A hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate drug and a preparation method thereof

Abstract

This invention discloses a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate and its preparation method in the field of drug preparation technology. First, hyaluronic acid is activated with carboxylic acid, then subjected to an amidation reaction with 3-aminophenylboronic acid to obtain a hyaluronic acid-phenylboronic acid conjugate. Next, methotrexate is activated and added to the hyaluronic acid-phenylboronic acid mixture, followed by stirring to obtain the hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate. The preparation method of this invention is simple. While the three drugs exert their respective effects, the introduction of phenylboronic acid enhances the targeted antitumor effect of the drug. The combined use of hyaluronic acid and phenylboronic acid further reduces the toxic side effects of methotrexate and improves the biocompatibility, bioavailability, and degradability of the resulting drug, making it suitable for application in the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of drug preparation technology, and in particular to a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate drug and its preparation method. Background Technology

[0002] Malignant tumors are a major threat to health, with incidence rates rising dramatically over the past few decades. Treatment for cancer patients is typically multimodal, including surgery, chemotherapy, radiotherapy, immunotherapy, and sometimes hormone therapy. All of these drugs have serious side effects, and their therapeutic effects often fall short of expectations. Nanomedicines can increase drug solubility, improve drug stability, prolong drug half-life, and enhance drug targeting, solving the problems of low efficiency and severe toxicity associated with traditional drug delivery methods.

[0003] Hyaluronic acid (HA) is a natural polysaccharide with good biocompatibility and degradability. HA and its derivatives can serve as sustained-release carriers for drugs, delaying drug release and providing a long-lasting effect. They can be used for the delivery of various drugs, including proteins, nucleic acids, and antitumor drugs. HA and its derivatives can specifically bind to multiple receptors on the cell surface, enabling targeted drug delivery, particularly for antitumor drugs.

[0004] Phenylboronic acid (PBA) and its derivatives have shown a strong ability to bind to sialic acid, a nine-carbon monosaccharide unit that is overexpressed as a terminal group of glycolipids and glycoproteins on the surface of tumor cells. The mechanism of this interaction is based on the reversible formation of five- and six-membered cyclic borate esters between the boron group of PBA and the outer ring polyol chain of sialic acid.

[0005] Methotrexate (MTX) exerts its antifolate effect through a structure similar to folate, allowing it to competitively inhibit dihydrofolate reductase (DHFR). MTX interferes with the synthesis of purines and pyrimidines, which are essential for DNA replication and cell proliferation. Inhibition of DHFR and other enzymes by MTX leads to the depletion of reduced dihydrofolate (DHF) and nucleotides, which strongly affects the proliferation of treated cell populations and induces cell death. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and provide a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate drug and its preparation method. The preparation method is simple, and the resulting drug has excellent targeting, good water solubility, and few toxic side effects.

[0007] The objective of this invention is achieved as follows: a method for preparing a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate, comprising the following steps:

[0008] Step 1: Hyaluronic acid (HA) was dissolved in morpholine ethanesulfonic acid (MES) buffer solution, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) was added. The mixture was stirred to activate the carboxylic acid, and then 3-aminophenylboronic acid (PBA) was added. The amidation reaction was carried out under light-protected conditions. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain the hyaluronic acid-phenylboronic acid (HA-PBA) conjugate.

[0009] Reaction formula:

[0010]

[0011] The mass ratio of hyaluronic acid, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and 3-aminophenylboronic acid is 1:(1.5-2.5):(0.8-1.2).

[0012] Step 2: Methotrexate (MTX) was dissolved in a dimethyl sulfoxide (DMSO) solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc) and 4-dimethylaminopyridine (DMAP), and the carboxyl group was activated by stirring. The hyaluronic acid-phenylboronic acid (HA-PBA) conjugate prepared in Step 1 was added, and the stirring was continued until the esterification reaction was complete. The mixture was dialyzed and freeze-dried to obtain the hyaluronic acid-phenylboronic acid-methopterin (HA-PBA-MTX) antitumor conjugate.

[0013] The reaction formula is:

[0014]

[0015] The mass ratio of methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine is 5:10:1, and the mass ratio of hyaluronic acid-phenylboronic acid coupling compound and methotrexate is 1:(0.8-1.2).

[0016] Furthermore, the activation time of the carboxylic acid in step 1 is 1 hour, the amidation reaction time is 24 hours, and the dialysis time is 3 days.

[0017] Furthermore, the pH value of the morpholine ethanesulfonic acid buffer solution described in step 1 is 5.5.

[0018] Furthermore, the carboxyl activation time in step 2 is 1 hour, the esterification reaction time is 24 hours, and the dialysis time is 3 days.

[0019] The hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate drug was obtained according to the preparation method of the present invention.

[0020] In this invention, a hyaluronic acid-phenylboronic acid-methotrexate (HA-PBA-MTX) antitumor conjugate is prepared through amidation and esterification reactions. This drug targets the CD44 receptor and sialic acid receptor overexpressed by cancer cells, improving the drug's targeting ability, reducing the toxic side effects of methotrexate, improving the water solubility of methotrexate, and exhibiting good biocompatibility, making it suitable for use in the preparation of antitumor drugs. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the synthesis of hyaluronic acid-phenylboronic acid in the examples.

[0022] Figure 2 The diagram below illustrates the synthesis of hyaluronic acid-phenylboronic acid-methopterin in the examples.

[0023] Figure 3 The NMR spectra are those of HA, PBA, MTX, HA-PBA, and HA-PBA-MTX in Example 1, and HA-MTX conjugate in the comparative example.

[0024] Figure 4 The Fourier transform infrared spectra of the HA, PBA, MTX, HA-PBA, and HA-PBA-MTX conjugates prepared in Example 2 are shown.

[0025] Figure 5 The particle size distribution and morphology of HA-PBA and HA-PBA-MTX NPs in Example 1 are shown.

[0026] Figure 6 Cell viability staining images of MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example after HEPG2 cancer cell culture.

[0027] Figure 7 The figure shows the results of the cck-8 assay after co-culturing HEPG2 cancer cells with MTX, HA-PBA-MTX prepared in Example 1 and HA-MTX prepared in the comparative example.

[0028] Figure 8 Cell viability and mortality staining images of MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example after co-culturing with normal HUVEC cells.

[0029] Figure 9 The image shows the results of the cck-8 assay after co-culturing normal HUVEC cells with MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example.

[0030] Figure 10The results show the blood compatibility assessment of the HA-PBA-MTX nanomedicine prepared in Example 1.

[0031] Figure 11 The images show mouse tumors in the MTX, HA-PBA-MTX and blank groups in Example 1.

[0032] Figure 12 The figure shows the results of the cck-8 assay after co-culturing HEPG2 cancer cells with MTX, HA-PBA-MTX prepared in Example 2, and HA-MTX prepared in the comparative example. Detailed Implementation

[0033] Example 1:

[0034] A method for preparing a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate, comprising:

[0035] Step 1, Preparation of HA-PBA conjugate: Dissolve 200 mg HA in 30 mL of MES buffer solution with pH 5.5, add 400 mg DMT-MM, stir for 1 h to activate the carboxyl group, then add 200 mg 3NH2-PBA, react under light-protected conditions for 24 h, after the reaction is complete, dialyze for 3 days and freeze dry to obtain HA-PBA conjugate;

[0036] Step 2, Preparation of HA-PBA-MTX conjugate: Dissolve 100 mg of methotrexate in 10 mL of DMSO solution containing 200 mg EDC and 20 mg DMAP, stir for 1 h to activate the carboxyl group, then add 10 mL of 10 mg / mL HA-PBA aqueous solution, react under light-protected conditions for 24 h, after the reaction is complete, dialyze for 3 days and freeze dry to obtain HA-PBA-MTX conjugate.

[0037] Example 2:

[0038] A method for preparing a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate, comprising:

[0039] Step 1, Preparation of HA-PBA conjugate: Dissolve 100 mg HA in 30 mL of MES buffer solution with pH 5.5, add 200 mg DMT-MM, stir for 1 h to activate the carboxyl group, then add 120 mg 3NH2-PBA, react under light-protected conditions for 24 h, after the reaction is complete, dialyze for 3 days and freeze dry to obtain HA-PBA conjugate;

[0040] Step 2, Preparation of HA-PBA-MTX conjugate: Dissolve 100 mg of methotrexate in 10 mL of DMSO solution containing 200 mg EDC and 20 mg DMAP, stir for 1 h to activate the carboxyl group, then add 10 mL of HA-PBA aqueous solution at 12 mg / mL, react under light-protected conditions for 24 h, after the reaction is complete, dialyze for 3 days and freeze dry to obtain HA-PBA-MTX conjugate.

[0041] When the mass ratio of hyaluronic acid to 3-aminophenylboronic acid is 1:(0.8-1.2), different mass ratios affect the grafting rate of 3-aminophenylboronic acid, thus affecting its targeting of sialic acid. Simultaneously, phenylboronic acid can reduce toxic side effects and promote cell proliferation; excessively high grafting rates can affect the toxic side effects of methotrexate on cancer cells, as demonstrated by the toxicity of HEPG 2 cells in Examples 1 and 2.

[0042] The mass ratio of hyaluronic acid-phenylboronic acid coupling compound to methotrexate was 1:(0.8–1.2). Different mass ratios affect the grafting rate of methotrexate. A low mass ratio results in a low grafting rate, while an excessively high mass ratio leads to incomplete reaction of methotrexate, resulting in waste of raw materials. In Example 1, the grafting rate of methotrexate was 14.52%, and in Example 2, the grafting rate was 14.54%.

[0043] Comparative Example: Preparation of HA-MTX Conjugates:

[0044] 75 mg EDC and 46 mg DMAP were added, and the mixture was stirred for 1 h to activate the carboxylic acid. Then, 50 mg HA and 3 mL DMSO were added to carry out the esterification reaction. The reaction was carried out in the dark for 24 h, dialyzed for 3 days, and then freeze-dried to obtain the HA-MTX conjugate.

[0045] Figure 1 This is a schematic diagram of the synthesis of hyaluronic acid-phenylboronic acid. The structural formula of the HA-PBA conjugate is shown below. Figure 1 As shown; Figure 2 The schematic diagram shows the synthesis of hyaluronic acid-phenylboronic acid-methopterin. The structural formula of the HA-PBA-MTX conjugate is shown below. Figure 2 As shown in the image.

[0046] Figure 3The NMR spectra of HA, PBA, MTX, HA-PBA, HA-MTX, and HA-PBA-MTX conjugates were obtained, which determined their chemical structures. The characteristic peak of the acetyl group (-NHCOCH3) in HA was at 2.04 ppm, and the characteristic peak of glycoside H was at 3.0-4.0 ppm. PBA showed characteristic peaks at 7.90 ppm, 6.90 ppm, 6.52 ppm, and 4.75 ppm. Compared with the PBA spectrum, the weak characteristic peak of HA-PBA at 7.0-8.0 ppm corresponded to the benzene ring of PBA. The spectrum also showed the acetyl group (2.08 ppm) and glycoside H (3.0-4.0 ppm) in HA, confirming the successful coupling of PBA and HA. The characteristic peak of MTX corresponding to the aromatic proton at 6.8-8.6 ppm indicated that MTX was successfully coupled to the HA-PBA conjugate, demonstrating the successful preparation of the HA-PBA-MTX conjugate.

[0047] Figure 4 The Fourier transform infrared spectra of the HA, PBA, MTX, HA-PBA, and HA-PBA-MTX conjugates prepared in Example 2 are shown. HA at 1625 cm⁻¹ -1 (-CO-), 1125cm -1 (COC), 1050cm -1 MTX exhibits characteristic transmittance at (-C-OH); MTX at 3500 cm⁻¹ -1 (-COOH), 2960cm -1 (-CH3), 1720cm -1 (-C=O), 1600cm -1 and 1500cm -1 and 820cm -1 (para-benzene); PBA at 3250cm -1 (-NH2), 1640cm -1 (-C=O), 1350cm -1 Characteristic transmittance is observed at (-BO). A 1600 cm⁻¹ region was found in the infrared spectrum of the HA-PBA-MTX conjugate. -1 and 1500cm -1 The FT-IR peaks, attributed to the stretching of the benzene ring, indicate that methotrexate (MTX) has been successfully coupled to hyaluronic acid (HA). The simultaneous appearance of characteristic peaks of MTX and PBA in the HA-PBA-MTX spectrum demonstrates the successful coupling of HA-PBA-MTX.

[0048] Figure 5The particle size distribution and morphology of the HA-PBA and HA-PBA-MTX NPs in Example 1 are shown in the images. Atomic force microscopy (AFM) was used to characterize the particle size distribution and morphology of the synthesized HA-PBA(A) and HA-PBA-MTX(C) NPs. These nanoparticles are spherical and approximately 100 nm in size. These self-assembled nanoparticles consist of an internal hydrophobic core and hydrophilic groups such as OH and COOH from the HA polysaccharide chains. The hydrophobic molecules of MTX form the core structure to act as a drug reservoir, while the outer hydrophilic shell of the glycosaminoglycan HA and phenylboronic acid can specifically target cancer cells through their CD44 and sialic acid binding capabilities.

[0049] Figure 6 The images show cell viability and mortality staining of MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example after co-culturing HEPG 2 cancer cells for 48 hours. The results show that MTX, HA-MTX, and HA-PBA-MTX can effectively inhibit the proliferation of HEPG 2 cancer cells, and HA-PBA-MTX has a stronger inhibitory effect on HEPG 2 cancer cells than MTX and HA-MTX.

[0050] Figure 7 The image shows the CCK-8 assay results of HEPG 2 cancer cells after co-culturing MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example for 48 hours. The results indicate that MTX, HA-MTX, and HA-PBA-MTX effectively inhibited the proliferation of HEPG 2 cancer cells, consistent with the live-death assay results. The inhibitory effect significantly increased with increasing drug concentration, and HA-PBA-MTX showed a stronger inhibitory effect on HEPG 2 cancer cells than MTX and HA-MTX. At a drug concentration of 100 μg / mL, HA-PBA-MTX inhibited HEPG 2 cancer cells by approximately 66%, while HA-MTX inhibited them by approximately 45%, and MTX by approximately 42%. Therefore, HA-PBA-MTX exhibits a stronger inhibitory effect on HEPG 2 cancer cells than MTX and HA-MTX.

[0051] Figure 8 Cell viability and mortality staining images of MTX, HA-PBA-MTX prepared in Example 1 and HA-MTX prepared in the comparative example after co-culturing normal HUVEC cells for 48 h; the results show that MTX, HA-MTX and HA-PBA-MTX can kill normal cells to some extent, but the toxic side effects of HA-PBA-MTX are less than those of MTX and HA-MTX.

[0052] Figure 9The image shows the CCK-8 assay results of MTX, HA-PBA-MTX prepared in Example 1, and HA-MTX prepared in the comparative example after co-culturing normal HUVEC cells for 48 h. When the drug concentration was 100 μg / mL, the cell viability of HA-PBA-MTX was 22%, 35%, and 55%, respectively. The results indicate that the toxic side effects of HA-PBA-MTX were less than those of MTX and HA-MTX.

[0053] Figure 10 This figure shows the blood compatibility evaluation results of the HA-PBA-MTX nanomedicine prepared in Example 1. In this study, the HA-PBA-MTX nanomedicine exhibited dose-dependent hemolytic effects on cells at different concentrations ranging from 10 to 1600 μg / mL. Figure 10 As shown, the hemolysis rate of HA-PBA-MTX was less than 5% at all concentrations. This phenomenon indicates that, according to national biosafety standards, HA-PBA-MTX nanomedicines can be classified as non-hemolytic substances.

[0054] Figure 11 The images show tumors in mice 15 days after drug injection. It can be seen that the tumors injected with MTX and HA-PBA-MTX are smaller than those in the control group, and the tumors injected with HA-PBA-MTX are smaller than those injected with MTX.

[0055] Figure 12 The image shows the CCK-8 assay results of HEPG 2 cancer cells after co-culturing the MTX, HA-PBA-MTX prepared in Example 2, and the HA-MTX prepared in the comparative example for 48 hours. The results indicate that MTX, HA-MTX, and HA-PBA-MTX effectively inhibited the proliferation of HEPG 2 cancer cells. The inhibitory effect significantly increased with increasing drug concentration, and HA-PBA-MTX showed a stronger inhibitory effect on HEPG 2 cancer cells than MTX and HA-MTX. At a drug concentration of 100 μg / mL, HA-PBA-MTX showed an inhibition rate of approximately 54% against HEPG 2 cancer cells, while HA-MTX showed an inhibition rate of approximately 45%, and MTX showed an inhibition rate of approximately 42%. Therefore, HA-PBA-MTX showed a stronger inhibitory effect on HEPG 2 cancer cells than MTX and HA-MTX.

[0056] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for preparing hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugate drug, characterized in that, The preparation method comprises the following steps: Step 1: dissolving hyaluronic acid in a morpholine ethanesulfonic acid buffer solution, adding 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, stirring to activate the carboxylic acid, then adding 3-aminobenzenboronic acid, and performing amidation reaction under light-proof condition; after the reaction is completed, dialysis, and freeze-drying to obtain a hyaluronic acid-phenylboronic acid conjugate; The reaction formula is as follows: The mass ratio of the hyaluronic acid, the 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride and the 3-aminobenzenboronic acid is 1: (1.5-2.5) : (0.8-1.2) ; Step 2: dissolving methotrexate in a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine DMSO solution, stirring to activate the carboxyl group, adding the hyaluronic acid-phenylboronic acid conjugate prepared in step 1, continuing to stir until the esterification reaction is completed, dialysis and freeze-drying to obtain a hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugated drug; The reaction formula is as follows: The mass ratio of the methotrexate, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the 4-dimethylaminopyridine is 5: 10: 1, and the mass ratio of the hyaluronic acid-phenylboronic acid conjugate and the methotrexate is 1: (0.8-1.2).

2. The preparation method of the hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugated drug according to claim 1, characterized in that: The carboxylic acid activation time in step 1 is 1 h, the amidation reaction time is 24 h, and the dialysis time is 3 days.

3. The method for preparing hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugated drug according to claim 1, characterized in that: The pH value of the morpholine ethanesulfonic acid buffer solution in step 1 is 5.

5.

4. The method for preparing hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugated drug according to claim 1, characterized in that: The carboxyl group activation time in step 2 is 1 h, the esterification reaction time is 24 h, and the dialysis time is 3 days.

5. The hyaluronic acid-phenylboronic acid-methotrexate antitumor conjugated drug obtained by the preparation method according to any one of claims 1-4.

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