CN106963732A - The preparation and its application in neoplasm tracing and treatment of a kind of sericin mating type paclitaxel nano medicine - Google Patents
The preparation and its application in neoplasm tracing and treatment of a kind of sericin mating type paclitaxel nano medicine Download PDFInfo
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- CN106963732A CN106963732A CN201710081863.XA CN201710081863A CN106963732A CN 106963732 A CN106963732 A CN 106963732A CN 201710081863 A CN201710081863 A CN 201710081863A CN 106963732 A CN106963732 A CN 106963732A
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- 108010013296 Sericins Proteins 0.000 title claims abstract description 73
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 66
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 title claims abstract description 66
- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 65
- 239000003814 drug Substances 0.000 title claims abstract description 62
- 230000013011 mating Effects 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 20
- 238000011282 treatment Methods 0.000 title claims abstract description 13
- 208000035269 cancer or benign tumor Diseases 0.000 title claims abstract description 8
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 claims abstract description 23
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- 239000000178 monomer Substances 0.000 claims description 8
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims 1
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Classifications
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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
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- 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
-
- 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
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0028—Oxazine dyes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0082—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The invention discloses a kind of preparation of sericin mating type paclitaxel nano medicine and its in neoplasm tracing and the application in treatment.The present invention prepares amphipathic sericin polymer using sericin as hydrophilic segment, using the poly- peptide of hydrophobicity as hydrophobic chain segment by alpha amino acid N carboxyanhydrides (NCA) ring-opening reaction approach.Simultaneously using amphipathic sericin as carrier, using hydrophobic interaction, the antitumor drug paclitaxel and Nile red fluorescer of non-aqueous are loaded, sericin mating type paclitaxel nano medicine (Sericin PBLG PTX Nile red) is prepared.The sericin mating type paclitaxel nano medicine is expected to be used in tumor area as function admirable, the novel modified release pharmaceutical of high efficiency anti-tumor.
Description
Technical field
The invention belongs to biomedical materials field, and in particular to a kind of sericin mating type paclitaxel nano medicine
(Sericin-PBLG-PTX-Nile-red) preparation and its application in neoplasm tracing and treatment.
Background technology
China is world population big country, and the morbidity total number of persons of its malignant tumour accounts for the significant proportion of global number, and this is serious
Influence the population health problem of China.The Traditional regimen of malignant tumour, mainly includes surgery excision, chemotherapy, radiation at present
Treatment, but the prognosis of current most of malignant tumours is still poor, and the death rate is higher.Because classic chemotherapy medicine is swollen in Locally Advanced
Knurl focus is difficult to the valid density for reaching control growth of cancer cells, causes the adverse consequences such as the easy Preventive of tumour, so it is traditional
Treatment produces little effect.In order to improve therapeutic effect of malignant tumour, extension the survival of patients cycle, clinically propose targeted therapy,
The kinds of schemes such as light power thermotherapy, immunization therapy and therapeutic alliance, but its limits it the shortcomings of medicine valency is high, curative effect is unstable
Use, be clinically difficult to low price, safe and reliable, efficient complex treatment.
Taxol (PTX) is the medicine extracted from peaceful red deal, Chinese yew for 1962, can be with intracellular canaliculus
Protein binding, the normal dynamic regeneration of micro-pipe net necessary to suppressing mitosis, prevents normal mitotic spindle
Formed, cause the fracture of chromosome, and then suppress the duplication of cell.Based on the mechanism of action, taxol is widely used in including
The treatment of the Several Kinds of Malignancy such as stomach cancer, breast cancer.But taxol due to poorly water-soluble, be orally difficult to absorption characteristics, often only
It is limited to intravenous injection；Taxol after intravenous in blood circulation lacks tumor-targeting, and not only focus is difficult to reach active drug
Concentration, and often cause the whole body system toxic side effect such as allergy, bone marrow suppression, neurotoxicity.Albumin combination type taxol this
New formulation, using nanoparticle albumin as carrier, optimizes taxol soluble, and born of the same parents way is worn by Albumin receptor (Gp60)
Acidic secretion protein (SPARC) approach of the outer interstitial of footpath and tumour cell improves tumor locus drug effect, and clinic is had been used at present,
But the price of albumin combination type taxol is high, strongly limit its clinical practice.Therefore, it is anti-swollen in order to maximize taxol
Knurl benefit, clinically needs a kind of inexpensive, safe and efficient, low toxicity side effect new taxol badly.
Sericin (Sericine) is a kind of viscous protein of the animal origin extracted in silk cocoon, rich with resource
The advantages of richness, low immunogenicity, biodegradable performance and high-biocompatibility, start be applied to tissue engineering material field and
Biomedicine field.In recent years, it is research to prepare amphipathic nature polyalcohol using hydrophilic protein segment and hydrophobic polypeptide segment
Focus, with the albuminoid structure that can be interacted with biologic artifact, is expected to be used as the more excellent albumin nano of a class performance
Ball alternative medicine.Up to the present, there is not yet the patent report of sericin-paclitaxel nano medicine.
The content of the invention
In energy Several Kinds of Malignancy therapeutic process of the taxol for including stomach cancer, breast cancer, there is poorly water-soluble, tumour
Targeting is low, many defects such as systemic toxic side effect after administration, and to solve current problem, the present invention provides a kind of new silk gum
The preparation method of protein binding type paclitaxel nano medicine (Sericin-PBLG-PTX-Nile-red) and its neoplasm tracing with
Application in treatment, we utilize hydrophilic sericin, the poly benzyl glutamate (PBLG) of hydrophobic peptide fragment, synthesizing amphipathic glue
Shu Zaiti (Sericin-PBLG), using the paclitaxel loaded chemotherapeutic of hydrophobic interaction, Nile red fluorescer, can efficiently, it is high
Activity contains medicine, hence into producing antitumor action in malignant tumour focus.The Nano medication can be by retaining Japanese yew
While alcohol antitumor properties, from many aspects optimization taxol active compound such as water-soluble, stability and tumor-targeting, it is assigned
Efficiently, the dual antitumor action of low toxicity, while carrying fluorescent drug, realizes visualization spike in tumor locus absorption process
Effect.
The primary and foremost purpose of the present invention is the preparation method for providing above-mentioned sericin mating type paclitaxel nano medicine.This
Similar ABI-007 nanometer can be achieved by synthesizing Sericin-PBLG-PTX-Nile-red novel nano micellas in invention
The antitumor action of particulate, can increase the high activity during the water solubility of taxol, tumor-targeting, and maintenance metabolism,
Toxic side effect is reduced, low cost, the high-efficiency low-toxicity chemotherapy of stomach cancer is finally completed.
Another object of the present invention is to provide a kind of sericin mating type paclitaxel nano medicine.
Still a further object of the present invention is to provide above-mentioned sericin mating type paclitaxel nano medicine in neoplasm tracing with controlling
Application in treatment.
The object of the invention is achieved through the following technical solutions：
A kind of preparation method of sericin mating type paclitaxel nano medicine, comprises the following steps：
(1) extraction of sericin：Silk cocoon boils degumming through aqueous slkali, is filtered to remove fibroin albumen and other insoluble matters,
Pure water dialysis removes ion salt, and freeze-drying obtains sericin；
(2) preparation of a-amino acid-N- carboxyanhydrides (NCA)：Under inert gas shielding, amino acid monomer is dissolved in
In anhydrous tetrahydro furan, add at the Triphosgene of 1.0-2.5 times of amino acid monomer quality, 45-60 DEG C and react 0.5-1.5h, it is cold
But to room temperature, vacuum distillation removes tetrahydrofuran, and obtained oily molten thing is dissolved in ethyl acetate, saturated sodium bicarbonate water is used
Solution is washed, and separates organic phase, is dried, and vacuum distillation removes solvent, obtains a-amino acid-N- carboxyanhydrides；
(3) preparation of the poly- peptide polymer of sericin-hydrophobicity：The sericin that step (1) is obtained is dissolved in organic
Solvent anhydrous dimethyl sulphoxide, adds a-amino acid-N- carboxyanhydrides prepared by step (2), reacts 2-4 days, is sunk in water
Form sediment and remove organic solvent, be dried to obtain the poly- peptide polymer of sericin-hydrophobicity；
(4) preparation of sericin mating type paclitaxel nano medicine：Sericin-hydrophobicity that step (3) is obtained
Poly- peptide polymer is dissolved in stirring in dimethyl sulfoxide (DMSO) (DMSO), adds Nile red and taxol drug is well mixed, Ran Hou
Stirring is lower to be instilled in deionized water, and finally mixed solution is fitted into bag filter, dialysed with deionized water, the silk is finally given
Glue protein mating type paclitaxel nano medicine (Sericin-PBLG-PTX-Nile-red).
Aqueous slkali described in step (1) be one kind in sodium acid carbonate, sodium carbonate, potassium carbonate, potassium bicarbonate solution or
A variety of, concentration is 0.01-0.05mol/L；Described usually time is 0.5-1.5h；The cellulose dialysis that described dialysis is used
Bag molecular cut off is 8000-14000Da.
Amino acid monomer described in step (2) is Pidolidone-r- benzyl esters, aspartic acid benzyl ester, phenylalanine and benzyl
One kind in oxygen carbonyl lysine.
Drying described in step (2) refers to be dried overnight with anhydrous magnesium sulfate, and drier is removed by filtration.
A-amino acid-N- carboxyanhydrides monomer and sericin quality ratio described in step (3) are 1：1-1：3.
Dialysis condition described in step (4) is：Bag filter for can molecular cut off be 3500KD specifications, dialysis temperature be
20-25 DEG C, dialysis time is 12-36h.
The mass ratio of the poly- peptide polymer of sericin-hydrophobicity, Nile red and taxol described in step (4) is
10mg：1-5μg：0.5-1mg.
Above-mentioned sericin mating type paclitaxel nano medicine can be applied to neoplasm tracing and treatment：The Nano medication has
High biological histocompatbility, can load the antitumor drug paclitaxel of non-aqueous, optimization taxol soluble deficiency, property are not
Stable the shortcomings of, the stable Nano medication of well dispersed, property is formed in aqueous, can quickly be absorbed by stomach cancer cell, is produced
The raw chemotherapy effect than single medicine taxol efficiently, safe；
The Nano medication carries strong fluorescence, is conducive to the stable spike of Nano medication in vivo, external in therapeutic process.
Room temperature of the present invention, normal temperature refer to 20-25 DEG C.
The present invention further verifies above-mentioned application by tests below：
(1) preparation of Nile red fluorescence labeling sericin nano-micelle carrier：The silk gum egg that above-mentioned steps (3) are obtained
The poly- peptide polymer of in vain-hydrophobicity is dissolved in stirring in dimethyl sulfoxide (DMSO) (DMSO), adds Nile red and is well mixed, then in stirring
In lower instillation deionized water, finally mixed solution is fitted into bag filter, dialysed at normal temperatures with deionized water, Buddhist nun is finally given
Luo Hong fluorescence labeling sericin nano-micelles carrier (i.e. Nile red mark Sericine-PBLG nano-micelles).
(2) cellular uptake and cytotoxicity of Nile red fluorescence labeling sericin nano-micelle carrier：Nile red is glimmering
Signal sericin nano-micelle carrier adds intracellular, different time points laser confocal microscope, CCK-8 experiment inspections
Survey the cellular uptake and in vitro toxicity of medicine.
(3) preparation of sericin mating type paclitaxel nano medicine：The sericin that above-mentioned steps (3) are obtained-dredge
Aqueous poly- peptide polymer is dissolved in stirring in dimethyl sulfoxide (DMSO) (DMSO), adds Nile red and taxol drug is well mixed, so
Instill under agitation in deionized water afterwards, finally mixed solution is fitted into bag filter, dialysed at 25 DEG C with deionized water, most
Sericin mating type paclitaxel nano medicine (Sericin-PBLG-PTX-Nile-red) is obtained eventually.
(4) the cellular uptake experiment of sericin mating type paclitaxel nano medicine：By stomach cancer cell AGS and sericin
Mating type paclitaxel nano medicine is incubated after certain time altogether, real with inverted microscope, laser confocal microscope, cell streaming
Test, observe the change of the time gradient absorbing rule and cellular morphology of Nano medication.
(5) toxicity test of sericin mating type paclitaxel nano medicine：By stomach cancer cell AGS respectively with single medicine Japanese yew
Alcohol, sericin mating type paclitaxel nano medicine are incubated after certain time altogether, with CCK-8 Germicidal efficacies and contrast different pharmaceutical
To stomach cancer cell AGS IC50 (median lethal dose) concentration.
Compared with prior art, the present invention has advantages below and beneficial effect：
(1) sericin mating type paclitaxel nano medicine of the present invention, using sericin as hydrophilic segment,
The amphiphilic nano carrier formed using poly- peptide as hydrophobic chain segment, with excellent biocompatibility, biodegradable and
Water solubility, efficiently quick, safe can enter stomach cancer focus.
(2) sericin mating type paclitaxel nano pharmaceutical properties of the present invention are stable, and preparation condition is gentle, required
Raw material is cheap, easy to operate, and technique is simple, highly effective.The Nano medication prepared, from water-soluble, stability and tumour
The broad aspect of targeting three optimizes taxol active compound, assigns its efficient, dual antitumor action of low toxicity, in the application of stomach cancer field
In have efficiently, low toxicity targeted chemotherapy advantage.
(3) sericin mating type paclitaxel nano medicine of the present invention, has benefited from Nile red and swashs in itself in 565nm
The characteristics of carrying fluorescence under hair wavelength, under the conditions of visualization is realized, can observe absorption site, absorption efficiency of medicine etc., enter
And possibility is provided for the precise positioning of stomach cancer, it is expected to realize tumour diagnosis and treatment integration, is that new antitumoral mechanism aspect is brought
New thinking, the further investigation for contributing to other malignant tumour mechanism regulatings to study.
(4) the sericin mating type paclitaxel nano medicine prepared of the present invention, not only surface there is abundant group,
Also there is height carrying capacity simultaneously, can be reacted with other drugs, nucleic, dyestuff equimolecular, carry out multiple chemotherapy, light power thermotherapy
Derive etc. multifunction, can be used as the novel pharmaceutical modified release carrier of function admirable.
Brief description of the drawings
Fig. 1 is the infrared spectrogram of the sericin of embodiment 1 and sericin-poly benzyl glutamate polymer.
Fig. 2 is the stomach cancer cell toxicity figure of 2 sericins of embodiment-poly benzyl glutamate polymer.
Fig. 3 is the Nile red of embodiment 2 mark Sericine-PBLG nano-micelles stomach cancer cell intake streaming figure.
Fig. 4 is sericin mating type paclitaxel nano medicine stomach cancer cell intake focused view altogether.
Fig. 5 is sericin mating type paclitaxel nano medicine stomach cancer cell cytotoxicity (MTT) figure.
Fig. 6 is that sericin mating type paclitaxel nano medicine promotes apoptosis in gastric cancer streaming figure.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.Unless stated otherwise, reagent involved in embodiment, method are reagent commonly used in the art and method.
Embodiment 1
(1) 60min is boiled in the sodium carbonate liquor that the silk cocoon shredded is added to 0.02mol/L, insoluble silk is filtered to remove
Fibroin and other insoluble matters, filtrate is loaded in cellulose dialysis bag of the molecular cut off for 8000-14000Da, pure water thoroughly
Analysis removes ion salt for three days, and freeze-drying obtains sericin.
(2) under argon gas protection, the 2g of protection Pidolidone-r- benzyl esters are dissolved in the tetrahydrofuran removed water through NaH, plus
Enter and react 1h at 1.4g Triphosgene, 55 DEG C, be cooled to room temperature, vacuum distillation removes tetrahydrofuran, by obtained oily molten thing
It is dissolved in 100mL ethyl acetate, is washed with saturated sodium bicarbonate aqueous solution, separates organic phase, it is dried with anhydrous magnesium sulfate
At night, drier is filtered to remove, vacuum distillation removes solvent, obtains benzyl glutamate ring inner-acid anhydride.
(3) weigh the sericin that 0.5g steps (1) obtain and be scattered in anhydrous dimethyl sulphoxide, be heated to 50 DEG C, stirring
2h makes it fully dissolve, and is cooled to room temperature, sets 3 groups, is separately added into the glutamic acid that 0.25g, 0.5g, 1.0g step (2) are obtained
Benzyl ester ring inner-acid anhydride, 25 DEG C are reacted 3 days, and precipitation removes DMSO in water, are dried to obtain in 3 groups of sericins and benzyl glutamate ring
The different sericin of acid anhydrides mass ratio-poly benzyl glutamate polymer (Sericine-PBLG).
In Fig. 1 infrared spectrogram, the visible special crest of benzyl glutamate graft of sericin, 1739cm-1 is paddy
The carbonylic stretching vibration absworption peak of propylhomoserin benzyl ester, 1654cm-1 is the carbonylic stretching vibration absworption peak (acyl of sericin secondary amide
Amine I peaks), 1538cm-1 is acid amides C-N-H flexural vibrations absworption peak on sericin (acid amides II peak), and 1162cm-1 is glutamic acid
The asymmetric stretching vibration characteristic absorption peaks of ester group C-O-C of benzyl ester, 750 and 696 is monosubstituted for the protection group on benzyl glutamate
The C-H out-of-plane bending stretching vibration absworption peaks of phenyl ring.
Embodiment 2
(1) by the sericin in 10mg embodiments 1-poly benzyl glutamate polymer (sericin and benzyl glutamate
Ring inner-acid anhydride mass ratio is 1：1) it is dissolved in 1mL DMSO, stirs 1h, going for 4mL is then instilled under magnetic stirring
Ion, mixed solution is fitted into the bag filter that molecular cut off is 3500, with deionized water dialysis 24h, dilution at 25 DEG C
To 10mL, filtered through 0.45 μm of syringe filter, remain detection.(i.e. Nile red is marked the micella of load Nile red
Sericine-PBLG nano-micelles) preparation method is similar, and difference is to dissolve sericin-poly benzyl glutamate polymerization
5 μ g Nile reds are added while thing.
(2) cell concentration is adjusted to 2 × 10 after stomach cancer cell (AGS) digestion is resuspended4/ L, per hole in 96 well culture plates
Addition nutrient solution volume is 200 μ L.After being separately added into the Sericine-PBLG carriers of various concentrations gradient after cell attachment (i.e.
Sericin carrier).24h after dosing, adds 20 μ L MTT working solutions (5g/L) in each group hole, continues 37 DEG C of incubation 2h.
Supernatant is sucked, dimethyl sulfoxide (DMSO) (DMSO) 200 μ L are added per hole, is put after plate shaker shakes to crystallization and be completely dissolved, enzyme mark
Instrument determines absorbance (A) value in each hole of 490nm wavelength.Experiment is repeated 3 times, and draws cytotoxicity curve, and then determines it
IC50 values.As a result it is as shown in Figure 2.
(3) culture stomach cancer cell (AGS) adds Nile red mark Sericine-PBLG nanometre glues to 70-80% density
Beam, is incubated after 24h altogether, the cytotoxicity of nano-micelle is detected using mtt assay, using flow cytomery AGS to nanometre glue
The intake ability of beam.As a result it is as shown in Figure 3.
In Fig. 2 MTT figures, ags cell can reach more than 70% survival rate under the micella effect of various concentrations,
This points out the micella to have high biological histocompatbility, so as to verify its safety low-poison characteristic.
In Fig. 3 streaming figure, compared with blank control group, Nile red marks the experiment of Sericine-PBLG nano-micelles
The visible PE of group cell ＞ 90% are positive, this prompting Sericine-PBLG micella can efficiently, rapidly enter in AGS.In Fig. 3
Nile red mark sericin refers to that Nile red marks Sericine-PBLG nano-micelles.
Embodiment 3
(1) by the sericin in 10mg embodiments 1-poly benzyl glutamate polymer (sericin and benzyl glutamate
Ring inner-acid anhydride mass ratio is 1：1), 5 μ g Nile reds, 1mg taxols are dissolved in 1mL DMSO, are stirred 1h, are then stirred in magnetic force
The lower deionization for being instilled 4mL is mixed, mixed solution is fitted into the bag filter that molecular cut off is 3500, spent at 25 DEG C
Ionized water dialysis 24h, is made sericin mating type paclitaxel nano medicine.10mL is diluted to, through 0.45 μm of pin type
Filter is filtered, and obtains Sericin-PBLG-PTX-Nile-red micellas, remains detection.
(2) stomach cancer cell (AGS) is cultivated to 70-80% density, plus Sericin-PBLG-PTX- in the burnt ware of copolymerization
Nile-red micellas, the accumulating capacity of micella in the cell is observed with Laser Scanning Confocal Microscope, inverted microscope；As a result such as Fig. 4 institutes
Show.
(3) culture stomach cancer cell (AGS) is divided into 2 groups, is separately added into Sericin-PBLG-PTX- to 70-80% density
The taxol of Nile-red micellas and same concentrations, is incubated after 24h altogether, and the cytotoxicity of nano-micelle is detected using mtt assay,
Utilize intake abilities of the flow cytomery AGS to nano-micelle.As a result it is as shown in Figure 5.
(4) culture stomach cancer cell (AGS) adds Sericin-PBLG-PTX-Nile-red glue to 70-80% density
Beam, 24h collects cell.After PBS is resuspended twice, 500 μ L Binding Buffer are added in the cell, 5 μ L are added thereafter
Annexin V-FITC are mixed, and after lucifuge, room temperature reaction 10min, flow cytomery apoptosis rate is used in 60min.
As a result it is as shown in Figure 6.
Fig. 4 is that confocal laser scanning microscope nano-micelle (arrow) enters intracellular process：Upper figure is that white light shows
Picture, figure below is that 488nm green glows excite imaging.In Fig. 4 cellular uptake figure, Laser Scanning Confocal Microscope and inverted fluorescence microscope result
It can be seen that：Sericin-PBLG-PTX-Nile-red micellas can efficiently enter cell, and part AGS stomach cancer cell forms are rounded (apoptosis
May), this prompting Sericin-PBLG-PTX-Nile-red micella can efficiently, to rapidly enter stomach cancer cell (AGS) interior, have
High biological histocompatbility and antitumor potential.
Fig. 5 refers to ags cell under conditions of identical PTX concentration, Sericin-PBLG-PTX-Nile-red micella groups
(black post), the cell survival rate with simple PTX groups (grey post).In Fig. 5 MTT figures, ags cell is in identical PTX concentration
Under the conditions of, Sericin-PBLG-PTX-Nile-red micella group cell survival rates are below PTX groups, and then point out taxol to exist
After micella parcel, antitumor action can be efficiently produced.
In Fig. 6 streaming figure, Sericin-PBLG-PTX-Nile-red micellas are added in ags cell and co-cultured, after 24h
Collect cell row flow cytometer detection：Blank control group apoptosis rate is 7.38%, and experimental group apoptosis rate is 24.5%.With blank control group
Compare, the apoptosis rate of Sericin-PBLG-PTX-Nile-red micellas seminar is higher, point out Sericin-PBLG-
The high efficiency anti-tumor effect of PTX-Nile-red micellas.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. a kind of preparation method of sericin mating type paclitaxel nano medicine, it is characterised in that comprise the following steps：
(1) extraction of sericin：Silk cocoon boils degumming through aqueous slkali, is filtered to remove fibroin albumen and other insoluble matters, pure water
Dialysis removes ion salt, and freeze-drying obtains sericin；
(2) preparation of a-amino acid-N- carboxyanhydrides：Under inert gas shielding, amino acid monomer is dissolved in anhydrous tetrahydrochysene
In furans, add at the Triphosgene of 1.0-2.5 times of amino acid monomer quality, 45-60 DEG C and react 0.5-1.5h, be cooled to room
Temperature, vacuum distillation；Obtained oily molten thing is dissolved in ethyl acetate, is washed, separated organic with saturated sodium bicarbonate aqueous solution
Phase, is dried, and vacuum distillation obtains a-amino acid-N- carboxyanhydrides；
(3) preparation of the poly- peptide polymer of sericin-hydrophobicity：The sericin that step (1) is obtained is dissolved in organic solvent
Anhydrous dimethyl sulphoxide, adds a-amino acid-N- carboxyanhydrides prepared by step (2), reacts 2-4 days, precipitates and removes in water
Organic solvent is removed, the poly- peptide polymer of sericin-hydrophobicity is dried to obtain；
(4) preparation of sericin mating type paclitaxel nano medicine：The poly- peptide of sericin-hydrophobicity that step (3) is obtained
Polymer is dissolved in dimethyl sulfoxide (DMSO) and stirred, and adds Nile red and taxol drug is well mixed, then instill under agitation
In deionized water, finally mixed solution is fitted into bag filter, dialysed with deionized water, the sericin mating type is obtained purple
China fir alcohol Nano medication.
2. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In, the aqueous slkali described in step (1) is the one or more in sodium acid carbonate, sodium carbonate, potassium carbonate, potassium bicarbonate solution,
Concentration is 0.01-0.05mol/L；Described usually time is 0.5-1.5h；The cellulose dialysis bag that described dialysis is used is cut
It is 8000-14000Da to stay molecular weight.
3. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In the amino acid monomer described in step (2) is Pidolidone-r- benzyl esters, aspartic acid benzyl ester, phenylalanine and benzyloxycarbonyl group
One kind in lysine.
4. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In the drying described in step (2) refers to be dried overnight with anhydrous magnesium sulfate, and drier is then removed by filtration.
5. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In a-amino acid-N- carboxyanhydrides monomer and sericin quality ratio described in step (3) are 1：1-1：3.
6. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In the dialysis condition described in step (4) is：Bag filter for can molecular cut off be 3500KD specifications, dialysis temperature is 20-25
DEG C, dialysis time is 12-36h.
7. a kind of preparation method of sericin mating type paclitaxel nano medicine according to claim 1, its feature exists
In the mass ratio of the poly- peptide polymer of sericin-hydrophobicity, Nile red and taxol described in step (4) is 10mg：1-5μ
g：0.5-1mg.
8. a kind of sericin mating type paclitaxel nano medicine, it is characterised in that it is as described in any one of claim 1 to 7
A kind of sericin mating type paclitaxel nano medicine preparation method be made.
9. the sericin mating type paclitaxel nano medicine described in claim 8 is in neoplasm tracing and the application in treatment.
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