Superparamagnetic nanoparticles as a contrast agent for magnetic resonance imaging (MRI) of magnetic susceptibility (T2*)

Abstract

The invention relates to the use of biocompatible superparamagnetic nanoparticles comprising an inorganic core and a coating including an electrically charged polymer, and having low tissue and vascular adhesion, for use as contrast agents in magnetic resonance imaging (MRI). The aforementioned nanoparticles have novel pharmacokinetic and relaxability T2* properties, with high potential for use in in vivo tissue imaging and tumour perfusion strategies based on parameter T2*.

Claims

1. A process for obtaining a T2*-weighed image a tumor in vivo in an animal or human comprising the operations of: (a) intravenously administering of a contrast agent to an animal or a human, the contrast agent comprising a superparamagnetic nanoparticle having a single core less than 15 nm in diameter and a surface net negative electrical charge, and comprising: 1) an inorganic core and 2) a water-soluble polymer coating, which does not accumulate in the liver or spleen, having serum relaxivity values r2* greater than 90 s.sup.−1 mM.sup.−1, characterized in that: i) the inorganic core is composed of magnetite —Fe.sub.3O.sub.4—; and ii) the water-soluble polymer coating is polyacrylic acid that is directly and covalently bound to the core by the use of carbodiimide; and (b) performing perfusion imaging by T2*-weighted magnetic resonance imaging (MRI) between 0 and 6 hours after operation (a).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a diagram representing the process used for coating the Fe3O.sub.4 magnetic nanoparticle with polyacrylic acid (PAA) by carbodiimide in Example 1. The presence of negatively charged carboxylic groups stands out, providing a net negative charge at physiological pH.

(2) FIG. 2 shows scanning electron microscope images of metal oxide nanoparticles coated with PAA. The inorganic core of the nanoparticles is composed of Fe3O.sub.4.

(3) FIG. 3 shows T1 and T2 relaxation properties of Nanotex in water (A, B) and fetal bovine serum (C, D) 0.5 to 1 Tesla, at concentrations ranging between 0 and 0.05 mM Fe. Values are the mean±standard deviation of the pixels observed in each condition.

(4) FIG. 4 shows T2 and T2* relaxation values in water at 7 Tesla (A, B) and fetal bovine serum (C, D) of Nanotex suspensions, at concentrations ranging between 0 and 0.05 mM Fe values are the mean standard deviation of the pixels observed in each condition.

(5) FIG. 5 shows the effects of increasing concentration of Nanotex on C6 cell viability detected by lactate dehydrogenase (LDH) released into the incubation medium after 1 hour (A) or 24 hours of incubation (B).

(6) FIG. 6 shows accumulation in the spleen detected by T2*-weighted MRI in four mouse spleens (A-D), obtained one hour after intravenous injection of Nanotex (15 micromoles Fe/Kg of body weight).

(7) FIG. 7 shows representative images T2*-weighted (A) of the thorax and abdomen of mice and T2* maps (B) obtained 24 hours after intravenous administration of Nanotex (15 micromoles Fe/Kg body weight).

(8) FIG. 8 shows the relative changes in hepatic T2* after injection of a single dose (15 micromoles Fe/Kg body weight) and a double dose (30 micromoles Fe/Kg body weight) of Nanotex in a tail vein of the mouse. The results are represented as the mean and standard deviation of four animals studied after administration of the contrast agent. The insert shows an enlarged view of the region between 0 and 6 hours for a better appreciation of the potentiation of the effect.

(9) FIG. 9 shows the determination of brain perfusion using the “bolus tracking” method in rats implanted with C6 glial tumors using Nanotex.

EXAMPLES

(10) The following examples are intended to be descriptive and should not be understood as limitations to the present invention.

Example 1 Preparation of Magnetic Iron Oxide Nanoparticles Used as a Contrast Agent in Tumor Perfusion

(11) Magnetite nanoparticles (Fe3O.sub.4) are prepared in an inert atmosphere at 25° C., by coprecipitation of Fe.sup.3+ and Fe.sup.2+ ions 0.3 M (molar ratio 2:1) with an ammonia solution (29.6%) up to pH=10, followed by a hydrothermal treatment at 80° C. for 30 minutes. The magnetic nanoparticles are washed several times with deionized water and ethanol, and allowed to dry at 70° C. in an oven for subsequent treatment. For the bonding of polyacrylic acid (PAA), 100 mg of Fe3O.sub.4 nanoparticles was firstly mixed with 2 ml of buffer A (0.003 M phosphate, pH 6) and 0.5 ml of carbodiimide solution (0.025 g.Math.mL.sup.−1 in the buffer A). After being sonicated for 10 minutes, 2.5 ml of the PAA solution (60 mg.Math.mL.sup.−1 in buffer A) are added and the mixture is sonicated for a further 30 minutes. Lastly, the PAA-coated Fe3O.sub.4 nanoparticles are magnetically recovered, washed twice with water and dialyzed against a buffered saline solution (FIG. 1 and FIG. 2A). Hereinafter, this nanoparticle shall be called Nanotex.

Example 2: Evaluation of Magnetic Relaxation Properties (T1, T2 and T2*) of the Nanotex Contrast Agent

(12) The evaluation of the magnetic relaxation properties (T1, T2 and T2*) of Nanotex developed in the present invention from the nanoparticles synthesized in Example 1 was performed at 1.5 Teslas, a clinical field strength, using a magnetic resonance spectrometer Bruker Minispec (Bruker Biospin, Ettligen, Germany), and at 7 Tesla using a Pharmascan Bruker scanner (Bruker Biospin, Ettlingen, Germany).

(13) T1 values at 1.5 Teslas were obtained using a spin-echo sequence with progressive saturation, TE: 10 ms, TR: 70-12000 ms (at least 9 values), T1 values were obtained at 7 Tesla using coronal sections (1.5 mm) along a set of capillaries (1 mm in diameter) each containing decreasing concentrations of Nanotex. The acquisition conditions were: FOV (display window): 30 mm, matrix: 256×256.

(14) T2 values at 1.5 Teslas were obtained using a spin-echo sequence (Carr-Purcell-Meiboom-Gill) independent of the diffusion with TR: 9000 s, TE: 10-2000 ms (at least 9 values). T2 values were determined at 7 Tesla in T2 maps of coronal sections of the capillary (1 mm in diameter) with FOV: 30 mm, Matrix: 256×256, coronal section 1.5 mm.

(15) T2* maps were obtained from 7 Tesla coronal sections (1.5 mm) along sets of capillaries (1 mm in diameter) containing increasing concentrations of Nanotex using a gradient echo sequence, TR: 300 s, TE: 2.3 to 40 ms (at least 9 values), FOV 30 mm, matrix: 256×256, coronal section: 1.5 mm. T2* values were calculated from T2* maps and are expressed as the mean±standard deviation.

(16) FIG. 3 shows the properties of T1 and T2 relaxation Nanotex at 1, 5 Tesla in water and serum at concentrations ranging between 0 and 0.05 mM Fe highest concentration tested reducing water T1 3200 ms to 2600 ms, and T2 from 2500 ms to 600 ms. The T2 effect is significantly higher than T1, as befits a superparamagnetic nanoparticle. In the case of the serum, there was a reduction in T1 from 1800 ms to 1600 ms, and a reduction in T2 of from 1000 ms to 700 ms. The effect remains higher in T2 than in T1, but the observed range is lower than in pure water.

(17) FIG. 4 shows the relationship between T2 and T2* at 7 Tesla in Nanotex suspensions prepared in deionized water and fetal bovine serum at concentrations ranging between 0 and 0.05 mM Fe. Nanotex reduces the value of T2 in water from 300 ms to 220 ms. The reductions of T2* by Nanotex are 18 ms. In the presence of serum, a slight reduction is observed in T2 (159 ms to 133 ms) for Nanotex, while reduction in T2* is 10 ms. The corresponding relaxivity values measured in serum are shown in Table 1.

(18) TABLE-US-00001 TABLE 1 Relaxivity values, r.sub.2 and r.sub.2* of Nanotex measured at 7 Tesla in serum r.sub.1 (mM.sup.−1, s.sup.−1) r.sub.2 (mM.sup.−1, s.sup.−1) r.sub.2{circumflex over ( )} (mM.sup.−1, s.sup.−1) Nanotex 0.49 94 119

(19) The relaxivity values were determined in nanoparticle suspensions in fetal calf serum at ambient temperature. The concentrations used for the measurement of relaxivity are based on the iron content of the nanoparticle.

Example 3: Determination of Cytotoxicity of the Nanotex Contrast Agent in Cultured C6 Glioma Cells

(20) The in vitro toxicity of Nanotex using C6 glioma cells was researched by testing the release of lactate dehydrogenase (LDH), a procedure that determines the integrity of the cell membrane. Cell death is detected by measuring the release of the enzyme into the incubation medium. Under these conditions, LDH release is associated with a dramatic alteration of the permeability of the cell membrane or breakage, so that the increase in LDH release indicates higher cell death and reduced viability.

(21) FIG. 5 shows the results of LDH release of C6 cells versus increasing concentrations of Nanotex. The changes in viability are not detectable in the concentration range studied, revealing low Nanotex toxicity in C6 glioma cells. A positive control (hydroxylamine cytotoxic concentration) was used to confirm that viable cells can be killed, and that this process can be detected by the release of LDH.

Example 4: Determination of In Vivo Toxicity, Accumulation of the Contrast Agent Nanotex in the Spleen

(22) Accumulation in vivo of Nanotex in the spleen is determined by measuring the values of T2* in the isolated spleens of mice sacrificed one hour after intravenous administration of Nanotex (15 micromoles Fe/Kg body weight). This dose corresponds to the clinical dose of nanoparticles recommended by commercial manufacturers and is used here as a reference dose. The spleens were isolated from mice sacrificed by cervical dislocation and placed in six plexiglass plates to allow reconstruction of the corresponding T2* maps. FIG. 6 shows representative results of this approach on a plate with isolated spleens of six animals that were administered Nanotex. The resolution and sensitivity achieved using this method allows very precise measurements of T2* in spleen ex vivo, not achieved in spleens in vivo.

(23) Table 2 shows the values of T2* in spleens isolated before and one hour after intravenous injection of Nanotex (15 micromoles of Fe/Kg of body weight). Nanotex does not induce a significant decrease in T2* in the spleen, suggesting a very low or no accumulation in the spleen and poor biological adhesion.

(24) TABLE-US-00002 TABLE 2 Accumulation in the spleen of Nanotex detected by the T2* value in the spleen one hour after intravenous administration of the nanoparticles. Condition T2* in spleen (ms) Control (saline) 4.78 ± 0.20 Nanotex 4.26 ± 0.26 (15 micromoles Fe/Kg body weight)

(25) During in vivo studies, appreciable toxicity is not detected in vivo after administration of Nanotex. Nanotex is also compatible with the anesthesia protocol employed (1-2% isoflurane) and no deaths due to the nanoparticle were detected in any of the healthy animals studied (n=12).

(26) In particular, the administration of Nanotex did not induce significant changes in breathing or heart rate, no outward signs of liver toxicity such as yellow skin were observed and Nanotex did not induce bald spots or hair colour, hyper- or hypo-activity (drowsiness), aggressiveness, hemiparesis or hemiplegia.

Example 5: Determination of Pharmacokinetics In Vivo of the Nanotex MRI Contrast Agent Nanotex

(27) In order to study the pharmacokinetics in vivo of Nanotex nanoparticles for MRI, T2*-weighted images were obtained and their corresponding maps in coronal sections through the thorax and abdomen of CD1 Swiss mice. Images were obtained prior to intravenous administration of Nanotex and at increased times following administration (1, 3, 6, 24, 48, 168 h). Nanotex nanoparticles were administered intravenously at a dose of 15 micromoles Fe/Kg body weight. This dose corresponds to the clinical dose of nanoparticles recommended by the producers of commercial nanoparticles and is used here as a reference dose.

(28) FIG. 7 shows a T2-weighted image representative of the abdomen and thorax before (A), and a representative of T2* map obtained 24 hours after (B), the intravenous administration of the same dose of Nanotex (15 micromoles Fe/Kg body weight). The T2* map (FIG. 7B) shows a significantly lower value of T2* in the liver of animals treated with Nanotex, confirming the previous results.

(29) FIG. 8 summarizes the results of measurements for T2* and hepatic accumulation and elimination after intravenous administration of a single dose (15 micromoles Fe/Kg body weight) and a double dose (30 micromoles of Fe/Kg body weight) of Nanotex. Nanotex induces a slight reduction in hepatic T2*, with a rapid decrease followed by a rapid elimination from liver tissue. The single dose of 15 micromoles Fe/Kg body weight of Nanotex was eliminated entirely in approximately 24 hours with a mean of approximately half-life (ti.sub.R) of hepatic elimination of 10 h. This mean half-life is significantly shorter than that of the dextran-coated nanoparticles, revealing a lower tissue and vascular adhesion and allowing administration protocols repeated at short time intervals. The study of the pharmacokinetics of Nanotex after injection of twice the recommended dose (30 micromoles of Fe/Kg body weight) demonstrates an increased relaxation effect, without modification to the rapid rate of elimination from the liver. The administration of a double dose does not show adverse symptoms and all the mice survived the study.

Example 6: Assessment of the Potential Use of Nanotex as a Contrast Agent in Perfusion Imaging in a Glioblastoma Multiforme Model by Magnetic Resonance Imaging (MRI)

(30) The evaluation procedures of microvascular perfusion are based on the monitoring of the kinetics of the transit of a “bolus”-type contrast. Basically, a rapid injection is administered so that the contrast agent flows through the vasculature as a grouped “bolus”, maintaining the initial concentration of the injected solution for each transit tissue (at least during the first transit tissue). When the bolus reaches the section of the plane of the MR image acquired, a decrease in image intensity which is proportional to the concentration of solute injected can be measured by magnetic resonance imaging (MRI).

(31) The kinetics of the contrast agent that passes through the image plane approaches a gamma function with an initial portion, a point of maximum intensity and a decrease until disappearing altogether. The area under the curve represents the cerebral blood volume (CBV, ml/100 g) in the image plane. The time between the start of the transit and the maximum concentration is known as the mean transit time (MTT) and measures the time (s) in which half of the contrast bolus has passed through the section. Lastly, cerebral blood flow (CBF) is the CBV/MTT ratio and represents the blood flow [(ml/100 g)/min] through the cerebral section studied.

(32) FIG. 9 illustrates the determination of cerebral perfusion in rats bearing C6 glial tumor implants using Nanotex. Basically, the figure illustrates the adaptation of gamma function (red) perfusion to the different contrast agent (blue) transit kinetics. Nanotex shows rapid transit time and virtually complete recovery of perfusion after injection, indicating that Nanotex does not remain fixed or adhered or interacts significantly with the endothelium or the cerebral microvasculature.

(33) Table 3 shows values for CBF [(ml/100 g)/min], CBV (ml/100 g) and MTT (s) Nanotex (15 micromoles Fe/Kg body weight) in tumor-bearing rats, calculated based on adjustments in the gamma transit function of the contrast agent. Nanotex has a short mean transit time in healthy brain tissue. In summary, Nanotex reflects very favorable first transit kinetics and recovery through the cerebral microvasculature.

(34) TABLE-US-00003 TABLE 3 Cerebral perfusion parameters determined by the magnetic resonance bolus tracking method using Nanotex (15 micromoles Fe/Kg body weight). Value Perfusion parameter (mean ± sd) CBF (mL/100 g/min) 22.86 ± 4.82  CBV (ml/100 g) 1.41 ± 0.06 MTT (s) 3.55 ± 1.00

(35) In order to investigate the effectiveness of the particles as probes for vascularization and angiogenesis, perfusion measurements were made in the centre of the gliomas (core), which contains mainly the necrotic area and its periphery, which contains the highly vascularized growth zone. Nanotex (dose 1× and 2×) have been used for comparison. Table 4 shows the results obtained with Nanotex in the three variables.

(36) TABLE-US-00004 TABLE 4 Tumor perfusion parameters determined using single and double doses of Nanotex by means of bolus tracking in C6 gliomas in a rat brain three weeks after implantation. Nanotex Nanotex Perfusion Tumor (15 micromoles/ (30 micromoles/ parameter region Kg body weight Kg body weight) CBF Interior 11.5 ± 1.8  45.85 ± 8.3  (mL/100 g/min) Periphery 47.5 ± 3.8  64.66 ± 9.6  CBV (mL/100 g) Interior  0.9 ± 0.12 1.11 ± 0.6  Periphery 4.4 ± 0.7 4.6 ± 0.8 MTT (s) Interior 4.3 ± 1.4 2.1 ± 0.4 Periphery 5.5 ± 0.9 4.4 ± 0.2

(37) Note how a single dose of Nanotex allows detection of perfusion heterogeneity of the centre and periphery of the tumor. This dose corresponds to the clinical dose recommended by the manufacturers of commercial nanoparticles and serves as a reference dose herein. Using a double dose increases confidence in the parameters due to increased sound signal in the images, without significant toxic effects in the animals.