CN105055318B - A kind of dual target liposome and its preparation method and application modified with MAN and WGA - Google Patents
A kind of dual target liposome and its preparation method and application modified with MAN and WGA Download PDFInfo
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Abstract
The dual target liposome and its preparation method and application that the invention discloses a kind of to modify with MAN and WGA.Dual target liposome provided by the invention is made of the trim of liposome and its surface, and the trim of the surface of liposome is 4 aminobenzene α D mannopyranoses glycosides and wheat germ agglutinin.The present invention also protects a kind of drug-loaded liposome, is to contain what drug obtained with the target liposomes.The drug concretely Farmorubine Hydrochloride and/or resveratrol.Dual target liposome use pair provided by the invention is ligand modified, contains two kinds of drugs, dual-targeting function is played, make drug across being targeted to glioma position after blood-brain barrier, and then it is enriched in tumor locus, the two kinds of drugs contained simultaneously can improve chemotherapy effect the effect of lesions position generates collaboration or is added.The present invention provides a new thinking to prepare treatment glioma drug, has important theory significance and clinical meaning.
Description
Technical field
The dual target liposome and its preparation method and application that the present invention relates to a kind of to modify with MAN and WGA.
Background technology
Clinically the treatment still faces enormous challenge of glioma, non-invasive diagnostic become brain colloid with treatment at present
One new direction of tumor treatment brings hope to improve therapeutic effect and extending the life span of patient.But it is most
There is no good therapeutic effects for medicine, the reason is as follows that：(1) due to the presence of blood-brain barrier, 98% small-molecule drug
The tumor locus that cannot all enter inside brain with most macromolecular drug plays therapeutic effect；(2) blood brain can be penetrated
The medicine of barrier cannot well tumor locus be enriched with, largely be diffused into normal brain tissue, cause it is serious not
Good reaction, even results in death.Therefore want effective treatment glioma to need to overcome two obstacles：Blood-brain barrier and
Brain-glioma barrier.To meet the Treatment need of glioma, a kind of new drug delivery system of research can make drug penetrate blood brain
Barrier can target glioma and is enriched with as far as possible in tumor locus again, can reduce medicine in this way to normal cerebral tissue
Toxic side effect, and therapeutic effect can be improved.
Epi-ADM (Epirubicin, EPI) is a kind of new anthracycline derivatives of adriamycin, is a kind of wide spectrum
Antitumor drug, inhibited to kinds of tumors, mechanism of action is mainly：Epi-ADM molecule be inserted into DNA chain to
Inhibit the synthesis of DNA and RNA；Epi-ADM triggers DNA cracking by topoisomerase II, leads to cell death.It is clinical at present
On by Epi-ADM for breast cancer, non-Hodgkin lymphoma, oophoroma, soft tissue sarcoma, cancer of pancreas, gastric cancer, cellule
Lung cancer and acute leukemia.Compared with adriamycin, under similar dosage, Epi-ADM shows smaller blood and Amplatzer duct occluder
Property.In some external research experiments, Epi-ADM has very strong inhibiting effect, however internal light to brain glioblastoma cell
It studies as observed result shows that Epi-ADM is not detected in the brain region protected by blood-brain barrier.The reason is that due to blood brain screen
The presence of barrier, Epi-ADM can not reach brain tissue and play therapeutic effect to glioma.Resveratrol (resveratrol,
RES) it is a kind of natural molecule being primarily present in grape, there is various biological effect, it is such as antitumor, anti-oxidant, anti-prominent
Change, anti-inflammatory and reduction blood glucose etc., it has apparent inhibiting effect to the generation of tumour, promotion and 3 stages of development.In vain
Veratryl alcohol antineoplastic action mechanism is by inhibiting cancer cell synthesis, arresting cell cycle, cancer cell specific induction of apoptosis, inhibiting enzyme
Class simultaneously interferes coherent signal conduction path and plays antitumor action, and In vivo study shows without apparent toxic side effect.
Invention content
The object of the present invention is to provide a kind of dual target liposome modified with MAN and WGA and preparation method thereof and
Using.
Dual target liposome provided by the invention is made of the trim of liposome and its surface, it is characterised in that：Institute
The trim for stating surface of liposome is 4- aminobenzene-α-D- mannopyranoses glycosides and wheat germ agglutinin.
4- aminobenzene-α-D- mannopyranose the glycosides may connect on the amino of the liposome；The wheat germ lectin
Element may connect on the carboxyl of the liposome.The amino of the liposome can be by DSPE-PEG2000-NH2It provides；The lipid
The carboxyl of body can be by DSPE-PEG2000- COOH is provided.
Specifically：When the liposome preparation, DSPE-PEG can be added in the feed2000-NH2And DSPE-PEG2000-
COOH, DSPE-PEG2000-NH2And DSPE-PEG2000- COOH is attached to surface of liposome, provides for connecting 4- aminobenzenes-α-
The amino and carboxyl of D- mannopyranoses glycosides and wheat germ agglutinin.
Egg yolk lecithin, cholesterol, DSPE-PEG specifically can be used in the liposome2000、DSPE-PEG2000- COOH and
DSPE-PEG2000-NH2It is prepared as raw material.Egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000-COOH
And DSPE-PEG2000-NH2Molar ratio can be (50-55)：(40-45)：4：(0.25-0.75)：(0.25-0.75).Yolk ovum
Phosphatide, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2Molar ratio concretely 52:
43:4:0.5:0.5。
In the target liposomes, the quality of the wheat germ agglutinin and egg yolk lecithin when preparing the liposome and courage
The proportioning of the quality sum of sterol can be (4-6)：30, concretely 5：30, the 4- aminobenzene-α-D- mannopyranose glycosides
The proportioning of quality and the quality sum of egg yolk lecithin when preparing the liposome and cholesterol can be (1-2):30, concretely
1.5:30。
The present invention also provides a kind of preparation methods of target liposomes, include the following steps：
1) with egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2For
Raw material, which prepares surface, has the liposome of amino and carboxyl；
2) amino of liposome prepared by 4- aminobenzene-α-D- mannopyranoses glycosides and step 1) is connect, wheat germ is coagulated
Collection element is connect with the carboxyl of liposome prepared by step 1)；
Obtain target liposomes.
In the step 1), the method for preparing liposome is specific as follows：
By egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2Dissolving
In methyl alcohol, rotary evaporation removes methanol, then 250mM ammonium sulfate solutions are added, so in 35 DEG C, 40rpm rotary evaporations film forming
After be ultrasonically treated (ultrasound parameter：The 10s that often works stops 30s, all times 10min, and protection temperature is 35 DEG C), then make
Crossing 400nm polycarbon resins films 3 times and 200nm polycarbon resins film respectively with liposome squeezer, 3 times (sequence is as follows：400nm polycarbon resin films
→ 400nm polycarbon resins film → 400nm polycarbon resins film → 200nm polycarbon resins film → 200nm polycarbon resins film → 200nm polycarbon resins film),
It is subsequently installed in the PBS buffer solution for being placed in pH7.4 in bag filter and dialyses, obtain liposome.
In the step 2), bridging agent can be made with glutaraldehyde, by 4- aminobenzene-α-D- mannopyranoses glycosides and liposome
Amino is (by DSPE-PEG2000-NH2There is provided) connection.
In the step 2), bridging agent can be made with EDCI, by the carboxyl of wheat germ agglutinin and liposome (by DSPE-
PEG2000- COOH is provided) connection.
In the step 1), egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-
PEG2000-NH2Molar ratio can be (50-55)：(40-45)：4：(0.25-0.75)：(0.25-0.75).In the step 1),
Egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2Molar ratio specifically may be used
It is 52:43:4:0.5:0.5.
The target liposomes can be applied to prepare pharmaceutical carrier.The drug concretely Farmorubine Hydrochloride and/
Or resveratrol.
The present invention also protects a kind of pharmaceutical carrier, its active constituent is the target liposomes.The drug is specific
Can be Farmorubine Hydrochloride and/or resveratrol.
The present invention also protects a kind of drug-loaded liposome, is to contain what drug obtained with the target liposomes.The medicine
Object concretely Farmorubine Hydrochloride and/or resveratrol.
The preparation method of the drug-loaded liposome is specific as follows：
(1) by resveratrol, egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-
PEG2000-NH2In methyl alcohol, rotary evaporation removes methanol for dissolving, then 250mM sulphur is added in 35 DEG C, 40rpm rotary evaporations film forming
Sour aqueous ammonium, is then ultrasonically treated (ultrasound parameter：The 10s that often works stops 30s, all times 10min, protects temperature
It is 35 DEG C), then use liposome squeezer to cross 400nm polycarbon resins films 3 times and 200nm polycarbon resins film 3 times respectively (sequentially such as
Under：400nm polycarbon resins film → 400nm polycarbon resins film → 400nm polycarbon resins film → 200nm polycarbon resins film → 200nm polycarbon resin films
→ 200nm polycarbon resins film), it is subsequently installed in the PBS buffer solution for being placed in pH7.4 in bag filter and dialyses, obtain liposome A0；
(2) make bridging agent with glutaraldehyde, by the amino of 4- aminobenzene-α-D- mannopyranoses glycosides and liposome A0 (by
DSPE-PEG2000-NH2There is provided) connection, obtain liposome B0；
(3) make bridging agent with EDCI, by the carboxyl of wheat germ agglutinin and liposome B0 (by DSPE-PEG2000- COOH is carried
For) connection, obtain liposome D0；
(4) Farmorubine Hydrochloride is mixed with liposome D0, reaction (specifically can 60 DEG C, left and right oscillation, 120 times/min shakes
Swing 20min) after obtain liposome D1 (drug-loaded liposome for containing sour Epi-ADM and resveratrol).
In step (1), the addition quality of resveratrol is that the proportioning of the quality sum of egg yolk lecithin and cholesterol is 1：
30。
In step (4), the addition quality of Farmorubine Hydrochloride and the quality of step (1) mesolecithal lecithin and cholesterol it
The proportioning of sum is=1：12.
In the step (1), egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-
PEG2000-NH2Molar ratio can be (50-55)：(40-45)：4：(0.25-0.75)：(0.25-0.75).In the step (1),
Egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2Molar ratio specifically may be used
It is 52:43:4:0.5:0.5.
The present invention also protects a kind of drug for treating brain tumor, its active constituent be contain Farmorubine Hydrochloride and/or
The drug-loaded liposome of resveratrol.The brain tumor can be glioma, concretely caused by C6 malignant glioma cells
Glioma.
The drug-loaded liposome for containing Farmorubine Hydrochloride and/or resveratrol can be applied to prepare treatment brain tumor
Drug.The brain tumor can be glioma, concretely glioma caused by C6 malignant glioma cells.
MAN has good effect in terms of assisting pharmaceutical carrier to penetrate blood-brain barrier.Since GLUT1 is in blood-brain barrier
It is expressed with high on brain tumor cell, and MAN has specific affinity to GLUT1, so the present invention is using MAN as targeting ligand
It is connected to surface of liposome, will be played a significant role in terms of assisting drug-loaded liposome to penetrate blood-brain barrier, while MAN can also
Enhance the ability of drug-loaded liposome targeting brain tumor cell.Wheat germ agglutinin WGA can be used as the drug of efficient targeting blood-brain barrier
Carrier ligand, main cause, which is WGA, to be had relative to other agglutinins in higher affinity and lower cerebral capillary
Chrotoplast toxicity.It is swollen as assisting to promote agglutinin for the height tendentiousness that wheat germ agglutinin shows malignant cell
Development in terms of tumor treatment.
The grain size of liposome can be with the influence interior medicine dynamics property of liposome of conspicuousness, toxicity and its antitumor
Activity.Itself with regard to grain size, average grain diameter possesses longest plasma half-life in the liposome of 100nm or so.The present invention obtains
To the liposome for containing Epi-ADM and resveratrol average grain diameter near 100nm, polydispersity coefficient is in 0.15-0.23
Left and right is that a particle size is suitable, the dispersion that is evenly distributed, and circulation time in blood is long, makes liposome vesicle
There are more chances to be enriched with to tumor tissues by " hole " in tumor locus new capillary vessel endothelial tissue, to real
Now better antitumor activity.Current potential be the physical stability for weighing liposome or lipid nano particle monodisperse system parameter it
One.The current potential that the present invention obtained contain the liposome of Epi-ADM and resveratrol shows slightly negativity, illustrate liposome vesicle it
Between have suitable electrostatic repulsion forces, liposome bin stability is more preferable, is not susceptible to coagulation.
Dual target liposome provided by the invention using it is double it is ligand modified (after MAN is modified, can be by blood-brain barrier
Glucose transporter identification, so that liposome transmembrane transport is passed through blood-brain barrier；Liposome is after WGA is modified, to tumour cell
The tendentiousness of specific height plays dual-targeting function to improve the drug concentration of tumor locus), contain two kinds of drug (hydrochloric acid
Epi-ADM and resveratrol), dual-targeting function has been played, has made drug across being targeted to glioma portion after blood-brain barrier
Position, and then be enriched in tumor locus, while the two kinds of drugs contained can carry the effect of lesions position generates collaboration or is added
High chemotherapy effect.The present invention provides a new thinking to prepare treatment glioma drug, has important theory significance
With clinical meaning.
Description of the drawings
Fig. 1 is HPLC chromatogram；1：RES goes out peak position；2：EPI goes out peak position；λ=306,254nm.
Fig. 2 is the photo for carrying out morphological observation to liposome D1 using transmission electron microscope.
Fig. 3 is the survival results of cell；A, P<0.05, to Farmorubine Hydrochloride；B, P<0.05, to liposome A1；C, P
<0.05, to liposome A2；D, P<0.05, to liposome B1；E, P<0.05, to liposome C1.
Fig. 4 is flow cytomery result.
Fig. 5 is laser confocal microscope testing result.
Fig. 6 is drug-loaded liposome in vitro to C6 malignant glioma cells apoptotic effect results.
Fig. 7 is C6 malignant glioma cells ball experimental results；A, P<0.01, to blank control；B, P<0.05, to hydrochloric acid
Epi-ADM；C, P<0.05, to liposome A1；D, P<0.05, to liposome A2；E, P<0.05, to liposome B1；F, P<
0.05, to liposome C1.
Fig. 8 is HRP content measuring standard curves.
Fig. 9 is the HRP Penetration ration measurement results of different time.
Figure 10 is across the in vitro blood-brain barrier Transport Rate result of Farmorubine Hydrochloride；A, P<0.05, to Farmorubine Hydrochloride；B,
P<0.05, to liposome A1；C, P<0.05, to liposome A2；D, P<0.05, to liposome B1；E, P<0.05, to liposome
C1。
Figure 11 is across the in vitro blood-brain barrier Transport Rate result of Farmorubine Hydrochloride.
Figure 12 is inhibiting rate of the different liposome to C6 malignant glioma cells；A, P<0.05, to Farmorubine Hydrochloride；
B, P<0.05, to liposome A1；C, P<0.05, to liposome A2；D, P<0.05, to liposome B1；E, P<0.05, to liposome
C1。
Figure 13 is the HE coloration results of the brain sample of tumor tissues.
Figure 14 is gross tumor volume inhibiting rate after one week of dosing；A, P<0.05, to Farmorubine Hydrochloride；B, P<0.05, to fat
Plastid A1；C, P<0.05, to liposome A2；D, P<0.05, to liposome B1；E, P<0.05, to liposome C1.
Figure 15 is the kaplan-Meier survival rate of the C6 glioma rats of different drug-loaded liposomes processing.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Egg yolk lecithin (EPC)：Germany Lipoid company,ShangHai local agent,China.Courage
Sterol (cholesterol)：Beijing bispin microbiological culture media products factory.Polyethylene glycol-distearoyl phosphatidyl ethanolamine
(DSPE-PEG2000)：NOF Corp (Japanese NOF companies).DSPE-PEG2000-COOH、DSPE-PEG2000-NH2、
P-aminophenyl- α-D-manno-pyranoside (4- aminobenzene-α-D- mannopyranose glycosides, MAN), wheat germ agglutinin
(WGA), sephadex (shephedex G-50), trinitrobenzene sulfonic acid (TNBS)：Sigma companies.Carbodiimide hydrochloride
(EDCI), N- hydroxy thiosuccinimides (NHS)：The Shanghai bio tech ltd Yuan Ye.Farmorubine Hydrochloride
(epirubicin, EPI), resveratrol (resveratrol, RES)：Nanjing celestial worthy Ze Zhong chemical reagents corporations.Ammonium sulfate
((NH4)2SO4), analysis is pure：Tianjin good fortune morning chemical reagent factory.DMEM(Dulbecco’s Modified Eagle Medium)
High glucose medium, 0.25% pancreatin：Gibico companies.Fetal calf serum：Hangzhou Chinese holly bio tech ltd.Penicillin-
The dual anti-reagent of streptomysin (100 ×)：Beijing bio tech ltd Suo Laibao.Sulforhodamine B albumen (SRB)：The U.S.
Sigma companies.(Horseradish peroxidase, HRP molecular weight is 40000 to horseradish peroxidase, enzymatic activity ＞
250U·mg-1)：Sigma Co., USA.C6 malignant glioma cells：Shanghai cell institute of the Chinese Academy of Sciences.Mouse brain endothelium is thin
Born of the same parents' (bEnd.3 cells)：Beijing North Na Chuanlian Bioisystech Co., Ltd.
Poly- carbon ester filter (aperture 400,200nm)：Millipore(Bedford,MA,USA).Shimadzu LC-15C high
Effect liquid phase chromatogram instrument：Japanese Shimadzu Corporation, UV detector.UV-2401PC type ultraviolet-visible spectrophotometers：Japanese Shimadzu
Company.Malvern particle size analyzer：Malvern instrument company of Britain.SartoriusBP211D electronic balances：German Sai Duolisi
Group.Ultrasonic cleaner：Its roc New Electronic Techniques Beijing Co., Ltd.EYELA-1000S type Rotary Evaporators：Japan
EYELA Tokyo Physico-chemical Apparatus Co., Ltd..JY92 cell Ultrasonic Cell Disruptors：NingBo XinZhi Biology Science Co., Ltd.It is permanent
Warm oscillator：Medical Instruments factory of Jintan City of Jiangsu Province.Bag filter：Molecular cut off 8000-12000.Liposome squeezer：Germany
Sai Duolisi groups.Cell inserter：Corning companies of the U.S. (Coring, NY, USA；O.4 μm aperture, 12mm diameters,
1.12cm2Surface area).Stereotaxic apparatus：Shenzhen Rui Wode Co., Ltds.CO2Constant incubator：The limited public affairs of Thermo science and technology
Department, the U.S..Enzyme-labeled immunity analyzer：Shanghai Gene Tech. Company Limited.Resistance determinator：EVOMk, Word Precision
Instruments, USA.
The assay method of WGA modification rates is following, and (WGA is protein, therefore can be used what Coomassie Brilliant Blue detection dissociated
WGA)：Use shephedex G-50 post separations free WGA (pillar internal diameter for 1.5cm, pillar height 18cm；Applied sample amount is
1ml；Elution process：First use PBS buffer solution balance pillar → loading of pH7.4 → eluted with the PBS buffer solution of pH7.4, flow velocity
It is present in the eluent that retention volume is 20ml-32ml for 1.0mL/min → free WGA), then use Coomassie Brilliant Blue
The amount of the free wheat germ agglutinin of detection (in terms of total protein)；CEWGA=(1-Cuncoupled/Ctotal) × 100%, CEWGAFor WGA
Joint efficiency, CuncoupledFor the amount of free wheat germ agglutinin, CtotalFor the initial incremental amount of WGA.
The assay method of MAN modification rates is as follows：Using DSPE-PEG on TNBS methods detection liposome2000-NH2Amino,
CEMAN=(the amino quantity that can be reacted with trinitrobenzene sulfonic acid on liposome after 1- connections MAN/connecting can on MAN proliposomes
The amino quantity reacted with trinitrobenzene sulfonic acid).
The content of Farmorubine Hydrochloride and resveratrol is detected using HPLC methods, relevant parameter is as follows：
Chromatographic condition：Chromatographic column is U.S.'s Féraud door Luna C18 columns (250mm × 4.6mm, 5 μm)；Mobile phase is " first
- 0.4% phosphate aqueous solution of alcohol-acetonitrile " (volume ratio 16:21:63)；Flow velocity is 1.0ml/min；Ultraviolet detection wavelength is
254nm (Farmorubine Hydrochloride) and 306nm (resveratrol)；Column temperature is room temperature；Sample size is 20 μ L；
Chromatographic behavior：Under above-mentioned chromatographic condition, demulsification solution (A), the resveratrol control of blank liposome are taken respectively
Product solution (B), Farmorubine Hydrochloride reference substance solution (C), be loaded with resveratrol and Epi-ADM liposome demulsification solution
(D) sample introduction is analyzed, as a result sees chromatogram 1, blank liposome is noiseless to drug monitoring as seen from the figure.Resveratrol chromatographic peak is protected
It is about 11.7min to stay the time, Farmorubine Hydrochloride chromatographic peak retention time is about 14.6min.
The preparation of embodiment 1, liposome
One, the preparation of blank liposome
1, the preparation of blank liposome
By egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2Massage
That ratio 52:43:4:0.5:In methyl alcohol, rotary evaporation removes methanol, 35 DEG C, 40rpm rotary evaporations in eggplant-shape bottle for 0.5 dissolving
Then film forming is added 250mM ammonium sulfate solutions, is then ultrasonically treated (ultrasound parameter：The 10s that often works stops 30s, whole
Time is 10min, and protection temperature is 35 DEG C), then liposome squeezer is used to cross 400nm polycarbon resins film 3 times and 200nm respectively
(sequence is as follows for polycarbon resin film 3 times：400nm polycarbon resins film → 400nm polycarbon resins film → 400nm polycarbon resins film → 200nm polycarbon resins
Film → 200nm polycarbon resins film → 200nm polycarbon resins film), it is subsequently installed in the PBS buffer solution for being placed in pH7.4 in bag filter thoroughly
Analysis, obtains liposome A.
2, the preparation of the liposome of blank MAN modifications
Make bridging agent by glutaraldehyde and MAN is chemically attached to DSPE-PEG on liposome A2000-NH2Amino on, specifically
Step is：By the liposome A of 2ml 15mg/ml, (egg yolk lecithin and courage are solid when the quality of liposome A is to prepare liposome A
The quality sum meter of alcohol) it is mixed with 1.5mg MAN, it is then slowly added into glutaraldehyde (glutaraldehyde concentration is made to reach 15mM), room
Temperature is incubated 5min, is subsequently installed in the PBS buffer solution for being placed in pH7.4 in bag filter and dialyses.Obtain liposome B.
3, the preparation of the liposome of blank WGA modifications
Make bridging agent by DSPE-PEG on the amino of WGA and liposome A by EDCI2000The carboxyl of-COOH connects, specifically
Step is：Taking the liposome A of 2ml 15mg/ml, (egg yolk lecithin and courage are solid when the quality of liposome A is to prepare liposome A
The quality sum meter of alcohol), 1.0mg EDCI and 1.44mg NHS and mixing is added, is subsequently placed in constant temperature oscillator (37 DEG C, a left side
It is right oscillation, 120 times/min) in vibrate 12h, then be added 5mgWGA be placed in constant temperature oscillator (37 DEG C, left and right oscillation, 120
Secondary/min) in vibrate 1h.Obtain liposome C.
4, the preparation of the liposome of blank MAN and WGA modifications
Make bridging agent by DSPE-PEG on the amino of WGA and liposome B by EDCI2000The carboxyl connection of-COOH (uses fat
Plastid B replaces liposome A, other same step 3).Obtain liposome D.
Liposome A, liposome B, liposome C and the liposome D of above-mentioned preparation are suspended in liquid phase.
Two, the preparation of drug-loaded liposome
Quality of the medicine fat than=drug：In liposome the quality sum of lecithin and cholesterol (lecithin and cholesterol
Quality to prepare liposome when the quality of lecithin and cholesterol that is added in terms of).
Drug in this step is Farmorubine Hydrochloride or resveratrol.
Difference with the 1 of step 1 is only that：Resveratrol (medicine fat ratio=1 is added when originating reaction：30).It is carried
There is the liposome A0 of RES.
Farmorubine Hydrochloride is mixed to (medicine fat ratio=1 with liposome A：12), be placed in constant temperature oscillator (60 DEG C, left and right shake
Swing, 120 times/min) in vibrate 20min.Obtain the liposome A1 for being loaded with EPI.
Farmorubine Hydrochloride is mixed to (medicine fat ratio=1 with liposome A0：12), be placed in constant temperature oscillator (60 DEG C, left and right shake
Swing, 120 times/min) in vibrate 20min.Obtain the liposome A2 for being loaded with EPI and RES.
Liposome A is replaced with liposome A0, the 2 of other same step 1.Obtain the liposome for being loaded with RES modified through MAN
B0。
Farmorubine Hydrochloride is mixed to (medicine fat ratio=1 with liposome B0：12), be placed in constant temperature oscillator (60 DEG C, left and right shake
Swing, 120 times/min) in vibrate 20min.Obtain the liposome B1 for being loaded with EPI and RES modified through MAN.
Liposome A is replaced with liposome A0, the 3 of other same step 1.Obtain the liposome for being loaded with RES modified through WGA
C0。
Farmorubine Hydrochloride is mixed to (medicine fat ratio=1 with liposome C0：12), be placed in constant temperature oscillator (60 DEG C, left and right shake
Swing, 120 times/min) in vibrate 20min.Obtain the liposome C1 for being loaded with EPI and RES modified through WGA.
Liposome A is replaced with liposome B0, the 3 of other same step 1.It obtains being loaded with RES through what MAN and WGA were modified jointly
Liposome D0.
Farmorubine Hydrochloride is mixed to (medicine fat ratio=1 with liposome D0：12), be placed in constant temperature oscillator (60 DEG C, left and right shake
Swing, 120 times/min) in vibrate 20min.Obtain the liposome D1 for being loaded with EPI and RES modified jointly through MAN and WGA.
Each liposome of above-mentioned preparation is suspended in liquid phase.
The CE of liposome C1WGAIt is 22.57 ± 0.12%.The CE of liposome D1WGAIt is 19.25 ± 0.087%.
The CE of liposome B1MANIt is 37.83 ± 1.04%.The CE of liposome D1MANIt is 36.98 ± 1.70%.
The performance measurement of embodiment 2, liposome
One, the measurement of encapsulation rate
Liposome A1 that embodiment 1 obtains, liposome A2, liposome B1, liposome C1, liposome D1 are carried out such as respectively
Under (1) or (2) operation.
(1) liposome A1, liposome A2, liposome B1, liposome C1, liposome D1 are proceeded as follows respectively：It will
Liposome is placed in 10mL volumetric flasks, is destroyed with the methanol of 10 times of volumes and (is obtained demulsification solution), detects resveratrol and Biao A
The concentration of mycin.
(2) liposome A1, liposome A2, liposome B1, liposome C1, liposome D1 are proceeded as follows respectively：It will
Liposome crosses shephedex G-50 columns, is eluted with the PBS buffer solution of pH7.4, to remove free resveratrol, is subsequently installed in
It is placed in dialysis in the PBS buffer solution of pH7.4 in bag filter and is subsequently placed in 10mL volumetric flasks to remove free Epi-ADM
In, it is destroyed with the methanol of 10 times of volumes and (is obtained demulsification solution), detect the concentration of resveratrol and Epi-ADM.
In encapsulation rate (%)=step (2) in concentration/step (1) of resveratrol or Epi-ADM resveratrol or table Ah
Concentration × 100% of mycin.It the results are shown in Table 1.
The encapsulation rate (n=3) of the different drug-loaded liposomes of table 1
| Epi-ADM encapsulation rate % | Resveratrol encapsulation rate % | |
| Liposome A1 | 92.04±1.5 | —— |
| Liposome A2 | 90.07±0.76 | 65.01±3.52 |
| Liposome B1 | 90.05±1.11 | 66.56±0.16 |
| Liposome C1 | 91.46±0.07 | 65.88±0.45 |
| Liposome D1 | 92.49±1.29 | 66.29±0.55 |
Two, grain size and potential measurement
Respectively by liposome A freshly prepared in 1ml embodiments 1, liposome A1, liposome A2, liposome B1, liposome
C1, liposome D1 are diluted with the PBS buffer solution of pH7.4, use Nano Series Zen 4003Zeta Sizer (Malvern
Instruments, Ltd, UK) measure liposome grain size, polydispersity coefficient and Zeta potential.It is the grain size of different liposome, more
The measurement result of the coefficient of dispersion (PDI) and zeta current potentials is shown in Table 2.Liposomal particle size is evenly distributed, and grain size is in 100nm or so.Respectively
Drug-loaded liposome all slightly negative electricity illustrate there is suitable electrostatic repulsion forces between liposome vesicle, and bin stability is more preferable, no
Coagulation easily occurs.
2 different liposome grain size of table, polydispersity coefficient and Zeta potential (n=3)
| Grain size (nm) | Polydispersity coefficient | Zeta potential (mv) | |
| Liposome A | 85.24±0.33 | 0.210±0.002 | -3.91±0.08 |
| Liposome A1 | 89.10±0.46 | 0.204±0.013 | -7.80±0.18 |
| Liposome A2 | 94.30±0.51 | 0.178±0.008 | -8.30±0.45 |
| Liposome B1 | 99.18±0.61 | 0.159±0.007 | -13.43±0.31 |
| Liposome C1 | 100.21±0.26 | 0.160±0.009 | -12.07±0.59 |
| Liposome D1 | 103.77±0.65 | 0.225±0.009 | -14.40±0.70 |
Three, transmission electron microscope (TEM)
Morphological observation is carried out to liposome D1 prepared by embodiment 1 using transmission electron microscope.As a result see Fig. 2, it is seen that lipid
Volume morphing more rounding, size are uniform.
Embodiment 3, drug-loaded liposome are in vitro to C6 brain glioblastoma cell inhibiting effect researchs
When the addition form of drug is liposome, the addition of Farmorubine Hydrochloride is multiplied by encapsulating for the addition of liposome
Rate.
1, the C6 malignant glioma cells of logarithmic growth phase are inoculated in 96 orifice plates with 3000/hole (190 holes μ L/)
In, it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
2, after completing step 1,96 orifice plate is taken, it is dense that the series that 10 μ L are prepared with the PBS buffer solution of pH7.4 is added per hole
(the addition form of drug is respectively the drug solution of degree：Farmorubine Hydrochloride, liposome A1, liposome A2, liposome B1, fat
Plastid C1 or liposome D1, it is respectively 0.05,0.1,0.5,1,2.5,5 or 10 μ to make the concentration of each test group Farmorubine Hydrochloride
M；Blank control with isometric PBS buffer solution alternatives to medication solution is set), it is placed in 37 DEG C, 5%CO2Under the conditions of culture it is 48 small
When, it then inhales and abandons culture supernatant, measured per hole trap OD values at 540nm using srb assay.
Cell survival rate=test group OD values/blank control group OD value × 100%.
It carries out three repetitions to test, results are averaged.
The survival results of cell are shown in Fig. 3.The results show that liposome D1 shows strongest anticol under each concentration
The effect of matter tumor cell proliferation.
IC50 values (Epi-ADM concentration when cell survival rate=50%) the results are shown in Table 3.The IC50 values of liposome D1 are bright
It is aobvious to be less than other groups, the antiproliferative effect of C6 glioma cells is greatly improved, hence it is evident that higher than free Epi-ADM and other fat
Plastid.
The IC50 values of 3 different liposome of table
| IC50(μM) | |
| Farmorubine Hydrochloride | 1.07 |
| Liposome A1 | 3.12 |
| Liposome A2 | 2.15 |
| Liposome B1 | 1.93 |
| Liposome C1 | 1.37 |
| Liposome D1 | 0.85 |
Embodiment 4, C6 malignant glioma cells act on the intake of drug-loaded liposome
When the addition form of drug is liposome, the addition of Farmorubine Hydrochloride is multiplied by encapsulating for the addition of liposome
Rate.
One, flow cytomery
1, the C6 malignant glioma cells of logarithmic growth phase, with 1 × 105A/hole (1500 holes μ L/) is inoculated in 12 holes
In plate, it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
2, after completing step 1,12 orifice plate is taken, it is dense that the series that 80 μ L are prepared with the PBS buffer solution of pH7.4 is added per hole
(the addition form of drug is respectively the drug solution of degree：Farmorubine Hydrochloride, liposome A2, liposome B1, liposome C1 or fat
Plastid D1 makes a concentration of 68 μ g/ml of each test group Farmorubine Hydrochloride；Setting is molten with isometric PBS buffer solution alternatives to medication
The blank control of liquid), it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate 4 hours.
3, after completing step 2,12 orifice plate, suction is taken to abandon culture supernatant, two is rinsed with the PBS buffer solution of cold pH7.4
Time, then digested with 0.25% pancreatin, then with the PBS buffer solution of pH7.4 wash cell twice, then using flow cytometer into
Row detection.
As a result see that (A is blank control to Fig. 4, and B is liposome A2, and C is liposome B1, and E is that liposome C1, F are liposome
D1, G are Farmorubine Hydrochloride).Compared with free Epi-ADM, liposome C1, B1, A2, liposome D1 shows strongest C6
Intake ability in malignant glioma cells.
Two, laser confocal microscope detects
1, the C6 malignant glioma cells of logarithmic growth phase, with 2 × 105A/hole (2000 holes μ L/) is inoculated in 6 holes
In plate, it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
2, after completing step 1, the 6 orifice plates are taken, it is dense that the series that 100 μ L are prepared with the PBS buffer solution of pH7.4 is added per hole
(the addition form of drug is respectively the drug solution of degree：Farmorubine Hydrochloride, liposome A2, liposome B1, liposome C1 or fat
Plastid D1 makes a concentration of 5 μ g/ml of each test group Farmorubine Hydrochloride；Isometric PBS buffer solution alternatives to medication solution is set
Blank control), be placed in 37 DEG C, 5%CO2Under the conditions of cultivate 2 hours.
3, after completing step 2, the 6 orifice plates, suction is taken to abandon culture supernatant, three is rinsed with the PBS buffer solution of cold pH7.4
It is secondary, 10min then is fixed with 4% paraformaldehyde, then Hoechst33258 is used to carry out nuclear targeting, it is then total with laser
Focusing microscope (LSM 510, Zeiss, Germany) excites Epi-ADM (excitation wavelength with the laser beam of 480nm wavelength
480nm, launch wavelength 560nm), Hoechst33258 (excitation wavelength 346nm, transmitted wave are excited with the laser beam of 346nm wavelength
Long 460nm), then image analysis is carried out with Aim Image Examiner softwares.
As a result see that (A is Farmorubine Hydrochloride to Fig. 5, and B is liposome A2, and C is liposome B1, and D is that liposome C1, E are lipid
Body D1；A1 display Farmorubine Hydrochlorides, A2 display nucleus, A3 display stacking charts, remaining figure and so on).Liposome is through C6
It is mainly distributed in nucleus after malignant glioma cells endocytosis.Compared with free Epi-ADM, liposome C1, B1, A2, fat
Plastid D1 abundances in C6 malignant glioma cells are most, and the intake that C6 malignant glioma cells are detected with streaming method is made
With consistent.
Embodiment 5, drug-loaded liposome in vitro study C6 malignant glioma cells apoptotic effects
When the addition form of drug is liposome, the addition of Farmorubine Hydrochloride is multiplied by encapsulating for the addition of liposome
Rate.
1, the C6 malignant glioma cells of logarithmic growth phase, with 2 × 105A/hole (2000 holes μ L/) is inoculated in 6 holes
In plate, it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
2, after completing step 1, the 6 orifice plates are taken, it is dense that the series that 100 μ L are prepared with the PBS buffer solution of pH7.4 is added per hole
(the addition form of drug is respectively the drug solution of degree：Farmorubine Hydrochloride, liposome A2, liposome B1, liposome C1 or fat
Plastid D1 makes a concentration of 5 μ g/ml of each test group Farmorubine Hydrochloride；Isometric PBS buffer solution alternatives to medication solution is set
Blank control), be placed in 37 DEG C, 5%CO2Under the conditions of cultivate 48 hours.
3, after completing step 2, the 6 orifice plates, suction is taken to abandon culture supernatant, is rinsed with the PBS buffer solution of cold pH7.4, so
It is digested afterwards with the pancreatin without EDTA, then cell is washed twice with the PBS buffer solution of pH7.4, then with the PBS of 300 μ LpH7.4
Buffer solution blows cell even, sequentially adds Binding Buffer, 5 μ L Annexin V-APC, 5 μ L 7- of 500 μ L later
AAD reacts 10min after mixing, through flow cytomery under conditions of room temperature is protected from light.
It carries out three repetitions to test, results are averaged.
As a result see Fig. 6 (a is liposome A2, and b is liposome B1, and c is liposome C1, and d is liposome D1, e be hydrochloric acid table Ah
Mycin, f are blank control).Drug concentration and drug incubation time under the same conditions, induce C6 malignant glioma cells
The sequence of the percentage of apoptosis from high to low is as follows successively：Liposome D1>Liposome C1>Liposome B1>Liposome A2>Free table
Adriamycin>Blank control.Q4-4 percentages are followed successively by 49.9 ± 0.19%, 47.4 ± 0.23%, 38.2 ± 0.41%,
29.5 ± 0.26%, 10.7 ± 0.29% and 0.5 ± 0.06%.The result shows that the dual-target that MAN and WGA are modified jointly is multiple
Square liposome D1 has the function of stronger induction C6 brain glioblastoma cell apoptosis compared with other preparation groups.
Embodiment 6, the experiment of C6 malignant glioma cells balls
1,96 orifice plates are taken, the serum-free DMEM culture solutions of 40 μ l agaroses containing 2g/100ml are added per hole.
2, after completing step 1,96 orifice plate is taken, it is (2000 thin that 150 μ l C6 malignant glioma cells are inoculated with per hole
Born of the same parents/hole), it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.
3, after completing step 2,96 orifice plate is taken, it is molten that the drug that 20 μ L are prepared with serum-free DMEM culture solutions is added per hole
(the addition form of drug is respectively liquid：Farmorubine Hydrochloride, liposome A1, liposome A2, liposome B1, liposome C1 or fat
Plastid D1 makes a concentration of 4 μM of each test group Farmorubine Hydrochloride；Isometric serum-free DMEM culture solution alternatives to medication is set
The blank control of solution), it is placed in 37 DEG C, 5%CO2Under the conditions of cultivate, before administration, the 1st day after administration, the 2nd day, the 3rd
Whether the form of it and the 5th day observation tumour ball under inverted microscope, ball have necrosis, and tumour ball is measured with eyepiece micrometer
Major diameter and minor axis, calculate tumour sphere volume.
V=(π × dmax × dmin)/6；
V is tumour sphere volume, and dmax is tumour ball major diameter, and dmin is tumour ball minor axis.
R=(Vday i/Vday 0) × 100%；
R is tumour sphere volume rate, and Vday i are i-th day after administration C6 malignant glioma cells tumour sphere volumes,
Vday 0 is the volume of C6 malignant glioma cells tumour balls before administration.
It carries out three repetitions to test, results are averaged.
As a result see that (1 is blank control to Fig. 7, and 2 be Farmorubine Hydrochloride, and 3 be liposome A1, and 4 be liposome A2, and 5 be lipid
Body B1,6 be liposome C1, and 7 be liposome D1).Liposome D1 processing group C6 malignant glioma cells balls are in size and volume
On compared compared with other groups, generate the reduction of conspicuousness.C6 malignant glioma cells sphere volume change rates at the 5th day：Blank
Control group is 2.56 ± 0.47, and the Epi-ADM that dissociates is 0.61 ± 0.09, and liposome A1 is 0.64 ± 0.12, and liposome A2 is
0.62 ± 0.08, liposome B1 are 0.61 ± 0.07, and liposome C1 is 0.57 ± 0.31, and liposome D1 is 0.46 ± 0.33.Knot
Fruit illustrates that double ligand modified liposomes more can effectively inhibit the growth of cell ball.
The outer dual-target evaluation of effect of embodiment 7, Via Liposomes
One, in vitro blood-brain barrier model evaluation
1, the measurement of blood-brain barrier external model cross-film resistance
By bEnd.3 cell inoculations plug-in type Tissue Culture Dish upper storage reservoir (5 × 104A cells/well), upper storage reservoir is added 500
1350 μ l DMEM culture solutions, culture 4 days (replacing a culture solution in every two days) is added in μ l DMEM culture solutions, lower pond.Pass through survey
The resistance of order layer blood-brain barrier (BBB) come judge BBB formed tightness degree, resistance value be higher than 150 Ω cm2(deduct background
It can be used for testing afterwards), the cross-film resistance (TEER) of blood-brain barrier external model is 163-181 Ω cm2。
2, the foundation of HRP content measuring standards curve
Use the DMEM of serum-free as solvent, compound concentration 12.5ng/ml, 6.25ng/ml, 3.125ng/ml,
The horseradish peroxidase series standard solution of 1.56ng/ml, 0.78ng/ml and 0.39ng/ml.96 orifice plates are taken, are added per hole
50 μ l standard solution add 90 μ l TMB developing solutions, and 50 μ l terminate liquids (1mol/L H are added after being incubated 15min2SO4), it is placed in
Absorbance is measured in microplate reader under 450nm wavelength, establish HRP content standards curve (see Fig. 8).
3, BBB permeabilities measure
(1) by bEnd.3 cell inoculations plug-in type Tissue Culture Dish upper storage reservoir (5 × 104A cells/well), upper storage reservoir is added
1350 μ l DMEM culture solutions, culture is added in 500 μ l DMEM culture solutions, lower pond.
(2) when step (1) proceeds to TEER values more than 150 Ω, the culture solution for abandoning upper storage reservoir and lower pond is inhaled, is added to upper storage reservoir
The complete DMEM culture mediums of 1350 μ l are added in the DMEM complete mediums of 500 μ l HRP containing 500ng, downward pond, respectively at 0.5,1,
Take out 50 μ l liquid detecting HRP concentration when 2, after 4,8,12,24 hours from lower pond, while to add 50 μ l DMEM complete in pond downwards
Full culture medium.
Penetration rationHRP=CPond under t/C0 upper storage reservoir× 100%；
CPond under t：Under t moment in pond HRP concentration, C0 upper storage reservoir：Test the concentration (1000ng/ml) of HRP in upper storage reservoir when starting.
The HRP Penetration ration measurement results of different time are shown in Fig. 9.After external BBB models are built up, the Penetration ration in 24 hoursHRP
Less than 7%, illustrate that the compactness of model is good, meets subsequent experimental requirement.
Two, different liposome preparation penetrates the measurement of blood-brain barrier ability
1, by bEnd.3 cell inoculations plug-in type Tissue Culture Dish upper storage reservoir (5 × 104A cells/well), upper storage reservoir is added
1350 μ l DMEM culture solutions, culture is added in 500 μ l DMEM culture solutions, lower pond.
2, when step 1 proceeds to TEER values more than 150 Ω, the culture solution for abandoning upper storage reservoir and lower pond is inhaled.
3, after completing step 2, first group is added 500 μ l plasma-free DMEM mediums (as blank control) to upper storage reservoir, the
Two groups the plasma-free DMEM medium that 500 μ l contain 5 μ g Farmorubine Hydrochlorides is added to upper storage reservoir, and 500 μ l are added to upper storage reservoir in third group
The plasma-free DMEM medium of A1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides), the 4th group is added 500 μ l to upper storage reservoir and contains liposome
The plasma-free DMEM medium of A2 (containing 5 μ g Farmorubine Hydrochlorides), the 5th group is added 500 μ l B1 containing liposome to upper storage reservoir and (contains 5 μ g
Farmorubine Hydrochloride) plasma-free DMEM medium, the 6th group to upper storage reservoir be added 500 μ l C1 containing liposome (contain 5 μ g hydrochloric acid table Ah
Mycin) plasma-free DMEM medium, the 7th group is added 500 μ l D1's containing liposome (containing 5 μ g Farmorubine Hydrochlorides) to upper storage reservoir
1000 μ l plasma-free DMEM mediums are added in plasma-free DMEM medium, the downward pond of each group.
4, after completing step 3, continue to cultivate, be taken from Xia Chi after cultivating 2 hours, 4 hours, 8 hours and 24 hours
Go out 0.5ml liquid detecting Farmorubine Hydrochloride concentration, while 0.5ml DMEM complete mediums are added in pond downwards.
Across the in vitro blood-brain barrier Transport Rate=C of Farmorubine HydrochloridePond under t/C0 upper storage reservoir× 100%；
CPond under t：Under t moment in pond Farmorubine Hydrochloride concentration, C0 upper storage reservoir：Farmorubine Hydrochloride in upper storage reservoir when experiment starts
Concentration (10 μ g/ml).
It carries out three repetitions to test, results are averaged.
Across the in vitro blood-brain barrier Transport Rate the result is shown in Figure 1 of Farmorubine Hydrochloride 0.Liposome D1 blood-brain barrier models in vitro
On Transport Rate be higher than other liposomes, and with the extension of transhipment time, advantage is further apparent.
Three, MAN competitive assays
1, by bEnd.3 cell inoculations plug-in type Tissue Culture Dish upper storage reservoir (5 × 104A cells/well), upper storage reservoir is added
1350 μ l DMEM culture solutions, culture is added in 500 μ l DMEM culture solutions, lower pond.
2, when step 1 proceed to TEER values more than 150 Ω when, to upper storage reservoir be added 200 μ gMAN, 30min after inhale abandon upper storage reservoir and
The culture solution in lower pond.
3, after completing step 2, first group of nothing that 500 μ l B1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides) is added to upper storage reservoir
Serum DMEM culture mediums, second group of serum-free DMEM that 500 μ l D1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides) is added to upper storage reservoir
1000 μ l plasma-free DMEM mediums are added in culture medium, the downward pond of each group.
4, when step 1 proceeds to TEER values more than 150 Ω, the culture solution for abandoning upper storage reservoir and lower pond is inhaled.
5, after completing step 4, the nothing of 500 μ l B1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides) is added to upper storage reservoir for third group
Serum DMEM culture mediums, the 4th group of serum-free DMEM that 500 μ l D1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides) is added to upper storage reservoir
1000 μ l plasma-free DMEM mediums are added in culture medium, the downward pond of each group.
6, after being respectively completed step 3 and step 5, continue to cultivate, respectively at culture 2 hours, 4 hours, 8 hours and 24 hours
0.5ml liquid detecting Farmorubine Hydrochloride concentration is taken out from lower pond afterwards, while 0.5ml DMEM complete mediums are added in pond downwards.
It carries out three repetitions to test, results are averaged.
Across the in vitro blood-brain barrier Transport Rate the result is shown in Figure 1 of Farmorubine Hydrochloride 1.After MAN is added, due to free MAN with
GLUT1 competitive bindings so that liposome B1, liposome D1 across blood-brain barrier ability substantially reduce.It should be the results show that MAN
After being combined with GLUT1 specific recognitions, the epi-doxorubicine liposome modified through MAN can be mediated across blood-brain barrier, and can be significantly
Improve turn-over capacity.
Four, external dual-target Journal of Sex Research
1, by bEnd.3 cell inoculations plug-in type Tissue Culture Dish upper storage reservoir (5 × 104A cells/well), upper storage reservoir is added
1350 μ l DMEM culture solutions are added in 500 μ l DMEM culture solutions, lower pond, cultivate 2 days.
2, after completing step 1, the plug-in type Tissue Culture Dish is taken, C6 malignant glioma cells (1 are inoculated in lower pond
×105A cells/well), it cultivates 1 day.
3, after completing step 2, the culture solution for abandoning upper storage reservoir and lower pond is inhaled.
4, after completing step 3, first group is added 500 μ l plasma-free DMEM mediums (as blank control) to upper storage reservoir, the
Two groups the plasma-free DMEM medium that 500 μ l contain 5 μ g Farmorubine Hydrochlorides is added to upper storage reservoir, and 500 μ l are added to upper storage reservoir in third group
The plasma-free DMEM medium of A1 containing liposome (containing 5 μ g Farmorubine Hydrochlorides), the 4th group is added 500 μ l to upper storage reservoir and contains liposome
The plasma-free DMEM medium of A2 (containing 5 μ g Farmorubine Hydrochlorides), the 5th group is added 500 μ l B1 containing liposome to upper storage reservoir and (contains 5 μ g
Farmorubine Hydrochloride) plasma-free DMEM medium, the 6th group to upper storage reservoir be added 500 μ l C1 containing liposome (contain 5 μ g hydrochloric acid table Ah
Mycin) plasma-free DMEM medium, the 7th group is added 500 μ l D1's containing liposome (containing 5 μ g Farmorubine Hydrochlorides) to upper storage reservoir
Plasma-free DMEM medium, the downward pond of each group are added 1000 μ l plasma-free DMEM mediums, cultivate 48 hours.
5, after completing step 4, culture supernatant is abandoned in suction, is measured per hole trap OD values at 540nm using srb assay.
Cell inhibitory rate=(1- test groups OD values/blank control group OD values) × 100%.
It carries out three repetitions to test, results are averaged.
The result is shown in Figure 12.Different liposome is respectively to the inhibiting rate of C6 malignant glioma cells：Liposome D1
(64.48±0.59)>Liposome B1 (58.88 ± 0.84)>Liposome C1 (39.95 ± 0.83)>Liposome A2 (29.11 ±
0.56)>Liposome A1 (27.02 ± 0.23)>Free Epi-ADM (17.23 ± 0.39).The result shows that MAN and WGA are repaiied jointly
The drug-loaded liposome of decorations shows " two level targeting ", and drug-loaded liposome can be mediated across blood-brain barrier, and then targets C6
Malignant glioma cells.
Evaluation on Its Targeting Performance in embodiment 8, dual target liposome body
Experimental animal is the male SD rat of 200-250g.
One, the foundation of rats with brain glioma model
Experimental animal, 20% urethane of intraperitoneal injection is taken to be anaesthetized, go inner suture hair to send out about 1.0cm × 1.5cm, be fixed on
On direction finder, the tincture of iodine, alcohol disinfecting.Along endocanthion line midpoint longitudinally slit rat scalp 1cm backward, exposure skull, according to big
Mouse brain stereotactic collection of illustrative plates is drilled on the skull of 0.4mm, sagittal suture side 3.5mm with dental burr after bregma, and dental burr is in addition
Plastic bushing, only allows drill bit to drill through the deep 1.0~1.5mm (prevent from puncturing endocranium) of skull, and micro syringe sucks 10 μ L
C6 malignant glioma cells suspension (contains 1 × 106A cell), vertical inserting needle depth 5mm injects caudate nucleus with 1 μ L/min rates
It is interior, needle is slowly pulled out, bone hole is closed with bacteria-free bone wax immediately, visual area normal saline flushing, and notch meets conjunction, postoperative muscle injection five
Its penicillin, 40000U/ is only.
C6 malignant glioma cells are inoculated with after 7 days, rat is taken and dissects, it is observed that having formed brain tumor.
Brain sample containing tumor tissues takes 8 μm of slices, HE normal dyeings to see Figure 13 after paraffin embedding.In Figure 13, A
For normal cerebral tissue's HE coloration results (× 40)；B is brain tumor HE coloration results (× 40).Visual tumors cell growth is active,
Dense clusters are simultaneously grown to brain tissue in wettability.Though brain tissue can see that tumour cell invades profit with tumour intersection, remain to see
To an obvious boundary.
Three, survivorship curve
The rat model after C6 malignant glioma cells 7 days will be inoculated with and be randomly divided into 7 groups, every group 9 (3 are served only for surveying
Gross tumor volume is measured, 6 are served only for drawing survivorship curve), packet transaction is as follows：
1st group：Tail vein gives liposome A1, and dosage is 5mg/kg (being calculated with the amount of Farmorubine Hydrochloride)；
2nd group：Tail vein gives liposome A2, and dosage is 5mg/kg (being calculated with the amount of Farmorubine Hydrochloride)；
3rd group：Tail vein gives liposome B1, and dosage is 5mg/kg (being calculated with the amount of Farmorubine Hydrochloride)；
4th group：Tail vein gives liposome C1, and dosage is 5mg/kg (being calculated with the amount of Farmorubine Hydrochloride)；
5th group：Tail vein gives liposome D1, and dosage is 5mg/kg (being calculated with the amount of Farmorubine Hydrochloride)；
6th group：Tail vein gives Farmorubine Hydrochloride, and dosage is 5mg/kg (with the gauge of Farmorubine Hydrochloride
It calculates)；
7th group：Tail vein gives physiological saline.
The brain tumor cut using 4%PBS paraformaldehyde liquid fixed solutions, vernier caliper measurement pathological anatomy tumour maximum layer
Face diameter, with maximum length, width and height diameter calculation gross tumor volume (mm3) (V=π × dmax×dmin/ 6), gross tumor volume inhibiting rate=examination
Test gross tumor volume × 100% of gross tumor volume/saline control group of group.
Gross tumor volume inhibiting rate is shown in Figure 14 after one week of dosing.Farmorubine Hydrochloride is 10.71 ± 1.27%, drug-loaded liposome
A1 is 14.64 ± 1.57%, and drug-loaded liposome A2 is 17.02 ± 1.41%, and drug-loaded liposome B1 is 35.7 ± 1.28%, carries medicine
Liposome C1 is 25.92 ± 1.33%, and drug-loaded liposome D1 is 46.06 ± 2.46%.As can be seen from the results, physiological saline
There is no inhibition to gross tumor volume, compared with other groups, the gross tumor volume conspicuousness of double ligand modified drug-loaded liposome D1
Reduce.
The death time of animal is recorded, every group is made survivorship curve.It is measured and is survived using SPSS software Kaplan-Meier methods
Rate draws survivorship curve.The result is shown in Figure 15,1 is physiological saline group, and 2 be liposome A1, and 3 be liposome A2, and 4 be liposome B1,
5 be liposome C1, and 6 be liposome D1, and 7 be free Epi-ADM group.The lipid modified as can be seen from the results through MAN, WGA
Body D1 liposomes greatly improve the antitumor action of lotus C6 glioma rats, and postoperative longest life span was up to 25 days, hence it is evident that
Higher than free medicine group and other liposome groups, illustrate that liposome D1 has obvious antitumous effect.The lotus of liposome D1 groups
Tumor mouse mean survival time has significant difference, knot compared with free medicine and the mean survival time of other liposome groups
Fruit is shown in Table 4.
4 each group life span of table compares
| Liposome A1 | Liposome A2 | Liposome B1 | Liposome C1 | Liposome D1 | Free Epi-ADM | Physiological saline | |
| The median survival time number of days | 11.9* | 13.5* | 16.3* | 16* | 19.5 | 14.3* | 10.5* |
| Maximum survival day | 16 | 18 | 23 | 22 | 25 | 18 | 14 |
*.P<0.05, drug-loaded liposome D1 is compared with other groups.
Claims (7)
1. a kind of target liposomes are made of the trim of liposome and its surface, it is characterised in that：The surface of liposome
Trim is 4- aminobenzene-α-D- mannopyranoses glycosides and wheat germ agglutinin；
The surface of the liposome has amino and carboxyl；4- aminobenzene-α-D- mannopyranose the glycosides is connected to the fat
On the amino of plastid；The wheat germ agglutinin is connected on the carboxyl of the liposome；The amino of the liposome is by DSPE-
PEG2000-NH2It provides；The carboxyl of the liposome is by DSPE-PEG2000- COOH is provided；
The liposome is with egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-
NH2It is prepared as raw material；Egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-
PEG2000-NH2Molar ratio be 52:43:4:0.5:0.5；
In the target liposomes, the quality of the wheat germ agglutinin and egg yolk lecithin when preparing the liposome and cholesterol
Quality sum proportioning be 5：30, the quality of the 4- aminobenzene-α-D- mannopyranose glycosides and when preparing the liposome
The proportioning of the quality sum of egg yolk lecithin and cholesterol is 1.5:30.
2. a kind of preparation method of target liposomes, includes the following steps：
1) with egg yolk lecithin, cholesterol, DSPE-PEG2000、DSPE-PEG2000- COOH and DSPE-PEG2000-NH2For raw material system
Standby surface has the liposome of amino and carboxyl；
2) amino of liposome prepared by 4- aminobenzene-α-D- mannopyranoses glycosides and step 1) is connect, by wheat germ agglutinin
It is connect with the carboxyl of the liposome of step 1) preparation；
Obtain target liposomes.
3. application of the target liposomes described in claim 1 in preparing pharmaceutical carrier.
4. a kind of pharmaceutical carrier, its active constituent is target liposomes described in claim 1.
5. a kind of drug-loaded liposome contains drug with target liposomes described in claim 1 and obtains.
6. a kind of drug for treating brain tumor, its active constituent is the drug-loaded liposome described in claim 5；The load medicine fat
The drug that plastid contains is Farmorubine Hydrochloride and/or resveratrol.
7. application of the drug-loaded liposome in the drug for preparing treatment brain tumor described in claim 5；The drug-loaded liposome
The drug contained is Farmorubine Hydrochloride and/or resveratrol.
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Non-Patent Citations (4)
| Title |
|---|
| Dual-Targeting Topotecan Liposomes Modified with Tamoxifen and Wheat Germ Agglutinin Significantly Improve Drug Transport across the Blood-Brain Barrier and Survival of Brain Tumor-Bearing Animals;Du,et al.;《MOLECULAR PHARMACEUTICS》;20091231;第6卷(第3期);第905-917页 * |
| Radiation-Guided P-Selectin Antibody Targeted to Lung Cancer;G.HARIRI,et al;《Annals of Biomedical Engineering》;20080531;第36卷(第5期);第821-830页 * |
| 脑胶质瘤靶向药物递送系统;李剑峰等;《国际药学研究杂志》;20131031;第40卷(第5期);第573-581页 * |
| 脑靶向脂质体的研究进展;黄向华等;《中国药业》;20121020;第21卷(第20期);第15-17页 * |
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