Liquid metal piezoelectric catalyst, nano-drug as well as preparation method and application of nano-drug

Abstract

The invention discloses a liquid metal piezoelectric catalyst, a nano-drug as well as a preparation method and application of the liquid metal piezoelectric catalyst and the nano-drug. The GaInZn liquid metal has excellent physicochemical properties, dynamic interfaces, movable active atoms, structural rearrangement and other characteristics, so that the GaInZn liquid metal shows excellent catalytic performance. The nano-drug disclosed by the invention has excellent tumor fibroblast and Golgi apparatus targeting ability, and can effectively reduce tumor interstitial pressure, remodel tumor microenvironment and clear obstacles for deep infiltration of immune cells. According to the nano-drug, secretion of immunosuppression related protein is reduced under the piezoelectric catalysis effect, the killing effect on tumor cells is improved, immune cell infiltration and drug delivery efficiency can be enhanced, immune response is improved, and the anti-tumor effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a liquid metal piezoelectric catalyst, a nano-drug, and their preparation methods and applications. Background Art

[0002] Immunotherapy enhances immune cell recognition and tumor elimination, showing powerful efficacy. However, the immunosuppressive tumor microenvironment characterized by high tumor interstitial fluid pressure (TIFP) and dense extracellular matrix (ECM) severely hinders immune infiltration. Abnormal angiogenesis, poor lymphatic drainage, and rapid tumor growth further increase TIFP and vascular tension. Cancer-associated fibroblasts (CAFs) secrete collagen and hyaluronic acid, hardening the ECM, increasing interstitial solid pressure, and inhibiting T cell function through TGF-β signaling. In addition, tumor cells rely on the Golgi apparatus to secrete growth factors and pro-angiogenic mediators, thus activating signal pathways that drive angiogenesis and tumor proliferation. Therefore, targeting Golgi function provides therapeutic potential by reducing tumor interstitial pressure, remodeling the tumor microenvironment (TME), and alleviating the barriers to immune cell infiltration into the tumor core.

[0003] In tumor targeted therapy research, new materials have attracted much attention, and liquid metals are a highly potential class among them. As a unique metal alloy, liquid metal has a liquid phase, spatial fluidity, and dynamic atomic configuration, with low viscosity, high electrical and thermal conductivity. Due to its excellent chemical stability and flexibility, it has been used in a variety of biomedical applications in recent years. Based on the dynamic interface and mobile active atoms of liquid metal, and being able to rearrange its surface atoms according to the charge and structure of the reactants to generate the most suitable active sites for the reaction, it can self-adjust during the catalytic process and optimize the reaction path. Although it has a high surface tension under physiological conditions, ultrasound can induce its topological reconstruction. Its atomic properties endow it with abundant catalytic sites, and its self-adaptability and deformability help it penetrate tumor cells, showing great potential in in vivo delivery and biochemical effects. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a liquid metal piezoelectric catalyst, a nano-drug, and their preparation methods and applications.

[0005] To achieve the above object, the technical scheme adopted by the present invention is as follows:

[0006] The present invention provides a preparation method of a liquid metal piezoelectric catalyst, which includes: after crushing the liquid metal into nano-particles, adding polyetherimide (PEI), and then adding a chondroitin sulfate solution containing EDC and NHS, and mixing evenly to obtain the liquid metal piezoelectric catalyst, denoted as LM / CS; wherein, the liquid metal is gallium-indium-zinc (GaInZn) liquid metal.

[0007] Furthermore, the atomic ratio of Ga, In, and Zn in the gallium-indium-zinc liquid metal is (84.2 - 84.4):(12.8 - 13):(2.4 - 2.6); the preparation method of the gallium-indium-zinc liquid metal includes: under an argon atmosphere, taking high-purity Ga, In, and Zn metals and sealing them in a vacuum container; heating the vacuum container to 500 - 600 °C to completely melt the metals; then placing the vacuum container in an ultrasonic cleaner and treating it at 70 °C for 20 - 60 min to obtain the gallium-indium-zinc liquid metal.

[0008] Furthermore, the preparation method of the chondroitin sulfate solution containing EDC and NHS includes: dissolving the chondroitin sulfate solution in a PBS buffer solution with pH = 6.0, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and mixing at room temperature to activate the carboxyl group of chondroitin sulfate to obtain the chondroitin sulfate solution containing EDC and NHS.

[0009] Furthermore, in this preparation method, the crushing method is ultrasonic crushing, the power is 300 - 500 W, and the time is 1 - 2 h; the mixing method is stirring, the rotation speed is 100 - 300 rpm, and the time is 0.5 - 1 h.

[0010] Furthermore, the mass ratio of the gallium-indium-zinc liquid metal to chondroitin sulfate in the chondroitin sulfate solution containing EDC and NHS is (6.1 - 6.3):(13 - 15).

[0011] The present invention also provides a liquid metal piezoelectric catalyst prepared by the above preparation method of the liquid metal piezoelectric catalyst.

[0012] The present invention also provides a piezoelectric catalytic nano-drug, the nano-drug includes the liquid metal piezoelectric catalyst as described above, and a tumor fibroblast cell membrane wrapped on the liquid metal piezoelectric catalyst; optionally, the mass ratio of the gallium-indium-zinc liquid metal, chondroitin sulfate in the chondroitin sulfate solution containing EDC and NHS, and the tumor fibroblast cell membrane is (6.1 - 6.3):(13 - 15):(2 - 4).

[0013] The present invention further provides a method for preparing the above piezoelectric catalytic nano-drug, which includes: preparing the liquid metal piezoelectric catalyst; washing the tumor fibroblasts 3 times with PBS buffer solution, expanding them with ultrapure water at 37°C, then breaking them and collecting by centrifugation to obtain tumor fibroblast cell membranes; resuspending the tumor fibroblast cell membranes with PBS buffer solution to obtain a tumor fibroblast cell membrane solution; adding the liquid metal piezoelectric catalyst to the tumor fibroblast cell membrane solution and repeatedly extruding it with a liposome extruder to obtain the piezoelectric catalytic nano-drug, denoted as LM / CS@M.

[0014] Further, in this preparation method, the breaking method uses a cell disruptor, the ultrasonic power is 100 - 200W, the ultrasonic time is 5 - 10min, and the centrifugation speed is 2000 - 3000rpm.

[0015] The present invention provides the application of the above liquid metal piezoelectric catalyst and the above piezoelectric catalytic nano-drug in the preparation of anti-tumor drugs.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] The present invention provides a liquid metal piezoelectric catalyst, a nano-drug, and their preparation methods and applications. The present invention utilizes the asymmetric structure and chemical reactivity of GaInZn liquid metal to design a unique piezoelectric catalytic nano-drug that can enhance drug and immune cell infiltration. In the preparation method of this piezoelectric catalytic nano-drug, GaInZn liquid metal is prepared, and after the GaInZn liquid metal is prepared into nanoparticles (LM) by ultrasonic treatment, chondroitin sulfate (CS) is used for surface modification to endow it with Golgi targeting (LM / CS), and finally, it is wrapped with CAFs membrane to obtain LM / CS@M, realizing the dual targeting function of CAF and Golgi. This nano-drug efficiently accumulates at the tumor fibroblast and Golgi sites through a dual targeting strategy. Subsequently, under ultrasonic stimulation, hydroxyl radicals (·OH) are generated to destroy the Golgi of CAFs, reduce the secretion of collagen and immunosuppressive factors, and reduce the tumor interstitial solid pressure (TISP). In addition, this nano-drug can also catalyze the decomposition of water in the tumor interstitial fluid, reduce its volume and TIFP, thereby enhancing the infiltration of immune cells. This dual mechanism not only improves the immunotherapy effect of liquid metal in tumor treatment, expands the treatment potential of liquid metal in tumor treatment, but also provides a new strategy for overcoming the tumor immune barrier and optimizing the treatment effect.

[0018] 1) The present invention utilizes the piezoelectric catalysis of liquid metal to catalyze the decomposition of water in the tumor interstitial fluid to generate H2, thereby reducing the tumor interstitial fluid pressure. Through physical and chemical dual mechanisms, the tumor matrix structure is reshaped, the immune barrier is reduced, and the immunotherapy effect is improved.

[0019] 2) The ultrasound-activated nano-drugs target CAFs and the Golgi apparatus, generating ·OH to reduce ECM stiffness and collagen secretion. The disruption of the Golgi apparatus induces immunogenic cell death, promotes matrix remodeling and T cell activation, and improves the efficacy of immunotherapy.

[0020] 3) In the present invention, GaInZn liquid metal has unique liquid properties, spatial fluidity and dynamic atomic structure to design nano-drugs with precise targeting. The liquid metal has high electrical conductivity, excellent deformability, adjustable surface tension and chemical activity, and has excellent catalytic performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transmission electron microscopy image of the piezoelectric catalytic nano-drug obtained in Example 1;

[0022] Figure 2 Hydrogen generation image of the piezoelectric catalytic nano-drug obtained in Example 2;

[0023] Figure 3 Methylene blue (MB) degradation image of the piezoelectric catalytic nano-drug obtained in Example 3;

[0024] Figure 4 CAFs Golgi targeting image of the piezoelectric catalytic nano-drug obtained in Example 3;

[0025] Figure 5 Inhibitory effect on CAFs activation image of the piezoelectric catalytic nano-drug obtained in Example 3;

[0026] Figure 6 Tumor inhibition rate image of the piezoelectric catalytic nano-drug obtained in Example 3;

[0027] Figure 7 In vivo anti-tumor effect image of the piezoelectric catalytic nano-drug obtained in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0028] Regarding the piezoelectric catalytic nano-drug involved in the present invention, the synthesized liquid metal is prepared into LM by ultrasonic fragmentation method, then functionalized with CS, and finally wrapped with CAFs membrane to obtain LM / CS@M, realizing dual targeting of CAF and the Golgi apparatus. When receiving precise ultrasonic stimulation at the tumor site, the surface of LM generates periodic charge distribution changes and instantaneous electric fields due to mechanical vibration, driving the accumulation of charges on the metal surface, forming a micro-electrostatic potential difference, promoting the decomposition of water molecules, generating H + and OH - , H + is reduced to H2, and OH -Oxidation generates cytotoxic ·OH. This not only reduces the volume of tumor interstitial fluid, decreases TIFP, weakens the physical resistance to immune cell infiltration, but also induces the disassembly of the Golgi apparatus in CAFs, reduces matrix stiffness, enhances immune cell penetration, and at the same time promotes tumor cell apoptosis, triggers immune responses, and enhances the effect of immunotherapy, providing strong evidence for the use of liquid metal-based nanomedicines in cancer treatment.

[0029] The present invention will be further described below in conjunction with embodiments, but these embodiments do not limit the scope of the present invention. The test methods without specific conditions noted in the embodiments of the present invention are generally carried out under conventional conditions; or according to the conditions recommended by the raw material or commodity manufacturers. Reagents without specific sources noted are conventional reagents that can be purchased on the market.

[0030] Example 1

[0031] High-purity Ga, In, and Zn metals were weighed in an argon atmosphere using a Partulob MRVS-1002 vacuum sealer at a vacuum level of 10 -2 Pa according to an atomic ratio of 84.2:12.8:2.4, and then sealed in a quartz tube. The quartz tube was heated to 500 °C using an alcohol lamp to achieve complete melting of the metal. Subsequently, to ensure sufficient mixing of the alloy components, the quartz tube was placed in an ultrasonic cleaner and treated at 70 °C for 20 min to obtain Ga 84.2 In 12.8 Zn 2.4 liquid metal. 8 μL of the prepared liquid metal was added to 5 mL of water and sonicated at a power of 300 W for 1 h (cycle 2 s, interval 2 s) to produce nanoscale liquid metal, which was then stored at 4 °C for later use. 30 mg of PEI (10 mg / mL) was added to the liquid metal suspension. The mixture was stirred on a magnetic stirrer for 30 min. 130 mg of chondroitin sulfate was dissolved in 15 mL of PBS buffer (pH 6.0). Subsequently, 24.26 mg of EDC (10 mM) and 17.44 mg of NHS (10 mM) were added to the chondroitin sulfate solution and stirred at room temperature for 0.5 h to activate the carboxyl groups of chondroitin sulfate. The liquid metal was mixed with the activated chondroitin sulfate solution and stirred at room temperature for 4 h. After the reaction was completed, it was washed three times with ultrapure water and anhydrous ethanol respectively to obtain LM / CS, which was stored at 4 °C for later use. The CAFs were washed three times with PBS, expanded in ultrapure water at 37 °C, and then disrupted using an ultrasonic cell disruption system. The cell membranes were collected by centrifugation and resuspended in PBS. Finally, LM / CS was added to the membrane solution and repeatedly extruded using a liposome extruder to obtain LM / CS@M. As Figure 1 The TEM image of shows that LM / CS@M presents a uniform spherical structure and is successfully wrapped by the cell membrane on the surface.

[0032] Example 2

[0033] High-purity Ga, In, and Zn metals were weighed in an argon atmosphere using a Partulob MRVS-1002 vacuum sealer at a vacuum level of 10 -2 Pa, and then sealed in a quartz tube. The quartz tube was heated to 550 °C using an alcohol lamp to achieve complete melting of the metals. Subsequently, to ensure sufficient mixing of the alloy components, the quartz tube was placed in an ultrasonic cleaner and treated at 70 °C for 40 min to obtain a Ga 84.3 In 12.9 Zn 2.5 liquid metal. 9 μL of the prepared liquid metal was added to 5 mL of water and sonicated at a power of 400 W for 1.5 h (cycle 2 s, interval 2 s) to produce nanoscale liquid metal, which was then stored at 4 °C for later use. 40 mg of PEI (10 mg / mL) was added to the liquid metal suspension. The mixture was stirred on a magnetic stirrer for 30 min. 140 mg of chondroitin sulfate was dissolved in 15 mL of PBS buffer (pH 6.0). Subsequently, 24.27 mg of EDC (10 mM) and 17.45 mg of NHS (10 mM) were added to the chondroitin sulfate solution and stirred at room temperature for 1 h to activate the carboxyl groups of chondroitin sulfate. The liquid metal was mixed with the activated chondroitin sulfate solution and stirred at room temperature for 5 h. After the reaction was completed, it was washed three times with ultrapure water and anhydrous ethanol respectively to obtain LM / CS, which was stored at 4 °C for later use. The CAFs were washed three times with PBS, expanded with ultrapure water at 37 °C, and then disrupted using an ultrasonic cell disruption system. The cell membranes were collected by centrifugation and resuspended in PBS. Finally, LM / CS was added to the membrane solution and repeatedly extruded using a liposome extruder to obtain LM / CS@M. As Figure 2 shown by the hydrogen detection results, the performance of the liquid metal in catalytically decomposing water to produce hydrogen was evaluated using gas chromatography. As the sonication time increased and the drug concentration increased, the hydrogen production increased significantly, indicating that the nanodrug has excellent piezoelectric catalytic performance in decomposing water to produce hydrogen.

[0034] Example 3

[0035] High-purity Ga, In, and Zn metals were weighed in an argon atmosphere using a Partulob MRVS-1002 vacuum sealer at a vacuum level of 10 -2 Pa, and then sealed in a quartz tube. The quartz tube was heated to 600 °C using an alcohol lamp to achieve complete melting of the metals. Subsequently, to ensure sufficient mixing of the alloy components, the quartz tube was placed in an ultrasonic cleaner and treated at 70 °C for 60 min. To obtain Ga 84.4 In13 Zn 2.6 Liquid metal. Add 10 μL of the prepared liquid metal into 5 mL of water, and ultrasonically disrupt it at a power of 500 W for 2 h (cycle 2 s, interval 2 s) to produce nano-scale liquid metal, and then store it at 4 °C for later use. Add 50 mg of PEI (10 mg / mL) to the liquid metal suspension. Stir the mixture on a magnetic stirrer for 30 min. Dissolve 150 mg of chondroitin sulfate in 15 mL of PBS buffer (pH 6.0). Subsequently, add 24.28 mg of EDC (10 mM) and 17.46 mg of NHS (10 mM) to the chondroitin sulfate solution, and stir at room temperature for 1.5 h to activate the carboxyl group of chondroitin sulfate. Mix the liquid metal with the activated chondroitin sulfate solution and stir at room temperature for 6 h. After the reaction is completed, wash it three times with ultrapure water and anhydrous ethanol respectively to obtain LM / CS, and store it at 4 °C for later use. Wash the CAFs three times with PBS, expand them with ultrapure water at 37 °C, and then disrupt them with an ultrasonic cell disruption system. Centrifuge to collect the cell membrane and resuspend it with PBS. Finally, add LM / CS to the membrane solution and repeatedly extrude it with a liposome extruder to obtain LM / CS@M. Figure 3 It shows the ability of the nano-drug to generate reactive oxygen species under piezoelectric catalysis. It can be seen that the degradation efficiency of MB increases significantly with the extension of ultrasonic time, proving its ability to catalytically generate ·OH.

[0036] Example 4

[0037] To verify the active targeting effect of adding CAFs membrane and chondroitin sulfate, mouse embryonic fibroblasts (NIH3T3) were treated with tumor cell supernatant to obtain CAFs, and LM / CS@M was stained with rhodamine B, and then co-incubated with CAFs, and the Golgi apparatus was labeled with Golgi-Tracker Green. Fluorescence is as Figure 4 shown. Bright red signals in LM / CS@M were gradually enhanced in the Golgi apparatus of CAFs with the extension of co-incubation time. This is because the homologous targeting of the cell membrane and the Golgi targeting effect of chondroitin sulfate promoted the Golgi aggregation of the nano-drug. The results confirmed that the liquid metal piezoelectric catalyst has excellent active targeting ability.

[0038] Example 5

[0039] As a key cytoskeletal protein, α-smooth muscle actin (α-SMA) is an important marker for evaluating the transformation and activation of CAFs, and the expression level of α-SMA can directly reflect the activation degree of CAFs. As Figure 5As shown, fluorescence staining of α-SMA revealed that compared with the other groups, the expression of α-SMA in CAFs was significantly decreased after LM / CS@M+US treatment. The morphological analysis was consistent with the molecular results. The CAFs in the control group had longer cell processes, indicating stronger migration ability; after treatment with LM / CS@M+US, the cell morphology became round and the processes decreased, indicating that the piezocatalytic effect could effectively inhibit the activation of CAFs.

[0040] Example 6

[0041] To evaluate the tumor cell killing ability of the nanodrug, a CCK8 kit was used to analyze the cell viability. Figure 6 The results showed that the cell inhibition rates of the groups without ultrasound and with ultrasound alone were relatively low, while the cell inhibition rate of the LM+US group was significantly enhanced, and the cell inhibition rate of the LM / CS+US group was the most obvious, showing a strong tumor cell killing effect. This result indicates that the piezocatalysis induced by the nanodrug improves the killing of tumor cells, which helps to enhance immune cell infiltration and drug delivery efficiency, and improve the immune response and anti-tumor effect.

[0042] Example 7

[0043] To study the in vivo anti-tumor effect of the nanodrug, a mouse tumor model was constructed. Seven days after inoculating PAN02 tumor cells subcutaneously into the right hip of mice, the mice were randomly divided into eight groups, and tail vein injection and ultrasound treatment were performed. As Figure 7 The tumor growth curve showed that compared with the Saline group, the individual treatments of LM, LM / CS, LM / CS@M, and US had no obvious inhibitory effect on tumor growth; while the inhibition rates of the LM+US, LM / CS+US, and LM / CS@M+US groups on the orthotopic tumor were 20.18%, 80.03%, and 98.71% respectively. It shows that the nanodrug can reshape the tumor microenvironment through piezocatalysis, enhance immune cell infiltration, and thus achieve a better ablation effect on tumors.

[0044] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present invention, and in practical applications, various changes can be made in form and details without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope defined by the claims.

Claims

1. A method for preparing a liquid metal piezoelectric catalyst, characterized in that: The preparation method comprises: breaking liquid metal into nanoparticles, adding polyetherimide, and then adding a chondroitin sulfate solution containing EDC and NHS, and mixing them evenly to obtain the liquid metal piezoelectric catalyst, which is denoted as LM / CS; wherein the liquid metal is gallium indium zinc liquid metal.

2. The method for preparing a liquid metal piezoelectric catalyst according to claim 1, characterized in that: The atomic ratio of Ga, In and Zn in the gallium indium zinc liquid metal is (84.2-84.4): (12.8-13): (2.4-2.6); The preparation method of the gallium indium zinc liquid metal comprises: taking high-purity Ga, In and Zn metals and sealing them in a vacuum container under an argon atmosphere; heating the vacuum container to 500-600° C. to completely melt the metals; and then placing the vacuum container in an ultrasonic cleaning machine and treating it at 70° C. for 20-60 minutes to obtain the gallium indium zinc liquid metal.

3. The method for preparing a liquid metal piezoelectric catalyst according to claim 1, characterized in that: The preparation method of the chondroitin sulfate solution containing EDC and NHS comprises: dissolving chondroitin sulfate in a PBS buffer solution with a pH of 6.0, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and mixing at room temperature to activate the carboxyl group of the chondroitin sulfate to obtain the chondroitin sulfate solution containing EDC and NHS.

4. The method for preparing a liquid metal piezoelectric catalyst according to claim 1, characterized in that: In the preparation method, the crushing method is ultrasonic crushing, the power is 300-500W, and the time is 1-2h; the mixing method is stirring, the speed is 100-300rpm, and the time is 0.5-1h.

5. The method for preparing a liquid metal piezoelectric catalyst according to claim 1, characterized in that: The mass ratio of the gallium indium zinc liquid metal to the chondroitin sulfate in the chondroitin sulfate solution containing EDC and NHS is (6.1-6.3): (13-15). 6 . The liquid metal piezoelectric catalyst obtained by the method for preparing the liquid metal piezoelectric catalyst according to claim 1 .

7. A piezoelectric catalytic nanomedicine, characterized in that: The nanomedicine comprises the liquid metal piezoelectric catalyst as claimed in claim 6, and a tumor fibroblast membrane wrapped on the liquid metal piezoelectric catalyst; Optionally, the mass ratio of the gallium indium zinc liquid metal, the chondroitin sulfate solution containing EDC and NHS, and the tumor fibroblast membrane is (6.1-6.3):(13-15):(2-4).

8. The method for preparing the piezoelectric catalytic nanomedicine according to claim 7, characterized in that: The preparation method comprises: preparing the liquid metal piezoelectric catalyst; The tumor fibroblasts were washed three times with PBS buffer, expanded with ultrapure water at 37°C, and then broken and collected by centrifugation to obtain tumor fibroblast membranes; the tumor fibroblast membranes were resuspended with PBS buffer to obtain a tumor fibroblast membrane solution; The liquid metal piezoelectric catalyst is added into the tumor fibroblast membrane solution, and is repeatedly extruded by a liposome extruder to obtain the piezoelectric catalytic nanomedicine, which is denoted as LM / CS@M.

9. The method for preparing the piezoelectric catalytic nanomedicine according to claim 8, characterized in that: In the preparation method, a cell disruptor is used for disruption, the ultrasonic power is 100-200W, the ultrasonic time is 5-10min, and the centrifugal speed is 2000-3000rpm.

10. Use of the liquid metal piezoelectric catalyst according to claim 6 and the piezoelectric catalytic nanomedicine according to claim 7 in the preparation of anti-tumor drugs.

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