CN113750255B - Environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle and preparation method and application thereof - Google Patents
Environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle and preparation method and application thereof Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 76
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Classifications
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
-
- 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
- A61K31/365—Lactones
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- 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
- 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
Abstract
The invention discloses an environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle, a preparation method and application thereof, which are respectively a pH-sensitive prodrug micelle and a pH-reduction double-sensitive prodrug micelle. Starting from hyaluronic acid and podophyllotoxin as raw materials, firstly, modifying the hyaluronic acid by using ethylenediamine or cystamine to synthesize aminated hyaluronic acid, then, carboxylating the podophyllotoxin by using succinic anhydride, and finally, carrying out condensation reaction on the aminated hyaluronic acid and carboxylated podophyllotoxin to obtain the polymer prodrug. The synthesized prodrug can form micelle in aqueous solution by self-assembly, has good blood compatibility, and is suitable for intravenous injection. The invention also provides application of the prodrug micelle in drug delivery, has controllable drug release behavior, has targeting capability on tumor cells, has good anti-tumor effect, and has wide application prospect.
Description
Technical Field
The invention discloses an environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle and a preparation method and application thereof, belonging to the technical field of novel hyaluronic acid nano drug carriers.
Background
Cancer has become one of the major diseases that seriously jeopardize human health, and its morbidity and mortality are high. Chemotherapy remains the most widely used treatment modality at present. However, the existing antitumor drugs have strong killing effect on tumor cells, and meanwhile, the existing antitumor drugs also cause serious damage to normal cells, thereby causing multiple complications and limiting the clinical application and development of the antitumor drugs. Currently, nanoparticle drug delivery vehicles have made tremendous progress in cancer treatment, particularly polymeric prodrug micelles. These polymeric prodrug micelles can passively accumulate in tumor tissue with high stability and prolonged blood circulation time. However, nonspecific targeting and low drug release efficiency in cells limit their antitumor effects. Therefore, it is necessary to develop environmentally responsive prodrug micelles with specific targeting. The pH-based and reduction-sensitive polymer prodrug micelles allow for controlled drug delivery and release depending on the apparent differences in pH and Glutathione (GSH) concentration between tumor and normal cells.
Hyaluronic Acid (HA) is a natural polymer material, can be specifically combined with CD44 receptor over-expressed on the surface of tumor cells, HAs no immunogenicity, and can be used as a comparatively ideal drug delivery carrier. Therefore, the hyaluronic acid targeting-based polymer prodrug micelle with environmental response can realize the specific distribution of the drug among different tissues, thereby achieving the purpose of high-efficiency drug administration and reducing the damage of the drug to normal tissues.
Podophyllotoxin (PPT) is a lignin compound with natural activity, can effectively inhibit the assembly of tumor cell microtubules, and has remarkable anti-tumor activity. However, the use of podophyllotoxins in tumor therapy is limited due to the off-target effect and serious toxic side effects caused by poor water solubility.
The invention synthesizes two environmental sensitive prodrugs, namely, the pH sensitive hyaluronic acid-podophyllotoxin prodrug (HA-NH-CO-PPT) and the pH and reduction double sensitive prodrug (HA-S-S-PPT) from the raw materials of hyaluronic acid and podophyllotoxin, can self-assemble in water to form micelles, is used for treating cancers, and provides technical reference for delivery and treatment of other similar drugs.
Disclosure of Invention
Aiming at the defects of poor water solubility, nonspecific accumulation and release, serious toxic and side effects and the like of the conventional chemotherapy drug podophyllotoxin, the invention provides an environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-S-PPT) and a preparation method and application thereof. The invention has the characteristics of specific targeting, controllable drug release, good biocompatibility and the like.
The technical scheme of the invention is as follows:
it is a first object of the present invention to provide a method for preparing an environmentally-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-PPT).
The preparation method of the environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-S-PPT) comprises the following steps:
(1)HA-NH 2 or HA-S-S-NH 2 Is prepared from the following steps: ethylenediamine or cystamine dihydrochloride is dissolved in dimethyl sulfoxide (DMSO), then solution 1 is added and reacted at 25 ℃ for 24-48 hours. After the reaction is finished, the obtained mixture is put into a dialysis bag and dialyzed by deionized water, filtered and freeze-dried to obtain HA-NH 2 Or HA-S-S-NH 2 A solid.
The preparation method of the solution 1 comprises the following steps: hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in PBS solution and reacted under ice bath conditions for 1-2 hours to activate the carboxyl groups of HA, to obtain solution 1.
(2) Preparation of PPT-COOH: succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) are dissolved in toluene solution, refluxed for 48-72 hours at 105-110 ℃, the solvent is removed, and dried in vacuum to obtain carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT or HA-S-S-PPT: HA-NH 2 Or HA-S-S-NH 2 The solid was dissolved in PBS solution, after which solution 2 was added and reacted at 25℃for 24-48 hours. After the reaction is finished, the obtained mixture is put into a dialysis bag and dialyzed by deionized water, filtered and freeze-dried to obtain HA-NH-CO-PPT or HA-S-S-PPT solid.
The preparation method of the solution 2 comprises the following steps: PPT-COOH, EDC and NHS were dissolved in N, N-Dimethylformamide (DMF) and reacted under ice bath conditions for 1-2 hours to give solution 2.
(4) Preparation of HA-NH-CO-PPT or HA-S-S-PPT prodrug micelle: dissolving the HA-NH-CO-PPT or the HA-S-S-PPT solid in ultrapure water, carrying out ultrasonic treatment for 10-20 minutes to uniformly disperse the HA-NH-CO-PPT or the HA-S-S-PPT solid, and standing for 3-6 hours to obtain the HA-NH-CO-PPT or the HA-S-S-PPT prodrug micelle.
Preferably, in step (1), the HA HAs a molecular weight of 3000-10000Da.
Preferably, in the step (1), the molar ratio of HA, EDC and NHS is 1:0.5:0.5-1:1:1; the molar ratio of HA to ethylenediamine is 1:0.5-1:2; the molar ratio of HA to cystamine dihydrochloride is 1:0.5-1:5.
Preferably, in the step (2), the molar ratio of PPT, SA and DMAP is 1:1:1-10:15:1.
Preferably, in the step (2), the method for removing the solvent is as follows: the mixture was distilled off and washed with anhydrous methanol.
Preferably, in the step (3), the molar ratio of PPT-COOH, EDC and NHS is 1:0.5:0.5-1:1:1; the HA-NH 2 Or HA-S-S-NH 2 And a PPT-COOH molar ratio of 1:0.5 to 1:30.
Preferably, in steps (1) and (3), the PBS solution has a pH of 8.0.
Preferably, in steps (1) and (3), the resulting mixture is placed in a dialysis bag and dialyzed against deionized water for 48-72 hours, with deionized water being replaced every 3-12 hours.
Preferably, in steps (1) and (3), the dialysis bag has a molecular weight cut-off of 1000-2000Da.
Preferably, the HA-NH-CO-PPT prodrug micelle is a pH-sensitive hyaluronic acid-podophyllotoxin prodrug micelle, and the HA-S-S-PPT prodrug micelle is a pH-reduced double-sensitive hyaluronic acid-podophyllotoxin prodrug micelle.
Specifically, when the environmentally-responsive hyaluronic acid-podophyllotoxin prodrug micelle is a pH-sensitive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT), the steps are as follows:
(1)HA-NH 2 is prepared from the following steps: hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in PBS solution at pH 8.0 and reacted under ice bath conditions for 1-2 hours to activate the carboxyl groups of HA to obtain solution 1. Then, ethylenediamine was dissolved in dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, followed by reaction at 25℃for 24 to 48 hours. After the reaction is finished, the obtained mixture is placed into a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and the deionized water is replaced every 3 to 12 hours. Finally, the solution is filtered and lyophilized to obtain HA-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) are dissolved in toluene solution and refluxed at 105-110 ℃ for 48-72 hours, and the mixture is distilled and washed with anhydrous methanol, and dried in vacuum to obtain carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH, EDC and NHS were dissolved in N, N-Dimethylformamide (DMF) and reacted under ice bath conditions for 1-2 hours to give solution 2. HA-NH is then added 2 After dissolving in PBS solution with pH 8.0, the solution was added to the above solution 2, and reacted at 25℃for 24 to 48 hours. After the reaction is finished, the obtained mixture is placed into a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and the deionized water is replaced every 3 to 12 hours. Finally, filtering and freeze-drying to obtain the HA-NH-CO-PPT solid.
(4) Preparation of HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT in ultrapure water, carrying out ultrasonic treatment for 10-20 minutes to uniformly disperse the HA-NH-CO-PPT, and standing for 3-6 hours to obtain the HA-NH-CO-PPT prodrug micelle.
When the environmental response type hyaluronic acid-podophyllotoxin prodrug micelle is a pH, reduction double-sensitive hyaluronic acid-podophyllotoxin prodrug micelle (HA-S-S-PPT), the following steps are carried out:
(1)HA-S-S-NH 2 is prepared from the following steps: hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in PBS solution at pH 8.0 and reacted under ice bath conditions for 1-2 hours to activate the carboxyl groups of HA to obtain solution 1. Then, cystamine dihydrochloride was dissolved in dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, and reacted at 25℃for 24 to 48 hours. After the reaction is finished, the obtained mixture is placed into a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and the deionized water is replaced every 3 to 12 hours. Finally, the solution is filtered and lyophilized to obtain HA-S-S-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) are dissolved in toluene solution and refluxed at 105-110 ℃ for 48-72 hours, and the mixture is distilled and washed with anhydrous methanol, and dried in vacuum to obtain carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH, EDC and NHS were dissolved in N, N-Dimethylformamide (DMF) and reacted under ice bath conditions for 1-2 hours to give solution 2. Then HA-S-S-NH 2 After dissolving in PBS solution with pH 8.0, the solution was added to the above solution 2, and reacted at 25℃for 24 to 48 hours. After the reaction is finished, the obtained mixture is placed into a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and the deionized water is replaced every 3 to 12 hours. Finally, filtering and freeze-drying to obtain the HA-S-S-PPT solid.
(4) Preparation of HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT in ultrapure water, carrying out ultrasonic treatment for 10-20 minutes to uniformly disperse the HA-S-S-PPT, and standing for 3-6 hours to obtain the HA-S-S-PPT prodrug micelle.
The second object of the present invention is to provide an environmentally-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-PPT) prepared by the above method.
A third object of the present invention is to provide an application of the above-mentioned environmental-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-PPT) in preparing a medicament for treating cancer, which is breast cancer, lung cancer or liver cancer.
The environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle prepared by the invention is prepared from a targeting material hyaluronic acid and an anticancer drug podophyllotoxin, is an amphipathic derivative synthesized through chemical bond connection, can self-assemble in an aqueous solution to form a micelle with smaller particle size and uniform dispersion, improves the water solubility and biocompatibility of the podophyllotoxin, can target to tumor tissues, is controlled to release the drug by responding to low pH in the tumor microenvironment, is controlled to release the drug by responding to low pH and is reduced to high concentration glutathione in the tumor microenvironment, and realizes efficient drug delivery and enhanced antitumor effect.
The invention has the beneficial effects that:
the invention provides two preparation methods of an environmentally-sensitive targeted prodrug micelle, namely a pH-sensitive prodrug micelle and a pH-sensitive and reduction-sensitive prodrug micelle. Starting from hyaluronic acid and podophyllotoxin, the hyaluronic acid is modified by ethylenediamine or cystamine to synthesize aminated hyaluronic acid (HA-NH) 2 And HA-S-S-NH 2 ) And then carboxylating podophyllotoxin by succinic anhydride, and finally carrying out condensation reaction on the aminated hyaluronic acid and the carboxylated podophyllotoxin to obtain polymer prodrugs (HA-NH-CO-PPT and HA-S-S-PPT). The synthesized prodrug can form micelle in aqueous solution by self-assembly, has good blood compatibility, and is suitable for intravenous injection. The invention also provides application of the HA-NH-CO-PPT and the HA-S-S-PPT prodrug micelle in drug delivery, HAs controllable drug release behavior, HAs targeting capability on tumor cells, HAs good anti-tumor effect and HAs wide application prospect.
Drawings
FIG. 1 is a synthetic route to HA-NH-CO-PPT and HA-S-S-PPT prodrugs;
FIG. 2 is a FT-IR spectrum of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug prepared in example 5;
FIG. 3 shows the HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug prepared in example 5 1 H NMR spectrum;
FIG. 4 is a TEM photograph of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5;
FIG. 5a is a graph showing the change in particle size of HA-NH-CO-PPT prodrug micelles prepared in example 1 in response to different pH values;
FIG. 5b is a graph showing the change in particle size of HA-S-S-PPT prodrug micelles prepared in example 5 by responding to different pH and GSH;
FIG. 6 is an in vitro drug release behavior of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5;
FIG. 7 is the blood compatibility of HA-NH-CO-PPT prepared in example 2 and HA-S-S-PPT prodrug micelles prepared in example 6;
FIG. 8 is a targeting of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5;
FIG. 9 shows cytotoxicity of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and all experimental equipment, materials, reagents and the like used can be purchased from chemical companies.
Example 1
(1)HA-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, ethylenediamine in an amount equal to the molar amount of HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the solution 1 was added thereto, followed by reaction at 25℃for 24Hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-NH 2 。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-NH is added in the equimolar amount with PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain the HA-NH-CO-PPT.
(4) Preparation of HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-NH-CO-PPT, and standing for 3 hours to obtain the HA-NH-CO-PPT prodrug micelle.
Example 2
(1)HA-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, ethylenediamine in an amount equal to that of HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the solution 1 was added thereto, followed by reaction at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-NH 2 。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-NH is added in the equimolar amount with PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain the HA-NH-CO-PPT.
(4) Preparation of HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (5 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-NH-CO-PPT, and standing for 3 hours to obtain the HA-NH-CO-PPT prodrug micelle.
Example 3
(1)HA-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, ethylenediamine in an amount equal to that of HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the solution 1 was added thereto, followed by reaction at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-NH 2 。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 72 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 in 5mLN, N-diIn methylformamide (DMF), the reaction was carried out for 1 hour in ice bath to obtain solution 2. Then the HA-NH is added in the equimolar amount with PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain the HA-NH-CO-PPT.
(4) Preparation of HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-NH-CO-PPT, and standing for 3 hours to obtain the HA-NH-CO-PPT prodrug micelle.
Example 4
(1)HA-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, ethylenediamine in an amount equal to that of HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the solution 1 was added thereto, followed by reaction at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-NH 2 。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-NH is added in the equimolar amount with PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 48 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with replacement every 6 hoursAnd (3) ionized water. Filtering and freeze-drying to obtain the HA-NH-CO-PPT.
(4) Preparation of HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-NH-CO-PPT, and standing for 3 hours to obtain the HA-NH-CO-PPT prodrug micelle.
Example 5
(1)HA-S-S-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, an equimolar amount of cystamine dihydrochloride to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, and reacted at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-S-S-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-S-S-NH with the same molar quantity as PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparation of HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-S-S-PPT, and standing for 3 hours to obtain the HA-S-S-PPT prodrug micelle.
Example 6
(1)HA-S-S-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, an equimolar amount of cystamine dihydrochloride to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, and reacted at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-S-S-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-S-S-NH with the same molar quantity as PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparation of HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (5 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-S-S-PPT, and standing for 3 hours to obtain the HA-S-S-PPT prodrug micelle.
Example 7
(1)HA-S-S-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054. Mu. Mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with molecular weight of 3000-10000DaRatio 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, an equimolar amount of cystamine dihydrochloride to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, and reacted at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-S-S-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 72 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-S-S-NH with the same molar quantity as PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 24 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparation of HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-S-S-PPT, and standing for 3 hours to obtain the HA-S-S-PPT prodrug micelle.
Example 8
(1)HA-S-S-NH 2 Is prepared from the following steps: hyaluronic acid (HA, 400mg, equivalent to 1054 μmol COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da in a molar ratio of 1:1:1 was dissolved in PBS solution (5 mL) having pH 8.0, and reacted under ice bath conditions for 1 hour to activate the carboxyl group of HA, to obtain solution 1. Then, an equimolar amount of cystamine dihydrochloride to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and then the above solution 1 was added thereto, and reacted at 25℃for 24 hours. After the reaction, the resulting mixture was reactedThe dialysis was performed with deionized water (MWCO 2000) for 72 hours, with deionized water being replaced every 6 hours. Finally, the solution is filtered and lyophilized to obtain HA-S-S-NH 2 A solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5 mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were combined in a molar ratio of 15:10:1 was dissolved in 20mL of toluene and refluxed at 110℃for 48 hours, and the mixture was distilled off and washed with anhydrous methanol, and dried in vacuo to give carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. Mu. Mol), EDC and NHS were combined in a molar ratio of 1:1:1 was dissolved in 5mLN, N-Dimethylformamide (DMF), and reacted under ice bath condition for 1 hour to obtain a solution 2. Then the HA-S-S-NH with the same molar quantity as PPT-COOH 2 After being dissolved in 5mL of PBS (pH 8.0), the solution was added to the above solution 2, and the reaction was carried out at 25℃for 48 hours. After the end of the reaction, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, with deionized water being changed every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparation of HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1 mg) in ultrapure water (1 mL), performing ultrasonic treatment for 10 minutes to uniformly disperse the HA-S-S-PPT, and standing for 3 hours to obtain the HA-S-S-PPT prodrug micelle.
Infrared characterization of HA-NH-CO-PPT and HA-S-S-PPT prodrugs
The HA-NH-CO-PPT prepared in example 1 or the HA-S-S-PPT prodrug prepared in example 5 (1-2 mg) was mixed with solid potassium bromide powder (100-200 mg) and ground to uniform, tabletted at 4000-500cm -1 Scanning in range and infrared characterization. FIG. 2 is an infrared spectrum at 1710cm of the HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug prepared in example 5 -1 Characteristic signal peak of ester bond (-CO-O-) appears at 1650cm -1 1560cm -1 The characteristic signal peak (-NH-CO-) of the amide bond shows that the target product is synthesized.
Nuclear magnetic characterization of HA-NH-CO-PPT and HA-S-S-PPT prodrugs
The HA-NH-CO-PPT prepared in example 1 or the HA-S-S-PPT prodrug prepared in example 5 (5 mg) was dissolved in deuterated water (0.5 mL) for nuclear magnetic hydrogen spectroscopy characterization.
FIG. 3 is a nuclear magnetic resonance spectrum of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug prepared in example 5, signal peak at chemical shift 2.76 (corresponding to 7) belonging to ethylenediamine (-CH) 2 The signal peak of-NH-CO-) methylene indicates the grafting of ethylenediamine onto HA. Chemical shifts 5.8-6.8 (corresponding to 2-6) are ascribed to the hydrogen signal peak on the PPT benzene ring, proving the presence of PPT in the obtained HA-NH-CO-PPT. Similarly, for HA-S-S-PPT, the signal peak of hydrogen on the PPT benzene ring with chemical shift of 5.8-6.8 is at position 7 is cystamine (-CH 2-S-S-) on CH 2 Is successful in introducing PPT into the obtained HA-S-S-PPT.
Characterization of the morphology of HA-NH-CO-PPT and HA-S-S-PPT prodrugs
The HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug micelle prepared in example 5 were subjected to transmission electron microscopy to characterize morphology and particle size.
FIG. 4 is a transmission electron micrograph of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5, the HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles being substantially spherical or ellipsoidal.
PH sensitivity of HA-NH-CO-PPT prodrug micelle
The HA-NH-CO-PPT prodrug micelles (1 mg/mL) prepared in example 1 were incubated in PBS at pH 7.4 and pH 5.0 for 0, 4, 8 and 12 hours, respectively, and the particle size distribution of the samples was measured using a nanoparticle size analyzer.
FIG. 5a is a graph showing that the HA-NH-CO-PPT prodrug micelle prepared in example 1 shows no significant change in the size of the HA-NH-CO-PPT prodrug micelle at pH 7.4 by responding to the change in particle size at different pH, whereas the HA-NH-CO-PPT prodrug micelle gradually increases in particle size (from 148.2nm to 506.9 nm) at pH 5.0. Indicating that the HA-NH-CO-PPT prodrug micelle HAs pH sensitivity.
PH and reduction double sensitivity of HA-S-S-PPT prodrug micelle
The HA-S-S-PPT prodrug micelles (1 mg/mL) prepared in example 5 were incubated with PBS for 0, 4, 8 and 12 hours at pH 7.4, pH 7.4+glutathione (GSH, 20 mM), pH 5.0 and pH 5.0+glutathione (GSH, 20 mM), respectively, and the particle size distribution of the samples was measured.
FIG. 5b is a graph showing that the particle size of HA-S-S-PPT prodrug micelles did not change significantly at pH 7.4 by responding to particle size changes of different pH and GSH, but gradually increased (from 93.0nm to 454.7 nm) at pH 5.0; especially in the presence of 20mM GSH at pH 5.0, the particle size increased significantly (from 96.2nm to 837.1 nm), indicating that HA-S-S-PPT prodrug micelles have pH, reducing dual sensitivity.
Drug release behavior of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles
The release behavior of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5 was studied using a dialysis method. 2mL of HA-NH-CO-PPT prodrug micelle was added to a dialysis bag (molecular weight cut-off 2000 Da), and the dialysis bag was immersed in 50mL of PBS centrifuge tubes at pH 5.0 and pH 7.4. Similarly, 2mL of HA-S-S-PPT prodrug micelle was added to a dialysis bag (molecular weight cut-off 2000 Da), and the dialysis bag was then immersed in a 50mLPBS (pH 5.0, pH 5.0+20mM GSH, pH 7.4 and pH 7.4+20mM GSH) centrifuge tube. Then placing the mixture in a constant temperature shaking table, wherein the shaking speed is 200r/min, and the temperature is controlled at 37 ℃. At 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h and 72h, 2.0mL of liquid was removed from the release medium and replaced with 2.0mL of PBS of the corresponding pH to maintain the volume unchanged. The concentration of podophyllotoxin released into the PBS solution was measured with an ultraviolet spectrophotometer.
FIG. 6 is a graph showing the release profiles of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5, with less than 25.6% of PPT being slowly released after 72h incubation at pH 7.4 and the cumulative release increasing to 53.6% after 72h incubation at pH 5.0 for HA-NH-CO-PPT micelles. For HA-S-S-PPT micelles, only 21.7% of PPT was released when incubated at pH 7.4, and the cumulative release of PPT increased from 66.2% when pH 5.0 was reached, and more particularly, the cumulative release of PPT increased significantly to 86.7% when 20mM GSH was added to PBS at pH 5.0.
Hemolysis evaluation of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles
Firstly, fresh mouse plasma is subjected to defibrination, 2mL of mouse blood is taken, and the blood is gently stirred by a glass rod in a clockwise direction for 15min to remove fibrin. Then 8mL of PBS solution was added, and the mixture was put into a centrifuge and centrifuged at 2500r/min for 10min. Skim the supernatant and repeat centrifugation three times to obtain a red blood cell suspension. The cell suspension was diluted with PBS and vortexed for 5min to homogenize the suspension, resulting in a red blood cell suspension having a concentration of about 5% (v/v). The red blood cell suspension was mixed with 5mg/mL of HA-NH-CO-PPT, HA-S-S-PPT prodrug micelles, tween 80 solution, respectively, such that the final concentration of the two types of prodrug micelles or Tween 80 in the mixture ranged from 0.1 to 2mg/mL (0.1, 0.25, 0.5, 1, 2mg/mL, respectively). Simultaneously, 1mL of red blood cell suspension is respectively mixed with ultrapure water and PBS in equal quantity to be used as positive and negative control. After incubation for 4h and 12h on a constant temperature shaker at 37℃the mixture was centrifuged at 2500r/min for 10min. The absorbance of the supernatant at 541nm was measured with a microplate reader, and the degree of hemolysis was calculated using the following equation:
wherein A is test ，A neg ，A pos Absorbance values for sample, negative control (PBS) and positive control (water), respectively.
FIG. 7 is a graph showing the rate of hemolysis of HA-NH-CO-PPT prepared in example 2, HA-S-S-PPT prodrug micelle prepared in example 6, and Tween 80, wherein the rate of Tween 80-induced hemolysis increased significantly from 10.48% to 72.33% when the concentration increased from 0.1 to 2.0 mg/mL. However, HA-NH-CO-PPT and HA-S-PPT prodrug micelles showed no more than 5% hemolysis at the same concentration, with results significantly lower than tween 80, indicating that the prepared micelles have good blood compatibility.
Targeted evaluation of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles
Human breast cancer cells (MCF-7) were plated at 6X 10 per well 4 Is plated on 12-well plates and after 24h incubation, the cells are observed to account for approximately 80% of the bottle wall. 10mg/mL of hyaluronic acid (molecular weight 3000-10000 Da) was added for 2 hours, and the mixture was used as a control without hyaluronic acid treatment. After that, the medium (DMEM) was removed, and fresh DMEM containing 5. Mu.g/mL of the HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug micelle prepared in example 5 was added thereto, and the culture was continued for 4 hours. Cells were washed three times with 1 XPBS (pH 7.4) byTrypsin digestion, blowing and harvesting cells, sieving. Flow cytometry was selected for detection.
FIG. 8 is a cell targeting study of HA-NH-CO-PPT, HA-S-S-PPT prodrug micelles prepared in example 1, showing a decrease in cell uptake from 93.7 and 97.4% to 32.4% and 30.7%, respectively, indicating HA-mediated specific endocytosis.
Cytotoxicity of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles
Toxicity of the HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug micelles prepared in example 5 to human breast cancer cells (MCF-7) was determined by the CCK-8 method. Cells were grown in 96-well cell culture plates, 5 wells in parallel, 5X 10 cells were grown per well 4 Individual cells, at 37 ℃,5% co 2 Culturing in a cell culture box until the cell density reaches more than 80%. DMEM was removed, fresh DMEM containing 0.01, 0.1, 1, 2, 4, 5, 10 and 20 μg/mL HA-NH-CO-PPT prepared in example 1 and HA-S-PPT prodrug micelles prepared in example 5, respectively, was added and cultured for 72 hours. Add 10. Mu.L of CCK-8 reagent, incubate for 1h, and move to the microplate reader to determine the absorbance at 450nm of each well. Cell viability was calculated (the percentage of absorbance values of the experimental group to the absorbance values of the control group).
FIG. 9 is a graph showing that HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5 have toxicity to MCF-7 cells, and that HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles show better inhibition effect on MCF-7 cells than free PPT, especially the HA-S-S-PPT prodrug micelles have the best inhibition effect.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. The preparation method of the prodrug micelle of the environment-responsive hyaluronic acid-podophyllotoxin is characterized by comprising the following steps:
(1) Dissolving ethylenediamine or cystamine dihydrochloride in dimethyl sulfoxide, adding the solution 1, and reacting at 25 ℃ for 24-48 hours; reactionAfter the completion, the obtained mixture is put into a dialysis bag and dialyzed by deionized water, and then filtered and freeze-dried to obtain HA-NH 2 Or HA-S-S-NH 2 A solid;
the preparation method of the solution 1 comprises the following steps: dissolving Hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in PBS solution, and reacting for 1-2 hours under ice bath condition to obtain solution 1;
(2) Dissolving succinic anhydride, podophyllotoxin (PPT) and 4-dimethylaminopyridine in toluene, refluxing at 105-110deg.C for 48-72 hr, removing solvent, and drying to obtain carboxylated PPT solid (PPT-COOH);
(3) HA-NH 2 Or HA-S-S-NH 2 Dissolving the solid in PBS solution, adding solution 2, and reacting at 25 ℃ for 24-48 hours; after the reaction is finished, the obtained mixture is put into a dialysis bag and dialyzed by deionized water, and then filtered and freeze-dried to obtain HA-NH-CO-PPT or HA-S-S-PP solid;
the preparation method of the solution 2 comprises the following steps: PPT-COOH, EDC and NHS are dissolved in N, N-dimethylformamide and reacted for 1 to 2 hours under the ice bath condition to obtain a solution 2;
(4) Dissolving the HA-NH-CO-PPT or the HA-S-S-PP solid in ultrapure water, carrying out ultrasonic treatment for 10-20 minutes, and standing for 3-6 hours to obtain the HA-NH-CO-PPT or the HA-S-S-PPT prodrug micelle.
2. The method of claim 1, wherein in step (1), the HA HAs a molecular weight of 3000-10000Da; in the steps (1) and (3), the pH of the PBS solution is 8.0.
3. The method according to claim 1, wherein in the steps (1) and (3), the obtained mixture is put into a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and deionized water is replaced every 3 to 12 hours; the molecular weight cut-off of the dialysis bag is 1000-2000Da.
4. The method of claim 1, wherein in step (1), the molar ratio of HA, EDC, and NHS is 1:0.5:0.5 to 1:1:1; the molar ratio of HA to ethylenediamine is 1:0.5-1:2; the molar ratio of HA to cystamine dihydrochloride is 1:0.5-1:5.
5. The method of claim 1, wherein in step (2), the molar ratio of PPT, SA and DMAP is 1:1:1 to 10:15:1.
6. The process of claim 1, wherein in step (3), the molar ratio of PPT to COOH, EDC to NHS is 1:0.5:0.5 to 1:1:1; the HA-NH 2 Or HA-S-S-NH 2 And a PPT-COOH molar ratio of 1:0.5 to 1:30.
7. The method of claim 1, wherein the HA-NH-CO-PPT prodrug micelle is a pH-sensitive hyaluronic acid-podophyllotoxin prodrug micelle, and the HA-S-PPT prodrug micelle is a pH, reduced double-sensitive hyaluronic acid-podophyllotoxin prodrug micelle.
8. An environmentally-responsive hyaluronic acid-podophyllotoxin prodrug micelle prepared by the method of any one of claims 1-7.
9. Use of an environmentally-responsive hyaluronic acid-podophyllotoxin prodrug micelle in accordance with claim 8 for the preparation of a medicament for the treatment of breast cancer.
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