Preparation method of black phosphorus loaded sacubitril valsartan and application thereof in treatment of myocardial fibrosis

Abstract

This invention relates to the field of biomaterials technology, specifically to a method for preparing sacubitril / valsartan loaded with black phosphorus and its application in the treatment of myocardial fibrosis. A black phosphorus-loaded sacubitril / valsartan comprises a black phosphorus nanosheet as a carrier and sacubitril / valsartan loaded on the black phosphorus nanosheet. This invention utilizes the unique structural characteristics and good biocompatibility of black phosphorus nanosheets to load the drug sacubitril / valsartan, achieving a sustained-release effect. In vivo, it can degrade into phosphate (PO4) ions. 3‑ ) and phosphate (HPO3) 2‑ The black phosphorus nanosheets contain small molecules such as sacubitril and valsartan, exhibiting good biodegradability. Simultaneously, the drug-loaded black phosphorus nanosheets possess a certain ability to scavenge reactive oxygen species (ROS), enabling them to release ROS-responsive drugs in the pathological microenvironment following myocardial infarction and slowing the development of myocardial fibrosis. This invention provides an effective therapeutic strategy for inhibiting fibrosis by loading sacubitril and valsartan onto black phosphorus nanosheets through non-covalent bonding.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a method for preparing black phosphorus-loaded sacubitril / valsartan and its application in the treatment of myocardial fibrosis. Background Technology

[0002] Cardiovascular disease has become a major threat to human health and safety, with myocardial infarction being one of the leading causes of death worldwide. Myocardial infarction is usually caused by coronary artery occlusion, leading to the death of a large number of myocardial cells and severe inflammation. Post-infarction myocardial fibrosis is a key pathological process following ischemic injury to the heart. When the heart suffers ischemia, mechanical injury, or inflammation, fibroblasts are activated and secrete large amounts of extracellular matrix. Excessive deposition of extracellular matrix and collagen ultimately leads to changes in myocardial structure and function. Although fibrosis initially helps maintain the integrity of the heart structure and reduces the risk of cardiac rupture, excessive fibrosis can lead to pathological cardiac remodeling and impair the heart's contractile ability, causing serious complications such as heart failure and arrhythmias that endanger life.

[0003] Current clinical treatments for myocardial infarction include: medication, coronary artery bypass surgery, and stent placement. These methods only restore blood supply to the infarcted area. While these treatments can slow the progression of heart failure to some extent, they cannot prevent excessive cardiac fibrosis or reverse cardiac remodeling. Therefore, finding effective strategies to inhibit or reverse cardiac fibrosis is crucial for improving the prognosis and quality of life of heart disease patients. Summary of the Invention

[0004] The purpose of this application is to provide a method for preparing sacubitril / valsartan loaded with black phosphorus and its application in the treatment of myocardial fibrosis. This invention combines the antifibrotic drug sacubitril / valsartan with black phosphorus, which has a high drug loading rate, to promote cellular uptake while delaying drug release and exerting a sustained drug effect.

[0005] To achieve the above objectives, this application provides the following technical solution: A black phosphorus-loaded sacubitril / valsartan comprising a black phosphorus nanosheet carrier and sacubitril / valsartan loaded on the black phosphorus nanosheet.

[0006] This invention provides a black phosphorus-loaded sacubitril / valsartan for inhibiting myocardial fibrosis and reversing ventricular remodeling after myocardial infarction. Sacubitril / valsartan (LCZ696), a novel angiotensin receptor neprilysin inhibitor, reduces the degradation of natriuretic peptides by inhibiting neprilysin, thereby increasing natriuretic peptide concentration, dilating blood vessels, lowering blood pressure, inhibiting myocardial hypertrophy, inhibiting the release of renin and aldosterone, reducing cardiac preload and afterload, improving ventricular remodeling, and exerting effects such as fibrosis prevention, anti-inflammation, and improvement of vascular endothelial function. Black phosphorus nanosheets (BP), as a novel two-dimensional nanomaterial, show broad application prospects in multiple fields due to their unique physical and chemical properties. Black phosphorus nanosheets have a high specific surface area and a unique wrinkled structure, which enables them to efficiently adsorb and load drug molecules. Simultaneously, under physiological conditions, black phosphorus nanosheets can degrade into non-toxic phosphate ions (PO4). 3- It exhibits low cytotoxicity and good biocompatibility, making it suitable as a drug carrier for targeted drug delivery. The black phosphorus nanosheets of this invention are loaded with sacubitril and valsartan via non-covalent bonding.

[0007] Preferably, the black phosphorus nanosheets have a particle size of 500nm±50nm and a thickness of 2 to 10 layers; the purity of the sacubitril / valsartan is >99%.

[0008] More preferably, the black phosphorus-loaded cubitril / valsartan contains carbon, oxygen, and phosphorus, and the black phosphorus-loaded cubitril / valsartan has a characteristic absorption peak at 252 nm.

[0009] A method for preparing black phosphorus-loaded sacubitril / valsartan as described above includes the following steps: mixing black phosphorus nanosheets with sacubitril / valsartan, centrifuging, and washing to obtain the final product.

[0010] Preferably, at room temperature, black phosphorus nanosheets and sacubitril / valsartan are dissolved in deionized water, stirred and mixed in a light-protected environment, and then centrifuged. The precipitate after the first centrifugation is washed with deionized water and centrifuged a second time to obtain the final product.

[0011] More preferably, the mass ratio of the black phosphorus nanosheets to sacubitril / valsartan is 1:2.5 to 20.

[0012] More preferably, the stirring speed is 400 rpm, and the speed of the first centrifugation and the second centrifugation are both 10000 g.

[0013] The use of black phosphorus-loaded sacubitril / valsartan as described above in the preparation of drugs for treating, inhibiting or reversing myocardial fibrosis.

[0014] Beneficial effects: This invention utilizes the unique structural characteristics and excellent biocompatibility of black phosphorus nanosheets to load the drug sacubitril / valsartan for sustained release, which can be degraded into phosphate (PO4) in vivo. 3- ) and phosphate (HPO3) 2- The black phosphorus nanosheets contain small molecules such as sacubitril and valsartan, exhibiting good biodegradability. Simultaneously, the drug-loaded black phosphorus nanosheets possess a certain ability to scavenge reactive oxygen species (ROS), enabling them to release ROS-responsive drugs in the pathological microenvironment following myocardial infarction and slowing the development of myocardial fibrosis. This invention provides an effective therapeutic strategy for inhibiting fibrosis by loading sacubitril and valsartan onto black phosphorus nanosheets through non-covalent bonding. Attached Figure Description

[0015] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. Wherein: Figure 1 The energy dispersive spectroscopy (EDS) diagram of LCZ696 loaded with black phosphorus in Experiment Example 1 is shown. Figure 2 The UV absorption spectrum of LCZ696 loaded with black phosphorus in Experiment Example 2; Figure 3 The Fourier transform infrared spectrum of LCZ696 loaded with black phosphorus in Experiment Example 3; Figure 4 This is an immunofluorescence staining image of Col-I, a marker protein of fibrosis, from Experiment Example 4. Figure 5 This is a quantitative immunofluorescence graph of the fibrosis marker protein Col-I from Experiment Example 4; Figure 6 Immunofluorescence staining image of Col-III, a fibrosis marker protein, in Experiment Example 4; Figure 7 This is a quantitative immunofluorescence graph of the fibrosis marker protein Col-III in Experiment Example 4; Figure 8 Immunofluorescence staining image of α-SMA, a fibrosis marker protein, in Experiment Example 4; Figure 9 This is a quantitative immunofluorescence graph of the fibrosis marker protein α-SMA in Experiment Example 4. Detailed Implementation

[0016] The present application will now be described in detail with reference to the accompanying drawings and embodiments. Various examples are provided by way of explanation and not by way of limitation. In fact, those skilled in the art will understand that modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, a feature shown or described as part of one embodiment may be used in another embodiment to produce yet another embodiment. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention should fall within the scope of protection of the embodiments of the present invention.

[0017] Examples 1-5 A method for preparing black phosphorus-loaded sacubitril / valsartan includes the following steps: (1) Mix 50 μg of black phosphorus nanosheets with sacubitril and valsartan at a feed ratio of 1:10.

[0018] (2) Black phosphorus nanosheets and sacubitril valsartan were added to deionized water at different feed ratios and stirred in the dark for 24 hours. The stirring speed was 400 rpm. After stirring, the mixture was centrifuged at 10000g for 20 min. Then, the supernatant was removed, and the precipitate after centrifugation was washed with deionized water and centrifuged three times to remove the sacubitril valsartan that was not loaded with black phosphorus nanosheets, thus obtaining black phosphorus-loaded sacubitril valsartan (BP@LCZ696). After washing and centrifugation, BP@LCZ696 was resuspended in 1 mL of deionized water for later use.

[0019] 1. Characterization methods (1) In order to evaluate whether LCZ696 is loaded on black phosphorus nanosheets, energy dispersive spectroscopy analysis was performed on BP@LCZ696 prepared in step 2 above.

[0020] First, the BP@LCZ696 obtained by centrifugation was dried in a freeze dryer for 24 hours to remove surface moisture and ensure that the surface of the BP@LCZ696 sample was clean and flat. Then, the content and distribution of each element in the sample were detected by energy dispersive spectroscopy (EDS) to determine whether LCZ696 was successfully loaded onto black phosphorus.

[0021] The dried sample was placed on the sample stage and fixed with conductive adhesive. The types of elements contained in samples BP, LCZ696 and BP@LCZ696 were compared by energy dispersive spectroscopy.

[0022] (2) The absorbance of sample BP@LCZ696 was measured using a UV spectrophotometer. Sample BP@LCZ696 was dissolved in deionized water to prepare a solution of appropriate concentration, which was then placed in a quartz cuvette. The cuvette was filled with deionized water and placed in the instrument to calibrate the absorbance. The sample to be tested was placed in the cuvette, and the absorbance at a wavelength of 252 nm was measured. The absorbance at corresponding wavelengths of samples BP, LCZ696, and BP@LCZ696 were compared.

[0023] (3) The characteristic peaks of sample BP@LCZ696 were analyzed by Fourier transform infrared spectroscopy to determine the loading of LCZ696. After drying, sample BP@LCZ696 was ground into fine powder and mixed with potassium bromide (KBr) at a ratio of 1:100. The mixture was then pressed into transparent sheets on a tablet press. Before testing the samples, background correction was required. A blank sheet was used as the background, and the background spectrum was recorded. The prepared sample was placed on the sample holder, ensuring that the sample surface was flat and free of contamination. The position of the sample holder was adjusted so that the sample was located at the center of the beam, ensuring that the beam could uniformly irradiate the sample. The scanning range was set to 4000-400 cm⁻¹. -1 The resolution is typically 4 cm. -1 Record the infrared spectrum of the sample.

[0024] (4) To investigate the inhibitory effect of BP@LCZ696 on in vitro myocardial fibrosis, primary rat myocardial fibroblasts in good growth condition were used at 2 x 10⁶ cells / plate. 4 Cells were seeded at a density of [number] cells in confocal dishes and cultured for 24 hours before being divided into four groups: The control group, model group, LCZ696 group, and BP@LCZ696 group were divided into four groups. The control group was treated with DMEM containing 1% FBS, the model group was treated with DMEM containing 10 ng / mL TGF-β (recombinant protein) and 1% FBS, the LCZ696 group was treated with DMEM containing LCZ696 and TGF-β and 1% FBS, and the BP@LCZ696 group was treated with DMEM containing BP@LCZ696 and TGF-β and 1% FBS. After being cultured in a cell culture incubator for 24 hours, the original culture medium was discarded, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde. α-smooth muscle actin (α-SMA) is a common feature of myofibroblasts and ECM proteins such as type I / III collagen (Col-I / III) are associated with fibrosis. The expression of these proteins was assessed by immunofluorescence staining.

[0025] 2. Results and Analysis (1) Energy dispersive spectroscopy analysis to determine whether LCZ696 is loaded on black phosphorus nanosheets Based on energy dispersive spectroscopy analysis, it was determined whether LCZ696 was successfully loaded onto black phosphorus nanosheets. Figure 1 As shown, the pure black phosphorus nanosheet group (BP group) contains only phosphorus, the pure drug group (LCZ696 group) contains carbon and oxygen, and the black phosphorus nanosheet drug-loaded group (BP@LCZ696 group) contains carbon, oxygen and phosphorus. Therefore, energy dispersive spectroscopy analysis proves that LCZ696 was successfully loaded onto black phosphorus nanosheets.

[0026] (2) Ultraviolet spectrophotometer analysis The wavelengths of the BP group, LCZ696 group, and BP@LCZ696 group were detected by ultraviolet light, respectively. Figure 2 As shown, the LCZ696 group has a characteristic absorption peak at 252 nm, and the BP@LCZ696 group also shows a characteristic absorption peak of the LCZ696 group at 252 nm. Therefore, the UV analysis proves that LCZ696 was successfully loaded onto black phosphorus nanosheets.

[0027] (3) Fourier transform infrared spectroscopy analysis of drug loaded on black phosphorus nanosheets Fourier transform infrared spectroscopy comparison of characteristic peaks of the BP group, LCZ696 group and BP@LCZ696 group, such as Figure 3 As shown, the BP@LCZ696 group exhibits characteristic absorption peaks of both the LCZ696 group and the BP group, proving that LCZ696 was successfully loaded onto black phosphorus nanosheets.

[0028] (4) Immunofluorescence staining to detect the inhibition of fibrosis-related protein expression by BP@LCZ696 The characteristic proteins Col-I, Col-III, and α-SMA secreted by activated myocardial fibroblasts were used as evaluation indicators to assess whether BP@LCZ696 could inhibit myocardial fibrosis. Figure 4-9 As shown, weak characteristic fluorescence signals were detected in the untreated control group of cardiac fibroblasts. Conversely, strong fluorescence was observed in the TGF-β-treated Model group, indicating the production of large amounts of α-SMA, Col-I, and Col-III. The fluorescence signals in the LCZ696 group and the BP@LCZ696 group were reduced to varying degrees. Furthermore, quantitative analysis of the fluorescence intensity of α-SMA, Col-I, and Col-III, which are involved in the fibrosis process, indicated that BP@LCZ696 has a certain inhibitory effect on myocardial fibrosis.

[0029] The above experiments demonstrate that BP@LCZ696 has an anti-myocardial fibrosis effect and has the potential to be developed into an anti-myocardial fibrosis drug.

[0030] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0031] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A black phosphorus-loaded sacubitril / valsartan, characterized in that, The invention comprises black phosphorus nanosheets as a carrier and sacubitril / valsartan loaded on the black phosphorus nanosheets. The black phosphorus nanosheets have a particle size of 500 nm ± 50 nm and a thickness of 2 to 10 layers. The purity of the sacubitril / valsartan is >99%, and the mass ratio of the black phosphorus nanosheets used to prepare the black phosphorus-loaded sacubitril / valsartan to the sacubitril / valsartan is 1:2.5 to 20.

2. The black phosphorus-loaded sacubitril / valsartan according to claim 1, characterized in that, The black phosphorus-loaded cubitril / valsartan contains carbon, oxygen, and phosphorus, and has a characteristic absorption peak at 252 nm.

3. A method for preparing black phosphorus-loaded sacubitril / valsartan as described in claim 1 or 2, characterized in that, Includes the following steps: The black phosphorus nanosheets were mixed with sacubitril and valsartan, centrifuged, and washed to obtain the final product.

4. The method for preparing black phosphorus-loaded sacubitril / valsartan according to claim 3, characterized in that, At room temperature, black phosphorus nanosheets and sacubitril / valsartan were dissolved in deionized water and stirred and mixed in a light-protected environment. After mixing, the mixture was centrifuged. The precipitate after the first centrifugation was washed with deionized water and centrifuged a second time to obtain the final product.

5. The method for preparing black phosphorus-loaded sacubitril / valsartan according to claim 4, characterized in that, The stirring speed is 400 rpm, and the speed of the first and second centrifugation is 10000 g.

6. The use of the black phosphorus-loaded sacubitril / valsartan as described in claim 1 or 2 in the preparation of a drug for treating, inhibiting or reversing myocardial fibrosis.

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