CN115227668B - Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof - Google Patents

Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof Download PDF

Info

Publication number
CN115227668B
CN115227668B CN202210698647.0A CN202210698647A CN115227668B CN 115227668 B CN115227668 B CN 115227668B CN 202210698647 A CN202210698647 A CN 202210698647A CN 115227668 B CN115227668 B CN 115227668B
Authority
CN
China
Prior art keywords
delivery system
ginsenoside
drug
drug delivery
mnps
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210698647.0A
Other languages
Chinese (zh)
Other versions
CN115227668A (en
Inventor
张云龙
陈华清
刘妍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Medical University
Original Assignee
Guangzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Medical University filed Critical Guangzhou Medical University
Priority to CN202210698647.0A priority Critical patent/CN115227668B/en
Publication of CN115227668A publication Critical patent/CN115227668A/en
Application granted granted Critical
Publication of CN115227668B publication Critical patent/CN115227668B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Psychology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a ginsenoside Rb1 drug delivery system targeting dopamine neurons, and a preparation method and application thereof. According to the invention, the nanometer preparation containing ginsenoside Rb1 is prepared by culturing primary dopamine neurons and extracting neuron cell membranes, rb1 can be delivered by targeted dopamine neurons, and PD can be effectively treated. Experiments prove that based on the targeting of the system, the motor function of a PD model mouse is obviously improved, and a novel drug administration treatment strategy for targeting dopamine neurons is provided.

Description

Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a ginsenoside Rb1 drug delivery system targeting dopamine neurons, and a preparation method and application thereof.
Background
Parkinson's Disease (PD) is a common neurodegenerative disease of middle-aged and elderly people, and clinical symptoms are mainly manifested by resting tremor, bradykinesia, increased muscle tone and dysgestion. The incidence of PD increases year by year as the population ages. Epidemiological data shows that about 200 ten thousand PD patients have a new year up to about 10 ten thousand years old, with a prevalence of 1.67% of the disease over 65 years old. PD seriously jeopardizes human health, and not only brings great pain to patients, but also causes heavy burden to families and society.
The etiology of PD is currently not completely understood, and the main pathology of PD is represented by progressive degeneration and death of the nigra dopaminergic neurons of the midbrain, and the presence of eosinophilic protein inclusion bodies in the remaining neurons. Traditional anti-parkinsonism is mainly levodopa or dopamine agonists, but the drugs can not prevent the progress of diseases, and can not effectively reduce the death rate of the diseases due to a plurality of serious adverse reactions such as switching effect, abnormal symptoms, heart disease induction and the like which are easy to cause after long-term use. In addition, the related technology shows that the death of the nigral dopaminergic neurons is at least more than 50% when the PD presents clinical symptoms, and the content of the striatal dopamine is reduced by more than 70-80%. Intracranial surgery, such as pallidotomy and hypothalamic electrical stimulation, can alleviate some PD symptoms, and neural stem cell transplantation can replace DA neurons by cell proliferation, differentiation and integration, however, the above method has not been able to alter the progression of nigrostriatal neurodegeneration. Recent studies have shown that some new synthetic compounds (e.g., NPT 100-18A) can reduce their toxic effects by targeting α -synuclein, thereby improving dyskinesia in the PD model, but they have not been applied in clinical treatment. Therefore, the etiology research of PD is enhanced, and effective targets for developing new PD therapeutic drugs are found, so that the method has great significance for improving the human health level.
Ginsenoside Rb1 is a panaxadiol saponin derived from Panax ginseng (Panax ginsengC.A.mey.), panax quinquefolium L, panax notoginseng (Panax notoginseng (Burt.) F.H. Chen) and other plants in Panax of Araliaceae, and belongs to dammarane type triterpene saponins compound, and has a structure composed of hydrophilic saccharide and hydrophobic aglycone, and molecular formula of C 54H92O23. Research shows that in ginseng grown for 5 years, the total ginsenoside content is about 3.03%, and the ginsenoside Rb1 content is about 0.67%. In American ginseng, the content of ginsenoside Rb1 is higher and is about 1.51-2.21%. Ginsenoside Rb1 has various pharmacological activities, and has been widely studied, especially in the aspects of central nervous system, cardiovascular system, immune system, anti-tumor, etc. In the aspect of the central nervous system, the ginsenoside Rb1 has central inhibition and tranquilization effects, and has the neuroprotection effects of improving learning and memory capacity, resisting cerebral ischemia injury and the like.
The traditional administration mode of ginsenoside Rb1 adopts the mode of intragastric injection or intraperitoneal injection, on one hand, the administration mode has lower drug efficiency reaching the brain because of the barrier of the blood brain barrier, and on the other hand, the drug entering the brain can not completely target dopamine neurons to play a role, thus leading to the difficult research and development of ginsenoside Rb1 in the field of anti-parkinsonism.
Therefore, the development of a novel drug delivery system suitable for ginsenoside Rb1 has great significance for the research of ginsenoside Rb1 in anti-Parkinson disease and related aspects.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a ginsenoside Rb1 drug delivery system for targeting dopamine neurons and a preparation method and application thereof. Experiments prove that based on the targeting of the system, the motor function of a PD model mouse is obviously improved, and a novel drug administration treatment strategy for targeting dopamine neurons is provided.
In a first aspect of the invention, a drug delivery system is provided, the drug delivery system consisting of a nanoparticle core and an envelope;
the nanoparticle core contains albumin and a drug molecule;
The envelope contains neuronal cell membranes.
In the invention, the nano drug-loading technology is adopted, so that the water solubility of the insoluble drug can be improved by the nano technology, and the dosage of drug administration can be effectively improved. Meanwhile, the dopamine neuron cell membrane is extracted to carry out homologous assembly, so that the targeted treatment of the dopamine neuron is realized. The medicine small molecules which are entrapped by the nano materials have less chance of contacting blood in the long circulation in vivo, so that the half life of the medicine small molecules in the blood circulation is obviously improved. Meanwhile, human serum albumin derived from human plasma is used, so that the composition has high bioavailability and biocompatibility.
The inventor combines a nano drug delivery strategy, solves the problem of low efficiency of drugs passing through blood brain barriers by applying human serum albumin-entrapped ginsenoside Rb1, and simultaneously improves the targeting effect of the drugs on dopamine neurons by preparing dopamine neuron membranes for homologous targeting, and provides high-efficiency targeting distribution of Rb1 in brain by combining two modes, thereby providing a new idea for synthesizing novel targeting nano particles of traditional Chinese medicine monomers and applying the novel targeting nano particles in anti-parkinsonism treatment.
According to a first aspect of the invention, in some embodiments of the invention, the albumin comprises human serum albumin.
Of course, those skilled in the art can reasonably select other albumins with similar effects according to the actual needs of use, including but not limited to the human serum albumin.
In some embodiments of the invention, the drug molecule comprises ginsenoside Rb1.
Of course, those skilled in the art can reasonably select other ginsenosides with similar effects according to practical requirements, including but not limited to the above ginsenoside Rb1.
Ginsenoside Rb1 has a broad neuroprotective effect, but is limited in its application due to its low efficiency of penetrating the blood brain barrier and lack of targeting against dopamine neurons. In the invention, the primary mouse substantia nigra dopamine neuron cell membrane is extracted to be wrapped on the surface of serum albumin particles containing ginsenoside Rb1, on one hand, the assembled nano particles can pass through the blood brain barrier relatively easily, and on the other hand, the primary dopamine neuron cell membrane has homology with the mouse, so that the dopamine neurons can be well targeted after entering the brain. After being ingested by dopamine neurons, the serum albumin particles containing ginsenoside Rb1 are cracked under the acidic condition of lysosomes to release the entrapped ginsenoside Rb1, thereby playing a role in protection. Thus, the homozygously assembled nano particles can well realize the targeted delivery treatment of the parkinsonism.
In some embodiments of the invention, the coating further comprises a lipid dispersion.
In some embodiments of the invention, the lipid dispersion is DSPE-PEG2000 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000).
Of course, other lipid dispersions with similar effects can be reasonably selected by those skilled in the art according to the actual needs of use, including but not limited to the above DSPE-PEG2000.
In some embodiments of the invention, the neuronal cell membrane comprises a dopamine neuronal cell membrane.
In some embodiments of the invention, the dopamine neuronal cell membrane is extracted from a dopamine neuronal cell.
In some embodiments of the invention, the dopamine neuron cell membrane extraction method is as follows: and (3) using neutral RIPA lysate to lyse the dopamine neuron cells in an ice bath, performing ultrasonic treatment in ice bath protection, and centrifuging to obtain the dopamine neuron cell membrane.
In some embodiments of the invention, the particle size of the delivery system is 30-145 nm.
In some embodiments of the invention, the drug delivery system has a particle size of 138.9± 5.186 nm.
In some embodiments of the invention, the PDI polydispersity of the drug delivery system is 0.42±0.06.
In some embodiments of the invention, the PDI polydispersity index of the drug delivery system is 0.4160 ± 0.04729.
In some embodiments of the invention, the drug loading rate of the drug delivery system is about 3.4%.
In a second aspect of the present invention, there is provided a method of preparing a drug delivery system according to the first aspect of the present invention, comprising the steps of:
mixing the drug molecules with albumin, and performing ultrasonic treatment to obtain nanoparticle cores;
mixing the coating solution with the nanoparticle cores, and adopting a film extrusion process to obtain a drug delivery system;
the coating solution contains neuron cell membranes.
According to a second aspect of the invention, in some embodiments of the invention, an organic solvent is also added to the nanoparticle core.
In some embodiments of the invention, the organic solvent comprises at least one of methanol, chloroform.
Of course, those skilled in the art can also reasonably select other organic solvents for dissolving the drug molecules according to practical requirements, including but not limited to the above-mentioned methanol, chloroform.
In some embodiments of the invention, the organic solvent is chloroform.
In the embodiment of the invention, the use amount of the organic solvent is 100-200 mu L, the concentration of the ginsenoside Rb1 can be controlled to be 0.5-2 mg/mL, and of course, the use amount of the organic solvent and the ginsenoside Rb1 can be adjusted by a person skilled in the art according to the use requirement so as to meet the load requirement.
In some embodiments of the invention, the sonication conditions are: 28~32 W,18~22 kHz,4.5~5.5 min.
In some embodiments of the invention, the sonication conditions are: 30 W,20 kHz,5 min.
In the drug delivery system, traditional Chinese medicine monomer ginsenoside Rb1 and human serum albumin are connected through covalent bonds, and meanwhile, dopamine neuron cell membranes are coated. In the synthesis of the whole nano-particles, toxic chemical connecting agents are not used, and the nano-particles are prepared by only ultrasonic treatment of traditional Chinese medicine monomers and human serum albumin for a few minutes under the protection of ice bath, so that the nano-particles have extremely high safety.
In some embodiments of the invention, a lipid dispersion is also included in the coating solution.
In some embodiments of the invention, the lipid dispersion is DSPE-PEG2000 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000).
Of course, other lipid dispersions with similar effects can be reasonably selected by those skilled in the art according to the actual needs of use, including but not limited to the above DSPE-PEG2000.
In some embodiments of the invention, the ratio of neuronal cell membrane to lipid dispersion is based on the amount of phospholipid in the lipid dispersion, wherein the ratio is 10 6~107 cell membranes: 100. mu g-300 mu g of phospholipid.
In some embodiments of the invention, the ratio of nanoparticle core to coating solution is based on the drug molecule content in nanoparticle core and the amount of lipid dispersion phospholipid in coating solution, wherein the ratio is 1 mg/mL drug molecule: 100. mu g-300 mu g of phospholipid.
According to a second aspect of the invention, in some embodiments of the invention, the preparation method specifically comprises: dissolving ginsenoside Rb1 in chloroform, adding human serum albumin aqueous solution, mixing, and performing ultrasonic treatment by cell ultrasonic breaker to form nanoparticle core (NPs@Rb1). The neuronal cell membrane, lipid dispersion were then mixed with nps@rb1 as described above and extruded through 220 nm polycarbonate membrane to give mnps@rb1 nanoparticles.
In some embodiments of the invention, the method of preparation further comprises purification of mnps@rb1 nanoparticles.
In some embodiments of the invention, the purifying comprises: dialyzing the obtained nano particles to remove the non-encapsulated ginsenoside Rb1 and neutral lysate, and ultrafiltering and concentrating.
The invention provides a preparation method of ginsenoside Rb1 human serum albumin nano-particles targeting dopamine neurons, which is used for preparing efficient nano-preparations with neuron targeting. The preparation method is simple and quick to operate, and the prepared human serum albumin nano-particles are controllable in size and stable in property, so that the problems of low permeability, poor targeting and the like of the traditional Chinese medicine monomer ginsenoside Rb1 blood brain barrier are solved. Meanwhile, the ginsenoside Rb1 human serum albumin nano-particles targeting the dopamine neurons, which are prepared by the invention, are only verified to have a good anti-parkinsonism effect, and the ginsenoside Rb1 is released by targeting the dopamine neurons, so that the dyskinesia of parkinsonism mice is improved, and the nano-particles can be used as novel neuroprotective drugs for anti-parkinsonism treatment.
In a third aspect of the invention, there is provided a medicament comprising the delivery system of the first aspect of the invention, as well as other pharmaceutically acceptable excipients.
According to a third aspect of the invention, in some embodiments of the invention, the pharmaceutically acceptable excipients include diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH buffers, antioxidants, bacteriostats.
Of course, those skilled in the art can also reasonably select other pharmaceutically acceptable excipients according to the actual application requirements, including but not limited to the diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH buffers, antioxidants, bacteriostats.
The drug delivery system solves the problems of blood brain barrier penetrability and targeting of ginsenoside Rb1, and performs high-efficiency targeting protection on dopamine neurons in Parkinson's disease.
In a fourth aspect, the invention provides the use of a delivery system according to the first aspect of the invention for the manufacture of a product for the treatment of neurodegenerative diseases or nerve damage.
According to a fourth aspect of the invention, in some embodiments of the invention, the neurodegenerative disease comprises parkinson's disease.
The invention discloses preparation of a novel preparation for delivering ginsenoside Rb1 by targeting dopamine neurons and application of the preparation in resisting parkinsonism. The novel targeted dopamine neuron albumin drug-carrying nano-particles are successfully prepared by adopting dopamine neuron cell membrane extraction and human serum albumin nano-particles. The method has simple operation, fewer steps and good repeatability, and the prepared albumin drug-loaded nano-particles solve the targeting problem of the ginsenoside Rb1 monomer of the traditional Chinese medicine, thereby further improving the drug effect of resisting parkinsonism; the homozygously assembled traditional Chinese medicine monomer ginsenoside Rb1 can be obviously absorbed by dopamine neurons, has the effect of resisting parkinsonism, and the homozygously assembled traditional Chinese medicine monomer ginsenoside Rb1 can be discovered for the first time to improve the dyskinesia of parkinsonism models, so that the targeted dopamine neurons for delivering the ginsenoside Rb1 have important application value in the field of parkinsonism treatment.
The beneficial effects of the invention are as follows:
The ginsenoside Rb1 drug delivery system targeting dopamine neurons has the advantages that the carrier has good biocompatibility and neuron targeting, and the blood concentration in the brain is improved by combining with human serum albumin; meanwhile, by combining with the cell membrane of the dopamine neuron, the targeting of the dopamine neuron to the neuron is improved, the motor function of a PD model mouse is obviously improved, and a novel drug administration treatment strategy for targeting the dopamine neuron is provided. The preparation method of the ginsenoside Rb1 drug delivery system targeting dopamine neurons is simple and quick to operate, the prepared human serum albumin nano-particles are controllable in size and stable in property, and the problems of low permeability, poor targeting and the like of the traditional Chinese medicine monomer ginsenoside Rb1 blood brain barrier are solved. Meanwhile, the ginsenoside Rb1 human serum albumin nano-particles targeting the dopamine neurons, which are prepared by the invention, are verified to have a good anti-parkinsonism effect, and the ginsenoside Rb1 is released by targeting the dopamine neurons, so that the dyskinesia of parkinsonism mice is improved, and the nano-particles can be used as a novel neuroprotective medicament for anti-parkinsonism treatment.
Drawings
Fig. 1 is a schematic diagram of a synthesis flow and a structure of mnps@rb1 nanoparticles in an embodiment of the invention.
FIG. 2 is a comparison of the characterization of NPs@Rb1 and MNPs@Rb1 nanoparticles in an embodiment of the invention, wherein A is the particle size distribution, B is the average particle size, and C is the PDI polydispersity.
FIG. 3 is a graph showing comparison of expression of MNPs@Rb1 nanoparticle proteins in an embodiment of the invention.
FIG. 4 is a graph showing comparison of cell viability of NPs@Rb1 and MNPs@Rb1 nanoparticles in the example of the invention, wherein A is cell viability and B is the number of surviving cells.
FIG. 5 is a graph comparing the cellular uptake capacity of NPs@Rb1 and MNPs@Rb1 nanoparticles in examples of this invention.
FIG. 6 is a comparison of the improvement effect of each experimental group on the movement disorder of the PD model mice in the embodiment of the invention, wherein A-F are respectively the total distance, total speed, exploring time in the center of open field, climbing time, hanging time and roller movement time of the mice in open field.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.
The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.
Preparation of ginsenoside Rb1 human serum albumin nano-particles
2 Mg ginsenoside Rb1 (CAS number: 41753-43-9) was dissolved in 100. Mu.L of methanol-chloroform mixture (methanol: chloroform volume ratio=1:3). Adding human serum albumin solution (40 mg HSA dissolved in 2mL deionized water), mixing, ultrasonic (batch) treating with 5min (30W, 20 kHz) by cell ultrasonic breaker, and removing organic solvent. Adding 150 mu L of 0.1M-mu m sodium carbonate aqueous solution, ultrasonically cleaning for 5-10 minutes to obtain transparent and clear nanoparticle core (NPs@Rb1) aqueous solution, removing free small molecules in a nano system by ultrafiltration/dialysis, filtering by a 0.22 mu m filtering membrane to obtain purified NPs@Rb1, and keeping away from light for later use.
Preparation of ginsenoside Rb1 human serum albumin nano-particles targeting dopamine neurons
Dissolving 2 mg ginsenoside Rb1 in 100 μl of chloroform, adding human serum albumin solution (40 mg HSA is dissolved in 2 mL deionized water), mixing, and performing ultrasonic treatment with cell ultrasonic breaker for 5 min (30W, 20 kHz) to form nanoparticle core (NPs@Rb1). The neuronal cell membrane, 100. Mu.L of DSPE-PEG2000 (10 mg/mL) lipid dispersion was then mixed with the NPs@Rb1 described above and extruded approximately 10 times through a 220 nm polycarbonate membrane to give MNPs@Rb1 nanoparticles. Dialyzing the obtained nanoparticle with PBS (pH 7.4) at room temperature for 24 hours to remove non-encapsulated ginsenoside Rb1 and neutral lysate, concentrating by ultrafiltration to obtain purified MNPs@Rb1 nanoparticle, and storing in dark for use.
The preparation flow chart is shown in figure 1.
The neuron cell membrane is pre-extracted, and the specific extraction method comprises the following steps:
(1) Preparation of primary dopamine neuronal cells:
The whole brain of 14 day pregnant foetus mice was aseptically stripped and midbrain isolated. The midbrain was cut into chyme, trypsin was added, and 8 min was digested at 37 ℃. After completion of digestion, pancreatin digestion was terminated using DMEM/F12 medium containing 10% fetal bovine serum. Centrifuging, discarding supernatant, adding neuron culture medium, and re-suspending and precipitating. Centrifuging again, discarding supernatant, re-suspending the precipitate in neuron culture medium, and sieving with 200 mesh cell sieve to obtain single cell suspension. Inoculating into a cell culture flask, and culturing at 37deg.C under 5% CO 2 to obtain primary dopamine neuron cells.
The composition of the neuron culture medium is as follows: on the basis of Neurobasal Medium, 2% (volume ratio) of B27 cell culture additive, 4mM glutamine and diabody were added, wherein the diabody was 200U/mL penicillin and 100U/mL streptomycin.
(2) Extraction of dopamine neuron cell membranes:
And (3) cracking the dopamine neuron cells obtained by culture by neutral RIPA lysate in ice bath for 30 min, performing ultrasound for 20: 20 s in ice bath protection, and centrifuging to obtain neuron cell membranes.
Characterization of NPs@Rb1 and MNPs@Rb1
The particle size distribution and polydispersity (polydispersity index, PDI) of nps@rb1 and mnps@rb1 prepared in the above examples were measured using Zetasizer Nano ZS instrument, respectively.
The results are shown in FIG. 2.
It was found that the particle sizes of nps@rb1 and mnps@rb1 were in the range of 30-145 nm (fig. 2A), wherein the average particle size of nps@rb1 was 113.2± 11.02 nm,MNPs@Rb1 and 138.9± 5.186 nm (fig. 2B). The PDI polydispersity numbers for nps@rb1 and mnps@rb1 are 0.3673 ± 0.09152 and 0.4160 ± 0.04729, respectively (fig. 2C).
Meanwhile, the dopamine neuron cell membrane and the MNPs@Rb1 in the above examples were extracted respectively, and the protein expression was detected by SDS-PAGE.
The specific operation of SDS-PAGE detection is as follows: the dopamine neuron cell, the cell membrane of the dopamine neuron cell and MNPs@Rb1 in the above examples are respectively extracted, a black tissue protein sample is used as a control, a commercial BCA quantitative detection kit is used for determining the protein concentration, a loading buffer is added according to the application instruction of the kit, and the solution is subjected to boiling water bath at 100 ℃ for 5 min. Proteins were transferred to PVDF membranes after electrophoresis. PVDF membrane 5min was rinsed with TBST buffer at room temperature, blocked with 5% bsa blocking solution for 2 h, 3 rinses of TBST buffer 5min each. Incubation was carried out overnight at 4 ℃. The membrane was rinsed 3 times with TBST buffer, 5min each, and the membrane was incubated with secondary antibody for 1h at room temperature and rinsed with TBST buffer. The chemiluminescent reagent A and the chemiluminescent reagent B in the kit are mixed in equal amounts, the PVDF membrane 1min is incubated, and exposure is performed on an exposure instrument.
The results are shown in FIG. 3.
It can be found that the dopamine neuron cell, the cell membrane of the dopamine neuron cell and MNPs@Rb1 all show similar protein expression, and the neuronal cell membrane in MNPs@Rb1 is proved to be successfully coated, so that the complete assembly of MNPs@Rb1 is realized.
Verification of the effects of NPs@Rb1 and MNPs@Rb1
(1) Cell viability and cell uptake capacity of nps@rb1 and mnps@rb1:
NPs@Rb1 and MNPs@Rb1 with different ginsenoside Rb1 coating concentrations (3.125-100 mug/mL) are prepared according to the above example, and are respectively added into a culture solution containing dopamine neuron cells, incubated for 24 h, and the cell viability is detected by using an MTT method.
The specific detection steps are as follows:
According to the instructions of MTT cell proliferation and cytotoxicity detection kit, 25mg MTT was dissolved in 5 mL MTT solvent to prepare 5 mg/ml MTT solution. The cell fluid (100. Mu.L) was transferred to each well in an amount of 2000 cells per well, 10. Mu.L MTT solution was added per well, and incubated in a cell incubator for 4 hours. Then 100 mu L Formazan of the solution was added to each well, mixed well and incubated in a cell incubator until the crystals of Formazan were found to be completely dissolved by observation under a normal light microscope (incubation at 37 ℃ C. For about 3-4 hours, the crystals of purple Formazan were all dissolved). Absorbance was measured at 570 nm.
The results are shown in FIG. 4.
It was found that in the range of ginsenoside Rb1 coating concentration from 3.125-100. Mu.g/mL, neither NPs@Rb1 nor MNPs@Rb1 was found to reduce cell viability, indicating that both were very toxic (FIG. 4).
Meanwhile, ginsenoside Rb1 in NPs@Rb1 and MNPs@Rb1 was further labeled with Cy5.5 in an amount of 0.5 μg/mL, and after incubation with dopamine neurons of 3 h, uptake capacity of NPs@Rb1 and MNPs@Rb1 on the dopamine neurons was detected by using flow cytometry.
The specific operation is as follows:
Dopamine neuronal cells were cultured according to the above examples and seeded into 6-well plates at a density of 1×10 6 cells/well. After 24 hours of incubation, the medium was replaced with fresh medium containing NPs@Rb1 and MNPs@Rb1, incubated for 3 hours, the cells were washed with PBS and collected for flow cytometry detection analysis (Ex: 638 nm, em:712/25 nm).
Wherein, the ginsenoside Rb1 coating concentration in NPs@Rb1 and MNPs@Rb1 is 200 mug/mL, and the labeling is carried out by using 0.5 mug/mL Cy5.5, and the specific preparation method is the same as the above example.
The results are shown in FIG. 5.
Compared with NPs@Rb1, the MNPs@Rb1 has stronger Cy5.5 fluorescence intensity in cells, so that the MNPs@Rb1 has better neuronal cell uptake capacity, and meanwhile, the MNPs@Rb1 drug delivery system coated on the basis of neuronal cell membranes has good neuronal targeting.
(2) Improvement effect of NPs@Rb1 and MNPs@Rb1 on motor behavior disorder of PD model mice
In this example, an α -Synuclein A T transgenic PD model mouse (purchased from Hainan model biotechnology Co., ltd.) was used as a PD animal model for the validation test. The alpha-Synuclein A T-related mouse model has the pathological characteristics of aggregation of alpha-Synuclein and TH cytopenia, and is a PD model conventional in the art.
The experiments were divided into 5 groups, namely, a control group (WT-PBS, normal wild type mice (WT), PBS administration), a model group (A53T. Alpha. -Syn-PBS, PD model mice, PBS administration), an NPs@Rb1 experimental group (A53T. Alpha. -Syn-NPs@Rb1, PD model mice, NPs@Rb1 administration), an MNPs experimental group (A53T. Alpha. -Syn-MNPs, PD model mice, human serum albumin particles MNPs entrapped in the cell membranes of dopamine neurons not carrying ginsenoside Rb1 administration), an MNPs@Rb1 experimental group (A53T. Alpha. -Syn-MNPs@Rb1, PD model mice, MNPs@Rb1 administration). PBS, NPs@Rb1, MNPs and MNPs@Rb1 were injected by tail vein in 200. Mu.L volume.
The feeding conditions of each group are consistent.
The tail vein was injected with PBS, NPs@Rb1, MNPs and MNPs@Rb1 once every other day, and after 10 times of tail vein injection, a behavioral experiment was performed to test the therapeutic effect of each group of mice.
The behavioural experiment comprises a roller experiment, a pole climbing experiment and a suspension experiment.
The settings for each behavioural experiment were as follows:
Roller test: the roller experiment requires that the experimental animal keeps balance and continuously moves on the roller, and is widely used for detecting motion coordination, in the embodiment, the diameter of the roller is 6 cm, the rotating speed is 20 r/min, after the mice are adapted for 5 times, each detection interval is 1 min, and the average value is continuously measured for 5 times.
Pole climbing experiment: a foam pellet of diameter 2 cm was secured to the top of a 50 a cm a 1a cm a wooden pole with 2 layers of gauze wrapped around to prevent slippage. The tail of the hand-held mouse is downwards arranged on the rod top (based on the fact that the two hind limbs of the mouse are arranged on the ball), so that the hand-held mouse naturally climbs down, and the mouse climbs the whole length from the rod top to the two forelimbs contact rod bottom platform. And respectively taking a certain number of mice from each experimental group to perform pole climbing experiments, observing the behavioral changes of the mice, and recording pole climbing time.
Suspension experiment: the test box is made of organic glass, a horizontally placed metal rod (with the diameter of 1.5 mm and the distance from the ground of 30 cm) is arranged, and a cover is arranged above the metal rod by 1cm so as to prevent the mice from being overturned on the metal rod. The mice were suspended on metal rods during the experiment and the time before scaling hours was recorded. The time interval between each detection is 1min, and the average value is taken for 5 times.
The results are shown in FIG. 6.
It can be seen that the mnps@rb1 experimental group significantly improved motor function of PD model mice after nps@rb1, MNPs, mnps@rb1 treatment, and was characterized by a significant increase in total distance moved in open field (fig. 6A), total velocity (fig. 6B), and exploration time in the center of open field (fig. 6C). In addition, the pole-climbing time of mnps@rb1 experimental mice was significantly reduced (fig. 6D), and both the suspension time and the roller movement time were significantly up-regulated (fig. 6E and F). The two NPs@Rb1 and MNPs have no obvious difference from the model group, and do not show obvious protective effect, which indicates that the NPs@Rb1 and MNPs cannot effectively treat PD. The results prove that the ginsenoside Rb1 can be effectively targeted to the dopamine neurons by using MNPs@Rb1, so that an effective treatment effect is realized, and the dyskinesia of a PD model mouse is remarkably improved.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (6)

1. A delivery system comprising a nanoparticle core and an envelope;
the nanoparticle core contains albumin and a drug molecule;
the envelope contains neuron cell membranes and a lipid dispersion, wherein the lipid dispersion is DSPE-PEG2000;
The albumin is human serum albumin;
the medicine molecule is ginsenoside Rb1;
the neuron cell membrane is a dopamine neuron cell membrane;
The ratio of neuronal cell membrane to lipid dispersion is based on the amount of phospholipid in the lipid dispersion, wherein the ratio is 10 6~107 cell membranes: 100. mu g-300 mu g of phospholipid;
The ratio of nanoparticle core to envelope is based on the drug molecule content in nanoparticle core and the amount of lipid dispersion phospholipid in envelope, wherein the ratio is 1mg/mL drug molecule: 100. mu g-300 mu g of phospholipid.
2. The drug delivery system of claim 1, wherein the drug delivery system has a particle size of 30-145 nm; the PDI polydispersity of the drug delivery system is 0.42+ -0.06.
3. The method of preparing a drug delivery system according to any one of claims 1-2, comprising the steps of:
mixing the drug molecules with albumin, and performing ultrasonic treatment to obtain nanoparticle cores;
Mixing the coating solution with the nanoparticle core, and adopting a film extrusion process to obtain the drug delivery system.
4. A method of preparation according to claim 3, wherein an organic solvent is also added to the nanoparticle core for dissolving the drug molecules;
the organic solvent comprises at least one of methanol, chloroform and chloroform.
5. A medicament, characterized in that it comprises the delivery system of any one of claims 1-2, as well as other pharmaceutically acceptable excipients.
6. Use of the delivery system according to any one of claims 1-2 for the preparation of a parkinson's disease treatment product.
CN202210698647.0A 2022-06-20 2022-06-20 Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof Active CN115227668B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210698647.0A CN115227668B (en) 2022-06-20 2022-06-20 Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210698647.0A CN115227668B (en) 2022-06-20 2022-06-20 Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115227668A CN115227668A (en) 2022-10-25
CN115227668B true CN115227668B (en) 2024-04-30

Family

ID=83669118

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210698647.0A Active CN115227668B (en) 2022-06-20 2022-06-20 Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115227668B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106806352A (en) * 2017-01-26 2017-06-09 中国人民解放军军事医学科学院毒物药物研究所 It is a kind of to be sustained, target the Nano medication for being applied to nerve degenerative diseases
CN114569576A (en) * 2022-04-21 2022-06-03 浙江中医药大学 Brain-targeted erythrocyte membrane-coated salvianolic acid B nanoparticles, preparation method and application thereof
CN114588275A (en) * 2022-02-21 2022-06-07 广州中医药大学(广州中医药研究院) Brain-targeting cell membrane biomimetic modification drug nanocrystal and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106806352A (en) * 2017-01-26 2017-06-09 中国人民解放军军事医学科学院毒物药物研究所 It is a kind of to be sustained, target the Nano medication for being applied to nerve degenerative diseases
CN114588275A (en) * 2022-02-21 2022-06-07 广州中医药大学(广州中医药研究院) Brain-targeting cell membrane biomimetic modification drug nanocrystal and preparation method and application thereof
CN114569576A (en) * 2022-04-21 2022-06-03 浙江中医药大学 Brain-targeted erythrocyte membrane-coated salvianolic acid B nanoparticles, preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
人参皂苷Rg1、Rb1、Rd、Re和帕金森病的研究进展;李冬等;《解剖学杂志》;第43卷(第6期);第517-521页，尤其是第519页左栏倒数第2段 *

Also Published As

Publication number Publication date
CN115227668A (en) 2022-10-25

Similar Documents

Publication Publication Date Title
CN111249449A (en) Extracellular vesicle-interleukin-10 nano targeted drug and preparation method and application thereof
Huang et al. Rat bone mesenchymal stem cell‐derived exosomes loaded with miR‐494 promoting neurofilament regeneration and behavioral function recovery after spinal cord injury
CN111346070A (en) Macrophage vesicle-loaded nano-drug preparation and application thereof in pharmacy
CN107875124B (en) Method for extracting and purifying cell vesicles wrapping medicine from cell suspension
Long et al. Extracellular vesicles from medicated plasma of Buyang Huanwu decoction-preconditioned neural stem cells accelerate neurological recovery following ischemic stroke
CN115227668B (en) Ginsenoside Rb1 drug delivery system targeting dopamine neurons and preparation method and application thereof
WO2024093149A1 (en) Method for preparing tumor-derived cell microparticles by microwave treatment
CN114652703A (en) Application of aerosol inhalation functional extracellular vesicles in improvement of acute lung injury
CN110279673B (en) AuNP @ PP/poly (I: C), preparation method thereof and application thereof in preparation of drugs for treating glioma
CN109528719B (en) Application of vinpocetine in preparation of medicine for preventing and/or treating altitude disease caused by acute altitude advancement
CN110638760A (en) Cxcr 4-modified osthole long-circulating liposome, and preparation method and application thereof
CN113713082B (en) Nanometer autophagy inducer for Alzheimer's disease and preparation method and application thereof
CN114288419B (en) Preparation of rifampicin nano drug delivery system and application thereof in preventing and treating parkinsonism
CN112826942B (en) Lipid nano drug delivery system targeting craniocerebral injury focus and preparation method and application thereof
CN114369575B (en) Brain glioma cell-derived exosome without tumor promotion function, and preparation method and application thereof
CN117942410A (en) Drug delivery carrier of polypeptide modified human umbilical cord mesenchymal cell source exosome and preparation method thereof
CN116334004A (en) Application of RVG-Exo-miR-3059-5p in treating cerebral apoplexy
CN102174513B (en) Mammal ribonucleic acid small molecular compound and application thereof
CN116459229A (en) Preparation method of nano particles and application of nano particles in preparation of anti-parkinsonism drugs
KR100332562B1 (en) The preparation of Urushiol-ethanol nanoparticle and anticancer composition containing the same
CN118121631A (en) Application of iron oxide nanoparticle engineering macrophage exosome in preparation of drugs for inhibiting retinal neovascularization
CN117363557A (en) Preparation method and application of engineering plant exosome for improving mitochondrial dysfunction
CN117919315A (en) Application of pepper exosomes in preparing medicines for preventing and treating inflammatory bowel disease
CN117205176A (en) Traditional Chinese medicine loaded engineering exosome nano-particles and preparation method and application thereof
CN117462701A (en) Liver-targeted deformed erythrocyte preparation and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant