EA031124B1

EA031124B1 - Contrast medium based on amorphous or amorphous-crystalline nanoparticles of manganese oxides or hydroxides for brain mri

Info

Publication number: EA031124B1
Application number: EA201790844A
Authority: EA
Inventors: Юрий Михайлович Мошкин; Дмитрий Валерианович Петровский; Александр Викторович Ромащенко
Original assignee: Деркач, Олег Вадимович
Priority date: 2017-05-15
Filing date: 2017-05-15
Publication date: 2018-11-30
Also published as: EA201790844A1

Abstract

The invention relates to a contract medium for magnetic resonance imaging, comprising amorphous or amorphous-crystalline nanoparticles, including manganese oxide or hydroxide of formula MnOH, where x is 1 or 3, y is 2, 3 or 4, z is 0 or 2, wherein if is x is 1, then z is 2; and a nonionic surfactant. The invention also includes a method of producing said medium, a diagnostic composition and a method of diagnostics using the same. The technical result provides improvement of neuronal uptake of hydrophilic amorphous or amorphous-crystalline nanoparticles according to the invention, which permits to carry out diagnostics of affections and accurate localization of space-occupying lesions in the central nervous system by intranasal administration of the nanoparticles.

Description

The invention relates to a contrast agent for magnetic resonance imaging, containing amorphous or amorphous-crystalline nanoparticles, including manganese oxide or hydroxide of the formula Mn _x O _y H _z , where x is 1 or 3, y is 2, 3 or 4, z is 0 or 2, and if x is 1, then z is 2; and non-ionic surfactant. The invention also includes a method of obtaining this tool, a diagnostic composition and a method of diagnosis with its use. The technical result consists in an improved neuronal capture of hydrophilic amorphous or amorphous-crystalline nanoparticles according to the invention, which allows diagnostics of lesions and precise localization of bulky structures in the central nervous system by intranasal administration of nanoparticles.

The technical field to which the invention relates.

This invention relates to the field of medicine and nanotechnology, and relates to the preparation of amorphous and amorphous-crystalline nanoparticles of manganese oxides and hydroxides and their use for MRI studies of the brain.

The level of technology

In the arsenal of modern diagnostic tools there is magnetic resonance imaging (MRI) with contrast agents (contrasts), in which organic gadolinium chelate complexes are used in practice. Known contrast agents for MRI are administered intravenously, which creates significant inconvenience for patients and increases the likelihood of complications. In addition, the contrast agents currently used do not allow for accurate topographic diagnostics of the lesions of the central nervous system and functional diagnostics of the state of the nervous pathways.

Attempts have been made to overcome the disadvantages associated with the use of gadolinium complexes. In particular, the authors of WO 2014035341 have developed bilayer nanoparticles containing gadolinium, manganese (P) or iron (III) in the form of oxides or chlorides, and the particles may contain lanthanides as dopants. From document WO 2014035341 it follows that this material, consisting of nanoparticles containing lanthanides as dopants, can be used as a contrast agent that improves the quality of the magnetic resonance signal.

Publication WO 2008093999 is considered as the closest analogue. It discloses the possibility of using nanoparticles of manganese oxide (MnO) in MRI diagnostics of tumor metastases in the brain, while solutions of these nanoparticles are injected intravenously, and accumulation in cancer cells occurs by non-specific capture of nanoparticles from the blood.

However, to date no low toxicity contrast agents have been found that could be effectively used for neurodiagnostic MRI studies. Thus, previous technical solutions in this area do not provide opportunities for accurate topographic diagnosis of pathological processes of the central nervous system, in particular, neurodegenerative and traumatic injuries, ischemic and hemorrhagic lesions, as well as voluminous formations, including tumors of the central nervous system.

Thus, the objective of this invention is to provide a new tool that provides the ability to solve these problems inherent in the technical solutions of the prior art.

To solve this problem, the present invention provides a contrast agent with increased neuronal uptake (in particular, it is captured by olfactory neuron receptors upon intranasal administration) and providing the ability to quantify capture and transmission by MRI.

Summary of Invention

The authors of the present invention developed nanoparticles, overcoming the disadvantages associated with the use of contrast agents of the prior art. It has been shown that with intranasal administration, hydrophilic amorphous and amorphous-crystalline nanoparticles in accordance with the invention are captured by olfactory receptors more efficiently than crystalline, and are transported along the neural pathways to the brain. Moreover, it was found that the trajectory of movement of nanoparticles in accordance with the invention is strictly localized within the conducting structures and nuclei of the limbic (olfactory) system. Based on the relationship between the number (concentration) of particles and the value of the T1-weighted MRI signal, the authors for the first time gave a quantitative description of the trajectory and dynamics of the passage of nanoparticles into the brain. The authors showed that the ability of nanoparticles containing Mn _x O _y H _z (also referred to as Mn _x O _y H _z - LF) to move along the nerve paths can be used for topographic diagnosis of pathological processes of the central nervous system, in particular, traumatic injuries. , ischemic, hemorrhagic lesions and neoplasms of the central nervous system relative to the nervous pathways by MRI.

The above technical results of the invention are achieved by the objects of the invention, given in the attached claims.

In particular, the invention is a contrast agent for magnetic resonance imaging, containing amorphous or amorphous-crystalline nanoparticles comprising manganese oxide or hydroxide of the formula Mn _x O _y H _z , where x is 1 or 3, y is 2, 3 or 4, z is 0 or 2, and if x is 1, then z is 2; and non-ionic surfactant.

In accordance with the invention, manganese oxide has the formula Mn ₃ O ₄ , and manganese hydroxide has the formula Mn (OH) ₂ or MnO (OH) ₂ .

The non-ionic surfactant may be selected from the group consisting of polysorbate, octylphenoxypolyethoxyethanol and octylphenol ethoxylate. In one of the embodiments of the implementation of the polysorbate is a polysorbate-20, octylphenoxypolyethoxyethanol is

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Nonidet P40, and ethoxylate octylphenol is Triton X100.

The nanoparticles in accordance with the invention have a diameter of from 20 to 50 nm (according to transmission electron microscopy) and a hydrodynamic diameter in the range from 100 to 150 nm (according to dynamic light scattering).

In one of the embodiments, the composition of the nanoparticles is characterized by the formula Mn _x O _y H _z · (polysorbate-20), while the polysorbate-20 is present in an amount of about 5-10 wt.% By weight of the nanoparticle.

The invention further relates to a diagnostic composition for magnetic resonance imaging, comprising the above agent and at least one targeted additive. The target additive may be a carrier, which is preferably water or saline containing 0.9% NaCl.

The final concentration of manganese [Mn] included in the nanoparticles. the composition is 0.1-1 mol / L.

The number of nanoparticles in the composition can range from 18.1 ± 3 to 321 ± 30.5 g / l.

In particular, the number of nanoparticles in the composition is from 32.1 ± 3 to 321 ± 30.5 g / l for nanoparticles prepared according to Protocol A);

from 18.1 ± 3 to 181 ± 30.5 g / l for nanoparticles prepared according to protocol B) and from 19.7 ± 3 to 197 ± 30.5 g / l (for nanoparticles prepared according to protocols D and D) .

The composition according to the invention may be a colloidal solution.

Preferably, the additional target additive is a mucolytic agent in an amount of from 0.1 to 10 wt.%, Inclusive of the weight of the composition. In this case, the mucolytic agent can be selected from acetylcysteine, erdostein, carbocysteine and ambroxol.

Preferably, magnetic resonance imaging is a magnetic resonance imaging of the central nervous system.

The invention also includes the use of said diagnostic composition as a diagnostic tool when conducting magnetic resonance imaging.

In addition, the invention includes a method for preparing the aforementioned agent, comprising preparing a first solution by adding to the initial solution of a non-ionic surfactant an initial solution of MnCl ₂ ;

preparing a second solution by adding to the initial KOH solution a solution of a non-ionic surfactant;

incubating the first solution and incubating the second solution;

mixing the first solution with the second solution;

homogenization of the resulting mixture and the deposition of nanoparticles.

After deposition, the obtained nanoparticles can be washed out from an excess of non-ionic surfactant and unreacted components.

In a preferred embodiment of the invention, the non-ionic surfactant is in the initial solution in an amount of 20% by weight.

In one of the embodiments of the invention, the non-ionic surfactant is a polysorbate, which can be a polysorbate-20.

The concentration of MnCl2 in the initial solution can be 0.5 mol / l, and the concentration of KOH can be 2 mol / l.

In one embodiment, the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes at room temperature, and the room temperature can be 2025 ° C.

In one embodiment, the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes with heating, and the heating can be carried out to a temperature of 80 ° C.

In an alternative embodiment, the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes while cooling, and cooling can be carried out to a temperature of 04 ° C.

Preferably, after mixing the first solution with the second solution, the mixture is divided into two parts in separate containers, wherein said containers are tubes.

In the private embodiment, the homogenization of the mixture is carried out using an ultrasonic homogenizer, and the deposition of nanoparticles is carried out by centrifugation.

Washing of the obtained nanoparticles from an excess of non-ionic surfactant and unreacted components can be carried out with water, and the obtained nanoparticles are washed 1-3 times.

In specific embodiments of the invention, the obtained nanoparticles are washed in ultrapure water, and centrifugation is carried out at 20,000 g and a temperature of 4 ° C. The finished particles are stored at room temperature.

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The invention further relates to a method for diagnosing a disease of the central nervous system, comprising intranasal administration of said composition and magnetic resonance imaging. The invention also includes the use of this composition as a means for diagnosing a disease of the central nervous system by conducting magnetic resonance imaging. According to the invention, the diagnosed disease of the central nervous system is a traumatic injury, ischemic damage, hemorrhagic damage or a tumor.

In one of the embodiments, the composition of the invention is further administered intravenously, and the magnetic resonance imaging of the central nervous system is carried out after the intravenous administration of said composition.

Preferably, magnetic resonance imaging of the central nervous system is magnetic resonance imaging of the brain.

List of figures

FIG. 1 shows the characteristics of the nanoparticles in accordance with the invention (see also Table 1, below).

FIG. 1A shows an image obtained by transmission electron microscopy of nanoparticles (NPs) of manganese oxide hydroxide (MnO (OH) ₂ ), manufactured according to Protocol D, below. The scale (50 nm) is indicated in the lower right corner.

FIG. 1B shows the results of EXAFS (Extended X-Ray absorption fine structure) spectroscopy of amorphous [am], amorphous-crystalline [am.-cr.] And crystalline [cr.] Nanoparticles: Mn (OH) ₂ [am] (protocol C), MnO (OH) ₂ [am.] (Protocol D), Mn ₃ O ₄ [am.cr.] (Protocol A) and Mn ₃ O ₄ [cr.] (Modified protocol A without the addition of polysorbate-20 ). Peaks marked as Mn-O correspond to the length of intramolecular interatomic bonds (<2.5 A). Peaks marked as Mn-O-Mn correspond to intermolecular distances (> 2.5 A). This figure confirms that, in contrast to crystalline nanoparticles for amorphous nanoparticles, there are no distinct peaks (> 2.5 A) corresponding to intermolecular bonds in the crystal. In amorphous-crystalline nanoparticles, the length of intermolecular bonds is longer than for crystalline ones, which implies a more loose packing.

FIG. 1B shows the determination of the solubility of nanoparticles containing Mn ₃ O ₄ (obtained in accordance with Protocol A, below) in water at a neutral pH and at an acidic pH of ~ 4.0. Characteristics of the dissolution parameters are derived by interpolating the logistic curve

Е = а / (1 + е ^_Ь '^ ^_с) ) _(g where X is the square root of time

Y is the square root of the% dissolved Mn ^{2+ ions} .

Nanoparticles obtained according to protocols C, D and D, below, have similar solubility properties.

FIG. 1G shows the dependence of the MRI signal on the concentration of manganese [Mn] (in molar concentration, μM) in the main olfactory bulbs (MOB). The MRI signal is expressed as binary logarithm (log ₂ ) of the intensity of the MRI signal before (S ⁰ ) and after (S ^[Mn] ) intranasal injection of nanoparticles containing Mn ₃ O ₄ . MRI analysis was performed 24 hours after the intranasal administration of nanoparticles containing Mn ₃ O ₄ (X: log ₂ (MPT: S ^[Mn] / S ⁰ )). After that, to determine the concentration of manganese [Mn] in MOB used the method of two-string arc atomic emission spectroscopy. The graph is based on data on the concentration of manganese [Mn] and the level of the MRI signal in the MOB male line of BALB / c mice (N = 7). The olfactory bulbs collected from each mouse were combined into one sample, and the concentration of manganese [Mn] was determined in 7 samples.

FIG. 1D shows the recalibration of the MRI signal (X; Nd ₂ (MRI: S ^[Mn] / S ⁰ )) depending on the accumulation of nanoparticles (NPs) containing Mn ₃ O ₄ in MOB, expressed in the number of nanoparticles containing Mn3O4, on neuron.

FIG. 2 shows an MRI analysis of nanoparticle accumulation in the brain.

FIG. 2A shows the accumulation of amorphous nanoparticles of manganese oxide-hydroxide (Mn (OH) 2), amorphous-crystalline nanoparticles of manganese oxide (Mn ₃ O ₄ ) obtained in accordance with the following protocols (G and A, respectively), and commercial crystalline nanoparticles (Gd ₂ O ₃ ) in the olfactory bulbs (MOB). MRI analysis of the accumulation of nanoparticles was carried out 24 hours after the intranasal introduction of nanoparticles into the right nostril. The relative intensity of the MRI signal is marked on the scale. It can be seen that hydrophilic amorphous oxide-hydroxide nanoparticles (Mn (OH) 2) and amorphous-crystalline manganese oxide (Mn3O4) are effectively captured by olfactory receptors from the surface of the olfactory epithelium and pass into the main olfactory bulb (MOB) by axonal transport.

Crystalline Gd ₂ O ₃ nanoparticles are not detected in the MOB, despite the similar parameters of r1 relaxivity (see Table 1 below), which indicates a predominant neuronal

- 3 031124 capture of amorphous and amorphous-crystalline nanoparticles in accordance with the invention, in contrast to the lack of capture of commercial Gd ₂ O ₃ nanoparticles.

FIG. 2B shows an MRI analysis of the accumulation in the brain of nanoparticles containing manganese oxide Mn ₃ O ₄ , with intranasal administration. Nanoparticles were injected into the right nostril, which led to their accumulation on the ipsilateral (right) side of the brain. The time scan of MRT images (24-48 hours, 48-96 hours, 168 hours, and 672 hours after intranasal administration) taken at different depth of sections (4, 6, 7 mm) is shown. The relative intensity of the MRI signal is marked on the scale. The sections show the structures of the olfactory tract and the limbic system (see diagram in Fig. 2B), characterized by the highest intensity of the MRI signal due to the accumulation of nanoparticles.

FIG. 2B is a schematic representation of the nuclei and pathways of the limbic system of the brain, starting from the olfactory epithelium (ОЕ), in which nanoparticles prepared according to protocols A, B, D, D are given below. The following structures are marked on the diagram: MOB is the main olfactory bulb; AON - front olfactory nucleus; Pir - pear bark; aPir almond-shaped pear-shaped transient field; AI is the agranular region of the insular cortex; LEnt / MEnt lateral entorhinal cortex / medial entorhinal cortex; DG - dentate gyrus; AMYG - amygdala; GP - pale ball; BNST is the core of the edge strip; Hip - hippocampus; LSD - dorsolateral septum; CIC / InCo - the central nucleus of the lower collic of the quadrilateral / intercollicular nucleus; SR — caudal part of the shell; SVZ - subventricular zone. Numbers indicate the number of synaptic transmissions overcome by the nanoparticles of the invention, ranging from olfactory receptors located in the OU.

FIG. 3 shows an example of constructing a mathematical model describing the quantitative dynamics of accumulation of nanoparticles (using as an example nanoparticles containing Mn3O4), as measured by MRI in the olfactory bulb (MOB). Left panel. The model is based on two functions:

MRI zoom function

and the function of decreasing MRI signal ^S deceit) = ^ex P

Right panel. The equation

5 (1) - 5 _max · · 5 ^ _eee y (t) describes the full dynamics of nanoparticle accumulation, where

V max reflects the asymptotic maximum of the MRI signal. The model coefficients are found by the method of nonlinear least squares of the experimental values of the MRI signal, measured after intranasal administration (0.25; 0.5; 1; 2; 4; 12; 24; 48; 96; 168; 336; 504; 672 h) . The calculated maximum accumulation is marked by a dot, the band corresponds to the 95% confidence interval of the model prediction. The coefficients of the models describing the accumulation of nanoparticles in the nuclei of the limbic system are shown in Table. 2 (given below), calculated for nanoparticles containing Mn3O4. In addition, the average time and the maximum rate of accumulation and decrease in the number of nanoparticles can be calculated from the models obtained (see Table 3 below).

FIG. 4 shows the relationship between the various parameters of the accumulation and elimination of nanoparticles containing Mn ₃ O ₄ and the number of synaptic gears overcome by nanoparticles. The obtained dependences were determined on the basis of quantitative analysis of the MRI signal (see Example 4, Tables 2 and 3) after intranasal administration of 10 μl of nanoparticles at a molar concentration of [Mn] in the final composition of 0.2 mol / L.

FIG. 4A shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system and the characteristic accumulation time ·

FIG. 4B shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system and the maximum accumulation rate (h ^-1 ).

FIG. 4B shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system and the maximum magnitude of the MRI signal < ^max > ·

FIG. 4G shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system, the maximum concentration of manganese [Mn] (molar concentration, mM), and the number of nanoparticles containing Mn ₃ O ₄ per cell.

FIG. 4D shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system and the characteristic time of elimination ( ^Tdeca v).

FIG. 4E shows the relationship between the number of synaptic transmissions connecting the nuclei of the limbic system and the maximum rate of elimination (h ^-1 ).

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All values are shown on a logarithmic scale and correlation coefficients (r) are indicated. The lines correspond to regression models calculated on a logarithmic scale.

FIG. 5 shows the MRI topography of a brain tumor (xenotransplanted human glioblastoma) relative to the neural pathways after intranasal administration of nanoparticles containing Mn ₃ O ₄ prepared in accordance with this invention. Human U-87 glioblastoma cancer cells were transplanted into SCID mice in the hippocampal region (left panel). Upon reaching significant development of glioblastoma (glioma, left panel), nanoparticles containing Mn ₃ O ₄ were intranasally injected into mice. The growth and general localization of the tumor were determined using a T2-weighted MRI of the brain (the tumor is circled in a gray oval). The dynamics of passage and accumulation of nanoparticles containing Mn ₃ O ₄ from the olfactory bulb (MOB) was investigated using T1-weighted MRI tomography (accumulation areas within the tumor are marked with a white oval). After 96 h, substantial accumulation of nanoparticles containing Mn ₃ O ₄ is found in those parts of the tumor that intersect the path of nanoparticles through the structures of the limbic system, for example, in the areas of the hippocampus (Hip), dorsolateral partition (LSD) and subventricular zone (SVZ). The relative intensity of the MRI signal is marked on the scale.

FIG. 6 shows the quantitative dynamics of the passage of nanoparticles containing Mn ₃ O ₄ through the limbic system in SCID mice with human xenografts. The block diagrams of the accumulation of nanoparticles containing Mn ₃ O ₄ in the structures of the limbic system: MOB, AON, Pir, Hip, and also in the tumor itself (glioma), after intranasal administration in SCID mice with developed glioblastomas (xenotransplant group) with respect to to reference models, expressed in the magnitude of the MRI signal. The diagrams (boxes) correspond to a 25-75% range of values on a sample of 6 mice, the lines indicate the median, and the whiskers are -1.5xIRQ (interquartile range).

FIG. 7 shows nonspecific accumulation of nanoparticles containing Mn3O4 in human glioblastomas when nanoparticles are introduced into the blood.

When administered intravenously, there is a rapid accumulation of nanoparticles containing Mn ₃ O ₄ in the glioblastoma (glioma, white oval) for already 10 minutes. MRI analysis after the intravenous injection of these nanoparticles allows to detect the development of a cancer tumor at early stages, but does not allow to identify the topographic location (map) of the tumor relative to the nerve pathways. The growth and general localization of the tumor were determined using a T2-weighted MRI of the brain. The dynamics of passage and accumulation of nanoparticles containing Mn ₃ O ₄ were examined using T1-weighted MRI tomography. The relative intensity of the MRI signal is marked on the scale.

The implementation of the invention

The invention is further illustrated by experimental examples, confirming the possibility of carrying out the invention with the achievement of the above technical results.

Example 1. The preparation and characterization of stable nanoparticles of manganese oxides and hydroxides.

For the synthesis of nanoparticles of manganese oxides and hydroxides, the following initial solutions were used.

Reagent 1: manganese chloride (MnCl ₂ ) at a concentration of 0.5 mol / l;

Reagent 2: Polysorbate-20 (commercial name Tween® 20) in an amount of 20% by weight;

Reagent 3: potassium hydroxide (KOH) at a concentration of 2 mol / l;

Reagent 4: hydrogen peroxide (H ₂ O ₂ ) in an amount of 10 wt.%.

All solutions were prepared in ultrapure water Mili-Q, corresponding to the international standard ISO 3696.

Next, the particles were obtained in accordance with the following protocols.

Protocols A, B, V.

1) From the initial reagents, 2 solutions were prepared in 50 ml Falcon ™ plastic tubes.

Solution 1: To 15 ml of reagent 2 solution (20 wt.% Tween® 20) 10 ml of reagent 1 solution (MnCl ₂ at a concentration of 0.5 mol / l) was added,

Solution 2: To 5 ml of reagent 3 solution (KOH at a concentration of 2 mol / l) was added 20 ml of reagent 2 solution (Tween® 20 in an amount of 20 wt.%).

Protocol A: solution 1 and solution 2 were incubated for 30-60 minutes at room temperature (20-25 ° C).

Protocol B: solution 1 and solution 2 were incubated for 30-60 minutes in a thermostat at 80 ° C.

Protocol B: solution 1 and solution 2 were incubated for 30-60 minutes on ice (0-4 ° C).

2) After this, solution 1 was added to solution 1 and, after stirring, the prepared mixture was divided into two equal parts (25 ml each) in Falcon ™ 50 ml tubes. The tubes were placed in a QSonica Q700 ultrasonic homogenizer bath (QSonica, USA) and the reaction was carried out for 60 minutes at room temperature (20-25 ° C) with the following ultrasonic homogenization parameters: frequency - 20 kHz, power - 300 W / l, 60 cycles (30 s with the device turned on and 30 s with the device turned off).

3) After completion of the reaction, the obtained nanoparticles were washed from an excess of polysorbate-20 and unreacted components. For this, nanoparticles were precipitated by centrifugation: 10 minutes at

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20000 g (relative strength of centrifugation - RCF) and a temperature of 4 ° C. After centrifugation, the supernatant was collected and the particles were diluted in ultrapure Mili-Q water. Water was added to 25 ml to each tube. The resulting solution was homogenized in an ultrasonic homogenizer QSonica Q700 (QSonica, USA) at room temperature (20-25 ° C) with the following parameters of ultrasonic homogenization: frequency - 20 kHz, power - 300 W / l, 60 cycles (30 s with the device turned on and 30 seconds when turned off).

4) Next, the particles were washed 3 times with water, as described above in step 3, and the particles were besieged each time by centrifugation for 40 minutes at 20,000 g and 4 ° C. After the last ultrasonic washing, the finished particles are stored at room temperature.

Nanoparticles obtained according to protocol (A) correspond to Mn ₃ O ₄ .

Nanoparticles obtained according to protocol (B) correspond to Mn ₃ O ₄ with a non-separable impurity of Mn (OH) ₂ nanoparticles.

After 2-3 weeks, the nanoparticles prepared according to protocol (B) are divided into 2 approximately equal phases by centrifuging at 20,000 g for 40 minutes at 4 ° C. The lower (dark) phase is represented by Mn ₃ O ₄ nanoparticles, the upper (whitish) Mn (OH) ₂ nanoparticles. Thus, the separation of the upper phase from the bottom after the implementation of the method according to the protocol (B) allows to obtain two types of manganese nanoparticles, namely nanoparticles containing Mn3O4, and nanoparticles containing Mn (OH) 2.

Protocol G.

1) From the initial solutions were prepared 2 solutions in plastic tubes Falcon ™ 50 ml.

Solution 1: To 15 ml of reagent 2 solution (20% Tween® 20 in the amount of 20 wt.%) 10 ml of reagent 1 solution (MnCl ₂ at a concentration of 0.5 mol / l) was added;

Solution 2: To 5 ml of reagent 3 solution (KOH at a concentration of 2 mol / l) were added 20 ml of reagent 2 solution (Tween® 20 in an amount of 20 wt.%) And 2 ml of reagent 4 (H ₂ O ₂ in an amount of 10 wt. %).

Solution 1 and solution 2 were incubated for 10-20 minutes at room temperature (20-25 ° C). Next, the reaction was carried out as described in Protocol (A).

The nanoparticles obtained by the protocol (G) correspond to MnO (OH) 2.

Protocol D.

Solution 1: To 15 ml of reagent 2 solution (Tween® 20 in the amount of 20 wt.%) 10 ml of reagent 1 solution (MnCl ₂ at a concentration of 0.5 mol / l) was added;

Solution 2: To 5 ml of reagent 3 solution (KOH at a concentration of 2 mol / l) was added 20 ml of reagent 2 solution (20% Tween® 20).

Solution 1 and solution 2 were incubated for 30-60 minutes at room temperature (20-25 ° C).

2) After that, the tube with solution 2 was placed in a QSonica Q700 ultrasonic homogenizer bath (QSonica, USA) and with the device turned on (frequency - 20 kHz, power - 300 W / l) solution 1 and 2 ml of reagent 4 solution (H ₂ O ₂ in the amount of 10 wt.%). After mixing by transferring the mixture from one tube to another (2 times), the prepared mixture was divided into two equal parts (25 ml each) in Falcon ™ 50 ml tubes and subjected to ultrasound treatment as described in protocol (A).

All subsequent steps were carried out as described in protocol (A).

Nanoparticles obtained according to protocol (D) correspond to MnO (OH) 2 with an admixture of Mn3O4 nanoparticles.

The volume of the reaction mixture can be reduced to 5 ml or increased in the presence of an ultrasonic homogenizer for large volumes.

It should be noted that polysorbate (in particular, polysorbate-20, which was used in Protocols A to D) can be replaced by other non-ionic surfactants (Nonidet P40 (Nonidet P40), Triton X100 (Triton X100), etc. ).

The size of the nanoparticles containing the above oxides and hydroxides of manganese, can vary depending on the concentration of nonionic surfactants, in particular polysorbate-20. Polysorbate-20 is present in nanoparticles in a concentration of from 5 to 10 wt.%. The concentration of the incorporated polysorbate-20 can be estimated by weighing the dried nanoparticles.

The temperature and time of incubation of solutions 1 and 2 can be varied to increase or decrease the degree of oxidation of manganese in nanoparticles.

Example 2. Obtaining a diagnostic composition containing nanoparticles according to the invention.

To obtain a diagnostic composition for intranasal administration after the last ultrasonic washing, the prepared particles are dissolved or

a) in water with or without added mucolytic agent; or

b) in physiological solution (0.9 wt.% NaCl) with or without the addition of mucolytic agent; or

c) in any other buffer medium compatible with intranasal administration, with or without the addition of mucolytic agent.

The volume of solvent according to the above options a), b) and C) preparation of the composition

- 6 031124 can vary from 5 to 50 ml depending on the desired concentration of nanoparticles and, in a preferred embodiment, is 25 ml. The molar concentration of manganese [Mn] included in the nanoparticles in the final composition is preferably 0.2 mol / l, but depending on the final volume of the composition it can vary in the range from 0.1 to 1 mol / l.

For the preparation of a composition intended for intravenous administration, the nanoparticles are dissolved in physiological solution (0.9 wt.% NaCl) or in any other buffer medium suitable for intravenous administration in a volume from 5 to 50 ml, without the addition of a mucolytic agent. In a preferred embodiment, the preparation of a composition intended for intravenous administration, the volume of solvent is 25 ml.

The resulting solution is homogenized in an ultrasonic homogenizer. Preferably, a QSonica Q700 homogenizer (QSonica, USA) is used, the homogenization is carried out at room temperature (20-25 ° C) with the following parameters of ultrasonic homogenization: frequency - 20 kHz, power - 300 W / l, 60 cycles (30 s with the device turned on and 30 seconds when turned off).

The technical result achieved by the addition of mucolytic agents is to improve the passage of nanoparticles according to the invention from the olfactory epithelium to the brain. Acetylcysteine (ACC, or N-acetyl-Gcysteine) is preferably used as a mucolytic agent; in other embodiments, carbocysteine, erdostein, ambroxol, or any other agent that depolymerizes and liquefies the mucoprotein and mucopolysaccharide fibers can be used. The amount of mucolytic agent can be from 0.1 to 10 wt.%, Inclusive of the weight of the composition. In particular, the amount of mucolytic agent can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wt.%. Alternatively, any amount may be used in the intervals between the indicated values. Thus, the diagnostic composition of the invention is a solution containing nanoparticles prepared in accordance with any of the above protocols A, B, D and / or D, water or saline and, optionally, a mucolytic agent.

Examples of obtaining diagnostic compositions for intranasal administration.

A ') Nanoparticles obtained according to Protocol A (or B, D, D), after the last wash, are dissolved in 25 ml of water containing an amount of acetylcysteine, carbocysteine, erdostein or ambroxol, selected from the following: 0.1, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 wt.%. While the molar concentration of manganese [Mn], part of the nanoparticles in the final composition is 0.2 mol / L. The number of nanoparticles in the finished composition can be 64.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol A); 36.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol C) and 39.4 ± 6.1 g / l (for nanoparticles prepared according to Protocols D and D). The correction to the mass of nanoparticles ± 6.1 g / l depends on the amount of polysorbate-20 included in the composition of the nanoparticles, which can vary from 5 to 10% by weight of the nanoparticles.

B ') Nanoparticles obtained according to Protocol A (or B, D, D), after the last wash, are dissolved in 25 ml of water containing 0.9 wt.% NaCl, with an amount of acetylcysteine, carbocysteine, erdosteine or ambroxol selected from the following: 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt.%. While the molar concentration of manganese [Mn], part of the nanoparticles in the final composition is 0.2 mol / L. The number of nanoparticles in the finished composition can be 64.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol A); 36.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol C) and 39.4 ± 6.1 g / l (for nanoparticles prepared according to Protocols D and D).

B ') Nanoparticles obtained according to Protocol A (or B, D, D), after the last wash, are dissolved in 25 ml of sodium phosphate buffer (8.00 g / l NaCl, 0.20 g / l KCl, 1.44 g / l Na ₂ HPO ₄ , 0.24 g / l KH ₂ PO ₄ , pH 7.4), with an amount of acetylcysteine, carbocysteine, erdosteine or ambroxol, selected from the following: 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt.%. While the molar concentration of manganese [Mn], part of the nanoparticles in the final composition is 0.2 mol / L. The number of nanoparticles in the finished composition can be 64.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol A); 36.2 ± 6.1 g / l (for nanoparticles prepared according to Protocol C) and 39.4 ± 6.1 g / l (for nanoparticles prepared according to Protocols D and D).

The resulting composition is homogenized in an ultrasonic homogenizer QSonica Q700 (QSonica, USA) at room temperature (20-25 ° C) with the following parameters of ultrasonic homogenization: frequency - 20 kHz, power - 300 W / l, 60 cycles (30 s with the device turned on and 30 seconds when turned off). The finished compositions are stored at room temperature or at + 4 ° C. Before use, after long storage (more than a month), it is desirable to rehomogenize the finished compositions in an ultrasonic homogenizer with the same parameters: frequency -20 kHz, power - 300 W / l, 60 cycles (30 s with the device turned on and 30 s with the device off).

Example 3. The study of the physico-chemical properties of the obtained nanoparticles.

The morphology and size of nanoparticles.

The morphology and dimensions (diameter) of nanoparticles containing manganese oxides, manganese oxide hydroxides and manganese hydroxides in accordance with the invention (Fig. 1A) were studied by means of

- 7 031124 scanning electron microscopy (TEM) on a JEM 1400 microscope (JEOL, Japan) with a Veleta digital camera (SIS, Germany).

Hydrodynamic diameter and zeta potential (Q nanoparticles containing oxides, oxides of hydroxides and manganese hydroxides in accordance with the invention, was determined in suspension by dynamic light scattering at 90 ° scattering angles and 22 ° C and electrophoresis, respectively, using Zetasizer NanoZS (Malvern, England ) in accordance with the manufacturer’s protocol (Table 1).

Among other things, the authors determined the structure of nanoparticles containing manganese oxides and manganese hydroxides in accordance with the invention (Table 1) using EXAFS spectroscopy, FIG. 1B). In accordance with the invention, nanoparticles prepared according to Protocol A have an amorphous-crystalline structure; according to protocols C, D, D - amorphous structure.

Table 1

Nanoparticle Characteristics

Nanoparticles Structure * ³ * cUq (nm) **> ς (mV) <®> rl, (mM1. s- ¹ ) <=> r2, (mM ^-1 s' ¹ ) <"> Accumulation ^{| r} > M113O4 (Protocol A) amorphous crystal 128.3 ± 11 -25, b ± 3 0.28 10.71 + MP (OH) 2 (Protocol B) amorphous 115.2 ± 15 -22.7 ± 7 0, 35 12.7 ++ MpO (OH) g amorphous 128.6 ± -22.3 ± 5 0.31 11.74 ++ (Protocol D) 24 MpO (OH) 2 (Protocol E) amorphous 132.5 ± 11 -19.8 ± 4 0, 35 13, 88 ++ Gd ₂ O ₄ (USNano) crystalline 152 ± 22 -48 ± 5.2 0.5 13.2 -

^(a) The structure of the nanoparticles (amorphous, amorphous-crystalline and crystalline) was determined by EXAFS spectroscopy. Nanoparticles prepared in accordance with protocols A, B, D and D, do not have a pronounced crystal lattice and have an amorphous or amorphous-crystalline structure.

^{(b) The} Idrodynamic diameter (d _aq ) and zeta potential were measured using a Zetasizer Nano ZS ™ (Malvern).

^{(c) The} r1 and r2 relaxation of nanoparticles were determined on a BioSpec 117/16 USR (11rub), 11.7 Tesla ultra-high-resolution tomograph.

^(d) Accumulation of nanoparticles in olfactory bulbs.

X-ray diffraction data (XRD, X-ray powder diffraction) confirm that nanoparticles prepared according to protocols A, C, D, D correspond to the indicated formulas. XRD was carried out on the VEPP-3 accelerator complex with the following synchrotron radiation parameters: a monochromatic beam wavelength λ = 0.1516 nm and an angle range of 2Θ from 45 to 75 °. The spectra were compared with the diffraction base of this Match! (Crystal Impact, Germany).

Manganese is a paramagnetic, therefore, is able to reduce the time of spin-lattice relaxation T1. For all manganese nanoparticles used, characteristic relaxation times were obtained on a BioSpec 117/16 USR (Bruker, Germany) -11.7 ultra-high-resolution tomograph (Table 1). The reciprocal of the relaxation value (T1 ^-1 , ms ^-1 ) was directly proportional to the concentration of nanoparticles containing oxides, oxides, hydroxides and manganese hydroxides in accordance with the invention, and inversely proportional to size. All nanoparticles obtained by the authors have increased R1 relaxivity, which makes them suitable for MRI studies as contrast agents.

Solubility of nanoparticles containing manganese oxides and manganese hydroxides in accordance with the invention.

One of the important properties of these nanoparticles is their low solubility, which reduces the toxicity associated with soluble manganese ions. To measure the solubility of nanoparticles containing Mn ₃ O ₄ prepared according to Protocol A, the authors used the property that adding Mn ^{2+ ions} to a solution of nanoparticles containing Mn ₃ O ₄ reduces T1 relaxation time. This allows you to directly evaluate the solubility of nanoparticles containing Mn ₃ O ₄ , based on the calibration

- 8,031,124 curve. Thus, to assess the solubility, a colloidal solution of nanoparticles containing Mn ₃ O ₄ was diluted in 10 volumes of citrate buffer (citric acid / sodium citrate; pH ~ 7.0 or pH ~ 4.0) and incubated at 22 ° C for 240 h with sampling (400 μl) to determine the T1 relaxation time. At neutral pH, the T1 relaxation time does not change, which indicates the insolubility of nanoparticles containing Mn ₃ O ₄ . At acidic pH (~ 4.0), characteristic of a number of cellular organelles, such as lysosomes, there is a decrease in T1 relaxation time, which indicates a partial dissolution of nanoparticles containing Mn ₃ O ₄ . The dissolution of nanoparticles containing Mn ₃ O ₄ at pH ~ 4.0 is described by a logistic curve, analysis of which shows that the maximum released concentration of Mn ^{2 + / 3 +} ions reaches ~ 8.7% of the total volume after 48 hours with a plateau ( Fig. 1B). Nanoparticles prepared according to protocols C, D and D, had comparable solubility characteristics.

Example 4. Diagnostic studies using nanoparticles containing manganese oxides and hydroxides as contrast agents for MRI.

Lines of mice, conditions of detention and research protocols in mice.

Studies of the passage of nanoparticles from the nasal cavity into the brain were carried out on male BALB / c SPF (Specific Pathogen Free) male status of 12-14 weeks old, weighing 28-32 g.

After jigging at 3 weeks of age and prior to the beginning of the experiments, the mice were kept in groups of 5 individuals of the same sex in individually ventilated cells of the OptiMice system (Animal Care Systems, RF) under controlled conditions, at a temperature of 22-26 ° C, relative humidity of 30-60% and light mode light / dark 14/10. Briquetted feed (SSNIFF, Bio Service, Germany) and mouse water were given ad libitum. Dust free sawdust was used as a litter material.

For studies of the passage of nanoparticles from the nose to the brain, colloidal solutions of nanoparticles containing Mn _x O _y (at a molar concentration of [Mn] 0.2 mol / l) were administered once into one or both nostrils (intranasally) 10 μl each.

Studies of the passage of nanoparticles containing Mn _x O _y from the nasal cavity to the brain during the development of a brain tumor were conducted on SCID males (SHO-Prkdc ^scid Hr ^hr ) (Charles River, USA) with SPF status at the age of 8-12 weeks. For this purpose, before starting the experiment, U87 glioblastoma cell cultures were cultured (5-7 passages) in DMEM-F12 (1: 1) medium containing 10% fetal serum (FBS). Before injection, the tumor cells were removed from the substrate with Trypsin-Versen solution, centrifuged for 5 min at 1000 rpm (rpm) and resuspended in serum-free medium at a concentration of 100 thousand cells in 1 μl. Before the operation, the animal was anesthetized with isoflurane gas anesthesia (Baxter Healthcare Corp., USA) using the Univentor 400 Anesthesia Unit (Univentor, Malta) on a heated operating table with a surface temperature of 37 ° C. The cell suspension was introduced into the subcortical structures of the brain in the region of the hippocampus to a point through an opening in the cranium of the animal. To do this, a 3-4 mm long skin incision was made in the bregma area on the head in the caudal-cranial direction and 5 μl of cell suspension (500 thousand cells) was injected through the opening in the skull. The growth dynamics of tumors and the accumulation of nanoparticles containing Mn ₃ O ₄ in them were investigated using magnetic resonance imaging (MRI).

Magnetic resonance imaging (MRI).

The distribution of MnxOy-containing nanoparticles by brain structures and mouse blood was investigated by MRI based on T1-weighted images using the FLASH (Fast Low Angle Shot) method using the BioSpec 117/16 USR Ultra-High Tomography (Bruker, Germany) -11 , 7 Tesla. Parameters of the pulse sequence of the method (TE = 2.5 ms, TR = 200 ms); image parameters (size 2.5x2.5 cm; matrix 512x512 pixels; slice thickness 0.5 mm; distance between slices 0.5 mm; number of slices 15; orientation of the slices is axial) total scan time 6 min. To obtain the numerical values of the MRI contrast for each of the investigated image structure were subjected to additional processing. To get rid of irregularities associated with the sensitivity of the surface coil, a Gaussian low-pass filter with a radius of 10 pixels was applied to the original image. The magnitude of the MRI contrast (S (t)) was calculated as the ratio of the MRI signal after the introduction of nanoparticles containing Mn _x O _y <> to the magnitude of the signal before the introduction of nanoparticles containing Mn _x O _y ₍ S ^O j. S (t) = 1 ₂ (MRI: S ^[W ^] / S °).

To calibrate the values of the MRI signal with the accumulation of nanoparticles containing Mn ₃ O ₄ in the brain, the method of two-stringed atomic emission spectroscopy was used in the brain. For this, 6 males of the BALB / c line were intranasally injected with 10 μl of a colloidal solution of Mn3O4 - NP (nanoparticles containing Mn3O4) prepared according to Protocol A into each nostril and after 24 hours the magnitude of the MRT signal

Immediately after MRI, olfactory bulbs were removed from mice to further determine the concentration of manganese using a two-stringed atomic-emitting plasmatron arc. Both olfactory bulbs from each animal were combined into one sample, thus, all

- 9 031124 6 samples were formed. Data on the concentration of Mn in the olfactory bulbs studied was highly reliably correlated with the MRI signal values (Fig. 1D).

Considering that the average diameter of nanoparticles containing Mn ₃ O ₄ is ~ 30 nm; spherical volume (V) -1,414-10 nm (1,414-10 ^- cm); density (ρ) Mn3O4 ~ 4.86 g / cm; mass nanoparticles containing Mn3O4 (m = Vp), is -0.1 femtogramm (1.0-10 ^-16 g) and the number of molecules per particle Mn3O ₄ is -263,2-10 ^3. From here it is possible to compare the concentration of manganese accumulated in the tissues with the number of nanoparticles. So, taking into account the fact that nanoparticles containing Mn _x O _y predominantly accumulate in the glomerular and mitral layers of the olfactory bulb, where the average number of neurons is -3.8-10 ⁸ / cm ³ , which is comparable to the average density of neurons in the brain component -10 ⁸ / cm ³ (or gram), the authors attributed the change in MRI signal to the number of particles per cell (Fig. 1D).

Improved passage of amorphous and amorphous-crystalline Mn _x O _y H _z nanoparticles - NP from the nose to the brain.

With the direct introduction of Mn _x O _y H _z solutions - NPs (nanoparticles containing Mn _x O _y H _z ) into the nasal cavity, there is a uniform sedimentation of nanoparticles on the surface of the olfactory epithelium (OE) and the capture of olfactory receptors by cells. After that, Mn _x O _y H _z - LFs are transferred to the olfactory bulb nucleus (MOB) by axonal transport, as shown by quantitative T1-weighted MRI analysis (Fig. 2A). At the same time, the passage of amorphous nanoparticles prepared according to protocols C, D, D, and amorphous-crystalline (protocol A) is significantly improved compared to crystalline Gd ₂ O ₃ nanoparticles (Fig. 2 A).

Neuronal transport of amorphous and amorphous-crystalline Mn _x O _y H _z nanoparticles - NP.

From MOB, nanoparticles can move deeper into the brain along the olfactory tract and the limbic system. Indeed, MRI analysis of the accumulation of Mn ₃ O ₄ - NP revealed a significant increase in the MRI signal compared to control (before the introduction of particles) for 168 h in the following structures: anterior olfactory nucleus (AON); pear-shaped bark (Piriform Cortex - Pir); Agranular region of the insular cortex (Agranular Insular Cortex - AI); Lateral entorhinal cortex / medial entorhinal cortex (Lateral and Medial Entorhinal Cortex - LEnt / MEnt); dentate gyrus (Dentate Gyrus - DG); the amygdala (Amygdala - AMYG); Pale Ball (Globus Pallidus - GP); the core core of the edge strip (Bed Nucleus of the Terminal Terminal - BNST); Hippocampus (Hippocampus Hip); dorsolateral septum (Dorsolateral Septum - LSD); nuclei of the thalamus: the central nucleus of the lower collar of the tetragonal / intercollicular nucleus (CIC / InCo); caudal shell (Caudoputamen - CP); as well as the subventricular Zone (SVZ), and a number of other brain structures (Fig. 2B, 2B). It should be noted that during the whole time, up to hatching, the Mn _x O _y H _z nanoparticles remain strictly localized within the limits of the designated brain structures. This indicates that the nanoparticles spread through the brain by neuronal transport intracellularly, while overcoming a number of synaptic transmissions. Thus, in order to reach the deepest regions represented by the CIC / InCo thalamus nuclei, at least 7 synaptic transmissions must be overcome from the surface of the OE nanoparticles (Fig. 1B).

Statistical and mathematical analysis of the dynamics of the accumulation of MnxOyHz - LF.

MR images were processed and counted using ImageJ using a set of standard tools (Schneideretal., 2012). Statistical data analysis, linear and non-linear regression models were performed in R (RCoreTeam, 2015). To search for the coefficients of nonlinear regression models, the Levenberg-Marquardt algorithm was used, aimed at solving least-squares problems with a restriction on the parameters.

Briefly, the dynamics of accumulation and elimination of nanoparticles containing Mn _x O _y H _z , according to time J) different brain regions, expressed by the magnitude of the MRI signal

S (t) = log ₂ (MRT: y1 ^Mp 1/5 °) can be empirically described as follows:

where corresponds to the asymptotic maximum of the MRI signal;

^t ass and ^{τ <ΙεΓα} ? - characteristic time of accumulation and decrease, respectively;

= describes an exponential increase in the MRI signal, depending on

time; and describes an exponential decrease in the MRI signal as a function of time (Fig. 3).

- 10 031124

Next, taking the derivatives of and you can determine the changes in the rate of increase and decrease ^(deca / \ MRI signal depending on time. So,

In addition, one can calculate the mean accumulation / decrease time 'as well as meMn _x OyHz-H4 τ τ · Γ (1 + 1 //?) (G is the gamma function) and the variance of the diane ( ^ίϊχ2 ) and the mode ^Μο ~ ^τ ¹ 4β for times of accumulation / decrease. Finally, the maximum rate of accumulation / decrease achieved with ^{tl = Mo} “^ · and ^t2 ~ ^WOctecay , respectively, will be equal to

Quantitative dynamics of the passage of nanoparticles Mn _x O _Y H _z - NP from the nose to the brain.

Thus, the determination of the coefficients of the model (1) from experimental data on the accumulation of Mn _x O _y H _z - LF in different parts of the brain, detected by MRI, gives a complete picture of the dynamics of the passage of nanoparticles along the nerve paths (Tables 2, 3).

table 2

Model ¹ parameters ^! changes in the MPT signal as a function of time after intranasal administration of Mn ₃ O ₄ - LF

Structure S _waKC ± SE ± SE pdeciy ± SE ± SE Tctacay ± SE MOV 3.31 ± 0.12 0, 65 ± 0.04 1.24 ± 0, 11 3.63 ± 0.47 333.74 ± 16, 67 AON 1.79 ± 0, 12 1, 68 ± 0.14 1.23 ± 0.15 18.30 ± 1.23 239.13 ± 19, 12 PIR 2.23 ± 0, 07 1, 90 ± 0.15 1.58 ± 0.14 16.23 ± 0.73 332.38 ± 13, 19 AI 1, 58 ± 0, 13 1, 48 ± 0.15 1.43 ± 0.21 21.54 ± 2, 14 279.19 ± 23, 50 LEnt 1.29 ± 0, 06 2, 11 ± 0,39 1.43 ± 0.21 11.70 ± 0.78 392.73 ± 25, 59 Ment 1, 17 + 0, 07 1.19 + 0.13 1.99 + 0.34 25.19 + 3.10 426.94 + 23.78 DG 0, 95 ± 0, 05 1, 63 ± 0.26 3.36 + 0.68 45.40 ± 4.83 463.11 ± 19, 05 Svz 1.58 ± 0, 11 1.01 ± 0.09 2.41 ± 0.35 39.14 ± 5.61 457.69 ± 19, 64 CP 0, 81 ± 0.23 1.49 ± 0.15 1.17 ± 0.35 46.39 ± 9.69 274.15 ± 77.27 Amygdala 1, 85 ± 0.20 1.29 ± 0.11 2.33 ± 0.37 72.16 ± 11.11 435.15 ± 25, 92 GP 1, 86 + 0.46 1.41 + 0.14 2.24 + 0.53 95.23 + 25.12 370.01 + 44.81 Bnst 1.29 ± 0, 12 1.13 ± 0.11 3.25 + 0.55 67.73 + 11.49 482.30 ± 19.24 Hip 1.36 ± 0, 10 1.00 ± 0.10 2.30 ± 0.39 33.28 ± 5.38 430.74 ± 22.30 LSD 0, 92 ± 0, 10 2.76 ± 0.63 1.63 ± 0.35 57.04 ± 5, 07 402.41 ± 37.48 CIC / InCo 1.24 ± 0, 38 1.40 ± 0, 17 1.56 ± 0.55 74.68 ± 21.92 394.04 ± 89, 61 SE - standard error coefficient ( model candidates

- 11 031124

Table 3

Average times and maximum rates of accumulation and elimination of Mn ₃ O ₄ - LF

The average time Maximum Average time Maximum accumulation speed deductions speed Structure ± accumulation (hour ^- deductions g clock) H < ^r ( ^T de _e ^) _{f Part 1)} (hour ^-1 )

mov 4.96 ± 7.91 NA 311.60 ± 253.36 -0,0022 AON 16.34 ± 10.02 0.0424 223.56 ± 182.74 -0,0031 PIR 14.40 ± 7.37 0.0512 298.44 ± 193.73 -0.0023 AI 19.47 ± 13.35 0.0345 253.55 ± 179.49 -0, 0026 LEnt 10.37 ± 5.16 0.0761 356.80 ± 253.22 -0,0019 Ment 23.76 ± 20.09 0.0301 378.39 ± 198.33 -0, 0020 DG 40.63 ± 25.54 0,0169 415, 78 ± 136, 63 -0, 0028 svz 36.91 ± 38.36 0.0241 405, 76 ± 179, 59 -0, 0021 CP 41.92 + 28.67 0,0160 259.35 ± 221.52 -0, 0028 Amygdala 66.79 ± 52.32 0,0102 385.56 ± 175.78 -0,0022 GP 86.68 ± 62.28 0,0077 327.72 ± 154.66 -0,0025 Bnst 64.80 ± 57.47 0,0116 432.32 ± 146, 10 -0, 0026 Hip 33.28 ± 33.27 0,0300 381.60 1 175.88 -0,0022 LSD 50.77 ± 19.85 0,0192 360, 19 ± 226, 57 -0,0019 CIC / InCo 68.06 ± 49.20 0,0098 354.16 ± 231.94 -0,0019

Thus, it was noted that with the overcoming of synaptic transmissions, the characteristic accumulation time of Mn ₃ O ₄ increases - LF < ^Gpge) ·

In this case, both the maximum magnitude of the MPT signal ^ ^max and the concentration [Mn], and the maximum rate of accumulation of nanoparticles (Tables 2, 3; Fig. 4A, 4B, 4B, 4G) decrease. Thus, the maximum MRI signal is detected in the MOB, and taking into account the ratio between the magnitude of the MRI signal and the number of particles in the neuron (Fig. 1D), MOB cells accumulate ~ 20,000 particles per cell at a manganese concentration greater than 2 mmol / L (Fig. 4D) . However, in deeper nuclei, such as, for example, BNST, LSD, CIC, the maximum number of Mn ₃ O ₄ - NP nanoparticles is ~ 500-1000 per cell and the total content of manganese contained in the particles is about 0.1 mmol / l and less (Fig. 4G). It is known that concentrations of Mn ^{2+ ions} above 0.1 mmol / l are neurotoxic, however, the authors found that the solubility of the nanoparticles according to the invention does not exceed 10% even at pH ~ 4.0 (Fig. 1B). In other words, the concentration of Mn ^{2+ ions} for most structures does not exceed the neurotoxic one.

In contrast to the time and rate of accumulation, the time and speed of removal of Mn _x O _y H _z nanoparticles is significantly lower (Tables 2, 3, Fig. 4D, 4E). So, if the time to reach maximum values of the MRT signal after intranasal administration of Mn ₃ O ₄ -NP is in the range from 24 to 168 h, then complete elimination is observed within 672 h (Fig. 2B).

The characteristic time of removal of MI ₃ O ₄ - LF " ^T " ^egau : varies from ~ 250 to ~ 500 h and is partly correlated with the relative distance from the OE (Fig. 4D). Moreover, the maximum rate of reduction of the MRI signal is an order of magnitude smaller and does not correlate with the relative distance from the OE (Fig. 4E). Hence the conclusion that, in contrast to accumulation, the elimination of manganese nanoparticles is in no way connected with axonal transport. One of the possible mechanisms for removing nanoparticles from cells can be either exocytosis, or, more likely, the slow dissolution of nanoparticles with the subsequent elimination of Mn ^{2 + / 3 +} ions.

Taken together, these results show that Mn _x O _y H _z nanoparticles are captured from the surface of the olfactory epithelium by the olfactory receptor cells and moving along the nerve pathways in strict accordance with the trajectory of the olfactory tract and the limbic system. The movement of nanoparticles most likely occurs through axonal transport at speeds corresponding to the lower limit of fast axonal transport (~ 1-2.5 mm / h). With the overcoming of synaptic transmissions, the maximum number of particles per cell falls and the characteristic accumulation time increases. The nanoparticles according to the invention containing Mn3O4 reach relatively deep structures of the limbic system, represented by the thalamic nuclei (CIC / InCo). Whole accumulation process

- 12 031124 nanoparticles according to the invention, containing Mn ₃ O ₄ , takes about 168 h, after which the nanoparticles are gradually removed from the brain. Given the low solubility of the nanoparticles according to the invention containing Mn ₃ O ₄ , complete elimination is observed after 28 days after the start of the experiment. At the same time, low solubility provides a low level of free Mn ^{2+ ions} with neurotoxic effects. Indeed, during the whole experiment, we did not observe any changes in the state and behavior of animals in the experimental group. The remaining Mn _x O _y H _z - NP nanoparticles obtained by protocols C, D, and D have similar characteristics.

Example 5. MRI diagnostics of lesions of the central nervous system and topographic diagnosis of brain tumors relative to the nervous pathways.

The ability of nanoparticles obtained by the authors of the invention to move strictly along the pathways of the nervous system, together with their paramagnetic properties and the possibility of detection using T1-weighted MRI, opens up broad prospects for their diagnostic use. To illustrate this diagnostic application, the inventors performed xenotransplantation of U87 human glioblastoma cells to SCID mice (SHO-Prkdc ^scld Hr ^hr ) in the hippocampus region (Hip). The growth dynamics of tumors was determined by T2-weighted MRI and, upon reaching a certain size, nanoparticles of the invention containing Mn3O4 (Fig. 5) were intranasally injected into mice with developed glioblastoma. The passage of nanoparticles containing Mn3O4 through the olfactory tract and the limbic system, and the accumulation of nanoparticles in these structures were examined using T1-weighted MRI for 12, 24, 48, and 96 h after injection (Fig. 5, 6). The average intensity of the MRI signal, determined for 6 mice and characterizing the accumulation of nanoparticles containing Mn ₃ O ₄ , was compared with the reference models of nanoparticle accumulation in MOB, AON, Pir and Hip (Fig. 6).

This analysis shows that if in MOB the magnitude of the MRI signal in mice with xenograft glioblastoma is comparable to the control, in other sections of AON and Pir there is a decrease in the accumulation of nanoparticles containing Mn3O4 (Fig. 6).

Analysis of the localization of nanoparticles containing Mn ₃ O ₄ within the glioblast, revealed a specific accumulation of nanoparticles only in those parts of the tumor that intersect with the trajectory of the nanoparticles in the limbic system (Fig. 5). Thus, nanoparticles containing Mn ₃ O ₄ predominantly accumulate in the areas of tumors bordering the hippocampus (Hip), LSD or SVZ and do not spread further. Thus, these results show that the introduction of magnetic-contrast Mn _x O _y H _z nanoparticles - NPs through the nose allows localizing the areas of cancer tumors adjacent to the olfactory tract and the limbic system.

In contrast to the neuronal pathways, nanoparticles injected into the blood freely spread along the bloodstream and accumulate throughout the entire volume of xenotransplanted tumors (Fig. 7). Such accumulation is possible due to damage to the blood-brain barrier caused by tumor growth, because in control animals, the passage of MnxOyHz nanoparticles from the blood to the brain is either impossible or difficult.

Based on the above properties of nanoparticles, the authors propose the following approach to MRI topography of brain cancers. Thus, the delivery of hydrophilic amorphous or amorphous-crystalline nanoparticles containing Mn _x O _y H _z to the nerve endings (through the nose, or by other methods described above) makes it possible to identify those areas of tumors that are at the intersection with the corresponding nerve paths T1-weighted MRI scanner. At the same time, the simultaneous delivery of nanoparticles or other contrasts that enhance T2-weighted MRI images through the bloodstream (in particular, through intravenous administration) allows the entire tumor to be detected. In combination, this allows for the detailed localization of cancer tumors with their topography relative to the nervous pathways.

Thus, the authors first obtained magnetic-contrast amorphous and amorphous-crystalline nanoparticles based on manganese oxides and hydroxides of the formula MnxOyHz, where x is 1 or 3, y is 2, 3 or 4, z is 2 or absent. The obtained nanoparticles practically do not aggregate and have low solubility at both neutral and acidic pH. The average diameter of the obtained nanoparticles is ~ 30 nm with a hydrodynamic diameter of from 100 to 150 nm. Nanoparticles according to the invention, containing Mn _x O _y H _z , reduce T1 relaxation time and have reduced toxicity, which makes them suitable for various diagnostic studies using MRI.

In addition, it has been experimentally confirmed that with the intranasal administration of these nanoparticles, their effective neuronal uptake and specific accumulation occurs only in those areas of central nervous system tumors that are at the intersection with the trajectory of neuronal transport of these nanoparticles. Given this feature, the delivery of nanoparticles to the nerve endings and receptors of the nerve paths is used to visualize the nerve pathways and to determine the topography of lesions of the central nervous system, in particular cancer tumors, relative to the nerve pathways by MRI.

It was further established that the passage of the nanoparticles according to the invention and their accumulation in the conductive

- 13,031,124 pathways and other structures of the central nervous system depends on the integrity of the nerve paths, which is used for MRI diagnostics of changes in the nervous functions caused by lesions, injuries, neurodegenerative processes, etc. Thus, microdamages of the brain lead to a statistically significant decrease in the accumulation of nanoparticles according to the invention, even in those brain regions that are remote from the site of damage. The authors also noted changes in the dynamics of the passage of nanoparticles containing Mn _x O _y H _z along the nerve pathways caused by microdamages of the brain. Thus, any abnormalities in the dynamics of the passage of nanoparticles in accordance with the invention along the nerve pathways, determined by MRI methods, can be used as a diagnostic marker to identify neurodegenerative processes and damage to the nerve fiber.

When introduced into the bloodstream, which is an additional diagnostic tool in accordance with the invention, the nanoparticles of the invention effectively penetrate brain tumors, providing contrast MRI images throughout the entire tumor volume. It follows that the combination of T1 and T2 of contrasting nanoparticles with their separate introduction through the blood and delivery to the nerves (where the diagnostic composition containing the nanoparticles in accordance with the invention is administered intranasally, and T2 contrasting agents are administered intravenously) is used according to the invention for complex topographic diagnosis bulk formations, in particular tumor processes.

All of these diagnostic tools and methods improve the accuracy of diagnosis and, therefore, provide the best opportunity for adequate treatment of traumatic injuries, ischemic, hemorrhagic lesions and volume structures of the central nervous system.

Claims

CLAIM

1 --------------------- 1 --------------------- 1 ----- ---------------- 1 --------------------- g

About 10 20

X log ₂ (MPT S ^ / S °) d

About 10 20

X log ₂ (MPT S "^ / S")

FIG. 1/27% -4 mm 'tEiSA - ⁶ MM

LOW

LOW * high

MRI signal

FIG. 2

1. Contrast agent for magnetic resonance imaging, containing amorphous or amorphous-crystalline nanoparticles consisting of manganese oxide or hydroxide of the formula Mn _x O _y H _z , where x is 1 or 3, y is 2, 3 or 4, z is 0 or 2, and if x is 1, then z is 2; and nonionic surfactants.

2. The tool according to claim 1, where the manganese oxide has the formula Mn ₃ O ₄ .

3. The tool according to claim 1, where the manganese hydroxide has the formula Mn (OH) ₂ .

4. The tool according to claim 1, where the manganese hydroxide has the formula MnO (OH) ₂ .

5. A tool according to any one of claims 1 to 4, where the non-ionic surfactant is a polysorbate.

6 12 24 48 96 240

20 40 60

Ί ------------'-----'------- 1 --------- ¹ ------------ --Ί ------------------------ G ^-

O 50 100 150

X (time, hours) ^1st

R-δ, (A)

6. The tool according to claim 5, where the polysorbate is a polysorbate-20.

7. The tool according to claim 1, where the nanoparticles have a diameter of from 20 to 50 nm according to transmission electron microscopy.

8. The tool according to claim 1, where the hydrodynamic diameter of the nanoparticles is in the range from 100 to 150 nm.

9. The tool according to claim 6, where the composition of the nanoparticles is characterized by the formula Mn _x O _y H _z · (polysorbate-20), while the polysorbate-20 is present in an amount of 5-10 wt.% By weight of the nanoparticles.

10. The agent according to claim 1, wherein the non-ionic surfactant is selected from the group consisting of octylphenoxypolyethoxyethanol and octylphenol ethoxylate.

11. The tool of claim 10, where octylphenoxypolyethoxyethanol is Nonidet R40.

12. The agent according to claim 10, wherein the octylphenol ethoxylate is Triton X100.

13. Diagnostic composition for magnetic resonance imaging, containing a tool according to any one of claims 1 to 12 and at least one targeted additive.

- 14 031124 preparation of the second solution by adding to the initial solution of KOH solution of a non-ionic surfactant;

incubating the first solution and incubating the second solution;

mixing the first solution with the second solution;

homogenization of the resulting mixture and the deposition of nanoparticles.

23. The method according to p. 22, where, after deposition, the resulting nanoparticles are washed from an excess of non-ionic surfactant and unreacted components.

24. The method according to p. 22, where the non-ionic surfactant is in the initial solution in an amount of 20 wt.%.

25. The method according to claim 22, wherein the non-ionic surfactant is polysorbate.

26. The method according to claim 25, wherein the polysorbate is polysorbate-20.

27. The method according to p. 22, where the concentration of MnCl ₂ in the initial solution is 0.5 mol / L.

28. The method according to p. 22, where the concentration of KOH is 2 mol / L.

29. The method according to p. 22, where the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes at room temperature.

30. The method according to clause 29, where the room temperature is 20-25 ° C.

31. The method according to p. 22, where the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes when heated.

32. The method according to p, where the heating is carried out to a temperature of 80 ° C.

33. The method according to p. 22, where the incubation of the first solution and the incubation of the second solution is carried out for 30-60 minutes with cooling.

34. The method according to p, where the cooling is carried out to a temperature of 0-4 ° C.

35. The method according to p. 22, where after mixing the first solution with the second solution, the mixture is divided into two parts in separate containers.

36. The method according to clause 34, where these containers are tubes.

37. The method according to p. 22, where the homogenization of the mixture is carried out using an ultrasonic homogenizer.

38. The method according to p. 22, where the deposition of nanoparticles is carried out by centrifugation.

39. The method according to item 23, where the washing of the obtained nanoparticles from an excess of non-ionic surfactants and unreacted components is carried out with water.

40. The method according to claim 39, where the washing of the obtained nanoparticles is carried out 1-3 times.

41. The method according to p, where the washing of the obtained nanoparticles is carried out in ultrapure water.

42. The method according to claim 38, where centrifugation is carried out at 20,000 g and a temperature of 4 ° C.

43. The method according to claim 22, where the finished particles are stored at room temperature.

44. A method for diagnosing a disease of the central nervous system, comprising intranasal administration of a composition according to any one of claims 13 to 21, and conducting magnetic resonance imaging.

45. The method of claim 44, wherein the disease of the central nervous system is a traumatic injury, ischemic injury, hemorrhagic damage, or a tumor.

46. The method of claim 45, further comprising intravenous administration of the composition of claim 16.

47. The method according to claim 46, in which the magnetic resonance imaging of the central nervous system is carried out after intravenous administration of the specified composition.

48. The method according to any of paragraphs.44-47, in which the magnetic resonance imaging of the central nervous system is a magnetic resonance imaging of the brain.

14. The composition according to item 13, where the target additive is a carrier.

- 15 031124

In the burden of measurements, hours

15. The composition according to claim 14, where the carrier is water.

- 16 031124

FIG. 3

FIG. four

(bit An '' Al MEANT h. · Hip • O ’. x ^ DG LSD • R = Ό.73 AMYG • BNSWX. a-, oc *

16. The composition according to p. 14, where the carrier is a saline solution containing 0.9% NaCl.

- 17 031124

T1-weighted MPTl T1-weighted MPT

T2-weighted MPT _i___________t.

/ ·, /

T2-weighted MPT or

T1-weighted MPT

T1-weighted MPT low

T1-weighted MPT high

MPT signal

FIG. 5 measurement time, hours

FIG. 6

17. Composition according to any one of p-16, where the final concentration of manganese [Mn] included in the nanoparticles in the composition is 0.1-1 mol / l, and the number of nanoparticles in the composition ranges from 18.1 ± 3 to 321 ± 30.5 g / l.

18. Composition according to any one of p-16, where the additional target additive is a mucolytic agent in an amount of from 0.1 to 10 wt.% Inclusive by weight of the composition.

19. The composition according to claim 18, wherein the mucolytic agent is selected from acetylcysteine, erdostein, carbocysteine and ambroxol.

20. Composition according to any one of p-16, which is a colloidal solution.

21. Composition according to any one of paragraphs.13-16, where magnetic resonance imaging is a magnetic resonance imaging of the central nervous system.

22. The method of obtaining an agent according to any one of claims 1-12, comprising preparing a first solution by adding to the initial solution of a non-ionic surfactant an initial solution of MnCl ₂ ;

- 18 031124

MPT signal

FIG. 7