利用ATR-FTIR研究脂质纳米粒的鼻黏液渗透性

Application of ATR-FTIR Spectroscopy to Evaluate the Permeability of Lipid Nanoparticles in Nasal Mucus

鼻黏液是影响疫苗和药物经鼻吸收的首要屏障。由于干扰因素复杂多变,在体评价黏液渗透性较难实施,多采用体外评价。现有的药物鼻黏液渗透性检测方法如细胞模型法、多粒子示踪技术等,存在细胞培养周期长、操作繁琐、成本高、可获得信息少且需要进行荧光标记等不足,对鼻黏膜制剂的体外评价具有很大的局限性,因此迫切需要建立一种快速、简便、灵敏的鼻黏膜制剂黏液渗透性评价方法。基于衰减全反射-傅里叶红外光谱(ATR-FTIR)对药物结构以及粘蛋白二级结构变化的敏感性,利用其对不同性质(粒径与电荷)脂质体的鼻黏液渗透性进行研究,通过FTIR图谱分析不同脂质纳米粒与黏液中粘蛋白的相互作用,建立了鼻黏膜制剂黏液渗透性的体外评价方法。方法学研究表明,对于聚乙二醇10000(PEG10000)、壳聚糖、海藻酸钠脂质体,该方法线性关系分别为Y=2.3866X+2.154、Y=1.8703X+0.2789、Y=1.13014X+0.0609,线性相关系数分别为0.9958、0.9945、0.9909,精密度RSD值分别为0.62%、0.73%、0.95%;重复性试验中RSD值分别为0.83%、0.97%、0.88%,说明该方法线性关系良好、精密度高、重复性好,可用于体外评价药物制剂在黏液中的渗透性。研究结果表明,利用ATR-FTIR在不同时间内扫描样品可得到不同脂质体制剂样品的强度增加的吸收带。对于不同粒径PEG脂质体而言,粒径越小其黏液渗透性越强;对于不同电荷脂质体而言,壳聚糖脂质体黏液渗透性最弱,海藻酸钠次之,PEG脂质体黏液渗透性最强。进一步研究表明,不同电荷脂质体黏液渗透性的差异源于其与粘蛋白相互作用,该结论可通过分析粘蛋白酰胺Ⅰ带所包含的各二级结构信息得到。综上,基于ATR-FTIR所建立的体外评价方法灵敏简便,可作为多种不同制剂的鼻黏液渗透性的快速测定,经改进后还可用于药物制剂在其他黏液中的渗透性评价,应用前景广阔。

Nasal mucus is the primary barrier affecting the nasal absorption of vaccines and drugs.In vivo assessments are difficult to perform due to the complexity and variability of the confounding factors,whereas in vitro evaluations are mostly used.The existing in vitro assays for monitoring nasal mucus permeability of drugs,such as cell model assays and multiparticle tracer techniques,have the disadvantages of a long cell culture cycle,cumbersome operation,high cost,little available information,and the need for fluorescent labelling,which have great limitations for the in vitro evaluation of nasal mucosal formulations.Therefore,there is an in urgent need to establish a rapid,simple and sensitive method for the evaluation of mucus permeability of nasal mucosal formulations.Based on the sensitivity of ATR-FTIR spectroscopy to changes in drug structure and mucin secondary structures,the nasal mucus permeability of liposomes,typical lipid nanoparticles,with different properties(particle size and charge)was studied in this paper,and the interaction of different liposomes with mucin in mucus by FTIR spectra to establish an in vitro evaluation method for mucus permeability of nasal mucosal preparations.Methodological studies showed that for PEG10000,chitosan,and sodium alginate liposomes,the linear relationships of the method were Y=2.3866X+2.154,Y=1.8703X+0.2789,Y=1.13014X+0.0609,the linear correlation coefficients were 0.9958,0.9945,0.9909,and the precision RSD values were 0.62%,0.73%,and 0.95%,respectively;the RSD values in the repeatability experiment were 0.83%,0.97%,and 0.88%,respectively.It is indicated that the method has a good linear relationship,high precision,and good repeatability and can be used to evaluate the permeability of pharmaceutical preparations in mucus in vitro.The results showed that the sample absorption bands with increasing intensity of different liposome formulations could be obtained by scanning the samples at different times in interaction with mucus using ATR-FTIR.For PEG liposomes with different particle sizes,the smaller the particle size,the stronger the mucus permeability;for liposomes with different charges,chitosan liposomes with positive charges have the weakest mucus permeability,followed by sodium alginate and PEG liposomes have the strongest mucus permeability.Further studies have shown that the difference in mucus permeability of liposomes with different charges stems from their interaction with mucins,and this conclusion can be obtained by analyzing the information on each secondary structure(α-helix,β-sheet,β-turn,irregular turn)contained in the mucinamideⅠband(1600~1700 cm-1).In summary,the in vitro evaluation method established in this paper based on ATR-FTIR is sensitive and simple and can be used as a rapid assay of nasal mucus permeability for various preparations.And with improved applicability,it can also be used to evaluate the permeability of pharmaceutical preparations in other mucus,which has a broad application prospect.