Material and methods

3.1. Chemical

Unlabelled DTPA, a calcium salt aqueous solution CaNa₃-DTPA (mw 497.4 g.mol^-1, 250 mg.ml^-1, pH 7.5, 2200 mOsm.l^-1), was provided by the Pharmacie Centrale des Armées (Orléans, France). [¹⁴C]-DTPA labelled at carbon-2 in the acetate moiety (mw 394.8 g.mol^-1) was from Amersham Pharmacia Biotech (Orsay, France). Its specific activity was 1.63 GBq.mmol^-1. [¹⁴C]-DTPA was dissolved in the unlabelled DTPA solution (250 mg.ml^-1) to give a specific activity of 3.7 x 10⁷ Bq.ml^-1 (active DTPA solution). Solutions of ²³⁹Pu phytate (87% of Pu[IV]) were prepared by dilution of a stock solution of ²³⁹Pu in nitric acid (2 N) in a 0.2mM solution of phytic acid to obtain a Pu activity of about 500 Bq/100 µl. Solutions of

238Pu citrate (97% of Pu[IV]; 0.1 M) had a Pu activity of about 442 Bq/100 µl. These solutions were filtered (porosity: 0.22 µm; Schleicher & Schuell (Mantes la Ville, France) FP 30/0.2 CA) before injection.

3.2. Lipids

Lipids were first dissolved in chloroform. Dioleoylphosphatidylcholine (DOPC, 20 mg.ml^-1), cholesterol (CH, 10 mg.ml^-1), phosphatidylglycerol (PG, 10 mg.ml^-1) (Sigma-Aldrich, St Quentin Fallavier, France) were used for the formulation of conventional liposomes. DOPC, CH and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG, 20 mg.ml^-1) (Avanti Polar Lipids USA obtained from Coger, Paris, France) were used for the formulation of stealth liposomes.

3.3. Liposome formulation

Both types of liposomes were formulated according to the lipid hydration method (Bangham et al. 1965).Conventional and stealth liposomes were composed of DOPC/CH/PG and DOPC/CH/DSPE-PEG of molar ratios 6/3/1 and 64/30/6, respectively. Several total lipid concentrations were assessed (10, 50, 75 and 100 mM). Lipids in chloroform were mixed and dried under vacuum. The resulting lipidic film was then hydrated with 2ml [¹⁴C]-DTPA solution (0.37 x 10⁷ Bq.ml^-1, 220 mOsm. l^-1, 25 mg. ml^-1). The resulting suspension was shaken and vortexed until a homogenous suspension containing the liposomes was obtained.

This suspension was then ultracentrifuged (Model L5-50 Ultracentrifuge Beckman, rotor 50 Ti No. 7E.2709, tubes Open-Top Ultra-Clear 2ml Beckman ref. 344091, Beckman Instruments, Carlsbad, CA, USA) four times (1 h, 193 000g, 4°C) to remove the non-encapsulated fraction. The same volume of 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES; Sigma-Aldrich)/NaCl buffer was added to the solid fraction before each ultracentrifugation. Free-DTPA control liposomes were also prepared and blended with an aqueous solution of [¹⁴C]-DTPA.

Rates of encapsulation of liposomes containing [¹⁴C]-DTPA were estimated by counting the radioactivity in the supernatant (=non-encapsulated fraction) and pellet. Four centrifugations (1 h, 193 000g, 4°C) were needed to remove the non-encapsulated fraction (ratio of the non-encapsulated fraction to the encapsulated fraction below 10%). Radioactivity was measured using liquid scintillation and a β counter (1214 Rackbeta LKB Wallac, Perkin Elmer Instruments, Courtaboeuf, France). Liposome sizes were measured using a submicron particle analyzer Coulter model N4 Plus (Beckman Instruments) and their stability was

checked on liposome preparations (2 ml) stored at 4°C under nitrogen by measuring both percentage and rate of encapsulation four times a month.

3.4. In vivo studies

The pharmacokinetic and decorporation studies were first conducted in rats over 48 h because of the well-known high rate of elimination of DTPA when administered as an aqueous solution. However, pharmacokinetic data evidenced an accumulation of DTPA in the body when encapsulated in liposomes. Other studies were therefore conducted over 16 days to allow DTPA to be completely removed from the body.

3.4.1 Animals

Animals use procedures were in accordance with the recommendations of the EEC (86/609/CEE) and the French National Committee (decret 87/848) for the care and use of laboratory animals. IFFA CREDO (Les Oncins, France) supplied the male Sprague–Dawley rats used. Rats were 12 weeks old when they arrived, with a weight range of 200–220 g.

Groups of two and four animals were used for the pharmacokinetic and decorporation studies, respectively. Rats were housed in glass metabolism cages. Food and water were given ad libitum. The animal room had a controlled temperature (22°C) and light cycle (light exposure from 06.00 to 20.00 h).

3.4.2. Pharmacokinetic studies

During the first pharmacokinetic study (=48-h study), the different treatments were compared using the same concentration of DTPA: 4.0 µmol.kg^-1 rat (corresponding to the highest rate of encapsulation with conventional liposomes). During the next study (=16-day study), and since the biodistribution of liposomes is known to be dependent on lipid concentration (Litzinger et al. 1996), the same volume of formulation was injected to maintain the same amount of lipid for both liposome formulations.

Three formulations were tested in the 48-h study: (1) a [¹⁴C]-DTPA solution in HEPES/NaCl buffer (43 x 10⁴ Bq.ml^-1, at 4.0 µmol unlabelled DTPA. kg^-1 rat), (2) a formulation of a [¹⁴C]-DTPA solution encapsulated in conventional liposome (37 x 10⁴ Bq.ml

-1, 4.0 µmol unlabelled DTPA.kg^-1) and (3) a formulation of a [¹⁴C]-DTPA solution encapsulated in stealth liposomes (56 x 10⁴ Bq.ml^-1, 4.0 µmol unlabelled DTPA.kg^-1). Both formulations were prepared as explained in Section 3.3.

Five formulations were assessed for the 16-day study: (1) a [¹⁴C]-DTPA solution in HEPES/NaCl buffer (43 x 10⁴ Bq.ml^-1, 4.0 µmol unlabelled DTPA.kg^-1), (2) a formulation of radiolabelled DTPA solution encapsulated in conventional liposome (56 x 10⁴ Bq.ml^-1, 6.0 µmol unlabelled DTPA.kg^-1), (3) a formulation of radiolabelled DTPA solution encapsulated in stealth liposome (89 x 10⁴ Bq.ml^-1, 9.6 µmol unlabelled DTPA.kg^-1), and (4, 5) two mixtures of labelled DTPA solution after a 10-fold dilution in distilled water along with DTPA-free control liposomes (conventional or stealth) containing 5 and 3% DTPA due to the simple mix, respectively. The specific activities of both formulations were 48.0 x 10⁴ and 88.8 x 10⁴ Bq.ml^-1, respectively. Doses were 5.2 and 9.6 µmol unlabelled DTPA.kg^-1 rat.

Rats were anaesthetized with 40 mg.kg^-1 pentobarbital (Sanofi Santé Nutrition Animale, Libourne, France) before injecting one of these preparations into the penile vein (200 µl/rat). Syringes for DTPA treatments were weighed before and after injection to determine the injected dose.

During the 48-h study, urine samples (0–4, >4–8, >8–24 and >24–48 h) and faeces samples (0–24 and >24–48 h) were collected and sampled for analysis. Animals were sacrificed at selected times: plasma and tissues (liver, spleen, kidney during the 48-h and 16- day studies), femur (during the 16-day study) were analyzed for their carbon-14 content.

Tissue samples were cleaned and stored at -20°C until analysis. Tissues were weighed then ground in distilled water (Ultra-turrax T25, Labo-modern, Paris, France), and aliquots (n=3) were oxidized in a Packard 307 Oxidizer (Packard Instruments, Rungis, France). Femurs were weighed and oxidized untreated. The carbon dioxide produced was absorbed in an organic base plus scintillant. The carbon-14 activity of each sample was determined by liquid scintillation counting (1214 Rackbeta liquid scintillation spectrometer, LKB Wallac, Perkin Elmer Instruments, Courtaboeuf, France). Retention of DTPA was expressed as a percentage of the administered activity.

The skeletal DTPA content was estimated to be the femur content x 22, considering that the ratio of the weights of one femur versus total skeleton in rat was 1/22 (Sontag 1991).

The blood/plasma weight was assumed to be 1/13 of the body weight (Litzinger and Huang 1992).

Studies are in progress to evaluate the in vivo metabolism of [¹⁴C]-DTPA. Preliminary results using a high-performance liquid chromatography method demonstrated that there was no or negligible DTPA metabolism in plasma (data not shown). Thus, the radioactivity monitored mainly represented the [¹⁴C]-DTPA and not a sum of [¹⁴C] radiolabelled inactive metabolites.

3.4.3. Decorporation studies

Five experiments were conducted. Anaesthetized rats were contaminated intravenously with ²³⁹Pu phytate or ²³⁸Pu citrate 2 h before treatment (syringes were weighed before and after injection). Rats (n=4) were then injected in the penile vein with 200 µl a saline serum solution, a calcium salt aqueous solution of unlabelled DTPA (4 or 30 µmol.kg^-1) or formulations of unlabelled DTPA encapsulated in liposomes with DTPA concentration of 6.6 and 9.6 µmol kg^-1 for conventional or stealth dosage, respectively.

Percentages of encapsulation were assumed to be equal to those obtained with liposomes containing [¹⁴C]-DTPA formulated at the same time (10.4% for conventional liposomes, 18.2% for stealth liposomes).

Rats were sacrificed 48 h and 16 days after treatment. Cumulative samples (n=2) of urine and faeces were collected. Tissue samples (n=3) corresponding to femurs, liver, kidneys and spleen were dissected and mineralized. Pu content was determined by liquid scintillation counting in Instagel-Plus medium using a Packard Tri-Carb 2500 TR/AB counter. Pu retention was expressed in terms of the percentage of the injected radioactivity.

The Pu skeletal content was estimated to be 20 times the femur content (Durbin et al.

1972, Sontag 1984). The blood weight was assumed to be 1/13 of the body weight (Litzinger and Huang 1992).