CN105457014A - Nanometer magnetic Fe3O4 particles compounded with hairtail tetrapeptide as well as preparation method and application of nanometer magnetic Fe3O4 particles - Google Patents

Nanometer magnetic Fe3O4 particles compounded with hairtail tetrapeptide as well as preparation method and application of nanometer magnetic Fe3O4 particles Download PDF

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CN105457014A
CN105457014A CN201510923310.5A CN201510923310A CN105457014A CN 105457014 A CN105457014 A CN 105457014A CN 201510923310 A CN201510923310 A CN 201510923310A CN 105457014 A CN105457014 A CN 105457014A
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tetrapeptide
magnetic
trichiurus haumela
granule
nano
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林慧敏
袁宁
邓尚贵
金图南
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Zhejiang Ocean University ZJOU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • 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
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds

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  • General Health & Medical Sciences (AREA)
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  • Inorganic Chemistry (AREA)
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Abstract

The invention discloses a preparation technology of nanometer magnetic Fe3O4 hairtail tetrapeptide. According to the preparation technology, magnetic Fe3O4 particles and hairtail tetrapeptide (with the amino acid sequence: Gln-Ala-Glu-Gly) are taken as the raw materials, and hairtail tetrapeptide is subjected to amination modification, and mixed with the magnetic Fe3O4 particles to be loaded to the surfaces of the magnetic Fe3O4 particles. The magnetic Fe3O4 particles are taken as the core, and loaded with hairtail tetrapeptide having the tumor proliferation inhabitation effect due to the surface amination modification, so that the magnetic nanometer particles have good water solubility, good dispersibility and good biocompatibility, and become ideal medicine supply carriers. The obtained product magnetic nanometer hairtail iron peptide particles have the superparamagnetism and the good dispersibility, and are suitable for being applied in a biological system. When an applied magnetic field achieves +/-10000Oe, the magnetization intensity of magnetic nanometer hairtail iron peptide achieves the saturation state, namely, 57.5emu.g<-1>.

Description

A kind of nano-magnetic Fe 3o 4compound Trichiurus haumela tetrapeptide granule and its preparation method and application
Technical field
The present invention relates to a kind of nano-magnetic Fe 3o 4compound Trichiurus haumela tetrapeptide granule and preparation method thereof, belongs to drug world.
Background technology
Nanotechnology (Nanotechnology) is the emerging technology of 21 century.Along with the fast development of nanotechnology, nano material because of the application of its peculiar property in the fields such as optics, magnetics, cosmetics, catalysis, pharmacy and medical science increasing, nano material is that the diagnosis and treatment of the various diseases comprising tumor provide brand-new solution route and reference value.And magnetic nanoparticle is widely used in the field such as medicament slow release, biomedicine because of the performance that its biocompatibility and stronger magnetic responsiveness etc. are excellent.In magnetic nanoparticle, due to Fe 3o 4there is hypotoxicity, superparamagnetism, preparation method is various, pattern is various and be easy to many advantages such as modification, make it be widely used in the biomedical sector such as nuclear magnetic resonance and drug release.
Summary of the invention
The technical problem to be solved in the present invention overcomes existing defect, provides a kind ofly to have superparamagnetism and the nano-magnetic Fe be evenly distributed 3o 4compound Trichiurus haumela tetrapeptide granule;
Another object of the present invention is to provide above-mentioned nano-magnetic Fe 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule;
Another object of the present invention is to provide above-mentioned nano-magnetic Fe 3o 4the application of compound Trichiurus haumela tetrapeptide granule.
Object of the present invention carrys out specific implementation by the following technical programs:
A kind of nano-magnetic Fe 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, with the Fe of magnetic 3o 4the raw material that granule and Trichiurus haumela tetrapeptide are, after the amination of Trichiurus haumela tetrapeptide is modified, then with the Fe of magnetic 3o 4granule mixes, and is carried on the Fe of magnetic 3o 4particle surface.
The aminoacid sequence of Trichiurus haumela tetrapeptide is: Gln-Ala-Glu-Gly.The encoding gene of Trichiurus haumela tetrapeptide is as shown in sequence in sequence table 1, and antibacterial Trichiurus haumela tetrapeptide is as shown in sequence in sequence table 2.
Preferably, above-mentioned nano-magnetic Fe 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, comprises the steps:
1) amination of Trichiurus haumela tetrapeptide is modified
Be dissolved in dehydrated alcohol by Trichiurus haumela tetrapeptide, obtain the tetrapeptide ethanol of surface amination, dilute with water is for subsequent use after obtaining tetrapeptide ethanol water;
2) coated
To step 1) instill Fe while stirring in the tetrapeptide ethanol water that obtains 3o 4in solution of magnetic nanoparticles, then after reacting 2h under magnetic stirring, purification.
Preferably, described step 1) in, Trichiurus haumela tetrapeptide: dehydrated alcohol is 5mg:0.07ml;
Preferably, described step 1) in, during dilution, the tetrapeptide ethanol that every 5mg Trichiurus haumela tetrapeptide is formed is diluted with water to 50ml.
Preferably, described step 2) in, described Fe 3o 4aqueous solution or the alcoholic solution of solution of magnetic nanoparticles to be concentration be 40-50wt%, further preferred concentration is the alcoholic solution of 45wt%.
Preferably, described step 2) in, described tetrapeptide alcoholic solution and Fe 3o 4proportionate relationship between solution of magnetic nanoparticles is: the corresponding 40mlFe of the tetrapeptide ethanol water that every 5mg Trichiurus haumela tetrapeptide is formed 3o 4solution of magnetic nanoparticles;
Preferably, described step 2) in, in 30min, by step 1) in the tetrapeptide ethanol water that formed of the 5mg Trichiurus haumela tetrapeptide that obtains dropwise be added dropwise to 40mlFe 3o 4in solution of magnetic nanoparticles.
Preferably, described step 2) in, the rotating speed of described magnetic agitation is 600rpm;
Preferably, described step 2) in, described purge process is: by reacting rear material with the centrifugal 40min of 3000rpm, outwell supernatant, add distilled water, ultrasonic disperse, repeats 3 times.
Nano-magnetic Fe prepared by said method 3o 4compound Trichiurus haumela tetrapeptide granule, with the magnetic Fe of tool 3o 4nano-particle is core, and Surface coating has Trichiurus haumela tetrapeptide, and grain diameter is 15-25nm, unbodied spherical in edge.
Preferably, described Trichiurus haumela tetrapeptide and Fe 3o 4the mass ratio of granule is 1:1.5.
Preferably, the methyl of described Trichiurus haumela tetrapeptide, methylene, ketone group and Fe 3o 4the combination of chemical bond is there is between magnetic nanoparticle.
A kind of nano-magnetic Fe 3o 4the application of compound Trichiurus haumela tetrapeptide granule, for the growth of anticancer;
Preferably, described nano-magnetic Fe 3o 4the application of compound Trichiurus haumela tetrapeptide granule, simultaneously with alternating magnetic field combination in cancerous cell;
Preferred further, described nano-magnetic Fe 3o 4compound Trichiurus haumela tetrapeptide granule uses pH scope to be 6.5 ~ 9.
The present invention is with the magnetic nanometer Fe of tool 3o 4granule is core, and load has the Trichiurus haumela tetrapeptide of Tumor suppression proliferation function after surface amination is modified, and makes this magnetic nanoparticle have good water solublity, dispersibility and biocompatibility, becomes more satisfactory confession drug carrier.Products obtained therefrom magnetic Nano Trichiurus haumela ferrum peptide particles has superparamagnetism, dispersibility is better, is suitable for applying in living things system.When externally-applied magnetic field reach ± 10000Oe time, the intensity of magnetization of magnetic Nano Trichiurus haumela ferrum peptide reaches capacity, and reaches 57.5emug -1.Specifically see the experimental analysis in following examples.
Accompanying drawing explanation
Fig. 1 is the AFM photo of Trichiurus haumela tetrapeptide;
Fig. 2 is the AFM photo of ferrum peptide microgranule (the concise and to the point statement of magnetic Nano Trichiurus haumela ferrum peptide of the present invention, as follows);
Fig. 3 is Fe 3o 4the hysteresis curve of nano-particle, ferrum peptide microgranule;
Fig. 4 is CW-2 collection of illustrative plates cell generation cycle (blank) of flow cytomery;
Fig. 5 is CW-2 collection of illustrative plates cell generation cycle under the effect of 0.2 μ g/mL Magnetic nano iron peptide under the non-magnetic field condition of flow cytomery;
Fig. 6 is CW-2 collection of illustrative plates cell generation cycle under the effect of 0.2 μ g/mL Magnetic nano iron peptide under the alternating magnetic field condition of flow cytomery.
Detailed description of the invention
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
Embodiment 1:
In order to essence of the present invention is described, applicant carried out following experiment.
1 experiment material and equipment
Trichiurus haumela tetrapeptide: Gln-Ala-Glu-Gly, purity is more than 95%, and the encoding gene of Gln-Ala-Glu-Gly is as shown in sequence in sequence table 1, and antibacterial Trichiurus haumela tetrapeptide is as shown in sequence in sequence table 2.；
Fe 3o 4magnetic nanoparticle: be purchased from Chongqing Wei Siteng company.
The preparation of 2 nanometer Trichiurus haumela ferrum peptide microgranules
The tetrapeptide getting 5mg is dissolved in the dehydrated alcohol of 0.07ml, obtains the tetrapeptide ethanol of surface amination, is diluted to 50ml and is placed in separatory funnel, be dropwise added dropwise to the 40mlFe of stirring in 30min 3o 4in solution of magnetic nanoparticles, be 600rpm stirring reaction 2h, finally with the centrifugal 40min of 3000rpm, outwell supernatant, add distilled water, ultrasonic disperse at magnetic agitation medium speed, repeat 3 times, the Nanoscale Iron peptide microparticulate of purification is in water, and 4 DEG C of cold preservations are for subsequent use.
The structural analysis of 3 nanometer Trichiurus haumela ferrum peptides
3.1 atomic force microscope test (AFM)
Tested height imaging (four-dimensional figure), phase image, the scanogram of every one deck by AFM, Fig. 1 is Trichiurus haumela tetrapeptide microgranule atomic force microscope test result, and Fig. 2 is Trichiurus haumela tetrapeptide and Fe 3o 4the atomic force microscope test result of mixed proportion when being 1:1.5, Trichiurus haumela tetrapeptide skewness as seen from the figure, granular sensation is comparatively strong, and sample is more coarse, and Trichiurus haumela tetrapeptide and Fe 3o 4microgranule is distribution uniform then, and very smooth, due to the gluing effect of tetrapeptide, and Fe 3o 4define reunion with tetrapeptide, clearly can observe Fe 3o 4about about the 15-25nm of-tetrapeptide nano-complex particle.
3.2 magnetic properties are measured
With vibrating specimen magnetometer (VSM) in magnetic field range-10kOe ~ 10kOe, under room temperature, determine the magnetic of sample.Fig. 3 is respectively Fe 3o 4the hysteresis curve of nano-particle, nanometer Trichiurus haumela ferrum peptide.As can be seen from the figure, the hysteresis curve of prepared sample is different from general magnetic material.Hysteresis curve normally two magnetization curves do not overlapped of general magnetic material, form a closed loop checking installation.Hysteresis curve shown in Fig. 3 is then two magnetization curves overlapped, and this is just embodying a key character of magnetic material: superparamagnetism.Along with the enhancing of externally-applied magnetic field, the intensity of magnetization of sample also grow thereupon.When externally-applied magnetic field reach ± 10000Oe time, the intensity of magnetization of magnetic Nano Trichiurus haumela ferrum peptide reaches capacity, and reaches 57.5emug -1.
The stability of 3.3 magnetic Nano Trichiurus haumela ferrum peptides
The magnetic Nano Trichiurus haumela ferrum peptide of this experiment preparation, under the condition of pH=6.5, deposits 3 time-of-weeks and does not produce any gathering; Adjustment pH to more than 9 time, a small amount of gathering can be produced.This is because the isoelectric point, IP of amino is at about pH=9, when alkalescence is crossed strong, can neutralize amino with positive charge; Change pH to less than 5 time, because nano-particle itself is made up of basic anhydride, therefore placement 2 days after, nano-particle can be dissolved.Therefore, the best of magnetic Nano Trichiurus haumela ferrum peptide particles uses pH scope to be 6.5 ~ 9, compares and is suitable for applying in living things system.
4 magnetic Nano Trichiurus haumela ferrum peptide antitumor action researchs
4.1 Nanoscale Iron peptides are on the impact of CW-2 cell proliferation
Table 1 Nanoscale Iron peptide is on the impact (x ± s) of CW-2 cell proliferation
* p<0.01 is compared with same time matched group
What table 1 showed is CW-2 cell through Magnetic nano iron peptide and or magnetic field process after the result that detects of MTT.As seen from the table, 0.1 and 0.2ug/mL concentration Magnetic nano iron peptide individual processing cell 24h observable impact is not caused on cell, the Magnetic nano iron peptide of respective concentration obviously can cause the growth inhibited (p<0.01) of cell in conjunction with alternating magnetic field processed group 24h, the prolongation in time of this depression effect significantly increases.0.4 and 0.8ug/mL concentration Magnetic nano iron peptide individual processing cell 24h activity capable of inhibiting cell, but the Magnetic nano iron peptide of respective concentration is compared in conjunction with alternating magnetic field processed group, inhibition rate of tumor cell significantly (p<0.01).Experimental result shows, and alternating magnetic field obviously can cause the growth inhibited of cell in conjunction with the combined effect of Magnetic nano iron peptide, and the synergy of the two demonstrates cooperative effect.4.2 impacts on CW-2 cell membrane fluidity
Table 2 magnetic Nano Trichiurus haumela ferrum peptide to CW-2 cell membrane P, the impact of η value
Note: * p<0.05 compared with same state matched group * * p<0.01 compared with same state matched group
4.3 impacts on CW-2 cell generation cycle
See Fig. 4-6, flow cytomery Magnetic nano iron peptide is on the impact of CW-2 cell cycle, under the process of alternating magnetic field and non-magnetic field condition, G0/G1 phase cell percentages drops to 45.25% (non-magnetic field) and 38.48% (alternating magnetic field) by 50.20% reducing gradually, the cell of S phase is under different disposal, there is no significant change, remain on about 40%, G2/M phase cells ratio and then present with concentration rising the trend risen gradually.Cell cycle generally experiences the G1-S-G2-M phase, and most of antitumorigenic substance has cell cycle specific, and it has two important check points, G1/S and G2/M.As can be seen from experimental result, G0/G1 phase cell proportion declines, G2/M phase cells ratio raises, this shows after Magnetic nano iron peptide is in conjunction with alternating magnetic field 60min process, CW-2 human colon cancer cell accumulates by being obstructed when entering mitotic phase post-synthetic phase, thus blocked cell and enter the next normal cell proliferation cycle, cause the Leukopenia being in the G0/G1 phase, cell death inducing.And the Leukopenia of Magnetic nano iron peptide individual processing cell G0/G1 phase is not obvious.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. a nano-magnetic Fe 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: with the Fe of magnetic 3o 4the raw material that granule and Trichiurus haumela tetrapeptide are, after the amination of Trichiurus haumela tetrapeptide is modified, then with the Fe of magnetic 3o 4granule mixes, and is carried on the Fe of magnetic 3o 4particle surface.
2. nano-magnetic Fe according to claim 1 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: comprise the steps:
1) amination of Trichiurus haumela tetrapeptide is modified
Be dissolved in dehydrated alcohol by Trichiurus haumela tetrapeptide, obtain the tetrapeptide ethanol of surface amination, dilute with water is for subsequent use after obtaining tetrapeptide ethanol water;
2) coated
To step 1) instill Fe while stirring in the tetrapeptide ethanol water that obtains 3o 4in solution of magnetic nanoparticles, then after reacting 2h under magnetic stirring, purification.
3. nano-magnetic Fe according to claim 2 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: described step 1) in, Trichiurus haumela tetrapeptide: dehydrated alcohol is 5mg:0.07ml;
And/or during dilution, the tetrapeptide ethanol that every 5mg Trichiurus haumela tetrapeptide is formed is diluted with water to 50ml.
4. nano-magnetic Fe according to claim 2 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: described step 2) in, described Fe 3o 4aqueous solution or the alcoholic solution of solution of magnetic nanoparticles to be concentration be 40-50wt%, preferred concentration is the alcoholic solution of 45wt%.
5. nano-magnetic Fe according to claim 2 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: described step 2) in, described tetrapeptide alcoholic solution and Fe 3o 4proportionate relationship between solution of magnetic nanoparticles is: the corresponding 40mlFe of the tetrapeptide ethanol water that every 5mg Trichiurus haumela tetrapeptide is formed 3o 4solution of magnetic nanoparticles;
And/or, in 30min, by step 1) in the tetrapeptide ethanol water that formed of the 5mg Trichiurus haumela tetrapeptide that obtains dropwise be added dropwise to 40mlFe 3o 4in solution of magnetic nanoparticles.
6. nano-magnetic Fe according to claim 2 3o 4the preparation method of compound Trichiurus haumela tetrapeptide granule, is characterized in that: described step 2) in,
The rotating speed of described magnetic agitation is 600rpm;
And/or described purge process is: by reacting rear material with the centrifugal 40min of 3000rpm, outwell supernatant, add distilled water, ultrasonic disperse, repeat 3 times.
7. the nano-magnetic Fe that according to any one of claim 1-6 prepared by method 3o 4compound Trichiurus haumela tetrapeptide granule, is characterized in that: with the magnetic Fe of tool 3o 4nano-particle is core, and Surface coating has Trichiurus haumela tetrapeptide, and grain diameter is 15-25nm, unbodied spherical in edge.
8. nano-magnetic Fe according to claim 7 3o 4compound Trichiurus haumela tetrapeptide granule, is characterized in that: described Trichiurus haumela tetrapeptide and Fe 3o 4the mass ratio of granule is 1:1.5.
9. the nano-magnetic Fe according to claim 7 or 8 3o 4compound Trichiurus haumela tetrapeptide granule, is characterized in that: the methyl of described Trichiurus haumela tetrapeptide, methylene, ketone group and Fe 3o 4the combination of chemical bond is there is between magnetic nanoparticle.
10. a nano-magnetic Fe 3o 4the application of compound Trichiurus haumela tetrapeptide granule, for the growth of anticancer;
Preferably, described nano-magnetic Fe 3o 4the application of compound Trichiurus haumela tetrapeptide granule, simultaneously with alternating magnetic field combination in cancerous cell;
Preferably, described nano-magnetic Fe 3o 4compound Trichiurus haumela tetrapeptide granule uses pH scope to be 6.5 ~ 9.
CN201510923310.5A 2015-12-11 2015-12-11 Nanometer magnetic Fe3O4 particles compounded with hairtail tetrapeptide as well as preparation method and application of nanometer magnetic Fe3O4 particles Pending CN105457014A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN107344731A (en) * 2017-07-07 2017-11-14 南京大学 A kind of preparation method of the water-soluble SPIO of individual layer cladding

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CN107344731A (en) * 2017-07-07 2017-11-14 南京大学 A kind of preparation method of the water-soluble SPIO of individual layer cladding
CN107344731B (en) * 2017-07-07 2019-06-21 南京大学 A kind of preparation method of the water-soluble Superparamagnetic Fe3O4 nanoparticles of single layer cladding

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