CN114010596B

CN114010596B - Targeted polymer micelle for active oxygen responsive drug release in inflammatory microenvironment and preparation method thereof

Info

Publication number: CN114010596B
Application number: CN202111257361.0A
Authority: CN
Inventors: 刘艳华; 李依凡; 曹永敬; 杨嘉玉; 朱溶月; 毕嘉威
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2023-01-13
Anticipated expiration: 2041-10-27
Also published as: CN114010596A

Abstract

The invention discloses a targeted polymer micelle for active oxygen responsive drug release in inflammatory microenvironment and a preparation method thereof. The invention has the characteristics of active oxygen response, CD44 active targeting and ELVIS mediated passive targeting cooperative drug delivery system applied to inflammation related diseases, makes the polymer micelle more intelligent aiming at the characteristic of active oxygen level rise in an inflammation microenvironment, increases the drug release speed of an inflammation part and the effective accumulation of the drug, and simultaneously reduces the toxic and side effects on normal tissues, thereby realizing accurate and efficient co-delivery of the drug to the focus part and achieving the purpose of efficiently and intelligently treating inflammation by the polymer micelle.

Description

Targeted polymer micelle for active oxygen responsive drug release in inflammatory microenvironment and preparation method thereof

Technical Field

The invention relates to the field of research of medicinal high polymer materials and novel medicinal preparations, in particular to a targeted polymer micelle for responsive active oxygen release in an inflammatory microenvironment and a preparation method thereof.

Background

Rheumatoid arthritis is a chronic autoimmune disease, and inflammatory reactions are one of the main pathological features. In the inflammatory microenvironment, the development of inflammation-related diseases can promote the overproduction of reactive oxygen species, which in turn damages protein, lipid, nucleic acid and matrix components in synovial tissue, breaking the balance between oxidants and antioxidants, leading to oxidative stress states and local tissue hypoxia that induce inflammation. Therefore, it is of great significance to timely and effectively regulate the reactive oxygen species level of the inflammatory microenvironment to maintain the normal level. Aiming at the characteristic of active oxygen overexpression in an inflammation microenvironment, a high molecular polymer material containing an active oxygen responsive structure is designed, and the high molecular polymer material not only can be used as a drug delivery carrier for targeted treatment, but also can be used as an intelligent responsive material for inhibiting inflammation and promoting normal repair of damaged tissues. The ketal thiol containing different active groups is a compound with symmetrical structure and good active oxygen responsiveness, so that the polymer material containing the structure can realize active oxygen responsiveness degradation in vivo.

In addition, monotherapy based on anti-inflammatory drugs has failed to meet the needs of clinical treatment of inflammatory diseases due to its severe toxic side effects and multidrug resistance. At present, the combination treatment of two or more medicaments with different anti-inflammatory action mechanisms can effectively overcome the defects of single treatment and become an effective strategy for clinically treating the rheumatoid arthritis. Artesunate as a small molecular Chinese medicine monomer can reduce the expression of proinflammatory cytokines IL-1 beta, TNF-alpha and the like, inhibit the activation of a nuclear transcription factor (NF-kB) signal channel and the expression of hypoxia inducible factor 1 alpha (HIF-1 alpha), and relieve the hypoxia state of an inflammation microenvironment. Therefore, the combination of artesunate as hypoxia inducible factor inhibitor and other insoluble anti-inflammatory drugs such as dexamethasone, methotrexate, indomethacin, betamethasone, leflunomide and the like is an important strategy for improving the clinical anti-inflammatory curative effect.

For the treatment of inflammation-related diseases at present, chinese patent CN 113181149 provides an external pharmaceutical preparation for relieving the pain and swelling of joints by smearing administration, but the obstruction of the skin stratum corneum greatly limits the curative effect of the medicine. Chinese patent CN 113412126 provides a metal nanoparticle medicament, and the preparation process of the metal nanoparticle is complex, high in cost, undefined in vivo safety, and causes environmental pollution, and is not suitable for being widely applied to clinic, so that how to provide a composition capable of meeting the requirements of effective co-loading and in vivo co-delivery of a hydrophobic hypoxia inducible factor inhibitor artesunate and other anti-inflammatory drugs for synergistically inhibiting inflammation is an urgent problem to be solved by technical personnel in the field.

Disclosure of Invention

In view of this, the invention provides an intelligent polymer micelle, namely a targeted polymer micelle for active oxygen-responsive drug release in an inflammation microenvironment, and a preparation method thereof, which are used for realizing efficient co-loading of artesunate and a combined anti-inflammatory drug.

In order to realize the purpose, the invention adopts the following technical scheme:

a targeted polymer micelle for active oxygen-responsive drug release in inflammatory microenvironment comprises a hyaluronic acid-ketothiol-artesunate polymer carrier and an anti-inflammatory drug encapsulated by the polymer carrier.

Preferably, the hyaluronic acid-ketone thioketal-artesunate polymer carrier is grafted with active oxygen-responsive linkage ketone thioketal and/or artesunate, and has the following structure:

wherein R is selected from one of-OH, thioketal or thioketal-artesunate, and n is a positive integer.

Preferably, the ketamine is one or more of the following structures:

the ketamine-artesunate is one or more of the following structures:

preferably, the grafting rate of the artesunate is 25-35%, the grafting rate of the thioketal is 60-70%, and the anti-inflammatory drug accounts for 10-20% of the total mass of the micelle.

Preferably, the anti-inflammatory drug is one or two or more of dexamethasone, methotrexate, indomethacin, betamethasone and leflunomide.

The invention also provides a preparation method of the targeted polymer micelle for the responsive release of the active oxygen in the inflammatory microenvironment, which comprises the following steps:

the method comprises the following steps: synthesis of ketals

(1.1) dissolving cysteamine hydrochloride, ethyl trifluoroacetate and triethylamine in methanol to react for 8-12 h, neutralizing the reaction solution with acetic acid and removing an organic solvent, extracting the obtained mixture with ethyl acetate and water, drying an organic layer with anhydrous magnesium sulfate for 6-24 h, and removing the solvent to obtain a compound 1 for later use;

(1.2) dissolving the compound 1 and acetone in acetonitrile under the protection of nitrogen, cooling to 0 ℃ in an ice bath, adding boron trifluoride diethyl etherate, reacting for 2-6 h at 0-4 ℃, then pouring the reaction system into a sodium carbonate solution with the mass fraction of 5-15%, extracting with ethyl acetate, combining organic layers, washing with the sodium carbonate solution, drying with anhydrous sodium sulfate for 6-24 h, and then mixing the organic layers according to the volume ratio of petroleum ether to n-hexane: separating and purifying by silica gel column chromatography by using ethyl acetate = 7:1-10 as an eluent to obtain a compound 2 for later use;

(1.3) dissolving the compound 2 in 6M sodium hydroxide solution, reacting for 2-6 h at room temperature, and extracting with dichloromethane to obtain a compound 3, namely the ketal thiol;

step two: synthesis of hyaluronic acid-ketodithiols

Dissolving hyaluronic acid in water, adding a catalyst, heating the reaction to room temperature, continuing to react for 1-4 h, dissolving ketamine in DMF solution, adding the solution into a hyaluronic acid reaction system, reacting for 12-24 h at room temperature, dialyzing in pure water for two days by using a 1000-4000 Da dialysis bag, and freeze-drying for later use;

step three: preparation of hyaluronic acid-ketone thiol-artesunate polymer carrier

Dissolving artesunate in DMF, adding a catalyst, reacting for 1-4 h under an ice bath condition, adding the hyaluronic acid-ketothiolane aqueous solution of 20-50 mg/mL obtained in the step two, reacting for 8-24h, dialyzing a dialysis bag of 1000-4000 Da in pure water for two days, and freeze-drying to obtain an active oxygen responsive polymer carrier;

step four: preparation of targeted polymer micelle for active oxygen responsive drug release in inflammatory microenvironment

And (3) dissolving the anti-inflammatory drug in an organic solvent, adding the anti-inflammatory drug into the hyaluronic acid-ketothiol-artesunate polymer carrier aqueous solution of 2-5 mg/mL obtained in the third step, stirring for 8-24 h to volatilize the organic solvent, and then performing ice bath ultrasound, wherein the ultrasound power is 100-500W, and the ultrasound time is 2-10 min, so as to obtain the active oxygen responsive drug release targeted polymer micelle.

Preferably, the molar ratio of the hyaluronic acid to the ketodithiol in the second step is 1:2-1:8, and the molar ratio of the hyaluronic acid to the catalyst is 1:1-1:5.

Preferably, the catalyst in the second step and the third step is any one or two of DMT-MM, EDCI, DMAP and NHS.

Preferably, the mol ratio of the artesunate to the hyaluronic acid-ketone dithiol in the third step is 2:1-8:1.

Preferably, the organic solvent in the fourth step is any one of methanol, ethanol, acetone and tetrahydrofuran.

Preferably, the mass ratio of the anti-inflammatory drug to the polymer carrier in the fourth step is 1:5-1.

According to the technical scheme, compared with the prior art, the invention discloses the targeted polymer micelle for the active oxygen responsive drug release in the inflammatory microenvironment and the preparation method thereof, and the preparation method has the following beneficial effects:

the invention aims at the characteristic of high active oxygen level of inflammatory microenvironment, uses the polymer carrier prepared by bonding hyaluronic acid and drugs, has simple preparation process, constructs a CD44 targeted polymer carrier with active oxygen responsive drug release to deliver anti-inflammatory drugs, has good biocompatibility and CD44 targeted action, can increase the selective accumulation of drugs at inflammatory sites, further improves the permeability and in vivo long circulation of insoluble drugs, and in addition, the addition of the thioketal further realizes the integral active oxygen response of the polymer carrier, can deliver the anti-inflammatory drugs to focus sites, and achieves breakthrough progress in the aspect of drug combination treatment of inflammatory diseases. The intelligent response drug delivery system jointly applies the receptor-mediated targeted drug delivery and the active oxygen responsive drug release strategy to the micelle system, shows good cooperative targeted co-delivery of the drugs, improves the treatment efficiency of the anti-inflammatory drugs, reduces the toxic and side effects of the anti-inflammatory drugs, and provides a new strategy for the targeted treatment of inflammation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a 1H NMR spectrum of (A) HA-TK-ART micelle of the present invention after active oxygen-responsive cleavage and (B) HA-TK-ART micelle;

FIG. 2 is an in vitro release profile of DEX/HA-TK-ART co-drug loaded micelles of the present invention in different media;

FIG. 3 is a transmission electron microscope image of HA-TK-ART and DEX/HA-TK-ART (B) according to the present invention (A);

FIG. 4 is a graph showing the effect of DEX/HA-TK-ART micelles of the present invention on the survival rate of (A) inactivated macrophage cells and (B) activated macrophages;

FIG. 5 shows the result of DEX/HA-TK-ART of the present invention inducing apoptosis of RAW264.7 cells;

FIG. 6 is a graph showing the in vivo anti-inflammatory pharmacodynamic results of DEX/HA-TK-ART micelles of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Preparation of hyaluronic acid-ketone thiol-artesunate polymer carrier

The method comprises the following steps: synthesis of ketals

(1.1) Synthesis of Compound 1

Dissolving 4g of cysteamine hydrochloride, 4.2mL of ethyl trifluoroacetate and 9.76mL of triethylamine in 100mL of methanol, reacting overnight under stirring, neutralizing the reaction solution with acetic acid and removing the solvent, dissolving the obtained mixture in ethyl acetate, extracting and washing the purified organic layer with water, drying the organic layer with anhydrous magnesium sulfate for 6 hours, and distilling under reduced pressure to obtain a compound 1 for later use;

(1.2) Synthesis of Compound 2

Dissolving 1g of compound 1,0.2mL of acetone in 10mL of acetonitrile under the protection of nitrogen, cooling to 0 ℃ in an ice bath, slowly dropwise adding 1mL of boron trifluoride diethyl etherate, adding the boron trifluoride diethyl etherate within 30min, reacting for 2h under continuous stirring at 0 ℃, then pouring a reaction system into a sodium carbonate solution with the mass fraction of 15%, extracting with ethyl acetate, combining organic layers, washing with a sodium carbonate solution with the mass fraction of 5%, drying with anhydrous sodium sulfate for 6h, distilling under reduced pressure to obtain a yellow liquid, separating and purifying by silica gel column chromatography (petroleum ether: ethyl acetate) =1 to obtain a transparent oily product, and crystallizing to obtain a white solid to obtain a compound 2;

(1.3) Synthesis of Compound 3

Dissolving the compound 2 in 6M sodium hydroxide solution, stirring at room temperature for 4h to react, generating yellow oil drops, extracting with dichloromethane, and distilling under reduced pressure to obtain amber oil, namely the thioketal;

step two: synthesis of hyaluronic acid-ketodithiols

Dissolving 170mg of hyaluronic acid in distilled water, adding 470mg of DMT-MM catalyst under an ice bath condition, heating the reaction to room temperature, continuing to react for 3 hours, dissolving 165mg of ketothiol in a DMF solution, slowly dropwise adding the ketothiol into the reaction system, stirring the mixture at the room temperature for reaction for 12 hours, filling the crude product into a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing the mixture in the distilled water for two days, and freeze-drying the mixture for later use;

step three: preparation of hyaluronic acid-ketal thiol-artesunate polymer carrier

Dissolving 500mg of artesunate in a DMF solution, adding 250mg of DECI and 159mg of NHS catalyst, reacting for 3h in ice bath, slowly dropwise adding 28mg/mL of hyaluronic acid-ketomercaptan aqueous solution into the reaction system, stirring and reacting for 24h, filling the crude product into a dialysis bag with molecular weight cut-off of 3500Da, dialyzing in distilled water for two days, and freeze-drying to obtain the polymer carrier.

Example 2

Preparation of hyaluronic acid-ketone thiol-artesunate polymer carrier

The method comprises the following steps: synthesis of ketals

(1.1) Synthesis of Compound 1

Dissolving 4g of cysteamine hydrochloride, 4.2mL of ethyl trifluoroacetate and 9.76mL of triethylamine in 100mL of methanol, reacting for 12 hours under stirring, neutralizing the reaction solution with acetic acid and removing the solvent, dissolving the obtained mixture in ethyl acetate, extracting and washing the purified organic layer with water, drying the organic layer with anhydrous sodium sulfate for 24 hours, and distilling under reduced pressure to obtain a compound 1 for later use;

(1.2) Synthesis of Compound 2

Dissolving 1g of compound 1,0.2mL of acetone in 10mL of acetonitrile under the protection of nitrogen, cooling to 0 ℃ in an ice bath, slowly dropwise adding 1mL of boron trifluoride diethyl etherate, completing the addition within 30min, reacting for 4h under the continuous stirring at 0 ℃, then pouring the reaction system into a sodium carbonate solution with the mass fraction of 15%, extracting with ethyl acetate, combining organic layers, washing with a sodium carbonate solution with the mass fraction of 5%, drying with anhydrous magnesium sulfate for 24h, distilling under reduced pressure to obtain a yellow liquid, separating and purifying by silica gel column chromatography (normal hexane: ethyl acetate) =7:2 to obtain a transparent oily product, and crystallizing to obtain a white solid to obtain a compound 2;

(1.3) Synthesis of Compound 3

Dissolving the compound 2 in 6M sodium hydroxide solution, stirring at room temperature for 5h to react to generate yellow oil drops, extracting with dichloromethane, and distilling under reduced pressure to obtain amber oil, namely the thioketal;

step two: synthesis of hyaluronic acid-ketodithiols

Dissolving 124mg of hyaluronic acid in distilled water, adding 276.72mg of EDCI catalyst under the ice bath condition, heating the reaction to room temperature, continuing to react for 4 hours, dissolving 183mg of TK in DMF solution, slowly dropwise adding the above reaction system, stirring the reaction system at room temperature for 24 hours, filling the crude product in a dialysis bag with the molecular weight cutoff of 3000Da, dialyzing in distilled water for two days, and freeze-drying for later use;

Dissolving 216mg of artesunate in a DMF solution, adding 215mg of DECI and 137mg of DMAP catalyst, reacting for 3h in an ice bath, then slowly dropwise adding 20mg/mL of HA-TK aqueous solution into the reaction system, stirring and reacting for 24h, filling the crude product into a dialysis bag with the cut-off molecular weight of 3000Da, dialyzing in distilled water for two days, and freeze-drying to obtain the polymer carrier.

Example 3

Preparation of dexamethasone-co-loaded hyaluronic acid-ketone thiol-artesunate polymer micelle

Precisely weighing 5mg of dexamethasone to dissolve in 1mL of methanol to prepare a dexamethasone solution (5 mg/mL), weighing 10mg of the hyaluronic acid-thioketal-artesunate polymer carrier prepared in the example 1 to dissolve in 5mL of water (2 mg/mL), and mixing the hyaluronic acid-thioketal-artesunate polymer carrier with the dexamethasone according to the mass ratio of 10:1, slowly and dropwise adding the dexamethasone solution into the hyaluronic acid-ketal mercaptan-artesunate polymer carrier solution, and stirring for 24 hours to volatilize the organic solvent. After ultrasonic treatment for 5min, centrifuging and taking the supernatant to prepare the 150-250 nm hyaluronic acid-ketothiolane-artesunate polymer micelle carrying dexamethasone.

The transmission electron microscope image of the hyaluronic acid-ketal thiol-artesunate polymer carrier HA-TK-ART (A) and the hyaluronic acid-ketal thiol-artesunate polymer micelle DEX/HA-TK-ART (B) co-carried with dexamethasone is shown in figure 3. As can be seen from the transmission electron microscope picture, they are smooth and round spheres and have good dispersibility.

Example 4

Determination of active oxygen responsiveness of hyaluronic acid-Ketone thiol-Artesunate Polymer Carrier

Dissolving HA-TK-ART micelles in a solution containing H ₂ O ₂ (10 mM) in heavy water, incubation at 37 ℃,2h later samples were collected and used ¹ H NMR was used to detect cleavage of the thioketal bond. The cleavage of the thioketal bond was roughly calculated by observing the disappearance of the dimethyl signal peak at 1.58ppm and comparing the integrated area of the dimethyl signal peak of the thioketal bond with the integrated area of the proton signal peak of the anomeric carbon of hyaluronic acid with reference to the degree of substitution of TK in HA-TK.

As can be seen in FIG. 1, HA-TK was treated with 10mM H ₂ O ₂ After treatment, about half of the ketal and thiol bonds are degraded, and a more obvious acetone methyl signal peak appears at 2.10ppm, which shows that the active oxygen can efficiently degrade the ketal and thiol bonds.

Effect example 1:

in vitro release of co-drug loaded micelles DEX/HA-TK-ART

Placing DEX/HA-TK-ART micelle solution containing 4mg DEX into dialysis bag with cut-off molecular weight of 3000-3500, and placing dialysis bag into dialysis bag containing 10Mm H ₂ O ₂ PBS buffer solution and no H ₂ O ₂ 4mL of the dialysate was taken each time, then the same release medium was added, samples were taken at 0.5, 1, 2, 4, 6, 8, 12, 24, 48h, and the UV absorption of the removed dialysate was measured with a UV-visible spectrophotometer.

As a result, as shown in FIG. 2, ART and DEX were slowly released in both media without burst release. At 12H, the cumulative release of ART and DEX is higher than 50%, which is far larger than that without H ₂ O ₂ The cumulative release amount of the drug in the PBS buffer solution shows that the DEX/HA-TK-ART co-drug-loaded micelle can release ART and DEX more quickly in the ROS environment. This demonstrates the ability of the DEX/HA-TK-ART co-loaded micelle to release ART and DEX in response to active oxygen.

Effect example 2:

cytotoxicity Studies

Macrophage RAW264.7 was cultured in a medium with or without lipopolysaccharide (1. Mu.g/ml) at a density of 1X 10 ⁴ Inoculating into 96-well cell culture plate, incubating in 37 deg.C cell culture box for 24 hr, discarding the old culture solution, and adding different concentrations of the aboveART solution group, DEX solution group, ART&The administration concentration of the DEX solution group, the HA-TK-ART micelle group and the DEX/HA-TK-ART micelle group is consistent with that of the drug-loading group. After 72h incubation, 50. Mu.L MTT solution at 5mg/mL was added to each well, and after 4h incubation, 100. Mu.L DMSO was added to each well. OD value was measured at 492nm using a microplate reader, and cell viability was calculated.

As a result, as shown in FIG. 4, macrophage inhibitory rate increased with the increase of DEX concentration by different treatment groups, wherein DEX/HA-TK-ART micelle group showed the strongest inhibitory effect on lipopolysaccharide-activated inflammatory macrophages because combined anti-inflammatory effects were exerted at cellular level by targeted co-delivery of ART and DEX to inflammatory macrophages through HA-TK-ART polymer micelles.

Effect example 3:

apoptosis study

Macrophage RAW264.7 is inoculated into a 6-hole cell culture plate (5 multiplied by 104 cells/hole), lipopolysaccharide is stimulated for 24 hours, old culture solution is discarded, then ART solution (5 mu g/ml), DEX solution (5 mu g/ml), ART & DEX solution, HA-TK-ART micelle and DEX/HA-TK-ART micelle are given, after 48 hours of incubation, the micelle group is stained by an Annexin V-FITC/PI apoptosis detection kit, and the capacity of the micelle group to induce the apoptosis of the macrophage RAW264.7 is detected by a flow cytometer.

As a result, as shown in FIG. 5, compared with other treatment groups, DEX/HA-TK-ART micelles exhibited the strongest ability to induce apoptosis of macrophage RAW264.7, and the apoptosis rate reached 53.9%, indicating that DEX/HA-TK-ART micelles can efficiently co-deliver ART and DEX to inflammatory macrophages, exerting a synergistic anti-inflammatory effect.

Effect example 4:

in vivo anti-inflammatory pharmacodynamics

The establishment of rat AIA model was carried out by selecting 160-180g of male SD rats. 100 μ L of complete Freund's adjuvant (10 mg/m L) was subcutaneously injected into the place of spat spathula in the right foot of rat. After 14 days, all indexes tend to be stable, and the modeling rats are randomly divided into seven groups, namely a normal saline group, an ART solution group, a DEX solution group and an ART group&DEX mixed solution group, HA-TK-ART group and DEX/HA-TK-ART micelle group. The other group of healthy rats was not treated with any treatment and was a negative control group. Administration on

days

14, 17, 20, 23, 26 and observationThe rats had toe changes and were scored by recording the degree of toe swelling. Swelling degree of joint (mm) ² ) = right paw swelling (mm) × right ankle swelling (mm). Representative photographs of the paw of each group of rats were taken before sacrifice on day 30 to study the anti-inflammatory effect of the co-loaded micelles in vivo.

As shown in FIGS. 6 (B) and (C), the minimum swelling degree of paw of rats in the DEX/HA-TK-ART micelle group was 50.67mm ² The score is 1.50 at the lowest, and is almost close to the normal group, which shows that the synchronous in-vivo co-delivery of ART and DEX can be realized through the co-drug-loaded micelle DEX/HA-TK-ART, the inflammation part is targeted, and the combined anti-inflammatory effect is exerted. FIG. 6 (A) is a photograph of rat paw of different treatment groups, the results are the same as those of FIGS. 6 (B) and (C), which further confirms that co-delivery of DEX with ART via the vector HA-TK-ART enhances accumulation of both drugs at the site of inflammation, and achieves optimal anti-inflammatory effect.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A targeted polymer micelle for active oxygen-responsive drug release in inflammatory microenvironment comprises a hyaluronic acid-ketothiol-artesunate polymer carrier and an anti-inflammatory drug encapsulated by the polymer carrier;

the preparation method of the targeted polymer micelle for the active oxygen responsive drug release in the inflammatory microenvironment comprises the following steps:

the method comprises the following steps: synthesis of ketals

(1.1) dissolving cysteamine hydrochloride, ethyl trifluoroacetate and triethylamine in methanol to react for 8-12h, neutralizing the reaction solution with acetic acid, removing the organic solvent, extracting the obtained mixture with ethyl acetate and water, drying the organic layer with anhydrous magnesium sulfate for 6-24h, and removing the solvent to obtain a compound 1 for later use;

(1.2) dissolving a compound 1 and acetone in acetonitrile under the protection of nitrogen, cooling to 0 ℃ in an ice bath, adding boron trifluoride diethyl etherate, reacting at 0~4 ℃ for 2 to 6 hours, then pouring a reaction system into a sodium carbonate solution with the mass fraction of 5 to 15 percent, extracting with ethyl acetate, combining organic layers, washing with the sodium carbonate solution, drying with anhydrous sodium sulfate for 6 to 24h, and then mixing the organic layers according to the volume ratio of petroleum ether to n-hexane: separating and purifying by silica gel column chromatography with ethyl acetate = 7;

step two: synthesis of hyaluronic acid-ketodithiols

Dissolving hyaluronic acid in water, adding a catalyst, heating the reaction to room temperature, continuing to react for 1 to 4 hours, dissolving ketamine in DMF solution, adding the solution into a hyaluronic acid reaction system, reacting for 12 to 24h at room temperature, dialyzing for two days in pure water by using a 1000 to 4000Da dialysis bag, and freeze-drying for later use;

Dissolving artesunate in DMF, adding a catalyst, reacting for 1 to 4 hours under an ice bath condition, adding the hyaluronic acid-ketothiolate aqueous solution of 20 to 50mg/mL obtained in the second step, reacting for 8 to 24h, dialyzing the dialysis bag of 1000 to 4000Da in pure water for two days, and freeze-drying to obtain an active oxygen responsive polymer carrier;

And (3) dissolving the anti-inflammatory drug in an organic solvent, adding the anti-inflammatory drug into the hyaluronic acid-ketothiolate-artesunate polymer carrier aqueous solution of 2-5 mg/mL obtained in the third step, stirring for 8-24h to volatilize the organic solvent, and then performing ice-bath ultrasound, wherein the ultrasound power is 100-500W, and the ultrasound time is 2-10min to obtain the active oxygen responsive drug release targeted polymer micelle.

2. The targeted polymeric micelle for the responsive release of active oxygen in inflammatory microenvironment, according to claim 1, is characterized in that the grafting rate of artesunate is 25-35%, the grafting rate of thioketal is 60-70%, and the anti-inflammatory drug accounts for 10-20% of the total mass of the micelle.

3. The targeted polymeric micelle for active oxygen-responsive drug release in inflammatory microenvironment of claim 1, wherein the anti-inflammatory drug is one or two or more of dexamethasone, methotrexate, indomethacin, betamethasone and leflunomide.

4. The targeted polymeric micelle for the responsive release of active oxygen in the inflammatory microenvironment according to claim 1, wherein the molar ratio of the hyaluronic acid to the ketothiol in the second step is 1 to 2 to 1, and the molar ratio of the hyaluronic acid to the catalyst is 1 to 1.

5. The targeted polymer micelle for responsive release of active oxygen in inflammatory microenvironment according to claim 1, wherein the catalyst in step two and step three is any one or two of DMT-MM, EDCI, DMAP and NHS.

6. The targeted polymer micelle for the responsive release of active oxygen in inflammatory microenvironment according to claim 1, wherein the molar ratio of artesunate to hyaluronic acid-ketothiolane in step three is 2 to 1.

7. The targeted polymer micelle of claim 1, wherein the organic solvent is any one of methanol, ethanol, acetone and tetrahydrofuran.

8. The targeted polymeric micelle for the responsive release of active oxygen in the inflammatory microenvironment according to claim 1, wherein the mass ratio of the anti-inflammatory drug to the polymeric carrier in the step four is 1 to 5-1.