CN108042805A - A kind of tumour carries medicine microparticle preparation and preparation method thereof - Google Patents
A kind of tumour carries medicine microparticle preparation and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of tumour and carries medicine microparticle preparation and preparation method thereof, including being derived from the cell vesicle of tumor stem cell apoptosis release and being wrapped in the chemotherapeutic in the cell vesicle as active ingredient；It is mixed and is made in culture medium with three-dimensional soft fibre protein adhesive and tumour cell.Tumour provided by the invention carries medicine microparticle and is more advantageous to tumor tissues are highly enriched, tumour deep penetrates and be more easy to effectively to be absorbed by Common tumors cell and tumor stem cell, improve killing of the chemotherapeutics to Common tumors cell and tumor stem cell, can solve the problems, such as Common tumors cell derived load medicine microparticle it is impermeable to tumour deep to kill more tumor stem cell, while reduce toxic side effect of the chemotherapeutics to body.
Description
Technical field
The present invention relates to drug targeting carrier technique fields more particularly to a kind of tumour to carry medicine microparticle preparation and its preparation
Method.
Background technology
Malignant tumour seriously endangers human health, and chemotherapy is one of the important means of its treatment.But classic chemotherapy drug master
To be directed to Common tumors cell, and the tumour deep of anoxic there are a group quantity it is few have self-renewal capacity, multidirectional point
Change the strong tumor stem cell (or tumour initiator cell, tumor regrowth cell) of potential, internal one-tenth knurl ability, due to its height expression
Anti-apoptotic proteins and abc transport albumen, extremely strong DNA repair abilities etc. and with height drug resistance, be cause tumor recurrence and
The main reason for transfer.
Nano medicament carrying system is due to that with EPR effects, can improve tumor-targeting, enhancing chemotherapeutical medicine curative effect and reduce
Toxic side effect and be widely used in oncotherapy.It is currently reported using artificial synthesized nano-carrier load antitumor drug or
Tumor stem cell signal path (such as WNT, TGF-β, Notch, Hedgehog) inhibitor kills tumor stem cell.Due to nanometer
Material is exogenous material, itself to living organism there are certain toxic side effect, while the production of many nano materials and
Technological requirement is high, and cost is big, is unfavorable for clinical application.
Extracellular vesica (extracellular vesicles, EVs) is to be discharged into extracellular film vesicles, in physiology
Or play a significant role under pathological conditions, and vesica has low immunogenicity, excellent body-internal-circulation stability and high cellular targets
The features such as tropism.Microparticle (Microparticles) is that the skeleton that cell is triggered under the stimulation of coherent signal under cell membrane becomes
Change, cell membrane caused to wrap up cellular content in local outside bulging, and be discharged into the form of vesica it is extracellular caused by
A kind of extracellular vesica, grain size is in 100-1000nm.Tumour cell source carries medicine microparticle can effectively killing tumor cell and energy
The drug resistance of reversing tumor stem cell opens new approach for tumor biotherapy.But the load medicine in this tumour cell source
Microparticle cannot deeper penetrate into tumour deep and more kill tumor stem cell.
The content of the invention
It is an object of the invention to solve to exist in the prior artThe load medicine microparticle of Common tumors cell derived cannot ooze Thoroughly to tumour deep to kill more tumor stem cellThe problem of, a kind of tumour is provided and carries medicine microparticle preparation and its preparation side
Method.
A kind of tumour provided by the invention carries medicine microparticle preparation, the cytocyst including being derived from the release of tumor stem cell apoptosis
It steeps and is wrapped in the chemotherapeutic in the cell vesicle as active ingredient.Numerous studies show nanofeature, as grain size,
The profound influences Nano medication physiological disposition such as surface potential, hardness, and then influence its antitumous effect.With hard nanoparticle phase
Than the hydraulic power in soft nanoparticle blood circulation makes it easier to deform, and is not easy to by reticuloendothelial system phagocytic, thus favourable
In its internal long circulating, increase the chance for reaching tumor tissues and tumour is contributed to be enriched with.Tumor stem cell is compared with differentiation
Tumour cell is softer, and secreted vesica is also softer, is more prone to deform and efficiently absorbs the load medicine microparticle of source of human stem cell,
More conducively its blood long circulating, targeting are stronger in vivo, deeper into penetrate to tumour deep, be enriched at tumour, so as to
Enhance the lethal effect to tumor stem cell, improve antitumous effect.
Preferably, the tumor stem cell is to be mixed with three-dimensional soft fibre protein adhesive with tumour cell in culture medium
It is made.Tumor stem cell that three-dimensional soft fibre protein adhesive culture obtains multiplication is good, cell survival rate is higher, and and Nostoc commune Vanch
The tumor stem cell that base culture obtains is compared, and the load medicine microparticle in tumor stem cell source provided by the invention can be by tumour cell
Huge uptake, the therapeutic effect of the chemotherapeutic as active ingredient are significantly improved.
Preferably, content ratio of the tumour cell with three-dimensional soft fibre protein adhesive in the medium is 1~1.5 × 104
It is a:1.5~2mg.The present invention is to screen tumor stem cell based on Mechanics of Machinery, and preferred proportioning is that most suitable mechanical force is formed
Condition with this condition, can more form tumor stem cell.
Preferably, the tumour cell is derived from acute leukemia, lymthoma, breast cancer, lung cancer, oophoroma, chorion
Tumour cell in epithelioma, cervix cancer, liver cancer, carcinoma of urinary bladder or cutaneum carcinoma；The chemotherapeutic include treatment acute leukemia,
Lymthoma, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma and various other
One or more in the chemotherapeutic of entity tumor.The maximum amount of the chemotherapeutic depends on used chemotherapeutic in microparticle
In maximum saturation, therefore the medicament of different size can be obtained in the range of highest.Microparticle for wrapping up chemotherapeutic can
Be with institute tumour cell source not of the same race to be treated or source of the same race, preferably with institute's tumour cell to be treated
Tumor stem cell of the same race.The dosage form for carrying medicine microparticle preparation can be ejection preparation or oral formulations.
Preferably, the granularity for carrying medicine microparticle preparation is 200~600nm.The load medicine microparticle preparation is nanometer
Size is more advantageous to carrying medicine microparticle preparation in tumor locus enrichment, through tumor vessel and permeate tumour deep, more effectively
Common tumors cell and tumor stem cell are killed by Common tumors cell and tumor stem cell intake, normal tissue will not produce
Raw any damage, avoid use foreign material as carrier and to the toxic action of body generation.
The present invention also provides the preparation methods that a kind of tumour carries medicine microparticle preparation, comprise the following steps：
S1：The screening of tumor stem cell：With the medium culture tumour cell containing three-dimensional soft fibre protein adhesive, filter out swollen
Knurl stem cell clone ball is to get to tumor stem cell；
S2：Chemotherapeutics is handled into the tumor stem cell and is allowed to apoptosis, collects micro- of the load medicine that apoptotic cell is discharged
Grain, the load medicine microparticle are that cell vesicle wraps up the tumour load medicine microparticle preparation formed after chemotherapeutic；Or
The tumor stem cell is irradiated using ultraviolet light and is allowed to Apoptosis, collects the cytocyst that apoptotic cell is discharged
The cell vesicle, then with chemotherapeutic is incubated, chemotherapeutics is made to be wrapped up by the cell vesicle by bubble, and it is micro- to collect load medicine
Particle carries medicine microparticle preparation to get to the tumour.
Preferably, in the step S1, the screening of tumor stem cell concretely comprises the following steps：In advance by tumour cell culture medium
Culture is diluted to 1~1.5 × 104A/mL obtains tumor cell suspension, then the three-dimensional soft fibre protein adhesive with 1.5~2mg/mL
With volume ratio 1:1 mixing adds in fibrin ferment and is uniformly mixed so as to obtain to add in culture medium after mixed liquor and cultivated, thereto by formation
Tumor stem cell clone ball is picked out to get to tumor stem cell.The culture medium can use not according to tumour source difference
Same culture medium, preferably 1640 culture mediums of RPMI are added in by mixed liquor in 1640 culture mediums of RPMI before culture, can be first by 1 μ
The fibrin ferment (0.1U/ μ L) of L is added in 96 orifice plates of precooling, is added to tumor cell suspension and three-dimensional soft fibre protein adhesive and is mixed
Mixed liquor is obtained in the 50 μ L mixtures closed, then the mixed liquor is added in 200 μ L RPMI, 1640 complete mediums and is cultivated.
The present invention is to screen tumor stem cell based on Mechanics of Machinery, and preferred proportioning is most suitable mechanical force formation condition, at this
Under part, tumor stem cell can be more formed.
Preferably, before 1640 culture mediums of RPMI are added in, the mixed liquor is incubated 10 at a temperature of 37 DEG C
~15min.The preferred time is to ensure that three-dimensional soft fibre protein adhesive is preferably formed by curing suitable mechanical force.
Preferably, further included before the tumor stem cell clone ball apoptosis is made with tumor stem cell culture medium to tumour
Stem cell is enlarged the step of culture；The tumor stem cell culture medium is：HLA-B27 containing 2g/mL (belongs to HLA to resist
It is former), the DMEM-F12 culture mediums of the glutamine of 20ng/mL mouse epidermal cells growth factor and 1g/mL.Preferred Tumor Stem
Cell culture medium be enable to screen the stem cell to be formed through three-dimensional soft fibre protein adhesive it is a large amount of in the case where keeping dryness feature
Amplification.
Preferably, in the step S2, micro- of medicine of load is collected under 4~6 DEG C of low temperature, with the centrifugal force of 200~20000g
Grain.Preferably vesica of the grain size of tumor stem cell secretion in 200~600nm can be more effectively enriched with by centrifugal condition.
Technical scheme has the advantages that：
(1) by micro- from the tumor stem cell source after being cultivated in the culture medium containing three-dimensional soft fibre protein adhesive
Carrier of the grain as the pharmaceutical preparation of the present invention solves foreign vector and is administered to the toxic side effect of body, while makes as having
The chemotherapeutic for imitating ingredient is penetrated in tumor locus enrichment, tumour deep and absorbed by tumour cell, makes micro- of the load medicine of the present invention
Grain improves fragmentation effect of the chemotherapeutic to tumour cell while toxic side effect of the reduction chemotherapeutic to normal cell.
(2) tumor stem cell obtained by the medium culture containing three-dimensional soft fibre protein adhesive, apoptosis discharge to obtain
Microparticle as carrier, compared to the microparticle of Common tumors cell derived, there is preferable tumor stem cell targeting, profit
Go deep into tumour deep in improving chemotherapeutic, enhance the fragmentation effect to tumor stem cell.
(3) tumour cell has the characteristic of infinite multiplication, and tumour cell has been cultivated in three-dimensional soft fibre protein adhesive
The ability of tumor stem cell is formed, the scheme recorded according to the present invention can obtain substantial amounts of tumor stem cell from tumour cell
The microparticle in source is used to prepare the pharmaceutical preparation of the present invention, and cost is small, easy to operate.
Description of the drawings
Fig. 1 is the load medicine microparticle laser co-focusing figure that tumor stem cell generates after chemotherapeutic is handled；
Fig. 2 is that the microparticle that tumor stem cell discharges after UV treatment is incubated the load medicine microparticle generated with chemotherapeutic
Laser co-focusing figure；
Fig. 3 is to carry accumulation of the medicine microparticle in tumor tissues to influence result figure；
Fig. 4 is the laser co-focusing figure for carrying medicine microparticle in tumour cell group；
Fig. 5 is that murine hepatocarcinoma cell influences result figure to the intake of the load medicine microparticle in tumor stem cell source；
Fig. 6 is that the medicine microparticle that carries in tumor stem cell source influences result figure to the lethal effect of murine hepatocarcinoma cell；
Fig. 7 is that tumor stem cell influences result figure to the intake of the load medicine microparticle in tumor stem cell source；
Fig. 8 is that the accumulation for carrying medicine microparticle tumor stem cell in vivo in tumor stem cell source influences result figure；
Fig. 9 is that the medicine microparticle that carries in tumor stem cell source influences result figure to the lethal effect of in-vivo tumour stem cell；
Figure 10 is application drawing of the load medicine microparticle to more tumour cells of the present invention；
Figure 11 A are that the load adriamycin microparticle of rat liver cancer source of human stem cell inhibits H22 subcutaneous tumors rat liver cancer grown junctions
Fruit is schemed；
Figure 11 B are that H22 subcutaneous tumors mouse influences result figure using the time-to-live for carrying adriamycin microparticle；
Figure 11 C are that the load adriamycin microparticle of rat liver cancer source of human stem cell inhibits Murine Hepatoma22 subcutaneous tumors tumor quality shadow
Ring result figure；
Figure 12 A are the growth for the microparticle inhibition Murine Hepatoma22 subcutaneous tumors that rat liver cancer source of human stem cell carries 5 FU 5 fluorouracil
Influence result figure；
Figure 12 B are the microparticle treatment that Murine Hepatoma22 subcutaneous tumors mouse carries 5 FU 5 fluorouracil using liver-cancer stem cell source
Time-to-live influences result figure；
Figure 12 C are the microparticle inhibition Murine Hepatoma22 subcutaneous tumors tumour matter that rat liver cancer source of human stem cell carries 5 FU 5 fluorouracil
Amount influences result figure；
Figure 13 A are that the load medicine microparticle of murine melanoma source of human stem cell inhibits melanoma mouse lung transforming growth
Figure；
Figure 13 B are the time-to-live influence knot for carrying medicine microparticle and extending tumor-bearing mice of murine melanoma source of human stem cell
Fruit is schemed；
Figure 14 A are that the creatine kinase content for the load medicine microparticle group that mouse injects tumor stem cell source influences result figure；
Figure 14 B are that the glutamic-pyruvic transaminase content for the load medicine microparticle group that mouse injects tumor stem cell source influences result
Figure；
Figure 14 C are that the mouse weight of the load medicine microparticle group in injection tumor stem cell source influences result figure.
Specific embodiment
It, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical solution be explicitly described, it is clear that described embodiment be part of the embodiment of the present invention rather than whole
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without making creative work
The every other embodiment obtained, belongs to the scope of protection of the invention.Unless otherwise specified, technological means used is in embodiment
Conventional means well-known to those skilled in the art.
Terminology used in the present invention " cell vesicle " is generated by apoptotic tumor cell, does not wrap up chemotherapeutic, " carries medicine
Microparticle " is formed after wrapping up chemotherapeutic by cell vesicle.Heretofore described " chemotherapeutic " that is wrapped in cell vesicle,
Can be by the active ingredient in the chemotherapeutic of clinical practice or the chemotherapeutics of clinical practice, it is involved in the present invention
And drug dose, should be understood as the active ingredient amount of the drug.
By the way that tumor stem cell apoptosis is induced to discharge to obtain cell vesicle, can by skilled person will appreciate that method
During chemotherapeutic is wrapped up into the cell vesicle so as to obtain carry medicine microparticle.It can be used at a high speed for the collection for carrying medicine microparticle
Centrifuge is separated under cryogenic.According to well known to a person skilled in the art criterion, tumour cell is such as observed
Diminution, refractive power are dimmed, you can think Apoptosis.Cell vesicle and chemotherapeutic, which be incubated, makes cell vesicle wrap up chemotherapeutic
Can by ambient temperature by the cell vesicle system that is with the addition of chemotherapeutic carry out 2~4 it is small when standing realize.
Various tumour cells, drug and the experimental animal used in following embodiments：
H22 murine hepatocarcinoma cells, human hepatoma cell line HepG2, mouse skin cancerous cell line B16, can be from U.S. ATCC
Company or Chinese Typical Representative object collection CCTCC purchases.
BALB/C mice, C57 mouse are purchased from Wuhan University's medical experiment animal center, 18-20 grams of weight；
Purchased from Sigma companies, Fibrinogen is purchased from for adriamycin, Fluoresceincarboxylic acid succinimide ester (CFSE)
Reagent proteins companies, 5 FU 5 fluorouracil are purchased from Aladdin company.
Embodiment 1：The tumour generated using chemotherapeutic inducing mouse liver-cancer stem cell apoptosis carries medicine microparticle preparation
1st, experiment material and reagent
H22 murine hepatocarcinoma cells, adriamycin (carrying red fluorescence), Fluoresceincarboxylic acid succinimide ester (CFSE) are (green
Color fluorescent dye).
2nd, experimental procedure
1) H22 murine hepatocarcinoma cells are cultivated in 1640 cell culture fluids of RPMI, H22 cells are trained in advance with RPMI 1640
Foster base is diluted to 1 × 104A/mL, obtains cell suspension, and soft fibre protein adhesive is diluted to the molten of 2mg/mL with T7 buffer solutions
Liquid, by the solution and H22 cell suspensions with volume ratio 1:1 mixing, obtains mixture；1 μ L are added in 96 orifice plates of precooling
Fibrin ferment (0.1U/ μ L), and mixing is blown and beaten with 50 μ L said mixtures, mixed liquor is obtained, 15min is incubated under the conditions of 37 DEG C
Afterwards, the mixed liquor is added in 1640 complete mediums of RPMI of 200 μ L, culture obtains tumor stem cell clone after 6 days.
It is taken out from 96 orifice plates, in serum-free DMEM/F12 culture mediums (HLA-B27 containing 2g/mL, 20ng/mL mouse epidermal cells
The DMEM-F12 culture mediums of growth factor and 1g/mL glutamine) in expand culture, cell concentration is made to reach 2 × 107。
2) above-mentioned H22 rat liver cancers stem cell is marked according to a conventional method using Green fluorescent dye CFSE.
3) adriamycin is applied to the H22 rat liver cancers stem cell (band cell culture fluid) of taking-up, makes adriamycin in culture solution
In final concentration of 200 μ g/mL.
4) after application adriamycin for 24 hours, apoptosis tumor stem cell is implemented to separate.Supernatant is taken progressively to be centrifuged, with
After the rotating speed of 200g, 600g respectively centrifuge 10min, with the centrifugal force 1min of 14000g, to remove cell and fragment, centrifugation is taken
Supernatant afterwards further with the centrifugal force 1h of 20000g, is obtained from load caused by H22 rat liver cancer stem cells apoptosis
Medicine microparticle.
3rd, experimental result
After load medicine microparticle obtained above is resuspended with the physiological saline of 0.9% (i.e. 0.9g/mL), smear, in laser
It is observed under Laser Scanning Confocal Microscope, it will be seen from figure 1 that micro- of the load medicine generated using the H22 rat liver cancers stem cell of adriamycin
Grain, it is observed that the fluorescence of red (Figure 1B) and green (Figure 1A) after smear, and red fluorescence and green fluorescence overlapping are shown
Yellow (Fig. 1 C).It since single Doxorubicin molecules are minimum, can not be differentiated using fluorescence microscope, could gathered after being only wrapped
Collect and be observed, the red observed under fluorescence microscope be exactly cell vesicle package chemotherapeutic adriamycin sent it is red
Light；Green is due to cell marking after green fluorescence, and the microparticle of secretion caught green；And green and red are fixed altogether
Position, illustrates that adriamycin has been wrapped in microparticle.It can be seen that tumor stem cell after chemotherapeutic is handled, form carry medicine it is micro-
Particle, size is in 500nm or so, it was demonstrated that microparticle has wrapped up chemotherapeutic.
Embodiment 2：It is obtained after being incubated using the microparticle that ultraviolet induction rat liver cancer stem cell apoptosis generates with chemotherapeutic
Medicine microparticle must be carried.
1st, experiment material and reagent
With embodiment 1, ultraviolet device owns the H22 murine hepatocarcinoma cells used for regular growth superclean bench, Ah
Mycin is the same as embodiment 1.
2nd, experimental procedure
1) H22 rat liver cancer stem cells are cultivated, cell concentration is made to reach 2 × 107, cultural method is the same as embodiment 1.
2) 60min is irradiated with ultraviolet light to H22 rat liver cancers stem cell (band cell culture fluid).
3) after uv irradiation for 24 hours, the supernatant of the tumor stem cell culture solution of apoptosis is progressively centrifuged, method
With embodiment 1, the microparticle of the tumor stem cell is obtained.
4) after the microparticle obtained is resuspended with the physiological saline of 0.9% (i.e. the 0.9g/mL) of 1mL, adriamycin is added in,
Make the final concentration of 200 μ g/mL of adriamycin, be then incubated at room temperature 2h again, afterwards with the centrifugal force 1h of 20000g, using PBS
It cleans 2 times and with the centrifugal force 1h of 20000g, obtains and carry medicine microparticle.
3rd, experimental result
After load medicine microparticle obtained above is resuspended with 0.9% physiological saline, smear, in laser confocal microscope
Lower observation carries medicine microparticle from figure 2 it can be seen that being generated using the H22 rat liver cancer stem cells of adriamycin, can after smear
To observe the fluorescence of red (Fig. 2 B) and green (Fig. 2A), and red fluorescence and green fluorescence overlapping show yellow (Fig. 2 C),
Confirm that tumor stem cell after ultraviolet light irradiates, releases microparticle, and after being incubated with chemotherapeutic, which has wrapped up change
Treat medicine.
Embodiment 3：Carry accumulation of the medicine microparticle in tumor tissues
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, chemotherapeutic adriamycin is commercially available, BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) H22 murine hepatocarcinoma cells and liver-cancer stem cell are cultivated, cell concentration is made to respectively reach 2 × 107, wherein H22 mouse
The cultural method of liver-cancer stem cell is the same as embodiment 1, H22 murine hepatocarcinoma cells 1640 medium cultures of RPMI.
2) 1 × 10 is taken from the cell of above-mentioned culture7A cell applies adriamycin, and the final concentration of adriamycin is made to reach 200 μ
G/mL, remaining cell continue to cultivate, without any processing.
3) after application chemotherapeutic for 24 hours, the load from H22 rat liver cancer stem cells is collected according to the method for embodiment 1
Medicine microparticle；Collect the load medicine microparticle from H22 murine hepatocarcinoma cells.
The foundation of rat liver cancer H22 subcutaneous tumors models：It is inoculated at the nearly right lower extremity in BALB/C mice back lower right side
(cell number is 1 × 10 to 100 μ L of Murine Hepatoma22 cell suspension5), establish rat liver cancer H22 subcutaneous tumors models.
When rat liver cancer H22 subcutaneous tumors grow to volume as 0.09~0.12cm3When, lotus there are into Murine Hepatoma22 subcutaneous tumors at random
Mouse (18g~22g) be divided into 3 groups, every group 6.By the load medicine microparticle of prepared H22 rat liver cancer source of human stem cell with
The dosage of 0.5mg (adriamycin equivalent)/kg, the dosage of 200 μ L are respectively through 6 BALB/C mices of tail vein injection as experiment
Group, by the load medicine microparticle in prepared H22 murine hepatocarcinoma cells source with the dosage of 0.5mg (adriamycin equivalent)/kg,
The dosage of 200 μ L is respectively through 6 BALB/C mices of tail vein injection as a control group 1；Other 6 BALB/C mices are noted simultaneously
The free adriamycin of same dose is penetrated, as a control group 2.Each group puts to death mouse after tail vein injection 48h, takes out tumour and prepares
It is homogenized into tissue, medicament contg therein is detected by fluorescence spectrophotometry.
3rd, experimental result
It was found from the Tissue distribution figure of Fig. 3, the concentration of the adriamycin after tail vein injection load medicine microparticle 48h in tumour will
Far above the free adriamycin group of injection；In the experimental group of load medicine microparticle for injecting tumor stem cell source, adriamycin is in tumour
The concentration at position is significantly higher than the load medicine microparticle control group of injection Common tumors cell derived, shows tumor stem cell source
Carrying medicine microparticle can more be enriched in tumor locus.
Embodiment 4：Carry medicine microparticle penetrating in tumour cell group.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and Fibrinogen is purchased from
Reagent proteins companies.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The preparation method of H22 tumour cells group treats that cell mass is grown to the 6th day with embodiment 1, and prepared H22 is small
Mouse liver-cancer stem cell source carries medicine microparticle, the load medicine microparticle in common liver cancer cells source and free adriamycin with 0.5 μ
The concentration of g/mL adds in cell mass and is incubated 6h.It is copolymerized after fixing 30min with 4% paraformaldehyde after washing 3 times by PBS with laser
Focusing microscope is imaged.Adriamycin is observed by the optical filter of Ex=488nm, Em=560nm.
3rd, experimental result
As shown in Figure 4 (in figure gray areas be adriamycin fluorescence distribution region), the load medicine in tumor stem cell source is micro-
Particle rolls into a ball the doxorubicin fluorescence intensity of different level in H22 tumour cells, and equal conspicuousness is higher than the load of Common tumors cell derived
Medicine microparticle and free adriamycin processing group.Show that the load medicine microparticle in tumor stem cell source can be in H22 tumour cells group
Deep penetrates, and illustrates the load medicine microparticle in tumor stem cell source and has good tumour deep penetration capacity.
Embodiment 5:Intake of the murine hepatocarcinoma cell to the load medicine microparticle in tumor stem cell source.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
By the load medicine microparticle of prepared H22 rat liver cancer source of human stem cell, the load medicine in H22 murine hepatocarcinoma cells source
Microparticle and free adriamycin are incubated different time with H22 murine hepatocarcinoma cells, are respectively experimental group, control group 2 and control
Group 1.By the content of doxorubicin fluorescence in flow cytometry analysis cell, intake of the tumour cell to load medicine microparticle is determined
Amount.
3rd, experimental result
As shown in figure 5, H22 cells are most to the load medicine microparticle intake in H22 liver-cancer stem cells source, and intake is shown
It writes and carries medicine microparticle and free drug more than common liver cancer cells source.Show tumor stem cell source provided by the invention
Load medicine microparticle can be by tumour cell huge uptake.
Embodiment 6：Tumor stem cell source carries lethal effect of the medicine microparticle to murine hepatocarcinoma cell.
1st, experiment material and reagent
For the H22 murine hepatocarcinoma cells used with embodiment 1, chemotherapeutic adriamycin is commercially available.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
4) by the load medicine microparticle (experimental group) in liver-cancer stem cell source and the load medicine microparticle in common liver cancer cells source
(control group 2) and tumour H22 cells co-incubation 48h in 96 orifice plates, observes the apoptosis of tumour cell, common with tumour cell
The load medicine microparticle of culture should be micro- of the load medicine formed after the microparticle package chemotherapeutic that same kind tumour cell obtains
Grain.Adriamycin (control group 1) free to tumour cell application simultaneously makes final concentration of 0.5 μ g/mL, and after handling 48h, observation is swollen
The death of oncocyte.
3rd, experimental result
As shown in fig. 6, free chemotherapeutic adriamycin can kill H22 murine hepatocarcinoma cells, but still there is a certain amount of tumour
Cell survival；And the load medicine microparticle in the tumor stem cell source containing chemotherapeutic can make most death of neoplastic cells, kill
Hinder the load medicine microparticle that cell effect is significantly higher than Common tumors cell derived.Show the present invention with tumor stem cell microparticle
Load medicine microparticle that chemotherapeutic is formed is wrapped up after tumour cell is administered to, significantly improves chemotherapeutic as active ingredient
Therapeutic effect.
Embodiment 7:Intake of the tumor stem cell to the load medicine microparticle in tumor stem cell source.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
By the load medicine microparticle of prepared H22 rat liver cancer source of human stem cell, the load medicine in H22 murine hepatocarcinoma cells source
Microparticle, free adriamycin and H22 tumor stem cells are incubated different time, are respectively experimental group, control group 2 and control group 1.It is logical
The content of doxorubicin fluorescence in overflow-type Cytometric Analysis H22 tumor stem cells determines tumor stem cell to carrying medicine microparticle
Intake.
3rd, experimental result
As shown in fig. 7, H22 tumor stem cells are most to the load medicine microparticle intake in H22 liver-cancer stem cells source, and take the photograph
Taken amount is significantly more than the load medicine microparticle of common H22 cell deriveds and free drug.Show that Tumor Stem provided by the invention is thin
The load medicine microparticle in born of the same parents source can be by tumor stem cell huge uptake.
Embodiment 8：Accumulation of the medicine microparticle in tumor stem cell is carried in vivo.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The foundation of rat liver cancer H22 subcutaneous tumors models：With embodiment 3.
Medication is with embodiment 3, by the load medicine microparticle tail vein injection 4 of prepared H22 rat liver cancer stem cells
BALB/C mice is as experimental group, by the load medicine microparticle tail vein injection 4 of prepared H22 murine hepatocarcinoma cells
BALB/C mice as a control group 2；Simultaneously to the free adriamycin of other 4 BALB/C mices injection same dose, as right
According to group 1.
The mouse of each group is put to death afterwards for 24 hours, take out tumor tissues and is prepared into tissue homogenate, passes through flow cytometry analysis
The content of doxorubicin fluorescence, determines storage of the chemotherapeutic in tumor stem cell wherein in the side population cell with tumor stem cell characteristic
Accumulated amount.
3rd, experimental result
As shown in figure 8, side population cell in tumor tissues to the load medicine microparticle intake in H22 liver-cancer stem cells source most
It is more, and intake is significantly more than the load medicine microparticle of common H22 cell deriveds and free drug.Show provided by the invention swollen
Load medicine microparticle energy targeting tumor stem cells of knurl source of human stem cell and can be by tumor stem cell huge uptake.
Embodiment 9：Lethal effect of the medicine microparticle to tumor stem cell is carried in vivo.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The foundation of rat liver cancer H22 subcutaneous tumors models：With embodiment 3.
Medication is the same as embodiment 3.By the load medicine microparticle tail vein injection 4 of prepared H22 rat liver cancer stem cells
BALB/C mice is as experimental group, by the load medicine microparticle tail vein injection 4 of prepared H22 murine hepatocarcinoma cells
BALB/C mice as a control group 3；Simultaneously other 8 BALB/C mices are injected respectively same dose free adriamycin and
0.9% physiological saline, respectively as control group 2 and 1.Same operation and dosage, every group of injection in every two days once, connect
It is 14 days continuous.
The mouse of each group was put to death in 16th day, takes out tumour and be simultaneously prepared into tissue homogenate, by flow cytometry analysis its
In side population cell ratio, determine the content of tumor stem cell.
3rd, experimental result
As shown in figure 9, after drug therapy, the load medicine microparticle group side population cell ratio in H22 liver-cancer stem cells source is most
It is small, illustrate that it has liver-cancer stem cell high killing rate, effect is considerably better than the load medicine microparticle of Common tumors cell derived
With simple free adriamycin.Show the load medicine microparticle energy targeting tumor stem cells in tumor stem cell source provided by the invention
And tumor stem cell is killed, obtains good chemotherapy effect.
Embodiment 10：The application of more tumour cells.
1st, experiment material and reagent
Different tumor cell lines include：Human hepatoma cell line HepG2, mouse skin cancerous cell line B16；Chemotherapeutic adriamycin
It is commercially available.
2nd, experimental procedure
1) tumour cell is cultivated in DMEM culture solutions, cell concentration is made to respectively reach 2 × 107It is a, while cultivate Tumor Stem
Cell makes cell concentration respectively reach 2 × 107A, cultural method is the same as embodiment 1.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
4) experimental method is the same as embodiment 6.
3rd, experimental result
As shown in Figure 10, the load medicine microparticle in various tumor stem cell sources has the tumour cell in its source high killing
Rate, and effect is intended to be considerably better than the load medicine microparticle of Common tumors cell derived and free drug, shows that the present invention carries
The preparation of confession is applicable in kinds of tumor cells, and can obtain good therapeutic effect.
Embodiment 11：Influence of the load adriamycin microparticle of rat liver cancer source of human stem cell to H22 subcutaneous tumors mouse growths
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The foundation of rat liver cancer H22 subcutaneous tumors models：With embodiment 3.
Medication is the same as embodiment 3.By the load medicine microparticle tail vein injection 16 of prepared H22 rat liver cancer stem cells
BALB/C mice is as experimental group, by the load medicine microparticle tail vein injection 16 of prepared H22 murine hepatocarcinoma cells
BALB/C mice as a control group 3；16 BALB/C mices are injected to the free adriamycin of same dose, as a control group 2.It is surplus
The PBS of next group of 16 BALB/C mice injection same volume, as a control group 1.Same operation and dosage, every group every
It injects once within two days, continuous 14 days.
3rd, experimental result
Mouse in experimental group and control group is all divided into etc. to two parts of quantity, a portion uses vernier caliper daily
The most strong point (L) of tumour and the widest part (W) are measured, calculates gross tumor volume V=L × W2/2.The 16th day mouse by each group is put to death,
It strips out subcutaneous tumors and weighs.Another part is used to observe the time-to-live.
The result shows that the load adriamycin microparticle of H22 rat liver cancers source of human stem cell can significantly inhibit mouse in experimental group
The growth (as shown in Figure 11 A and 11C) of H22 liver cancer, and extend the time-to-live (as shown in Figure 11 B) of mouse, wherein Tumor Stem
The load medicine microparticle effect of cell derived will be significantly better than the load medicine microparticle of Common tumors cell derived and free adriamycin.
Embodiment 12：The load 5 FU 5 fluorouracil microparticle of rat liver cancer source of human stem cell is to H22 subcutaneous tumors mouse growths
It influences
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic 5 FU 5 fluorouracil is commercially available, BALB/C mice purchase
From Wuhan University's medical faunae center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The foundation of rat liver cancer H22 subcutaneous tumors models：With embodiment 3.
Dosage regimen is the same as embodiment 9.
3rd, experimental result
Mouse in experimental group and control group is all divided into etc. to two parts of quantity, a portion uses vernier caliper daily
The most strong point (L) of tumour and the widest part (W) are measured, calculates gross tumor volume V=L × W2/2, the 16th day mouse by each group is put to death,
It strips out subcutaneous tumors and weighs.In addition a part is used to observe the time-to-live.
The result shows that the microparticle of the load 5 FU 5 fluorouracil of H22 rat liver cancers source of human stem cell can significantly inhibit in experimental group
The growth (as shown in Figure 12 A and 12C) of mouse H22 liver cancer, and extend the time-to-live (as shown in Figure 12 B) of mouse, wherein swollen
The load medicine microparticle of knurl source of human stem cell the drug with obvious effects that carry medicine microparticle and dissociate for being better than Common tumors cell derived.
Embodiment 13：It carries medicine microparticle and inhibits pulmonary metastases growth, the time-to-live for extending tumor-bearing mice.
1st, experiment material and reagent
The B16-F10 mouse melanin tumor cells used are with embodiment 8, and chemotherapeutic adriamycin is commercially available, and C57BL/6 is small
Mouse is purchased from Wuhan University's medical faunae center.
2nd, experimental procedure
1) B16-F10 mouse melanin tumor cells and its stem cell are cultivated, cell concentration is made to respectively reach 3 × 107, culture side
Method is the same as embodiment 8.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
The foundation of mouse B16-F10 pulmonary metastases models：It is hanged in C57BL/6 mouse tails intravenous injection B16-F10 cells
(cell number is 5 × 10 to 100 μ L of liquid5), establish mouse pulmonary metastases model.
After injecting B16-F10 cells for 24 hours, C57BL/6 mouse are divided into 4 groups, every group 16 at random.Medication is the same as implementation
Example 3, by load medicine microparticle 16 C57BL/6 mouse of tail vein injection of prepared B16-F10 murine melanoma stem cells
As experimental group；The load medicine microparticle of prepared B16-F10 mouse melanin tumor cells is injected 16 C57BL/6 mouse to make
For control group 3；16 C57BL/6 mouse are injected to the adriamycin of same dose, as a control group 2；It is one group 16 remaining
C57BL/6 mouse inject same volume PBS, and as a control group 1.Same operation and dosage, every group of injection one in every two days
It is secondary, continuous 14 days.
3rd, experimental result
Mouse in experimental group and control group is all divided into etc. to two parts of quantity, a portion is at 21 days by each group
Mouse is put to death, and takes out lung tissue, records tubercle number, and another part is used to observe the time-to-live.
The result shows that the load medicine microparticle of B16-F10 murine melanomas source of human stem cell can significantly inhibit in experimental group
Mouse tumor lung shifts (as shown in FIG. 13A), and extends the time-to-live (as shown in Figure 13 B) of mouse, and wherein Tumor Stem is thin
The load medicine microparticle in born of the same parents source the drug with obvious effects that carry medicine microparticle and dissociate for being better than Common tumors cell derived.
Embodiment 14：Carry the biological safety of medicine microparticle.
1st, experiment material and reagent
The H22 murine hepatocarcinoma cells used are with embodiment 1, and chemotherapeutic adriamycin is commercially available, and BALB/C mice is purchased from force
Chinese University Hospital animal center.
2nd, experimental procedure
1) cultural method of H22 rat liver cancers stem cell is cultivated with embodiment 1, H22 murine hepatocarcinoma cells with RPMI 1640
Base culture.
2) chemotherapeutic processing method is the same as embodiment 3.
3) medicine microparticle collection method is carried with embodiment 1.
4) foundation of rat liver cancer H22 subcutaneous tumors model：With embodiment 3.
Medication is the same as embodiment 3.By the load adriamycin microparticle of prepared H22 rat liver cancer stem cells through tail vein
6 BALB/C mices are injected as experimental group, the load adriamycin microparticle tail vein of prepared H22 murine hepatocarcinoma cells is noted
Penetrate 6 BALB/C mices as a control group 1；Inject free Ah mould of same dose respectively to other 12 BALB/C mices simultaneously
Element and 0.9% physiological saline, respectively as control group 2 and 3.Same operation and dosage, every group of injection one in every two days
It is secondary, continuous 14 days.
5) the 15th day, respectively to experimental group, 3 mouse extracting vein blood of control group 1, control group 2 and control group, detect in serum
Glutamic-pyruvic transaminase and creatine kinase content, and weigh mouse weight.
3rd, experimental result
The result shows that compared to the control group mice of injecting normal saline, injection carries the paddy of adriamycin microparticle group mouse
Pyruvic transaminase content does not have significant change (as shown in Figure 14B), and weight is also without significant change (as shown in Figure 14 C), and inject
The creatine kinase of the load medicine microparticle group in tumor stem cell source illustrates its heart less than all control groups (as shown in Figure 14 A)
Toxicity is very low.As it can be seen that with the load medicine microparticle preparation in tumor stem cell source to body substantially without toxic side effect.
To sum up, load medicine microparticle provided by the invention be more advantageous to tumor tissues are highly enriched, tumour deep penetrates and
It is more easy to effectively be absorbed by Common tumors cell and tumor stem cell, improves chemotherapeutics to Common tumors cell and tumor stem cell
Killing, enhance the therapeutic effect of tumour, while reduce toxic side effect of the chemotherapeutics to body.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that：It still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent substitution to which part technical characteristic；
And these modification or replace, do not make appropriate technical solution essence depart from various embodiments of the present invention technical solution spirit and
Scope.
Claims (10)
1. a kind of tumour carries medicine microparticle preparation, which is characterized in that includingCell vesicle from the release of tumor stem cell apoptosis With the chemotherapeutic being wrapped in the cell vesicle as active ingredient。
2. tumour as described in claim 1 carries medicine microparticle preparation, which is characterized in that the tumor stem cell is with three-dimensional soft
Fibrin Glue is mixed in culture medium with tumour cell and is made.
3. tumour as claimed in claim 2 carries medicine microparticle preparation, which is characterized in that the tumour cell and three-dimensional soft fibre
The content ratio of protein adhesive in the medium is 1~1.5 × 104It is a:1.5~2mg.
4. tumour as described in claim 1 carries medicine microparticle preparation, which is characterized in that the tumor stem cell is derived from acute
In leukaemia, lymthoma, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, liver cancer, carcinoma of urinary bladder or cutaneum carcinoma
Tumor stem cell；It is described to carry chemotherapeutic included in medicine microparticle preparation as the chemotherapeutic containing treatment function of tumor；Institute
Stating chemotherapeutic includes treatment acute leukemia, lymthoma, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, liver
One or more in cancer, carcinoma of urinary bladder, the chemotherapeutic of cutaneum carcinoma and various other entity tumors.
5. tumour as described in claim 1 carries medicine microparticle preparation, which is characterized in that the grain size for carrying medicine microparticle preparation
For 200~600nm.
6. a kind of preparation method that medicine microparticle preparation is carried such as Claims 1 to 5 any one of them tumour, which is characterized in that
Comprise the following steps：
S1：The screening of tumor stem cell：It is mixed in culture medium, filtered out with tumour cell with three-dimensional soft fibre protein adhesive
Tumor stem cell clone ball is to get to tumor stem cell；
S2：Chemotherapeutics is handled into the tumor stem cell and is allowed to apoptosis, collects the load medicine microparticle that apoptotic cell is discharged, it should
It is that cell vesicle wraps up the tumour load medicine microparticle preparation formed after chemotherapeutic to carry medicine microparticle；Or
The tumor stem cell is irradiated using ultraviolet light and is allowed to Apoptosis, collects the cell vesicle that apoptotic cell is discharged, so
The cell vesicle with chemotherapeutic is incubated afterwards, chemotherapeutics is made to be wrapped up by the cell vesicle, collects and carries medicine microparticle,
It obtains the tumour and carries medicine microparticle preparation.
7. tumour as claimed in claim 6 carries the preparation method of medicine microparticle preparation, which is characterized in that in the step S1,
The screening of tumor stem cell concretely comprises the following steps：Tumour cell is diluted to 1~1.5 × 10 with culture medium in advance4A/mL, obtains
Tumor cell suspension, then with the three-dimensional soft fibre protein adhesive of 1.5~2mg/mL with volume ratio 1:1 mixing, adds in blood coagulation thereto
Enzyme is uniformly mixed so as to obtain to add in 1640 culture mediums of RPMI after mixed liquor and be cultivated, and the tumor stem cell clone ball of formation is selected
Go out to get to tumor stem cell.
8. tumour as claimed in claim 7 carries the preparation method of medicine microparticle preparation, which is characterized in that by the mixed liquor
It adds in before 1640 culture mediums of RPMI, the mixed liquor is incubated 10~15min at a temperature of 37 DEG C.
9. tumour as claimed in claim 6 carries the preparation method of medicine microparticle preparation, which is characterized in that makes the Tumor Stem
The step of culture is enlarged to tumor stem cell with tumor stem cell culture medium is further included before Apoptosis；
The tumor stem cell culture medium is：HLA-B27 containing 2g/mL, 20ng/mL mouse epidermal cells growth factor and 1g/mL
The DMEM-F12 culture mediums of glutamine.
10. tumour as claimed in claim 6 carries the preparation method of medicine microparticle preparation, which is characterized in that in the step S2,
Load medicine microparticle is collected under 4~6 DEG C of low temperature, with the centrifugal force of 200~20000g.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109893515A (en) * | 2019-02-26 | 2019-06-18 | 华中科技大学 | A kind of macrophage carries medicine microparticle preparation and preparation method thereof |
| CN111228221A (en) * | 2020-02-25 | 2020-06-05 | 湖北盛齐安生物科技股份有限公司 | Method for improving productivity of drug-loaded vesicle |
| CN111265491A (en) * | 2020-03-25 | 2020-06-12 | 江苏省中医药研究院 | Cell microparticle drug delivery system for encapsulating allicin and preparation method and application thereof |
| CN112569205A (en) * | 2019-09-27 | 2021-03-30 | 湖北盛齐安生物科技股份有限公司 | Antitumor drug carrier, preparation method thereof and pharmaceutical preparation containing antitumor drug carrier |
| CN113116927A (en) * | 2019-12-30 | 2021-07-16 | 北京大学人民医院(北京大学第二临床医学院) | Preparation method of vesicle-coated arsenic trioxide derived from mesenchymal stem cells |
| CN113383749A (en) * | 2020-03-12 | 2021-09-14 | 中国医学科学院基础医学研究所 | Method for constructing mouse tumor model by using single tumor cell |
| CN114395531A (en) * | 2021-12-29 | 2022-04-26 | 镇江市中西医结合医院（镇江市第二人民医院） | Preparation method and application of bioactive substance or drug delivery microvesicle |
| CN114686417A (en) * | 2020-12-25 | 2022-07-01 | 中国人民解放军海军军医大学第三附属医院 | Cell vesicle preparation for efficiently inhibiting liver cancer, preparation method and application thereof |
| WO2022237247A1 (en) * | 2021-05-12 | 2022-11-17 | 中国医学科学院基础医学研究所 | Use of cellular microparticles in treatment of respiratory viral pneumonia |
| WO2022257849A1 (en) * | 2021-06-10 | 2022-12-15 | 中山大学 | Preparation method for engineered vesicle and application thereof |
| WO2023178810A1 (en) * | 2022-03-25 | 2023-09-28 | 华中科技大学 | Co-drug-loaded cell microparticle formulation and preparation method therefor |
| WO2023197901A1 (en) * | 2022-04-11 | 2023-10-19 | 华中科技大学 | Tumor cell-derived microparticle loaded with succinic acid, preparation method therefor, and use thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302784A (en) * | 2011-08-22 | 2012-01-04 | 湖北盛齐安生物科技有限公司 | Tumor chemotherapeutic medicinal preparation and preparation method thereof |
| WO2015052526A1 (en) * | 2013-10-09 | 2015-04-16 | Reneuron Limited | Stem cell microparticles and mirna |
| CN104703609A (en) * | 2012-07-19 | 2015-06-10 | 兰诺龙有限公司 | Stem cell microparticles |
| CN105142646A (en) * | 2013-02-12 | 2015-12-09 | 兰诺龙有限公司 | Method of producing microparticles |
| WO2017172769A1 (en) * | 2016-03-28 | 2017-10-05 | The Johns Hopkins University | Biomimetic anisotropic polymeric particles with naturally derived cell membranes for enhanced drug delivery |
-
2017
- 2017-11-20 CN CN201711156593.0A patent/CN108042805B/en active Active
- 2017-11-20 CN CN202110322231.4A patent/CN113209026A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302784A (en) * | 2011-08-22 | 2012-01-04 | 湖北盛齐安生物科技有限公司 | Tumor chemotherapeutic medicinal preparation and preparation method thereof |
| CN104703609A (en) * | 2012-07-19 | 2015-06-10 | 兰诺龙有限公司 | Stem cell microparticles |
| CN105142646A (en) * | 2013-02-12 | 2015-12-09 | 兰诺龙有限公司 | Method of producing microparticles |
| WO2015052526A1 (en) * | 2013-10-09 | 2015-04-16 | Reneuron Limited | Stem cell microparticles and mirna |
| WO2017172769A1 (en) * | 2016-03-28 | 2017-10-05 | The Johns Hopkins University | Biomimetic anisotropic polymeric particles with naturally derived cell membranes for enhanced drug delivery |
Non-Patent Citations (2)
| Title |
|---|
| ICHIRONAKANOA等: "Extracellular vesicles in the biology of brain tumour stem cells – Implications for inter-cellular communication, therapy and biomarker development", 《SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY》 * |
| 王琎: "细胞外囊泡研究新进展", 《中国组织工程研究》 * |
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| CN111265491B (en) * | 2020-03-25 | 2022-05-20 | 江苏省中医药研究院 | Cell microparticle drug delivery system for entrapping allicin and preparation method and application thereof |
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| CN114686417A (en) * | 2020-12-25 | 2022-07-01 | 中国人民解放军海军军医大学第三附属医院 | Cell vesicle preparation for efficiently inhibiting liver cancer, preparation method and application thereof |
| WO2022237247A1 (en) * | 2021-05-12 | 2022-11-17 | 中国医学科学院基础医学研究所 | Use of cellular microparticles in treatment of respiratory viral pneumonia |
| WO2022257849A1 (en) * | 2021-06-10 | 2022-12-15 | 中山大学 | Preparation method for engineered vesicle and application thereof |
| CN114395531A (en) * | 2021-12-29 | 2022-04-26 | 镇江市中西医结合医院（镇江市第二人民医院） | Preparation method and application of bioactive substance or drug delivery microvesicle |
| WO2023178810A1 (en) * | 2022-03-25 | 2023-09-28 | 华中科技大学 | Co-drug-loaded cell microparticle formulation and preparation method therefor |
| WO2023197901A1 (en) * | 2022-04-11 | 2023-10-19 | 华中科技大学 | Tumor cell-derived microparticle loaded with succinic acid, preparation method therefor, and use thereof |
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| CN113209026A (en) | 2021-08-06 |
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