Construction of a bionic nano traditional Chinese medicine and its application in anti-pulmonary fibrosis

Abstract

Construction of a bionic nano traditional Chinese medicine and its application in anti-pulmonary fibrosis, belonging to the field of pharmaceutical preparations. In this invention, two natural products, formononetin (FMN) and baicalein (BE), are used as the preferred drug combination. The drug conjugate bFB bonded by borate ester bond is designed and synthesized by using phenylboronic acid-glycol coupling strategy. The drug conjugate bFB self-assembles in aqueous solution and undergoes biomimetic modification on it. The obtained bionic nano traditional Chinese medicine PbFB@PM actively targets the damaged site, accumulates, and then responds to the high ROS and acidic environment to rapidly release drugs, effectively scavenging free radicals, alleviating pulmonary oxidative stress, and inhibiting endothelial cell activation. PbFB@PM remodels the oxidative stress and angiogenesis pathological microenvironment in the lungs of mice with bleomycin (BLM)-induced pulmonary fibrosis (PF) model during the injury stage, prevents the progression of fibrosis, and prolongs the survival period of mice.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and particularly relates to the construction of a bionic nano traditional Chinese medicine and its application in anti-pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF), as a progressive interstitial lung disease, has a median survival of 2 - 4 years. There is a lack of available drug therapies. Most patients can only receive palliative treatment with two FDA-approved drugs, pirfenidone and nintedanib. Lung transplantation remains the only cure for end-stage patients. There is an urgent need to develop effective drug products to improve the clinical benefits of patients.

[0003] The development of PF treatment drugs is difficult due to the complex microenvironment and multiple pathological changes of the disease. As the main barrier against pathogen damage, macrophages resident in the lung are activated and release a large amount of toxic substances such as nitric oxide, peroxides, especially reactive oxygen species (ROS), which damage lung tissue, leading to oxidative stress reactions and aggravating biological damage. In addition, inflammatory exudation hinders gas exchange, resulting in local relative oxygen deficiency, and releases a large amount of pro-angiogenic factors. Along with the proliferation of endothelial cells, malformed alveolar capillaries are derived, providing necessary nutritional support for the proliferation of mesenchymal cells while also hindering the effective infiltration of therapeutic drugs. The dysregulation of angiogenesis continues to progress to the fibrotic proliferation stage, accompanied by poor prognosis. Single drug therapy cannot address all obstacles simultaneously, emphasizing the necessity of strategic design and rational drug selection to break the physical barrier and achieve the purpose of remodeling the pathological microenvironment at the same time.

[0004] Drug combination therapy provides a new idea. Effective synergy will significantly reduce the drug dosage, achieve the ideal therapeutic potency, and avoid damage to normal tissues at the same time. The rapid development of nanotechnology provides more opportunities for the rational design of advanced drug co-delivery formulations and corresponding targeting strategies. Self-assembled prodrug nanoformulations based on stimulus response, as a new paradigm of controllable drug delivery, have received extensive attention. By releasing the loaded drugs under specific stimulus conditions, more precise therapeutic effects can be achieved, reducing off-target side effects. Phenylboronic acid (PBA) and its derivatives are a class of artificial synthetic diol substance recognition bodies that can form covalent complexes with polyhydroxy compounds containing adjacent diol structures and have reversibility. Due to the drug release behavior in response to ROS and acidic environment, they have been widely studied in drug delivery. Summary of the Invention

[0005] The present invention provides a bionic nano traditional Chinese medicine capable of achieving multi-drug targeted delivery and release, and a construction method thereof, aiming to efficiently treat pulmonary fibrosis by regulating the pathological microenvironment.

[0006] The present invention is realized through the following technical solutions:

[0007] The present invention provides a bionic nano traditional Chinese medicine, PbFB@PM.

[0008] The bionic nano traditional Chinese medicine is prepared from a drug conjugate bFB and a platelet membrane (PM);

[0009] wherein, the drug conjugate bFB is a drug conjugate bFB prepared by using a ROS-responsive phenylboronic acid-glycol coupling strategy with two natural products, formononetin (FMN) and baicalein (BE), as the preferred drug combination.

[0010] The present invention provides a preparation route for the drug conjugate bFB, and specifically synthesizes the drug conjugate bFB by a three-step method:

[0011] i) In the medium of tetrahydrofuran, triethylamine and trifluoromethanesulfonyl chloride are added and reacted with FMN. After the reaction is completed, it is quenched and extracted to obtain an intermediate fsF;

[0012] ii) fsF is mixed with palladium acetate and bis(pinacolato)diboron in tetrahydrofuran and refluxed with stirring until the reaction is complete, then separated and purified to obtain an intermediate, and the intermediate is reacted with an aqueous hydrochloric acid solution to obtain borylated formononetin (bF);

[0013] iii) bF and BE are mixed in a DMSO solvent to form a drug conjugate bFB.

[0014] Further, in the step i), the molar ratio of FMN to trifluoromethanesulfonyl chloride is 4:5; in the step ii), the molar ratio of fsF:palladium acetate:bis(pinacolato)diboron = 20:1:10, and the concentration of the hydrochloric acid is 6 mol / L; in the step iii), the molar ratio of bF:BE = 1:1.

[0015] The present invention provides a preparation method for the bionic nano traditional Chinese medicine, and the specific steps are as follows:

[0016] 1) Prepare PbFB: A DMSO solution containing the drug conjugate bFB is dropped into an aqueous solution of polyvinylpyrrolidone (PVP), stirred, centrifuged, the supernatant is discarded, and the precipitate is collected. Subsequently, it is washed, freeze-dried, and weighed to obtain PbFB.

[0017] 2) Prepare the platelet membrane (PM): The platelet membrane (PM) is prepared by gradient centrifugation and liquid nitrogen freeze-thaw method.

[0018] 3) Prepare the bionic nano traditional Chinese medicine (PbFB@PM): The PbFB suspension is mixed with the PM suspension, and passed through 1 μm, 400 nm, and 200 nm polycarbonate porous membranes in a liposome extruder to obtain the bionic nano traditional Chinese medicine PbFB@PM.

[0019] The obtained bionic nano traditional Chinese medicine PbFB@PM targets to the inflammatory lesion site, remodels the pathological microenvironment in the injury stage, and delays the progression of fibrosis.

[0020] Further, in the step 1), the mass ratio of the drug conjugate bFB to PVP is W bFB / W PVP = 3:1, and the obtained PbFB exists in the form of nanocrystals;

[0021] Further, in the step 3), the PbFB suspension is a suspension obtained by mixing PbFB with water, and the concentration is 3.5 mg / mL; the PM suspension is a suspension obtained by mixing PM with water, and the concentration is 1 mg / mL of membrane protein.

[0022] The present invention provides the application of the bionic nano traditional Chinese medicine in the preparation of drugs for treating pulmonary fibrosis.

[0023] The present invention provides a pharmaceutical composition, comprising the bionic nano traditional Chinese medicine and pharmaceutically acceptable excipients.

[0024] The present invention provides the application of the pharmaceutical composition in the preparation of drugs for treating pulmonary fibrosis.

[0025] The beneficial effects of the present invention:

[0026] The present invention adopts a responsive phenylboronic acid - diol coupling strategy, uses borated formononetin and baicalein as raw materials, and synthesizes a drug conjugate bFB with dual ROS and pH responsive functions through a one-step method, which can ensure that the drug conjugate bFB releases the original drug molecules FMN and BE in an inflammatory environment with high ROS and an acidic environment.

[0027] The present invention adopts an anti-solvent precipitation method to induce the drug conjugate bFB to self-assemble into dense nanocrystals PbFB in an aqueous solution in a specific ratio by using intermolecular hydrophobic interactions and π-π stacking interactions, significantly improving the drug loading content.

[0028] The present invention adopts an extrusion method to prepare the bionic nano traditional Chinese medicine PbFB@PM coated with PM, retains the function of platelet-related membrane proteins, improves the in vivo stability of the nano traditional Chinese medicine, and endows the damaged inflammatory site with active targeting ability, avoiding the rapid clearance of small molecules in the body and systemic side effects.

[0029] The present invention describes the preparation method of the bionic nano traditional Chinese medicine, as well as the related preparation characterization, in vivo targeting evaluation and pharmacodynamic evaluation.

[0030] The present invention proves the feasibility of achieving pulmonary targeted drug delivery in vivo by tail vein injection of the bionic nano traditional Chinese medicine by virtue of the inflammatory tropism of PM through in vivo distribution experiments.

[0031] The present invention proves through immunofluorescence experiments on frozen sections of lung tissues that the bionic nano traditional Chinese medicine effectively scavenges ROS at the lesion site, alleviates pulmonary oxidative stress, inhibits endothelial cell activation, and effectively regulates the oxidative stress / angiogenesis pathological microenvironment.

[0032] The present invention proves through comprehensive pharmacodynamic experiments that the bionic nano traditional Chinese medicine significantly improves the treatment effect of pulmonary fibrosis, effectively inhibits pulmonary inflammation and collagen deposition, and significantly prolongs the survival period of mice. Brief Description of the Drawings

[0033] Figure 1 It is the structural formula of the synthesis of the drug conjugate bFB and its important intermediates.

[0034] Figure 2 It is the characterization of the bionic nano traditional Chinese medicine prepared in Example 1:

[0035] Figure A is the particle size distribution diagram and photo of PbFB and PbFB@PM;

[0036] Figure B is the quantitative diagram of the hydrodynamic diameter and Zeta potential of PM, PbFB, and PbFB@PM;

[0037] Figure C is the SDS-PAGE picture of PbFB, PM, and PbFB@PM;

[0038] Figure D is the ultraviolet-visible absorption spectrum indicating the scavenging of free radicals by bF, PbFB, and PbFB@PM in the methylene blue solution system.

[0039] Figure 3 It is the in vivo targeting result of the bionic nano traditional Chinese medicine in Example 2:

[0040] Figure A is the fluorescence change image of DID in the main organs of the model mice in vivo after treatment with DID-PbFB and DID-PbFB@PM;

[0041] Figure B is the fluorescence quantitative analysis of DID in the main organs of the model mice in vivo after treatment with DID-PbFB and DID-PbFB@PM.

[0042] Figure 4 It is the result of the bionic nano traditional Chinese medicine regulating the oxidative stress / angiogenesis pathological microenvironment in Example 3:

[0043] Figure A is the qualitative result of the ROS level in the lung tissue of the model mice after treatment;

[0044] Figure B is the quantitative analysis result of the MDA level in the lung tissue of the model mice after treatment;

[0045] Figure C is the qualitative analysis result of the VEGF level in the lung tissue of the model mice after treatment;

[0046] Figure D shows the semi - quantitative analysis results in the lung tissue of model mice after treatment.

[0047] Figure 5 This is the pharmacodynamic evaluation of the bionic nano - traditional Chinese medicine for anti - pulmonary fibrosis in Example 4:

[0048] Figure A shows the hematoxylin - eosin (H&E) staining of the lung tissues of mice in the sham - operation group, model group, and PbFB@PM treatment group;

[0049] Figure B shows the Masson staining of the lung tissues of mice in the sham - operation group, model group, and PbFB@PM treatment group;

[0050] Figure C shows the body weight change curves of mice in the sham - operation group, model group, and PbFB@PM treatment group;

[0051] Figure D shows the survival curve analysis of mice in the sham - operation group, model group, and PbFB@PM treatment group within 40 days. Detailed implementation manners

[0052] The following further illustrates the solutions in the present invention through examples. The examples shall not be construed as limiting the present invention. Unless otherwise specified, the experimental operations in the examples all adopt the mature existing technologies in the biological field or chemical field, and the raw materials and reagents used can all be obtained through commercial purchase.

[0053] The mice used in the present invention are male C57BL / 6 mice, which are well - recognized in the art and obtained through commercial purchase. The bleomycin (BLM) - induced mouse PF model selected is a classic modeling method.

[0054] Example 1: Preparation and characterization of bionic nano - traditional Chinese medicine

[0055] Adopt Figure 1 the shown preparation route. First, in the medium of tetrahydrofuran, add triethylamine and trifluoromethanesulfonyl chloride to react with FMN, stir at room temperature for 2 h. After the reaction is completed, quench and extract to obtain the intermediate fsF (yield 80%); add palladium acetate and bis(pinacolato)diboron to fsF, stir and reflux for 2 hours to obtain C 22 H 23 BO5 with a yield of 44%. After hydrochloric acid acidification, borated formononetin (bF) is obtained. Then, bF and BE are complexed in DMSO in a 1:1 equimolar ratio to form the drug conjugate bFB. Then, PbFB is prepared by the anti - solvent precipitation method. First, weigh 4 mg of polyvinylpyrrolidone (PVP) and dissolve it in 2 mL of ddH2O and place it in a 10 - mL vial, stir at room temperature, and then drop the DMSO solution containing bFB into the PVP aqueous phase (W bFB / W PVP= 3:1), stir at room temperature for 2 h, then centrifuge to discard the supernatant and collect the precipitate. Repeat centrifugation to wash the preparation 3 times in ddH2O to remove DMSO and unloaded drugs, and then lyophilize and weigh to obtain PbFB nanocrystals, which are stored at 4 °C for later use. The preparation method of DiD-labeled PbFB is to dissolve DiD and the drug conjugate bFB in DMSO solution and slowly add it dropwise to the PVP aqueous phase. The remaining preparation steps are the same as above to obtain DiD-labeled PbFB nanocrystals. After extracting mouse platelets by gradient centrifugation, freeze-thaw them repeatedly in liquid nitrogen 7 times, centrifuge at 10000×g for 10 min, and discard the supernatant. The precipitate is the platelet membrane (PM). Mix 100 μL of PbFB suspension (3.5 mg / mL) with 150 μL of PM suspension (1 mg / mL membrane protein), and use a liposome extruder to pass through 1 μm, 400 nm, and 200 nm polycarbonate porous membranes at room temperature to obtain the biomimetic nano traditional Chinese medicine PbFB@PM.

[0056] The results are as Figure 2 shown. The results show that the formed nanocrystals PbFB have a high drug loading rate and encapsulation rate, which are 81.6% and 95.42% respectively. PbFB@PM appears as a light yellow liquid and has an obvious Tyndall effect under laser irradiation. The particle size of PbFB@PM is 88.5 ± 6.3 nm, and the size distribution is uniform, increasing by about 20 nm compared with PbFB. The potential of PbFB@PM is -27.3 mV, showing no significant difference from PM. The SDS-PAGE analysis results show no significant difference in the protein bands between PbFB@PM and PM, indicating that PbFB@PM completely retains the protein characteristics of PM. In addition, the phenylboronic acid or phenylboronic acid ester structure can undergo an oxidative addition reaction with ROS (including superoxide anion, hydrogen peroxide, ·OH, etc.). Based on the methylene blue (MB) fading experiment, it was found that bF, PbFB, or PbFB@PM (40 μg / mL) can all quickly restore the color of the MB indicator by reacting with ·OH, indicating that PbFB@PM has a certain ROS depletion ability.

[0057] Example 2: In vivo targeting results of biomimetic nano traditional Chinese medicine

[0058] Male C57 mice at 6 to 8 weeks old were used to establish a bleomycin (BLM)-induced pulmonary fibrosis model. One day before the surgery, the hair on the throat, lungs, and abdomen of the mice was removed with depilatory cream. A BLM solution of 1 mg / mL prepared with PBS was placed on ice for standby. After the mice were anesthetized, tracheotomy was performed, and 50 μL of the BLM solution was instilled into the trachea using a micro-liquid atomization device. Twenty-four hours after the surgery, the model mice were randomly divided into two groups, and DiD-PbFB and DiD-PbFB@PM (DiD 1 mg / kg) were injected into the tail vein, respectively. The fluorescence distribution in the mice was collected at 1, 2, 4, 8, 12, and 24 h through a small animal in vivo imaging system (PerkinElmer, USA). After the last imaging time point, the main organs such as the liver, heart, spleen, lungs, and kidneys of each group of mice were taken out for ex vivo imaging analysis.

[0059] The results showed that: as Figure 3 shown, in the figure ** P < 0.01. After the PF model mice were injected with DiD-PbFB@PM via the tail vein, the fluorescence signal in the lung tissue gradually increased with time, reached the peak at 12 h, and still had strong accumulation 24 h later. Although DiD-PbFB had a certain accumulation in the lungs, it mainly accumulated and was cleared in the liver. The enhanced fluorescence of DiD-PbFB@PM in the lungs in the imaging results of the main organs (liver, heart, spleen, lungs, and kidneys) further verified the good targeting of PbFB@PM to the lung tissue of the model mice. The above results indicated that PbFB@PM achieved targeted delivery to the lung injury site by virtue of the inflammatory targeting of the platelet membrane and long blood circulation.

[0060] Example 3: Results of bionic nano traditional Chinese medicine regulating oxidative stress / angiogenesis pathological microenvironment

[0061] The PF model mice were randomly divided into 6 groups with 5 mice in each group. PBS, FMN, BE, PbFB, PbFB@PM (10 mg / kg) were injected into the tail vein (i.v.) respectively, and the sham operation group was used as a control. Twenty-four hours after the sixth administration, that is, on the 7th day of the injury stage, the mice were sacrificed and lung tissue samples were collected. After preparing frozen sections from the middle lobe of the left lung, the staining area was circled with an immunohistochemical pen, and DCFH-DA staining solution (20 μmol / L) was used as a ROS indicator probe or Anti-VEGFA and Alexa Immunofluorescence staining was performed with 647 antibody, and 4% paraformaldehyde solution was added dropwise for fixation in the dark at room temperature for 20 min. Subsequently, Hoechst 33258 staining solution was added dropwise to the staining area and incubated at room temperature for 7 min. After sealing the slices with immunofluorescence mounting medium, the slices were placed under a laser confocal microscope for imaging analysis. An equal amount of cryopreserved lung tissue was weighed and added to 1 mL of PBS solution, and the sample was crushed in a tissue homogenizer. After high-speed centrifugation for 10 min, the supernatant was collected. The MDA detection working solution was prepared according to the instructions of the MDA detection kit, and the absorbance values of each sample at 532 nm were detected with an enzyme-linked immunosorbent assay (ELISA) reader.

[0062] The results showed that, as Figure 4 shown, in the figure *** P < 0.001, the DCFH fluorescence signal was significantly enhanced in the lungs of BLM-induced PF model mice. PbFB and PbFB@PM could effectively weaken the DCFH fluorescence signal, and the PbFB@PM group had the weakest fluorescence signal, showing the best ROS scavenging ability. Lipid oxidation often occurs during the process of oxidative stress. Therefore, the MDA content was detected as an index to evaluate oxidative stress. The MDA content in the lungs of model mice increased to 1.5 times that of the Sham group, while PbFB and PbFB@PM could significantly decrease the MDA content, and there was no significant difference between the PbFB@PM group and the Sham group. VEGF has the functions of promoting the proliferation and differentiation of endothelial cells and is one of the important cytokines involved in the repair of vascular endothelial cells and angiogenesis. Through immunofluorescence staining experiments, it was observed that the VEGF fluorescence signal was significantly enhanced in the lungs of BLM-induced PF model mice. The nanocrystal groups (PbFB and PbFB@PM) and the single-drug groups (FMN and BE) could all reduce the VEGF fluorescence signal to a certain extent. Among them, the PbFB@PM group had the weakest fluorescence signal, showing the best ability to inhibit VEGF. The above results indicate that PbFB@PM can effectively regulate the oxidative stress / angiogenesis pathological microenvironment at the injury stage.

[0063] Example 4: Pharmacodynamic evaluation of bionic nano traditional Chinese medicine against pulmonary fibrosis

[0064] To evaluate the efficacy of bionic nano traditional Chinese medicine in anti-pulmonary fibrosis, we conducted a detailed study on the therapeutic effect of PbFB@PM in a BLM-induced PF mouse model. On the first day after modeling, the mice were randomly divided into three groups of 13 mice each. PBS or PbFB@PM (10 mg / kg) was injected into the tail vein for 6 consecutive days. The sham operation group was injected with 50 μL PBS solution into the trachea as a control. During the treatment period, the activity status of each group of mice was recorded and the body weight change of the mice was recorded every 3 days. On the 21st day, 5 mice from each group were sacrificed to collect lung tissues, and the middle lobe of the left lung was fixed with 4% paraformaldehyde solution for 48 h, and then paraffin sections were prepared and stained with H&E and Masson. The obtained sections were observed under a whole slide scanner. The remaining 8 mice were continuously cultured and observed until 40 days after modeling, and the survival period was statistically analyzed.

[0065] The results showed that as Figure 5 shown, in the figure *** P<0.001. In the pathological sections, inflammatory damage pathological changes such as severe alveolar hemorrhage, edema, alveolar septum thickening and inflammatory cell infiltration were observed in the lung tissues stimulated by BLM, and a large amount of collagen deposition was present in the lung interstitium. However, no obvious inflammatory pathological changes were found in the PbFB@PM group, no obvious collagen deposition was seen, and the structure of the lung interstitium and alveolar tissue were both intact, showing no obvious changes compared with the Sham group. During the monitoring of the survival period, the body weight of the model mice decreased rapidly due to impaired lung function and all died at 35 days. There was no significant difference in the body weight change between the PbFB@PM group and the Sham group, and only 1 mouse died at the end of the survival period monitoring, with the survival rate significantly increased to 87.5%. The above results indicate that PbFB@PM can exert an effective anti-pulmonary fibrosis therapeutic effect and can be used as a potential new anti-pulmonary fibrosis drug.

Claims

1. A bionic nano traditional Chinese medicine, characterized in that, The bionic nano traditional Chinese medicine, hereinafter referred to as PbFB@PM, is prepared from the drug conjugate bFB, polyvinylpyrrolidone PVP and platelet membrane; The drug conjugate bFB is prepared by the following method: i) In the medium of tetrahydrofuran, triethylamine and trifluoromethanesulfonyl chloride are added and reacted with formononetin. After the reaction is completed, it is quenched and extracted to obtain the intermediate fsF; ii) fsF is mixed with palladium acetate and bis(pinacolato)diboron in tetrahydrofuran and refluxed with stirring until the reaction is complete. After separation and purification, the intermediate is obtained, and the intermediate is reacted with hydrochloric acid aqueous solution to obtain boric acid formononetin; iii) Boric acid formononetin and baicalein are mixed in DMSO solvent to form the drug conjugate bFB; The preparation method of PbFB@PM specifically adopts the following steps: 1) Preparation of PbFB: The DMSO solution containing the drug conjugate bFB is dropped into the aqueous solution of polyvinylpyrrolidone PVP, stirred, centrifuged, and the supernatant is discarded to collect the precipitate. Subsequently, it is washed, freeze-dried, and weighed to obtain PbFB; 2) Preparation of platelet membrane, PM: The platelet membrane, PM, is prepared by gradient centrifugation and liquid nitrogen freeze-thaw method; 3) Preparation of bionic nano traditional Chinese medicine: The PbFB suspension is mixed with the PM suspension, and passed through 1 μm, 400 nm, and 200 nm polycarbonate porous membranes in a liposome extruder to obtain the bionic nano traditional Chinese medicine.

2. The bionic nano traditional Chinese medicine according to claim 1, wherein In step 1) of the preparation method of the PbFB@PM, the mass ratio of the drug conjugate bFB to polyvinylpyrrolidone is W bFB :W PVP = 3:1; The obtained PbFB exists in the form of nanocrystals.

3. The bionic nano traditional Chinese medicine according to claim 1, wherein, In step 3) of the preparation method of PbFB@PM, the PbFB suspension is a suspension obtained by mixing PbFB and water, with a concentration of 3.5 mg / mL; the PM suspension is a suspension obtained by mixing PM and water, with a concentration of 1 mg / mL of membrane protein.

4. Use of the bionic nano traditional Chinese medicine according to claim 1 in the preparation of a drug for treating pulmonary fibrosis.

5. A pharmaceutical composition comprising the bionic nano traditional Chinese medicine according to claim 1 and pharmaceutically acceptable excipients.

6. Use of the pharmaceutical composition according to claim 5 in the preparation of a drug for treating pulmonary fibrosis.

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