CN103720658B - Heparin modified Evacet preparation and preparation method thereof - Google Patents
Heparin modified Evacet preparation and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to field of pharmaceutical preparations, be specifically related to a kind of heparin modified Evacet (Hep-DOX-Lip) preparation, it is characterized in that, by amycin 1 part, heparin 1 ~ 4 part, soybean lecithin 5 ~ 30 parts, 0.5 ~ 4 part, cholesterol, cationic materials 0.5 part, the invention also discloses the preparation method of this heparin modified Evacet.The heparin modified Evacet of the present invention, has the effect of similar PEGization, can significantly improve the stability of Evacet, the Half-life in vivo of prolong drug, improve the bioavailability of medicine, significantly can reduce the toxic and side effects of chemotherapeutics simultaneously, improve the compliance of patient.
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, be proved to be can safely, extensively, the concurrent phlebothrombosis of effective prophylaxis of cancer.Researcheres also excitedly find that the auxiliary heparin that uses is treated, can life-span of 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.Such as: a large amount of zoopery confirms, the cell-cell adhesion that heparin can be mediated by selectin inhibition thus the transfer of inhibition tumor cell.Heparin also can promote the release of tissue factor alternative pathway mortifier (Tissue Factor PathwayInhibitor, TFPI) and intervene tumor blood vessels new life.
Moreover, heparin can also replace PEG as the Hydrophilic modification material of pharmaceutical carrier.Time general nano-medicament carrier circulates in vivo, can very soon engulf by the macrophage of liver spleen, thus lose curative effect.In order to reduce engulfing carrier, hinder plasma protein to the absorption of nanoparticle, extend nanoparticle circulation time in blood, to improve Targeting Effect further, people have carried out large quantifier elimination.Found that, modify by hydrophilic polymer material and drug administration carrier can be made to 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 in vivo in also deposit that all kinds of corrupt practices creep in and some problems that can not ignore, (accelerated blood clearance is removed as vein duplicate injection PEGization liposome can accelerate blood, 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 rapidly cleared in humans in blood circulation, and the distribution of liver spleen significantly increases, thus greatly limit its clinical practice.Heparin is a kind of natural mucopolysaccharide, is with a large amount of negative charge to be highly acid, and extremely water-soluble.The people such as Byung Cheol Shin find, heparin, as Hydrophilic modification material, has the effect of class PEGization, and the carriers display after modification goes out more outstanding long circulating effect and serum stability.
The model drug that the present invention selects is amycin (Doxorubicin, DOX).DOX be the earliest 1969 from loose Streptothrix light grey mutant (Str.Peucetius var caesius) extraction and isolation to, for orange red crystalline powder, another name doxorubicin, doxorubicin, 14-Adriamycin, fusing point 204-205 DEG C, molecular weight is 579.98.With hydroxyl in its structure, in alkalescence.Be insoluble in water comparatively by force because himself is fat-soluble, be often prepared into doxorubicin hydrochloride, to increase its water solubility.Amycin has very strong active anticancer, be widely used in treatment 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 while performance therapeutical effect, also can produce a lot of toxic and side effects as vomiting, feel sick, alopecia, the common adverse reactions such as bone marrow depression and gastrointestinal toxicity.In addition, because amycin compounds and myocardium affinity are apparently higher than its hetero-organization, and semiquinone metabolite infringement myocardial cell can be produced, thus serious dose dependent cardiac toxicity is caused, show as various arrhythmia, 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 points out nano-carrier parcel amycin, greatly can reduce the toxic and side effects of amycin, improve 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 on the basis building amycin cationic-liposome (DOX-Lip), by the heparin of a large amount of negative charge of mode combined belt of Electrostatic Absorption.The heparin modified Evacet of the present invention, finds the effect with similar PEGization.The experiment of SD rat Internal pharmacokinetics proves, the Half-life in vivo of Hep-DOX-Lip energy significant prolongation amycin, bioavailability in the body of raising amycin, reduces the toxic and side effects of amycin simultaneously.Mouse tail vein acute toxicity testing result proves that the safety of Hep-DOX-Lip improves greatly, shows that preparation can reduce the toxic and side effects of chemotherapeutics, improves the compliance of patient.
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, containing 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, first constructs amycin cationic-liposome (DOX-Lip).Wherein, the proportioning of amycin, soybean lecithin and cholesterol three directly has influence on the quality of cationic-liposome.
Ratio involved in the present invention is weight ratio.
When cholesterol gets a fixed value, when soybean lecithin gets 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 (n=3) of liposomal particle size and envelop rate
As can be seen from the table, when amycin and soybean lecithin weight ratio are less than 1: 5, particle diameter is comparatively large, 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%, and therefore the ratio of Chinese medicine/soybean lecithin of the present invention is preferably 1: 10 ~ 20.
When soybean lecithin gets a fixed value, when cholesterol gets 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 (n=3) of liposomal particle size and envelop rate
As can be seen from the table, when the weight ratio of medicine and cholesterol is less than 1: 0.5, particle diameter is comparatively large, and envelop rate is very low; When the weight ratio of medicine and cholesterol is greater than 1: 4, envelop rate is lower, and namely placement flocculates 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, particle diameter and envelop rate the best, therefore the ratio of Chinese medicine/cholesterol of the present invention is preferably 1: 0.8 ~ 2.
The present invention build DOX-Lip basis on, by the heparin of a large amount of negative charge of mode combined belt of Electrostatic Absorption.When the cationic-liposome of 1mL, with the heparin of 1mL, (medicine: heparin=1: after 2 ~ 18) hatching 30min, ultrafiltration separated free heparin, then measured the heparin content be adsorbed on liposome by toluidines blue laws draw heparin adsorption curve.The results are shown in Figure 1.
As can be seen from the figure, along with the raising of hatching heparin concentration, the binding capacity of heparin also increases.Reach capacity after in conjunction with 4 parts of heparin, DOX-Lip no longer heparin-binding 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, and therefore 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, significantly can reduce the toxic and side effects of chemotherapeutics simultaneously, improve the compliance of patient.
One, rat Internal pharmacokinetics experimentation
Body internally-powered result of study shows, and the half-life of Hep-DOX-Lip significant prolongation amycin in SD rat body, bioavailability improves greatly.
Experimental technique: rat 9, is divided into 3 groups, often organizes 3, weighs with after etherization.Tail vein injection DOX solution, DOX-Lip and Hep-DOX-Lip respectively, dosage is 3.5mg/kg.After administration respectively at 0.5,1,2,3,4,6,8,12,24,36, the capillary tube of 48h heparinization gets blood 2mL 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, the centrifugal 10min of vortex 5min, 3000r/min.Quantitative absorption lower floor chloroform layer blowing air stream volatilizes solvent, and residue adds people's methanol 120 μ L and dissolves, (vortex 1min, centrifugal 3000r/min, 5min), gets the analysis of supernatant 20 μ L sample introduction.Plasma drug concentration data adopts pharmacokinetics Win Nonlin software (China Medicine University) to carry out pharmacokinetic model matching, calculates pharmacokinetic parameters, according to AIC minimum principle determination compartment model.
Experimental result: after rat intravenous injection administration, blood concentration-time curve 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.Namely disappear from large metabolism in mice fast in amycin aqueous solution group 2h, this is the half-life short (T very due to amycin
1/2=0.18h).The contrary Half-life in vivo that significantly can be extended liposome by liposome entrapment medicine.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 ensure 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 investigating DOX solution, DOX-Lip and Hep-DOX-Lip tri-groups of preparations.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 tries to achieve r=1.112.Therefore by 50 male white mouses (20+2g), be divided into 5 groups at random, often organize 10.With intravenous mode administration, 5 dosage component Wei 34.21mg/kg, 30.78mg/kg, 27.69mg/kg, 24.91mg/kg, 22.41mg/kg.Identical, 5 dosage component that can obtain DOX-Lip group Wei 39.49mg/kg, 36.21mg/kg, 33.21mg/kg, 30.45mg/kg, 27.92mg/kg.5 dosage component of Hep-DOX-Lip group Wei 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.Observe the behavior of animal, state and death time after administration immediately, and observe raising 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 in the 2nd day to occur death, and death is mainly distributed in 3 ~ 5 days; Animal present walking unstable, repose, rapid breathing, finally dead, obduction other main organs change without exception except heart is pale asphyxia and be in relaxed state by the naked eye.Two liposome group mices all started in the 4th day to occur death, and death all mainly concentrates on 5th ~ 8 days; Animal presents walking shakiness, repose rapid breathing, finally dead, obduction other main organs change without exception except heart is pale asphyxia and be in relaxed state by the naked eye.The LD of DOX solution, DOX-Lip and Hep-DOX-Lip 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, be 1.11 times of DOX-Lip group, illustrate that the safety of Hep-DOX-Lip is higher.Specific experiment the results are shown in Table 4.
Table 4 Mouse Acute Toxicity experimental 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 different freeze drying protectants wherein, lyophilization, redissolves, the results are shown in Table 5:
Table 5 freeze drying protectant is on the impact (n=3) of Hep-DOX-Lip particle diameter and envelop rate
"+" common " ++ " good " +++ " is very good
As can be seen from Table 5, in various common freeze drying protectant, the effect of sucrose, lactose, glucose, mannitol is better, and the indices of liposome all meets the demands; And trehalose, sorbitol are as freeze drying protectant, although its outward appearance is qualified, envelop rate has comparatively significantly reduction and it is not good enough to the control of liposomal particle size.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 organic solvent, evaporation under reduced pressure removed organic solvent, and the amycin that ammonium sulphate gradient is loaded 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.After the DOX-Lip of preparation is hatched 30min with heparin solution, the heparin that ultrafiltration removing is free, obtains Hep-DOX-Lip suspension.When being prepared into lyophilized injectable powder, freeze drying protectant is dissolved in liposome turbid liquor, 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, the preparation method of the Hep-DOX-Lip provided 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 compared to DOX-Lip.
Accompanying drawing explanation
Fig. 1 is heparin adsorption curve of the present invention
Fig. 2 is amycin blood concentration-time figure 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 Hep-DOX-Lip of the present invention to the inhibition of B16F10 melanoma metastasis and growth
Detailed description of the invention
Embodiment 1
Take the soybean lecithin 10mg of recipe quantity, cholesterol 1mg respectively, DDA 0.5mg is placed in eggplant-shape bottle, add q. s. methylene chloride, ultrasonic making it is dissolved completely; Rotating pressure-decreasing evaporating organic solvent, bottle wall is formed the lipid membrane of uniform drying.The ammonium sulfate 1mL adding 300mmol/L washes film, hydration 1h at 37 DEG C, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome with 10% sucrose solution dialyse after 3h (every 1h changes a dialysis solution) for medium, add Doxorubicin solution (1mg/mL), 30min is hatched in 60 DEG C of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic Enoxaparin solution (4mg/mL) hatches 30min, 25 DEG C.Ultrafiltration removes free heparin, obtains Hep-DOX-Lip suspension.Add suitable amount of sucrose by above-mentioned solution again, after aseptic filtration (0.2 μm, membrane filter aperture), subsequent filtrate is sub-packed 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 is slightly shaken in solvent for injection is as normal saline, can form rapidly homogeneous, stable, that light transmission is good red suspension in 10s.
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, drops to special purpose copper online, naturally volatilizes rear transmission electron microscope observation liposome and take pictures.Further, by the spatial shape of atomic force microscope observation liposome.The form (Fig. 3) of liposome is have studied by transmission electron microscope and atomic force microscope technology.Observe and find to be wrapped in (Fig. 3 B) by brushy thing around Hep-DOX-Lip, and the periphery of DOX-Lip smooth (Fig. 3 A), show 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 observed by transmission electron microscope and atomic force microscope is similar to the measured value of laser particle size analyzer, greatly in the scope of 100-200nm.
By freeze-dried lipidosome at 4 DEG C of ambient storage after 3 months, doxorubicin content is 97.8%, and adding the liposome encapsulation obtained after injection stage normal saline redissolves is 93.1%, and particle diameter is 156.4nm.
Embodiment 2
Take the soybean lecithin 15mg of recipe quantity, cholesterol 2mg respectively, stearylamine 0.5mg is placed in eggplant-shape bottle, add appropriate chloroform, ultrasonic making it is dissolved completely; Rotating pressure-decreasing evaporating organic solvent, bottle wall is formed the lipid membrane of uniform drying.The ammonium sulfate 1mL adding 300mmol/L washes film, hydration 1h at 37 DEG C, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome with 10% glucose solution dialyse after 3h (every 1h changes a dialysis solution) for medium, add Doxorubicin solution (1mg/mL), 30min is hatched in 60 DEG C of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic unfractionated heparin solution (4mg/mL) hatches 30min, 25 DEG C.Ultrafiltration removes free heparin, obtains Hep-DOX-Lip suspension.Add appropriate glucose by above-mentioned solution again, after aseptic filtration (0.2 μm, membrane filter aperture), subsequent filtrate is sub-packed in cillin bottle, lyophilization.
By freeze-dried lipidosome at 4 DEG C of ambient storage after 3 months, doxorubicin content is 95.8%, and adding the liposome encapsulation obtained after injection stage normal saline redissolves is 90.1%, and particle diameter is 171.4nm.
Embodiment 3
Take the soybean lecithin 20mg of recipe quantity, cholesterol 1.5mg respectively, trimethylcetyl base ammonium 0.5mg is placed in eggplant-shape bottle, add proper amount of methanol, ultrasonic making it is dissolved completely; Rotating pressure-decreasing evaporating organic solvent, bottle wall is formed the lipid membrane of uniform drying.The ammonium sulfate 1mL adding 300mmol/L washes film, hydration 1h at 37 DEG C, forms liposome colostric fluid.Probe Ultrasonic Searching (200W, 100 times), obtains blank liposome.Get above-mentioned blank liposome with 10% lactose solution dialyse after 3h (every 1h changes a dialysis solution) for medium, add Doxorubicin solution (1mg/mL), 30min is hatched in 60 DEG C of water-baths, obtains DOX-Lip.Get DOX-Lip solution and isopyknic dalteparin sodium solution (4mg/mL) hatches 30min, 25 DEG C.Ultrafiltration removes free heparin, obtains Hep-DOX-Lip suspension.Add appropriate lactose by above-mentioned solution again, after aseptic filtration (0.2 μm, membrane filter aperture), subsequent filtrate is sub-packed in cillin bottle, lyophilization.
By freeze-dried lipidosome at 4 DEG C of ambient storage after 3 months, doxorubicin content is 97.1%, and adding the liposome encapsulation obtained after injection stage normal saline redissolves is 93.1%, and particle diameter is 168.2nm.
Embodiment 4
Mtt assay investigates Hep-DOX-Lip preparation to the in vitro toxicity of B16F10 Tumor cell vaccine.In vitro cell experiment research is carried out to the product of embodiment 1.
MTT full name is 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt, and commodity are called tetrazolium bromide.Its Cleaning 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, measure its absorbance value with enzyme-linked immunosorbent assay instrument in certain wave strong point, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to cell number.
Preparation group: Hep-DOX-Lip group
Matched group: DOX solution group, DOX-Lip group
Empty vectors 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 complete medium 37 DEG C cultivates 24h, removes culture fluid, with PBS cleaning 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.After hatching 48h, discard Incubating Solution, wash once with PBS.Getting 25 μ .L Methyl thiazoly tetrazolium assay (MTT, 5mg/mL) adds in each hole, hatches 4h, abandoning supernatant for 37 DEG C, adds 100 μ L DMSO and dissolves the crystallization of first a ceremonial jade-ladle, used in libation, measure light absorption value (OD by microplate reader in 570nm
sample).And measure the light absorption value of matched group (n.s) in the same way, 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 empty vectors by mtt assay.Empty vectors Lip and Hep-Lip be equal no cytotoxicity in the concentration range of setting.This guarantees that the liposome prepared can as the antineoplastic drug carrier of safety.Be investigated by external mtt assay be loaded with amycin preparation to the inhibit activities (Fig. 4 B) of B16F10 cell.IC
50evaluate medicine or the Cytotoxic important indicator of preparation, according to each preparation of improvement Kou Shi formulae discovery to the IC of B16F10 cell
50value.Corresponding to DOX solution, the corresponding IC of DOX-Lip and Hep-DOX-Lip
50value is respectively 0.070,0.076 and 0.091 μ g/mL.The cytotoxicity of DOX-Lip is close with free DOX solution, 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 the risk combined by reticuloendothelial system identification and opsonin with strong positive charge, finally cause nanometer to be removed fast in vivo.It should be noted that Hep-DOX-Lip inhibits Growth of Cells when being greater than 90% when 3.397 μ g/mL.Even if this explanation only has a few micrograms per millilitre, Hep-DOX-Lip still can suppress the growth of B16F10 cell well.
Embodiment 5
Investigate the inhibition that Hep-DOX-Lip shifts B16F10 melanoma cell and grows in C57BL/6 Mice Body, anti-tumor in vivo transfer Pharmacodynamic experimentation is carried out to the product of embodiment 1.
Preparation group: Hep-DOX-Lip group
Matched group: DOX solution group, Hep solution group, DOX-Lip group, Hep-Lip group
Experimental technique
B16F10 tumor cell (3 × 10 is inoculated to mice (C57BL/6) by tail vein
5individual, 100 μ LPBS).Often organize 10 mices.20min before injection tumor cell, gives respectively and often organizes mouse tail vein injection normal saline, DOX solution group, Hep solution, DOX-Lip, Hep-Lip and Hep-DOX-Lip.Every three days, tail vein gave identical medicine again afterwards, and put to death the mice of half after 11 days, within 21 days, put to death residue mice afterwards.The lung obtained is weighed, immerses in paraffin afterwards, slice thickness 5 μm, and dye with Lignum Sappan Yihong (H & E) staining.The pathological change of optical microphotograph Microscopic observation.Get the mean number (× 40, n=10) of 10 visual field branch on count stoves, concrete outcome is shown in Fig. 5.
Experimental result
In order to study the ability of Hep-DOX-Lip Tumor suppression transfer, 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 stoves.DOX solution group shows certain therapeutic effect, carries this inhibitory action improve further by the bag of liposome.At the 11st day, compared to 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 of Hep solution and Hep-Lip two groups is slightly different, 11 days time 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 (see Fig. 5 B) that metastatic tumor generates effect.After treatment is finished, the meansigma methods sequence of each group 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 to grow at secondary tissue organ implantation.
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. the heparin modified Evacet preparation of claim 1 or 2, wherein heparin select unfractionated heparin, dalteparin sodium, Enoxaparin, receive in calciparine, Parnaparin Sodium, spit of fland heparin sodium one or more.
4. the heparin modified Evacet preparation of claim 1 or 2, wherein cationic materials selects chlorination trimethyl-2, 3-bis-oily alkene oxygen base propyl ammonium, bromination trimethyl-2, the oily acyloxy propyl ammonium of 3-bis-, trifluoroacetic acid dimethyl-2, 3-bis-oily alkene oxygen base propyl group-2-(2-spermine formamido group) ethyl ammonium, bromination trimethyldodecane base ammonium, Tetradonium Bromide, trimethylcetyl base ammonium, DDA, N-(2-spermine formoxyl)-N ', N '-bis-octadecyl Aminoacetamide, 1, 2-bis-oleoyl-3-succinyl-sn-glycerolcholine ester, 3 β-[N-(N ', N '-dimethyl amine ethyl) amido formacyl] cholesterol, lipid poly-l-lysine, one or more in stearylamine.
5. the heparin modified Evacet preparation of claim 1 or 2, also containing 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.
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| 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 |
| CN111001011B (en) * | 2019-11-05 | 2021-07-20 | 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 |
| CN115645546B (en) * | 2022-10-28 | 2024-05-28 | 中国药科大学 | Preparation and application of membrane modified doxorubicin liposome |
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| 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 |
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|---|---|---|---|---|
| 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 |
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