Diclofenac self-assembled nano prodrug connected with diselenide as well as preparation method and application of diclofenac self-assembled nano prodrug

Abstract

The invention discloses a diclofenac self-assembly nano prodrug based on diselenide connection and application of the diclofenac self-assembly nano prodrug in osteoarthritis treatment, and belongs to the technical field of biological medicine. According to the nano prodrug, diclofenac is coupled through a diselenide bond to construct a prodrug unit (DSED), the prodrug unit (DSED) is further cooperatively assembled with a cartilage regeneration accelerant carbogenin, cartilage targeting peptide WYRGRL is subjected to surface modification, and multifunctional nano particles W / KGN and DSED NPs with responsive release capacity are formed. The nano prodrug system integrates triple treatment functions of oxidative stress resistance, cartilage repair and analgesia, has good tissue targeting, stability and biocompatibility, can realize accurate delivery and combined treatment of osteoarthritis focuses, and is suitable for relieving joint symptoms, improving joint microenvironment, promoting cartilage regeneration and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a diselenide-bond-connected diclofenac self-assembled nano-prodrug as well as a preparation method and application thereof. The technology relates to the fields of organic chemistry, nanomaterials, biomedical engineering and the like, and aims to construct an intelligent prodrug platform for multifunctional synergistic therapy. BACKGROUND

[0002] Osteoarthritis (OA) is a common degenerative joint disease, and its pathological features mainly include joint cartilage degeneration, synovial inflammation and subchondral bone remodeling, and it is clinically manifested as chronic pain, stiffness and movement disorders. At present, the treatment of OA mainly includes non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoid injection and physical rehabilitation, but most of the methods can only relieve symptoms and are difficult to effectively delay the course of disease or promote cartilage repair.

[0003] Current OA drug therapy faces multiple challenges, including short retention time of drugs in the joint cavity, low bioavailability, non-specific distribution and large potential side effects. Therefore, it is urgent to develop a new treatment strategy with targeting, controlled release and multifunctional synergistic effect.

[0004] Diselenide has good oxidative stress responsiveness because it can break the diselenide bond (Se-Se) in the presence of reactive oxygen species (ROS), and the released selenium element (Se) can activate the Nrf2 pathway and induce antioxidant defense response, which has been proven to have a positive effect on cartilage cell protection. However, free diselenide has poor stability, easy leakage and poor targeting in in vivo delivery, which greatly limits its practical application.

[0005] Therefore, a nanodrug system integrating responsive release, targeted delivery and synergistic therapy functions is urgently needed to improve treatment efficiency and reduce side effects, so as to effectively solve the treatment problems of OA. SUMMARY

[0006] In order to solve the problems of low drug release efficiency, lack of targeting and single treatment method in the prior art for treating osteoarthritis, the present application provides a preparation method of a diselenide-connected self-assembled nano-prodrug for enhancing the treatment of osteoarthritis. The nano-prodrug is constructed by a cartilage-targeting peptide (WYRGRL), a cartilage formation agent KGN and a diclofenac prodrug (DSeD) connected by a diselenide compound.

[0007] The present application utilizes the diselenide bond to covalently connect diclofenac (DIC) molecules, synthesizes small molecule prodrug DSeD with self-assembly ability, which can spontaneously form stable nanoparticles in aqueous solution. During the assembly process, the hydrophobic KGN is co-encapsulated in the interior of the nanoparticles, at the same time, the particle surface is functionalized and modified by the DSPE-PEG coupled WYRGRL peptide, so as to obtain the composite nanoparticle prodrug W / KGN@DSeD nanoparticles with responsive release, antioxidant activity and targeted delivery ability.

[0008] The nanoparticle prodrug preparation described in the present application mainly consists of three effective components: first, KGN can promote mesenchymal stem cells to differentiate into chondrocytes and play the function of cartilage regeneration; second, DIC is a commonly used non-steroidal anti-inflammatory drug, which can relieve inflammation and analgesia; third, the WYRGRL peptide has the ability to specifically bind to collagen II in cartilage, effectively increasing the accumulation of drugs in cartilage tissue and reducing the risk of clearance in synovial fluid. Among them, the structural formula of DIC and DIC is: .

[0009] The nanoparticle prodrug preparation method provided by the present application is based on the diselenide bridging structure to construct the prodrug unit DSeD of DIC, to form a nanostructure with KGN through a self-assembly process, and then to target modify the nanoparticles by using the DSPE-PEG coupled WYRGRL peptide, so as to obtain the final W / KGN@DSeD nanoparticles.

[0010] The preparation method of the diclofenac self-assembled nanoparticle prodrug connected by diselenide compound of the present application comprises the following steps: S1, covalently connecting diclofenac by diselenide to form a small molecule prodrug (DSeD); S2, using the self-assembly performance of the small molecule prodrug (DSeD) to spontaneously form nanoparticles in aqueous solution, loading the chondrogenic small molecule KGN to form a combined nanoparticle prodrug (KGN@DSeD NPs); S3, modifying the surface of the combined nanoparticle prodrug with a protein targeting peptide to obtain a nanoparticle prodrug (W / KGN@DSeD NPs) with targeting and responsive release functions.

[0011] The preparation method of the diclofenac self-assembled nanoparticle prodrug connected by diselenide compound of the present application comprises the following steps: S1, covalently connecting diclofenac by diselenide to form a small molecule prodrug: s101, dissolve sodium hydroxide in water, add hydrazine hydrate dropwise, then gradually add selenium powder, react at 90°C for 2 hours; after cooling to 50°C, slowly add 2-bromoethanol and react overnight. After cooling to room temperature, filter through diatomite, extract with ethyl acetate, dry the organic layer and purify by silica gel column chromatography to obtain yellow solid HO-Se-Se-OH; s102, dissolve diclofenac (DIC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in dichloromethane, add N,N-diisopropylethylamine (DIPEA), then add HO-Se-Se-OH, stir at room temperature overnight; after the reaction is completed, extract, dry, concentrate, and purify by silica gel column to obtain small molecule prodrug DSeD; S2, using the self-assembly properties of the small molecule prodrug to spontaneously form nanoparticles in an aqueous phase, loading the cartilage generating small molecule KGN to form a combination type nano prodrug particle: s201, dissolve DSeD in DMSO, add dropwise to deionized water after ultrasonic treatment for 10 minutes, then dialyze in a nanodialysis bag with a molecular weight cutoff of 1000 Da for 24 hours to obtain self-assembled DSeD nanoparticles; s202, dissolve phthalic anhydride in glacial acetic acid, add 1,1'-biphenyl-4-amine solution dropwise at 60°C, continue stirring for 24 hours. After the reaction is completed, cool and filter, and recrystallize the crude product from ethanol to obtain white solid KGN; s203, dissolve the self-assembled DSeD nanoparticles and KGN in different proportions in DMSO, add dropwise to the aqueous phase, ultrasonic treatment and dialysis to obtain KGN@DSeD nano prodrug particles; S3, surface modification of the combination type nano prodrug with protein targeting peptide; Dissolve KGN@DSeD nano prodrug and protein targeting peptide in DMSO, add dropwise to water, ultrasonic treatment, then dialyze to remove organic solvents and freeze-dry to obtain the final multifunctional nano prodrug W / KGN@DSeD nanoparticles.

[0012] Further, the specific reaction equations of DSeD and KGN are as follows (A) and (B) respectively:

[0013] In the reaction equation, Se is selenium powder, EDCI is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP is 4-dimethylaminopyridine, DIPEA is N,N-diisopropylethylamine, HAc is glacial acetic acid, and EtOH is ethanol.

[0014] The complete preparation process of the W / KGN@DSeD nanoparticles is as follows.

[0015] .

[0016] Further, the protein targeting peptide is a type II collagen targeting peptide-polyethylene glycol-phospholipid.

[0017] Further, the mass ratio of the self-assembled small molecule prodrug nanoparticles and the Katojin in step S203 is 4:1 Further, the amount of the protein targeting peptide added in step S3 is 5% of the mass of the combined nanoparticle prodrug. Another object of the present application claims a diselenide compound linked diclofenac self-assembled nanoparticle prepared by the method of preparing the diselenide compound linked diclofenac self-assembled nanoparticle.

[0018] Another object of the present application is to claim the use of the diselenide compound linked diclofenac self-assembled nanoparticle in the preparation of a drug for treating osteoarthritis. The nanoparticle has the triple functions of responsive drug release, antioxidant defense and targeted delivery, and can synergistically regulate the microenvironment of the osteoarthritis lesion, achieve the comprehensive treatment effect of relieving symptoms, inhibiting disease progression and promoting cartilage tissue regeneration. The preparation can be prepared into a water-soluble injection form for clinical application.

[0019] The diselenide compound linked diclofenac self-assembled nanoparticle and the preparation method and application thereof have the following beneficial effects: (1) DSeD is synthesized through a diselenide bridging structure to realize the construction of a small molecule prodrug and self-assembly into stable nanoparticles.

[0020] (2) During the formation of the nanoparticles, KGN and DSeD are co-assembled to form KGN@DSeD nanoparticles; in the microenvironment of osteoarthritis, the increase of ROS level can specifically cleave the diselenide bond, induce the depolymerization of the nanoparticles and realize the controlled release of DIC, KGN and Se elements.

[0021] (3) The introduced DSPE-PEG-WYRGRL is coated on the surface of the nanoparticles through hydrophobic interaction, which endows the nanoparticles with the ability of targeting cartilage tissue.

[0022] (4) The W / KGN@DSeD nanoparticles have achieved remarkable results in reducing cartilage structure damage and improving symptoms in vivo due to their targeting property and fusion of multiple treatment mechanisms.

[0023] (5) The nano-carrier system provided by the application has the advantages of targeting, multi-drug synergistic delivery, responsive release and antioxidant protection, not only enhances the treatment effect and reduces toxic side reactions, but also significantly improves the accumulation and utilization efficiency of drugs in the joint cartilage part, and provides a new scheme for the combined treatment of osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0025] Figure 1 NMR hydrogen spectrum of DSeD in Example 1; Figure 2 Transmission electron micrograph of the self-assembled nano-prodrug W / KGN@DSeD nanoparticles in Example 1, scale: 2.0 μm; Figure 3 Katojin release diagram of the self-assembled nano-prodrug W / KGN@DSeD nanoparticles in Example 1 under the stimulation of the simulated microenvironment; Figure 4 Western blot diagram of the effect of the self-assembled nano-prodrug W / KGN@DSeD nanoparticles in Example 1 on the protein expression of chondrocytes; Figure 5 Therapeutic effect diagram of the self-assembled nano-prodrug W / KGN@DSeD nanoparticles in Example 1 on mouse cartilage tissue. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with specific embodiments and drawings.

[0027] Example 1

[0028] A preparation method of a self-assembled nano-prodrug of diclofenac connected by a diselenide compound, comprising the following steps: S1, covalently connecting diclofenac by diselenide to form a small molecule prodrug (DSeD) s101, Dissolve sodium hydroxide (1.72 g, 30 mmol) in 80 mL of deionized water; slowly add hydrazine hydrate (85%, 0.52 g, 28 mmol) and heat the solution at 60-70 °C. Then add selenium powder (2.21 g, 28 mmol) gradually, heat to 90 °C and continue stirring for 2 hours. After the solution is cooled to about 50 °C, slowly add 2-bromoethanol (3.5 g, 28 mmol) and let the reaction stand overnight. After the reaction is complete, cool to room temperature and remove unreacted selenium powder by filtration through diatomite. The resulting solution is extracted with ethyl acetate and the combined organic layers are dried over anhydrous sodium sulfate. Finally, concentrate under vacuum and purify by silica gel column chromatography (petroleum ether / ethyl acetate = 2:1, v / v) to obtain the yellow product HO-Se-Se-OH, 248.1 mg in yield; s102, Dissolve diclofenac (592.3 mg, 2.0 mmol), EDCI (421.7 mg, 2.2 mmol) and DMAP (24.4 mg, 0.2 mmol) in 30 mL of dichloromethane. After adding DIPEA (517.0 mg, 4.0 mmol) and stirring for 30 min, add HO-Se-Se-OH (248.1 mg, 1.0 mmol) and stir the reaction at room temperature overnight. After the reaction is complete, extract with ethyl acetate, dry the combined organic layers over anhydrous sodium sulfate, concentrate under vacuum and purify by silica gel column chromatography (PE / EA = 4:1, v / v) to obtain the yellow product small molecule prodrug (DSeD); 1H NMR (500 MHz, CDCl3) δ (ppm): 7.34 (dd, J = 8.0, 6.0 Hz, 4 H),7.24 - 7.21 (m, 2 H), 7.13 - 7.10 (m, 2 H), 6.99 - 6.93 (m, 4 H), 6.55 (dd, J= 8.0, 6.5 Hz, 4 H), 4.49 (t, J = 7.5 Hz, 1 H), 4.41 (t, J = 7.5 Hz, 3 H),3.81 (s, 4 H), 3.29 (t, J = 7.5 Hz, 1 H), 4.41 (t, J = 7.5 Hz, 3 H).

[0029] S2, Utilize the self-assembly properties of the small molecule prodrug to spontaneously form nanoparticles in an aqueous phase, load the cartilage generating small molecule KGN to form a combination nanoparticle prodrug particle KGN@DSeD S201. Dissolve 5.0 mg DSeD in 1.0 mL DMSO, sonicate for 10 min, and then slowly add it dropwise to 9.0 mL deionized water. Dialyze the resulting mixture using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 hours to remove residual solvent, and finally freeze-dry to obtain DSeD nanoparticles. S202. Phthalic anhydride (786.0 mg, 5.3 mmol) was dissolved in 50 mL of glacial acetic acid. The mixture was heated to 60°C, and a solution of 1,1'-biphenyl-4-amine (1.0 g, dissolved in 20 mL of glacial acetic acid) was slowly added dropwise over 30 min, while stirring continuously for 24 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to obtain the crude product, and then purified by recrystallization from ethanol to give a white solid, KGN. 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.81 - 7.98 (m, 2 H), 7.94 - 7.92(m, 2 H), 7.83 (d, J = 8.5 Hz, 2 H), 7.75 (d, J = 7.5 Hz, 2 H), 7.56 (d, J =8.0 Hz, 2 H), 7.51 (t, J = 7.5 Hz, 2 H), 7.41 (t, J = 7.5 Hz, 1 H); s203. Dissolve 5.0 mg DSeD and KGN in different mass ratios in 1.0 mL DMSO, add dropwise to 9.0 mL deionized water, react under vigorous sonication for 10 min, dialyze to remove DMSO and freeze dry to obtain KGN@DSeD nanoparticles.

[0030] S3 modifies the surface of a combinatorial prodrug nanoparticle with protein-targeting peptides to obtain a prodrug nanoparticle with targeting and responsive release functions, W / KGN@DSeD. KGN@DSeD and type II collagen-targeting peptide-polyethylene glycol-phospholipid (DSPE-PEG-WYRGRL) were dissolved together in 1.0 mL DMSO, added dropwise to 9.0 mL deionized water, sonicated for 10 min, dialyzed and freeze-dried to obtain diselenide-linked self-assembled nanoprodrug nanoparticles W / KGN@DSeD.

[0031] Transmission electron microscopy images of the W / KGN@DSeD nanoparticles prepared in Example 1 are attached. Figure 3 As shown, the particle size and morphology of the prepared nanoprodrug are clearly displayed.

[0032] The captogin release curves of W / KGN@DSeD nanoparticles under redox stimulation conditions are attached. Figure 4As shown, it is shown that the nano-prodrug constructed by the present application can realize the responsive release of the drug under the stimulation of ROS.

[0033] The expression results of the effect of W / KGN@DSeD nanoparticles on chondrocyte protein expression in in vitro experiments are shown in the following table: Figure 5 As shown, it is shown that the nanoparticles of the present application have excellent therapeutic effect.

[0034] The in vivo treatment experiment results of W / KGN@DSeD nanoparticles are shown in the following table: Figure 5 , showing excellent effect of reducing cartilage structure damage and improving symptoms.

[0035] The above describes the technical solutions provided by the embodiments of the present application in detail. This article applies specific examples to describe the principles and implementation manners of the embodiments of the present application. The above description of the embodiments is only applicable to help understand the principles of the embodiments of the present application; meanwhile, for those skilled in the art, the specific implementation manners and application ranges of the embodiments of the present application will be changed, and the above description of the present application should not be understood as a limitation of the present application.

Claims

1. A method for preparing a diclofenac self-assembled nanoprodrug linked to a diselenoside compound, characterized in that, Includes the following steps: S1. Diclofenac is covalently linked to diselenide to form a small molecule prodrug; S2. Utilizing the self-assembly properties of small molecule prodrugs, nanoparticles are spontaneously formed in the aqueous phase, and cartilage is loaded to generate small molecule captogine to form combined nanoprodrug nanoparticles. S3. Modify the surface of the combined nanoprodrug with protein-targeting peptides to obtain nanoprodrugs with targeting and responsive release functions.

2. The method for preparing diclofenac self-assembled nanoprodrugs linked to diselenylene compounds according to claim 1, characterized in that, The diselenide mentioned in step S1 is diselenol.

3. The method for preparing diclofenac self-assembled nanoprodrug linked to the diselenoside compound according to claim 2, characterized in that, Step S1 includes the following steps: S101. Sodium hydroxide was dissolved in water at 60–70℃, hydrazine hydrate was added dropwise, followed by selenium powder. The mixture was heated to 90℃ and stirred for 2 hours. After cooling to 50℃, 2-bromoethanol was slowly added dropwise and allowed to stand overnight. After the reaction was completed, the mixture was cooled to room temperature, filtered through diatomaceous earth, extracted with ethyl acetate, dried, concentrated, and purified by silica gel column chromatography to obtain diselenool compound HO-Se-Se-OH. s102. Diclofenac, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine were dissolved in dichloromethane. N,N-diisopropylethylamine was added dropwise and the mixture was stirred for 30 minutes. HO-Se-Se-OH obtained in step (1) was added and the mixture was reacted overnight at room temperature. The mixture was extracted with ethyl acetate, dried, concentrated, and purified by silica gel column chromatography to obtain a small molecule prodrug linked to a diselenide compound.

4. The method for preparing diclofenac self-assembled nanoprodrug linked to the diselenoside compound according to claim 3, characterized in that, Step S2 includes the following steps: S201. Dissolve the small molecule prodrug obtained in step S102 in DMSO, sonicate for 10 minutes, add dropwise to deionized water, and then dialyze through a nanodialysis bag for 24 hours to obtain self-assembled small molecule prodrug nanoparticles. s202. Phthalic anhydride was dissolved in glacial acetic acid and heated to 60°C. A 1,1'-biphenyl-4-amine solution was added dropwise and stirred at a constant temperature for 24 hours. After cooling, the crude product was obtained by filtration and recrystallization from ethanol to obtain captogin. s203. The self-assembled small molecule prodrug nanoparticles obtained in step s201 are mixed with the captogjinin obtained in step s202 and dissolved in DMSO. The mixture is then added dropwise to deionized water. After ultrasonic treatment and dialysis to remove the organic solvent, combined nanoprodrug nanoparticles are obtained.

5. The method for preparing diclofenac self-assembled nanoprodrug linked to the diselenoside compound according to claim 4, characterized in that, The protein-targeting peptide modification in step S3 is as follows: the combined nano-prodrug particles obtained in step S203 are mixed with the protein-targeting peptide and dissolved in DMSO, added to deionized water, sonicated, then dialyzed to remove the organic solvent and freeze-dried to obtain the final multifunctional nano-prodrug.

6. The method for preparing diclofenac self-assembled nanoprodrugs linked to diselenylene compounds according to claim 1, characterized in that, The protein-targeting peptide is a type II collagen-targeting peptide-polyethylene glycol-phospholipid.

7. The method for preparing diclofenac self-assembled nanoprodrugs linked to diselenylene compounds according to claim 4, characterized in that, In step S203, the mass ratio of self-assembled small molecule prodrug nanoparticles to captogine is 4:

1.

8. A diclofenac self-assembled nanoprodrug linked to a diselenide compound obtained by the preparation method according to any one of claims 1-7.

9. The use of the diselenide compound-linked diclofenac self-assembled nanoprodrug of claim 8 in the preparation of a drug for treating osteoarthritis.

10. The application according to claim 9, characterized in that, The drug is formulated as an aqueous injection.

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