A modified polymer nanoparticle loaded with jph203, its preparation method and application

Abstract

The application belongs to the field of medicine, and particularly relates to a modified polymer nanoparticle loaded with JPH203, a preparation method and application thereof. The preparation method is as follows: JPH203, polylactic acid-glycolic acid copolymer and polylactic acid-glycolic acid copolymer-maleimide are dissolved in an organic solvent to obtain a blending liquid; the blending liquid is added into a emulsifier solution and then sequentially subjected to ultrasonic emulsification, rotary evaporation, room temperature stirring to obtain a polymer nanoparticle solution loaded with JPH203; the polymer nanoparticle solution loaded with JPH203 is subjected to centrifugation, and the supernatant is discarded to obtain a precipitate; the precipitate is purified, redissolved, mixed with thiolated hyaluronic acid and subjected to a reaction, and the modified polymer nanoparticle loaded with JPH203 is obtained. The application synthesizes a nano JPH203 delivery system based on PLGA polymer through a microemulsion method, and then performs surface targeting modification of hyaluronic acid (HA), so that the targeting delivery of small molecule JPH203 is realized, and the serious toxic side effects caused by drug systemic exposure are reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a modified polymer nanoparticle loaded with JPH203, a preparation method and application thereof. BACKGROUND

[0002] L-type amino acid transporter 1 (LAT1 / SLC7A5) is a kind of amino acid transmembrane protein. It is reported that targeting LAT1-mediated amino acid uptake is an effective strategy for inhibiting inflammation. JPH203 is one of the inhibitors with the highest affinity to LAT1 currently developed. However, JPH203 has very poor solubility in a physiological environment and lacks targeting in application, so that in clinical experimental patients, systemic side effects such as fever, hypertension, diarrhea, dizziness, dermatitis, liver dysfunction and the like are prone to occur, thereby hindering its clinical application.

[0003] Therefore, the technical scheme of the present application is proposed. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present application provides a modified polymer nanoparticle loaded with JPH203, a preparation method and application thereof. The present application develops a poly(lactic-co-glycolic acid) (PLGA) targeted nano system for delivering JPH203. In the system, hyaluronic acid has the ability to target inflammation cells overexpressing CD44, thereby achieving targeted delivery of JPH203.

[0005] The technical scheme of the present application is to provide a preparation method of a modified polymer nanoparticle loaded with JPH203, which comprises the following steps:

[0006] (1) dissolving JPH203, poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-maleimide (PLGA-MAL) in an organic solvent to obtain a blending solution;

[0007] (2) adding the blending solution to a emulsifier solution to obtain an oil-in-water emulsion;

[0008] (3) ultrasonically emulsifying the oil-in-water emulsion to fully fuse the organic phase and the aqueous phase, rotary evaporating the organic reagent, and gradually obtaining a polymer nanoparticle solution loaded with JPH203 by slowly stirring at room temperature;

[0009] (4) centrifuging the polymer nanoparticle solution loaded with JPH203, discarding the supernatant, and obtaining a precipitate;

[0010] (5) After the purification of the precipitate, the maleimide groups on the surface of the nanoparticles due to the presence of PLGA-MAL can form covalent bonds with the thiol groups of the thiolated hyaluronic acid (HA-SH) through a mixed reaction, so that the HA-SH is successfully modified on the surface, that is, the modified polymer nanoparticles loaded with JPH203 are obtained.

[0011] Preferably, in step (1), the weight ratio of the JPH203, the polylactic acid-glycolic acid copolymer and the polylactic acid-glycolic acid copolymer-maleimide is 0.6-1:45:5-6.

[0012] Preferably, in step (1), the organic solvent is one of dichloromethane, acetonitrile, methanol or chloroform.

[0013] Preferably, in step (2), the emulsifier solution is one of a polyvinyl alcohol aqueous solution, a polyvinylpyrrolidone aqueous solution or Tween-80.

[0014] Preferably, in step (3), the power of the ultrasonic emulsification is 20-30%, and the time of the ultrasonic emulsification is 4-5 min.

[0015] And / or, the time of the rotary evaporation is 15-20 min.

[0016] And / or, the time of the stirring at room temperature is 4-12 h, and the stirring speed is 350-400 r / min.

[0017] Preferably, in step (4), the speed of the centrifugation is 15000-16000 r / min, the time of the centrifugation is 15-20 min, and the temperature of the centrifugation is 4-6℃.

[0018] Preferably, in step (5), the temperature of the reaction is 20-25℃, and the time of the reaction is 2-3 h.

[0019] Based on the same technical concept, the scheme of the present application is to provide a modified polymer nanoparticle loaded with JPH203 obtained by the above preparation method.

[0020] Still another scheme of the present application is to provide an application of the modified polymer nanoparticle loaded with JPH203 in the preparation of a medicine for treating ulcerative colitis.

[0021] Preferably, the medicine is an oral medicine.

[0022] The present application has the following beneficial effects:

[0023] 1、The preparation method is simple and easy to implement, the nano JPH203 delivery system based on the PLGA polymer is synthesized through a microemulsion method, and then the surface targeting modification HA is carried out, so that the targeted delivery of the small molecule JPH203 is realized, and the serious toxic side effects caused by the whole body exposure of the drug are reduced.

[0024] Specifically, by loading JPH203 into the PLGA nano carrier, the solubility of JPH203 is increased, and by coupling HA-SH on the surface through a chemical bond, the inflammation targeting ability of JPH203 is further improved, so that JPH203 has better bioavailability in the subsequent further disease application process, and has good biological safety.

[0025] 2, After oral administration, the JPH203-loaded modified polymer nanoparticles can use active and passive targeting to efficiently transfer the drug to the colon, thereby effectively relieving the ulcerative colitis of the mice. DETAILED DESCRIPTION

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0027] Figure 1 is the PLGA NPs obtained in Example 1. JPH203 is the real object diagram of the chitosan / alginic acid hydrogel of the HA NPs.

[0028] Figure 2 is the transmission electron microscope diagram of the PLGA NPs obtained in Example 1.

[0029] Figure 3 is the magnified diagram of Figure 2 .

[0030] Figure 4 is the transmission electron microscope diagram of the PLGA JPH203 -HA NPs obtained in Example 1.

[0031] Figure 5 is the magnified diagram of the PLGA JPH203 -HA NPs obtained in Example 1.

[0032] Figure 6 is the particle size distribution diagram of different kinds of nanoparticles.

[0033] Figure 7 is the average particle size and polydispersity coefficient (PDI) statistical diagram of different kinds of nanoparticles.

[0034] Figure 8 Figure 11 is a zeta potential graph of different kinds of nanoparticles.

[0035] Figure 9 Figure 12 is PLGA JPH203 Figure 13 is a result graph of cytotoxicity characterization of HA nanoparticles.

[0036] Figure 10 Figure 14 is a graph of cell targeting endocytosis mediated by nanoparticles.

[0037] Figure 11 Figure 15 is a result graph of nanoparticles alleviating oxidative stress caused by lipopolysaccharide at cell level.

[0038] Figure 12 Figure 16 is a graph of body weight change of mouse DSS model of ulcerative colitis after oral administration.

[0039] Figure 13 Figure 17 is a statistical graph of colon length of mouse DSS model of ulcerative colitis after oral administration.

[0040] Figure 14 Figure 18 is an endoscopy graph and HE staining graph of mouse DSS model of ulcerative colitis after oral administration.

[0041] Figure 15 Figure 19 is a graph of body weight change of mouse after oral administration of PLGA JPH203 -HA.

[0042] Figure 16 Figure 20 is a graph of blood biochemical indicators of mouse after oral administration of PLGA JPH203 -HA.

[0043] Figure 17 Figure 21 is a graph of HE staining of major organs pathological sections of mouse after oral administration of PLGA JPH203 -HA. DETAILED DESCRIPTION

[0044] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0045] Embodiment 1

[0046] The present embodiment provides a preparation method of JPH203-loaded modified polymer nanoparticles, which comprises the following steps:

[0047] (1) 1 mg JPH203, 45 mg PLGA, 5 mg PLGA-MAL were dissolved in 2 mL dichloromethane to obtain a blending solution;

[0048] (2) The blending solution was added dropwise into 4 mL of 2.5 wt.% PVA aqueous solution under vortex to form an oil-in-water emulsion;

[0049] (3) The oil-in-water emulsion was ultrasonically emulsified for 4 min at a power of 20%; then water was gradually added to the system, and residual dichloromethane was removed by rotary evaporation for 15 min; finally, the system was transferred to a beaker and stirred at room temperature at a speed of 350 r / min for 4 h to make the polymer into balls, thereby obtaining a JPH203-loaded polymer nanoparticle solution;

[0050] (4) The JPH203-loaded polymer nanoparticle solution was centrifuged at 4°C at a speed of 15000 r / min for 15 min using a refrigerated centrifuge, after which the supernatant was discarded, and the precipitate was resuspended with ultrapure water, and the centrifugation step was repeated 3 times to purify the JPH203-loaded polymer nanoparticles (PLGA JPH203 NPs);

[0051] (5) The purified PLGA JPH203 NPs were resuspended with a small amount of ultrapure water, and an equal volume of cryoprotective solution was added and stored in a -80°C refrigerator for 24 h, after which the sample was freeze-dried into a powder and stored in a -20°C refrigerator;

[0052] (6) The resuspended PLGA JPH203 NPs were mixed with HA-SH and stirred at 20°C for 2 h, so that the thiol groups of HA-SH were connected to the maleimide groups of PLGA JPH203 , so that the surface of the PLGA JPH203 NPs obtained targeting property, i.e., JPH203-loaded modified polymer nanoparticles (PLGA -HA NPs) were obtained.

[0053] Example 2

[0054] The present embodiment provides a preparation method of JPH203-loaded modified polymer nanoparticles, which comprises the following steps:

[0055] (1) 0.6 mg JPH203, 45 mg PLGA, 6 mg PLGA-MAL were dissolved in 2 mL methanol to obtain a blending solution;

[0056] (2) The blending solution was added dropwise into 4 mL of 2.5 wt.% PVA aqueous solution under vortex to form an oil-in-water emulsion;

[0057] (3) The oil-in-water emulsion was ultrasonically emulsified for 5 min at a power of 30%; water was gradually added to the system and the residual methanol was removed by rotary evaporation for 20 min; finally, the system was transferred to a beaker and stirred for 12 h at room temperature and a speed of 400 r / min to form polymer spheres, thereby obtaining a polymer nanoparticle solution loaded with JPH2O3.

[0058] (4) The JPH203-loaded polymer nanoparticle solution was centrifuged at 16000 r / min for 20 min at 4 °C using a refrigerated centrifuge. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in ultrapure water. The centrifugation step was repeated three times to purify the JPH203-loaded polymer nanoparticles (PLGA). JPH203 NPs);

[0059] (5) Use a small amount of ultrapure water to rinse the purified PLGA JPH203 Resuspend the sample and add an equal volume of cryoprotectant. Store the sample in a -80°C freezer for 24 hours. Then freeze-dry the sample into powder using a freeze dryer and store it in a -20°C freezer.

[0060] (6) Reconstituted PLGA JPH203 Mix with HA-SH and stir at 25°C for 3 hours to allow the thiol groups of HA-SH to react with PLGA. JPH203 By linking it to maleimide, it gains targeting on the surface, thus obtaining modified polymer nanoparticles (PLGA) loaded with JPH2O3. JPH203 -HA NPs).

[0061] Application examples

[0062] The modified polymer nanoparticles loaded with JPH203 obtained in Example 1 were applied to mice, specifically as follows:

[0063] (1) Dissolve chitosan powder in acetic acid, add 0.1 mol / L sodium hydroxide solution to neutralize, and obtain a chitosan solution with a final concentration of 0.6% (wt / vol).

[0064] (2) Dissolve sodium alginate in 0.15 mol / L sodium chloride to obtain a sodium alginate solution with a final concentration of 1.4% (wt / vol).

[0065] (3) The chitosan solution and the sodium alginate solution were mixed in a 1:1 ratio and then the modified polymer nanoparticles loaded with JPH203 obtained in Example 1 were added to obtain chitosan / alginate hydrogel.

[0066] (4) First, administer 100 μL of the chitosan / alginate hydrogel to the mice by gavage. Then, prepare a chelation solution with a volume ratio of 2:1 using 30 mM Na2SO4 and 70 mM CaCl2. Administer 50 μL of this chelation solution to the mice.

[0067] The actual image of the chitosan / alginate hydrogel is shown below. Figure 1 As shown.

[0068] Note: Since PLGA is not tolerant of gastric acid during oral administration, this application example utilizes a chitosan / sodium alginate hydrogel that is tolerant of gastric acid for oral delivery of this carrier.

[0069] Verification Comparison

[0070] (I) Nanoparticle morphology

[0071] Figure 2 This is a transmission electron microscope image of the PLGA NPs obtained in Example 1. Figure 3 for Figure 2 Enlarged image, Figure 4 The PLGA obtained in Example 1 JPH203 -Transmission electron microscopy image of HA NPs Figure 5 The PLGA obtained in Example 1 JPH203 - Enlarged view of HANPs.

[0072] Depend on Figures 2 to 5 It can be seen that PLGA JPH203 NPs and PLGA JPH203 - HA NPs are all spherical, with regular morphology and relatively uniform particle size, indicating that the nanoparticles were successfully prepared.

[0073] (II) Nanoparticle size and zeta potential

[0074] Figure 6 The particle size distribution diagrams show the particle size distribution of different types of nanoparticles. Figure 7 Statistical graphs showing the average particle size and polydispersity index (PDI) of different types of nanoparticles. Figure 8 Zeta potential diagrams for different types of nanoparticles.

[0075] exist Figures 6 to 8 In this context, PLGA NPs represent polylactic acid-glycolic acid copolymer nanoparticles. JPH203 This refers to polymer nanoparticles loaded with JPH2O3, PLGA JPH203 -HA indicates polymer nanoparticles loaded with JPH203 grafted with hyaluronic acid.

[0076] Depend on Figure 6 , Figure 7 It can be seen that PLGA JPH203The average size is approximately 279.33 ± 4.07 nm, after HA surface modification (PLGA) JPH203 -HA), with the average particle size increasing to 288±2.4nm.

[0077] Depend on Figure 8 It can be seen that PLGA JPH203 The zeta potential is -22.23 ± 0.23 mV, while PLGA JPH203 The measured value of -HA was -18.27 ± 2.13 mV.

[0078] That is, combination Figures 6 to 8 It can be seen that HA in PLGA has been proven. JPH203 Grafting was successfully performed on the NPs surface.

[0079] (III) Cytotoxicity of Nanoparticles

[0080] Figure 9 For PLGA JPH203 The cytotoxicity of HA nanoparticles, as a result of... Figure 9 It is known that even at higher doses (400 μg / mL), PLGA... JPH203 -HA nanoparticles did not exhibit significant cytotoxicity in either cell line. The results indicate that these nanoparticles showed excellent biocompatibility at the cellular level in both FHC and RAW264.7 cell lines, demonstrating no significant cytotoxicity.

[0081] (iv) Nanoparticle-mediated targeted endocytosis

[0082] Figure 10 For PLGA JPH203 -The results of targeted enhancement of endocytosis by HA in RAW264.7 cells were investigated by pretreating RAW264.7 cells with lipopolysaccharide to simulate an inflammatory state, and then loading fluorescent dyes into PLGA nanoparticles to study their endocytic effects. Figure 10 It can be seen that compared to PLGA JPH203 In terms of group, PLGA JPH203 - The HA group exhibited higher levels of nanoparticle endocytosis within cells. The results indicate that HA modification of the nanoparticle surface, due to HA's targeting effect on inflammation, further mediates efficient nanoparticle endocytosis.

[0083] (V) Nanoparticles alleviate oxidative stress caused by inflammation at the cellular level.

[0084] Figure 11 For PLGA JPH203 -HA alleviates cellular oxidative stress induced by lipopolysaccharide. The DCFH probe was used to verify intracellular reactive oxygen species levels. Figure 11It can be seen that the level of intracellular reactive oxygen species is significantly increased after LPS treatment compared with the control group. However, the level of intracellular reactive oxygen species is significantly decreased after nanoparticle treatment. The results show that the nanoparticle treatment can significantly alleviate the oxidative stress caused by inflammation at the cellular level.

[0085] (VI) Oral nanoparticle treatment of DSS-induced colitis in mice

[0086] By giving mice 2% DSS water for seven days to construct a mouse colitis model, Figures 12 to 14 Oral nanoparticle treatment for alleviating DSS-induced colitis in mice. By Figure 12 、 Figure 13 It can be seen that the body weight of mice is significantly reduced and the colon length is significantly shortened after the construction of mouse colitis, and these related symptoms of mice are significantly alleviated after oral nanoparticle treatment. Figure 14 The intestinal endoscopic images and colon HE pathological sections of each group of mice are shown by Figure 14 It can be seen that the intestinal mucosa of mice is damaged and the goblet cells are missing after DSS treatment, while the intestinal mucosa of the nanoparticle treatment group is almost complete and the HE pathological section is similar to the normal group. The above results all show that the nanoparticle treatment can effectively alleviate the symptoms of colitis in mice.

[0087] (VII) Biological safety after oral nanoparticle treatment

[0088] Figures 15 to 17 The biological safety data of mice after oral administration for seven days, including body weight, blood indicators, and pathological tissue sections of major organs. It can be seen that the body weight, blood biochemical indicators, and pathological sections of the heart, liver, spleen, lung, and kidney of mice after oral administration for seven days have no significant changes compared with the control group. The above results all show that the nanoparticle has good biological safety at the oral level in mice.

[0089] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any skilled person in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing JPH203-loaded modified polymer nanoparticles, characterized by, The preparation method comprises the following steps: (1) dissolving JPH203, polylactic acid-glycolic acid copolymer and polylactic acid-glycolic acid copolymer-maleimide in an organic solvent to obtain a blending solution; wherein: The weight ratio of the JPH203, the polylactic acid-glycolic acid copolymer and the polylactic acid-glycolic acid copolymer-maleimide is 0.6-1:45:5-6; (2) adding the blending solution into an emulsifier solution to obtain an oil-in-water emulsion; (3) sequentially ultrasonic emulsifying, rotary evaporating and stirring at room temperature the oil-in-water emulsion to obtain a JPH203-loaded polymer nanoparticle solution; (4) centrifuging the JPH203-loaded polymer nanoparticle solution, discarding the supernatant and obtaining a precipitate; (5) purifying and redissolving the precipitate, mixing and reacting with thiolated hyaluronic acid to obtain modified JPH203-loaded polymer nanoparticles.

2. The method of claim 1, wherein the modified polymer nanoparticles loaded with JPH203 are prepared by the method comprising the steps of: In step (1), the organic solvent is one of dichloromethane, acetonitrile, methanol or chloroform.

3. The method of claim 1, wherein the modified polymer nanoparticles loaded with JPH203 are prepared by the method comprising the steps of: In step (2), the emulsifier solution is one of polyvinyl alcohol aqueous solution, polyvinylpyrrolidone aqueous solution or Tween-80.

4. The method of claim 1, wherein the modified polymer nanoparticles loaded with JPH203 are prepared by the method comprising the steps of: In step (3), the power of ultrasonic emulsification is 20-30%, and the time of ultrasonic emulsification is 4-5 min; And / or, the time of rotary evaporation is 15-20 min; And / or, the time of stirring at room temperature is 4-12 h, and the stirring speed is 350-400 r / min.

5. The method for preparing modified polymer nanoparticles supported on JPH2O3 according to claim 1, characterized in that, In step (4), the speed of centrifugation is 15000-16000 r / min, the time of centrifugation is 15-20 min, and the temperature of centrifugation is 4-6℃.

6. The method of claim 1, wherein the modified polymer nanoparticles loaded with JPH203 are prepared by the method comprising the steps of: In step (5), the temperature of reaction is 20-25℃, and the time of reaction is 2-3 h.

7. Modified JPH203-loaded polymer nanoparticles obtained by the preparation method of any one of claims 1-6.

8. Application of the modified JPH203-loaded polymer nanoparticles obtained by the preparation method of any one of claims 1-6 in the preparation of a medicine for treating ulcerative colitis.

9. Use according to claim 8, characterized in that, The medicine is an oral medicine.

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