CN101716348B - Construction and application of gold-magnetic nanoparticle-based medicament carrying platform - Google Patents

Construction and application of gold-magnetic nanoparticle-based medicament carrying platform Download PDF

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CN101716348B
CN101716348B CN2009102323406A CN200910232340A CN101716348B CN 101716348 B CN101716348 B CN 101716348B CN 2009102323406 A CN2009102323406 A CN 2009102323406A CN 200910232340 A CN200910232340 A CN 200910232340A CN 101716348 B CN101716348 B CN 101716348B
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CN101716348A (en
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胥传来
徐乃丰
陈伟
徐丽广
朱颖越
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Jiangnan University
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Abstract

The invention relates to the construction and application of a gold-magnetic nanoparticle-based medicament carrying platform, which belong to the technical field of magnetic nanophase materials. The construction comprises the synthesis of magnetic nanoparticles, the synthesis of an Fe3O4-SiO2 nuclear shell structure, the amine surface modification of the Fe3O4-SiO2 nuclear shell structure, protonation and the electrostatic adherence of gold nanoparticles after the surface modification, the coupling of curcumin and the coupling of mercapto polyethylene glycol PEG-SH so as to prepare a multifunctional gold-magnetic nano-platform. The construction and the application have the following advantages that: the gold-magnetic nanoparticle-based medicament carrying platform is a magnetic carrier with a modified surface and has the characteristics of relatively stronger magnetism, good chemical inertness, biocompatibility, easy functionalization and the like, and can be prepared by a simple method; and the magnetic nanophase materials can be quickly separated with a magnetic field in a suspension system, are highly concentrated and act on tumor cells under the guidance of an external magnetic field, and can be applied in biological fields, such as bioseparation, medicament transportation, biological tags, cell imaging and developing and study on the separation of cells or other biomolecules.

Description

A kind of structure and application of the medicine carrying platform based on gold-magnetic nanoparticle
Technical field
A kind of structure and application of the medicine carrying platform based on gold-magnetic nanoparticle the invention belongs to field of nanometer material technology, more specifically, relate to be applied to the nano material of the transportation of life sciences Chinese medicine and oncotherapy.
Background technology
The biological function formed material is the popular domain of materialogy research; Nano material wherein since the eighties in last century because its special physicochemical character; Cause that scientist pays close attention to widely, thereby the significant problem of utilizing nanotechnology to study and solve field of biology also becomes one of the research field in forward position.
Superparamagnetic nano particle is with a wide range of applications at biomedical sectors such as bio-separation, immune detection, targeted drugs owing to its unique character (no remanent magnetism).But shortcomings such as himself chemical stability, colloidal stability and biocompatibility difference, its surface often needs to modify some polymer or silicon dioxide etc.Because having chemistry and advantages such as colloidal stability, good biocompatibility, silicon dioxide make it become one of ideal material of protecting magnetic particle.Be applicable to bio-separation behind the magnetic particle finishing silicon dioxide, but the ferrite of gained-silicon dixoide nucleocapsid structure diameter of particle distributes and check figure is all wayward, needs mature technique.
Golden nanometer particle not only has quantum size effect, skin effect, macro quanta tunnel effect, but also has unique electricity, optics and catalytic performance, and these characteristics are applicable to the sign of nano-particle behind cytophagy.
Curcumin is a kind of natural Phenolic Antioxidant of from zingiberaceous plant turmeric stem, extracting, and color and luster is stable and toxicity is very low, and can suppress the growth of kinds of tumor cells system in the body, and tumor is had the chemoprophylaxis effect.But because curcumin is water insoluble, therefore need a kind of carrier to strengthen its biocompatibility, and can be carried into corresponding tumor locus effectively and play effectiveness.The curcumin molecular structural formula
Figure G2009102323406D00011
The current technical problem that presses for solution provides a kind of core-shell type nano platform with surface biological functionalization of biocompatibility; To overcome extremely low dissolubility of simple medicine and very poor biocompatibility; Be difficult to effectively absorbed, can not bring into play the deficiency of anticancer function by organism.
Summary of the invention
The construction method that the purpose of this invention is to provide a kind of nanometer platform based on magnetic nano-particle and golden nanometer particle, and it is applied to transport cancer therapy drug, inducing tumor cell generation apoptosis, and then suppress its growth, reach the performance anticancer function.
Technical scheme of the present invention: a kind of construction method of the medicine carrying platform based on gold-magnetic nanoparticle comprises the synthetic of magnetic nano-particle, Fe 3O 4-SiO 2Nucleocapsid structure synthetic, Fe 3O 4-SiO 2The surface amino groups of nucleocapsid structure is modified, protonated and Electrostatic Absorption golden nanometer particle after the finishing, and coupling curcumin and coupling band sulfydryl Polyethylene Glycol PEG-SH make the multifunctional nano platform, and cell culture absorbs and characterizes; Processing step is:
(1) magnetic nano-particle is synthetic: with the synthetic magnetic nano-particle with superparamagnetism of coprecipitation;
(2) Fe 3O 4-SiO 2Synthesizing of nucleocapsid structure: the magnetic nano-particle that utilizes step (1) to obtain, according to reverse microemulsion process synthetic Fe in water in oil environment 3O 4-SiO 2Nucleocapsid structure;
(3) Fe 3O 4-SiO 2The surface amino groups of nucleocapsid structure is modified: with the resulting Fe of step (2) 3O 4-SiO 2Nucleocapsid structure is with its outer field silicon ball surface amination;
(4) protonated and Electrostatic Absorption golden nanometer particle after the finishing: it is protonated in the phosphate buffer of pH 6.0 that surface amino groups is modified the back; Products therefrom Electrostatic Absorption golden nanometer particle;
(5) coupling curcumin and coupling band sulfydryl Polyethylene Glycol PEG-SH: first conjugation coupling curcumin; Coupling PEG-SH forms stable golden sulfide linkage with golden nanometer particle again, makes the nanometer platform of gold-magnetic nanoparticle;
(6) cell culture absorbs and characterizes: characterize with the medicine carrying platform delivery entering cell of gold-magnetic nanoparticle and with multiple detection means:
Magnetic resonance video picture MRI characterize cells is engulfed the multifunctional nano platform;
Tetramethyl azo azoles MTT characterize cells vigor;
Flow cytometer FCM, the characteristic of laser confocal scanning microscope CLSM phenetic analysis tumor cell early apoptosis;
The cell electron microscopic section characterize the multifunctional nano platform by cytophagy after in cell the position.
One, magnetic nano-particle is synthetic:
1. take by weighing Fe respectively 3+, Fe 2+Be dissolved in the 80mL ultra-pure water, Fe 3+And Fe 2+Total concentration be 0.08mol/L, Fe 3+: Fe 2+Mol ratio 1-1.2: 2,80 ℃, 400r/min stirring reaction 10min;
2. dropwise add 5mL ammonia, and keep pH 10.0, dropwise afterreaction 30min;
3. ultrasonic mixing is washed 2～3 times;
4. magnetic separates dry powder.
Two, Fe 3O 4-SiO 2Synthesizing of nucleocapsid structure:
1. add TritonX, each 7mL of hexanol mixes ultrasonic about 20min to there not being oily nematic liquid;
2. the ultrasonic 45min of 28mL cyclohexane extraction pours in the 100mL three-neck flask, and 400r/min stirs 30min;
3. 4mg magnetic nano-particle MNP/2mLH 2O is scattered in the 2mL ammonia, and mixed liquor dropwise adds in the step three-neck flask 2., and rate of addition 50 μ L/min dropwise continued and stir 30min;
4. add the positive tetraethyl orthosilicate TEOS of 3mL, stir 24h;
5. add 8mL acetone, stop to stir, pour into and leave standstill 60min, precipitated and separated in the beaker;
6. the centrifugal 10min of 4000r/min, deposition adds dissolve with ethanol, and ultrasonic 10min is centrifugal again, repeats 6～8 times;
7. 55 ℃ of vacuum dryings are 7 hours;
8. cool off in the exsiccator, subsequent use.
Three, Fe 3O 4-SiO 2The surface amino groups of nucleocapsid structure is modified:
1. add 15mL glycerin and 25mL methanol, ultrasonic mixing in the 100mL tool plug conical flask;
2. add 60mg Fe 3O 4-SiO 2Continue ultra-sonic dispersion;
3. add 200 μ L ultra-pure waters, the ultrasonic homodisperse that makes;
4. add 2mL 3-aminopropyl triethoxysilane APTS, 60 ℃ of water-bath magnetic agitation 6h;
5. after reaction finishes, with absolute ethanol washing 2～3 times, vacuum drying.
Four, protonated after the finishing, the Electrostatic Absorption golden nanometer particle:
1. get above-mentioned amido modified magnetic microsphere 30mg and join among the phosphate buffer PB of 4mL 0.01mol/L pH6.0, amino cation is with on amido modified magnetic microsphere surface;
2. the 0.3mmol/L golden nanometer particle that adds 4mL behind the 5min;
3. 3000r/min is centrifugal behind the Electrostatic Absorption 20min, and ultraviolet characterizes, and gets the carrier of Electrostatic Absorption golden nanometer particle.
Five, conjugation coupling curcumin and band sulfydryl Polyethylene Glycol PEG-SH:
1. the carrier with above-mentioned Electrostatic Absorption golden nanometer particle is made into the suspension A that concentration is 6mg/mL with ultra-pure water, take by weighing curcumin be dissolved among the dimethyl sulfoxine DMSO B, concentration 2mg/mL;
2. it is centrifugal behind the reaction 15min A/B to be put together by 2: 1 volume ratios, and filter cake adds ultra-pure water and recovers volume, and ultraviolet characterizes;
3. add PEG-SH reaction 20min, centrifugal, filter cake adds ultra-pure water and recovers volume.
The application based on the medicine carrying platform of gold-magnetic nanoparticle with said method makes up is applied to biological field: the separate study of medicament transport, biomarker, cell imaging and development, cell or other biological molecule.
Beneficial effect of the present invention: the medicine carrying platform of gold-magnetic nanoparticle of the present invention is the magnetic carrier of finishing, has both had stronger magnetic, have characteristics such as good chemical inertness, biocompatibility and easy functionalization again, and method for preparing is simple.This magnetic Nano material can carry out sharp separation with magnetic field in suspension system; Under the guiding of externally-applied magnetic field, highly concentrate, act on tumor cell; Can be widely used in biological field, like the separate study of bio-separation, medicament transport, biomarker, cell imaging and development, cell or other biological molecule.
Description of drawings
Magnetic nano-particle Electrostatic Absorption golden nanometer particle after Fig. 1 modifies, coupling curcumin ultraviolet characterizes.
Fig. 2 cell culture and magnetic resonance video picture (MRI) characterize, and wherein the left side is experimental group: the multifunctional nano platform MNPS-Au-Curcumin-PEG that makes; The right side is a blank control group.
A B C D
Mean/SD 1164.9/434 1299.3/284 3042.5/1580 2391.8/1937
Fig. 3 flow cytometer detects apoptosis.
The specific embodiment
Embodiment 1
(1) coprecipitation synthesizing magnetic nanoparticle
1. take by weighing Fe respectively 3+, Fe 2+Be dissolved in the 80mL ultra-pure water, Fe 3+And Fe 2+Total concentration be 0.08mol/L, Fe 3+: Fe 2+Mol ratio 1-1.2: 2,80 ℃, 400r/min stirring reaction 10min;
2. dropwise add 5mL ammonia, and keep pH10.0, dropwise afterreaction 30min;
3. ultrasonic mixing is washed 2～3 times;
4. magnetic separates dry powder, makes magnetic nano-particle.
(2) SiO 2Coated magnetic nanoparticle
Utilize reverse microemulsion process, in water in oil environment, positive tetraethyl orthosilicate is dissolved into the silicon ball, magnetic nano-particle is wrapped up.
1. add TritonX, each 7mL of hexanol mixes ultrasonic about 20min to there not being oily nematic liquid;
2. the ultrasonic about 45min of 28mL cyclohexane extraction pours in the 100mL three-neck flask, 400r/min stir about 30min;
3. 4mg magnetic nano-particle (MNP)/2mLH 2O is scattered in the 2mL ammonia, and mixed liquor dropwise adds in the three-neck flask 2., and the about 50 μ L/min of rate of addition dropwise continued and stir 30min
4. add 3mL TEOS (positive tetraethyl orthosilicate) and stir 24h;
5. add 8mL acetone and stop simultaneously stirring, pour into and leave standstill 60min, precipitated and separated in the beaker;
6. the centrifugal 10min of 4000r/min, deposition adds dissolve with ethanol, and ultrasonic 10min is centrifugal again, repeats 6～8 times;
7. 55 ℃ of vacuum drying 7h;
8. cool off in the exsiccator, make the nanoparticle of nucleocapsid structure, subsequent use.
(3) surface amino groups is modified
1. add 15mL glycerin and 25mL methanol, ultrasonic mixing in the 100mL tool plug conical flask;
2. add 60mg MNP and continue ultra-sonic dispersion;
3. add 200 μ L ultra-pure waters, the ultrasonic homodisperse that makes;
4. add 2mL 3-aminopropyl triethoxysilane (APTS), 60 ℃ of water-bath magnetic agitation 6h;
5. after reaction finishes,, make the core-shell structure nanometer particle of surface amino groups modification with absolute ethanol washing 2～3 times, vacuum drying.
(4) protonated, the Electrostatic Absorption golden nanometer particle
1. the core-shell structure nanometer particle 30mg that gets the above-mentioned surface amino groups modification that makes joins among the PB of 4mL0.01mol/L pH6.0, and the silicon dioxide meter of amido modified core-shell nano is worn amino cation;
2. the 0.3mmol/L golden nanometer particle liquid that adds 4mL behind the 5min;
3. 3000r/min is centrifugal behind the Electrostatic Absorption 20min, and ultraviolet characterizes.
(5) conjugation coupling curcumin and PEG-SH
1. above-mentioned conjugate is made into the suspension A that concentration is 6mg/mL with ultra-pure water, take by weighing curcumin be dissolved in the dimethyl sulfoxine (DMSO) B, concentration 2mg/mL;
2. it is centrifugal behind the reaction 15min A/B to be put together by 2: 1 volume ratios, and filter cake adds ultra-pure water and recovers volume, and ultraviolet characterizes;
3. add PEG-SH (MW3000) reaction 20min, centrifugal, add ultra-pure water and recover volume, make the multifunctional nano platform.
Embodiment 2
(1) cell culture
After the leukaemia HL-60 recovery, to contain the IMDM culture fluid of 20% hyclone, 37 ℃, 5%CO 2Environment is cultivated down, gets into exponential phase of growth (logarithmic (log) phase) to cell.
(2) sign of multifunctional nano platform
1. (Magnetic Resonance Imaging, MRI) characterize cells is engulfed the multifunctional nano platform in magnetic resonance video picture
Siemens Avanto 1.5T magnetic resonance system is adopted in experiment.Cell culture is collected the logarithmic (log) phase cell, the experimental group cell with synthetic multifunctional nano platform action 24h after, collecting cell is regulated concentration of cell suspension 1 * 10 in 1.5mL PE pipe 6/ mL.Cellular control unit does not add the multifunctional nano platform, and all the other processing are identical.
2. tetramethyl azo azoles (MTT) characterize cells vigor
Collect the logarithmic (log) phase cell, regulate concentration of cell suspension 1 * 10 6/ mL; Get 100uL and join 96 orifice plates (edge hole is filled with the PBS of pH7.4); Zeroing hole (culture fluid, MTT, dimethyl sulfoxide) is set simultaneously, control wells (the medicine dissolution medium dimethyl sulfoxine DMSO of cell, same concentrations, culture fluid, MTT, DMSO), every settings 4 multiple holes.Put 37 ℃, 5%CO 2Hatch 48h, inverted microscope is observed down.Add synthetic multifunctional nano platform in the experimental group, establish 9 time gradients, set concentration 3.2mg/mL, 10uL is added in every hole.
Colour generation: after cultivating 36h, every hole adds 10uL MTT solution (5mg/mL, promptly 0.5%MTT prepares with pH7.4PBS), continues to cultivate 4h
Every hole adds the 100uL dimethyl sulfoxide, puts low-speed oscillation 10min on the shaking table, and crystal is fully dissolved.Enzyme-linked immunosorbent assay instrument OD450nm measure each hole light absorption value (Absorbance, Ab)
3. flow cytometer (FCM), the characteristic of laser confocal scanning microscope (CLSM) phenetic analysis tumor cell early apoptosis
Leukaemia HL60 is added after synthetic multifunctional nano platform carries out apoptotic stimulus, and the centrifugal 5min of 1000g abandons supernatant, collecting cell, pH7.4PBS re-suspended cell and counting; Get 50000～100000 resuspended cells, the centrifugal 5min of 1000g abandons supernatant, adds 195uL Annexin V-FITC and combines liquid (1X) re-suspended cell gently; Add 5uL Annexin V-FITC, gently mixing; Room temperature (20～25 ℃) lucifuge is hatched 10min; The centrifugal 5min of 1000g abandons supernatant, adds 190uL AnnexinV-FITC and combines liquid (1X) re-suspended cell gently; Add 10uL propidium iodide (PI), mixing gently, the ice bath lucifuge is carried out FCM and CLSM immediately and is detected
4. the cell electron microscopic section characterizes behind the multifunctional nano platform cytophagy position in the cell
100kV Hitachi-600 transmission electron microscope is adopted in experiment.Experiment is established three groups: blank control group; Experimental group 1: make apoptotic stimulus with curcumin merely, curcumin concentration 2mg/mL; Experimental group 2: make apoptotic stimulus, concentration 3.2mg/mL with synthetic multifunctional nano platform.36h behind the apoptotic stimulus collects the 2mL cell, with 2.5% glutaraldehyde solution adjustment concentration of cell suspension 1-1.2 * 10 7/ mL, fixedly 1h.After pH 7.4PBS washed twice, reuse 1% Osmic acid. is fixed.Alcoholic solution with gradient concentration carries out the cell sample dehydration, and the ethanol gradient concentration is: 50%, 70%, 90%, 95%, 100%.Then, sample soaks into 30min in the acetic acid isoamyl oxide, triplicate.After taking-up was dried, sample was cut into lamellar, carried out transmission electron microscope observing subsequently.

Claims (1)

1. the construction method based on the medicine carrying platform of gold-magnetic nanoparticle is characterized in that comprising the synthetic of magnetic nano-particle, Fe 3O 4-SiO 2Nucleocapsid structure synthetic, Fe 3O 4-SiO 2The surface amino groups of nucleocapsid structure is modified, protonated and Electrostatic Absorption golden nanometer particle after the finishing, and coupling curcumin and coupling band sulfydryl Polyethylene Glycol PEG-SH make the multifunctional nano platform, and cell culture absorbs and characterizes; Step is:
(1) magnetic nano-particle is synthetic: with the synthetic magnetic nano-particle with superparamagnetism of coprecipitation;
1. take by weighing Fe respectively 3+, Fe 2+Be dissolved in the 80mL ultra-pure water, Fe 3+And Fe 2+Total concentration be 0.08mol/L, Fe 3+: Fe 2+Mol ratio 1-1.2: 2,80 ℃, 400r/min stirring reaction 10min;
2. dropwise add 5mL ammonia, and keep pH 10.0, dropwise afterreaction 30min;
3. ultrasonic mixing is washed 2～3 times;
4. magnetic separates dry powder;
(2) Fe 3O 4-SiO 2Synthesizing of nucleocapsid structure: the magnetic nano-particle that utilizes step (1) to obtain, according to reverse microemulsion process synthetic Fe in water in oil environment 3O 4-SiO 2Nucleocapsid structure;
1. add TritonX, each 7mL of hexanol mixes ultrasonic 20min to there not being oily nematic liquid;
2. the ultrasonic 45min of 28mL cyclohexane extraction pours in the 100mL three-neck flask, and 400r/min stirs 30min;
3. 4mg magnetic nano-particle MNP/2mLH 2O is scattered in the 2mL ammonia, and mixed liquor dropwise adds in the step three-neck flask 2., and rate of addition 50 μ L/min dropwise continued and stir 30min;
4. add the positive tetraethyl orthosilicate TEOS of 3mL, stir 24h;
5. add 8mL acetone, stop to stir, pour into and leave standstill 60min, precipitated and separated in the beaker;
6. the centrifugal 10min of 4000r/min, deposition adds dissolve with ethanol, and ultrasonic 10min is centrifugal again, repeats 6～8 times;
7. 55 ℃ of vacuum dryings are 7 hours;
8. cool off in the exsiccator, subsequent use;
(3) Fe 3O 4-SiO 2The surface amino groups of nucleocapsid structure is modified: with the resulting Fe of step (2) 3O 4-SiO 2Nucleocapsid structure is with its outer field silicon ball surface amination;
1. add 15mL glycerin and 25mL methanol, ultrasonic mixing in the 100mL tool plug conical flask;
2. add 60mg Fe 3O 4-SiO 2Continue ultra-sonic dispersion;
3. add 200 μ L ultra-pure waters, the ultrasonic homodisperse that makes;
4. add 2mL 3-aminopropyl triethoxysilane APTS, 60 ℃ of water-bath magnetic agitation 6h;
5. after reaction finishes, with absolute ethanol washing 2～3 times, vacuum drying;
(4) protonated and Electrostatic Absorption golden nanometer particle after the finishing: it is protonated in the phosphate buffer of pH 6.0 that surface amino groups is modified the back; Products therefrom Electrostatic Absorption golden nanometer particle;
1. get above-mentioned amido modified magnetic microsphere 30mg and join among the phosphate buffer PB of 4mL 0.01mol/L pH6.0, amino cation is with on amido modified magnetic microsphere surface;
2. the 0.3mmol/L golden nanometer particle liquid that adds 4mL behind the 5min;
3. 3000r/min is centrifugal behind the Electrostatic Absorption 20min, and ultraviolet characterizes, and gets the carrier of Electrostatic Absorption golden nanometer particle;
(5) coupling curcumin and coupling band sulfydryl Polyethylene Glycol PEG-SH: first conjugation coupling curcumin; Coupling PEG-SH forms stable golden sulfide linkage with golden nanometer particle again, makes the nanometer platform of gold-magnetic nanoparticle;
Conjugation coupling curcumin and band sulfydryl Polyethylene Glycol PEG-SH:
1. the carrier with above-mentioned Electrostatic Absorption golden nanometer particle is made into the suspension A that concentration is 6mg/mL with ultra-pure water, take by weighing curcumin be dissolved among the dimethyl sulfoxine DMSO B, concentration 2mg/mL;
2. it is centrifugal behind the reaction 15min A/B to be put together by 2: 1 volume ratios, and filter cake adds ultra-pure water and recovers volume, and ultraviolet characterizes;
3. add PEG-SH reaction 20min, centrifugal, filter cake adds ultra-pure water and recovers volume;
(6) cell culture absorbs and characterizes: characterize with the medicine carrying platform delivery entering cell of gold-magnetic nanoparticle and with multiple detection means:
Magnetic resonance video picture MRI characterize cells is engulfed the multifunctional nano platform;
Tetramethyl azo azoles MTT characterize cells vigor;
Flow cytometer FCM, the characteristic of laser confocal scanning microscope CLSM phenetic analysis tumor cell early apoptosis;
The cell electron microscopic section characterize the multifunctional nano platform by cytophagy after in cell the position.
CN2009102323406A 2009-12-07 2009-12-07 Construction and application of gold-magnetic nanoparticle-based medicament carrying platform Expired - Fee Related CN101716348B (en)

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