Functional polyurea nanogel with universal drug loading capacity and preparation method and application thereof

Abstract

The application relates to the technical field of nanogel, and discloses a functional polyurea nanogel with universal drug loading capacity as well as a preparation method and application thereof. A functional modified polyurea compound comprises the following raw materials: a double primary amine compound, a double (2-oxoazepan-1-formamide alkyl) amine, a polyethylene glycol-amino compound, N,N'-carbonyl bis (caprolactam), an acid anhydride and an organic solvent. The functional modified polyurea compound provided by the application is free of isocyanate groups and has high biological safety; the functional polyurea nanogel synthesized from the functional modified polyurea compound has high biological safety, charge flipping performance, multiple intermolecular non-covalent interactions including hydrogen bond interaction, electrostatic interaction and hydrophobic interaction, can be used for universal drug loading, and has pH value responsive swelling properties and can be used for pH responsive drug release.

Description

Technical Field

[0001] This invention relates to the field of nanogel technology, and in particular to a functional polyurea nanogel with universal drug loading capacity, its preparation method, and its application. Background Technology

[0002] Nanogels are intramolecularly cross-linked polymer gels existing in the form of nanoparticles, combining the superior properties of both nanomaterials and hydrogels. Their nanoscale size enables them to exhibit highly efficient permeability into pathological environments such as bacterial biofilms. Simultaneously, as a soft gel polymer material, their internal network structure gives them high water content, allowing them to swell without dissolving, thus possessing the ability to encapsulate bioactive molecules and exhibiting excellent biocompatibility.

[0003] Currently, nanogels are mainly prepared through two methods: chemical covalent crosslinking and physical non-covalent crosslinking. Non-covalent crosslinking, with its hydrogen bonding interactions, offers advantages in environmental friendliness and biocompatibility. It can occur between various functional groups such as hydroxyl, amino, and carboxyl groups, exhibits reversibility, and can undergo breakage and reorganization, making it easy to design, modify, or control the structure, thus facilitating the preparation of controllable, multifunctional nanogels. Aliphatic polyureas (aPUs), composed of urea groups and alkyl chains, possess abundant hydrogen bonding and hydrophobic interactions, and can interact with drug molecules of different physicochemical properties, endowing nanogels with highly efficient drug loading capacity.

[0004] Polyurea nanogels are a high-performance drug delivery carrier, but current polyurea preparation relies on the reaction between isocyanates and amino compounds. Although this reaction is highly reactive, isocyanates can react with most groups (such as hydroxyl, amino, carboxyl, and thiol groups), making it impossible to directly introduce functional groups into the molecular chain (main chain or side chain) during polyurea synthesis. This results in polyurea nanogels lacking effective functional groups and thus hindering downstream functionalization modifications. Furthermore, isocyanate groups that do not participate in the polymerization reaction remain in the material. Therefore, traditional isocyanate-based polyurea preparation processes often lead to materials with significant toxicity and safety hazards, thereby limiting their applications. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a functionally modified polyurea compound; a second objective is to provide a method for preparing such a functionally modified polyurea compound; a third objective is to provide a functional polyurea nanogel; and a fourth objective is to provide a product.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

[0008] The primary 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-oxoazonicyclic heptane-1-carboxamide alkyl)amine is selected from at least one of the compounds of formula (1) to formula (3):

[0010]

[0011] Where 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 secondary amine, tertiary amine, acryloyl, methacryl, maleimide, norbornene, and allyl.

[0013] Preferably, the synthesis of the bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine is based on existing technology (Chem. Mater. 2023, 35, 4705-4716), specifically by reacting a bis(primary amine) small molecule compound with N,N′-carbonylbis(caprolactam) (CBC) via a nucleophilic substitution reaction of the terminal group. By changing the structure of the bis(primary amine) small molecule compound raw material, the corresponding bis(2-oxoazacycloheptane-1-carboxamide alkyl)amine monomer can be obtained.

[0014] Specifically, the bis(2-oxoazonicycloheptan-1-carboxamide alkyl)amines of formulas (1) to (3) are respectively made from bis-primary amine small molecule compounds having structures of formulas (4) to (6):

[0015]

[0016] Where 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), hydroxy-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 anhydride is selected from at least one of methacrylic anhydride, acrylic anhydride, 4-pentene anhydride, norbornene anhydride, and maleic anhydride.

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

[0021] S1. The bis(2-oxozylidene-1-carboxamide)amine compound and bis(2-oxozylidene-1-carboxamide)amine were dissolved in organic solvent I, and after deoxygenation, a first-stage heating reaction was carried out; N,N′-carbonylbis(caprolactam) was added, and after deoxygenation, a second-stage heating reaction was carried out to obtain product I;

[0022] S2. Dissolve the product I with the polyethylene glycol-amino compound in organic solvent II, remove oxygen, and then heat to react to obtain product II.

[0023] S3. Dissolve product II in organic solvent III, add acid anhydride, and react to obtain the functionally modified polyurea compound.

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

[0025] Preferably, organic solvents I and II are N,N-dimethylformamide (DMF); and 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 bis(2-oxozyracycloheptane-1-carboxamide alkyl)amine is (0.5-2):1;

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

[0029] 3) The temperature of the first-stage heating reaction is 120-140℃, and the time is 6-72h;

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

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

[0032] 1) The molar ratio of the bis(2-oxozyracycloheptane-1-carboxamide alkyl)amine is (0.5-1.5):1;

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

[0034] 3) The temperature of the first-stage heating reaction is 120-130℃, and the time is 6-24h;

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

[0036] Preferably, in step S1, the N,N′-carbonylbis(caprolactam) is added after being dissolved in an excess of the organic solvent I.

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

[0038] 1) The molar ratio of 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℃ for 48-80 hours.

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

[0041] 1) The molar ratio of 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℃ for 48-72 hours.

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

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

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

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

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

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

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

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

[0051] Preferably, in step S1, after the two-stage heating reaction is completed, the process further includes the steps of removing the solvent by rotary evaporation, dissolving the remaining substances with N,N-dimethylformamide, settling with ice-cold ether, and drying the precipitate at 40-60°C for 10-20 hours to obtain product I.

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

[0053] Preferably, in step S3, the process of dissolving 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 process further includes rotary evaporation to remove the solvent, precipitation with ice-cold ether, and drying the precipitate at 40-60°C for 10-20 hours to obtain the functionally modified polyurea compound.

[0056] Specifically, the preparation principle of functionally modified polyurea compounds is as follows: linear polyureas with a main chain containing a secondary or tertiary amine and modified with hydrophilic polymer polyethylene glycol at both ends are prepared by selectively temperature-controlled nucleophilic substitution reaction of primary or secondary amines with N-carbonylcaprolactam.

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

[0058] Preferably, the crosslinking 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 polyamine 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'-tetra(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, ethylenediaminetetraethanolamine, 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, norbornene anhydride, 5-norbornene-2-acyl chloride, maleic anhydride, and furanoyl chloride.

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

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

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

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

[0066] Preferably, the process of adding the carboxylic acid derivative to the small molecule diamine solution is carried out under ice bath and stirring conditions.

[0067] Preferably, the organic solvent used for 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 extraction.

[0070] Preferably, the extraction process includes the following steps: collecting the organic phase, adding an excess of anhydrous sodium sulfate powder, drying for 10-15 hours, filtering, collecting the filtrate and removing the solvent by rotary evaporation, and drying at 40-60°C for 10-15 hours to obtain the crosslinking agent.

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

[0072] Preferably, the amount of photoinitiator added is 2-10 mg / mL; more preferably, the amount of 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-hydroxycyclohexylphenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone, 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 the mixture to obtain the functional polyurea nanogel.

[0075] Preferably, the preparation of the functional polyurea nanogel further includes the use of 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 crosslinking agent and a photoinitiator in a solvent, removing the solvent by rotary evaporation, adding water and sonicating, curing, centrifuging with an ultrafiltration tube, washing with ultrapure water and collecting the concentrate, and freeze-drying to obtain the functional polyurea nanogel.

[0077] Specifically, the preparation principle of functional polyurea nanogels is as follows: starting from the secondary or tertiary amine of the functionally modified polyurea compound, functional modification is carried out to introduce crosslinkable reactive groups, and then functional polyurea nanogels that can be stably dispersed in aqueous solution are formed through the self-assembly of linear polyurea in a selective solvent and subsequent crosslinking methods.

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

[0079] Preferably, the ultrasound duration is 10-30 minutes; more preferably, the ultrasound duration is 20-30 minutes.

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

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

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

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

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

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

[0086] A 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; said pharmaceutically active ingredient is selected from at least one of rifampin, 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 drugs, reagents, and additives.

[0090] Compared with the prior art, the beneficial effects of the present invention are:

[0091] 1) The functionally modified polyurea compound provided by this invention does not contain isocyanate groups and has high biosafety;

[0092] 2) The method for preparing functionally modified polyurea compounds provided by this invention does not rely on the polymerization process of traditional isocyanate monomers. While solving the toxicity problem of traditional polyurea materials and improving the biosafety 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 steps are simple, the conditions are mild, the synthesis efficiency is high, and it is suitable for widespread use.

[0093] 3) The functional polyurea nanogel provided by this invention uses functionally modified polyurea compounds as raw materials, has high biosafety, has charge-flipping properties, and exhibits multiple intermolecular non-covalent interactions, including hydrogen bonding, electrostatic interaction and hydrophobic interaction. It can be used for universal drug loading and has pH-responsive swelling properties, which can be used for pH-responsive drug release.

[0094] 4) The drug-loaded functional polyurea nanogel products provided by this invention include functional polyurea nanogels loaded with active pharmaceutical ingredients such as rifampin, curcumin, vancomycin, doxorubicin hydrochloride, aztreonam, lysozyme, and homoserine lactone inhibitors. These nanogels can exert antibacterial, bactericidal, antimicrobial, inhibit or eliminate bacterial biofilm formation, prevent and / or inhibit distal colonization of biofilms, kill cancer cells, inhibit tumor growth, or have anticancer effects, and have a wide range of applications. Attached Figure Description

[0095] Figure 1 The 1H NMR spectrum of the intermediate product aPU(BHT) in Example 1;

[0096] Figure 2 The 1H NMR spectrum of the intermediate product PEG-aPU(BHT)-PEG in Example 1;

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

[0098] Figure 4 The image shows the NMR structure of the crosslinking agent PMA in Example 2.

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

[0100] 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.

[0101] Figure 7 The UV-Vis 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 under pH = 7.4 and pH = 5.0 conditions;

[0103] Figure 9 The UV-Vis spectrum of the curcumin-loaded functional polyurea nanogel in Example 4;

[0104] Figure 10The UV-Vis spectrum of the vancomycin-loaded functional polyurea nanogel in Example 5;

[0105] Figure 11 The UV-Vis spectrum of the functional polyurea nanogel supported on doxorubicin hydrochloride in Example 6;

[0106] Figure 12 The UV-Vis spectrum of the functional polyurea nanogel supported on aztreonam in Example 7;

[0107] Figure 13 The UV-Vis spectrum of the lysozyme-loaded functional polyurea nanogel in Example 8;

[0108] Figure 14 This demonstrates the bacterial biofilm removal effect of rifampicin-loaded functional polyurea nanogels in an application example. Detailed Implementation

[0109] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0110] Example 1

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

[0112] S11. Weigh 2.15 g (10 mmol) of bis(hexamethylene)triamine and the corresponding bis(2-oxozyracycloheptan-1-carboxamide alkyl)amine monomer BHT (compound of formula (1), 4.93 g, 10 mmol) into a clean double-necked flask. Add 100 mL of DMF to dissolve it completely. Then, circulate argon gas for 30 min to remove oxygen from the system. Under the protection of argon gas, heat the reaction system to 130 °C and stir continuously for 12 h. After the collected solution is cooled, add DMF with excess CBC dissolved in it to the reaction system. After purging argon gas to remove oxygen again, heat the reaction system to 80 °C and stir continuously for 8 h. After the reaction is completed, remove the organic solvent from the obtained solution by rotary evaporation. After the remaining substance is cooled to room temperature, dissolve it in a small amount of dichloromethane and slowly add it dropwise to a large amount of ice-cold anhydrous diethyl ether to settle. Collect the precipitate and dry it under vacuum 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, purge with argon for 30 min to remove oxygen, heat the reaction system to 130 °C, and stir for 72 h. After the reaction is complete, 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 ice-cold diethyl ether. Collect the precipitate and dry it under vacuum 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. Under ice bath and stirring conditions, slowly add 5 mL of methacrylic anhydride solution (28 mg / mL, solvent: anhydrous dichloromethane) to the reaction system. Then remove the ice bath and continue stirring the reaction at room temperature for 24 h. Finally, remove most of the organic solvent from the obtained solution by rotary evaporation and slowly add it to a large amount of ice-cold anhydrous diethyl ether for precipitation. Collect the precipitate and dry it under vacuum at 50 °C for 15 h to obtain the functionally modified polyurea compound, abbreviated as PEG-PU / MA-PEG.

[0115] Figure 1 The image shows the 1H NMR spectrum of the intermediate product aPU(BHT) from Example 1. Figure 1 It can be seen that step S11 successfully prepared a polyurea with a main chain containing a secondary amine and 2-oxoazonicycloheptan-1-carboxamide at both ends; Figure 2 The image shows the 1H NMR spectrum of the intermediate product PEG-aPU(BHT)-PEG from Example 1. Figure 2 It can be seen that step S21 successfully prepared a polyurea with a main chain containing secondary amine and polyethylene glycol at both ends; Figure 3 The image shows the 1H NMR spectrum of the functionally modified polyurea compound PEG-PU / MA-PEG from Example 1. Figure 3 It can be seen that step S31 successfully prepared a functionally modified polyurea compound with methacryloyl groups on the side chain and polyethylene glycol at both ends.

[0116] Example 2

[0117] This embodiment uses the polyurea compound prepared in Example 1 as a raw material to prepare a functional polyurea nanogel. The steps are as follows:

[0118] (1) Preparation of crosslinking agent: Weigh 1,4-bis(3-aminopropyl)piperazine (2g, 10mmol) and 3mL of anhydrous triethylamine, dissolve in 20mL of anhydrous dichloromethane, then place the reaction system in an ice bath, and add 5mL of methacrylic anhydride solution (0.65g / mL, solvent is anhydrous dichloromethane) dropwise under stirring. After the addition is complete, remove the ice bath and continue stirring at room temperature for 24h. After the reaction is completed, adjust the pH to 8.0 with 0.1mol / L NaOH solution, then extract with saturated NaCl aqueous solution 5 times, collect the organic phase, add excess anhydrous sodium sulfate powder to it, dry for 12h and filter, collect the filtrate and remove dichloromethane by rotary evaporation, and dry under vacuum at 50℃ for 15h to obtain crosslinking agent PMA.

[0119] The reaction route is shown below:

[0120]

[0121] Figure 4 The image shown is the NMR structure characterization diagram of the crosslinking agent PMA in Example 2. Figure 4 It can be concluded that the crosslinking agent has been successfully synthesized.

[0122] (2) Weigh PEG-PU / MA-PEG and PMA at a mass ratio of 1:1, dissolve them in dichloromethane, add 0.5wt% of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (IC2959), mix thoroughly, and rotary evaporate at room temperature to remove the solvent and form a solid film at the bottom of the flask. Then add ultrapure water, sonicate at a frequency of 30kHz for 30min, irradiate under a UV curing light source (wavelength of 365nm) for 10min, place the above dispersion in an ultrafiltration centrifuge tube with a cutoff size of 3000Da, centrifuge at a speed of 5000g for 20min, and wash 5 times with ultrapure water to remove uncrosslinked PMA. Collect the concentrate in the ultrafiltration tube to obtain functional polyurea nanogel.

[0123] Figure 5 The image shown is a transmission electron microscope (TEM) image of the functional polyurea nanogel in Example 2. Figure 5 (a) is a transmission electron microscope image under pH = 7.4 conditions. Figure 5 (b) is a transmission electron microscope image under pH=5.0 conditions. As shown in Table 5, the functional polyurea nanogel is in the form of spherical particles, and the particle size increases under acidic pH conditions, indicating that the functional polyurea nanogel provided by the present invention has pH responsiveness.

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

[0125] Example 3

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

[0127] Rifampicin loading: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinking agent PMA prepared in Example 2, and rifampicin were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, and 0.5 wt% of photoinitiator IC2959 was added. The mixture was thoroughly mixed, and the solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Then, ultrapure water was added, and the mixture was sonicated at a frequency of 30 kHz for 30 min. The mixture was then 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 5000 g for 20 min, and washed 5 times with ultrapure water to remove uncrosslinked PMA and unloaded rifampicin. The concentrate in the ultrafiltration tube was collected to obtain rifampicin-loaded functional polyurea nanogel.

[0128] Rifampicin release: Rifampicin-loaded polyurea nanogel solution was placed in a dialysis bag with a cutoff size of 3500 Da and dialyzed in a constant temperature shaking incubator at 37℃. The dialysate was phosphate-buffered saline (PBS) with pH = 7.4 or pH = 5.0. At time points of 0.5h, 1h, 2h, 3h, 5h, 8h, 10h, and 12h, 1 mL of dialysate was taken out and 1 mL of fresh dialysate was added back to the system. The absorbance of the dialysate taken out at each time point was measured at 474 nm, and the cumulative release rate of rifampicin was calculated based on the standard curve of absorbance at 474 nm versus concentration of rifampicin.

[0129] Figure 7 The UV-Vis spectrum of the rifampicin-loaded functional polyurea nanogel in Example 3 is shown below. Figure 7 It can be seen that rifampicin has been successfully loaded into functional polyurea nanogels.

[0130] Figure 8 The in vitro drug release performance of the rifampicin-loaded functional polyurea nanogel in Example 3 under pH = 7.4 and pH = 5.0 conditions was determined by... Figure 8It can be seen that at each test time point within 0-12, the cumulative drug release rate of the rifampicin-loaded functional polyurea nanogel under pH=5.0 conditions is higher than that under pH=7.4 conditions, and is 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 pharmaceutical active ingredient curcumin. The preparation steps are as follows:

[0133] Curcumin loading: The functionally modified polyurea compound PEG-PU / MA-PEG prepared in Example 1, the crosslinking agent PMA prepared in Example 2, and curcumin were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, and 0.5 wt% of photoinitiator IC2959 was added. The mixture was thoroughly mixed, and the solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Then, ultrapure water was added, and the mixture was sonicated at a frequency of 30 kHz for 30 min. The mixture was then 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 5000 g for 20 min, and washed 5 times with ultrapure water to remove uncrosslinked PMA and unloaded curcumin. The concentrate in the ultrafiltration tube was collected to obtain curcumin-loaded functional polyurea nanogel.

[0134] Figure 9 The UV-Vis spectrum of the curcumin-loaded functional polyurea nanogel in Example 4 is shown below. Figure 9 It can be seen that curcumin has been successfully loaded into functional polyurea nanogels.

[0135] Example 5

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

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

[0138] Figure 10 The UV-Vis spectrum of the vancomycin-loaded functional polyurea nanogel in Example 5 is shown below. Figure 10 It can be seen that vancomycin was successfully loaded into functional polyurea nanogel.

[0139] Example 6

[0140] This embodiment provides a product, which is a functional polyurea nanogel loaded with the active pharmaceutical ingredient doxorubicin hydrochloride. 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 crosslinking agent PMA prepared in Example 2, and doxorubicin hydrochloride were weighed in a mass ratio of 1:1:1, dissolved in dichloromethane, and 0.5 wt% of photoinitiator IC2959 was added. The mixture was thoroughly mixed, and the solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Then, ultrapure water was added, and the mixture was sonicated at a frequency of 30 kHz for 30 min. The mixture was then irradiated under a UV curing light source (wavelength of 365 nm) for 10 min. The dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000 Da and centrifuged at a speed of 5000 g for 20 min. The mixture was washed five times with ultrapure water 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] Figure 11 The UV-Vis spectrum of the functional polyurea nanogel supported on doxorubicin hydrochloride in Example 6 is shown below. Figure 11 It can be seen that doxorubicin hydrochloride was successfully loaded into functional polyurea nanogels.

[0143] Example 7

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

[0145] Aztreonam loading: 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, and 0.5 wt% of photoinitiator IC2959 was added. The mixture was thoroughly mixed, and the solvent was removed by rotary evaporation at room temperature to form a solid film at the bottom of the flask. Then, ultrapure water was added, and the mixture was sonicated at a frequency of 30 kHz for 30 min. The mixture was then irradiated under a UV curing light source (wavelength of 365 nm) for 10 min. The dispersion was placed in an ultrafiltration centrifuge tube with a cutoff size of 3000 Da and centrifuged at a speed of 5000 g for 20 min. The mixture was washed 5 times with ultrapure water to remove uncrosslinked PMA and unloaded aztreonam. The concentrate in the ultrafiltration tube was collected to obtain aztreonam-loaded functional polyurea nanogel.

[0146] Figure 12 The UV-Vis spectrum of the functional polyurea nanogel supported on aztreonam in Example 7 is shown below. Figure 11 It can be seen that aztreonam has been successfully loaded into functional polyurea nanogels.

[0147] Example 8

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

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

[0150] Figure 13 The UV-Vis spectrum of the lysozyme-loaded functional polyurea nanogel in Example 8 is shown below. Figure 13 It can be seen that lysozyme was successfully loaded into functional polyurea nanogel.

[0151] Furthermore, the functional polyurea nanogel provided by this invention has universal drug loading properties. In addition to loading the active pharmaceutical 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, macromolecule drugs, and protein drugs. It can also swell in an acidic microenvironment to promote drug release.

[0152] Application examples

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

[0154] Will contain 1×10 6 200 μL of TSB medium containing CFU / mL *Pseudomonas aeruginosa* was added to each well of a 96-well plate and incubated statically in a constant temperature incubator. The medium was replaced after 24 hours. After another 24 hours of incubation, a mature biofilm formed in the 96-well plate. A series of solutions containing rifampin (Rfp) or rifampin-loaded polyurea nanogel were prepared using fresh tryptic soy broth (TSB) with rifampin equivalent concentrations of 4, 8, 16, 32, or 64 μg / mL. PBS was set up as a control group. The mature biofilm in the 96-well plate was covered with the above solutions and incubated in a constant temperature shaking incubator at 37°C for 48 hours. The biomass of the remaining biofilm in each well was assessed by crystal violet staining, which served as the criterion for evaluating the bacterial biofilm removal effect.

[0155] Figure 14 To demonstrate the bacterial biofilm removal effect of rifampicin-loaded functional polyurea nanogels in the application example, [the following text is incomplete and requires further context to translate accurately]. Figure 14 It has been observed that rifampicin, a broad-spectrum antibiotic loaded in polyurea nanogels, exhibits a more efficient bacterial biofilm removal effect than free antibiotics. Furthermore, it has been verified that the functional polyurea nanogel products prepared in Examples 4-8, loaded with different pharmacologically active ingredients such as curcumin, vancomycin, doxorubicin hydrochloride, aztreonam, and lysozyme, also effectively remove bacterial biofilms. These bacteria include, but are not limited to, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, and Enterococcus faecalis. Therefore, the drug-loaded functional polyurea nanogels provided by this invention possess excellent bacterial biofilm removal effects. Moreover, it is anticipated that these drug-loaded functional polyurea nanogel products can be in different forms such as drugs, reagents, or additives, and can serve as antibacterial, bactericidal, or antimicrobial products; products that inhibit or remove bacterial biofilm formation; products that prevent and / or treat diseases caused by bacterial infections; products that kill cancer cells or inhibit tumor growth; or anticancer products, exerting antibacterial, bactericidal, antimicrobial, and antimicrobial effects, inhibiting bacterial biofilm formation, preventing and / or inhibiting distal colonization of biofilms, killing cancer cells, inhibiting tumor growth, or anticancer effects.

Claims

1. A method for producing a function-modified polyurea compound, characterized by, The method comprises the following steps: S1, dissolving a bis-primary amine compound and bis(2-oxoazepan-1-formamide alkyl) amine in an organic solvent I, carrying out a first heating reaction after deoxygenation; adding N,N'-carbonylbis(caprolactam), carrying out a second heating reaction after deoxygenation to obtain product I; S2, dissolving the product I and a polyethylene glycol-amino compound in an organic solvent II, carrying out a heating reaction after deoxygenation to obtain product II; S3, dissolving the product II in an organic solvent III, adding an anhydride, and reacting to obtain the functional modified polyurea compound; wherein the bis-primary amine compound is at least one selected from bis(hexamethylene)triamine, tetraethylenepentamine, triethylenetetramine, pentaethylenehexamine, spermine, diethylenetriamine, bis(3-aminopropyl)amine, and 1,4-bis(3-aminopropyl)piperazine; the bis(2-oxoazepan-1-formamide alkyl) amine is at least one selected from compounds of formula (1) to formula (3): ； ； ； in formula (1) to formula (3), x = 2, 3 or 6; n = 1, 2 or 3; the organic solvents I and II are each one selected from N,N-dimethylformamide and dimethyl sulfoxide; and the solvent III is selected from C1-C2 chloroalkanes.

2. The method for preparing a functionally modified polyurea compound according to claim 1, characterized by, the polyethylene glycol-amino compound is at least one selected from methoxy-polyethylene glycol-amino, hydroxy-polyethylene glycol-amino, carboxyl-polyethylene glycol-amino, tert-butyl-amino-polyethylene glycol-amino, biotin-polyethylene glycol-amino, azido-polyethylene glycol-amino, and diphenylcyclooctyne-polyethylene glycol-amino; and / or, the anhydride is at least one selected from methacrylic anhydride, acrylic anhydride, 4-pentene anhydride, norbornene diacid anhydride, and maleic anhydride.

3. The method for preparing a functionally modified polyurea compound according to claim 1, characterized by, The reaction conditions of step S1 include at least one of the following: 1) the molar ratio of the bis-primary amine compound to bis(2-oxoazepan-1-formamide alkyl) amine is (0.5-2):1; 2) the molar ratio of the bis-primary amine compound to N,N'-carbonylbis(caprolactam) is 1:(2-5); 3) the temperature of the first heating reaction is 120-140℃, and the time is 6-72h; 4) the temperature of the second heating reaction is 70-100℃, and the time is 6-24h; and / or, the reaction conditions of step S2 include at least one of the following: 1) the amount-of-substance ratio of the product I to the polyethylene glycol-amino compound is 1:(2-4); 2) the temperature of the heating reaction is 120-140℃, and the time is 48-80h; 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 anhydride is 1:(50-150); 2) the temperature of the reaction is 20-25℃, and the time is 6-30h.

4. A functional polyurea compound, characterized by, The functional modified polyurea compound is prepared by the method of any one of claims 1-3.

5. A functional polyurea nanogel, characterized in that, The method comprises the following raw materials: the functional modified polyurea compound of claim 4, a crosslinking agent, and a photoinitiator.

6. The functional polyurea nanogel according to claim 5, wherein, The crosslinking 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; The small molecule polyamino compound is at least one selected from 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; The small molecule polyol compound is at least one selected from 1,4-bis(2-hydroxyethyl)piperazine, ethylenediamine tetraethanol, triisopropanolamine, N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine, N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, and N-butyl diethanolamine; The carboxylic acid derivative is at least one selected from methacrylic anhydride, methacryloyl chloride, acrylic anhydride, acryloyl chloride, 4-pentene anhydride, norbornene diacid anhydride, 5-norbornene-2-carbonyl chloride, maleic anhydride, and furan formyl chloride.

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

8. The functional polyurea nanogel according to claim 5, wherein, The mass ratio of the functional modified polyurea compound to the crosslinking agent is (1-3):1; The addition amount of the photoinitiator is 2-10mg / mL.

9. The functional polyurea nanogel according to any one of claims 5-8, wherein, The functional polyurea nanogel is prepared by a method comprising the following steps: mixing the functional modified polyurea compound, the crosslinking agent, and the photoinitiator, and solidifying to obtain the functional polyurea nanogel.

10. A product characterized by, The functional polyurea nanogel comprises at least one active pharmaceutical ingredient selected from rifampicin, curcumin, vancomycin, doxorubicin hydrochloride, amikacin, lysozyme, and fumagillin inhibitor.

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