Self-assembling nanoformulations based on hyaluronate-fatty acid conjugate prodrugs

Abstract

The application discloses a kind of self-assembled nano-preparations based on hyaluronic acid-fatty acid coupling prodrug, the self-assembled nano-preparations use hyaluronic acid-fatty acid coupling prodrug as active ingredient, the hyaluronic acid-fatty acid coupling prodrug has structure shown in formula I, wherein, R is saturated or unsaturated fatty acyl;N=1~900, m=0~899.The application also discloses the preparation method of the above-mentioned self-assembled nano-preparations, and the application of the above-mentioned self-assembled nano-preparations in preparing anti-colitis drugs.The application avoids using pharmaceutical adjuvant, can avoid the allergic reaction and toxicity caused by adjuvant, has good biocompatibility and can be biodegraded, has good clinical conversion prospect.And it can realize intestinal inflammatory site and M1 macrophage target enrichment, eliminate excess ROS while inhibiting inflammatory response, has better anti-intestinal inflammation efficacy compared with clinical 5-amino salicylic acid, dexamethasone.

Description

Technical Field

[0001] This invention belongs to the field of anti-enteritis drugs, specifically relating to a self-assembled nano-formulation based on hyaluronic acid-fatty acid conjugated prodrug. Background Technology

[0002] The development and progression of inflammatory bowel disease (IBD) are closely related to innate immune dysregulation, with macrophage polarization imbalance considered a key driving factor. Studies have shown that excessive activation of M1 macrophages can produce excessive reactive oxygen species (ROS) and drive the NF-κB pathway to induce the secretion of large amounts of pro-inflammatory factors, leading to intestinal mucosal damage and immune microenvironment dysregulation. While existing anti-inflammatory drugs such as 5-aminosalicylic acids, glucocorticoids, immunomodulators, and biologics can alleviate symptoms, they still have limitations in lesion targeting, retention, and safety, affecting long-term efficacy. Therefore, improving the lesion targeting of anti-inflammatory drugs and achieving targeted and controlled drug release based on the characteristics of the inflammatory site microenvironment, thereby inhibiting M1 macrophage function, can effectively improve the treatment efficacy of IBD.

[0003] Hyaluronic acid (HA), as a natural polysaccharide, possesses high stability, safety, low toxicity, hydrophilicity, ease of chemical modification, and biodegradability. It can specifically bind to the CD44 receptor highly expressed on the surface of pro-inflammatory M1 macrophages and has been widely used in the treatment of enteritis and targeted delivery. For example, Reference 1 (Choi KY, ChungH, Min KH, et al. Self-assembled hyaluronic acid nanoparticles for active tumor targeting[J], Biomaterials, 2010, 31(1):106-114) discloses a novel micellar drug delivery system based on hyaluronic acid derivatives. Cholanic acid is chemically linked to the hyaluronic acid backbone to form an amphiphilic hyaluronic acid derivative, which can self-assemble into micelles in aqueous solution. By using this carrier to encapsulate drugs, the treatment of colon cancer has been achieved.

[0004] Current research primarily focuses on modifying hyaluronic acid with anti-inflammatory or antioxidant components (non-clinically used anti-inflammatory drugs) to improve therapeutic efficacy. For example, Reference 2 (Lee, Y., Sugihara, K., Gillilland, MG et al. Hyaluronic acid–bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome and immune responses in colitis, Nat. Mater., 2020, 19, 118-126) discloses the use of hyaluronic acid-bilirubin nanomedicine to target dysregulated intestinal barrier, microbiome and immune responses in colitis, achieving anti-colitis efficacy through the preparation of self-assembled nanoformulations via covalent coupling of bilirubin. However, the grafting rate of these covalent modifications is generally low, requiring increased dosage and frequency of administration to achieve better anti-inflammatory effects, thus posing safety challenges.

[0005] Therefore, in order to reduce the toxic side effects caused by high-dose and high-frequency administration, improve the targeting of lesions, and prolong the action time of anti-inflammatory components at the site of inflammation, it is necessary to develop efficient modification schemes for hyaluronic acid and prepare corresponding nano-formulations. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention provides a self-assembled nanoformulation based on hyaluronic acid-fatty acid conjugated prodrugs.

[0007] A self-assembled nanoformulation based on a hyaluronic acid-fatty acid conjugated prodrug, wherein the self-assembled nanoformulation uses the hyaluronic acid-fatty acid conjugated prodrug as the active ingredient, and the structure of the hyaluronic acid-fatty acid conjugated prodrug is shown in Formula I:

[0008]

[0009] Formula I

[0010] Where R is a saturated or unsaturated fatty acyl group; n=1~900, m=0~899, n+m≤1800.

[0011] Preferably, n = 139~726, 25 ≤ n + m ≤ 1800.

[0012] Preferably, n=262~726, 25≤n+m≤1800; n=139~422, 25≤n+m≤1800; n=262~422, 25≤n+m≤1800; n=422~726, 25≤n+m≤1800; n=405~675, 25≤n+m≤1800; n=405~422, 25≤n+m≤1800.

[0013] More preferably, n=422, n+m=844.

[0014] Preferably, R is a saturated C1~C24 alkyl acyl group; or, R is a C1~C24 unsaturated fatty acyl group with 1~6 double bonds.

[0015] More preferably, R is a saturated C1~C18 alkyl group; R is a saturated C1~C7 alkyl group; R is a saturated C7~C18 alkyl group.

[0016] More preferably, R is a C1~C18 unsaturated fatty acyl group with one double bond; R is a C1~C16 unsaturated fatty acyl group with one double bond; R is a C1~C18 unsaturated fatty acyl group with two double bonds; R is a C1~C18 unsaturated fatty acyl group with three double bonds; R is a C1~C20 unsaturated fatty acyl group with four double bonds; R is a C1~C22 unsaturated fatty acyl group with six double bonds.

[0017] Preferably, R is selected from acetyl, n-heptyl, stearoyl, tetracosyl, oleyl, ricinoleyl, palmitoyl, linoleyl, linolenic acid, linolenic acid, arachidonic acid, eicosapentaenoyl, and docosahexaenoyl.

[0018] A method for preparing a self-assembled nanoformulation based on a hyaluronic acid-fatty acid conjugated prodrug includes the following steps:

[0019] Step 1: Under the action of a condensing agent, N-hydroxysuccinimide undergoes an esterification reaction with the saturated or unsaturated fatty acid corresponding to R to obtain a carboxyl-activated fatty acid product.

[0020] Step 2: Under the action of the first catalyst, the carboxyl-activated fatty acid product undergoes a condensation reaction with dopamine hydrochloride to obtain a dopamine-fatty acid coupling prodrug.

[0021] Step 3: Under the action of a second catalyst, hyaluronic acid undergoes an affinity substitution reaction with 4-(bromomethyl)phenylboronic acid to obtain a hyaluronic acid-phenylboronic acid prodrug;

[0022] Step 4: The dopamine-fatty acid conjugated prodrug is dissolved in dimethyl sulfoxide and then ultrasonically injected into an aqueous solution containing hyaluronic acid-phenylboronic acid prodrug. Dialysis is then performed to obtain a self-assembled nano-formulation.

[0023] Preferably, in step 1, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; in step 2, the catalyst is N,N-diisopropylethylamine; and in step 3, the catalyst is cesium carbonate.

[0024] More preferably, the molar ratio of the condensing agent, N-hydroxysuccinimide, and saturated or unsaturated fatty acid in step 1 is (1~2):(1~1.5):1.

[0025] Preferably, in step 1, the saturated fatty acid has the following structure: Wherein, n=0~24; the unsaturated fatty acids are ω-9, ω-6, ω-3 series unsaturated fatty acids.

[0026] More preferably, the saturated or unsaturated fatty acid is selected from one or more of acetic acid, n-heptanoic acid, stearic acid, tetracosanoic acid, oleic acid, ricinoleic acid, palmitoleic acid, linoleic acid, linolenic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid.

[0027] More preferably, the molar ratio of the catalyst, the carboxyl-activated fatty acid product, and dopamine hydrochloride in step 2 is (2~2.5):(1~2):1.

[0028] More preferably, the molar ratio of the catalyst, 4-(bromomethyl)phenylboronic acid, and hyaluronic acid in step 3 is (2000~3000):(4000~6000):1.

[0029] More preferably, the molar ratio of the dopamine-fatty acid prodrug to the phenylboronic acid portion of the hyaluronic acid-phenylboronic acid prodrug in step 4 is (0.001~1):1.

[0030] More preferably, the molar ratio of the dopamine-fatty acid prodrug to the phenylboronic acid portion of the hyaluronic acid-phenylboronic acid prodrug is 1:1.

[0031] Preferably, the reaction solvent in step 1 is dichloromethane or chloroform; the reaction solvent in step 2 is N,N-dimethylformamide; the reaction solvent in step 3 is dimethyl sulfoxide; and the reaction solvent in step 4 is a mixed solution of dimethyl sulfoxide and water, wherein the volume ratio of dimethyl sulfoxide to water is 1:9.

[0032] Preferably, the reaction temperature of step 1 is 40~50℃ and the reaction time is 2~4 hours; the reaction temperature of step 2 is room temperature and the reaction time is 4~8 hours; the reaction temperature of step 3 is 75℃ and the reaction time is 12~24 hours; and the reaction temperature of step 4 is room temperature and the reaction time is 0.2~1 hour.

[0033] Preferably, the concentration of the hyaluronic acid-phenylboronic acid prodrug in water is 0.25~5 mg / mL, and the concentration of the dopamine-fatty acid prodrug in dimethyl sulfoxide is 0.1~11 mg / mL.

[0034] On the other hand, the present invention also provides the application of the self-assembled nanoformulation based on hyaluronic acid-fatty acid conjugated prodrug in the preparation of anticolitis drugs.

[0035] Preferably, the colitis model includes an acute colitis model induced by dextran sulfate sodium salt.

[0036] To better leverage the advantages of nanotechnology for enhanced efficacy and reduced toxicity, this invention covalently couples hyaluronic acid with saturated or unsaturated fatty acids using ROS-responsive borate ester bonds to construct self-assembled nano-formulations. The resulting hyaluronic acid-fatty acid self-assembled nano-formulations target and accumulate at inflammatory sites and are selectively endocytosed by M1 macrophages, efficiently eliminating intracellular ROS and inhibiting the NF-κB pathway, thus restoring intestinal immune microenvironment homeostasis. Compared with clinical 5-aminosalicylic acid and dexamethasone, it exhibits superior anti-enteritis effects.

[0037] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0038] The hyaluronic acid and saturated or unsaturated fatty acids used in this invention are both substances required by the human body, biodegradable and biocompatible; in addition, the phenylboronic acid structure is the core functional unit of the boron neutron capture therapy (BNCT) drug Steboronine®, so the hyaluronic acid-fatty acid prodrug self-assembled nanoformulation developed in this invention has good prospects for clinical translation.

[0039] This invention uses a saturated or unsaturated fatty acid modification strategy to endow the aqueous phase of hyaluronic acid pre-drug with self-assembly ability, avoiding the use of pharmaceutical excipients and thus better avoiding allergic reactions and toxicity caused by excipients.

[0040] The self-assembled nano-formulation based on hyaluronic acid-fatty acid conjugated prodrug prepared in this invention can be targeted and enriched at the site of inflammation and can be selectively internalized by M1 macrophages, achieving efficient co-delivery of multiple anti-inflammatory components to target cells, thereby enhancing efficacy and reducing toxicity.

[0041] The phenylboronic acid ester bond in the hyaluronic acid-fatty acid prodrug synthesized in this invention is ROS responsive. While eliminating excessive ROS in inflammatory sites and M1 macrophages, it can achieve targeted and controlled release of drugs at the target site or in the target cells, further ensuring the in vivo safety of the hyaluronic acid-fatty acid prodrug self-assembled nanoformulation.

[0042] The self-assembled nano-formulation based on hyaluronic acid-fatty acid conjugated prodrug constructed in this invention can exert a synergistic effect and has a better anti-inflammatory effect compared with clinical 5-aminosalicylic acid and dexamethasone. Attached Figure Description

[0043] Figure 1 The synthetic routes for products 1-26 in Examples 1-26 are shown below.

[0044] Figure 2 This is the synthetic route for the HA-PBA conjugated prodrug in Example 27.

[0045] Figure 3 The synthetic routes for products 28-34 in Examples 28-34 are shown below.

[0046] Figure 4 The synthetic routes for products 35-40 in Examples 35-40 are shown below.

[0047] Figure 5 The NMR spectrum is shown for the OA-NHS conjugated prodrug in Example 6.

[0048] Figure 6 The NMR spectrum of the LA-NHS conjugated prodrug in Example 8 is shown.

[0049] Figure 7 The NMR spectrum of the LNA-NHS conjugate prodrug in Example 9 is shown.

[0050] Figure 8 The NMR spectrum of the OA-DA conjugated prodrug in Example 19 is shown.

[0051] Figure 9 The NMR spectrum of the LA-DA conjugated prodrug in Example 21 is shown.

[0052] Figure 10 The NMR spectrum of the LNA-DA conjugate prodrug in Example 22 is shown.

[0053] Figure 11 This is the mass spectrum of the OA-DA conjugated prodrug in Example 19.

[0054] Figure 12 This is the mass spectrum of the LA-DA conjugated prodrug in Example 21.

[0055] Figure 13 This is the mass spectrum of the LNA-DA conjugated prodrug in Example 22.

[0056] Figures 14-17 The NMR spectra of HA-PBA conjugate prodrugs with different linkage rates in Example 27 are shown.

[0057] Figure 18 The infrared spectra of the conjugated prodrugs in Examples 27 and 33 are shown.

[0058] Figure 19 The particle size distribution of the nano-formulations in Examples 33, 35, and 36 is shown.

[0059] Figure 20 Transmission electron microscopy of the nano-formulations in Examples 33, 35, and 36.

[0060] Figure 21 The results are the determination results of the binding constants of OA-DA and HP-PBA in Example 19.

[0061] Figure 22 This is a diagram from the in vitro ROS elimination experiment of Example 33.

[0062] Figure 23 This is a diagram from Example 33, showing the experiment to eliminate intracellular ROS in M1 macrophages.

[0063] Figure 24 Example 33 compares the experimental protocol and colon length and spleen weight diagrams of clinical 5-aminosalicylic acid and dexamethasone.

[0064] Figure 25 Example 33 shows intestinal H&E staining images comparing clinical 5-aminosalicylic acid and dexamethasone.

[0065] Figure 26 This is an experimental diagram showing the determination of MPO, TNF-α and IL-6 levels in the intestinal inflammatory sites of mice after treatment in Example 33. Detailed Implementation

[0066] All raw materials used in this invention are commercially available.

[0067] Example 1: Synthesis of Ac-NHS conjugated prodrug, the specific reaction route is as follows: Figure 1 As shown: N-hydroxysuccinimide (1054.0 mg, 9.16 mmol) and glacial acetic acid (500 mg, 8.33 mmol) were added sequentially to a 100 mL round-bottom flask and dissolved in 5 mL of anhydrous dichloromethane. Then, EDC (1541.4 mg, 10.00 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 3 hours, and the reaction was observed using thin-layer chromatography. When the reaction was essentially complete, the reaction solution was cooled and washed with 5% citric acid solution, saturated sodium bicarbonate, and saturated saline solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and the solvent removed under reduced pressure. After purification by column chromatography (pure DCM), product 1 (1088.4 mg, yield 83.2%, [C6H8NO4]) was obtained. + [M+H] + =158.0448), the structural formula is as follows:

[0068] .

[0069] Example 2: The synthesis process of the Hep-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (486.3 mg, 4.23 mmol) and n-heptanoic acid (500 mg, 3.84 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (710.6 mg, 4.61 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0070] Product 2 (694.2 mg, yield 79.6%), [C 11 H 18 NO4] + [M+H] + =228.1230), the structural formula is as follows:

[0071] .

[0072] Example 3: The synthesis process of the Ste-NHS-coupled prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (445.1 mg, 3.87 mmol) and stearic acid (1000 mg, 3.52 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (651.3 mg, 4.22 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0073] Product 3 (1183.8 mg, yield 88.2%), [C 22 H 40 NO4] + [M+H] + =382.2952), the structural formula is as follows:

[0074] .

[0075] Example 4: The synthesis process of the t-cos-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (343.5 mg, 2.99 mmol) and tetracosanoic acid (1000 mg, 2.71 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (501.2 mg, 3.25 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0076] Product 4 (942.1 mg, yield 74.7%), [C28 H 52 NO4] + [M+H] + =466.3891), the structural formula is as follows:

[0077] .

[0078] Example 5: The synthesis process of the PA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (497.3 mg, 4.32 mmol) and palmitoleic acid (1000 mg, 3.93 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (727.2 mg, 4.72 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0079] Product 5 (1260.3 mg, yield 91.3%), [C 20 H 34 NO4] + [M+H] + =352.2482), the structural formula is as follows:

[0080] .

[0081] Example 6: The synthesis process of the OA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (448.3 mg, 3.90 mmol) and oleic acid (1000 mg, 3.54 mmol) were dissolved in 2 mL of anhydrous dichloromethane, and then EDC (655.6 mg, 4.25 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0082] Product 6 (1240.7 mg, yield 92.4%) has the following structural formula:

[0083] ;

[0084] Product 6 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 5 As shown: 1 H NMR (400 MHz, CDCl3): δ5.39-5.32 (m, 2H), 2.83 (s, 4H), 2.60 (t, J = 8.0, 2H), 2.02 (d, J= 4.0,4H), 1.78-1.71 (m, 2H), 1.42-1.27 (m, 20H), 0.88 (t, J = 8.0, 3H).

[0085] Example 7: The synthesis process of the RA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (424.3 mg, 3.69 mmol) and ricinoleic acid (1000 mg, 3.35 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (620.4 mg, 4.02 mmol) was added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0086] Product 7 (1089.4 mg, yield 82.2%), [C 22 H 38 NO5] + [M+H] + =396.2744), the structural formula is as follows:

[0087] .

[0088] Example 8: The synthesis process of the LA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (451.5 mg, 3.93 mmol) and linoleic acid (1000 mg, 3.57 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (660.3 mg, 4.28 mmol) was rapidly added dropwise. The mixture was stirred at 43 °C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0089] Product 8 (1130.2 mg, yield 83.9%) has the following structural formula:

[0090] ;

[0091] Product 8 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 6 As shown: 1 H NMR (400 MHz, CDCl3): δ5.40-5.28 (m, 4H), 2.82 (s, 4H), 2.76 (t, J = 8.0, 2H), 2.59 (t, J= 8.0,2H), 2.06-2.01 (m, 4H), 1.77-1.69 (m, 2H), 1.41-1.24 (m, 14H), 0.88 (t, J =8.0, 3H).

[0092] Example 9: The synthesis process of the LNA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (454.8 mg, 3.95 mmol) and linolenic acid (1000 mg, 3.59 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (665.1 mg, 4.31 mmol) was rapidly added dropwise. The mixture was stirred at 43 °C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0093] Product 9 (1037.2 mg, yield 77.0%) has the following structural formula:

[0094] ;

[0095] Product 9 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 7 As shown: 1 H NMR (400 MHz, CDCl3): δ5.42-5.26 (m, 6H), 2.82-2.74 (m, 8H), 2.59 (t, J = 8.0, 2H), 2.10-2.02 (m,4H), 1.77-1.69 (m, 3H), 1.36-1.29 (m, 8H), 0.98-0.87 (m, 3H).

[0096] Example 10: The synthesis process of the GLA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (454.8 mg, 3.95 mmol) and linolenic acid (1000 mg, 3.59 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (665.1 mg, 4.31 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0097] Product 10 (1103.5 mg, yield 81.8%), [C 22 H 34 NO4] + [M+H] + =376.2482), the structural formula is as follows:

[0098] .

[0099] Example 11: The synthesis process of the AA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (415.9 mg, 3.62 mmol) and arachidonic acid (1000 mg, 3.29 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (608.2 mg, 3.94 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0100] Product 11 (1066.3 mg, yield 80.8%), [C 24 H 36 NO4] + [M+H] + =402.2639), the structural formula is as follows:

[0101] .

[0102] Example 12: The synthesis process of the EPA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference is that N-hydroxysuccinimide (418.7 mg, 3.64 mmol) and eicosapentaenoic acid (1000 mg, 3.31 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (612.3 mg, 3.97 mmol) was rapidly added dropwise. The mixture was stirred at 43 °C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0103] Product 12 (1088.9 mg, yield 82.4%), [C 24 H 34 NO4] + [M+H] + =400.2482), the structural formula is as follows:

[0104] .

[0105] Example 13: The synthesis process of the DHA-NHS conjugated prodrug is the same as in Example 1, and the specific reaction route is as follows: Figure 1 As shown, the difference lies in the following: N-hydroxysuccinimide (385.5 mg, 3.35 mmol) and cis-4,7,10,13,16,19-docosahexaenoic acid (1000 mg, 3.05 mmol) were dissolved in 5 mL of anhydrous dichloromethane, and then EDC (563.7 mg, 3.66 mmol) was rapidly added dropwise. The mixture was stirred at 43°C for 4 hours, and the reaction was observed using thin-layer chromatography.

[0106] Product 13 (1102.6 mg, yield 85.1%), [C 26 H 36 NO4] + [M+H]+=426.2639), the structural formula is as follows:

[0107] .

[0108] Example 14: Synthesis of Ac-DA conjugated prodrug, the specific reaction route is as follows: Figure 1 As shown:

[0109] In a 100 mL round-bottom flask, Ac-NHS (300 mg, 1.91 mmol) and dopamine hydrochloride (301.0 mg, 1.59 mmol) were added sequentially and dissolved in 4 mL of anhydrous N,N-dimethylformamide. Then, DIEA (411.0 mg, 3.18 mmol) was rapidly added dropwise. The mixture was stirred at room temperature for 6 hours under nitrogen protection, and the reaction was observed using thin-layer chromatography. When the reaction was essentially complete, the reaction solution was cooled and washed with 5% citric acid solution and saturated saline solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and the solvent was removed under reduced pressure. After separation and purification by column chromatography (DCM:MeOH = 20:1), product 14 (250.6 mg, yield 80.8%) was obtained. 10 H 14 NO3] + [M+H] + =196.0968), the structural formula is as follows:

[0110] .

[0111] Example 15: The synthesis process of the Hep-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: Hep-NHS (300 mg, 1.32 mmol) and dopamine hydrochloride (208.1 mg, 1.10 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (284.3 mg, 2.20 mmol) was added dropwise.

[0112] Product 15 (236.5 mg, yield 81.1%), [C 15 H 24 NO3] + [M+H] + =266.1751), the structural formula is as follows:

[0113] .

[0114] Example 16: The synthesis process of the Ste-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: Ste-NHS (300 mg, 0.79 mmol) and dopamine hydrochloride (124.0 mg, 0.66 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (170.6 mg, 1.32 mmol) was added dropwise.

[0115] Product 16 (240.6 mg, yield 86.9%), [C 26 H 46 NO3] + [M+H] + =420.3472), the structural formula is as follows:

[0116] .

[0117] Example 17: The synthesis process of the t-cos-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is that t-cos-NHS (300 mg, 0.65 mmol) and dopamine hydrochloride (101.6 mg, 0.54 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (138.9 mg, 1.07 mmol) was added dropwise.

[0118] Product 17 (232.6 mg, yield 85.6%), [C 32 H 58 NO3] + [M+H] + =504.4411), the structural formula is as follows:

[0119] .

[0120] Example 18: The synthesis process of the PA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: PA-NHS (300 mg, 0.85 mmol) and dopamine hydrochloride (134.6 mg, 0.71 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (183.5 mg, 1.42 mmol) was added dropwise.

[0121] Product 18 (239.2 mg, yield 86.5%), [C 24 H 40 NO3] + [M+H] +=390.3003), the structural formula is as follows:

[0122] .

[0123] Example 19: The synthesis process of the OA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: OA-NHS (300 mg, 0.79 mmol) and dopamine hydrochloride (124.9 mg, 0.66 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (170.3 mg, 1.32 mmol) was added dropwise.

[0124] Product 19 (257.2 mg, yield 93.5%), [C 26 H 44 NO3] + [M+H] + =418.3309, mass spectrum as shown Figure 11 As shown), the structural formula is as follows:

[0125] ;

[0126] Product 19 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 8 As shown: 1 H NMR (400 MHz, DMSO-d6): δ 8.68 (d, J = 40.0, 2H), 7.78 (t, J = 4.0, 1H), 6.62-6.40 (m, 3H), 5.32(t, J = 8.0, 2H), 3.17-3.12 (m, 2H), 2.03-1.96 (m, 6H), 1.45 (t, J = 8.0,2H), 1.24 (s, 22H), 0.85 (t, J = 8.0, 3H).

[0127] Example 20: The synthesis process of RA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: RA-NHS (300 mg, 0.76 mmol) and dopamine hydrochloride (119.6 mg, 0.63 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (163.5 mg, 1.27 mmol) was added dropwise.

[0128] Product 20 (236.9 mg, yield 86.4%), [C 26 H 44 NO4] + [M+H] + =434.3265), the structural formula is as follows:

[0129] .

[0130] Example 21: The synthesis process of the LA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: LA-NHS (300 mg, 0.80 mmol) and dopamine hydrochloride (125.3 mg, 0.66 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (171.3 mg, 1.32 mmol) was added dropwise.

[0131] Product 21 (260.6 mg, yield 94.7%), [C 26 H 42 NO3] + [M+H] + =416.3159, mass spectrum as shown Figure 12 As shown), the structural formula is as follows:

[0132] ;

[0133] Product 21 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 9 As shown: 1 H NMR (400 MHz, DMSO-d6): δ 8.66 (d, J = 40.0, 2H), 7.77 (t, J = 4.0, 1H), 6.62-6.40 (m, 3H), 5.38-5.27 (m, 4H), 3.18-3.12 (m, 2H), 2.73 (t, J = 8.0, 2H), 2.01 (t, J = 4.0,6H), 1.46 (t, J =8.0, 2H), 1.35-1.24 (m, 16H), 0.86 (t, J = 8.0, 3H).

[0134] Example 22: The synthesis process of the LNA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1As shown, the difference is: LNA-NHS (300 mg, 0.80 mmol) and dopamine hydrochloride (125.9 mg, 0.67 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (172.2 mg, 1.33 mmol) was added dropwise.

[0135] Product 22 (262.6 mg, yield 95.4%), [C 26 H 40 NO3] + [M+H] + =414.2997, mass spectrum as shown Figure 13 As shown), the structural formula is as follows:

[0136] ;

[0137] Product 22 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figure 10 As shown: 1 H NMR (400 MHz, DMSO-d6): δ 8.68 (d, J = 40.0, 2H), 7.78 (t, J = 4.0, 1H), 6.62-6.40 (m, 3H), 5.39-5.24 (m, 6H), 3.17-3.12 (m, 2H), 2.79-2.72 (m, 4H), 2.07-2.00 (m, 6H), 1.49-1.42 (m, 2H), 1.32-1.19 (m, 10H), 0.94-0.84 (m, 3H).

[0138] Example 23: The synthesis process of the GLA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: GLA-NHS (300 mg, 0.80 mmol) and dopamine hydrochloride (126.0 mg, 0.67 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (173.2 mg, 1.34 mmol) was added dropwise.

[0139] Product 23 (226.3 mg, yield 81.7%), [C 26 H 40 NO3] + [M+H] + =414.3003), the structural formula is as follows:

[0140] .

[0141] Example 24: The synthesis process of the AA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: AA-NHS (300 mg, 0.75 mmol) and dopamine hydrochloride (117.8 mg, 0.62 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (160.3 mg, 1.24 mmol) was added dropwise.

[0142] Product 24 (230.1 mg, yield 84.5%), [C 28 H 42 NO3] + [M+H] + =440.3159), the structural formula is as follows:

[0143] .

[0144] Example 25: The synthesis process of EPA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: EPA-NHS (300 mg, 0.75 mmol) and dopamine hydrochloride (118.4 mg, 0.63 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (162.8 mg, 1.26 mmol) was added dropwise.

[0145] Product 25 (230.9 mg, yield 83.8%), [C 28 H 40 NO3] + [M+H] + =438.3003), the structure is as follows:

[0146] .

[0147] Example 26: The synthesis process of the DHA-DA conjugated prodrug is the same as in Example 14, and the specific reaction route is as follows: Figure 1 As shown, the difference is: DHA-NHS (300 mg, 0.71 mmol) and dopamine hydrochloride (111.1 mg, 0.59 mmol) were added, dissolved in 4 mL of anhydrous N,N-dimethylformamide, and then DIEA (152.5 mg, 1.18 mmol) was added dropwise.

[0148] Product 26 (226.5 mg, yield 82.9%), [C 30 H 42 NO3] + [M+H] +=464.3159), the structural formula is as follows:

[0149] .

[0150] Example 27: Synthesis of HA-PBA-conjugated prodrug, the specific reaction route is as follows: Figure 2 As shown:

[0151] In a 100 mL round-bottom flask, HA (100 mg, 0.0003125 mmol), 4-(bromomethyl)phenylboronic acid (340 mg, 1.5825 mmol), and cesium carbonate (Cs₂CO₃, 257.8 mg, 0.7913 mmol) were added sequentially and dissolved in 25 mL of anhydrous dimethyl sulfoxide. The mixture was stirred at 75 °C for 24 hours, and the reaction was observed using thin-layer chromatography. When the reaction was essentially complete, the reaction solution was cooled and dialyzed against a methanol-water mixture (v / v, 1:1, 1:3) and pure water (MW 3500) for 24 hours, respectively. After freeze-drying, products HA-PBA (108.1 mg, 91.8%) with PBA linkage equivalences of 16.5%, 31.0%, 50%, and 86.0%, respectively, were obtained. The structural formula is as follows:

[0152] ;

[0153] Product 27 1 The H NMR data are as follows: [NMR spectrum image would be inserted here] Figures 14-17 As shown:

[0154] 1 H NMR (400 MHz, D2O): δ 7.71-7.64 (m, 0.34H), 7.39 (t, J = 8.0,0.33H), 5.31-5.12 (m, 0.18H), 4.37 (d, J = 24.0, 2H), 3.69-3.20 (m, 10H), 1.88-1.85 (m, 3H).

[0155] 1 H NMR (400 MHz, D2O): δ 7.71-7.66 (m, 0.63H), 7.40 (t, J = 10.0,0.62H), 5.22 (d, J = 60.0, 0.54H), 4.43-4.37 (m, 2H), 3.71-3.36 (m, 10H), 1.89-1.88 (m, 3H).

[0156] 1 H NMR (400 MHz, D2O): δ 7.80-7.73 (m, 1H), 7.47 (t, J = 8.0, 1H), 5.28 (d, J = 64.0, 1H), 4.44 (d, J = 36.0, 2H), 3.77-3.27 (m, 10H), 1.95-1.87 (m, 3H).

[0157] 1 H NMR(400 MHz, D2O): δ 7.73-7.68 (m, 1.77H), 7.44-7.35 (m, 1.72H), 5.32-5.13 (m, 1.92H), 4.45-4.34 (m, 2H), 3.72-3.36 (m, 10H), 1.91-1.88 (m,3H).

[0158] The infrared spectrum of product 27 is as follows Figure 18 As shown.

[0159] Example 28: Preparation of Ac-HA-conjugated prodrugs and self-assembled nanoformulations, the synthetic route is as follows: Figure 3 As shown:

[0160] HA-PBA (10 mg, 0.0000266 mmol) was dissolved in 5 mL of purified water in a 20 mL sample vial, and Ac-DA (2.19 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing Ac-DA was rapidly injected into the water containing HA-PBA. Finally, the organic solvent was removed by dialyzing (MW 3500) to obtain uniformly dispersed nanoparticles. The structural formula of product 28 is as follows:

[0161] .

[0162] Example 29: The preparation process of Hep-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: Hep-DA (2.97 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing Hep-DA was rapidly injected into water containing HA-PBA. The structural formula of product 29 is as follows:

[0163] .

[0164] Example 30: The preparation process of Ste-HA-conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: Ste-DA (4.70 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing Ste-DA was rapidly injected into water containing HA-PBA. The structural formula of product 30 is as follows:

[0165] .

[0166] Example 31: The preparation process of t-cos-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: t-cos-DA (5.64 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing t-cos-DA was rapidly injected into water containing HA-PBA. The structural formula of product 31 is as follows:

[0167] .

[0168] Example 32: The preparation process of PA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: PA-DA (4.36 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing PA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 32 is as follows:

[0169] .

[0170] Example 33: The preparation process of OA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: OA-DA (4.67 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing OA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 33 is as follows, and the infrared spectrum of the OA-HA conjugated prodrug is as follows. Figure 18 As shown, the particle size distribution and transmission electron microscopy are as follows: Figure 19 A and Figure 20 As shown:

[0171] .

[0172] Example 34: The preparation process of RA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 28, and the synthetic route is as follows: Figure 3 As shown, the difference lies in the following: RA-DA (4.85 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing RA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 34 is as follows:

[0173] .

[0174] Example 35: Preparation of LA-HA-conjugated prodrugs and self-assembled nanoformulations, the synthetic route is as follows: Figure 4 As shown:

[0175] HA-PBA (10 mg, 0.0000266 mmol) was dissolved in 5 mL of purified water in a 20 mL sample vial, and LA-DA (4.65 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the LA-DA-containing dimethyl sulfoxide solution was rapidly injected into the HA-PBA-containing water. Finally, dialysis (MW 3500) was performed to remove the organic solvent, yielding uniformly dispersed nanoparticles. The structural formula of product 35 is shown below, and its particle size distribution and transmission electron microscopy are as follows. Figure 19 B and Figure 20 As shown:

[0176] .

[0177] Example 36: The preparation process of LNA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 35, and the synthetic route is as follows: Figure 4 As shown, the difference lies in the following: LNA-DA (4.63 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing LNA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 36 is as follows, and its particle size distribution and transmission electron microscopy are shown below. Figure 19 C and Figure 20 As shown:

[0178] .

[0179] Example 37: The preparation process of GLA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 35, and the synthetic route is as follows: Figure 4 As shown, the difference lies in the following: GLA-DA (4.63 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing GLA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 37 is as follows:

[0180] .

[0181] Example 38: The preparation process of AA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 35, and the synthetic route is as follows: Figure 4 As shown, the difference lies in the following: AA-DA (4.92 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing AA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 38 is as follows:

[0182] .

[0183] Example 39: The preparation process of EPA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 35, and the synthetic route is as follows: Figure 4 As shown, the difference lies in the following: EPA-DA (4.90 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing EPA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 39 is as follows:

[0184] .

[0185] Example 40: The preparation process of DHA-HA conjugated prodrug and self-assembled nanoformulation is the same as in Example 35, and the synthetic route is as follows: Figure 4 As shown, the difference lies in the following: DHA-DA (5.20 mg, 0.0112 mmol) was dissolved in 500 μL of dimethyl sulfoxide solution. Under ultrasonic conditions, the dimethyl sulfoxide solution containing DHA-DA was rapidly injected into water containing HA-PBA. The structural formula of product 40 is as follows:

[0186] .

[0187] Example 1: Affinity test of hyaluronic acid-phenylboronic acid prodrug with OA-DA

[0188] Alizarin ARS and HAPBA (7.6×10) -4 The hyaluronic acid-phenylboronic acid prodrug (HAPBA) was mixed in PBS solution (pH 7.4) at a molar ratio of 1:1, and then OADA (based on 0.1-2.5 equivalents of HAPBA) was added to the mixture. The fluorescence intensity at an emission wavelength of 596 nm was recorded using a fluorescence spectrophotometer, and the binding constant between the hyaluronic acid-phenylboronic acid prodrug and OA-DA was calculated accordingly. Figure 21 As shown, OA-DA can displace ARS from the ARS / HA-PBA complex with a binding constant of 0.713 µM. -1 This indicates that OA-DA readily reacts with HA-PBA to generate hyaluronic acid-fatty acid conjugated prodrugs.

[0189] Example 2: Experiment on the elimination of excess ROS by hyaluronic acid-phenylboronic acid prodrug

[0190] A hyaluronic acid-phenylboronic acid prodrug solution with a final drug concentration of 1 mg / mL was prepared and incubated with AAPH (a free radical generator) for 5 or 30 minutes, followed by mixing with indocyanine green (ICG, a ROS indicator). After thorough mixing, the UV-Vis absorption spectrum of ICG in the above solution was measured. ICG or ICG / AAPH was used as a control. Figure 22 As shown, AAPH can induce ICG degradation and significantly reduce UV-Vis absorbance, while self-assembled nano-formulations can effectively remove ROS and protect ICG from degradation.

[0191] Example 3: Experiment on the elimination of intracellular ROS in M1 macrophages by self-assembled nanoformulation of hyaluronic acid-fatty acid prodrugs.

[0192] A self-assembled nanoformulation with a final drug concentration of 2.5 mg / mL was prepared and co-cultured with macrophages at a concentration of 0.05 mg / mL for 3 hours. Subsequently, intracellular ROS levels were measured using the H2DCFDA probe after 5 hours of stimulation with LPS / IFN-γ. Figure 23 As shown, self-assembled nanoformulations significantly downregulated intracellular ROS levels in M1 macrophages.

[0193] Example 4: Anti-enteritis experiment of self-assembled nano-formulation of hyaluronic acid-fatty acid prodrug.

[0194] An acute enteritis model was established in male C57BL / 6 mice by feeding them an aqueous solution containing 3% DSS. Oral administration was given on day 3 after DSS administration, once daily for a total of 7 times (dosing regimen see below). Figure 24 (As shown in Figure A). Mice were fed 3% DSS for 6 consecutive days. During the treatment period, the activity status of the mice was observed and recorded. On the 8th day after administration, mice in each group were sacrificed, and the colon and spleen were dissected and the colon length and spleen weight were measured to evaluate the efficacy of the self-assembled nano-formulation.

[0195] like Figure 24 As shown in Figures B-C, compared with clinical 5-aminosalicylic acid and dexamethasone, the self-assembled nano-formulation of hyaluronic acid-fatty acid prodrug effectively alleviated intestinal inflammation, resulting in a longer colon and no spleen swelling, similar to the healthy group. Histological staining results showed that the self-assembled nano-formulation significantly reduced colonic damage. Figure 25 ).

[0196] In addition, such as Figure 26As shown, the self-assembled nanoformulation of hyaluronic acid-fatty acid prodrug significantly downregulated the levels of myeloperoxidase (MPO) and pro-inflammatory factors such as TNF-α and IL-6 in intestinal inflammatory sites, resulting in a more potent anti-enteritis effect.

[0197] In summary, the hyaluronic acid-fatty acid conjugated prodrug self-assembled nanoformulation of this invention avoids the use of pharmaceutical excipients, thus effectively mitigating allergic reactions and toxicity caused by excipients. Furthermore, the hyaluronic acid and saturated or unsaturated fatty acids used are all substances required by the human body, biodegradable, and biocompatible. The phenylboronic acid structure contained within is also a core functional unit of Steboronine®, thus demonstrating promising prospects for clinical translation. Notably, the self-assembled nanoformulation eliminates excess ROS at the site of inflammation and within M1 macrophages while achieving targeted and controlled drug release at the target site or within target cells, exhibiting superior anti-inflammatory efficacy and in vivo safety compared to clinical 5-aminosalicylic acid and dexamethasone.

Claims

1. A self-assembled nanoformulation based on a hyaluronic acid-fatty acid conjugated prodrug, characterized in that, The self-assembled nanoformulation uses a hyaluronic acid-fatty acid conjugated prodrug as its active ingredient, and the structure of the hyaluronic acid-fatty acid conjugated prodrug is shown in Formula I: ， Formula I, where R is a saturated C1~C24 fatty acyl group; or, R is a C1~C24 unsaturated fatty acyl group with 1~6 double bonds; n=1~900, m=0~899.

2. The self-assembled nanoformulation according to claim 1, characterized in that, n=139~726, 25≤n+m≤1800.

3. The self-assembled nanoformulation according to claim 1, characterized in that, R is selected from acetyl, n-heptyl, stearoyl, tetracosyl, oleyl, castor oil oleyl, palm oil oleyl, linoleyl, linolenic acid, linolenic acid, arachidonic acid, eicosapentaenoyl, and docosahexaenoyl.

4. The method for preparing the self-assembled nano-formulation according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1: Under the action of a condensing agent, N-hydroxysuccinimide undergoes an esterification reaction with the saturated or unsaturated fatty acid corresponding to R to obtain a carboxyl-activated fatty acid product. Step 2: Under the action of the first catalyst, the carboxyl-activated fatty acid product undergoes a condensation reaction with dopamine hydrochloride to obtain a dopamine-fatty acid coupling prodrug. Step 3: Under the action of a second catalyst, hyaluronic acid undergoes a nucleophilic substitution reaction with 4-(bromomethyl)phenylboronic acid to obtain a hyaluronic acid-phenylboronic acid prodrug; Step 4: The dopamine-fatty acid conjugated prodrug is dissolved in dimethyl sulfoxide and then ultrasonically injected into an aqueous solution containing hyaluronic acid-phenylboronic acid prodrug. Dialysis is then performed to obtain a self-assembled nano-formulation.

5. The method for preparing self-assembled nano-formulations according to claim 4, characterized in that, In step 1, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; In step 2, the first catalyst is N,N-diisopropylethylamine; In step 3, the second catalyst is cesium carbonate.

6. The method for preparing self-assembled nano-formulations according to claim 5, characterized in that, The molar ratio of the condensing agent, N-hydroxysuccinimide, and saturated or unsaturated fatty acid in step 1 is (1~2):(1~1.5):1; In step 2, the molar ratio of the catalyst, the carboxyl-activated fatty acid product, and dopamine hydrochloride is (2~2.5):(1~2):1; In step 3, the molar ratio of the catalyst, 4-(bromomethyl)phenylboronic acid, and hyaluronic acid is (2000~3000):(4000~6000):1; In step 4, the molar ratio of the dopamine-fatty acid prodrug to the phenylboronic acid portion of the hyaluronic acid-phenylboronic acid prodrug is (0.001~1):

1.

7. The method for preparing self-assembled nano-formulations according to claim 4, characterized in that, The concentration of hyaluronic acid-phenylboronic acid prodrug in water is 0.25~5 mg / mL, and the concentration of dopamine-fatty acid prodrug in dimethyl sulfoxide is 0.1~11 mg / mL.

8. The method for preparing self-assembled nano-formulations according to claim 4, characterized in that, The reaction temperature in step 1 is 40~50 ℃, and the reaction time is 2~4 hours; The reaction temperature in step 2 is room temperature, and the reaction time is 4 to 8 hours. The reaction temperature in step 3 is 75 ℃, and the reaction time is 12~24 hours; The reaction temperature in step 4 is room temperature, and the reaction time is 0.2 to 1 hour.

9. The use of the self-assembled nanoformulation according to any one of claims 1 to 3 in the preparation of anti-colitis drugs.

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