Application of sodium copper chlorophyllin in the preparation of drugs for treating osteoarthritis

Abstract

This invention discloses the application of sodium copper chlorophyllin in the preparation of drugs for treating osteoarthritis, belonging to the field of biomedical technology. The sodium copper chlorophyllin of this invention can block excessive activation of osteoclasts and subsequent angiogenesis, and successfully reprograms macrophage polarization, exhibiting significant anti-inflammatory and repairing effects. Furthermore, this invention constructs a dual-targeted sustained-release drug delivery system. After encapsulating sodium copper chlorophyllin into MOF material, the MOF material is then modified with MMP9 responsive peptides and bone-targeting peptides via click chemistry, achieving the goal of continuously releasing sodium copper chlorophyllin to the subchondral bone region of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of sodium copper chlorophyllin in the preparation of drugs for treating osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a highly prevalent and complex disease affecting the entire joint, leading to chronic pain, functional impairment, and psychological distress. Cartilage degeneration, joint inflammation, and abnormal subchondral bone remodeling are often referred to as the "triad" in the development of OA. Of these three pathological markers, the subchondral bone, located beneath the articular cartilage, supports the cartilage by providing nutrition and structural stability, and its bone immune microenvironment may be a key initiating factor for the degeneration of the superior cartilage during the onset and progression of OA. A growing body of research reveals that overactive osteoclasts and inflammatoryally polarized macrophages in the subchondral bone immune microenvironment are closely associated with the development of OA. However, the role of the subchondral bone immune microenvironment in the progression of OA remains unclear.

[0003] In fact, the subchondral bone immune microenvironment is a complex and dynamic environment, containing not only osteoblasts, osteoclasts, and macrophages, but also various cellular components such as vascular endothelial cells. Furthermore, it contains a large number of receptors, signaling molecules, and transcription factors. Recent research has identified a specific vascular subtype characterized by increased expression of CD31 and endomucin, termed the H-type or CD31 subtype. high Emcn high Blood vessels. Furthermore, vascular-associated osteoclasts (VAOs) located near the H-shaped vascular arch in the metaphysis can secrete angiogenic factors and trigger the release of matrix metalloproteinase-9 (MMP-9) from the H-shaped vessels to degrade the cartilage matrix, promoting H-shaped vessel invasion into articular cartilage. It has been demonstrated that inflammatory polarized macrophages in the subchondral bone microenvironment secrete large amounts of inflammatory cytokines, which amplify the inflammatory response throughout the joint and degrade the cartilage matrix. This degradation process creates a favorable environment for H-shaped vessels to invade and proliferate into the growth plate region. Subchondral bone and cartilage lacking blood vessels are in a hypoxic state, which is crucial for chondrocyte homeostasis. Hypervascularization of the subchondral bone is a hallmark and a new breakthrough in OA progression.

[0004] To better understand the pathogenesis of osteoarthritis (OA) at the molecular level and to find corresponding targeted therapies, transcription factors involved in the OA process have been a research hotspot in recent decades. ETS family transcription factors (TFs) are one of the largest transcription factor families, playing a crucial role in cell proliferation and differentiation, angiogenesis, and inflammation. PU.1 is a major regulator guiding the directed differentiation of hematopoietic and immune progenitor cells into myeloid cells and is considered essential for the differentiation of myeloid progenitor cells into osteoclasts. Therefore, the purpose of this invention is to provide a drug for treating osteoarthritis by inhibiting the expression of transcription factor PU.1. Summary of the Invention

[0005] Based on the technical problems existing in the background art, the present invention proposes the application of sodium copper chlorophyllin in the preparation of drugs for treating osteoarthritis.

[0006] This invention proposes the application of sodium copper chlorophyllin in the preparation of drugs for treating osteoarthritis.

[0007] Preferably, the osteoarthritis is a disease that affects the entire joint.

[0008] Preferably, the osteoarthritis is caused by the degeneration of cartilage into the subchondral bone immune microenvironment.

[0009] Preferably, the sodium copper chlorophyllin is encapsulated in MOF material to inhibit the expression of transcription factor PU.1.

[0010] Preferably, the MOF material is UIO-66-NH2.

[0011] Preferably, the dosage form of the drug is any pharmacologically acceptable dosage form, such as tablets, capsules, drops, granules, oral liquids, and solutions.

[0012] Preferably, the dosage of the drug is any pharmacologically acceptable dosage.

[0013] This invention proposes a method for preparing a dual-targeted sustained-release drug delivery system. The drug delivery system is used to prepare drugs for treating osteoarthritis. The method steps are as follows: S1: UIO-66-NH2, sodium copper chlorophyllin, and MAL-PEG are added. 2000 -NHS and deionized water were stirred at room temperature for 22-26 h to obtain sodium copper chlorophyllin encapsulated in MOF material, denoted as C-NPs; S2: The sodium copper chlorophyllin encapsulated in the MOF material prepared in S1 was magnetically stirred with the MMP9 responsive peptide and the bone-targeting peptide Asp8-SH, and a drug delivery system, denoted as C-PANPs, was obtained through maleimide-thiol click chemistry reaction.

[0014] Preferably, S1 contains UIO-66-NH2, sodium copper chlorophyllin, and MAL-PEG. 2000 The molar ratio of -NHS is 1-1.5:1:15-20.

[0015] Preferably, the molar ratio of sodium copper chlorophyllin, MMP9 responsive peptide, and bone-targeting peptide Asp8-SH encapsulated in MOF material in S2 is 1:1-3:1-3.

[0016] Preferably, the reaction conditions in S2 are 3-5 hours at room temperature.

[0017] The present invention proposes a dual-targeted sustained-release drug delivery system, which is prepared using the above-described preparation method.

[0018] Beneficial technical effects of the present invention: The sodium copper chlorophyllin of this invention can block excessive activation of osteoclasts and subsequent angiogenesis, and successfully reprograms macrophage polarization, exhibiting significant anti-inflammatory and repair effects.

[0019] The C-PANPs nanotherapy system developed in this invention encapsulates sodium copper chlorophyllin into MOF material, and then modifies the MOF material with MMP9 responsive peptide and bone-targeting peptide through click chemistry, thereby achieving the goal of continuously releasing sodium copper chlorophyllin to the subchondral bone site in OA. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the construction process of the C-PANPs drug delivery system proposed in this invention. Figure 2 TEM images of UIO-66-NH2 and the drug delivery system C-PANPs proposed in this invention; Figure 3 Infrared images of each component and the total of the C-PANPs drug delivery system proposed in this invention; Figure 4 Images of C-PANPs synthesis observed by confocal microscopy as proposed in this invention; wherein Chlorophyllin was stained with DIO, Asp8-SH was stained with CY3, and MMP9 cleavable peptide-SH (MMP9-SH) was stained with CY5.5, scale bar 20 nM; Figure 5 The cell viability diagram of primary macrophages after incubation with different doses of Chlorophyllin, Asp8-SH, UIO-66-NH2, and MMP9-SH as proposed in this invention; Figure 6The particle size distribution diagrams of UIO-66-NH2 (red) and C-PANPs (blue) proposed in this invention are shown. Figure 7 This invention provides a mouse in vivo imaging experiment. Figure 8 HE staining of mouse knee joint cartilage as proposed in this invention; scale bar 100 μm; Figure 9 The figure shows the results of Sham, DMM, DMM+UIO-66-NH2 and DMM+C-PANPs from left to right for the quantification of mouse knee osteoarthritis scores proposed in this invention. Figure 10 Immunohistochemical staining of the subchondral bone trap of the mouse knee joint proposed in this invention. The attached figures, from left to right, show the results of Sham, DMM, DMM+UIO-66-NH2, and DMM+C-PANPs. Figure 11 The attached images, from left to right, show the results of Sham, DMM, DMM+UIO-66-NH2, and DMM+C-PANPs for the quantitative analysis of the trap-positive region of the subchondral bone of the mouse knee joint proposed in this invention. Figure 12 The CD31 of the subchondral bone of the mouse knee joint proposed in this invention high Emcn high Vascular status, the attached images from left to right are the results of Sham, DMM, DMM+UIO-66-NH2 and DMM+C-PANPs; Figure 13 Immunohistochemical staining of CD86 in the subchondral bone of the mouse knee joint as proposed in this invention. The attached figures, from left to right, show the results of Sham, DMM, DMM+UIO-66-NH2, and DMM+C-PANPs; scale bar 100μm. Figure 14 The quantitative analysis of the CD86-positive region in the subchondral bone of the mouse knee joint proposed in this invention is shown in the attached figures from left to right, which are the results of Sham, DMM, DMM+UIO-66-NH2 and DMM+C-PANPs. Figure 15 Immunohistochemical staining of MMP13 in mouse knee joint cartilage tissue as proposed in this invention. The attached figures, from left to right, show the results of Sham, DMM, DMM+UIO-66-NH2, and DMM+C-PANPs; scale bar 100 μm. Figure 16The quantitative analysis of the MMP13 positive region in mouse knee cartilage tissue proposed in this invention is shown in the figure from left to right, which are the results of Sham, DMM, DMM+UIO-66-NH2 and DMM+C-PANPs. Figure 17 The attached figure is a top view of the mouse knee joint Micro-CT scan proposed in this invention. The figures from left to right are the results of Sham, DMM, DMM+UIO-66-NH2 and DMM+C-PANPs; scale bar 1 mm. Figure 18 The figures below, from left to right, show the results of Sham, DMM, DMM+UIO-66-NH2, and DMM+C-PANPs for quantitative analysis of mouse knee joint bone volume proposed in this invention. Detailed Implementation

[0021] The present invention will be further explained below with reference to specific embodiments. Example

[0022] Preparation of UIO-66-NH2: Dissolve 50 mg of dimethylsiloxane-polyoxy-polypropylene ether (F127) in 3 mL of deionized water, add 0.4 mL of acetic acid and 150 mg of NaClO4·H2O, stir thoroughly to form a homogeneous solution, add 115.6 mg of ZrO(NO3)2·2H2O and 50 mg of BDC-NH2 to the above mixture, stir magnetically at 40 °C for 12 hours, centrifuge the solution at 10000 rpm to obtain the precipitate, wash the precipitate once with deionized water and centrifuge again, resuspend the product in anhydrous ethanol and store it.

[0023] Preparation of UIO-66-NH2-encapsulated Chlorophyllin (C-NPs): 10 mg UIO-66-NH2 was mixed with 5 mL DI-water and 4 mg sodium copper chlorophyllin and stirred at room temperature for 24 h to obtain solution A; 1 mg / mL UIO-66-NH2 loaded with sodium copper chlorophyllin was mixed with 10 mmol / L Mal-PEG. 2000 The PBS solution of -NHS was reacted at room temperature (RT) for 4 hours. After 4 hours of reaction, it was centrifuged at 12000g for 10 minutes using an ultrafiltration tube and washed 3 times with PBS to remove unreacted Mal-PEG2000-NHS.

[0024] Preparation of PANPs: A PBS solution containing MOF-encapsulated sodium copper chlorophyllin, MMP9-responsive peptide, and bone-targeting peptide Asp8-SH in a molar ratio of 1:2:2 was reacted at room temperature (RT) for 4 hours. Afterward, the mixture was centrifuged at 12000g for 10 minutes using an ultrafiltration tube, and washed three times with PBS to remove unreacted MMP9-responsive peptide and bone-targeting peptide Asp8-SH. The construction process is as follows. Figure 1 As shown.

[0025] UIO-66-NH2 and C-PANPs were observed and photographed using an electron microscope. The results are as follows: Figure 2 As shown, the loose porous structure of UIO-66-NH2 and the porous crystal structure of C-PANPs filled with the drug can be observed. This porous crystal structure can improve the sustained-release performance of the drug.

[0026] The peaks of C-PANPs and the peaks of each component were detected by Fourier transform infrared spectroscopy, and the results are as follows: Figure 3 As shown, C-PANPs exhibit characteristic peaks for each component.

[0027] To verify the loading of Chlorophyllin and the conjugation of Asp8-SH and MMP9-SH, three different fluorescent dyes were used for labeling: CY3 for Asp8, CY5.5 for Chlorophyllin, and DIO-Green for MMP9. The results are as follows: Figure 4 As shown, the three dyes almost completely overlap, proving that Chlorophyllin was successfully loaded into UIO66-NH2, and Asp8-SH and MMP9-SH perfectly conjugated to both ends of UIO66-NH2.

[0028] The effects of different concentrations of UIO-66-NH2, Asp8-SH, MMP9-SH, and Chlorophyllin on the viability of primary macrophages were detected using a CCK8 assay. After 12 h of treatment, the toxic effects of the materials on primary macrophages were assessed. Some optimal concentrations were... Figure 5 As shown.

[0029] The particle size of UIO-66-NH2 and C-PANPs was measured. The diameter of UIO-66-NH2 was concentrated at 100 nm, while that of C-PANPs was concentrated at 110 nm. The results are as follows. Figure 6 As shown.

[0030] Histologically, DMM was induced in mice by inducing meniscus injury, and the mice were randomly divided into groups. Seven days after the operation, different treatment methods were administered to different groups. After 90 days of treatment, the joints of the mice were removed to further evaluate the effects of C-PANP nanoparticles on osteoarthritis.

[0031] To verify the retention time of C-PANPs in the joint cavity of mice, CY5.5-labeled Chlorophyllin was used. In vivo imaging was employed to observe the drug retention time in the joint cavity after tail vein injection of C-PANPs. The results are as follows: Figure 7 As shown, in vivo imaging revealed that, with the help of Asp8-SH, MMP9-SH, and UIO-66-NH2, C-PANPs could reach the inflamed knee joint and achieve a sustained release time of 7 days, thus maintaining drug concentration to a great extent and increasing the duration of drug action.

[0032] HE staining was performed on the joints of all euthanized mice to observe cartilage wear. In the sham-operated group, the cartilage structure was clear, with no depressions or cracks after modeling, a relatively smooth surface, and normal cartilage thickness. The effect of UIO-66-NH2 was almost invisible, and the cartilage tissue remained uneven. Due to modifications of UIO-66-NH2, Asp8-SH, and MMP9-SH, C-PANP injection achieved the best therapeutic effect. The cartilage structure was significantly similar to the sham-operated group, with no depressions or cracks, a relatively smooth surface, and normal cartilage thickness. These results are as follows: Figure 8 As shown in the figure. We used the OARSI Score as the scoring standard. The score of the sham surgery group was close to 0, while the scores of the DMM group and the DMM+UIO-66-NH2 group were significantly higher. The injection of C-PANPs achieved the best effect, and the score was significantly reduced. The results are as follows. Figure 9 As shown.

[0033] Trape tissue staining was performed on the subchondral bone of the knee joint of all euthanized mice, and the results are as follows: Figure 10 As shown, you can see the trap of the DMM group. + The number of osteoclasts increased significantly, indicating a significant effect of C-PANPs. The addition of C-PANPs significantly reduced trapping. + The number of osteoclasts; its quantitative analysis is as follows: Figure 11 As shown. In addition, CD31 / EMCN staining was also performed, and the results are as follows. Figure 12 As shown, CD31 of the DMM group can be seen. + EMCN + The number of cells increased significantly, demonstrating the remarkable effect of C-PANPs. The addition of C-PANPs significantly reduced CD31 levels. + EMCN + Cell count. CD86 staining was also performed, and the results are as follows. Figure 13 As shown, you can see CD86 of the DMM group. +The number of cells increased significantly, demonstrating the remarkable effect of C-PANPs. The addition of C-PANPs significantly reduced CD86 levels. + The number of cells; its quantification is as follows Figure 14 As shown. MMP13 staining was performed on the knee cartilage of all sacrificed mice, and the results are as follows. Figure 15 As shown, MMP13 of the DMM group can be seen. + The number of cells increased significantly, demonstrating the remarkable effect of C-PANPs. The addition of C-PANPs significantly reduced MMP13. + The number of cells; its quantification is as follows Figure 16 As shown.

[0034] The subchondral bone of the mice was also scanned, and the results were as follows: Figure 17 As shown, the subchondral bone structure in the sham-operated group was porous and loose, while the cartilage in the DMM group showed significant sclerosis, and the porous and loose structure disappeared. Furthermore, quantitative calculations revealed a significant reduction in bone volume in the DMM group. After the action of C-PANPs, the trabeculae were arranged in an orderly manner, and osteophytes were reduced, very similar to the sham-operated group. The above quantitative results are as follows: Figure 18 As shown.

[0035] C-PANPs can restore normal joint parameters and have a certain effect on subchondral bone. Therefore, they can be used as an effective method for preparing drugs to treat OA, which also shows that C-PANPs have a certain prospect in the clinical application of OA treatment.

[0036] This invention elucidates for the first time the role of Chlorophyllin in osteoarthritis. It explores the mechanism by which subchondral bone alleviates the progression of osteoarthritis and demonstrates that Chlorophyllin improves vascular endothelial cell invasion of the cartilage matrix by reducing subchondral bone osteoclast activation and reduces inflammatory factors eroding the cartilage matrix by alleviating subchondral bone macrophage polarization. Furthermore, this invention constructs a dual-targeting sustained-release MOF material, C-PANPs, loaded with Chlorophyllin to target the subchondral bone, achieving progress in alleviating osteoarthritis in a mouse DMM model. In this invention, UIO-66-NH2 modified with Asp8-SH and MMP9-SH exhibits inflammatory responsiveness and bone targeting. C-PANPs can be injected via the tail vein to the subchondral bone of the knee joint cavity. Chlorophyllin is effectively conjugated to the surface of the MOF material UIO-66-NH2. Due to the loose and porous natural structure of the MOF material, the Chlorophyllin inside UIO-66-NH2 can achieve long-term slow release, maintaining the local drug concentration. Furthermore, it exhibits good biocompatibility and has no impact after degradation, providing a good foundation for the therapeutic effect of C-PANPs.

[0037] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. The scope of this application is defined by the appended claims and their equivalents, all of which should be included within the protection scope of this application.

Claims

1. The use of sodium copper chlorophyllin in the preparation of a drug for treating osteoarthritis.

2. The use of sodium copper chlorophyllin according to claim 1 in the preparation of a medicament for treating osteoarthritis, characterized in that, The sodium copper chlorophyllin is encapsulated in MOF material to inhibit the expression of transcription factor PU.

1.

3. The use of sodium copper chlorophyllin according to claim 2 in the preparation of a medicament for treating osteoarthritis, characterized in that, The MOF material is UIO-66-NH2.

4. The use of sodium copper chlorophyllin according to claim 1 in the preparation of a medicament for treating osteoarthritis, characterized in that, The dosage form of the drug is any pharmacologically acceptable dosage form.

5. The use of sodium copper chlorophyllin according to claim 1 in the preparation of a medicament for treating osteoarthritis, characterized in that, The dosage of the drug is any pharmacologically acceptable dosage.

6. A method for preparing a dual-targeted sustained-release drug delivery system, the drug delivery system being used to prepare a drug for treating osteoarthritis, characterized in that, The steps are as follows: S1: UIO-66-NH2, sodium copper chlorophyllin, and MAL-PEG are added. 2000 - NHS and deionized water were stirred at room temperature for 22-26 h to obtain sodium copper chlorophyllin encapsulated in MOF material; S2: The sodium copper chlorophyllin encapsulated in the MOF material prepared in S1 was magnetically stirred with the MMP9 responsive peptide and the bone-targeting peptide Asp8-SH, and a drug delivery system was obtained through maleimide-thiol click chemistry reaction.

7. The method for preparing the dual-targeted sustained-release drug delivery system according to claim 6, characterized in that, S1 contains UIO-66-NH2, sodium copper chlorophyllin, and MAL-PEG. 2000 The molar ratio of -NHS is 1-1.5:1:15-20.

8. The method for preparing the dual-targeted sustained-release drug delivery system according to claim 6, characterized in that, In S2, the molar ratio of sodium copper chlorophyllin, MMP9 responsive peptide, and bone-targeting peptide Asp8-SH encapsulated by MOF material is 1:1-3:1-3.

9. The method for preparing the dual-targeted sustained-release drug delivery system according to claim 6, characterized in that, The reaction conditions in S2 are 3-5 hours at room temperature.

10. A dual-targeted sustained-release drug delivery system, characterized in that, It is prepared by the preparation method according to any one of claims 6-8.

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