Novel cascaded targeted selenium-based hydrogel microspheres for multi-stage antioxidant treatment of osteoarthritis and preparation method of novel cascaded targeted selenium-based hydrogel microspheres

Abstract

The invention discloses novel cascaded targeted selenium-based hydrogel microspheres for multi-stage antioxidant treatment of osteoarthritis and a preparation method of the novel cascaded targeted selenium-based hydrogel microspheres. The preparation method comprises the following steps: step S1, preparing an LAP solution; s2, preparing a hydrogel precursor solution; s3, preparing a hydrogel oil phase for later use; step S4, preparing hydrogel microspheres with a porous structure; and step S5, preparing the hydrogel microspheres (AHAMA-HMs) loaded with the novel selenium nanoparticles (HA-SeNPs). The AHAMA hydrogel microspheres loaded with the HA-SeNPs can enter an articular cavity through a 5mL injector, and the cartilage is adhered and abraded in a targeted manner by virtue of a Schiff base reaction of-CHO on the surface of the microspheres and-NH2 on the surface of the cartilage, so that micron-level targeting is realized; then, HA-SeNPs released from the AHAMA-HMs is combined with CD44 on the surface of the abraded cartilage cell through hyaluronic acid (HA), so that nano-scale targeting is realized. Subsequently, after entering the cartilage cells, the HA-SeNPs can participate in removal of excess reactive oxygen species (ROS) in the cells in multiple stages, so that multi-stage antioxidant treatment is realized, and further osteoarthritis is treated.

Description

Technical Field

[0001] The present invention belongs to the technical field of injectable drug-loaded hydrogels, and in particular, relates to a novel cascade-targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis and a preparation method thereof. Background Art

[0002] Essential trace elements for the human body, such as selenium, iodine, and iron, play an indispensable role in maintaining normal physiological functions and anti-oxidation by acting as structural components or cofactors of enzymes. As an important member of the system, selenium (Se) is well-known for its powerful antioxidant effect and is a key element of the human antioxidant defense system. In the body, Se plays a vital role in scavenging oxygen free radicals and alleviating oxidative stress, mainly through the synthesis of antioxidant selenoproteins, such as glutathione peroxidases (GPXs) and thioredoxin reductases (TXNRDs). However, when Se is deficient, the function of the body's antioxidant defense system declines, and redox homeostasis is unbalanced, leading to the occurrence of many diseases. For example, Se deficiency is closely related to osteoarthritis (OA), and clinical evidence shows that the serum Se level of OA patients is significantly lower than that of healthy volunteers of the same age. In addition, Se deficiency weakens the synthesis of antioxidant selenoproteins in chondrocytes, leading to impaired redox homeostasis in chondrocytes, mitochondrial dysfunction, and increased levels of reactive oxygen species (ROS), ultimately promoting the occurrence of OA. Therefore, existing evidence shows that Se deficiency plays an important role in OA pathology, and Se supplementation may be a potential new direction for the treatment of OA.

[0003] The latest studies have shown that Se supplementation can improve the antioxidant capacity of chondrocytes and effectively reduce the progression of OA. Whether it is an inorganic Se supplement (such as sodium selenite, Na2SeO3) or an organic Se supplement (such as selenomethionine, SeMet), after entering the body, it will be converted into H2Se and further participate in the synthesis of antioxidant selenoproteins as a raw material. However, it is worth noting that Se is actually a "double-edged sword". Se deficiency or Se excess will lead to adverse outcomes - decreased antioxidant defense function, or Se poisoning, or even the promotion of ROS generation. At the same time, the safety range of Se supplements is very narrow, which greatly limits the application of Se supplements. In addition, articular cartilage lacks blood vessels, and Se supplements are difficult to reach and maintain effective concentrations. Therefore, a new Se supplementation strategy is urgently needed to achieve targeted delivery to Se-deficient chondrocytes while efficiently supplementing Se to OA cartilage tissue and maintaining an effective therapeutic concentration in the joint cavity.

[0004] With the development of nanoscience, nanoselenium (SeNPs) has emerged as a new type of selenium supplement. Compared with traditional inorganic / organic selenium supplements, SeNPs are zero-valent single-substance Se particles with low toxicity and high biological activity. It is particularly noteworthy that SeNPs have outstanding advantages in multi-link antioxidant defense: 1) On the one hand, due to the particularity of its electronic structure, quantum size effect and surface area effect of nanoparticles, SeNPs have enzyme-like activity and can directly undergo redox reactions with ROS in vitro without relying on the enzyme system in the body, generating selenite (SeO3 2- ); 2) On the other hand, the generated SeO3 2- It can be used as a synthetic raw material to further promote the synthesis of antioxidant selenoproteins in the body, thereby exerting its antioxidant function again. Recent studies have shown that local injection of SeNPs into the knee joint cavity of mouse and rat OA models can effectively remove ROS, reduce local inflammation, inhibit cell apoptosis and promote cartilage repair. However, SeNPs have poor stability and may undergo nanoparticle aggregation in a short period of time to form inert particles larger than micrometers, while losing the properties of directly removing ROS, being taken up by cells and promoting selenoprotein synthesis. In addition, SeNPs injected directly into the joint cavity lack targeting, and additional Se may cause potential adverse effects on unworn cartilage that is not lacking in Se (such as causing increased ROS levels and cell apoptosis). It is worth noting that some polysaccharides (such as hyaluronic acid) can form hydrogen bonds or Se-O bonds with the surface of SeNPs through a large number of hydroxyl groups on their molecular skeletons to achieve the stability of dispersed nanoselenium particles. In addition, Lei et al. found that the level of CD44 in cartilage of OA patients was positively correlated with the severity of the disease, and CD44 is one of the main specific receptors of hyaluronic acid (HA). Therefore, we believe that encapsulating SeNPs with HA modification can improve the stability of SeNPs while providing SeNPs with precise navigation for targeting worn chondrocytes, thereby avoiding the potential adverse effects of intra-articular injection.

[0005] As nano-sized particles, SeNPs can easily penetrate the dense cartilage matrix, but they are also easily cleared by the synovial lymphatic system, and nanoparticle aggregation may occur in a short period of time. Once SeNPs aggregate, they will form inert particles larger than micrometers, and lose their properties including direct scavenging of ROS, cell uptake, and promotion of selenoprotein synthesis. Therefore, local injection of SeNPs has the limitation of short half-life, requiring frequent injections or increased dosing concentration, which may increase patient pain, potential toxicity risks, and other related costs. Therefore, it is urgent to take measures to prolong the residence time of SeNPs in the joint cavity and delay their aggregation. It is worth noting that hydrogel microspheres (HMs) are micrometer-sized injectable porous carriers that are widely used as "drug storage depots". Based on the nano-micro composite strategy, HMs can load and continuously release a variety of nano-sized therapeutic agents, such as exosomes, liposomes, and nanoparticles. Therefore, the porous structure of HMs provides sufficient "seats" for SeNPs, and physical barriers can delay the aggregation of SeNPs. In addition, as micron-sized "big balls", HMs can prevent the loaded SeNPs from being cleared prematurely by the synovial lymphatic system while slowly releasing SeNPs, thereby prolonging their retention time in the joint cavity and enhancing the therapeutic effect. Recent studies have found that the surface of articular cartilage, especially the surface of cartilage on the worn side, is rich in amino residues (-NH2) due to the exposure of a large amount of type II collagen. Therefore, we introduced aldehyde groups (-CHO) into methacrylated HA microspheres (HAMA-HMs) to synthesize AHAMA-HMs, which can enhance the adhesion of HMs to the worn side cartilage based on the Schiff base reaction of -CHO and -NH2, thereby improving the targeting and utilization of SeNPs.

[0006] In view of this, the present invention is proposed. Based on the nano-micro composite strategy, the present invention introduces a cascade-targeted nano-micro composite hydrogel microsphere (HA-SeNPs@AHAMA-HMs), which aims to target and adhere to worn cartilage and release SeNPs with multi-link antioxidant defense functions to reduce chondrocyte ROS, improve mitochondrial dysfunction, and inhibit OA progression. Specifically, hyaluronic acid aldehyde (AHA) and methacrylated hyaluronic acid aldehyde (AHAMA) were prepared, and injectable AHAMA-HMs loaded with HA-SeNPs were prepared by microfluidic technology, relying on the Schiff base reaction of microsphere surface-CHO and cartilage surface-NH2 to target and adhere to worn cartilage, achieving the first layer of targeting. In addition, HA-encapsulated SeNPs (HA-SeNPs) were synthesized by redox reaction, which, after entering the joint cavity, combined with CD44 on the surface of worn chondrocytes through HA to achieve the second layer of targeting; at the same time, after entering the cells, HA-SeNPs exerted their multi-link antioxidant defense function to achieve the purpose of treating OA. Based on the above advantages, this cascade-targeted nano-microcomposite hydrogel microspheres show therapeutic potential to alleviate oxidative stress and delay the progression of OA. Summary of the invention

[0007] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is: A novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis and a preparation method thereof, comprising the following steps: Step S1: weigh 0.05 g of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt powder (LAP powder), add 20 mL of PBS solution, and stir at room temperature (350 rpm) until completely dissolved to obtain a 0.25% (w / v) LAP solution; Step S2: Weigh 80 mg of AHAMA solid, add 10 mL of LAP solution, stir (350 rpm) in a dark place until completely dissolved, and obtain an AHAMA hydrogel precursor solution, which is temporarily stored in a dark place; Step S3: Take 45 mL of light paraffin, add 5 mL of Span 80, shake and mix thoroughly to obtain an oil phase, and let it stand for later use; Step S4: using a microfluidic device, the AHAMA hydrogel precursor solution is used as the water phase, and the water phase and the oil phase are pushed at a uniform speed to obtain AHAMA hydrogel droplets, which are then cross-linked by ultraviolet light (UV) after freezing to obtain hydrogel microspheres (AHAMA-HMs) with uniform particle size and porous structure, which are then freeze-dried for use; Step S5: Add the above freeze-dried AHAMA-HMs to the HA-SeNPs solution and stir at room temperature for 24 hours (rotation speed 250 rpm) to obtain hydrogel microspheres (HA-SeNPs@AHAMA-HMs) loaded with novel selenium nanoparticles (HA-SeNPs).

[0008] As a preferred embodiment of the present invention, the synthesis method of the novel selenium nanoparticles (HA-SeNPs) is as follows: a: Prepare 50 mL of 0.3% w / v HA solution, 5 mL of 100 mM sodium selenite solution, and 5 mL of 100 mM ascorbic acid solution; b: Add 1 mL of sodium selenite solution to the HA solution and stir at room temperature for 10 minutes at a speed of 350 r / min; c: Add 4 mL of ascorbic acid solution to the reaction system and stir at room temperature for 24 hours at a speed of 350 r / min; d: The reaction system was transferred to a 3500Da dialysis bag and dialyzed at room temperature for 24 hours at a rotation speed of 350 r / min to obtain new HA-SeNPs wrapped with HA.

[0009] As a preferred embodiment of the present invention, the synthesis method of the novel hydrogel material (AHAMA) is as follows: a: Weigh 1g HA powder and dissolve it in 100mL deionized water, and stir mechanically until the solution is clear and transparent (speed 350rpm); b: Add 5 mL of 0.5 M sodium periodate solution to the HA solution and stir for 2 hours in the dark (speed 250 rpm); c: Add 1 mL of ethylene glycol to the reaction system to inactivate the sodium periodate, and stir for 1 hour in the dark (speed 250 rpm); d: After the reaction is completed, the reaction system is transferred to a 8-14 kDa dialysis bag and dialyzed at room temperature for 72 hours (rotation speed 250 rpm), followed by freeze-drying to obtain hyaluronic acid aldehyde (AHA) solid; e: Weigh 1g AHA solid and dissolve it in 50mL deionized water, and stir mechanically until the solution is clear and transparent (speed 350rpm); f: Add 20 mL of methacrylic anhydride (MA) to the AHA solution at a uniform rate and stir evenly (speed 250 rpm); g: Place the reaction system in an ice-water bath and use a microsyringe pump to uniformly add 5 M NaOH (20 mL) to the reaction system over 2 hours, then place the entire reaction system at 4 °C overnight; h: After the reaction, the reaction system was centrifuged at 7000 rpm for 15 minutes, the precipitate was discarded, and the supernatant was transferred to a 8-14 kDa dialysis bag and dialyzed at room temperature for 72 hours (speed 250 rpm), followed by freeze-drying to obtain methacrylated hyaluronic acid aldehyde (AHAMA) solid.

[0010] As a preferred embodiment of the present invention, a novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis is prepared by the method.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. Modified synthesis of new selenium nanoparticles (HA-SeNPs): In the redox reaction system for preparing traditional selenium nanoparticles (SeNPs), hyaluronic acid (HA) was added, and a layer of HA shell was coated around the traditional SeNPs to synthesize new selenium nanoparticles with a shell-core structure (HA shell, SeNPs core, diameter of about 90nm). Compared with normal chondrocytes, the chondrocyte membrane at the worn part highly expresses CD44 protein, and CD44 protein is a specific receptor for HA. Therefore, this new selenium nanoparticle (HA-SeNPs) can target worn chondrocytes in osteoarthritis. At the same time, HA can form hydrogen bonds or Se-O bonds with the surface of SeNPs through a large number of hydroxyl groups on its molecular skeleton to achieve the stability of dispersed nano-selenium particles. Therefore, the stability of this new selenium nanoparticle (HA-SeNPs) is better than that of traditional SeNPs.

[0012] 2. A two-step method was used to construct a novel hydrogel (AHAMA) that can adhere to worn cartilage: ① Sodium periodate (NaIO4) was used to oxidize two adjacent hydroxyl groups (-OH) in the hyaluronic acid (HA) molecular chain and form two aldehyde groups (-CHO) to synthesize hyaluronic acid aldehyde (AHA); ② Methacrylic anhydride (C8H 10 O3) replaces the remaining part of the hydroxyl groups (-OH) in AHA under alkaline conditions to synthesize methacrylated hyaluronic acid aldehyde (AHAMA).

[0013] 3. Synthesis of a new type of injectable hydrogel microspheres (AHAMA-HMs) with uniform particle size and targeted adhesion to worn cartilage: Based on microfluidic technology, AHAMA hydrogel microspheres (AHAMA-HMs, with a diameter of approximately 280 μm) were synthesized by cutting the water phase with the oil phase. After UV curing, they can be locally injected into the joint cavity through a 5 mL syringe. At the same time, since the microsphere molecular skeleton contains aldehyde groups (-CHO), they can undergo Schiff base reactions with the amino groups (-NH2) exposed on the worn cartilage to form Schiff base bonds, allowing AHAMA-HMs to adhere to the worn cartilage surface in a targeted manner.

[0014] 4. Based on the nano-micro composite strategy, AHAMA hydrogel microspheres loaded with new selenium nanoparticles (HA-SeNPs) were prepared to achieve cascade targeting and multi-stage antioxidant therapy: AHAMA hydrogel microspheres loaded with HA-SeNPs can enter the joint cavity through a 5mL syringe, relying on the Schiff base reaction between the microsphere surface-CHO and the cartilage surface-NH2 to target and adhere to the worn cartilage, achieving micron-level targeting; HA-SeNPs released from AHAMA-HMs then bind to CD44 on the surface of worn chondrocytes through HA, achieving nano-level targeting. Subsequently, after entering the chondrocytes, HA-SeNPs can participate in the removal of excess reactive oxygen species (ROS) in the cells at multiple stages, achieving multi-stage antioxidant therapy.

[0015] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the attached picture: Figure 1 Schematic diagram of the synthesis of the novel hydrogel material (AHAMA) in the present invention; Figure 2 Schematic diagram of the synthesis of nano-micro composite hydrogel microspheres of the present invention; Figure 3 Schematic diagram of the nano-micro composite hydrogel microspheres of the present invention adhering to cartilage; Figure 4 This is an experimental diagram of HA, AHA and AHAMA in the solid state of the present invention; Figure 5 This is an experimental diagram of the new selenium nanoparticles synthesized by the present invention; Figure 6 This is a microscope image of the novel nano-micro composite hydrogel microsphere prepared by the present invention; Figure 7 This is an actual experimental diagram of the ability of the novel hydrogel microspheres of the present invention to adhere to cartilage; Figure 8 This is an experimental diagram of the injectability of the new hydrogel microspheres. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention.

[0018] like Figures 1 to 8 As shown, a novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis and a preparation method thereof include the following steps: Step S1: weigh 0.05 g of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt powder (LAP powder), add 20 mL of PBS solution, and stir at room temperature (350 rpm) until completely dissolved to obtain a 0.25% (w / v) LAP solution; Step S2: Weigh 80 mg of AHAMA solid, add 10 mL of LAP solution, stir (350 rpm) in a dark place until completely dissolved, and obtain an AHAMA hydrogel precursor solution, which is temporarily stored in a dark place; Step S3: Take 45 mL of light paraffin, add 5 mL of Span 80, shake and mix thoroughly to obtain an oil phase, and let it stand for later use; Step S4: using a microfluidic device, the AHAMA hydrogel precursor solution is used as the water phase, and the water phase and the oil phase are pushed at a uniform speed to obtain AHAMA hydrogel droplets, which are then cross-linked by ultraviolet light (UV) after freezing to obtain hydrogel microspheres (AHAMA-HMs) with uniform particle size and porous structure, which are then freeze-dried for use; Step S5: Add the above freeze-dried AHAMA-HMs to the HA-SeNPs solution and stir at room temperature for 24 hours (rotation speed 250 rpm) to obtain hydrogel microspheres (HA-SeNPs@AHAMA-HMs) loaded with novel selenium nanoparticles (HA-SeNPs).

[0019] Furthermore, the synthesis method of the novel selenium nanoparticles (HA-SeNPs) is as follows: a: Prepare 50 mL of 0.3% w / v HA solution, 5 mL of 100 mM sodium selenite solution, and 5 mL of 100 mM ascorbic acid solution; b: Add 1 mL of sodium selenite solution to the HA solution and stir at room temperature for 10 minutes at a speed of 350 r / min; c: Add 4 mL of ascorbic acid solution to the reaction system and stir at room temperature for 24 hours at a speed of 350 r / min; d: The reaction system was transferred to a 3500Da dialysis bag and dialyzed at room temperature for 24 hours at a rotation speed of 350 r / min to obtain new HA-SeNPs wrapped with HA.

[0020] Furthermore, the synthesis method of the novel hydrogel material (AHAMA) is as follows: a: Weigh 1g HA powder and dissolve it in 100mL deionized water, and stir mechanically until the solution is clear and transparent (speed 350rpm); b: Add 5 mL of 0.5 M sodium periodate solution to the HA solution and stir for 2 hours in the dark (speed 250 rpm); c: Add 1 mL of ethylene glycol to the reaction system to inactivate the sodium periodate, and stir for 1 hour in the dark (speed 250 rpm).

Claims

1. A method for preparing a novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis, characterized in that: The steps include: Step S1: weigh 0.05 g of phenyl phosphate lithium salt powder, add 20 mL of PBS solution, and stir at room temperature until completely dissolved to obtain a 0.25% w / v LAP solution; Step S2: Weigh 80 mg of hydrogel solid, add 10 mL of LAP solution, stir in dark place until completely dissolved, obtain hydrogel precursor solution, and store in dark place temporarily; Step S3: Take 45 mL of light paraffin, add 5 mL of Span 80, shake and mix thoroughly to obtain an oil phase, and let it stand for later use; Step S4: using a microfluidic device, with a hydrogel precursor solution as the water phase, the water phase and the oil phase are pushed at a uniform speed to obtain droplets of a novel hydrogel material, and then UV ultraviolet light cross-linking is performed after freezing to obtain hydrogel microspheres with uniform particle size and porous structure, which are then freeze-dried for standby use; Step S5: adding the above freeze-dried hydrogel microspheres into the novel selenium nanoparticle solution, stirring at room temperature for 24 hours, to obtain hydrogel microspheres loaded with novel selenium nanoparticles.

2. The method for preparing a novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis according to claim 1, characterized in that: The synthesis method of the novel selenium nanoparticles is as follows: a: Prepare 50 mL of 0.3% w / v HA solution, 5 mL of 100 mM sodium selenite solution, and 5 mL of 100 mM ascorbic acid solution; b: Add 1 mL of sodium selenite solution to the HA solution and stir at room temperature for 10 minutes at a speed of 350 r / min; c: Add 4 mL of ascorbic acid solution to the reaction system and stir at room temperature for 24 hours at a speed of 350 r / min; d: The reaction system was transferred to a 3500Da dialysis bag and dialyzed at room temperature for 24 hours at a rotation speed of 350 r / min to obtain new HA-coated selenium nanoparticles.

3. The method for preparing a novel cascade targeted selenium-based hydrogel microspheres for multi-stage antioxidant treatment of osteoarthritis according to claim 1, characterized in that: The synthesis method of the novel hydrogel material is as follows: a: Weigh 1g HA powder and dissolve it in 100mL deionized water. Stir mechanically until the solution is clear and transparent at a speed of 350rpm. b: Add 5 mL of 0.5 M sodium periodate solution to the HA solution, stir for 2 hours in the dark at 250 rpm; c: Add 1 mL of ethylene glycol to the reaction system to inactivate the sodium periodate, and stir for 1 hour in the dark at 250 rpm; d: After the reaction is completed, the reaction system is transferred to a 8-14 kDa dialysis bag and dialyzed at room temperature for 72 hours with a stirring speed of 250 rpm, followed by freeze-drying to obtain hyaluronic acid aldehyde solid; e: Weigh 1g AHA solid and dissolve it in 50mL deionized water, and stir mechanically until the solution is clear and transparent (speed 350rpm); f: Add 20 mL of methacrylic anhydride to the AHA solution at a uniform rate and stir evenly at a speed of 250 rpm; g: Place the reaction system in an ice-water bath and use a microsyringe pump to uniformly add 5M NaOH to the reaction system over 2 hours in a volume of 20 mL. Then place the entire reaction system at 4°C overnight. h: After the reaction, the reaction system was centrifuged at 7000 rpm for 15 minutes, the precipitate was discarded, and the supernatant was transferred to a 8-14 kDa dialysis bag and dialyzed at room temperature for 72 hours at a speed of 250 rpm, followed by freeze-drying to obtain methacrylated hyaluronic acid aldehyde solid.

4. The method for preparing a novel cascade targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis according to claim 1, characterized in that: The phenyl group in the phenyl phosphate lithium salt powder is specifically 2,4,6-trimethylbenzoyl.

5. The method for preparing a novel cascade targeted selenium-based hydrogel microspheres for multi-stage antioxidant treatment of osteoarthritis according to claim 1, characterized in that: The stirring speed in step S1 and step S2 is 350 rpm, and the stirring speed in step S5 is 250 rpm.

6. A novel cascade-targeted selenium-based hydrogel microsphere for multi-stage antioxidant treatment of osteoarthritis, characterized in that: It is prepared by the method according to any one of claims 1 to 3.

Images