Traditional Chinese medicine monomer nanoparticle oral delivery system based on intestinal liver circulation and preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine monomer nanoparticle oral delivery system based on intestinal liver circulation and a preparation method thereof, spherical nanoparticles (SA NPs) with the particle size of 194 + / -20 nm are formed through self-assembly of threat resin acid (SA), and the limitation of a traditional nano carrier is broken through: the oral bioavailability reaches 34.7% by utilizing an ASBT-mediated intestinal liver circulation mechanism; the gastric acid environment is tolerated (the pH is stable at 1.5), and after penetrating through the intestinal epithelium, the drug is targeted to ischemic brain tissues; the medicine has carrier-medicine dual functions, and the cerebral infarction volume is reduced by 34.1% when the medicine is singly used; after the neuroprotective peptide NA1 is loaded (the drug loading capacity is 5.8 wt%), the infarct volume is reduced by 68.7% through synergism. The system is suitable for pre-hospital first aid of cerebral apoplexy, and the freeze-dried powder can be stored at normal temperature for 12 months. The delivery system has a great application prospect in preparation of drugs for treating acute ischemic stroke needing rapid brain targeting.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to an oral delivery system for traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation and its preparation method. Background Technology

[0002] Most traditional Chinese medicine monomers suffer from significant first-pass metabolism in the liver, low intestinal absorption, and poor chemical stability, resulting in oral bioavailability generally below 10%. Traditional formulations (tablets / capsules) struggle to overcome the dual degradation caused by the intestinal mucosal barrier and liver metabolism, limiting their clinical application value. However, some natural small molecules possess modifiable groups, multiple action sites, hydrophobic side chains, and rigid frameworks, along with self-assembly properties. Therefore, they can be used to construct self-assembled nanoparticles (NPs), which exhibit superior therapeutic effects compared to their monomeric components. Currently, no research has been published on sumaresinolic acid nanoparticles (SA NPs).

[0003] While existing NPs (such as liposomes and polymer micelles) can improve drug solubility, they are difficult to overcome a series of physiological barriers in the gastrointestinal tract, including the highly acidic environment, enzymatic degradation, and intestinal epithelium. Moreover, the passive targeting mechanism leads to the capture of NPs by the reticuloendothelial system.

[0004] Therefore, it is necessary to develop a method for preparing an oral delivery system of traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation to meet the timeliness requirements of emergency treatment for acute ischemic stroke (AIS). Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an oral delivery system of Chinese medicine monomer nanoparticles based on enterohepatic circulation. SA NPs are stable in a strongly acidic environment simulating the stomach and can be rapidly entered into the bloodstream through enterohepatic circulation to target ischemic brain tissue, meeting the timeliness requirements of AIS emergency treatment.

[0006] To achieve the aforementioned objective, the present invention adopts the following technical solution:

[0007] In a first aspect of the invention, an oral delivery system for traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation is provided, the delivery system comprising spherical nanoparticles formed by the self-assembly of the traditional Chinese medicine monomer sumaric acid.

[0008] Furthermore, the particle size of the nanoparticles is 194±20 nm.

[0009] In a second aspect of the present invention, a method for preparing the oral delivery system of traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation is provided, the method comprising:

[0010] Sumatran resin acid was dissolved in acetone to form an organic phase, which was then added dropwise to a first aqueous solution of polyvinyl alcohol to obtain an emulsion.

[0011] The emulsion was subjected to ultrasonic treatment in an ice bath and then added to an aqueous solution of the second polyvinyl alcohol, stirred, and centrifuged.

[0012] Nanoparticles were obtained by collecting the heart, which is the oral delivery system of Chinese medicine monomer nanoparticles based on enterohepatic circulation.

[0013] In a preferred embodiment, the mass concentration of the first polyvinyl alcohol aqueous solution is 2%-3%. If the concentration is too high, the emulsion viscosity will be too high, affecting the dispersibility of nanoparticles; if the concentration is too low, the emulsion interface will not be stable, resulting in uneven particle size.

[0014] In a preferred embodiment, the mass concentration of the second polyvinyl alcohol aqueous solution is 0.2%-0.4%. When used to dilute the primary emulsion, excessively high concentrations may encapsulate residual PVA, increasing the risk of nanoparticle toxicity; excessively low concentrations may not effectively prevent nanoparticle aggregation.

[0015] In a preferred embodiment, the mass-to-volume ratio of sumaric acid (SA) dissolved in acetone is 4-8 mg / mL. The solubility of sumaric acid (SA) in acetone affects the drug loading efficiency of the nanoparticles. At this mass-to-volume ratio, complete self-assembly is possible.

[0016] In a third aspect of the invention, a composite nanoparticle is provided, which is obtained by loading a hydrophobic drug into the oral delivery system of traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation.

[0017] Furthermore, the hydrophobic drug includes IR780, IR676, Cy5.5, coumarin 6, and protective peptide NA1.

[0018] IR780, a fat-soluble cyanocyanine dye, is commercially available from Sigma-Aldrich under the code [IR780 iodide, 1154472-49-7], with a purity >95%.

[0019] IR676, with a structure similar to IR780, has excitation / emission wavelengths of 676 / 689 nm and is commercially available from Lumiprobe under catalog number [B-3337].

[0020] Cy5.5, a sulfonated cyanocyanine dye, with excitation / emission wavelengths of 675 / 694 nm, is commercially available from Cytiva (product number PA15401).

[0021] Coumarin 6, a green fluorescent probe (excitation / emission: 466 / 504 nm), is a commonly used cellular uptake tracer with logP=5.8. It is commercially available from Sigma-Aldrich under catalog number [442631].

[0022] NA1 (trade name Tat-NR2B9c), amino acid sequence: YGRKKRRQRRR (Tat transmembrane peptide)-KLSSIESDV (PSD-95 inhibitory peptide), target: blocking PSD-95 / nNOS protein interaction, inhibiting excitotoxicity, commercially available, such as MedChemExpress catalog number [HY-P0273A].

[0023] In a fourth aspect of the invention, a method for preparing the aforementioned composite nanoparticles is provided, the method comprising:

[0024] Sumatramic acid and a hydrophobic drug are dissolved together in acetone to form an organic phase, which is then added dropwise to a first aqueous solution of polyvinyl alcohol to obtain an emulsion.

[0025] The emulsion was subjected to ultrasonic treatment in an ice bath and then added to an aqueous solution of the second polyvinyl alcohol, stirred, and centrifuged.

[0026] Composite nanoparticles were obtained by heart collection.

[0027] In a fifth aspect of the invention, the application of the aforementioned enterohepatic circulation-based oral delivery system for traditional Chinese medicine monomer nanoparticles in the preparation of nanocarriers for delivering hydrophobic drugs is provided.

[0028] In a sixth aspect of the invention, the use of the aforementioned composite nanoparticles in the preparation of an oral medicament for treating ischemic stroke is provided.

[0029] Furthermore, the dosage of the oral medication is 1-5 mg SA NPs / kg body weight.

[0030] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0031] The purpose of this invention is to provide an oral delivery system for traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation, achieving the following breakthroughs through integrated carrier-drug design and a multi-level targeting mechanism of gut-liver-brain:

[0032] 1. Overcoming the first-pass effect bottleneck and improving bioavailability: Utilizing the enterohepatic circulation characteristics of self-assembled nanoparticles of traditional Chinese medicine monomer (sumaric acid), oral bioavailability is improved through intestinal absorption mediated by bile acid transporter protein (ASBT), ultimately achieving an oral bioavailability of 34.7%.

[0033] 2. Carrier-Drug Synergistic Effect: The nanoparticles possess the dual functions of drug active ingredients and delivery carriers, avoiding the immunogenicity risks introduced by exogenous carrier materials, while achieving sustained drug release in the ischemic brain region; enhancing brain targeting, and the carrier-drug synergistic effect; SA NPs themselves can reduce infarct volume by 34.1%, and the combined loading of neuroprotective agents (NA1) can produce a synergistic effect (the combined drug group reduced infarct volume by 68.7%, which is better than the single drug group).

[0034] 3. Suitable for acute treatment scenarios: SA NPs are stable in the highly acidic environment simulating the stomach and can rapidly enter the bloodstream via enterohepatic circulation to target ischemic brain tissue, meeting the timeliness requirements for AIS emergency treatment. The oral administration method is convenient, meeting the home oral administration needs within the golden time window for diseases such as stroke, overcoming the dependence on medical conditions for intravenous injection formulations.

[0035] 4. Safety and accessibility dual advantages: (1) Biocompatibility: single-component self-assembly avoids the toxicity of exogenous carriers, and the hemolysis rate is <5% (compliant with ISO 10993 standard); (2) Convenience of administration: the lyophilized powder can be stored at room temperature for 12 months and can be reconstituted in water within 10 seconds, making it suitable for pre-hospital emergency scenarios. Attached Figure Description

[0036] Figure 1 Mechanisms of SA NP penetration into the gastrointestinal tract, enterohepatic circulation, and brain targeting. (A, B) SEM images of SA NPs after 24 hours of incubation in solutions at pH 7.4, 1.5, and 8.5. Scale bars are 5 μm and 1 μm, respectively. (C) Images of IR780-labeled SA NPs in major organs 24 hours after oral administration. (D) Average level of IR676 fluorescence in blood-time curves after oral administration of free IR676 and SA NPs loaded with IR676. (E) Average level of Cy5.5 in blood-time curves after pretreatment with 500 mg / kg UDCA, indicating that UDCA inhibits SA NP uptake. (F) IVIS images and semi-quantitative (left) and fluorescence images (right) of SA NPs isolated from the gastrointestinal tract of tMCAO mice. (G, H) Semi-quantitative (right) images of IR780-loaded SANPs and free IR780 in the brain isolated from tMCAO mice. (****P<0.0001).

[0037] Figure 2SA NPs enter the enterohepatic circulation after oral administration to tMCAO mice; (A) In vivo images of IR780-labeled SA NPs throughout the body of mice 20 minutes and 24 hours after oral administration. (B) Image of IR780-labeled SA NPs in the gastrointestinal tract shown by IVIS. GItract: gastrointestinal tract. (C) Images of IR780-labeled SANPs and free IR780 isolated from various organs of tMCAO mice at different time points after oral administration. The level is high at 2 hours and then decreases. (D) Images of IR780-labeled SA NPs and free IR780 isolated from the gastrointestinal tract of tMCAO mice at different time points after oral administration. The level is low at 2 hours and then increases. (E) Time-semi-quantitative curves of IR780-loaded SA NPs and free IR780 in the small intestine and pancreas isolated from tMCAO mice. (F) Time-history variation of radiation efficiency of IR780-SA NPs and free IR780 in pancreatic tissue.

[0038] Figure 3 To investigate the accumulation of SA NPs in ischemic brain tissue of tMCAO mice and their reduction of infarct volume. (A) Scanning electron microscopy image of SA NPs. Scale bar: 1 μm. (B, C) In vivo and ex vivo IVIS imaging of mouse brain tissue after oral administration of IR780 / IR780-SA NPs and quantitative analysis in tMCAO mouse brain tissue (n=5). (DF) TTC staining, infarct volume, and neurological scores of tMCAO mice treated with different doses of SA NPs (n=4). (*P<0.05, **P<0.01, ***P<0.001).

[0039] Figure 4 SA NPs can be orally delivered to deliver NA1 in combination with tMCAO. (A) Scanning electron microscopy of NA1-SA NPs. Scale bar: 1 μm. (B) NA1 release from SA nanoparticles in PBS at pH 7.4. (C) Representative images of TTC-stained brain sections from mice that received the specified treatment. tMCAO mice were randomly assigned to three groups: oral PBS (n = 4), oral NA1 (n = 5), oral SA NPs (n = 4), and oral NA1-SA NPs (n = 7). (D) Neurological function scores and (E) infarct area of ​​MCAO mice that received the specified treatment. (*P<0.05, ***P<0.001, ****P<0.0001).

[0040] Figure 5SA NPs treatment reduces blood-brain barrier (BBB) ​​leakage. (A, B) SA NPs reduce Evansblue extravasation in ischemic ipsilateral brain tissue (n=3). (C, D) Representative images of ZO-1 (red) expression in ischemic ipsilateral brain tissue of mice receiving the specified treatment (n=3).

[0041] Figure 6 To overcome the incompatibility between NA1 and tPA by delivering NA1 to SA NPs. (A) Experimental design. ig, gastric administration; iv, intravenous injection. (B) Representative images of general appearance and cerebral infarction in the combined treatment group. (C) Quantification of plasma MMP-9 levels in control (normal) mice and tMCAO mice receiving the specified treatment at 24 hours. (D) Neurological function scores and (E) infarct area in stroke mice receiving the specified treatment. Data are presented as mean ± standard error (*P < 0.05, **P < 0.01, ****P < 0.0001).

[0042] Figure 7 TTC staining of the brains of tMCAO mice at different time points after oral administration of NA1-SA NPs. Detailed Implementation

[0043] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0044] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0045] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0046] The following will provide a detailed description of an oral delivery system for traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation and its preparation method, in conjunction with embodiments and experimental data.

[0047] Example 1: Preparation and characterization of SA NPs, an oral delivery system for traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation.

[0048] 1. SA NPs were prepared using the standard emulsification method.

[0049] SA was dissolved in acetone (1 ml) and added dropwise to 2 ml of 2.5% polyvinyl alcohol (PVA) (aqueous phase). The resulting emulsion was sonicated on ice for 40 seconds (10 seconds on, 10 seconds off) and then added to a 0.3% PVA solution in water (aqueous phase, 50 ml) with stirring. After evaporation overnight at 4°C, SA NPs were collected by centrifugation at 18,000 rpm for 30 minutes. The particles were then suspended in 40 ml of water to remove residual PVA and centrifuged at 18,000 rpm for 30 minutes to obtain NPs. The particles were then suspended in 5 ml of water, sonicated for 3 minutes, and then lyophilized for storage.

[0050] 2. Characterization

[0051] like Figure 3 As shown in Figure A, the particle size was determined to be 194 ± 20 nm by dynamic light scattering (DLS) and SEM, and the SEM showed a spherical monodisperse structure.

[0052] Example 2: Preparation of composite nanoparticles NA1-SA NPs and characterization of drug loading and release.

[0053] 1. Preparation of composite nanoparticles NA1-SA NPs

[0054] The preparation of NA1-SA NPs employs a double emulsion method. The following is the synthetic procedure for SA NPs loaded with hydrophobic drugs (including IR780, IR676, Cy5.5, coumarin 6 (C6), and NA1): The above-mentioned drugs and 5 mg of SA are dissolved together in 1 mL of acetone to form an organic phase solution. Subsequent steps are the same as for the preparation of SA NPs. In this embodiment of the invention, NA1 is specifically loaded.

[0055] 2. Characterization of drug loading and release rate of composite nanoparticles NA1-SA NPs

[0056] (1) Determination of drug loading

[0057] SA NPs loaded with NA1 were dissolved in dimethyl sulfoxide (DMSO) to release the drug components. The concentration of NA1 was quantitatively analyzed using the Pierce™ BCA Protein Quantification Kit (catalog number 23225) and a SpectraMax microplate reader (detection wavelength 562 nm).

[0058] (2) In vitro release experiment

[0059] NA1-SA NPs were placed in a dialysis bag with a molecular weight cutoff of 3000 Da and immersed in a test tube containing 30 mL of phosphate-buffered saline (PBS) (preset leak conditions). Release was carried out at 37°C and a shaking rate of 100 times / min. At selected time points, 1 mL of the extratubation solution was aspirated (and an equal volume of fresh medium was added simultaneously). The NA1 concentration was quantitatively detected using a SpectraMax microplate reader, the cumulative release rate was calculated, and a release curve was plotted.

[0060] like Figure 4 As shown in Figure A, NA1-supported SA nanoparticles (NA1-SA NPs) composite nanoparticles were successfully prepared via a dual emulsification method. These nanoparticles retained the original morphology of SA NPs, had a drug loading of 5.8% (weight percentage), and achieved 77% sustained drug release within 3 days. Figure 4 B).

[0061] Example 3: Validation of oral administration targeting the brain and entry into the enterohepatic circulation (tMCAO model)

[0062] Blood supply to the right central cerebrovascular artery (MCA) of approximately 23g male C57BL / 6 mice (8 weeks old) was blocked using a suture occlusion method. After 1.5 hours of cerebral ischemia, the suture was removed, and the ligatures of the internal carotid and common carotid arteries were opened to achieve reperfusion after ischemia. Then, at 0, 24, and 48 hours after suture removal, mice were randomly assigned to four groups (n=10) via gavage to receive PBS, 3 mg SA NPs, 3 mg NA1-SA NPs (containing NA1 3nM / g), and NA1 (3nM / g), respectively. Twenty-four hours after the last treatment, the mice were euthanized, and the brains were isolated, sectioned, and stained with TTC. Neurological function was assessed using the modified neurological severity score (mNSS). After tMCAO mice were gavaged with IR780-SA NPs and an equal dose of IR780, IVIS imaging was performed at different time points. SA NPs were observed to accumulate in ischemic brain tissue. When gastrointestinal tissue and other organs were separated, IR780-labeled SA NPs were observed to enter the enterohepatic circulation.

[0063] SA NPs can rapidly reach ischemic areas (within 1 hour) after oral administration. They possess both antioxidant therapeutic effects and can serve as oral drug delivery carriers. Utilizing this convenient oral administration method allows AIS patients to receive treatment more quickly, providing an option for securing the "golden time" and pre-hospital treatment. We evaluated the stability of SA NPs in the highly acidic environment of the stomach (where the extremely high pH is one of the main obstacles to oral drug delivery).

[0064] 1. Verification of gastric acid stability

[0065] SA NPs were suspended in simulated gastric fluid (0.1 M HCl containing 1% pepsin, pH 1.2) and shaken at 37°C for 6 h. Particle size changes were monitored in real time using DLS. The morphology, particle integrity, and aggregation of SA NPs were observed using SEM.

[0066] To simulate different pH conditions in vivo, SA NPs were incubated at various pH values. Scanning electron microscopy (SEM) analysis revealed that the SA NPs were spherical with a diameter of 194 ± 20 nm. After 24 hours of incubation at pH 1.5 or 7.4, their morphology showed no significant change; however, when incubated at pH 8.5, the NPs aggregated and fused. Figure 1 (A, B). These findings indicate that SA NPs can tolerate the highly acidic environment of the stomach. Oral administration of SA NPs significantly improved neurological function in tMCAO mice, demonstrating that SA NPs can effectively penetrate the gastrointestinal tract and blood-brain barrier.

[0067] 2. Bioavailability

[0068] Our research group observed that SA NPs loaded with IR780 (a near-infrared dye that can be used for in vivo imaging) accumulate in the brain, and that NPs preferentially accumulate in ischemic brain tissue. Figure 1 C). Over time, the accumulation of SA NPs in ischemic brain tissue remained high 72 hours after a single dose. By encapsulating IR676 into SA NPs and quantifying the fluorescence intensity of IR676 in the blood, we determined the oral bioavailability of SA NPs to be 42%. Figure 1 D).

[0069] 3. Intestinal absorption

[0070] Furthermore, our research group observed that SA NPs are absorbed by the intestine through interaction with the apical sodium-dependent bile acid transporter (ASBT). Pretreatment with ursodeoxycholic acid (UDCA, a substrate of ASBT) significantly reduced serum SA NP concentration by 62.1%. Figure 1 E). Furthermore, a large number of SA NPs were observed to accumulate in the ileum, primarily in the mucosa, muscle layer, and serosa, indicating that SA NPs can penetrate the intestinal epithelium and enter the bloodstream. Figure 1 F). Analysis showed that serum SA NP concentrations peaked at multiple time points after oral administration. Similar trends were observed in the brain and other major organs such as the liver and pancreas. We also found that the small intestine showed multiple peaks after a single oral dose of SA NPs, with concentrations opposite to those in other organs. Figure 1 G, H). In contrast, when the free fluorescent dye was orally administered, it showed a single peak in all organs (G, H).Figure 1 H).

[0071] The above-mentioned use of fluorescently loaded SA NPs confirmed that SA NPs enter the enterohepatic circulation after oral administration. Figure 2 SA NPs, after being administered to tMCAO mice via gavage, enter the bloodstream from the ileum in the gastrointestinal tract and eventually accumulate significantly in the brain. Figure 2 A, B). The changes in the levels in the liver, spleen, lungs, kidneys, and pancreas at different time points after gavage were similar to those in brain tissue. Figure 2 C). The changes in intestinal content are different, especially at the 2-hour time point after oral administration, where SA NPs were observed to be high in organs but decreased in the intestines. Figure 2 D). SA NPs metabolism exhibits a multi-peak phenomenon, while free dyes show a single peak (D). Figure 2 E, F). Figure 2 F confirmed that SA NPs accumulate in the pancreas via lymphatic circulation (non-first-pass effect), providing evidence for targeted drug delivery in diabetes / pancreatic cancer and further supporting the entry of SA NPs into the enterohepatic circulation after oral administration. This is a multi-stage process involving hepatic metabolism, bile secretion, and reabsorption from the intestine to systemic circulation, which can prolong the duration of plasma drug action and improve therapeutic efficacy. Furthermore, liver function was tested at different time points after administration, and no abnormalities were found.

[0072] Example 4: Targeting Ischemic Cerebral Regions and Improving Function in Ischemic Stroke After Oral Administration of SA NPs. Blood supply to the right middle cerebral artery (MCA) of approximately 23g male mice was blocked using a suture occlusion method. After 1.5 hours of cerebral ischemia, the suture was removed, and the ligatures of the internal carotid and common carotid arteries were opened to achieve reperfusion after ischemia. SA NPs were then administered by gavage at 0, 24, and 48 hours after suture removal, using PBS and different doses, respectively. Twenty-four hours after the last treatment, the mice were euthanized, and the brains were isolated, sectioned, and stained with TTC. Neurological function was assessed using a scoring system: 1 = No neurological dysfunction; 2 = Bending of the trunk and contralateral forelimb when the tail is raised; 3 = Circling to the contralateral side; 4 = Tilt to the contralateral side at rest; 5 = No spontaneous movement. The scoring was double-blind.

[0073] Scanning electron microscopy showed that SA NPs were uniformly sized spheres with a particle size of approximately 200 nm. Figure 3 A). After gavage administration of IR780-SA NPs to tMCAO mice, IVIS imaging showed accumulation of SA NPs in ischemic brain tissue, while in vivo imaging of normal mouse heads showed accumulation of SA NPs in scalp tissue. Figure 3 B, C). Treatment with different doses of SA NPs improved cerebral infarction volume and neurological function in tMCAO mice. Figure 3(D, E, F). Oral administration of 3 mg SA NPs reduced infarct area by 34.1% and improved neurological function.

[0074] Furthermore, experiments have shown that treatment with SA NPs reduces blood-brain barrier (BBB) ​​leakage. Figure 5 ), determined by the standard EvansBlue assay, and with increased expression of the blocking band-1 (ZO-1) ( Figure 5 C), which is one of the transmembrane and cytoplasmic proteins that form tight junctions (TJs) between endothelial cells.

[0075] Example 5: SA NPs can orally deliver NA1 in combination with tMCAO

[0076] NA1 (also known as Tat-NR2B9c) is a peptide drug that exerts its neuroprotective effect by inhibiting the interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 (PSD-95). Nitric oxide, as a key cellular signaling molecule, participates in physiological processes such as synaptic plasticity, vasomotor regulation, and inflammatory responses, while PSD-95 is a crucial postsynaptic scaffold protein in excitatory neurons. Therefore, blocking their interaction can alleviate neuronal damage caused by stroke. NA1 recently demonstrated significant efficacy in stroke patients who did not receive tPA thrombolysis in the Phase III clinical trial ESCAPE-NA1. However, as a hydrophilic peptide, NA1 is intolerant to gastric acid and poorly absorbed when administered orally. We evaluated the potential of SA NPs as an oral delivery carrier for NA1.

[0077] As shown in Example 2, the NA1-loaded SA nanoparticles (NA1-SA NPs) prepared by the double emulsification method retain the original morphology of SANPs, and the drug loading is 5.8% (weight percentage). Therefore, in the in vivo experiment, we controlled the drug loading at 5.8% to ensure that when the SA NPs were administered at a dose of 3 mg, the delivery concentration of NA1 was maintained at 3 nM / g, which has been proven to be an ineffective therapeutic dose in previous studies.

[0078] Experimental results showed that the infarct volume was reduced by 68.7% in the NA1-SA NPs treatment group, significantly better than the 34.1% reduction in the blank SA NPs group. Consistent with this, the treatment significantly improved neurological function scores. Figure 3 C, D, E). As a control, free NA1 had almost no therapeutic effect due to intolerance to the gastrointestinal environment. Figure 3 (C, D, E). The above data indicate that SA NPs not only possess therapeutic activity themselves, but can also serve as carriers for oral brain-targeted delivery of NA1, enabling previously ineffective low-dose NA1 to exert significant stroke treatment effects.

[0079] Example 6: SA NPs transport NA1 to overcome the incompatibility between NA1 and tPA.

[0080] In current clinical practice, tPA is the only thrombolytic drug approved by the US Food and Drug Administration (FDA) and must be administered within 4.5 hours of symptom onset. Beyond this time window, tPA infusion significantly increases the risk of hemorrhagic transformation (HT) because tPA-induced reperfusion exacerbates already partially damaged blood-brain barrier (BBB) ​​damage, leading to the infiltration of harmful blood components into brain tissue. Since oral SA NPs significantly improve the functional and structural integrity of the blood-brain barrier (Figure 5), this treatment may help reduce the occurrence of hemorrhagic transformation after tPA treatment and prolong the thrombolytic time window. To verify this hypothesis, a tMCAO mouse model was established and treated with oral NA1-SA NPs. tPA was infused into mice at different time points (1.5, 3.5, 5.5, 7.5, and 9 hours) after nanoparticle treatment. The mice were euthanized three days later. Brain imaging, sectioning, and TTC staining were performed (Figure 6A). Figure 7 Brain imaging showed that tPA infusion only began to induce hemorrhagic transformation 9 hours after stroke (Figure 5B). Sustained treatment with SANPs significantly reduced serum matrix metalloproteinase-9 (MMP-9) concentrations 24 hours after stroke (Figure 6C), a biomarker associated with thrombolysis risk and hemorrhagic transformation.

[0081] The recently completed ESCAPE clinical trial has confirmed that NA1 is incompatible with tPA, as the efficacy of NA1 is reduced in patients receiving concurrent tPA infusion. This incompatibility may not be surprising, as the positively charged TAT peptide in NA1 can bind nonspecifically to tPA. Due to this binding, NA1 may not only lose its pharmacological activity but also its ability to penetrate the brain. We found that the incompatibility between NA1 and tPA is overcome when NA1 is delivered via SA NPs, as tPA infusion does not reduce the efficacy of NA1-SA NPs (Figures 6D, E, Figure 7).

[0082] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0083] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0084] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. An oral delivery system of traditional Chinese medicine monomer nanoparticles based on enterohepatic circulation, characterized in that, The delivery system comprises spherical nanoparticles formed by self-assembly of traditional Chinese medicine monomer gummiferan.

2. The oral delivery system of claim 1, wherein the system is based on enterohepatic circulation. The nanoparticle has a particle size of 194±20 nm.

3. A preparation method of the traditional Chinese medicine monomer nanoparticle oral delivery system based on enterohepatic circulation according to any one of claims 1-2, characterized in that, The method comprises: Dissolving gummiferan in acetone to form an organic phase, and adding it dropwise into a first polyvinyl alcohol aqueous solution to obtain an emulsion; Ultrasonically treating the emulsion in an ice bath, adding it into a second polyvinyl alcohol aqueous solution, stirring, and collecting the nanoparticles by centrifugation, namely the traditional Chinese medicine monomer nanoparticle oral delivery system based on enterohepatic circulation. The first polyvinyl alcohol aqueous solution has a mass concentration of 2%-3%, and the second polyvinyl alcohol aqueous solution has a mass concentration of 0.2%-0.4%.

4. The preparation method of claim 3, wherein the preparation method of the traditional Chinese medicine monomer nanoparticle oral delivery system based on enterohepatic circulation is characterized by, The composite nanoparticles are obtained after loading the hydrophobic drug on the traditional Chinese medicine monomer nanoparticle oral delivery system based on enterohepatic circulation according to any one of claims 1-2.

5. A composite nanoparticle, characterized in that, The hydrophobic drug comprises one or more of IR780, IR676, Cy5.5, coumarin 6, or protective peptide NA1.

6. The composite nanoparticle of claim 5, wherein, The method comprises:

7. A method of preparing the composite nanoparticle of any one of claims 5-6, wherein, Dissolving gummiferan and a hydrophobic drug in acetone to form an organic phase, and adding it dropwise into a first polyvinyl alcohol aqueous solution to obtain an emulsion; Ultrasonically treating the emulsion in an ice bath, adding it into a second polyvinyl alcohol aqueous solution, stirring, and collecting the composite nanoparticles by centrifugation.

8. Use of the traditional Chinese medicine monomer nanoparticle oral delivery system based on enterohepatic circulation according to any one of claims 1-2 in the preparation of a nanocarrier for delivering a hydrophobic drug.

9. Use of the composite nanoparticles according to any one of claims 5-6 in the preparation of an oral drug for treating ischemic stroke. The oral drug has a dosage of 1-5 mg SANPs per kilogram of body weight. ​ 10. Use according to claim 9, characterized in that, ​

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