CN112569363A - Lead compound of tumor treatment electric field, preparation method thereof and sensitization tumor treatment electric field - Google Patents
Lead compound of tumor treatment electric field, preparation method thereof and sensitization tumor treatment electric field Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
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Abstract
The invention discloses a lead compound of a tumor treatment electric field, a preparation method thereof and a sensitization tumor treatment electric field, comprising the following steps: the tumor antibody, the graphene oxide and the ferroferric oxide are connected through a non-covalent bond; the tumor antibody is cetuximab. The lead compound of the tumor treatment electric field is formed by connecting a tumor antibody, graphene oxide and ferroferric oxide through a non-covalent bond. The mitosis of glioma cells is effectively blocked, the proliferation of glioma cells is inhibited, the death of glioma cells is promoted, and the glioma cells are killed and killed by the medium-frequency low-strength electric field. The formed lead compound can effectively and directly kill most tumor cells and greatly enhance biological reaction. In addition, the lead compound of the tumor treatment electric field can enhance the electric field, reduce the adverse reaction of the scalp and improve the safety and effectiveness of killing glioma cells.
Description
Technical Field
The invention relates to the field of tumor treatment electric fields, in particular to a lead compound of a tumor treatment electric field. In addition, the invention also relates to a preparation method of the lead compound comprising the tumor treatment electric field and a sensitization tumor treatment electric field.
Background
Tumor treatment electric fields are a new tumor treatment means which is newly appeared in recent years. At present, a plurality of in vitro and in vivo experiments prove that the tumor treatment electric field has strong clinical treatment effect on tumors and is approved by Food and Drug Administration (FDA). However, the most common adverse reaction of the application of the tumor treatment electric field is the skin adverse reaction at the contact part of the electrode slice, and the adverse reaction is usually applied to patients with surgical incisions and scars left on the scalp, which causes scalp infection, and the adverse reaction becomes a new research hotspot gradually for the next difficulty needing to be overcome continuously.
Disclosure of Invention
The invention provides a lead compound of a tumor treatment electric field, a preparation method thereof and a sensitization tumor treatment electric field, and aims to solve the technical problem that the application of the existing tumor treatment electric field has adverse reaction.
The technical scheme adopted by the invention is as follows:
a lead compound for an electric field for tumor therapy, comprising: the tumor antibody, the graphene oxide and the ferroferric oxide are connected through a non-covalent bond; the tumor antibody is cetuximab.
Furthermore, the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is (4-10) to (0.5-1.5) to (1-5).
Furthermore, the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is 5: 1-5.
Further, the ferroferric oxide adopts nano-scale ferroferric oxide; the particle size of the ferroferric oxide is 100 nm-200 nm.
According to another aspect of the present invention, there is also provided a method for preparing the lead compound for an electric field for tumor therapy, comprising the following steps:
adding cetuximab and graphene oxide into 10-50% by volume of phosphate buffer solution, shaking and shaking uniformly at 4-40 ℃, reacting for 2-24 h to obtain graphene oxide modified cetuximab mixed solution, centrifuging the mixed solution for 2-2.5 h at 20000-25000 r/min, removing supernatant, and then re-suspending with water to obtain graphene oxide modified cetuximab suspension;
adding ferroferric oxide into graphene oxide modified cetuximab suspension, shaking up the mixture at 4-40 ℃, reacting for 8-12 h, centrifuging for 2-2.5 h at 20000-25000 r/min, and removing supernatant to obtain the lead compound.
Furthermore, the dosage of water is 200-1000 mug of ferroferric oxide and 1-2 ml of water is added.
According to another aspect of the invention, a sensitization electric field for tumor therapy is also provided, which comprises the lead compound of the electric field for tumor therapy.
Further, at least one electric field is applied to the lead compound and the glioma cells.
Further, the intensity of the electric field is 1V to 10V.
Further, the frequency of the electric field is 100 kHz-500 kHz, and the interval time is 1 ms-100 ms.
The invention has the following beneficial effects:
the lead compound of the tumor treatment electric field is formed by connecting a tumor antibody, graphene oxide and ferroferric oxide through a non-covalent bond. The mitosis of glioma cells is effectively blocked, the proliferation of glioma cells is inhibited, the death of glioma cells is promoted, and the glioma cells are killed and killed by the medium-frequency low-strength electric field. The antibody of the formed lead compound is specifically targeted, and the ferroferric oxide is a sensitization electric field, so that the ferroferric oxide and the antibody are linked on graphene oxide through non-covalent, the graphene oxide is used as a carrier for connecting the graphene oxide and the graphene oxide, glioma cells can be directly reached, most of the glioma cells can be effectively and directly killed, and biological reaction can be greatly enhanced. After the effect of the ferroferric oxide sensitization electric field is achieved, the effect of killing tumor cells can be achieved under the condition of reducing the field intensity, the field intensity is reduced, the adverse reaction of the scalp is reduced, and the safety and the effectiveness of killing glioma cells are improved.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph showing the results of a cell culture experiment according to a preferred embodiment of the present invention;
FIG. 2 is a graph showing the result of measuring the proliferation activity of MTS glioma cells in accordance with a preferred embodiment of the present invention; and
fig. 3 is a confocal microscopic image of a preferred embodiment of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
FIG. 1 is a graph showing the results of a cell culture experiment according to a preferred embodiment of the present invention; FIG. 2 is a graph showing the result of measuring the proliferation activity of MTS glioma cells in accordance with a preferred embodiment of the present invention; fig. 3 is a confocal microscopic image of a preferred embodiment of the present invention.
The lead compound of the tumor treatment electric field of the embodiment is used for the lead compound comprising: the tumor antibody, the graphene oxide and the ferroferric oxide are connected through a non-covalent bond; the tumor antibody is cetuximab.
The lead compound of the tumor treatment electric field is formed by connecting a tumor antibody, graphene oxide and ferroferric oxide through a non-covalent bond. The mitosis of glioma cells is effectively blocked, the proliferation of glioma cells is inhibited, the death of glioma cells is promoted, and the glioma cells are killed and killed by the medium-frequency low-strength electric field. The antibody of the formed lead compound is specifically targeted, and the ferroferric oxide is a sensitization electric field, so that the ferroferric oxide and the antibody are linked on graphene oxide through non-covalent, the graphene oxide is used as a carrier for connecting the graphene oxide and the graphene oxide, glioma cells can be directly reached, most of the glioma cells can be effectively and directly killed, and biological reaction can be greatly enhanced. After the effect of the ferroferric oxide sensitization electric field is achieved, the effect of killing tumor cells can be achieved under the condition of reducing the field intensity, the field intensity is reduced, the adverse reaction of the scalp is reduced, and the safety and the effectiveness of killing glioma cells are improved.
The cetuximab has specific targeting, ferroferric oxide is used as a sensitization electric field, and graphene oxide is a carrier for connecting the cetuximab and the ferroferric oxide and is non-covalent bond. Preferably, the tumor antibody, graphene oxide and ferroferric oxide are bound by pi-pi bonds.
The graphene oxide contains sp2 hybridized benzene rings with hydroxyl and carboxyl, has stable property and good biocompatibility, and can be non-covalently combined with antibodies, drug molecules, metal ions, fluorescent groups or cells through pi bonds, hydrophobic interaction, hydrogen bonds and ionic bonds. Secondly, the surface of the graphene oxide with the two-dimensional structure is higher than that of any other nano material by at least 4 orders of magnitude, and each atom of the graphene oxide is exposed on the surface and is suitable for carrying drugs. The graphene oxide is a basic building block of other graphite materials, can be wrapped into a zero-dimensional sphere, rolled into a one-dimensional carbon nanotube, and laminated into graphite with a three-dimensional structure.
Graphene, although a recognized nanomaterial, is not necessarily suitable as a drug for intravenous use. Because the nano material concept means that any three-dimensional structure can be called as a nano material as long as one dimension reaches the nanometer level. For example, unmodified graphene (available from Sigma) has a thickness on the order of nanometers, but has a larger diameter that does not meet the nanometer-scale requirements. The applicant believes that as a drug for intravenous use, it must reach the nanoscale of all dimensions to reduce toxicity and maximize the advantages of nanomaterials. Graphene is of a two-dimensional structure, and two-dimensional nano graphene with the thickness and the diameter both conforming to the nanometer level is selected or prepared as a raw material, so that the graphene oxide modified cetuximab is more suitable for in vitro experiments and in vivo experiments, especially in vivo experiments.
The particle size of the graphene oxide is less than 0.22 μm. The graphene oxide with larger particles is easy to aggregate in a physiological salt solution to generate precipitates, and the properties are unstable. The graphene oxide with the particle size of less than 0.22 mu m can be stably and uniformly dispersed in human serum. The graphene oxide with the particle size of less than 0.22 μm can be purchased directly or prepared by crushing large-particle graphene oxide, such as ultrasonic crushing.
In addition, although graphene oxide is dispersible in water, such as physiological saline, graphene oxide is susceptible to aggregation due to the charge shielding effect of the salt. The good drug carrier has excellent surface chemical properties, good biocompatibility and controllable biological change. Therefore, the surface modification of graphene oxide is imperative, and at present, the surface modification is mainly divided into two types: covalent and non-covalent, the former generally being techniques of atomic doping or reaction formed by the destruction of the unsaturated structure of graphene oxide; the latter does not change the natural structure of graphene oxide, and is more flexible in combination only through non-covalent bonds, van der waals forces and charge attraction. In the lead compound, cetuximab, ferroferric oxide and graphene oxide are connected through non-covalent bonds, the combination is flexible, and the effect of killing lymphoma cells is strong.
In the embodiment, the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is (4-10) to (0.5-1.5) to (1-5). The graphene oxide is used as a carrier and is combined with a tumor antibody and ferroferric oxide through non-covalent bonds, the molecular structure of the tumor antibody is larger than that of the ferroferric oxide, the graphene oxide is firstly combined with the tumor antibody to ensure that stable graphene oxide modified cetuximab is formed, and then the rest covalent bonds of the graphene oxide are combined with the ferroferric oxide, so that the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is (4-10): (0.5-1.5): (1-5) to meet the stability of the spatial structure of the lead compound.
Preferably, the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is 5: 1-5.
In the embodiment, the ferroferric oxide adopts nano-scale ferroferric oxide; the particle size of the ferroferric oxide is 100 nm-200 nm. The ferroferric oxide is also in a nanometer level, so that the ferroferric oxide is stably combined with the graphene oxide modified cetuximab.
According to another aspect of the present invention, there is also provided a method for preparing the lead compound for an electric field for tumor therapy, comprising the following steps:
adding cetuximab and graphene oxide into 10-50% by volume of phosphate buffer solution, shaking and shaking uniformly at 4-40 ℃, reacting for 2-24 h to obtain graphene oxide modified cetuximab mixed solution, centrifuging the mixed solution for 2-2.5 h at 20000-25000 r/min, removing supernatant, and then re-suspending with water to obtain graphene oxide modified cetuximab suspension;
adding ferroferric oxide into graphene oxide modified cetuximab suspension, shaking up the mixture at 4-40 ℃, reacting for 8-12 h, centrifuging for 2-2.5 h at 20000-25000 r/min, and removing supernatant to obtain the lead compound.
The graphene oxide and the ferroferric oxide are both in a nanometer level, and the nanometer particles of the ferroferric oxide are water-soluble. The graphene oxide is combined with the cetuximab to obtain the graphene oxide modified cetuximab, and then the graphene oxide modified cetuximab is combined with the ferroferric oxide, and the cetuximab is large in molecular structure, and needs to be combined with the graphene oxide after reaching a stable combination state, so that the cetuximab and the ferroferric oxide can exist stably and can be substituted into cells.
The graphene oxide modified cetuximab is prepared firstly, the obtained graphene oxide modified cetuximab and ferroferric oxide are combined in a non-covalent bond mode, shaking and shaking are carried out uniformly under the condition of controlling the temperature to be 4-40 ℃, the reaction is carried out for 8-12 hours, the reaction time is too short, the combination time of the graphene oxide modified cetuximab and the ferroferric oxide is not enough, and the combination amount of the graphene oxide modified cetuximab and the ferroferric oxide is small and the combination is unstable. And if the reaction time is too long, the risk of antibody variation is increased, and the subsequent binding stability of ferroferric oxide is possibly reduced. Therefore, the reaction time may preferably be 8 to 12 hours. And the higher the preparation temperature is, the more serious the high-temperature denaturation of antibody molecules is, the weaker the activity of the antibody is, and when the temperature is too high, the structure of a lead compound of a tumor treatment electric field is also damaged, so that the antibody is shaken and shaken uniformly to be combined under the condition of 4-40 ℃.
The graphene oxide modified cetuximab is prepared by adopting Phosphate Buffered Saline (PBS) as a common reagent in the field, and the pH value of the PBS is 7.35-7.45. Phosphate buffer solution (PBS solution) with the volume percentage of 10-50% is added with water in an amount adjusted based on the original formula and diluted. Preferably, the volume percentage of the phosphate buffer solution is 10%, and the amount of the phosphate buffer solution is 1mg of graphene oxide added into 0.5-1.5 ml of the phosphate buffer solution, preferably 1ml of the phosphate buffer solution. Under the condition of 10% PBS, the binding amount of CTX is higher, and more importantly, the 10% PBS has a relatively stable buffer system and is closer to the physiological condition. Preferably, the reaction temperature is 37 ℃. The reason is two: firstly, the killing effect is best at the temperature; secondly, the temperature is also physiological temperature, which is beneficial to ensuring the stability of in vitro and in vivo experimental results and lays a foundation for obtaining consistent in vitro and in vivo results. Preferably, the reaction time is 12-24 h, the amounts of cetuximab and graphite oxide are large, the combination is stable, the reaction time is continuously increased, and the combined amount of cetuximab and graphite oxide is not obviously increased. The reaction time may be preferably 12 hours in view of time cost.
The ferroferric oxide is added into graphene oxide modified cetuximab suspension, the mixture is shaken and shaken evenly at the temperature of 4-40 ℃, the reaction lasts for 8-12 h, then the mixture is centrifuged for 2-2.5 h at 20000-25000 r/min, and the free ferroferric oxide can be removed through centrifugal treatment, so that the obtained lead compound has high purity and can be effectively applied to an electric field for treating tumors.
In the embodiment, the amount of water is 200 to 1000 mu g of ferroferric oxide, and 1 to 2ml of water is added.
According to another aspect of the invention, a sensitization electric field for tumor therapy is also provided, which comprises the lead compound of the electric field for tumor therapy.
In this example, at least one electric field is applied to the lead compound and the glioma cells. The lead compound and the glioma cells are placed in an electric field, and the glioma cells can be placed at the intersection points of a plurality of electric fields so as to obtain more remarkable capability of inhibiting or killing the glioma cells. The at least one electric field may be a vertical electric field with 2 anodes and 2 cathodes, or an electric field with 1 anode, a plurality of cathodes, or an electric field with 1 cathode and a plurality of anodes.
In this example, the intensity of the electric field is 1V to 10V.
In this embodiment, the frequency of the electric field is 100 kHz-500 kHz, and the interval time is 1 ms-100 ms. Preferably, the frequency is 100 kHz-300 kHz, and the interval time is 1 ms-20 ms.
Examples
Cetuximab was purchased from Sigma, usa.
Graphene oxide was purchased from Sigma company, usa.
Ferroferric oxide was purchased from thunder corporation, Beijing.
The electric field therapeutic apparatus adopts ASCLU-300 of Nanantangkang Biotechnology GmbH.
Example 1
The graphene oxide was purified by dialysis to remove alcohol, prepared to a concentration of 1mg/ml, and sonicated for 2 hours, 10 seconds in mode, and 5 seconds at a sonication cell sonifier intensity of 3.5, Misonix, usa. Note that the whole process is carried out in an ice-water mixture. After the ultrasonic treatment is finished, filtering the solution by using a 0.22-micron filter to prepare graphene oxide, measuring the final concentration by using a Nanodrop, and refrigerating the solution for later use;
adding 1mg of cetuximab and 0.2mg of graphene oxide into 1ml of 10 volume percent phosphate buffer solution, shaking and shaking uniformly at 4 ℃, reacting for 12 hours to obtain a mixed solution of the graphene oxide modified cetuximab, centrifuging the mixed solution for 2 hours at 20000 revolutions per minute, removing supernatant, and then re-suspending with 1ml of deionized water to obtain a graphene oxide modified cetuximab suspension;
adding 1mg of ferroferric oxide into graphene oxide modified cetuximab suspension, shaking and shaking the ferroferric oxide with the particle size of 100nm at 4 ℃, reacting for 12 hours, centrifuging for 2 hours at 20000 rpm, removing supernatant, and resuspending with 1ml of deionized water to obtain the lead compound solution.
Example 2
The difference from example 1 is that 1mg of cetuximab, 0.2mg of graphene oxide and 0.6mg of ferroferric oxide are added.
Example 3
The difference from example 1 is that 1mg of cetuximab, 0.2mg of graphene oxide and 0.2mg of ferroferric oxide are added.
Example 4
The difference from example 1 is that shaking was carried out at 37 ℃.
Example 5
The difference from example 1 is that shaking at 25 ℃ is employed.
Example 6
The difference from example 1 is that the reaction is carried out for 8 h.
Example 7
The difference from example 1 is that the reaction is carried out for 10 h.
The sensitization tumor treatment electric field comprises the lead compound prepared in the embodiment 1-7, and a vertical electric field with 2 anodes and 2 cathodes is applied to the lead compound and glioma cells by an electric field treatment instrument, wherein the strength of the electric field is 1V, the frequency of the electric field is 100kHz, and the interval time is 20 ms. Can inhibit or kill glioma cells.
Comparative example 1
Only graphene oxide modified cetuximab suspensions were prepared.
Experimental data
The pilot compound used in the following test data was prepared as in example 1. The lead compound is abbreviated as CTX/GO/Fe3O4The graphene oxide-modified cetuximab is abbreviated as CTX/GO, the graphene oxide is abbreviated as GO, and the cetuximab is abbreviated as CTX.
(1) Cell culture experiments
Culturing glioma cells in a round-bottom 96-well plate at a cell concentration of 4x104200. mu.l/ml, divided into 7 groups. PBS solution without glioma cells, PBS solution without glioma cells is not applied with electric field (TTF-free group for short), and PBS solution, GO solution and Fe with the same concentration are added into the other 6 groups3O4CTX solution, CTX/GO/Fe from example 13O4The solution and the CTX/GO suspension liquid of the comparative example 1 are cultured for 2-3 days under the conditions of constant temperature, constant humidity and constant carbon dioxide concentration and under the intervention of an electric field applied by an electric field therapeutic apparatus, the strength of the electric field is 1-10V, the frequency is 100-500 kHz, and the interval time is 1-20 ms. The survival number of glioma cells in each group was observed under a microscope (5 × 20 × ═ 100 ×).
As shown in FIG. 1, the observation revealed that TTF group (A), PBS group (B), GO group (C) and Fe were absent3O4The groups (D) and (E) showed higher survival rate of glioma cells, the group (F) showed lower survival rate of glioma cells, and the group (F) showed lower survival rate of glioma cells, CTX/GO/Fe3O4The survival number of glioma cells of group (G) was much smaller than that of the other control groups.
The experimental results prove that:
CTX group and Fe3O4The group had no or weak ability to inhibit or kill glioma cells in vitro. This is consistent with the well-known knowledge. Because of CTX and Fe3O4When applied to human body, it acts on human body immune system to stimulate immune system to eliminate glioma cell, so that in vitro experiment, because there is no immune system or complement, the concentration selected by us is much lower than that of clinical application, and CTX and Fe3O4Has weak effect on glioma cells.
CTX/GO/Fe3O4Groups and CTX/GO have the ability to inhibit or kill glioma cells. The control group had a greatly reduced number of surviving glioma cells. After CTX is combined with GO, the action mechanism of CTX antitumor drugs is changed, so that the CTX antitumor drugs still have the capability of inhibiting or killing glioma cells in vitro cell tests. Under the action of electric field, CTX/GO/Fe3O4The group had much higher ability to inhibit or kill glioma cells than the CTX/GO group.
(2) MTS glioma cell proliferation activity assay
Culturing glioma cells in a round-bottom 96-well plate at a cell concentration of 1X104200 μ l/ml, divided into 7 groups corresponding to PBS group, GO group, and Fe group3O4Group, CTX + GO + Fe3O4Group (CTX + GO + Fe)3O4Group (2): mixing 1mg cetuximab, 0.2mg graphene oxide and 1mg ferroferric oxide, standing for 12h at 4 ℃, centrifuging for 2h at 20000 revolutions per minute, removing supernatant, and adopting 1ml deionized water for resuspension), CTX/GO group and CTX/GO/Fe3O4And (4) grouping. Wherein the PBS group is PBS solution, the GO group is 1 microgram/ml GO solution, and Fe3O4The composition was 1. mu.g/ml Fe3O4The solution, CTX group of 5 mug/ml CTX and CTX/GO group of 5 mug/ml CTX/GO solution are processed into corresponding intervention groups. 5 mug/ml CTX + GO + Fe3O4Mixed liquor and CTX/GO/Fe3O4The solution was the experimental group. Culturing for 2-3 days under the conditions of constant temperature, constant humidity and constant carbon dioxide concentration and under the intervention of an electric field applied by an electric field therapeutic apparatus, wherein the strength of the electric field is 1V-10V, the frequency is 100 kHz-500 kHz, and the interval time is 1 ms-20 ms. And (3) detecting the glioblastoma cell DBTRG-05MG of each group, adding 20 mu l of MTS reagent into each hole of a 96-hole plate, slightly shaking and uniformly mixing to avoid splashing of liquid. The culture medium is put into an incubator for further 2 hours, and then the absorbance at 450nm is read in a full-function microplate reader Synergy H1 instrument. The absorbance value can reflect the cell proliferation activity and is used for evaluating the curative effect of the intervention drug.
As shown in fig. 2, the experimental results show that,the absorbance value in MTS represents cell proliferation activity, such as PBS group, GO group, CTX group and Fe3O4Group, CTX + GO + Fe3O4The absorbance of the group is centered around 100, while the CTX/GO group and CTX/GO/Fe3O4The absorbance of the group gradually increased with decreasing gradient concentration, but was still much lower than that of the other groups, indicating CTX/GO and CTX/GO/Fe3O4Can obviously inhibit the activity of glioma cell strain DBTRG.
Experimental results prove that CTX/GO/Fe3O4Under the action of the electric field, the effect of the electric field can be effectively enhanced, and the effect of killing glioma cells is stronger.
(3) Confocal microscope
And (3) combining an FITC group on the cetuximab to prepare a cetuximab-FITC antibody (CTX-FITC) for later use. CTX-FITC is combined with two-dimensional nano graphene oxide to form CTX-FITC/GO, and then the CTX-FITC/GO is combined with Fe3O4Combining, preparing to obtain CTX-FITC/GO/Fe3O4. With Fe3O4FITC as control group (Fe)3O4Binding to the FITC group). Fe3O4FITC includes A-D, CTX-FITC/GO/Fe3O4Including E to H.
A DBTRG cell line was prepared by the following procedure: the cells were resuspended in medium RPMI1640 at a density of 1X1051ml of the solution was placed in a 12-well plate, and Fe was added to each well3O4FITC 50. mu.g or CTX-FITC/GO/Fe3O450 μ g, mixed well and cultured for 2 hours.
Mixing CTX-FITC/GO/Fe3O4Preparing cell slide of 1-10 × 104The cell density of the cell is 16-20 mm, the cell is taken out after the wall is completely adhered at night, the cell is placed in an electric field therapeutic apparatus and cultured for 2-3 days, the strength of an electric field is 1V-10V, the frequency is 100 kHz-500 kHz, the interval time is 1 ms-20 ms, and the cell is taken out after 48 hours. And (3) keeping the control group of creeping slide for normal culture, treating in an electric field therapeutic apparatus after 48 hours, and observing under a micro-confocal microscope.
As shown in FIG. 3, in the confocal microscopy images, A and E are Fe, respectively3O4FITC group and CTX-FITC/GO/Fe3O4DAPI staining of nuclei of the group, the location of the nuclei. B and F are respectively Fe3O4FITC group and CTX-FITC/GO/Fe3O4Staining of phalloidin from the cytoskeleton of the cells was the location of the cytoplasm. And, Fe3O4The basic form of the cytoskeleton of the group (B) is normal and is in a silk shape, the cell synapse is obvious, and the active invasiveness for prompting the cell growth is strong; and CTX/GO/Fe3O4The cytoskeleton of group (F) contractures into a conglomeration without obvious synapses, suggesting insufficient cell viability and invasiveness. C and G are respectively Fe3O4FITC group and CTX-FITC/GO/Fe3O4Fe of group3O4The location of FITC. D and H are respectively Fe3O4FITC group and CTX-FITC/GO/Fe3O4Fused images of the groups. It can be seen that Fe3O4FITC of FITC group (D) is relatively low, dispersed mainly on the surface of the cells and hardly incorporated into the cytoplasm; and CTX-FITC/GO/Fe3O4FITC is present in a large proportion in group (H), and is distributed both on the surface of tumor cells and in the cytoplasm, and even at the periphery of the nucleus. CTX/GO/Fe prompt3O4The group can effectively enter the interior of cells under the action of an electric field and an antibody, and cause cytoskeleton disorder and induce cell death. The above-mentioned micro-confocal image can explain CTX/GO/Fe3O4Better effect of inhibiting glioma cells, CTX/GO/Fe3O4With free Fe3O4Compared with the traditional Chinese medicine, the traditional Chinese medicine can more effectively enter the interior of cells and induce the micro-disorder of cell microtubules, thereby causing apoptosis.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A lead compound of an electric field for treating tumor is characterized in that,
the method comprises the following steps: the tumor antibody, the graphene oxide and the ferroferric oxide are connected through a non-covalent bond;
the tumor antibody is cetuximab.
2. Lead compound of an electric field for tumor therapy according to claim 1,
the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is (4-10) to (0.5-1.5) to (1-5).
3. Lead compound of an electric field for tumor therapy according to claim 2,
the mass ratio of the tumor antibody to the graphene oxide to the ferroferric oxide is 5: 1-5.
4. Lead compound of an electric field for tumor therapy according to claim 2,
the ferroferric oxide adopts nano-scale ferroferric oxide;
the particle size of the ferroferric oxide is 100 nm-200 nm.
5. A method for preparing a lead compound of an electric field for tumor therapy according to any one of claims 1 to 4, comprising the steps of:
adding cetuximab and graphene oxide into 10-50% by volume of phosphate buffer solution, shaking and shaking uniformly at 4-40 ℃, reacting for 2-24 h to obtain graphene oxide modified cetuximab mixed solution, centrifuging the mixed solution for 2-2.5 h at 20000-25000 r/min, removing supernatant, and then re-suspending with water to obtain graphene oxide modified cetuximab suspension;
adding ferroferric oxide into graphene oxide modified cetuximab suspension, shaking up the mixture at 4-40 ℃, reacting for 8-12 h, centrifuging for 2-2.5 h at 20000-25000 r/min, and removing supernatant to obtain the lead compound.
6. The method for preparing a lead compound for an electric field for tumor therapy according to claim 5,
the dosage of the water is 200-1000 mug of ferroferric oxide, and 1-2 ml of water is added.
7. A sensitized tumor treatment electric field comprising a lead compound of the tumor treatment electric field according to any one of claims 1 to 4.
8. The sensitized tumor therapy electric field according to claim 7,
at least one electric field is applied to the lead compound and the glioma cells.
9. The sensitized tumor therapy electric field according to claim 8,
the strength of the electric field is 1V-10V.
10. The sensitized tumor therapy electric field according to claim 8,
the frequency of the electric field is 100 kHz-500 kHz, and the interval time is 1 ms-100 ms.
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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 |
| WO2019100016A1 (en) * | 2017-11-17 | 2019-05-23 | Abbvie Inc. | Methods of treating glioblastoma |
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