Nanometer titanium dioxide fluorescence imaging agent, preparation method thereof and application of nanometer titanium dioxide fluorescence imaging agent in tumor imaging

Abstract

The invention discloses a nano titanium dioxide fluorescence imaging agent, a preparation method of the nano titanium dioxide fluorescence imaging agent, and an application of the nano titanium dioxide fluorescence imaging agent in tumor imaging. The preparation method comprises the following steps: dispersing fluorescein isothiocyanate and a polylactic acid-glycolic acid copolymer FITC-PLGA-FA modified by folic acid in ethyl acetate to serve as an oil phase, preparing a stable TiO2FPs emulsion by adopting a Pickering emulsion method, and filtering bacteria through a filter membrane to obtain the composite material. The method is based on an OS-RC-2 kidney cancer cell line, and a 3D organ-like cell model and a tumor-bearing mouse model are constructed. The result shows that the TiO2FPs can realize fluorescence imaging of tumors on a 3D cell sphere model and a tumor-bearing mouse model. The invention provides a preparation method of a fluorescence imaging agent based on nano titanium dioxide and realizes tumor fluorescence imaging. The method is reasonable in design and high in repeatability.

Description

Technical Field

[0001] The invention belongs to the field of materials and biotechnology, and relates to a nano titanium dioxide fluorescent imaging agent and a preparation method thereof and application in tumor imaging. The nano titanium dioxide fluorescent imaging agent is TiO2FPs, which can be used in tumor fluorescence imaging. Background Art

[0002] At present, a variety of imaging diagnostic techniques have been applied to clinical medicine to reveal potential diseases, evaluate prognosis and efficacy, including magnetic resonance imaging (MRI), X-ray photography, computed tomography (CT), positron emission tomography (PET), ultrasound imaging (US) and optical imaging. However, due to relatively low target cell concentration in vivo, background tissue noise, tissue metabolism and limited resolution and signal penetration depth and other factors. Traditional imaging methods such as CT, MRI and US lack sufficient specificity and sensitivity, and although PET has higher sensitivity, there are problems such as poor resolution and radioactive safety (CA Boswell, MW Brechbiel, Development of radioimmunotherapeutic and diagnostic antibodies: an inside-out view. Nuclear Medicine & Biology. 2007, 34 (7), 757-778.). Compared with traditional CT, MRI and radioisotope imaging methods, optical-based imaging methods can improve target and background ratio by using optical probes, and its unique characteristics include the ability to simultaneously perform multicolor imaging and real-time signal activation of target sites. Therefore, optical imaging, such as fluorescence and bioluminescence imaging, can achieve accurate tumor diagnosis through non-invasive, real-time and high-resolution imaging, which is very beneficial for use in surgical treatment (AL. Vahrmeijer, M. Hutteman, DVJR. Van, DVJH Van, JV. Frangioni, Image-guided cancer surgery using near-infraredfluorescence. Nature Reviews Clinical Oncology. 2013, 10(9), 507-518.).

[0003] Fluorescence bioimaging is an emerging non-invasive, real-time, high-resolution detection method that can be used for early diagnosis of tumors. Fluorescence signals can provide molecular information about tumor tissues related to tumor anatomy and metabolism. There is an urgent need to establish stable, efficient, safe and multifunctional fluorescence systems to accelerate the realization of accurate and personalized medicine. Successful fluorescent probes must meet several requirements for medical imaging, including wavelength, brightness, biostability, photostability, specific tissue accumulation and pharmacokinetics. One approach is to simply and directly combine fluorophores with ligands that can bind to specific cell surface receptors: so-called active imaging probes. The imaging mechanism relies on the accumulation and retention of the probe as a contrast agent at the target site. This approach facilitates tumor imaging, but non-bound probes must be rapidly cleared from the circulation by excretion. Activatable imaging probes can be used to achieve highly specific delivery; such probes are activated only within targeted tumor cells, for example by tumor-specific enzymes. Such probes are non-fluorescent or weakly fluorescent in the unactivated state, but become strongly fluorescent after activation by relevant molecular triggers. The target-to-background ratio is higher than that of active probes. The activation mechanism of activatable probes mainly depends on enzymatic digestion and quenching effects. After removing the quencher part, the released fluorophore will immediately emit fluorescence. However, the fluorescent probes currently used in intraoperative imaging systems for clinical fluorescence imaging are non-targeted fluorophores, including indocyanine green, methylene blue, 5-aminolevulinic acid, and fluorescein (LG. Wang, CW. Barth, CH. Kitts, MD. Mebrat, SL. Gibbs, Near-infrared nerve-binding fluorophores for buried nerve tissue imaging. Science Translational Medicine. 2020, 12 (542), eaay0712.). Therefore, the development of fluorescent probes with active targeting function and specific activation imaging is of great value for auxiliary surgical treatment. Precision medicine requires advances in treatment methods, and the development of in vivo medical imaging technology for visualizing tumor lesions provides new technical means for diagnosis, auxiliary surgical resection, research on treatment effects and improved prognosis. Among them, fluorescence imaging has high specificity and sensitivity and has been clinically applied in medical imaging (M.Gao, F.Yu, C.Lv, J.Choo, L.Chen, Fluorescent chemical probes for accurate tumor diagnosis and targeting therapy. Chemical Society Reviews, 2017, 46(8): 2237-2271.); it has also spawned the development of various new targeted fluorescent probes.

[0004] Titanium dioxide (TiO2) nanoparticles have antibacterial and photocatalytic activities attributed to their ability to produce reactive oxygen species (ROS). The positive charge of TiO2 nanoparticles attracts negatively charged cell membranes, and ROS react with the cell surface proteins they come into contact with, affecting cell function and causing cell death. This is the nature basis for the application of TiO2 nanoparticles in cancer treatment. At the same time, the high specific surface area of ​​TiO2 nanoparticles can accommodate more microorganisms in contact with nanoparticles, increasing the destruction of cancer cells and causing cell death (P. Maheswari, S. Harish, S. Ponnusamy, C. Muthamizhchelvan, A novel strategy of nanosized herbal Plectranthusamboinicus, Phyllanthus niruri and Euphorbia hirta treated TiO2 nanoparticles for antibacterial and anticancer activities. Bioprocess Biosyst Eng, 2021, 44 (8): 1593-1616.). In addition, nano-TiO2 also has the characteristics of good biocompatibility, low phototoxicity, and stable performance. At present, cancer treatment with TiO2 nanoparticles mainly focuses on drug carriers, photodynamic therapy, sonodynamic therapy, multimodal synergistic therapy, and visual diagnosis and treatment. Fluorescein isothiocyanate (FITC) is the most widely used green fluorescent derivative in biology. FITC has the characteristics of high absorption rate and excellent fluorescence quantum yield, with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 520nm. If FITC can be combined with nano-TiO2, it is expected to further promote the development of tumor fluorescence imaging or the integration of imaging and treatment.

[0005] The basic components of Pickering emulsion are water phase, oil phase and solid particles, and all components are ultrasonically emulsified or homogenized at high speed. Pickering emulsion refers to the use of solid particles to replace traditional chemical emulsifiers, so that the solid particles form a thin film on the surface of the dispersed phase droplets. Among them, Pickering solid particles are solid particles with moderate hydrophilicity and lipophilicity ranging from tens of nanometers to several microns in size. They prevent the dispersed phase from agglomerating by irreversible adsorption at the oil-water interface to stabilize the emulsion (Liu Haozhe, Ye Meifang. Study on modified SiO2 Pickering particles and their emulsion stability. Colloids and Polymers, 2023, 41(01): 29-32.). In recent years, Pickering emulsions have attracted widespread attention from researchers at home and abroad because they can avoid the toxicity and side effects of surfactants and have a unique interfacial particle self-assembly effect. For Pickering emulsions under given conditions, the concentration of solid particles is one of the important factors affecting the droplet size and emulsion stability of Pickering emulsions, just like for traditional emulsions, as the concentration of surfactants increases, the droplet size decreases while the stability gradually improves. Unlike traditional emulsions, Pickering emulsions have the smallest droplet size and the highest stability when the oil-water volume ratio is near the phase transition point. At the same time, the pH value of the continuous phase can change the electrical properties or wetting characteristics of solid particles, thereby affecting the adsorption behavior of solid particles.

[0006] Based on this, the present invention proposes a new nano titanium dioxide fluorescent imaging agent (TiO2FPs), which has simple preparation, good repeatability, and excellent tumor fluorescence imaging effect. It is prepared by using FITC-PLGA-FA based on nano TiO2 and the Pickering method. Through experiments, the key parameters in the preparation process, such as aqueous phase nano TiO2 concentration, aqueous phase pH, water-oil ratio, etc., are studied. And the optimal preparation parameters are determined by characterization by particle size, zeta potential, transmission electron microscopy, etc.; OS-RC-2 renal cancer cell line is used, and 3D cell spheroid model and tumor-bearing mouse model are constructed to investigate the tumor fluorescence imaging effect of TiO2FPs. Experiments show that the optimal aqueous phase pH for preparing TiO2FPs is 3.0±0.5, m(H2O) / m(Monomers) is 9:1, and the optimal aqueous phase TiO2 nanoparticle concentration is 40μg / mL; TiO2FPs has good tumor fluorescence imaging effect in both renal cancer 3D organoid cell model and tumor-bearing mouse model. Summary of the invention

[0007] The purpose of the present invention is to provide a nano titanium dioxide fluorescent imaging agent and a preparation method thereof and its application in tumor imaging in view of the deficiencies of the prior art. The fluorescent imaging agent (TiO2FPs) is prepared by the Pickering method using FITC-PLGA-FA based on nano TiO2. Experiments have confirmed that TiO2FPs has good tumor fluorescence imaging effects in both renal cancer 3D organoid cell models and tumor-bearing mouse models.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect, a nano titanium dioxide fluorescent imaging agent of the present invention is prepared by dispersing nano titanium dioxide in deionized water as the aqueous phase, dispersing fluorescein isothiocyanate and folic acid modified polylactic acid-glycolic acid copolymer FITC-PLGA-FA in ethyl acetate as the oil phase, adopting the Pickering emulsion method to prepare a stable TiO2FPs emulsion, and filtering the obtained product through a membrane filter.

[0010] In the above technical solution, further, the nano titanium dioxide is rutile titanium dioxide with a particle size of 25 nm.

[0011] Furthermore, the concentration of titanium dioxide in the aqueous phase is 40 μg / mL, and the pH is adjusted to 3.0±0.5.

[0012] Furthermore, the FITC-PLGA-FA is a polylactic acid-glycolic acid copolymer PLGA modified with fluorescein isothiocyanate FITC and folic acid FA, wherein FITC, PLGA, and FA are interconnected through a condensation reaction between carboxylic acid and hydroxyl groups.

[0013] Furthermore, the concentration of FITC-PLGA-FA in the oil phase is 10 mg / mL.

[0014] Furthermore, the Pickering emulsion method is to add the water phase and the oil phase into a centrifuge tube, place it in an ultrasonic water bath for ultrasonic heating, and after the reaction is complete, centrifuge the obtained emulsion at high speed to separate the supernatant and the precipitate, add deionized water to dissolve the precipitate, filter the bacteria with a filter membrane, and obtain the fluorescent imaging agent TiO2FPs.

[0015] Furthermore, the ratio of the aqueous phase to the oil phase satisfies the mass ratio of water in the aqueous phase to FITC-PLGA-FA in the oil phase, m(H2O) / m(Monomers), of 9:1.

[0016] Furthermore, the parameters of the ultrasonic water bath are: ultrasonic power 0.5 kW, water bath temperature 25±5° C., and treatment time 15±5 min.

[0017] Furthermore, the high-speed centrifugation parameters are: 10000 rpm for 10 min.

[0018] In the second aspect, the nano titanium dioxide fluorescent imaging agent can be applied to tumor imaging. It can be directly used as an imaging agent, and can also be used to prepare tumor imaging fluorescent probes.

[0019] The beneficial effects of the present invention are at least:

[0020] (1) A nano-titanium dioxide imaging agent with fluorescent imaging function was constructed by using 25 nm golden red titanium dioxide as the basis and poly (D,L-lactic acid-co-glycolic acid) copolymer (PLGA, 50:50) modified with fluorescein isothiocyanate (FITC) and folic acid (FA); the nano-titanium dioxide fluorescent imaging agent (TiO2FPs) was prepared by the Pickering emulsion method.

[0021] (2) By controlling the conditions in the preparation process including the aqueous phase pH, 25 nm titanium dioxide concentration, and the m(H2O) / m(Monomers) ratio, and analyzing the zeta potential and particle size, the optimal preparation parameters were achieved: the optimal aqueous phase pH was 3.0±0.5, the m(H2O) / m(Monomers) was 9:1, and the optimal 25 nm TiO2 nanoparticle concentration was 40 μg / mL.

[0022] (3) TiO2FPs were used to achieve fluorescence imaging of tumor cells on a 3D organoid model of renal cancer (OS-RC-2) tumor cells.

[0023] (4) Achieve the effect of TiO2FPs tumor fluorescence imaging in experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effect curve of different 25 nm titanium dioxide concentrations in the aqueous phase on the zeta potential of aqueous phase particles.

[0025] Figure 2 Effect of different pH water phases on particle size in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0026] Figure 3 Effect of different pH water phase on zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0027] Figure 4 Effect of different m(H2O) / m(Monomers) ratios on particle size in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0028] Figure 5Effect of different m(H2O) / m(Monomers) ratios on zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0029] Figure 6 The effect of different concentrations of 25 nm titanium dioxide in aqueous phase on particle size in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs). Figure 7 Effect of different concentrations of 25 nm titanium dioxide in aqueous phase on zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0030] Fig. 8A Transmission electron microscopy image of nano-titanium dioxide fluorescent imaging agent (TiO2FPs).

[0031] Figure 8B Particle size analysis of nano-titanium dioxide fluorescent imaging agent (TiO2FPs).

[0032] Fig. 9 Fluorescence imaging effect of nano-titanium dioxide fluorescent imaging agents (TiO2FPs) on renal cancer cell 3D organoids.

[0033] Fig.10 Tumor fluorescence imaging effect of nano-titanium dioxide fluorescent imaging agent (TiO2FPs) on tumor-bearing mouse model (right); blank control injected with normal saline (left).

[0034] Fig.11 A synthetic route of FITC-PLGA-FA. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0036] According to a specific example of the present invention, a method for preparing a nano titanium dioxide fluorescent imaging agent (TiO2FPs) is provided, wherein the imaging agent is prepared by the following construction steps:

[0037] (1) Nano-titanium dioxide (golden red type, 25 nm) was dispersed in the aqueous phase (deionized water) and the concentrations were controlled to be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL for later use. The solutions were named (A-1T, A-2T, A-3T, A-4T, and A-5T), respectively. The zeta potential was measured by a zeta potential meter to determine the optimal aqueous phase particle concentration range.

[0038] (2) Nano-titanium dioxide (golden red type, 25 nm) was dispersed in an aqueous phase (deionized water) and the concentration was controlled to be 50 μg / mL. 0.01 mol / L hydrochloric acid solution and 0.01 mol / L sodium hydroxide solution were used to adjust the pH to 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, and the solutions were named (A-1, A-2, A-3, A-4, and A-5), respectively.

[0039] (3) Fluorescein isothiocyanate and folic acid modified poly(lactic acid-co-glycolic acid) (FITC-PLGA-FA) were dissolved in ethyl acetate to prepare a 10 mg / mL solution, named solution B. FITC-PLGA-FA is a product in which FITC, PLGA, and FA are interconnected through a condensation reaction of carboxylic acid and hydroxyl groups. Its chemical structure and synthesis route are shown in Fig.11 As shown, it can also be purchased directly.

[0040] (4) Solutions A-1, A-2, A-3, A-4, A-5 and solution B were added into microcentrifuge tubes with a ratio of solution A to solution B of 9:1. The microcentrifuge tubes were placed in an ultrasonic water bath for ultrasonic heating for 10 min. After the reaction was complete, the resulting Pickering emulsion was subjected to high-speed centrifugation (10,000 rpm, 10 mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered using a 0.2 μm filter membrane to obtain TiO2FPs named P-1, P-2, P-3, P-4, and P-5, respectively. The particle size and zeta potential were measured using a dynamic light scattering (DSL) particle size analyzer and a zeta potential meter to determine the optimal aqueous phase pH during the preparation.

[0041] (5) Solution A-1 and solution B were added into a microcentrifuge tube, and the ratio of solution A to solution B (m(H2O) / m(Monomers)) was controlled to be 6:1, 7:1, 8:1, and 9:1, respectively. The microcentrifuge tube was placed in an ultrasonic water bath and ultrasonically heated for 10 min. After the reaction was complete, the resulting Pickering emulsion was centrifuged at high speed (10,000 rpm, 10 mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered using a 0.2 μm filter membrane to obtain TiO2FPs named P-1M, P-2M, P-3M, P-4M, and P-5M, respectively. The particle size and zeta potential were measured by a dynamic light scattering (DSL) particle size analyzer and a zeta potential meter to determine the optimal m(H2O) / m(Monomers) in the preparation.

[0042] (6) Solutions A-1T, A-2T, A-3T, A-4T, and A-5T were prepared; the pH value was controlled to 3.0 using 0.01 mol / L hydrochloric acid solution, solution B was added into a microcentrifuge tube, and the ratio of solution A to solution B (m(H2O) / m(Monomers)) was controlled to be 9:1. The microcentrifuge tube was placed in an ultrasonic water bath and ultrasonically heated for 10 min. After the reaction was complete, the resulting Pickering emulsion was subjected to high-speed centrifugation (10,000 rpm, 10 mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered using a 0.2 μm filter membrane to obtain TiO2FPs named P-1T, P-2T, P-3T, P-4T, and P-5T. The particle size and zeta potential were measured using a dynamic light scattering (DSL) particle size analyzer and a zeta potential meter to determine the optimal aqueous phase 25 nm TiO2 concentration in the preparation.

[0043] (7) P-4T was prepared as a transmission electron microscopy sample, and the morphological characteristics of nano-titanium dioxide fluorescent imaging agents (TiO2FPs) were investigated by TEM.

[0044] According to another specific embodiment of the present invention, the prepared nano titanium dioxide fluorescent imaging agent (TiO2FPs) is used in tumor fluorescence imaging. This is achieved by the following steps: (1) In the 3D cell spheroid model of OS-RC-2 renal cancer cells, a certain concentration of TiO2FPs is added to the cell culture incubator for a certain period of time, and the cell spheroid is placed under a fluorescence confocal microscope for CLSM imaging, and the Z stack image data of the 3D cell spheroid in the range of 5 to 50 μm is analyzed, and a three-dimensional graph is constructed by Imaris9.3.1 software. (2) Through a tumor-bearing mouse model, a certain concentration of nano titanium dioxide fluorescent imaging agent (TiO2FPs) is injected, and the fluorescence imaging effect is investigated by small animal in vivo imaging technology.

[0045] Example 1 Determination of the optimal preparation parameters of nano-titanium dioxide fluorescent imaging agent (TiO2FPs).

[0046] 1.1 Zeta potential of different 25 nm titanium dioxide concentrations in water

[0047] (1) Disperse nano-titanium dioxide (golden red type, 25nm) in the aqueous phase (deionized water) and control the concentration to 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, and 50μg / mL respectively for use. Name them as solutions (A-1T, A-2T, A-3T, A-4T, and A-5T) respectively. Use a zeta potential meter to measure the zeta potential and determine the optimal aqueous phase particle concentration range. When the absolute value of the zeta potential is very high, the electrostatic repulsion between particles is very large, and the particles are not prone to flocculation, so they are very stable. The reason for this phenomenon is that when solid particles are wrapped in water molecules, the particles will be charged. The net charge on the surface of the particles will attract an equal amount of counter ions (ions with opposite charge to the particles) close to the surface to affect the ion distribution in the area around the particles, thus forming a double electrical layer between the solid and liquid phases. Figure 1 It can be seen that when the concentration of TiO2 nanoparticle aqueous dispersion is 20μg / mL, the zeta potential is close to 0, which is extremely unstable and more inclined to agglomeration; when the concentration is greater than 40μg / mL, the absolute value is greater than 10mV, which is conducive to the subsequent preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0048] 1.2 Effects of different pH water phases on particle size and zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs)

[0049] (1) Nano-titanium dioxide (gold red type, 25 nm) was dispersed in an aqueous phase (deionized water) to a concentration of 50 μg / mL. A 0.01 mol / L hydrochloric acid solution and a 0.01 mol / L sodium hydroxide solution were used to adjust the pH to 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, and the solutions were named (A-1, A-2, A-3, A-4, and A-5), respectively. Fluorescein isothiocyanate and folic acid-modified polylactic acid-glycolic acid copolymer (FITC-PLGA-FA) were dissolved in ethyl acetate to prepare a 10 mg / mL solution, named solution B. Solutions A-1, A-2, A-3, A-4, A-5 and solution B were added into microcentrifuge tubes with a ratio of solution A to solution B of 9:1. The microcentrifuge tubes were placed in an ultrasonic water bath and ultrasonically heated for 10 min. After the reaction was complete, the obtained Pickering emulsion was centrifuged at high speed (10000 rpm, 10 mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered using a 0.4 μm filter membrane to obtain TiO2FPs named P-1, P-2, P-3, P-4, and P-5, respectively.

[0050] (2) The particle size and zeta potential were measured by a dynamic light scattering (DSL) particle size analyzer and a zeta potential analyzer. Figure 2It can be seen that the emulsion particle size first increases with the increase of pH, reaches the maximum when the pH of the aqueous phase is 5.0, and then decreases with the increase of pH. Figure 2 and Figure 3 It can be seen that when the pH is 5.0, not only the emulsion particle size is the largest but also the absolute value of the zeta potential is the smallest. This shows that the TiO2 nanoparticle-stabilized Pickering emulsion is not stable at this time. Except for when the pH is 5.0, the absolute value of the emulsion zeta potential is greater than 20 and is in a relatively stable state. At the same time, when the pH is 3.0, the emulsion particle size is the smallest at 191.42nm. The smaller the particle size, the more stable the Pickering emulsion. Therefore, the pH range of 3.0±0.5 when the particle size is the smallest is considered to be the optimal aqueous phase pH.

[0051] 1.3 Effect of different m(H2O) / m(Monomers) ratios on particle size and zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs)

[0052] (1) Nano-titanium dioxide (golden red type, 25 nm) was dispersed in an aqueous phase (deionized water) to a concentration of 50 μg / mL, and a 0.01 mol / L hydrochloric acid solution was used to adjust the pH to 3.0 for later use, which was named solution A. Fluorescein isothiocyanate and folic acid-modified polylactic acid-glycolic acid copolymer (FITC-PLGA-FA) were dissolved in ethyl acetate to prepare a 10 mg / mL solution, which was named solution B. Solution A and solution B were added into a microcentrifuge tube, and the ratio of solution A to solution B (m(H2O) / m(Monomers)) was controlled to be 6:1, 7:1, 8:1, and 9:1, respectively. The microcentrifuge tube was placed in an ultrasonic water bath and ultrasonically heated for 10 minutes. After the reaction was complete, the obtained Pickering emulsion was centrifuged at high speed (10000rpm, 10mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered using a 0.4μm filter membrane to obtain TiO2FPs named P-1M, P-2M, P-3M, P-4M, and P-5M, respectively.

[0053] (2) The particle size and zeta potential are measured by a dynamic light scattering (DSL) particle size analyzer and a zeta potential analyzer. Generally, the water-oil volume ratio does not have a significant effect on the stability of the emulsion, but it will have a significant effect on the average particle size and type of the emulsion. Figure 4It can be seen that the emulsion particle size is generally larger when m(H2O) / m(Monomers) is 6:1 to 8:1 than when m(H2O) / m(Monomers)>8:1. As the volume of the internal phase increases, the particle size of the emulsion droplets increases accordingly. If the volume of the internal phase increases to a certain extent, the solid particles adsorbed on the water-oil interface will not be sufficient to prevent the aggregation and merging of the droplets, resulting in a phase inversion phenomenon. TiO2 nanoparticles are hydrophilic and form an oil-in-water emulsion system. The smaller the m(H2O) / m(Monomers), the less water content in the system, the larger the volume of the internal phase, i.e. the oil phase, the more intense the aggregation and merging reaction, and the particles are more likely to aggregate. As m(H2O) / m(Monomers) increases, the volume of the oil phase in the entire system decreases, and the particle size of the emulsion droplets decreases. For oil-in-water emulsions, when the monomer concentration is higher, polymer microspheres with larger particle sizes tend to be formed. Figure 5 It can be seen that when m(H2O) / m(Monomers) is 9:1, the zeta potential is -15.2mV, which has a large absolute value, indicating electronegativity, which is beneficial for circulation in the body. Therefore, the optimal m(H2O) / m(Monomers) is determined to be 9:1.

[0054] 1.4 Effect of different concentrations of 25nm titanium dioxide in aqueous phase on particle size and zeta potential in the preparation of nano-titanium dioxide fluorescent imaging agents (TiO2FPs).

[0055] (1) Solutions A-1T, A-2T, A-3T, A-4T, and A-5T were prepared; the pH value was controlled to 3.0 with 0.01 mol / L hydrochloric acid solution, solution B was added into a microcentrifuge tube, and the ratio of solution A to solution B (m(H2O) / m(Monomers)) was controlled to be 9:1, and the microcentrifuge tube was placed in an ultrasonic water bath for ultrasonic heating for 10 min. After the reaction was complete, the obtained Pickering emulsion was subjected to high-speed centrifugation (10000 rpm, 10 mins) to separate the supernatant and the precipitate. The supernatant was stored for later use, and the precipitate was re-dissolved in deionized water. The bacteria were filtered with a 0.4 μm filter membrane to obtain TiO2FPs named P-1T, P-2T, P-3T, P-4T, and P-5T, ​​respectively.

[0056] (2) The particle size and zeta potential were measured by a dynamic light scattering (DSL) particle size analyzer and a zeta potential meter to determine the optimal aqueous phase 25 nanometer TiO2 concentration in the preparation. Under certain emulsification conditions, the initial solid particle concentration has an important influence on the droplet size and the stability of the emulsion. The effect of solid particle concentration on the stability of the Pickering emulsion is almost the same as that of the surfactant. When the solid particle concentration is relatively low, the droplet size is larger and demulsification is prone to occur. As the solid particle concentration increases, the droplet size becomes smaller and the stability is also improved. Figure 6 It can be seen that when the concentration of TiO2 nanoparticles is 10μg / mL and 20μg / mL, the particle size of the emulsion is relatively large. As the concentration of TiO2 nanoparticles increases to 30μg / mL, 40μg / mL, and 50μg / mL, the particle size of the emulsion tends to be gentle; among them, the particle size of 246nm is relatively the smallest when the concentration of TiO2 nanoparticles is 40μg / mL. Figure 7 Judging from the zeta potential of the Pickering emulsions prepared at different 25 nm TiO2 concentrations (the zeta potential of 25 nm TiO2 concentration of 40 μg / mL is -27.3 mV), 40 μg / mL was selected as the optimal concentration for preparing 25 nm TiO2 in the aqueous phase.

[0057] (3) Under the optimal preparation parameter conditions (aqueous phase 25 nm TiO2 concentration 40 μg / mL, aqueous phase pH 3.0±0.5, m(H2O) / m(Monomers) 9:1), the prepared nano-titanium dioxide fluorescent imaging agent (TiO2FPs) P-4T was characterized by TEM. Fig. 8A It can be seen that the spherical morphology is conducive to in vivo application, and the particle size is Figure 8B The particle size is consistent with that measured by DSL shown.

[0058] Example 2 Fluorescence imaging effect of nano-titanium dioxide fluorescent imaging agent (TiO2FPs) on 3D cell spheroids of renal cancer cells. 3D cell spheroids are a cell culture model between the two-dimensional monolayer cell model and the solid tumor model. OS-RC-2 cells were cultured in DMEM / F-12 culture medium containing 2mM glutamine, 100U / mL penicillin-streptomycin, 1×B27, 20ng / mL EGF, and 10ng / mL FGF in an ultra-low adsorption culture dish (Corning) at 37°C and 5% C02 cell culture incubator for 4 days. The culture medium was replaced, and a certain concentration of nano-titanium dioxide fluorescent imaging agent (TiO2FPs) was added. After incubation for a certain period of time, the cells were observed under a confocal fluorescence microscope, and the Z stack images of the 3D cell spheroids in the range of 5 to 50μm were analyzed, and three-dimensional graphics were constructed using Imaris 9.3.1 software. By Fig. 9 It can be seen that, except for a few free TiO2FPs, most of them can effectively target tumors and achieve fluorescence imaging of tumor morphology.

[0059] Example 3 Tumor fluorescence imaging effect of nano-titanium dioxide fluorescent imaging agent (TiO2FPs) on mouse tumor-bearing model. Renal cell carcinoma OS-RC-2 cells were used to establish a tumor-bearing model by subcutaneous injection. Logarithmic phase OS-RC-2 cells were collected, digested and the culture medium was removed, washed with PBS and resuspended to prepare 1×10 8 / mL of cell suspension, 100 μL of cell suspension was injected subcutaneously in the armpit of nude mice, and the mice were fed for about 30 days until the tumor volume reached 100 mm 3 . Nano-titanium dioxide fluorescent imaging agent (TiO2FPs) physiological saline solution was injected into the tail vein of nude mice 4 hours later, and then observed by small animal in vivo imaging system. Fig.10 It can be seen that there is a strong fluorescence signal at the tumor site in the mouse armpit, realizing in vivo fluorescence imaging of the tumor.

[0060] The above-described embodiments are only some of the preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A nano titanium dioxide fluorescent imaging agent, characterized in that: The method comprises the following steps: dispersing nano-titanium dioxide in deionized water as the aqueous phase, dispersing fluorescein isothiocyanate and polylactic acid-glycolic acid copolymer FITC-PLGA-FA modified with folic acid in ethyl acetate as the oil phase, adopting the Pickering emulsion method to prepare a stable TiO2FPs emulsion, and filtering the emulsion with a membrane to obtain the obtained product.

2. The nano titanium dioxide fluorescent imaging agent according to claim 1, characterized in that: The nano titanium dioxide is rutile titanium dioxide with a particle size of 25 nm.

3. The nano titanium dioxide fluorescent imaging agent according to claim 1, characterized in that: The concentration of titanium dioxide in the aqueous phase was 40 μg / mL, and the pH was adjusted to 3.0±0.

5.

4. The nano titanium dioxide fluorescent imaging agent according to claim 1, characterized in that: The FITC-PLGA-FA is a polylactic acid-glycolic acid copolymer PLGA modified with fluorescein isothiocyanate FITC and folic acid FA, wherein FITC, PLGA and FA are interconnected through a condensation reaction between carboxylic acid and hydroxyl.

5. The nano titanium dioxide fluorescent imaging agent according to claim 1, characterized in that: The concentration of FITC-PLGA-FA in the oil phase is 10 mg / mL.

6. The nano titanium dioxide fluorescent imaging agent according to claim 1, characterized in that: The Pickering emulsion method is to add the water phase and the oil phase into a centrifuge tube, place it in an ultrasonic water bath for ultrasonic heating, centrifuge the obtained emulsion at high speed after the reaction is complete, separate the supernatant and the precipitate, add deionized water to redissolve the precipitate, filter the bacteria with a filter membrane, and obtain the fluorescent imaging agent TiO2FPs.

7. The nano titanium dioxide fluorescent imaging agent according to claim 6, characterized in that: The ratio of the aqueous phase to the oil phase satisfies the mass ratio of water in the aqueous phase to FITC-PLGA-FA in the oil phase, m(H2O) / m(Monomers), which is 9:

1.

8. The nano titanium dioxide fluorescent imaging agent according to claim 6, characterized in that: The parameters of the ultrasonic water bath are: ultrasonic power 0.5 kW, water bath temperature 25±5°C, and treatment 15±5 min.

9. The nano titanium dioxide fluorescent imaging agent according to claim 6, characterized in that: The high-speed centrifugation parameters are: 10000 rpm for 10 min.

10. Use of the nano titanium dioxide fluorescent imaging agent according to any one of claims 1 to 9 in the preparation of a tumor fluorescent imaging agent.

Images