A drug delivery system and its preparation method and application

Abstract

The present invention provides a drug delivery system and a preparation method and application thereof, wherein the drug delivery system uses magnetic nanoparticles as a core layer and graphene quantum dot-modified silica as a shell layer, and the graphene quantum dots are loaded with doxorubicin. The drug delivery system provided by the present invention can be used to monitor drug delivery by detecting FRET signals; GQDs act as both a drug carrier and a donor in the FRET model, while DOX acts as an anticancer drug and a receptor in the FRET model; and effective drug loading and controlled release are performed through the FRET model. The drug delivery system provided by the present invention can not only be used as a biomarker to track targeted tumors, but also can realize the monitoring and delivery of drugs, thereby improving diagnostic accuracy. The drug delivery system provided by the present invention can not only be used to monitor drug delivery and fluorescence / MRI bimodal cell imaging, but also be used for simultaneous cancer diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a drug delivery system and a preparation method and application thereof. Background Art

[0002] In recent years, the advancement of nanotechnology has driven its growing demand in the biomedical field, thanks to the contribution of multifunctional nanoplatforms to nanoscale cancer diagnosis and treatment. Multifunctional nanoplatforms are a system that integrates medical targeting, bioimaging, diagnosis and treatment, and have broad prospects for future cancer diagnosis and treatment. Currently, multifunctional nanoplatforms still need to reduce artifacts and significantly improve diagnostic accuracy, reduce harmful nonspecific side effects, and aim to overcome the limitations associated with conventional anticancer drugs, such as rapid clearance, insolubility in aqueous solution, and lack of selectivity.

[0003] Currently, fluorescence imaging is one of the most sensitive technologies in monitoring strategies, which can provide sensitive and rapid whole-body imaging and can even detect microscopic tumors. Magnetic resonance imaging (MRI) is widely used in the diagnosis and treatment of cancer due to its advantages of good contrast between different soft tissues, no ionizing radiation, and high air resolution, but it requires long scanning times, which prevents in vivo and in vitro detection. Since each imaging modality has its advantages and disadvantages, there is great interest in the combination of imaging modalities to improve the treatment effect through synergistic multimodal imaging, which cannot be achieved by a single modality.

[0004] CN103571493A discloses a magnetic fluorescent nano-microsphere, which is obtained by coating polyaniline macromolecules on the surface of magnetic nanoparticles and successfully connecting fluorescent nanomaterial quantum dots through the functional amino groups at the ends of the polyaniline molecules. The magnetic fluorescent nano-microsphere retains the excellent fluorescence characteristics of quantum dots and has the superparamagnetism of magnetic particles. It can be collected and separated by a simple external magnetic field and can be applied to the fields of immune detection, targeted therapy and cell separation.

[0005] CN103571494A discloses a magnetic fluorescent nanocomposite microsphere, which has the following composition: magnetic nanoparticles and fluorescent quantum dots; the magnetic nanoparticles and the fluorescent quantum dots are chemically coupled via the functional hydroxyl groups at the ends of mannooligosaccharide molecules and the carboxyl groups of the fluorescent quantum dots. The magnetic fluorescent nanomicrosphere has both the superparamagnetism of magnetic particles and the excellent fluorescent properties of quantum dots. It can be collected and separated by a simple external magnetic field, and can be applied to chemical analysis detection, immunoassay, fluorescence tracking and magnetic separation.

[0006] Based on the above research, it can be seen that the use of magnetic and fluorescent dual-functional nanocomposite microspheres can make the microspheres have both fluorescent properties and magnetic properties, and can achieve fluorescence and MRI synergistic imaging. However, at present, the selection of magnetic particles and fluorescent particles needs to be further optimized. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a drug delivery system and a preparation method and application thereof, wherein the drug delivery system can realize the monitoring of drug delivery and fluorescence / MRI dual-modality cell imaging.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a drug delivery system, wherein the drug delivery system has magnetic nanoparticles as a core layer and silicon dioxide modified with graphene quantum dots as a shell layer, wherein the graphene quantum dots are loaded with doxorubicin.

[0010] Graphene Quantum Dots (GQDs) are a new type of zero-dimensional fluorescent carbon-based nanomaterials that can be used as labeling biomarkers and anticancer drug carriers. They have the characteristics of large surface area of ​​delocalized electrons, ideal photoluminescence, stable fluorescence, solubility in a variety of solvents, excellent biocompatibility and low toxicity. In addition, their small lateral size and abundant peripheral carboxyl groups can bind to targeting molecules, providing support for the potential application of tumor-targeted imaging in cancer diagnosis.

[0011] Magnetic nanoparticles tend to aggregate and dissolve under acidic conditions, while the silica coating can effectively protect the internal magnetite core of the magnetic nanoparticles and maintain the stability of the magnetic nanoparticles.

[0012] In the present invention, the magnetic nanoparticles include any one of cobalt ferrite, ferroferric oxide or ferrous oxide, or a combination of at least two thereof, preferably cobalt ferrite.

[0013] Cobalt ferrite (CoFe2O4) is a spinel ferrite with high saturation magnetization and coercivity, large specific surface area, good chemical stability, and easy synthesis. It is an excellent candidate as a drug carrier in cancer treatment.

[0014] Preferably, the magnetic nanoparticles are connected to silicon dioxide via Si—O bonds.

[0015] Preferably, the graphene quantum dots are connected to silicon dioxide via amide bonds.

[0016] The graphite quantum dots interact with doxorubicin via π-π bonds.

[0017] Preferably, the drug loading of the drug delivery system is 60-70% (for example, it may be 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, etc.).

[0018] In the present invention, the graphene quantum dots are also loaded with tumor targeting molecules.

[0019] Preferably, the tumor targeting molecule comprises folic acid.

[0020] The targeting molecules are all capable of achieving a targeting effect in the drug delivery process, wherein folic acid (FA) as an active tumor localization signal has a high affinity for tumor cells that overexpress folate receptors.

[0021] Preferably, the tumor targeting molecule and the graphene quantum dots are connected via an amide bond.

[0022] Preferably, the loading amount of the tumor targeting molecule accounts for 60-70% of the total mass of the drug delivery system (for example, it may be 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, etc.).

[0023] In the present invention, the drug delivery system has cobalt ferrite as a core layer and silicon dioxide modified with graphene quantum dots as a shell layer, and the graphene quantum dots are loaded with doxorubicin and folic acid;

[0024] The cobalt ferrite is connected to silicon dioxide via Si-O bonds, and the graphene quantum dots are connected to silicon dioxide via amide bonds; the doxorubicin is loaded onto the surface of the graphene quantum dots via π-π stacking; and the graphene quantum dots are connected to folic acid via amide bonds.

[0025] In a second aspect, the present invention provides a FRET model, wherein the FRET model comprises the drug delivery system described in the first aspect.

[0026] Preferably, the graphene quantum dots serve as drug carriers and / or donors in the FRET model.

[0027] Preferably, the doxorubicin is used as an anticancer drug and / or receptor in the FRET model.

[0028] The DOX / GQD-CFO@SiO2 / FA nanoplatform is used in a controlled drug delivery system, allowing real-time monitoring of the drug release process through FRET signals. GQDs act as both drug carriers and donors in the FRET model, while DOX acts as an anticancer drug and receptor in the FRET model. The FRET "on" / FRET "off" changes can be adjusted by loading / releasing DOX onto / from GQDs, allowing drug delivery to be monitored through FRET signals.

[0029] In the present invention, drug delivery is monitored by corresponding changes in FRET. GQD-CFO@SiO2 / FA was tested by MRI; the reduced brightness of the T2-weighted MRI image indicated the potential of GQD-CFO@SiO2 / FA as a negative MRI contrast agent. Fluorescence / MRI bimodal cell imaging can promote the transport of drug delivery. In addition, cytotoxicity experiments confirmed the ideal biocompatibility of the GQD-CFO@SiO2 / FA nanoplatform, and DOX / GQD-CFO@SiO2 / FA showed significant cytotoxicity to HeLa cells, which helped to enhance the targeting effect of FA-modified DOX / GQD-CFO@SiO2 / FA. These results indicate that the therapeutic efficiency of the drug delivery system provided by the present invention is significantly improved. Therefore, the drug delivery system provided by the present invention can monitor drug delivery and fluorescence / MRI bimodal cell imaging for simultaneous diagnosis and treatment of cancer.

[0030] In a third aspect, the present invention provides a method for preparing the drug delivery system according to the first aspect, the preparation method comprising the following steps:

[0031] (1) modifying silica with magnetic nanoparticles by emulsion polymerization to obtain magnetic nanoparticles@silica;

[0032] (2) subjecting the magnetic nanoparticles@silica to an amination reaction, and then reacting the magnetic nanoparticles@silica with graphene quantum dots to obtain graphene quantum dots-magnetic nanoparticles@silica;

[0033] (3) Mixing graphene quantum dots-magnetic nanoparticles@silica with doxorubicin to obtain doxorubicin / graphene quantum dots-magnetic nanoparticles@silica.

[0034] In the present invention, in step (1), the magnetic nanoparticles are cobalt ferrite, and the method for preparing the cobalt ferrite comprises the following steps: mixing a cobalt source, an iron source, oleic acid, 1,2-tetradecanediol, oleylamine and a solvent to obtain the cobalt ferrite.

[0035] Preferably, the cobalt source comprises Co(acac)2, and the iron source comprises Fe(acac)3.

[0036] Preferably, the molar ratio of the cobalt source to the iron source is 1:(1.5-2.5);

[0037] Among them, "1.5-2.5" can be 1.5, 1.7, 1.9, 2.1, 2.3, 2.5, etc.

[0038] Preferably, the solvent comprises phenyl ether.

[0039] Preferably, the molar ratio of oleic acid, 1,2-tetradecanediol and oleylamine is (4-8):(8-12):(4-8);

[0040] Among them, "4-8" can be 4, 5, 6, 7, 8, etc.;

[0041] "8-12" can be 8, 9, 10, 11, 12, etc.

[0042] Preferably, the mixing temperature is 180-300°C (for example, 180°C, 200°C, 220°C, 240°C, 260°C, 280°C, 300°C, etc.), and the mixing time is 1-5h (for example, 1h, 2h, 3h, 4h, 5h, etc.).

[0043] Preferably, in step (1), the preparation method by emulsion polymerization comprises the following steps: dispersing the magnetic nanoparticles in a mixed solution containing deionized water and anhydrous ethanol, ultrasonicating, and then adding ammonia water; and then mixing and reacting with tetraethoxysilane to obtain the magnetic nanoparticles@silicon dioxide.

[0044] Preferably, the volume ratio of the deionized water, anhydrous ethanol and aqueous ammonia is (8-12):(30-50):(0.5-1.5);

[0045] Among them, "8-12" can be 8, 9, 10, 11, 12, etc.;

[0046] "30-50" can be 30, 35, 40, 45, 50, etc.;

[0047] "0.5-1.5" can be 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, etc.

[0048] Preferably, the ultrasonication time is 0.5-1.5 h (for example, it can be 0.5 h, 0.7 h, 0.9 h, 1.1 h, 1.3 h, 1.5 h, etc.).

[0049] Preferably, the mixing reaction time is 0.5-1.5 h (for example, it can be 0.5 h, 0.7 h, 0.9 h, 1.1 h, 1.3 h, 1.5 h, etc.).

[0050] In the present invention, in step (2), the amination agent used in the amination reaction is 3-aminopropyltriethoxysilane.

[0051] Preferably, in step (2), the method for preparing the graphene quantum dots comprises the following steps: heating citric acid and then mixing it with a base to obtain the graphene quantum dots.

[0052] Preferably, the heating temperature is 180-200°C (for example, 180°C, 185°C, 190°C, 195°C, 200°C, etc.), and the heating time is 10-40min (for example, 10min, 20min, 30min, 40min, etc.).

[0053] Preferably, the alkali comprises an aqueous sodium hydroxide solution, and the molar concentration of the aqueous sodium hydroxide solution is 0.05-0.2M (for example, it can be 0.05M, 0.07M, 0.09M, 0.11M, 0.13M, 0.15M, 0.18M, ​​0.2M, etc.).

[0054] Preferably, in step (2), the graphene quantum dots need to be activated before reacting, and the activation reagents used for the activation include 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0055] Preferably, in step (3), before mixing the graphene quantum dots-magnetic nanoparticles@silica with doxorubicin, the method further comprises the following steps: subjecting the graphene quantum dots-magnetic nanoparticles@silica to a condensation reaction with folic acid.

[0056] Preferably, the condensation reagent used in the condensation reaction includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0057] As a preferred technical solution of the present invention, the preparation method of the drug delivery system comprises the following steps:

[0058] (a) modifying silica with magnetic nanoparticles through an inverse emulsion phase to obtain magnetic nanoparticles@silica;

[0059] (b) subjecting the magnetic nanoparticles@silica to an amination reaction, and then subjecting the magnetic nanoparticles@silica to an amide condensation reaction with graphene quantum dots to obtain graphene quantum dots-magnetic nanoparticles@silica;

[0060] Wherein, the graphene quantum dots need to be activated before reacting, and the activation reagents used in the activation include 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide;

[0061] (c) subjecting the graphene quantum dots-magnetic nanoparticles@silica to an amide condensation reaction with folic acid to obtain graphene quantum dots-magnetic nanoparticles@silica / folic acid;

[0062] (d) π-π stacking of graphene quantum dots-magnetic nanoparticles@silica / folic acid and doxorubicin to obtain doxorubicin / graphene quantum dots-magnetic nanoparticles@silica / folic acid.

[0063] In a fourth aspect, the present invention provides a use of the drug delivery system according to the first aspect or the FRET model according to the second aspect in monitoring drug delivery and / or fluorescence / MRI bimodal cell imaging.

[0064] Explanation of terms

[0065] CoFe2O4 / CFO------------Cobalt ferrite

[0066] NPs - nanoparticles

[0067] SiO2------------Silicon dioxide

[0068] GQDs------------Graphene quantum dots

[0069] FA------------Folic acid (FA)

[0070] DOX------------Adriamycin

[0071] FRET Resonance Energy Transfer

[0072] FR------------Folic acid receptor

[0073] MRI------------Magnetic resonance imaging

[0074] Iron(III)acetylacetonate------------Acetylacetonate iron(III), Fe(acac)3

[0075] Cobalt(II)acetylacetonate------------Cobalt(II)acetylacetonate, Co(acac)2

[0076] Aqueous ammonia-----------Ammonia (25%),

[0077] 3-aminopropyl triethoxysilane (APTES, 99%) 3-aminopropyl triethoxysilane (APTES, 99%)

[0078] Polyoxyethylene(10)octylphenyl ether(Triton X-100)-----------Polyoxyethylene(10)octylphenyl ether

[0079] Tetraethyl orthosilicate (TEOS, 98%) ------------Tetraethyl orthosilicate (TEOS, 98%),

[0080] Citric acid (99.5%)------------Citric acid (99.5%) was purchased from Sigma.

[0081] Oleic acid------------Oleic acid (90%)

[0082] Oleylamine------------Oleylamine (>70%),

[0083] Phenyl ether (99%) ------------ Phenyl ether (99%),

[0084] 1,2-tetradecanediol(97%)------------1,2-tetradecanediol(97%).

[0085] 1-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(EDC,99%),------------1-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(EDC,99%),

[0086] N-hydroxysuccinimide (NHS, 98%) ------------N-hydroxysuccinimide (NHS, 98%),

[0087] Methyl thiazolyl tetrazolium(MTT)

[0088] Dimethyl sulfoxide(DMSO)------------Dimethyl sulfoxide(DMSO) and

[0089] Phosphate buffered saline (PBS, 7.4) Phosphate buffered saline (PBS, 7.4)

[0090] Dulbecco's modified Eagle's medium-----------Eagle medium (DMEM),

[0091] Penicillin-streptomycin------------Penicillin-streptomycin,

[0092] Fetal bovine serum (FBS)

[0093] Trypsin-EDTA------------Trypsin-EDTA.

[0094] Compared with the prior art, the present invention has the following beneficial effects:

[0095] (1) The drug delivery system provided by the present invention can be used to monitor drug delivery by detecting FRET signals. GQDs act as both drug carriers and donors in the FRET model, while DOX acts as an anticancer drug and receptor in the FRET model; effective drug loading and controlled release are performed through the FRET model.

[0096] (2) The drug delivery system provided by the present invention can not only be used as a biomarker to track targeted tumors, but also can monitor the delivery of drugs, thereby improving diagnostic accuracy.

[0097] (3) The drug delivery system provided by the present invention exhibits significant cytotoxicity to HeLa cells and can significantly enhance the therapeutic effect.

[0098] (4) The drug delivery system provided by the present invention can not only be used to monitor drug delivery and fluorescence / MRI bimodal cell imaging, but also be used for simultaneous cancer diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 The morphology of CFO@SiO2 NPs and GQDs;

[0100] Among them, Figure a is the electron microscope image of CFO@SiO2 NPs, and Figure b is the electron microscope image of GQD.

[0101] Figure 2 Schematic diagram of the synthesis of DOX / GQD-CFO@SiO2 / FA.

[0102] Figure 3This is the standard curve of DOX.

[0103] Figure 4 This is the loading capacity diagram of GQD-CFO@SiO2 / FA.

[0104] Figure 5 This is the in vitro DOX release diagram of DOX / GQD-CFO@SiO2 / FA.

[0105] Figure 6 Hysteresis loops of CFO, CFO@SiO2 and GQD-CFO@SiO2 / FA at 300K;

[0106] Among them, 1 is CFO, 2 is GQD-CFO@SiO2, and 3 is GQD-CFO@SiO2 / FA.

[0107] Figure 7 T2-weighted MRI of GQD-CFO@SiO2 / FA at different CFO concentrations.

[0108] Figure 8 is the relaxation rate of GQD-CFO@SiO2 / FA at different CFO concentrations. DETAILED DESCRIPTION

[0109] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0110] Example 1

[0111] This embodiment provides a CFO, and the preparation method of the CFO comprises the following steps:

[0112] Co(acac)2 (1 mmol) and Fe(acac)3 (2 mmol) were added to a solution containing 6 mmol oleic acid, 10 mmol 1,2-tetradecanediol, 6 mmol oleylamine and 20 mL phenyl ether, and magnetic stirring was performed under N2. The resulting mixture was then heated to 200°C for 1.5 h and refluxed at 280°C for 1 h. After removing the heat source and cooling, the black mixture was washed with acetone and chloroform solvents to obtain CFO.

[0113] Example 2

[0114] This embodiment provides a CFO@SiO2 NPs, and the preparation method of the CFO@SiO2 NPs comprises the following steps:

[0115] 4 mL of CFO prepared in Example 1 was redispersed in a mixed solution containing 10 mL of deionized water and 40 mL of anhydrous ethanol. After ultrasonic stirring for 1 h, 1 mL of NH3·H2O was added. Then 2 mL of TEOS (tetraethyl orthosilicate) was added and the reaction was continued for 10 h to obtain CFO@SiO2 NPs.

[0116] Example 3

[0117] This embodiment provides NH2-CFO@SiO2 NPs, and the preparation method of the NH2-CFO@SiO2 NPs comprises the following steps:

[0118] The CFO@SiO2 NPs (brown mixture) prepared in Example 2 were redispersed in 6 mL of anhydrous ethanol, and 4 mL of the suspension was dispersed in a mixed solution containing 10 mL of deionized water and 40 mL of anhydrous ethanol. Then, a solution containing 3-aminopropyltriethoxysilane (APTES) (0.75 mL) dissolved in ethanol (10 mL) was added dropwise to the mixture, and then gently stirred for 8 h. After multiple washings, NH2-CFO@SiO2 NPs were obtained and set aside.

[0119] Example 4

[0120] This embodiment provides a GQD-CFO@SiO2 nanoparticle, and the preparation method of the GQD-CFO@SiO2 nanoparticle includes the following steps:

[0121] (1) Preparation of GQDs: 0.5 g of citric acid was heated to 190 °C for 25 min, and the resulting yellow solution was added to 50 mL of 0.1 M NaOH solution. NaOH and citric acid were neutralized to obtain an aqueous solution of GQDs.

[0122] (2) Preparation of GQD-CFO@SiO2 / FA nanoparticles: EDC (50 mg) and NHS (10 mg) were added to 10 mL of the GQDs solution (prepared in step (1)) and stirred for 30 min to obtain a suspension. Then, NH2-CFO@SiO2 NPs prepared in Example 3 were added to the above suspension and conjugated with GQDs to obtain GQD-CFO@SiO2. The carboxyl groups of GQDs and the amine groups of CFO@SiO2 formed a classic amide bond, and the GQDs were modified with amine-modified CFO@SiO2 NPs.

[0123] Example 5

[0124] This embodiment provides a GQD-CFO@SiO2 / FA nanoparticle, and the preparation method of the GQD-CFO@SiO2 / FA nanoparticle comprises the following steps:

[0125] FA was covalently bound to GQD-CFO@SiO2 nanoparticles via EDC / NHS reaction: 10 mL of MES buffer containing 50 mg of FA pretreated with EDC and NHS was added to the GQD-CFO@SiO2 nanoparticle solution prepared in Example 4, and gently shaken for 24 h to obtain GQD-CFO@SiO2 / FA nanoparticles. Finally, the obtained GQD-CFO@SiO2 / FA nanoparticles were purified and redispersed in PBS solution.

[0126] like Figure 1 As shown, the surface morphology of CFO@SiO2 NPs and GQDs was characterized using a Philips CM-100 transmission electron microscope;

[0127] Among them, Figure (a) is an electron microscope image of CFO@SiO2 NPs, from which it can be seen that the average size is about 25nm, with a shell of nearly 5nm; Figure (b) is an electron microscope image of GQD particles, from which it can be seen that the GQD particles are spherical, with an average size of 3.5nm and a standard deviation of 0.6nm.

[0128] Example 6

[0129] This embodiment provides a DOX / GQD-CFO@SiO2 / FA, and the preparation method of the DOX / GQD-CFO@SiO2 / FA comprises the following steps:

[0130] Drug loading was performed by mixing DOX (4.5 mg / mL, PBS) with the PBS buffer (2 mg / mL) of GQD-CFO@SiO2 / FA NPs prepared in Example 5. The mixture was sonicated for 30 min and then stirred at room temperature in the dark for 24 h to obtain DOX / GQD-CFO@SiO2 / FA.

[0131] like Figure 2As shown in the figure, the synthesis schematic diagram of DOX / GQD-CFO@SiO2 / FA, it can be seen from the figure that Co(acac)2 and Fe(acac)3 first synthesize CFO, then react with TEOS to generate CFO@SiO2 NPs; then generate NH2-CFO@SiO2 NPs through APTES amination; then react with GQD through amide condensation to generate GQD-CFO@SiO2; then react with folic acid through amide condensation to generate GQD-CFO@SiO2 / FA; then through π-π stacking to obtain DOX / GQD-CFO@SiO2 / FA. GQDs serve as drug carriers and donors in the FRET model, while DOX serves as an anticancer drug and receptor. After DOX / GQD-CFO@SiO2 / FA enters cancer cells, it can bind to the folate receptors on cancer cells and release drugs.

[0132] Test Example 1

[0133] Drug loading and release experiments of GQD-CFO@SiO2 / FA nanoplatform

[0134] Test method: DOX (0-0.9 mg / mL, PBS) was added to GQD-CFO@SiO2 / FA NPs in PBS buffer (2 mg / mL) for drug loading. The mixture was sonicated for 30 min and then stirred at room temperature in the dark for 24 h.

[0135] The DOX loading on GQD-CFO@SiO2 / FA NPs was determined by the absorbance at 480 nm by UV / visible spectroscopy. The amount of DOX loaded on GQD-CFO@SiO2 / FA NPs was quantified by an improved method reported in the literature (Dr. Liu published in Adv. Mater. in 2008. Literature: Liu, Z.; Liu, Q.; Huang, Y.; Ma, Y.; Yin, S.; Zhang, X.; Sun, W.; Chen, YJAM, Organic photovoltaic devices based on a novel acceptor material: graphene. Adv. Mater.,, 2008, 20 (20), 3924-3930.).

[0136] To further explore the FRET-based drug delivery, drug loading measurements were performed by mixing DOX with GQD-CFO@SiO2 / FA in PBS buffer. The UV-VIS standard curve of DOX was determined by UV / Vis spectroscopy using the absorbance at 480 nm.

[0137] like Figure 3The standard curve of DOX is shown in Figure 1, Y = 18.2077X-0.0079, R 2 =0.9997, good linear relationship.

[0138] like Figure 4 As shown in the GQD-CFO@SiO2 / FA loading capacity diagram, the loading capacity of GQD-CFO@SiO2 / FA increases with the increase of initial DOX concentration, and the maximum loading amount of GQD-CFO@SiO2 / FA is 65.4wt%.

[0139] In order to obtain the release curve of DOX, DOX / GQD-CFO@SiO2 / FA was dispersed in PBS solutions with different pH values ​​at 37°C with continuous stirring. At predetermined time intervals, the DOX / GQD-CFO@SiO2 / FA samples were separated from the buffer, and the concentration of DOX released in the supernatant was determined based on the standard curve of DOX. The cumulative amount of DOX released from DOX / GQD-CFO@SiO2 / FA within 48 h was calculated as follows:

[0140] Cumulative DOX release [%] = (Mt / M0) × 100%; where Mt is the total amount of DOX released from DOX / GQD-CFO@SiO2 / FA at time t, and M0 is the initial amount of DOX loaded into GQD-CFO@SiO2 / FA.

[0141] like Figure 5 As shown, the release graph of DOX in vitro from DOX / GQD-CFO@SiO2 / FA. At pH 7.4, less than 18% of DOX was released within 6 hours and showed great stability. In contrast, up to 42.6% of DOX was rapidly released within 6 hours at pH 5.5. After 48 hours, nearly 71% of DOX was released in acidic buffer at pH = 5.5, while less than 30% of DOX was released at pH = 7.4. pH-controlled release relies on the protonation of DOX in low pH media, which can make DOX more hydrophilic and water-soluble, resulting in increased release of DOX from the nanoplatform in aqueous solution. The experimental results are in good agreement with other literature describing similar pH-responsive drug release behaviors.

[0142] Test Example 2

[0143] MRI experiment of GQD-CFO@SiO2 / FA nanoplatform

[0144] Test methods: In order to study the magnetic properties of the obtained nanoparticles and evaluate the performance of GQD-CFO@SiO2 / FA as a diagnostic MR contrast agent, MRI tests were performed on GQD-CFO@SiO2 / FANPs. The prepared GQD-CFO@SiO2 / FA NPs with different CFO concentrations were dispersed and suspended in 200μL 1.0% agarose gel. All samples were then quickly transferred to a 6-well plate for T2-weighted MRI imaging. In addition, HeLa cells were incubated with GQD-CFO@SiO2 / FA NPs overnight and then detached by trypsin digestion. After the HeLa cells were washed with PBS, they were dispersed and suspended in 200μL 1.0% agarose gel and quickly transferred to a test tube for MRI experiments.

[0145] like Figure 6 As shown, the hysteresis loops of CFO, CFO@SiO2 and GQD-CFO@SiO2 / FA at 300K; the results show that the saturated nuclear magnetization (Ms) of CFO is 69.4emu / g, while the saturated nuclear magnetization (Ms) of CFO@SiO2 and CFO@SiO2-NH2 decreases to 22.1 and 15.3emu / g, respectively. The silica coating leads to a significant decrease in the saturated nuclear magnetization due to the reduction of the CFO fraction in the nanocomposite and the non-magnetic contribution of the shell. Although the silica coating and GQDs modification reduce the saturated nuclear magnetization of CFO, GQD-CFO@SiO2 / FA exhibits a nearly superparamagnetic behavior, which is similar to CFO NPs.

[0146] like Figure 7 As shown, T2-weighted MRI of GQD-CFO@SiO2 / FA at different CFO concentrations. The brightness of the T2-weighted image directly corresponds to the value of T2. Due to the dipole interaction between the magnetic moment of CFO and the protons in the surrounding medium, the signal intensity decreases with increasing CFO concentration. Since GQD-CFO@SiO2 / FA can shorten T2 and the transverse relaxation time, it leads to darker MR images.

[0147] The relaxation rate was extracted by a single exponential curve fitting the signal intensity versus echo time at different concentrations using the following equation:

[0148] Mxy(t)=Mxy(0)et / T2

[0149] where Mxy(0) and Mxy(t) are the initial and time equal to t transverse magnetization, respectively.

[0150] like Figure 8As shown in Figure 2, the relaxation rate of GQD-CFO@SiO2 / FA at different CFO concentrations, T2 relaxation time is linearly proportional to particle concentration. The R2 relaxation coefficient value of GQD-CFO@SiO2 / FA is 75.6mM -1 S -1 MRI results showed that GQD-CFO@SiO2 / FA exhibited an ideal contrast effect, indicating that GQD-CFO@SiO2 / FA is suitable for use as a negative MRI contrast agent.

[0151] Test Example 3

[0152] Cytotoxicity test of GQD-CFO@SiO2 / FA nanoplatform

[0153] The cytotoxicity of CFO@SiO2, GQD, GQD-CFO@SiO2, and GQD-CFO@SiO2 / FA on HeLa cells was determined by MTT assay. Briefly, HeLa cells were seeded into 96-well plates and grown overnight. The medium in each well was then replaced with medium (150 μL) containing free DOX, DOX / GQD-CFO@SiO2, or DOX / GQD-CFO@SiO2 / FA treatment. After 24 h of incubation, the medium in each well was replaced with fresh medium containing MTT reagent at a concentration of 0.5 mg / mL and incubated for 4 h. After careful washing and removal of the medium, 100 μL of DMSO was added to the wells to dissolve the formazan crystals. The absorbance of each well at 490 nm was recorded on a microplate reader to determine cell viability. MTT assay was performed to evaluate the tumoricidal potential of the fabricated nanoplatform.

[0154] HeLa cells were incubated with CFO@SiO2, GQD-CFO@SiO2, GQD-CFO@SiO2 / FA, GQDs, free DOX, DOX / GQD-CFO@SiO2, and DOX / GQD-CFO@SiO2 / FA, respectively.

[0155] The results showed that CFO@SiO2, GQD-CFO@SiO2, GQD-CFO@SiO2 / FA, and GQDs had no significant effect on the viability of HeLa cells, indicating that GQD-CFO@SiO2 / FA had ideal biocompatibility.

[0156] Compared with HeLa cells incubated with DOX / GQD-CFO@SiO2 and free DOX, DOX-loaded DOX / GQD-CFO@SiO2 / FA showed higher toxicity to HeLa cells. Obviously, DOX / GQD-CFO@SiO2 / FA showed significantly enhanced cytotoxicity to HeLa cells (84.7%), which was much higher than that of non-FA nanoplatform DOX / GQD-CFO@SiO2 (67.2%); at 10μg / mL DOX concentration, the DOX / GQD-CFO@SiO2 / FA nanoplatform brought HeLa cells through targeted molecular modification due to the improved targeting effect. These results indicate that FRET-based DOX / GQD-CFO@SiO2 / FA significantly improves the therapeutic effect.

[0157] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A drug delivery system, characterized in that The drug delivery system has cobalt ferrite as a core layer and silicon dioxide modified with graphene quantum dots as a shell layer, and the graphene quantum dots are loaded with doxorubicin and tumor targeting molecules; The cobalt ferrite and silicon dioxide are connected via Si—O bonds; The graphene quantum dots interact with doxorubicin via π-π bonds; The loading amount of the tumor targeting molecule accounts for 60-70% of the total mass of the drug delivery system; The cobalt ferrite is prepared by the following method: a cobalt source, an iron source, oleic acid, 1,2-tetradecanediol, oleylamine and a solvent are mixed to obtain the cobalt ferrite; the solvent comprises phenyl ether.

2. The drug delivery system according to claim 1, characterized in that The graphene quantum dots are connected to silicon dioxide via amide bonds.

3. The drug delivery system according to claim 1, characterized in that The drug loading of the drug delivery system is 60-70%.

4. The drug delivery system according to claim 1, characterized in that The tumor targeting molecule includes folic acid.

5. The drug delivery system according to claim 1, characterized in that The tumor targeting molecule and the graphene quantum dots are connected via an amide bond.

6. The drug delivery system according to claim 1, characterized in that The drug delivery system has cobalt ferrite as a core layer and silicon dioxide modified with graphene quantum dots as a shell layer, wherein the graphene quantum dots are loaded with doxorubicin and folic acid; The cobalt ferrite is connected to the silicon dioxide via a Si-O bond, and the graphene quantum dots are connected to the silicon dioxide via an amide bond; the doxorubicin is loaded onto the surface of the graphene quantum dots via π-π stacking; the graphene quantum dots are connected to folic acid via an amide bond; The loading amount of folic acid accounts for 60-70% of the total mass of the drug delivery system.

7. A FRET model, characterized in that: The FRET model includes the drug delivery system according to any one of claims 1-6.

8. The FRET model according to claim 7, characterized in that The graphene quantum dots serve as drug carriers and / or donors in the FRET model.

9. The FRET model according to claim 7, characterized in that The doxorubicin is used as an anticancer drug and / or receptor in the FRET model.

10. A method for preparing a drug delivery system according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) cobalt ferrite is modified with silica by emulsion polymerization to obtain cobalt ferrite@silica; (2) subjecting the cobalt ferrite@silica to an amination reaction, and then reacting it with graphene quantum dots to obtain graphene quantum dots-cobalt ferrite@silica; (3) condensing graphene quantum dots-cobalt ferrite@silica with folic acid to obtain graphene quantum dots-cobalt ferrite@silica / folic acid, and mixing graphene quantum dots-cobalt ferrite@silica / folic acid with doxorubicin to obtain doxorubicin / graphene quantum dots-cobalt ferrite@silica / folic acid.

11. The preparation method according to claim 10, characterized in that: In step (1), the method for preparing the cobalt ferrite comprises the following steps: mixing a cobalt source, an iron source, oleic acid, 1,2-tetradecanediol, oleylamine and a solvent to obtain the cobalt ferrite; the solvent comprises phenyl ether.

12. The preparation method according to claim 11, characterized in that: The cobalt source includes Co(acac)2, and the iron source includes Fe(acac)3.

13. The preparation method according to claim 11, characterized in that: The molar ratio of the cobalt source to the iron source is 1:(1.5-2.5).

14. The preparation method according to claim 11, characterized in that: The molar ratio of oleic acid, 1,2-tetradecanediol and oleylamine is (4-8):(8-12):(4-8).

15. The preparation method according to claim 11, characterized in that: The mixing temperature is 180-300° C. and the mixing time is 1-5 h.

16. The preparation method according to claim 10, characterized in that: In step (1), the preparation method by emulsion polymerization comprises the following steps: dispersing cobalt ferrite into a mixed solution containing deionized water and anhydrous ethanol, ultrasonicating, and then adding ammonia water; and then mixing and reacting with tetraethoxysilane to obtain the cobalt ferrite@silicon dioxide.

17. The preparation method according to claim 16, characterized in that: The volume ratio of the deionized water, anhydrous ethanol and aqueous ammonia is (8-12):(30-50):(0.5-1.5).

18. The preparation method according to claim 16, characterized in that: The ultrasonic time is 0.5-1.5 h.

19. The preparation method according to claim 16, characterized in that: The mixing reaction time is 0.5-1.5 h.

20. The preparation method according to claim 10, characterized in that: In step (2), the amination reagent used in the amination reaction is 3-aminopropyltriethoxysilane.

21. The preparation method according to claim 10, characterized in that: In step (2), the method for preparing graphene quantum dots comprises the following steps: heating citric acid and then mixing it with a base to obtain the graphene quantum dots.

22. The preparation method according to claim 21, characterized in that: The heating temperature is 180-200° C. and the heating time is 10-40 min.

23. The preparation method according to claim 21, characterized in that: The alkali comprises a sodium hydroxide aqueous solution, and the molar concentration of the sodium hydroxide aqueous solution is 0.05-0.2M.

24. The preparation method according to claim 10, characterized in that: In step (2), the graphene quantum dots need to be activated before the reaction, and the activation reagent used in the activation includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N -Hydroxysuccinimide.

25. The preparation method according to claim 10, characterized in that: In step (3), the condensation reagent used in the condensation reaction includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N -Hydroxysuccinimide.

26. The preparation method according to claim 10, characterized in that: The preparation method comprises the following steps: (a) Cobalt ferrite is modified with silica through an inverse emulsion phase to obtain cobalt ferrite@silica; (b) subjecting cobalt ferrite@silica to an amination reaction, and then subjecting it to an amide condensation reaction with graphene quantum dots to obtain graphene quantum dots-cobalt ferrite@silica; The graphene quantum dots need to be activated before the reaction, and the activation reagent used in the activation includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N -Hydroxysuccinimide; (c) subjecting graphene quantum dots-cobalt ferrite@silica to an amide condensation reaction with folic acid to obtain graphene quantum dots-cobalt ferrite@silica / folic acid; (d) Graphene quantum dots-cobalt ferrite@silica / folic acid are π-π stacked with doxorubicin to obtain doxorubicin / graphene quantum dots-cobalt ferrite@silica / folic acid.

27. Use of the drug delivery system according to any one of claims 1 to 6 in preparing a delivery system for monitoring drug delivery and / or fluorescence / MRI bimodal cell imaging.

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