Preparation method and application of nanoparticles for destroying hepatoma cell ferritin

Abstract

The invention relates to the field of biological medicine manufacturing, in particular to a preparation method and application of nanoparticles for destroying hepatoma cell ferritin, and provides ultrasonic response type nanoparticles (NPs) composed of polylactic acid-glycolic acid (PLGA) modified by ferritin homing peptide (HKN15) and acoustic droplet vaporization (ADV) material perfluorohexane (PFH). Through the positive feedback effect of overexpression of the HKN15 and the tumor cell ferritin, the NPs (HKN15-coated PLGA-PFH) specifically gathers around the ferritin in the HCC cell. Tumor cells can be directly killed by a cavitation effect generated by ADV through ultrasonic irradiation of a tumor area; more importantly, along with collapse of ferritin, the concentration of iron in cells is increased, so that inevitable ferroptosis is caused. The scheme can destroy hepatoma cell ferritin, increase the concentration of free iron in the hepatoma cell ferritin and induce a ferroptosis effect to achieve the purpose of tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical manufacturing, and in particular to a method for preparing nanoparticles for destroying ferritin in liver cancer cells and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. The vast majority of HCC patients are in the advanced stage when they first visit the doctor. The liver is one of the important organs in the human body, involved in digestion, excretion, detoxification and immune regulation, so the liver is also one of the most vulnerable organs in the human body. However, diseases such as liver cancer have caused a large number of deaths and seriously threatened human health.

[0003] At present, the main methods for treating liver cancer include chemotherapy, radiotherapy, surgery, photodynamic therapy, etc. The treatment methods that were developed relatively early and widely used are chemotherapy and radiotherapy. Their main disadvantages are that the treatment methods are not highly targeted to cancer cells and cannot effectively distinguish cancer cells from normal cells. In particular, chemotherapy and some protein drugs have high cytotoxicity, which also cause great damage to normal organs during treatment. According to the traditional method of drug administration, small molecule drugs are randomly distributed in the body, and only a small number of drugs are actually accumulated in the diseased area and exert their efficacy. Most of them are metabolized and excreted from the body, and their bioavailability is very low.

[0004] The invention patent with application number 202211083048.4 discloses a pH and GSH dual-responsive nano drug carrier targeting liver cancer cells and its preparation method. A homemade hydrophilic galactose-containing polymer is used as a macromolecular chain transfer agent for a reversible addition-fragmentation chain transfer (RAFT) reaction or a macromolecular initiator for an atom transfer radical polymerization (ATRP) reaction, and a nanocarrier with a core-shell structure and a particle size of less than 100 nm is prepared by soap-free emulsion active polymerization assisted by 12-crown ether-4. The galactose groups on the shell polymer chain can actively target liver cancer cells, and the core layer is further hydrolyzed to obtain a pH / GSH dual-responsive nano drug carrier. The nanocarrier can be stored as a lyophilized powder. The size of the nanocarrier not only facilitates the endocytosis of cells, but also the core layer and shell layer have unique functions: the galactose groups on the shell polymer chain will actively recognize the asialoprotein (ASGP) receptors overexpressed on the surface of liver cancer cells, realizing the active targeting function of the nano drug carrier to liver cancer cells; the poly (meth) acrylic acid in the core layer will shrink rapidly from the swollen state at the acidic pH of the tumor site, and the GSH in the tumor cells will quickly degrade the disulfide cross-linking agent in the core layer. The synergistic effect of the two allows the embedded drugs to be released quickly. The nano drug carrier has great application potential in drug controlled release, especially in chemotherapy of malignant liver cancer.

[0005] The manufacturing process of the above technology is relatively complicated. In order to meet its own high proliferation and metabolic needs, hepatocellular carcinoma (HCC) will increase its iron intake. Part of the absorbed iron is consumed for growth, and the excess iron will be transported and stored in ferritin to avoid oxidative damage to itself by excess free iron. Now a technology is needed to destroy liver cancer cell ferritin and increase the free iron concentration in it to induce ferroptosis to achieve the purpose of tumor treatment. Summary of the invention

[0006] The present invention provides a method for preparing nanoparticles for destroying liver cancer cell ferritin and an application thereof, which can destroy liver cancer cell ferritin and increase the free iron concentration therein to induce ferroptosis effect to achieve the purpose of tumor treatment.

[0007] In order to solve the above technical problems, this application provides the following technical solutions: A method for preparing nanoparticles for destroying ferritin in liver cancer cells comprises the following steps: S1: PP NPs were constructed by encapsulating the liquid-gas phase change material PFH in PLGA via an improved double emulsion method; S2: Construction of P NPs by PLGA via a modified double emulsion method; S3: Ferritin homing peptide HKN was synthesized by carbodiimide coupling reaction 15 covalently bonded to the surface of PP NPs to obtain KPP NPs; S4: The obtained KPP NPs were washed with deionized water to remove the micro-reactive substances.

[0008] Furthermore, the prepared nanoparticles need to be labeled by adding DiI or DiR in steps S1 and S2.

[0009] Furthermore, the mass of typical protons of the prepared KPP NPs was detected, in which the chemical shift was evaluated by F-NMR, the changes in chemical bonds were evaluated by FTIR, DLS was used to measure the average hydrodynamic diameter and zeta potential of the NPs, PP and KPP NPs were suspended in PBS to observe the changes in particle size, and the morphology of KPP NPs was observed by SEM and TEM.

[0010] Furthermore, the prepared nanoparticles need to be tested, and the test steps are as follows: A1: HCC cells for culture experiment: A2: Select mice aged 6 to 8 weeks as test subjects; A3: 5 × 10 suspended in 100 μL PBS were injected subcutaneously in the right lower limb. 6 HCC cells were cultured into C57BL / 6J mice with HCC; A4: The subcutaneous tumor size of mice injected with HCC cells was continuously observed, and after the injection threshold time, the mice were randomly divided into different groups.

[0011] Furthermore, the liver cancer cell culture method in step A1 is as follows: HCC cells are cultured in a humidified 5% CO2 environment at 37°C with 10% fetal bovine serum, 100 mg mL −1 Streptomycin and penicillin in DMEM.

[0012] Furthermore, it also includes the intracellular uptake of nanoparticles and ferritin targeting: first, the cellular internalization of NPs in liver cancer cells was detected; then the co-localization of ferritin with PP and KPP NPs was observed; DiR-labeled PP and KPP NPs suspensions were injected into tumor-bearing mice, and the fluorescence signals were monitored and analyzed at 2, 6, 10, 24, and 48 h after injection using an in vivo imaging system.

[0013] Furthermore, it also includes ferritin destruction and ferroptosis induction: HSF was used to simulate ferritin in vitro, and the iron content discharged from HSF was evaluated using a colorimetric iron assay kit; the ferritin expression levels in HCC cells treated with three types of PBS, KPP, and KPP + LIFU were evaluated by WB; and the microstructural morphology of cells after LIFU + KPP treatment at different power intensities was observed using SEM.

[0014] An application of nanoparticles for destroying ferritin in liver cancer cells, comprising nanoparticles prepared by the above preparation method.

[0015] The basic principle and beneficial effects of this scheme: In this scheme, a NP precisely targeting tumor ferritin and controlled by low-intensity focused ultrasound (LIFU) was constructed, namely HKN 15 @PLGA-PFH (KPP) is a novel nanostructured polymer that selectively destroys ferritin in HCC cells in a spatiotemporal manner to induce ferroptosis. Poly(lactic-co-glycolic acid) (PLGA) is used as a carrier, which has excellent nanoscale biological effects, high biocompatibility, and easy surface modification. The ADV-responsive material perfluorohexane (PFH) is encapsulated in PLGA as the core. The ferritin homing peptide HKN 15 (HKNKGKKNGKHNGWK) grafted onto the PLGA shell as a navigator to recognize and target ferritin. HKN 15 It has been demonstrated to have significant ferritin targeting ability, and lysosomal escape can be achieved through caveolin-mediated endocytosis, helping NPs reach the cytoplasm. 15Due to the positive feedback effect of tumor ferritin and the overexpression of ferritin, KPP NPs accumulate around ferritin in large quantities. Then, the ADV effect is precisely controlled by external LIFU, resulting in the degradation of tumor cells and ferritin. As the iron pool is destroyed, the enhanced aggregation of cellular iron induces ferroptosis. In general, this protocol proposes a new controllable method to target the disruption of ferritin homeostasis, providing a potential innovative method for the clinical treatment of HCC. This protocol successfully constructed nanoparticles that precisely target tumor ferritin and are regulated by spatiotemporal LIFU. In addition to directly killing tumor cells, with the disintegration of tumor ferritin, the aggregation of cellular iron is enhanced, and ferroptosis is induced through the disturbance of iron-catalyzed redox homeostasis to achieve the purpose of tumor treatment. Therefore, the combination of KPP NPs and LIFU is feasible and proposes a promising clinical strategy for the treatment of HCC without systemic toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For the characterization of nanoparticles; Figure 2 for intracellular uptake and targeting to ferritin; Figure 3 for ferritin destruction and ferroptosis induction; Figure 4 For biosafety assessment. DETAILED DESCRIPTION

[0017] The following is further described in detail through specific implementation methods: Embodiment 1 A method for preparing nanoparticles for destroying ferritin in liver cancer cells, characterized in that it comprises the following steps: S1: Materials for preparing nanoparticles were selected. PLGA-COOH was from Xi’an Ruixi Biotechnology Co., Ltd. (Xi’an, China). Ferritin targeting peptide NH2-HKN 15 Synthesized by QYAOBIO (Shanghai Zhongpeptide Technology Co., Ltd.). PFH was from Sigma-Aldrich (St. Louis, MO, USA). HSF (100 mg mL-1) was purchased from MacLean Biochemicals (Shanghai, China). Anti-ferritin antibody (ab75973) was purchased from Abcam (Shanghai, China).

[0018] The liquid-gas phase change material PFH is encapsulated in PLGA to construct PP NPs through an improved double emulsion method; the improved double emulsion method is a method for preparing nanoparticles. In this process, PFH will be encapsulated in PLGA to form small particles, which are PP nanoparticles; due to the stirring effect, the mixture of PFH and PLGA will be dispersed into many small droplets, each of which is the core of a nanoparticle.

[0019] S2: Construction of P NPs by PLGA via a modified double emulsion method; S3: Ferritin homing peptide HKN was synthesized by carbodiimide coupling reaction 15 covalently bonded to the surface of PP NPs to obtain KPP NPs; S4: The obtained KPP NPs were washed with deionized water to remove the micro-reactive substances.

[0020] The synthesis of KPP NPs was carried out in two steps: preparation of PP NPs and surface modification. The liquid-gas phase change material PFH was encapsulated in PLGA by an improved double emulsion method to construct PP. P was also prepared by this method, except that PFH was not added. Then, the ferritin homing peptide HKN was added to the PLGA by a carbodiimide coupling reaction. 15 covalently bonded to the surface of PP NPs. Finally, the obtained KPP NPs were washed three times with deionized water to eliminate the unreacted substances. The synthesis method of NPs labeled with DiI ` or 1,1′-dioctadecyl-3,3,3,3′-tetramethylindotricarbocyanine iodide (DiR) was similar to the above, except that DiI or DiR was added in the first step. It is worth noting that all the above steps were carried out in an ice bath. Finally, we prepared various P, PP and KPP NPs and DiI or DiR labeled NPs for different purposes.

[0021] The chemical shift of typical protons of KPP NPs was evaluated by F-NMR (Bruker AVANCE III, Germany). The changes in chemical bonds were evaluated by FTIR (iS10; Thermo Nicolet, USA). DLS (Malvern Instruments, Ltd., USA) was used to measure the average hydrodynamic diameter and zeta potential of NPs. PP and KPP NPs were suspended in PBS to observe the changes in particle size. The morphology of KPP NPs was observed by SEM (Hitachi S-3400N, Japan) and TEM (Hitachi H-7600, Japan).

[0022] It also includes liver cancer cell culture and tumor-bearing mouse model construction A1: HCC cell line was obtained from Boster (Wuhan). It was cultured in DMEM containing 10% fetal bovine serum, 100 mg mL−1 streptomycin and penicillin at 37°C in a humidified 5% CO2 environment. A2: All animal research procedures were in accordance with the ethical guidelines of Chongqing Medical University (No. (2022) 224). C57BL / 6J mice (6–8 weeks old, male) were purchased from Enswell Biotechnology Co., Ltd. (Chongqing, China). A3: 5×10 suspended in 100 μL PBS were injected subcutaneously in the right lower limb. 6 HCC cells were cultured into C57BL / 6J mice with HCC. The tumor volume was calculated as 0.5×length×width 2 A4: Ten days after HCC cell injection, subcutaneous tumors reached a measurable size and the mice were randomly divided into different groups for subsequent studies.

[0023] Also included are the intracellular uptake of nanoparticles and ferritin targeting: CLSM (Nikon, Japan) was used to detect the cellular internalization of NPs in liver cancer cells. HCC cells (2 × 105 cells per well) were seeded into laser confocal cell culture dishes for 24 h and then treated with DiI-labeled NPs (PP and KPP) for 2 h and 6 h, respectively. Finally, the cells were fixed with 4% paraformaldehyde and then stained with DAPI for 15 min before CLSM observation. The red fluorescence was attributed to the PP or KPP NPs inside the cells, while the blue fluorescence represented the cell nuclei stained with DAPI.

[0024] CLSM was used to observe the colocalization of ferritin with PP and KPP NPs. Cells were seeded in laser confocal cell culture dishes, and after attachment, PP and KPP NPs labeled with DiI were added to the culture medium and incubated for another 6 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Intracellular ferritin was detected with specific rabbit anti-ferritin primary antibody (1:100) and goat anti-rabbit secondary antibody (1:500). Images of the colocalization results were observed by CLSM with red and green fluorescence. In addition, colocalization analysis of the images of the red and green channels was performed using ImageJ software.

[0025] The DiR-labeled PP and KPP NPs suspensions were injected into tumor-bearing mice, and the fluorescence signals at 2, 6, 10, 24, and 48 h after injection were monitored and analyzed using an in vivo imaging system (Guangzhou Biolight Biotechnology Co., Ltd.).

[0026] Also included: ferritin destruction and ferroptosis induction; To investigate whether KPP NPs under LIFU can destroy ferritin, we used HSF to simulate ferritin in vitro. The iron content discharged from HSF was evaluated using a colorimetric iron assay kit. HSF (0.5 mg mL -1 ) and KPP NP solution (1 mg mL -1 ) and then irradiated the mixture with LIFU (3 W / cm2) for 1 min. The supernatant was obtained after centrifugation at 16,000 × g for 10 min. The reducing solution and the probe solution were then poured into the above mixture and the absorbance at a wavelength of 593 nm was measured.

[0027] The expression level of ferritin in HCC cells with three treatments (PBS, KPP, KPP + LIFU) was evaluated by WB. The cells were lysed with RIPA buffer containing protease inhibitors and centrifuged at 10,000 rpm for 10 min at 4°C. The cell supernatant was harvested and the protein concentration was quantified by BCA protein assay kit. Equal amounts of protein were loaded on 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and separated. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk powder for 2 h. After incubation with anti-ferritin primary antibody (1:1000) at 4°C overnight, the secondary antibody (ab150077, 1:2000) was subsequently incubated with the membrane for 1 h. Immunoblots were visualized by enhanced chemiluminescence (ECL) WB detection reagent. PBS or KPP NPs (5 mg kg −1 ) were intravenously injected into each mouse bearing HCC cells (n = 3), and 10 h later, LIFU (3 W / cm 2 ; 10 min). The mice were killed at the end of the experiment. The isolated tumors were fixed, paraffin-embedded and subjected to immunofluorescence staining.

[0028] The microstructure of cells treated with LIFU + KPP at different power intensities was observed by SEM. After incubation with KPPNPs for 6 h, the cells were exposed to LIFU (0-3 W / cm 2, 1 min, 1 MHz, 50% duty cycle). The cells were then gently scraped off with a cell scraper and placed in a 1.5 mL pointed EP tube. After centrifugation at 1,000 rpm for 5 min, the supernatant was aspirated and 2.5% glutaraldehyde fixative (1 mL; 4°C) was slowly added to fix the cells. The samples were dehydrated and dried before SEM observation. To evaluate ferroptosis caused by ferritin degradation, we used TEM to observe the morphology of mitochondria after different treatments. HCC cells (2 × 10 cells per well) were placed in the 10-well plate. 5 Cells (100 μg / mL) were seeded into KPP NPs and incubated for 6 h before LIFU irradiation. After treatment, samples were taken and fixed with 2.5% glutaraldehyde at 4°C overnight. Samples were dehydrated, dried, embedded, and sectioned before observation by TEM.

[0029] It also includes biosafety assessment; To evaluate the in vivo toxicity of KPP NPs, 15 healthy mice were intravenously injected with 200 μL of KPP solution (5 mg mL -1 ). Three healthy control mice were intravenously injected with normal saline. Retro-orbital blood was collected at different time points (1, 3, 7, 14, and 28 days) for biochemical analysis and routine blood tests. Finally, major organs were obtained, fixed, and stained with H&E for pathological analysis.

[0030] It also includes statistical analysis; Statistical data were analyzed using the Statistical Package for Social Sciences (SPSS; IBM, New York, NY, USA). Quantitative data were expressed as mean ± SD. Statistical estimates were performed using one-way ANOVA and Student's t test. Significance levels were expressed as *P < 0.05, **P < 0.01, and ***P < 0.001.

[0031] As attached Figure 1 Shown are the characterization of the nanoparticles, A) F-NMR spectra of P, PFH, and PP (label numbers a, b, c, and d represent the characteristic peaks of fluorine). B) FTIR spectra of PP, HKN15, and KPP. C) Zeta potential of P, PP, and KPP NPs. D) Representative TEM image of KPP. E) Representative SEM image of KPP. F) Average diameters of P, PP, and KPP NPs. G) 7-day stability of particle size of PP and KPP. Data are expressed as mean ± SD (n = 3). ***P < 0.001. Scale bar = 200 nm. PLGA, PLGA-PFH, and HKN15@PLGA-PFH NPs are abbreviated as P, PP, and KPP, respectively.

[0032] As shown in Figure 1A, by fluorine nuclear magnetic resonance (F-NMR) spectroscopy, characteristic peaks of fluorine (labeled numbers a, b, c, and d) were found in PLGA-PFH (PP), and their positions were consistent with those in PFH. In addition, no characteristic peaks of fluorine were observed in PLGA (P). This indicates that PP was successfully prepared. Fourier transform infrared spectroscopy (FTIR) showed that: 15 Compared with PP, the KPP spectrum at 3059 cm −1 (-NH2) and 1445 cm −1 The peak at 1674 cm-1 in KPP disappears, which may be attributed to the formation of amide bonds. −1 The obvious characteristic peak at -CONH- directly indicates the successful synthesis of the amide bond (Figure 1B). Dynamic light scattering (DLS) results show that compared with P or PP, HKN 15 The zeta potential of modified NPs is due to HKN 15 Positive charge increases to a positive value ( Figure 1 C). These findings further confirm that HKN 15 Successful peptide attachment. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images ( Figure 1 D, E) Visually show that KPP was successfully synthesized and has a spherical structure with relatively consistent size. The diameters of P, PP, and KPP are 140.90 ± 0.55 nm, 218.70 ± 1.60 nm, and 258.60 ± 2.96 nm, respectively ( Figure 1 F). These sizes facilitate the preferential aggregation of NPs to the tumor site through the enhanced permeability and retention (EPR) effect. With the addition of other components, the size of these NPs gradually increased, indirectly verifying that the above elements had been successfully inserted. In addition, Figure 1G shows the stability of PP and KPP, which exhibited a nanoscale effect and showed significant stability over 7 days. The successful establishment and surface modification of NPs laid the foundation for further research on the regulation of KPP and ferritin metabolism.

[0033] As attached Figure 2Shown: Intracellular uptake and ferritin targeting. A) CLSM images of HCC cells incubated with PP or KPP NPs for 2 h or 6 h. B) CLSM images and colocalization analysis of PP, KPP, and ferritin. C) Fluorescence imaging of HCC tumor-bearing mice after tail vein injection of targeted (DiR-KPP) and non-targeted (DiR-PP) groups at different time points in vivo. D) Quantitative analysis of fluorescence signals in tumor sites at corresponding time points. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01. Scale bar = 50 μm. Pearson correlation coefficient is abbreviated as PCC.

[0034] As shown in Figure 2A, PP and KPP NPs tended to be distributed in the cytoplasm after endocytosis. In addition, both PP and KPP showed increased uptake and accumulation over time. Notably, the red fluorescence signal of NPs in the KPP group was always higher than that in the PP group. These results suggest that KPP NPs are more easily taken up and internalized into the cytoplasm of HCC cells, which may be attributed to the HKN 15 modification.

[0035] like Figure 2 As shown in B, green fluorescence in HCC cells represents ferritin located in the cytoplasm. In the KPP NP incubation group, the red fluorescence (KPP) overlapped with the green fluorescence (ferritin) to produce clear yellow fluorescence, indicating that the KPP NPs in the cells were distributed around the ferritin. In the PP NP incubation group, the NPs were widely distributed in the cytoplasm. In addition, the overlap between the two fluorescence channels in the KPP NPs group was large, and the Pearson correlation coefficient (PCC) (0.75) was also higher than that in the PP NP group (0.43), indicating that the co-localization level of KPP NPs and ferritin in HCC cells was significantly higher than that of PP NPs.

[0036] In addition, at the tumor site, the fluorescence signals of both types of NPs (PP and KPP) gradually increased within 10 hours after injection and decreased after 24 hours (Figure 2C). Therefore, 10 hours was selected as the time point for LIFU irradiation to activate the NPs in vivo, thereby achieving time control. It is worth noting that the average fluorescence signal of the KPP NP group at each time point was higher than that of the PP NP group as determined by quantitative methods (Figure 2D). The efficient accumulation and persistent retention of KPP NPs may be due to their suitable size, positive charge, and HKN 15 These findings indicate that KPP NPs can be effectively internalized into HCC cells and have good tumor and ferritin targeting abilities, laying the foundation for the subsequent ferritin degradation.

[0037] As attached Figure 3 Shown: A) Safe LIFU intensity and duration of action for ferritin destruction and ferroptosis induction. B) Bio-SEM images of cells incubated with KPP NPs at different LIFU intensities. C) In vitro quantitative analysis of iron released from horse spleen ferritin after different treatments. D, E) Western blot (D) and grayscale analysis (E) of ferritin levels in HCC cells after different treatments. F) Immunofluorescence staining of ferritin in tumor tissues resected from HCC tumor-bearing mice after various treatments. G) Bio-TEM images of mitochondrial morphology of HCC cells incubated with KPP NPs and LIFU (red arrows indicate mitochondrial morphological features of ferroptosis, i.e., mitochondrial atrophy, increased membrane density, and reduced cristae). Data are expressed as mean ± SD (n = 5 in A; n = 3 in C and E). ***P < 0.001, ns: not significant To investigate whether KPP NPs under LIFU would destroy ferritin, horse spleen ferritin (HSF) was used to simulate ferritin in vitro. First, the appropriate LIFU intensity and duration were evaluated by CCK-8 assay. The parameters of safe LIFU are as follows ( Figure 3 A): 1 W / cm 2 (1, 2, 3 minutes), 2 W / cm 2 (1, 2, 3 minutes) and 3 W / cm 2 (1 minute). Therefore, 3 W / cm 2 (1 min) was used as a LIFU parameter for in vitro study. As shown in the biological SEM image ( Figure 3 B) As the irradiation power intensity increases, the holes on the cell surface and the damage to the cells gradually become obvious, and the cavitation effect can be detected intuitively. The iron analysis kit was used to determine ( Figure 3 C), the total iron content in the KPP NP incubation solution after LIFU activation was significantly higher than that in the KPP NP solution without LIFU irradiation, verifying the destruction of ferritin by ADV. In HCC cells, the ferritin content in the group incubated with KPP and LIFU was lower compared with the control group and the group using KPP only, providing direct evidence for ferritin destruction (Figure 3D, E). Interestingly, there was no significant difference in ferritin content between the control group and the group using KPP only, indicating that LIFU is an important factor in the disintegration of ferritin (Figure 3E).

[0038] To investigate the degradation of ferritin in vivo by KPP NPs and LIFU activation, ferritin immunofluorescence staining was performed. The results showed that the ferritin level in the KPP NPs plus LIFU group was significantly lower than that in the control group or the KPP-only group, indicating that LIFU-activated KPP NPs had a significant ability to destroy ferritin (Figure 3F). In addition, the bioTEM images shown in Figure 3G showed that HCC cells in the KPP + LIFU group presented morphological features of mitochondrial ferroptosis (mitochondrial atrophy, increased membrane density, and reduced cristae), which were significantly different from cells treated with PBS. In short, under LIFU irradiation, KPP NPs can destroy tumor cells and their ferritin and induce HCC cell ferroptosis.

[0039] As attached Figure 4 , indicating biosafety assessment. A, B) Routine blood analysis (A) and blood biochemical analysis (B) of mice at different days after intravenous injection of KPP NPs (n = 3). C) H&E staining of major organs (heart, liver, spleen, lung, and kidney) of mice sacrificed at different days after intravenous injection of KPP NPs (n = 3). Scale bar = 100 μm.

[0040] Confirming the biosafety of nanomaterials is crucial for clinical translation. Compared with the control group, there was little difference in blood biochemical indicators after intravenous injection of the test dose ( Figure 4 A, B). In addition, H&E staining of the major organs in the group injected with KPP NPs did not show abnormal inflammatory response or significant histopathological changes compared with the control group ( Figure 4 C). These results indicate that KPPNPs do not cause acute (1 day), short-term (3 or 7 days), or long-term (14 or 28 days) toxic side effects.

[0041] In summary, this protocol successfully constructed nanoparticles that precisely targeted tumor ferritin and were regulated by spatiotemporal LIFU. In addition to directly killing tumor cells, with the disintegration of tumor ferritin, the aggregation of cellular iron was enhanced, and ferroptosis was induced through the disturbance of iron-catalyzed redox homeostasis to achieve the purpose of tumor treatment. Therefore, the combination of KPP NPs and LIFU is feasible and proposes a promising clinical strategy for the treatment of HCC without systemic toxic side effects.

[0042] In this study, we constructed a nanoparticle HKN that can be controlled by low-intensity focused ultrasound (LIFU) and precisely targeted to tumor ferritin. 15@PLGA-PFH (KPP). Polylactic acid glycolic acid (PLGA) is used as a carrier, which has excellent nanoscale biological effects, high biocompatibility and easy surface modification. The ADV responsive material perfluorohexane (PFH) is encapsulated in PLGA as the core, and the ferritin homing peptide HKN 15 (HKNKGKKNGKHNGWK) grafted onto the PLGA shell, acting as a navigator to recognize and target ferritin. HKN 15 It has been demonstrated to have significant ferritin targeting ability, enabling lysosomal escape through caveolin-mediated endocytosis, allowing nanoparticles to reach the cytoplasm. 15 Due to the positive feedback effect of ferritin and the overexpression of ferritin, KPP nanoparticles accumulated around ferritin in large quantities. Subsequently, the ADV effect was precisely regulated by external LIFU, resulting in the degradation of tumor cells and ferritin. As the iron pool was destroyed, the accumulation of intracellular iron was enhanced, thereby inducing ferroptosis. Overall, this protocol proposed a new controllable method to target the disruption of ferritin homeostasis, providing a potential innovative approach for the clinical treatment of hepatocellular carcinoma (HCC).

[0043] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or the priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. A method for preparing nanoparticles for destroying ferritin in liver cancer cells, characterized in that: The following steps are involved: S1: PP NPs were constructed by encapsulating the liquid-gas phase change material PFH in PLGA via an improved double emulsion method; S2: Construction of P NPs by PLGA via a modified double emulsion method; S3: Ferritin homing peptide HKN was synthesized by carbodiimide coupling reaction 15 covalently bonded to the surface of PP NPs to obtain KPPNPs; S4: The obtained KPP NPs were washed with deionized water to remove the micro-reactive substances.

2. The method for preparing nanoparticles for destroying ferritin in liver cancer cells according to claim 1, characterized in that: The prepared nanoparticles also need to be labeled by adding DiI or DiR in steps S1 and S2.

3. The method for preparing nanoparticles for destroying ferritin in liver cancer cells according to claim 1, characterized in that: The mass of typical protons of the prepared KPP NPs was detected, where the chemical shift was evaluated by F-NMR, the changes in chemical bonds were evaluated by FTIR, and DLS was used to measure the average hydrodynamic diameter and zeta potential of the NPs. PP and KPPNPs were suspended in PBS to observe the changes in particle size. The morphology of KPP NPs was observed by SEM and TEM.

4. A method for preparing nanoparticles for destroying ferritin in liver cancer cells according to any one of claims 1 to 3, characterized in that: The prepared nanoparticles also need to be tested, and the test steps are as follows: A1: HCC cells for culture experiment: A2: Select mice aged 6 to 8 weeks as test subjects; A3: 5 × 10 suspended in 100 μL PBS were injected subcutaneously in the right lower limb. 6 HCC cells were cultured into C57BL / 6J mice with HCC; A4: The subcutaneous tumor size of mice injected with HCC cells was continuously observed, and after the injection threshold time, the mice were randomly divided into different groups.

5. The method for preparing nanoparticles for destroying ferritin in liver cancer cells according to claim 4, characterized in that: The method for culturing liver cancer cells in step A1 is as follows: HCC cells are cultured in a humidified 5% CO2 environment at 37°C with 10% fetal bovine serum, 100 mg mL −1 Streptomycin and penicillin in DMEM.

6. The method for preparing nanoparticles for destroying ferritin in liver cancer cells according to claim 4, characterized in that: It also includes the intracellular uptake of nanoparticles and ferritin targeting: first, the cellular internalization of NPs in liver cancer cells was detected; then the co-localization of the observation protein with PP and KPP NPs was detected; DiR-labeled PP and KPP NPs suspensions were injected into tumor-bearing mice, and the fluorescence signals were monitored and analyzed at 2, 6, 10, 24, and 48 h after injection using an in vivo imaging system.

7. The method for preparing nanoparticles for destroying ferritin in liver cancer cells according to claim 6, characterized in that: It also includes ferritin destruction and ferroptosis induction: HSF was used to simulate ferritin in vitro, and a colorimetric iron assay kit was used to evaluate the iron content excreted from HSF; WB was used to evaluate the ferritin expression levels in HCC cells treated with PBS, KPP, and KPP + LIFU; SEM was used to observe the microstructural morphology of cells after LIFU + KPP treatment at different power intensities.

8. An application of nanoparticles for destroying ferritin in liver cancer cells, characterized in that: The invention comprises nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

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