Liposome preparation

Liposomes were prepared by using thin layer evaporation method with some modifications as previously reported [19, 31]. Briefly, DPPC, DPPS, Chol, GM1 and DSPEmPEG2000-mal, final molar ratio of 3:3:3:0.8:0.2, were co-dissolved by using an organic solvent mixture (chloroform: methanol, 3:1 v/v) in a round glass vial. The organic solvent was removed by using a rotavapor Büchi R-210 at 45 °C (Büchi, Milan, Italy) connected to a vacuum pump. The thin lipid film was hydrated with an aqueous solution of 40 mg/mL of CDP-choline (PBS 10 mM, pH 6.8), and a final lipid concentration of 50 mg/mL was obtained. Three minutes of warming at 60 °C and three minutes of vigorous stirring at 800 rpm were carried out for three times. The resulting multilamellar liposomes were warmed at 60 °C for 1 h and then frozen and thawed (5 min in liquid nitrogen and 15 min at 60 °C) ten-folds to improve the entrapment efficiency of the payload. The liposomal suspension was then extruded through polycarbonate filters with a pore size from 800 to 50 nm (Whatman® Nuclepore™ Track-Etched Membranes, Merk Life Science S.r.l., Milan, Italy), by using a stainless-steel extrusion device (Lipex Biomembranes, Northern Lipids Inc., Vancouver, BC, Canada). The un-entrapped drug was removed by using Amicon® Ultra centrifugal filters (cut-off 50 kDa).

The OX26 antibody was finally conjugated to the surface of small unilamellar liposomes as following reported.

Untargeted CDP-choline-loaded liposomes (CDP-choline/Lip) were prepared by replacing DSPEmPEG2000-mal with the same molar ratio of DSPEmPEG2000 during the lipid film preparation, while empty liposomes were made up by hydrating lipid film with PBS (pH 7.4, 10 mM). Radiolabeled liposomes were prepared by using [3H]-cholesteryl hexadecyl ether ([3H]CHE, 0.003% w/w) at a final concentration of 0.5 µCi/mL.

Antibody conjugation

A sulfhydryl group was added to the N-terminal portion of OX26 antibody by using SATA reagent as previously reported elsewhere with some modifications [32]. A stock solution of SATA was prepared by dissolving this compound (6 mg) in 1 mL of DMF. 1 μL of resulting solution was added to 1 mL of OX26 solution (0.5 mg/mL in PBS at pH 7.4) to have a final molar ratio between SATA and antibody of at least 8:1. The resulting mixture was incubated for 30 min at room temperature and then purified by using amicon ultracentrifuge tube (cut-off 3 kDa, Merck Millipore S.A.S., France). The acetylated OX26 was stored at -80 °C upon their use, de-acetylated by incubation with de-acetylating solution (0.5 M hydroxylamine.HCl, 25 mM EDTA in PBS, pH 7.4) at room temperature for 2 h, and then conjugated to liposomes (Fig. 1). The thiolate antibody was purified with desalting columns pre-equilibrated with PBS buffer (PBS 10 mM, 10 mM EDTA, pH 6.8) and concentrated up to 1 mL by using amicon ultracentrifuge tube. The presence of thiol group on modified OX26 was carried out by the Ellman reaction (data not shown) as previously published [33].

Fig. 1

Schematic representation of OX26 conjugation on liposome surface. The conjugation of OX26 to liposomes was obtained between thiol group of OX26 and maleimide group of DSPE-PEG2000-mal

Thiolate OX26 antibody (800 μL, 0.5 mg/mL) was conjugated to the surface of liposomes (lipid concentration 50 mg/mL) through the reaction between thiol group in the backbone of antibody and maleimide residual of DSPEmPEG2000-mal on the surface of liposomes by incubation at room temperature for 3 h, followed by an overnight incubation at 4 °C under continuous magnetic stirring (250 rpm) (Fig. 1). Ethanethiol was used at the end of reaction to block un-reacted maleimide residuals. The resulting OX26 liposomes were purified with exclusion chromatography by using the Akta Prime apparatus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a glass column packed with Sephadex G-25 and equipped with a spectrophotometer at fixed wavelength of 280 nm. When required, after purification, liposomes were concentrated by using total recovery amicon ultracentrifuge tube with a pore size of 50 kDa (Merck Millipore S.A.S., France).

Physicochemical characterization

The average size, size distribution and zeta potential of liposomes were analyzed by using dynamic light scattering (DLS) technique as previously reported with some modifications [34]. Briefly, samples were diluted with isosmotic pyrogen free solution (1:50 v/v) to avoid multiscattering phenomena, and the analysis was carried out at 25 °C by using Zetasizer Nano ZS (Malvern Panalytical Ltd, UK). Results are the average of three independent analyses ± standard deviation (S.D.). The conjugated OX26 was indirectly quantified as previously reported with some modifications [35]. OX26-conjugated liposomes were centrifuged at 90,000 × g (1 h, 4 °C) and the concentration of OX26 in the supernatant was quantified by Mouse IgG2a ELISA quantification kit (Sigma Aldrich, Milan, Italy). The amount of OX26 conjugated on the liposome surface was then calculated by deleting the amount of unconjugated monoclonal antibody in the supernatant from total amount of OX26 that are used during the liposome preparation.

The morphology of nanovesicles was also studied by using TEM analysis, as previously published [36]. Samples were properly diluted in isosmotic buffer and dropped into coated grids. Uranyl acetate solution (2% w/v) was used to stain the samples after drying. TEM images were acquired by using a Veleta (Olympus Soft Imaging System) digital camera, operating at 100 kV with Tecnai G2 (FEI) transmission electron microscope (TEM).

Drug entrapment efficiency and release kinetic of liposomes

The purified CDP-choline-loaded liposomes were dried under vacuum by using a ThermoScientific™ Savant™ SpeedVac™ (Fisher Scientific Italia, Rodano (MI), Italy) for 12 h. Dried liposomes were dissolved by using cooled methanol (+4 °C) and the CDP-choline entrapment efficiency was evaluated by using HPLC apparatus as previously reported by Lin et al. with some modification [37]. Samples were analyzed by using a C18 column (4.6 × 100 mm, 3 μm, Gemini-Nx plus C18; Phenomenex, CA, USA) at 25 °C. The mobile phase was methanol and KH2PO4 0.5 M (10:90 v/v ratio, respectively) with a flow rate of 1 mL/min. CDP-choline detection was carried out by using a UV-detector connected to HPLC apparatus at 280 nm. Empty liposomes were used as blank and an external calibration curve of CDP-choline, in the range from 1 to 25 μg/mL, was used to quantify the drug inside liposomes. The drug loading percentage (D.L.%) (Eq. 1) and the entrapment efficiency percentage (E.E.%) (Eq. 2) were calculated by using the following equations:

$$D.L.\%=\frac_}_}*100$$

(1)

$$E.E.\%=\frac_}_}*100$$

(2)

where, Den is the amount of drug loaded inside liposomes, while Liptot and Dtot are the total amount of lipid, and the total amount of drug that are used during the preparation procedure of liposomes.

The CDP-choline release from liposome was studied in vitro by using the bag dialysis method as previously published with some modification [38]. Briefly, liposomes were filled inside a cellulose acetate dialysis tube (Spectra/Por 1 Standard RC Dry Dialysis Tubing, 50 kDa, Spectrum Labs, USA) and hold in the receptor medium to have a final ratio between liposomes and receptor medium of 1:100 v/v. The release study was carried out at 37 ± 0.5 °C under a slow and continuous stirring (200 rpm) up to 24 h. At fixed time points (30 min, 1, 2, 3, 4, 6, 8, 10, 24 h), 1 mL of receptor medium was withdrawn and replaced with the same volume of fresh medium. Two different media, i.e. PBS (10 mM, pH 7.4) and PBS supplemented with 50% of human plasma, were used to study the kinetic release profile of CDP-choline. To avoid potential interference during the analysis, proteins of human plasma were removed. Briefly, a slight acid methanol solution was mixed with different samples (methanol/sample 3:1 v/v ratio), centrifuged at 11,000 × g for 10 min and the supernatant was then analyzed.

The percentage of CDP-choline released from liposomes was quantify by using the following equation (Eq. 3):

$$\mathrm=\left(\frac}}_}}}}}_}}}*}.}.\right)\times 100$$

(3)

where, Drel is the amount of drug released at specific time point, Den is the amount of drug loaded inside liposomes and d.f. is the dilution factor between the volume of liposomes loaded in the dialysis tube and the volume of receptor medium. Any further dilutions, before the analysis, were considered to calculate the amount of CDP-choline released from liposomes. Empty liposomes were used as negative control.

Stability in human plasma

The physical stability of liposomes was tested in a human plasma/PBS mixture (50:50 v/v ratio) (HP/PBS) as previously published with some modifications [39]. Briefly, 400 µL of CDP-choline-loaded OX26-conjugated liposomes (CDP-choline/OX26Lip) were incubated with 2 mL of HP/PBS medium (50% v/v) at 37 °C and then gently stirred up to 24 h. At fixed time points, 100 µL of the resulting mixture were analyzed by using DLS, and the average sizes of liposomes incubated with HP/PBS medium were measured. Liposomes incubated at the same conditions with saline solution (NaCl 0.9% w/v) were used as negative control during the experiment.

Turbiscan lab expert analysis

Turbiscan Lab expert (Formulaction, L’Union, France) was used to test the long-term stability of liposomes at 37 and 25 °C as previously published [40]. Briefly, CDP-choline/Lip (control) and CDP-choline/OX26Lip were hold in a glass vial tube and diluted ten-times with PBS (10 mM, pH 7.4) up to a final volume of 6 mL. The analysis was carried out for the full height of samples (~ 10 mm) for 1 h. A pulsed infrared LED (wavelength of 880 nm) was used for different measurements and the results were reported as transmitted and backscattered lights through and by liposomes. Backscattering and transmittance were measured with optical detectors at 45° and 180°, for evaluating the long-term stability of liposomes. Potential sedimentation, creaming and/or flocculation of colloidal nanoparticles, like liposomes, did not occur with an instrument threshold below or equivalent to 5%. The results were reported for sample height ranging between 2.5 and 10 mm, because variations of backscattering and transmittance profiles over 5% at the sample height of 2 mm and/or over 10 mm are related to the presence of bubbles air at the bottom and top of glass holder, and they are not related to the occurrence of destabilization phenomena [40]. The global destabilization profiles (TSI) of liposomes were also recorded as a function of time up to 1 h of incubation. Moreover, the mean diameter of liposomes was also evaluated during the study and the potential variations have been reported as a function of time.

Animals

Animal studies were carried in accordance with the Guide for the Care and Use of Laboratory Animals from directive 2010/63/EU of the European Parliament and protocols approved by the National Directorate of Veterinary Services (Italy, Permit No. 235 on June 30, 2011). Adult Wistar rats (250–300 g, body weight) were used for these studies and housed at 25 °C, 65% relative humidity, 12 h dark/12 h light cycle, with water and food ad libitum.

Biodistribution studies

Long-circulating properties of CDP-choline/OX26Lip and the relative uptake in the main RES organs (i.e. liver and spleen) were studied by injecting [3H]-labeled liposomes in the tail vain of rat (average weight of ~ 270 g). At fixed time points after injection (3, 12 and 24 h) the animals were sacrificed, and the tissues were collected for the analysis. Three animals were used at different time points for each independent experiment, and three independent experiments were carried out. Briefly, organs were hold into polypropylene-based liquid scintillation cylinder vials (Sigma-Aldrich Chemie, GmbH, Steinheim, Germany) and incubated (4 h at 60 °C under continuous stirring) with 2 mL of quaternary ammonium hydroxide solution (Sigma-Aldrich Chemie, GmbH, Steinheim, Germany) to have a complete dissolution of tissues. Hydrogen peroxide (2 mL at 24% v/v) was used to decolorize the mixture and 7 mL of liquid scintillation cocktail (Ready Organic™, Beckman Coulter Inc., Fullerton, USA) was further added to samples and vigorously mixed. The resulting samples were quantified by using Wallac Win Spectral™ 1414 liquid scintillation counter coulter (PerkinElmer Life and Analytical Sciences, Inc. Waltham, MA, USA) and data were analyzed by 1414 Win Spectral Wallac LCS Software. The quantification of radio-labeled liposomes accumulated in different tissues was performed as previously published [41]. The signal intensity of endothelium and blood, which interfered with collected samples, was corrected and the following equation (Eq. 4) was used for the analysis:

$$_=_-\left(_* _\right)$$

(4)

where, Rtissue is the corrected radioactivity, Rorgan is the level of radioactivity measured in the different samples, V0 is the total volume of interstitial fluid and vasculature calculated as a ratio between the whole organ radioactivity levels and the blood concentration 1 min after the injection of radio-labeled liposomes, and Ct is the blood concentration at time t. The radioactive intensity of organs collected from control (untreated rats) was used as a further correction factor.

Induction of ischemic stroke in rats

The ischemic stroke in adult male Wistar rats (250–300 g) was induced according to experimental protocol previously reported with some modification [19]. Briefly, the animals were anesthetized [42] by isoflurane inhalation (2.5% in 100% oxygen) and then the ischemic stroke was induced by the bilateral occlusion of the common carotid arteries. 30 min after occlusion, the blood flow was restored, and the ischemic animals were split in different groups and injected with different formulations for the evaluation of the therapeutic activity.

In vivo therapeutic activity

Therapeutic efficacy of CDP-choline/OX26Lip was carried out by injecting liposomes in Wistar rats, during the reperfusion process and once a day for six days. The survival rate of treated animals was studied up to 8 days after the induction of ischemic event. The survival rate percentage (%) was calculated according to the following equation (Eq. 5):

$$Survival \;rate\; \left(\%\right)=\frac*100$$

(5)

Liposomes were intravenously injected into the tail vein at a CDP-choline dose of 20 mg/kg. CDP-choline/Lip were injected at the same drug dose. Saline solution was used as a control. For each group a total number of 15 animals were used (5 animals in each group for every independent experiment, with a total number of 3 independent experiments).

For the study of lipid peroxidation and lactate levels, 1 h after reperfusion, the animals were sacrificed and then the analysis was carried out as previously published [19].

Statistical analysis

The statistical significance was carried out by One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Analysis was performed by using SigmaPlot v.12 and Excel (Office 2010) and the significance levels was carried out for *p < 0.05, **p < 0.01 and ***p < 0.001.