Sonodynamic therapy combined with copper death nano platform and preparation method and application thereof

Abstract

The application discloses a kind of acoustic power combined copper death nano platform and preparation method thereof, the nano platform includes Cu@Mg-Al LDH nanoparticle, the Cu@Mg-Al LDH nanoparticle includes Mg-Al layered double hydroxide (Mg-Al LDH) nanosheet as matrix, and Cu ion is doped in the lattice of Mg-Al LDH nanosheet;Wherein, by previously synthesized Mg-Al LDH nanosheet is contacted with the solution containing Cu ion, and by ion isomorphism substitution reaction makes Cu ion be inserted into the lattice of Mg-Al LDH nanosheet, so as to complete the doping of Cu ion.The nano platform of the application is prepared by first skeleton and then copper substitution, avoids the generation of impurity phase and introduces lattice distortion to form high active site, has the functions of oxygen release, oxygen improvement, ultrasonic excitation ROS generation and copper release induced copper death, and efficient antitumor is realized by acoustic power and copper death combination.

Description

Technical Field

[0001] This invention relates to the field of medical nanomaterials technology, and more specifically to a sonodynamically coupled copper death nanoplatform, its preparation method, and its applications. Background Technology

[0002] Cancer immunotherapy and physiochemical therapy are currently hot research topics in the field of tumor treatment. Among them, sonodynamic therapy (SDT), as a non-invasive treatment, utilizes the high tissue penetration of ultrasound to activate sonosensitive agents at the tumor site to generate highly toxic reactive oxygen species (ROS), thereby achieving the goal of precisely killing tumor cells. However, the hypoxic characteristics prevalent in the tumor microenvironment (TME) severely limit the efficiency of oxygen-dependent SDT, resulting in limited clinical efficacy in the treatment of deep tumors.

[0003] In recent years, layered bimetallic hydroxides (LDHs) have shown promise in drug delivery and synergistic therapy due to their unique two-dimensional layered structure, good biocompatibility, and tunable composition. Studies have shown that introducing transition metal ions (such as Cu ions) into LDHs can endow them with enzyme-like activity or induce copper death in cells, thereby producing a synergistic effect with SDTs. However, existing Cu-based LDHs are usually synthesized using a one-step co-precipitation method. Due to the difference in hydrolysis constants between Cu ions and the carrier metal ions, impurities such as copper hydroxide are easily precipitated during this process, resulting in low product purity and uncontrollable lattice defects, which in turn weakens their catalytic activity and biosafety.

[0004] Therefore, how to develop a structurally stable, precisely composed, and effective acoustic-dynamically combined copper death nanoplatform that can overcome the limitations of tumor hypoxia is a key technical problem that urgently needs to be solved in the field of nanomedicine. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a sonodynamically coupled copper death nanoplatform and its preparation method. This nanoplatform is prepared by first building a framework and then replacing copper, which avoids the generation of impurity phases and introduces lattice distortion to form highly active sites. It also has the functions of releasing oxygen to improve hypoxia, generating ROS through ultrasound excitation, and releasing copper to induce copper death. The combination of sonodynamics and copper death achieves highly efficient anti-tumor effects.

[0006] According to a first aspect of the present invention, a sonic-dynamically coupled copper death nanoplatform is provided, the nanoplatform comprising Cu@Mg-Al LDH nanoparticles, the Cu@Mg-Al LDH nanoparticles comprising Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets as a matrix, and Cu ions doped in the lattice of the Mg-Al LDH nanosheets;

[0007] In this process, Cu ion doping is achieved by contacting pre-synthesized Mg-Al LDH nanosheets with a solution containing Cu ions and then subjecting them to an ion isomorphic substitution reaction to embed Cu ions into the lattice of the Mg-Al LDH nanosheets.

[0008] As an optional implementation, the Cu ions exist in a form where multiple valence states coexist, including Cu... + and Cu 2+ .

[0009] As an optional implementation, the molar ratio of magnesium, copper, and aluminum in the Cu@Mg-Al LDH nanoparticles is (1.06~1.08):(0.24~0.30):1.

[0010] As an optional implementation, the Cu@Mg-Al LDH nanoparticles have an average particle size of 99.2±10.7 nm and a Zeta potential of 47.5±2.8 mV.

[0011] According to a second aspect of the present invention, a method for preparing the aforementioned acoustic-dynamically coupled copper death nanoplatform is provided, comprising the following steps:

[0012] Mg-Al layered double hydroxide (Mg-AlLDH) nanosheets were prepared by hydrothermal method using magnesium salts, aluminum salts and alkaline solutions as raw materials.

[0013] At room temperature, Mg-Al LDH nanosheets were mixed with an aqueous solution of copper salt and stirred to allow Cu ions to embed into the lattice of the Mg-Al LDH nanosheets. After centrifugation and washing, Cu@Mg-Al LDH nanoparticles were collected.

[0014] As an optional implementation method, the specific process for preparing Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets using a hydrothermal method includes:

[0015] At room temperature, a mixed aqueous solution of MgCl2·6H2O and AlCl3·6H2O was added dropwise to an aqueous solution of NaOH. The homogeneous solution was stirred, and the precipitate was collected by centrifugation. The precipitate was then washed and redispersed in deionized water.

[0016] The redispersed precipitate was subjected to a hydrothermal reaction. After natural cooling to room temperature, the resulting solution was collected, centrifuged, and Mg-Al LDH nanosheets were collected. The Mg-Al LDH nanosheets were then redispersed in deionized water for later use.

[0017] As an optional implementation, the molar ratio of MgCl2·6H2O, AlCl3·6H2O and NaOH is (6~8):(2~4):(16~20).

[0018] As an optional implementation, the hydrothermal reaction conditions are: temperature 100℃~110℃, reaction time 4 h~5 h.

[0019] As an optional implementation method, Mg-Al LDH nanosheets and Cu in copper salts 2+ The molar ratio is 1.7:1 to 2.1:1, and the copper salt includes CuCl2·2H2O.

[0020] In a third aspect of the present invention, the application of the aforementioned acoustic-dynamic combined copper death nanoplatform in the preparation of antitumor drugs is provided.

[0021] As can be seen from the above technical solutions of the present invention, the acoustic-dynamic combined copper death nanoplatform proposed in this invention has the following significant beneficial effects:

[0022] (1) Precise control of crystal structure was achieved, significantly improving product purity.

[0023] This invention employs an ion isomorphic substitution strategy of "building the framework first, then adding Cu," which helps overcome the technical bottleneck of Cu ions easily forming impurity phases such as copper hydroxide due to differences in hydrolysis constants in the traditional one-step coprecipitation method. This strategy ensures that Cu ions can be stably and uniformly embedded in the Mg-Al LDH lattice, obtaining high-purity single-phase nanocrystals while maintaining the integrity of the layer structure, thus laying the structural foundation for subsequent catalytic activity.

[0024] (2) By introducing lattice distortion effect, highly active catalytic sites were constructed.

[0025] Through ionic isomorphic substitution, Cu ions with different atomic radii induce local lattice distortion and internal stress in the LDH layer. This change in microstructure breaks the original symmetry, optimizes the electron distribution, and thus forms a large number of highly active catalytic centers. These active sites not only enhance the enzyme-like catalytic efficiency, but also significantly reduce the energy barrier of the sonodynamic reaction.

[0026] (3) It has the ability to produce oxygen independently, which effectively overcomes the drug resistance of tumor hypoxia.

[0027] The Cu@Mg-Al LDH nanoparticles prepared in this invention have excellent catalase-like (CAT) activity, which can convert hydrogen peroxide highly expressed in the tumor microenvironment into oxygen in situ. This autonomous oxygen production mechanism directly alleviates the hypoxic state inside the tumor, continuously providing raw materials for sonodynamic therapy and solving the problem of low efficiency or even failure of traditional sonosensitive agents in hypoxic environments.

[0028] (4) It achieves efficient ROS increase under both aerobic and hypoxic mechanisms.

[0029] Thanks to the introduction of Cu ions and the special crystal structure, the nanoplatform of this invention exhibits excellent acoustic-dynamic performance under ultrasonic excitation. It can not only generate ROS through an oxygen-dependent pathway, but also generate sufficient reactive oxygen species through the direct interaction of electron-hole pairs in an oxygen-deficient environment, thus achieving efficient ROS generation in all environments and significantly improving the anti-tumor killing efficiency.

[0030] (5) Synergistic copper death and sonodynamic therapy to construct a new multimodal combined treatment approach.

[0031] The nanoplatform of this invention can controllably release Cu ions in the tumor microenvironment, inducing intracellular copper overload and triggering abnormal mitochondrial metabolism, thereby causing copper death in tumor cells. This novel cell death mode has a significant synergistic effect with ROS damage generated by sonodynamics. Through the dual attack of biochemical toxicity and physicochemical killing, it provides an efficient and precise comprehensive solution for the complete eradication of tumors. Attached Figure Description

[0032] Figure 1 This is a transmission electron microscope image of Cu@Mg-Al LDH nanoparticles in the example of this invention. Figure 1 Part A of the image), scanning electron microscope image ( Figure 1 Part B of the diagram) and the layer thickness measurement diagram ( Figure 1 (Part C of the text).

[0033] Figure 2 This is the elemental mapping diagram of Cu@Mg-Al LDH nanoparticles in the example of this invention. Figure 2 Part A of the analysis), XPS analysis ( Figure 2 Part B of the diagram and the mass ratio of metal ions detected by ICP (in the diagram). Figure 2 (Part C of the text).

[0034] Figure 3 This is an inset showing the DLS particle size distribution and Zeta potential diagram of Cu@Mg-Al LDH nanoparticles in the example of this invention.

[0035] Figure 4 This is the XRD pattern of Cu@Mg-Al LDH nanoparticles in the example of this invention.

[0036] Figure 5 This is the Nyquist plot of Cu@Mg-Al LDH nanoparticles in the example of this invention.

[0037] Figure 6 This refers to the catalase-like ability of the Cu@Mg-Al LDH nanoparticles in the example of this invention; wherein, Figure 6Part A in the figure represents the catalase-like ability of Cu@Mg-Al LDH in hydrogen peroxide solution; Figure 6 Part B in the text describes the catalase-like ability of Cu@Mg-Al LDH nanoparticles in mouse 4T1 breast cancer cells.

[0038] Figure 7 The ultrasonic stimulation in this invention example promotes the ability of Cu@Mg-Al LDH nanoparticles to generate reactive oxygen species (ROS).

[0039] Figure 8 This refers to the ability of Cu@Mg-Al LDH nanoparticles to release copper ions in mouse 4T1 breast cancer cells under ultrasonic stimulation, as described in this invention example.

[0040] Figure 9 This invention demonstrates the ability of Cu@Mg-Al LDH nanoparticles, under ultrasonic stimulation, to induce increased aggregation of dihydrolipoic acid S-acetyltransferase (DLAT) in mouse 4T1 breast cancer cells.

[0041] Figure 10 This is a diagram illustrating the therapeutic effect of Cu@Mg-Al LDH nanoparticles on mouse 4T1 breast cancer cells under ultrasonic stimulation, as shown in this invention example.

[0042] Figure 11 This is an in vivo therapeutic effect diagram of Cu@Mg-Al LDH nanoparticles on 4T1 breast cancer in mice under ultrasonic stimulation, as shown in the example of this invention. Detailed Implementation

[0043] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0044] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0045] Acoustic dynamics combined with copper death nanoplatform

[0046] In one embodiment of the present invention, a nanoplatform for co-electro-copper death is provided, the nanoplatform comprising Cu@Mg-Al LDH nanoparticles, wherein the Cu@Mg-Al LDH nanoparticles comprise Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets as a matrix, and Cu ions doped in the lattice of the Mg-Al LDH nanosheets.

[0047] In this process, Cu ion doping is achieved by contacting pre-synthesized Mg-Al LDH nanosheets with a solution containing Cu ions and then subjecting them to an ion isomorphic substitution reaction to embed Cu ions into the lattice of the Mg-Al LDH nanosheets.

[0048] In some embodiments, Cu ions exist in a form where multiple valence states coexist, including Cu... + and Cu 2 + The two valence states of Cu ions synergistically enhance enzyme-like catalytic activity and sonodynamic performance.

[0049] In some embodiments, the molar ratio of magnesium, copper, and aluminum in Cu@Mg-Al LDH nanoparticles is (1.06~1.08):(0.24~0.30):1.

[0050] In some embodiments, the average particle size of Cu@Mg-Al LDH nanoparticles is 99.2 ± 10.7 nm.

[0051] In some embodiments, the Zeta potential of Cu@Mg-Al LDH nanoparticles is 47.5 ± 2.8 mV.

[0052] Preparation method of nanoplatform for acoustic dynamics combined with copper death

[0053] In another embodiment of the present invention, a method for preparing the aforementioned acoustic-dynamic combined copper death nanoplatform is provided, comprising the following steps:

[0054] Mg-Al layered double hydroxide (Mg-AlLDH) nanosheets were prepared by hydrothermal method using magnesium salts, aluminum salts and alkaline solutions as raw materials.

[0055] At room temperature, Mg-Al LDH nanosheets were mixed with an aqueous solution of copper salt and stirred to allow Cu ions to embed into the lattice of the Mg-Al LDH nanosheets. After centrifugation and washing, Cu@Mg-Al LDH nanoparticles were collected.

[0056] In some embodiments, the specific process for preparing Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets using a hydrothermal method includes:

[0057] At room temperature, a mixed aqueous solution of MgCl2·6H2O and AlCl3·6H2O was added dropwise to an aqueous solution of NaOH. The homogeneous solution was stirred, and the precipitate was collected by centrifugation. The precipitate was then redispersed in deionized water.

[0058] The redispersed precipitate was subjected to a hydrothermal reaction. After natural cooling to room temperature, the resulting solution was collected, centrifuged, and Mg-Al LDH nanosheets were collected. The Mg-Al LDH nanosheets were then redispersed in deionized water for later use.

[0059] In some embodiments, the molar ratio of MgCl2·6H2O, AlCl3·6H2O and NaOH is (6~8):(2~4):(16~20), and is particularly preferred to be 7:3:18.

[0060] In some embodiments, the hydrothermal reaction conditions are: temperature 100℃~110℃, reaction time 4 h~5 h.

[0061] In some embodiments, Mg-Al LDH nanosheets and Cu in copper salt 2+ The molar ratio is 1.7:1 to 2.1:1, and more preferably 1.9:1 to 2.0:1; the copper salt includes CuCl2·2H2O.

[0062] Below, using MgCl2·6H2O as the magnesium salt, AlCl3·6H2O as the aluminum salt, CuCl2·2H2O as the copper salt, and NaOH aqueous solution as the alkaline solution, an exemplary method for preparing a sonodynamically coupled copper death nanoplatform is given, including the following specific steps:

[0063] {Preparation of Mg-Al LDH nanosheets using a hydrothermal method}

[0064] At room temperature, a mixed aqueous solution of MgCl2·6H2O and AlCl3·6H2O (molar ratio of MgCl2·6H2O, AlCl3·6H2O and NaOH is 7:3:18) was added dropwise to NaOH aqueous solution. After stirring vigorously for 10 min, the resulting homogeneous solution was centrifuged to collect the precipitate, the precipitate was washed and redispersed in deionized water.

[0065] The redispersed product was transferred to a polytetrafluoroethylene-lined stainless steel autoclave, sealed, and placed in an oven. It was allowed to stand at 100℃~110℃ for 4 h~5 h. After naturally cooling to room temperature, the resulting solution was collected, centrifuged, and Mg-Al LDH nanosheets were collected. The Mg-Al LDH nanosheets were then redispersed in deionized water for later use.

[0066] {Ionic isomorphic substitution with copper ions}

[0067] At room temperature, a suspension of Mg-Al LDH nanosheets was added dropwise to an aqueous solution of CuCl2·2H2O (Mg-Al LDH nanosheets and Cu in copper salt). 2+The molar ratio of Cu to Mg-Al was 1.9:1. After stirring vigorously for 4 h, the mixture was centrifuged and washed with deionized water to obtain Cu@Mg-Al LDH nanoparticles.

[0068] application

[0069] In other embodiments of the present invention, the application of the aforementioned acoustic-dynamic combined copper death nanoplatform in the preparation of antitumor drugs is provided.

[0070] In other embodiments, an antitumor drug is also provided, comprising the aforementioned sonodynamically coupled copper death nanoplatform. When using this drug for antitumor treatment, the ultrasonic excitation process parameters are as follows:

[0071] The ultrasonic frequency is 1 MHz to 3 MHz, and the ultrasonic intensity is 1 W / cm². 2 ~3 W / cm 2 Under the condition of ultrasound duty cycle of 40%~50%, ultrasound time is 2 min~5 min.

[0072] Given the high heterogeneity of tumor cell populations, single-modality treatment often leads to adaptive resistance due to compensatory mechanisms. This invention combines the strong oxidative damage induced by sonodynamic therapy (SDT) with copper ion-induced metabolic collapse (copper death), achieving cell death through two completely different biological pathways. This combined strategy can effectively overcome tumor heterogeneity and drug resistance.

[0073] Ultrasound can penetrate deep tissues without damage, avoiding damage to surrounding normal tissues, thus achieving truly precise treatment and overcoming the off-target toxicity problem that may exist in traditional ROS therapy; at the same time, the catalase-like activity of the material alleviates the hypoxic environment of the tumor and improves the level of SDT.

[0074] The advantage of this combined anti-tumor strategy lies in its ability to achieve efficient and precise treatment results through the synergy of multiple mechanisms.

[0075] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0076] Unless otherwise specified, the following embodiments are all conventional methods.

[0077] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0078] For ease of description, Cu@Mg-Al LDH nanoparticles in the examples and accompanying drawings are abbreviated as Cu-LDH, and Mg-Al LDH nanosheets are abbreviated as LDH.

[0079] The quantitative method for Mg-Al LDH nanosheet suspension is as follows: take 1 mL of the prepared Mg-Al LDH nanosheet suspension, centrifuge at 10000 rpm, discard the supernatant, place it in an oven for 12 h, weigh it, calculate the mass concentration, the sample size is 3, and take the average value.

[0080] Example 1

[0081] Synthesis and Characterization of Cu@Mg-Al LDH Nanoparticles

[0082] {Preparation of Mg-Al LDH Nanosheets}

[0083] At room temperature, 10 mL of a mixed aqueous solution of 0.7 mol / L MgCl2·6H2O and 0.3 mol / L AlCl3·6H2O was added dropwise to 40 mL of 0.45 mol / L NaOH aqueous solution. After stirring at 1000 rpm for 10 min, the resulting homogenized solution was centrifuged at 8000 rpm for 15 min to collect the precipitate. The product was washed three times with deionized water and then redispersed in 40 mL of deionized water.

[0084] The redispersed product was transferred to a polytetrafluoroethylene-lined stainless steel autoclave, sealed, and placed in an oven. It was allowed to stand at 100 °C for 4 h. After naturally cooling to room temperature, the resulting solution was collected. After centrifugation at 10,000 rpm for 15 min, Mg-Al LDH nanosheets were collected and redispersed in 10 mL of deionized water for later use.

[0085] {Preparation of Cu@Mg-Al LDH Nanoparticles}

[0086] At room temperature, 10 mL of Mg-Al LDH nanosheet suspension was added dropwise to 40 mL of 20 mmol / L CuCl2·2H2O aqueous solution, stirred at 1000 rpm for 4 h, centrifuged at 10000 rpm for 15 min, washed three times with deionized water, and Cu@Mg-Al LDH nanoparticles were collected and resuspended in 40 mL of deionized water.

[0087] {Representation}

[0088] The obtained solution was characterized by transmission electron microscopy, atomic force microscopy, layer thickness measurement, elemental analysis, XRD, DLS particle size distribution and zeta potential, and EIS measurement. The results are as follows: Figures 1-5 As shown.

[0089] Figure 1Part A in the figure shows the morphology of the synthesized Cu@Mg-Al LDH nanoparticles. Figure 1 Part B in the image shows an atomic force microscope image of the synthesized material. Figure 1 Part C in the image shows that the thickness of the nanosheet is approximately 0.645 nm.

[0090] Figure 2 The elemental distribution diagram in section A shows the distribution of Cu, Mg, and Al elements. Figure 2 The XPS image in section B shows that Cu is present in the synthesized Cu@Mg-Al LDH nanoparticles. + and Cu 2+ Copper in two valence states, Cu 2+ A stronger signal indicates a higher relative content of the substance in the sample. Figure 2 Section C shows the final mass ratio of the three metal cations after ICP detection. The calculated final molar ratio of magnesium, copper, and aluminum is Mg:Cu:Al=(1.07±0.01):(0.27±0.03):1, (Mg+Cu):Al=(1.34±0.03):1. This ratio is within the stable composition range of LDH material, indicating that the prepared Cu@Mg-Al LDH conforms to the compositional characteristics of typical hydrotalcite materials.

[0091] Figure 3 The average particle size of the Cu@Mg-Al LDH nanoparticles is approximately 99.2 ± 10.7 nm, and the surface charge is 47.5 ± 2.8 mV.

[0092] Figure 4 XRD patterns show the crystal structure of the synthesized Cu@Mg-Al LDH nanoparticles. The isomorphic substitution of copper ions leads to local lattice deformation, which indirectly affects the interlayer stacking mode. The diffraction peaks of the crystal planes shift to lower angles, which directly proves that the interlayer spacing is increased, further indicating the incorporation of copper ions.

[0093] Figure 5 The Nyquist plot shows that Cu@Mg-Al LDH nanoparticles have a smaller capacitive arc diameter, reflecting their lower charge transfer resistance and greater potential as acoustic sensors.

[0094] Therefore, it can be seen that the present invention successfully embedded Cu ions stably and uniformly into the lattice of Mg-Al LDH nanosheets, obtaining high-purity single-phase nanocrystals while maintaining the integrity of the layer structure, and introducing lattice distortion, indicating that the desired Cu@Mg-Al LDH nanoparticles were successfully synthesized.

[0095] Example 2

[0096] [Detection of the catalase-like ability of Cu-LDH]

[0097] Using Ru(dpp)3Cl2 (Luminescent oxygen sensor) as a probe to measure oxygen production, the fluorescence intensity of [Ru(dpp)3]Cl2 was significantly reduced due to dynamic quenching caused by molecular oxygen. Therefore, molecular oxygen was detected by measuring light intensity: the experimental groups were US group, Cu-LDH group, and Cu-LDH + US group; the ultrasonic frequency was 3 MHz and the ultrasonic intensity was 3 W / cm. 2 The ultrasound duty cycle was 50%, and the ultrasound time was 3 min; the Cu-LDH concentration was 2 mg / mL; oxygen generation was determined by reacting with 3% H2O2; and luminescence was recorded using a small animal fluorescence imaging system (IVIS, λex = 488 nm, λem = 620 nm).

[0098] Improvement in hypoxia was detected in 4T1 cells: cells were inoculated at a concentration of 1×10⁻⁶ cells / cells. ^4 Cells were seeded at a density of / wells into 6-well plates and cultured overnight in an incubator under hypoxia. After treatment with Ru(dpp)3Cl2 (50 μM) for 4 h, Cu-LDH was added for 12 h. The cells were washed 3 times to remove residual nanomaterials, sonicated, and then cultured under hypoxia for another 4 h. Cell fluorescence was then observed at Ex 488 nm and Em 610 nm, and images were recorded using a fluorescence microscope.

[0099] The results are as follows Figure 6 As shown, Figure 6 Part A of the results showed a significant decrease in the fluorescence intensity of [Ru(dpp)3]Cl2 in the Cu-LDH group, and the same was true in the Cu-LDH + US group; the results of cell experiments... Figure 6 Part B also shows a decrease in the red fluorescence of [Ru(dpp)3]Cl2 in the Cu-LDH group and the Cu-LDH + US group, indicating that the Cu-LDH of the present invention has catalase-like ability and can alleviate the hypoxic environment of tumors to a certain extent.

[0100] Example 3

[0101] [Detection of Cu-LDH-induced reactive oxygen species (•OH) generation under ultrasound (US) stimulation]

[0102] The TMB detection reagent is used as a probe to measure the generation of hydroxyl radicals (•OH). Hydroxyl radicals can cause TMB to undergo a color reaction (changing from colorless to blue), and it can be detected by measuring the absorbance at 650 nm using ultraviolet-visible spectroscopy.

[0103] The experimental groups were the US group, the Cu-LDH group, and the Cu-LDH + US group, with an ultrasonic frequency of 3 MHz and an ultrasonic intensity of 3 W / cm. 2 The ultrasonic duty cycle was 50%, and the ultrasonic time was 3 min; the Cu-LDH concentration was 2 mg / mL; the formation of •OH was determined by reacting Cu-LDH with H₂O₂. The results are as follows: Figure 7 As shown.

[0104] Figure 7 The results showed that the absorption peak of TMB at 650 nm of Cu-LDH increased significantly in the ultrasonic treatment group, indicating an increase in the generation of •OH, which shows that Cu-LDH of the present invention can effectively generate reactive oxygen species (•OH) under ultrasonic stimulation.

[0105] Example 4

[0106] [Test on the ability of ultrasound to promote copper ion release and induce copper death]

[0107] Detection of increased copper ions in 4T1 cells: Cells were inoculated at a concentration of 1×10⁻⁶ cells / cells. ^4 The cells were seeded at a density of / wells into 6-well plates and cultured overnight in an incubator; after treatment with Cu-LDH for 12 h, washed 3 times to remove residual nanomaterials, and sonicated, the cells were cultured for another 12 h; Coppersonsor 1 solution (50 μM) was added and incubated for 30 min; after washing 3 times with PBS, images were recorded using a fluorescence microscope, and the results are as follows. Figure 8 As shown.

[0108] Increased accumulation of dihydrolipoamide S-acetyltransferase (DLAT) in 4T1 cells was detected: DLAT levels were assessed using immunofluorescence analysis. Cell culture and treatment were as described previously. After fixation, permeabilization, and blocking, 4T1 cells were co-incubated overnight with anti-DLAT antibody. Subsequently, fluorescence imaging was performed using AF488-labeled secondary antibody and DAPI. The results are as follows: Figure 9 As shown.

[0109] Figure 8 This is a fluorescence change graph of the probe Coppersonsor 1. After sonication in Cu-LDH, obvious red fluorescence of Coppersonsor 1 can be observed, indicating that a large number of copper ions were produced in the cell.

[0110] Figure 9 This is an immunofluorescence image of DLAT. The abnormal aggregation of DLAT under the condition of increased copper ion release indicates cellular metabolic disorder, making cells more sensitive to copper death. The observation results are consistent with the characteristic phenotype of copper death.

[0111] Example 5

[0112] [Therapeutic effects of Cu-LDH on 4T1 breast cancer at both cellular and in vivo levels]

[0113] 4T1 cells were loaded at a rate of 1×10⁴ cells per well. ^4 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated for 12 h. Then, fresh medium containing different concentrations (0-200 µg / mL) of Cu-LDH was added to each well, and the cells were co-incubated for another 12 h. Following sonication, the cells were cultured for another 12 h. Subsequently, 100 μL of CCK-8 solution was added to each well, and the cells were cultured in the dark at 37°C for 2 h. The absorbance at 450 nm was then recorded using a microplate reader.

[0114] 4T1 cells (1×10) ^6 (Number of tumors) were subcutaneously injected into the right back of Balb / c mice to establish a tumor model. When the tumor volume reached approximately 50-100 mm... 3 Mice carrying tumors were randomly divided into two groups (n=5 in each group) and treated intravenously with the following: (1) control group, (2) Cu-LDH group (20 mg / kg), and (3) Cu-LDH + US group (20 mg / kg). The ultrasound frequency was 3 MHz and the ultrasound intensity was 3 W / cm. 2 The ultrasound duty cycle was 50%, and the ultrasound duration was 3 minutes. Tumor volume was measured in all groups every two days. The formula for calculating tumor volume was [(length × width)]. ^2 ) / 2], the result is as follows Figures 10-11 As shown.

[0115] Figure 10 The results showed that Cu-LDH + US had a significant killing effect on tumor cells at the cellular level. Figure 11 This is an in vivo treatment effect diagram, showing that Cu-LDH + US can significantly inhibit tumor growth compared with the control group.

[0116] Therefore, it can be proven that the Cu-LDH of the present invention can effectively kill tumor cells under the action of ultrasound.

[0117] As can be seen from the above, the present invention, through the ion substitution strategy of "first building the framework and then adding Cu", can effectively avoid the generation of impurity phases. On the other hand, copper ions will also introduce lattice distortion and stress in the LDH layer. These local structural changes are usually highly active catalytic sites.

[0118] The obtained sonodynamic combined with copper death nanoplatform possesses catalase-like activity. Through catalase-like activity, it catalyzes hydrogen peroxide in situ to generate oxygen, thereby alleviating tumor hypoxia. It also utilizes the EPR effect to precisely enrich the tumor site, achieving efficient generation of reactive oxygen species (ROS) under ultrasound excitation in all environments. Simultaneously, by releasing copper ions, it induces intracellular copper overload, promotes the aggregation of dihydrolipoamide S-acetyltransferase (DLAT) and the loss of iron-sulfur cluster protein expression, triggering protein toxicity stress and copper death. Ultimately, through the dual synergistic mechanism of physicochemical damage and metabolic collapse, it achieves efficient and precise anti-tumor therapy.

[0119] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A nanoplatform for acoustic-dynamic combined copper death, characterized in that, The nanoplatform includes Cu@Mg-Al LDH nanoparticles, which include Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets as the matrix and Cu ions doped in the Mg-Al LDH nanosheet lattice. In this process, Cu ion doping is achieved by contacting pre-synthesized Mg-Al LDH nanosheets with a solution containing Cu ions and then subjecting them to an ion isomorphic substitution reaction to embed Cu ions into the lattice of the Mg-Al LDH nanosheets.

2. The acoustodynamically coupled copper death nanoplatform according to claim 1, characterized in that, The Cu ions exist in a form where multiple valence states coexist, including Cu + and Cu 2+ .

3. The acoustodynamically coupled copper death nanoplatform according to claim 1, characterized in that, In the Cu@Mg-AlLDH nanoparticles, the molar ratio of magnesium, copper, and aluminum is (1.06~1.08):(0.24~0.30):

1.

4. The acoustodynamically coupled copper death nanoplatform according to claim 1, characterized in that, The Cu@Mg-AlLDH nanoparticles have an average particle size of 99.2±10.7 nm and a Zeta potential of 47.5±2.8 mV.

5. A method for preparing a nanoplatform for acoustic-dynamic combined copper death as described in any one of claims 1-4, characterized in that, Includes the following steps: Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets were prepared by hydrothermal method using magnesium salts, aluminum salts and alkaline solutions as raw materials. At room temperature, Mg-Al LDH nanosheets were mixed with an aqueous solution of copper salt and stirred to allow Cu ions to embed into the lattice of the Mg-Al LDH nanosheets. After centrifugation and washing, Cu@Mg-Al LDH nanoparticles were collected.

6. The method for preparing the acoustodynamically coupled copper death nanoplatform according to claim 5, characterized in that, The specific process for preparing Mg-Al layered double hydroxide (Mg-Al LDH) nanosheets using the hydrothermal method includes: At room temperature, a mixed aqueous solution of MgCl2·6H2O and AlCl3·6H2O was added dropwise to an aqueous solution of NaOH. The homogeneous solution was stirred, and the precipitate was collected by centrifugation. The precipitate was then washed and redispersed in deionized water. The redispersed precipitate was subjected to a hydrothermal reaction. After natural cooling to room temperature, the resulting solution was collected, centrifuged, and Mg-Al LDH nanosheets were collected. The Mg-Al LDH nanosheets were then redispersed in deionized water for later use.

7. The method for preparing the acoustodynamically coupled copper death nanoplatform according to claim 6, characterized in that, The molar ratio of MgCl2·6H2O, AlCl3·6H2O and NaOH is (6~8):(2~4):(16~20).

8. The method for preparing the acoustodynamically coupled copper death nanoplatform according to claim 6, characterized in that, The conditions for the hydrothermal reaction are: temperature 100℃~110℃, reaction time 4 h~5 h.

9. The method for preparing the acoustodynamically coupled copper death nanoplatform according to claim 5, characterized in that, Mg-Al LDH nanosheets and Cu in copper salt 2+ The molar ratio is 1.7:1 to 2.1:1, and the copper salt includes CuCl2·2H2O.

10. The use of the sonodynamically coupled copper death nanoplatform as described in any one of claims 1-4 in the preparation of antitumor drugs.

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