CN107157930A - A kind of mithramycin nano granule suspension and preparation method thereof - Google Patents
A kind of mithramycin nano granule suspension and preparation method thereof Download PDFInfo
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- CN107157930A CN107157930A CN201710436381.1A CN201710436381A CN107157930A CN 107157930 A CN107157930 A CN 107157930A CN 201710436381 A CN201710436381 A CN 201710436381A CN 107157930 A CN107157930 A CN 107157930A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
Abstract
The present invention relates to a kind of mithramycin nano granule suspension and preparation method thereof, the preparation method includes diblock copolymer solution, prepares mithramycin organic phase, prepares emulsifier solution, mixing and post-treating and other steps.Mithramycin nano particle prepared by the present invention can preferably retain the pharmacological activity of mithramycin, and the material that the present invention is used is biocompatible materials, with application security feature, shows good application prospect.Meanwhile, the mithramycin nano particle effectively can be enriched with transplanted tumor in nude mice position, and be presented to the transplanted tumor in nude mice of human pancreatic cancer cell BxPC 3 significantly therapeutic effect.
Description
【Technical field】
The invention belongs to Drug delivery technology field.More particularly it relates to which a kind of mithramycin nano particle is outstanding
Supernatant liquid, further relates to the preparation method of the mithramycin nano granule suspension.
【Background technology】
Mithramycin (MithramycinA, MIT), also known as plicamycin, are that a kind of polyketone class naturally occurred is antitumor
Antibiotic, belongs to aureolic acid family.Mithramycin has gone through since clinical practice, and it is successively used for as a kind of chemotherapeutics
Treat the malignant tumours such as embryonal carcinoma of testis, glioma.Mithramycin can pass through the ditch knot with GC enrichment regions on DNA
Close, topmost mechanism of action is the combination of Reverse transcriptase transcription factor Spl and gene regulatory elements, then influence DNA- eggs
The formation of white compound, and regulate and control the expression of related gene.Sp1 is a kind of transcription factor with zinc fingers, and it is a variety of
The gene expression that many aspects in evolution occur for cancer is overexpressed and participated in tumour cell, including is expanded, divided
Change, DNA damage reparation, apoptosis, angiogenesis, the invasion and attack and transfer of cancer cell.Mithramycin can be by suppressing Sp1 transcription tune
Function is saved, regulates and controls to include the oncogene c-Myc and c-Src of Sp1 binding sites in the expression of downstream gene, such as promoter in radiance
Expression quantity is decreased obviously after mycin processing.Meanwhile, mithramycin can also lower p53 negative regulatory factors MDM2 transcriptional level,
So as to activate p53 paths and cause cell-cycle arrest and apoptotic process.Mithramycin is also proved to be able to by multiple approach
The medicine impedance of inverse cancer cell, such as reduce the expression quantity of different first biomass pump ABCG2 and Multidrug resistance gene MDR1.It is existing
Research shows that mithramycin can influence apparent by suppressing DNA methylation transferase DNMT and acetylation of histone enzyme HDAC
Heredity.Preclinical study it has been proved that mithramycin can suppress lung cancer, sarcoma, breast cancer, cancer of pancreas, glioma, stomach cancer and
The occurrence and development of the kinds of tumors such as prostate cancer.In recent years, the preclinical study achievement of mithramycin also promoted it to be used to control
The multiple I/II clinical trial phases for treating cancer are carried out in succession.As can be seen here, clinical practice of the mithramycin in terms of oncotherapy
It might have a bigger development.However, mithramycin needs higher dosage when being clinically used for oncotherapy, and it is produced
Systemic toxicity limit therapeutic effect.In order to strengthen its security and curative effect, developing new mithramycin delivery system is
A kind of feasible method.
Nanometer technology has been widely used in the exploitation of drug delivery system by its exclusive characteristic, particularly in cancer
In terms of disease treatment.Using nano particle as carrier, targeting conveying chemotherapeutics is believed to improve bioavilability well
And reduce system toxicity.With small-molecule drug in itself compared with, drug-loading nanoparticles can avoid kidney remove then extend in blood
Half-life period in slurry.Simultaneously as wall of micrangium abnormal in tumor tissues and the outer row function of repressed lymphatic vessel so that
The material of nanoscale can be accumulated by EPR effects (enhancedpermeability and retention) in tumor locus
It is tired.The polymer material that natural molecule copolymerization is formed in body has the characteristic of biodegradable and bio-compatible, and
It is used for the clinical treatment of various ways.Polymer, which can be protected, to be contained thing and not to be degraded and controlled release, is widely used in preparation
Multi-medicament delivery system.In pharmaceutical carrier prepared by it drug releasing rate by polymer degradation in vivo speed and
Medicine diffusion velocity in matrix influences, so as to play the effect of control release.Nano particle can be extended by modification of surfaces
Plasma half-life, so as to preferably play passive target effect.
Based on above prior art situation, the present inventor completes this finally by lot of experiments and analysis and summary
Invention.
【The content of the invention】
[technical problem to be solved]
It is an object of the invention to provide a kind of mithramycin nano granule suspension.
It is a further object to provide the preparation method of the mithramycin nano granule suspension.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of preparation method of mithramycin nano granule suspension.
The preparation method step of mithramycin nano granule suspension of the present invention is as follows：
A, prepare diblock copolymer solution
Diblock copolymer is dissolved in ethyl acetate solvent, the diblock copolymer that concentration is 30~40mg/mL is obtained
Ethyl acetate solution；
B, prepare mithramycin organic phase
According to the ratio 1 of ethyl acetate solution and the mithramycin in terms of milligram in terms of milliliter：1.0~3.0, mithramycin plus
Into above-mentioned diblock copolymer ethyl acetate solution, vibration dissolving obtains a kind of organic phase containing mithramycin；
C, prepare emulsifier solution
Emulsifying agent is soluble in water, and it is by weight 0.5~5% emulsifier solution to obtain concentration；
D, mixing
Under conditions of the emulsifier solution prepared using shearing force homogenizer to step C carries out homogeneous, it is added dropwise in step B
Mixed liquor, is placed in ice bath by obtained organic phase immediately after completion of dropwise addition, is continuing with Probe Ultrasonic Searching instrument and carries out ultrasonic emulsification；
E, post processing
The emulsion that step D is obtained removes the organic solvent wherein contained using vacuum rotary evaporator at room temperature；Connect
The unentrapped free drug wherein contained for 30000Da super filter tube removing using Millipore companies molecular cut off, is made
Continuous ultrafiltration is carried out to suspension with distilled water to clean, then filtered by 0.22 μm of syringe needle filter, obtain mithramycin nanometer
Grain suspension.
A preferred embodiment of the invention, in step, described diblock copolymer are selected from by two kinds
PLGA, polyethylene glycol, PLLA, dextrorotation PLA, poly-dl-lactide, polyamide, poly- isopropyl
Base acrylamide, polyethyleneimine, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly- amino chitosan, polyoxyethylene
Alkene, cellulose acetate, poly- [(R) -3-hydroxybutyrate], the biocompatibility polymerization of poly- polysaccharide or poly 2-hydroxyethyl methacrylate
Thing composition.
According to another preferred embodiment of the present invention, in step, described ethyl acetate is by selected from dichloromethane
The organic solvent of alkane, chloroform or ether is replaced.
According to another preferred embodiment of the present invention, in stepb, mithramycin is by selected from mithramycin SK, light
Brightness mycin SDK or EC-8042 mithramycin structure of modification compound are replaced.
According to another preferred embodiment of the present invention, in step C, described emulsifying agent be selected from PLURONICS F87,
Poloxamer188, PVA, Emulsifier EL-60, vitamin E polyethylene glycol succinic acid ester, polyethylene maleic anhydride, sodium taurocholate,
Sodium docusate, cetyl trimethylammonium bromide, saponin(e, sugar ester, polysorbas20 or Tween 80.
According to another preferred embodiment of the present invention, in step D, described mixed liquor is in 30~50% amplitudes
Under the conditions of 2~6min of ultrasonic emulsification.
According to another preferred embodiment of the present invention, in step D, described mithramycin nano granule suspension
Stored at 4 DEG C of temperature.
The invention further relates to the mithramycin nano granule suspension that the preparation method is prepared.
A preferred embodiment of the invention, mithramycin nano particle has following physical characteristics：
Average grain diameter is 20.4~29.6nm；Polymer monodispersity index is 0.252~0.282；Surface charge is -15.0
~24.2mV.
According to another preferred embodiment of the present invention, the mithramycin nano particle has following chemical characteristics：
The drugloading rate of the mithramycin nano particle is 1.60~2.62%；Envelop rate is 22.28~36.68%；Saturating
Analyse in pipe, in 100mLpH 7.4PBS solution, 5 days total release rates are under 37 DEG C of magnetic agitations with rotating speed 150rpm of temperature
59.4~65.4%.
The present invention is described in more detail below.
The present invention relates to a kind of preparation method of mithramycin nano granule suspension.
The preparation method step of mithramycin nano granule suspension of the present invention is as follows：
A, prepare diblock copolymer solution
Diblock copolymer is dissolved in ethyl acetate solvent, the diblock copolymer that concentration is 30~40mg/mL is obtained
Ethyl acetate solution；
In the present invention, the main function of diblock copolymer is to contain medicine as carrier material and control medicine to release
Put, extend the plasma half-life of medicine.
According to the present invention, described diblock copolymer is to be selected from PLGA [poly by two kinds
(lactide-co-glycolide), PLGA], polyethylene glycol [poly (ethylene glycol), PEG], PLLA
[poly (L-lactic acid), PLLA], dextrorotation PLA [poly (D-lactic acid), PDLA], poly-dl-lactide
[poly (D, L-lactic acid), PDLLA], polyamide (polyamide, PA), PNIPAM [Poly (N-
Isopropylacrylamide), PNIPA], polyethyleneimine (polyethylenimine, PEI), polyvinyl alcohol
(polyvinyl alcohol, PVA), polyvinyl acetate (polyvinyl acetate, PVAc), polycaprolactone [poly (e-
Caprolactone), PCL], poly- amino chitosan (chitosan), polyethylene glycol oxide [poly (ethylene oxide),
PEO], cellulose acetate (cellulose acetate, CA), poly- [(R) -3-hydroxybutyrate] { Poly [(R) -3-
Hydroxybutyric acid], PHB, poly- polysaccharide (polysaccharide), poly 2-hydroxyethyl methacrylate [Poly (2-
Hydroxyethyl methacrylate), PHEMA] biocompatible polymer composition.For example, the present invention use two
Block copolymer is all the product sold in the market, such as by Jinan Dai Gang bioengineering Co., Ltd with the poly- second of trade name
It is the PEG-PLGA diblock copolymers of glycol PLGA sale, public by Jinan Dai Gang bioengineering Co., Ltd
Take charge of with the PEG-PCL diblock copolymers of trade name polyethylene glycol polycaprolactone diblock copolymer sale, by a Jinan Mount Tai handle of the Big Dipper and give birth to
PEG-PLLA diblock copolymers that thing Engineering Co., Ltd is sold with trade name polyethylene glycol PLLA, by a Jinan Mount Tai handle of the Big Dipper
The PEG-PDLA diblock copolymers that bioengineering Co., Ltd is sold with trade name polyethylene glycol dextrorotation PLA.
According to the present invention, the ethyl acetate solvent used can be by selected from the organic of dichloromethane, chloroform or ether
Solvent is replaced.The solvent that the present invention is used can be the mixture of the above-mentioned solvent of one of which or a variety of above-mentioned solvents, and these are molten
Agent is all the product sold in the market.Certainly, it is every have with the organic solvent similar performance, while to mithramycin
Other organic solvents that nano granule suspension is not adversely affected can also be applied to the present invention, and these solvents are also all in this hair
Within bright protection domain.
According to the present invention, if the concentration of diblock copolymer ethyl acetate solution is less than under 30mg/mL, envelop rate
Drop；If the concentration of diblock copolymer ethyl acetate solution is more than 40mg/mL, particle diameter increase and drugloading rate decline；Cause
This, the concentration of diblock copolymer ethyl acetate solution is that 30~40mg/mL is rational；Preferably 32~38mg/mL.
B, prepare mithramycin organic phase
According to the ratio 1 of ethyl acetate solution and the mithramycin in terms of milligram in terms of milliliter：1.0~3.0, mithramycin plus
Into above-mentioned diblock copolymer ethyl acetate solution, vibration dissolving obtains a kind of organic phase containing mithramycin；
The mithramycin that the present invention is used is the product sold in the market, for example by Sigma-Aldrich companies with
The mithramycin of trade name mithramycin sale.
The mithramycin that the present invention is used can by selected from mithramycin SK, mithramycin SDK or EC-8042 it is brilliant mould
Plain structure of modification compound is replaced.These mithramycin structure of modification compounds are that mithramycin sugar chain and sugared aglucon group are repaiied
Decorations and transformation product, they are the compounds for having similar structurally and functionally mechanism to mithramycin.Such as mithramycin SK, light
Brightness mycin SDK is by document J Am Chem Soc.2003May 14；125(19):What 5745-53 was reported has with mithramycin
The derivative of similar action；Mithramycin EC-8042 is by Clin Cancer Res.2016Aug15；22(16):4105-18
The derivative with mithramycin with similar action of report.
In the present invention, if the ratio of ethyl acetate solution and mithramycin is more than 1：1.0, then drugloading rate reduction；If
The ratio of ethyl acetate solution and mithramycin is less than 1：3.0, then envelop rate reduction；Therefore, ethyl acetate solution and mithramycin
Ratio be 1：1.0~3.0 be it is appropriate, preferably 1：1.4~2.6；More preferably 1：1.8~2.2.
C, prepare emulsifier solution
Emulsifying agent is soluble in water, and it is by weight 0.5~5% emulsifier solution to obtain concentration；
In the present invention, the main function of emulsifying agent is the surface tension reduced between organic phase and aqueous phase and reduces organic
The size of the drop formed in aqueous phase, therefore, the emulsifying agent that the present invention is used are the emulsifying agents of oil-in-water type.In this hair
It is every that there is these effects in bright, while dysgenic emulsifying agent will not be produced all to mithramycin nano granule suspension
The present invention is can apply to, also all within protection scope of the present invention.
According to the present invention, described emulsifying agent is selected from PLURONICS F87, poloxamer188, PVA, Emulsifier EL-60
(Cremophor EL), vitamin E polyethylene glycol succinic acid ester (Vitamin E-TPGS), polyethylene maleic anhydride [poly
(ethylene-alt-maleic anhydride), PEMA], sodium taurocholate (cholic acid sodium salt, CHA), sulphur
Base dioctyl succinate sodium (dioctyl sulfosuccinate sodium, DSS), cetyl trimethylammonium bromide
(hexadecyltrimethylammoniumbromide, CTAB), saponin(e (saponin), sugar ester (sugar
EsterD1216), polysorbas20 or Tween 80.
These emulsifying agents that the present invention is used all are the products sold in the market, such as by Gibco companies with Pu Lang
Buddhist nun gram F-68 trade names sale PLURONICS F87, sold by lark prestige company with polyvinyl alcohol trade name PVA, by
Vitamin E-TPGS that MedChemExpress companies are sold with vitamin E polyethylene glycol succinic acid ester trade name, by Germany
The Cremophor EL of BASF AG's sale.
D, mixing
Under conditions of the emulsifier solution prepared using shearing force homogenizer to step C carries out homogeneous, it is added dropwise in step B
Mixed liquor, is placed in ice bath by obtained organic phase immediately after completion of dropwise addition, is continuing with Probe Ultrasonic Searching instrument and carries out ultrasonic emulsification；
The effect that the present invention carries out homogeneous first by shearing force homogenizer is organic phase is preliminarily formed emulsion droplet；Then make
The effect for carrying out ultrasonic emulsification with Probe Ultrasonic Searching instrument is further to emulsify and emulsion droplet is reduced and is uniformed；
Homogeneous is carried out to emulsifying agent prepared by step C under the conditions of 22000rpm using shearing force homogenizer.The present invention makes
Shearing force homogenizer is, for example, with trade name hand-held homogenizer pin by Dragon Laboratory Instruments (Beijing) Co., Ltd.
The product sold.
Preferably, using Probe Ultrasonic Searching instrument allow described mixed liquor under conditions of 30~50% amplitudes ultrasonic emulsification 2~
6min.The Probe Ultrasonic Searching instrument that the present invention is used is, for example, to be sold by Sonics＆materials companies with trade name Probe Ultrasonic Searching instrument
Product.
According to the present invention, described mithramycin nano granule suspension is stored at 4 DEG C of temperature, because avoiding medicine
Thing discharges during storage and the nanoparticle suspension is stable at 4 DEG C.
E, post processing
The emulsion that step D is obtained removes the organic solvent wherein contained using vacuum rotary evaporator at room temperature；Connect
The unentrapped free drug wherein contained for 30000Da super filter tube removing using Millipore companies molecular cut off, is made
Continuous ultrafiltration is carried out to suspension with distilled water to clean, then filtered by 0.22 μm of syringe needle filter, obtain mithramycin nanometer
Grain suspension.
Organic solvent is removed under the conditions of reduced pressure at room temperature using vacuum rotary evaporator, it is organic molten in emulsion to reach
Agent content is by weight less than 0.01%.
The vacuum rotary evaporator that the present invention is used is, for example, with trade name rotary evaporation by Shanghai Yarong Biochemical Instrument Plant
The product of device sale.
When the present invention removes unentrapped free drug using super filter tube, in emulsion the content of unentrapped free drug be with
Weight meter is below 0.01%.
The purpose that the present invention is cleaned using distilled water continuous ultrafiltration is the emulsifying agent for removing free drug and excess.
The invention further relates to the mithramycin nano granule suspension that the preparation method is prepared.
Particle diameter, surface potential and the form of mithramycin nano particle of the present invention are detected by the following method：
The mithramycin nano granule suspension of preparation is diluted ten times using distilled water, is added in sample cell, then be placed in
In the ZetaPlus particle size analyzers sold by Brooker Hai Wen companies, particle is detected using dynamic light scattering method at 25 DEG C of temperature
Footpath and surface potential.
In order to observe particle shape, the μ g/mL mithramycin nano granule suspensions of 50uL 10 are taken to be added dropwise on aluminium foil,
Volatilize at room temperature, be then fixed on copper billet and metal spraying film, then detected using ESEM.
It is another to be added dropwise to 10 μ g/mL mithramycin nano granule suspensions on 200 mesh copper mesh of covering carbon film, inhaled with filter paper
Surplus liquid is taken, one layer of liquid film is retained on copper mesh, is spontaneously dried.Use 2% Salkowski's solution negative staining sample by weight
15min, then washes away free phosphotungstic acid and dries with distilled water, is placed in transmission electron microscope detection.Testing result is shown (referring to accompanying drawing
2) the mithramycin nano particle average grain diameter that, prepared by the inventive method is 20.4~29.6nm；Polymer monodispersity index is
0.252~0.282；Surface charge is -15.0~24.2mV.Electromicroscopic photograph shows that nano particle is subsphaeroidal.
The drugloading rate and envelop rate of mithramycin nano particle of the present invention are detected by the following method：
Take 1mL mithramycin nano particle suspensions to be placed in -70 DEG C of pre-freezes of temperature, then freeze 24h.Lyophilized nano particle claims
Weight, and add 1mL dmso solutions.15min is centrifuged using bath-sonicated 30min, then under the conditions of 12000xg, on
Clear liquid is detected using HPLC, and calculates institute's bag medicament contg in nano particle by mithramycin standard curve.Drugloading rate and encapsulating
Rate calculation formula is as follows：
Testing result shows that the drugloading rate of mithramycin nano particle is 1.60~2.62%；Envelop rate be 22.28~
36.68%.
The external drug release rate detection of mithramycin nano particle：
Take the mithramycin nano particle suspension of 1mL ultrafiltration to be placed in dialysis tubing, dialysis tubing is then put into 100mLPBS
In (pH 7.4), with 150rpm low speed magnetic agitations PBS at 37 DEG C of temperature.Taken out at multiple time points on the outside of 1mL dialysis tubings
Liquid carries out HPLC and detects its mithramycin concentration, while being supplemented the fresh PBS of 1mL.Testing result is listed in Fig. 3.
Fig. 3 results show that prepared mithramycin nano particle has one to discharge speed quickly in initial 48h
Degree, then mithramycin with keeping a relative durations slow release, total release rate is 59.4~65.4% after 5 days.
The present invention establishes a kind of method for preparing mithramycin nano particle, this method by emulsified solvent volatilization technology
The drugloading rate and envelop rate of amphoteric compound mithramycin can be effectively improved, and there is the characteristics of particle diameter is small, is effectively improved
Intravenously administrable problem.At present, there is not yet preparing the report of mithramycin nano particle using similar manner.
[beneficial effect]
The beneficial effects of the invention are as follows：The average grain diameter of mithramycin nano particle of the present invention is 20.4~29.6nm；It is many
Polymers monodispersity index is 0.252~0.282；Surface charge is -15.0~24.2mV.The load medicine of the mithramycin nano particle
Measure as 1.60~2.62%；Envelop rate is 22.28~36.68%；In dialysis tubing, in 100mLpH 7.4PBS solution,
37 DEG C of temperature and rotating speed 150rpm lower 5 days total release rates of magnetic agitation are 59.4~65.4%.
The present invention prepares the nano particle for containing mithramycin by emulsion-solvent evaporation method, improves the release power of medicine
Learn, slow down the internal degradation process of medicine, change the drug cycles time, increase medicine is enriched with tumor locus, and avoids machine
The medicine impedance mechanism of body.The pharmacology that mithramycin nano particle prepared by the present invention can preferably retain mithramycin is lived
Property, and the material that the present invention is used is biocompatible materials, with application security feature, is shown before good application
Scape.Meanwhile, the mithramycin nano particle effectively can be enriched with transplanted tumor in nude mice position, and be presented thin to human pancreas cancer
Born of the same parents BxPC-3 transplanted tumor in nude mice significantly therapeutic effect.
【Brief description of the drawings】
Fig. 1 is mithramycin nano granule suspension preparation flow figure of the present invention；
Fig. 2 is mithramycin nano particle form electron microscopic observation figure of the present invention；
Fig. 3 is the external insoluble drug release testing result figure of mithramycin nano particle of the present invention；
Fig. 4 is the confocal laser scanning microscope figure that tumour cell absorbs mithramycin nano particle of the present invention；
Fig. 5 is the Flow cytometry result figure that tumour cell absorbs mithramycin nano particle of the present invention；
In figure：
5A- relative intensity of fluorescence-cell number curve map；M- relative intensity of fluorescence block diagram during 5B-.
Fig. 6 is to suppress swollen using srb assay detection mithramycin (MIT) and mithramycin nano particle of the present invention (MIT-NP)
Tumor cell proliferation exercising result figure；
Fig. 7 is living imaging Germicidal efficacy mithramycin nano particle of the present invention dividing in vivo in Nude Mouse Model
Cloth situation；
In figure：
The fluoroscopic image of 7A- different time points tumor-bearing mices；
In vitro tumor mass and a variety of organs (1- tumor mass after 7B- injections 168h；2- livers；The 3- hearts；4- lungs；
5- stomaches；6- small intestines；7- colons；8- kidneys；9- pancreas；10- spleens；11- femurs) fluoroscopic image；
The numerical value of scale from bottom to top is 1.0,1.2,1.4,1.6 respectively on the right side of BxPC-3 columns in Fig. 7 A
(×1010)
In Fig. 7 A on the right side of MIAPaca-2 columns the numerical value of scale from bottom to top be 2.0 respectively, 2.5,3.0,
3.5、4.0(×1010)
In Fig. 7 B on the right side of BxPC-3 columns the numerical value of scale from bottom to top be 4.0 respectively, 4.5,5.0,5.5,
6.0、6.5、7.0(×1010)
In Fig. 7 B on the right side of MIAPaca-2 columns the numerical value of scale from bottom to top be 0.8 respectively, 1.0,1.2,
1.4、1.6、1.8、2.0(×1010)；
Fig. 8 is the growing state that mithramycin nano particle of the present invention suppresses human pancreas cancer BxPC-3 transplanted tumor in nude mice；
In figure：
8A- growth of transplanted human curve maps；8B- tumor-bearing mice body weight curve maps.
Fig. 9 is the H＆E dyes of mithramycin nano particle group (2mg/kg) of the present invention and a variety of slices of organs of nude mice of control group
Chromatic graph (pancreas, marrow, spleen, liver, lung, the heart, kidney, small intestine).
Figure 10 is Ki-67 expression condition diagrams in SABC detection tumor tissues.
【Embodiment】
The present invention is will be better understood that by following embodiments.
Embodiment 1：Prepare mithramycin nano granule suspension
Mithramycin nano particle basic process is prepared referring to accompanying drawing 1 using emulsion-solvent evaporation method.
The implementation steps of the embodiment are as follows：
A, prepare diblock copolymer solution
MPEG-PLGA diblock copolymers (the PEG molecular weight that will be sold by Jinan Dai Gang bioengineering Co., Ltd
5000Da；PLGA molecular weight 15000Da；Lactic acid and glycolic mol ratio：50/50) it is dissolved in ethyl acetate solvent, obtains concentration
For 30mg/mL diblock copolymer ethyl acetate solution；
B, prepare mithramycin organic phase
According to the ratio 1 of ethyl acetate solution and the mithramycin in terms of milligram in terms of milliliter：2.2, by Sigma-Aldrich
Company is added in above-mentioned diblock copolymer ethyl acetate solution with the trade name mithramycin mithramycin sold, vibration
Dissolving, obtains a kind of organic phase containing mithramycin；
C, prepare emulsifier solution
PLURONICS F87 emulsifying agent is soluble in water, and it is by weight 2.0% PLURONICS F87 emulsification to obtain concentration
Agent solution；
D, mixing
Using the shearing sold by Dragon Laboratory Instruments (Beijing) Co., Ltd. with trade name hand-held homogenizer
Power homogenizer is carried out under the conditions of 5 grades (22000rpm) to emulsifier solution prepared by step C under conditions of homogeneous, is added dropwise in step
Mixed liquor, is placed in ice bath by the organic phase that rapid B is obtained immediately after completion of dropwise addition, is continuing with public by Sonics＆materials
The Probe Ultrasonic Searching instrument of department's sale carries out ultrasonic emulsification 2min under conditions of 30% amplitude；
E, post processing
The rotary evaporator that the emulsion that step D is obtained is sold using Shanghai Yarong Biochemical Instrument Plant removes it at room temperature
In the organic solvent that contains；Then removed and wherein contained for 30000Da super filter tube using Millipore companies molecular cut off
Unentrapped free drug, continuous ultrafiltration cleaning is carried out to suspension using distilled water, then filtered by 0.22 μm of syringe needle filter,
Obtain mithramycin nano granule suspension.Described mithramycin nano granule suspension is stored at 4 DEG C of temperature.
The method described according to this specification have detected the thing of the mithramycin nano granule suspension of embodiment preparation
Reason and chemical property.
In mithramycin nano granule suspension prepared by the embodiment, the average grain diameter of mithramycin nano particle is
27.3nm；Polymer monodispersity index is 0.252；Surface charge is -21.9mV.The drugloading rate of the mithramycin nano particle is
1.83%；Envelop rate is 36.68%；In dialysis tubing, in 100mLpH 7.4PBS solution, in 37 DEG C of temperature and rotating speed
150rpm lower 5 days total release rates of magnetic agitation are 59.4%.
Embodiment 2：Prepare mithramycin nano granule suspension
The implementation steps of the embodiment are as follows：
A, prepare diblock copolymer solution
PEG-PLLA diblock copolymers (the PEG molecular weight that will be sold by Jinan Dai Gang bioengineering Co., Ltd
3000Da；PLLA molecular weight 15000Da) it is dissolved in dichloromethane solvent, obtain the diblock copolymer two that concentration is 40mg/mL
Chloromethanes solution；
B, prepare mithramycin organic phase
According to the ratio 1 of dichloromethane solution and the mithramycin SK in terms of milligram in terms of milliliter：1.0, by by document JAm
Chem Soc.2003May 14；125(19):The mithramycin SK of 5745-53 reports is added to above-mentioned diblock copolymer dichloromethane
In alkane solution, vibration dissolving obtains a kind of organic phase containing mithramycin SK；
C, prepare emulsifier solution
Emulsifying agent PVA is soluble in water, and it is by weight 5% PVA emulsifier solutions to obtain concentration；
D, mixing
Using the shearing sold by Dragon Laboratory Instruments (Beijing) Co., Ltd. with trade name hand-held homogenizer
Power homogenizer is carried out under the conditions of 6 grades (30000rpm) to emulsifier solution prepared by step C under conditions of homogeneous, is added dropwise in step
Mixed liquor, is placed in ice bath by the organic phase that rapid B is obtained immediately after completion of dropwise addition, is continuing with public by Sonics＆materials
The Probe Ultrasonic Searching instrument of department's sale carries out ultrasonic emulsification 4min under conditions of 50% amplitude；
E, post processing
The rotary evaporator that the emulsion that step D is obtained is sold using Shanghai Yarong Biochemical Instrument Plant removes it at room temperature
In the organic solvent that contains；Then removed and wherein contained for 30000Da super filter tube using Millipore companies molecular cut off
Unentrapped free drug, continuous ultrafiltration cleaning is carried out to suspension using distilled water, then filtered by 0.22 μm of syringe needle filter,
Obtain mithramycin nano granule suspension.Described mithramycin nano granule suspension is stored at 4 DEG C of temperature.
The method described according to this specification have detected the thing of the mithramycin nano granule suspension of embodiment preparation
Reason and chemical property.
In mithramycin nano granule suspension prepared by the embodiment, the average grain diameter of mithramycin nano particle is
20.4nm；Polymer monodispersity index is 0.282；Surface charge is -15.0mV.The drugloading rate of the mithramycin nano particle is
1.60%；Envelop rate is 33.08%；In dialysis tubing, in 100mLpH 7.4PBS solution, in 37 DEG C of temperature and rotating speed
150rpm lower 5 days total release rates of magnetic agitation are 63.9%.
Embodiment 3：Prepare mithramycin nano granule suspension
The implementation steps of the embodiment are as follows：
A, prepare diblock copolymer solution
PEG-PDLA diblock copolymers (the PEG molecular weight that will be sold by Jinan Dai Gang bioengineering Co., Ltd
5000Da；PDLA molecular weight 20000Da) it is dissolved in chloroform solvent, obtain the diblock copolymer three that concentration is 34mg/mL
Chloromethanes solution；
B, prepare mithramycin organic phase
According to the ratio 1 of chloroform soln and the mithramycin SDK in terms of milligram in terms of milliliter：1.6, by document JAm
Chem Soc.2003May 14；125(19):The mithramycin SDK of 5745-53 reports is added to above-mentioned diblock copolymer trichlorine
In dichloromethane, vibration dissolving obtains a kind of organic phase containing mithramycin SDK；
C, prepare emulsifier solution
Emulsifying agent sodium taurocholate is soluble in water, and it is by weight 1.0% sodium taurocholate emulsifier solution to obtain concentration；
D, mixing
Using the shearing sold by Dragon Laboratory Instruments (Beijing) Co., Ltd. with trade name hand-held homogenizer
Power homogenizer is carried out under the conditions of 4 grades (18000rpm) to emulsifier solution prepared by step C under conditions of homogeneous, is added dropwise in step
Mixed liquor, is placed in ice bath, is continuing with by .Sonics＆materials by the organic phase that rapid B is obtained immediately after completion of dropwise addition
The Probe Ultrasonic Searching instrument of company's sale carries out ultrasonic emulsification 6min under conditions of 36% amplitude；
E, post processing
The rotary evaporator that the emulsion that step D is obtained is sold using Shanghai Yarong Biochemical Instrument Plant removes it at room temperature
In the organic solvent that contains；Then removed and wherein contained for 30000Da super filter tube using Millipore companies molecular cut off
Unentrapped free drug, continuous ultrafiltration cleaning is carried out to suspension using distilled water, then filtered by 0.22 μm of syringe needle filter,
Obtain mithramycin nano granule suspension.Described mithramycin nano granule suspension is stored at 4 DEG C of temperature.
The method described according to this specification have detected the thing of the mithramycin nano granule suspension of embodiment preparation
Reason and chemical property.
In mithramycin nano granule suspension prepared by the embodiment, the average grain diameter of mithramycin nano particle is
29.6nm；Polymer monodispersity index is 0.274；Surface charge is -24.2mV.The drugloading rate of the mithramycin nano particle is
2.62%；Envelop rate is 25.88%；In dialysis tubing, in 100mLpH 7.4PBS solution, in 37 DEG C of temperature and rotating speed
150rpm lower 5 days total release rates of magnetic agitation are 65.4%.
Embodiment 4：Prepare mithramycin nano granule suspension
The implementation steps of the embodiment are as follows：
A, prepare diblock copolymer solution
PEG-PCL diblock copolymers (the PEG molecular weight that Jinan company of Dai Gang bioengineering Co., Ltd is sold
5000Da；PCL molecular weight 10000Da) it is dissolved in ether solvent, obtain the diblock copolymer ether that concentration is 38mg/mL molten
Liquid；
B, prepare mithramycin organic phase
According to the ratio 1 of diethyl ether solution and the mithramycin EC-8042 in terms of milligram in terms of milliliter：3.0, by Clin
CancerRes.2016Aug 15；22(16):The mithramycin EC-8042 of 4105-18 reports is added to above-mentioned diblock copolymer
In diethyl ether solution, vibration dissolving obtains a kind of organic phase containing mithramycin；
C, prepare emulsifier solution
Polyethylene maleic anhydride emulsifying agent is soluble in water, and it is 3.5% polyethylene maleic anhydride by weight to obtain concentration
Emulsifier solution；
D, mixing
Using the shearing sold by Dragon Laboratory Instruments (Beijing) Co., Ltd. with trade name hand-held homogenizer
Power homogenizer is carried out under the conditions of 5 grades (22000rpm) to emulsifier solution prepared by step C under conditions of homogeneous, is added dropwise in step
Mixed liquor, is placed in ice bath by the organic phase that rapid B is obtained immediately after completion of dropwise addition, is continuing with public by Sonics＆materials
Take charge of the Probe Ultrasonic Searching instrument sold with trade name Probe Ultrasonic Searching instrument and ultrasonic emulsification 4min is being carried out under conditions of 42% amplitude；
E, post processing
The emulsion that step D is obtained uses the vacuum sold by Shanghai Yarong Biochemical Instrument Plant with trade name rotary evaporator
Rotary Evaporators remove the organic solvent wherein contained at room temperature；Then it is using Millipore companies molecular cut off
30000Da super filter tube removes the unentrapped free drug wherein contained, and continuous ultrafiltration cleaning is carried out to suspension using distilled water,
Then filtered by 0.22 μm of syringe needle filter, obtain mithramycin nano granule suspension.Described mithramycin nano particle
Suspension is stored at 4 DEG C of temperature.
The method described according to this specification have detected the thing of the mithramycin nano granule suspension of embodiment preparation
Reason and chemical property.
In mithramycin nano granule suspension prepared by the embodiment, the average grain diameter of mithramycin nano particle is
25.0nm；Polymer monodispersity index is 0.259；Surface charge is -17.3mV.The drugloading rate of the mithramycin nano particle is
1.86%；Envelop rate is 22.28%；In dialysis tubing, in 100mLpH 7.4PBS solution, in 37 DEG C of temperature and rotating speed
150rpm lower 5 days total release rates of magnetic agitation are 60.9%.
Test example 1：The cell in vitro endocytosis experiment of mithramycin nano particle of the present invention
I, confocal laser scanning microscope cellular uptake mithramycin nano particle ability
Take the mithramycin nanometer that confocal laser scanning microscope tumour cell is marked to fluorescent dye Cy5 in vitro
The endocytic processes of particle.Its step is as follows：Human pancreatic cancer cell BxPC-3 and MIAPaca-2 in exponential phase is thin
Born of the same parents are digested to single cell suspension with pancreatin, and cell count simultaneously adjusts its concentration, by every hole 2 × 104The density of cell is inoculated in 8 holes
Chamber cover glass (Thermo Fisher companies).24h is cultivated at 37 DEG C of temperature, culture medium is abandoned in suction, and add 300 μ L and trained
The mithramycin nano particle (1mg/mL) of the present invention of base dilution is supported, when continuing to cultivate 0.5-2h, culture medium is abandoned in suction, is moistened with PBS
Wash 3 times, each 5min.Add DiI (cell membrane red fluorescence probe, green skies biotechnology research institute) lucifuges at room temperature
15min is handled, then with PBS rinses 3 times, each 5min.30min is fixed using 4% paraformaldehyde by weight, with PBS rinses 3
Secondary, each 5min is subsequently added into the anti-cancellation mountant comprising DAPI.Use confocal laser scanning microscope.It observes knot
Fruit shows that BxPC-3 and MIAPaca-2 cells can quickly absorb the mithramycin nano particle of this method preparation (referring to accompanying drawing
4)。
The ability of II, flow cytomery cellular uptake mithramycin nano particle of the present invention
Take the logarithm BxPC-3 the and MIAPaca-2 cells in growth period, digest and count, be configured to single cell suspension, with every
Hole 3 × 105Cell adds 6 orifice plates, cellar culture to cell confluency degree about 70%.Culture medium is abandoned in suction, is added 2mL and is included 1mg/mL
Cy5 marks the fresh culture of mithramycin nano particle.Lucifuge handles 0.25-2h, reuses pancreatin and digests and collect, and uses
PBS is washed 3 times.Then FACSCalibur flow cytomeries are used.Testing result shows that the present invention is brilliant after processing 15min
Mycin nano particle can enter cell, quickly increase with time lengthening intensity of cellular fluorescence, illustrate that tumour cell can be quick
Absorb nano particle (referring to accompanying drawing 5).
Test example 2：External inhibitory activity of the mithramycin nano particle of the present invention to tumour cell
The inhibitory action of mithramycin and its nano particle in vitro for tumour cell is detected using srb assay.Its step
It is as follows：Take the logarithm human pancreatic cancer cell BxPC-3, MIAPaca-2, AsPC-1 and PANC-1 cell in growth period, pancreatin digestion
And count, it is inoculated in 3000-7000/ holes in 96 orifice plates, in cultivating 24h at 37 DEG C of temperature.Then, the light of various concentrations is added
Brightness mycin and mithramycin nano particle of the present invention, each drug concentration set 5 parallel holes.In being incubated 24- at 37 DEG C of temperature
72h, inhales the solution of trichloroacetic acid for abandoning that culture medium and 100 μ L concentration of addition are by weight 10%.In 4 DEG C of fixed 1h of temperature, use
Distilled water flushing 5 times, dries naturally.Add 100 μ L concentration by weight 0.4% SRB solution (contain 1% acetic acid), dyeing
5min, with distilled water flushing 5 times, dries naturally.Finally, 100 μ L 10mM Tris solution (pH 10.5) dissolving knot is added per hole
The SRB of conjunction, 570nm absorbance is measured using ELIASA.
The survival rate of cell is calculated according to the following equation：
Cell survival rate=(dosing group A570Value-blank group A570Value)/(control group A570Value-blank group A570Value) ×
100%
In formula：
Dosing group A570Value represents the 570nm absorbances of measurement dosing group；
Control group A570Value represents the 570nm absorbances of measurement control group；
Blank group A570Value represents the 570nm absorbances of measurement blank group；
These result of the tests show that mithramycin and mithramycin nano particle of the present invention have for 4 kinds of tumour cells
There is the inhibitory action of concentration dependent and time dependence (referring to accompanying drawing 6).And the mithramycin of the equal drug dose of contrast
Influence with mithramycin nano particle of the present invention to cell survival rate can be seen that both inhibitory action have no significant difference,
Show that nano particle preparation process does not influence the tumors inhibition activity of mithramycin.
Test example 3：The ability of mithramycin nano particle target tumor tissue in live body of Cy5 marks
By human pancreas cancer BxPC-3 and MIAPaca-2 cell with 1 × 107Armpit on the right side of/a subcutaneous vaccination to BALB/c nude mices
Under.When gross tumor volume to 300-400mm3, the nano particle that Cy5 is marked is entered in Mice Body by tail vein injection.Utilize
IVIS living imaging systems (PerkinElmer companies) are observed.In different time points, entered using Medical anesthetic agent isoflurane
Row processing, then observes the situation of mithramycin nano particle target tumor tissue in tumor-bearing mice body.
Accompanying drawing 7 shows that mithramycin nano particle prepared by the present invention can preferably target BxPC-3 and MIAPaca-2 is naked
Mouse transplantable tumor position.The 2h after injection, tumor locus be appearance stress signal, 12h or so fluorescence intensity is most strong, gradually subtracts thereafter
It is weak, until 168h still has the fluorescence signal higher than other positions in tumor locus.Mouse is put to death and separated in 168h after injection
Go out major organs progress fluorescence imaging and show that tumor tissues have the fluorescence signal for being significantly stronger than other organs.Therefore, above knot
Fruit illustrates that the mithramycin nano particle prepared by the present invention can be enriched with tumor locus.
Test example 4：Growth inhibition effect of the mithramycin nano particle of the present invention to transplanted tumor in nude mice BxPC-3
By the BxPC-3 cell tryptases enzymic digestion in exponential phase and count, be configured to 1 × 108/ mL cell suspensions.Take
Body weight sets up Transplanted tumor model, every inoculation 1 × 10 for 18-20g female BAl BIc/c nude mices7Individual cell.Treat that tumor mass reaches about
100mm3When, it is grouped according to the knurl volume of nude mice, every group 6.Handled according to following dosage regimens：Physiological saline group, it is empty
White nano particle (non-drug containing) group, mithramycin group (1mg/kg) and mithramycin nano particle group (1mg/kg and 2mg/
kg).Tail vein injection, once in a week, is administered for continuous 4 weeks.After the administration of first pin, nude mice body weight and gross tumor volume were entered in every 4 days
Row measurement.According to formula V=ab2/ 2 calculate gross tumor volume (a：Tumour major diameter, b：Tumour minor axis).In 29 days after inoculated tumour,
Put to death mouse and dissect taking-up tumor mass, pancreas, femur, spleen, liver, lung, the heart, kidney and small intestine.Then 10% formalin is used
Fixed, then FFPE, section carries out hematoxylin-eosin (H＆E) dyeing respectively, and anti-to tumor tissue progress core increment
Former Ki-67 SABC detection.As a result show：Mithramycin is weaker to the rejection ability of BxPC-3 growth of transplanted human, suppresses
Rate is 51%, and the inhibiting rate of isodose mithramycin nano particle group (1mg/kg) of the present invention is 86% (Fig. 8).Meanwhile,
2mg/kg mithramycin nano particle group inhibiting rates of the present invention are 96%, and have no that mouse main organs damage occur (referring to attached
Fig. 9).Showed by immune group result, mithramycin nano particle of the present invention can reduce tumor tissue center increment antigen Ki-67's
Expression is (referring to accompanying drawing 10).
Claims (10)
1. a kind of preparation method of mithramycin nano granule suspension, it is characterised in that as follows the step of the preparation method：
A, prepare diblock copolymer solution
Diblock copolymer is dissolved in ethyl acetate solvent, the diblock copolymer acetic acid that concentration is 30~40mg/mL is obtained
Ethyl ester solution；
B, prepare mithramycin organic phase
According to the ratio 1 of ethyl acetate solution and the mithramycin in terms of milligram in terms of milliliter：1.0~3.0, mithramycin is added to
State in diblock copolymer ethyl acetate solution, vibration dissolving obtains a kind of organic phase containing mithramycin；
C, prepare emulsifier solution
Emulsifying agent is soluble in water, and it is by weight 0.5~5% emulsifier solution to obtain concentration；
D, mixing
Under conditions of the emulsifier solution prepared using shearing force homogenizer to step C carries out homogeneous, dropwise addition is obtained in step B
Organic phase, mixed liquor is placed in ice bath immediately after completion of dropwise addition, be continuing with Probe Ultrasonic Searching instrument carry out ultrasonic emulsification；
E, post processing
The emulsion that step D is obtained removes the organic solvent wherein contained using vacuum rotary evaporator at room temperature；Then make
The super filter tube for being 30000Da with Millipore companies molecular cut off removes the unentrapped free drug wherein contained, using double
Steam water and continuous ultrafiltration cleaning is carried out to suspension, then filtered by 0.22 μm of syringe needle filter, obtain mithramycin nano particle and hang
Supernatant liquid.
2. preparation method according to claim 1, it is characterised in that in step, described diblock copolymer be by
Two kinds are selected from PLGA, polyethylene glycol, PLLA, dextrorotation PLA, poly-dl-lactide, polyamides
Amine, PNIPAM, polyethyleneimine, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly- amino chitosan,
Polyethylene glycol oxide, cellulose acetate, poly- [(R) -3-hydroxybutyrate], the biofacies of poly- polysaccharide or poly 2-hydroxyethyl methacrylate
Capacitive polymer composition.
3. preparation method according to claim 1, it is characterised in that in step, described ethyl acetate is by selected from two
The organic solvent of chloromethanes, chloroform or ether is replaced.
4. preparation method according to claim 1, it is characterised in that in stepb, mithramycin is by selected from mithramycin
SK, mithramycin SDK or EC-8042 mithramycin structure of modification compound are replaced.
5. preparation method according to claim 1, it is characterised in that in step C, it is husky that described emulsifying agent is selected from pool Lip river
Nurse 188, poloxamer188, PVA, Emulsifier EL-60, vitamin E polyethylene glycol succinic acid ester, polyethylene maleic anhydride,
Sodium taurocholate, Sodium docusate, cetyl trimethylammonium bromide, saponin(e, sugar ester, polysorbas20 or Tween 80.
6. nano granule suspension according to claim 1, it is characterised in that in step D, described mixed liquor is 30
2~6min of ultrasonic emulsification under conditions of~50% amplitude.
7. nano granule suspension according to claim 1, it is characterised in that in step D, described mithramycin is received
Rice grain suspension is stored at 4 DEG C of temperature.
8. the mithramycin nano particle that the preparation method according to any one of claim 1-7 claim is prepared hangs
Supernatant liquid.
9. mithramycin nano granule suspension according to claim 1, it is characterised in that mithramycin nano particle has
There are following physical characteristics：
Average grain diameter is 20.4~29.6nm；Polymer monodispersity index is 0.252~0.282；Surface charge be -15.0~
24.2mV。
10. mithramycin nano granule suspension according to claim 1, it is characterised in that mithramycin nano particle has
There are following chemical characteristics：
The drugloading rate of the mithramycin nano particle is 1.60~2.62%；Envelop rate is 22.28~36.68%；In dialysis tubing
In, in 100mLpH 7.4PBS solution, 5 days total release rates are 59.4 under 37 DEG C of magnetic agitations with rotating speed 150rpm of temperature
~65.4%.
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