靶向E-选择素的工程化脂质体的设计与炎症靶向评价

doi:10.12122/j.issn.1673-4254.2025.05.14

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (5): 1013-1022.doi: 10.12122/j.issn.1673-4254.2025.05.14

靶向E-选择素的工程化脂质体的设计与炎症靶向评价

叶雨濛¹(), 余波³^,⁴, 陆沙沙¹, 周于¹, 丁美红¹^,²(), 程桂林¹^,²()

^1.浙江中医药大学，药学院，浙江杭州 310035
^2.浙江中医药大学，中医药科学院，浙江杭州 310035
^3.绍兴文理学院医学院，浙江绍兴 312000
^4.浙江普施康生物科技有限公司，浙江绍兴 312000

收稿日期:2024-12-27 出版日期:2025-05-20 发布日期:2025-05-23
通讯作者: 丁美红,程桂林 E-mail:yeyumeng2024@163.com;dingmeihong0125@163.com;20151035@ zcmu.edu.cn
作者简介:叶雨濛，在读硕士研究生，E-mail: yeyumeng2024@163.com
基金资助:
国家自然科学基金(82004250);浙江中医药大学校级科研项目(2021FSYYZY31)

Design and inflammation-targeting efficiency assessment of an engineered liposome-based nanomedicine delivery system targeting E-selectin

Yumeng YE¹(), Bo YU³^,⁴, Shasha LU¹, Yu ZHOU¹, Meihong DING¹^,²(), Guilin CHENG¹^,²()

^1.School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
^2.Academy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
^3.School of Medicine, Shaoxing University, Shaoxing 312000, China
^4.Zhejiang PushKang Biotechnology Co. Ltd. , Shaoxing 312000, China

Received:2024-12-27 Online:2025-05-20 Published:2025-05-23
Contact: Meihong DING, Guilin CHENG E-mail:yeyumeng2024@163.com;dingmeihong0125@163.com;20151035@ zcmu.edu.cn
Supported by:
National Natural Science Foundation of China(82004250)

摘要/Abstract

摘要：

目的设计合成一种纳米药物递送系统，靶向E-选择素，可竞争性地抑制E-选择素与中性粒细胞表面的配体结合，阻断中性粒细胞在血管的黏附，进而抑制中性粒细胞向病变部位的募集和浸润。方法采用后插入法开发了基于E-选择素的亲和短肽IELLQARC标记的盐酸多柔比星（DOX）脂质体（IEL-Lip/DOX）。分别制备2-1P配方组［Mol（PC）：Mol（DPI）=100∶1］和2-3P配方组［Mol（PC）：Mol（DPI）=100∶3］，对其修饰密度、体外释放特性进行测定。并且通过脂多糖（LPS）诱导建立体外细胞炎症模型、小鼠急性肺损伤（ALI）模型、大鼠股动脉物理损伤炎症模型、斑马鱼局部炎症模型，尾静脉或躯干定点分别注射不同配方的PEG化DOX脂质体（P、2-1P、2-3P）测定其炎症靶向能力。结果不同配方（2-1P、2-3P）IEL-Lip/DOX的表面短肽修饰密度分别为4.76、7.57 pmoL/cm²。与未修饰脂质体相比，IEL-Lip/DOX在pH 5.5条件下的48 h累积释放速率均降低（P<0.05）；在体外细胞炎症模型中，活化炎症内皮细胞对IEL-Lip/DOX的摄取量增加（P<0.0001），且2-1P配方穿过内皮屏障的能力高于2-3P配方（P<0.01）；在小鼠ALI模型中，IEL-Lip/Cy5.5在肺组织中的荧光强度增加（P<0.05），Z-（2-1P）和Z-（2-3P）配方分别提高了53.71%和93.41%，且2-1P配方在肺中的分布相比于正常小鼠增加（P<0.05），提升幅度为24.19%；在大鼠股动脉物理损伤炎症模型中，2-1P配方的损伤侧/健康侧血管荧光强度值升高（P<0.05）；在斑马鱼局部炎症模型中，各配方在炎症部位的聚集程度均增加（P<0.001）。结论提出了一种靶向E-选择素的纳米药物递送系统，高效靶向活化炎症内皮细胞，增加药物在炎症部位的蓄积，为中性粒细胞不适当激活引起的炎症性疾病提供了一种新的治疗策略。

关键词: E-选择素, IEL短肽, 盐酸多柔比星脂质体, 靶向递送

Abstract:

Objective To develop an E-selectin-targeting nanomedicine delivery system that competitively inhibits E-selectin-neutrophil ligand binding to block neutrophil adhesion to vessels and suppress their recruitment to the lesion sites. Methods Doxorubicin hydrochloride (DOX)-loaded liposomes (IEL-Lip/DOX) conjugated with E-selectin-affinity peptide IELLQARC were developed using a post-insertion method. Two formulations [2-1P: Mol(PC): Mol(DPI)=100:1; 2-3P: 100:3] were prepared and their modification density and in vitro release characteristics were determined. Their targeting efficacy was assessed in a cell model of LPS-induced inflammation, a mouse model of acute lung injury (ALI), a rat femoral artery model of physical injury-induced inflammation, and a zebrafish model of local inflammation. Results The prepared IEL-Lip/DOX 2-1P and 2-3P had peptide modification densities of 4.76 and 7.57 pmoL/cm², respectively. Compared with unmodified liposomes, IEL-Lip/DOX exhibited significantly reduced 48-h cumulative release rates at pH 5.5. In the inflammation cell model, IEL-Lip/DOX showed increased uptake by activated inflammatory endothelial cells, and 2-1P exhibited a higher trans-endothelial ability. In ALI mice, the fluorescence intensity of IEL-Lip/Cy5.5 increased significantly in lung tissues by 53.71% [Z-(2-1P)] and 93.41% [Z-(2-3P)], and 2-1P had an increased distribution by 24.19% in the inflammatory lung tissue compared to normal mouse lung tissue. In rat femoral artery models, 2-1P had greater injured/normal vessel fluorescence intensity contrast. In the zebrafish models, both 2-1P and 2-3P showed increased aggregation at the site of inflammation. Conclusion This E-selectin-targeting nanomedicine delivery system efficiently targets activated inflammatory endothelial cells to increase drug concentration at the inflammatory site, which sheds light on new strategies for treating neutrophil-mediated inflammatory diseases and practicing the concept of "one drug for multiple diseases".

Key words: E-selectin, IEL short peptides, doxorubicin hydrochloride-loaded liposomes, targeted delivery

叶雨濛, 余波, 陆沙沙, 周于, 丁美红, 程桂林. 靶向E-选择素的工程化脂质体的设计与炎症靶向评价[J]. 南方医科大学学报, 2025, 45(5): 1013-1022.

Yumeng YE, Bo YU, Shasha LU, Yu ZHOU, Meihong DING, Guilin CHENG. Design and inflammation-targeting efficiency assessment of an engineered liposome-based nanomedicine delivery system targeting E-selectin[J]. Journal of Southern Medical University, 2025, 45(5): 1013-1022.

图/表 6

表1 各个配方修饰脂质体的粒径、多分散指数和电位

Tab.1 Particle size, polydispersity index (PDI), and potential of modified liposomes for each formulation (n=3)

Groups	Size (nm)	PDI	Zeta (mV)
Control	93.84±1.96	0.11±0.01	-1.66±0.41
2-1P	81.36±0.28	0.08±0.05	-25.30±0.40
2-3P	83.21±0.52	0.04±0.03	-23.87±0.12

表2 脂质体表面短肽修饰密度

Tab.2 Peptide modification density on liposome surface

Groups	Moles of DPI (μmol)	Liposome's area (cm²)	DPI modification density (pmol/ cm²)
2-1P	0.04	8966.09	4.76
2-3P	0.13	17718.10	7.57

图1 各个配方在pH 5.5, pH 6.5和pH 7.4条件下的释放情况(37 ℃)

Fig.1 Release profiles of each formulation at pH 5.5, pH 6.5, and pH 7.4. A: Cumulative release rate of PEGylated DOX liposomes (P) within 48 h. B: Cumulative release rate of 2-1P formulation of IEL-Lip/DOX within 48 h. C: Cumulative release rate of 2-3P formulation of IEL-Lip/DOX within 48 h. D: Release of the different formulations at different pH values after 48 h. *P<0.05, ****P<0.0001 vs P group.

图2 IEL-Lip/DOX的体外靶向评价

Fig.2 In vitro targeting evaluation of IEL-Lip/DOX. A: Representative fluorogram images (×400) of E-selectin expression and DOX uptake in bEnd.3 inflammatory cells with different formulations of IEL-Lip/DOX. Green represents E-selectin labeling on the cell membrane, blue represents the DAPI-stained cell nucleus, and red represents DOX. B: Fluorogram (×400) of DOX uptake by HUVEC inflammatory cells. Blue represents the DAPI-stained cell nucleus, and red represents DOX. C: Flow cytometry analysis of DOX uptake by activated bEnd.3 and HUVEC cells in response to different formulations of liposomes (****P<0.0001 vs P group). D: Percentage bar chart of variables for drug penetration into the lower chamber in the Transwell simulating an invitro endothelial barrier model (**P<0.01 vs 2-3P group). P: Unmodified PEGylated DOX liposomes; 2-1P: IEL-Lip/DOX prepared in the ratio of Mol(PC): Mol(DPI)=100:1; 2-3P: IEL-Lip/DOX prepared in the ratio of Mol(PC): Mol(DPI)=100:3.

图3 小鼠ALI模型的体内靶向考察

Fig.3 In vivo targeting efficiency of the prepared liposomes in mouse models of acute lung injury (ALI). A: Representative immunofluorescence images (×200) of E-selectin expression in lung tissue sections at different time points after LPS challenge. CK represents lung tissue sections from normal mice, green represents E-selectin labeling, and blue represents nuclear staining with DAPI. B: Quantitative plots of E-selectin immunofluorescence expression and positive area in lung tissue sections at different time points (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CK group). C: Representative tissue fluorescence images of ALI mice. D: Quantitative histogram of lung tissue fluorescence imaging in ALI mice. The upper panel shows DOX fluorescence intensity and the bottom panel shows Cy5.5 fluorescence intensity. (*P<0.05, **P<0.01 vs Z-Cy5.5 group, n=4). E: Comparison of DOX distribution in tissues and plasma after 2-1P injection in normal and ALI mice (*P<0.05, **P<0.01, ***P<0.001 vs Normal group, n=4). F: Quantification of tissue distribution of differently formulated liposomes in ALI mice (**P<0.01 vs P group, n=4).

图4 大鼠股动脉物理损伤炎症模型的体内靶向考察

Fig.4 In vivo targeting efficiency of the prepared liposomes in rat femoral artery physical injury inflammation model. A: Representative in vivo imaging fluorescence maps of femoral artery vasculature after injection of each formulation. B: DOX fluorescence intensity ratio of the injured side vessel to the normal side vessel for each formulation. *P<0.05 vs P group (n=4).

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靶向E-选择素的工程化脂质体的设计与炎症靶向评价

Design and inflammation-targeting efficiency assessment of an engineered liposome-based nanomedicine delivery system targeting E-selectin

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