Preparation of Lip-TTO-PHMB and evaluation of its in vitro antibacterial activity and biocompatibility
-
摘要:
目的 制备卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物（Lip-TTO-PHMB）,探讨其体外抗菌活性及生物相容性。 方法 该研究为包含成组设计、析因设计及重复测量设计的实验研究。采用薄膜水合法制备Lip-TTO-PHMB。通过单因素实验结合Box-Behnken响应面法明确Lip-TTO-PHMB的最优制备条件为卵磷脂与胆固醇质量比3.77∶1.00、茶树精油体积分数0.64%、聚山梨酯80体积分数0.13%,模型预测包封率为51.035%。按最优制备条件制备Lip-TTO-PHMB,采用超速离心紫外法测量其包封率。取小鼠成纤维细胞L929,分为分别采用卵磷脂脂质体-聚六亚甲基双胍盐酸盐（Lip-PHMB）、Lip-TTO-PHMB、聚六亚甲基双胍盐酸盐（PHMB）处理的Lip-PHMB组、Lip-TTO-PHMB组、PHMB组,均依次将其中的PHMB终质量浓度调整为62.5、31.3、15.6 μg/L,培养24 h后,采用噻唑蓝法检测细胞存活率；另取L929细胞,同前分组并采用相应材料处理（PHMB终质量浓度均为15.0 μg/L）,培养24 h后,采用活/死细胞染色法检测细胞存活率。前述实验样本数均为4。取人永生化角质形成细胞（HaCaT细胞）,分为仅用无血清MEM培养基培养的对照组及分别用PHMB、Lip-TTO-PHMB处理的PHMB组和Lip-TTO-PHMB组（PHMB终质量浓度均为15.0 μg/L）,行划痕试验检测划痕后24、48 h的细胞迁移率（样本数为3）。采用微量肉汤二倍稀释法测定PHMB对金黄色葡萄球菌ATCC 6538和大肠埃希菌ATCC 8739的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）,样本数为5。 结果 Lip-TTO-PHMB的实测包封率为（52.35±0.24）%,与模型预测包封率相近。噻唑蓝法检测结果显示,培养24 h后,PHMB终质量浓度为62.5、31.3、15.6 μg/L时,Lip-PHMB组与Lip-TTO-PHMB组L929细胞存活率均显著高于PHMB组（P<0.05）,Lip-PHMB组L929细胞存活率均显著高于Lip-TTO-PHMB组（P<0.05）。活/死细胞染色法检测结果显示,培养24 h后,PHMB组L929细胞存活率显著低于Lip-PHMB组和Lip-TTO-PHMB组（P值均<0.05）。划痕后24、48 h,Lip-TTO-PHMB组HaCaT细胞迁移率均显著高于PHMB组（P<0.05）；划痕后48 h,对照组HaCaT细胞迁移率显著高于PHMB组（P<0.05）。Lip-PHMB、Lip-TTO-PHMB及PHMB对金黄色葡萄球菌的MIC分别为31.3、15.6、15.6 μg/mL,对大肠埃希菌的MIC分别为31.3、15.6、7.8 μg/mL。Lip-PHMB、Lip-TTO-PHMB及PHMB对金黄色葡萄球菌的MBC分别为20、10、15 μg/mL,对大肠埃希菌的MBC分别为30、20、20 μg/mL。 结论 成功制备Lip-TTO-PHMB,该材料在保留优异抗菌活性的同时,具备良好生物相容性。 Abstract:Objective To prepare a lecithin liposome-tea tree oil-polyhexamethylene biguanide hydrochloride complex (Lip-TTO-PHMB) and to investigate its in vitro antibacterial activity and biocompatibility. Methods This study was an experimental research including group design, factorial design, and repeated measurement design. Lip-TTO-PHMB was prepared by the thin-film hydration method. Through single-factor experiments combined with the Box-Behnken response surface methodology, the optimal preparation condition for Lip-TTO-PHMB was determined as follows: mass ratio of lecithin to cholesterol of 3.77: 1.00, volume fraction of tea tree oil of 0.64%, and volume fraction of polysorbate 80 of 0.13%. The model-predicted encapsulation efficiency was 51.035%. Lip-TTO-PHMB was prepared under the optimal preparation condition and its encapsulation efficiency was measured. Mouse fibroblasts L929 were divided into lecithin liposome-polyhexamethylene biguanide hydrochloride (Lip-PHMB) group, Lip-TTO-PHMB group, and polyhexamethylene biguanide hydrochloride (PHMB) group treated with the corresponding materials, with the final mass concentrations of PHMB adjusted to 62.5, 31.3, and 15.6 μg/L, and the cell viability was detected by the thiazolyl blue tetrazolium bromide assay. Another batch of L929 cells were divided into the same groups as before and treated with the corresponding materials with the final mass concentration of PHMB at 15.0 μg/L, and the cell viability was detected by live/dead cell staining. The sample size for the above experiments was 4. Human immortalized keratinocytes (HaCaT) were divided into control group cultured with serum-free MEM medium, and PHMB group and Lip-TTO-PHMB group treated with the corresponding materials with the final mass concentration of PHMB at 15.0 μg/L. The scratch test was performed to detect the cell migration rates at 24 and 48 h after scratch (with the sample size of 3). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PHMB against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739 were determined by the broth microdilution twofold dilution method, with the sample size of 5. Results The measured encapsulation efficiency of Lip-TTO-PHMB was (52.35±0.24)%, which was close to the model-predicted value. After 24 h of culture, at PHMB final mass concentrations of 62.5, 31.3, and 15.6 μg/L, the viability of L929 cells in Lip-PHMB group and Lip-TTO-PHMB group was significantly higher than that in PHMB group (P<0.05), and the viability of L929 cells in Lip-PHMB group was significantly higher than that in Lip-TTO-PHMB group (P<0.05). After 24 h of culture, the viability of L929 cells in PHMB group was significantly lower than that in Lip-PHMB group and Lip-TTO-PHMB group (with P values both <0.05). At 24 and 48 h after scratch, the migration rates of HaCaT cells in Lip-TTO-PHMB group were significantly higher than those in PHMB group (P<0.05); at 48 h after scratch, the migration rate of HaCaT cells in control group was significantly higher than that in PHMB group (P<0.05). The MICs of Lip-PHMB, Lip-TTO-PHMB, and PHMB against Staphylococcus aureus were 31.3, 15.6, and 15.6 μg/mL, respectively, and those against Escherichia coli were 31.3, 15.6, and 7.8 μg/mL, respectively. The MBCs of Lip-PHMB, Lip-TTO-PHMB, and PHMB against Staphylococcus aureus were 20, 10, and 15 μg/mL, respectively, and those against Escherichia coli were 30, 20, and 20 μg/mL, respectively. Conclusions The prepared Lip-TTO-PHMB can retain excellent antibacterial activity and possess favorable biocompatibility. -
参考文献
(44) [1] Antimicrobial Resistance Collaborators.Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis[J].Lancet,2022,399(10325):629-655.DOI: 10.1016/S0140-6736(21)02724-0. [2] YeC, LiW, YangY, et al. Inappropriate use of antibiotics exacerbates inflammation through OMV-induced pyroptosis in MDR Klebsiella pneumoniae infection[J]. Cell Rep, 2021, 36(12):109750. DOI: 10.1016/j.celrep.2021.109750. [3] DeusenberyC,WangY,ShuklaA.Recent innovations in bacterial infection detection and treatment[J].ACS Infect Dis,2021,7(4):695-720.DOI: 10.1021/acsinfecdis.0c00890. [4] IslamMF, ArkaPB, RohmanM, et al. Pooling the complex survey data across the 64 lower and middle-income countries: a study on antibiotic usage in under-five children[J]. Heliyon, 2024, 11(1):e41470. DOI: 10.1016/j.heliyon.2024.e41470. [5] 孟青,周林涛,陈运生,等.2015-2021年CHINET儿童患者临床分离菌的分布特征和耐药性变迁[J].中国感染与化疗杂志,2025,25(1):48-58.DOI: 10.16718/j.1009-7708.2025.01.008. [6] 金剑 PHMB修饰的人工真皮的抗菌能力检测 上海 海军军医大学 2018 金剑. PHMB修饰的人工真皮的抗菌能力检测[D]. 上海:海军军医大学,2018.
[7] SchuchLF,KirschnickLB,de ArrudaJAA,et al.Malignant peripheral nerve sheath tumour of the oral and maxillofacial region-a systematic review[J].Oral Dis,2022,28(8):2072-2082.DOI: 10.1111/odi.13982. [8] WorsleyA,VassilevaK,TsuiJ,et al.Polyhexamethylene biguanide:polyurethane blend nanofibrous membranes for wound infection control[J].Polymers (Basel),2019,11(5):915.DOI: 10.3390/polym11050915. [9] AhaniE, MontazerM, MianehroA, et al. Preparation of long-lasting antibacterial wound dressing through diffusion of cationic-liposome-encapsulated polyhexamethylene biguanide [J]. React Funct Polym, 2021, 169: 105092. DOI: 10.1016/j.reactfunctpolym.2021.105092. [10] 赵玉浩,蒋恩社,刘梦瑶,等.含聚己双胍抗菌剂在慢性伤口治疗与护理中的应用研究进展[J].中华医院感染学杂志,2025,35(21):3355-3360.DOI: 10.11816/cn.ni.2025-246665. [11] Sowlati-HashjinS,CarboneP,KarttunenM.Insights into the polyhexamethylene biguanide (PHMB) mechanism of action on bacterial membrane and DNA: a molecular dynamics study[J].J Phys Chem B,2020,124(22):4487-4497.DOI: 10.1021/acs.jpcb.0c02609. [12] 杨娜,杨彬,孙文魁,等.皮肤消毒剂开发应用研究进展[J].中国消毒学杂志,2018,35(4):297-300.DOI: 10.11726/j.issn.1001-7658.2018.04.018. [13] 王奎涛,王玉亚,任欢欢,等. 聚六亚甲基双胍消毒剂含量测定方法及消毒效果的研究[J]. 应用化工,2019,48(6):1506-1509. DOI: 10.3969/j.issn.1671-3206.2019.06.056. [14] 俞鑫,王超,夏建明.聚六亚甲基双胍和季铵盐在羊毛抗菌整理中的应用[J].印染,2024,50(5):51-55.DOI: 10.3969/j.yinran.202405006. [15] BaggaB,GowthamL,AhirwarLK,et al.Outcome of photodynamic therapy with Rose Bengal in conjunction with topical PHMB and chlorhexidine combination in Acanthamoeba keratitis[J].J Ophthalmic Inflamm Infect,2025,15(1):18.DOI: 10.1186/s12348-025-00466-w. [16] FranchA,KnutssonKA,PedrottiE,et al.Treatment of Acanthamoeba keratitis with high dose PHMB (0.08%) monotherapy in clinical practice: a case series[J].Eur J Ophthalmol,2025,35(4):1235-1241.DOI: 10.1177/11206721241299470. [17] WangWY, HuHW, ChiouJC, et al. Poly(hexamethylene biguanide) hydrochloride (PHMB)-based materials: synthesis, modification, properties, determination, and application [J]. Polymer Chemistry, 2023, 14(48): 5226. DOI: 10.1039/d3py01148h [18] SzokaFJr,PapahadjopoulosD.Comparative properties and methods of preparation of lipid vesicles (liposomes)[J].Annu Rev Biophys Bioeng,1980,9:467-508.DOI: 10.1146/annurev.bb.09.060180.002343. [19] PardiN,HoganMJ,PorterFW,et al.mRNA vaccines - a new era in vaccinology[J].Nat Rev Drug Discov,2018,17(4):261-279.DOI: 10.1038/nrd.2017.243. [20] WeissigV. From olive oil emulsions to COVID-19 vaccines: liposomes came first [M].New York: Humana,2023: 1-19. DOI: 10.1007/978-1-0716-2954-3_1. [21] ŠturmL,Poklar UlrihN.Basic methods for preparation of liposomes and studying their interactions with different compounds, with the emphasis on polyphenols[J].Int J Mol Sci,2021,22(12):6547.DOI: 10.3390/ijms22126547. [22] YangJ, BoerJC, KhongkowM, et al. The development of surface-modified liposomes as an intranasal delivery system for group a streptococcus vaccines[J]. Vaccines (Basel), 2023, 11(2):305. DOI: 10.3390/vaccines11020305. [23] ThiTTH,SuysEJA,LeeJS,et al.Lipid-based nanoparticles in the clinic and clinical trials: from cancer nanomedicine to COVID-19 vaccines[J].Vaccines (Basel),2021,9(4):359.DOI: 10.3390/vaccines9040359. [24] HouX,ZaksT,LangerR,et al.Lipid nanoparticles for mRNA delivery[J].Nat Rev Mater,2021,6(12):1078-1094.DOI: 10.1038/s41578-021-00358-0. [25] GhoshR,DeM.Liposome-based antibacterial delivery: an emergent approach to combat bacterial infections[J].ACS Omega,2023,8(39):35442-35451.DOI: 10.1021/acsomega.3c04893. [26] 孙艺文 基于自组装纳米颗粒增强的纤维素基敷料抗菌止血性能研究 淄博 山东理工大学 2025 孙艺文. 基于自组装纳米颗粒增强的纤维素基敷料抗菌止血性能研究[D]. 淄博:山东理工大学,2025.
[27] LamNS,LongX,SuXZ,et al.Melaleuca alternifolia (tea tree) oil and its monoterpene constituents in treating protozoan and helminthic infections[J].Biomed Pharmacother,2020,130:110624.DOI: 10.1016/j.biopha.2020.110624. [28] YuanX,CaoD,XiangY,et al.Antifungal activity of essential oils and their potential synergistic effect with amphotericin B[J].Sci Rep,2024,14(1):31125.DOI: 10.1038/s41598-024-82380-0. [29] 李炳霞,张亮.茶树精油的提取及抗耐甲氧西林金黄色葡萄球菌(MRSA)的活性研究[J].山东畜牧兽医,2021,42(6):11-13.DOI: 10.3969/j.issn.1007-1733.2021.06.004. [30] 张瑜,何华名,何聪芬.植物精油抗病毒研究概况[J].中国野生植物资源,2021,40(7):48-54.DOI: 10.3969/j.issn.1006-9690.2021.07.010. [31] 吴克刚,黄洁虹,柴向华,等.植物精油气相清除DPPH自由基的研究[J].中国食品添加剂,2013(3):110-113.DOI: 10.3969/j.issn.1006-2513.2013.03.011. [32] CulasSM, KaurL, PopovichDG, et al. Physicochemical properties, colloidal stability, and encapsulation efficiency of lecithin-based and chitosan-coated liposomes loaded with Cinnamomum zeylanicum bioactives [J]. Appl Sci, 2026, 16(8):3754. DOI: 10.3390/app16083754. [33] GadaniM, BadakS, UpadhyayR. Sunflower lecithin-based liposomal formulations of sesbania grandiflora and acerola cherry extracts: enhancing stability and controlled release [J]. International Journal of Biochemistry Research & Review, 2025, 34(5): 186-198. DOI: 10.9734/ijbcrr/2025/v34i51049. [34] ManY,TangH,WangY,et al.Casein-liposome complex loading basil essential oil: preparation, antibacterial effect, and preservative effect on braised beef[J].Food Chem,2026,511:148807.DOI: 10.1016/j.foodchem.2026.148807. [35] ZamanW,UllahN,AminA.Rationally engineered essential oil-loaded nanocarriers for acne vulgaris: integrating multiscale molecular modeling, machine learning, and response surface optimization[J].J Microencapsul,2026,43(3):282-313.DOI: 10.1080/02652048.2026.2695092. [36] KalantariM, AziziK, ShahheidariR, et al. A stable nanoliposomal carboxymethyl cellulose hydrogel of Pelargonium essential oil: formulation, characterization, and enhanced repellent activity against Anopheles stephensi[J]. Parasite Epidemiol Control, 2026, 33:e00490. DOI: 10.1016/j.parepi.2026.e00490. [37] 王洁,陆俊梅,黄晓辰,等.微量MIC检测判断结核分枝杆菌药敏的方法学研究[J].中华检验医学杂志,2010,33(4):315-319.DOI: 10.3760/cma.j.issn.1009-9158.2010.04.006. [38] AhaniE, MontazerM, MianehroA, et al. Encapsulation of the PHMB with nanoliposome and attachment to wound dressing for long-term antibacterial activity and biocompatibility[J]. World J Microbiol Biotechnol, 2024,40(11):361. DOI: 10.1007/s11274-024-04170-0. [39] NguyenHL,NguyenHMX,NguyenTBN.Nano-enabled advances in tea tree essential oil (Melaleuca alternifolia): composition, bioactivity, and emerging roles in food protection[J].Materials (Basel),2026,19(13):2915.DOI: 10.3390/ma19132915. [40] WangH,ZhangH,WangJ,et al.Interfacial co-assembly of CO2-responsive rosin-based surfactant and CNF in gel emulsions for encapsulation and release of tea tree oil[J].Food Chem,2026,509:148580.DOI: 10.1016/j.foodchem.2026.148580. [41] LinB, WeiW, CaoC, et al. Mechanistic analysis for the inhibition of Staphylococcus aureus by terpinen-4-ol[J]. Nat Prod Commun, 2026, 21(1):1-13. DOI: 10.1177/1934578X251414816. [42] de AssisKMA,deA Rêgo RI,de MeloDF,et al.Therapeutic potential of Melaleuca alternifolia essential oil in new drug delivery systems[J].Curr Pharm Des,2020,26(33):4048-4055.DOI: 10.2174/1381612826666200305124041. [43] AlegunO,PandeyaA,CuiJ,et al.Donnan potential across the outer membrane of Gram-negative bacteria and its effect on the permeability of antibiotics[J].Antibiotics (Basel),2021,10(6):701.DOI: 10.3390/antibiotics10060701. [44] YousefiR, RezaeeY, BayatF, et al. Molecular docking, molecular dynamics simulation, preparation, and characterization of naltrexone-phospholipid complex: a novel cargo with improved loading into multivesicular liposomes [J]. Results in Chemistry, 2024, 9: 101656. DOI: 10.1016/j.rechem.2024.101656. -
图 1 空白卵磷脂脂质体和Lip-TTO-PHMB的粒径分布。1A.空白卵磷脂脂质体；1B.Lip-TTO-PHMB
注：Lip-TTO-PHMB为卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物；该图为横坐标数据经过lg处理后形成的描记图
图 2 Lip-TTO-PHMB的微观形貌及3种材料的傅里叶变换红外光谱。2A.Lip-TTO-PHMB的微观形貌 透射电子显微镜 ×50 000；2B.3种材料的红外光谱
注：Lip-TTO-PHMB为卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物,PHMB为聚六亚甲基双胍盐酸盐,Lip-PHMB为卵磷脂脂质体-PHMB
图 3 3组小鼠成纤维细胞L929培养24 h经含不同终质量浓度PHMB的材料处理后的细胞存活情况。3A、3B、3C.分别为用PHMB终质量浓度为62.5、31.3、15.6 μg/L的Lip-PHMB处理的Lip-PHMB组 噻唑蓝 ×100；3D、3E、3F.分别为用PHMB终质量浓度为62.5、31.3、15.6 μg/L的Lip-TTO-PHMB处理的Lip-TTO-PHMB组,图3A、3B、3C存活细胞多于图3D、3E、3F 噻唑蓝 ×100；3G、3H、3I.分别为用终质量浓度为62.5、31.3、15.6 μg/L的PHMB处理的PHMB组,图3A、3B、3C与图3D、3E、3F存活细胞多于图3G、3H、3I 噻唑蓝 ×100；3J.细胞存活率条图（
注：PHMB为聚六亚甲基双胍盐酸盐；分组因素主效应,F=151.206,P<0.001；浓度因素主效应,F=36.667,P<0.001；两者交互效应,F=33.277,P<0.001；与卵磷脂脂质体-PHMB（Lip-PHMB）组比较,aP<0.05；与卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物（Lip-TTO-PHMB）组比较,bP<0.05
图 4 3组小鼠成纤维细胞L929培养24 h后细胞存活情况。4A、4B、4C.分别为Lip-PHMB组、Lip-TTO-PHMB组、PHMB组,图4B中活细胞数与图4A相近,图4C中活细胞数显著少于图4A、4B 钙黄绿素-乙酰氧基甲酯/碘化丙啶 ×20；4D.细胞存活率条图（
注：卵磷脂脂质体-聚六亚甲基双胍盐酸盐（Lip-PHMB）组、卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物（Lip-TTO-PHMB）组、聚六亚甲基双胍盐酸盐（PHMB）组细胞分别用PHMB终质量浓度均为15.0 μg/L的Lip-PHMB、Lip-TTO-PHMB、PHMB处理；3组细胞存活率总体比较,差异有统计学意义（F=18.289,P=0.001）；与Lip-PHMB组比较,aP<0.05；与Lip-TTO-PHMB组比较,bP<0.05
图 5 3组HaCaT细胞划痕后各时间点迁移情况。5A、5B、5C.分别为对照组划痕后0（即刻）、24、48 h 倒置显微镜 ×200；5D、5E、5F.分别为PHMB组划痕后0、24、48 h 倒置显微镜 ×200；5G、5H、5I.分别为Lip-TTO-PHMB组划痕后0、24、48 h,图5C剩余划痕面积小于图5F,图5H、5I剩余划痕面积分别小于图5E、5F 倒置显微镜 ×200；5J.细胞迁移率条图（
注：对照组细胞仅用无血清MEM培养基培养,聚六亚甲基双胍盐酸盐（PHMB）组和卵磷脂脂质体-茶树精油-聚六亚甲基双胍盐酸盐复合物（Lip-TTO-PHMB）组细胞分别用PHMB终质量浓度为15.0 μg/L的PHMB和Lip-TTO-PHMB培养；分组因素主效应,F=28.166,P=0.013；时间因素主效应,F=23.838,P=0.014；两者交互效应,F=2.608,P=0.221；与PHMB组比较,aP<0.05；与对照组比较,bP<0.05
-
下载:
图(6)
计量
- 文章访问数: 4
- HTML全文浏览量: 4
- PDF下载量: 1
- 被引次数: 0
