CN113368079A - Cancer cell membrane-coated drug-loaded lignin nanoparticle and preparation method and application thereof - Google Patents
Cancer cell membrane-coated drug-loaded lignin nanoparticle and preparation method and application thereof Download PDFInfo
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- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
The invention discloses a cancer cell membrane coated drug-loaded lignin nanoparticle and a preparation method and application thereof, belonging to the fields of chemical materials and biomedicine. The preparation method of the lignin nanoparticle coated with the drug-loaded by the cancer cell membrane comprises the following steps: (1) uniformly mixing gamma-valerolactone/water system solution containing lignin and gamma-valerolactone/water system solution containing medicaments to obtain mixed solution; diluting the mixed solution in water, stirring, reacting completely, centrifuging, and collecting to obtain gambogic acid/lignin nanoparticles; (2) mixing gambogic acid/lignin nanoparticles with the cell membrane dispersion liquid, and extruding to obtain the lignin nanoparticles coated with the cancer cell membrane. The invention realizes the cancer cell membrane wrapping (GA-LNPs @ CCM) of the lignin drug-loaded nanoparticles, so that the lignin drug-loaded nanoparticles have the advantages of good biocompatibility, specific tumor targeting property, small toxic and side effects on organisms and the like.
Description
Technical Field
The invention belongs to the field of chemical materials and biomedicine, and particularly relates to a drug-loaded lignin nanoparticle wrapped by a cancer cell membrane, and a preparation method and application thereof.
Background
Cancer is one of the leading causes of abnormal human death today. Chemotherapy remains the primary means of treating cancer. However, the chemotherapy has poor performance and strong toxic and side effects, and can treat cancer without differential damage to normal tissue cells, damage to the immune system of a human body and increase the risk of cancer recurrence. The use of the nano-carrier can improve the performance of the patent medicine and reduce the toxic and side effects. For example, the macromolecule self-assembly nano material can effectively improve the loading efficiency of the common hydrophobic drug, and the proper modification can improve the targeting property of the drug so as to reduce the toxic and side effects. However, if the nanocarrier is to be put to clinical transformation, it must be safe, efficient, cost effective and capable of mass production. However, most of the currently used nanocarriers have difficulty in simultaneously solving the mentioned influencing factors of clinical transformation.
Lignin is a natural polymer with phenylpropane as a basic unit, which is renewable and biodegradable. As one of main components in plant cell walls which are renewable resources, the reserves of lignin are second to cellulose, which is the second most abundant biomass resource on the earth, and the lignin has a mature extraction process and lower production cost. The nano Lignin (LNPs) is used as a lignin self-assembly body, can be stably dispersed in a physiological environment, has a high specific surface area, a large number of pi bonds, a branched molecular structure and a hydrophobic core, and is easy to load bioactive molecules or drugs in a high flux, so that the treatment efficiency is improved. However, at present, the nano lignin is often used as a carrier to load drugs, so that the safety risk of production is undoubtedly increased, and meanwhile, the toxic organic solvent cannot avoid the residual in the nano carrier, so that the toxic and side effects of the carrier are also increased. In addition, the currently used method of loading the drug with the nano lignin usually uses a low-concentration dialysis method, and requires a large amount of water and a long time; or the nano lignin is prepared by a method of synthesizing nano lignin step by step and adsorbing the loaded drug, and the method not only reduces the production efficiency, but also has low drug loading efficiency. Finally, the nano-lignin carrier itself does not have the ability to actively target cancer cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims at providing a preparation method of lignin nanoparticles with drug loading wrapped by cancer cell membranes, which is a green and environment-friendly preparation method of nano lignin synchronously loading natural drugs, and simultaneously, the lignin is wrapped by the cancer cell membranes to realize active targeting of cancer cells. The method uses industrial water-insoluble lignin as a raw material, and dissolves the lignin by a gamma-valerolactone/water binary solvent system (GWS). Gamma-valerolactone (GVL) is a green organic solvent, which can be degraded from cellulose in plants, and the material is widely available and renewable. The GVL has a higher boiling point, is not easy to volatilize and cannot form an azeotrope with water, so that the GVL is easy to separate from water, and the GVL is recycled; GVL also has a distinctive herbal smell, so its leakage is very noticeable. In addition, the Hildebrand Solubility Parameter (HSP) of GVL was 22.7MPa1/2And HSP of water is 47.6MPa1/2Therefore, the ratio between the two can be adjusted to ensure that the HSP of the GVL/water system is between 22.7 and 47.6MPa1/2The excellent dissolving performance of the system is exerted. According to the method, high-concentration lignin and a medicinal solution are obtained by dissolving lignin by GWS, and the high-concentration lignin medicinal solution is dripped into water to be diluted to form lignin nanoparticles by a nano precipitation method. In addition, the obtained nano lignin medicine is wrapped by cancer cell membrane to obtain the productActive targeting of homologous cancer cells is now possible.
The invention also aims to provide the cancer cell membrane-coated drug-loaded lignin nanoparticles prepared by the preparation method.
The invention further aims to provide application of the cancer cell membrane wrapped drug-loaded lignin nanoparticles.
The purpose of the invention is realized by the following scheme:
a preparation method of lignin nanoparticles coated with drug-loaded by cancer cell membranes comprises the following steps:
(1) uniformly mixing gamma-valerolactone/water system solution containing lignin and gamma-valerolactone/water system solution containing medicaments to obtain mixed solution; diluting the mixed solution with water, stirring, reacting completely, centrifuging, and collecting to obtain Gambogic acid/lignin nanoparticles (GA-LNPs);
(2) mixing gambogic acid/lignin nanoparticles with cell membrane (CCM) dispersion, and extruding to obtain drug-loaded lignin nanoparticles (GA-LNPs @ CCM) wrapped by cancer cell membrane.
The lignin in the step (1) is preferably water-insoluble lignin; more preferably alkali lignin.
The gamma-valerolactone/water system solution in the step (1) contains 20 vol% -100 vol% gamma-valerolactone (GVL); more preferably, it contains 80 vol% of Gamma Valerolactone (GVL).
The medicament in the step (1) is preferably a water-insoluble medicament; more preferably Gambogic Acid (GA).
The mass ratio of the lignin to the medicine in the step (1) is preferably 45-55: 1; more preferably in a mass ratio of 50: 1.
The uniform mixing in the step (1) is preferably uniformly mixed at room temperature in a dark place; the room temperature is 25-35 ℃.
The operation of standing overnight after the uniform mixing in the step (1) is also included.
The water in the step (1) is preferably deionized water.
The stirring in the step (1) is preferably carried out for 0.5 to 3 hours at 4 to 40 ℃; further preferably, the mixture is gently stirred for 1 to 3 hours at the temperature of 25 to 35 ℃; more preferably, the mixture is gently stirred at 30 ℃ for 2 hours.
The centrifugation condition in the step (1) is preferably 8000-12000 g for 8-12 min; more preferably 10000g, 10 min.
The average particle size of the gambogic acid/lignin nanoparticles (GA-LNPs) is 50-300 nm, and the gambogic acid/lignin nanoparticles have good dispersibility.
The cell membrane (CCM) dispersion described in step (2) is preferably obtained by the following method: centrifuging the cells, and taking the precipitate to obtain 4T1 cancer cell membrane protein; adding the 4T1 cancer cell membrane protein into the cell lysate, and standing at low temperature to obtain a 4T1 cancer cell lysate; breaking cells by repeated freeze thawing, centrifuging at low temperature, collecting supernatant, and centrifuging to obtain cell membrane (CCM) debris precipitate; dispersing the cell membrane fragment precipitate in water to obtain cell membrane (CCM) dispersion.
The preferable mass ratio of the gambogic acid/lignin nanoparticles to the cell membrane (CCM) dispersion liquid in the step (2) is 1: 1-2.
The method for uniformly mixing and extruding the gambogic acid/lignin nanoparticles and the cell membrane (CCM) dispersion liquid in the step (2) is preferably as follows: uniformly mixing gambogic acid/lignin nanoparticles with a cell membrane (CCM) dispersion solution, and then filtering and extruding by adopting a polycarbonate microporous filter membrane.
The blending is preferably performed by stirring.
The filter membrane is preferably a polycarbonate microporous filter membrane.
The aperture of the filter membrane is preferably 0.45-1 μm; more preferably at least one of 1 μm, 0.85 μm and 0.45 μm.
A lignin nanoparticle coated with cancer cell membrane and carrying medicine is prepared by the above preparation method.
The cancer cell membrane coated drug-loaded lignin nanoparticles are applied to preparation of drugs for treating and/or preventing cancers.
Compared with the prior art, the invention has the following advantages and effects:
(1) the lignin related by the invention has the advantages of wide raw material source, mature extraction process, low cost, reproducibility, degradability and good biocompatibility.
(2) The method has the advantages of simple process conditions, normal temperature and pressure, simple equipment and simple and convenient operation, and the adopted green organic solvent gamma-valerolactone is nontoxic and can be repeatedly utilized.
(3) The invention realizes the one-pot method of the lignin nanoparticles (GA-LNPs) carrying the medicine in the green solvent for the first time, so that the medicine is loaded efficiently, and the invention has important inspiration significance for the efficient utilization of the medicine and the high-valued application of the lignin in the field of biomedicine.
(4) The invention realizes the cancer cell membrane wrapping (GA-LNPs @ CCM) of the lignin drug-loaded nanoparticles, so that the lignin drug-loaded nanoparticles have the advantages of good biocompatibility, specific tumor targeting property, small toxic and side effects on organisms and the like.
Drawings
FIG. 1 is a graph of the particle size and zeta potential of GA-LNPs obtained from nano-lignin and gambogic acid of different mass ratios in example 1.
FIG. 2 is a graph showing the results of SDS-PAGE protein gel electrophoresis of the standard protein, 4T1 cancer cell lysate, 4T1 cancer cell membrane protein and GA-LNPs @ CCM in example 3.
FIG. 3 is a graph of the particle size and zeta potential of GA-LNPs @ CCM obtained in example 3, with different mass ratios of GA-LNPs to CCM.
FIG. 4 is a graph showing the effect of LNPs and GA-LNPs in example 4 on the cell survival rate of mouse breast cancer cells (4T 1).
FIG. 5 is a graph showing the effect of GA in GA-LNPs @ CCM of example 5 on cell viability of mouse breast cancer cells (4T1) and mouse fibroblasts (3T 3).
FIG. 6 is a graph showing the results of tumor volume changes with time after 4T1 tumor cells were treated with different drugs in example 6.
FIG. 7 is a graph showing the results of active targeting of GA-LNPs @ CCM-Cy5 on different cancer cells in example 7.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The lignin used in the examples was alkali lignin, which was purchased from Sigma Aldrich; cell membrane extraction reagents were purchased from Biyuntian Biotechnology Ltd.
Example 1: preparation of loaded gambogic acid lignin nanoparticles (GA-LNPs)
In order to bind the lignin and the drug well, 100. mu.L of a 40mg/mL lignin gamma-valerolactone/water system (GWS) solution (containing 80 vol% gamma-valerolactone (GVL)) and 80. mu.L of a 1mg/mL Gambogic Acid (GA) GWS solution (containing 80 vol% GVL) were mixed at room temperature (30 ℃ C.) and left to stand overnight in the dark to obtain a mixed solution. Then, the mixed solution was added dropwise to 5mL of deionized water, gently stirred at 30 ℃ for 2 hours, after the reaction was completed, gambogic acid/lignin nanoparticles (GA-LNPs) were collected by centrifugation (10000g, 10min), and washed with deionized water for 2 times. The input mass ratio of lignin to Gambogic Acid (GA) in example 1 was 50: 1.
The resulting GA-LNPs had a particle size of about 130nm and a zeta potential of about-28 mV. The entrapment rate of the nano lignin to Gambogic Acid (GA) is up to 90%.
The inventor tries to add lignin and Gambogic Acid (GA) according to the mass ratio of 25:1, 4:1, 100:1, 10:1, 2:1, 5:1 and 1:1 respectively. The size and potential of the obtained gambogic acid nanoparticles (GA-LNPs) are shown in FIG. 1.
As can be seen from fig. 1, as the mass ratio of lignin to gambogic acid decreased, their particle size increased gradually.
In addition, the inventors carried out the above experiments by replacing gamma-valerolactone (GVL) with tetrahydrofuran, dimethyl sulfoxide, acetone, methanol and dimethyl amide, respectively, and determined the encapsulation efficiency of the nano-lignin to Gambogic Acid (GA) in different solvent systems.
As a result, it was found that the entrapment rate of the nano lignin obtained by the above method using Gamma Valerolactone (GVL) as a solvent to Gambogic Acid (GA) was higher than that of the nano lignin obtained by other solvents to Gambogic Acid (GA).
Example 2: extraction of Cancer Cell Membrane (CCM) fragments
Culturing mouse breast cancer cells (4T1 cells, purchased from ATCC company) in a culture dish with a diameter of 15cm, collecting the cells by using a cell scraper, and centrifuging for 10min at 700g to obtain a cell precipitate, namely 4T1 cancer cell membrane protein; the cell membrane is resuspended in hypotonic cell lysis solution (containing cell membrane extraction reagent and phenylmethylsulfonyl fluoride PMSF, purchased from Shanghai Biyunshi Biotech, and kept at 4 ℃ for 10-15 minutes to obtain 4T1 cancer cell lysate, then the cells are disrupted by repeated freeze-thawing, centrifuged at 4 ℃ for 10min at 700g, the supernatant is carefully collected, then centrifuged at 14000g for 30min to obtain cell membrane (CCM) debris precipitate, and the cell membrane debris precipitate is dispersed in water to prepare 1.5mg/mL cell membrane (CCM) dispersion for later use.
Example 3: preparation of lignin nanoparticles (GA-LNPs @ CCM) coated with drug-loaded on cancer cell membrane
The GA-LNPs (1mg/mL, 1mL) from example 1 and the CCM dispersion (1.5mg/mL, 1mL) from example 2 were first mixed well with stirring. Then, the mixture is extruded through polycarbonate microporous filter membranes with different pore diameters (1 μm, 0.8 μm and 0.45 μm) by using an Avanti micro extruder, and the drug-loaded lignin nanoparticles (GA-LNPs @ CCM) wrapped by the cancer cell membranes can be obtained. Standard proteins (bovine serum albumin from Aladdin), 4T1 cancer cell lysate, 4T1 cancer cell membrane protein, and GA-LNPs @ CCM were then verified separately by SDS-PAGE protein gel electrophoresis, and the results are shown in FIG. 2.
The SDS-PAGE protein gel electrophoresis result of FIG. 2 shows that the membrane protein component of GA-LNPs @ CCM is cancer cell membrane protein, and at the same time, the successful encapsulation of GA-LNPs by cancer cell membrane is demonstrated.
The inventors tried to perform the above-described tests with the mass ratios of CCM and GA-LNPs of 0.5:1, 1:1, 1.5:1, 0:1 (i.e., GA-LNPs only), and 1:0 (i.e., CCM only), respectively, and the results are shown in fig. 3.
As shown in FIG. 3, the particle size of GA-LNPs @ CCM was increased compared to non-enveloped GA-LNPs; when the above test was carried out with the mass ratio of the CCM to the GA-LNPs 1.5:1, the grain size of the obtained GA-LNPs @ CCM reached about 250nm, which was still a desirable size.
Example 4: cancer cell inhibitory Effect of GA-LNPs
Cytotoxicity of LNPs and GA-LNPs against mouse breast cancer cells (4T1 cells, purchased from ATCC) was examined by CCK-8 assay. First 4T1 cells were cultured at 8X 103Cell/well Density seeded into 96-well plates, and the plates were placed in CO2The incubator was overnight. Subsequently, the original medium was removed and replaced with fresh complete medium (purchased from warrior and seikagaku life technologies limited) containing LNPs at a concentration of 10.1 μ g/mL, 20.2 μ g/mL, 40.3 μ g/mL, 60.5 μ g/mL, 70.6 μ g/mL, respectively, and further incubated at 37 ℃ for 24 hours, the cells were washed with PBS buffer (pH 7.4, 50mM), then 100 μ L of fresh complete medium containing 10% CCK-8 was added to each well, the cells were incubated again for about 20 minutes, and finally the cell viability was measured and calculated using a microplate reader.
The results are shown in FIG. 4. FIG. 4 shows that GA-LNPs have excellent anticancer effects, whereas LNPs themselves have no inhibitory effect on cancer cells in the range of use.
Example 5: in vitro anti-cancer cell effect of GA-LNPs @ CCM
The cytotoxicity of GA-LNPs @ CCM against mouse breast cancer cells (4T1) and mouse fibroblasts (3T3 cells, purchased from ATCC) was examined by the CCK-8 method, respectively. After plating 4T1 cells and 3T3 cells, the cells were plated and cultured, and then added with complete medium having GA concentration of 0.261. mu.g/mL, 0.348. mu.g/mL, 0.435. mu.g/mL, 0.522. mu.g/mL, 0.696. mu.g/mL, 0.87. mu.g/mL, and 1.74. mu.g/mL in GA-LNPs @ CCM, respectively, and incubated for 24 hours, after removing the old complete medium and rinsing with PBS buffer solution several times, 100. mu.L of fresh complete medium containing 10% CCK-8 was added to each well, and after incubation and color development, absorbance at 450nm of each well was measured with a microplate reader and the relative cell survival rate was calculated.
The results are shown in FIG. 5, and FIG. 5 shows that GA-LNPs @ CCM has selective toxicity to cancer cells.
Example 6: in vivo anticancer cell effect of GA-LNPs @ CCM
Healthy BALB/c female mice (purchased from Tokyo Wintolite laboratory animal technologies, Inc.) aged 4-5 weeks were randomly divided into 5 groups of 5 in parallel, each group was designated PBS, LNPs, GA-LNPs and GA-LNPs @ CCM, 4T1 tumor cells (1mg GA per kg of mouse, as day 0) were subcutaneously injected into the left hind dorsal aspect of each mouse, and then drugs (1mg GA per kg of mouse) were injected again on days 1, 4 and 7, respectively, and the volume of the tumor was measured every two days, and the results are shown in FIG. 6.
FIG. 6 shows that the GA loading of LNPs significantly improves the antitumor effect, and the GA-LNPs @ CCM encapsulating the 4T1 cell membrane almost completely inhibits the growth of 4T1 tumor cells.
Example 7: homologous targeting effect of GA-LNPs @ CCM
First, 4T1 cell membrane fragments were labeled with Cy 5-N-hydroxysuccinimide ester (Cy 5). Subsequently, the labeled cancer cell membranes (designated as CCM-Cy5) were mixed with the prepared GA-LNPs to obtain GA-LNPs @ CCM-Cy 5. For cell uptake studies, 4T1 cells, B16F10 cells (purchased from ATCC) and 3T3 cells were used at 8X 104The density of cells/well was inoculated into a cell culture dish and cultured in the respective culture medium for 24 hours, then, the culture medium was discarded and replaced with fresh complete medium containing GA-LNPs and GA-LNPs @ CCM-Cy5(LNPs concentration 8. mu.g/mL), and the cells were incubated for 4 hours, respectively. Thereafter, the cells were rinsed several times with PBS buffer, and then 1mL of PBS buffer containing 1. mu.L of Hoechst 33342(10mmol/L) and 1. mu.L of green cell membrane dye (5mg/mL) was added to co-incubate the cells for 15 minutes, respectively, to stain the cell nuclei and cell membranes, respectively. After staining, cells were washed several times with PBS buffer to remove excess dye, and then exposed to a confocal laser scanning microscope (CLSM, Zeiss, LSM 880). The results were analyzed with ZEN software.
For Flow Cytometry Analysis (FCA), 4T1 cells, B16F10 cells (purchased from ATCC company) and 3T3 cells were used at 1X 105The density of cells/well was seeded in 24-well plates. After incubating GA-LNPs @ CCM-Cy5 (LNPs: 40. mu.g/mL. times.20.17%) with the cells for 4 hours, the cells were washed three times with PBS buffer, digested with trypsin (purchased from Gibco, USA), and collected by centrifugation at 1000rpm for 5 minutes. The bottom cells were washed twice with PBS buffer and then suspendedCells were filtered and examined by flow cytometry. Similar experiments with CLSM and FCA were performed on GA-LNPs @ CCM-Cy5 in 4T1 cells, B16F10 cells, and 3T3 cell line to verify homotypic targeting. To protect the fluorescent dye, the treatment was kept in the dark throughout the experiment. The results are shown in FIG. 7.
FIG. 7 shows that the efficiency of endocytosis of GA-LNPs encapsulated by 4T1 cancer cell membranes by 4T1 cancer cells is as high as 90%, which is much higher than the efficiency of phagocytosis by other cells, i.e., the encapsulation of cancer cell membranes achieves the homologous targeting of GA-LNPs.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of lignin nanoparticles coated with drug-loaded by cancer cell membranes is characterized by comprising the following steps:
(1) uniformly mixing gamma-valerolactone/water system solution containing lignin and gamma-valerolactone/water system solution containing medicaments to obtain mixed solution; diluting the mixed solution in water, stirring, reacting completely, centrifuging, and collecting to obtain gambogic acid/lignin nanoparticles;
(2) mixing gambogic acid/lignin nanoparticles with the cell membrane dispersion liquid, and extruding to obtain the lignin nanoparticles coated with the cancer cell membrane.
2. The preparation method according to claim 1, wherein the mass ratio of the lignin to the medicine in the step (1) is 45-55: 1.
3. The method according to claim 1, wherein the gamma valerolactone/water system solution of step (1) comprises 20 vol% to 100 vol% gamma valerolactone.
4. The method according to claim 1, wherein the cell membrane dispersion in the step (2) is obtained by: centrifuging the cells, and taking the precipitate to obtain 4T1 cancer cell membrane protein; adding the 4T1 cancer cell membrane protein into the cell lysate, and standing at low temperature to obtain a 4T1 cancer cell lysate; breaking cells by repeated freeze thawing, centrifuging at low temperature, collecting supernatant, and centrifuging to obtain cell membrane debris precipitate; dispersing the cell membrane fragment precipitate in water to obtain cell membrane dispersion.
5. The production method according to claim 1,
and (3) calculating the gambogic acid/lignin nano-particles and the cell membrane dispersion liquid in the step (2) according to the mass ratio of 1: 1-2.
6. The method according to claim 1, wherein the lignin in step (1) is a water-insoluble lignin; further alkali lignin;
the medicament in the step (1) is a water-insoluble medicament; further gambogic acid.
7. The production method according to claim 1,
uniformly mixing in the step (1) in a dark place at room temperature;
the operation of standing overnight after uniformly mixing in the step (1) is also included;
the water in the step (1) is deionized water;
the stirring in the step (1) is carried out for 0.5-3 h at 4-40 ℃; further stirring gently at 25-35 ℃ for 1-3 h;
the centrifugation condition in the step (1) is 8000-12000 g for 8-12 min.
8. The method for preparing the cell membrane dispersion liquid according to claim 1, wherein the step (2) of mixing and extruding the gambogic acid/lignin nanoparticles with the cell membrane dispersion liquid comprises the following steps: uniformly mixing gambogic acid/lignin nanoparticles with the cell membrane dispersion liquid, and then filtering and extruding by adopting a polycarbonate microporous filter membrane;
the uniform mixing is carried out by stirring and mixing;
the filter membrane is a polycarbonate microporous filter membrane;
the aperture of the filter membrane is 0.45-1 μm.
9. A lignin nanoparticle coated with a drug and coated by a cancer cell membrane, which is characterized by being prepared by the preparation method of any one of claims 1-8.
10. The use of the cancer cell membrane-encapsulated drug-loaded lignin nanoparticle of claim 9 in the preparation of a medicament for the treatment and/or prevention of cancer.
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