CN111450061A - Hybrid mesenchymal stem cell exosome drug delivery system and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a hybrid mesenchymal stem cell exosome drug delivery system which is constructed by hybrid fusion of mesenchymal stem cell exosomes and liposomes. The system has both passive targeting and active targeting of the tumor, obviously improves the tumor targeting efficiency, reduces the toxic and side effects of the whole body, and can be applied to the aspect of medicine preparation, in particular to anticancer medicines.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a tumor-targeted medicine delivery system, in particular to a hybrid mesenchymal stem cell exosome medicine delivery system and a preparation method and application thereof.
Background
According to the latest statistical data of the international agency for research on cancer (IARC), in 2018, 1810 ten thousand new tumor cases and 960 ten thousand death cases all over the world exist, and the incidence and the death rate of tumors in China are the first in the world. In tumor chemotherapy, the low targeting efficiency of anticancer drugs and strong systemic toxic and side effects are the most prominent problems, even the direct or indirect cause of death of one third of patients, so that the difference between tumor and normal tissues needs to be fully utilized to carry out tumor specific treatment, and the toxicity to the normal tissues is reduced.
The liposome (liposome) is a phospholipid bilayer closed vesicle prepared manually, the particle size is 60-200 nm, and the liposome can utilize the high permeation and retention effect (EPR effect) of tumor tissues, namely the tumor passive targeting effect, so that the in-vivo distribution of the drug is improved, the enrichment amount of the drug at tumor parts is increased, and the toxic and side effects of the whole body are reduced. To date, a number of liposomal formulations have been marketed at home and abroad and have been established for clinical use, such as doxorubicin liposomes
Vincristine liposome
And irinotecan liposome
And the like. The liposome is also a carrying form commonly adopted in the research of combined medicaments, such as vincristine and adriamycin double-carrying liposome, adriamycin and P-gp inhibitor quercetin double-carrying liposome and the like. The water-soluble medicine can be encapsulated in the water phase in the liposome, and the fat-soluble medicine is encapsulated between the phospholipid bimolecular layers. However, liposomes do not have the ability to actively target tumor tissue, and they readily bind Apo-E after entering the systemic circulation, and are subsequently bound by Apo-EThe reticuloendothelial system phagocytizes and distributes preferentially to the liver and spleen. The target head which is artificially synthesized and can be specifically combined with a tumor high-expression receptor is modified on the surface of the liposome, so that the capability of actively targeting tumor tissues can be endowed to the target head, but only a few types of target heads which have definite research effects can be artificially synthesized, and the method is very limited.
Exosomes (exosomes) are vesicles secreted by cells with biological activity, typically 30-150 nm in diameter, containing various biomolecules in the maternal cells. Research has shown that exosome plays an important role in human physiological and pathological processes, participates in processes such as angiogenesis, cell migration, immune response and tumor generation and development, and plays roles in molecular delivery and information exchange among cells. Because of their nature, many of the exosomes have been unknown, and have become a hot international research spot in the life science field in recent years. The exosome is used as an endogenous vesicle and has natural molecular transport characteristics and good biocompatibility, and researches show that the exosome can be used for delivering various medicaments such as chemical medicaments (such as adriamycin, gemcitabine, carboplatin, 5-fluorouracil and the like), proteins, peptide ligands, RNA and the like. The use of exosomes for drug delivery also presents certain technical challenges. At present, the technology that exosomes carry RNA drugs by transfecting mother cells is mature, however, for cytotoxic drugs, high-concentration drugs and the mother cells are cultured together, the cytotoxic drugs have a killing effect on the mother cells, and the secretion of the drug-containing exosomes is obviously influenced; direct loading of drugs into exosomes often involves electroporation, which often affects the integrity of the exosome membrane or drug. The low drug-loading capacity of cytotoxic drugs is a bottleneck problem to be solved for the research of exosomes as tumor drug delivery systems.
How to take advantage of liposomes and exosomes to overcome their limitations? The research finds that the phospholipid molecules forming the membrane structure can be rearranged under specific conditions, and hybrid fusion between vesicles is realized. The liposome and the exosome are phospholipid bilayer membrane closed vesicles with similar particle size ranges, Raw 264.7 cell exosomes, CMS7 exosomes highly expressing HER2 receptors and PEG chain modified liposomes are subjected to hybrid fusion through freeze-thaw cycles by Yuko T.Sato successfully prepare hybrid vectors, and the cell uptake capacity is evaluated. This study demonstrated that it is technically feasible to form hybrid vectors using liposomes and exosomes, however, the study has not been directed to encapsulation and targeted delivery of anti-tumor drugs using hybrid vectors, nor the exosome species selected have tumor tropism.
Exosomes secreted by different cells have different functional properties. The mesenchymal stem cells are non-hematopoietic stem cells which exist in adults and contain a plurality of differentiation potentials, the surfaces of the mesenchymal stem cells express CD105, CD73, CD44, MHC molecules and the like, and the mesenchymal stem cells have the characteristics of low immunogenicity, strong tumor tropism and solid tumor penetrating capacity and the like. The exosome derived from the mesenchymal stem cells also has certain characteristics of the mesenchymal stem cells, such as capability of avoiding interaction with opsonin, antibodies, blood coagulation factors and the like, thereby avoiding phagocytosis of a reticuloendothelial system, prolonging the circulation time in vivo, and approaching to the primary and metastatic parts gathered on tumors. At present, the research on the tumor tropism mechanism of the exosome derived from the mesenchymal stem cell and the research on the targeted delivery of the antitumor drug by utilizing the tumor tropism are not available.
The combination of multiple drugs is applied to the field of cancer treatment, such as drug combination of different action targets, drug combination of different action mechanisms and the like, the FDA approved in 2014 that Dabrafenib (Dabrafenib, BRAF inhibitor) and Trametinib (MEK inhibitor) are combined to treat BRAF V600 mutation positive melanoma, and the combination becomes the development trend of cancer treatment.
Through searching, the following publications related to the patent application of the invention are found:
CN107913408A discloses an exosome-aptamer liposome composite drug delivery system, a preparation method and application thereof, CN107980004A discloses application of exosome for treating diseases, Journal documents are Mei L u, et al, Complex of exosome-mimetic liposomes with associated lipids for intracellular delivery of siRNA. International Journal of pharmaceuticals, 2018(550): 100. 113. liposomes for preparing mimetic exosomes deliver VEGF to epithelial cells, which can improve transfection efficiency, Masaton Nishio, et al, real-time assay for exosome fusion with extracellular membrane of biological specific lipid binding membrane, 11918 is disclosed as a real-time enhanced conductance channel A, and 11918 is disclosed as a real-time detection technology for extracellular membrane fusion.
By contrast, the present patent application is substantially different from the above publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a hybrid mesenchymal stem cell exosome drug delivery system and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a hybrid mesenchymal stem cell exosome drug delivery system is constructed by hybrid fusion of mesenchymal stem cell exosomes and liposomes.
And, the mesenchymal stem cell exosome is derived from bone marrow mesenchymal stem cells, skeletal muscle mesenchymal stem cells, periosteum mesenchymal stem cells, trabecular bone mesenchymal stem cells, umbilical cord mesenchymal stem cells or placenta mesenchymal stem cells of the animal.
Moreover, the liposome is a phospholipid bilayer closed vesicle composed of cholesterol components and phospholipid materials;
wherein the cholesterol component is selected from cholesterol or derivatives thereof;
the phospholipid material is selected from one or more of egg yolk lecithin, soybean lecithin, 1-stearoyl-lysophosphatidylcholine, egg yolk phosphatidylglycerol, dipalmitoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, pelotted phosphatidylethanolamine, erucyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine and (2, 3-dioleoyl-propyl) -trimethylamine.
Moreover, the hybrid mesenchymal stem cell exosome drug delivery system is in a vesicle shape, the particle size distribution of the drug delivery system is that the median diameter is 60-200 nm;
or the multi-drug-loading hybrid mesenchymal stem cell exosome drug delivery system can comprise several fat-soluble drugs, or the fat-soluble drugs are combined with water-soluble drugs, or several water-soluble drugs.
A preparation method of the hybrid mesenchymal stem cell exosome drug delivery system comprises the following steps:
s1, mixing the cholesterol component and the phospholipid material according to the weight ratio of 5-20: 80-95 dissolving in organic solvent, adding fat-soluble antitumor drug, and dissolving cholesterol and phospholipid material in water bath at 35-55 deg.C;
s2, under the condition of water bath, rotating and vacuumizing to remove the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
s3, adding the film obtained in the step S2 into a pH buffer solution dissolved with the water-soluble anti-tumor drug, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
s4, carrying out ultrasonic or homogenization, and preparing a drug-loaded liposome with a median diameter of 60-200 nm;
s5, continuously loading the water-soluble drug with strong acid and weak base salt characteristics or strong base and weak acid salt characteristics on the basis of the drug-loaded liposome obtained in the step S4 by utilizing the preparation technology of gradient change of the internal and external pH values of the carrier to prepare the multiple drug-loaded liposome;
s6, culturing the mesenchymal stem cells, collecting the residual liquid culture medium in the culturing process, carrying out centrifugal operation on the liquid culture medium to obtain an exosome precipitate, and preparing an exosome stock solution;
s7, mixing the exosome stock solution obtained in the step S6 with the drug-loaded liposome obtained in the step S5, and repeatedly freezing and thawing for several times to obtain the hybrid mesenchymal stem cell exosome drug delivery system.
Moreover, the preparation technology of the gradient change of the pH inside and outside the carrier in the step S5 is as follows: aiming at strong acid and weak base salt, the pH value of the inner aqueous phase of the liposome needs to be adjusted to 2.5-4.0, and the pH value of the outer aqueous phase needs to be adjusted to 6.0-8.5;
for strong alkali and weak acid salt, the pH value of the liposome internal water phase is required to be adjusted to 8.5-10.5, and the pH value of the external water phase is required to be adjusted to 5.5-8.0.
Further, step S6 is specifically:
taking mesenchymal stem cells into a cell culture bottle, adding a DMEM/F12 culture medium containing 10-20% fetal calf serum by mass concentration, culturing in a constant-temperature culture box, removing non-adherent cells, carrying out primary culture for 5-15 days, carrying out primary passage, carrying out secondary passage culture when the growth of adherent mesenchymal stem cells reaches 70-90%, taking mesenchymal stem cells of 3-6 generations, collecting cell supernatant, subpackaging in a centrifuge tube, centrifuging for 1-30min at 200-.
Further, step S7 is specifically:
mixing the exosome stock solution and multiple drug-loaded liposome with the volume 1-10 times of the volume of the exosome stock solution at room temperature, stirring and incubating for 10-30min at 30-40 ℃, placing the mixture in liquid nitrogen for freezing for 5-10min, unfreezing at room temperature, stirring and incubating for 15-30min at 30-40 ℃, then placing the mixture in liquid nitrogen for freezing for 5-10min, and performing multiple freeze-thaw cycles to fully fuse and hybridize the exosome membrane and the liposome membrane to prepare the multiple drug-loaded hybrid mesenchymal stem cell exosome drug delivery system.
The application of the hybrid mesenchymal stem cell exosome drug delivery system in the aspect of drug preparation.
Moreover, the applications are: the drug delivery system can entrap and deliver antitumor drugs which are two or more of drugs inhibiting the activity of enzymes in tumor cells, drugs inhibiting the formation of tubulin, drugs inhibiting the formation of DNA or RNA, drugs causing hypoxia of tumor tissues, and drugs causing the generation of oxygen radicals in tumor tissues.
The invention has the advantages and positive effects that:
1. the system has both passive targeting and active targeting of the tumor, obviously improves the tumor targeting efficiency, reduces the toxic and side effects of the whole body, and can be applied to the aspect of medicine preparation, in particular to anticancer medicines.
2. The system of the invention simultaneously entraps and delivers a plurality of antitumor drugs with different targets or mechanisms, well inhibits the drug resistance of the tumor and improves the prognosis of patients.
3. The system is a novel nano-carrier, and compared with liposome, the tumor targeting property is greatly improved; compared with exosome, the drug loading capacity is obviously improved, and the defects of the two existing nano-carriers are overcome by utilizing the advantages of the two existing nano-carriers.
4. Aiming at the problems of low drug targeting efficiency and strong systemic toxic and side effects in tumor chemotherapy, the invention firstly performs hybrid fusion on the liposome and the mesenchymal stem cell exosome to construct a hybrid exosome. The liposome has the advantages of strong liposome drug-loading capacity and tumor passive targeting function, has the specific tumor tropism of mesenchymal stem cell exosomes, and delivers chemotherapeutic drugs to tumor parts under the dual targeting function. The generation of drug resistance is the main reason of poor tumor prognosis, and the hybrid exosome prepared by the invention can simultaneously entrap two or more anti-tumor drugs, has synergistic effect and can be delivered to tumor parts to play a role. The invention improves the tumor target inhibition effect from the perspective of the carrier and reduces the systemic toxicity; from the perspective of combined medication, the drug resistance of the tumor is reduced, and the prognosis of the patient is improved; meanwhile, the technical bottleneck problems of low drug-loading capacity of exosomes, low liposome targeting efficiency, simultaneous entrapment of various drugs and the like are solved, and a new thought and approach are provided for the research of a tumor targeting nano-drug delivery system.
5. The invention firstly utilizes the hybridization and fusion of the mesenchymal stem cell exosome and the liposome, and can simultaneously entrap a plurality of medicines, thereby realizing the synergistic interaction, reducing the drug resistance of the tumor and improving the prognosis of patients. The invention mainly contributes to the application of the mesenchymal stem cell exosome in tumor targeted drug delivery, combines the advantages of liposome, fuses and hybridizes to construct a brand new nano-carrier, combines combined drug, provides a new thought for the research and development of a tumor targeted nano-drug delivery system, and has no research report at present.
6. The invention aims to blend and hybridize a multi-drug-loaded liposome and a mesenchymal stem cell exosome to prepare a hybrid carrier, improve the capacity of simultaneously encapsulating various drugs, improve the tumor targeting efficiency of a drug delivery system from two angles of passive targeting and active targeting, reduce toxicity and improve efficiency.
Drawings
FIG. 1 is a morphological diagram of mesenchymal stem cells of 3 rd to 6 th generations in accordance with the present invention;
FIG. 2 is a particle size distribution diagram (A) and a Transmission Electron Microscope (TEM) morphology diagram (B) of a hybrid mesenchymal stem cell exosome drug delivery system of tirapazamine and hydralazine in the invention;
FIG. 3 is a graph of tumor volume inhibition in mice of the present invention;
FIG. 4 is a graph showing the change in body weight of a mouse according to the present invention;
FIG. 5 is a graph (A) showing the results of the study of tissue distribution in mice after injection of the test agent for 0.5 hour and a graph (B) showing the results of the study of tissue distribution in mice after injection of the test agent for 8 hours.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A hybrid mesenchymal stem cell exosome drug delivery system is constructed by hybrid fusion of mesenchymal stem cell exosomes and liposomes.
Preferably, the mesenchymal stem cell exosome is derived from bone marrow mesenchymal stem cells, skeletal muscle mesenchymal stem cells, periosteum mesenchymal stem cells, trabecular bone mesenchymal stem cells, umbilical cord mesenchymal stem cells or placenta mesenchymal stem cells of animals.
Preferably, the liposome is a phospholipid bilayer closed vesicle composed of cholesterol components and phospholipid materials;
wherein the cholesterol component is selected from cholesterol or derivatives thereof;
the phospholipid material is selected from one or more of egg yolk lecithin, soybean lecithin, 1-stearoyl-lysophosphatidylcholine, egg yolk phosphatidylglycerol, dipalmitoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, pelotted phosphatidylethanolamine, erucyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine and (2, 3-dioleoyl-propyl) -trimethylamine.
Preferably, the hybrid mesenchymal stem cell exosome drug delivery system is in a vesicle shape, and the median diameter in the particle size distribution is 60-200 nm;
or the multi-drug-loading hybrid mesenchymal stem cell exosome drug delivery system can comprise several fat-soluble drugs, or the fat-soluble drugs are combined with water-soluble drugs, or several water-soluble drugs.
A preparation method of the hybrid mesenchymal stem cell exosome drug delivery system comprises the following steps:
s1, mixing the cholesterol component and the phospholipid material according to the weight ratio of 5-20: 80-95 dissolving in organic solvent, adding fat-soluble antitumor drug, and dissolving cholesterol and phospholipid material in water bath at 35-55 deg.C;
s2, under the condition of water bath, rotating and vacuumizing to remove the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
s3, adding the film obtained in the step S2 into a pH buffer solution dissolved with the water-soluble anti-tumor drug, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
s4, carrying out ultrasonic or homogenization, and preparing a drug-loaded liposome with a median diameter of 60-200 nm;
s5, continuously loading the water-soluble drug with strong acid and weak base salt characteristics or strong base and weak acid salt characteristics on the basis of the drug-loaded liposome obtained in the step S4 by utilizing the preparation technology of gradient change of the internal and external pH values of the carrier to prepare the multiple drug-loaded liposome;
s6, culturing the mesenchymal stem cells, collecting the residual liquid culture medium in the culturing process, carrying out centrifugal operation on the liquid culture medium to obtain an exosome precipitate, and preparing an exosome stock solution;
s7, mixing the exosome stock solution obtained in the step S6 with the drug-loaded liposome obtained in the step S5, and repeatedly freezing and thawing for several times to obtain the hybrid mesenchymal stem cell exosome drug delivery system.
Preferably, the preparation technology of the gradient change of the pH inside and outside the carrier in the step S5 is as follows: aiming at strong acid and weak base salt, the pH value of the inner aqueous phase of the liposome needs to be adjusted to 2.5-4.0, and the pH value of the outer aqueous phase needs to be adjusted to 6.0-8.5;
for strong alkali and weak acid salt, the pH value of the liposome internal water phase is required to be adjusted to 8.5-10.5, and the pH value of the external water phase is required to be adjusted to 5.5-8.0.
Preferably, the step S6 specifically includes:
taking mesenchymal stem cells into a cell culture bottle, adding a DMEM/F12 culture medium containing 10-20% fetal calf serum by mass concentration, culturing in a constant-temperature culture box, removing non-adherent cells, carrying out primary culture for 5-15 days, carrying out primary passage, carrying out secondary passage culture when the growth of adherent mesenchymal stem cells reaches 70-90%, taking mesenchymal stem cells of 3-6 generations, collecting cell supernatant, subpackaging in a centrifuge tube, centrifuging for 1-30min at 200-.
Preferably, the step S7 specifically includes:
mixing the exosome stock solution and multiple drug-loaded liposome with the volume 1-10 times of the volume of the exosome stock solution at room temperature, stirring and incubating for 10-30min at 30-40 ℃, placing the mixture in liquid nitrogen for freezing for 5-10min, unfreezing at room temperature, stirring and incubating for 15-30min at 30-40 ℃, then placing the mixture in liquid nitrogen for freezing for 5-10min, and performing multiple freeze-thaw cycles to fully fuse and hybridize the exosome membrane and the liposome membrane to prepare the multiple drug-loaded hybrid mesenchymal stem cell exosome drug delivery system.
The application of the hybrid mesenchymal stem cell exosome drug delivery system in the aspect of drug preparation.
Preferably, the application is: the drug delivery system can entrap and deliver antitumor drugs which are two or more of drugs inhibiting the activity of enzymes in tumor cells, drugs inhibiting the formation of tubulin, drugs inhibiting the formation of DNA or RNA, drugs causing hypoxia of tumor tissues, and drugs causing the generation of oxygen radicals in tumor tissues.
More specifically, the preparation and detection are as follows:
example 1 preparation of tirapazamine-hydralazine double drug-loaded hybrid mesenchymal stem cell exosomes
(1) Preparation of exosome stock solutions
Taking 1ml of a fresh animal mesenchymal stem cell sample into a cell culture bottle, adding DMEM/F12 culture medium containing 10% fetal calf serum, culturing in a constant-temperature incubator at 37 ℃ and 5% CO2, respectively changing the total amount of culture solution of the culture bottle at 24, 48 and 72 hours, removing nonadherent cells such as red blood cells and fat cells, changing the solution once every three days, carrying out primary culture for 10-15 days, carrying out primary passage, observing that the growth of the adherent mesenchymal stem cells reaches 80-90% after 4-7 days, carrying out secondary passage culture, taking the mesenchymal stem cells of 3 rd to 6 th generation (figure 1), collecting 100ml of cell supernatant, subpackaging in a centrifuge tube, centrifuging for 10min at 300 × g, taking supernatant, centrifuging for 10min at 2000 × g, taking supernatant, centrifuging for 30min at 10000 × g, taking supernatant, centrifuging for 70min at 10000 × g, obtaining exosome precipitate, cleaning by PBS solution, and obtaining exosome stock solution.
(2) Preparation of tirapazamine-hydralazine double-drug-loading liposome
Firstly, dissolving cholesterol, dipalmitoyl phosphatidylcholine and yolk lecithin in an organic solvent according to the weight ratio of 10:60:30, then adding fat-soluble tumor hypoxia medicament tirapazamine, and uniformly dissolving in water bath at 35-55 ℃;
then, under the condition of water bath, rotating and vacuumizing, removing the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
then, adding citric acid-sodium citrate buffer solution with the pH value of 3.0 into the film, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
then, ultrasonically treating to prepare a tirapazamine liposome with the median diameter of 90-100 nm;
finally, the pH of the external water phase is slowly adjusted to 7.0 by utilizing the preparation technology of gradient change of the internal and external pH values of the carrier, and a water-soluble drug with the characteristics of strong acid and weak base salt, hydralazine hydrochloride, is continuously loaded on the basis of the tirapazamine liposome, and the tirapazamine-hydralazine double drug-loaded liposome has the effects of reducing the tumor blood perfusion and increasing the hypoxia degree of the tumor blood perfusion.
(3) Exosome-liposome hybrid fusion
Mixing exosome stock solution with 1-fold volume and tirapazamine-hydralazine double-drug-loading liposome with 5-fold volume at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, thawing at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, and performing multiple freeze-thaw cycles to fully fuse and hybridize an exosome membrane and a liposome membrane to prepare the hybrid mesenchymal stem cell exosome drug delivery system (the particle size distribution and the shape are shown in figure 2) simultaneously encapsulating tirapazamine and hydralazine.
Comparative example 1 preparation of tirapazamine hybrid mesenchymal stem cell exosomes
(1) Preparation of exosome stock solutions
The same as in example 1.
(2) Preparation of tirapazamine liposome
Firstly, dissolving cholesterol, dipalmitoyl phosphatidylcholine and yolk lecithin in an organic solvent according to the weight ratio of 10:60:30, then adding fat-soluble tumor hypoxia medicament tirapazamine, and uniformly dissolving in water bath at 35-55 ℃;
then, under the condition of water bath, rotating and vacuumizing, removing the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
then, adding citric acid-sodium citrate buffer solution with the pH value of 3.0 into the film, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
finally, carrying out ultrasonic treatment to prepare the tirapazamine liposome with the median diameter of 90-100 nm.
(3) Exosome-liposome hybrid fusion
Mixing exosome stock solution with 1-fold volume with tirapazamine liposome with 5-fold volume at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, thawing at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, and performing multiple freeze-thaw cycles to fully fuse and hybridize an exosome membrane and a liposome membrane, thereby preparing the tirapazamine-encapsulated hybrid mesenchymal stem cell exosome drug delivery system.
Comparative example 2 preparation of hydralazine hydrochloride hybrid mesenchymal stem cell exosomes
(1) Preparation of exosome stock solutions
The same as in example 1.
(2) Preparation of hydralazine hydrochloride double-drug-loading liposome
Firstly, dissolving cholesterol, dipalmitoylphosphatidylcholine and egg yolk lecithin in an organic solvent according to the weight ratio of 10:60:30, and uniformly dissolving in a water bath at 35-55 ℃;
then, under the condition of water bath, rotating and vacuumizing, removing the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
then, adding citric acid-sodium citrate buffer solution with the pH value of 3.0 into the film, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
then, carrying out ultrasonic treatment to prepare blank liposome with the median diameter of 90-100 nm;
and finally, slowly adjusting the pH value of the external water phase to 7.0 by utilizing a preparation technology of gradient change of the internal and external pH values of the carrier, and continuously loading a water-soluble drug with the characteristics of strong acid and weak base salt, namely hydralazine hydrochloride on the basis of the blank liposome, wherein the hydralazine hydrochloride has the effects of reducing the blood perfusion of the tumor and increasing the anoxic degree of the tumor, so that the hydralazine hydrochloride liposome is prepared.
(3) Exosome-liposome hybrid fusion
Mixing exosome stock solution with 1-fold volume of exosome stock solution and tirapazamine-hydralazine double-drug-loading liposome with 5-fold volume at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, thawing at room temperature, stirring and incubating for 20min at 35 ℃, freezing for 5-10min in liquid nitrogen, performing multiple freeze-thaw cycles, and fully fusing and hybridizing an exosome membrane and a liposome membrane to prepare the hydralazine hydrochloride-entrapped hybrid mesenchymal stem cell exosome drug delivery system.
Comparative example 3 preparation of tirapazamine-hydralazine double drug-loaded liposomes
Firstly, dissolving cholesterol, dipalmitoyl phosphatidylcholine and yolk lecithin in an organic solvent according to the weight ratio of 10:60:30, then adding fat-soluble tumor hypoxia medicament tirapazamine, and uniformly dissolving in water bath at 35-55 ℃;
then, under the condition of water bath, rotating and vacuumizing, removing the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
then, adding citric acid-sodium citrate buffer solution with the pH value of 3.0 into the film, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
then, ultrasonically treating to prepare a tirapazamine liposome with the median diameter of 90-100 nm;
finally, the pH of the external water phase is slowly adjusted to 7.0 by utilizing the preparation technology of gradient change of the internal and external pH values of the carrier, and a water-soluble drug with the characteristics of strong acid and weak base salt, hydralazine hydrochloride, is continuously loaded on the basis of the tirapazamine liposome, and the tirapazamine-hydralazine double drug-loaded liposome has the effects of reducing the tumor blood perfusion and increasing the hypoxia degree of the tumor blood perfusion.
Comparative example 4 preparation of tirapazamine-hydralazine double drug loaded exosomes
(1) Preparation of exosome stock solutions
The same as in example 1.
(2) Drug loading
Mixing tirapazamine, hydralazine hydrochloride and exosome stock solution in electroporation buffer solution at 4 ℃, carrying out electroporation drug loading by using a Gene Pulser II electroporator under the conditions of 350V and 150mF, and then incubating for 30min at 37 ℃ to recover an exosome membrane, thereby preparing the tirapazamine-hydralazine double-drug-loading exosome.
Comparative example 5 preparation of aqueous tirapazamine-hydralazine solution
The tirapazamine and hydralazine are added into water for dissolving, and a little RH40 surfactant is added for helping dissolve the tirapazamine.
To better illustrate the beneficial effects of the present invention, the present invention performs a verification test on example 1 and comparative examples 1 to 5:
experiment on tumor inhibition effect and systemic toxicity reduction effect
A nude mouse xenograft tumor model is constructed by HepG-2, 18-22g (female parent) of the nude mouse xenograft tumor model is randomly divided into 4 groups, and 3 mice in each group. The combined effect of tirapazamine and hydralazine was characterized by the administration of saline (control), comparative example 1, comparative example 2 and example 1, respectively, to each experimental group. The drug is administered once in 3 days for 6 times, and a tumor inhibition curve and a body weight change curve are drawn.
As can be seen from the tumor inhibition curve (fig. 3), hydralazine alone has a slight tumor inhibition effect, and tirapazamine and hydralazine are combined, so that the tumor inhibition capacity is remarkably improved and is superior to that of the tirapazamine single-use group. The hydralazine is shown to reduce the oxygen content of solid tumors, improve the cytotoxicity of tirapazamine in an anoxic environment, and the two medicines have synergistic effect.
From the body weight change curve (fig. 4), it can be seen that the systemic toxicity of comparative example 1 is strongest, the systemic toxicity of comparative example 2 is basically not obvious, the body weight increase is mainly caused by tumor growth, the body weight of the mice in the example group is slightly reduced, but is obviously better than that in the comparative example 1, and the systemic toxicity effect is reduced.
Second, tissue distribution experiment
Constructing a nude mouse xenograft tumor model by HepG-2, randomly dividing the nude mouse xenograft tumor model into 4 groups by 18-22g (female parent), 6 mice in each group, respectively injecting tail vein into each group, comparing with 4, comparing with 5 and example 1, removing cervical vertebra at 0.5 and 8 hours, killing the mice (each group kills 3 mice at each time point), taking heart, liver, spleen, lung, kidney and tumor, using HP L C-MS/MS to perform tissue distribution research, inspecting the drug accumulation condition of the tumor part, comparing with other organs (blood, heart, liver, spleen, lung and kidney), and explaining the tumor targeting capability and the drug in vivo release condition of each preparation group.
The chromatographic conditions are that the chromatographic column is AgelaVenusal XBP C18(2.1mm × 30mm, 3.0 μm), the mobile phase is a phase A which is methanol and a water phase (5mmol L)-1Ammonium acetate + 1% acetonitrile, pH 9.8) 90:10, methanol-water phase 30:70, flow rate 0.4m L. min-1The sample introduction amount is 10 mu L, the column temperature is 30 ℃, the mobile phase adopts gradient elution, the ratio of [ 0-0.2 min A to B is 60:40, the ratio of 0.2-1.48 min A to B is increased from 60:40 to 100:0, the ratio of 1.48-1.5 min A to B is decreased to 60:40, the ratio of A to B is maintained to be 60:40 for 1.5-5 min]The total analysis time was 5 min.
Selecting pneumatic auxiliary electrospray ionization (ESI), and adopting mass spectrum scanning mode of multi-reaction monitoring (MRM) under positive ionization mode, wherein nitrogen is used as drying gas and the flow rate is 9L min-1Temperature 350 deg.C, atomization chamber pressure 25 psi.
From the results of the tissue distribution study (fig. 5), it can be seen that the tumor targeting accumulation capacity of the drug of example 1 is the best, and has no significant difference from that of comparative example 4, the tumor targeting of comparative example 5 is the worst, and the tumor targeting capacity of comparative example 3 is between the two.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (10)
1. A hybrid mesenchymal stem cell exosome drug delivery system, characterized in that: the drug delivery system is constructed by hybrid fusion of mesenchymal stem cell exosomes and liposomes.
2. The hybrid mesenchymal stem cell exosome drug delivery system according to claim 1, characterized in that: the mesenchymal stem cell exosome is derived from bone marrow mesenchymal stem cells, skeletal muscle mesenchymal stem cells, periosteum mesenchymal stem cells, trabecular bone mesenchymal stem cells, umbilical cord mesenchymal stem cells or placenta mesenchymal stem cells of animals.
3. The hybrid mesenchymal stem cell exosome drug delivery system according to claim 1, characterized in that: the liposome is a phospholipid bilayer closed vesicle composed of cholesterol components and phospholipid materials;
wherein the cholesterol component is selected from cholesterol or derivatives thereof;
the phospholipid material is selected from one or more of egg yolk lecithin, soybean lecithin, 1-stearoyl-lysophosphatidylcholine, egg yolk phosphatidylglycerol, dipalmitoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, pelotted phosphatidylethanolamine, erucyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine and (2, 3-dioleoyl-propyl) -trimethylamine.
4. The hybrid mesenchymal stem cell exosome drug delivery system according to claim 1, characterized in that: the hybrid mesenchymal stem cell exosome drug delivery system is in a vesicle shape, the particle size distribution of the drug delivery system is that the median diameter is 60-200 nm;
or the multi-drug-loading hybrid mesenchymal stem cell exosome drug delivery system can comprise several fat-soluble drugs, or the fat-soluble drugs are combined with water-soluble drugs, or several water-soluble drugs.
5. A method for preparing a hybrid mesenchymal stem cell exosome drug delivery system according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:
s1, mixing the cholesterol component and the phospholipid material according to the weight ratio of 5-20: 80-95 dissolving in organic solvent, adding fat-soluble antitumor drug, and dissolving cholesterol and phospholipid material in water bath at 35-55 deg.C;
s2, under the condition of water bath, rotating and vacuumizing to remove the organic solvent to form a honeycomb-shaped film on the inner wall of the container, and placing the container in a vacuum drying oven for 8-12 h;
s3, adding the film obtained in the step S2 into a pH buffer solution dissolved with the water-soluble anti-tumor drug, carrying out water bath at 40-60 ℃, and rotating to completely dissolve the film;
s4, carrying out ultrasonic or homogenization, and preparing a drug-loaded liposome with a median diameter of 60-200 nm;
s5, continuously loading the water-soluble drug with strong acid and weak base salt characteristics or strong base and weak acid salt characteristics on the basis of the drug-loaded liposome obtained in the step S4 by utilizing the preparation technology of gradient change of the internal and external pH values of the carrier to prepare the multiple drug-loaded liposome;
s6, culturing the mesenchymal stem cells, collecting the residual liquid culture medium in the culturing process, carrying out centrifugal operation on the liquid culture medium to obtain an exosome precipitate, and preparing an exosome stock solution;
s7, mixing the exosome stock solution obtained in the step S6 with the drug-loaded liposome obtained in the step S5, and repeatedly freezing and thawing for several times to obtain the hybrid mesenchymal stem cell exosome drug delivery system.
6. The method for preparing a hybrid mesenchymal stem cell exosome drug delivery system according to claim 5, characterized in that: the preparation technology of the gradient change of the pH inside and outside the carrier in the step S5 is as follows: aiming at strong acid and weak base salt, the pH value of the inner aqueous phase of the liposome needs to be adjusted to 2.5-4.0, and the pH value of the outer aqueous phase needs to be adjusted to 6.0-8.5;
for strong alkali and weak acid salt, the pH value of the liposome internal water phase is required to be adjusted to 8.5-10.5, and the pH value of the external water phase is required to be adjusted to 5.5-8.0.
7. The method for preparing a hybrid mesenchymal stem cell exosome drug delivery system according to claim 5, characterized in that: the step S6 specifically includes:
taking mesenchymal stem cells into a cell culture bottle, adding a DMEM/F12 culture medium containing 10-20% fetal calf serum by mass concentration, culturing in a constant-temperature culture box, removing non-adherent cells, carrying out primary culture for 5-15 days, carrying out primary passage, carrying out secondary passage culture when the growth of adherent mesenchymal stem cells reaches 70-90%, taking mesenchymal stem cells of 3-6 generations, collecting cell supernatant, subpackaging in a centrifuge tube, centrifuging for 1-30min at 200-.
8. Preparation method of hybrid mesenchymal stem cell exosome drug delivery system according to any one of claims 5 to 7, characterized in that: the step S7 specifically includes:
mixing the exosome stock solution and multiple drug-loaded liposome with the volume 1-10 times of the volume of the exosome stock solution at room temperature, stirring and incubating for 10-30min at 30-40 ℃, placing the mixture in liquid nitrogen for freezing for 5-10min, unfreezing at room temperature, stirring and incubating for 15-30min at 30-40 ℃, then placing the mixture in liquid nitrogen for freezing for 5-10min, and performing multiple freeze-thaw cycles to fully fuse and hybridize the exosome membrane and the liposome membrane to prepare the multiple drug-loaded hybrid mesenchymal stem cell exosome drug delivery system.
9. Use of a hybrid mesenchymal stem cell exosome drug delivery system according to any one of claims 1 to 4 in the preparation of a medicament.
10. The use of claim 9, wherein: the application is as follows: the drug delivery system can entrap and deliver antitumor drugs which are two or more of drugs inhibiting the activity of enzymes in tumor cells, drugs inhibiting the formation of tubulin, drugs inhibiting the formation of DNA or RNA, drugs causing hypoxia of tumor tissues, and drugs causing the generation of oxygen radicals in tumor tissues.
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