Hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate drug and preparation method thereof

Abstract

The invention discloses a hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate and a preparation method thereof. The method first activates hyaluronic acid with carboxylic acid, then adds mangiferin, and the reaction obtains a hyaluronic acid-mangiferin conjugate, and then reacts the carboxylic acid-activated methotrexate with the hyaluronic acid-mangiferin conjugate to obtain a hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate. The preparation method of the present invention is simple, and by introducing mangiferin, while the three drugs each play their own role, the combination of hyaluronic acid and mangiferin can improve the targeted anti-tumor effect of the drug, and further reduce the toxic and side effects of methotrexate, and improve the biocompatibility, bioavailability and degradability of the drug.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and relates to a hyaluronic acid-mangiferin-methotrexate anti-tumor coupling drug and a preparation method thereof. Background Art

[0002] Chemotherapy is the main method for treating cancer. Although it can relieve pain and achieve certain therapeutic effects, it is accompanied by inevitable side effects. Not all cancer patients can survive from malignant tumors and the side effects of chemotherapy. Polymer-drug conjugates can overcome the above shortcomings of chemical anticancer drugs. Polymers are used as carriers and connected to chemical anticancer drugs. Polymer macromolecules can be retained in tumor cells and enhance the permeability of tumor cells, thereby achieving targeted treatment of tumors.

[0003] Methotrexate (MTX) is an anti-metabolite tumor drug with a structure similar to folic acid (FA) and is an inhibitor of dihydrofolate reductase (DHFR). MTX has a high affinity for DHFR. When it is internalized into cells, it can participate in the FA metabolic cycle and inhibit the formation of DHFR, preventing the reduction of dihydrofolate to tetrahydrofolate, and can terminate the biosynthesis of DNA and RNA. MTX penetrates into the cytoplasm, causing tumor cell death and achieving an anti-tumor effect. However, MTX has the characteristics of most anti-metabolite drugs, namely, no tissue selectivity, high dose, and large toxic side effects. The use of nano-drug carriers to actively target MTX to lesions has always been the focus of researchers. This method of administration provides a new way for MTX targeted therapy.

[0004] Hyaluronic acid (HyaluronicAcid, HA) has good biocompatibility, is certified by the U.S. FDA as non-toxic and harmless, is often used as a filler inside the human body, and its derivatives are often used as drug carrier materials, and hyaluronic acid can participate in many key and complex biological functions through its receptor protein CD44 mediation on the cell membrane. According to research reports, many tumor cell surfaces will overexpress CD44 to varying degrees, providing a new development direction for the design of targeted drug delivery systems. CD44 is a ubiquitous glycoprotein on the surface of mammalian cells, overexpressed in a variety of solid tumors (such as pancreatic cancer, breast cancer and lung cancer, etc.). Hyaluronic acid is the main CD44 binding molecule, and hyaluronic acid targeting ability can be used to effectively deliver drugs to tumor tissue (Hyaluronic acid targeting of CD44 for cancer therapy:from receptorbiology to nanomedicine, Journal of Drug Targeting, Volume 23, 2015, 605-618). Chinese patent CN 108888775 A discloses a hyaluronic acid-methotrexate self-assembly nano-micelle and its preparation method, hyaluronic acid and methotrexate are coupled and linked by bridge (cystamine link of disulfide bond). On the one hand, it utilizes hyaluronic acid to specifically bind to tumor cells, so that polymer micelles have targeting, and can make drugs loaded into lesion sites with a target, and on the other hand, utilizes the glutathione responsiveness of disulfide bonds to realize the controlled release of drugs. Chinese patent CN 107049955A has good multi-level targeting by hyaluronic acid encapsulating polyphthalamide-amine to carry out methotrexate drug loading, but toxic and side effects are large and complicated to prepare. Chinese patent CN 110237266 A discloses a method for preparing hyaluronic acid-alendronate-methotrexate nanoparticles, wherein a hyaluronic acid-alendronate-methotrexate conjugate is prepared by covalently linking amide bonds and ester bonds. The conjugate can target cancer cells for treatment. However, since alendronate is easily adsorbed to bone tissue, the conjugate is limited to targeting bone tumors and has a limited scope of application.

[0005] Mangiferin is a pharmacologically active natural polyphenol compound with good anti-inflammatory, anti-allergic, anti-tumor and antioxidant activities. Mangiferin has a wide range of applications and can treat diabetes, neurodegenerative diseases and prevent aging symptoms. At present, there is no literature report on the preparation of anti-tumor drugs by coupling mangiferin with methotrexate in China. Summary of the invention

[0006] The object of the present invention is to provide a hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate drug with excellent targeting and low toxicity and side effects and a preparation method thereof.

[0007] The technical solution for achieving the purpose of the present invention is as follows:

[0008] The preparation method of hyaluronic acid-mangiferin-methotrexate anti-tumor drug conjugate comprises the following steps:

[0009] Step 1, dissolving hyaluronic acid (HA) in dimethyl sulfoxide (DMSO), adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) in DMSO, stirring to activate the carboxylic acid, then adding mangiferin (MA), continuing to stir until the reaction is complete, dialyzing and freeze-drying to obtain a hyaluronic acid-mangiferin (HA-MA) conjugate;

[0010] Step 2, dissolving methotrexate in dimethyl sulfoxide (DMSO), adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine in DMSO, stirring to activate the carboxylic acid, then adding hyaluronic acid-mangiferin conjugate, continuing to stir until the reaction is complete, dialyzing and freeze-drying to obtain hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate drug (HA-MA-MTX).

[0011] Preferably, in step 1, the mass ratio of hyaluronic acid to mangiferin is 1:2.

[0012] Preferably, in step 1, the stirring reaction time is 1 hour, the stirring reaction time is continued for 24 hours, and the freeze-drying time is 24 hours.

[0013] Preferably, in step 2, the mass ratio of hyaluronic acid-mangiferin conjugate to methotrexate is 1:4.

[0014] Preferably, in step 2, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 4-dimethylaminopyridine is 2:1.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) In the present invention, mangiferin, methotrexate and hyaluronic acid are combined through an ester bond. Hyaluronic acid can combine with CD44 receptors overexpressed in cancer cells and has tumor cell targeting. At the same time, its macromolecular structure can also be used as a drug carrier to carry methotrexate. In addition to exerting its own medicinal value, mangiferin also synergizes with hyaluronic acid to improve the targeted anti-tumor effect of the drug, while further reducing the toxic and side effects of methotrexate, thereby improving the biocompatibility, bioavailability and degradability of the drug.

[0017] (2) In the present invention, the structures of the three raw materials, hyaluronic acid, mangiferin and methotrexate, do not need to be coupled through a bridge (cystamine link of a disulfide bond), and the coupled drug structure is stable and the preparation method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthesis of the hyaluronic acid-mangiferin-methotrexate system in the embodiment;

[0019] Figure 2 The NMR spectra of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX conjugates prepared in the examples;

[0020] Figure 3 : The Fourier transform infrared spectra of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX conjugates prepared in the examples;

[0021] Figure 4 The particle size distribution and morphology of HA-MA, HA-MTX and HA-MA-MTX NPs in the examples;

[0022] Figure 5 The live and dead staining images of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX prepared in the examples after culturing K7 cancer cells;

[0023] Figure 6 This is a graph showing the cck-8 experimental results of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX prepared in the example after co-culture with K7 cancer cells;

[0024] Figure 7 Cell live-death staining images of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX prepared in the examples after co-culture with MC3T3-E1 normal cells;

[0025] Figure 8 This is a graph showing the CCK-8 experimental results of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX prepared in the examples after co-culture with MC3T3-E1 normal cells. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0027] Example

[0028] 1. Preparation of HA-MTX conjugate: 42 mg MTX was dissolved in 3 mL DMSO, 75 mg EDC and 46 mg DMAP were added, and the mixture was stirred to activate the carboxylic acid; 50 mg HA and 3 mL DMSO were then added for esterification. The solution was wrapped with aluminum foil to avoid light, and the reaction was allowed to proceed for 48 h, dialyzed for 2 d, and freeze-dried for 24 h to obtain the HA-MTX conjugate.

[0029] 2. Preparation of HA-MA conjugate and HA-MA-MTX conjugate:

[0030] (1) 200 mg HA was dissolved in 60 mL DMSO mixed with 800 mg EDC and 400 mg DMAP, and the mixture was stirred for 1 h. Then 400 mg MA was added to the reaction mixture, stirred for 24 h and dialyzed for 2 days, and freeze-dried to obtain HA-MA conjugate.

[0031] (2) 400 mg of MTX was dissolved in 60 mL of DMSO mixed with 800 mg of EDC and 400 mg of DMAP. After stirring for reaction, 100 mg of HA-MA was added, and stirring was continued for 24 h. The mixture was dialyzed for 2 d and freeze-dried for 24 h to obtain a HA-MA-MTX conjugate.

[0032] Figure 1 It is a schematic diagram of the synthesis of the hyaluronic acid-mangiferin-methotrexate system, and the structural formula of the HA-MA-MTX conjugate is shown in the figure.

[0033] Figure 2 The nuclear magnetic resonance spectra of HA, MA, MTX, HA-MA, HA-MTX and HA-MA-MTX conjugates can be used to determine their chemical structures. The acetyl (-NHCOCH3) content in HA is 1.85ppm, and the glycoside H content is 3.0-4.0ppm. MA has characteristic peaks at 7.04ppm, 3.38ppm and 0.97ppm. Compared with the MTX spectrum, the weak characteristic peaks of the HA-MTX spectrum at 6.8-8.6ppm correspond to the aromatic protons of MTX. The spectrum also shows the N-acetyl (1.85ppm) and glycoside H (3.0-4.0ppm) of HA, confirming the successful connection between MTX and HA. The characteristic peaks of 6.8-8.6ppm corresponding to the aromatic protons of MTX indicate that MTX is successfully coupled to the HA-MA conjugate, indicating that the HA-MA-MTX conjugate is successfully prepared.

[0034] Figure 3 This is the Fourier infrared spectrum of HA-MA-MTX conjugate. HA is at 1625cm -1 (-CO - )、1125cm -1(COC), 1050cm -1 (-C-OH) has a characteristic transmittance; MA at 2920cm -1 (-CH2), 1720cm -1 (-C=O), 1250cm -1 (Ar-O-Ar) and 1050cm -1 (-C-OH) has a characteristic transmittance; MTX at 3500cm -1 (-COOH), 2960cm -1 (-CH3), 1720cm -1 (-C=O), 1600cm -1 and 1500cm -1 and 820cm -1 The characteristic transmittance at 1600 cm was found in the infrared spectra of MTX and HA-MTX conjugates. -1 and 1500cm -1 The FT-IR peaks of HA-MA-MTX were attributed to the stretching of the benzene ring, indicating that MTX has been successfully coupled to hyaluronic acid. The characteristic peaks of MTX and MA appeared simultaneously in the spectrum of HA-MA-MTX, indicating that HA-MA-MTX was successfully coupled.

[0035] Figure 4 The particle size distribution and morphology of the synthesized HA-MA (A, B), HA-MTX (C, D) and HA-MA-MTX (E, F) NPs were characterized using atomic force microscopy (AFM). These nanoparticles are spherical and are around 100 nm in size. These self-assembled nanoparticles consist of an internal hydrophobic core of MA, MTX or a combination and hydrophilic groups such as OH, COOH of the HA polysaccharide chain. The hydrophobic molecules of MA and MTX form the inner core of the structure to act as a drug reservoir. The outer hydrophilic shell of the glycosaminoglycan HA can specifically target cancer cells through its CD44 binding ability.

[0036] Figure 5 These are the live-dead staining images of HA (A), MA (B), MTX (C), HA-MA (D), HA-MTX (E), and HA-MA-MTX (F) after co-culture of K7 cancer cells. The results show that MTX, HA-MTX, and HA-MA-MTX can effectively inhibit the proliferation of K7 cancer cells.

[0037] Figure 6The cck-8 test results of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX after co-culture of K7 cancer cells are shown in Figure 1. The results show that HA-MTX and HA-MA-MTX conjugate nanoparticles have the same inhibitory effect on the growth of tumor cells as free MTX, which is consistent with the results of the cck-8 experiment. As the concentration increases, the inhibitory effect is significantly enhanced, and HA-MA-MTX has a stronger inhibitory effect than free MTX and HA-MTX. When the given drug concentration is 100 μg / mL, the inhibitory effect of HA-MA-MTX can reach about 72%, while the inhibition rate of MTX is about 60%, and the inhibition rate of HA-MTX is about 65%. Therefore, for K7 cancer cells, the inhibitory effect of HA-MA-MTX is higher than that of free MTX and HA-MTX, which shows that the addition of MA can further enhance the tumor-suppressing effect of HA-MTX conjugates.

[0038] Figure 7 These are the live-dead staining images of HA (A), MA (B), MTX (C), HA-MA (D), HA-MTX (E) and HA-MA-MTX (F) after co-culture in MC3T3-E1 normal cells. The results show that MTX, HA-MTX and HA-MA-MTX can kill normal cells to a certain extent, but the HA-MA-MTX conjugate kills fewer normal cells, that is, the toxic side effects are small, which proves that compared with HA-MTX, the addition of MA can further reduce the toxic side effects of HA-MA-MTX on normal tissue cells.

[0039] Figure 8 The cck-8 test results of HA, MA, MTX, HA-MA, HA-MTX, and HA-MA-MTX after co-culture in MC3T3-E1 normal cells are shown. The results show that free MTX significantly reduces the viability of normal cells and has great toxic side effects. When the given drug concentration is 100 μg / mL, the survival rates of MC3T3-E1 cells are about 57%, 62%, and 68%, respectively. Among them, the cell survival rate under the action of HA-MA-MTX conjugate nanoparticles is higher than that of free MTX and HA-MTX drugs. The above results show that compared with HA-MTX, HA-MA-MTX nanoparticles can further reduce the toxic side effects of MTX on normal cells.

Claims

1. A method for preparing a hyaluronic acid-mangiferin-methotrexate anti-tumor drug conjugate, characterized in that: The steps include: Step 1, dissolving hyaluronic acid in dimethyl sulfoxide, adding a dimethyl sulfoxide solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, stirring to activate the carboxylic acid, and then adding mangiferin, the mass ratio of hyaluronic acid to mangiferin is 1:2, continuing to stir until the reaction is complete, dialyzing and freeze-drying to obtain a hyaluronic acid-mangiferin conjugate; Step 2, dissolving methotrexate in dimethyl sulfoxide, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine in dimethyl sulfoxide solution, stirring to activate the carboxylic acid, and then adding hyaluronic acid-mangiferin conjugate, the mass ratio of hyaluronic acid-mangiferin conjugate to methotrexate is 1:4, continuing to stir and react until complete, dialyzing and freeze-drying to obtain hyaluronic acid-mangiferin-methotrexate anti-tumor conjugate drug.

2. The preparation method according to claim 1, characterized in that In step 1, the stirring reaction time is 1 h, the stirring reaction time is continued for 24 h, and the freeze-drying time is 24 h.

3. The preparation method according to claim 1, characterized in that: In step 2, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 4-dimethylaminopyridine is 2:

1.

4. The hyaluronic acid-mangiferin-methotrexate anti-tumor drug conjugate prepared according to the preparation method according to any one of claims 1 to 3.

Images