Nano composite material for treating drug resistance of non-small cell lung cancer

Abstract

The invention discloses a composite nano material for treating drug resistance of non-small cell lung cancer as well as a preparation method and application of the composite nano material, and belongs to the technical field of biological medicines. The material is of a core-shell structure, zinc / copper double-monatomic nano-enzyme is taken as a core, a mesoporous silica shell layer is coated outside to load YAP-siRNA, and glucose oxidase and an endoplasmic reticulum targeting agent TsG are adsorbed on the outermost layer. According to the application disclosed by the invention, active oxygen outbreak is realized in tumor cells, a YAP drug-resistant pathway is inhibited and strong endoplasmic reticulum stress is induced through the coordination of double-monatomic nano-enzyme catalysis, YAP gene silencing and glucose oxidase mediated metabolism deprivation, so that the EGFR-TKI drug resistance of the non-small cell lung cancer is effectively reversed. The preparation method of the material is mild in condition and good in biocompatibility, shows a remarkable anti-tumor effect in an in-vitro cell experiment and an in-vivo animal model, and provides a new solution for clinically solving the drug resistance problem of non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanocomposite material, and more particularly to a composite nanomaterial based on dual single-atom metal nanozymes, capable of inducing endoplasmic reticulum stress by combining gene silencing and metabolic intervention, for reversing drug resistance to targeted therapy of non-small cell lung cancer (NSCLC), as well as its preparation method and application. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. For NSCLC patients with epidermal growth factor receptor (EGFR) mutations, targeted therapy with EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib, gefitinib, or osimertinib is an important treatment approach. However, due to factors such as secondary gene mutations in tumor cells, activation of bypass signaling pathways, and changes in the tumor microenvironment (TME), the vast majority of patients develop resistance to EGFR-TKIs in the later stages of treatment, leading to treatment failure. Although changing targeted drugs or using multi-drug combination strategies can prolong patient survival to some extent, they cannot fundamentally solve the problem of drug resistance, and the overall cure rate has not been significantly improved. Therefore, overcoming EGFR-TKI resistance has become a core challenge in the clinical treatment of NSCLC.

[0003] In recent years, nanozyme technology, especially metal single-atom nanozymes (such as zinc, manganese, and copper-zinc diatomic materials), has shown great potential in tumor therapy due to its highly efficient catalytic activity. They can catalyze the generation of highly toxic hydroxyl radicals (·OH) from hydrogen peroxide (H2O2) in the tumor microenvironment via a Fenton-like reaction, enabling chemokinetic therapy (CDT). However, the killing effect of reactive oxygen species (ROS) alone is often insufficient to reverse complex drug resistance mechanisms. Studies have shown that the overexpression of YAP, a key effector in the Hippo pathway, is closely related to EGFR-TKI resistance in NSCLC. Simultaneously, the induction of endoplasmic reticulum stress (ERS) can trigger immunogenic cell death (ICD) and affect the expression of multidrug resistance-related proteins (such as MRP1).

[0004] Currently, no technology has been found that can synergistically integrate highly efficient dual single-atom catalysis, YAP gene silencing, and endoplasmic reticulum-targeted stress into a single nanoplatform to systematically address the drug resistance problem in NSCLC. Therefore, developing a novel therapeutic strategy that can synergistically achieve ROS burst, drug resistance gene silencing, and intense endoplasmic reticulum stress simultaneously has urgent clinical needs and significant application value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composite nanomaterial (Zn / Cu-BSRGTNPs) that can synergistically combine chemokinetic therapy, sonodynamic therapy, gene silencing and metabolic intervention to efficiently generate reactive oxygen species through multiple pathways, while reversing erlotinib resistance in non-small cell lung cancer and significantly improving the therapeutic effect.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned composite nanomaterials.

[0007] Another object of the present invention is to provide the application of the above-mentioned composite nanomaterials in the preparation of a medicament for treating drug-resistant non-small cell lung cancer.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composite nanomaterial with a core-shell structure, comprising: Core: Zinc / copper dual single-atom nanozymes (Zn / Cu-BSAN NPs); Middle layer: Mesoporous silica shell (SiO2) surrounding the core, which is used to load YAP-siRNA; Outer layer: Glucose oxidase (GOx) and endoplasmic reticulum targeting molecule TsG adsorbed outside the mesoporous silica shell.

[0009] Preferably, the zinc / copper dual single-atom nanoenzyme particles have a particle size of 120-130 nm, and the final composite nanomaterial (Zn / Cu-BSRGT NPs) has a particle size of 150-160 nm; the silica shell has a thickness of 10-25 nm. This particle size range facilitates the accumulation of nanomaterials in tumor tissue through enhanced penetration and retention effects, and their effective uptake by tumor cells.

[0010] Secondly, the present invention provides a method for preparing the composite nanomaterial, comprising the following steps: (1) Preparation of Zn / Cu-BSANs@SiO2: Zn / Cu-BSAN NPs were dispersed in methanol, and then added sequentially. Hexadecyltrimethylammonium bromide (CTAB) and tetraethyl silicate (TEOS) were added, and the mixture was stirred at room temperature. After stirring, the mixture was centrifuged, washed, and dried to obtain nanoparticles coated with silica.

[0011] (2) Synthesis of endoplasmic reticulum targeting agent TsG: Glycine was dissolved in Na2CO3 solution, cooled and p-methylbenzenesulfonyl chloride was added in batches. After the reaction, it was purified by thin-layer chromatography, acidified with hydrochloric acid, filtered, washed and crystallized to obtain TsG.

[0012] (3) Loading YAP-siRNA: Zn / Cu-BSANs@SiO2 nanoparticles were dispersed in a mixed solution of guanidine hydrochloride and methanol, and a DEPC-treated YAP-siRNA aqueous solution was added. After shaking and incubation, centrifugation was performed to obtain Zn / Cu-BSAN@SiO2@siRNA loaded with siRNA.

[0013] (4) Adsorption of glucose oxidase: Dissolve GOx in water, add Zn / Cu-BSAN@SiO2@siRNA obtained in step (3), stir the reaction, centrifuge, wash, freeze dry to obtain Zn / Cu-BSANs@SiO2@siRNA@GOx (denoted as Zn / Cu-BSRG NPs).

[0014] (5) Coupling the endoplasmic reticulum targeting agent TsG: TsG was dissolved in DMSO and then diluted in PBS buffer. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to activate the carboxyl group of TsG. Then Zn / Cu-BSRG NPs were added and the reaction was stirred overnight at 4°C. Finally, the product was obtained by ultrafiltration, centrifugation, washing, and freeze-drying to obtain the final product Zn / Cu-BSANs@SiO2@siRNA@GOx@TsG (denoted as Zn / Cu-BSRGT NPs).

[0015] Thirdly, this invention provides the application of the aforementioned composite nanomaterial in the preparation of a medicament for treating non-small cell lung cancer, particularly EGFR-TKI-resistant non-small cell lung cancer. The medicament can be administered via intravenous injection or other methods, and can be supplemented with ultrasound irradiation at the tumor site to enhance the therapeutic effect. Beneficial effects

[0016] 1. Synergistic Reversal of Drug Resistance through Multiple Mechanisms: This invention innovatively integrates dual single-atom nanozyme catalysis, YAP gene silencing, and GOx-mediated metabolic deprivation. Through the synergistic effect of "ROS burst + YAP pathway inhibition + endoplasmic reticulum stress", it effectively reverses erlotinib resistance in NSCLC, solving the problem of limited efficacy of single therapy.

[0017] 2. Highly Efficient Catalysis and Targeted Delivery: Zn / Cu dual-single-atom nanozymes exhibit higher catalytic efficiency than single-atom or traditional nanomaterials, enabling efficient generation of ·OH. A mesoporous silica shell facilitates the effective loading and protection of YAP-siRNA. TsG modification ensures that the nanomaterials can precisely target the endoplasmic reticulum of tumor cells, achieving localized killing.

[0018] 3. Self-supplied cascade reaction: GOx consumes glucose within tumor cells, while simultaneously producing gluconic acid and H2O2. The former lowers the local pH, accelerating the Fenton-like reaction of the nanozyme; the latter provides additional substrate for the Fenton-like reaction, achieving self-supplied and self-enhanced chemokinetic therapy.

[0019] 4. Good biocompatibility and clinical translation potential: The components of the material have good biocompatibility, the preparation method is mild and reproducible, laying a solid foundation for subsequent clinical translation.

[0020] 5. Significant in vitro and in vivo efficacy: Both in vitro cell experiments and in vivo animal models have confirmed that Zn / Cu-BSRGT NPs, under ultrasound assistance, can effectively induce tumor cell apoptosis, significantly inhibit the growth of drug-resistant tumors, and regulate the tumor immune microenvironment, demonstrating remarkable therapeutic effects. Attached Figure Description

[0021] Figure 1 The images shown are transmission electron microscope (TEM) images of the nanomaterials prepared in each stage in Example 1, where (a) is ZIF-8 nanoparticles; (b) is ZIF-8@SiO2 nanoparticles; (c) is Zn / Cu-BSAN nanoparticles; and (d) is a high-magnification image of the final product Zn / Cu-BSRGT nanoparticles.

[0022] Figure 2 The diagrams show the structural characterization of zinc and copper single atoms in Example 1, where (ac) is the XANES spectrum, R-space, and K-space Fourier transform diagram of zinc single atoms; (df) is the corresponding spectrum of copper single atoms; and (g,h) are the wavelet transform diagrams of zinc and copper single atoms and their controls, respectively.

[0023] Figure 3 This is a graph showing the performance verification of reactive oxygen species generated in vitro by Zn / Cu-BSRGT NPs in Example 1.

[0024] Figure 4 This diagram illustrates the in vitro molecular mechanism by which Zn / Cu-BSRGT NPs reverse drug resistance in non-small cell lung cancer, as shown in Example 1.

[0025] Figure 5 This is a graph evaluating the antitumor effect of Zn / Cu-BSRGT NPs in tumor-bearing mice in Example 1.

[0026] Figure 6 This is a diagram showing the TUNEL apoptosis detection and H&E staining analysis of tumor tissue after treatment in Example 1. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] Example 1: Preparation of composite nanomaterials (Zn / Cu-BSRGT NPs) (1) 0.5 g Zn / Cu-BSAN NPs were dispersed in 240 mL methanol, and 0.1487 g CTAB and 1.5 mL TEOS were added sequentially. The mixture was stirred at room temperature for 2 h. After centrifugation at 10000 rpm for 5 min, the mixture was washed three times with methanol and dried under vacuum at 60 °C to obtain Zn / Cu-BSANs@SiO2 NPs.

[0029] (2) Dissolve 0.75 g g of glycine in 15 mL of Na2CO3 solution (concentration 1.27 mg·mL⁻¹).

[0030] (3) After stirring the above solution for 5 min, cool it to -5℃, and add 2.28 g of p-toluenesulfonyl chloride in 4 portions over 2 hours. Heat the mixture to room temperature and stir vigorously for 3 h. After the reaction is complete, purify by thin-layer chromatography and then acidify with 20% HCl to pH=2.0. Filter the mixture through a Buchner funnel, and wash the resulting crystals repeatedly with cold water to obtain white TsG crystals.

[0031] (4) Mix 10 mL of guanidine hydrochloride aqueous solution with 40 mL of methanol, add 50 mg of Zn / Cu-BSANs@SiO2, and ultrasonically disperse evenly.

[0032] (5) Add 0.1 mL of YAP-siRNA (dissolved in DEPC water) with a concentration of 60 μg·mL⁻¹ to the above mixture, shake at room temperature for 1 h, and collect by centrifugation to obtain Zn / Cu-BSAN@SiO2@siRNA.

[0033] (6) Dissolve 30 mg GOx in 30 mL of distilled water and stir for 30 min. Add 50 mg Zn / Cu-BSAN@SiO2@siRNA and stir for 24 h. Centrifuge at 10000 rpm for 5 min, wash three times with distilled water, and freeze dry to obtain Zn / Cu-BSRGNPs.

[0034] (7) Dissolve 2.28 mg TsG in 10 mL DMSO and then slowly add it dropwise to 240 mL PBS buffer (pH=7.4).

[0035] (8) Add 1.552 mg EDC and 1.15 mg NHS to the above TsG solution and stir at room temperature for 30 min to activate the carboxyl groups. Then add Zn / Cu-BSRG NPs and stir at 4 °C for 12 h. After the reaction is complete, remove unreacted TsG by ultrafiltration centrifugation, wash three times with PBS, and finally freeze-dry to obtain the final product Zn / Cu-BSRGT NPs.

[0036] Example 2: Material Characterization and Performance Testing 1. Morphological and structural characterization: The morphology of the products at each stage was observed using transmission electron microscopy. For example... Figure 1 As shown, ZIF-8, ZIF-8@SiO2, Zn / Cu-BSAN, and Zn / Cu-BSRGT NPs with regular morphology and uniform size were successfully prepared. X-ray absorption fine structure spectra (...) Figure 2 This confirms that both Zn and Cu exist in monatomic form.

[0037] 2. Catalytic performance test: Peroxidase-like activity: Under acidic conditions (pH 6.5), with the addition of H2O2 and GSH, Zn / Cu-BSRGT NPs can effectively oxidize TMB (… Figure 3 (d) indicates that it has sustained POD-like activity.

[0038] ·OH generation: Electron spin resonance spectroscopy shows that Zn / Cu-BSRGT NPs can generate a significant ·OH signal in the presence of H2O2. Figure 3 f).

[0039] Acoustodynamic activity: Under ultrasonic irradiation, Zn / Cu-BSRGT NPs can effectively generate singlet oxygen (¹O2), degrading SOSG probes (…). Figure 3 g, e).

[0040] Glucose consumption and pH adjustment: The loading of GOx enabled Zn / Cu-BSRGT NPs to effectively catalyze glucose oxidation, leading to a continuous decrease in the solution pH. Figure 3 c), which creates a favorable acidic environment for CDT.

[0041] Example 3: Evaluation of Anti-drug Resistance Efficacy at the In Vitro Cellular Level Evaluation was conducted using the A549 cell line, which is resistant to erlotinib.

[0042] Endoplasmic reticulum stress induction: Western blot results showed ( Figure 4a) Compared with the control group, the expression of endoplasmic reticulum stress marker proteins (p-PERK, p-eIF2α, ATF4, CHOP, GRP78) was significantly upregulated in cells treated with Zn / Cu-BSRGTNPs+US, indicating that strong endoplasmic reticulum stress was successfully induced.

[0043] ATP consumption: Due to GOx consuming glucose, the intracellular ATP level in cells treated with Zn / Cu-BSRGT NPs was significantly reduced. Figure 4 (b) weakens the cell's energy supply and drug efflux capacity.

[0044] Example 4: In vivo anti-tumor experiment A nude mouse model of A549 erlotinib-resistant tumors was established, and mice were randomly assigned to groups and treated.

[0045] Histological analysis: TUNEL and H&E staining was performed on the tumor tissue after treatment. Figure 6 The results showed that the Zn / Cu-BSRGT NPs+US group exhibited the largest area of ​​apoptotic region (green fluorescence) and the most severe tumor cell necrosis / damage, further confirming its strong in vivo antitumor activity.

[0046] Biocompatibility: Throughout the experiment, the weight of mice in each group did not decrease significantly, and no obvious pathological damage was found in the H&E staining of major organs, indicating that Zn / Cu-BSRGT NPs have good in vivo biocompatibility.

[0047] In summary, the composite nanomaterials, their preparation methods, and applications provided by this invention can efficiently and synergistically reverse targeted therapy resistance in non-small cell lung cancer, and have broad clinical application prospects.

[0048] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A composite nanomaterial for treating drug-resistant non-small cell lung cancer, characterized in that, It has a core-shell structure, comprising: a core composed of zinc / copper dual single-atom nanozymes; a mesoporous silica shell surrounding the core, wherein YAP-siRNA is loaded in the silica shell; and glucose oxidase and endoplasmic reticulum targeting agent TsG adsorbed outside the silica shell.

2. The composite nanomaterial according to claim 1, characterized in that, The zinc / copper dual single-atom nanozyme particles have a particle size of 120-130 nm.

3. The composite nanomaterial according to claim 1, characterized in that, The overall particle size of the composite nanomaterial is 150-160 nm.

4. The composite nanomaterial according to claim 1, characterized in that, The thickness of the silica shell is 10-25 nm.

5. A method for preparing composite nanomaterials as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Disperse zinc / copper dual single-atom nanozymes in methanol, add hexadecyltrimethylammonium bromide and tetraethyl silicate to react, and after centrifugation, washing and drying, obtain Zn / Cu-BSANs@SiO2 nanoparticles with silica coating on the surface. (2) Glycine was reacted with p-toluenesulfonyl chloride to synthesize the endoplasmic reticulum targeting agent TsG; (3) The Zn / Cu-BSANs@SiO2 nanoparticles obtained in step (1) were incubated with YAP-siRNA in a mixed solution of guanidine hydrochloride and methanol, and centrifuged to obtain Zn / Cu-BSAN@SiO2@siRNA loaded with siRNA. (4) Mix the glucose oxidase solution with the Zn / Cu-BSAN@SiO2@siRNA obtained in step (3), stir the reaction, centrifuge, wash, freeze dry to obtain Zn / Cu-BSRG NPs; (5) The TsG obtained in step (2) is activated by EDC / NHS and reacted with the Zn / Cu-BSRG NPs obtained in step (4) in a buffer solution. After centrifugation, washing and freeze-drying, the final product Zn / Cu-BSRGT NPs is obtained.

6. The use of a composite nanomaterial as described in any one of claims 1-4 in the preparation of a medicament for treating non-small cell lung cancer.

7. The application according to claim 6, characterized in that, The non-small cell lung cancer mentioned is EGFR-TKI resistant non-small cell lung cancer.

8. The application according to claim 6 or 7, characterized in that, Ultrasound irradiation is used as an adjunct to the treatment.

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