CN108079297A - A kind of application of up-conversion luminescence-thermochemotherapy composite Nano probe and preparation method thereof and therapeutic alliance Programmed control - Google Patents

Abstract

The invention belongs to the technical field of nanoprobes, and specifically provides an upconversion luminescence-thermotherapy composite nanoprobe, a preparation method thereof, and an application of programmed control of combined therapy. Among them, the upconversion luminescence-thermal chemotherapy composite nanoprobe has two layers of rare earth fluoride as the core, the middle layer is a hollow silica shell loaded with photothermal materials, and the outer layer is an organic molecular film loaded with small molecule chemotherapeutic drugs. Its general structural formula is: NaL _1‑x‑y Yb _x Er _y F ₄ @NaLF ₄ @SiO ₂ ‑M@N‑P. In the nanocomposite material of the present invention, under the irradiation of near-infrared light, the photothermal molecules generate heat energy to promote the dissociation of the thermosensitive coating, the chemotherapy drug molecules are released, and cancer chemotherapy is realized. At the same time, the heat energy generated by the photothermal molecules can realize cancer treatment. The thermal killing of cells can realize the programmatic step-by-step process of drug release and photothermal therapy in combined cancer therapy. Using this strategy can reduce the dose of chemotherapeutic drugs and thermal energy.

Description

An upconversion luminescence-thermotherapy composite nanoprobe and its preparation method and combination Application of treatment programming control

technical field

The invention belongs to the technical field of nanoprobes, and specifically relates to a preparation method of an upconversion luminescence-thermal chemotherapy composite nanoprobe and the control of the operation sequence of different treatment modes in combined cancer treatment, so as to optimize the effect of cancer treatment and reduce the administration of drugs Increase the amount.

Background technique

Combination therapy for cancer is a treatment strategy that combines two or more treatment modalities. Since the anti-cancer mechanisms of different treatment modes are different, combination therapy can often show cumulative killing effects, thereby reducing the probability of cancer progression and recurrence. Recently, the concept of combined therapy has been adopted by researchers to design some nanomaterial-based probes for cancer therapy. The current development of nanoprobes with combined therapeutic functions has involved the combination of many cancer treatment modalities, including chemotherapy, radiotherapy, photothermal therapy, photodynamic therapy, immunotherapy and gene therapy, etc. Among them, the combination of chemotherapy and photothermal therapy is more common. However, the current combination of chemotherapy and photothermal therapy cannot control the sequence of these two treatment modes, and chemotherapy and photothermal therapy are often triggered simultaneously. However, the order in which the treatment modes are implemented is of great importance for the final efficacy. In the traditional hyperthermia regimen (wherein the chemotherapeutic drug is doxorubicin), administration of doxorubicin before hyperthermia can obtain better curative effect than administration of both at the same time. In a typical chemotherapy-photothermal therapy combined nanosystem, the chemotherapeutic drug will be loaded in some components with thermal response function, and the drug will be released by the thermal energy generated by the photothermal nanomaterial. In view of the current temperature monitoring of the photothermal process, macroscopic instruments such as thermometers or thermal imagers are commonly used. Therefore, in order to ensure that the drug can be released as completely as possible, the apparent temperature is generally raised to a higher level. In this way, chemotherapy and hyperthermia will be triggered at the same time, and cannot be performed separately. In order to realize the ordering of the various treatment modes, it is first necessary to use a suitable temperature measurement method to control the temperature rise of the light and heat. Since the photothermal effect originates from microscopic nanomaterials, it is necessary to monitor the temperature of these nanomaterials themselves. If the intrinsic temperature can be controlled to only trigger drug release without generating excess heat, then a stepwise progression of the therapeutic modality can be achieved.

Contents of the invention

The object of the present invention is to provide a class of optical probe materials with the functions of chemotherapy, photothermal therapy and real-time temperature feedback, which can emit near-infrared light of 800-1200nm under the excitation of 700-1000nm laser, and generate 500-580nm upper Convert fluorescence emission, photothermal organic small molecules convert light energy into thermal energy, realize photothermal tumor killing and dissociation of heat-sensitive organic molecular membranes, and realize the release of chemotherapeutic drugs loaded therein. Among them, the up-conversion fluorescence emission can detect the temperature of the nanoprobe itself, and through the orderly control of the temperature of the nanoprobe itself, the procedural control of cancer photothermal therapy-chemotherapy combined therapy can be realized.

Another object of the present invention is to provide a method for preparing the above-mentioned optical probe material.

The third object of the present invention is to provide an application of the above-mentioned optical probe material in the programmed control of combined cancer therapy.

The purpose of the present invention can be achieved through the following measures:

An upconversion luminescence-thermal chemotherapy composite nanoprobe, which has two layers of rare earth fluoride as the core, the middle layer is a hollow silica shell loaded with photothermal materials, and the outer layer is an organic compound loaded with small molecule chemotherapeutic drugs. Molecular membrane, its general structural formula is:

NaL _1-xy Yb _x Er _y F ₄ @NaLF ₄ @SiO ₂ -M@NP

in,

L is Y or Lu atom;

x is 0.1~0.5, y is 0.01~0.20, and x+y≤1;

M is photothermal material;

N is an organic molecular film;

P is a chemotherapeutic small molecule drug.

The novel optical probe material of the present invention has the functions of combined cancer treatment and real-time temperature feedback. It is a kind of up-conversion light-emitting composite structure material with a three-layer structure, with two layers of rare earth fluoride as the core, and the outer package is loaded with light and heat. The hollow silica shell of the material is wrapped with an organic molecular film loaded with small molecule chemotherapeutic drugs.

In a preferred embodiment, in the general structural formula, x is 0.15-0.40, y is 0.01-0.10, and x+y≤1.

In a more preferred solution, in the general structural formula, x is 0.2-0.4, y is 0.01-0.05, and x+y≤1.

In a preferred embodiment, M is metal phthalocyanine compound, porphyrin compound or indocyanine green.

In a preferred embodiment, N is dipalmitoylphosphatidylcholine or poly-N-isopropylacrylamide.

In a preferred embodiment, P is doxorubicin, paclitaxel, gemcitabine or cisplatin.

Typical composite materials are shown in Table 1, but this does not limit the present invention.

Table 1

The invention provides a method for preparing an upconversion luminescence-thermotherapy composite nanoprobe, which comprises the following steps:

(1) Take the trifluoroacetate salts of L, Yb, Er and Na elements corresponding to the first layer of rare earth fluoride, stir in a solvent at 80-100°C to form a uniform solution, and then evaporate part of the water in the open ;

(2) The solution obtained in step (1) is heated up to 290-330° C. for reaction under nitrogen protection, and cooled after the reaction;

(3) adding ethanol to the reaction solution obtained in step (2), then separating the obtained solid and washing;

(4) Take the trifluoroacetate salts of L and Na elements corresponding to the second layer of rare earth fluorides, stir in a solvent at 80-100° C. to form a uniform solution, and then evaporate part of the water in the open;

(5) Mix the solution obtained in step (4) with the solid obtained in step (3), stir at 80-100°C, then heat up to 290-330°C under nitrogen protection to react, and cool after the reaction;

(6) adding ethanol to the reaction solution obtained in step (5), then separating the obtained solid and washing, and finally ultrasonically dispersing the obtained solid in cyclohexane to obtain a cyclohexane solution of rare earth nanomaterials;

(7) Add the cyclohexane solution of the rare earth nanomaterial into the CO-520 cyclohexane solution, add ammonia water and stir evenly, then add tetraethoxysilane, N-(2-aminoethyl) -3-Aminopropyltrimethoxysilane and tetraethoxysilane were added and stirred for 10-14 hours respectively; then acetone was added, the precipitated solid was separated by centrifugation, washed and dispersed in ethanol; water and hydrogen were added Hydrofluoric acid aqueous solution, after stirring evenly, centrifuge to collect solids, wash again and disperse in ethanol to obtain ethanol solution;

(8) adding the ethanol solution obtained in step (7) into the chloroform solution containing the photothermal material, stirring evenly and then evaporating the chloroform, and washing the obtained solid and dispersing it in water;

(9) Mix the film-forming organic molecules and chemotherapeutic small molecule drugs in chloroform to form a film in the container; add the solution obtained in step (8) into the container containing the film, and use ultrasonic treatment for 10-20 minutes , use centrifugation to collect the precipitated solid, wash the separated solid, or disperse it in water.

Preferably, in the above step (1), the amount of each element is calculated according to the stoichiometric ratio in the general structural formula, and the water is evaporated for 30 to 60 minutes in the open; the solvent is oleic acid, oleylamine, 1-octadecyl alkenes or stearic acid;

Preferably, in the above step (2), the reaction time is 30 to 90 minutes;

Preferably, in the above step (3), the amount of ethanol is the same as the volume of the reaction solution, and the obtained solid is washed 2 to 3 times with a mixed solution of ethanol and cyclohexane;

Preferably, in the above step (4), the amount of each element is calculated according to the stoichiometric ratio in the general structural formula, and the water is evaporated for 30 to 60 minutes in the open; the solvent is oleic acid, oleylamine, 1-octadecyl Alkene or stearic acid; The consumption of each element adopted between step (1) and (4), also calculates with the stoichiometric ratio in the general structural formula.

Preferably, in the above step (5), the reaction time is 30-90 minutes.

Preferably, in the above step (6), the amount of ethanol is the same as the volume of the reaction solution, and the resulting solid is washed 2-3 times with a mixed solution of ethanol and cyclohexane; cyclohexane in the cyclohexane solution of rare earth nanomaterials The dosage is 2-6ml/mmol in terms of L element.

Preferably, in the above step (7), cyclohexane solution of rare earth nanomaterials, CO-520 cyclohexane solution, ammonia water, tetraethoxysilane added for the first time, N-(2-aminoethyl)-3 - The volume ratio of aminopropyltrimethoxysilane, tetraethoxysilane added again, acetone, ethanol for first dispersion, water, hydrofluoric acid aqueous solution, and ethanol for second dispersion is 1:5～20:0.02～ 0.1: 0.01～0.05: 0.01-0.02: 0.05～0.1: 3～15: 5～10: 5～10: 5～10: 5～10, during the centrifugation process, use 10000-15000 rpm to separate 10～ After 20 minutes, the solid separated by centrifugation was washed with ethanol, and the mass content of the hydrofluoric acid aqueous solution was 0.5-2%.

Preferably, in the above step (8), the volume ratio of the ethanol solution to the chloroform solution and the dispersion water is 1-10:5-10:5-10; the content of the photothermal material in the chloroform solution is 0.1-3mg/5-10mL, the solid obtained after distilling off chloroform was washed with ethanol.

Preferably, in the above step (9), the mass ratio of the film-forming organic molecule to the chemotherapeutic small molecule drug is 10-20:0.1-1, and the centrifugation process adopts a rotational speed of 10000-15000 rpm for 10-20 minutes , the solid separated by centrifugation was washed with deionized water three times and the precipitate was dispersed in deionized water.

The present invention also provides a more specific preparation method of the above-mentioned photoluminescence-photothermal nanocomposite structure material (i.e. upconversion luminescence-thermotherapy composite nanoprobe), the specific steps of which are as follows:

(1) According to the rare earth metal species and the stoichiometric ratio of the first layer in the nanocomposite structure material, weigh the corresponding rare earth and sodium trifluoroacetate, add a solvent, and heat and stir at 80 to 100° C. After 30 minutes, dissolve to form a uniform solution, and then evaporate the water for 30 to 60 minutes; the solvent is at least one of oleic acid, oleylamine, 1-octadecene, and stearic acid;

(2) Warm up the solution obtained in step (1) to 290-330° C. under nitrogen protection, react for 30-90 minutes, and then cool to room temperature;

(3) Add the same volume of ethanol to the solution obtained in step (2), obtain a solid by centrifugation, and then wash the obtained solid with a mixed solution of ethanol and cyclohexane for 2-3 times;

(4) According to the rare earth metal element type and the stoichiometric ratio of the second layer in the nanocomposite structure material, weigh the corresponding rare earth metal or sodium trifluoroacetate, add a solvent, and heat and stir at 80-100°C Dissolve for 5-30 minutes to form a homogeneous solution, and then evaporate the water for 30-60 minutes; the solvent is at least one of oleic acid, oleylamine, 1-octadecene, and stearic acid;

(5) Add the solid obtained in step (3) to the solution obtained in step (4), heat and stir at 80-100°C for 10-20 minutes, then heat up to 290-330°C under nitrogen protection, and react for 30-90 minutes, and then cooled to room temperature;

(6) Add the same volume of ethanol to the solution obtained in step (5), obtain a solid by centrifugation, then wash the resulting solid with a mixed solution of ethanol and cyclohexane for 2-3 times, and finally the solid is ultrasonically dispersed in 5-10mL in cyclohexane;

(7) Add 5 to 20 mL of CO-520 cyclohexane solution to the cyclohexane solution obtained in step (6), then add 0.02 to 0.1 mL of ammonia water and stir for 30 to 90 minutes, then add 0.01 to 0.01 mL of tetraethoxysilane Stir 0.05 mL for 12 hours, then add 0.01-0.02 mL of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, stir for 12 hours, then add 0.05-0.1 mL of tetraethoxysilane and stir for 12 hours . Then add 3-15 mL of acetone, centrifuge at 10000-15000 rpm for 10-20 minutes, collect the precipitated solid, add 10-20 mL of ethanol to wash the precipitate three times, and disperse the precipitate in 5-10 mL of ethanol. Then add 5-10 mL of deionized water and 5-10 mL of 1% by mass hydrofluoric acid aqueous solution. After stirring for 30-60 minutes, centrifuge at 10000-15000 rpm, collect the centrifuged solid after 10-20 minutes, wash three times with 5-10mL ethanol, and finally disperse in 5-10mL ethanol.

(8) Measure 1-10 mL of the ethanol solution obtained in step (7), add 5-10 mL of chloroform solution containing 0.1-3 mg of photothermal material, stir at room temperature for 10-20 minutes, and then rotary evaporate the chloroform Finally, wash the solid three times with 5-20 mL of ethanol, and finally disperse the solid in 5-10 mL of water;

(9) Mix 10-20 mg of film-forming organic molecules and 0.1-1 mg of chemotherapeutic drug molecules in 5-20 mL of chloroform, and use rotary evaporation to form a film in a flask. Add the solution obtained in step (8) into the flask containing the membrane, use ultrasonic treatment for 10-20 minutes, use centrifugation at 10000-15000 rpm for 10-20 minutes, collect the precipitated solid, add 10-20mL deionized water to wash Precipitate three times and disperse the precipitate in 10 mL of deionized water.

The photoluminescence-photothermal nanocomposite structural material provided by the present invention (that is, the composite nanoprobe of upconversion luminescence-thermotherapy) can be applied in the program control of combined cancer treatment. The present invention constructs an up-conversion luminescent nanocomposite material with a combined therapeutic function, and the rare-earth-doped up-conversion luminescent nano-core is used to detect the temperature of the nano-particles, and the load in the hollow-structured silica shell has the photothermal conversion function A small molecule with a silica shell coated with a heat-sensitive coating containing chemotherapy drugs. Under the irradiation of near-infrared light, the photothermal molecules generate heat energy to promote the dissociation of the thermosensitive coating, and the chemotherapeutic drug molecules are released to achieve cancer chemotherapy. At the same time, the thermal energy generated by photothermal molecules can realize thermal killing of cancer cells. Under the guidance of temperature-sensitive upconversion luminescence, the action range of thermal energy can be precisely controlled, thereby realizing the programmed step-by-step process of drug release and photothermal therapy in combined cancer therapy. The present invention utilizes this step-by-step combined cancer treatment strategy to reduce the dosage of chemotherapeutic drugs and heat energy. Therefore, the present invention will greatly promote the development of new cancer treatment strategies to achieve milder treatment conditions and lower side effects.

A method for applying the up-conversion luminescence-thermotherapy composite nanoprobe of the present invention in the programmed control of different treatment modes of cancer combination therapy is: dispersing the up-conversion luminescence-thermotherapy composite nanoprobe in water to form a concentration of 0.5-5mg/mL water dispersion liquid is irradiated with a near-infrared laser with a power density of 20-200mW/cm ² and a wavelength of 700-1000nm to achieve a microscopic temperature rise of 3-10°C for the probe material in the aqueous solution; the composite nano When the probe material is irradiated by a near-infrared laser of 700-1000nm, the integrated intensity ratio of the two emission bands conforms to the Boltzmann distribution, that is, ln(I _a /I _b )=C+(-ΔE/kT) . Among them, I _a is the integrated fluorescence intensity of one emission band a, I _b is the integrated fluorescence intensity of one emission band b, C is a constant, ΔE is the energy difference between emission band a and emission band b, and T is temperature. C and ΔE are fitted according to the temperature change curve, and k is the Boltzmann constant. Using this mathematical relationship can realize the microscopic temperature detection of the nanocomposite structure.

Another method of applying the up-conversion luminescence-thermotherapy composite nanoprobe of the present invention in the programmed control of different treatment modes of cancer combination therapy is: the up-conversion luminescence-thermotherapy composite nanoprobe or the concentration of 0.5～ 5 mg/mL up-conversion luminescence-thermochemotherapy compound nanoprobe phosphate buffer dispersion liquid, when co-incubating with cancer cells, use the near-infrared laser with a power density of 20-200mW/cm ² and a wavelength of 700-1000nm for step-by-step irradiation , through scanning confocal fluorescence microscopy, imaging of the microscopic temperature distribution in the cells labeled with the composite nanoprobe and step-by-step control of the temperature rise of the composite nanoprobe itself by 3-10°C are realized, and the step-by-step progress of chemotherapy and photothermal therapy is realized.

Another method for applying the up-conversion luminescence-thermotherapy composite nanoprobe of the present invention in the programmed control of different treatment modes of cancer combination therapy is: compound the up-conversion luminescence-thermochemotherapy with a concentration of 0.1-10 mg/mL The phosphate buffer solution of the nanoprobe was injected into the vein of the tumor-bearing mice. After 2-24 hours, according to the guidance results of the photothermal treatment temperature monitoring, the tumors of the mice were treated with 700-1000nm laser light of 20-200mW/ ^cm2 . The area is irradiated step by step, and under the monitoring of up-conversion luminescence, the control of the temperature rise of the nanoprobe itself by 3-10°C is realized, and the step-by-step progress of chemotherapy and photothermal therapy is realized.

Compared with the prior art, the present invention has the following advantages:

1. The up-conversion luminescence-thermotherapy composite nanoprobe of the present invention can absorb near-infrared laser light of 700-1000nm, emit up-conversion luminescence of 500-580nm, realize the microscopic temperature rise of the nanocomposite structure in aqueous solution at 3-10°C, and The microscopic temperature rise is regulated by changing the relative intensity of the spectrum.

2. The present invention can realize the step-by-step programming of chemotherapy-photothermal therapy by regulating the microscopic temperature, which will greatly promote the development of new cancer treatment strategies, and achieve milder treatment conditions and lower side effects.

3. The present invention provides an effective method for compounding photothermal therapy and chemotherapy, and realizes the release of chemotherapeutic drugs through photothermal action. It has great application prospects in the field of biomedicine.

Description of drawings

FIG. 1 is a transmission electron micrograph of Example 1.

FIG. 2 is a powder X-ray diffraction pattern of Example 1. FIG.

FIG. 3 is an absorption spectrum diagram of Example 1. FIG.

Fig. 4 shows that the fluorescence intensity of Example 4 shows a linear correlation trend with temperature rise.

Figure 5 is the release behavior of doxorubicin in Example 5 at mild microscopic temperatures.

FIG. 6 is a schematic diagram of the principle of programmed combined therapy in Example 6. FIG.

Fig. 7 is the combination therapy effect of cell-level programming in Example 6.

Fig. 8 shows the effect of combined treatment of small living animal level programming in Example 7.

Fig. 9 is a schematic diagram of combined cancer therapy using the composite nanoprobe of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the specific implementation modes of the present invention will be described in detail below with reference to the drawings and examples. However, those of ordinary skill in the art can understand that in each implementation manner of the present invention, many technical details are proposed in order to enable readers to better understand the present invention. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solution claimed by each claim of the present invention can also be realized.

Example 1: Synthesis of NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX

Weigh Lu(CF ₃ COO) ₃ 0.78mmol, Yb(CF ₃ COO) ₃ 0.2mmol, Er(CF ₃ COO) ₃ 0.02mmol, Na(CF ₃ COO) 3mmol, add solvent oleylamine 3mL, oleic acid 3mL, 15 mL of 1-octadecene was dissolved by heating and stirring at 90°C for 30 minutes to form a homogeneous solution, and then the water was evaporated for 30 minutes. Under the protection of nitrogen atmosphere, the temperature was raised to 330° C., kept for 60 minutes, and then cooled to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v).

Then weigh Lu(CF ₃ COO) ₃ 1mmol, Na(CF ₃ COO) 3mmol, add solvent oleic acid 6mL, 1-octadecene 15mL, heat and stir at 90°C for 30 minutes to dissolve to form a uniform solution, Then open and steam for 30 minutes. Add the solid obtained by centrifugation to the solution, heat and stir at 80-100°C for 20 minutes, then raise the temperature to 330°C under nitrogen protection, react for 60 minutes, and then cool to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v), and finally the solid was ultrasonically dispersed in 10 mL of cyclohexane.

Take 1 mL of cyclohexane solution of rare earth nanomaterials, add 9 mL of CO-520 cyclohexane solution, then add 0.05 mL of ammonia water and stir for 90 minutes, then add 0.02 mL of tetraethoxysilane and stir for 12 hours, then add N-(2 -Aminoethyl)-3-aminopropyltrimethoxysilane 0.01mL, stirred for 12 hours, then added tetraethoxysilane 0.05mL and stirred for 12 hours. Then add 5 mL of acetone, centrifuge at 15,000 rpm for 10 minutes, collect the precipitated solid, add 10 mL of ethanol to wash the precipitate three times, and disperse the precipitate in 10 mL of ethanol. Then 5 mL of deionized water and 5 mL of 1% by mass hydrofluoric acid aqueous solution were added. After stirring for 30 minutes, centrifuge at 15,000 rpm, collect the centrifuged solid after 10 minutes, wash with 5mL ethanol three times, and finally disperse in 10mL ethanol.

Measure 2 mL of the obtained ethanol solution, add it to 5 mL of chloroform solution containing 0.5 mg PdPc, stir at room temperature for 20 minutes, then rotary evaporate the chloroform, wash the solid three times with 10 mL of ethanol, and finally disperse the solid in 5 mL in water;

Mix 10 mg of DPPC and 0.1 mg of DOX molecules in 10 mL of chloroform, and form a film in a flask using rotary evaporation. Add the obtained solution into the flask containing the membrane, use ultrasonic treatment for 10 minutes, and use centrifugation at 15,000 rpm for 10 minutes. After collecting the precipitated solid, add 10 mL of deionized water to wash the precipitate three times, and disperse the precipitate in 10 mL of deionized water.

The morphology, crystal structure and absorption spectrum results of the upconversion luminescence-thermotherapy composite nanoprobe are shown in Figures 1-3.

The size of the NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX material is 48nm, and the rare earth up-conversion luminescent material is a hexagonal crystal phase, and there is palladium phthalocyanine absorption in the near infrared 600-900nm peak.

Example 2: Synthesis of NaY _0.78 Yb _0.2 Er _0.02 F ₄ @NaYF ₄ @SiO ₂ -ICG@PNIPAm-PTX

Weigh Y(CF ₃ COO) ₃ 0.78mmol, Yb(CF ₃ COO) ₃ 0.2mmol, Er(CF ₃ COO) ₃ 0.02mmol, Na(CF ₃ COO) 3mmol, add solvent oleylamine 3mL, oleic acid 3mL, 15 mL of 1-octadecene was dissolved by heating and stirring at 90°C for 30 minutes to form a homogeneous solution, and then the water was evaporated for 30 minutes. Under the protection of nitrogen atmosphere, the temperature was raised to 330° C., kept for 60 minutes, and then cooled to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v).

Then weigh Y(CF ₃ COO) ₃ 1mmol, Na(CF ₃ COO) 3mmol, add solvent oleic acid 6mL, 1-octadecene 15mL, heat and stir at 90°C for 30 minutes to dissolve to form a uniform solution, Then open and steam for 30 minutes. Add the solid obtained by centrifugation to the solution, heat and stir at 80-100°C for 20 minutes, then raise the temperature to 330°C under nitrogen protection, react for 60 minutes, and then cool to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v), and finally the solid was ultrasonically dispersed in 10 mL of cyclohexane.

Take 1 mL of cyclohexane solution of rare earth nanomaterials, add 9 mL of CO-520 cyclohexane solution, then add 0.05 mL of ammonia water and stir for 90 minutes, then add 0.02 mL of tetraethoxysilane and stir for 12 hours, then add N-(2 -Aminoethyl)-3-aminopropyltrimethoxysilane 0.01mL, stirred for 12 hours, then added tetraethoxysilane 0.05mL and stirred for 12 hours. Then add 5 mL of acetone, centrifuge at 15,000 rpm for 10 minutes, collect the precipitated solid, add 10 mL of ethanol to wash the precipitate three times, and disperse the precipitate in 10 mL of ethanol. Then 5 mL of deionized water and 5 mL of 1% by mass hydrofluoric acid aqueous solution were added. After stirring for 30 minutes, centrifuge at 15,000 rpm, collect the centrifuged solid after 10 minutes, wash with 5mL ethanol three times, and finally disperse in 10mL ethanol.

Measure 2 mL of the obtained ethanol solution, add it to 5 mL of chloroform solution containing 0.5 mg ICG, stir at room temperature for 20 minutes, then rotary evaporate the chloroform, wash the solid three times with 10 mL of ethanol, and finally disperse the solid in 5 mL in water;

PNIPAm 10 mg and PTX molecule 0.2 mg were mixed in 10 mL of chloroform, and a film was formed in a flask using rotary evaporation. Add the obtained solution into the flask containing the membrane, use ultrasonic treatment for 10 minutes, and use centrifugation at 15,000 rpm for 10 minutes. After collecting the precipitated solid, add 10 mL of deionized water to wash the precipitate three times, and disperse the precipitate in 10 mL of deionized water.

Example 3: Synthesis of NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -CuPc@DPPC-GEM

Weigh Lu(CF ₃ COO) ₃ 0.78mmol, Yb(CF ₃ COO) ₃ 0.2mmol, Er(CF ₃ COO) ₃ 0.02mmol, Na(CF ₃ COO) 3mmol, add solvent oleylamine 3mL, oleic acid 3mL, 15 mL of 1-octadecene was dissolved by heating and stirring at 90°C for 30 minutes to form a homogeneous solution, and then the water was evaporated for 30 minutes. Under the protection of nitrogen atmosphere, the temperature was raised to 330° C., kept for 60 minutes, and then cooled to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v).

Then weigh Lu(CF ₃ COO) ₃ 1mmol, Na(CF ₃ COO) 3mmol, add solvent oleic acid 6mL, 1-octadecene 15mL, heat and stir at 90°C for 30 minutes to dissolve to form a uniform solution, Then open and steam for 30 minutes. Add the solid obtained by centrifugation to the solution, heat and stir at 80-100°C for 20 minutes, then raise the temperature to 330°C under nitrogen protection, react for 60 minutes, and then cool to room temperature. The same volume of ethanol was added to the cooled solution, the solid was obtained by centrifugation, and the obtained solid was washed three times with a solution of ethanol:cyclohexane (1:1 v/v), and finally the solid was ultrasonically dispersed in 10 mL of cyclohexane.

Take 1 mL of cyclohexane solution of rare earth nanomaterials, add 9 mL of CO-520 cyclohexane solution, then add 0.05 mL of ammonia water and stir for 90 minutes, then add 0.02 mL of tetraethoxysilane and stir for 12 hours, then add N-(2 -Aminoethyl)-3-aminopropyltrimethoxysilane 0.01mL, stirred for 12 hours, then added tetraethoxysilane 0.05mL and stirred for 12 hours. Then add 5 mL of acetone, centrifuge at 15,000 rpm for 10 minutes, collect the precipitated solid, add 10 mL of ethanol to wash the precipitate three times, and disperse the precipitate in 10 mL of ethanol. Then 5 mL of deionized water and 5 mL of 1% by mass hydrofluoric acid aqueous solution were added. After stirring for 30 minutes, centrifuge at 15,000 rpm, collect the centrifuged solid after 10 minutes, wash with 5mL ethanol three times, and finally disperse in 10mL ethanol.

Measure 2 mL of the obtained ethanol solution, add it to 5 mL of chloroform solution containing 0.5 mg CuPc, stir at room temperature for 20 minutes, then rotate the chloroform to evaporate, wash the solid three times with 10 mL of ethanol, and finally disperse the solid in 5 mL in water;

10 mg of DPPC and 0.5 mg of GEM molecules were mixed in 10 mL of chloroform, and a film was formed in a flask using rotary evaporation. Add the obtained solution into the flask containing the membrane, use ultrasonic treatment for 10 minutes, and use centrifugation at 15,000 rpm for 10 minutes. After collecting the precipitated solid, add 10 mL of deionized water to wash the precipitate three times, and disperse the precipitate in 10 mL of deionized water.

Example 4:

Standard curve of fluorescence emission of NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX as a function of temperature determined by temperature-variable fluorescence spectroscopy

Disperse NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX in water and prepare 2 mL of a 0.5 mg/mL solution. Use the circulating water system to change the temperature of the solution from 0°C to 90°C, and at the same time use a 980nm laser to excite, collect the emission spectrum, take two emission bands at 525nm and 545nm for emission intensity integration and ratio, and put it into the formula ln(I ₈₁₅ /I ₈₄₀ )=C+(-ΔE/kT) to obtain a standard curve of fluorescence emission as a function of temperature. As shown in Figure 4, the fluorescence intensity shows a linear correlation trend with the temperature rise.

Example 5: Drug release from NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX under mild microscopic temperature rise

Disperse NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX in water and prepare 2 mL of a 0.5 mg/mL solution. Using 50mW/cm ² of 730nm laser to continuously irradiate the solution, adjust the intrinsic temperature of NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX at 41.5°C to release DOX, at this time The apparent temperature of the solution was 37.8°C. The release observation time is 10 minutes. Also according to the fluorescence observation results of DOX, the cumulative release amount changes with time. The time points of fluorescence collection are 0, 1, 2, 3, 4, 6, 8, and 10 minutes respectively. In addition, the release of DOX from the solution of YSUCNP-PdPc@DPPC-DOX at 37°C and 37.8°C was measured as a control experiment. As shown in Fig. 5, under mild laser irradiation, the release of DOX can be achieved when the solution phase temperature rises by 0.8 °C.

Example 6: NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX for cell-level programmed combination therapy

Tetramethylazole (MTT) method was used to evaluate the effect of cell-level programmed combination therapy. MIAPaCa-2 cells were seeded into 96-well plates, and the number of cells per well was about 5× ¹⁰⁴ . After 24 hours, the cells were completely attached to the wall, and were incubated with NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX, and the cells were irradiated with 730nm laser light of different powers. After the treatment was completed, 5 μl of MTT solution with a concentration of 5 mg ml-1 was added to each well and incubated for another 4 hours. Finally, 50 μl of 10% SDS solution was added to each well. Then use the Tecan InfiniteM200 microplate reader to measure the absorbance value at 570nm in each well, and use the following formula to calculate the cell survival rate, cell survival rate (%)=(absorbance value at 570nm at the treatment group/absorbance at 570nm at the control group value) 100%. As shown in the schematic diagram of programmed combined therapy in Figure 6, cells are given two instructions, instruction 1 is to receive chemotherapy, and instruction 2 is to receive photothermal therapy. By detecting the microscopic temperature, the 730nm laser is adjusted. Under low laser power , realize the operation of instruction 1, and realize the operation of instruction 2 under high laser power. As shown in Figure 7, under the conditions of the 730nm laser irradiation power density involved in the present invention, the best cancer cell killing effect can only be achieved under the condition of low-power and then high-power irradiation, that is, programmed combined therapy.

Example 7: NaLu _0.78 Yb _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX for small animal hierarchical programmed combination therapy

MIA PaCa-2 cells were injected into the subcutaneous 2mm tissue of four-week-old Balb/c mice at a dose of 10 ⁸ cells per mouse, and after 15 days a large tumor of 0.6 cm was formed, 0.2 mL of NaLu _0.78 Yb with a concentration of 2 mg/mL _0.2 Er _0.02 F ₄ @NaLuF ₄ @SiO ₂ -PdPc@DPPC-DOX phosphate buffer was injected into the mouse vein, and after 12 hours, according to the guidance results of photothermal treatment temperature monitoring, the tumor of the mouse was treated with 730nm laser The area was irradiated step by step, and the regression of the tumor was observed. As shown in Figure 8, only when the 730nm laser of 46mW/cm ² and 140mW/cm ² irradiated the tumor area of the mouse step by step, it was programmed. Tumor regression occurred in mice treated with the combination.

Although the invention has been described in detail with preferred embodiments, it is not intended to limit the invention. Any person skilled in the art should be able to make various modifications and changes without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be regarded as the scope defined by the appended claims.

Claims (10)

1. The up-conversion luminescence-thermochemical chemical therapy composite nanoprobe is characterized in that two layers of rare earth fluorides are taken as cores, the middle layer is a hollow silicon dioxide shell loaded with a photothermal material, and the outer layer is an organic molecular membrane loaded with a micromolecular chemical therapy drug, and the structural general formula of the up-conversion luminescence-thermochemical chemical therapy composite nanoprobe is as follows:

NaL_1-x-yYb_xEr_yF₄@NaLF₄@SiO₂-M@N-P

wherein,

l is Y or Lu atom;

x is 0.1-0.5, y is 0.01-0.20, and x + y is less than or equal to 1;

m is a photo-thermal material;

n is an organic molecular film;

p is chemotherapy micromolecular drug.

2. The upconversion luminescence-thermal chemotherapy composite nanoprobe according to claim 1, wherein in the structural general formula, x is 0.15-0.40, y is 0.01-0.05, M is a metal phthalocyanine compound, a porphyrin compound or indocyanine green, N is dipalmitoylphosphatidylcholine or poly-N-isopropylacrylamide, and P is doxorubicin, paclitaxel, gemcitabine or cisplatin.

3. The method for preparing the upconversion luminescence-thermal chemotherapy composite nanoprobe according to claim 1, comprising the following steps of:

(1) taking trifluoroacetate of L, Yb, Er and Na elements corresponding to the first layer of rare earth fluoride, stirring the trifluoroacetate in a solvent at 80-100 ℃ to form a uniform solution, and then steaming part of water in an open port;

(2) heating the solution obtained in the step (1) to 290-330 ℃ under the protection of nitrogen for reaction, and cooling after the reaction;

(3) adding ethanol into the reaction liquid obtained in the step (2), and then separating and washing the obtained solid;

(4) taking trifluoroacetate of L and Na elements corresponding to the second layer of rare earth fluoride, stirring the trifluoroacetate in a solvent at 80-100 ℃ to form a uniform solution, and then, opening the solution to evaporate part of water;

(5) mixing the solution obtained in the step (4) and the solid obtained in the step (3), stirring at 80-100 ℃, heating to 290-330 ℃ under the protection of nitrogen for reaction, and cooling after reaction;

(6) adding ethanol into the reaction liquid obtained in the step (5), separating out the obtained solid, washing, and finally ultrasonically dispersing the obtained solid in cyclohexane to obtain a cyclohexane solution of the rare earth nano material;

(7) adding the cyclohexane solution of the rare earth nano material into a CO-520 cyclohexane solution, adding ammonia water, uniformly stirring, then sequentially adding tetraethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane and tetraethoxysilane, and stirring for 10-14 hours respectively after adding; then adding acetone, centrifugally separating precipitated solid, washing and dispersing in ethanol; adding water and hydrofluoric acid aqueous solution, stirring uniformly, performing centrifugal separation to collect solid, washing again, and dispersing in ethanol to obtain ethanol solution;

(8) adding the ethanol solution obtained in the step (7) into a trichloromethane solution containing a photo-thermal material, uniformly stirring, evaporating to remove trichloromethane, and washing the obtained solid and dispersing in water;

(9) mixing the film-forming organic molecules and the chemotherapy micromolecular medicines in chloroform, and forming a film in a container; adding the solution obtained in the step (8) into a container containing a membrane, performing ultrasonic treatment for 10-20 minutes, collecting precipitated solid by centrifugal separation, and washing the separated solid, or dispersing the solid in water.

4. The production method according to claim 3,

in the step (1), the dosage of each element is calculated according to the stoichiometric ratio in the structural general formula, and the water is steamed for 30-60 minutes in an open way; the solvent is oleic acid, oleylamine, 1-octadecene or stearic acid;

in the step (2), the reaction time is 30-90 minutes;

in the step (3), the using amount of ethanol is the same as the volume of the reaction solution, and the obtained solid is washed for 2-3 times by adopting a mixed solution of ethanol and cyclohexane;

in the step (4), the dosage of each element is calculated according to the stoichiometric ratio in the structural general formula, and the water is steamed for 30-60 minutes in an open way; the solvent is oleic acid, oleylamine, 1-octadecene or stearic acid;

in the step (5), the reaction time is 30-90 minutes;

in the step (6), the using amount of ethanol is the same as the volume of the reaction solution, and the obtained solid is washed for 2-3 times by adopting a mixed solution of ethanol and cyclohexane; the dosage of cyclohexane in the cyclohexane solution of the rare earth nano material is 2-6 ml/mmol calculated by the element L.

5. The preparation method according to claim 3, wherein in the step (7), the volume ratio of the cyclohexane solution of the rare earth nanomaterial, the CO-520 cyclohexane solution, the ammonia water, the tetraethoxysilane added for the first time, the N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, the tetraethoxysilane added for the second time, the acetone, the ethanol used for the first dispersion, the water, the hydrofluoric acid aqueous solution, and the ethanol used for the second dispersion is 1: 5-20: 0.02-0.1: 0.01-0.05: 0.01-0.02: 0.05-0.1: 3-15:

5-10: 5-10: 5-10: 5-10, adopting a rotation speed of 10000-.

6. The production method according to claim 3,

in the step (8), the volume ratio of the ethanol solution to the chloroform solution to the dispersing water is 1-10: 5-10: 5-10; the content of the photothermal material in the trichloromethane solution is 0.1-3 mg/5-10 mL, and the solid obtained after the trichloromethane is evaporated is washed by ethanol;

in the step (9), the mass ratio of the film-forming organic molecules to the chemotherapeutic micromolecular drugs is 10-20: 0.1-1, adopting 10000-15000 r/min rotation speed to separate for 10-20 min in the centrifugal separation process, washing the centrifugally separated solid with deionized water for three times to precipitate, and dispersing the precipitate in the deionized water.

7. Use of the upconversion luminescence-thermal chemotherapy composite nanoprobe of claim 1 in combination with the procedural manipulation of cancer treatment.

8. The use of claim 7, wherein the upconversion luminescence-thermal chemotherapy composite nanoprobe is dispersed in water to form an aqueous dispersion with a concentration of 0.5-5 mg/mLThe power density is 20-200 mW/cm²Irradiating near-infrared laser with the wavelength of 700-1000nm to realize microscopic temperature rise of the probe material in an aqueous solution at 3-10 ℃; under the irradiation of 700-1000nm near-infrared laser, the integrated intensity ratio of two emitted emission bands of the composite nano probe material conforms to the Boolean distribution.

9. The use according to claim 7, wherein the upconversion luminescence-thermochemical therapy composite nanoprobe or the upconversion luminescence-thermochemical therapy composite nanoprobe phosphate buffer solution dispersion with the concentration of 0.5-5 mg/mL is incubated with cancer cells with the power density of 20-200 mW/cm²And step-by-step irradiation is carried out on near-infrared laser with the wavelength of 700-1000nm, and the microscopic temperature distribution imaging in the cells marked by the composite nano probe and the step-by-step control of the temperature rise of the composite nano probe by 3-10 ℃ are realized through a scanning confocal fluorescence microscope, so that the step-by-step implementation of chemotherapy and photothermal therapy is realized.

10. The use according to claim 7, wherein the phosphate buffer solution of the upconversion luminescence-thermochemical therapy composite nanoprobe with the concentration of 0.1-10 mg/mL is injected into the vein of the tumor-bearing mouse, and after 2-24 hours, according to the guidance result of monitoring the photothermal therapy temperature, 20-200 mW/cm is used²The 700-1000nm laser irradiates the tumor area of the mouse step by step, the temperature of the nanoprobe is controlled to rise by 3-10 ℃ under the monitoring of up-conversion luminescence, and the step by step of chemotherapy and photothermal therapy is realized.