CN115177742B - Preparation method and application of drug-loaded brain-targeted exosome - Google Patents
Preparation method and application of drug-loaded brain-targeted exosome Download PDFInfo
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- CN115177742B CN115177742B CN202210597989.3A CN202210597989A CN115177742B CN 115177742 B CN115177742 B CN 115177742B CN 202210597989 A CN202210597989 A CN 202210597989A CN 115177742 B CN115177742 B CN 115177742B
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Classifications
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- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
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- General Chemical & Material Sciences (AREA)
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Abstract
The invention particularly relates to a preparation method and application of a drug-loaded brain-targeted exosome. The drug-loaded brain-targeted exosomes comprise brain-targeted peptide molecules, exosomes and drugs entrapped in the exosomes. The medicine is losartan which is an angiotensin II receptor antagonist. Brain targeting peptides are co-cultured to bind to the surface of exosomes via the exosome marker protein CD 63. The exosomes are derived from the cell supernatant of mouse mononuclear macrophage leukemia cells. The brain-targeted exosome drug-carrying system can carry drugs into exosome without obvious biotoxic effect. In addition, small amounts of administration to the tail vein can penetrate the blood brain barrier into the hub to effect inhibition of angiotensin (RAS) expression. In doxorubicin-induced chronic kidney disease mouse models, brain-targeted exosome-loaded losartan drug complexes are capable of significantly improving renal function, renal tissue injury, and renal interstitial collagen fiber deposition by down-regulating central RAS expression.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a preparation method and application of a drug-loaded brain-targeted exosome.
Background
Chronic Kidney Disease (CKD) is a disease characterized by a gradual decline in renal function caused by hypertension, diabetes, glomerulonephritis, polycystic kidney disease, etc. High incidence rate of CKD, low early detection rate, high mortality disability rate, high treatment cost and serious influence on the life quality of people. If the kidney disease is not effectively treated in early stage, the kidney disease will progress to the end-stage kidney disease, and kidney replacement therapy has to be accepted, which not only seriously affects the life quality of patients, but also brings about a heavy economic burden to the patients and family members. At present, the clinical drug treatment is very limited, mainly comprises an angiotensin enzyme inhibitor, a glucocorticoid, an immunosuppressant, a traditional Chinese medicine and the like, but the effect on the aspect of delaying the disease progression is still limited.
It was found that lateral ventricle injection of antisense oligonucleotides inhibited brain renin angiotensin system (RAS axis) down-regulates hypertension in chronic kidney-double-clamp kidney disease rats. In a model of renal fibrosis in which high salt promotes CKD, brain-renal RAS is involved with the interactive axis, and centrally administered small doses (about 1/500 of an effective dose for peripheral administration) of RAS inhibitors (such as losartan) significantly reduce chronic kidney injury. However, such small doses of the drug are not effective in treating chronic kidney injury by peripheral administration. Thus, if the drug can be targeted to the central system, better therapeutic results can be achieved with lower doses of the drug. However, in view of the dangers and high difficulty of central administration, it is difficult to popularize it into clinical application. Thus, there is a need for a low immunogenicity, brain-targeted and highly effective drug-loaded drug carrier to deliver RAS inhibitors through central targeting, ultimately achieving kidney protection.
In recent years, nanomaterials have demonstrated great medical advantages, and can be used for disease treatment and drug delivery through surface modification or their own characteristics. For example, the targeting of the drug to the damaged organ is improved, the toxicity of the drug is reduced, the blood concentration is increased, and the like. However, most of artificially synthesized nano-drug carriers have the defects of high preparation difficulty, poor compatibility, undefined toxicity and the like.
Exosomes are a class of nanoscale disc vesicles with diameters of 30-200nm, naturally occurring in a variety of cells and body fluids, and have many advantages as drug carriers such as better low biotoxicity, stability and low immunogenicity. However, in the field of kidney disease treatment, brain-targeted drug delivery technology is greatly limited in this field due to the specificity of kidney disease and its therapeutic agents.
Disclosure of Invention
The invention provides a drug-loaded brain-targeted exosome, a preparation method and application thereof. In view of the defects in the prior art, the research inventor obtains the main technical scheme of the invention through repeated practice, and mainly provides a brain-targeted exosome drug-carrying system loaded with drugs such as losartan, which is used for repairing and treating chronic kidney diseases. The invention also aims to provide the application of the drug-loaded brain-targeted exosome in chronic kidney disease repair. The drug-loaded brain-targeted exosomes comprise brain-targeted peptide molecules, exosomes derived from macrophages and a drug angiotensin II receptor antagonist losartan loaded in the exosomes, and can penetrate through the blood-brain barrier, and the drug-loaded brain-targeted exosomes can transport drugs to brain tissues in a targeted manner and down regulate RAS expression at hippocampal parts. Can achieve the effect of protecting chronic kidney disease by using a very small drug dose (about 1/500 of the effective dose for peripheral administration).
In order to achieve the technical effects of the invention, the technical scheme adopted by the invention comprises the following steps:
Firstly, the drug-loaded brain-targeted exosome of the present invention comprises a brain-targeted peptide, an exosome, and a drug loaded in the exosome, wherein:
the exosomes are derived from mouse mononuclear macrophage leukemia cells RAW264.7.
The source exosomes have certain orientation to brain tissues with improved inflammatory response, which is different from the characteristics of other source exosomes, and the encephalitis is found to be significantly up-regulated in the animal model of chronic kidney disease, which is helpful for enrichment of the source exosomes in the brain tissues of the animal model of CKD, so that the source exosomes can achieve the effect of carrying the specific drugs of the application to the administration sites and regulating the corresponding expression, and other source exosomes cannot achieve the effect.
The brain targeting peptide is Angiopep-2-CP05, and the preparation method is a solid phase synthesis method.
Compared with the existing brain targeting technology, the brain targeting peptide has the advantages that the synthesis method is simple, and the brain targeting peptide can be specifically combined with the exosome surface markers adopted by the invention, so that the exosome and the medicine carried by the exosome can be carried into the central nervous system through the blood brain barrier to play a role.
The drug is an angiotensin II receptor antagonist, such as but not limited to losartan, valsartan, irbesartan, telmisartan and the like.
Further, the drug concentration in the drug-loaded brain-targeted exosome is 10-70 mug/100 mug, but is optimally 70 mug/100 mug.
The drug concentration is the effective concentration of the drug-loaded brain-targeted exosome, especially when the drug concentration is 1mg/kg, the optimal combination of drug effect and drug dosage can be realized, and the drug has the lowest biotoxicity.
A great deal of research experiments of the inventor show that only the combination of the brain targeting peptide Angiopep-2-CP05 and exosomes derived from mouse mononuclear macrophage leukemia cells RAW264.7 and related drugs (especially losartan) can achieve the technical effects and have low biotoxicity.
Further, the exosome size (diameter) distribution is predominantly 140±50nm.
The invention also provides a preparation method of the drug-loaded brain-targeted exosome, which comprises the following steps:
Co-culturing the brain targeting peptide and the exosome, and linking the brain targeting peptide to the surface of the exosome to form a brain targeting peptide modified exosome complex; and co-culturing the medicine and the brain targeting peptide modified exosome complex to obtain the medicine carrying brain targeting exosome system.
Further, the brain targeting peptide modified exosome complex is subjected to ultrafiltration centrifugation to remove the non-encapsulated drug.
Further, the mouse mononuclear macrophage leukemia cell RAW264.7 derived exosome is obtained by the following method:
Culturing mouse mononuclear macrophage leukemia cell RAW264.7 cells by adopting a conventional culture method, changing into an exosome-free serum culture medium in the logarithmic growth phase, and collecting cell supernatants after culturing for 48 hours to obtain mouse mononuclear macrophage leukemia cell RAW 264.7-derived exosomes.
The invention also provides application of the drug-loaded brain-targeted exosome in treating kidney diseases or preparing drugs for treating kidney diseases.
The beneficial effects of the invention are as follows:
Firstly, no related technical scheme for treating kidney diseases through brain targeting and transporting a very small amount of drugs is currently studied, the technical scheme of a drug-loaded brain targeting exosome drug-loaded system for treating kidney diseases is provided for the first time, and exosomes derived from macrophages have a certain targeting effect on inflammatory brain tissues of a chronic kidney disease animal model, so that enrichment of loaded drugs in brain tissues of a CKD model is promoted, and finally, effective effects on the brain tissues are ensured on the basis of reducing the drug consumption. The invention provides support for treating CKD by using specific exosomes and specific brain targeting peptides and specific binding kidney disease therapeutic drugs, thereby opening up a new therapeutic direction.
Research experiments show that the drug-loaded brain-targeted exosome has the characteristic of extremely small drug dosage, which is about 1/500 of the effective dosage of conventional peripheral administration; in addition, on the basis of ensuring the drug effect, the drug has the special characteristic of extremely low biological toxicity, and the drug safety is greatly ensured. When the doxorubicin chronic kidney disease mice are applied, the renal function, inflammatory cell infiltration of kidney tissues and interstitial collagen deposition can be improved, and the repair of chronic kidney disease can be promoted.
In conclusion, compared with the prior art, the drug-loaded brain-targeted exosome prepared by the invention can load small-molecule compound drugs into the exosome, has lower cytotoxicity and systematic toxicity, and has the capacity of down-regulating the expression of hippocampal RAS components by brain-targeted delivery drugs, and can promote the repair of chronic kidney diseases.
Drawings
FIG. 1 is a flow chart of the preparation of drug-loaded brain-targeted exosomes according to the present invention;
FIG. 2A is a transmission electron microscope of the exosomes and brain-targeted exosomes described in test example 1;
FIG. 2B is an immunoblot of exosome marker proteins from test example 1;
FIG. 2C is a graph showing the particle size distribution of NTA-analyzed exosomes and brain-targeted exosomes in test example 1;
FIG. 3A is a graph showing 24-hour survival rate results in test example 2;
FIG. 3B is a graph showing the results of the 48-hour survival rate in test example 2;
FIG. 4 is a fluorescence imaging of major organs of test example 3 brain-targeted exosomes in normal mice;
FIG. 5 is an H & E diagram of the drug-loaded brain-targeted exosomes of test example 3 for each major organ in a normal mouse;
FIG. 6 is a graph comparing the effect of drug-loaded brain-targeted exosomes on kidney function in a chronic kidney disease mouse model in test example 3;
FIG. 7 is a graph of H & E staining of drug-loaded brain-targeted exosomes against chronic kidney disease mouse model kidney tissue in test example 3;
FIG. 8 is a map of drug-loaded brain-targeted exosomes versus chronic kidney disease mouse model kidney tissue Masson staining in test example 3;
FIG. 9 is an immunohistochemical view of drug-loaded brain-targeted exosomes versus renin (AGT) in the hippocampus of a mouse model of chronic kidney disease in test example 3.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the technical gist of the present invention is further described by the following specific embodiments, and the embodiments can help those skilled in the relevant art to better understand the present invention. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Based on the above disclosure, those skilled in the art may make various modifications and adjustments to the present invention without essential details, and still fall within the scope of the present invention.
The brain-targeted exosome drug-carrying system, the preparation thereof and the therapeutic effect on chronic kidney disease of mice are further described below by specific examples.
The brain targeting peptide Angiopep-2-CP05 (amino acid sequence: CRHSQMTVTSRLGSGTFFYGGSRGKRNNFKTEEY) was used in the following examples.
In some preferred embodiments, the drug includes losartan (hereinafter may be abbreviated as Los), but is not limited thereto, and may be other angiotensin ii receptor antagonists such as valsartan and the like. Wherein the medicine enters the medicine carrying exosome in a co-culture mode.
The source of the exosomes is mouse mononuclear macrophage leukemia cells RAW264.7.
Example 1
1. Extraction and identification of mouse mononuclear macrophage leukemia cell exosomes:
culturing RAW264.7 cells by adopting a conventional culture method, changing into an exosome-free serum culture medium in the logarithmic growth phase, culturing for 48 hours, and collecting the supernatant to obtain the exosome from macrophage. The specific process is as follows:
RAW264.7 cells in the cryopreservation tube were transferred to a 15mL centrifuge tube containing 6mL of complete medium, centrifuged at 1,000rpm for 5min; the supernatant was discarded, and the pellet was resuspended in 6mL complete medium, inoculated into 10cm 2 dishes, and cultured in a 5% CO 2 cell incubator at 37℃to log phase; 3mLPBS, washing once, replacing the culture medium with 10% exosome-free serum culture medium, culturing for 48h, and collecting the supernatant; centrifuging at 4 ℃,3,000g for 10min, collecting supernatant liquid, discarding cell debris into a new 15mL centrifuge tube; adding exosome extract (volume ratio is 4:1) into the supernatant centrifuge tube (cell supernatant exosome extraction kit is purchased from Shanghai Meihai Meihao biological technology Co., ltd., model is UR 52121), shaking for 1min, and fully mixing the solution. Standing for 12h at 4 ℃; centrifuging the mixture at 4deg.C for 10,000g for 60min, gently discarding supernatant, concentrating exosomes on the tube wall, and centrifuging for 2min to remove supernatant as much as possible; the exosomes were resuspended with 200 μl of PBS and then collected in 1.5mL EP tubes; the supernatant was collected by centrifugation at 4℃and 12,000g for 2min, the supernatant being enriched in exosomes.
The obtained exosomes were characterized by transmission electron microscopy, particle size analyzer, western blotting, as shown in fig. 2A-2C.
2. Preparation of brain-targeted exosomes
2Mg of brain targeting peptide (Angiopep-2-CP 05) was dissolved in 2mL of deionized water to prepare a concentration of 1. Mu.g/. Mu.L; according to the mass 1:1 synthesis. Namely, after 50 mug exosomes and 50 mug gAngiopep-2-CP05 are fully mixed, shaking and evenly mixing, sealing by sealing glue, and incubating for 12-16 hours at 4 ℃; centrifuging at 4deg.C for 20min at 5,000rpm in a 100kDa ultrafiltration tube to remove free polypeptide; repeating centrifugation for three times, adding 200 mu L PBS into the ultrafiltration tube after each centrifugation is finished, and centrifuging after uniformly mixing; after centrifugation, the inner tube was aspirated, and then the inner tube was rinsed with 50. Mu.L of PBS, and repeated 2 times, and the total liquid in the collected inner tube was the total synthetic Exo-Angiopep-2-CP05 (Exo ACP).
3. Preparation of drug-loaded brain-targeted exosomes
100 Μg of Exo ACP solution and 110 μg of losartan solution were mixed uniformly, and the exosome membrane structure was restored with "0.25" tip,20% intensity, 30s on/off for 3 minutes with 2 minute cooling time intervals to properly increase the outer membrane permeability of the line exosome to promote drug permeation into the exosome, followed by 37 ℃ for 60 min. Transferring to a 100kDa ultrafiltration centrifuge tube, 5,000rpm,20min,4 ℃, removing the drug which is not loaded, ultrafiltering for 3 times, and collecting inner tube liquid, namely the drug-loaded brain-targeted exosome (Exo ACP @ Los).
The losartan concentration was measured by high performance liquid chromatography, and the concentration of losartan loaded therein was measured to be about 70 μg/100 μg of exosomes.
Example 2
1. Extraction and identification of mouse mononuclear macrophage leukemia cell exosomes:
culturing RAW264.7 cells by adopting a conventional culture method, changing into an exosome-free serum culture medium in the logarithmic growth phase, culturing for 48 hours, and collecting the supernatant to obtain the exosome from macrophage. The specific process is as follows:
RAW264.7 cells in the cryopreservation tube were transferred to a 15mL centrifuge tube containing 6mL of complete medium, centrifuged at 1,000rpm for 5min; the supernatant was discarded, and the pellet was resuspended in 6mL complete medium, inoculated into 10cm 2 dishes, and cultured in a 5% CO 2 cell incubator at 37℃to log phase; 3mLPBS, washing once, replacing the culture medium with 10% exosome-free serum culture medium, culturing for 48h, and collecting the supernatant; centrifuging at 4 ℃,3,000g for 10min, collecting supernatant liquid, discarding cell debris into a new 15mL centrifuge tube; adding exosome extract (volume ratio is 4:1) into the supernatant centrifuge tube (cell supernatant exosome extraction kit is purchased from Shanghai Meihai Meihao biological technology Co., ltd., model is UR 52121), shaking for 1min, and fully mixing the solution. Standing for 12h at 4 ℃; centrifuging the mixture at 4deg.C for 10,000g for 60min, gently discarding supernatant, concentrating exosomes on the tube wall, and centrifuging for 2min to remove supernatant as much as possible; the exosomes were resuspended with 200 μl of PBS and then collected in 1.5mL EP tubes; the supernatant was collected by centrifugation at 4℃and 12,000g for 2min, the supernatant being enriched in exosomes.
2. Preparation of brain-targeted exosomes
2Mg of brain targeting peptide (Angiopep-2-CP 05) was dissolved in 2mL of deionized water to prepare a concentration of 1. Mu.g/. Mu.L; according to the mass 1:1 synthesis. Namely, after 50 mug exosomes and 50 mug Angiopep-2-CP05 are fully mixed, shaking and evenly mixing, sealing by sealing glue, and incubating for 12-16 hours at 4 ℃; centrifuging at 4deg.C, 5,000rpm, and 20min in a 100kDa ultrafiltration tube to remove free polypeptide; repeating centrifugation for three times, adding 200 mu L PBS into the ultrafiltration tube after each centrifugation is finished, and centrifuging after uniformly mixing; after centrifugation, the liquid in the inner tube is sucked out, then the inner tube is washed by 50 mu LPBS, and the inner tube is repeated for 2 times, and all the collected liquid in the inner tube is the synthesized total Exo-Angiopep-2-CP05 (Exo ACP).
3. Preparation of drug-loaded brain-targeted exosomes
100 Μg of Exo ACP solution and 90 μg of losartan solution were mixed uniformly, and the Exo membrane structure was restored with "0.25" tip,20% intensity, 30s on/off for 3 minutes with 2 minute cooling time intervals to properly increase the outer membrane permeability of the line exosomes to promote drug permeation into the exosomes, followed by 37 ℃ for 60 min. Transferring to a 100kDa ultrafiltration centrifuge tube, 5,000rpm,20min,4 ℃, removing the drug which is not loaded, ultrafiltering for 3 times, and collecting inner tube liquid, namely the drug-loaded brain-targeted exosome (Exo ACP @ Los).
The losartan concentration was measured by high performance liquid chromatography, and the concentration of losartan loaded therein was measured to be about 40 μg/100 μg of brain-targeted exosomes.
Example 3
1. Extraction and identification of mouse mononuclear macrophage leukemia cell exosomes:
culturing RAW264.7 cells by adopting a conventional culture method, changing into an exosome-free serum culture medium in the logarithmic growth phase, culturing for 48 hours, and collecting the supernatant to obtain the exosome from macrophage. The specific process is as follows:
RAW264.7 cells in the cryopreservation tube were transferred to a 15mL centrifuge tube containing 6mL of complete medium, centrifuged at 1,000rpm for 5min; the supernatant was discarded, and the pellet was resuspended in 6mL complete medium, inoculated into 10cm 2 dishes, and cultured in a 5% CO 2 cell incubator at 37℃to log phase; 3mLPBS, washing once, replacing the culture medium with 10% exosome-free serum culture medium, culturing for 48h, and collecting the supernatant; centrifuging at 4 ℃,3,000g for 10min, collecting supernatant liquid, discarding cell debris into a new 15mL centrifuge tube; adding exosome extract (volume ratio is 4:1) into the supernatant centrifuge tube (cell supernatant exosome extraction kit is purchased from Shanghai Meihai Meihao biological technology Co., ltd., model is UR 52121), shaking for 1min, and fully mixing the solution. Standing for 12h at 4 ℃; centrifuging the mixture at 4deg.C for 10,000g for 60min, gently discarding supernatant, concentrating exosomes on the tube wall, and centrifuging for 2min to remove supernatant as much as possible; the exosomes were resuspended with 200 μl of PBS and then collected in 1.5mL EP tubes; the supernatant was collected by centrifugation at 4℃and 12,000g for 2min, the supernatant being enriched in exosomes.
2. Preparation of brain-targeted exosomes
2Mg of brain targeting peptide (Angiopep-2-CP 05) was dissolved in 2mL of deionized water to prepare a concentration of 1. Mu.g/. Mu.L; according to the mass 1:1 synthesis. Namely, after 50 mug exosomes and 50 mug Angiopep-2-CP05 are fully mixed, shaking and evenly mixing, sealing by sealing glue, and incubating for 12-16 hours at 4 ℃; centrifuging at 4deg.C, 5,000rpm, and 20min in a 100kDa ultrafiltration tube to remove free polypeptide; repeating centrifugation for three times, adding 200 mu L PBS into the ultrafiltration tube after each centrifugation is finished, and centrifuging after uniformly mixing; after centrifugation, the inner tube was aspirated, and then the inner tube was rinsed with 50. Mu.L of PBS, and repeated 2 times, and the total liquid in the collected inner tube was the total synthetic Exo-Angiopep-2-CP05 (Exo ACP).
3. Preparation of drug-loaded brain-targeted exosomes
100 Μg of Exo ACP solution and 70ug of losartan solution were mixed well, and the Exo membrane structure was restored with "0.25" tip,20% intensity, 30s on/off for 3min, 2min cooling time intervals to properly increase the outer membrane permeability of the line exosome to promote drug permeation into the exosome, followed by 37 ℃,60 min. Transferring to a 100kDa ultrafiltration centrifuge tube, 5,000rpm,20min,4 ℃, removing the drug which is not loaded, ultrafiltering for 3 times, and collecting inner tube liquid, namely the drug-loaded brain-targeted exosome (Exo ACP @ Los).
The losartan concentration was measured by high performance liquid chromatography, and the concentration of losartan loaded therein was measured to be about 20 μg/100 μg of brain-targeted exosomes.
Comparative example
The method for preparing the drug-loaded brain-targeted exosomes comprises the following specific preparation steps of:
1. Extracting mouse blood-derived exosomes: blood from the inner canthus venous plexus of mice was used, and after standing overnight at 4℃at 3,000rpm for 10min, supernatant was collected, and the sample was centrifuged again at 4℃at 10,000g for 10min to remove impurities, and the supernatant was transferred to a new centrifuge tube. According to serum: 1 XPBS solution: blood PureExo Solution = 1ml:3 ml: 1ml of a proportional vortex shaker were mixed for 1min (serum exosome extraction kit was purchased from Shanghai Yufubo biotechnology Co., ltd., model number UR 52136) and allowed to stand at 4℃for 2h. The exosome extract mixture was centrifuged at 12,000g at 4℃for 60min to precipitate exosomes, which were Blood-derived exosomes (Blood-Exo).
2. Preparation of brain-targeted exosomes: the specific procedure was as in example 1 to give Blood-Exo ACP.
3. Preparation of drug-loaded brain-targeted exosomes: the specific procedure was as in example 1 to give Blood-Exo ACP @los.
Test example 1
The exosomes, brain-targeted exosomes, drug-loaded brain-targeted exosomes in example 1 were observed and identified as follows:
(1) As shown in the transmission electron microscope result of fig. 2A, the exosomes and the brain-targeted exosomes of example 1 both have disc-like structures (exosomes on the left side of the figure and brain-targeted exosomes on the right side).
(2) As shown in fig. 2B, the identification of the exosome marker protein using the western blot method revealed that the exosomes and the brain-targeted exosomes CD63, CD9 and TSG101 of example 1 were significantly highly expressed (exosomes on the left side of the figure and brain-targeted exosomes on the right side).
(3) As shown in fig. 2C, the size distribution of exosomes and brain-targeted exosomes was analyzed using NTA, the latter were found to be slightly larger in size than the former (particle size mean: 135.9nmvs.125.9 nm), indicating successful modification of brain-targeted peptide to the surface of exosomes, the exosome size (diameter) distribution being predominantly 140±50nm.
Test example 2
Biotoxicity validation assay of brain-targeted exosomes obtained in example 1:
HCMECD3 cells (immortalized human brain microvascular endothelial cells) were grown to 80% confluency, and then pancreatin digested cells were prepared into a cell suspension at a concentration of 5×10 3/mL, and the cell suspension was inoculated in 100 μl to 96 well plates, incubated at 37 ℃ in 5% co 2 for 24 hours, and incubated in serum-free medium for 12 hours to allow the cells to stand still, 10 μl of brain-targeted exosomes at different concentrations (0.1,1,5, 10, 15 μg/mL, respectively) were added, respectively, while the culture solution alone was used as a blank, and the cells containing but not the brain-targeted exosomes were used as a normal control. 3 duplicate wells were made for each concentration and the plates were incubated in the incubator for 24h and 48h, respectively.
After the incubation, the cell supernatant was discarded, 10. Mu.L of CCK-8 solution and 90. Mu.L of DMEM basal medium were added to each well, and after 2 hours the absorbance at 450nm was measured with an ELISA reader. Experiments were repeated 3 times.
Cell survival percentage= (brain-targeted exosome treated group OD 450 value-blank OD 450 value)/(normal control OD 450 value-blank OD 450 value) ×100%.
The 24 hour survival results of cells co-cultured with brain-targeted exosomes are detailed in fig. 3a and the 48 hour survival is detailed in fig. 3B.
The results prove that: when the material concentration is up to 15 mug/mL, the brain targeting exosome has no obvious effect on the activity of human brain microvascular endothelial cells, which proves that the brain targeting exosome obtained by the invention has very low biotoxicity.
Test example 3
The drug-loaded brain-targeted exosomes of example 1 and comparative example were applied to treatment trials in mice with chronic kidney disease, specifically as follows:
healthy adult male C57BL/6 mice, 6 weeks old, weighing 20-25g, were selected and adapted for one week. A mouse model of chronic kidney disease was constructed using tail vein injection of doxorubicin (10 mg/kg). Intervention was initiated at the third week after molding, once every other day, and all mice were sacrificed at the fifth week.
(1) When the C57BL/6 mice were subjected to doxorubicin intravenous injection and molding for the third week, the mice were injected with Cy5.5 and Exo ACP-Cy5.5、Blood-ExoACP -Cy5.5 through tail veins, 5mg/kg of each group of brain-targeted exosomes was injected (Cy5.5 groups were injected with the same amount of Cy5.5 as other treatment groups), the mice were sacrificed after 24 hours, and the main organs (heart, liver, spleen, lung, kidney, brain) of the mice were left, and fluorescence imaging was performed on a small animal imager. The imaging results of the blank control group with cy5.5 injection are shown in fig. 4:
Example 1 the fluorescence intensity of the brain of mice with drug-loaded brain-targeted exosomes after stem prognosis is obviously stronger than that of mice with Cy5.5 alone, and the brain-targeted effect of the drug-loaded brain-targeted exosomes is better than that of the mice with drug-loaded brain-targeted exosomes of comparative example, which shows that the drug-loaded brain-targeted exosomes of the invention have obvious targeting to the brain, and the targeting is better than that of the blood-derived exosomes with brain targeting reported in the prior literature.
(2) 30C 57BL/6 mice were randomly divided into 6 groups:
The first group is a control group, and the tail vein is injected with equivalent physiological saline;
the second group is the ADR group: the same amount of physiological saline is injected into the tail vein every other day after the doxorubicin molding is started at the 3 rd week;
the third group is adr+los: losartan (1 mg/kg/d) is infused every other day after doxorubicin molding, at week 3;
The fourth group is adr+exo ACP: the brain-targeted exosomes in example 1 were injected equally beginning at week 3 after doxorubicin molding and beginning at the time of the tail vein;
the fifth group is ADR+ Boold-Exo ACP @ Los: drug-loaded brain-targeted exosomes (losartan: 1 mg/kg/d) in the comparative example were injected intravenously at the beginning of the day 3 post-molding week;
The sixth group is ADR+Exo ACP @los: drug-loaded brain-targeted exosomes (losartan: 1 mg/kg/d) in example 1 were injected intravenously at the beginning of day 3 weeks after doxorubicin molding.
All mice were sacrificed at the fifth week after molding in the above grouping experiments, and mouse blood specimens, kidney and vital organ tissue specimens were collected. Renal function assays were performed using mouse serum. After the kidneys of the mice are fixed, dehydrated, embedded and the like, the kidneys of the mice are cut into 2 mu m slices for hematoxylin-eosin (H & E) staining and collagen fiber trichromatic (Masson) staining respectively, and important organ tissues (heart, liver, spleen and lung) are left for H & E staining.
The results of H & E staining of important organs of mice with different groups of dry prognosis are detailed in fig. 5: the important organ histomorphology of the mice with different intervention groups was not significantly different, indicating that the drug-loaded brain-targeted exosomes of example 1 were less biotoxic for ADR mice.
Serum creatinine and urea nitrogen results for mice with different groups of dry prognosis are detailed in fig. 6: serum creatinine and urea nitrogen were significantly elevated in the ADR group, indicating successful modeling. The small dose of losartan can not reduce the kidney function injury of mice after being infused with stomach, the Boold-Exo ACP @ Los of the comparative example also has no obvious kidney function improvement effect, and the blood creatinine and urea nitrogen level of the drug-loaded brain-targeted exosome Exo ACP @ Los treatment group of the embodiment 1 of the invention are obviously reduced. The brain-targeted exosome Exo ACP @ Los loaded with losartan has a better effect of improving the kidney injury of mice with doxorubicin nephropathy, and the same dose of losartan cannot be used for intervention through intragastric administration.
The results of kidney H & E staining and Masson staining of mice from different intervention groups are shown in fig. 7 and 8:
the H & E staining results of fig. 7 show that ADR model mice exhibited significant kidney damage, including tubular lumen expansion, tubular formation, tubular cell shedding necrosis, and interstitial inflammatory cell infiltration, among others. The relevant damage to the tubule cells was significantly reduced after administration of the drug-loaded brain-targeted exosomes of example 1, and the effect was significantly better than the comparative example.
The Masson staining results of fig. 8 show that tubular dilation, tubular cell vacuolation and interstitial collagen deposition are all obvious in the ADR group, and there is no obvious reduction in inflammatory cell and interstitial collagen deposition after administration of oral losartan and blood-derived drug-loaded brain-targeted exosome Boold-Exo ACP @los, whereas these lesions are significantly improved after administration of the drug-loaded brain-targeted exosome Exo ACP @los of example 1 of the present invention.
All mice were sacrificed fifth week after molding according to the above grouping experiment, and Angiotensinogen (AGT) immunohistochemical staining of brain tissue of all mice was collected on hippocampal sites. As shown in fig. 9, AGT expression at the hippocampal site of the ADR group was significantly upregulated compared to the control group, there was no significant downregulation of AGT expression following treatment with a small dose of losartan and blood-derived drug-loaded brain-targeted exosome Boold-Exo ACP @ Los whereas AGT expression was significantly reduced following administration of the drug-loaded brain-targeted exosome Exo ACP @ Los of example 1 of the present invention, indicating that the drug-loaded brain-targeted exosome of the drug-loaded brain of the present invention was able to significantly downregulate RAS component expression at the brain tissue, thereby downregulating central RAS activity, which is probably the likely mechanism of the drug-loaded brain-targeted exosome to ameliorate kidney injury in chronic kidney disease mice.
From the above results, the drug-loaded brain-targeted exosomes of the present invention include macrophage-derived exosomes, brain-targeted peptide molecules and angiotensin II receptor inhibitors (especially losartan). The exosome size (diameter) is 140+/-50 nm, and the surface of the exosome is modified with brain targeting polypeptide molecules; the brain-targeted exosome complex is capable of loading an angiotensin II receptor inhibitor. The brain targeting exosome drug-carrying system has specific targeting to brain tissues on one hand and can release drugs in the brain through blood brain barrier; on the other hand, the exosome has no obvious toxicity to cells, and the medicine can be wrapped in the exosome to increase the stability of the medicine. The brain-targeted exosome drug-carrying system prepared by the invention has the characteristics of low biotoxicity, high drug loading rate, certain targeting property on brain tissues and the like. Animal experiments prove that the brain-targeted exosome drug-carrying system prepared by the invention can be specifically enriched in brain, has good brain targeting property, can obviously improve the drug utilization rate, can realize the treatment effect on the chronic kidney disease of mice by downregulating the activity of central RAS by loading small dose of angiotensin II receptor inhibitor, and is an ideal drug-carrying system for treating the chronic kidney disease.
Claims (8)
1. A drug-loaded brain-targeted exosome, which comprises a brain-targeted peptide molecule, an exosome and a drug loaded in the exosome, and is characterized in that the exosome is derived from mouse mononuclear macrophage leukemia cell RAW264.7, the brain-targeted peptide is Angiopep-2-CP05, and the drug is an angiotensin II receptor antagonist; the angiotensin II receptor antagonist is losartan.
2. The drug-loaded brain-targeted exosome of claim 1, wherein the exosome size distribution is 140±50nm.
3. The drug-loaded brain-targeted exosome of claim 1, wherein the drug-loaded brain-targeted exosome has a drug concentration of 10-70 μg/100 μg brain-targeted exosome.
4. The drug-loaded brain-targeted exosome of claim 3, wherein the drug-loaded brain-targeted exosome has a drug concentration of 70 μg/100 μg brain-targeted exosome.
5. The method for preparing the drug-loaded brain-targeted exosome of claim 1, wherein the brain-targeted peptide is co-cultured with the exosome, and the brain-targeted peptide is linked to the surface of the exosome to form a brain-targeted peptide modified exosome complex; and co-culturing the medicine and the brain-targeted exosome complex to obtain the medicine-carrying brain-targeted exosome system.
6. The method for preparing a drug-loaded brain-targeted exosome according to claim 5, wherein the complex is subjected to ultrafiltration centrifugation to remove the non-encapsulated drug.
7. The method for preparing a drug-loaded brain-targeted exosome according to claim 5, wherein the RAW264.7 source exosome is obtained by the following method:
And culturing RAW264.7 cells, changing into an exosome-free serum culture medium in the logarithmic growth phase, culturing for 48 hours, and collecting cell supernatant to obtain the exosome from macrophage.
8. The use of a drug-loaded brain-targeted exosome according to claim 1 for the preparation of a medicament for the treatment of kidney disease.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011005098A1 (en) * | 2009-07-10 | 2011-01-13 | Abbott Healthcare Products B.V. | Peptide ligands for targeting to the blood-brain barrier |
| CN110115769A (en) * | 2019-04-08 | 2019-08-13 | 浙江大学 | A kind of Brain targeting excretion body and its preparation method and application |
| WO2020247675A1 (en) * | 2019-06-06 | 2020-12-10 | Spiritus Therapeutics, Inc. | Methods for attenuating viral infection and for treating lung injury |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011005098A1 (en) * | 2009-07-10 | 2011-01-13 | Abbott Healthcare Products B.V. | Peptide ligands for targeting to the blood-brain barrier |
| CN110115769A (en) * | 2019-04-08 | 2019-08-13 | 浙江大学 | A kind of Brain targeting excretion body and its preparation method and application |
| WO2020247675A1 (en) * | 2019-06-06 | 2020-12-10 | Spiritus Therapeutics, Inc. | Methods for attenuating viral infection and for treating lung injury |
Non-Patent Citations (4)
| Title |
|---|
| Angiotensin-converting enzyme inhibitors and receptorblockers in heart failure and chronic kidney disease –Demystifying controversies;Kader Muneer等;《Indian Heart Journal》;第69卷(第3期);第371-374页 * |
| Crossing the Blood–Brain Barrier: RecentAdvances in Drug Delivery to the Brain;Mayur M. Patel等;《CNS Drugs》;第31卷;第109-133 * |
| 脂肪源性外泌体对肾脏RAAS活化的作用研究;李映燕;《中国优秀硕士论文全文数据库 医药卫生科技辑》(2021年第2期);第1-46页 * |
| 负载姜黄素的巨噬细胞衍生外泌体的制备及其对阿尔茨海默病模型小鼠改善认知能力的机制研究;王浩;《中国优秀博士论文全文数据库 医药卫生科技辑》(2021年第5期);第1-71页 * |
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