Flexible liposomes represent a new opportunity to enhance the skin penetration of compounds1,2. In this work, we investigated the possible relationships between liposome elastic modulus (Y) and/or liposome resistance to extrusion (σ) and the extent and depth of vesicle penetration. With this aim, we exploited the feasibility to modulate the flexibility of cationic vesicles by loading glycosaminoglycans (GAGs) with different physico-chemical characteristics, namely hyaluronan (F1), unfractionated heparin (F2), enoxaparin sodium (F3) or nadroparin calcium (F4). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was selected as main lipid constituent, since it can favour the GAG encapsulation by electrostatic interactions and, being a non-physiological compound, it can be detected in the skin. Plain DOTAP cationic liposomes (F0) and vesicles prepared replacing DOTAP with phosphatidylcholine (PC) (empty or loaded with enoxaparin) were also prepared and used as controls. All formulations were characterized in terms of particle size (dH), z-potential (ζ), drug content, morphology and deformability. The last one was assessed by both AFM and an extrusion assay appositely developed to measure the resistance offered by liposomes that underwent deformation under constant load or at constant rate. The skin penetration of the vesicles was qualitatively evaluated by TEM and quantitatively by assaying the DOTAP retained into the epidermis by HPLC-ESI-MS/MS. To this purpose, DOTAP was extracted from the epidermis with methanol and the diluted extract was analysed and quantified using a TSQ™ Triple Quadrupole (Thermo Scientific). The acquisitions were performed by a Selected Reaction Monitoring (SRM) mode and the quantitation was obtained by the use of an internal standard (trimethyloctadecylammonium bromide). Unexpectedly, after ultracentrifugation all GAG loaded cationic liposomes formed two populations, the former in the supernatant (Fs), the latter precipitated as a pellet (Fp). This anomalous phenomenon was attributed to the different density since the entrapment efficiency (GAG/DOTAP μg/mg) of Fp populations was at least 2.7 fold higher than the corresponding Fs populations. TEM and AFM images showed unilamellar and almost spherical vesicles having an electrodense core (proof of drug encapsulation) and a shell consistent with the presence of GAG bound to the surface, as confirmed by the reduction of ζ-values. Fs formulations showed a dH close to that of F0 (about 130 nm), with exception of F1s (dH = 114.6 ± 0.7 nm), whereas Fp were significantly larger (from 148 ± 0.7 nm, F1, to 193.4 ± 3.8 nm, F3). The Fp formulations were more rigid (higher elastic modulus, Y, and higher resistance to extrusion) than the corresponding Fs, as a result of the larger dH and lower ζ-values. In the case Fs series, the rigidity was influenced by the GAG content (r= 0.94) and the and the ζ-values (r= -0.99). The flexibility of the Fp series was mainly influenced by the dH (r= -0.98). The formulations F0 (Y= 3.6 MPa), F3s (Y= 39 MPa) and F3p (Y= 66 MPa) were selected to evaluate the impact of deformation on the in vitro penetration of liposomes in human epidermis since they represent the whole range of deformability. All the tested formulations penetrated the skin. The flexible liposome were clearly detectable in the spinosum and granulosum layers, independently of their elastic modulus. Conversely, the quantification of DOTAP evidenced a relationship between the amount of liposomes penetrated the human epidermis (F0= 3.33 ± 0.02 μg/mg; F3s= 1.18 ± 0.26 μg/mg; F3s= 0.53 ± 0.33 μg/mg) and their elasticity (r=-0.82) confirming the relevance of such parameter in determining the extent of liposome penetration.

Dotap LC-MS/MS analysis to assess the flexibility and the skin permeability of elastic vesicles / S. Franzé, A. Podestà, P. Procacci, M. Orioli, M. Carini, P. Minghetti, F. Cilurzo. ((Intervento presentato al 24. convegno National Meeting on Medicinal Chemistry tenutosi a Perugia nel 2016.

Dotap LC-MS/MS analysis to assess the flexibility and the skin permeability of elastic vesicles

S. Franzé;A. Podestà;P. Procacci;M. Orioli;M. Carini;P. Minghetti;F. Cilurzo
2016

Abstract

Flexible liposomes represent a new opportunity to enhance the skin penetration of compounds1,2. In this work, we investigated the possible relationships between liposome elastic modulus (Y) and/or liposome resistance to extrusion (σ) and the extent and depth of vesicle penetration. With this aim, we exploited the feasibility to modulate the flexibility of cationic vesicles by loading glycosaminoglycans (GAGs) with different physico-chemical characteristics, namely hyaluronan (F1), unfractionated heparin (F2), enoxaparin sodium (F3) or nadroparin calcium (F4). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was selected as main lipid constituent, since it can favour the GAG encapsulation by electrostatic interactions and, being a non-physiological compound, it can be detected in the skin. Plain DOTAP cationic liposomes (F0) and vesicles prepared replacing DOTAP with phosphatidylcholine (PC) (empty or loaded with enoxaparin) were also prepared and used as controls. All formulations were characterized in terms of particle size (dH), z-potential (ζ), drug content, morphology and deformability. The last one was assessed by both AFM and an extrusion assay appositely developed to measure the resistance offered by liposomes that underwent deformation under constant load or at constant rate. The skin penetration of the vesicles was qualitatively evaluated by TEM and quantitatively by assaying the DOTAP retained into the epidermis by HPLC-ESI-MS/MS. To this purpose, DOTAP was extracted from the epidermis with methanol and the diluted extract was analysed and quantified using a TSQ™ Triple Quadrupole (Thermo Scientific). The acquisitions were performed by a Selected Reaction Monitoring (SRM) mode and the quantitation was obtained by the use of an internal standard (trimethyloctadecylammonium bromide). Unexpectedly, after ultracentrifugation all GAG loaded cationic liposomes formed two populations, the former in the supernatant (Fs), the latter precipitated as a pellet (Fp). This anomalous phenomenon was attributed to the different density since the entrapment efficiency (GAG/DOTAP μg/mg) of Fp populations was at least 2.7 fold higher than the corresponding Fs populations. TEM and AFM images showed unilamellar and almost spherical vesicles having an electrodense core (proof of drug encapsulation) and a shell consistent with the presence of GAG bound to the surface, as confirmed by the reduction of ζ-values. Fs formulations showed a dH close to that of F0 (about 130 nm), with exception of F1s (dH = 114.6 ± 0.7 nm), whereas Fp were significantly larger (from 148 ± 0.7 nm, F1, to 193.4 ± 3.8 nm, F3). The Fp formulations were more rigid (higher elastic modulus, Y, and higher resistance to extrusion) than the corresponding Fs, as a result of the larger dH and lower ζ-values. In the case Fs series, the rigidity was influenced by the GAG content (r= 0.94) and the and the ζ-values (r= -0.99). The flexibility of the Fp series was mainly influenced by the dH (r= -0.98). The formulations F0 (Y= 3.6 MPa), F3s (Y= 39 MPa) and F3p (Y= 66 MPa) were selected to evaluate the impact of deformation on the in vitro penetration of liposomes in human epidermis since they represent the whole range of deformability. All the tested formulations penetrated the skin. The flexible liposome were clearly detectable in the spinosum and granulosum layers, independently of their elastic modulus. Conversely, the quantification of DOTAP evidenced a relationship between the amount of liposomes penetrated the human epidermis (F0= 3.33 ± 0.02 μg/mg; F3s= 1.18 ± 0.26 μg/mg; F3s= 0.53 ± 0.33 μg/mg) and their elasticity (r=-0.82) confirming the relevance of such parameter in determining the extent of liposome penetration.
set-2016
Settore CHIM/08 - Chimica Farmaceutica
Settore CHIM/09 - Farmaceutico Tecnologico Applicativo
Settore BIO/17 - Istologia
Settore FIS/03 - Fisica della Materia
Dotap LC-MS/MS analysis to assess the flexibility and the skin permeability of elastic vesicles / S. Franzé, A. Podestà, P. Procacci, M. Orioli, M. Carini, P. Minghetti, F. Cilurzo. ((Intervento presentato al 24. convegno National Meeting on Medicinal Chemistry tenutosi a Perugia nel 2016.
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