TECHNICAL FIELD

The present invention relates to the sphere of obtaining stable suspensions of nano-size particles of heterocrystal minerals and the methods of their obtaining.

Industry is highly interested in suspensions of titanium dioxide and silicon dioxide nanoparticles because of diversity of their properties and a great number of fields where they can be used.

BACKGROUND OF THE INVENTION

Preferably for nanoparticles preparation minerals with heterocrystal structure are used, especially rutile (TiO ₂ ; (Ti, Nb, Fe)O ₂ ), anatase (TiO ₂ ,(Ti, Nb, Fe)O ₂ ), quartzite (SiO ₂ ). These minerals are widely extended in the nature and could be easily dispersed.

Rutile is a natural mineral, being a principal titanic ore as a metal source, used in high-technology alloys thanks to its low density, high strength and corrosive resistance. Microscopical inclusions of rutile could be found in quartz, tourmaline, ruby, sapphire. This stone in the process of formation at high temperatures and pressures forms in stable in these conditions form n(SiO ₂ )-n(TiO ₂ ), but at lowering of temperature and decrease of pressure these two components separate forming crystals of rutile captured by the quartz crystals. In particular, because of their physical-chemical characteristics nanoparticles become highly useful in different fields: treatment of different diseases, for example, infectious, bacterial or viral infections, cancer therapy, wounds healing, anemia treatment etc.

W02007048634 «NANOPARTICLES OF HETEROCRYSTAL FOR USE AS A MEDICAL AGENT AND METHOD OF THEIR OBTAINING».

Synthesis optimized at low concentration is hard to repeat at higher concentration without problems in stability and aggregation, that is why it is necessary to emphasis, that troubles arising in industry adaptation are in synthesis of systems stable in time with high concentration and controlled particles sizes.

From the technics area the patent EP2462915A1 is known Publication 24.12.2014 Priority PCT/JP2010/062958 from 2010-07- 30. «Organic-inorganic composite particles and process for production thereof, dispersion containing the particles, and cosmetic containing the particles».

Organo-inorganic composition particles for cosmetics, including particles of inorganic oxide, every one having cationic charge on the particle surface, and molecules of polymeric gel, obtained of a natural substance and having anionic functional group and one or several hydroxyl groups in a molecule, at that said molecules of polymeric gel are electrostatically bound with surfaces of inorganic oxide particles. Inorganic oxide particles are the particles of oxide or complex oxide of at least one metallic element, chosen from caesium, magnesium, calcium, barium, cerium, titanium, zirconium, vanadium, iron, zinc, aluminium and silicon (excepting silicon oxide particles). Average diameter of organic-inorganic composite particles desirable to lay in the range from 0.1 to 300 mcm, preferably from 0.15 to 280 mcm. But the particles of organic-inorganic composite could be also used, having average diameter of particles less than 0.1 mcm, a force of particles dispersion increases, that complicates particles treatment, therefore such particles are undesirable. If the average particles diameter exceeds 300 mcm, the force of spontaneous deposition increases, that makes difficult to prepare homogenous dispersion of particles in a dispersion medium. At that at the final stage after filtration and drying of organic-inorganic composite particles water is added for final dispersion.

For a cosmetic agent the characteristic of homogenous flowability of the obtained suspension is given.

From the technical field the European patent No. 016541 WO 2007/118884 2007.10.25 (PCT PCT/EP2007/053761) is known, Compositions of magnetic nanoparticles and their use.

The invention relates to use of biocompatible nanoparticle or nanoparticles aggregate in combination with external non-oscillating magnetic field, where said nanoparticle includes: a) a core containing magnetic material; b) biocompatible cover, surrounding the core; and, optionally, c) a marking agent, where the external diameter of the cover is less than about 100 nm, for preparation of composition, where said composition does not contain any other means for targeting on the cell. The present invention also relates to obtained compositions and their use in the healthcare area, for cancer treatment or in diagnostics (for example, visualization), for monitoring of the tumor development. Ferromagnetic material is chosen from the group consisting of iron, nickel, cobalt, gadolinium, samarium, neodymium, boron, aluminium and any mixture of them. Material of ferromagnetic core is in form of oxide, hydroxide or metal.

Compositions may exist in solid or liquid form (suspended nanoparticles), for example, in the form of paste or aerosol.

«The method of obtaining multicomponent particles» is known according to the patent US 2010/0262115 including the stage of obtaining inorganic nanoparticles.

Said invention is destined for an agent containing nanoparticles, for cancer treatment, pharmaceutical compositions containing them, and the methods of their use for medicines delivery and cancer treatment by ultrasound or light.

Nanoparticle containing internal volume containing derivative of hypocrelline-B and polyvinylpyrolidone cover, encapsulating internal volume of hypocrelline-B is SL052. Nanoparticle is bound with a marking agent being detected. Nanoparticle where the detected marking agent is chosen of fluorescent or other light-emitting marker, radioactive indicator or contrasting agent. Nanoparticle is used in combination with one or more pharmaceutically admissible carriers for formation of pharmaceutical composition for tumors treatment.

Polyvinylpyrolidone (PVP) (having average molecular mass of 40 000 Da) is a derivative of hypocrelline-B (designated as «SL052»). The method of deposition was used for obtaining SL052-NPS. 1.5 ml 0.5% (7.5 mg/ml) of PVP water solution were added to 6 ml of water when mixing at the room temperature. Ten minutes later 1.59 ml 4.6 mm SL052 in dimethyl sulfoxide (DMSO, Fisher Scientific) were added to this mixture. The obtained solution was mixed during ten minutes in the darkness in order to obtain nanodispersion with nanoparticles size of 136 nm.

Suspensions reported in the literature are obtained either immediately in the course of synthesis or by the way of synthesis and deposition of nanopowder with subsequent redispersion in a solvent in presence of additives [P K Khanna et al. Mater. Lett. 61 (2007) 3366]; however, it is necessary to note that for improvement of the system stability in time, for facilitation of scaling and in order to avoid the stage of extraction, washing out and repeated dispersion of powder nanoparticles are preferably converted in suspension immediately after synthesis.

The methods of obtaining stable suspensions of metallic nanoparticles Au, Ag, Cu, Pd, Pt, Fe are known, realized in aqueous medium at low temperature, under pressure and in atmosphere of environment at heating using microwave apparatus, as well as stable suspensions of nanoparticles obtained in such a way. Publication of the application PCT/EP2010/052534WO 2010/100107 (10.09.2010).

The term «metallic nanoparticles» designates particles of Au, Ag, Cu, Pd, Pt, Fe with sizes in the range from 1 up to 250 nm, preferably from 1 to 100 nm; in particular, the invention relates to nanometric suspensions of Ag and Au.

The elaborated method consists of adding saline precursor of metal to reactionary medium, already heated up to the reaction temperature and containing reducing agent, chelating agent and a catalyst in strictly determined molar ratios chelating agent/metal, reducing agent/metal, catalyst/metal. The method ensures obtaining of colloidal suspensions having high concentration and being stable in time (during time periods more than 7 months).

Suspensions obtained in such a way are characterized by the rate of average dynamic light scattering (the DLS method) about 20- 30 nm and show monodisperse distribution before concentrations, and for higher concentrations bimodal distributions are noted in presence of the basic particles population in the size range of 5-10 nm.

Use of suspensions of metallic nanoparticles of Au, Ag, Cu, Pd, Pt, Fe is restricted in view of complexity of withdrawal from the human body because of unstable suspension condition. In connection with particles agglomeration setting of sediment occurs, and it is not isolated/sampled from organism.

Au is chemically inert, does not interact with acids, aqueous medium of organism and oxygen and practically is not isolated/sampled from organism.

Study of biological properties of nanoparticles of silver shown that in aqueous medium these particles are essentially in the oxide form. Silver oxide has positive biological activity (antimicrobial and antitumoral) in particular, thanks to the mechanism of photocatalysis of passage of dissolved atmospheric oxygen PO ₂ in the active form AFO.

Moreover, silver in suspension has tendency to aggregation, loss of oxide form (passage in catalytically inactive quasimetal particles), that is negatively estimated by the toxicologists. The technical problem for solving of which the present invention is intended, is obtaining of stable suspensions on the base of crystals, bio-dissociable, ceramic, potentially applied in medical forms composition, namely, two products of titanium dioxide or silicon dioxide, acquiring expressed biological activity in the case of their special structure at obtaining suspensions from them, possibility of the crystals surface to enter into chemical-physical bonds with the energy, reaction centers.

The technical result is obtaining of long time stability suspensions of heterocrystals of titanium dioxide or silicon dioxide with activated particles characterized by presence of oxygen on their surface, ensuring initiation of an active form of oxygen AFO in the human body.

The product representing heterocrystals of titanium dioxide is mentioned hereinafter as activated titanium dioxide. The internal part of particles of activated (nanocrystalline) titanium dioxide still has crystalline structure of pigmental titanium dioxide, used as excipient in pharmaceutical compositions. Improvement of interphase properties (physical, technological and catalytic characteristics) due to activation of nanoparticles do not result in formation of a new chemical compound, but to the same compound with changed colloidal and surface properties, namely, presence of oxygen in the structure, presence of certain Zeta-potential on the crystals surface, ensuring initiating of the active form of oxygen in a live organism.

The chemical precondition of the reaction of activation of heterocrystals of titanium dioxide or particles of silicon dioxide is modulation of electrostatic properties of the surface (increase of Zeta- potential due to increase of the surface charge density) due to chemisorption H+ and formation of photocatalytic domains (lattice imperfections) due to aggressive acid treatment of the surface.

The ratio of particles and crystals charge is one of the factors determining physical stability of suspensions.

By determining Zeta potential in preparation of suspension it is possible to quantify the magnitude of electrostatic repulsion between particles and so the higher is the zeta potential, the higher is the physical stability. Usually the particle charge is defined in terms of quantity by Zeta-potential, being measured, for example, using electrophoretic mobility of particles in the electric field.

SUMMARY OF THE INVENTION

The method of obtaining stable suspensions of heterocrystal of titanium dioxide and particles of silicon dioxide is characterized by the fact that the starting material is in the form of aggregates with size more than 0.5 micrometer is mixed with an aqueous solution of inorganic acid, with subsequent direction to homogenizing for the first stage of mixing, after that the obtained aqueous suspension is subjected to thermal treatment, then aqueous suspension is directed to the rotary evaporator working under pressure lower than 100 kPa at temperature no more than 70°C for evaporation of inorganic acid with suspension expense through the rotary evaporator no more than 25 l/min and then the obtained activated particles are mixed with water in hydrodynamical cavitational homogenizer with regulated pulsating wave mode until obtaining stable suspension of heterocrystal of titanium dioxide or particles of silicon dioxide with size less than 450 nm, and presence on the lattice surface up to 80% of electronically-excited triplet oxygen ³ O ₂ in the energy centers, namely, in the quantum dots (QD) - zones of local overheating, ensuring catalytic activity for formation of Active forms of oxygen (AFO) in the live organism.

For obtaining stable suspensions of heterocrystals TiO ₂ as starting material, the powder TiO ₂ is used, containing modification of anatase and rutile in ratio preferably from 10:90 up to 90:10, more preferably from 60:40 up to 40:60.

Initial Starting material is correspondingly mixed with Pharmaceutically approved, for example, hydrochloric or sulfuric one acid in the form of 0.0001 N-0.1 N aqueous solution.

At the expense of using aggressive acid treatment the level of microbial, pyrogenic and other hydrolyzable organic contaminations decrease, that is highly important for subsequent elimination of toxicity of obtained stable suspensions and their application in medical agents composition.

Pharmaceutically approved acids may be also chosen from the following acid range: chiefly hydrochloric acid, sulfuric acid, as well as metaphosphoric acid, methanesulfonic acid, nitric acid, phosphoric acid, sulfuric acid, tartaric acid.

Stability of obtained suspensions, in particular, is significantly influenced by pH of a medium obtained as a result of acid treatment of starting material.

Stability of obtained suspensions is ensured by low or neutral value of the pH of the suspension medium. The obtained mixture is directed to the primary stage of mixing into the homogenizer, being, for example, an apparatus provided with supplying nozzle, that trough the ejection channels is interconnected with coaxial auxiliary chamber, attached to it by a vortical chamber and a chamber of mixing, connected with the discharging nozzle for output of homogenous aqueous dispersion after the first stage of mixing.

Then obtained after homogenizing aqueous suspension of heterocrystals of titanium dioxide or particles of silicon dioxide is directed to the ultrasonic bath with ultrasonic frequency 20-90 kHz, where it is kept at the temperature no more than 70°C during no more than 2 hours.

Dispersive medium is evaporated in the vacuum rotary evaporator under pressure lower than 100 kPa at the temperature no more than 70°C for evaporation of pharmaceutically approved acid with suspension expense through the rotary evaporator no more than 25 l/min.

During evaporation of a dispersion medium volatile acid vapors are released. At this time the surface of particles and crystals is activated. After that activated powder TiO ₂ or SiO ₂ is obtained, having developed surface and special structure of crystals and particles.

The activated crystals TiO ₂ or particles SiO ₂ are obtained with presence in oxygen structure up to 80% in metastable electronically- excited of third stage triplet condition ³ O ₂ at that the TiO ₂ particles have Zeta-potential +30 - +15 mV, and the SiO ₂ particles have Zetapotential - 20 - -15 mV, and the TiO ₂ crystals or SiO ₂ particles are characterized by presence of sorption properties. Zeta potential is measured by Malvern Zetasizer Nano ZS instrument (Phase Analysis Light Scattering method). The range of Zeta-potential is 10-100 mV.

A powder of activated TiO ₂ or SiO ₂ particles is directed into the hydrodynamical cavitational homogenizer, where water is added for obtaining from 0.0001 % - 10% of stable suspension of TiO ₂ heterocrystals or SiO ₂ . particles.

The process of treatment in the hydrodynamical cavitational homogenizer lasts from 10 minutes depending on specified indices required for suspension.

The homogenizer contains the blocks for treating fluid medium sequentially connected with one another along the flow of mixed medium, at that the first block of preliminary mixing contains the inlet nozzle of basic medium, the inlet nozzle of admixed medium and the outlet channel of mixed medium, through which the last one overflows in the homogenization block and then overflows in the block of controlled output of homogenized product, at that, according to the invention, in the homogenization block the cavitational mixing chamber is made with the reflector of flow mix at the output of the chamber, fulfilled in the form of narrowing along the flow chamber walls, at that in the chamber the insertion with the central channel is installed, on the external surface of the insertion the spiral groove is fulfilled, the insertion contacts with the internal wall of the chamber with possibility of forming the open-end spiral channel along the spiral groove and mixing of flows of spiral and central channels in the cavitational chamber before the reflector, and the central channel is provided with narrowing and widening sections, at that the block of the controlled output is provided with the controlling rod of the flow medium reflector, the projection of which is located in the communication channel with the outlet of the homogenizing block, and the controlling rod is connected with the means of changing location of its projection in the communication channel.

In the cavitational chamber the insertion with the central channel is installed, on the external surface of the insertion the spiral groove is fulfilled. The insertion contacts with the internal wall of the chamber with possibility of forming the open-end spiral channel along the spiral groove and mixing of flows of spiral and central channels in the cavitational chamber before the regulated reflector.

A fluid product fed to the chamber is twisted in the spiral channel, formed by the spiral groove, and forms pulsating intensive vortical layer.

In the homogenizing chamber the intensive vortical layer is formed on the flow surface, that at hitting against the reflector excites turbulent wave pulsations considering thermophysical influence of hitting, where temperature of treated fluid medium/product rises.

The structure of the cavitational homogenizer ensures high degree of homogenizing the obtained suspension.

The suspension stability is determined by presence of procedures and operations of the method, use of starting material, namely, combination of anatase and rutile in obtaining suspension of titanium dioxide heterocrystals, use of an acid solution, application of different types homogenizers at the first stage of mixing and hydrodynamical cavitational homogenizer for final mixing and obtaining stable suspensions. Stable suspension of the titanium dioxide crystals and silicon dioxide particles, obtained in accordance with the elaborated method is characterized by distribution of activated titanium dioxide crystals with size up to 1 nm, that is 0.3 vol%, up to 20 nm is 5-40 vol%, particles with size up to 80 nm are 10-80 vol%, particles with size up to 150 nm are 5-30 vol%, particles with size up to 250 nm are 5-20 vol%, particles with size more than 250 nm are no more than 10 vol%, and distribution of activated silicon dioxide particles with size 40-80 nm are 10-80 vol%, particles with size 80-150 nm are 10-80 vol%, particles with size 150-250 nm are less than 30 vol%, particles with size more than 250 nm are no more than 15 vol% on the base of the data obtained in the analysis with the photon correlation spectroscopy instrument Malvern Zetasizer Nano ZS and is characterized by presence on the lattice surface up to 80% of electronically-excited triplet oxygen ³ O ₂ in the energy centers, namely, in the quantum dots - zones of local overheating, ensuring catalytic activity for formation of active forms of oxygen in the living organism.

The Fig. 1 shows distribution of titanium dioxide particles with different sizes in suspension volume.

The Fig. 2 shows distribution of silicon dioxide particles with different sizes in suspension volume.

Polydispersity of obtained suspensions determines their unique properties at forming medical agents, that ensures not only targeted delivery into the area of pathological processes, but also unique mechanisms of action for use in the medical agents.

The obtained polydisperse suspension has biological activity due to not only photocatalytic properties, but also thermal activation, for the first time achieved at the expense of the human body temperature 36.6°C.

In the case of variations of regulated modulations of electrostatic properties, achieved thanks to the defects of lattices of extended crystals surface, the crystals depending on the size and structure surfaces and, correspondingly, charge (Zeta-potential) acquire possibility of use as photocatalysts in the wide range of waves, from ultraviolet to infrared irradiation.

One more advantage of heterocrystals and particles, in using them as photocatalysts, is possibility of their activation with a help of visible light.

An additional advantage is that in one therapeutic dose the ratios of different sizes of crystals and particles are modulated in photodynamic therapy (PDT) depending not only on the character of influence on pathology.

Stability in time for suspension obtained in accordance with the elaborated method is from 24 months and during no less than 15 years, in the tests for stability no changes are noted in optical properties (spectroscopy in UV visible range, Fig. 3, Fig. 4) and in the particles size (DLS), that indicates absence of changes in quantity and sizes of particles in the course of time.

Hereinafter the tests of stability examination for the QD series suspension ADAM QD/T (TiO ₂ ) are shown.

The Tables 1 and 2 show Results obtained during normal (long term) stability study (+25 ± 1 °C). Table 1

Table 2

The obtained data showed that: pH of these samples did not show significant tendentious alterations.

Dynamic viscosity of these samples did not change significantly upon storage.

The colloidal stability of the product was found outstanding. No tendentious changes were found in the particle size of the product.

The titanium dioxide content of the product did not change during storage. Sterility and endotoxin content investigations were only prescribed to be performed at the end of the storage study. These samples were found sterile and containing lower concentration of endotoxin than specified.

In accelerated, and also long-time examinations of stability Appearance of the samples stored at different storage conditions did not show alterations from the specification.

The Tables 3 and 4 show Results obtained during normal (long term) stability study (QD) ADAM QD/S (SiO ₂ ), t +25 ± 1 °C.

Table 3

Table 4

The obtained data showed that: pH of these samples did not show significant tendentious alterations.

Dynamic viscosity of these samples did not change significantly upon storage.

The colloidal stability of the product was found outstanding. No tendentious changes were found in the particle size of the product.

The silicon dioxide content of the product did not change during storage.

Sterility and endotoxin content investigations were only prescribed to be performed at the end of the period planned for the storage study and found to meet the product specifications.

In accelerated, and also long-time examinations of stability Appearance of the samples stored at different storage conditions did not show alterations from the specification.

Thereby, continuous stability of suspensions is ensured as a result of a complex of procedures and operations of the elaborated method, in particular due to use of different special homogenizers at the process stages.

Presence on the crystal surface of oxygen O ₂ , occupying up to 80% of structure and presence in the ruptures of the developed surface of crystals lattices of a great number of ionic groups of ligands ensures formation of excitonic structures, ionic bonds and zones of local energy overheating, i.e. the quantum dots, where O ₂ is in metastable electronically-excited triplet condition ( ² T ³⁺ ) with unique characteristics of conversion into biological activity - singlet condition (S' ¹ ' ³ ).

Illustration of pharmacological study of measuring quantity of electronically-excited triplet oxygen ( ² T ³⁺ ), on particles surface. Executed as follows. Researches executed on a rabbit, male with 0.75 kg weight (without injuries).

Through the needle 5 ml of suspension was injected, where 5 ml is injection water and 3 mg TiO ₂ , at the entry inside the needle opening there was a light guide for feeding laser photons and supplying injection suspension simultaneously.

The photonic-thermic procedure was executed with the laser IR 960 nm with 3 Wt power, dose 20 J regulating synchronously.

In the local treated part of the examined rabbit, with a help of an additional needle the sensor for measuring O ₂ was installed, and the analyser of automatic chemiluminiscent gas analyser of singlet oxygen ³ O ₂ (the model FOMS - 200 OXYGEN, made in FRG).

Presence of conversion of triplet oxygen with presence in oxygen structure up to 80% in metastable electronically-excited condition into singlet condition (S' ¹ ' ³ ) was fixed.

The surface of heterocrystals of titanium dioxide and silicon dioxide particles has sorption ability, that is an important factor for use in the medical forms.

Active chemical groups in the places of ruptures of crystal lattice easily form the chemical bonds with the same active molecules of environment.

The existing mechanisms of sorption interaction between different substances molecules are:

• nonspecific binding with porous structures; • ionic sorption;

• covalent one.

As a result of the fact that the surface of titanium dioxide crystals and silicon dioxide particles, containing up to 80% of oxygen, and presence in the ruptures of developed lattices surface of a great number of ionic groups of ligands, formation of excitonic structures, ionic bonds and a range of zones of local energy overheating is ensured, all the types of sorption are possible, that determines detoxication possibilities of the obtained stable suspensions.

• The researches showed, that in one dose of the preparation 1 mg/ml including stable suspension of titanium dioxide crystals or silicon dioxide particles their sorption capacity as a result of said sorption properties of crystals and particles is two times higher in comparison with the existing preparations.

Fig. 5 shows the data of comparative efficiency of sorption of exogenous Creatinine in the experiment.

The experiment was executed on a rabbit, 3 kg weight. The sorption capacity of Activated carbon 10 g samples was studied, arteriovenous perfusion 50 ml/min - 20 min, TiO ₂ I 0.6 mg in 1 ml aqueous suspension, intramuscular injection, at the level of Creatinine sorption. The tests were executed in identical illumination conditions.

The images on Fig. 5:

1 - before use (mg %). a - without use, b - Activated carbon sample, c - stable suspension TiO ₂ or SiO ₂ 2- after injection;

3 - one hour later;

4 - 3 hours later.

From the experimental data a conclusion can be made, that sorption capacity of the obtained stable suspensions of titanium dioxide and silicon dioxide in the medical forms would ensure organism detoxication.

Every neutral inorganic object found in a living tissue, inevitably gets in the category of the “foreign bodies”, i.e. relates to formations with principally pathological character.

Biocompatible TiO ₂ crystals and SiO ₂ particles are the only most indifferent in use in different forms of medical agents.

Absence of general and local toxicity is confirmed by the results of toxicological researches.

The side effects in examinations of toxicity and examination of the repeated dosed were not revealed, and the results of researches also confirm absence of mutagenicity and clastogenicity. Parenteral tolerated dose for TiO ₂ is 21.72 mg/kg bw, and for SiO ₂ 11.39 mg/kg bw. Peroral tolerated dose for TiO ₂ is 2172 mg/kg bw, and for SiO ₂ 1 139 mg/kg bw (Dossier No. 488.729.21 17; 488.729.21 19 from 06.07.2010, TOXI COOP Zrt).

For example, compounds of metals, in particular, Fe automatically get in the forbidden area for use in pharmaceutics and obtaining pharmaceutical receipts of preparation. Metals of iron Fe are of the “heavy” group, are considered «xenobiotic», and also are not released from the body of living organism.

A certain exception is a group of precious metals. But in fact only silver is regarded in this group. Examination of biological properties of silver nanoparticles showed, that in aqueous medium (such is it in the organism tissues) these particles are essentially in the oxide form. Silver oxide has positive biological activity (antimicrobial, antitumoral), in particular, thanks to the mechanism of photocatalysis of conversion of dissolved atmospheric oxygen (PO ₂ ) into active form of oxygen (AFO). However, silver has a number of imperfections: aggregation arises in suspension, losses of oxide form (conversion into catalytically inactive particles of quasimetal).

Biological inertness of TiO ₂ crystals and SiO ₂ particles is well- known, and expressed and regulated biological activity they acquire in the course of a special method of their obtaining, as a result of which the local zones of energy overheating appear (de la Hoz A, Diaz-Ortiz A, Moreno A. Microwaves in organic synthesis. Thermal and nonthermal microwave effects. Chem Soc Rev. 2005 Feb;34(2): 164-78. doi: 10.1039/b411438h. Epub 2005 Jan 12. PMID: 15672180), ensuring catalytic activity for formation of active forms of oxygen in the body of living organism.

Catalytic properties of crystals and particles are unique in the field of creation of different forms of medical agents for influence on a wide range of pathogens.

At normal conditions atmospheric oxygen is in principle ^1Q 2 condition, such molecule is not capable for participation in oxidative biological processes, for its activation heterogenous, energically stimulated “triplet-singlet” conversion ³ TO ₂ — ¹ SO ₂ must occur, i.e. this process can only be triggered at the expense of catalytic properties of crystals and particles (TiO ₂ , SiO ₂ ) by the way of transferring additional energy of 130-180 kJ/mole, i.e. at the expense of endothermic reaction with enthalpy (Janbazi H, Schulz C, Wlokas I, Peukert S. Thermochemistry of Oxygen-Containing Organosilane Radicals and Uncertainty Estimations of Organosilane Group- Additivity Values. J Phys Chem A. 2021 Oct 7; 125(39):8699-871 1. doi: 10.1021/acs.jpca.1c06941. Epub 2021 Sep 24. PMID: 34559967).

Obtaining of active, singlet oxygen in gaseous medium is easier to execute, for example, by the way of direct absorption of photons. In fluid medium such conversion is impossible. Another, special catalyst is necessary - photocatalyst - (photosense PS) in the form of crystals and particles TiO ₂ and SiO ₂ (QD).

Conversion ³ TO ₂ ¹ so ₂ conditions:

• Photosense PS must be in heterophase condition (polymer, suspension) and for “triplet - singlet” conversion ³ TO ₂ —> ¹ SO ₂ it must have additional energy from without (E), no less than 130-180 kJ/mole.

• Photocatalyst Photosense PS must have electronically- excited form of oxygen molecule.

• In the solution it is only possible with catalysis reaction through the contact photocatalyst - photosense PS, containing crystals and particles of TiO ₂ and SiO ₂ (QD).

• At the surface of heterocrystals and particles the energy centers (zones of local overheating) must be present, where oxygen molecule is in triplet condition;

• At contact of zones said energy become sufficient for ³ TO ₂ 1SO ₂ conversion;

• Catalysis is thermally dependent: rising temperature also initiates a process of ³ TO ₂ - ¹ SO ₂ conversion. Said reaction in the proposed invention is for the first time achieved from energy of environment of the human body itself 36.6 °C, and also in particular cases of diseases (pathologies) for reaching complex therapeutic effect, when it is necessary to carry out photon hyperthermia, with the electromagnetic wave emitter.

A mediator in liquid, aqueous medium, namely, in the organism tissues, may be dense, not dissolved substance with maximum large developed surface, on which there are sufficient number of active energy centers such as crystals and particles TiO ₂ and SiO ₂ (QD).

One more advantage of heterocrystals and particles, using them as photocatalysts, is possibility of their activation in a wide range from ultraviolet to infrared irradiation.

Table 5 shows the comparative data for sensibilize substances and crystals and particles of TiO ₂ , SiO ₂ (QD).

Table 5

The table shows that the crystals and particles TiO ₂ and SiO ₂ have fundamental advantages over sensibilize substances (photocatalysts) pigments:

• Absence of general toxicity;

• Colourlessness, transparency, inherent optical density;

• Insolubility (creation of heterogenous suspensional structure);

• Large surface of contact with a medium;

• Presence on it of active energy centers (zones of local overheating);

• High thermal stability - protection of destruction at laser influence;

• Maximum of catalytic activity in the zone of low optical density of tissues;

• Wide range of spectral photosensitivity;

Possibility of activation of AFO synthesis by thermic method at the temperature 36.6°C.

Action of suspension on the base of said crystals of titanium dioxide and particles of silicon dioxide is aimed to stimulation of natural processes at the cell level. AFO synthesized by the QD crystals or particles initiates the process of influence upon problem cells, when using in the medical forms.

Stable suspension of titanium dioxide crystals or silicon dioxide particles, due to synthesis of regulated active oxygen AFO, in the first place, is selectively directed into the problem cells of an organism, targeting the areas of inflammation, with infectious and non-infectious nature, in particular, interacting with membrane-associated ferment NADF - H, the immune cells of phagocytes and macrophages, causing in them numerous cytomorphologic changes (vacuoles in cytoplasm, fragmentation of membrane, abnormality of mitosis), that launches apoptosis or necrosis type of death of pathogenic cells (because the pathogenic cells have no effective antioxidant ferments).

Many works showed, that all the cells, forming intima of the blood vessels, enough intensively generate AFO as a response both to physical irritants (for example, to pulsating blood flow), and to hormonal factors, in particular, angiotensin. On this model the consequences of intensification of AFO production induced by regulatory factors were in detail studied.

Hishikawa K., Oemar B. S., Yang Z., Luscher T. F. (1997). "Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor- kappa B in Human Coronary Smooth Muscle." Circulation Research, 81 , 797-803.

Krieger-Brauer H.I., Medda P.K., Sattel B., Kather H. (2000). inhibitory effect of isoproterenol on NADPH-dependent O ₂ generation in human adipocyte plasma membranes is mediated by betagamma-subunits derived from G(s).» J Biol Chem, 275(4), 2486- 90.

Target action of penetration in problem cells of organism, reaching inflammation areas, of infectious and non-infectious nature is conditioned by multiple increase of oxygen necessity in inflammation focuses.

Example of targeted delivery of stable dispersions. Fig. 6 shows an example of studying penetration into the cell of luminescent stable dispersions of heterocrystals of titanium dioxide crystals and silicon dioxide particles marked with rhodamine on the fibroblast cells of mice. Absorption of fluorescent QD by tumor cells of mice fibroblasts (NIH-3T3). From above, from the left to right, downwards in the course of time: confocal laser fluorescent microscopic images show, that a tumor absorbs QD, and then the QD crystals go away from the cells.

The executed research with confocal laser microscope show that luminescent crystals of the QD series marked with rhodamine penetrate inside the cell. Accumulation of the crystals initially occurs in cytoplasm. Cell nuclei accept the crystals in a less degree. In the top left section of the combined picture a normal transmission microscopic image of the treated cell with visible (deep black colour) crystals inside cytoplasm is visible. The images from the microscope were sequentially recorded, the treated cells were cut into layers with one micron thickness. It is important to note, that cutting of apical basal parts of the cell showed, that a number of luminescent crystals drops until total removal.

EXAMPLES

Examples of obtaining stable suspension TiO ₂

Example 1

Initial powder of nanosized titanium-dioxide in the form of aggregates with size 0.5 micrometer (modification of anatase and rutile taken in ratio 10:90) is white powder with character smell of butanol and isobutanol. Preparation of solution 500 ml 0.1 N initial sulition of HCI is prepared of aqueous solution HCI.

Dispertion.

100.0 g of TiO ₂ with water adsorbed (recalculated for dry mass) is weighed. Dispersion of TiO ₂ is executed in 300 ml 0.1 N solution of HCI.

Irradiation is ultrasonic bath

For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60°C, irradiation time: from 10 - 90 minutes).

Evaporation of dispersion medium.

Dispersion medium is steamed in vacuum-rotary evaporator.

In the process of evaporation of dispersion medium, volatile vapors of hydrochloric acid are finally discharged.

Filtration.

Dispersion suspension is filtered through filter fabric. IPC 2: Integrity of a filter is ensured.

Evaporation of dispersion medium.

Dispersion medium is evaporated in vacuum-rotary evaporator under pressure lower than 100 kPa at temperature no more than 70°C. In the process of evaporation of low pH dispersion medium volatile vapors of hydrochloric acid are discharged. At the same time surface of nanoparticles TiO ₂ is activated, since chemical properties at the surface undergo significant changes. After evaporation of dispersion medium, dry activated powder TiO ₂ is obtained.

Powder of activated particles TiO ₂ is directed into the hydrodynamical cavitational homogenizer, in which water is added for obtaining 0.001 % - 10% stable suspension TiO ₂ and expose to additional treatment during 10-90 minutes.

Stability in time for suspension obtained in such a way is at least 24 months. During not less than 15 years changes of optical properties were not indicated.

Example 2

Initial powder of nanosized titanium dioxide (modification of anatase and rutile taken in ratio 10:90) is white powder in the form of aggregates with size 0.5 micrometer with character smell of butanol and isobutanol.

Preparation of solution -500 ml 0.1 N initial sulition of HCI is prepared of aqueous solution HCI.

Dispertion.

100.0 g of TiO ₂ (recalculated for dry mass) is weighed. Dispersion of TiO ₂ is executed in 300 ml 0.1 N solution of HCI.

Irradiation is ultrasonic bath

For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60°C, irradiation time: 1 .2 hours).

Filtration. Dispersion suspension is filtered through filter fabric. I PC 2: Integrity of a filter is ensured.

Evaporation of dispersion medium.

Dispersion medium is evaporated in vacuum-rotary evaporator under pressure lower than 100 kPa at temperature no more than 70°C. In the process of evaporation of pH dispersion medium volatile vapors of hydrochloric acid are discharged. At the same time surface of nanoparticles TiO ₂ is activated, since chemical properties at the surface undergo significant changes. After evaporation of dispersion medium, dry activated powder TiO ₂ is obtained.

Powder of activated particles TiO ₂ is directed into the hydrodynamical cavitational homogenizer, in which water is added for obtaining 0.01% - 10% stable suspension TiO ₂ and expose to additional treatment during from 10 to 90 minutes (1 minutes from 10 liters of treated suspension, where content depends on the specified indices, i.e. 0.001 - 10%).

Stability in time for suspension obtained in such a way is at least 24 months. During not less than 15 years changes of optical properties were not indicated.

Example of obtaining stable suspension SiO ₂

Example 3

255 g of initial nano-powder of silicon dioxide is dispersed in 10 I of water adding 0.0001 N of HCI solution. Then dissolved nanopowder is placed into a reaction vessel with volume of 5000 ml, where dispersion is executed under pressure 900 kPa - 1200 kPa with speed 10 l/min.

Irradiation is ultrasonic bath.

For preparation suspension is irradiated in ultrasonic bath working at frequency 20-90 kHz (bath temperature: 60°C, irradiation time: 1 .2 hours).

Suspension of nanoparticles SiO ₂ is obtained with particles size in 95.0 number % < 250 nm. Content of silicon dioxide is 50 mg/ml.

Reactionary mixture is filtered through a mesh filter 10 micrometer (Sefar Medifab). After filtration all the volume of suspension is pumped into a glass container and corked up thoroughly.

Obtained suspension is placed into an autoclave at temperature 121 °C, exposure time: 30 min.

Evaporation of dispersion medium.

Dispersion medium is evaporated in vacuum-rotary evaporator under pressure lower than 10 kPa at temperature no more than 70°C.

In the process of evaporation of low pH dispersion medium volatile vapors of hydrochloric acid are discharged. At the same time surface of nanoparticles SiO ₂ is activated, since chemical properties at the surface undergo significant changes. After evaporation of dispersion medium, dry activated powder SiO ₂ is obtained.

Circulation of mixed aqueous mixture of silicon dioxide through a suspending device in the hydrodynamical cavitational homogenizer with input pressure 900 kPa -1200 kPa (provided by SCHALI/Prommet/SA) during up to 90 minutes at room temperature.

Appearance - white or gray-white suspension, being shaken for transparency orange tint, easily resuspended.

Size of particles EP 2.9.31 min 90.0 number % < 250 nm. Content of silicon dioxide 50 mg/ml, pH of the product 5.0 - 7.5, dynamic viscosity 0.94 - 1 .14 mPas.

Stability in time for suspension obtained in such a way is at least 24 months. During not less than 15 years changes of optical properties were not indicated.

The present invention relates to stable suspensions of titanium dioxide or silicon dioxide heterocrystals, methods of obtaining said nano-suspensions and use of said nano-suspension, ensuring more efficient delivery of biologically active agents in blood flow of a subject. At contact of such nano-suspensions with a body organ, for example, with oral cavity area, including mucous membrane of cheeks, a compound is absorbed into blood flow in amount sufficient for causing a specified biological reaction. Correspondingly, nanosuspensions may be delivered with a help of usual micro-fluidized spray, hydrogel, topical products, aerosol or liquid. Delivery may be executed by parenteral, intrathecal, intravenous, through mucous membranes or every other generally recognized method of medical agents delivery.

Thus use of stable suspensions of heterocrystals of titanium dioxide and silicon dioxide particles, obtained in accordance with the elaborated method for medical agents, used intravenously, intramuscularly, orally, nasally, vaginally, rectally, topically and locally (ENT) is claimed.

The crystals peculiarity is that on the surface an acceptor, i.e. O ₂ , is initially in excited triplet condition, with regulated possibility of catalysis in AFO, even in the cases where the human body temperature 36.6°C is sufficient, for use in medical forms.

Heterocrystals of titanium dioxide or silicon dioxide particles are characterized by presence on the surface of oxygen crystal O ₂ , which occupies up to 80% of structure, they have biological activity due to not only photocatalytic properties, but also the principle of hyperthermia, achieved for the first time due to the human body temperature 36.6°C.

Medical forms on the base of titanium dioxide or silicon dioxide crystals, at the expense of synthesis of regulated active oxygen, AFO, firstly, selectively penetrate into the pathologenic cells of organism, targeting the areas of inflammation, with infectious and non-infectious nature, in particular, the cells using a ferment NADF - H for AFO formation, phagocytes and macrophages, causing in them numerous cytomorphologic changes (vacuoles in cytoplasm, fragmentation of membrane, abnormality of mitosis), that launches apoptosis or necrosis type of death of pathogenic cells (because the pathogenic cells haven’t effective antioxidant ferments). Regulated active oxygen, AFO become firstly claimed in the pathologies focuses (respiratory explosion), where oxygen necessity increases many times.

NADF-H oxydases membrane-bound enzymatic complex, inversed into extracellular space of plasmatic membrane, and also in membranes of phagosomes, used by neutrophilic leukocytes (immune cells, white blood corpuscles) for absorption of microorganisms is executed with the immune response.

NADF-G oxidase (NOX) is one of the main sources of cellular active forms of oxygen (AFO) and is formed by the way of catalysis.

Stable suspension of titanium dioxide crystals and silicon dioxide particles ensures induction of immune response of vertebrate by means of physical or chemical interaction with antigens.

Photocatalytic properties of titanium dioxide or silicon dioxide heterocrystals - mobilizing at immute reaction to pathogens and normalization of AFO production in the human body or an animal body.

Laboratory and clinical researches, when oxygen, O ₂ , on the crystal lattices is initially in excited condition ² T ³ in the case of the crystals-sensitizers use in composition of medicines hydrogels (forms of use: topically, nose, throat, mucous, vaginally, rectally, orally), and also medical articles of topical use in the form of hydrocolloidal medical plasters, impregnation for tampons, absorbent pads, daipers, tapes, bandages etc., having antimicrobial and antiseptic influence, anti-inflammatory action, immunomodulatory and adsorbing action, depending on form and method of their use.

Mechanism of action in composition of a medical article or gel is that in interaction of electronically excited O ₂ on the surface of crystals or particles with membrane cellular enzymatic complex NADF-H AFO is formed.

Efficiency of using stable suspensions of TiO ₂ heterocrystals and SiO ₂ particles for adjuvant therapy of cancer diseases is also experimentally proven in combination with cytostatic agents (Doxorubicin, Lenalidomide (Revlimid), Nivolumab, Ibrutinib).

A positive result was demonstrated on the model of metastasis into spleen and liver, primary tumor significantly reduces after intraabdominal administration, after administration together with cytostatic agent. In addition, a tendency to decrease of metastatical nodes was observed.

The experiments have proven use of stable suspension of heterocrystals of titanium dioxide or silicon dioxide particles, for formulation of medical agents, used intravenously, intramuscularly, orally, nasally, vaginally, rectally, locally (ENT) use or topically, at that therapeutically important amount of an active substance is for intravenous use from 0.006 mg/ml, intramuscular use from 0.01 mg/ml, orally from 0.01 mg/ml, nasally from 0.01 mg/ml, vaginally from 0.01 mg/ml, rectally from 0.01 mg/ml, local ENT use from 0.01 mg/ml, ocular use from 0.001 mg/ml, and also medical articles of topical use in the form of hydrocolloidal medical plasters from 0.0003 mg/g. Amount of stable suspension included into medical forms is chosen in accordance with well-known principles of pharmacy, clinical medicine and pharmacology. Usually, therapeutically effective amount of a medical means is meant. The term “therapeutically effective amount” means that pharmaceutically efficient amount is considered in relation of, for example, pharmaceutical preparations. Pharmaceutically efficient amount is amount of a medical remedy or pharmaceutically active substance that is sufficient for achieving acceptable biological reaction in its use.

A medical remedy may be executed in the form of rectal-vaginal suppositories, gel, ointment, liquid.

INDUSTRIAL APPLICABILITY

Used elaborated stable suspensions in composition of medical remedies have the following advantages:

• It is catalyst of energy-exchange reactions;

• Absence of general toxicity;

• Absence of allergenic properties, i.e. hypoallergen;

• Absence of harmful toxicological compounds;

• Heterogenous suspension structures are photocatalysts having catalytic properties;

• Have anti-pathogenous antiviral activity, preventing penetration of virus into the cells and promotes destruction of viruses with lipid membrane due to damaging of the latter.

• It is possible to effectively influence onto pathogens.

• It is possible to use high light capacities (5 and more Wt/sq.cm). • It is possible to accelerate process of influence (up to 1 -3 min).

• Absence of toxic action of photocatalyst with/without photodynamic therapy (PDT); Use of stable suspensions, obtained by the elaborated method, ensures antiviral therapeutic effect, antipathogen therapeutic effect, sorption capacity of crystals and particles ensures detoxication of organism, ensures elimination of underoxidation processes of in the body of living organism.

USE OF SUSPENSIONS IN PHARMACEUTICS FOR MODERN MEDICINE