CN106727309A - Polymer micelle solution containing Quercetin and its preparation method and application - Google Patents

Abstract

The invention relates to a polymer micelle solution containing quercetin, a preparation method and application thereof. The polymer micelle solution comprises quercetin, an amphiphilic polymer micelle material, a stabilizer and water, and the mass percentages of each component are respectively 0.3% to 1.5% of quercetin and 10% of micelle material. Agent 0.5% ~ 3%, water 85.5% ~ 89.2%. The polymer micelle solution containing quercetin overcomes the defect that quercetin is insoluble in water, and at the same time crosses the transmembrane barrier of quercetin during oral absorption, which can significantly prolong the release time of quercetin in vitro and improve degree of complete release. The polymer micelle of the invention has good biocompatibility, can increase bioavailability, and improve the stability of quercetin.

Description

Polymer micelle solution containing quercetin and its preparation method and application

technical field

The invention relates to the technical field of biopharmaceuticals, in particular to an amphiphilic polymer enzyme solution containing quercetin and its preparation method and application.

Background technique

Quercetin, also known as quercetin and quercetin, can be used as a medicine, which has good expectorant and cough-relieving effects, and has a certain anti-asthma effect. In addition, it has the effects of lowering blood pressure, enhancing capillary resistance, reducing capillary fragility, lowering blood lipids, expanding coronary arteries, and increasing coronary blood flow. Quercetin is widely used in the treatment of chronic bronchitis, and also has adjuvant therapeutic effect on patients with coronary heart disease and hypertension. Modern medical research shows that quercetin also has a good anti-tumor effect, and it has been used in clinical treatment of tumors abroad. But the stability of quercetin is poor, the half-life of circulation in the body is short, it is difficult to effectively reach the lesion site, and quercetin is insoluble in water, and the oral bioavailability is poor.

Contents of the invention

Based on this, it is necessary to provide a polymer micelle solution containing quercetin capable of improving the bioavailability of quercetin, its preparation method and application.

A polymer micelle solution containing quercetin, comprising the following components in mass percent:

Wherein, the amphiphilic polymer micelle material coats the quercetin to form a core-shell structure.

In one of the embodiments, the polymer micelle solution includes the following components in mass percentage:

Quercetin 0.5%～1.0%;

Amphiphilic polymer micellar material 10%; and

Stabilizer 1.5% ~ 2.5%;

The balance is water.

In one of the embodiments, the amphiphilic polymer micelle material is selected from polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer, sodium deoxycholate, polyoxyethylene, polyethylene glycol chitosan , povidone, imitation cell membrane phosphorylcholine, polyamino acid and polylactic acid-glycolic acid copolymer at least one.

In one embodiment, the stabilizer is at least one selected from natural water-soluble vitamin E, poloxamer 407 and Tween 80.

In one of the embodiments, the mass percentage of quercetin is 0.7%; the amphiphilic polymer micelle material is polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer; the stable The stabilizer is poloxamer 407, and the mass percentage of the stabilizer is 2%.

A preparation method of a quercetin-containing polymer micelle solution described in any of the above-mentioned embodiments, comprising the steps of:

Step 1: dissolving the quercetin and the amphiphilic polymer micelle material in an organic solvent according to the corresponding mass percentage ratio, stirring to make the quercetin and the amphiphilic polymer The micellar material is completely dissolved until mixed uniformly to obtain an organic solution containing quercetin and amphiphilic polymer micellar material;

Step 2: completely remove the organic solvent in the solution by volatilization to obtain a polymer film material comprising the quercetin and the amphiphilic polymer micelle material;

Step 3: Add the polymer film material into the aqueous solution containing the stabilizer according to the corresponding mass percentage ratio, vortex and stir to obtain the polymer micelle solution containing quercetin.

In one embodiment, the organic solvent is at least one of acetone, methanol, ethyl acetate and glacial acetic acid.

In one of the embodiments, the volume-to-mass ratio of the organic solvent to the amphiphilic polymer micelle material is (0.1-0.3) mL:100 mg.

In one of the embodiments, the stirring in the step 1 is magnetic stirring in a water bath, and the temperature of the water bath is 40-60°C;

The stirring in the step 3 is magnetic stirring, the stirring speed is 500-700 rpm, and the stirring time is 0.5-2 h.

Application of the quercetin-containing polymer micelle solution described in any of the above embodiments in the preparation of antitumor drugs.

The above-mentioned amphiphilic polymer micelle material in the polymer micelle solution containing quercetin can spontaneously form a stable polymer micelle in water, which has a hydrophobic core-hydrophilic shell structure, and the hydrophobic core can serve as a hydrophobic The reservoir of the drug quercetin, solubilizing the poorly soluble drug quercetin in the inner core, can increase the stability of quercetin, improve its bioavailability, and promote its absorption; the hydrophilic shell not only stabilizes the polymer micelles Enhanced properties, and affect the interaction between polymer micelles and the external environment, thereby affecting the behavior of polymer micelles in vivo. Polymer micelles have very high thermodynamic stability, and there are hydrophobic interactions between multiple points in the structure, so that polymer micelles have high dynamic stability, so their good thermodynamic stability and dynamic stability , can prevent the drug from being separated out in the body, and ensure the stability of the packaged drug.

The preparation method of the polymer micelle solution containing quercetin is simple in process, high in encapsulation efficiency, and low in production cost, is conducive to industrial production, has good application prospects, and is conducive to promoting the industrial development of liquid crystal nanometers.

Description of drawings

Fig. 1 is the particle diameter and the potential of the polymer micelle containing quercetin containing different stabilizing agents in embodiment 2;

Fig. 2 is the particle diameter of the polymer micelle containing quercetin with different stabilizing agent concentrations in embodiment 3;

Fig. 3 is the particle size, polydispersity coefficient, encapsulation efficiency, drug loading and potential of the polymer micelles containing quercetin in different dosages in Example 4;

Fig. 4 is the particle diameter and encapsulation efficiency of the polymer micelle containing quercetin under the different magnetic stirring time of embodiment 5;

Fig. 5 is the scanning electron micrograph of the polymer micelle containing quercetin in embodiment 6;

Fig. 6 is the differential scanning calorimetry figure in embodiment 7 (A: quercetin; B: polymer micelle containing quercetin; C: blank polymer micelle);

Fig. 7 is the powder X-ray diffraction figure (A: quercetin in embodiment 7; B: the physical mixture of quercetin and blank polymer micelle; C: the polymer micelle containing quercetin; D: blank polymer micelles);

Fig. 8 is the infrared absorption spectrogram in embodiment 8 (A: quercetin; B: the physical mixture of quercetin and blank polymer micelle; C: blank polymer micelle; D: the polymerization containing quercetin object micelles);

9 is a schematic diagram of in vitro release of polymer micelles containing quercetin in Example 9;

Figure 10 is a schematic diagram of the influence of artificially simulated gastrointestinal fluid on the particle size of Qu-PMs in Example 10;

Figure 11 is a schematic diagram of the effect of artificially simulated gastrointestinal fluid on the particle size of Qu-PMs in Example 10 (A: pH 1.2, 2h; B: pH 7.4, 4h; C: pH 7.4, 6h; D: pH 7.4, 8h) ;

Figure 12 is a schematic diagram of the stability of the particle size and encapsulation efficiency of the Qu-PMs colloidal solution stored at room temperature in Example 11;

Fig. 13 is the average plasma concentration-time curve (n=3) of quercetin crude drug and quercetin-containing polymer micelles in beagle dogs administered orally in Example 12.

detailed description

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the associated drawings. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The polymer micelle solution containing quercetin of one embodiment, it comprises each component that has following mass percentage content:

Among them, the amphiphilic polymer micelle material coats quercetin to form a core-shell structure.

Preferably, the polymer micelle solution includes each component with the following mass percentages:

Quercetin 0.5%～1.0%;

Amphiphilic polymer micellar material 10%; and

Stabilizer 1.5% ~ 2.5%;

The balance is water.

The amphiphilic polymer micelle material is selected from polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer (soluplus), sodium deoxycholate, polyoxyethylene (PEO), polyethylene glycol chitosan (PEG- CS), povidone (PVP), imitation cell membrane phosphorylcholine, polyamino acid and polylactic-co-glycolic acid (PLGA).

Quercetin has phenolic hydroxyl groups in its structure, which are easy to form intermolecular hydrogen bonds with carbonyl groups. Preferably, the amphiphilic polymer micelle material is polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer. Polyethylene glycol-vinylcaprolactam-vinyl acetate copolymer has an amphiphilic structure with a hydrophilic segment and a hydrophobic segment of polyvinylcaprolactam-vinyl acetate, and its CMC value (6.44×10 ^-8 mol·L ^-1 ) is very Low, can spontaneously form stable polymer micelles in water. The hydrophobic segment of polyvinylcaprolactam-vinyl acetate forms a hydrophobic inner core, embeds insoluble drugs, improves solubility and enhances its stability. The hydrophobic inner core of polymer micelles formed by polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer has the carbonyl structure of amide and acetyl groups, which can form hydrogen bonds with the phenolic hydroxyl groups of quercetin, thus forming a stable coating core-shell structure.

The stabilizer is at least one selected from natural water-soluble vitamin E (TPGS), poloxamer 407 (F127) and Tween 80 (Tween80).

Preferably, in the polymer micelle solution containing quercetin in this embodiment, the mass percentage of quercetin is 0.7%; the amphiphilic polymer micelle material is polyethylene glycol-vinyl caprolactam-acetic acid Vinyl ester copolymer; the stabilizer is poloxamer 407, and the mass percentage of the stabilizer is 2%.

This embodiment also provides a method for preparing the above-mentioned polymer micelle solution containing quercetin, which includes the following steps:

Step 1: Dissolving the quercetin and the amphiphilic polymer micelle material in an organic solvent according to the corresponding mass percentage ratio, stirring to completely dissolve the quercetin and the amphiphilic polymer micelle material until evenly mixed , to obtain an organic solution containing quercetin and amphiphilic polymer micelle material;

Step 2: completely remove the organic solvent in the solution by volatilization, and obtain a polymer film material comprising quercetin and amphiphilic polymer micelle material;

Step 3: Add the polymer film material into the aqueous solution containing the stabilizer according to the corresponding mass percentage ratio, vortex and stir to obtain a polymer micelle solution containing quercetin.

The organic solvent is preferably but not limited to acetone, methanol, ethyl acetate and glacial acetic acid, and the volume-to-mass ratio of the organic solvent to the amphiphilic polymer micelle material is (0.1-0.3) mL:100 mg.

The stirring in step 1 is magnetic stirring in a water bath, and the temperature of the water bath is 40-60°C.

The stirring in step 3 is magnetic stirring, the stirring speed is 500-700 rpm, and the stirring time is 0.5-2 h. Preferably, the stirring speed is 650rpm, and the stirring time is 2h.

The above-mentioned polymer micelle solution containing quercetin can be widely used in the preparation of antitumor drugs. The obtained antitumor drug has good stability, and the bioavailability is greatly improved compared with single quercetin, and the absorption efficiency is also greatly improved.

The following is the specific embodiment part.

Example 1: Preparation of polymer micelle solution containing quercetin.

Weigh a certain amount of quercetin (Qu) and 100mg carrier soluplus (polyethylene glycol-vinyl caprolactam-vinyl acetate copolymer) and dissolve in 0.2mL acetone, magnetically stir to dissolve completely, mix well, and then place in Magnetic stirring in a water bath at 50°C, volatilization and removal of acetone, to obtain a polymer film material containing quercetin and amphiphilic polymer micelles; weigh the stabilizer and dissolve it in water to prepare an aqueous solution of a certain concentration; The aqueous solution was added to the prepared polymer film material to make the total weight of the system 1 g, vortexed, and magnetically stirred at 650 rpm to obtain a polymer micelle solution (Qu-NPs) containing quercetin.

Example 2: Effects of stabilizer types on polymer micelles containing quercetin (hereinafter referred to as "polymer micelles").

The influence of different types of stabilizers on the polymer micelles was investigated. The preparation method was the same as in Example 1. The polymer micelles solution used 1% TPGS, 1% F127, 1% Tween80 and deionized water respectively. Wherein the amount of quercetin is 0.7% (7mg), and magnetically stirred for 2h. The polymer micelle particle size, Zeta potential and encapsulation efficiency were used as the investigation indexes.

Particle size and potential measurement method, after diluting the polymer micelle solution with ultrapure water, take 0.5mL of the polymer micelle solution and add it to the sample cell, equilibrate at 25°C for 2min, and use the Zetasizer Nano ZS90 particle size analyzer to measure the polymer gel Beam size, polydispersity index (PDI) and Zeta potential.

The encapsulation efficiency of quercetin was determined by membrane filtration method. Membrane filtration method means that the unencapsulated drug exists in two forms: free microcrystals (mostly) and dissolved in the medium (very small part), so microcrystals can be used. The pore filter retains free microcrystals, and the dissolved drug concentration can be approximately considered as the solubility of the drug.

Accurately measure 0.5mL of the diluted Qu-PMs colloidal solution, filter the unencapsulated drug with a 0.45μm microporous membrane, demulsify the filtrate with methanol, set the volume to a 10mL volumetric flask, and measure the content by HPLC; Put 0.5mL of the diluted Qu-PMs colloidal solution in a 10mL volumetric flask, add methanol to break the emulsification, make up to volume, measure the drug content by HPLC, and calculate the entrapment efficiency (EE).

The HPLC conditions are as follows: chromatographic column: Odyssil C18 (250×4.6mm, 5μm); mobile phase: methanol: 0.2% phosphoric acid (60:40); column temperature: 35°C; detection wavelength: 375nm; flow rate: 1.0mL min- 1; Injection volume: 20 μL.

As shown in Figures 1A and 1B, the results show that under the conditions of adding different stabilizers TPGS, F127, and Tween80, polymer micelles smaller than 100 nm can be obtained, and the encapsulation efficiencies are all higher than 80%. Using 1% TPGS as a stabilizer, the particle size (62.11±0.53nm) and PDI (0.081±0.02) of the prepared polymer micelles were small, but the encapsulation efficiency was relatively lowest (83.02±3.46%), and the Zeta potential was -5.19 ±1.83mV, easy to gather during placement. Using 1% F127 as a stabilizer, the particle size (61.64±0.35nm) and PDI (0.017±0.02) of the prepared polymer micelles were smaller, and the encapsulation efficiency was the highest (95.85±2.86), Zeta(-13.63±1.45) minimum. The characteristics of polymer micelles prepared by using 1% Tween80 as a stabilizer are similar to those of using 1% F127 as a stabilizer, but it is found that they are easy to aggregate during the standing process, and the particle size and PDI of polymer micelles prepared without stabilizers are relatively large.

Example 3: Effect of stabilizer concentration on polymer micelles.

To investigate the influence of different stabilizer concentrations on polymer micelles, the preparation method was the same as in Example 1, and the polymer micelles solutions were used with mass percentages of 0.5% F127, 1% F127, 2% F127 and 3% F127. Wherein the amount of quercetin is 0.7% (7mg), and magnetically stirred for 2h. The polymer micelle particle size and encapsulation efficiency are used as the investigation index, and the method is the same as in Example 2.

As shown in Figure 2, the results show that the concentration of 0.5-2% F127 has little effect on the particle size and encapsulation efficiency of polymer micelles, and the encapsulation efficiency is greater than 85%. The particle size of the polymer micelle prepared by 3% F127 was larger (107nm), and the particle size of the polymer micelle prepared by 0.5%, 1% and 2% F127 was smaller.

Example 4: Effect of dosage on polymer micelles.

Investigate the impact of different dosages on polymer micelle, preparation method is the same as embodiment 1, and the dosage of quercetin is respectively 0.3% (3mg), 0.5% (5mg), 0.7% ( 7mg), 1.0% (10mg), 1.5% (15mg). Among them, the stabilizer is 2% F127, and magnetically stirred for 2 hours. The polymer micelle particle size, PDI, drug loading and encapsulation efficiency are used as the investigation indicators, and the method is the same as in Example 2.

The results are shown in Figure 3. The particle size results in Fig. 3A show that with the increase of dosage, the particle size of polymer micelles gradually increases, and when the dosage is 10 mg, the particle size of polymer micelles containing quercetin increases significantly. When the dosage was 15mg, the particle size increased sharply to 559.23±19.41nm, and the PDI was 0.427±0.104, which indicated that the unencapsulated drug formed microcrystals in the solution.

The results of encapsulation efficiency in Figure 3B show that as the dosage increases, the encapsulation efficiency of the drug is on the rise. When the dosage is 0.7%, the encapsulation efficiency of the drug is the largest, and when the dosage is 1.0%, the encapsulation efficiency Less than 60%, half of the medicine is not encapsulated, and the utilization rate of the medicine is low. When the dosage is 1.5%, the encapsulation efficiency is only 2.29%.

The results of drug loading in Figure 3B show that as the dosage increases, the drug loading of the drug shows an upward trend. When the dosage is 0.7%, the drug loading is the largest. When the dosage is 1.0%, the drug loading It began to decline, and the drug loading was 5.01%. When the dosage was 1.5%, the drug loading was only 0.34%.

The potential results in Figure 3C show that as the dosage increases, the Zeta potential of the drug shows an upward trend. When the dosage reaches 7%, the Zeta potential of the polymer micelle is the smallest, and then as the dosage increases, the Zeta potential changes. Trend.

Comprehensive analysis of the above results, with the increase of quercetin dosage, the particle size, drug loading, encapsulation efficiency and absolute value of Zeta potential all increased. The increase of the drug loading of the polymer micelle means that the amount of drug entrapped in the inner core of the polymer micelle increases, and more drugs are included in the inner core, causing the diameter of the inner core to become larger, so the particle size of the polymer micelle increases. At the same time, due to the limited space of the hydrophobic inner core, increasing the input amount of quercetin, when the drug loading capacity of the inner core is exceeded, some quercetin must not be included in the inner core, which will reduce the utilization rate of raw materials. When the dosage is 1.0%, the particle size of the polymer micelles containing quercetin exceeds 100nm, which may be due to the increase in the diameter of the hydrophobic inner core when the dosage increases. When the dosage is 1.5%, it exceeds the ability of the polymer micelle to carry the drug, and the drug forms microcrystals in the aqueous solution. Therefore, considering the particle size, encapsulation efficiency, drug loading and Zeta potential of the particles, the appropriate dosage should be controlled at 0.7% (7mg).

Example 5: Effect of magnetic stirring time on polymer micelles.

The influence of different magnetic stirring times on the micelles was investigated. The preparation method was the same as in Example 1, and the stirring times were 0.5, 1, 1.5 and 2 hours respectively. The stabilizer is 2% F127, and the dosage is 0.7% (7mg). The polymer micelle particle size and encapsulation efficiency are used as the investigation index, and the method is the same as in Example 2.

As shown in Figure 4, the results show that the particle size of the polymer micelles becomes smaller with the prolongation of the magnetic stirring time, and there is no significant change, and the PDI becomes smaller with the prolongation of the magnetic stirring time. Magnetic stirring time had little effect on encapsulation efficiency. When the magnetic stirring time is 2h, the particle size of the polymer micelles is small, and the distribution range is narrow, and the encapsulation efficiency is also good. Taking into account the choice of magnetic stirring for 2h.

Based on the results of each example of the above prescription and process, the best preparation process and prescription were optimized: the dosage was 7 mg, 2% F127 was used as a stabilizer, and magnetic stirring was performed at 650 rpm for 2 hours.

Example 6: Morphological characterization of polymer micelles.

In order to investigate the morphology of polymer micelles and the size of particles, scanning electron microscopy (SEM) was used to observe.

Sample preparation of polymer micellar lyophilized powder containing quercetin. Take the polymer micelle colloidal solution, add 5% mannitol freeze-drying protective agent, put it into -80 ℃ refrigerator for 12 hours, and then place it in a freeze dryer for 24 hours to obtain the quercetin-containing quercetin. Polymer micelles lyophilized powder.

Place the quercetin-containing polymer micelle freeze-dried powder sample prepared in Example 1 on a copper plate adhered with insulating glue, spray gold for 2.5 minutes, dry in vacuum, scan and image with a scanning electron microscope, observe and take pictures. The results are shown in FIG. 5 , and the scanning electron microscope showed that the prepared polymer micelles containing quercetin were spherical or quercetin-like, with an average particle diameter of about 100 nm.

Example 7: Study on the crystal form of quercetin in a polymer micelle system.

In order to investigate the existence state of quercetin in polymer micelles, DSC and X-ray diffraction (XRD) were used for analysis and determination.

DSC: An appropriate amount of 5 mg quercetin (Qu), the blank polymer micelle lyophilized powder and the drug-loaded polymer micelle lyophilized powder (Qu-PMs) lyophilized powder prepared in Example 1 were respectively taken for DSC analysis. The DSC conditions are as follows: the heating rate is 10°C/min, the measurement range is 30-400°C, the empty aluminum pan is used as the blank control, and the gas in the furnace is nitrogen.

The DSC results are shown in Figure 6. The results show that the quercetin raw material drug exists in the form of crystalline forms. The DSC chart shows two peaks at 134°C and 325°C. The peak at 134°C is a dehydration peak of the drug, and the peak at 325°C is a peak of the drug. Obvious melting endothermic peaks; four endothermic peaks appear in the blank polymer micelles, and the endothermic peaks of the quercetin-containing polymer micelles are consistent with the blank polymer micelles, which is consistent with the glass transition temperature of the carrier Related to the relationship between them, there is no endothermic peak of the raw material drug at 134°C and 325°C. DSC results showed that the dispersion of quercetin in polymer micelles was different from that of the bulk drug. It can also be seen from the DSC figure that the peak position of the polymer micelles containing quercetin at about 350 ° C has shifted compared with that of the blank polymer micelles, which shows that quercetin and amphiphilic polymers Some forces may exist between micellar materials.

X-ray diffraction (XRD): take an appropriate amount of quercetin (Qu), blank polymer micelle lyophilized powder (PMs), the physical mixture of blank polymer micelle lyophilized powder and raw materials, and the drug-loading prepared in the examples Polymer micelles lyophilized powder (Qu-PMs) samples were analyzed by XRD. The XRD conditions are as follows: the scanning angle is 3°≤2θ≤50°, the step size is 0.9min-1, the dwell time is 2 seconds, the voltage is 40KV, and the current is 25mA.

The results of the X-ray diffraction analysis are shown in FIG. 7 . The results show that the quercetin bulk drug presents the diffraction peak of the crystal structure; in the physical mixture of the quercetin bulk drug and the blank polymer micellar freeze-dried powder, quercetin still presents the diffraction peak of the crystal structure of the drug , indicating that quercetin still exists in the crystal form in the physical mixture; and there are many peaks in the XRD of the polymer micelle containing quercetin, but they are different from the crystal form diffraction peaks of the raw material drug or the drug in the physical mixture, This indicates that quercetin has a crystal structure different from quercetin bulk drug in the polymer micelle. The movement of diffraction peaks from physical mixing, blank polymer micelles and polymer micelles containing quercetin shows that there is a certain interaction between quercetin and polymers. XRD results also indicated that quercetin may exist in molecular state or amorphous state in polymer micelles.

Example 8: Interaction of quercetin with amphiphilic polymer micellar materials (FTIR).

In order to investigate the interaction between quercetin and amphiphilic polymer micelles, quercetin (Qu), blank polymer micelles freeze-dried powder (PMs), blank polymer micelles freeze-dried The physical mixture of the dry powder and the bulk drug and the freeze-dried powder of drug-loaded polymer micelles (Qu-PMs) prepared in Example 1 were analyzed for the structural characteristics of the samples.

First, press the freeze-dried sample into powder, add a certain amount of potassium bromide (KBr), mix well, dry under infrared light to remove excess water, press into thin slices, and place it in an infrared spectrometer in the range of 400 to 4000 cm ^-1 Measure the infrared spectrum of the sample.

The infrared spectrum results are shown in Fig. 8A, Fig. 8B, Fig. 8C and Fig. 8D. Whether there is interaction between molecules can be observed through infrared spectral shift and intensity. The results showed that there were many characteristic absorption peaks in the infrared spectrum of quercetin: OH stretching vibration peak (3700-3300cm ^-1 ); C=O absorption peak (1670cm ^-1 ); CC stretching vibration peak (1612cm ^-1 ); CH bending vibration peaks (1456, 1383, and 866cm ^-1 ); CO stretching vibration peaks (1272cm ^-1 ) and CO stretching vibration peaks (1070-1050cm ^-1 ) on the ring structure, which are consistent with the reports in the literature . Many characteristic absorption peaks also appeared in blank micelles: OH stretching vibration peak (3500-3250cm ^-1 ); sp3CH stretching vibration peak (2932cm ^-1 , methylene); C=O absorption (1742cm ^-1 , ester group), C =O absorption (1641 cm ^-1 , amide group). It can be seen from the infrared absorption diagram of the physical mixture of blank polymer micelles and quercetin raw materials that both the characteristic absorption of the blank micelles and the characteristic absorption peak of quercetin appeared; the polymerization containing quercetin The characteristic absorption peaks of the micelles appeared: C=O absorption (1737cm ^-1 , 1636cm ^-1 ), compared with the C=O absorption peaks (1742cm ^-1 , 1641cm ^-1 ) in the blank micelles, the displacement changed, C= The O absorption (1636cm ^-1 ) peak became broad, which indicated that hydrogen bonds were formed between the quercetinol hydroxyl groups in the quercetin micelles and the carbonyl groups of the polymer.

Example 9: Release behavior and release model of polymer micelles containing quercetin.

The drug release behavior of quercetin-containing polymer micelles was studied by dialysis bag method. Because quercetin is insoluble in water, and its solubility in gastrointestinal fluid is very small, it is difficult to achieve the sink condition. Therefore, 35% ethanol is selected as the release medium to meet the sink condition. Compared with the raw material quercetin and The release of polymer micelles containing quercetin provides a reference for in vivo experimental research.

According to the optimal prescription, prepare the Qu-PMs solution with dosages of 5%, 7% and 9%, and prepare the propylene glycol solution of quercetin at the same time. Take a certain sample and place it in a dialysis bag (molecular weight cut-off 14000), and tighten it for dialysis. At both ends of the bag, place the drug-containing dialysis bag in 100mL of 35% ethanol release medium, release at 37±0.5°C, and rotate at 100rpm, sample 4mL at regular intervals, and replenish the corresponding amount of release medium at the same temperature in time, after 0.45 The microporous membrane was filtered to obtain the filtrate, and the content of the drug was determined by HPLC. Calculate the cumulative release percentage of the drug at different time points, and draw the cumulative release curve.

HPLC conditions: chromatographic column: Odyssil C18 (250×4.6mm, 5μm); mobile phase: methanol: 0.2% phosphoric acid (60:40); column temperature: 35°C; detection wavelength: 375nm; flow rate: 1.0mL· min ^-1 ; injection volume: 20 μL.

The release results are shown in Figure 9. The results showed that the quercetin bulk drug was basically released completely within 24h, reaching 96.13%; while the polymer micelles containing quercetin only released 26.22% within 24h and 57.78% within 240h, compared with the bulk drug , The polymer micelles containing quercetin have obvious sustained-release effect. The experimental results show that soluplus polymer micelles can be used as the carrier of sustained-release preparations.

It can also be seen from the results that within 240 hours, the Qu-PMs with dosages of 7mg and 9mg released about 70%, while the Qu-PMs with dosage of 5mg released less than 60%. The larger the dosage, the faster the release, and the faster the early release. Later release becomes slower.

The in vitro release characteristics of sustained-release preparations can basically be evaluated according to the following equation and its correlation coefficient:

Zero-order drug release model: y=k ₁ t+a ₁

First order drug release model: ln(100-y)=k ₂ t+a ₂

Higuchi equation (diffusion equation): y=k ₃ t0.5+a ₃

Wherein, y is the cumulative release percentage, t is the sampling time, a ₁ to a ₃ are constants, and k ₁ to k ₃ are release constants. The release parameters of quercetin were fitted according to the above equation to investigate the release mechanism of the drug from the polymer micelle, and the best fitting model of the formulation was determined by the correlation coefficient r. The regression equation obtained by fitting is shown in Table 1.

Table 1 Fitting equations of different release models of Qu-PMs

Note: y, cumulative release percentage; t, sampling time; R, correlation coefficient.

The results showed that according to the Higuchi equation fitting, the correlation coefficient (R ² >0.98) was large, and the fitting effect was good, indicating that the release of Qu-PMs was mainly in the form of diffusion, and the drug molecules were released slowly from the hydrophobic core of the micelles. Diffused slowly into the release medium. Due to the possible hydrogen bond between the hydroxyl group on the molecular structure of the drug and the carbonyl group of the hydrophobic core, the drug release is slower. The release of quercetin-containing polymer micelles with different dosages showed a similar trend, and the release of quercetin tended to accelerate slightly with the increase of dosage. Compared with the quercetin bulk drug, the polymer micelles containing quercetin generally exhibited an obvious sustained-release effect.

Example 10: Stability of quercetin-containing polymer micelles in simulated gastrointestinal fluid.

In order to ensure that nanocarriers can deliver drugs to the absorption site, it is necessary to investigate the stability of nanocarriers in artificially simulated gastrointestinal fluids at different pH values, as follows.

Precisely take 9 mL of artificial simulated gastric juice (without enzyme), add 1 mL of Qu-PMs solution prepared in Example 1, shake and mix, let stand for 2 hours, and measure the particle size of Qu-PMs with Zetasizer Nano ZS90 particle size analyzer.

Precisely take 9mL of artificial simulated intestinal juice (without enzyme), add Example 1 to prepare 1mL Qu-PMs solution, shake and mix, let stand for 4h, 6h, 8h, use Zetasizer Nano ZS90 particle size analyzer to measure the particle size of Qu-PMs.

The results are shown in Table 2, Figure 10 and Figures 11A-11D, and the results show that the changes in the drug-loaded polymer micelles in artificially simulated gastrointestinal fluids with different pH values are different. Incubate in the artificial simulated gastric juice of pH 1.2 for 2 hours, the particle size changes from 61.54±0.494nm to 70.72±2.77nm; incubate in the artificial simulated intestinal juice of pH7.4 for 4h, 6h, 8h, the particle size is 60.38±0.64nm , 61.07±0.47nm and 61.03±0.94nm. In conclusion, although the particle size in the artificial simulated gastrointestinal fluid varies, they are all less than 100nm, which indicates that the polymer micelles containing quercetin are relatively stable in the artificial simulated gastrointestinal fluid. This lays a good foundation for the absorption of the polymer micelles containing quercetin in the body.

Table 2 Effect of artificial simulated gastrointestinal fluid on the particle size of Qu-PMs

Example 11: Stability of quercetin-containing polymer micelles at simulated room temperature.

The stability of Qu-PMs at simulated room temperature was investigated. The polymer micelle colloid solution containing quercetin prepared in Example 1 was placed at room temperature for 3 months, and samples were taken at 30 days, 60 days and 90 days respectively, and the particle size of the polymer micelles containing quercetin was measured. Diameter and encapsulation efficiency (method is the same as embodiment 5). The results are shown in Figure 12 and Table 3. The results showed that the particle size, encapsulation efficiency and appearance of Qu-PMs did not change significantly after 90 days, and the prepared Qu-PMs colloidal solution had good stability.

Table 3 Storage stability of Qu-PMs colloidal solution at room temperature

Example 12: Pharmacokinetic study of quercetin-containing polymer micelles.

To investigate the pharmacokinetic properties of the prepared Qu-PMs colloidal solution, the method is as follows.

Take 6 healthy male Beagle dogs, fast for 12 hours before administration, and drink water freely. Six Beagle dogs were randomly divided into two groups: the first group was administered intragastrically with 16 mg·kg ^-1 quercetin micelles; the second group was given 16 mg·kg ^-1 quercetin suspension (0.3% CMCNa) was administered to Beagle dogs as a control group. At 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 hours after administration, about 3 mL of blood was collected from the veins of the hind limbs. The whole blood was placed in a heparin tube and centrifuged at 5000 rpm for 10 minutes to separate the plasma. Store at ℃; accurately draw 0.1mL of blank plasma into a 1.5mL centrifuge tube, add 0.3mL of methanol, 0.1mL of 25% hydrochloric acid, vortex mix for 2min, heat in a water bath at 50℃ for 10min, centrifuge at 12000rpm for 10min, and take 100μL of the supernatant for injection. The content was determined by HPLC method.

HPLC conditions are as follows: chromatographic column: Odyssil C18 (250×4.6mm, 5μm), pre-column (4.6×12.5mm, 5μm); mobile phase: methanol: 0.2% phosphoric acid (60:40); column temperature: 35 ℃; detection wavelength: 375nm, flow rate: 1.0mL·min ^-1 ; injection volume: 100μL.

3P87 program was used to process the single-dose plasma concentration data. According to the plasma concentration, the pharmacokinetic parameters such as C _max , T _max , T _1/2 , and AUC _0-∞ of quercetin after oral administration were calculated. The relative bioavailability of the Qu-PMs colloidal solution was calculated according to the following formula: F=AUCQu-PMs/AUCQu×100%. The two preparations were fitted with the single-compartment model and the weight was 1/C ² , which was the most consistent with the actual curve.

The average drug-time curves after intragastric administration of the two preparations are shown in Figure 13. The pharmacokinetic parameters and relative bioavailability results of the model are shown in Table 4, where Cmax and Tmax are measured values, and other parameters are calculated by 3p87 software. The results of blood drug concentration in Beagle dogs showed that the quercetin API (Qu) group had a shorter time to peak (T _max was 5.31±1.08h), and the peak concentration (C _max ) was 5.24±1.32μg·mL ^-1 , Then the concentration decreased rapidly with large fluctuations, and no drug could be detected by 24 hours. The biological half-life (T _1/2 ) of the drug was 4.94±2.03h, and the area under the drug-time curve (AUC _0～∞ ) was 37.68±16.8μg·h ^-1 ·mL ^-1 . The mean residence time (MRT) is 7.18±2.25h, and it has been completely eliminated from the body after 24h, indicating that it is rapidly metabolized in the body. After oral administration of quercetin-containing polymer micelles (Qu-PMs), the T _max was 7.02±2.02h, the C _max was 7.56±3.28μg·mL ^-1 , and the T _1/2 was 10.81±3.7 h, MRT was 7.18±2.25h, AUC _0～∞ was 107.84±54.4μg·h ^-1 ·mL ^-1 . The T _1/2 and MRT of Qu-PMs are 2.19 times and 3.77 times that of Qu, respectively, indicating that the polymer micelles slow down the elimination of drugs in the body and prolong the residence time, so that they maintain a high concentration at 48h, indicating that Qu-PMs PMs have good sustained-release characteristics in vivo, and the bioavailability is significantly improved (AUC _0～∞ is 2.86 times that of quercetin). The experimental results show that oral administration of Qu-PMs solution can significantly improve the absorption process of quercetin in beagle dogs, promote the absorption of the drug, and effectively improve the bioavailability of the drug, which provides a theoretical basis for the study of oral preparations of quercetin.

Table 4 The pharmacokinetic parameters of quercetin after oral administration (n=3)

Note: AUC, area under the drug-time curve; T _1/2 , elimination half-life; C _max , peak plasma concentration; T _max , time to peak plasma concentration.

The substances and their concentrations used in the above embodiments are only examples, and it can be understood that in other embodiments, the corresponding substances and their concentrations are not limited to those described in the examples, such as the amphiphilic polymer micelle materials can also be selected from One of sodium deoxycholate, polyoxyethylene, pegylated chitosan, povidone, imitation cell membrane phosphorylcholine, polyamino acid or polylactic acid-glycolic acid copolymer, or selected from soluplus and these polymers A mixture of two or more materials, etc.

The various technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. a kind of polymer micelle solution containing Quercetin, it is characterised in that including with each of following weight/mass percentage composition Component：

Wherein, the amphipathic nature polyalcohol micellar material coats the Quercetin and forms core shell structure.

2. the polymer micelle solution containing Quercetin as claimed in claim 1, it is characterised in that the polymer micelle is molten Liquid includes each component with following weight/mass percentage composition：

Quercetin 0.5%~1.0%；

Amphipathic nature polyalcohol micellar material 10%；And

Stabilizer 1.5%~2.5%；

Balance of water.

3. the polymer micelle solution containing Quercetin as claimed in claim 1 or 2, it is characterised in that the amphipathic Compound micellar material be selected from polyethylene glycol-caprolactam-vinyl acetate co-polymer, NaTDC, polyoxyethylene, In Pegylation shitosan, PVP, imitative cell membrane phosphocholine, polyaminoacid and Poly(D,L-lactide-co-glycolide It is at least one.

4. the polymer micelle solution containing Quercetin as claimed in claim 1 or 2, it is characterised in that the stabilizer choosing It is at least one from water-soluble vitamin E, POLOXAMER 407 and Tween 80.

5. the polymer micelle solution containing Quercetin as claimed in claim 1 or 2, it is characterised in that the Quercetin Weight/mass percentage composition is 0.7%；The amphipathic nature polyalcohol micellar material is polyethylene glycol-caprolactam-acetic acid second Enoate copolymer；The stabilizer is POLOXAMER 407, and the weight/mass percentage composition of the stabilizer is 2%.

6. the preparation method of a kind of polymer micelle solution containing Quercetin as any one of Claims 1 to 5, its It is characterised by, comprises the following steps：

Step one：The Quercetin and the amphipathic nature polyalcohol micellar material are matched according to corresponding weight/mass percentage composition molten In organic solvent, stirring makes the Quercetin and the amphipathic nature polyalcohol micellar material be completely dissolved to well mixed, obtains To the organic solution containing Quercetin and amphipathic nature polyalcohol micellar material；

Step 2：Volatilization removes the organic solvent in the solution completely, obtains comprising the Quercetin and the amphiphilic The polymer thin-film material of property polymer latex beam material；

Step 3：The polymer thin-film material is added containing the stabilizer according to corresponding weight/mass percentage composition proportioning In the aqueous solution, it is vortexed and stirs, obtains the polymer micelle solution containing Quercetin.

7. the preparation method of the polymer micelle solution containing Quercetin as claimed in claim 6, it is characterised in that described to have Machine solvent is at least one in acetone, methyl alcohol, ethyl acetate and glacial acetic acid.

8. the preparation method of the polymer micelle solution containing Quercetin as claimed in claim 7, it is characterised in that described to have Machine solvent is (0.1-0.3) mL with the volume mass ratio of the amphipathic nature polyalcohol micellar material:100mg.

9. the preparation method of the polymer micelle solution containing Quercetin as any one of claim 6~8, its feature It is that the stirring in the step one is the magnetic agitation in water-bath, and the temperature of water-bath is 40~60 DEG C；

Stirring in the step 3 is magnetic agitation, and mixing speed is 500~700rpm, and mixing time is 0.5~2h.

10. the polymer micelle solution containing Quercetin as any one of Claims 1 to 5 is preparing antineoplastic In application.