A stable and controllable multifunctional oxygen-carrying nanoparticle preparation and preparation method thereof

Abstract

The present invention discloses a stable and controllable multifunctional oxygen-carrying nanoparticle and a preparation method thereof, wherein the multifunctional oxygen-carrying nanoparticle comprises a hollow mesoporous nanoparticle as a core, wherein the hollow mesoporous nanoparticle is encapsulated with oxygen-saturated perfluoropolyether, and the outer surface of the hollow mesoporous nanoparticle is coated with biocompatible polydopamine, and the outer layer of polydopamine is grafted with gold nanoparticles to form a gold nano-modified polydopamine-coated hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether. The multifunctional oxygen-carrying nanoparticle of the present invention can achieve a high drug loading of gold nanoparticles, Fenton-like reagents and oxygen, and can be synchronously delivered to tumor tissues to improve the hypoxia of the tumor microenvironment, which is conducive to significantly improving the efficacy of starvation / photothermal / chemodynamic therapy.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, in particular to a stable and controllable multifunctional oxygen-carrying nano preparation and a preparation method thereof. Background Art

[0002] Tumor starvation / chemodynamic therapy has attracted much attention in the research of malignant tumor treatment. Usually, starvation / chemodynamic therapy can first catalyze glucose into H2O2 aerobically under the combined action of enzyme / Fenton reagent, and then convert it into highly toxic hydroxyl free radicals, thereby inducing tumor cell death. Compared with traditional tumor treatment methods, starvation / chemodynamic therapy has the advantages of being non-invasive, specific and having fewer side effects.

[0003] However, starvation / chemodynamic therapy is affected by the hypoxic microenvironment of the tumor, which limits its efficacy. Improving the hypoxic microenvironment of the tumor is crucial to improving the therapeutic effect of tumors. Perfluorocarbon (PFC) liquids are often used as oxygen carriers to alleviate tumor hypoxia due to their high dissolved oxygen content. However, low molecular weight PFCs are volatile at room temperature, resulting in partial oxygen leakage before reaching the hypoxic area, thereby reducing the efficiency of oxygen delivery. Therefore, it is necessary to increase the stability of oxygen delivery. In addition, the entire oxygen diffusion process is mainly driven by the concentration gradient, which is slow and uncontrollable, and it is difficult to maintain high oxygen levels in the lesion area. Therefore, it is urgent to develop a stable and controllable oxygen delivery system to improve the hypoxic tumor microenvironment and improve the therapeutic effect of tumors. Summary of the invention

[0004] The purpose of the present invention is to provide a stable and controllable multifunctional oxygen-carrying nanoformulation and a preparation method thereof to solve the problems raised in the above-mentioned background technology. The multifunctional oxygen-carrying nanoformulation can achieve a high drug loading of gold nanoparticles, Fenton-like reagents and oxygen, and can be synchronously delivered to tumor tissues to improve the hypoxia of the tumor microenvironment, which is beneficial to significantly improve the efficacy of starvation / photothermal / chemodynamic therapy, thereby improving the tumor treatment effect.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A stable and controllable multifunctional oxygen-carrying nanoparticle preparation comprises hollow mesoporous nanoparticles as a core, wherein oxygen-saturated perfluoropolyether is encapsulated inside the hollow mesoporous nanoparticles, and biocompatible polydopamine is coated on the outer surface of the hollow mesoporous nanoparticles, and the outer layer of the polydopamine is grafted with gold nanoparticles to form a gold-nano-modified polydopamine-coated hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether.

[0007] As a further solution of the present invention, the hollow mesoporous copper sulfide nanoparticles are cage-like hollow mesoporous structures, and the particle diameter is 80 to 200 nm.

[0008] As a further embodiment of the present invention, the hollow mesoporous nanoparticles are hollow mesoporous ferrosoferric oxide nanoparticles, hollow mesoporous copper oxide nanoparticles, hollow mesoporous copper sulfide nanoparticles, hollow mesoporous zinc oxide nanoparticles, hollow mesoporous cerium oxide nanoparticles and / or hollow mesoporous manganese oxide nanoparticles.

[0009] The multifunctional oxygen-carrying nanoparticle of the present invention is a hollow mesoporous nanoparticle drug loaded with oxygen-saturated perfluoropolyether coated with polydopamine modified by gold nanoparticles, wherein the hollow mesoporous nanoparticles have a uniform pore structure and a high drug encapsulation surface area, and are a potential drug delivery carrier. Under near-infrared light, both gold nanoparticles and polydopamine (PDA) have good photothermal conversion properties, and work together to control oxygen release. In addition, the metal ions released by the hollow mesoporous nanoparticles can also participate in the Fenton / Fenton-like reaction to produce active oxygen.

[0010] In addition, dopamine can form a polydopamine (PDA) photothermal adhesive film on the surface of hollow mesoporous nanoparticles under slightly alkaline conditions. During the in vivo transportation of hollow mesoporous nanoparticles as drug delivery carriers, since their surfaces are modified with polydopamine (PDA) as a coating, the leakage of oxygen loaded with perfluorocarbon (PFC) during circulation in the body can be effectively avoided. At the same time, the surface of polydopamine (PDA) has abundant amino, quinone and phenolic hydroxyl groups, which provides convenience for modifying thiol groups and high-loaded gold nanoparticles.

[0011] In the present invention, perfluoropolyether has the characteristics of being non-volatile and having a high oxygen-carrying capacity. After oxygen adsorption is saturated, it is transported through the multifunctional oxygen-carrying nanoformulation of the present invention, which can effectively deliver oxygen to tumor tissue and controllably release oxygen under the tumor-specific microenvironment and photothermal stimulation, thereby improving the hypoxic state and enhancing the tumor treatment effect.

[0012] As a further solution of the present invention, the diameter of the gold nanoparticles is 1 to 5 nm. The gold nanoparticles have a glucose oxidase effect, which is beneficial to improving the hunger effect.

[0013] In summary, the multifunctional oxygen-carrying nanoformulation of the present invention comprehensively utilizes the advantageous properties of various materials, bringing new breakthroughs and possibilities to the field of tumor treatment.

[0014] A method for preparing a stable and controllable multifunctional oxygen-carrying nanoparticle preparation comprises the following steps:

[0015] S1. Preparation of hollow mesoporous nanoparticles;

[0016] S2. Encapsulating perfluoropolyether in hollow mesoporous nanoparticles by ultrasonic emulsification, adding dopamine solution, and reacting at room temperature in the dark to prepare a hollow mesoporous composite nanomaterial coated with polydopamine and loaded with perfluoropolyether;

[0017] S3. Grafting gold nanoparticles on the outer layer of the polydopamine-coated perfluoropolyether-loaded hollow mesoporous composite nanomaterial, and then filling with pure oxygen until the internal perfluoropolyether reaches oxygen saturation, to prepare a gold nano-modified polydopamine-coated oxygen-saturated perfluoropolyether-loaded hollow mesoporous nanomedicine.

[0018] As a preferred solution of the present invention, the hollow mesoporous nanoparticles in step S1 are specifically hollow mesoporous copper sulfide nanoparticles, and the specific preparation steps of the hollow mesoporous copper sulfide nanoparticles are:

[0019] S1-1, weigh polyvinyl pyrrolidone, add it to the solvent, and dissolve it under stirring at room temperature;

[0020] S1-2, under stirring at room temperature, gradually add cupric chloride solution and anhydrous hydrazine solution;

[0021] S1-3, under stirring at room temperature, gradually add sodium hydroxide solution to react;

[0022] S1-4, gradually add sodium sulfide solution under stirring at room temperature, then transfer to a water bath for heating, raise the temperature of the mixed solution to 60°C, and react under magnetic stirring;

[0023] S1-5. After the reaction is completed, the solution is naturally cooled to room temperature, washed several times by centrifugation with deionized water, and freeze-dried to obtain hollow mesoporous copper sulfide nanoparticles.

[0024] As a preferred solution of the present invention, the specific preparation steps of the hollow mesoporous composite nanomaterial loaded with perfluoropolyether coated with polydopamine in step S2 are:

[0025] S2-1. Weigh the hollow mesoporous nanoparticles, add perfluoropolyether, and disperse them by ultrasonic in an ice-water bath to form an emulsion.

[0026] S2-2, add deionized water, and disperse by ultrasonic in an ice water bath to form a dispersion;

[0027] S2-3, add dopamine solution, add Tris-HCl buffer under stirring, and react at room temperature in the dark;

[0028] S2-4. After the reaction is completed, centrifugation is performed, and then the mixture is washed three times with deionized water and resuspended in deionized water to obtain a hollow mesoporous composite nanomaterial coated with polydopamine and loaded with perfluoropolyether.

[0029] As a preferred embodiment of the present invention, the specific preparation steps of the hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether coated with gold nanoparticles modified polydopamine in step S3 are:

[0030] S3-1, adding aminopolyethylene glycol mercaptopyridine to the hollow mesoporous composite nanomaterial loaded with perfluoropolyether coated with polydopamine, stirring for reaction, centrifuging after the reaction, and then resuspending in deionized water;

[0031] S3-2, adding excess dithiothreitol to react, and after the reaction is completed, centrifuging, and then resuspending in deionized water;

[0032] S3-3, adding gold nanoparticle solution to react, after the reaction is completed, centrifuging, then resuspending in deionized water, filling with pure oxygen until the internal perfluoropolyether reaches oxygen saturation, to prepare a gold nano-modified polydopamine-coated hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Good biocompatibility: The present invention uses polydopamine (PDA) to coat biodegradable hollow mesoporous nanoparticles. Polydopamine (PDA) can be grafted with gold nanoparticles, so that the multifunctional oxygen-carrying nanoformulation has good biocompatibility.

[0035] 2. Intelligent and controllable oxygen release: On the surface of hollow mesoporous nanoparticles loaded with oxygen-saturated perfluoropolyether, the multifunctional oxygen-carrying nanoformulation of the present invention is coated with a biodegradable polydopamine (PDA) shell. Both gold nanoparticles and polydopamine (PDA) have good photothermal effects at 808nm, which is conducive to achieving controlled oxygen release in response to tumor microenvironment and photothermal stimulation, and reducing or avoiding leakage during oxygen delivery.

[0036] 3. Enhanced catalytic effect of glucose: The gold nanoparticles in the present invention also have the catalytic function of glucose oxidase. The combination of gold nanoparticles and oxygen-saturated perfluoropolyether is beneficial to improving the catalytic effect of glucose, significantly increasing the yield of hydrogen peroxide, and cascadingly amplifying hydroxyl radicals.

[0037] 4. Better oxygen loading capacity: The present invention adopts perfluoropolyether as a carrier for loading oxygen. Compared with the prior art, it can load more oxygen. When coated with a polydopamine (PDA) shell, it can stably deliver more oxygen to tumor tissue, which is beneficial to improving the tumor treatment effect.

[0038] 5. Synergistic enhancement of multiple therapeutic effects: The multifunctional oxygen-carrying nanoparticle preparation of the present invention cleverly combines the starvation therapy of gold nanoparticles, the photothermal therapy under the cooperation of gold nanoparticles and polydopamine (PDA), and the chemodynamic therapy effect of hollow mesoporous nanoparticles to form a synergistically enhanced tumor treatment effect.

[0039] 6. Simple operation, safety and high efficiency: The present invention has the characteristics of simple operation, high safety, mild reaction conditions, controllable reaction process and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the preparation process of the multifunctional oxygen-carrying nanoformulation of the present invention, taking mesoporous copper sulfide as an example.

[0041] Figure 2 This is a particle size diagram of the CuS and CFOPA materials synthesized in the present invention.

[0042] Figure 3 This is the Zeta potential diagram of the CuS and CFOPA materials synthesized in the present invention.

[0043] Figure 4 This is a transmission electron microscope image of the hollow mesoporous copper sulfide synthesized by the present invention.

[0044] Figure 5 This is a transmission electron microscopy image of the Au NP synthesized in the present invention.

[0045] Figure 6 This is a transmission electron microscope image of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0046] Figure 7 This is an elemental imaging diagram of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0047] Figure 8 This is the EDS image of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0048] Fig. 9 This is the XPS graph of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0049] Fig.10 This is an infrared thermal imaging image of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0050] Fig.11 This is the photothermal stability curve of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention.

[0051] Fig.12 This is the stability diagram of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluoropolyether at 30°C.

[0052] Fig.13 This is the oxygen loading capacity diagram of the multifunctional oxygen-carrying nanoformulation CFOPA when it contains perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluoropolyether respectively.

[0053] Fig.14 Graph showing the oxygen loading capacity of the oxygen-carrying nanoformulation when it contains perfluoropolyether or not.

[0054] Fig.15 This is a photothermal oxygen release diagram of the multifunctional oxygen-carrying nanoparticle CFOPA after stabilization for 12 hours, where: Fig.15 (a) is a schematic diagram of the operation; Fig.15 (b) is the oxygen release curve.

[0055] Fig.16 This is a test diagram of the multifunctional oxygen-carrying nanoparticle CFOPA catalyzing glucose to produce gluconic acid and H2O2, wherein: Fig.16 (a) is the pH change of glucose solution catalyzed by CFOPA after 30 days of storage; Fig.16 (b) is the graph showing the pH change of glucose solution catalyzed by CFOPA when it contains gold nanoparticles or not; Fig.16 (c) is the test diagram of CFOPA catalyzing glucose to produce H2O2.

[0056] Fig.17 The flow cytometer test chart shows that the multifunctional oxygen-carrying nanoparticle CFOPA of the present invention produces reactive oxygen species in 4T1 tumor cell lines under normoxic and hypoxic environments.

[0057] Fig.18 The killing effect of the multifunctional oxygen-carrying nanoparticle preparation CFOPA of the present invention on the 4T1 tumor cell line is shown.

[0058] Fig.19 This is the in vivo distribution diagram of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention in 4T1 tumor-bearing mice.

[0059] Fig. 20 This is the in vivo infrared thermal imaging image of the multifunctional oxygen-carrying nanoformulation CFOPA of the present invention in 4T1 tumor-bearing mice.

[0060] Fig.21 This is a diagram showing the therapeutic effect of the multifunctional oxygen-carrying nanoparticle CFOPA on 4T1 tumor-bearing mice, wherein: Fig.21 (a) is a schematic diagram of treatment; Fig.21 (b) is a tumor volume growth curve; Fig.21 (c) is a photograph of an in vitro tumor; Fig.21 (d) is the tumor mass map; Fig.21 (e) is a graph showing the changes in mouse body weight.

[0061] Fig. 22 This is a HE staining image of organs in 4T1 tumor-bearing mice after being treated with the multifunctional oxygen-carrying nanoparticle CFOPA of the present invention. DETAILED DESCRIPTION

[0062] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Embodiment: A stable and controllable multifunctional oxygen-carrying nanoformulation comprises hollow mesoporous nanoparticles as a core, wherein the hollow mesoporous nanoparticles are hollow mesoporous ferroferric oxide nanoparticles, hollow mesoporous copper oxide nanoparticles, hollow mesoporous copper sulfide nanoparticles, hollow mesoporous zinc oxide nanoparticles, hollow mesoporous cerium oxide nanoparticles and / or hollow mesoporous manganese oxide nanoparticles, preferably hollow mesoporous copper sulfide nanoparticles (CuS), wherein the hollow mesoporous copper sulfide nanoparticles (CuS) are encapsulated with oxygen-saturated perfluoropolyether (PFC, molecular weight 1500DA). The surface of hollow mesoporous copper sulfide nanoparticles (CuS) is coated with biocompatible polydopamine (PDA), and the outer layer of polydopamine (PDA) is grafted with gold nanoparticles (AuNP) to form gold nano-modified polydopamine-coated hollow mesoporous copper sulfide nanodrugs loaded with oxygen-saturated perfluoropolyether (CuS-PFC-O2@PDA-PEG-Au, abbreviated as CFOPA).

[0064] In this embodiment, the hollow mesoporous copper sulfide nanoparticles (CuS) are cage-shaped hollow mesoporous structures with a particle diameter of 120 nm, and the diameter of the gold nanoparticles (AuNP) is 3 nm.

[0065] A stable and controllable method for preparing multifunctional oxygen-carrying nanoparticles, please refer to Figure 1 As shown, the following steps are included:

[0066] S1. Preparation of hollow mesoporous copper sulfide nanoparticles (CuS):

[0067] S1-1, weigh 1.20 g of polyvinyl pyrrolidone (PVP-K30), add it to 140 mL of neutral deionized water, and dissolve it under stirring at room temperature;

[0068] S1-2, under stirring at room temperature, gradually add 0.6 mL of 67.5 mg / mL CuCl2·2H2O and 38.4 μL of anhydrous hydrazine (85%) solution;

[0069] S1-3, under stirring at room temperature, gradually add 120 mL of 0.5 mg / mL NaOH solution (pH = 9) and react for 5 min;

[0070] S1-4, under stirring at room temperature, gradually add 2.4 mL of 160 mg / mL Na2S solution, then transfer to a water bath for heating, heat the mixed solution to 60°C, and react for 2 h under magnetic stirring;

[0071] S1-5. After the reaction is completed, the solution is naturally cooled to room temperature, washed several times by centrifugation with deionized water, and freeze-dried to obtain hollow mesoporous copper sulfide nanoparticles (CuS).

[0072] The prepared hollow mesoporous copper sulfide nanoparticles were characterized by transmission electron microscopy, such as Figure 4 As shown in the figure, the structure of copper sulfide nanoparticles (CuS) is a hollow mesoporous structure. Figure 3 is the potential value of the products in each stage. Among them, the potential value of copper sulfide is -15mV, indicating that its surface is negatively charged. Figure 2 This is a test chart of the hydrated particle size of copper sulfide nanoparticles (CuS). The particle size of copper sulfide is 120nm.

[0073] S2. Preparation of hollow mesoporous copper sulfide composite nanomaterials loaded with perfluoropolyether coated with polydopamine (CuS-PFC@PDA):

[0074] S2-1. Weigh 4 mg of hollow mesoporous copper sulfide nanoparticles, add 200 μL of perfluoropolyether, and disperse by ultrasonication in an ice-water bath for 10 min to form an emulsion.

[0075] S2-2, add 4 mL of neutral deionized water and vortex to disperse to form a dispersion;

[0076] S2-3, add 1 mL of 2 mg / mL dopamine solution, add Tris-HCl buffer under stirring, and react at room temperature in the dark for 1 h;

[0077] S2-4, after the reaction is completed, centrifuge at 12000 rpm for 15 min, then wash three times with deionized water, and resuspend in neutral deionized water to obtain a polydopamine-coated perfluoropolyether-loaded hollow mesoporous copper sulfide composite nanomaterial (CuS-PFC@PDA, referred to as CFP);

[0078] S3. Preparation of hollow mesoporous copper sulfide nanoparticles (CFOPA) coated with gold nanoparticles modified with polydopamine and loaded with oxygen-saturated perfluoropolyether:

[0079] S3-1, add 2 mL of 1 mg / mL aminopolyethylene glycol thiopyridine (NH2-PEG 2000-OPSS), stirred and reacted for 2 h to prepare CuS-PFC@PDA-PEG-OPSS; after the reaction, centrifuged at 12000 rpm for 15 min, and then resuspended in neutral deionized water;

[0080] S3-2, adding excess dithiothreitol (DTT) and reacting for 2 hours. After the reaction, centrifuging at 12000 rpm for 15 minutes, and then resuspending in neutral deionized water to obtain CuS-PFC@PDA-PEG-SH;

[0081] S3-3. Add 20 mL of ultrapure water to 200 μL of 50 mM HAuCl4·4H2O (4 mg) solution, stir for 4 min, then add 2 mL of 1.75 mg / mL NaBH4 solution (0.092 M), stir for 5 min; centrifuge at 12000 rpm for 15 min, wash with ethanol and ultrapure water to obtain gold nanoparticles (AuNP). Figure 5 This is a transmission electron microscope image of gold nanoparticles (AuNPs). The particle size of gold nanoparticles (AuNPs) is about 3nm.

[0082] S3-4, at room temperature, add 400 μL of 50 μg / mL gold nanoparticle (Au NP) solution to 1 mL of 1 mg / mL CuS-PFC@PDA-PEG-SH solution, react for 2 hours, centrifuge at 12000 rpm for 15 minutes after the reaction, then resuspend in 1 mL of neutral deionized water, fill with pure oxygen, O2 flow rate of 1 L / min, for 30 minutes, so that the perfluorocarbon reaches oxygen saturation, thereby preparing a gold nano-modified polydopamine-coated hollow mesoporous copper sulfide nanodrug loaded with oxygen-saturated perfluoropolyether (CuS-PFC-O2@PDA-PEG-Au, abbreviated as CFOPA), that is, the stable and controllable multifunctional oxygen-carrying nanoformulation of the present invention. The prepared gold nano-modified polydopamine-coated hollow mesoporous copper sulfide nanodrug loaded with oxygen-saturated perfluoropolyether was characterized by transmission electron microscopy, as shown in FIG. Figure 6 As shown, the structure of the nano drug (CFOPA) in the figure is a hollow mesoporous structure, and the surface is modified with gold nanoparticles; Figure 7 This is the elemental mapping of the CFOPA nanoformulation, where evenly distributed elements such as Cu, S, F, and Au can be seen in the nanoformulation; Figure 8 Energy dispersive X-ray spectrum of CFOPA nanoformulation CFOPA nanoformulation mainly contains Cu, S, F and Au elements; X-ray photoelectron spectroscopy analysis of CFOPA was also performed, such as Fig. 9 As shown, it indicates the successful preparation of CFOPA.

[0083] 1. In vitro photothermal detection

[0084] The multifunctional oxygen-carrying nanoparticle preparation of the present invention was dispersed in deionized water to form a dispersion with a concentration of 100 μg / mL. 200 μL was transferred to a 1.5 mL centrifuge tube and irradiated with an 808 nm laser at 1.00 W / cm 2 The power density was irradiated for 5 min, and the temperature change of the solution was observed and recorded with a thermal imager at 1 min intervals. Under the same experimental conditions, the temperature change of the same volume of phosphate buffer (PBS) was used as the experimental control. Fig.10 As shown in the figure, compared with the control group, after 5 minutes of illumination, the temperature of the multifunctional oxygen-carrying nanoparticle (CFOPA) increased significantly to 53°C, proving that the multifunctional oxygen-carrying nanoparticle has a good photothermal effect. Fig.11 As shown, under continuous laser irradiation, after four times of heating and cooling, the multifunctional oxygen-carrying nanoformulation (CFOPA) still has good photothermal stability.

[0085] 2. Oxygen carrying capacity and oxygen controlled release testing

[0086] First, the stability of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluoropolyether was investigated at 30°C. Fig.12 As shown in the figure, at 30°C, perfluoropolyether remained stable within 2 hours with almost no mass loss, while other low molecular weight perfluorocarbons were completely volatilized, indicating that perfluoropolyether has good stability at room temperature. Then, the oxygen carrying capacity of the multifunctional oxygen-carrying nanoformulation (CFOPA) for different PFCs was evaluated. In the experiment, 20 mL of deoxygenated water was added to a 25 mL flask sealed with a rubber stopper, and the oxygen electrode probe was inserted into the flask through the rubber stopper to measure the oxygen concentration in the solution in real time. Subsequently, 0.00, 0.25, 0.50 and 1.00 mg / mL of CFOPA were injected into the closed system with a syringe, and the oxygen concentration in the solution was recorded after 12 hours. The oxygen carrying capacity of CFOPA loaded with perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluoropolyether was investigated respectively. As Fig.13 As shown, the oxygen loading capacity of CFOPA loaded with perfluoropolyether is 3.44±0.15 mg / g, which is significantly higher than the oxygen loading capacity of perfluoropentane (0.57±0.05 mg / g), perfluorohexane (0.84±0.08 mg / g), perfluoroheptane (1.96±0.13 mg / g) and perfluorooctane (2.02±0.12 mg / g). Finally, in order to verify whether the high oxygen loading capacity of the nanoformulation is attributed to the perfluoropolyether contained therein, the oxygen loading capacity of the multifunctional oxygen-carrying nanoformulation without perfluoropolyether was investigated after saturation with oxygen and equilibration for 12 hours (expressed as CuS-O2@PDA-Au). As Fig.14 As shown, the oxygen concentration hardly increased, further proving that the high oxygen carrying capacity of CFOPA is due to the perfluoropolyether it contains.

[0087] In addition, in order to investigate the oxygen release of the photothermally triggered nanoformulation, a solution of 1.00 mg / mL of the multifunctional oxygen-carrying nanoformulation (CFOPA) that had been stabilized for 12 h was irradiated with near-infrared light for 5 min (808 nm, 1.00 W / cm 2 ), real-time monitoring of dissolved oxygen changes. Fig.15 As shown, compared with the non-irradiated multifunctional oxygen-carrying nanoformulation group (CFOPA), the oxygen concentration of the multifunctional oxygen-carrying nanoformulation group after illumination (CFOPA+NIR) was significantly increased, proving that photothermal can further control and trigger the oxygen release of the multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention.

[0088] 3. Evaluation of in vitro catalytic production of gluconic acid and H2O2 from glucose

[0089] The catalytic activity of the multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention is detected by monitoring the change of pH value and the generation of H2O2. 0.50 mg of CFOP (without gold nanoparticles) and 0.5 mg of CFOPA are dispersed in 10 mL of ultrapure water, and then the pH value change of the solution is monitored every 1 min. At the 5th minute, 1 mL of glucose (Glu, 200 mM / L) is added, and the pH value is continuously monitored for 30 minutes. At the same time, the catalytic stability of the CFOPA synthesized as it is and stored for 30 days is compared. Fig.16 As shown in the figure, after the addition of glucose, a similar pH decrease trend was observed on the 1st and 30th days, indicating that CFOPA still has good catalytic activity after long-term storage; in addition, due to the lack of Au NPs, CFOP has no catalytic activity, while the CFOPA treatment group has a significant decrease in pH and an increase in H2O2 concentration, confirming that CFOPA can be triggered by Au NPs and exhibit stable and effective catalytic performance.

[0090] 4. Evaluation of Cellular Reactive Oxygen Species Production in Vitro

[0091] The multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention was used to detect the production of reactive oxygen species in tumor cells by flow cytometry. 4T1 cells in good culture state were inoculated into 6-well plates and placed in normoxia incubator and hypoxia incubator for 24 hours respectively; CuS, CuS-PFC@PDA (abbreviated as CFP), CuS-PFC@PDA-Au (abbreviated as CFP A) and CuS-PFC-O2@PDA-Au (abbreviated as CFOPA) were diluted to 100μg / mL with culture medium; the cells were divided into four groups, namely Control+NIR, CFP+NIR, CFPA+NIR and CFOPA+NIR treated cells; the culture medium was removed after 12 hours of incubation, the cells were rinsed 3 times with PBS, DCFH-DA (ROS indicator, 10μM) was added and incubated for 20 minutes, and then 808nm laser (1.00W / cm 2 , 5min), and continue to culture for 24h; flow cytometry was used to test the production of reactive oxygen species in the cells. Fig.17 As shown, the CFOPA+NIR group can better promote the production of reactive oxygen species in hypoxic cells.

[0092] 5. Evaluation of in vitro cell killing effect

[0093] The toxicity of the multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention to tumor cells was detected by MTT experiment. 4T1 cells in good culture state were inoculated into 96-well plates and cultured in a hypoxic incubator for 24 hours; CFOPA was diluted to different concentrations with culture medium and acted for 24 hours. The cells were divided into three groups, namely, CuS-PFC@PDA-Au+NIR (abbreviated as CFPA+NIR, which was not oxygenated) group, CuS-PFC-O2@PDA-Au (abbreviated as CFOPA) group and CuS-PFC-O2@PDA-Au+NIR (abbreviated as CFOPA+NIR) group. After 12 hours of administration, the laser group was treated with 808nm laser (1.00W / cm 2 , 5min), and continue to culture for 24h; use a multifunctional microplate reader to detect cell viability. Fig.18 As shown, the CFOPA+NIR group had excellent anti-tumor effect.

[0094] 6. Material biodistribution in vivo and tumor photothermal imaging assessment

[0095] In order to evaluate whether the multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention can be effectively enriched in tumors, a subcutaneous transplant tumor model of BALB / c mice was constructed using the 4T1 cell line, and CFOPA-PEG-Cy5 (dose: 10 mg / kg) was injected through the tail vein. The in vivo fluorescence intensity at 0, 4, 8, 12, 24 and 36 h was monitored using a small animal in vivo imaging device. Fig.19 As shown in the figure, after the injection of CFOPA-PEG-Cy5, the fluorescence signal at the tumor site gradually increased, reaching the maximum value at about 12 hours, and the fluorescence began to weaken at 24 hours, indicating that CFOPA-PEG-Cy5 has good tumor enrichment ability. In order to evaluate the in vivo warming effect of the multifunctional oxygen-carrying nanoformulation (CFOPA) of the present invention, a BALB / c mouse subcutaneous transplant tumor model was constructed using the 4T1 cell line, and the in vivo photothermal effect of the multifunctional oxygen-carrying nanoformulation (CFOPA) was studied by tail vein injection (dose: 10 mg / kg) combined with laser. The mice were randomly divided into two groups and injected with PBS and CFOPA, respectively. After 3, 6, 12 and 18 hours, 808nm laser (1.00W / cm 2 ) irradiate the tumor area for 5 minutes and record the temperature changes in the tumor area. Fig. 20 As shown in the figure, the temperature of the tumor of mice injected with CFOPA increased to 52°C after 12 hours. The temperature increase was mainly attributed to the high permeability and long retention effect of the nanoparticles and the high photothermal effect of gold nanoparticles, hollow mesoporous copper sulfide nanoparticles (CuS) and polydopamine (PDA).

[0096] 7. Tumor killing effect in vivo

[0097] In order to evaluate the anti-tumor effect of the multifunctional oxygen-carrying nanoparticle (CFOPA) of the present invention in vivo, a BALB / c mouse subcutaneous transplant tumor model was constructed using the 4T1 cell line. The anti-tumor effect of the multifunctional oxygen-carrying nanoparticle (CFOPA) was studied by tail vein injection (dose: 10 mg / kg) combined with laser. The tumor size of the mice was observed every day. When the tumor volume was about 100 mm 3 Five groups of mice were given drugs at different times: Control group, CuS group, CuS-PFC@PDA (referred to as CFP) group, CuS-PFC@PD A-Au (referred to as CFPA) group, and CuS-PFC-O2@PDA-Au (referred to as CFOPA) group. The drugs were given every other day for two times, and the weight and tumor volume of mice were measured at the same time. 12 hours after the drug administration, the mice were illuminated with 808nm laser (1.00W / cm 2 , 5min) to irradiate the tumor area. Fig.21As shown in the figure, compared with the control group, the CuS+NIR group and the CFP+NIR group showed a certain degree of inhibition on tumor growth, but the inhibitory effect was slightly inferior to that of the CFPA+NIR group. In addition, the CFOPA+NIR group had the best inhibitory effect, with a tumor weight of only 0.14g, much lower than the 1.16g of the control group. The tumor volume of mice in the CFOPA+NIR group was only 322mm 3 , much lower than the control group 1885mm 3 , indicating that the multifunctional oxygen-carrying nanoparticle preparation (CFOPA) of the present invention has a significant inhibitory effect on tumor growth under light. In addition, the HE staining results of the mouse organs after treatment in the PBS group (Con ntrol) and the CFOPA group showed ( Fig. 22 ), no obvious tissue damage was found in the CFOPA group, indicating that the multifunctional oxygen-carrying nanoformulation has good biosafety in vivo.

[0098] In summary, the multifunctional oxygen-carrying nanoparticle preparation (CFOPA) of the present invention has excellent anti-tumor effect.

[0099] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A stable and controllable multifunctional oxygen-carrying nanoparticle preparation, characterized in that: The invention comprises hollow mesoporous nanoparticles as a core, wherein the hollow mesoporous nanoparticles are hollow mesoporous copper sulfide nanoparticles, wherein oxygen-saturated perfluoropolyether is encapsulated inside the hollow mesoporous nanoparticles, and the outer surface of the hollow mesoporous nanoparticles is coated with biocompatible polydopamine, and the outer layer of the polydopamine is modified with thiol groups, and the thiol groups are grafted with gold nanoparticles in a covalent bond manner, so as to form hollow mesoporous nanomedicines loaded with oxygen-saturated perfluoropolyether and coated with polydopamine modified with gold nanoparticles.

2. A stable and controllable multifunctional oxygen-carrying nanoformulation according to claim 1, characterized in that: The hollow mesoporous nanoparticles are cage-shaped hollow mesoporous structures, and the particle diameter is 80-200nm.

3. A stable and controllable multifunctional oxygen-carrying nanoformulation according to claim 1, characterized in that: The diameter of the gold nanoparticles is 1-5 nm.

4. A method for preparing a stable and controllable multifunctional oxygen-carrying nanoparticle preparation, characterized in that: The following steps are involved: S1. preparing hollow mesoporous nanoparticles, wherein the hollow mesoporous nanoparticles are hollow mesoporous copper sulfide nanoparticles; S2. Encapsulating perfluoropolyether in hollow mesoporous nanoparticles by ultrasonic emulsification, adding dopamine solution, and reacting at room temperature in the dark to prepare a hollow mesoporous composite nanomaterial coated with polydopamine and loaded with perfluoropolyether; S3, grafting gold nanoparticles on the outer layer of the hollow mesoporous composite nanomaterial loaded with perfluoropolyether coated with polydopamine, and then filling with pure oxygen until the internal perfluoropolyether reaches oxygen saturation, to prepare a hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether coated with gold nanoparticles; The specific preparation steps of the gold nano-modified polydopamine-coated hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether in step S3 are as follows: S3-1, adding aminopolyethylene glycol mercaptopyridine to the hollow mesoporous composite nanomaterial loaded with perfluoropolyether coated with polydopamine, stirring for reaction, centrifuging after the reaction, and then resuspending in deionized water; S3-2, adding excess dithiothreitol to react, and after the reaction is completed, centrifuging, and then resuspending in deionized water; S3-3, adding gold nanoparticle solution to react, after the reaction is completed, centrifuging, then resuspending in deionized water, filling with pure oxygen until the internal perfluoropolyether reaches oxygen saturation, to prepare a gold nano-modified polydopamine-coated hollow mesoporous nanomedicine loaded with oxygen-saturated perfluoropolyether.

5. The method for preparing a stable and controllable multifunctional oxygen-carrying nanoparticle preparation according to claim 4, characterized in that: The hollow mesoporous nanoparticles in step S1 are preferably hollow mesoporous copper sulfide nanoparticles. The specific preparation steps of the hollow mesoporous copper sulfide nanoparticles are: S1-1, weigh polyvinyl pyrrolidone, add it to the solvent, and dissolve it under stirring at room temperature; S1-2, under stirring at room temperature, gradually add cupric chloride solution and anhydrous hydrazine solution; S1-3, under stirring at room temperature, gradually add sodium hydroxide solution to react; S1-4, gradually add sodium sulfide solution under stirring at room temperature, then transfer to a water bath for heating, raise the temperature of the mixed solution to 60°C, and react under magnetic stirring; S1-5. After the reaction is completed, the solution is naturally cooled to room temperature, washed several times by centrifugation with deionized water, and freeze-dried to obtain hollow mesoporous copper sulfide nanoparticles.

6. The method for preparing a stable and controllable multifunctional oxygen-carrying nanoparticle preparation according to claim 4, characterized in that: The specific preparation steps of the hollow mesoporous composite nanomaterial loaded with perfluoropolyether coated with polydopamine in step S2 are as follows: S2-1, weighing hollow mesoporous nanoparticles, adding perfluoropolyether, and ultrasonically dispersing in an ice-water bath to form an emulsion; S2-2, add deionized water, vortex disperse to form a dispersion; S2-3, add dopamine solution, add Tris-HCl buffer under stirring, and react at room temperature in the dark; S2-4. After the reaction is completed, centrifugation is performed, and then the mixture is washed three times with deionized water and resuspended in deionized water to obtain a hollow mesoporous composite nanomaterial coated with polydopamine and loaded with perfluoropolyether.

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