CN109908358B

CN109908358B - Ursolic acid polymer drug-loaded nanoparticle and preparation method and application thereof

Info

Publication number: CN109908358B
Application number: CN201910245556.XA
Authority: CN
Inventors: 吴钧; 康洋; 区坤勇
Original assignee: National Sun Yat Sen University
Current assignee: National Sun Yat Sen University
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2021-02-26
Anticipated expiration: 2039-03-28
Also published as: CN109908358A

Abstract

The invention discloses an ursolic acid polymer drug-loaded nanoparticle and a preparation method and application thereof. The invention prepares the poly ursolic acid by esterification reaction of hydroxyl at the 3-position and carboxyl at the 17-position of a plurality of ursolic acid molecules; and the nanometer carrier with the target delivery of the antitumor drug is successfully constructed by using the micelle type nanometer particle with the polyruvant as a hydrophobic inner core and the hydrophilic chain segment PEG of the DSPE-PEG2000 as a shell through a nanometer precipitation method by the polyruvant and a proper amount of DSPE-PEG 2000. The invention uses the ursolic acid as a new drug carrier for loading hydrophobic anti-tumor drugs, the obtained nanoparticles have stable structure and small particle size, the drug loading rate can reach 10 percent, the selectivity of the anti-tumor drugs is improved, the effective delivery is realized, and the acute toxic and side effects are reduced. The invention has the advantages of simple preparation method, environmental protection, economy and the like, and has great application value in the field of biological medicine.

Description

Ursolic acid polymer drug-loaded nanoparticle and preparation method and application thereof

Technical Field

The present invention belongs to the field of biomedical polymer material and medicine technology. More particularly, relates to ursolic acid polymer drug-loaded nanoparticles and a preparation method and application thereof.

Background

The focus of the current nano-drugs is mainly the research on the theoretical transmission route and the targeting function of pharmaceutical preparations, therapeutic preparations and diagnostic preparations. In particular, it is aimed at tumor targeting, including accurate identification of tumor sites and reduction of side effects. After intravenous administration, the antitumor nano-drug will have complex interaction (nano-bio interaction) with biological systems (such as protein, cells, body fluid, tissues and organs, etc. therein), which will greatly affect the antitumor effect of the nano-drug. After the ideal anti-tumor nano material enters an organism, the interaction between the ideal anti-tumor nano material and a biological system can be effectively and self-regulated along with the difference of the reached tissue parts; in the blood circulation process, the nano-drug should avoid or reduce the interaction with phagocytes such as macrophages and the like as much as possible; when the nano-drug reaches the tumor tissue, the nano-drug can enhance the interaction with the tumor cells; in tumor cells, the nanomedicines should be able to rapidly release active drug molecules to enhance the interaction with the drug target. Although this design principle is consistently recognized by researchers, the challenges of how to implement such a principle are still great.

The traditional drug delivery mode has certain defects: firstly, the accumulation concentration of the drug in tumor tissues is insufficient due to the over-short circulation time of the drug, and the preset effect is not achieved or the dosage is increased; secondly, common antitumor drugs have certain cytotoxicity and generate toxic and side effects on normal tissue cells in vivo, and the defects greatly limit the clinical application of the antitumor drugs. Therefore, it is a problem to be solved how to prolong the circulation time of the drug in vivo, increase the concentration of the drug in the tumor tissue, release the drug in the target of the tumor tissue, and reduce the toxic and side effects to the normal tissue. And the selection of an appropriate carrier for drug delivery is an effective strategy. The materials often used as the drug carrier include natural polymer materials such as lipids, saccharides, proteins and the like, while the non-toxic carrier materials approved by the FDA in the United states for injection at present include polylactic acid (PLA), polylactic-polyglycolic acid copolymer (PLGA) and the like which belong to synthetic polyester polymer materials, and the carrier materials have good biocompatibility and degradability, and the prepared nanoparticles can wrap small-molecule drugs so as to prolong the action time of the small-molecule drugs and prolong the retention time in vivo, reduce the drug toxicity, have targeting property and the like, and are common carrier materials in a drug controlled release system.

The Chinese patent with the application number of CN201210250588.7 discloses a preparation method and application of a tree-type polyester-polyglycidyl segmented copolymer, and the segmented copolymer used for drug loading is obtained by regulating and controlling a molecular configuration structure. However, the preparation process is complex, and various reagents with high toxicity, such as sodium azide and the like, are used in the preparation, so that the purification is not facilitated to obtain the material with good biocompatibility for the human body. The Chinese patent with the application number of CN201410851243.6 discloses a drug sustained and controlled release type polyester drug-loaded nanoparticle and a preparation method thereof, unsaturated polyester is prepared from aliphatic diol and aliphatic diacid or anhydride, drug encapsulation is realized through radiation crosslinking, the prepared nanoparticle has the particle size of 50-1000 nm, the drug can be released at regular time and in a quantitative manner, and is embedded in the polyester nanoparticle so that the stability of the drug is high, but the drug-loaded nanoparticle has wide particle size distribution, is not beneficial to guiding target cells, and influences the quantitative release of the drug and the targeted therapy of the drug. The invention discloses a Chinese patent with application number CN200810051452.7, which discloses a biodegradable polyester drug-carrying microsphere of a peroxidase simulant and a preparation method thereof, and introduces the preparation of the drug-carrying microsphere with a coating material of polylactic acid or polylactic acid-glycolic acid, the used peroxidase simulant is a polyethylene glycol conjugate of heme hexapeptide or heme hexapeptide, the microsphere has the size of 10-35 mu m and uniform diameter distribution, but the transportation of the larger particle size (more than 200nm) of the microsphere in blood has certain difficulty and is not beneficial to keeping in vivo for a long time.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art, and provide an ursolic acid polymer drug-loaded nanoparticle which is simple in preparation method, small in particle size, uniform and stable, capable of realizing positioning and controllable release of drugs in tumor cells, has the characteristics of efficient tumor targeting and efficient tumor cell growth inhibition, and can effectively solve the problems of wide particle size distribution, poor drug targeting property, poor biological safety and the like of the drug-loaded nanoparticle.

It is a first object of the present invention to provide a drug carrier material.

The second purpose of the invention is to provide a preparation method of ursolic acid polymer micelle type targeting nanoparticles containing the medicine carrier material.

The third purpose of the invention is to provide the application of the ursolic acid polymer micelle type targeting nanoparticles in the preparation of the nano-drug carrier with the tumor targeting function.

The fourth purpose of the invention is to provide a drug-loaded nanoparticle of ursolic acid polymer micelle type targeting nanoparticles containing the drug carrier material.

The above purpose of the invention is realized by the following technical scheme:

a drug carrier material is poly ursolic acid PUA obtained by esterification of a plurality of ursolic acid molecules, and the structural formula of the poly ursolic acid PUA is shown as the following formula (I):

wherein n is 3-10.

The Poly Ursolic Acid (PUA) is prepared by esterification reaction of hydroxyl at the 3-position and carboxyl at the 17-position of a plurality of ursolic acid molecules, the preparation method is simple, the synthesized poly ursolic acid has good biocompatibility and degradability, and can wrap and load different kinds of anti-tumor drugs, so that the anti-tumor drugs can be released at regular time and quantity, meanwhile, the drug-loaded nanoparticles prepared from the poly ursolic acid have small particle size and uniform and stable particle size distribution, and are beneficial to guiding target cells, so that the quantitative release of the drugs and the targeted therapy of the drugs are realized.

In the invention, the preparation method of the drug carrier material comprises the following steps: anhydrous pyridine is used as an acid-binding agent, after stirring in an ice bath, thionyl chloride and ursolic acid are added, the ursolic acid changes carboxyl on C-17 into acyl chloride through the thionyl chloride, and then the acyl chloride and hydroxyl on C-3 of the ursolic acid are subjected to esterification reaction, and the ursolic acid is used as a polymerization monomer to generate ester.

In anhydrous pyridine, the ursolic acid changes carboxyl on C-17 into acyl chloride through thionyl chloride, then the acyl chloride reacts with hydroxyl on C-3 to generate ester, the pyridine serves as an acid-binding agent and can generate pyridinium salt with the acyl chloride, the pyridine hydrochloride generated by hydrochloric acid is absorbed to promote the forward reaction, and finally the product is obtained by precipitating in water to remove organic solvent and freeze drying.

Further, in a preferred embodiment of the present invention, the molar ratio of thionyl chloride to ursolic acid is 1.2 to 4.8: 1. the molar ratio of thionyl chloride to ursolic acid may be 1.2: 1. 1.5: 1. 2: 1. 2.5: 1. 3: 1. 3.5: 1. 4.2: 1 or 4.8: 1.

furthermore, in a preferred embodiment of the present invention, the molar ratio of thionyl chloride to ursolic acid is 3: 1.

further, in a preferred embodiment of the present invention, the time of the esterification reaction is 0.55 to 6 hours. For example, the esterification reaction time can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6 h.

Furthermore, in a preferred embodiment of the present invention, the time for the esterification reaction is 1 to 2 hours.

Further, in a preferred embodiment of the present invention, the volume ratio of the anhydrous pyridine to the thionyl chloride is 10: 0.1 to 0.3.

Further, in a preferred embodiment of the present invention, the volume ratio of the anhydrous pyridine to the thionyl chloride is 10: 0.2.

the invention also relates to a preparation method of the ursolic acid polymer micelle type targeting nanoparticles, which is characterized in that after the poly ursolic acid PUA is dissolved in an organic solvent, the poly ursolic acid PUA and a stabilizer DSPE-PEG2000 (distearoyl phosphatidyl ethanolamine-polyethylene glycol) are stirred and react in water, and the organic solvent is removed through ultrafiltration, thus obtaining the ursolic acid polymer micelle type targeting nanoparticles.

The poly ursolic acid molecule can be self-assembled in water to form micelle type nano particles taking the self as a core and PEG as a shell in the presence of a proper amount of stabilizer DSPE-PEG2000, so that the anti-tumor drug is stably wrapped or loaded, more accurate and controllable delivery of the anti-tumor drug is realized, and the bioavailability of the hydrophobic anti-tumor drug is improved.

Further, in a preferred embodiment of the present invention, the amount of the stabilizer DSPE-PEG2000 is 0.1 to 30% (wt%) of the amount of the ursolic acid PUA. For example, the stabilizer DSPE-PEG2000 may be used in an amount of 0.1%, 15%, 20%, 25%, 30% of the amount of the ursolic acid PUA.

Further, in a preferred embodiment of the present invention, the amount of the stabilizer DSPE-PEG2000 is 25% of the amount of the ursolic acid PUA.

Further, in a preferred embodiment of the present invention, the organic solvent is DMSO.

Further, in a preferred embodiment of the present invention, the particle size of the ursolic acid polymer micelle-type targeting nanoparticle is 100-200 nm, and the drug loading is 10% (wt%).

Correspondingly, the application of the ursolic acid polymer micelle type targeting nanoparticles prepared by the preparation method in the preparation of the nano-drug carrier with the tumor targeting function is also within the protection scope of the invention.

The invention also relates to a drug-loaded nanoparticle, which comprises the ursolic acid polymer micelle type targeted nanoparticle and a drug loaded by the micelle type targeted nanoparticle.

Preferably, the micelle-type targeting nanoparticle-loaded drug is a hydrophobic anti-tumor drug.

Preferably, the micelle-type targeting nanoparticle-loaded drug includes but is not limited to one or more of adriamycin, paclitaxel and camptothecin.

In a preferred embodiment of the present invention, the preparation method of the drug-loaded nanoparticle comprises the following steps:

s1, dissolving the poly ursolic acid PUA, the anti-tumor drug and the DSPE-PEG2000 in dimethyl sulfoxide (DMSO) to obtain a mixture solution, and fully swirling;

s2, dropwise adding the mixed solution in the step S1 into the stirring ultrapure water, continuously stirring for a moment, carrying out centrifugal ultrafiltration for 2-3 times to remove the organic solvent and the unencapsulated free drug, and re-dispersing the concentrated nanoparticle solution into the ultrapure water to obtain the nanoparticle liquid;

further, in a preferred embodiment of the present invention, in step S1, the mass ratio of the anti-tumor drug to the ursolic acid PUA is 1-3: 15; preferably, the ratio of 2: 15.

further, in the preferred embodiment of the present invention, in step S1, the amount of DMSO is 1% to 3% (v/v) of the amount of ultrapure water; preferably 2% (v/v).

Further, in the preferred embodiment of the present invention, in step S1, the mass ratio of DSPE-PEG2000 to the PUA is 21% to 29%; preferably 25%.

Further, in a preferred embodiment of the present invention, in step S2, the dropping speed of the mixed solution is 0.2 to 0.4 mL/min; preferably 0.3 mL/min.

Further, in the preferred embodiment of the present invention, in step S2, the first stirring speed of the ultrapure water is 800 to 1200 rpm; preferably 1000 rpm.

The invention takes the ursolic acid as a new drug carrier, a hydrophobic anti-tumor drug is loaded to form a hydrophobic inner core, and the ursolic acid and a shell formed by a hydrophilic chain segment PEG of DSPE-PEG2000 form nanoparticles, the obtained micelle type nanoparticles have stable structure, small particle size and good tissue penetration, can be accumulated in tumor tissues through high permeability and high retention (EPR effect) of the tumor tissues to realize the passive targeting effect, and can be disintegrated by utilizing the subacidity (pH 6.5-6.8) environment of the tumor tissues to release the drugs to act on tumor cells (figure 1), thereby finally realizing the controlled release of the drugs and achieving the treatment purpose.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention synthesizes high molecular polymer Poly Ursolic Acid (PUA) by using ursolic acid as a raw material, self-assembles the puA into nanoparticles, has a nanoparticle shell-core structure form, is nontoxic and good in biocompatibility, can be used as an advantageous carrier of a drug loading system, and can enhance the targeting property of the drug at a tumor part by utilizing an EPR effect.

(2) The particle sizes of the ursolic acid polymer micelle type targeting nanoparticles and the drug-loaded nanoparticles are concentrated in the range of 100-200 nm, the particle sizes are small, the particle size distribution is uniform and stable, the targeting of target cells is facilitated, the problem that a tumor drug carrier is too small in size and can enter vascular gaps of normal tissues in vivo and cannot enter the vascular gaps of tumor tissues if too large is solved, a stronger targeting treatment effect can be exerted under the condition that no or few toxic and side effects are generated, and more accurate and controllable delivery of the anti-tumor drug is realized.

(3) The drug-loaded nanoparticles can be accumulated in tumor tissues through high permeability and high retention (EPR effect) of the tumor tissues, so that the passive targeting effect is realized; after the nanoparticles are taken up by tumor cells, the nanoparticles can be disintegrated by utilizing the slightly acidic (pH 6.5-6.8) environment of tumor tissues, so that the drug is released to act on the tumor cells, and finally the controllable release of the drug is realized, thereby efficiently inhibiting the proliferation of the tumor cells and achieving the purpose of treatment.

(4) The method has the advantages of simple reaction process, few reaction steps, short reaction period, good repeatability and the like, and has good application prospect and wide development space in the field of medicine.

Drawings

Fig. 1 is a schematic diagram of ursolic acid polymer micelle type targeting nanoparticles and their release in tumor cells according to the present invention.

FIG. 2 is a nuclear magnetic resonance spectrum of Poly Ursolic Acid (PUA) prepared in example 1 of the present invention, wherein a is of PUA¹H-NMR (400HZ, DMSO-d6) spectrum, b is of PUA¹³C-NMR (500HZ, DMSO-d6) spectrum.

FIG. 3 is an infrared spectrum of PUA prepared in example 1 of the present invention and of UA as a starting material.

FIG. 4 is a Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) result diagram of the ursolic acid polymer micelle type targeting nanoparticle of the present invention;

wherein, a and b are Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) result graphs of the ursolic acid polymer micelle targeting nanoparticles (PUA-NPs); c and d are graphs of Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) results for drug-loaded nanoparticles (PUA-NPs @ PTX).

FIG. 5 is a graph showing the effect of drug-loaded nanoparticles (PUA-NPs @ PTX), ursolic acid polymer micelle-type targeting nanoparticles (PUA-NPs) and free PTX on mouse colon cancer cells CT 26.

FIG. 6 is a graph showing the distribution of the PUA-NPs @ PTX cell membrane-loaded red fluorescent probe Dil in CT26 cells; wherein a-c is a fluorescence micrograph cultured for 1h by using PUA-NPs @ PTX loaded with Dil, a is a picture of carrying out fluorescence labeling on cell nucleus by using a fluorescent dye, b is a fluorescence signal picture of Dil loaded with nanoparticles when the time is 1h, c is a picture of carrying out fluorescence labeling on cytoskeleton by using the fluorescent dye, and d is a coincidence picture of the pictures a, b and c; e-h is a fluorescence micrograph of 4h cultured by PUA-NPs @ PTX loaded with Dil, e is a graph of fluorescence labeling of cell nucleus by a fluorescent dye, f is a graph of fluorescence signal of Dil loaded with nanoparticles at 4h, g is a graph of fluorescence labeling of cytoskeleton by a fluorescent dye, and h is a coincidence graph of the e graph, the f graph and the g graph.

FIG. 7 is a graph of the release profiles of drug-loaded nanoparticles (PUA-NPs @ PTX) of the present invention at different pH conditions.

FIG. 8 is a product of the poly-ursolic acid of comparative example 1 of the present invention which has not been successfully prepared¹H-NMR (400HZ, DMSO-d6) spectrum.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The terms used herein:

as used herein, the term "PTX" refers to paclitaxel.

As used herein, the term "UA" refers to ursolic acid.

As used herein, the term "PUA" refers to polyaspartic acid.

As used herein, the term "PUA-NPs" refers to ursolic acid polymer micelle-type targeting nanoparticles (polymeric ursolic acid nanoparticles).

As used herein, the term "PUA-NPs @ PTX" refers to paclitaxel-loaded polyaspartic acid nanoparticles.

The detection methods of the particle size, TEM, DLS, drug loading rate, release degree and the like all adopt the conventional detection method in the prior art.

Example 1 Synthesis of PUA

1. The following examples, using pyridine and thionyl chloride as catalysts, prepare the poly-ursolic acid (PUA) through the esterification condensation of ursolic acid, comprising the following steps:

(1) under a good ventilation environment, adding 10mL of anhydrous pyridine into a clean and dry 25mL round-bottom flask, and stirring in an ice bath for 5 min;

(2) add 200. mu.L (2.76mmol) of thionyl chloride so that the volume ratio of anhydrous pyridine to thionyl chloride is 10: 0.2, stirring in an ice bath for 15 min;

(3) 0.42g (0.92mmol) of UA was added so that the molar ratio of thionyl chloride to ursolic acid was 3: 1, the reaction solution is stirred and reacted for 1h at normal temperature, the reaction solution is transferred to a 150mL beaker after the reaction is finished, the reaction solution is washed by 250mL of water and filtered, the filtering is repeated for 3 times to obtain a white filter cake, and the product PUA is obtained after freeze drying, wherein the amount of the product PUA is 0.35g (yield: 83.3%).

The reaction formula of PUA is as follows (n ═ 10):

2. results

Method for carrying out PUA on NMR apparatus¹H-NMR and¹³obtaining a nuclear magnetic resonance spectrogram by C-NMR measurement¹H-NMR and¹³the nuclear magnetic resonance spectrum obtained by the C-NMR measurement is shown in FIG. 2. After infrared spectrum analysis, the obtained comparison spectrogram of the infrared spectrum of the ursolic acid standard substance and the ursolic acid polymer is shown in figure 3. The above results indicate that ursolic acid is successfully polymerized to obtain ursolic acid.

Example 2 Synthesis of PUA

The other conditions were the same as in example 1, with the only difference that: controlling the volume ratio of anhydrous pyridine to thionyl chloride to be 10: 0.1, the molar ratio of the thionyl chloride to the ursolic acid is 1.2: 1, stirring the reaction solution at normal temperature for 0.55h to successfully prepare the ursolic acid.

Example 3 Synthesis of PUA

The other conditions were the same as in example 1, with the only difference that: controlling the volume ratio of anhydrous pyridine to thionyl chloride to be 10: 0.3, the molar ratio of the thionyl chloride to the ursolic acid is 4.8: 1, stirring the reaction solution at normal temperature for 6 hours to successfully prepare the ursolic acid.

Example 4 Synthesis of Ursolic acid Polymer micelle-type targeting nanoparticles (PUA-NPs)

The preparation method of ursolic acid polymer micelle type targeting nanoparticles (PUA-NPs) by a nano precipitation method comprises the following steps:

dissolving PUA and a stabilizer DSPE-PEG2000 in 1mL of DMSO, and vortexing to obtain a mixed solution containing 15mg/mL of PUA, wherein the dosage of the stabilizer DSPE-PEG2000 is 25% (wt%) of that of the PUA; slowly dripping (0.3mL/min) 200 mu L of mixed solution into 10mL of ultrapure water at the stirring speed of 1000rpm, stirring for 30s after dripping, carrying out centrifugal ultrafiltration for 10min at 2500rpm to remove DMSO, and repeating for 3 times to obtain the poly-ursolic acid nanoparticles (PUA-NPs), namely the ursolic acid polymer micelle type targeting nanoparticles.

Example 5 Synthesis of Ursolic acid Polymer micelle-type targeting nanoparticles (PUA-NPs)

The other conditions were the same as in example 4, with the only difference that: the dosage of the stabilizer DSPE-PEG2000 is controlled to be 0.1 percent (wt percent) of the dosage of the poly ursolic acid PUA, and the ursolic acid polymer micelle type targeting nanoparticles are successfully prepared.

Example 6 preparation of paclitaxel-loaded Polyursolic acid nanoparticles PUA-NPs @ PTX

The preparation method of the paclitaxel-loaded ursolic acid nanoparticles by a nano precipitation method comprises the following steps:

s1, dissolving PUA, PTX and a stabilizer DSPE-PEG2000 in 1mL of DMSO, and vortexing, wherein the mass ratio of PUA to PTX in the obtained mixed solution is 2: 15, wherein the final concentration of PUA is 15mg/mL, the final concentration of PTX is 2mg/mL, and the mass ratio of DSPE-PEG2000 to PUA is 25% (wt%);

s2, slowly dropping (0.3mL/min) 200 mu L of mixed liquor into 10mL of ultrapure water at a stirring speed of 1000rpm, stirring for 30s after dropping, performing centrifugal ultrafiltration for 10min by using the ultrapure water at 2500rpm to remove DMSO and free PTX, and repeating for 3 times to obtain the paclitaxel-loaded polyabutic acid nanoparticles (PUA-NPs @ PTX), wherein the drug loading is 10%.

Example 7 preparation of paclitaxel-loaded Polyursolic acid nanoparticles PUA-NPs @ PTX

s1, dissolving PUA, PTX and a stabilizer DSPE-PEG2000 in 1mL of DMSO, and vortexing, wherein the mass ratio of PUA to PTX in the obtained mixed solution is 1: 15, wherein the final concentration of PUA is 15mg/mL, the final concentration of PTX is 1mg/mL, and the mass ratio of DSPE-PEG2000 to PUA is 21% (wt%);

s2, slowly dropping (0.2mL/min) 200 mu L of mixed liquor into 10mL of ultrapure water at the stirring speed of 800rpm, stirring for 30s after dropping, carrying out centrifugal ultrafiltration for 10min by using the ultrapure water at 2500rpm to remove DMSO and free PTX, and repeating for 3 times to obtain the paclitaxel-loaded polyabutic acid nanoparticles (PUA-NPs @ PTX).

Example 8 preparation of paclitaxel-loaded Polyursolic acid nanoparticles PUA-NPs @ PTX

s1, dissolving PUA, PTX and a stabilizer DSPE-PEG2000 in 1mL of DMSO, and vortexing, wherein the mass ratio of PUA to PTX in the obtained mixed solution is 3: 15, wherein the final concentration of PUA is 15mg/mL, the final concentration of PTX is 3mg/mL, and the mass ratio of DSPE-PEG2000 to PUA is 29% (wt%);

s2, slowly dropping (0.4mL/min) 200 mu L of mixed liquor into 10mL of ultrapure water at the stirring speed of 1200rpm, stirring for 30s after dropping, carrying out centrifugal ultrafiltration for 10min by using the ultrapure water at 2500rpm to remove DMSO and free PTX, and repeating for 3 times to obtain the paclitaxel-loaded polyabutic acid nanoparticles (PUA-NPs @ PTX).

Example 9 Property testing

The following property tests were carried out on the PUA-NPs and PUA-NPs @ PTX prepared in the above examples of the present invention:

1. particle size and characterization

(1) At 25 ℃ PUA-NPs formed in water with an average size of 115nm, and TEM and DLS results are shown in FIG. 4.

(2) At 25 ℃ PUA-NPs @ PTX formed in water with an average size of 173nm, and TEM and DLS results are shown in FIG. 4.

The results show that the particle sizes of the PUA-NPs and the PUA-NPs @ PTX are concentrated in the range of 100-200 nm, the particle sizes are small, the particle size distribution is uniform and stable, the targeting of target cells is facilitated, the problem that the tumor drug carrier is too small in size, can enter the vascular clearance of normal tissues in vivo and cannot enter the vascular clearance of tumor tissues if too large is avoided, the strong targeting treatment effect can be exerted under the condition that no or few toxic and side effects are generated, and more accurate and controllable antitumor drug delivery is realized.

2. In vitro anti-CT 26 tumor cell activity test

The in vitro anti-CT 26 tumor cell activity experiments were performed on PUA-NP, PUA-NPs @ PTX and free PTX prepared in the above examples, and the effects are shown in FIG. 5.

PUA-NP showed no significant inhibition of the growth of CT26 cells, indicating that PUA has good biocompatibility. Meanwhile, the medicine nano PUA-NPs @ PTX under the action of EPR not only maintains the anticancer activity of PTX, but also has better inhibition effect compared with free PTX.

3. Intracellular distribution profile

The PUA-NP prepared in the above example was used to load Dil of a cell membrane red fluorescent probe, and the intracellular distribution of drug-loaded nanoparticles was observed under a laser scanning confocal microscope, as shown in FIG. 6. CT26 cytoskeleton was stained with FITC-phalloidin for green fluorescence and nuclei were stained with DAPI for blue fluorescence.

The result shows that only a small amount of Dil-loaded nanoparticles enter cells at 1h, and a large amount of Dil-loaded nanoparticles enter cells after 4h, which indicates that PUA-NPs have better drug delivery performance, and the uptake of paclitaxel by CT26 cells has certain time dependence.

4. Different pH Release profiles

Drug release behavior experiments were performed on the drug-loaded nanoparticles PUA-NPs @ PTX prepared in the above examples, and the results are shown in FIG. 7.

At different pH conditions: the drug-loaded nanoparticles are released under pH 5.5, pH 6.5 and pH 7.4, and show long-term slow release effect in all release media; wherein the cumulative release percentage of PTX after 72h in PBS (pH 7.4) medium is 20%; but the cumulative percent drug release increased significantly in PBS (pH 5.5) medium, approximately 75%. The result shows that the drug release of the drug-loaded nanoparticles PUA-NPs @ PTX in the simulated slightly acidic environment is more obvious, the long-term slow release of the drug-loaded nanoparticles PUA-NPs @ PTX also provides guarantee for achieving a long-acting anti-tumor effect, and the drug-loaded nanoparticles PUA-NPs @ PTX have better tumor targeting property.

EXAMPLE 10 Effect of the amount of stabilizer used on the particle size and drug load of PUA-NPs @ PTX

The effect of different amounts of stabilizer on the particle size and drug loading of the PUA-NPs @ PTX was examined and the results are shown in Table 1.

TABLE 1 Effect of the amount of stabilizer used on the particle size and drug load of PUA-NPs @ PTX

As can be seen from Table 1, when the dosage of the stabilizer DSPE-PEG2000 is 15-30% of the dosage of the ursolic acid, the drug loading is 5.3-10.1%, wherein when the dosage of the stabilizer DSPE-PEG2000 is 25% of the dosage of the ursolic acid, the drug loading is the highest.

Comparative example 1

The other conditions were the same as in example 1, with the only difference that: controlling the volume ratio of anhydrous pyridine to thionyl chloride to be 10: 0.2, the molar ratio of the thionyl chloride to the ursolic acid is 6: 1, stirring the reaction solution at normal temperature for 1 hour to react, and failing to successfully prepare the ursolic acid. As shown in FIG. 8, this condition synthesized the product¹H-NMR spectrogram shows that the ursolic acid has more impurities and does not have the characteristics of the ursolic acid.

The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it is not intended that the present invention necessarily depends on the above embodiment for implementation. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A drug carrier material is characterized in that the drug carrier material is poly ursolic acid PUA obtained by esterification of a plurality of ursolic acid molecules, and the structural formula of the poly ursolic acid PUA is shown as the following formula (I):

wherein n is 3-10;

the drug carrier material is prepared by taking anhydrous pyridine as an acid-binding agent, stirring in an ice bath, adding thionyl chloride and ursolic acid, converting carboxyl on C-17 into acyl chloride by the ursolic acid through the thionyl chloride, and then carrying out esterification reaction with hydroxyl on C-3 of the acyl chloride, and taking the ursolic acid as a polymerization monomer to generate ester; the mol ratio of the thionyl chloride to the ursolic acid is 1.2-4.8: 1; the volume ratio of the anhydrous pyridine to the thionyl chloride is 10: 0.1 to 0.3.

2. The drug carrier material of claim 1, wherein the esterification reaction time is 0.55 to 6 hours.

3. A preparation method of ursolic acid polymer micelle type targeting nanoparticles is characterized in that the medicine carrier material in any one of claims 1 or 2 is dissolved in an organic solvent, then is stirred and reacts with a stabilizer DSPE-PEG2000 in water, and is ultrafiltered to remove the organic solvent, so that the ursolic acid polymer micelle type targeting nanoparticles are obtained.

4. The method according to claim 3, wherein the amount of the stabilizer DSPE-PEG2000 is 0.1 to 30% (wt%) of the amount of the PUA.

5. The method according to claim 4, wherein the amount of the stabilizer DSPE-PEG2000 is 25% of the amount of the PUA.

6. The application of the ursolic acid polymer micelle type targeting nanoparticles prepared by the preparation method of any one of claims 3 to 5 in preparing a nano drug carrier with a tumor targeting function.

7. A drug-loaded nanoparticle is characterized by comprising the ursolic acid polymer micelle type targeting nanoparticle prepared by the preparation method of any one of claims 3-5 and a drug loaded by the ursolic acid polymer micelle type targeting nanoparticle.

8. The drug-loaded nanoparticle according to claim 7, wherein the micelle-type targeting nanoparticle-loaded drug is one or more of adriamycin, paclitaxel and camptothecin.

9. The drug-loaded nanoparticle according to claim 8, wherein the preparation method of the drug-loaded nanoparticle comprises the following steps:

s1, dissolving the poly ursolic acid PUA, the anti-tumor drug and the DSPE-PEG2000 in dimethyl sulfoxide to obtain a mixture solution, and fully swirling;

s2, dropwise adding the mixed solution in the step S1 into the stirring ultrapure water, continuously stirring for a moment, carrying out centrifugal ultrafiltration for 2-3 times to remove the organic solvent and the unencapsulated free drug, and re-dispersing the concentrated nanoparticle solution into the ultrapure water to obtain the nanoparticle.