CN114376963A - Rhein and metal ion coordination hydrogel, preparation method thereof and application thereof in osteoarthritis - Google Patents

Rhein and metal ion coordination hydrogel, preparation method thereof and application thereof in osteoarthritis Download PDF

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CN114376963A
CN114376963A CN202111482169.1A CN202111482169A CN114376963A CN 114376963 A CN114376963 A CN 114376963A CN 202111482169 A CN202111482169 A CN 202111482169A CN 114376963 A CN114376963 A CN 114376963A
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hydrogel
rhein
coordination
metal ions
drug
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王杨
杨曌宇
唐涛
胡恩
张翼
郑俊
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Xiangya Hospital of Central South University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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Abstract

The invention discloses rhein and metal ion coordination hydrogel, a preparation method thereof and application thereof in osteoarthritis, and belongs to the technical field of supramolecular hydrogel. Rhein is coordinated with metal ions to form supermolecule hydrogel. The hydrogel components are natural products with good biocompatibility, the hydrogel has low toxicity and excellent biocompatibility, chemical modification and other carriers are not needed, the drug loading capacity and safety are improved, the drug can be rapidly delivered to a target position in an injection mode, the hydrogel can be self-delivered and self-sustained-released, the hydrogel has good biocompatibility and biodegradability, meanwhile, the microstructure of the hydrogel is a three-dimensional network, rhein drug molecules can be slowly released, the hydrogel has long-acting release capacity, the drug can be prevented from being rapidly removed, the utilization degree of the drug is improved, the hydrogel has good anti-inflammation and cartilage repair promotion effects, and the hydrogel has a good application prospect in the fields of osteoarthritis and the like. The preparation method is simple, low in cost, capable of being commercialized and suitable for large-scale production.

Description

Rhein and metal ion coordination hydrogel, preparation method thereof and application thereof in osteoarthritis
Technical Field
The invention relates to the technical field of supermolecule hydrogel, in particular to a preparation method of rhein and metal ion coordination hydrogel and application of rhein and metal ion coordination hydrogel in osteoarthritis diseases.
Background
Osteoarthritis is a persistent chronic disease, primarily manifested by synovial inflammation, cartilage degeneration, subchondral bone sclerosis, and osteophyte formation. Up to 7% of the world's population is affected by osteoarthritis, with an increase in the number of patients of 48% from 1990 to 2019. By 2032, it was estimated that 29.5% of people aged 45 years or older had osteoarthritis. Although the pathophysiology of osteoarthritis is well understood, few effective treatments are available. Current therapies include systemic administration (oral or intravenous), physical therapy, and surgical therapy. However, none of them showed efficacy in ameliorating diseases and inhibiting long-term disability. Even some clinical trials have not produced positive results. The FDA ranks osteoarthritis as "a serious disease with unmet medical needs". Thus, there is an urgent need for promising biomedical drugs to alleviate, arrest or reverse the progression of osteoarthritis.
Targeting articular cartilage is the best option to achieve a positive effect in order to reduce or prevent osteoarthritis progression. Since according to the osteoarthritic pathology the marker is articular cartilage loss, this predicts the risk of future joint replacement. As the disease develops and progresses, an imbalance of cartilage matrix anabolism and catabolism persists. As a first line of defense against the onset of osteoarthritis, the outermost surface layer of articular cartilage begins with a dysfunction in the production of lubricin, the inclusion of chondroprogenitors, and shear resistance. In this case, chondrocytes appear to be helped by mesenchymal stem cells, which migrate from the subchondral region to the defect region to regenerate cartilage. The complex and lengthy repair process, coupled with the repeated exposure to mechanical loads, can result in cartilage destruction that is susceptible to arthritic degeneration. In addition, the biomechanical microenvironment contributes to the development of osteoarthritis. The extracellular matrix synthesized by chondrocytes accounts for 90% of the dry weight of cartilage and is critical for the physiological stability of the microenvironment. During osteoarthritis, the extracellular matrix undergoes remodeling and loss of flexibility, leading to abnormal behavior of the plantar and chondrocytes, further exacerbating cartilage function. These extracellular matrix fragments can trigger inflammatory processes in the joint environment due to cartilage destruction and complement pathway activation.
Successful cartilage repair requires that the drug enter the cartilage to the full depth to reach the chondrocytes and extracellular matrix. However, there is no vascularity, nerves and lymphatic tissue in the cartilage, which results in minimal diffusion of the drug to the damaged joint through systemic administration. Thus, targeting cartilage by intra-articular injection tends to be a better therapeutic strategy. Although osteoarthritic cartilage site injections have been reported (such as analgesics, glucocorticoids, hyaluronic acid and other unproven alternative therapeutic compounds), these small molecules and biological macromolecules may encounter rapid elimination of the drug, resulting in inadequate retention and reduced concentrations. In addition, most compounds have an intra-articular half-life as short as 2 to 4 hours. Therefore, exploring the great potential for good drug penetration and retention in cartilage to achieve osteoarthritis treatment remains a great challenge.
Over the past decades, scientists have created a series of innovative drug delivery systems for sustained release into cartilage. Among these systems, injectable hydrogels are one of the most commonly used biofunctional materials. In addition, the technique enables the embedding of the target site by minimally invasive surgery. In the aspect of cartilage repair, hydrogels are soft materials with an open porous structure and high water content; therefore, they are attractive candidates for extracellular matrix simulation. In addition, self-assembled hydrogels form three-dimensional nanofiber network immobilized water that can provide advantages such as cellular biocompatibility, natural biodegradability, non-immunogenicity, and low cytotoxicity.
Despite extensive research, the injected hydrogels of the invention are far from clinical transformed cartilage repair in osteoarthritis. Polymeric hydrogels provide superior mechanical strength that synovial fluid in the joint cavity cannot withstand. In addition, these hydrogels increase the incidence of systemic toxicity due to undefined in vivo breakdown and degradation. Self-assembled hydrogels composed of small molecules have better properties than polymer hydrogels, including responsiveness, reversibility, tunability, biomimetic, modularity, and adaptability. However, the vast majority of small molecule-based hydrogels involve delivery vehicles, which can result in poor biocompatibility and biodegradability, low loading efficiency, and unexpected side effects. Therefore, it is imperative to explore on-demand hydrogel injections that overcome the existing obstacles.
To design hydrogels for on-demand therapy, we attempted to prepare a supramolecular hydrogel without cargo delivery. The hydrogel is formed by combining self-assembled rhein and zinc for coordination. Rhein is a bioactive metabolite of diacerein, is an anthraquinone small molecule, and is used for treating osteoarthritis. Since rhein self-assembles through non-covalent interactions, supramolecular nanofibers form as a three-dimensional network of the hydrogel to immobilize water. The subsequent addition of zinc ions enhances the cross-linking of the biomaterial system to mimic the native extracellular matrix. In addition, zinc ions produce beneficial effects on cartilage repair and bone formation through metalloprotease activation and bone metabolic homeostasis. Therefore, the customized biofunctional self-assembled nano hydrogel not only improves injectability, slow release performance, biocompatibility and biodegradability, but also plays a great potential in osteoarthritis cartilage repair with almost no side effect.
Disclosure of Invention
The invention aims to provide a preparation method of rhein and metal ion coordination hydrogel and application of the rhein and metal ion coordination hydrogel in osteoarthritis diseases, so as to solve the problems of low bioavailability, short effective blood concentration maintaining time and low safety of the existing rhein pharmaceutical dosage form.
In order to achieve the above object, according to one aspect of the present invention, there is provided a coordination hydrogel of rhein and metal ions, the microstructure of the coordination hydrogel is a three-dimensional network structure, and the coordination hydrogel of rhein and metal ions is a hydrogel formed by coordination self-assembly of rhein and metal ions in an alkaline aqueous solution.
The invention utilizes carboxyl in a small molecular drug rhein to form carboxylate ions in an alkaline aqueous solution, and then the carboxylate ions and metal ions are self-assembled to form the injectable hydrogel through non-covalent acting forces such as hydrogen bonds, pi-pi accumulation, hydrophobic action, coordination action and the like. In the system, a dynamic and reversible coordination bond is formed between rhein drug molecules and metal ions, the hydrogel can be quickly sheared and thinned under certain stress, and the original gel state can be quickly recovered after injection.
The rhein and metal ion coordination hydrogel can be directly injected and slowly release rhein drug molecules. The hydrogel is formed by self-assembly of drug molecules, a carrier is not required to be added, toxic and side effects caused by the adoption of liposome, polymer microspheres, micelles, nanoparticles, macromolecules and the like as drug carriers in the prior art are avoided, and the drug loading capacity is improved. The rhein injectable hydrogel solves the problems of poor solubility, easy and rapid removal and the like of rhein, and has very important clinical significance.
Furthermore, the concentration of the rhein is 5mg/mL-8mg/mL, and the content is 0.38 wt% -0.8 wt%. Through experimental research, when the content of the rhein is lower than the minimum gelling concentration of 0.38 wt%, hydrogel cannot be formed; when the content of rhein is more than 0.8 wt% of the maximum concentration, it may cause the hydrogel to be non-uniformly transparent. Therefore, the content of rhein in the hydrogel is preferably in the range of 0.38 wt% to 0.8 wt%.
Furthermore, the mole ratio of the rhein to the metal ions is 1 (0.03-0.25). Research shows that the molar ratio of the rhein to the metal ions is better than that of the hydrogel obtained in the range; if too many metal ions are used, the hydrogel is not uniform, is not transparent and can cause cytotoxicity; too little metal ion can result in failure to self-assemble to form a hydrogel.
Further, the metal ion is Zn2+、Ca2+、Mg2+One or more of (a).
Further, the alkaline aqueous solution is a PBS buffer solution, a sodium hydroxide solution or a potassium hydroxide solution.
Further, the pH value of the alkaline aqueous solution is 8.35-9.2, and the pH value of the rhein and the metal ion coordination hydrogel is 7.0-7.4.
According to another aspect of the present invention, a preparation method of the coordination hydrogel of rhein and metal ions is provided, wherein rhein is dissolved in an alkaline aqueous solution, a metal ion solution is added, and rhein and metal ion coordination hydrogel is obtained after ultrasonic mixing.
Further, the time of ultrasonic mixing is 2min-30 min.
Further, the concentration of the metal ion solution is 0.01mol/L-0.5 mol/L. The concentration of the metal ion solution in the range is the best effect, the concentration of the metal ions is too low, so that the metal ions cannot be coordinated with rhein to form hydrogel by self-assembly, and if the concentration is too high, the gel is not uniform and transparent.
According to another aspect of the present invention, there is provided an application of the coordination hydrogel of rhein and metal ions or the coordination hydrogel of rhein and metal ions obtained by the above preparation method, wherein the coordination hydrogel of rhein and metal ions is used for the development of an injection formulation for osteoarthritis.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes the coordination self-assembly of clinical medicine rhein and metal ions to form injectable hydrogel, and chemical modification and other carriers are not needed for medicine molecules, so that the medicine-loading rate and the safety are improved; the hydrogel can rapidly deliver the drug to a target position through an injection mode; the hydrogel can be self-delivered and self-sustained released, and has good biocompatibility and biodegradability. The microstructure of the coordination hydrogel of rhein and metal ions prepared by the invention is a three-dimensional network, and the coordination hydrogel can slowly release rhein drug molecules, has long-acting release capacity, can prevent the drug from being rapidly removed, and improves the utilization rate of the drug. The rhein and metal ion coordination hydrogel does not need chemical modification and additional carriers, has high drug utilization rate, has shear thinning and self-repairing performance, can be directly injected for administration, and has good slow release property and biocompatibility; the preparation method is green, environment-friendly and simple. The preparation method is simple, low in cost, capable of being commercialized and suitable for large-scale production. The rhein and metal ion coordination hydrogel prepared by the invention has good anti-inflammatory and cartilage repair promotion effects, and has good application prospects in the fields of osteoarthritis and the like.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) image of a hydrogel;
FIG. 2 is a transmission microscope (TEM) image of a hydrogel in example 1 of the present invention;
FIG. 3 is a rheological diagram of a hydrogel in example 2 of the present invention;
FIG. 4 is a graph showing that hydrogel inhibits inflammation expression in example 3 of the present invention;
FIG. 5 is a photograph of immunofluorescence promoting cartilage repair in example 4 of the present invention;
FIG. 6 is a graph showing the staining patterns of safranin-fast green, eosin-hematoxylin, and toluidine blue for promoting cartilage repair in mice according to example 5 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
Weighing 5.0mg rhein in a screw bottle, adding 0.1mol/L zinc acetate solution, adding 1mL PBS buffer solution (pH 8.5), and performing ultrasonic treatment for 60s to ensure that the molar ratio of rhein to zinc ions is 8: 1; obtaining the chrysophanic acid and zinc coordination injectable hydrogel with the orange color. The microstructure of the rhein and zinc coordination injectable hydrogel is observed by using a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM).
Preparing a scanning electron microscope sample: 10 μ L of hydrogel was placed on a clean silicon wafer, lyophilized for 24h, and then tested. And carrying out gold spraying treatment before scanning due to poor conductivity of the sample. Fig. 1 is a scanning electron microscope image of the rhein and zinc coordination injectable hydrogel obtained in this example. As shown in figure 1, the micro-morphology of the rhein and zinc coordination hydrogel is a three-dimensional network structure.
Preparation of a transmission electron microscope sample: 3ul of hydrogel was placed on a clean copper mesh, air dried naturally, and then tested. Fig. 2 is a transmission electron microscope image of the rhein and zinc coordination injectable hydrogel obtained in the example. As shown in FIG. 2, the micro-morphology of the rhein and zinc coordination hydrogel is a fibrous structure.
Example 2
Weighing 10.0mg rhein in a screw bottle, adding 0.1mol/L zinc acetate solution, adding 2mL PBS buffer solution (pH 8.4), and performing ultrasonic treatment for 60s to ensure that the molar ratio of rhein to zinc ions is 12: 1; obtaining the chrysophanic acid and zinc coordination injectable hydrogel with the orange color.
The shear thinning and self-repairing capability of the rhein hydrogel is tested by a rheometer. We reacted to the state of the hydrogel by detecting changes in storage modulus (G ') and loss modulus (G'). When G '> G', the sample is in a gel state; when G '< G', the sample is in a solution state.
The specific testing steps are as follows: placing the prepared hydrogel on a rheometer, setting experiment parameters, and dividing the experiment into three stages: in the first stage, under low stress, the stress is set to be 0.1 percent, and the time is 180 s; in the second stage, under high stress, the stress is set to be 35%, and the time is kept for 60 s; the third stage was returned to a low stress of 0.1% stress for a period of 180 seconds from a high stress, and the change in each of stages G' and G "was observed.
As shown in fig. 3, in the first stage, at a low stress of 0.1%, the storage modulus (G') is always greater than the loss modulus (G ″), indicating that the sample is in a gel state; in the second stage, when the stress is increased to 35%, the storage modulus (G ') is always smaller than the loss modulus (G'), which indicates that the sample is in a solution state: in the third stage, the storage modulus (G ') is greater than the loss modulus (G') when returning from high stress to low stress, indicating that the sample changes from the solution state back to the gel state when the stress is reduced. The experiments show that the rhein hydrogel has good shear thinning and self-repairing capabilities.
Example 3
Weighing 10.0mg rhein in a screw bottle, adding 0.1mol/L zinc acetate solution, adding 2mL PBS buffer solution (pH 8.4), and performing ultrasonic treatment for 60s to ensure that the molar ratio of rhein to zinc ions is 12: 1; obtaining the chrysophanic acid and zinc coordination injectable hydrogel with the orange color.
Primary chondrocytes were pre-treated for 30min and stimulated with IL-1 β (10 ng/mL). After 48h of treatment, the supernatant was collected. The levels of TNF-. alpha.IL-1. beta., IL-6 and IL-10 were determined by ELISA kits (clumsy, Shanghai, China) according to the manufacturer's instructions.
As shown in FIG. 4, the hydrogel group significantly reduced the levels of TNF-. alpha.IL-6 and IL-1. beta. and increased the level of IL-10 as compared to the solution group.
Example 4
Weighing 10.0mg rhein in a screw bottle, adding 0.1mol/L zinc acetate solution, adding 2mL PBS buffer solution (pH 8.4), and performing ultrasonic treatment for 60s to ensure that the molar ratio of rhein to zinc ions is 12: 1; obtaining the chrysophanic acid and zinc coordination injectable hydrogel with the orange color.
An immunofluorescence step: rehydrated sagittal sections were heated in sodium citrate buffer (10mM trisodium citrate, pH6.0, 92 ℃) for 20 minutes and then blocked with 3% Bovine Serum Albumin (BSA) plus 0.02% triton-X100 for 1 hour. Rabbit anti-collagen II (1: 400; ab 34712; Abcam; USA) and rabbit anti-SOX 9(1: 800; 82630S; Cell Signaling Technology; USA) were then incubated for 14 hours at 4 ℃. Positive antigens were visualized using Cy 3-conjugated donkey anti-rabbit IgG as secondary antibody. Fluorescence was captured by a zeiss microscope (carl zeiss, germany) at 200 x magnification.
We isolated primary chondrocytes from mouse joints and induced osteoarthritis cell models in vitro with IL-1 β. Immunofluorescence results indicated that the expression levels of SOX9 and COII were reduced in the model group. Treatment with hydrogels and solutions increased the expression levels of SOX9 and COII. Among them, the hydrogel group increased the expression of COII and SOX9 compared to the solution group (FIG. 5).
Example 5
Weighing 10.0mg rhein in a screw bottle, adding 0.1mol/L zinc acetate solution, adding 2mL PBS buffer solution (pH 8.4), and performing ultrasonic treatment for 60s to ensure that the molar ratio of rhein to zinc ions is 12: 1; obtaining the chrysophanic acid and zinc coordination injectable hydrogel with the orange color.
Morphological staining step: the whole joint was decalcified in 10% EDTA buffer (pH7.4) at 4 ℃ for 2 weeks with gentle shaking. The joints were embedded in paraffin and sagittal sections of 3 microns were made through the entire medial joint at 40 micron intervals. Sections were then deparaffinized and rehydrated for further histological evaluation. For the safranin O-fast green staining, hematoxylin (G1371; Solarbio; China), fast green (G1053-2; Servicobio; China) and safranin O (G1053-1; Servicobio) were used for 3 minutes, and 5 minutes in this order. For hematoxylin and eosin staining (H & E), sections were immersed in hematoxylin solution (G1004; ServiceBio) for 5 minutes and eosin solution (G1001; ServiceBio) for 20 seconds. For toluidine blue staining, incubate in toluidine blue for 3 minutes. To visualize tartrate-resistant acid phosphatase (TRAP) -positive cells, sections were performed according to the TRAP staining kit (G1050; ServiceBio) manufacturer's protocol. 30 seconds 1% methyl green (C0115; Solambio) was used to reveal the nuclei. After dehydration and transparentization, the sections were sealed and photographed. Standard OARIS was used to assess the severity of knee joint injury.
Structural and functional impairment of cartilage and bone is a major pathology of osteoarthritis. Histologically, the hydrogel group significantly inhibited cartilage erosion and proteoglycan loss as assessed by safranin-fast green, eosin-hematoxylin and toluidine blue staining in FIG. 6.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. A rhein and metal ion coordination hydrogel is characterized in that: comprises rhein, metal ions and alkaline aqueous solution.
2. The coordination hydrogel of rhein and metal ions according to claim 1, wherein: the concentration of the rhein is 5mg/mL-8 mg/mL.
3. The coordination hydrogel of rhein and metal ions according to claim 1, wherein: the molar ratio of the rhein to the metal ions is 1 (0.03-0.25).
4. The coordination hydrogel of rhein and metal ions according to claim 1, wherein: the metal ion is Zn2+、Ca2+、Mg2+One or more of (a).
5. The coordination hydrogel of rhein and metal ions according to claim 1, wherein: the alkaline aqueous solution is PBS buffer solution, sodium hydroxide solution or potassium hydroxide solution.
6. The coordination hydrogel of rhein and metal ions according to claim 1, wherein: the pH value of the alkaline aqueous solution is 8.35-9.2, and the pH value of the rhein and the metal ion coordination hydrogel is 7.0-7.4.
7. A method for preparing the coordination hydrogel of rhein and metal ions according to any one of claims 1 to 6, wherein: dissolving rhein in an alkaline aqueous solution, adding a metal ion solution, and performing ultrasonic mixing to obtain the rhein and metal ion coordination hydrogel.
8. The method for preparing the coordination hydrogel of rhein and metal ions according to claim 7, wherein the coordination hydrogel comprises: the ultrasonic mixing time is 2min-30 min.
9. The method for preparing the coordination hydrogel of rhein and metal ions according to claim 7, wherein the coordination hydrogel comprises: the concentration of the metal ion solution is 0.01-0.5 mol/L.
10. Use of the complex hydrogel of rhein and metal ions according to any one of claims 1 to 6 or the complex hydrogel of rhein and metal ions obtained by the preparation method according to any one of claims 7 to 9, wherein: the rhein and metal ion coordination hydrogel is used for the development of an injection formulation of osteoarthritis.
CN202111482169.1A 2021-12-06 2021-12-06 Rhein and metal ion coordination hydrogel, preparation method thereof and application thereof in osteoarthritis Pending CN114376963A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115531291A (en) * 2022-09-02 2022-12-30 南京医科大学 Medicine-carrying lysine rhein self-assembled hydrogel and preparation method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115531291A (en) * 2022-09-02 2022-12-30 南京医科大学 Medicine-carrying lysine rhein self-assembled hydrogel and preparation method and application thereof

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