High-capacity drug-loaded multi-responsive hydrogel and its preparation method and application

Abstract

The present invention provides a method for preparing a multi-responsive hydrogel with high-capacity drug loading. The method promotes the cross-linking of a hydrogel precursor into a gel by promoting boron-nitrogen coordination between phenylboronic acid groups and hydrophobic drugs, or directly promoting the formation of boronate bonds between phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups under the action of hydrophilic basic drug molecules. This method successfully solves the disadvantage of pH adjustment faced by constructing hydrogels through boronate bonds, does not introduce impurities into the hydrogel material, and can achieve high-capacity loading of hydrophobic drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a multi-responsive hydrogel with high-capacity drug loading, and a preparation method and application thereof. Background Art

[0002] Hydrogels have high biocompatibility and the ability to deliver drugs continuously, and have broad application prospects in the field of drug delivery. However, hydrogels are limited to delivering hydrophilic drugs. This is because the polymer matrix in the hydrogel is hydrophilic, so hydrophobic drugs are usually incompatible with hydrogels, and the loading amount and uniformity of hydrophobic drugs in the hydrogel matrix are limited. However, currently about 40% of marketed drugs and 60% of drugs in the development stage show poor water solubility. Hydrophobic drugs play an important role in current drug treatments. Therefore, developing new methods to encapsulate hydrophobic drugs into hydrogels is of great value to expanding the application field of hydrogel drug delivery.

[0003] There are three main approaches to improving the compatibility of hydrogels with hydrophobic compounds: 1. Incorporating molecules capable of forming inclusion complexes, such as cyclodextrin; 2. Incorporating hydrophobic moieties into the hydrogel structure; and 3. Incorporating micelles / nanoparticles into the hydrogel system to encapsulate the hydrophobic compound. However, current methods for encapsulating hydrophobic compounds impose high requirements on the hydrogel's process and also result in low drug loading (typically less than 7%). Summary of the Invention

[0004] The present invention aims to address the deficiencies of the prior art and provide a multi-responsive hydrogel with high-capacity drug loading and a preparation method thereof. The invention promotes the cross-linking of the hydrogel precursor into a gel while simultaneously achieving the loading of drug molecules, and the drug loading capacity of the hydrophobic drug can reach 40%, by promoting boron-nitrogen coordination between the phenylboronic acid group and the hydrophobic drug, or directly promoting the formation of boronate ester bonds between the phenylboronic acid group and the 1,2- or 1,3-diol / phenol group under the action of hydrophilic basic drug molecules.

[0005] According to a first aspect of the present invention, a method for preparing a multi-responsive hydrogel with high drug loading capacity is provided, comprising the following steps:

[0006] The first substance and the drug are mixed in a biocompatible medium to obtain a multi-responsive hydrogel with high drug loading capacity;

[0007] Wherein, the drug is a hydrophobic drug containing at least one group of primary amine, secondary amine, and tertiary amine, or a hydrophilic basic drug;

[0008] The first substance comprises a first component, or comprises a second component and a third component;

[0009] The first component is a polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups;

[0010] The second component is a small molecule compound or polymer derivative containing a phenylboronic acid group, or a small molecule compound or polymer derivative containing both a phenylboronic acid group and a 1,2- or 1,3-diol / phenol group;

[0011] The third component is a small molecule compound or polymer derivative containing a 1,2- or 1,3-diol / phenol group, or a small molecule compound or polymer derivative containing both a phenylboronic acid group and a 1,2- or 1,3-diol / phenol group;

[0012] At least one of the second component and the third component is a polymer derivative;

[0013] When the drug is a hydrophobic drug containing at least one of primary amine, secondary amine, and tertiary amine groups, the boron-nitrogen coordination reaction between the phenylboronic acid group and the hydrophobic drug is promoted to form a boronate ester bond with the phenylboronic acid group and the 1,2- or 1,3-diol / phenol group, thereby promoting the cross-linking of the hydrogel precursor to form a gel and simultaneously achieving the loading of the hydrophobic drug molecules;

[0014] When the drug is a hydrophilic alkaline drug, under the action of the hydrophilic alkaline drug molecules, the phenylboronic acid group is promoted to form a borate ester bond with the 1,2- or 1,3-diol / phenol group, thereby promoting the cross-linking of the hydrogel precursor into a gel while simultaneously achieving the loading of hydrophobic drug molecules.

[0015] In an optional embodiment, the general formula of the first component is as shown in Formula I:

[0016] (OH) 2n -P1-(B(OH)2) n Formula I;

[0017] Wherein, n≥2, P1 is a water-soluble or hydrophilic polymer derivative.

[0018] In an optional embodiment, the first component is any one of the structures shown in Formulas I-1 to I-11:

[0019]

[0020] Wherein, n, x, y are all degrees of polymerization, and n≥2, x≥2, y≥2.

[0021] In an optional embodiment, the general formula of the second component is as shown in Formula II:

[0022] P2-(B(OH)2) n Formula II;

[0023] Wherein, n≥2, P2 is a water-soluble or hydrophilic polymer derivative, or a small molecule compound.

[0024] In an optional embodiment, the second component is any one of the structures shown in Formula II-1 to II-15:

[0025]

[0026] Wherein, n, x, y are all degrees of polymerization, and n≥2, x≥2, y≥2.

[0027] In an optional embodiment, the general formula of the third component is as shown in Formula III:

[0028] P3-(OH) 2n Formula III;

[0029] Wherein, n≥2, P3 is a water-soluble or hydrophilic polymer derivative, or a small molecule compound.

[0030] In an optional embodiment, the third component is any one of the structures shown in Formulas III-1 to III-15:

[0031]

[0032] Or any one of alginic acid, chondroitin sulfate, heparin, aminodextran, pullulan, sodium alginate, and cellulose;

[0033] Wherein, n is the degree of polymerization, and n≥2, x≥2, y≥2.

[0034] In an alternative embodiment, the pKa of the drug is ≥8.81.

[0035] According to a second aspect of the present invention, a multi-responsive hydrogel with high drug loading capacity prepared by the above-mentioned preparation method is provided.

[0036] According to the third aspect of the present invention, there is provided an application of the aforementioned high-capacity drug-loaded multi-responsive hydrogel in the preparation of hemostatic drugs or materials, drug-loaded burn and scald dressings, chronic wound care drugs or materials, bone repair drugs or materials, tissue engineering scaffold materials, 3D printing-bio-ink, cells, proteins or drug carriers.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The method for preparing a high-capacity drug-loaded multi-responsive hydrogel of the present invention promotes the cross-linking of a hydrogel precursor into a gel by promoting boron-nitrogen coordination between phenylboronic acid groups and hydrophobic drugs, or directly promoting the formation of boronate bonds between phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups under the action of hydrophilic basic drug molecules. This successfully solves the disadvantage of pH adjustment faced in constructing hydrogels through boronate bonds, and does not introduce impurities into the hydrogel material. The resulting hydrogel has excellent performance and wider application. In addition, gelation and drug loading of the hydrogel are completed simultaneously. The prepared hydrogel has an ultra-high drug loading capacity and has great potential for clinical transformation.

[0039] The high-capacity drug-loaded multi-responsive hydrogel of the present invention can load not only water-soluble drugs but also hydrophobic drugs, effectively achieving substantial solubilization of poorly soluble drugs, and the loading capacity can be precisely adjusted, with a maximum drug loading of up to 40%.

[0040] The high-capacity drug-loaded multi-responsive hydrogel of the present invention has stimulus (pH, glucose, ROS) responsive controlled release performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a diagram of the reaction process mechanism when the present invention uses a hydrophobic drug.

[0042] Figure 2 This is a diagram of the reaction process mechanism when the present invention uses a hydrophilic drug.

[0043] Figure 3 This is the H NMR spectrum of HA-PBA with different phenylboronic acid grafting rates in Example 1 of the present invention (400M, D2O)

[0044] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the aldehyde-modified hyaluronic acid in Example 5 of the present invention.

[0045] Figure 5 These are photos of HA-PBA-Sum hydrogels formed after HA-PBA was loaded with the drug sumatriptan in Example 15 of the present invention (the first row, from left to right, shows 5, 10, 15, and 20 mg of Sum dissolved in 1 mL of 5% (w / v) HA-PBA (grafting rate 20.1%) solution to form hydrogels; the second row, from left to right, shows 15 mg of Sum dissolved in 1 mL of HA-PBA solutions with grafting rates of 9.0%, 20.1%, 35.5%, and 5% (w / v) to form hydrogels).

[0046] Figure 6These are photos of 5 mg of hydrophobic sumatriptan added to 1 mL of water and a 5% (w / v) HA-PBA (grafting rate 20.1%) aqueous solution in Example 15 of the present invention at 0, 5 min, 15 min, 30 min, 24 h and after shaking, respectively. The left side of each picture is water, and the right side is the HA-PBA aqueous solution.

[0047] Figure 7 This is the NMR boron spectrum in Example 18 of the present invention; wherein, from bottom to top, they are the NMR boron spectra of phenylboric acid (PBA), a mixture of PBA and dopamine in a molar ratio of 1:2 (PBA:Dop(1:2)), a mixture of sumatriptan and phenylboric acid in a molar ratio of 1:2 (Sum:PBA(1:2)), and a mixture of dopamine, sumatriptan and phenylboric acid in a molar ratio of 1:1:2 (Dop:Sum:PBA(1:1:2)).

[0048] Figure 8 a and Figure 8 b are the rheological curves of sample 15-1 and sample 15-10 in Example 15 of the present invention.

[0049] Figure 9 9a and 9b are scanning electron microscope (SEM) images at different magnifications of sample 15-1 in Example 15 of the present invention, i.e., HA-PBA-Sum hydrogel formed after loading the drug sumatriptan and freeze-drying.

[0050] Figure 10 These are the swelling diagrams of samples 15-2 and 15-7 in Example 15 of the present invention, i.e., HA-PBA-Sum hydrogels formed after loading the drug sumatriptan. The PBA grafting ratios of the HA-PBA used are 20.1% and 35.5%, respectively.

[0051] Figure 11 The graph shows the changes in swelling and dissolution over time in water of HA-PBA-Sum (sample 15-2) and HA-PBA-Azi (sample 15-10) hydrogels formed after loading sumatriptan and azithromycin, respectively, and HA-PBA (NaOH) hydrogel (sample 14-6) formed after adjusting the alkalinity by NaOH.

[0052] Figure 12 From top to bottom are the PXRD patterns of sumatriptan (Sum), a mixture of sumatriptan and HA-PBA, HA-PBA, and a freeze-dried sample of sumatriptan-loaded hydrogel (HA-PBA-Sum).

[0053] Figure 13 These are the PXRD patterns of freeze-dried samples of sumatriptan-loaded hydrogels (HA-PBA-Sum) prepared with different molar ratios of PBA groups and Sum in HA-PBA.

[0054] Figure 14 From bottom to top, the PXRD patterns of azithromycin-loaded hydrogel freeze-dried samples (HA-PBA-Azi) prepared by combining PBA groups in HA-PBA with azithromycin in different molar ratios, a mixture of HA-PBA and azithromycin in a molar ratio of 1:1.5 (physical mixture (1:1.5)), and azithromycin (Azi) are shown.

[0055] Figure 15 This is a graph of the reversible change of the pH response sol-gel using phenolphthalein as an indicator, for sample 15-2 in Example 15 of the present invention, i.e., HA-PBA-Sum hydrogel formed after loading the drug sumatriptan.

[0056] Figure 16 This is a graph showing the hemostatic effect of sample 15-10 in Example 15 of the present invention, i.e., the HA-PBA-Azi hydrogel formed after loading azithromycin (left figure). The middle figure and the right figure are graphs showing the effects of the gauze-treated group and the untreated group, respectively.

[0057] Figure 17 This is the in vitro drug release curve of sample 15-2 in Example 15 of the present invention, i.e., the HA-PBA-Sum hydrogel formed after loading sumatriptan in the presence of different pH values ​​and different concentrations of hydrogen peroxide. DETAILED DESCRIPTION

[0058] In order to better understand the technical content of the present invention, specific embodiments are given and described below with reference to the accompanying drawings.

[0059] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to be comprehensive. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of a number of ways.

[0060] pH-responsive biomaterials are expected to store drugs at normal pH and release them under specific pH conditions. To this end, pH-sensitive chemical bonds can be anchored within drug carriers, and the configuration of the carrier can be deformed by dynamic chemical bond cleavage, thereby controlling the release of payload drugs; glucose-responsive systems can continuously and automatically regulate the release of payloads by changes in blood glucose levels, and have potential applications in self-regulated drug delivery; reactive oxygen species (ROS) are present in many types of tumor cells and can also be induced during photodynamic, sonodynamic, or chemodynamic therapy.

[0061] pH, glucose, and ROS (reactive oxygen species) responsive hydrogel drug carriers are highly stable under physiological conditions and can effectively prevent premature drug release.

[0062] In recent years, boronic acid derivatives have been frequently incorporated into hydrogel systems. Their unique boronic acid dihydroxyl groups can form dynamic boronate ester bonds with dihydroxyl groups in polymer structures under specific conditions. As reversible covalent bonds, boronate ester bonds offer simple construction and excellent biocompatibility. They can respond to microenvironmental changes in the body, such as pH, glucose, and ROS (reactive oxygen species), without the need for additional induction conditions, making them excellent stimulus-responsive sites. The formation of boronate ester bonds imparts pH, glucose, and ROS (reactive oxygen species) responsiveness to hydrogels.

[0063] However, borate ester bond formation occurs when the pH is greater than or equal to the pKa value of phenylboronic acid (e.g., the pKa value of phenylboronic acid is approximately 8.81). Therefore, borate ester bond hydrogels prepared via this crosslinking mechanism generally require adjusting the pH to an alkaline level to form a hydrogel. However, using other compounds such as sodium hydroxide to adjust the pH not only introduces impurities into the hydrogel material but also makes it difficult to precisely adjust the pH, seriously affecting the hydrogel's performance and applications.

[0064] The present invention provides a method for preparing a multi-responsive hydrogel with high-capacity drug loading. The method promotes the cross-linking of a hydrogel precursor into a gel by promoting boron-nitrogen coordination between phenylboronic acid groups and hydrophobic drugs, or directly promoting the formation of boronate bonds between phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups under the action of hydrophilic basic drug molecules. This successfully solves the drawback of pH adjustment faced in constructing hydrogels through boronate bonds, does not introduce impurities into the hydrogel material, and can achieve high-capacity loading of hydrophobic drugs.

[0065] In a preferred embodiment of the present invention, a method for preparing a multi-responsive hydrogel with high drug loading capacity is provided, comprising the following steps:

[0066] The first substance and the drug are mixed in a biocompatible medium to obtain a multi-responsive hydrogel with high capacity for loading drugs.

[0067] Wherein, the drug is a hydrophobic drug containing at least one group of primary amine, secondary amine, and tertiary amine, or a hydrophilic basic drug.

[0068] The first substance comprises a first component, or comprises a second component and a third component.

[0069] The first component is a polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups.

[0070] The second component is a small molecule compound or a polymer derivative containing a phenylboronic acid group, or a small molecule compound or a polymer derivative containing both a phenylboronic acid group and a 1,2- or 1,3-diol / phenol group.

[0071] The third component is a small molecule compound or a polymer derivative containing a 1,2- or 1,3-diol / phenol group, or a small molecule compound or a polymer derivative containing both a phenylboronic acid group and a 1,2- or 1,3-diol / phenol group.

[0072] At least one of the second component and the third component is a polymer derivative, so that the hydrogel precursor can form a gel.

[0073] like Figure 1 As shown, when the drug is a primary amine containing ( Figure 1 A), secondary amine ( Figure 1 B), tertiary amine ( Figure 1 C) contains a hydrophobic drug having at least one group, the phenylboronic acid group undergoes a boron-nitrogen coordination reaction with the hydrophobic drug, promoting the formation of a boronate ester bond between the phenylboronic acid group and the 1,2- or 1,3-diol / phenol group, thereby promoting the cross-linking of the hydrogel precursor into a gel and simultaneously achieving the loading of the hydrophobic drug molecules.

[0074] like Figure 2 As shown, when the drug is a hydrophilic alkaline drug, under the action of the hydrophilic alkaline drug molecules, the phenylboronic acid group is promoted to form a borate ester bond with the 1,2- or 1,3-diol / phenol group, thereby promoting the cross-linking of the hydrogel precursor into a gel while simultaneously achieving the loading of hydrophobic drug molecules.

[0075] In an optional embodiment, the general formula of the first component is as shown in Formula I:

[0076] (OH) 2n -P1-(B(OH)2) n Formula I;

[0077] Wherein, n≥2, P1 is a water-soluble or hydrophilic polymer derivative.

[0078] In an optional embodiment, the first component is any one of the structures shown in Formulas I-1 to I-11:

[0079]

[0080] Wherein, n, x, y are all degrees of polymerization, and n≥2, x≥2, y≥2.

[0081] In an optional embodiment, the general formula of the second component is as shown in Formula II:

[0082] P2-(B(OH)2)n Formula II;

[0083] Wherein, n≥2, P2 is a water-soluble or hydrophilic polymer derivative, or a small molecule compound.

[0084] In an optional embodiment, the second component is any one of the structures shown in Formula II-1 to II-15:

[0085]

[0086] Wherein, n, x, y are all degrees of polymerization, and n≥2, x≥2, y≥2.

[0087] In an optional embodiment, the general formula of the third component is as shown in Formula III:

[0088] P3-(OH) 2n Formula III;

[0089] Wherein, n≥2, P3 is a water-soluble or hydrophilic polymer derivative, or a small molecule compound.

[0090] In an optional embodiment, the third component is any one of the structures shown in Formulas III-1 to III-15:

[0091]

[0092] Or any one of alginic acid, chondroitin sulfate, heparin, aminodextran, pullulan, sodium alginate, and cellulose;

[0093] Wherein, n is the degree of polymerization, and n≥2.

[0094] It should be understood that the first component, the second component and the third component include but are not limited to the aforementioned optional structures.

[0095] It is understandable that the first component, the second component or the third component may contain one or more hydrophilic or water-soluble polymers of different groups, or a mixture of one or more hydrophilic or water-soluble polymers of different groups.

[0096] In an optional embodiment, in the aforementioned Formula I, Formula II and Formula III, when P1, P2, and P3 are water-soluble or hydrophilic polymer derivatives, they include water-soluble or hydrophilic natural polymers or water-soluble or hydrophilic synthetic polymers, and P1, P2, and P3 are independently selected from the same or different substances.

[0097] It can be understood that water-soluble or hydrophilic natural polymers include polysaccharides and their modifications or degradation products, proteins and their modifications, modified products or degradation products.

[0098] In one exemplary embodiment, the polysaccharide comprises hyaluronic acid, cellulose, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, aminodextran, pullulan, ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan or chitosan quaternary ammonium salt;

[0099] In another exemplary embodiment, the protein includes various hydrophilic or water-soluble animal and plant proteins, collagen, serum protein, silk fibroin, and elastin; and the degradation products of the protein include gelatin or polypeptide.

[0100] In another exemplary embodiment, the water-soluble or hydrophilic synthetic polymer includes two-arm or multi-arm polyethylene glycol, polyethyleneimine, dendrimers, synthetic polypeptides, polylysine, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone.

[0101] The first component, the second component and the third component of the present invention can be prepared by methods commonly used in the art and are not further limited herein.

[0102] In an optional embodiment, the pKa of the drug is ≥8.81; that is, when a hydrophilic drug is loaded, a hydrophilic basic drug with a pKa ≥8.81 is selected; when a hydrophobic drug is loaded, it is a hydrophobic drug containing at least one of primary amine, secondary amine, and tertiary amine groups; or it is other hydrophobic materials containing at least one of primary amine, secondary amine, and tertiary amine groups, for example, a basic bioactive factor or a basic protein.

[0103] In an exemplary embodiment, the affinity drug is any one or a combination of arginine, lysine, baking soda, and Levamisole.

[0104] In another exemplary embodiment, the hydrophobic drug is any one or a combination of Sumatriptan, Buprenorphine, Vinblastine, Auristatin E, Carfizomi, Duocarmycin, Retapamulin, Doxycycline and Azithromycin.

[0105] In another exemplary embodiment, the basic bioactive factor is bFGF.

[0106] In another exemplary embodiment, the basic protein is any one or a combination of protamine, histone and protamine.

[0107] In a preferred embodiment of the present invention, a multi-responsive hydrogel with high drug loading capacity prepared by the aforementioned preparation method is provided, which can form gel without adjusting the pH value by adding external substances, and gelation and drug loading are completed simultaneously. Drug loading is not limited to hydrophilic drugs, and has a high drug loading capacity for hydrophobic drugs.

[0108] In a preferred embodiment of the present invention, there is provided an application of the aforementioned high-capacity drug-loaded multi-responsive hydrogel in the preparation of hemostatic drugs or materials, drug-loaded burn and scald dressings, chronic wound care drugs or materials, bone repair drugs or materials, tissue engineering scaffold materials, 3D printing-bio-ink, cells, proteins or drug carriers.

[0109] The above multi-responsive hydrogel with high drug loading capacity and its preparation method are described below with reference to specific examples, as well as experimental tests.

[0110] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0111] The molecular weights described in the following examples, unless otherwise specified, refer to the weight average molecular weight of the polymer.

[0112] Preparation of polymer derivatives containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups

[0113] Example 1

[0114] [Preparation of Hyaluronic Acid Derivatives Functionalized with Phenylboronic Acid Groups at Different Grafting Rates]

[0115] According to the reactants listed in Table 1, 2000 mg of hyaluronic acid (HA) was dissolved in 200 mL of 0.1 M MES buffer solution, and the pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the HA solution. The reaction was activated for approximately 30 minutes. 3-Aminophenylboronic acid was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), and the pH of the solution was maintained at 5.5 during the purification process. After dialysis, the solution was freeze-dried to obtain hyaluronic acid derivatives (HA-PBA) functionalized with phenylboronic acid groups at different grafting rates. The reaction process is shown in Equation (1).

[0116]

[0117] Each HA-PBA obtained was 1 H NMR characterization: HA-PBA was dissolved in D2O and TMS was used as the internal standard to test its 1 H NMR spectrum, the results are as follows Figure 3 shown.

[0118] The peaks with chemical shifts between 7.44 ppm and 7.73 ppm correspond to the chemical shifts of the protons on the benzene ring in PBA, while the proton peak at 1.99 ppm corresponds to the methyl group on the hyaluronic acid backbone. This demonstrates the successful preparation of a phenylboronic acid-modified hyaluronic acid derivative (HA-PBA).

[0119] The grafting rate of phenylboronic acid can be calculated by integral area calculation, and the results are shown in Table 1.

[0120] Table 1 Synthesis ratio of hyaluronic acid derivatives functionalized with phenylboronic acid groups with different grafting rates

[0121]

[0122] Example 2

[0123] [Preparation of Hyaluronic Acid Derivatives with Different Molecular Weights Functionalized with Phenylboronic Acid Groups]

[0124] According to the reactants listed in Table 2, 2000 mg of hyaluronic acid was dissolved in 200 mL of 0.1 M MES buffer and the pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the hyaluronic acid solution. The reaction was allowed to activate for approximately 30 minutes. 3-Aminophenylboronic acid was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da), maintaining a pH of 5.5 during purification. After dialysis, the solution was freeze-dried to obtain hyaluronic acid derivatives of varying molecular weight functionalized with phenylboronic acid groups.

[0125] through 1 After H NMR characterization, its structure can be verified and its phenylboronic acid grafting rate can be calculated, as shown in Table 2.

[0126] Table 2 Synthesis ratio of hyaluronic acid derivatives with different molecular weight functionalized with phenylboronic acid groups

[0127]

[0128] Example 3

[0129] [Preparation of polymer derivatives containing carboxyl groups and 1,2- or 1,3-diol / phenol groups functionalized with 3-aminophenylboronic acid groups]

[0130] The reactants listed in Table 3 were added to a polymer containing carboxyl groups and 1,2- or 1,3-diol / phenol groups in 200 mL of 0.1 M MES buffer. The pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the polymer solution containing carboxyl groups and 1,2- or 1,3-diol / phenol groups. The reaction was activated for approximately 30 minutes. 3-Aminophenylboronic acid was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis tubing with a molecular weight cutoff of 3500 Da) and the pH was maintained at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative containing carboxyl groups and 1,2- or 1,3-diol / phenol groups functionalized with 3-aminophenylboronic acid groups.

[0131] Taking the alginate derivative functionalized with 3-aminophenylboronic acid group (sample 3-2) as an example, the reaction process is shown in formula (2).

[0132]

[0133] After 1H NMR characterization, its structure can be verified and its phenylboronic acid grafting rate can be calculated, as shown in Table 3.

[0134] Table 3 Feed ratios for the synthesis of polymer derivatives functionalized with 3-aminophenylboronic acid groups containing carboxyl groups and 1,2- or 1,3-diol / phenol groups

[0135]

[0136] Example 4

[0137] [Preparation of Polymers Containing Amino Groups and 1,2- or 1,3-Diol / Phenol Groups Functionalized with 4-Carboxyphenylboronic Acid Groups]

[0138] According to the reactants listed in Table 4, 385 mg of 4-carboxyphenylboronic acid, 1400 mg of EDC, and 200 mg of NHS were dissolved in water and reacted for 30 minutes. The polymer containing amino groups and 1,2- or 1,3-diol / phenol groups was then dissolved in 200 mL of 0.1 M MES buffer at a concentration of 1% (w / v). The two were mixed, the pH of the reaction system was controlled at approximately 5.5, and the reaction was allowed to proceed at room temperature for 12 hours. After completion of the reaction, the resulting solution was dialyzed (dialysis bag with a molecular weight cutoff of 3500 Da), maintaining the pH at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative containing amino groups and 1,2- or 1,3-diol / phenol groups functionalized with 4-carboxyphenylboronic acid groups.

[0139] Taking the ethylene glycol chitosan derivative functionalized with 4-carboxyphenylboronic acid group (sample 4-1) as an example, the reaction process is shown in formula (3).

[0140]

[0141] After 1H NMR characterization, its structure can be verified and its phenylboronic acid grafting rate can be calculated, as shown in Table 4.

[0142] Table 4 Feed ratios for the synthesis of polymer derivatives functionalized with 4-carboxyphenylboronic acid groups containing amino groups and 1,2- or 1,3-diol / phenol groups

[0143]

[0144] Example 5

[0145] [Preparation of polymers containing aldehyde groups and 1,2- or 1,3-diol / phenol groups functionalized with 2-aminophenylboronic acid groups]

[0146] Dissolve 3.28 g of NaIO4 in 100 mL of deionized water. Add the NaIO4 solution dropwise to 400 mL (1.25 g / mL) of an aqueous solution of a polymer containing 1,2- or 1,3-diol / phenol groups. Stir for 24 hours at room temperature in the dark. After the reaction, add ethylene glycol in an amount equimolar to the NaIO4 to quench any unreacted NaIO4. The resulting reaction solution is dialyzed (dialysis bag with a molecular weight cutoff of 3500 Da) and freeze-dried to yield aldehyde-modified polymer derivatives containing 1,2- or 1,3-diol / phenol groups (polymers shown in Table 5).

[0147] Taking the aldehyde-modified alginic acid derivative as an example, the reaction process is shown in formula (4).

[0148]

[0149] The aldehyde content of polymer derivatives containing 1,2- or 1,3-diol / phenol groups can be calculated by 1H NMR characterization. Figure 4 As shown in Table 5 (aldehyde-modified hyaluronic acid), the three peaks at 4.8-5.2 ppm are the aldehyde peaks after oxidation of hyaluronic acid, and the single peak at 2.0 ppm is the methyl peak of hyaluronic acid itself. The aldehyde degree can be calculated according to the ratio. The aldehyde degree of each substance is shown in Table 5.

[0150] According to the reactants listed in Table 5, the aldehyde-functionalized polymer containing 1,2- or 1,3-diol / phenol groups and 2-aminophenylboronic acid were dissolved in deionized water to a total concentration of 5% (w / v). The reaction was stirred at room temperature for 24 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), and the pH of the solution was maintained at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative containing aldehyde groups and 1,2- or 1,3-diol / phenol groups functionalized with 2-aminophenylboronic acid groups.

[0151] Taking the aldehyde-modified alginate derivative functionalized with 2-aminophenylboronic acid group (sample 5-4) as an example, the reaction process is shown in formula (5).

[0152]

[0153] Table 5 Feed ratios for the synthesis of polymer derivatives functionalized with 2-aminophenylboronic acid groups containing aldehyde groups and 1,2- or 1,3-diol / phenol groups

[0154]

[0155] Example 6

[0156] [Preparation of polymer derivatives containing amino groups and 1,2- or 1,3-diol / phenol groups functionalized with aldehyde phenylboronic acid groups]

[0157] Following the reactants listed in Table 6, formaldehyde-phenylboronic acid and a polymer derivative containing amino groups and 1,2- or 1,3-diol / phenol groups were dissolved in deionized water to a total concentration of 5%. The reaction was stirred at room temperature for 24 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), maintaining a pH of 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative containing amino groups and 1,2- or 1,3-diol / phenol groups functionalized with formaldehyde-phenylboronic acid groups.

[0158] Taking the glycol chitosan derivative functionalized with aldehyde phenylboronic acid group (sample 6-1) as an example, the reaction process is shown in formula (6).

[0159]

[0160] Table 6 Synthesis ratio of polymer derivatives containing amino groups and 1,2- or 1,3-diol / phenol groups functionalized with aldehyde phenylboronic acid groups

[0161]

[0162] Example 7

[0163] [Preparation of polymer derivatives containing 1,2- or 1,3-diol / phenol groups functionalized with 4-carboxyphenylboronic acid groups]

[0164] According to the reactants listed in Table 7, 1540 mg of 4-carboxyphenylboronic acid and 1507 mg of 1,1'-carbonyldiimidazole (CDI) were dissolved in anhydrous DMSO and reacted for 0.5 h. Then, a solution of a polymer containing 1,2- or 1,3-diol / phenol groups dissolved in anhydrous DMSO was added to the reaction mixture under a nitrogen atmosphere. The reaction was allowed to proceed at room temperature for 24 hours. After completion of the reaction, the resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), maintaining the pH at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative containing 1,2- or 1,3-diol / phenol groups functionalized with 4-carboxyphenylboronic acid groups.

[0165] Taking the hyaluronic acid derivative functionalized with 4-carboxyphenylboronic acid group (sample 7-1) as an example, the reaction process is shown in formula (7).

[0166]

[0167] Table 7 Feed ratios for the synthesis of polymer derivatives functionalized with 4-carboxyphenylboronic acid groups containing 1,2- or 1,3-diol / phenol groups

[0168]

[0169] Example 8

[0170] [Polymer derivatives functionalized with phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups]

[0171] Polymer 1 was dissolved in 200 mL of 0.1 M MES buffer according to the reactants listed in Table 8. The pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the polymer solution. The reaction was activated for approximately 30 minutes. 3-Aminophenylboronic acid was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da), maintaining a pH of 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative functionalized with phenylboronic acid groups.

[0172] Polymer 2 was dissolved in 200 mL of 0.1 M MES buffer according to the reactants listed in Table 8. The pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the polymer solution. The reaction was activated for approximately 30 minutes. Dopamine was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), and the pH of the solution was maintained at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative functionalized with phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups.

[0173] Table 8 Synthesis ratio of polymer derivatives functionalized with phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups

[0174]

[0175] Preparation of Polymer Derivatives Containing Phenylboronic Acid Groups

[0176] Example 9

[0177] [Preparation of carboxyl-containing polymer derivatives functionalized with 3-aminophenylboronic acid groups]

[0178] The reactants listed in Table 9 were added, and the polymer was dissolved in 200 mL of 0.1 M MES buffer. The pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the polymer solution. The reaction was activated for approximately 30 minutes. 3-Aminophenylboronic acid or dopamine was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da), maintaining a pH of 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a carboxyl-containing polymer derivative functionalized with 3-aminophenylboronic acid groups.

[0179] Table 9 Feed ratios for the synthesis of carboxyl-containing polymer derivatives functionalized with 3-aminophenylboronic acid groups

[0180]

[0181] Example 10

[0182] [Preparation of amino-containing polymer derivatives functionalized with 4-carboxyphenylboronic acid groups]

[0183] According to the reactants listed in Table 10, 385 mg of 4-carboxyphenylboronic acid, 1400 mg of EDC, and 200 mg of NHS were dissolved in water and reacted for 30 minutes. The amino-containing polymer was then dissolved in 200 mL of 0.1 M MES buffer at a concentration of 1% (w / v). The two were mixed, the pH of the reaction system was controlled at approximately 5.5, and the reaction was allowed to proceed at room temperature for 12 hours. After completion of the reaction, the resulting reaction solution was dialyzed (dialysis bag with a molecular weight cut-off of 3500 Da), maintaining the pH at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain the amino-containing polymer functionalized with 4-carboxyphenylboronic acid groups.

[0184] Table 10 Feed ratios for the synthesis of amino-containing polymer derivatives functionalized with 4-carboxyphenylboronic acid groups

[0185]

[0186] Example 11

[0187] [Preparation of aldehyde-containing polymer derivatives functionalized with 3-aminophenylboronic acid groups]

[0188] Dissolve 3.28 g of NaIO4 in 100 mL of deionized water. Add the NaIO4 solution dropwise to 400 mL of a 12.5 mg / mL aqueous polymer solution (polymers listed in Table 11). Stir for 24 hours at room temperature in the dark. After the reaction, add ethylene glycol in an amount equimolar to the NaIO4 to quench any unreacted NaIO4. The resulting reaction solution is dialyzed (dialysis bag with a molecular weight cutoff of 3500 Da) and freeze-dried to yield the aldehyde-modified polymer derivative.

[0189] According to the reactants listed in Table 11, the aldehyde-functionalized polymer and 2-aminophenylboronic acid were dissolved in deionized water to a total concentration of 5% (w / v). The reaction was stirred at room temperature for 24 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cut-off: 3500 Da), and the pH of the solution was maintained at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymeric derivative functionalized with 2-aminophenylboronic acid groups and containing aldehyde groups.

[0190] Table 11 Feed ratios for the synthesis of aldehyde-containing polymer derivatives functionalized with 3-aminophenylboronic acid groups

[0191]

[0192] Example 12

[0193] [Preparation of amino-containing polymer derivatives functionalized with aldehyde phenylboronic acid groups]

[0194] The reactants listed in Table 11 were added: formaldehyde phenylboronic acid and an amino-containing polymer derivative were dissolved in deionized water to a total concentration of 5%. The reaction was stirred at room temperature for 24 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cut-off: 3500 Da), maintaining a pH of 5.5 during purification. After dialysis, the solution was freeze-dried to obtain an amino-containing polymer derivative functionalized with formaldehyde phenylboronic acid groups.

[0195] Table 12 Synthesis of amino-containing polymer derivatives functionalized with aldehyde phenylboronic acid groups

[0196]

[0197] Preparation of polymer derivatives containing 1,2- or 1,3-diol / phenol groups

[0198] Example 13

[0199] [Preparation of polymer derivatives functionalized with 1,2- or 1,3-diol / phenol groups]

[0200] Polymer 1 was dissolved in 200 mL of 0.1 M MES buffer according to the reactants listed in Table 13. The pH of the solution was adjusted to 5.5 using 0.1 M HCl. 1400 mg of EDC and 200 mg of NHS were then added to the polymer solution. The reaction was activated for approximately 30 minutes. Polymer 2 was then added to the reaction mixture and allowed to react at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500 Da), and the pH of the solution was maintained at 5.5 during purification. After dialysis, the solution was freeze-dried to obtain a polymer derivative functionalized with 1,2- or 1,3-diol / phenol groups.

[0201] Table 13 Feed ratios for the synthesis of polymer derivatives functionalized with 1,2- or 1,3-diol / phenol groups

[0202]

[0203] Preparation of multi-responsive hydrogels with high drug loading capacity

[0204] Example 14

[0205] [Gel formation from a single-component material (a polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups) and a hydrophilic alkaline (pKa > 8.81) drug]

[0206] The reactants listed in Table 14 were added to prepare a polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups at a concentration of 50 mg / mL in water. An appropriate amount of a hydrophilic alkaline drug was added and stirred until the drug dissolved and formed a gel to prepare a multi-stimulus-responsive drug-loaded hydrogel with a drug loading of 40%.

[0207] Table 14 Feed ratio of single-component materials and hydrophilic alkaline (pKa>8.81) drugs to form gel

[0208]

[0209] Example 15

[0210] [Gel formation from a single-component material (a polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups) and a hydrophobic alkaline (pKa > 8.81) drug]

[0211] The reactants listed in Table 15 were added to dissolve a polymer derivative (polymer) containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups in water at a concentration of 30 to 100 mg / mL. An appropriate amount of a hydrophobic drug was added and stirred to uniformly disperse the drug. The mixture was then slowly stirred until sumatriptan was completely dissolved and formed into a gel (effectively solubilizing the hydrophobic drug), thereby preparing a multi-stimulus-responsive drug-loaded hydrogel.

[0212] according to Figure 5 、 Figure 6 As shown, the drug-loaded hydrogel was successfully prepared, and the increase of drugs affected the stability of the hydrogel; BN coordination successfully increased the solubility of the drug in aqueous solution.

[0213] Taking sample 15-1 as an example, the cross-linking mechanism of the hydrogel is shown in formula (8).

[0214]

[0215] Table 15 Feed ratios of single-component materials and hydrophobic (pKa ≥ 8.81) drug molecules containing primary, secondary, and tertiary amines for gel formation

[0216]

[0217] Example 16

[0218] [Gel formation of two-component materials and hydrophilic alkaline (pKa>8.81) drugs]

[0219] The reactants listed in Table 16 were added, and the polymer derivatives functionalized with phenylboronic acid groups in Examples 9-12 and the polymer derivatives containing 1,2- or 1,3-diol / phenol groups were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophilic drug was added and stirred until the drug dissolved and formed a gel to prepare a multi-stimulus-responsive drug-loaded hydrogel, thereby obtaining samples 16-1 and 16-2.

[0220] The reactants listed in Table 16 were added, and the polymer derivatives functionalized with phenylboronic acid groups in Examples 9-12 and the polymer derivatives containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups in Example 1-8 were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophilic drug was added, and the mixture was stirred until the drug dissolved and formed a gel to prepare a multi-stimulus-responsive drug-loaded hydrogel, thereby obtaining samples 16-3 and 16-4.

[0221] The reactants listed in Table 16 were added, and the polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups in Example 1-8 and the polymer derivative containing 1,2- or 1,3-diol / phenol groups in Example 13 were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophilic drug was added, and the mixture was stirred until the drug dissolved and formed a gel to prepare a multi-stimulus-responsive drug-loaded hydrogel, thereby obtaining samples 16-5 and 16-6.

[0222] Table 16 Feed ratio of two-component materials and hydrophilic alkaline (pKa>8.81) drugs to form gel

[0223]

[0224] Example 17

[0225] [Gel formation of two-component materials and hydrophobic (pKa ≥ 8.81) drugs containing primary, secondary, and tertiary amines]

[0226] The materials listed in Table 17 were added, and the polymer derivatives functionalized with phenylboronic acid groups in Examples 9-12 and the polymer derivatives containing 1,2- or 1,3-diol / phenol groups were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophobic drug was added and stirred to uniformly disperse the drug. The solution was then slowly stirred until sumatriptan was completely dissolved and gelled (effectively achieving solubilization of the hydrophobic drug) to prepare multi-stimulus-responsive drug-loaded hydrogels, and samples 17-1 and 17-2 were obtained.

[0227] The materials listed in Table 17 were added, and the polymer derivatives functionalized with phenylboronic acid groups in Examples 9-12 and the polymer derivatives containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups in Example 1-8 were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophobic drug was added and stirred to uniformly disperse the drug. The solution was then slowly stirred until sumatriptan was completely dissolved and gelled (effectively achieving solubilization of the hydrophobic drug), to prepare multi-stimulus-responsive drug-loaded hydrogels, and samples 17-3 and 17-4 were obtained.

[0228] The materials listed in Table 17 were added, and the polymer derivative containing both phenylboronic acid groups and 1,2- or 1,3-diol / phenol groups in Example 1-8 and the polymer derivative containing 1,2- or 1,3-diol / phenol groups in Example 13 were added in a mass ratio of 1:1 to prepare an aqueous solution with a total concentration of 25 mg / mL. An appropriate amount of hydrophobic drug was added and stirred to uniformly disperse the drug. The solution was then slowly stirred until sumatriptan was completely dissolved and gelled (effectively achieving solubilization of the hydrophobic drug) to prepare multi-stimulus-responsive drug-loaded hydrogels, and samples 17-5, 17-6, and 17-7 were obtained.

[0229] Table 17 Feed ratio of two-component materials and hydrophobic alkaline (pKa>8.81) drug to form gel

[0230]

[0231] Example 18

[0232] [Studying the Gelation and Drug-Loading Mechanism of Drug-Loaded Hydrogels Using Boron Nuclear Magnetic Spectroscopy]

[0233] The 11B NMR spectra were measured at room temperature using a Varian UNITY-plus 400MNMR spectrometer. From bottom to top, they are the NMR boron spectra of phenylboronic acid (PBA), a mixture of PBA and dopamine in a molar ratio of 1:2 (PBA:Dop(1:2)), a mixture of sumatriptan and phenylboronic acid in a molar ratio of 1:2 (Sum:PBA(1:2)), and a mixture of dopamine, sumatriptan and phenylboronic acid in a molar ratio of 1:1:2 (Dop:Sum:PBA(1:1:2)). The results are as follows: Figure 7 shown.

[0234] The results showed that boron-nitrogen coordination and boronate ester bonds can coexist in the blend of ethylene glycol, sumatriptan and phenylboronic acid. The signal peak of sumatriptan at 26.2 ppm and the signal peak of dopamine at 8.3 ppm shifted to the left by 3.5 ppm at the same time, reaching 29.7 ppm and 11.8 ppm, respectively. That is, the boronate ester bonds constitute the entire hydrogel network, while the boron-nitrogen coordination plays a drug-carrying role.

[0235] Example 19

[0236] [Rheological Analysis of Drug-Loaded Hydrogel]

[0237] Rheological properties of hydrogels: The rheological properties of hydrogels were measured using a rheometer with cone-plate geometry (Antongpa MCR302, Austria).

[0238] The prepared hydrogels (samples 15-1 and 15-10) were sized to fit the size of the rotator plate and placed on the plate. An oscillatory strain amplitude sweep was performed at 25°C with a fixed frequency of 1 Hz and a strain range of 10-1 to 10-3%. The storage modulus (G′) and loss modulus (G″) were measured at 25°C and 1% strain using a sweep mode. The results are shown in Figure 2. Figure 8 shown.

[0239] The results showed that sample 15-1( Figure 8 a) and sample 15-10( Figure 8 b) The storage modulus (G′) of the hydrogel is greater than the loss modulus (G″), indicating that the hydrogel is successfully prepared and has good stability and high modulus.

[0240] Example 20

[0241] [Microscopic morphology of the prepared drug-loaded hydrogel studied by scanning electron microscopy]

[0242] Study on the micromorphology of hydrogels: Scanning electron microscopy (SEM) was used to observe the micromorphology of multi-stimulus-responsive drug-loaded hydrogels, and to observe their surface micromorphology, three-dimensional structure, pore size distribution and other characteristics.

[0243] The prepared hydrogel (sample 15-1) was freeze-dried, and the freeze-dried gel was placed on a copper plate, vacuumed and sprayed with gold, and the microscopic morphology of the multi-stimulus responsive drug-loaded hydrogel was observed by SEM.

[0244] from Figure 9 As can be seen in a and 9b, the pores of the hydrogel are uniform and about 5 μm in size, indicating that a multi-responsive hydrogel with high-capacity drug loading was successfully prepared.

[0245] Example 2 1

[0246] [Swelling and degradation of hydrogels]

[0247] The hydrogel swelling rate test was performed by placing the freeze-dried samples 15-2 and 15-7 hydrogels in a PBS solution (pH 7.2, 37°C). The hydrogels were taken out at time points of 10 min, 20 min, 30 min, 60 min, 2 h, 4 h, and 6 h, and the moisture on the surface of the hydrogels was removed with filter paper. The weight was then weighed and recorded. Three parallel hydrogel samples were set under each condition.

[0248] The swelling rate is calculated using the formula: Wherein, w0 is the weight of the hydrogel sample after freeze-drying, w t is the mass of swollen hydrogel at different time points.

[0249] The hydrogel degradation rate test was performed by immersing the freeze-dried dyed samples 15-2, 15-10, and 14-6 hydrogels in PBS solution (pH 7.2-7.4, 0.1 M) and placing them in a shaker at 37°C and 100 rpm. The samples were photographed and compared at time intervals of 10 min, 20 min, 30 min, 60 min, 2 h, 4 h, and 6 h.

[0250] like Figure 10 and Figure 11 As shown in the figure, the swelling rate of sample 15-2 is greater than that of sample 15-7. This is because the grafting rate of phenylboronic acid is high and the valence bond distribution of the hydrogel is more dense. The final swelling rate of the hydrogel is about 800%. Samples 15-2, 15-10 and 14-6 hydrogels all have a very fast degradation rate and are completely degraded within 6 hours, which is suitable for rapid drug delivery.

[0251] Example 22

[0252] [Powder X-ray Diffraction (PXRD) Analysis]

[0253] The physical state of Sum in the multi-stimuli-responsive drug-loaded hydrogel was analyzed using an X-ray diffractometer (Bruker AXS, D8 Advance, Karlsruhe, Germany), using Cu Ka line as the radiation source and operating at 40 kV voltage and 40 mA current.

[0254] The samples used for powder X-ray diffraction (PXRD) studies were measured with a scan angle range of 5-40° (2θ) with a step size of 0.02° and a scan rate of 3° / min.

[0255] Figure 12 From top to bottom are the PXRD patterns of sumatriptan (Sum), the mixture of sumatriptan and sample 1-3, sample 1-3, and sample 15-2.

[0256] Figure 13The PXRD patterns of freeze-dried samples of sumatriptan-loaded hydrogels prepared with different molar ratios of PBA groups and Sum in samples 1-3 are shown respectively.

[0257] Figure 14 From bottom to top are the freeze-dried samples of azithromycin-loaded hydrogels prepared by different molar ratios of PBA groups and azithromycin (Azi) in samples 1-3, the mixture of samples 1-3 and azithromycin in a molar ratio of 1:1.5 (physical mixture (1:1.5)) and the PXRD patterns of azithromycin (Azi).

[0258] like Figure 12 、 13 As shown in Figures 14 and 15, neither Sum nor Azi exists as crystals in the hydrogel, that is, they are not physically dispersed in the hydrogel medium. Instead, they are successfully loaded into the hydrogel via boron-nitrogen coordination. Hydrogels loaded with Sum or Azi at a drug loading of 40% show no crystalline peak, while hydrogels loaded with Sum or Azi at a drug loading greater than 40% do show a crystalline peak. Therefore, the results indicate that the maximum drug loading of Sum or Azi, i.e., hydrophobic drugs, can reach 40%.

[0259] Example 23

[0260] [Study on the pH-responsive properties of the prepared drug-loaded hydrogel]

[0261] pH stimulus responsiveness of the hydrogel: Using phenolphthalein as an indicator, 50 μl of a 0.5% phenolphthalein ethanol solution was added to 1 mL of a 5% aqueous solution of sample 1-3. 20 mg of sumatriptan was added and dispersed evenly. The mixture was then slowly stirred until the sumatriptan was completely dissolved and gelled (effectively solubilizing hydrophobic drugs). This produced a multi-stimulus responsive drug-loaded hydrogel (sample 15-2). The acid and base were adjusted with 1 M HCl and 1 M NaOH, respectively, and the changes in color and state were observed.

[0262] like Figure 15 As shown, using phenolphthalein as a pH indicator, acid-induced cleavage of the boronate bonds caused the hydrogel sample 15-2 to transition from a gel to a liquid state, with the hydrogel turning from red to white. Alkaline-induced cleavage of the boronate bonds caused the hydrogel sample 15-2 to transition from a liquid to a gel state, with the hydrogel turning from white to red. This demonstrates that multi-stimuli-responsive hydrogel materials capable of high-capacity loading of hydrophilic and hydrophobic drugs exhibit excellent pH responsiveness.

[0263] Example 24

[0264] [Application of multi-responsive hydrogel with high drug loading capacity in rat liver hemostasis]

[0265] 8-10-week-old Sprague-Dawley rats were randomly divided into groups (10 per group) based on body weight before the experiment: a) Sample 15-2 hydrogel group; b) Commercial hemostatic agent; c) Model blank group. During surgery, bleeding volume and duration were recorded for each group. Histological sections were then stained and analyzed.

[0266] like Figure 16 As shown, it can be seen that the multi-responsive hydrogel with high capacity loading of drugs of the present invention has a good hemostatic effect, and the hemostatic effect of commercial hemostatic agents is obviously slower than that of the multi-stimulus responsive hydrogel material with high capacity loading of hydrophilic / hydrophobic drugs.

[0267] Example 25

[0268] [Application of high-capacity drug-loaded multi-responsive hydrogels for temporary protection of second-degree burns in rats]

[0269] 8-10 week-old Sprague-Dawley rats were treated with second-degree burns covering 5% of their skin surface. Some rats also underwent bacterial infection. Before the experiment, rats were randomly divided into groups (10 per group) based on body weight: a) burn group; b) burn group + bacterial infection group; and c) burn group + Sample 17-1 hydrogel + bacterial infection group. After 72 hours, the burn areas of the different groups were observed for infection.

[0270] The results showed that the rats in the burn group (Group A) had some degree of infection; the burn + bacterial infection group (Group B) developed severe bacterial infection; and the rats in the experimental hydrogel-treated group (Group C) had virtually no infection on their burn surfaces. The hydrogel in the experimental group was then washed off with a lysine solution, providing temporary protection for the burn surface.

[0271] These results indicate that the multi-responsive hydrogel with high drug loading capacity of the present invention has a good protective effect on the tissue of the burn surface.

[0272] Example 26

[0273] [Application of high-capacity drug-loaded multi-responsive hydrogels in the sealing and care of dorsal wounds in SD rats]

[0274] 8-10 week old SD rats were used and each rat received abdominal trauma to establish a traumatic bleeding model. Before the experiment, the rats were randomly divided into 10 groups according to body weight: a: sample 14-4 hydrogel group; b: model blank group.

[0275] During surgery, the hydrogel precursor was applied to the bleeding site of the wound. As the hydrogel gradually cross-linked, the wound was sealed. The amount and duration of bleeding were recorded for each group. Excess hydrogel was covered with gauze soaked in a lysine solution and washed away after half an hour. Histological sections were then stained to analyze the hydrogel's adhesion to the wound surface and the inflammatory response.

[0276] The results showed that the multi-responsive hydrogel with high drug loading capacity of the present invention had good hemostatic and sealing effects on wounds. The analysis results of tissue staining of sections also showed that the hydrogel could adhere well to the tissue and achieve a sealing effect.

[0277] Example 27

[0278] [Application of high-capacity drug-loaded multi-responsive hydrogels in repairing rabbit radial bone defects]

[0279] New Zealand male white rabbits underwent surgical treatment of the radial bone to establish a bone defect model. Before the experiment, the rabbits were randomly divided into 10 groups based on body weight: a) Sample 16-1 hydrogel group; b) Sample 16-1 hydrogel + cell activation factor (bone morphogenetic protein-2, abbreviated as BMP-2) group; and c) blank group.

[0280] During the surgery, the hydrogel precursor solution was mixed evenly to form a plastic gel, which was then applied to the defect. As the hydrogel gradually cross-linked, it transformed from a plastic gel to an elastic gel, providing mechanical support for the defect. One month after the surgery, the rabbits were sacrificed via intravenous injection of air, and samples were collected to evaluate the repair results.

[0281] The results showed that the hydrogel-treated defect sites had a moderate repair effect, with complete bone repair achieved in group B, while no bone repair was observed in the blank group. Therefore, the high-capacity, drug-loaded, multi-responsive hydrogels of the present invention demonstrated a promising bone repair effect.

[0282] Example 28

[0283] [High-capacity drug-loaded multi-responsive hydrogel for vertebroplasty in sheep]

[0284] Sheep were used as animal models. Each sheep underwent vertebral surgery to establish a vertebral defect model. Before the experiment, the sheep were randomly divided into three groups based on body weight: a) Sample 16-1 hydrogel group; b) Sample 16-1 hydrogel + BMP-2 group; and c) Model blank group.

[0285] During the surgery, the hydrogel precursor solution was mixed evenly to form a plastic gel, which was then applied to the defect. As the hydrogel gradually cross-linked, it transformed from a plastic gel to an elastic gel, providing mechanical support for the defect. Three months after the surgery, the sheep were sacrificed via intravenous injection of air, and samples were collected to evaluate the repair results.

[0286] The results showed that the hydrogel-treated vertebrae showed a moderate repair effect, with complete repair achieved in group B, while no repair was observed in the blank control group. Therefore, the high-capacity, drug-loaded, multi-responsive hydrogels of the present invention demonstrate promising results for vertebroplasty.

[0287] Example 29

[0288] [Multiple responsive hydrogels with high drug loading capacity for 3D printing bioinks]

[0289] The multi-responsive hydrogel with high-capacity drug loading of the present invention is conducive to the extrusion process of the gel in the syringe and can be applied to the fused deposition printing process. At the same time, the printed hydrogel can be washed away with a solution. It is an ideal sacrificial material and can solve many printing technology problems of complex structures without support, providing a more ideal material and method for the development of hydrogel material 3D printing technology.

[0290] Sample 14-4 hydrogel was used for printing, and its printability was quickly evaluated using a 0.5 mm diameter nozzle attached to a 10 mL syringe. Once the hydrogel properties were deemed suitable for printing, the test continued with the printing device. The hydrogel was inserted into a 10 mL syringe, which was then placed in a speed mixer for approximately 5 minutes to remove bubbles and ensure sample homogeneity. The hydrogel was then dispensed through a pipette tip based on air pressure control. The 3D structure was printed on a plastic substrate in a layer-by-layer method using a CAD-controlled xyz motion control system.

[0291] The results show that the multi-responsive hydrogel with high drug loading capacity of the present invention can be applied to 3D printing bio-ink.

[0292] Example 30

[0293] [Multi-responsive hydrogels with high drug loading capacity for drug encapsulation and release]

[0294] Hydrogels are cross-linked polymer networks. Because they are mostly composed of water, they possess excellent biocompatibility, making them particularly suitable as carriers for drugs and bioactive macromolecules. Drugs or bioactive macromolecules encapsulated in hydrogels achieve sustained release through molecular diffusion and material degradation.

[0295] The in vitro drug release evaluation of sample 15-2 hydrogel was performed.

[0296] The drug release in the solution was analyzed by UV test to evaluate the drug release effect of the material. Figure 17 As shown in the figure, in the environment of pH 5.5 PBS and 0.01M H2O2, Sum is released most rapidly; in the environment of pH 5.5 PBS alone, the Sum release rate is similar to that in the environment of 0.01M H2O2 alone; in the environment of pH 7.4 PBS alone, the Sum release rate is the slowest.

[0297] The experimental results show that the multi-responsive hydrogel with high drug loading capacity of the present invention can achieve sustained release of drugs, and the pH and ROS responsiveness of the boronate bond can further enhance the drug release rate.

[0298] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing a multi-responsive hydrogel with high drug loading capacity, characterized in that: The following steps are involved: The first substance and the drug are mixed in a biocompatible medium to obtain a multi-responsive hydrogel with high drug loading capacity; The first substance comprises a first component, which is a hyaluronic acid derivative functionalized with a phenylboronic acid group, and has the following structural formula: ； Wherein, n is the degree of polymerization, and n≥2; The drug is a hydrophobic drug containing at least one of a primary amine, a secondary amine, and a tertiary amine group, including sumatriptan or azithromycin; The boron-nitrogen coordination effect between the phenylboronic acid group and the hydrophobic drug promotes the formation of a boronate ester bond between the phenylboronic acid group and the 1,2- or 1,3-diol / phenol group, thereby promoting the cross-linking of the hydrogel precursor into a gel and simultaneously achieving the loading of the hydrophobic drug molecules.

2. The method for preparing a multi-responsive hydrogel with high drug loading capacity according to claim 1, characterized in that: The pKa of the drug is ≥8.

81.

3. A multi-responsive hydrogel with high drug loading capacity prepared by the preparation method according to any one of claims 1 to 2.

4. Use of the high-capacity drug-loaded multi-responsive hydrogel according to claim 3 in the preparation of hemostatic drugs or materials, drug-loaded burn and scald dressings, chronic wound care drugs or materials, bone repair drugs or materials, tissue engineering scaffold materials, and 3D printing bio-inks.

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