CN110054660A - A kind of preparation and application of the breast cancer targeting lipids material of fructose modification - Google Patents
A kind of preparation and application of the breast cancer targeting lipids material of fructose modification Download PDFInfo
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Abstract
The invention discloses a kind of novel lipid materials, transmitting and extension drug treating time for realizing breast cancer targeted drug.The novel lipid material adds connection chain, is connected with the fructose with breast cancer target function, the ligand molecular with breast cancer targeting is made by modifying endogenous cholesterol.Matrix material is modified using the ligand molecular, the affinity between novel lipid material and receptor can be improved, realizes stronger tumor-targeting, plays significantly more efficient breast cancer treatment effect.The different dosage forms that the novel lipid material can be used for including liposome, nanoparticle, micella etc., made load Paclitaxel liposome have apparent breast cancer targeting, gather around and have broad application prospects.
Description
Technical field
The present invention relates to a kind of novel lipid material and its applications in drug delivery system, and there is breast cancer to target medicine
Object transmitting and the function of extending circulation time in vivo, the preparation including the material, and its as pharmaceutical carrier in drug delivery
Application, belong to pharmaceutical technology field.
Background technique
Breast cancer is a kind of height heterogeneity disease, is women common cancer, is to typically occur in breast galandular epithelium tissue
Malignant tumour.The factors such as heredity, environment, age, life style and eating habit make the HER2 of patient, BRCA1, BRCA2, RB
Equal genes mutate, and then lead to the generation of breast cancer.Clinical manifestation is relieving mammary gland gall, has lymph under lump, armpit
Bear existing enlargement etc..According to statistics, 99% breast cancer is women, and breast cancer is one of big malignant tumour of women three, although China
It is the low hair area of breast cancer, but disease incidence rapid increase, annual morbidity are about 12/,100,000, women suffers from breast cancer among all one's life
Probability be about 12.3%, and patient tends to rejuvenation.The breast cancer new cases of China about 12.1 ten thousand in 2000,
2005 are 16.8 ten thousand, and Chinese tumour Register annual report middle finger in 2015 goes out, and breast cancer still occupies female cancer disease incidence
First place seriously affects the health and lives of China women.It expects 2021, Chinese breast cancer patients will be up to 2,500,000.Illness
In early days, operative treatment, middle and advanced stage, more options radiotherapy or chemotherapy are given more.However operative treatment on patient body not only to bringing
Considerable distress, and poor prognosis, easy infection, while certain injury is also had on patients ' psychological.Though radiotherapy has certain curative effect,
But it can not equally eradicate, and side effect is big.Although chemotherapy plays a very important role in the complex treatment of breast cancer, energy
The whole body of enough control breast cancer early stage generations is sent out, and the relapse and metastasis of Mammary cancer is alleviated in prevention, and it is raw effectively to extend patient
It deposits the phase, but because of its inorganization and organ specificity, drug is distributed in whole body, normal tissue also imitate by toxic side effect, treatment
Fruit is less desirable.The targeted therapy treatment completely new as one kind except operation, radiotherapy, three great tradition treatment means of chemotherapy
Method, mechanism of action and toxicity are different from traditional cytotoxic chemotherapies, usually have better tolerance than classic chemotherapy, can
More effectively for the selection of clinical treatment provide with further reference to.Along with the depth of pharmacology and molecular biology research
Enter, the research and application of targeted drug also achieve breakthrough, and the research and development of new therapy target drug also have become people pass
The hot spot of note.
In recent years, researcher utilizes ring-type RGD (cRGD), folic acid (FA), biotin (biotin), human epidermal growth
The different types of targeting ligands such as factor acceptor -2 (Her2), galactolipin, glycyrrhizic acid (glycyrrhizin), fructose carry out
The research of tumor-targeting drug delivery system.Compared with normal cell, in order to meet fast breeding and spread necessary energy
Demand, tumour cell can all over-express one or more glucose transporter (GLUT) generally to transport some high energy
Glucide, for example, glucose, fructose, galactolipin etc..Fructose is another important bottom of tumour energetic supersession in addition to glucose
Object.In 12 kinds of hypotypes (GLUT1-12) of glucose transporter, GLUT5 is the exclusive transport protein of fructose specificity.
GLUT5 is also one of three kinds of glucose transporter (GLUT1, GLUT2 and GLUT5) that tumour is mainly expressed, especially in cream
It is highly expressed in adenoncus oncocyte, and does not find GLUT5 in normal galactophore tissue.Therefore, the transhipment using GLUT5 to fructose
Characteristic and the high expressivity in breast cancer cell, it is possible to develop selective development of target medicines for treatment of breast cancer.Furthermore have
Document report shows the overexpression due to surface GLUT5 receptor, and breast cancer cell has one to the intake of fructose modified polymer
Determine the raising of degree.Moreover, modified by the different location to fructose, research find the position C1 of fructose carry out coupling with
GLUT5 receptor has affinity well.Therefore, it is controlled by the breast cancer targeting with fructose (Fru) recognition reaction of mediation
Treatment method will be a kind of very promising research direction.
Summary of the invention
Based on the studies above and it is assumed that object of this investigation is the matrix material for synthesizing a kind of modification of fructose, it is used in combination
In the preparation of liposome.Using the high expressivity of breast cancer cell GLUT5 and to the stronger affinity of fructose, make to repair by fructose
The matrix material of decorations has breast cancer targeting.This material is applied to specific preparation, the drug orientation that it can be made to encapsulate
Breast tumor tissue is acted on, drug concentration is improved, plays significantly more efficient breast cancer treatment effect.It is targeted simultaneously by it
Function reduces drug in the distribution of peripheral organ, reduces the toxic side effect of drug.Therefore, we devise as shown in logical formula (I)
A kind of matrix material, the cholesterol moiety of the matrix material is embedded into liposomal phospholipids bilayer, has breast cancer target
To fructose moiety be then exposed to the surface of liposome, thus make liposome have breast cancer target function.This matrix material
The different dosage forms that can be used for including liposome, nanoparticle, micella etc., while there is long circulating, breast cancer targeting and reduce poison
Property function, by this matrix material be applied to drug delivery system, will have great application prospect, with this matrix material
Made load Paclitaxel liposome has apparent breast cancer target function.
The present invention provides the compound for leading to structure shown in formula (I) or its pharmaceutically acceptable salt or hydrate:
Wherein, the acid anhydrides in used connection chain is one of succinic anhydride, malonic anhydride and glutaric anhydride, polyethylene glycol
For one of triethylene glycol or PEG200,400,600,800,1000,1500,2000,4000.
The specific preparation method of structure shown in logical formula (I) is as follows:
Novel lipid material of the present invention can be used as the liposome that ligand is used to prepare breast cancer targeting.
The liposome is it is characterized in that include phosphatide, cholesterol, Fru-Chol and activating agent.
The liposome is mainly made of membrane material and activating agent, and membrane material is phospholipid bilayer, by lecithin, cholesterol
And liposomal ligand composition, wherein each component proportion relation is as follows: the molar ratio of cholesterol and phosphatide is 1 ~ 2:1 ~ 10, rouge
The molar content of plastid ligand is the 1 ~ 25% of the total mole number of cholesterol and phosphatide.Activating agent preferred therapeutic of the present invention
Agent or developer, as known in the art, the dosage of activating agent can be adjusted according to including the activating agent in steroidal, wherein
In percentage by weight, activating agent accounts for the 0.1% ~ 50% of total lipid.
Phosphatide in the liposome includes all types of phosphatide, including but not limited to soybean lecithin, lecithin, phosphorus
Acyl ethanol amine, phosphatidyl serine, phosphatidylinositols, phosphatidyl glycerol, diphosphatidylglycerol；Preferably lecithin.
Activating agent in the liposome can be anti-tumor drug, including but not limited to alkylating agent, antimetabolite, anti-
Anti-neoplastic antibiotic, anthracycline antibiotic, plant alkaloid, paclitaxel derivatives, Topoisomerase inhibitors, monoclonal antibody,
Photosensitizer, kinase inhibitor and compound containing platinum.Antiepileptic, including but not limited to barbiturates, second propionyl ureas, double-strand
Fatty acid, succinimide class, benzodiazepine class, imino group glycoside, sulfamido, oxazolidine diketone class, pepper bases, skin
Matter steroids, immunoglobulin etc..Antidepressant, including but not limited to norepinephrine reuptake inhibitors, monoamine oxygen
Change enzyme inhibitor, serotonin reuptake inhibitor.
The preparation method of breast cancer target liposomes of the present invention, comprising the following steps:
(1) phosphatide, cholesterol, taxol are weighed in eggplant type flask, is dissolved with appropriate solvent, the lipid of corresponding proportion is added
Body ligand (blank liposome is not added) removes organic solvent in 20-40 DEG C of water bath with thermostatic control rotary evaporation；
(2) eggplant type bottle is placed in vacuum desiccator again and is dried in vacuum overnight removing residual solvent；
(3) hydrating fluids such as phosphate buffer or ammonium sulfate are added into eggplant type bottle, with 20 DEG C of constant temperature air bath shaking tables
After aquation about 0.5-2 hours, ice-water bath Probe Ultrasonic Searching is existed liposomal particle size control with the methods of film or ultrasound was squeezed
110nm or so.
Taxol in preferred step (1): matrix material ratio is 1:30.
Solvent in preferred step (2) is chloroform, lipid molar ratios 1:2(cholesterol: soybean lecithin).
The 0.01M phosphate buffer (PBS) that hydrating fluid in preferred step (3) is pH 7.4.
Above-mentioned purpose that the invention is realized by the following technical scheme:
Specific implementation method
Following embodiment is intended to illustrate invention rather than limitation of the invention further.It is further detailed referring to embodiment
Thin to illustrate the present invention, however, the present invention is not limited to these examples and the preparation method that uses.Moreover, those skilled in the art
Description according to the present invention can be equivalently replaced the present invention, combines, improves or modify, but these are intended to be included in this hair
In bright range.
The novel lipid material is specifically prepared by the following steps:
Embodiment 1
The preparation of compound 2
Cholesterol 1(32.00 g, 82.76 mmol) is dissolved in 100 mL anhydrous pyridines, tolysulfonyl is added dropwise at 0 DEG C
The pyridine solution (50 mL) of chlorine (TsCl, 23.67 g, 124.14 mmol).After being added dropwise, reaction solution is moved to 50 DEG C,
Continue stirring 5 hours, solvent pyridine is removed under reduced pressure, ethyl acetate (300 mL) is added into residue, and successively use dilute hydrochloric acid
The washing of (1 mol/L, 100 mL × 2), saturated sodium-chloride water solution (100 mL × 2), anhydrous sodium sulfate is dry, and decompression removes
Solvent is gone to obtain 42.35 g of white solid, yield 94.62%, product can directly carry out next step reaction without purifying.Mp:
129-132 °C. 1H NMR (400 MHz, CDCl3, ppm) δ: 0.66 (s, 3H), 0.85 (d, 6H, J = 6.4
Hz), 0.91 (d, 3H, J=6.4 Hz), 0.99 (s, 3H), 0.66-2.38 (remaining cholesterol
protons), 3.16-3.21 (m, 1H), 3.59-3.75 (m, 12H), 5.34 (s, 1H)。
Embodiment 2
The preparation of compound 3
Compound 2(22.48 g, 41.56 mmol) is dissolved in 130 mL dioxane, triethylene-glycol (27.86 is added
ML, 207.82 mmol), it is warming up to back flow reaction 6 hours, solvent is removed under reduced pressure, residue is molten with methylene chloride (200 mL)
Xie Hou is washed with saturated sodium-chloride water solution (100 mL × 2), and organic layer is dry with anhydrous sodium sulfate, and solvent is removed under reduced pressure,
Residue obtains 12.27 g of colorless oil, yield 56.92% through silica gel column chromatography (petroleum ether/acetone=8/1) purifying.1H
NMR (600 MHz, CDCl3, ppm) δ: 0.66 (s, 3H), 0.85 (d, 6H, J = 6.4 Hz), 0.91 (d,
3H, J = 6.4 Hz), 0.99 (s, 3H), 0.67-2.38 (remaining cholesterol protons),
3.16-3.21 (m, 1H), 3.59-3.75 (m, 12H), 5.32 (m, 1H)。
Embodiment 3
The preparation of compound 5
, will be without fruit drops 10(2g, 11.11mmol under argon gas protection) it is dissolved in 0 DEG C of 39ml anhydrous propanone and 1.95ml sulfuric acid
It in mixed solution, finishes, reaction solution is moved into room temperature reaction 2h.TLC monitors fully reacting, under ice bath slowly into reaction solution
0 DEG C of sodium hydroxide (6.1g, 152.5mmol) aqueous solution of 55ml is added.It filters, solvent in filtrate is removed under reduced pressure, by residue
It is dissolved in 50ml methylene chloride, successively uses water (100ml × 2), saturated salt solution (100ml × 2) washing reaction liquid, anhydrous sodium sulfate
Dry, concentration, petroleum ether recrystallizes to obtain white solid 2.1g, yield 72.7%.Fusing point: 94-96 DEG C.
Embodiment 4
The preparation of compound 6
Compound 4(3.00 g, 4.74 mmol) is dissolved in 20 mL toluene, and addition p-methyl benzenesulfonic acid (0.13 g, 0.95
Mmol), 110 DEG C of return stirrings are warming up to react 8 hours.It is cooled to room temperature, reaction solution washs (30 with saturated sodium-chloride water solution
ML × 2), after toluene layer anhydrous sodium sulfate drying, concentration, residue is through silica gel column chromatography (methylene chloride/methanol=10/1)
Purifying obtains 2.45 g of light yellow oil, yield 89.65%.1H NMR (600 MHz, CDCl3, ppm) δ:0.67 (s,
3H), 0.86 (d, 6H, J = 6.4 Hz), 0.92 (d, 3H, J = 6.6 Hz), 0.99 (s, 3H), 0.66-
2.39 (remaining cholesterol protons), 3.19-3.23 (m, 1H), 3.62-3.80 (m, 12H),
4.17 (s, 2H), 5.34 (d, 1H, J = 2.4 Hz)。
Embodiment 5
The preparation of compound 7
Compound 6(1.042g, 2.892mmol) is dissolved in 30ml methylene chloride, DCC(795mg is added at -5 DEG C,
3.856mmol), DMAP(47mg, 0.3856mmol), it activates 30 minutes.By compound 3(1.0g, 1.928mmol) 20ml bis-
Chloromethanes dissolution, is added dropwise in above-mentioned activating solution.Reaction 3h is stirred at room temperature in drop Bi Yizhi.TLC monitors fully reacting, by reaction solution
It is spin-dried for, is dissolved with ethyl acetate, filtered, filtrate obtains faint yellow sticky oil object 1.284g, yield 77% through silica gel column chromatography.1H-
NMR (400MHz, CDCl3,ppm) δ: 0.68 (s, 3H), 0.87 (d, 6H, J=6.4Hz), 0.92 (d, 3H,J=6.0Hz), 1.00 (s, 3H), 1.35 (s, 4H), 1.42 (s, 3H), 1.49 (s, 3H), 1.55(s,
3H), 0.87-2.38 (remaining cholesterol protons), 2.70 (s, 4H), 3.17-3.21 (m,
1H), 3.65 (d, 8H, J=10Hz), 3.71 (t, 2H, J=3.2Hz), 3.76-3.78 (m, 1H), 3.90-
3.92 (m, 1H), 4.06-4.08 (m, 1H), 4.24-4.27 (m, 3H), 4.32 (s, 1H), 4.43-4.45
(m, 1H), 4.61-4.62 (m, 1H), 5.35 (s, 1H). ESI-MS calculated for C49H80O12Na+ [M
+Na]+ 883.5542, found 883.5550。
Embodiment 6
The preparation of ligand Fru-Chol
Compound 7(3.49g, 4mmol) is added to 40ml CF3COOH and H2In the mixed solution of O (V/V=1:1), room temperature
It is stirred to react for 24 hours, TLC monitors fully reacting.Solvent C F is removed under reduced pressure3Saturation Na is added in COOH2CO3It neutralizes, EA extraction, concentration
Organic layer obtains faint yellow sticky oil object 1.197g, yield 44% through silica gel column chromatography.1H-NMR (600MHz, CDCl3,ppm)
δ: 0.67(s, 3H), 0.86 (d, 6H, J=8.0Hz), 0.91 (d, 3H, J=8.0Hz), 0.99 (s, 3H),
0.85-2.48 (remaining cholesterol protons), 2.69 (s, 4H), 3.15-3.21 (m, 1H),
3.41 (br, 8H), 3.65 (d, 8H, J=9.2Hz), 3.70 (t, 2H, J=4Hz), 4.23-4.25 (m, 3H),
4.36 (br, 2H), 5.34 (d, 1H, J=8.0Hz). ESI-MS calculated for C43H76NO12 + [M+NH4]+
798.5362, found 798.1278。
The specific preparation method of the breast cancer target liposomes:
Embodiment 7
Film hydration method is most widely used, easy to operate, the liposome knot prepared as classical method for preparing lipidosome
Structure is typical.Therefore, present invention selection prepares Paclitaxel liposome using film hydration method.
Grope according to the preparation for carrying Paclitaxel liposome, final we choose optimized prescription: lipid molar ratios 1:2
(cholesterol: soybean lecithin), hydrating fluid are the phosphate buffer (PBS) (0.01M) of pH7.4, taxol: matrix material quality
Than for 1:30.We are prepared for blank with above-mentioned prescription respectively and carry Paclitaxel liposome PTX-Lip and Fru-Chol modification
Carry Paclitaxel liposome PTX-Fru-Lip.
Accurately weigh recipe quantity matrix material (by soybean lecithin: cholesterol=2:1 molar ratio), taxol (matrix material
It than being 1:30) in eggplant type flask, is dissolved with appropriate chloroform, the liposomal ligand of corresponding proportion is added, and (blank liposome is not
Add), 37 ± 1 DEG C of water bath with thermostatic control rotary evaporations obtain uniform film after removing chloroform, are dried in vacuum overnight removing residual solvent.It is added
PH 7.4(0.01M) PBS buffer solution, 20 DEG C of constant temperature air bath shaking tables, under the conditions of 180r/min after aquation 0.5h, ice-water bath is visited
Head ultrasonic (80W, 5S, 5S) the 3 minutes liposome solutions to get slightly opalescence.Then to load Paclitaxel liposome obtained
Vitro characterization is carried out.
Embodiment 8
The encapsulation rate and partial size of liposome and the measurement of current potential
Using the method separation free paclitaxel and load Paclitaxel liposome of refrigerated centrifuge.Blank, which is prepared, by embodiment 8 carries Japanese yew
The load Paclitaxel liposome PTX-Fru-Lip of alcohol liposome PTX-Lip and Fru-Chol modification, respectively takes two parts of (0.5 ml/
Part), under the conditions of 4 DEG C, 10000 rpm are centrifuged 20 minutes a liposome solutions, and supernatant is to be free of free paclitaxel
Liposome.The liposomal samples carried before PTX liposome and centrifugation after taking 40 μ l centrifugation respectively, are added 960 μ l methanol, vortex 10
After minute, 10000 rpm are centrifuged 10 minutes again, and 20 μ l supernatants injection high performance liquid chromatograph is taken to be analyzed respectively,
HPLC chromatogram condition are as follows:
Chromatographic column: SinoChrom ODS-C18 column (200 × 4.6 mm, 5 μm)；
Mobile phase: methanol-water (67:33)；
Column temperature: 35 DEG C；
Flow velocity: 1.0 ml/min；
Sample volume: 20 μ l；
Detection wavelength: 227 nm.
And calculate carry Paclitaxel liposome encapsulation rate (encapsulation efficiency, EE%): EE%=
AAfter centrifugation/ABefore centrifugation× 100%, wherein AAfter centrifugationRefer to the peak area of liposomal samples after being centrifuged, ABefore centrifugationRefer to centrifugation proliposome sample
The peak area of product.The encapsulation rate of each group liposome is shown in Table 1.In addition, also to this two kinds load Paclitaxel liposomes carried out partial size andZetaThe measurement (table 1) of current potential.The result shows that the encapsulation rate in Paclitaxel liposome reaches 86% or more, encapsulation rate is good；Two
Group group carry the partial size of Paclitaxel liposome in 110 nm hereinafter, monodispersity index (polymer dispersity index,
PDI it) is respectively less than 0.2, liposome presentation is uniformly distributed；Zeta potential is in elecrtonegativity.
1 two kinds of the table load encapsulation rates of Paclitaxel liposome, partial size andZetaCurrent potential (n=3)
Embodiment 10
Serum stability evaluation
Light transmittance of the Paclitaxel liposome modified by Nephelometric Determination different ligands in 50% fetal calf serum.
It takes each group to carry Paclitaxel liposome to be uniformly mixed with isometric fetal calf serum respectively, slowly shake in constant-temperature table
(37 DEG C, 50 rpm) are shaken, in scheduled time point (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h), pass through enzyme mark
Instrument measures absorbance value of all samples at 750 nm, and is converted into light transmittance.It is stood after liposome is mixed with fetal calf serum
When the light transmittance measured is 0 h as a result, mix with buffer PBS after the light transmittance that measures immediately as negative control, tie
Fruit sees Fig. 1.The result shows that all liposomes after being incubated for 48 hours with fetal calf serum altogether, light transmittance is still greater than 90%, without bright
Aobvious clustering phenomena shows that prepared liposome has preferable serum stability, for the later period is external, experiments are established in vivo etc.
Basis.
Embodiment 11
Tablets in vitro evaluation
It is investigated by vitro drug release of the dialysis to the liposome that different ligands are modified.Each group is taken to carry taxusol-lipid
Body and the free drug taxol of equivalent are respectively placed in the bag filter of 8000-12000 Da, and sealing is placed on 40 ml dialysis
In medium (PBS solution of 1%Tween 80, v/v), (37 DEG C, 50 rpm) slowly are shaken in constant-temperature table, when scheduled
Between point (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h) sample 0.1 ml, and isometric release is added and is situated between
The sample of matter, taking-up is analyzed by above-mentioned HPLC chromatogram condition.And the release of each sample of various time points is calculated, it draws
Its release behavior curve, is as a result shown in Fig. 2.The result shows that the release of free paclitaxel is more rapid, discharged being incubated in 12 hours
90%, and the liposome release that other each groups carry PTX is more slow, 60% or so are discharged in 48 hours, without being obviously released
Phenomenon, and the release characteristic no significant difference between each group liposome, show that liposome can be obviously improved the release of drug
Slow releasing function is played in behavior.
Embodiment 12
Hemolytic evaluation
Kunming mice (Sichuan University's Experimental Animal Center) is taken, takes blood through eye socket, blood is collected and is placed in the centrifugation for being coated with heparin sodium
Guan Zhong, at 4 DEG C, 10000 rpm are centrifuged 10 minutes, are removed supernatant, and cleaned red blood cell three times with physiological saline, finally will
Red blood cell is resuspended as the solution of 2%(w/v).By the two kinds of load Paclitaxel liposomes that will be prepared, it is gradually diluted with physiological saline
At 600 nmol/ml of lipid concentration, 400 nmol/ml, 200 nmol/ml, 100 nmol/ml, 50 nmol/ml, 25
Nmol/ml and 5 nmol/ml.After the liposome of above-mentioned various concentration is mixed with isometric red cell suspension respectively, in 37
It DEG C is incubated for 1 hour in constant-temperature table, 10000 rpm are centrifuged 10 minutes, and supernatant is taken to detect blood red egg at the nm of λ=540
White release.The result that 1% Qula logical (Triton X-100) and red blood cell are incubated for altogether is as positive control, by its hemolysis rate
It is set as 100%, the result being incubated for jointly using PBS and red blood cell sets 0% for its hemolysis rate as negative control.Each group rouge
The hemolysis rate calculation formula of plastid are as follows: hemolysis rate (hemolysis rate) %=(ASample – ANegative)/(APositive –
ANegative) × 100%.As a result see Fig. 3.The result shows that within the scope of the lipid concentration of setting, the lipid of different ligands modification
The equal < 10% of the hemolysis rate of body, does not cause the release of apparent hemoglobin, there is preferable biological safety.
Embodiment 13
The preparation of the liposome of CFPE label
By the preparation method of liposome in above-described embodiment 7, the liposome of CFPE label is prepared.Weigh the soybean phosphorus of recipe quantity
Ligand is not added in rouge, cholesterol and the unmodified liposome of ligand Fru-chol() it is dissolved in the mixed of chloroform-methanol (v/v=2/1) respectively
In bonding solvent, then a certain amount of fluorescein-labeled phosphatide CFPE is added thereto, making the concentration of final CFPE is about 20 μ g/
Solvent is removed under reduced pressure to form uniform lipid film in ml, continues to place 4 hours under vacuum.The phosphate that pH 7.4 is added is slow
Fliud flushing (PBS), 180 rpm aquation 30 minutes in 20 DEG C of constant-temperature tables, then with ultrasonic cell disruptor ultrasound (80W, 5S,
5S) 3 minutes liposome CFPE-Lip, CFPE-Fru-Lip to get CFPE label.
Embodiment 14
Cellular uptake experiment 1
For the ability into cell for the liposome modified from cellular level verifying fructose, respectively by three negative breasts of source of people
Cancer cell MDA-ME-231 and the triple negative breast cancer cell 4T1 of source of mouse are with 1 × 105The concentration of a cells/well is inoculated in 12 holes
In plate, at 37 DEG C, 5% CO2Cell incubator in be incubated for 24 hours, discard culture medium, by CFPE mark different ligands repair
The liposome of decorations is added separately in orifice plate after being diluted with culture medium, makes the 2 μ g/ml of concentration of final CFPE, and lipid concentration is
0.3 μm of ol/ml continues to be incubated for 2 hours in 37 DEG C of incubator, discards culture solution and is cleaned twice with PBS, digestion is collected thin
Born of the same parents are simultaneously cleaned three times with PBS again, measure the fluorescence intensity of cell after resuspension by flow cytometer.Ingestion result such as Fig. 4 institute
Show.By ingestion result as it can be seen that the liposomal ligand of fructose modification can increase by three negative shape breast cancer cell MDA-MB- to a certain extent
Intake of the 231 and 4T1 to liposome has respectively reached 1.422 times and 1.281 compared with without ligand modified liposome Lip
Times.
Cellular uptake experiment 2
In order to more intuitively observe MDA-ME-231 and 4T1 cell to the intake situation of the CFPE liposome marked, we
Qualitative research is carried out to cell with laser confocal microscope, concrete operations are as follows: MDA-ME-231 and 4T1 cell has been distinguished
With 1 × 106Concentration be inoculated in the 6 orifice plates for being equipped with coverslip, in 37 DEG C, 5% CO2Cell incubator in be incubated for 24 hours,
Culture medium is discarded, is added separately in orifice plate, makes after the liposome of the different ligands modification of CFPE label is diluted with culture medium
The concentration of final CFPE is 2 μ g/ml, and lipid concentration is 0.3 μm of ol/ml, continues to be incubated for 2 hours in 37 DEG C of incubator, abandon
It removes culture solution and is cleaned twice with PBS, 4% paraformaldehyde fixes 30 minutes, discards fixer, PBS is cleaned 3 times, with 5 μ g/ml
DAPI contaminates core 5 minutes, dyestuff is discarded, after PBS cleaning three times, with glycerol mounting.Slice is placed under laser confocal microscope
Shooting, as a result such as Fig. 5.The result shows that the liposomal ligand of fructose modification can increase triple negative breast cancer cell to a certain extent
Intake of the MDA-MB-231 and 4T1 to liposome.
Internal Evaluation on Its Targeting Performance
After targeting evaluation, we have further investigated the cancer target of Fru-Lip in lotus 4T1 breast cancer mouse model
Ability.
Embodiment 16
Carry the preparation of nir dye DiD liposome
By the preparation method of liposome in embodiment 6, preparation carries nir dye DiD liposome.Weigh the soybean phosphorus of recipe quantity
Rouge, cholesterol and ligand Fru-Chol(blank liposome are not added) it is dissolved in the mixed solvent of chloroform-methanol (v/v=2/1) respectively
In, then with lipid: a certain amount of nir dye DiD is added thereto, makes the concentration of final DiD about for the ratio of DiD=30:1
For 50 μ g/ml, solvent is removed under reduced pressure to form uniform lipid film.The phosphate buffer (PBS) of pH7.4 is added, in 20 DEG C
180 rpm aquation 30 minutes in constant-temperature table, then with ultrasonic cell disruptor ultrasonic (80W, 5S, 5S) 3 minutes to get load
The liposome solutions DiD-Lip and DiD-Fru-Lip of DiD.
Embodiment 17
Living imaging
It is small to model by the dosage of 500 μ g/kg of DiD through tail vein injection after lotus 4T1 breast cancer mouse model establishes 14 days
Mouse give carry DiD liposome DiD-Lip, DiD-Fru-Lip 2 h, 6 h, 12 h, 16h and 24 h are small by each group upon administration
Mouse is placed in small animal living body imager and observes, after immediately the mouse heart perfusion at each time point is put to death, by the heart, liver,
Spleen, lung, kidney and tumour are taken out, and are equally placed in living imaging instrument and observe, and carry out quantitative place with software to Ex vivo Tumor picture
Reason.As a result such as Fig. 6.
The result shows that (Fig. 6), shows stronger tumor target in the liposome of various time points, Fru-chol modification
To ability, tumor locus has reached highest drug concentration in 12h.2 h, 6 h, 12 h, 16h and 24 h, Fru-chol after administration
Drug concentration of the liposome of modification in tumour is 2.04,1.22,1.68,1.78, the 1.49 of unmodified liposome respectively
Times.It is mainly enriched in metabolic organ's liver in internal organ, and reaches Cmax in 6h, then most of for 24 hours to be eliminated, other are dirty
Device enrichment is less, and the especially heart and kidney is almost without enrichment.These the result shows that the liposome of Fru-Chol modification compared to not repairing
The lipid physical efficiency of decorations significantly improves liposome targeting breast cancer ability.
Detailed description of the invention
Fig. 1: the variation (n=3) of light transmittance after the load Paclitaxel liposome of different ligands modification is incubated in 50% serum
Fig. 2: the tablets in vitro behavior (n=3) of the load Paclitaxel liposome of different ligands modification
Fig. 3: the hemolysis rate (n=3) of the load Paclitaxel liposome of different ligands modification
The liposome of Fig. 4: CFPE label is in MDA-MB-231 cell (left side) and in the intake situation (n=3) of 4T1 cell
The liposome of Fig. 5: CFPE label is in MDA-MB-231 cell (left side) and in the intake situation of 4T1 cell (right side)
Fig. 6: the living imaging figure at each time point after the liposome of (A) lotus 4T1 breast cancer mouse injection load DiD；(B) isolated viscus
Living imaging figure, the respectively heart, liver, spleen, lung and kidney from left to right；(C) the living imaging figure of Ex vivo Tumor；(D) in vitro swollen
The fluorescence intensity of tumor is quantitatively schemed.A=DiD-Lip, b=DiD-Fru-Lip, n=3.
Claims (6)
1. a kind of breast cancer targeting lipids material of fructose modification, for structure or its pharmaceutically acceptable salt shown in logical formula (I)
Or hydrate, matrix material (I) are characterized in that: using triethylene glycol and succinic anhydride as connection chain, one end connects endogenous cholesterol,
Other end connection has the fructose of breast cancer target function:
Wherein, the acid anhydrides in used connection chain is one of succinic anhydride, malonic anhydride and glutaric anhydride, polyethylene glycol
For one of triethylene glycol or PEG200,400,600,800,1000,1500,2000,4000.
2. Novel breast gland cancer targeting lipids material according to claim 1 targets medicine in preparation breast cancer as pharmaceutical carrier
Application in object.
3. breast cancer target liposomes made by Novel breast gland cancer targeting lipids material according to claim 1, special
Sign is, including membrane material and activating agent, and the membrane material is phospholipid bilayer, and by lecithin, cholesterol and liposome are matched
Body composition, wherein each component proportion relation is as follows: the molar ratio of cholesterol and phosphatide is 1 ~ 2:1 ~ 10, and liposomal ligand rubs
Your content is the 1 ~ 25% of the total mole number of cholesterol and phosphatide；Activating agent of the present invention uses therapeutic agent or developer, living
The dosage of property agent can be adjusted according to including the activating agent in steroidal, wherein in percentage by weight, activating agent Zhan is total
The 0.1%-50% of lipid；Hydrating fluid is the 0.01M phosphate buffer (PBS) of pH 7.4.
4. breast cancer target liposomes made by Novel breast gland cancer targeting lipids material according to claim 1, special
Sign is, according to said components proportion relation, prepares breast cancer target liposomes using membrane process, can be prepared partial size and
The stable breast cancer target liposomes of Zeta potential, liposome particle size are 110nm or so, and encapsulation rate is greater than 86%.
5. breast cancer target liposomes made by Novel breast gland cancer targeting lipids material according to claim 1, special
Sign is, uses lecithin in the phosphatide present invention.
6. breast cancer target liposomes made by Novel breast gland cancer targeting lipids material according to claim 1, special
Sign is that the therapeutic agent in the activating agent present invention uses taxol, and developer uses CFPE or DiD.
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