(32^{\circ} C)$	$y = 8.0595 x + 47.641$	0.9835	8.0595
	Liposome $(32^{\circ} C)$ 脂质体 $(32^{\circ} C)$	$y = 8.4650 x + 58.338$	0.9841	8.4650
	Solution $(36^{\circ} C)$ 解决方案 $(36^{\circ} C)$	$y = 14.477 x + 31.419$	0.9940	14.477
	Liposome $(36^{\circ} C)$ 脂质体 $(36^{\circ} C)$	$y = 15.463 x + 40.058$	0.9987	15.463
Nude mouse skin 裸鼠皮肤	Solution $(32^{\circ} C)$ 解决方案 $(32^{\circ} C)$	$y = 19.910 x + 30.516$	0.9922	19.910
	Liposome $(32^{\circ} C)$ 脂质体 $(32^{\circ} C)$	$y = 31.002 x + 43.670$	0.9808	31.002
	Solution $(36^{\circ} C)$ 解决方案 $(36^{\circ} C)$	$y = 22.130 x + 28.981$	0.9898	22.130
	Liposome $(36^{\circ} C)$ 脂质体 $(36^{\circ} C)$	$y = 33.689 x + 41.713$	0.9887	33.689

(Continued) （续）

Table I (Continued). 表 I（续）.

Group 组别		$Q_{t}$	$R^{2}$	$J_{s s} / [μ g / ({cm}^{2} \cdot h)]$
BALB/c normal mouse skin BALB/c 正常小鼠皮肤	Solution ( $32^{\circ} C$ ) 解决方案 ( $32^{\circ} C$ )	$y = 9.0889 x + 16.038$	0.9932	9.0889
	Liposome ( $32^{\circ} C$ ) 脂质体 ( $32^{\circ} C$ )	$y = 10.692 x + 23.502$	0.9984	10.692
	Solution ( $36^{\circ} C$ ) 解决方案 ( $36^{\circ} C$ )	$y = 9.9542 x + 17.001$	0.9968	9.9542
	Liposome ( $36^{\circ} C$ ) 脂质体 ( $36^{\circ} C$ )	$y = 11.828 x + 24.074$	0.9967	11.828
BALB/c injury mouse skin BALB/c 损伤小鼠皮肤	Solution ( $32^{\circ} C$ ) 解决方案 ( $32^{\circ} C$ )	$y = 10.790 x + 17.512$	0.9980	10.790
	Liposome ( $32^{\circ} C$ ) 脂质体 ( $32^{\circ} C$ )	$y = 14.838 x + 18.416$	0.9739	14.838
	Solution ( $36^{\circ} C$ ) 解决方案 ( $36^{\circ} C$ )	$y = 12.137 x + 15.977$	0.9971	12.137
	Liposome ( $36^{\circ} C$ ) 脂质体 ( $36^{\circ} C$ )	$y = 16.591 x + 17.802$	0.9935	16.591

Table 2 Skin Accumulation and Accumulation Rate of Collagen and Liposomes
表 2 皮肤胶原蛋白和脂质体的累积量和累积率

Group 组别

累积量

/ (μ g \cdot {cm}^{- 2})

Accumulation

Volume

/ (μ g \cdot {cm}^{- 2})

Accumulation Rate

/ %

累积率

/ %

Tonguefishes skin 舌鱼皮肤

溶液

(32^{\circ} C)

脂质体 (

32^{\circ} C

) 溶液

(36^{\circ} C)

脂质体 (

36^{\circ} C

)

Solution

(32^{\circ} C)

Liposome (

32^{\circ} C

)

Solution

(36^{\circ} C)

Liposome (

36^{\circ} C

)

\begin{aligned} 35.342 \pm 0.973 \\ 91.249 \pm 1.362 \\ 29.428 \pm 0.973 \\ 80.629 \pm 0.852 \end{aligned}

\begin{aligned} 0.164 \pm 0.002 \\ 0.287 \pm 0.005 \\ 0.109 \pm 0.003 \\ 0.256 \pm 0.003 \end{aligned}

Nude mouse skin 裸鼠皮肤

溶液

(32^{\circ} C)

脂质体

(32^{\circ} C)

溶液

(36^{\circ} C)

脂质体 (

36^{\circ} C

)

Solution

(32^{\circ} C)

Liposome

(32^{\circ} C)

Solution

(36^{\circ} C)

Liposome (

36^{\circ} C

)

\begin{aligned} 50.850 \pm 1.609 \\ 65.529 \pm 0.750 \\ 44.318 \pm 1.160 \\ 60.630 \pm 0.696 \end{aligned}

\begin{aligned} 0.148 \pm 0.001 \\ 0.199 \pm 0.001 \\ 0.129 \pm 0.003 \\ 0.184 \pm 0.002 \end{aligned}

BALB/c normal mouse skin
BALB/c 正常小鼠皮肤

溶液

(32^{\circ} C)

脂质体 (

32^{\circ} C

) 溶液

(36^{\circ} C)

脂质体 (

36^{\circ} C

)

Solution

(32^{\circ} C)

Liposome (

32^{\circ} C

)

Solution

(36^{\circ} C)

Liposome (

36^{\circ} C

)

\begin{aligned} 45.23 I \pm 0.608 \\ 77.399 \pm 1.081 \\ 40.806 \pm 0.677 \\ 69.532 \pm 0.843 \end{aligned}

\begin{aligned} 0.151 \pm 0.002 \\ 0.256 \pm 0.001 \\ 0.127 \pm 0.002 \\ 0.232 \pm 0.003 \end{aligned}

BALB/c injury mouse skin
BALB/c 损伤小鼠皮肤

溶液

(32^{\circ} C)

脂质体

(32^{\circ} C)

溶液

(36^{\circ} C)

脂质体

(36^{\circ} C)

Solution

(32^{\circ} C)

Liposome

(32^{\circ} C)

Solution

(36^{\circ} C)

Liposome

(36^{\circ} C)

\begin{aligned} 39.261 \pm 1.216 \\ 68.584 \pm 1.054 \\ 28.024 \pm 0.760 \\ 59.770 \pm 0.877 \end{aligned}

\begin{aligned} 0.122 \pm 0.001 \\ 0.217 \pm 0.001 \\ 0.093 \pm 0.003 \\ 0.199 \pm 0.003 \end{aligned}

Observation of Skin Permeation by Fluorescence Microscopy
用荧光显微镜观察皮肤渗透情况

The penetration of cross-sections of BALB/c normal mouse skin tissue was observed by fluorescence microscopy, and the qualitative penetration was evaluated by a multi-fluorescent compound technique. Figure 9 shows the fluorescence images of a cross-section of the BALB/c normal mouse skin after 18 h of infiltration of fluoresceinlabeled collagen and collagen liposomes with the mouse skin and FITC/coumarin-6 represented by blue fluorescence and green fluorescence, respectively. After 18 h of infiltration, collagen liposomes gradually penetrated into the epidermis and dermis with high fluorescence intensity. There was a large number of liposomes in the hair follicle, and collagen was deposited in the cuticle. Collagen liposomes were attached to the hair follicle, and the embedded collagen was released and diffused. In contrast, the liposomes that did not penetrate the hair follicle may penetrate the skin through intercellular and transcellular pathways, similar to the results of Subongkot et al

^{64}

In addition, these results were confirmed by the skin retention results.
用荧光显微镜观察了 BALB/c 正常小鼠皮肤组织横截面的渗透情况，并用多荧光化合物技术评估了定性渗透情况。图 9 显示了荧光素标记的胶原蛋白和胶原蛋白脂质体浸润小鼠皮肤 18 小时后 BALB/c 正常小鼠皮肤横截面的荧光图像，FITC/香豆素-6 分别用蓝色荧光和绿色荧光表示。浸润 18 小时后，胶原蛋白脂质体逐渐渗入表皮和真皮，荧光强度很高。毛囊中有大量脂质体，胶原蛋白沉积在角质层中。胶原蛋白脂质体附着在毛囊上，嵌入的胶原蛋白被释放并扩散。相比之下，没有穿透毛囊的脂质体可能通过细胞间和跨细胞途径穿透皮肤，这与 Subongkot 等人的研究结果类似

^{64}

此外，这些结果也得到了皮肤保留结果的证实。

Figure 9 FM image of a cross section of BALB/c normal mouse skin cultured on a V-C diffusion tank containing fluorescein collagen and liposomes (scale bar is

200 μ m

).
图 9 在含有荧光素胶原和脂质体的 V-C 扩散槽上培养的 BALB/c 正常小鼠皮肤横截面的调频图像（比例尺为

200 μ m

）。

Conclusion 结论

In the present study, we confirmed that collagen extracted from bovine Achilles tendon was type I and that its structure was not damaged during the extraction process according to FTIR, UV and SDS-PAGE analysis. We also evaluated the relative solubility of the extracted collagen. The results showed that the lowest solubility of collagen was at

pH = 8

, which is the isoelectric point of collagen. Under the guidance of the above results, bovine type I collagen nanoliposomes were prepared for the first time. DLS measurement and TEM observation showed that the collagen liposomes were spherical with an average diameter and high stability. Physical stability studies showed that low temperature

(4^{\circ} C)

prolonged the storage time of collagen liposomes, and SDS-PAGE analysis showed that collagen was completely encapsulated in liposomes. The MTT assay showed that collagen liposomes were biologically safe. Finally, permeability studies indicated that the collagen-loaded liposomes more easily penetrated the skin compared to collagen itself and that they increased the retention amount of collagen in the skin to delay the release of collagen.
在本研究中，我们通过傅立叶变换红外光谱（FTIR）、紫外光谱（UV）和 SDS-PAGE 分析证实，从牛跟腱中提取的胶原蛋白是 I 型胶原蛋白，其结构在提取过程中未受到破坏。我们还评估了提取胶原蛋白的相对溶解度。结果表明，胶原蛋白在

pH = 8

处的溶解度最低，而

pH = 8

处正是胶原蛋白的等电点。在上述结果的指导下，首次制备了牛Ⅰ型胶原蛋白纳米脂质体。DLS 测量和 TEM 观察表明，胶原蛋白脂质体呈球形，直径平均，稳定性高。物理稳定性研究表明，低温

(4^{\circ} C)

延长了胶原蛋白脂质体的储存时间，SDS-PAGE分析表明胶原蛋白被完全包裹在脂质体中。MTT 试验表明，胶原蛋白脂质体对生物是安全的。最后，渗透性研究表明，与胶原蛋白本身相比，负载胶原蛋白的脂质体更容易穿透皮肤，而且脂质体增加了胶原蛋白在皮肤中的保留量，延缓了胶原蛋白的释放。

In conclusion, the present study demonstrated the feasibility of encapsulating collagen by liposomes to improve its bioavailability and absorption efficiency, thereby providing a reference for the study of protein preparations that are unfavorable for external use due to poor water solubility and skin permeability, suggesting an optimal application for biomedicine, health products, cosmeceuticals and pharmaceutical industries.
总之，本研究证明了用脂质体包裹胶原蛋白以提高其生物利用度和吸收效率的可行性，从而为研究因水溶性和皮肤渗透性差而不利于外用的蛋白质制剂提供了参考，为生物医学、保健品、药妆和制药行业提供了最佳应用。

Declaration of Transparency and Scientific Rigour
透明度和科学严谨性宣言

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.
本声明确认，本文遵守 BJP《设计与分析和动物实验指南》中规定的临床前研究透明报告和科学严谨性原则，以及资助机构、出版商和其他参与支持研究的组织的建议。

Funding 资金筹措

The research was supported by Tianjin Excellent science and Technology Commissioner Project Fund [22YDTPJC00520] and Open Fund of Tianjin Enterprise Key Laboratory on Hyaluronic Acid Application Research [no. KTRDHA-Y201906] provided by Tianjin Kangting Bioengineering Group Corp. Ltd.
该研究得到了天津市优秀科技特派员项目基金[22YDTPJC00520]和天津康婷生物工程集团股份有限公司提供的 "透明质酸应用研究天津市企业重点实验室开放基金"[编号：KTRDHA-Y201906]的支持。天津康婷生物工程集团股份有限公司

Disclosure 信息披露

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
作者不存在利益冲突。本文的内容和写作仅由作者本人负责。

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Preparation, Characterization and ex vivo Skin Permeability Evaluation of Type I Collagen-Loaded Liposomes I 型胶原负载脂质体的制备、表征和体内外皮肤渗透性评估

Abstract 摘要

Introduction 导言

Graphical Abstract 图表摘要

Materials and Methods 材料与方法

Materials 材料

Physical-Chemical Properties of Collagen Measurement of Collagen Solubility胶原蛋白的物理化学特性 测量胶原蛋白的溶解度

UV-Vis Spectroscopy of Collagen胶原蛋白的紫外可见光谱分析

Fourier Transform Infrared Spectroscopy of Collagen胶原蛋白的傅立叶变换红外光谱分析

Preparation of Collagen Liposomes胶原蛋白脂质体的制备

Preparation of Collagen Liposome胶原蛋白脂质体的制备

Preparation of Fluorescein-Labeled Collagen Liposomes荧光素标记胶原脂质体的制备

Characterization of Liposomes脂质体的表征

Size, Size Distribution and ζ ζ zeta\zeta-Potential Measurements尺寸、尺寸分布和 ζ ζ zeta\zeta 电位测量

Determination of Encapsulation Rate确定封装率

Morphological Analysis 形态分析

Physical Stability Studies物理稳定性研究

SDS-PAGE

Cell Viability Detection (MTT)细胞活力检测（MTT）

Ex vivo Skin Permeability Assessment of Collagen and Collagen Liposomes胶原蛋白和胶原蛋白脂质体的体外皮肤渗透性评估

Skin Preparation 皮肤准备

The Skin of Tonguefishes舌鱼的皮肤

Nude Mouse Skin 裸鼠皮肤

BALB/c Mouse Skin BALB/c 小鼠皮肤

Microscopic Observation of Skin Permeation Phenomenon皮肤渗透现象的显微镜观察

Fluorescein-Labeled Collagen荧光素标记的胶原蛋白

Fluorescence Microscope Observation of Skin Permeation荧光显微镜观察皮肤渗透情况

Statistical Analysis 统计分析

Results and Discussion 结果与讨论

Physical-Chemical Properties of Collagen胶原蛋白的物理化学特性

Measurement of Collagen Solubility胶原蛋白溶解度测量

UV-Vis Spectroscopy of Collagen胶原蛋白的紫外可见光谱分析

FTIR Analysis of Collagen胶原蛋白的傅立叶变换红外分析

Characterization of Liposomes脂质体的表征

Particle Size, Size Distribution and ζ ζ zeta\zeta-Potential Measurements粒度、粒度分布和 ζ ζ zeta\zeta 电位测量

Measurement of Encapsulation Rate封装速率的测量

Morphological Analysis 形态分析

Physical Stability Studies物理稳定性研究

SDS-PAGE

Cell Viability Detection (MTT)细胞活力检测（MTT）

Permeability of Collagen and Collagen Liposomes胶原蛋白和胶原蛋白脂质体的渗透性

Skin Retention ( Q s ) Q s (Q_(s))\left(Q_{s}\right) and Retention Rate ( A s ) A s (A_(s))\left(A_{s}\right) of Collagen皮肤保留 ( Q s ) Q s (Q_(s))\left(Q_{s}\right) 和胶原蛋白保留率 ( A s ) A s (A_(s))\left(A_{s}\right)

Observation of Skin Permeation by Fluorescence Microscopy用荧光显微镜观察皮肤渗透情况

Conclusion 结论

Declaration of Transparency and Scientific Rigour透明度和科学严谨性宣言

Funding 资金筹措

Disclosure 信息披露

References 参考资料

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Preparation, Characterization and ex vivo Skin Permeability Evaluation of Type I Collagen-Loaded Liposomes
I 型胶原负载脂质体的制备、表征和体内外皮肤渗透性评估

Physical-Chemical Properties of Collagen Measurement of Collagen Solubility
胶原蛋白的物理化学特性测量胶原蛋白的溶解度

UV-Vis Spectroscopy of Collagen
胶原蛋白的紫外可见光谱分析

Fourier Transform Infrared Spectroscopy of Collagen
胶原蛋白的傅立叶变换红外光谱分析

Preparation of Collagen Liposomes
胶原蛋白脂质体的制备

Preparation of Collagen Liposome
胶原蛋白脂质体的制备

Preparation of Fluorescein-Labeled Collagen Liposomes
荧光素标记胶原脂质体的制备

Characterization of Liposomes
脂质体的表征

Size, Size Distribution and $ζ$ -Potential Measurements
尺寸、尺寸分布和 $ζ$ 电位测量

Determination of Encapsulation Rate
确定封装率

Physical Stability Studies
物理稳定性研究

Cell Viability Detection (MTT)
细胞活力检测（MTT）

Ex vivo Skin Permeability Assessment of Collagen and Collagen Liposomes
胶原蛋白和胶原蛋白脂质体的体外皮肤渗透性评估

The Skin of Tonguefishes
舌鱼的皮肤

Microscopic Observation of Skin Permeation Phenomenon
皮肤渗透现象的显微镜观察

Fluorescein-Labeled Collagen
荧光素标记的胶原蛋白

Fluorescence Microscope Observation of Skin Permeation
荧光显微镜观察皮肤渗透情况

Physical-Chemical Properties of Collagen
胶原蛋白的物理化学特性

Measurement of Collagen Solubility
胶原蛋白溶解度测量

UV-Vis Spectroscopy of Collagen
胶原蛋白的紫外可见光谱分析

FTIR Analysis of Collagen
胶原蛋白的傅立叶变换红外分析

Characterization of Liposomes
脂质体的表征

Particle Size, Size Distribution and $ζ$ -Potential Measurements
粒度、粒度分布和 $ζ$ 电位测量

Measurement of Encapsulation Rate
封装速率的测量

Physical Stability Studies
物理稳定性研究

Cell Viability Detection (MTT)
细胞活力检测（MTT）

Permeability of Collagen and Collagen Liposomes
胶原蛋白和胶原蛋白脂质体的渗透性

Skin Retention $(Q_{s})$ and Retention Rate $(A_{s})$ of Collagen
皮肤保留 $(Q_{s})$ 和胶原蛋白保留率 $(A_{s})$

Observation of Skin Permeation by Fluorescence Microscopy
用荧光显微镜观察皮肤渗透情况

Declaration of Transparency and Scientific Rigour
透明度和科学严谨性宣言

Publish your work in this journal
在本期刊上发表您的作品