CN113384552A - Double-drug-loading nano preparation for neurodegenerative diseases and preparation method thereof - Google Patents
Double-drug-loading nano preparation for neurodegenerative diseases and preparation method thereof Download PDFInfo
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- CN113384552A CN113384552A CN202110529193.XA CN202110529193A CN113384552A CN 113384552 A CN113384552 A CN 113384552A CN 202110529193 A CN202110529193 A CN 202110529193A CN 113384552 A CN113384552 A CN 113384552A
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Abstract
The invention discloses a double-drug-loading nano preparation for neurodegenerative diseases and a preparation method thereof, wherein the preparation comprises the following components in parts by weight: a cell membrane fraction having amphiphilicity, and a drug containing a benzene ring and a drug containing a guanidine group loaded in the cell membrane fraction. The preparation uses cell membrane components with high biocompatibility and amphiphilicity as carriers to load drugs containing benzene rings and drugs containing guanidyl, can effectively improve the treatment effect, obviously improve cognition, protect neurons, inhibit the expression of inflammasome and delay the disease progression on the premise of not bringing other side effects.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a double-drug-loading nano preparation for neurodegenerative diseases and a preparation method thereof.
Background
Neurodegenerative diseases are caused by the loss of neurons and/or their myelin sheaths, which worsen over time and become dysfunctional. Such diseases put heavy stress on patients, families and society; however, the etiology is complex and numerous hypotheses exist. Effective therapeutic means are not yet available.
Donepezil (Don) was approved by the FDA in the united states for the treatment of neurodegenerative diseases in 1996. It is a reversible non-competitive inhibitor of acetylcholinesterase. Can increase the level of neurotransmitter acetylcholine in brain, thereby enhancing cholinergic neurotransmission and regulating cognitive function. The curative effect of the long-term use of donepezil has been clinically verified. However, prolonged use, especially at high doses, can produce a variety of side effects including nausea, difficulty falling asleep, aggression, diarrhea, tiredness, muscle spasms, and serious side effects may include cardiac arrhythmias, bladder dysuria, seizures.
Metformin (Met) is a hydrophilic drug of choice for the treatment of type 2 diabetes and has been shown to exert an anti-inflammatory effect in neurological disorders, but has a short half-life and insignificant neural enrichment.
Therefore, the side effect of the medicine is reduced, the half-life period of the medicine in vivo is prolonged, the bioavailability of the medicine is improved, and the medicine has important clinical application significance.
Disclosure of Invention
In order to solve the problems, the invention provides a double-drug-loading nano preparation for neurodegenerative diseases and a preparation method thereof, and the double-drug-loading nano preparation effectively improves the treatment effect, obviously improves cognition, protects neurons, inhibits the expression of inflammatory bodies and delays the disease progress on the premise of not bringing other side effects.
To achieve the above objects, embodiments of the present invention in one aspect propose a double drug-loaded nano-formulation for neurodegenerative diseases, which includes: a cell membrane fraction having amphiphilicity, and a drug containing a benzene ring and a drug containing a guanidine group loaded in the cell membrane fraction.
According to the double-drug-loading nano preparation for neurodegenerative diseases, the cell membrane component with high biocompatibility and amphiphilicity is used as a carrier to load the drug containing the benzene ring and the drug containing the guanidyl, so that the safety is high, the influence of the barrier action in a receptor is small by the advantage of the cross-in-vivo barrier of the nano particle, the accumulation of a phospholipid delivery system in a nervous system is increased, and the distribution of the two drugs in the nervous system can be improved; and the clinical drugs (drugs containing benzene rings) such as enzyme inhibitors or receptor blockers are innovatively provided as 'probes' to promote the enrichment of drugs containing guanidine groups in the brain and treat nervous system diseases, so that the anti-dementia effect of the drugs containing benzene rings is enhanced by relatively increasing the content of the drugs containing guanidine groups in diseased parts, the purpose of synergistically treating the nervous system diseases is achieved, and the nervous system function can be enhanced and the neuronal injury and inflammatory reaction can be inhibited at the same time. Therefore, on the premise of no other side effects, the traditional Chinese medicine composition effectively improves the treatment effect, obviously improves cognition, protects neurons, inhibits the expression of inflammatory bodies and delays the disease progression.
The double drug-loaded nano preparation for the neurodegenerative diseases provided by the embodiment of the invention also comprises the following characteristics:
optionally, the membrane fraction has one or more hydrophilic polar heads, two or more hydrophobic tails.
Further, the hydrophilic polar head is a phosphate group, and the hydrophobic tail is a saturated fatty acid or an unsaturated fatty acid.
Optionally, the drug containing a benzene ring is one of donepezil, chlorimipramine, edaravone, galantamine, ketamine, imipramine, fluoxetine, penicillin, metronidazole, clozapine, and amoxicillin.
Optionally, the drug containing guanidine groups is one of metformin, guanethidine, guanidyl chlorophenol, aciclovir, moroxydine, arginine and guanidinoacetic acid.
Optionally, the drug loading rate of the drug containing the benzene ring is 5-20%; the drug loading rate of the drug containing guanidyl is 5-20%.
Optionally, the weight ratio of drug loaded in the cell membrane fraction to the cell membrane fraction is 1: 2.
The embodiment of the invention provides a preparation method of the double drug-loaded nano preparation, which comprises the following steps: dissolving a medicine containing a benzene ring, a medicine containing guanidyl and a cell membrane component containing amphipathy in an organic solvent by adopting an ultrasonic thin film hydration method, heating and stirring for 12 hours, and removing the organic solvent by rotary evaporation to form a first thin film; ultrasonically dissolving the first film in an organic solvent in which the two single drugs are insoluble, filtering, and removing the organic solvent by rotary evaporation to form a second film; and adding the second film into the aqueous solution, and performing room temperature ultrasonic treatment to obtain the double-drug-loaded nano preparation.
According to the preparation method of the double-drug-loading nano preparation, disclosed by the embodiment of the invention, a cell membrane component with high biocompatibility and amphiphilicity is used as a carrier to wrap a drug containing a benzene ring and a drug containing guanidyl through an ultrasonic thin film hydration method, so that the accumulation of a phospholipid delivery system in a nervous system is increased and the content of the phospholipid delivery system in the nervous system is increased based on the advantage of the nano particle in vivo crossing barrier; the distribution of the two drugs in the nervous system can be improved; and the anti-dementia effect of the medicament containing the benzene ring is enhanced by relatively increasing the content of the medicament containing guanidyl in the pathological part, thereby playing the aim of cooperatively treating nervous system diseases. Therefore, on the premise of no other side effects, the traditional Chinese medicine composition effectively improves the treatment effect, obviously improves cognition, protects neurons, inhibits the expression of inflammatory bodies and delays the disease progression.
Optionally, the organic solvent is one or more of methanol, n-hexane, tetrahydrofuran, ethanol, chloroform, and acetone.
Optionally, the aqueous solution is one or more of normal saline, phosphate buffer, and glucose solution.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
Fig. 1 is an electron microscope scan of dual drug-loaded nano-formulation MDP NPs according to example 1 of the present invention;
fig. 2 is a hydrated particle size plot of dual drug-loaded nano-formulation MDP NPs according to example 1 of the present invention;
fig. 3 is an appearance diagram of dual drug-loaded nano-formulation MDP NPs according to example 1 of the present invention;
FIG. 4 is a graph showing the toxic effect of different concentrations of MDP NPs on red blood cells according to example 2 of the present invention;
FIG. 5 is a graph of the BBB-penetrating and neuronal and microglial cell-targeting effects of MDP NPs according to example 3 of the present invention;
fig. 6 shows the distribution and brain targeting of the dual drug-loaded nano-formulation MDP NPs according to example 4 of the present invention in vivo;
FIG. 7 is a positioning voyage experiment according to embodiment 5 of the present invention;
FIG. 8 is a space exploration experiment according to example 5 of the present invention;
FIG. 9 is a graph showing the effect of MDP NPs according to example 6 of the present invention on neurons in a model mouse with Alzheimer's disease of scopolamine;
FIG. 10 is a graph showing the effect of MDP NPs according to example 7 of the present invention on the activation of microglia in hippocampal and cortical regions and the expression of inflammasome NLRP3 in a mouse model of Alzheimer's disease with scopolamine.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1 metformin (Met)5mg, donepezil (Don)5mg and Phospholipid (PC)20mg, 30mg, 40mg were weighed in one portion each.
The above PC was dissolved in 10mL methanol in a reaction flask, and a magnetic rotor was added, and the mixture was placed on a constant temperature magnetic stirrer set at 50 ℃ and 900 rpm.
Dissolving Met 5mg and Don 5mg in 10mL of methanol together and shaking to obtain a mixed solution; and then dripping the mixture into the methanol dissolved with the PC by using a dropper (dripping while stirring), sealing and stirring overnight, and obtaining the methanol mixed solution containing the metformin-donepezil-phospholipid (MDP, Met-Don-PC) compound the next day.
Transferring the methanol mixed solution into a distillation flask for later use; checking the tightness of condensing equipment of the rotary evaporator, and tightly connecting the liquid receiving flask, the buffer bottle and the vacuum pump; opening condensed water, connecting a distillation flask filled with a target solution with a buffer bottle, closing a vacuum valve, opening a vacuum pressure pump, setting a proper water temperature, treating for 12 hours, carrying out rotary evaporation to remove the solvent, dissolving a substance adhered to the bottom of the bottle by using n-hexane under the action of an ultrasonic instrument, filtering a 0.45 mu m organic solvent by using a filter to remove undissolved drug monomers, and continuously removing the organic solvent from the obtained n-hexane solution by using a rotary evaporator; obtaining a light yellow paste adhered to the bottom of the bottle, namely a double-drug phospholipid (Met-Don-PC) compound, adding a proper amount of 1mL PBS into the double-drug phospholipid (Met-Don-PC) compound for dissolving, and shaking the mixture to obtain a milky suspension while carrying out ultrasonic treatment, thus obtaining a nano particle solution. Pouring into a tube, sealing with tin foil paper, and standing at-20 deg.C for more than 12 hr; and (3) taking out the solid-state double-drug-loaded nano preparation (MDP NPs) after the solid-state double-drug-loaded nano preparation is observed, pricking 20 small holes on the tin foil paper by using a 5mL syringe needle, and placing the tin foil paper into a freeze dryer for drying (the freeze dryer is precooled for 30min, and the parameter is set to be-50 ℃) for more than 24 h.
The double drug-loaded nano-preparation (MDP NPs) prepared by the embodiment is placed for 5 days and then scanned by an electron microscope, and the result is shown in figure 1, and no precipitation still occurs at room temperature for 5 days, which shows that the double drug-loaded nano-preparation prepared by the embodiment improves the drug solubility; in addition, compared with the rest phospholipid ratio composition, when the mass ratio of the double drugs to the PC is 1:2, the nano structure of the double drug phospholipid nano preparation is the most complete and clear, the particle size is smaller, the structure is more uniform, and no adhesion exists.
The measurement result of the laser dynamic light scattering method is shown in fig. 2, the particle size of the three double-drug-loaded nano preparations is about 180nm, which indicates that the particle size of the three double-drug-loaded nano preparations is relatively stable in different solvents (pure water, PBS, DMEM medium, 10% FBS DMEM medium).
The total dosage is as follows: the phospholipid mass is 1:2(20mg phospholipid) which is a nano solution prepared by the optimal drug carrier ratio, and the nano solution has obvious light beams when being irradiated by laser, namely has obvious Tyndall effect and has the property of a nano structure.
Example 2
Biocompatibility experiments (hemolysis experiments) of double drug-loaded Nanoparticulates MDP NPs (20mg phospholipid prepared in example 1)
C57 mouse eyeball blood 4mL, placed in an anticoagulation centrifuge tube, 4 ℃ 2500r centrifugation for 3min, the obtained precipitate washed twice with PBS, separated to obtain the bottom red precipitate substance, namely red blood cells. The red blood cells were diluted to a 10% suspension with PBS. Simultaneously, 8 1.5mL EP tubes were prepared, and 200. mu. mol of pure water, 200. mu. mol of PBS, 100. mu. mol of MDP NPs, 50. mu. mol of MDP NPs, 25. mu. mol of MDP NPs, 12.5. mu. mol of MDP NPs, and 6.25. mu. mol of MDP NPs (the MDP NPs are calculated as the number of moles of donepezil contained) were added thereto. Adding 200 μ L10% erythrocyte suspension into each tube), incubating at 37 deg.C for 8h, photographing, centrifuging at 4 deg.C 2500r for 5min, collecting supernatant, adding into 96-well plate, adding 3 multiple wells, and measuring absorbance at 540nm and 577nm with microplate reader.
The results are shown in FIG. 4, which is a photograph of hemolysis experiment of nanoparticles with different concentrations and absorbance at corresponding wavelengths. The test result shows that the liquid in the positive control tube is red, no red precipitate is left at the bottom of the tube, which indicates that the positive control tube is completely hemolyzed, the red cells in the sample tube and the negative control tube sink, and the upper layer is clear liquid; the absorbance measurement of the sample tube supernatant shows that when the concentration of the nanoparticles is 6.25, 12.5, 25, 50, 100 and 200 mu mol (taking the concentration of donepezil in the metformin-donepezil-phospholipid nanoparticles as a standard), the solutions with the concentrations at 540nm and 577nm of the characteristic hemoglobin absorption peak have no absorption peak, namely the supernatant has no hemoglobin or only trace hemoglobin, and can be considered as having no hemolysis. When the concentration of the drug reaches 200 mu mol, hemolysis does not occur, which indicates that the nano preparation has good biocompatibility.
Example 3
MDP NPs were labeled with ICG.
The Tranwell dish is a 24-well plate with a chamber (not more than 5 μm membrane pore size) embedded therein, the xend.3 cells are cultured in the upper layer, and the neuronal cells (N2A) or microglia cells (BV2) are cultured in the lower layer. The cell amount per well in the chamber was 5X 104The cell amount of each hole in the 24-hole plate is 4-5 multiplied by 105. And (3) changing the liquid every day after the cells in the chamber are plated, culturing for seven days, and detecting, wherein the 24-hole plate cell plating is carried out 24 hours in advance. During experimental operation, 50 mu M MDPNPs are added into the chamber at different time points of 1h, 6h and 12h respectively, and CO is added2After incubation in the incubator, the ICG fluorescence intensity in N2A or BV2 was measured by flow.
The results are shown in fig. 5, where the cells (N2A or BV2) had greater uptake of nanoparticle-encapsulated ICG than free ICG: MDP NPs have strong blood brain barrier penetrating capability, and can penetrate blood brain barrier to target neurons and microglia.
Example 4
The same dose (0.1mg) of free ICG and ICG-labeled MDP NPs (obtained in example 3) were administered intraperitoneally to BALB/C nude mice (mice aged about 6 weeks, 18-20g), respectively. The distribution of the two formulations was determined under isoflurane anesthesia in nude mice placed in a mouse in vivo imaging system at 0h, 1h, 3h, 6h, 12h, 24h, 36h of administration. The in vivo living body imaging animal is obtained at the end of the process, and in vitro fluorescence imaging is carried out to determine the fluorescence aggregation condition in the brain.
The results are shown in fig. 6, compared with the control group, the double drug-loaded nano preparation MDP NPs have obvious tissue penetration capacity, long in-vivo circulation time and strong brain enrichment effect, and the MDP NPs delivery system can improve the bioavailability of double drug loading and the capacity of double drug-loaded targeting brain.
Example 5
Alzheimer's disease model mouse Water maze test
1. Positioning navigation experiment; the water maze experiment is divided into two parts to be carried out in sequence, and the first part is a positioning navigation experiment stage. The positioning sailing test is carried out for 5 days, and in the process of the test, a small circular platform with the diameter of about 10cm placed at the middle point position of a target quadrant is kept unchanged, and markers distributed on the walls of the four quadrant pools are also kept unchanged. Each mouse was trained twice daily. Because the experimental process is greatly influenced by the experimental mouse, the physical strength of the experimental mouse needs to be ensured, and the body temperature of the experimental mouse needs not to be too low, the moisture of the hair of the experimental mouse needs to be wiped off immediately after each water-entering training is finished, the experimental mouse is placed into a breeding cage with wood chips, and the experimental mouse needs to rest at proper intervals (generally more than 6min), so that the next training cannot be influenced. In the experiment, the head of the mouse is required to face the wall of the pool and is gently put into water, the training time of each time is 90s, and in the 90s, the mouse clicks a test button after entering the water to start recording. When the mouse finds the round platform and stays on the platform for 15s, the recording software automatically ends recording, the data is automatically stored, and then the next experiment is started. If the mouse fails to find the small round platform and does not stay on the small round platform for 15s within a period of 90s, intervening the swimming behavior of the tested mouse, slowly introducing the mouse to the platform, and staying the mouse on the platform for 15 s; in this case, the latency of the mouse was recorded as 90 s. After training or teaching is finished, the mouse is fished out from the water maze and wiped to be dry and put back into the wood chip feeding cage.
2. The second part of the space exploration experiment, mouse behavioural-water maze experiment, is the space exploration experiment. And removing the platform at the central position on the target quadrant the next day after the positioning navigation experiment is finished, and starting a second partial space exploration experiment. The experimental mice still had their heads towards the pool walls and in the space exploration experiments the point of entry was opposite to the quadrant where the target platform was located. The mice were placed in the water maze and allowed to swim freely for 90 s. The tracking software can automatically record in these 90s the swimming trajectory in the water maze, the length of time to swim in the target quadrant, the number of times to cross the target platform, and the swimming distance in the target quadrant, reflecting the memory ability of the experimental mouse after learning training.
As a result, as shown in FIG. 7, it was observed that the swimming trajectories of the mice in each group were significantly different, and the MDP nano-drug group was more concentrated in the target quadrant than the model group. In the localization experiment, the incubation period of the mice in the MDP nano-drug group was shortened compared to the mice in the AD model group on the 5 th day of training. The residence time of a target quadrant and the swimming distance of the target quadrant of the mouse in the AD model group are obviously reduced; compared with the AD model group, the target quadrant residence time of the donepezil group and MDP nano-drug group mice is significantly increased, while the target quadrant swimming distance is also significantly increased; and the residence time of the MDP nano-drug group experimental mice in the target quadrant is obviously longer than that of the physical mixed group, and the nano-particles are supposed to act on the hippocampus which plays a role in spatial memory. The long-term administration of MDP NPs is shown to improve the cognitive dysfunction of AD mice, and the dual drug-loaded nano delivery system can enhance the effect of donepezil on improving the cognitive function of the AD mice through metformin.
Example 6
Materials are taken for dementia models, toluidine blue and NeuN immunofluorescence staining are carried out, survival of hippocampal CA1 neurons is observed, and the experimental process is as follows:
toluidine blue staining
The main components of the Nile body are rough endoplasmic reticulum and free ribosomes; there are relatively significant differences among neurons of different functions. Toluidine blue is a basic dye, can be used for Nisshin body dyeing, and can better react to the Nisshin body state. The specific experimental steps are as follows:
1. airing sheet 2, washing 3, degreasing 5, washing 6, dyeing 7, washing 8, differentiation 9, washing 10, dehydration 11, transparence 12, sealing sheet 13, photographing
Immunofluorescence staining
(1) Selecting brain slice of hippocampus, soaking in PBS for 5min to remove OCT embedding medium
(2) Membrane permeation: then putting into PBST containing 0.3% TritonX-100 for 10min at room temperature, penetrating the membrane, then air drying and drawing circles with immunohistochemical pen.
(3) And (3) sealing: one drop of PBST (0.3% TritonX-100) containing 2% normal goat serum was added to each brain slice, placed in a wet box, and sealed at room temperature for 2 h.
(4) Primary anti-reaction: the blocking serum was discarded with filter paper and the corresponding primary antibody reaction solution was added: NeurN (1: 200), preferably slightly over the brain, was incubated overnight at 4 ℃ in a refrigerator.
(5) And (3) fluorescent secondary antibody reaction: the brain slice is soaked and washed in PBST for 3 times the next day, the following operations are carried out at room temperature in a dark place, the brain slice is incubated by corresponding fluorescent secondary antibody (1: 300), and the brain slice is reacted for 2 hours in the dark place; after the secondary antibody is incubated, PBST is used for immersion washing for 3 times, filter paper is used for suction drying, DAPI working solution is used for dark incubation for about 6min, and PBST is used for immersion washing for 3 times.
(7) Sealing: removing water from periphery of brain slice, adding appropriate amount of anti-fluorescence quenching liquid, covering with cover glass from one end, preventing bubble generation, storing in a wet box at 4 deg.C, observing under a common inverted fluorescence microscope, and taking a picture.
As shown in FIGS. 8 and 9, in order to further study the brain protection effect of MDP nano-drugs on dementia model mice, Niger staining on Niger bodies in a hippocampal vulnerable area CA1 area and immunofluorescence are used for displaying the NeuN expression in the area, and the neuron survival in the hippocampal CA1 area of each group of mice is observed. The results show that: the edges of the normal group of neuron cells are regularly and closely arranged, and the cell morphology is good; compared with the normal group, the number of the neuron cells of the AD model group is obviously reduced, the arrangement is sparse, and the cells are seriously damaged. The donepezil group and the metformin group showed local cell looseness, the phospholipid group and the AD model group, and the physical mixed group showed similar performance to the donepezil group and the metformin group; and the MDP nano-drug group neuron cells are regularly and closely arranged, have good cell morphology, are large and round, and almost have no difference with the normal group. NeuN expression corresponds approximately to this. The double-drug-loaded nano preparation is shown to play an obvious role in protecting hippocampus and cortical neurons of brains of AD mice.
Example 7
The detection of cerebral microglia and inflammatory corpuscle NLRP3 of a model mouse with the senile dementia induced by scopolamine comprises the following steps:
immunofluorescence similar to example 6, the primary antibody was Iba-1 (1: 200)
Western blot assay: 1. extracting protein 2, measuring protein concentration of brain tissue protein extract (BCA method) 3, concentration matching and denaturation treatment of protein sample 4, preparing SDS polyacrylamide gel 5, gel electrophoresis 6, transferring membrane 7, blocking non-specific protein 8, incubating primary antibody (NLRP3)9, washing membrane 10, incubating secondary antibody 11, washing membrane 12 again, developing and screening
As shown in FIG. 10, the hippocampal cortex microglia (labeled Iba-1) was stained by immunofluorescence, and preliminary observation showed that the expression of hippocampus and cortex Iba-1 in the AD model group was increased and that the expression of Iba-1 in donepezil group was not significantly decreased compared to the normal group. The expression of the Iba-1 is increased in the metformin group and the phospholipid group, and the expression of the Iba-1 is not obviously reduced in the physical mixed group. The expression of the nanometer medicine group Iba-1 has a descending trend compared with the model group. WB detection shows that the expression of NLRP3 is obviously reduced after nano-drug treatment compared with the AD model group. MDP NPs can obviously treat inflammatory reaction in the brain of AD mice.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A double drug-loaded nano-preparation for neurodegenerative diseases, which is characterized by comprising: a cell membrane fraction having amphiphilicity, and a drug containing a benzene ring and a drug containing a guanidine group loaded in the cell membrane fraction.
2. The double-drug-loaded nanoformulation according to claim 1, wherein the membrane fraction has one or more hydrophilic polar heads, two or more hydrophobic tails.
3. The double drug-loaded nano-formulation of claim 2, wherein the hydrophilic polar head is a phosphate group and the hydrophobic tail is a saturated fatty acid or an unsaturated fatty acid.
4. The dual drug-loaded nano-formulation of claim 1, wherein the drug containing a benzene ring is one of donepezil, chlorimipramine, edaravone, galantamine, ketamine, imipramine, fluoxetine, penicillin, metronidazole, clozapine, and amoxicillin.
5. The dual drug-loaded nano-formulation of claim 1, wherein the drug containing guanidine group is one of metformin, guanethidine, guanylchlorophene, argonavir, moroxydine, arginine and guanidinoacetic acid.
6. The double drug-loaded nano-preparation of claim 1, wherein the drug-loading rate of the drug containing benzene ring is 5-20%; the drug loading rate of the drug containing guanidyl is 5-20%.
7. The double-drug-loaded nano-formulation of claim 1, wherein the weight ratio of the drug loaded in the cell membrane fraction to the cell membrane fraction is 1: 2.
8. The method of preparing a double drug-loaded nano-formulation according to any one of claims 1 to 7, comprising:
dissolving a medicine containing a benzene ring, a medicine containing guanidyl and a cell membrane component containing amphipathy in an organic solvent by adopting an ultrasonic thin film hydration method, heating and stirring for 12 hours, and removing the organic solvent by rotary evaporation to form a first thin film; ultrasonically dissolving the first film in an organic solvent in which the two single drugs are insoluble, filtering, and removing the organic solvent by rotary evaporation to form a second film; and adding the second film into the aqueous solution, and performing room temperature ultrasonic treatment to obtain the double-drug-loaded nano preparation.
9. The method according to claim 8, wherein the organic solvent is one or more selected from methanol, n-hexane, tetrahydrofuran, ethanol, chloroform, and acetone.
10. The method according to claim 8, wherein the aqueous solution is one or more of normal saline, phosphate buffer, and glucose solution.
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| CN104623671A (en) * | 2015-02-09 | 2015-05-20 | 徐云根 | Compound medicine composition containing acetylcholinesterase inhibitor and metformin |
| CN108452303A (en) * | 2018-03-06 | 2018-08-28 | 中国药科大学 | It is a kind of to carry double medicine nanometer formulations and preparation method thereof |
| CN108685875A (en) * | 2018-07-30 | 2018-10-23 | 中国药科大学 | A kind of natural nano grain-pharmaceutical composition of anti-alzheimer's disease and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104623671A (en) * | 2015-02-09 | 2015-05-20 | 徐云根 | Compound medicine composition containing acetylcholinesterase inhibitor and metformin |
| CN108452303A (en) * | 2018-03-06 | 2018-08-28 | 中国药科大学 | It is a kind of to carry double medicine nanometer formulations and preparation method thereof |
| CN108685875A (en) * | 2018-07-30 | 2018-10-23 | 中国药科大学 | A kind of natural nano grain-pharmaceutical composition of anti-alzheimer's disease and its preparation method and application |
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