CN110743013A - Up-conversion nano composite material for dual-power cooperative treatment, preparation method and application - Google Patents

Abstract

The invention discloses a preparation method for dual-dynamic synergistic treatment of up-conversion nano-composite materials, which comprises the following steps: (1) synthesizing oil-soluble rare earth up-conversion luminescent nanocrystals coated with an inert _NaGdF layer on the surface to obtain azelaine Acid-terminated hydrophilic upconverting nanoparticles, forming the first dispersion; ( ₂ ) adding KMnO to the first dispersion until a brown colloid forms, forming the second dispersion; (3) preparing the hydrophilic polymer ligands , and stir with the second dispersion to obtain the third dispersion; (4) C ₃ N ₄ acidified with nitric acid is prepared, and the third dispersion is activated and coupled with the third dispersion under the action of the activator to obtain the side growth MnO ₂ and Covalently linked _{C3N4 upconversion} nanocomposites. The invention also discloses the prepared material and application. The material also has multiple application values such as synergistic dual-dynamic therapy, up/down conversion fluorescence imaging, magnetic resonance imaging, etc., and has potential application prospects in the biomedical fields such as tumor diagnosis and treatment.

Description

Upconversion nanocomposite for dual-dynamic synergistic therapy, preparation method and same application

technical field

The invention belongs to the technical field of nano-biological materials, and in particular relates to an up-conversion nano-composite material for dual-dynamic synergistic therapy and a preparation method and application thereof.

Background technique

In recent years, cancer has become one of the major diseases that endanger human health. As one of the main methods of cancer diagnosis, bioimaging has gradually become a research hotspot. Compared with other imaging techniques, bio-optical imaging has the advantages of simple operation, intuitive and easy to accept, signal strength and relatively low cost, and has broad prospects in the diagnosis and treatment of various diseases. Among them, rare earth upconversion luminescent nanomaterials have attracted more and more attention due to their unique luminescent properties. This is because it can be excited by near-infrared light to emit ultraviolet, visible and near-infrared light, which is an inverse Stokes luminescence process. As the excitation light source, near-infrared light has almost no damage to biological tissues, and is expected to achieve a centimeter-level tissue penetration depth without causing background fluorescence. As a new type of imaging agent, upconversion nanomaterials have been widely used in a series of biological imaging fields, such as cell imaging, in vivo imaging of small animals, magnetic resonance imaging (MRI) and so on.

In view of the current clinical diagnosis and treatment of cancer are independent of each other, the treatment cycle is long, the cost is high, and chemotherapy, radiotherapy, surgery and other treatment methods have serious side effects, etc. Materials have become a new direction for cancer development.

In the prior art, the reported composite materials based on up-conversion nanomaterials and PDT/CDT reagents are mostly composites of a PDT/CDT reagent and up-conversion nanomaterials alone. The effect of stimulation is limited, and the therapeutic effect has great limitations. Among them, PDT is an abbreviation for photodynamic therapy, and CDT is an abbreviation for chemodynamic therapy.

Magnetic resonance imaging (MRI) is one of the most respected non-invasive, high-resolution and high-flexibility diagnostic techniques in medicine today. MRI contrast agents are playing an increasingly important role in clinical diagnosis. Photodynamic therapy (PDT) with photosensitizer as the core is a recently developed new method for the treatment of tumors with selective destruction and less toxic and side effects. In view of the obvious application advantages and development prospects of the two, if a new class of reagents that can combine the dual functions of magnetic resonance imaging contrast agents and photosensitizers for photodynamic therapy can be developed, the combination of diagnosis and treatment of the two can be effectively promoted, and the It may bring revolutionary changes to the diagnosis and treatment of tumors.

Photodynamic therapy (PDT) is a new technology for the diagnosis and treatment of diseases based on the interaction of light, photosensitizers and oxygen. Excited state and transfer its energy to surrounding oxygen molecules, thereby generating singlet oxygen ( ¹ O ₂ ) or reactive oxygen species (ROS) such as free radicals, and thereby causing tissue and organ damage and destroying the target tissue to achieve the purpose of treatment . Compared with traditional cancer treatments including surgery, chemotherapy and radiotherapy, photodynamic therapy (PDT) has significant advantages such as less trauma, low toxicity, good selectivity, high precision, reproducible treatment and synergistic surgery.

At present, compared with traditional cancer treatment methods (radiotherapy, chemotherapy, etc.), which are more harmful to human body, photodynamic therapy (PDT) is a non-invasive and mild medical technology, especially in anti-cancer treatment. , can use appropriate photosensitizers to generate reactive oxygen species (ROS) when irradiated at specific wavelengths to achieve the effect of killing cancer cells. As an emerging and promising cancer treatment modality, PDT can also be used to improve the shortcomings of other traditional anticancer therapies.

However, some unresolved challenges seriously hinder the further clinical application of PDT: First, the conventionally used organic photosensitizers have low water solubility and low stability, and are poorly soluble in physiological environments, so they are prone to severe agglomeration, which is often It has high toxicity to normal tissues; secondly, its photosensitization efficiency is low, resulting in low photodynamic efficiency and limited therapeutic effect. Finally, the low penetration depth of UV-Vis makes it impossible to reach deep pathological tissues or organs, and can only be used for the treatment of very shallow epidermal layers and areas where photons can reach. Therefore, other innovative methods and materials need to be developed to improve treatment efficiency.

Chemodynamic therapy (CDT) may be a good way to improve the efficiency of PDT, especially with photosensitizers, which can achieve two therapeutic approaches in the tumor microenvironment.

In the prior art, the document of Chinese Patent Application No. 201410826953.3 discloses a nanocomposite material applied to photodynamic therapy and a preparation method thereof, which is to use Au ₂₅ to modify UCNPs@SiO ₂ to obtain nanoparticles, so that the nanoparticles The photodynamic performance has been significantly improved. The invention prepares a kind of uniform, monodisperse, size-controllable (50-100 nm), mesoporous _SiO2 -coated nanocarriers with up-conversion nanoparticle core-shell structure, and the photosensitizer drug molecule _Au25 is prepared by a suitable adsorption method. (Capt) ₁₈ -(Au ₂₅ ) is connected to the mesoporous SiO ₂ framework to construct multifunctional composite materials, which are used to solve the current problems of photodynamic therapy (PDT). However, it is necessary to prepare NaGdF ₄ :20%Yb/1%Er(UCNPs) by high temperature pyrolysis method, then prepare UCNPs by continuous coating method, then synthesize core-shell UCNPs@SiO ₂ nanomaterials, and then synthesize Au ₂₅ , and finally modified Au ₂₅ with UCNPs@SiO ₂ nanoparticles. The whole process is complicated and requires high conditions.

Therefore, to develop a new upconversion nanocomposite that can simultaneously satisfy the integration of clinical diagnosis and treatment, namely fluorescence imaging, magnetic resonance imaging, and dual-dynamic synergy, and make it break through the existing treatment limitations, realize dual-dynamic synergistic therapy, and achieve a large It is very meaningful to improve its efficacy.

SUMMARY OF THE INVENTION

The present invention aims at the deficiencies of the prior art, and provides a preparation method of an up-conversion nano-composite material for the integration of dual-power synergistic therapy and diagnosis and treatment. The two-dimensional material method for preparing the upconversion nanocomposite material has few steps, simple operation, mild reaction, and can be completed at room temperature, which is beneficial to mass production.

The invention also provides an up-conversion nanocomposite material and application for the integration of dual-dynamic synergistic therapy and diagnosis and treatment. The material is compounded with two different therapeutic agents, and the nanomaterial is small in size, stable in structure, and has good biocompatibility. It can greatly improve its efficacy and meet the needs of integrated clinical diagnosis and treatment, namely, fluorescence imaging, magnetic resonance imaging, and dual-dynamic synergistic therapy.

For achieving the above object, the technical scheme adopted in the present invention is:

A preparation method of an up-conversion nanocomposite for dual-power synergistic therapy, characterized in that it comprises the following

step:

(1) Oil-soluble rare earth upconversion luminescent nanomaterials dispersed in cyclohexane coated with an inert _NaGdF layer on the surface were added to an aqueous solution of cyclohexane, tert-butanol, water and K ₂ CO ₃ , and added thereto Lemieux-von Rudloff reagent, stirring, centrifuging, washing, and collecting the product; then, treating the product with HCl to obtain azelaic acid-terminated hydrophilic upconverting nanoparticles to form a first dispersion;

( ₂ ) KMnO was added to the first dispersion, and sonicated in an acidic environment until a brown colloid was formed, allowing _MnO to grow on the sides of the up-conversion nanoparticles; then, the MnO _- modified up-conversion nanoparticles were collected, forming a second dispersion;

(3) Prepare a hydrophilic polymer (SH-PEG-NH ₂ ) ligand, and stir it with the second dispersion liquid at room temperature to further improve the water solubility and biocompatibility of the material, and finally disperse the material in a weak In the alkaline buffer, the third dispersion is obtained;

(4) Prepare the nitric acid-acidified C ₃ N ₄ dispersed in the weakly acidic buffer solution, and carry out carboxyl amine activation coupling with the third dispersion solution under the action of an activator, so that C ₃ N ₄ and growth on the side of the upconversion nanoparticles The upper MnO ₂ is covalently linked, that is, the up-conversion nanocomposite that grows on the side with good water solubility and covalently connects two different two-dimensional materials, and makes it capable of magnetic resonance imaging (MRI), down-conversion fluorescence imaging ( DSL), upconversion fluorescence imaging (UCL) action layer, and chemical/photodynamic (CDT+PDT) synergistic therapeutic composite structure.

In the step (1), the oil-soluble rare-earth up-conversion luminescent nanomaterial whose surface is coated with an inert _NaGdF layer is:

NaYF ₄ : Yb,Tm@NaGdF ₄ ; NaYF ₄ :Yb,Tm,Er@NaGdF ₄ ; NaYF ₄ :Yb,Tm,Ho@NaGdF ₄ .

The step (1) is specifically as follows: adding the upconversion luminescent nanomaterials dispersed in cyclohexane, cyclohexane, tert-butanol, water and K ₂ CO ₃ aqueous solution into a two-necked flask, and stirring at room temperature for 20 hrs. -30min, then the Lemieux-von Rudloff reagent was added dropwise to the solution, the resulting mixture was stirred at 40-50°C for 48 hours, centrifuged, the product was collected, and washed several times with deionized water and ethanol. Subsequently, the product was treated with HCl, and the mixture was stirred at room temperature for 30-40 min; finally, the product was centrifuged and washed, and then dispersed in deionized water to obtain a first dispersion; the Lemieux-von Rudloff reagent was 5.7 mM KMnO ₄ and 0.105 M NaIO ₄ in water.

The step (2) is specifically as follows: adding an aqueous solution containing azelaic acid-terminated hydrophilic up-conversion nano-luminescent material to a buffer solution (0.1M, 2-(N-morpholino)ethanesulfonic acid (MES) containing pH=6.0) into the centrifuge tube, add _KMnO to the tube, sonicate for 20-30 min, until a brown colloid is formed, allowing _MnO to grow _on the side of the upconversion nanoparticles; The nanoparticles were upconverted, washed three times with deionized water to remove excess potassium and free manganese ions, and redispersed in deionized water.

The step (4) is specifically as follows: preparing the nitric acid acidified C ₃ N ₄ dispersed in the weakly acidic buffer MES, adding EDC and NHS, ultrasonicating for 15-30 s, and at the same time, heating the oil bath to stabilize at 37° C. ; After ultrasonication, immediately put it into an oil bath, and stir for 15-20min; then centrifuge quickly, add the _third dispersion into it, quickly ultrasonicate for _30-50s , and then stir in an oil bath overnight to make C3N4 and The upper MnO ₂ grown on the side of the upconversion nanoparticle is covalently linked, and finally centrifuged and washed to obtain an upconversion nanocomposite with good water solubility on the side and covalently linked two different two-dimensional materials, and make it simultaneously With MRI, DSL, UCL action layer, and CDT+PDT dual dynamic synergistic treatment composite structure.

An upconversion nanocomposite material for dual-dynamic synergistic therapy prepared by the aforementioned method, characterized in that it is obtained by growing _MnO2 on the side of an oil-soluble rare earth upconversion luminescent nanomaterial coated with an inert NaGdF4 layer _on the surface. , and then covalently connected with C ₃ N ₄ , which is a layered structure with an oil-soluble rare earth upconversion luminescent nanomaterial coated with an inert NaGdF ₄ layer on the surface as the core, and MnO ₂ is grown on the side of the core. layer, which is covalently linked by C ₃ N ₄ on the MnO ₂ layer; that is, two different therapeutic agents are compounded on the oil-soluble rare earth upconversion luminescent nanomaterial coated with an inert NaGdF ₄ layer on the surface, so that the At the same time, it has MRI, DSL, and UCL action layers, as well as a composite structure that connects two different two-dimensional materials and implements CDT+PDT dual-dynamic synergistic therapy.

An application of the aforementioned up-conversion nano-composite material for the integration of dual-dynamic therapy and diagnosis and treatment is characterized in that, using it to grow _{MnO 2 ,} The MRI, DSL, and UCL action layers formed by covalently connecting C ₃ N ₄ are used as contrast agents for preparing fluorescence imaging or magnetic resonance imaging.

An application of the aforementioned up-conversion nanocomposite for the integration of dual-dynamic therapy and diagnosis and treatment, characterized in that the CDT+PDT dual-dynamic synergistic therapeutic composite formed by the MnO ₂ layer and the C ₃ N ₄ covalently connected to it is used. structure, overcome the limitation of the therapeutic effect of a single photosensitizer, and use it as a synergistic therapeutic agent for the preparation of photo/chemical dynamics.

The advantages of the present invention are:

(1) The preparation method of the up-conversion nanocomposite material of side growth and covalent connection of two different two-dimensional materials provided by the present invention adopts the method of side growth and covalent connection, which overcomes the high temperature and complexity of the existing method. The method is easy to operate, the preparation process is efficient, stable, and has high repeatability, and is easy to industrialize;

(2) The upconversion nanocomposite material provided by the present invention, which grows on the side and covalently connects two different two-dimensional materials, breaks through the technological and structural limitations of similar products, and can realize nanoscale structure, small size, and biocompatibility. Good, stable quality, no toxic and side effects;

(3) The up-conversion nanocomposite material provided by the present invention, which is grown on the side and covalently connected with two different two-dimensional materials, breaks through the performance and effect of the existing materials, overcomes the limitation of the therapeutic effect of a single photosensitizer, and has magnetic properties at the same time. Resonance Imaging (MRI), Down-Conversion Fluorescence Imaging (DSL), Up-Conversion Fluorescence Imaging (UCL) Action Layer, and Chemical/Photodynamic (CDT+PDT) Synergistic Therapeutic Composite Structures with Fluorescence/Magnetic Resonance Multimodality Imaging, and Synergy Photodynamic therapy and chemodynamic therapy have been tested to have a dual-dynamic synergistic therapeutic effect on cancer that is 2 to 3 times that of the existing non-synergistic therapy technology, and its biomedical application prospect is very broad.

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

Description of drawings

FIG. 1 is a TEM photograph of a rare earth thulium-doped upconversion luminescent nanocomposite of dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in an embodiment of the present invention;

2 is a schematic diagram of the lateral growth, covalent connection process and structure of a rare earth thulium-doped upconversion luminescent nanocomposite of dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) according to an embodiment of the present invention;

3 is the up/down conversion fluorescence spectrum of the rare earth thulium doped upconversion luminescent nanocomposite of the dual dynamic therapeutic agent (C ₃ N ₄ , MnO ₂ ) prepared in the embodiment of the present invention under the excitation of 980 nm and 405 nm lasers, respectively ;

4 is a magnetic resonance imaging image of a rare earth thulium-doped upconversion luminescent nanocomposite of dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in an embodiment of the present invention;

5 is a monitoring diagram of ROS of the rare earth thulium-doped upconversion luminescent nanocomposite of dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in the embodiment of the present invention;

FIG. 6 is the cell survival rate of the rare earth thulium doped upconversion luminescent nanocomposite of dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ) prepared in the embodiment of the present invention for dual-dynamic therapy of cancer cells.

7 is the DCF relative fluorescence intensity of the rare earth thulium-doped upconversion luminescent nanocomposite of dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ) prepared in the embodiment of the present invention for dual-dynamic therapy of cancer cells, that is, the therapeutic effect Comparison diagram.

Detailed ways

Example 1

Referring to Figures 1 to 7 , the present embodiment provides an upconversion nanocomposite for the integration of dual-dynamic therapy and diagnosis and treatment, which adopts side growth and covalent connection method to combine two therapeutic agents (C ₃ N _{4 ).} , MnO ₂ ) is connected to the surface of the up-conversion nanocrystal, and the up-conversion nanocrystal is NaYF ₄ :Yb,Tm@NaGdF ₄ . The method for preparing the rare earth thulium doped up-conversion luminescent nanomaterial of the composite dual-dynamic therapy agent (C ₃ N ₄ , MnO ₂ ) comprises the following steps:

(1) 20 mg of NaYF ₄ :Yb,Tm@NaGdF ₄ upconversion luminescent nanomaterials dispersed in cyclohexane, 20 mL of cyclohexane, 14 mL of tert-butanol, 2 mL of water and 1M aqueous K ₂ CO ₃ solution were added to both necks The flask was stirred at room temperature for 20 min, then ₄ mL of Lemieux-von Rudloff reagent (5.7 mM KMnO and 0.105 M _NaIO in water) was added dropwise to the solution, the resulting mixture was stirred at 40 °C for 48 hours, centrifuged to collect the product , and washed several times with deionized water and ethanol. Subsequently, the product was treated with an equal volume of HCl (PH=4-5), and the mixture was stirred at room temperature for 30 min; finally, the product was centrifuged and washed, and then dispersed in 1 mL of deionized water to obtain a first dispersion.

(2) 100 μL of the aqueous solution containing the azelaic acid-terminated hydrophilic upconversion nanoluminescent material was added to 250 μL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH=6.0) into a centrifuge tube, and 250 μL of KMnO ₄ was added to the tube and sonicated for 30 min until a brown colloid formed. Subsequently, the _MnO2 -modified upconversion nanoparticles were collected by centrifugation, washed three times with deionized water to remove excess potassium and free manganese ions, and redispersed in 1 mL of deionized water;

(3) Prepare a hydrophilic polymer (SH-PEG-NH ₂ ) ligand, and stir it with the second dispersion at room temperature, and finally disperse 20 mg of the material in 10 mL of weakly alkaline PBS buffer to obtain a third dispersion liquid;

(4) Prepare 20 mg of nitric acid-acidified C ₃ N ₄ dispersed in 10 mL of weakly acidic buffer MES, add 80 mg of EDC and 120 mg of NHS, and sonicate for 15 s. At the same time, the temperature of the oil bath is stabilized at 37 °C. , immediately put it into the oil bath, stirred for 15min, and then centrifuged quickly, and added the third dispersion liquid to it, supersonicated rapidly for 30s, and then stirred in the oil bath overnight. After centrifugation and washing, rare earth thulium-doped upconversion luminescent nanocomposites with good water-soluble dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ) are obtained, and the nanocomposite material is capable of magnetic resonance imaging (MRI) and down-conversion at the same time. Fluorescence imaging (DSL), upconversion fluorescence imaging (UCL) action layer, and chemical/photodynamic (CDT+PDT) synergistic therapeutic composite structure.

An upconversion nanocomposite for dual-dynamic synergistic therapy prepared by the aforementioned method, which is by growing _MnO2 on the side of an oil-soluble rare earth upconversion luminescent nanomaterial coated with an inert NaGdF4 layer _on the surface, and then covalently connecting C Made _{of 3N4} , it is a layered structure with an oil-soluble rare earth up-conversion luminescent nanomaterial coated with an inert NaGdF4 layer _on the surface as the core, and a _MnO2 layer is grown on the side of the core, and the _MnO2 layer is The upper covalent connection is made of C ₃ N ₄ ; that is, two different therapeutic agents are compounded on the oil-soluble rare earth up-conversion luminescent nanomaterial coated with an inert NaGdF ₄ layer on the surface, so that it has MRI, DSL, UCL action layer, and a composite structure connecting two different two-dimensional materials to implement CDT+PDT dual-dynamic synergistic therapy.

An application of the aforementioned upconversion nanocomposite for dual-dynamic therapy and diagnosis and treatment integration, utilizing the oil-soluble rare earth upconversion luminescent nanomaterial coated with an inert NaGdF4 layer _on the surface to grow MnO ₂ on the side and covalently connect it The MRI, DSL, and UCL action layers formed by C ₃ N ₄ are used as contrast agents for preparing fluorescence imaging or magnetic resonance imaging.

An application of the aforementioned up-conversion nanocomposite for the integration of dual-dynamic therapy and diagnosis and treatment, utilizing the CDT+PDT dual-dynamic synergistic therapeutic composite structure formed by the MnO ₂ layer and the C ₃ N ₄ covalently connected to it, to overcome the single Due to the limitations of the therapeutic effect of photosensitizers, using them as synergistic therapeutic agents for the preparation of photo/chemical dynamics can achieve outstanding dual-dynamic synergistic efficacy.

FIG. 1 is a TEM image of the rare earth thulium-doped upconversion luminescent nanocomposite modified with dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1, and it can be seen that MnO ₂ has been connected around the upconversion nanoparticles , C ₃ N ₄ photodynamic therapy reagents, indicating that the method can obtain nanomaterials with good morphology and uniform growth; and the final nanomaterials have an average particle size of about 180-200 nm and a positive surface. Therefore, nanoparticles are easier to be endocytosed by cells, which is beneficial to circulation in the body, and can meet the needs of the integration of clinical diagnosis and treatment of fluorescence imaging, magnetic resonance imaging and synergistic photodynamic therapy.

2 is a schematic diagram of the lateral growth, covalent connection process and structure of the rare earth thulium-doped upconversion luminescent nanocomposite of the dual-dynamic therapeutic agent (C ₃ N ₄ , MnO ₂ ) of the present embodiment. It is made by growing MnO ₂ on the side of the oil-soluble rare earth up-conversion luminescent nanomaterial coated with an inert NaGdF ₄ layer, and then covalently connecting C ₃ N ₄ , which is an oil-soluble rare earth coated with an inert NaGdF ₄ layer on the surface. The layered structure with the up-conversion luminescent nanomaterial as the core, a MnO ₂ layer is grown on the side of the core, and the MnO ₂ layer is covalently connected by C ₃ N ₄ ; that is, the surface is wrapped with an inert NaGdF The ₄ -layer oil-soluble rare-earth upconversion luminescent nanomaterial is compounded with two different therapeutic agents, so that it has MRI, DSL, and UCL action layers at the same time, as well as connecting two different two-dimensional materials and implementing CDT+PDT dual power synergy The composite structure of treatment.

Example 2

The present embodiment provides an up _- conversion nanocomposite material for dual _- dynamic therapy, a preparation method and an application. ₂ ) The rare earth erbium and thulium co-doped up-conversion luminescent nanomaterials are basically the same as those in Example 1, except that they include the following steps:

(1) 25 mg of NaYF ₄ : Yb, Tm, Er@NaGdF ₄ upconversion luminescent nanomaterials dispersed in cyclohexane, 20 mL of cyclohexane, 14 mL of tert-butanol, 2 mL of water and 1 M aqueous K ₂ CO ₃ solution were added In a two-necked flask, stirred at room temperature for 30 min, then 5 mL of Lemieux-von Rudloff reagent (5.7 mM KMnO ₄ and 0.105 M NaIO ₄ aqueous solution) was added dropwise to the solution, the resulting mixture was stirred at 45 ° C for 40 hours, centrifuged, The product was collected and washed several times with deionized water and ethanol. Subsequently, the product was treated with an equal volume of HCl (PH=4-5), and the mixture was stirred at room temperature for 40 min. Finally, the product was centrifuged and washed, and then dispersed in 1.5 mL of deionized water to obtain a first dispersion;

(2) 100 μL of the aqueous solution containing the azelaic acid-terminated hydrophilic upconversion nanoluminescent material was added to 200 μL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH=6.0) into a centrifuge tube, and 200 μL of KMnO ₄ was added to the tube and sonicated for 50 min until a brown colloid formed. Subsequently, the _MnO2 -modified upconversion nanoparticles were collected by centrifugation, washed three times with deionized water to remove excess potassium and free manganese ions, and redispersed in 1.5 mL of deionized water;

(3) Prepare a hydrophilic polymer (SH-PEG-NH ₂ ) ligand, and stir it with the second dispersion at room temperature, and finally disperse 25 mg of the material in 15 mL of weakly alkaline PBS buffer to obtain a third dispersion liquid;

(4) Prepare 25 mg of nitric acid-acidified C ₃ N ₄ dispersed in 15 mL of weakly acidic buffer MES, add 85 mg of EDC and 125 mg of NHS, and sonicate for 15 s. At the same time, the temperature of the oil bath is stabilized at 37 °C. , immediately put it into the oil bath, stirred for 15min, and then centrifuged quickly, and added the third dispersion liquid to it, supersonicated rapidly for 40s, and then stirred in the oil bath overnight. After centrifugation and washing, the rare earth erbium and thulium co-doped upconversion luminescent nanocomposite material with good water solubility of dual kinetic therapeutic agents (C ₃ N ₄ , MnO ₂ ) is obtained.

An up-conversion nano-composite material for dual-dynamic therapy prepared by the above-mentioned method, which adopts side growth and covalent connection method to connect two therapeutic agents (C ₃ N ₄ , MnO ₂ ) on the up-conversion nanocomposite. The up-conversion nanocrystals are NaYF ₄ :Yb,Tm,Er@NaGdF ₄ .

Example 3

The present embodiment provides an up _- conversion nanocomposite material for dual _- dynamic therapy, a preparation method, and an application. ₂ ) The rare earth holmium and thulium co-doped up-conversion luminescent nanomaterials are basically the same as those in Embodiments 1 and 2, except that they include the following steps:

(1) 22 mg of NaYF ₄ : Yb, Tm, Ho@NaGdF ₄ upconversion luminescent nanomaterials dispersed in cyclohexane, 20 mL of cyclohexane, 14 mL of tert-butanol, 2 mL of water and 1 M aqueous K ₂ CO ₃ solution were added In a two-necked flask, stirred at room temperature for 35 min, then 5 mL of Lemieux-von Rudloff reagent (5.7 mM KMnO ₄ and 0.105 M aqueous NaIO ₄ solution) was added dropwise to the solution, the resulting mixture was stirred at 45 ° C for 40 hours, centrifuged, The product was collected and washed several times with deionized water and ethanol. Subsequently, the product was treated with an equal volume of HCl (PH=4-5), and the mixture was stirred at room temperature for 35 min. Finally, the product was centrifuged and washed, and then redispersed in 2 mL of deionized water to obtain a first dispersion.

(2) 100 μL of the aqueous solution containing the azelaic acid-terminated hydrophilic upconversion nanoluminescent material was added to 230 μL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH=6.0) into a centrifuge tube, and 230 μL of KMnO ₄ was added to the tube and sonicated for 60 min until a brown colloid formed. Subsequently, the _MnO2 -modified upconverting nanoparticles were collected by centrifugation, washed three times with deionized water to remove excess potassium and free manganese ions, and redispersed in 2 mL of deionized water.

(3) Prepare a hydrophilic polymer (SH-PEG-NH ₂ ) ligand, and stir it with the second dispersion at room temperature, and finally disperse 22 mg of the material in 13 mL of weakly alkaline PBS buffer to obtain a third dispersion liquid.

(4) Prepare 22 mg of nitric acid-acidified C ₃ N ₄ dispersed in 13 mL of weakly acidic buffer MES, add 83 mg of EDC and 123 mg of NHS, and sonicate for 15 s. At the same time, the temperature of the oil bath is stabilized at 37 °C. , immediately put it into an oil bath, stirred for 15 minutes, and then centrifuged quickly, and added the third dispersion liquid to it, quickly ultrasonicated for 50s, and then stirred overnight in the oil bath. After centrifugation and washing, the rare earth holmium and thulium co-doped upconversion luminescent nanocomposite material with good water solubility of dual kinetic therapeutic agents (C ₃ N ₄ , MnO ₂ ) is obtained.

An up-conversion nano-composite material for dual-dynamic therapy prepared by the above-mentioned method, which adopts side growth and covalent connection method to connect two therapeutic agents (C ₃ N ₄ , MnO ₂ ) on the up-conversion nanocomposite. The up-conversion nanocrystal is NaYF ₄ :Yb,Tm,Ho@NaGdF ₄ .

Example 4

The up-conversion nanocomposite material, preparation method and application provided in this embodiment are basically the same as those in Embodiments 1-3, and the differences are:

It is specifically a method for applying the rare earth thulium doped upconversion luminescence nanocomposite material modified with dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1 to cell fluorescence imaging, including the following steps:

(1) Prepare 0-15 mg of rare earth thulium-doped upconversion luminescent nanocomposites modified with dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ), cell culture medium, and HeLa cells cultured in 96-well plates;

(2) The prepared nanomaterial medium is prepared to 3-4 mg/mL, and ultrasonically dispersed for 5 minutes to form a mixed dispersion;

(3) Different concentrations of the dispersion in (2) were added to a 96-well plate, then placed in a constant temperature and humidity cabinet for 2 hours, and observed under a laser confocal microscope.

3 is the up/down conversion fluorescence spectrum of the rare earth thulium doped upconversion luminescent nanocomposite modified with dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in the embodiment of the present invention under the excitation of 980 nm and 405 nm lasers, respectively From the figure, the emission peaks at 375nm and 475nm can be observed, which is conducive to the imaging of deeper penetration levels, and 980nm is just located in the "optical window" of biological tissues, indicating that this material is very suitable for cells and small animals. imaging. At the same time, due to the good down-conversion luminescence effect of C ₃ N ₄ , it can exhibit the imaging effect of down-conversion fluorescence under the excitation of 405 nm laser. In cells and animals, it can achieve up-conversion dual-mode imaging and more accurate tumor localization. .

Example 5

The up-conversion nanocomposite material, preparation method and application provided in this embodiment for dual dynamic therapy are basically the same as those in Embodiments 1-4, and the differences are:

The rare earth thulium-doped upconversion luminescence nanocomposite material modified with dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) provided in this embodiment is applied to a method for magnetic resonance imaging, which includes the following steps:

(1) Prepare 15-30 mL of dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ) modified rare earth thulium-doped upconversion luminescent nanocomposites with a concentration of 10-15 mg/mL, and phosphate buffer solution (PBS) It is formulated into 1-2 mg/mL, and dispersed by ultrasonic for 5 minutes to form a mixed dispersion;

(2) Take the mixed dispersion liquid in (1) with different concentrations, and measure its magnetic resonance imaging on a magnetic resonance apparatus respectively.

4 is a magnetic resonance imaging image of a rare earth thulium-doped upconversion luminescent nanocomposite modified with dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1 of the present invention. It can be seen from the comparison that Mn ⁴ The existence of ⁺ further improves the magnetic resonance imaging effect of the material. The relaxation constant R ₁ is obtained by plotting the concentration of Gd ³⁺ against 1/T ₁ and fitting a straight line, which confirms that the nanomaterial can be used for magnetic resonance imaging.

Example 6

The upconversion nanocomposite material for dual-dynamic therapy, which is grown on the side and covalently connected to two different two-dimensional materials, the preparation method and the application provided in this example are basically the same as those in Examples 1-5, and the difference is that :

Specifically, the method for applying the rare earth thulium-doped upconversion luminescent nanocomposite material modified by dual kinetic therapeutic agents (C ₃ N ₄ , MnO ₂ ) to in vitro photo/chemical kinetic experiments includes the following steps:

(1) Prepare 400 μg/mL of nanomaterials with deionized water;

(2) Take 10 mg of the material and mix it with 2 mL of 0.5 mg·mL ^-1 DPBF by ultrasonication, and protect from light, and the solution is excited by 980 nm laser (0.5 cm ^-1 ) irradiation;

(3) The UV absorption curves of the samples at different time points were measured, and at the same time, H ₂ O ₂ was added to the last set of samples for 10 minutes to study the effect of MnO ₂ on CDT.

5 is the ultraviolet absorption curve (i.e. ROS monitoring chart) of the rare earth thulium-doped upconversion luminescent nanocomposite modified with dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1 of the present invention, irradiated by laser Then, with the increase of time, the UV-vis absorption band of DPBF at 410nm wavelength decreased with the prolongation of irradiation time, indicating the increase of ROS amount, which also confirmed that the nanomaterial has a good dual dynamic treatment effect, which is a good follow-up It is possible to conduct biological experiments.

Example 7

The upconversion nanocomposite for dual-dynamic therapy, which is grown on the side and covalently connected to two different two-dimensional materials, the preparation method and the application provided in this example are basically the same as those in Examples 1-6, and the difference is that :

Specifically, the rare-earth thulium-doped upconversion luminescent nanocomposite material modified by dual-dynamic therapy reagents (C ₃ N ₄ , MnO ₂ ) is applied to the dual-dynamic therapy of cancer cells, including the following steps:

(1) Prepare the material in Example 1 and prepare it into DMEM medium of 0, 25, 50, 100, 200, 400 μg/mL;

(2) HeLa cells were cultured in the above-mentioned culture medium for 24h respectively;

(3) Rinse three times with PBS buffer solution, and wash away the material that is not absorbed by cells;

(4) The cultured cells were irradiated with a 980nm laser of 0.5W/cm ² for 6 minutes at intervals (irradiated for 2 minutes and stopped for 2 minutes), and then continued to be cultured in the medium for 24 hours, and the CCK-8 method was used to measure the survival rate of the cells; At the same time, live and dead cells were stained with calcein and propidium iodide, respectively, and the emitted fluorescence was collected with a fluorescence microscope.

Figure 6 shows the cell viability after culture of the rare earth thulium-doped upconversion luminescent nanocomposite modified with dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1 of the present invention. From the figure, we can It was seen that UCNPs@MnO ₂ -C ₃ N ₄ exhibited excellent concentration-dependent toxicity to HeLa cells under 980 nm laser irradiation. When the concentration of UCNPs@MnO ₂ -C ₃ N ₄ was 400 μg/mL, the cell viability decreased to 23% of the control. These results fully demonstrate that the UCNPs@MnO ₂ -C ₃ N ₄ nanocomposite exhibits a good dual-dynamic therapy (CDT+PDT) effect under laser irradiation.

Example 8

The up-conversion nanocomposite material, preparation method and application provided by the side growth and covalent connection of two different two-dimensional materials are basically the same as those in Examples 1-7, and the differences are:

Specifically, the rare-earth thulium-doped upconversion luminescent nanocomposite material modified by dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) is applied to the monitoring of singlet oxygen, thereby detecting the therapeutic effect, including the following steps:

(1) HeLa cells were seeded in the plate and cultured overnight, UCNPs@MnO ₂ and UCNPs@MnO ₂ -C ₃ N ₄

(400 μg/mL) after co-incubation, washed with D-Hanks solution;

(2) DCFH-DA was added to the medium and incubated, and then washed three times.

(3) Irradiated with a 980 nm near-infrared laser with a power of 0.5 W/cm ² , and blank holes without irradiation were also detected as a control. DCF fluorescence intensity was detected using a microplate reader (485nm excitation/528nm emission).

Figure 7 shows the effect of DCFH-DA on the effect of DCFH-DA on cell singlet oxygen after culturing the rare earth thulium-doped upconversion luminescent nanocomposite modified with dual dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) prepared in Example 1 of the present invention. From the graph, we can see that the content of singlet oxygen in the CDT material alone is almost doubled compared to the control group, while the content of singlet oxygen in the final material group is nearly triplet. The results further demonstrate the potential diagnostic and therapeutic value of the UCNPs@MnO ₂ -C ₃ N ₄ nanocomposite, whose synergistic therapeutic efficacy is 2-3 times higher than the state-of-the-art.

The key point of the present invention is that the present invention adopts the method of direct lateral growth and covalent connection, unique and simple process method and component ratio, and obtains the nanocomposite based on the direct growth of nanocrystal surface for dual dynamic therapy, and the preparation process is simple, The process is easy to control, the product structure is stable, the size is uniform, the water solubility is good, the repeatability is high, and it is easy to industrialize.

The present invention is not limited to the above-mentioned embodiments, and the method of using the rare earth up-conversion luminescent nanocomposite modified by other dual-dynamic therapeutic agents (C ₃ N ₄ , MnO ₂ ) obtained by the same or similar method, such as the method of up-conversion luminescent nanocomposites doped with different rare earth ions, Converted nanocrystals (NaYF ₄ : Yb, Tm@NaGdF ₄ ; NaYF ₄ : Yb, Tm, Er@NaGdF ₄ ; NaYF ₄ : Yb, Tm, Ho@NaGdF ₄ ), different water-soluble polymers, etc. are all in the present invention within the scope of protection.

Claims (8)

1. A preparation method of an up-conversion nano composite material for dual-power cooperative therapy is characterized by comprising the following steps:

(1) coating inert NaGdF on the surface dispersed in cyclohexane₄The oil-soluble rare earth up-conversion luminescent nano material of the layer is added into cyclohexane,tert-Butanol, Water and K₂CO₃Adding a Lemieux-von Rudloff reagent into the aqueous solution, stirring, centrifuging, washing, and collecting a product; subsequently, the product was treated with HCl to obtain azelaic acid-capped hydrophilic upconversion nanoparticles, forming a first dispersion;

(2) mixing KMnO₄Adding into the first dispersion, and ultrasonic treating in acidic environment until forming brown colloid and MnO₂Growing on the sides of the upconversion nanoparticles; then, MnO was collected₂(ii) modified upconverting nanoparticles to form a second dispersion;

(3) preparing hydrophilic polymer (SH-PEG-NH)₂) Ligand, and stirring with the second dispersion liquid at normal temperature, thereby further improving the water solubility and biocompatibility of the material, and finally dispersing the material in a weak alkaline buffer solution to obtain a third dispersion liquid;

(4) preparation of nitric acidified C dispersed in weakly acidic buffer₃N₄And performing carboxyl-ammonia activated coupling with the third dispersion liquid under the action of an activating agent to obtain C₃N₄With upper MnO grown on the side of the upconversion nanoparticles₂And (3) covalent connection, namely the up-conversion nano composite material which grows laterally and connects two different two-dimensional materials with good water solubility is obtained, and the up-conversion nano composite material simultaneously has Magnetic Resonance Imaging (MRI), down-conversion fluorescence imaging (DSL), up-conversion fluorescence imaging (UCL) action layers and a chemical/photodynamic (CDT + PDT) synergistic treatment composite structure.

2. The method of claim 1, wherein the inert NaGdF is coated on the surface of step (1)₄The oil-soluble rare earth up-conversion luminescent nano material of the layer is as follows:

NaYF₄:Yb,Tm@NaGdF₄；NaYF₄:Yb,Tm,Er@NaGdF₄；NaYF₄:Yb,Tm,Ho@NaGdF₄。

3. preparation of up-conversion nanocomposite for hybrid cotherapy according to claim 1The preparation method is characterized in that the step (1) is specifically as follows: mixing up-conversion luminescent nano-material dispersed in cyclohexane, tert-butyl alcohol, water and K₂CO₃The aqueous solution was added to both flasks, stirred at room temperature for 20-30min, then Lemieux-von Rudloff reagent was added dropwise to the solution, the resulting mixture was stirred at 40-50 ℃ for 48 hours, centrifuged, the product collected, and washed several times with deionized water and ethanol. Subsequently, the product was treated with HCl and the mixture was stirred at room temperature for 30-40 min; finally, centrifuging and washing the product, and then dispersing in deionized water to obtain a first dispersion liquid; the Lemieux-von Rudloff reagent is 5.7mM KMnO₄And 0.105M NaIO₄An aqueous solution of (a).

4. The method for preparing the upconversion nanocomposite material for hybrid synergistic therapy according to claim 1, wherein the step (2) is specifically as follows: an aqueous solution containing azelaic acid-terminated hydrophilic up-converting nanophosphors was added to a centrifuge tube containing 2- (N-morpholino) ethanesulfonic acid (MES) buffer (0.1M, pH 6.0) and KMnO was added₄Adding into tube, and ultrasonic treating for 20-30min until brown colloid is formed to allow MnO₂Growing on the sides of the upconversion nanoparticles; subsequently, MnO was collected by centrifugation₂The modified upconverting nanoparticles were washed three times with deionized water to remove excess potassium and free manganese ions and redispersed in deionized water.

5. The method for preparing the upconversion nanocomposite material for hybrid synergistic therapy according to claim 1, wherein the step (4) is specifically as follows: preparation of nitric acidified C dispersed in weakly acidic buffer MES₃N₄Adding EDC and NHS, and carrying out ultrasonic treatment for 15-30s, and simultaneously, heating the oil bath kettle to be stable at 37 ℃; immediately putting the mixture into an oil bath pot after the ultrasonic treatment is finished, and stirring for 15-20 min; then quickly centrifuging, adding the third dispersion, quickly performing ultrasonic treatment for 30-50s, and stirring in oil bath overnight to allow for C₃N₄With upper MnO grown on the side of the upconversion nanoparticles₂Covalent attachment; and finally, centrifuging and washing to obtain the up-conversion nano composite material which grows on the side surface and is covalently connected with two different two-dimensional materials and has good water solubility, and the up-conversion nano composite material simultaneously has MRI, DSL and UCL action layers and a CDT + PDT dual-power synergistic treatment composite structure.

6. An upconversion nanocomposite for hybrid synergistic therapy prepared by the method of any one of claims 1 to 5, wherein the nanocomposite is coated with inert NaGdF₄MnO is grown on the side surface of oil-soluble rare earth up-conversion luminescent nano material of the layer₂Then covalently linking C₃N₄Is prepared by coating inert NaGdF on the surface₄The oil-soluble rare earth up-conversion luminescent nano material of the layer is a layered structure with a core, and MnO grows on the side surface of the core₂Layer in MnO₂Covalently bound on the layer by C₃N₄(ii) a Namely, the surface is wrapped with inert NaGdF₄Two different therapeutic agents are compounded on the oil-soluble rare earth up-conversion luminescent nano material of the layer, so that the layer has the functions of MRI, DSL and UCL at the same time, and a composite structure which is connected with two different two-dimensional materials and implements CDT + PDT dual-power synergistic treatment.

7. The use of the hybrid treatment and diagnosis-treatment integrated up-conversion nanocomposite material as claimed in claim 6, wherein the nanocomposite material is coated with inert NaGdF₄MnO is grown on the side surface of oil-soluble rare earth up-conversion luminescent nano material of the layer₂And covalently bound to C₃N₄The formed MRI, DSL and UCL action layer is used as a contrast agent for preparing fluorescence imaging or magnetic resonance imaging.

8. Use of the hybrid treatment and theranostic upconversion nanocomposite material of claim 6 in a hybrid therapy and therapy application₂Layer and C covalently linked thereto₃N₄The formed CDT + PDT dual-power synergistic treatment composite structure is used for preparing light/chemical kineticsA synergistic agent for treating diseases.

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