CN107375952A - The double relaxation platinum oxidation iron gold nano grains of T1/T2 and preparation method - Google Patents
The double relaxation platinum oxidation iron gold nano grains of T1/T2 and preparation method Download PDFInfo
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Abstract
The present invention discloses a kind of double relaxation platinum oxidation iron gold nano grain preparation methods of T1/T2, utilize the double relaxation platinum oxidation iron gold coupled nanosecond particles of the peptide modified T1/T2 for targetting CXCR4, Pt Fe3O4 Au nano-particles prepared by the present invention are by controlling the mol ratio of oleic acid and iron pentacarbonyl, the controlled syntheses of isomer can be realized, the stability of the double dumbbell structures of nano-particle can be improved, and realize the double enhancing mode of stable T1/T2, the CXCR4 Pt Fe3O4 Au heterotrimer nano-particles prepared on this basis have outstanding magnetic performance, good biocompatibility and the high targeting to triple negative breast cancer, new means are provided for early diagnosis triple negative breast cancer, available for preparation triple negative breast cancer targeted contrast agent.
Description
Technical field
The present invention disclose a kind of T1/T2 pair relaxation platinum-iron oxide-gold nano grain preparation methods, using targetting CXCR4's
The golden coupled nanosecond particle of the double relaxation platinum-iron oxide-of peptide modified T1/T2, belong to the technical field of contrast agent preparation.
Background technology
Breast cancer is one of most common malignant tumour of women, and serious threat female pathology is healthy.At present, to estrogen by
Body (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2（HER2）The diagnosis and treatment of positive breast cancer are
Through there is breakthrough progress, by targetting these acceptors, its specific diagnostic and individualized treatment can be achieved, and obtained good
Effect.But for lacking the triple negative breast cancer of hormone receptor and HER2 expression（TNBC）, because it lacks specific molecular mark
Will thing, then be difficult to Accurate Diagnosis in early days, causes its survival not high simultaneously because lacking specific treatment medicine.Before
Phase research discovery, C-X-C Chemokine receptor4s（CXCR4）There is higher expression and pre- with it in triple negative breast cancer
There is substantial connection afterwards.
At present, MRI possesses substantial worth in breast cancer is early diagnosed, but existing contrast agent is remained at some not
Foot, present contrast agent synthesis strategy products therefrom is mostly that the different isomers of same product are formed, and lacks orientation and closes
Into the scheme of single isomer product, while contrast agent lacks targeting, and only T1WI contrast medium used by clinic,
Lack T2WI contrast imaging, higher soft tissue contrast is lacked to the micro-structural of tumour, therefore to micro- calcium in breast cancer
Change and its pathological is difficult to make correct diagnosis, it is impossible to meet the clinical diagnosis demand to triple negative breast cancer.Therefore, at present
Active demand exploitation is a kind of to realize that isomer is controllable, while has T1/T2 enhancing effects, realizes high-affinity and Gao Min
The chemical synthesis strategy of the novel nano particle of the targeting CXCR4 acceptors imaging of perception, to realize the morning to triple negative breast cancer
Phase Precise Diagnosis.
The content of the invention：
The invention provides a kind of golden coupled nanosecond particle of the double relaxation platinum-iron oxide-of T1/T2（Abbreviation DB-HNT）（Fig. 1）, pass through
Synthesize the golden coupled nanosecond particle of the double relaxation platinum-iron oxide-of T1/T2 of target CXCR4 peptide modified double dumbbell structures（Referred to as
CM-DB-HNT）It can be used for the targeting diagnosis of breast cancer, new thinking and foundation provided for early diagnosis triple negative breast cancer.
The present invention further discloses the golden coupled nanosecond of the double relaxation platinum-iron oxide-of controlled syntheses isomer T1/T2
The preparation method of grain, the double golden coupled nanosecond particles of relaxation platinum-iron oxide-of T1/T2 are modified using the polypeptide for targetting CXCR4
Particle.
A kind of preparation method of the double relaxation platinum-iron oxide-gold nano grains of T1/T2 of the present invention, it is characterised in that
Comprise the following steps：
1）Synthesize Pt nano particles：
Under nitrogen atmosphere, by 1- octadecylenes, oleic acid amine, oleic acid by volume 10:1:1-20:1:1 ratio is mixed；Add
Enter mass ratio for 1:1 divalence acetylacetone,2,4-pentanedione platinum and divalence acetyl oleyl amine；Heated under vacuum is to 100-120 DEG C, argon atmospher
Continue to be heated to 180-200 DEG C under enclosing, add iron pentacarbonyl solution（It is 1 with divalence acetylacetone,2,4-pentanedione mol ratio：4）Mixed
Liquid, 190-220 DEG C is heated to, reacts 1h；Room temperature is cooled to, is centrifuged, isopropyl alcohol cleans to obtain nano particle, n-hexane solvent two
Secondary purifying, ethanol cleaning；
2）The double dumbbell structure nano particles of Pt-Fe3O4 in synthetic iron oxide ion blocking group closing Pt areas：
1- octadecylenes and oleic acid are pressed 20：1-40：1 volume ratio is well mixed, and under the conditions of 90-120 DEG C, is persistently taken off with nitrogen stream
Gas 20-30 minutes；Under nitrogen atmosphere, iron pentacarbonyl is added（Oleic acid/the mol ratio without carbonyl iron：1.5/1-3/1）, 10-
After 20min, injection mol ratio is 1:1-2:1 oleic acid amine and Pt nano particle seeds, resulting solution is under the conditions of 250-300 DEG C
15-20min is reacted, is cooled to room temperature；Ethanol and n-hexane purifies and separates obtain nano particle；
3）Controlled syntheses isomer Pt-Fe3O4-Au heterotrimer nano particles：
1- octadecylenes and oleyl amine are pressed 20:1-20：3 volume ratio is well mixed, and is added gold chloride stirring, is heated up to 60-100
℃；Under nitrogen protection, step 2 is added）Platinum-ferric oxide nanometer particle of preparation（It is 1 with gold chloride mol ratio:1-1:2）, heating
1.5-2.5 hours, it is cooled to room temperature；Purified and cleaned using isopropanol, obtain Pt-Fe3O4-Au nano-particles.
A kind of platinum-iron oxide-golden coupled nanosecond particle system of the double relaxation CXCR4 monoclonal antibodies modifications of T1/T2 provided by the invention
Preparation Method, specific steps include：
1）By Pt-Fe3O4-Au nano-particle water solubility carboxyl-functionals：
Pt-Fe3O4-Au nano-particles prepared by the present invention are dissolved in chloroform；By mass ratio 1:1 dopamine D MF solution, nothing
Aqueous sodium carbonate mixes, and adds DB-HNT chloroformic solutions（With dopamine D MF liquor capacities than 1:1-1:2）Stir at room temperature；
It is 1 by mass ratio:2:25-1:2:50 EDAC, NHS and the nitrine polyethylene glycol chloroformic solution of carboxyl-functional mix equal
It is even, it is added to Pt-Fe3O4-Au nano-particle chloroformic solutions；It is dry under nitrogen stream to be dissolved in after n-hexane purifying washes twice
In water, centrifuge after being filtered by micron membrane filter, be dissolved in the water after thickening and washing, add mass ratio 1:20:2-1:20:4 sulphur
The alkynyl PEG of sour copper, ascorbic acid and amino functional, after stirring 2h, centrifuge, be dissolved in sodium bicarbonate buffer liquid after washing concentrating
In, it is 1 to add with the alkynyl PEG mass ratioes of ascorbic acid and amino functional:1 succinimide ester-gadolinium-DOTA, stirring 2
After hour, centrifugation, washing concentrating, the Pt-Fe3O4-Au nano-particles of water-soluble carboxyl-functional are obtained, it is standby；
2）Prepare targeting CXCR4-Pt-Fe3O4-Au heterotrimer nano-particles：
CXCR4 targeting peptides by EDC and NHS activation after obtain active ester, then in aqueous phase with step 1）The water solubility of preparation
The amino of the Pt-Fe3O4-Au nanoparticle surfaces of carboxyl-functional is attached, and is carried out after completion of the reaction with PD-10 gel columns
Purifying, obtains targeting CXCR4-Pt-Fe3O4-Au heterotrimer nano-particles.
Pt-Fe3O4-Au nano-particles of the present invention purposes in the double enhancing contrast agent of T1/T2 are prepared.
CXCR4- Pt-Fe3O4-Au heterotrimers nano-particle of the present invention is preparing triple negative breast cancer targeting
Purposes in contrast agent.
The positive effect of the present invention is：
The method for providing controlled syntheses isomer and CXCR4- Pt-Fe3O4-Au heterotrimer nano-particles, the present invention
Prepared Pt-Fe3O4-Au nano-particles are by controlling the mol ratio of oleic acid and iron pentacarbonyl, it is possible to achieve isomer
Controlled syntheses, it is possible to increase the stability of the double dumbbell structures of nano-particle, and realize the double enhancing mode of stable T1/T2,
The CXCR4- Pt-Fe3O4-Au heterotrimer nano-particles prepared on the basis of this have outstanding magnetic performance, good biology
Compatibility and the high targeting to triple negative breast cancer, new means are provided for early diagnosis triple negative breast cancer, can be used for
Prepare triple negative breast cancer targeted contrast agent.
Brief description of the drawings
Fig. 1 is CM-DB-HNT schematic diagrames of the present invention；
Fig. 2 is CM-DB-HNT transmission electron microscope images of the present invention；
Fig. 3 nano particles are evenly distributed, and particle diameter is 12.45 ± 1.21nm；
Fig. 4 is ZETA potentials of the present invention and hydration particle diameter test result：DB-HNT nano grain surfaces potential and hydration particle diameter point
Wei not -11.8mV ± 1.25mV, 20nm ± 0.98nm；
Fig. 5 tests for magnetic performance of the present invention：Relaxation rate r1 values are respectively 61.67 ± 0.82mM with relaxation rate r2 values-1s-1、
181.49±1.57 mM-1s-1；
Fig. 6 is CXCR4 target polypeptides Mass Spectrometric Identification result of the present invention；
Fig. 7 is external T1 and T2 imaging tests of the various concentrations C-DB-HNT in 96 orifice plates of the present invention（Nano particle is dense with Fe
Spend for standard）；
Fig. 8 tests for In vitro cell experiment phase contrast microscope of the present invention：The CM-DB-HNT nano particles processing 24 of various concentrations is small
When after cellular morphology PBS processing cell compare, do not distinguish significantly；
Fig. 9 tests for prussian blue staining of the present invention：CM-DB-HNT nano particles are substantially swallowed by human breast cancer cells,
And as phagocytosis quantity is as the concentration (0.05-0.8mg/L) of CM-DB-HNT nano particles increases and increases；
Figure 10 is perspective electron microscope of the present invention（TEM）Test：CM-DB-HNT nano particles are phagocytized by cells into cell
In matter.
Embodiment
By following examples further illustrate description the present invention, do not limit the invention in any way, without departing substantially from
On the premise of the technical solution of the present invention, what those of ordinary skill in the art made for the present invention easily realized any changes
Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1
1st, under nitrogen atmosphere, in the three-neck flask of 100ml volumes by magnetic agitation by 1- octadecylenes（20ml）, oleic acid amine
（20ml）, oleic acid（2ml）And divalence acetylacetone,2,4-pentanedione platinum（0.2g）Preparation is in mixed solution, by divalence acetyl oleyl amine（0.2g）Add
Wherein.Solution is heated to 120 DEG C under vacuum, then continues to be heated to 180 DEG C under argon atmosphere, in this condition
Under, add 0.2ml iron pentacarbonyl solution（Concentration 0.1Fe (CO)5/ 1ml n-hexanes）.Obtained mixed liquor is heated to 190 DEG C,
1h is reacted on this condition.After being cooled to room temperature, cleaned by centrifugation and isopropyl alcohol, obtain nano particle.Then, will
The particle of acquisition is dispensed into n-hexane solvent again, is then centrifuged for separating nano-particles, and cleaned with ethanol.Product is suspended in
In 4ml hexane solutions comprising 1% oleic acid amine and 1% oleic acid.
2nd, 1- octadecylenes（20ml）And oleic acid（1ml）It is sufficiently stirred after mixing, gained mixed liquor is used under the conditions of 120 DEG C
Nitrogen stream persistently deaerates 30 minutes.Under nitrogen atmosphere, iron pentacarbonyl is added（0.14ml, 1mmol）, after 10min, in this thermosol
Oleic acid amine is injected in liquid（97%, Acros）（1ml）With step 1）Gained Pt nano particle seeds（20mg, be dispersed in 2ml just oneself
In alkane solution）, resulting solution reacts 20min under the conditions of 300 DEG C, removes heating mantles afterwards and is cooled to room temperature.By the pure second of 60ml
Alcohol is added to above-mentioned resulting solution and centrifuged, and obtains nanoparticle precipitate.By gained nano particle be dispensed into again just oneself
Alkane, nanoparticle precipitate is obtained by adding ethanol and centrifugation.This process is repeated 2 times with purified nanotubes particle.Final gained is received
Rice grain is dispensed into 10ml hexane solutions.
3rd, 1- octadecylenes are added in 20ml vial（10ml）And oleyl amine（0.5ml）, in strength after fully mixing
By gold chloride under magnetic stirring（40mg）Add, temperature is risen to 60 DEG C after stirring 1min.Then under nitrogen protection by before 1ml
Synthetic platinum-ferric oxide nanometer particle is rapidly added in above-mentioned solution and heated 2.5 hours, after obtaining purplish grey colloidal solution,
Mixture is cooled to room temperature.Then 40ml isopropanols are added and are centrifuged（5000 turns, 5 minutes）Collect nano particle, so with
Isopropanol washes the DB-HNT for being afterwards dissolved in nano particle 10mg/ml is obtained in chloroform twice.
4th, 1ml dopamines（2mg）DMF solution and 2mg natrium carbonicum calcinatums, above-mentioned preparation is rapidly added in the case where being sufficiently stirred
In DB-HNT chloroformic solutions（1ml）And continue to stir 2 hours at room temperature.Then by 1mg EDAC, 2mgNHS and 2ml (50mg)
The nitrine polyethylene glycol of carboxyl-functional（Azide-PEG-5000-COOH）Chloroformic solution adds after being stirred at room temperature 30 minutes
To above-mentioned nano particle chloroformic solution, continue stirring 24 hours at room temperature.Then it is same to add 15ml n-hexanes change solution polarity
When centrifuge to separate out nano particle, collect nano particle.After being washed twice again with n-hexane, then dry under nitrogen flowing, and
It is dissolved in water.After nano particle aqueous solution is filtered by 0.22 micron membrane filter, the centrifugation of 30kDa super filter tubes is added, concentration is washed
It is dissolved in the water after washing, adds the alkynyl PEG of 1mg copper sulphate, 20mg ascorbic acid and 2mg amino functionals（ALK-PEG2-
NH2）, after 2h is stirred at room temperature, 30kDa super filter tubes centrifuge, and are dissolved in sodium bicarbonate buffer liquid after washing concentrating（pH8.5）In, add
1mg succinimide esters-gadolinium-DOTA（NHS-Gd-DOTA）, after being stirred at room temperature 2 hours, the centrifugation of 30kDa super filter tubes, wash
It is standby to wash concentration.
5th, by 50ulCXCR4（Concentration 0.5mg/ml）Monoclonal antibody is dissolved in 100mlPH6.8 PBS cushioning liquid, is added
30mg NHS and 200mg EDC, activate 30min clocks.Then 4 are added）Products therefrom, at room temperature PD-10 gel columns purifying 4h,
The CM-DB-HNT of CXCR4 modifications is made.
Embodiment 2：
1st, under nitrogen atmosphere, in the three-neck flask of 100ml volumes by magnetic agitation by 1- octadecylenes（40ml）, oleic acid amine
（20ml）, oleic acid（2ml）And divalence acetylacetone,2,4-pentanedione platinum（0.2g）Preparation is in mixed solution, by divalence acetyl oleyl amine（200mg）Add
Enter wherein.Solution is heated to 120 DEG C under vacuum, then continues to be heated to 190 DEG C under argon atmosphere, in this condition
Under, add 0.2ml iron pentacarbonyl solution（Concentration 0.1Fe (CO)5/ 1ml n-hexanes）.Obtained mixed liquor is heated to 160 DEG C,
1h is reacted on this condition.After being cooled to room temperature, cleaned by centrifugation and isopropyl alcohol, obtain nano particle.Then, will
The particle of acquisition is dispensed into n-hexane solvent again, is then centrifuged for separating nano-particles, and cleaned with ethanol.Product is suspended in
In 4ml hexane solutions comprising 1% oleic acid amine and 1% oleic acid.
2nd, 1- octadecylenes（20ml）And oleic acid（0.5ml）Be sufficiently stirred after mixing, gained mixed liquor under the conditions of 120 DEG C,
Persistently deaerated with nitrogen stream 30 minutes.Under nitrogen atmosphere, iron pentacarbonyl is added（0.14ml, 1mmol）, it is hot herein after 10min
Oleic acid amine is injected in solution（97%, Acros）（1ml）With step 1）The nano particle seed of gained（20mg, it is being dispersed in 2ml just
In hexane solution）, resulting solution reacts 20min under the conditions of 300 DEG C, removes heating mantles afterwards and is cooled to room temperature.60ml is pure
Ethanol is added to above-mentioned resulting solution and centrifuged, and obtains nanoparticle precipitate.Gained nano particle is dispensed into just again
Hexane, nanoparticle precipitate is obtained by adding ethanol and centrifugation.This process is repeated 2 times with purified nanotubes particle.Final gained
Nano particle is dispensed into 10ml hexane solutions.
3rd, 1- octadecylenes are added in 20ml flask（5ml）And oleyl amine（1.5ml）, stirred after fully mixing in strong magnetic
It is lower to add 25mg gold chlorides, temperature is risen to 80 DEG C after stirring 1min.Then under nitrogen protection will be synthetic before 1ml
Platinum-ferric oxide nanometer particle is rapidly added in above-mentioned solution and heated 2 hours, after obtaining purplish grey colloidal solution, mixture is cold
But room temperature is arrived.Then 40ml isopropanols are added and are centrifuged（5000 turns, 5 minutes）Nano particle is collected, so washes two with isopropanol
Nano particle is dissolved in the DB-HNT that 10mg/ml is obtained in chloroform after secondary.
4th, 1ml dopamines（2mg）DMF solution and 2mg natrium carbonicum calcinatums, the above-mentioned systems of 1ml are rapidly added in the case where being sufficiently stirred
In standby DB-HNT chloroformic solutions and continue to stir 2 hours at room temperature.Then by 1mg EDAC, 2mgNHS and 2ml (50mg) carboxylic
The nitrine polyethylene glycol of base functionalization（Azide-PEG-5000-COOH）Chloroformic solution is added to after being stirred at room temperature 30 minutes
Above-mentioned nano particle chloroformic solution, continue stirring 24 hours at room temperature.Then 15ml n-hexanes are added and change solution polarity simultaneously
Centrifugation collects nano particle to separate out nano particle.After being washed twice again with n-hexane, then dry under nitrogen flowing, and it is molten
In Xie Shui.After nano particle aqueous solution is filtered by 0.22 micron membrane filter, the centrifugation of 30kDa super filter tubes, thickening and washing are added
After be dissolved in the water, add 1mg copper sulphate, the alkynyl PEG of 20mg ascorbic acid and 2mg amino functionals（ALK-PEG2-
NH2）, after 2h is stirred at room temperature, 30kDa super filter tubes centrifuge, and are dissolved in sodium bicarbonate buffer liquid after washing concentrating（pH8.5）In, add
2mg succinimide esters-gadolinium-DOTA（NHS-Gd-DOTA）, after being stirred at room temperature 2 hours, the centrifugation of 30kDa super filter tubes, wash
It is standby to wash concentration.
5th, by 50ulCXCR4（Concentration 0.5mg/ml）Monoclonal antibody is dissolved in 100mlPH7.2 PBS cushioning liquid, is added
30mg NHS and 200mg EDC, activate 40min clocks.Then 4 are added）Products therefrom, at room temperature PD-10 gel columns purifying 5h,
The CM-DB-HNT of CXCR4 modifications is made.
Embodiment 3：
1st, under nitrogen atmosphere, in the three-neck flask of 100ml volumes by magnetic agitation by 1- octadecylenes（20ml）, oleic acid amine
（20ml）, oleic acid（2ml）And divalence acetylacetone,2,4-pentanedione platinum（0.2g）Preparation is in mixed solution, by divalence acetyl oleyl amine（200mg）Add
Enter wherein.Solution is heated to 120 DEG C under vacuum, then continues to be heated to 200 DEG C under argon atmosphere, in this condition
Under, add 0.2ml iron pentacarbonyl solution（Concentration 0.1Fe (CO)5/ 1ml n-hexanes）.Obtained mixed liquor is heated to 120 DEG C,
1h is reacted on this condition.After being cooled to room temperature, cleaned by centrifugation and isopropyl alcohol, obtain nano particle.Then, will
The particle of acquisition is dispensed into n-hexane solvent again, is then centrifuged for separating nano-particles, and cleaned with ethanol.Product is suspended in
In 4ml hexane solutions comprising 1% oleic acid amine and 1% oleic acid.
2nd, 1- octadecylenes（20ml）And oleic acid（1.2ml）It is sufficiently stirred after mixing, gained mixed liquor is used under the conditions of 90 DEG C
Nitrogen stream persistently deaerates 30 minutes.Under nitrogen atmosphere, iron pentacarbonyl is added（0.14ml, 1mmol）, after 10min, in this thermosol
Oleic acid amine is injected in liquid（97%, Acros）（1ml）With step 1）The nano particle seed of gained（20mg, be dispersed in 2ml just oneself
In alkane solution）, resulting solution reacts 20min under the conditions of 300 DEG C, removes heating mantles afterwards and is cooled to room temperature.By the pure second of 60ml
Alcohol is added to above-mentioned resulting solution and centrifuged, and obtains nanoparticle precipitate.By gained nano particle be dispensed into again just oneself
Alkane, nanoparticle precipitate is obtained by adding ethanol and centrifugation.This process is repeated 2 times with purified nanotubes particle.Final gained is received
Rice grain is dispensed into 10ml hexane solutions.
3rd, 1- octadecylenes are added in 20ml flask（5ml）And oleyl amine（0.75ml）, stirred after fully mixing in strong magnetic
Mix lower by gold chloride（25mg）Add, temperature is risen to 100 DEG C after stirring 1min.Then will be closed under nitrogen protection before 1ml
It is rapidly added in above-mentioned solution and heats 1.5 hours into good platinum-ferric oxide nanometer particle, will after obtaining purplish grey colloidal solution
Mixture is cooled to room temperature.Then 40ml isopropanols are added and are centrifuged（5000 turns, 5 minutes）Nano particle is collected, so with different
Propyl alcohol washes the DB-HNT for being afterwards dissolved in nano particle 10mg/ml is obtained in chloroform twice.
4th, 1ml dopamines（2mg）DMF solution and natrium carbonicum calcinatum（2mg）, it is above-mentioned in the case where being sufficiently stirred to be rapidly added 1ml
In the DB-HNT chloroformic solutions of preparation and continue to stir 2 hours at room temperature.Then by 1mg EDAC, 2mgNHS and 4ml
The nitrine polyethylene glycol of (100mg) carboxyl-functional（Azide-PEG-5000-COOH）Chloroformic solution is stirred at room temperature 30 points
Above-mentioned nano particle chloroformic solution is added to after clock, continues stirring 24 hours at room temperature.Then it is molten to add the change of 15ml n-hexanes
Liquid polarity centrifuges to separate out nano particle simultaneously, collects nano particle.After being washed twice again with n-hexane, then under nitrogen flowing
Dry, and be dissolved in water.After nano particle aqueous solution is filtered by 0.22 micron membrane filter, the centrifugation of 30kDa super filter tubes is added,
It is dissolved in the water after thickening and washing, adds the alkynyl PEG of 1mg copper sulphate, 20mg ascorbic acid and 2mg amino functionals（ALK-
PEG2-NH2）, after 2h is stirred at room temperature, 30kDa super filter tubes centrifuge, and are dissolved in sodium bicarbonate buffer liquid after washing concentrating（pH8.5）In,
Add 1mg succinimide esters-gadolinium-DOTA（NHS-Gd-DOTA）, after being stirred at room temperature 2 hours, 30kDa super filter tubes from
The heart, washing concentrating are standby.
5th, by 50ulCXCR4（Concentration 0.5mg/ml）Monoclonal antibody is dissolved in 100mlPH6.8 PBS cushioning liquid, is added
30mg NHS and 200mg EDC, activate 40min clocks.Then 4 are added）Products therefrom, at room temperature PD-10 gel columns purifying 6h,
The CM-DB-HNT of CXCR4 modifications is made.
Test example 1：
The sign and In vitro cell experiment of CM-DB-HNT nano particles
1st, CM-DB-HNT nano particles are prepared using the method for the present invention, in preparation procedure, Pt nano particles are by 1- 18
Alkene（20ml）, oleic acid amine（20ml）, oleic acid（2ml）And divalence acetylacetone,2,4-pentanedione platinum（0.2g）, divalence acetyl oleyl amine（0.2g）True
120 DEG C are heated under sky, after be warming up under argon gas 185 DEG C again with 0.2ml iron pentacarbonyls solution at 190 DEG C in n-hexane it is anti-
Answer prepared by 1h, with this Pt nano particle and 1- octadecylenes（20ml）And oleic acid（1.2ml）And iron pentacarbonyl（0.14ml）Reaction
Platinum-ferric oxide nanometer particle is prepared, adds gold chloride on this basis（25mg）, 4h is reacted at 60 DEG C and prepares Pt-Fe3O4-Au
Nano-particle, gained nano-particle carry out CXCR4 monoclonal antibody modifications.Utilize perspective electron microscope（TEM）, Zeta Plus current potentials
Analyzer, mass spectrometer analyze the particle diameter distribution of gained nanoparticle respectively（Fig. 2, Fig. 3）, nano-particle ZETA potentials and
It is hydrated particle diameter（Fig. 4）And the molecular weight of CXCR4 target polypeptides（Fig. 6）.As a result prepared CM-DB-HNT nano-particle is shown
12.45 ± 1.21nm, particle diameter is smaller, has well water-soluble and dispersed, easily by cellular uptake, and the nano particle table
Face is negatively charged, by the nonspecific attraction of the materials such as reduction and protein, can preferably ensure its biocompatibility, scheme simultaneously
6 results show the CXCR4 monoclonal antibody structural integrities modified on nano-particle, possess targeting ability.
2nd, using the Skyra magnetic resonance of 3.0T Siemens, sample is swept under the conditions of 25 DEG C using inversion-recovery sequence
Retouch, calculate r1 relaxation rates corresponding to the particle concentration of series concentration diluted sample；R2 is measured using more echo spin-echo sequences
Relaxation rate（Fig. 5 and Fig. 7）, Fig. 5 results show that CM-DB-HNT can have very high relaxation rate, Fig. 7 tables at very low concentrations
Bright CM-DB-HNT possesses T1/T2 enhancing effects.
3rd, people's normal mammary epithelial is set to pass through PBS（a）, various concentrations（10-100mg/L）CM-DB-
HNT nano particles and the carefully bag bath altogether of people's normal breast epithelial（b-f）, tested after 24 hours using phase contrast microscope, assess CM-
DB-HNT biocompatibility（Fig. 8）.As a result it is existing to show that people's normal mammary epithelial phagocytosis does not occur to CM-DB-HNT
As, while people's normal mammary epithelial is complete, shows that prepared CM-DB-HNT has good biocompatibility.
4th, the CM-DB-HNT nanoparticles solutions in concentration gradient (0.05-0.8mg/L) are prepared（a-f）, with human breast carcinoma
After tumour cell bathes 24h altogether, prussian blue staining experiment, situation about being absorbed for assessing nano particle by tumour cell are carried out
（Fig. 9, Figure 10）.As a result find that prepared CM-DB-HNT can be swallowed by people's triple negative breast cancer cell, show CM-DB-
HNT possesses the ability of targets identification people's triple negative breast cancer cell, while people's triple negative breast cancer cell gulps down to CM-DB-HNT
Bite with the increase of CM-DB-HNT concentration and increase, show prepared nano-particle in people's triple negative breast cancer cell
There is good enrichment.
Conclusion：
The isomer Pt-Fe3O4-Au nano-particles of controlled syntheses can be used for preparing the double enhancing contrast agent of T1/T2；
CXCR4-Pt-Fe3O4-Au heterotrimer nano-particles can be used in preparing triple negative breast cancer targeted contrast agent.
Claims (4)
1. the preparation method of the double relaxation platinum-iron oxide-gold nano grains of a kind of T1/T2, it is characterised in that comprise the following steps：
1）Synthesize Pt nano particles：
Under nitrogen atmosphere, by 1- octadecylenes, oleic acid amine, oleic acid by volume 10:1:1 ratio is mixed；Add quality
Than for 1:1 divalence acetylacetone,2,4-pentanedione platinum and divalence acetyl oleyl amine；Heated under vacuum to 100-120 DEG C, under argon atmosphere after
It is continuous to be heated to 180-200 DEG C, add iron pentacarbonyl solution（It is 1 with divalence acetylacetone,2,4-pentanedione mol ratio：4）Mixed liquor is obtained, is added
Heat reacts 1h to 190-220 DEG C；Room temperature is cooled to, is centrifuged, isopropyl alcohol cleans to obtain nano particle, and n-hexane solvent is secondary pure
Change, ethanol cleaning；
2）The double dumbbell structure nano particles of Pt-Fe3O4 in synthetic iron oxide ion protection blocking group closing Pt areas：
1- octadecylenes and oleic acid are pressed 20：1-40：1 volume ratio is well mixed, and under the conditions of 90-120 DEG C, is persistently taken off with nitrogen stream
Gas 20-30 minutes；Under nitrogen atmosphere, iron pentacarbonyl is added（Oleic acid/the mol ratio without carbonyl iron：1.5/1-3/1）, 10-
After 20min, injection mol ratio is：1:1-2:1 oleic acid amine and Pt nano particle seeds, resulting solution is in 250-300 DEG C of condition
Lower reaction 15-20min, is cooled to room temperature；Ethanol and n-hexane purifies and separates obtain nano particle；
3）Controlled syntheses isomer Pt-Fe3O4-Au heterotrimer nano particles：
1- octadecylenes and oleyl amine are pressed 20:1-20：3 volume ratio is well mixed, and is added gold chloride stirring, is heated up to 60-100
℃；Under nitrogen protection, step 2 is added）Platinum-ferric oxide nanometer particle of preparation（It is 1 with gold chloride mol ratio:1）, heat 1.5-
2.5 hours, it is cooled to room temperature；Purified and cleaned using isopropanol, obtain Pt-Fe3O4-Au nano-particles.
2. a kind of platinum-iron oxide-golden coupled nanosecond preparation method of granules of the double relaxation CXCR4 monoclonal antibodies modifications of T1/T2, specific steps
Including：
1）By the Pt-Fe3O4-Au nano-particle water solubility carboxyl-functionals prepared by right 1：
Pt-Fe3O4-Au nano-particles prepared by right 1 are dissolved in chloroform；By mass ratio 1:1 dopamine D MF solution, nothing
Aqueous sodium carbonate mixes, and adds DB-HNT chloroformic solutions（With dopamine D MF liquor capacities than 1:1-1:2）Stir at room temperature；
It is 1 by mass ratio:2:25-1:2:50 EDAC, NHS and the nitrine polyethylene glycol chloroformic solution of carboxyl-functional mix equal
It is even, it is added to Pt-Fe3O4-Au nano-particle chloroformic solutions；It is dry under nitrogen stream to be dissolved in after n-hexane purifying washes twice
In water, centrifuge after being filtered by micron membrane filter, be dissolved in the water after thickening and washing, add mass ratio 1:20:2-1:20:4 sulphur
The alkynyl PEG of sour copper, ascorbic acid and amino functional, after stirring 2h, centrifuge, be dissolved in sodium bicarbonate buffer liquid after washing concentrating
In, it is 1 to add with the alkynyl PEG mass ratioes of ascorbic acid and amino functional:1 succinimide ester-gadolinium-DOTA, stirring 2
After hour, centrifugation, washing concentrating, the Pt-Fe3O4-Au nano-particles of water-soluble carboxyl-functional are obtained, it is standby；
2）Prepare targeting CXCR4-Pt-Fe3O4-Au heterotrimer nano-particles：
CXCR4 targeting peptides by EDC and NHS activation after obtain active ester, then in aqueous phase with step 1）The water solubility of preparation
The amino of the Pt-Fe3O4-Au nanoparticle surfaces of carboxyl-functional is attached, and is carried out after completion of the reaction with PD-10 gel columns
Purifying, obtain the C-DB-HNT of CXCR4 modifications.
3. Pt-Fe3O4-Au nano-particles as claimed in claim 1 purposes in the double enhancing contrast agent of T1/T2 are prepared.
4. CXCR4- Pt-Fe3O4-Au heterotrimers nano-particle as claimed in claim 2 is preparing triple negative breast cancer target
The purposes into contrast agent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110940808A (en) * | 2019-11-18 | 2020-03-31 | 汕头大学医学院 | Establishment method of CXCR4 targeted drug therapy triple negative breast cancer platform |
| CN110960695A (en) * | 2019-12-09 | 2020-04-07 | 浙江大学 | Gold/mesoporous silicon/iron oxide nano composite material and preparation method and application thereof |
| CN112206824A (en) * | 2020-10-30 | 2021-01-12 | 江西维邦生物科技有限公司 | Preparation method of polydopamine-mediated magnetic bimetallic nanoenzyme |
-
2017
- 2017-07-24 CN CN201710605059.7A patent/CN107375952A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| KAI CHENG等: ""Hybrid Nanotrimers for Dual T1 and T2-Weighted Magnetic Resonance Imaging"", 《ACS NANO》 * |
| MATTHEW R. BUCK等: ""A total-synthesis framework for the construction of high-order colloidal hybrid nanoparticles"", 《NATURE CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110940808A (en) * | 2019-11-18 | 2020-03-31 | 汕头大学医学院 | Establishment method of CXCR4 targeted drug therapy triple negative breast cancer platform |
| CN110960695A (en) * | 2019-12-09 | 2020-04-07 | 浙江大学 | Gold/mesoporous silicon/iron oxide nano composite material and preparation method and application thereof |
| CN112206824A (en) * | 2020-10-30 | 2021-01-12 | 江西维邦生物科技有限公司 | Preparation method of polydopamine-mediated magnetic bimetallic nanoenzyme |
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