CN106039321A - Rituximab/graphene oxide composite antibody as well as preparation method and application thereof - Google Patents
Rituximab/graphene oxide composite antibody as well as preparation method and application thereof Download PDFInfo
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- CN106039321A CN106039321A CN201610364619.XA CN201610364619A CN106039321A CN 106039321 A CN106039321 A CN 106039321A CN 201610364619 A CN201610364619 A CN 201610364619A CN 106039321 A CN106039321 A CN 106039321A
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- graphene oxide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
Abstract
The invention discloses a rituximab/graphene oxide composite antibody as well as a preparation method and an application thereof. The rituximab/graphene oxide composite antibody consists of rituximab and graphene oxide, wherein the rituximab and the graphene oxide are linked by virtue of a non-covalent bonds, so that the composite antibody is formed; and an antineoplastic biological reaction can be enhanced greatly. Compared with the rituximab which has a slight inhibitory effect in vitro, the rituximab/graphene oxide composite antibody can directly kill lymphoma cells.
Description
Technical field
The present invention relates to antibody art, especially, relate to a kind of Rituximab/graphene oxide compound antibody.Additionally,
The invention still further relates to a kind of preparation method and application including above-mentioned Rituximab/graphene oxide compound antibody.
Background technology
Targeted therapy refers to act on special molecule (molecular targeted) thus stops tumor growth, the medicine bred, spread
Or small-molecule substance, also referred to as molecular targeted therapy, molecular targeted agents and accurate medical science.Targeted therapy is with chemotherapy not
With: (1) targeted therapy is only acting exclusively on the molecule that tumor is relevant, and chemotherapy suppresses cellular metabolism, affects cell cycle, effect
Cell in all quick divisions had both included tumor, also included normal cell, and in other words, the specificity of chemotherapy is relatively poor；
(2) targeted therapy is aiming target cell or target spot is well-designed and selects, and the selection of chemotherapy is only because can kill
Dead or suppression tumor cell；(3) targeted therapy be suppression cell proliferation and differentiation (Cytostatic), with cell cycle without
Close, and chemotherapeutics is to utilize the sensitive stage of cell cycle to cause cytotoxic to kill cell (Cytotoxic).
Rituximab is the target therapeutic agent of a kind of CD20 antibody having used 20 years, and curative effect is the most micro-
Weak.Along with immune infringement, drug resistance are recurred by chemotherapy, the appearance of the situations such as congenital reaction is low, need badly more efficient
CD20 antibody.
Summary of the invention
The invention provides a kind of Rituximab/graphene oxide compound antibody, to solve existing Rituximab
The technical problem that curative effect is weak.
The technical solution used in the present invention is as follows:
One aspect of the present invention provides a kind of Rituximab/graphene oxide compound antibody, Rituximab/oxidation stone
Ink alkene compound antibody includes that Rituximab and graphene oxide, Rituximab and graphene oxide are bonded by non-covalent
Connect.
Further, the mass ratio of appropriate former times monoclonal antibody and graphene oxide is 50～5:1.
Further, the particle diameter of graphene oxide is less than 22 μm.
Another aspect of the present invention provides the preparation method of Rituximab/graphene oxide compound antibody, including following
Step: by Rituximab, graphene oxide add percentage by volume be 10～50% phosphate buffer in, 37～42
At DEG C, concussion shakes up, and reacts and within 4～24 hours, obtains Rituximab/graphene oxide compound antibody.
Further, the percentage by volume of phosphate buffer is 10%, and the consumption of phosphate buffer is that 1mg aoxidizes stone
Ink alkene adds 1000ml phosphate buffer.
Further, reaction temperature is 37 DEG C.
Further, the response time is 12～24 hours, preferably 12 hours.
Further, the preparation of graphene oxide comprises the following steps:
By graphene oxide raw material under conditions of 0～4 DEG C, by ultrasonic disruption, through the filter of aperture 0.22 μm
Net filtration, obtains graphene oxide.
Further, the operation of ultrasonic disruption comprises the following steps:
Graphene oxide raw material is configured to 1～2mg/ml, under conditions of the intensity of ultrasonic disruption is 3.5, super
Sound 2 hours, ultrasound mode is work 10s, rest 5s.
Present invention also offers a kind of Rituximab/graphene oxide compound antibody in preparation treatment lymphoid tumor medicament
Application.
The method have the advantages that above-mentioned Rituximab/graphene oxide compound antibody, Rituximab and
Graphene oxide is connected by non-covalent bond, forms compound antibody, can significantly strengthen biologically.Compare rituximab list
Anti-only have inhibitory action in vitro, and above-mentioned Rituximab/graphene oxide compound antibody can directly kill lymphoma cell.
In addition to objects, features and advantages described above, the present invention also has other objects, features and advantages.
Below with reference to figure, the present invention is further detailed explanation.
Accompanying drawing explanation
The accompanying drawing of the part constituting the application is used for providing a further understanding of the present invention, and the present invention's is schematic real
Execute example and illustrate for explaining the present invention, being not intended that inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is the raw-material structural representation of graphene oxide；
Fig. 2 is the graphene oxide schematic diagram of the supersound process of the preferred embodiment of the present invention；
Fig. 3 is the graphene oxide of graphene oxide raw material and supersound process stability comparison figure in human serum；
Fig. 4 is the RTX/GO of comparative example 1 preparation scattergram in mouse lung；
Fig. 5 is the RTX/GO of embodiment 1 preparation scattergram in mouse lung；
Fig. 6 is the RTX/GO of comparative example 1 preparation scattergram in Mouse Liver；
Fig. 7 is the RTX/GO of embodiment 1 preparation scattergram in Mouse Liver；
Fig. 8 is the fragmentation effect figure of the different RTX/GO of preparation temperature；
Fig. 9 is the fragmentation effect figure of the different RTX/GO of preparation time；
Figure 10 is the fragmentation effect figure of the different RTX/GO of solvent.
Detailed description of the invention
Below in conjunction with accompanying drawing, embodiments of the invention are described in detail, but the present invention can be defined by the claims
Implement with the multitude of different ways covered.
The preferred embodiments of the present invention provide one aspect of the present invention, and to provide a kind of Rituximab/graphene oxide multiple
Closing antibody (RTX/GO), Rituximab/graphene oxide compound antibody includes Rituximab (RTX) and graphene oxide
(GO), Rituximab and graphene oxide are connected by non-covalent bond.
Graphene oxide contains the hydroxyl phenyl ring with carboxyl of sp2 hydridization, not only stable in properties, good biocompatibility, goes back
π key, hydrophobic interaction, hydrogen bond and ionic bond can be passed through non-with antibody, drug molecule, metal ion, fluorophor or cell
Covalent bond.Secondly, any other nano material of the surface ratio of the GO of two-dimensional structure all exceeds at least 4 magnitudes, it each
Atom is all exposed to surface, is suitable for drug delivery.GO is the basic building block of other graphite materials, can wrap up in into the ball of 0 dimension
Body, is rolled into the CNT of 1 dimension, is laminated into the graphite of 3-dimensional structure.
Although GO is dissolved in water, such as normal saline, the electron screening effect yet with salt makes GO easily assemble.Well
Pharmaceutical carrier there is excellent surface chemical property, not only good biocompatibility, and biological change is controlled.Therefore, GO
It is imperative that surface is modified, and currently mainly divides two kinds: covalency and non-covalent, and the former is typically by the unsaturation of graphene oxide
Structural deterioration and formed atom doped or the technology of reaction；The latter does not change the natural structure of GO, only by non-covalent bond, and model
De Huali, charge attraction, in conjunction with more flexible.In Rituximab in the application/graphene oxide compound antibody, rituximab
Monoclonal antibody (RTX) and graphene oxide are connected by non-covalent bond, and in conjunction with flexible, and the effect killing lymphoma cell is strong.
The method have the advantages that above-mentioned Rituximab/graphene oxide compound antibody, Rituximab and
Graphene oxide is connected by non-covalent bond, forms compound antibody, can significantly strengthen biologically.Compare rituximab list
Anti-only have inhibitory action in vitro, and above-mentioned Rituximab/graphene oxide compound antibody can directly kill lymphoma cell.
Alternatively, the mass ratio of appropriate former times monoclonal antibody and described graphene oxide is 50～5:1.
Alternatively, the particle diameter of graphene oxide is less than 22 μm.The graphene oxide that granule is bigger holds under physiological solt solution
Easily assembling, generate precipitation, character is unstable.Use particle diameter can stablize all in human serum less than the graphene oxide of 22 μm
Even dispersion.Particle diameter can directly be bought less than the graphene oxide of 22 μm or by oarse-grained graphene oxide through ultrasonic disruption
Produce.
Another aspect of the present invention provides the preparation method of a kind of Rituximab/graphene oxide compound antibody, including
Following steps: by Rituximab, graphene oxide add percentage by volume be 10～50% phosphate buffer in, 37
～at 42 DEG C, concussion shakes up, and reacts and within 4～24 hours, obtains described Rituximab/graphene oxide compound antibody.
Phosphate buffer (PBS) is reagent commonly used in the art, and its pH value range is 7.35～7.45.Percentage by volume is
10～50% phosphate buffer i.e. PBS solution on the basis of original formulation, adjust the addition of water, diluted.
Rituximab, graphene oxide react under conditions of vibration shakes up, and the two is combined by non-covalent bond and obtains
Rituximab/graphene oxide compound antibody.And Rituximab/graphene oxide compound antibody can be from as solvent
Phosphate buffer is separated, it is possible to not separate and still keep in phosphate buffer.
Rituximab/graphene oxide the compound antibody obtained in above-mentioned condition still has certain after dilution one times
Fragmentation effect.
Response time is too short, and Rituximab and graphite oxide binding time are inadequate, cause the two binding capacity less, in conjunction with
Unstable.And the response time is long, then add the risk of antibody variation.Therefore the response time is preferably 4～24 hours.?
Along with the concentration dilution of RTX/GO in research, the ability of the RTX/GO killing cell of preparation under major part different temperatures progressively subtracts
Weak, but the RTX/GO only 37 DEG C and 42 DEG C synthesis not substantially do not lessen the curative effect along with concentration dilution, point out 37 DEG C with
The therapeutic effect of the RTX/GO of 42 DEG C of preparations is best.And preparation temperature is the highest, antibody molecule is high-temperature denatured the most serious, and antibody is lived
Property is the most weak, and when temperature is too high, the structure of graphene oxide also can be destroyed, and the factor of these two aspects compromises RTX/GO
Killing ability.
Alternatively, the percentage by volume of phosphate buffer is 10%, and the consumption of phosphate buffer is 1mg graphite oxide
Alkene adds 1000ml phosphate buffer.Under conditions of 10%PBS, the binding capacity of RTX is higher, it is often more important that 10%PBS has
More stable buffer system, closer to physiological conditions.
Alternatively, reaction temperature is 37 DEG C.
Reaction temperature selects 37 DEG C.Reason has two: the first, and at a temperature of this, fragmentation effect is best；Second, this temperature is also
It is physiological temp, is conducive to ensureing stablizing of in vitro and in vivo experimental result, and the result for obtaining in vitro and in vivo consistent is established
Determine basis.
Alternatively, the response time is 12～24 hours, preferably 12 hours.Response time is 12～24 hours, rituximab
Monoclonal antibody and graphite oxide amount are big, in conjunction with stable, continue to increase the response time, and Rituximab and graphite oxide combined amount are not
Can substantially increase.In view of time cost, the response time optional excellent be 12 hours.
Alternatively, the preparation of graphene oxide comprises the following steps:
By graphene oxide raw material under conditions of 0～4 DEG C, by ultrasonic disruption, through the filter of aperture 0.22 μm
Net filtration, obtains graphene oxide.
Preparing of graphene oxide is critically important, strict control size and thickness.The former material of graphene oxide can be buied from Sigma
Material, dialysis purification removes ethanol.Graphene oxide raw material is broken into fine particle at sonicator.Then will pass through
The strainer filtering of aperture 0.22 μm.Whole process can be used to carry out operating to ensure that reaction temperature is at 0～4 DEG C in mixture of ice and water
Condition.
Alternatively, the operation of ultrasonic disruption comprises the following steps:
Graphene oxide raw material is configured to 1～2mg/ml, under conditions of the intensity of ultrasonic disruption is 3.5, super
Sound 2 hours, ultrasound mode is work 10s, rest 5s.
In U.S. Misonix sonicator Sonicator 3000, intensity 3.5, continual ultrasonic 2 hours, pattern work
Make 10 seconds, have a rest 5 seconds.Under this condition, the particle diameter of graphene oxide is less than 50nm, and thickness is 1～3nm.With reference to Fig. 1, ultrasonic
The GO that filtration treatment is crossed stable and uniform can disperse in human serum, but (the most general granule is bigger without ultrasonic general GO
Graphene oxide, such as the graphene oxide raw material buied from Sigma, its particle diameter > 0.5-1 μm and thickness > 10nm) little 4
Just precipitate time within.For the difference of the GO after the general GO of direct visual comparison and process, we use Germany JPK
NanoWizard atomic force microscope shooting GO, with reference to Fig. 3.Visible, the GO thinner than general GO (about 8.6 times) after process is more
Little, stability is more preferable, and bio distribution is more preferably.
Embodiment 1
Buying graphene oxide raw material from Sigma, dialysis purification is removed ethanol, is formulated as concentration 1mg/ml, in the U.S.
Under conditions of Misonix sonicator Sonicator 3000 intensity 3.5, continual ultrasonic 2 hours, pattern works 10 seconds,
Have a rest 5 seconds.Notice that whole process is carried out in mixture of ice and water.After ultrasonic end, filter with the filter of 0.22 μm, prepare oxidation stone
Ink alkene, Nanodrop measures ultimate density, and cold preservation is standby.
It is the phosphate buffer of 10% by 1mg Rituximab, 0.2mg graphene oxide addition 1ml percentage by volume
In, shake at 4 DEG C and shake up, react 12 hours, obtain the mixed liquor containing Rituximab/graphene oxide compound antibody.
Embodiment 2
Difference with embodiment 1 is, temperature when preparing Rituximab/graphene oxide compound antibody is 37 DEG C.
Embodiment 3
Difference with embodiment 1 is, temperature when preparing Rituximab/graphene oxide compound antibody is 42 DEG C.
Embodiment 4
Difference with embodiment 1 is, response time when preparing Rituximab/graphene oxide compound antibody is 0
Hour.
Embodiment 5
Difference with embodiment 1 is, response time when preparing Rituximab/graphene oxide compound antibody is 24
Hour.
Embodiment 6
Difference with embodiment 1 is, solvent when preparing Rituximab/graphene oxide compound antibody is water.
Embodiment 7
Difference with embodiment 1 is, solvent when preparing Rituximab/graphene oxide compound antibody is PBS.
Comparative example 1
Difference with embodiment 1 is, graphene oxide raw material directly reacts with Rituximab for broken.
Experimental data
1, about stability
After intravenous injection RTX/GO, the RTX/GO of comparative example 1 preparation is only found in pulmonary vascular, and lung tissue or remote
Place's internal organs such as liver fails to record.And the RTX/GO of embodiment 1 preparation there is no Ink vessel transfusing retention, but the most uniformly divide
Cloth, with reference to Fig. 4～7.
2, the determination of Rituximab/graphene oxide compound antibody optimum temperature is prepared
Round bottom 96 orifice plate cultivates Raji cell, and cell concentration is 4x106/ ml, 200 μ l.Respectively Example 1～3 is made
Standby mixed liquor mixes with Raji cell (i.e. lymphoma cell), and its final chemical equivalent is GO2.5 μ g/ml, RTX12.5 μ g/
Ml, and according to the dilution proportion of 2 times, totally 5 Concentraton gradient, PBS is as a control group.At constant temperature, constant humidity, constant dense carbon dioxide
Cultivate 12 hours in the case of degree.Raji cell, after cold PBS washs, is fixed through Flow buffer (1% formalin), then
Through flow cytometry analysis cell number, disposal data respectively organizes number of viable cells or ratio.INPUT group is that cell is cultivated
The cell number of 96 orifice plates is added, it is simply that directly take same concentrations and the cell of volume cultivating Raji cell, directly during beginning
PBS washs, and 1% formalin buffer is fixed, and flow cytometry analysis gathers data subsequently.
As described in Figure 8, wherein transverse axis is Concentraton gradient to experimental data, and vertical pivot is cell survival rate.The RTX/GO of 4 DEG C of synthesis
Therapeutic effect the most weak, it may be possible to because temperature is low, molecule activity slows down, and the chance of intermolecular mutual collision reduces, RTX and GO
In conjunction with probability reduce, cause the growing amount of RTX-GO to reduce.And along with the concentration dilution of RTX/GO, make under different temperatures
Standby most of RTX/GO kills the ability of cell progressively to be weakened.And do not have along with dense at the RTX/GO of 37 DEG C and 42 DEG C synthesis
Degree dilution substantially lessens the curative effect, and the therapeutic effect pointing out 37 DEG C and the 42 DEG C RTX/GO prepared is best.Therefore rituximab list is prepared
The temperature of anti-/ graphene oxide compound antibody is preferably 37 DEG C～42 DEG C.
In view of fragmentation effect at 37 DEG C, preferably and this temperature is physiological temp, is conducive to ensureing in vitro and in vivo experiment knot
Stablizing of fruit, and be that the result obtaining in vitro and in vivo consistent is laid a good foundation.The most more preferably, Rituximab/oxygen is prepared
The temperature of functionalized graphene compound antibody is 37 DEG C.
3, the determination of Rituximab/graphene oxide compound antibody optimum reacting time is prepared
Round bottom 96 orifice plate cultivates Raji cell, and cell concentration is 4x106/ ml, 200 μ l.Respectively in Example 1,4 and 5
The mixed liquor of preparation mixes with Raji cell (i.e. lymphoma cell), and its final chemical equivalent is GO2.5 μ g/ml, RTX12.5 μ
G/ml, and according to the dilution proportion of 2 times, totally 5 Concentraton gradient, PBS is as a control group.At constant temperature, constant humidity, constant carbon dioxide
Cultivate 12 hours in the case of concentration.Raji cell, after cold PBS washs, is fixed through 1% formalin buffer, then through stream
Formula Cytometric Analysis cell number, disposal data respectively organizes number of viable cells or ratio.INPUT group is that cell cultivation starts
Time add the cell number of 96 orifice plates, it is simply that directly taking same concentrations and the cell of volume cultivating Raji cell, direct PBS washes
Washing, 1% formalin buffer is fixed, and flow cytometry analysis gathers data subsequently.
As described in Figure 9, wherein transverse axis is Concentraton gradient to experimental data, and vertical pivot is cell survival rate.In addition to PBS control group,
Remaining each group has reduction along with the lethal effect of RTX/GO concentration reduction Raji cell.Wherein, 12 hours groups of reaction are little with 24
Time group to kill Raji cell effect optimal, and 0 hour group effect is the most weak, along with the dilution of concentration, does not has any killing soon
Acting on, in prompting preparation process, the enough response time is the important guarantee of fragmentation effect.This experiment is pointed out, and individually distinguishes
RTX and GO added, does not has lethal effect in the presence of not having RTX-GO.Why 0 hour high concentration has
A little lethal effect, it may be possible to because RTX and GO being separately added into forms RTX/GO during cell is cultivated, thus produce
Lethal effect.But in the case of low concentration, RTX and GO molecule amount is on the low side, the chance of reaction impinging one another is little, adds reaction
Condition such as salinity, be not formed RTX/GO optimum condition, cause the fragmentation effect of RTX/GO to be lost rapidly.Cause
This, the response time preparing Rituximab/graphene oxide compound antibody is preferably 12～24 hours.More preferably, it is contemplated that
Time cost, finally selects 12 hours as optimum reacting time.
4, the determination of Rituximab/optimal salinity of graphene oxide compound antibody is prepared
Take the mixed liquor prepared of embodiment 1,6 and 7.In order to detect the amount of RTX on GO that is attached to, respectively by RTX-GO
Centrifugation, washing, at SDS glue (sds page) loading containing Dithiothreitol (DTT)
Buffer boils 10 minutes, and with the RTX 25 μ g of concentration known, 50 μ g and 100 μ g as comparison, be separately added into 12%SDS
Polyacrylamide gel electrophoresis (PAGE), electrophoresis seals running gel scanning result up for safekeeping after terminating.
Sample speed in electrophoresis or relative mobility (Mr) and protein nature (such as molecular size), gel pore
Footpath and deposition condition (such as electric current, voltage) etc. are closely related, after protein bound SDS Polyacrylamide, it will offset each
Shape between group and charge differences, now the mobility speed of protein is only relevant with the size of respective molecular weight.
As shown in Figure 10, compared with PBS, the RTX amount combining GO in embodiment 6 i.e. pure water group is the highest, and embodiment 1 is i.e. for result
10%PBS (0.09%NaCl) group is taken second place.RTX matched group in conjunction with concentration known, it is possible to estimation is at 10%PBS
(0.09%NaCl) RTX combining GO in group is about 100 μ g.In like manner can be evaluated whether, the RTX combining GO in pure water group is about 200 μ
G, embodiment 7 i.e. PBS group is about 50 μ g.Thus it is obvious that can be incorporated into the amount of RTX and salinity on GO is inverse ratio, salt is dense
Spend the highest, in conjunction with RTX amount the lowest.Because salinity can affect the stability of GO, so inventor speculates, salinity height can
Charge property and the distribution on GO surface can be affected, be affected with the binding ability of RTX, cause the growing amount of RTX-GO to reduce,
Thus color depth that PBS group is on protein electrophoresis is minimum.Although the binding capacity of RTX is the highest in pure water group, but itself and organism
Practical situation differ farther out.Though and PBS is closer to the practical situation of organism, but the binding capacity of PBS group RTX is too low.10%
The binding capacity of PBS group RTX is higher, it is often more important that 10%PBS has more stable buffer system, closer to physiological conditions.Cause
Under this considers, 10%PBS is preferred scheme as solvent.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. Rituximab/graphene oxide compound antibody, it is characterised in that described Rituximab/graphene oxide
Compound antibody includes that Rituximab and graphene oxide, described Rituximab and graphene oxide are bonded by non-covalent
Connect.
Rituximab the most according to claim 1/graphene oxide compound antibody, it is characterised in that described appropriate former times is single
Anti-and described graphene oxide mass ratio is 50～5:1.
Rituximab the most according to claim 1/graphene oxide compound antibody, it is characterised in that described oxidation stone
The particle diameter of ink alkene is less than 22 μm.
4. a preparation method for Rituximab according to any one of claims 1 to 3/graphene oxide compound antibody, its
It is characterised by, comprises the following steps: Rituximab, graphene oxide are added the phosphate that percentage by volume is 10～50%
In buffer, shake at 37～42 DEG C and shake up, react within 4～24 hours, obtain described Rituximab/graphene oxide be combined
Antibody.
The preparation method of Rituximab the most according to claim 4/graphene oxide compound antibody, it is characterised in that
The percentage by volume of described phosphate buffer is 10%, and the consumption of described phosphate buffer is graphene oxide described in 1mg
Add phosphate buffer described in 1000ml.
The preparation method of Rituximab the most according to claim 4/graphene oxide compound antibody, it is characterised in that
Reaction temperature is 37 DEG C.
The preparation method of Rituximab the most according to claim 4/graphene oxide compound antibody, it is characterised in that
Response time is 12～24 hours, preferably 12 hours.
8. according to the preparation method of the Rituximab/graphene oxide compound antibody according to any one of claim 4～7,
It is characterized in that, the preparation of described graphene oxide comprises the following steps:
By graphene oxide raw material under conditions of 0～4 DEG C, by ultrasonic disruption, through the filter screen mistake of aperture 0.22 μm
Filter, obtains graphene oxide.
The preparation method of Rituximab the most according to claim 4/graphene oxide compound antibody, it is characterised in that
The operation of described ultrasonic disruption comprises the following steps:
Graphene oxide raw material is configured to 1～2mg/ml, under conditions of the intensity of ultrasonic disruption is 3.5, ultrasonic 2 little
Time, ultrasound mode is work 10s, rest 5s.
10. the Rituximab described in an any one of claims 1 to 3/graphene oxide compound antibody drenches in preparation treatment
Application in bar tumor medicine.
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| CN106474483A (en) * | 2016-12-23 | 2017-03-08 | 中南大学湘雅医院 | The Cetuximab of graphene oxide modification, preparation method and application |
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