CN110152015A - Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes - Google Patents
Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes Download PDFInfo
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Abstract
The present invention relates to human pluripotent stem cells excretion body of load anti-tumor drug and preparation method thereof and purposes.The present invention is using human pluripotent stem cells excretion body as the nano carrier of anti-tumor drug, and it is manually modified by carrying out RGD peptide to excretion body surface face, make it with more tumor-targeting, so that building is easy to by blood-brain barrier and with the natural drug delivery system of tumor-targeting.The present invention is provided simultaneously with the antitumor function of special efficacy that is antitumor, adjusting the functions such as immune, anti-inflammatory and anti-tumor drug of multipotential stem cell excretion body；The dual function of multipotential stem cell excretion body and anti-tumor drug can preferably play anti-tumor effect, while greatly reduce the dosage of anti-tumor drug, thus greatly reduce the toxic side effect of anti-tumor drug；There can be unique advantage by blood-brain barrier, the treatment of Central nervous system tumor using its nanometer of level characteristics；It carries medicine human pluripotent stem cells excretion body to modify through RGD peptide, substantially increases its tumor-targeting.
Description
Technical field
The invention belongs to cell biology, molecular biology and medicament research and development technical fields, more particularly, to a kind of load
Human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes.
Background technique
Excretion body is the extracellular vesica of a kind of diameter about 30-150nm secreted by cell, carries donor cell sources
Various bioactivators include the entrance effects cell such as DNA, RNA, albumen, Effective Regulation body cell signal transduction.It grinds
The excretion body for showing stem cell secretion is studied carefully by the bioactive substance to effector cell's transmitting source of human stem cell, and physiological is repaired
Cell that is multiple or removing body injury, lesion and aging plays the functions such as anti-inflammatory, adjusting is immune.In recent years studies have reported that, lead to
It crosses into the culture medium of liver cancer cells, lymphoblastoid cells and glioma cell and adds a certain number of liver stem cells sources
Microvesicles (extracellular vesica), the proliferative capacity of three kinds of oncocytes obviously weakens, and apoptotic cell quantity dramatically increases.
Also studies have reported that, the exosome (excretion body) of mesenchymal stem cell secretion is able to suppress U87 glioma cell, 4T1 cream
The proliferation of adenocarcinoma cell and multiple myeloma cells.Umbilical cord mesenchymal stem cells secretion excretion body be able to suppress glioma and
The growth of kidney.Although stem cell excretion body has shown certain effect in anti-tumor aspect, the change with direct killing tumour
It treats drug to compare, act on or relatively limited.
Excretion body is the natural nano carrier of cell secretion, and excretion body has obtained widely as the nano-carrier of drug
Concern and research.Compared with artificial synthesized nano-carrier, excretion body has unique advantage in the application of field of medicine release.
The constituent of excretion body derives from cell, avoids the use of the materials such as artificial synthesized liposome, macromolecule, nano-silicon
The problems such as bring cytotoxicity, biocompatibility.Since excretion body inherits the substances such as phosphatide, the surface protein of cell, tool
Have physiological function abundant, the transmission efficiency of drug can be improved, realize the specific delivery of drug, across biological barrier transmitting with
And the functions such as Immune privilege.The substances such as the self-contained albumen of excretion body, gene have cell ability of regulation and control, can be to specific
Disease plays therapeutic effect.
Currently, the Nano medication delivery system of various kinds of cell excretion body building has been used to the exploration of disease treatment.Wood
Seminar utilizes the excretion body in the source immature Dendritic Cells (immature dendritic cells, iDC) negative at first
It carries siRNA and treats alzheimer's disease.The excretion body surface face of iDC secretion lacks t cell activation albumen (such as MHC-I, MHC-
II, CD86), there is immunologic inertia.The method that they are transfected by gene, modifies acetyl on the albumen Lamp2b of excretion body surface face
Choline receptor Recognition polypeptide segment RVG, and then assign the ability of iDC excretion body positioning brain neuroblastoma cell；Then, turned using electricity
Method, the siRNA of BACE-1 is imported into iDC excretion body, by mouse model, demonstrate these carry medicine excretion body
The potentiality for treating alzheimer's disease.However, iDC cell extraction process is cumbersome, cell quantity is limited, with high costs, it is difficult to produce
Industry metaplasia produces.Equally, the excretion body in tumour cell source is also used for the building of drug delivery system.Although tumour cell can be with
Unlimited amplification prepares a large amount of excretion body, but their potential security risks limit it in clinical use.Recently, it grinds
Study carefully personnel and reports the Nano medication delivery system constructed using milk origin excretion body.Compared with the excretion body of cell origin,
The excretion body extraction of milk origin is easy, at low cost, and realization can meet clinical demand with large scale preparation.But due to milk
The substances such as gene and protein containing a large amount of non-source of people in excretion body, are still needed by the safety that intravenous systemic is injected
It further confirms that, and the emphasis of the research focuses primarily upon the pharmaceutical carrier function of excretion body, does not give full play to excretion
Treatment potentiality of the substance entrained by body itself to disease and damage.Therefore, not only it had been convenient for industrialization production but also had carried cell certainly
The excretion body of body function is only the pharmaceutical carrier with Transformation Application prospect.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of load antineoplastics
Human pluripotent stem cells excretion body of object and preparation method thereof and purposes.
The present invention is using human pluripotent stem cells excretion body as the nano carrier of anti-tumor drug, and by excretion body
Surface progress RGD peptide is manually modified, makes it with more tumor-targeting, so that building is easy to pass through blood-brain barrier and has tumour
The natural drug delivery system of targeting.
The purpose of the present invention can be achieved through the following technical solutions:
First aspect present invention:
The human pluripotent stem cells excretion body of load anti-tumor drug is provided, including human pluripotent stem cells excretion body and is wrapped in
The intracorporal drug of human pluripotent stem cells excretion.
In an embodiment of the invention, the human pluripotent stem cells excretion body is the outer of people's derived from embryonic stem cells
It secretes body or people induces multi-potent stem cell the excretion body in source.
In an embodiment of the invention, the human pluripotent stem cells excretion body is prepared by the following:
Culture medium is collected in serum free culture system culture people ESCs or iPSCs using no feeder layer cultural method, is received
Collect the excretion body in pure medium, as human pluripotent stem cells excretion body.
In an embodiment of the invention, purifying culture is collected using rotation combining ultrafiltration low temperature Ultracentrifugation Method
Excretion body in base.
Rotation combining ultrafiltration low temperature Ultracentrifugation Method has main steps that: by the culture of a constant volume be based on 4 DEG C of 400g from
Heart 10min removes free cell, and obtained supernatant moves into another pipe, 4 DEG C of 15000g 20min, removes cell fragment, obtains
Supernatant pour into millipore ultrafiltration apparatus, with PBS elute collect filter membrane (100KD) on concentrated liquid, i.e., again plus
Enter PBS, through the ultrafiltration again of millipore ultrafiltration apparatus, obtained concentrate is transferred to 30% sucrose/heavy water density pad
(1.210g/cm3), 4 DEG C of 100800g are centrifuged 210 minutes, collect bottom sucrose/heavy water density pad, and two volumes PBS is added, and are turned
It moves in the ultrafiltration carefulness pipe that can retain 100KD molecule, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, finally presses subsequent experimental
It is required that being settled to certain volume, Exosomes suspension is obtained, packing is saved to -80 DEG C.
In an embodiment of the invention, serum free culture system select commercially available model TeSRTM-E8TM or
The culture medium of mTeSR1 or mTeSR2.
In an embodiment of the invention, after collecting excretion body, pass through the side such as Electronic Speculum, granularmetric analysis, immunoblotting
Method identifies the characteristic of excretion body, as a result meets excretion body characteristics: diameter range 50-150nm, under transmission electron microscope
In duplicature cystic structures, CD9, the markers such as CD63 are contained in membrane structure surface.
In an embodiment of the invention, the package concentration of the anti-tumor drug is 0.1-1000 μ g/mL, preferably
For 20-200 μ g/mL.
Second aspect of the present invention:
The preparation method of the human pluripotent stem cells excretion body of the load anti-tumor drug is provided, by being incubated for, electricity turn, is squeezed
One of pressure, ultrasound, freeze thawing or method of saponin processing wrap up the anti-tumor drug of doses into human pluripotent stem cells excretion
Body.
In an embodiment of the invention, the processing technique of incubation mainly carries out by the following method: by a certain concentration
Excretion liquid suspension and drug solution mixing, be placed under certain temperature and be incubated for certain time.Above-mentioned solution is then placed in one
Determine in the super filter tube of molecular cut off, three times using biocompatible media ultrafiltration washing, obtains the excretion body for carrying medicine.This method
In, the concentration range of excretion body suspension is 1 × 106/mL–1×1012/ mL, preferably 1 × 1010/mL；The range of incubation temperature is
4 DEG C -50 DEG C, preferably 37 DEG C；Incubation time range is 1h -48h, preferably 4h；The molecular cut off range of super filter tube is
10KDa -5000KDa, preferably 100KDa；Biocompatible media is physiological saline, physiological buffer, cell culture medium etc..
In an embodiment of the invention, the processing technique of freeze thawing mainly carries out by the following method: by a certain concentration
Excretion liquid suspension and drug solution mixing, be placed in the refrigerator of certain temperature and freezed.It then thaws, then freezes, such as
This recycles certain number.Finally, above-mentioned solution is placed in the super filter tube of certain molecular cut off, it is situated between using biocompatibility
The excretion body for obtaining carrying medicine three times is washed in matter ultrafiltration.In this method, the concentration range of excretion body suspension is 1 × 106/mL–1×
1012/ mL, preferably 1 × 1010/mL；Cryogenic temperature range is -10 DEG C -- 200 DEG C, preferably -80 DEG C；Cycle-index is 1-
20 times, preferably 3 times；The molecular cut off range of super filter tube is 10KDa -5000KDa, preferably 100KDa；Biocompatibility
Medium is physiological saline, physiological buffer, cell culture medium etc..
In an embodiment of the invention, the processing technique that electricity turns mainly carries out by the following method: by a certain concentration
Excretion liquid suspension turn liquid with drug solution and electricity and mix, be placed in electroporation, electricity turns certain time under certain condition.With
Afterwards, above-mentioned solution is placed in the super filter tube of certain molecular cut off, three times using biocompatible media ultrafiltration washing, is obtained
To the excretion body for carrying medicine.In this method, the concentration range of excretion body suspension is 1 × 106/mL–1×1012/ mL, preferably 1 ×
1010/mL；It is KCl, K3PO4 and OptiPrep that electricity, which turns liquid,TMThe mixed solution of cell gradient centrifugate, wherein the concentration model of KCl
It encloses for 1mM -1M, preferably 25mM, the concentration range of K3PO4 is 0.01mM-1M, preferably 1.15mM, OptiPrepTMCell ladder
Spend centrifugate volume fraction range be 80%-0.1%, preferably 21%；The range that electricity turns voltage in parameter is 10KV-
0.1V, preferably 400V, the range of capacitor are 0.1-1000F, preferably 150F；It is 0.1s -1min that electricity, which turns time range, preferably
For 10s；The molecular cut off range of super filter tube is 10KDa -5000KDa, preferably 100KDa；Biocompatible media is physiology
Salt water, physiological buffer, cell culture medium etc..
In an embodiment of the invention, ultrasonic processing technique mainly carries out by the following method: by a certain concentration
Excretion liquid suspension mixed with drug solution, it is certain to above-mentioned mixed solution ultrasound under certain condition using pin type Ultrasound Instrument
Time.Then, above-mentioned solution is placed in the super filter tube of certain molecular cut off, is washed using biocompatible media ultrafiltration
Three times, obtain carrying the excretion body of medicine.In this method, the concentration range of excretion body suspension is 1 × 106/mL–1×1012/ mL, preferably
It is 1 × 1010/mL；In ultrasound condition, the range of voltage is 1KV -1V, and preferably 500V, the range of frequency is 1Hz -20KHz, excellent
It is selected as 2KHz, the range of cycle-index is 1-100 time, and preferably 6 times, the ultrasound output time is 1s-60s in circulation, preferably
5s, the range of interval time are 1s -300s, preferably 5s；The molecular cut off range of super filter tube is 10KDa -5000KDa, excellent
It is selected as 100KDa；Biocompatible media is physiological saline, physiological buffer, cell culture medium etc..
In an embodiment of the invention, it will be wrapped by means such as conventional ultrafiltration, ultracentrifugation or desalting columns
The excretion body for the anti-tumor drug wrapped up in is separated with free anti-tumor drug.
In an embodiment of the invention, pass through high performance liquid chromatography (High Performance Liquid
Chromatography, HPLC) efficiency of human pluripotent stem cells excretion body carrying medicament is tested and analyzed, finally obtain people
The Nano medication delivery system of multipotential stem cell excretion body load anti-tumor drug.
Third aspect present invention:
On the basis of first aspect present invention and second aspect, RGD peptide is carried out to human pluripotent stem cells excretion body surface face
It is manually modified, make it with more tumor-targeting, obtains the human pluripotent stem cells excretion body of targeting sex modification.
In an embodiment of the invention, the RGD peptide includes cRGD and iRGD, wherein cRGD is valine-
Arginine-glycine-aspartic acid-glutamic acid cyclic peptide, iRGD are ring (cysteine-arginine-glycine-aspartic acid-
Lysine-glycine-Pro-Asp-cysteine).
In an embodiment of the invention, it is main by method that functional motor ability plastid merges to stem cell excretion body
Surface is modified.The specific method is as follows:
Pass through method reported in the literature (Yifei Zhang, et, al., European Journal of first
Pharmaceutics and Biopharmaceutics, 2010,467-473) synthesized phosphatide-polyethylene glycol-iRGD or
Liposome-polyethylene glycol-cRGD functional motor ability plastid molecule.Then, by these modification functional motor ability plastid molecule according to
Certain concentration, is dissolved in biocompatible media, forms nano-micelle.Then, by these micellas and stem cell excretion body
Suspension is sufficiently mixed according to a certain percentage, and is incubated for a period of time at a certain temperature.Finally, mixing liquid is down to
Room temperature purifies stem cell excretion body by size exclusion chromatograph, obtains the stem cell excretion body of targeting sex modification, can refer to Fig. 1.
In an embodiment of the invention, phosphatide is selected from phosphatide glyceride, sphingomyelins, glycolipid, two palmityl phosphatide
The natural and artificial synthesized liposome such as phatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine
Molecule；The molecular weight ranges of polyethylene glycol are as follows: 0.5KDa -100KDa, preferably 0.2KDa；The concentration range of liposome is
0.01mg/mL -100mg/mL, preferably 1mg/mL；Biocompatible solvent includes physiological saline, physiological buffer, water, cell
Culture medium etc.；The quantitative proportion range that micella is mixed with stem cell excretion body is 100:1-1:100, preferably 1:1；Incubation temperature
Range is 4 DEG C -60 DEG C, preferably 40 DEG C；The time range of incubation is 1min -48h, preferably 2h.
Fourth aspect present invention:
On the basis of third aspect present invention, using the human pluripotent stem cells excretion body of targeting sex modification, load anti-swollen
Tumor medicine obtains load anti-tumor drug and targets the human pluripotent stem cells excretion body of sex modification.
The method for targeting the human pluripotent stem cells excretion body load anti-tumor drug of sex modification is referred to without targeting sex modification
Human pluripotent stem cells excretion body load anti-tumor drug method.
By through targeting modification loaded anti-tumor drug human pluripotent stem cells excretion body treat tumour can significantly reduce
The dosage of anti-tumor drug thus greatly reduces the toxic side effect of anti-tumor drug.
Fifth aspect present invention:
On the basis of the above summary of the invention, provide a batch specific anti-tumor drug, with antitumor for obtaining loading
The human pluripotent stem cells excretion body of drug, or load anti-tumor drug and the human pluripotent stem cells excretion body for targeting sex modification.
The anti-tumor drug that human pluripotent stem cells excretion body is loaded, includes the following categories:
(1) alkylating agents: common drug have Chlorambucil, cyclophosphamide, Dacarbazine, ifosfamide, melphalan,
It is mustargen, nitrosoureas (such as lomustine), procarbazine, procarbazine, streptozotocin, Temozolomide, thiotepa, suitable
Platinum, carboplatin, oxaliplatin etc.；
(2) antimetabolite class: common drug have azacitidine, carat benefit guest, cytarabine, Decitabine, fludarabine,
Fluorouracil, formyl tetrahydrofolic acid, capecitabine, gemcitabine, hydroxycarbamide, mercaptopurine, methotrexate (MTX), methyl-GAG, training U.S. are bent
Plug, Pentostatin, Raltitrexed, 6-thioguanine, Trimetrexate (Trimetrexate), uracil/Tegafur etc.；
(3) antitumor antibiotics class: common drug has actinomycin D, bleomycin, daunorubicin, Doxorubicin (Ah mould
Element, Hydroxydaunomycin), liposomal doxorubicin, epirubicin, idarubicin, plicamycin, mitomycin, mitoxantrone etc.；
(4) influence the drug of mitotic spindle: common drug has taxol, protein binding type taxol (taxol
Nanometer formulation), Docetaxel, Estramustine, Ipsapirone (Ixempra epothilones breast cancer new drug), vincaleukoblastinum, length
Spring new alkali, eldisine, vinorelbine etc.；
(5) topoisomerase enzyme inhibitor class: common drug has Etoposide, Irinotecan, Teniposide, Hycamtin
Deng；
(6) family tyrosine kinase inhibitor class: common drug has CYP3A4 inhibitor and inducer, Imatinib (lattice
Column defend), Dasatinib (Sprycel), Erlotinib (Erlotinib), Gefitinib (Iressa), Lapatinib (Tykerb), Buddhist nun
Sieve replaces Buddhist nun (nilotinib), Sorafenib (Nexavar), Sunitinib malate (sotan) etc.；
(7), other medicament classes: common drug has anagrelide, L-Asparaginasum, bortezomib, denileukin (Bai Jie
Plain 3 fusion toxins), hemel, interferon-' alpha ', interleukins, lenalidomide (thalidomide derivatives), retinoic acid receptors
Inhibitor (Tretinoin), suramin, temsirolimus (temsirolimu injection), Thalidomide etc..
Sixth aspect present invention:
The human pluripotent stem cells excretion body for loading anti-tumor drug or load anti-tumor drug are provided and targeting is repaired
One or more of the application, including following purposes of the human pluripotent stem cells excretion body of decorations:
(1) medicinal application as treatment central nerve neuroma:
In an embodiment of the invention, the human pluripotent stem cells excretion body of application load taxol treats brain glue
Matter tumor.Nude mouse lotus knurl model is established with the glioma cell subcutaneous injection of in vitro culture, through tumor internal injection doses
The human pluripotent stem cells excretion body of taxol or the taxol of human pluripotent stem cells excretion body or various dose have been loaded, ratio is observed
Compared with human pluripotent stem cells excretion body and loaded the human pluripotent stem cells excretion body of taxol to the therapeutic effect of glioma and
The dose-effect relationship of taxol.
In an embodiment of the invention, it has been loaded outside the human pluripotent stem cells of taxol using through RGD peptide modification
Secrete body treatment glioma.Nude mouse lotus knurl model is established with the glioma cell subcutaneous injection of in vitro culture, is infused through tail vein
Penetrate doses through RGD peptide modification loaded taxol human pluripotent stem cells excretion body or human pluripotent stem cells excretion body or
The physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of taxol to colloid through RGD peptide modification
Tumor has significant therapeutic effect.
In an embodiment of the invention, dry using the people's multipotency for having loaded Temozolomide (TMZ) through RGD peptide modification
Cell excretion body treats glioma.Nude mouse lotus knurl model is established with the glioma cell subcutaneous injection of in vitro culture, through tail
The human pluripotent stem cells excretion body or human pluripotent stem cells excretion that TMZ has been loaded through RGD peptide modification of intravenous injection doses
The physiological saline of body or same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of TMZ to colloid through RGD peptide modification
Tumor has significant therapeutic effect.
In an embodiment of the invention, using the human pluripotent stem cells for having loaded vincristine through RGD peptide modification
Excretion body treats medulloblastoma.Nude mouse lotus knurl model is established with the medulloblastoma cell subcutaneous injection of in vitro culture, is passed through
Tail vein injection doses through RGD peptide modification loaded vincristine human pluripotent stem cells excretion body or people's multipotency it is dry thin
The extracellular physiological saline for secreting body or same dose, as a result, it has been found that having loaded the human pluripotent stem cells of vincristine through RGD peptide modification
Excretion body has significant therapeutic effect to medulloblastoma.
(2) medicinal application as treatment lung cancer:
In an embodiment of the invention, using the human pluripotent stem cells excretion for having loaded cis-platinum through RGD peptide modification
Body treats lung cancer.Nude mouse lotus knurl model is established with the lung carcinoma cell subcutaneous injection of in vitro culture, through the certain agent of tail vein injection
The human pluripotent stem cells excretion body or human pluripotent stem cells excretion body or same dose that cis-platinum has been loaded through RGD peptide modification of amount
Physiological saline, as a result, it has been found that the human pluripotent stem cells excretion body for having loaded cis-platinum through RGD peptide modification has significant treatment effect to lung cancer
Fruit.
In an embodiment of the invention, using the human pluripotent stem cells for having loaded Irinotecan through RGD peptide modification
Excretion body treats lung cancer.Nude mouse lotus knurl model is established with the lung carcinoma cell subcutaneous injection of in vitro culture, through tail vein injection one
That determines dosage modifies the human pluripotent stem cells excretion body for having loaded Irinotecan or human pluripotent stem cells excretion body or phase through RGD peptide
With the physiological saline of dosage, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of Irinotecan to lung cancer through RGD peptide modification
There is significant therapeutic effect.
(3) medicinal application as treatment gastroenteric tumor:
In an embodiment of the invention, more using the people for having loaded 5 FU 5 fluorouracil (5-FU) through RGD peptide modification
It can dry cell excretion body treatment gastric cancer.Nude mouse lotus knurl model is established with the stomach cancer cell subcutaneous injection of in vitro culture, it is quiet through tail
The human pluripotent stem cells excretion body or human pluripotent stem cells excretion body that 5-FU has been loaded through RGD peptide modification of arteries and veins injection doses
Or the physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of 5-FU to gastric cancer through RGD peptide modification
There is significant therapeutic effect.
In an embodiment of the invention, more using the people for having loaded 5 FU 5 fluorouracil (5-FU) through RGD peptide modification
It can the dry cell excretion body treatment cancer of the esophagus.Nude mouse lotus knurl model is established with the esophageal cancer cell subcutaneous injection of in vitro culture, is passed through
Tail vein injection doses have loaded outside the human pluripotent stem cells excretion body or human pluripotent stem cells of 5-FU through RGD peptide modification
The physiological saline of body or same dose is secreted, as a result, it has been found that having loaded the human pluripotent stem cells excretion body pair of 5-FU through RGD peptide modification
The cancer of the esophagus has significant therapeutic effect.
In an embodiment of the invention, using the human pluripotent stem cells for having loaded capecitabine through RGD peptide modification
Excretion body treats colon cancer.Nude mouse lotus knurl model is established with the colon cancer cell subcutaneous injection of in vitro culture, is infused through tail vein
Penetrate the human pluripotent stem cells excretion body or human pluripotent stem cells excretion body that capecitabine has been loaded through RGD peptide modification of doses
Or the physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body pair of capecitabine through RGD peptide modification
Colon cancer has significant therapeutic effect.
In an embodiment of the invention, using the human pluripotent stem cells for having loaded gemcitabine through RGD peptide modification
Excretion body treats cancer of pancreas.Nude mouse lotus knurl model is established with the pancreatic cancer cell subcutaneous injection of in vitro culture, is infused through tail vein
Penetrate the human pluripotent stem cells excretion body or human pluripotent stem cells excretion body that gemcitabine has been loaded through RGD peptide modification of doses
Or the physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body pair of gemcitabine through RGD peptide modification
Cancer of pancreas has significant therapeutic effect.
In an embodiment of the invention, using the human pluripotent stem cells for having loaded Sorafenib through RGD peptide modification
Excretion body treats liver cancer.Nude mouse lotus knurl model is established with the liver cancer cells subcutaneous injection of in vitro culture, through tail vein injection one
That determines dosage modifies the human pluripotent stem cells excretion body for having loaded Sorafenib or human pluripotent stem cells excretion body or phase through RGD peptide
With the physiological saline of dosage, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of Sorafenib to liver cancer through RGD peptide modification
There is significant therapeutic effect.
(4) medicinal application as treatment breast cancer:
In an embodiment of the invention, using the human pluripotent stem cells for having loaded epirubicin through RGD peptide modification
Excretion body treats breast cancer.Nude mouse lotus knurl model is established with the breast cancer cell subcutaneous injection of in vitro culture, is infused through tail vein
Penetrate the human pluripotent stem cells excretion body or human pluripotent stem cells excretion body that epirubicin has been loaded through RGD peptide modification of doses
Or the physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body pair of epirubicin through RGD peptide modification
Breast cancer has significant therapeutic effect.
(5) medicinal application as treatment gynecological tumor:
In an embodiment of the invention, it has been loaded outside the human pluripotent stem cells of taxol using through RGD peptide modification
Secrete body treatment oophoroma.Nude mouse lotus knurl model is established with the ovarian cancer cell subcutaneous injection of in vitro culture, through tail vein injection
Doses modify the human pluripotent stem cells excretion body for having loaded taxol or human pluripotent stem cells excretion body or phase through RGD peptide
With the physiological saline of dosage, as a result, it has been found that having loaded the human pluripotent stem cells excretion body of taxol to oophoroma through RGD peptide modification
There is significant therapeutic effect.
(6) medicinal application as treatment Patients with Urinary System Tumors:
In an embodiment of the invention, using the human pluripotent stem cells for having loaded docetaxel through RGD peptide modification
Excretion body treats prostate cancer.Nude mouse lotus knurl model is established with the prostate gland cancer cell subcutaneous injection of in vitro culture, it is quiet through tail
Arteries and veins injection doses have loaded outside the human pluripotent stem cells excretion body or human pluripotent stem cells of docetaxel through RGD peptide modification
The physiological saline of body or same dose is secreted, as a result, it has been found that having loaded the human pluripotent stem cells excretion of docetaxel through RGD peptide modification
Body has significant therapeutic effect to prostate cancer.
In an embodiment of the invention, using the human pluripotent stem cells excretion for having loaded sotan through RGD peptide modification
Body treats kidney.Nude mouse lotus knurl model is established with the kidney cancer cell subcutaneous injection of in vitro culture, it is certain through tail vein injection
Dosage modifies the human pluripotent stem cells excretion body for having loaded sotan or human pluripotent stem cells excretion body or same dose through RGD peptide
Physiological saline, as a result, it has been found that through RGD peptide modification loaded sotan human pluripotent stem cells excretion body have significant treatment to kidney
Effect.
(7) medicinal application as treatment skin neoplasin:
In an embodiment of the invention, more using the people for having loaded interleukins (IL-2) through RGD peptide modification
It can dry cell excretion body treatment melanoma.Nude mouse lotus knurl mould is established with the melanoma cells s injection of in vitro culture
Type, through tail vein injection doses through RGD peptide modification loaded IL-2 human pluripotent stem cells excretion body or people's multipotency it is dry
The physiological saline of cell excretion body or same dose, as a result, it has been found that having been loaded outside the human pluripotent stem cells of IL-2 through RGD peptide modification
Secreting body has significant therapeutic effect to melanoma.
(8) medicinal application as treatment sarcoma:
In an embodiment of the invention, using the human pluripotent stem cells for having loaded Doxorubicin through RGD peptide modification
Excretion body treats osteosarcoma.Nude mouse lotus knurl model is established with the osteosarcoma cell subcutaneous injection of in vitro culture, is infused through tail vein
Penetrate the human pluripotent stem cells excretion body or human pluripotent stem cells excretion body that Doxorubicin has been loaded through RGD peptide modification of doses
Or the physiological saline of same dose, as a result, it has been found that having loaded the human pluripotent stem cells excretion body pair of Doxorubicin through RGD peptide modification
Osteosarcoma has significant therapeutic effect.
It should be understood that above-mentioned application is described according to the classification of different system tumors, each specific
Under the classification of different system tumors, the embodiment provided is the high kinds of tumor of some disease incidence and usually used
Carrying medicament can any combination as long as a certain anti-tumor drug has therapeutic effect for a certain tumour.
Seventh aspect present invention:
The preparation of the human pluripotent stem cells excretion body based on the load anti-tumor drug is provided, the preparation selection is following
Any one of form:
A, suspending agent: the human pluripotent stem cells excretion body of the load anti-tumor drug is dissolved in solvent, with suspending agent
Form exist；
B, it is sustained the compound of excretion body: by the human pluripotent stem cells excretion body and carrier knot of the load anti-tumor drug
The compound for forming sustained release excretion body is closed, the carrier includes various hydrogels, biomembrane, bioceramic material, nanostructure
Biomaterial；
C, to load the human pluripotent stem cells excretion body of anti-tumor drug as additive: with the load anti-tumor drug
Additive of the human pluripotent stem cells excretion body as functional component.
Eighth aspect present invention:
The preparation of human pluripotent stem cells excretion body based on load anti-tumor drug and targeting sex modification, the preparation are provided
Select any one of following form:
A, the human pluripotent stem cells excretion body of the load anti-tumor drug and targeting sex modification suspending agent: is dissolved in solvent
In, exist in the form of suspending agent；
B, it is sustained the compound of excretion body: by outside the human pluripotent stem cells of the load anti-tumor drug and targeting sex modification
The compound that body forms sustained release excretion body in conjunction with carrier is secreted, the carrier includes various hydrogels, biomembrane, bioceramic material
The biomaterial of material, nanostructure；
C, to load anti-tumor drug and target the human pluripotent stem cells excretion body of sex modification as additive: with described negative
Additive of the human pluripotent stem cells excretion body of carrying anti-tumor drug and targeting sex modification as functional component.
In an embodiment of the invention, the solvent of suspending agent is that physiological saline or phosphate buffer or basis are thin
Born of the same parents' culture medium.The suspending agent can be made by oral, intravenous injection, or directly in forms such as sufferer tissue site injections
With.
Human pluripotent stem cells excretion body of the invention is from embryonic stem cell or induction human pluripotent stem cells.Embryo is dry thin
Born of the same parents (embryonic stem cell, ESCs) and induction human pluripotent stem cells (induced pluripotent stem
Cells, iPSCs) characteristic in vitro culture infinite multiplication, self-renewing and Multidirectional Differentiation.ESCs and iPSCs has multipotency
Property (Pluripotency), i.e. ESCs and iPSCs can be divided into tridermic arbitrary cell, have the energy for developing into Various Tissues
Power, in vitro can be with Immortalization.
Multipotential stem cell can Immortalization convenient for industrialization production enrich excretion body, and shown certain antitumor effect
It answers, thus multipotential stem cell can be used as the outstanding seed cell in drug natural nano grade carrier source.
Glioma is the most common Primary intracranial tumor, and glioma accounts for about all central nerve neuromas
30%, the glioblastoma of moderately-highly malignant accounts for about the half of glioma.Due to being limited by blood-brain barrier, it is some have it is bright
The chemotherapeutics of true curative effect can not play a role in the treatment of central nervous system malignant tumour.Excretion body diameter is in 30-
150nm is the natural nano grade carrier that may span across blood-brain barrier, thus, have in terms of pivot nervous system disease in the treatment
Unique advantage.
Although excretion body can be used as the delivery vector of anti-tumor drug, including the excretion body of source of human stem cell
Most excretion body pharmaceutical carriers do not have the specific enrichment abilities of tumor tissues.Correlative study shows, intravenous injection
Excretion body is mainly enriched in the organs such as liver and spleen, and the excretion body quantity for reaching tumor tissues is extremely limited.Therefore, simple benefit
The carrier for using stem cell excretion body as anti-tumor drug is difficult to realize enrichment and tumor locus of the drug at tumor tissues
The performance of stem cell excretion body function.The present invention is manually modified by carrying out RGD peptide to excretion body surface face, makes it with more tumour
Targeting, iRGD and cRGD can identify the α of tumor endothelial cell surface specific expressionvβ3And αvβ5Integrin receptor, in turn
CendR access is activated, the encytosis of tumour cell is enhanced.By can be effective in pharmaceutical carrier surface modification iRGD and cRGD
Increase carrier to the specific recognition capability of tumour cell, promotes carrier in the enrichment of tumor tissues and to inside tumor cells
Infiltration.Therefore, increase it in tumor group by permeating polypeptide in stem cell excretion body surface modification tumour and having great potential
The specific enrichment ability knitted.
Compared with prior art, the present invention has the following advantages and beneficial effects:
1, be provided simultaneously with multipotential stem cell excretion body it is antitumor, adjust the functions such as immune, anti-inflammatory and anti-tumor drug
The antitumor function of special efficacy；
2, the dual function of multipotential stem cell excretion body and anti-tumor drug can preferably play anti-tumor effect, simultaneously
The dosage for greatly reducing anti-tumor drug thus greatly reduces the toxic side effect of anti-tumor drug；
3, can have unique excellent by blood-brain barrier, the treatment of Central nervous system tumor using its nanometer of level characteristics
Gesture；
4, it carries medicine human pluripotent stem cells excretion body to modify through RGD peptide, substantially increases its tumor-targeting.
Detailed description of the invention
Fig. 1: the acquisition process of the human pluripotent stem cells excretion body of sex modification is targeted.
The particle diameter distribution of Fig. 2: qNano detection ES-exo.
The surface marker of Fig. 3: WB detection ES-exo.
The particle diameter distribution of Fig. 4: qNano detection iPSC-exo.
The surface marker of Fig. 5: WB detection iPS-exo.
Fig. 6: PTX quantitation curves.
Fig. 7: HPLC testing result.
Fig. 8: endocytosis situation of the glioma cell to cRGD-ES-Exos or ES-Exos.
Fig. 9: fluorescent vital imaging.
Figure 10: after different modes are treated 14 days and 28 days, the subcutaneous glioma volume of each group mouse.
Figure 11: after different modes are treated 21 days in embodiment 6, each group mouse subcutaneous transplanting knurl product.
Figure 12: after different modes are treated 21 days in embodiment 7, each group mouse subcutaneous transplanting knurl product.
Figure 13: after different modes are treated 21 days in embodiment 8, each group mouse subcutaneous transplanting knurl product.
Figure 14: after different modes are treated 21 days in embodiment 9, each group mouse subcutaneous transplanting knurl product.
Figure 15: after different modes are treated 21 days in embodiment 10, each group mouse subcutaneous transplanting knurl product.
Figure 16: after different modes are treated 21 days in embodiment 11, each group mouse subcutaneous transplanting knurl product.
Figure 17: after different modes are treated 21 days in embodiment 12, each group mouse subcutaneous transplanting knurl product.
Figure 18: after different modes are treated 21 days in embodiment 13, each group mouse subcutaneous transplanting knurl product.
Figure 19: after different modes are treated 21 days in embodiment 14, each group mouse subcutaneous transplanting knurl product.
Figure 20: after different modes are treated 21 days in embodiment 15, each group mouse subcutaneous transplanting knurl product.
Figure 21: after different modes are treated 21 days in embodiment 16, each group mouse subcutaneous transplanting knurl product.
Figure 22: after different modes are treated 21 days in embodiment 17, each group mouse subcutaneous transplanting knurl product.
Figure 23: after different modes are treated 21 days in embodiment 18, each group mouse subcutaneous transplanting knurl product.
Figure 24: after different modes are treated 21 days in embodiment 19, each group mouse subcutaneous transplanting knurl product.
Figure 25: after different modes are treated 21 days in embodiment 20, each group mouse subcutaneous transplanting knurl product.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
The culture of human embryo stem cell (ESCs) and the extraction and identification of excretion body
In one layer of embryonic stem cell matrigel of culture dish middle berth (ESC-Qualified BD Matrigel, BDSparks, MD, USA), ESCs is moved into the culture dish, mTeSR1 serum free medium is added
(StemCell Vancouver, BC, Canada), (37 DEG C, 5%CO2, the saturated humidity) trainings in incubator
It supports, collects the culture medium replaced daily.Culture medium is centrifuged 30 points by 0.22 micron pore size membrane filtration and 4 DEG C of 10000g
Clock removes cell fragment；Using the super filter tube of 100KD molecular weight, the excretion being centrifuged in (3500g, 15min) retention concentration supernatant
Body obtains excretion body concentrate；Concentrate is transferred to 30% sucrose/heavy water density pad (1.210g/cm3), 4 DEG C of 100000g
Bottom 5ml sucrose/heavy water density pad is collected in centrifugation 210 minutes, PBS dilution is added, 100KD molecular weight can be retained by being transferred to
In ultra-filtration centrifuge tube, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, is finally settled to certain volume by subsequent experimental requirement, is obtained
To excretion body suspension ES-exo, packing is saved to -80 DEG C.
The form of ES-exo is observed by transmission electron microscope (TEM).Load sample copper mesh (aperture 2nm) is fixed on bracket, it will
20 μ L samples are added drop-wise on copper mesh, after being stored at room temperature 3 minutes, are sucked liquid with filter paper in copper mesh side, and 3% phosphotungstic acid is added dropwise
30 μ L of solution carries out negative staining (room temperature, 5 minutes) to sample.Negative staining liquid is sucked with filter paper, copper mesh is transferred to transmission electron microscopy
Under mirror, excretion volume morphing is observed.
The partial size and concentration of ES-exo is examined by nanoparticle analysis system (iZon qNano, New Zealand)
It surveys, according to operational manual conditioning instrumentation parameters: Nanopore (NP100) being installed to below well, is adjusted
45 μ L PBS are added into well by Stretch to 47mm, stablize electric current within the scope of 100-120nA.PBS is sucked, is added
The 45 diluted CPC100 standard items (partial size 114nm) of μ L 1:1000 measure population and concentration, obtain standard song by software
Line.Standard items are sucked, PBS is washed 3 times, and the 45 diluted samples to be tested of μ L1:500 are added, and measures population and concentration, duplicate measurements 3
It is secondary.Data analysis is carried out by software, obtains analysis report.Particle size range is 50-150nm (attached drawing 2) as the result is shown.
The expression of ES-exo specific surfaces marker CD9 and CD63 are detected by western blot.Specific steps: outer
Body total protein extraction is secreted, sample protein concentration is detected by BCA protein assay kit, glue prepares 10% separation gel, electricity
Swimming, transferring film, closing and antibody incubation, chemiluminescence imaging analyzer observe band development situation.As a result extracted excretion body
Equal expression specificity surface marker CD9 and CD63 (attached drawing 3).
Embodiment 2:
People induces the culture of human pluripotent stem cells (iPSCs) and the extraction and identification of excretion body
In one layer of embryonic stem cell matrigel of culture dish middle berth (ESC-Qualified BD Matrigel, BDSparks, MD, USA), iPSC is moved into the culture dish, mTeSR1 serum free medium is added
(StemCell Vancouver, BC, Canada), (37 DEG C, 5%CO2, the saturated humidity) trainings in incubator
It supports, collects the culture medium replaced daily.Culture medium is centrifuged 30 points by 0.22 micron pore size membrane filtration and 4 DEG C of 10000g
Clock removes cell fragment；Using the super filter tube of 100KD molecular weight, the excretion being centrifuged in (3500g, 15min) retention concentration supernatant
Body obtains excretion body concentrate；Concentrate is transferred to 30% sucrose/heavy water density pad (1.210g/cm3), 4 DEG C of 100000g
Bottom 5ml sucrose/heavy water density pad is collected in centrifugation 210 minutes, PBS dilution is added, 100KD molecular weight can be retained by being transferred to
In ultra-filtration centrifuge tube, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, is finally settled to certain body by subsequent experimental requirement with PBS
Product, obtains excretion body suspension iPS-exo, and packing is saved to -80 DEG C.
The form of iPS-exo is observed by transmission electron microscope (TEM).Load sample copper mesh (aperture 2nm) is fixed on bracket, it will
20 μ L samples are added drop-wise on copper mesh, after being stored at room temperature 3 minutes, are sucked liquid with filter paper in copper mesh side, and 3% phosphotungstic acid is added dropwise
30 μ L of solution carries out negative staining (room temperature, 5 minutes) to sample.Negative staining liquid is sucked with filter paper, copper mesh is transferred to transmission electron microscopy
Under mirror, excretion volume morphing is observed.
The partial size and concentration of iPS-exo is examined by nanoparticle analysis system (iZon qNano, New Zealand)
It surveys, according to operational manual conditioning instrumentation parameters: Nanopore (NP100) being installed to below well, is adjusted
45 μ L PBS are added into well by Stretch to 47mm, stablize electric current within the scope of 100-120nA.PBS is sucked, is added
The 45 diluted CPC100 standard items (partial size 114nm) of μ L 1:1000 measure population and concentration, obtain standard song by software
Line.Standard items are sucked, PBS is washed 3 times, and the 45 diluted samples to be tested of μ L1:500 are added, and measures population and concentration, duplicate measurements 3
It is secondary.Data analysis is carried out by software, obtains analysis report (attached drawing 4).
The expression of iPS-exo specific surfaces marker CD9 and CD63 are detected by western blot.Specific steps:
Excretion body total protein extraction detects sample protein concentration by BCA protein assay kit, and glue prepares 10% separation gel,
Electrophoresis, transferring film, closing and antibody incubation, chemiluminescence imaging analyzer observe band development situation.As a result extracted excretion
The equal expression specificity surface marker CD9 and CD63 (attached drawing 5) of body.
Embodiment 3
The source people ESC excretion body (ESC-Exos) by being incubated for method package taxol (PTX) altogether
ESC-Exos solution comes from embodiment 1.The quantitation curves of PTX measure: the PTX powder for accurately weighing 1mg is molten
The solution is then diluted by solution in the methanol of 1mL with methanol, is configured to 50 μ g/mL, 20 μ g/mL, the PTX of 10 μ g/mL
Methanol standard solution, HPLC sample introduction.Test condition is as follows: flow velocity 1mL/min；10 μ L of sample volume；Detection wavelength 227nm；Column
30 DEG C of temperature, splitter: Zorbax Extend-C18Analytical, 4.6 × 150mm 5-micron.
After obtaining corresponding experimental result, the function by the peak area (PA) of chromatographic peak as PTX concentration (C, μ g/mL) is made
Scheme (as shown in Fig. 6), obtains the quantitation curves of Res under the following chromatographic separation condition: PA=26.7388C+2.6119
The package of PTX: preparing the normal saline solution (pH=4.0) of the PTX of 1mg/mL, then uses the sodium hydroxide water of 1N
Solution adjusts pH value of solution to 6 or so.Take 100 μ L (concentration 1.0*10 of ESC-Exos solution12/ mL), the Res of 1mL is added thereto
Solution.After 37 DEG C of incubation 1h, solution is washed twice with the physiological saline ultrafiltration of 5mL, leaves and takes 200 μ L liquid, obtains load PTX's
ESC-Exos solution.Then, the 50 μ L solution are taken, is diluted using the acetonitrile of 200 μ L, is centrifuged, removes under 12000rad/min
Deproteinized precipitating, supernatant carry out HPLC detection under the determination condition of standard curve.Shown in testing result attached drawing 7, PTX's
Package concentration is 25 μ g/mL.
Embodiment 4
The modification of derived from embryonic stem cells excretion body (ESC-Exos) surface cRGD
Distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol 2000-cRGD is synthesized by method reported in the literature
(DSPE-PEG-cRGD) liposome.It accurately weighs DSPE-PEG-cRGD to be dissolved in physiological saline, prepares the solution of 1mg/mL.
Then, by solution under ultrasonic wave 5-10min of ultrasound, promote the formation of micella, using nanoparticle analyzer to micellar solution
It is counted.It takes a certain amount of excretion body suspension and micellar solution to be sufficiently mixed according to the ratio of population 1:1, will mix
Liquid is placed in 40 DEG C of shaking table and is incubated for 2h.Finally, mixed liquor is concentrated into 0.5mL, and separated using size exclusion chromatograph,
Obtain the excretion body (cRGD-ESC-Exos) of DSPE-PEG-cRGD modification.Then, recycle DiR dyestuff to cRGD-ESC-
Exos and ESC-Exos has carried out fluorescent marker.CRGD-ESC-Exos or ESC-Exos are then added to the glue of culture again
In matter oncocyte culture medium.Experimental result shows that glioma cell is apparently higher than ESC- to the endocytosis efficiency of cRGD-ES-Exos
Exos (attached drawing 8).The result confirms that cRGD modification has successfully been arrived the surface ESC-Exos by the present invention, and improves stem cell
Accumulation ability of the source excretion body in tumour cell.
Embodiment 5
The paclitaxel loaded treatment glioma of cRGD-ESC-Exos
Prepare cRGD-ES-Exos referring to the method for example 4, and according to the method for example 4 using cRGD-ESC-Exos or
Person ESC-Exos is paclitaxel loaded, and carries out fluorescent marker to excretion body using DiI dyestuff.Glioma cell U87 is injected into
The hind leg armpit lower part of nude mice constructs animal model.Then, nude mice similar in gross tumor volume is randomly divided into four groups: experimental group
There is the cRGD-ES-Exos suspension of 25 μ g/mL PTX by tail vein injection load；Control group is contained by tail vein injection respectively
There are the ES-Exos suspension of equivalent PTX, the PTX solution of equivalent and PBS.The small time-division of 1,2,4,6 and 8 after first time injects
It is other to there is every mouse of excretion body to carry out fluorescent vital image checking injection.The 14th day and the 28th day difference after administration is intervened
It takes out the subcutaneous transplantation tumor of every group of mouse and carries out volume detection.Experimental result is shown, compared to ESC-Exos, cRGD-ESC-
After Exos is by intravenous injection, can effectively it be enriched in tumor tissues position (attached drawing 9), and significantly more efficient inhibition tumour
Growth (attached drawing 10).The specific enrichment in tumor locus may be implemented in the results show cRGD-ESC-Exos, thus
Improve the therapeutic effect to tumour.
Embodiment 6
CRGD-ESC-Exos loads TMZ and treats glioma
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By glioma
Cell U87 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is divided at random
At four groups: experimental group loads the cRGD-ES-Exos suspension of TMZ by tail vein injection；Control group passes through tail vein injection respectively
Load the ES-Exos suspension of equivalent TMZ, the TMZ solution and PBS of equivalent.After administration is intervened every group is taken out respectively within the 21st day
The subcutaneous transplantation tumor of mouse simultaneously carries out volume detection.Experimental result is shown: compared with each control group, loading the cRGD-ESC- of TMZ
After Exos is by intravenous injection, the growth (attached drawing 11) of tumour can be significantly inhibited.The results show cRGD-ESC-Exos
The specific enrichment in tumor locus may be implemented, to improve the therapeutic effect to tumour.
Embodiment 7
CRGD-ESC-Exos loads vincristine treatment medulloblastoma
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Marrow is female thin
Born of the same parents' oncocyte Daoy is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, by nude mice similar in gross tumor volume with
Machine is divided into four groups: experimental group loads the cRGD-ES-Exos suspension of vincristine by tail vein injection；Control group passes through respectively
Tail vein injection loads the vincristine solution and PBS of the ES-Exos suspension of equivalent vincristine, equivalent.Intervene in administration
It takes out within the 21st day the subcutaneous transplantation tumor of every group of mouse respectively afterwards and carries out volume detection.Experimental result is shown: with each control group phase
Than the growth (attached drawing 12) of tumour can be significantly inhibited after loading the cRGD-ESC-Exos of vincristine by intravenous injection.
The specific enrichment in tumor locus may be implemented in the results show cRGD-ESC-Exos, controls to improve tumour
Therapeutic effect.
Embodiment 8
CRGD-ESC-Exos loads Cisplatin Treating Lung Cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Lung cancer is thin
Born of the same parents A549 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is randomly divided into
Four groups: experimental group loads the cRGD-ES-Exos suspension of cis-platinum by tail vein injection；Control group passes through tail vein injection respectively
Load the ES-Exos suspension of equivalent cis-platinum, the cisplatin solution and PBS of equivalent.It takes out respectively within the 21st day after administration is intervened every
The subcutaneous transplantation tumor of group mouse simultaneously carries out volume detection.Experimental result is shown: compared with each control group, loading the cRGD- of cis-platinum
After ESC-Exos is by intravenous injection, the growth (attached drawing 13) of tumour can be significantly inhibited.The results show cRGD-ESC-
The specific enrichment in tumor locus may be implemented in Exos, to improve the therapeutic effect to tumour.
Embodiment 9cRGD-ESC-Exos loads irinotecan lung cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Lung cancer is thin
Born of the same parents A549 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is randomly divided into
Four groups: experimental group loads the cRGD-ES-Exos suspension of Irinotecan by tail vein injection；Control group passes through tail vein respectively
Injection loads the Irinotecan solution and PBS of the ES-Exos suspension of equivalent Irinotecan, equivalent.The 21st after administration is intervened
It takes out the subcutaneous transplantation tumor of every group of mouse respectively and carries out volume detection.Experimental result is shown: compared with each control group, load
After the cRGD-ESC-Exos of Irinotecan is by intravenous injection, the growth (attached drawing 14) of tumour can be significantly inhibited.The experiment knot
Fruit proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 10cRGD-ESC-Exos loads 5-FU and treats gastric cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Gastric cancer is thin
Born of the same parents SGC-7901 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of 5-FU by tail vein injection；Control group passes through tail vein respectively
Injection loads the 5-FU solution and PBS of the ES-Exos suspension of equivalent 5-FU, equivalent.It is taken respectively within the 21st day after administration is intervened
The subcutaneous transplantation tumor of every group of mouse and carry out volume detection out.Experimental result is shown: compared with each control group, loading 5-FU's
After cRGD-ESC-Exos is by intravenous injection, the growth (attached drawing 15) of tumour can be significantly inhibited.The results show
The specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 11cRGD-ESC-Exos loads 5-FU and treats the cancer of the esophagus
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By the cancer of the esophagus
Cell EC109 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of 5-FU by tail vein injection；Control group passes through tail vein respectively
Injection loads the 5-FU solution and PBS of the ES-Exos suspension of equivalent 5-FU, equivalent.It is taken respectively within the 21st day after administration is intervened
The subcutaneous transplantation tumor of every group of mouse and carry out volume detection out.Experimental result is shown: compared with each control group, loading 5-FU's
After cRGD-ESC-Exos is by intravenous injection, the growth (attached drawing 16) of tumour can be significantly inhibited.The results show
The specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 12cRGD-ESC-Exos loads capecitabine and treats colon cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By colon cancer
Cell SW480 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of capecitabine by tail vein injection；Control group passes through tail respectively
Intravenous injection loads the capecitabine solution and PBS of the ES-Exos suspension of equivalent capecitabine, equivalent.After administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21st day respectively and carries out volume detection.Experimental result is shown: compared with each control group,
After the cRGD-ESC-Exos of load capecitabine is by intravenous injection, the growth (attached drawing 17) of tumour can be significantly inhibited.The reality
Testing result proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, so that the treatment improved to tumour is imitated
Fruit.
Embodiment 13cRGD-ESC-Exos loads gemcitabine and treats cancer of pancreas
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By cancer of pancreas
Cell SW1990 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of gemcitabine by tail vein injection；Control group passes through tail respectively
Intravenous injection loads the Jixitabin solution and PBS of the ES-Exos suspension of equivalent gemcitabine, equivalent.After administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21st day respectively and carries out volume detection.Experimental result is shown: compared with each control group,
After the cRGD-ESC-Exos of load gemcitabine is by intravenous injection, the growth (attached drawing 18) of tumour can be significantly inhibited.The reality
Testing result proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, so that the treatment improved to tumour is imitated
Fruit.
Embodiment 14cRGD-ESC-Exos loads Sorafenib and treats liver cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Liver cancer is thin
Born of the same parents HepG2 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is divided at random
At four groups: experimental group loads the cRGD-ES-Exos suspension of Sorafenib by tail vein injection；It is quiet that control group passes through tail respectively
Arteries and veins injection loads the Sorafenib solution and PBS of the ES-Exos suspension of equivalent Sorafenib, equivalent.The after administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21 days respectively and carries out volume detection.Experimental result is shown: compared with each control group, being born
After the cRGD-ESC-Exos of load Sorafenib is by intravenous injection, the growth (attached drawing 19) of tumour can be significantly inhibited.The experiment
As a result prove that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 15cRGD-ESC-Exos loads epirubicin and treats breast cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By breast cancer
Cell MCF-7 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of epirubicin by tail vein injection；Control group passes through tail respectively
Intravenous injection loads the epirubicin solution and PBS of the ES-Exos suspension of equivalent epirubicin, equivalent.After administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21st day respectively and carries out volume detection.Experimental result is shown: compared with each control group,
After the cRGD-ESC-Exos of load epirubicin is by intravenous injection, the growth (attached drawing 20) of tumour can be significantly inhibited.The reality
Testing result proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, so that the treatment improved to tumour is imitated
Fruit.
Embodiment 16cRGD-ESC-Exos loads PTX and treats oophoroma
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By oophoroma
Cell SK-OV-3 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, by nude mice similar in gross tumor volume with
Machine is divided into four groups: experimental group loads the cRGD-ES-Exos suspension of PTX by tail vein injection；Control group passes through tail vein respectively
Injection loads the PTX solution and PBS of the ES-Exos suspension of equivalent PTX, equivalent.It is taken out respectively within the 21st day after administration is intervened
The subcutaneous transplantation tumor of every group of mouse simultaneously carries out volume detection.Experimental result is shown: compared with each control group, loading the cRGD- of PTX
After ESC-Exos is by intravenous injection, the growth (attached drawing 21) of tumour can be significantly inhibited.The results show cRGD-ESC-
The specific enrichment in tumor locus may be implemented in Exos, to improve the therapeutic effect to tumour.
Embodiment 17cRGD-ESC-Exos loads docetaxel and treats prostate cancer
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By prostate
Cancer cell PC-3 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of docetaxel by tail vein injection；Control group passes through tail respectively
Intravenous injection loads the docetaxel solution and PBS of the ES-Exos suspension of equivalent docetaxel, equivalent.After administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21st day respectively and carries out volume detection.Experimental result is shown: compared with each control group,
After the cRGD-ESC-Exos of load docetaxel is by intravenous injection, the growth (attached drawing 22) of tumour can be significantly inhibited.The reality
Testing result proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, so that the treatment improved to tumour is imitated
Fruit.
Embodiment 18
CRGD-ESC-Exos loads sotan and treats kidney
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.Kidney is thin
Born of the same parents 786-O is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is divided at random
At four groups: experimental group loads the cRGD-ES-Exos suspension of sotan by tail vein injection；Control group is infused by tail vein respectively
Penetrate the ES-Exos suspension of load equivalent sotan, the sotan solution and PBS of equivalent.It is taken out respectively within the 21st day after administration is intervened
The subcutaneous transplantation tumor of every group of mouse simultaneously carries out volume detection.Experimental result is shown: compared with each control group, loading sotan
After cRGD-ESC-Exos is by intravenous injection, the growth (attached drawing 23) of tumour can be significantly inhibited.The results show
The specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 19
CRGD-ESC-Exos loads IL-2 and treats melanoma
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By melanin
Oncocyte B-16 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of IL-2 by tail vein injection；Control group passes through tail vein respectively
Injection loads the IL-2 solution and PBS of the ES-Exos suspension of equivalent IL-2, equivalent.It is taken respectively within the 21st day after administration is intervened
The subcutaneous transplantation tumor of every group of mouse and carry out volume detection out.Experimental result is shown: compared with each control group, loading IL-2's
After cRGD-ESC-Exos is by intravenous injection, the growth (attached drawing 24) of tumour can be significantly inhibited.The results show
The specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, to improve the therapeutic effect to tumour.
Embodiment 20
CRGD-ESC-Exos loads Doxorubicin and treats osteosarcoma
Prepare same the embodiment described above of mode of cRGD-ES-Exos and cRGD-ES-Exos carrying medicament.By osteosarcoma
Cell MG-63 is injected into the hind leg armpit lower part of nude mice, constructs animal model.Then, nude mice similar in gross tumor volume is random
Be divided into four groups: experimental group loads the cRGD-ES-Exos suspension of Doxorubicin by tail vein injection；Control group passes through tail respectively
Intravenous injection loads the Doxorubicin solution and PBS of the ES-Exos suspension of equivalent Doxorubicin, equivalent.After administration is intervened
The subcutaneous transplantation tumor of every group of mouse is taken out within 21st day respectively and carries out volume detection.Experimental result is shown: compared with each control group,
After the cRGD-ESC-Exos of load Doxorubicin is by intravenous injection, the growth (attached drawing 25) of tumour can be significantly inhibited.The reality
Testing result proves that the specific enrichment in tumor locus may be implemented in cRGD-ESC-Exos, so that the treatment improved to tumour is imitated
Fruit.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of human pluripotent stem cells excretion body for loading anti-tumor drug, which is characterized in that including human pluripotent stem cells excretion
Body and it is wrapped in the intracorporal drug of human pluripotent stem cells excretion；
Preferably, the human pluripotent stem cells excretion body is that the excretion body of people's derived from embryonic stem cells or people induce multi-potent stem cell
The excretion body in source.
2. a kind of human pluripotent stem cells excretion body for loading anti-tumor drug according to claim 1, which is characterized in that institute
Human pluripotent stem cells excretion body is stated to be prepared by the following:
Culture medium is collected in serum free culture system culture people ESCs or iPSCs using no feeder layer cultural method, is collected pure
Change the excretion body in culture medium, as human pluripotent stem cells excretion body.
3. the preparation method of the human pluripotent stem cells excretion body of load anti-tumor drug as described in claim 1, which is characterized in that
By being incubated for, electricity turn, squeeze, ultrasound, one of the method for freeze thawing or saponin processing by the anti-tumor drug of doses wrap up into
Human pluripotent stem cells excretion body.
4. a kind of human pluripotent stem cells excretion body for targeting sex modification, which is characterized in that human pluripotent stem cells excretion body surface face
Progress RGD peptide is manually modified, obtains the human pluripotent stem cells excretion body of targeting sex modification, the RGD peptide includes cRGD and iRGD；
Preferably, stem cell excretion body surface face is modified by the method that functional motor ability plastid merges,
It is further preferred that phosphatide-polyethylene glycol-iRGD or liposome-polyethylene glycol-cRGD are dissolved in biocompatibility
In medium, nano-micelle is formed, these micellas are mixed with human pluripotent stem cells excretion body suspension, and is incubated for, then by mixed liquor
Body is down to room temperature, purifies stem cell excretion body by size exclusion chromatograph, obtains the stem cell excretion body of targeting sex modification.
5. a kind of load anti-tumor drug and the human pluripotent stem cells excretion body for targeting sex modification, utilize target described in claim 3
To the human pluripotent stem cells excretion body of sex modification, anti-tumor drug is loaded, obtain load anti-tumor drug and targets sex modification
Human pluripotent stem cells excretion body.
6. according to claim 1, human pluripotent stem cells excretion body described in 4 or 5, which is characterized in that human pluripotent stem cells excretion
The anti-tumor drug that body is loaded, includes the following categories:
(1) alkylating agents: Chlorambucil, cyclophosphamide, Dacarbazine, ifosfamide, melphalan, mustargen, nitroso ureas
Class, procarbazine, procarbazine, streptozotocin, Temozolomide, thiotepa, cis-platinum, carboplatin, oxaliplatin；
(2) antimetabolite class: azacitidine, carat benefit guest, cytarabine, Decitabine, fludarabine, fluorouracil, formyl
Tetrahydrofolic acid, capecitabine, gemcitabine, hydroxycarbamide, mercaptopurine, methotrexate (MTX), methyl-GAG, pemetrexed, Pentostatin, thunder
For Qu Sai, 6-thioguanine, Trimetrexate, uracil/Tegafur；
(3) antitumor antibiotics class: actinomycin D, bleomycin, daunorubicin, Doxorubicin, liposomal doxorubicin, table are soft
Than star, idarubicin, plicamycin, mitomycin, mitoxantrone；
(4) influence mitotic spindle drug: taxol, protein binding type taxol, Docetaxel, Estramustine,
Ipsapirone, vincaleukoblastinum, vincristine, eldisine, vinorelbine；
(5) topoisomerase enzyme inhibitor class: Etoposide, Irinotecan, Teniposide, Hycamtin；
(6) family tyrosine kinase inhibitor class: CYP3A4 inhibitor and inducer, Imatinib, Dasatinib, angstrom sieve replace
Buddhist nun, Gefitinib, Lapatinib, nilotinib, Sorafenib, Sunitinib malate；
(7), other medicament classes: anagrelide, L-Asparaginasum, bortezomib, denileukin, hemel, interferon-' alpha ',
Interleukins, lenalidomide, retinoic acid receptors inhibitor, suramin, temsirolimus, Thalidomide.
7. according to claim 1, human pluripotent stem cells excretion body described in 4 or 5, which is characterized in that the anti-tumor drug
Package concentration is 0.1-1000 μ g/mL.
8. the application of the human pluripotent stem cells excretion body as described in claim 1,4 or 5, which is characterized in that including following purposes
One or more of:
(1) application as the drug of preparation treatment central nerve neuroma；
(2) application as the drug of preparation treatment lung cancer；
(3) application as the drug of preparation treatment gastroenteric tumor；
(4) application as the drug of preparation treatment breast cancer；
(5) application as the drug of preparation treatment gynecological tumor；
(6) application as the drug of preparation treatment Patients with Urinary System Tumors；
(7) application as the drug of preparation treatment skin neoplasin；
(8) application as the drug of preparation treatment sarcoma.
9. the use as claimed in claim 7, which is characterized in that
Medicine of the human pluripotent stem cells excretion body described in the claim 1,4 or 5 as preparation treatment central nerve neuroma
Object in application,
Application of the human pluripotent stem cells excretion body of application load taxol as preparation treatment glioma drug, or,
Glioma drug is treated as preparation using the human pluripotent stem cells excretion body for having loaded taxol through RGD peptide modification
Using, or,
Brain colloid is treated as preparation using the human pluripotent stem cells excretion body for having loaded Temozolomide (TMZ) through RGD peptide modification
The application of tumor medicine, or,
Medulloblastoma medicine is treated as preparation using the human pluripotent stem cells excretion body for having loaded vincristine through RGD peptide modification
The application of object；
The human pluripotent stem cells excretion body described in the claim 1,4 or 5 as preparation treatment lung cancer drug in application,
Using the application for having loaded the human pluripotent stem cells excretion body of cis-platinum as preparation through RGD peptide modification and treating lung-cancer medicament,
Or,
Using human pluripotent stem cells excretion body the answering as preparation treatment lung-cancer medicament for having loaded Irinotecan through RGD peptide modification
With；
The human pluripotent stem cells excretion body described in the claim 1,4 or 5 being answered as the drug of preparation treatment gastroenteric tumor
Used time,
Gastric cancer is treated as preparation using the human pluripotent stem cells excretion body for having loaded 5 FU 5 fluorouracil (5-FU) through RGD peptide modification
The application of drug, or,
Oesophagus is treated as preparation using the human pluripotent stem cells excretion body for having loaded 5 FU 5 fluorouracil (5-FU) through RGD peptide modification
The application of cancer drug, or,
Colon cancer drug is treated as preparation using the human pluripotent stem cells excretion body for having loaded capecitabine through RGD peptide modification
Using, or,
Pancreatic cancer drug is treated as preparation using the human pluripotent stem cells excretion body for having loaded gemcitabine through RGD peptide modification
Using, or,
Using human pluripotent stem cells excretion body the answering as preparation treatment liver-cancer medicine for having loaded Sorafenib through RGD peptide modification
With, or,
The human pluripotent stem cells excretion body described in the claim 1,4 or 5 as preparation treatment breast cancer drug in application,
Breast cancer medicines are treated as preparation using the human pluripotent stem cells excretion body for having loaded epirubicin through RGD peptide modification
Using；
Application of the human pluripotent stem cells excretion body described in the claim 1,4 or 5 as the drug of preparation treatment gynecological tumor
When,
Using human pluripotent stem cells excretion body the answering as preparation treatment ovarian cancer for having loaded taxol through RGD peptide modification
With；
Drug of the human pluripotent stem cells excretion body described in the claim 1,4 or 5 as preparation treatment Patients with Urinary System Tumors
In application,
Prostate cancer drug is treated as preparation using the human pluripotent stem cells excretion body for having loaded docetaxel through RGD peptide modification
Application, or,
Using the application for having loaded the human pluripotent stem cells excretion body of sotan as preparation through RGD peptide modification and treating kidney drug,
Or,
Application of the human pluripotent stem cells excretion body described in the claim 1,4 or 5 as the drug of preparation treatment skin neoplasin
When,
Black is treated as preparation using the human pluripotent stem cells excretion body for having loaded interleukins (IL-2) through RGD peptide modification
The application of plain tumor medicine；
The human pluripotent stem cells excretion body described in the claim 1,4 or 5 as preparation treatment sarcoma drug in application,
Osteosarcoma is treated as preparation using the human pluripotent stem cells excretion body for having loaded Doxorubicin through RGD peptide modification.
10. the preparation of the human pluripotent stem cells excretion body as described in claim 1,4 or 5, which is characterized in that the preparation selection
Any one of following form:
A, suspending agent: human pluripotent stem cells excretion body described in claim 1,4 or 5 is dissolved in solvent, with the shape of suspending agent
Formula exists；
B, it is sustained the compound of excretion body: human pluripotent stem cells excretion body shape in conjunction with carrier as described in claim 1,4 or 5
At the compound of sustained release excretion body, the carrier includes the life of various hydrogels, biomembrane, bioceramic material, nanostructure
Object material；
C, using human pluripotent stem cells excretion body described in claim 1,4 or 5 as the additive of functional component.
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| CN201810141463.8A CN110152015A (en) | 2018-02-11 | 2018-02-11 | Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes |
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