Hyaluronic acid-polyethylene glycol-curcumin polymer, preparation method thereof, nano-micelle and application thereof

Abstract

The invention provides a hyaluronic acid-polyethylene glycol-curcumin polymer, a preparation method of the hyaluronic acid-polyethylene glycol-curcumin polymer, nano-micelles and application of the nano-micelles. In the hyaluronic acid-polyethylene glycol-curcumin polymer, polyethylene glycol is four-arm polyethylene glycol, curcumin and polyethylene glycol are combined through an ester bond, and hyaluronic acid and polyethylene glycol are combined through an amido bond. The hyaluronic acid-polyethylene glycol-curcumin polymer can be self-assembled to form the hyaluronic acid-polyethylene glycol-curcumin polymer nano-micelle, so that the anti-tumor medicine can be passively transported to tumor tissues by utilizing an EPR effect, and the anti-tumor medicine can be targeted to a CD44 receptor through hyaluronic acid, so that more medicine molecules enter focus tissues, and the anti-tumor effect is enhanced. The absorption and residence time of the anti-tumor medicine in tumors is prolonged. Meanwhile, the solubility and the anti-tumor targeting property of curcumin are improved, the drug metabolism time is prolonged, the bioavailability is improved, and the drug suitability is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hyaluronic acid-polyethylene glycol-curcumin polymer and a preparation method thereof, a nano micelle and an application thereof. Background Art

[0002] Chemotherapy drugs are a type of drug used to treat tumors. Chemotherapy drugs can kill tumor cells. These drugs can act on different stages of tumor cell growth and reproduction, inhibiting or killing tumor cells. Chemotherapy drug treatment is currently one of the main means of treating tumors.

[0003] However, chemotherapy drugs are non-targeted and can inhibit and kill both tumor cells and normal cells, causing serious toxic side effects on the body. Summary of the invention

[0004] In order to solve the technical problem that existing chemotherapy drugs cannot be accurately targeted for administration, the present invention provides a hyaluronic acid-polyethylene glycol-curcumin polymer and a preparation method thereof, a nano-micelle and an application thereof.

[0005] The first aspect of the present invention provides a hyaluronic acid-polyethylene glycol-curcumin polymer, wherein the polyethylene glycol is a four-arm polyethylene glycol, the curcumin is bonded to the polyethylene glycol via an ester bond, and the hyaluronic acid is bonded to the polyethylene glycol via an amide bond.

[0006] The second aspect of the present invention provides a hyaluronic acid-polyethylene glycol-curcumin nano-micelle prepared from a hyaluronic acid-polyethylene glycol-curcumin polymer.

[0007] The third aspect of the present invention provides a use of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles provided by the second aspect of the present invention in the preparation of a drug for treating drug-resistant tumors.

[0008] The fourth aspect of the present invention provides a method for preparing a hyaluronic acid-polyethylene glycol-curcumin polymer, comprising the following steps:

[0009] Esterification: mixing polyethylene glycol, curcumin and an esterification agent to react to obtain polyethylene glycol-curcumin, wherein the polyethylene glycol is a four-arm polyethylene glycol;

[0010] Activation: activating the carboxyl end of the polyethylene glycol-curcumin with an activation reagent to undergo an amidation reaction to obtain an activated form;

[0011] Cross-linking esterification: The activated body is mixed with deacetylated hyaluronic acid to react, thereby obtaining the hyaluronic acid-polyethylene glycol-curcumin polymer.

[0012] Compared with the existing technology, hyaluronic acid-polyethylene glycol-curcumin polymer can self-assemble to form hyaluronic acid-polyethylene glycol-curcumin polymer nano-micelles, which can not only passively transport anti-tumor drugs to tumor tissues by using the EPR effect, but also target CD44 receptors through hyaluronic acid, allowing more drug molecules to enter the lesion tissues, increasing the absorption and retention time of anti-tumor drugs in tumors. At the same time, it is used to increase the solubility and anti-tumor targeting of curcumin, prolong drug metabolism time, improve bioavailability, and improve drug suitability. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A schematic flow chart of a method for preparing a hyaluronic acid-polyethylene glycol-curcumin polymer provided in the fourth aspect of an embodiment of the present invention;

[0014] Figure 2 for Figure 1 The synthetic route of HA-PEG-Curcumin polymer is shown;

[0015] Figure 3 FT-IR spectra of hyaluronic acid, polyethylene glycol, curcumin, a physical mixture of polyethylene glycol / curcumin / hyaluronic acid, and the hyaluronic acid polyethylene glycol-curcumin polymer prepared in Example A1 of the present invention;

[0016] Figure 4A , 4B 4C and 4D are hyaluronic acid, polyethylene glycol, curcumin and the hyaluronic acid polyethylene glycol-curcumin polymer prepared in Example A1 of the present invention, respectively. 1 H-NMR spectrum;

[0017] Figure 5 This is a particle size distribution diagram of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles prepared in Example A2 of the present invention;

[0018] Figure 6 This is a potential diagram of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles prepared in Example A2 of the present invention;

[0019] Figure 7 The hyaluronic acid-polyethylene glycol-curcumin nanomicelles provided in the embodiment of the present invention were subjected to an in vitro anti-tumor cell effect curve using the MTT method. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0022] In the present invention, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e. a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0023] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "an" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0024] The weight of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass in the description of the embodiments of the present invention may be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0025] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the embodiments of the present invention, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0026] For the numerical ranges disclosed in the present invention, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0027] The first aspect of the embodiment of the present invention provides a hyaluronic acid-polyethylene glycol-curcumin polymer, wherein the polyethylene glycol (PEG) is a four-arm polyethylene glycol, curcumin (CUR) is bonded to the polyethylene glycol via an ester bond, and hyaluronic acid (HA) is bonded to the polyethylene glycol via an amide bond. The molar ratio of the four-arm polyethylene glycol to the curcumin can be 0.5:1-1:5, preferably 1:2.

[0028] It should be noted that hyaluronic acid, also known as uronic acid, is a large polysaccharide composed of two disaccharide units D-glucuronic acid and N-acetylglucosamine. It is a biocompatible and biodegradable targeting molecule. Hyaluronic acid can specifically bind to a large number of hyaluronic acid receptors CD44 receptors on the surface of some tumor cells, which can enable more drug molecules to enter the lesion tissue, increase the accumulation of anti-tumor drugs in the tumor tissue, and inhibit tumor growth. PEG is a hydrophilic material of polymer micelles. The hydrophilic shell formed by PEG has stealthiness, can reduce drug enzymolysis, can avoid protein adsorption such as opsonin and avoid being engulfed by the reticuloendothelial system (RES), thereby increasing the efficacy and reducing the toxic side effects of the drug. At the same time, PEG can change the fluidity of the cell membrane, making it easy for P-glycoprotein (P-gp) substrates to enter cells and inhibit P-gp efflux. Curcumin is a small molecule plant polyphenol extracted from the root lotus of the ginger family, which is a diketone compound. Curcumin has pharmacological effects such as anti-cancer, anti-oxidation, anti-inflammatory, lowering blood sugar, inhibiting angiogenesis, and anti-tumor migration. Curcumin can inhibit the growth of various tumor cells and prevent the formation of various chemical and radioactive induced tumors in experimental animals. It has an inhibitory effect on the generation, proliferation, and metastasis of cancer cells such as breast cancer, cervical cancer, colon cancer, gastric cancer, liver cancer, oral epithelial cell cancer, pancreatic cancer, and prostate cancer.

[0029] The second aspect of the embodiment of the present invention provides a hyaluronic acid-polyethylene glycol-curcumin nano-micelle. The hyaluronic acid-polyethylene glycol-curcumin polymer (HA-PEG-CUR) provided in the first embodiment of the present invention is dissolved in water as a prodrug, and can self-assemble to form hyaluronic acid-polyethylene glycol-curcumin nano-micelles, thereby increasing the solubility and anti-tumor targeting of curcumin and improving its drug suitability. The particle size of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles can be 50 to 200 nm, and the concentration of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles is 0.1 mg / ml-10 mg / ml, which can be used to prepare drug-resistant tumor drugs.

[0030] In some embodiments, the potential of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles is -40 to -10 mV.

[0031] It should be noted that tumor cells are for rapid uptake of nutrients, and the tumor area will produce loose capillaries, which are loosely arranged compared to the epithelial cells of normal capillaries, and the pores between the epithelial cells are larger. The hyaluronic acid-polyethylene glycol-curcumin nano micelles of the present invention have a small particle size, can smoothly pass through loose capillaries, and passively target the tumor area based on high permeability and retention effect (Enhanced permeability and retention effect, EPR effect). Meanwhile, the micelles with a small particle size are not easily captured by the reticuloendothelial system in the blood circulation system, and the hydrophilic shell formed by PEG has stealth. Therefore, the hyaluronic acid-polyethylene glycol-curcumin nano micelles of the present invention can stably exist for a long time in the blood, which is beneficial to improve the passive targeting effect to the tumor site realized by the EPR effect. Meanwhile, the particle size is small and also helps to improve the production yield.

[0032] In addition, the smaller the particle size of the micelle, the stronger the permeability and the weaker the retention capacity. The present invention combines the passive targeting of the EPR effect with the active targeting of hyaluronic acid to improve the targeting and retention capacity of hyaluronic acid-polyethylene glycol-curcumin nanomicelles, while taking into account the targeting, permeability and retention capacity.

[0033] Compared with the prior art, the present invention uses hyaluronic acid-polyethylene glycol-curcumin polymer as a prodrug, which can be self-assembled into hyaluronic acid-polyethylene glycol-curcumin polymer nano-micelles. It has the following beneficial effects:

[0034] 1) Hyaluronic acid-polyethylene glycol-curcumin polymer nano-micelles can not only passively transport anti-tumor drugs to tumor tissues by using the EPR effect, but also target CD44 receptors through hyaluronic acid, allowing more drug molecules to enter the lesion tissues, increasing the absorption and retention time of anti-tumor drugs in tumors. At the same time, it is used to increase the solubility and anti-tumor targeting of curcumin, prolong drug metabolism time, improve bioavailability, and improve drug suitability.

[0035] 2) The preparation method of hyaluronic acid-polyethylene glycol-curcumin nano-micelles is simple, and the hyaluronic acid-polyethylene glycol-curcumin nano-micelles are easy to preserve and can be used to protect the activity of genes and protein drugs, and the injection of biomacromolecule drugs or anti-tumor drugs.

[0036] 3) The polyethylene glycol in the hydrophilic shell of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles can prevent the adsorption of plasma opsonins, which can effectively prolong the blood circulation time of the drug, achieve a long circulation effect, and thus increase the efficacy; at the same time, the hydrophobic core acts as a reservoir for poorly soluble drugs, which can increase the solubility and stability of the drug and reduce the drug's toxic side effects.

[0037] 4) Hyaluronic acid-polyethylene glycol-curcumin polymer can encapsulate poorly soluble drugs, and the prepared polymer micelles have small particle size and narrow distribution, and the preparation is stable.

[0038] The third aspect of the embodiment of the present invention provides a use of a hyaluronic acid-polyethylene glycol-curcumin nano-micelle in preparing a drug for treating drug-resistant tumors, wherein the hyaluronic acid-polyethylene glycol-curcumin nano-micelle is the hyaluronic acid-polyethylene glycol-curcumin nano-micelle provided in the second aspect of the embodiment of the present invention. Specifically, when preparing the hyaluronic acid-polyethylene glycol-curcumin nano-micelle, anti-tumor drugs can be encapsulated.

[0039] The fourth aspect of the present invention provides a method for preparing a hyaluronic acid-polyethylene glycol-curcumin polymer, which is used to prepare the hyaluronic acid-polyethylene glycol-curcumin polymer of the first aspect of the present invention. Figure 1 and Figure 2 ,in, Figure 1 A schematic flow chart of a method for preparing a hyaluronic acid-polyethylene glycol-curcumin polymer provided in the fourth aspect of an embodiment of the present invention, Figure 2 for Figure 1 The synthetic route of hyaluronic acid-polyethylene glycol-curcumin polymer shown in FIG. 1 comprises the following steps:

[0040] Step 101, esterification: polyethylene glycol, curcumin and an esterification agent are mixed and reacted to obtain polyethylene glycol-curcumin, wherein the polyethylene glycol is four-arm polyethylene glycol;

[0041] In the present embodiment, four-arm polyethylene glycol (PEG-COOH), curcumin and esterification agent are mixed in organic solution for reaction, and esterification agent includes at least one of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The organic solvent can be dichloromethane, the mol ratio of esterification agent to four-arm polyethylene glycol can be 1: 1-1: 10, the mol ratio of curcumin to four-arm polyethylene glycol can be 1: 1-1: 10, the molecular weight of four-arm polyethylene glycol is 2000-8000Da, and the molecular weight of hyaluronic acid is 5000-20000Da.

[0042] In this embodiment, the esterification step may specifically include the following steps:

[0043] Dissolve four-arm polyethylene glycol in dichloromethane, add DCC, DMAP and curcumin, and react under the protection of inert gas at room temperature to obtain a reaction mixture;

[0044] separating the reaction mixture to obtain a first filtrate;

[0045] The first filtrate is subjected to a crystallization operation to obtain polyethylene glycol-curcumin.

[0046] Wherein, when carrying out esterification reaction, inert gas can be nitrogen, and can monitor by thin layer chromatography (chloroform: methanol 20: 1, fluorescence at 365nm) reaction progress to reaction completion, during the reaction, continuous magnetic stirring for more than 24h. When separating reaction mixture, solvent can be evaporated to about half of original volume on rotary evaporator, and the generated dicyclohexyl urea (DCU) precipitation is removed by suction filtration to obtain the first filtrate. During crystallization, the filtrate can be added to cold ether to separate out crystals, suction filtration, and vacuum drying to obtain product polyethylene glycol-curcumin (HOOC-PEG-CUR).

[0047] It can be understood that the value of n in four-arm polyethylene glycol four-arm (CH2CH2O)n can be the same or different.

[0048] In one embodiment, the structural formula of polyethylene glycol-curcumin is:

[0049]

[0050] Step 102, activation: using an activation reagent to activate the carboxyl end of polyethylene glycol-curcumin, and undergoing an amidation reaction to obtain an activated form.

[0051] In this embodiment, the activation reagent includes N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide, and the molar ratio of polyethylene glycol-curcumin to the activator can be 1:2. The hydroxyl end of N-hydroxysuccinimide and the carboxyl end of polyethylene glycol-curcumin undergo an amination reaction to generate an activated body N-hydroxysuccinimide-polyethanol-curcumin (NHS-PEG-CUR). The activation step can specifically include the following steps:

[0052] Dissolve polyethylene glycol-curcumin in water, add NHS and DCC, and react by stirring at room temperature to obtain a first reaction solution;

[0053] washing and drying the first reaction liquid to obtain a second filtrate;

[0054] The second filtrate is concentrated, an organic solvent is added to dissolve the precipitate, and the third filtrate is obtained by filtering;

[0055] The third filtrate was subjected to a crystallization operation to obtain NHS-PEG-CUR.

[0056] Wherein, when the amidation reaction is carried out, the stirring time can be 2h, and the reaction time can be 24h to ensure that the reaction is complete. When washing and drying, a saturated sodium chloride solution can be used for washing, and then anhydrous magnesium sulfate can be used for drying for one hour and then filtered to obtain a second filtrate. The organic solvent can be dichloromethane. During crystallization, the third filtrate can be crystallized with dry ether to precipitate the product, filter with suction, wash with a small amount of ether to obtain a white solid, and vacuum dry to constant weight to obtain NHS-PEG-CUR.

[0057] In one embodiment, the structural formula of NHS-PEG-CUR is:

[0058]

[0059] Step 103, cross-linking esterification: the activated body is mixed with the deacetylated hyaluronic acid to react, thereby obtaining a hyaluronic acid-polyethylene glycol-curcumin polymer.

[0060] In this embodiment, the molar ratio of the activated body to the deacetylated hyaluronic acid (HA-NH2) can be 1:1-1:5. The cross-linking esterification step can specifically include the following steps:

[0061] Dissolve NHS-PEG-CUR and deacetylated hyaluronic acid in water, mix well, and react at room temperature to obtain a second reaction solution;

[0062] adding ethanol to the second reaction solution, and filtering to obtain a precipitate;

[0063] The precipitate was washed and dried to obtain a hyaluronic acid-polyethylene glycol-curcumin polymer.

[0064] Wherein, when the cross-linking esterification reaction is carried out, the reaction time can be 24 hours. The ethanol added to the second reaction solution can be 95% ethanol by mass. When washing and drying, ethanol washing can be used for 3 times, and the washed precipitate is dried in a vacuum drying oven to obtain a light yellow solid, which is a hyaluronic acid-polyethylene glycol-curcumin polymer (HA-PEG-CUR).

[0065] In one embodiment, the structural formula of the hyaluronic acid-polyethylene glycol-curcumin polymer is:

[0066]

[0067] In one embodiment, the preparation method of the hyaluronic acid-polyethylene glycol-curcumin polymer further comprises:

[0068] Deacetylation: Hyaluronic acid is treated with a deacetylation agent to obtain deacetylated hyaluronic acid.

[0069] Wherein, the deacetylation agent comprises at least one of sodium hydroxide solution and hydrazine hydrate. The deacetylation step may specifically comprise the following steps:

[0070] Hyaluronic acid is dissolved in a NaOH solution and stirred to react to obtain deacetylated hyaluronic acid.

[0071] The mass fraction of the NaOH solution may be 20%, and the stirring may be performed by magnetic stirring.

[0072] Compared with the prior art, the method for synthesizing HA-PEG-CUR in the present invention adopts simple procedures such as filtration, is suitable for industrial large-scale production, has simple process and is convenient to prepare.

[0073] The above description of the hyaluronic acid-polyethylene glycol-curcumin polymer and its preparation method, nano micelle and its application provided by the present invention is described. In order to make the details and operation of the above embodiments of the present invention clearly understood by those skilled in the art, and to embody the significant improvement performance of the hyaluronic acid-polyethylene glycol-curcumin polymer and its preparation method, nano micelle and its application in the embodiments of the present invention, the above technical scheme is illustrated by the following examples.

[0074] Example A1. Preparation of hyaluronic acid-polyethylene glycol-curcumin polymer

[0075] The four-arm polyethylene glycol was dissolved in dichloromethane, and a DCC / DMAP esterification reagent was added in an amount of 1.5 times the molar ratio of PEG-COOH, and curcumin was added in an amount of 1.2 times the molar ratio of PEG-COOH. Under nitrogen protection, magnetic stirring was carried out at room temperature for more than 24 hours, and thin layer chromatography monitoring (chloroform: methanol 20:1, fluorescence at 365nm) was performed until the reaction was complete. After the reaction was completed, the solvent was evaporated to about half of the original volume on a rotary evaporator, and the generated DCU precipitate was removed by suction filtration. The filtrate was added to cold ether to precipitate crystals, suction filtered, and vacuum dried to obtain the product COOH-PEG-CUR.

[0076] The COOH-PEG-CUR obtained after drying was dissolved in water, and NHS and DCC in a 2-fold molar ratio were added to the solution, stirred for 2 hours, and then reacted at room temperature for 24 hours. After the reaction was completed, it was washed with a saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered after one hour, and the filtrate was concentrated. 30 ml of dichloromethane was added and heated until the precipitate was dissolved, and then filtered. The filtrate was crystallized with dry ether to precipitate the product, filtered, washed with a small amount of ether to obtain a white solid, and vacuum dried to constant weight to obtain NHS-PEG-CUR.

[0077] Hyaluronic acid was dissolved in 20% NaOH solution at room temperature and stirred magnetically overnight to obtain HA-NH2.

[0078] Take the above NHS-PEG-CUR and HA-NH2 and dissolve them in water at a molar ratio of 1:2, mix them evenly, and react at room temperature. After reacting for 24 hours, add 95% ethanol by mass to the reaction solution, filter and collect the precipitate, and wash the precipitate with ethanol three times. Dry the washed precipitate in a vacuum drying oven to obtain HA-PEG-CUR polymer.

[0079] The prepared HA-PEG-CUR was characterized as follows.

[0080] 1. Measurement by infrared spectrometer

[0081] The polyethylene glycol, curcumin, hyaluronic acid, physical mixture of polyethylene glycol / curcumin / hyaluronic acid, and the hyaluronic acid-polyethylene glycol-curcumin polymer prepared above were mixed with dry potassium bromide (KBr) and ground into a fine powder mixture in an agate mortar. An appropriate amount was taken and compressed into tablets by a tablet press and loaded into an infrared spectrometer for measurement. The measurement results can be found in Figure 3 , which are FT-IR spectra of hyaluronic acid, polyethylene glycol, curcumin, a physical mixture of polyethylene glycol / curcumin / hyaluronic acid and Example A1 of the present invention. As can be seen from the figure, functional groups such as amide groups and ester bonds exist in HA-PEG-CUR, which helps to illustrate that the final product is successfully synthesized.

[0082] 2. Nuclear Magnetic Resonance Spectrometer Determination

[0083] Dissolve the polyethylene glycol, curcumin, hyaluronic acid, physical mixture of polyethylene glycol / curcumin / hyaluronic acid, and the above hyaluronic acid-polyethylene glycol-curcumin polymer in 0.5 ml of heavy water (D2O) respectively. After complete dissolution, add the sample solution into the NMR tube and measure it with a nuclear magnetic resonance spectrometer at room temperature. The integration processing software is NUTS software. Please refer to the measurement results. Figure 4A , 4B 4C, 4D are hyaluronic acid, polyethylene glycol, curcumin and the hyaluronic acid polyethylene glycol-curcumin polymer prepared in Example A1 of the present invention, respectively. 1 H-NMR spectrum. As can be seen from the figure, the prepared hyaluronic acid-polyethylene glycol-curcumin polymer has characteristic peaks of polyethylene glycol, curcumin and hyaluronic acid at the same time, confirming the structure of HA-PEG-CUR.

[0084] Example A2. Preparation of hyaluronic acid-polyethylene glycol-curcumin nanomicelles

[0085] The hyaluronic acid-polyethylene glycol-curcumin polymer prepared in Example A1 was dissolved in ultrapure water at a concentration of 5 mg / ml, and self-assembled to form hyaluronic acid-polyethylene glycol-curcumin nano-micelles.

[0086] Take an appropriate amount of hyaluronic acid-polyethylene glycol-curcumin nano-micelles, add ultrapure water to dilute, and use Malvern Zetasizer Nano ZS-90 particle size analyzer to measure the particle size and Zeta potential value of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles. Each experiment is repeated three times and the average value is taken for statistical analysis. Figure 5 , is the particle size distribution diagram of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles prepared in Example A2 of the present invention. The particle size (Size) of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles ranges from 50 to 200 nm, showing a normal distribution, with a small particle size and a narrow distribution. Please refer to Figure 6 , is the potential diagram of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles prepared in Example A2 of the present invention. The potential (ApparentZeta Potential) of the hyaluronic acid-polyethylene glycol-curcumin nanomicelles is -13.87±2.50 mV.

[0087] The preparation of Examples A1 and A2 is described above. The following is an evaluation of the in vitro anti-tumor cell effect of hyaluronic acid-polyethylene glycol-curcumin nanomicelles through experiments.

[0088] MTT method was used to 5 MCF-7 (non-resistant cell line) and MCF-7 / ADR (resistant cell line) were inoculated into 96-well culture plates and cultured in a 37°C, CO2 incubator for 12 hours. After the cells adhered to the wall, 20μl of hyaluronic acid-polyethylene glycol-curcumin nano-micelles of different concentrations were added to the two types of cells. Three replicate wells were made for each gradient. After 12 hours of action, 20μl of MTT solution (5mg / ml) was added to each well. The cells were cultured at 37°C for 4 hours, the supernatant was discarded, 150μl of dimethyl sulfoxide was added to each well, and the cells were shaken for 10 minutes. The absorbance of each well at 570nm (A) was measured by enzyme-linked immunosorbent assay. The serum-free culture medium was used as the blank control. The average value was taken and the cell growth inhibition rate was calculated, which was expressed as relative cell ability %. Please refer to Figure 7 , which is the in vitro anti-tumor cell effect curve of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles provided in the embodiment of the present invention using the MTT method. As can be seen from the figure, the hyaluronic acid-polyethylene glycol-curcumin nano-micelles have a good inhibitory effect on both non-resistant cells and resistant cells.

[0089] The above descriptions are only some embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A hyaluronic acid-polyethylene glycol-curcumin polymer, characterized in that: The polyethylene glycol is four-arm polyethylene glycol, the curcumin is bonded to the polyethylene glycol via an ester bond, and the hyaluronic acid is bonded to the polyethylene glycol via an amide bond.

2. The hyaluronic acid-polyethylene glycol-curcumin polymer according to claim 1, characterized in that The molar ratio of the polyethylene glycol to the curcumin is 0.5:1-1:

5.

3. A hyaluronic acid-polyethylene glycol-curcumin nanomicelle prepared by using the hyaluronic acid-polyethylene glycol-curcumin polymer described in any one of claims 1 to 2.

4. The hyaluronic acid-polyethylene glycol-curcumin nano-micelle according to claim 3, wherein the hyaluronic acid-polyethylene glycol-curcumin nano-micelle has a concentration of 0.1 mg / ml-10 mg / ml, a particle size of 50 to 200 nm, and a potential of -40 to -10 mV.

5. Use of the hyaluronic acid-polyethylene glycol-curcumin nano-micelles as claimed in claim 3 in the preparation of a drug for treating drug-resistant tumors.

6. A method for preparing a hyaluronic acid-polyethylene glycol-curcumin polymer, characterized in that: The steps include: Esterification: mixing polyethylene glycol, curcumin and an esterification agent to react to obtain polyethylene glycol-curcumin, wherein the polyethylene glycol is a four-arm polyethylene glycol; Activation: activating the carboxyl end of the polyethylene glycol-curcumin with an activation reagent to undergo an amidation reaction to obtain an activated form; Cross-linking esterification: The activated body is mixed with deacetylated hyaluronic acid to react, thereby obtaining the hyaluronic acid-polyethylene glycol-curcumin polymer.

7. The preparation method according to claim 6, characterized in that: Also includes: Deacetylation: hyaluronic acid is treated with a deacetylation agent to obtain the deacetylated hyaluronic acid.

8. The preparation method according to claim 7, characterized in that: The deacetylation agent includes at least one of a sodium hydroxide solution and hydrazine hydrate.

9. The preparation method according to claim 6, characterized in that: The polyethylene glycol, the curcumin and the esterification reagent are mixed and reacted in an organic solution, and the esterification reagent includes dicyclohexylcarbodiimide and 4-dimethylaminopyridine.

10. The preparation method according to claim 6, characterized in that: The activation reagent comprises N-hydroxysuccinimide and dicyclohexylcarbodiimide, and the hydroxyl end of the N-hydroxysuccinimide undergoes an amidation reaction with the carboxyl end of the polyethylene glycol-curcumin to generate the activated body.

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