CN115177742A - 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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- CN115177742A CN115177742A CN202210597989.3A CN202210597989A CN115177742A CN 115177742 A CN115177742 A CN 115177742A CN 202210597989 A CN202210597989 A CN 202210597989A CN 115177742 A CN115177742 A CN 115177742A
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- 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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Abstract
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. The drug-loaded brain targeting exosome comprises a brain targeting peptide molecule, an exosome and a drug encapsulated in the exosome. The drug is an angiotensin II receptor antagonist losartan. The brain targeting peptide is bound to the surface of the exosome through the exosome-marker protein CD63 in a co-culture manner. The exosome is derived from the cell supernatant of mouse monocyte macrophage leukemia cells. The brain targeting exosome drug-loading system can load drugs into exosomes and has no obvious biotoxicity effect. In addition, small amounts of tail vein administration can penetrate the blood brain barrier into the center to achieve inhibition of angiotensin (RAS) expression. In a mouse model of adriamycin-induced chronic kidney disease, a brain-targeted exosome loaded losartan drug complex can obviously improve renal function, renal tissue injury and renal interstitial collagen fiber deposition by down-regulating the expression of a central RAS.
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, and the like. The CKD incidence rate is high, the early detection rate is low, the lethal disability rate is high, the treatment cost is high, and the life quality of people is seriously influenced. If the kidney disease cannot be effectively treated in the early stage, the kidney disease will progress to the end-stage kidney disease, and the kidney replacement therapy has to be received, which not only seriously affects the life quality of the patient, but also brings heavy economic burden to the patient and family members. The current clinical drug therapy is very limited, mainly comprises angiotensin enzyme inhibitor, glucocorticoid, immunosuppressant, traditional Chinese medicine and the like, but has limited effect on delaying the disease progression.
The research shows that the lateral ventricle injection of antisense oligonucleotide can inhibit the renin angiotensin system (RAS axis) of the brain to down-regulate the hypertension caused by chronic kidney disease rats. In the renal fibrosis model of high-salt CKD promotion, the brain-renal RAS interaction axis is involved, and chronic renal injury can be significantly reduced by centrally administering a small dose (about 1/500 of the peripherally administered effective dose) of a RAS inhibitor (e.g., losartan). However, such small doses of the drug have little therapeutic effect on chronic kidney injury by peripheral routes. Thus, if a drug can be targeted to the central system, better therapeutic results can be achieved with fewer drug doses. However, it is difficult to popularize central administration for clinical application in view of the risk and high difficulty of central administration. Therefore, there is a need for a low immunogenic, brain-targeted and drug-loaded drug carrier that delivers RAS inhibitors centrally for ultimate kidney protection.
In recent years, nanomaterials have shown great advantages in medicine, and can provide assistance for disease treatment and drug delivery through surface modification or characteristics of nanomaterials. 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 the artificially synthesized nano-drug carriers have the defects of high preparation difficulty, poor compatibility, undefined toxicity and the like.
Exosomes are nanoscale disc-shaped vesicles with diameters of 30-200nm, naturally exist in various cells and body fluids, and have various advantages such as better low biotoxicity, stability and low immunogenicity as drug carriers. However, in the field of kidney disease treatment, due to the particularity of kidney diseases and therapeutic chemicals thereof, the brain targeting drug delivery technology is greatly limited in the field.
Disclosure of Invention
The invention provides a drug-loaded brain-targeted exosome and a preparation method and application thereof. In view of the defects in the prior art, the inventor of the present invention obtains the main technical scheme of the present invention through repeated practice, and mainly provides a brain-targeted exosome drug carrier system loaded with drugs such as losartan, which is used for repairing and treating chronic kidney diseases. The invention also aims to provide application of the drug-loaded brain targeting exosome in chronic kidney disease repair. The drug-loaded brain-targeted exosome comprises a brain-targeted peptide molecule, an exosome derived from macrophage and a drug angiotensin II receptor antagonist losartan loaded in the exosome, and the drug-loaded brain-targeted exosome can transport drugs to brain tissues in a targeted manner through a blood brain barrier and down-regulate RAS expression of a hippocampus. Can achieve the effect of protecting the chronic kidney diseases by using a very small drug dose (about 1/500 of the effective dose of the peripheral administration).
In order to achieve the technical effects, the technical scheme adopted by the invention comprises the following steps:
firstly, the drug-loaded brain targeting exosome comprises a brain targeting peptide, an exosome and a drug loaded in the exosome, wherein:
the exosome is derived from mouse mononuclear macrophage leukemia cell RAW264.7.
The source exosome has certain tropism to brain tissues with improved inflammatory response, which is different from characteristics of exosomes from other sources, and brain inflammation can be found to be obviously up-regulated in a chronic kidney disease animal model, so that the source exosome is beneficial to enrichment of the brain tissues of a CKD animal model, and the effect that the source exosome carries a specific medicine to reach a dosing site and regulates corresponding expression can be realized, and the effect cannot be realized by the exosomes from other sources.
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 a simple synthesis method, can be specifically combined with the exosome surface marker adopted by the invention, and further carries the exosome and the drug carried by the exosome to enter the center through the blood brain barrier to play a role.
The drug is angiotensin II receptor antagonist, such as losartan, valsartan, irbesartan, telmisartan, and the like, but is not limited thereto.
Further, the drug concentration in the drug-loaded brain-targeted exosome is 10-70 μ g/100 μ g of brain-targeted exosome, but is optimally 70 μ g/100 μ g of brain-targeted exosome.
The drug concentration is the effective concentration of the drug-loaded brain targeting exosome, and especially when the drug concentration is 1mg/kg, the optimal combination of drug effect and drug dosage can be realized, and the lowest biotoxicity is realized.
A large number 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 realize the technical effect and simultaneously has low biological toxicity.
Further, the exosome size (diameter) distribution is mainly at 140 ± 50nm.
The invention also provides a preparation method of the drug-loaded brain target exosome, which comprises the following steps:
co-culturing the brain targeting peptide and the exosome, linking the brain targeting peptide to the surface of the exosome to form a brain targeting peptide modified exosome compound; and co-culturing the medicine and the exosome compound modified by the brain targeting peptide to obtain a medicine-carrying brain targeting exosome system.
Further, the brain targeting peptide modified exosome complex removes the drug which is not coated by means of ultrafiltration and centrifugation.
Further, the mouse mononuclear macrophage leukemia cell RAW 264.7-derived exosome is obtained by the following method:
culturing mouse mononuclear macrophage leukemia cell RAW264.7 cells by a conventional culture method, changing the cell into an exosome-free serum culture medium in logarithmic growth phase, and collecting cell supernatant after culturing for 48h to obtain mouse mononuclear macrophage leukemia cell RAW 264.7-derived exosomes.
The invention also provides application of the drug-loaded brain-targeted exosome, which is used for treating kidney diseases or preparing drugs for treating kidney diseases.
The invention has the beneficial effects that:
firstly, at present, no relevant technical scheme for treating the kidney disease by brain-targeted delivery of a very small amount of drugs is available for relevant research, the drug-loaded brain-targeted exosome of the invention firstly provides a technical scheme of a brain-targeted exosome drug-loading system for treating the kidney disease, and the exosome derived from macrophages has a certain targeting property on inflammatory brain tissues of a chronic kidney disease animal model, so that the enrichment of the loaded drugs in the brain tissues of a CKD model is promoted, and the effective action on the brain tissues is finally ensured on the basis of reducing the drug dosage. The invention utilizes the corresponding characteristic specificity of the specific exosome and the specific brain targeting peptide to combine with the kidney disease treatment drug, provides support for central targeting drug-loaded treatment of CKD and opens up a new treatment direction.
Research experiments show that the drug-loaded brain target exosome has the characteristic of extremely small dosage which is about 1/500 of the conventional peripheral administration effective dosage; in addition, on the basis of ensuring the drug effect, the compound preparation also has the characteristics of extremely low biotoxicity, and greatly ensures the drug safety. When the adriamycin is applied to mice with chronic kidney diseases, the adriamycin can improve renal functions, infiltration of inflammatory cells of renal tissues and interstitial collagen deposition, and promote the repair of the chronic kidney diseases.
In conclusion, compared with the prior art, the drug-loaded brain-targeted exosome prepared by the invention can load a small molecular compound drug into the exosome, has lower cell and system toxicity, has the capacity of down-regulating hippocampal RAS component expression by the brain-targeted delivery drug, and promotes the repair of chronic kidney disease.
Drawings
FIG. 1 is a flow chart of the preparation of a drug-loaded brain-targeted exosome according to the present invention;
FIG. 2A is a transmission electron micrograph of exosomes and brain-targeted exosomes described in experimental example 1;
FIG. 2B is an immunoblot of an exosome-marker protein in experimental example 1;
FIG. 2C is a graph showing the particle size distribution of the NTA-analyzed exosomes and brain-targeted exosomes in experimental example 1;
FIG. 3A is a graph showing the results of the 24-hour survival rate 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 fluorescent image of each major organ of the brain-targeted exosomes in the normal mouse in test example 3;
FIG. 5 is an H & E diagram of each major organ of the drug-loaded brain-targeted exosome in the normal mouse in experimental example 3;
FIG. 6 is a graph comparing the effect of drug-loaded brain-targeted exosomes in experimental example 3 on kidney function in a mouse model of chronic kidney disease;
FIG. 7 is a graph of H & E staining of chronic kidney disease mouse model kidney tissue by drug-loaded brain-targeted exosomes in experimental example 3;
FIG. 8 is a Masson staining graph of drug-loaded brain-targeted exosomes on chronic kidney disease mouse model kidney tissue in test example 3;
FIG. 9 is an immunohistochemistry of drug-loaded brain-targeted exosomes of experimental example 3 against Angiotensinogen (AGT) of hippocampus of a mouse model of chronic renal disease.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects of the present invention more clearly apparent, the technical gist of the present invention is further described below by specific embodiments, which 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 merely illustrative of the invention and are not intended to limit the invention. The present invention may be embodied with non-essential modifications and adaptations by those skilled in the art in light of the foregoing disclosure, and yet fall within the scope of the present invention.
The brain-targeting exosome drug-loading system and the preparation thereof and the treatment effect on the chronic kidney disease of the mouse are further described 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 also 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 exosome is mouse mononuclear macrophage leukemia cell RAW264.7.
Example 1
1. Extracting and identifying mouse mononuclear macrophage leukemia cell exosome:
culturing RAW264.7 cells by a conventional culture method, changing into an exosome-free serum culture medium in logarithmic growth phase, and collecting supernatant after culturing for 48h to obtain exosomes derived from macrophages. The specific process is as follows:
transferring RAW264.7 cells in the frozen tube to a 15mL centrifuge tube containing 6mL of complete culture medium, centrifuging at 1,000rpm for 5min; discard the supernatant, precipitate with 6mL complete mediumResuspending and inoculating to 10cm 2 Petri dish, 5% CO at 37% 2 Culturing in a cell culture box to logarithmic growth phase; washing with 3ml PBS once, changing culture medium to 10% exosome-free serum culture medium, culturing for 48h, and collecting supernatant; centrifuging at 4 ℃ for 3,000g and 10min, collecting supernatant liquid, and discarding cell debris to a new 15mL centrifuge tube; adding exosome extract (4:1 in volume ratio) into supernatant centrifuge tube (cell supernatant exosome extraction kit is purchased from Shanghai Yubo Biotechnology Co., ltd., type UR 52121), and shaking for 1min to mix the solution thoroughly. Standing at 4 deg.C for 12h; centrifuging the mixed solution at 4 deg.C for 10,000g,60min, slightly discarding supernatant, enriching exosome in tube wall, centrifuging for 2min, and removing supernatant as much as possible; resuspend exosomes with 200 μ Ι _ of PBS, then collect in 1.5mL EP tubes; centrifuging at 4 deg.C for 2min to collect supernatant rich in exosome, and centrifuging at 12,000g.
The obtained exosome was characterized by transmission electron microscopy, particle size analyzer, western blot, as shown in fig. 2A-2C.
2. Preparation of brain-targeted exosomes
Dissolving 2mg of brain targeting peptide (Angiopep-2-CP 05) in 2mL of deionized water to prepare a concentration of 1 mug/muL; according to the mass ratio of 1:1, synthesizing. Namely, 50 mu g of exosome and 50 mu g of angiopep-2-CP05 are mixed fully, vibrated and mixed evenly, sealed by sealing glue and incubated for 12 to 16 hours at the temperature of 4 ℃; centrifuging the 100kDa ultrafiltration tube at 4 ℃ for 5,000rpm for 20min to remove free polypeptide; centrifuging for three times, adding 200 μ L PBS into the ultrafiltration tube after each centrifugation, mixing, and centrifuging; after the centrifugation is finished, firstly sucking out the liquid in the inner tube, then flushing the inner tube by 50 mu L PBS (phosphate buffer solution), repeating for 2 times, and collecting all the liquid in the inner tube to obtain the synthesized total Exo-Angiopep-2-CP05 (Exo) ACP )。
3. Preparation of drug-loaded brain-targeted exosomes
100 μ g of Exo ACP The solution and 110 μ g of losartan solution were mixed well, with "0.25" tip,20% strength, 30s on/off, for 3 minutes, with 2 minute intervals of cooling time, to increase the outer membrane permeability of the line exosomes appropriately to promote the drug permeation into the exosomes, followed by 37 deg.c,the exosome membrane structure is recovered in 60 min. Transferring to 100kDa ultrafiltration centrifuge tube, ultrafiltering at 5,000rpm for 20min and 4 deg.C for 3 times, and collecting inner tube liquid as drug-loaded cerebral targeting exosome (Exo) ACP @Los)。
The concentration of the losartan is measured by high performance liquid chromatography, and the concentration of the loaded losartan is measured to be about 70 mug/100 mug of exosome.
Example 2
1. Extracting and identifying mouse mononuclear macrophage leukemia cell exosome:
culturing RAW264.7 cells by a conventional culture method, changing into an exosome-free serum culture medium in logarithmic growth phase, and collecting supernatant after culturing for 48h to obtain exosomes derived from macrophages. The specific process is as follows:
transferring RAW264.7 cells in the frozen tube to a 15mL centrifuge tube containing 6mL of complete culture medium, centrifuging at 1,000rpm for 5min; the supernatant was discarded, the pellet was resuspended in 6mL of complete medium and inoculated to 10cm 2 Petri dish, 5% CO at 37% 2 Culturing in a cell culture box to logarithmic growth phase; washing with 3ml PBS once, changing culture medium to 10% exosome-free serum culture medium, culturing for 48h, and collecting supernatant; centrifuging at 4 ℃ for 3,000g and 10min, collecting supernatant liquid, and discarding cell debris to a new 15mL centrifuge tube; adding exosome extract (4:1 in volume ratio) into supernatant centrifuge tube (cell supernatant exosome extraction kit from Shanghai Yubo Biotechnology Co., ltd., type UR 52121), and shaking for 1min to mix the solution thoroughly. Standing at 4 ℃ for 12h; centrifuging the mixed solution at 4 deg.C for 10,000g,60min, slightly discarding supernatant, enriching exosome in tube wall, centrifuging for 2min, and removing supernatant as much as possible; resuspend exosomes with 200 μ Ι _ of PBS, then collect in 1.5mL EP tubes; centrifuging at 4 deg.C for 2min to collect supernatant rich in exosome, and centrifuging at 12,000g.
2. Preparation of brain-targeted exosomes
Dissolving 2mg of brain targeting peptide (Angiopep-2-CP 05) in 2mL of deionized water to prepare a concentration of 1 mug/muL; according to the mass ratio of 1:1, synthesizing. Namely 50 mu g of exosome and 50 mu g of Angiopep-2-CP05, fully mixing, shaking and uniformly mixing, and sealingSealing with glue, and incubating at 4 deg.C for 12-16h; centrifuging in a 100kDa ultrafiltration tube at 4 ℃,5,000rpm and 20min to remove free polypeptide; centrifuging for three times, adding 200 μ L PBS into the ultrafiltration tube after each centrifugation, mixing, and centrifuging; after the centrifugation is finished, firstly sucking out the liquid in the inner tube, then flushing the inner tube with 50 mu LPBS (L-propyl butyrate) for 2 times, and collecting all the liquid in the inner tube to obtain the synthesized total Exo-Angiopep-2-CP05 (Exo) ACP )。
3. Preparation of drug-loaded brain-targeted exosome
The concentration of the losartan is determined by high performance liquid chromatography, and the loaded losartan concentration is determined to be about 40 mug/100 mug of brain-targeted exosome.
Example 3
1. Extracting and identifying mouse mononuclear macrophage leukemia cell exosome:
culturing RAW264.7 cells by a conventional culture method, changing into an exosome-free serum culture medium in logarithmic growth phase, and collecting supernatant after culturing for 48h to obtain exosomes derived from macrophages. The specific process is as follows:
transferring RAW264.7 cells in the frozen tube to a 15mL centrifuge tube containing 6mL complete culture medium, centrifuging at 1,000rpm for 5min; the supernatant was discarded, the pellet was resuspended in 6mL of complete medium and inoculated to 10cm 2 Petri dish, 5% CO at 37% 2 Culturing in a cell culture box to logarithmic growth phase; washing with 3ml PBS once, changing culture medium to 10% exosome-free serum culture medium, culturing for 48h, and collecting supernatant; centrifuging at 4 ℃ for 3,000g and 10min, collecting supernatant liquid, and discarding cell debris to a new 15mL centrifuge tube; adding secretion into the supernatant centrifuge tubeShaking the body extractive solution (volume ratio is 4:1) (cell supernatant exosome extraction kit from Shanghai Yubo Biotechnology Co., ltd., type UR 52121) for 1min to mix the solution thoroughly. Standing at 4 deg.C for 12h; centrifuging the mixed solution at 4 deg.C for 10,000g,60min, slightly discarding supernatant, enriching exosome in tube wall, centrifuging for 2min, and removing supernatant as much as possible; resuspend exosomes with 200 μ Ι _ of PBS, then collect in 1.5mL EP tubes; centrifuging at 4 deg.C for 2min to collect supernatant rich in exosome, and centrifuging at 12,000g.
2. Preparation of brain-targeted exosomes
Dissolving 2mg of brain targeting peptide (Angiopep-2-CP 05) in 2mL of deionized water to prepare a concentration of 1 mug/muL; according to the mass ratio of 1:1, synthesizing. Namely, 50 mu g of exosome and 50 mu g of Angiopep-2-CP05 are mixed fully, shaken and mixed evenly, sealed by sealing glue and incubated for 12 to 16 hours at 4 ℃; centrifuging the 100kDa ultrafiltration tube at 4 ℃ for 5,000rpm for 20min to remove free polypeptide; centrifuging for three times, adding 200 μ L PBS into the ultrafiltration tube after each centrifugation, mixing, and centrifuging; after the centrifugation is finished, firstly sucking out the liquid in the inner tube, then flushing the inner tube by 50 mu L PBS (phosphate buffer solution), repeating for 2 times, and collecting all the liquid in the inner tube to obtain the synthesized total Exo-Angiopep-2-CP05 (Exo) ACP )。
3. Preparation of drug-loaded brain-targeted exosomes
The concentration of losartan is determined by high performance liquid chromatography, and the loaded losartan concentration is determined to be about 20 mug/100 mug of brain-targeted exosome.
Comparative example
By taking a mouse blood source exosome as a contrast, preparing a corresponding drug-loaded brain target exosome, and the specific preparation steps are as follows:
1. extracting a mouse blood source exosome: the blood of the mouse inner canthus venous plexus is adopted, kept stand at 4 ℃ overnight at 3,000rpm for 10min, the supernatant is reserved and taken, centrifuged at 4 ℃ for 10,000g for 10min to remove impurities in the sample, and the supernatant is transferred to a new centrifuge tube. According to the serum: 1 × PBS solution: blood pure exo Solution =1ml in a proportional vortex shaker of 1ml (serum exosome extraction kit from shanghai meibo biotechnology limited, model No. UR 52136) for 1min, and left to stand at 4 ℃ for 2h. Centrifuging the exosome extract mixture at 4 ℃ for 60min at 12,000g to precipitate exosomes, wherein the exosomes are Blood-derived exosomes (Blood-Exo).
2. Preparation of brain-targeted exosomes: the specific procedure was the same as in example 1 to obtain Blood-Exo ACP 。
3. Preparing a drug-loaded brain targeting exosome: the specific procedure was the same as in example 1 to obtain Blood-Exo ACP @Los。
Test example 1
The characteristics of the exosome, the brain targeting exosome and the drug-loaded brain targeting exosome in example 1 are observed and identified, and the results are as follows:
(1) As shown in fig. 2A transmission electron microscopy results, both exosomes of example 1 and brain-targeted exosomes had 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 exosome-tagged protein was identified by using the western blot method, and the results showed that the exosome and brain-targeted exosomes of example 1, CD63, CD9 and TSG101, were significantly highly expressed (exosome on the left side of the figure, brain-targeted exosome on the right side).
(3) As shown in FIG. 2C, size distributions of exosomes and brain-targeted exosomes were analyzed using NTA, and the latter was found to be slightly larger than the former (mean particle size: 135.9nmvs.125.9 nm), indicating successful modification of the brain-targeted peptide to the surface of exosomes, which were mainly distributed in size (diameter) of 140. + -.50 nm.
Test example 2
Example 1 biotoxicity validation test of the brain-targeted exosomes obtained:
cells grown to 80% confluence by HCMECD3 cells (immortalized human brain microvascular endothelial cells) are prepared into 5 × 10 concentration by trypsinization 3 Cell suspension/mL, seeding cell suspension 100. Mu.L into 96-well plate, setting at 37 ℃,5% CO 2 Culturing for 24h, changing to serum-free culture solution to culture for 12h to make the cells in a stationary phase, adding 10 μ L of brain target exosomes with different concentrations (the concentrations of the added brain target exosomes obtained in example 1 are 0.1,1,5, 10 and 15 μ g/mL respectively), setting a blank control by adding only the culture solution, and setting a normal control by adding cells but not adding the brain target exosomes. 3 multiple wells were set for each concentration and the plates were incubated in incubators for 24h and 48h, respectively.
After incubation, 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, absorbance at 450nm was measured using a microplate reader. The experiment was repeated 3 times.
Percent cell survival = (brain-targeted exosome treated group OD) 450 Value-blank OD 450 value)/(Normal control OD 450 Value-blank OD 450 Value) × 100%.
The results of 24-hour survival of cells co-cultured with brain-targeting exosomes and cells are detailed in fig. 3a, and 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 still has no obvious influence on the activity of human brain microvascular endothelial cells, which shows 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 the comparative example are applied to a treatment test of a mouse with chronic kidney disease, and the specific operation is as follows:
healthy adult male C57BL/6 mice, 6 weeks old, 20-25g in weight, were selected and fed ad-hoc for one week. A mouse model of chronic kidney disease is constructed by tail vein injection of adriamycin (10 mg/kg). Intervention was initiated the third week after molding, injected once every other day, and all mice were sacrificed at the fifth week.
(1) When C57BL/6 mice were molded by intravenous doxorubicin injection for the third week,mice were injected with Cy5.5, exo via tail vein separately ACP -Cy5.5、Blood-Exo ACP Cy5.5, injecting 5mg/kg of each group of exosomes according to brain targets (Cy5.5 groups inject Cy5.5 with the same amount as other treatment groups), sacrificing the mice after 24h, reserving main organs (heart, liver, spleen, lung, kidney and brain) of the mice, and carrying out fluorescence imaging on a small animal imager. The imaging results of the blank control group injected with Cy5.5 are detailed in FIG. 4:
in the embodiment 1, the fluorescence intensity of the mouse brain after the drug-loaded brain-targeted exosome is dried is obviously stronger than that of a pure Cy5.5 treatment group, and the brain targeting effect of the drug-loaded brain-targeted exosome is better than that of a comparative drug-loaded brain-targeted exosome, which indicates that the drug-loaded brain-targeted exosome has obvious targeting property to the brain, and the targeting property is better than that of a blood-source exosome with brain targeting reported in the existing 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 the same amount of normal saline;
the second group is ADR group: injecting equivalent physiological saline into tail vein every other day from 3 weeks after adriamycin molding;
the third group is ADR + Los: performing intragastric administration of losartan (1 mg/kg/d) every other day starting from 3 weeks after doxorubicin molding;
fourth group is ADR + Exo ACP : the same amount of the brain-targeted exosomes of example 1 was injected into the tail vein every other day starting at week 3 after doxorubicin molding;
the fifth group is ADR + Boold-Exo ACP @ Los: starting from 3 weeks after doxorubicin molding, alternate-day tail vein injection of the drug-loaded brain-targeted exosomes (losartan: 1 mg/kg/d) in the comparative example;
the sixth group is ADR + Exo ACP @ Los: the drug-loaded brain-targeted exosomes of example 1 (losartan: 1 mg/kg/d) were injected into the tail vein every other day starting at week 3 after doxorubicin fabrication.
All mice were sacrificed in the fifth week after the modeling of the above grouping experiment, and blood samples, kidney and vital organ tissue samples of the mice were collected. Mouse serum was used for renal function tests. Mouse kidney, fixed, dehydrated and embedded, cut into 2 μm slices, stained with hematoxylin-eosin (H & E) and collagen fiber trichrome (Masson), and important organ tissues (heart, liver, spleen and lung) were left for H & E staining.
The results of H & E staining of important organs of mice after different group interventions are detailed in fig. 5: the morphology of important organ tissues of different groups of intervening mice has no obvious difference, which indicates that the drug-loaded brain targeting exosome of example 1 has less biological toxicity to ADR mice.
The serum creatinine and urea nitrogen results for different groups of dry prognosis mice are detailed in fig. 6: serum creatinine and urea nitrogen of the ADR group are obviously increased, which shows that the model building is successful. After small dose of losartan is perfused into the stomach, the injury of the kidney function of a mouse cannot be reduced, and the Boold-Exo of the comparative example ACP @ Los also has no significant renal function improving effect, while the drug-loaded brain targeting exosome Exo of the invention example 1 ACP The blood creatinine and urea nitrogen levels were significantly reduced in the @ Los treated group. Illustrating the losartan-loaded brain-targeted exosome Exo ACP @ Los has a good improvement effect on kidney injury of doxorubicin nephropathy mice, while the same dose of losartan cannot take effect when intervention is performed through intragastric administration.
The results of H & E staining and Masson staining of the kidneys of mice in different intervention groups are shown in fig. 7 and fig. 8:
the H & E staining results in fig. 7 show that the ADR model mice exhibited significant kidney damage, including luminal dilatation of the tubules, cast formation, tubular cell exfoliation and necrosis, and interstitial inflammatory cell infiltration. After the drug-loaded brain targeting exosome in example 1 is administered, the related damage of small-tube cells is obviously reduced, and the effect is obviously better than that of a comparative example.
FIG. 8 shows Masson staining results, the ADR group shows that renal tubular dilation, tubular cell vacuolation and interstitial collagen deposition are obvious, and the targeted exosome of brain Boold-Exo is given after oral administration of losartan and blood-source drug-loaded ACP No significant reduction in inflammatory cell and interstitial collagen deposition was observed following @ Los intervention, whereas Exo, a drug-loaded brain-targeted exosome of example 1 of the present invention, was administered ACP These lesions are significantly improved after @ Los.
Sacrifice all mice at the fifth week post-molding according to the above grouping experimentAnd Angiotensinogen (AGT) immunohistochemical staining of hippocampal region of brain tissue of all mice was collected. The results are shown in fig. 9, the AGT expression of the hippocampal part of the ADR group is obviously up-regulated compared with that of the control group, and small dose of losartan and blood source drug-loaded brain-targeted exosome Boold-Exo are administered ACP After treatment with @ Los, there was no significant downregulation of AGT expression, whereas Exo, a drug-loaded brain-targeted exosome of example 1 of the present invention, was administered ACP After @ Los, the expression of AGT is obviously reduced, which shows that the drug-loaded brain-targeted exosome can obviously reduce the expression of local RAS components of brain tissues, thereby reducing the activity of central RAS, and is probably a possible mechanism for improving the kidney injury of chronic kidney disease mice by the drug-loaded brain-targeted exosome.
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 size (diameter) of the exosome is 140 +/-50 nm, and the surface of the exosome is modified with brain-targeting polypeptide molecules; the brain-targeting exosome complex is capable of loading angiotensin II receptor inhibitors. The brain targeting exosome drug-loading system has specific targeting property on the one hand to brain tissues, and can release drugs in the brain through a blood brain barrier; on the other hand, the exosome has no obvious toxicity to cells, and the stability of the medicine can be improved by wrapping the medicine in the exosome. The brain targeting exosome drug-loading 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-targeting exosome drug-loading system prepared by the invention can be specifically enriched in the brain, has good brain targeting property, can remarkably improve the utilization rate of the drug, can realize the treatment effect on the chronic kidney diseases of mice by down-regulating the central RAS activity by loading a small-dose angiotensin II receptor inhibitor, and is an ideal drug-loading system for treating the chronic kidney diseases.
Claims (9)
1. The drug-loaded brain-targeted exosome 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 a mouse mononuclear macrophage leukemia cell RAW264.7, the brain-targeted peptide is Angiopep-2-CP05, and the drug is an angiotensin II receptor antagonist.
2. The drug-loaded brain-targeting exosome according to claim 1, wherein the exosome size distribution is at 140 ± 50nm.
3. The drug-loaded brain-targeted exosome according to claim 1, wherein the angiotensin ii receptor antagonist is losartan, valsartan, irbesartan, telmisartan.
4. The drug-loaded brain-targeting exosome according to claim 1, wherein the drug concentration in the drug-loaded brain-targeting exosome is 10-70 μ g per 100 μ g of brain-targeting exosome.
5. The drug-loaded brain-targeting exosome according to claim 4, wherein the drug concentration in the drug-loaded brain-targeting exosome is 70 μ g per 100 μ g of brain-targeting exosome.
6. The method for preparing the drug-loaded brain-targeted exosome according to claim 1, characterized in that 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 targeting exosome compound to obtain a medicine-carrying brain targeting exosome system.
7. The method of making a drug-loaded brain-targeted exosome according to claim 6, wherein the complex is subjected to ultrafiltration centrifugation to remove unencapsulated drug.
8. The method for preparing a drug-loaded brain-targeted exosome according to claim 6, wherein the macrophage RAW 264.7-derived exosome is obtained by the following method:
culturing RAW264.7 cells, changing into an exosome-free serum culture medium in logarithmic growth phase, and collecting cell supernatant after culturing for 48h to obtain exosomes derived from macrophages.
9. The use of the drug-loaded brain-targeted exosome of claim 1 for the treatment of renal disease or for the preparation of a medicament for the treatment of renal disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117838682A (en) * | 2024-03-07 | 2024-04-09 | 北京国卫生物科技有限公司 | Method for exosome targeted removal of chronic nephrotic toxins |
| CN117838682B (en) * | 2024-03-07 | 2024-06-04 | 北京国卫生物科技有限公司 | Method for exosome targeted removal of chronic nephrotic toxins |
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