CN116515011A - Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof - Google Patents

Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof Download PDF

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CN116515011A
CN116515011A CN202310503281.1A CN202310503281A CN116515011A CN 116515011 A CN116515011 A CN 116515011A CN 202310503281 A CN202310503281 A CN 202310503281A CN 116515011 A CN116515011 A CN 116515011A
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hyaluronic acid
acid
graft copolymer
catalyst
amphiphilic
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梁娜
孙少平
李刚
王胜
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Harbin Normal University
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Harbin Normal University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Abstract

The invention particularly relates to an amphiphilic graft copolymer based on hyaluronic acid, and a preparation method and application thereof. The copolymer takes hyaluronic acid as a main framework, and methoxy polyethylene glycol, octadecanoic acid and 2, 4-dinitrobenzenesulfonic acid are grafted on the copolymer; the invention also relates to a preparation method of the copolymer, and a drug carrier and a drug composition prepared from the copolymer. The polymer prepared by the invention has better biocompatibility,has active and passive dual targeting capability, redox conversion and SO release 2 Therefore, the method can be used for preparing the release carrier of the anticancer drug, realizes high uptake and quick release of the drug in vivo, and has wide application prospect.

Description

Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof
Technical Field
The invention belongs to the technical field of high polymer materials, and particularly relates to an amphiphilic graft copolymer based on hyaluronic acid, and a preparation method and application thereof.
Background
Cancer has been the primary killer of life safety hazard to humans, and its therapeutic and diagnostic protocols have been the focus of research. The main treatments for cancer at present include surgical treatment, radiation treatment and chemical drug treatment (chemotherapy), wherein chemotherapy is currently the most clinically prominent treatment. However, chemotherapy has a number of problems such as poor water solubility, low biocompatibility and large toxic and side effects of chemotherapeutic drugs. Therefore, there is a need to develop a drug carrier that ameliorates many of the shortcomings of chemotherapeutic drugs.
The patent number is 201910046763.2, the invention relates to a hyaluronic acid derivative, a preparation method and application thereof, and discloses a hyaluronic acid derivative which is prepared by modifying hyaluronic acid with fatty alcohol by taking hyaluronic acid as a matrix skeleton, and on the basis, disulfide bonds and mPEG are modified to enable the hyaluronic acid derivative to have the characteristics of tumor targeting, long circulation and reduction sensitivity drug release, and when the hyaluronic acid derivative is used as an antitumor drug carrier, the directional and accurate release of drugs at tumor positions can be realized, and the synergistic and toxicity reduction effects can be achieved; the patent has remarkable effect on improving the tumor inhibition rate and has high clinical application value.
Patent No. 202010280704.4, entitled "an amphiphilic graft copolymer based on hyaluronic acid, a method for preparing the same, and use thereof", comprising: (1) hyaluronic acid as a parent skeleton; (2) Deoxycholate groups grafted onto the first primary alcohol group of hyaluronic acid; (3) Methoxy polyethylene glycol units grafted on the carboxyl groups of hyaluronic acid; (4) An N-acetylcysteine group grafted onto a second primary alcohol group of hyaluronic acid; the copolymer has good biocompatibility, active and passive dual targeting and redox conversion, can be used for releasing carriers of anticancer drugs, realizes prolonged blood circulation, high ingestion and quick drug release of the drugs, effectively improves the concentration of the drugs in tumor cells, and has wide application prospect.
However, the above research results cannot achieve the effect of assisting the chemotherapeutic drugs in inhibiting tumor growth by releasing sulfur dioxide, and further result in a large dosage, wherein the dosage of the drugs of the derivatives prepared by the above patent technology in the experimental process is 15mg/kg, and the person skilled in the art knows that the larger the dosage, the side effects, the treatment cost and the organism drug resistance are increased.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an amphiphilic graft copolymer based on hyaluronic acid, and a preparation method and application thereof.
The method is realized by the following technical scheme:
a first object of the present invention is to provide: an amphiphilic hyaluronic acid-based graft copolymer comprising:
(1) Hyaluronic acid as a parent skeleton;
(2) Methoxy polyethylene glycol units grafted on the carboxyl groups of hyaluronic acid;
(3) 2, 4-dinitrobenzenesulfonic acid groups grafted onto the first primary alcohol group of hyaluronic acid;
(4) An octadecanoic acid group grafted onto a second primary alcohol group of hyaluronic acid.
An amphiphilic graft copolymer based on hyaluronic acid, having the structural formula:
wherein m is 7-323, and n is 12-1241.
The average molecular weight of the methoxy polyethylene glycol unit is 370-20000 Da.
The average molecular weight of the hyaluronic acid is 5100-500000 Da.
A second object of the present invention is to provide: the preparation method of the amphiphilic graft copolymer based on hyaluronic acid comprises the following steps:
(1) Dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst, uniformly mixing, stirring for 0.5-24 hours at the temperature of 0-60 ℃, adding methoxy polyethylene glycol, stirring for reacting for 2-96 hours at the temperature of 0-60 ℃, and freeze-drying a dialysis product by using distilled water to obtain a first reaction product methoxy polyethylene glycol-hyaluronic acid derivative, namely mPEG-HA;
(2) Dissolving 2, 4-dinitrobenzenesulfonic acid in a second reaction solvent, adding a second catalyst, uniformly mixing, stirring for 3-9 hours at 20-100 ℃, then adding mPEG-HA dissolved in a DMSO/pyridine mixed solution, continuously stirring for reacting for 6-24 hours to obtain a second reaction product, and freeze-drying the second reaction product to obtain a freeze-dried methoxypolyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid derivative, which is counted as mPEG-HA-DNs;
(3) Dissolving octadecanoic acid in a third reaction solvent, adding a third catalyst, carrying out catalytic reaction for 0.5-24h at 0-60 ℃, adding mPEG-HA-DNs, continuing to react for 2-48h to obtain a third reaction product, and carrying out freeze drying on the third reaction product to obtain a freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid-octadecanoic acid derivative, namely mPEG-HA (SA) -DNs, namely the amphiphilic graft copolymer based on hyaluronic acid.
The first reaction solvent, the second reaction solvent and the third reaction solvent are independently selected from at least one of formamide, thionyl chloride, N-dimethylformamide, N-dimethylacetamide, water, DMSO and pyridine.
The first catalyst and the third catalyst are independently selected from at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl), N-hydroxysuccinimide (NHS), 4-Dimethylaminopyridine (DMAP) and Dicyclohexylcarbodiimide (DCC).
The first catalyst is a combination of EDC, HCl and NHS; the molar ratio of EDC, HCl and NHS in the first catalyst is (1:10) - (10:1); the molar ratio of the first catalyst to hyaluronic acid is (1:100) - (100:1); the molar ratio of methoxy polyethylene glycol to hyaluronic acid is (1:10) - (100:1).
The second catalyst is N, N-Dimethylformamide (DMF); the molar ratio of the second catalyst to the 2, 4-dinitrobenzenesulfonic acid is (1:20) - (20:1); the molar ratio of 2, 4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10) - (110:1).
The third catalyst is a composition of EDC, HCl and NHS; the molar ratio of EDC, HCl and NHS in the third catalyst is (1:10) - (10:1); the molar ratio of the third catalyst to the octadecanoic acid is (1:5) - (5:1); the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10) - (200:1).
A third object of the present invention is to provide: the application of the amphiphilic graft copolymer based on hyaluronic acid or the amphiphilic graft copolymer based on hyaluronic acid prepared by the method in preparing anticancer drugs.
Further, the aforementioned hyaluronic acid-based amphiphilic graft copolymer or the hyaluronic acid-based amphiphilic graft copolymer prepared by the method thereof is used as an anticancer drug carrier.
Furthermore, the application of the amphiphilic graft copolymer based on hyaluronic acid or the amphiphilic graft copolymer based on hyaluronic acid prepared by the method in preparing anticancer drugs, in particular to the application of the amphiphilic graft copolymer based on hyaluronic acid as a coating for encapsulating anticancer active ingredients to form the anticancer drugs with tumor targeted release.
The anticancer active component is at least one of docetaxel, paclitaxel, epirubicin and camptothecine.
The beneficial effects are that:
(1) The amphiphilic graft copolymer based on hyaluronic acid has the characteristics of high drug loading capacity, high encapsulation efficiency, good biocompatibility, high safety and in vivo degradability.
(2) The amphiphilic graft copolymer based on hyaluronic acid is used as a drug carrier, the particle size of the carrier is small, and passive targeting can be realized through the EPR effect; modification of hyaluronic acid allows the copolymer to specifically bind to the CD44 receptor overexpressed on tumor cells, thereby achieving active targeting. Due to the modification of the 2, 4-dinitrobenzenesulfonic acid, the polymer can selectively release the loaded drug and SO under the condition of high glutathione concentration 2 Greatly increase the medicineThe utilization rate reduces the toxic and side effects and the dosage of the medicine. The copolymer carrier can quickly generate oxidation reduction in tumor cells, accurately release medicines and sulfur dioxide, and assist the treatment of chemotherapy medicines by the sulfur dioxide, so that the long circulation, high ingestion and quick release of the medicines in the body are realized, the high-efficiency treatment effect is maintained on the basis of reducing the dosage of the medicines, and the copolymer carrier has great application potential in the controlled release of the medicines.
(3) The amphiphilic graft copolymer based on hyaluronic acid takes octadecanoic acid and 2, 4-dinitrobenzene sulfonic acid groups as hydrophobic ends, and the 2, 4-dinitrobenzene sulfonic acid groups can be used for opening sulfonate bonds and releasing SO (SO) by using a redox mechanism in tumor tissue microenvironment 2 The characteristics of the anti-cancer drug are that the anti-cancer drug can be rapidly and targeted released in the organism, and the capability of the anti-cancer drug for inhibiting tumor is improved; and meanwhile, the stability of the micelle is improved by utilizing the octadecanoic acid group.
(4) According to the invention, polyethylene glycol and hyaluronic acid are used as main hydrophilic ends of the amphiphilic polymer carrier, wherein the hyaluronic acid is used as a target head, is combined with a CD44 receptor on the surface of a tumor cell and enters the tumor cell through endocytosis of the cell, and the problem of low cell uptake capacity of a common carrier micelle is solved.
(5) The amphiphilic graft copolymer based on hyaluronic acid disclosed by the invention is simple and convenient to prepare, wide in raw material source, easy to modify in structure and low in preparation condition requirement.
Drawings
FIG. 1 is a synthetic scheme of methoxypolyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid-octadecanoic acid derivative of example 1;
FIG. 2 is an infrared spectrum of the third product of example 1;
FIG. 3 is a nuclear magnetic resonance spectrum of the third product of example 1.
Detailed Description
The following detailed description of the invention is provided in further detail, but the invention is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the invention as claimed.
A first object of the present invention is to provide: an amphiphilic hyaluronic acid-based graft copolymer comprising:
(1) Hyaluronic acid as a parent skeleton;
(2) Methoxy polyethylene glycol units grafted on the carboxyl groups of hyaluronic acid;
(3) 2, 4-dinitrobenzenesulfonic acid groups grafted onto the first primary alcohol group of hyaluronic acid;
(4) An octadecanoic acid group grafted onto a second primary alcohol group of hyaluronic acid.
In the present invention, the positions of the first primary alcohol group grafted with a 2, 4-dinitrobenzenesulfonic acid group, the carboxyl group grafted with a methoxypolyethylene glycol unit, and the second primary alcohol group grafted with an octadecanoic acid group on the skeleton are not particularly limited, and the first primary alcohol group and the second primary alcohol group are only used to distinguish one from the other, and there is no limitation on the sequential positions and importance thereof on the skeleton.
In some preferred embodiments, the hyaluronic acid-based amphiphilic graft copolymer has the following structural formula:
wherein m is 7 to 323 (e.g., 50, 65, 80), and n is 12 to 1241 (e.g., 100, 150, 200).
In some preferred embodiments, the value of m is such that the average molecular weight of the methoxypolyethylene glycol units is 370 to 20000Da; for example 100, 1500, 3000, 4000, 15000 or 20000Da.
In some preferred embodiments, the value of n is such that the average molecular weight of the hyaluronic acid is 5100 to 500000Da; for example 20000, 25000, 30000 or 35000Da.
A second object of the present invention is to provide: the preparation method of the amphiphilic graft copolymer based on hyaluronic acid comprises the following steps:
(1) Dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst, uniformly mixing, stirring for 0.5-24h (for example, 0.5, 1, 2, 5, 8, 10, 12, 15, 18, 21 or 24 h) at 0-60 ℃ (for example, 0, 10, 20, 30, 40, 50 or 60 ℃), adding methoxypolyethylene glycol, stirring for 2-96h (for example, 2, 3, 6, 10, 14, 18, 24, 36, 72, 84 or 96 h) at 0-60 ℃ (for example, 0, 10, 20, 30, 40, 50 or 60 ℃), and freeze-drying by using distilled water to obtain a first reaction product methoxypolyethylene glycol-hyaluronic acid derivative, namely mPEG-HA;
(2) Dissolving 2, 4-dinitrobenzenesulfonic acid in a second reaction solvent, adding a second catalyst, uniformly mixing, stirring for 3-9h (for example, 3, 4, 6, 7, 8 or 9 h) at 20-100 ℃ (for example, 20, 30, 40, 60, 80, 90 or 100 ℃), then adding mPEG-HA dissolved in a DMSO/pyridine mixed solution, continuously stirring for reacting for 6-24h (for example, 6, 7, 9, 12, 18, 21 or 24 h), obtaining a second reaction product, and freeze-drying the second reaction product to obtain a freeze-dried methoxypolyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid derivative, which is calculated as mPEG-HA-DNs;
(3) Dissolving octadecanoic acid in a third reaction solvent, adding a third catalyst, carrying out catalytic reaction for 0.5-24h (for example, 0.5, 1, 2, 5, 8, 10, 12, 15, 18, 21 or 24 h) under the condition of 0-60 ℃ (for example, 0, 10, 20, 30, 40, 50 or 60 ℃), adding mPEG-HA-DNs, continuing to react for 2-48h (for example, 2, 3, 6, 10, 14, 18, 24, 36, 45 or 48 h), obtaining a third reaction product, and carrying out freeze drying on the third reaction product to obtain the freeze-dried methoxypolyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid-octadecanoic acid derivative which is calculated as mPEG-HA (SA) -DNs, namely, the amphiphilic graft copolymer based on hyaluronic acid.
In some preferred embodiments, the first, second, and third reaction solvents are independently selected from at least one of formamide, thionyl chloride, N-dimethylformamide, N-dimethylacetamide, water, DMSO, and pyridine.
In some preferred embodiments, the first catalyst and the third catalyst are independently selected from at least one of EDC HCl, NHS, DMAP, DCC.
In other preferred embodiments, the first catalyst is preferably a combination of EDC HCl and NHS; the molar ratio of edc.hcl to NHS in the first catalyst is (1:10) - (10:1), e.g., 1:2, 1:5, 1:10, 8:1, 5:1, 2:1, or 1:1; the molar ratio of the first catalyst to hyaluronic acid is (1:100) - (100:1), for example 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of methoxypolyethylene glycol to hyaluronic acid is (1:10) - (100:1), for example 1:2, 1:5, 1:8, 1:1, 2:1, 5:1, 6:1 or 8:1.
In some preferred embodiments, the second catalyst is N, N-Dimethylformamide (DMF); the molar ratio of the second catalyst to 2, 4-dinitrobenzenesulfonic acid is (1:20) - (20:1), for example 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of 2, 4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10) - (110:1), for example 1:2, 1:5, 1:10, 1:1, 2:1, 5:1, 6:1 or 8:1.
In other preferred embodiments, the third catalyst is preferably a combination of EDC HCl and NHS; the molar ratio of edc.hcl to NHS in the third catalyst is (1:10) - (10:1), e.g., 1:3, 1:6, 1:1, 8:1, 5:1, or 2:1; the molar ratio of the third catalyst to octadecanoic acid is (1:5) - (5:1), for example 1:2, 1:5, 5:1, 2:1 or 1:1; the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10) - (200:1) e.g. 1:2, 1:5, 1:8, 1:1, 2:1, 5:1, 6:1 or 8:1.
In a specific embodiment, the first and third reaction solvents are used in an amount of at least the minimum amount sufficient to dissolve hyaluronic acid; the second reaction solvent is used in an amount at least a minimum amount sufficient to dissolve the 2, 4-dinitrobenzenesulfonic acid; the third reaction solvent is used in an amount at least a minimum amount sufficient to dissolve the octadecanoic acid; the DMSO/pyridine mixed solution is used in an amount of at least the minimum amount sufficient to dissolve the first product mPEG-HA, wherein DMSO and pyridine are in any volume ratio.
A third object of the present invention is to provide: the application of the amphiphilic graft copolymer based on hyaluronic acid or the amphiphilic graft copolymer based on hyaluronic acid prepared by the method in preparing anticancer drugs.
Further, amphiphilic graft copolymer based on hyaluronic acid is used as a coating to encapsulate anticancer active ingredients, so that the anticancer drug with tumor targeted release is formed.
The anticancer active component is at least one of docetaxel, paclitaxel, epirubicin and camptothecine.
Further, a method for preparing an anticancer drug preparation based on the amphiphilic graft copolymer of hyaluronic acid comprises the following steps:
(1) Dissolving paclitaxel in acetone solution to obtain paclitaxel solution;
(2) Slowly dripping paclitaxel solution into amphiphilic graft copolymer based on hyaluronic acid by ultrasonic method, removing non-entrapped paclitaxel by dialysis bag, and lyophilizing to obtain anticancer medicinal preparation.
In other preferred embodiments, in step (1), the concentration of paclitaxel in the paclitaxel solution is between 0.01mg/mL and 10mg/mL (e.g., 0.1, 0.5, 1.0, 5.0 mg/mL).
In other preferred embodiments, in step (2), the ultrasound time is 1-8min (e.g., 1, 2,4, 7, or 8 min).
In other preferred embodiments, in step (2), the mass ratio of hyaluronic acid-based amphiphilic graft copolymer to paclitaxel is from 1:20 to 20:1, for example 1:7, 1:5, 1:2, 1:1, 2:1, 5:1 or 7:1.
The amphiphilic graft copolymer based on hyaluronic acid or the amphiphilic graft copolymer based on hyaluronic acid prepared by the method has excellent biocompatibility and can be used for drug entrapment. The invention utilizes CD44 receptor active targeting, EPR mediated receptor passive targeting and SO in the delivery process for anticancer drugs 2 The auxiliary treatment is released, so as to achieve the purposes of improving the uptake of the medicine in tumor tissues and increasing the curative effect of the medicine. Therefore, the novel material has wide application prospect.
Example 1
A method for preparing an amphiphilic graft copolymer based on hyaluronic acid, comprising the following steps:
(1) Synthesis of first product mPEG-HA
88.3mgEDC, HCl, 53.0mgNHS and 183mg hyaluronic acid are respectively weighed, in a round bottom flask, the hyaluronic acid is dissolved in 50mL of first reaction solvent, then EDC, HCl and NHS are added and uniformly mixed, and stirring and activating are carried out for 5 hours at room temperature; after the reaction is completed, 550mg of methoxy polyethylene glycol is added, and stirring is continued for 14 hours at room temperature; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a first product;
when the first product is synthesized, the molecular weight of the adopted hyaluronic acid is 20000, and the first reaction solvent is formamide; the molecular weight of the methoxy polyethylene glycol is 1200;
(2) Synthesis of second product mPEG-HA-DNs
Measuring 240mg of 2, 4-dinitrobenzenesulfonic acid and 180mg of the first product, dissolving the 2, 4-dinitrobenzenesulfonic acid in 9mL of a second reaction solvent in a round bottom flask, adding 1mL of a second catalyst DMF, and stirring at 60 ℃ for reaction for 5 hours; subsequently, 180mg of the first product is dissolved in 61.5mL of a DMSO/pyridine (60:1.5, v/v) mixed solution, slowly dropped into the reaction solution under ice bath, stirred at normal temperature for reaction for 24 hours, dialyzed for 48 hours by distilled water, and freeze-dried to obtain a second product;
in the synthesis of the second product, the second reaction solvent is thionyl chloride, which is used for activating the sulfonic acid group with a second catalyst DMF;
(3) Synthesis of third product mPEG-HA (SA) -DNs
192 mgEDC.HCl, 115mgNHS and 190mg octadecanoic acid are respectively weighed; in a round bottom flask, stearic acid is dissolved in 50mL of a third reaction solvent, EDC, HCl and NHS are added and mixed uniformly, and stirring and activating are carried out for 10h at room temperature; after the reaction was completed, 150mg of the second product was added and stirring was continued at room temperature for 14h; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a third product;
in the synthesis of the third product, the third reaction solvent is dimethyl sulfoxide;
the inventors finally carried out infrared detection and nuclear magnetic detection on the finally obtained product mPEG-HA (DNs) -SA, and the results are shown in figures 2 and 3.
In the infrared spectrum shown in FIG. 2, at 1616cm -1 The absorption peak at (a) is assigned to the absorption peak of C=O (carboxyl) on Hyaluronic Acid (HA), 1738cm -1 An absorption peak belonging to c=o (ester carbonyl) on the second product, at 934cm -1 The absorption peak at DNs is that of S-O at 2985cm -1 at-CH 2 The telescopic vibration absorption peak belongs to the characteristic absorption peak of SA, thereby proving that mPEG-HA (SA) -DNs HAs been successfully synthesized.
Example 2
A method for preparing an amphiphilic graft copolymer based on hyaluronic acid, comprising the following steps:
(1) Synthesis of first product mPEG-HA
Respectively weighing 94mgDCC and 183mg hyaluronic acid, dissolving hyaluronic acid in 50mL of first reaction solvent in a round bottom flask, adding DCC, mixing uniformly, stirring and activating at room temperature for 5h; after the reaction is completed, 550mg of methoxy polyethylene glycol is added, and stirring is continued for 14 hours at room temperature; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a first product;
when the first product is synthesized, the molecular weight of the adopted hyaluronic acid is 20000, and the first reaction solvent is formamide; the molecular weight of the methoxy polyethylene glycol is 1200;
(2) Synthesis of second product mPEG-HA-DNs
Measuring 240mg of 2, 4-dinitrobenzenesulfonic acid and 180mg of the first product, dissolving the 2, 4-dinitrobenzenesulfonic acid in 9mL of a second reaction solvent in a round bottom flask, adding 1mL of a second catalyst DMF, and stirring at 60 ℃ for reaction for 5 hours; dissolving 180mg of the first product in 61.5mL of DMSO/pyridine (60:1.5, v/v) mixed solution, slowly dripping the solution into the reaction solution under ice bath, stirring at normal temperature for reaction for 24h, dialyzing for 48h by using distilled water, and freeze-drying to obtain a second product;
in the synthesis of the second product, the second reaction solvent is thionyl chloride, which is used for activating the sulfonic acid group with a second catalyst DMF;
(3) Synthesis of third product mPEG-HA (SA) -DNs
Respectively weighing 207mg DCC and 190mg octadecanoic acid; in a round bottom flask, octadecanoic acid is dissolved in 50mL of a third reaction solvent, DCC is added and mixed uniformly, and stirring and activating are carried out for 10h at room temperature; after the reaction was completed, 150mg of the second product was added and stirring was continued at room temperature for 14h; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a third product;
in synthesizing the third product, the third reaction solvent is dimethyl sulfoxide.
Example 3
A method for preparing an amphiphilic graft copolymer based on hyaluronic acid, comprising the following steps:
(1) Synthesis of first product mPEG-HA
56mgDMAP and 183mg hyaluronic acid are weighed respectively; dissolving hyaluronic acid in 50mL of a first reaction solvent in a round-bottom flask, then adding DMAP, uniformly mixing, and stirring and activating for 5h at room temperature; after the reaction is completed, 550mg of methoxy polyethylene glycol is added, and stirring is continued for 14 hours at room temperature; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a first product;
when the first product is synthesized, the molecular weight of the adopted hyaluronic acid is 20000, and the first reaction solvent is formamide; the molecular weight of the methoxy polyethylene glycol is 1200;
(2) Synthesis of second product mPEG-HA-DNs
Measuring 240mg of 2, 4-dinitrobenzenesulfonic acid and 180mg of the first product; dissolving 2, 4-dinitrobenzenesulfonic acid in 9mL of a second reaction solvent in a round bottom flask, adding 1mL of a second catalyst, and stirring at 60 ℃ for reaction for 5h; dissolving 180mg of the first product in 61.5ml of mixed solution of LDMSO/pyridine (60:1.5, v/v), slowly dripping the solution into the reaction solution under ice bath, stirring at normal temperature for reaction for 24 hours, dialyzing for 48 hours by using distilled water, and freeze-drying to obtain a second product;
in the synthesis of the second product, the second reaction solvent is thionyl chloride, which is used for activating the sulfonic acid group with a second catalyst DMF;
(3) Synthesis of third product mPEG-HA (SA) -DNs
122mgDMAP and 190mg octadecanoic acid are respectively weighed; in a round bottom flask, stearic acid is dissolved in 50mL of a third reaction solvent, DMAP is added and mixed uniformly, and stirring and activating are carried out for 10h at room temperature; after the reaction was completed, 150mg of the second product was added and stirring was continued at room temperature for 14h; after the reaction is finished, dialyzing the mixture by using distilled water, and freeze-drying to obtain a third product;
in synthesizing the third product, the third reaction solvent is dimethyl sulfoxide.
Comparative example 1
Step (1) was performed in the same manner as in example 1 to obtain mPEG-HA.
Comparative example 2
Step (1) and step (3) were performed in the same manner as in example 1 to obtain mPEG-HA-SA.
Comparative example 3
The procedure of example 1 was followed to give mPEG-HA-DNs.
Comparative example 4
Step (2) was performed in the same manner as in example 1, except that the first product was replaced with hyaluronic acid to prepare HA-DNs.
Application examples 1 to 9
These application examples relate to the use of the hyaluronic acid-based amphiphilic graft copolymer of the present invention as an anticancer drug release carrier, wherein an anticancer drug release composition comprising the amphiphilic graft copolymer and an anticancer drug entrapped by the amphiphilic graft copolymer is prepared. The anticancer drug release composition is nanoparticle containing mPEG-HA (SA) -DNs entrapped Paclitaxel (PTX). Firstly, dissolving PTX with different mass into 0.5mL of acetone to prepare PTX solution; the mPEG-HA (SA) -DNs prepared in examples 1-3 (10 mg each) was dissolved in 10mL distilled water, and the PTX solution was added dropwise under ultrasound conditions for 5min to allow PTX to fully enter the mPEG-HA (SA) -DNs. Thereafter, the above suspension was dialyzed using a dialysis bag (molecular weight cut-off: 3500 Da) to remove excess PTX, and finally the dialyzed product was lyophilized, thereby producing an anticancer drug releasing composition. The particle size, encapsulation efficiency and drug loading rate of the drug-loaded nanoparticles in the anticancer drug-releasing composition and the weight percentage of the anticancer drug in the anticancer drug-releasing composition were then measured, and the results are shown in table 1.
Comparative application example 1
An anticancer drug releasing composition was prepared in substantially the same manner as in application example 1, except that the mPEG-HA prepared in comparative application example 1 was used in place of mPEG-HA (SA) -DNs.
Comparative application example 2
An anticancer drug releasing composition was prepared in substantially the same manner as in application example 1, except that the mPEG-HA-SA prepared in comparative application example 2 was used instead of mPEG-HA (SA) -DNs.
Comparative application example 3
An anticancer drug releasing composition was prepared in substantially the same manner as in application example 1, except that the mPEG-HA-DNs prepared in comparative application example 3 was used instead of mPEG-HA (SA) -DNs.
Comparative application example 4
An anticancer drug releasing composition was prepared in substantially the same manner as in application example 1, except that comparative application example 4 used HA-DNs prepared in comparative example 4 instead of mPEG-HA (SA) -DNs.
Safety test
To evaluate whether the prepared anticancer drug release composition was safe for intravenous administration, an in vitro hemolysis test was performed on the prepared anticancer drug release composition. Collecting New Zealand white rabbit ear venous blood, removing fibrinogen, adding normal saline, washing, centrifuging, and making into 2% (v/v) suspension; taking 2.5mL of erythrocyte suspension and 2.5mL of anticancer drug release composition (1 mg/mL), and uniformly mixing; after incubating the mixture at 37 ℃ for 4 hours, centrifuging for 10 minutes at a rotating speed of 3000r/min, collecting supernatant, and measuring the absorbance of the sample at 540 nm; physiological saline and distilled water are additionally taken, and the normal saline and distilled water are operated in the same way and respectively used as negative and positive controls to calculate the percentage of hemolysis; percent hemolysis = (sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance) ×100%. The results are shown in Table 1 below.
TABLE 1 anticancer drug entrapping effect and safety of the prepared anticancer drug releasing composition
The results show that all the formulations in application examples 1 to 9 have higher encapsulation efficiency and drug loading, and the particle size is smaller than 200nm, and the encapsulation efficiency decreases and the particle size increases with increasing initial drug loading.
In addition, when the concentration of paclitaxel is in the range of 0-200. Mu.g/mL, the hemolytic activity of the anticancer drug releasing composition is almost negligible, and the percentage of hemolysis is not more than 3.5%, which indicates that the preparation has good hemolysis safety.
Antitumor effect test:
cervical cancer U14 tumor-bearing mice model was constructed, tumor-bearing mice were randomly divided into 4 groups of 5 animals, each group of animals was labeled with a number and started to be administered with physiological saline, the anticancer drug release compositions (dose 10 mg/kg) prepared in application example 1 and comparative application examples 1 and 2, respectively (treatment group), and this day was designated as administration day 1. The preparation was administered once every 3 days by tail vein injection, and 4 times in succession. Animals were sacrificed, tumor masses were removed, weighed, and tumor inhibition rates were calculated over 4 dosing cycles. Tumor inhibition rate= (mean tumor weight in saline group-mean tumor weight in treatment group)/mean tumor weight in saline group x 100%. The result shows that the tumor inhibition rate of the anticancer drug release composition is 82.60%, so that the anticancer drug release composition has obvious tumor growth inhibition effect on cervical cancer.
In addition, the present inventors performed experiments using H22 liver cancer mice. First, mice were randomly divided into 5 groups of 5 animals each, each group being individually labeled with a number. Then, administration was started, wherein physiological saline (control group), the anticancer drug releasing compositions (dose 10 mg/kg) prepared in application example 1 and comparative application examples 1 to 4 were administered respectively (treatment group), and this day was noted as day 1 of administration. The administration was carried out once every 3 days by tail vein injection, and 4 times in succession. Animals were sacrificed, tumor masses were removed, weighed, and tumor inhibition rates were calculated over 4 dosing cycles. As a result, it was found that the average tumor suppression rate of the anticancer drug delivery composition prepared in application example 1 was 75.10%, and thus it was found that it had an obvious tumor growth suppression effect on liver cancer.
In addition, lewis lung carcinoma tumor bearing mice were also used for the test. Wherein the mice were randomly divided into 5 groups of 5 animals each, each group of animals was individually assigned a marker number. When administered, physiological saline, the anticancer drug releasing compositions (dose 10 mg/kg) prepared in application example 1 and comparative application examples 1 to 4, respectively (treatment group), were administered, and this day was designated as day 1 of administration. The administration was carried out once every 3 days by tail vein injection, and 4 times in succession. Animals were sacrificed, tumor masses were removed, weighed, and tumor inhibition rates were calculated over 4 dosing cycles. As a result, it was found that the average tumor suppression rate of the anticancer drug delivery composition prepared in application example 1 was 77.20%, and thus it had an obvious tumor growth suppression effect on lung cancer.
TABLE 2 tumor inhibition rate of anticancer drug delivery composition
From the data shown in table 2, it can be seen that the anticancer drug release composition prepared by the invention has remarkable inhibition effect (the tumor inhibition rate is 82.60%) on tumor cells of the U14 mice, and also has remarkable inhibition effect on other cancer cells such as liver cancer and lung cancer, and the tumor inhibition rates are 75.10% and 77.20%, respectively.
In addition, the invention effectively reduces the dosage of the anticancer drug, which is probably due to the auxiliary treatment of the released sulfur dioxide and the effect result of accurate release, thus obviously reducing the dosage of the drug and greatly reducing the side effect of the drug on the basis of ensuring the tumor inhibition rate.
It is noted that the above examples and test examples are only limited to further explanation and understanding of the technical solutions of the present invention, and are not to be construed as further limiting the technical solutions of the present invention, and the invention without significant essential features and significant improvements made by those skilled in the art still falls within the scope of protection of the present invention.

Claims (9)

1. An amphiphilic graft copolymer based on hyaluronic acid, characterized in that it comprises:
(1) Hyaluronic acid as a parent skeleton;
(2) Methoxy polyethylene glycol units grafted on the carboxyl groups of hyaluronic acid;
(3) 2, 4-dinitrobenzenesulfonic acid groups grafted onto the first primary alcohol group of hyaluronic acid;
(4) An octadecanoic acid group grafted onto a second primary alcohol group of hyaluronic acid.
2. The amphiphilic hyaluronic acid-based graft copolymer of claim 1, having the structural formula:
wherein m is 7-323, n is 12-1241;
the average molecular weight of the methoxy polyethylene glycol unit is 370-20000 Da;
the average molecular weight of the hyaluronic acid is 5100-500000 Da.
3. The method for preparing the amphiphilic graft copolymer based on hyaluronic acid according to claim 1 or 2, comprising the following steps:
(1) Dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst, uniformly mixing, stirring for 0.5-24 hours at the temperature of 0-60 ℃, adding methoxy polyethylene glycol, stirring for reacting for 2-96 hours at the temperature of 0-60 ℃, and freeze-drying a dialysis product by using distilled water to obtain a first reaction product methoxy polyethylene glycol-hyaluronic acid derivative, namely mPEG-HA;
(2) Dissolving 2, 4-dinitrobenzenesulfonic acid in a second reaction solvent, adding a second catalyst, uniformly mixing, stirring for 3-9 hours at 20-100 ℃, then adding mPEG-HA dissolved in a DMSO/pyridine mixed solution, continuously stirring for reacting for 6-24 hours to obtain a second reaction product, and freeze-drying the second reaction product to obtain a freeze-dried methoxypolyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid derivative, which is counted as mPEG-HA-DNs;
(3) Dissolving octadecanoic acid in a third reaction solvent, adding a third catalyst, carrying out catalytic reaction for 0.5-24h at 0-60 ℃, adding mPEG-HA-DNs, continuing to react for 2-48h to obtain a third reaction product, and carrying out freeze drying on the third reaction product to obtain a freeze-dried product methoxy polyethylene glycol-hyaluronic acid-2, 4-dinitrobenzenesulfonic acid-octadecanoic acid derivative, namely mPEG-HA (SA) -DNs, namely the amphiphilic graft copolymer based on hyaluronic acid.
4. The method for preparing the amphiphilic graft copolymer based on hyaluronic acid according to claim 3, wherein the first reaction solvent, the second reaction solvent and the third reaction solvent are independently selected from at least one of formamide, thionyl chloride, N-dimethylformamide, N-dimethylacetamide, water, DMSO and pyridine;
the first catalyst and the third catalyst are independently selected from at least one of EDC HCl, NHS, DMAP, DCC;
the second catalyst is DMF.
5. The method for preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 4, wherein the first catalyst is a combination of EDC, HCl and NHS; the molar ratio of EDC, HCl and NHS in the first catalyst is (1:10) - (10:1); the third catalyst is a composition of EDC, HCl and NHS; the molar ratio of EDC & HCl to NHS in the third catalyst is (1:10) - (10:1).
6. The method of preparing an amphiphilic graft copolymer based on hyaluronic acid according to claim 4, wherein the molar ratio of the first catalyst to hyaluronic acid is (1:100) - (100:1); the molar ratio of the methoxy polyethylene glycol to the hyaluronic acid is (1:10) - (100:1); the molar ratio of the second catalyst to the 2, 4-dinitrobenzenesulfonic acid is (1:20) - (20:1); the molar ratio of the 2, 4-dinitrobenzenesulfonic acid to mPEG-HA is (1:10) - (110:1); the molar ratio of the third catalyst to the octadecanoic acid is (1:5) - (5:1); the molar ratio of octadecanoic acid to mPEG-HA-DNs is (1:10) - (200:1).
7. Use of the amphiphilic hyaluronic acid-based graft copolymer according to claim 1 or 2 or the amphiphilic hyaluronic acid-based graft copolymer prepared by the method according to any of claims 3-6 in the manufacture of an anticancer drug.
8. The use of the amphiphilic hyaluronic acid-based graft copolymer for preparing anticancer drugs according to claim 7, wherein the amphiphilic hyaluronic acid-based graft copolymer is used as a coating to encapsulate anticancer active ingredients, thereby forming anticancer drugs with tumor targeted release.
9. The use of the amphiphilic hyaluronic acid-based graft copolymer for preparing anticancer drugs according to claim 8, wherein the anticancer active ingredient is at least one of docetaxel, paclitaxel, epirubicin, camptothecine.
CN202310503281.1A 2023-05-06 2023-05-06 Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof Pending CN116515011A (en)

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