CN107913408A - Composite drug-loaded system of a kind of excretion body aptamer liposome and its preparation method and application - Google Patents
Composite drug-loaded system of a kind of excretion body aptamer liposome and its preparation method and application Download PDFInfo
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- CN107913408A CN107913408A CN201711133862.1A CN201711133862A CN107913408A CN 107913408 A CN107913408 A CN 107913408A CN 201711133862 A CN201711133862 A CN 201711133862A CN 107913408 A CN107913408 A CN 107913408A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
Abstract
The invention discloses a kind of composite drug-loaded system of excretion body aptamer liposome, including antitumor drug, the surface of the antitumor drug is coated with nano liposomes, the surface of the nano liposomes is connected with aptamer by chemical bond, the aptamer liposome of load antitumor drug is formed, excretion body is compounded with the aptamer liposome of the load antitumor drug.The invention also discloses the preparation method and applications of above-mentioned composite drug-loaded system.The composite drug-loaded system targeting is good, immune evasion is good, and no cytotoxicity, it is not easy to be degraded by macrophage, it can be applied to the cancer therapy drug chemotherapy of cancer patient, greatly improve cancer therapy drug curative effect, low chemotherapy effect and recurrence caused by drug resistance are reduced, improves the bottleneck problem of current cancer patients undergoing chemotherapy, there is huge application value and application prospect in terms for the treatment of of cancer effect is strengthened.
Description
Technical field
The present invention relates to the targeting drug delivery system technical field of cancer, and in particular to a kind of excretion body-aptamer fat
Composite drug-loaded system of plastid and its preparation method and application.
Background technology
The R and D of nano-medicament carrier have received widespread attention in recent years.Nano-medicament carrier has many excellent
Gesture, such as：Nano particle is easily prepared, modification processing and controllability are good, and medicine, gene or functional molecular can be protected from machine
Degradation of body or some enzymes etc..Some nano particles are while " carrier " role is served as, also with immunologic adjuvant
Function.
Liposome is a kind of artificial imitated vesicle structure, liposome in water when, in the hydrophilic head insertion water of phospholipid molecule, fat
Plastid hydrophobic tail stretches to air, forms the spherical liposomes of double-deck fat molecule after agitation, and diameter can control 25~
In the range of 1000nm, the cholesterol of certain mass would generally be adulterated when preparing liposome, makes its stable structure.Since liposome is excellent
Good hydrophilic and hydrophobic performance, therefore gene, albumen and small-molecule drug carrier can be used as.By the use of natural grease plastid as
Advantage with following several respects during pharmaceutical carrier：Size is controllable, easy to operate；Cost is low, and waste is few；Carrier medicine carrying efficiency is high, can
Carry hydrophilic, dewatering medicament.However, natural grease plastid lacks function, although and artificial synthesized liposome is easy to rhetorical function base
Group, but cost is significantly increased, and building-up process is also more cumbersome.
Aptamers are a kind of through screening obtained single stranded DNA or RNA, are specifically bound by three-dimensional structure with high-affinity
On target, also referred to as " chemical antibody ".Compared with monoclonal antibody, adaptor molecules amount is small, is easy to modification synthesis, low toxicity
Low immunogenicity, tissue permeability are strong etc., and therefore, aptamers are widely used in the necks such as drug targeting treatment and lesion detection
Domain.
Excretion body (Exosome) is the vesica corpusculum secreted by a variety of living cells, wherein a variety of containing protein and RNA etc.
Key component, excretion body plays an important role in a variety of physiology and pathology engineering, including malignant tumour.For some diseases
Disease, especially now threatens the mankind maximum malignant tumour, and the targeting transport of medicine and the resistance to the action of a drug of tumour are faced all the time
The problem of bed.
The content of the invention
For the deficiency and defect mentioned in background above technology, it is an object of the present invention to provide a kind of targeting it is good,
The good excretion body of the immune evasion-composite drug-loaded system of aptamer liposome, and provide a kind of above-mentioned composite drug-loaded
The preparation method and applications of system.
In order to solve the above technical problems, technical solution proposed by the present invention is：
A kind of excretion body-composite drug-loaded system of aptamer liposome, including antitumor drug, the antitumor drug
Surface be coated with nano liposomes, the surface of the nano liposomes is connected with aptamer by chemical bond, is formed negative
The aptamer liposome of carrying anti-tumor medicine, is compounded with the aptamer liposome of the load antitumor drug outer
Secrete body.
The excretion body in tumour cell source can play the role of suppressing tumour growth by the DC immune responses mediated；And
And since excretion body diameter is smaller, can pass through blood-brain barrier and enter brain blood circulation, excretion body can by antitumor RNA with
Protein is brought into, and therapeutic effect is played to brain primary tumor.Compared with the immune cell therapy of tumour, excretion body has bright
Aobvious advantage, under the slightly acidic environment of tumour cell, immunocyte can not give full play to its effect.For excretion body, acid
Property environment be more conducive to excretion body by tumour cell absorb absorb, further to play the antitumor activity energy of excretion body.But outside
Secrete body and do not possess targeting in itself, limit its antineoplaston effect.Excretion body and surface are passed through chemistry by the present invention first
The liposome of key connection aptamer is mutually compound, forms the composite drug-loaded system of " excretion body-aptamer-liposome ".
Antitumor drug is coated using nano liposomes, on the surface of nano liposomes by chemical key connection aptamer, and
The compound excretion body on aptamer liposome, obtains the complex of excretion body-aptamer liposome, as compound load
Medicine system.Excretion body and aptamer and lipid bluk recombination, aptamer liposome are improved into the targeting of excretion body,
By the synergistic effect between excretion body, aptamer, liposome, make the composite drug-loaded system of gained that there is good targeting
With immune evasion, being formed has the function of cancer cell targets identification and the cancer therapy drug system with powerful lethality.It can incite somebody to action
It is applied to the cancer therapy drug chemotherapy of cancer patient, greatly improves cancer therapy drug curative effect, reduces due to drug resistance and cause
Low chemotherapy effect and recurrence, improve the bottleneck problem of current cancer patients undergoing chemotherapy, have in terms for the treatment of of cancer effect is strengthened
Huge application value.
As preferable scheme, the excretion body can be the excretion body by being extracted after various cancer cell cultures, such as
A549 cells, MCF7 cells or Hep2 cells.
As preferable scheme, the excretion body is mixed by the aptamer liposome with loading antitumor drug
Afterwards, react, merged on the aptamer liposome of load antitumor drug through thawing.
As preferable scheme, the composite drug-loaded system is in granular form, its particle diameter is 100nm~150nm.
As preferable scheme, the aptamer is aptamer AS1411, aptamer S6, nucleic acid adaptation
Any one in body sgc8, aptamer A10, aptamer S2.2 or aptamer TLS11a.
As preferable scheme, the antitumor drug is adriamycin, fluorouracil, oxaliplatin, carboplatin, ring phosphinylidyne
It is any one in amine, vincaleukoblastinum, cytarabine, mustargen, phosphinothioylidynetrisaziridine, methotrexate (MTX), Tegafur, mitomycin and daunorubicin
Kind.
As preferable scheme, the nano liposomes include phosphatide, cholesterol component and feature phosphatide.
Wherein, the phosphatide is selected from one or both of soybean lecithin and egg yolk lecithin；
The one kind or more of the cholesterol component in cholesterol and its derivative, cholestane, cholic acid and bile acid
Kind；
The feature phosphatide is selected from distearoylphosphatidylethanolamine-polyethylene glycol-maleimide cross-linking agent, two
Stearoyl phosphatidyl monoethanolamine-polyethylene glycol-carboxyl cross-linking agent, distearoylphosphatidylethanolamine-polyethylene glycol-N- ambers
One or more in amber acid imide and distearoylphosphatidylethanolamine-polyethylene glycol-amino cross-linking agent.
The technical concept total as one, another aspect of the present invention provide a kind of above-mentioned excretion body-aptamer fat
The preparation method of the composite drug-loaded system of plastid, comprises the following steps：
S1, by phosphatide, cholesterol component and feature phosphatide in molar ratio (50~60)：(25~30)：(10~25)
Dissolving in organic solvent, prepares blank liposome；
S2, prepare the liposome for wrapping up antitumor drug using blank liposome obtained by step S1 with antitumor drug；
Aptamer, be connected to obtained by step S2 on the liposome of parcel antitumor drug by S3, and it is anti-swollen to prepare load
The aptamer liposome of tumor medicine；
S4, culture excretion body cell, collect incubation in remaining fluid nutrient medium, by remaining fluid nutrient medium into
Excretion body precipitation is obtained after row ultracentrifugation, then configures excretion body stock solution；
S5, the aptamer fat by excretion body stock solution obtained by step S4 and load antitumor drug obtained by step S3
Plastid mix, multigelation several times, up to excretion body-composite drug-loaded system of aptamer liposome.
As preferable scheme, the step S1 is specially：By phosphatide, cholesterol component and feature phosphatide by mole
Than for (50~60)：(25~30)：(10~25) are dissolved in the mixed solution of methanol and chloroform, at 37 DEG C using rotating speed as
100rpm/min~200rpm/min rotates and vacuumizes to form dry lipid membrane, removes organic solvent；The lipid is thin
Film is dialysed in ammonium sulfate after aquation, is redispersed in ultra-pure water solution and is obtained blank liposome.
As preferable scheme, the step S3 specifically, parcel antitumor drug liposome solution according to
The molar ratio of aptamer and feature phosphatide is 1：(5~10) aptamer is added, room temperature is anti-under nitrogen environmental protection
Should, and dialyse washing unnecessary feature phosphatide, antitumor drug and aptamer, obtain the nucleic acid of load antitumor drug
Aptamers liposome.
As preferable scheme, the step S4 is specially：By excretion body cell in 36 DEG C~38 DEG C, 4%~5%CO2
Under the conditions of cultivate, serum-concentration be 9%~10%, collect incubation in remaining fluid nutrient medium, 250g~350g centrifugation
Power centrifugation 9min~10min removes cell, then takes supernatant to continue 2000g~2100g centrifugation 9min~10min removings dead thin
Born of the same parents, then take supernatant to remove cell fragment with 10000g~11000g centrifugal forces 30min~32min, finally take supernatant
Liquid obtains the protein of excretion body precipitation and pollution with 100000g~110000g centrifugal force ultracentrifugations 70min~75min,
After PBS buffer is cleaned, 10000g~11000g centrifugal force high speed centrifugation obtains excretion body precipitation again, is finally delayed with PBS
Fliud flushing configures excretion body stock solution, and the centrifugal process is maintained at 4 DEG C~5 DEG C and carries out, and keeps sterile environment, described
Serum is the serum for removing excretion body.
As preferable scheme, the step S5 is specially：Excretion body stock solution is taken with loading the core of antitumor drug
Sour aptamers liposome mixing, with liquid nitrogen cryopreservation 5min~10min, then room temperature ultrasound 30min~32min thawings, circulation is frozen
Melt several times, obtain excretion body-composite drug-loaded system of aptamer liposome.
The technical concept total as one, another aspect of the present invention provide a kind of above-mentioned excretion body-aptamer
The composite drug-loaded system of liposome or the excretion body-aptamer lipid bluk recombination being prepared by above-mentioned preparation method carry
Application of the medicine system in cancer therapy drug.
Compared with prior art, the advantage of the invention is that：Excretion body is applied to antitumor drug and carries medicine system by the present invention
In system, the nano liposomes prepared using phosphatide coat antitumor drug, pass through chemical key connection on the surface of nano liposomes
Aptamer, and the compound excretion body on aptamer liposome, obtain " excretion body-aptamer-liposome "
Complex, as composite drug-loaded system.Act synergistically between excretion body, aptamer, liposome, make gained composite drug-loaded
System has good biocompatibility, targeting and immune evasion, and no cytotoxicity, is not easy to be degraded by macrophage,
The cancer therapy drug chemotherapy of cancer patient is can be applied to, cancer therapy drug curative effect is greatly improved, reduces due to drug resistance and cause
Low chemotherapy effect and recurrence, improve the bottleneck problem of current cancer patients undergoing chemotherapy, have in terms for the treatment of of cancer effect is strengthened
Huge application value and application prospect.
Brief description of the drawings
Fig. 1 is the transmission electron microscope figure (TEM) of the composite drug-loaded system of the present invention.
Fig. 2 is the grain size distribution of the composite drug-loaded system of the present invention.
Fig. 3 is that the fluorescence distribution curve of the composite drug-loaded system frozen-thaw process of the present invention (is respectively freeze thawing 0 time, 5 times, 10 times
With 15 times).
Embodiment
For the ease of understanding the present invention, the present invention is made below in conjunction with Figure of description and preferred embodiment more complete
Face, meticulously describe, but protection scope of the present invention is not limited to embodiment in detail below.
Unless otherwise defined, all technical terms used hereinafter and the normally understood implication of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection domain.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1：
A kind of preparation method of excretion body of the invention-composite drug-loaded system of aptamer liposome, including following step
Suddenly：
(1) culture of excretion body cell
By A549 cells in 37 DEG C, 5%CO2Under the conditions of be incubated at 75cm2Blake bottle in, serum-concentration 10%, thin
When born of the same parents' degrees of fusion reaches 50%~60%, the serum free culture system of no excretion body is replaced with 2~3 days, collect cell culture medium at 4 DEG C
300g centrifugal forces 10min removes cell；
(2) extraction of excretion body
Cell culture medium 2000g (centrifugal force) is centrifuged into 10min, to remove dead cell；Centrifuged supernatant is taken to be placed in
In 15mL centrifuge tubes, 20000g is centrifuged 30 minutes, removes cell fragment and microcapsule bubble；Supernatant is taken to use 0.22 μm of filtering
Device is filtered, and further removes impurity；Using PBS buffer trim centrifuge tube, 120000g centrifugations 90min (60min≤from
The heart time≤120min)；Carefully remove supernatant, it is seen that excretion body ball；Cleaned using 1mL PBS buffer, 100000g
Centrifuge 70min；Topple over supernatant, the μ L PBS buffer of 50 μ L~100 is resuspended, and obtains excretion body stock solution；All of above centrifugation
Operation is in 4 DEG C of progress；Excretion body protein concentration is surveyed using BCA methods；The excretion body of acquisition is identified：10 μ g excretion bodies are molten
Add 4XSDS albumen sample-loading buffers in 20 μ L PBS, carry out WB detection excretion body marker proteins, TSG101, Alix or CD81.
(3) preparation of aptamer
1mL methanol/chloroform mixed solution is added in the centrifuge tube equipped with 8OD (optical density) aptamer S6 powder,
Ice-water bath 10min after boiling water bath 10min, obtains aptamer S6 stock solutions；Aptamer S6 stock solutions are shifted again
Into the screw socket bottle of 5.0mL lucifuges processing for cleaning drying, 460 μ L DSPE-PEG2000-NHS storing solution (distearyls are added
Phosphatidyl acetyl amine-n-hydroxysuccinimide-polyethylene glycol 2000), ultrasonic disperse is uniform, magnetic agitation bonding 2h~3h；
Then dialysis removes the aptamer S6 not being bonded, obtains pure DSPE-PEG2000-S6.
(4) preparation of the aptamer liposome of antitumor drug is loaded
The μ L of the HSPC stock solutions (soybean lecithin) of about 1mL, 400 μ L~600 are added in the 5mL heart bottles for cleaning drying
Cholesterol stock solution, 2mL~3mL DSPE-PEG2000-S6 stock solutions, 0.5min~1min ultrasonic mixings are uniform, 37 DEG C
Slow rotary evaporation is into film down；Then 5mL ammonium sulfate stock solution aquations are used, ultrasound some minutes obtain to clear
Aptamer liposome；5mL aptamers liposome is taken to be mixed with 5mL adriamycin stock solutions, water-bath 2h at 65 DEG C~
3h, then non-encapsulated adriamycin is removed with ultra-pure water dialysis 2h~3h, obtain the aptamer liposome of load adriamycin.
(5) preparation of excretion body-composite drug-loaded system of aptamer liposome
Take excretion body stock solution and load adriamycin aptamer liposome mix, with liquid nitrogen cryopreservation 5min~
10min, then room temperature ultrasound thawing 30min, circulating freezing resistance several times, obtain excretion body-aptamer of load adriamycin
The heterozygote of liposome, is excretion body-composite drug-loaded system of aptamer liposome of the present invention.(used in aptamers
Fluorophor containing rhodamine and NBD on phosphatide, both fluorophors formation FRET (fluorescence resonance energy transfer) is right, uses
To carry out deciding degree during freeze thawing).
Transmissioning electric mirror test, its transmission electron microscope are carried out to the composite drug-loaded system of gained excretion body-aptamer liposome
Figure is as shown in Figure 1.As seen from Figure 1, the composite drug-loaded system graininess spherical in shape.Fig. 2 is the particle diameter of the composite drug-loaded system of gained
Distribution map, from figure 2 it can be seen that the particle diameter of composite drug-loaded system is mainly distributed on 100nm~150nm sections.Fig. 3 is compound
The fluorescence distribution curve of drug-loading system frozen-thaw process, as seen from Figure 3, with the increase of number of freezing and thawing, fluorescence resonance phenomenon weakens,
Rhodamine and the increase of NBD groups relative distance, illustrate in liposome successful fusion into excretion body.
Embodiment 2：
A kind of preparation method of excretion body of the invention-composite drug-loaded system of aptamer liposome, including following step
Suddenly：
(1) culture of excretion body cell
By MCF7 cells in 37 DEG C, 5%CO2Under the conditions of be incubated at 75cm2Blake bottle in, serum-concentration 10%, thin
When born of the same parents' degrees of fusion reaches 50%~60%, the serum free culture system of no excretion body is replaced with 2~3 days, collect cell culture medium at 4 DEG C
300g centrifugal forces 10min removes cell；
(2) extraction of excretion body
Cell culture medium 2000g (centrifugal force) is centrifuged into 10min, to remove dead cell；Centrifuged supernatant is taken to be placed in
In 15mL centrifuge tubes, 20000g centrifugation 30min, remove cell fragment and microcapsule bubble；Supernatant is taken to use 0.22 μm of filtering
Device is filtered, and further removes impurity；Using PBS buffer trim centrifuge tube, 120000g centrifugations 90min (60min≤from
The heart time≤120min)；Carefully remove supernatant, it is seen that excretion body ball；Cleaned using 1mL PBS buffer, 100000g
Centrifuge 70min；Topple over supernatant, the μ L PBS buffer of 50 μ L~100 is resuspended, and obtains excretion body stock solution；All of above centrifugation
Operation is in 4 DEG C of progress；Excretion body protein concentration is surveyed using BCA methods；The excretion body of acquisition is identified：10 μ g excretion bodies are molten
Add 4XSDS albumen sample-loading buffers in 20 μ L PBS, carry out WB detection excretion body marker proteins, TSG101, Alix or CD81.
(3) preparation of aptamer
It is molten that 1mL methanol/chloroform mixing is added in the centrifuge tube equipped with 8OD (optical density) aptamer AS1411 powder
Liquid, ice-water bath 10min after boiling water bath 10min, obtains aptamer AS1411 stock solutions；Aptamer AS1411 is stored up again
Standby solution is transferred in the screw socket bottle for the 5.0mL lucifuges processing for cleaning drying, adds 460 μ L DSPE-PEG2000-NHS deposits
Liquid (distearoylphosphatidyl acetyl amine-n-hydroxysuccinimide-polyethylene glycol 2000), ultrasonic disperse is uniform, magnetic agitation
It is bonded 2h~3h；Then dialysis removes the aptamer AS1411 not being bonded, obtains pure DSPE-PEG2000-
AS1411。
(4) preparation of the aptamer liposome of antitumor drug is loaded
The μ L of the HSPC stock solutions (soybean lecithin) of about 1mL, 400 μ L~600 are added in the 5mL heart bottles for cleaning drying
Cholesterol stock solution, 2mL~3mL DSPE-PEG2000-AS1411 stock solutions, 0.5min~1min ultrasonic mixings are uniform,
Slow rotary evaporation is into film at 37 DEG C；Then 5mL ammonium sulfate stock solution aquations are used, ultrasound some minutes to clear,
Obtain aptamer liposome；5mL aptamers liposome is taken to be mixed with 5mL fluorouracil stock solutions, water at 65 DEG C
2h~3h is bathed, then non-encapsulated fluorouracil is removed with ultra-pure water dialysis 2h~3h, obtains the nucleic acid adaptation of load fluorouracil
Body fat plastid.
(5) preparation of excretion body-composite drug-loaded system of aptamer liposome
Take excretion body stock solution and load fluorouracil aptamer liposome mix, with liquid nitrogen cryopreservation 5min~
10min, then room temperature ultrasound thawing 30min, circulating freezing resistance several times, obtain excretion body-nucleic acid adaptation of load fluorouracil
The heterozygote of body fat plastid, is excretion body-composite drug-loaded system of aptamer liposome of the present invention.
Embodiment 3：
A kind of preparation method of excretion body of the invention-composite drug-loaded system of aptamer liposome, including following step
Suddenly：
(1) culture of excretion body cell
By Hep2 cells in 37 DEG C, 5%CO2Under the conditions of be incubated at 75cm2Blake bottle in, serum-concentration 10%, thin
When born of the same parents' degrees of fusion reaches 50%~60%, the serum free culture system of no excretion body is replaced with 2~3 days, collect cell culture medium at 4 DEG C
300g centrifugal forces 10min removes cell；
(2) extraction of excretion body
Cell culture medium 2000g (centrifugal force) is centrifuged into 10min, to remove dead cell；Centrifuged supernatant is taken to be placed in
In 15mL centrifuge tubes, 20000g centrifugation 30min, remove cell fragment and microcapsule bubble；Supernatant is taken to use 0.22 μm of filtering
Device is filtered, and further removes impurity；Using PBS buffer trim centrifuge tube, 120000g centrifugations 90min (60min≤from
The heart time≤120min)；Carefully remove supernatant, it is seen that excretion body ball；Cleaned using 1mL PBS buffer, 100000g
Centrifuge 70min；Topple over supernatant, the μ L PBS buffer of 50 μ L~100 is resuspended, and obtains excretion body stock solution；All of above centrifugation
Operation is in 4 DEG C of progress；Excretion body protein concentration is surveyed using BCA methods；The excretion body of acquisition is identified：10 μ g excretion bodies are molten
Add 4XSDS albumen sample-loading buffers in 20 μ L PBS, carry out WB detection excretion body marker proteins, TSG101, Alix or CD81.
(3) preparation of aptamer
1mL methanol/chloroform mixed solution is added in the centrifuge tube equipped with 8OD (optical density) aptamer A10 powder,
Ice-water bath 10min after boiling water bath 10min, obtains aptamer A10 stock solutions；Aptamer A10 stock solutions are turned again
Move in the screw socket bottle for the 5.0mL lucifuges processing for cleaning drying, add 460 μ L DSPE-PEG2000-NHS storing solution (distearyls
Acyl phosphatidyl acetyl amine-n-hydroxysuccinimide-polyethylene glycol 2000), ultrasonic disperse is uniform, magnetic agitation bonding 2h~
3h；Then dialysis removes the aptamer A10 not being bonded, obtains pure DSPE-PEG2000-A10.
(4) preparation of the aptamer liposome of antitumor drug is loaded
The μ L of the HSPC stock solutions (soybean lecithin) of about 1mL, 400 μ L~600 are added in the 5mL heart bottles for cleaning drying
Cholesterol stock solution, 2mL~3mL DSPE-PEG2000-A10 stock solutions, 0.5min~1min ultrasonic mixings are uniform, and 37
Slow rotary evaporation is into film at DEG C；Then 5mL ammonium sulfate stock solution aquations are used, ultrasound some minutes obtain to clear
To aptamer liposome；5mL aptamers liposome is taken to be mixed with 5mL oxaliplatin stock solutions, water-bath at 65 DEG C
2h~3h, then non-encapsulated oxaliplatin is removed with ultra-pure water dialysis 2h~3h, obtain the aptamer of load oxaliplatin
Liposome.
(5) preparation of excretion body-composite drug-loaded system of aptamer liposome
Take excretion body stock solution and load oxaliplatin aptamer liposome mix, with liquid nitrogen cryopreservation 5min~
10min, then room temperature ultrasound thawing 30min, circulating freezing resistance several times, obtain excretion body-nucleic acid adaptation of load oxaliplatin
The heterozygote of body fat plastid, is excretion body-composite drug-loaded system of aptamer liposome of the present invention.
Claims (10)
1. a kind of excretion body-composite drug-loaded system of aptamer liposome, including antitumor drug, the antitumor drug
Surface is coated with nano liposomes, it is characterised in that the surface of the nano liposomes is connected with nucleic acid by chemical bond and is adapted to
Body, forms the aptamer liposome of load antitumor drug, the aptamer liposome of the load antitumor drug
On be compounded with excretion body.
2. the excretion body according to claim 1-composite drug-loaded system of aptamer liposome, it is characterised in that described
Excretion body is obtained by being extracted after A549 cells, MCF7 cells or Hep2 cell culture.
3. the excretion body according to claim 1-composite drug-loaded system of aptamer liposome, it is characterised in that described
After excretion body with the aptamer liposome of load antitumor drug by mixing, reacted through thawing, merge and loading
On the aptamer liposome of antitumor drug.
4. the excretion body according to claim 1-composite drug-loaded system of aptamer liposome, it is characterised in that described
Composite drug-loaded system is in granular form, its particle diameter is 100nm~150nm.
5. the excretion body according to claim 1-composite drug-loaded system of aptamer liposome, it is characterised in that described
Aptamer is aptamer AS1411, aptamer S6, aptamer sgc8, aptamer A10, nucleic acid are fitted
Any one in ligand S2.2 or aptamer TLS11a；The antitumor drug is adriamycin, fluorouracil, Ao Shali
Platinum, carboplatin, endoxan, vincaleukoblastinum, cytarabine, mustargen, phosphinothioylidynetrisaziridine, methotrexate (MTX), Tegafur, mitomycin and soft red mould
Any one in element.
6. according to excretion body according to any one of claims 1 to 5-composite drug-loaded system of aptamer liposome, it is special
Sign is that the nano liposomes include phosphatide, cholesterol component and feature phosphatide；
Wherein, the phosphatide is selected from one or both of soybean lecithin and egg yolk lecithin；
One or more of the cholesterol component in cholesterol and its derivative, cholestane, cholic acid and bile acid；
The feature phosphatide is selected from distearoylphosphatidylethanolamine-polyethylene glycol-maleimide cross-linking agent, distearyl
Acylphosphatidyl ethanolamine-polyethylene glycol-carboxyl cross-linking agent, distearoylphosphatidylethanolamine-polyethylene glycol-N- succinyls
One or more in imines and distearoylphosphatidylethanolamine-polyethylene glycol-amino cross-linking agent.
It is 7. a kind of such as the system of excretion body according to any one of claims 1 to 6-composite drug-loaded system of aptamer liposome
Preparation Method, comprises the following steps：
S1, by phosphatide, cholesterol component and feature phosphatide in molar ratio (50~60)：(25~30)：(10~25) dissolve
In organic solvent, blank liposome is prepared；
S2, prepare the liposome for wrapping up antitumor drug using blank liposome obtained by step S1 with antitumor drug；
Aptamer, be connected to obtained by step S2 on the liposome of parcel antitumor drug by S3, prepares load antineoplastic
The aptamer liposome of thing；
S4, culture excretion body cell, collect remaining fluid nutrient medium in incubation, remaining fluid nutrient medium are surpassed
Excretion body precipitation is obtained after high speed centrifugation, then configures excretion body stock solution；
S5, the aptamer liposome by excretion body stock solution obtained by step S4 and load antitumor drug obtained by step S3
Mixing, multigelation several times, up to excretion body-composite drug-loaded system of aptamer liposome.
8. preparation method according to claim 7, it is characterised in that the step S4 is specially：Excretion body cell is existed
36 DEG C~38 DEG C, 4%~5%CO2Under the conditions of cultivate, serum-concentration be 9%~10%, collect incubation in remaining liquid
Culture medium, 250g~350g centrifugal forces 9min~10min remove cell, then take supernatant continue 2000g~2100g from
Heart 9min~10min removes dead cell, then takes supernatant to be removed with 10000g~11000g centrifugal forces 30min~32min
Cell fragment is removed, finally takes supernatant to obtain excretion with 100000g~110000g centrifugal force ultracentrifugations 70min~75min
Body precipitates and the protein of pollution, and after PBS buffer is cleaned, 10000g~11000g centrifugal force high speed centrifugation obtains outer again
Body precipitation to be secreted, finally configures excretion body stock solution with PBS buffer, the centrifugal process is maintained at 4 DEG C~5 DEG C and carries out,
And sterile environment is kept, the serum is the serum for removing excretion body.
9. preparation method according to claim 7, it is characterised in that the step S5 is specially：Take excretion body deposit molten
Liquid is mixed with loading the aptamer liposome of antitumor drug, and with liquid nitrogen cryopreservation 5min~10min, then room temperature is ultrasonic
30min~32min melts, and circulating freezing resistance several times, obtains excretion body-composite drug-loaded system of aptamer liposome.
10. such as excretion body according to any one of claims 1 to 6-composite drug-loaded system of aptamer liposome or such as
The excretion body that preparation method any one of the claim 7~9 is prepared-composite drug-loaded system of aptamer liposome
Application of the system in cancer therapy drug.
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| CN110917215A (en) * | 2019-12-04 | 2020-03-27 | 陕西佰傲再生医学有限公司 | Complex, tissue repair material, and preparation method and application thereof |
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