CN110743013A - Up-conversion nano composite material for dual-power cooperative treatment, preparation method and application - Google Patents
Up-conversion nano composite material for dual-power cooperative treatment, preparation method and application Download PDFInfo
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Abstract
The invention discloses a preparation method of an up-conversion nano composite material for dual-power cooperative therapy, which comprises the following steps: (1) the synthesized surface is coated with inert NaGdF4The oil-soluble rare earth of the layer is converted into luminescent nanocrystalline on the surface to obtain azelaic acid-terminated hydrophilic upconversion nanoparticles, and a first dispersion liquid is formed; (2) mixing KMnO4Adding the first dispersion until a brown colloid is formed, forming a second dispersion; (3) preparing hydrophilic polymersThe ligand is stirred with the second dispersion liquid to obtain a third dispersion liquid; (4) c for preparatory nitric acid acidification3N4Under the action of an activating agent, performing carboxyl-ammonia activated coupling with the third dispersion liquid to obtain MnO growing on the side surface2And covalently bound to C3N4The upconverting nanocomposite of (1). The invention also discloses a material prepared by the method and application of the material. The material has multiple application values of synergistic dual-power treatment, up/down conversion fluorescence imaging, magnetic resonance imaging and the like, and has potential application prospects in the biomedical fields of tumor diagnosis, treatment and the like.
Description
Technical Field
The invention belongs to the technical field of nano biomaterials, and particularly relates to an up-conversion nano composite material for dual-power cooperative therapy, and a preparation method and application thereof.
Background
In recent years, cancer has become one of the major diseases that endanger human health. Biological imaging is also becoming a focus of research as one of the major means of cancer diagnosis. Compared with other imaging technologies, the bio-optical imaging has the advantages of simple and convenient operation, intuition, easy acceptance, relatively low signal intensity and cost and the like, and has wide prospects in the aspects of diagnosis and treatment of various diseases. Among them, rare earth up-conversion luminescent nano materials have attracted much 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 anti-Stokes luminescence process. And the near infrared light is used as an excitation light source, almost has no damage to biological tissues, is expected to realize tissue penetration depth of centimeter level and does not cause background fluorescence. The up-conversion nano material as a novel imaging agent is widely applied to a series of biological imaging fields, such as: cellular imaging, small animal in vivo imaging, Magnetic Resonance Imaging (MRI), and the like.
Aiming at the problems that the diagnosis and treatment of the cancer are mutually independent, the treatment period is long, the cost is high, the side effect of the treatment means such as chemical treatment, radiation treatment, operation treatment and the like is large and the like in the current clinic, the combination of imaging and treatment is adopted, and the preparation of the diagnosis and treatment integrated multifunctional nano material becomes a new direction for the development of the cancer.
In the prior art, most of reported composite materials based on upconversion nanomaterials and PDT/CDT reagents are independently compounded by one PDT/CDT reagent and the upconversion nanomaterials, the upconversion nanoparticles obtained by combining the PDT/CDT reagents and the upconversion nanomaterials have limited excitation effect, and the treatment effect has great limitation. Where PDT is an abbreviation for photodynamic therapy and CDT is an abbreviation for chemokinetic therapy.
Magnetic Resonance Imaging (MRI) is one of the most advocated non-invasive, high resolution and highly flexible diagnostic techniques in medicine today, and magnetic resonance imaging contrast agents are playing an increasingly important role in clinical diagnosis. Photodynamic therapy PDT with photosensitizer as a core is a new method for treating tumors with selective destruction and small toxic and side effects, which is developed recently and is considered as a good choice for avoiding surgical operations in the aspect of clinical treatment of cancers. In view of the obvious application advantages and development prospects of the two, if a new class of reagents which can have double functions of a magnetic resonance imaging contrast agent and a photosensitizer for photodynamic therapy can be researched, the diagnosis-treatment combination of the two can be effectively promoted, and revolutionary changes can be brought to the diagnosis and treatment of tumors.
Photodynamic therapy (PDT) is a new technique for the diagnosis and treatment of disease based on the interaction of light, a photosensitizer and oxygen, based on the principle that, under illumination of a specific wavelength and intensity, the photosensitizer molecules are excited to an excited state and transfer their energy to the surrounding oxygen molecules, thereby generating singlet oxygen: (1O2) Or Reactive Oxygen Species (ROS) such as free radicals, and the like, and the target tissues are damaged by the ROS, so that the aim of treatment is fulfilled. Compared with traditional cancer treatment means including surgery, chemotherapy and radiotherapy, photodynamic therapy (PDT) has the remarkable advantages of small wound, low toxicity, good selectivity, high precision, repeatable treatment, cooperative surgery and the like.
At present, compared with the traditional cancer treatment means (radiotherapy, chemotherapy, etc.) which have great harm to human body, the photodynamic therapy (PDT) is a non-invasive and mild medical technology, and especially in the anticancer treatment, the photosensitizer can generate Reactive Oxygen Species (ROS) when irradiated under a specific wavelength, so as to achieve the effect of killing cancer cells. PDT, as an emerging and promising treatment for cancer, can also be used to ameliorate the shortcomings of other traditional anti-cancer therapies.
However, some of the unresolved challenges severely hamper 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, and thus are susceptible to severe agglomeration, which often produces high toxicity to normal tissues; secondly, the photosensitization efficiency is low, so that the photodynamic efficiency is low and the treatment effect is limited. Finally, the low penetration depth of uv-visible light results in failure to reach deep pathological tissues or organs, and can only be used for treatment of very shallow epidermal layers and sites where photons can reach. Therefore, there is a need to develop additional innovative methods and materials to improve the efficiency of treatment.
While chemokinetic therapy (CDT) may be a good way to increase the efficiency of PDT, in particular to interact with photosensitizers, allowing two treatments in the tumor microenvironment.
In the prior art, the document of Chinese patent application No. 201410826953.3 discloses a nano composite material applied to photodynamic therapy and a preparation method thereof, wherein Au is adopted25Modified UCNPs @ SiO2The nanometer particle is prepared, and the photodynamic performance of the nanometer particle is obviously improved. The invention prepares uniform, monodisperse, size-controllable (50-100nm) mesoporous SiO2Coating a nano carrier with an up-conversion nano particle core-shell structure, and adsorbing a photosensitizer drug molecule Au by a proper adsorption method25(Capt)18-(Au25) Is connected to mesoporous SiO2A multifunctional composite material is constructed in the framework, and the material is adopted to solve the existing problems of photodynamic therapy (PDT). But firstly, the NaGdF is prepared by adopting a high-temperature pyrolysis method 420% Yb/1% Er (UCNPs), preparing UCNPs by adopting a continuous coating method, and synthesizing the UCNPs @ SiO with a core-shell structure2Nano material, and then synthesizing Au25And finally using UCNPs @ SiO2Nanoparticle modified Au25The whole process is complex to operate and high in required conditions.
Therefore, it is very significant to develop a new upconversion nanocomposite material which can simultaneously satisfy clinical diagnosis and treatment integration, i.e. fluorescence imaging, magnetic resonance imaging and dual-power synergistic, and break through the existing treatment limitation, realize dual-power synergistic treatment and greatly improve the curative effect.
Disclosure of Invention
The invention provides a preparation method of an up-conversion nano composite material for dual-power cooperative treatment and diagnosis and treatment integration aiming at the defects of the prior art, the up-conversion nano composite material is prepared by growing on the side surface of the up-conversion nano material and covalently connecting two different two-dimensional materials, the steps are few, the operation is simple and convenient, the reaction is mild, the up-conversion nano composite material can be prepared at normal temperature, and the method is favorable for batch production.
The invention also provides an up-conversion nano composite material for dual-power cooperative treatment and diagnosis and treatment integration and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of an up-conversion nano composite material for dual-power synergistic treatment is characterized by comprising the following steps
The method comprises the following steps:
(1) coating inert NaGdF on the surface dispersed in cyclohexane4Adding oil-soluble rare earth up-conversion luminescent nano material of layer into cyclohexane, tert-butyl alcohol, 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.
The surface in the step (1) is wrapped by inert NaGdF4The 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。
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).
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 centrifugation2Modified upconversion nanoparticles washed three times with deionized water to remove excess potassium and free manganeseIonized and redispersed in deionized water.
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 nanoparticles2Covalently linked; 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.
The upconversion nanocomposite material prepared by the method for dual-power synergistic treatment is characterized in that the surface of the upconversion 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 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.
The application of the up-conversion nano composite material for the integration of dual-power treatment and diagnosis and treatment is characterized in that the surface of the up-conversion nano composite 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 device for preparing fluorescence imaging or magnetic resonance imagingAnd (3) a contrast agent.
The application of the up-conversion nano composite material for dual-power treatment and diagnosis and treatment integration is characterized in that MnO is utilized2Layer and C covalently linked thereto3N4The formed CDT + PDT dual-power cooperative treatment composite structure overcomes the limitation of the treatment effect of a single photosensitizer, and is used for preparing a light/chemical power cooperative diagnosis and treatment agent.
The invention has the advantages that:
(1) the preparation method of the up-conversion nano composite material with the side growth and the covalent connection of two different two-dimensional materials adopts the side growth and the covalent connection method, overcomes the defects of high temperature and complexity of the existing method, is convenient to operate, has high efficiency, stability and repeatability in the preparation process, and is easy to industrialize;
(2) the up-conversion nano composite material which grows on the side surface and is covalently connected with two different two-dimensional materials breaks through the process and structure limitations of the similar products, can realize a nano-scale structure, and has the advantages of small size, good biocompatibility, stable quality and no toxic or side effect;
(3) the up-conversion nano composite material which grows on the side surface and is covalently connected with two different two-dimensional materials breaks through the defects of performance and effect of the existing material, overcomes the limitation of treatment effect of a single photosensitizer, simultaneously has an action layer of Magnetic Resonance Imaging (MRI), down-conversion fluorescence imaging (DSL), up-conversion fluorescence imaging (UCL) and a chemical/photodynamic (CDT + PDT) cooperative treatment composite structure, has fluorescence/magnetic resonance multi-mode imaging and cooperative photodynamic and chemical power treatment, has the double-power cooperative treatment effect on cancer 2-3 times of that of the existing non-cooperative treatment technology through testing, and has very wide biomedical application prospect.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) TEM illumination of rare earth thulium doped up-conversion luminescent nano composite materialSlicing;
FIG. 2 shows a dual kinetic therapeutic agent (C) according to an embodiment of the present invention3N4、MnO2) The rare earth thulium-doped up-conversion luminescent nano composite material has the side growth, covalent connection process and structural schematic diagram;
FIG. 3 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) The rare earth thulium-doped up-conversion luminescence nano composite material has up/down conversion fluorescence spectrograms under the excitation of 980nm and 405nm lasers respectively;
FIG. 4 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) The rare earth thulium-doped up-conversion luminescence nano composite material is used for magnetic resonance imaging;
FIG. 5 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) Monitoring graph of ROS of rare earth thulium doped up-conversion luminescence nanometer composite material;
FIG. 6 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) The rare earth thulium-doped up-conversion luminescent nanocomposite is used for the cell survival rate of the double dynamical therapy of cancer cells.
FIG. 7 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) The comparative schematic diagram of DCF relative fluorescence intensity, namely treatment effect, of the rare earth thulium-doped up-conversion luminescent nano composite material used for double-power treatment of cancer cells is shown.
Detailed Description
Example 1
Referring to fig. 1 to 7, in the embodiment, two therapeutic agents (C) are added to the upconversion nanocomposite for dual-power therapy and diagnosis and treatment by using a lateral growth and covalent bonding method3N4、MnO2) Connected to the surface of the up-conversion nanocrystal, wherein the up-conversion nanocrystal is NaYF4:Yb,Tm@NaGdF4. The compound dual-power therapeutic agent (C)3N4、MnO2) Method for preparing rare earth thulium doped up-conversion luminescent nano materialThe preparation method comprises the following steps:
(1) 20mg NaYF dispersed in cyclohexane4:Yb,Tm@NaGdF4Upconversion luminescent nanomaterial, 20mL cyclohexane, 14mL tert-butanol, 2mL water and 1M K2CO3The aqueous solution was added to both flasks, stirred at room temperature for 20min, and then 4mL of Lemieux-von Rudloff reagent (5.7mM KMnO)4And 0.105M NaIO4Aqueous solution) was added dropwise to the solution, and the resulting mixture was stirred at 40 ℃ for 48 hours, centrifuged, and 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 30 min; finally, the product was centrifuged and washed, and then redispersed in 1mL of deionized water to give a first dispersion.
(2) 100 μ L of an aqueous solution containing azelaic acid-terminated hydrophilic up-converting nanophosphors was added to a centrifuge tube containing 250 μ L of 2- (N-morpholino) ethanesulfonic acid (MES) buffer (0.1M, pH 6.0), and 250 μ L of KMnO was added4Added to the tube and sonicated for 30min until a brown gel formed. Subsequently, MnO was collected by centrifugation2The modified upconverted nanoparticles were washed three times with deionized water to remove excess potassium and free manganese ions and redispersed in 1mL of deionized water;
(3) preparing hydrophilic polymer (SH-PEG-NH)2) Ligand, and stirring with the second dispersion solution at normal temperature, and finally dispersing 20mg of the material in 10mL of weak alkaline PBS buffer solution to obtain a third dispersion solution;
(4) preparation of 20mg of nitric acidified C dispersed in 10mL of weakly acidic buffer MES3N4Adding 80mg of EDC and 120mg of NHS, performing ultrasonic treatment for 15s, simultaneously, heating the oil bath kettle to be stable at 37 ℃, immediately putting the oil bath kettle into the oil bath kettle after the ultrasonic treatment is finished, stirring for 15min, then performing rapid centrifugation, adding the third dispersion into the oil bath kettle, performing rapid ultrasonic treatment for 30s, and then stirring the oil bath kettle overnight. Then centrifugating and washing to obtain the dual-power therapeutic agent (C) with good water solubility3N4、MnO2) The rare earth thulium-doped up-conversion luminescent nano composite material simultaneously has Magnetic Resonance Imaging (MRI), down-conversion fluorescence imaging (DSL),An up-conversion fluorescence imaging (UCL) active layer, and a chemo/photodynamic (CDT + PDT) co-therapy complex structure.
The upconversion nanocomposite material prepared by the method and used for dual-power synergistic treatment is prepared by wrapping inert NaGdF on the surface4MnO 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.
The application of the up-conversion nano composite material for dual-power treatment and diagnosis and treatment integration is to wrap inert NaGdF on the surface of the up-conversion nano composite material4MnO 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.
The application of the up-conversion nano composite material for dual-power treatment and diagnosis and treatment integration utilizes MnO2Layer and C covalently linked thereto3N4The formed CDT + PDT dual-power cooperative treatment composite structure overcomes the limitation of treatment effect of a single photosensitizer, and can achieve outstanding dual-power cooperative treatment effect by using the photosensitizer as a synergistic diagnosis and treatment agent for preparing light/chemical power.
FIG. 1 shows the dual-powered therapeutic agent (C) obtained in example 13N4、MnO2) TEM photograph of modified rare earth thulium doped up-conversion luminescent nano composite material shows that MnO is connected around the up-conversion nano particles2、C3N4Two kinds of light movementThe force treatment reagent shows that the method can obtain the nano material with good appearance and uniform growth; and the average particle size of the final nano material is about 180-200 nm, and the surface of the final nano material is electropositive. Therefore, the nano particles are easier to be endocytosed by cells, are beneficial to circulation in organisms, and can meet the requirements of clinical diagnosis and treatment integration of fluorescence imaging, magnetic resonance imaging and cooperative photodynamic therapy.
FIG. 2 shows the dual-powered therapeutic agent (C) of this embodiment3N4、MnO2) The lateral growth, covalent connection process and structure schematic diagram of the rare earth thulium doped up-conversion luminescent nano composite material can be seen from the figure, the inert NaGdF is wrapped on the surface of the rare earth thulium doped up-conversion luminescent nano composite material4MnO 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.
Example 2
The present embodiment provides an upconversion nanocomposite material for dual-powered therapy, a preparation method and an application thereof, and specifically, a lateral growth and covalent attachment method is adopted to compound a dual-powered therapeutic agent (C)3N4、MnO2) The rare earth erbium and thulium co-doped up-conversion luminescent nano-material is basically the same as that in the embodiment 1, and the difference is that the method comprises the following steps:
(1) 25mg NaYF dispersed in cyclohexane4:Yb,Tm,Er@NaGdF4Upconversion luminescent nanomaterial, 20mL cyclohexane, 14mL tert-butanol, 2mL water and 1M K2CO3The aqueous solution was added to a two-necked flask and stirred at room temperatureStirring for 30min, then 5mL of Lemieux-von Rudloff reagent (5.7mM KMnO)4And 0.105M NaIO4Aqueous solution) was added dropwise to the solution, and the resulting mixture was stirred at 45 ℃ for 40 hours, centrifuged, and 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, centrifuging and washing the product, and then dispersing in 1.5mL of deionized water to obtain a first dispersion liquid;
(2) 100 μ L of an aqueous solution containing azelaic acid-terminated hydrophilic up-converting nanophosphoric material was added to a centrifuge tube containing 200 μ L of 2- (N-morpholino) ethanesulfonic acid (MES) buffer (0.1M, pH 6.0), and 200 μ L of KMnO was added4Added to the tube and sonicated for 50min until a brown gel formed. Subsequently, MnO was collected by centrifugation2The modified upconverted nanoparticles were washed three times with deionized water to remove excess potassium and free manganese ions and redispersed in 1.5mL of deionized water;
(3) preparing hydrophilic polymer (SH-PEG-NH)2) Ligand, stirring with the second dispersion solution at normal temperature, and finally dispersing 25mg of material in 15mL of weak alkaline PBS buffer solution to obtain a third dispersion solution;
(4) preparation of 25mg of Nitratated C dispersed in 15mL of weakly acidic buffer MES3N4Adding 85mg of EDC and 125mg of NHS, performing ultrasonic treatment for 15s, simultaneously, heating the oil bath kettle to 37 ℃, immediately putting the oil bath kettle into the oil bath kettle after the ultrasonic treatment is finished, stirring for 15min, then performing rapid centrifugation, adding the third dispersion into the oil bath kettle, performing rapid ultrasonic treatment for 40s, and then stirring the oil bath kettle overnight. Then centrifugating and washing to obtain the dual-power therapeutic agent (C) with good water solubility3N4、MnO2) The rare earth erbium and thulium co-doped up-conversion luminescence nanometer composite material.
The up-conversion nano composite material prepared by the method for double dynamical therapy adopts a lateral growth and covalent connection method to combine two therapeutic agents (C)3N4、MnO2) Connected to the surface of the up-conversion nanocrystal, wherein the up-conversion nanocrystal is NaYF4:Yb,Tm,Er@NaGdF4。
Example 3
The present embodiment provides an upconversion nanocomposite material for dual-powered therapy, a preparation method and an application thereof, and specifically, a lateral growth and covalent attachment method is adopted to compound a dual-powered therapeutic agent (C)3N4、MnO2) The rare earth holmium and thulium co-doped up-conversion luminescent nano material is basically the same as the materials in the embodiments 1 and 2, and the difference is that the method comprises the following steps:
(1) 22mg NaYF dispersed in cyclohexane4:Yb,Tm,Ho@NaGdF4Upconversion luminescent nanomaterial, 20mL cyclohexane, 14mL tert-butanol, 2mL water and 1M K2CO3The aqueous solution was added to both flasks, stirred at room temperature for 35min, and then 5mL of Lemieux-von Rudloff reagent (5.7mM KMnO)4And 0.105M NaIO4Aqueous solution) was added dropwise to the solution, and the resulting mixture was stirred at 45 ℃ for 40 hours, centrifuged, and 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 2mL of deionized water to give a first dispersion.
(2) 100 μ L of an aqueous solution containing azelaic acid-terminated hydrophilic up-converting nanophosphoric material was added to a centrifuge tube containing 230 μ L of 2- (N-morpholino) ethanesulfonic acid (MES) buffer (0.1M, pH 6.0), and 230 μ L of KMnO was added4Added to the tube and sonicated for 60min until a brown gel formed. 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 2mL of deionized water.
(3) Preparing hydrophilic polymer (SH-PEG-NH)2) Ligand, and stirring with the second dispersion at room temperature, and finally dispersing 22mg of the material in 13mL of weakly alkaline PBS buffer to obtain a third dispersion.
(4) Preparation of 22mg of nitric acidified C dispersed in 13mL of weakly acidic buffer MES3N483mg EDC and 123mg NHS were added, sonicated for 15s, and at the same time the oil was addedAnd (3) after the temperature of the bath kettle is stably raised to 37 ℃, immediately putting the bath kettle into the oil bath kettle after the ultrasonic treatment is finished, stirring for 15min, then quickly centrifuging, adding the third dispersion into the bath kettle, quickly performing ultrasonic treatment for 50s, and then stirring the bath kettle overnight. Then centrifugating and washing to obtain the dual-power therapeutic agent (C) with good water solubility3N4、MnO2) The rare earth holmium and thulium co-doped up-conversion luminescent nano composite material.
The up-conversion nano composite material prepared by the method for double dynamical therapy adopts a lateral growth and covalent connection method to combine two therapeutic agents (C)3N4、MnO2) Connected to the surface of the up-conversion nanocrystal, wherein the up-conversion nanocrystal is NaYF4:Yb,Tm,Ho@NaGdF4。
Example 4
The upconversion nanocomposite material for dual-power therapy, the preparation method and the application provided in this embodiment are basically the same as those in embodiments 1 to 3, and the differences are as follows:
specifically, the dual-powered therapeutic agent (C) prepared in example 13N4、MnO2) The modified rare earth thulium-doped up-conversion luminescent nano composite material is applied to a method for cell fluorescence imaging, and comprises the following steps:
(1) preparing dual-power therapeutic agent (C)3N4、MnO2) 0-15 mg of modified rare earth thulium doped up-conversion luminescent nano composite material, a cell culture medium and HeLa cells cultured by a 96-well plate;
(2) preparing the prepared nano material into 3-4 mg/mL by using a culture medium, and dispersing for 5 minutes by using ultrasonic waves to form a mixed dispersion liquid;
(3) the dispersions in (2) with different concentrations are added into a 96-well plate, and then the plate is placed in a constant temperature and humidity cabinet for culturing for 2 hours and observed under a laser confocal microscope.
FIG. 3 shows a dual kinetic therapeutic agent (C) prepared according to an embodiment of the present invention3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescence nano composite material has up/down conversion fluorescence spectrograms under the excitation of 980nm and 405nm lasers respectively, and the up/down conversion fluorescence spectrograms can be observed from the up/down conversion fluorescence spectrogramsEmission peaks at 375nm and 475nm are favorable for imaging deeper penetration levels, while 980nm is located just above the "optical window" of the biological tissue, indicating that the material is well suited for in vivo imaging of cells and small animals. At the same time, due to C3N4The good down-conversion luminescence effect can show the imaging effect of down-conversion fluorescence under the excitation of 405nm laser, and up-and-down conversion dual-mode imaging is realized in cells and animal bodies, so that tumor positioning is more accurately carried out.
Example 5
The upconversion nanocomposite material for dual-power therapy, the preparation method and the application provided in this embodiment are basically the same as those in embodiments 1 to 4, and the differences are as follows:
this example provides a dual-powered therapeutic agent (C)3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescent nano composite material is applied to a magnetic resonance imaging method and comprises the following steps:
(1) preparing 15-30 mL of dual-power therapeutic agent (C) with concentration of 10-15 mg/mL3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescent nano composite material is prepared into 1-2 mg/mL by Phosphate Buffer Solution (PBS), and is dispersed by ultrasound for 5 minutes to form mixed dispersion liquid;
(2) taking the mixed dispersion liquid in the step (1) with different concentrations, and respectively measuring the magnetic resonance imaging of the mixed dispersion liquid on a magnetic resonance instrument.
FIG. 4 shows a dual kinetic therapeutic agent (C) prepared in example 1 of the present invention3N4、MnO2) Magnetic resonance imaging graph of modified rare earth thulium doped up-conversion luminescence nano composite material, and comparison shows that Mn4+The existence of the Gd further improves the magnetic resonance imaging effect of the material3+Concentration pair 1/T1Drawing and fitting straight line to obtain relaxation constant R1The nano material can be used for magnetic resonance imaging.
Example 6
The up-conversion nanocomposite for dual-power therapy, which is laterally grown and covalently connected with two different two-dimensional materials, the preparation method and the application provided in this embodiment are basically the same as those in embodiments 1 to 5, except that:
which particularly comprises a dual-powered therapeutic agent (C)3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescence nano composite material is applied to a method of in vitro light/chemical dynamic experiment, and comprises the following steps:
(1) preparing 400 mu g/mL of nano material by using deionized water;
(2) taking 10mg of the material and 2mL of 0.5 mg/mL-1The DPBF is mixed well by ultrasonic treatment, protected from light, the solution is treated by 980nm laser (0.5 cm)-1) Irradiation excitation;
(3) the UV absorption curves of the samples at different time points were measured, and H was added to the last 10 min group of samples2O2To study MnO2Impact on CDT.
FIG. 5 shows a dual kinetic therapeutic agent (C) prepared in example 1 of the present invention3N4、MnO2) The ultraviolet absorption curve graph (namely ROS monitoring graph) of the modified rare earth thulium-doped up-conversion luminescent nano composite material is characterized in that after laser irradiation, the UV-vis absorption band of DPBF at the wavelength of 410nm is reduced along with the increase of irradiation time along with the increase of the irradiation time, the increase of ROS is shown, the nano material is also proved to have a good dual-power treatment effect, and the possibility is provided for the subsequent biological experiment.
Example 7
The up-conversion nanocomposite for dual-power therapy, which is laterally grown and covalently connected with two different two-dimensional materials, the preparation method and the application provided in this example are basically the same as those in examples 1 to 6, and the differences are as follows:
which particularly comprises a dual-powered therapeutic agent (C)3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescent nano composite material is applied to double-power treatment of cancer cells and comprises the following steps:
(1) the material of example 1 was prepared and prepared into DMEM medium at 0,25,50,100,200, 400. mu.g/mL;
(2) culturing HeLa cells in the culture medium for 24h respectively;
(3) washing with PBS buffer solution three times to wash away materials not absorbed by cells;
(4) the cultured cells were incubated at a concentration of 0.5W/cm2Irradiating with 980nm laser at interval for 6min (2 min, stopping for 2min), culturing in culture medium for 24 hr, and determining cell survival rate by CCK-8 method; meanwhile, live and dead cells were stained with calcein and propidium iodide, respectively, and emission fluorescence was collected with a fluorescence microscope.
FIG. 6 shows a dual kinetic therapeutic agent (C) prepared in example 1 of the present invention3N4、MnO2) The survival rate of the cultured cells of the modified rare earth thulium-doped up-conversion luminescent nanocomposite can be seen from the figure, UCNPs @ MnO is irradiated by 980nm laser2-C3N4Exhibits excellent concentration-dependent toxicity to HeLa cells. When UCNPs @ MnO2-C3N4At a concentration of 400. mu.g/mL, cell viability decreased to 23% of control. These results fully indicate that UCNPs @ MnO2-C3N4The nanocomposite material shows good dual power treatment (CDT + PDT) effect under laser irradiation.
Example 8
The up-conversion nanocomposite material with two different two-dimensional materials grown laterally and covalently connected, the preparation method and the application provided in this example are substantially the same as those in examples 1 to 7, and the differences are as follows:
which particularly comprises a dual-powered therapeutic agent (C)3N4、MnO2) The modified rare earth thulium-doped up-conversion luminescent nano composite material is applied to monitoring singlet oxygen so as to detect the treatment effect, and comprises the following steps:
(1) HeLa cells were seeded onto plates and cultured overnight, UCNPs @ MnO2And UCNPs @ MnO2-C3N4
(400. mu.g/mL) after co-incubation, washing with D-Hanks solution;
(2) DCFH-DA was added to the medium and incubated, then washed three times.
(3) The power consumption is 0.5W/cm2980nm near infrared laser irradiation, blank wells without irradiation were also examined as controls. DCF fluorescence intensity was measured using a microplate reader (485nm excitation/528 nm emission).
FIG. 7 shows a dual kinetic therapeutic agent (C) prepared in example 1 of the present invention3N4、MnO2) After the modified rare earth thulium-doped up-conversion luminescent nanocomposite material is cultured, a detection graph of cell singlet oxygen is obtained by DCFH-DA, and from the detection graph, the content of singlet oxygen in a single CDT material is almost turned over by 2 times, and the content of singlet oxygen in a final material group is close to 3 times compared with a control group. The results further indicate UCNPs @ MnO2-C3N4The potential diagnosis and treatment value of the nano composite material is 2-3 times of the synergistic treatment effect of the nano composite material in the prior art.
The key point of the invention is that the nano-composite for dual-power treatment based on direct growth of the surface of the nanocrystal is obtained by adopting a direct lateral growth and covalent connection method, a unique and simple process method and a component proportion, 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 the industrialization is easy.
The present invention is not limited to the above-mentioned embodiments, and other dual-powered therapeutic agents (C) obtained by the same or similar method as that of the above-mentioned embodiments can be used3N4、MnO2) Methods for modifying rare earth up-conversion luminescent nanocomposites, such as different rare earth ion-doped up-conversion nanocrystals (NaYF)4:Yb,Tm@NaGdF4；NaYF4:Yb,Tm,Er@NaGdF4；NaYF4:Yb,Tm,Ho@NaGdF4) Different water soluble polymers, etc. are within the scope of the present invention.
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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