Nano-drug of electroresponsive controlled-release fenfluramine as well as preparation method and application of nano-drug

Abstract

The invention relates to the technical field of medicine preparation, in particular to an electric response controlled release fenfluramine nano-medicine and a preparation method and application thereof.The preparation method at least comprises the following steps that S1, fenfluramine and polyethylene glycol are heated and stirred and then cooled to the room temperature, the pH value of a solution is adjusted to be alkalescent, and a solution A is obtained; s2, sequentially adding pyrrole, dopamine hydrochloride and an ammonium persulfate aqueous solution into the solution A, and uniformly stirring and reacting to obtain a solution B; and S3, adding ethanol into the solution B, and centrifuging to obtain a final product, namely the nano-drug of the electric response controlled release fenfluramine. According to the invention, an optimal electric response release drug system is obtained through specific reaction temperature and optimization of the ratio of dopamine to pyrrole monomer to drug molecules, so that epileptic neuron membrane potential is effectively responded, epileptic drugs are controllably released, and epileptic seizure frequency and degree are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, and in particular to a nano drug for electrically responsive controlled-release fenfluramine and a preparation method and application thereof. Background Art

[0002] Epilepsy is a neurological disease caused by abnormal discharges of brain neurons. It usually recurs multiple times, and the duration of the seizures is often short. The repeated attacks of epilepsy cause great mental stress and heavy psychological burden to patients and their families. Therefore, it is very important to achieve efficient treatment of epilepsy. Fenfluramine is an emerging epilepsy treatment drug that can be used to treat Dravet syndrome (a rare childhood epilepsy) in children aged 2 years and above. Its new mechanism of action is to regulate the activity of serotonin receptors and sigma-1 receptors to reduce the frequency of epileptic seizures. However, if fenfluramine is used inappropriately, it may have side effects such as causing cardiovascular diseases such as heart valves. Therefore, how to achieve controlled release of epilepsy drugs is of great significance for the efficient treatment of epilepsy.

[0003] Electric field responsive materials usually have the characteristics of high conductivity, small electrical impedance and good electrochemical stability, and are widely used in drug delivery, sensing applications and microelectronic devices. When epilepsy occurs, it is accompanied by abnormal discharge of neurons. Prior art CN115350193A discloses a method for preparing an electric field responsive lamotrigine composite preparation and its application, which is made of electric field responsive nanocarriers and lamotrigine, wherein the electric field responsive nanocarrier uses FeCl3 as an oxidant, and the diaminopyridine monomer is polymerized, and the nanogel freeze-dried sample obtained after subsequent treatment; and the electric field responsive nanocarrier can be used to prepare an electric field responsive lamotrigine composite preparation and its use to prepare anti-epileptic drugs, thereby improving the anti-epileptic efficacy of lamotrigine. However, there is little research on the controlled release of fenfluramine in the prior art. Therefore, if an electric responsive material that responds to epileptic seizures can be developed for the controlled release of fenfluramine, it has great potential for exerting the efficacy of the drug while reducing side effects. Summary of the invention

[0004] In order to solve the problems in the prior art, the first aspect of the present invention provides a method for preparing an electrically responsive controlled-release nanomedicine of fenfluramine, comprising at least the following steps:

[0005] S1: heating fenfluramine and polyethylene glycol and stirring them, cooling them to room temperature, and adjusting the pH value of the solution to be weakly alkaline to obtain solution A;

[0006] S2: adding pyrrole, dopamine hydrochloride and ammonium persulfate aqueous solution to solution A in sequence, stirring and reacting uniformly to obtain solution B;

[0007] S3: After adding an equal volume of ethanol to solution B, centrifugation was performed to obtain the final product, i.e., the nanomedicine with electrically responsive controlled release of fenfluramine.

[0008] In the present invention, the nanomedicine for electrically responsive controlled release of fenfluramine is denoted as PPY-PDA-FFM.

[0009] In one embodiment, the molecular weight of the polyethylene glycol is 2000.

[0010] In one embodiment, the mass ratio of fenfluramine to polyethylene glycol in step S1 is 1-10:20-100.

[0011] In one embodiment, the mass ratio of fenfluramine to polyethylene glycol in step S1 is 1:10.

[0012] In one embodiment, the heating condition in step S1 is: stirring at 40-60° C. for 1 h.

[0013] In one embodiment, the heating condition in step S1 is: stirring at 50° C. for 1 h.

[0014] In one embodiment, in the step S1, a hydrochloric acid solution is used to adjust the pH value, the concentration of the hydrochloric acid solution is 1.0 mol / L, and the amount added is 100 μL.

[0015] In one embodiment, the pH value of solution A in step S1 is 8.0.

[0016] In one embodiment, the step S2 is specifically as follows:

[0017] Pyrrole and dopamine hydrochloride were first added to solution A and stirred evenly, and then an aqueous solution of ammonium persulfate was added and stirred evenly, and the mixture was reacted for 12 hours in a light-proof condition to obtain solution B.

[0018] In one embodiment, the mass concentration of the ammonium persulfate aqueous solution is 10 mg / mL

[0019] In one embodiment, the usage ratio of the pyrrole, dopamine hydrochloride, and ammonium persulfate aqueous solution is 10-100 μL: 1-10 mg: 100 mg.

[0020] In one embodiment, the usage ratio of pyrrole, dopamine hydrochloride, and ammonium persulfate aqueous solution is 50 μL: 5 mg: 100 mg.

[0021] In one embodiment, the dosage ratio of fenfluramine, pyrrole, and dopamine hydrochloride is 5-50 mg: 10-100 μL: 1-10 mg.

[0022] In one embodiment, the dosage ratio of fenfluramine, pyrrole, and dopamine hydrochloride is 25 mg: 50 μL: 5 mg.

[0023] In one embodiment, the centrifugal speed in step S3 is 18000 r / min.

[0024] The second aspect of the present invention provides a nano drug obtained according to the preparation method of the above-mentioned nano drug for electrically responsive controlled release of fenfluramine.

[0025] The third aspect of the present invention provides an application of the method for preparing the above-mentioned nanomedicine for electrically responsive controlled release of fenfluramine in the preparation of anti-epileptic drugs.

[0026] Beneficial Effects

[0027] 1. The present invention optimizes the ratio of dopamine to pyrrole monomers and drug molecules to obtain an optimal electrically responsive drug release system, which can electrically respond to and controllably release epilepsy drugs.

[0028] 2. The present invention explores the effect of temperature on material morphology and drug loading. Through a specific reaction temperature, especially when the temperature is 40-60°C, the morphology and drug loading of the prepared drug material are optimal.

[0029] 3. The present invention controls the dosage ratio of dopamine, pyrrole monomers and drug molecules, especially when the dosage ratio of fenfluramine, pyrrole and dopamine hydrochloride is 5-50 mg: 10-100 μL: 1-10 mg, the prepared electrically responsive controllable release epilepsy drug can effectively respond to the membrane potential of epileptic neurons and reduce the frequency and severity of epileptic seizures.

[0030] 4. The present invention controls the pH value of the reaction system to be weakly alkaline to reduce the influence on the subsequent polymerization rate of pyrrole and dopamine and the conductivity of polypyrrole.

[0031] 5. The present invention adopts a one-pot reaction, and the preparation process is simple. It is expected to be widely used in the preparation of anti-epileptic drugs and has good practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the transmission electron microscopy image of PPY-PDA-FFM prepared in Example 1.

[0033] Figure 2 This is the scanning electron microscope image of PPY-PDA-FFM prepared in Example 1.

[0034] Figure 3 Infrared spectra of PPY-PDA-FFM, PPY and PPY-PDA prepared in Example 1.

[0035] Figure 4 This is the UV-visible absorption spectrum of fenfluramine released over time from PPY-PDA-FFM prepared in Example 1 at a voltage of 120 mV.

[0036] Figure 5 This is a UV-visible absorption spectrum of the PPY-PDA-FFM prepared in Example 1, with the maximum absorption value at 255 nm varying with time.

[0037] Figure 6 NMR spectrum of fenfluramine released over time from the PPY-PDA-FFM prepared in Example 1 in the brain of epileptic mice.

[0038] Figure 7 The PPY-PDA-FFM prepared in Example 1 was used to investigate the effect of PPY-PDA-FFM on the brain of mice with epilepsy. 19 F-MRI imaging atlas.

[0039] Figure 8 The graphs are for testing the membrane potential excitability of PPY-PDA-FFM, FFM and PPY-PDA prepared in Example 1, wherein A is a graph showing changes in the cell membrane potential of nerve cells; B is a graph showing changes in the influx of calcium ions into nerve cells; and C is a graph showing changes in the efflux of potassium ions into nerve cells.

[0040] Fig. 9 The graphs are test graphs of the anti-epileptic effect of PPY-PDA-FFM prepared in Example 1 in the brains of mice with epilepsy, wherein AD is the expression levels of MCP-1, TNF-α, IL-1β and IL-6 in brain tissue after the QPCR detection of the material; EG is the behavioral observation of mice after the action of different materials; H is the survival rate analysis of mice after the action of different materials; I is the fluorescence graph of GFAP and FJB in the DG area of ​​the hippocampus after the action of the material; JK is the quantitative result analysis graph of GFAP and FJB. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods for which specific conditions are not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially.

[0042] Example 1

[0043] The first aspect of the present embodiment provides a method for preparing a nanomedicine for electrically responsive controlled-release of fenfluramine, comprising the following steps:

[0044] S1: 25 mg of fenfluramine and 250 mg of polyethylene glycol were heated and stirred, then cooled to room temperature, and the pH value of the solution was adjusted to 8.0 to obtain solution A;

[0045] S2: First, add 50 μL of pyrrole and 5 mg of dopamine hydrochloride to solution A and stir evenly, then add 100 mg of ammonium persulfate aqueous solution and stir evenly, and react for 12 hours in a dark environment to obtain solution B;

[0046] S3: After adding ethanol to solution B, centrifugation was performed to obtain the final product, i.e., the nanomedicine with electrically responsive controlled release of fenfluramine.

[0047] The heating condition in step S1 is: stirring at 50° C. for 1 h.

[0048] In the step S1, a hydrochloric acid solution is used to adjust the pH value. The concentration of the hydrochloric acid solution is 1.0 mol / L, and the amount added is 100 μL.

[0049] The centrifugal speed in step S3 is 18000 r / min.

[0050] The second aspect of this embodiment provides a nano drug obtained according to the preparation method of the above-mentioned nano drug for electrically responsive controlled release of fenfluramine.

[0051] The third aspect of this embodiment provides an application of the above-mentioned method for preparing the nanomedicine with electrically responsive controlled release of fenfluramine in the preparation of anti-epileptic drugs.

[0052] Performance Testing

[0053] In the present application: PPY is polypyrrole; PPY-PDA is polypyrrole-polydopamine; FFM is fenfluramine.

[0054] 1. Electron microscope image: Figure 1 This is the transmission electron microscopy image of PPY-PDA-FFM prepared in Example 1. Figure 2 This is a scanning electron microscopy image of PPY-PDA-FFM prepared in Example 1. Transmission electron microscopy and scanning electron microscopy images can show the successful preparation of the drug.

[0055] 2. Infrared spectrum: Figure 3 The infrared spectra of PPY-PDA-FFM and PPY-PDA prepared in Example 1 are shown in FIG. 1 . As can be seen from the figure, the PPY-PDA-FFM prepared in this example has the same characteristic peaks as fenfluramine, indicating the successful loading of fenfluramine.

[0056] 3. Ultraviolet absorption spectrum: Figure 4 This is a UV-visible absorption spectrum of fenfluramine released over time by PPY-PDA-FFM prepared in Example 1 at a voltage of 120 mV; Figure 5 The maximum absorption value of PPY-PDA-FFM prepared in Example 1 at 255 nm changes with time. As can be seen from the figure, the nano drug prepared according to this example can controllably release fenfluramine under 120mV voltage stimulation, indicating that the nano drug prepared in the present invention has the effect of releasing fenfluramine in electrical response.

[0057] 4. NMR F spectrum: Figure 6 The NMR spectrum of fenfluramine released over time in the brain of epileptic mice by PPY-PDA-FFM prepared in Example 1, the drug in the figure is pure fenfluramine, and the material is PPY-PDA-FFM prepared in Example 1. As can be seen from the figure, 10 minutes after PPY-PDA-FFM (5 μL, 20 mg / mL) was injected into the brain of epileptic mice, F-MRI imaging was obvious, proving that the PPY-PDA-FFM prepared in this example can respond to the brain voltage of epileptic mice and successfully release fenfluramine.

[0058] 5. Magnetic resonance spectrum: Figure 7 The PPY-PDA-FFM prepared in Example 1 was used to investigate the effect of PPY-PDA-FFM on the brain of mice with epilepsy. 19 F-MRI imaging spectrum, from which it can be seen that the PPY-PDA-FFM prepared in this example can be used for epilepsy treatment under imaging monitoring.

[0059] 6. Membrane potential excitability test:

[0060] Figure 8 A is a graph showing changes in the cell membrane potential of neurons: primary neurons were seeded in a 24-well plate at 2×105 / well, 100 ng / μL of kainic acid was added to activate the cells, different materials (PPY-PDA-FFM, FFM, PPY-PDA prepared in Example 1) were pre-incubated with the cells for 12 h, DiSBAC2(3) was added to the culture medium at a final concentration of 100 nM, incubated for 30 min, and the changes in cell fluorescence intensity were recorded using a flow cytometer;

[0061] Figure 8 B is a graph showing changes in calcium ion influx in nerve cells: primary nerve cells were seeded in a 24-well plate at 2×105 / well, 100 ng / μL of kainic acid was added to activate the cells, different materials (PPY-PDA-FFM, FFM, PPY-PDA prepared in Example 1) were pre-incubated with the cells for 12 h, nerve cells were labeled with a calcium ion probe and flow cytometry was performed to record changes in cell fluorescence intensity;

[0062] Figure 8C is the change of potassium ion outflow of nerve cells: primary nerve cells were seeded in a 24-well plate, 2×105 / well, 100 ng / μL of kainic acid was added to activate the cells, different materials (PPY-PDA-FFM, FFM, PPY-PDA prepared in Example 1) were pre-incubated with the cells for 12 h, the nerve cells were labeled with potassium ion probes and flow analysis was performed to record the changes in cell fluorescence intensity;

[0063] It can be seen from the figure that the fenfluramine with electrically responsive controlled release of PPY-PDA-FFM prepared by the present invention can effectively reduce the excitability of membrane potential, reduce calcium influx and potassium efflux, and effectively reduce the membrane potential of nerve cells.

[0064] 7. Behavioral experiments and brain slice tests:

[0065] Establishment of acute epilepsy rat model: Rats that need surgery were intraperitoneally injected with the same dose (5 μL, 20 mg / mL) of different materials (FFM, PPY-PDA-FFM, PPY-PDA) and then anesthetized with intraperitoneal injection of chloral hydrate (400 mg / kg). After anesthesia, they were fixed on a stereotaxic instrument, and the electrodes were correctly positioned to implant the right cortex (AP: -3.2 mm, L: -3.0 mm, V: -1.8 mm), and the cannula was implanted into the left lateral ventricle (AP: -1 mm, L: -1.2 mm, V: -3.6 mm). The electrode was made of a double-strand stainless steel wire (d: 0.05 mm) with a polytetrafluoroethylene insulation layer on the outer layer, and the lower end was forked 0.6 mm, and about 0.5 mm of the insulation layer was stripped. The upper end of the electrode was welded to a micro three-hole socket, as close to the skull as possible, and then fixed with red and white dental cement. The rats recovered for one week after surgery. One week after the surgery, a microinjection needle was used to slowly push kain (KA) (1.25 g / mL, 0.65 uL / rat) into the lateral ventricle within 10 minutes. After administration, the needle was placed in the cannula for 5 minutes before slowly withdrawing the needle. After KA administration, each rat was placed in a (50 cm × 30 cm × 30 cm) transparent resin observation box to observe the animal's epileptic behavior and record EEG changes. The animal's epileptic behavior was divided into IV grades according to Racine's criteria: Grade I showed non-selective chewing; Grade II showed head shaking; Grade III showed unilateral forelimb lifting; Grade IV showed bilateral forelimb lifting accompanied by wet dog shaking; Grade V showed convulsions and falling to the ground. Grades I to III were focal seizures; Grades IV and V were grand mal seizures.

[0066] Animal sampling and histopathological sections: Tissue sampling was performed 30 days after the epilepsy model was established. After the mice were anesthetized, the chest cavity was opened to expose the heart. The thick injection needle was inserted from the apex of the heart through the incision of the left ventricle and fixed. Then the right atrial appendage was cut open and 250 mL of 0.9% saline was quickly perfused. After the liver turned white, 250 mL of 4% neutral paraformaldehyde fixative was perfused until the rat was stiff. The perfused rat was then decapitated, the cranial cavity was opened, and the whole brain tissue was carefully removed. It was fixed with 4% paraformaldehyde at 4°C overnight and immersed in 30% sucrose solution until the brain tissue sank. After sinking, the brain tissue was embedded with an embedding agent and placed in a -86°C ultra-low temperature refrigerator for 2 hours before frozen sections were started. The tissue after sectioning was subjected to pathological staining.

[0067] Test results see Fig. 9 ,in Fig. 9 AD is the test graph of the expression of MCP-1, TNF-α, IL-1β and IL-6 in brain tissue after the action of QPCR detection materials. It can be seen from the figure that after the action of PPY-PDA-FFM, the levels of related expression factors were significantly lower than those of the FFM and PPY-PDA groups, indicating that PPY-PDA-FFM has a certain immunosuppressive effect on the production of cytokines, and further indicates that PPY-PDA-FFM can significantly inhibit the onset of epilepsy.

[0068] Fig. 9 EG is a behavioral observation test chart of mice after the action of different materials. It can be seen from the figure that FFM, PPY-PDA and PPY-PDA-FFM can all reduce the frequency of epileptic seizures, among which PPY-PDA-FFM prepared in this example has the effect of significantly reducing the frequency of epileptic seizures and shortening the duration of epileptic seizures.

[0069] Fig. 9 H is a survival rate analysis diagram of mice after being treated with different materials. It can be seen from the figure that the electric response material PPY-PDA-FFM prepared in this example is relatively safe.

[0070] Fig. 9 I is the fluorescence image of GFAP and FJB in the hippocampal DG area after PPY-PDA-FFM and FFM treatment. Fig. 9 JK is a quantitative analysis chart of GFAP and FJB. It can be seen from the figure that the expression of GFAP and FJB in the hippocampal DG region was significantly reduced after the electroresponsive material PPY-PDA-FFM prepared in the embodiment was used, which further indicates that PPY-PDA-FFM can significantly inhibit the onset of epilepsy.

Claims

1. A method for preparing a nano drug for electrically responsive controlled release of fenfluramine, characterized in that: At least the following steps are included: S1: heating fenfluramine and polyethylene glycol and stirring them, cooling them to room temperature, and adjusting the pH value of the solution to be weakly alkaline to obtain solution A; S2: adding pyrrole, dopamine hydrochloride and ammonium persulfate aqueous solution to solution A in sequence, stirring and reacting uniformly to obtain solution B; S3: After adding an equal volume of ethanol to solution B, centrifugation was performed to obtain the final product, i.e., the nanomedicine with electrically responsive controlled release of fenfluramine.

2. The method for preparing the nano drug of electrically responsive controlled release of fenfluramine according to claim 1, characterized in that: The mass ratio of fenfluramine to polyethylene glycol in step S1 is 1-10:20-100.

3. The method for preparing the nano drug of electrically responsive controlled release of fenfluramine according to claim 2, characterized in that: The heating condition in step S1 is: stirring at 40-60° C. for 1 h.

4. The method for preparing the nano drug of electrically responsive controlled release of fenfluramine according to claim 1, characterized in that: The pH value of solution A in step S1 is 8.

0.

5. The method for preparing the nano drug of electrically responsive controlled release of fenfluramine according to claim 1, characterized in that: The S2 step is specifically as follows: Pyrrole and dopamine hydrochloride were first added to solution A and stirred evenly, and then an aqueous solution of ammonium persulfate was added and stirred evenly, and the mixture was reacted for 12 hours in a light-proof condition to obtain solution B.

6. The method for preparing the electrically responsive controlled-release fenfluramine nanomedicine according to claim 5, characterized in that: The usage ratio of the pyrrole, dopamine hydrochloride and ammonium persulfate aqueous solution is 10-100 μL: 1-10 mg: 100 mg.

7. The method for preparing the nano drug of electrically responsive controlled release of fenfluramine according to claim 1, characterized in that: The dosage ratio of fenfluramine, pyrrole and dopamine hydrochloride is 5-50 mg: 10-100 μL: 1-10 mg.

8. The method for preparing the electrically responsive controlled-release fenfluramine nanomedicine according to claim 1, characterized in that: The centrifugal speed in step S3 is 18000 r / min.

9. A nano drug obtained according to the method for preparing the nano drug for electrically responsive controlled release of fenfluramine according to any one of claims 1 to 8.

10. Use of the method for preparing the nano drug for electrically responsive controlled release of fenfluramine according to any one of claims 1 to 8 in preparing anti-epileptic drugs.

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