Chiral metal-organic framework material and preparation method and application thereof

Abstract

The application relates to a chiral metal organic framework material and a preparation method and application thereof, relates to the technical field of medical nanomaterials, and solves the problems of insufficient target specificity and poor stability of existing Parkinson's disease treatment drugs. ZrOCl2.8H2O, Cu-TCCP and benzoic acid are dissolved in DMF, a cysteine aqueous solution is added, reaction is carried out at 30-70 DEG C, then the temperature is increased to 80-120 DEG C for reaction; the precipitate is collected by centrifugation, the precipitate is washed by DMF and ultrapure water and centrifuged, and the chiral metal organic framework material is obtained. The material has good stability, high biocompatibility, simple preparation, low cost, easy enlargement and industrialized production, overcomes the defects of poor stability, low bioavailability and strong non-specificity of traditional alpha-syn inhibitors, and provides a new strategy and high-efficiency candidate material for early intervention and treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of medical nanomaterials technology, specifically to a chiral metal-organic framework material, its preparation method, and its application. Background Technology

[0002] Parkinson's disease is a common neurodegenerative disease that causes progressive dysfunction in the midbrain and basal ganglia, leading to core motor symptoms such as bradykinesia, resting tremor, rigidity, and postural instability. Parkinson's disease has also become the fastest-growing neurological disease burden globally, with the number of patients worldwide projected to exceed 12 million by 2040.

[0003] The core pathological markers of Parkinson's disease are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the abnormal aggregation of α-synuclein (α-syn) forming Lewy bodies. Currently, the abnormal aggregation of α-syn and its pathological propagation in the central nervous system are considered the core mechanisms driving disease progression. This process can trigger neuroinflammation, mitochondrial dysfunction, and oxidative stress, ultimately leading to neuronal degeneration. In recent years, strategies aimed at inhibiting the aggregation and deaggregation of α-syn monomers into fibrils have become key research directions, hoping to delay or even reverse the pathological process at its source. In recent years, regulators targeting α-syn aggregation have emerged, such as small molecule compounds, peptides, antibodies, and natural extracts. However, these materials generally suffer from low bioavailability, insufficient targeting specificity, or poor stability, limiting their practical application in the treatment of Parkinson's disease.

[0004] Advances in nanomaterials have offered new possibilities for overcoming the aforementioned challenges. Benefiting from their tunable surface properties, ease of functionalization, and high specific surface area, nanomaterials have become a promising class of α-syn aggregation inhibitors, expected to achieve efficient, stable, and targeted regulatory effects. Among them, metal-organic frameworks (MOFs) are a class of porous crystalline nanomaterials formed by the self-assembly of inorganic metal centers and organic ligands through coordination bonds. They possess advantages such as tunable structure, high porosity, high modifiability, good biocompatibility, and outstanding chemical stability. Summary of the Invention

[0005] To address the issues of insufficient targeting specificity and poor stability of existing Parkinson's disease treatments, this invention proposes a chiral metal-organic framework material, its preparation method, and its applications.

[0006] The specific technical solution of the present invention is as follows:

[0007] A method for preparing a chiral metal-organic framework material includes the following steps:

[0008] ZrOCl2·8H2O, Cu-TCCP (copper porphyrin organic framework) and benzoic acid were dissolved in DMF, and cysteine ​​aqueous solution was added. The mixture was reacted at 30~70 ℃ for 1~5 h, and then the temperature was raised to 80~120 ℃ for 1~5 h.

[0009] After the reaction was completed, the precipitate was collected by centrifugation at 8000~10000 rpm for 5~10 min. The precipitate was then washed with DMF (N,N-dimethylformamide) and ultrapure water and centrifuged 3~6 times to obtain the chiral metal-organic framework material.

[0010] Preferably, the cysteine ​​is selected from cysteine ​​with L, D or DL ​​configurations.

[0011] More preferably, the cysteine ​​is L-configuration cysteine.

[0012] Preferably, the mass ratio of Cu-TCCP, ZrOCl2·8H2O and benzoic acid is 1:1~10:20~50.

[0013] Preferably, the final concentration of the cysteine ​​aqueous solution is 0.1~3 mg / mL.

[0014] Preferably, the Cu-TCCP is synthesized through the following steps:

[0015] Cu(NO3)2·3H2O, trichloroacetic acid and PVP were dissolved in a mixed solution of DMF and ethanol. A solution of DMF and ethanol containing dissolved TCCP (tetracarboxyphenylporphyrin) was added dropwise. The reaction solution was heated to 60~90 °C and reacted for 1~5 h.

[0016] After the reaction was completed, the product was centrifuged at 6000-9000 rpm for 5-10 min, collected, and washed 2-5 times with ethanol by centrifugation. The product was then dried to obtain solid Cu-TCCP.

[0017] Preferably, the mass ratio of Cu(NO3)2·3H2O, trichloroacetic acid, PVP and TCCP is 1:1~5:1~5:1~3.

[0018] Preferably, the ratio of the DMF and ethanol mixture is 1 to 5:1.

[0019] The present invention also provides a chiral metal-organic framework material, which is prepared by the above-described preparation method.

[0020] The present invention also provides an application of the above-mentioned chiral metal-organic framework material, specifically in the preparation of a drug for treating Parkinson's disease.

[0021] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0022] This invention enables the efficient synthesis of nanoscale chiral metal-organic frameworks using a mild solvothermal method. The prepared materials exhibit an approximately rice-grain-like morphology with good particle dispersibility, regular morphology, no obvious agglomeration, and uniform particle size. The materials demonstrate clear chiral characteristics, with L-PCN (L-cysteine ​​chiral metal-organic framework) and D-PCN (D-cysteine ​​chiral metal-organic framework) exhibiting typical mirror-symmetric chiral signals and good chiral enantiometry. Simultaneously, the crystal structure is complete, the characteristic diffraction peaks are sharp and free of impurities, and the structure has high stability, providing a reliable structural basis for biomedical applications.

[0023] The chiral materials of this invention can simultaneously inhibit α-synergistic aggregation and depolymerize existing α-synergistic fibers, synergistically reducing α-synergistic fiber accumulation from a two-dimensional perspective. This overcomes the limitations of traditional inhibitors that only have a single effect, enabling intervention at the pathological source of Parkinson's disease. The materials exhibit a significant chirality-dependent effect, with L-PCN showing significantly better performance than D-PCN and DL-PCN (DL-cysteine ​​chiral metal-organic framework materials) in inhibiting α-synergistic fibrosis, providing a new and preferred system for achieving precise anti-Parkinson's disease treatment.

[0024] The material of this invention has good stability, excellent water solubility, and high biocompatibility. Moreover, the preparation process is simple, the conditions are mild, and the cost is low. It is easy to scale up and industrialize. It overcomes the defects of traditional α-syn inhibitors, such as poor stability, low bioavailability, and strong non-specific effects. It provides a new strategy and efficient candidate material for the early intervention and precision treatment of Parkinson's disease. Attached Figure Description

[0025] Figure 1 Transmission electron microscopy (TEM) image of the chiral metal-organic framework material prepared in Example 1;

[0026] Figure 2 The circular dichroism spectrum of the chiral metal-organic framework material prepared in Example 1;

[0027] Figure 3 The X-ray diffraction pattern of the chiral metal-organic framework material prepared in Example 1;

[0028] Figure 4 The results of the thiosulfate T (ThT) fluorescence experiment of the chiral metal-organic framework material prepared in Example 1 are shown.

[0029] Figure 5 The results of the cytotoxicity test of the chiral metal-organic framework material prepared in Example 1;

[0030] Figure 6 The results are from animal experiments on the chiral metal-organic framework material prepared in Example 1. Detailed Implementation

[0031] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0032] Example 1.

[0033] This embodiment prepared chiral metal-organic framework nanomaterials through the following steps:

[0034] (1) Dissolve Cu(NO3)2·3H2O (9 mg), trichloroacetic acid (25 µL), and PVP (25 mg) in a 30 mL mixture of DMF and ethanol (V:V=3:1). Then, add TCCP (10 mg) dissolved in a 10 mL mixture of DMF and ethanol (V:V=3:1) dropwise with stirring, keeping the reaction solution transparent throughout the addition. After the addition is complete, sonicate the reaction solution for 10 min. Heat the reaction solution to 80 °C and maintain the reaction for 3 h. After the reaction is complete, collect the product by centrifugation at room temperature (8000 rpm, 10 min) and wash three times by centrifugation with ethanol (8000 rpm, 10 min). Dry the product in a 37 °C oven to obtain solid Cu-TCPP.

[0035] (2) Dissolve 90 mg ZrOCl2·8H2O, 30 mg Cu-TCCP, and 810 mg benzoic acid in 30 mL DMF, add 0.3 mL of ultrapure water, and sonicate until the reaction solution is clear and transparent. Divide the reaction solution into three equal portions, add 0.2 mL of L, D, and DL configuration cysteine ​​aqueous solutions (50 mg / mL) to each portion, and mix thoroughly. Then, place the solution at 60 ℃ and 200 rpm for 2 h, and then raise the temperature to 90 ℃ and continue the reaction for 2 h. After the reaction is completed, collect the product by centrifugation at room temperature (10000 rpm, 10 min), and centrifuge twice with DMF and ultrapure water (10000 rpm, 10 min each) and wash twice to obtain the product. The products are named L-PCN (L-cysteine ​​chiral metal-organic framework material), D-PCN (D-cysteine ​​chiral metal-organic framework material), and DL-PCN (DL-cysteine ​​chiral metal-organic framework material), respectively.

[0036] To investigate the morphology, chiral characteristics, and crystal structure of the prepared chiral metal-organic framework material, transmission electron microscopy (TEM), circular dichroism spectroscopy (CD), and X-ray diffraction (XRD) were performed on the product. The results are as follows:

[0037] Transmission electron microscopy observation results as follows Figure 1 As shown in the figure, the chiral metal-organic framework material synthesized by the above method exhibits an approximately rice-grain-like morphology, with good particle dispersion, regular morphology, no obvious agglomeration, and good particle size uniformity, with an average particle size of about 200 nm. This indicates that the preparation process can successfully prepare nanoscale chiral MOF materials.

[0038] Figure 2 The circular dichroism spectra of the three products are shown. The spectral curves clearly show that L-PCN and D-PCN exhibit a strict mirror symmetry relationship at their characteristic wavelengths. However, DL-PCN does not show a significant chiral absorption peak because the chiral signals of the two configurations cancel each other out. This indicates that by introducing cysteine ​​residues with different configurations, chiral information has been successfully and precisely imparted to the metal-organic framework.

[0039] Figure 3 The X-ray diffraction pattern of the product is shown. Comparison with the standard diffraction peaks of MOF materials reveals that the prepared L-PCN, D-PCN, and DL-PCN all exhibit characteristic diffraction peaks of MOF materials. Furthermore, the diffraction peaks are of high intensity and sharp shape, with no obvious impurity peaks. This indicates that regardless of whether L-, D-, or DL-type cysteine ​​is introduced, the crystal framework structure of the obtained product remains unchanged, maintaining a good crystal phase structure, thus providing structural stability for its subsequent applications.

[0040] Example 2.

[0041] To demonstrate that the chiral metal-organic framework obtained in Example 1 can inhibit α-syn aggregation and depolymerize the formed fibrous material, thereby synergistically reducing the accumulation of α-syn fibers from both the inhibition of aggregation and depolymerization dimensions, this example conducted a thiosulfate T (ThT) fluorescence experiment.

[0042] (1) α-syn aggregation inhibition experiment:

[0043] α-syn was dissolved in 50 mM phosphate-buffered saline (PBS, pH=7.4) to prepare a protein solution with a final concentration of 40 μM. The protein solution was aliquoted into centrifuge tubes, divided into a blank group (α-syn only) and an experimental group (α-syn + chiral metal-organic framework, final framework concentration 35 μg / mL). The tubes were placed in a 37°C water bath and stirred at 200 rpm. Samples were taken at different time points for later use.

[0044] (2) α-syn fiber depolymerization experiment:

[0045] The α-syn fibers, which had been pre-incubated in vitro, were diluted in 50 mM PBS buffer (pH=7.4) to prepare a fiber suspension with a final concentration of 30 μM. The suspension was aliquoted into centrifuge tubes, divided into a blank group (α-syn fibers only) and an experimental group (α-syn fibers + chiral metal-organic framework, with a final framework concentration of 100 μg / mL). The tubes were placed in a 37°C water bath and stirred at 200 rpm. Samples were taken at different time points for later use.

[0046] (3) ThT fluorescence detection:

[0047] The β-sheet structure formed by α-syn aggregation can specifically bind to ThT dye, significantly enhancing the fluorescence intensity of the system. This allows for the quantitative reflection of the aggregation rate and extent of α-syn and the depolymerization effect of fibers. Protein samples at each time point were mixed with ThT solution, and the final concentrations of ThT and α-syn were adjusted to 22.5 μM and 4 μM, respectively. After incubation at 37°C for 30 min, fluorescence detection was performed. Fluorescence spectroscopy parameters: excitation wavelength (Ex) 440 nm, emission wavelength (Em) 480 nm, and excitation and emission slit widths of 5 nm.

[0048] The results are as follows Figure 4 As shown, chiral metal-organic frameworks have a significant inhibitory effect on the aggregation of α-synuclein and can effectively promote the depolymerization of existing α-synuclein fibers. This effect is chiral dependent. In the aggregation inhibition experiment, L-PCN is significantly more effective than D-PCN and DL-PCN, and has a stronger ability to inhibit α-synuclein fibrillation.

[0049] Example 3.

[0050] To investigate whether the chiral metal-organic framework prepared in Example 1 can alleviate the neurotoxicity caused by the accumulation of α-syn fibrosis by regulating the α-syn fibrosis process, this example used SH-SY5Y neuroblastoma cells as a model and the MTT assay was used to detect cell viability.

[0051] Cell seeding and culture: SH-SY5Y cells were seeded at a rate of 1×10⁻⁶ cells / year. 4 The cells were seeded at a density of 1 cell per well in 96-well plates and incubated in a 5% CO2, 37°C incubator for 24 hours until the cells adhered to the walls and covered the bottom of the wells.

[0052] Treatment: The culture medium was changed, and the experimental groups were given 200 μL of α-syn monomer medium containing different configurations of chiral metal-organic frameworks, α-syn fiber (incubated in vitro for 48 h) medium, or an incubation mixture of α-syn fiber and chiral metal-organic frameworks, respectively; the blank control group was given 200 μL of DMEM basal medium. The mixture was incubated in the incubator for another 24 h.

[0053] MTT assay for cell viability: After incubation, the culture medium in the wells was aspirated and the cells were gently washed 2-3 times with PBS to remove the material. 100 μL of PBS solution containing 0.5 mg / mL MTT was added to each well and the cells were cultured for another 4 h. Then 100 μL of dimethyl sulfoxide (DMSO) was added and the cells were shaken at low speed for 10 min to fully dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader, and the cell viability was calculated.

[0054] Figure 5 Cell viability results showed that α-syn fibers significantly reduced the viability of SH-SY5Y cells, while intervention with chiral metal-organic frameworks effectively reversed this toxic effect. Among them, L-PCN, through its dual action of inhibiting α-syn aggregation and depolymerizing existing fibers, showed the most significant effect in alleviating α-syn fiber-induced neurotoxicity, far superior to D-PCN and DL-PCN.

[0055] Example 4.

[0056] To verify the in vivo therapeutic effect of the chiral metal-organic framework prepared in Example 1 on Parkinson's disease (PD), this example constructs a PD model of C57BL / 6J mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The therapeutic efficacy and biocompatibility are explored through weight monitoring and behavioral assessment.

[0057] (1) Construction of PD animal model:

[0058] Male C57BL / 6J mice weighing 23-25g were selected and a PD model was established by intraperitoneal injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The injection dose was 30mg / kg / day, and the injection was repeated for 5 consecutive days.

[0059] (2) Experimental grouping and dosing regimen:

[0060] Mice were randomly divided into 6 groups of 6 mice each, and the grouping and treatment were as follows:

[0061] Control group: Intraperitoneal injection of an equal volume of normal saline, without MPTP injection;

[0062] Model group: MPTP was injected intraperitoneally, and no other treatment drugs were given;

[0063] Sham surgery group: MPTP injected intraperitoneally, and saline injected into the brain stereotactically;

[0064] L-PCN treatment group: MPTP was injected intraperitoneally and L-PCN was injected stereotactically into the brain;

[0065] D-PCN treatment group: MPTP was injected intraperitoneally and D-PCN was injected stereotactically into the brain;

[0066] DL-PCN treatment group: MPTP was injected intraperitoneally, and DL-PCN was injected stereotactically into the brain.

[0067] During the experiment, mouse weight was recorded every 3 days to assess the biocompatibility of the material in vivo.

[0068] (3) Behavioral assessment:

[0069] Five days after treatment, the mice's spontaneous activity ability was assessed using an open field test. The experimental setup included an open field test chamber and an automated data acquisition and processing system. Before the formal test, the mice were placed in the test room for 30 minutes to acclimatize. Then, each mouse was placed in the center of the open field, and its free movement over 6 minutes was recorded using a digital camera. The total distance traveled and the average speed were analyzed as two key indicators. Each mouse was tested individually. During the intervals between tests, the inner walls and bottom of the test chamber were wiped with 75% ethanol to eliminate odor interference.

[0070] Figure 6 The results showed that there was no significant difference in body weight between the treatment groups and the control and sham-operated groups, indicating that chiral metal-organic frameworks have good biocompatibility in vivo. The total movement distance and average movement speed of mice in the model group were significantly shortened, while the L-PCN treatment group significantly improved MPTP-induced motor dysfunction in mice, and its effect was significantly better than that of the D-PCN and DL-PCN treatment groups.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a chiral metal-organic framework material, characterized in that, Includes the following steps: ZrOCl2·8H2O, Cu-TCCP and benzoic acid were dissolved in DMF, and an aqueous solution of L-configured cysteine ​​was added. The mixture was reacted at 30~70 ℃ for 1~5 h, and then the temperature was raised to 80~120 ℃ for 1~5 h. After the reaction was completed, the product was collected by centrifugation at 8000~10000 rpm for 5~10 min. The product was then washed with DMF and ultrapure water and centrifuged 3~6 times to obtain chiral metal-organic framework materials.

2. The method for preparing the chiral metal-organic framework material according to claim 1, characterized in that, The mass ratio of Cu-TCCP, ZrOCl2·8H2O and benzoic acid is 1:1~10:20~50.

3. The method for preparing the chiral metal-organic framework material according to claim 1, characterized in that, The final concentration of the cysteine ​​aqueous solution is 0.1~3 mg / mL.

4. The method for preparing the chiral metal-organic framework material according to claim 1, characterized in that, The Cu-TCCP is synthesized through the following steps: Cu(NO3)2·3H2O, trichloroacetic acid and PVP were dissolved in a mixed solution of DMF and ethanol. A solution of DMF and ethanol containing dissolved TCCP was added dropwise. The reaction solution was heated to 60~90 ℃ and reacted for 1~5 h. After the reaction was completed, the product was centrifuged at 6000-9000 rpm for 5-10 min, collected, and washed 2-5 times with ethanol by centrifugation. The product was then dried to obtain solid Cu-TCCP.

5. The method for preparing the chiral metal-organic framework material according to claim 4, characterized in that, The mass ratio of Cu(NO3)2·3H2O, trichloroacetic acid, PVP and TCCP is 1:1~5:1~5:1~3.

6. The method for preparing the chiral metal-organic framework material according to claim 4, characterized in that, The ratio of the DMF and ethanol mixture is 1 to 5:

1.

7. A chiral metal-organic framework material, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. An application of the chiral metal-organic framework material as described in claim 7, characterized in that, Preparation of drugs for the treatment of Parkinson's disease.

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