CN110819339B - Cu-amino acid composite up-conversion nano material and preparation method thereof - Google Patents

Cu-amino acid composite up-conversion nano material and preparation method thereof Download PDF

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CN110819339B
CN110819339B CN201910972424.7A CN201910972424A CN110819339B CN 110819339 B CN110819339 B CN 110819339B CN 201910972424 A CN201910972424 A CN 201910972424A CN 110819339 B CN110819339 B CN 110819339B
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李春霞
陈晴
吕光磊
王曼
沈扬
王冬梅
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Zhejiang Normal University CJNU
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Abstract

The invention belongs to the field of nano biomedicine, and relates to an upconversion fluorescence imaging Cu-amino acid composite upconversion nano material capable of simultaneously realizing chemical kinetic treatment and near infrared light activation and a preparation method thereof. The material can simultaneously realize chemical kinetic treatment and near-infrared light activated up-conversion fluorescence imaging, is uniform and spherical in shape, and has an obvious core-shell structure. The core is rare earth fluoride nano particles, and the shell is a Cu-L-cysteine complex. The Cu-amino acid composite up-conversion nano material can be enriched at a tumor part through an EPR effect, and is specifically activated by a tumor microenvironment without damaging normal tissues. Can consume the reduced glutathione excessively expressed in cells, release cuprous ions with fenton-like reaction activity, and simultaneously generate efficient fenton reaction to generate OH with strong cytotoxicity. Under the irradiation of near infrared light, ultraviolet light and visible light can be emitted, and the imaging function which is not available in the common chemical kinetic reagent is realized.

Description

Cu-amino acid composite up-conversion nano material and preparation method thereof
Technical Field
The invention belongs to the field of nano biomedicine, and relates to an upconversion fluorescence imaging Cu-amino acid composite upconversion nano material capable of simultaneously realizing chemical kinetic treatment and near infrared light activation and a preparation method thereof.
Background
Malignant tumors are one of the important diseases threatening human health, and the traditional treatment means mainly comprises surgical treatment, radiotherapy and chemotherapy. Chemotherapy is a treatment means of systemic administration, which aims to kill tumor cells, but because it has no targeting property, it can cause damage to normal tissues and organs, causing serious side effects. Meanwhile, the problems of high metabolism speed, multi-drug resistance and the like exist. High permeability and retention at the tumor site (EPR effect), and milder, higher concentration of Glutathione (GSH) and hydrogen peroxide (H) 2 O 2 ) Has attracted a great deal of attention.
The nano-carrier capable of being specifically activated by the tumor microenvironment is widely applied to the diagnosis and treatment of diseases, and the material has the greatest advantages that the nano-carrier can be enriched on the tumor part through the EPR effect and is specifically activated by the tumor microenvironment without damaging normal tissues and organs. The chemokinetic therapy (CDT) is to kill tumor cells by utilizing nano materials with Fenton reaction or Fenton-like reaction activity, such as ferroferric oxide, antiferromagnet, metal silicate or iron-containing nano particles to catalyze hydrogen peroxide over-expressed at tumor sites to generate highly toxic OH. OH and biological macromolecules of surrounding organisms generate oxidation reactions, which damage intracellular lipids and DNA, and further induce apoptosis or necrosis of cells, and the low efficiency caused by nonspecific side effects of traditional chemotherapy and limited light transmission depth and oxygen dependence in photodynamic therapy is basically avoided. Such nanomaterials capable of specifically responding to the Tumor Microenvironment (TME) have been widely used in the treatment of tumors.
The key to the chemokinetic treatment is the use of Fenton's reagentThe rate of reaction. However, the iron-based Fenton reaction is only effective under strictly controlled acidic conditions (pH)<4). In the weakly acidic microenvironment of tumor, the efficiency of Fenton's reaction is low and the concentration of H in the lesion tissue is low 2 O 2 And high concentrations of reduced Glutathione (GSH) have largely limited the use of CDT. Biological imaging plays an important role in promoting the development of life science and medicine, and can clearly display and monitor the occurrence and development processes of various physiological and pathological phenomena in real time. Unfortunately, most fenton reagents do not have the ability to image. These all pose significant limitations to the widespread use of CDT. The rare earth doped up-conversion nano material is a typical non-invasive imaging probe, and has special advantages which other nano materials do not have, such as: no flicker, high stability, negligible autofluorescence, etc. In addition, UCNPs can be used as an energy converter to convert low-energy near-infrared light with a deep tissue penetration depth into high-energy ultraviolet and visible light, can accurately position cancer treatment of deep tissues and can provide high-resolution imaging guidance.
In order to improve the efficiency of CDT and expand the application range of CDT, the invention compounds the up-conversion nano material Cu-L-cysteine complex nano material and glucose oxidase to form a multifunctional cascade catalysis nano preparation, thereby realizing high-efficiency chemokinetic treatment, and simultaneously the material can be activated by a tumor microenvironment and has the up-conversion biological imaging function triggered by near infrared light.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a Cu-amino acid composite upconversion nano material capable of simultaneously realizing chemical kinetics treatment and near infrared light activated fluorescence imaging and a preparation method thereof.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the Cu-amino acid composite up-conversion nano material simultaneously realizes chemo-kinetic treatment and near infrared light activated up-conversion fluorescence imaging, is uniform and spherical in shape, has an obvious core-shell structure, and has an inner core of rare earthThe fluoride nano-particle has a Cu-L-cysteine complex as a shell; the chemical expression is as follows: naYF 4 :Yb 3+ ,A 3+ @NaBF 4 @NaYF 4 :Yb 3+ ，C 3+ @NaDF 4 Wherein A is Er, tm or Ho; b is Gd, Y or Lu; c is Er, tm or Ho; d is Gd, Y or Lu, the upconversion nano particle is prepared by a thermal decomposition method, and the surface ligand of the upconversion nano particle is OA.
Replacing the ligand on the surface of the upconversion nanoparticles with L-cysteine by Cu 2+ And inducing the Cu-amino acid complex to coat the surface of the UCNPs by the coordination between the Cu-amino acid complex and the L-cysteine to obtain the Cu-amino acid complex up-conversion nano material UCNPs @ Cu-Cys.
Further converting the surface of the nano material on the Cu-amino acid composite to modify HS-PEG-NH 2 . Then, the amino and the carboxyl on the glucose oxidase form an amido bond to fix the glucose oxidase on the surface of the composite material.
In the technical scheme, the composite nano material is spherical, has an obvious core-shell structure, and has a particle size of about 57nm. The related up-conversion nano particles are provided with an element A which is one or more of Er, tm and Ho, an element B which is one of Gd, Y and Lu, an element C which is one or more of Er, tm and Ho but different from the element A, and an element D which is one of Gd, Y and Lu.
A preparation method of a Cu-amino acid composite up-conversion nano material capable of simultaneously realizing enhanced chemical kinetics therapy and near infrared light activated up-conversion fluorescence imaging comprises the following steps:
(1) Preparation of Up-converting nanoparticle beta-NaYF 4 :Yb 3+ ,A 3+ Upconversion nanoparticles:
mixing YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ACL 3 ·6H 2 Mixing O with OA (OA) and ODE (ODE), and heating to obtain a uniform solution; after the solution is cooled to room temperature, naOH and NH are added 4 Stirring the solution of F in methanol, heating and vacuumizing to remove methanol, oxygen and moisture in the solution; introducing nitrogen into the reaction system, andraising the temperature to 300 ℃ for 1h; after the solution is cooled to room temperature, precipitating the obtained nanocrystal by absolute ethyl alcohol, and alternately cleaning by cyclohexane and ethanol to obtain the up-conversion nano particles beta-NaYF 4 :Yb 3+ ,A 3+ 。
(2) Preparation of NaYF 4 :Yb 3+ ,A 3+ @NaBF 4 @NaCF 4 :Yb 3+ ,Tm 3+ @NaDF 4 Upconversion nanoparticles:
uniformly mixing the up-conversion nanoparticles obtained in the step (1) with OA and ODE; then, raising the temperature and vacuumizing to remove cyclohexane, oxygen and moisture in the solution; introducing nitrogen into the reaction system, and raising the temperature to 310 ℃; will contain CF 3 COONa and B (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after 1h, will contain CF 3 COONa、Yb(CF 3 COO) 3 、C(CF 3 COO) 3 And Y (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after a further 1h, will contain CF 3 COONa and D (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after reacting for 1h, cooling the solution to room temperature, adding ethanol to precipitate the nanoparticles, and washing with cyclohexane and ethanol alternately to obtain the upconversion nanoparticles (UCNPs).
(3) Preparing the L-cysteine modified up-conversion nanoparticles:
dissolving L-cysteine in deionized water, adding the chloroform solution of the up-conversion nanoparticles prepared in the step (2), performing ultrasonic treatment, heating to reflux, and keeping for 12 hours; after cooling to room temperature, the product was centrifuged and then washed repeatedly with deionized water to obtain L-cysteine modified upconversion nanoparticles (UCNPs-Cys).
(4) Preparing a Cu-amino acid composite up-conversion nano material:
adding CuCl 2 Mixing the water solution with the aqueous solution of UCNPs-Cys, and stirring for 5min; then, dropwise adding an aqueous solution containing NaOH and L-cysteine into the solution, and stirring for 5min; the product was separated by centrifugation and washed alternately with water and ethanol to give Cu-amino groupsAcid-recombination upconversion nanometer material (UCNPs @ Cu-Cys).
(5) Preparing a Cu-amino acid composite up-conversion nano material with a surface fixed with GOX:
coupling UCNPs @ Cu-Cys and HS-PEG-NH 2 Adding into ethanol and stirring for 12h, collecting solid product by centrifugation, and washing with deionized water several times to remove free HS-PEG-NH 2 To obtain HS-PEG-NH 2 The modified Cu-amino acid composite up-conversion nanometer material (UCNPs @ Cu-Cys-PEG). Stirring glucose oxidase, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in PBS for 40min; adding the up-conversion nano particle Cu-amino acid composite nano material, and continuously stirring for 1h; the product was centrifuged, and washed 2 times with PBS to remove free glucose oxidase, resulting in a glucose oxidase-loaded nanocomposite (UCNPs @ Cu-Cy-GOX).
The technical scheme is as follows:
(1) Preparation of Up-converting nanoparticle beta-NaYF 4 :Yb 3+ ,A 3+ Upconversion nanoparticles: 1.0mmol of RECl in a 100mL three-necked flask 3 (RE =0.78Y +0.2Yb + 0.02A) was mixed with 15mL ODE and 10mL OA. The solution was heated to 140 ℃ to give a homogeneous solution, which was then cooled to room temperature. To the above solution was added 10mL of a solution containing 0.15g NH 4 F and 0.1g of NaOH in methanol, the temperature is raised to 70 ℃ and stirring is carried out at this temperature for 10min. After that, the solution was heated to 110 ℃ and degassed for 30min. Then in N 2 Heating to 300 ℃ under protection, and reacting for 1h. Cooling the reaction system to room temperature, adding ethanol to precipitate the nanoparticles, centrifugally separating, and washing with ethanol for several times to obtain NaYF 4 Yb, A nanoparticles.
(2) Synthesis of NaYF 4 :Yb,Er@NaGdF 4 @NaYF 4 :Yb,Tm@NaGdF 4 Nanoparticle: naYF is added 4 Yb, A nanoparticles in cyclohexane were mixed with 10mL OA and 10mL ODE. The resulting mixed solution was evacuated at 110 ℃ for 30min to remove cyclohexane, and then N 2 Heated to 310 ℃ under protection. Thereafter, 4mL of a solution containing 0.25mmol of CF 3 COONa and 0.25mmol B (CF) 3 COO) 3 OA and ODE (V) ofThe mixed solution of/V = 1) was injected into the reaction system. After 1h, it contained 1mmol of CF 3 COONa，0.4mmol Yb(CF 3 COO) 3 ,0.05mmol C(CF 3 COO) 3 And 0.595mmol of Y (CF) 3 COO) 3 4mL of a mixed solution of OA and ODE (V/V = 1). After another 1h, 4mL of a solution containing 0.5mmol of CF 3 COONa，0.5mmol D(CF 3 COO) 3 The mixed solution of OA and ODE (V/V = 1) was injected into the reaction system, and the reaction was continued for 1 hour. Finally, the reaction was cooled to room temperature, the nanoparticles were precipitated by adding ethanol, collected by centrifugation, washed several times with ethanol to obtain NaYF 4 :Yb,Er@NaGdF 4 @NaYF 4 :Yb,Tm@NaGdF 4 Nanoparticles.
(3) Preparing the L-cysteine modified upconversion nanoparticles: 0.3g of L-cysteine was weighed and added to 30mL of deionized water and 10mL of the above UCNP solution. Ultrasonic treating for 3min, heating to 100 deg.C, and reflux stirring for 12 hr. After cooling to room temperature, the solid product was collected by centrifugation (10000 rpm,5 min) and washed 3 times with deionized water to obtain L-cysteine modified upconverting nanoparticles.
(4) Preparing a Cu-amino acid composite up-conversion nano material: 10mL of 10 mmol. L -1 CuCl of 2 1mL of the UCNPs-Cys solution was added to the aqueous solution, and the mixture was stirred for 5min. Then, 10mL of a solution containing 10 mmol. L was added dropwise -1 NaOH and 20 mmol. L -1 An aqueous solution of L-cysteine. Stirring for 5min, centrifuging (8000rpm, 3min) to obtain a solid product, and washing once by using deionized water and ethanol to obtain the up-conversion nano material Cu-amino acid complex composite nano material.
(5) The amino group on the surface of the composite nano material and the carboxyl group on the glucose oxidase form an amido bond to connect the amino group and the carboxyl group together: 45mg UCNPs @ Cu-Cys and 10mg HS-PEG-NH 2 Add to 20mL ethanol and stir for 12h. The solid product was collected by centrifugation (8000rpm, 3min). Washing twice with deionized water to remove free HS-PEG-NH 2 . Obtaining HS-PEG-NH 2 The modified composite nano material UCNPs @ Cu-Cys-PEG. 0.1mg EDC,0.3mg NHS and 0.2mg GOX were added to 5mL PBS, stirred for 40min, then 20mg UCNPs @ Cu-Cys-PEG was added, followed byStirring was continued for 1h, and the solid product was collected by centrifugation (8000rpm, 3min) and washed twice with PBS. Obtaining the nanocomposite material UCNPs @ Cu-Cys-GOX loaded with glucose oxidase.
Compared with the prior art, the invention has the following main advantages:
(1) The Cu-amino acid composite up-conversion nano material prepared by the method can be enriched on a tumor part through an EPR effect, and is specifically activated by a tumor microenvironment without damaging normal tissues.
(2) Can consume the reduced glutathione excessively expressed in cells, release cuprous ions with fenton-like reaction activity, and simultaneously generate efficient fenton reaction to generate OH with strong cytotoxicity.
(3) H produced by reaction of glucose oxidase with glucose after glucose oxidase is immobilized on the surface of the substrate 2 O 2 The method can further enhance the Fenton-like reaction rate and realize the enhanced chemical kinetics effect.
(4) The Cu-amino acid composite up-conversion nano material prepared by the method can emit ultraviolet light and visible light under the irradiation of near infrared light, and realizes the imaging function which is not available in common chemical kinetic reagents.
Drawings
FIG. 1 is a schematic representation of UCNP @ Cu-Cys-GOX for enhanced chemokinetic therapy and near infrared light activated up-conversion fluorescence imaging.
FIG. 2 is a transmission electron micrograph a and a scanning transmission electron micrograph d of UCNP @ Cu-Cys, and corresponding elemental analysis plots b-f representing Cu, S, er and Tm elements, respectively.
FIG. 3 is a graph showing the upconversion fluorescence spectra of UCNP @ Cu-Cys-GOX of the present invention under the excitation of 980nm light after the reaction with different amount of 10mM glutathione.
FIG. 4 is an XPS spectrum of UCNP @ Cu-Cys-GOX before reaction with glutathione.
FIG. 5 is an XPS spectrum of UCNP @ Cu-Cys-GOX after reaction with glutathione.
FIG. 6 shows an electron spin resonance spectrum measured in the presence of glucose after reaction of UCNP @ Cu-Cys-GOX with glutathione.
FIG. 7 shows UCNP @ Cu-Cys at H 2 O 2 In the presence of the catalyst, terephthalic acid reacts with the generated OH-induced fluorescence enhancement.
Fig. 8 is a fluorescence enhancement reaction induced by terephthalic acid and generated OH in the presence of glucose in a composite nanomaterial loaded with glucose oxidase.
FIG. 9 shows the survival rate of cancer cells and normal cells co-cultured with the nanocomposite material of the present invention for 24 hours.
FIG. 10 is a graph showing the effect of UCNP @ Cu-Cys-GOX for in vivo imaging.
FIG. 11 shows the time-dependent change of total fluorescence intensity at a tumor site when UCNP @ Cu-Cys-GOX is used for in vivo imaging.
FIG. 12 is a graph showing the body weight change a and tumor growth curve b of mice treated with the upconversion nanoparticles and Cu-amino acid complex composite nanocherapy according to the present invention.
Detailed Description
The invention relates to an upconversion fluorescence imaging Cu-amino acid composite upconversion nanometer material capable of simultaneously realizing chemical kinetics treatment and near infrared light activation and a preparation method thereof. The invention can simultaneously realize chemical kinetic treatment and near infrared light activated up-conversion fluorescence imaging, and the material is uniform and spherical and has an obvious core-shell structure. The core is rare earth fluoride nano particles, and the shell is a Cu-L-cysteine complex. The Cu-amino acid composite up-conversion nano material can be enriched at a tumor part through an EPR effect, and is specifically activated by a tumor microenvironment without damaging normal tissues. Can consume the reduced glutathione excessively expressed in cells, release cuprous ions with fenton-like reaction activity, and simultaneously generate efficient fenton reaction to generate OH with strong cytotoxicity. Under the irradiation of near infrared light, ultraviolet light and visible light can be emitted, and the imaging function which is not available in the common chemical kinetic reagent is realized.
In order to clearly explain the technical features of the present invention, the present invention will be explained below with reference to specific embodiments. The scope of protection of the invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1: and (3) preparing the up-conversion nano particle and Cu-amino acid complex composite nano material.
Step 1, preparing up-conversion nano particle beta-NaYF 4 :Yb 3+ ,Er 3+ Upconversion nanoparticles:
in a 100mL three-necked flask, 1.0mmol of ErCl 3 (RE =0.78Y +0.2Yb + 0.02Er) was mixed with 15mL ODE and 10mL OA. Heating to 140 deg.C to obtain a homogeneous solution, cooling to room temperature, and adding 10mL of a solution containing 0.15g NH 4 F and 0.1g of NaOH in methanol, the temperature is raised to 70 ℃ and stirring is carried out at this temperature for 10min. After that, the stirred solution was heated to 110 ℃ and degassed for 30min. Then in N 2 Heating to 300 ℃ under protection, and reacting for 1h. After cooling the reaction system to room temperature, adding absolute ethyl alcohol to precipitate the nano particles, centrifugally separating, washing with absolute ethyl alcohol for a plurality of times, and finally dissolving in 10mL of cyclohexane for later use. The transmission electron micrograph of the obtained upconverting nanoparticles is shown in fig. 2 a.
Step 2, preparing NaYF 4 :Yb 3+ ,Er 3+ @NaGdF 4 @NaYF 4 :Yb 3+ ,Tm 3+ @NaGdF 4 Upconversion nanoparticles:
synthesis of NaYF 4 :Yb,Er@NaGdF 4 @NaYF 4 :Yb,Tm@NaGdF 4 (UCNPs): 2.5mL NaYF 4 A cyclohexane solution of Yb, er nanoparticles was mixed with 10mL OA and 10mL ODE. The resulting mixed solution was evacuated at 110 ℃ for 30min to remove cyclohexane, and then under N 2 Heated to 310 ℃ under protection. Thereafter, 2mL of a solution containing 0.25mmol of CF 3 COONa and 0.25mmol Gd (CF) 3 COO) 3 The mixed solution of OA and ODE (V/V = 1). After 1h, it contained 1mmol of CF 3 COONa，0.4mmol Yb(CF 3 COO) 3 ，0.05mmol Tm(CF 3 COO) 3 And 0.595mmol of Y (CF) 3 COO) 3 4mL of a mixed solution of OA and ODE (V/V = 1). After another 1h, 2mL of a solution containing 0.5mmol of CF 3 COONa，0.5mmol Gd(CF 3 COO) 3 The mixed solution of OA and ODE (V/V = 1) was injected into the reaction system, and the reaction was continued for 1 hour. Finally, cooling the reaction system to room temperature, precipitating the nanoparticles by adding absolute ethyl alcohol, centrifugally collecting, washing with absolute ethyl alcohol for several times to obtain the up-conversion nanoparticles UCNPs, and finally dissolving in 20mL of chloroform for later use. The transmission electron micrograph of the resulting upconverted nanoparticles is shown in fig. 2 b.
Step 3, preparing the L-cysteine modified up-conversion nanoparticles:
0.3g of L-cysteine was weighed and added to 30mL of deionized water and 10mL of the above UCNPs in chloroform. Ultrasonic treating for 3min, heating to 100 deg.C, and reflux stirring for 12 hr. After cooling to room temperature, the solid product was collected by centrifugation (10000 rpm,5 min), washed 3 times with deionized water to obtain L-cysteine modified up-conversion nanoparticles UCNPs-Cys, and finally dispersed in 20mL of deionized water for further use.
Step 4, preparing the Cu-amino acid composite up-conversion nano material:
10mL of 10 mmol. L -1 CuCl of 2 1mL of the UCNPs-Cys solution was added to the aqueous solution, and the mixture was stirred for 5min. Then, 10mL of a solution containing 10 mmol. Multidot.L was added dropwise -1 NaOH and 20 mmol. L -1 An aqueous solution of L-cysteine. Stirring for 5min, obtaining a solid product by centrifugation (8000rpm, 3min), washing with deionized water and absolute ethyl alcohol once to obtain the up-conversion nanoparticle Cu-amino acid complex composite nanomaterial UCNPs @ Cu-Cys, and finally dispersing in absolute ethyl alcohol and storing at 4 ℃. The transmission electron microscope and scanning transmission electron microscope photographs of the obtained composite nanomaterial are shown in fig. 2c and d. FIGS. 2e-f are the corresponding elemental analysis diagrams, representing the Cu, S, er and Tm elements, respectively.
Step 5, preparing HS-PEG-NH 2 Modified Cu-amino acid composite up-conversion nano material:
45mg UCNPs @ Cu-Cys and 10mg HS-PEG-NH 2 Added to 20mL of absolute ethanol and stirred for 12h. The solid product was collected by centrifugation (8000rpm, 3min). Washing twice with deionized water to remove free HS-PEG-NH 2 . Obtaining HS-PEG-NH 2 Modified up-conversion nanoparticle Cu-amino acid composite nanomaterial UCNPs @ Cu-Cys-PEG. Finally, it was dispersed in 10mL KH 2 PO 4 Buffer (pH 6.0, 50 mM) for use.
After obtaining the material UCNPs @ Cu-Cys-PEG, we tested the ability of the material to generate hydroxyl free radicals. Terephthalic acid is selected as a probe to detect the generation of OH, the terephthalic acid can react with the OH to generate hydroxylation product 2-hydroxy terephthalic acid, and the 2-hydroxy terephthalic acid can emit fluorescence of about 440nm under the excitation of light with the wavelength of 312nm so as to indirectly detect the amount of hydroxyl radicals formed in the solution. To 1mL of 1mmol. L -1 1mL of glutathione with the same concentration is added into the nano composite material, the mixture is evenly stirred, and after 15min of reaction, a certain amount of hydrogen peroxide and terephthalic acid are added into the mixture to ensure that the final concentrations of the hydrogen peroxide and the terephthalic acid are respectively 10mM and 5mM. The fluorescence intensity at 440nm under excitation at a wavelength of 312nm was measured at different times. As shown in FIG. 7, the fluorescence at 440nm gradually increased with time, demonstrating the generation of hydroxyl radicals.
Example 2: preparing a Cu-amino acid composite up-conversion nano material with glucose oxidase fixed on the surface.
Step 1 to step 5 reference case one.
And 6, forming an amide bond by amino on the surface of the composite nano material and carboxyl on the glucose oxidase to connect the amino and the glucose oxidase together to finally obtain the material UCNPs @ Cu-Cys-GOX.
0.1mg EDC,0.3mg NHS and 0.2mg GOX were added to 3mL PBS, stirred for 40min, then 20mg UCNPs @ Cu-Cys was added, stirring was continued for 1h, the solid product was collected by centrifugation (8000rpm, 3min) and washed twice with PBS. Composite nanomaterial ucnps @ cu-Cys-GOX loaded with glucose oxidase was obtained, finally dispersed in PBS (pH =6.0, 50 mM) and stored at 4 ℃.
After taking the material UCNPs @ Cu-Cys-GOX, it is firstly reacted with glutathione, and the change of the copper ion valence state in Cu-Cys and the recovery capability of the upconversion fluorescence are tested. Respectively and uniformly mixing UCNPs @ Cu-Cys-GOX with glutathione (10 mM) with different volumes, reacting for 20min, and detecting XPS spectra of materials treated under different conditions. The XPS spectra of Cu before reaction with GSH are shown in figure 4,peaks in the Cu XPS spectrum with Binding Energies (BE) of 932.9 and 952.8eV were assigned as Cu 2p by comparison with carbon calibrated CuS spectra 3/2 And Cu 2p 1/2 Peak of (2). XPS spectra of Cu after reaction with GSH (theoretical equivalent reaction) are shown in FIG. 5, cu 2p 1/2 And Cu 2p 3/2 Slightly shifted from 952.8 and 932.9eV to 951.9 and 932.3eV, respectively. It was demonstrated that GSH reduces copper from a +2 to a +1 valence in the reaction. Meanwhile, the upconversion fluorescence intensity of the upconversion nanoparticles before and after the reaction under the excitation of 980nm laser is detected. As shown in FIG. 3, the up-conversion fluorescence is enhanced along with the increase of the added glutathione, which indicates that the Cu-Cy is decomposed under the action of the glutathione, and indicates that UCNPs @ Cu-Cys-GOX has the potential of being applied to biological imaging and providing high-resolution imaging guidance.
Then, the ability of UCNPs @ Cu-Cys-GOX to generate active oxygen was examined. To 1mL of 1mmol. L -1 1mL of glutathione with the same concentration is added into UCNPs @ Cu-Cys-GOX, the mixture is stirred evenly, after 15min of reaction, a certain amount of beta-D-glucose and terephthalic acid are added into the mixture to ensure that the final concentration of hydrogen peroxide and the final concentration of terephthalic acid are 10mM and 5mM respectively. The fluorescence intensity at 440nm under excitation at a wavelength of 312nm was measured at different times. As shown in FIG. 8, the fluorescence at 440nm gradually increased with time, demonstrating the generation of hydroxyl radicals. UCNPs @ Cu-Cys-GOX, beta-D-glucose and glutathione are mixed evenly, after reacting for 20min, BMPO is added and mixed evenly, and after reacting for 3h, the electron spin spectrum is detected. As shown in fig. 6, a quadruple characteristic peak with an intensity of 1.
Next, we examined the chemokinetic killing ability of UCNPs @ Cu-Cys-GOX on 4T1 cells. The killing capacity of UCNPs @ Cu-Cys-GOX to 4T1 cells is detected by using an MTT method. Cells were cultured using 96-well plates, with a cell number of 5000 per well. 200ug/mL,100ug/mL,50ug/mL and 25ug/mL of the composite were added to cultured 4T1 cells using the method of reciprocal dilution, three wells were repeated for each concentration, and cells without the composite were used as a control experiment. And continuously culturing the cells in the cell culture box for 4h, discarding the culture medium, replacing the culture medium with a new one, and continuously culturing in the culture box for 20h. After 4h 10. Mu.L MTT (5 mg/mL) was added, the supernatant was removed and 150. Mu.L DMSO was added. The absorbance (OD) at 570nm was recorded by a microplate reader. Cell viability was calculated according to the following formula: cell viability (%) = OD (sample)/OD (control) × 100%. As can be seen from fig. 9, the survival rate of the cells gradually decreased as the concentration of the composite material increased. This indicates that the composite material can generate active oxygen in cells and kill the cells.
Encouraged by the in vitro up-conversion fluorescence recovery capability, the in vivo fluorescence imaging capability activated by UCNPs @ Cu-Cys-GOX near infrared light is detected. Tumor-bearing Balb/c mice injected intratumorally with UNCPs @ Cu-Cy-GOX (0.2mL, 10mg/mL) were imaged on an in vivo Maestro whole-body imaging system equipped with an external 980nm laser as an excitation source for in vivo UCL imaging at different time periods (10, 30, 60 and 90 min). And the total signal intensity of the UCL at different time periods (10, 30, 60, and 90 min) was recorded. As shown in fig. 10 and 11, about 2-fold enhancement of UCL was observed from injection 10min to 90min, a phenomenon attributed to the well-designed core-shell structure of ucnps @ cu-Cys-GOX. The results indicate that our invented UCNPs @ Cu-Cys-GOX as an ideal imaging agent is applicable to recoverable UCL bioimaging.
Finally, the detection of the in vivo anti-tumor capability of UCNPs @ Cu-Cys-GOX is carried out. BalB/C mice (6 weeks) were used to test the antitumor effect of UCNPs @ Cu-Cys-GOX composite nanoparticles. 4T1 cells were injected subcutaneously into the axilla to allow tumor growth to about 100mm 3 Of the substrate is uniform. Mice bearing 4T1 tumors were randomized into 2 groups (n = 5). The first group was injected with normal saline (control group), and the second group was injected with UCNPs @ Cu-Cys-GOX composite nanoparticles. The solution of 10mg/mL of UCNPs @ Cu-Cys-GOX composite nano particles is administrated by tail vein injection, and the administration dosage is 10mg/kg. According to Vr = V/V 0 ×100％(V 0 : tumor volume on day one, vr: relative tumor volume) was calculated. Mice body weight and tumor volume were observed to see the effect of treatment. The body weight change of the mice is shown in FIG. 12a, and the tumor growth curve is shown in FIG. 12 b.
The experimental results show that the tumor growth of the mice in the experimental group is effectively inhibited, which indicates that the composite nano-material prepared by the invention has obvious tumor treatment effect. The weight of the mouse does not obviously and abnormally reduce in the treatment period, which shows that the composite nano particles prepared by the invention have no toxic effect on the mouse. The tumor growth of the control mice was not inhibited and increased rapidly.
The Cu-amino acid composite up-conversion nano material and the preparation method thereof provided by the invention have the following characteristics:
1. provides a concept of combining up-conversion nanoparticles with Cu-amino acid complex, and forms a composite nano material which can realize both chemokinetic treatment and near-infrared activated up-conversion fluorescence imaging.
2. Provides a method for coating a Cu-amino acid complex on an upconversion nano particle, namely, a surface ligand on the upconversion nano particle is replaced by UCNPs-Cys of L-cysteine through ligand exchange, and then the UCNPs-Cys and Cu are coated 2+ Mixing and stirring in the water solution for 5min to make Cu 2+ Coordinating with L-cysteine to induce Cu-amino acid complex to form on the surface of UCNPs, and finally obtaining the Cu-amino acid complex up-conversion nano material.
3. Glucose Oxidase (GOX) is fixed on the surface of the conversion nano material on the Cu-amino acid composite, and the GOX can react with glucose to generate gluconic acid and H 2 O 2 The hydrogen peroxide is a raw material of Fenton-like reaction, and the large amount of hydrogen peroxide generated enhances the effect of chemokinetic treatment (enhanced chemokinetic treatment effect). (1) Cu (copper) 2+ +GSH→Cu + +GSSH ②GOX+glucose→H 2 O 2 +gluconic acid ③Cu + +H 2 O 2 →Cu + +·OH+OH - 。

Claims (10)

1. A Cu-amino acid composite up-conversion nano material is characterized in that chemical kinetic treatment and near-infrared light activated up-conversion fluorescence imaging can be realized simultaneously, the material is uniform and spherical, and has an obvious core-shell structure, the inner core is rare earth fluoride nano particles, and the shell is a Cu-L-cysteine complex; the chemical expression is as follows: naYF 4 :Yb 3+ ,A 3+ @NaBF 4 @NaYF 4 :Yb 3+ ,C 3+ @NaDF 4 @ Cu-Cys, wherein A is Er, tm or Ho; b is Gd, Y or Lu; c is Er, tm or Ho; d is Gd, Y or Lu.
2. A preparation method of a Cu-amino acid composite up-conversion nano material is characterized in that the up-conversion nano material is prepared by a thermal decomposition method, the prepared up-conversion nano material surface ligand is OA, then the ligand exchange method is adopted to replace the surface ligand to L-cysteine, then the Cu-amino acid complex is induced to coat the up-conversion nano material surface through the coordination effect between copper ions and the L-cysteine to obtain the composite nano material, and HS-PEG-NH is modified on the surface of the composite nano material 2 (ii) a And finally, forming an amido bond by using the amino group on the surface of the composite nano material and the carboxyl group on the glucose oxidase to connect the amino group and the glucose oxidase together, and finally obtaining UCNPs @ Cu-Cys-GOX.
3. The method of claim 2, comprising the steps of:
(1) Preparation of Up-conversion nanoparticle beta-NaYF 4 :Yb 3+ ,A 3+ ：
Mixing YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ACL 3 ·6H 2 Mixing O, oleic acid OA and octadecene ODE uniformly, and heating to obtain a uniform solution; after the solution is cooled to room temperature, naOH and NH are added 4 Stirring the solution of F in methanol, heating and vacuumizing to remove methanol, oxygen and moisture in the solution; introducing nitrogen into the reaction system, and raising the temperature to 300 ℃ and keeping the temperature for 1h; after the solution is cooled to room temperature, precipitating the obtained nanocrystal with absolute ethyl alcohol, and alternately cleaning with cyclohexane and ethanol to obtain the up-conversion nano particle beta-NaYF 4 :Yb 3+ ,A 3+ ；
(2) Preparation of NaYF 4 :Yb 3+ ,A 3+ @NaBF 4 @NaCF 4 :Yb 3+ ,Tm 3+ @NaDF 4 Upconversion nanoparticles:
uniformly mixing the up-conversion nanoparticles obtained in the step (1) with OA and ODE; then, raising the temperature and vacuumizing to remove cyclohexane, oxygen and moisture in the solution; introducing nitrogen into the reaction system, and raising the temperature to 310 ℃; will contain CF 3 COONa and B (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after 1h, will contain CF 3 COONa、Yb(CF 3 COO) 3 、C(CF 3 COO) 3 And Y (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after a further 1h, will contain CF 3 COONa and D (CF) 3 COO) 3 The mixed solution of OA and ODE of (1) is injected into the reaction system; after reacting for 1h, cooling the solution to room temperature, adding ethanol to precipitate nanoparticles, and alternately washing with cyclohexane and ethanol to obtain the up-conversion nanoparticles UCNPs;
(3) Preparing the L-cysteine modified up-conversion nanoparticles:
dissolving L-cysteine in deionized water, adding the chloroform solution of the up-conversion nanoparticles prepared in the step (2), performing ultrasonic treatment, heating to reflux, and keeping for 12 hours; cooling to room temperature, centrifugally separating the product, and repeatedly washing with deionized water to obtain L-cysteine-modified upconversion nanoparticles UCNPs-Cys;
(4) Preparing a Cu-amino acid composite up-conversion nano material:
adding CuCl 2 Mixing the water solution with the aqueous solution of UCNPs-Cys, and stirring for 5min; then, dropwise adding an aqueous solution containing NaOH and L-cysteine into the solution, and stirring for 5min; centrifugally separating a product, and alternately washing with water and ethanol to obtain a Cu-amino acid composite up-conversion nano material UCNPs @ Cu-Cys;
(5) Preparing a Cu-amino acid composite up-conversion nano material with a surface fixed with GOX:
coupling UCNPs @ Cu-Cys and HS-PEG-NH 2 Adding into ethanol and stirring for 12h, collecting solid product by centrifugation, and washing with deionized water several times to remove free HS-PEG-NH 2 To obtain HS-PEG-NH 2 Modified Cu-amino acid composite up-conversion nano material UCNPs @ Cu-Cys-PEG; glucose oxidase, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS were stirred in PBS for 40min; adding the up-conversion nano particle Cu-amino acid composite nano material, and continuously stirring for 1h; and centrifuging and separating the product, and washing the product for 2 times by using PBS (phosphate buffer solution) to remove free glucose oxidase to obtain the glucose oxidase-loaded nanocomposite UCNPs @ Cu-Cys-GOX.
4. The method of claim 2, wherein the UCNPs prepared in step (2) are subjected to surface ligand substitution; weighing 0.3g of L-cysteine, adding 30mL of deionized water and 10mL of the UCNP solution, performing ultrasonic treatment for 3min, heating to 100 ℃, performing reflux stirring for 12h, cooling to room temperature, centrifuging at 10000rpm/5min, collecting a solid product, and then washing with deionized water for 3 times to obtain the L-cysteine modified upconversion nanoparticles.
5. The method of claim 2, wherein the L-cysteine-modified upconversion nanoparticles are coated with a Cu-amino acid; 10mL of 10 mmol. L -1 CuCl of 2 Adding 1mL of UCNPs-Cys into the aqueous solution, and stirring for 5min; then, 10mL of a solution containing 10 mmol. Multidot.L was added dropwise -1 NaOH and 20 mmol. L -1 And stirring the aqueous solution of the L-cysteine for 5min, centrifuging at 8000rpm/3min to obtain a solid product, and washing with deionized water and ethanol once to obtain the Cu-amino acid composite up-conversion nano material.
6. The preparation method of claim 2, wherein the amino group on the surface of the composite nanomaterial and the carboxyl group on the glucose oxidase form an amide bond to link the amino group and the carboxyl group together; adding 0.1mg EDC,0.3mg NHS and 0.2mg GOX into 5mL PBS, stirring for 40min, then adding 20mg UCNPs @ Cu-Cys, continuing stirring for 1h, centrifuging at 8000rpm/3min to collect a solid product, and washing twice with PBS to obtain the nanocomposite loaded with glucose oxidase.
7. The method of claim 3The preparation method is characterized in that the method in the steps (1) and (2) specifically comprises the following steps: in a 100mL three-necked flask, 1.0 mmoleRECl was placed 3 Mixed with 15mL ODE and 10mL OA, RE =0.78Y +0.2Yb +0.02Er; the mixed solution was heated to 140 ℃ to obtain a homogeneous solution, and after cooling to room temperature, 10mL of a solution containing 0.15g of NH was added 4 F and 0.1g NaOH in methanol, heated to 70 ℃ and stirred at this temperature for 10min, heated to 110 ℃ and degassed for 30min, then filtered under N 2 Heating to 300 ℃ under protection, reacting for 1h, cooling to room temperature, adding ethanol to precipitate the nanoparticles, centrifugally separating, washing with ethanol for several times to obtain beta-NaYF 4 :Yb 3+ ,A 3+ Dispersing the nano particles in 10mL of cyclohexane for later use;
the method in the step (2) specifically comprises the following steps: 2.5mL of beta-NaYF 4 A cyclohexane solution of Yb, er nanoparticles was mixed with 10mL OA and 10mL ODE, followed by evacuation at 110 ℃ for 30min to remove cyclohexane, followed by N 2 Heating to 310 ℃ under protection; then 4mL of a solution containing 0.25 mmoleCF 3 COONa and 0.25mmol B (CF) 3 COO) 3 Injecting the mixed solution of 1mL of OA and 1mL of ODE into the reaction system; after 1h, it contained 1mmol of CF 3 COONa，0.4mmol Yb(CF 3 COO) 3 ,0.05mmol C(CF 3 COO) 3 And 0.595mmol of Y (CF) 3 COO) 3 2mL of OA and 2mL of ODE was injected into the reaction system, and after 1 hour, 4mL of a solution containing 0.5mmol of CF 3 COONa，0.5mmol D(CF 3 COO) 3 Injecting the mixed solution of 1mL of OA and 1mL of ODE into the reaction system, and continuing to react for 1h; finally, the reaction was cooled to room temperature, and the nanoparticles were precipitated by adding ethanol, collected by centrifugation, and washed several times with ethanol to obtain the upconversion nanoparticles (UCNPs) described in step (2).
8. The preparation method according to claim 3, wherein the method of step (3) is specifically: weighing 0.3g of L-cysteine, adding 30mL of deionized water and 10mL of the UCNP solution, performing ultrasonic treatment for 3min, heating to 100 ℃, performing reflux stirring for 12h, cooling to room temperature, centrifuging at 10000rpm/5min, collecting a solid product, and then washing with deionized water for 3 times to obtain the L-cysteine modified up-conversion nanoparticles UCNPs-Cys.
9. The preparation method according to claim 3, wherein the method of step (4) is specifically: 10mL of 10 mmol. L -1 CuCl of 2 Adding 1mL of UCNPs-Cys into the aqueous solution, and stirring for 5min; then, 10mL of a solution containing 10 mmol. L was added dropwise -1 NaOH and 20 mmol. L -1 And stirring the aqueous solution of the L-cysteine for 5min, centrifuging at 8000rpm/3min to obtain a solid product, and washing with deionized water and ethanol once to obtain the Cu-amino acid composite up-conversion nano material UCNPs-Cu-Cys-GOX.
10. The preparation method according to claim 3, wherein the method of step (5) is specifically:
45mg UCNPs @ Cu-Cys and 10mg HS-PEG-NH 2 Adding into 20mL ethanol and stirring for 12h; collecting the solid product by centrifugation at 8000rpm for 3 min; washing twice with deionized water to remove free HS-PEG-NH 2 (ii) a Obtaining HS-PEG-NH 2 The modified composite nano material UCNPs-Cu-Cys-PEG; then, 0.1mg EDC,0.3mg NHS and 0.2mg GOX were added to 5mL PBS, stirred for 40min, then 20mg UCNPs @ Cu-Cys-PEG was added, stirring was continued for 1h, the solid product was collected by centrifugation at 8000rpm for 3min, and washed twice with PBS, to obtain glucose oxidase-loaded nanocomposite UCNPs @ Cu-Cys-GOX.
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