An antibody functionalized nanodelivery system MET@Fe3O4@BSA-TZ, and a preparation method and application thereof

Abstract

The application discloses an antibody functionalized nano delivery system MET@Fe3O4@BSA-TZ and a preparation method and application thereof. A four-iron oxide (Fe3O4) nanoparticle having a chemical dynamic therapy (CDT), a photothermal therapy (PTT) and an iron death induction capacity is prepared by using a one-step hydrothermal synthesis method as a nano carrier core, metformin (MET) capable of improving a tumor microenvironment and enhancing NK cell infiltration is loaded by physical adsorption, bovine serum albumin (BSA) is wrapped on the surface to improve biocompatibility and reduce the clearance efficiency of the body, and finally, trastuzumab (TZ) is covalently coupled and modified to realize specific targeting of Her-2 positive breast cancer cells, so that a multifunctional nano delivery system MET@Fe3O4@BSA-TZ (MFBT) is successfully constructed. In-vivo and in-vitro experiments prove that the MFBT can significantly inhibit the proliferation of drug-resistant tumors, improve the immunosuppressive microenvironment of the tumor and improve the anti-tumor treatment effect, and is safe and reliable, thereby providing a new and efficient treatment strategy for the precise treatment of Her-2 positive breast cancer.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and more specifically, to an antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ, its preparation method, and its applications. Background Technology

[0002] Breast cancer is one of the leading causes of cancer-related deaths among women worldwide. Studies show that on average, one in 20 women globally will be diagnosed with breast cancer in their lifetime. Breast cancer expressing human epidermal growth factor receptor 2 (Her-2+) accounts for 15% to 20% of all breast cancer cases, and its aggressive nature and poor prognosis pose a serious threat to women's health and lives.

[0003] Trastuzumab (TZ), as the first humanized monoclonal antibody targeting Her-2, has become a core drug for the treatment of Her-2 positive breast cancer, playing an important role in both metastatic breast cancer and adjuvant therapy, and significantly prolonging progression-free survival and overall survival. However, the clinical limitations of TZ monotherapy are becoming increasingly apparent. Its objective response rate (ORR) is low, and primary and acquired resistance are frequent, especially for patients with advanced metastatic Her-2 positive breast cancer, where TZ treatment has limited efficacy and tumor recurrence is common, becoming a key bottleneck restricting treatment effectiveness. Therefore, there is an urgent need to explore effective solutions to overcome resistance and improve treatment efficacy.

[0004] Recent studies have shown that one of the key mechanisms underlying the reduced responsiveness of tumors to tumor toxicity (TZ) is the significant decrease in the infiltration and functional activity of natural killer (NK) cells in the tumor microenvironment (TME). NK cells, as an important component of the body's innate immunity, can directly recognize and kill tumor cells, playing a crucial role in the anti-tumor immune response. Abnormal NK cell function can lead to tumor immune escape, thereby exacerbating the development of TZ resistance.

[0005] Therefore, it is of great significance to provide a new, efficient, and precise treatment strategy for Her-2 positive breast cancer, especially for TZ-resistant cases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ, its preparation method and application, which reverses the resistance of Her-2 positive breast cancer to trastuzumab (TZ) and achieves synergistic treatment of chemodynamics, photothermal activity, ferroptosis and immune enhancement.

[0007] To address the above technical problems, according to one aspect of the present invention, an antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ is provided, comprising a nanocarrier and a targeted therapeutic drug; The nanocarrier comprises iron(III) oxide (Fe3O4) nanoparticles, metformin (MET), and bovine serum albumin (BSA); the iron(III) oxide (Fe3O4) nanoparticles serve as the core of the nanodelivery system; metformin (MET) is loaded onto the surface of the iron(III) oxide (Fe3O4) nanoparticles; and bovine serum albumin (BSA) is wrapped around the iron(III) oxide (Fe3O4) nanoparticles and metformin (MET) to form a shell. The targeted therapeutic drug is trastuzumab (TZ), which is covalently coupled to the shell layer.

[0008] As a preferred embodiment, the maximum drug loading of trastuzumab (TZ) in the nanocarrier is 22.4% by mass percentage.

[0009] According to another aspect of the present invention, a method for preparing the antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ described above is provided, comprising: Step 1: Prepare Fe3O4NPs; FeCl3·6H2O and trisodium citrate were dissolved in ethylene glycol, and then anhydrous sodium acetate was added. Fe3O4NPs were obtained by hydrothermal reaction. Step 2: Prepare MET@Fe3O4NPs; Fe3O4NPs were dissolved in PBS, and then a PBS solution containing metformin (MET) was added. After sonication, centrifugation, stirring, washing, and drying, MET@Fe3O4NPs were obtained. Step 3: Preparation of MET@Fe3O4@BSANPs MET@Fe3O4NPs were dissolved in distilled water and added dropwise to an aqueous solution of bovine serum albumin (BSA). After stirring and dialysis, the mother liquor obtained by dialysis was freeze-dried to obtain MET@Fe3O4@BSANPs. Step 4: Prepare MET@Fe3O4@BSA-TZNPs; Trastuzumab (TZ) was activated with NHS and EDC. The activated trastuzumab (TZ) was then mixed with MET@Fe3O4@BSANPs and reacted. After centrifugation, washing, and freeze-drying, MET@Fe3O4@BSA-TZNPs were obtained.

[0010] In a preferred embodiment, in step one, the hydrothermal reaction temperature is 180-220℃, and the reaction time is 8-12 hours. In some preferred embodiments, the hydrothermal reaction temperature is 180℃, 200℃, or 220℃; in some preferred embodiments, the reaction time is 8 hours, 10 hours, or 12 hours.

[0011] In a preferred embodiment, in step two, the mass ratio of Fe3O4NPs to metformin (MET) is 10:1.

[0012] In a preferred embodiment, in step three, the mass ratio of MET@Fe3O4 to bovine serum albumin (BSA) is 1:5.

[0013] In a preferred embodiment, in step four, the mass ratio of trastuzumab (TZ) to MET@Fe3O4@BSANPs is 1:(0.5-3). In some preferred embodiments, the mass ratio of trastuzumab (TZ) to MET@Fe3O4@BSANPs is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3.

[0014] As a preferred embodiment, the mass ratio of trastuzumab (TZ) to MET@Fe3O4@BSANPs is 1:2.

[0015] According to another aspect of the invention, the use of the antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ in a drug for treating Her-2 positive breast cancer is provided.

[0016] The treatment described in the above applications includes: achieving specific targeted recognition of Her-2 positive breast cancer cells through trastuzumab (TZ), enhancing biocompatibility with bovine serum albumin (BSA), and utilizing the responsive release characteristics of Fe3O4 in the acidic environment of the TME to achieve slow, targeted release of iron ions and trastuzumab (TZ); Fe3O4-mediated PTT and CDT synergistically trigger tumor cell ferroptosis, which further enhances the cytotoxic function of NK cells, while metformin (MET) promotes NK cell infiltration. The two synergistically improve the immunosuppressive state of the TME, ultimately reversing the resistance of Her-2 positive breast cancer to trastuzumab (TZ), and achieving synergistic treatment of chemodynamics, photothermal activity, ferroptosis, and immune enhancement.

[0017] This invention employs a one-step hydrothermal synthesis method to prepare iron(III) oxide (Fe3O4) nanoparticles with chemodynamic therapy (CDT), photothermal therapy (PTT), and ferroptosis-inducing capabilities as the core of the nanocarrier. Metformin (MET), which can improve the tumor microenvironment and enhance NK cell infiltration, is loaded onto the surface through physical adsorption. Bovine serum albumin (BSA) is then coated on the surface to improve biocompatibility and reduce the body's clearance efficiency. Finally, trastuzumab (TZ) is covalently coupled and modified to achieve specific targeting of Her-2 positive breast cancer cells, thus successfully constructing the MET@Fe3O4@BSA-TZ (MFBT) multifunctional nanodelivery system.

[0018] The nanodelivery system provided by this invention achieves precise targeting of Her-2 positive breast cancer based on trastuzumab. It efficiently induces ferroptosis in drug-resistant tumor cells through the photothermal and chemodynamic effects of Fe3O4, while simultaneously regulating the tumor microenvironment and promoting NK cell infiltration via metformin. These multiple mechanisms synergistically reverse the trastuzumab resistance of tumors. In vitro and in vivo experiments have confirmed that MFBT can significantly inhibit the proliferation of drug-resistant tumors, improve the tumor immunosuppressive microenvironment, enhance the anti-tumor therapeutic effect, and has reliable safety. The multifunctional nanoplatform constructed in this study integrates the advantages of targeted therapy, chemodynamic therapy, ferroptosis therapy, and immunomodulatory therapy, providing a novel and efficient treatment strategy for the precise clinical treatment of Her-2 positive breast cancer, especially advanced metastatic cases and trastuzumab-resistant cases, and promoting the development of synergistic treatment strategies combining targeted therapy, ferroptosis, and immunotherapy. Attached Figure Description

[0019] Figure 1 These are TEM images of (a) Fe3O4, (b) MF, (c) MFB, (d) MFBT, and (e) energy-dispersive X-ray (EDX) elemental mapping images of MFBT. Scale bar: 100 nm. Figure 2 These are the XRD patterns of Fe3O4, MF, MFB, and MFBT; In Figure 3, (a) is the XPS total spectrum of Fe3O4, MF, MFB and MFBT; fine spectrum of MFBT: (b) C1s, (c) N1s, (d) O1s, (e) Fe2p, (f) S2p; Figure 4 shows the ultraviolet absorption spectra of Fe3O4, MF, MFB and MFBT; Figure 5 shows the FTIR infrared spectra of Fe3O4, MF, MFB and MFBT; Figure 6 shows the Zeta potential and particle size of Fe3O4, MF, MFB and MFBT; Figure 7In the table, (a) represents the drug loading of MFB at different drug loading ratios; and (b) represents the drug release of MFBT under different pH conditions. Figure 8 In the figures, (a) shows the in vitro ·OH generation of the TMB+H2O2 group, MFBT+TMB group, MFBT+H2O2 group, and MFBT+TMB+H2O2 group; (b) shows the ·OH generation of MFBT under different pH conditions; (c) shows the in vitro ·OH generation of MFBT under different H2O2 concentrations; and (d) shows the in vitro ·OH generation of MFBT over time. Figure 9 In the figure, (a) shows the temperature changes of H2O, Fe3O4, MF, MFB and MFBT under 808 nm laser irradiation (1.8 W, 10 min); (b) shows the temperature changes of MFBT at different concentrations under 808 nm laser irradiation (1.8 W, 10 min); (c) shows the temperature changes of MFBT under 808 nm laser irradiation at different powers (0.5-1.8 W, 10 min); and (d) shows the heating and cooling cycle diagram of MFBT. Figure 10 The graphs show the single-cycle heating and cooling curves of MFBT under 808 nm laser irradiation and the linear relationship between cooling time and -ln(θ). Figure 11 This is a photothermal imaging image showing the changes of H2O, Fe3O4, MF, MFB, and MFBT over time under 808 nm laser irradiation. Figure 12 In the table, (a) is the cell survival rate of HUVEC cells and SKBR-3 cells after incubation with different concentrations of MFBT; (b) is the cell survival rate of SKBR-3 cells after incubation with MFBT, MFBT+H2O2, MFBT+NIR, and MFBT+H2O2+NIR for 24 h; (*p<0.05, **p<0.01, ***p<0.001). Figure 13 This is a fluorescence imaging image of SKBR-3 cells after incubation with PBS and MFBT-RhB for 4 hours (scale bar = 20 μm). Figure 14 In the figure, (a) shows the ROS production of SKBR-3 cells after incubation with PBS, H2O2, MFBT and MFBT+H2O2 groups (scale bar = 30 μm); (b) shows the quantitative analysis of fluorescence intensity (*p<0.05, **p<0.01, ***p<0.001). Figure 15In the figure, (a) shows the killing effect of PBS group, H2O2+NIR group, MFBT+H2O2 group, MFBT+NIR group, and MFBT+H2O2+NIR group on SKBR-3 cells (scale bar = 200 μm); (b) is the quantitative analysis graph (*p<0.05, **p<0.01, ***p<0.001). Figure 16 Mitochondrial membrane potential (scale bar = 20 μm) of SKBR-3 cells after treatment with PBS, H2O2+NIR, MFBT, MFBT+H2O2, MFBT+NIR and MFBT+H2O2+NIR groups. Figure 17 The changes in HMGB1 (a) and CRT (b) in SKBR-3 cells after treatment with PBS, H2O2+NIR, MFBT, MFBT+H2O2, MFBT+NIR and MFBT+H2O2+NIR groups (scale bar = 20 μm). Figure 18 The graph shows the apoptosis status and quantitative analysis of apoptosis rate of SKBR-3 cells after incubation in the PBS group, H2O2+NIR group, MFBT group, MFBT+H2O2 group, MFBT+NIR group and MFBT+H2O2+NIR group (*p<0.05, **p<0.01, ***p<0.001). Figure 19 This is an experiment on the hemolysis rate of different concentrations of MFBT; Figure 20 In the figure, (a) shows the distribution of MFBT in mice at different time points after injection; (b) shows the fluorescence images of major organs and tumor tissues in mice 9 hours after MFBT injection. Figure 21 These are infrared thermal images of mice at different time points after being injected via the tail vein with Saline, Fe3O4, MF, MFB, and MFBT, respectively. Figure 22 This is a basic flowchart of an in vitro antitumor therapy protocol for mice; Figure 23 The images show (a) mouse body weight change curves, (b) relative tumor volume, (c) tumor weight changes in different treatment groups during the treatment period, and (d) mouse images after 14 days of treatment in different treatment groups, (e) images of ex vivo tumor tissue, and (f) HE staining of mouse tumor tissue sections. Figure 24 These are the routine blood tests and biochemical indicators of each group of mice; Figure 25 HE staining of the major organs (heart, liver, spleen, lung, and kidney) of each group of mice; Figure 26This is a schematic diagram of the preparation (a) and application (b) of MFBT. Detailed Implementation

[0020] Ferroplasmosis, a novel programmed cell death mechanism dependent on iron ions, is triggered by the massive accumulation of lipid peroxides and differs significantly from traditional cell death mechanisms such as apoptosis and necrosis. It plays an important role in tumor immune regulation—ferroplasmosis can activate immune cell responses and enhance the cytotoxic function of NK cells by releasing damage-associated molecular patterns, providing a new approach to reversing TZ resistance.

[0021] Nanomaterials, with their unique tumor microenvironment-responsive properties, targeted delivery capabilities, and multifunctional integration advantages, have become important carriers for precision cancer treatment. Among them, tumor microenvironment-responsive nanoformulations can selectively release drugs into target organs, promote tumor cell uptake, improve drug tissue distribution, and reduce side effects, providing an effective strategy for overcoming chemotherapy and targeted therapy resistance. Iron(III) oxide (Fe3O4) nanoparticles, as a typical Fenton reaction reagent, possess excellent photothermal properties, chemodynamic catalytic activity, and ferroptosis-inducing ability. They are also non-toxic, biocompatible, and can selectively release iron ions in an acidic TME environment, making them an ideal core for constructing multifunctional nanotherapeutic systems. Furthermore, Fe3O4 nanoparticles readily form superparamagnetic structures, allowing for targeted delivery and multifunctional integration through surface modification, demonstrating enormous application potential in the field of cancer diagnosis and treatment.

[0022] Metformin (MET) has been shown in recent years to have potential anti-tumor and immunomodulatory effects. It can create an immune-stimulating environment and significantly enhance the infiltration ability of NK cells in tumor tissues, providing a feasible approach to improve the immunosuppressive state of tumor mesenchymal exchange (TME) and enhance NK cell function. Bovine serum albumin (BSA) has good biocompatibility, biodegradability, and low immunogenicity. Using it to encapsulate nanoparticles can effectively improve their biocompatibility, reduce in vivo clearance, prolong blood circulation time, and improve the bioavailability of nanosystems.

[0023] Based on this, this invention designs and constructs an antibody-functionalized multifunctional nanodelivery system, MET@Fe3O4@BSA-TZ (MFBT), which organically integrates the targeted therapy of TZ, the photothermal therapy (PTT) and chemodynamic therapy (CDT) of Fe3O4, and the ferroptosis-inducing effect with the immune-enhancing effect of MET. This system achieves specific targeted recognition of Her-2 positive breast cancer cells through TZ, enhances biocompatibility with BSA, and utilizes the responsive release characteristics of Fe3O4 in the acidic environment of the tumor microenvironment (TME) to achieve slow, targeted release of iron ions and TZ. Fe3O4-mediated PTT and CDT synergistically trigger ferroptosis in tumor cells, which further enhances the cytotoxic function of NK cells. Simultaneously, MET promotes NK cell infiltration. These two mechanisms synergistically improve the immunosuppressive state of the TME, ultimately reversing the TZ resistance of Her-2 positive breast cancer, achieving a synergistic therapeutic effect of chemodynamics, photothermal therapy, ferroptosis, and immune enhancement. This study aims to provide a new, efficient, and precise treatment strategy for Her-2 positive breast cancer, especially for TZ-resistant cases, and to promote the synergistic development of targeted therapy, immunotherapy, and ferroptosis therapy.

[0024] 1. Preparation of MET@Fe3O4@BSA-TZNPs (MFBT) Preparation of Fe3O4NPs: Fe3O4NPs were synthesized via a hydrothermal one-step method. 1.0 g of FeCl3·6H2O and 0.415 g of trisodium citrate were dissolved in 35 mL of ethylene glycol to obtain a homogeneous yellow solution. Then, 2.4 g of anhydrous sodium acetate was added to the solution, and the mixture was stirred at room temperature for 10 min. The resulting brown solution was transferred to a stainless steel autoclave and heated at 200 °C for 10 h to obtain a black solution, which was Fe3O4NPs. The Fe3O4NPs were washed six times with ethanol and deionized water and dried in a vacuum oven at 50 °C for 10 h to obtain black Fe3O4NPs powder.

[0025] Preparation of MET@Fe3O4NPs (MF): 200 mg of the prepared Fe3O4NPs powder was dissolved in 20 mL of PBS. Then, 20 mL of PBS solution containing 20 mg of MET was added, and the mixture was sonicated for 5 minutes. The obtained MET@Fe3O4NPs were extracted by centrifugation at 12000 rpm for 40 min, stirred overnight, washed with distilled water, and dried in an oven at 50 °C to obtain MET@Fe3O4NPs powder.

[0026] Preparation of MET@Fe3O4@BSANPs (MFB): 10 mL of distilled water was added to dissolve 50 mg of BSA. Then, 10 mg of MET@Fe3O4NPs was dissolved in 10 mL of distilled water and added dropwise to the prepared BSA solution. The mixture was stirred with a magnetic stirrer for two hours. The prepared solution was dialyzed for 2 hours to remove impurities from the dialysis bag with a molecular weight cutoff of 12 kDa. The dialysis bag was immersed in a 600 mL beaker containing distilled water, and the water in the beaker was changed every 20 minutes. The dialyzed mother liquor was freeze-dried to obtain MET@Fe3O4@BSANPs powder.

[0027] Preparation of MET@Fe3O4@BSA-TZNPs (MFBT): 1 mL of trastuzumab (TZ) (1 mg / mL) was mixed with 1 mL of NHS (5 mg / mL) and EDC (5 mg / mL), and stirred for 15 minutes to activate the carboxyl groups on TZ. The activated TZ was then mixed with MET@Fe3O4@BSANPs (10 mg / mL) and reacted. After stirring the mixture for 6 hours, it was centrifuged, washed, and freeze-dried to obtain MET@Fe3O4@BSA-TZNPs powder.

[0028] 2. Characterization of MFBT The morphology of Fe3O4, MF, MFB, and MFBT was characterized using transmission electron microscopy. Figure 1(a) shows that the prepared Fe3O4 exhibits a spherical structure with a particle size of approximately 100 nm. Figure 1 As shown in (bd), MF, MFB, and MFBT all exhibit regular spherical structures, indicating that loading MET, encapsulating BSA, and modifying TZ did not alter the surface morphology of Fe3O4. After loading small molecule MET, the TEM image of MF shows a spherical morphology similar to that of Fe3O4. Figure 1 (b) After being coated with BSA, a tiny and transparent shell was observed around Fe3O4, indicating that BSA was successfully coated on the Fe3O4 surface. Figure 1 (c)); Figure 1 (d) It can be seen that the surface morphology of MFBT did not change significantly after TZ modification. Figure 1 (e) The energy dispersive X-ray (EDX) elemental mapping image of MFBT shows that the nanocomposite is composed of five elements: Fe, S, C, N and O.

[0029] X-ray diffraction was used to analyze the crystal structure characteristics of the MFBT nanocomposite. Figure 2The XRD patterns of Fe3O4, MF, MFB, and MFBT are shown in the figure. As shown, Fe3O4 exhibits strong diffraction peaks at 30.1°, 35.5°, 43.1°, 53.4°, 57.0°, and 62.6°, corresponding to the (220), (311), (400), (422), (511), and (440) crystal planes of Fe3O4, respectively. These characteristic peaks are consistent with the standard Fe3O4 crystal structure (JCPDS No. 19-0629), proving the successful synthesis of highly crystalline Fe3O4 nanoparticles. After loading small molecule metformin (MET) onto the Fe3O4 surface, the characteristic diffraction peaks of Fe3O4 are still clearly preserved, indicating that the MET loading process did not destroy the crystal structure of Fe3O4, suggesting that MET is loaded onto the Fe3O4 surface in an amorphous or low-crystallinity state. In MFB, a broad diffraction peak appeared in the 10–30° range, which is a typical characteristic diffraction peak of the amorphous organic macromolecule BSA, proving that BSA was successfully encapsulated on the MF surface and that the encapsulation process did not change the crystal structure of Fe3O4. In MFBT, after surface modification with the targeted molecule TZ, the crystal structure did not change significantly, indicating that the modification of TZ did not have a significant impact on the crystal structure of the material, and a structurally stable MFBT nanocomposite was successfully prepared.

[0030] Elemental and valence states of nanomaterials were analyzed using XPS. The XPS spectra of Fe3O4, MF, MFB, and MFBT showed that ( Figure 3 (a) Fe3O4 contains three elements: C, O, and Fe. The C and O elements originate from sodium citrate, a stabilizer introduced during the synthesis process. MF, MFB, and MFBT contain five elements: C, N, O, Fe, and S. To understand the valence states of each element in MFBT, a detailed elemental spectrum of MFBT was collected. Figure 3 (bf)). In the fine spectrum of C1s ( Figure 3 (b) The peaks at 284.5 eV, 285.7 eV, and 288.5 eV correspond to C=C bonds, CO bonds, and C=O bonds, respectively; in the fine spectrum of N1s ( Figure 3 (c) The peaks at 401.5 eV and 399.5 eV correspond to CN and NH bonds, respectively; Figure 3 In (d), the peaks at 531.9 eV and 531.2 eV correspond to the CO bond and C=O bond, respectively; in the fine spectrum of Fe2p ( Figure 3 (e) shows two absorption peaks at 711.4 eV and 725.3 eV, which are characteristic peaks of Fe2p3 / 2 and Fe2p1 / 2, respectively. In the fine spectrum of S2p, absorption peaks at 163.5 eV and 164.5 eV are observed, corresponding to characteristic peaks of S2p3 / 2 and S2p1 / 2, respectively. Figure 3 (f)).

[0031] The UV absorption spectra of Fe3O4, MF, MFB and MFBT are as follows: Figure 4 As shown, Fe3O4 exhibits a broad absorption band around 400 nm. Since MET is a small molecule, its absorption peak did not change significantly after loading. The UV spectrum of MFB shows a distinct characteristic absorption peak at 280 nm, and a similar absorption band to Fe3O4 still exists at 400 nm. After TZ modification, MFBT exhibits a strong absorption peak in the UV-Vis wavelength range of 260 nm.

[0032] like Figure 5 The FTIR spectroscopic analysis results of Fe3O4, MF, MFB and MFBT shown indicate that Fe3O4 at 580 cm⁻¹ -1 The characteristic peak at 3431 cm⁻¹ corresponds to the stretching vibration of the Fe-O bond, proving the successful synthesis of the magnetic core; simultaneously, the characteristic peak at 3431 cm⁻¹ corresponds to the stretching vibration of the Fe-O bond, proving the successful synthesis of the magnetic core; -1 The broad absorption peak at 1575 cm⁻¹ corresponds to the stretching vibration of hydroxyl (OH) groups on the material surface. MF is located at 1575 cm⁻¹. -1 The new peak at 1648 cm⁻¹ is attributed to the stretching vibration of the NH bond, proving that MET was successfully loaded onto the Fe₃O₄ surface via chemical bonding. In the infrared spectrum of MFB obtained by encapsulating BSA, the peak is at 1648 cm⁻¹. -1 and 1542 cm -1 The presence of characteristic absorption peaks at these locations, attributed to the amide I band (stretching vibration of the C=O bond) and amide II band (bending vibration of the NH bond) in the BSA molecule, directly proves that BSA was successfully coated onto the MF surface. Finally, after modifying with TZ to obtain MFBT, its spectrum retained the characteristic absorption peaks of MFB, and the characteristic absorptions of the TZ molecule appeared at 1631 cm⁻¹ and 1537 cm⁻¹, merging and enhancing with the amide band of BSA. This indicates that TZ was successfully modified onto the MFB surface, and the entire composite process did not damage the chemical structure of the original organic components.

[0033] The zeta potential and particle size results for Fe3O4, MF, MFB, and MFBT are as follows: Figure 6 As shown, the surface potential of Fe3O4 is -20.1 mV. After loading with positively charged MET, its negative surface charge is consumed, so the potential of MF is -8.2 mV. Since the surface of BSA is negatively charged, the potential of MF after being modified with BSA is -16.9 mV. After introducing positively charged TZ, its potential changes from -16.9 mV to -9 mV. The particle size of Fe3O4 is 138.8 nm. After loading with small molecule MET, the particle size of MF is 142.1 nm. After encapsulating BSA, the particle size of MFB is 333.6 nm. After TZ modification, the particle size of MFBT is 363.2 nm.

[0034] All the above experimental results demonstrate the successful preparation of MFBT.

[0035] 3. In vitro experiments of MFBT 3.1 Drug Loading Determination Drug loading refers to the proportion of drug content in a nanocarrier, usually expressed as a percentage by mass. It reflects the nanocarrier's ability to encapsulate drugs.

[0036] Trastuzumab (TZ) was dissolved in deionized water to prepare TZ standard solutions with concentrations ranging from 10 to 100 μg / mL. The absorbance (A) of each concentration of standard solution was measured at 260 nm using a UV-Vis spectrophotometer, and a TZ standard curve was plotted. According to the mass ratios of TZ to MFB (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3), the TZ aqueous solutions were pre-activated using the carbodiimide method (EDC / NHS). 5 mL of each TZ solution was mixed with 5 mL of MFB aqueous solution, stirred overnight, and then centrifuged at 12000 r / min for 10 min. The absorbance (A) of the supernatant at 260 nm was measured, and the drug loading (DLC) of MFB was calculated using the following formula: Formula (1-1) Where M1 is the TZ mass of the load and M0 is the mass of the MFB nanoparticles.

[0037] According to Formula 1-1, when the drug loading ratio is 2:1, the maximum drug loading is 22.4% ( Figure 7 (a)).

[0038] 3.2 Drug Release Study MFBT nanoparticles were packed into dialysis bags with a molecular weight cutoff of 3500 Da and placed in 500 mL beakers containing PBS buffer at pH 5.5, 6.5, and 7.4, respectively. The dialysis apparatus was operated at 37 °C and 100 r / min. The solution outside the dialysis bag was collected at different time points, and its absorbance (A) was measured. The release amount was calculated based on a standard curve. Figure 7 (b) It can be seen that the drug release rates of MFBT in PBS with pH=7.4 / 6.5 / 5.5 are 24.8%, 45.5% and 63.1% respectively, which indicates that the drug release behavior of MFBT is pH responsive.

[0039] 3.3 In vitro chemokinetic effects 3,3',5,5'-Tetramethylbenzidine (TMB) was used as a fluorescent probe to evaluate the chemokinetic properties of MFBT. Five experimental groups were set up: ①TMB + H₂O₂ group, ②MFBT + TMB group, ③MFBT + H₂O₂ group, and ④MFBT + TMB + H₂O₂ group, with TMB and H₂O₂ concentrations of 5 mM and 10 mM, respectively, and the nanomaterial concentration of 100 μg / mL. After shaking and reacting the solutions at 37 ℃ for 10 min, the absorbance at 650 nm was measured using a UV-Vis spectrophotometer. To further investigate the effect of pH on hydroxyl radical generation, mixed solutions of TMB (5 mM), H₂O₂ (10 mM), and MFBT (100 μg / mL) were prepared in PBS with different pH values ​​(5.5, 6.5, and 7.4), and different H₂O₂ concentrations and time points were used to evaluate the ability of each group to generate ·OH.

[0040] like Figure 8 As shown in (a), no obvious fluorescence signal was observed in the TMB+H2O2 group, the MFBT+TMB group, and the MFBT+H2O2 group, while the MFBT+TMB+H2O2 group produced a significant fluorescence signal at 650 nm. This indicates that the MFBT+TMB+H2O2 group generates hydroxyl radicals (·OH); Figure 8 (b) pH gradient experiments (7.4 / 6.5 / 5.5) showed that, under the same time conditions, MFBT generated more ·OH under acidic conditions (pH=5.5). This result indicates that an acidic environment can significantly promote the catalytic generation of ·OH from H2O2 by MFBT, thereby enhancing its chemodynamic therapeutic effect; Figure 8 (c) Further analysis by increasing the H2O2 concentration showed that more ·OH was generated with increasing H2O2 concentration. Finally, Figure 8 (d) The generation of ·OH by MFBT in vitro gradually increased over time. In summary, MFBT exhibits good chemokinetic properties in vitro and can effectively catalyze the generation of ·OH from H2O2.

[0041] 3.4 In vitro photothermal performance measurement To investigate the in vitro photothermal effects of different materials, an 808 nm laser was selected as the light source, and thermocouple probes were used to detect the temperature change curves of different materials after laser irradiation. Figure 9(a) Experimental results show that the temperature of H2O did not increase significantly after 10 min of laser irradiation, while the temperatures of Fe3O4, MF, MFB, and MFBT increased to 44℃, 42.2℃, 40.3℃, and 39.1℃, respectively, after 10 min of laser irradiation. This result indicates that MFBT has good photothermal performance, and its photothermal heating capacity was not affected by the loading of MET, BSA, and TZ. Figure 9 (b) and Figure 9 (c) shows that the temperature rise of MFBT increases significantly with increasing concentration and laser power, indicating that the material exhibits concentration- and laser power-dependent temperature rise. To verify the photothermal stability of MFBT, five heating and cooling cycles were conducted. Figure 9 (d) The results showed that the temperature of the MFBT solution remained stable throughout the multiple cycles, demonstrating that the nanocomposite has good photothermal stability.

[0042] Through the above photothermal stability experiment, a single heating and cooling curve and a linear relationship between time and -ln(θ) can be obtained. Figure 10 By fitting the cooling curve, the τs of MFBT was found to be 198. Combined with formulas (1-2) to (1-6), the photothermal conversion efficiency was calculated to be 38.68%, which proves that MFBT has good photothermal effect.

[0043] Formula (1-2) Formula (1-3) Formula (1-4) Formula (1-5) Formula (1-6) Where h is the heat transfer coefficient, T max and T surr These represent the highest solution temperature and ambient temperature, respectively, and S and Q. dis τ represents the surface area of ​​the quartz cuvette and the heat absorbed by the cuvette itself, respectively. s Let I be the system heat transfer time constant, I be the laser power, A be the absorbance of the material at 808 nm, m be the mass of the solution, and C be the mass of the solution. water Let t be the specific heat capacity of water, and t be time.

[0044] Figure 11Photothermal images of H2O, Fe3O4, MF, MFB, and MFBT under 808 nm laser irradiation. During the 10-minute laser irradiation process, no significant color change was observed in the H2O group. Compared with the other groups, the MFBT group still showed a significant heating effect after surface modification, indicating a significant deepening of the solution color, demonstrating that MFBT has good in vitro photothermal imaging capabilities.

[0045] 4. In vitro therapeutic performance study of MFBT 4.1 Dark cytotoxicity The MTT assay was used to determine the dark cytotoxicity of MFBT on HUVEC and SKBR-3 cells. SKBR-3 cells were seeded in 96-well plates and cultured for 24 h, then divided into four groups: ① MFBT group, ② MFBT+H2O2 group, ③ MFBT+NIR group, and ④ MFBT+NIR+H2O2 group. Each group was further incubated with different concentration gradients of MFBT (0, 10, 20, 40, 60, 80, and 100 μg / mL) for 24 h. Groups ③ and ④ were irradiated with an 808 nm laser for 20 min. After incubation, the supernatant was removed, and MTT was added. After 4 h of incubation, the MTT solution was removed, and 150 μL of DMSO was added to each well. The absorbance of each group was measured using a microplate reader, and cell viability was calculated.

[0046] Formula (1-7) Figure 12(a) shows that when HUVEC cells and SKBR-3 cells were incubated with different concentrations (0-100 μg / mL) of MFBT, the survival rate of both HUVEC cells and SKBR-3 cells remained above 80% when the MFBT concentration reached 100 μg / mL, indicating that MFBT has good biosafety.

[0047] 4.2 Cellular phototoxicity To evaluate the killing effect of different treatment groups on SKBR-3 cells, the MTT assay was used to detect cell viability in each group. Figure 12 As shown in (b), the cell viability rates of the MFBT group, MFBT+H2O2 group, and MFBT+NIR group at a concentration of 100 μg / mL were 80.15%, 61.45%, and 61.12%, respectively, indicating that single treatment modality (CDT / PTT) has a certain inhibitory effect on breast cancer cell proliferation. However, the cell viability rate of the MFBT+H2O2+NIR group (CDT+PTT) at a concentration of 100 μg / mL was significantly reduced to 23.56%. This data indicates that the synergistic effect of chemodynamic therapy and photothermal therapy can more effectively inhibit the proliferation of SKBR-3 cells, exhibiting a strong anti-tumor effect and demonstrating significant therapeutic advantages.

[0048] 4.3 Cell uptake experiment To analyze the uptake process of the nanocomposite by SKBR-3 cells, Rhodamine B (RhB) was used as a fluorescent probe to covalently label the MFBT nanocomposite, obtaining MFBT-RhB with fluorescent tracking function. SKBR-3 cells were seeded in confocal culture dishes and cultured adherently for 24 h. Experimental treatments were performed: ① the control group was treated with PBS; ② the experimental group was treated with medium containing MFBT-RhB (100 μg / mL). After incubation for another 4 h, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with propidium iodide solution (DAPI) in the dark for 15 min, and finally images were acquired using a laser confocal microscope.

[0049] like Figure 13 As shown, in the PBS group, only the cell nuclei showed blue fluorescence of DAPI, while the cytoplasm and cell nuclei showed no red fluorescence. In the SKBR-3 group incubated with MFBT-RhB, there was strong red fluorescence in the cells, and the cell nuclei showed blue fluorescence of DAPI, indicating that MFBT can be taken up by tumor cells.

[0050] 4.4 Intracellular reactive oxygen species detection DCFH-DA was used as a fluorescent probe to investigate the level of intracellular ROS. SKBR-3 cells were seeded in confocal culture dishes and incubated overnight. The cells were divided into four groups: ①PBS, ②H2O2, ③MFBT, and ④MFBT+H2O2. After co-incubation with the corresponding materials for 4 h, the culture medium was discarded, and then DCFH-DA (10 μM) was added and incubated for 20 min. The cells were washed three times with PBS, and images were acquired using a laser confocal microscope. The fluorescence intensity was analyzed using ImageJ software.

[0051] ROS levels in cells were detected by DCFH-DA, and the intensity of green fluorescence in the cells was positively correlated with the level of reactive oxygen species in the cells. Figure 14 As shown in (a), no obvious green fluorescence signal was observed in the PBS group, H2O2+NIR group, and MFBT group, indicating that there was essentially no reactive oxygen species (ROS) production. Weak green fluorescence was observed in the MFBT+H2O2 group and the MFBT+NIR group, indicating that CDT and PTT induced a small amount of ROS production. The green fluorescence was significantly enhanced in the MFBT+H2O2+NIR group, indicating that the synergistic effect of CDT and PTT significantly increased the intracellular ROS level. (Quantitative analysis diagram) Figure 14(b) shows that the fluorescence intensity of the MFBT+ H2O2+NIR group was significantly enhanced compared with other control groups, indicating that the MFBT nanocomposite has a good ability to generate intracellular reactive oxygen species. This experiment verifies the high efficiency of MFBT in the synergistic effect of chemodynamic therapy and photothermal therapy.

[0052] 4.5 Double staining experiment for live and dead cells Using SKBR-3 as a cell model, AM-PI staining was performed on cells in each group. Figure 15 As shown, cells in the PBS group and the H2O2+NIR group both exhibited green fluorescence, while cells in the MFBT group, MFBT+H2O2 group, MFBT+NIR group, and MFBT+H2O2+NIR group simultaneously exhibited varying degrees of red and green fluorescence. Among them, the red fluorescence in the MFBT single group was the weakest, followed by the MFBT+H2O2 group and the MFBT+NIR group, and the red fluorescence in the MFBT+H2O2+NIR group was the strongest, indicating that most cells underwent apoptosis, suggesting that the combined action of CDT and PTT in this group had the best therapeutic effect on tumor cells.

[0053] 4.6 Cellular Mitochondrial Damage Experiment To further investigate the mechanism of action of MFBT on apoptosis, the degree of damage to MMPs by MFBT was assessed using the fluorescent probe JC-1. Figure 16 As shown, the PBS, H2O2+NIR, and MFBT groups exhibited strong red fluorescence, indicating that they did not damage the mitochondrial membrane potential of the cells. However, the MFBT+H2O2, MFBT+NIR, and MFBT+H2O2+NIR groups showed significantly enhanced green fluorescence, indicating that these groups led to a decrease in mitochondrial membrane potential and impaired mitochondrial function, ultimately resulting in apoptosis. This confirms that the induction of apoptosis during treatment is achieved through the mitochondrial pathway.

[0054] 4.7 Immunogenic cell death study Immunogenic cell death (ICD) represents a unique form of programmed cell death that triggers a strong immune response against tumor cells. This process is mediated by the release of damage-associated molecular patterns (DAMPs) from cancer cells, including calreticulin (CRT) and high-mobility group box 1 (HMGB1). These DAMPs act as potent immune stimuli, promoting the recruitment and activation of dendritic cells (DCs), thereby initiating T cell-mediated anti-tumor immunity. Therefore, changes in immune-related indicators after nanoparticle platform intervention were examined. HMGB1, as a danger signal, can activate pattern recognition receptors on antigen-presenting cells (APCs) and other immune cells. In the control group, HMGB1 is mainly localized in the nucleus of SKBR-3 cells. However, after MFBT treatment, as shown in the image, HMGB1 is more readily localized in the nucleus of SKBR-3 cells. Figure 17 As shown, the green fluorescence signal of HMGB1 in the cell nucleus was significantly reduced, indicating that HMGB1 almost completely translocated from the nucleus to the cytoplasm, triggering an in vitro anti-tumor immune response. Similarly, MFBT treatment significantly increased the exposure of calreticulin (CRT) on the surface of SKBR-3 cells. In summary, MFBT anti-tumor therapy induced HMGB1 nuclear translocation and CRT surface exposure, indicating that the MFBT nanoplatform can activate an anti-tumor immune response in vitro.

[0055] 4.8 Flow Cytometry Experiment Cell apoptosis in each group was detected using flow cytometry. Results are as follows: Figure 18 As shown in (a), the apoptosis rates in the PBS group, NIR+H2O2 group, and MFBT group were all below 10%, indicating that single laser irradiation and MFBT failed to significantly induce apoptosis. However, the MFBT+H2O2 (CDT) group, MFBT+NIR (PTT) group, and MFBT+H2O2+NIR (CDT / PTT) group exhibited different degrees of apoptosis, with apoptosis rates of 35%, 35.64%, 46.9%, and 81.04%, respectively (Figure 18(b)). Among them, the apoptosis rate in the MFBT+H2O2+NIR (CDT / PTT) group was as high as 84.1%, demonstrating that the synergistic effect of CDT and PTT treatment was significantly better than single treatment, verifying that MFBT has a good apoptosis-inducing ability.

[0056] 4.9 Hemolysis test The hemocompatibility of MFBT was assessed through a hemolysis test. If the hemolysis rate was greater than 5%, the material was considered to cause a hemolytic reaction. Figure 19As shown, by observing the color of red blood cells in the EP tube, no hemolysis was observed in the red blood cells of the negative control group, while obvious hemolysis was observed in the red blood cells of the positive control group. No obvious hemolysis was observed in any of the experimental groups with different concentrations of MFBT. Further calculation using formula (1-8) showed that the hemolysis rate of each experimental group was less than 5%. This verifies that MFBT nanomaterials have good blood compatibility.

[0057] Formula (1-8) 5. Evaluation of the in vivo antitumor effect of MFBT 5.1 Mouse fluorescence imaging The tumor targeting and tissue biodistribution of MFBT nanoparticles in tumor-bearing mice were evaluated using a small animal in vivo imaging system. Fluorescence intensity at the tumor site was measured as follows: Figure 20 As shown in (a), after MFBT was injected into mice via the tail vein, it gradually accumulated at the tumor site from 0 h to 24 h, with the fluorescence intensity of each group continuously increasing. Starting from 3 h, due to the tumor-targeting properties of TZ, MFBT nanoparticles showed obvious fluorescence signals at the tumor site; the fluorescence signal at the tumor site reached its strongest at 9 h, indicating that MFBT accumulation in the tumor region was most significant at this time; the fluorescence signal at the tumor site gradually weakened at 12 h; at 24 h, the fluorescence signal at the tumor site became weak, with only the liver still showing obvious fluorescence signals; at 48 h, the fluorescence signal at the liver site basically disappeared. To further verify the enrichment of MFBT at different sites, mice were dissected at 9 h when the tumor fluorescence signal was strongest, and tumor tissue and major organs were collected for in vitro fluorescence imaging analysis. Figure 20 The results in (b) show that the fluorescence intensity of the major organs (heart, liver, spleen, lung, and kidney) in each group is lower than that of the tumor site. The results of ex vivo imaging are consistent with those of in vivo imaging, further confirming that MFBT mainly accumulates in the tumor site.

[0058] 5.2 Mouse photothermal imaging Infrared thermal imaging was used to evaluate the photothermal effects of each group of materials in vivo. Photothermal imaging, such as... Figure 21 As shown, under 808 nm laser irradiation, the temperature of the tumor site in the Saline group of mice did not change significantly, while the temperature of mice in the Fe3O4, MF, MFB, and MFBT groups all increased to varying degrees under laser irradiation. Among them, the mice injected with MFBT showed a significant enhancement of photothermal signal at the tumor site, with the most significant temperature increase. This may be due to the targeting effect of TZ, which causes MFBT to accumulate in large quantities at the tumor site, thereby achieving efficient photothermal conversion, resulting in a more significant temperature increase in this group than in the other groups. Therefore, MFBT has good photothermal imaging capabilities and can achieve effective accumulation and photothermal conversion at the mouse tumor site.

[0059] 5.3 Evaluation of antitumor therapeutic effects in mice To evaluate the in vivo antitumor effect of MFBT, a tumor-bearing mouse model was established. Figure 22 When the tumor volume in mice reaches 100 mm... 3 Different groups of materials were injected via tail vein to treat tumors, and mouse body weight and tumor size were monitored every other day. Figure 23 (a) It can be seen that there was no significant difference in the body weight of mice during the treatment period, indicating that MFBT has no significant systemic toxic side effects. For example... Figure 23 As shown in (b), tumors in mice in the Saline and NIR groups grew rapidly during treatment. Tumor growth in the Fe3O4 group was somewhat inhibited by CDT treatment. The MFBT and MFB+NIR groups showed more significant inhibition of tumor growth compared to the Fe3O4 group due to the intervention of the chemotherapy drug TZ targeted therapy and photothermal therapy, respectively. The MFBT+NIR group showed the most significant inhibition of tumor growth. On day 14 of the treatment period, tumors from each group of mice were collected and weighed. The results are as follows: Figure 23 As shown in (c), compared to the other five groups, the MFBT+NIR group showed significantly limited tumor growth due to the intervention of the targeted agent and near-infrared light, demonstrating that the combined treatment group had the most significant therapeutic effect. Figure 23 (d) and Figure 23 (e) It was observed that tumors in the MFBT+NIR (combination therapy) group were significantly suppressed at the end of treatment. Finally, histopathological analysis of tumors in different groups was performed using HE-stained sections. Figure 23 (f) No tumor ablation was observed in the saline group and the single laser group. Tumors in the Fe3O4 group and the MFB+NIR group showed some degree of damage. The MFBT+NIR group exhibited significant tumor cell ablation. In conclusion, MFBT can effectively inhibit tumor cell growth and possesses good in vivo anti-tumor activity.

[0060] 5.4 In vivo biosafety evaluation Blood was collected from each group of mice after treatment, and the biosafety of MFBT was assessed by blood routine and biochemical index testing, such as... Figure 24 The results showed that the blood routine and liver and kidney indicators of mice in the control group and each treatment group remained at normal levels with no significant abnormalities, confirming that the synthesized nanocomposite has good biocompatibility. Finally, the major organs of mice in each group were collected, and the biocompatibility of MFBT in vivo was further investigated by HE staining. Figure 25 The experimental results showed that the major organs of mice in different treatment groups did not have obvious morphological abnormalities, indicating that MFBT has good biocompatibility during treatment.

[0061] This invention utilizes TZ targeting to effectively aggregate MFBT nanomaterials at the tumor site. The photothermal effect generated under NIR laser irradiation effectively kills tumor cells. Simultaneously, the Fenton reaction of ferroptosis promotes the decomposition of H2O2, generating a large amount of •OH, achieving a synergistic therapeutic effect of CDT / PTT / ferroptosis, thereby significantly inhibiting tumor growth. HE staining results show no significant toxicity to normal tissues. In summary, the MFBT nanocomposite material designed and developed in this study achieves synergistic CDT / PTT / ferroptosis therapy, providing a new research strategy and direction for multimodal targeted therapy and immunotherapy of Her-2 positive breast cancer, highlighting the enormous application potential of nanomedicine delivery systems in the treatment of Her-2 positive breast cancer.

Claims

1. An antibody-functionalized nanodelivery system, MET@Fe3O4@BSA-TZ, characterized in that, Including nanocarriers and targeted therapeutic drugs; The nanocarrier comprises iron(III) oxide (Fe3O4) nanoparticles, metformin (MET), and bovine serum albumin (BSA); the iron(III) oxide (Fe3O4) nanoparticles serve as the core of the nanodelivery system; metformin (MET) is loaded onto the surface of the iron(III) oxide (Fe3O4) nanoparticles; and bovine serum albumin (BSA) is wrapped around the iron(III) oxide (Fe3O4) nanoparticles and metformin (MET) to form a shell. The targeted therapeutic drug is trastuzumab (TZ), which is covalently coupled to the shell layer.

2. The antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ according to claim 1, characterized in that: Based on the mass percentage of trastuzumab (TZ) in the nanocarrier, the maximum drug loading of trastuzumab (TZ) is 22.4%.

3. The method for preparing the antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ according to any one of claims 1 or 2, characterized in that, include: Step 1: Prepare Fe3O4NPs; FeCl3·6H2O and trisodium citrate were dissolved in ethylene glycol, and then anhydrous sodium acetate was added. Fe3O4NPs were obtained by hydrothermal reaction. Step 2: Prepare MET@Fe3O4NPs; Fe3O4NPs were dissolved in PBS, and then a PBS solution containing metformin (MET) was added. After sonication, centrifugation, stirring, washing, and drying, MET@Fe3O4NPs were obtained. Step 3: Preparation of MET@Fe3O4@BSANPs MET@Fe3O4NPs were dissolved in distilled water and added dropwise to an aqueous solution of bovine serum albumin (BSA). After stirring and dialysis, the mother liquor obtained by dialysis was freeze-dried to obtain MET@Fe3O4@BSANPs. Step 4: Prepare MET@Fe3O4@BSA-TZNPs; Trastuzumab (TZ) was activated with NHS and EDC. The activated trastuzumab (TZ) was then mixed with MET@Fe3O4@BSANPs and reacted. After centrifugation, washing, and freeze-drying, MET@Fe3O4@BSA-TZNPs were obtained.

4. The method according to claim 3, characterized in that: In step one, the hydrothermal reaction temperature is 180-220℃, and the reaction time is 8-12 h.

5. The method according to claim 3, characterized in that: In step two, the mass ratio of Fe3O4NPs to metformin (MET) is 10:

1.

6. The method according to claim 3, characterized in that: In step three, the mass ratio of MET@Fe3O4 to bovine serum albumin (BSA) is 1:

5.

7. The method according to claim 3, characterized in that: In step four, the mass ratio of trastuzumab (TZ) to MET@Fe3O4@BSANPs is 1:(0.5-3).

8. The method according to claim 7, characterized in that: The mass ratio of trastuzumab (TZ) to MET@Fe3O4@BSANPs is 1:

2.

9. The use of the antibody-functionalized nanodelivery system MET@Fe3O4@BSA-TZ according to claim 1 or 2 in the preparation of a drug for treating Her-2 positive breast cancer.

10. The application according to claim 9, characterized in that: The treatment includes: achieving specific targeted recognition of Her-2 positive breast cancer cells through trastuzumab (TZ), enhancing biocompatibility with bovine serum albumin (BSA), and utilizing the responsive release characteristics of Fe3O4 in the acidic environment of the TME to achieve slow, targeted release of iron ions and trastuzumab (TZ); Fe3O4-mediated PTT and CDT synergistically trigger tumor cell ferroptosis, which further enhances the cytotoxic function of NK cells, while metformin (MET) promotes NK cell infiltration. The two synergistically improve the immunosuppressive state of the TME, ultimately reversing the resistance of Her-2 positive breast cancer to trastuzumab (TZ), achieving synergistic treatment of chemodynamics, photothermal activity, ferroptosis, and immune enhancement.

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