CN113750255A - 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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- 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
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
Abstract
The invention discloses an environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle as well as a preparation method and application thereof, wherein the environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle is a pH-sensitive prodrug micelle and a pH-reduction double-sensitive prodrug micelle. Starting from hyaluronic acid and podophyllotoxin as raw materials, firstly, ethylenediamine or cystamine is used for modifying hyaluronic acid to synthesize aminated hyaluronic acid, then succinic anhydride is used for carrying out carboxylation treatment on podophyllotoxin, and finally, the aminated hyaluronic acid and the carboxylated podophyllotoxin are subjected to condensation reaction to obtain the polymer prodrug. The prodrug synthesized by the invention can be self-assembled in aqueous solution to form micelle, has good blood compatibility and is suitable for intravenous injection. The invention also provides the application of the prodrug micelle in drug delivery, has controllable drug release behavior, targeting capability on tumor cells, good anti-tumor effect and 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 seriously harming human health, and the morbidity and mortality of cancer are high. Chemotherapy continues to be the most widely used treatment currently. However, the existing antitumor drugs have strong killing effect on tumor cells, and can also cause serious damage to normal cells, cause multiple complications and limit the clinical application and development of the antitumor drugs. Currently, nanoparticulate drug delivery vehicles have made tremendous progress in cancer therapy, especially in polymeric prodrug micelles. These polymeric prodrug micelles can passively accumulate in tumor tissues with high stability and prolonged blood circulation time. However, their anti-tumor effect is limited by non-specific targeting and low drug release efficiency in cells. Therefore, it is necessary to develop an environmentally responsive prodrug micelle having specific targeting. The pH and reduction sensitive polymer prodrug micelle can realize controllable drug delivery and release according to the obvious difference between the pH value of tumor cells and normal cells and the concentration of Glutathione (GSH).
Hyaluronic Acid (HA) is a natural polymer material, can be specifically combined with a CD44 receptor over-expressed on the surface of a tumor cell, HAs no immunogenicity, and can be used as an ideal drug delivery carrier. Therefore, the hyaluronic acid-targeted polymer prodrug micelle with environmental response can realize the specific distribution of the drug among different tissues, and reduce the damage of the drug to normal tissues while achieving the aim of high-efficiency drug delivery.
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, due to severe toxic and side effects caused by off-target effects, poor water solubility, and the like, the application of podophyllotoxin in tumor therapy is limited.
The invention starts from hyaluronic acid and podophyllotoxin as raw materials, synthesizes two environment-sensitive prodrugs, namely a pH-sensitive hyaluronic acid-podophyllotoxin prodrug (HA-NH-CO-PPT) and a pH and reduction double-sensitive prodrug (HA-S-S-PPT), can be self-assembled in water to form micelles, is used for treating cancers, and provides technical reference for the delivery and treatment of other similar medicaments.
Disclosure of Invention
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, aiming at the defects of poor water solubility, non-specific accumulation and release, serious toxic and side effects and the like of the current chemotherapeutic drugs podophyllotoxin. 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:
the first purpose of the invention is to provide a preparation method of an environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-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-NH2or HA-S-S-NH2The preparation of (1): ethylenediamine or cystamine dihydrochloride is dissolved in dimethyl sulfoxide (DMSO), and then solution 1 is added and reacted at 25 ℃ for 24 to 48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag, dialyzing with deionized water, filtering and freeze-drying to obtain HA-NH2Or HA-S-S-NH2And (3) 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 a PBS solution and reacted for 1-2 hours under ice bath conditions to activate carboxyl groups of HA, obtaining solution 1.
(2) Preparation of PPT-COOH: dissolving Succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) in a toluene solution, refluxing for 48-72 hours at the temperature of 105-110 ℃, removing the solvent, and drying in vacuum to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT or HA-S-S-PPT: reacting HA-NH2Or HA-S-S-NH2The solid was dissolved in PBS solution, then solution 2 was added and reacted at 25 ℃ for 24-48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag, dialyzing with deionized water, filtering and freeze-drying 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 for 1-2 hours under ice bath conditions to obtain solution 2.
(4) Preparing a HA-NH-CO-PPT or HA-S-S-PPT prodrug micelle: dissolving HA-NH-CO-PPT or 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 HA-S-S-PPT solid, and standing for 3-6 hours to obtain the HA-NH-CO-PPT or HA-S-PPT prodrug micelle.
Preferably, in step (1), the HA molecular weight is 3000-10000 Da.
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 the HA to the 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 solvent is removed by: spin-evaporate and wash 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-NH2Or HA-S-S-NH2And PPT-COOH in a molar ratio of 1:0.5-1: 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 to 72 hours, with deionized water being replaced every 3 to 12 hours.
Preferably, in steps (1) and (3), the cut-off molecular weight of the dialysis bag is 1000-2000 Da.
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 environment-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-NH2the preparation of (1): hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in a PBS solution at pH 8.0, and reacted for 1-2 hours under ice bath conditions to activate carboxyl groups of HA, obtaining solution 1. Then, ethylenediamine was dissolved in dimethyl sulfoxide (DMSO), and the solution 1 was added to the solution to react at 25 ℃ for 24 to 48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag for dialysis for 48-72 hours by using deionized water, and replacing the deionized water every 3-12 hours. Finally, the solution was filtered and lyophilized to obtain HA-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) are dissolved in a toluene solution, refluxed for 48 to 72 hours at the temperature of 105-110 ℃, rotary evaporated, washed by anhydrous methanol and dried in vacuum to obtain a 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 for 1-2 hours under ice bath conditions to obtain solution 2. Then adding HA-NH2Dissolved in PBS solution of pH 8.0, and added to the above solution 2, and reacted at 25 ℃ for 24 to 48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag for dialysis for 48-72 hours by using deionized water, and replacing the deionized water every 3-12 hours. And finally, filtering and freeze-drying to obtain HA-NH-CO-PPT solid.
(4) Preparing HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT in ultrapure water, performing 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 environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle is a pH and reduction double-sensitive hyaluronic acid-podophyllotoxin prodrug micelle (HA-S-S-PPT), the steps are as follows:
(1)HA-S-S-NH2the preparation of (1): hyaluronic Acid (HA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in a PBS solution at pH 8.0, and reacted for 1-2 hours under ice bath conditions to activate carboxyl groups of HA, obtaining solution 1. Then, cystamine dihydrochloride was dissolved in dimethyl sulfoxide (DMSO) and added to the above solution 1, and reacted at 25 ℃ for 24 to 48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag for dialysis for 48-72 hours by using deionized water, and replacing the deionized water every 3-12 hours. Finally, the solution was filtered and lyophilized to obtain HA-S-S-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic Anhydride (SA), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) are dissolved in a toluene solution, refluxed for 48 to 72 hours at the temperature of 105-110 ℃, rotary evaporated, washed by anhydrous methanol and dried in vacuum to obtain a 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 for 1-2 hours under ice bath conditions to obtain solution 2. Then HA-S-S-NH2Dissolved in PBS solution of pH 8.0, and added to the above solution 2, and reacted at 25 ℃ for 24 to 48 hours. After the reaction is finished, putting the obtained mixture into a dialysis bag for dialysis for 48-72 hours by using deionized water, and replacing the deionized water every 3-12 hours. And finally, filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparing HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT in ultrapure water, performing 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 purpose of the invention is to provide the environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-S-PPT) prepared by the method.
The third objective of the invention is to provide an application of the environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT or HA-S-S-PPT) in preparation of a drug for treating cancer, wherein the cancer is breast cancer, lung cancer or liver cancer.
The environment-responsive hyaluronic acid-podophyllotoxin prodrug micelle prepared by the invention starts from targeted material hyaluronic acid and anticancer drug podophyllotoxin, the amphiphilic derivative is synthesized by chemical bond connection, can form micelles with smaller particle size and uniform dispersion in aqueous solution by self-assembly, improves the water solubility and biocompatibility of podophyllotoxin, the pH-sensitive hyaluronic acid-podophyllotoxin prodrug micelle (HA-NH-CO-PPT) can controllably release the drug by responding to the low pH in a tumor microenvironment, and the pH-reduced double-sensitive hyaluronic acid-podophyllotoxin prodrug micelle (HA-S-S-PPT) can controllably release the drug by responding to the low pH and the high-concentration glutathione in the tumor microenvironment, so that the efficient drug delivery and the enhanced anti-tumor effect are realized.
The invention has the beneficial effects that:
the invention provides two preparation methods of an environment-sensitive targeted prodrug micelle, namely a pH-sensitive prodrug micelle and a pH-reduction double-sensitive prodrug micelle. Starting from hyaluronic acid and podophyllotoxin as raw materials, firstly, ethylenediamine or cystamine is used for modifying hyaluronic acid to synthesize aminated hyaluronic acid (HA-NH)2And HA-S-S-NH2) Then carboxylation treatment is carried out on the podophyllotoxin by succinic anhydride, and finally condensation reaction is carried out on the aminated hyaluronic acid and the carboxylated podophyllotoxin to obtain the polymer prodrug (HA-NH-CO-PPT and HA-S-S-PPT). The prodrug synthesized by the invention can be self-assembled in aqueous solution to form micelle, has good blood compatibility and is suitable for intravenous injection. The invention also provides application of the HA-NH-CO-PPT and HA-S-S-PPT prodrug micelle in drug delivery, and the micelle HAs controllable drug release behavior, targeting capability on tumor cells, good anti-tumor effect and wide application prospect.
Drawings
FIG. 1 is a scheme for the synthesis of 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 is a graph of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug prepared in example 51H NMR spectrum;
FIG. 4 is a TEM photograph of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelle 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;
FIG. 5b is a graph of the particle size change of HA-S-S-PPT prodrug micelle prepared in example 5 by responding to different pH and GSH;
FIG. 6 is the in vitro drug release behavior of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelle prepared in example 5;
FIG. 7 is a graph showing 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 shows targeting of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelle prepared in example 5;
FIG. 9 is a graph showing the 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 figures and the specific examples, without limiting the scope of protection of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be purchased from chemical companies.
Example 1
(1)HA-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, the amount will be equimolar to HAThe ethylene diamine (D) was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution (1) was added thereto to conduct a reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-NH2。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-NH-CO-PPT.
(4) Preparing HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1mg) in ultrapure water (1mL), carrying out 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, ethylenediamine in an amount equimolar to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added to the solution to react at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-NH2。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-NH-CO-PPT.
(4) Preparing HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (5mg) in ultrapure water (1mL), carrying out 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, ethylenediamine in an amount equimolar to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added to the solution to react at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-NH2。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 72 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3)HA-NPreparation of H-CO-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-NH-CO-PPT.
(4) Preparing HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1mg) in ultrapure water (1mL), carrying out 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, ethylenediamine in an amount equimolar to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added to the solution to react at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-NH2。
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-NH-CO-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 48 hours. Reaction ofAfter completion, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, replacing the deionized water every 6 hours. Filtering and freeze-drying to obtain HA-NH-CO-PPT.
(4) Preparing HA-NH-CO-PPT prodrug micelle: dissolving HA-NH-CO-PPT (1mg) in ultrapure water (1mL), carrying out 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, cystamine dihydrochloride in an equimolar amount to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added and reacted at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-S-S-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-S-S-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparing HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1mg) in ultrapure water (1mL), 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, cystamine dihydrochloride in an equimolar amount to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added and reacted at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-S-S-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-S-S-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparing HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (5mg) in ultrapure water (1mL), 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-bis) with a molecular weight of 3000-10000DaMethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, cystamine dihydrochloride in an equimolar amount to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO), and the solution 1 was added and reacted at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-S-S-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 72 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-S-S-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparing HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1mg) in ultrapure water (1mL), 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-NH2The preparation of (1): hyaluronic acid (HA, 400mg, corresponding to 1054. mu. mol of COOH groups), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molecular weight of 3000-10000Da were reacted in a molar ratio of 1:1:1 was dissolved in a PBS solution (5mL) at pH 8.0 and reacted for 1 hour under ice-bath conditions to activate the carboxyl group of HA, yielding solution 1. Then, cystamine dihydrochloride in an equimolar amount to HA was dissolved in 5mL of dimethyl sulfoxide (DMSO)) After neutralization, the above solution 1 was added and reacted at 25 ℃ for 24 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Finally, the solution was filtered and lyophilized to obtain HA-S-S-NH2And (3) a solid.
(2) Preparation of PPT-COOH: succinic anhydride (SA, 1.5mmol), podophyllotoxin (PPT) and 4-Dimethylaminopyridine (DMAP) were mixed in a molar ratio of 15: 10:1 was dissolved in 20mL of toluene, refluxed at 110 ℃ for 48 hours, rotary evaporated and washed with anhydrous methanol, and dried in vacuo to obtain a carboxylated PPT solid (PPT-COOH).
(3) Preparation of HA-S-S-PPT: PPT-COOH (200. mu. mol), EDC and NHS were mixed in a molar ratio of 1:1:1 was dissolved in 5ml of N-Dimethylformamide (DMF) and reacted for 1 hour under ice bath conditions to obtain a solution 2. Then, the molar quantity of HA-S-S-NH which is equal to that of PPT-COOH is added2Dissolved in 5mL of PBS (pH 8.0), and added to the above solution 2, followed by reaction at 25 ℃ for 48 hours. After the reaction was completed, the resulting mixture was dialyzed against deionized water (MWCO 2000) for 72 hours, and the deionized water was replaced every 6 hours. Filtering and freeze-drying to obtain HA-S-S-PPT solid.
(4) Preparing HA-S-S-PPT prodrug micelle: dissolving HA-S-S-PPT (1mg) in ultrapure water (1mL), 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 (1-2mg) and potassium bromide solid powder (100-200mg) were mixed, ground uniformly, tableted and placed at 4000 cm 500cm-1Scanning in range and infrared characterization. FIG. 2 is an IR spectrum of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug prepared in example 5 at 1710cm-1The characteristic signal peak of ester bond (-CO-O-) appears at position, 1650cm-1And 1560cm-1The peak is a characteristic signal peak (-NH-CO-) of amido bond, which indicates 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 (5mg) was dissolved in deuterated water (0.5mL) for nuclear magnetic hydrogen spectroscopy characterization.
FIG. 3 is nuclear magnetic hydrogen spectra of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug prepared in example 5, with a signal peak at chemical shift 2.76 (corresponding to 7) belonging to ethylenediamine (-CH)2Signal peak of-NH-CO-) methylene indicating ethylene diamine grafting to HA. Chemical shifts 5.8-6.8 (corresponding to 2-6) are assigned to the hydrogen signal peak on the PPT benzene ring, which proves that PPT exists in the obtained HA-NH-CO-PPT. Similarly, for HA-S-S-PPT, except for the signal peak of hydrogen on PPT benzene ring with chemical shift of 5.8-6.8, the position 7 is CH on cystamine (-CH2-S-S-)2The signal peak of (a) successfully introduces PPT in the obtained HA-S-S-PPT.
Morphological characterization of HA-NH-CO-PPT and HA-S-S-PPT prodrugs
The appearance and the particle size of the HA-NH-CO-PPT prepared in the example 1 and the HA-S-S-PPT prodrug micelle prepared in the example 5 are represented by a transmission electron microscope.
FIG. 4 is a transmission electron micrograph of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles prepared in example 1 and 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 size distribution of the samples was measured using a nanometer particle size analyzer after incubating the HA-NH-CO-PPT prodrug micelles (1mg/mL) prepared in example 1 in PBS at pH 7.4 and pH 5.0 for 0, 4, 8, and 12 hours, respectively.
FIG. 5a is a graph showing that the size of HA-NH-CO-PPT prodrug micelle prepared in example 1 does not change significantly at pH 7.4 by responding to the particle size change at different pH values, while the size of HA-NH-CO-PPT prodrug micelle increases gradually (from 148.2nm to 506.9nm) at pH 5.0. Indicating that the HA-NH-CO-PPT prodrug micelle HAs pH sensitivity.
PH, reduction double sensitivity of HA-S-S-PPT prodrug micelle
The HA-S-S-PPT prodrug micelle (1mg/mL) prepared in example 5 was incubated in PBS at pH 7.4, pH 7.4+ glutathione (GSH, 20mM), pH 5.0 and pH 5.0+ glutathione (GSH, 20mM) for 0, 4, 8 and 12 hours, respectively, to measure the particle size distribution of the sample.
FIG. 5b is a graph showing that the HA-S-S-PPT prodrug micelle HAs no significant change in particle size at pH 7.4 and gradually increases in particle size (from 93.0nm to 454.7nm) at pH 5.0 by responding to particle size changes at different pH and GSH; especially at pH 5.0 in the presence of 20mM GSH, the particle size increased significantly (from 96.2nm to 837.1nm), indicating that the HA-S-S-PPT prodrug micelle HAs pH, reducing double 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 micelle prepared in example 5 were investigated using dialysis method. 2mL of HA-NH-CO-PPT prodrug micelle was added to a dialysis bag (molecular weight cut-off 2000Da) which was then immersed in 50mL 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 2000Da) which was then immersed in a 50mL PBS (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 oscillation 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 at the corresponding pH to maintain the volume constant. 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 micelle prepared in example 5, wherein for HA-NH-CO-PPT micelle, less than 25.6% of PPT is slowly released after incubation for 72h at pH 7.4, and the cumulative release increases to 53.6% after incubation for 72h at pH 5.0. 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 was 5.0, especially by adding 20mM GSH to PBS at pH 5.0, to 86.7%.
Evaluation of hemolysis of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles
Fresh mouse plasma was first defibrated, and 2mL of mouse blood was gently stirred clockwise with a glass rod for 15min to remove fibrin from the blood. Then 8mL of PBS solution was added, and the mixture was centrifuged at 2500r/min for 10min in a centrifuge. And (4) skimming the supernatant, and repeatedly centrifuging three times until the supernatant is colorless to obtain the red blood cell suspension. The cell suspension was diluted with PBS and mixed well by vortexing for 5min to obtain a suspension of red blood cells at a concentration of about 5% (v/v). The red blood cell suspension was mixed with 5mg/mL HA-NH-CO-PPT, HA-S-S-PPT prodrug micelles, Tween 80 solutions, respectively, to give a final concentration of the two types of prodrug micelles or Tween 80 in the mixture ranging from 0.1 to 2mg/mL (0.1, 0.25, 0.5, 1, 2mg/mL, respectively). Meanwhile, 1mL of erythrocyte suspension is respectively mixed with ultrapure water and PBS in equal amount to be used as positive and negative controls. After incubation for 4h and 12h on a constant temperature shaker at 37 ℃ centrifugation at 2500r/min for 10 min. 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 istest，Aneg，AposThe absorbance values for the sample, negative control (PBS) and positive control (water), respectively.
FIG. 7 is a graph showing the hemolysis rates of HA-NH-CO-PPT prepared in example 2, HA-S-S-PPT prodrug micelle prepared in example 6, and Tween 80, wherein the hemolysis rate induced by Tween 80 was significantly increased from 10.48% to 72.33% when the concentration was increased from 0.1 to 2.0 mg/mL. However, the HA-NH-CO-PPT and HA-S-S-PPT prodrug micelle shows a hemolysis rate of not more than 5% at the same concentration, and the result is obviously lower than that of Tween 80, which indicates that the prepared micelle HAs 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 well4The cells were seeded in 12-well plates and after 24h incubation, the cells were observed to account for approximately 80% of the vial wall. Adding 10mg/mL hyaluronic acid (molecular weight 3000-10000Da) to culture for 2h, and using the hyaluronic acid-free solution as a control. Thereafter, the medium (DMEM) was removed, and fresh HA-NH-CO-PPT prepared in example 1 and fresh HA-NH-CO-PPT prepared in example 5 were added thereto at a concentration of 5. mu.g/mLDMEM of HA-S-S-PPT prodrug micelle is continuously cultured for 4 hours. Cells were washed three times with 1 × PBS (pH 7.4), trypsinized, blown and harvested, and sieved. Flow cytometry was selected for detection.
FIG. 8 is a cell targeting study of HA-NH-CO-PPT and HA-S-S-PPT prodrug micelles prepared in example 1, showing that cellular uptake rates were reduced 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
The toxicity of the HA-NH-CO-PPT prepared in example 1 and the HA-S-S-PPT prodrug micelle prepared in example 5 on human breast cancer cells (MCF-7) was determined by the CCK-8 method. Cells were plated in 96-well cell culture plates in 5 parallel wells of 5X 10 cells per well4Individual cells, 5% CO at 37 ℃2Culturing in a cell culture box until the cell density reaches more than 80%. DMEM was removed and fresh DMEM containing 0.01, 0.1, 1, 2, 4, 5, 10 and 20 μ g/mL HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5 was added and incubated for 72 h. 10 μ L of CCK-8 reagent was added, incubated for 1h, and transferred to a microplate reader to determine the absorbance at 450nm in each well. Cell viability (the percentage of absorbance values for the experimental group to the control group) was calculated.
FIG. 9 is the toxicity of HA-NH-CO-PPT prepared in example 1 and HA-S-S-PPT prodrug micelles prepared in example 5 on MCF-7 cells, showing better inhibition of MCF-7 cells, especially the best inhibition of HA-S-S-PPT prodrug micelles, compared to free PPT.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The preparation method of the prodrug micelle of the environment-responsive hyaluronic acid-podophyllotoxin is characterized by comprising the following steps:
(1) di-salts of ethylenediamine or cystamineDissolving the acid salt in dimethyl sulfoxide, adding the solution 1, and reacting at 25 ℃ for 24-48 hours; after the reaction is finished, putting the obtained mixture into a dialysis bag, dialyzing the mixture by using deionized water, filtering and freeze-drying the mixture to obtain HA-NH2Or HA-S-S-NH2A 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 a PBS solution, and reacting for 1-2 hours under an ice bath condition to obtain a solution 1;
(2) dissolving succinic anhydride, podophyllotoxin (PPT) and 4-dimethylaminopyridine in toluene, refluxing for 48-72 hours at the temperature of 105-110 ℃, removing the solvent, and drying to obtain a carboxylated PPT solid (PPT-COOH);
(3) reacting HA-NH2Or HA-S-S-NH2Dissolving the solid in PBS solution, adding solution 2, and reacting at 25 deg.C for 24-48 hr; after the reaction is finished, putting the obtained mixture into a dialysis bag, dialyzing the mixture by using deionized water, filtering and freeze-drying the mixture to obtain HA-NH-CO-PPT or HA-S-S-PP solid;
the preparation method of the solution 2 comprises the following steps: dissolving PPT-COOH, EDC and NHS in N, N-dimethylformamide, and reacting for 1-2 hours under an ice bath condition to obtain a solution 2;
(4) dissolving HA-NH-CO-PPT or 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 HA-S-S-PPT prodrug micelle.
2. The method according to claim 1, wherein in the step (1), the HA HAs a molecular weight of 3000-10000 Da; in the steps (1) and (3), the pH of the PBS solution is 8.0.
3. The method according to claim 1, wherein in steps (1) and (3), the resulting mixture is placed in a dialysis bag and dialyzed with deionized water for 48 to 72 hours, and the deionized water is replaced every 3 to 12 hours; the cut-off molecular weight of the dialysis bag is 1000-2000 Da.
4. The method according to claim 1, wherein in step (1), the molar ratio of HA, EDC and NHS is 1:0.5:0.5-1:1: 1; the molar ratio of the HA to the ethylenediamine is 1:0.5-1: 2; the molar ratio of HA to cystamine dihydrochloride is 1:0.5-1: 5.
5. The method according to claim 1, wherein in step (2), the molar ratio of PPT, SA and DMAP is 1:1:1-10:15: 1.
6. The method according to claim 1, wherein in the step (3), the molar ratio of PPT-COOH, EDC and NHS is 1:0.5:0.5-1:1: 1; the HA-NH2Or HA-S-S-NH2And PPT-COOH in a molar ratio of 1:0.5-1: 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-reducing 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 the environmentally responsive hyaluronic acid-podophyllotoxin prodrug micelle of claim 8 for the preparation of a medicament for the treatment of cancer.
10. The use of claim 9, wherein the cancer is breast cancer, lung cancer or liver cancer.
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