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J. Phys. Chem. B 2002, 106, 10468-10473
Electron Spin Resonance and Differential Scanning Calorimetry as Combined Tools for the Study of Liposomes in the Presence of Long Chain Nitroxides Ilaria Perissi, Sandra Ristori, Simona Rossi, Luigi Dei, and Giacomo Martini* Dipartimento di Chimica, UniVersita` di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy ReceiVed: January 30, 2002; In Final Form: April 18, 2002
Macroscopic and molecular techniques such as differential scanning calorimetry (DSC) and electron spin resonance (ESR), respectively, were used to characterize both structure and dynamics of liposomes built up with dioleoyltrimethylammonium propane (DOTAP) and dioleoylphosphatidylethanolamine (DOPE) lipids. These liposomes are good candidates to act as molecular carriers in cancer therapy, such as, for instance, boron neutron capture therapy (BNCT). DSC revealed that neither Tm nor ∆Hm of the gel-liquid crystal phase transition depended on the presence of relatively concentrated exogenous molecules as they are the n-DSA and n-DPC spin probes used in this work. The results suggested that the structure and the internal dynamics of the liposomes did no change after small loading of the paramagnetic probes. This was proved at a molecular level by the computer analysis of the ESR spectra of the above probes, which revealed discontinuities of the motional parameters (correlation times, τ⊥ and τ|, and order parameter S20) in the same range of temperatures of the endothermic events observed by DSC on probe-free liposomes. A dependence of τ⊥, τ|, and S20 on the localization of the probe was found, and the activation energies for the motion were consistent with the mobility of the domains sensed by the different probes. The results reported in this work may be considered as a useful basis for the characterization of both plain and loaded liposomes when these aggregates are used as drug carriers.
Introduction Liposomes are typically spherical or quasispherical closed structures built up with one or more curved bilayers of amphiphilic molecules.1-4 Because of their structural and dynamic features, they have been used as models for natural membranes able to entrap in their interior chemical species to which liposome bilayers are selectively permeable. In the last 30 years, liposomes have therefore been studied as potential drug carriers.5 Since then, pharmacologically and biologically active molecules have been carried with opportunely tailored liposomes, and in the early 90s, the first commercial formulations for drug delivery were patented.6 In the past few years, some of the present authors studied the carrier properties of hydrocarbon/fluorocarbon vesicles, and they thoroughly characterized these mixed systems from a physicochemical point of view by means of magnetic resonance spectroscopies, scattering techniques, and Cryo-TEM, which allowed them to establish the perturbations induced by the presence of either molecules mimicking biological compounds or paramagnetic probes.7-12 In this work, the results obtained on cationic liposomes containing long chain nitroxides with a technique that gives mostly macroscopic information (differential scanning calorimetry, DSC) are compared with the results obtained with a technique that gives details mostly at a molecular level (electron spin resonance, ESR). The liposomes investigated were typical mixed liposomes formed with the neutral dioleoylphosphatidylethanolamine (DOPE) lipid and the cationic 1,2-dioleoyltrimethylammoniumpropane (DOTAP) lipid. These liposomes * To whom correspondence should be addressed.
may be used as carriers for borocompounds in the boron neutron capture therapy (BNCT).13 Experimental Section Materials. Aqueous dispersions of liposomes built up with DOPE (1, MW ) 744.04) and DOTAP (2, MW ) 698.55), in the 1:1 weight ratio, were purchased from Northern Lipids, Vancouver (Canada) or extruded directly from a mixture of the individual components with the Liposofast apparatus (Avestin Inc., Ottawa, Canada).
Total lipid content was10 mg/mL, pH ) 6.5, and mean liposome diameter was 86 nm (data given by the manufacturer and checked by quasielastic light scattering). These dispersions were stored at 277 K. Their stability is given for at least 6 months.
10.1021/jp0202845 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/12/2002
Liposomes in the Presence of Long Chain Nitroxides Nitroxide radicals n-doxylphosphatidylcholine (n-DPC, 3) and n-doxylstearic acid (n-DSA, 4) were purchased from Sigma Chemicals, Mu¨nchen, Germany, and used as received.
J. Phys. Chem. B, Vol. 106, No. 40, 2002 10469 TABLE 1: Best-Fit Magnetic Parameters Used to Reproduce the 310 K ESR Spectra of n-DPC and n-DSA in Water Solutiona and after Insertion into DOTAP/DOPE Liposomesb gxx
gyy
gzz
τ⊥, Axx, Ayy, Azz, G G G sx1010
Pure Water 5-DSA 2.0080 2.0062 2.0029 6.2 5.8 35.4 16-DSA 2.0080 2.0062 2.0029 6.2 5.8 35.4
1.9 5.2
DOTAP/DOPE Liposomes 2.0058 2.0025 5.5 5.7 33 2.0058 2.0025 5.5 5.7 33 2.0060 2.0029 6.2 6.3 33 2.0060 2.0024 5.9 6 32.5
7.2 6.7 6.4 5.0
5-DPC 16-DPC 5-DSA 16-DSA
2.0087 2.0087 2.0089 2.0093
n
S20
2 1.5 10 1 10 1
0.49 0.03 0.5 0.03
a Accuracy: gii ) (0.0003; Axx, Ayy ) (0.2 G, Azz ) (0.5 G; τ⊥ ) (0.2 × 10-10 s; S20) (0.03; n ) τ⊥/τ|. b Accuracy: gii ) (0.0003; Axx, Ayy ) (0.2 G, Azz ) (0.5 G; τ⊥ ) (0.2 × 10-10 s; S20) (0.03; n ) τ⊥/τ|.
The surface per polar head is approximately 0.65 nm2 for both DOTAP and DOPE.14,15 On the basis of this value, samples were prepared with spin probe/liposome ratios in the range of 3500-7000 for DSC runs and 700-3500 for ESR runs. Methods. DSC curves were obtained with the Perkin-Elmer DSC-7 calorimeter equipped with the TAC 7/DX interface and the software PE Pyris 3.52. Aluminum sealed containers were used for the measurements. Thermograms were recorded in the temperature range of 234-313 K with a scanning rate of 0.5 K/min. Data handling and treatment were as reported in ref 16. ESR spectra were obtained with the aid of the Bruker model 200D spectrometer. Data acquisition was performed with the STELAR software. The temperature was controlled with Bruker VTB 3000 accessory. The accuracy was (0.5 K. Quartz tubes of inner diameter
