WO2021094820A1

WO2021094820A1 - Method of manufacturing zinc oxide nanoparticles

Info

Publication number: WO2021094820A1
Application number: PCT/IB2019/060584
Authority: WO
Inventors: Jolanta Pulit-Prociak; Anita Staroń; Marcin BANACH
Original assignee: Politechnika Krakowska im. Tadeusza Kościuszki
Priority date: 2019-11-11
Filing date: 2019-12-09
Publication date: 2021-05-20
Also published as: PL243247B1; PL431747A1

Abstract

A method of manufacturing zinc oxide nanoparticles coated with a coating selected from the group of peptides or aldohexoses, where in the first phase zinc hydroxide nanoparticles are obtained by precipitation in aqueous or alcoholic solution, and the source of zinc ions are zinc salts, while the precipitation agent is group I hydroxides or sodium carbonate consists in that, in the II phase, the dehydration of zinc hydroxide is carried out in the presence of an organic modifying substance belonging to the group of peptides or aldohexoses, and then in the III phase the obtained suspension is subjected to centrifugation or filtration and washing, and after separating the supernatant or filtrate, the obtained precipitate is dried and ground.

Description

METHOD OF MANUFACTURING ZINC OXIDE NANOPARTICLES

TECHNICAL FIELD

The object of the present invention is a method of manufacturing zinc oxide nanoparticles coated with molecules selected from the group of peptides or aldohexoses, intended especially for use in medicine.

BACKGROUND ART

Nanoparticles have been used in nanomedicine as carriers of active substances, as well as in diagnostics. In chemotherapy polymer nanoparticles, liposomes, polymer micelles, dendrimers or carbon nanotubes are used. Among the polymer nanoparticles in medicines transporting systems, the most important role play: poly (lactic-co-gly colic acid) (PLGA) copolymer, methoxypoly (ethylene glycol) (mPEG), poly (lactic acid) (PLA), poly (ε-caprolactone) (PCL), poly(N- isopropylacrylamice)-b-poly(ε-caprolactone) (PNPCL, chitosan and poly(amidoamine) (PAMAM) [CHEMIK 2012, 66, 8, 868-88], Quantum dots with a diameter of 2 to 8 nm play the role of fluorescent markers used in imaging of cancerous tumors. Due to their magnetic properties, iron oxide nanoparticles have been used in magnetic resonance imaging [NOWOTWORY Journal of Oncology

2013, volume 63, number 4, 320-330],

The authors of the US20060204438A1 patent describe a method for obtaining a biocompatible colloidal system comprising iron oxide nanoparticles.

Nanoparticles coated with polyvinylpyrrolidone have great contrasting properties, thus they may be used in MRI diagnostics. In addition, they can be used in targeted drug delivery, cell and tissue treatment, thermotherapy, etc. The method of their manufacturing consists in introducing an iron oxide precursor at a temperature of

120-600° C into a polyvinylpyrrolidone solution. Polyvinylpyrrolidone is dissolved in a polar organic solvent. The whole is mixed at a speed of 300 to 500 rpm. within

30 minutes to 72 hours. A dispersion of iron oxide nanoparticles coated with polyvinylpyrrolidone is obtained, which is a known stabilizing substance.

Patents application US20120128781 A1 discloses a method of functionalizing nanoparticles used in molecular imaging as biosensors and/or medicines delivery systems and/or for the manufacturing of such systems. The essence of the invention is the use of low molecular weight oligosaccharide derivatives comprising glucosamine moieties, of which one amino group is substituted by an anchoring group by which the molecule is chemisorbed on the surface of nanoparticles, which leads to their encapsulation. In addition, this derivative, together with the surfactant, can form a bilayer that surrounds nanoparticles. In the process e.g. oligo- glucosamine or chitosan oligomers may be used. Thiol, amine, hydroxylamine, hydrazine, sulfide, sulfoxide, sulfone, phosphine, phosphite, phosphine oxide, carboxylate, thiocarboxylate, alcohol, carbene, imidazole, thiazole, or triazole are used as the anchor group supplier. In order to obtain chitosan functionalized gold nanoparticles, an aqueous solution of the chitosan derivative is introduced into their cyclohexane suspension and the whole is subjected to ultrasound for 1 min. The particles are precipitated by the addition of ethanol. The precipitated particles are separated, washed with chloroform and ethanol and then dispersed in water. Patent application US20130302508A1 discloses a method of functionalizing magnetic nanoparticles acting as a medicine carrier used for sustained delivery of active substances. The system consists of iron oxide coated with a long-chain polymer that acts as a stabilizer for nanoparticles in water. The method involves heating to 80° C an iron (II) and (ΙΠ) precursors mixture under continuous mixing under a nitrogen atmosphere. Then ammonium hydroxide and glycerol monooleate are added to the mixture. The product is washed several times with organic solvents to remove excess of glycerol monooleate. The solid product separated as a result of centrifugation is subjected to a freeze-drying process to obtain a powdery substance.

Patent CN109806277 discloses a method of obtaining nanocomposites consisting of a glycolipid coating metallic or metal oxide nanoparticles. The glycolipid is the ester of erythritol and mannose, and the nanocomposite core may be silver, gold or zinc oxide nanoparticles. First, under vigorous stirring, zinc acetate is introduced to the water-diluted ester dissolved in methanol, and after its dissolution, an aqueous solution of potassium hydroxide is added. The whole is heated to 90° C and stirred for 1 h. The resulting suspension is filtered, the precipitate washed with water and ethanol, and after drying calcined at 500° C for

2 h. The authors state that as a result zinc oxide nanoparticles coated with erythritol and mannose ester are obtained. The particles size does not exceed 50 nm, and the product may be used as a reducing, antibacterial and anti-cancer agent. In-vitro studies confirmed that the products inhibited the growth of HepG2 liver cancer cells. In the patent specifictation PL224432B1, the authors disclose a method of obtaining zinc oxide nanoparticles stabilized with amide ligands. The method involves subjecting the zinc oxide nanoparticle precursor to a supramolecular ligand and to air, steam, water or oxygen and water simultaneously. Modified or unmodified alpha-, beta- or gamma-cyclodextrin is used as the ligand. The process is carried out in an organic solvent. The authors state that the product may be used for medical, biomedical and biochemical applications, in particular for diagnostic purposes.

Patent application CN107325808A describes a method of manufacturing mannose-coated quantum dots. First, quantum dots are functionalized with amide groups, and mannose with imide groups. The coupling of mannose with the surface of quantum dots occurs through an electrophilic addition reaction between amide and imide groups. The authors state that in this way stable nanoparticles are obtained, which can easily be subjected to further bio-modification processes. The product may be used in bioluminescent labelling.

Patent application W02018141940A1 discloses a method of obtaining a metal-ligand complex, where the ligand is galactose. The core of the complex consists of nanoparticles of gold, silver, copper, platinum, palladium, iron, cobalt, gadolinium and/or zinc and/or a combination thereof. The core size does not exceed

5 nm, and the complex size - 50 nm. In addition, the complex can be encapsulated in a biocompatible polymer. The authors state that the complex may find application for transporting the active substance, which is coupled to the complex. The product is intended for transporting the active substance combating liver cancer. The methods of manufacturing coated zinc oxide nanoparticles - nanoparticles having a coating selected from the group of peptides or aldohexoses known from the prior art are complicated and time consuming. Unexpectedly, it turned out that it is possible to develop a method, which is much simpler and environmentally friendly.

DISCLOSURE OF THE INVENTION

A method of manufacturing zinc oxide nanoparticles coated with a coating selected from the group of peptides or aldohexoses, where in the first phase zinc hydroxide nanoparticles are obtained by precipitation in aqueous or alcoholic solution, and the source of zinc ions are zinc salts, while the precipitation agent is group I hydroxides or sodium carbonate according to the invention is characterized by this, that in the Π phase, the dehydration of zinc hydroxide is carried out in the presence of an organic modifying substance belonging to the group of peptides or aldohexoses, and then in the ΙΠ phase the obtained suspension is subjected to centrifugation or filtration and washing, and after separating the supernatant or filtrate, the obtained precipitate is dried and ground. The zinc salts used in the method are zinc nitrate (V), zinc sulfate (VI), zinc chloride or zinc acetate. The hydroxides used are sodium hydroxide or potassium hydroxide. The molar ratio of precipitation agent to zinc ions is from 1 : 1 to 6: 1. Preferably, the product suspension is separated by filtration or centrifugation. Preferably, centrifugation of the product suspension is carried out at a speed of from 2000 to 10,000 rpm. Preferably the solid residue is washed with deionized water or ethyl alcohol. The organic modifying substance is selected from the group consisting of glutathione, mannose or galactose.

In the precipitation process, an organic stabilizing substance is introduced from the group of polyethers or polyphenols in order to inhibit too rapid dehydration of zinc hydroxide to zinc oxide - inhibition of zinc oxide crystallites aggregation and maintaining their size at the nanometric level.

The organic stabilizing substance is polyethylene glycol or tannic acid.

The mass ratio of polyethylene glycol to zinc oxide is from 0 to 1.

The molar ratio of tannic acid to zinc ions is from 0 to 0.1.

Phase I is carried out using an ultrasonic homogenizer, which increases the efficiency of zinc hydroxide precipitation. Preferably, sonication is carried out for

1 to 10 minutes. Preferably, the precipitation agent solution is added dropwise to the zinc salt solution at a rate of 0.1 to 2 mL/sec. Preferably the ultrasound power is from 40 to 320 W.

Phase II is carried out in a microwave field in a pressure microwave reactor or in a pressure reactor with conventional heating. The use of a pressure microwave reactor allows the temperature of the reaction mixture to be reached above 100° C, while the pressure increases. By using polar (water) or medium polar solvent

(ethanol) in the microwave field, it is possible to quickly and efficiently transfer heat to the entire volume of the reaction mixture in a short time, which significantly speeds up the process. By using a pressure reactor with conventional (electric) heating, equipped with an automatically controlled mixing system, it is possible to eliminate the error related to the intensity of mixing the substrates. The set temperature is achieved by means of a heating jacket, which ensures that the desired value is maintained with an accuracy of 1°C. In the case of using microwave reactor, a solution of the organic modifying substance is introduced into the reaction mixture under continuous mixing, and the whole is transferred to a Teflon vessel, which is placed in a microwave reactor. In the case of using a pressure reactor, the mixture of zinc salt, precipitation agent (and stabilizer) solutions is transferred to a stainless reaction vessel and, under continuous mixing, the whole is heated by means of jacket heating. After reaching the set temperature, a modifying substance solution is introduced into the reaction mixture by means of a pumping system.

Thanks to the possibility of changing the flow rate of the introduced agent in the pressure reactor, and thanks to the changes in temperature, pressure and time of the process, as well as due to changes in molar ratios of individual reagents, it is possible to control the size of zinc oxide nanoparticles in the obtained products.

Phase Π is carried out at a temperature of 100 to 250° C for 3 to 60 minutes.

The pressure in the reaction vessel is from 1 to 40 bar.

The microwaves power is from 150 to 300 W.

The volumetric flow rate of the modifying agent solution introduced into the pressure reactor is from 2 to 10 mL/min.

The obtained precipitate is dried at a temperature of 70 to 120° C.

The method being the object of the invention consists in the chemical modification of zinc oxide nanoparticles in order to reduce their toxic properties to living tissues. Explanations of the phenomenon of limiting toxic properties due to the attachment of modifiers to nanoparticles should be seen in limiting the release of zinc ions from nanoparticles due to the attachment of coating particles. Coating particles sorb on the oxide surface and/or form a bilayer interlayer with a layer of stabilizing agent coating the oxide. The released metallic ions affect the formation of reactive oxygen species (ROS), which in turn cause intracellular oxidative stress.

Limiting the release of ions is tantamount to reducing the toxic properties of nanoparticles. Also, nanoparticles captured by the cell may dissolve inside the cell, releasing metallic ions. Due to the presence of modifiers on the zinc oxide nanoparticles surface, it is possible to significantly reduce their penetration into the cells, as well as inhibit their dissolution.

The obtained modified nanostructured zinc oxide can be used in drug delivery systems and/or medicinal excipients. The undoubted advantage of the present invention is the size of the zinc oxide nanoparticles obtained. It is in the range of

50 to 800 nm. This is particularly important in the use of products in passive targeted therapy. In the passive cancer therapy the anatomical and physiological properties of the tumour tissues are utilized. Tumour tissue is characterized by increased vascular permeability (a leaky network of blood vessels). Scientists have found out that the diameter of the fractures is from 100 to 800 nm, while in healthy tissues only 2 - 6 nm. The average size of most of anti-cancer drugs is small and does not exceed 10 nm. Their use in independent form would cause their diffusion into both healthy and diseased tissues. Combining them with nanocaniers (50-800 nm) will significantly reduce and/or even eliminate the penetration of medicinal substances into the structure of healthy tissues. Similarly, the developed nanocaniers can find application in the treatment of rheumatoid arthritis and other rheumatoid-related diseases.

EXAMPLES

The subject of the invention is illustrated by the following examples:

Example 1

0.23 mL of polyethylene glycol is introduced into 11.0 mL of an aqueous solution of 1.136 mol/L zinc nitrate (V). Using an ultrasonic homogenizer, 15.0 mL of a 5 mol/L aqueous sodium hydroxide solution are added dropwise to the resulting mixture at a rate of 0.125 mL/sec. The whole is homogenized for 2 min with an ultrasonic power of 100 W. Then, under continuous mixing, 4.0 mL of an aqueous solution of reduced L-glutathione at a concentration of 0.625 mol/L is introduced into the system. The whole is transferred to a Teflon vessel, which is placed in a microwave reactor. The mixture is subjected to microwave irradiation for 15 min using microwave power of 300 W. The temperature of 120° C and the pressure of

8 bar is obtained. The product is filtered using a filter with a pore diameter of 0.45 pm. The precipitate is washed with 100 mL of deionized water. The product is dried at 85°C and then ground to obtain a fine crystalline precipitate of zinc oxide nanoparticles coated with glutathione with an average particle size of 782 nm and an electrokinetic potential of 19 mV. The release of zinc ions from the obtained product and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a stabilizing and modifying substance, was checked. Solutions of these substances were replaced with the same volume of deionized water. The test involved mixing the powder with a leaching agent, which was deionized water or Ringer's solution. The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower on average by

49.6% and 44.9% in water and Ringer's fluid, respectively.

Example 2

In the conditions of ultrasonic homogenizing, 5.0 mL of 5 mol/L aqueous sodium hydroxide solution are added dropwise to 23.1 mL of 0.5411 mol/L aqueous zinc nitrate (V) solution at a rate of 0.1 mL/sec. The whole is homogenized for 1 min with an ultrasonic power of 200 W. Then, under continuous mixing, 6.9 mL of 0.2 mol/L aqueous D (+) - mannose solution is introduced into the system. The whole is transferred to a Teflon vessel, which is placed in a microwave reactor. The mixture is subjected to microwave irradiation for 5 min using microwave power of

300 W. The temperature of 120°C and the pressure of 8 bar is obtained. The product is centrifuged at 9000 rpm. The precipitate is washed with deionized water. The product is dried at 104°C and then ground to obtain a fine crystalline precipitate of zinc oxide nanoparticles coated with mannose with an average particle size of 254 nm and an electrokinetic potential of 24.5 mV. The release of zinc ions from the obtained product and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a modifying substance, was checked. The solution of this substance was replaced with the same volume of deionized water. The test consisted of mixing the powder with a leaching agent, which was deionized water. The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower on average by 39.3%.

Example 3

In the conditions of ultrasonic homogenizing, 10.0 mL of 10 mol/L aqueous potassium hydroxide solution are added dropwise to 12.5 mL of 1.0 mol/L aqueous zinc sulfate (VI) solution at a rate of 0.1 mL/sec. The whole is homogenized for 3 min with an ultrasonic power of 80 W. Then, under continuous mixing, 12.5 mL of

0.2 mol/L of aqueous D (+) - mannose solution is introduced into the system. The whole is transferred to a Teflon vessel, which is placed in a microwave reactor. The mixture is subjected to microwave irradiation for 5 min using microwave power of

300 W. The temperature of 120°C and the pressure of 8 bar is obtained. The product is filtered using a filter with a pore diameter of 0.45 pm. The precipitate is washed with 100 mL of deionized water. The product is dried at 90° C and then ground to obtain a fine crystalline precipitate of zinc oxide nanoparticles coated with mannose with an average particle size of 649 nm and an electrokinetic potential of 21.2 mV.

The release of zinc ions from the product obtained and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a modifying substance, was checked. The solution of this substance was replaced with the same volume of deionized water. The test consisted of mixing the powder with a leaching agent, which was deionized water. The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower on average by

77.9%.

Example 4

In the conditions of ultrasonic homogenizing, 15.0 mL of 5 mol/L aqueous sodium hydroxide solution is added dropwise to 17.3 mL of 0.7225 mol/L aqueous zinc chloride solution at a rate of 0.125 mL/sec. The whole is homogenized for 4 min with an ultrasonic power of 75 W. Then, under continuous mixing, 5.0 mL of 0.5 mol/L aqueous galactose solution are introduced into the system. The whole is transferred to a Teflon vessel, which is placed in a microwave reactor. The mixture is subjected to microwave radiation for 5 min using microwave power of 300 W. A temperature of 150°C and a pressure of 11 bar are obtained. The product is centrifuged at 9000 rpm. The precipitate is washed with deionized water. The product is dried at 104° C and then ground to obtain a fine crystalline precipitate of galactose-coated zinc oxide nanoparticles with an average particle size of 371 nm and an electrokinetic potential of 23.8 mV. The release of zinc ions from the product obtained and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a modifying substance, was checked.

The solution of this substance was replaced with the same volume of deionized water. The test consisted of mixing the powder with a leaching agent, which was deionized water or simulated body fluid (SBF). The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower by 29.5% and

7.5% on average in water and SBF, respectively.

Example 5

In the conditions of ultrasonic homogenizing, 12.5 mL of 2 mol/L aqueous sodium carbonate solution is added dropwise to 13.5 mL of 0.9259 mol/L aqueous zinc nitrate solution at a rate of 0.1 mL/sec. The whole is homogenized for 2 min with an ultrasonic power of 75 W. Then, under continuous mixing, 4.0 mL of an aqueous solution of reduced L-glutathione at a concentration of 0.625 mol/L is introduced into the system. The whole is transferred to a Teflon vessel, which is placed in a microwave reactor. The mixture is subjected to microwave irradiation for 15 min using microwave power of 300 W. The temperature of 150°C and the pressure of

11 bar is obtained. The product is centrifuged at 9000 rpm. The precipitate is washed with deionized water. The product is dried at 104°C and then ground to obtain a fine crystalline precipitate of zinc oxide nanoparticles coated with glutathione with an average particle size of 228 nm and an electrokinetic potential of 20.2 mV. The release of zinc ions from the product obtained and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a modifying substance, was checked. The solution of this substance was replaced with the same volume of deionized water. The test consisted of mixing the powder with a leaching agent, which was deionized water. The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower by 48.2% on average.

Example 6

25 mL of 0.05 mol/L aqueous tannic acid solution is introduced into 170.0 mL of

0.7353 mol/L aqueous zinc chloride solution. In the conditions of ultrasonic homogenizing, 150.0 mL of a 5 mol/L aqueous sodium hydroxide solution are added dropwise to the resulting mixture at a rate of 1.25 mL/sec. The whole is homogenized for 6 min with an ultrasonic power of 100 W. The whole is transferred to a stainless steel vessel, which is placed in a pressure reactor. The mixture, under constant stirring (1000 rpm), is heated by means of a heating jacket to the temperature of 150° C and after reaching it, 5.0 mL of a 0.5 mol/L aqueous galactose solution is pumped into the reaction mixture. A pump with a flow rate of

5 mL/min is used for this purpose. After dosing, the whole is kept at the set temperature for another 5 min. A pressure of 4 bar is obtained. The product is filtered using a filter with a pore diameter of 0.45 pm. The precipitate is washed with 1000 mL deionized water. The product is dried at 104° C and then ground to obtain a fine crystalline precipitate of zinc oxide coated galactose nanoparticles with an average particle size of 671 nm and an electrokinetic potential of -20.9 mV.

The release of zinc ions from the product obtained and from the reference product, i.e. pure zinc oxide obtained under the same conditions, but without the addition of a stabilizing and modifying substance, was checked. Solutions of these substances were replaced with the same volume of deionized water. The test consisted of mixing the powder with the leaching agent, which was SBF fluid. The test was carried out for 80 min taking samples at set intervals. The samples were filtered and the concentration of zinc was determined by atomic absorption spectroscopy. The study showed that compared to the reference material, the zinc release rate is lower by an average of 21.3%.

Claims

PATENT CLAIMS

1. A method of manufacturing zinc oxide nanoparticles coated with a coating selected from the group of peptides or aldohexoses, where in the first phase zinc hydroxide nanoparticles are obtained by precipitation in aqueous or alcoholic solution, and the source of zinc ions are zinc salts, while the precipitation agent is group I hydroxides or sodium carbonate, characterized in that, in the Π phase, the dehydration of zinc hydroxide is carried out in the presence of an organic modifying substance belonging to the group of peptides or aldohexoses, and then in the ΙΠ phase the obtained suspension is subjected to centrifugation or filtration and washing, and after separating the supernatant or filtrate, the obtained precipitate is dried and ground.

2. The method according to claim 1, characterized in that, the molar ratio of precipitation agent to zinc ions is from 1 : 1 to 6: 1.

3. The method according to claims 1 or 2, characterized in that, the organic modifying substance is selected from the group of consisting of glutathione, mannose or galactose.

4. The method according to any of the preceding claims, in the precipitation process characterized in that, an organic stabilizing substance from the group of polyethers or polyphenols is introduced.

5. The method according to claim 4, characterized in that, the organic stabilizing substance is polyethylene glycol and/or tannic acid.

6. The method according to claim 5, characterized in that, the mass ratio of polyethylene glycol to zinc oxide is from 0 to 1.

7. The method according to claim 5, characterized in that, the molar ratio of tannic acid to zinc ions is from 0 to 0.1.

8. The method according to any of the preceding claims, characterized in that, the phase I is carried out using an ultrasonic homogenizer.

9. The method according to any of the preceding claims, characterized in that, the phase Π is carried out in the microwave field in a pressure microwave reactor or in a pressure reactor with conventional heating.

10. The method according to claim 9, characterized in that, the phase Π is carried out at a temperature of from 100 to 250°C for from 3 to 60 minutes.

11. The method according to claims 9 or 10, characterized in that, the pressure in the reaction vessel is from 1 to 40 bar.

12. The method according to claims 9 or 10, characterized in that, the microwaves power is from 150 to 300 W.

13. The method according to any of the preceding claims, characterized in that, the volumetric flow rate of the modifying agent solution introduced into the pressure reactor is from 2 to 10 mL/min.

14. The method according to any of the preceding claims, characterized in that, the obtained precipitate is dried at a temperature of 70 to 120°C.