新型中性粒细胞响应性发光纳米胶束的构建及其在小鼠烧伤模型成像中的应用

胡涛; 郭嘉伟; 张建祥

doi:10.16016/j.2097-0927.202405117

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新型中性粒细胞响应性发光纳米胶束的构建及其在小鼠烧伤模型成像中的应用

基础医学 | 更新时间：2025-02-27

- 新型中性粒细胞响应性发光纳米胶束的构建及其在小鼠烧伤模型成像中的应用
- Preparation of novel luminescent nanomicelles responsive to neutrophils and their imaging application in mouse burn model
- 陆军军医大学学报 2025年47卷第4期页码：335-349
- 作者机构：
  
  1.重庆大学生物工程学院，重庆；陆军军医大学（第三军医大学）药学与检验医学系：2.药物分析学教研室
  2.药剂学教研室，重庆
  3.Department of Pharmaceutics， Faculty of Pharmacy and Laboratory Medicine， Army Medical University （Third Military Medical University）， Chongqing， 400038， China
- 作者简介：
  
  郭嘉伟， E-mail：breakhp@16.com
  张建祥， E-mail：jxzhang@tmmu.edu.cn
- 基金信息：
  
  ‍基‍金‍项‍目‍］‍ ‍重庆市研究生导师团队建设项目2022;GUO Jiawei, E-mail： breakhp@16.com
- DOI：10.16016/j.2097-0927.202405117
  中图分类号： R363.2;R644
- 收稿日期：2024-05-31，
  
  修回日期：2025-01-05，
  
  纸质出版日期：2025-02-28
- 稿件说明：
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胡涛,郭嘉伟,张建祥.新型中性粒细胞响应性发光纳米胶束的构建及其在小鼠烧伤模型成像中的应用[J].陆军军医大学学报,2025,47(04):335-349.

HU Tao,GUO Jiawei,ZHANG Jianxiang.Preparation of novel luminescent nanomicelles responsive to neutrophils and their imaging application in mouse burn model[J].J Army Med Univ,2025,47(04):335-349.
胡涛,郭嘉伟,张建祥.新型中性粒细胞响应性发光纳米胶束的构建及其在小鼠烧伤模型成像中的应用[J].陆军军医大学学报,2025,47(04):335-349. DOI：10.16016/j.2097-0927.202405117.

HU Tao,GUO Jiawei,ZHANG Jianxiang.Preparation of novel luminescent nanomicelles responsive to neutrophils and their imaging application in mouse burn model[J].J Army Med Univ,2025,47(04):335-349. DOI：10.16016/j.2097-0927.202405117.

摘要

目的

基于发光材料鲁米诺（Luminol）构建中性粒细胞响应性发光纳米胶束，并考察其在小鼠烧烫伤模型早期炎症中的成像能力。

方法

以六氯三聚磷腈（hexachlorotripolyphosphazene，HCCP）为骨架材料，化学键合鲁米诺（Luminol）和聚乙二醇（polyethylene Glycol，PEG）合成两亲性发光材料LHP，并采用红外光谱、核磁氢谱等方法对LHP的化学结构进行表征；将LHP溶于去离子水自组装形成纳米胶束LHP NM，同时测定其粒径大小和电位，并采用透射电镜（transmission electron microscope， TEM）观察纳米胶束的形态；使用光纤光谱仪、微弱发光测量仪和小动物活体成像仪对纳米胶束的光谱学性质、化学发光规律和体外发光成像能力进行详细评价。使用70、80和90 ℃热水按简单随机抽样法分组对雌性Balb/c小鼠进行烫伤处理以建立Ⅰ度至深Ⅱ度小鼠烧烫伤模型，各组样本数

=10，通过H

E染色结果判断模型小鼠皮肤烫伤深度，采用荧光定量qPCR测定烫伤皮肤组织TNF-α，IL-1β、IL-6和髓过氧化物酶（myeloperoxidase，MPO）的mRNA表达水平，ROS检测试剂盒考察烧伤小鼠皮肤组织活性氧（reactive oxygen species，ROS）水平随烫伤时间和深度变化的情况；采用FITC-Ly6G抗体标记中性粒细胞，观察模型小鼠皮肤组织中性粒细胞数量的变化；使用小动物活体成像系统考察纳米胶束LHP NM在小鼠烧烫伤模型中的成像能力，分析发光强度改变与中性粒细胞募集数量变化的相关性，对发光纳米胶束在小鼠烧烫伤模型早期炎症反应监测及烧烫伤深度能力诊断中的效果进行评价。

结果

核磁谱图解析证实一个HCCP分子上约键合了5个鲁米诺单元和一个聚乙二醇（PEG）链，TEM和粒径测定结果表明所制备纳米胶束形态呈空心球形，粒径约为120.000 nm。体外发光实验中，在不同浓度活性氧和MPO条件下，纳米胶束能实现高亮且持续的化学发光，发光强度与活性氧水平及纳米胶束浓度呈现良好的剂量依赖性。细胞实验表明纳米胶束能够实现中性粒细胞响应性成像，发光强度与中性粒细胞数量和LHP NM剂量呈正相关，线性相关系数分别为

=0.98、0.99。动物实验结果表明，烧烫伤模型小鼠皮肤损伤组织中的中性粒细胞数量和活性氧水平显著升高（

0.05）。实验发现，在24 h时间点，80 ℃与70 ℃处理组对比，中性粒细胞募集数量增加约86.4%，发光成像强度增加约71.5%，证明烧伤深度与损伤局部中性粒细胞募集数量具有相关性，且LHP NM可在模型小鼠中实现中性粒细胞响应性成像，其成像强度变化与皮肤损伤组织中的中性粒细胞数量和活性氧水平密切相关。

结论

成功构建中性粒细胞响应性发光纳米胶束LHP NM，基于LHP NM可实现小鼠烧烫伤模型皮肤损伤组织的响应性成像，其发光强度可准确反映烧烫伤模型中的中性粒细胞浸润程度及ROS水平变化，从而实现小鼠烧烫伤模型早期炎症反应的实时监测，并为烧烫伤深度的诊断提供方法和策略。

Abstract

Objective

To prepare neutrophil-responsive luminescent nanomicelles based on self-luminating compound

luminol

and investigate their imaging capability for a mouse burn model at early stage of inflammation.

Methods

Hexachlorotripolyphosphazene (HCCP) was utilized as the skeleton material to synthesize amphiphilic LHP luminescence materials through chemical synthesis of luminol and polyethylene glycol (PEG). The chemical structure of LHP was characterized using infrared spectrum and nuclear magnetic hydrogen spectrum. Nanomicelle LHP NM was formed by self-assembly of LHP in deionized water

and then its particle size and potential were measured. Transmission electron microscopy (TEM) was applied to observe the morphology of nanomicelles. Optical fiber spectrometer

weak luminescence measuring instrument

and small animal living imager were employed to evaluate the spectroscopic properties

chemiluminescence rules

and

in vitro

luminescence imaging ability of the nanomicelles. After that

female Balb/c mice were subjected to scalding with hot water at temperatures of 70 ℃

80 ℃

and 90 ℃

respectively to establish a mouse burn model ranging from degree I to degree II burns. The depth of skin scalds in the model mice was determined through HE staining

while the expression levels of TNF-α

IL-1β

IL-6 and myeloperoxidase (MPO) in the scalded skin tissues were assessed with fluorescence quantitative qPCR. The changes in reactive oxygen species (ROS) levels over time and burn depth in the skin tissues were determined with ROS detection kit. Additionally

the neutrophils within the skin tissues of model mice were labeled with FITC-Ly6G antibody to count the neutrophil number. Finally

a small animal imaging system was utilized to examine the imaging capability of nanomicelle LHP NM in a mouse burn model to analyze the correlation between

luminous intensity and number of recruited neutrophil in order to evaluate the effectiveness of luminous nanomicelles for monitoring early inflammatory response and diagnosing burn depth in a mouse burn model.

Results

Nuclear magnetic resonance (NMR) spectroscopy confirmed the bonding of approximately 5 luminol units and a polyethylene glycol (PEG) chain to 1 HCCP molecule. TEM and particle size determination results demonstrated that the prepared nanomicelles were spherical

hollow structures in a diameter of around 120 nm.

In vitro

luminescence experiments revealed that the nanomicelles exhibited high brightness and sustained chemiluminescence under varying concentrations of ROS and MPO levels

with luminescence intensity dependent on both ROS level and nanomicelle concentration. The

in vitro

cellular experiments demonstrated that the nanomicelles exhibited neutrophil-responsive imaging capability. The luminescence intensity was positively correlated with both the number of neutrophils and the dose of LHP NM

with a linear correlation coefficient (r) of 0.98 and 0.99

respectively.

In vivo

animal study revealed a significant increase (

0.05) in the count of neutrophils and ROS level in the skin tissue of burned and scalded mice. Notably

at the time point of 24 h

compared to the 80 ℃ and 70 ℃ treatment groups

the number of recruited neutrophils was increased by 86.4%

and the luminescent imaging intensity rose by 71.5%. These findings indicated that the severity of burns was correlated with the extent of neutrophil recruitment in the injured area

and LHP NM could effectively achieve neutrophil-responsive imaging in model mice. The changes in imaging intensity were closely associated with the number of neutrophils and the level of ROS in the injured skin tissue.

Conclusion

The neutrophil-responsive luminescent nanomicelle LHP NM is successful prepared

and the nanomicelles enable responsive imaging of skin damage in mouse burn model. The luminescence intensity accurately reflects the neutrophil infiltration and ROS level

allowing for real-time monitoring of early inflammatory responses in mouse burn model. Additionally

this study provides methods and strategies for diagnosing burn depth.

关键词

Keywords

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