CN112402619A - Medicine carrying system for treating tumors based on near-infrared carbon quantum dot chemical-photothermal synergistic effect and preparation method thereof - Google Patents
Medicine carrying system for treating tumors based on near-infrared carbon quantum dot chemical-photothermal synergistic effect and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared carbon quantum dot-based medicine carrying system for chemical-photothermal synergistic treatment of tumors, which comprises a nano-carrier, a chemotherapeutic medicine and a targeting functional molecule, wherein the chemotherapeutic medicine is a cis-platinum derivative, the nano-carrier is a near-infrared carbon quantum dot, the targeting functional molecule is a polyethylene glycol composite nano-material with a pH response charge reversal characteristic, and the medicine carrying system is prepared by coupling the chemotherapeutic medicine and the nano-carrier and then complexing with the targeting functional molecule. The invention can play a synergistic role of chemical treatment and photothermal treatment in tumor treatment, can completely eliminate the tumor in a nude mouse, has no tumor recurrence and small toxic and side effects on organisms, and has a treatment effect remarkably superior to that of single chemical treatment or photothermal treatment. The chemical-photothermal synergistic treatment scheme has the clinical integrated application potential from tumor imaging and drug tracking to anti-tumor treatment, and has great prospect in the high-efficiency clinical tumor treatment application.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biological medicines, in particular to a medicine carrying system for treating tumors by using near-infrared carbon quantum dots in a chemical-photothermal synergistic manner and a preparation method thereof.
[ background of the invention ]
Over the past decade, cancer theranostics, a combination of bio-imaging diagnostics and cancer therapy, has been an emerging area that provides personalized cancer therapy for patients. In the course of cancer treatment, chemophotothermal therapy is considered as one of the most effective methods, in which hyperthermia caused by photothermal therapy enhances cellular uptake and release of chemotherapeutic drugs and even suppresses multidrug resistance of tumors, and chemotherapy is a continuous treatment without spatial limitation and can make up for the deficiency of photothermal therapy.
The selection of photothermal agents is critical to achieving high efficiency photothermal therapy. The existing photothermal reagent mainly comprises a noble metal nano material and an organic near-infrared chromophore, wherein the noble metal nano material has the defects of high cost, potential long-term toxicity, nondegradable property and the like, for example, a gold nanoshell packaged by polyethylene glycol-5000 is proved to be difficult to be discharged out of a body through a renal gap; the existing organic near-infrared chromophore has the defects of poor hydrophilicity, poor light stability, long-term toxicity and the like. Carbon Quantum Dots (CQDs) are a new star in the field of carbon materials, and are gaining favor of more researchers due to their unique intrinsic properties, such as near infrared absorption, long fluorescence lifetime, low toxicity, good light stability and biocompatibility. Carbon dots have been reported to be currently used as photothermal agents for photothermal therapy. On the other hand, the nano-carrier has been used as an imaging-guided nano-carrier for transporting chemotherapeutic drugs, photosensitizers, therapeutic genes and the like by utilizing the excellent biocompatibility, good water solubility and sensitive fluorescence imaging characteristics of the nano-carrier. Infrared emitting fluorescent carbon dots (RCQDs) can provide important components for full color emission, can be excited under long wavelength light irradiation and have better organ penetration depth for transporting drugs and PTT, and are very beneficial for application in the biomedical field.
While highly effective antineoplastic drugs are the key to chemotherapy. The existing antitumor drug is mainly cisplatin (Pt (II)), but has the defects of large toxic and side effects, difficulty in enrichment in tumor tissues, easiness in nonspecific combination of other biomolecules and the like. In order to reduce the toxic and side effects, researchers use other cisplatin derivatives such as leplatin and oxaliplatin for treatment. However, these cisplatin derivatives may show a decrease in drug load or a decrease in antitumor activity. In addition, the carrier used as the tumor drug carrier also has the capability of controllable drug release. The existing drug delivery carrier is adsorbed on the surface of the nano probe through simple physics, and after the nano probe enters into a living body, some loaded drug molecules can be quickly released from the nano probe in blood, namely are released into the blood or are retained in peripheral tissues of a tumor part before reaching the tumor part, and can not be effectively gathered at the tumor part, so that the treatment effect is influenced.
Therefore, if the carbon-based quantum dots can be used as a carrier, and a chemical photo-thermal synergistic tumor treatment drug-loading system is prepared by selecting a high-efficiency cisplatin derivative anti-tumor drug and a carrier for actively transporting and releasing the drug in a targeted manner, the carbon-based quantum dots have a wide application prospect.
[ summary of the invention ]
The invention aims to solve the problems and provides a chemical photothermal synergistic tumor treatment drug loading system based on near-infrared carbon quantum dots.
The invention also provides a preparation method of the medicine carrying system for treating tumors by using the near-infrared carbon quantum dots in a chemophotothermal synergistic manner.
In order to achieve the purpose of the invention, the invention provides a near-infrared carbon quantum dot-based chemical-photothermal synergistic tumor treatment drug carrier system, which comprises a nano carrier, a chemotherapeutic drug and a targeting functional molecule, wherein the chemotherapeutic drug is a cis-platinum derivative, the nano carrier is a near-infrared emission fluorescent carbon quantum dot, the targeting functional molecule is a polyethylene glycol-chitosan polymer with pH response charge reversal characteristics, and the drug carrier system is prepared by coupling the chemotherapeutic drug and the nano carrier and then complexing with the targeting functional molecule.
The near-infrared carbon quantum dots are aminated infrared emission fluorescent carbon quantum dots, and the carbon quantum dots are prepared by taking osmanthus fragrans seed coats as a carbon source.
The targeting functional molecule is polyethylene glycol-chitosan-dimethyl maleic anhydride polymer.
The invention also provides a preparation method of the medicine carrying system for treating tumors by using the near-infrared carbon quantum dots in a chemical-photothermal synergistic manner, which comprises the following steps:
a. preparation of aminated infrared emission fluorescent carbon quantum dot
Placing the osmanthus fragrans seed peels and absolute ethyl alcohol into a reaction container, heating to 100 ℃ under the protection of nitrogen, stirring, reacting for 12 hours, cooling the obtained reaction product to room temperature, filtering supernate, and obtaining filtrate, namely the infrared emission fluorescent carbon quantum dots; reacting the infrared emission fluorescent carbon quantum dots with an amino-polyethylene glycol-amino absolute ethyl alcohol solution at 120 ℃, filtering supernate, purifying filtrate by using a dialysis membrane to obtain the aminated infrared emission fluorescent carbon quantum dots, performing rotary evaporation and drying to obtain a solid sample, and storing the solid sample at 4 ℃ for later use;
b. amination infrared emission fluorescent carbon quantum dot modified cisplatin derivative
Uniformly stirring the cisplatin derivative, hydrogen peroxide and 1- (3- (dimethylamino) propyl) -3-ethyl diimine hydrochloride at room temperature, adding the aminated infrared emission fluorescent carbon quantum dots, uniformly stirring, dialyzing, and freeze-drying to obtain a modified infrared emission fluorescent carbon quantum dot-cisplatin derivative;
c. synthesis of targeting functional molecules
Reacting chitosan with active ester of polyethylene glycol to obtain polyethylene glycol-chitosan, dissolving the polyethylene glycol-chitosan in phosphate buffer solution with the pH value of 8.0, adding dimethyl maleic anhydride, and reacting to obtain a targeting functional molecule polyethylene glycol-chitosan-dimethyl maleic anhydride polymer;
d. polyethylene glycol-chitosan-dimethyl maleic anhydride is complexed with infrared emission fluorescent carbon quantum dot-cisplatin derivative
Dripping the infrared emission fluorescent carbon quantum dot-cisplatin derivative solution into a polyethylene glycol-chitosan-dimethyl maleic anhydride solution, stirring at room temperature, and freeze-drying to obtain the drug-carrying system infrared emission fluorescent carbon quantum dot-cisplatin derivative/polyethylene glycol-chitosan-dimethyl maleic anhydride.
In the step a, 2ml of absolute ethyl alcohol is added into each gram of osmanthus fragrans seed husk, the supernatant is filtered by a 0.22 micron organic filter membrane, and the molecular weight cut-off of the dialysis membrane is 1000.
In the step b, the synthetic method of the cisplatin derivative comprises the following steps: suspending cis-diamminedichloroplatinum in water, adding hydrogen peroxide at 50 ℃, stirring, cooling to room temperature, performing rotary evaporation, concentrating the solvent to 2mL, washing and filtering the crystals to obtain an intermediate dihydroxydiamminedichloroplatinum, performing vacuum drying on the intermediate, dissolving the intermediate and succinic anhydride in dimethylformamide, stirring at room temperature, performing freeze drying, and washing the freeze-dried solid to obtain the cisplatin derivative.
Further, the weight ratio of the cis-diammine dichloroplatinum to the hydrogen peroxide is 1:1.05, and the weight ratio of the intermediate to the succinic anhydride is 10: 3.
In the step b, the molecular weight cut-off of the dialysis membrane is 1kDa, the dialysis time is 48h, and the load of platinum in the modified infrared emission fluorescent carbon quantum dot-cisplatin derivative is 8.8%.
In step c, the pH of the reaction system is adjusted to pH8.5 by using 0.2mol of NaOH solution, and a product is dialyzed by using a phosphate buffer solution and then is freeze-dried.
In the step d, the weight ratio of the infrared emission fluorescent carbon quantum dot-cisplatin derivative to the polyethylene glycol-chitosan-dimethyl maleic anhydride is 2:5, and the medicine carrying system is stored at the temperature below-20 ℃.
The invention has the beneficial effects that: the invention obtains the red-emitting nitrogen self-doped carbon quantum dots with excellent performance by using the natural biomass-osmanthus fragrans seed husk through a solvothermal method, and has the advantages of easy surface functionalization, good biocompatibility, low toxicity, higher photothermal conversion efficiency, wider absorption band in the region from visible light to NIR light and the like. Based on the infrared emission fluorescent carbon quantum dot, a novel drug-carrying system for releasing a pH response charge reversal drug, namely the infrared emission fluorescent carbon quantum dot-cis-platinum derivative/polyethylene glycol-chitosan-dimethyl maleic anhydride, is successfully designed, and is applied to the chemical-photothermal synergistic efficient treatment of tumors of nude mice. The infrared emission fluorescent carbon quantum dot-cisplatin derivative/polyethylene glycol-chitosan-dimethyl maleic anhydride drug-loaded system prepared by the invention has the functions of transporting Pt (IV) drug molecules in a stable form to reduce the advanced degradation of the drug molecules and finally realizing the targeted release of high-toxicity Pt (II) at the tumor site, can play the synergistic effect of chemotherapy and photothermal therapy in tumor therapy, can completely eliminate the tumor in a nude tumor mouse, has no tumor recurrence and small toxic and side effects on organisms, and has a treatment effect remarkably superior to that of single chemotherapy or photothermal therapy. The chemical-photothermal synergistic treatment scheme has the clinical integrated application potential from tumor imaging and drug tracking to anti-tumor treatment, and has great prospect in the high-efficiency clinical tumor treatment application.
[ detailed description ] embodiments
FIG. 1 is a synthetic scheme of cisplatin derivatives of the present invention.
FIG. 2 is a schematic of the PEG-CS-DA synthesis scheme of the present invention.
FIG. 3 is a synthetic scheme of RCQDs-Pt (IV)/PEG-CS-DA according to the present invention.
FIG. 4 is a FTIR spectrum of RCQDs (a), RCQDs-Pt (IV) (b), and RCQDs-Pt (IV)/PEG-CS-DA (c) according to the present invention.
FIG. 5 is a spectrum of ultraviolet-visible absorption of RCQDs-Pt (IV) of the present invention.
FIG. 6 is a graph showing the change of Zeta potential of RCQDs-Pt (IV)/PEG-CS-DA of the present invention under different conditions.
FIG. 7 is a graph of the effect of GSH and pH on drug release from RCQDs-Pt (IV)/PEG-CS-DA in accordance with the present invention.
FIG. 8 shows the cell viability of T24 cells of the invention after 24h incubation under different conditions with RCQDs and RCQDs-Pt (IV)/PEG-CS-DA.
FIG. 9A is a graph showing the temperature change of aqueous solutions of PBS, Pt (II), RCQDs and RCQDs-Pt (IV)/PEG-CS-DA with time under NIR laser irradiation according to the present invention; 9B are stained CLSM images of cancer cells incubated with different combination drugs, wherein (a) PBS control group, (B) NIR group, (c) RCQDs 20mg/mL, (d) RCQDs 20mg/mL + NIR group, (e) RCQDs 105 mg/mL-Pt (IV)/PEG-CS-DA, (f) RCQDs 105 mg/mL-Pt (IV)/PEG-CS-DA + NIR group.
FIG. 10A is a drug delivery in vivo imaging and tracing of the present invention: RCQDs-Pt (IV)/PEG-CS-DA intratumoral (a) and intravenous (b) in vivo fluorescence imaging images taken at different time periods injected into nude mice; FIG. 10B is in vivo fluorescence imaging taken 8h after intravenous injection into nude mice with PBS (a), RCQDs-Pt (IV) (B), and RCQDs-Pt (IV)/PEG-CS-DA (c); FIG. 10C shows injection of PBS, Pt (II), RCQDs and RCQDs-Pt (IV)/PEG-CS-DA to the tumor site of mice using a 680nm laser (1.5W/cm)2) Thermal imaging at different times of irradiation.
FIG. 11A is a graph showing the growth of tumors in nude mice in PBS + -NIR group, Pt (II) + -NIR group, RCQDs and RCQDs-Pt (IV)/PEG-CS-DA + -NIR group according to the present invention; FIG. 11B are photographs of tumors in nude mice after 14 days of treatment with Pt (II). + -. NIR group, RCQDs + -NIR group, and RCQDs-Pt (IV)/PEG-CS-DA + -NIR group; FIG. 11C is a photograph of a representative nude mouse after one week of treatment in each experimental group; FIG. 11D shows the H & E staining results (scale: 200 μm) of tumor tissue sections of nude mice of each experimental group.
FIG. 12 shows the H & E staining results (scale: 200 μm) of tissue sections of major organs of nude mice of PBS group (a), RCQDs-Pt (IV)/PEG-CS-DA + NIR group (b) and Pt (II) group (c) according to the present invention.
FIG. 13 is a graph showing the change of body weight of nude mice over time in different experimental groups according to the present invention.
[ detailed description ] embodiments
The following examples are further illustrative and supplementary to the present invention and do not limit the present invention in any way.
Example 1
Synthesis of aminated infrared-emitting fluorescent carbon quantum dots (RCQDs)
Adding 15g of cut osmanthus fragrans seed husk into 30mL of absolute ethyl alcohol, adding into a 50mL round-bottom flask, adopting a nitrogen protection device, uniformly mixing, sealing, pumping, fixing in a heating jacket, heating to 100 ℃, stirring for reaction for 12 hours, naturally cooling a reaction product solution to room temperature, and filtering the obtained supernatant through a 0.22-micrometer organic filter membrane. 30mL of the filtrate was taken in a 50mL beaker, and 0.2g of H was added2N-PEG-NH2And (3) fully and uniformly mixing the anhydrous ethanol solution, refluxing and heating to 120 ℃, and stirring for 10 hours in a nitrogen atmosphere. The resulting supernatant was filtered through a 0.22 μm inorganic filter, and the filtrate was purified by dialysis against a dialysis membrane (1000MWCO) for 48 hours to obtain pure aminated RCQDs, and finally, the purified product was subjected to rotary evaporation under vacuum conditions, dried at 65 ℃ for 24 hours to obtain a solid sample, and stored in a refrigerator at 4 ℃ in the dark for later use.
Example 2
Synthesis of cisplatin derivative (Pt (IV))
300.0mg Cis-Pt (NH)3)2Cl2Suspended in 7.5mL of water, 10.5mL of 30 wt% H was added dropwise at 50 deg.C2O2. After stirring for 2h, the mixture was cooled to room temperature. The solvent was concentrated to about 2mL by rotary evaporation and the resulting solution was stored at 0 ℃ overnight. The light yellow crystals are washed by cold water and cold ether and collected by filtration to obtain an intermediate c, t, c- [ PtCl ]2(OH)2(NH3)2]And (5) drying in vacuum. 120.0mg of c, t, c- [ PtCl ]2(OH)2(NH3)2]And 36.0mg of succinic anhydride were dissolved in 2.0mL of DMF and stirred at room temperature for 24 h. The mixture was freeze dried and the resulting solid was washed with cold acetone and ether to give the pale yellow Pt (IV) prodrug c, c, t- [ PtCl ]2(OH)(NH3)2(O2CCH2CH2CO2H)]The synthetic route is shown in figure 1.
Example 3
Amination infrared emission fluorescent carbon quantum dot modified cisplatin derivative
20mg of Pt (IV), 3.5mg of hydrogen peroxide (NHS) and 4.5mg of 1- (3- (dimethylamino) propyl) -3-ethyldiimine hydrochloride (EDC. HCl) were stirred in water at room temperature for 30 min. Then, 40.0mg of aminated RCQDs were added to the above solution, and the resulting mixture was stirred for another 24 hours. The mixture was dialyzed in ultrapure water for 48 hours through the dialysis membrane (MWCO: 1kDa) used, and the ultrapure water was replaced every 3 hours to remove the unreacted compound. After freeze drying, the modified infrared emission fluorescent carbon quantum dot-cisplatin derivative (RCQDs-Pt (IV)) is obtained, and the RCQDs-Pt (IV) is stored at room temperature for further use. The percentage loading of Pt was 8.8% as measured by ICP-MS.
Example 4
Synthesis of polyethylene glycol-chitosan-dimethylmaleic anhydride (PEG-CS-DA)
Reacting Chitosan (CS) with an active ester of polyethylene glycol (PEG) (PEG-NHS) to obtain PEG-CS. As shown in FIG. 2, first, 0.2g of CS was dissolved in 20mL of ultrapure water, 1.0g of PEG-NHS was added thereto, the mixture was magnetically stirred for reaction for 24 hours, dialyzed with ultrapure water, and lyophilized to obtain PEG-CS. 0.2g of PEG-CS was dissolved in 20mL of Phosphate Buffered Saline (PBS) having pH8.0, and 3-fold molar amount of dimethylaminoethyl methacrylate (DMMA) was slowly added thereto to react to obtain PEG-CS-DA. The pH of the reaction system was adjusted to about pH8.5 using 0.2mol NaOH solution, magnetically stirred at room temperature for 8h, dialyzed against PBS (pH8.0), and lyophilized.
Example 5
PEG-CS-DA complexation with RCQDs-Pt (IV)
60.0mL of a 1mg/mL solution of RCQDs-Pt (IV) was added dropwise to 150.0mL of 1mg/mL PEG-CS-DA solution. The mixed solution is stirred at room temperature overnight and then freeze-dried to obtain a drug-loaded system RCQDs-Pt (IV)/PEG-CS-DA which is stored at-20 ℃ for the next experiment. The specific synthetic route is shown in figure 3.
Example 6
Characterization of nanomaterials
The surface functional groups of RCQDs and the complexes thereof are characterized by FT-IR spectrum, and the characteristic structure is shown in FIG. 4 (a). As can be seen from FIG. 4(a), 1055cm-1The peak observed here is attributed to the bending vibration of the C — O bond, showing the main absorption band associated with the carboxyl group. 1380cm-1The peaks appearing there belong to the characteristic stretches of N-H bonds. At 1640cm-1The peak at (a) is attributable to the typical stretching mode of C ═ O stretching vibration; at 3443cm-1Where large absorption bands are considered to be O-H and N-H bonds, FT-IR spectra indicate the presence of many amino and carboxyl groups on the surface of RCQDs, which functional groups improve the hydrophilicity, stability and modifiability of RCQDs in aqueous systems.
Because the covalent coupling of RCQDs to Pt (IV) is via the amino group on RCQDs and the carboxyl group on Pt (IV) with a typical EDC/NHS amidation reaction. After modification of Pt (IV) on RCQDs, as shown by FTIR spectrum of FIG. 4(b), the relative ratio between the oscillation intensities of C ═ O and N-H in RCQDs-Pt (IV) was increased as compared with that of RCQDs, indicating that Pt (IV) had been successfully modified on RCQDs, and the FTIR spectrum after combination of RCQDs-Pt (IV) and PEG-CS-DA as shown in FIG. 4(C), it can be seen that many peaks were enhanced, indicating that RCQDs-Pt (IV) and PEG-CS-DA had been successfully combined. The potential difference between the sliding layer of the nanoparticles and the surrounding solution environment is called Zeta potential, which reflects the average value of the overall charge condition of the nanoparticles. The Zeta potential of the prepared aminated RCQDs was measured to be 18.0mV, and the reason for the positive charge should be the presence of a large number of positively charged amino groups on the surface. After loading Pt (IV) on RCQDs, the Zeta potential of the RCQDs is measured to be 10.7mV, the Zeta potential is reduced because some positively charged amino groups on the RCQDs are coupled with Pt (IV) prodrug during the conjugation and combination of Pt (IV) and the RCQDs, so that the Zeta potential of the RCQDs-Pt (IV) is lower than that of the original RCQDs, and the Zeta potential measurement result further indicates that Pt (IV) is successfully coupled to the RCQDs. As shown in FIG. 5, the UV-visible absorption of RCQDs-Pt (IV) is similar to that of RCQDs, indicating that the covalent coupling of Pt (IV) to RCQDs does not affect the UV absorption optical properties of RCQDs, and shows strong absorption in the NIR region of 600 to 800nm, which is suitable for the photothermal therapy as PTT reagent. The QY of RCQDs-Pt (IV) is calculated to be 19.8% by taking quinine sulfate as a reference, and the feasibility of the RCQDs-Pt (IV) in biological imaging application is disclosed.
PEG-CS-DA is a PEG material that undergoes charge reversal in response to stimulation by pH. Under physiological conditions (pH 7.4), the PEG-CS-DA with negative charges can coat the RCQDs-Pt (IV) with positive charges through the attraction of the positive charges and the negative charges to obtain the stably existing RCQDs-Pt (IV)/PEG-CS-DA.
After reaching tumor tissue by EPR effect, the pH value is slightly acidic in tumore(6.8) upon stimulation, the PEG-CS-DA charges change from negative to positive, and thus are shed from the RCQDs-Pt (IV) by charge repulsion, exposing the positively charged RCQDs-Pt (IV). Positively charged RCQDs-Pt (IV) are more readily taken up into cells, and Pt (IV) is reduced to highly toxic Pt (II) for treatment under intracellular GSH stimulation. As shown in FIG. 6, the change in Zeta potential of RCQDs-Pt (IV)/PEG-CS-DA at extracellular pH of 6.8 in tumor cells and pH of 7.4 in normal physiological conditions confirms this property. At pH6.8, RCQDs-Pt (IV)/PEG-CS-DA incubated at 37 ℃ for various periods showed a marked charge reversal from negative to positive with an increase in Zeta potential from-17 to +10.9 mV. At pH7.4, RCQDs-Pt (IV)/PEG-CS-DA showed charge-non-reversible characteristics after incubation at 37 ℃ for 4h, with a more constant negative charge.
Example 7
pH responsive in vitro drug release study
In order to further research the pH value of RCQDs-Pt (IV)/PEG-CS-DA and the release characteristics of Pt (II) drug molecules triggered by a reducing agent, the invention carries out drug molecule release experiments under different pH values and reducing agent conditions. The method for the drug molecule release experiment comprises the following steps:
15.0mg of RCQDs-Pt (IV)/PEG-CS-DA were dissolved in 1.0mL of PBS buffer (10mmol/L, pH7.4 or 6.8) containing 0mmol/L, 5. mu. mol/L and 10mmol/L of GSH, respectively, and transferred to a dialysis tube (MWCO: 1kDa), followed by dialysis with PBS buffer (69mL, pH7.4 or 6.8, containing 0mmol/L, 5. mu. mol/L and 5mmol/L of GSH) in the dark at 37 ℃. At a predetermined point in time (0, 1, 2, 4, 6, 8, 10h), a certain released volume of dialysate is removed and the same amount is added to the original released dialysate with a new buffer solution. At each predetermined time point, PBS buffer was removed by centrifugation for analysis. The amount of Pt in the PBS solution was determined by inductively coupled plasma mass spectrometry (ICP-MS) characterization and detection.
It is concluded from the principle that when RCQDs-Pt (IV)/PEG-CS-DA is in acid environment, the PEG-CS-DA layer of the drug delivery system will immediately hydrolyze and release RCQDs-Pt (IV), RCQDs-Pt (IV) under the action of intracellular reducing substances such as Glutathione (GSH) or ascorbic acid, Pt (IV) removes two axial derivatization groups to release Pt (II). In this example, Glutathione (GSH), which is ubiquitous in the organism, and the pH of the extracellular microenvironment (pH 6.8) and normal physiological environment (pH 7.4) of tumors were selected as influencing factors to examine the behavior of RCQDs-Pt (IV)/PEG-CS-DA for in vitro drug release. First, 15mg of RCQDs-Pt (IV)/PEG-CS-DA was added to PBS buffer (pH 7.4 or 6.8) containing GSH (0, 5. mu. mol/L and 5mmol/L) at different concentrations, and the drug release profile of Pt (II) over 10h was analyzed, as shown in FIG. 7. The results of the drug molecule release experiments are consistent as expected, and it can be seen from fig. 7 that the concentration and pH of GSH have a greater effect on the release rate of pt (ii), and when the concentration of GSH approaches the concentration of 5 μmol/L in normal extracellular plasma, only about 20% of pt (ii) is released at pH6.8 after 6h, and the release rate is lower at pH 7.4. And when the concentration of the GSH is increased to 5mmol/L, the concentration is similar to the concentration of the GSH in the tumor cells, the release rate of Pt (II) from RCQDs-Pt (IV)/PEG-CS-DA is remarkably increased, almost 61 percent of the Pt (II) medicament can be released from a medicament-carrying system under the condition of pH6.8 after 6h, and the release amount of the Pt (II) medicament exceeds 85 percent after 10h of incubation. At pH7.4, however, only a small amount of Pt (II) drug was released from the drug loading system at GSH concentrations of 5. mu. mol/L and 5 mmol/L. The experiment result shows that the concentration and the pH value of the GSH reducing agent have great influence on the controlled release of Pt (II) medicaments by a medicament carrying system RCQDs-Pt (IV)/PEG-CS-DA.
The behavior of RCQDs-Pt (IV)/PEG-CS-DA, which is dependent and sensitive to GSH concentration and pH value, can effectively prevent the loss of Pt (II) medicaments in the blood circulation process, and has the advantages of releasing a large amount of Pt (II) medicaments to reach tumor parts in a targeted and self-targeted manner at fixed points, avoiding the premature release of the medicaments in the transportation process, not only enhancing the tumor treatment effect, but also reducing the toxic and side effects of the medicaments in the whole body in the medicament releasing process.
Example 8
Cytotoxicity Studies
1) Cell culture and cytotoxicity
At 37 deg.C, the density is 1X 105Individual cell/well T24 cells placed in 5% CO2Incubate under atmosphere for 24 h. Thereafter, the cells were further cultured with DMEM containing RCQDs and RCQDs-Pt (IV)/PEG-CS-DA (containing the same amount of 5. mu.g/mL RCQDs) in a cell culture medium of pH6.8 or 7.4 containing 5% CO at 37 ℃2Incubations were carried out for an additional 4h in a humidified environment. A laser confocal fluorescence microscope (CLSM) is used for observing the uptake of RCQDs and RCQDs-Pt (IV)/PEG-CS-DA by T24 cells under different pH values and the distribution of nanoprobes in the cells. T24 cells were washed 3 times with PBS before CLSM imaging.
The increase of the temperature of the surrounding environment of the tumor can increase the permeability of the surrounding blood vessel wall and the tumor cell membrane, so that the medicine can more easily enter the tumor cells, and in the photothermal therapy, the photothermal effect can increase the toxicity of the chemical medicine Pt (II). Based on RCQDs, strong absorption in the 600 to 800nm NIR region, i.e. good photothermal effect. It is presumed that the photothermal effect of RCQDs increases the toxicity of RCQDs-Pt (IV) @ PEG-CS-DA to tumor cells, and for this reason, the cytotoxicity of RCQDs-Pt (IV) @ PEG-CS-DA was confirmed in the control experiment. MTT assay was used to evaluate the toxicity of RCQDs and RCQDs-Pt (IV)/PEG-CS-DA. T24 cells were cultured at a density of 1X 104Individual cell/well 96-well plates containing 5% CO at 37 deg.C2In a humidified incubator, in DMEM for 24 h. Then, DMEM at pH6.8 containing RCQDs and RCQDs-Pt (IV)/PEG-CS-DA at different concentrations was used in place of the original medium. At 37 ℃ 5% CO2And incubated for a further 2h in a humid environment. After all media had been cleared, 100. mu.L of dimethyl sulfoxide was added and shaking continued for 15 min. Then, absorbance of each well was measured at 570nm with an enzyme-linked immunosorbent assay using pure dimethylsulfoxide as a blank.
T24 cells were experimentally associated with RCQDs and RCQDs-Pt (IV), respectively) @ PEG-CS-DA incubation with near infrared light (680nm, 1.5W/cm)23min) controls with and without NIR light were performed to compare cell viability in different experimental groups. As shown in FIG. 8, without NIR light irradiation, the toxicity of RCQDs to T24 cells was negligible in the concentration range of 0.1 to 100. mu. mol/L, while the cell survival rate of other groups gradually decreased with the increase of the sample concentration, and the toxicity of RCQDs-Pt (IV) @ PEG-CS-DA was greatly increased by the light irradiation heat induced after NIR light irradiation, which was much higher than that of the single photothermal therapy (RCQDs) and the single chemotherapy RCQDs-Pt (IV) @ PEG-CS-DA groups. The experimental results fully show that the illuminated functionalized nano drug-loaded system RCQDs-Pt (IV) @ PEG-CS-DA can effectively combine photothermal therapy with chemotherapy, can achieve the effect of synergistically enhancing cytotoxicity, and has huge potential for efficient treatment of tumor cells.
Example 9
Photothermal effect and in vitro synergistic therapeutic effect of drug-loaded system
As the RCQDs-Pt (IV)/PEG-CS-DA drug delivery system is designed for chemical-photothermal co-therapy, the photothermal effect of RCQDs-Pt (IV)/PEG-CS-DA in an acid environment needs to be determined respectively in the experiment. The test method comprises the following steps: mu.g of RCQDs, 105. mu.g of RCQDs-Pt (IV)/PEG-CS-DA (equal load of RCQDs), 155. mu.g of RCQDs-Pt (IV)/PEG-CS-DA dissolved in 1mL of PBS (pH 6.8), Pt (II) and PBS as blank controls were placed in 1.5mL centrifuge tubes, respectively, and then a 680nm laser (1.5W/cm)2) The irradiation was continued for 10min and the change in temperature of the solution at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10min time was recorded with a temperature tester.
As shown in fig. 9, it can be seen from fig. 9A that the temperatures of the PBS group and the pt (ii) group did not increase significantly at each test time point and under laser irradiation. However, between 0 and 5min, the temperature of the experimental groups of 20 μ g/mL RCQDs, 105 μ g/mLRCQDs-Pt (IV)/PEG-CS-DA (equivalent load RCQDs) and 155 μ g/mL RCQDs-Pt (IV)/PEG-CS-DA increases with the time during the laser irradiation, and the temperature reaches about 60 ℃ after the irradiation for 5min, and from the experimental results, no large difference appears between 20 μ g/mL RCQDs and 105 μ g/mL RCQDs-Pt (IV)/PEG-CS-DA (equivalent load RCQDs), this indicates that the photothermal conversion capabilities of RCQDs-Pt (IV)/PEG-CS-DA and RCQDs are substantially the same, and furthermore, the photothermal effect of RCQDs-Pt (IV)/PEG-CS-DA solutions is highly dependent on their concentrations. The above experimental results show that the photothermal effect of the RCQDs is not destroyed after the RCQDs are modified with Pt (IV) and PEG-CS-DA, and the RCQDs-Pt (IV)/PEG-CS-DA also causes the temperature of the surrounding environment to be obviously increased after being hydrolyzed under the irradiation of near infrared light. This means that RCQDs-Pt (IV)/PEG-CS-DA has great potential in the field of chemical-photothermal combination therapy of tumor cells in weak acid tumor environment.
In order to observe the in vitro site-directed treatment effect of RCQDs-Pt (IV)/PEG-CS-DA more intuitively, we also investigate the photo-thermal toxicity of the drug-loaded system on cancer cells through a cell staining experiment. The experimental method comprises the following steps of designing a PBS group, a near infrared laser irradiation (NIR) group, an RCQDs + NIR group, an RCQDs-Pt (IV)/PEG-CS-DA + NIR group for experiment, and specifically comprising the following steps: first, A549 cells were cultured at 1X 105The density of individual cells/well was seeded in 6-well plates. After a conventional 24h incubation, the medium was replaced with fresh medium at pH6.8, PBS, 20 μ g/mL RCQDs (NIR), 105 μ g/mL RCQDs-pt (iv)/PEG-CS-DA, and 105 μ g/mL RCQDs-pt (iv)/PEG-CS-DA (NIR) were added to the medium for 24h incubation, followed by careful three washes with PBS to remove nanoprobes not taken up into a549 cells, and finally an equal amount of fresh medium was added. Adjusting a 680nm NIR laser at 1.5W/cm2The power of (2) was applied to A549 cells in the above-mentioned NIR group, RCQDs + NIR group and RCQDs-Pt (IV)/PEG-CS-DA + NIR group for 3 min. After the laser irradiation treatment is completed, all the combined A549 cells are placed in a cell culture box for further incubation for 4 hours, then Calcein-AM (Calcein-AM/Propidium Iodide (PI) double staining method is adopted to identify the activity of the A549 cells, under the field of confocal microscope, the surviving cells emit green fluorescence, and the dead cells emit red fluorescenceThe effect is based on Pt (II) chemotherapy, and the treatment effect of RCQDs-Pt (IV)/PEG-CS-DA + NIR group is based on chemo-photothermal synergistic treatment effect. After incubation of cancer cells with cell culture medium (pH 6.8) containing different combinations of drugs for 4h, live cells were stained green by Calcein-AM and dead cells red by PI. The experimental results are shown in fig. 9B, and it can be seen from fig. 9B that no cell death occurred in the PBS group, the NIR group, and the RCQDs group, indicating that the intensity of the near-infrared laser light or the concentration of the RCQDs did not cause cell death, and further indicating that the laser irradiation power and irradiation time selected by us are safe. However, the RCQDs + NIR group, RCQDs-Pt (IV)/PEG-CS-DA group and RCQDs-Pt (IV)/PEG-CS-DA + NIR group all suffered from cell death. And the number of dead cells in the RCQDs-Pt (IV)/PEG-CS-DA + NIR group is more than that in the RCQDs + NIR group and the RCQDs-Pt (IV)/PEG-CS-DA group, no living cells are observed at all, which shows that the toxicity of the RCQDs-Pt (IV)/PEG-CS-DA is greatly increased by the light radiation heat caused by the near infrared laser irradiation, and the toxicity is much higher than that of the single chemotherapy (RCQDs-Pt (IV)/PEG-CS-DA group) and the photothermal therapy (RCQDs + NIR group). The experimental results show that the charge reversal drug loading system RCQDs-Pt (IV)/PEG-CS-DA can exert the effect of stronger cytotoxicity than single photothermal therapy and chemotherapy, can realize the fixed-point killing of tumor cells and exert the efficient chemical-photothermal synergistic treatment effect.
Example 10
Nude mouse tumor model establishment, living body imaging and thermal imaging experiment
To verify the tumor targeting transport and image tracking ability of RCQDs-Pt (IV)/PEG-CS-DA in nude mice, the image analysis was performed on the different treated nude mice groups by using a live body imager. Wherein BALB/c nude mice (5-6 weeks old) were purchased from Schlekschada laboratory animals Co., Ltd, Hunan, and bred in animal laboratories of university of Guangxi Master and West. The use of all nude mice in this example is in accordance with current ethical considerations and all comply with the guidelines set forth in the "guidelines for care and use of laboratory animals". In the experiment, a subcutaneous nude mouse tumor model is established by injecting T24 cell suspension subcutaneously on the ventral side of a nude mouse. T24 cells were first cultured at 1X 106The density of individual cells/dish was seeded in cell culture dishes, conventionalAfter 48h incubation, the concentration was prepared by cell counting after digesting the cells to 2X 107Individual cells/mL of physiological saline cell suspension. Nude mice were randomly grouped and anesthetized with isoflurane, and then the mice were injected ventrally with suspension in 100 μ L of the above T24 cell suspension. The nude mice injected with the T24 cell suspension are revived and then placed in the animal room for feeding. Until the tumor volume reaches 60-70mm3In the experiment, the time points of fluorescent picture shooting in the experiment are set at 30min, 1h, 4h, 6h, 10h and 48h after injection. In addition, nude mice were injected with 200. mu.L of RCQDs-Pt (IV)/PEG-CS-DA, RCQDs-Pt (IV), or PBS by intravenous injection. The dose of RCQDs injected into each nude mouse is guaranteed to be 10 mg/kg. After 8h, after T24 cell tumor model mice are anesthetized by isoflurane, based on the characteristic that RCQDs have near-infrared fluorescence, a Bruker in-vivo FPRO imaging system is used for carrying out in-vivo imaging analysis on nude tumor mice of each experimental group by a drug-carrying system (excitation: 630 nm; emission: 700 nm).
As shown in FIG. 10A (a), RCQDs-Pt (IV)/PEG-CS-DA injection-treated nude mice can observe obvious and concentrated fluorescence at the tumor site soon, reach the peak value 4h after injection, and can detect strong red fluorescence signal at the tumor site within 48 h. FIG. 10A (b) shows that the visible near infrared fluorescence signal is mainly concentrated at the liver part of the nude mice 30min after the intravenous injection of RCQDs-Pt (IV)/PEG-CS-DA, the fluorescence signal at the tumor part is gradually increased along with the time, and after 1h, a weaker red fluorescence signal can be observed at the tumor part and reaches a peak value 10h after the injection. While no significant fluorescence signal was observed after 2h at the liver site, which is probably because the neutral environment of the liver did not cause the release of RCQDs-Pt (IV)/PEG-CS-DA in large amounts from RCQDs-Pt (IV)/PEG-CS-DA to RCQDs and Pt (II) drug molecules, although RCQDs-Pt (IV)/PEG-CS-DA would be temporarily retained at the liver site. The result shows that RCQDs-Pt (IV)/PEG-CS-DA has excellent tumor targeted transportation and fluorescence tracking capability.
In order to further verify the tumor targeting transport capacity of the charge reversal RCQDs-Pt (IV)/PEG-CS-DA system, PBS, RCQDs-Pt (IV) and RCQDs-Pt (IV)/PEG-CS-DA are injected into a nude tumor mouse intravenously, and after 8 hours after injection, the nude tumor mouse is anesthetized by isoflurane and then a living body imaging system is used for observing a fluorescence signal at a tumor part. As shown in FIG. 10B, the strongest fluorescence was detected in the tumor sites of nude mice injected with RCQDs-Pt (IV)/PEG-CS-DA, while the tumor sites of nude mice injected with RCQDs-Pt (IV) showed weaker fluorescence signals. The positive RCQDs-Pt (IV) is easy to be captured by the liver in the in-vivo transportation process and cannot be transported to a tumor site in a passive targeting manner, the negatively charged RCQDs-Pt (IV)/PEG-CS-DA in blood can avoid nonspecific interaction with blood components to achieve the effect of prolonging the circulation time, when the tumor tissue is reached through the EPR effect, the PEG-CS-DA layer is decomposed by the weak acid environment of the tumor, and the loaded RCQDs and Pt (II) drug molecules are gradually released by the charge reversal of the RCQDs-Pt (IV)/PEG-CS-DA retained at the tumor site. The results of in vivo fluorescence imaging of nude mice show that RCQDs-Pt (IV)/PEG-CS-DA has the potential of enhancing the stability of the drug in blood circulation, improving the aggregation capability and the targeted transport capability of drug molecules at tumor sites and realizing biological fluorescence tracing as diagnosis and treatment integration.
We also studied the photothermal effects of PBS, Pt (II), RCQDs, and RCQDs-Pt (IV)/PEG-CS-DA in vivo using thermography. FIG. 10C shows the use of a 680nm laser (1.5W/cm)2) Thermal imaging pictures of mouse tumor sites at different time points after irradiation. As can be seen from FIG. 10C, the temperature of the injected PBS and Pt (II) groups did not change, while the temperature of the tumor sites of the mice injected with RCQDs and RCQDs-Pt (IV)/PEG-CS-DA (equal loading of RCQDs) groups increased significantly (increased to about 52 ℃ after 3min), which indicates that the temperature of the tumor sites of the mice increased not from PBS and reduced Pt (II), but RCQDs and RCQDs-Pt (IV)/PEG-CS-DA have excellent photothermal effect in vivo, can be used as PTT reagent to induce high temperature to kill tumor cells in vivo, and the two groups did not have great difference during the light heating process, again indicating that the photothermal conversion capability of RCQDs-Pt (IV)/PEG-CS-DA and RCQDs is basically the same.
Example 11
In-vivo chemical-photothermal synergistic tumor treatment effect of drug-loaded system
To further examine the chemo-photothermal synergistic therapeutic effect of RCQDs-Pt (IV)/PEG-CS-DA, 8 experimental groups were set for this example, each with and without light control. Experimental selection implant 2X 107About 7 days after each T24 cell/mL, the resulting volume was about 60-70mm3Female nude tumor mice (18-20g) of tumors were the subjects of investigation. On the 0 th day and the 2 nd day from the start of the experiment, 200. mu.L of PBS group, 200. mu.L of Pt (II) group, 200. mu.L of RCQDs group, and RCQDs-Pt (IV)/PEG-CS-DA group were injected into the body of mice via tail vein, respectively, while ensuring the same Pt content (10mg/kg) in the nude mice. According to the distribution imaging result of the nanoprobe in vivo, the tumor part of the nude tumor mouse of the light irradiation group is irradiated by near infrared light (680nm, 1.5W/cm) 10h after the nanoprobe is injected by tail vein2) Irradiating for 3min for chemical-photothermal synergistic treatment. Tumor size of nude mice was measured once a day using a digital vernier caliper after completion of the treatment, and the entire measurement experiment was continued from the irradiation treatment for 14 days. Length (a) and width (b) of the tumor were measured daily with a vernier caliper and the body weight was weighed with an electronic balance, the tumor volume calculation formula: v1/2 × a × b2Wherein a and b are respectively the length and width of tumor body. The calculation formula of the relative tumor volume is V/V0(V0As the pre-injection volume).
FIG. 11A is a graph of the relative volume of tumors in nude mice over time for each experimental group. As shown in fig. 11A, the tumor growth of the PBS nude mice was fast, however, the tumor growth of pt (ii) group was only slightly inhibited, because free pt (ii) did not target autonomously, and it was difficult to reach the tumor site to exert the drug effect to inhibit the tumor growth. In contrast, the tumor volume of the RCQDs-Pt (IV)/PEG-CS-DA group (i.e., the chemotherapy group alone) nude mice was also significantly affected. Because RCQDs-Pt (IV)/PEG-CS-DA are stable in normal physiological environment, the EPR effect can realize the targeted transportation of the drug, and no side reaction occurs when the drug is combined with biomolecules such as proteins in the blood circulation process, the RCQDs-Pt (IV)/PEG-CS-DA group has better chemical treatment effect on tumor cells, but can not completely inhibit the growth of tumors. On the other hand, the RCQDs + NIR group (i.e., photothermal treatment group alone) nude mice showed a decrease in tumor volume in the first 4 days, but then became gradually larger. It is feared that the tumors of the RCQDs-Pt (IV)/PEG-CS-DA + NIR group of nude mice completely disappeared 7 days after NIR laser irradiation, and no more tumors were observed at the later treatment time. Figure 11B shows photographs of tumors from nude mice with and without NIR irradiation on day 14 of experiment, the trend of tumor growth is consistent with the curve depicted in figure 11A. Experiments show that the chemotherapy and the photothermal therapy alone can not completely kill tumor cells and can cause the recurrence of the tumor. FIG. 11C is a photograph of a representative nude mouse after one week of treatment in each experimental group, and it can be seen that the tumors of RCQDs-Pt (IV)/PEG-CS-DA + NIR group nude mice left only one black burned scar after one week of treatment. The charge reversal drug-loaded therapeutic system enables drug molecules to be released in a targeting manner at a tumor tissue part, achieves targeting delivery to tumors and simultaneously minimizes side effects on non-treatment parts, and the photothermal effect can increase the toxicity of Pt (II), and can prolong the circulation time of negatively charged RCQDs-Pt (IV)/PEG-CS-DA in blood; the EPR effect is enhanced at high temperature, so that the medicament can easily enter tumor cells, and more accumulation of the medicament at tumor parts is promoted; therefore, the charge-reversible drug-loading system RCQDs-Pt (IV)/PEG-CS-DA + NIR group realizes targeted drug transportation and release, can play a synergistic effect of chemotherapy and photothermal therapy in treatment, can completely eliminate the tumor of a nude mouse, and improves the tumor treatment effect.
In addition, in order to evaluate pathological changes of tumor tissues after the chemo-photothermal synergistic treatment, tumor tissues of nude mice of each experimental group were subjected to cryo-sectioning and H & E staining after NIR laser irradiation treatment, and pathological observation was performed using an optical microscope. The method comprises the following steps: after completion of the tumor growth monitoring experiment, mice were sacrificed after anesthesia, tumor tissue was removed, and tumors were washed three times with PBS buffer and fixed with 3% paraformaldehyde for further study of H & E staining. Histopathological experiments were performed according to standard laboratory procedures. After H & E staining, the pathological sections were observed using an optical microscope and photographs were taken.
As shown in FIG. 11D, it can be clearly observed that the tumor cells of nude mice in RCQDs + NIR and RCQDs-Pt (IV)/PEG-CS-DA + -NIR groups showed large area of rupture, especially in RCQDs-Pt (IV)/PEG-CS-DA + NIR group, the cell nuclei were significantly reduced, indicating that the tumor cells were apoptotic and necrotic. In contrast, no significant damage was observed to cells from PBS, RCQDs and pt (ii) controls without NIR laser irradiation, cells were still very tightly aligned as seen from sections, and nuclei were also seen. Therefore, from the pathological angle, the chemical-photothermal synergistic treatment group has obvious killing effect on tumor tissue cells, so that the tumor cells are subjected to apoptosis and necrosis, and finally, the tumor of a nude tumor mouse is completely eliminated, so that the tumor treatment effect is obviously enhanced.
Example 12
Histological analysis
When the chemical-photothermal synergistic treatment effect of the pH response charge reversible drug-loaded system is examined, the toxic and side effects of the treatment system on normal tissues are also considered. The experiment was divided into 3 groups, PBS group, Pt (II) group and RCQDs-Pt (IV)/PEG-CS-DA + NIR group. The specific method comprises the following steps: when the size of the tumor reaches about 70mm3(about 7 days after transplantation), 200. mu.L of PBS, Pt (II) and RCQDs-Pt (IV)/PEG-CS-DA were injected into nude mice via tail vein injection, respectively, to make the Pt content in the nude mice the same. After the injection of the drug for 10h, the tumor part of the nude mice in the group was irradiated with near infrared light (680nm, 1.5W/cm)2) Irradiating for 3 min. After 8H, the tumor and major organ tissues (heart, liver, spleen, lung and kidney) in nude mice were taken out, fixed by soaking in 3% paraformaldehyde solution for 24H, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H)&E) Staining and final histological analysis of the major organ tissues of nude mice using fluorescence microscopy. As shown in FIG. 12, no significant damage was observed in the major organs of nude mice in both PBS group (a) and RCQDs-Pt (IV)/PEG-CS-DA + NIR group (b). However, more obvious lesions were observed in the liver, spleen and kidney of mice injected with pt (ii) group (c) nude tumors. Furthermore, nude mice from different experimental groups of FIG. 13 weighed timeThe change curve shows that the body weight of the mice in the Pt (II) group is obviously reduced after the administration, while the body weight of the mice in the RCQDs-Pt (IV)/PEG-CS-DA + NIR group is not obviously changed, compared with the toxic and side effects of the mice in the Pt (II) group and the Pt (II) and NIR group, the toxic and side effects are obviously less, and the light-irradiation drug-loaded system has obvious treatment effect and small toxic and side effects in tumor treatment and has great application potential in tumor treatment.
Although the present invention has been described with reference to the above embodiments, the scope of the present invention is not limited thereto, and modifications, substitutions and the like of the above members are intended to fall within the scope of the claims of the present invention without departing from the spirit of the present invention.
Claims (10)
1. The utility model provides a medicine carrying system of chemistry-light and heat concurrent therapy tumour based on near-infrared carbon quantum dot which characterized in that, this medicine carrying system includes nano-carrier, chemotherapy medicine and targeting function molecule, the chemotherapy medicine is cisplatin derivative, the nano-carrier is near infrared emission fluorescence carbon quantum dot, targeting function molecule is for having the polyethylene glycol-chitosan polymer of pH response charge upset characteristic, the medicine carrying system by chemotherapy medicine and nano-carrier coupling are again made with targeting function molecule complex.
2. The near-infrared carbon quantum dot-based drug delivery system for chemical-photothermal synergistic tumor treatment according to claim 1, wherein the near-infrared carbon quantum dots are aminated infrared emission fluorescent carbon quantum dots prepared by taking osmanthus fragrans seed coats as a carbon source.
3. The near-infrared carbon quantum dot-based drug delivery system for chemo-photothermal synergistic tumor treatment according to claim 1, wherein the targeting functional molecule is polyethylene glycol-chitosan-dimethylmaleic anhydride polymer.
4. A preparation method of a medicine carrying system for treating tumors by using a chemical-photothermal synergistic manner based on near-infrared carbon quantum dots is characterized by comprising the following steps:
a. preparation of carbon amide quantum dots
Placing the osmanthus fragrans seed peels and absolute ethyl alcohol into a reaction container, heating to 100 ℃ under the protection of nitrogen, stirring, reacting for 12 hours, cooling the obtained reaction product to room temperature, filtering supernate, and obtaining filtrate, namely the infrared emission fluorescent carbon quantum dots; reacting the infrared emission fluorescent carbon quantum dots with an amino-polyethylene glycol-amino absolute ethyl alcohol solution at 120 ℃, filtering supernate, purifying filtrate by using a dialysis membrane to obtain the aminated infrared emission fluorescent carbon quantum dots, performing rotary evaporation and drying to obtain a solid sample, and storing the solid sample at 4 ℃ for later use;
b. amination infrared emission fluorescent carbon quantum dot modified cisplatin derivative
Uniformly stirring the cisplatin derivative, hydrogen peroxide and 1- (3- (dimethylamino) propyl) -3-ethyl diimine hydrochloride at room temperature, adding the aminated infrared emission fluorescent carbon quantum dots, uniformly stirring, dialyzing, and freeze-drying to obtain a modified infrared emission fluorescent carbon quantum dot-cisplatin derivative;
c. synthesis of targeting functional molecules
Reacting chitosan with active ester of polyethylene glycol to obtain polyethylene glycol-chitosan, dissolving the polyethylene glycol-chitosan in phosphate buffer solution with the pH value of 8.0, adding dimethyl maleic anhydride, and reacting to obtain a targeting functional molecule polyethylene glycol-chitosan-dimethyl maleic anhydride polymer;
d. polyethylene glycol-chitosan-dimethyl maleic anhydride is complexed with infrared emission fluorescent carbon quantum dot-cisplatin derivative
Dripping the infrared emission fluorescent carbon quantum dot-cisplatin derivative solution into a polyethylene glycol-chitosan-dimethyl maleic anhydride solution, stirring at room temperature, and freeze-drying to obtain the drug-carrying system infrared emission fluorescent carbon quantum dot-cisplatin derivative/polyethylene glycol-chitosan-dimethyl maleic anhydride.
5. The method of claim 4, wherein in the step a, 2ml of absolute ethyl alcohol is added into each gram of osmanthus fragrans seed husks, the supernatant is filtered by a 0.22 micron organic filter membrane, and the molecular weight cut-off of the dialysis membrane is 1000.
6. The method of claim 4, wherein in step b, said cisplatin derivative is synthesized by: suspending cis-diamminedichloroplatinum in water, adding hydrogen peroxide at 50 ℃, stirring, cooling to room temperature, performing rotary evaporation, concentrating the solvent to 2mL, washing and filtering the crystals to obtain an intermediate dihydroxydiamminedichloroplatinum, performing vacuum drying on the intermediate, dissolving the intermediate and succinic anhydride in dimethylformamide, stirring at room temperature, performing freeze drying, and washing the freeze-dried solid to obtain the cisplatin derivative.
7. The method of claim 6, wherein the weight ratio of cis-diamminedichloroplatinum to hydrogen peroxide is 1:1.05, and the weight ratio of the intermediate to succinic anhydride is 10: 3.
8. The method of claim 4, wherein in step b, the molecular weight cut-off of the dialysis membrane is 1kDa, the dialysis time is 48h, and the loading amount of platinum in the modified infrared emission fluorescent carbon quantum dot-cisplatin derivative is 8.8%.
9. The method of claim 4, wherein in step c, the pH of the reaction system is adjusted to pH8.5 using 0.2mol NaOH solution, and the product is dialyzed against phosphate buffer and lyophilized.
10. The method of claim 4, wherein in step d, the weight ratio of the infrared-emitting fluorescent carbon quantum dot-cisplatin derivative to the polyethylene glycol-chitosan-dimethylmaleic anhydride is 2:5, and the drug-loaded system is stored below-20 ℃.
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