首页 > 分享 > 松萝酸纳米胶束的构建及其抗菌活性与药代动力学特征

松萝酸纳米胶束的构建及其抗菌活性与药代动力学特征

花匠小妙招
2025-02-02 14:37

摘要: 松萝酸(usnic acid, UA)具有优良的抗菌活性，但水溶性差、生物利用度低极大地限制了其临床应用。本研究采用薄膜水合法利用聚己内酯-聚乙二醇(polycaprolactone-polyethylene glycol, PCL-PEG)高分子材料构建UA胶束，利用单因素试验及Box-Behnken响应面法优化制备条件，从而提高UA的水溶性及生物利用度。表征及评价UA胶束的粒径、zeta电位、微观形貌、包埋效果、水溶性、体外释药、稳定性及安全性。并明确UA胶束对四种常见细菌的体外抗菌活性及其对金黄色葡萄球菌感染小鼠的体内治疗效果。考察UA胶束在大鼠体内的药代动力学特征。结果显示制得的UA胶束呈现为大小均匀、表面光滑的球体，粒径为120.4 nm，zeta电位为-7.8 mV，包封率与载药量分别为80.66%和9.47%，且UA被成功包埋在胶束的疏水性内核中，水溶性提高19.11倍。UA胶束具备一定的缓释性能，48 h药物累计释放度为76.26%，在4℃贮存60天后，稳定性良好，用药安全性良好，对多种常见细菌均具有一定抗菌活性，且UA胶束对金黄色葡萄球菌感染小鼠的治疗效果比UA原药更好。与UA原药相比，UA胶束在大鼠体内的相对生物利用度提高156%，Cmax提高1.34倍，半衰期延长21.38%，清除率降低32.39%。本研究可为UA胶束的临床应用提供一定理论依据。

Abstract: Usnic acid (UA) has excellent antibacterial activity, but its poor water solubility and low bioavailability greatly limit its clinical application. In this study, polycaprolactone-polyethylene glycol (PCL-PEG) polymer material was used to construct UA micelles by film dispersion method. Single-factor test and Box-Behnken response surface method were used to optimize the preparation conditions, so as to improve the water solubility and bioavailability of UA. The particle size, zeta potential, microstructure, embedding effect, water solubility, drug release in vitro, stability and safety of UA micelles were characterized. The antibacterial activity of UA micelles against four common bacteria in vitro and the therapeutic effect on Staphylococcus aureus infected mice in vivo were investigated. The pharmacokinetic characteristics of UA micelles in rats were clarified. The results show that UA micelles are uniform size and smooth surface spheres with a particle size of 120.4 nm, zeta potential of −7.8 mV, encapsulation rate and drug loading of 80.66% and 9.47%, respectively. UA is successfully embedded in the hydrophobic core of the micelles, and the water solubility is increased by 19.11 times. UA micelles have a certain slow-release performance, and the cumulative drug release rate is 76.26% after 48 h. After 60 days of storage at 4℃, it shows good stability and good drug safety, and has certain antibacterial activity against many common bacteria. In addition, the therapeutic effect of UA micelles on Staphylococcus aureus infected mice is better than that of the original UA drug. Compared with the original UA drug, the relative bioavailability of UA micelles in rats is improved by 156%, Cmax of UA micelles is increased by 1.34 times, the half-life of UA micelles is extended by 21.38%, and clearance rate of UA micelles is decreased by 32.39%. This study can provide a theoretical basis for the clinical application of UA micelles.

图 1 190~600 nm 全波长扫描结果(a)及 UA 标准曲线(b)

Figure 1. Full wavelength scanning results from 190 to 600 nm (a) and standard curve of UA (b)

图 2 四种因素对UA胶束包封率影响的交互作用

Figure 2. Interaction of the influence of four factors on the encapsulation efficiency of UA micelles

图 3 四种因素对UA胶束载药量影响的交互作用

Figure 3. Interaction of the influence of four factors on the drug loading of UA micelles

图 4 UA胶束的部分表征结果

(a) UA胶束粒径分布图；(b) TEM观察UA胶束的微观形貌(2.5 k×)；(c) 1H NMR结果；(d) 红外光谱测定结果；(e) UA胶束体外释放行为；(f) UA胶束溶血试验结果

Figure 4. Partial characterization of UA micelles

(a) Particle size distribution of UA micelles; (b) The morphology of UA micelles observed by TEM; (c) 1H NMR results; (d) Infrared spectroscopic results; (e) In vitro release behavior of UA micelles; (f) Hemolysis test results of UA micelles

图 5 金黄色葡萄球菌感染小鼠不同脏器的菌落数

Figure 5. Number of colonies in different organs of mice infected by Staphylococcus aureus

图 6 金黄色葡萄球菌感染小鼠的肺脏、脾脏组织病理学改变情况(200×)

Figure 6. Histopathological changes of lung and spleen in mice infected by Staphylococcus aureus (200×)

图 7 HPLC法检测血浆中UA的结果及血浆药物浓度-时间曲线

(a) 不同血浆样品的HPLC色谱图；(b) 标准曲线；(c) 血浆药物浓度-时间曲线

Figure 7. Determination of UA in plasma by HPLC and plasma drug concentration-time curve

(a) HPLC chromatograms of different plasma samples; (b) Standard curve; (c) Plasma drug concentration-time curve

表 1 松萝酸(UA)的HPLC检测梯度洗脱程序

Table 1 HPLC gradient elution procedure for detection of usnic acid (UA)

Time/min Methanol 15 mmol/L KH2PO4∶triethylamine=500∶1 0 60% 40% 3 70% 30% 7 75% 25% 15 60% 40%

表 2 Box-Behnken响应面法的试验设计及结果

Table 2 Experimental design and results of Box-Behnken response surface design

Formula PBS volume UA dosage Trichloromethane :
Methanol PBS pH value Encapsulation
Efficiency/% Drug loading/% 1 25 mL 3 mg 1∶1 8.0 72.25 8.39 2 25 mL 3 mg 2∶1 7.4 68.71 8.03 3 25 mL 5 mg 2∶1 7.4 72.35 8.9 4 25 mL 3 mg 1∶1 6.5 42.55 6.21 5 25 mL 4 mg 1∶2 6.5 47.36 6.33 6 20 mL 4 mg 2∶1 7.4 74.54 8.76 7 30 mL 3 mg 1∶1 7.4 67.4 7.98 8 25 mL 3 mg 1∶2 7.4 72.72 8.43 9 25 mL 5 mg 1∶2 7.4 75.48 9.02 10 20 mL 3 mg 1∶1 7.4 68.7 8.02 11 25 mL 4 mg 2∶1 6.5 48.62 6.66 12 20 mL 4 mg 1∶1 8.0 72.01 8.71 13 25 mL 4 mg 1∶1 7.4 79.59 9.36 14 30 mL 4 mg 1∶1 8.0 66.18 8.44 15 20 mL 5 mg 1∶1 7.4 70.37 8.62 16 30 mL 4 mg 1∶1 6.5 49.45 6.70 17 30 mL 4 mg 1∶2 7.4 70.35 8.56 18 20 mL 4 mg 1∶2 7.4 72.16 8.68 19 20 mL 4 mg 1∶1 6.5 43.55 6.28 20 25 mL 4 mg 1∶1 7.4 78.4 9.25 21 25 mL 5 mg 1∶1 6.5 49.92 6.98 22 25 mL 4 mg 1∶1 7.4 78.62 9.27 23 25 mL 4 mg 1∶2 8.0 73.33 8.69 24 25 mL 5 mg 1∶1 8.0 67.67 8.59 25 25 mL 4 mg 2∶1 8.0 71.04 8.62 26 30 mL 4 mg 2∶1 7.4 72.97 8.72 27 30 mL 5 mg 1∶1 7.4 70.26 8.60 28 25 mL 4 mg 1∶1 7.4 77.48 9.11 29 25 mL 4 mg 1∶1 7.4 78.31 9.24

表 3 UA胶束在不同温度下避光贮存不同时间的包封率和载药量

Table 3 Encapsulation efficiency and drug loading of UA micelles stored at different temperatures for different times and away from light

Storage temperature 0 day 15 day 30 day 45 day 60 day Encapsulation efficiency/% 4℃ 80.60±0.31 72.88±0.78 68.13±1.76 64.72±2.06 61.11±3.36 25℃ 80.60±0.31 65.81±1.13 60.06±1.77 58.64±2.31 55.90±2.89 Drug loading/% 4℃ 9.10±0.35 8.43±0.92 7.96±1.12 7.46±1.43 7.17±1.19 25℃ 9.10±0.35 7.53±0.66 7.01±2.02 6.77±0.62 6.60±1.15

表 4 UA胶束和UA原药对四种细菌的最小抑菌浓度(minimal inhibitory concentration, MIC)和最小杀菌浓度(minimal bactericidal concentration, MBC)[29]

Table 4 MIC and MBC of UA micelles and original UA against four bacteria

Staphylococcus aureus ATCC 1007/s13205-018-1409-6

[6]

GALANTY A, ZAGRODZKI P, GDULA-ARGASIŃSKA J, et al. A comparative survey of anti-melanoma and anti-inflammatory potential of usnic acid enantiomers-a comprehensive in vitro approach[J]. Pharmaceuticals (Basel), 2021, 14(9): 945. DOI: 10.3390/ph14090945

[7]

KUMAR K, MISHRA JPN, SINGH RP. Usnic acid induces apoptosis in human gastric cancer cells through ROS generation and DNA damage and causes up-regulation of DNA-PKcs and γ-H2A. X phosphorylation[J]. Chemico-Biological Interactions, 2020, 315: 108898-108910. DOI: 10.1016/j.cbi.2019.108898

[8] 苏祖清. 松萝酸对小鼠急性肺损伤以及肺纤维化的保护作用[D]. 广东: 广州中医药大学, 2015.

SU Zuqing. The protective effects of Usnic acid on lipopolysaccharide-induced acute lung injury and bleomycin-induced pulmonary fibrosis in mice[D]. Guangdong: Guangzhou University of Chinese Medicine, 2015. (in Chinese).

[9] 李德. 松萝酸钠在大鼠皮肤伤口愈合过程中对成纤维细胞的影响[D]. 黑龙江: 东北农业大学, 2016.

LI De. Effect of sodium usnic acid for the fibroblasts on rat skin wound healing[D]. Heilongjiang: Northeast Agricultural University, 2016. (in Chinese).

[10] 郑宇. 松萝酸钠对皮肤创伤愈合的影响及临床应用实验[D]. 黑龙江: 东北农业大学, 2016.

ZHENG Yu. The effects of sodium usnic acid on skin wound healing and clinical experiments[D]. Heilongjiang: Northeast Agricultural University, 2016. (in Chinese).

[11] 杨帆. 松萝酸纳米给药系统的制备及减毒作用的初步研究[D]. 重庆: 重庆理工大学, 2020.

YANG Fan. Preliminary study on preparation and attenuation of usnic acid nano-drug delivery system[D]. Chongqing: Chongqing University of Technology, 2020. (in Chinese).

[12]

BATTISTA S, BELLIO P, CELENZA G, et al. Correlation of physicochemical and antimicrobial properties of liposomes loaded with (+)-usnic acid[J]. Chempluschem, 2020, 85(5): 1014-1021. DOI: 10.1002/cplu.202000125

[13]

FRANCOLINI I, GIANSANTI L, PIOZZI A, et al. Glucosylated liposomes as drug delivery systems of usnic acid to address bacterial infections[J]. Colloids and Surfaces B-Biointerfaces, 2019, 181: 632-638. DOI: 10.1016/j.colsurfb.2019.05.056

[14]

ZUGIC A, TADIC V, SAVIC S. Nano- and microcarriers as drug delivery systems for usnic acid: review of literature[J]. Pharmaceutics, 2020, 12(2): 156-180. DOI: 10.3390/pharmaceutics12020156

[15] 宋婷, 宋丹, 管海燕, 等. 松萝酸磷脂复合物在大鼠体内的药动学及组织分布研究[J]. 中草药, 2018, 49(6): 1358-1364. DOI: 10.7501/j.issn.0253-2670.2018.06.019

SONG Ting, SONG Dan, GUAN Haiyan, et al. Pharmacokinetics and tissue distribution of usnic acid phospholipid complex in rats[J]. Chinese Traditional and Herbal Drugs, 2018, 49(6): 1358-1364(in Chinese). DOI: 10.7501/j.issn.0253-2670.2018.06.019

[16] 王永中, 方晓玲. 聚合物胶束作为肿瘤靶向给药载体的研究[J]. 中国新药杂志, 2005, 14(10): 1127-1131. DOI: 10.3321/j.issn:1003-3734.2005.10.003

WANG Yongzhong, FANG Xiaoling. Polymeric micelles and targeting drug delivery systems for cancer therapy[J]. Chinese Journal of New Drugs, 2005, 14(10): 1127-1131(in Chinese). DOI: 10.3321/j.issn:1003-3734.2005.10.003

[17]

CHO HK, CHO JH, JEONG SH, et al. Polymeric vehicles for topical delivery and related analytical methods[J]. Archives of Pharmacal Research, 2014, 37(4): 423-434. DOI: 10.1007/s12272-014-0342-4

[18]

MATSUMURA Y. The drug discovery by nanomedicine and its clinical experience[J]. Japanese Journal of Clinical Oncology, 2014, 44(6): 515-525. DOI: 10.1093/jjco/hyu046

[19]

YOKOYAMA M. Polymeric micelles as drug carriers: their lights and shadows[J]. Journal of Drug Targeting, 2014, 22(7): 576-583. DOI: 10.3109/1061186X.2014.934688

[20]

GOU M, WEI X, MEN K, et al. PCL/PEG copolymeric nanoparticles: potential nanoplatforms for anticancer agent delivery[J]. Current Drug Targets, 2011, 12(8): 1131-1150. DOI: 10.2174/138945011795906642

[21] 苑莹芝. pH响应型抗肿瘤纳米药物载体的合成及药物控释[D]. 甘肃: 西北师范大学, 2017.

YUAN Yingzhi. Synthesis and drug controlled release of pH-responsive antitumor nano-drug delivery systems[D]. Gansu: Northwest Normal University, 2017. (in Chinese).

[22]

GHEZZI M, PESCINA S, PADULA C, et al. Polymeric micelles in drug delivery: an insight of the techniques for their characterization and assessment in biorelevant conditions[J]. Journal of Controlled Release, 2021, 332: 312-336. DOI: 10.1016/j.jconrel.2021.02.031

[23]

MAJUMDER N, G DAS N, DAS SK. Polymeric micelles for anticancer drug delivery[J]. Therapeutic Delivery, 2020, 11(10): 613-635. DOI: 10.4155/tde-2020-0008

[24] 蔡杰慧, 杨英全, 郑燕菲. 载姜黄素类化合物PCL-PEG-PCL微球的制备及释药性、抗氧化性研究[J]. 现代化工, 2022, 42(7): 201-206.

CAI Jiehui, YANG Yingquan, ZHENG Yanfei. Preparation of curcuminoids-loaded PCL-PEG-PCL microspheres, and study on their drug delivery and antioxidant activity[J]. Modern Chemical Industry, 2022, 42(7): 201-206(in Chinese).

[25] 苏晓梅, 张丹参, 赵凯燕, 等. 以PCL-PEG为载体的人参皂苷Rg3纳米粒的制备及表征[J]. 应用化工, 2018, 47(3): 525-529. DOI: 10.3969/j.issn.1671-3206.2018.03.026

SU Xiaomei, ZHANG Danshen, ZHAO Kaiyan, et al. Preparation and characterization of ginsenoside Rg3 nanoparticles based on PCL-PEG[J]. Applied Chemical Industry, 2018, 47(3): 525-529(in Chinese). DOI: 10.3969/j.issn.1671-3206.2018.03.026

[26]

LI J, DU YT, SU HT, et al. Interfacial properties and micellization of triblock poly(ethylene glycol)-poly(ε-caprolactone)-polyethyleneimine copolymers[J]. Acta Pharmaceutica Sinica B, 2020, 10(6): 1122-1133. DOI: 10.1016/j.apsb.2020.01.006

[27]

REDDY BP, YADAV HK, NAGESHA DK, et al. Polymeric micelles as novel carriers for poorly soluble drugs-a review[J]. Journal of Nanoscience and Nanotechnology, 2015, 15(6): 4009-4018. DOI: 10.1166/jnn.2015.9713

[28]

LI Q, LAI KL, CHAN PS, et al. Micellar delivery of dasatinib for the inhibition of pathologic cellular processes of the retinal pig-ment epithelium[J]. Colloids and Surfaces B-Biointerfaces, 2016, 140: 278-286. DOI: 10.1016/j.colsurfb.2015.12.053

[29]

Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing: M100S[S]. New York: Infobase Publishing, 2017.

目的

松萝酸(usnic acid, UA)具有抗菌、抗炎等多种药理活性，且价廉易得，不易产生耐药性，具有较大的临床应用前景，但其水溶性差导致生物利用度低，严重制约了其临床应用。本文利用聚合物胶束(polymeric micelle, PM)包载UA，从而提高其水溶性及生物利用度。

方法

采用薄膜水合法利用聚己内酯-聚乙二醇(polycaprolactone-polyethylene glycol, PCL-PEG)高分子材料构建UA胶束。利用单因素试验及Box-Behnken响应面法优化四种制备条件(三氯甲烷与甲醇的体积比、UA的投药量、PBS体积、PBS的pH值)。利用紫外-可见分光光度法测定UA胶束的包封率及载药量，利用纳米粒度仪测定UA胶束的粒径及zeta电位，利用TEM观察UA胶束的微观形貌，结合NMR及FTIR判断胶束对UA的包埋效果，利用紫外-可见分光光度计测定UA胶束的饱和溶解度，采用透析法测定UA胶束的体外释药性能，利用包封率及载药量反映UA胶束的贮存稳定性，利用溶血试验考察UA胶束的用药安全性。利用微量肉汤稀释法测定UA胶束对金黄色葡萄球菌、化脓隐秘杆菌、大肠杆菌及肺炎链球菌的体外抑菌活性。构建金黄色葡萄球菌感染的小鼠模型，利用微生物培养计数明确UA胶束的体内治疗效果。利用HPLC考察UA胶束在大鼠体内的药代动力学特征。

结果

UA胶束的最佳制备条件为：三氯甲烷:甲醇=1:1，UA投药量为4.15 mg，PBS体积为24.38 mL，PBS pH值为7.4。UA胶束的包封率及载药量分别为80.66%和9.47%，粒径约为120.4 nm，zeta电位为-7.8 mV。UA胶束的微观形貌为大小均匀、表面光滑的球体，胶束粒子之间无粘连。NMR检测结果显示，UA胶束与UA原药的图谱相比特征峰发生了改变。FTIR检测结果显示，UA胶束与UA原药及UA与PCL-PEG的物理混合物相比，红外吸收光谱的官能团区发生变化，观察不到明显峰形。饱和溶解度测定结果显示，与UA原药相比，UA胶束的水溶性提高了19.11倍。体外释药结果显示UA胶束48 h时释放度为76.26%。稳定性考察结果显示，UA胶束在4℃、25℃避光贮存不同时间后，包封率和载药量均有少量下降，其中4℃下降更缓慢。溶血试验考察结果显示，UA胶束在8 mg/mL浓度时，溶血率小于2%。药敏试验结果显示，UA胶束对四种细菌的MIC和MBC基本与UA原药相同。体内抗感染研究结果显示，UA胶束可显著下调金黄色葡萄球菌感染小鼠不同脏器的菌落数，改善小鼠肺脏及脾脏组织的病理损伤。药动学研究结果显示，与UA原药相比，UA胶束的相对生物利用度提高了156%，C提高了1.34倍，半衰期延长了21.38%，清除率降低了32.39%，平均驻留时间MRT也明显延长。

结论

采用薄膜水合法可成功将UA包载进PCL-PEG的PM内部，显著提高UA的水溶性，且具有药物缓释能力，贮存稳定性及用药安全性良好。UA胶束对四种常见细菌具有体外抗菌活性，对金黄色葡萄球菌感染小鼠的治疗效果比UA原药更好。UA胶束在大鼠体内具有较强的缓释作用，相对生物利用度得以提升，半衰期延长，清除率降低。