CN116850294A - Composite photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof - Google Patents

Composite photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof Download PDF

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CN116850294A
CN116850294A CN202310771134.2A CN202310771134A CN116850294A CN 116850294 A CN116850294 A CN 116850294A CN 202310771134 A CN202310771134 A CN 202310771134A CN 116850294 A CN116850294 A CN 116850294A
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CN116850294B (en
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侯智尧
汪晓照
谭美玲
高智敏
万宇驰
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Guangzhou Medical University
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Abstract

The application relates to the technical field of biology, and particularly discloses a compound photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof. The compound photo-thermal reagent comprises a glucose transporter (GLUT) inhibitor and a photo-thermal material with photo-thermal conversion performance. The GLUT inhibitor can inhibit heat shock protein over-expression induced by heat stress by regulating cell metabolism, so that the ablation of the mild photothermal therapy on tumors is realized, the photothermal material can further promote the curative effect of the mild photothermal therapy by consuming the existing heat shock protein in cells, the GLUT inhibitor is combined with the photothermal nanomaterial to regulate the heat shock protein level of tumor cells, the defect of poor effect of the existing mild photothermal therapy technology is overcome, and the medication scheme for realizing heat shock protein silencing has important significance for promoting clinical transformation of the mild photothermal therapy.

Description

Composite photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof
Technical Field
The application relates to the technical field of biology, in particular to a compound photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof.
Background
Cancer is one of the most serious diseases threatening human health, and traditional methods for treating cancer include surgical excision, chemotherapy, radiotherapy, immunotherapy and the like, but the methods have certain drawbacks or limitations that make the treatment effect unexpected. Photothermal therapy (photothermal therapy, PTT) has attracted considerable attention in the field of application-based research as a highly space-time selective specific site-directed therapeutic strategy. PTT is prepared by using a nanocomposite material with a photothermal effect as a photothermal therapeutic agent (photothermal agents, PTAs), delivering the material to a solid tumor, irradiating the solid tumor with an external light source with a proper wavelength, converting light energy into heat energy to cause local overheating of the focus, causing protein denaturation, DNA damage and cell membrane destruction, realizing tumor ablation and triggering immune response to inhibit metastasis. Studies show that PTT has remarkable killing effect on solid tumors, naomi J.Halas, a nano scientist of Lesi university, reported a clinical research result of photothermal therapy on the journal of national academy of sciences in 2019 for the first time, 15 men suffering from prostate cancer with low to moderate risk received the treatment with Au@SiO 2 As photothermal ablation therapy for PTAs (NCT 02680535), 13 patients were followed one year later with no signs of cancer recurrence. The United states Food and Drug Administration (FDA) ablates this locally The Therapy is classified as an AuroLase Therapy treatment method and is standardized as a medical device, which has a milestone significance for photothermal Therapy into clinical treatment of tumors.
In order to achieve complete ablation of the tumor, PTT requires that the local temperature of the cancerous tissue be raised above 50 ℃. For safety of clinical transformations, light-induced nonspecific thermal diffusion can cause irreversible thermal damage to normal tissue surrounding the tumor, and more serious, thermal ablation can cause some adverse biological effects in cancer treatment. On the one hand, when the temperature is higher than 50 ℃, the main way in which PTT is effective is to force cell death by necrosis, which process leads to release of cell debris and intracellular biomolecules, thereby creating severe local inflammation, further damaging normal tissues and increasing the risk of tumor metastasis. PTT, on the other hand, induces dead cancer cells in the primary lesion to release tumor-associated antigens and initiate T cell-mediated immune responses, but excessive hyperthermia in the tumor microenvironment risks damaging the immune antigens, thereby making it difficult to evoke immune cells to perform immune responses. Therefore, the use of mild photothermal therapy (43-45 ℃) is of great importance for pushing PTT into clinical treatment of tumors. However, even a small temperature rise can place cancer cells in a thermally stressed state, affecting the killing effect of mild light and heat on the cells. The role of heat shock proteins (heat shock proteins, HSPs) is a major reason for their development of heat tolerance, and their expression in cancer cells is rapidly up-regulated when tumors are exposed to hyperthermia, protecting cancer cells from injury, resulting in heat resistance of the tumor. Thus, modulation of HSPs expression provides an effective strategy for enhancing the mildly PTT tumor-inhibiting effect.
Currently, various HSPs inhibitors, including small molecule inhibitors and small interfering RNAs, are used to enhance mild PTT. However, many small molecule HSPs inhibitors have been found in clinical trials to cause systemic toxicity, such as 2-phenylacetylsulfonamide, geldanamycin, VER-155008, and the like. Meanwhile, the small molecular HSPs inhibitor has the characteristics of poor solubility, serum instability and the like, so that the expression regulation and control of the HSPs are difficult to realize safely in a complex physiological environment. Nanosystems prepared by combining PTAs with HSPs inhibitors have also been used to enhance low temperature PTT, but their complex process may increase pharmacokinetic uncertainty and reduce reproducibility of clinical transformations. Reactive oxygen species (reactive oxygen species, ROS) can crosslink with primary amino structures of proteins, disrupting the structure and function of HSPs, and thus modulating intracellular ROS stress levels can reduce the heat tolerance of cancer cells. However, due to the inherent characteristics of limited ROS transmission distance and short lifetime, heat stress up-regulated HSPs cannot be thoroughly removed, and a single strategy for inhibiting HSPs is difficult to realize the regulation of HSPs when facing a complex tumor microenvironment (tumor microenvironment, TME), so that the treatment effect is poor. Thus, developing a drug regimen that achieves complete silencing of HSPs by a combination strategy is of great importance to promote clinical transformation of mild PTT.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide a compound photo-thermal reagent for tumor mild photo-thermal treatment (mPT) and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
the first object of the application is to provide a compound photo-thermal agent for tumor mild photo-thermal treatment, which comprises a glucose transporter inhibitor and a photo-thermal material with photo-thermal conversion performance.
Glucose transporter (glucose transporter, GLUT) inhibitors can inhibit glucose uptake by cancer cells and cannot provide energy for Adenosine Triphosphate (ATP) synthesis. Heat Shock Proteins (HSPs) are up-regulated by stress and the energy required for their synthesis is supplied by ATP.
ATP is an important high-energy phosphate compound that functions to store, transfer, release and utilize energy in cells and organisms. By shutting off the supply of energy-derived ATP and blocking downstream pathway hydrolytic ATP from releasing energy, inhibiting energy metabolism by cancer cells provides an efficient energy deprivation strategy for reducing cancer cell viability. However, the intended therapeutic effect cannot be achieved by inhibiting intracellular energy supply alone. Fortunately, some studies have shown that a decrease in intracellular ATP levels may sensitize tumors to other therapeutic approaches, e.g., decreasing ATP levels may reverse multi-drug resistance of chemotherapeutic drugs. More importantly, the inhibition of glucose metabolism can be caused by specific deprivation of intracellular glucose, thereby reducing ATP production. Under heat stress conditions, the power of cells expressing HSPs is derived from ATP, meaning that the efficacy of mild PTT is not affected by HSPs if ATP is produced by the glycolysis process is inhibited from its source. Most cancer cells are often glucose "addicts", which achieve high uptake and high utilization of glucose compared to normal cells, and subsequently produce large amounts of ATP via anaerobic glycolysis, enabling rapid growth and proliferation of cancer cells, which is largely dependent on their upregulated glucose transporter (glucose transporters, GLUTS). Thus, GLUTS targeting tumor cells is considered an effective strategy to inhibit tumor progression. Glucose uptake by tumor cells can be reduced by specific inhibition of GLUTS, which is the main raw material for intracellular ATP synthesis, and a decrease in glucose content necessarily results in a decrease in ATP content. Under heat stress conditions, the power of cells expressing HSPs is derived from ATP, so that the decrease of intracellular ATP will prevent HSPs synthesis, which means that if GLUTS can be effectively inhibited, HSPs synthesis can be inhibited from the source, and the efficacy of mild PTT will be further improved.
In contrast, the present application has found that the combination of a glucose transporter inhibitor and a photothermal material (either) having photothermal conversion properties can prevent the expression of heat shock proteins from increasing under the stimulation of mild light and heat (at a photothermal temperature lower than 45 ℃), thereby realizing the inhibition of tumor by mild light and heat treatment.
As a preferred embodiment of the composite photo-thermal agent for the mild photo-thermal treatment of tumors according to the application, the glucose transporter inhibitor is a molecule or material which inhibits the uptake of glucose by cells through a glucose transporter signaling pathway, preferably, the glucose transporter inhibitor comprises at least one of KL-11743, IMX-120, BAY-876, STF-31, WZB117, fatentin, DRB18, GLUT inhibitor-1, GLUT1-IN-1, GLUT4-IN-2 and Phloretin (phlorizin).
In the present application we selected a number of GLUT inhibitors to conduct experiments aimed at the inhibition of different types of GLUT proteins (GLUT 1, GLUT2, GLUT3 and GLUT 4), respectively, including WZB-117 (acting on GLUT 1), fasentin (acting on GLUT1 and GLUT 4), DRB18 (acting on GLUT1, GLUT2, GLUT3, GLUT 4), filoretin (acting on GLUT 1), BAY-876 (acting on GLUT1, GLUT2, GLUT3, GLUT 4) and KL-11743 (acting on GLUT1, GLUT2, GLUT3, GLUT 4). More preferably, KL-1174 having various GLUT inhibition functions is selected for the cell and animal level warming and photothermal experiments. KL-11743 is an effective, orally active glucose competitive class I glucose transporter inhibitor, inhibiting GLUT1, GLUT2, GLUT3 and GLUT 4. The research shows that KL-11743 has obvious inhibition effect on various cancer cells including lung cancer, can specifically block glucose metabolism and reduce tumor growth activity. All GLUT drugs which can regulate and control the energy metabolism of tumor cells can be used as GLUT inhibitors of the application.
The GLUT inhibitor KL-11743 and a photo-thermal material with photo-thermal conversion performance (not only can generate ROS, but also has photo-thermal conversion performance) are combined, so that the expression of HSPs can be more completely inhibited, and the efficient mild photo-thermal treatment effect is realized. Specifically, the GLUT inhibitor KL-11743 can reduce the uptake and utilization of glucose by tumor cells by inhibiting glucose transport proteins, so that the ATP content of the cell energy is reduced, the normal functions of the cell are interfered, the expression of Heat Shock Proteins (HSP) is inhibited, and then the tumor mild photothermal treatment effect of the photothermal material is promoted.
As a preferable embodiment of the compound photo-thermal reagent for the moderate photo-thermal treatment of tumors, the mass ratio of the glucose transporter inhibitor to the photo-thermal material with photo-thermal conversion performance is 1 (40-200).
Preferably, in the cell experiment, the mass ratio of the glucose transporter inhibitor to the photothermal material having photothermal conversion properties is 1:40. In animal experiments, the mass ratio of the glucose transporter inhibitor to the photothermal material with photothermal conversion performance is 1:200.
As a preferred embodiment of the compound photo-thermal agent for the mild photo-thermal treatment of tumors, the photo-thermal material with photo-thermal conversion performance is a material capable of converting light energy with a wavelength range of 650-1700nm into heat energy. Preferably, the photo-thermal material with photo-thermal conversion performance comprises a composite structure constructed by at least one of manganese-containing oxide, indocyanine green ICG, noble metal nanocrystalline, iron-containing oxide, carbon dots, rare earth nanocrystalline, transition metal sulfide and conjugated polymer.
Preferably, the photo-thermal material with photo-thermal conversion performance comprises the following two components: mnO in hollow mesoporous form x And rare earth nanocrystalline modified on the surface thereof; x is more than or equal to 1 and less than or equal to 2; the rare earth nanocrystalline comprises doped Nd 3+ NaYF of (F) 4 Nanocrystal cores and coating on Nd-doped material 3+ NaYF of (F) 4 CaF on the surface of nanocrystal core 2 A housing.
The photo-thermal material with photo-thermal conversion performance of the application shows catalase-like activity (CAT), oxidases-like activity (OXD) and glutathione oxidase-like activity (GSHOx) in the tumor microenvironment, and can generate Fenton-like reaction. The photothermal material induces accumulation of Lipid Peroxide (LPO) and Reactive Oxygen Species (ROS) in tumor microenvironment, depleting HSPs already present in cancer cells, promoting efficacy of mild photothermal therapy. The preparation of the protective material and the promotion of the enzyme activity thereof to mild light and heat.
MnO in the present application x The ultra-small rare earth nanocrystalline has the functions of simulating enzyme activity and medicine carrier, and has the functions of photo-thermal effect and fluorescence imaging.
As a preferred embodiment of the compound photo-thermal agent for the mild photo-thermal treatment of tumor according to the application, the Nd-doped compound photo-thermal agent 3+ NaYF of (F) 4 Nd in nanocrystal core 3+ The doping ratio of (a) is 30-80% (the amount of substance). When Nd 3+ When the doping ratio is in the above range, the photo-thermal properties are improved.
As a preferred embodiment of the compound photothermal agent for the mild photothermal treatment of tumor according to the application, the MnO having a hollow mesoporous structure x The preparation method of the (C) comprises the following steps:
1) Silicon ball SiO 2 Dispersed inAdding permanganate into pure water, and ultrasonic mixing to obtain SiO 2 @MnO x (manganese oxide coated silica) particles;
2) SiO obtained in step 1) is reacted with 2 @MnO x Adding carbonate into the particle dispersed pure water, mixing uniformly, condensing and refluxing to obtain hollow mesoporous MnO x 。
MnO in hollow mesoporous form in the photo-thermal material x Has POD nano-enzyme activity, and can catalyze high-expression hydrogen peroxide (H) 2 O 2 ) Generates hydroxyl free Radicals (ROS) with high cytotoxicity, and further denatures Heat Shock Proteins (HSP) up-regulated by heat stress in tumor cells, thereby reducing heat resistance of the tumor cells.
As a preferred embodiment of the composite photo-thermal reagent for the mild photo-thermal treatment of tumors, the preparation method of the rare earth nanocrystalline comprises the following steps:
1) Adding rare earth trifluoroacetate and sodium trifluoroacetate into a mixed solution of oleic acid, oleylamine and octadecene, heating, adding absolute ethanol for precipitation, and centrifuging to obtain Nd-doped alloy 3+ NaYF of (F) 4 A nanocrystal core;
2) Adding calcium trifluoroacetate into the mixed solution of oleic acid and oleylamine, and heating to obtain a precursor solution;
3) Will be doped with Nd 3+ NaYF of (F) 4 Mixing and heating the nanocrystal core, oleic acid and octadecene, and injecting the precursor solution obtained in the step 2) to finally obtain the rare earth nanocrystal.
The rare earth nanocrystalline has photo-thermal conversion performance, can convert 808nm near infrared light (NIR) into heat energy, can effectively raise the temperature of the environment inside and outside tumor cells to 43-45 ℃, causes killing effect on the tumor cells, and can simultaneously cause the rise of heat shock protein expression in the tumor cells.
As a preferred embodiment of the compound photo-thermal reagent for the mild photo-thermal treatment of tumors, the preparation method of the photo-thermal material with photo-thermal conversion performance comprises the following steps:
1) Will take the form of a middle schoolMnO of hollow mesoporous x Dispersing in absolute ethanol, adding 3-aminopropyl triethoxysilane to obtain-NH 2 Modified MnO x A solution;
2) Fully and uniformly mixing 2-bromoisobutyric acid, citric acid, chloroform and N, N-dimethylformamide, dropwise adding a chloroform solution of rare earth nanocrystalline, and carrying out ultrasonic mixing to obtain a-Br modified nanocrystalline solution;
3) the-NH produced in step 1) is reacted with 2 Modified MnO x Mixing the solution with the-Br modified nanocrystalline solution prepared in the step 2) to prepare the photo-thermal material with photo-thermal conversion performance.
The photo-thermal material (H-MnO) prepared by the application x @ Nd-NCs) is abbreviated as MN, which comprises MnO in the form of hollow mesopores x And nanocrystalline, proved by experiments, in GSH and H 2 O 2 In the coexistent condition, the photo-thermal reagent can generate ROS to fade MB solution, has POD nano-enzyme activity, and the generated ROS content is related to GSH content; and the MN can detect a characteristic signal under the action of a specific capture agent of the hydroxyl radical, which indicates that the specific type of ROS generated by the MN is the hydroxyl radical.
As a preferred embodiment of the method for producing a composite photo-thermal reagent of the present application, the-NH group 2 Modified MnO x The mass ratio of the solution to the solution of the Br modified nanocrystalline is 2:1.
The second purpose of the application provides application of the compound photo-thermal reagent in preparing a heat shock protein inhibitor.
As a preferred embodiment of the use according to the application, the glucose transporter inhibitor in the complex photothermal agent inhibits heat shock protein overexpression induced by heat stress by modulating cellular metabolism.
Glucose transporter (GLUT) inhibitors can inhibit Heat Shock Protein (HSPs) overexpression induced by heat stress by regulating cell metabolism, so as to realize ablation of the ptt on tumors.
The third object of the application is to provide the application of the compound photo-thermal reagent in preparing medicaments for promoting tumor mild photo-thermal treatment.
As a preferred embodiment of the application of the application, the temperature of the gentle photo-thermal is 42-45 ℃.
The photothermal material (with enzyme activity) with the composite structure can further promote the curative effect of mild photothermal therapy (mPT) by consuming the existing heat shock protein in cells. The combined strategy regulates the level of the heat shock protein of the cancer cells, overcomes the defect of poor curative effect caused by the prior single technology, and has important significance for promoting the clinical transformation of the mPT.
The photothermal material prepared by the application has nano enzyme activity and is used for mild photothermal treatment of tumors. The independent 808nm laser has no killing effect on tumor cells, the killing effect of the tumor cells of the photo-thermal material is obviously improved under the irradiation of 808nm laser, and the killing effect of the tumor cells of the photo-thermal material is further improved after the GLUT inhibitor KL-11743 is combined, which proves that the GLUT inhibitor KL-11743 is effectively promoted for the mild photo-thermal treatment of tumors.
And the GLUT inhibitor KL-11743 and the photo-thermal material are combined, so that the generation of HSP70 in tumor cells is greatly inhibited, and the GLUT inhibitor KL-11743 and the photo-thermal material are very important for promoting the low-temperature photo-thermal treatment effect of tumors.
Compared with the prior art, the application has the following beneficial effects:
the application provides a compound photo-thermal reagent for mild photo-thermal treatment of tumors and application thereof, GLUT inhibitor can inhibit Heat Shock Protein (HSPs) over-expression induced by heat stress by regulating cell metabolism, so as to realize ablation of mPT on the tumors, photo-thermal material can further promote mPT curative effect by consuming existing HSPs in cells, GLUT inhibitor is combined with photo-thermal material to regulate and control the HSPs level of cancer cells, the defect of poor single treatment effect in the prior art is overcome, and the drug scheme for realizing complete silencing of HSPs has important significance for promoting clinical transformation of mild PTT.
Drawings
FIG. 1 is H-MnO x A crystalline phase of (2);
FIG. 2 is a crystalline phase of Nd-NCs;
FIG. 3 is H-MnO x XPS test chart of medium manganese element;
FIG. 4 is H-MnO x A preparation flow chart of Nd-NCs (MN);
FIG. 5 shows a photo-thermal material H-MnO x Transmission electron microscopy image (TEM, japanese electronics Co., ltd., scale bar 100 nm) of Nd-NCs (MN), wherein FIG. 5-a is H-MnO x The method comprises the steps of carrying out a first treatment on the surface of the FIG. 5-b is Nd-NCs; FIG. 5-c is H-MnO x @Nd-NCs)；
FIG. 6 shows a photo-thermal material H-MnO x Element profile of @ Nd-NCs (MN);
FIG. 7 is a graph showing the results of POD nano-enzyme activity test and EPR test of the photo-thermal material H-MnOx@Nd-NCs (MN) of example 2;
FIG. 8 shows a photo-thermal material H-MnO x Graph of temperature change over time for an aqueous solution of Nd-NCs (MN) under 808nm laser irradiation;
FIG. 9 is a graph showing the results of changes in intracellular glucose content and intracellular ATP content of tumor cells CT26 under the action of GLUT inhibitors at different concentrations;
FIG. 10 is a graph showing the results of the survival of tumor cells in the different experimental groups of example 5;
FIG. 11 is a graph showing the results of the death of the tumor cells of example 5 after various treatments;
FIG. 12 is a graph showing the results of Western Blot (Western Blot) assay of intracellular HSP70 expression levels in example 5;
FIG. 13 shows that GLUT inhibitor KL-11743 of example 6 faces H-MnO in vivo x The results of the experimental results are shown in the verification of the promotion of mild photothermal treatment of Nd-NCs (MN) tumors.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
The following examples relate to the main pharmaceutical agents including: tetraethyl orthosilicate (TEOS), ammonia water, pure water, absolute ethyl alcohol, potassium permanganate (KMnO) 4 ) Sodium carbonate, oleic Acid (OA), oilAmine (OM), octadecene (ODE), neodymium oxide (Nd) 2 O 3 ) Yttria (Y) 2 O 3 ) Trifluoroacetic acid, sodium trifluoroacetate (CF) 3 COONa), calcium trifluoroacetate (Ca (CF) 3 COO) 2 ) 3-aminopropyl triethoxysilane (APTES), 2-bromoisobutyric acid (BMPA), citric acid, chloroform, N-Dimethylformamide (DMF), cyclohexane, methylene Blue (MB), naHCO 3 Reduced Glutathione (GSH), H 2 O 2 (9.8M), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), PBS buffer (ph=6.5), liperfluo probe, glucose detection kit, ATP detection kit, BCA protein quantification kit, thiazole blue (MTT), dimethyl sulfoxide (DMSO), calcein AM/PI probe.
Example 1 photothermal material H-MnO having nanoenzyme Activity x Synthesis of @ Nd-NCs (MN)
(1)H-MnO x (MnO in hollow mesoporous form) x ) Is synthesized by the following steps:
a. silicon ball SiO 2 Is synthesized by the following steps:
25mL of absolute ethyl alcohol, 0.5mL of ultrapure water and 1.8mL of ammonia water are taken and stirred uniformly in a single-neck flask, then stirred uniformly at 40 ℃ through a magnetic stirring water bath, 0.75mL of tetraethyl orthosilicate (TEOS) is rapidly injected, and stirring is continued overnight. Alternately washing with absolute ethanol and ultrapure water to obtain uniform SiO with particle size of about 150nm 2 。
b.SiO 2 @MnO x Is synthesized by the following steps:
the SiO obtained above is treated 2 Dispersing in 5mL ultrapure water, adding into KMnO solution 600mg 4 Ultrasonic for 2h in 100mL of ultrapure water, stirring overnight, and centrifugally washing with deionized water for three times to obtain SiO 2 @MnO x 。
c.H-MnO x Is synthesized by the following steps:
dispersing the particles in 20mL of ultrapure water, taking 10mL of the ultrapure water, adding 25mL of pure water and 6.36g of sodium carbonate, fully dissolving and uniformly mixing, keeping the temperature at 60 ℃ for condensation reflux for 12 hours, and finally washing with deionized water for three times for standby.
(2)NaYF 4 :Nd 3+ @CaF 2 Synthesis of (Nd-NCs) nanocrystals: first, synthesizing monodisperse NaYF in a system where Oleic Acid (OA), oleylamine (OM) solvent provides high temperature environment for surface ligand, octadecene (ODE) 4 :Nd 3+ Nanocrystal core (doped Nd) 3+ Is a NaYF4 nanocrystal core). Weigh 10mmol Nd 2 O 3 And 10mmol Y 2 O 3 Respectively placing the mixture into a 250mL three-neck flask, respectively adding 10mL deionized water and 10mL trifluoroacetic acid, heating the solution to 100 ℃, and introducing nitrogen when the solution is clear and transparent, so that the mixed solution is completely volatilized, and obtaining white solid powder, namely the rare earth trifluoroacetate (Nd-TFA and Y-TFA). Subsequently, 0.8mmol of Nd-TFA, 0.2mmol of Y-TFA and 1mmol of Na-TFA (CF) 3 COONa) was dissolved in a mixed solution of 3.2mL OA, 3.2mL OM and 6.4mL ODE, and the solution was heated to 120 ℃ for 30min to remove oxygen and moisture. Then heating to 310 ℃ for reaction for 30min, and continuously introducing nitrogen to prevent the reactants from being oxidized in the whole reaction process. After the reaction is finished, naturally cooling to room temperature, adding excessive absolute ethanol for precipitation, and centrifuging to obtain NaYF 4 :Nd 3+ The nanocrystalline is dispersed in 10mL of cyclohexane for standby; then, ca (CF 3 COO) 2 Precursor coated inert CaF 2 A layer. Ca (CF) 3 COO) 2 The precursor synthesis method comprises the following steps: after synthesizing Ca-TFA solid powder in the same manner as above, 4mmol of Ca-TFA was dissolved in a mixed solution of 3.2mL of OA and 3.2mL of ODE, and nitrogen was introduced and the temperature was raised to 120℃for 30 minutes to obtain Ca-TFA-OA precursor solution (0.625 mol/mL). 5mL of the above synthesized NaYF was taken 4 :Nd 3+ Nanocrystalline, 10mL OA and 10mL ODE to three-neck flask, the solution was heated to 120℃and held for 30min. Then the temperature of the solution is increased to 310 ℃, after the temperature is stabilized, 1mL of Ca-TFA-OA precursor solution is injected for reaction for 30min, and then excessive absolute ethyl alcohol is added for centrifugation to obtain NaYF 4 :Nd 3+ @CaF 2 (Nd-NCs) nanocrystals.
(3)H-MnO x Synthesis of @ Nd-NCs (MN):
(a)H-MnO x of (2) NH 2 Modification: 10mg of H-MnO is taken x After washing with alcohol, the mixture was dispersed in 25mL of absolute ethanol, 1mL of APTEs was added thereto, and the mixture was stirred overnight, and the mixture was washed with absolute ethanol three timesStandby;
(b) -Br modification of Nd-NCs: 0.5g BMPA,0.05g citric acid, 6mL chloroform and 7mL DMF are taken and fully dissolved and evenly mixed, then 1mL Nd-NCs chloroform solution (0.1M) is added dropwise, the mixture is stirred overnight after ultrasonic treatment for 20min, and then the mixture is added with excessive cyclohexane for centrifugal washing and dispersed in 1mL absolute ethanol to obtain Nd-Br alcohol solution.
(c)H-MnO x Compounding with Nd-NCs: 5mg of H-MnO was added to 15mL of absolute ethanol x -NH 2 And 0.25mL Nd-Br alcohol solution, and then dispersing in ultrapure water after washing with absolute ethyl alcohol three times to obtain H-MnO x The @ Nd-NCs (MN) water dispersed solution. The flow is as in fig. 4.
H-MnO x The Nd-NCs crystal phase is shown in figures 1-2, H-MnO x XPS test patterns of the medium manganese element are shown in FIG. 3.
Referring to FIGS. 5-6, the photothermal material is morphologically characterized, FIG. 5-a being H-MnO x Is a transmission electron microscope image; FIG. 5-b is a transmission electron microscope image of Nd-NCs; FIG. 5-c is H-MnO x Transmission electron microscopy at Nd-NCs; FIG. 6 shows a photo-thermal material H-MnO x Element distribution diagram of Nd-NCs (MN), the above diagram shows successful synthesis of photothermal material MN.
Example 2 photothermal Material H-MnO x Nano-enzymatic Activity analysis of Nd-NCs (MN)
This example demonstrates the photothermal material H-MnO from the solution and cell layer x Nano-enzyme activity of Nd-NCs (MN) is mainly demonstrated by the Reactive Oxygen Species (ROS) generating ability of nano-enzymes in simulated tumor microenvironment. ROS production capacity was characterized by POD nanoenzyme activity assay.
(1) POD nano enzyme activity test: detection of photo-thermal Material H-MnO at the solution level by using Methylene Blue (MB) as an indicator x Nano-enzyme activity of Nd-NCs (MN), and judging the ROS production capacity. 350 μg of photothermal material H-MnO x Nd-NCs (MN) (by H-MnO) x To quantify) and 5mg NaHCO 3 Dissolving in 2mL deionized water, sequentially adding reduced Glutathione (GSH) (0,1,2,4,8, 16 mM) with different concentrations, and adding 1 μl H after complete reaction 2 O 2 (9.8M) and 1mL of MB in water (80 mg/L)) Stirring for 20min, detecting MB solution absorbance change with ultraviolet/visible light spectrophotometer (Shimadzu UV-3600 plus), and verifying photo-thermal material H-MnO x ROS production capability of Nd-NCs (MN).
(2) EPR test: ROS were detected qualitatively by paramagnetic resonance spectroscopy (ESR/EPR), and hydroxyl radicals generated in the reaction system were detected using the specific ROS scavenger DMPO (test company completed). 300 mu g of photo-thermal material H-MnO x Nd-NCs (MN) (by H-MnO) x To quantify) and 5mg NaHCO 3 Dissolving in 2mL deionized water, adding 3mg reduced Glutathione (GSH) for complete reaction, and adding 0.5 μl H 2 O 2 (9.8M) uniformly blowing, and detecting the generation of hydroxyl radicals in the system in three time periods of 0min,5min and 15min respectively.
Referring to FIG. 7, it can be seen from FIG. 7-a that in GSH and H 2 O 2 In the coexistent condition, the photo-thermal material can generate ROS to fade MB solution, has POD nano-enzyme activity, and the generated ROS content is related to GSH content; as can be seen from FIG. 7-b, the photo-thermal material H-MnOx@Nd-NCs (MN) can be detected as a characteristic signal under the action of a specific capturing agent of hydroxyl radicals, indicating that the photo-thermal material H-MnO x A specific type of ROS generated by Nd-NCs (MN) is hydroxyl radical.
Example 3 photothermal Material H-MnO x Photo-thermal performance test of Nd-NCs (MN)
This example demonstrates the photothermal material H-MnO x Nd-NCs (MN) have light-heat conversion properties.
The experimental steps are as follows:
1mL of photothermal material H-MnO was taken x Aqueous solutions of Nd-NCs (MN) (200. Mu.g/mL, passed through H-MnO) x Quantitative) in a transparent container under the irradiation of a 808nm laser (vincristine industry) (1W/cm) 2 ) Recording the temperature rise condition of the material by using a thermal infrared imager (FLIR); after the temperature is raised to the highest temperature and stabilized, the laser is turned off until the photo-thermal material H-MnO x The aqueous solution of Nd-NCs (MN) was returned to room temperature and the cooling process was recorded. Repeated three times.
The photothermal conversion efficiency η is calculated by the formula (1):
wherein T is max Representing the highest temperature; t (T) surr Representing ambient temperature; q (Q) dis Indicating heat loss due to absorption of light by the container, which is about 0mW; i represents the laser power density; a is that 808 Representing the absorbance of the sample at 808 nm; h represents a heat transfer coefficient; s represents the surface area of the container, and hs can be obtained from equation (2) according to a cooling fit curve (blue curve of fig. a):
wherein, the liquid crystal display device comprises a liquid crystal display device,τ s is a time constant, T is the temperature of MN aqueous solution, m D And c D Is the solvent mass (1 g) and heat capacity (4.2 Jg -1 ℃ -1 )。
Referring to FIG. 8, it can be seen from FIG. 8-a that the photo-thermal material H-MnO x After the aqueous solution of @ Nd-NCs (MN) is irradiated by a 808nm laser for 600s, the temperature reaches the maximum value and tends to be stable, the temperature reaches the room temperature after the laser is turned off for 1000s, and the photo-thermal conversion efficiency eta is 27.4%; as can be seen from FIG. 8-b, the photo-thermal material H-MnO x The aqueous solution of Nd-NCs (MN) can be repeatedly heated and cooled, and has good photo-thermal stability.
Example 4 tumor cell energy modulation experiments with GLUT inhibitors
This example discusses the energy modulation effects of WZB-117, fatentin, DRB18, phloretin, BAY-876, KL-11743 on tumor cells, including its inhibition of glucose uptake by tumor cells and reduction of ATP content within tumor cells.
Experimental procedure (KL-11743 is taken as an example for illustration, and other medicines are the same):
(1) Intracellular glucose content detection of tumor cells:
first, CT26 cells were seeded into 100mm dishes in four groups of 500X 10 each 4 After the inoculation, the cells are put into 37 ℃ and 5 percent CO 2 Incubation in a sterile incubator (sameira); after cell attachment, the treatments were respectively performed according to the following groups: (1) Control; (2) KL-11743 (0.5 μm); (3) KL-11743 (1 μm); (4) KL-11743 (2 μm); after the treatment, the dishes were again placed at 37℃with 5% CO 2 Is incubated for 12h in a sterile incubator.
Then, the glucose content of each group of cells was detected by a glucose detection kit. The experimental procedure is summarized as follows: (1) Sucking the complete culture medium of each group of cells, and slightly washing each group of cells with PBS buffer solution once to remove redundant culture medium and medicines; (2) Adding 1mL of pancreatin into each group of cells for digestion, standing for 2min, adding 3mL of complete culture medium, shaking, beating and collecting; (3) The collected cell fluid was centrifuged at low speed (1000 rpm,5 min), and the supernatant was discarded and resuspended in 3mL PBS buffer; (4) The resuspended cell fluid is centrifuged again at low speed, then 3mL PBS buffer solution is added for resuspension, and the step is repeated three times to fully remove the cell culture medium; (5) Each group of cells was added with 500. Mu.L of distilled water and sonicated to obtain test samples.
Finally, the test samples are detected according to a glucose detection kit (G264, DOJINDO) using method, three compound holes are arranged, the absorption values of all groups of samples are obtained through an enzyme-labeled instrument (BioTek Instruments, inc.), and the glucose content of all groups of samples can be calculated according to a standard curve of glucose standard solution.
(2) Intracellular ATP content detection of tumor cells:
first, CT26 cells were seeded into 24-well plates in four groups of 1X 10 each 4 After the inoculation, the cells are put into 37 ℃ and 5 percent CO 2 Incubation in a sterile incubator (sameira); after the cells had attached and reached 50% density, treatments were performed separately according to the following groupings: (1) Control; (2) KL-11743 (0.5 μm); (3) KL-11743 (1 μm); (4) KL-11743 (2 μm); after the treatment, the 24-well plate is put into 37 ℃ again, 5 percent CO 2 Is incubated for 12h in a sterile incubator.
The ATP content of each group of cells was then detected by the ATP detection kit. The experimental procedure is summarized as follows: (1) After the complete media of each group of cells was aspirated, each group of cells was gently rinsed with PBS buffer once to remove excess media and drug, and then the remaining PBS buffer was blotted with filter paper; (2) Adding 100 mu L of lysate into each group of cells to carry out cell lysis, and lightly shaking and blowing, wherein the operation is carried out by using an ice bath; (3) Pre-cooling the high-speed centrifuge at 4 ℃ in advance, then centrifuging the collected cells at a high speed (12000 rpm,10 min), and taking the supernatant for later use; (4) Preparing an ATP detection working solution according to the proportion of 1:4 of the ATP detection liquid and the ATP detection diluent, detecting by using a black opaque 96-well plate, setting three compound wells, adding 100 mu L of the ATP detection working solution and 10 mu L of samples (the samples comprise supernatant after cell lysis of each group and ATP standard liquid matched with an ATP detection kit) into each well, obtaining fluorescent signals through a biochemical luminescence detector (BioTek Instruments, inc.), and finally calculating the ATP content of each group of samples through the standard curve of the ATP standard liquid.
Finally, protein quantification is carried out on each group of cells by using the BCA protein quantification kit, so that the ATP content of each group of cells is normalized, and the ATP concentration is converted into nmol/mg protein form, thereby eliminating ATP content errors caused by protein content difference. The experimental procedure is summarized as follows: (1) Preparing BCA protein detection working solution according to the ratio of 1:50 of the reagent A to the reagent B (the reagent A and the reagent B are matched reagents in the BCA protein quantitative kit); (2) Protein content detection is carried out through a 96-well plate, three compound wells are arranged, 100 mu L of BCA protein detection working solution and 10 mu L of samples (the samples comprise supernatant after cell lysis of each group and protein standard solution matched with a BCA protein quantitative kit) are added into each well, the samples are evenly mixed in an oscillating way and then are placed in a 37 ℃ oven for incubation for 30min, then the absorbance value of each sample at 562nm is detected through an enzyme-labeling instrument (BioTek), finally the protein content of each group of samples can be calculated through the standard curve of the protein standard solution, and the ATP content of each group of cells can be divided by the protein content to obtain the ATP content condition of each group of cells after normalization.
Referring to FIG. 9, FIGS. 9-a through 9-f show the changes in intracellular glucose and ATP levels of tumor cell CT26 at various concentrations of GLUT inhibitor WZB-117, faentin, DRB18, phloretin, BAY-876 and KL-11743, respectively. With the increase of the drug concentration, the intracellular glucose content of tumor cells is gradually reduced, and the intracellular ATP content is gradually reduced, which indicates that the ability of inhibiting the glucose uptake of cells can effectively inhibit the synthesis of intracellular ATP.
EXAMPLE 5 GLUT inhibitor KL-11743 faces H-MnO at the cell layer x Verification experiment of the acceleration effect of Nd-NCs (MN) tumor Mild photothermal therapy
This example discusses KL-11743 as a photothermal material H-MnO at the cellular level x The promotion of mild photothermal treatment of Nd-NCs (MN) tumors reflects cell survival mainly by MTT and live dead cell staining, and intracellular HSP70 protein expression is detected by western immunoblotting (WB).
The experimental steps are as follows:
(1) Cell activity was measured by MTT assay: (a) CT26 cells were seeded into 96-well plates in six groups of five duplicate wells each, 6X 10 each 3 After the inoculation, the cells are put into 37 ℃ and 5 percent CO 2 Incubation overnight in a sterile incubator (zemoeid); after cell attachment and after a density of 70%, treatments were performed separately according to the following groupings: (1) Control; (2) control+NIR; (3) MN (50. Mu.g/mL); (4) KL-11743 (0.5. Mu.M) +MN (50. Mu.g/mL); (5) MN (50. Mu.g/mL) +NIR (44.5 ℃,6 min); (6) KL-11743 (0.5. Mu.M) +MN (50. Mu.g/mL) +NIR (44.5 ℃,6 min). Wherein Control means incubating the cells for 7h with complete medium without any drug and without any treatment; control+NIR means irradiation with 808nm laser, irradiation time and power were consistent with other experimental groups; MN (50. Mu.g/mL) +NIR (44.5 ℃ C., 6 min) means that after incubating cells for 7h with complete medium containing 50. Mu.g/mL MN, irradiation was performed with 808nm laser to raise the average temperature of the cells to about 44.5 ℃ C., irradiation time was 6min; the rest of the packets and so on. After the treatment, the 96-well plate was again placed at 37℃with 5% CO 2 Incubation for 17h in a sterile incubator (zemoeid); (b) Preparing 5mg/mL MTT solution, pre-cooling at 4deg.C, adding 10 μl MTT solution into each well, gently shaking, standing at 37deg.C, and 5% CO 2 Incubate in sterile incubator (zemoeid) for 4h. After incubation, the supernatant was aspirated, 150 μl DMSO was added to each well, and the wells were shaken in the shaker for 15min under light-shielding conditions, and the absorbance of each cell group was measured at 490nm by an enzyme-labeled instrument (BioTek) to calculate cell viability. Survival = absorbance per Control group x 100% for each experimental group.
(2) Cell viability and death staining assay: cells were stained with Calcein AM/PI probe and fluorescence was observed under a Zeiss confocal microscope (Carl Zeiss AG). The experimental procedure is summarized as follows: CT26 cells were seeded in confocal dishes 5X 10 each 4 Individual cells were placed at 37℃in 5% CO 2 Culturing in a sterile incubator (Siemens) and grouping after the cell density reaches about 80%, wherein the grouping is as follows: (1)
Control；(2)Control+NIR；(3)KL-11743(0.5μM)；(4)MN(50μg/mL)；
(5) KL-11743 (0.5. Mu.M) +MN (50. Mu.g/mL); (6) MN (50. Mu.g/mL) +NIR (44.5 ℃,6 min); (7) KL-11743 (0.5. Mu.M) +MN (50. Mu.g/mL) +NIR (44.5 ℃,6 min). Wherein Control means incubating the cells for 7h with complete medium without any drug and without any treatment;
control+NIR means that after incubating the cells for 7h with complete medium without any drug, irradiation with 808nm laser was performed with consistent irradiation time and power with other experimental groups; KL-11743 (0.5. Mu.M) represents incubating cells for 7h with complete medium containing 0.5. Mu.M KL-11743; MN (50. Mu.g/mL) +NIR (44.5 ℃ C., 6 min) means that after incubating cells for 7h with complete medium containing 50. Mu.g/mL MN, irradiation was performed with 808nm laser to raise the average temperature of the cells to about 44.5 ℃ C., irradiation time was 6min; the rest of the packets and so on. After the treatment, the supernatant was aspirated, the cells were gently rinsed with PBS buffer to remove excess complete medium and drug reagents, and then the prepared Calcein AM/PI solution was added at 37deg.C, 5% CO 2 Incubate in sterile incubator (zemoeid) for 1h protected from light and finally perform fluorescence imaging by Zeiss confocal microscopy (Carl Zeiss AG).
Referring to FIG. 10, the 808nm laser alone has no killing effect on tumor cells at 808nm laser irradiation, photo-thermal material H-MnO x The tumor cell killing effect of the @ Nd-NCs (MN) is obviously improved, and after the GLUT inhibitor KL-11743 is combined, the photo-thermal material H-MnO x Tumor cell killing by Nd-NCs (MN) was further enhanced, demonstrating the effective promotion of GLUT inhibitor KL-11743 on tumor mild photothermal therapy (note: P in significance analysis <0.05; * Represents P<0.01; * Represents P<0.001; * Represents P<0.0001)。
Referring to fig. 11, the tumor cells were treated differently and the live cells were green fluorescent and the dead cells were red fluorescent. From the figure, it can be seen more intuitively that GLUT inhibitor KL-11743 and photo-thermal material H-MnO x The combined use of Nd-NCs (MN) can indeed improve the mild photothermal treatment effect of tumor cells.
(3) Western Blot (Western Blot) to detect intracellular HSP70 expression levels:
1) Cell treatment: CT26 cells were plated at 3X 10 per well 5 Is inoculated into 6-well plates and divided into 5 groups: (a) Control; (b) control+nir; (c) MN+NIR (100. Mu.g/mL, 44.5 ℃); (d) KL-11743+NIR (1. Mu.M); (e) MN-KL+NIR (100. Mu.g/mL, 44.5 ℃). Wherein Control means incubating the cells for 8h with complete medium without any drug and without any treatment; control+NIR means that after 8h incubation of cells with complete medium without any drug, irradiation with 808nm laser was performed with consistent irradiation time and power with other experimental groups; KL-11743+nir (1 μm) means that cells were incubated for 8h with complete medium containing 1 μm KL-11743, irradiated with 808nm laser, with irradiation time and power consistent with other experimental groups; MN-KL+NIR (100. Mu.g/mL, 44.5 ℃) means that after 8h incubation of cells with complete medium containing 100. Mu.g/mL MN, irradiation was performed with 808nm laser to raise the average temperature of the cells to about 44.5℃for 5min.
2) Cell total protein extraction: after the cell treatment, the residual culture medium was washed off with PBS buffer pre-chilled in advance, the cells were lysed by adding an appropriate amount of RIPA lysate (containing protease inhibitor), then rapidly harvested in a 1.5mL EP tube with a spatula, lysed by vortexing for 15s, allowed to stand for 2min, repeated 16 times, centrifuged at 4℃for 30min (12000 rpm), and the protein supernatant was taken in a fresh 1.5mL EP tube and stored in a refrigerator at-80 ℃. Note the whole ice bath.
3) Protein quantification: protein quantification was performed using BCA protein quantification kit, and protein standard solution and BCA working solution were prepared as required in the specification. After thawing the samples on ice, 10-fold dilution with ultrapure water was performed and protein detection was performed by 96-well plates: 5 mu L of sample (or protein standard solution) and 95 mu L of BCA working solution are added into each hole, three compound holes are arranged, the mixture is fully and evenly shaken, and then incubated for 30min in a 37 ℃ incubator, and then the absorbance at the wavelength of 562nm is measured by a multifunctional enzyme-labeled instrument. And drawing a standard curve by taking the absorbance value as an abscissa and the concentration of the protein standard solution as an ordinate, and substituting the absorbance value into the absorbance value of the sample to calculate the concentration of the sample. Multiplying the calculated sample concentration by the volume of each sample to obtain the total protein of each sample, then adding 5×loading Buffer to dilute to 1×loading Buffer according to the equal mass and equal volume Loading mode, uniformly mixing, and heating in boiling water for 10min to denature the protein, thus obtaining the quantitative loaded sample. Note that the loading volume per well is generally not more than 20 μl, and the loading mass is about 30 μg.
4) SDS-PAGE gel electrophoresis: fresh electrophoretic fluid is prepared and added into an electrophoresis tank, and then the sample is carefully added into a gel sample adding hole by a liquid-transferring gun without bubbles. Running for 30min at constant voltage of 80V at the beginning of electrophoresis, and after all bands are observed to be compressed into linearity, electrophoresis is carried out at 120V until bromophenol blue migrates to the bottom of gel.
5) Transferring: and (3) putting the soaked and balanced sponge, filter paper, gel, PVDF film (methanol activated for 2 min), filter paper and sponge into a protein transfer tank splint, carefully removing bubbles among the layers, closing the splint, transferring into a transfer tank, and transferring into an ice constant flow membrane for 100min, wherein the current is kept at 260/270A, and the voltage is between 90 and 110V.
6) Closing: the PVDF membrane obtained by membrane transfer is rinsed once by PBST, and after positive and negative marks are made, the PVDF membrane is soaked in 5% skimmed milk (2 g skimmed milk powder+40 mL PBST) and gently shaken on a horizontal shaker at room temperature for 2h.
7) Incubating primary antibody: after the end of the sealing, the PVDF membrane is taken out and washed twice by PBST, then the membrane is cut according to the target protein required by the PVDF membrane, the membrane is marked, the PVDF membrane is put into an antibody incubation box, the concentration of the antibody is diluted by an anti-diluent according to the requirement, and the PVDF membrane is incubated on a shaking table of a refrigerator at 4 ℃ for overnight.
8) Incubating the secondary antibody: after the incubation of the primary antibody was completed, the primary antibody was recovered, washed three times with PBST, and then the secondary antibody was diluted with skimmed milk as needed, taking care that the species sources of the primary and secondary antibodies had to be identical, and gently swirled on a horizontal shaker at room temperature for 1h.
9) Exposure: and after the secondary antibody incubation is finished, recovering the secondary antibody, washing the secondary antibody for three times by using PBST, fully contacting the membrane with ECL working solution to perform chemiluminescence, exposing the membrane by using an ultrahigh sensitivity chemiluminescence instrument, and finally performing gray value analysis on the target protein band by using Image J software.
Referring to fig. 12, in the control group, exogenous thermal stimulation can up-regulate intracellular HSP70 expression, whereas in the experimental group, due to the glucose inhibition effect of KL-11743 and the enzyme activity of MN, the HSP70 expression content in tumor cells is not increased and decreased under exogenous thermal stimulation, especially the combined use of KL-11743 and MN greatly inhibits the generation of HSP70 in tumor cells, which is important for promoting the low-temperature photothermal treatment effect of tumors. (remark: in significance analysis) * Representing P<0.05； ** Representing P<0.01； *** Representing P<0.001； **** Representing P<0.0001)。
EXAMPLE 6 GLUT inhibitor KL-11743 faces H-MnO in vivo x Experiment for verifying promotion of moderate photothermal treatment of Nd-NCs (MN) tumor
The present example discusses the promotion of KL-11743 on the cellular level to moderate photothermal treatment of a tumor of a photothermal material MN, and reflects the cell survival condition mainly by MTT method and living and dead cell staining method.
The experimental steps are as follows:
SPF-class female Balb/c mice (4 weeks old) were purchased from Guangdong Kangdong Biotechnology Inc., and used by the laboratory under license number SYXK (Guangdong) 2021-0168. All animal studies were conducted in accordance with guidelines of the experimental animal administration regulations.
CT26 tumor cells resuspended in PBS buffer were assayed at 100. Mu.L/min (2X 10) 6 Individual) was subcutaneously injected into the back of mice to construct a mouse transplantation tumor model. When the tumor grows to about 100mm 3 Time (tumor volume v=length x width) 2 2) mice were randomized into 6 groups (5 per group) for treatment: (1) Control; (2)
Control+NIR；(3)MN；(4)KL-11743+MN；(5)MN+NIR；(6)
KL-11743+MN+NIR. Control group indicates intratumoral injection of PBS (50. Mu.L/mouse) into mice; control+NIR groups represent 808nm laser irradiation (irradiation time and power consistent with other experimental groups) 8h after intratumoral injection of PBS into mice; MN group represents intratumoral injection of PBS buffer containing photothermal material MN (MN concentration 4mg/mL,50 μl/mouse); group KL-11743+MN indicated that mice were intratumorally injected with PBS buffer containing GLUT inhibitor KL11743 and photothermal material MN (KL-11743 concentration 20. Mu.g/mL, MN concentration 4mg/mL, 50. Mu.L/min); MN+NIR group indicates that mice were intratumorally injected with PBS buffer containing photothermal material MN (MN concentration 4mg/mL, 50. Mu.L/min.) for 8h and then subjected to 808nm laser irradiation (44.5 ℃ C., 5 min); the KL-11743+MN+NIR group indicates that mice were intratumorally injected with PBS buffer (KL-11743 concentration 20. Mu.g/mL, MN concentration 4mg/mL, 50. Mu.L/min) containing GLUT inhibitor KL11743 and photothermal material MN for 8 hours and then subjected to 808nm laser irradiation (44.5 ℃ C., 5 min). The above treatments were repeated on days 3 and 7, respectively. The effect of each group was assessed by measuring tumor volume and body weight of mice on alternate days. On day 14, each group of mice was euthanized, dissected, tumor removed, and weighed for photography.
Referring to fig. 13, fig. 13-a shows tumor volume increase in each group of mice over 14 days of treatment. From the graph, the tumor inhibition effect of the photo-thermal material MN is obviously enhanced under the irradiation of 808nm laser, and the tumor inhibition effect of the photo-thermal material MN is further enhanced after the GLUT inhibitor KL-11743 is combined; FIGS. 13-b and 13-c show tumor physical plots and tumor weights of groups of mice after 14 days of treatment, with open circles in the plots showing complete tumor disappearance in the treated mice with 14; FIG. 13-d shows the weight change of each group of mice within 14 days of treatment, and the overall weight of the mice is seen to be increased, which indicates that the GLUT inhibitor KL-11743 and the photothermal material MN and the 808nm laser have no obvious toxic and side effects on the health of the mice.
From the above experiments, KL-11743 and photothermal material H-MnO having nano enzyme activity were found x The combination of Nd-NCs (MN) overcomes the defect of poor monotherapy effect in the prior art, and the drug proposal for realizing complete silencing of HSPs has important significance for promoting clinical transformation of mild PTT.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.

Claims (15)

1. A compound photo-thermal reagent for mild photo-thermal treatment of tumors, which is characterized by comprising a glucose transporter inhibitor and a photo-thermal material with photo-thermal conversion performance.
2. The composite photo-thermal reagent of claim 1, wherein the glucose transporter inhibitor is a molecule or material that inhibits glucose uptake by cells through a glucose transporter signaling pathway, preferably the glucose transporter inhibitor comprises at least one of KL-11743, IMX-120, BAY-876, STF-31, WZB117, fasentin, DRB18, GLUT inhibitor-1, GLUT1-IN-1, GLUT4-IN-2, and piloretin.
3. The compound photo-thermal reagent according to claim 1, wherein the mass ratio of the glucose transporter inhibitor to the photo-thermal material having photo-thermal conversion property is 1 (40-200).
4. The compound photo-thermal reagent according to claim 1, wherein the photo-thermal material having photo-thermal conversion property is a material capable of converting light energy having a wavelength region of 650-1700nm into heat energy.
5. The compound photo-thermal reagent according to claim 4, wherein the photo-thermal material with photo-thermal conversion performance comprises a composite structure constructed by at least one of manganese-containing oxide, indocyanine green ICG, noble metal nanocrystalline, iron-containing oxide, carbon dots, rare earth nanocrystalline, transition metal sulfide, and conjugated polymer.
6. The composite photo-thermal reagent according to claim 1, wherein the photo-thermal material having photo-thermal conversion property comprises the following two components: mnO in hollow mesoporous form x And rare earth nanocrystalline modified on the surface thereof; x is more than or equal to 1 and less than or equal to 2; the rare earth nanocrystalline comprises doped Nd 3+ NaYF of (F) 4 Nanocrystal cores and coating on Nd-doped material 3+ NaYF of (F) 4 CaF on the surface of nanocrystal core 2 A housing.
7. The compound photothermal reagent of claim 6 wherein said Nd doped 3+ NaYF of (F) 4 Nd in nanocrystal core 3+ The doping ratio of (2) is 30-80%.
8. The compound photo-thermal reagent as defined in claim 6, wherein the MnO is hollow mesoporous x The preparation method of the (C) comprises the following steps:
1) Silicon ball SiO 2 Dispersing in pure water, adding permanganate, and ultrasonic mixing to obtain SiO 2 @MnO x Particles;
2) SiO obtained in step 1) is reacted with 2 @MnO x Adding carbonate into the particle dispersed pure water, mixing uniformly, condensing and refluxing to obtain hollow mesoporous MnO x 。
9. The composite photo-thermal reagent according to claim 6, wherein the preparation method of the rare earth nanocrystalline comprises the following steps:
1) Oleic acid and oleylamine are used as surfacesAdding rare earth trifluoroacetate and sodium trifluoroacetate into a mixed solution of oleic acid, oleylamine and octadecene, heating, adding absolute ethanol for precipitation, and centrifuging to obtain Nd-doped compound 3+ NaYF of (F) 4 A nanocrystal core;
2) Adding calcium trifluoroacetate into the mixed solution of oleic acid and oleylamine, and heating to obtain a precursor solution;
3) Will be doped with Nd 3+ NaYF of (F) 4 Mixing and heating the nanocrystal core, oleic acid and octadecene, and injecting the precursor solution obtained in the step 2) to finally obtain the rare earth nanocrystal.
10. The compound photo-thermal reagent according to claim 6, wherein the preparation method of the photo-thermal material with photo-thermal conversion performance comprises the following steps:
1) MnO to be hollow mesoporous x Dispersing in absolute ethanol, adding 3-aminopropyl triethoxysilane to obtain-NH 2 Modified MnO x A solution;
2) Fully and uniformly mixing 2-bromoisobutyric acid, citric acid, chloroform and N, N-dimethylformamide, dropwise adding a chloroform solution of rare earth nanocrystalline, and carrying out ultrasonic mixing to obtain a-Br modified nanocrystalline solution;
3) the-NH produced in step 1) is reacted with 2 Modified MnO x Mixing the solution with the-Br modified nanocrystalline solution prepared in the step 2) to prepare the photo-thermal material with photo-thermal conversion performance.
11. The composite photo-thermal reagent of claim 10, wherein the-NH 2 Modified MnO x The mass ratio of the solution to the solution of the Br modified nanocrystalline is 2:1.
12. Use of a compound photo-thermal reagent according to any one of claims 1 to 11 in the preparation of a heat shock protein inhibitor.
13. The use of claim 12, wherein the glucose transporter inhibitor in the composite photo-thermal agent inhibits heat stress induced overexpression of heat shock proteins by modulating cellular metabolism.
14. Use of a compound photo-thermal agent according to claims 1-9 in the manufacture of a medicament for promoting a gentle photo-thermal treatment of a tumour.
15. The use according to claim 14, wherein the temperature of the gentle photo-thermal is 42 to 45 ℃.
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