A polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle and preparation method thereof

Abstract

The present invention belongs to the technical field of stimulus-responsive drug formulations, and particularly relates to a stimulus-responsive biomimetic drug-loaded nanoparticle based on polypyrrole and a preparation method thereof. The preparation method is as follows: First, pyrrole is reacted with an oxidant to obtain a polypyrrole skeleton, then the polypyrrole skeleton is coupled with halomethylphenylboronic acid, and then through a dehydration reaction, the phenylboronic acid group is coupled with a drug containing an ortho-diol or propylene glycol structure; Next, the polypyrrole skeleton can adsorb charged drugs, and finally, the cell membrane is wrapped to obtain a biomimetic drug-loaded nanoparticle. The biomimetic nanoparticle can recognize the relevant receptors expressed on the blood-brain barrier, target and internalize into the brain at the blood-brain barrier, accumulate in the brain lesions, and selectively release relevant drugs under the stimulation of high-concentration reactive oxygen species, abnormal bioelectricity, and acidic pH of lysosomes in neurons in the lesions, exerting the curative effects of protecting neurons, inhibiting reactive gliosis, and repairing the damaged blood-brain barrier; At the same time, the phenylboronic acid group can scavenge the reactive oxygen species in the lesions, synergistically reduce oxidative stress, and relieve the inflammatory response.

Description

Technical Field

[0001] The present invention belongs to the technical field of stimulus-responsive pharmaceutical preparations, and particularly relates to a stimulus-responsive biomimetic 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 lesions, central injuries, or infections, causing diseases such as stroke, traumatic brain injury, and acute seizure of epilepsy. During the development of such diseases, the injury leads to local neuronal ischemia and hypoxia. On the one hand, it causes the mitochondrial electron transport chain of neurons to be overly active, generating reactive oxygen species, which diffuse from intracellular to extracellular, further damaging other neurons in the lesion and producing more reactive oxygen species. On the other hand, reactive oxygen species simultaneously stimulate microglia and astrocytes, causing reactive gliosis, secreting pro-inflammatory factors, and clearing damaged neurons. Dead neurons themselves also release pro-inflammatory factors, exacerbating the inflammatory response in the lesion. In addition, activated astrocytes lose their role as a glutamate "reservoir", resulting in an abnormal increase in glutamate concentration in the lesion, directly stimulating abnormal neuronal discharge and causing excitotoxic damage to neurons. Therefore, the oxidative stress of neurons and the inflammatory response of glial cells form a vicious cycle, continuously deteriorating the microenvironment of the brain lesion. At the same time, factors such as ischemia and hypoxia induce compensatory hyperplasia of cerebral blood vessels, but the newly formed blood vessels do not have a complete blood-brain barrier function, causing exogenous immune cells and albumin to leak into the brain parenchyma, further stimulating the immune inflammatory response in the lesion.

[0003] Therefore, for drug therapies for acute brain injury diseases, it should start from the "neurovascular unit" as a whole. On the one hand, it is necessary to rescue 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 also necessary to repair damaged cerebral blood vessels and block the leakage of exogenous substances. At the same time, when delivering drugs to the brain lesion, it is also necessary to consider overcoming the obstacle of the blood-brain barrier, improving the efficiency of drug entry into the brain; and enabling the drug to be fully enriched in the lesion to achieve targeted accumulation, on-demand drug release, and long-acting release effects. Summary of the Invention

[0004] The present invention provides a stimulus-responsive biomimetic drug-loaded nanoparticle based on polypyrrole and a preparation method thereof. On the premise of fully considering the physical and chemical factors related to acute brain injury (including high concentrations of reactive oxygen species and abnormally active bioelectricity), first, a polypyrrole skeleton with electrostimulus responsiveness is prepared based on a conductive polymer; then, a reactive oxygen species-responsive and scavenging phenylboronic acid group is introduced and coupled with a drug containing a vicinal diol or propylene glycol structure; then, a charged drug is adsorbed, and finally, a cell membrane is wrapped to obtain a biomimetic drug-loaded nanoparticle. The biomimetic nanoparticle can recognize the relevant receptors expressed on the blood-brain barrier, target and internalize into the brain, accumulate in the brain lesions, and selectively release relevant drugs under the stimulation of high concentrations of reactive oxygen species, abnormal bioelectricity, and acidic pH in lysosomes of neuronal cells at the lesions, so as to play the curative effects of protecting neurons, inhibiting reactive gliosis, and repairing the damaged blood-brain barrier; at the same time, the phenylboronic acid group can scavenge the reactive oxygen species at the lesions, synergistically reduce oxidative stress, and relieve the inflammatory response.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] A preparation method of a stimulus-responsive biomimetic drug-loaded nanoparticle based on polypyrrole, comprising the following steps:

[0007] (1) Dissolve a suspending agent in an aqueous hydrochloric acid solution, add pyrrole and an oxidant, stir and react at room temperature for 24 h, place the product in a dialysis bag with a cut-off molecular weight of 3500, immerse it in water, dialyze to remove impurities, and freeze-dry to obtain a polypyrrole skeleton;

[0008] The reaction process is shown in Formula I:

[0009]

[0010] Formula I

[0011] (2) S1. Disperse the polypyrrole skeleton in an organic solvent, add 4-halomethylphenylboronic acid and a deacidifying agent, stir and react at 25-50 °C under the protection of an inert gas until the precipitation no longer increases, filter and collect the filtrate; S2. Add a drug containing a vicinal diol or propylene glycol structure to the filtrate, add a dehydrating agent, stir and react at room temperature for 6 h-24 h under the protection of an inert gas, filter and collect the filtrate, dialyze in water to remove impurities, and freeze-dry to obtain a polypyrrole loaded with a single drug;

[0012] The reaction process is shown in Formula II:

[0013]

[0014] Formula II

[0015] Wherein, X represents a halogen atom, represents a drug containing a vicinal diol structure, A drug containing a propylene glycol structure;

[0016] (3) Disperse the single-drug-loaded polypyrrole in water, add a charged drug, and under the protection of an inert gas, stir and react at room temperature for 6 h to 24 h, and dialyze in water to remove impurities to obtain a double-drug-loaded polypyrrole;

[0017] The reaction process is shown in Formula III:

[0018]

[0019] Formula III

[0020] Or,

[0021]

[0022] Wherein, represents a charged drug;

[0023] (4) Mix the aqueous solution of the double-drug-loaded polypyrrole with the cell membrane solution, ultrasonicate at low temperature, and successively pass through 0.45 μm and 0.22 μm filter membranes to obtain the biomimetic drug-loaded nanoparticles.

[0024] Preferably, in the step (1), the molar ratio of the suspending agent, pyrrole to the oxidant is 0.5: 1 to 1.5: 2 to 2.5.

[0025] Preferably, in the step (1), the suspending agent is sodium dodecyl sulfate, and the oxidant is selected from hydrogen peroxide or ferric chloride.

[0026] Preferably, in the step (1), the concentration of the hydrochloric acid aqueous solution is 20 mmol / L to 100 mmol / L.

[0027] Preferably, in the step (2), the molar ratio of 4-halomethylphenylboronic acid, the acid-binding agent, the drug containing an o-diol or propylene glycol structure to the dehydrating agent is 1: 1.2 to 1.5: 1.2 to 1.5: 2.

[0028] Preferably, 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.

[0029] Preferably, in the step (3), the molar ratio of the phenylboronic acid group contained in the single-drug-loaded polypyrrole to the charged drug is 1: 0.5 to 10.

[0030] Preferably, in the step (3), the charged drug is a quaternary ammonium salt drug.

[0031] Preferably, in the step (4), the cell membrane is extracted from RAW 264.7 cells or SH-SY5Y cells.

[0032] More preferably, in step (4), the method for preparing the cell membrane solution is as follows: Cells are cultured in a 10-cm petri dish until the confluence exceeds 100%. The cell suspension is collected with a cell scraper, centrifuged at 250 g for 4 min, and washed three times with PBS 7.4. The cells are resuspended in a hypotonic solution, which is 0.1×TM buffer containing 1 mmol / L PMSF. Ultrasonic treatment is performed at low temperature for 1 h, and the cell nuclei are removed by centrifugation at 3200 g for 5 min, and the supernatant is extracted. Then, the cell organelles are removed by centrifugation at 20000 g for 25 min, and the supernatant is extracted. Then, the precipitate is extracted by ultracentrifugation at 100000 g for 60 min to obtain an oily substance, which is the cell membrane. Water is added, and the precipitate is resuspended by ultrasonic treatment at low temperature to obtain the cell membrane solution, and the concentration of the cell membrane is determined by the BCA method.

[0033] Even more preferably, in step (4), the low-temperature ultrasonic treatment temperature is 2 - 8°C.

[0034] Preferably, in step (4), the mass ratio of the double-drug-loaded polypyrrole to the cell membrane is 2:0.5 - 1.5; the low-temperature ultrasonic treatment time is 0.2 - 1 h.

[0035] In the present invention, the room temperature refers to 25°C.

[0036] The present invention also provides a polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle prepared by the above method, and the diameter of the nanoparticle is 40 - 150 nm.

[0037] The present invention also provides the application of the polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle in the preparation of a drug for treating acute brain injury diseases.

[0038] Compared with the prior art, the beneficial effects of the present invention include:

[0039] (1) The biomimetic drug-loaded nanoparticles prepared by the present invention have a small particle size, uniform morphology, high drug-loading efficiency, are easy to store, and have good biocompatibility.

[0040] (2) The biomimetic drug-loaded nanoparticles prepared by the present invention can be used as nano-drugs for treating acute brain injury diseases and are suitable for intravascular injection. The biomimetic nanoparticles can recognize the related receptors expressed on the blood-brain barrier, target to the blood-brain barrier and internalize into the brain, accumulate in the brain lesions, and selectively release related drugs under the stimulation of high-concentration reactive oxygen species, abnormal bioelectricity, and acidic pH of lysosomes in neurons in the lesions, so as to play the curative effects of protecting neurons, inhibiting reactive gliosis, and repairing the damaged blood-brain barrier. At the same time, the phenylboronic acid group can scavenge the reactive oxygen species in the lesions and synergistically reduce oxidative stress and relieve the inflammatory response. Description of the Drawings

[0041] Figure 1Synthesis route of fingolimod- and citicoline sodium-loaded nanoparticles in Example 1;

[0042] Figure 2 Synthesis route of quercetin- and carbachol-loaded nanoparticles in Example 2;

[0043] Figure 3 Infrared spectra of pyrrole monomer and polypyrrole;

[0044] Figure 4 Transmission electron microscopy observation of fingolimod- and citicoline sodium-loaded nanoparticles before and after coating, and dynamic light scattering particle size distribution curve of the coated nanoparticles;

[0045] Figure 5 Curves of the release of drugs by the biomimetic fingolimod- and citicoline sodium-loaded nanoparticles in response to reactive oxygen species, electricity, and acid stimuli. Detailed implementation manners

[0046] 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 progresses. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0047] Example 1

[0048] A polypyrrole-based stimulus-responsive biomimetic fingolimod- and citicoline sodium-loaded nanoparticle and its preparation method, according to the Figure 1 synthesis route in, includes the following steps:

[0049] (1) Dissolve 0.5 mmol of sodium dodecyl sulfate in 5 mL of 40 mmol / L hydrochloric acid aqueous solution, add 1.5 mmol of pyrrole and 250 μL of 30% hydrogen peroxide solution (2.5 mmol), stir and react at room temperature for 24 h, place the product in a dialysis bag with a molecular weight cut-off of 3500, immerse it in water, dialyze to remove impurities, and lyophilize to obtain a polypyrrole skeleton.

[0050] (2)S1. Disperse 100 mg of polypyrrole framework (about 1.5 mmol) in 20 mL of anhydrous dimethyl sulfoxide, add 0.05 mmol of 4-bromomethylphenylboronic acid and 0.075 mmol of anhydrous potassium carbonate, and under argon protection, stir and react at 30 °C until the precipitation no longer increases. Filter and collect the filtrate; S2. Add 0.075 mmol of fingolimod to this filtrate, add 0.1 mmol of anhydrous sodium sulfate, under argon protection, stir and react at room temperature for 16 h, filter and collect the filtrate, dialyze in water to remove impurities, and lyophilize to obtain polypyrrole loaded with a single drug.

[0051] (3)Redisperse all of the above-mentioned polypyrrole loaded with a single drug in 20 mL of water, add 0.2 mmol of cytidine choline sodium, under argon protection, stir and react at room temperature for 16 h, dialyze in water to remove impurities, and obtain polypyrrole loaded with two drugs.

[0052] (4)Culture RAW 264.7 cells in a 10 cm petri dish until the confluence exceeds 100%. Scrape to collect the cell suspension, centrifuge at 250 g for 4 min, and wash 3 times with PBS 7.4. Resuspend the cells with a hypotonic solution (the hypotonic solution is 0.1×TM buffer containing 1 mmol / L PMSF). Sonicate at 4 °C for 1 h, centrifuge at 3200 g for 5 min to remove the cell nuclei, and extract the supernatant. Then centrifuge at 20000 g for 25 min to remove the cell organelles, and extract the supernatant. Then ultracentrifuge at 100000 g for 60 min to extract the precipitate, and obtain an oily substance, which is the cell membrane. Add water, sonicate and resuspend the precipitate at 4 °C to obtain the RAW 264.7 cell membrane solution, and determine the cell membrane concentration by the BCA method.

[0053] (5)Mix the aqueous solution of polypyrrole loaded with two drugs and the RAW 264.7 cell membrane solution according to the mass ratio of the dispersed substance of 2:1, sonicate at 4 °C for 0.5 h, and successively pass through 0.45 μm and 0.22 μm filter membranes to obtain biomimetic nanoparticles loaded with fingolimod and cytidine choline sodium.

[0054] Example 2

[0055] A polypyrrole-based stimulus-responsive biomimetic nanoparticle loaded with quercetin and carbachol and a preparation method, according to the Figure 2 synthesis route therein, comprising the following steps:

[0056] (1)Dissolve 0.5 mmol of sodium dodecyl sulfate in 5 mL of 40 mmol / L hydrochloric acid aqueous solution, add 1.5 mmol of pyrrole and 1.35 mL of 30% ferric chloride solution (2.5 mmol), stir and react at room temperature for 24 h, place the product in a dialysis bag with a molecular weight cut-off of 3500, immerse it in water, dialyze to remove impurities, and lyophilize to obtain the polypyrrole framework.

[0057] (2) S1. Disperse 100 mg of polypyrrole framework (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, under argon protection, stir and react at 30 °C 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 and react at room temperature for 16 h, filter and collect the filtrate, dialyze in water to remove impurities, and lyophilize to obtain polypyrrole loaded with a single drug.

[0058] (3) Redisperse all of the above single-drug-loaded polypyrrole in 20 mL of water, add 0.2 mmol of carbachol, under argon protection, stir and react at room temperature for 16 h, dialyze in water to remove impurities, and obtain polypyrrole loaded with two drugs.

[0059] (4) Culture SH-SY5Y cells in a 10 cm petri dish until the confluence exceeds 100%, scrape to collect the cell suspension, centrifuge at 250 g for 4 min, and wash 3 times with PBS 7.4. Resuspend the cells with a hypotonic solution (the hypotonic solution is 0.1×TM buffer containing 1 mmol / L PMSF). Sonicate at 4 °C for 1 h, centrifuge at 3200 g for 5 min to remove the cell nuclei, and extract the supernatant. Then centrifuge at 20000 g for 25 min to remove the cell organelles, and extract the supernatant. Then ultracentrifuge at 100000 g for 60 min to extract the precipitate, and obtain an oily substance, which is the cell membrane. Add water, sonicate and resuspend the precipitate at 4 °C to obtain the SH-SY5Y cell membrane solution, and determine the cell membrane concentration by the BCA method.

[0060] (5) Mix the aqueous solution of the polypyrrole loaded with two drugs and the SH-SY5Y cell membrane solution according to the mass ratio of the dispersed substances of 2:1, sonicate at 4 °C for 0.5 h, and successively filter through 0.45 μm and 0.22 μm filters to obtain the biomimetic nanoparticles loaded with quercetin and carbachol.

[0061] The following tests were performed on the above biomimetic drug-loaded nanoparticles:

[0062] Figure 3 are the infrared spectra of pyrrole monomer and polypyrrole. In the fingerprint region around 1000 cm -1 nearby, it can be observed that the =CH– bending vibration signal decreases significantly, proving successful polymerization.

[0063] Figure 4For observing the fingolimod- and citicoline-loaded nanoparticles before and after coating by transmission electron microscopy, the samples were negatively stained with phosphotungstic acid. After coating, high-contrast substances appeared around the nanoparticles, indicating successful coating. The particle size was consistent with the results of dynamic light scattering measurement, approximately 55 nm.

[0064] Figure 5 It is the curve of the biomimetic fingolimod- and citicoline-loaded nanoparticles for decomposing and releasing drugs in response to reactive oxygen species, electricity, and acid stimuli.

[0065] Hydrogen peroxide is one of the most typical reactive oxygen species in vivo. Therefore, hydrogen peroxide solution was selected to simulate reactive oxygen species at a concentration of 50 μmol / L and co-incubated with the 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 bioelectricity stimulation. In addition, considering that the pH of lysosomes in neuron 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, more and more 4-hydroxybenzyl alcohol and fingolimod were produced by the biomimetic nanoparticles under hydrogen peroxide stimulation; without hydrogen peroxide stimulation, the biomimetic nanoparticles hardly released 4-hydroxybenzyl alcohol and fingolimod. In addition, electrical stimulation could further accelerate the release of 4-hydroxybenzyl alcohol and fingolimod from the biomimetic nanoparticles. Acidic pH could also promote the release of citicoline from the 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, and dialyzing in water to remove impurities to obtain dual drug-loaded polypyrrole; The reaction process is shown in Formula III: or, Formula III in, Indicates charged drugs; (4) The dual-drug-loaded polypyrrole aqueous solution is 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; The drug containing a vicinal diol or propylene glycol structure is fingolimod or quercetin; The charged drug is citicoline sodium or clocarbamazepine; Cell membranes were extracted from RAW 264.7 cells or SH-SY5Y cells.

2. The method for preparing a polypyrrole-based stimulus-responsive biomimetic 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.

3. The method for preparing a polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle according to claim 1, characterized in that: In step (1), the suspending agent is sodium lauryl sulfate, and the oxidant is selected from hydrogen peroxide or ferric chloride.

4. The method for preparing a stimulus-responsive bionic drug-loaded nanoparticle based on polypyrrole according to claim 1, characterized in that: The concentration of the aqueous hydrochloric acid solution in step (1) is 20 mmol / L to 100 mmol / L.

5. The method for preparing a polypyrrole-based stimulus-responsive biomimetic 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.

6. The method for preparing a stimulus-responsive bionic drug-loaded nanoparticle based on polypyrrole 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.

7. The method for preparing a polypyrrole-based stimulus-responsive biomimetic 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.

8. The method for preparing a polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle according to claim 1, characterized in that: In the step (4), the preparation method of 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 with a 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.

9. The method for preparing a polypyrrole-based stimulus-responsive biomimetic drug-loaded nanoparticle according to claim 8, characterized in that: In the step (4), the low-temperature ultrasonic temperature is 2 to 8°C.

10. The method for preparing a stimulus-responsive bionic drug-loaded nanoparticle based on polypyrrole 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.

11. Stimuli-responsive bionic drug-loaded nanoparticles based on polypyrrole prepared according to the preparation method according to any one of claims 1 to 10.

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