FIELD OF TECHNOLOGY

The invention relates to a method for production of ferrimagnetic chitosan/poly(sodium aspartate)/Fe ₃ 0 ₄ nanocomposite having negative surface charge, which is designed for use as an element of scaffolds for cell culture, as controlled drug delivery and release systems and as a contrast agent in magnetic resonance imaging or in computer tomography.

THE PRIOR ART

Ferrimagnetic nanoparticles of iron(II, III) oxide are particles of size between 1 and 100 nm with the chemical formula Fe ₃ 0 ₄ or FeO Fe ₂ 0 ₃ , where iron is present on both the second and the third oxidation states. The oxides occur in nature in the form of magnetite. They have unique optical, electrical and biological properties. Therefore, they are widely used in various technical fields, such as electronics (ferrofluids), medicine (controlled drug delivery and release systems, contrast agents for computer tomography and magnetic resonance), chemistry (modern catalysts), or environmental protection (industrial wastewater treatment).

The most well known methods for obtaining magnetite are:

1 . Laux process, in which magnetite is a by-product in the process of aniline production as a result of the reaction of nitrobenzene with iron in the presence of FeCl ₂ as a catalyst:

C ₆ H ₅ N0 ₂ + 3 Fe + 2 H ₂ 0 -» C ₆ H ₅ NH ₂ + Fe ₃ 0 ₄ 2. Oxidation of Fe(II) compounds: iron(II) salts are converted to hydroxides and then subjected to the oxidation process while maintaining strict pH control.

3. Controlled incomplete reduction of iron(III) oxide with hydrogen gas or carbon monoxide:

3 Fe ₂ 0 ₃ + H ₂ -> 2 Fe ₃ 0 ₄ + H ₂ 0

3 Fe ₂ 0 ₃ + CO -> 2 Fe ₃ 0 ₄ + C0 ₂

The reactions of magnetite synthesis described above are multistage and they make it impossible to obtain nanometer-size particles.

The method that enables to obtain nanometer- size Fe ₃ 0 ₄ particles is the Massart reaction:

FeCl ₂ + 2 FeCl ₃ + 8 NaOH = Fe ₃ 0 ₄ + 8 NaCl + 4 H ₂ 0

Due to their ferrimagnetic properties, the nanoparticles of iron(II, III) oxide are often used in medicine and pharmacy. It is well-known that ferrimagnetic nanoparticles for medical and pharmaceutical purposes must have the size greater than 10 nm, which prevents their accumulation in a body. For this reason, in order to obtain nanoparticles with increased biocompatibility and bioactivity, not only solutions enabling simple and quick synthesis of stable Fe ₃ 0 ₄ nanoparticles having controlled diameters, but also effective and environmentally friendly stabilizing agents are sought.

Chitosan is a biopolymer obtained by deacetylation of chitin.

Chitosan is characterized by:

- high water retention value after crosslinking;

- ability to form polymer films directly from the solution;

- high adhesiveness;

- controlled bioactivity, especially antibacterial activity; - non-toxicity, biocompatibility, biodegradability;

- high stability in the form of a suspension;

- good miscibility with many substances, including polymers;

- high chemical reactivity;

- high sorption capacity and chelating abilities;

Chitosan is widely used in medicine and industry. Its source is exoskeletons of crustaceans and insects, as well as some fungi.

Poly(sodium aspartate) is a polymer obtained by polycondensation of aspartic acid to poly(succinimide), followed by its hydrolysis with NaOH, or by polycondensation of monosodium salt of aspartic acid. Poly(succinimide) can also be obtained by polycondensation of maleic anhydride with urea at a molar ratio of 2 : 1, but the obtained polymer is contaminated with by-products and has a very irregular structure, which makes it impossible to use for medical purposes.

Among the known methods for obtaining ferrimagnetic nanocomposites based on chitosan, the closest to the solution of the present invention is the method described in the patent application CN10556132. The application discloses a method for obtaining Fe ₃ 0 ₄ nanoparticles through drop-wise addition of NaOH solution to the mixture of solutions of iron(II) chloride and iron(III) chloride at the temperature of 50 °C under nitrogen atmosphere, until a black product having magnetic properties is formed. Then the nanoparticles are purified and dried and introduced into 2% aqueous solution of chitosan in 2% acetic acid solution. The solution undergoes the action of ultrasounds in order to disperse the nanoparticles and then glutaraldehyde is introduced into the solution in order to crosslink the polymer and to obtain magnetic chitosan hydrogel in which the content of chitosan is min. 25% and the content of ferrimagnetic nanoparticles is min. 20%. Unfortunately, the crosslinking agent used in this method (glutaraldehyde) has a neurotoxic effect and therefore the material introduced into a living organism will cause undesirable immunological reactions and will act toxic to nerve cells. The article "Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells", Yeast 2015; 32: 239-243 discloses the method for microwave synthesis of magnetic chitosan microspheres with positive surface charge, which are intended for immobilization of yeast cells having negative surface charge. The authors described obtaining the microspheres as a result of microwave treatment of the suspension containing chitosan and Fe ₃ 0 ₄ obtained by adding NaOH solution to the solution of iron(II) sulphate(VI) until solution became alkaline. Then the resulting composite undergoes crosslinking reaction. The described method also proposes using glutaraldehyde as a crosslinking agent, which is known to have a neurotoxic effect, whereby the material introduced into the living organism will cause undesirable immune reactions and will act toxic to nerve cells.

As it is reported in the professional and patent literature, currently the method for obtaining ferrimagnetic chitosan/poly(sodium aspartate)/Fe ₃ 0 ₄ nanocomposite with a negative surface charge without using harmful reagents and using microwave radiation for heating reagents, is not known.

THE AIM OF THE INVENTION

The aim of the invention is to develop a method for production of ferrimagnetic chitosan/poly(sodium aspartate)/! ⁷ e ₃ 0 ₄ nanocomposite which has negative surface charge. According to the method of the invention, the chemical structure of chitosan is modified during the production process to such an extent, that it allows biodegradation of the nanocomposite, increases its biocompatibility and tissue affinity and the nanocomposite does not show toxicity to the cells.

NATURE OF THE INVENTION

According to the invention, the method for production of ferrimagnetic chitosan poly(sodium aspartate)/Fe ₃ 0 ₄ nanocomposite having negative surface charge and containing Fe ₃ 0 ₄ nanoparticles with the size from 10 to 100 nm, wherein the method uses iron(II) and iron(III) salts, a precipitating agent which increases the pH, chitosan dissolved in an aqueous environment in the presence of acid, a crosslinking agent and an organic stabilizer, is characterized in that aqueous solutions of iron(II) salt and iron(III) salt are introduced to a vessel transparent for microwaves. During intensive stirring and simultaneous microwave irradiation, the substance that increases the pH of the mixture is introduced. The substance is selected from the group consisting of organic and/or inorganic bases and said substance is introduced until black color of the solution is obtained. Then the composition containing chitosan dissolved in the aqueous solution of an organic acid or an amino acid, a crosslinker in the form of glycol having 2 to 8 carbon atoms and poly(sodium aspartate) as a stabilizer, are introduced into the suspension. Next, the mixture undergoes the reaction of crosslinking with microwave irradiation for at least 10 minutes at the temperature of at least 100 °C, preferably for 25-45 minutes at the temperature of 130-160 °C, and then the nanocomposite is separated from the post- reaction solution.

According to the preferred embodiment of the method of the invention, in the reaction solution of iron(II) salt and iron(III) salt is used and its molar concentration value is from 0.01 M to 3 M, the preferred concentration is 0.5 M.

Preferably, the volume ratio of equimolecular solutions of iron(II) and iron(III) salts is from 1.5 : 1 to 1 : 1.5, the preferred ratio is 1.5 : 1.

Preferably, a substance that increases the pH of the solution of iron(II) and iron(III) salts, which has the form of an organic and/or inorganic base, is introduced into the reaction medium at once or in portions.

Preferably, during the synthesis of the ferrimagnetic nanoparticles (Fe ₃ 0 ₄ ), the reaction mixture has pH value above 9.

Preferably, the process of obtaining ferrimagnetic nanoparticles is carried out at the temperature of 20 °C to 300 °C and at the pressure of at least 900 hPa.

Preferably, the resulting suspension of ferrimagnetic nanoparticles is stabilized in the field of microwave radiation for at least 5 minutes and then chitosan dissolved in the organic acid or amino acid, glycol and poly(sodium aspartate) are introduced to the suspension.

Preferably, in the process of chitosan dissolution at least one organic acid is used. The acid is selected from the group consisting of: acids with one carboxyl group and no other functional groups, acids with one carboxylic group and at least one other functional group, acids with more than one carboxylic group and no other functional groups, acids with more than one carboxylic group and at least one other functional group.

Preferably, the pH value of the chitosan solution is below 6.3, the most preferably from 4 to 6.

Preferably, per 1 g of chitosan at least 5 cm ³ of glycol is used, the most preferably from 10 to 20 cm ^' , and 0.5 g of poly( sodium aspartate).

Preferably, poly(sodium aspartate) is the product of polycondensation of aspartic acid to poly(succinimide) and subsequently its basic hydrolysis, or it is the product of polycondensation of monosodium aspartate.

Preferably, chitosan has the average molecular mass of 200 000 to 1 200 000 g/mol, preferably 400 000 g/mol, and poly(sodium aspartate) has the average molecular mass of at least 5 000 g/mol.

BENEFITS OF TFE INVENTION

The ferrimagnetic chitosan/poly(sodium aspartate)/Fe ₃ 0 ₄ nanocomposite having negative surface charge produced according to the method of the invention contains ferrimagnetic nanoparticles with the size of 10 to 100 nm covered with a porous layer of crosslinked chitosan having negative surface charge, resulting from the partial degradation of chitosan in the microwave radiation field and due to the presence of poly(sodium aspartate). The method enables to modify the pore structure by changing the amount and type of the acid used for decreasing the pH value during the process of chitosan dissolving and by changing the length of the crosslinking process.

According to the method of the invention, the step of chemical crosslinking of chitosan is carried out with non-toxic crosslinking agents (acids). Thanks to this, it is possible to obtain a high-porous nanocomposite having chemically modified structure that has no negative effect on living cells.

The nanocomposite obtained by the method according to the invention has more favorable ferrimagnetic properties and increased biodegradability, chemical and mechanical resistance and biocompatibility, compared to nanocomposites obtained by known methods.

Thanks to controlled degree of crosslinking of chitosan (biopolymer), the ferrimagnetic nanocomposite produced according to the method of the invention may biodegrade more quickly, it has increased tissue affinity and still has antibacterial properties which is particularly advantageous when used, for example, as a part of controlled drug or other active substances delivery and release system.

Microwave irradiation during the crosslinking process causes partial surface degradation of chitosan, which results in the formation of carboxyl groups on its surface, thanks to which cell adhesion is possible.

Furthermore, beneficial feature of the invention is the absence of harmful by-products of the ferromagnetic nanocomposite preparation method described above.

The method of the invention is illustrated by the following examples, which do not limit the scope of its protection.

EXAMPLES

Example 1

10 cm ³ of 0.5 M aqueous solution of FeCl ₃ and 15 cm ³ of 0.5 M aqueous solution of FeCl ₂ contained in a reaction vessel placed in a microwave reactor was exposed to 100 W microwave irradiation at the temperature of 50 °C and the pressure of 5 atm. Next, 15% ammonia solution was introduced in portions, under intense stirring with a magnetic stirrer and still under microwave irradiation, until the color of the reaction mixture changed to black. The next stage was the initial stabilization of the obtained ferrimagnetic nanoparticles by exposing the suspension to microwave irradiation at the unchanged temperature for 30 minutes. The obtained nanoparticles were characterized by magnetic properties and the size of 10 - 20 nm.

Fig.1 of the attached drawings shows the SEM microphotograph of the obtained ferrimagnetic nanoparticles.

The next step was to add to the suspension containing ferrimagnetic nanoparticles 20 cm ³ of aqueous solution of biopolymer having pH value of 4.2, which contained 0.8 g of aspartic acid, 1 g of chitosan, 0.5 g of poly(sodium aspartate) as well as 20 cm ^" of propylene glycol. The whole mixture was subjected to 200 W microwave irradiation for 45 minutes at the temperature of 140 °C. The obtained nanocomposite was filtered and washed with 20 cm ³ of ethanol in a known manner and dried at room temperature.

Fig. 2 of the attached drawings shows the SEM microphotograph of the obtained nanocomposite.

Example 2

10 cm ³ of 0.5 M aqueous solution of FeCl ₃ and 10 cm ³ of 0.5 M aqueous solution of FeS0 ₄ contained in a reaction vessel placed in a microwave reactor was exposed to 100 W microwave irradiation at the temperature of 100 °C and the pressure of 2 atm. Next, a 10% solution of sodium hydroxide was introduced in portions under intense stirring with a magnetic stirrer and still under microwave irradiation, until the color of the reaction mixture changed to black. The next stage was the initial stabilization of the obtained ferrimagnetic nanoparticles by subjecting the suspension to microwave irradiation at unchanged temperature for 15 minutes.

The obtained nanoparticles were characterized by magnetic properties and the size of 20 - 30 nm.

The next step was to add to the suspension containing ferrimagnetic nanoparticles 20 cm ³ of aqueous solution of biopolymer having pH value of 5, which contained 0.6 g of adipic acid, 1 g of chitosan, 0.5 g of poly(sodium aspartate) as well as 10 cm ³ of ethylene glycol. The whole mixture was exposed to 200 W microwave irradiation for 30 minutes at the temperature of 145 °C. The obtained nanocomposite was filtered and washed with 20 cm ³ of ethanol in a known manner and dried at room temperature.

Example 3

10 crri of 1 M aqueous solution of FeCl ₃ and 15 cm of 1 M aqueous solution of FeS0 ₄ contained in a reaction vessel placed in a microwave reactor was exposed to microwave irradiation at the temperature of 80 °C and the pressure of 1 atm. Next, a concentrated sodium ethylate solution was added in portions under intense stirring with a magnetic stirrer and still under microwave irradiation, until the pH value was over 9 and the color of the reaction mixture changed to black.

The next stage was the initial stabilization of the obtained ferrimagnetic nanoparticles by exposing the suspension to microwave irradiation at unchanged temperature for ] 0 minutes.

The obtained nanoparticles were characterized by magnetic properties and the size of 30 - 40 nm.

The next step was to add 20 cm ³ of aqueous solution of biopolymer having pH value of 6, which contained 0.5 g of levulinic acid, 1 g of chitosan, 0.5 g of poly(sodium aspartate) as well as 20 cm ³ of propylene glycol. The whole mixture was exposed to 200 W microwave irradiation for 20 minutes at the temperature of 135 °C.

The obtained nanocomposite was then filtered and washed with 20 cm ³ of ethanol in a known manner and dried at room temperature. Example 4

15 cm ³ of 2 M aqueous solution of FeCl ₃ and 10 cm of 2 M aqueous solution of FeS0 ₄ contained in a reaction vessel placed in a microwave reactor was exposed to microwave irradiation at the temperature of 100 °C and the pressure of 1 atm. Next, concentrated potassium hydroxide solution was introduced in portions under intense stirring with a magnetic stirrer, and still under microwave irradiation, until the pH value was over 9 and the color of the reaction mixture changed to black.

The next stage was the initial stabilization of the obtained ferrimagnetic nanoparticles by exposing the suspension to microwave irradiation at unchanged temperature for

20 minutes.

The obtained nanoparticles were characterized by magnetic properties and the size of 30 - 40 nm.

The next step was to add 20 cm ³ of aqueous solution of biopolymer having pH value of 5.5, which contained 0.5 g of glycolic acid, 1 g of chitosan, 0.5 g of poly(sodium aspartate) as well as 15 cm ³ of butyl ene glycol. The whole mixture was exposed to 200 W microwave irradiation for 25 minutes at the temperature of 150 °C.

The obtained nanocomposite was filtered and washed with 20 cm ³ of ethanol in a known manner and dried at room temperature.