Abstract

Introduction

Oil body (OB), a subcellular organelle that stores oil in plant seeds, is considered a new transdermal drug delivery system. With the increasing understanding of the OB and its main protein (oleosin), numerous studies have been conducted on OB as “carrier” for the expression of exogenous proteins. In our previous study, oil body fused with aFGF (OLAF) was obtained using a plant oil body expression system that had been preliminarily proven to be effective in accelerating the healing of skin wounds. However, no dermal toxicological information on OLAF is available.

Objective

To ensure the dermal safety of OLAF, a series of tests (the acute dermal toxicity test, 21-day repeat dermal toxicity test, dermal irritation test and skin sensitisation test) were conducted after optimising the extraction protocol of OLAF.

Materials and methods

To improve the extraction rate of OLAF, response surface methodology (RSM) was first employed to optimise the extraction conditions. Then, Wistar rats were exposed to OLAF (400 mg·kg⁻¹ body weight) in two different ways (6 hours/time for 24 hours and 1 time/day for 21 days) to evaluate the acute dermal toxicity and 21-day repeated dermal toxicity of OLAF. In the acute dermal toxicity test, clinical observations were conducted to evaluate the toxicity, behaviour, and health of the animals for 14 consecutive days. Similarly, the clinical signs, body weight, haematological and biochemical parameters, histopathological changes and other indicators were also detected during the 21 days administration. For the dermal irritation test, single and multiple doses of OLAF (125 mg·kg⁻¹ body weight) were administered to albino rabbits for 14 days (1 time/day). The irritation reaction on the skin of each albino rabbit was recorded and scored. Meanwhile, skin sensitisation to OLAF was conducted using guinea pigs for a period of 28 days.

Results

Suitable extraction conditions for OLAF (PBS concentration 0.01, pH of PBS 8.6, solid–liquid ratio 1:385 g·mL⁻¹) were obtained using RSM. Under these conditions, the extraction rate and particle size of OLAF were 7.29% and 1290 nm, respectively. In the tests of acute dermal toxicity and 21-day repeated dermal toxicity, no mortality or significant differences were observed in terms of clinical signs, body weight, haematological parameters, biochemical parameters and anatomopathological analysis. With respect to the dermal irritation test and skin sensitisation test, no differences in erythema, oedema or other abnormalities were observed between treatment and control groups on gross and histopathological examinations.

Conclusions

The results of this study suggest that OLAF does not cause obvious toxicity, skin sensitisation or irritation in animals.

中文翻译：

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酸性成纤维细胞生长因子（OLAF）融合油体提取条件及皮肤毒性的优化

摘要

介绍

油体（OB）是一种在植物种子中储存油脂的亚细胞器，被认为是一种新型的透皮给药系统。随着人们对OB及其主要蛋白（油质蛋白）认识的不断深入，大量研究将OB作为外源蛋白表达的“载体”。在我们之前的研究中，使用植物油体表达系统获得了与 aFGF (OLAF) 融合的油体，该系统已初步证明可有效加速皮肤伤口的愈合。但是，没有关于 OLAF 的皮肤毒理学信息。

客观的

为确保OLAF的皮肤安全，在优化OLAF提取方案后，进行了一系列试验（急性皮肤毒性试验、21天重复皮肤毒性试验、皮肤刺激试验和皮肤致敏试验）。

材料和方法

为了提高OLAF的提取率，首先采用响应面法（RSM）优化提取条件。然后，Wistar大鼠以两种不同的方式（6小时/次24小时和1次/天21天）暴露于OLAF（400 mg·kg ^-1体重），以评估急性皮肤毒性和21天重复OLAF 的皮肤毒性。在急性经皮毒性试验中，连续 14 天进行临床观察以评估动物的毒性、行为和健康状况。同样，在21天的给药过程中，还检测了临床体征、体重、血液学和生化参数、组织病理学变化等指标。对于皮肤刺激试验，单剂量和多剂量 OLAF (125 mg·kg ^-1体重）给予白化兔 14 天（1 次/天）。记录并评分每只白化兔皮肤的刺激反应。同时，使用豚鼠进行为期28天的对OLAF的皮肤致敏。

结果

使用 RSM 获得了适合 OLAF 的提取条件（PBS 浓度 0.01，PBS 的 pH 值 8.6，固液比 1:385 g·mL ^{-1 ）。}在这些条件下，OLAF 的提取率和粒径分别为 7.29% 和 1290 nm。在急性经皮毒性和21天反复经皮毒性试验中，临床体征、体重、血液学参数、生化参数和解剖病理学分析均未观察到死亡或显着差异。关于皮肤刺激试验和皮肤致敏试验，在肉眼和组织病理学检查中，治疗组和对照组之间没有观察到红斑、水肿或其他异常情况的差异。

结论

这项研究的结果表明，OLAF 不会对动物造成明显的毒性、皮肤过敏或刺激。