CN106750252B - Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid and its preparation method and application - Google Patents
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid and its preparation method and application Download PDFInfo
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- 239000002253 acid Substances 0.000 title claims abstract description 66
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 49
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 73
- 239000011724 folic acid Substances 0.000 claims abstract description 49
- 235000019152 folic acid Nutrition 0.000 claims abstract description 48
- 229960000304 folic acid Drugs 0.000 claims abstract description 26
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000002961 echo contrast media Substances 0.000 claims abstract description 18
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 14
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- UHUSDOQQWJGJQS-QNGWXLTQSA-N 1,2-dioctadecanoyl-sn-glycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](CO)OC(=O)CCCCCCCCCCCCCCCCC UHUSDOQQWJGJQS-QNGWXLTQSA-N 0.000 claims abstract description 10
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- 239000000047 product Substances 0.000 claims description 12
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- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 claims description 10
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
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- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 5
- HVUMOYIDDBPOLL-UHFFFAOYSA-N 2-(3,4-Dihydroxyoxolan-2-yl)-2-hydroxyethyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)C1OCC(O)C1O HVUMOYIDDBPOLL-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 229960001760 dimethyl sulfoxide Drugs 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 150000003462 sulfoxides Chemical class 0.000 claims description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G2650/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterized by the type of post-polymerisation functionalisation
- C08G2650/04—End-capping
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- Acoustics & Sound (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid and preparation method thereof；The present invention synthesizes 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-N- hydroxysuccinimide first, is connected by aminoadipic acid with the Amino End Group folic acid of activation, that is, has synthesized the lipid of double leaf acid molecule.The present invention also provides distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid to prepare the application in double leaf acid targeted ultrasound contrast agent.Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid can significantly improve the targeting binding ability of acoustic contrast agent breast cancer MCF-7.
Description
Technical field
The invention belongs to the crossing domain of chemistry and medicine, in particular to a kind of distearoylphosphatidylethanolamine-is poly-
Ethylene glycol 2000- double leaf acid and preparation method thereof and preparing the application in acoustic contrast agent.
Background technique
Morbidity and mortality of the breast cancer in women are higher, and the whole world annual about 500,000 people dies of breast cancer.Mammary gland
The early detection of cancer and making a definite diagnosis is conducive to select corresponding treatment in time, reduces the death rate, is the research mesh that everybody strongly pursues
Mark.
With the fast development of ultrasound molecular imaging technique and bio-nanotechnology, nanoscale ultrasound contrast agents development is fast
Speed has higher scattering properties, can significantly increase the substance of ultrasound detection signal.With conventional ultrasound contrast agent (blood pool imaging agent)
Difference, nanoscale ultrasound contrast agents have very strong penetration power, can pass through vascular wall, and development and application also helps further
Develop targeting, high efficiency, miniaturization and the new contrast-enhanced ultrasound agent with auxiliary therapeutic action.And targeted nano grade ultrasound is micro-
Appearances of bubble contrast agent makes it possible the targeted imaging of lesion, is the important symbol that ultrasound molecular iconography develops.
Recent study shows：Tumor tissues need more folic acid, folacin receptor compared with normal tissue during the growth process
It is hardly expressed in most normal tissues, and abnormal expression increases on most of cancer cell.Report display：Ovary
Cancer, carcinoma of endometrium, breast cancer, colon cancer, the folacin receptor in nasopharyngeal carcinoma are all in significant high level expression；And height is undifferentiated
Metastatic carcinoma expression folacin receptor level it is more taller than non-metastatic cancer.Folate-targeted Na Pao (NB) can significantly improve Na Pao
With the passive binding ability of the highly expressed tumour cell of folacin receptor.The combination energy of folic acid and tumor surface folacin receptor specificity
Power is related with following two factor：1) amount of different tumor tissue cells surface folacin receptor；2) folic acid connected on carrier
Content.There are two types of the targeted approach for being directed to folacin receptor reported at present：One is utilize the monoclonal such as mAb Mov18
Antibody, but since mAb Mov18 has stronger immunogenicity and one microvesicle composite molecular weight of antibody is big, tissue penetration
The weak equal limitation of power, limits its development；Another kind is with folic acid itself, and folic acid is relatively stable, cheap and be not likely to produce pair
Effect, therefore application is than wide.PEG2000 has good biocompatibility, hydrophilic, is most normal as group connection
One of high molecular polymer seen.
It does not find to make more using L-2-Aminoadipic acid aminoadipic acid as intermediate linking group in the prior art
More folic acid is connect with the surface lipid DSPE, to realize Na Pao in the enrichment of tumour target area.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of distearoylphosphatidylethanolamine-polyethylene glycol
2000- double leaf acid (DSPE-PEG2000-AD- (FOL)2) and preparation method thereof；The present invention also provides distearyl acyl group phosphatidyls
Ethanol amine-polyethylene glycol 2000-double leaf acid is preparing the application in double leaf acid targeted ultrasound contrast agent.
The technical scheme is that：
One, distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, has structure as follows：
Two, the preparation of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
The preparation method of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, preparation route are as follows：
(1) it is sub- to synthesize 1,2- distearoyl-SN- glycerol-3- phosphatidyl ethanolamine-polyethylene glycol 2000-N- maloyl
Amine；
(2) 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-aminoadipic acid is synthesized；
(3) Amino End Group folic acid is synthesized；
(4) synthesis distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid；
It is preferred according to the present invention, the system of the distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
Preparation Method, preparation step are as follows：
(1) it is sub- to synthesize 1,2- distearoyl-SN- glycerol-3- phosphatidyl ethanolamine-polyethylene glycol 2000-N- maloyl
Amine
280mg1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol 2000 (c-terminus) is dissolved in anhydrous
In methylene chloride 8mL, 17.5mg N- hydroxysuccinimide and 62mg dicyclohexylcarbodiimide are sequentially added.Mixture is normal
3h is protected from light under temperature；It is filtered to remove the N of generation, N- dichloro urethanes；Filtrate pours into the ether 200mL pre-cooled
In, ether mixtures are placed in -20 DEG C of 4h；Filtering, is dried to obtain required product；
(2) 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-aminoadipic acid is synthesized
49mgL-2 aminoadipic acid is dissolved in the 0.1M borate buffer 10mL that pH is 8, it is hard that 300mg bis- is then added
Acyl phosphatidyl-ethanolamine-polyethylene glycol 2000-N- hydroxysuccinimide, is protected from light 30min；By reaction solution 0.2N salt
Acid for adjusting pH value is 4-4.5, is extracted 3 times, each 80mL with chloroform, merges organic phase, is dried, filtered with anhydrous sodium sulfate, dense
Contracting, is subsequently poured into the ether that 200mL is pre-cooled, 4h is stirred at -20 DEG C, filters, is dried to obtain required product；
(3) Amino End Group folic acid is synthesized
441mg folic acid is dissolved in 20mL anhydrous dimethyl sulphoxide, 428mgN- tertbutyloxycarbonyl -1,3- third is sequentially added
Diamines and 572mg dicyclohexylcarbodiimide, react 4h at room temperature.Reaction solution is poured into the ether that 300mL is pre-cooled,
In -20 DEG C of stirring 4h, filtering, filter cake is washed with tetrahydrofuran, dry, obtains folic acid-N- tertbutyloxycarbonyl (yield：90%)；It will
654mg Amino End Group folic acid is dissolved in anhydrous methylene chloride, and 50mL trifluoroacetic acid is added under ice bath；2h is reacted at room temperature；Decompression rotation
Evaporation removes methylene chloride and trifluoroacetic acid；Gained residue is poured into the ether pre-cooled, filters, is dried to obtain institute
Need product；
(4) synthesis distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-the amino that will be synthesized in 300mg step (2)
Adipic acid is dissolved in the dry n,N-Dimethylformamide of 5mL, and the Amino End Group folic acid synthesized in 55mg step (3) is dissolved in 1mL bis-
In methyl sulfoxide, then it is added into system；Reacting at normal temperature without light is stayed overnight；Then be added 8mL water, the hydrochloric acid of 6mL 0.1N and
2mL saturated salt solution is extracted 4 times, each 80mL with chloroform；Merge organic phase, anhydrous sodium sulfate dries, filters, and is concentrated；Gained
Residue pours into the ether that 200mL is pre-cooled, and 3h is stirred at -20 DEG C, and filtering is dried to obtain required product.
Three, the application of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
The application of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid of the present invention, feature exist
In being used to prepare double leaf acid targeted ultrasound contrast agent.
According to the present invention, the preparation method of the double leaf acid targeted ultrasound contrast agent, steps are as follows：
0.5mg sorbester p18 is weighed, 0.5ml Tween 80 is placed in 1.5mLEP pipe, 1mg dipalmitoylphosphatidylcholine is added,
1mg distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, PBS are settled to 1mL, are passed through perfluoropropane gas and set
Swap out air, and centrifuge tube is placed in thermostat water bath and heats 50min and is completely dissolved to lipid, is protected from light and DiI dyestuff is added, then
The secondary perfluoropropane gas that is filled with replaces air therein, takes out after mechanical oscillation 90s, and 300rpm is centrifuged 3min, takes lower liquid,
As prepared double leaf acid targeted ultrasound contrast agent.
The present invention also provides a kind of double leaf acid targeted ultrasound contrast agents, which is characterized in that by distearyl of the present invention
Acylphosphatidyl ethanolamine-polyethylene glycol 2000-double leaf acid is prepared.
It is not specified according to the prior art in the above preparation method of the present invention.
The present invention synthesizes 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-N- maloyl first
Imines is connected with the Amino End Group folic acid of activation by aminoadipic acid, that is, has synthesized the lipid of double leaf acid molecule.The present invention also mentions
Supply distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid in preparing double leaf acid targeted ultrasound contrast agent
Using.Double leaf acid targeted ultrasound is further prepared by distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid to make
Shadow agent.
Beneficial effects of the present invention are as follows：
(1) it is super to be not apparent from increase for distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid prepared by the present invention
Sound contrast agent partial size；
(2) the external development capability of acoustic contrast agent is not influenced；
(3) distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid can be shown under the premise of above-mentioned (1), (2)
Write the targeting binding ability for improving acoustic contrast agent breast cancer MCF-7.
Detailed description of the invention
Fig. 1 is the nuclear-magnetism spectrum for prompting distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid successfully to synthesize
Figure；
Fig. 2 is the light microscopic figure of the acoustic contrast agent of three groups of different folate contents；
Fig. 3 is the acoustic contrast agent grain-size graph of three groups of different folate contents；
Fig. 4 is the acoustic contrast agent development figure in vitro of three groups of different folate contents；
Fig. 5 is the different lipid concentrations for the acoustic contrast agent that CCK-8 method detects three groups of different folate contents to breast cancer
MCF-7 cytotoxicity figure；
Fig. 6 is fluorogram of the acoustic contrast agent to breast cancer MCF-7 binding ability of three groups of different folate contents；
Fig. 7 is flow cytometry of the acoustic contrast agent to breast cancer MCF-7 binding ability of three groups of different folate contents
Figure.
Specific embodiment
The present invention will be further explained with reference to the examples below, but not limited to this.
Embodiment 1：The preparation of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
The preparation method of distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, includes the following steps：
(1) it is sub- to synthesize 1,2- distearoyl-SN- glycerol-3- phosphatidyl ethanolamine-polyethylene glycol 2000-N- maloyl
Amine
280mg1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol 2000 (c-terminus) is dissolved in anhydrous
In methylene chloride (8mL), 17.5mg N- hydroxysuccinimide and 62mg dicyclohexylcarbodiimide are sequentially added.Mixture
3h is protected from light under room temperature.It is filtered to remove the N of generation, N- dichloro urethanes；Filtrate pours into the ether pre-cooled
In (200mL), ether mixtures are placed in -20 DEG C of 4h.Filtering, is dried to obtain required product.
(2) 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-aminoadipic acid is synthesized
49mgL-2 aminoadipic acid is dissolved in the 0.1M borate buffer (10mL) that pH is 8,300mg bis- is then added
Stearyl phosphatidyl ethanol amine-polyethylene glycol 2000-N- hydroxysuccinimide, is protected from light 30min.By reaction solution 0.2N
Salt acid for adjusting pH value is 4-4.5, extracts (3*80mL) with chloroform, merges organic phase, dried, filtered with anhydrous sodium sulfate, is concentrated,
It is subsequently poured into the ether that 200mL is pre-cooled, 4h is stirred at -20 DEG C, filter, be dried to obtain required product.
(3) Amino End Group folic acid is synthesized
441mg folic acid is dissolved in 20mL anhydrous dimethyl sulphoxide, 428mgN- tertbutyloxycarbonyl -1,3- third is sequentially added
Diamines and 572mg dicyclohexylcarbodiimide, react 4h at room temperature.Reaction solution is poured into the ether that 300mL is pre-cooled,
In -20 DEG C of stirring 4h, filtering, filter cake is washed with tetrahydrofuran, dry, obtains folic acid-N- tertbutyloxycarbonyl (yield：90%)；It will
654mg Amino End Group folic acid is dissolved in anhydrous methylene chloride, and 50mL trifluoroacetic acid is added under ice bath.2h is reacted at room temperature.Decompression rotation
Evaporation removes methylene chloride and trifluoroacetic acid.Gained residue is poured into the ether pre-cooled, filters, is dried to obtain institute
Need product.
(4) synthesis distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
The synthesis 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-amino that will be synthesized in 300mg (2)
Adipic acid is dissolved in the dry n,N-Dimethylformamide of 5mL, and the Amino End Group folic acid synthesized in 55mg (3) is dissolved in 1mL dimethyl
In sulfoxide, then it is added into system.Reacting at normal temperature without light is stayed overnight.Then 8mL water is added, the hydrochloric acid and 2mL of 6mL 0.1N is full
And saline solution, (4*80mL) is extracted with chloroform.Merge organic phase, anhydrous sodium sulfate dries, filters, and is concentrated.Gained residue falls
Enter in the ether that 200mL is pre-cooled, 3h is stirred at -20 DEG C, filters, be dried to obtain required product.
In the present embodiment, the yield of gained distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid is
70%, purity 90%.
The nuclear magnetic spectrogram that distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid successfully synthesizes is shown in Fig. 1.?
The hydrogen of position six that chemical shift is 0.8, each DSPE molecule have eight between chemical shift 6~8 in the position there are two methyl
A hydrogen is the hydrogen on folic acid phenyl ring, and each folate molecule contains 4 hydrogen, so corresponding two folate molecules.
Embodiment 2：The preparation of double leaf acid targeted ultrasound contrast agent
0.5mg sorbester p18 is weighed, 0.5ml Tween 80 is placed in 1.5mLEP pipe, 1mg dipalmitoylphosphatidylcholine is added,
1mg distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, PBS are settled to 1mL, are passed through perfluoropropane gas and set
Swap out air, and centrifuge tube is placed in thermostat water bath and heats 50min and is completely dissolved to lipid, is protected from light and DiI dyestuff is added, then
The secondary perfluoropropane gas that is filled with replaces air therein, takes out after mechanical oscillation 90s, and 300rpm is centrifuged 3min, takes lower liquid,
As prepared double leaf acid targeted ultrasound contrast agent.
Below with reference to experimental example, the present invention is described further, but not limited to this.
The preparation of the acoustic contrast agent of three kinds of different folate contents：
0.5mg sorbester p18 is weighed, 0.5ml Tween 80 is placed in 1.5mLEP pipe, 1mg dipalmitoylphosphatidylcholine is added,
It is separately added into 1mg1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol (A group), 1mg1,2- distearoyl -
SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-folic acid (B group), 1mg distearoylphosphatidylethanolamine-polyethylene glycol
2000- double leaf is sour (C group), and PBS is settled to 1mL, is passed through perfluoropropane gas and displaces air, and centrifuge tube is placed in thermostatted water
50min is heated in bath to be completely dissolved to lipid, is protected from light and DiI dyestuff is added, and it is therein to be filled with perfluoropropane gas displacement again
Air takes out after mechanical oscillation 90s, and 300rpm is centrifuged 3min, takes lower liquid, as prepared acoustic contrast agent.
A group is blank liposomes group, and B group is single folic acid group, and C group is leafy sour group.
Experimental example 1：The characterization experiment of the acoustic contrast agent of three kinds of different folate contents
Light microscopic observation nanoscale UCAs size, form, the Na Pao of visible different folate contents is under 1000 × light microscopic
Spherical shape, uniform particle diameter, be uniformly dispersed no aggregation, sees Fig. 2.It is made using nanometer laser granularity and Zeta potential analyzer detection ultrasound
The partial size of shadow agent, all indexs measure be averaged in triplicate, and particle diameter distribution difference, A, B, C are steeped in receiving for different folate contents
Three groups of partial sizes are respectively 129.43 ± 12.33nm, and 244.1 ± 35.09nm, 286.87 ± 22.96nm are shown in Fig. 2.Single folic acid group,
Double leaf acid group receive bubble partial size (P is equal for significant difference compared to the blank group<0.05), single folic acid group is with double leaf acid group partial size without obvious
Difference (P>0.05), three groups of acoustic contrast agent partial sizes are respectively less than 400nm, see Fig. 3.
Experimental example 2：The external development capability measurement experiment of acoustic contrast agent of three kinds of different folate contents
Three groups of NBs are placed in circular hole made of agar gel, using the contrast mode of GElogiq E9 diasonograph,
Frequency 9.0MHz, mechanical index (M I) 0.12, two dimension are observed with ultrasonic contrast pattern synchronization, adjustment parameter setting, so that tube sealing
Occur uniformly enhancing inside needle, parameter remains unchanged, and stores image document, Image J software point with Ultrasound Instrument inter workstation
Noise ratio is analysed, the external development capability development capability of the NBs of different folate contents is different, sees Fig. 4, is consistent with partial size difference.
A, tri- groups of B, C of gray value is respectively 205.28 ± 2.10,216.13 ± 1.16,218.17 ± 2.09, wherein single folic acid group,
Double leaf acid group receive bubble partial size (P is equal for significant difference compared to the blank group<0.01), single folic acid group is with double leaf acid group partial size without obvious
Difference (P>0.05), see Fig. 4.
Experimental example 3：The acoustic contrast agent of three kinds of different folate contents is to MCF-7 Breast Cancer Cell cytotoxicity test experience
With 10000/hole by MCF-7 cell inoculation in 96 orifice plates, be added culture medium, 37 DEG C, in 5%CO2 incubator
For 24 hours, being separately added into 10ul concentration is 0 μ g/mL, 100 μ g/mL, 120 μ g/mL, 150 μ g/mL, 200 μ g/mL, 300 μ g/ for culture
The lipid of mL, 600 μ g/mL after overnight incubation, change culture medium into fresh culture containing 10ulCCK-8, continue to be incubated for 1-
4h detects its absorbance value with microplate reader at 450nm wavelength.Lipid concentration non-table when less than 50 μ g/mL as the result is shown
Reveal apparent cytotoxicity, prompting to carry out suitable lipid concentration when cell experiment is 50 μ g/mL, sees Fig. 5.
Experimental example 4：The acoustic contrast agent of three kinds of different folate contents is real to the external binding ability of MCF-7 Breast Cancer Cell
It tests
Be inoculated with 6 × 105 cells in culture bottle, add culture solution 5mL, 37 DEG C, train in 5%CO2 incubator it is adherent for 24 hours,
Equivalent list folic acid, double leaf acid NBs are separately added into experimental group and control group, blank NBs is added in control group, is placed in incubator altogether
It is taken out after culture 1h, cell is rinsed 3 times with PBS, is centrifuged after 0.25% trypsin digestion, 200 μ LPBS are added and mix carefully
Born of the same parents stick ratio with flow cytomery nanoparticles on tumor cells.Flow cytometry tests find double leaf acid group MCF-7
Ratio 96.76% of the cell with fluorescence, single folic acid group and the ratio without targeting group cell with fluorescence be respectively 33.33%,
0.52%, show that the more mono- folic acid group of double leaf acid group Na Pao and blank group and tumour cell have higher combination effect, sees Fig. 6.
The MCF-7 cell of logarithmic growth phase takes 10 μ L cell suspensions to count with 0.25% trypsin digestion, with containing
For the DMEM culture solution adjustment cell concentration of 10% fetal calf serum to 5 × 104/mL, every hole 1mL, which is inoculated in, has been placed in coverslip
Creep plate in 12 orifice plates, 37 DEG C, 5%CO2Adherent in incubator to be separately added into 100 μ L blank liposomes, single folic acid for 24 hours, double leaf acid is received
Bubble, is placed in incubator and co-cultures 1h.Coverslip is taken out, PBS is rinsed 3 times, acetone/methanol mixed liquor fixed 10min, PBS
It rinses 2 times, Hoechst dyes 10min, and PBS is rinsed 3 times, and magazine saves after mounting, under the microscope in fluorescence microscopy.Fluorescence is aobvious
Nucleus fluorescence blue in the visible control group of micro- microscopic observation, it is visible blue in single folic acid group without other fluorescence phenomenons
Red fluorogen is surrounded around the nucleus of fluorescence, and is largely attached on cell membrane, and double leaf acid group also shows blue
Red fluorogen is surrounded around the nucleus of fluorescence, and is largely attached on cell membrane, compared to single folic acid group fluorescence intensity
It is relatively strong, see Fig. 7.
Claims (7)
1. distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, which is characterized in that have knot as follows
Structure：
2. the preparation method of compound as described in claim 1, which is characterized in that preparation route is as follows：
(1) 1,2- distearoyl-SN- glycerol-3- phosphatidyl ethanolamine-polyethylene glycol 2000-N- hydroxysuccinimide is synthesized；
(2) 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-aminoadipic acid is synthesized；
(3) Amino End Group folic acid is synthesized；
(4) synthesis distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid；
3. the preparation method of compound as claimed in claim 2, which is characterized in that preparation step is as follows：
(1) 1,2- distearoyl-SN- glycerol-3- phosphatidyl ethanolamine-polyethylene glycol 2000-N- hydroxysuccinimide is synthesized
280mg1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol 2000 is dissolved in anhydrous methylene chloride 8mL
In, sequentially add 17.5mg N- hydroxysuccinimide and 62mg dicyclohexylcarbodiimide；It is protected from light under mixture room temperature
3h；It is filtered to remove the N of generation, N- dichloro urethanes；Filtrate is poured into the ether 200mL pre-cooled, and ether is mixed
It closes object and is placed in -20 DEG C of 4h；Filtering, is dried to obtain required product；
(2) 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-aminoadipic acid is synthesized
49mgL-2 aminoadipic acid is dissolved in the 0.1M borate buffer 10mL that pH is 8,300mg distearyl is then added
Phosphatidyl-ethanolamine-polyethylene glycol 2000-N- hydroxysuccinimide, is protected from light 30min；By reaction solution 0.2N hydrochloric acid tune
Section pH value is 4-4.5, is extracted 3 times, each 80mL with chloroform, merges organic phase, is dried, filtered with anhydrous sodium sulfate, is concentrated, so
It is poured into the ether that 200mL is pre-cooled afterwards, 4h is stirred at -20 DEG C, filtered, be dried to obtain required product；
(3) Amino End Group folic acid is synthesized
441mg folic acid is dissolved in 20mL anhydrous dimethyl sulphoxide, 428mgN- tertbutyloxycarbonyl -1,3- propane diamine is sequentially added
With 572mg dicyclohexylcarbodiimide, 4h is reacted at room temperature；Reaction solution is poured into the ether that 300mL is pre-cooled, -20
DEG C stirring 4h, filtering, filter cake washed with tetrahydrofuran, dry, obtains folic acid-N- tertbutyloxycarbonyl, which is up to 90%；It will
654mg Amino End Group folic acid is dissolved in anhydrous methylene chloride, and 50mL trifluoroacetic acid is added under ice bath；2h is reacted at room temperature；Decompression rotation
Evaporation removes methylene chloride and trifluoroacetic acid；Gained residue is poured into the ether pre-cooled, filters, is dried to obtain institute
Need product；
(4) synthesis distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid
By the 1,2- distearoyl-SN- glycerol -3- phosphatidyl ethanolamine-polyethylene glycol-amino synthesized in 300mg step (2) oneself two
Acid is dissolved in the dry n,N-Dimethylformamide of 5mL, and the Amino End Group folic acid synthesized in 55mg step (3) is dissolved in 1mL dimethyl
In sulfoxide, then it is added into system；Reacting at normal temperature without light is stayed overnight；Then 8mL water is added, the hydrochloric acid and 2mL of 6mL 0.1N is full
And saline solution, it is extracted 4 times with chloroform, each 80mL；Merge organic phase, anhydrous sodium sulfate dries, filters, and is concentrated；Gained residual
Object pours into the ether that 200mL is pre-cooled, and 3h is stirred at -20 DEG C, and filtering is dried to obtain required product.
4. the application of compound as described in claim 1, which is characterized in that be used to prepare double leaf acid targeted ultrasound contrast agent.
5. the application of compound as claimed in claim 4, which is characterized in that the system of the double leaf acid targeted ultrasound contrast agent
Preparation Method, steps are as follows：
0.5mg sorbester p18 is weighed, 0.5ml Tween 80 is placed in 1.5mLEP pipe, and 1mg dipalmitoylphosphatidylcholine, 1mg is added
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, PBS are settled to 1mL, are passed through perfluoropropane gas displacement
Centrifuge tube is placed in thermostat water bath and heats 50min and be completely dissolved to lipid, is protected from light and DiI dyestuff is added, again by air out
It is filled with perfluoropropane gas and replaces air therein, taken out after mechanical oscillation 90s, 300rpm is centrifuged 3min, takes lower liquid, i.e.,
For prepared double leaf acid targeted ultrasound contrast agent.
6. a kind of double leaf acid targeted ultrasound contrast agent, which is characterized in that by distearyl acyl group phosphatidyl second described in claim 1
Hydramine-polyethylene glycol 2000-double leaf acid is prepared.
7. the preparation method of double leaf acid targeted ultrasound contrast agent as claimed in claim 6, which is characterized in that steps are as follows：
0.5mg sorbester p18 is weighed, 0.5ml Tween 80 is placed in 1.5mLEP pipe, and 1mg dipalmitoylphosphatidylcholine, 1mg is added
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- double leaf acid, PBS are settled to 1mL, are passed through perfluoropropane gas displacement
Centrifuge tube is placed in thermostat water bath and heats 50min and be completely dissolved to lipid, is protected from light and DiI dyestuff is added, again by air out
It is filled with perfluoropropane gas and replaces air therein, taken out after mechanical oscillation 90s, 300rpm is centrifuged 3min, takes lower liquid, i.e.,
For prepared double leaf acid targeted ultrasound contrast agent.
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| WO2016014337A1 (en) * | 2014-07-25 | 2016-01-28 | Nemucore Medical Innovations, Inc. | Drug delivery nanoemulsion systems |
| CN105535996A (en) * | 2016-01-08 | 2016-05-04 | 贵州医科大学 | Novel fluorescence lipidosome nano probe and preparing method thereof |
| CN106794164A (en) * | 2014-08-14 | 2017-05-31 | L.E.A.F.控股集团公司 | Liposomal encapsulated compatibility medicine |
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| WO2016014337A1 (en) * | 2014-07-25 | 2016-01-28 | Nemucore Medical Innovations, Inc. | Drug delivery nanoemulsion systems |
| CN106794164A (en) * | 2014-08-14 | 2017-05-31 | L.E.A.F.控股集团公司 | Liposomal encapsulated compatibility medicine |
| CN105535996A (en) * | 2016-01-08 | 2016-05-04 | 贵州医科大学 | Novel fluorescence lipidosome nano probe and preparing method thereof |
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