Nanoparticles for treating knee joint cartilage degeneration and preparation method thereof

Abstract

The invention discloses a nano particle for treating knee joint cartilage degeneration and a preparation method thereof, and relates to the technical field of knee joint cartilage degeneration inhibition, the nano particle is Ce (at) DTP NPs, and is obtained by assembling deferasirox, cerium chloride and pterostilbene, and the nano particle for treating knee joint cartilage degeneration and the preparation method thereof have the advantages that the nano particle can be used for treating knee joint cartilage degeneration; when the nanoparticles are applied to treatment of knee joint cartilage degeneration, drug toxicity and singleness are reduced, drug resistance possibility is reduced, the nanoparticles can inhibit cell ferroptosis in specific tissues, repair of cartilage cells can be promoted by using the nanoparticles, metabolic imbalance of extracellular matrixes can be corrected, and the nanoparticles have an ROS response function and can be used for treating knee joint cartilage degeneration. Conditional release can be achieved in a specific environment, the functions of the nanoparticles can still be kept after the nanoparticles are repeatedly frozen and thawed, and the nanoparticles have the advantages in the aspects of storage, transportation and use.

Description

Technical Field

[0001] The present invention relates to the technical field of inhibiting knee cartilage degeneration, and in particular to a nanoparticle for treating knee cartilage degeneration and a preparation method thereof. Background Art

[0002] Knee cartilage degeneration is an age-related disease. Currently, there is no effective and thorough treatment plan, and the only way to alleviate clinical symptoms is through medication. Its pathogenesis is related to multiple factors, such as joint load, trauma, etc. In the degenerated articular cartilage tissue, it can be observed that chondrocyte activity decreases or dies, and the content of collagen in the extracellular matrix decreases.

[0003] Ferroptosis is a new type of cell death that is involved in promoting a variety of diseases. Its mechanism is mainly that under the action of divalent iron and esteroxygenase, intracellular ROS catalyzes the oxidation of highly expressed unsaturated fatty acids on the cell membrane, causing lipid peroxidation and accumulation in the form of lipid ROS, thereby inducing cell death. In addition, it is also manifested as a decrease in the expression of GPX4, the core enzyme regulating the antioxidant system (glutathione system). Regulation of the above targets can promote or inhibit the occurrence of ferroptosis, but previous basic research often only focuses on one of the targets and lacks a holistic perspective. By simultaneously regulating multiple targets to inhibit ferroptosis, the efficacy can be better achieved and drug resistance can be reduced through synergistic effects.

[0004] There is now research evidence that chondrocyte ferroptosis is an important phenotype in knee cartilage degeneration. When cartilage degenerates, intracellular ROS is pathologically upregulated, and the sensitivity of chondrocyte ferroptosis increases significantly. In addition, in order to be able to selectively release in degenerated cells, responsive release under specific conditions should be considered. Currently, there is no clinical drug that inhibits ferroptosis to treat knee cartilage lesions. Therefore, a nanoparticle for treating knee cartilage degeneration and a preparation method thereof are proposed. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a nanoparticle for treating knee cartilage degeneration and a preparation method thereof, which solves the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: a nanoparticle for treating knee cartilage degeneration, wherein the nanoparticle is Ce@DTP NPs, which is obtained by assembling deferasirox, cerium chloride and pterostilbene.

[0007] A method for preparing nanoparticles for treating knee cartilage degeneration comprises the following steps:

[0008] Step 1: Connection of pterostilbene to ROS response bond: 40 μmol (10 mg) of pterostilbene was dissolved in 1 ml of dichloromethane (DCM), excess chloroformate and diisopropylethylamine (DIPEA) were added to the solution, 2,2'-(propane-2,2-diylbis(thiodiyl))diethanol was added to the DCM mixed solution, stirred at room temperature for a certain period of time, and then triethylamine (TEA) was added to react to form a connection product;

[0009] Step 2: Introduction of Deferasirox: 40 umol (15 mg) of Deferasirox was dissolved in DCM, and amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA were added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of Deferasirox;

[0010] Step 3: DCM was evaporated using a rotary evaporator to leave a nanoparticle suspension, which was centrifuged in a centrifuge to collect the nanoparticles. The washing process was repeated three times to remove excess PVA.

[0011] Step 4: Collect the washed nanoparticles, freeze-dry or dry at room temperature, prepare a 10 mM cerium chloride (CeCl3) solution, dissolve it in ultrapure water, resuspend the dried nanoparticles in the cerium chloride solution, and stir at room temperature for a period of time;

[0012] Step 5: Centrifuge the incubated suspension in a centrifuge, remove the supernatant, wash the nanoparticles three times with ultrapure water to remove unbound cerium ions, collect the washed nanoparticles again, and dry them.

[0013] Optionally, the step 2: introduction of deferasirox: 40 umol (15 mg) of deferasirox is dissolved in DCM, and amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA are added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of deferasirox. The mixed solution is reacted at room temperature for 12-16 hours.

[0014] Optionally, the step 1: connection of pterostilbene to ROS response bond: 40 μmol (10 mg) of pterostilbene is dissolved in 1 ml of dichloromethane (DCM), excess chloroformate and diisopropylethylamine (DIPEA) are added to the solution, 2,2'-(propane-2,2-diylbis(sulfadiyl))diethanol is added to the DCM mixed solution, stirred at room temperature for a certain time, and then triethylamine (TEA) is added to react. The stirring reaction time at room temperature in the process of forming a connection product is 4-6 hours.

[0015] Optionally, the step four: collect the washed nanoparticles, freeze-dry or dry them at room temperature, prepare a 10mM cerium chloride (CeCl3) solution, dissolve it in ultrapure water, resuspend the dried nanoparticles in the cerium chloride solution, and stir gently at room temperature for 2-4 hours.

[0016] Optionally, the step five is: centrifuging the incubated suspension in a centrifuge, removing the supernatant, washing the nanoparticles three times with ultrapure water to remove unbound cerium ions, collecting the washed nanoparticles again, and centrifuging the incubated suspension in a centrifuge at 10,000 rpm for 10 minutes in the drying process.

[0017] Optionally, the step five is: centrifuging the incubated suspension in a centrifuge, removing the supernatant, washing the nanoparticles three times with ultrapure water to remove unbound cerium ions, and collecting the washed nanoparticles again. There are two ways to dry the washed nanoparticles in the drying process, namely, freeze-drying and room temperature drying.

[0018] The present invention provides a nanoparticle for treating knee cartilage degeneration and a preparation method thereof, which has the following beneficial effects:

[0019] The nanoparticles for treating knee cartilage degeneration and the preparation method thereof, use the nanoparticles in the treatment of knee cartilage degeneration to reduce drug toxicity and monotony, reduce the possibility of drug resistance, the nanoparticles can inhibit cell ferroptosis in specific tissues, use of the nanoparticles can promote chondrocytes and repair, correct the metabolic imbalance of the extracellular matrix, the nanoparticles have ROS response function, can achieve conditional release under specific conditions, the nanoparticles can still maintain their functions after repeated freezing and thawing, and have advantages in storage, transportation and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The transmission electron microscope, particle size, Zeta potential and Tyndall effect test results of the present invention are shown in the figure;

[0021] Figure 2 It is one of the structural representation diagrams of the compound of the present invention;

[0022] Figure 3 This is the second structural representation diagram of the compound of the present invention;

[0023] Figure 4 This is the third structural representation diagram of the compound of the present invention;

[0024] Figure 5 This is the fourth structural representation diagram of the compound of the present invention;

[0025] Figure 6This is the fifth structural representation diagram of the compound of the present invention;

[0026] Figure 7 This is the sixth structural characterization diagram of the compound of the present invention;

[0027] Figure 8 This is the seventh structural representation diagram of the compound of the present invention;

[0028] Fig. 9 This is a working state diagram of the ROS response key of the present invention;

[0029] Fig.10 This is a flow cytometry result diagram of the present invention;

[0030] Fig.11 It is a protein immunoblot and quantitative indication diagram of the present invention;

[0031] Fig.12 The protein immunoblot and quantitative graph of the present invention are shown;

[0032] Fig.13 This is the mouse experiment of the present invention and the Micro-CT three-dimensional reconstruction image;

[0033] Fig.14 It is a flow chart of the present invention;

[0034] Fig.15 This is a composition diagram of the nanoparticles of the present invention. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0036] See also Fig.15 The present invention provides a technical solution: a nanoparticle for treating knee cartilage degeneration, the nanoparticle is Ce@DTP NPs, which is obtained by assembling deferasirox, cerium chloride and pterostilbene. Deferasirox is a clear iron chelator that has passed FDA certification and can chelate powerful iron ions in cells. Cerium ions have ROS enzyme activity and can catalyze the decomposition of ROS in cells. Pterostilbene has multiple antioxidant functions and can balance and repair the extracellular matrix of cartilage cells.

[0037] A method for preparing nanoparticles for treating knee cartilage degeneration comprises the following steps:

[0038] Step 1: Connection of pterostilbene to ROS response bond: 40 μmol (10 mg) of pterostilbene was dissolved in 1 ml of dichloromethane (DCM), excess chloroformate and diisopropylethylamine (DIPEA) were added to the solution, 2,2'-(propane-2,2-diylbis(thiodiyl))diethanol was added to the DCM mixed solution, stirred at room temperature for a certain period of time, and then triethylamine (TEA) was added to react to form a connection product;

[0039] Step 2: Introduction of Deferasirox: 40 umol (15 mg) of Deferasirox was dissolved in DCM, and amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA were added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of Deferasirox;

[0040] Step 3: DCM was evaporated using a rotary evaporator to leave a nanoparticle suspension, which was centrifuged in a centrifuge to collect the nanoparticles. The washing process was repeated three times to remove excess PVA.

[0041] Step 4: Collect the washed nanoparticles, freeze-dry or dry at room temperature, prepare a 10 mM cerium chloride (CeCl3) solution, dissolve it in ultrapure water, resuspend the dried nanoparticles in the cerium chloride solution, and stir at room temperature for a period of time;

[0042] Step 5: Centrifuge the incubated suspension in a centrifuge, remove the supernatant, wash the nanoparticles three times with ultrapure water to remove unbound cerium ions, collect the washed nanoparticles again, and dry them.

[0043] Furthermore, those skilled in the art will know that step 2: introduction of deferasirox: 40 umol (15 mg) of deferasirox is dissolved in DCM, amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA are added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of deferasirox. The mixed solution is reacted at room temperature for 12-16 hours.

[0044] Further, those skilled in the art can know that step 1: connection of pterostilbene to ROS response bond: 40 μmol (10 mg) of pterostilbene is dissolved in 1 ml of dichloromethane (DCM), excess chloroformate and diisopropylethylamine (DIPEA) are added to the solution, 2,2'-(propane-2,2-diylbis(thiodiyl))diethanol is added to the DCM mixed solution, stirred at room temperature for a certain time, and then triethylamine (TEA) is added to react. The stirring reaction time at room temperature in the process of forming a connection product is 4-6 hours.

[0045] Furthermore, those skilled in the art will know that step four: collect the washed nanoparticles, freeze-dry or dry at room temperature, prepare a 10 mM cerium chloride (CeCl3) solution, dissolve it with ultrapure water, re-suspend the dried nanoparticles in the cerium chloride solution, and stir at room temperature for a period of time, and then gently stir at room temperature for 2-4 hours.

[0046] Furthermore, those skilled in the art will appreciate that, in step five, the incubated suspension is centrifuged in a centrifuge, the supernatant is removed, the nanoparticles are washed three times with ultrapure water to remove unbound cerium ions, the washed nanoparticles are collected again, and the incubated suspension is centrifuged in a centrifuge at 10,000 rpm for 10 minutes in the drying process.

[0047] Furthermore, those skilled in the art will know that step five: centrifuging the incubated suspension in a centrifuge, removing the supernatant, washing the nanoparticles three times with ultrapure water to remove unbound cerium ions, and collecting the washed nanoparticles again. There are two ways to dry the washed nanoparticles in the drying process, namely, freeze-drying and room temperature drying.

[0048] Performance testing of the obtained Ce@DTP NPs:

[0049] S1: Structural characterization of Ce@DTP NPs.

[0050] The prepared Ce@DTP NPs were tested by transmission electron microscopy, particle size, Zeta potential and Tyndall effect. The experimental results are shown in Figure 1 The prepared Ce@DTP NPs have regular spherical morphology, uniform particle size, and a peak value of particle size distribution at about 104.9 nm. They have a significant Tyndall effect. 4+ During the process, the potential reverses, which indicates that Ce 4+ be effectively carried;

[0051] In order to clarify the synthetic product, the product and intermediate product were characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. The final product was characterized by hydrogen nuclear magnetic resonance and carbon-13 nuclear magnetic resonance to provide a more comprehensive characterization of the compound structure. The results are as follows Figure 2 - Figure 8 As shown;

[0052] S2: Verify the stability of Ce@DTP NPs and the working status of ROS response key;

[0053] In order to verify the working status of the ROS response bond, different concentrations of ROS mimics (H2O2) were added to the Ce@DTP NPs solution, such as Fig. 9As shown, the results indicate that with the addition of H2O2, the content of free PTE in the solution increased significantly, while no obvious change in PTE content was found in the group without the addition of ROS mimics within 24 hours, which means that in a ROS-free environment, Ce@DTP NPs can maintain good stability, but in a high ROS environment, Ce@DTP NPs can respond and release quickly.

[0054] S3: Ce@DTP NPs can improve ferroptosis in multiple ways;

[0055] like Fig.10 and Fig.11 As shown in the figure, the results of ROS fluorescence probes indicate that Ce@DTP NPs can significantly reduce the ROS content in chondrocytes. The results of flow cytometry showed that Ce@DTP NPs can improve cell apoptosis. Protein immunoblotting and its quantification indicated that Ce@DTP NPs significantly increased the expression of the antioxidant index GPX4 and reduced the expression of ferroptosis-promoting factors such as ACSL4 and P53. The polymerase chain reaction obtained the same trend at the gene level.

[0056] S4: Ce@DTP NPs improve the metabolism of cartilage extracellular matrix in in vitro experiments;

[0057] like Fig.12 As shown in the figure, protein immunoblotting and its quantification revealed that Ce@DTP NPs significantly increased the expression of matrix proteins (Col2 and Aggrecan) and decreased the expression of matrix metalloproteinases (MMP13 and ADAMTS5). The polymerase chain reaction obtained the same trend at the gene level.

[0058] S5: Ce@DTP NPs repair cartilage in knee joint degeneration model mice;

[0059] In order to verify the repair effect of Ce@DTP NPs on knee cartilage degeneration in vivo, we established a mouse model by destabilization of the medial meniscus (DMM). Fig.13 As shown in the figure, the quantitative index used the number of osteophytes, and further pathological examination of the knee joint by H&E staining and immunohistochemistry showed that the expression of Col2 decreased in the model group and rebounded after Ce@DTP NPs treatment, while MMP13 showed the opposite trend. This means that Ce@DTP NPs played a clear role in repair, such as Fig.14 shown.

[0060] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A nanoparticle for treating knee cartilage degeneration, characterized in that: The nanoparticles are Ce@DTP NPs, which are obtained by assembling deferasirox, cerium chloride and pterostilbene.

2. A method for preparing nanoparticles for treating knee cartilage degeneration, characterized in that: The following steps are involved: Step 1: Connection of pterostilbene to ROS response bond: 40 μmol (10 mg) of pterostilbene was dissolved in 1 ml of dichloromethane (DCM), excess chloroformate and diisopropylethylamine (DIPEA) were added to the solution, 2,2'-(propane-2,2-diylbis(thiodiyl))diethanol was added to the DCM mixed solution, stirred at room temperature for a certain period of time, and then triethylamine (TEA) was added to react to form a connection product; Step 2: Introduction of Deferasirox: 40 umol (15 mg) of Deferasirox was dissolved in DCM, and amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA were added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of Deferasirox; Step 3: Use a rotary evaporator to evaporate the DCM, leaving a nanoparticle suspension, centrifuge the suspension in a centrifuge to collect the nanoparticles, and repeat the washing process 3 times to remove excess PVA; Step 4: Collect the washed nanoparticles, freeze-dry or dry at room temperature, prepare a 10 mM cerium chloride (CeCl3) solution, dissolve it in ultrapure water, resuspend the dried nanoparticles in the cerium chloride solution, and stir at room temperature for a period of time; Step 5: Centrifuge the incubated suspension in a centrifuge, remove the supernatant, wash the nanoparticles three times with ultrapure water to remove unbound cerium ions, collect the washed nanoparticles again, and dry them.

3. The nanoparticles for treating knee cartilage degeneration and the preparation method thereof according to claim 2, characterized in that: The second step: introduction of deferasirox: 40 umol (15 mg) of deferasirox was dissolved in DCM, and amino protecting agents N-Boc-ethylenediamine, EDCI, N-hydroxysuccinimide (NHS) and TEA were added, reacted at room temperature for a period of time, and deprotected using trifluoroacetic acid (TFA) to obtain the active form of deferasirox. The mixed solution was reacted at room temperature for 12-16 hours.

4. The nanoparticles for treating knee cartilage degeneration and the preparation method thereof according to claim 2, characterized in that: The step 1: connecting pterostilbene to the ROS response bond: dissolving 40 μmol (10 mg) of pterostilbene in 1 ml of dichloromethane (DCM), adding excess chloroformate and diisopropylethylamine (DIPEA) to the solution, adding 2,2'-(propane-2,2-diylbis(thiodiyl))diethanol to the DCM mixed solution, stirring at room temperature for a certain time, and then adding triethylamine (TEA) to react, and the stirring reaction time at room temperature in the process of forming the connection product is 4-6 hours.

5. The nanoparticles for treating knee cartilage degeneration and the preparation method thereof according to claim 2, characterized in that: The fourth step is to collect the washed nanoparticles, freeze-dry or dry them at room temperature, prepare a 10 mM cerium chloride (CeCl3) solution, dissolve it in ultrapure water, re-suspend the dried nanoparticles in the cerium chloride solution, and stir at room temperature for a period of time, and gently stir at room temperature for 2-4 hours.

6. The nanoparticles for treating knee cartilage degeneration and the preparation method thereof according to claim 2, characterized in that: The fifth step is to centrifuge the incubated suspension in a centrifuge, remove the supernatant, wash the nanoparticles three times with ultrapure water to remove unbound cerium ions, collect the washed nanoparticles again, and perform a drying process in which the incubated suspension is centrifuged in a centrifuge at 10,000 rpm for 10 minutes.

7. The nanoparticles for treating knee cartilage degeneration and the preparation method thereof according to claim 2, characterized in that: The step five is: centrifuging the incubated suspension in a centrifuge, removing the supernatant, washing the nanoparticles three times with ultrapure water to remove unbound cerium ions, collecting the washed nanoparticles again, and drying the washed nanoparticles in two ways, namely, freeze drying and room temperature drying.

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