Resonance Imaging

Field of the Invention. This invention relates to chemistry, more specifically, to methods of synthesizing iron oxide magnetic nanoparticles which can be used in medicine as a contrast agent for magnetic resonance imaging (MRI), e.g. for the diagnosis of various neoplasms.

Prior Art. The reliability of MRI diagnosis of various disorders is currently increased by means of various contrast agents which can be subdivided in two main types, one of which includes Tl contrast agents which are paramagnetic metallic ions containing large numbers of unpaired electrons (Gd ³⁺ , Eu ³⁺ , Cr ³⁺ , Mn ²⁺ , Fe ³⁺ ), while the second one includes T2 contrast agents which are iron oxide magnetic nanoparticles stabilized with various biocompatible coatings.

The invention claimed herein is intended to provide magnetic nanoparticles of iron oxide and a T2 contrast agent on their basis, stabilized with human serum albumin and/or bovine serum albumin with the size of the nanoparticles (or globules) being up to 50 nm. T2 contrast agents are negative contrasts: their accumulation reduces signal intensity in operation modes with signal weighing by magnetic field homogeneity during MRI. Importantly, T2 contrast agents, with their by several orders of magnitude higher magnetic susceptibility due to their constituent iron oxide nanosized crystals, have a T2 relaxivity level that is 50-100 times that for Tl contrast agents, which reduces the dose required for efficient contrasting.

Currently, the main methods of synthesizing magnetic nanoparticles are thermal decomposition of melatorganic precursors in high-boiling organic solvents in the presence of surfactants (Method 1 : A.H. Lu, E.L. Salabas, F. Schiith, Magnetic nanoparticles: Synthesis, protection, functionalization, and application, Angew. Chemie - Int. Ed. 46 (2007) 1222-1244. doi: 10.1002/anie.200602866.) and co-deposition of metal salts in stoichiometric ratios (Method 2: A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomaterials. 26 (2005) 3995^1021. doi: 10.1016/j.biomaterials.2004.10.012). Method 1 allows obtaining monodispersed nanoparticles, but the introduction of surfactants makes the resultant nanoparticles hyfrophobic, preventing their dissolution in aqueous media and hence making them unsuitable for injection into human body. Method 2 allows obtaining hydrophilic nanoparticles, but its specific feature is the production of a mixture of heterogeneously sized nanoparticles, hindering its further use due to the composition and particle size inhomogeneities.

Other methods exist, e.g. pyrolysis, microwave synthesis and sonochemical synthesis, but they cannot provide iron oxide nanoparticles with the required properties making them suitable for MRI diagnosis.

The use of magnetic nanoparticles in clinical practice implies adding their preparations with biocompatible, non-toxic and biodegradable components for improving the stability of the preparation and reducing its toxicity.

The prior art discloses the Resovist® preparation from Bayer Schering Pharma AG, which is in the form of dextrane coated magnetic nanoparticles with a size of above 100 nm.

However, this latter preparation has a very short circulation time in blood flow (2-5 min) due to the nanoparticle size exceeding 100 nm and the tendency to accumulate only in the liver, which significantly hinders its applications in the diagnosis of tumors in other organs.

The prior art also discloses a magnetic nanocomposite material for a contrast agent, a contrast agent itself, and a transport agent for the delivery of a diagnostic and therapeutic means (Invention Application US20130045160A1 as of 17.04.2013). Said reference discloses coating of nanoparticles with amphiphilic compounds, including albumin, to produce a magnetic nanocomposite material comprising one or multiple magnetic nanoparticles distributed in a hydrophobic domain of said amphiphilic compounds, and a coating comprising a hydrophilic domain of said amphiphilic compounds, wherein said hydrophobic domain of said amphiphilic compounds is conjugated with the surface of said magnetic nanoparticles with a physical bond rather than a chemical bond.

A disadvantage of said known nanocomposite material is that physical bonds are several orders of magnitude weaker compared with chemical bonds, which makes said coatings unstable. Furthermore, if after injection in blood the agent is physically immobilized due to hydrophobic interactions, the coating of said amphiphilic compound will be replaced by blood serum proteins, and the amphiphilic polymers themselves may also be absorbed on the surface of blood cells, changing the pharmacokinetics of the preparation and detracting from its deliverability to the tumor nidus.

The prior art further discloses a technical solution contained in Invention Application US20080206146A1 as of 09.11.2007, describing magnetic nanoparticles and methods of their synthesis and use. The Application discloses magnetic nanoparticles comprising a functional group providing for differential conjugation with brain, vessel and bone tissues and intended for use as a diagnostic means for MRI and as a medicine transport agent. A functionalized magnetic nanoparticle is incapsulated into the core from albumin. Albumin incapsulation comprises the following stages: 200 mg of human serum albumin is dissolved in 2.0 ml of water containing magnetic nanoparticles (e.g., magnetite particles). The solution pH is adjusted to 8.4 by the drop-wise addition of 0.01M and 0.1 M NaOH solution while constantly stirring. Further, 8.0 ml of ethanol is added drop-wise while constantly stirring so 10% human serum albumin solution is obtained. Ethanol addition is followed by the addition of 235 μΐ of 8% glutaraldehyde solution. In 24 h the resultant nanoparticles are purified by 3 -cycle centrifugation (16,100 g, 8 min) and repeated, re-suspension by sonication in a water bath. Functionalized magnetic nanoparticles coated with human serum albumin have an average diameter of 60 nm to 990 nm depending on preparation pH and particle type used.

However, the performance characteristics of the preparation of these nanoparticles are impaired by the large polydispersity of the nanoparticles (the particle size scatter is 60 nm to 990 nm) which restricts its practical applications, since large nanoparticles have dramatically shorter half-excretion times which prevents their efficient delivery to the tumor nidus.

A difference of the solution disclosed herein from the known counterpart is the use of thermal decomposition of iron acetylacetonate for obtaining monodispersed magnetic nanoparticles with an initial hydrodynamic size of up to 20 nm, preferably, up to 12 nm, capable of dissolving in water with subsequent immobilization by human/bovine serum albumin, stabilization by glutaraldehyde and the synthesis of preparations comprising magnetic nanoparticles with an initial hydrodynamic size of within 50 nm, which allows eliminating the disadvantages that are typical of the above cited counterparts.

The prior art discloses a magnetic nanoparticle synthesis method (Hilda T.R. Wiogo, May Lim, Volga Bulmus, Jimmy Yun, and Rose Amal Stabilization of Magnetic Iron Oxide Nanoparticles in Biological Media by Fetal Bovine Serum (FBS) // Langmuir 2011, Vol.27, ½2, P.843-850.) for synthesizing nanoparticles having carboxyl groups on the surface, by mixing with the fetal calf serum. It is disclosed that said particles have an average hydrodynamic size of 180 nm and retain it for 16 h of incubation in an RPMI nutrient medium. It is further disclosed that protein immobilization occurs in a noncovalent manner. However, the large nanoparticles size (180 nm) may hinder their diffusion through vessels to the tumor thus reducing their efficiency as a contrast agent. Moreover, the noncovalent modification of the surface may cause coating desorption upon injection into the blood flow and hence lead to the loss of all the components conjugated to the coating. Furthermore, said reference does not disclose any data on the value of T2 relaxivity which are required for assessing the efficiency of the resultant contrast agent for MRI diagnosis.

The closest counterpart of the invention disclosed herein is an MRI contrast agent, its synthesis method and MRI diagnostic method of multimorphic glioblastoma (Patent RU2530762 οτ 14.12.2012; Abakumov MA, et al, Nanomedicine. May 2015, Volume 11, Issue 4, pp. 825-833). According to the cited work, magnetic nanoparticles were synthesized by thermal decomposition of iron (III) acetylacetonate in benzyl alcohol. Magnetic nanoparticles were synthesized in an inert gas flow with constant stirring of the reactant mixture by gradual heating to 383 K, exposing 1 h to that latter temperature for water evaporation from the solution, raising the temperature of the reaction mixture to 473 K at a 25 K/h rate, the heating time totaling to 9 h, and exposing to that latter temperature for 40 h. In 40 h the reaction mixture was gradually cooled to room temperature, added with 90 ml of water-free acetone and separated from the magnetic nanoparticles by centrifugation at 2000 g for 10 min. The residue was twice washed in an excess of acetone and dried on a rotor evaporator for complete acetone removal. The diameter of the resultant nanoparticles was (14±4) nm according to transmission electron microscopy. At the next process stage the water colloidal solutions of iron oxide nanoparticles were stabilized using bovine serum albumin (BSA). To this end, 10 mg of particles was added with 5 ml of distilled water and titrated to pH 11 with 1M NaOH solution. The resultant dispersion was then sonicated for 10 min and added with 40 mg of polymer dissolved in 5 ml of water. The resultant mixture was incubated for 4 h at room temperature with constant stirring, dialyzed against distilled water and then added with 500 μΐ of 1M NaOH and further with 2.3 ml of 25% glutaraldehyde water solution drop-wise while stirring. The resultant mixture was incubated for 15 min while stirring and then added with 500 μΐ of 3 M glycine (pH 9.2) for binding unreacted aldehyde groups. The resultant solution was added with 1 ml of sodium boron hydride solution in PBS with a 2 mg/ml concentration and incubated for 60 min. For separating the bovine serum albumin coated magnetic nanoparticles (BSA-MNP) from excess protein, the nanoparticle solution was filtered through cellulose centrifuge filters with a 100 kDa pore size. The residue was re-suspended in water and re-filtered. The procedure was repeated until the complete absence of protein in the washing fluid. Protein was cleaned from molecular cross-linking products by gel filtration on Sepharose CL-6B (column height 50 cm, diameter 2.5 cm, flow rate 0.7 ml/min). At the next stage magnetic particle were conjugated with monoclonal vessel endothelium growth factor antibodies with a hydrodynamic size of within 150 nm which were further used as a contrast agent for MRI diagnosis of multimorphic glioblastoma.

In accordance with the method disclosed in RU2530762, MRI visalization was carried out before and after intravenous injection of the resultant contrast agent, the MRI visualization mode providing a magnetic susceptibility weighed image of the area being visualized, and the judgment as to the existence of multimorphic glioblastoma was made by comparing the MRI images taken before and after the injection of the contrast agent on the basis of image brightness reduction in selected areas.

Said method increases the authenticity and information value of diagnosis by increasing the contrast of the areas corresponding to multimorphic glioblastoma tissues and vessels and neoangiogenesis nidus in MRI images. However, the size of the nanoparticles in accordance with said method was 92 nm. Therefore a disadvantage of said method is the impossibility of obtaining nanoparticles with human serum albumin coatings having a size of less than 50 nm due to the use of long-term boiling in the process (40 h at 200 °C). Boiling leads to chemical aging of nanoparticle surfaces (transformation of the OH groups to oxo bridges) which reduces the quantity of charged groups on the nanoparticle surface thus impairing the stabilization of electrostatic repulsion and reducing the charge density required for protein sorption. Moreover, chemical aging increases the initial size of the nanoparticles which deleteriously affects their stability during re-suspension in water media. The combination of these two processes results in the nanoparticles synthesized after long-term heating being less stable to water media and more susceptible to aggregation. It should be borne in mind that the aggregation of the source magnetic cores increases the sizes of the final nanoparticles (globules) already coated with human serum albumin.

Disclosure of the Invention. The technical task solved by the invention disclosed herein is providing a method of synthesizing serum albumin stabilized albumin magnetic nanoparticles suitable as contrast agents for the MRI visualization of tumors.

The technical result of the invention disclosed herein is the possibility of synthesizing magnetic nanoparticles with an initial hydrodynamic size (before HSA/BSA stabilization) of up to 20 nm and HSA/BSA stabilized nanoparticles (globules) with a size of up to 50 nm, due to changing the mode of iron (III) acetylacetonate solution boiling in benzyl alcohol in the course of the synthesis of the initial magnetic nanoparticles so the time of boiling is within 4 h. Boiling for more than 4 h causes irreversible ageing processes on the surfaces of the magnetic nanoparticles which hinder their dissolution in water and HSA sorption on their surface.

The preparation obtained using the method claimed herein provides for an increase in the efficiency of the MRI visualization of neoplasms by improving the penetrability of the preparation into the tumor nidus upon its injection in the course of MRI. This enables efficient penetration through the pores of the defective vessels of the tumor tissue.

Said technical result is achieved by using the MRJ tumor diagnosis preparation synthesis method comprising the preparation of iron (III) acetylacetonate solution in benzyl alcohol with a concentration of 75-200 g/1 followed by heating in an inert gas flow to the benzyl alcohol boiling point for 4-8 h and boiling said solution for 30 min to 4 h to obtain suspension, cooling the suspension, washing with a polar organic solvent to obtain Fe ₃ 0 ₄ iron oxide nanoparticles which are then coated with human serum albumin and/or bovine serum albumin, and stabilizing the resultant coating by intermolecular cross- linking with glutaraldehyde.

For coating with human serum albumin and/or bovine serum albumin, the nanoparticles are dissolved in water with pH 10 - 11 to a 2-8 mg/ml concentration, following which the resultant solution is added with HSA and/or BSA in the form of a water solution with an HSA and/or BSA concentration of 4-16 mg/ml in a 1: 1 volume ration, and is dialyzed.

After coating stabilization by intermolecular cross-linking the solution is additionally cleaned from by-products by ultra-filtration and filtration sterilization. Ultra-filtration can be achieved with filters having a pore diameter of 100-300 kDa.

In the preferred embodiment of the invention heating carried out is at a constant rate.

Said polar organic solvent can be selected from the group consisting of water-soluble aliphatic monoatomic alcohols, acetones, ketones and nitriles. Said water-soluble aliphatic monoatomic alcohols can be selected from the group consisting of ethyl alcohol, methyl alcohol or propyl alcohol, said ketones can be e.g. acetone or butanon-2, and said nitriles can be e.g. acetonitrile.

The solution is washed until the removal of benzyl alcohol traces. In a specific embodiment of the invention, washing can be carried out by portions at least equal to the volume of the suspension, for at least 3 cycles. Washing can otherwise be achieved using settling by centrifugation.

The preparation obtained using the method claimed herein can be in the form of a solution or a lyophilizate. For the MRI diagnosis of tumors, an MRI study of the object (human or animal) is carried out before the injection of said preparation in modes providing for Tl and T2 weighing, then the preparation is injected intravenously in a quantity of 1-10 mg/kg body weight by iron concentration, and MRI visualization is carried out again in modes providing for Tl and T2 within 2 h after preparation injection, followed by comparison of the intensities in the visualized areas of the MR images taken before and after preparation injection. If hypointensity areas are identified, conclusion is drawn on the presence of tumor and tumor limits are outlined.

The method claimed herein allows reducing the time required for obtaining magnetic nanoparticles and the contrast agent on their basis, i.e. magnetic nanoparticles with albumin, specified by the presence of aggregates (nanoparticles) sized less than 50 nm. This simplifies the technology of the contrast agent preparation, in particular, by excluding the gel filtration stage (as compared with the counterpart technology). The contrast agent synthesis method claimed herein requires from 5-6 to 10 h compared to prior art methods requiring up to 46 h. Furthermore, technologies disclosed in other cited works do not provide stable albumin mixtures with magnetic nanoparticles due to chemical bonding. Still another advantage of this invention is that the preparation claimed herein is excreted through kidney within several hours after the injection, unlike other known preparations which are accumulated in liver (Majumdar S, Zoghbi SS, Gore JC. Pharmacokinetics of superparamagnetic iron-oxide MR contrast agents in the rat. Invest Radiol. 1990;25:771-777), and therefore side effects of preparation injection are herein avoided. In addition, the preparation claimed herein is stable during storage for one year in the form of lyophilizate and can be re-suspended while retaining all the parameters.

Brief Description of the Drawings.

This invention will be further illustrated with drawings, wherein Figure 1 shows a micrograph of iron oxide nanoparticles obtained using the claimed method, wherein said micrograph was obtained under a transmission electron microscope; Figure 2 shows data on the hydrodynamic sizes of the magnetic nanoparticles obtained after 30 min boiling in benzyl alcohol; Figure 3 shows data on the hydrodynamic sizes of the magnetic nanoparticles obtained after 1 h boiling in benzyl alcohol; Figure 4 shows data on the hydrodynamic sizes of the magnetic nanoparticles obtained after 4 h boiling in benzyl alcohol; Figure 5 shows data on the hydrodynamic sizes of the magnetic nanoparticles obtained after 20 h boiling in benzyl alcohol; Figure 6 shows an MRI image of a brain tumor in rat C6 (A) before and (B) 5 min after the injection of the preparation based on iron oxide magnetic nanoparticles obtained using the claimed method; Figure 7 shows an MRI image of mucinous carcinoma of liver RS-1 (A) before and (B) 5 min after the injection of the preparation based on iron oxide magnetic nanoparticles obtained using the claimed method; Figure 8 shows an MRI image of mammary gland adenocarcinoma in mouse (A) before and (B) 5 min after the injection of the preparation based on iron oxide magnetic nanoparticles obtained using the claimed method.

Embodiments of the Invention. The method of obtaining Fe3C>4 iron oxide nanoparticles, preferably, for use as a contrast agent in the MRI diagnosis of tumors, is implemented as follows.

Disclosed below is a detailed description of the invention showing specific quantitative parameters of component contents and parameters of the process for obtaining magnetic nanoparticles and a preparation for MRI diagnosis; however, this description does not restrict possible embodiments of this invention to the values/parameters specified herein, and is only intended to demonstrate the possibility of its implementation providing for the achievement of the claimed result.

For synthesis, an iron (III) acetylacetonate solution in benzyl alcohol with a 75-200 g/1 concentration is prepared. The iron (III) acetylacetonate solution in benzyl alcohol with a 75-200 g/1 concentration is heated in an inert gas flow to the solvent boiling point for 4-8 h, preferably 6 h, and further boiled for 30 min to 4 h. The reaction mixture is cooled to room temperature, centrifuged to separate the nanoparticle residue from the solution, washed in acetone/ethanol or methyl alcohol, or propyl alcohol / acetonitnle, and iron oxide magnetic nanoparticles are obtained after solvent evaporation.

The magnetic nanoparticles (80±20 mg) are diluted in 20 ml of distilled water, added with 500 μΐ of 1M NaOH to produce an alkaline media, and the solution is stirred to remove the residue. Then human serum albumin / bovine serum albumin solution (160±50 mg in 20 ml of water) is added. The resultant mixture is stirred for 15 min following which large particle aggregations are removed by filtration with 0.22-0.45 pore size syringe filters. The mixture is then added with 500-1500 μΐ of 25% glutaraldehyde water solution. The reaction mixture is incubated for 10-60 min while constantly stirring, added with 0.5-2 ml of 3M glycine solution and further stirred for 30 min to 2 h. Then the reaction mixture is added with 0.5-4 ml of NaBH4 (10 mg/ml) and incubated for 30 min to 4 h.

Surplus low molecular weight substances and free HSA molecules are removed by washing in a phosphate/salt buffer solution in centrifuge filters with the permission edge 100-1000 kDa for globular proteins.

The nanoparticles obtained as described above will dissolve in distilled water at pH 10-11, and when covered with human serum albumin they form nanoparticles consisting of crystalline magnetic iron oxide cores and human serum albumin shells, with an overall size of within 40 nm.

Experiments were carried out with animals for glioma or other tumor experimental setups. For glioma visualization in animals, animals were placed in , a MRI apparatus, and MR scanning in T2 or T2* weighed mode was carried out. The animal was then intravenously injected with the HSA stabilized magnetic nanoparticle solution. The injected magnetic nanoparticle dose reduced to iron concentration was 12,010 mg/kg. MRI scanning was carried out on a Bruker Clinscan 7T tomograph. MRI scanning in T2 or T2* weighed mode was carried out 0-24 h after the intravenous injection.

The advantage of using nanoparticles less than 50 nm in diameter for MRI diagnosis is accounted for by the increased time of nanoparticle (globule) circulation in blood as a result of their smaller size, which provides for efficient visualization without increasing injected doses. Furthermore, tumor vessels possess higher penetrability and retention due to the hyperexpression of pro- angiogenic factors causing accelerated growth of the vessel network. The pores forming in the vessels are 50-200 nm in size, and only nanoparticles less than 50 nm in diameter can efficiently penetrate through these pores to tumor tissue.

Example 1. Synthesis of Fe ₃ 0 ₄ magnetic nanoparticles.

10.5 g iron (III) acetylacetonate and 220 g benzyl alcohol are heated while stirring in a glass retort to 50-70 °C for 1 h. The heating rate is 25 °C/h. Heating is stopped 30 min after the reaction mixture temperature reaches the boiling point. The reaction mixture is cooled to room temperature and added with 90 ml of acetone, and nanoparticles are settled down by centrifugation at 900g for 19 min.

Example 2-5. Synthesis of Fe ₃ 0 ₄ magnetic nanoparticles.

Magnetic nanoparticles are synthesized as described above in Example 1 except that acetone is replaced for methyl alcohol, ethyl alcohol, propyl alcohol, butanol-2 or acetonitrile.

Example 6. Synthesis of HSA/BSA coated Fe ₃ 0 ₄ magnetic nanoparticles.

20 iron oxide magnetic nanoparticles are added with 5 ml of distilled water, adjusted to pH 11 and stirred until complete dissolution. Then 5 ml of HSA/BSA water solution is added with a 8 mg/ml concentration at the same pH. The resultant mixture is incubated for 15 min at room temperature with constant stirring and filtered through a 0.2-0.5 μηι pore filter. 20 mg of the resultant solution having a protein and Fe concentrations of 4 mg/ml and 2 mg/ml, respectively, is added with 250 μΐ of 1M alkali and then with 230 μΐ of 25% glutaraldehyde water solution drop-wise. The resultant mixture is incubated while stirring for 15 min and then added with 250 μΐ of 3M glycine water solution (pH 9.2) and incubated for 1 h. The solution is then added with 332 μΐ of sodium borhydride in a 10 mg/ml phosphate/salt buffer solution and incubated for 2 h following which the reaction mixture is washed with the buffer solution and the iron concentration is measured.

Example 7. MRI visualization of experimental brain tumor in rat C6.

The experiments are carried out with animals having experimental model of glioma C6 or another type of tumor. For glioma visualization in animals, animals are placed in an MRI apparatus, and MR scanning in T2* weighed mode is carried out with the following parameters: TE/TR =19/50 ms, cut thickness 0.5 mm, FOV = 30 mm, resolution 256/176. The animal is then intravenously injected with the HSA stabilized magnetic nanoparticle solution. The injected magnetic nanoparticle dose reduced to iron concentration is 5 mg/kg. MRI scanning is carried out on a Bruker Clinscan 7T tomograph. MRI scanning in T2* weighed mode is carried out 5 min after the intravenous injection with the following parameters: TE/TR =19/50 ms, cut thickness 0.5 mm, FOV = 30 mm, resolution 256/176.

The nanoparticles obtained in the course of the experiments according to Examples 1 - 5 were within 10 nm in size (Figure 1).

Increasing the time of boiling leads to a gradual increase in the size of the nanoparticles from 7.5 nm to 50 nm (Figure 2 - 5).

Moreover, upon injection of magnetic nanoparticles into animals with experimental tumors and sunsequent MRI visualization allow visualizing several types of tumors, including brain glioblastoma C6, mucinous carcinoma of liver RS-1 and mammary gland adenocarcinoma in mouse 4T1 (Figures 6 - 8).