CN103520207A - Targeting cisplatin sodium nano-alginate liposome - Google Patents
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Abstract
The invention relates to a targeting cisplatin sodium nano-alginate liposome. The targeting performance of the targeting cisplatin sodium nano-alginate liposome is embodied by that the liposome is modified by targeting molecules, and the liposome has the grain diameter of about 100nm and is composed of phosphatidylcholine, cholesterol, sodium alginate, an anti-tumor drug cisplatin and targeting molecules. The liposome has a high systematic cisplatin carrying rate (15.24%) and a high encapsulation rate (89.92%), can be subjected to specific combination with targeting molecules on surfaces of tumor cells so as to achieve toxicity attenuation and synergism, and is locally and specifically applied to the preparation of drugs for tumor treatment. Particularly, the liposome has more excellent anti-tumor activity to oophoroma resisting.
Description
Technical field
The present invention relates to medical technical field, specifically, relate to a kind of preparation and the application in oophoroma tumor treatment thereof of targeting cisplatin nano sodium alginate liposome.
Background technology
Ovarian cancer is the highest disease of grade malignancy in gynecological tumor, occupies gynecologic malignant tumor mortality rate the first, and women's healthy reproduction in serious threat., very easily there is invasion and attack and metastasis in ovarian tumors concealment, approximately 70%～80% ovarian cancer patients belongs to III/IV phase while diagnosing, and its five year survival rate is only 30% left and right.The treatment of ovarian cancer be take tumor thoroughly and is subtracted the art of going out as main, but because its branch mode shows as the extensive plantation that intraperitoneal is dispersed in more, therefore rely on surgery resection tumor still impossible, the remaining tumor nodule that is dispersed in, must rely on postoperative chemotherapy.
The most frequently used is to take platinum class as a main line Combination chemotherapy.But because chemotherapeutics lacks selectivity and has toxic reaction, it has often also killed normal cell when killing tumor cell, cause patient to occur the situations such as bone marrow depression, renal insufficiency, digestive tract reaction, cardiac conduction obstacle, quality of life degradation, the infection that some patients even cause because of chemotherapy and the complication such as hemorrhage are dead.Cause drug utilization degree limited, using dosage is limited.In addition, also have some old or intentionally, the patient of liver, renal insufficiency cannot tolerate the toxic reaction of chemotherapy and can not carry out chemotherapy, causes ovarian cancer progress and dead rapidly.Therefore, need a kind of effective targeting drug delivery system to make medicine arrive tumor area.This system not only can improve medicine in the concentration of tumor area, killing tumor cell more effectively, and can reduce the infringement to normal cell, tissue.
The generation of known cancer, development are the extremely complicated processes that the polygenes that regulated and controled by Cellular signalling network participates in, EGF-R ELISA (Epidermal growth factor receptor, EGFR) signal path is closed condition more in normal structure, and the opening that is activated in tumor tissues, and its active degree and tumor disease progression are proportionate.High expressed EGFR in the inventor and much research equal prompter ovarian cancer tissue both domestic and external and cell strain.The nanoparticulate carriers that can utilize the ligands, EGF modification medicine carrying of EGFR, will make undoubtedly the ingestion of medicines amount of the ovarian cancer cell of surface of cell membrane high expressed EGFR increase, thereby make drug specificity act on ovarian cancer cell, and performance drug effect, realizes targeted therapy.Article and patent (Wang Y, Biomaterials, 2013,34:4068-4077 in our early stage; CN201210181369.8) Targeting Effect of this having been reported EGF decorated nanometer grain in obviously strengthens.
Cisplatin, CDDP (II), belongs to cell cycle nonspecific agent (CCNSA), has higher cytotoxicity, is the first-line drug for the treatment of of ovarian cancer.Yet cisplatin lacks selectivity and tool toxic reaction, and it has often also killed normal cell when killing tumor cell, causes patient to occur serious toxic and side effects, quality of life degradation.Simultaneously because cisplatin belongs to non-fat-soluble medicine, and be slightly soluble in water (Chinese Pharmacopoeia, 2010), therefore limited the exploitation to its preparation, so at present the cisplatin liposome ubiquity of report envelop rate or the low phenomenon of drug loading (envelop rate of CN201210037920.1 report is 51%, and drug loading is 4.0%; The envelop rate of CN201010530655.1 report is 71.25%, and drug loading is 8.81%).The present invention, according to the construction features of cisplatin, has made the prodrug of cisplatin, i.e. cisplatin-sodium alginate connector.This prodrug is water-soluble, has increased the molecular weight of cisplatin, is more conducive to liposome coated, and the prepared envelop rate carrying along targeted nano sodium alginate liposome of the present invention is 89.92%, and carrying drug ratio is 15.24%.In addition, sodium alginate, is a kind of natural polysaccharide, has the required stability of pharmaceutical preparation adjuvant, dissolubility, viscosity and safety, and in pharmaceutical preparation, food additive etc. various aspects are widely used.
At present, U.S. FDA approved Evacet preparation is for the therapeutic alliance of multiple myeloma, and there have multiple antitumor Liposomal formulation to be also applied to be clinical.The present invention, with a most frequently used clinically line cisplatin model drug (making cisplatin prodrug by modification), internationally recognized liposome material (safety non-toxic, good biocompatibility) be carrier, EGF is targeting part, year cisplatin targeted nano sodium alginate liposome that the final EGF of formation modifies, thereby active targeted therapy ovarian cancer.
Summary of the invention
The invention provides a kind of targeting cisplatin nano sodium alginate liposome, this cisplatin liposome is convenient to preparation, and cost is low, but carrying drug ratio and envelop rate are high, is conducive to more effectively delivering medicament.Utilize human ovarian cancer high expressed EGFR and normal structure is not expressed or express very low feature, introduce EGF, modified liposome, thereby more targeting specific act on tumor tissues, reach the object of attenuation synergistic.
Another object of the present invention is to provide the preparation of above-mentioned cisplatin prodrug and liposome.
Another object of the present invention is to provide the application of above-mentioned liposome in ovarian cancer.
The drug-loaded liposome of target tumor cell, its structure generally includes: 1) part, can identify and in conjunction with tumor cell surface specific receptor, easily by cell endocytic; 2) liposome that prepared by the matrix material of biocompatibility, wraps up and delivers antitumor drug; 3) linking group, connects described part and liposome.
The present invention connects hEGF EGF on nanoparticle surface, and cell endocytic can is combined and occur to this part with its Receptor EGFR of tumor cell surface.By the guiding function of ligand-receptor, the liposome that EGF modifies can be applied to the tumor of all EGFR overexpressions in principle.
The present invention selects to have the matrix material of good biocompatibility: i.e. EPC(egg phosphatidylcholine, Ovum Gallus domesticus Flavus lecithin), CHOL(cholesterol, cholesterol), mPEG
2000-DSPE(1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine[methoxy (polyethyleneglycol)-2000] methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE), Macrogol 2000-distearyl L-ALPHA-GPE that amine-PEG2000-DSPE(is aminated) and as the biocompatibility matrix material of preparing liposome.
The invention provides the preparation method of a kind of cisplatin modification or prodrug, i.e. cisplatin-sodium alginate connector.
The bag that the invention provides a kind of hEGF EGF modification carries the liposome (targeting cisplatin nano sodium alginate liposome) of cisplatin, comprises following structure:
Part, is hEGF EGF, and the human epidermal growth factor acceptor EGFR of tumor cell surface can optionally be identified and be incorporated into this part;
Liposome prepared by biocompatibility matrix material, wraps up and delivers antitumor drug in liposome, described biocompatibility matrix material EPC, CHOL, amine-PEG2000-DSPE be take certain mass percent as mixing; And
Linking ligand, is connected to amine-PEG by EGF
2000on-DSPE.
Wherein said targeting cisplatin nano sodium alginate liposome, it is as follows that corresponding preparation forms the preferential range of choice:
Phospholipid, quality percentage composition is 50～70%;
Cholesterol, quality percentage composition is 5～15%;
Sodium alginate, quality percentage composition is 5～20%;
Antitumor drug cisplatin, quality percentage composition is 5～20%;
Targeted molecular, quality percentage composition is 0.1～0.8%;
Methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE mPEG2000-DSPE, quality percentage composition is 1～5%;
Aminated Macrogol 2000-distearyl L-ALPHA-GPE amine-PEG2000-DSPE, quality percentage composition is 0.5～1%.
In addition, the size of liposome also can affect the targeting of medicine, if particle diameter is excessive, in blood circulation, is easy to be engulfed by reticuloendothelial system (RES), causes medicine to be removed rapidly, cannot realize target tumor cell.The particle diameter of the drug-loaded liposome of selectively targeted tumor cell of the present invention, preferably at 80～100nm, is added PEGization, can effectively reduce engulfing of RES, extends circulation time, improves the targeting of pharmaceutical preparation.
Targeted nanometer sodium alginate method for preparing lipidosome, concrete steps are as follows:
(a) cisplatin and sodium alginate coupling are prepared to cisplatin prodrug.Concrete grammar is:
Mass ratio that cisplatin be take with sodium alginate mixes as 2:1～1:2, be dissolved in distilled water, wherein cisplatin and the sodium alginate quality percentage composition in aqueous solution is respectively 33.33%～66.67% and 33.33%～66.67%, reacts 24h and be connected to form SA-CDDP under 37 ℃ of constant temperature; Described SA represents sodium alginate; CDDP represents cisplatin;
(b) prepare targeting cisplatin nano sodium alginate liposome, concrete grammar is:
Adopt film dispersion method, lecithin, cholesterol, amine-PEG2000-DSPE are dissolved in its mixing in chloroform soln, rotary evaporation is made lipid membrane at 30～45 ℃, and vacuum drying spends the night.Then the aqueous solution that contains cisplatin prodrug SA-CDDP is joined in lipid membrane, mix aquation 30min, the ultrasonic 3min of water-bath (50W), probe is interrupted ultrasonic 1min (100W), forms and carries cisplatin nano sodium alginate liposome.Then add cross-linking agent EDC and NHS 1～2mg respectively, room temperature lower magnetic force stirs after 0.5～1h, the hEGF EGF that gets 50～100 μ g joins in above-mentioned solution, under room temperature, lucifuge continues to stir 4～6h, then put it in 10K super filter tube, centrifugal 5～10min under 4000～7500rpm, removes EDC and NHS and unreacted EGF, obtains targeting cisplatin nano sodium alginate liposome.The method of the preparation of non-targeted property cisplatin nano sodium alginate liposome is the same, only amine-PEG2000-DSPE need to be changed into mPEG2000-DSPE.The mass ratio of described lecithin, cholesterol, amine-PEG2000-DSPE or mPEG2000-DSPE, cisplatin prodrug SA-CDDP is 50～70%:5～15%:0.5～1%:1～5%:15～25%.Wherein said EPC is represented as Ovum Gallus domesticus Flavus lecithin, and CHOL is cholesterol; Described amine-PEG2000-DSPE is aminated Macrogol 2000-distearyl L-ALPHA-GPE; Described DDW is distilled water; Described EDC is carbodiimides; Described NHS is N-hydroxy-succinamide; Described mPEG2000-DSPE is methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE.
Targeted nanometer sodium alginate liposome of the present invention can be in preparing antitumor drug application, described tumor is ovarian cancer, compares this target liposomes of each matched group and has more excellent anti-tumor activity.
Accompanying drawing explanation
Fig. 1 is Fourier's infrared spectrogram of cisplatin sodium alginate provided by the invention and connector SA-CDDP thereof;
Fig. 2 is the particle size distribution figure of targeting cisplatin nano sodium alginate liposome provided by the invention;
Fig. 3 is the potential image of targeting cisplatin nano sodium alginate liposome provided by the invention;
Fig. 4 is the transmission electron microscope picture of targeting cisplatin nano sodium alginate liposome provided by the invention;
Fig. 5 is the Release Performance comparison diagram of targeting cisplatin nano sodium alginate liposome provided by the invention;
Fig. 6 is the cellular uptake comparison diagram of non-targeted property provided by the invention (PEG-Lip) and targeting (EGF-Lip) liposome;
Fig. 7 is the Study of cytotoxicity comparison diagram of targeting cisplatin nano sodium alginate liposome provided by the invention to ovarian cancer cell SKOV3;
Fig. 8 is the therapeutic effect comparison diagram of targeting cisplatin nano sodium alginate liposome provided by the invention to ovarian cancer tumor bearing nude mice;
The comparison diagram of Fig. 9 targeting cisplatin nano provided by the invention sodium alginate liposome on the impact of ovarian cancer tumor bearing nude mice body weight.
Symbol description
Fig. 1: wave number is abscissa, % transmitance is vertical coordinate, and wherein a is cisplatin, and b is the connector of cisplatin and sodium alginate, and c is sodium alginate.
Fig. 2: particle diameter is abscissa, intensity is vertical coordinate, wherein Size (d.nm) is size (diameter, unit nanometer), Intensity(percent) is intensity (percentage ratio)
Fig. 3: Zeta potential is abscissa, intensity is vertical coordinate, and wherein Apparent Zeta Potential (mV) is surperficial Zeta potential (unit millivolt), and Total Counts is grand total, represents intensity
Fig. 5: Cumulative release of CDDP (%) represents cisplatin preparation, Time (h) express time (hour), Free CDDP represents free drug cisplatin, CS-PEG-Lip represents to carry a cisplatin nano sodium alginate liposome, and CS-EGF-Lip represents that targeting carries cisplatin nano sodium alginate liposome
Fig. 6: PEG-Lip and EGF-Lip represent respectively non-targeted property and targeted hposome, and White represents white light, and Rh represents rhodamine fluorochrome, and Merge represents white light and fluorescence stack
Fig. 7: Free CDDP represents free drug cisplatin, PEG-Lip represents to carry cisplatin nano sodium alginate liposome, EGF-Lip represents that targeting carries cisplatin nano sodium alginate liposome, Cell viability(%) represent cells survival rate, Concentration(μ g/ml) indicated concentration (ug/ml), A, B, C represent respectively 24,48,72h
Fig. 8: Saline is normal saline, PEG-Lip is nanometer sodium alginate liposome empty carrier, EGF-Lip is targeted nanometer sodium alginate liposome empty carrier, Free CDDP represents free drug cisplatin, CS-PEG-Lip represents to carry cisplatin nano sodium alginate liposome, CS-EGF-Lip represents that targeting carries cisplatin nano sodium alginate liposome, and a, b, c, d, e, f are respectively above each group, Tumor volume (mm
3) represent gross tumor volume (unit cubic millimeter), the weight (unit gram) that Tumor weight (g) is tumor, Days after xenograft model was established represents from setting up the natural law of lotus tumor model
Fig. 9: Saline is normal saline, PEG-Lip is nanometer sodium alginate liposome empty carrier, EGF-Lip is targeted nanometer sodium alginate liposome empty carrier, Free CDDP represents free drug cisplatin, CS-PEG-Lip represents to carry cisplatin nano sodium alginate liposome, CS-EGF-Lip represents that targeting carries cisplatin nano sodium alginate liposome, and vertical coordinate %Weight loss represents the percentage rate of weight loss, and abscissa Day represents to treat natural law
The specific embodiment
Below in conjunction with embodiment, describe the present invention, but enforcement of the present invention is not limited only to this.
Embodiment 1: the preparation of the cisplatin prodrug of invention (cisplatin sodium alginate connector)
Precision measures 20mg cisplatin and 20mg sodium alginate, i.e. mass ratio 1:1.Then be dissolved in 20mL DDW, under 37 ℃ of constant temperature, react 24h, be connected to form SA-CDDP.SA represents sodium alginate, and CDDP represents cisplatin, and DDW is distilled water.
Fourier infrared spectrograph FTIR analyzes: pure CDDP, SA and graft copolymer SA-CDDP carry out infrared spectrum measurement by Thermo Nicolet380 Fourier infrared spectrograph method after KBr tabletting.Fig. 1 is that the infrared spectrum of CDDP, SA and SA-CDDP compares SA(c) and infrared spectrum SA-CDDP(b), by the visible 3445.90cm of c
-1the absworption peak at place is the stretching vibration peak of OH base; At 1621.74cm
-1and 1417.86cm
-1near there will be two absworption peaks, the former is-COO
-antisymmetric stretching vibration, peak shape is wide, very strong; The latter is-COO
-symmetrical stretching vibration peak, intensity slightly a little less than, peak shape is sharp-pointed.This is all characteristic absorption peaks of sodium alginate.Compare with Fig. 1 c in Fig. 1 b except retaining the key band of SA, due to antisymmetric stretching vibration-COO
-in oxonium ion (as a kind of highly basic) and platinum Pt(as a kind of weak lewis acid) react, absworption peak has increased 9.85cm
-1(from 1621.74cm
-1to 1631.59cm
-1).Such bond polarization, be applied to-carbon atom of COO-group on, can cause a little positive charge, and the carbonyl therefore having increased has larger intensity and the probability of wave number.In addition, at SA-CDDP, also increased a 3285.17cm
-1absworption peak, this is the stretching vibration peak of the N-H group of connected cisplatin.
Embodiment 2: the preparation of the targeting cisplatin nano sodium alginate liposome of invention
Adopt film dispersion method, precision measures EPC10mg, CHOL2mg, and amine-PEG2000-DSPE0.3mg, by its mixed dissolution, in 3mL chloroform, rotary evaporation is made lipid membrane at 35 ℃, and vacuum drying spends the night.Then get above-mentioned prepared cisplatin prodrug SA-CDDP2mL and 3mL DDW and join in lipid membrane, mix aquation 30min, the ultrasonic 3min of water-bath (50W), probe is interrupted ultrasonic 1min (100W), forms and carries cisplatin nano sodium alginate liposome.Then add cross-linking agent EDC and NHS 2mg respectively, room temperature lower magnetic force stirs after 30min, the EGF100 μ L that gets 0.5mg/mL joins in above-mentioned solution, under room temperature, lucifuge continues to stir 4～6h, then put it in 10K super filter tube, centrifugal 10min under 4000rpm, removes EDC and NHS and unreacted EGF, obtains targeting cisplatin nano sodium alginate liposome.The method of the preparation of non-targeted property cisplatin nano sodium alginate liposome is the same, only amine-PEG2000-DSPE need to be changed into mPEG2000-DSPE(0.8mg).Wherein said EPC is represented as Ovum Gallus domesticus Flavus lecithin, and CHOL is cholesterol; Described amine-PEG2000-DSPE is aminated Macrogol 2000-distearyl L-ALPHA-GPE; Described DDW is distilled water; Described EDC is carbodiimides; Described NHS is N-hydroxy-succinamide; Described mPEG
2000-DSPE is methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE.
This liposome solutions, is typical light blue opalescence shape, at 4 ℃, preserves after January, without precipitation, produces.Through Ma Erwen ZETA current potential Particle Size Analyzer, measure this nano particle diameter in 100nm left and right.Mean diameter Z-Average size (d.nm) is 112.37, and particle size distribution index PdI is 0.069, and as Fig. 2, Zeta potential Zeta Potential (mV) is-23.83, as Fig. 3.Get after the appropriate dilution of this liposome, drop on the copper mesh that is coated with carbon film, uranium acetate aqueous solution negative staining with 4%, siphons away unnecessary liquid with filter paper, after natural drying, under transmission electron microscope, observes, for size distribution even, smooth surface, form is regular, ellipse or spheroidal particle, can see characteristic dactylotype, as Fig. 4.
The targeting cisplatin nano sodium alginate liposome extracorporeal releasing experiment of embodiment 3 inventions
Precision takes cisplatin (CDDP), carries cisplatin sodium alginate liposome (CS-PEG-Lip), year cisplatin sodium alginate liposome that EGF modifies ((CS-EGF-Lip) each three parts, 2mL.Be placed in the bag filter of anticipating, tighten bag filter two ends, be suspended in tool plug conical flask, in conical flask, add 18mL PBS (pH=7.4) or MES (pH=5.5) buffer, be placed on (temperature is 37 ℃ ± 0.5 ℃, 100rpm persistent oscillation) in water bath with thermostatic control agitator.Respectively at 0.5,1,2,3,4,5,6,12,24,36,48,72 (h), 4,5,6,7 (d) sampling, draw the outer solution 2mL of bag filter at every turn, immediately fill into temperature and be PBS or the MES buffer solution 2mL of 37 ℃.The 2mL solution taking out is measured for inductive coupling plasma emission spectrograph ICP-OES.Percentage rate (%) with cumulative release medicine was mapped to the time (hours), drew the release curve of liposome nano granule.Vitro drug release experiment shows, the prepared cisplatin nano sodium alginate liposome that carries has good slow-releasing, along with the prolongation of release time, carries the release medicine that cisplatin nano sodium alginate liposome nano granule can be gradually, and pH is lower, and slow-releasing is strong, sees Fig. 5.
The targeted nanometer sodium alginate liposome cellular uptake experiment of embodiment 4 inventions
At 37 ℃, 5%CO
2in incubator, hatch Proliferation of Human Ovarian Cell SKOV3, the trophophase cell of taking the logarithm is tested, and spreads 24 orifice plates, and the density of every porocyte is 2 * 10
4individual, 1mL trains liquid, and overnight incubation in 37 ℃ of cell culture incubators, after cell attachment, is removed culture fluid.In different holes, add respectively culture fluid 50,150, the 300 μ L containing the liposome of equivalent rhodamine B labelling non-targeted (PEG-Lip) and targeting (EGF-Lip), in 37 ℃ of cell culture incubators, continue to cultivate 2h.Draw out culture fluid, with PBS rinsing 3 times.Then the picked-up situation of observation of cell to liposome nano granule under fluorescence microscope.For the situation that better observation of cell absorbs, we have carried out picked-up quantitatively by flow cytometer simultaneously.Lucifuge operation is carried out in above-mentioned experiment.Result shows, compares with PEG-Lip liposome, and EGF-Lip liposome can more effectively be absorbed by cell, thereby causes red fluorescence intensity to strengthen, and this picked-up increases along with the increase of concentration.This also confirms, we have better Targeting Effect by prepared targeted nanometer sodium alginate liposome nano granule, see Fig. 6.
The external killing tumor cell experiment of the targeting cisplatin nano sodium alginate liposome of implementation column 5 inventions
The main CCK-8 of employing method is tested.SKOV3 ovarian cancer cell spreads 96 orifice plates, and the density of every porocyte is 5 * 10
3individual, 100 μ L, at 37 ℃, 5%CO
2in incubator, cultivate after 24h, remove culture fluid, adding respectively the culture fluid 100 μ L(that carry cisplatin sodium alginate liposome nano granule and free cisplatin that cisplatin sodium alginate liposome, EGF modify of carrying that contain different cisplatin medicine concentration, to take the groups of cells that only adds culture fluid be matched group), each experiment condition arranges 5 multiple holes.Then hatch altogether respectively cultivation 24,48,72h, add CCK-810 μ L, continue to cultivate 1.5h, finally shake 30s, in microplate reader, survey 450nm place light absorption value.The survival rate of cell (%)=A
experiment/ A
contrast* 100%(A experiment---the average light absorption value of experimental group, A contrast---the average light absorption value of matched group).Result shows that year cisplatin sodium alginate liposome and free drug cisplatin that a year cisplatin sodium alginate liposome, EGF modify can be along with the increase of concentration or the prolongations of incubation time, cell survival rate and reducing gradually, show the killing effect in vitro very strong to tumor cell, wherein with prepared targeting carry along nanometer sodium alginate liposome effect the most obvious, see Fig. 7.
The experiment of the targeting cisplatin nano sodium alginate liposome of embodiment 6 inventions to the therapeutic effect of lotus SKOV3 ovarian cancer mice
Female nude mice, 4～6 week age, 20g left and right, Proliferation of Human Ovarian Cell SKOV3, cell is in In vitro culture, and culture medium is 1640, adds 10% hyclone, 37 ℃, 5%CO
2incubator in cultivate.Nude mice is in subcutaneous injection ovarian cancer SKOV3 cell, every 2 * 10
6individual cell, became tumor after 10 days, divided at random four groups, 6 every group.First group is normal saline Saline matched group, second, three groups is empty carrier group, be PEG-Lip/EGF-Lip, the 4th group is free drug CDDP group, the 5th group is a year cisplatin nano sodium alginate liposome group CS-PEG-Lip, and the 6th group is that targeting is carried cisplatin nano sodium alginate liposome group CS-EGF-Lip.Medication is tail vein injection, and injected dose is 5mg/kg (amounting to into cisplatin concentration), is every other day administered once, and totally 16 days, within after administration finishes the 3rd day, puts to death mice.Record the gross tumor volume situation of change of nude mice every day, survival condition and body weight change situation.Result shows, it is good that targeting is carried cisplatin nano sodium alginate liposome tumor-targeting, and tumor is had to obvious inhibitory action, can obviously extend the time-to-live of tumor bearing nude mice, at the bottom of toxicity etc., sees Fig. 8, Fig. 9.
Claims (8)
1. a targeting cisplatin nano sodium alginate liposome, the targeting that it is characterized in that described targeting cisplatin nano sodium alginate liposome is embodied in the modification of targeted molecular to nanometer sodium alginate liposome, the particle diameter of liposome is at 80～120nm, and its preparation formation comprises:
Phospholipid, quality percentage composition is 15～80%;
Cholesterol, quality percentage composition is 5～60%;
Sodium alginate, quality percentage composition is 0.5～50%;
Antitumor drug cisplatin, quality percentage composition is 0.5～50%;
Targeted molecular, quality percentage composition is 0.001～20%;
Methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE mPEG
2000-DSPE, quality percentage composition is 0.001～20%;
Aminated Macrogol 2000-distearyl L-ALPHA-GPE amine-PEG
2000-DSPE, quality percentage composition is 0.001～20%;
The targeted molecular of described nano-lipid surface is a kind of in epidermal growth factor EGF, VEGF VEGF, " arginine-glycine-aspartic acid " tripeptide sequence RGD, fibroblast growth factor tbFGF, monoclonal antibody Herceptin Trastuzumab or Cetuximab Cetuximab or two or more.
2. targeting cisplatin nano sodium alginate liposome as claimed in claim 1, is characterized in that described preparation forms mass percent scope as follows:
Phospholipid, quality percentage composition is 50～70%;
Cholesterol, quality percentage composition is 5～15%;
Sodium alginate, quality percentage composition is 5～20%;
Antitumor drug cisplatin, quality percentage composition is 5～20%;
Targeted molecular, quality percentage composition is 0.1～0.8%;
Methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE mPEG
2000-DSPE, quality percentage composition is 1～5%;
Aminated Macrogol 2000-distearyl L-ALPHA-GPE amine-PEG
2000-DSPE, quality percentage composition is 0.5～1%.
3. targeted nanometer sodium alginate liposome as claimed in claim 1, it is characterized in that described phospholipid is selected from one or more of soybean lecithin, hydrogenated soy phosphatidyl choline, Ovum Gallus domesticus Flavus lecithin, hydrogenated yolk lecithin, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, or one or more of above-mentioned any derivant.
4. targeted nanometer sodium alginate liposome as claimed in claim 1, is characterized in that described sodium alginate is pharmaceutical grade sodium alginate.
5. targeted nanometer sodium alginate liposome as claimed in claim 1, the targeted molecular that it is characterized in that nano-lipid surface is epidermal growth factor EGF.
6. a preparation method for targeted nanometer sodium alginate liposome as claimed in claim 1, is characterized in that obtaining by following steps:
(a) cisplatin and sodium alginate coupling are prepared to cisplatin prodrug:
Mass ratio that cisplatin be take with sodium alginate mixes as 2:1～1:2, be dissolved in distilled water, wherein cisplatin and the sodium alginate quality percentage composition in aqueous solution is respectively 33.33%～66.67% and 33.33%～66.67%, reacts 24h and be connected to form SA-CDDP under 37 ℃ of constant temperature; Described SA represents sodium alginate; CDDP represents cisplatin;
(b) prepare targeting cisplatin nano sodium alginate liposome:
Adopt film dispersion method, lecithin, cholesterol, amine-PEG2000-DSPE are dissolved in its mixing in chloroform soln, rotary evaporation is made lipid membrane at 30～45 ℃, and vacuum drying spends the night; Then the aqueous solution that contains cisplatin prodrug SA-CDDP is joined in lipid membrane, mix aquation 30min, the ultrasonic 3min of water-bath 50W, probe is interrupted the ultrasonic 1min of 100W, forms and carries cisplatin nano sodium alginate liposome; Then add cross-linking agent EDC and NHS 1～2mg respectively, room temperature lower magnetic force stirs after 0.5～1h, the hEGF EGF that gets 50～100 μ g joins in above-mentioned solution, under room temperature, lucifuge continues to stir 4～6h, then put it in 10K super filter tube, centrifugal 5～10min under 4000～7500rpm, removes EDC and NHS and unreacted EGF, obtains targeting cisplatin nano sodium alginate liposome;
The method of the preparation of non-targeted property cisplatin nano sodium alginate liposome is the same, only amine-PEG2000-DSPE need to be changed into mPEG2000-DSPE;
The mass ratio of described lecithin, cholesterol, amine-PEG2000-DSPE or mPEG2000-DSPE, cisplatin prodrug SA-CDDP is respectively 50～70%:5～15%:0.5～1%:1～5%:15～25%.
Wherein said EPC is represented as Ovum Gallus domesticus Flavus lecithin, and CHOL is cholesterol; Described amine-PEG2000-DSPE is aminated Macrogol 2000-distearyl L-ALPHA-GPE; Described DDW is distilled water; Described EDC is carbodiimides; Described NHS is N-hydroxy-succinamide; Described mPEG2000-DSPE is methoxy poly (ethylene glycol) 2000-distearyl L-ALPHA-GPE.
7. a targeted nanometer sodium alginate liposome as claimed in claim 1 application in preparing antitumor drug.
8. the application of targeted nanometer sodium alginate liposome as claimed in claim 6 in preparing antitumor drug, is characterized in that described tumor is ovarian cancer.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017046416A1 (en) | 2015-09-18 | 2017-03-23 | Technische Universität München | LIGANDS FOR INTEGRIN AVß6, SYNTHESIS AND USES THEREOF |
| CN106551917A (en) * | 2016-11-29 | 2017-04-05 | 哈尔滨工业大学 | A kind of preparation method of utilization pH regulation and control cancerous cell gel microcapsules and the detection method using microcapsule kill cancerous cell |
| CN106798730A (en) * | 2017-03-09 | 2017-06-06 | 苏州大学 | Cis-platinum prodrug Liposomal formulation that a kind of weary oxygen improves and preparation method and application |
| CN107998080A (en) * | 2017-11-21 | 2018-05-08 | 东南大学 | A kind of active targeting of coupled antibody carries medicine long circulating liposome and preparation method thereof |
| WO2018167295A1 (en) | 2017-03-17 | 2018-09-20 | Technische Universität München | LIGANDS FOR INTEGRIN αvβ8, SYNTHESIS AND USES THEREOF |
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| CN114652682A (en) * | 2022-03-07 | 2022-06-24 | 河南省医药科学研究院 | New houttuynin sodium and cisplatin co-loading acidic sensitive liposome preparation and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017046416A1 (en) | 2015-09-18 | 2017-03-23 | Technische Universität München | LIGANDS FOR INTEGRIN AVß6, SYNTHESIS AND USES THEREOF |
| CN106551917A (en) * | 2016-11-29 | 2017-04-05 | 哈尔滨工业大学 | A kind of preparation method of utilization pH regulation and control cancerous cell gel microcapsules and the detection method using microcapsule kill cancerous cell |
| CN106551917B (en) * | 2016-11-29 | 2020-06-16 | 哈尔滨工业大学 | Preparation method of microcapsule for controlling cancer cell gelation by using pH and detection method for killing cancer cells by using microcapsule |
| CN106798730A (en) * | 2017-03-09 | 2017-06-06 | 苏州大学 | Cis-platinum prodrug Liposomal formulation that a kind of weary oxygen improves and preparation method and application |
| CN106798730B (en) * | 2017-03-09 | 2020-02-21 | 苏州杰纳生物科技有限公司 | Cisplatin prodrug liposome preparation with improved hypoxia as well as preparation method and application thereof |
| WO2018167295A1 (en) | 2017-03-17 | 2018-09-20 | Technische Universität München | LIGANDS FOR INTEGRIN αvβ8, SYNTHESIS AND USES THEREOF |
| CN107998080A (en) * | 2017-11-21 | 2018-05-08 | 东南大学 | A kind of active targeting of coupled antibody carries medicine long circulating liposome and preparation method thereof |
| CN113081964A (en) * | 2021-04-14 | 2021-07-09 | 广州市力鑫药业有限公司 | Penehyclidine hydrochloride nano-drug and preparation method thereof |
| CN114652682A (en) * | 2022-03-07 | 2022-06-24 | 河南省医药科学研究院 | New houttuynin sodium and cisplatin co-loading acidic sensitive liposome preparation and preparation method thereof |
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