Functional polyurea nanogel with universal drug loading capacity as well as preparation method and application of functional polyurea nanogel

Abstract

The invention relates to the technical field of nanogel, and discloses functional polyurea nanogel with universal drug loading capacity as well as a preparation method and application of the functional polyurea nanogel. The invention relates to a functionally modified polyurea compound, which is prepared from the following raw materials: a double primary amine compound, double (2-oxo azacycloheptane-1-formamido alkyl) amine, a polyethylene glycol-amino compound, N, N '-carbonyl double (caprolactam), acid anhydride and an organic solvent. The functionally modified polyurea compound provided by the invention does not contain an isocyanate group and is high in biological safety; the functional polyurea nanogel synthesized by taking a functional modified polyurea compound as a raw material is high in biological safety, has charge overturning performance, has multiple intermolecular non-covalent interaction including hydrogen bond interaction, electrostatic interaction and hydrophobic interaction, can be used for universal drug loading, has pH value responsive swelling property, and can be applied to drug loading. The compound can be used for releasing pH-responsive drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of nanogels, and in particular to a functional polyurea nanogel with universal drug loading capacity, and a preparation method and application thereof. Background Art

[0002] Nanogel is a kind of intramolecular cross-linked polymer gel in the form of nanoparticles, which has the superior properties of both nanomaterials and hydrogel materials. The nanoscale size enables it to show efficient penetration into pathological environments such as bacterial biofilms. At the same time, as a soft gel polymer material, its internal network structure makes it have a high water content, can swell without dissolving, and has the ability to encapsulate bioactive molecules and excellent biocompatibility.

[0003] At present, nanogels are mainly prepared by chemical covalent crosslinking and physical non-covalent crosslinking. The hydrogen bond interaction of non-covalent crosslinking has the advantages of environmental friendliness and biocompatibility. It can occur between many functional groups such as hydroxyl, amine, and carboxyl. It is reversible and can undergo breakage and reorganization. It is easy to carry out structural design, modification or regulation, which provides convenience for the preparation of controllable multifunctional nanogels. Aliphatic polyureas (aPUs) composed of urea groups and alkyl chains have abundant hydrogen bonds and hydrophobic interactions, and can interact with drug molecules of different physical and chemical properties to give nanogels efficient drug loading capacity.

[0004] Polyurea nanogel is a drug delivery carrier with excellent comprehensive performance, but the current preparation of polyurea relies on the reaction of isocyanate and amine compounds. Although the reaction is highly active, isocyanate can react with most groups (such as hydroxyl, amine, carboxyl, and thiol), resulting in the inability to directly introduce functional groups into its molecular chain (main chain or side chain) while synthesizing polyurea. As a result, the polyurea nanogel lacks effective functional groups and cannot be functionalized downstream. In addition, the isocyanate groups that do not participate in the polymerization reaction will remain in the material. Therefore, the traditional process for preparing polyurea based on isocyanate often results in the material having greater toxic side effects and greater safety hazards, thereby limiting its application. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, one of the purposes of the present invention is to provide a functionally modified polyurea compound; the second purpose of the present invention is to provide a method for preparing the functionally modified polyurea compound; the third purpose of the present invention is to provide a functional polyurea nanogel; the fourth purpose of the present invention is to provide a product.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a functionally modified polyurea compound, the polyurea compound comprising the following preparation raw materials: a diprimary amine compound, a bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine, a polyethylene glycol-amino compound, N,N′-carbonylbis(caprolactam), an acid anhydride and an organic solvent;

[0008] The diprimary amine compound is selected from at least one of bis(hexamethylene)triamine, tetraethylenepentamine, triethylenetetramine, pentaethylenehexamine, spermine, diethylenetriamine, bis(3-aminopropyl)amine, and 1,4-bis(3-aminopropyl)piperazine;

[0009] The bis(2-oxoazacycloheptane-1-carboxamidoalkyl)amine is selected from at least one of the compounds of formula (1) to formula (3):

[0010]

[0011] Wherein, x=2, 3 or 6; n=1, 2 or 3.

[0012] Specifically, the functionally modified polyurea compound is a polyethylene glycol-polyurea-polyethylene glycol triblock polymer modified with a functional group (R); the functional group (R) is selected from at least one of a secondary amine, a tertiary amine, an acryloyl group, a methacryloyl group, a maleimide group, a norbornene group, and an allyl group.

[0013] Preferably, the synthesis of the bis(2-oxoazacycloheptane-1-carboxamidealkyl)amine refers to the prior art (Chem. Mater. 2023, 35, 4705-4716), specifically, it is prepared by a nucleophilic substitution reaction of the end group of a diprimary amine small molecule compound and N,N′-carbonylbis(caprolactam) (CBC), and the corresponding bis(2-oxoazacycloheptane-1-carboxamidealkyl)amine monomer can be obtained by changing the structure of the diprimary amine small molecule compound raw material.

[0014] Specifically, the bis(2-oxoazacycloheptane-1-carboxamidealkyl)amines of formula (1) to (3) are respectively prepared from diprimary amine small molecule compounds having structures of formula (4) to (6):

[0015]

[0016] Wherein, x=2, 3 or 6; n=1, 2 or 3.

[0017] Preferably, the average molecular weight of the polyethylene glycol-amino compound is 1000-10000 Da.

[0018] Preferably, the polyethylene glycol-amino compound is selected from at least one of methoxy-polyethylene glycol-amino (mPEG-NH2), hydroxyl-polyethylene glycol-amino (HO-PEG-NH2), carboxyl-polyethylene glycol-amino (COOH-PEG-NH2), tert-butyl-amino-polyethylene glycol-amino (Boc-NH-PEG-NH2), biotin-polyethylene glycol-amino (Biotin-PEG-NH2), azide-polyethylene glycol-amino (N3-PEG-NH2), and diphenylcyclooctyne polyethylene glycol amino (DBCO-PEG-NH2).

[0019] Preferably, the acid anhydride is selected from at least one of methacrylic anhydride, acrylic anhydride, 4-pentene anhydride, nadic anhydride and maleic anhydride.

[0020] The second aspect of the present invention provides a method for preparing the functionally modified polyurea compound according to the first aspect of the present invention, comprising the following steps:

[0021] S1. Dissolve a diprimary amine compound and bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine in an organic solvent I, and perform a first-stage heating reaction after deoxygenation; add N,N′-carbonylbis(caprolactam), and perform a second-stage heating reaction after deoxygenation to obtain product I;

[0022] S2, dissolving the product I and the polyethylene glycol-amino compound in an organic solvent II, removing oxygen and heating for reaction to obtain a product II;

[0023] S3, dissolving the product II in an organic solvent III, adding anhydride, and reacting to obtain the functionally modified polyurea compound;

[0024] Wherein, the organic solvents I and II are respectively selected from one of N,N-dimethylformamide and dimethyl sulfoxide; and the solvent III is selected from C1-C2 chloroalkanes.

[0025] Preferably, the organic solvents I and II are N,N-dimethylformamide (DMF); and the organic solvent III is dichloromethane (DCM).

[0026] Preferably, the reaction conditions of step S1 include at least one of the following:

[0027] 1) The molar ratio of the diprimary amine compound to bis(2-oxoazepane-1-carboxamidoalkyl)amine is (0.5-2):1;

[0028] 2) the molar ratio of the diprimary amine compound to N,N′-carbonylbis(caprolactam) is 1:(2-5);

[0029] 3) The temperature of the first stage heating reaction is 120-140° C. and the time is 6-72 hours;

[0030] 4) The temperature of the second stage heating reaction is 70-100°C and the time is 6-24h.

[0031] Further preferably, the reaction conditions of step S1 include at least one of the following:

[0032] 1) The molar ratio of the diprimary amine compound to bis(2-oxoazepane-1-carboxamidoalkyl)amine is (0.5-1.5):1;

[0033] 2) the molar ratio of the diprimary amine compound to N,N′-carbonylbis(caprolactam) is 1:(2-5);

[0034] 3) The temperature of the first stage heating reaction is 120-130° C. and the time is 6-24 hours;

[0035] 4) The temperature of the second stage heating reaction is 70-90°C and the time is 6-15h.

[0036] Preferably, in the step S1, the N,N′-carbonylbis(caprolactam) is excessively dissolved in the organic solvent I and then added.

[0037] Preferably, the reaction conditions of step S2 include at least one of the following:

[0038] 1) The molar ratio of the product I to the polyethylene glycol-amino compound is 1:(2-4);

[0039] 2) The heating reaction is carried out at a temperature of 120-140° C. and for a time of 48-80 hours.

[0040] Further preferably, the reaction conditions of step S2 include at least one of the following:

[0041] 1) The molar ratio of the product I to the polyethylene glycol-amino compound is 1:(2-3);

[0042] 2) The heating reaction is carried out at a temperature of 120-130° C. and for a time of 48-72 hours.

[0043] Preferably, the reaction conditions of step S3 include at least one of the following:

[0044] 1) The molar ratio of the product II to the acid anhydride is 1:(50-150);

[0045] 2) The reaction temperature is 20-25°C and the reaction time is 6-30h.

[0046] Further preferably, the reaction conditions of step S3 include at least one of the following:

[0047] 1) The molar ratio of the product II to the acid anhydride is 1:(100-150);

[0048] 2) The reaction temperature is 20-25°C and the reaction time is 12-24h.

[0049] Preferably, in step S1, the solid-liquid ratio of the diprimary amine compound to N,N-dimethylformamide is 1 g:(30-80) mL; further preferably, the solid-liquid ratio of the diprimary amine compound to N,N-dimethylformamide is 1 g:(40-70) mL.

[0050] Preferably, in step S1, the deoxygenation time is selected from 20-50 min; further preferably, the deoxygenation time is selected from 20-40 min.

[0051] Preferably, in step S1, after the second stage heating reaction is completed, the steps of removing the solvent by rotary evaporation, dissolving the remaining substance by N,N-dimethylformamide, settling by glacial ether, and drying the precipitate at 40-60° C. for 10-20 hours are further included to obtain the product I.

[0052] Preferably, in step S2, after the heating reaction is completed, the steps of removing the solvent by rotary evaporation, dissolving the remaining substance by N,N-dimethylformamide, settling by glacial ether, and drying the precipitate at 40-60° C. for 10-20 hours are further included to obtain the product II.

[0053] Preferably, in step S3, the process of dissolving the product II in dichloromethane is carried out under ice bath and stirring conditions.

[0054] Preferably, in step S3, the acid anhydride is added dropwise.

[0055] Preferably, in step S3, after the reaction is completed, the steps of removing the solvent by rotary evaporation, settling with glacial ether, and drying the precipitate at 40-60° C. for 10-20 h to obtain the functionally modified polyurea compound are further included.

[0056] Specifically, the preparation principle of the functionally modified polyurea compound is as follows: by means of a selective temperature-controlled nucleophilic substitution reaction of a primary amine or a secondary amine with N-carbonyl caprolactam, a linear polyurea having a main chain containing a secondary amine or a tertiary amine and modified with a hydrophilic polymer polyethylene glycol at both ends is prepared.

[0057] The third aspect of the present invention provides a functional polyurea nanogel, comprising the following preparation materials: the functionally modified polyurea compound described in the first aspect of the present invention, a crosslinking agent and a photoinitiator.

[0058] Preferably, the cross-linking agent comprises the following raw materials: a small molecule diamine solution and a carboxylic acid derivative; the small molecule diamine solution is prepared by dissolving a small molecule polyamino compound or a small molecule polyol compound in dichloromethane.

[0059] Preferably, the small molecule polyamino compound is selected from at least one of 1,4-bis(3-aminopropyl)piperazine, N,N-bis(3-aminopropyl)methylamine, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, and N,N,N',N'-tetrakis(3-aminopropyl)-1,4-butanediamine.

[0060] Preferably, the small molecule polyol compound is selected from at least one of 1,4-bis(2-hydroxyethyl)piperazine, ethylenediaminetetraethanol, triisopropanolamine, N,N,N',N'-tetra(2-hydroxyethyl)ethylenediamine, N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine, and N-butyldiethanolamine.

[0061] Preferably, the carboxylic acid derivative is selected from at least one of methacrylic anhydride, methacryloyl chloride, acrylic anhydride, acryloyl chloride, 4-pentene anhydride, nadic anhydride, 5-norbornene-2-yl chloride, maleic anhydride and furanoyl chloride.

[0062] Preferably, the cross-linking agent is prepared by a method comprising the following steps: dropping the carboxylic acid derivative into a small molecule diamine solution, reacting, and extracting to obtain the cross-linking agent.

[0063] Preferably, the molar ratio of the solute to the carboxylic acid derivative in the small molecule diamine solution is 1:(1-2); further preferably, the molar ratio of the solute to the carboxylic acid derivative in the small molecule diamine solution is 1:(1-1.5).

[0064] Preferably, the reaction temperature is 20-30°C; further preferably, the reaction temperature is 20-25°C.

[0065] Preferably, the reaction time is 6-15 h; further preferably, the reaction time is 8-12 h.

[0066] Preferably, the process of adding the carboxylic acid derivative dropwise to the small molecule diamine solution is carried out in an ice bath with stirring.

[0067] Preferably, the organic solvent used for the extraction includes dichloromethane and chloroform;

[0068] Preferably, the aqueous phase of the extraction is a saturated sodium chloride solution.

[0069] Preferably, the step of adjusting the pH of the reaction system to 7.5-8.5 is included before the extraction.

[0070] Preferably, after the extraction, the organic phase is collected, an excess of anhydrous sodium sulfate powder is added, the mixture is dried for 10-15 hours and then filtered, the filtrate is collected and the solvent is removed by rotary evaporation, and the cross-linking agent is obtained by drying at 40-60° C. for 10-15 hours.

[0071] Preferably, the mass ratio of the functionally modified polyurea compound to the crosslinking agent is (1-3):1; further preferably, the mass ratio of the functionally modified polyurea compound to the crosslinking agent is (1-2):1.

[0072] Preferably, the amount of the photoinitiator added is 2-10 mg / mL; further preferably, the amount of the photoinitiator added is 2-7 mg / mL.

[0073] Preferably, the photoinitiator is selected from at least one of 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0074] Preferably, the functional polyurea nanogel is prepared by a method comprising the following steps: mixing a functionally modified polyurea compound, a crosslinking agent and a photoinitiator, and curing to obtain the functional polyurea nanogel.

[0075] Preferably, the preparation of the functional polyurea nanogel further comprises using a solvent; the solvent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, ethanol, acetone and water.

[0076] Preferably, the functional polyurea nanogel is prepared by a method comprising the following steps: dissolving a functionally modified polyurea compound, a crosslinker and a photoinitiator in a solvent, removing the solvent by rotary evaporation, adding water and ultrasound, solidifying, centrifuging in an ultrafiltration tube, washing with ultrapure water, collecting the concentrated solution, and freeze-drying to obtain the functional polyurea nanogel.

[0077] Specifically, the preparation principle of functional polyurea nanogel is as follows: starting from the secondary amine or tertiary amine of the functionally modified polyurea compound, functional modification is carried out to introduce cross-linkable reactive groups, and then through the self-assembly of linear polyurea in a selective solvent and its subsequent cross-linking means, a functional polyurea nanogel that can be stably dispersed in an aqueous solution for a long time is formed.

[0078] Preferably, the frequency of the ultrasound is 20-40 kHz; further preferably, the frequency of the ultrasound is 30-40 kHz.

[0079] Preferably, the ultrasonic treatment time is 10-30 min; further preferably, the ultrasonic treatment time is 20-30 min.

[0080] Preferably, the curing method is UV-visible light curing.

[0081] Preferably, the wavelength of the curing light source is selected from one of 365 nm and 405 nm.

[0082] Preferably, the curing time is 10-20 min; more preferably, the curing time is 10-15 min.

[0083] Preferably, the molecular weight cut-off of the ultrafiltration tube is selected from one of 500Da, 1000Da and 3000Da.

[0084] Preferably, the centrifugation condition is (4500-5500) g×(15-25) min.

[0085] Preferably, the washing is performed 3-6 times.

[0086] The fourth aspect of the present invention provides a product, comprising the functional polyurea nanogel described in the third aspect of the present invention, and a pharmaceutically active ingredient; the pharmaceutically active ingredient is selected from at least one of rifampicin, curcumin, vancomycin, doxorubicin hydrochloride, aztreonam, lysozyme, and homoserine lactone inhibitors.

[0087] Preferably, the mass ratio of the active pharmaceutical ingredient in the product to the functionally modified polyurea compound in the functional polyurea nanogel is (1-5):1.

[0088] Preferably, the content of the active pharmaceutical ingredient in the product is 4-248 μg / mL.

[0089] Preferably, the product includes medicines, reagents, additives.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] 1) The functionally modified polyurea compound provided by the present invention does not contain isocyanate groups and has high biological safety;

[0092] 2) The preparation method of the functionally modified polyurea compound provided by the present invention does not rely on the polymerization process of traditional isocyanate monomers. While solving the toxicity problem of traditional polyurea materials and improving the biological safety of the products, it promotes the further functionalization modification of aliphatic polyurea materials and realizes the introduction of functional groups without the complex process of protection / deprotection. The method has simple steps, mild conditions, high synthesis efficiency, and is suitable for popularization and use;

[0093] 3) The functional polyurea nanogel provided by the present invention uses functionally modified polyurea compounds as raw materials, has high biological safety, has charge reversal properties, has multiple intermolecular non-covalent interactions including hydrogen bonding interactions, electrostatic interactions and hydrophobic interactions, can be used for universal drug loading, and has pH-responsive swelling properties, and can be used for pH-responsive drug release;

[0094] 4) The drug-loaded functional polyurea nanogel product provided by the present invention, including functional polyurea nanogel loaded with active drug ingredients such as rifampicin, curcumin, vancomycin, doxorubicin hydrochloride, aztreonam, lysozyme, homoserine lactone inhibitors, etc., can exert the effects of antibacterial, bactericidal, antibacterial, inhibiting or removing bacterial biofilm formation, preventing and / or inhibiting remote colonization of biofilm, killing cancer cells, inhibiting tumor growth or anti-cancer, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the intermediate product aPU (BHT) in Example 1;

[0096] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the intermediate product PEG-aPU(BHT)-PEG in Example 1;

[0097] Figure 3 is the H NMR spectrum of the functionally modified polyurea compound PEG-PU / MA-PEG in Example 1;

[0098] Figure 4 This is the NMR structure characterization diagram of the cross-linking agent PMA in Example 2;

[0099] Figure 5 is a transmission electron microscopy image of the functional polyurea nanogel in Example 2;

[0100] Figure 6 Surface potential diagrams of the functional polyurea nanogel in Example 2 at pH = 7.4 (a) and pH = 5.0 (b);

[0101] Figure 7 is the UV-visible spectrum of the rifampicin-loaded functional polyurea nanogel in Example 3;

[0102] Figure 8 The in vitro drug release performance of the rifampicin-loaded functional polyurea nanogel in Example 3 at pH = 7.4 and pH = 5.0;

[0103] Fig. 9 is the UV-visible spectrum of the curcumin-loaded functional polyurea nanogel in Example 4;

[0104] Fig.10is the UV-visible spectrum of the vancomycin-loaded functional polyurea nanogel in Example 5;

[0105] Fig.11 is the UV-visible spectrum of the functional polyurea nanogel loaded with doxorubicin hydrochloride in Example 6;

[0106] Fig.12 is the UV-visible spectrum of the functional polyurea nanogel loaded with aztreonam in Example 7;

[0107] Fig.13 is the UV-visible spectrum of the functional polyurea nanogel loaded with lysozyme in Example 8;

[0108] Fig.14 This is the bacterial biofilm removal effect of rifampicin-loaded functional polyurea nanogel in the application example. DETAILED DESCRIPTION

[0109] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0110] Example 1

[0111] This example prepares a functionally modified polyurea compound, and the steps are as follows:

[0112] S11, weigh bis(hexamethylene)triamine (2.15 g, 10 mmol) and the corresponding bis(2-oxoazacycloheptane-1-carboxamidoalkyl)amine monomer BHT (compound of formula (1), 4.93 g, 10 mmol) into a clean two-necked flask, add 100 mL of DMF to fully dissolve, then circulate and ventilate with argon for 30 min to remove oxygen in the system, heat the reaction system to 130° C. under the protection of argon, and continue stirring and reacting for 12 h; after the collected solution is cooled, DMF dissolved with excessive CBC is added to the reaction system, and after argon is re-vented for deoxygenation, the reaction system is heated to 80° C. and continued stirring and reacting for 8 h; after the reaction is completed, the organic solvent in the obtained solution is removed by rotary evaporation, the remaining material is cooled to room temperature and dissolved with a small amount of dichloromethane, and slowly added dropwise to a large amount of ice anhydrous ether for precipitation, the obtained precipitate is collected, and vacuum dried at 50° C. for 15 h to obtain product I, abbreviated as aPU(BHT);

[0113] S21, weigh aPU (BHT) (1.1 g, 0.5 mmol) and mPEG-NH2 (2.2 g, 1.1 mmol), dissolve in 30 mL of anhydrous DMF, pass argon for 30 min to remove oxygen, heat the reaction system to 130 ° C, and stir the reaction for 72 h; after the reaction, remove most of the organic solvent by rotary evaporation, dissolve the residue with a small amount of dichloromethane, and precipitate with a large amount of glacial ether, collect the precipitate, and vacuum dry at 50 ° C for 15 h to obtain product II, abbreviated as PEG-aPU (BHT) -PEG;

[0114] S31. Weigh PEG-aPU(BHT)-PEG (1.85 g, 0.3 mmol) and dissolve it in 20 mL of anhydrous dichloromethane. Slowly drop 5 mL of methacrylic anhydride solution (28 mg / mL, solvent: anhydrous dichloromethane) into the reaction system under ice bath and stirring conditions. Then remove the ice bath and continue stirring the reaction at room temperature for 24 h. Finally, remove most of the organic solvent of the resulting solution by rotary evaporation, slowly drop the solution into a large amount of ice anhydrous ether for precipitation, collect the precipitate, and vacuum dry it at 50°C for 15 h to obtain a functionally modified polyurea compound, abbreviated as PEG-PU / MA-PEG.

[0115] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the intermediate product aPU (BHT) in Example 1, Figure 1 It can be seen that step S11 successfully prepared a polyurea having a secondary amine in the main chain and 2-oxoazacycloheptane-1-carboxamide at both ends; Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the intermediate product PEG-aPU(BHT)-PEG in Example 1, Figure 2 It can be seen that step S21 successfully prepared a polyurea with a secondary amine in the main chain and polyethylene glycol at both ends; Figure 3 is the H NMR spectrum of the functionally modified polyurea compound PEG-PU / MA-PEG in Example 1, Figure 3 It can be seen that step S31 successfully prepares a functionally modified polyurea compound having a methacryloyl group in the side chain and polyethylene glycol at both ends.

[0116] Example 2

[0117] In this example, the polyurea compound prepared in Example 1 is used as a raw material to prepare a functional polyurea nanogel, and the steps are as follows:

[0118] (1) Preparation of cross-linking agent: 1,4-bis(3-aminopropyl)piperazine (2 g, 10 mmol) and 3 mL of anhydrous triethylamine were weighed and dissolved in 20 mL of anhydrous dichloromethane. The reaction system was then placed in an ice bath and, under stirring, 5 mL of methacrylic anhydride solution (0.65 g / mL, solvent: anhydrous dichloromethane) was added dropwise. After the addition was complete, the ice bath was removed and the reaction was continued under stirring at room temperature for 24 h. After the reaction was completed, the pH was adjusted to 8.0 with a 0.1 mol / L NaOH solution, and then extracted with a saturated NaCl aqueous solution for 5 times. The organic phase was collected and an excess of anhydrous sodium sulfate powder was added thereto. The organic phase was dried for 12 h and then filtered. The filtrate was collected and dichloromethane was removed by rotary evaporation. The cross-linking agent PMA was obtained by vacuum drying at 50° C. for 15 h.

[0119] The reaction route is shown below:

[0120]

[0121] Figure 4 is the nuclear magnetic structure characterization diagram of the cross-linking agent PMA in Example 2, Figure 4 It can be seen that the cross-linking agent has been successfully synthesized.

[0122] (2) PEG-PU / MA-PEG and PMA were weighed in a mass ratio of 1:1, dissolved in dichloromethane, and 0.5 wt% of a photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (IC2959), was added, mixed thoroughly, and rotary evaporated at room temperature to remove the solvent and form a solid film at the bottom of the flask. Ultrapure water was then added, and the mixture was ultrasonicated at a frequency of 30 kHz for 30 min, and irradiated under a UV curing light source (wavelength of 365 nm) for 10 min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000 Da, centrifuged at a speed of 5000 g for 20 min, and washed repeatedly with ultrapure water for 5 times to remove uncrosslinked PMA. The concentrate in the ultrafiltration tube was collected to obtain a functional polyurea nanogel.

[0123] Figure 5 is a transmission electron microscopy image of the functional polyurea nanogel in Example 2, wherein: Figure 5 (a) is the transmission electron microscopy image under pH=7.4. Figure 5 (b) is a transmission electron microscopy image under the condition of pH = 5.0. It can be seen from Table 5 that the functional polyurea nanogel is in the form of spherical particles, and under acidic pH conditions, the particle size of the particles increases, indicating that the functional polyurea nanogel provided by the present invention has pH responsiveness.

[0124] Figure 6 The surface potential diagrams of the functional polyurea nanogel in Example 2 at pH = 7.4 (a) and pH = 5.0 (b) are shown in FIG. Figure 6It can be seen that under the condition of pH = 7.4, the Zeta potential of the functional polyurea nanogel is negative, and under the condition of pH = 5.0, the Zeta potential of the functional polyurea nanogel is positive, indicating that the functional polyurea nanogel provided by the present invention has charge reversal performance.

[0125] Example 3

[0126] This embodiment provides a product, which is a functional polyurea nanogel loaded with a broad-spectrum antibiotic rifampicin as an active pharmaceutical ingredient. The preparation steps are as follows:

[0127] Loading of rifampicin: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinker PMA prepared in Example 2, and rifampicin were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, and 0.5wt% of photoinitiator IC2959 was added, mixed thoroughly, and rotary evaporated at room temperature to remove the solvent and form a solid film at the bottom of the flask, then ultrapure water was added, ultrasonicated at a frequency of 30kHz for 30min, and irradiated under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water 5 times to remove uncrosslinked PMA and unloaded rifampicin, and the concentrate in the ultrafiltration tube was collected to obtain rifampicin-loaded functional polyurea nanogel.

[0128] Release of rifampicin: Take the rifampicin-loaded polyurea nanogel solution, place it in a dialysis bag with a cutoff size of 3500Da, and dialyze it in a constant temperature oscillating box at 37°C. The dialysate is a phosphate buffer solution (PBS) with a pH of 7.4 or pH of 5.0; 1 mL of the dialysate is taken out at the time points of 0.5h, 1h, 2h, 3h, 5h, 8h, 10h, and 12h, respectively, and 1 mL of fresh dialysate is replenished into the system; the absorbance of the dialysate taken out at each time point at 474nm is measured, and the cumulative release rate of rifampicin is calculated based on the standard curve of the absorbance and concentration of rifampicin at 474nm.

[0129] Figure 7 is the UV-visible spectrum of the functional polyurea nanogel loaded with rifampicin in Example 3, Figure 7 It can be seen that rifampicin was successfully loaded into the functional polyurea nanogel.

[0130] Figure 8 The in vitro drug release performance of the functional polyurea nanogel loaded with rifampicin in Example 3 at pH = 7.4 and pH = 5.0 is shown in FIG. Figure 8It can be seen that at each test time point between 0 and 12, the in vitro cumulative drug release rate of the rifampicin-loaded functional polyurea nanogel under pH = 5.0 was higher than the cumulative release rate under pH = 7.4, and was close to 100%, indicating that the rifampicin-loaded functional polyurea nanogel provided by the present invention has good in vitro drug release performance and pH-responsive drug release properties.

[0131] Example 4

[0132] This embodiment provides a product, which is a functional polyurea nanogel loaded with the active pharmaceutical ingredient curcumin, and the preparation steps are as follows:

[0133] Loading of curcumin: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinker PMA prepared in Example 2, and curcumin were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, 0.5wt% of photoinitiator IC2959 was added, and mixed thoroughly. The solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask, and then ultrapure water was added. Ultrasonication was performed at a frequency of 30kHz for 30min, and irradiation was performed under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water 5 times to remove uncrosslinked PMA and unloaded curcumin, and the concentrate in the ultrafiltration tube was collected to obtain a functional polyurea nanogel loaded with curcumin.

[0134] Fig. 9 is the UV-visible spectrum of the functional polyurea nanogel loaded with curcumin in Example 4, Fig. 9 It can be seen that curcumin was successfully loaded into the functional polyurea nanogel.

[0135] Example 5

[0136] This embodiment provides a product, which is a functional polyurea nanogel loaded with the active pharmaceutical ingredient vancomycin, and the preparation steps are as follows:

[0137] Vancomycin loading: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinker PMA prepared in Example 2, and vancomycin were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, 0.5wt% of photoinitiator IC2959 was added, and mixed thoroughly. The solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Ultrapure water was then added, and the mixture was ultrasonicated at a frequency of 30kHz for 30min and irradiated under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water for 5 times to remove uncrosslinked PMA and unloaded vancomycin. The concentrate in the ultrafiltration tube was collected to obtain vancomycin-loaded functional polyurea nanogel.

[0138] Fig.10 is the UV-visible spectrum of the vancomycin-loaded functional polyurea nanogel in Example 5, Fig.10 It can be seen that vancomycin was successfully loaded into the functional polyurea nanogel.

[0139] Example 6

[0140] This embodiment provides a product, which is a functional polyurea nanogel loaded with an active pharmaceutical ingredient, doxorubicin hydrochloride, and the preparation steps are as follows:

[0141] Loading of doxorubicin hydrochloride: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinker PMA prepared in Example 2, and doxorubicin hydrochloride were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, 0.5wt% of photoinitiator IC2959 was added, and mixed thoroughly. The solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Ultrapure water was then added, and the mixture was ultrasonicated at a frequency of 30kHz for 30min and irradiated under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water 5 times to remove uncrosslinked PMA and unloaded doxorubicin hydrochloride. The concentrate in the ultrafiltration tube was collected to obtain functional polyurea nanogel loaded with doxorubicin hydrochloride.

[0142] Fig.11 is the UV-visible spectrum of the functional polyurea nanogel loaded with doxorubicin hydrochloride in Example 6, Fig.11 It can be seen that doxorubicin hydrochloride is successfully loaded into the functional polyurea nanogel.

[0143] Example 7

[0144] This embodiment provides a product, which is a functional polyurea nanogel loaded with aztreonam, an active pharmaceutical ingredient, and the preparation steps are as follows:

[0145] Loading of aztreonam: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinking agent PMA prepared in Example 2, and aztreonam were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, 0.5wt% of photoinitiator IC2959 was added, and fully mixed. The solvent was removed by rotary evaporation at room temperature and a solid film was formed at the bottom of the flask. Ultrapure water was then added, and the mixture was ultrasonicated at a frequency of 30kHz for 30min, and irradiated under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water 5 times to remove uncrosslinked PMA and unloaded aztreonam, and the concentrated solution in the ultrafiltration tube was collected to obtain aztreonam-loaded functional polyurea nanogel.

[0146] Fig.12 is the UV-Vis spectrum of the functional polyurea nanogel loaded with aztreonam in Example 7, Fig.11 It can be seen that aztreonam was successfully loaded into the functional polyurea nanogel.

[0147] Example 8

[0148] This embodiment provides a product, which is a functional polyurea nanogel loaded with a drug active ingredient lysozyme, and the preparation steps are as follows:

[0149] Loading of lysozyme: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinker PMA prepared in Example 2, and lysozyme were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, 0.5wt% of photoinitiator IC2959 was added, and mixed thoroughly. The solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Ultrapure water was then added, and the mixture was ultrasonicated at a frequency of 30kHz for 30min, and irradiated under a UV curing light source (wavelength of 365nm) for 10min. The above dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuged at a speed of 5000g for 20min, and washed repeatedly with ultrapure water 5 times to remove uncrosslinked PMA and unloaded lysozyme, and the concentrate in the ultrafiltration tube was collected to obtain a functional polyurea nanogel loaded with lysozyme.

[0150] Fig.13 is the UV-visible spectrum of the functional polyurea nanogel loaded with lysozyme in Example 8, Fig.13 It can be seen that lysozyme was successfully loaded into the functional polyurea nanogel.

[0151] In addition, the functional polyurea nanogel provided by the present invention has universal drug loading performance. In addition to loading active drug ingredients in Examples 3-8, it can effectively load drugs with different physical properties such as small molecule hydrophilic drugs, small molecule hydrophobic drugs, charged drugs, macromolecular drugs, protein drugs, etc., and can swell in an acidic microenvironment to promote the release of drugs.

[0152] Application Examples

[0153] In this application example, the rifampicin-loaded functional polyurea nanogel prepared in Example 3 is used to remove bacterial biofilms, and the steps are as follows:

[0154] Containing 1×10 6 CFU / mL TSB culture medium of Pseudomonas aeruginosa was added to a 96-well plate, 200 μL per well, and placed in a constant temperature incubator for static incubation, and the culture medium was replaced after 24 hours; after continuing to culture for 24 hours, a mature biofilm was formed in the 96-well plate; rifampicin (Rfp) or rifampicin-loaded polyurea nanogel was prepared into a series of solutions dissolved in TSB culture medium with rifampicin equivalent concentrations of 4, 8, 16, 32 or 64 μg / mL, respectively, using fresh tryptic soy casein broth (TSB); and PBS was set as a control group; the above solutions were respectively covered with the mature biofilms in the 96-well plate, and after incubation in a constant temperature shaking box at 37°C for 48 hours, the biomass of the remaining biofilm in each well plate was evaluated by crystal violet staining as a basis for judging the bacterial biofilm removal effect.

[0155] Fig.14 The bacterial biofilm removal effect of rifampicin-loaded functional polyurea nanogel in the application example is shown in Figure 2. Fig.14 It can be seen that the broad-spectrum antibiotic rifampicin loaded in the polyurea nanogel exhibits a more efficient bacterial biofilm removal effect than free antibiotics, and it has been verified that the functional polyurea nanogel products loaded with different active ingredients of curcumin, vancomycin, doxorubicin hydrochloride, aztreonam and lysozyme prepared in Examples 4-8 can also efficiently remove bacterial biofilms, and bacteria include but are not limited to Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Enterococcus faecalis. It can be seen that the drug-loaded functional polyurea nanogel provided by the present invention has an excellent bacterial biofilm removal effect. In addition, it is expected that the drug-loaded functional polyurea nanogel product can be in different forms such as drugs, reagents or additives, and can be used as antibacterial, bactericidal or antibacterial products, products that inhibit or remove bacterial biofilm formation, prevent and / or treat bacterial infections and diseases caused by products, kill cancer cells or inhibit tumor growth or anticancer products, and play antibacterial, bactericidal, antibacterial, inhibit bacterial biofilm formation, prevent and / or inhibit biofilm remote colonization, kill cancer cells, inhibit tumor growth or anticancer effects.

Claims

1. A functionally modified polyurea compound, characterized in that: The method comprises the following preparation raw materials: a diprimary amine compound, bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine, a polyethylene glycol-amino compound, N,N′-carbonylbis(caprolactam), anhydride and an organic solvent; The diprimary amine compound is selected from at least one of bis(hexamethylene)triamine, tetraethylenepentamine, triethylenetetramine, pentaethylenehexamine, spermine, diethylenetriamine, bis(3-aminopropyl)amine, and 1,4-bis(3-aminopropyl)piperazine; The bis(2-oxoazacycloheptane-1-carboxamidoalkyl)amine is selected from at least one of the compounds of formula (1) to formula (3): Wherein, x=2, 3 or 6; n=1, 2 or 3.

2. The polyurea compound according to claim 1, characterized in that The polyethylene glycol-amino compound is selected from at least one of methoxy-polyethylene glycol-amino, hydroxy-polyethylene glycol-amino, carboxyl-polyethylene glycol-amino, tert-butyl-amino-polyethylene glycol-amino, biotin-polyethylene glycol-amino, azide-polyethylene glycol-amino, and diphenylcyclooctyne polyethylene glycol amino; and / or, the acid anhydride is selected from at least one of methacrylic anhydride, acrylic anhydride, 4-pentene anhydride, nadic anhydride, and maleic anhydride.

3. The method for preparing the functionally modified polyurea compound according to claim 2, characterized in that: The following steps are involved: S1. Dissolve a diprimary amine compound and bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine in an organic solvent I, and perform a first-stage heating reaction after deoxygenation; add N,N′-carbonylbis(caprolactam), and perform a second-stage heating reaction after deoxygenation to obtain product I; S2, dissolving the product I and the polyethylene glycol-amino compound in an organic solvent II, removing oxygen and heating for reaction to obtain a product II; S3, dissolving the product II in an organic solvent III, adding anhydride, and reacting to obtain the functionally modified polyurea compound; Wherein, the organic solvents I and II are respectively selected from one of N,N-dimethylformamide and dimethyl sulfoxide; and the solvent III is selected from C1-C2 chloroalkanes.

4. The preparation method according to claim 3, characterized in that: The reaction conditions of step S1 include at least one of the following: 1) The molar ratio of the diprimary amine compound to bis(2-oxoazepane-1-carboxamidoalkyl)amine is (0.5-2):1; 2) the molar ratio of the diprimary amine compound to N,N′-carbonylbis(caprolactam) is 1:(2-5); 3) The temperature of the first stage heating reaction is 120-140° C. and the time is 6-72 hours; 4) The temperature of the second stage heating reaction is 70-100°C and the time is 6-24h. And / or, the reaction conditions of step S2 include at least one of the following: 1) The molar ratio of the product I to the polyethylene glycol-amino compound is 1:(2-4); 2) The heating reaction is carried out at a temperature of 120-140° C. and for a time of 48-80 hours. And / or, the reaction conditions of step S3 include at least one of the following: 1) The molar ratio of the product II to the acid anhydride is 1:(50-150); 2) The reaction temperature is 20-25°C and the reaction time is 6-30h.

5. A functional polyurea nanogel, characterized in that: The preparation method comprises the following raw materials: the functionally modified polyurea compound according to any one of claims 1 to 2, a crosslinking agent and a photoinitiator.

6. The functional polyurea nanogel according to claim 5, characterized in that: The cross-linking agent comprises the following raw materials: a small molecule diamine solution and a carboxylic acid derivative; the small molecule diamine solution comprises a small molecule polyamino compound or a small molecule polyol compound dissolved in dichloromethane; Preferably, the small molecule polyamino compound is selected from at least one of 1,4-bis(3-aminopropyl)piperazine, N,N-bis(3-aminopropyl)methylamine, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, and N,N,N',N'-tetrakis(3-aminopropyl)-1,4-butanediamine; Preferably, the small molecule polyol compound is selected from at least one of 1,4-bis(2-hydroxyethyl)piperazine, ethylenediaminetetraethanol, triisopropanolamine, N,N,N',N'-tetra(2-hydroxyethyl)ethylenediamine, N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine, and N-butyldiethanolamine; Preferably, the carboxylic acid derivative is selected from at least one of methacrylic anhydride, methacryloyl chloride, acrylic anhydride, acryloyl chloride, 4-pentene anhydride, nadic anhydride, 5-norbornene-2-yl chloride, maleic anhydride and furanoyl chloride.

7. The functional polyurea nanogel according to claim 6, characterized in that: The cross-linking agent is prepared by a method comprising the following steps: dropping the carboxylic acid derivative into a small molecule diamine solution, reacting, and extracting to obtain the cross-linking agent; Preferably, the molar ratio of the solute to the carboxylic acid derivative in the small molecule diamine solution is 1:(1-2); Preferably, the reaction temperature is 20-30°C and the reaction time is 6-15h.

8. The functional polyurea nanogel according to claim 5, characterized in that: The mass ratio of the functionally modified polyurea compound to the crosslinking agent is (1-3):1; And / or, the added amount of the photoinitiator is 2-10 mg / mL.

9. The functional polyurea nanogel according to any one of claims 5 to 8, characterized in that: The functional polyurea nanogel is prepared by a method comprising the following steps: mixing a functionally modified polyurea compound, a crosslinking agent and a photoinitiator, and curing the mixture to obtain the functional polyurea nanogel.

10. A product, characterized in that The invention comprises the functional polyurea nanogel according to any one of claims 5 to 9, and a pharmaceutically active ingredient; the pharmaceutically active ingredient is selected from at least one of rifampicin, curcumin, vancomycin, doxorubicin hydrochloride, aztreonam, lysozyme, and a homoserine lactone inhibitor.

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