CN105496961A - Targeted lipidosome drug-loading system containing aptamers, preparation method and application - Google Patents
Targeted lipidosome drug-loading system containing aptamers, preparation method and application Download PDFInfo
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Abstract
The invention relates to a targeted lipidosome drug-loading system containing aptamers and a preparation method of the system. The targeted lipidosome drug-loading system containing the aptamers comprises the aptamers, an antitumor drug and nano-liposomes prepared from sphingomyelin, wherein the antitumor drug is wrapped with the nano-liposomes and the aptamers are connected onto the nano-liposomes through chemical bonds for specifically identifying tumor cells. The nano-liposomes are prepared from sphingomyelin and are connected with an appropriate quantity of the aptamers after the antitumor drug is wrapped with the nano-liposomes, sphingomyelin contains more amido bonds and can resist chemical and biological degradation better, protect stability of a lipidosome structure and increase the drug accumulation content of the tumor cells, and accordingly, the antitumor effect is improved.
Description
Technical field
The present invention relates to the targeting drug delivery system for cancer, more particularly, relate to a kind of aptamers target liposomes drug-loading system and preparation method and application.
Background technology
Liposome (Liposome), the vesicle with bilayer structure formed by phospholipid or other lipids is a kind of stable in properties, in blood circulation, have the non-virus carrier of long-acting half-life.Antitumor drug is encapsulated in liposome, not only can protects medicine decomposition from body enzyme before entering lesions position, prolong drug circulation time, increase medicine in the concentration of lesions position and action time, improve curative effect, reduce toxicity; And compare with other transmission system, liposome can provide superior biocompatibility, biodegradability, hypotoxicity, dimensional controllability and function of surface modifiability.
Although the Liposomal formulation of antitumor drug transmits the high efficiency of medicine, reduce the toxic and side effects of antitumor drug, enhance the therapeutic effect of cancer.But the conventional liposome prepared with traditional lecithin, cholesterol is many by reticuloendothelial system (reticuloendothelialsystem in vivo, RES) engulf, in blood circulation, residence time is shorter, and its physical and chemical stability is poor, store and easily occur to merge in application process to assemble, entrapped drug easily leaks, surface functional group is few, not easily carry out multifunction modification, and without targeting, function singleness etc.Therefore, the stability of antitumor medicinal liposome preparation, targeting and drug delivery efficiency have much room for improvement.
Aptamers is a class through screening the single stranded DNA or RNA that obtain, by three dimensional structure with high-affinity specific binding on target, also referred to as " chemical antibody ".Compare with monoclonal antibody, adaptor molecules amount is little, to be easy to modify synthesis, low toxicity reduced immunogenicity, tissue permeability strong etc., therefore, aptamers is widely used in drug targeting and treats and the field such as lesion detection.
In prior art, aptamer is received on antitumor medicinal liposome by chemical bond-linking by existing research, to prepare aptamers target liposomes drug-loading system, strengthens its stability, targeting and drug delivery efficiency.But these aptamers target liposomes drug-loading systems still adopt conventional phospholipid as raw material preparations such as lecithin, be difficult to the chemistry in opposed body and biological degraded, be thus also unsuitable for the less nano-particle of preparation size.
Summary of the invention
The technical problem to be solved in the present invention is, the defect that the aptamers target liposomes drug-loading system stability prepared for existing conventional phospholipid is not high, provide a kind of use sphingomyelins to prepare aptamers target liposomes drug-loading system and preparation method and application.
The technical solution adopted for the present invention to solve the technical problems is: construct a kind of aptamers target liposomes drug-loading system, the nanometer liposome prepared by aptamer, antitumor drug and use sphingomyelins forms, wherein said antitumor drug is wrapped in described nanometer liposome, and described aptamer is connected on specially recognizing tumor cells on described nanometer liposome by chemical bond-linking.
According in aptamers target liposomes drug-loading system of the present invention, nanometer liposome prepared by described use sphingomyelins comprises sphingomyelins, phospholipid stabilizing agent, cholesterol composition and functional phospholipid; Wherein, described phospholipid stabilizing agent is selected from one or more in DSPE-PEG, DMPEA-Polyethylene Glycol and PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol; Described cholesterol composition be selected from cholesterol and derivant, cholestane, cholic acid and bile acid one or more; Described functional phospholipid be selected from DSPE-PEG-maleimide cross-linking agent, DSPE-PEG-carboxyl cross-linking agent, DSPE-PEG-N-butanimide and DSPE-PEG-amino cross-linking agent one or more.
According in aptamers target liposomes drug-loading system of the present invention, the particle diameter of described aptamers target liposomes drug-loading system is 100-120nm.
According in aptamers target liposomes drug-loading system of the present invention, described aptamer is sgc8, A10, S2.2, AS1411 or TLS11a; Described antitumor drug is vincristine sulfate, amycin, daunorubicin, cisplatin or paclitaxel.
According in aptamers target liposomes drug-loading system of the present invention; preferably; the nanometer liposome that described aptamers target liposomes drug-loading system is prepared by aptamer sgc8, vincristine sulfate and use sphingomyelins forms, and the nanometer liposome wherein using sphingomyelins to prepare comprises following composition: sphingomyelins, DSPE-PEG, cholesterol and DSPE-PEG-maleimide cross-linking agent.
Present invention also offers a kind of preparation method of aptamers target liposomes drug-loading system, this preparation method comprises:
S1, by sphingomyelins, cholesterol composition, phospholipid stabilizing agent and functional phospholipid in molar ratio for 50-60:35-45:4-5:0.2-0.4 dissolve prepare blank liposome in organic solvent;
The liposome of the blank liposome preparation parcel antitumor drug of S2, use step S1;
S3, aptamer is connected on the liposome of parcel antitumor drug, preparation aptamers target liposomes drug-loading system.
According in the preparation method of aptamers target liposomes drug-loading system of the present invention, described step S1 is specially: sphingomyelins composition, cholesterol composition, phospholipid stabilizing agent and functional phospholipid are dissolved in molar ratio the mixing in chloroform for 50-60:35-45:4-5:0.2-0.4, at 50-70 DEG C, with rotating speed be that 100-200rpm/min rotates and evacuation forms dry lipid membrane, remove organic solvent; Described lipid membrane is dispersed in citrate buffer, at liquid nitrogen and 50-60 DEG C of water-bath multigelation repeatedly, and obtains blank liposome after particle diameter screening.
According in the preparation method of aptamers target liposomes drug-loading system of the present invention; when adopting aptamer to be sgc8 in described step S3; be that 1:5-1:10 adds aptamer sgc8 according to the mol ratio of aptamer sgc8 and functional phospholipid in the solution of the liposome of parcel antitumor drug; nitrogen protection atmosphere at room temperature reacts; and functional phospholipid, antitumor drug and aptamer that dialysis washing is unnecessary, obtain described aptamers target liposomes drug-loading system.
Present invention also offers one and treat and/or prevent tumour medicine, this active component treating and/or preventing tumour medicine is foregoing aptamers target liposomes drug-loading system.
Present invention also offers foregoing aptamers target liposomes drug-loading system and treat and/or prevent the application in tumor.
Implement aptamers target liposomes drug-loading system of the present invention and preparation method and application; there is following beneficial effect: the present invention uses sphingomyelins to prepare nanometer liposome; appropriate aptamer is connected after parcel antitumor drug; its sphingomyelin contains more amido link can resist chemistry and biological degraded better; stablizing of protection liposome structure; improve the drug-rich amount of tumor cell, thus improve antitumous effect.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is the preparation method figure of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention;
Fig. 2 is the structural representation of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention;
Fig. 3 A-C is respectively the transmission electron microscope picture of each stage particle of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention;
Fig. 4 is the grain size distribution of each stage particle of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention;
Fig. 5 is the Binding experiment figure of cem cell and sgc8/VCR-Lipo and VCR-Lipo;
Fig. 6 is the variation diagram of the gross tumor volume of the tumor-bearing mice of different disposal group;
Fig. 7 is tumor-bearing mice time diagram apart from inoculated tumour when different survival rates;
Fig. 8 is the tumor size figure of the lotus tumor BalB/c nude mice of different disposal;
Fig. 9 is the targeting in vivo figure of aptamers target liposomes drug-loading system.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.
The invention provides a kind of aptamers target liposomes drug-loading system, the nanometer liposome prepared by aptamer, antitumor drug and use sphingomyelins forms, wherein antitumor drug is wrapped in nanometer liposome, and aptamer is connected on by chemical bond-linking can specially recognizing tumor cells on nanometer liposome.In the present invention, main sphingomyelins prepares nanometer liposome, and its composition comprises sphingomyelins, phospholipid stabilizing agent, cholesterol composition and functional phospholipid.Its sphingomyelin, as a kind of phospholipid, cooperatively forms the basic substance of liposome with cholesterol composition.This cholesterol composition be selected from cholesterol and derivant, cholestane, cholic acid and bile acid one or more.Cholesterol composition has the effect regulating membrane fluidity, is therefore called as " the mobility buffer agent " of liposome.Phospholipid is primarily of the hydrophilic radical of a phosphate group and quaternary ammonium salt group composition, and the lipophilic group be made up of two longer alkyl.The phospholipid used can be natural phospholipid, also can be synthetic phospholipid.Natural phospholipid is mainly with lecithin, and it is main for having another name called phosphatidylcholine (Phosphatidylcholine is called for short PC), is mainly derived from egg yolk lecithin and soybean phospholipid, aobvious neutral.Synthetic phospholipid mainly contains dipalmitoyl phosphatidyl choline (DPPC), DPPE (DPPE), distearoyl phosphatidylcholine (DSPC) etc., and these all belong to hydrogenated phospholipid class.Select sphingomyelins (sphingomyelin) to substitute conventional phospholipid in the present invention, this is because sphingomyelins contains the aliphatic chain of more amido link connection, chemistry and biological degraded can be resisted better, stablizing of protection liposome structure.
Aforementioned phospholipid stabilizing agent is also referred to as mPEGization phospholipid or mPEG phospholipid, and it adds can make the conventional liposome be made up of sphingomyelins and cholesterol composition become space surely to transport liposome, hidden liposome or long circulating liposomes.This phospholipid stabilizing agent be selected from DSPE-PEG (DSPE-PEG), DMPEA-Polyethylene Glycol (DMPE-PEG) and PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol (PE-PEG) one or more.
Functionalization phospholipid of the present invention is also referred to as functionalization PEG phospholipid, its PEG one end is connected with sphingomyelins, the other end by dimaleoyl imino, complete the modifications such as base, except making liposome have except spatial stability, stealth or macrocyclic feature, functionalization PEG phospholipid modify dimaleoyl imino, complete base etc. and can be connected with nucleic acid aptamer target head and make liposome have active targeting.This functional phospholipid is selected from DSPE-PEG-maleimide cross-linking agent (DSPE-PEG-Mal), DSPE-PEG-carboxyl cross-linking agent (DSPE-PEG-COOH), DSPE-PEG-N-butanimide (DSPE-PEG-NHS) and DSPE-PEG-amino cross-linking agent (DSPE-PEG-NH
2) in one or more, the aptamer according to required connection is determined.
In the present invention, the antitumor drug of aptamers target liposomes drug-loading system parcel can be vincristine sulfate, amycin, daunorubicin, cisplatin or paclitaxel etc.
The aptamer that the present invention connects is sgc8, A10, S2.2, AS1411 or TLS11a.Wherein aptamer sgc8 can targets identification leukemia CCRF-CEM tumor cell, and aptamer A10 can targets identification prostatic neoplasms cell; Aptamer S2.2 and aptamer AS1411 can targets identification breast cancer cell; Aptamer TLS11a can targets identification hepatoma carcinoma cell.
Present invention also offers one and treat and/or prevent tumour medicine, this active component treating and/or preventing tumour medicine is foregoing aptamers target liposomes drug-loading system.Aptamers target liposomes drug-loading system of the present invention has using value treating and/or preventing in tumor.
Present invention also offers the reagent set for the preparation of aforementioned aptamers target liposomes drug-loading system, for three kinds in nanometer liposome these three kinds prepared by aforementioned nucleic acid aptamers, antitumor drug, use sphingomyelins or any two.In addition, present invention also offers aforementioned reagent set and prepare the application treated and/or prevented in tumour medicine.
The present invention is special preparation process and the parameter providing applicable aptamers target liposomes drug-loading system for sphingomyelins also, this is because the chemical constitution of different phospholipid and hydrophilic etc. are different, in prior art, other phospholipid is prepared the step of aptamers target liposomes drug-loading system and parameter and is not exclusively applicable to the present invention.Refer to Fig. 1, be the preparation method of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention, foregoing aptamers target liposomes drug-loading system can be prepared.As shown in the figure, this preparation method comprises:
First, in step sl, sphingomyelins composition, cholesterol composition, phospholipid stabilizing agent and functional phospholipid are prepared blank liposome in organic solvent for 50-60:35-45:4-5:0.2-0.4 dissolves in molar ratio.Preferably, this step S1 is specially: sphingomyelins composition, cholesterol composition, phospholipid stabilizing agent and functional phospholipid are dissolved in molar ratio the mixing in chloroform for 50-60:35-45:4-5:0.2-0.4, at 50-70 DEG C, with rotating speed be that 100-200rpm/min rotates and evacuation forms dry lipid membrane, remove organic solvent; Described lipid membrane is dispersed in citrate buffer, at liquid nitrogen and 50-60 DEG C of water-bath multigelation repeatedly, and obtains blank liposome after particle diameter screening, namely uses nanometer liposome prepared by sphingomyelins.More preferably, the mol ratio of sphingomyelins composition, cholesterol composition, phospholipid stabilizing agent and functionalization phospholipid is 55:40:4.7:0.3.
Subsequently, in step s 2, the liposome of the blank liposome preparation parcel antitumor drug of step S1 is used.Preferably, this step S2 is specially: antitumor drug and described blank liposome are dissolved according to mass ratio 1:9-11, and regulates aqueous phase pH for neutral, is placed on 50-65 DEG C of water-bath 5-10 minute, the solution of the liposome of obtained parcel antitumor drug.
Finally, in step s3, aptamer is connected on the liposome of parcel antitumor drug, preparation aptamers target liposomes drug-loading system.Preferably; it is that 1:5-1:10 adds aptamer according to the mol ratio of aptamer and functional phospholipid that this step is specially in the solution of the liposome of parcel antitumor drug; nitrogen protection atmosphere at room temperature reacts; and wash unnecessary functional phospholipid, antitumor drug and aptamer, obtain aptamers target liposomes drug-loading system.The nanometer liposome prepared for sphingomyelins especially in the present invention, have studied the optimization connection amount of aptamer, this is another unique distinction of the present invention.This is because with liposome in conjunction with time, very few aptamer can affect the targeting of nano medicament carrying system, and too much aptamers can affect the stability of nano medicament carrying system.Therefore the input amount of the present invention to aptamer is optimized, and the mol ratio of result show nucleic acid aptamers and functional phospholipid is within the scope of 1:5-1:10, and the liposome formed meets the targeted drug delivery system that subsequent experimental requires more.In addition, the present invention also obtains the connection amount of optimization for different aptamer especially sgc8 etc., wherein the mol ratio of aptamer and functional phospholipid is preferably 1:9.
The present invention is by experimental results demonstrate, adopt the inventive method, use aptamers target liposomes drug-loading system prepared by sphingomyelins, compared with the aptamers target liposomes drug-loading system using conventional phospholipid to prepare, its performance is more stable, the drug level arriving tumor by local is higher, and effectively can extend the life span of tumor-bearing mice.The performance of the aptamers target liposomes drug-loading system that the nanometer liposome wherein prepared with aptamer sgc8, vincristine sulfate and sphingomyelins is formed is the most stable, and circulation time is in vivo long.As example, aptamers target liposomes drug-loading system of the present invention and preparation method are specifically described below.The aptamers target liposomes drug-loading system that this embodiment provides is the vincristine sulfate liposome system of targeting Pancytopenia, and its title is abbreviated as sgc8/VCR-Lipo.The nanometer liposome wherein using sphingomyelins to prepare adopts following composition to form: sphingomyelins, DSPE-PEG (DSPE-PEG), cholesterol and DSPE-PEG-maleimide cross-linking agent (DSPE-PEG-Mal).
Referring to Fig. 2, is the structural representation of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention.This aptamers target liposomes drug-loading system sgc8/VCR-Lipo is made up of blank liposome (Lipo), aptamer sgc8, vincristine sulfate (VCR); This blank liposome is the nanometer liposome using sphingomyelins to prepare, i.e. PEG long circulating liposomes.In Fig. 2, label 1 represents phospholipid (Lipid) i.e. sphingomyelins, and label 2 represents cholesterol (Cholesterol), and label 3 represents DSPE-PEG2000, and label 4 represents DSPE-PEG2000-MAL.As shown in the figure, after the blank liposome using sphingomyelins to prepare and vincristine sulfate (VCR) react, VCR is wrapped up, and be connected with aptamer sgc8 by the MAL group of functionalization phospholipid DSPE-PEG2000-MAL, sgc8 energy specific recognition Pancytopenia cell, the sequence of aptamer sgc8 is: 5 '-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAG (T) 10-SH-3 '.
The concrete preparation method of sgc8/VCR-Lipo is as follows:
The preparation of S1, PEG long circulating liposomes (Lipo):
Egg sphingomyelins (sphingomyelin in chloroform will be dissolved in, SM), cholesterol (Cholesterol), DSPE-PEG2000 and DSPE-PEG2000-MAL in molar ratio 50-60:35-45:4-5:0.2-0.4 add in flask at the bottom of garden, with Rotary Evaporators at 50-70 DEG C, be that 100-200rpm/min rotates and opens vacuum pump evacuation 10-20min formation dry film with rotating speed, vacuum drying instrument dried overnight is to remove residual organic solvent.
The citrate buffer (pH=4.0) of the lipid membrane lml300mM at the bottom of bottle is carried out aquation, after 50-60 DEG C of water bath sonicator 20-30min, liquid nitrogen, 50-60 DEG C water-bath multigelation 4-6 time.Product is loaded liposome and squeezes thruster, successively by each 15-25 time of the polycarbonate membrane of 200nm, 100nm, finally cross polycarbonate membrane 15-25 time of 50nm, the blank liposome (Lipo) that formation diameter is modified at the PEG of about 100nm.
The preparation of S2, packaging medicine liposome (VCR-Lipo)
According to antitumor drug: blank liposomes body mass ratio 1:9-11, get 0.5mg vincristine solution (1mg/ml) and corresponding blank liposome, add 1mlNa
2hPO
4solution (500mmol/L, pH9.0) regulates outer aqueous phase pH to be 7.0-7.2, places 50-65 DEG C of water-bath 5-10 minute, i.e. the nanometer liposome VCR-Lipo of preparation parcel vincristine sulfate.
S3, connection aptamer sgc8, preparation sgc8/VCR-Lipo;
Mol ratio according to aptamer and MAL-PEG2000-DSPE is within the scope of 1:5-1:10, gets appropriate VCR-Lipo, adds suitable quantities of nucleic acid aptamers sgc8, react 24 hours at nitrogen protection atmosphere at room temperature.β-the cysteamine adding 2mM removes unreacted MAL group.With ultra-filtration centrifuge tube to sgc8/VCR-Lipo carry out ultrafiltration centrifugal after, after the centrifugal 10min of 10000rpm, add 1mL deionized water, the centrifugal 10min of 10000rpm, wash away free vincristine sulfate, β-cysteamine and aptamer sgc8, obtain aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
embodiment 1
This embodiment 1 prepares aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
The preparation of S1, PEG long circulating liposomes (Lipo):
Egg sphingomyelins (sphingomyelin in chloroform will be dissolved in, SM), cholesterol (Cholesterol), DSPE-PEG2000 and DSPE-PEG2000-MAL in molar ratio 55:40:4.7:0.3 add in flask at the bottom of garden, with Rotary Evaporators at 60 DEG C, be that 200rpm/min rotates and opens vacuum pump evacuation 10min formation dry film with rotating speed, vacuum drying instrument dried overnight is to remove residual organic solvent.
Wherein SM is Avantipolarlipids Products, and article No. is 860061P; Cholesterol is Avantipolarlipids Products, and article No. is 700100P; DSPE-PEG2000 is Avantipolarlipids Products, and article No. is 880128P; DSPE-PEG2000-MAL is Avantipolarlipids Products, and article No. is 880126P.
The citrate buffer (pH=4.0) of the lipid membrane lml300mM at the bottom of bottle is carried out aquation, after 50 DEG C of water bath sonicator 20min, liquid nitrogen, 60 DEG C of water-bath multigelations 5 times.Product is loaded liposome and squeezes thruster, successively by each 20 times of the polycarbonate membrane of 200nm, 100nm, finally cross the polycarbonate membrane 21 times of 50nm, form the blank liposome (Lipo) that diameter is modified at the PEG of about 100nm.
The preparation of S2, packaging medicine liposome (VCR-Lipo)
According to antitumor drug: blank liposomes body mass ratio 1:10, get 0.5mg vincristine solution (1mg/ml) and 5mg blank liposome, add 1mlNa
2hPO
4solution (500mmol/L, pH9.0) regulates outer aqueous phase pH to be 7.0, places 60 DEG C of water-baths 10 minutes, i.e. the nanometer liposome VCR-Lipo of preparation parcel vincristine sulfate.Wherein, vincristine sulfate is Haizheng Medicine Stock Co., Ltd., Zhejiang Prov's product, and lot number is 130501.
S3, connection aptamer sgc8, preparation sgc8/VCR-Lipo;
Mol ratio according to aptamer and MAL-PEG2000-DSPE is within the scope of 1:9, gets appropriate VCR-Lipo, adds suitable quantities of nucleic acid aptamers sgc8, react 24 hours at nitrogen protection atmosphere at room temperature.This aptamer sgc8 is the synthesis of Shanghai bio-engineering corporation, and the nucleotides sequence of sgc8 is classified as 5 '-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAG (T) 10-SH-3 '.β-the cysteamine adding 2mM again removes unreacted MAL group.With ultra-filtration centrifuge tube to sgc8/VCR-Lipo carry out ultrafiltration centrifugal after, after the centrifugal 10min of 10000rpm, add 1mL deionized water, the centrifugal 10min of 10000rpm, wash away free vincristine sulfate, β-cysteamine and aptamer sgc8, obtain aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
embodiment 2
This embodiment 2 prepares aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
The preparation of S1, PEG long circulating liposomes (Lipo):
Egg sphingomyelins (sphingomyelin in chloroform will be dissolved in, SM), cholesterol (Cholesterol), DSPE-PEG2000 and DSPE-PEG2000-MAL in molar ratio 50:45:5:0.2 add in flask at the bottom of garden, with Rotary Evaporators at 70 DEG C, be that 100rpm/min rotates and opens vacuum pump evacuation 20min formation dry film with rotating speed, vacuum drying instrument dried overnight is to remove residual organic solvent.
The product that its Raw SM, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL select is in the same manner as in Example 1.
The citrate buffer (pH=4.0) of the lipid membrane lml300mM at the bottom of bottle is carried out aquation, after 60 DEG C of water bath sonicator 30min, liquid nitrogen, 50 DEG C of water-bath multigelations 6 times.Product is loaded liposome and squeezes thruster, successively by each 25 times of the polycarbonate membrane of 200nm, 100nm, finally cross the polycarbonate membrane 15 times of 50nm, form the blank liposome (Lipo) that diameter is modified at the PEG of about 100nm.
The preparation of S2, packaging medicine liposome (VCR-Lipo)
According to antitumor drug: blank liposomes body mass ratio 1:9, get 0.5mg vincristine solution (1mg/ml) and corresponding blank liposome, add 1mlNa
2hPO
4solution (500mmol/L, pH9.0) regulates outer aqueous phase pH to be 7.2, places 65 DEG C of water-baths 5 minutes, i.e. the nanometer liposome VCR-Lipo of preparation parcel vincristine sulfate.Wherein, the product of vincristine sulfate use is in the same manner as in Example 1.
S3, connection aptamer sgc8, preparation sgc8/VCR-Lipo;
Mol ratio according to aptamer and MAL-PEG2000-DSPE is 1:10, gets appropriate VCR-Lipo, adds suitable quantities of nucleic acid aptamers sgc8, reacts 24 hours at nitrogen protection atmosphere at room temperature.β-the cysteamine adding 2mM removes unreacted MAL group.With ultra-filtration centrifuge tube to sgc8/VCR-Lipo carry out ultrafiltration centrifugal after, after the centrifugal 10min of 10000rpm, add 1mL deionized water, the centrifugal 10min of 10000rpm, wash away free vincristine sulfate, β-cysteamine and aptamer sgc8, obtain aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
embodiment 3
This embodiment 3 prepares aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
The preparation of S1, PEG long circulating liposomes (Lipo):
Egg sphingomyelins (sphingomyelin in chloroform will be dissolved in, SM), cholesterol (Cholesterol), DSPE-PEG2000 and DSPE-PEG2000-MAL in molar ratio 60:35:4:0.4 add in flask at the bottom of garden, with Rotary Evaporators at 50 DEG C, be that 200rpm/min rotates and opens vacuum pump evacuation 15min formation dry film with rotating speed, vacuum drying instrument dried overnight is to remove residual organic solvent.
The product that its Raw SM, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL select is in the same manner as in Example 1.
The citrate buffer (pH=4.0) of the lipid membrane lml300mM at the bottom of bottle is carried out aquation, after 55 DEG C of water bath sonicator 25min, liquid nitrogen, 55 DEG C of water-bath multigelations 4 times.Product is loaded liposome and squeezes thruster, successively by each 15 times of the polycarbonate membrane of 200nm, 100nm, finally cross the polycarbonate membrane 25 times of 50nm, form the blank liposome (Lipo) that diameter is modified at the PEG of about 100nm.
The preparation of S2, packaging medicine liposome (VCR-Lipo)
According to antitumor drug: blank liposomes body mass ratio 1:11, get 0.5mg vincristine solution (1mg/ml) and corresponding blank liposome, add 1mlNa
2hPO
4solution (500mmol/L, pH9.0) regulates outer aqueous phase pH to be 7.0, places 50 DEG C of water-baths 8 minutes, i.e. the nanometer liposome VCR-Lipo of preparation parcel vincristine sulfate.Wherein, the product of vincristine sulfate use is in the same manner as in Example 1.
S3, connection aptamer sgc8, preparation sgc8/VCR-Lipo;
Mol ratio according to aptamer and MAL-PEG2000-DSPE is 1:5, gets appropriate VCR-Lipo, adds suitable quantities of nucleic acid aptamers sgc8, reacts 24 hours at nitrogen protection atmosphere at room temperature.β-the cysteamine adding 2mM removes unreacted MAL group.With ultra-filtration centrifuge tube to sgc8/VCR-Lipo carry out ultrafiltration centrifugal after, after the centrifugal 10min of 10000rpm, add 1mL deionized water, the centrifugal 10min of 10000rpm, wash away free vincristine sulfate, β-cysteamine and aptamer sgc8, obtain aptamers target liposomes drug-loading system sgc8/VCR-Lipo.
embodiment 4
This embodiment 4 prepares aptamers target liposomes drug-loading system A10/ADM-Lipo.Wherein, ADM is amycin.This embodiment 4 is identical with embodiment 1, and only its Chinese medicine need be replaced by amycin ADM, aptamer is replaced by A10.
embodiment 5
This embodiment 5 prepares aptamers target liposomes drug-loading system S2.2/IDA-Lipo.Wherein, IDA is daunorubicin.This embodiment 5 is identical with embodiment 1, and only its Chinese medicine need be replaced by daunorubicin (IDA), aptamer is replaced by S2.2.
embodiment 6
This embodiment 6 prepares aptamers target liposomes drug-loading system AS1411/DDP-Lipo.Wherein, DDP is cisplatin.This embodiment 6 is identical with embodiment 1, and only its Chinese medicine need be replaced by cisplatin (DDP), aptamer is replaced by AS1411.
embodiment 7
This embodiment 7 prepares aptamers target liposomes drug-loading system TLS11a/TAXOL-Lipo.Wherein, TAXOL is paclitaxel.This embodiment 7 is identical with embodiment 1, and only its Chinese medicine need be replaced by paclitaxel (TAXOL), aptamer is replaced by TLS11a.
The present invention has carried out drug loading, envelop rate, particle diameter and to the targeting of tumor cell and the experiment of therapeutical effect to aptamers target liposomes drug-loading system prepared by each embodiment above-mentioned, and result shows that the particle diameter of final obtained aptamers target liposomes drug-loading system in the present invention is also 100-120nm.These aptamers target liposomes drug-loading systems have good stability, in vivo not easily by chemistry and biodegradation, to target site, and good therapeutic effect can be had to the tumor cell of targeting by targeting well, especially adopt product best results prepared by embodiment 1.The aptamers target liposomes drug-loading system sgc8/VCR-Lipo prepared for embodiment 1 is below described experiment effect of the present invention.
1, the envelop rate of vincristine sulfate medicine, drug loading calculate
Get 0.3mLsgc8/VCR-Lipo sample and add ultra-filtration centrifuge tube, after the centrifugal 10min of 10000rpm, add 1mL deionized water, the centrifugal 10min of 10000rpm, washes away free VCR.
Get the upper liquid of ultra-filtration centrifuge tube filter, put into 5ml volumetric flask, add 250 μ L10% Triton X-100s (TritonX-100), room temperature places 20min, adds secondary deionized water (MillQ) and is settled to 5ml.Draw 20 μ L, under above-mentioned chromatographic condition, inject chromatograph of liquid, record peak area, calculates the medicament contg wrapping into liposome according to standard curve.Drug loading and envelop rate according to following formulae discovery liposome:
DLC (%)=[lipidosome Chinese traditional medicine thing amount/(lipidosome Chinese traditional medicine thing+carrier total amount)] × 100%;
Envelop rate DLE (%)=(the drug/lipid body Chinese medicine total amount encapsulated in liposome) × 100%.
The drug loading that experiment records the obtained sgc8/VCR-Lipo of the embodiment of the present invention 1 is 14.16% envelop rate is 90.63%.
2, droplet measurement
Referring to Fig. 3 A-C, is the transmission electron microscope picture of each stage particle of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention.Hitachi's transmission scanning electron microscope that the transmission electron microscope used in this experiment is H-7650 for model, accelerating potential is 80kv, and enlargement ratio is 200000x.Fig. 3 A is blank liposome Lipo, Fig. 3 B is the nanometer liposome VCR-Lipo wrapping up vincristine sulfate, and Fig. 3 C is aptamers target liposomes drug-loading system sgc8/VCR-Lipo.Incorporated by reference to consulting Fig. 4, it is the grain size distribution of each stage particle of aptamers target liposomes drug-loading system according to the preferred embodiment of the invention.Wherein, A is blank liposome Lipo; B is VCR-Lipo; C is sgc8/VCR-Lipo.Result shows, and the particle size distribution of often kind of nanoparticle all presents normal distribution, and the particle diameter of blank liposome Lipo is at about 90nm; The particle diameter of VCR-Lipo is at about 100nm; The particle diameter of sgc8/VCR-Lipo is at about 110nm.
3, aptamers target liposomes drug-loading system sgc8/VCR-Lipo is to the targeting of tumor cell
By labelling cell membrane red fluorescence probe (DiI) fluorescent dye in the phospholipid bilayer of embodiment 1, labelling Fluorescein isothiocyanate (FITC) fluorescence on aptamer sgc8.Obtain the sgc8/VCR-Lipo of FITC and DiI with tense marker respectively, and the blank liposome Lipo of DiI labelling.
3 × 10 are added in 15ml centrifuge tube
6cem cell, often pipe adds Lipo, sgc8/VCR-Lipo (final concentration of aptamer controls at 200nM) of DiI labelling respectively, hatches 20-30min for 37 DEG C.Centrifugal collecting cell also washes 3 times with PBS.3 times are washed with PBS with after the fixing 30min of paraformaldehyde (4%).Cell 1mg/mlDAPI contaminates core 5 minutes, washes 3 times and centrifugal collecting cell with PBS.Under inverted fluorescence microscope observation of cell fluorescence intensity and take pictures.
Referring to Fig. 5, is the Binding experiment figure of cem cell and sgc8/VCR-Lipo and VCR-Lipo.Wherein DAPI is 4', 6-diamidino-2-phenylindone, for transfect cell core, in blue-fluorescence; DiI is used for labelling liposome, and take on a red color fluorescence; FITC is used for labeling nucleic acid aptamers sgc8, in green fluorescence; Merge represents the fluorescence that the nano material with fluorescence merges from different Cell binding.In Fig. 5, A is the fluorogram after sgc8/VCR-Lipo and cem cell are hatched; B is the fluorogram after VCR-Lipo and cem cell are hatched; C is the fluorogram after con/VCR-Lipo and cem cell are hatched; Con representative contrast aptamers, itself and aptamer sgc8 have the DNA sequence of identical base number, but are not combined with target cell.
4, aptamers target liposomes drug-loading system sgc8/VCR-Lipo is to the therapeutical effect of tumor
Respectively VCR, VCR-Lipo and sgc8/VCR-Lipo of embodiment 1 dissolved with PBS or suspend, obtaining VCR concentration and be the VCR injection of 1.0ng/ μ l, VCR-Lipo injection and sgc8/VCR-Lipo injection.
Get the inbred line Female nude mice (BALB/cNudeMice) in 4-6 age in week, every only in left fore axillary fossa subcutaneous vaccination 1 × 10
7individual CCRF-CEM (people's acute lymphoblastic leukemia T lymphocyte).This inbred line Female nude mice (BALB/cNudeMice) is Shanghai Bang Yao Bioisystech Co., Ltd product.CCRF-CEM tumor cell is presented by Hunan University professor Tan Weihong.Measure tumor major diameter and minor axis, according to formula TV=1/2 × a × b twice weekly
2calculate gross tumor volume, wherein a is the longest diameter of tumor, and b is the shortest diameter of tumor.Treat that tumor average volume grows to about 100mm
3time, obtain lotus tumor BalB/c nude mice.Get 25 tumor bearing nude mices, be divided into five groups at random, inject 200 μ Lsgc8/VCR-Lipo in the tail vein of wherein one group every lotus tumor BalB/c nude mice to treat, first time injection is designated as treatment the 0th day, the above-mentioned sgc8/VCR-Lipo injection of 200 μ L within 9th day, is all injected, coprocessing 5 lotus tumor BalB/c nude mices respectively in treatment the 3rd day, treatment the 6th day and treatment.
According to the method described above, respectively sgc8/VCR-Lipo injection is replaced with PBS, VCR injection, VCR-Lipo injection and sgc8-Lipo injection, other steps are all constant, obtain the tumor-bearing mice of PBS treatment, the tumor-bearing mice of VCR treatment, the tumor-bearing mice of VCR-Lipo treatment, the tumor-bearing mice of sgc8-Lipo treatment respectively.
Within 12nd day, measure the gross tumor volume often organizing lotus tumor BalB/c nude mice in treatment the 2nd day, treatment the 4th day, treatment the 6th day, treatment the 8th day, treatment the 10th day and treatment respectively, its result is see Fig. 6 and form 1.Form 1 is the change (mm of the gross tumor volume of the tumor-bearing mice of different disposal group
3).
Form 1
Result shows, and inoculation the 12nd day, the gross tumor volume after sgc8/VCR-Lipo treatment was respectively 0.25 times, 0.26 times, 0.58 times and 0.57 times of PBS, sgc8/Lipo, VCR and VCR-Lipo.Show, sgc8/VCR-Lipo has obvious inhibitory action to tumor, can reduce the speed of growth of tumor.
Get a collection of 30 tumor bearing nude mices in addition, be divided into five groups at random, inject 200 μ Lsgc8/VCR-Lipo in the tail vein of wherein one group every lotus tumor BalB/c nude mice to treat, first time injection is designated as treatment the 0th day, the above-mentioned sgc8/VCR-Lipo injection of 200 μ L within 9th day, is all injected, coprocessing 6 lotus tumor BalB/c nude mices respectively in treatment the 3rd day, treatment the 6th day and treatment.
According to the method described above, respectively sgc8/VCR-Lipo injection is replaced with PBS, VCR injection, VCR-Lipo injection and sgc8-Lipo injection, other steps are all constant, obtain the tumor-bearing mice of PBS treatment, the tumor-bearing mice of VCR treatment, the tumor-bearing mice of VCR-Lipo treatment, the tumor-bearing mice of sgc8-Lipo treatment respectively.Record from treatment the time-to-live often organizing lotus tumor BalB/c nude mice 1st day, statistics survival rate is see Fig. 7 and form 2.Form 2 be tumor-bearing mice when different survival rates apart from inoculated tumour time (my god).
Form 2
Result shows, and sgc8/VCR-Lipo obviously can extend the life span of lotus tumor BalB/c nude mice.Apart from 2.18 times, 2.0 times, 1.32 times and 1.22 times of the time of the 1st treatment when the tumor-bearing mice survival rate being respectively the treatment of PBS, sgc8/Lipo, VCR, VCR-Lipo apart from the time of the 1st treatment when the lotus tumor BalB/c nude mice survival rate of sgc8/VCR-Lipo treatment is 50% is 50%.
Fig. 8 is the tumor size of the lotus tumor BalB/c nude mice of different disposal.For starting the tenth day after treating in Fig. 8, put to death mouse, take out the sample drawing after the taking pictures of tumor, often group gets 5 samples, by its gross tumor volume of vernier caliper measurement.Fig. 9 is the targeting in vivo figure of aptamers target liposomes drug-loading system, carry out living imaging shooting to the lotus tumor BalB/c nude mice after process respectively at 0.5h, 2h, 6h, 12h, 24h, 36h, 48h, 72h and 96h respectively, tumor region is right upper extremity back.Experiment proves, the aptamers target liposomes drug-loading system sgc8/VCR-Lipo that sgc8 of the present invention modifies has targeting to tumor: the lotus tumor BalB/c nude mice of fluorescently-labeled sgc8/VCR-Lipo process in the fluorescence intensity of tumor site apparently higher than the lotus tumor BalB/c nude mice of fluorescently-labeled VCR-Lipo process in the fluorescence intensity of tumor site.
Above-mentioned experimental result shows, aptamers target liposomes drug-loading system sgc8/VCR-Lipo of the present invention has good antitumous effect, obviously can grow by Tumor suppression, extend the life span of lotus tumor BalB/c nude mice.
In sum, the aptamers target liposomes drug-loading system that prepared by the present invention has following characteristics:
(1) the present invention is being prepared in liposome process, have selected the sphingomyelins connecting aliphatic chain containing more amido link, and compared with the phospholipid commonly used with other, it can resist chemistry and biological degraded better, stablizing of protection liposome structure; Experimental result shows, even if also can maintain the drug level arriving targeting moiety when the aptamers target liposomes drug-loading system particle diameter of preparation is about 100nm;
(2) the present invention is to the optimization of the connection concentration of aptamer such as sgc8, not only ensure that the stable of liposomal delivery system, and at utmost adds the targeting of this system;
(3) synthesis of the targeted molecular such as single-chain nucleic acid aptamers sgc8 used in the preparation of aptamers target liposomes drug-loading system is with low cost, and method is simple;
(4) preparation method of aptamers target liposomes drug-loading system of the present invention is simple, can prepare on a large scale;
(5) the present invention adds PEG2000 when preparing nanometer liposome, makes Nano medication transmission system circulation time in vivo long;
(6) aptamers target liposomes drug-loading system of the present invention can realize the targeting to tumor, improves the drug-rich amount of tumor cell, thus improves antitumous effect.Experiment proves, the growth of Nano medication transmission system energy Tumor suppression of the present invention, and can extend the time-to-live of tumor animal, can be used to treat tumor.
The present invention is described according to specific embodiment, but it will be understood by those skilled in the art that when not departing from the scope of the invention, can carry out various change and equivalent replacement.In addition, for adapting to specific occasion or the material of the technology of the present invention, can many amendments be carried out to the present invention and not depart from its protection domain.Therefore, the present invention is not limited to specific embodiment disclosed herein, and comprises all embodiments dropping into claims.
Claims (10)
1. an aptamers target liposomes drug-loading system, it is characterized in that, the nanometer liposome prepared by aptamer, antitumor drug and use sphingomyelins forms, wherein said antitumor drug is wrapped in described nanometer liposome, and described aptamer is connected on specially recognizing tumor cells on described nanometer liposome by chemical bond-linking.
2. aptamers target liposomes drug-loading system according to claim 1, is characterized in that, nanometer liposome prepared by described use sphingomyelins comprises sphingomyelins, phospholipid stabilizing agent, cholesterol composition and functional phospholipid;
Wherein, described phospholipid stabilizing agent is selected from one or more in DSPE-PEG, DMPEA-Polyethylene Glycol and PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol;
Described cholesterol composition be selected from cholesterol and derivant, cholestane, cholic acid and bile acid one or more;
Described functional phospholipid be selected from DSPE-PEG-maleimide cross-linking agent, DSPE-PEG-carboxyl cross-linking agent, DSPE-PEG-N-butanimide and DSPE-PEG-amino cross-linking agent one or more.
3. aptamers target liposomes drug-loading system according to claim 1, is characterized in that, the particle diameter of described aptamers target liposomes drug-loading system is 100-120nm.
4. aptamers target liposomes drug-loading system according to claim 1, is characterized in that, described aptamer is sgc8, A10, S2.2, AS1411 or TLS11a; Described antitumor drug is vincristine sulfate, amycin, daunorubicin, cisplatin or paclitaxel.
5. aptamers target liposomes drug-loading system according to claim 1; it is characterized in that; the nanometer liposome that described aptamers target liposomes drug-loading system is prepared by aptamer sgc8, vincristine sulfate and use sphingomyelins forms, and the nanometer liposome wherein using sphingomyelins to prepare comprises following composition: sphingomyelins, DSPE-PEG, cholesterol and DSPE-PEG-maleimide cross-linking agent.
6. a preparation method for aptamers target liposomes drug-loading system, is characterized in that, described preparation method comprises:
S1, by sphingomyelins, cholesterol composition, phospholipid stabilizing agent and functional phospholipid in molar ratio for 50-60:35-45:4-5:0.2-0.4 dissolve prepare blank liposome in organic solvent;
The liposome of the blank liposome preparation parcel antitumor drug of S2, use step S1;
S3, aptamer is connected on the liposome of parcel antitumor drug, preparation aptamers target liposomes drug-loading system.
7. the preparation method of aptamers target liposomes drug-loading system according to claim 6, it is characterized in that, described step S1 is specially: sphingomyelins composition, cholesterol composition, phospholipid stabilizing agent and functional phospholipid are dissolved in molar ratio the mixing in chloroform for 50-60:35-45:4-5:0.2-0.4, at 50-70 DEG C, with rotating speed be that 100-200rpm/min rotates and evacuation forms dry lipid membrane, remove organic solvent; Described lipid membrane is dispersed in citrate buffer, at liquid nitrogen and 50-60 DEG C of water-bath multigelation repeatedly, and obtains blank liposome after particle diameter screening.
8. the preparation method of aptamers target liposomes drug-loading system according to claim 7; it is characterized in that; when adopting aptamer to be sgc8 in described step S3; be that 1:5-1:10 adds aptamer sgc8 according to the mol ratio of aptamer sgc8 and functional phospholipid in the solution of the liposome of parcel antitumor drug; nitrogen protection atmosphere at room temperature reacts; and functional phospholipid, antitumor drug and aptamer that dialysis washing is unnecessary, obtain described aptamers target liposomes drug-loading system.
9. treat and/or prevent a tumour medicine, it is characterized in that, described in treat and/or prevent the active component of tumour medicine for the aptamers target liposomes drug-loading system in claim 1-5 described in any one.
10. treating and/or preventing the application in tumour medicine according to the aptamers target liposomes drug-loading system in claim 1-5 described in any one.
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