CN103720658A - Heparin-modified adriamycin liposome preparation and preparation method thereof - Google Patents
Heparin-modified adriamycin liposome preparation and preparation method thereof Download PDFInfo
- Publication number
- CN103720658A CN103720658A CN201410005857.2A CN201410005857A CN103720658A CN 103720658 A CN103720658 A CN 103720658A CN 201410005857 A CN201410005857 A CN 201410005857A CN 103720658 A CN103720658 A CN 103720658A
- Authority
- CN
- China
- Prior art keywords
- heparin
- dox
- lip
- preparation
- hep
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of medicinal preparations, and in particular relates to a heparin-modified adriamycin liposome (Hep-DOX-Lip) preparation. The preparation is characterized by consisting of 1 part of adriamycin, 1-4 parts of heparin, 5-30 parts of soybean lecithin, 0.5-4 parts of cholesterol and 0.5 part of a cationic material. The invention further discloses a preparation method of the heparin-modified adriamycin liposome preparation. The heparin-modified adriamycin liposome preparation has an effect similar to pegylation, and can remarkably enhance the stability of an adriamycin liposome, prolong the in-vivo half-life period of a medicament, and enhance the bioavailability of the medicament. Meanwhile, the heparin-modified adriamycin liposome preparation can remarkably lower the toxic and side effects of chemotherapeutic drugs and enhance the compliance of patients.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, be specifically related to a kind of heparin modified Evacet (Hep-DOX-Lip) preparation, the invention also discloses the preparation method of this Hep-DOX-Lip preparation.
Background technology
Heparin (heparin, Hep) is a kind of mucopolysaccharide of acidity, being proved to be can be safely, extensively, the effective concurrent phlebothrombosis of prophylaxis of cancer.Researcheres also excitedly find that the auxiliary heparin that uses treats, life-span that can significant prolongation malignant tumor patient.Therefore, heparin is not only a kind of useful clinically antithrombotic, and other biological activity of heparin makes it have using value widely.For example: a large amount of zooperies confirm, thereby the iuntercellular that heparin can mediate by selectin inhibition sticks the transfer of inhibition tumor cell.Heparin also can promote the release of tissue factor alternative pathway mortifier (Tissue Factor Pathway Inhibitor, TFPI) and intervene tumor tissues angiogenesis.
Moreover, heparin can also replace PEG as the Hydrophilic modification material of pharmaceutical carrier.When general nano-medicament carrier circulates in vivo, can by the macrophage of liver spleen, be engulfed very soon, thereby lose curative effect.In order to reduce the engulfing of carrier, hinder the absorption of plasma protein to nanoparticle, extend the circulation time of nanoparticle in blood, further to improve Targeting Effect, people have carried out a large amount of research.Found that, by hydrophilic polymer material, modify and can make drug administration carrier obtain satisfied long circulating effect (being also referred to as stealthy effect).Hydrophilic material PEG is usually used in carrier modification, to reach macrocyclic object.But, the carrier that PEG modifies also deposits that all kinds of corrupt practices creep in and some problems that can not ignore in vivo, as can accelerating blood, vein duplicate injection PEGization liposome removes (accelerated blood clearance, ABC), when this ABC phenomenon refers to that interval several days is to same animal duplicate injection PEGization liposome, the PEGization liposome of the 2nd injection is removed rapidly in blood circulation, and liver spleen distributes significantly to be increased, thereby has greatly limited its clinical practice.Heparin is a kind of natural mucopolysaccharide, is with a large amount of negative charges to be highly acid, and extremely water-soluble.People's discoveries such as Byung Cheol Shin, heparin, as Hydrophilic modification material, has the effect of class PEGization, and the carrier after modification shows more outstanding long circulating effect and serum stability.
The model drug that the present invention selects is amycin (Doxorubicin, DOX).DOX extracts and is separated to for 1969 from the light grey mutant of loose Streptothrix (Str.Peucetius var caesius), for orange red crystalline powder, another name doxorubicin, doxorubicin, 14-Adriamycin, fusing point 204-205 ℃, molecular weight is 579.98.In its structure, with hydroxyl, be alkalescence.Because himself is fat-soluble compared with being insoluble in water by force, be often prepared into doxorubicin hydrochloride, to increase its water solubility.Amycin has very strong active anticancer, be widely used in treating melanoma, acute leukemia, non_hodgkin lymphoma, sarcoma, multiple myeloma and breast carcinoma, the Several Kinds of Malignancies such as hepatocarcinoma, gastric cancer, Small Cell Lung Cancer With Selective, cervical cancer, ovarian cancer.But, amycin in performance therapeutical effect, also can produce a lot of toxic and side effects as vomiting, feel sick, the common adverse reactions such as alopecia, bone marrow depression and gastrointestinal toxicity.In addition, because amycin compounds and myocardium affinity are apparently higher than its hetero-organization, and can produce semiquinone metabolite infringement myocardial cell, thereby cause serious dose dependent cardiac toxicity, show as various arrhythmias, or even irreversible myocardial damage and congestive heart failure.These toxic and side effects make its clinical practice be subject to great restriction.In recent years, lot of documents report is pointed out nano-carrier parcel amycin, can greatly reduce the toxic and side effects of amycin, improves the targeting of Drug therapy.Evacet more becomes the study hotspot in this field, at present existing multiple Evacet preparation list marketing.
The present invention is building on the basis of amycin cationic-liposome (DOX-Lip), the heparin of a large amount of negative charges of mode combined belt by Electrostatic Absorption.The heparin modified Evacet of the present invention, discovery has the effect of similar PEGization.In SD rat body, pharmacokinetics experiment showed, the Half-life in vivo of Hep-DOX-Lip energy significant prolongation amycin, improves the interior bioavailability of body of amycin, has reduced the toxic and side effects of amycin simultaneously.The safety of mouse tail vein acute toxicity testing result proof Hep-DOX-Lip improves greatly, shows that preparation can reduce the toxic and side effects of chemotherapeutics, improves patient's compliance.
Summary of the invention
The invention discloses a kind of clinical applicable heparin modified Evacet (Hep-DOX-Lip) and injection lyophilized powder thereof, this Hep-DOX-Lip lyophilized powder can again disperse after adding injection normal saline.
Hep-DOX-Lip preparation of the present invention, contains following component and weight ratio:
Amycin 1
Heparin 1～4
Soybean lecithin 5～30
Cholesterol 0.5～4
Cationic materials 0.5
Hep-DOX-Lip preparation of the present invention, the preferred weight ratio of each component is:
Amycin 1
Heparin 4
Soybean lecithin 10～20
Cholesterol 0.8～2
Cationic materials 0.5
The present invention, has first built amycin cationic-liposome (DOX-Lip).Wherein, amycin, soybean lecithin and cholesterol three's proportioning directly has influence on the quality of cationic-liposome.
Ratio involved in the present invention is weight ratio.
When cholesterol is got a fixed value, when soybean lecithin is got different weight, the mean diameter of DOX-Lip and envelop rate are also different.The results are shown in Table 1.
Table 1: the ratio of medicine and soybean lecithin is on the impact of liposome particle diameter and envelop rate (n=3)
As can be seen from the table, when amycin and soybean lecithin weight ratio are less than 1: 5, particle diameter is larger, and envelop rate is very low; When medicine: when phospholipid weight ratio is greater than 1:30, the light transmission of liposome solutions declines, and particle diameter increases gradually, places more unstable more for a long time; When medicine and soybean lecithin weight ratio are in 1: 5～30 scopes, envelop rate is higher, and particle diameter meets the requirements; When amycin and phospholipid weight ratio are in 1: 10～20 scopes, particle diameter is less, preparation stabilization, and envelop rate is greater than 95%, so the ratio of Chinese medicine/soybean lecithin of the present invention be preferably 1: 10～20.
When soybean lecithin is got a fixed value, when cholesterol is got different weight, the mean diameter of DOX-Lip and envelop rate are also different.The results are shown in Table 2.
Table 2: the ratio of medicine and cholesterol is on the impact of liposome particle diameter and envelop rate (n=3)
As can be seen from the table, the weight ratio of medicine and cholesterol is less than at 1: 0.5 o'clock, and particle diameter is larger, and envelop rate is very low; When the weight ratio of medicine and cholesterol is greater than 1: 4, envelop rate is lower, and place i.e. flocculation in a moment, when the part by weight of medicine and cholesterol is in 1: 0.5～4 scopes, particle diameter and envelop rate are all better, and ratio is in 1: 0.8～2 scopes, and particle diameter and envelop rate are best, so the ratio of Chinese medicine/cholesterol of the present invention is preferably 1: 0.8～2.
The present invention is building on the basis of DOX-Lip, the heparin of a large amount of negative charges of mode combined belt by Electrostatic Absorption.When the cationic-liposome of 1mL is with the heparin of 1mL (medicine: heparin=1: 2～18) hatch after 30min, ultrafiltration separated free heparin, then measured and be adsorbed on the heparin content on liposome by toluidines blue laws, draws heparin adsorption curve.The results are shown in Figure 1.
As can be seen from the figure,, along with hatching the raising of heparin concentration, the binding capacity of heparin also increases.After in conjunction with 4 parts of heparin, reach capacity, DOX-Lip heparin-binding no longer when Heparin-binding amount is greater than 4 parts.Therefore the ratio of Chinese medicine of the present invention and heparin is 1: 1～4, and maximum combined ratio is 1: 4.Heparin-binding amount is more, and slow release effect is more obvious, so the ratio of preferred agents and heparin is also 1: 4.
The heparin modified Evacet of the present invention, not only has the effect of similar PEGization, can significantly improve the stability of DOX-Lip, the Half-life in vivo of prolong drug, improve the bioavailability of medicine, can significantly reduce the toxic and side effects of chemotherapeutics simultaneously, improve patient's compliance.
One, pharmacokinetics experimentation in rat body
The demonstration of body internally-powered result of study, the half-life of Hep-DOX-Lip significant prolongation amycin in SD rat body, bioavailability improves greatly.
Experimental technique: 9 of rats, be divided into 3 groups, 3 every group, with weighing after etherization.Difference tail vein injection DOX solution, DOX-Lip and Hep-DOX-Lip, dosage is 3.5mg/kg.After administration respectively at 0.5,1,2,3,4,6,8,12,24,36,48h gets blood 2mL with the capillary tube of heparinization in eye rear vein beard.Be loaded on the centrifuge tube of heparinization.6000r/min, centrifugal 5min separated plasma.Get plasma sample 0.3mL, add 30 μ L methanol and 30 μ L inner mark solutions, mix homogeneously.Add chloroform-methanol (4: 1) 2.0mL, vortex 5min, the centrifugal 10min of 3000r/min.Quantitatively draw lower floor's chloroform layer blowing air stream and volatilize solvent, residue adds people's methanol 120 μ L and dissolves, and (vortex 1min, centrifugal 3000r/min, 5min), gets supernatant 20 μ L sample introduction analyses.Blood drug level data acquisition carries out pharmacokinetics models fitting with pharmacokinetics Win Nonlin software (China Medicine University), calculates pharmacokinetic parameters, according to AIC minimum principle, determines compartment model.
Experimental result: after rat intravenous injection administration, blood drug level-time graph is shown in Fig. 2.Utilize pharmacokinetics software Win-Nonlin to carry out matching and find that the pharmacokinetics behavior in rat body meets non-compartment model, concrete outcome is in Table 3.In amycin aqueous solution group 2h, from large metabolism in mice, disappear fast, this is due to half-life of amycin short (T very
1/2=0.18h).By liposome entrapment medicine, can significantly extend on the contrary the Half-life in vivo of liposome.And the biological half-life of Hep-DOX-Lip group is 2.5 times of DOX-Lip group.
The pharmacokinetic parameters (n=3) of table 3 amycin in SD rat body
Two, mouse tail vein acute toxicity testing
In order to guarantee the clinical safety of Hep-DOX-Lip, we carry out safety evaluatio by mouse tail vein acute toxicity testing to Hep-DOX-Lip drug-supplying system from the angle of pharmaceutical research, for the safety of clinical practice provides foundation.
Experimental technique: the acute toxicity of investigating DOX solution, DOX-Lip and tri-groups of preparations of Hep-DOX-Lip.According to prerun, DOX solution group fatal dose scope exists
22.41 between～38.03mg/kg.By (the ratio between r=adjacent doses group; Dm=maximal dose; Dn=minimum dose; The pre-packet count of n=) formula is tried to achieve r=1.112.Therefore by 50 male white mouses (20+2g), be divided at random 5 groups, 10 every group.With intravenous mode administration, 5 dosage groups are respectively 34.21mg/kg, 30.78mg/kg, 27.69mg/kg, 24.91mg/kg, 22.41mg/kg.Identical, 5 dosage groups that can obtain DOX-Lip group are respectively 39.49mg/kg, 36.21mg/kg, 33.21mg/kg, 30.45mg/kg, 27.92mg/kg.5 dosage groups of Hep-DOX-Lip group are respectively 41.96mg/kg, 38.52mg/kg, 35.36mg/kg, 32.46mg/kg, 29.80mg/kg.Mice single tail intravenously administrable, injection speed is about 0.5mL/min.After administration, observe immediately behavior, state and the death time of animal, and observe and raise 21 days, record each treated animal symptom and death condition, adopt Bliss method to calculate LD
50and 95% fiducial limit.
Experimental result: amycin group mice started to occur dead in the 2nd day, death is mainly distributed in 3～5 days; Animal present walking unstable, repose, rapid breathing, finally dead, obduction by the naked eye except heart is pale asphyxia and in relaxed state other main organs without ANOMALOUS VARIATIONS.Two liposome group mices all started appearance death in the 4th day, death all mainly concentrates on 5th～8 days; Animal presents that walking is unstable, the rapid breathing that reposes, finally dead, obduction by the naked eye except heart is pale asphyxia and in relaxed state other main organs without ANOMALOUS VARIATIONS.The LD that DOX solution, DOX-Lip and Hep-DOX-Lip are tri-groups
50respectively be 26.36,33.71,37.41mg/kg.Therefore, the LD of Hep-DOX-Lip
50for 1.42 times of free DOX, for DOX-Lip group 1.11 times, illustrate that the safety of Hep-DOX-Lip is higher.Specific experiment the results are shown in Table 4.
Table 4 chmice acute toxicity test result
Hep-DOX-Lip of the present invention also can add freeze drying protectant and be prepared into lyophilized formulations.Test finds that different freeze drying protectants is also different to the quality influence of Hep-DOX-Lip, first prepares Hep-DOX-Lip of the present invention, then adds therein different freeze drying protectants, and lyophilization, redissolves, and the results are shown in Table 5:
Table 5 freeze drying protectant is on the impact of Hep-DOX-Lip particle diameter and envelop rate (n=3)
"+" common " ++ " good " +++ " is very good
As can be seen from Table 5, in various common freeze drying protectants, the effect of sucrose, lactose, glucose, mannitol is better, and the indices of liposome all meets the demands; And trehalose, sorbitol be as freeze drying protectant, although its outward appearance is qualified, envelop rate have significantly reduce and its control to liposome particle diameter not good enough.Therefore as one or more in the freeze drying protectant preferably sucrose of Hep-DOX-Lip, lactose, glucose, mannitol.
The preparation method of Hep-DOX-Lip of the present invention comprises: soybean lecithin, cholesterol, cationic materials are dissolved in to organic solvent, and evaporation under reduced pressure removed organic solvent, the amycin that ammonium sulphate gradient is written into recipe quantity forms DOX-Lip suspension.Described organic solvent is selected from one or more in chloroform, dichloromethane, methanol, ether, ethanol, acetone.Preferred dichloromethane, chloroform, methanol.The DOX-Lip of preparation is hatched after 30min with heparin solution, and free heparin is removed in ultrafiltration, obtains Hep-DOX-Lip suspension.While being prepared into lyophilized injectable powder, freeze drying protectant is dissolved in liposome turbid liquor to lyophilization.
Hep-DOX-Lip of the present invention, not only envelop rate is greater than 95%, and particle diameter, at 100nm～200nm, has again good stability.Feature of the present invention is that the preparation method of the Hep-DOX-Lip providing is simple, is easy to industrialized great production; Heparin hydrophilic is good, can replace PEG prolong drug carrier biological half-life in vivo, improves the bioavailability of antitumor drug; And experiment also confirms that Hep-DOX-Lip preparation is a kind of safe and reliable intravenous formulations, safer than DOX-Lip.
Accompanying drawing explanation
Fig. 1 is heparin adsorption curve of the present invention
Fig. 2 is amycin blood drug level-time diagram after rat intravenous injection of the present invention
Fig. 3 is transmission electron microscope and the atomic force microscopy of Hep-DOX-Lip of the present invention
Fig. 4 is the Cytotoxic evaluation result of Hep-DOX-Lip of the present invention to B16F10 melanoma cell strain
Fig. 5 is the inhibition of Hep-DOX-Lip of the present invention to B16F10 melanoma metastasis and growth
The specific embodiment
Embodiment 1
Soybean lecithin 10mg, the cholesterol 1mg, the DDA 0.5mg that take respectively recipe quantity are placed in eggplant-shape bottle, add appropriate dichloromethane, and ultrasonic making it dissolved completely; Organic solvent is removed in rotating pressure-decreasing evaporation, forms the lipid membrane of uniform drying on bottle wall.Add the ammonium sulfate 1mL of 300mmol/L to wash film, hydration 1h at 37 ℃, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome and take 10% sucrose solution after medium dialysis 3h (every 1h changes dialysis solution one time), add amycin solution (1mg/mL), 30min is hatched in 60 ℃ of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic Enoxaparin solution (4mg/mL) is hatched 30min, 25 ℃.Ultrafiltration is removed free heparin, obtains Hep-DOX-Lip suspension.To in above-mentioned solution, add appropriate sucrose again, after aseptic filtration, (membrane filter aperture 0.2 μ m), is sub-packed in subsequent filtrate in cillin bottle lyophilization.
Finished product character: this product is red fine and smooth, loose the solid-state of adhesion of not subsiding.
Rehydration dispersibility: this product solvent for injection as normal saline in slightly concussion, in 10s, can form rapidly homogeneous, the red suspension that stable, light transmission is good.
Particle size determination: get this product in right amount with adopting laser particle size analyzer to measure particle diameter, mean diameter is 137.9nm, and polydispersity coefficient is 0.214.
Morphological observation: get appropriate liposome turbid liquor, by 1% phosphotungstic acid negative staining, drop to special purpose copper online, naturally volatilize rear by transmission electron microscope observation liposome form and take pictures.Further, use the spatial shape of atomic force microscope observation liposome.By transmission electron microscope and atomic force microscope technology, studied the form (Fig. 3) of liposome.Observe and find that Hep-DOX-Lip is wrapped in (Fig. 3 B) around by brushy thing, and the periphery of DOX-Lip smooth (Fig. 3 A) shows that heparin is present in the Evacet surface of modification.Use atomic force microscope technology to observe the 3D form (Fig. 3 C) of Hep-DOX-Lip, liposome becomes nearly spheroid, the smooth of the edge.The particle size values observing with atomic force microscope by transmission electron microscope is similar to the measured value of laser particle size analyzer, large in the scope of 100-200nm.
Freeze-dried lipidosome is stored after 3 months at 4 ℃ of environment, and amycin content is 97.8%, and adding the liposome encapsulation obtaining after the redissolution of injection stage normal saline is 93.1%, and particle diameter is 156.4nm.
Soybean lecithin 15mg, the cholesterol 2mg, the stearylamine 0.5mg that take respectively recipe quantity are placed in eggplant-shape bottle, add appropriate chloroform, and ultrasonic making it dissolved completely; Organic solvent is removed in rotating pressure-decreasing evaporation, forms the lipid membrane of uniform drying on bottle wall.Add the ammonium sulfate 1mL of 300mmol/L to wash film, hydration 1h at 37 ℃, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome and take 10% glucose solution after medium dialysis 3h (every 1h changes dialysis solution one time), add amycin solution (1mg/mL), 30min is hatched in 60 ℃ of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic unfractionated heparin solution (4mg/mL) is hatched 30min, 25 ℃.Ultrafiltration is removed free heparin, obtains Hep-DOX-Lip suspension.To in above-mentioned solution, add appropriate glucose again, after aseptic filtration, (membrane filter aperture 0.2 μ m), is sub-packed in subsequent filtrate in cillin bottle lyophilization.
Freeze-dried lipidosome is stored after 3 months at 4 ℃ of environment, and amycin content is 95.8%, and adding the liposome encapsulation obtaining after the redissolution of injection stage normal saline is 90.1%, and particle diameter is 171.4nm.
Soybean lecithin 20mg, the cholesterol 1.5mg, the bromination trimethyl cetyltrimethyl ammonium 0.5mg that take respectively recipe quantity are placed in eggplant-shape bottle, add appropriate methanol, and ultrasonic making it dissolved completely; Organic solvent is removed in rotating pressure-decreasing evaporation, forms the lipid membrane of uniform drying on bottle wall.Add the ammonium sulfate 1mL of 300mmol/L to wash film, hydration 1h at 37 ℃, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome and take 10% lactose solution after medium dialysis 3h (every 1h changes dialysis solution one time), add amycin solution (1mg/mL), 30min is hatched in 60 ℃ of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic dalteparin sodium solution (4mg/mL) is hatched 30min, 25 ℃.Ultrafiltration is removed free heparin, obtains Hep-DOX-Lip suspension.To in above-mentioned solution, add appropriate lactose again, after aseptic filtration, (membrane filter aperture 0.2 μ m), is sub-packed in subsequent filtrate in cillin bottle lyophilization.
Freeze-dried lipidosome is stored after 3 months at 4 ℃ of environment, and amycin content is 97.1%, and adding the liposome encapsulation obtaining after the redissolution of injection stage normal saline is 93.1%, and particle diameter is 168.2nm.
Mtt assay is investigated the in vitro toxicity of Hep-DOX-Lip preparation to B16F10 Tumor cell vaccine.The product of embodiment 1 is carried out to In vitro cell experiment research.
MTT full name is 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt, and commodity are called tetrazolium bromide.Its detection principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.Then by the first a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) dissolved cell, with enzyme-linked immunosorbent assay instrument, in certain wave strong point, measure its absorbance value, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization forms is directly proportional to cell number.
Preparation group: Hep-DOX-Lip group
Matched group: DOX solution group, DOX-Lip group
Blank vehicle group: blank unmodified liposome group (Lip), blank heparin modified liposome group (Hep-Lip)
Experimental technique
Take the logarithm the B16F10 cell of trophophase with 1 * 10
4individual/hole is inoculated in 96 orifice plates, and 37 ℃ of cultivation 24h of complete medium, remove culture fluid, with PBS, clean twice.With the dilution of DMEM culture medium Lip, Hep-Lip, DOX solution, DOX-Lip and Hep-DOX-Lip to 0.005～3.397 μ g/mL.Hatch after 48h, discard Incubating Solution, with PBS washing once.Get 25 μ .L tetramethyl azo azoles blue (MTT, 5mg/mL) and add in each hole, hatch 4h for 37 ℃, abandoning supernatant, adds 100 μ L DMSO to dissolve the crystallization of first a ceremonial jade-ladle, used in libation, by microplate reader, in 570nm, measures light absorption value (OD
sample).And measure in the same way the light absorption value of matched group (n.s), be designated as OD
control.And calculate the half-inhibition concentration IC of each preparation to B16F10 cell according to result
50.
Experimental result
As Fig. 4 A, first experiment evaluates the safety of blank carrier by mtt assay.Blank carrier Lip and Hep-Lip be equal no cytotoxicity in the concentration range of setting.This liposome of guaranteeing preparation can be used as safe antineoplastic drug carrier.By external mtt assay, also studied and be loaded with the inhibition activity (Fig. 4 B) of the preparation of amycin to B16F10 cell.IC
50be to evaluate medicine or the Cytotoxic important indicator of preparation, according to improvement Kou Shi formula, calculate the IC of each preparation to B16F10 cell
50value.Corresponding to the corresponding IC of DOX solution, DOX-Lip and Hep-DOX-Lip
50value is respectively 0.070,0.076 and 0.091 μ g/mL.The cytotoxicity of DOX-Lip and free DOX solution are close, and a little more than Hep-DOX-Lip.This is positively charged due to DOX-Lip surface, more easily by B16F10 cellular uptake.But nanoparticle also faces by the risk of reticuloendothelial system identification and opsonin combination with strong positive charge, finally cause nanometer to be removed fast in vivo.It should be noted that Growth of Cells when Hep-DOX-Lip has suppressed to be greater than 90% when 3.397 μ g/mL.Even if this explanation only has every milliliter of several microgram, Hep-DOX-Lip still can suppress the growth of B16F10 cell well.
Investigate the inhibition that Hep-DOX-Lip shifts and grows in C57BL/6 Mice Body B16F10 melanoma cell, the product of embodiment 1 is carried out to anti-tumor in vivo and shift Pharmacodynamic experimentation.
Preparation group: Hep-DOX-Lip group
Matched group: DOX solution group, Hep solution group, DOX-Lip group, Hep-Lip group
Experimental technique
By tail vein, to mice (C57BL/6), inoculate B16F10 tumor cell (3 * 10
5individual, 100 μ LPBS).Every group of 10 mices.20min before injection tumor cell, gives respectively every group of mouse tail vein injection normal saline, DOX solution group, Hep solution, DOX-Lip, Hep-Lip and Hep-DOX-Lip.Every three days, tail vein was given identical medicine again afterwards, and at 11 days, put to death afterwards the mice of half, within 21 days, put to death afterwards residue mice.The lung of obtaining is weighed, immerse afterwards in paraffin, slice thickness 5 μ m, and dye with Lignum Sappan Yihong (H & E) staining.The pathological change of optical microphotograph Microscopic observation.(* 40, n=10), concrete outcome is shown in Fig. 5 to get the mean number of 10 visual field branch on count kitchen ranges.
Experimental result
The ability that suppresses neoplasm metastasis in order to study Hep-DOX-Lip, B16F10 murine melanoma metastasis model has been selected in this experiment, and carried out physiology H & E tissue section strain, get the mean number (Fig. 5 A) of 10 visual field branch on count kitchen ranges.DOX solution group shows certain therapeutic effect, and the bag by liposome carries this inhibitory action and further improves.At the 11st day, than DOX solution group (P<0.001vs.Hep-DOX-Lip group) or DOX-Lip group (P<0.01vs.Hep-DOX-Lip group), Hep-DOX-Lip group can suppress the generation of metastasis more significantly.At 21 days, also observe similar trend.The performance that Hep solution and Hep-Lip are two groups is slightly different, in the time of 11 days two groups show good inhibition, but in 21 days, this inhibition obviously weakens, there is obvious metastasis in the pulmonary of mice.And the therapeutic effect of Hep-Lip group is slightly better than Hep solution group.
Early stage result of study proves, for metastatic tumor model, the weight of lung is proportional to the generation quantity of metastasis.Therefore, the tumor of lung also can be used as and evaluates the index (seeing Fig. 5 B) that metastatic tumor generates effect.After treatment finishes, each meansigma methods sequence of organizing mouse lung weight is Hep-DOX-Lip group <DOX-Lip group <DOX solution group <Hep-Lip group <Hep solution group < normal saline group.Generally speaking, the above results proves: the lung weight of Hep-DOX-Lip group small mouse increases the slowest, and melanoma is difficult in secondary histoorgan implantation growth.
Claims (6)
1. a heparin modified Evacet, is characterized in that containing following component and weight ratio:
2. the heparin modified Evacet preparation of claim 1, is characterized in that the weight ratio of each component is:
3. claim 1 or 2 heparin modified Evacet preparation, wherein heparin select unfractionated heparin, dalteparin sodium, Enoxaparin, receive in calciparine, Parnaparin Sodium, spit of fland heparin sodium one or more.
4. claim 1 or 2 heparin modified Evacet preparation, wherein cationic materials selects chlorination trimethyl-2, the oily alkene oxygen of 3-bis-base propyl ammonium, bromination trimethyl-2, the oily acyloxy propyl ammonium of 3-bis-, trifluoroacetic acid dimethyl-2, the oily alkene oxygen of 3-bis-base propyl group-2-(2-spermine formamido group) ethyl ammonium, bromination trimethyldodecane base ammonium, Tetradonium Bromide, bromination trimethyl cetyltrimethyl ammonium, DDA, N-(2-spermine formoxyl)-N ', N '-bis-octadecyl Aminoacetamides, 1, 2-bis-oleoyls-3-succinyl-sn-glycerolcholine ester, 3 β-[N-(N ', N '-dimethyl aminoethyl) amido formacyl] cholesterol, lipid poly-l-lysine, one or more in stearylamine.
5. claim 1 or 2 heparin modified Evacet preparation, also contain freeze drying protectant.
6. the heparin modified Evacet preparation of claim 5, wherein freeze drying protectant is selected from one or more in sucrose, lactose, glucose, mannitol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410005857.2A CN103720658B (en) | 2014-01-07 | 2014-01-07 | Heparin modified Evacet preparation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410005857.2A CN103720658B (en) | 2014-01-07 | 2014-01-07 | Heparin modified Evacet preparation and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103720658A true CN103720658A (en) | 2014-04-16 |
| CN103720658B CN103720658B (en) | 2015-10-07 |
Family
ID=50445125
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410005857.2A Expired - Fee Related CN103720658B (en) | 2014-01-07 | 2014-01-07 | Heparin modified Evacet preparation and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103720658B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104666247A (en) * | 2015-01-29 | 2015-06-03 | 中国药科大学 | Heparin-modified cleavable adriamycin liposome preparation and preparation method thereof |
| CN106729747A (en) * | 2016-12-27 | 2017-05-31 | 中国药科大学 | A kind of heparin modified cationic-liposome and preparation method thereof |
| CN111001011A (en) * | 2019-11-05 | 2020-04-14 | 中国药科大学 | Low-molecular-weight heparin-modified bone targeting liposome and preparation method thereof |
| CN111870701A (en) * | 2020-07-10 | 2020-11-03 | 中国药科大学 | Heparin-modified liposome preparation and preparation method and application thereof |
| CN111939126A (en) * | 2019-05-15 | 2020-11-17 | 上海交通大学医学院附属第九人民医院 | Cationic liposome, dispersion liquid containing same, and preparation method and application thereof |
| CN115645546A (en) * | 2022-10-28 | 2023-01-31 | 中国药科大学 | Preparation and application of cell membrane modified adriamycin liposome |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998563A (en) * | 2006-12-21 | 2007-07-18 | 华中科技大学同济医学院附属协和医院 | Magnetic pegylated liposomal doxorubicin and preparation method and application |
| CN101048138A (en) * | 2004-09-09 | 2007-10-03 | 耶路撒冷希伯来大学伊萨姆研发公司 | Use of liposomal glucocorticoids for treating inflammatory states |
| CN101244038A (en) * | 2007-10-26 | 2008-08-20 | 吴念 | Stable adriablastina lipoid plastid and preparation thereof |
-
2014
- 2014-01-07 CN CN201410005857.2A patent/CN103720658B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101048138A (en) * | 2004-09-09 | 2007-10-03 | 耶路撒冷希伯来大学伊萨姆研发公司 | Use of liposomal glucocorticoids for treating inflammatory states |
| CN100998563A (en) * | 2006-12-21 | 2007-07-18 | 华中科技大学同济医学院附属协和医院 | Magnetic pegylated liposomal doxorubicin and preparation method and application |
| CN101244038A (en) * | 2007-10-26 | 2008-08-20 | 吴念 | Stable adriablastina lipoid plastid and preparation thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104666247A (en) * | 2015-01-29 | 2015-06-03 | 中国药科大学 | Heparin-modified cleavable adriamycin liposome preparation and preparation method thereof |
| CN106729747A (en) * | 2016-12-27 | 2017-05-31 | 中国药科大学 | A kind of heparin modified cationic-liposome and preparation method thereof |
| CN111939126A (en) * | 2019-05-15 | 2020-11-17 | 上海交通大学医学院附属第九人民医院 | Cationic liposome, dispersion liquid containing same, and preparation method and application thereof |
| CN111001011A (en) * | 2019-11-05 | 2020-04-14 | 中国药科大学 | Low-molecular-weight heparin-modified bone targeting liposome and preparation method thereof |
| CN111870701A (en) * | 2020-07-10 | 2020-11-03 | 中国药科大学 | Heparin-modified liposome preparation and preparation method and application thereof |
| CN115645546A (en) * | 2022-10-28 | 2023-01-31 | 中国药科大学 | Preparation and application of cell membrane modified adriamycin liposome |
| CN115645546B (en) * | 2022-10-28 | 2024-05-28 | 中国药科大学 | Preparation and application of membrane modified doxorubicin liposome |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103720658B (en) | 2015-10-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103720658B (en) | Heparin modified Evacet preparation and preparation method thereof | |
| US11260032B2 (en) | Method for preparing biofilm-coated drug nanocrystal and application thereof | |
| Zhang et al. | Construction of a tumor microenvironment pH-responsive cleavable PEGylated hyaluronic acid nano-drug delivery system for colorectal cancer treatment | |
| CN104983716B (en) | The double target tumor Nano medication slow-released systems of tumor cell membrane/nuclear membrane and its preparation and application | |
| KR20180120220A (en) | Biodegradable amphiphilic polymers specifically targeting ovarian cancer, polymeric vesicle made therefrom and uses thereof | |
| CN105287383A (en) | Application of novel liposome-entrapped mitoxantrone combined chemotherapeutic drug in antineoplastic treatment | |
| WO2019158060A1 (en) | Polyionic complex formed by polysialic acid and cationic compound, preparation method therefor and application thereof | |
| CN106344924B (en) | It is a kind of combine metabolic block nano-formulation and its drug resistance inversion application | |
| CN104042567A (en) | Ampelopsin nano-micelle and application thereof | |
| CN107019673A (en) | A kind of Paclitaxel liposome preparation with tumor-targeting function and its preparation method and application | |
| CN106420604B (en) | A kind of nano suspension and preparation method thereof of kind of litchi element class drug | |
| CN111000828A (en) | Emodin lipid polymer nanoparticles and preparation method and application thereof | |
| CN102188439B (en) | Nanoparticle liposome for releasing different medicines in programmed manner and preparation and use thereof | |
| CN102139113A (en) | Novel pharmaceutical solubilization carrier and preparation method and application thereof | |
| CN104666247A (en) | Heparin-modified cleavable adriamycin liposome preparation and preparation method thereof | |
| CN108451906A (en) | A kind of nanometer formulation and preparation method for antitumor and anti-metastatic therapy cholesterol-low molecular weight heparin | |
| CN104324007A (en) | Preparation technology and application of natural recombinant nanostructured lipid carrier | |
| CN102631678A (en) | Triblock polymer carrier containing polyarginine as well as preparation method and application thereof | |
| CN112618558B (en) | Erianin-adriamycin-containing double-drug co-carried liposome as well as preparation and application thereof | |
| CN108309940A (en) | Beta-elemene carries liposome and preparation method thereof altogether with platinum medicine | |
| CN104274413A (en) | Nanoparticles of camptothecin drugs and preparation method of nanoparticles | |
| CN105168134A (en) | Galactose ester modified traditional Chinese and western medicine jointly-loaded orally-taking liver-targeting microemulsion delivery system | |
| CN115025244A (en) | Preparation method and application of transferrin-modified double-targeting triptolide magnetic nanoparticles | |
| CN107669637A (en) | A kind of injection Artemether liposome and its preparation method and application | |
| You et al. | Nanocrystals of a new camptothecin derivative WCN-21 enhance its solubility and efficacy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151007 Termination date: 20190107 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |