RU2721562C1 - Method of loading inorganic nanoparticles or organic molecules into porous particles of micron or submicron size - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to loading porous particles of micron or submicron size with inorganic nanoparticles or organic molecules. Described is a method of loading inorganic nanoparticles or organic molecules into porous particles of micron or submicron size, involving obtaining a suspension of inorganic nanoparticles or organic molecules and porous particles in an aqueous medium or in an organic solvent medium; controlled suspension freezing with rate of crystallization front lower than critical speed, at which particles are captured by front, wherein freezing is carried out while stirring with speed, at which there is no sedimentation of particles; thawing of suspension; and recovering loaded porous particles, as well as a method of producing micron or submicron-size polymer capsules, comprising obtaining porous micronized or submicron sized particles, forming a polymer shell on the surface of said loaded porous particles of micron or submicron size, dissolving porous particles of micron or submicron size by processing with a reagent which dissolves porous particles, as well as a polymer capsule containing inorganic nanoparticles and/or organic molecules.

EFFECT: technical result is higher efficiency of loading inorganic nanoparticles or organic molecules into porous particles of micron or submicron size or polymer capsules.

13 cl, 3 ex, 1 tbl, 6 dwg

Description

FIELD OF THE INVENTION

The invention relates to the loading of porous micron or submicron particles with inorganic nanoparticles or organic molecules. The method also allows to obtain polymer capsules loaded with inorganic nanoparticles or organic molecules.

State of the art

Currently, there is a problem of manufacturing new innovative products for the pharmaceutical, aerospace, photonics, ecology and food industries. Progress in this direction is constrained by the lack of methods for producing multifunctional smart materials. The multifunctionality of materials and the control of their properties can be realized using multicomponent composite structures with strictly defined mass fractions and distribution of each component. Crystallization-induced adsorption (ICA) is a new approach to the controlled loading of inorganic nanoparticles and organic molecules, in particular proteins, into porous particles. The ICA method allows the adsorption of components quantitatively, with high reproducibility for each cycle.

There is a known method of pressing materials under the influence of directional freezing of ice crystals, which is used to compress the precursors of ceramic materials (Qian, L. & Zhang, H. Controlled freezing and freeze drying: a versatile route for porous and micro- / nano-structured materials. J Chem. Technol. Biotechnol. 86, 172-184 (2011). Encapsulation of fluorescent dyes or fluorescently labeled polymers and active drugs into theranostic carriers with multifunctional properties is also known. In vivo navigation of such carriers can be carried out using magnetic field gradients (Voronin, DV et al. In Vitro and in Vivo Visualization and Trapping of Fluorescent Magnetic Microcapsules in a Bloodstream. ACS

Appl. Mater. Interfaces 9, 6885-6893 (2017) and Galanzha, E. I. et al. In vivo magnetic enrichment, photoacoustic diagnosis, and photothermal purging of infected blood using multifunctional gold and magnetic nanoparticles. PLoS One 7, e45557 (2012)), and they provide the highest photoacoustic signal in undiluted blood, and also demonstrate laser activation and release by ultrasound. The most widely used types of templates for such carriers are porous material particles of micron and submicron sizes. These particles have significant advantages, such as low cost, easily scalable production, biocompatibility, biodegradability, solubility under mild pH conditions (6.5), high porosity and stability at high temperature and pressure.

Examples of encapsulated substances include anticancer drugs cisplatin and doxorubicin, tetracycline hydrochloride and photodynamic dyes. Most published articles on vaterite particles have reported studies of internalization efficacy, toxicity (cell viability), and in vitro kinetic drug release profiles. A significant achievement in the efficiency of particle internalization by cells and drug delivery is associated with the transition from the micron to submicron range.

Substrate carriers began to be used in vivo more than eight years ago (Genina, E. A. et al. In vivo optical monitoring of transcutaneous delivery of calcium carbonate microcontainers. Biomed. Opt. Express 7, 2082-2087 (2016)). Currently, they are used in biomedicine as templates and carriers for drug delivery systems. They are administered in various ways, including transdermal administration, inhalation, intertumoral and oral administration. Vaterite particles are also used in the preparation of food polymer additives and in water treatment. However, their transition from research and development to industrial applications was complicated by the lack of an effective approach to loading. Traditional methods allow you to enter a maximum of only 4 wt.% Magnetite in seven stages, and the reproducibility of the load during one stage is low. Among the mentioned methods, it should be noted, such as adsorption from a solution (De Cock, LJ et al. Polymeric multilayer capsules in drug delivery. Angew. Chemie Int. Ed. 49, 6954-6973 (2010)), encapsulation during the preparation of particles (method coprecipitation) (Svenskaya, YI et al. Photodynamic therapy platform based on localized delivery of photosensitizer by vaterite submicron particles. Colloids Surfaces B Biointerfaces 146, 171-179 (2016)), chemical vapor deposition (Christian, P., Ehmann, HMA, Coclite, AM & Werzer, O. Polymer Encapsulation of an Amorphous Pharmaceutical by initiated Chemical Vapor Deposition for Enhanced Stability. ACS Appl. Mater. Interfaces 8, 21177-21184 (2016)) and supercritical fluid technology (Liu, H., Finn, N. & Yates, MZ Encapsulation and sustained release of a model drug, indomethacin, using CO2-based microencapsulation. Langmuir 21, 379-385 (2005)).

Improving the efficiency of loading functional nanoparticles or molecular substances into porous particles is an urgent task. The solution to this problem will improve the carriers of bioactive substances intended for targeted delivery and develop effective functional materials based on nanoparticles with superparamagnetic or plasmonic properties.

SUMMARY OF THE INVENTION

The freeze / thaw approach has been widely used for many purposes, including the creation of lamellar and porous structures, micro and nanowires, as well as micro and nanoparticles. This approach is based on controlled directional freezing so that ice crystals grow in one direction. In the direction “a” of the hexagonal base, the crystals grow 100 times faster than in the perpendicular direction “c”. As a result, ice crystals grow in the form of a lamellar microstructure parallel to the “a” direction (Wegst, UGK, Schecter, M., Donius, AE & Hunger, PM Biomaterials by freeze casting. Philos. Trans. R. Soc. London A Math.Phys Eng. Sci. 368, 2099-2121 (2010)).

Until now, the mentioned approach has not been applied for loading inorganic nanoparticles or organic molecules into micron and submicron porous particles. In the present invention, the authors propose to use solvent crystallization (IKA) to load inorganic nanoparticles or organic molecules into porous matrices.

The solution to this problem is achieved by the method of loading inorganic nanoparticles or organic molecules into porous particles of micron or submicron size, including obtaining a suspension of inorganic nanoparticles or organic molecules and porous particles in an aqueous medium or in an organic solvent; controlled freezing of the suspension with a speed of crystallization front of a lower speed at which particles are captured by the front, and freezing is carried out with stirring at a speed at which sedimentation of particles does not occur; thawing the suspension; and the selection of loaded porous particles. Preferably, after thawing, an additional amount of inorganic nanoparticles or organic molecules is introduced into the suspension and re-freezing and thawing are performed.

If necessary, the introduction of an additional amount of inorganic nanoparticles or organic molecules, repeated freezing and thawing is carried out several times.

Preferably, the organic molecules are proteins, in particular bovine serum albumin, or dyes, in particular indocyanine green, cyanine dyes, phthalocyanines.

Typically, micron or submicron sized porous particles contain CaCO₃, in particular, one of its polymorphic modifications is waterite.

Preferably, the inorganic nanoparticles contain magnetite or gold.

In addition, the use of the ICA method allows to obtain polymer capsules of micron or submicron size, by obtaining loaded porous particles of micron or submicron size in the manner described above;

the formation of a polymer shell on the surface of these loaded porous particles of micron or submicron size;

dissolving porous particles of micron or submicron size by treatment with a reagent that dissolves the porous particles.

Preferably, the polymer shell further comprises protein.

Typically, a polymeric multilayer shell is formed by layer-by-layer adsorption of polymers. To do this, obtain a suspension of loaded porous particles of micron or submicron size, polymer and, if necessary, protein, in water; carry out controlled freezing of the suspension with a speed of the crystallization front of a lower critical speed at which particles are captured by the crystallization front, and freezing is carried out with stirring at a speed at which sedimentation of particles does not occur; thaw the suspension and isolate the particles. The shell can be formed by various pairs of polymers, for example: bovine serum albumin and tannic acid, poly-L-arginine and dextransulfate, polyallylamine hydrochloride and sodium polystyrene sulfonate.

The invention also discloses porous micron or submicron particles loaded with inorganic nanoparticles or organic molecules obtained by the method of the invention and a polymer capsule obtained by the method of the invention.

The invention is further disclosed in view of the following drawings:

Figure 1. (a) an outline of an ICA method;

(b) (I) loading magnetite nanoparticles (MNPs) by coprecipitation;

(b) (II, III), the 1st and 7th loading of MNPs through adsorption;

(IV, V), 1st and 7th loading of MNP by ICA method;

(c) the dependence of the mass fraction of MNP (determined by weighing the solid residue after each freeze-thaw cycle) on the number of freeze-thaw cycles.

Figure 2. Optical microscopy of the ICA process;

(a) Suspension of microparticles;

(b) Movement of the crystallization front;

(c) Pressing nanoparticles into microparticles with a crystallization front;

(d-f) Scheme of MNP adsorption caused by advancement of the crystallization front.

Figure 3. SEM images of micron (a-f) and submicron (g-i) particles of vaterite;

(a, d, g) before implementing the ICA method;

(b, e, h) after one ICA cycle;

(c, f, i) after the maximum number of ICA cycles. The insets show SEM images at higher magnification.

Figure 4. (a) An SEM image of a microparticle loaded with gold nanoparticles (NPP) using the ICA method;

(b) Image obtained by confocal laser scanning microscopy (CLSM) image of unloaded control microparticles;

(c) The KLSM image of a microparticle loaded with a BSA-RITC complex using the ICA method;

(d) Dependence of the mass fraction of the BSA-RITC complex on the number of freeze / thaw cycles or adsorption steps. The arrow indicates the mass fraction of the BSA-RITC complex achieved by coprecipitation; (e) photographs of tubes containing CaCO ₃ / BSA-RITC microparticles obtained by performing (I) adsorption, (II) coprecipitation, and (III) the ICA method.

Figure 5. (a) Formation of polymer capsules;

(b-d) SEM images of microcapsules with various amounts of magnetite;

(e-g) SEM images of submicron capsules with various amounts of magnetite.

6. Temporal dependence of the extinction of a solution of microcapsules in an external magnetic field.

The implementation of the invention

In the general case, the ICA method involves several successive stages: adding suspensions of micro- and nanoparticles to the container, freezing the samples with gentle stirring, thawing the samples, and separating the suspension. This process can be repeated several times, which makes it possible to achieve high efficiency loading of nanoparticles (figa). The number of freeze / thaw cycles is determined by the required load.

The continuous phase of the suspension is water or aqueous solutions, in addition, organic solvents such as dimethyl sulfoxide can be used.

To press nanoparticles into the porous matrix of microparticles, both types of particles must be pushed out by the crystallization front until the entire liquid phase of the solvent in which the particles were initially dispersed crystallizes.

Particles are displaced by the crystallization front if the current front velocity is less than the critical speed at which particles are captured during crystallization.

From the work (Wegst, UG K., Schecter, M., Donius, A.E. & Hunger, P. M. Biomaterials by freeze casting. Philos. Trans. R. Soc. London A Math.Phys. Eng. Sci. 368, 2099-2121 (2010)) it is known that the critical crystallization rate is calculated by the formula:

- свободная энергия,

- среднее расстояние между молекулами в жидкости, η - вязкость жидкости, r - радиус частицы,

где

и

- теплопроводность частиц и жидкости соответственно.Where

- free energy,

is the average distance between the molecules in the liquid, η is the viscosity of the liquid, r is the radius of the particle,

Where

and

- thermal conductivity of particles and liquid, respectively.

The ratio of the current velocity of the crystallization front to its critical velocity depends on the chemical composition of the crystallizing liquid and particles, the radius of the particles, and freezing conditions. Suspensions of CaCO ₃ microparticles and magnetite nanoparticles (MNPs) were individually frozen to verify compliance with the above conditions for each type of particle. Nano- and microparticles were displaced from the walls of the tube by the crystallization front to its center, which can be easily verified by visual observation. This means that the current velocity of the crystallization front is less than the critical velocity of the crystallization front.

The presence of particles near the crystallization front leads to its curvature. The solid-liquid interface undergoes a convex bend in the direction of the particle if the thermal conductivity of the particle is lower than the thermal conductivity of the liquid. If the thermal conductivity of the particle is higher than the thermal conductivity of the liquid, the solid-liquid interface undergoes a concave bend in the direction opposite to the particle. In addition to this, regions are formed ahead of the crystallization front, which are characterized by high concentrations of nano- and microparticles and a low crystallization temperature (concentration supercooling). The crystallization temperature of water in pores less than 100 nm is lower than in bulk water, and decreases with decreasing pore diameter. As a result, this effect can lead to the expulsion of nanoparticles into the pores of the laterite under the action of a crystallization front.

Images of vaterite microparticles obtained by the SEM method before the implementation of the ICA method with respect to the MNP (Fig. 3a), as well as after the 1st (Fig. 3b) and 7th (Fig. 3c) freeze / thaw cycles, are shown in FIG. 3. MNPs formed shells on the surface of the laterite (Fig. 3b, c, dashed lines).

As shown in FIG. 3b, c, e, f, h, i, the shape of micron and submicron particles of vaterite was maintained after each freeze / thaw cycle with magnetite nanoparticles.

In this way, inorganic nanoparticles with a diameter of 5 to 40 nm, for example, gold or iron oxide (magnetite), as well as titanium dioxide, and organic molecules with high (proteins, such as bovine serum albumin) and low molecular weight (organic dyes - green indocyanin, cyanine dyes, phthalocyanines). Choice of the loaded substance (sorbate): inorganic particles and / or organic molecules, depends on the specific tasks for which carriers are created. In addition, the main criterion is the ejection of the loaded particles by the crystallization front of the dispersion medium. For the experiments, CaCO ₃ was used as porous particles (sorbent), in particular, vaterite with a size of 400-700 nm (submicron particles) and 3-5 microns (micron particles), since vaterite particles have several advantages: high porosity, biocompatibility, the technological simplicity of their dissolution after the formation of the polymer shell (if required), the low cost of the reagents for their preparation. However, the choice of sorbent is not limited only to vaterite. The main thing for both the sorbent and the sorbate should be the ability to push particles out by the crystallization front of the dispersion medium.

The invention can be illustrated by the following examples.

Example 1

As an example of the method, an experiment was carried out on the loading of vaterite particles by magnetite particles, carried out in various ways.

Microparticles of CaCO _{3 were} loaded by coprecipitation as follows:

0.615 ml of a 1 M solution of CaCl ₂ and 0.615 ml of a 1 M solution of Na ₂ CO _{3 were} injected into 2.5 ml of a suspension of MNP in water with vigorous stirring. After one minute, stirring was stopped, and the resulting dispersion of particles was separated by centrifugation and washed twice with deionized water.

Microparticles of CaCO _{3 were} charged by adsorption as follows:

to 40 mg of CaCO ₃ particles, 2 ml of MNP suspension was added and the mixture was shaken for 15 minutes. After that, the particles were separated by centrifugation and washed twice with deionized water. For various samples, this procedure was repeated up to seven times.

Microparticles of CaCO _{3 were} loaded by the ICA method as follows:

to 40 mg of CaCO ₃ particles, 2 ml of a suspension of MNP in water was added. The microcentrifuge tube with the reaction mixture was kept in the freezer at -20 ° C for 2 hours with slow and constant stirring at a speed of 4 rpm in a rotary mixer.

After that, the samples were thawed at room temperature and washed. Defrosting conditions are not significant. It is carried out at a temperature of not more than 35 ° C in order to reduce the likelihood of recrystallization of calcium carbonate from a polymorphic modification of vaterite to a more thermodynamically stable calcite.

Some samples were dried in an oven, while others were re-frozen / thawed. For various samples, freezing / thawing was repeated up to seven times.

The number of MNP loaded by the ICA method was measured as the difference between the mass of particles before and after loading. During coprecipitation, the amount of MNPs was determined by colorimetric titration, since in this case, MNPs were loaded during the preparation of vaterite microparticles.

The experimental results are illustrated in FIG. 1b and 1s.

A comparative image of a vaterite suspension with MNPs loaded in various ways is shown in FIG. 1b. The image shows the method of ICA (fig.1b (IV, V)) in comparison with other methods used (fig.1b (I-III)). The ICA method allows more significant amounts of magnetite (13%) to be adsorbed into the vaterite microparticles compared with adsorption (4%) and coprecipitation (3%). You can add an additional amount of nanoparticles of the same or another type by coprecipitation before the implementation of the ICA method, or you can adsorb an additional amount of nanoparticles of the same or different type on a polyelectrolyte shell after the implementation of the ICA method. This allows you to achieve even higher load or create multifunctional media. Samples of vaterite particles were washed, dried and weighed after each loading to estimate the mass fraction of adsorbed MNPs. The dependence of the mass of adsorbed MNPs on the number of freezing cycles is shown in Fig. 1c. In FIG. It was shown that, using the same mass ratio between the MNP and vaterite particles for submicron and micron vaterite particles, the number of adsorbed MNPs was greater on submicron particles (mass fraction 31%) than on micron particles (mass fraction 13%). Presumably, this is due to the fact that submicron particles have a more developed porous surface. Moreover, with the same mass of vaterite particles, the total surface area of micron-sized particles was eight times smaller than the area of submicron-sized particles.

Freezing a suspension containing microparticles of CaCO ₃ and MNP is shown in Fig.2. During crystallization, MNPs and CaCO ₃ microparticles advanced under the action of a crystallization front (Fig. 2b, e;), and MNPs were concentrated around the surface of the laterite particles. At the final stage of the process (Fig. 2c, f), the MNPs were pressed under the action of the growing pressure of the formed ice on the surface of CaCO ₃ particles.

Example 2

The ICA method was tested on gold nanoparticles (NPP) to assess the efficiency of adsorption of particles of 10, 20 and 40 nm in size. The loading of vaterite by freezing / thawing was repeated three times. It was found that the adsorption efficiency increased with increasing size of the nanoparticles. NPPs were aggregated during the loading process, and the aggregates were placed on the surface of microparticles (Fig. 4a). The efficiency of the NPP loading was determined by optical spectroscopy (Synergy H1 Multi-Mode Reader, BioTek Instruments, USA) by measuring the absorption of light in the range 300–900 nm. After each freeze / thaw cycle, the amount of unloaded NPF in the supernatant was measured. The number of loaded NPPs (Δm) was determined as the difference between the initial and unloaded particles. As a result of the experiment, it turned out that the number of NPPs loaded with the ICA method was three times larger than the amount achieved during adsorption. The results are presented in table 1

Table 1.

The adsorption efficiency of gold nanoparticles of 10, 20 and 40 nm in size.

Download Count ICA Solution adsorption 10 nm 20 nm 40 nm 10 nm 20 nm 40 nm Δm, mcg Δm, mcg Δm, mcg Δm, mcg Δm, mcg Δm, mcg 1 390 660 700 130 580 210 2 285 650 620 100 220 250 3 300 600 700 80 twenty 270 Total qty 980 1920 2010 310 820 730

The ICA method can be used for adsorption of polymers on the surface of vaterite. This allows you to get structures with a large number of complex bovine serum albumin - rhodamine-B-isothiocyanate (BSA-RITZ; fig.4e). The BSA-RITC complex was adsorbed on microparticles from phosphate buffered saline (PBS). The formation of BSA-RITC shells on the vaterite surface was confirmed by confocal laser scanning microscopy (KLSM; Fig. 4b, c), and the amount of adsorbed BSA-RITC complex was determined by spectrophotometry (the dependence of the mass fraction of the BSA-RITC complex determined by optical spectroscopy on the number ICA / adsorption loading cycles are shown in fig.4d). The ICA method allows you to adsorb four times more BSA-RITC complex (13.5%) than adsorption methods (3.7%) and coprecipitation (3.6%). The freeze-thaw process can affect the tertiary or quaternary structure of albumin. It can be controlled by factors such as freezing rate and protein concentration. An increase in the freezing rate leads to a reduction in the time period of the strong influence of the cooler on the molecules (Yu, Z., Garcia, AS, Johnston, KP & Williams, RO III Spray freezing into liquid nitrogen for highly stable protein nanostructured microparticles. Eur. J. Pharm. Biopharm 58, 529-537 (2004)). In addition to this, an increase in protein concentration increases its stability. This can be explained by a decrease in the relative number of protein molecules at the ice / concentrated solution interface. Thus, there are many possibilities for eliminating the negative effects of freezing on the tertiary or quaternary albumin structure.

The obtained micron and submicron composite particles were used as a template for polymer magnetic capsules (Fig. 5a). The formation of polymer capsules consisted of four stages. The first stage was the synthesis of micron or submicron particles of vaterite. The second stage was the loading of various concentrations of MNP onto the surface of vaterite particles by the ICA method. At the third stage, multilayer shells consisting of BSA and tannic acid were formed on the surface of composite particles. These shells were formed by a complex of polyphenols and proteins, interacting mainly due to hydrogen bonds. They can be strong enough to be stable, although the electrostatic forces that activate the interlayer interactions are not involved in the formation of the mentioned microspheres. In the fourth stage, the lateritic nuclei were slowly dissolved by adding a 0.2 M EDTA solution, followed by a washing step. This procedure resulted in the production of micron and submicron sized polymer capsules with different amounts of magnetite (Fig. 5b-g). The concentration of the BSA-RITC complex was determined by measuring the fluorescence intensity (A _ex = 560 nm, A _em = 590 nm) of the supernatant after each stage of freezing / thawing. All measurements were performed at 24 ° C in single-use 96-well plates.

In FIG. 5d, g show that capsules formed on the basis of vaterite microparticles loaded with a large amount of magnetite did not shrink after drying. This directly indicates that the bulk of the magnetite particles was distributed over the surface of the vaterite particles and formed magnetite shells / crusts that retained their shape after the core was dissolved. Unlike micron-sized capsules, submicron capsules did not retain their shape when dried.

The speed of movement of the obtained capsules in an external magnetic field was determined by the shape of the time dependence of the extinction of the sample suspension at 660 nm when a permanent magnet was applied to one of the walls of the cell. These dependencies are shown in graph form in FIG. 6. As shown in FIG. 6, the ICA method allows to obtain polymer nanocomposite capsules with large quantities of magnetite and, as a result, with a large magnetic moment for better magnetic navigation in drug delivery applications.

The release of the BSA-RITC complex from the obtained microcapsules was measured for 9 days. The release of the BSA-RITC complex was slow and steady. The loaded material can also be released in a controlled manner as a result of enzymatic degradation of the membrane by laser radiation or ultrasonic treatment.

The preparation of polymer capsules is illustrated by the following example.

Example 3

The template for polymer capsules was vaterite particles loaded with MNPs as described in Example 1. A layered assembly was used to make micron and submicron capsules. A method of producing microcapsules using layer-by-layer assembly by sequential adsorption of polyelectrolytes is disclosed in (GB Sukhorukov, E. Donath, S. Davis, H. Lichtenfeld, F. Caruso, VI Popov, H. Möhwald, Polym. Adv. Technol. 1998, 9, , 759-767, E. Donath, GB Sukhorukov, F. Caruso, SA Davis, H. Möhwald, Angew. Chem. Int. Ed. 1998, 37, 2201-2205) and in EP1064087A2.

Microcapsules were prepared by sequential adsorption of 1 ml of a suspension of BSA (concentration in water 2 mg / ml) and tannic acid (concentration in water 2 mg / ml) on the spherical surfaces of CaCO ₃ nuclei. Each cycle of polymer adsorption was carried out for 15 min. Then the nuclei were slowly dissolved by treatment with EDTA (concentration in water 0.2 M, pH 7.3), and the reaction products were removed by washing twice in deionized water. To slowly dissolve the nuclei, the EDTA reagent was slowly added to the particle suspension with shaking until the nuclei were completely dissolved. After each adsorption cycle, as well as after dissolution of CaCO ₃ nuclei, the suspension of microparticles was centrifuged (at 240 g in the case of micron capsules and at 1300 g in the case of submicron capsules) and washed twice with deionized water. As a result, the capsules were shells with three double layers of BSA-tannic acid. Unlike vaterite particles, polymer composite capsules are very stable. All composite capsule samples were stored in a refrigerator at 4 ° C. After 15 months of storage, the capsules retained their shape and zeta potential (-45 ± 5 mV) and did not aggregate.

In all cases, loading in% is the ratio of the mass of the loaded substance to the total mass of the resulting carrier together with the loaded substance.

As demonstrated in this document with the help of NPP, MNP and the BSA-RITC complex in the case of micron-sized waterite particles, the ICA method achieved at least three times the load of magnetite nanoparticles and four times the protein load compared to traditional methods such as adsorption and coprecipitation. For submicron particles, the ICA method allows five times the loading of magnetite nanoparticles in comparison with traditional methods.

The specified cheap and easy for subsequent scaling method can be used to manufacture composites fillers for new alloys, multifunctional contrast agents, sensors and carriers for the delivery of bioactive substances with adjustable sensitivity to external stimulants (magnetic field gradient, laser radiation or ultrasound).

Claims (20)

1. The method of loading inorganic nanoparticles or organic molecules into porous particles of micron or submicron size, including obtaining a suspension of inorganic nanoparticles or organic molecules and porous particles in an aqueous medium or in an organic solvent; controlled freezing of the suspension with a crystallization front velocity lower than the critical speed at which the particles are captured by the front, and freezing is carried out with stirring at a speed at which particle sedimentation does not occur; thawing the suspension; and Isolation of loaded porous particles. 2. The method according to p. 1, characterized in that after thawing, an additional amount of inorganic nanoparticles or organic molecules is introduced into the suspension and repeated freezing and thawing are performed. 3. The method according to p. 2, characterized in that the introduction of an additional amount of inorganic nanoparticles or organic molecules, repeated freezing and thawing is carried out several times. 4. The method according to any one of paragraphs. 1-3, in which the organic molecules are proteins, in particular bovine serum albumin. 5. The method according to any one of paragraphs. 1-3, in which the organic molecules are dyes, in particular indocyanine green, cyanine dyes, phthalocyanines. 6. The method according to any one of paragraphs. 1-5, in which the porous particles of micron or submicron size contain CaCO3 in particular vaterite. 7. The method according to any one of paragraphs. 1-6, in which the inorganic nanoparticles are composed of magnetite or gold. 8. Porous particles of micron or submicron size loaded with inorganic nanoparticles or organic molecules obtained by the method according to any one of paragraphs. 1-7. 9. A method of producing polymer capsules of micron or submicron size, including obtaining loaded porous particles of micron or submicron size by the method according to any one of paragraphs. 1-7; the formation of a polymer shell on the surface of these loaded porous particles of micron or submicron size; and the dissolution of porous particles of micron or submicron size by treatment with a reagent that dissolves the porous particles. 10. The method according to p. 9, characterized in that the polymer shell further comprises a protein. 11. The method according to p. 9 or 10, characterized in that the polymer shell is formed by obtaining a suspension of loaded porous particles of micron or submicron size, polymer and, if necessary, protein, in water; controlled freezing of the suspension with a speed of the crystallization front of a lower critical speed at which particles are captured by the crystallization front, and freezing is carried out with stirring at a speed at which sedimentation of particles does not occur; thawing the suspension and isolating particles. 12. The method according to p. 10 or 11, characterized in that the polymer shell contains a pair of polymers selected from the series: bovine serum albumin and tannic acid, poly-L-arginine and dextransulfate, polyallylamine hydrochloride and sodium polystyrenesulfonate. 13. A polymer capsule containing inorganic nanoparticles and / or organic molecules obtained by the method according to any one of paragraphs. 9-12.

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