金黄色葡萄球菌噬菌体脂质体的制备及胞内抗菌活性

doi:10.11843/j.issn.0366-6964.2025.09.041

摘要/Abstract

摘要：

本试验旨在探讨脂质体作为载体，包封递送噬菌体入胞及噬菌体脂质体对牛乳腺上皮细胞内金黄色葡萄球菌的抗菌作用。本试验采用薄膜水合法制备脂质体，用于包封噬菌体SDQ制备噬菌体脂质体，并测定其酸碱稳定性和热稳定性。使用激光粒度仪测定噬菌体脂质体粒径大小、多分散系数和Zeta电位，结合透射电镜观察噬菌体脂质体结构特征。采用CCK-8法测定噬菌体脂质体对MAC-T细胞活力的影响。通过平板计数法测定噬菌体脂质体入胞效率和胞内杀菌活性。最终，选择大豆卵磷脂、胆固醇、吐温80、十八胺以8∶2∶1∶0.5比例制备脂质体，包封噬菌体SDQ后制备噬菌体脂质体(Lip-p-7)。Lip-p-7包封率大于50%，在温度(4~60 ℃)和pH值(2~11)范围内活性稳定。Lip-p-7平均粒径为1 262.5 nm，Zeta电位从中性转向带负电荷，表明脂质体成功包封噬菌体SDQ；多分散系数(polydispersity index，PDI)小于0.7，表明其均匀分散。制备后的Lip-p-7储存于4 ℃条件下，可在90 d内保持完整活性，具有良好的稳定性。脂质体可有效提升噬菌体SDQ入胞数量，降低MAC-T细胞内金黄色葡萄球菌数量。综上，本试验为脂质体作为载体递送噬菌体入胞，为治疗胞内细菌感染提供参考。

关键词: 脂质体, 噬菌体, 金黄色葡萄球菌, 牛乳腺上皮细胞, 胞内抗菌活性

Abstract:

The aim of this study was to investigate the use of liposomes as a carrier to deliver phage into the cell and the antibacterial effect of phage liposomes on Staphylococcus aureus in bovine mammary epithelial cells. In this study, liposome-encapsulated phage SDQ was prepared using the thin-film hydration method. The acid-base stability and thermal stability of the phage liposomes were determined using the double-layer plate method. The particle size, polydispersity coefficient, and Zeta potential of phage liposomes were measured using a laser particle size analyzer, while the structural characteristics of the phage liposomes were observed through transmission electron microscopy. The effect of phage liposomes on the viability of MAC-T cells was determined using the CCK-8 assay. The efficiency of cell entry and the intracellular and bactericidal activity of phage liposomes were measured using the plate counting method. The results showed that liposomes made from soybean lecithin, cholesterol, Tween 80, and octadecylamine were prepared in a ratio of 8:2:1:0.5, and phage liposomes (Lip-p-7) were created by encapsulating phage SDQ. The encapsulation rate of Lip-p-7 was greater than 50%, and its activity is stable in the range of temperature (4-60 ℃) and pH value (2-11). The average particle size of Lip-p-7 was 1 262.5 nm, and the shift of Zeta potential from neutral to negatively charged signified the successful encapsulation of phage SDQ by the liposome. The polydispersity index (PDI) was less than 0.7, suggesting a uniform dispersion. When stored at 4 ℃, the prepared Lip-p-7 could maintain its intact activity for up to 90 days, demonstrating good stability. Liposomes can effectively increase the number of phage SDQ entering cells and decrease the number of Staphylococcus aureus in MAC-T cells. In conclusion, this study provides a reference for the treatment of intracellular bacterial infections by using liposomes as a carrier to deliver bacteriophages into the cells.

Key words: liposomes, bacteriophage, Staphylococcus aureus, MAC-T cells, intracellular antibacterial activity

中图分类号:

Q939.48

杨书博, 苑庆欣, 陈麒百, 王培, 高东阳, 李鹤, 宋军. 金黄色葡萄球菌噬菌体脂质体的制备及胞内抗菌活性[J]. 畜牧兽医学报, 2025, 56(9): 4638-4645.

YANG Shubo, YUAN Qingxin, CHEN Qibai, WANG Pei, GAO Dongyang, LI He, SONG Jun. Preparation and Intracellular Antibacterial Activity of Liposomes Encapsulating Staphylococcus aureus Bacteriophage[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4638-4645.

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参考文献 28

1	G ABRILA,G VILLAT,BARROS-VELÁZQUEZJ,et al.Staphylococcus aureus exotoxins and their detection in the dairy industry and mastitis[J].Toxins (Basel),2020,12(9):537. doi: 10.3390/toxins12090537
2	AHMAD-MANSOURN,LOUBETP,POUGETC,et al.Staphylococcus aureus toxins: An update on their pathogenic properties and potential treatments[J].Toxins (Basel),2021,13(10):677. doi: 10.3390/toxins13100677
3	YANL,YANGY,MAX,et al.Effect of two different drug-resistant Staphylococcus aureus strains on the physiological properties of MAC-T cells and their transcriptome analysis[J].Front Vet Sci,2022,9,818928. doi: 10.3389/fvets.2022.818928
4	ZHANGL,HOUX,SUNL,et al.Staphylococcus aureus bacteriophage suppresses LPS-induced inflammation in MAC-T bovine mammary epithelial cells[J].Front Microbiol,2018,9,1614. doi: 10.3389/fmicb.2018.01614
5	ZHUX Y,WANGM L,CAIM,et al.Protein expression profiles in exosomes of bovine mammary epithelial cell line MAC-T infected with Staphylococcus aureus[J].Appl Environ Microbiol,2023,89(4):e0174322. doi: 10.1128/aem.01743-22
6	GÓRSKIA,BORYSOWSKIJ,MIDZYBRODZKIR.Phage therapy: Towards a successful clinical trial[J].Antibiotics (Basel),2020,9(11):827. doi: 10.3390/antibiotics9110827
7	WUN,DAIJ,GUOM,et al.Pre-optimized phage therapy on secondary Acinetobacter baumannii infection in four critical COVID-19 patients[J].Emerg Microbes Infect,2021,10(1):612-618. doi: 10.1080/22221751.2021.1902754
8	DEDRICKR M,GUERRERO-BUSTAMANTEC A,GARLENAR A,et al.Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus[J].Nat Med,2019,25(5):730-733. doi: 10.1038/s41591-019-0437-z
9	BODNERK,MELKONIANA L,COVERTM W.The enemy of my enemy: New insights regarding bacteriophage-mammalian cell interactions[J].Trends Microbiol,2021,29(6):528-541. doi: 10.1016/j.tim.2020.10.014
10	彭颖,雷晓萌,吴玉欢,等.维生素A修饰柴胡皂苷a/d脂质体的制备及体内外释药评价[J].中国中医基础医学杂志,2024,30(10):1751-1760.
	PENGY,LEIX M,WUY H,et al.Preparation and in vitro drug release evaluation of vitamin A-modified saikosaponin a/d liposomes[J].Journal of Basic Chinese Medicine,2024,30(10):1751-1760.
11	叶钰滢,王鸿鑫,刘栩沂,等.黄芪多糖脂质体的制备及其在Caco-2细胞模型的转运特性研究[J].中国畜牧兽医,2024,51(10):4606-4615.
	YEY Y,WANGH X,LIUZ J,et al.Preparation of astragalus polysaccharide liposomes and its transport characteristics in Caco-2 cell model[J].China Animal Husbandry & Veterinary Medicine,2024,51(10):4606-4615.
12	ZYLBERBERGC,MATOSEVICS.Pharmaceutical liposomal drug delivery: a review of new delivery systems and a look at the regulatory landscape[J].Drug Deliv,2016,23(9):3319-3329. doi: 10.1080/10717544.2016.1177136
13	SONGJ,RUANH,CHENL,et al.Potential of bacteriophages as disinfectants to control of Staphylococcus aureus biofilms[J].BMC Microbiol,2021,21(1):57. doi: 10.1186/s12866-021-02117-1
14	NINGH,ZHANGJ,ZHAOQ,et al.Development of the phage lysin-loaded liposomes as preservatives for live clams[J].Int J Food Microbiol,2023,387,110059. doi: 10.1016/j.ijfoodmicro.2022.110059
15	郭少波,吴迎花,陈惠惠,等.超小粒径的纳米Ag协同Cu基铁氧体复合材料的制备及其对金黄色葡萄球菌抑菌机制研究[J].中国科学: 生命科学,2024,54(2):338-348.
	GUOS B,WUY H,CHENH H,et al.Preparation of ultrasmall-sized Ag in synergy with Cu-based ferrite composite and its antibacterial activity against Staphylococcus aureus[J].Scientia Sinica(Vitae),2024,54(2):338-348.
16	ZHANGL,SUNL,WEIR,et al.Intracellular Staphylococcus aureus control by virulent bacteriophages within MAC-T bovine mammary epithelial cells[J].Antimicrob Agents Chemother,2017,61(2):e01990-16.
17	KAMALF,DENNISJ J.Burkholderia cepacia complex phage-antibiotic synergy (PAS): antibiotics stimulate lytic phage activity[J].Appl Environ Microbiol,2015,81(3):1132-1138. doi: 10.1128/AEM.02850-14
18	KWIATEKM,PARASIONS,NAKONIECZNAA.Therapeutic bacteriophages as a rescue treatment for drug-resistant infections-an in vivo studies overview[J].J Appl Microbiol,2020,128(4):985-1002. doi: 10.1111/jam.14535
19	崇倩,赵学慧,曹青,等.产单核细胞李氏杆菌噬菌体的应用研究进展[J].畜牧兽医学报,2024,55(5):1819-1826. doi: 10.11843/j.issn.0366-6964.2024.05.001
	CHONGQ,ZHAOX H,CAOQ,et al.Research progress of applied research on Listeria monocytogenes phage[J].Acta Veterinaria et Zootechnica Sinica,2024,55(5):1819-1826. doi: 10.11843/j.issn.0366-6964.2024.05.001
20	HODYRA-STEFANIAKK,MIERNIKIEWICZP,DRAPAŁAJ,et al.Mammalian host-versus-phage immune response determines phage fate in vivo[J].Sci Rep,2015,5,14802. doi: 10.1038/srep14802
21	TIANY,WUM,LIUX,et al.Probing the endocytic pathways of the filamentous bacteriophage in live cells using ratiometric pH fluorescent indicator[J].Adv Healthc Mater,2015,4(3):413-419. doi: 10.1002/adhm.201400508
22	NGUYENS,BAKERK,PADMANB S,et al.Bacteriophage transcytosis provides a mechanism to cross epithelial cell layers[J].mBio,2017,8(6):e01874-17.
23	ALMEIDAB,NAGO K,ROGERSK E,et al.Recent progress in bioconjugation strategies for liposome-mediated drug delivery[J].Molecules,2020,25(23):5672. doi: 10.3390/molecules25235672
24	王震,刘昱璇,张梦奇,等.包埋青蛤免疫活性肽脂质体的制备及性质表征[J].食品科学技术学报,2024,42(5):104-114.
	WANGZ,LIUY X,ZHANGM Q,et al.Preparation and characterization of liposomes encapsulating Cyclina sinensis immunoreactive peptides[J].Journal of Food Science and Technology,2024,42(5):104-114.
25	SHAHS,DHAWANV,HOLMR,et al.Liposomes: Advancements and innovation in the manufacturing process[J].Adv Drug Deliv Rev,2020,154-155,102-122. doi: 10.1016/j.addr.2020.07.002
26	曹启栋,亢淑梅,鲁宸硕,等.十八胺微胶囊搭载环氧涂层的制备及耐蚀机理研究[J].电镀与精饰,2024,46(6):25-33.
	CAOQ D,KANGS M,LUC S,et al.Properties and corrosion resistance mechanism of octadecylamine microcapsules loaded with epoxy resin[J].Plating and Finishing,2024,46(6):25-33.
27	CUIH,YUANL,LINL.Novel chitosan film embedded with liposome-encapsulated phage for biocontrol of Escherichia coli O157:H7 in beef[J].Carbohydr Polym,2017,177,156-164. doi: 10.1016/j.carbpol.2017.08.137
28	SINGLAS,HARJAIK,KATAREO P,et al.Encapsulation of bacteriophage in liposome accentuates its entry in to macrophage and shields it from neutralizing antibodies[J].PLoS One,2016,11(4):e0153777. doi: 10.1371/journal.pone.0153777

样本 Sample	大豆卵磷脂: 胆固醇: 吐温80:十八胺 PC: Chol: Tw-80: ODA	温度/℃ Temperature	酸碱度 pH value	包封率/% Encapsulation rate
Lip-p-1	6:4:2:0	4~70	4~10	10.98
Lip-p-2	8:4:2:0	4~60	3~10	22.66
Lip-p-3	8:4:2:0.5	4~70	3~11	35.51
Lip-p-4	6:4:2:0.5	4~50	2~10	23.65
Lip-p-5	6:2:1:0.5	4~60	2~11	38.72
Lip-p-6	8:2:1:1	4~50	3~11	53.28
Lip-p-7	8:2:1:0.5	4~60	2~11	50.37
Lip-p-8	6:1:1:1	4~60	3~11	19.73
Lip-p-9	8:1:1:1	4~60	3~11	22.48

脂质体 Liposomes	平均粒径(${\bar x}$±s)/nm Average particle size /nm	多分散系数 Polydispersity coefficient	Zeta电位(${\bar x}$±s)/mV Zeta potential /mV
Lip	656.75±127.40	0.505	44.40±9.38
Lip-p-7	1 262.50± 200.70	0.675	-13.70±7.20

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