CN102657872B - Multifunctional graphene oxide/ polyamide-amine (PAMAM)/ diethylene triamine pentaacetic-gadolinium (DTPA-Gd)/ prostate stem cell antigen (PSCA) antibody material and preparation method and application thereof - Google Patents

Multifunctional graphene oxide/ polyamide-amine (PAMAM)/ diethylene triamine pentaacetic-gadolinium (DTPA-Gd)/ prostate stem cell antigen (PSCA) antibody material and preparation method and application thereof Download PDF

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CN102657872B
CN102657872B CN2012101380846A CN201210138084A CN102657872B CN 102657872 B CN102657872 B CN 102657872B CN 2012101380846 A CN2012101380846 A CN 2012101380846A CN 201210138084 A CN201210138084 A CN 201210138084A CN 102657872 B CN102657872 B CN 102657872B
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pamam
graphene oxide
dtpa
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psca
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CN102657872A (en
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吴惠霞
侍海丽
王雪
杨仕平
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Shanghai Normal University
University of Shanghai for Science and Technology
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Abstract

The invention discloses a multifunctional graphene oxide/ polyamide-amine (PAMAM)/ diethylene triamine pentaacetic-gadolinium (DTPA-Gd)/ prostate stem cell antigen (PSCA) antibody material. PAMAM dendrimers are covalently modified on nano graphene oxide flakes, and DTPA-Gd and PSCA antibodies are modified by using amino on the PAMAM dendrimers. The obtained material has very high biocompatibility and stability, can be used as a good T1 contrast medium, and has a good targeting property for prostatic cancer cells; and doxorubicin hydrochloride can be adsorbed to the graphene oxide/ PAMAM/ DTPA-Gd/ PSCA antibody material under coaction of phi-phi bonds, and the material is high in medicament loading rate and has an obvious targeting effect. The multifunctional graphene oxide/ PAMAM/ DTPA-Gd/ PSCA antibody material has targeting medicament loading and magnetic resonance imaging (MRI) functions, and has a very wide biological application prospect.

Description

Graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material and its preparation method and application
Technical field
The present invention relates to a kind of graphene oxide/PAMAM/DTPA-Gd/ doxorubicin hydrochloride/psca antibody multifunctional material and preparation method thereof, belong to nano-medicament carrier and nano-contrast agent technical field.
Background technology
Carbon is the most magical element of nature, in Nanometer World, the performance of carbon is amazing too, the strong K sea nurse of the peace moral of Univ Manchester UK in 2004 (Andre K.Geim) produces Graphene first, it is in the world the thinnest New Two Dimensional nano material, and its thickness is only 0.35nm.Its peculiar property has attracted countless scientists, can say that it is that in many new materials, tool is potential, is called as " miracle material ", all has great application prospect in industry, power industry and electronic industry.
With the graphite-phase ratio, in graphene oxide, contain the chemical groups such as carboxyl, hydroxyl, carbonyl and epoxy radicals, wherein carboxyl and hydroxyl modified are in the edge of oxidized graphite flake, carbonyl and epoxy radicals are between the graphene oxide layer, graphite is water-fast material originally, oxidation rear oxidation Graphene has extremely strong hydrophilic, easily is scattered in the stable colloidal sol of formation in polarizable medium, and this is the existence due to these active groups of graphene oxide surface.
The 1950's, scientist has proposed the structure of graphene oxide in succession, is mainly three large basic models, Ruess model, Hofmann model and Scholz-Boehm model.It is generally acknowledged, graphene oxide is two-dimensional layered structure, and interlayer contains a large amount of hydroxyls and the acid active group of carboxyl.At present, the research of graphene oxide starts to pay close attention to its application aspect biological medicine, and such as medicine, gene transportation, particularly the research for the treatment of of cancer targeted drug transmission system has caused very large concern.
Summary of the invention
The purpose of this invention is to provide a kind of graphene oxide/PAMAM/DTPA-Gd/ doxorubicin hydrochloride/psca antibody multifunctional material.This pharmaceutical carrier good dispersion in water, good biocompatibility, the toxicity of material own is little, and the drug loading ability is strong, in nuclear magnetic resonance, has good application.
Another object of the present invention is for the preparation method of this difunctional pharmaceutical carrier is provided, and that the method has is simple to operate, raw material is easy to get and the advantage such as with low cost.
The object of the present invention is achieved like this:
The preparation method of graphene oxide/PAMAM/DTPA-Gd/ doxorubicin hydrochloride/psca antibody multifunctional material, comprise the steps:
(1) on the nano graphene oxide surface, connect PAMAM: nano graphene oxide is also activation in being dispersed in alcohol, with contain the third generation and take trimesic acid and be the alcoholic solution mix and blend 18～30hr of the PAMAM of kernel, obtain the GO/PAMAM nano material; Nano graphene oxide and third generation PAMAM weight ratio are 1:20～40;
(2) the GO/PAMAM nano material is dissolved in organic solvent, add triethylamine and diethylene-triamine pentaacetic acid dianhydride, heat 20～60min under 70～85 ℃, after stopped heating, continue reaction 10～18hr, get washing of precipitate, obtain the GO/PAMAM/DTPA nano material; The weight ratio of GO/PAMAM nano material and diethylene-triamine pentaacetic acid dianhydride is 4～8:1;
(3) the GO/PAMAM/DTPA nano material is with after solubility gadolinium salt mixes, and 35～50 ℃ of insulation 10～15hr under the condition of isolated air, get precipitation water dialysis 12～30hr, obtains graphene oxide/PAMAM/DTPA-Gd nano material; Gadolinium element and GO/PAMAM/DTPA nano material amount ratio are 0.002～0.01mmol/mg;
(4) connect anti-psca antibody: graphene oxide/PAMAM/DTPA-Gd nano material, after glutaraldehyde is processed, is reacted 10～16hr washing with anti-prostate stem cell antigen-antibody PSCA under 0～6 ℃ in the phosphate buffer of pH=7.2～7.5; Graphene oxide/PAMAM-DTPA-Gd nano material and anti-prostate stem cell antigen-antibody weight ratio are 10000:1～10000:5.
Nano graphene oxide thickness in step (1) is 1～2nm, and surface is with carboxyl.
The nano graphene oxide preparation method comprises the steps: to add 95wt%～98wt% concentrated sulphuric acid to stir 6～12hr after graphene powder expands with saline solution, then adds KMnO 4, mixture, under 35～40 ℃, stirring 25～40min, stir 40～60min under 65～80 ℃, then stirs 20～35min under 98～105 ℃; Use the hydrogen peroxide cessation reaction, washing is got solid, ultrasonic dispersion in water.
In step (1), with 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide DEC and N-hydroxy succinic acid imines NHS activation nano graphene oxide, nano graphene oxide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy succinic acid imines weight ratio are 1:3～8:3～8; Alcohol described in step (1) is methanol.
In step (2), described organic solvent is dimethyl formamide DMF; The weight ratio of GO/PAMAM nano material and triethylamine is 1:4～8.
By said method, can obtain graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material.
This graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material, for treatment cancer targeting and the nuclear magnetic resonance, NMR T of controlled pharmacy release 1Contrast agent.
Further can prepare graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride composite, step comprises:
By graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material with contain doxorubicin hydrochloride, pH=7.2～7.5 phosphate buffers stirring reaction 16～30hr under the lucifuge condition; Doxorubicin hydrochloride and graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material weight ratio is 1:1～1:2, is preferably 2:3.
At first the present invention adopts the standby graphene oxide of improved Hummers legal system, then by the dendritic macromole (PAMAM) of this graphene oxide finishing 3G, graphene oxide and the trimesic acid of take are connected by generating the amido link covalency between the dendritic macromole (PAMAM) of kernel; Utilize amido modified DTPA-Gd and psca antibody above dendroid, doxorubicin hydrochloride interacts and is adsorbed onto on graphene oxide by pi-pi bond.Graphene oxide has bilateral structure, and there are a lot of active groups on surface, and extraordinary biocompatibility and stability are arranged, and the drug loading ability is strong, can be good at carrying out drug loading and transportation, in modification, can be used as good T after DTPA-Gd 1Contrast agent, thus realize synthetic purpose with multifunctional material of targeted therapy and MRI imaging.Resulting materials has good biocompatibility simultaneously, is conducive to the application of material at biomedicine field.
Essential implementation of the present invention is: while adopting Hummers method and supercritical ultrasonics technology to prepare graphene oxide, note adding the amount of potassium permanganate and the grasp of time, to add potassium permanganate with the time of about 1 hour, the last ultrasonic time will be grown, the graphene oxide lamella of preparation is thinner like this, and evenly.When meeting DTPA-Gd, temperature is crucial, guarantee that the temperature of each step is accurate, and heating and cooling rapidly are all unfavorable to the preparation of material.The material that our this method is prepared can be scattered in water well, and has good biocompatibility.To equipment require low; The needed raw material price is low; By-product is nuisanceless, environmentally safe.The present invention is creative, novelty and extensive practicality is being arranged aspect cancer and oncotherapy.
Advantage of the present invention is:
1. the graphene oxide made/PAMAM/DTPA-Gd/ doxorubicin hydrochloride/psca antibody multifunctional material, the drug loading amount is large, and Targeting Effect is obvious, and the kill cancer cell effect is remarkable.
2. preparation facilities is simple;
3. thinner, the good dispersion of graphene oxide lamella, the good water solubility of preparation;
Raw material be easy to get, cheap;
5. simple to operate; Environmentally safe.
6. be a kind of multifunctional material with carcinoma of prostate tumor cell specific target tropism, drug loading and transmission and NMR (Nuclear Magnetic Resonance)-imaging, large in field using values such as medicine and biologies.
The accompanying drawing explanation
Fig. 1 is the infrared spectrogram (IR figure) of the graphene oxide of preparation in the embodiment of the present invention 1; A in Fig. 1 is graphite, and b is graphene oxide;
Fig. 2 is the atomic force microscope picture (AFM) of the graphene oxide of preparation in the embodiment of the present invention 1;
Fig. 3 is the transmission electron microscope photo (TEM) of the graphene oxide of preparation in the embodiment of the present invention 1;
Fig. 4 is graphene oxide/PAMAM(a) and the amino density analysis of graphene oxide/PAMAM/DTPA material (b) of preparation in the embodiment of the present invention 2;
Fig. 5 be graphene oxide in the embodiment of the present invention 3/PAMAM/DTPA-Gd/PSCA dispersion of materials in water T1 weighted imaging figure (a) and relaxation rate R1 (1/T1) and sample in linear relationship chart (b) between gadolinium concentration;
Fig. 6 be graphene oxide in the embodiment of the present invention 3/PAMAM/DTPA-Gd/PSCA dispersion of materials in water T2 weighted imaging figure (a) and relaxation rate R2 (1/T2) and sample in linear relationship chart (b) between gadolinium concentration;
Fig. 7 is graphene oxide in the embodiment of the present invention 3/PAMAM/DTPA-Gd/PSCA material and PC-3(a) and MCF-7(b) the MTT toxicity data of two kinds of cells;
Fig. 8 is the T1 weighted imaging figure (a) of graphene oxide in the embodiment of the present invention 3/PAMAM/DTPA-Gd/PSCA antibody material and PC-3 and two kinds of cells of MCF-7, the amount (c) of signal strength map (b) and cytophagy Gd ion;
Fig. 9 is respectively ultraviolet-visible light spectrogram (a) and the drug loading scattergram (b) of graphene oxide in the embodiment of the present invention 4/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride material drug carrier amount.Through calculating drug loading as can be known, it is 0.7415 milligram/milligram;
Figure 10 is graphene oxide in the embodiment of the present invention 4/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride material medicine cumulative release figure when pH=7.4 and pH=5.3;
Figure 11 is respectively the MTT toxicity data of embodiment 4 graphene oxides/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride and doxorubicin hydrochloride and PC-3 and two kinds of cells of MCF-7; (a) and (b) being respectively the MTT that graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride and doxorubicin hydrochloride and PC-3 cell are hatched, is (c) and (d) respectively the MTT that graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride and doxorubicin hydrochloride and MCF-7 cell are hatched.
The specific embodiment
In order to understand better essence of the present invention, below by embodiment, describe technology contents of the present invention in detail, but content of the present invention is not limited to this.
Embodiment 1
1) take the 1g graphene powder in beaker, then add 50g sodium chloride, stir 10min; In beaker, add appropriate distilled water, carry out sucking filtration and collect the graphene powder expanded;
2) graphene powder of the expansion of collection is placed in to single neck flask of 250mL, adds 98% the concentrated sulphuric acid of 23mL, magnetic agitation 8h, slowly progressively add 3g KMnO 4, keep temperature to be less than 20 ℃ therebetween;
3) mixture stirs 30min at 35～40 ℃ of lower magnetic forces, then at 65～80 ℃ of lower magnetic forces, stirs 45min.After reacting by heating finished, toward the distilled water that adds 46mL in single neck flask, mixture was at 98～105 ℃ of lower magnetic forces stirring 30min; 30% the H that adds the distilled water of 140mL and 10mL 2O 2, cessation reaction;
4) with 5% HCl and distilled water by mixture centrifugal and filtering and washing repeatedly, product is dispersed in water to ultrasonic 1h, obtain nano graphene oxide, solution colour is brown.
Fig. 1 is the infrared spectrogram (IR figure) of the graphene oxide of preparation in embodiment 1, from infrared spectrogram, seeing, at 1723cm -1C=O stretching vibration on carboxyl appears in place, illustrates that graphite powder is oxidized to graphene oxide, and surface is with carboxyl; A in Fig. 1 is graphite, and b is graphene oxide.
Fig. 2 is the atomic force microscope picture (AFM) of the graphene oxide of preparation in embodiment 1, from on atomic force microscope figure, finding out very clearly that graphene oxide sheet is level and smooth, and the thickness that advanced Measurement and analysis graphene platelet as can be known is approximately 1.246nm, the thickness of the single-layer graphene of reporting with document coincide, and shows the successfully preparation of graphene oxide thin slice of monolayer;
Fig. 3 is the transmission electron microscope photo (TEM) of the graphene oxide of preparation in embodiment 1, on scheming, can find out that the structure of graphene platelet shape is very obvious, and on the whole Graphene is not fully smooth, but lamella is very thin, in edge, have comparatively significantly fold and Fold generation, this is because the thermodynamics fluctuation of two dimensional crystal is caused.
Embodiment 2
1) take graphene oxide GO40mg in beaker, add the 100mL absolute methanol, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC and each 0.2g of N-hydroxy succinic acid imines NHS, ultrasonic activation 1h;
Take in addition PAMAM (3G) 1g in the 50mL beaker, add the 30ml absolute methanol, ultrasonic dispersion 1h;
Ultrasonic dispersion slowly is added drop-wise to PAMAM (3G) solution in GO solution after finishing, and stirs 24h under room temperature;
After reaction finished, centrifugal (12000rpm, 12min) washing, made GO/PAMAM (3G) nano material;
2) take 20mg GO/PAMAM nano material, be dissolved in the 8mL dry DMF;
Add 0.18mL triethylamine (130mg), then add 4mg diethylene-triamine pentaacetic acid dianhydride (DTPA), be placed in the 25mL flask, then slowly be heated to 80 ℃, continue 30min, remove thermal source, one night of stirring at normal temperature;
Reacted material is separated with centrifugal method, first with the DMF washing that contains TEA1% 3 times, wash 3 times, acetone is washed 3 times again, after drying, be stored in the environment of low temperature, obtain connecting the GO/PAMAM nano material of divinyl five amine acetic acid parts (DTPA), i.e. GO/PAMAM (3G)-DTPA nano material;
3) 20mg GO/PAMAM-DTPA nano material and 10ml pH=8 buffer are mixed in the 25mL flask, then add 40mg Gd (NO 3) 36H 2O(0.09mmol), fully mix, then, by the reactant liquor good seal, be heated to lentamente 40 ℃, one night of constant temperature;
Reactant liquor is centrifugal, and precipitate is dialysed in distilled water 24h, remove responseless gadolinium ion, obtains the GO/PAMAM-DTPA-Gd nano material.
Fig. 4 is graphene oxide/PAMAM(a) and the amino density analysis of graphene oxide/PAMAM/DTPA material (b) of preparation in embodiment 2, records GO/PAMAM nano-material surface amino density and is about 1.152 * 10 -4Mol/g, the surface amino groups density that records the GO/PAMAM-DTPA nano material is 6.17 * 10 -5Mol/g, experimental result can be found out thus, nano-material surface is amino also has residue, can be used for connecing targeted molecular.
Embodiment 3
1) weigh the resulting graphene oxide of 5mg embodiment 2/PAMAM-DTPA-Gd nano material dispersion of materials in the PBS that contains 5% glutaraldehyde (pH=7.4) buffer solution, standing 2h, the material centrifugalize out, with PBS washing three times, then again be dispersed in PBS (pH=7.4) buffer solution, add 10 μ L (concentration 0.13 μ g/ μ L) psca antibody, graphene oxide/PAMAM-DTPA-Gd nano material is cultivated together with antibody;
2) at 4 ℃, stir 12h;
3) reaction is washed for several times with PBS (pH=7.4) buffer solution after finishing, and is stored in PBS (pH=7.4) buffer solution of 4 ℃.The graphene oxide obtained/PAMAM/DTPA-Gd/PSCA antibody multifunctional material has good water solublity.
Fig. 5 is the linear relationship chart (b) between gadolinium concentration in the T1 weighted imaging figure (a) that is dispersed in water of graphene oxide in embodiment 3/PAMAM/DTPA-Gd/PSCA antibody multifunctional material and relaxation rate R1 (1/T1) and sample;
Fig. 6 is the linear relationship chart (b) between gadolinium concentration in the T2 weighted imaging figure (a) that is dispersed in water of graphene oxide in embodiment 3/PAMAM/DTPA-Gd/PSCA antibody multifunctional material and relaxation rate R2 (1/T2) and sample, T 1And T 2Imaging signal intensity changes along with the increase of Gd ion concentration, is embodied in: T 1Imaging signal intensity is along with the increase image of Gd ion concentration obviously brightens; T 2Imaging signal intensity is along with the increase image of Gd ion concentration is obviously dimmed.Relaxation rate is along with the increase of sample concentration is linear growth, and the longitudinal relaxation coefficient is r 1For 8.34Gd Mm -1s -1, transverse relaxation coefficient r 2For 12.47Gd Mm -1s -1, and the r under 0.5T 2/ r 1Value is 1.5, is applicable to being T 1Contrast agent.These data also illustrate that the GO/PAMAM/DTPA-Gd/PSCA nano material can be used as a good T 1Contrast agent;
Fig. 7 is the MTT toxicity data of graphene oxide in embodiment 3/PAMAM/DTPA-Gd/PSCA antibody multifunctional material and PC-3 and two kinds of cells of MCF-7, along with the increase of the concentration of GO/PAMAM/DTPA-Gd/PSCA antibody multifunctional material, the vigor of PC-3 and MCF-7 cell does not occur significantly to reduce.After concentration was hatched 24 hours up to the sample of 200 μ g/mL and PC-3 and two kinds of cells of MCF-7, two kinds of cells still kept being greater than 85% cell viability.These results show, GO/PAMAM/DTPA-Gd/PSCA antibody multifunctional material cytotoxicity to PC-3 and two kinds of cells of MCF-7 under such concentration is lower.
Fig. 8 is graphene oxide in the embodiment 3/PC-3 of PAMAM/DTPA-Gd/PSCA antibody nano material labelling and the T1 weighted imaging figure (a) of two kinds of cells of MCF-7, the amount (c) of signal strength map (b) and cytophagy Gd ion, along with the T1 weighted imaging of two kinds of cells of increase of Gd ion concentration brightens gradually, signal intensity increases gradually, analysis-by-synthesis finds out that the T1 weighted imaging reinforced effects of high expressing cell PC-3 is more obvious, this illustrates the good targeting of psca antibody on the one hand, and illustrative material is well suited for the contrast agent as T1 on the one hand.
Embodiment 4
1) the GO/PAMAM-DTPA-Gd/PSCA antibody multifunctional material of getting 30mg embodiment 3 preparation in the PBS of the doxorubicin hydrochloride of 20mL solution under (pH=7.4) room temperature lucifuge stir 24h;
2) centrifugalize, be colourless with the centrifugal resulting solid of PBS solution washing to centrifugal liquid, and under room temperature, vacuum drying obtains GO/PAMAM-DTPA-Gd/PSCA antibody/doxorubicin hydrochloride medicine transmission material;
3) remaining medicament contg is by measuring the uv absorption of centrifugal liquid (containing cleaning mixture), and the absorption value that is taken at the 490nm place is calculated.Thereby also can calculate the amount that is loaded in the medicine on graphene oxide/PAMAM-DTPA-Gd/PSCA material.Parallel three experiments, drug loading is averaged.
The calculating of doxorubicin hydrochloride load capacity is by measuring the uv absorption of centrifugal liquid (containing cleaning mixture) and original solution, and the absorption value that is taken at the 490nm place is calculated.
Fig. 9 is respectively ultraviolet figure and the drug loading scattergram of graphene oxide in embodiment 4/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride material drug carrier amount.The difference absorbed according to the UV-vis before and after medicine and graphene oxide/PAMAM/DTPA-Gd effect can calculate drug loading.Through calculating drug loading as can be known, it is 0.74 milligram/milligram;
Figure 10 is graphene oxide in embodiment 4/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride material medicine cumulative release 100% figure when pH=7.4 and pH=5.3, as can be seen from the figure, the release of the DOX of medicament-carried GO/PAMAM/DTPA-Gd/PSCA hybrid material is the pH value response and lasting, can accomplish that controllability discharges the effect of gentle Slow release.When pH=7.4, the burst size of DOX is slow, and seldom, during 24h, medicine has only discharged 6.5%, during 72h drug release 8%, yet when pH=5.3, the burst size of DOX increases, and during 24h, medicine has discharged 38.5%, after 72h, the release of medicine has reached 40%, and this is that hydrophilic and the dissolubility of DOX increase greatly because under acid condition;
Figure 11 is respectively the MTT toxicity data after graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride material and doxorubicin hydrochloride and PC-3 and two kinds of cells of MCF-7 are hatched altogether; (a) and (b) being respectively the MTT data that graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride and doxorubicin hydrochloride and PC-3 cell are hatched, is (c) and (d) respectively the MTT data that graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride and doxorubicin hydrochloride and MCF-7 cell are hatched.
From result, can see, graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride multifunctional material is better than simple doxorubicin hydrochloride to the fragmentation effect of cancerous cell, and, high expressed and low cancerous cell of expressing are had to fragmentation effect, but obvious especially to the cancerous cell fragmentation effect of high expressed.

Claims (6)

1. the preparation method of graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material, is characterized in that, comprises the steps:
(1) on the nano graphene oxide surface, connect PAMAM: nano graphene oxide is also activation in being dispersed in alcohol, with contain the third generation and take trimesic acid and be the alcoholic solution mix and blend 18～30hr of the PAMAM of kernel, obtain the GO/PAMAM nano material; Nano graphene oxide and third generation PAMAM weight ratio are 1:20～40; Described alcohol is methanol;
With 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide activation nano graphene oxide, nano graphene oxide, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide weight ratio are 1:3～8:3～8;
Described nano graphene oxide thickness is 1～2nm, and surface is with carboxyl; Preparation method comprises the steps: to add 95wt%～98wt% concentrated sulphuric acid to stir 6～12hr after graphene powder expands with saline solution, then adds KMnO 4, mixture, under 35～40 ℃, stirring 25～40min, stir 40～60min under 65～80 ℃, then stirs 20～35min under 98～105 ℃; Use the hydrogen peroxide cessation reaction, washing is got solid, ultrasonic dispersion in water;
(2) the GO/PAMAM nano material is dissolved in organic solvent, add triethylamine and diethylene-triamine pentaacetic acid dianhydride, heat 20～60min under 70～85 ℃, after stopped heating, continue reaction 10～18hr, get washing of precipitate, obtain the GO/PAMAM/DTPA nano material; The weight ratio of GO/PAMAM nano material and diethylene-triamine pentaacetic acid dianhydride is 4:1～8:1; Organic solvent is dimethyl formamide; The weight ratio of GO/PAMAM nano material and triethylamine is 1:4～8;
(3) the GO/PAMAM/DTPA nano material is with after solubility gadolinium salt mixes, and 35～50 ℃ of insulation 10～15hr under the condition of isolated air, get precipitation water dialysis 12～30hr, obtains graphene oxide/PAMAM/DTPA-Gd nano material; Gadolinium element and GO/PAMAM/DTPA nano material amount ratio are 0.002～0.01mmol:1mg;
(4) connect the anti-prostate stem cell antigen-antibody: the GO/PAMAM/DTPA-Gd nano material, after glutaraldehyde is processed, is reacted 10～16hr washing under 0～6 ℃ with the anti-prostate stem cell antigen-antibody in the phosphate buffer of pH=7.2～7.5; The weight ratio of graphene oxide/PAMAM-DTPA-Gd nano material and anti-prostate stem cell antigen-antibody is 10000:1～10000:5.
2. graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material, is characterized in that, by method preparation claimed in claim 1.
3. graphene oxide/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride multifunctional material, it is characterized in that, preparation method comprises the steps: the described graphene oxide of claim 2/PAMAM/DTPA-Gd/PSCA antibody multifunctional material and the phosphate buffer stirring reaction 16～30hr under the lucifuge condition that contains doxorubicin hydrochloride, pH=7.2～7.5; Doxorubicin hydrochloride and graphene oxide/PAMAM/DTPA-Gd/PSCA antibody multifunctional material weight ratio is 1:1～1:2.
4. the described graphene oxide of claim 2/PAMAM/DTPA-Gd/PSCA antibody multifunctional material is for the preparation of NMR contrast agent.
5. the described graphene oxide of claim 2/PAMAM/DTPA-Gd/PSCA antibody multifunctional material is for the preparation of the carrier for the treatment of carcinoma of prostate medicine.
6. the described graphene oxide of claim 3/PAMAM/DTPA-Gd/PSCA antibody/doxorubicin hydrochloride multifunctional material is for the preparation for the treatment of carcinoma of prostate medicine.
CN2012101380846A 2012-05-05 2012-05-05 Multifunctional graphene oxide/ polyamide-amine (PAMAM)/ diethylene triamine pentaacetic-gadolinium (DTPA-Gd)/ prostate stem cell antigen (PSCA) antibody material and preparation method and application thereof Expired - Fee Related CN102657872B (en)

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