CN108926567A - Nano medication and preparation based on fibroin albumen for breast cancer targeting combined chemotherapy - Google Patents
Nano medication and preparation based on fibroin albumen for breast cancer targeting combined chemotherapy Download PDFInfo
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- CN108926567A CN108926567A CN201810941953.6A CN201810941953A CN108926567A CN 108926567 A CN108926567 A CN 108926567A CN 201810941953 A CN201810941953 A CN 201810941953A CN 108926567 A CN108926567 A CN 108926567A
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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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
- A61K31/12—Ketones
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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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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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
- A61K47/42—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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses the Nano medication and the preparations that target combined chemotherapy for breast cancer based on fibroin albumen.Using regenerated silk fibroin as carrier, the Nano medication of curcumin (CUR) and 5-Fluorouracil (5-FU) is prepared while is loaded with solvent evaporation method, and in its surface modification targeted molecular hyaluronic acid (HA).The nanoparticle has lesser hydration partial size (198.0 ± 1.8nm), preferable dimensional homogeneity (PDI:0.097), higher drugloading rate (CUR:2.1%；5-FU:1.7%), good dispersion, not easy to reunite facilitates the multi-functional load medicine fibroin albumen nanoparticle of building.The targeted nano drug based on fibroin that the present invention obtains can effectively inhibit tumor cell proliferation, improve the concentration of anticancer drug in the cell.The hyaluronic acid (HA) of surface modification, enhances single medicine to the therapeutic effect of breast cancer, realizes synergistic effect in cancer treatment.
Description
Technical field
The invention belongs to nano-medicament carrier technical fields, and in particular to a kind of two kinds of anti-tumor drugs of load based on fibroin
For breast cancer targeting combined chemotherapy Nano medication preparation method, characterization and application.
Background technique
It is generally known that cancer has been acknowledged as one of most challenging and destructive disease in the world at present, account for complete
The 13% of all death tolls of ball.More seriously, Cancer in China disease incidence and human mortality continue on sharply in recent years
It rises, National Cancer Center is analyzed according to case data in 2014 and issues newest " Chinese Incidence and Study on mortality
Report " display, national de novo malignancy case about 380.4 ten thousand in 2014, and death is 229.6 ten thousand.China point
Do not account for about the 21.8% and 27% of global malignant tumour new cases and death.Clinical at present various treatment methods (such as hand
Art, radiotherapy and immunotherapy) it can be used for treatment of cancer, but chemotherapy is most common strategy (1. Sud, A.;
Kinnersley, B.; Houlston, R. S. Genome-wide association studies of cancer:
current insights and future perspectives. Nat. Rev. Cancer 2017, 17 (11),
692-4.; 2.Yoo, W.; Yoo, D.; Hong, E.; Jung, E.; Go, Y.; Singh, S. V. B.;
Khang, G.; Lee, D. Acid-activatable oxidative stress-inducing polysaccharide
nanoparticles for anticancer therapy. J. Control. Release 2018, 269, 235-44.;
3. Xiao, B.; Zhang, M. Z.; Viennois, E.; Zhang, Y. C.; Wei, N.; Baker, M. T.;
Jung, Y. J.; Merlin, D. Inhibition of MDR1 gene expression and enhancing
cellular uptake for effective colon cancer treatment using dual-surface-
Functionalized nanoparticles. Biomaterials 2015,48,147-60.).
The multidrug resistance (Multidrug resistance, MDR) of tumour is maximum obstacle in chemotherapy at present, is swollen
Oncocyte not only generates drug resistance to the chemotherapeutics, also to other function and structure after the phase of generation contacts with chemotherapeutics
Different drugs generates intercrossing drug resistance.Clinically, about half of patients undergoing chemotherapy generates drug resistance to chemotherapeutics completely
Property, or it is effective to chemotherapeutics in a short time, it generates drug resistance again immediately, then brings extreme difficulties to chemotherapy.(1.
Dylla SJ; Beviglia L; Park IK; Chartier C; Raval J; Ngan L; Pickell K;
Aguilar J; Lazetic S; Smith-Berdan S; Clarke MF; Hoey T; Lewicki J; Gurney
AL. Correction: Colorectal Cancer Stem Cells Are Enriched in Xenogeneic
Tumors Following Chemotherapy[J]. Plos One, 2008, 3(6):e2428.; 2. Dean M,
Fojo T, Bates S. Dean M, Fojo T, Bates S. Tumor stem cells and drug
resistance. Nat Rev Cancer 5: 275-284[J]. Nature Reviews Cancer, 2005, 5(4):
275-284.; 3. Tian W, Liu J, Guo Y, et al. Self-assembled micelles of
amphiphilic PEGylated rapamycin for loading paclitaxel and resisting
multidrug resistant cancer cells[J]. J Mater Chem B Mater Biol Med, 2015, 3
(7): 1204-1207.).In order to overcome these problems, clinician is using the combined chemotherapy side based on combining a variety of drugs
Case, this is a kind of effective way for capturing tumour multidrug resistance.By adjusting the dosage of every kind of drug, a variety of drugs are given simultaneously
Medicine can not only delay the adaptation process and relevant tumor cell mutations, reduction side effects of pharmaceutical drugs of cancer, moreover it is possible to reach association
Same therapeutic effect.
Curcumin (Curcumin, CUR) is a kind of plant polyphenol, has anti-oxidant, anti-inflammatory, pre- preventing tumor and anticancer
Ability.Due to its unique property, cause more and more to pay close attention in field of cancer treatment.It can inhibit a variety of and send out with tumour
Relevant cell pathway is opened up in hair tonic；Reciprocation can be generated by molecule inside and outside various kinds of cell relevant with tumorigenesis, thus
Inhibit the generation and development of tumour.In addition, curcumin is smaller to the side effect of human body, in clinical test, safe dose can
Up to 8000 mg/ days.Curcumin is a kind of Chinese medicine chemotherapy resistance reversal agent, and action target spot is extensive, can be mentioned through a variety of ways
Rise sensibility of the mdr cell to drug, the multidrug resistance of reversing tumor.5-Fluorouracil (5-Fluorouracil, 5-FU)
It is first according to certain antimetabolite imagined and synthesized, and is clinically most widely used anti-miazines drug at present,
There is good efficacy to digestive system cancer and other solid tumors, is occupied an important position in Internal Medicine-Oncology treatment.Therefore, comprehensively consider
The different antitumor actions of CUR and 5-FU, it is new that the combination of both drugs may provide a kind of available chemotherapy for treatment of cancer
Dosage form.
Currently, Nano medication delivering field is more and more paid close attention to by scientific circles, since it can be by nano-carrier pair
Drug is wrapped up and is controlled release, thus improve drug to the therapeutic effect of disease and reduce toxic side effect (Xiao B,
Zhang M, Viennois E, Zhang Y, Wei N, Baker MT, et al. Inhibition of MDR1 gene
expression and enhancing cellular uptake for effective colon cancer treatment
using dual-surface-functionalized nanoparticles. Biomaterials. 2015;48:147-
60.).Nano medication has been widely used in the research of the clinical treatment of disease as a result,.In addition, so far, largely
Polymer is used as pharmaceutical carrier, such as lipid, PLGA and dendrimers, but for polymer, and fibroin albumen is made
For a kind of natural biological macromolecular material of abundance, there is the good biocompatibility of biodegradable and easy to process
And the features such as chemical modification, have as drug delivery vehicle material potentiality (Mottaghitalab F, Farokhi M,
Shokrgozar MA, Atyabi F, Hosseinkhani H. Silk fibroin nanoparticle as a novel
drug delivery system. J. Controlled Release. 2015;206:161-76.).Although CUR and 5-FU
With lot of advantages, but because the defects of its oral administration biaavailability is low, poorly water-soluble limits their application.At us
Research in, fibroin albumen is proved that the hydrophily of hydrophobic type drug can be improved, and the encapsulation rate of material is improved, so studying
In, we select fibroin albumen as carrier material, it is expected to improve the hydrophilicity and anticancer effect of drug-loading system.
Summary of the invention
In view of this, one of the objects of the present invention is to provide target combined chemotherapy for breast cancer based on fibroin albumen
Nano medication, with good biocompatibility, stability, stimulating responsive can be used for the treatment of breast cancer, of the invention
The second purpose is to provide the preparation method of the above-mentioned Nano medication based on fibroin albumen for breast cancer targeting combined chemotherapy.
For achieving the above object, technical solution are as follows:
1. the Nano medication based on fibroin albumen for breast cancer targeting combined chemotherapy, which is characterized in that the Nano medication
Drug comprising two kinds of anti-tumor drugs, fibroin albumen and target tumor position, two kinds of anti-tumor drugs are curcumin
(Curcumin, CUR) and 5-Fluorouracil (5-Fluorouracil, 5-FU), the fibroin albumen are from fresh silk cocoon
The regenerated silk fibroin that extracts and in Nano medication as the carrier material with good biocompatibility, the target
It is hyaluronic acid (Hyaluronan acid, HA) to the drug of tumor locus.
The preparation method based on fibroin albumen for the Nano medication of breast cancer targeting combined chemotherapy described in 2., including
Following steps:
(1) silk cocoon is completely dissolved after weak caustic solution degumming with lithium-bromide solution, and regenerated silk is being obtained after deionized water dialysis
Fibroin；
(2) regenerated silk fibroin is dissolved in secondary water, room temperature aquation is stayed overnight, the regenerated silk fibroin after obtaining aquation；
(3) appropriate curcumin (Curcumin, CUR) and 5-Fluorouracil (5-Fluorouracil, 5-FU) are weighed, it is then complete
Fully dissolved in methyl alcohol, obtains the methanol solution dissolved with CUR and 5-FU；
(4) regenerated silk fibroin after aquation that step (2) obtains is added drop-wise to the dissolution that step (3) obtains under vortex conditions
In the methanol solution for having CUR and 5-FU, solution becomes cloudy into emulsion at this time, obtains the emulsion containing CUR and 5-FU；Described
The volume ratio of regenerated silk fibroin after aquation and the methanol solution dissolved with CUR and 5-FU is 1:5；
(5) 3 min of emulsion vortex containing CUR and 5-FU for obtaining step (4)；
(6) emulsion that step (5) obtains is put into ice bath, 1 min of Probe Ultrasonic Searching, 10 s of ultrasound at once, suspends 2 s；
(7) the emulsion rotary evaporation 30min for obtaining step (6), evaporates most of organic solvent methanol；
(8) emulsion that step (7) obtains is stirred into draught cupboard 3 h, organic solvent is made sufficiently to volatilize completely；
(9) supernatant will be collected after emulsion low-speed centrifugal that step (8) obtains；
(10) after the supernatant high speed centrifugation for obtaining step (9), liquid is discarded supernatant, collects particle；
(11) particle that step (10) are collected is resuspended in acetate buffer solution, hyaluronic acid (Hyaluronan is added
Acid, HA) decorating liquid, 2 h of reaction, then Probe Ultrasonic Searching 1 min/ times are stirred at room temperature, ultrasound 3 times, 10 s of ultrasound suspends 2 s,
Obtain the fibroin nanoparticle suspension of HA modification；
(12) supernatant, supernatant will be collected after the fibroin nanoparticle suspension low-speed centrifugal for the HA modification that step (11) obtains
1 min of Probe Ultrasonic Searching again, 10 s of ultrasound suspend 2 s；
(13) after the supernatant high speed centrifugation for obtaining step (12), liquid is discarded supernatant, collects particle；
(14) the secondary aqueous suspension of the particle for obtaining step (13), the trehalose (Trehalose) that 0.1 grams per milliliter is added are molten
Liquid, secondary water and aqueous trehalose volume ratio are 1:1；
(15) -20 DEG C are stored in after being freeze-dried the nanoparticle of step (14), is obtained based on fibroin albumen for breast cancer
Target the Nano medication of combined chemotherapy.
Further, in the step (1), lithium-bromide solution concentration is 9.5 M.
Further, in the step (3), CUR concentration is 0.5 milligram/milli in the methanol solution dissolved with CUR and 5-FU
It rises, 5-FU concentration is 0.5 mg/ml.
Further, in the step (6), (11) and step (12), the amplitude of Probe Ultrasonic Searching is 60%.
Further, in the step (9) and step (12), the rate of low-speed centrifugal is 6000 rpm, low-speed centrifugal when
Between be 10 min.
Further, in the step (10) and step (13), ultracentrifugal rate is 13000 rpm, ultracentrifugal
Time is 20 min.
Further, in the step (11), acetate buffer solution pH is 6.0.
Further, in the step (15), freeze-drying is to carry out in accordance with the following methods: in -80 DEG C of freeze overnight postpositions
24~36 h are lyophilized in freeze dryer.
3. the nanometer based on fibroin albumen for breast cancer targeting combined chemotherapy is prepared according to above-mentioned preparation method
Drug.
4. being answered with medicine of the targeting combined chemotherapy Nano medication based on fibroin being prepared in terms for the treatment of breast cancer
With.
Compared with the prior art, the present invention has the advantages that
1. the invention discloses a kind of Nano medication based on fibroin albumen for breast cancer targeting combined chemotherapy and its preparation sides
Method, the fibroin albumen Nano medication, can with excellent biocompatibility and biology as a kind of completely new anti-tumor drug
Degradability, drug slowly and can regulate and control from the release in carrier material, and the concentration of drug is stablized, and have good inhibition swollen
Tumor cell growth kills the effect of cancer cell.70% or more can reach to the inhibitory effect of 4-T1 breast cancer cancer cell.
2. the present invention guarantees the bio-safety of nano-carrier using the excellent natural silk of bio-compatible performance as raw material
Performance, and entire preparation method and preparation process are simple and fast, and silk is from a wealth of sources, nanoparticle short preparation period, yield
Height, it is easy to operate, it is environmentally friendly.
3. the present invention plays CUR and 5-FU by the way that fibroin albumen is prepared nanoparticle with solvent evaporation method self assembly
Protective effect, reduces its oxygenolysis before reaching lesions position, and it is dynamic to improve the medicine generation of Nano medication in vivo
Mechanics improves its half-life period in vivo, and nanoparticle is easier to be phagocytized by cells, and can preferably be transported to lesion portion
Position increases curative effect.
Detailed description of the invention
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to the present invention make into
The detailed description of one step, in which:
Fig. 1 is the Flied emission scanning electricity based on fibroin albumen for the Nano medication of breast cancer targeting combined chemotherapy of embodiment 1
Mirror figure.
Fig. 2 is the XRD spectra based on fibroin albumen for the Nano medication of breast cancer targeting combined chemotherapy of embodiment 1.
Fig. 3 be embodiment 1 based on fibroin albumen for breast cancer targeting combined chemotherapy Nano medication to breast cancer cancer
The rejection of cell.
Specific embodiment
Hereinafter reference will be made to the drawings, and a preferred embodiment of the present invention will be described in detail.
Embodiment 1
Embodiment 1 is Nano medication of the preparation based on fibroin albumen for breast cancer targeting combined chemotherapy, including following preparation step
It is rapid:
(1) silk cocoon is completely dissolved after weak caustic solution degumming with lithium-bromide solution, and regenerated silk is being obtained after deionized water dialysis
Fibroin；
(2) regenerated silk fibroin is dissolved in secondary water, room temperature aquation is stayed overnight, the regenerated silk fibroin after obtaining aquation；
(3) appropriate curcumin (Curcumin, CUR) and 5-Fluorouracil (5-Fluorouracil, 5-FU) are weighed, it is then complete
Fully dissolved in methyl alcohol, obtains the methanol solution dissolved with CUR and 5-FU；
(4) regenerated silk fibroin after aquation that step (2) obtains is added drop-wise to the dissolution that step (3) obtains under vortex conditions
In the methanol solution for having CUR and 5-FU, solution becomes cloudy into emulsion at this time, obtains the emulsion containing CUR and 5-FU；Described
The volume ratio of regenerated silk fibroin after aquation and the methanol solution dissolved with CUR and 5-FU is 1:5；
(5) 3 min of emulsion vortex containing CUR and 5-FU for obtaining step (4)；
(6) emulsion that step (5) obtains is put into ice bath, 1 min of Probe Ultrasonic Searching, 10 s of ultrasound at once, suspends 2 s；
(7) the emulsion rotary evaporation 30min for obtaining step (6), evaporates most of organic solvent methanol；
(8) emulsion that step (7) obtains is stirred into draught cupboard 3 h, organic solvent is made sufficiently to volatilize completely；
(9) supernatant will be collected after emulsion low-speed centrifugal that step (8) obtains；
(10) after the supernatant high speed centrifugation for obtaining step (9), liquid is discarded supernatant, collects particle；
(11) particle that step (10) are collected is resuspended in acetate buffer solution, hyaluronic acid (Hyaluronan is added
Acid, HA) decorating liquid, 2 h of reaction, then Probe Ultrasonic Searching 1 min/ times are stirred at room temperature, ultrasound 3 times, 10 s of ultrasound suspends 2 s,
Obtain the fibroin nanoparticle suspension of HA modification；
(12) supernatant, supernatant will be collected after the fibroin nanoparticle suspension low-speed centrifugal for the HA modification that step (11) obtains
1 min of Probe Ultrasonic Searching again, 10 s of ultrasound suspend 2 s；
(13) after the supernatant high speed centrifugation for obtaining step (12), liquid is discarded supernatant, collects particle；
(14) the secondary aqueous suspension of the particle for obtaining step (13), the trehalose (Trehalose) that 0.1 grams per milliliter is added are molten
Liquid, secondary water and aqueous trehalose volume ratio are 1:1；
(15) -20 DEG C are stored in after being freeze-dried the nanoparticle of step (14), obtains one kind based on fibroin albumen for cream
The Nano medication of gland cancer targeting combined chemotherapy.
Test 1: the pattern of nanoparticle is observed by field emission scanning electron microscope.First by freeze-drying based on fibroin
Nano medication of the albumen for breast cancer targeting combined chemotherapy is suspended in secondary water, and is diluted to concentration appropriate, water-bath
After the ultrasonic several seconds, by suspension take it is micro be added drop-wise to natural air drying on silicon wafer, before analysis, nanoparticle need to by vacuum spray platinum
Processing.
As shown in Figure 1, for the Flied emission scanning of the Nano medication of combined chemotherapy is targeted for breast cancer based on fibroin albumen
Electron microscope.Can be seen that nanoparticle in spherical from obtained electromicroscopic photograph, dense accumulation be presented, particle diameter distribution than more uniform,
This is also the obvious feature of fibroin albumen nanoparticle.
Test 2: interaction passes through XRD spectrum analysis between drug and carrier in the Nano medication based on fibroin.By appropriate sample
Product are placed on clean objective table, are put into instrument, and XRD spectrum is tested.At room temperature, using Cu target Ka ray (λ=
0.15418 nm), tube voltage is 40 kV, and tube current is 30 mA, and 2 θ of scanning angle is 10~80 °, and sweep speed is 5 °/point
Clock.
As shown in Fig. 2, being the XRD spectra of the heterogeneity Nano medication based on fibroin.(i) free CUR, (ii) are free
5-FU, (iii) pure silk fibroin SF-NPs, (iv) based on fibroin albumen for breast cancer targeting combined chemotherapy Nano medication
(HA-SF-CUR-5-FU-NPs).In order to further elucidate the interaction between drug and fibroin albumen carrier, we are studied
Their XRD spectra.CUR and 5-FU has sharp peak between 10~50 °, which reflects their highly crystalline property,
It is existing for crystal form.However, two kinds of drugs of load of pure fibroin albumen nanoparticle SF-NPs and HA targeting modification
Fibroin albumen nanoparticle HA-SF-CUR-5-FU-NPs is but without this characteristic peak, it may be possible to because of fibroin albumen and above-mentioned two
The intermolecular interaction of kind anticancer drug (CUR and 5-FU) complexity hinders the formation of crystal.Therefore, the clear earth's surface of our result
Bright drug molecule (CUR and 5-FU) is dispersed in carrier matrix in the form of molecule, exactly because amorphous drug can be with
Prevent Ostwald ripening phenomenon.
Test 3: inhibition of the fibroin albumen nanoparticle of two kinds of drugs to source of people breast cancer 4-T1 cell in-vitro growth is carried
Effect test:
Subject cell: source of people breast cancer 4-T1 cell；
Subject material: the fibroin albumen nanoparticle of two kinds of drugs is carried；
Experimental method: after the 4-T1 cell in logarithmic growth phase is digested with pancreatin, cell concentration is adjusted with complete medium
It is 1 × 105/ mL, 200 holes μ L/ are added in 96 orifice plates, in 37 degree, 5 % CO2Incubator in cultivate 12 h.It is incubated overnight
It (is observed under inverted microscope, the bottom 60%-70% of single hole covers with cell) afterwards and sucks culture medium, contain Ca with cold2+、Mg2+
PBS wash one time, drug is suspended in basal medium, be diluted to various concentration (drug concentration is set as 1.0 μM, 2.0
μM, 4.0 μM, 8.0 μM, 16.0 μM, 32.0 μM, 64.0 μM), the concentration of each drug set 5 groups it is parallel, every hole adds 200 μ
The drug suspension of L.Furthermore each orifice plate requires two groups of control groups of setting, and Negative Control group adds 200 bases μ L
Culture medium, Positive Control group, add 200 μ L with sterile water-reducible 1% Triton X-100.24 h are cultivated respectively
Or 48 h, contain Ca with cold2+、Mg2+ PBS is washed one time, and 100 μ L, 0.5 mg/mL MTT solution is added and continues to be protected from light 4 h of incubation
Afterwards, remove supernatant, each hole is separately added into 50 μ L DMSO, and oscillator shakes 150 rpm, 15 min, 570 nm of microplate reader measurement
OD value calculates separately the relative activity of tumour cell according to OD value.
As shown in figure 3, for Nano medication and the training of 4-T1 cell of combined chemotherapy are targeted for breast cancer based on fibroin albumen
Antitumor cell performance after supporting 12 h and 48 h.The results showed that targeting amalgamation for breast cancer based on fibroin albumen
The Nano medication for the treatment of has apparent inhibiting effect to humanized's breast cancer 4-T1 cell, and two kinds of drugs of CUR and 5-FU have preferable
Therapeutic effect, better than the therapeutic effect of single medicine.
Finally, it is stated that above embodiments are only used to illustrate technical solution of the present invention rather than limit, although passing through ginseng
According to the preferred embodiment of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can
To make various changes to it in the form and details, without departing from the present invention defined by the appended claims
Spirit and scope.
Claims (10)
1. the Nano medication based on fibroin albumen for breast cancer targeting combined chemotherapy, which is characterized in that the Nano medication
Drug comprising two kinds of anti-tumor drugs, fibroin albumen and target tumor position, two kinds of anti-tumor drugs are curcumin
(Curcumin, CUR) and 5-Fluorouracil (5-Fluorouracil, 5-FU), the fibroin albumen are from fresh silk cocoon
The regenerated silk fibroin that extracts and in Nano medication as the carrier material with good biocompatibility, the target
It is hyaluronic acid (Hyaluronan acid, HA) to the drug of tumor locus；Described is used for breast cancer target based on fibroin albumen
To the preparation method of the Nano medication of combined chemotherapy, comprising the following steps:
(1) silk cocoon is completely dissolved after weak caustic solution degumming with lithium-bromide solution, and regenerated silk is being obtained after deionized water dialysis
Fibroin；
(2) regenerated silk fibroin is dissolved in secondary water, room temperature aquation is stayed overnight, the regenerated silk fibroin after obtaining aquation；
(3) appropriate curcumin (Curcumin, CUR) and 5-Fluorouracil (5-Fluorouracil, 5-FU) are weighed, it is then complete
Fully dissolved in methyl alcohol, obtains the methanol solution dissolved with CUR and 5-FU；
(4) regenerated silk fibroin after aquation that step (2) obtains is added drop-wise to the dissolution that step (3) obtains under vortex conditions
In the methanol solution for having CUR and 5-FU, solution becomes cloudy into emulsion at this time, obtains the emulsion containing CUR and 5-FU；Described
The volume ratio of regenerated silk fibroin after aquation and the methanol solution dissolved with CUR and 5-FU is 1:5；
(5) 3 min of emulsion vortex containing CUR and 5-FU for obtaining step (4)；
(6) emulsion that step (5) obtains is put into ice bath, 1 min of Probe Ultrasonic Searching, 10 s of ultrasound at once, suspends 2 s；
(7) 30 min of emulsion rotary evaporation for obtaining step (6), evaporates most of organic solvent methanol；
(8) emulsion that step (7) obtains is stirred into draught cupboard 3 h, organic solvent is made sufficiently to volatilize completely；
(9) supernatant will be collected after emulsion low-speed centrifugal that step (8) obtains；
(10) after the supernatant high speed centrifugation for obtaining step (9), liquid is discarded supernatant, collects particle；
(11) particle that step (10) are collected is resuspended in acetate buffer solution, hyaluronic acid (Hyaluronan is added
Acid, HA) decorating liquid, 2 h of reaction, then Probe Ultrasonic Searching 1 min/ times are stirred at room temperature, ultrasound 3 times, 10 s of ultrasound suspends 2 s,
Obtain the fibroin nanoparticle suspension of HA modification；
(12) supernatant, supernatant will be collected after the fibroin nanoparticle suspension low-speed centrifugal for the HA modification that step (11) obtains
1 min of Probe Ultrasonic Searching again, 10 s of ultrasound suspend 2 s；
(13) after the supernatant high speed centrifugation for obtaining step (12), liquid is discarded supernatant, collects particle；
(14) the secondary aqueous suspension of the particle for obtaining step (13), the trehalose (Trehalose) that 0.1 grams per milliliter is added are molten
Liquid, secondary water and aqueous trehalose volume ratio are 1:1；
(15) -20 DEG C are stored in after being freeze-dried the nanoparticle of step (14), is obtained based on fibroin albumen for breast cancer
Target the Nano medication of combined chemotherapy.
2. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (1), lithium-bromide solution concentration is 9.5 M.
3. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (3), CUR concentration is 0.5 milligram/milli in the methanol solution dissolved with CUR and 5-FU
It rises, 5-FU concentration is 0.5 mg/ml.
4. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (6), (11) and step (12), the amplitude of Probe Ultrasonic Searching is 60%.
5. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (9) and step (12), the rate of low-speed centrifugal is 6000 rpm, low-speed centrifugal when
Between be 10 min.
6. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (10) and step (13), ultracentrifugal rate is 13000 rpm, when ultracentrifugal
Between be 20 min.
7. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (11), acetate buffer solution pH is 6.0.
8. the preparation side of the Nano medication according to claim 1 based on fibroin albumen for breast cancer targeting combined chemotherapy
Method, which is characterized in that in the step (15), freeze-drying is to carry out in accordance with the following methods: being placed in -80 DEG C of freeze overnights
24~36 h are lyophilized in freeze dryer.
9. preparation method according to claim 1-8 is prepared based on fibroin albumen for breast cancer targeting connection
The Nano medication that combination is treated.
10. doctor of the targeting combined chemotherapy Nano medication according to claim 9 based on fibroin in terms for the treatment of breast cancer
Learn application.
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