CN117323444A - Intelligent drug delivery system and preparation method and application thereof - Google Patents
Intelligent drug delivery system and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Hematology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
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- Heart & Thoracic Surgery (AREA)
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Abstract
The invention discloses an intelligent drug delivery system, a preparation method and application thereof, in particular to application in treating acute myocardial infarction, and further the intelligent drug delivery system and a preparation for inhibiting passive absorption of liver and spleen form an intelligent drug system. The intelligent drug system can obviously block the endocytic function of MPS in spleen and liver, thereby obviously improving the in vivo distribution of therapeutic exosomes injected subsequently in other target organs.
Description
Technical Field
The invention belongs to the medicine technology, and relates to an intelligent medicine delivery system, in particular to application of the intelligent medicine delivery system in medicines for treating acute myocardial infarction, aiming at promoting active heart targeting and blocking passive liver and spleen interception.
Background
Myocardial infarction is a cardiovascular disease seriously harming human health, and along with the continuous improvement of the living standard of people in China, the incidence rate of ischemic myocardial infarction is also continuously increased. Ischemic myocardial infarction can lead to myocardial cell necrosis and scarring, which in turn affect cardiac function. Most of the current drug or device treatments only can relieve symptoms, but can not reverse heart tissue damage. Although heart transplantation can thoroughly improve heart conditions, it is difficult to be widely used clinically due to donor-derived scarcity, immune rejection, and expensive treatment costs.
In recent years, the use of exosomes as drug carriers for delivering drugs is becoming a very promising approach. Compared with other carriers, the exosome has the characteristics of low immunogenicity, biodegradability, low toxicity, encapsulation of endogenous bioactive molecules, strong protection effect on the entrapped content and the like. However, the biodistribution studies of mouse tail intravenous exosomes showed that exosomes rapidly aggregate in organs of the Megaloblastic Phagocytic System (MPS), such as liver and spleen, with few exosomes delivered to the heart. Thus, there is a need for an improvement in exosomes that allows for better treatment of myocardial infarction. In addition, exosomes are biosafety, but their therapeutic effects need to be improved.
Disclosure of Invention
The invention discloses an application of an intelligent drug delivery system in preparing drugs for treating myocardial infarction, which weakens the rapid uptake of a mononuclear macrophage system (MPS) to exosomes and improves the efficiency of drug delivery to the heart.
The invention adopts the following technical scheme:
an intelligent drug delivery system is prepared by the steps of carrying molecular drugs on exosomes of a surface-modified targeting molecule to obtain an intelligent drug delivery system; or the exosome surface of the loaded molecular medicine is modified with the targeting molecule to obtain the intelligent medicine delivery system. Preferably, the molecular drug comprises curcumin; targeting molecules include polypeptides; the exosomes are extracellular exosomes, such as stem cell exosomes.
A smart drug system for improving the efficiency of exosome delivery to the heart comprising the smart drug delivery system described above and a formulation that inhibits passive absorption by the liver and spleen, such as a clathrin (Cltc) inhibitor.
A medicine for treating myocardial infarction comprises the intelligent medicine delivery system or intelligent medicine system as active ingredient.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing myocardial infarction treatment drugs; preferably, the myocardial infarction is acute myocardial infarction.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing a drug with excellent targeting to an ischemic heart or in preparing a drug for reducing the aggregation of targeted exosomes in the liver and spleen.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing drugs with improved affinity with myocardial cells.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing drugs for improving cardiac function, in particular to an application in preparing drugs for improving cardiac function after myocardial infarction.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing an myocardial infarction fibrosis inhibitor.
The invention discloses an application of the intelligent drug delivery system or the intelligent drug system in preparing drugs for relieving myocardial excessive oxidative stress.
In the invention, the medicine is liquid and is solution, and the specific administration mode can adopt tail vein injection and the like.
The invention uses DOPE-PEG-NHS to target the ischemia heart to peptide (CSSMLKAC) is modified on the exosome membrane of the mesenchymal stem cells of the mice, and phenotype identification is carried out on the modified exosome by adopting a Transmission Electron Microscope (TEM), nanoparticle Tracking Analysis (NTA), electrophoresis Light Scattering (ELS), an atomic force microscope and the like; targeting exosome-loaded curcumin is further utilized as a smart drug delivery system. Next, a mouse myocardial infarction model was constructed, and DiR-labeled targeted exosomes (Exo CTP ) And disordered peptide exosomes (Exo Scr ) Observing the fluorescence intensity of the in vitro tissue by an In Vivo Imaging System (IVIS); the endocytic exosomes were then observed by tail vein injection of DiI-labeled targeted exosomes and unordered peptide exosomes by tissue slice fluorescence co-localization. Simultaneously observing the affinity of the cells and the exosomes in vitro through co-incubation of the cells and the exosomes; PBS, free curcumin (cur) and Exo are respectively injected into tail vein after establishment of myocardial infarction of mice CTP 、Cur@Exo CTP The change of the cardiac function of the mice is observed, and the heart tissue and serum of the mice on 3 days of myocardial infarction are detected, so that the reinforcing capability of the targeted exosomes on the antioxidant effect of curcumin is observed. Further, cur@Exo was injected in the tail vein CTP Pre-injection of a siCltc-loaded exosome to inhibit Cltc expression, thereby inhibiting the expression of Cur@Exo by liver and spleen macrophages CTP Is observed by an in vivo imaging system to inhibit macrophage phagocytosis and then liver, spleen and heart are marked with DiR CTP Absorption conditions; finally, injecting the exosomes loaded with the siCltc after the establishment of the myocardial infarction of the mice to inhibit the expression of the Cltc, and observing the change of the therapeutic targeted exosomes on the cardiac function of the mice. The intelligent drug system can obviously block the endocytic function of MPS in spleen and liver, thereby obviously improving the in vivo distribution of therapeutic exosomes injected subsequently in other target organs.
Drawings
FIG. 1 is an identification of targeted exosomes and unordered peptide exosomes.
Figure 2 shows that targeting exosomes has excellent targeting to the ischemic heart.
Figure 3 shows that the targeted exosomes have significant affinity for cardiomyocytes in vitro.
Fig. 4 is a diagram of a smart drug delivery system with improved biosafety.
Fig. 5 is a diagram of a smart drug delivery system accelerating cardiac recovery after myocardial infarction.
Fig. 6 is a diagram of a smart drug delivery system for relieving myocardial excessive oxidative stress.
FIG. 7 is Exo siCltc Pretreatment reduces targeted exosome aggregation in the liver and spleen.
FIG. 8 is Exo siCltc Pretreatment improves the therapeutic effect of the intelligent drug delivery system on myocardial infarction.
Detailed Description
The invention relates to application of an intelligent drug delivery system in a drug for treating acute myocardial infarction, in particular to application of a targeting exosome loaded with curcumin in the drug for treating acute myocardial infarction, aiming at promoting active heart targeting and blocking passive liver and spleen interception. The following examples are set forth only to aid those of ordinary skill in the art in a more complete understanding of the present invention and are not intended to limit the present invention in any way. The raw materials involved in the invention are existing substances, and the specific preparation operation and performance test are conventional technologies; the related animal experiments meet the related requirements of the university of Suzhou; the experimental results are well understood by those skilled in the art based on the common general knowledge in the art.
The invention discloses an intelligent drug delivery system, which is prepared by the following steps that exosomes of a targeting molecule are modified on the surface to load molecular drugs, so that the intelligent drug delivery system is obtained; preferably, the molecular drug comprises curcumin; targeting molecules include polypeptides; the exosomes are extracellular exosomes, preferably stem cell exosomes, such as bone marrow mesenchymal stem cell exosomes. Specifically, the targeting molecule is modified on the surface of the exosome and then the molecular drug is loaded by using a compound, so as to obtain the intelligent drug delivery system, wherein the compound comprises functionalized polyethylene glycol, and the functionalization refers to functionalization which can react with primary amine, such as DOPE-PEG-NHS.
By way of example, the invention modifies the ischemia heart targeting peptide (CSTSMLKAC) to mouse bone marrow mesenchymal stem cell exosome membrane by DOPE-PEG-NHS, and wraps curcumin to obtain curcumin-loaded targeting exosome for treating acute myocardial infarction.
The invention selects P3-P6 generation BMSCs culture supernatant with typical uniform morphology and good growth state to extract exosomes, and the specific extraction operation process is as follows: after the cell fusion degree reaches 60-70%, the original culture medium in the culture dish is sucked and removed, the bottom of the culture dish is washed for 3 times by using preheated PBS, and then the cells are continuously cultured for 48 hours by using BMSCs complete culture medium without exosome serum. After 48 hours, the cell supernatant was collected for subsequent extraction of exosomes (the supernatant could also be stored temporarily in a-80 ℃ freezer). Centrifugation was performed at 2000 g for 30 minutes at room temperature to remove dead cells and cell debris from the supernatant, which was then filtered through a 0.22 mm filter. The filtered supernatant was transferred to a special centrifuge tube for a super high speed centrifuge, after balancing, the supernatant was sucked off by centrifugation at 110000 g for 90 minutes at 4℃and the exosomes on the tube wall were resuspended in PBS. After detecting the exosome concentration by using the BCA enhanced kit, the exosomes are split-packed and stored.
Comparative example 1:
and respectively dissolving DOPE-PEG-NHS and polypeptide with the molar quantity of 100 times in water, adding the DOPE-PEG-NHS solution into the polypeptide solution after the two medicines are completely dissolved, uniformly mixing, and reacting for 2 hours at room temperature to obtain the DOPE-polypeptide solution. After exosomes are dissolved in water, adding DOPE-polypeptide solution and exosome solution into a round-bottom flask to be uniformly mixed according to a lipid to exosome ratio of 6000:1 (molar ratio, namely, 6000 molecules of DOPE-polypeptide are arranged in each exosome), and then placing the mixture in a water bath at 30 ℃ to incubate for 1 hour to obtain polypeptide modified exosomes; further, a nano dialysis device using 200 nm dialysis membrane was used to obtain a modified exosome with high purity by dialysis.
The polypeptide is ischemia heart targeting peptide (CSTSMLKAC), and the obtained modified exosome is called Exo CTP The method comprises the steps of carrying out a first treatment on the surface of the The polypeptide is disordered peptide (CSKTALSMC), and the modified exosome is called Exo Scr 。
Identification of targeted exosomes and unordered peptide exosomes. The ischemic heart targeting peptide and the disordered peptide are respectively modified on the mouse bone marrow mesenchymal stem cell exosome membrane through DOPE-PEG-NHS, and then the modified exosome is subjected to phenotype identification by adopting a Transmission Electron Microscope (TEM), a Nanoparticle Tracking Analyzer (NTA), a dynamic light scattering particle size analyzer (DLS), an Atomic Force Microscope (AFM), a full-automatic exosome fluorescence detection analysis system (ExoView) and the like, see figure 1.
Example 1:
an intelligent drug delivery system is a targeted exosome loaded with curcumin, and the preparation method comprises the following steps: 1, the method comprises the following steps: 4 (mg/mg, solute), curcumin was dissolved in DMSO and then mixed with modified exosomes (Exo) CTP ) Mixing and incubating for 15 minutes at room temperature; unbound curcumin was then removed by centrifugation twice at 5000 g and ultracentrifuged at 110000 g for 3 hours to take the pellet as a curcumin-loaded targeted exosome, called Cur@Exo CTP 。
Modified exosomes (Exo) CTP ) Replacement with a disordered peptide (Exo) Scr ) Referring to the method, cur@Exo is obtained Scr 。
Load efficiency calculation: curcumin loading efficiency (%, w/w) = (amount of curcumin loaded by modified exosomes/amount of curcumin initially added) ×100. The supernatant was collected and the free curcumin content in the supernatant was measured using a microplate reader (excitation wavelength=420 nm, emission wavelength=530 nm). Based on the content of free curcumin in the supernatant, the content of curcumin entrapped into the exosomes was calculated, and the entrapment efficiency was calculated to be 49.8% based on this.
Example 2: the targeted exosome has excellent targeting to ischemic heart
The main materials and sources used are as follows: c57BL/6J mice (supplied by biological medicine Co., ltd., hengnew morning, suzhou, approved by the university of Suzhou ethical Committee); small animal breathing machine (Shanghai Ornithoech Co., ltd., shanghai); a small animal ultrasound imaging system (Visual Sonics Vevo 2100).
1. Establishment of mouse myocardial infarction model
About 25 g C57BL/6J male mice are selected as experimental objects, and a myocardial infarction model is manufactured by adopting a left coronary anterior descending ligation method. After the intraperitoneal injection anesthesia, the air respirator is connected through an oral tracheal cannula, the breathing frequency is 110 times/min, the tidal volume is 3 ml, and the inhalation-exhalation ratio is 1:1.3. In the right lateral position, the left chest longitudinal incision cuts the outer skin, the pectoral major muscle is peeled off, the third and fourth intercostal transverse incisions open the chest, expose the heart, and the pericardium is torn with forceps. The left coronary artery is seen to be approximately walking by means of a surgical microscope. At the lower left atrial appendage edge 1-2 mm, the anterior descending coronary artery (LAD) was ligated along with a small amount of myocardial tissue, with a needle penetration depth of about 1 mm, and a width controlled to within 3 mm. Layer by layer, close chest. After ligation, the ligation part is visible to the naked eye until the apex of the heart becomes white, which proves that the myocardial infarction model is successfully built.
2. DiR fluorescent dye marked exosomes
DiR iodide [1,1-dioctadecyl-3, 3-tetramethylindotricarbocyanine iodide ] is highly lipophilic and widely used for cell membrane staining; infrared light emitted by DiR can pass through cells and tissues efficiently and is therefore commonly used for living imaging and tracking. The dye can uniformly mark cells through transverse diffusion in plasma membranes, and the DiR fluorescent dye has the characteristics of high specificity for marking the membranes, very low dyeing background and the like, and can be used for most applications requiring visual marking of exosome research.
(1) Preparing a storage solution: preparing DiR stock solution (2 mM) by DMSO, and sub-packaging and storing;
(2) Preparing a working solution: diluting the storage solution into a working solution of 50 mM by using 1' -PBS according to a certain proportion;
(3) The polypeptide modified exosomes were added to the 50 mM working fluid described above, with a polypeptide modified exosomes content of 100 mg. Placing the prepared solution into a water bath kettle to incubate for 30 minutes, wherein the water temperature of the water bath kettle is 37 ℃;
(4) After 30 minutes of incubation, washing with 10 mL' PBS, and ultracentrifugation according to 110000 g for 2 hours standard, the purpose of removing excess fluorescent dye is achieved by the method described above;
(5) After centrifugation, the purpose of re-suspending and precipitating is achieved by adding 200 mL of 1' -PBS;
(6) Detecting exosome concentration: BCA-enhanced kits were used to detect exosome concentrations.
3. Living animal imaging
Disordered peptide exosomes (Exo) Scr ) And targeted exosomes (Exo CTP ) Respectively carrying out DiR fluorescent marking, after a myocardial infarction model of a mouse is successfully built, respectively injecting 200 mug of DiR marked two modified exosomes and PBS through tail veins after 24 hours of myocardial infarction model establishment, killing the mouse after 6 hours of injection, respectively taking out heart, liver, spleen, lung and kidney of the mouse, and analyzing the fluorescence intensity of each tissue by adopting an IVIS animal tissue imaging system; experimental results: observing the fluorescence intensity of each tissue by IVIS imaging (figure 2), and researching the retention condition of DiR marked exosomes in the tissue; * P (P)<0.05，***P<0.001。
Example 3: targeting exosomes has significant affinity for cardiomyocytes in vitro
To find Exo CTP DiI-labeled Exo was injected into the tail vein of myocardial infarction mice separately from target cells at ischemic heart Scr Exo CTP Mice were sacrificed 12 hours later (as above), heart tissue was frozen and the DiI dye-labeled Exo was immunofluorescence detected Scr Exo CTP . Exo was determined by in vivo cardiac tissue immunostaining and in vitro cell experiments CTP The primary target cells at the infarcted heart were cardiomyocytes (fig. 3).
Example 4: intelligent drug delivery systems with better biosafety
Intelligent drug delivery system Cur@Exo CTP Adverse effects on normal organs and the whole body are the primary concern for therapy, for which Cur@Exo was studied separately CTP Short-term and long-term biosafety of (a). In the short-term biosafety study, normal mice were injected with PBS, free Cur, exo on day 0, day 1, day 2, and day 7, respectively CTP Or Cur@Exo CTP (injection dose: 0.1 mL PBS, 0.1 mL/20 mg Free Cur, 0.1 mL/160 mg Exo) CTP Or 0.1 mL/180 mg Cur@Exo CTP ) On day 3 or 8, mice were removed from their eyeballs and serum was subjected to Elisa assay.
In the exploration of the long-term biosafety part of Cur@ExoCTP, H was performed&E staining, normal mice were injected with PBS, fr on day 0, day 1, day 2 and day 7, respectivelyee Cur、Exo CTP Or Cur@Exo CTP (injection dose: 0.1 mL PBS, 0.1 mL/20 mg Free Cur, 0.1 mL/160 mg Exo) CTP Or 0.1 mL/180 mg Cur@Exo CTP ) After 1 month, the important organs of the mice, such as liver, spleen, lung, kidney, brain, were fixed in 4% paraformaldehyde for 24 hours and then H was performed&E staining to evaluate Cur@Exo CTP And the like, to the damage of tissue and organs in the body.
Hematology parameter analysis showed that alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen (BUN), creatinine (CRE) levels were normal between groups, both on day 3 and day 8, with no statistical differences. Indicating that the biological functions of the liver and the kidney of the mice are not affected by Free Cur and Exo CTP Or Cur@Exo CTP Effect of treatment. H&E staining results showed that the major organs of each group of mice were not significantly changed or damaged, thereby further confirming Cur@Exo CTP Referring to fig. 4, ns has no statistical difference.
Example 5: intelligent drug delivery system for accelerating heart recovery after myocardial infarction
1. Mouse myocardial infarction model was established as in example 2, and 0.1 mL PBS, 0.1 mL/20 mg Free Cur, 0.1 mL/160 mg Exo were injected via tail vein on day 0, day 1, day 2, day 7 and day 14, respectively, after establishment of mouse myocardial infarction model CTP Or 0.1 mL/180 mg Cur@Exo CTP ；
2. Heart function after myocardial infarction is detected by heart ultrasound.
The mice were anesthetized, left lateral decubitus after dehairing, the heart ultrasonic diagnostic apparatus probe was placed on the anterior wall of the heart, a two-dimensional section of the left ventricle was taken at the papillary muscle level, and simultaneously an M-scan was recorded, and left ventricular Ejection Fraction (EF) and shortening Fraction (FS) were measured.
Experimental results: the cardiac function is detected 28 days after myocardial infarction operation, the intelligent drug delivery system obviously improves the cardiac function after myocardial infarction, and the ejection fraction and the short-axis shortening rate are obviously improved. Cur@Exo CTP The minimum area of fibrosis is only 19.44+ -1.19%, which is smaller than that of Free Cur group (19.44+ -1.19% vs 29.05+ -4.11%, p<0.001 At the same time is smaller than Exo CTP The group of the components is arranged in a group,see fig. 5, P<0.05，**P<0.01，***P<0.001。
Example 6: intelligent drug delivery system for relieving myocardial excessive oxidative stress
To detect oxidative stress in myocardial tissue following myocardial infarction, 0.1 mL PBS, 0.1 mL/20 mg Free Cur, 0.1 mL/160 mg Exo were injected via tail vein on day 0, day 1 and day 2, respectively, after myocardial infarction model establishment in mice CTP Or 0.1 mL/180 mg Cur@Exo CTP After that, the ROS content in the heart frozen section of the mice on the 3 rd day after myocardial infarction is detected by using a DHE fluorescent probe, eyeballs of the mice on the 3 rd day of myocardial infarction are subjected to blood collection, and the related index of oxidative stress in serum is subjected to Elisa detection. Experimental results: curcumin can exert its antioxidant capacity better by targeting exosome delivery, and can lower the level of oxidative stress in myocardial infarction, improve its oxidative stress state (fig. 6),/P<0.001。
Example 7: exo (Exo) siCltc Pretreatment reduces targeted exosome aggregation in liver and spleen
siCltc is siRNA that silences cell Cltc, siNC is a negative control.
After the siCltc was dissolved in DEPC water, the exosomes were electroporated with 1 OD siRNA at a concentration of 1 mg/mL in 0.4 cm wide electroporation tubes at 700V/150 mF, followed by treatment of the exosomes with RNase A and RNase inhibitor, respectively, to remove siRNA that may bind to the exosome membrane and inactivate RNase A. The exosomes were then washed with cold PBS, centrifuged for 30 min at 12000 g to give siCltc loaded exosomes Exo siCltc 。
The same method prepares exosomes Exo loaded with siNC siNC 。
After successful construction of the myocardial infarction model, myocardial infarction mice were divided into two groups, one group was injected into the tail vein with the siCltc-loaded Exo siCltc An additional group of mice were injected i.v. with siNC-loaded Exo siNC As a control, after injection (immediately after myocardial infarction) of the exosomes with siRNA for 24 hours,injection of equivalent amounts of DiR fluorescent labelled Exo CTP Mice were sacrificed 3 hours after injection, hearts, livers and spleens of the mice were taken out, and the distribution of the two exosomes in each tissue and organ was fluorescently tracked by in vitro tissue imaging of the mice.
Experimental results: pre-injection of siCltc loaded Exo siCltc Can be used for treating Exo by reducing liver and spleen CTP Indirectly increases Exo in blood circulation CTP Content of (C) such that Exo in heart and blood CTP Can have more action time and finally lead to more Exo absorbed by heart CTP (FIG. 7). * P (P)<0.05，**P<0.01，***P<0.001。
Example 8: exo (Exo) siCltc Pretreatment to improve the therapeutic effect of intelligent drug delivery system on myocardial infarction
Injection of siCltc-loaded Exo via tail vein on day 0 and day 6, respectively, after mouse myocardial infarction model establishment siCltc Or exosomes Exo loaded with siNC siNC Smart drug delivery System by Tail vein injection 0.1 mL/180 mg Cur@Exo for each mouse on days 1, 2 and 7 CTP . Mouse heart function was assessed by cardiac ultrasound in mice at various time points such as 3, 7, 14 and 28 days post-surgery with preoperative day 1 mouse heart function as baseline. The mouse hearts on day 28 after myocardial infarction were selected, sectioned and Masson stained for myocardial fibrosis area. Three (3) sections were taken every 500 mm below the ligature for Masson staining.
Experimental results: exo (Exo) siCltc Pretreatment can promote the heart to Cur@Exo CTP Absorption is obviously improved CTP Improving myocardial contractile function of myocardial infarction mice and improving Cur@Exo CTP Therapeutic effect on myocardial infarction (fig. 8). * P (P)<0.05，**P<0.01，***P<0.001。
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. The preparation method of the intelligent drug delivery system is characterized by comprising the following steps of carrying molecular drugs on exosomes of the surface-modified targeting molecules to obtain the intelligent drug delivery system; or the exosome surface of the loaded molecular medicine is modified with the targeting molecule to obtain the intelligent medicine delivery system.
2. The method of preparing a smart drug delivery system of claim 1, wherein the molecular drug comprises curcumin; targeting molecules include polypeptides; the exosomes are extracellular exosomes.
3. A smart drug delivery system prepared according to the method of preparing a smart drug delivery system of claim 1.
4. A smart drug system comprising the smart drug delivery system of claim 3 and a formulation that inhibits passive absorption of the liver and spleen.
5. The intelligent pharmaceutical system according to claim 4, wherein the agent that inhibits passive absorption of liver and spleen is a clathrin inhibitor.
6. Use of the smart drug delivery system of claim 3 or the smart drug system of claim 4 for the manufacture of a medicament for improving the efficiency of exosome delivery to the heart.
7. Use of the smart drug delivery system of claim 3 or the smart drug system of claim 4 for the preparation of a medicament having excellent targeting to the ischemic heart; or in the preparation of medicaments for improving cardiac function; or in the preparation of a medicament with enhanced affinity to cardiomyocytes; or in the preparation of myocardial infarction fibrosis inhibitors.
8. Use of the smart drug delivery system of claim 3 or the smart drug system of claim 4 for the manufacture of a medicament for myocardial infarction treatment.
9. The use according to any one of claims 6 to 8, wherein the medicament is a solution.
10. A medicament for treating myocardial infarction, characterized in that the active ingredient is the smart drug delivery system of claim 3 or the smart drug system of claim 4.
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