CN111000826A - Medicine for synergistic chemical photothermal therapy and targeted treatment of liver cancer and preparation method - Google Patents
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- CN111000826A CN111000826A CN201911414848.8A CN201911414848A CN111000826A CN 111000826 A CN111000826 A CN 111000826A CN 201911414848 A CN201911414848 A CN 201911414848A CN 111000826 A CN111000826 A CN 111000826A
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Abstract
The invention discloses a medicine for synergistic chemophotothermal therapy and targeted therapy of liver cancer and a preparation method thereof, belonging to the technical field of biological medicines. According to the invention, graphene oxide is modified by using a connecting structure, Lactobionic Acid (LA) is modified by an amide reaction, and Curcumin (CUR) is successfully loaded by pi-pi accumulation, so that the obtained GO-linker/LA-CUR has good near infrared light absorption performance at 808nm, and the temperature change shows concentration dependence and excitation intensity dependence; it can trace in vivo transport and distribution by using fluorescence imaging technology; meanwhile, the stability of the compound is verified, and in vivo and in vitro experiments prove that the compound has low biotoxicity and high targeting effect, can be used for improving the curative effect by cooperating with photo-thermal, and has very good market application prospect.
Description
Technical Field
The invention relates to a medicine for synergistic chemophotothermal therapy and targeted therapy of liver cancer and a preparation method thereof, belonging to the technical field of biological medicines.
Background
Liver cancer ranks sixth in the incidence of cancer worldwide and fourth in the cause of tumor-related death, new cases and mortality of men are higher than those of women, the cases of men develop rapidly, are highly invasive, are easy to transfer, are difficult to treat, have poor prognosis, and pose great threat to the life health of patients, and nearly about one million people get lost due to the liver cancer every year. At present, the treatment means aiming at the malignant tumor of the liver mainly comprises operation and drug treatment, however, clinically confirmed liver cancer is mostly in middle and late stages, donors which can be used for transplantation are scarce, and conventional drugs always have some serious defects, such as high cytotoxicity and low tumor specificity. Although the currently clinically approved targeted drugs for liver cancer treatment achieve excellent clinical results in the aspect of treatment, the serious hand-foot skin disease side reaction is a challenge for the wide application of the targeted drugs. The nano drug-loaded complex is rapidly developed as a new research means, and is combined with drugs in a certain way through a proper carrier to prepare the nano-sized drugs, the particle size of the nano-sized drugs is within the range of 1-1000 nm, and the nano drug-loaded complex has high permeability and retention effect on tumors. However, the development of the nano-carrier in clinical application is limited due to the disadvantages of low drug loading rate, high biological toxicity and the like. And the treatment effect of the existing nano material for treating liver cancer in a tumor-bearing animal model is not ideal. Furthermore, although there have been many studies related to targeted delivery of chemotherapeutic drugs to liver cancer, systems for liver cancer treatment have not been fully explored. The target synergetic chemo-photothermal therapy can trace in vivo and in vitro, and the nano drug-loaded complex with the drug irritation response release function is rarely reported.
Therefore, how to construct a medicament which has low toxicity, better target synergistic chemical and photothermal curative effects, can trace in vivo and in vitro is urgent and has great challenges.
Disclosure of Invention
The technical problem is as follows:
the invention provides a novel integrated therapeutic drug for treating liver cancer by designing and preparing a biological safety nano complex integrating the functions into a whole; the obtained medicine has good medicine carrying effect and can realize low toxicity and excellent treatment effect.
The technical scheme is as follows:
the graphene oxide is used for constructing a carrier in the medicine, the carrier has a lamellar structure with a large specific surface area, and a surface is modified with a biocompatible connecting structure (such as polyethylene glycol (PEG)) and a targeting part of Lactobionic Acid (LA); the material has good near infrared light absorption performance of 808nm, and the temperature change shows concentration dependence and excitation intensity dependence; it can be used to trace in vivo transport and distribution using fluorescence imaging techniques.
Graphene oxide belonging to sp2The hybrid carbon nanomaterial has a large specific surface area, is rich in functional groups and is easy to further modify. The color of the material is brown yellow, the material can be used as a photothermal material, and under the irradiation of near infrared light with strong penetrating power to human tissues, the material is actively or passively enriched on an affected part to generate heat by combining various targeting technologies, so that the purposes of destroying tumor tissues and treating tumors are achieved. Studies have shown that the hepatic cell surface-specifically expressed asialoglycoprotein receptor (ASGPR) can mediate drug delivery as a targeted receptor, which can recognize and bind to molecules with exposed galactose, N-acetylgalactosamine or glucose residues and is present on the surface of liver cancer cell linesAnd (4) expressing.
The invention aims to provide a medicine (GO-linker/LA-CUR) for synergistic chemo-photothermal therapy and targeted therapy of liver cancer, which is prepared by loading Curcumin (CUR) by using a nano-carrier material as a carrier; the preparation method of the nano carrier material comprises the following steps:
(1) dispersing graphene oxide in water, adding an alkali reagent to adjust the pH value to 7.8-8.5, then adding a connecting reagent and a condensing agent to react, filtering after the reaction is completed, taking a precipitate, and washing to obtain graphene oxide (GO-linker) modified by a connecting group; the connecting reagent is polyethylene glycol or polyethylene glycol (NH) modified by terminal amino2-PEG2000-NH2) Or polydopamine;
(2) dispersing Lactobionic Acid (LA) and a condensing agent in water, and activating at 0-5 ℃; and (2) adding the polyethylene glycol functionalized graphene oxide obtained in the step (1), carrying out condensation reaction at room temperature, and after the reaction is finished, carrying out solid-liquid separation to obtain the nano carrier material GO-linker/LA.
In one embodiment of the present invention, the method for preparing the medicament is: the nano carrier material and curcumin are mixed according to the mass ratio of 1: (2-4) dissolving in a medium, adjusting the pH to be alkaline, and reacting at room temperature. Wherein, the mass ratio is preferably 1: 3. the medium is absolute ethyl alcohol. The pH is preferably 7.5 to 9.5. The reaction time is 20-30 h.
In one embodiment of the present invention, the structure of the nano-carrier material is represented by formula (1):
wherein A is selected from one or more of the following chemical structures, which may be the same or different, having biocompatibility: polyethylene glycol, terminal amino-modified polyethylene glycol or polydopamine; b is selected from one or more of the same or different monosaccharide or oligosaccharide molecular structures with galactose or galactosamine residues, and the structures can specifically recognize the asialoglycoprotein receptor overexpressed on the surface of the liver cancer cell; graphene oxide has a nanosheet structure.
In one embodiment of the present invention, the mass ratio of the graphene oxide to the linking reagent in the step (1) is 2.5: (2-3). Preferably 2.5: 2.
in one embodiment of the present invention, the condensing agent in the step (1) is EDC and NHS. Wherein the mass ratio of EDC to graphene oxide is (12-18): 1, preferably 16: 1. the mass ratio of NHS to graphene oxide is (12-18): 1, preferably 16: 1.
in one embodiment of the present invention, the reaction in step (1) is continuously stirred at room temperature for 40 to 50 hours.
In one embodiment of the present invention, the step (1) further comprises performing dialysis after the washing. The dialysis is carried out in double distilled water for 72h, and the water is changed every 12 h.
In an embodiment of the invention, the step (1) further comprises performing ultrasonic dispersion on the obtained graphene oxide with linker modification (GO-linker).
In one embodiment of the present invention, the mass ratio of lactobionic acid to graphene oxide with linker modification in step (2) is (0.6-0.8): 1. preferably 0.7: 1.
in one embodiment of the present invention, the condensing agent in the step (2) is EDC and NHS. Wherein the mass ratio of EDC to graphene oxide modified by a linking group is (1-1.5): 1, preferably 1.15: 1. The mass ratio of NHS to graphene oxide is (1-1.5): 1, preferably 1.38: 1.
In one embodiment of the present invention, the step (2) further comprises washing and dialysis after the reaction is completed. The dialysis is carried out in double distilled water for 72h, and the water is changed every 12 h.
The invention also aims to provide a GO-linker/LA-CUR medicine for the chemical-near infrared photothermal combined targeted therapy of liver cancer, wherein the structure of the medicine is shown as the following formula II:
wherein,
is curcumin; a is selected from one or more of the following structures of compounds with biocompatibility, which are the same or different: polyethylene glycol or polyethylene glycol derivatives, polydopamine; b is selected from one or more of the same or different monosaccharide or oligosaccharide molecular structures with galactose or galactosamine residues, and the structures can specifically recognize the asialoglycoprotein receptor overexpressed on the surface of the liver cancer cell.
An acid-sensitive medicine for treating liver cancer by combining chemotherapy medicine and photothermal targeting, which has a lamellar structure with a large specific surface area, and is modified with biocompatible polyethylene glycol (PEG) and targeting part Lactobionic Acid (LA) on the surface; the material has good near infrared light absorption performance of 808nm, and the temperature change shows concentration dependence and excitation intensity dependence; it can be used to trace in vivo transport and distribution using fluorescence imaging techniques.
In one embodiment of the present invention, the preparation method of the drug specifically comprises the following steps:
step one, preparing PEG functionalized graphene oxide (GO-PEG)
1) 5mL of graphene oxide (0.5mg/mL) was adjusted to pH 8.0 with triethylamine, and 40mg of EDC,40mg of NHS and 2mL of PEG (NH) were added thereto2-PEG2000-NH2) (1mg/mL), the reaction was stirred at room temperature for 48 hours;
2) washing the obtained crude product with ultrapure water for three times (9000r/min, 30min), dialyzing for 72h, and changing water once every 12h to obtain polyethylene glycol functionalized graphene oxide (GO-PEG NPs);
3) carrying out ultrasonic dispersion by using a cell ultrasonic crusher to obtain a carrier stock solution;
step two, preparing lactobionic acid modified targeting nano composite material (GO-PEG/LA)
1) Dissolving 0.8mM lactobionic acid, 2.4mM EDC & HCl and 4.8mM NHS in 100mL ultrapure water, placing the solution in an ice water bath, and stirring and activating for 2 h; adding 400mg of the GO-PEG obtained in the step one after ultrasonic dispersion, and stirring for 24 hours at room temperature;
2) centrifuging and washing the obtained crude product, dialyzing in double distilled water for 72 hours, and changing water once every 12 hours to obtain the target nano composite material GO-PEG/LA;
step three, preparation of targeting drug-loaded complex (GO-PEG/LA-CUR)
1) Weighing 600mg curcumin, fully dissolving the curcumin in 100mL absolute ethyl alcohol, then adding 200mg GO-PEG/LA, adding NaOH to adjust the pH value to 8.0, and reacting for 24 hours at room temperature;
2) centrifuging the mixed solution at 13000r/min for multiple times, discarding supernatant, placing in a dialysis bag for dialysis for 72h, and changing the dialysate once every 12h to finally obtain the target product GO-PEG/LA-CUR.
Has the advantages that:
according to the invention, graphene oxide is modified by using a connecting structure, Lactobionic Acid (LA) is modified by an amide reaction, Curcumin (CUR) is successfully loaded by pi-pi accumulation, and the amount of curcumin loaded in the GO-linker/LA-CUR is about 56%; the material has good near infrared light absorption performance of 808nm, and the temperature change shows concentration dependence and excitation intensity dependence; it can trace in vivo transport and distribution by using fluorescence imaging technology; the particle size of the nano drug-loaded complex applied to liver cancer photothermal chemotherapy is about 260 nm.
The stability of GO-linker/LA-CUR is verified; in vivo and in vitro experiments prove that the compound has low biological toxicity, can specifically act on ASGPR high-expression liver cancer cells, has good thermochemical synergistic treatment effect, and can trace in vivo transport and distribution by using a fluorescence imaging technology. The GO-linker/LA-CUR is cooperated with photo-thermal to improve curative effect, and meanwhile, no obvious toxic or side effect is caused to a tested mouse, so that the GO-linker/LA-CUR has a very good market application prospect.
Drawings
FIG. 1: a synthetic circuit diagram of a medicine for cooperating with chemophotothermal therapy and targeted therapy of liver cancer.
FIG. 2: particle size distribution of the nano-carrier. A. Atomic force electron microscopy images of nanocarrier GO; B. thickness of nanocarrier GO; C. transmission electron microscopy of the nanocarrier GO; D. particle size distribution of the nano-carrier.
FIG. 3: A. ultraviolet-visible light characteristic spectrum of the medicine obtained in the example; B. an infrared elemental characterization map of the nanocomposite; C. a plot of hydrated particle size versus potential change for the nanocomposite; D. long-term hydrodynamic diameter change profile of the nanocomposite.
FIG. 4: A. in-vitro photothermal performance characterization chart of graphene oxide before and after modification (5min, 1.5W/cm)2) (ii) a B. In vitro photothermal performance characterization of nanocomplexes of different concentrations (5min, 1.5W/cm)2) (ii) a C, in-vitro photo-thermal performance characterization graphs of the nano-composite under different near-infrared irradiation intensities; D. in vitro release profile of the drug at different pH conditions at 37 deg.C/with or without near infrared irradiation.
FIG. 5: A. hemolysis rate under different concentration nanocomplex conditions; B. the cell survival rates of GO-PEG/LA with different concentrations and L02, HEK293T, HepG2 and Huh7 cells cultured for 24h respectively; C. the cell survival rates of the CURs with different concentrations in the L02, HEK293T, HepG2 and Huh7 cells after 24h culture respectively; D. the cell survival rates of GO-PEG/LA mixed with CURs with different concentrations and cultured with L02, HEK293T, HepG2 and Huh7 cells for 24h respectively; E. the cell survival rates of GO-PEG/LA-CUR with different concentrations and L02, HEK293T, HepG2 and Huh7 cells cultured for 24h respectively; F. under the condition of near infrared irradiation (5min, 1.5W/cm)2) Cell viability in HepG2 and Huh7 cells cultured for 24 h.
FIG. 6: HepG2 and Huh7 cells were cultured in medium (control), GO-PEG/LA, CUR, GO-PEG/LA-CUR, GO-PEG/LA-CUR + near Infrared light (NIR, 5min, 1.5W/cm)2) The staining pattern of live and dead cells was incubated for 24h in the presence of the enzyme, live cells were stained with Calcein-AM and dead cells were stained with PI. A scale: 75 μm.
FIG. 7: l02, HEK293T, Hepg2 and Huh7(A, B, C and D) were incubated with GO-PEG/LA/CUR in the presence or absence of galactose (GAL +) for 3 h.
FIG. 8: confocal laser mapping of hepg2, Huh7, L02 and HEK293T cells after incubation with GO-PEG/LA-CUR in the presence or absence of galactose (GAL +) for 3h, scale: 20 μm. B.Z-stack technology showed GO-PEG/LA-CUR in HepG2(a) and Huh7(b) cells, nuclei were stained with DAPI, and lysosome was stained with LysoTckerGreen DND-26.
FIG. 9: A. b is the distribution of the in vivo and in vitro tissues of the tumor-bearing mice after 4h, 8h, 12h, 18h and 24h of respectively injecting GO-PEG/LA-RhB and GO-PEG-RhB into tail veins. C. Volume change curves of tumor tissues of different treatment groups; D. mean body weight change curves for different treatment groups of mice. Data are presented as mean ± standard deviation (n ═ 5); p values were calculated by t-test (× P < 0.001).
FIG. 10: h & E staining of isolated tumors and major organ sections of different treatment groups (microscope magnification 200 ×), scale: 100 μm.
Detailed Description
Example 1: preparation of nano-carrier materials
Preparation of graphene oxide
Graphene oxide was prepared by Hummers method. Respectively weighing 1g of graphite powder, 0.5g of sodium nitrate and 3g of potassium permanganate, and grinding the potassium permanganate into powder. Adding 23mL of concentrated sulfuric acid into a 250mL beaker carefully, placing the beaker in an ice bath, slowly adding weighed graphite powder and sodium nitrate under magnetic stirring, reacting for 1h, and then carefully and slowly adding KMnO4And (3) performing reaction on the powder for 2 hours at a temperature not exceeding 10 ℃. The ice water bath was removed and placed in a constant temperature oil bath at 38 ℃ with constant stirring for 30 minutes. Adding 46mL of ultrapure water for dilution, keeping the temperature for reaction for half an hour when the oil temperature is increased to 98 ℃, adding a proper amount of warm water for further dilution, and using 30% by mass of H2O2The solution was treated until the mixture turned from tan to light yellow. Centrifuging while hot and mixing the mixture by volume ratio of 1: and (3) repeatedly washing the hydrochloric acid aqueous solution 10 for several times, discarding colorless transparent supernatant, and resuspending the precipitate in ultrapure water, putting the ultrapure water into a dialysis bag, and dialyzing for one week to remove ionic impurities, so as to obtain a brown-yellow graphene oxide aqueous solution.
Preparation of PEG-functionalized graphene oxide
5mL of graphene oxide (0.5mg mL) was treated with triethylamine-1) Adjusted to pH 8.0, 40mg EDC,40mg NHS and 2mL PEG (NH) were added2-PEG2000-NH2)(1mg mL-1) The reaction was stirred at room temperature for 48 hours to obtainThe crude product of (2) was washed three times with ultra pure water (9000rpm, 30min) and dialyzed for three days to obtain polyethylene glycol functionalized graphene oxide (GO-PEG NPs).
Preparation of lactobionic acid modified nanocomposites
0.8mmol lactobionic acid (286.64mg), 2.4mmol EDC & HCl (460.08mg), 4.8mmol NHS (552.4mg) were dissolved in 100mL ultrapure water and activated by stirring in an ice-water bath for 2 h. Adding 400mg of GO-PEG dispersed by ultrasonic, stirring for 24h at room temperature, centrifuging and washing the obtained crude product, and dialyzing in ultrapure water for three days to obtain the target lactobionic acid modified nano carrier material GO-PEG/LA.
Example 2: preparation of medicine GO-PEG/LA-CUR
Weighing 600mg curcumin, fully dissolving in 100mL absolute ethyl alcohol, adding 200mg GO-PEG/LA, adding NaOH to adjust the pH value to 8.0, reacting overnight at room temperature, centrifuging for 13000r/min for multiple times, removing supernatant, placing in a dialysis bag, and dialyzing for three days to obtain the target product medicine-carrying nano complex GO-PEG/LA-CUR. In order to study the drug loading capacity, the gradient drug proportion is set for overnight reaction, centrifugation is carried out at 13000r/min, supernatant is taken out, the absorbance of the supernatant at 425nm position is obtained by an ultraviolet spectrophotometer, the absorbance is substituted into a standard curve to obtain the loaded drug amount, and a series of calculations are carried out by a drug loading rate encapsulation rate formula shown below to obtain the drug loading rate and the encapsulation rate. Calculating the formula:
the drug loading rate (DLC) ═ actual mass of drug entrapped in drug-loaded nanocomposite/mass of drug-loaded nanocomposite × 100%
The encapsulation efficiency (DEE) is the actual mass of drug encapsulated in the drug-loaded nanocomposite/total mass of drug put into reaction × 100%.
Example 3: characterization of material morphology and particle size:
the synthetic route for GO-PEG/LA-CUR is shown in FIG. 1, where drug loading is designed as the final step to avoid unnecessary losses.
To verify the target product, a series of tests were performed. Dropping a sample on a copper mesh, and shooting by using a transmission electron microscope to obtain the appearance; dripping a sample on a silicon wafer, naturally drying, and shooting by using an atomic force electron microscope to analyze the thickness; adding the sample into a quartz vessel, and obtaining an ultraviolet-visible absorption curve through an ultraviolet-visible spectrophotometer; centrifuging the sample solution, dissolving the precipitate in an organic solvent, dripping the organic solvent on a sample loading position, and acquiring an infrared characteristic spectrum by using an infrared spectrum measuring instrument; the sample is placed in a sample cell, the hydrated particle size is measured by dynamic light scattering, and the zeta potential analysis is used for obtaining the potential. To verify the long-term hydration kinetic stability of the nanocomplex, the particle size change of GO-PEG/LA-CUR was monitored over a period of 7 days in pure water, phosphate buffer solution with a ph of 7.4, and cell culture medium containing 10% fetal bovine serum, respectively.
The atomic force electron microscope (fig. 2A) result shows that the thickness of the graphene oxide sheet is about 1.7nm (fig. 2B); the transmission electron microscope shows that the morphology of the graphene oxide is a lamellar structure (fig. 2C), and the size is about 200 nm; the electron microscope results preliminarily prove that the graphene oxide is successfully prepared. The dispersion coefficients of GO and GO-PEG/LA are 0.601 and 0.199 respectively, and the dispersion of the nano-composite in water is improved by the modification of PEG and LA through the combination of particle size distribution (figure 2D). From the uv-vis spectrum results (fig. 3A), it can be seen that the characteristic peak of GO at 230nm (related to the electronic transition of C ═ C double bond) and the characteristic peak shoulder at 300nm (related to the electronic transition of C ═ O) are clearly visible all the time after it modifies PEG and LA; it is worth noting that the GO-PEG/LA-CUR increases a distinct characteristic peak (a characteristic peak of curcumin) around 425nm, which can prove that the drug has been successfully loaded. The C-O bond (1382 cm) of GO can be seen in the Fourier transform infrared spectrum (FIG. 3B)-1) C ═ O bond (1730 cm)-1) Stretching vibration peak and O-H (3440 cm)-1) The stretching peak of (1). GO-PEG spectrum is 1460cm-1C-N tensile peak at and 1650cm-1C ═ O stretching vibration indicates that PEG was successfully covalently bound to GO through an amide bond; at 1090cm-1Broad C-O-C band, associated with ether linkage of PEG fragment. GO-PEG/LA spectrum at 1405cm-1The newly appeared peak in the vicinity is attributed to the O-H moiety, and 3024cm-1The nearby bands are related to amino and carboxylic acids. In addition, in the GO-PEG/LA-CUR spectrum, 1600cm-1、1280cm-1The peaks at (a) are associated with the ketone group and the hydroxyl group in curcumin, respectively. The infrared spectroscopy results further confirmed the successful preparation of the nanocomplexes. The hydrated particle size data (figure 3C) shows that,the average hydrated particle sizes from GO to GO-PEG/LA-CUR are 227.4 + -3.2 nm,238.1 + -7.3 nm,255.5 + -9.0 nm and 257.5 + -4.5 nm, and the particle sizes are slightly increased. zeta potential (figure 3C) shows that the potential of graphene oxide is-36.5 mv ± 2.2mv, consistent with previous reports, the nanocomposite shows magnitude changes of potential after modification of polyethylene glycol and lactobionic acid, namely-11.5 mv ± 0.4mv and-18.7 mv ± 0.5mv respectively, due to amino and carboxyl groups, suggesting a successful reaction; because the graphene oxide and the curcumin both have benzene ring structures, pi-pi accumulation is easy, the graphene oxide has large specific surface area, the curcumin can be physically adsorbed, and the experimental potential measurement result is-29.8 mv +/-1.19 mv, which indicates that the curcumin is effectively adsorbed. Figure 3D shows that the nanocomposite has stable hydration kinetic dimensions.
Example 4: in vitro photothermal study
To study the effect of GO-PEG/LA-CUR concentration on the photo-thermal effect, GO-PEG/LA-CUR (0-100. mu.g/mL) aqueous solutions with different concentrations, GO aqueous solution with 50. mu.g/mL, and GO/LA aqueous solution with 50. mu.g/mL received 1.5W/cm2808nm near infrared light for 300 s; to investigate the effect of the intensity of the incident infrared radiation power on the photo-thermal effect, experiments were conducted on GO-PEG/LA-CUR at different intensities (0W/cm) at 10. mu.g/mL2-1.5W/cm2)。
The results of the experiment (FIGS. 4A, B) are shown at 1.5W/cm2Under the condition that the near-infrared light continuously irradiates for 5min, the temperatures of GO, GO-PEG/LA and GO-PEG/LA-CUR solutions with the concentration of 50 mu g/mL are increased by about 8 ℃ after the irradiation is finished, and meanwhile, the temperature change of the GO-PEG/LA-CUR solution is increased along with the increase of the concentration; meanwhile, the temperature change of the GO-PEG/LA-CUR aqueous solution of 10 mu g/mL is found to increase along with the increase of the near infrared irradiation intensity, and the temperature change shows concentration dependence and intensity dependence (figure 4C). Studies have shown that moderate temperature changes of 3-6 ℃ can induce apoptosis by interfering with intracellular enzyme activity; higher temperature changes will destroy the cell membrane and kill the cells in 4-6 minutes. The GO-PEG/LA-CUR has good near infrared thermal effect and can be used as a photo-thermal agent for tumor ablation.
Example 5: in vitro drug Release study
In order to simulate the release conditions of curcumin in a normal body fluid environment and a tumor acid environment in GO-PEG/LA-CUR, phosphate buffer solution systems with the pH values of 7.4 and 5.6 and containing 0.5 % Tween 80 and 25% ethanol are respectively prepared, and the specific method comprises the following steps: adding 20mL of buffer solution into a 250mL beaker; taking 5mLGO-PEG/LA-CUR reaction solution, placing the reaction solution in a dialysis bag with the cut-off molecular weight of 3.5Kda, and immersing the dialysis bag in buffer solution; magnetic stirring was continued for 72h at 37 ℃ and 1mL of buffer in the beaker was aspirated at regular intervals while 1mL of phosphate buffer at the corresponding pH was replenished. The collected sample is measured by a microplate reader for fluorescence emission intensity at 530nm, and the curcumin content in the supernatant at each time point is calculated according to a curcumin standard curve established on the microplate reader. The curcumin release condition under the near infrared excitation condition is researched, and on the basis of the method, near infrared light (1.5W/cm) is applied every 1h2) The irradiation was continued for 5 min.
Curcumin is a traditional Chinese medicine with an anti-tumor effect, and the inherent yellow fluorescence of curcumin can be used for tracking the transportation release and the absorption distribution of a medicine in vitro. Curcumin was therefore selected as a hydrophobic anticancer drug to study drug loading and release behavior. The drug loading rate and the encapsulation rate of GO-PEG/LA are 56.82 +/-1.38% and 56.34 +/-2.05% by ultraviolet visible spectrophotometry. Drug release in GO-PEG/LA-CUR was studied by measuring the fluorescence intensity at 530nm (excitation light 425 nm). The results show (fig. 4D) that the drug release effect was significant in the first 12h, and the slow release phenomenon was exhibited thereafter, and the pH dependence and near infrared light excitation dependence were exhibited. In a pH 7.4 buffer solution, the drug release is less than 30% after 72 h; in the buffer at pH5.6, the release of the drug after 72h was close to 60%. When the reaction is carried out at 1.5W/cm per hour2After 5min of the near infrared light intervention, the release amount of the drug in the buffer solution with the pH value of 7.4 and the buffer solution with the pH value of 5.6 is respectively increased by about 10 percent and 30 percent. The pH dependent release pattern of CUR may be due to a reduction in the electrostatic interaction of PEG, LA with GO-PEG/LA-CUR under acidic conditions, resulting in the shedding and dissociation of PEG, LA from the GO-PEG/LA-CUR surface. Meanwhile, as the pH decreases, the electrostatic attraction between GO-PEG/LA-CUR and CUR weakens, resulting in a pH triggered release pattern of CUR. The enhancement of the CUR release under the irradiation of the near-infrared laser can be attributed toThis effect may weaken the hydrophobic interaction between CUR and GO-PEG/LA-CUR due to the photothermal effect of GO-PEG/LA-CUR. These results suggest that the drug release of GO-PEG/LA-CUR exhibits a dual response, with pH triggered drug release suitable for tumor treatment, while in combination with photothermal therapy can significantly improve the sensitivity of chemotherapy.
Example 6: toxicity test
Firstly, cytotoxicity of carrier material
The toxicity of the nano-carrier material is obtained by analyzing a cell survival rate analysis method (thiazole bromide blue tetrazole, namely an MTT method), and the detection principle is that the thiazole bromide blue tetrazole (MTT) can be reduced into blue-purple crystalline formazan substance which is insoluble in an aqueous solution by succinic dehydrogenase in mitochondria of living cells, so that the detection can be carried out by a colorimetric method, and MTT cannot be reduced and developed by dead cells. L02, HEK293T, HepG2 and Huh7 cell suspensions were diluted to a cell concentration of 5X 10, respectively4one/mL. Taking out a sterile 96-well plate, adding 100 mu L of cell suspension into each well, after cell growth in the well plate is stable in a 37 ℃ cell culture box for 24 hours, respectively adding nano materials with different concentrations (0 mu g/mL-60 mu g/mL), continuously incubating for 24 hours, washing for three times by using a sterile phosphate buffer solution (PBS, pH 7.4), respectively adding 100 mu L of culture medium which is not added with phenol red and contains 0.5mg/mL MTT into each well, placing in the incubator, continuously incubating for 4 hours, carefully sucking out the culture medium containing MTT, adding 100 mu L of dimethyl sulfoxide into each well, and uniformly mixing by shaking. The absorbance values (absorption wavelength 550nm) of all the wells in the plate were then detected with a multifunctional microplate reader.
The viability of the cells in each well was calculated as follows: cell survival rate ═ AbSample (I)/AbControl)×100％
In the formula, AbSample (I)Is the absorbance, Ab, of a set of samples of materials of different concentrationsControlIs the absorbance of a pure cell culture without material action.
Second, hemolytic toxicity test
Red blood cell hemolysis experiments are often used to detect the risk of hemolysis of biological materials. In general, lysis of erythrocytes in aqueous solutions is the test of haemotoxicity.
The target to be detected is prepared into leaching liquor according to the GB/T16886.5-2011 standard, the hemolysis rate is less than 5%, which indicates that the target meets the hemolysis requirement of the medical material, and the hemolysis risk is greater than or equal to 5%. The experimental negative control group adopts sterilized phosphate buffer solution (pH is 7.4), the experimental positive control group adopts sterilized double distilled water, and the experimental groups respectively comprise blank nano-carriers GO-PEG/LA, free drugs CUR and drugs GO-PEG/LA-CUR (the concentration range is 0-100 mug/mL, and the concentration of GO-PEG/LA-CUR is based on the drug concentration). Adding 200L of fresh goat red blood cell suspension into different groups, placing in a cell culture box at 37 ℃ for 2h, separating out mixed liquor of each group, centrifuging for 5min at 2500r/min, slightly absorbing 100 mu L of supernatant of each group to a 96-well plate, and detecting the absorbance value of each well under 545nm by using a multifunctional microplate reader. The hemolysis rate is calculated as follows:
hemolysis rate (Ab)Experimental group－AbNegative control group)/(AbPositive control group－AbNegative control group) X 100% blood, especially when exposed to exogenous substances, may exacerbate hemolysis.
The experimental results (FIG. 5A) show that 0-100 μ g/mL GO-PEG/LA, free curcumin and GO-PEG/LA-CUR have hemolysis rate lower than 5%, i.e. no hemolytic toxicity. Meanwhile, it can be easily seen that GO-PEG/LA-CUR containing the same concentration of CUR has better hemolytic toxicity than the simple CUR. The GO-PEG/LA has good biocompatibility, can be used as a biological carrier, and can reduce the hemolytic toxicity of free drugs. The detection of the biological toxicity of the blank vector is a key link for designing the nano complex, the MTT experiment result (figure 5B) of GO-PEG/LA shows that the cell toxicity is low in the concentration range of 0-60 mu g/mL, and the cell viability is maintained at about 80% after L02, HEK293T, HepG2 and Huh7 cells are respectively incubated with the blank vector of 60 mu g/mL for 24 hours. It is shown that graphene oxide has different cytotoxicity in different cells, and the cytotoxicity of graphene oxide with the concentration of 80 μ g/mL or less is negligible. This is in mutual support with our findings.
Example 7: study of the effects of in vitro drug therapy
To investigate the influence of GO-PEG/LA-CUR on cell viabilityL02, HEK293T, HepG2 and Huh7 cell suspensions were diluted to a cell concentration of 5X 10, respectively4one/mL. Taking out a sterile 96-well plate, adding 100 mu L of cell suspension into each well, after cells grow stably in the well plate after 24 hours at 37 ℃, simply and physically mixing the free drug, the free drug and the nano-carrier with different concentrations and incubating the drug-loaded nano-complex in the cells for 24 hours respectively, wherein the concentration range is 0 mu M-30 mu M according to the concentration of curcumin. After washing three times with sterile phosphate buffered saline (PBS, pH 7.4), 100 μ L of medium containing 0.5mg/ml MTT without phenol red was added to each well, and the wells were incubated in an incubator for 4 hours, followed by carefully aspirating the medium containing MTT, adding 100 μ L of dimethylsulfoxide to each well, and mixing well. The absorbance values (absorption wavelength 550nm) of all the wells in the plate were then detected with a multifunctional microplate reader. The calculation was performed in the same manner as in example 6.
To investigate the in vitro photothermal efficacy of GO-PEG/LA-CUR, HepG2 and Huh7 cell suspensions were diluted to a cell concentration of 5X 10, respectively4one/mL. Setting a series of photothermal groups (a control group, a nano-carrier group, a dissociative drug group and nano-carrier physical mixed group and a nano-carrier group according to experiment needs, taking out a sterile 96-well plate, adding 100 mu L of cell suspension into each well, placing the plate in an incubator at 37 ℃ for incubation for 24 hours, discarding an old culture medium, washing the plate with PBS for three times, then respectively adding 100 mu L of fresh culture medium and 20 mu g/mL of nano-carrier into each group (the concentration of the carrier in the nano-carrier complex used is not more than 20ug/mL, so 20ug/mL is used as a control), 10 mu M of dissociative drug, 20 mu M of dissociative drug, 30 mu M of dissociative drug, 10, 20 and 30 mu M of dissociative drug, respectively serving as final concentrations, namely GO-PEG/LA + CUR of the nano-carrier group, and GO-PEG/LA-CUR of the nano-carrier complex with respectively 10, 20 and 30 mu M of drug, placing in an incubator at 37 ℃ for further incubation for 24h (wherein, concentrations of drug and carrier in GO-PEG/LA + CUR and GO-PEG/LA-CUR are consistent). After 3h incubation, 808nm near-infrared irradiation (1.5W/cm) was performed25 min). Cell viability was measured by the MTT assay as in example 6.
The results show that: compared with GO-PEG/LA-CUR, free CUR can directly contact cells to exert curative effect without passing through a drug release stage, so that the inhibition effect on normal cell tissues cannot be ignored. In order to better understand the in vitro cell curative effect of GO-PEG/LA-CUR, two hepatoma cells HepG2 and Huh7 and two normal cells L02 and HEK293T are selected to carry out MTT experiments of CUR, physically mixed CUR + GO-PEG/LA and GO-PEG/LA-CUR, and it can be seen that the cell viability shows a trend of decreasing with the increase of the drug concentration, when the cells are respectively incubated for 24h by using free drugs with the concentration of 30 mu mol/L (figure 5C), the cell viability of the normal cells is about 50%, and the hepatoma cell viability is about 60%; after the cells were incubated with the mixture of the free drug and the nanocarrier for 24 hours (fig. 5D), the cell viability of the normal cells of the group with the drug concentration of 30 μmol/L was lower than about 50%, and the liver cancer cell viability was about 55%; (ii) a After the GO-PEG/LA-CUR cells are respectively incubated for 24 hours by the drug-containing concentration of 30 mu mol/L (shown in figure 5E), the normal cell viability is 60 percent, and the viability of the liver cancer cell line is about 50 percent; the GO-PEG/LA-CUR group demonstrated the best cellular efficacy after near infrared light treatment (FIG. 5F). The GO-PEG/LA-CUR can reduce the toxic and side effect of free CUR on normal cells, the CUR + GO-PEG/LA cannot reduce the toxic and side effect on the normal cells, meanwhile, the near infrared excitation can more obviously improve the cancer cell treatment effect of GO-PEG/LA-CUR, and the phenomenon can be related to the drug-loaded nano complex body having the capability of active and passive transport and the drug release condition.
Example 8: dyeing by living and dead
In order to further evaluate the treatment effect of GO-PEG/LA-CUR on liver cancer cells, a living and dead cell staining experiment is carried out by selecting a drug-containing concentration of 20 mu mol/L, a control group, a nano-carrier group, a dissociative drug group, a nano-carrier drug group and a nano-carrier drug + near infrared group are arranged according to the experiment requirements, and HepG2 and Huh7 cell suspensions are diluted to a cell concentration of 1 multiplied by 105And (2) culturing cells directly by using confocal dishes, slowly adding 1.5mL of cell suspension into the groove of each dish, incubating for 24 hours in an incubator at 37 ℃, discarding the old culture medium, respectively adding 1mL of fresh culture medium, 20 mu g/mL of nano-carrier, 20 mu M of free drug and 20 mu M of drug-loaded nano-complex, and continuing to incubate for 24 hours. Wherein the last group is irradiated by infrared light (5min, 1) when adding drug-loaded nano complex and incubating for 3h.5W/cm2). And after 24 hours, slightly sucking the supernatant in each cuvette, centrifuging for 5min at 300g, discarding the supernatant, respectively adding 200 mu L of 1 xAssay Buffer and 100 mu L of Calcein-AM/PI staining working solution, uniformly mixing, completely transferring to the cuvette groove, continuously incubating for 15min at 37 ℃, and using the cuvette for operation and observation under a laser confocal microscope. Live cells were detected at 490 nm. + -. 10nm and dead cells were detected at 545 nm.
In order to visually observe the treatment effects of the nano drug-loaded complex and the near-infrared irradiation, the survival condition of the cells is observed under a low-power microscope through a laser confocal microscope, and the results (figure 6) can visually see that the blank control and the GO-PEG/LA have no significant difference in the density and survival state of the liver cancer cells and have no influence on the cell viability; the curative effect of GO-PEG/LA-CUR cells containing 20 mu mol/L of medicine is better than that of free CUR, and 1.5W/cm2The GO-PEG/LA-CUR shows the optimal cell treatment effect after 5min of near-infrared irradiation. These results are in disagreement with MTT experimental results, further demonstrating that nanocomplexes can be used in conjunction with thermochemical therapy for cancer treatment.
Example 9: targeted validation assay
In order to verify the targeting property of GO-PEG/LA-CUR, a flow cytometry quantitative experiment and a laser confocal qualitative experiment are carried out. Selecting hepatoma cell lines HepG2 and Huh7 as positive cells of high expression ASGPR; l02 (liver normal cells) and HEK293T (kidney normal cells) served as ASGPR negative control cells. After the GO-PEG/LA-CUR cells containing 20 mu mol/L of medicine are incubated for 3h, the fluorescence intensity of cells without adding galactose (GAL-) in HepG2 and Huh7 is respectively 4 times and 2 times of that of cells with 1mM galactose (GAL +), while the fluorescence intensity of cells with control cells is not greatly influenced by the incubation with galactose; it was observed by confocal laser microscopy (fig. 8A) that there was significant yellow fluorescence in ASGPR positive cells, whereas yellow fluorescence was not significant in liver cancer cells and ASGPR negative control cells in which galactose competition was added. The GO-PEG/LA-CUR modified by lactobionic acid can specifically recognize ASGPR positive cells, thereby promoting endocytic uptake of the cells, which is consistent with flow-type results. The Z-stack technology is used for co-localization research of GO-PEG/LA-CUR in cells, red represents lysosome, blue represents cell nucleus and yellow is curcumin, and the result (figure 8B) shows that GO-PEG/LA-CUR enters cells and then is distributed in cytoplasm and cell nucleus, which is probably related to the anti-tumor action mechanism of curcumin. The in vitro transfer experiment result proves that the GO-PEG/LA-CUR has good targeting performance, can target and identify ASGPR on the surface of a liver cancer cell line, and enhances the active transfer of liver cancer cells to drugs, thereby promoting the drugs to further play a role in the cells.
The specific process comprises the following steps:
flow cytometry: in order to verify the targeting ability of GO-PEG/LA-CUR, a flow cytometer detection experiment was performed. L02, HEK293T, HepG2 and Huh7 cell suspensions were diluted to a cell concentration of 2X 105one/mL. Taking out a sterile 48-well plate, setting a control cell hole, a competition administration hole (galactose +) and a target administration hole (galactose-), adding 500 mu L of cell suspension into each hole according to the experiment requirement, putting the cell suspension into an incubator at 37 ℃ for incubation, adding galactose with the final concentration of 1mmol/L into the competition holes after overnight adherence, continuing the culture for 24 hours, carefully sucking the culture medium in the holes until the cell density exceeds 90%, and carefully washing the cells twice by using PBS (phosphate buffered saline) with the pH value of 7.4. To a blank cell well, 500. mu.L of medium was added, and the remaining two wells were added with drug-loaded nanocomplexes, wherein the final concentration of galactose was maintained at 1mmol/L throughout the competing dosing wells. Putting a 48-pore plate into an incubator, after incubating for 3 hours, firstly sucking the supernatant into a centrifuge tube, digesting the rest adherent cells by 40 mu L of pancreatin, adding 500L of phosphate buffer solution containing 10% fetal calf serum after 2 minutes, transferring the successfully digested cells into the centrifuge tube, centrifuging for 4 minutes at 1040rpm, carefully discarding the supernatant, then re-suspending by using the phosphate buffer solution, repeating the centrifugal washing process for three times, finally re-suspending by using 0.2mL of the phosphate buffer solution containing 10% fetal calf serum for detection and analysis by using a flow cytometer.
Laser confocal qualitative experiment: to further validate the targeting ability of GO-PEG/LA-CUR, L02, HEK293T, HepG2 and Huh7 cell suspensions were diluted to a cell concentration of 5X 10 as required for the experiments4one/mL. Remove sterile 12-well plate and set controlCell holes, competitive administration holes (galactose +) and targeted administration holes (galactose-), adding 100 microliter culture medium into each hole, carefully clamping a cell slide by using tweezers sterilized by flame, placing the cell slide into a pore plate, carefully pressing the cell slide into the bottom of the pore plate by using a pipette tip to enable the cell slide and the pore plate to be tightly attached without bubbles, and then uniformly and slowly adding 2mL cell suspension into the holes. After overnight adherence, galactose was added to the competitive dosing wells to a final concentration of 1mmol/L and incubation was continued for 24 hours. Taking out the 12-hole plate, sucking out the culture medium of each hole, simultaneously supplementing the fresh culture medium into the blank hole, adding GO-PEG/LA-CUR and galactose into the competition hole, adding the loaded nano complex into the targeting hole, and ensuring that the final concentration of the galactose is 1mM and the concentration of the curcumin is 20 mu M. After incubation for 3h, the medium was removed from each well, washed three times with 500 μ L PBS and shaken on a shaker for 5 min/time; adding 500 μ L of 4% paraformaldehyde, fixing at room temperature for 15min, removing the fixing solution, washing with 500 μ L of PBS for three times, and shaking with shaking table for 5 min/time; carefully taking out the climbing film in the pore plate by using tweezers, enabling the climbing film to be reversely buckled on a glass slide on which a drop of glycerol DAPI is dropped, coating colorless nail polish on the edge of the climbing film for sealing, placing the prepared film in a ventilation position, and drying in the dark place for operation and observation under a laser confocal microscope.
Example 10: biodistribution of nanocomplexes in a transplanted tumor mouse model
In order to understand the targeting performance and the transport distribution condition of a nano carrier in a living body, two blank carriers including GO-PEG and GO-PEG/LA are designed, and a RhB fluorescent dye is used as a mark to carry out directional tracing on the transport of the nano carrier in a tumor-bearing mouse. The results show (fig. 9A) that the carrier injected via tail vein is transported into the body of the mouse with blood, the fluorescence intensity of the tumor part of the two groups of mice shows an enhancement trend with the time, and reaches the strongest 12h after injection, and the fluorescence intensity of the GO-PEG/LA group is obviously higher than that of the GO-PEG group, which suggests that the former has good targeting performance in vivo, so that the former can be effectively accumulated at the tumor part. Although the fluorescence intensity at the tumor site decreased after the injection, stronger fluorescence was still detected in the GO-PEG/LA group compared to the other group 24h after the injection. This means that GO-PEG/LA still has good in vivo retention capacity. It was found from ex vivo tumors and organs obtained at different time points (fig. 9B) that 4 hours after injection, fluorescence signals from the liver could be observed; obvious fluorescent signals appear at the kidney part after 8 hours; fluorescence signals from the liver and kidney parts disappear after 24 hours; in addition, no fluorescence signals from heart, spleen and lung were observed within 24 h. These phenomena may be attributed to the uptake properties of phagocytes by the liver and the excellent metabolic clearance function of the liver and kidney. Imaging results strongly suggest that GO-PEG/LA has excellent liver cancer cell targeted delivery characteristics and long-acting accumulation retention performance.
The specific process comprises the following steps: the research is approved by the ethical committee of experimental animals of the south of the Yangtze river university, conforms to the principles of animal protection, animal welfare and ethics, and strictly complies with the relevant regulations of the ethics of the national experimental animals welfare. For the establishment of the transplant tumor nude mouse model, 5-week-old female BALB/c nude mice with average body weight of 18-20g are inoculated with 100 μ L of 6 × 106one/mL suspension of Huh7 cells, closely focused on the neoplastic condition, measured tumor volume every other day until the tumor volume exceeds 100mm3Time (volume ═ tumor tissue length x tumor tissue width)2X 0.5), dividing the mice into two groups, injecting 5 mice each with GO-PEG/LA-RhB and GO-PEG-RhB which are fluorescently labeled with rhodamine B (RhB) for 4h, 8h, 12h, 18h and 24h respectively, and observing in vivo distribution by using a living body imaging system of the small animals. Dissecting out the main organs, such as heart, liver, spleen, lung, kidney and subcutaneous tumor, and analyzing and recording by using a small animal living body imager.
Example 11: study of therapeutic Effect in vivo in mouse model with transplanted tumor
To study the in vivo therapeutic effect of GO-PEG/LA-CUR, a 2-week tumor-bearing mouse treatment experiment was performed. FIG. 9C shows the tumor volume changes in mice in each group during the treatment period, and the mean tumor volume increases with time in the saline group, approaching 600mm at 14 days after injection3(ii) a The GO-PEG/LA group mice have no influence on tumor growth, and the trend and the degree of the GO-PEG/LA group mice are the same as those of a normal saline group, which indicates that the GO-PEG/LA has no biological toxicity; the tumor growth curve of the simple CUR group is below the normal saline group, indicating that tumor growth is inhibited; the tumor growth difference between the CUR + NIR irradiation group and the simple CUR group is not obvious,the radiation does not influence the physicochemical property of the medicine in the body; the tumor growth of the mice treated by GO-PEG/LA-CUR is slower than that of the mice treated by CUR, which is related to the targeting property and EPR effect of the nano drug-loaded complex; tumors of mice treated with GO-PEG/LA-CUR + NIR grew slowly throughout the treatment period, and differences from the other groups became significant at the end of treatment (p <0.001), probably due to the good targeted photochemical co-therapeutic properties of GO-PEG/LA-CUR. Tumor Inhibition Ratios (TIRs) of CUR, CUR + NIR, and GO-PEG/LA-CUR 48%, 49% and 62%, respectively, and GO-PEG/LA-CUR + NIR 90% of TIRs in mice were then calculated, indicating that GO-PEG/LA-CUR has excellent tumor growth inhibition and potential for reversal of growth under near infrared excitation. After 14 days of treatment, the body weight of each group of mice increased slightly (fig. 9D), indicating that the side effects of the treatment were not significant. H from ex vivo tumors&E staining results (FIG. 10) it can be observed that the GO-PEG/LA-CUR + NIR group has significant tumor necrosis, revealing that GO-PEG/LA-CUR + NIR has the best anti-tumor effect. Simultaneous ex vivo organ H&No obvious tissue damage appeared in the E staining (figure 10), further indicating that the GO-PEG/LA-CUR has the properties of low biological toxicity and good biocompatibility.
The specific process comprises the following steps: in a mouse model with subcutaneous tumor transplantation, the tumor volume is monitored every other day until the volume reaches 50mm3Randomly dividing mice into 6 groups, injecting physiological saline, blank nano-carrier, free drug and drug-loaded nano-complex respectively, wherein the 4 th group and the 6 th group are subjected to photothermal treatment (1.5W/cm) 12h after the injection of the free drug and the drug-loaded nano-complex25 min). The injection dose is 9.8mg/kg, and the injection is injected once every other day for 14 days continuously. The body weight and tumor volume of each group of mice were recorded every other day from the day before injection, and the main organs (heart, liver, spleen, lung and kidney) of the mice were dissected and isolated the last day, and the subcutaneous tumor tissue was peeled off and weighed. After each major organ and tumor tissue were immersed in 4% paraformaldehyde for 2 days, paraffin sections of 5 μm thickness were prepared, and hematoxylin and eosin staining was performed, and then the morphology of each tissue cell was observed under an optical microscope and photographed and recorded.
In summary, the GO-PEG/LA-CUR nanocomposite constructed by the method can realize targeted photochemical cooperative therapy. The biocompatible nanosheet layer is prepared through PEG modification, and the targeting part lactobionic acid is modified through an amide reaction, so that the prepared GO-PEG/LA-CUR has good photo-thermal performance under 808nm near-infrared radiation. In vitro experiment results show that the GO-PEG/LA-CUR nano complex has good biological safety and liver cancer cell targeting characteristics. The compound has excellent cancer cell inhibiting effect under near infrared laser irradiation, which is attributed to high drug loading and drug dual-phase response release behavior and inherent photothermal conversion performance. In vivo experiments show that the chemophotothermal synergistic therapy has obvious effect of inhibiting tumor growth and has no obvious toxic or side effect on normal organs. These results indicate that GO-PEG/LA based nanocomposites are promising candidate systems for directed light chemo-synergistic cancer therapy, providing a valuable approach to building a therapeutic platform for clinical applications.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A medicine for synergistic chemophotothermal therapy and targeted therapy of liver cancer is characterized in that the medicine is prepared by taking a nano-carrier material as a carrier and loading curcumin; the preparation method of the nano carrier material comprises the following steps:
(1) dispersing graphene oxide in water, adding an alkali reagent to adjust the pH value to 7.8-8.5, then adding a connecting reagent and a condensing agent to react, filtering after the reaction is completed, taking a precipitate, and washing to obtain graphene oxide GO-linker modified by a connecting group; the connecting reagent is polyethylene glycol, polyethylene glycol modified by terminal amino or polydopamine;
(2) dispersing Lactobionic Acid (LA) and a condensing agent in water, and activating at 0-5 ℃; and (2) adding the polyethylene glycol functionalized graphene oxide obtained in the step (1), carrying out condensation reaction at room temperature, and after the reaction is finished, carrying out solid-liquid separation to obtain the nano carrier material GO-linker/LA.
2. The drug as claimed in claim 1, which is prepared by using a nano-carrier material and curcumin in a mass ratio of 1: (2-4) dissolving in a medium, adjusting the pH to be alkaline, and reacting at room temperature.
3. The drug of claim 1, wherein the structure of the nanocarrier material is represented by formula (1):
wherein A is selected from one or more of the following chemical structures, which may be the same or different, having biocompatibility: polyethylene glycol, terminal amino-modified polyethylene glycol or polydopamine; b is selected from one or more of the same or different monosaccharide or oligosaccharide molecular structures with galactose or galactosamine residues, and the structures can specifically recognize the asialoglycoprotein receptor overexpressed on the surface of the liver cancer cell; graphene oxide has a nanosheet structure.
4. The drug according to claim 1, wherein the mass ratio of the graphene oxide to the linking reagent in the step (1) is 2.5: (2-3).
5. The pharmaceutical of claim 1, wherein the condensing agent in step (1) is EDC and NHS.
6. The pharmaceutical according to claim 1, wherein the reaction in step (1) is carried out under stirring at room temperature for 40 to 50 hours.
7. The medicament of claim 1, wherein step (1) further comprises dialysis after washing.
8. The medicament according to claim 1, wherein the mass ratio of lactobionic acid to graphene oxide with linker modification in step (2) is (0.6-0.8): 1.
9. the pharmaceutical of claim 1, wherein the condensing agent in step (2) is EDC and NHS.
10. The medicine for treating liver cancer by combining chemistry and near infrared photothermal targeting is characterized by having a structure shown in a formula (II):
wherein,
is curcumin; a is selected from one or more of the following structures of compounds with biocompatibility, which are the same or different: polyethylene glycol or polyethylene glycol derivatives, polydopamine; b is selected from one or more of the same or different monosaccharide or oligosaccharide molecular structures with galactose or galactosamine residues, and the structures can specifically recognize the asialoglycoprotein receptor overexpressed on the surface of the liver cancer cell.
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