Near-infrared light response fatty acid copper nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared light response fatty acid copper nano-drug as well as a preparation method and application thereof. According to the near-infrared light response fatty acid copper nano-drug, copper nanoparticles serve as a core, the outer layer of the copper nanoparticles is wrapped with a phase-change material organic shell layer, meanwhile, a copper ion carrier is loaded in the organic shell layer, and mitochondrial targeting lipid is modified on the shell layer. The nano-drug constructed by the invention can target tumor cells with high expression of CD36 and increase the dosage in the cells, has good mitochondrial targeting and space-time controllable release characteristics, shows a good photo-thermal treatment effect, can kill the tumor cells more accurately and controllably, and establishes a new strategy for cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a near-infrared light-responsive fatty acid copper nanomedicine and a preparation method and application thereof. Background Art

[0002] To mitigate the high costs, significant risk of failure, and lengthy clinical trials associated with developing new chemotherapeutic agents, repurposing clinically approved drugs as innovative anti-tumor agents is a viable strategy. Currently, the pharmacokinetics and biosafety of many existing drugs (such as disulfiram (DSF), chloroquine, and metformin) have been extensively studied and incorporated into nanodelivery systems to enhance their anti-tumor efficacy. Furthermore, these drugs possess unique chemical properties that enable synergistic interactions with other components of nanomedicines, enabling tumor-specific treatment through a transition from non-toxic to toxic properties.

[0003] Disulfiram (DSF) can directly chelate with copper ions to form a dithiocarbamate-copper complex (CuET), which significantly increases the concentration of copper ions in tumor cells. It shows more significant tumor therapeutic effects than simple DSF and copper ions. To further increase the concentration of copper ions in tumor sites, the co-loading method of copper-based nanoparticles and DSF is usually used to release DSF and copper ions to generate CuET. However, the above method cannot overcome the problem of asynchronous release of copper ions and DSF in time, nor can it quickly generate a large amount of CuET. In particular, for larger copper-based nanoparticles, their slow decomposition and short residence time in the tumor site may weaken their anti-tumor ability. In order to achieve the independence and synchronous release of DSF and copper-based nanoparticles in the same space during delivery, it is crucial to construct a new nano-drug delivery platform with rapid release function. However, the currently commonly used nanocarriers usually lack targeting, which greatly limits their enrichment in tumor sites.

[0004] Fatty acids exhibit a natural affinity for CD36 on the surface of tumor cell membranes. This property can be exploited to design fatty acid-based nanocarriers that can selectively enrich tumor cells overexpressing CD36, thereby improving therapeutic efficacy. Furthermore, fatty acids are temperature-sensitive and can be used as phase change materials (PCMs), enabling controlled drug release. However, different PCMs have different phase transition temperatures, necessitating the identification of PCMs that meet clinical requirements.

[0005] As core organs for cellular energy metabolism, apoptosis regulation, and redox balance, mitochondria play a key role in the development of many diseases, especially tumors. By precisely delivering drugs to the interior of mitochondria, the metabolic homeostasis and survival mechanism of cancer cells can be destroyed at the source. Compared with traditional treatments, mitochondrial targeting strategies can significantly enhance therapeutic selectivity and reduce systemic toxic side effects, especially in inducing cell apoptosis, interfering with oxidative phosphorylation, and triggering immune responses. Therefore, the construction of a nanodrug platform with mitochondrial targeting capabilities is becoming a new direction for precision anti-cancer treatment.

[0006] In summary, it can be seen that the development of a near-infrared light-responsive fatty acid copper nanodrug can effectively target tumor cell mitochondria and has great potential application value in the specific treatment of malignant tumors. Summary of the Invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a near-infrared light-responsive fatty acid copper nanomedicine.

[0008] Another object of the present invention is to provide a method for preparing the near-infrared light-responsive fatty acid copper nanomedicine.

[0009] Another object of the present invention is to provide an application of the near-infrared light-responsive fatty acid copper nanomedicine.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A near-infrared light-responsive fatty acid copper nanomedicine, which uses copper nanoparticles as the core, and is coated with an organic shell of phase-change material (the shell is an organic phase-change shell composed of fatty acids or fatty alcohols) on the outer layer of the copper nanoparticles. At the same time, a copper ion carrier is loaded in the organic shell, and mitochondrial-targeting lipids are modified on the shell.

[0012] The copper nanoparticles are copper-containing compounds; preferably, the copper-containing compounds have a particle size of about 10 to 100 nm; more preferably, Cu3BiS3, Cu 2-x At least one of Se, CuCl2, Cu(NO3)2, CuSO4, CuO and CuS; further preferably Cu3BiS3 or Cu 2-x Se.

[0013] The Cu3BiS3 is preferably prepared by the following method:

[0014] Bismuth salt and copper salt are dispersed in oleylamine, degassed and stirred at 60-160°C for uniform mixing, then heated to 160-500°C and maintained for 5-15 minutes, and then a sulfur source is rapidly injected for reaction. After the reaction is completed, the reaction temperature is cooled to 70±5°C, cyclohexane is added, the product is collected by centrifugation, and washed with ethanol to obtain Cu3BiS3 nanoparticles.

[0015] The bismuth salt is at least one of Bi(NO3)3·5H2O, Bi(NO3)3, Bi(OAc)3 and bismuth octanoate; preferably Bi(NO3)3·5H2O.

[0016] The copper salt is at least one of Cu(CH3COO)2, Cu(NO3)2·3H2O and CuCl2·2H2O; preferably Cu(CH3COO)2.

[0017] The sulfur source is at least one of thioacetamide (TAA), elemental sulfur, Na2S·9H2O and sodium diethyldithiocarbamate; preferably thioacetamide (TAA); before being added to the reaction system, the sulfur source can be dispersed in oleylamine and then added to the reaction system.

[0018] The molar ratio of the bismuth salt, the copper salt and the sulfur source is 1:(1-6):(3-12); preferably 1:1:3.

[0019] The amount of oleylamine used can be adjusted as needed, preferably calculated based on 10 to 15 mL of oleylamine per millimole of bismuth salt.

[0020] The degassing treatment is performed by vacuuming with a vacuum pump.

[0021] The degassing treatment time is 3 to 8 minutes, preferably 5 minutes.

[0022] The temperature for stirring and mixing uniformly is preferably 100°C.

[0023] The stirring speed is 500-2000 rpm / min, preferably 1500 rpm / min.

[0024] The stirring time can be adjusted as needed, preferably 0.5 to 1.5 hours, more preferably 1 hour.

[0025] The heating is preferably increased to 300° C. and maintained for 10 minutes.

[0026] The reaction time is 20 to 40 minutes, preferably 30 minutes.

[0027] The amount of cyclohexane used is preferably 1 to 2 times the volume of the reaction system.

[0028] The Cu3BiS3 nanoparticles can be dispersed in cyclohexane and stored to prevent the Cu3BiS3 nanoparticles from agglomerating.

[0029] The Cu 2-x Se is preferably prepared by the following method:

[0030] Sodium selenite solution was added to Tween-80 solution, ascorbic acid solution was added dropwise and stirred at room temperature for reaction, then a mixed solution of CuCl2 and ascorbic acid was added and stirred for reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain copper nanoparticles Cu. 2-x Se.

[0031] The molar ratio of the sodium selenite, ascorbic acid and CuCl2 is 1:8:1.1.

[0032] The concentration of the sodium selenite solution is preferably 1000 mmol / L.

[0033] The amount of the Tween-80 solution can be added according to actual needs, and preferably the volume ratio of the Tween-80 solution to the sodium selenite solution is 1:100.

[0034] The concentration of the ascorbic acid solution is preferably 4000 mmol / L.

[0035] The concentration of CuCl2 in the mixed solution of CuCl2 and ascorbic acid is preferably 550 mmol / L, and the concentration of ascorbic acid is preferably 2000 mmol / L.

[0036] The stirring reaction time at room temperature is 0.5 to 1.5 hours, preferably 1 hour.

[0037] The stirring reaction time is 10 to 15 hours, preferably 12 hours.

[0038] The dialysis is performed using a dialysis bag with a molecular weight cut-off of 50,000; preferably, the dialysis is performed using a dialysis bag with a molecular weight cut-off of 50,000 for 3 days.

[0039] The dialysate used in the dialysis is deionized water.

[0040] The phase change material is an organic phase change material with near-infrared light response characteristics, including at least one of palmitic acid, stearic acid, capric acid, lauric acid, myristic acid, linoleic acid, trans oleic acid, arachidic acid, behenic acid, tetradecanol, dodecanol, 1-tridecanol, 1-tetradecanol, 1-pentadecanol, hexadecanol and 1-octadecanol; further preferably, it is a eutectic mixture obtained by mixing lauric acid and stearic acid in a mass ratio of (1 to 4):1, or a eutectic mixture obtained by mixing tetradecanol and stearic acid in a mass ratio of (1 to 4):1; further preferably, it is a eutectic mixture obtained by mixing lauric acid and stearic acid in a mass ratio of 4:1, or a eutectic mixture obtained by mixing tetradecanol and stearic acid in a mass ratio of 4:1.

[0041] The copper ion carrier includes at least one of disulfiram (DSF), elisimol (Es) and 8-hydroxyquinoline (8-HQ), etc.; preferably disulfiram (DSF).

[0042] The mitochondrial-targeted liposomes include at least one of triphenylphosphine cations (TPP), imidazolium salts, and arginine-rich peptide (SS-31)-modified DSPE-PEG5000 liposomes, wherein the imidazolium salts are 1,3-dimethylimidazolium chloride and / or 1,3-dimethylimidazolium tetrafluoroborate; preferably at least one of DSPE-PEG5000-TPP and SS-31-DSPE-PEG5000; further preferably DSPE-PEG5000-TPP.

[0043] The preparation method of the near-infrared light-responsive fatty acid copper nanomedicine specifically comprises the following steps:

[0044] (1) dissolving the phase change material in methanol (making the phase change temperature reach about 40°C) to obtain a fatty acid PCM solution; then adding a copper ion carrier to obtain a mixed solution I;

[0045] (2) dissolving the mitochondria-targeted liposomes and lecithin (excipient) in an ethanol aqueous solution, and then uniformly mixing with the copper nanoparticle aqueous solution to obtain a mixed solution II;

[0046] (3) Adding the mixed solution I to the mixed solution II under vigorous stirring, reacting at 50-95° C. to obtain a mixed solution III; then cooling the mixed solution III in ice water until turbidity occurs to obtain a mixed solution IV;

[0047] (4) The mixed solution IV is filtered to remove unencapsulated molecules and organic solvents, and then collected by centrifugation, washed, and purified to obtain near-infrared light-responsive fatty acid copper nanomedicine.

[0048] The concentration of the fatty acid PCM solution in step (1) is 1 to 40 mg / mL, preferably 4 mg / mL.

[0049] The concentration of the copper ion carrier in the mixed solution I in step (1) is 0.1-10 mg / mL, preferably 0.5 mg / mL.

[0050] The mass ratio of the mitochondria-targeted liposomes to phosphatidylcholine in step (2) is (1-20):1; preferably 3:1.

[0051] The concentration of the ethanol aqueous solution in step (2) is 1 to 10% by mass, preferably 4% by mass.

[0052] In step (2), the mitochondria-targeted liposomes are dissolved in an ethanol aqueous solution to a final concentration of 1 to 10 mg / mL, preferably 1 mg / mL.

[0053] The concentration of the copper nanoparticle aqueous solution in step (2) is 0.1-10 mg / mL, preferably 0.5 mg / mL.

[0054] The volume ratio of the mixed solution I and the mixed solution II in step (2) is 1:(1-10); preferably 1:5.

[0055] The rotation speed of the vigorous stirring in step (3) is 100 to 1000 rpm / min, preferably 500 rpm / min.

[0056] The reaction temperature in step (3) is preferably 50°C to 60°C; more preferably 50°C.

[0057] The reaction time in step (3) is 5 to 10 minutes, preferably 5 minutes.

[0058] The cooling time in step (3) is 5 to 10 minutes, preferably 5 minutes.

[0059] The filtration described in step (4) is performed using a 0.2 μm surfactant-free cellulose acetate membrane.

[0060] The centrifugal speed in step (4) is 1000-10000 g, preferably 5000 g.

[0061] The purification described in step (4) is performed using a VIVASPIN 6 centrifugal concentrator (molecular weight cut-off MWCO = 10 kDa).

[0062] The washing in step (4) is performed with deionized water; preferably, the washing is performed with deionized water for more than 3 times.

[0063] Application of the near-infrared light-responsive fatty acid copper nanomedicine in the preparation of medicines for treating tumors.

[0064] The drugs include near-infrared photothermal therapy agents (drugs), copper death-photothermal therapy drugs, etc.

[0065] The tumor is a tumor expressing CD36, including breast cancer, etc.; preferably, it is breast cancer that highly expresses CD36.

[0066] The present invention has the following advantages and effects compared to the prior art:

[0067] The nanomedicine in this invention uses copper nanoparticles as its core, with fatty acids containing a phase-change material constructed on the outer layer of the copper nanoparticles, and mitochondrial-targeting liposomes modified on the shell. By forming a copper nanoparticle outer layer with phase-change and mitochondrial-targeting properties, the nanomedicine overcomes the serious toxic side effects caused by premature drug release after entering the bloodstream, as well as the difficulty in accumulation and uptake at the tumor site and the low drug release efficiency after entering tumor cells. This allows for more precise and controllable killing of tumor cells.

[0068] 2. Based on the reaction mechanism between copper ions and disulfiram (DSF), that is, the property that monovalent copper ions (Cu(I)) can react with DSF more directly and rapidly, the present invention uses Cu3BiS3 nanoparticles (NPs) with rich surface defects and high activity as a copper source, which can quickly reduce DSF to dithiocarbamate (DTC) and quickly generate CuET; and because Cu3BiS3 nanoparticles have a good surface plasmon resonance effect in the near-infrared region II (NIR-II), they exhibit excellent photothermal conversion efficiency, enabling them to exhibit good photothermal treatment effects.

[0069] 3. The present invention constructs a fatty acid nanodrug DCP-TPP with a photocontrolled cascade burst release. DSF and Cu3BiS3 nanoparticles are co-encapsulated in PCMs containing two fatty acids (lauric acid and stearic acid), and modified with a TPP liposome coating to enhance its mitochondrial targeting. The surface of DCP-TPP is rich in fatty acids and mitochondrial targeting ligands (TPP groups), which can be specifically enriched in the mitochondria of tumor cells overexpressing CD36. Below the eutectic point, DCP-TPP is in a solid state, which prevents premature reaction between Cu3BiS3 and DSF and leakage during delivery, ensuring their biocompatibility. After DCP-TPP accumulates at the tumor site, Cu3BiS3 exhibits good photothermal conversion efficiency after irradiation with near-infrared (NIR) light. At the phase transition temperature, DCP-TPP is in a solid state, which can prevent premature reaction between Cu3BiS3 and DSF and leakage of copper ions during transport, thereby ensuring its biocompatibility and significantly increasing its relative concentration at the tumor site. When the temperature exceeds the phase transition temperature, the phase change material (PCM) melts, resulting in the instantaneous release of Cu3BiS3 and DSF. Simultaneously, chelation occurs, rapidly generating CuET, thereby enhancing the therapeutic effect of in situ tumors. This innovative drug delivery system design overcomes the critical issue of low CuET concentration at the tumor site and enables precise control of the non-toxic-to-toxic transformation of DCP-TPP at the tumor site.

[0070] 4. The nanodrug constructed in this invention can target tumor cells that highly express CD36, increasing the intracellular drug delivery. It also exhibits excellent mitochondrial targeting and spatiotemporally controlled release. During drug delivery, the nanodrug remains "inactivated" while circulating in the body. Upon entering tumor tissue and / or cells, it exhibits a specific spatiotemporal response, "activating" the drug's toxicity, thereby reducing the drug's toxic side effects on other normal tissues and achieving precise tumor treatment. This effectively addresses the issues of premature release of nanodrugs in the bloodstream, making them difficult to be taken up by tumor cells, and low drug release efficiency after entering tumor cells.

[0071] 5. The nanomedicine DCP-TPP prepared in the present invention shows good photothermal conversion efficiency of Cu3BiS3 after near-infrared (NIR) light irradiation, triggering the synchronous release of disulfiram (DSF) and Cu3BiS3. This promotes the dissociation of copper ions in Cu3BiS3 and promotes the accumulation of copper ions in mitochondria. Excessive copper ions induce the oligomerization of mitochondrial DLAT proteins, destroying mitochondrial bioenergy metabolism (disrupting the tricarboxylic acid (TCA) cycle), oxidative phosphorylation collapse, and lipid metabolism, thereby triggering immunogenic cell death (ICD). Subsequently, immunogenic cell death recruits dendritic cells and cytotoxic T lymphocytes, initiating a systemic immune response to enhance the anti-tumor therapeutic effect. By combining copper death-driven metabolic destruction with immunogenic cell death-mediated immune activation, DCP-TPP establishes a new therapeutic strategy for breast cancer that overexpresses CD36.

[0072] 6. This invention facilitates the spatiotemporal controlled release of DSF and Cu3BiS3 nanoparticles through temperature control of phase change materials (PCMs). When incorporated into nanocarriers, these PCMs enable controlled drug release in response to external thermal stimuli, facilitating precise, on-demand delivery of the payload. The synergistic effect between CD36-mediated targeting and temperature-responsive drug release enhances therapeutic precision and efficacy, making fatty acid-based nanocarriers a versatile platform for precision oncology.

[0073] 7. The present invention combines metabolic therapy of copper death control with immunotherapy, which not only solves the problem of tumor cells' dependence on metabolism, but also breaks the immunosuppressive barrier, which will effectively improve the side effects of nanomedicines on the whole body and improve the survival rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic diagram of the synthesis process of DCP-TPP and killing cancer cells in Example 1 of the present invention; wherein, a is the synthesis process of DCP-TPP; b is a schematic diagram of DCP-TPP killing cancer cells.

[0075] Figure 2 This is the X-ray diffraction energy spectrum (XRD) of Cu3BiS3 in Example 1 of the present invention.

[0076] Figure 3 3BiS3, Bi and Cu in Example 1 of the present invention are X-ray photoelectron spectra (XPS); wherein a is the XPS spectrum of Cu3BiS3; b is the XPS spectrum of Bi; and c is the XPS spectrum of Cu.

[0077] Figure 4This is a transmission electron microscopy (TEM) characterization and particle size distribution result diagram of Cu3BiS3 in Example 1 of the present invention.

[0078] Figure 5 The heating and cooling curves of the photothermal conversion of Cu3BiS3 in Example 1 of the present invention and the calculation results of the photothermal conversion rate are shown; wherein, a is the UV-Vis spectrum of Cu3BiS3 in aqueous solution with different concentrations (0-500 μg / mL); b is the UV-Vis spectrum of Cu3BiS3 with different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 1.5 W / cm 2 c is the temperature change of Cu3BiS3 with different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 1 W / cm 2 d is the temperature change of Cu3BiS3 with different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 0.5 W / cm 2 , 10 minutes) under irradiation; e is 250μg / mL Cu3BiS3 under 808nm laser (power density: 1W / cm 2 ) irradiated with 5 laser on / off cycles; f is 250μg / mL Cu3BiS3 under 808nm laser (power density: 1W / cm 2 ) for 10 minutes, and then the laser is turned off when the temperature stabilizes; g is the heat transfer time constant calculated from the cooling cycle, and water is used as the control group.

[0079] Figure 6 This is the X-ray diffraction spectrum (XRD) of DCP-TPP in Example 1 of the present invention.

[0080] Figure 7 This is the infrared spectrum of DCP-TPP in Example 1 of the present invention.

[0081] Figure 8 This is the Zeta potential diagram of Cu3BiS3, DCP, DCP-TPP and DCP-TPP+L in PBS in Example 1 of the present invention.

[0082] Figure 9 This is a scanning electron microscope characterization result diagram of DCP-TPP in Example 2 of the present invention before and after light irradiation.

[0083] Figure 10The heating and cooling curves and the calculation results of the photothermal conversion rate of DCP-TPP in Example 1 of the present invention are shown; wherein, a is the UV-Vis spectrum of DCP-TPP in aqueous solution at different concentrations (0-500 μg / mL); b is the UV-Vis spectrum of DCP-TPP at different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 1.5 W / cm 2 c is the temperature change of DCP-TPP with different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 1 W / cm 2 d is the temperature change of DCP-TPP with different concentrations (0-500 μg / mL) under NIR laser (808 nm, power density: 0.5 W / cm 2 , 10 minutes) irradiation temperature change; e is 250μg / mL DCP-TPP under 808nm laser (power density: 1W / cm 2 ) for five laser on / off cycles; f is 250 μg / mL DCPTPP under 808 nm laser (power density: 1 W / cm 2 ) for 10 minutes, and then the laser is turned off when the temperature stabilizes; g is the heat transfer time constant calculated from the cooling cycle, and water is used as the control group.

[0084] Figure 11 1 is a graph showing the release curves of DSF and copper ions from DCP-TPP at 37° C. and 45° C. in Example 1 of the present invention; wherein a is a graph showing the release curve of DSF; and b is a graph showing the release curve of copper ions.

[0085] Figure 12 The figure shows the statistical results of DCP-TPP (concentration of 50 μg / mL, including FITC: 5 μg / mL) uptake by 4T1 cells after being treated with different cell uptake inhibitors (chlorpromazine, cytochalasin B, MβCD or SSO).

[0086] Figure 13 Figure 2 is the expression of CD36 in MCF-7, MDA-MB-231 and 3T3 cells.

[0087] Figure 14 The mean fluorescence intensity of DCP-TPP (50 μg / mL, containing 5 μg / mL FITC) was analyzed by flow cytometry in MDA-MB-231, MCF-7, and 3T3 cells.

[0088] Figure 15 The graph shows the cell viability of 4T1 cells after treatment with DCP-TPP or DCP-TPP+L for 12 hours.

[0089] Figure 16 Confocal microscopic images of 4T1 cells treated with DCP-TPP (50 μg / mL, containing FITC: 5 μg / mL) for 12 hours.

[0090] Figure 17 Figure 2 is a graph showing the tumor inhibition and survival curves of 4T1 tumor-bearing mice after treatment with different drugs; a is the statistical result of tumor volume; b is the survival curve.

[0091] Figure 18 Cu in Example 3 of the present invention 2-x X-ray diffraction (XRD) spectrum of Se.

[0092] Figure 19 Cu in Example 3 of the present invention 2-x X-ray photoelectron spectroscopy (XPS) of Se; wherein, a is Cu 2- x a is the XPS spectrum of Se; b is the XPS spectrum of Se; c is the XPS spectrum of Cu.

[0093] Figure 20 Graph showing the changes in solid and liquid states of the phase change material (PCM) of Example 4 of the present invention at 37°C and 39°C.

[0094] Figure 21 This is a graph showing the uptake of DCP-SS31 (at a concentration of 50 μg / mL, including FITC: 5 μg / mL) by 4T1 cells after treatment with different cell uptake inhibitors (chlorpromazine, cytochalasin B, MβCD, or SSO) in Example 5 of the present invention. DETAILED DESCRIPTION

[0095] The present invention will be described in further detail below in conjunction with Examples, but embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise specified, methods that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0096] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0097] The first aspect of the present invention discloses a nanomedicine, which uses copper nanoparticles as the core, a phase-transition organic shell wrapped around the outer layer of the copper nanoparticles, and a copper ion carrier loaded in the organic shell; the shell is an organic phase-transition shell composed of fatty acids or fatty alcohols, and the shell is modified with a lipid having a mitochondrial targeting group (mitochondrial targeting lipid).

[0098] The shell of the nanomedicine has multiple targeting activation properties. The outermost fatty acid / fatty alcohol organic shell has good specificity for tumor cells that highly express CD36. The mitochondrial targeting group modified on its surface further promotes the mitochondrial localization of the nanomedicine.

[0099] The nanomedicine is an intelligent responsive nanomedicine that stimulates the carrier to rupture by near-infrared (NIR) light, effectively promoting the release of drugs in tumor cells, reducing toxic side effects and improving therapeutic effects. It has good targeting and spatiotemporal controllable release, thereby increasing the amount of drug administered into the cell. Most importantly, during the drug delivery process, the loaded drug can be kept "inactivated" during the body's circulation process, and after entering the tumor tissue / cell, it exhibits a specific spatiotemporal response to "activate" the drug's toxicity, thereby reducing the toxic side effects of drug molecules on other normal tissues and achieving precise tumor treatment.

[0100] The copper nanoparticles described in the present invention refer to copper-containing compounds with a particle size of about 10 to 100 nm. Specific types can be copper oxides, sulfides, etc. Specific examples include CuO, Cu 2-x Se, CuS or Cu3BiS3, but not limited thereto. Preferably, in some specific embodiments of the present invention, the copper nanoparticles are Cu3BiS3.

[0101] The copper ion carrier described in the present invention includes at least one of disulfiram (DSF), elisimol (Es) and 8-hydroxyquinoline (8-HQ); preferably, in some specific embodiments of the present invention, the copper ion carrier is disulfiram (DSF).

[0102] The phase change material described in the present invention refers to a material that can change from a solid state to a liquid state when heated. Specific examples include, but are not limited to, at least one of palmitic acid, stearic acid, capric acid, lauric acid, myristic acid, linoleic acid, trans-oleic acid, arachidic acid, behenic acid, tetradecanol, dodecanol, 1-tridecanol, 1-tetradecanol, 1-pentadecanol, hexadecanol, and 1-octadecanol. Preferably, the phase change material is a eutectic mixture of lauric acid and stearic acid. Compared to other types of phase change materials, while having the effects of other temperature-sensitive phase transitions, the ratio of stearic acid to lauric acid can be adjusted to obtain a phase change carrier that meets clinical temperature requirements. Furthermore, the material can promote the uptake of nanomedicines in tumor cells that highly express CD36, thereby improving the specificity of the nanomedicine and reducing its toxic side effects.

[0103] The mitochondrial targeting lipid described in the present invention refers to a lipid that can effectively target the mitochondrial matrix. Specifically, the mitochondrial targeting liposomes described in the examples that can be mentioned are at least one of DSPE-PEG5000 liposomes modified with triphenylphosphine cation (TPP), imidazolium salts (1,3-dimethylimidazolium chloride and 1,3-dimethylimidazolium tetrafluoroborate) and arginine-rich peptide (SS-31); preferably at least one of DSPE-PEG5000-TPP (distearoylphosphatidylethanolamine-polyethylene glycol 5000-triphenyl phosphate) and SS-31-DSPE-PEG5000 (distearoylphosphatidylethanolamine-polyethylene glycol-mitochondrial targeting peptide ss31); further preferably DSPE-PEG5000-TPP.

[0104] The second aspect of the present invention provides a method for preparing the nanomedicine according to the first aspect of the present invention, the main steps of which are as follows:

[0105] S1. Preparation of copper nanoparticles

[0106] The copper nanoparticles of the present invention can be prepared or obtained using methods known in the art. Using the preparation of Cu3BiS3 as an example, a bismuth salt and a copper salt are dispersed in an oleylamine solution, degassed, and stirred to mix thoroughly. A sulfur source is then rapidly injected into the reaction mixture and stirred to produce Cu3BiS3 nanoparticles.

[0107] The bismuth salt may be at least one of Bi(NO3)3·5H2O, Bi(OAc)3 or bismuth octanoate.

[0108] The copper salt may be at least one of Cu(CH3COO)2, Cu(NO3)2·3H2O or CuCl2·2H2O.

[0109] The sulfur source can be at least one of thioacetamide, elemental sulfur, Na2S·9H2O or sodium diethyldithiocarbamate.

[0110] The molar ratio of the bismuth salt, the copper salt and the sulfur source is 1:(2-6):(3-12); the preferred molar ratio is 1:1:3.

[0111] The stirring and mixing temperature is 60-160°C, and the stirring speed is 500-2000 rpm / min. The time can be adjusted as needed; preferably, the temperature is 100°C and the stirring speed is 1500 rpm / min. Before injecting the sulfur source into the reaction mixture, the temperature of the reaction mixture needs to be rapidly raised to 160-500°C and maintained for 10 minutes; preferably, the temperature is rapidly raised to 300°C and maintained for 10 minutes.

[0112] S2. The outer layer of the copper nanoparticles is coated with liposomes having mitochondrial targeting and phase transition properties to form a shell layer. The specific steps are as follows:

[0113] ① Dissolve the copper ion carrier and the phase change material in methanol and stir to mix evenly to obtain a mixed solution.

[0114] The copper ion carrier is at least one of disulfiram (DSF), elisimol (Es) and 8-hydroxyquinoline (8-HQ), etc.; preferably disulfiram (DSF).

[0115] The phase change material is at least one of palmitic acid, stearic acid, capric acid, lauric acid, myristic acid, linoleic acid, trans oleic acid, arachidic acid, behenic acid, tetradecanol, dodecanol, 1-tridecanol, 1-tetradecanol, 1-pentadecanol, hexadecanol and 1-octadecanol; preferably, it is a eutectic mixture of lauric acid and stearic acid, and the mass ratio thereof is (1-4):1, preferably 4:1.

[0116] ② Disperse copper nanoparticles in deionized water, disperse mitochondrial targeting lipids and lecithin in ethanol aqueous solution, and then add them dropwise to the above mixed solution and stir to react.

[0117] The copper nanoparticles are used as copper ion donors and photothermal agents for photothermal conversion. Specific examples include CuO, Cu 2-x Se, CuS or Cu3BiS3, etc.; preferably Cu3BiS3.

[0118] The mitochondrial targeting lipid is at least one of DSPE-PEG5000 liposomes modified with triphenylphosphine cation (TPP), imidazolium (1,3-dimethylimidazolium chloride and 1,3-dimethylimidazolium tetrafluoroborate) and arginine-rich peptide (SS-31); preferably at least one of DSPE-PEG5000-TPP and SS-31-DSPE-PEG5000; further preferably DSPE-PEG5000-TPP.

[0119] The stirring reaction is carried out at a temperature maintained at 50-95°C, preferably at 50-60°C.

[0120] ③ The obtained solution is cooled in ice water until a turbid precipitate appears; finally, the unencapsulated molecules and organic solvent are removed by filtration, and the solution is washed and concentrated to obtain nanoparticles.

[0121] The cooling time is 5 to 10 minutes, preferably 5 minutes.

[0122] The centrifugal force for collecting the precipitate is 1000-10000 g, preferably 5000 g.

[0123] The copper ion carrier refers to a compound known in the art that can combine with copper ions to induce copper cell death in tumor cells or a pharmaceutically acceptable salt thereof, and specific examples include disulfiram or ilisimol.

[0124] The specific method for loading the nano drug with drugs can be carried out using methods known in the art, such as dispersing the nano carrier in a solution containing a copper ion carrier and stirring to obtain the nano drug.

[0125] The third aspect of the present invention provides use of the nanomedicine described in the second aspect of the present invention in the preparation of a drug for treating tumors.

[0126] Example 1 Preparation of copper nanoparticles (Cu3BiS3)

[0127] This embodiment provides a nanocarrier and a method for preparing the same. Figure 1 , the specific steps are as follows:

[0128] (1) Bi(NO3)3·5H2O (1 mmol) and Cu(CH3COO)2 (1 mmol) were placed in a 50 mL three-necked flask containing 12 mL of oleylamine (OM) and degassed using a vacuum pump for 5 min. The temperature was raised to 100°C and the mixture was gently stirred for 1 hour. Subsequently, the temperature of the reaction mixture was rapidly raised to 300°C and maintained for 10 min.

[0129] (2) Thioacetamide (3 mmol, TAA) was dispersed in OM (2.5 mL), and then the TAA solution was quickly injected into the above reaction mixture; after 30 minutes, the reaction system was cooled to 70 °C and 20 mL of cyclohexane was added; the product was collected by centrifugation and then washed three times with ethanol; finally, the prepared Cu3BiS3 nanoparticles were dispersed in 10 mL of cyclohexane to avoid agglomeration of the Cu3BiS3 nanoparticles.

[0130] Example 2 Preparation of near-infrared light-responsive fatty acid copper nanoparticles

[0131] (1) The mass ratio of lauric acid (LA) to stearic acid (SA) was adjusted to 4:1, and then dissolved in methanol (concentration of 4 mg / mL) until the phase transition temperature reached about 40°C to obtain a fatty acid PCM solution;

[0132] (2) Add disulfiram (DSF) powder to the above fatty acid PCM solution to maintain its concentration at 500 μg / mL to obtain mixed solution I;

[0133] (3) Phosphatidylcholine and DSPE-PEG5000-TPP (Xi'an Ruixi Biotechnology Co., Ltd., catalog number: R-9981) were dissolved in a 4% ethanol aqueous solution at a mass ratio of 3:1 to a final concentration of 1 mg / mL (12 mL), and mixed with a Cu3BiS3 aqueous solution (3 mL, 500 μg / mL) (the Cu3BiS3 stored in cyclohexane in Example 1 was first collected by centrifugation, then washed with water and redissolved in water to form a Cu3BiS3 aqueous solution) to obtain a mixed solution II;

[0134] (4) Under vigorous stirring (500 rpm / min), 3 mL of mixed solution I was slowly added to the mixed solution II, heated to 50°C, and maintained for 5 minutes; the resulting mixed solution III was cooled in ice water for 5-10 minutes until turbidity appeared, to obtain mixed solution IV;

[0135] (5) Finally, the mixed solution IV was filtered through a 0.2 μm surfactant-free cellulose acetate membrane (Thermo Fisher Scientific) to remove unencapsulated molecules and organic solvent. The encapsulated DCP-TPP nanoparticles were then collected by centrifugation (5000 g). After washing three times with deionized water, the solution was purified using a VIVASPIN 6 centrifugal concentrator (Sartorius, MWCO = 10 kDa), washed three times with deionized water, and freeze-dried to obtain purified DCP-TPP nanoparticles for later use.

[0136] Example 3 Characterization Test and Effect Experiment of Cu3BiS3 and DCP-TPP

[0137] 1. Test Characterization

[0138] 1. Characterization and testing of copper nanoparticles Cu3BiS3

[0139] (1) The crystal diffraction peaks of the copper nanoparticles Cu3BiS3 prepared in Example 1 were analyzed using an X-ray diffractometer. It can be seen that the crystal diffraction peaks of Cu3BiS3 correspond one to one with the crystal diffraction peaks on the PDF standard card, indicating that the copper nanoparticles Cu3BiS3 were successfully prepared ( Figure 2 ).

[0140] (2) X-ray photoelectron spectrometer was used to characterize the X-ray photoelectron spectrum of the copper nanoparticles Cu3BiS3 prepared in Example 1. The results are as follows: Figure 3 As shown: The two peaks of Bi 4f core level spectrum (mainly concentrated at 158.5eV and 163.6eV, respectively, are attributed to Bi 3+ Bi 4f ions 7 / 2 and Bi 4f 5 / 2 ); In addition, the other two peaks at 931 and 951 eV mainly correspond to Cu + Cu 2p 3 / 2 and Cu 2p 1 / 2 ; The peak at 161eV is assigned to S2p. Based on Figure 3 From the characterization results, it can be concluded that the valence states of S, Cu and Bi are -2, +1 and +3, respectively, which further proves the successful preparation of copper nanoparticles Cu3BiS3.

[0141] (3) The particle size and morphology of the copper nanoparticles Cu3BiS3 prepared in Example 1 were characterized using a transmission electron microscope ( Figure 4 ). It can be seen that Cu3BiS3 is a distinct spherical particle with uniform particle size. In addition, the particle size of the synthesized Cu3BiS3 was statistically analyzed, and the results showed that the average particle size of Cu3BiS3 was about 16.6±4.4nm.

[0142] (4) In order to examine the photothermal conversion efficiency of Cu3BiS3, a photothermal imager was used to record the temperature rise and fall of Cu3BiS3 at different powers and concentrations, and the photothermal conversion efficiency was calculated. An 808nm laser was used at 0.5, 1 and 1.5W / cm 2 The laser power density was used to irradiate a series of Cu3BiS3 solutions with a concentration gradient (0, 62.5, 125, 250, 500 μg / mL) in a test tube (continuous irradiation for 10 minutes), and the photothermal temperature rise was recorded at 30-second intervals using an infrared thermal imager ( Figure 5). Cu3BiS3 exhibits concentration-dependent photothermal properties under 808nm laser irradiation. After 5 cycles of heating and cooling, the heating curve and maximum temperature of Cu3BiS3 at 808nm remained stable, indicating that it has good cyclic photothermal heating ability and excellent photothermal stability. Finally, a 250μg / mL Cu3BiS3 aqueous solution was used as the research object, and the heating and cooling data were processed based on the calculation method reported by Roper et al., and the photothermal conversion efficiency of Cu3BiS3 was found to be 52.04%. These results indicate that Cu3BiS3 has strong light absorption and excellent photothermal heating performance in the near-infrared region, and has good photothermal stability, and can be used as a general near-infrared photothermal therapeutic agent.

[0143] 2. Characterization and testing of DCP-TPP nanoparticles

[0144] (1) The crystal structure of the purified DCP-TPP in Example 2 was analyzed by X-ray diffraction (XRD). The results showed that the main characteristic X-ray diffraction peaks of DCP-TPP were consistent with those of Cu3BiS3, and the positions of the diffraction peaks matched those of the standard JCPDS database (71-2115) of Cu3BiS3 ( Figure 6 ), proving that Cu3BiS3 was successfully encapsulated in DCP-TPP.

[0145] (2) The successful synthesis of DCP-TPP was further verified by Fourier transform infrared spectroscopy (FT-IR). FT-IR spectrum showed the presence of DSF and Cu3BiS3 in DCP-TPP. DSF was at 1504 cm -1 (CN stretching vibration) and 675cm -1 (CS stretching vibration) has an obvious characteristic peak, and Cu3BiS3 also shows its characteristic signal ( Figure 7 ). The above results show that DCP-TPP fat nanomedicine was successfully prepared.

[0146] (3) Zeta potential analysis was performed to analyze Cu3BiS3, DCP (prepared as in Example 2, except that DSPE-PEG5000-TPP was replaced with DSPE-PEG5000 (Xi'an Ruixi Biotechnology Co., Ltd., product number: R-1028-5K), DCP-TPP, and NIR irradiation (808 nm laser, 1 W / cm 2 The Zeta potential of the nanoparticles was measured after the addition of DCP-TPP (each substance concentration was 250 μg / mL) with irradiation for 10 minutes. The results showed that after TPP modification, the surface Zeta potential of the nanoparticles gradually increased from -11.27±0.81mV to -7.34±1.79mV ( Figure 8Meanwhile, the zeta potential further increased to -2.93 ± 0.42 mV after NIR irradiation. This change indicates that the introduction of TPP significantly altered the surface charge of the nanoparticles, while NIR irradiation further disrupted the nanoparticle structure, promoting the exposure of the Cu3BiS3 and TPP components.

[0147] (4) Scanning electron microscopy (SEM) was used to analyze the DCP-TPP (250 μg / mL) under NIR irradiation (808 nm laser, 1 W / cm 2 , continuous irradiation for 10 minutes) before and after morphological changes ( Figure 9 SEM images show the spherical morphology and uniform size of DCP-TPP. Statistical analysis of these images revealed an average diameter of 62.50 ± 5.20 nm. Upon light treatment, the DCP-TPP structure was significantly disrupted, with a gradual reduction in the number of monodisperse round particles and significant cross-linking.

[0148] (5) In order to examine the photothermal conversion efficiency of DCP-TPP fat nanocarriers, a photothermal imager was used to record the temperature rise and fall of DCP-TPP at different powers and concentrations, and the photothermal conversion efficiency was calculated ( Figure 10 ). 808nm laser was used at 0.5, 1 and 1.5W / cm 2 A series of DCP-TPP solutions with a concentration gradient (0, 62.5, 125, 250, and 500 μg / mL) in a test tube were irradiated with a laser power density of 100 μg / mL (continuous irradiation for 10 minutes), and the photothermal temperature rise was recorded at 30-second intervals using an infrared thermal imager. At a given laser power, the temperature increase of DCP-TPP was positively correlated with the concentration, and the temperature rise increased with increasing laser power. Five cycles of heating and cooling curve tests were conducted, each lasting 30 minutes. The maximum heating temperature of DCP-TPP in the second cycle was significantly lower than that in the first cycle by 4.5°C. This phenomenon fully demonstrates that the Cu3BiS3 nanoparticles contained in DCP-TPP can undergo instant photothermal conversion. After photothermal conversion, the structure of the phase-transition fatty acid carrier in DCP-TPP is destroyed, promoting the explosive release of Cu3BiS3 and DSF. Finally, we used a 250 μg / mL aqueous solution of DCP-TPP nanomaterials as the research object, and calculated the heating and cooling data according to the calculation principle reported by Roper. The calculated result showed that the photothermal conversion efficiency of DCP-TPP was 50.28%.

[0149] (6) To further demonstrate that DCP-TPP has the characteristics of temperature-sensitive controllable release, the release of DSF and copper ions was characterized by UV-Vis-NIR spectrophotometer and ICP-Ms mass spectrometer. When the temperature is higher than 39 °C, the fatty acid components in DCP-TPP will change from solid to liquid. To further verify the release of copper ions and DSF after exceeding the phase transition temperature, we tested the release of copper ions and DSF from DCP-TPP at 37 °C and 45 °C ( Figure 11 At 37 °C, the release amounts of DSF and Cu3BiS3 were negligible within 240 h, whereas at 45 °C, their release amounts increased by 1.17-fold and 7.11-fold, respectively, indicating that DCP-TPP has good thermally triggered drug release properties.

[0150] 2. In vitro cell assay

[0151] 1. Mouse 4T1 breast cancer cells (Baidi Biotech Ltd., C5009) were inoculated into 96-well plates (5×10 3 Each well contained 2 mL of R1640 growth medium. The cells were cultured in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was aspirated. The experimental grouping and specific procedures were as follows:

[0152] R1640 growth medium: RPMI-1640 (PM150110, Procell) + 10% (v / v) FBS (164210-50, Procell) + 1% P / S (PB180120, Procell, penicillin-streptomycin solution (double antibody)).

[0153] Culture conditions: Gas phase: air, 95%; CO2, 5%; Temperature: 37°C.

[0154] 4T1 cells were treated with drugs that block different endocytic pathways for 2 hours at 37°C. These drugs included: MβCD (methyl-β-cyclodextrin, a caveolin-mediated endocytosis inhibitor) at a final concentration of 5 mg / mL; cytochalasin B (a cell-permeable actin polymerization inhibitor that inhibits monosaccharide transport across the cell membrane) at a final concentration of 10 μg / mL; chlorpromazine (a clathrin-mediated endocytosis inhibitor) at a final concentration of 5 μg / mL; and sodium sulfosuccinimidyl oleate (SSO, a CD36 inhibitor) at a final concentration of 5 μg / mL. Subsequently, the treated cells were incubated with DCP-TPP (FITC-loaded DCP-TPP, obtained by mixing DCP-TPP with a fluorescent dye; the concentration of DCP-TPP was 50 μg / mL, and the FITC-loaded DCP-TPP was 50 μg / mL) for 4 hours at 37°C. After washing the treated cells three times with PBS, 100 μL of cell lysis buffer was added for 10 minutes. Finally, the mean fluorescence intensity of the cells after treatment with different drugs was analyzed using a fluorescence microplate reader (SYNERGY H1, BioTek, USA). The experiment was repeated three times.

[0155] The results are as follows Figure 12 Shown: Through Figure 12 It can be seen that the addition of SSO inhibitor significantly inhibited the uptake of DCP-TPP by tumor cells, and the uptake was reduced by 83.4% compared with the untreated group, indicating that CD36 plays a major role in the uptake of DCP-TPP.

[0156] 2. Mouse breast cancer cells 3T3 (Baidi Biotech Ltd., C5133), MCF-7 (Baidi Biotech Ltd., C5103) and MDA-MB-231 (Baidi Biotech Ltd., C6291) were cultured in 6-well plates (5 × 10 3 Each well contained 2 mL of R1640 growth medium and was cultured in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was aspirated. The experimental grouping and specific procedures were as follows:

[0157] R1640 growth medium: RPMI-1640 (PM150110, Procell) + 10% (v / v) FBS (164210-50, Procell) + 1% P / S (PB180120, Procell).

[0158] Culture conditions: Gas phase: air, 95%; CO2, 5%; Temperature: 37°C.

[0159] Subsequently, the cells were washed three times with PBS, and the above cells were lysed with RIPALysis Buffer (Medium, 20115ES60). After the lysis was completed, the supernatant was collected by centrifugation. After the protein concentration of different cells was determined by Bradford Protein Quantification Kit (20202ES76), 12% SDS-PAGE gel was used for protein separation, and 25 μg of protein sample was added to each lane. After the gel run was completed, the separated protein samples were tested with CD36 antibodies, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with rapid blocking solution for 2 hours at room temperature. After blocking, the PVDF membrane was incubated with CD36 primary antibody (ab252922, abcam) at 4°C overnight. Subsequently, the PVDF membrane was washed 3 times with TBST for 15 minutes each time to remove unbound primary antibody. After washing, the treated PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (A0208, Beyotime) at 4°C for 2 hours to bind the secondary antibody. β-actin (ab8226, abcam) was used as an internal control protein to ensure consistent protein loading and to correct experimental results. Finally, the PVDF membrane was washed to remove unbound secondary antibody. After washing, protein band signals were detected using an enhanced chemiluminescence (ECL) kit (170-5061, Bio-Rad) and a chemiluminescence system. The experiment was repeated three times.

[0160] The results are as follows Figure 13 Shown: Through Figure 13 It can be seen that the expression level of CD36 in MCF-7 cells is higher than that in MDA-MB-231 and 3T3 cells.

[0161] 3. Mouse breast cancer cells 3T3 (Baidi Biotech Ltd., C5133), MCF-7 (Baidi Biotech Ltd., C5103) and MDA-MB-231 (Baidi Biotech Ltd., C6291) were cultured in 6-well plates (5 × 10 3 Each well contained 2 mL of R1640 growth medium and was cultured in an incubator for 24 hours. After the cells adhered to the wall, the original culture medium was aspirated. The experimental grouping and specific procedures were as follows:

[0162] R1640 growth medium: RPMI-1640 (PM150110, Procell) + 10% (v / v) FBS (164210-50, Procell) + 1% P / S (PB180120, Procell).

[0163] Culture conditions: Gas phase: air, 95%; CO2, 5%; Temperature: 37°C.

[0164] The cells were co-cultured with 50 μg / mL DCP-TPP (labeled with fluorescein isothiocyanate: 5 μg / mL) for 3, 6, and 12 hours, respectively. Subsequently, the cells were washed three times with PBS and then treated with trypsin for 3 minutes to collect the cells. Finally, the mean fluorescence intensity of DCP-TPP-FITC in the three cell lines was analyzed by flow cytometry (Beckman Coulter, CRT). The experiment was repeated three times.

[0165] The results are as follows Figure 14 Shown: Through Figure 14 As can be seen, compared with cancer cells with low CD36 expression, MCF-7 cells with high CD36 expression increased their uptake of DCP-TPP-FITC by 2.58-fold, with significant time-dose-dependent internalization. This suggests that DCP-TPP can be effectively taken up by tumor cells with high CD36 expression.

[0166] 4. Culture mouse breast cancer cells 4T1 in 96-well plates, with 10 4 Each well contained 100 μL of R1640 growth medium. After the cells were cultured in an incubator for 24 hours and attached to the wall, the original culture medium was aspirated. The experimental grouping and specific procedures were as follows:

[0167] R1640 growth medium: RPMI-1640 (PM150110, Procell) + 10% (v / v) FBS (164210-50, Procell) + 1% P / S (PB180120, Procell).

[0168] Culture conditions: Gas phase: air, 95%; CO2, 5%; Temperature: 37°C.

[0169] DCP-TPP group: add 100 μL of fresh R1640 growth medium containing 0, 3.125, 6.25, 12.5, 25, 50, and 100 μg / ml of DCP-TPP and incubate for 12 h;

[0170] DCP-TPP+L group: 100 μL of DCP-TPP+L (pretreated with NIR, laser at 808 nm, 1 W / cm 2 , 10 minutes) in fresh R1640 growth medium and incubate for 12 hours.

[0171] The cell viability of the experimental group was detected by CCK-8. The experiment was repeated three times.

[0172] The experimental results are as follows Figure 15 :from Figure 15 It can be seen that the cell viability of DCP-TPP+L was significantly reduced compared with that of DCP-TPP, further indicating that NIR irradiation promoted the phase transformation of the DCP-TPP phase transformation shell, resulting in a large amount of release and direct contact of Cu3BiS3 and DSF, enhancing the synergistic cell spatiotemporally controllable copper death-photothermal therapy.

[0173] 5. Place 4T1 cells (10 cells per well) 4 Cells were inoculated in confocal microplates and cultured for 24 hours. Subsequently, DCP-TPP containing the fluorescent dye FITC (final concentration 100 μg / ml) was added and cultured for another 12 hours. To further observe the colocalization of DCP-TPP in different organelles, cells were stained with Hoechst 33342 (5 μM, for nuclear staining, Ex / Em = 346 / 460 nm), MitoTracker Red FM (0.5 μM, for mitochondrial labeling, Ex / Em = 579 / 599 nm), LysoTracker Red DND-99 (0.5 μM, for lysosomal labeling, Ex / Em = 577 / 590 nm), or ER-Tracker Red (1 μM, for endoplasmic reticulum labeling, Ex / Em = 587 / 615 nm). After staining, the cells were washed three times with PBS and the distribution of DCP-TPP in different organelles was observed under a confocal microscope. Finally, the colocalization coefficients of DCP-TPP in different organelles were calculated using Image J. The experiment was repeated three times.

[0174] The results are as follows Figure 16 Shown: From Figure 16 As can be seen, DCP-TPP highly overlaps with the mitochondrial fluorescence signal, exhibiting significant spatial colocalization. Quantitative analysis revealed a Manders colocalization coefficient (MCC) of 0.97 with mitochondria, significantly higher than its colocalization with lysosomes (MCC = 0.094) and the endoplasmic reticulum (MCC = 0.311). Therefore, DCP-TPP is primarily enriched in mitochondria within the cell and exhibits excellent mitochondrial targeting.

[0175] 3. Animal Model Experiment

[0176] Tumor-bearing mouse model was established by injecting 1×10 7 A 4T1 cell suspension was prepared at 400 μg / ml to establish a tumor-bearing Balb / c model. Fifty-six female BALB / c mice bearing tumors were randomly divided into seven groups (n=8), as follows:

[0177] (1) PBS group (200 μL); (2) PBS+L group (200 μL, 808 nm near-infrared light, 1 W / cm 2 , 10 minutes); (3) Cu3BiS3 group (20 mg / kg); (4) DSF group (5 mg / kg); (5) DCP group (without TPP) (20 mg / kg, containing DSF 5 mg / kg); (6) DCP-TPP group (20 mg / kg, containing DSF 5 mg / kg); (7) DCP-TPP+L group (20 mg / kg, containing DSF 5 mg / kg; 808 nm near-infrared light, 1 W / cm 2 For the PBS+L and DCP-TPP+L groups, the laser intensity was set to 1 W / cm 2 The irradiation time was 10 minutes to ensure uniform irradiation and standardization of experimental conditions. The drug was administered once every 2 days for a total of three times. After injection treatment, the body weight and tumor volume of the mice were monitored every other day. Tumor volume was calculated using the following formula:

[0178] V=0.5×L×W 2

[0179] Where V is the tumor volume, L is the tumor length, and W is the tumor width. The body weight and tumor volume of all mice were recorded regularly throughout the experiment until its conclusion. The efficacy of DCP-TPP in anti-tumor therapy was evaluated by comparing changes in tumor volume between treatment groups.

[0180] The results are as follows Figure 17 Shown: From Figure 17 As can be seen, the tumor growth inhibition rates in the PBS+L group, DSF group, Cu3BiS3 group, DCP group, and DCP-TPP group were 24.86%, 34.57%, 38.47%, 42.69%, and 58.60%, respectively. These groups all had limited inhibitory effects on tumor growth. The DCP-TPP+L group demonstrated the best effect in inhibiting tumor growth, with a tumor inhibition rate of up to 90%, significantly superior to the other treatment groups. Furthermore, survival monitoring over 10 weeks showed that the median survival period with DCP-TPP+L treatment was extended to 74 days, compared to 35 days in the PBS group.

[0181] Example 4

[0182] This embodiment provides another method for preparing copper nanoparticles, which adopts the same method as that of embodiment 1, except that the copper nanoparticles used are Cu 2-x Se. Other process steps and conditions are the same as those in Example 1. 2-x The preparation method of Se is:

[0183] 0.1mL sodium selenite (1000mM) is added in the Tween-80 solution (5mg / mL) of 10mL, then 0.1mL ascorbic acid (4000mM) is added dropwise, and mixed solution is stirred at room temperature for 1 hour.Then, 0.1mL CuCl2 (1100mM) is mixed with 0.1mL ascorbic acid (4000mM), is added dropwise in the above-mentioned mixed solution after standing for 5 minutes, and continues to stir.After 12 hours, the product is dialyzed 3 days with the deionized water of 50000 and 100 times of volume by molecular weight.Carry out freeze drying again, obtain copper nanoparticles Cu 2-x Se.

[0184] Copper nanoparticles Cu 2-x Se was characterized (the method is the same as in Example 3), and the results are as follows Figure 18 and Figure 19 As shown:

[0185] Figure 18 The crystal diffraction peaks of the copper nanoparticles Cu2-xSe prepared in this embodiment show that the synthesized Cu 2-x Se and single phase Cu 2-x The monoclinic structure of Se is completely consistent with the standard card JCPDS card number 06-0680. No other mass peaks were found, indicating that Cu 2-x Se nanoparticles were prepared with good purity.

[0186] Figure 19 The copper nanoparticles Cu prepared in this example 2-x X-ray photoelectron spectroscopy of Se. From the perspective of molecular formula, non-stoichiometric Cu 2-x Se can be considered as a mixture of stoichiometric Cu2Se and CuSe, and the valence states of Cu and Se can be determined by X-ray photoelectron spectroscopy (XPS). The binding energy of Se 3d in the XPS spectrum is 54.6 eV, which is attributed to Se 2- , while the peak at 58.8 eV is due to the oxidation of Se ( Figure 19 b) High-resolution XPS spectra of Cu 2p orbitals confirm that Cu + and Cu 2 + The existence of Cu + With Cu 2+ The ratio is 4.21( Figure 19 c) The Cu:Se ratio calculated from XPS results is 1.78, further proving that the Cu nanoparticles Cu 2-x Se NPs were successfully prepared.

[0187] Example 5

[0188] This example provides another method for preparing nanomedicines, using the same method as Example 2, except that the phase change material used is a mixture of tetradecanol and stearic acid (mass ratio 4:1). Other steps and conditions are the same as Example 2.

[0189] Observe the state changes of the mixture of tetradecyl alcohol and stearic acid at 37℃ and 39℃. The results are as follows Figure 20 As shown. Figure 20 It can be seen that at 39° C., the state thereof changes from solid to liquid, indicating that the phase change material in this embodiment has good temperature-sensitive phase change characteristics.

[0190] Example 6

[0191] This example provides another method for preparing a nanodrug, using the same method as Example 2, except that the mitochondrial targeting lipids used were phosphatidylcholine and SS-31-DSPE-PEG5000 (Xi'an Ruixi Biotechnology Co., Ltd., Catalog No. R-ROS-0240) (mass ratio 3:1). All other steps and conditions were the same as in Example 2 to prepare DCP-SS31 nanoparticles.

[0192] The liposomes (DCP-SS31) constructed by using SS-31-DSPE-PEG5000 in this example were tested for mitochondrial targeting (the specific method is the same as that in Example 3). Figure 21 As shown. Figure 21 As can be seen, DCP-SS31 highly overlaps with the mitochondrial fluorescence signal, exhibiting significant spatial colocalization. Quantitative analysis revealed a high Manders colocalization coefficient (MCC) of 0.907 with mitochondria, significantly higher than its colocalization with lysosomes (MCC = 0.306) and the endoplasmic reticulum (MCC = 0.375). Therefore, DCP-SS31 nanoparticles are primarily concentrated in mitochondria within cells, demonstrating excellent mitochondrial targeting.

[0193] In general, the present invention uses copper nanoparticles as the core, and the outer layer is wrapped with a thermosensitive phase-transition fatty acid nanocarrier. The surface of the nanocarrier is modified with mitochondrial-targeted lipids, which can effectively promote the accumulation of drugs in mitochondria. When the carrier enters the tumor tissue, it can be effectively taken up by tumor cells that highly express CD36 and then enriched in the mitochondria. After the fatty acid nanodrug is taken up by the tumor cells, the NIR near-infrared light is remotely triggered to promote the carrier from solid to liquid, resulting in the rapid release of copper nanoparticles and copper ion carriers, and the in situ generation of CuET, leading to cell apoptosis. In particular, the formation of CuET is accompanied by the formation of Cu 2+ Converted to Cu +, oligomerizes with mitochondrial proteins in the tricarboxylic acid cycle, and causes copper death in tumor cells. This solves the problems of existing nanoparticles, such as the serious side effects caused by premature release of drugs after entering the blood circulation, the difficulty in accumulation and uptake at the tumor site, and the low efficiency of drug release after entering the tumor cells, thereby achieving more precise and controllable killing of tumor cells.

[0194] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A near-infrared light-responsive fatty acid copper nanoparticle drug, characterized in that: The nanomedicine uses copper nanoparticles as the core, wraps the outer layer of the copper nanoparticles with a phase change material organic shell, loads copper ion carriers in the organic shell, and modifies mitochondrial targeting lipids on the shell.

2. The near-infrared light-responsive fatty acid copper nanomedicine according to claim 1, characterized in that: The copper nanoparticles are copper-containing compounds; The phase change material is an organic phase change material with near-infrared light response characteristics, including at least one of palmitic acid, stearic acid, capric acid, lauric acid, myristic acid, linoleic acid, trans-oleic acid, arachidic acid, behenic acid, tetradecanol, dodecanol, 1-tridecanol, 1-tetradecanol, 1-pentadecanol, hexadecanol and 1-octadecanol; The copper ion carrier includes at least one of disulfiram, ilisimol and 8-hydroxyquinoline; The mitochondria-targeted liposomes include at least one of triphenylphosphine cations, imidazolium salts and arginine-rich peptide-modified DSPE-PEG5000 liposomes; wherein the imidazolium salts are 1,3-dimethylimidazolium chloride and / or 1,3-dimethylimidazolium tetrafluoroborate.

3. The near-infrared light-responsive fatty acid copper nanomedicine according to claim 2, characterized in that: The copper nanoparticles are Cu3BiS3, Cu 2-x At least one of Se, CuCl2, Cu(NO3)2, CuSO4, CuO and CuS; The phase change material is a eutectic mixture obtained by mixing lauric acid and stearic acid in a mass ratio of 1 to 4:1, or a eutectic mixture obtained by mixing tetradecanol and stearic acid in a mass ratio of 1 to 4:1; The mitochondria-targeted liposome is at least one of DSPE-PEG5000-TPP and SS-31-DSPE-PEG5000.

4. The near-infrared light-responsive fatty acid copper nanomedicine according to claim 3, characterized in that: The Cu3BiS3 is prepared by the following method: bismuth salt and copper salt are dispersed in oleylamine, and after degassing, the mixture is stirred and mixed uniformly at 60-160°C, and then the temperature is raised to 160-500°C and maintained for 5-15 minutes, and then a sulfur source is quickly injected to react. After the reaction is completed, the reaction temperature is cooled to 70±5°C, cyclohexane is added, the product is collected by centrifugation, and washed with ethanol to obtain Cu3BiS3 nanoparticles; The Cu 2-x Se was prepared by the following method: sodium selenite solution was added to Tween-80 solution, ascorbic acid solution was added dropwise and stirred at room temperature, then a mixed solution of CuCl2 and ascorbic acid was added, stirring was continued, after the reaction was completed, dialyzed, and freeze-dried to obtain copper nanoparticles Cu 2-x Se.

5. The near-infrared light-responsive fatty acid copper nanomedicine according to claim 4, characterized in that: The bismuth salt is at least one of Bi(NO3)3·5H2O, Bi(NO3)3, Bi(OAc)3 and bismuth octanoate; The copper salt is at least one of Cu(CH3COO)2, Cu(NO3)2·3H2O and CuCl2·2H2O; The sulfur source is at least one of thioacetamide, elemental sulfur, Na2S·9H2O and sodium diethyldithiocarbamate; The molar ratio of the bismuth salt, the copper salt and the sulfur source is 1:1-6:3-12; The molar ratio of the sodium selenite, ascorbic acid and CuCl2 is 1:8:1.

1.

6. The method for preparing the near-infrared light-responsive fatty acid copper nanomedicine according to any one of claims 1 to 5, characterized in that: The specific steps include: (1) dissolving the phase change material in methanol to obtain a fatty acid PCM solution; then adding a copper ion carrier to obtain a mixed solution I; (2) dissolving mitochondria-targeted liposomes and lecithin in an ethanol aqueous solution, and then uniformly mixing with an aqueous solution of copper nanoparticles to obtain a mixed solution II; (3) adding the mixed solution I to the mixed solution II under vigorous stirring, and reacting at 50-95° C. to obtain a mixed solution III; The mixed solution III is then cooled in ice water until it becomes turbid to obtain a mixed solution IV; (4) The mixed solution IV is filtered to remove unencapsulated molecules and organic solvents, and then collected by centrifugation, washed, and purified to obtain near-infrared light-responsive fatty acid copper nanomedicine.

7. The preparation method according to claim 6, characterized in that: The concentration of the fatty acid PCM solution in step (1) is 1 to 40 mg / mL; The concentration of the copper ion carrier in the mixed solution I described in step (1) is 0.1 to 10 mg / mL; The mass ratio of the mitochondria-targeted liposomes to phosphatidylcholine in step (2) is 1 to 20:1; The concentration of the ethanol aqueous solution in step (2) is 1 to 10% by mass; In step (2), the mitochondria-targeted liposomes are dissolved in an ethanol-water solution to a final concentration of 1 to 10 mg / mL; The concentration of the copper nanoparticle aqueous solution in step (2) is 0.1 to 10 mg / mL; The volume ratio of the mixed solution I and the mixed solution II in step (2) is 1:1-10.

8. The preparation method according to claim 6, characterized in that: The rotation speed of the vigorous stirring in step (3) is 100 to 1000 rpm / min; The reaction temperature in step (3) is 50°C to 60°C; The reaction time in step (3) is 5 to 10 minutes; The cooling time in step (3) is 5 to 10 minutes; The filtration in step (4) is performed using a 0.2 μm surfactant-free cellulose acetate membrane; The centrifugal speed in step (4) is 1000-10000g; The purification described in step (4) is performed using a VIVASPIN 6 centrifugal concentrator with a molecular weight cut-off of 10 kDa; The washing in step (4) is performed using deionized water.

9. Use of the near-infrared light-responsive fatty acid copper nanomedicine according to any one of claims 1 to 5 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that: The drug includes a near-infrared photothermal therapy agent or a copper death-photothermal therapy drug; The tumor is a tumor expressing CD36.

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