Franzé Silvia, Donadoni Giulia, Podestà Alessandro, Procacci Patrizia, Orioli Marica, Carini Marina, Minghetti Paola, Cilurzo Francesco
Department of Pharmaceutical Sciences, Università degli Studi di Milano , via G. Colombo 71, 20133 Milano, Italy.
Department of Physics and CIMaINa, Università degli Studi di Milano , via Celoria 16, 20133 Milano, Italy.
Mol Pharm. 2017 Jun 5;14(6):1998-2009. doi: 10.1021/acs.molpharmaceut.7b00099. Epub 2017 Apr 24.
In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log(YM) values perfectly correlated (R = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC-MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ = 0.65 ± 0.10 MPa → R = 3.33 ± 0.02 μg/mg; σ = 0.95 ± 0.04 MPa → R = 1.18 ± 0.26 μg/mg; σ = 1.89 ± 0.30 MPa → R = 0.53 ± 0.33 μg/mg).
在这项工作中，我们试图评估药物诱导的柔韧性变化对可变形囊泡渗透进入人体皮肤的影响。通过包封未分级肝素、依诺肝素和那屈肝素，获得了八种具有不同柔韧性程度的阳离子脂质体。通过一种新开发并经囊泡弹性模量（log(YM)）的定量纳米级机械原子力显微镜测量验证的新颖、简便且可靠的挤压试验研究了其可变形性。所提出的挤压试验通过确定囊泡变形过程中涉及的力，对双层刚性（σ）和囊泡变形（K）的微小变化非常敏感。药物负载导致脂质体相对于参考空白脂质体的柔韧性降低，且与肝素类型、药物与阳离子脂质（DOTAP）的比例以及药物在囊泡内的分布情况一致。有趣的是，σ和log(YM)值具有完美的相关性（R = 0.935），证明了两种方法获得的可变形性数据的可靠性。透射电子显微镜（TEM）和液相色谱 - 串联质谱（LC-MS/MS）光谱法的结合使得能够追踪整个囊泡渗透进入皮肤的模式。在所有情况下，均观察到表皮层中有完整的脂质体，并且发现了渗透深度与脂质体柔韧性之间的关系，支持了整个囊泡渗透机制的假设。此外，人体皮肤样品中囊泡渗透程度（R）的结果显示与柔韧性值有直接关系（σ = 0.65 ± 0.10 MPa → R = 3.33 ± 0.02 μg/mg；σ = 0.95 ± 0.04 MPa → R = 1.18 ± 0.26 μg/mg；σ = 1.89 ± 0.30 MPa → R = 0.53 ± 0.33 μg/mg）。
