药学学报

Principle	Method	Advantage	Shortcoming	Ref.
Settlement coefficient difference	Differential ultracentrifugation	Low cost; large sample capacity; currently recognized as the gold standard for PDEVs separation	May damage PDEVs structure; high speeds cause contaminant sedimentation; complex and time-consuming process	[7, 8]
Buoyancy density difference of different EVs on density gradient	Density gradient ultracentrifugation
Size differences	Ultrafiltration	Simple operation; low cost; better maintain EVs structure	Prone to membrane blockage; unsuitable for large samples	[9]
Size exclusion chromatography	Maintain EVs structure; high separation purity	Requires complex process and specialized equipment	[10]
Asymmetric field flow separation	Highly automated; wide particle size distribution	Professional equipment required
Electrophoretic coupled dialysis	Maintain EVs structure	Membrane pollution leads to low yield; may cause EVs to accumulate	[11, 12]
Solubility differences	PEG precipitation	Simple operation; suitable for handling large quantities of samples	Prone to PDEVs aggregation and impurity co-precipitation	[13]
EVs surface specific proteins	Immunoaffinity	High specificity; fast separation speed	Limited knowledge of surface markers hinders effective PDEVs purification
The distribution coefficients of EVs are different in the two-phase system	Two aqueous system	Better maintain EVs structure; suitable for handling large quantities of samples	Higher cost	[14, 15]
Physical characteristics such as size, density, surface charge, etc	Microfluidic chip	Mild separation conditions; enables precise control; high repeatability and controllability	Requires skilled operation; unsuitable for large-scale samples

Principle	Method	Advantage	Shortcoming	Ref.
Settlement coefficient difference	Differential ultracentrifugation	Low cost; large sample capacity; currently recognized as the gold standard for PDEVs separation	May damage PDEVs structure; high speeds cause contaminant sedimentation; complex and time-consuming process	[7, 8]
Buoyancy density difference of different EVs on density gradient	Density gradient ultracentrifugation
Size differences	Ultrafiltration	Simple operation; low cost; better maintain EVs structure	Prone to membrane blockage; unsuitable for large samples	[9]
Size exclusion chromatography	Maintain EVs structure; high separation purity	Requires complex process and specialized equipment	[10]
Asymmetric field flow separation	Highly automated; wide particle size distribution	Professional equipment required
Electrophoretic coupled dialysis	Maintain EVs structure	Membrane pollution leads to low yield; may cause EVs to accumulate	[11, 12]
Solubility differences	PEG precipitation	Simple operation; suitable for handling large quantities of samples	Prone to PDEVs aggregation and impurity co-precipitation	[13]
EVs surface specific proteins	Immunoaffinity	High specificity; fast separation speed	Limited knowledge of surface markers hinders effective PDEVs purification
The distribution coefficients of EVs are different in the two-phase system	Two aqueous system	Better maintain EVs structure; suitable for handling large quantities of samples	Higher cost	[14, 15]
Physical characteristics such as size, density, surface charge, etc	Microfluidic chip	Mild separation conditions; enables precise control; high repeatability and controllability	Requires skilled operation; unsuitable for large-scale samples

Loading method	Loaded drug	Vesicle source	Ref.
Co-incubation	Chemicals	DOX	Celery	[8]
ICG	Aloe	[55]
MTX	Grapefruit	[56]
Nucleic acid	ApoB-siRNA	Acerola	[57]
Electroporation	Chemicals	DexP	Orange	[62]
Nucleic acid	DDHD1-siRNA	Tangerine	[63]
Sonication	Chemicals	5-Fluorouracil	Bitter melon	[12]
Nucleic acid	miR17	Grapefruit	[46]
Protein	BSA; HSP70	Grapefruit	[16]
HSP70	Grapefruit; Tomato	[64]
Sonication and coextrusion	Nucleic acid	siRNA-CD98	Ginger	[17]
Dmt1-siRNA	Ginger	[18]

Loading method	Loaded drug	Vesicle source	Ref.
Co-incubation	Chemicals	DOX	Celery	[8]
ICG	Aloe	[55]
MTX	Grapefruit	[56]
Nucleic acid	ApoB-siRNA	Acerola	[57]
Electroporation	Chemicals	DexP	Orange	[62]
Nucleic acid	DDHD1-siRNA	Tangerine	[63]
Sonication	Chemicals	5-Fluorouracil	Bitter melon	[12]
Nucleic acid	miR17	Grapefruit	[46]
Protein	BSA; HSP70	Grapefruit	[16]
HSP70	Grapefruit; Tomato	[64]
Sonication and coextrusion	Nucleic acid	siRNA-CD98	Ginger	[17]
Dmt1-siRNA	Ginger	[18]

Vesicle source	Engineering transformation mode	Loaded drug	Cell line/animal model	Administration route	Treated disease	Ref.
Grapefruit	Lipid recombination	Chemicals	MTX	DSS-induced mouse model of ulcerative colitis	Oral administration	Ulcerative colitis	[56]
Celery	None	DOX	A549 subcutaneous lung cancer xenograft model	Intravenous injection	Lung cancer	[8]
Ginger	Lipid recombination; FA surface modification	DOX	Subcutaneous colon cancer xenograft model	Intravenous injection	Colon cancer	[42]
Orange	None	DexP	Mouse model of IgA nephropathy	Oral administration	IgA nephropathy	[62]
Grapefruit	Activated leukocyte membrane coating	DOX	CT26 mouse colon cancer xenograft model; 4T1 mouse breast cancer xenograft model	Intravenous injection	Colon cancer; breast cancer	[44]
Grapefruit	Activated leukocyte membrane coating	Curcumin	DSS-induced mouse model of ulcerative colitis	Intravenous injection	Ulcerative colitis	[44]
Citrus	None	Nucleic acid	DDHD1-siRNA	SW480 colon cancer cell line	Co-incubation	Colon cancer	[63]
Acerola	None	ApoB-siRNA	Luciferase transgenic mouse	Oral administration	Effectively down-regulate target gene expression	[57]
Ginger	Lipid recombination; FA surface modification	Dmt1-siRNA	Intestine-specific Dmt1 knockout mouse; Hepc knockout mouse; double knockout mouse	Oral administration	Hereditary hemochromatosis	[18]
Grapefruit	Bionic synthesis; FA surface modification	miR17	GL-26 glioma orthotopic model in nude mice	Intranasal administration	Glioma	[46]
Grapefruit	None	Protein	HSP70	Human colon cancer HCT 116 and DLD-1 cells	Co-incubation	Colon cancer	[16]
Grapefruit	None	BSA	CD-1 IGS female mice	Intravenous injection	-	[16]
Grapefruit	None	HSP70	Glioma cell line (Gl-Tr)	Co-incubation	Glioma	[64]

Vesicle source	Engineering transformation mode	Loaded drug	Cell line/animal model	Administration route	Treated disease	Ref.
Grapefruit	Lipid recombination	Chemicals	MTX	DSS-induced mouse model of ulcerative colitis	Oral administration	Ulcerative colitis	[56]
Celery	None	DOX	A549 subcutaneous lung cancer xenograft model	Intravenous injection	Lung cancer	[8]
Ginger	Lipid recombination; FA surface modification	DOX	Subcutaneous colon cancer xenograft model	Intravenous injection	Colon cancer	[42]
Orange	None	DexP	Mouse model of IgA nephropathy	Oral administration	IgA nephropathy	[62]
Grapefruit	Activated leukocyte membrane coating	DOX	CT26 mouse colon cancer xenograft model; 4T1 mouse breast cancer xenograft model	Intravenous injection	Colon cancer; breast cancer	[44]
Grapefruit	Activated leukocyte membrane coating	Curcumin	DSS-induced mouse model of ulcerative colitis	Intravenous injection	Ulcerative colitis	[44]
Citrus	None	Nucleic acid	DDHD1-siRNA	SW480 colon cancer cell line	Co-incubation	Colon cancer	[63]
Acerola	None	ApoB-siRNA	Luciferase transgenic mouse	Oral administration	Effectively down-regulate target gene expression	[57]
Ginger	Lipid recombination; FA surface modification	Dmt1-siRNA	Intestine-specific Dmt1 knockout mouse; Hepc knockout mouse; double knockout mouse	Oral administration	Hereditary hemochromatosis	[18]
Grapefruit	Bionic synthesis; FA surface modification	miR17	GL-26 glioma orthotopic model in nude mice	Intranasal administration	Glioma	[46]
Grapefruit	None	Protein	HSP70	Human colon cancer HCT 116 and DLD-1 cells	Co-incubation	Colon cancer	[16]
Grapefruit	None	BSA	CD-1 IGS female mice	Intravenous injection	-	[16]
Grapefruit	None	HSP70	Glioma cell line (Gl-Tr)	Co-incubation	Glioma	[64]

植物细胞外囊泡作为药物载体的研究进展

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刘怡君 ¹^,² , 王红娟 ¹^,² , 张靖彬 ² , 陈欢 ¹^,²^,^* , 侯宏卫 ¹^,²^,^* , 胡清源 ¹^,²

药学学报 | 综述 2025,60(3): 721-730

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药学学报 | 综述 2025, 60(3): 721-730

植物细胞外囊泡作为药物载体的研究进展

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刘怡君¹^,², 王红娟¹^,², 张靖彬², 陈欢¹^,²^,^*, 侯宏卫¹^,²^,^*, 胡清源¹^,²

作者信息

1.国家烟草质量监督检验中心, 河南郑州 450001

2.北京生命科技研究院, 北京 102200

通讯作者:

^*陈欢, Tel: 86-371-67672597, E-mail: hunny_ch@163.com

侯宏卫, Tel: 86-371-67672727, E-mail: qsfctc@163.com

Research progress of plant-derived extracellular vesicles as drug carriers

Yi-jun LIU¹^,², Hong-juan WANG¹^,², Jing-bin ZHANG², Huan CHEN¹^,²^,^*, Hong-wei HOU¹^,²^,^*, Qing-yuan HU¹^,²

Affiliations

1. China National Tobacco Quality Supervision & Test Center, Zhengzhou 450001, China

2. Beijing Life Science Academy, Beijing 102200, China

出版时间: 2025-03-12 doi: 10.16438/j.0513-4870.2024-1174

文章导航

摘要

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植物来源的细胞外囊泡(plant-derived extracellular vesicles, PDEVs) 是由植物细胞分泌的具有脂质双分子层结构的纳米级囊泡, 具有安全性高、稳定性好、易于修饰等优势, 是良好的药物载体。作为一种新兴药物递送载体, PDEVs越来越受到人们的关注。本文综述了PDEVs的制备、表征、工程改造、药物装载方式和PDEVs作为药物载体应用及优势的最新研究进展, 展望了PDEVs应用于药物载体的未来发展方向。

关键词

植物细胞外囊泡 / 制备 / 表征 / 工程改造 / 药物递送

Abstract

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Plant-derived extracellular vesicles (PDEVs) are lipid bilayer-structured nanovesicles secreted by plant cells, recognized as excellent drug carriers due to their high stability, safety, and modifiability. As an emerging drug delivery system, PDEVs are gaining increasing attention. This review systematically summarizes the latest research progress on the preparation, characterization, engineering transformation, drug loading methods, and the applications and advantages of PDEVs as drug carriers. Finally, the future development directions of PDEVs in drug delivery are discussed.

Key words

plant-derived extracellular vesicle / preparation / characterization / engineering transformation / drug delivery

引用本文

刘怡君, 王红娟, 张靖彬, 陈欢, 侯宏卫, 胡清源. 植物细胞外囊泡作为药物载体的研究进展. 药学学报, 2025 , 60 (3) : 721 -730 . DOI: 10.16438/j.0513-4870.2024-1174

Yi-jun LIU, Hong-juan WANG, Jing-bin ZHANG, Huan CHEN, Hong-wei HOU, Qing-yuan HU. Research progress of plant-derived extracellular vesicles as drug carriers[J]. Acta Pharmaceutica Sinica, 2025 , 60 (3) : 721 -730 . DOI: 10.16438/j.0513-4870.2024-1174

正文

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细胞外囊泡(extracellular vesicles, EVs) 是指由细胞分泌的具有脂质双层膜结构的颗粒, 包括外泌体、微囊泡、凋亡小体^[1]。EVs广泛存在于各种生物体中, 在细胞间通讯和生物活性分子(如蛋白质、RNA及其他信号分子) 运输方面发挥着重要作用^[2]。近年来, EVs作为药物载体的研究受到了广泛关注, 特别是在肿瘤治疗、基因治疗和免疫调节等领域, EVs作为药物载体展现出巨大的应用潜力。

在以往的研究中, EVs主要来源于动物细胞。然而, 动物源EVs的应用存在诸多限制, 如产量低、可能携带病原体、易引起人体免疫反应, 以及加速血液清除现象等^{[3, 4]}。为克服这些问题, 研究者开始将目光转向其他来源的EVs。其中, 植物来源的细胞外囊泡(plant-derived extracellular vesicles, PDEVs) 因其生产成本低、无动物源性病原体风险、稳定性好等优势, 逐渐引起了研究者的兴趣。研究表明, PDEVs不仅能够稳定地携带并保护活性分子, 还具有良好的生物相容性和低免疫原性, 是一种理想的药物递送天然载体^[5]。除此之外, PDEVs存在于多种植物中, 来源丰富。鉴于这些独特优势, 深入探讨PDEVs在药物传递中的应用潜力, 对提高药物递送效率、减少药物不良反应以及克服药物耐受性具有重要意义。

1 PDEVs的制备

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植物组织通过研磨、榨汁、浸润等方式获得含有PDEVs的植物汁液后, 进一步纯化可采用超离心、超滤、聚乙二醇(polyethylene glycol, PEG) 沉淀、尺寸排阻色谱、非对称场流分离、免疫亲合法、微流控芯片法等方法。上述方法在Zhu等^[6]的文章中已有综述, 它们的原理、优缺点总结至表 1^[7-15], 本部分重点介绍电泳耦合透析、双水相体系法。

电泳耦合透析法是结合电泳和透析技术, 通过电场驱动小分子杂质透过透析袋, 而PDEVs被截留, 进而实现纯化。通过改变电流方向和更换电泳缓冲液可进一步提高分离效率。Yang等^{[11, 12]}利用此法从柠檬和苦瓜中分离出EVs, 证明了此法的可行性。双水相体系法的原理是利用两种不混溶的聚合物溶液(例如PEG和葡聚糖溶液) 形成双水相体系, 通过离心分离PDEVs。Savcı等^[14]利用此法从葡萄柚中分离出了EVs。

目前用于PDEVs分离的方法虽然多样, 但仍存在提取效率低、纯度不高、成本较高、难以实现规模化生产的问题。适当的技术组合可能更有利于获得高纯度和高产率的PDEVs^[16-18]。PDEVs的分离纯化是进行相关研究和应用的基础环节, 未来技术的不断进步和新方法的开发将继续提升PDEVs分离纯化的效率和应用潜力。

2 PDEVs的表征

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PDEVs的表征是其作为药物载体应用的关键一步。一方面, 对PDEVs的物理特性和成分进行表征可验证PDEVs的成功分离; 另一方面, PDEVs的组成成分与其生物功能密切相关, 准确分析PDEVs的组分对于优化其在药物递送中的应用具有重要参考价值。PDEVs的表征涵盖形态、粒径、脂质等方面。

2.1 物理表征

物理表征是将PDEVs与其他物质快速区分开来的重要指标。PDEVs的形态和粒径是其关键物理特性^[19]。电子显微镜如透射电子显微镜(transmission electron microscope, TEM)、扫描电子显微镜(scanning electron microscope, SEM) 和冷冻电子显微镜(cryo-electron microscopy, Cryo-EM) 常用于观察其形态^[20]。其中, Cryo-EM无需固定处理, 能更真实反映PDEVs形态^[21]。通常, Cryo-EM捕获的PDEVs呈现近似球形的形态, 而TEM和SEM捕获的PDEVs呈现杯状形态^[22]。不同植物来源的EVs直径不同, 但范围大多在50~500 nm^[1]。动态光散射(dynamic light scattering, DLS) 和纳米粒子跟踪分析(nanoparticle tracking analysis, NTA) 是测量粒径的常用方法。DLS可以快速测量小体积EVs的流体动力学直径, 但分辨率较低^[23]。NTA相比DLS精度更高, 可以识别和测量单个颗粒, 提供PDEVs粒径和浓度的详细信息, 是目前使用最广泛的EVs粒径表征技术^[24]。可调谐电阻脉冲传感(tunable resistance pulse sensing, TRPS) 作为一种新兴检测技术, 也已被用于EVs的表征^{[25, 26]}。TRPS可根据EVs通过孔洞时产生的阻力脉冲的变化来确定EVs的大小、电位和浓度。

2.2 脂质鉴定

脂质作为PDEVs的主要成分, 其组成对PDEVs的功能具有重要作用。脂质组学分析显示, PDEVs中存在的主要脂质种类包括磷脂酸(phosphatidic acid, PA)、磷脂酰乙醇胺和磷脂酰胆碱(phosphatidylcholine, PC)^[27]。脂类成分差异和组装结构会影响PDEVs在体内的分布。例如富含PA脂类的姜源性EVs倾向定位于肠道, PC脂类则促进PDEVs从肠道到肝脏的迁移^[28]。天然葡萄EVs很少被小鼠肠道干细胞摄取, 但其重组脂质纳米载体可以聚集在肠道并被肠道干细胞摄取^[29]。为了阐明PDEVs的功能特性并优化其在药物递送中的应用, 未来仍需对PDEVs的脂质功能进行进一步的研究。

2.3 其他成分鉴定

PDEVs的蛋白质含量较低, 主要由细胞质蛋白(如肌动蛋白、蛋白水解酶) 和少量膜蛋白组成^[30]。膜蛋白在PDEVs的摄取和内化中起重要作用。研究发现, 大蒜EVs表面的凝集素类蛋白可与细胞CD98受体结合, 促进其内化^[31]。PDEVs中的细胞质蛋白可能参与细胞壁重塑和病原防御^{[32, 33]}。PDEVs富含miRNA, 具有抗炎作用并能够调节肿瘤^[34]。如生姜EVs的miRNA能改善结肠炎^[28]。此外, PDEVs携带的生物活性小分子如6-姜酚、萝卜硫素等, 也具有重要的生物学功能^{[35, 36]}。例如, 西兰花EVs中的萝卜硫素通过激活AMPK通路抑制结肠炎^[36]。

目前, PDEVs的表征和鉴定主要集中在物理表征和脂质组成上, 对其他组分的分析尚不深入, 研究者仍需深入探究不同PDEVs的组成或聚焦于特定植物建立完整的数据库。阐明PDEVs的组成及成分功能将有助于优化其作为药物载体的靶向性和治疗效果。

3 PDEVs的工程改造

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工程改造可进一步扩大天然PDEVs作为药物载体的应用, 在保持原有功能的基础上赋予PDEVs额外的功能, 例如增强PDEVs的靶向能力、延长血浆半衰期、增加载药量、对PDEVs进行荧光成像等。PDEVs的工程改造方法主要包括表面修饰、脂质重组、膜包覆、膜融合、仿生合成、修饰EVs来源的亲代细胞(图 1)。

3.1 表面修饰

表面修饰指用蛋白质、肽、PEG、叶酸(folic acid, FA)、荧光染料等外源分子通过各种化学和物理方法修饰PDEVs表面, 赋予它们特定的功能。Sasaki等^[37]用PEG修饰玉米EVs, 发现其比未经修饰的玉米EVs具有更长的血液循环时间、更强的肿瘤组织靶向性和更低的肝脏蓄积毒性。Chen等^[38]以牛奶来源的EVs作为模型系统, 设计了一种温和的表面改性方法, 并在葡萄柚和生姜EVs中验证了该方法的普遍性。该方法通过将EVs表面蛋白的二硫键还原为二硫醇, 随后进行硫醇-马来酰亚胺缀合反应来实现表面改性。使用此法将转铁蛋白缀合至牛奶EVs, 增强了牛奶EVs的肿瘤靶向和药物递送能力。荧光示踪剂可以与PDEVs表面的一些配体结合。亲脂性碳菁染料(如DiR) 标记已广泛用于PDEVs的体内追踪。而FA等小分子已被用作靶向癌症组织的配体^[39-41]。

3.2 脂质重组

从PDEVs中提取脂质并将其重组为半人工的纳米载体是PDEVs工程改造的常用方法。Zhang等^[42]从生姜EVs中提取脂质并将其重组为纳米载体(ginger-derived lipid vehicles, GDLV), 用此载体封装化疗药物多柔比星(doxorubicin, DOX), 实现了DOX的缓释。Wang等^[43]从葡萄柚EVs中提取脂质将其重组为纳米载体(grapefruit-derived nanocarriers, GDN), 并用FA对其修饰(以下简称该载体为FA-GDN), 用于药物递送。在肿瘤动物模型中, FA-GDN装载的紫杉醇显示出比游离紫杉醇更强的抗肿瘤特性。

3.3 膜包覆、膜融合

将具有特殊受体的其他细胞膜与PDEVs的膜融合, 或用其他特殊功能的细胞膜包覆PDEVs是增强它们靶向特异性的可行途径。膜包覆可以使EVs具有源细胞的特性, 可增强EVs的靶向性。Wang等^[44]用富集炎症相关受体的活化白细胞膜包裹GDN, 增强了GDN靶向炎症组织的能力。Huang等^[45]将负载抗癌药物CX5461的葡萄柚EVs与来自牙龈的间充质干细胞(gingiva-derived mesenchymal stem cells, GMSCs) 的过表达CCR6⁺的纳米囊泡融合, 获得多功能融合囊泡。该融合囊泡兼具葡萄柚EVs、CX5461、GMSCs的生物活性, 且通过CCR6的作用, 融合囊泡可集中于富含趋化因子CCL20的炎症组织, 发挥免疫调节功能。

3.4 仿生合成

随着仿生合成技术的普及, 将PDEVs与其他无机纳米载体结合成共递送载体提高递送效率变得越来越普遍。Zhuang等^[46]用FA修饰GDN, 再用其封装聚乙烯亚胺(polyethylenimine, PEI) 制成复合药物载体, 既提高了RNA的载药量, 又降低了PEI的毒性。Niu等^[47]制备了负载DOX的pH敏感性肝素纳米颗粒(doxorubicin-loaded heparin-based nanoparticles, DN), 并通过肝素羧基和膜分子氨基的共价组合将其结合到葡萄柚EVs表面, 制成EV-DN纳米颗粒。其独特的设计赋予仿生EV-DN高于传统EVs 4倍的载药能力, 且实现酸性环境病变部位的特异性靶向, 显著提高了递送效率。Feng等^[48]将羟基磷灰石晶体靶向肽(targeting peptide, TP) 结合至负载硫代硫酸钠(sodium thiosulfate, STS, 一种治疗血管钙化的药物) 的葡萄柚EVs表面, 制备了一种仿生纳米载体用于STS的递送(TP-EVs-STS, ESTP)。在体外, ESTP被钙化血管平滑肌细胞高效摄取, 随后抑制血管平滑肌细胞的钙化。在血管钙化小鼠模型中, 与游离STS组和单独葡萄柚EVs装载STS的组相比, ESTP组药物在钙化动脉中显示出最高的积累。Qiao等^[49]提出了一种集成电动Pd-Pt纳米片和生姜EVs的仿生纳米平台(EV-Pd-Pt) 用于体内抗感染。生姜EVs优异的生物相容性和较长的血液循环时间保证了EV-Pd-Pt在感染部位的积累而避免免疫清除。更重要的是, EV-Pd-Pt能够以生姜EVs脂质依赖性的方式进入细菌细胞内部发挥抗感染作用。除此之外, 脂质体和水凝胶也是用于仿生合成的良好无机纳米载体, 这在哺乳动物来源的EVs中已得到验证^[50-52], 未来有望应用于PDEVs。

3.5 修饰EVs来源的亲代细胞

EVs来源细胞的基因修饰是EVs表面功能化的一种重要方法。通过将靶细胞受体基因转染到亲代细胞中, 这些受体蛋白在EVs生物发生过程中与EVs跨膜蛋白融合, 形成EVs表面表达的靶向部分。Alvarez-Erviti等^[53]首次将编码Lamp2b蛋白的质粒转染入树突状细胞, Lamp2b在树突细胞来源的EVs膜表面显著积累。用此EVs装载siRNA, 能够实现神经元、小胶质细胞和少突胶质细胞的靶向递送, 从而特异性发挥作用, 且在其他组织中未观察到EVs的非特异性摄取。Du等^[54]将CD47基因转染到供体细胞中, 实现了CD47在供体细胞源EVs上的有效积累。CD47过表达的EVs有效逃避了单核吞噬系统的吞噬, 延长了循环时间, 增加了在肿瘤组织中的分布。基因工程技术是实现EVs改造的可行方法, 但因其操作复杂且成本高昂, 目前尚未应用于PDEVs的工程改造, 未来仍需进一步发展。

4 药物装载

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PDEVs的载药方法与哺乳动物EVs类似, 分为被动加载和主动加载两种方式。被动加载如共孵育是最简单的载药方法, 分离的EVs直接与药物溶液孵育, 药物分子通过被动扩散和亲脂扩散装载至PDEVs中。共孵育法已被用于DOX、吲哚菁绿(indocyanine green, ICG)、甲氨蝶呤(methotrexate, MTX)、ApoB-siRNA等药物的PDEVs负载^{[8, 55-57]}。共孵育法方便且成本低, 然而, 由于缺乏促进药物扩散的外部动力, 共孵育法通常包封率较低^[58]。此外, 由于EVs膜的脂质双层结构, 亲脂性药物比亲水性药物更易通过孵育法装载。

主动加载如电穿孔、超声、共挤出、冻融循环等也是将药物转移到EVs中的有效方法^{[59, 60]}。电穿孔法是利用瞬态弱电脉冲打开脂质双分子层膜, 形成可逆小孔。通过这些小孔, 药物分子得以进入EVs的腔体。电击时间仅为几毫秒, 不会破坏PDEVs的固有结构^[61]。研究人员已通过电穿孔法成功将地塞米松磷酸钠(dexamethasone sodium phosphate, DexP)、DDHD1-siRNA装载至PDEVs中^{[62, 63]}。超声也是常用的载药方法之一, 通过超声提供的能量和机械力暂时改变脂质双分子层的结构, 药物能够穿透EVs。超声已被用于化学药物(如5-氟尿嘧啶)、基因药物(如miR17)、蛋白质药物[如牛血清白蛋白(bovine serum albumin, BSA)、热休克蛋白70 (heat shock protein 70, HSP70)] 等多种药物的PDEVs负载^{[12, 16, 46, 64]}。超声处理对EVs的脂质和蛋白质含量没有显著影响, 但超声提供的外力可能会破坏EVs的结构和完整性^[65]。共挤出技术是通过将EVs与药物混合后共同通过狭窄的通道或孔隙进行挤压, 产生强烈的机械剪切力, 从而使药物分子进入EVs的脂质双层或内部。超声和共挤出技术结合已被用于核酸药物的装载^{[17, 18]}。共挤出法的优势在于能够均匀、有效地实现药物装载, 适用于多种类型的药物。但共挤出过程的机械剪切力可能损伤EVs的结构, 影响其生物活性。冻融循环也是一种常用的载药方法。通过反复冻融, EVs的脂质双层结构轻微受损而形成孔隙, 药物分子得以扩散进入。Hajipour等^[66]利用该方法成功将人绒毛膜促性腺激素加载到子宫来源的外泌体中。此方法的优势在于对EVs结构损伤较小、操作简便, 然而反复的冻融过程可能导致蛋白质失活^[67]、EVs聚集及颗粒变大^[68]。相比于共孵育法, 主动加载法通常包封率较高。

PDEVs载药方法多种多样, 如表 2所示。研究者在实际应用中需谨慎考虑每种方法的优缺点, 根据自己的需求和目的选择合适的载药方法。

5 PDEVs作为药物载体的应用

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PDEVs的脂质双分子层结构使其能够将亲水药物封装在腔内, 并将疏水药物装载到脂质双分子层中。研究表明, PDEVs可携带化学、基因、蛋白质等多种药物。

5.1 化学药物载体

研究表明, PDEVs负载可以提高化学药物的稳定性、靶向性和生物利用度。GDN已被用于封装抗炎药物MTX。GDN封装在保留MTX功能的同时增强了肠道巨噬细胞对MTX的摄取^[56]。在葡聚糖硫酸钠(dextran sulfate sodium salt, DSS) 诱导的结肠炎小鼠模型中, 口服GDN-MTX显著缓解了结肠炎症状, 减少了促炎因子的分泌, 表现出比游离MTX更强的抗炎作用, 且显著降低了MTX的不良反应^[56]。用芹菜来源的EVs封装DOX, 通过静脉注射至A549人肺腺癌皮下移植瘤小鼠模型中^[8]。芹菜EV-DOX在抑制肿瘤生长方面的效果优于脂质体-DOX和游离DOX, 同时显著降低了DOX引起的毒性反应^[8]。Zhang等^[42]用FA修饰的GDLV封装化疗药物DOX, 显著增强了DOX的肿瘤靶向能力, 并延长了其在小鼠体内的循环时间。用橙汁来源的EVs封装DexP, 口服给药后, EV-DexP被回盲部黏膜下区域的淋巴细胞有效吸收^[62]。体内实验表明, EV-DexP在减轻肾脏病理病变方面效果优于游离DexP。Wang等^[44]用活化白细胞膜包覆的葡萄柚EVs封装DOX, 静脉注射至小鼠体内后, 延长了DOX在体内的循环时间, 增加了其在靶组织中的积累。用此载体封装姜黄素, 静脉注射至DSS诱导的结肠炎小鼠模型后, 提高了姜黄素在结肠组织的积累并显著降低了炎症因子水平, 治疗效果优于游离姜黄素^[44]。

5.2 基因药物载体

寻找合适的药物载体是基因治疗的主要问题。病毒和非病毒载体目前均用于递送基因药物, 但它们通常伴随着高免疫原性和低安全性的问题^[5]。研究发现用PDEVs携带siRNA, 可以保护siRNA不被体内环境降解, 同时实现靶向递送和治疗。Rabienezhad Ganji等^[63]用柑橘EVs递送DDHD1-siRNA, 成功将其靶向至人结直肠癌细胞, 抑制靶标表达约60%。用针叶樱桃来源的EVs负载ApoB-siRNA, 通过口服给药递送至小鼠体内^[57]。实验结果显示, 口服EVs-ApoB-siRNA复合物后, 小肠和肝脏中的靶基因表达显著下调, 尤其在小肠中抑制效果最为显著。Wang等^[18]用FA修饰的GDLV负载Dmt1-siRNA, 口服给药后, 药物能够靶向小鼠十二指肠上皮, 导致肠道Dmt1 mRNA表达减弱。Zhuang等^[46]用FA修饰的GDN与PEI制成的仿生复合药物载体装载miR17, 鼻内给药后miR17可快速递送至大脑被GL-26肿瘤细胞选择性摄取并延缓小鼠脑肿瘤的生长。

5.3 蛋白质药物载体

蛋白质药物在体内易被酶降解, 稳定性差, 半衰期短。用PDEVs负载可提高蛋白质药物的稳定性, 提高递送效率。Garaeva等^[16]在体外细胞试验中证明了天然葡萄柚来源的EVs是高效载体, 可用于递送外源BSA和HSP70进入人外周血单核细胞和结肠癌细胞。与游离蛋白质相比, 葡萄柚EVs负载显著改善了人体细胞对外源蛋白质的吸收, 且不影响HSP70的功能^[16]。静脉注射葡萄柚EV-BSA或游离BSA至小鼠体内, EV-BSA能够被大多数器官有效吸收, 吸收效率显著高于游离BSA^[16]。Kilasoniya等^[64]从葡萄柚和番茄中提取EVs并比较它们作为递送载体的潜力, 结果表明, 葡萄柚EVs对HSP70的载药量高于番茄EVs, 且葡萄柚EVs负载的HSP70比游离HSP70、番茄EVs负载的HSP70具有更高的神经胶质瘤细胞递送效率。

PDEVs作为药物载体在化学、基因、蛋白质等多种药物递送中展现出良好的应用潜力(表 3)。通过PDEVs负载可提高药物稳定性、增强靶向性以及降低药物毒副作用。然而, 目前相关研究仍处于基础研究阶段, 仅有极少数案例推进至临床试验。例如, 临床试验NCT01294072探讨了植物外泌体递送姜黄素用于结肠炎和结肠癌治疗的可能性。尽管试验尚未完成, 但这一探索验证了PDEVs在改善药物递送效率方面的潜力, 同时也反映出其临床转化的挑战, 包括标准化生产、质量控制等。

6 PDEVs作为药物载体的优势

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PDEVs在药物递送领域展现出独特的优势, 主要体现在安全性、稳定性和协同治疗能力方面, PDEVs的这些特性使其成为一种有前景的药物载体。

6.1 安全性

大多数PDEVs因源自可食用植物, 通常被认为具有较高的安全性。体内外实验也证实了PDEVs的安全性。如Wang等^[18]发现GDLV毒性低于合成脂质体, 100 mmol·L^-1浓度下不会破坏肠黏膜屏障。Feng等^[48]评价了仿生复合纳米药物ESTP的生物安全性, 发现其对小鼠器官无损害, 且在50 mg·L^-1浓度下未见溶血。小鼠口服10 mg·kg^-1 GDN后, 未见血清IFN-γ、肝酶及免疫细胞组成变化^[56], 60 µg·mL^-1的GDN不影响巨噬细胞活力, 静脉注射至孕鼠体内也不通过胎盘屏障^[43]。以上研究结果均验证了PDEVs的安全性。

6.2 稳定性

PDEVs能够耐受胃肠环境, 具有良好的稳定性。Wang等^[56]的研究证实, GDN在体外酸碱溶液和模拟胃肠溶液中均保持稳定, 用GDN封装MTX可防止其在胃肠环境中被破坏。生姜EVs在模拟胃肠液中高度稳定, 口服后被肠上皮细胞和巨噬细胞吸收^[42]。GDLV能够携带并保护siRNA递送至肠道, 并下调结肠细胞CD98基因的表达^[17]。然而, 目前对于PDEVs抵抗胃肠环境的机制仍不明确, 可能与其有序的质膜结构和脂质成分有关^[5]。

6.3 协同治疗

PDEVs保留了来源植物的生物功能, 如抗炎、抗癌等。PDEVs可通过多种机制发挥抗炎作用, 包括调节抗促炎细胞因子的释放、通过AhR途径产生抗菌肽以及抑制NLRP3炎症小体的激活等。例如, 大蒜来源的EVs能够通过抑制NF-κB信号通路, 降低肝脏中IL-6、IL-1β和TNF-α等炎症介质的水平, 从而改善脂多糖/D-氨基半乳糖诱导的小鼠肝损伤^[69]。桑椹来源的EVs则通过激活AhR信号通路诱导抗菌肽的表达, 有效预防DSS诱导的小鼠结肠炎^[70]。生姜EVs可通过抑制NLRP3炎症小体及其下游通路的激活, 减少caspase-1自裂解以及IL-1β和IL-18等促炎细胞因子的释放, 进而发挥抗炎作用^[71]。类似地, PDEVs在抗癌方面也表现出良好的潜力, 能够通过增加活性氧(reactive oxygen species, ROS) 水平、诱导细胞凋亡、调节肿瘤微环境及改变巨噬细胞极化等多种机制发挥抗癌作用。例如, 茶叶来源的EVs可通过增加细胞内ROS的生成, 触发线粒体损伤和细胞周期阻滞, 有效诱导乳腺癌细胞4T1的凋亡^[72]。人参EVs则通过促进巨噬细胞从M2型向M1型极化, 抑制肿瘤生长和转移, 并调节肿瘤微环境^[73]。作为药物载体时, PDEVs可与药物发挥协同治疗作用, 增强治疗效果。Wang等^[56]在其研究中做出了成功的尝试。研究发现GDN能够被肠道巨噬细胞摄取, 通过上调HO-1表达并抑制IL-1β和TNF-α的产生来改善DSS诱导的小鼠结肠炎。用GDN包封MTX可显著提高其治疗效果。PDEVs具有与药物进行协同治疗的潜力, 但不同来源的PDEVs所含成分不同, 导致其与药物的相互作用不同。在进行协同治疗时, 研究者还应首先分析PDEVs包含的具体成分, 再做出假设和研究验证。

7 展望与挑战

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PDEVs作为一种新兴的药物递送载体, 展现出巨大的应用潜力。通过多种纯化技术的结合, 可获得较高纯度的PDEVs。电子显微镜、DLS、NTA等物理表征手段结合脂质、蛋白质等成分鉴定可帮助研究者快速了解PDEVs的形态、尺寸和表面特性等信息, 这为进一步的研究提供了基础。通过脂质重组、表面修饰等工程改造方法, PDEVs的药物载体功能得到了进一步增强。PDEVs作为药物载体有主动和被动两种药物装载方式, 可用于化学、基因、蛋白质药物的递送, 具有安全性高、稳定性好、可与药物发挥协同治疗作用的优势。

然而, 目前PDEVs作为药物载体的应用仍受到以下限制: ①分离方法的标准化: PDEVs缺乏统一、标准化的分离方法。超离心、密度梯度离心、超滤和尺寸排阻色谱等方法在实验室中广泛应用, 但在产业转化过程中存在耗时长、收率低的问题。且超离心结果可能受离心速度、离心力和转子类型影响而不一致^[74]。因此, 有必要探索和建立一种理想的分离方法以获得均匀、稳定、高产的PDEVs。②工程改造的需求: 工程改造可赋予PDEVs更灵活的靶向特性, 是未来PDEVs作为药物载体应用的一个重要方向。例如, FA或肽配体修饰可增强PDEVs的靶向能力。膜融合技术可将PDEVs与其他具有特殊功能的膜进行融合, 从而提升其生物学特性和免疫逃逸能力。然而, 随着对PDEVs研究的深入, 仍需开发进一步的工程改造方法以拓宽PDEVs的应用范围。③天然活性成分的复杂性: PDEVs携带的天然活性成分也是一个不确定因素, 可能与药物发挥协同治疗作用, 但也可能会影响药物疗效^[75]。因此, 需要广泛深入的研究来阐明PDEVs中天然活性成分的组成以及天然活性成分与药物之间的作用机制, 以实现有效的负载或协同治疗作用。

总的来说, 基于PDEVs的药物递送系统具有安全、稳定、来源丰富、易于修饰等优势, 这使其成为潜在优异的药物递送载体。尽管目前PDEVs的生物医学研究仍处于起步阶段, 但通过跨学科的合作和技术创新, 这些障碍终将被克服, PDEVs有望在药物递送和其他生物医学领域实现更广泛的应用。

作者贡献: 刘怡君负责撰写并修改论文; 王红娟、张靖彬、陈欢、胡清源负责指导论文修改; 侯宏卫负责指导论文撰写并定稿。

利益冲突: 本文所有作者声明不存在利益冲突关系。

基金

收起

北京生命科技研究院自立项目(2023000CC0070)
北京生命科技研究院自立项目(2023000CC0160)

参考文献

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参考文献引证文献

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2025年第60卷第3期

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文章信息

doi: 10.16438/j.0513-4870.2024-1174

接收时间：2024-11-27
首发时间：2025-11-06
出版时间：2025-03-12

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文章信息

作者

出版历史

收稿日期：2024-11-27
修回日期：2025-01-09

基金

北京生命科技研究院自立项目(2023000CC0070)

北京生命科技研究院自立项目(2023000CC0160)

作者信息

1.国家烟草质量监督检验中心, 河南郑州 450001

2.北京生命科技研究院, 北京 102200

通讯作者:

^*陈欢, Tel: 86-371-67672597, E-mail: hunny_ch@163.com

侯宏卫, Tel: 86-371-67672727, E-mail: qsfctc@163.com

参考文献

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Principle	Method	Advantage	Shortcoming	Ref.
Settlement coefficient difference	Differential ultracentrifugation	Low cost; large sample capacity; currently recognized as the gold standard for PDEVs separation	May damage PDEVs structure; high speeds cause contaminant sedimentation; complex and time-consuming process	[7, 8]
Buoyancy density difference of different EVs on density gradient	Density gradient ultracentrifugation
Size differences	Ultrafiltration	Simple operation; low cost; better maintain EVs structure	Prone to membrane blockage; unsuitable for large samples	[9]
Size exclusion chromatography	Maintain EVs structure; high separation purity	Requires complex process and specialized equipment	[10]
Asymmetric field flow separation	Highly automated; wide particle size distribution	Professional equipment required
Electrophoretic coupled dialysis	Maintain EVs structure	Membrane pollution leads to low yield; may cause EVs to accumulate	[11, 12]
Solubility differences	PEG precipitation	Simple operation; suitable for handling large quantities of samples	Prone to PDEVs aggregation and impurity co-precipitation	[13]
EVs surface specific proteins	Immunoaffinity	High specificity; fast separation speed	Limited knowledge of surface markers hinders effective PDEVs purification
The distribution coefficients of EVs are different in the two-phase system	Two aqueous system	Better maintain EVs structure; suitable for handling large quantities of samples	Higher cost	[14, 15]
Physical characteristics such as size, density, surface charge, etc	Microfluidic chip	Mild separation conditions; enables precise control; high repeatability and controllability	Requires skilled operation; unsuitable for large-scale samples

Principle

Method

Advantage

Shortcoming

Ref.

Settlement coefficient difference

Differential ultracentrifugation

Low cost; large sample capacity; currently recognized as the gold standard for PDEVs separation

May damage PDEVs structure; high speeds cause contaminant sedimentation; complex and time-consuming process

[7, 8]

Buoyancy density difference of different EVs on density gradient

Density gradient ultracentrifugation

Size differences

Ultrafiltration

Simple operation; low cost; better maintain EVs structure

Prone to membrane blockage; unsuitable for large samples

[9]

Size exclusion chromatography

Maintain EVs structure; high separation purity

Requires complex process and specialized equipment

[10]

Asymmetric field flow separation

Highly automated; wide particle size distribution

Professional equipment required

Electrophoretic coupled dialysis

Maintain EVs structure

Membrane pollution leads to low yield; may cause EVs to accumulate

[11, 12]

Solubility differences

PEG precipitation

Simple operation; suitable for handling large quantities of samples

Prone to PDEVs aggregation and impurity co-precipitation

[13]

EVs surface specific proteins

Immunoaffinity

High specificity; fast separation speed

Limited knowledge of surface markers hinders effective PDEVs purification

The distribution coefficients of EVs are different in the two-phase system

Two aqueous system

Better maintain EVs structure; suitable for handling large quantities of samples

Higher cost

[14, 15]

Physical characteristics such as size, density, surface charge, etc

Microfluidic chip

Mild separation conditions; enables precise control; high repeatability and controllability

Requires skilled operation; unsuitable for large-scale samples

Loading method	Loaded drug	Vesicle source	Ref.
Co-incubation	Chemicals	DOX	Celery	[8]
ICG	Aloe	[55]
MTX	Grapefruit	[56]
Nucleic acid	ApoB-siRNA	Acerola	[57]
Electroporation	Chemicals	DexP	Orange	[62]
Nucleic acid	DDHD1-siRNA	Tangerine	[63]
Sonication	Chemicals	5-Fluorouracil	Bitter melon	[12]
Nucleic acid	miR17	Grapefruit	[46]
Protein	BSA; HSP70	Grapefruit	[16]
HSP70	Grapefruit; Tomato	[64]
Sonication and coextrusion	Nucleic acid	siRNA-CD98	Ginger	[17]
Dmt1-siRNA	Ginger	[18]

Loading method

Loaded drug

Vesicle source

Ref.

Co-incubation

Chemicals

DOX

Celery

[8]

ICG

Aloe

[55]

MTX

Grapefruit

[56]

Nucleic acid

ApoB-siRNA

Acerola

[57]

Electroporation

Chemicals

DexP

Orange

[62]

Nucleic acid

DDHD1-siRNA

Tangerine

[63]

Sonication

Chemicals

5-Fluorouracil

Bitter melon

[12]

Nucleic acid

miR17

Grapefruit

[46]

Protein

BSA; HSP70

Grapefruit

[16]

HSP70

Grapefruit; Tomato

[64]

Sonication and coextrusion

Nucleic acid

siRNA-CD98

Ginger

[17]

Dmt1-siRNA

Ginger

[18]

Vesicle source	Engineering transformation mode	Loaded drug	Cell line/animal model	Administration route	Treated disease	Ref.
Grapefruit	Lipid recombination	Chemicals	MTX	DSS-induced mouse model of ulcerative colitis	Oral administration	Ulcerative colitis	[56]
Celery	None	DOX	A549 subcutaneous lung cancer xenograft model	Intravenous injection	Lung cancer	[8]
Ginger	Lipid recombination; FA surface modification	DOX	Subcutaneous colon cancer xenograft model	Intravenous injection	Colon cancer	[42]
Orange	None	DexP	Mouse model of IgA nephropathy	Oral administration	IgA nephropathy	[62]
Grapefruit	Activated leukocyte membrane coating	DOX	CT26 mouse colon cancer xenograft model; 4T1 mouse breast cancer xenograft model	Intravenous injection	Colon cancer; breast cancer	[44]
Grapefruit	Activated leukocyte membrane coating	Curcumin	DSS-induced mouse model of ulcerative colitis	Intravenous injection	Ulcerative colitis	[44]
Citrus	None	Nucleic acid	DDHD1-siRNA	SW480 colon cancer cell line	Co-incubation	Colon cancer	[63]
Acerola	None	ApoB-siRNA	Luciferase transgenic mouse	Oral administration	Effectively down-regulate target gene expression	[57]
Ginger	Lipid recombination; FA surface modification	Dmt1-siRNA	Intestine-specific Dmt1 knockout mouse; Hepc knockout mouse; double knockout mouse	Oral administration	Hereditary hemochromatosis	[18]
Grapefruit	Bionic synthesis; FA surface modification	miR17	GL-26 glioma orthotopic model in nude mice	Intranasal administration	Glioma	[46]
Grapefruit	None	Protein	HSP70	Human colon cancer HCT 116 and DLD-1 cells	Co-incubation	Colon cancer	[16]
Grapefruit	None	BSA	CD-1 IGS female mice	Intravenous injection	-	[16]
Grapefruit	None	HSP70	Glioma cell line (Gl-Tr)	Co-incubation	Glioma	[64]

Vesicle source

Engineering transformation mode

Loaded drug

Cell line/animal model

Administration route

Treated disease

Ref.

Grapefruit

Lipid recombination

Chemicals

MTX

DSS-induced mouse model of ulcerative colitis

Oral administration

Ulcerative colitis

[56]

Celery

None

DOX

A549 subcutaneous lung cancer xenograft model

Intravenous injection

Lung cancer

[8]

Ginger

Lipid recombination; FA surface modification

DOX

Subcutaneous colon cancer xenograft model

Intravenous injection

Colon cancer

[42]

Orange

None

DexP

Mouse model of IgA nephropathy

Oral administration

IgA nephropathy

[62]

Grapefruit

Activated leukocyte membrane coating

DOX

CT26 mouse colon cancer xenograft model; 4T1 mouse breast cancer xenograft model

Intravenous injection

Colon cancer; breast cancer

[44]

Grapefruit

Activated leukocyte membrane coating

Curcumin

DSS-induced mouse model of ulcerative colitis

Intravenous injection

Ulcerative colitis

[44]

Citrus

None

Nucleic acid

DDHD1-siRNA

SW480 colon cancer cell line

Co-incubation

Colon cancer

[63]

Acerola

None

ApoB-siRNA

Luciferase transgenic mouse

Oral administration

Effectively down-regulate target gene expression

[57]

Ginger

Lipid recombination; FA surface modification

Dmt1-siRNA

Intestine-specific Dmt1 knockout mouse; Hepc knockout mouse; double knockout mouse

Oral administration

Hereditary hemochromatosis

[18]

Grapefruit

Bionic synthesis; FA surface modification

miR17

GL-26 glioma orthotopic model in nude mice

Intranasal administration

Glioma

[46]

Grapefruit

None

Protein

HSP70

Human colon cancer HCT 116 and DLD-1 cells

Co-incubation

Colon cancer

[16]

Grapefruit

None

BSA

CD-1 IGS female mice

Intravenous injection

[16]

Grapefruit

None

HSP70

Glioma cell line (Gl-Tr)

Co-incubation

Glioma

[64]