CN107998394B

CN107998394B - Novel nanoparticle-photosensitizer coupling system for X-ray excitation photodynamic therapy of deep tumor and preparation method thereof

Info

Publication number: CN107998394B
Application number: CN201711340433.1A
Authority: CN
Inventors: 卢虹冰; 张文立; 石峰; 范黎; 沈颖莉; 张晓峰; 戎军艳; 刘天帅; 高鹏
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2020-09-11
Anticipated expiration: 2037-12-14
Also published as: CN107998394A

Abstract

The invention provides a novel nanoparticle-photosensitizer coupling system for treating deep tumor by X-ray excitation photodynamic therapy, which converts β -NaGdF4 into X% Tb³⁺The nanoparticles are modified by AEP, EDC is added as an activator, and the luminescent nanoparticles and a hydrophilic photosensitizer RB are covalently coupled in a single step to obtain the fluorescent nanoparticles, wherein X is 3-18. The result of co-culture of the coupling system and the liver cancer cells shows that the coupling system can enter the cells in a classical endocytosis mode and stably stay in the small bubble structures such as endosomes/lysosomes and the like, and when low-dose X rays are used for irradiation, the coupling system plays a very remarkable inhibiting role in the growth of tumors, so that the targeted treatment of deep tumors is realized on the premise of reducing toxic and side effects.

Description

Novel nanoparticle-photosensitizer coupling system for X-ray excitation photodynamic therapy of deep tumor and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a novel Nanoparticle (NPs) -photosensitizer coupling system for X-ray excitation photodynamic treatment of deep tumors, and a preparation method and application of the system.

Background

Photodynamic Therapy (PDT) is performed by administering a photosensitizer intravenously, allowing the photosensitizer to selectively accumulate in a cancer lesion site after 4-48 hours, and exciting the photosensitizer by a light source with a specific wavelength, wherein the photosensitizer molecules absorb photons to generate a large amount of Reactive Oxygen Species (ROS) to kill cells at the lesion site. Compared with the traditional surgery, radiotherapy and chemotherapy, the photodynamic therapy has controllable local phototoxicity, is safe and noninvasive, has small side effect, and is widely used for clinical treatment of various malignant solid tumors. However, because the penetration depth of ultraviolet-visible light in human tissues is limited, the ultraviolet-visible light is difficult to develop in large or deep tumors at present, and the clinical application is greatly limited.

In recent years, with the development of X-ray Excited nanophosphors (XLNPs), a new type of photodynamic therapy, X-ray Excited photodynamic therapy (XE-PDT) has brought a new era for PDT of deep tumors. The principle is that XLNP enriched at the tumor part generates visible light after being excited by X-ray, and the visible light is used as an internal light source to excite a photosensitizer coupled with the XLNP and generate ROS to promote the apoptosis of tumor cells, thereby realizing the treatment of cancer. Therefore, XLNP with high matching of luminescence spectrum and photosensitizer absorption spectrum, good biosafety and high light yield is developed, and the coupling of XLNP and the mature photosensitizer is a key problem in X-ray excitation photodynamic therapy.

Rare earth fluorides of alkali metals (AReF)₄A is an alkali metal, and Re is a rare earth metal) compared with other X-ray excited nanomaterials, because of the characteristics of more stable lattice structure, controllable size, lower photon Energy state, wider Energy Gap (EG), insusceptibility to the influence of the surrounding biological environment, and the like, the materials have high-efficiency and stable luminescent performance, and adjustable high Permeability and Retention (EPR) effect. In addition, the rare earth metal ions have rich energy level structures, and the purpose of regulating and controlling the light-emitting spectrum can be achieved by changing the doped rare earth ions. Nano Letters in 2015The research on the method is that rare earth luminescent nano-particles are combined with photosensitizer MC540 approved by FDA, local X-ray irradiation is adopted for 0.5 hour for 0.5Gy radiation dose (16.7mGy/min), the photosensitizer can be effectively excited to generate ROS, 60% of tumor cells (Hongmin Chen, etc. Nanoscintet-medical X-ray induced photosensitizer Therapy for In Vivo Cancer treatment. Nanoleters.2015, 15,2249-₄:Eu³⁺Successfully applied to experimental animals and used for in vivo X-ray excitation luminescence imaging (Wenli Zhang, etc. sub-10 nm Water-Dispersible β -NaGdF4: X% Eu)³⁺Nanoparticles with EnhancedBiocompatibility for in Vivo X-ray Luminescence Computed Tomography.ACSApplied materials&Interface.2017,9(46), pp 39985-₄:X％Eu³⁺The emission spectrum of the nanoparticles is not matched with the absorption spectrum of RB, so that efficient XE-PDT treatment cannot be realized. The proposal of the invention effectively solves the practical problem.

Disclosure of Invention

In view of the deficiencies of the prior art, it is a primary object of the present invention to provide a novel Nanoparticle (NPs) -photosensitizer coupled system for low dose X-ray excitation photodynamic therapy of deep tumors.

To achieve the object of the present invention, the inventors have conducted NaGdF₄The intensive research on the series of luminescent nanoparticles surprisingly found that Tb is doped by changing the doped rare earth ions³⁺The emission spectrum of the red fluorescent material is highly matched with the absorption spectrum of the mature second-generation photosensitizer Rose Bengal (RB), so that more ROS are generated, the radiation dose of X rays is greatly reduced, and the aim of the invention is fulfilled.

Specifically, the technical scheme of the invention is summarized as that a novel nanoparticle-photosensitizer coupling system for treating deep tumor by X-ray excitation photodynamic therapy is characterized in that the system is a down-conversion β -NaGdF4, and X% Tb is used³⁺The nano-particles are modified by AEP, EDC is added as an activator, and the luminescent nano-particles and the hydrophilic photosensitizer are covalently coupled in a single step to obtain the nano-particles,wherein X is 3-25, AEP is 2-Aminoethylphosphonic Acid (AEP), EDC is 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (N- (3-dimethylamino propyl) -N-ethylcarbodiimide hydrochloride, EDC), and the hydrophilic photosensitizer is a photosensitizer having absorption at 490-540 nm.

It should be noted that, in the experiment, it was found that β -NaGdF was used₄:X％Tb³⁺The nanoparticles are modified after ligand exchange by the surface oleic acid group AEP, so that the nanoparticles have good water solubility and biological safety, and cancer cells can be inhibited or killed more safely and efficiently under the irradiation of lower-dose X rays.

Further preferably, the novel nanoparticle-photosensitizer coupled system for deep tumor treatment by X-ray excitation photodynamic therapy is described above, wherein X is preferably 3-18, and further preferably 7-15.

In a most preferred test treatment group of the present invention, a novel nanoparticle-photosensitizer coupled system for deep tumor treatment by X-ray excitation photodynamic therapy is described above, wherein X is 15.

In a most preferred test treatment group of the present invention, a novel nanoparticle-photosensitizer coupled system for deep tumor treatment by X-ray excitation photodynamic as described above, wherein the hydrophilic photosensitizer is Rose Bengal, MC540 or/and other photosensitizer with absorption at 490-540 nm.

Further preferred is the novel nanoparticle-photosensitizer coupled system for X-ray excitation photodynamic therapy of deep tumors as described above, wherein β -NaGdF₄:X％Tb³⁺The mass ratio of AEP to EDC is 1: (3-5): (0.8-2).

Still further preferably, the novel nanoparticle-photosensitizer coupled system for X-ray excitation photodynamic therapy of deep tumors as described above, wherein β -NaGdF₄:X％Tb³⁺The mass ratio of AEP to EDC to rose bengal is 1: (3-5): (0.8-2): (0.04-0.06).

In addition, the invention also provides a preparation method of the novel nanoparticle-photosensitizer coupling system for treating deep tumors by X-ray excitation photodynamic, which comprises the following steps:

(1) taking β -NaGdF₄:X％Tb³⁺Dispersing the nanoparticles and AEP in an alcohol solution, mixing to form a uniform solution, stirring at room temperature for reaction for 12-36 hours, and centrifuging to obtain AEP modified nanoparticles;

(2) mixing the AEP modified nanoparticles and a photosensitizer rose bengal in water, adding EDC as an activating agent, and stirring and reacting at room temperature for 6-18 hours;

(3) and (3) centrifuging the reaction solution obtained in the step (2) at a high speed of 12000-14000 rpm, separating out precipitates in the residual solution, and purifying the precipitates to obtain a target product.

Further preferably, the method for preparing the novel nanoparticle-photosensitizer coupled system for deep tumor X-ray excitation photodynamic therapy as described above, wherein the alcohol solution in step (1) is an ethanol solution with a volume fraction of 50% -70%.

The NaGdF of the invention₄:15％Tb³⁺The co-culture of the luminescent nanoparticle-RB coupled system and the liver cancer cell (HepG-2) shows that the coupled system can enter the cell through a classical endocytosis mode and stably stay in the vesicular structures such as endosome/lysosome and the like. When NaGdF₄:15％Tb³⁺The concentration of the luminescent nanoparticle-RB coupling system solution is 500 mug/mL, the singlet oxygen generation amount reaches a peak value when the X-ray is irradiated for 100 seconds, and the singlet oxygen generation amount is maintained at the peak value level along with the extension of the irradiation time. Through tumor-bearing nude mouse experiments, the nanoparticle-photosensitizer coupled system can be detected to show obvious treatment effects under different X-ray irradiation times, and plays a very obvious role in inhibiting the growth of tumors. Therefore, the invention also provides the application of the novel nanoparticle-photosensitizer coupling system in the preparation of a medicament for treating tumors by X-ray excitation photodynamic therapy. Further preferably, the tumor is a deep tumor including liver cancer, and a superficial tumor.

Compared with the prior art, the novel nanoparticle-photosensitizer coupling system has the following advantages and progressiveness when being used for treating tumors by X-ray excitation photodynamic therapy:

(1) β -NaGdF of the invention₄:X％Tb³⁺The emission spectrum of the nanoparticles is highly matched with the absorption spectrum of a mature second-generation photosensitizer RB, more ROS can be generated after the nanoparticles are irradiated by X rays, and more than 80% of liver cancer cells can be effectively killed, so that the growth of tumors is effectively inhibited, and the targeted therapy of the deep tumors by applying the coupling system is realized for the first time.

(2) β -NaGdF of the invention₄:X％Tb³⁺The RB coupled system requires very low doses of X-ray radiation in the photodynamic treatment of deep tumours, with an average dose of 0.25Gy per hour measured using an ionization chamber.

(3) β -NaGdF of the invention₄:X％Tb³⁺The RB coupling system has better biological safety, and the in vitro cell experiment and the in vivo mouse experiment both verify that the system has no obvious toxicity.

Drawings

FIG. 1 shows β -NaGdF₄:X％Tb³⁺Schematic diagram of construction of RB coupling system and its therapeutic mechanism.

FIG. 2 shows β -NaGdF₄:X％Tb³⁺NPs preparation scheme is shown schematically.

FIG. 3 shows β -NaGdF₄:15％Tb³⁺TEM image of NPs.

FIG. 4 shows β -NaGdF₄:X％Tb³⁺XRD pattern of NPs.

FIG. 5 shows different Tb³⁺Doping ratio of β -NaGdF₄:X％Tb³⁺Graph comparing light yield.

FIG. 6a shows the measurement of β -NaGdF by fluorescence spectroscopy₄:15％Tb³⁺The emission spectrum and RB absorption spectrum of the luminescent nanoparticles FIG. 6b is β -NaGdF measured by fluorescence spectrophotometer₄:15％Tb³⁺Comparison of spectra before and after covalent/physical coupling of luminescent nanoparticles with RB FIG. 6c is β -NaGdF₄:15％Tb³⁺And (3) comparing the luminescent nanoparticles with RB physically (left)/covalently (right) coupled real objects.

In FIG. 7, (a) is a DPBF method for determining different concentrations of β -NaGdF₄:15％Tb³⁺-graph comparing singlet oxygen production in solution of RB coupled system; (b-e) confocal laser microscope Observation coupling System and microscopeThe retention positions of the cells in the cells after the co-culture are shown in the specification, wherein (b) is blue fluorescence labeling cell nucleus, (c) is lysotraker labeling small bubble structures such as lysozyme or endosome, and the like, (d) is coupling system fluorescence, and (e) is an overlay graph of b, c and d; (f-j) detecting the cell entering condition and the cell position of the coupling system by a transmission electron microscope method.

FIG. 8 is a histogram of cell survival rate of co-culture of nanoparticle-photosensitizer coupled system and cells without X-ray irradiation for determining cytotoxicity by MTT method.

FIG. 9 is a graph showing the survival rate of cells after X-ray irradiation after the administration of the nanoparticle-photosensitizer coupled system is detected by MTT method.

FIG. 10 shows tumor weight comparison of tumor-bearing nude mice after tumor administration 4 times. Wherein, (a) is a tumor body weight comparison histogram of different administration groups; (b) the tumor bodies of different administration groups are compared with an intuitive picture.

FIG. 11 is a pathological section of the tumor cell apoptosis in each experimental group measured by the TUNNEL method.

FIG. 12 is a pathological section view of the main organs of each experimental group detected by HE staining.

Detailed Description

The invention develops a nanoparticle (β -NaGdF) with high spectral and RB coincidence, small size, regular shape, uniform particle size distribution and high light yield₄:X％Tb³⁺) And coupled with a mature photosensitizer (defined by a certain Tb)³⁺Doped β -NaGdF₄The nanometer luminescent material is taken as a substrate material and is in covalent coupling with RB), and a nanometer particle-photosensitizer coupling system with the emission spectrum of the luminescent core and the absorption spectrum of RB being highly matched is prepared. The nano luminescent material is excited by X-ray, the photosensitizer generates ROS after absorbing emitted light, and induces the apoptosis of tumor cells, the overall design is shown in figure 1, and the whole preparation and test thought is as follows:

x-ray excited down-conversion NaGdF₄:X％Tb³⁺And (3) preparing the luminescent nanoparticles.

Mainly adopts an oil heating method to prepare NaGdF₄:X％Tb³⁺The luminescent nano-particles are subjected to TEM and XRD detection, and the lattice structure, the particle morphology and the particle diameter of the luminescent nano-particles are analyzedDiameter; irradiating luminescent nanoparticles with X-rays, analyzing and researching different Tb³⁺Light yield performance of doping ratio.

2.NaGdF₄:Tb³⁺The luminescent nanoparticle is coupled with RB.

By adding prepared NaGdF₄:X％Tb³⁺The surface of the luminescent nanoparticle is subjected to AEP hydrophilic modification, so that the whole system has good biocompatibility under the condition of high light yield. EDC is added as an activating agent, and RB is stably coupled on the surface of the nano-particles in a covalent coupling (amido bond) mode.

3.NaGdF₄:X％Tb³⁺And analyzing the singlet oxygen generation amount of the luminescent nanoparticle-RB coupling system.

Irradiation of NaGdF by X-rays₄:15％Tb³⁺The nanoparticle-RB coupling system solution quantitatively analyzes the generation amount of singlet oxygen in the solution by using 1,3-diphenylisobenzofuran (1,3-diphenylisobenzofuran, DPBF).

4. And (3) testing the biological safety and endocytosis of the nanoparticle-photosensitizer coupling system.

Based on NaGdF₄:15％Tb³⁺And (4) analyzing the singlet oxygen production of the RB coupling system, co-culturing the coupling system and the liver cancer cell (HepG-2), researching the cell-entering capability of the coupling system by a confocal microscope and a cell transmission electron microscope method, and observing the cell-entering process. And (3) verifying the cytotoxicity of the coupling systems with different concentrations by adopting an MTT method.

5. And (3) evaluating a nanoparticle-photosensitizer coupling system in vitro.

The in vitro XE-PDT treatment effect of the nanoparticle-photosensitizer coupling system is detected by liver cancer cells HepG 2. The liver cancer cell HepG2 and the coupling system are co-cultured for 24 hours, so that the coupling system can effectively enter the cancer cell through the EPR effect, and the XE-PDT in-vitro treatment is carried out by irradiating X rays with different durations. Then, the survival rate of the cells is measured by using an MTT method, so that the in-vitro curative effect of the coupling system on the liver cancer cells is judged.

6. Mouse in vivo evaluation of nanoparticle-photosensitizer coupled system.

Detection of sodium by tumor-bearing nude miceThe rice granule-photosensitizer coupled system has in vivo XE-PDT treatment effect. Injecting the tumor-bearing nude mice into a coupling system in a tumor body administration mode, irradiating X rays for different time lengths in sequence, comparing the size and the quality of a tumor body, carrying out TUNNEL staining on a tumor body paraffin section, and observing the apoptosis condition of tumor cells in the tumor body of each control group. The conditions of the main organs of the nude mice in each group were observed by HE staining method to evaluate NaGdF₄:15％Tb³⁺The system toxicity and the biological safety of the luminescent nanoparticle-RB coupling system.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to be implemented according to the content of the description and the conventional technical solutions in the field, the present invention is further described in detail with reference to the following specific embodiments, which are intended to explain the present invention rather than to limit the present invention.

Example 1X-ray excited Down-conversion β -NaGdF₄:X％Tb³⁺Preparation of luminescent nanoparticles

0.3159g of GdCl were weighed out₃·6H₂O, 0.056g of TbCl₃·6H₂O and 6mL of Oleic Acid (OA), 15mL of 1-Octadecene (1-Octadecene, ODE) were added to a 100mL round bottom flask. The mixture was heated to 160 ℃ under an argon atmosphere for 60 minutes to form a uniform, transparent solution with a slight yellowish color. The heating equipment is closed, and NH dissolved in the methanol is generated when the temperature of the system is reduced to room temperature₄Slowly dropwise adding the mixed solution of F (4mmol, 0.1481g) and NaOH (2.5mmol, 0.1g) into a flask, controlling the time to be preferably 15-20 min, vigorously stirring for 60 min at room temperature, heating to 110-120 ℃, vacuumizing for 10 min, rapidly heating to 290 ℃ after the end of reaction, keeping the temperature for 1 h, cooling to room temperature after the end of reaction, centrifugally washing for 3 times by using absolute ethyl alcohol and methanol, dispersing in cyclohexane, and obtaining β -NaGdF₄:X％Tb³⁺The preparation process of the nanoparticles is shown in figure 2, and figure 3 shows β -NaGdF₄:15％Tb³⁺In a Transmission Electron Microscope (TEM) image of NPs, the NPs are seen to be uniform spheres with good dispersibility and sizes of 7-10 nm. FIG. 4 is NaGdF₄:X％Tb³⁺X-ray diffraction (XRD) pattern of (3, 5, 7,9, 12, 15, 18, 20, 22, 25), from which NaGdF was observed₄:Tb³⁺Good crystallinity, β phase crystal structure and hexagonal phase NaGdF₄The JCPDS cards of (1) are completely matched. When Tb³⁺The doping ratio of 15% is the highest light yield (as shown in fig. 5), so it is used as the material basis for the subsequent in vitro and in vivo studies.

Example 2 β -NaGdF₄:15％Tb³⁺Covalent coupling of luminescent nanoparticles with RB

Adding 6ml of absolute ethyl alcohol and 4ml of distilled water into a 50ml round-bottom flask, and weighing β -NaGdF₄:15％Tb³⁺Nanoparticles 10mg, dissolved in 1ml cyclohexane, were added to the round bottom flask. Weighing 40mg of AEP, adding the AEP into a round-bottom flask, violently stirring for 24 hours at room temperature, centrifugally washing for 3 times by using distilled water, re-dispersing in 1ml of distilled water, adding 0.05mg of photosensitizer RB and 10mg of activator EDC, stirring for 12 hours at room temperature, centrifugally washing for 3 times at high speed by using distilled water, and obtaining the nanoparticle-photosensitizer coupling system at the centrifugal speed of 13000 rpm.

Mixing RB with β -NaGdF₄:15％Tb³⁺NPs were coupled and observed β -NaGdF after X-ray excitation₄:15％Tb³⁺Respectively measuring the difference of RB absorption peak intensities of electrostatic adsorption and covalent coupling, calculating RB binding rate, selecting the optimal RB coupling mode, and obtaining β -NaGdF after X-ray excitation through experimental analysis of fluorescence spectrophotometry₄:15％Tb³⁺The emission spectrum of the NPs was highly matched to the absorption spectrum of RB (as shown in FIG. 6 a), and the measurement of RB binding rate by fluorescence spectroscopy showed that the amount of RB bound by covalent coupling was much higher than that by electrostatic adsorption, and that the bound RB amount could completely absorb β -NaGdF₄:15％Tb³⁺The NPs emitted light (as shown in fig. 6 b), and as can also be seen by visual colorimetry, the amount of covalently coupled RB was much higher than electrostatic adsorption (as shown in fig. 6 c).

Example 3 β -NaGdF₄:15％Tb³⁺Singlet oxygen production analysis of luminescent nanoparticle-RB coupled system

Coupling β -NaGdF by X-ray irradiation₄:15％Tb³⁺The method for quantitatively analyzing the generation amount of singlet oxygen in a solution of a luminescent nanoparticle-RB coupling system solution by using a 1,3-diphenylisobenzofuran (1,3-diphenylisobenzofuran, DPBF) method has the advantages that the experimental result shows that when the RB concentration in the coupling system solution is 500 mug/mL and X-ray irradiation is carried out for 100 seconds, the generation amount of singlet oxygen reaches a peak value, and the generation amount of singlet oxygen is maintained at the peak value level along with the extension of the irradiation time (as shown in figure 7 a).

Example 4: human hepatoma cell line HepG-2 cell recovery, culture and passage

The human hepatoma cell line HepG-2 was purchased from Thermo Fisher Scientific, Inc. Taking out the frozen tube, placing into 37 deg.C water bath, thawing for 2min under shaking, sterilizing the outside of the tube wall with alcohol, transferring into a superclean bench, transferring the cell in the tube into a centrifuge tube, adding 5ml of 37 deg.C preheated DMEM medium containing 10% fetal calf serum, cleaning the frozen tube for 1 time, centrifuging the centrifuge tube (1000rpm,5min), discarding the supernatant, adding 2ml of 37 deg.C preheated DMEM medium containing 10% fetal calf serum, transferring into a culture bottle, placing in 37 deg.C and 5% CO₂Incubate in a saturated humidity incubator, and digest with 0.5% trypsin and passage routinely.

Example 5: liver cancer cell in-vitro evaluation of nanoparticle-photosensitizer coupled system

Based on β -NaGdF₄:15％Tb³⁺And (3) analyzing the singlet oxygen production of the luminescent nanoparticle-RB coupling system, co-culturing the coupling system and the liver cancer cell (HepG-2), researching the cell entry capability of the coupling system by a confocal microscope and a cell transmission electron microscope method, and observing the cell entry process. The experimental results show that the coupled system can enter cells through a classical endocytosis mode and stably stay in small bubble structures such as endosome/lysosome and the like (as shown in figures 7 b-j). The MTT method verifies the cytotoxicity of the coupled system, and the experimental result shows that the coupled system is co-cultured with cells without X-ray irradiation, and even a high-concentration administration group does not show obvious cytotoxicity (as shown in figure 8). And the irradiation dose measured by using an ionization chamber is 1.17Gy after the X-ray irradiation is carried out for 1 hour,the administration group of the coupling system with the concentration of more than 500 mug/mL shows obvious cell killing effect, and the cell survival rate is less than 10 percent (as shown in figure 9). The results prove the treatment effect of XE-PDT in vitro, and provide parameters such as medicament dosage, X-ray irradiation dosage, irradiation time and the like for in vivo experiments.

Example 6: mouse in-vivo evaluation of nanoparticle-photosensitizer coupling system

The treatment effect of the nanoparticle-photosensitizer XE-PDT in vivo is investigated by tumor-bearing nude mice. The tumor-bearing nude mice are injected into a coupling system in a tumor body administration mode (1 injection is carried out every 3 days, administration with different concentrations is carried out, 100 mu L is injected into each tumor body every time), X-ray irradiation is carried out for 1 hour 12 hours after administration, the total administration is carried out for 4 times, the X-ray irradiation is carried out for 4 hours, the total irradiation dose of 4 hours is measured by using an ionization chamber and is 1Gy, and the average irradiation dose of each hour is 0.25 Gy.

Nude mice were sacrificed after 4 doses, tumor size and tumor weight were compared, and the in vivo XE-PDT treatment effect was examined. The experimental result shows that the administration group (20mg/mL) of the nanoparticle-photosensitizer coupling system has a remarkable treatment effect under different X-ray irradiation times, and the tumor weight is less than 1/10 of the normal saline control group (figure 10). Tumor paraffin sections were subjected to TUNNEL staining, and tumor cell apoptosis in the tumor of each control group was observed. The experimental results were consistent with the tumor inhibition experiments, and the administration of NP-RB to the X-ray-irradiated group resulted in the most severe apoptosis of tumor cells and the appearance of a large necrotic area (FIG. 11).

The main visceral organs of each group of nude mice are observed and treated by an HE staining method, and the system toxicity and the biological safety of the nanoparticle-coupling system are evaluated. As shown in FIG. 12, the treatment groups without X-ray irradiation were good in each organ, and no significant abnormality was observed. The spleen germinal center was slightly damaged in the nanoparticle-only group, which also occurred in the NPs-RB administration + X-ray irradiation group.

Claims

1. A nanoparticle-photosensitizer coupling system for treating deep tumor by X-ray excitation photodynamic is characterized in that the system converts down β -NaGdF4: X% Tb³⁺The nanoparticles are modified by AEP, and EDC is addedThe light-emitting nanoparticle is used as an activator and obtained by covalently coupling the light-emitting nanoparticle and a hydrophilic photosensitizer in a single step, wherein X = 3-25, AEP is 2-aminoethylphosphonic acid, EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the hydrophilic photosensitizer is a photosensitizer with absorption at the position of 490-540 nm.

2. The nanoparticle-photosensitizer coupling system for deep tumor X-ray excitation photodynamic therapy according to claim 1, wherein X = 3-18.

3. The nanoparticle-photosensitizer coupling system for photodynamic X-ray therapy according to claim 2, wherein X = 15.

4. The nanoparticle-photosensitizer coupling system for photodynamic X-ray therapy according to claim 1, wherein the hydrophilic photosensitizer is rose bengal, MC540 or/and other photosensitizers that absorb at 490-540 nm.

5. The nanoparticle-photosensitizer coupling system for deep tumor therapy with X-ray excitation photodynamic therapy according to claim 1, characterized by β -NaGdF₄:X% Tb³⁺The mass ratio of AEP to EDC is 1: (3-5): (0.8-2).

6. The nanoparticle-photosensitizer coupling system for deep tumor therapy with X-ray excitation photodynamic therapy according to claim 4, characterized in that β -NaGdF₄:X% Tb³⁺The mass ratio of AEP to EDC to rose bengal is 1: (3-5): (0.8-2): (0.04-0.06).

7. The method for preparing nanoparticle-photosensitizer coupled system for deep tumor treatment with X-ray excitation photodynamic therapy according to claim 1, wherein the method comprises the following steps:

(1) taking β -NaGdF₄:X% Tb³⁺The nanoparticles and AEP are dispersed in an alcoholic solutionMixing to form a uniform solution, stirring at room temperature for reaction for 12-36 hours, and centrifuging to obtain AEP modified nanoparticles;

(2) mixing the AEP modified nanoparticles and a hydrophilic photosensitizer in water, adding EDC as an activating agent, and stirring and reacting at room temperature for 6-18 hours;

8. The method for preparing nanoparticle-photosensitizer coupled system for deep tumor therapy with X-ray excitation photodynamic therapy according to claim 7, wherein the alcohol solution in step (1) is 50-70% by volume of ethanol solution.

9. The nanoparticle-photosensitizer coupled system of claim 1, for use in the preparation of a medicament for X-ray excitation photodynamic therapy of tumors.

10. The nanoparticle-photosensitizer coupled system of claim 9, wherein the tumor is a deep tumor including liver cancer or a superficial tumor.