Stimulus-responsive bionic drug-loaded nanoparticles based on polypyrrole and preparation method thereof

Abstract

The invention belongs to the technical field of stimuli-responsive pharmaceutical preparations, and particularly relates to a stimuli-responsive bionic drug-loaded nanoparticle based on polypyrrole and a preparation method thereof. The preparation method comprises the following steps: reacting pyrrole with an oxidizing agent to obtain a polypyrrole skeleton, coupling the polypyrrole skeleton with halogenated methylphenylboronic acid, and coupling a phenylboronic acid group with a drug containing a vicinal diol or propylene glycol structure through a dehydration reaction, then, the polypyrrole skeleton can adsorb charged drugs, and finally, the biomimetic drug-loaded nanoparticles are obtained by wrapping cell membranes. The bionic nanoparticles can recognize related receptors expressed by a blood brain barrier, reach the blood brain barrier in a targeting manner, are internalized into the brain, are enriched to a brain lesion, and selectively release related drugs under the stimulation of high-concentration active oxygen, abnormal bioelectricity and acidic pH of lysosome in neuronal cells of the lesion; the curative effects of protecting neurons, inhibiting reactive glial hyperplasia and repairing damaged blood-brain barriers are achieved; meanwhile, phenylboronic acid groups can remove active oxygen of focuses, oxidative stress is relieved synergistically, and inflammatory response is relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of stimulus-responsive drug preparations, and in particular relates to a stimulus-responsive bionic drug-loaded nanoparticle based on polypyrrole and a preparation method thereof. Background Art

[0002] Acute brain injury diseases usually originate from factors such as cerebrovascular disease, central nervous system damage or infection, causing diseases such as stroke, traumatic brain injury, and acute epileptic seizures. During the development of such diseases, damage leads to local neuronal ischemia and hypoxia, which, on the one hand, causes overactivity of the neuronal mitochondrial electron transport chain, produces reactive oxygen species, and diffuses from the intracellular to the extracellular, further damaging other neurons in the lesion and producing more reactive oxygen; on the other hand, reactive oxygen simultaneously stimulates microglia and astrocytes, causing reactive gliosis, secreting proinflammatory factors, and clearing damaged neurons. Dead neurons themselves also release proinflammatory factors, exacerbating the inflammatory response of the lesion. In addition, activated astrocytes lose their glutamate "storage" function, resulting in abnormally increased glutamate concentration in the lesion, directly stimulating abnormal discharge of neurons and causing excitotoxic damage to neurons. Therefore, the oxidative stress of neurons and the inflammatory response of glial cells form a vicious circle, causing the microenvironment of brain lesions to continue to deteriorate. At the same time, factors such as ischemia and hypoxia induce compensatory proliferation of cerebral blood vessels, but the new blood vessels do not have complete blood-brain barrier function, causing exogenous immune cells and albumin to leak into the brain parenchyma, further stimulating the immune inflammatory response of the lesions.

[0003] Therefore, drug therapy for acute brain injury diseases should start from the overall "neurovascular unit". On the one hand, it is necessary to save damaged neurons and reduce neuronal death; on the other hand, it is necessary to inhibit reactive gliosis and restore glial homeostasis; at the same time, it is necessary to repair damaged cerebral blood vessels and block the leakage of exogenous substances. At the same time, when delivering drugs to brain lesions, it is also necessary to consider overcoming the obstacles of the blood-brain barrier and improving the efficiency of drug entry into the brain; and to fully enrich the drugs in the lesions to achieve targeted accumulation, on-demand drug release and long-term release effects. Summary of the invention

[0004] The present invention provides a stimulation-responsive bionic drug-loaded nanoparticle based on polypyrrole and a preparation method thereof. Under the premise of fully considering the physical and chemical factors related to acute brain injury (including high concentration of reactive oxygen and abnormally active bioelectricity), a polypyrrole skeleton with electrical stimulation responsiveness is first prepared based on a conductive polymer; then, reactive oxygen responsive and scavenging phenylboronic acid groups are introduced, and coupled with drugs containing vicinal diol or propylene glycol structures; then, charged drugs are adsorbed, and finally, the cell membrane is wrapped to obtain bionic drug-loaded nanoparticles. The bionic nanoparticles can recognize the relevant receptors expressed by the blood-brain barrier, target the blood-brain barrier and internalize into the brain, and are enriched in brain lesions. Under the stimulation of high concentration of reactive oxygen in the lesions, abnormal bioelectricity and acidic pH of lysosomes in neuronal cells, the relevant drugs are selectively released, and the efficacy of protecting neurons, inhibiting reactive gliosis and repairing damaged blood-brain barriers is exerted; at the same time, the phenylboronic acid group can remove reactive oxygen in the lesions, synergistically reduce oxidative stress, and relieve inflammatory reactions.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: A method for preparing polypyrrole-based stimuli-responsive biomimetic drug-loaded nanoparticles comprises the following steps: (1) The suspending agent is dissolved in a hydrochloric acid aqueous solution, and pyrrole and an oxidant are added, and the mixture is stirred at room temperature for 24 h. The product is placed in a dialysis bag with a molecular weight cutoff of 3500, immersed in water, dialyzed to remove impurities, and freeze-dried to obtain a polypyrrole skeleton; The reaction process is shown in Formula I:

[0006] Formula I (2) S1. Disperse the polypyrrole skeleton in an organic solvent, add 4-halogenated methylphenylboronic acid and an acid-binding agent, and react at 25 to 50°C under the protection of an inert gas until the precipitation no longer increases, and filter and collect the filtrate; S2. Add a drug containing a vicinal diol or propylene glycol structure to the filtrate, and add a dehydrating agent, and react at room temperature under the protection of an inert gas for 6 h to 24 h, filter and collect the filtrate, dialyze in water to remove impurities, and freeze-dry to obtain a single drug-loaded polypyrrole; The reaction process is shown in Formula II:

[0007] Formula II Wherein, X represents a halogen atom, Indicates drugs containing vicinal diol structures, Indicates drugs containing propylene glycol structure; (3) dispersing the single drug-loaded polypyrrole in water, adding charged drugs, stirring and reacting at room temperature for 6 h to 24 h under the protection of inert gas, dialyzing in water to remove impurities, and obtaining dual drug-loaded polypyrrole; The reaction process is shown in Formula III:

[0008] Formula III or,

[0009] in, Indicates charged drugs; (4) The aqueous solution of dual-drug loaded polypyrrole was mixed with the cell membrane solution, subjected to low-temperature ultrasound, and filtered through 0.45 μm and 0.22 μm filter membranes to obtain bionic drug-loaded nanoparticles.

[0010] Preferably, in step (1), the molar ratio of the suspending agent, pyrrole and oxidant is 0.5:1~1.5:2~2.5.

[0011] Preferably, in step (1), the suspending agent is sodium dodecyl sulfate, and the oxidant is selected from hydrogen peroxide or ferric chloride.

[0012] Preferably, the concentration of the aqueous hydrochloric acid solution in step (1) is 20 mmol / L to 100 mmol / L.

[0013] Preferably, in step (2), the molar ratio of 4-halomethylphenylboronic acid, the acid binding agent, the drug containing a vicinal diol or propylene glycol structure, and the dehydrating agent is 1:1.2~1.5:1.2~1.5:2.

[0014] Preferably, in step (2), the organic solvent is anhydrous dimethyl sulfoxide; the acid binding agent is anhydrous potassium carbonate; and the dehydrating agent is anhydrous sodium sulfate.

[0015] Preferably, in step (3), the molar ratio of the phenylboronic acid groups contained in the single drug-loaded polypyrrole to the charged drug is 1:0.5-10.

[0016] Preferably, in step (3), the charged drug is a quaternary ammonium salt drug.

[0017] Preferably, in step (4), the cell membrane is extracted from RAW 264.7 cells or SH-SY5Y cells.

[0018] Further preferably, in the step (4), the method for preparing the cell membrane solution is as follows: cells are cultured in a 10 cm dish until the confluence exceeds 100%, the cell suspension is collected by scraper, centrifuged at 250 g for 4 min, and washed three times with PBS 7.4; hypotonic solution is added to resuspend the cells, the hypotonic solution is 0.1×TM buffer containing 1 mmol / L PMSF; low-temperature ultrasonication for 1 h, centrifuged at 3200 g for 5 min to remove the cell nucleus, and the supernatant is extracted; then centrifuged at 20,000 g for 25 min to remove the cell organelles, and the supernatant is extracted; then ultracentrifuged at 100,000 g for 60 min to extract the precipitate to obtain an oily substance, which is the cell membrane; water is added, and the precipitate is resuspended by low-temperature ultrasonication to obtain a cell membrane solution, and the cell membrane concentration is determined by the BCA method.

[0019] More preferably, in step (4), the low-temperature ultrasonic temperature is 2 to 8°C.

[0020] Preferably, in step (4), the mass ratio of dual-drug loaded polypyrrole to cell membrane is 2:0.5-1.5; and the low-temperature ultrasonic time is 0.2-1 h.

[0021] The room temperature in the present invention refers to 25°C.

[0022] The present invention also provides polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticles prepared by the above method, wherein the diameter of the nanoparticles is 40 to 150 nm.

[0023] The present invention also provides the use of the above-mentioned stimulation-responsive bionic drug-loaded nanoparticles based on polypyrrole in the preparation of drugs for treating acute brain injury diseases.

[0024] Compared with the prior art, the beneficial effects of the present invention include: (1) The bionic drug-loaded nanoparticles prepared by the present invention have small particle size, uniform morphology, high drug loading efficiency, easy storage, and good biocompatibility.

[0025] (2) The bionic drug-loaded nanoparticles prepared by the present invention can be used as nanomedicines for the treatment of acute brain injury diseases and are suitable for intravascular injection. The bionic nanoparticles can recognize the relevant receptors expressed by the blood-brain barrier, target the blood-brain barrier and internalize into the brain, and accumulate in the brain lesions. Under the stimulation of high concentrations of reactive oxygen species in the lesions, abnormal bioelectricity and acidic pH of lysosomes in neuronal cells, the relevant drugs are selectively released, thereby exerting the therapeutic effects of protecting neurons, inhibiting reactive gliosis and repairing damaged blood-brain barrier. At the same time, the phenylboronic acid group can remove reactive oxygen species in the lesions, synergistically reduce oxidative stress and alleviate inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthetic route of the nanoparticles loaded with fingolimod and citicoline sodium in Example 1; Figure 2 The synthetic route of the quercetin and clocarbamazepine nanoparticles in Example 2; Figure 3 is the infrared spectrum of pyrrole monomer and polypyrrole; Figure 4 Transmission electron microscopy observation of fingolimod and citicoline sodium loaded nanoparticles before and after encapsulation, and dynamic light scattering particle size distribution curve of the nanoparticles after encapsulation; Figure 5 The drug release curves of biomimetic nanoparticles loaded with fingolimod and citicoline sodium in response to reactive oxygen species, electrical and acid stimulation. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0028] Example 1 A stimuli-responsive bionic fingolimod and citicoline sodium-loaded polypyrrole nanoparticle and a preparation method thereof, Figure 1 The synthetic route comprises the following steps: (1) Dissolve 0.5 mmol sodium dodecyl sulfate in 5 mL 40 mmol / L hydrochloric acid aqueous solution, add 1.5 mmol pyrrole and 250 μL 30% hydrogen peroxide solution (2.5 mmol), stir at room temperature for 24 h, place the product in a dialysis bag with a molecular weight cutoff of 3500, immerse it in water, dialyze to remove impurities, and freeze-dry to obtain a polypyrrole skeleton.

[0029] (2) S1. Disperse 100 mg of polypyrrole skeleton (about 1.5 mmol) in 20 mL of anhydrous dimethyl sulfoxide, add 0.05 mmol 4-bromomethylphenylboronic acid and 0.075 mmol anhydrous potassium carbonate, and stir the reaction at 30 °C under argon protection until the precipitation no longer increases. Filter and collect the filtrate; S2. Add 0.075 mmol fingolimod to the filtrate, and add 0.1 mmol anhydrous sodium sulfate. Under argon protection, stir the reaction at room temperature for 16 h. Filter and collect the filtrate, dialyze in water to remove impurities, and lyophilize to obtain single drug-loaded polypyrrole.

[0030] (3) The above-mentioned single drug-loaded polypyrrole was redispersed in 20 mL of water, and 0.2 mmol of citicoline sodium was added. Under argon protection, the mixture was stirred at room temperature for 16 h. Impurities were removed by dialyzing in water to obtain dual drug-loaded polypyrrole.

[0031] (4) Cultivate RAW 264.7 cells in a 10 cm dish until the confluence exceeds 100%. Collect the cell suspension with a scraper, centrifuge at 250 g for 4 min, and wash three times with PBS 7.4. Add hypotonic solution (hypotonic solution is 0.1×TM buffer containing 1 mmol / L PMSF) to resuspend the cells. Ultrasonicate at 4°C for 1 h, centrifuge at 3200 g for 5 min to remove the cell nucleus, and extract the supernatant. Centrifuge at 20,000 g for 25 min to remove the cell organelles, and extract the supernatant. Ultrasonicate at 100,000 g for 60 min to extract the precipitate to obtain an oily substance, which is the cell membrane. Add water and resuspend the precipitate by ultrasonication at 4°C to obtain the RAW 264.7 cell membrane solution, and determine the cell membrane concentration by BCA method.

[0032] (5) The aqueous solution of dual-drug loaded polypyrrole was mixed with the RAW 264.7 cell membrane solution at a mass ratio of 2:1, ultrasonicated at 4°C for 0.5 h, and filtered through 0.45 μm and 0.22 μm filter membranes to obtain biomimetic nanoparticles loaded with fingolimod and citicoline sodium.

[0033] Example 2 A kind of stimulation responsive bionic quercetin and clocarbamazepine nanoparticles based on polypyrrole and preparation method thereof, according to Figure 2 The synthetic route comprises the following steps: (1) Dissolve 0.5 mmol sodium dodecyl sulfate in 5 mL of 40 mmol / L hydrochloric acid aqueous solution, add 1.5 mmol pyrrole and 1.35 mL of 30% ferric chloride solution (2.5 mmol), stir at room temperature for 24 h, place the product in a dialysis bag with a molecular weight cutoff of 3500, immerse it in water, dialyze to remove impurities, and freeze-dry to obtain a polypyrrole skeleton.

[0034] (2) S1. Disperse 100 mg of polypyrrole skeleton (about 1.5 mmol) in 20 mL of anhydrous dimethyl sulfoxide, add 0.05 mmol of 4-chloromethylphenylboronic acid and 0.075 mmol of anhydrous potassium carbonate, and stir the reaction at 30 °C under argon protection until the precipitation no longer increases. Filter and collect the filtrate; S2. Add 0.075 mmol of quercetin to the filtrate, and add 0.1 mmol of anhydrous sodium sulfate. Under argon protection, stir the reaction at room temperature for 16 h. Filter and collect the filtrate, dialyze in water to remove impurities, and lyophilize to obtain single drug-loaded polypyrrole.

[0035] (3) The above-mentioned single drug-loaded polypyrrole was completely redispersed in 20 mL of water, and 0.2 mmol of clocarbachol was added. Under argon protection, the reaction was stirred at room temperature for 16 h. The impurities were removed by dialyzing in water to obtain dual drug-loaded polypyrrole.

[0036] (4) SH-SY5Y cells were cultured in a 10 cm dish until the confluence exceeded 100%. The cell suspension was collected with a scraper, centrifuged at 250 g for 4 min, and washed three times with PBS 7.4. Hypotonic solution (hypotonic solution is 0.1×TM buffer containing 1 mmol / L PMSF) was added to resuspend the cells. Ultrasonication was performed at 4 °C for 1 h, and the cell nuclei were removed by centrifugation at 3200 g for 5 min, and the supernatant was extracted. The cell organelles were removed by centrifugation at 20,000 g for 25 min, and the supernatant was extracted. The precipitate was extracted by ultracentrifugation at 100,000 g for 60 min to obtain an oily substance, which is the cell membrane. Water was added and the precipitate was resuspended by ultrasonication at 4 °C to obtain the SH-SY5Y cell membrane solution, and the cell membrane concentration was determined by the BCA method.

[0037] (5) The aqueous solution of dual-drug loaded polypyrrole was mixed with the SH-SY5Y cell membrane solution at a mass ratio of 2:1, ultrasonicated at 4 °C for 0.5 h, and filtered through 0.45 μm and 0.22 μm filter membranes to obtain biomimetic quercetin and clocarbamide nanoparticles.

[0038] The bionic drug-loaded nanoparticles were subjected to the following tests: Figure 3 The infrared spectra of pyrrole monomer and polypyrrole are shown in the fingerprint region at 1000 cm -1 Nearby, it can be observed that the =CH– bending vibration signal is significantly reduced, proving that the polymerization is successful.

[0039] Figure 4 To observe the nanoparticles loaded with fingolimod and citicoline sodium before and after encapsulation using transmission electron microscopy, the samples were negatively stained with phosphotungstic acid, and it was seen that after encapsulation, high-contrast material appeared around the nanoparticles, proving that the encapsulation was successful, and the particle size was consistent with the dynamic light scattering measurement result, which was about 55 nm; Figure 5 The drug release curves of biomimetic nanoparticles loaded with fingolimod and citicoline sodium in response to reactive oxygen species, electrical and acid stimulation.

[0040] Hydrogen peroxide is one of the most typical reactive oxygen species in the body, so hydrogen peroxide solution was selected to simulate reactive oxygen with a concentration of 50 μmol / L, and incubated with biomimetic nanoparticles at 37 °C. Considering that the electric field strength of abnormal bioelectricity in brain lesions can reach 20 mV / m, an electric field strength of 50 mV / m was set to simulate abnormal bioelectric stimulation. In addition, considering that the pH of lysosomes in neuronal cells can be as low as 4.0, pH 6.0 and pH 5.0 were selected to simulate the acidic lysosomal environment. It can be seen that: over time, biomimetic nanoparticles produce more and more 4-hydroxybenzyl alcohol and fingolimod under the stimulation of hydrogen peroxide; while without hydrogen peroxide stimulation, biomimetic nanoparticles hardly release 4-hydroxybenzyl alcohol and fingolimod. In addition, electrical stimulation can further accelerate the release of 4-hydroxybenzyl alcohol and fingolimod from biomimetic nanoparticles. Acidic pH can also promote the release of citicoline sodium from biomimetic nanoparticles.

Claims

1. A method for preparing stimuli-responsive bionic drug-loaded nanoparticles based on polypyrrole, characterized in that: The following steps are involved: (1) The suspending agent is dissolved in a hydrochloric acid aqueous solution, and pyrrole and an oxidant are added, and the mixture is stirred at room temperature for 24 h. The product is placed in a dialysis bag with a molecular weight cutoff of 3500, immersed in water, dialyzed to remove impurities, and freeze-dried to obtain a polypyrrole skeleton; The reaction process is shown in Formula I: Formula I (2) S1. Disperse the polypyrrole skeleton in an organic solvent, add 4-halogenated methylphenylboronic acid and an acid-binding agent, and stir the reaction at 25 to 50° C. under the protection of an inert gas until the precipitation no longer increases, and filter and collect the filtrate; S2. Adding a drug containing a vicinal diol or propylene glycol structure to the filtrate, and adding a dehydrating agent, reacting at room temperature for 6 h to 24 h under the protection of an inert gas, filtering and collecting the filtrate, dialyzing in water to remove impurities, and freeze-drying to obtain a single drug-loaded polypyrrole; The reaction process is shown in Formula II: Formula II Wherein, X represents a halogen atom, Indicates drugs containing vicinal diol structures, Indicates drugs containing propylene glycol structure; (3) dispersing the single drug-loaded polypyrrole in water, adding charged drugs, stirring and reacting at room temperature for 6 h to 24 h under the protection of inert gas, dialyzing in water to remove impurities, and obtaining dual drug-loaded polypyrrole; The reaction process is shown in Formula III: or, Formula III in, Indicates charged drugs; (4) The aqueous solution of dual-drug loaded polypyrrole was mixed with the cell membrane solution, subjected to low-temperature ultrasound, and filtered through 0.45 μm and 0.22 μm filter membranes to obtain bionic drug-loaded nanoparticles.

2. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (1), the molar ratio of the suspending agent, pyrrole and oxidant is 0.5:1-1.5:2-2.5; Preferably, in step (1), the suspending agent is sodium dodecyl sulfate, and the oxidant is selected from hydrogen peroxide or ferric chloride; Preferably, the concentration of the aqueous hydrochloric acid solution in step (1) is 20 mmol / L to 100 mmol / L.

3. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (2), the molar ratio of 4-halomethylphenylboronic acid, the acid-binding agent, the drug containing a vicinal diol or propylene glycol structure and the dehydrating agent is 1:1.2~1.5:1.2~1.5:

2.

4. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (2), the organic solvent is anhydrous dimethyl sulfoxide; the acid binding agent is anhydrous potassium carbonate; and the dehydrating agent is anhydrous sodium sulfate.

5. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (3), the molar ratio of the phenylboronic acid groups contained in the single drug-loaded polypyrrole to the charged drug is 1:0.5-10.

6. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (3), the charged drug is a quaternary ammonium salt drug.

7. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (4), the cell membrane is extracted from RAW 264.7 cells or SH-SY5Y cells; Preferably, in step (4), the preparation method of the cell membrane solution is as follows: culturing cells in a 10 cm dish until the confluence exceeds 100%, collecting the cell suspension with a scraper, centrifuging at 250 g for 4 min, and washing three times with PBS 7.4; adding hypotonic solution to resuspend the cells, the hypotonic solution being 0.1×TM buffer containing 1 mmol / L PMSF; low-temperature ultrasonication for 1 h, centrifuging at 3200 g for 5 min to remove the cell nucleus, and extracting the supernatant; centrifuging at 20,000 g for 25 min to remove the cell organelles, and extracting the supernatant; and then ultracentrifuging at 100,000 g for 60 min to extract the precipitate to obtain an oily substance, which is the cell membrane; adding water, and resuspending the precipitate by low-temperature ultrasonication to obtain a cell membrane solution, and determining the cell membrane concentration by the BCA method; Further preferably, in step (4), the low-temperature ultrasonic temperature is 2 to 8°C.

8. The method for preparing a polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (4), the mass ratio of the dual-drug loaded polypyrrole to the cell membrane is 2:0.5-1.5; and the low-temperature ultrasonic time is 0.2-1 h.

9. Stimuli-responsive bionic drug-loaded nanoparticles based on polypyrrole prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the polypyrrole-based stimulus-responsive bionic drug-loaded nanoparticles according to claim 9 in the preparation of drugs for treating acute brain injury diseases.

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