Platelet-loaded bionanomedicines containing ginkgolide and butylphthalide, their preparation methods and applications

Abstract

This invention discloses a platelet-loaded bio-nanomedicine containing ginkgolide and butylphthalide, its preparation method, and its application, relating to the field of pharmaceutical formulation technology. The bio-nanomedicine comprises an active ingredient, a core drug carrier, a biological shell, and a target-seeking peptide. The active ingredient includes at least ginkgolide B and butylphthalide. The core drug carrier includes a lipid carrier, and the target-seeking peptide is a CITP peptide. The mass ratio of the active ingredient to the core drug carrier is 1:5 to 1:10. The amino acid sequence of the CITP peptide is PHSSHIGTTEIGKL. The biological shell includes at least one of platelet membrane and exosome membrane. The bio-nanomedicine of this invention exhibits good targeting and therapeutic efficacy, providing a new approach for the treatment of thrombotic diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to platelet-loaded ginkgolide and butylphthalide bio-nanomedicine, its preparation method, and its application. Background Technology

[0002] Cerebral infarction (iron deficiency stroke), a common and serious cerebrovascular disease, is characterized by high incidence, high disability rate, and high mortality rate, placing a heavy burden on society and families. Currently, treatment methods for cerebral infarction mainly include drug therapy and surgical treatment, but drug therapy remains the primary clinical approach. However, existing drug therapies face many challenges, such as the difficulty of drugs effectively crossing the blood-brain barrier to reach the lesion site, resulting in low drug concentrations in the brain and poor therapeutic effects; furthermore, the non-specific distribution of drugs may cause systemic adverse reactions, limiting their clinical application.

[0003] Ginkgolide and butylphthalide are two drugs with potential neuroprotective effects. Ginkgolide has various pharmacological effects, including antioxidant, anti-inflammatory, and antiplatelet aggregation effects, and can improve neurological deficits after cerebral ischemia. Butylphthalide can improve microcirculation in the ischemic area of ​​the brain, increase cerebral blood flow, and reduce cerebral edema. The two have synergistic pharmacological effects, and their combined use is expected to improve the treatment effect of cerebral infarction.

[0004] There are currently studies on the combined use of ginkgolide and butylphthalide for the treatment of mild to moderate acute cerebral infarction (Liu Fangzhen. Efficacy of Ginkgolide for Injection Combined with Butylphthalide Soft Capsules in the Treatment of Mild to Moderate Acute Cerebral Infarction [J]. Journal of Aerospace Medicine, 2022, 33(06):701-703.). In these studies, although the treatment effect was improved to some extent, ginkgolide was administered via intravenous infusion, while butylphthalide is a capsule preparation administered orally, which causes inconvenience in the treatment of cerebral infarction. Although butylphthalide can also be administered via intravenous infusion, studies have shown that the half-life of butylphthalide administered via intravenous infusion is greatly shortened (about 6 to 10 hours), significantly shorter than the half-life of oral administration (about 7 to 16 hours), usually requiring multiple administrations to maintain a stable therapeutic concentration (twice daily intravenous infusion). Intravenous infusion is time-consuming and extremely inconvenient.

[0005] Platelet-based biomimetic carriers have attracted widespread attention in recent years as a novel drug delivery system. The platelet membrane surface contains various membrane proteins that endow platelets with unique biological properties, such as targeted recognition and immune escape capabilities. Platelets can specifically recognize damaged vascular endothelial cells and thrombi, thereby achieving targeted drug delivery; simultaneously, the natural origin of platelets gives them good biocompatibility and low toxicity, reducing the occurrence of immune responses. This provides a new approach for the combined use of ginkgolide and butylphthalide. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention discloses a bio-nanomedicine loaded with ginkgolide and butylphthalide on platelets, its preparation method, and its applications.

[0007] Ginkgolide and butylphthalide exhibit significant complementary effects in the treatment of cerebral infarction. Ginkgolide can improve blood rheological parameters, and its mechanism of action mainly includes inhibiting platelet aggregation and scavenging free radicals. Platelet aggregation is a key step in thrombus formation, and ginkgolide can improve blood flow by inhibiting the activity of platelet-activating factor (PAF) and reducing platelet aggregation. Simultaneously, ginkgolide also has a free radical scavenging effect, which can alleviate the damage of oxidative stress to brain tissue.

[0008] Butylphthalide primarily exerts its neuroprotective effects. It can protect nerve cells through multiple pathways, such as improving mitochondrial function and inhibiting nerve cell apoptosis. In terms of free radical scavenging, butylphthalide can increase the activity of antioxidant enzymes, enhance the body's antioxidant capacity, and reduce free radical damage to nerve cells.

[0009] The first aspect of this invention provides a bio-nanomedicine for treating iron deficiency stroke. The bio-nanomedicine comprises an active ingredient, a core drug carrier, a biological shell, and a target-seeking peptide. The active ingredient comprises at least ginkgolide B and butylphthalide. The core drug carrier comprises a lipid carrier. The target-seeking peptide is a CITP peptide. The mass ratio of the active ingredient to the core drug carrier is 1:5 to 1:10. The amino acid sequence of the CITP peptide is PHSSHIGTTEIGKL (SEQ ID NO: 1). The biological shell comprises at least one of platelet membrane and exosome membrane.

[0010] Furthermore, the active ingredient also includes other drugs for treating cardiovascular diseases. Preferably, the active ingredient also includes at least one of aspirin, clopidogrel, argatroban, alteplase, batroxobin, rosuvastatin, atorvastatin, tanshinone, and ligustrazine.

[0011] Furthermore, the mass ratio of ginkgolide B to butylphthalide is 1:1 to 1:3.

[0012] Furthermore, the lipid carrier comprises phospholipids and cholesterol, with a phospholipid to cholesterol mass ratio of 3:1 to 5:1.

[0013] Furthermore, the total drug loading of the bio-nanomedicine is 5% to 8%.

[0014] Furthermore, the encapsulation rate of the bio-nanomedicine is 75%~90%.

[0015] In a second aspect, the present invention provides a composition comprising the aforementioned bionanomedicine and pharmaceutically acceptable additives. Preferably, the additives comprise at least one of a stabilizer, a surfactant, an antioxidant, and a preservative.

[0016] Furthermore, the application of the described bionanomedicine or the described composition in the preparation of drugs for treating cardiovascular diseases. Further, the cardiovascular diseases include thrombotic diseases, preferably, the cardiovascular diseases include at least one of myocardial infarction, iron deficiency stroke, and peripheral artery disease.

[0017] In a third aspect, the present invention provides a method for preparing the aforementioned bionanomedicine, the method comprising the following steps:

[0018] S1: Constructing the drug-loaded core: The core drug carrier and active ingredient are co-dissolved in a chloroform solution containing 3% to 7% ethanol at a mass ratio of 1:5 to 1:10. The solution is then rotary evaporated at a temperature of 35 to 45°C to form a uniform lipid film. After vacuum drying and reconstitution, the film is ultrasonically treated and extruded through a 100-200nm polycarbonate film to obtain nanoparticles, thus obtaining the core drug carrier.

[0019] S2: Targeted modification of biological shell: Platelet membrane is treated with a low-concentration alkaline solution to increase the number of free carboxyl groups and then activated. The carboxyl-activated platelet membrane is then coupled with CITP peptide to obtain a targeted modified platelet membrane.

[0020] S3: The lipid nanoparticles obtained in S1 and the targeted modified platelet membrane obtained in S2 are mixed in a volume ratio of 1:1 to 1:5 and then co-extruded to obtain a "core-shell" structure and thus a bio-nanomedicine.

[0021] Furthermore, the core drug carrier in step S1 includes phospholipids and cholesterol, wherein the mass ratio of phospholipids to cholesterol is 3:1 to 5:1, and the active ingredient in step S1 includes ginkgolide B and butylphthalide, wherein the mass ratio of ginkgolide B to butylphthalide is 1:1 to 1:3.

[0022] Further, the carboxyl activation step in step S2 is to activate the carboxyl group using EDC / NHS, wherein the molar ratio of EDC:NHS:membrane carboxyl group is (3~6):(1~3):1.

[0023] Furthermore, in step S3, the concentration of the core drug carrier is 2-4 mg / mL; and the concentration of the targeted modified platelet membrane is 0.5-1.5 mg / mL.

[0024] Furthermore, the method also includes a platelet membrane extraction step, wherein the extraction step includes at least a method of obtaining platelet membranes using repeated freeze-thaw cycles or hypotonic lysis.

[0025] In one embodiment of the present invention, platelet membranes are obtained by repeated freeze-thaw cycles, specifically including the steps of pre-cooling treatment, freezing, thawing, and repeated freeze-thaw cycles 3-5 times.

[0026] In one embodiment of the present invention, the hypotonic lysis method includes the steps of preparing hypotonic buffer solution, ice bath lysis, and isotonic termination reaction.

[0027] The beneficial effects of the present invention include, but are not limited to:

[0028] The platelet-loaded bio-nanomedicine containing ginkgolide and butylphthalide provided by this invention is modified by CITP peptide coupling with platelet membrane to increase its targeting; at the same time, the combined use of ginkgolide B and butylphthalide enhances the efficacy of cardiovascular drugs; and the platelet membrane also increases the biocompatibility of ginkgolide B, providing a new approach for the treatment of iron deficiency stroke. Attached Figure Description

[0029] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0030] Figure 1 This is a flowchart illustrating the preparation process of the platelet-loaded bio-nanomedicine containing ginkgolide and butylphthalide according to the present invention. Detailed Implementation

[0031] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0032] Ginkgolide B: purchased from Nantong Feiyu Biotechnology Co., Ltd.;

[0033] Butylphthalide: Nantong Feiyu Biotechnology Co., Ltd.;

[0034] Phospholipids: DSCP

[0035] Example 1 Platelet Membrane Extraction

[0036] Following strict blood collection standards for SD rats, whole blood was collected from the heart of SD rats and placed in centrifuge tubes containing anticoagulant. The whole blood was transferred to lysis buffer containing 5 mL of 0.9% NaCl, 5 mM EDTA, 0.1 g / ml PGE1, and 1 U / mlapyrase (pH 7.4), diluted with 5 mL of 0.9% NaCl solution, and centrifuged at low speed (200–300 ×g, 10–15 min) to remove red blood cells and white blood cells, obtaining platelet-rich plasma (PRP). The PRP was then centrifuged at high speed (1000–2000 ×g, 10–20 min) to precipitate the platelets, the supernatant was discarded, and the platelets were washed 2–3 times with PBS buffer. The obtained platelet suspension was repeatedly frozen and thawed (frozen at -80°C and thawed at room temperature, repeated 3–5 times), and then centrifuged at a differential speed (first centrifuged at 1000 ×g for 10 min to remove unlysed cells, then centrifuged at 100,000 ×g for 1–1.5 h to collect the membrane precipitate) to obtain purified platelet membrane fragments. The membrane precipitate was resuspended in PBS, centrifuged to remove impurities, and finally the platelet membrane suspension was obtained.

[0037] Example 2: Construction and Optimization of Core Drug Carrier

[0038] According to research (Liu Changya. Clinical observation on the efficacy of ginkgolide combined with butylphthalide in the treatment of acute cerebral infarction [J]. China Continuing Medical Education, 2018, 10(26): 142-144.), when the injections are used together according to specifications, the ratio of ginkgolide B (25 mg / day) to butylphthalide (50 mg / day) is approximately 1:2. Therefore, the study was conducted with the ratio of ginkgolide B to butylphthalide at a mass ratio of 1:2.

[0039] (1) Optimization of phospholipid to cholesterol ratio

[0040] Phospholipid DSCP and cholesterol were co-dissolved with the drug (ginkgolide B: butylphthalide = 1:2) in a chloroform solution containing 5% ethanol according to the ratio in Table 1. The solution was then rotary evaporated at 40°C to form a uniform lipid film. After vacuum drying, the film was hydrated with PBS (containing 0.01% Tween 80) and sonicated at a probe ultrasonic power of 200W, a temperature of 50°C, and an ultrasonic time of 10 minutes. Nanoparticles were obtained by extrusion through a 100-200 nm polycarbonate membrane. Drug loading and stability were calculated.

[0041] Stability calculation method: Calculate the leakage rate at 37°C for 24 hours.

[0042] Calculation formula:

[0043] Encapsulation efficiency (EE%) = (Drug content in nanoparticles / Initial drug dosage) × 100%;

[0044] Drug loading (DL%) = (Drug content in nanoparticles / Total mass of nanoparticles) × 100%.

[0045] Table 1. Effect of phospholipid to cholesterol ratio on drug loading.

[0046]

[0047] As shown in Table 1, the drug loading increases with the increase of the ratio of phospholipids to cholesterol, but the stability decreases. Considering both drug loading and stability, a phospholipid to cholesterol mass ratio of 3:1 to 5:1 was selected for further research.

[0048] (2) Optimization of drug-to-lipid ratio

[0049] Repeat the experimental steps in (1) according to the proportions in Table 2, and calculate the encapsulation efficiency, drug loading, and stability.

[0050] Table 2. Effect of drug-to-lipid ratio on encapsulation efficiency and drug loading.

[0051]

[0052] As shown in Table 2, when the total drug to total lipid mass ratio is 1:5 to 1:10, the total drug loading, encapsulation efficiency, and leakage rate are all suitable.

[0053] Example 3 Platelet Membrane Targeted Modification

[0054] The following ratios were selected for optimization: phospholipid:cholesterol (mass ratio) 4:1, total drug:total lipid (mass ratio) 3:20, and ginkgolide B:butylphthalide (mass ratio) 1:2.

[0055] Platelet membrane pretreatment: The membrane surface was treated with low concentration NaOH (0.1 M, 5 min) to increase the number of free carboxyl groups, then centrifuged (100000 × g, 1 h) to remove impurities, and resuspended in MES buffer (0.1 M, pH 6.0).

[0056] EDC / NHS activated carboxyl groups: EDC : NHS : membrane carboxyl groups were dissolved in MES buffer (pH: 5.0–6.0) at a molar ratio of EDC : NHS : membrane carboxyl groups = 5 : 2 : 1, and activated at room temperature (25°C) for 20–30 min under light-protected conditions.

[0057] CITP peptide conjugation: CITP peptide (SEQ ID NO: 1: PHSSHIGTTEIGKL) and activated platelet membrane were dissolved in PBS buffer (pH=7.4) at a molar ratio of 5:1-10:1 and reacted at 4°C for 2–4 h. Unreacted NHS ester was quenched with glycine (10 mM, 15 min) to terminate the reaction. Free peptides were removed by ultracentrifugation (100,000 × g, 1 h), and the membrane was washed three times with PBS to obtain the targeted-modified platelet membrane.

[0058] The prepared drug-loaded core (3 mg / mL) and activated platelet membrane (1 mg / mL) were mixed together at a volume ratio of 1:1 to 1:5 using a micro-extruder (Avanti Polar Lipids' Mini-Extruder). The mixture was repeatedly extruded (10-20 times) through a 100-200 nm polycarbonate membrane at an extrusion pressure of 500 psi. This caused the platelet membrane and core to form a "core-shell" structure under mechanical force, with hydrophobic interactions driving the directional extrusion of membrane proteins. During co-extrusion, the platelet membrane and drug-loaded core gradually fused, forming a bio-nanomedicine.

[0059] The bio-nanomedicine was reconstituted in PBS solution (pH 7.4) overnight and then purified by adding it to a protein dialysis bag with a pore size of 1000–3500 Da.

[0060] Example 4: Detection of Nanomedicine Properties

[0061] Test 1: Particle Size Detection

[0062] The bio-nanomedicine was diluted to 0.5 mg / mL with PBS buffer and the particle size distribution was measured using a dynamic light scattering instrument with the following parameters: temperature: 25°C; detection angle: usually 173°; solvent parameters: input the viscosity (η≈0.89cP) and refractive index (n≈1.33) of the buffer.

[0063] The results are shown in Table 3.

[0064] Table 3. Detection results of platelet-loaded ginkgolide and butylphthalide particle size.

[0065]

[0066] Test 2: Zeta potential detection

[0067] The surface charge was measured using a Zeta potentiometer, and the results are shown in Table 4.

[0068] Table 4. Zeta potential detection results of platelets carrying ginkgolides and butylphthalide.

[0069]

[0070] Test 3: Coupling Efficiency Measurement

[0071] FITC was reacted with the N-terminal amino group of the CITP peptide (pH 8.5 borate buffer, reaction in the dark for 2 h) to purify and remove free FITC. FITC-CITP was then used instead of regular CITP and coupled to the platelet membrane according to the EDC / NHS procedure. The mixture was ultracentrifuged at 100,000 ×g for 1 h, and the precipitate (coupled peptide) and supernatant (free peptide) were collected. The fluorescence intensity of the precipitate (resuspended in PBS) and supernatant was measured (Ex / Em = 495 / 520 nm).

[0072] Coupling efficiency (%) = (precipitated fluorescence intensity / total fluorescence intensity) × 100%.

[0073] The coupling efficiency was measured to be 78%.

[0074] Example 5: Optimization of the ratio of ginkgolide B and butylphenol

[0075] Animal model establishment: Before the experiment, adult mice were fasted for 12 hours but allowed free access to water. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1 mL / 10 g body weight). After anesthesia took effect, the mice were fixed supine on the operating table. A midline incision was made in the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. The ECA was ligated proximal to the heart, and sutures were threaded under the CCA and ICA for later use. A small incision was made in the ECA approximately 3-4 mm from the bifurcation point. A pre-treated suture plug (approximately 0.26-0.28 mm in diameter) was inserted into the ICA through the ECA incision to a depth of approximately 18-20 mm, occluding blood flow to the middle cerebral artery and inducing cerebral ischemia. The ischemia time was controlled to 60 minutes, after which the suture plug was slowly removed, allowing blood reperfusion.

[0076] Drug administration: PBS and platelet nanomedicine groups (ginkgolide B and butylphenol mass ratios are shown in Table 5) were prepared. During cerebral ischemia-reperfusion, each group was injected via tail vein at a relative dose of 20 mg / kg body weight. 24 hours after reperfusion, the rats were anesthetized with 5% chloral hydrate, decapitated, and the brain was harvested. Brain slices were placed in 2% TTC staining solution and stained in a 37°C incubator for 8 minutes in the dark. Images were taken, and infarct volume was calculated.

[0077] Percentage of infarct volume = (Volume of contralateral hemisphere - Volume of non-infarcted area in ipsilateral hemisphere) / Volume of contralateral infarcted hemisphere × 100%.

[0078] The results are shown in Table 5.

[0079] Table 5

[0080]

[0081] The results showed that when the mass ratio of ginkgolide B to butylphthalide was 1:1 to 1:3, the therapeutic effect on cerebral infarction was higher than that of single-drug therapy.

[0082] Example 6: Validation of the therapeutic effect of bio-nanomedicine

[0083] The platelet-loaded nanomedicine and free drug were tested using ginkgolide B: butylphthalide (mass ratio) of 1:1 and 1:3, respectively, following the experimental steps in Example 5. The effective drug concentrations in the platelet-loaded nanomedicine and free drug groups were the same. The results are shown in Table 6.

[0084] Table 6

[0085]

[0086] The results showed that platelet membrane-loaded ginkgolide B and butylphthalide were more effective than free ginkgolide B and butylphthalide in combination, and the platelet membrane-loaded drug group conjugated with CITP peptide had better therapeutic effects than the unmodified platelet membrane-loaded ginkgolide B and butylphthalide.

[0087] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A bionanomedicine for treating iron deficiency stroke, characterized in that, The bio-nanomedicine comprises an active ingredient, a core drug carrier, a biological shell, and a target-seeking peptide. The active ingredient is ginkgolide B and butylphthalide. The core drug carrier is a lipid carrier, and the target-seeking peptide is a CITP peptide. The mass ratio of the active ingredient to the core drug carrier is 1:5 to 1:

10. The amino acid sequence of the CITP peptide is PHSSHIGTTEIGKL. The biological shell includes at least one of platelet membrane and exosome membrane. The mass ratio of ginkgolide B to butylphthalide is 1:1 to 1:

3. The lipid carrier is phospholipid and cholesterol, and the mass ratio of phospholipid to cholesterol is 3:1 to 5:

1.

2. A composition, characterized in that, The composition includes at least the bionanomedicine of claim 1 and additives acceptable in the pharmaceutical field.

3. The use of the bionanomedicine of claim 1 or the composition of claim 2 in the preparation of a medicament for treating iron deficiency stroke.

4. A method for preparing the bionanomedicine of claim 1, characterized in that, The method includes the following steps: S1: Constructing the drug-loaded core: The core drug carrier and active ingredient are co-dissolved in a chloroform solution containing 3% to 7% ethanol at a mass ratio of 1:5 to 1:

10. The solution is then rotary evaporated at a temperature of 35 to 45°C to form a uniform lipid film. After vacuum drying and reconstitution, the solution is ultrasonically treated and then extruded through a 100 to 200 nm polycarbonate film to obtain lipid nanoparticles. S2: Targeted modification of biological shell: Platelet membranes are treated with a low-concentration alkaline solution to increase the number of free carboxyl groups and then activate the carboxyl groups. The carboxyl-activated platelet membranes are then coupled with CITP peptides to obtain targeted modified platelet membranes. S3: The lipid nanoparticles obtained in S1 and the targeted modified platelet membrane obtained in S2 are mixed at a volume ratio of 1:1 to 1:5 and then co-extruded to obtain a core-shell structured bionanomedicine.

5. The method according to claim 4, characterized in that, The core drug carrier in step S1 is phospholipids and cholesterol, wherein the mass ratio of phospholipids to cholesterol is 3:1 to 5:

1. The active ingredients in step S1 are ginkgolide B and butylphthalide, wherein the mass ratio of ginkgolide B to butylphthalide is 1:1 to 1:

3.

6. The method according to claim 4, characterized in that, The carboxyl activation step in step S2 involves activating the carboxyl group using EDC / NHS, where the molar ratio of EDC:NHS: membrane carboxyl group is (3~6):(1~3):

1.

7. The method according to claim 4, characterized in that, In step S3, the concentration of lipid nanoparticles is 2-4 mg / mL; the concentration of the targeted modified platelet membrane is 0.5-1.5 mg / mL.

8. The method according to claim 4, characterized in that, The method further includes a platelet membrane extraction step, wherein the extraction step includes at least a method of obtaining platelet membranes using repeated freeze-thaw cycles or hypotonic lysis.

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