CN115177600B - Protein phospholipid nano preparation, application and preparation method thereof - Google Patents
Protein phospholipid nano preparation, application and preparation method thereof Download PDFInfo
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- CN115177600B CN115177600B CN202210510621.9A CN202210510621A CN115177600B CN 115177600 B CN115177600 B CN 115177600B CN 202210510621 A CN202210510621 A CN 202210510621A CN 115177600 B CN115177600 B CN 115177600B
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- 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
- A61K9/51—Nanocapsules; Nanoparticles
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- A61K9/513—Organic macromolecular compounds; Dendrimers
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- 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
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- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention provides a protein phospholipid nano preparation, application and a preparation method thereof. Wherein the protein phospholipid nano preparation is a protein phospholipid complex based on combination of ApoA1 protein and phospholipid; the ApoA1 protein is an ApoA1 protein which does not contain a conserved sequence shown in SEQ ID No.1 or a homologous sequence of the conserved sequence. The protein-phospholipid complex provided by the invention can be used as an anti-CNS disease drug and also can be used as a brain targeting delivery system, and becomes a brain drug delivery system solution for preventing and treating CNS diseases of the central nervous system.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a protein phospholipid nano preparation, application and a preparation method thereof.
Background
Central Nervous System (CNS) diseases severely affect human health, for example, neurodegenerative diseases (e.g., alzheimer's disease, parkinson's disease), meningitis, brain tumors (e.g., glioblastoma, central lymphoma, etc.).
Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases, and about 10-12% of the population 65 years and older is affected by AD. The international conference on AAIC held in 2021 published a number of report data (World Alzheimer Report 2021), and it was expected that the total number of dementia patients in 2050 would be 1.52 million people, of which about 60-70% are Alzheimer's patients. At present, 1000 ten thousand Alzheimer's disease patients in China are in the first world, and the estimated number of people can possibly burst to 4000 ten thousand people in 2050. As the population of china ages, alzheimer's disease and other forms of dementia and major neurocognitive disorders are increasing dramatically. And current prevalence of Parkinson's Disease (PD) over 65 years of china is approximately 1.7%.
The Blood Brain Barrier (BBB) severely limits the treatment of Central Nervous System (CNS) diseases (e.g., neurodegenerative diseases, brain tumors, brain infections, and strokes) because it prevents 98% of small molecule drugs and macromolecules (e.g., peptide, genetic drugs, and protein drugs) from entering the brain.
Therefore, aiming at the bottleneck of the current Central Nervous System (CNS) diseases, there is a need to develop a protein system which can efficiently pass through the blood brain barrier, has the functions of brain targeting, safety, no toxicity, effective prevention and treatment of early neurodegenerative diseases and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a protein phospholipid nano-preparation which is a protein phospholipid complex based on the combination of ApoA1 protein and phospholipid;
wherein, the ApoA1 protein is an ApoA1 protein which does not contain a conserved sequence shown in SEQ ID No.1 or a homologous sequence of the conserved sequence.
Preferably, the amino acid sequence of the protein phospholipid nano-preparation comprises:
one or two of the 1 st amino acid sequence shown as SEQ ID No.2 and the 2 nd amino acid sequence shown as SEQ ID No. 3.
Preferably, the amino acid sequence of the protein phospholipid nano-preparation further comprises:
the 3 rd amino acid sequence shown as SEQ ID No. 4.
Preferably, the amino acid sequence of the protein phospholipid nano preparation is arranged in sequence of the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence.
Preferably, each nanoparticle of the protein phospholipid nanofabrication has a diameter of 5-130nm.
Preferably, the amount of internally embedded phospholipids per nanoparticle of the protein phospholipid nanofabrication is 100-1000.
Preferably, the protein phospholipid nano-preparation is applied to the preparation of products for preventing and/or treating early neurodegenerative diseases.
Preferably, the use of said protein phospholipid nanoformulation for the preparation of a product for the treatment and/or prevention of CNS disorders of the central nervous system.
In addition, in order to solve the above problems, the present application also provides a brain drug delivery system, including the protein phospholipid nano-preparation as described above.
In addition, in order to solve the above problems, the present application further provides a preparation method of the protein phospholipid nano-preparation, which comprises the following steps:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
assembling the purified protein with dimyristoyl phosphatidylcholine to obtain the protein phospholipid nano preparation.
The invention provides a protein phospholipid nano preparation, application and a preparation method thereof. The protein phospholipid nano preparation is a protein phospholipid complex based on the combination of ApoA1 protein and phospholipid by using genetic engineering, and has the following beneficial effects:
1. the protein phospholipid nanometer preparation can effectively cross the blood brain barrier on one hand, and can obviously show obvious treatment effect on early-stage neurodegenerative diseases (including AD diseases) on the other hand.
2. The phospholipid in the protein phospholipid nano preparation belongs to natural phospholipid, and the ApoA1 protein has good biocompatibility, so that the protein phospholipid nano preparation can achieve better stability in vivo;
3. the protein phospholipid nanometer preparation can load corresponding small molecules and can be used as a drug carrier.
In conclusion, the protein phospholipid nano preparation can be used as a medicine for resisting CNS diseases and also can be used as a brain targeting delivery system, and becomes a brain medicine delivery system solution for preventing and treating CNS diseases of a central nervous system.
Drawings
FIG. 1 is a band structure diagram of an ApoA1 protein containing a conserved sequence;
FIG. 2 is a SDS-PAGE map of purified ApoA1 protein;
FIG. 3a is a Size Exclusion (SEC) diagram of ApoA 1-ND;
FIG. 3 (b-1) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for large size particles (125 nm);
FIG. 3 (b-2) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for medium sized particles (38 nm);
FIG. 3 (b-3) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for small size particles (4.6 nm);
FIG. 3 (c-1) is an atomic force microscope nanoparticle topography;
FIG. 3 (c-2) is an atomic force microscope nanoparticle height diagram;
FIG. 4 is a brain profile of Bodipy-labeled ApoA1-ND following intravenous administration;
FIG. 5 is a graph of the accumulation of ApoA1-ND around Abeta aggregates after intravenous injection;
FIG. 6A-ApoA 1-ND brain tissue section with reduced Abeta deposition;
FIG. 6 (a) is that ApoA1-ND reduces amyloid deposition in cerebral cortex and hippocampus of APP/PS1 mice;
FIG. 6 (b) is a statistical chart of quantitative analysis of amyloid deposition in cerebral cortex of APP/PS1 mice;
FIG. 6 (c) is a statistical plot of amyloid deposition quantification in hippocampus of APP/PS1 mice;
FIG. 7 shows a photograph of a brain tissue section of ApoA1-ND with reduced microglial proliferation;
FIG. 7 (a) is that ApoA1-ND reduces reduced microglial proliferation in the cerebral cortex and hippocampus of APP/PS1 mice;
FIG. 7 (b) is a statistical plot of quantitative analysis of apoA1-ND with reduced microglial proliferation in the cerebral cortex of APP/PS1 mice;
FIG. 7 (c) is a statistical plot of quantitative analysis of apoA1-ND with reduced microglial proliferation in the hippocampus of APP/PS1 mice;
FIG. 8 is a graph of Nile staining results for tissue sections;
FIG. 9 (a) is a schedule of behavioral trace analysis (Morse water maze) treatment, pathology monitoring, and treatment assessment for mice with Alzheimer's disease model;
fig. 9 (b) is an escape latency graph at different time points;
FIG. 9 (c) is a swimming speed comparison chart;
FIG. 9 (d) is the number of times the mouse passed the platform the last day the platform was removed;
fig. 9 (e) is a representative swim path of MWM mice after different treatments;
FIG. 10 is a view of tissue sections of organs.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The term "about" in the present invention means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following is provided merely to aid in the understanding of the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
The technical solution of the present invention is further described in detail below with reference to specific embodiments, but the present invention is not limited thereto, and any modifications made by anyone within the scope of the claims of the present invention are still within the scope of the claims of the present invention.
The invention provides a protein phospholipid nano preparation, which is a protein phospholipid compound based on the combination of ApoA1 protein and phospholipid; wherein, the ApoA1 protein is an ApoA1 protein which does not contain a conserved sequence shown in SEQ ID No.1 or a homologous sequence of the conserved sequence.
Above, apoA1 is a main component of high density lipoprotein, and can be used as a scaffold of phospholipid nano-discs to assemble into phospholipid nano-discs, which is an apolipoprotein.
The periphery of the phospholipid nano-disc is wrapped by amphiphilic membrane scaffold protein, so that a stable bilayer membrane structure can be formed, the structural composition is stable, and the phospholipid nano-disc is not influenced by the concentration change of phospholipid.
The above-mentioned phospholipids, which are natural phospholipids in the present invention, have very good stability and biocompatibility. It should be noted that, according to the report in the literature (Human apolipoprotein A-I bonds amyoid-beta and precursors Abeta-induced neurotoxicity, INT J BIOCHEM CELL B,41 (2009) 1361-1370), a fragment of the conserved sequence "GNLLTLD" has homology with the ApoA1 protein, and the sequences of the sequence GNLLTLD and the homology thereof can be specifically bound with aggregated Abeta, and the homologous sequence is proved to be the binding site of Abeta. Referring to FIG. 1, the spatial band structure of the ApoA1 protein containing a fragment of a conserved sequence is shown. Table 1 shows homologous sequences with similar fragments to the conserved sequence, and sequences 2-18 all have the same partial fragments of "GNLLTLD" as the "GNLLTLD" fragment of conserved sequence 1, i.e., homologous sequences (shown in bold and italic letters). For example, "LNLKLLD" in sequence 2 has corresponding fragments "NL" and "LD" identical to a conserved sequence, which, although not identical, belong to homologous sequences close to the conserved sequence.
TABLE 1 homologous sequences with similar fragments to the conserved sequences
The ApoA1 related proteins of the conventional high density lipoprotein contain the above homologous sequences, namely, fragments of the conserved sequences, so that the ApoA1 related proteins can target aβ and promote degradation of aβ after crossing the blood brain barrier.
The protein phospholipid nano preparation provided by the invention is a protein phospholipid complex formed by combining ApoA1 protein and phospholipid. As one of ApoA 1-related proteins, a fragment interval which does not contain the same conserved sequence in ApoA 1-related proteins of conventionally disclosed high density lipoproteins, but shows an ability to interact with aβ, particularly a remarkable therapeutic effect on early neurodegenerative diseases AD, has not been reported in the literature.
The protein phospholipid nano preparation provided by the invention can target Abeta through a Blood Brain Barrier (BBB) and promote degradation of Abeta, reduce Abeta aggregation level, reduce microglial cell proliferation, reduce loss of hippocampus, namely cerebral cortex nerve cells, improve spatial learning and memory capacity, and can show remarkable early neurodegenerative disease prevention and treatment effects.
The protein phospholipid nano preparation provided by the invention can effectively cross the blood brain barrier on one hand, and can obviously show a remarkable treatment effect on early-stage neurodegenerative diseases (including AD diseases) on the other hand; the phospholipid in the protein phospholipid nano preparation belongs to natural phospholipid, and the ApoA1 protein has good biocompatibility, so that the protein phospholipid nano preparation can achieve better stability in vivo; the protein phospholipid complex can load corresponding small molecules and can be used as a drug carrier; the protein-phospholipid complex can be used as a medicine for resisting CNS diseases and a brain targeting delivery system, and becomes a brain medicine delivery system solution for preventing and treating CNS diseases of a central nervous system.
Further, the amino acid sequence of the protein phospholipid nano preparation comprises:
one or two of the 1 st amino acid sequence shown as SEQ ID No.2 and the 2 nd amino acid sequence shown as SEQ ID No. 3.
The amino acid sequence of the protein phospholipid nano preparation is ApoA1 protein after being expressed and modified in E.coli BL21 (DE 3), and at least one or two of the 1 st amino acid sequence (a) and the 2 nd amino acid sequence (b) are contained.
Here, the protein phospholipid nano-preparation may include the following cases, which may be: 1. a, a; 2. b; 3. a+b. Wherein the arrangement order is not limited between the sequences a and b.
Further, the amino acid sequence of the protein phospholipid nano preparation further comprises:
the 3 rd amino acid sequence shown as SEQ ID No. 4.
Further, the amino acid sequence of the protein phospholipid nano preparation is arranged in sequence of the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence.
In the above, the expression of the modified ApoA1 protein in e.collbl21 (DE 3), i.e. the protein phospholipid nano-preparation, comprises the following sequences in sequence: the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence are arranged in sequence.
Further, the diameter of each nano-disc particle of the protein phospholipid nano-preparation is 5-130nm.
In the protein phospholipid nano preparation, after the ApoA1 protein accords with the natural phospholipid, the particle size can be kept fixed, and the particle size specification can be as follows: the particle size can be kept between 5 and 130nm.
Preferably, the amount of internally embedded phospholipids per particle of the protein phospholipid nanofabrication is 100-1000.
The ApoA1 protein can also show good stability in a series of in vivo experiments, is an ideal carrier platform for drug delivery applicable to imaging diagnosis and treatment, and can be embedded into lipid molecules with the number of 100-1000 to form a complex after being combined with phospholipid, so that a complex biological macromolecule system is formed.
Further, the application of the protein phospholipid nano-preparation in preparing products for preventing and/or treating early neurodegenerative diseases.
Further, the use of said protein phospholipid nano-formulations for the preparation of a product for the treatment and/or prevention of CNS disorders of the central nervous system.
In addition, in order to solve the above problems, the present application also provides a brain drug delivery system, including the protein phospholipid nano-preparation as described above.
Drug delivery systems (Drug Delivery System, DDS) refer to a technical system that comprehensively regulates the distribution of drugs in an organism in terms of space, time and dose. The aim is to deliver a proper amount of medicine to the correct position at the right moment, thereby increasing the utilization efficiency of the medicine, improving the curative effect, reducing the cost and reducing the toxic and side effects. Drug delivery systems are a medical, ergonomic (material, mechanical, electronic) and pharmaceutical fusion disciplines, and the subject of investigation includes both drugs themselves, drug-loaded carrier materials, devices, and related techniques for physicochemical modification of drugs or carriers, etc. In this application, a brain drug delivery system is provided, and the brain drug delivery system has a certain therapeutic and preventive function for brain diseases.
The invention uses the genetically engineered apoA1 protein as the basic protein phospholipid nanometer preparation, which is a carrier platform for drug delivery, and after being combined with phospholipid, 100-1000 protein nanometer particles can be embedded into each apoA1 protein. The lipid molecules form a complex to form a complex biological macromolecule system, so that the carrier function of the drug delivery system is realized, and the lipid molecules can be used as a drug brain targeting delivery system for resisting CNS diseases and become a solution for the drug delivery system of the brain for CNS diseases of the central nervous system.
In addition, in order to solve the above problems, the present application further provides a preparation method of the protein phospholipid nano-preparation, which comprises the following steps:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
assembling the purified protein with dimyristoyl phosphatidylcholine to obtain the protein phospholipid nano preparation.
In the above-mentioned assembly of the purified protein and dimyristoyl phosphatidylcholine, the molar ratio of the purified protein to dimyristoyl phosphatidylcholine may be 1: (50-150).
Further, the molar ratio of the purified protein to dimyristoyl phosphatidylcholine may be 1: (60-120).
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1: inducible expression of the ApoA1 protein.
The preparation method comprises the following steps: and taking ApoA1 protein, and obtaining a crude protein sample after induced expression.
The experimental method comprises the following steps:
(1) Taking a recombinant plasmid containing an ApoA1 protein gene sequence, and converting the recombinant plasmid into an escherichia coli competent BL21 (DE 3) strain;
(2) After overnight incubation, the monoclonal was picked up into 50mL LB medium containing 100. Mu.g/mL kanamycin, placed on a shaker at 220r/min, and incubated overnight at 37 ℃.
(3) The bacterial solution was transferred to 1000mL of LB medium containing 100. Mu.g/mL kanamycin, so that the initial OD600 = 0.1. Shake-culturing at 37℃until the OD600 = 0.8, and adding 1mL of IPTG to a final concentration of 1mM.
(4) Placing on a shaking table at 37 ℃ and 220r/min for induction for 4 hours. Bacterial solutions (crude protein samples) were taken before and after induction and stored for subsequent SDS-PAGE gel electrophoresis.
Example 2: purification of proteins.
The preparation method comprises the following steps: purifying the crude protein sample to obtain purified protein.
The experimental method comprises the following steps:
(1) Induced bacteria (crude protein samples) were collected by centrifugation, washed 2 times with pre-chilled TBS buffer, the supernatant removed, lysate 20mM Tris-HCl ph= 8.0,1% Triton X was added to the pellet, the cells were resuspended thoroughly, and 1mg lysozyme was added. Low temperature ultrasound (ice bath) lyses bacteria. The lysate was centrifuged at 12000rpm for 45min at 4℃and the supernatant was collected for filtration and purified by Ni-NTA affinity column.
The buffers used were as follows: loading buffer solution: 1) ph=8.0, 20mm Tris-HCl,500mM NaCl,1% triton x (loading); 2) ph=8.0, 20mm Tris-HCl,500mM NaCl,50mM Na-cholate (flush); elution buffer: 3) ph=8.0, 20mm Tris-HCl,500mM NaCl,20mM imidazole (impurity-removing protein); 4) ph=8.0, 20mm Tris-HCl,500mMNaCl,50mM imidazole (impurity-removing protein); 5) ph=8.0, 20mm Tris-HCl,500mM NaCl,500mM imidazole (eluting the protein of interest);
(2) After elution of the protein of interest, it was dialyzed overnight against pH=8.0, 20mM Tris-HCl,50mM NaCl buffer. Purification then continues with an anion exchange column. Elution was performed with different concentrations of NaCl dissolved in 10mm Tris-HCl, ph=8.0. The eluted target protein was analyzed by SDS-PAGE in a small amount, and the remainder was dialyzed overnight against 20mM PBS buffer, pH=7.4.
(3) After being purified by nickel ion affinity chromatography and anion exchange chromatography, single ApoA1 protein can be obtained.
Experimental results: referring to FIG. 2, successful purification of the protein was shown by SDS-PAGE results. It should be noted that the yield of ApoA1 in this embodiment can be widely adjusted according to practical needs. Wherein, the yield of the ApoA1 protein is 1000mg of target protein can be obtained by expressing and purifying every 100L of liquid culture medium.
Example 3: assembly of DMPC with ApoA1 protein.
The preparation method comprises the following steps: assembling the purified protein with dimyristoyl phosphatidylcholine to obtain the protein phospholipid nano preparation.
The experimental method comprises the following steps: (1) Dimyristoyl phosphatidylcholine (DMPC) was dispersed in sodium cholate-containing PBS buffer (20 mm pH 7.4); (2) The ApoA1 protein solution was dropped drop-wise into DMPC and the experiment was performed in ultrasound until the solution became clear, with a molar ratio of ApoA1 protein to DMPC of 1:80. (3) The ApoA1 protein and DMPC mixture was dialyzed overnight against normal saline and the supernatant was centrifuged for characterization by Size Exclusion Chromatography (SEC), transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM).
Experimental results: referring to the AFM images of FIG. 3 (c-1) and FIG. 3 (c-2), the following table is consulted, and the size of the assembled individual protein phospholipid nanoparticles was measured in the AFM results. TEM shows that the diameter of the biological macromolecular system assembled by mixing the ApoA1 protein and the DMPC (hereinafter referred to as ApoA 1-ND) is about 10nm, and the successful construction of the ApoA1-ND complex is verified by the SEC chart of FIG. 3 (a) and the peak time of SEC, so that the successful assembly of the ApoA1-ND is proved.
Materials and instruments in examples 4-11:
1. experimental materials: (1) Male BALB/C nude mice, 4-5 weeks old, 20+ -2 g. Purchased from Fukang Biotechnology Co., ltd. (2) Male APP/PS1 double transgenic mice, 7 months old, 30+ -2 g. Purchased from Fukang Biotechnology Co., ltd. (3) Male C57BL/6J mice, 7 months old, 30+ -2 g. Purchased from Fukang Biotechnology Co., ltd.
2. Experimental reagent: bodipy, normal saline, GSH, rabbit anti-APP polyclonal antibody, rabbit anti-PECAM 1 polyclonal antibody, alexaFluor488 labeled goat anti-rabbit IgG, 4% paraformaldehyde, embedded paraffin, citric acid, methanol, hydrogen peroxide, triton X-100, concentrated normal goat serum (blocked), rabbit anti-PTRPC polyclonal antibody, immunohistochemical kit, nissl stain, absolute ethanol, xylene, hematoxylin, hydrochloric acid, neutral gum, dako REAL EnVision Detection System, toluidine blue, eosin Y (water soluble), anti-fluorescence quenching tablet.
3. Experimental instrument: dehydrator, ultrapure water system, pathological section machine, slicing knife, tissue spreading and baking machine, slide glass and cover glass, electroceramic stove for antigen repair, microscope and embedding machine.
Animal feeding methods in examples 4-11: the experiment adopts SPF mice, and the raising temperature of all mice is controlled at 25+/-1 ℃; clean grade pellet feed and sterile water can be obtained at any time by experimental mice; pad, squirrel cage material and drinking water replacement were performed every 5 d.
Example 4: in vivo fluorescence imaging analysis of ApoA1-ND.
The purpose of the experiment is as follows: to confirm that the protein phospholipid complex ND solution was able to cross the blood brain barrier into the center after intravenous administration, in vivo fluorescence imaging analysis was performed using fluorescence Bodipy labeled ApoA1-ND.
The experimental method comprises the following steps: the 12 nude mice were randomly divided into two groups, 200. Mu.L of physiological saline and Bodipy-Labeled ApoA1-ND, respectively, by tail vein injection. Image acquisition was performed 15min, 1h, 4h, 10h and 20h after intravenous administration.
Experimental results: (1) 15 minutes: the image acquisition results at 15min of solution injection showed that the saline-only control group was non-fluorescent and that Bodipy-labeled ApoA1-ND was mainly concentrated at the tail. (2) 1 hour: 1h image acquisition shows that Bodipy labeled ApoA1-ND enters the blood circulation, enters the heart after entering the inferior vena cava, reaches the brain through the aorta and its various branches, and can enter the brain through the blood brain barrier, and fluorescence is distributed throughout the brain. (3) 4 hours: the apoA1-ND reaches all parts of the whole body along with the blood circulation through the aorta and all the branches of the aorta after 4 hours, and the quantity of the apoA1-ND reaches the maximum, and the apoA1-ND pumped into the brain is greatly increased. (4) 10 hours: after 10 hours, the fluorescence intensity gradually decreased with the metabolism of the mice.
Summarizing: referring to FIG. 4, it can be seen from the trend of in vivo fluorescence imaging of ApoA1-ND for 15 minutes, 1 hour, 4 hours and 10 hours that ApoA1-ND can clearly demonstrate the ability to effectively cross the BBB.
Example 5: aβ immunofluorescence assay.
The purpose of the experiment is as follows: to demonstrate that ApoA1-ND can enter the brain parenchyma following tail vein administration, we used immunofluorescence techniques in combination with confocal microscopy analysis to assess the ability of ApoA1-ND to target aβ in vivo using aβ immunofluorescence analysis.
The experimental method comprises the following steps: 1) 200 μL of Bodipy-labeled ApoA1-ND tail at a concentration of 20 μM was injected intravenously into APP/PS1 mice; 2) After 4 hours, mice were treated and two frozen brain sections were prepared. To determine whether ApoA1-ND was delivered to the brain parenchyma and bound to aβ aggregates in the brain, the first set of sections labeled capillary blood with rabbit anti-PECAM 1 polyclonal antibody (primary antibody) and the second set labeled aβ aggregates in the brain with rabbit anti-APP polyclonal antibody (primary antibody). 3) Taking out the frozen slices from the refrigerator, recovering to room temperature, and soaking in PBS for several minutes; 4) Serum blocking: wiping the slide with absorbent paper, making an immune painting brush to circle around the tissue, dripping diluted normal goat serum, and sealing for 30min at room temperature to reduce nonspecific staining; 5) Adding an antibody: removing excessive liquid without washing, then dripping diluted primary antibody, and incubating overnight in a wet box at 4 ℃ after the primary antibody is added; 6) Secondary antibody (with fluorescence) was added: washing the slices with PBST for 3 times, each time for 3min, dripping diluted fluorescent secondary antibodies after the slices are wiped by absorbent paper, incubating for 1h at 37 ℃ in a wet box, washing the slices with PBST for 4 times, each time for 3min, and performing the steps and all the following operation steps in a dark place as far as possible; 7) Counterstaining and checking: dripping DAPI, incubating for 5min in dark place, and washing off redundant DAPI by PBST for 5min×4 times; 8) The liquid on the slice was wiped dry with a blotter paper, blocked with a blocking liquid containing an anti-fluorescence quencher, and then the image was collected under a fluorescence microscope.
Experimental results: referring to fig. 5, immunofluorescence signals (highlight positions of the aβ panels) by rabbit anti-APP polyclonal antibodies show that Bodipy-labeled ApoA1-ND (highlight positions of the ApoA1-ND panels) are highly aggregated around aβ in the major sites of aβ aggregation, i.e., brain cortex (Cotex) and Hippocampus (Hippocampus).
Summarizing: after 4h of tail vein administration, bodipy-labeled ApoA1-ND (red) showed high aggregation around Abeta in the major site of Abeta aggregation, i.e., cerebral cortex and hippocampus. The results show that ApoA1-ND can definitely target Abeta deposition after penetrating through cerebral blood vessels into brain parenchyma.
Example 6: anti-aβ immunostaining experiments.
The purpose of the experiment is as follows: to evaluate the targeting effect of ApoA1-ND on aβ and its disease modifying effect in AD.
The experimental method comprises the following steps: 7 month old male APP/PS1 mice were divided into three groups of three mice each, and tail vein injections were performed respectively: normal Saline (NS), 20 μΜ ApoA1-ND, 5 μΜ ApoA1-ND, 200 μl of the injection was performed for 2 consecutive weeks, and Normal Saline (NS) alone was injected under the same conditions as C57BL/6J male mice, specifically grouped as follows, and after two weeks, the mice were anesthetized, cold saline was perfused into the heart and brain tissue was collected.
| Group of | Test procedure |
| 1(Control) | C57BL/6J mice+intravenous physiological saline for 2 weeks continuously |
| 2 | APP/PS1 mice+intravenous physiological saline, 2 weeks in succession |
| 3 | APP/PS1 mice+intravenous ApoA1-ND (20. Mu.M concentration) for 2 weeks |
| 4 | APP/PS1 mice+intravenous ApoA1-ND (5. Mu.M concentration) for 2 weeks |
The operation flow is as follows: tissue dehydration, tissue transparency, wax impregnation, embedding, slicing and baking, slice dewaxing, antigen retrieval, endogenous peroxidase blocking, serum blocking, primary antibody adding, enzyme-labeled secondary antibody adding, color development agent adding, counterstaining, dehydration, sealing and microscopic photographing.
Experimental results: as can be seen from fig. 6 (a), apoA1-ND reduced amyloid deposition (brown plaques) in the cerebral cortex and hippocampus of APP/PS1 mice. When the concentration of ApoA1-ND was increased from 5. Mu.M to 20. Mu.M, the inhibition of the amount of amyloid plaques was significantly enhanced, and the amount of amyloid deposition was significantly reduced. As can be seen from FIG. 6 (b), apoA1-ND reduced amyloid deposition in the cerebral cortex of APP/PS1 mice (statistical plot). As can be seen from FIG. 6 (c), apoA1-ND reduced amyloid deposition in the hippocampus of APP/PS1 mice (statistical plot).
In summary, from figures 6 (a), 6 (b) and (c) anti-aβ immunostaining can clearly show a significant reduction in amyloid plaque burden in cortex and hippocampus of ApoA1-ND treated APP/PS1 double transgenic mice compared to saline treated APP/PS1 double transgenic mice.
Example 7: microglial activation assessment experiments.
The purpose of the experiment is as follows: abnormal activation of microglia was observed in AD patients and in the amyloidosis mouse model. Previous studies have shown that both aβ oligomers and fibrils trigger the neuroinflammatory cascade. In this example, activation of microglia was assessed using PTRPC as a marker.
The experimental method comprises the following steps: APP/PS1 mice 7 months old (n=8-9 per group) received ApoA1-ND treatment, 200 μl of 5 μΜ ApoA1-ND, were intravenously injected daily via the tail vein for 2 weeks, and physiological saline was administered as negative and normal controls using age-matched APP/PS1 and C57BL/6J mice, respectively. Brain sections (4 μm) were immunostained with rabbit anti-PTRPC polyclonal antibodies.
Experimental results: as can be seen from fig. 7 (a), apoA1-ND reduced microglial proliferation (brown signal) in the cerebral cortex and hippocampus of APP/PS1 mice. As can be seen from FIG. 7 (b), apoA1-ND reduced microglial proliferation in the cerebral cortex of APP/PS1 mice (statistical plot). As can be seen from FIG. 7 (c), apoA1-ND reduced microglial proliferation in the hippocampus of APP/PS1 mice (statistical plot).
In summary, apoA1-ND reduced amyloid (aβ) deposition in different tissues in mice model for alzheimer's disease compared to APP/PS1 double transgenic mice treated with physiological saline, and positively activated microglial load was significantly reduced in ApoA1-ND treated mice.
Given the central role of aβ aggregates in AD brain microglial activation, the significant reduction in activated microglial cells observed in ApoA1-ND treated mice may be attributed to the activity of ApoA1-ND in promoting aβ clearance.
Example 8: nissl staining analysis: neuronal status observations of cortex and hippocampus.
The purpose of the experiment is as follows: neuronal loss of the cortex and hippocampus is one of the main features of AD. It is possible to respond to the therapeutic effects of ApoA1-ND by observing neuronal states of cortex and hippocampus.
The experimental method comprises the following steps: the brain was examined for histological changes using Nissl staining.
1) Dehydrating the tissue; 2) The tissue is transparent; 3) Wax dipping; 4) Embedding; 5) Slicing and baking; 6) Dewaxing slices; 7) Slicing, preheating with 1% toluidine blue dye solution (Nib dye solution) at 60deg.C, dyeing for 40min, and washing with distilled water for 3 times; 8) Rapidly differentiating 95% alcohol, decolorizing, and microscopic examination until the background is clear; and (3) rapidly dehydrating the dried resin with absolute ethyl alcohol, enabling the dimethylbenzene to be transparent, and sealing the resin in a ventilation cabinet after air drying.
Experimental results:
from the Nissl staining analysis in fig. 8, both cortical and hippocampal of saline-treated APP/PS1 double transgenic mice showed neuronal subcellular reduction and neuronal nuclear atrophy compared to C57BL/6J mice. In contrast, apoA1-ND treatment significantly reduced neuronal integrity impairment and neuronal loss in APP/PS1 double transgenic mice.
Example 9: behavioral trace analysis of mice with Alzheimer's disease model: morris water maze experiment.
The purpose of the experiment is as follows: spatial learning and memory capacity of APP/PS1 double transgenic mice was assessed.
The experimental method comprises the following steps: male APP/PS1 mice of 7 months of age were randomly divided into 2 groups, and physiological saline or ApoA1-ND solution was injected into the tail vein, respectively, for 4 weeks, 200. Mu.L per day. Normal control groups were prepared by injecting normal saline into 7 month old male C57BL/6J mice, and the specific groups are as follows.
| Group of | Test procedure |
| 1(Control) | C57BL/6J mice+intravenous physiological saline for 4 weeks |
| 2 | APP/PS1 mice+intravenous physiological saline, 4 weeks continuously |
| 3 | APP/PS1 mice+intravenous ApoA1-ND (20. Mu.M concentration) for 4 weeks |
The experiment was performed for a total of 5 days, the first four days being the positioning navigation experiment: at the beginning of the experiment, the mice are put into a pool (without a platform) to swim freely for 2min so as to be familiar with the labyrinth environment, and the mice are put on the platform for a plurality of seconds so that the mice know that the escape platform exists in the water. Then the mice are put into a pool from the wall of the quadrant (I quadrant) farthest from the platform, timing is started, the timing is stopped 5s after the mice ascend the platform, the swimming time is allowed to be 120s, if the mice do not ascend the platform after 120s, the latency period is recorded as 120s, the mice stand on the platform for a plurality of seconds, and finally the mice are wiped dry and put into a cage. The mice were placed in the remaining three quadrants in this order (i.e., the experiments were performed sequentially in the order of I, II, III, IV), 4 quadrants per day for a total of 4 days. The video analysis system is trained to automatically record escape latency, trajectory (path), total distance, total time, average speed, swimming distance in each quadrant, time spent in each quadrant, etc. to the underwater platform. And the original platform is removed on the 5 th day, and the environment and the water temperature are the same as those of the positioning navigation test. The subsurface platform was dusted off and the mice were subjected to a single test with water from the wall of the quadrant furthest from the platform (quadrant i), the remaining quadrants not requiring testing. The swim track of the mice was recorded for 120s, after which the mice were taken out, dried with water and placed in cages. The measurement indexes include the number of times of crossing the original platform position (the number of times of entering and leaving the platform), the percentage of time in the quadrant of the original platform to the total time, the swimming distance of each quadrant, and the like.
Experimental results: as can be seen from FIG. 9 (a), 7 month old male APP/PS1 mice received ApoA1-ND treatment. FIG. 9 (b) is escape latency, which was significantly reduced in ApoA1-ND treated APP/PS1 double transgenic mice during four days of water maze training. FIG. 9 (c) shows that the swimming speed of ApoA1-ND treated APP/PS1 double transgenic mice is improved to a certain extent in the four-day water maze training process. FIG. 9 (d) shows the number of times each group of mice passed the platform one day after platform removal, and it can be seen that the number of times that APP/PS1 mice receiving ApoA1-ND treatment passed the platform is significantly greater than the number of times that APP/PS1 mice not receiving ApoA1-ND treatment passed the platform.
In summary, reference is made to fig. 9 (a), 9 (b), 9 (C) and 9 (d), wherein they exhibit defects in learning ability compared to C57BL/6J mice. In contrast, apoA1-ND treated APP/PS1 double transgenic mice exhibited significantly improved spatial learning and memory capacity during four days of water maze training, both escape latency and swimming speed.
After the escape platform is removed, the C57BL/6J mice concentrate on the quadrant where the search platform is located, and APP/PS1 double-transgenic mice treated by normal saline show a non-concentrated search strategy. After ApoA1-ND treatment, the searching strategy of APP/PS1 double-transgenic mice is obviously improved, has more positive exploratory performance and is more frequently appeared around the platform position.
Example 10: biosafety assessment experiment for ApoA1-ND treatment of AD.
The purpose of the experiment is as follows: evaluation of biological safety of ApoA1-ND for treatment of AD
The experimental method comprises the following steps:
male APP/PS1 mice of 7 months of age were randomly divided into 2 groups, and physiological saline or ApoA1-ND solution was injected into the tail vein, respectively, for 4 weeks, 200. Mu.L per day. Normal control groups were prepared by injecting normal saline into 7 month old male C57BL/6J mice, and the specific groups are as follows.
| Group of | Test procedure |
| 1(Control) | C57BL/6J mice+intravenous physiological saline for 4 weeks |
| 2 | APP/PS1 mice+intravenous physiological saline, 4 weeks continuously |
| 3 | APP/PS1 mice+intravenous ApoA1-ND (20. Mu.M concentration) for 4 weeks |
Experimental mice were sacrificed and major organs were collected, fixed, dehydrated, embedded in paraffin, serially sectioned, hematoxylin and eosin stained and finally assessed with an optical microscope.
Experimental results:
in comparison to animals treated with physiological saline, apoA1-ND treated animals had no apparent pathological changes in heart (a), liver (B), spleen (C) and kidney (E). In the lung (D), it was reported that inflammatory responses such as interalveolar hemorrhage, perialveolar capillary congestion and interstitial focal lymphocyte infiltration were observed in APP/PS1 mice treated with physiological saline.
This pathological change was not seen in ApoA1-ND treated animals. Since HDL (high-density lipoprotein, high density lipoprotein) has anti-inflammatory activity, it is presumed that ApoA1-ND may alleviate inflammatory responses in the lungs of APP/PS1 mice by the same mechanism.
As shown in fig. 10, A, B, C, D and E correspond to heart, liver, spleen, lung and kidney in this order. Treatment with ApoA1-ND reduced microglial proliferation and improved nervous system changes in APP/PS1 mice without causing other significant damage to brain tissue. Experimental data indicate that in vivo application of ApoA1-ND is safe under current dosing regimens. For anti-AD treatment, further evaluation of long-term in vivo safety is of significant commercial value.
Experimental results show that ApoA1-ND has higher binding affinity to Abeta monomers and oligomers, and accelerates degradation of Abeta in microglia and liver cells. More importantly, it can be seen from the experimental results that after intravenous injection in the tail of the mice, apoA1-ND can pass through the blood brain barrier and enter the brain.
And after continuous tail vein injection treatment, the model mice can reduce amyloid deposition, relieve microglial hyperplasia, improve nervous system change and save memory defects.
Because of the protein and the natural phospholipid which are derived from the ApoA1-ND component, the protein system developed by the invention can pass through the blood brain barrier with high efficiency, has high brain targeting property, is safe and nontoxic, can effectively eliminate the Alzheimer's symptoms, and can be applied to preventing and treating early neurodegenerative diseases.
While the preferred embodiments and examples of the present invention have been described, it should be noted that those skilled in the art may make various modifications and improvements without departing from the inventive concept, including but not limited to, adjustments of proportions, procedures, and amounts, which fall within the scope of the present invention. While the preferred embodiments and examples of the present invention have been described, it should be noted that those skilled in the art may make various modifications and improvements without departing from the inventive concept, including but not limited to, adjustments of proportions, procedures, and amounts, which fall within the scope of the present invention.
SEQUENCE LISTING
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Claims (5)
1. Use of a protein phospholipid nano-preparation for the preparation of a product for the prevention and/or treatment of CNS disorders of the central nervous system, characterized in that,
the CNS disorders include early stage neurodegenerative disorders;
the protein phospholipid nano preparation is a protein phospholipid complex based on the combination of ApoA1 protein and phospholipid;
wherein, the ApoA1 protein is an ApoA1 protein which does not contain a conserved sequence shown in SEQ ID No.1 or a homologous sequence of the conserved sequence;
the amino acid sequence of the protein phospholipid nano preparation comprises:
the 1 st amino acid sequence shown as SEQ ID No.2, the 2 nd amino acid sequence shown as SEQ ID No.3 and the 3 rd amino acid sequence shown as SEQ ID No. 4;
the amino acid sequence of the protein phospholipid nanometer preparation is sequentially arranged in the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence.
2. The use according to claim 1, wherein each nanoparticle of the protein phospholipid nanofabrication has a diameter of 5-130nm.
3. The use according to claim 1, wherein the amount of internally intercalated phospholipids per nanoparticle of the protein phospholipid nanofabrication is 100-1000.
4. The use according to claim 1, wherein,
the protein phospholipid nano preparation is prepared by the following method:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
assembling the purified protein with dimyristoyl phosphatidylcholine to obtain the protein phospholipid nano preparation.
5. A brain drug delivery system comprising a protein phospholipid nanoformulation in an application according to any one of claims 1-4.
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