CN114642736B - Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof - Google Patents
Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof Download PDFInfo
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- CN114642736B CN114642736B CN202011502081.7A CN202011502081A CN114642736B CN 114642736 B CN114642736 B CN 114642736B CN 202011502081 A CN202011502081 A CN 202011502081A CN 114642736 B CN114642736 B CN 114642736B
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Abstract
The invention relates to a blood-brain barrier drug delivery system, a preparation method and application thereof, wherein the blood-brain barrier drug delivery system comprises a neutrophil exosome and a drug entrapped in the neutrophil exosome; the medicine is a medicine for treating central nervous system diseases. The invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention provides a new strategy with quite transformation application prospect for non-invasive inflammatory microenvironment targeted drug delivery of glioma and other central nervous system diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a blood-brain barrier drug delivery system, a preparation method and application thereof.
Background
Gliomas are the most common primary malignancy of the central nervous system, with a high degree of invasiveness and a very poor prognosis. Traditional surgical removal of tumors is greatly limited by their infiltration and invasiveness. Meanwhile, the blood brain barrier system formed by closely connecting brain capillary endothelial cells, pericytes, astrocyte peripheria and the like prevents toxic substances from invading the brain and prevents most of medicaments from entering the brain, so that glioma and therapeutic medicaments for a plurality of central system diseases (such as Parkinson disease, alzheimer disease and the like) are limited in application. Therefore, designing a safe carrier that can assist the drug to efficiently penetrate the blood brain barrier and effectively target the focus is an important research direction for glioma and intracranial disease treatment.
Currently, strategies for the blood brain barrier mainly include invasive and non-invasive, but most invasive methods present the risk of potential nerve damage and intracranial infection. With the development of nanotechnology, intracranial administration of nanocarriers of non-invasive blood-brain barrier has been rapidly developed. However, it is worth noting that most nanomaterials have the problems of undegraded accumulation in brain tissues, undefined degradation metabolic pathways and the like, and the nanomaterials have the problems of safety and stability in the tedious preparation process.
In recent years, with the continuous and deep knowledge of tumor inflammation microenvironment, targeting tumor inflammation microenvironment becomes a research hotspot. Research shows that the tumor inflammation microenvironment is characterized by the presence of leukocytes in both the supporting stroma and the tumor area. In tumor microenvironments, tumor cells and non-tumor cells (such as immune cells and stromal cells) may release cytokines and chemokines, etc. By responding to specific chemokines, different immune cell subsets migrate to the tumor microenvironment and modulate the immune response of the tumor.
Although more and more blood-permeable brain barrier drug delivery systems are being developed for glioma and brain disease treatment, such as: liposome, polymer/inorganic material nanoparticle, nano-carrier of dendrimer and other materials, self-assembled micelle and the like. However, the availability, safety, feasibility of large-scale preparation and stability of the carrier materials constituting the aforementioned drug delivery systems are still to be further improved and clarified, and most of the carrier materials are in laboratory research stage, so that clinical transformation is difficult. On the other hand, if immune cells are used as drug delivery vehicles, however, since chemotherapeutic drugs tend to have some toxicity to cells, and immune cells play a multifaceted role in tumor microenvironments, it makes direct use of immune cells as drug carriers challenging.
Exosomes are lipid bilayer extracellular vesicles of 30-150nm diameter secreted endogenously by cells, as nanovesicles of natural origin, which are considered as potential good endogenous drug carriers for drug delivery due to their nanosize effects, stability in circulation and ability to naturally penetrate physiological barriers.
CN111690600a discloses an engineering human umbilical cord mesenchymal stem cell exosome, a transdermal preparation and application, wherein the engineering human umbilical cord mesenchymal stem cell exosome extracting solution is obtained by culturing 2-5 generation human umbilical cord mesenchymal stem cells and then enriching; lycopene encapsulated in the exosome of the engineered human umbilical cord mesenchymal stem cells; the lycopene and the exosome of the engineering human umbilical cord mesenchymal stem cells act on the skin cells together, so that the newly generated cells of the epidermis and dermis layers under promotion of the exosome have better cell states, and the effects of improving the skin quality, preventing the skin cells from aging, recovering the normal structure and physiological functions of the skin and improving the skin state are achieved.
CN110652492a discloses a drug-loaded infrared exosome and application thereof, and a liver disease drug. The medicine-carrying erythrocyte exosome is obtained by introducing medicine for treating liver diseases into the erythrocyte exosome. The research shows that the erythrocyte exosomes have liver chemotaxis without any modification, the problem of exosome yield is solved by using the erythrocyte-derived exosomes, and the erythrocyte does not contain DNA and RNA, is safer than exosomes derived from other cells in clinical blood transfusion for decades, and has good application prospect.
CN110917173a discloses an active anti-inflammatory engineering exosome and a preparation method thereof, wherein the exosome takes an exosome derived from M2 type macrophages as a carrier, HAL is loaded into the carrier in an electroporation manner, and the average particle size of the exosome is 120-250nm. The exosomes can actively target to an inflammation position by utilizing the anti-inflammatory and anti-inflammatory properties of the vector, and can evade a plurality of biological barriers such as a mononuclear phagocyte system and the like.
Disclosure of Invention
Aiming at the defects of the existing intracranial administration technology, the invention aims to provide a blood-brain barrier drug delivery system, and a preparation method and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a blood-brain barrier drug delivery system comprising a neutrophil exosome and a drug entrapped in the neutrophil exosome; the medicine is a medicine for treating central nervous system diseases.
The invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention takes doxorubicin as an anti-tumor model drug, and the neutrophil exosome drug-carrying system obtained by in-vivo evaluation test of a glioma in-situ transplantation model has very excellent anti-glioma effect.
Preferably, the central nervous system disorder comprises any one of glioma, pituitary tumor, meningioma, stroke, parkinson or alzheimer's disease.
The blood-permeable brain barrier drug delivery system disclosed by the invention can be used for targeted drug delivery of the central nervous system by utilizing the chemotaxis and blood-permeable brain barrier effect of neutrophil exosomes, and can be used for preventing or treating glioma, pituitary tumor, meningioma, cerebral apoplexy, parkinson or Alzheimer disease.
Preferably, the neutrophil exosomes comprise bone marrow or peripheral blood derived neutrophil exosomes.
Preferably, the central nervous system disease treatment drug comprises any one or a combination of at least two of doxorubicin, rapamycin, paclitaxel, docetaxel, hydroxycamptothecin, isovinblastine, amantadine or rivastigmine.
In a second aspect, the present invention provides a method for preparing a blood-brain barrier drug delivery system according to the first aspect, the method comprising the steps of:
and mixing the neutrophil exosome with the medicine, performing ultrasonic treatment, and incubating after the treatment is completed to recover the plasma membrane, so as to obtain the blood-permeable brain barrier medicine delivery system.
The preparation method of the blood-brain barrier drug delivery system is relatively simple to operate, is suitable for industrial production, and has a good transformation application prospect.
Preferably, the neutrophil exosomes are dissolved in a solvent at a concentration of 0.1-1.0mg/mL, for example 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL or 1.0mg/mL, etc., and other specific values within this range of values are selected and will not be described in detail herein. The concentration of neutrophil exosomes was measured using BCA kit.
Preferably, the solvent is a PBS solution. And the PBS solution of neutrophil exosomes was filtered with a 0.22 μm sterile filter before use.
Preferably, the drug is dissolved in a solvent at a concentration of 0.1-1.0mg/mL, for example, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1.0mg/mL, etc., and other specific values within this range of values are selected and will not be described in detail herein.
Preferably, the solvent is a PBS solution containing 5% DMSO and filtered through a 0.22 μm sterile filter.
Preferably, the mass ratio of the neutrophil exosome to the drug is 5:1-1:5, for example, 5:1, 2:1, 3:2, 1:1, 2:3, 1:2 or 1:5, and other specific point values within the numerical range can be selected, and will not be described in detail herein.
Preferably, the conditions of the ultrasound are: 15-25% amplitude, 4-10 cycles, 2-6s on, 1-4s off, and cooling the sample at 2-4deg.C for 1-5min for each cycle gap.
The ultrasonic conditions are specifically selected in the invention because the medicine can be more stably and efficiently packed in the neutrophil exosome without affecting the pharmacodynamic activity of the medicine.
The amplitude may be selected to be 15%, 20%, 25%, or the like; the number of the above cycles may be 4, 5, 6, 7, 8, 9 or 10; the on state may be maintained for 2s, 3s, 4s, 5s, 6s, or the like; the off state may be maintained for 1s, 2s, 3s, 4s, or the like; the cooling temperature can be 2 ℃, 3 ℃ or 4 ℃ and the like; the cooling time can be 1min, 2min, 3min, 4min, 5min, etc.; other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
Preferably, the incubation temperature is 35-40deg.C, such as 35deg.C, 36deg.C, 37deg.C, 38deg.C, 39deg.C or 40deg.C, etc., and the incubation time is 40-120min, such as 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min or 120min, etc., and other specific values within the above numerical ranges are selected, which will not be described in detail herein.
Preferably, after the incubation has ended, the sample is centrifuged (60-90) at (90000-120000) Xg for a min and washed with PBS.
The centrifugal force can be 90000g, 95000g, 100000g, 105000g, 110000g, 115000g or 120000g, the centrifugal time can be 60min, 70min, 80min or 90min, and other specific point values in the numerical ranges can be selected, and will not be described in detail here.
In the invention, the method for extracting the neutrophil exosome comprises the following steps:
(1) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) for 8-12 min at (800-1200) x g, centrifuging for 8-12 min at (1500-2500) x g, and centrifuging for 20-40 min at (9000-12000) x g to obtain supernatant;
(3) Filtering the supernatant obtained in the step (2), centrifuging (60-90) for a period of minutes at a concentration of (90000-120000) x g, and collecting the precipitate to obtain the neutrophil exosome.
The specific method is adopted to extract the neutrophil exosomes, so that the extraction rate is higher, and the extracted neutrophil exosomes can keep the physiological activity of the high-efficiency blood-brain-penetrating barrier and the inflammation trend to the greatest extent.
Preferably, the temperature of the culture in the step (1) is 36-38 ℃, such as 36 ℃, 37 ℃ or 38 ℃, and the like, and the time is 12-36 hours, such as 12 hours, 18 hours, 24 hours, 30 hours or 36 hours, and the like, and other specific point values in the numerical ranges can be selected, and will not be described in detail herein.
Preferably, the supernatant of step (2) is filtered with a 0.22 μm filter.
Preferably, after the precipitate is collected in the step (3), the precipitate is resuspended in PBS solution, and centrifuged at (90000-120000). Times.g for 60-90 min, and the precipitate is collected to obtain the neutrophil exosome.
In the present invention, the method for extracting neutrophils comprises the steps of:
(1') filtering the bone marrow with a 70 μm filter, centrifuging (400-500) ×g for 8-15 min, and re-suspending the pellet in PBS solution;
(2') lysing erythrocytes using an erythrocyte lysate, (400-500) ×g centrifuged (4-10) min, and resuspension of the pellet in PBS solution to obtain a single cell suspension;
(3') adding the single cell suspension into a mixed solution consisting of 58%,70% and 78% of Percoll working solution, (460-520). Times.g is centrifuged (25-40) for min, and the cells at the interface of 58% and 70% of Percoll working solution are taken to obtain the neutrophils.
The specific method for extracting the neutrophils enables the extraction rate to be higher, and the exosomes obtained by secretion of the extracted neutrophils can keep the physiological activities of high-efficiency blood-brain barrier permeation and inflammation trend to the greatest extent.
In a third aspect, the present invention provides the use of a blood-brain barrier drug delivery system according to the first aspect for the manufacture of a medicament for the prevention or treatment of a central nervous system disorder.
In a fourth aspect, the present invention provides the use of a blood-brain barrier drug delivery system according to the first aspect for the manufacture of a medicament for the prevention or treatment of intracranial tumors;
preferably, the intracranial tumor is a glioma.
Compared with the prior art, the invention has the following beneficial effects:
the invention constructs a neutrophil exosome drug delivery system with good biocompatibility, which has biological characteristics similar to neutrophils, can efficiently permeate blood brain barrier, effectively respond to inflammation, targets inflammation parts, and is a drug carrier with great application prospect for treating glioma and other central nervous system diseases. The invention takes doxorubicin as an anti-tumor model drug, and the neutrophil exosome drug-carrying system obtained by in-vivo evaluation test of a glioma in-situ transplantation model has very excellent anti-glioma effect.
Drawings
FIG. 1 is a transmission electron micrograph of the neutrophil exosomes prepared in example 1;
FIG. 2 is a transmission electron microscope image of the blood-brain barrier drug delivery system prepared in example 2;
FIG. 3 is a Western blot analysis of neutrophil exosomes prepared in example 1 and of the blood-brain barrier drug delivery system prepared in example 2;
FIG. 4 is a graph showing the results of observing the absorption of NEs-Exos/PKH67 of underlying C6 cells by a laser confocal microscope in the presence or absence of fMLP;
FIG. 5 is a live imaging view of the distribution of DiR fluorescence in tumor-bearing mice;
fig. 6 is a graph showing survival of each group of C6 glioma-bearing mice in example 6.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The Kunming mice referred to in the examples below were purchased from Chongqing medical university and BALB/c nude mice were purchased from Beijing Fukang Biotechnology Co., ltd. The reagents and raw materials required for other experiments were all available commercially.
Example 1
The method for extracting the neutrophil exosome in the embodiment comprises the following steps:
(1) Taking 25g of Kunming mice, killing the mice after anesthesia and neck breakage, putting the mice into 75% ethanol for sterilization for 5min, and transferring the mice into an ultra-clean workbench;
(2) Carefully separating leg skin of the mice with sterilized surgical equipment, cutting out whole thighs, putting into precooled PBS, peeling leg muscles of the mice, taking out femur and tibia, and putting into a culture dish containing RPMI 1640;
(3) Washing bone marrow with fresh appropriate amount of RPMI1640, filtering cells from bone marrow with 70 μm filter screen, centrifuging 450 Xg for 10min, and suspending precipitate in PBS solution;
(4) Using erythrocyte lysate to lyse erythrocytes, centrifuging for 5min at 450 Xg, and re-suspending the precipitate in PBS solution to obtain single cell suspension;
(5) Preparing a percoll stock solution by using 10 XPBS and a percoll cell separation solution in a volume ratio of 1:9, and preparing a percoll working solution with a volume ratio of 58%,70% and 78% by using 1 XPBS;
(6) Adding single cell suspension into mixed solution composed of 58%,70% and 78% of Percoll working solution, centrifuging for 30min at 490 Xg, and collecting cells at the interface of 58% and 70% of Percoll working solution to obtain neutrophils;
(7) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(8) Centrifuging the supernatant at 1000 Xg for 10min, centrifuging at 2000 Xg for 10min, and centrifuging at 10000 Xg for 30min to obtain supernatant;
(9) Filtering the supernatant, centrifuging at 100000 Xg for 70min, and collecting precipitate to obtain the neutrophil exosome.
Example 2
The preparation method of the drug delivery system for the blood-brain barrier comprises the following steps:
(1) Measurement of neutrophil exosome protein concentration using BCA kit, 0.4mg/mL of neutrophil exosome extracted according to the method of example 1 (exosome was diluted with 1 x PBS, used after filtration through 0.22 μm sterile filter) was added with an equivalent concentration of 0.4mg/mL of DOX solution (DOX was formulated with 1 x PBS containing 5% dmso);
(2) Placing the mixed solution of the neutrophil exosome and DOX into a centrifuge tube, and performing ultrasonic treatment in an ultrasonic cell disruption instrument; ultrasonic conditions: 20% amplitude, 6 cycles, 4s on, 2s off, cooling the sample on ice for 2min with each cycle gap;
(3) After the treatment was completed, the samples were incubated at 37℃for 1h to ensure recovery of the plasma membrane of NEs-Exos;
(4) After incubation at 37 ℃ is completed, the sample is centrifuged at 100000 Xg for 70min, washed with PBS, centrifuged again at 100000 Xg for 70min, and resuspended in PBS to obtain the blood-brain barrier drug delivery system.
Example 3
Examination of the neutrophil exosomes and the blood-brain barrier drug delivery systems prepared in example 1 and example 2 by transmission electron microscopy: the neutrophil exosomes or the blood-penetrating brain barrier drug delivery system were mixed with an equal amount of 4% paraformaldehyde and immobilized on a sample-carrying copper mesh (200 mesh, coated with a formaldehyde metal film). After 20min, the PBS was washed and the copper mesh was then placed on 1% glutaraldehyde drops for 5min. The copper mesh was then rinsed in ultra pure water. Each time for 2min, 8 times. Images were captured using a JEM 1200EX transmission electron microscope as shown in fig. 1 and 2, respectively, from which it can be seen that: the neutrophil exosomes appear as round cups or saucers (fig. 1), whereas the particle size of the DOX loaded neutrophil exosomes increases, but this morphology also conforms to the exosome morphology (fig. 2).
Western blot analysis was performed on neutrophil exosomes and the blood-brain barrier drug delivery systems prepared in example 1 and example 2: BCA measured protein concentration and Loading buffer was boiled for 10min after mixing. Using a 1.5mm SDS-PAGE gel, loading 40 mu L, and transferring to a 110V voltage for electrophoresis after 75V voltage reaches a separation position of a concentrated gel separation gel; transfer film was performed at 200mA for 90 min. After transfer, PVDF membranes were placed in a defatted protein solution prepared with 5% TBST, blocked for 2h, not washed after blocking, incubated overnight at 4℃with primary antibodies (CD 63, TSG101, ALIX), and washed 3 times with 1 XTBE on a shaker for 10min each. Incubation with the corresponding secondary antibody was carried out for 2h, and incubated at room temperature in 1 XTBST for 3 times each for 10min on a shaker. And (3) preparing ECL luminous liquid, mixing the liquid A and the liquid B in equal proportion, dripping the mixture onto the PVDF film, and developing. As shown in FIG. 3, the sample was shown to be rich in Exos-associated protein markers (TSG 101, alix and CD 63).
The transmission electron microscope, the particle size analysis and the western blotting result are combined to prove that the neutrophil exosome and the blood-brain barrier drug delivery system are successfully prepared.
Example 4
This example observes the response chemotactic peptide (fMLP) effect of the blood-brain barrier drug delivery system of the present invention, replaces the drug with PKH67 dye according to the method in example 2, loads PKH67 dye in the same loading manner, and establishes a transwell cell model to simulate the blood-brain barrier in vitro.
the transwell in vitro blood brain barrier model was established as follows: the matrigel is diluted by pre-cooled DMEM according to the ratio of 1:3, 50 mu L of diluted matrigel is absorbed and evenly spread on a basement membrane of an upper chamber of a 24-hole transwell chamber (bubbles are avoided), the liquid is spread fully through slight shaking, and after the glue spreading is completed, the transwell chamber is placed into an incubator. After 3h, the solution remaining in the chamber was carefully aspirated, and 30. Mu.L of serum-free DMEM medium was added to each well to hydrate the basement membrane for 30min. bEnd.3 cells were digested in advance and seeded in the upper chamber of a 24-well transwell chamber (1X 10) 5 ) Measurement of the TEER value of the monolayer bEnd.3 cells was performed using a resistance meter when the TEER value was greater than 300. Omega. Cm 2 The transwell chamber can be used as an in vitro permeabilization BBB assay. For detection of the blood-brain barrier permeabilized drug delivery System being carcinomatous after permeation through monolayer bEnd.3 cellsIn the case of uptake, a cell slide was plated and C6 cells were seeded in the lower chamber of a 24-well transwell chamber.
Experiments were performed with the addition of PKH67 loaded neutrophil exosomes (DMEM dilution) in the upper chamber and DMEM or DMEM diluted fMLP (100. Mu.M) in the lower chamber. Taking out the corresponding transwell cells after 4h, 8h and 12h respectively, sucking and removing liquid in the lower chamber, gently washing the climbing plate 3 times by using PBS, adding DAPI and incubating the cells for 15min, washing the cells 3 times by using PBS after incubation, and observing the absorption of the lower layer C6 cells to NEs-Exos/PKH67 under a laser confocal microscope.
The results are shown in FIG. 4: in the presence of fMLP, the uptake of neutrophil exosome drug-loaded particles by C6 cells increased over time, and reached the highest at 12h, with evident green fluorescence. In contrast, in the fMLP-free group, uptake of neutrophil exosome drug-loaded particles by lower chamber C6 cells was not apparent. The presence of fMLP increases the efficiency of penetration of the drug-loaded particles of the neutrophil exosomes through the transwell upper chamber, demonstrating the ability of the blood-brain barrier drug delivery system of the present invention to respond to chemokines similar to neutrophils.
Example 5
The present example explores the blood-brain barrier efficiency of the blood-brain barrier drug delivery system according to the present invention, and the method is as follows: modeling of tumor-bearing mice was performed using about 20g of male BALB/C nude mice as experimental model animals with C6-Luc glioma cells stably transfected with luciferase. Nude mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. Mice were fixed using a brain stereotactic apparatus, suspended in 2 μl PBS 2×10 6 The C6-Luc glioma cells were implanted into the right striatum (1.8 mm right, 1mm backward, 3mm downward with bregma as origin) of the mouse brain. The cell implantation process needs to slowly insert the needle, and the needle extraction process after implantation needs to be slowly carried out, so that the death of the mice caused by intracranial pressure mutation is avoided.
Neutrophil exosomes loaded with DiR fluorochromes were prepared as in example 2. The free DiR fluorochrome was administered by tail vein injection on day 15 after C6 cell seed tumor as a control. After injection was completed, the in vivo distribution of DiR fluorescence in tumor bearing mice (free DiR as control group) was observed and recorded under a small animal in vivo imager at set time points (30 min, 1h, 2h, 4h, 12h and 24 h).
The results are shown in FIG. 5: after 2h tail vein injection, diR loaded in neutrophil exosomes showed fluorescent signals in the brain of mice, and the fluorescence intensity of DiR in the brain was continuously increased with time. Whereas free DiR groups did not observe significant fluorescence in the brain during up to 24h of monitoring. Experimental results prove that the neutrophil exosome can effectively improve the blood-brain barrier efficiency of the wrapped medicine.
Example 6
The inhibition effect of the drug delivery system of the blood-brain barrier to glioma is explored in the embodiment, and the method is as follows: after the C6-Luc in situ glioma model was established as in example 5, the C6-Luc mice were randomly divided into 3 groups of 10 mice each according to the physiological saline group, the pure DOX group and the DOX-loaded neutrophil exosomes group. The corresponding medicines were injected into the tail vein on days 10, 12, 14, 16, 18, and 20, respectively, wherein the DOX amount was 3 mg.kg -1 . Death data of C6-Luc mice were recorded daily after brain engrafting of tumor cells, and Kaplan-Meier survival curves were plotted for 3 groups of tumor-bearing mice using GraphPad 7.00 software.
The results are shown in FIG. 6: the mice treated with the blood-brain-barrier drug delivery system of the present invention had significantly improved survival, with a median survival of 27 days, which was significantly longer than the free DOX group (22 days) and the physiological saline group (19 days), and these results confirmed that the blood-brain-barrier drug delivery system of the present invention was effective in inhibiting tumor growth and prolonging survival of mice.
The applicant states that the present invention is described by way of the above examples as a blood-brain barrier drug delivery system, and methods of making and using the same, but the present invention is not limited to, i.e., it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (17)
1. The blood-brain barrier drug delivery system is characterized by being prepared from a neutrophil exosome and doxorubicin entrapped in the neutrophil exosome; the blood-brain barrier drug delivery system is used for treating glioma;
the blood-brain barrier drug delivery system is prepared by a method comprising the following steps:
and mixing the neutrophil exosome with doxorubicin, performing ultrasonic treatment, and incubating after the treatment is finished to recover a plasma membrane to obtain the blood-brain-barrier drug delivery system.
2. The blood-permeable brain barrier drug delivery system of claim 1, wherein the neutrophil exosomes comprise bone marrow or peripheral blood derived neutrophil exosomes.
3. A method of preparing a blood-brain barrier drug delivery system according to claim 1 or 2, comprising the steps of:
and mixing the neutrophil exosome with doxorubicin, performing ultrasonic treatment, and incubating after the treatment is finished to recover a plasma membrane to obtain the blood-brain-barrier drug delivery system.
4. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the neutrophil exosomes are dissolved in the solvent at a concentration of 0.1-1.0 mg/mL.
5. The method of preparing a blood-brain barrier drug delivery system according to claim 4, wherein the solvent is a PBS solution and is filtered through a 0.22 μm sterile filter.
6. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the doxorubicin is dissolved in a solvent at a concentration of 0.1-1.0 mg/mL.
7. The method of preparing a blood-brain barrier drug delivery system according to claim 6, wherein the solvent is a PBS solution containing 5% dmso and filtered through a 0.22 μm sterile filter.
8. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the mass ratio of neutrophil exosomes to doxorubicin is 5:1-1:5.
9. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the ultrasound conditions are: 15-25% amplitude, 4-10 cycles, 2-6s cycles, 1-4s cycles, and cooling the sample at 2-4deg.C for 1-5min for each cycle gap.
10. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the incubation is performed at a temperature of 35-40 ℃ for a time of 40-120 min.
11. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the sample is centrifuged (60-90) at (90000-120000) ×g for a min after the incubation is completed, and washed with PBS.
12. The method of preparing a blood-brain barrier drug delivery system according to claim 3, wherein the method of extracting neutrophil exosomes comprises the steps of:
(1) Culturing neutrophils with exosome-free serum, and centrifugally collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) for 8-12 min at (800-1200) x g, centrifuging for 8-12 min at (1500-2500) x g, and centrifuging for 20-40 min at (9000-12000) x g to obtain supernatant;
(3) Filtering the supernatant obtained in the step (2), centrifuging (60-90) for a period of minutes at a concentration of (90000-120000) x g, and collecting the precipitate to obtain the neutrophil exosome.
13. The method of claim 12, wherein the culturing in step (1) is performed at a temperature of 36-38 ℃ for a time of 12-36 h.
14. The method of preparing a blood-brain barrier drug delivery system according to claim 12, wherein the supernatant of step (2) is filtered with a 0.22 μm filter.
15. The method of claim 12, wherein the collecting the precipitate in step (3) is followed by re-suspending in PBS solution, and centrifuging (60-90) for a period of time of (90000-120000) ×g, and collecting the precipitate to obtain the neutrophil exosome.
16. The method of preparing a blood-brain barrier drug delivery system according to claim 12, wherein the method of extracting neutrophils comprises the steps of:
(1') filtering the bone marrow with a 70 μm filter, centrifuging (400-500) ×g for 8-15 min, and re-suspending the pellet in PBS solution;
(2') lysing erythrocytes using an erythrocyte lysate, (400-500) ×g centrifuged (4-10) min, and resuspension of the pellet in PBS solution to obtain a single cell suspension;
(3') adding the single cell suspension into a mixed solution consisting of 58%,70% and 78% of Percoll working solution, (460-520). Times.g is centrifuged (25-40) for min, and the cells at the interface of 58% and 70% of Percoll working solution are taken to obtain the neutrophils.
17. Use of a blood-permeable brain barrier drug delivery system according to claim 1 or 2 for the preparation of a medicament for the treatment of glioma.
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