Multifunctional nanometer delivery system based on targeted ferritin and preparation method and application thereof

Abstract

The invention discloses a multifunctional nano delivery system based on targeted ferritin as well as a preparation method and application of the multifunctional nano delivery system. According to the system, traditional Chinese medicine active ingredients, namely neogambogic acid GNA, a photosensitizer Ce6 and ferritin targeting peptide HKN15 are integrated through a self-assembly technology, a synergistic treatment system integrating photodynamic therapy PDT and ferroptosis induction is constructed, and the treatment effect on non-small cell lung cancer NSCLC can be remarkably enhanced. According to the nano delivery system, precise targeting of a tumor site is achieved through the ferritin targeting peptide HKN15, and a double anti-tumor mechanism is formed by utilizing the photodynamic effect of the photosensitizer Ce6 and the ferroptosis induction effect of GNA. The preparation process is based on a molecular self-assembly principle, and has the advantages of high tumor accumulation efficiency, low system toxicity, remarkable tumor inhibition effect and the like, and an innovative solution is provided for precise treatment of the non-small cell lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial technology and relates to a multifunctional nano-delivery system based on targeted ferritin, a preparation method and application thereof, and in particular to a multifunctional nano-delivery system based on targeted ferritin, a preparation method thereof and application thereof in the preparation of drugs for treating non-small cell lung cancer. Background Art

[0002] Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Traditional treatments (such as chemotherapy, targeted therapy, and immunotherapy) face challenges such as drug resistance, toxic side effects, and tumor microenvironment heterogeneity. Ferroptosis, as a new type of programmed cell death, can specifically kill tumor cells through lipid peroxidation accumulation, but its inducers have problems such as poor water solubility, insufficient stability, and weak tumor targeting. In addition, although photodynamic therapy (PDT) has spatiotemporal controllability, its efficacy is limited by the hypoxic tumor microenvironment and interference from antioxidants (such as glutathione). In existing technologies, although traditional Chinese medicine monomers (such as neogamoic acid GNA) have the potential to induce ferroptosis, their low bioavailability and non-targeted distribution limit their clinical application.

[0003] CN110028553A discloses an antibacterial nanoprobe AuPEGAMPCe6. An antimicrobial peptide with the sequence GKRWWKWWRRC labeled with Ce6 is connected to Au via an AuS bond, and PEG is added as a stabilizer. The photosensitizer Ce6 is introduced for PDT antibacterial treatment, and it is used to generate reactive oxygen species (ROS) under laser irradiation, leading to oxidation of microbial molecules and cell damage and death, and producing a synergistic effect with the antimicrobial peptide (AMP). The antibacterial nanoprobe AuPEGAMPCe6 prepared by this invention has better antibacterial properties than traditional antimicrobial peptides, and has good biocompatibility with nanogold, has little interference with the normal physiological activities of body cells, has low toxicity, and is highly safe. However, the above-mentioned nanoprobe is mainly targeted at microorganisms, and high concentrations of Au are toxic to the body to a certain extent, making it unsuitable for anti-tumor treatment.

[0004] CN117503705A prepares a novel PEGOEL block copolymer for targeted drug delivery. First, a novel PEGOEL block copolymer is synthesized using lutein (L), orthoester (OE), and polyethylene glycol (PEG) as raw materials. PEGOEL and lecithin are then mixed in a specific mass ratio, and dual-responsive liposomes (Drlips) are prepared by ultrasonic thin-film hydration. Drlips has a high encapsulation efficiency for paclitaxel (PTX) and chlorin E6 (Ce6). Furthermore, under 660nm near-infrared (NIR) light, Ce6 accelerates the photodegradation rate of lutein and PEGOEL. Drlips loaded with Ce6 and PTX not only exhibit excellent pH and light dual responsiveness in targeted delivery and release, but also demonstrate significant reactive oxygen species (ROS) production and good antitumor activity in vitro. This patent primarily addresses the delivery of paclitaxel via liposomes, but the stability of liposome carriers is significantly affected by the microenvironment.

[0005] In summary, existing technologies suffer from low bioavailability of traditional Chinese medicine monomers, rapid systemic clearance, and a lack of targeting; single-modality ferroptosis therapy is susceptible to tumor microenvironmental constraints; photodynamic therapy relies on local oxygen concentration, exacerbating hypoxia during treatment, and reactive oxygen species are easily neutralized by reducing substances; and traditional nanocarriers suffer from low encapsulation efficiency, poor stability, and potential toxicity. Therefore, there is an urgent need to develop a multifunctional nanodelivery system that can overcome these shortcomings and achieve a comprehensive treatment approach with precise targeting, synergistic efficacy, and reduced toxicity. Summary of the Invention

[0006] Based on the problems existing in the prior art, the present invention provides a multifunctional nano-delivery system based on targeted ferritin, and a preparation method and application thereof.

[0007] Notably, ferroptosis and PDT are mechanistically complementary: ROS generated by PDT can serve as substrates for the Fenton reaction of ferroptosis, while the iron ion release and transporter regulation induced by PDT can provide raw materials for ferroptosis. Furthermore, ferroptosis-induced oxidative stress can enhance the immune synergistic effect of PDT. This bidirectional mechanistic coupling lays the theoretical foundation for the development of multimodal synergistic treatment strategies.

[0008] Although the existing technology focuses on the single therapy of PDT or ferroptosis, it ignores the intrinsic biochemical coupling between the two. This patent proposes for the first time that the ROS generated by PDT directly serves as the "substrate fuel" of the Fenton reaction of ferroptosis, and the photodynamic destruction of Fe released by ferritin 2+ By reversely feeding the ferroptosis chain reaction, the Chinese herbal monomer GNA inhibits the antioxidant pathway (GCH1) and forms a "double-pincer attack" of oxidative stress with PDT. This self-reinforcing closed loop of PDT producing oxygen and iron, which then amplifies oxidative damage through ferroptosis, which then weakens the antioxidant system and feeds back to PDT, goes beyond simple therapeutic stacking.

[0009] To achieve synergistic subcellular localization of a photosensitizer, a ferroptosis inducer, and a targeting element within a single nanostructure, we constructed a core-shell nanostructure via hydrophilic-hydrophobic self-assembly. Ce6 forms a hydrophobic core, PEG-HKN15 forms a hydrophilic shell, and GNA is loaded onto the core-shell structure's surface. The core uniqueness of this design lies in leveraging the unique characteristics of the tumor microenvironment, characterized by ferritin overexpression, to construct a novel "three-in-one" nanodelivery system, Ce6-PEG-HKN15-GNA, through molecular self-assembly. This system elevates the physical coexistence of PDT and ferroptosis to a biochemical cascade reaction, achieving a synergistic effect exceeding 1+1. This system integrates the photosensitizer Ce6 and GNA through a carrier-free nanostructured strategy. Ce6's photoactivation properties disrupt iron pool homeostasis, promoting iron overload and ·OH-mediated lipid peroxidation. Furthermore, its self-oxygenation function alleviates TME hypoxia, enhancing PDT efficacy. GNA further disrupts cellular redox homeostasis by inhibiting the GCH1 pathway. This multi-mechanism synergistic strategy not only circumvents the defects of traditional ferroptosis inducers, but also provides a new perspective for expanding the application of ferroptosis in tumor treatment.

[0010] The purpose of the present invention can be achieved by the following technical solutions: One of the technical solutions of the present invention provides a multifunctional nano-delivery system based on targeting ferritin, which is represented by Ce6-PEG-HKN15-GNA (CHG); the components of the nano-delivery system include: photosensitizer Ce6, ferritin targeting peptide HKN15, polyethylene glycol (PEG) and traditional Chinese medicine active ingredient GNA; the nano-delivery system self-assembles to form a core-shell structure through hydrophilic-hydrophobic interactions, wherein Ce6 is the hydrophobic core and the PEG-HKN15 complex is the hydrophilic shell. The PEG-HKN15 complex is formed by coupling polyethylene glycol PEG and ferritin targeting peptide HKN15 through EDC / NHS, and GNA is loaded on the surface of the core-shell structure.

[0011] The nanodelivery system has a regular spherical shape, an average particle size of 155.3±3.2 nm, a hydrated particle size of 156.4 nm, a polydispersity index (PDI) ≤ 0.2 (e.g., 0.172), a uniform particle size distribution, and a zeta potential of -10 to 0 mV (e.g., -5.49 mV); The nano-delivery system has characteristic UV-visible absorption peaks at 400 nm and 670 nm due to the loading of Ce6, and can efficiently generate singlet oxygen ( 1 O2); The nanodelivery system uses the ferritin-targeting peptide HKN15 (HKN15 peptide, amino acid sequence HKNKGKKNGKHNGWK, purity ≥98%) to specifically bind to ferritin overexpressed in tumor cells, achieving efficient accumulation at the tumor site and prolonged retention time. The system also exhibits excellent blood compatibility with a red blood cell hemolysis rate of less than 0.1% within a concentration range of 1-100 μg / mL. In vivo experiments have shown no pathological damage such as inflammatory infiltration or necrosis in major organs, demonstrating high safety. The nano-delivery system integrates photodynamic therapy and ferroptosis induction functions. Ce6 generates reactive oxygen species, triggers oxidative stress, and promotes Fe 2+ GNA inhibits the GCH1 pathway and weakens antioxidant defense, and the two synergistically enhance lipid peroxidation accumulation to achieve multi-mechanism anti-tumor effects.

[0012] The second technical solution of the present invention provides a method for preparing a multifunctional nano-delivery system based on targeting ferritin: S1: PEG and HKN15 were coupled via EDC / NHS to form a PEG-HKN15 complex; S2: Ce6 and PEG-HKN15 are self-assembled through hydrophobic interaction to form a Ce6-PEG-HKN15 complex (CH complex); S3: GNA was loaded onto the surface of the CH complex by solvent replacement method to obtain the multifunctional nanodelivery system Ce6-PEG-HKN15-GNA (CHG).

[0013] Furthermore, in S1, the dosage relationship of PEG and HKN15 is (80-120) mg: (10-30) mg, preferably 100 mg: 20 mg.

[0014] Furthermore, in S2, the dosage of the Ce6 and PEG-HKN15 complex is (5-20) mg: (10-30) mg, preferably 10 mg: 20 mg.

[0015] Furthermore, in S3, the dosage ratio of the GNA and CH complex is (1-3) mg: (5-20) mg, preferably 1 mg: 10 mg.

[0016] Furthermore, in S1, the specific operation of the EDC / NHS coupling is: S1.1. Activate PEG carboxyl groups: Dissolve PEG-2000 in deionized water, add a mixture of EDC and NHS, adjust the pH, and stir at room temperature. S1.2. Peptide coupling: Add ferritin-targeting peptide HKN15 and stir at room temperature under inert gas protection; S1.3, Purification: After stirring, the mixed reaction solution was placed in a dialysis bag and dialyzed against deionized water at room temperature. After dialysis, the mixture was freeze-dried to obtain a white flocculent PEG-HKN15 complex. In step S1.1, the molar ratio of EDC to NHS is (1-1.5):1, preferably 1.2:1; the molar amount of NHS to the molar amount of carboxyl groups in PEG-2000 is 1:1; calculation example: if PEG-2000 contains 0.05 mmol carboxyl groups, then EDC = 0.06 mmol, NHS = 0.05 mmol; the pH is adjusted to 5-6 (optimal activation pH, preferably 5.5), and the stirring is carried out at room temperature for 30-120 min, preferably 30 min.

[0017] The stirring time at room temperature in step S1.2 is 24-30 hours, preferably 24 hours.

[0018] The molecular weight cut-off of the dialysis bag in step S1.3 is 3500 kDa; the dialysis time is preferably 48 h, with water changed every 8 h.

[0019] Furthermore, the specific operation of the hydrophobic interaction self-assembly in step S2 is: S2.1. Dissolution of organic phase: dissolve Ce6 in anhydrous DMF in the dark and ultrasonicate. S2.2. Complex assembly: Add PEG-HKN15 solution dropwise and sonicate in the dark to form a homogeneous mixture; S2.3. Self-assembly reaction: introduce inert gas to remove oxygen and stir at room temperature in the dark; S2.4. Purification: The stirred reaction solution was placed in a dialysis bag, dialyzed against pure water, and freeze-dried to obtain a dark green powder of Ce6-PEG-HKN15 complex (CH complex).

[0020] Ultrasound in steps S2.1 and S2.2 at 300 W for 5 min; In step S2.3, the stirring speed is 800-1200 rpm, preferably 800 rpm, at room temperature in the dark, for 24-48 h, preferably 24 h; The molecular weight cut-off of the dialysis bag in step S2.4 is 12,000 kDa; the dialysis time is preferably 36 h, with water changed every 8 h; The inert gas in steps S1.2 and S2.3 is preferably nitrogen.

[0021] Furthermore, the specific operation of the solvent replacement method in S3 is: S3.1, organic phase dispersion: dissolve CH complex and GNA in anhydrous ethanol; S3.2, Solvent replacement: Add deionized water dropwise, stirring in the dark, until the solution changes from clear to turbid; S3.3. Remove solvent: Rotary evaporate to a volume < 1 mL. Add deionized water and continue rotary evaporation until the ethanol is completely removed. S3.4. Solidification: Freeze-dry the multifunctional nanodelivery system Ce6-PEG-HKN15-GNA (CHG) into yellow-green powder.

[0022] In step S3.2, the volume ratio of deionized water to anhydrous ethanol in step S3.1 is (0.8-1.2):1, preferably 1:1; the deionized water is dripped at a rate of 1 mL / min; the stirring speed is 500-1000 rpm, preferably 500 rpm; the solution changes from clear to turbid, indicating that GNA has precipitated and embedded in the nanoparticle interface; The temperature of the rotary evaporation in step S3.3 is 40-45° C., preferably 45° C.; the vacuum degree of the rotary evaporation is preferably 0.09 MPa; the volume of the added deionized water is 10-15 mL, preferably 10 mL.

[0023] The mechanism of action of the multifunctional nano-delivery system Ce6-PEG-HKN15-GNA (prodrug) in vivo is as follows: the nano-delivery system specifically binds to the ferritin-targeting peptide HKN15 overexpressed in tumor cells to achieve precise delivery; the photosensitizer Ce6 generates singlet oxygen ( 1 O2), triggering oxidative stress and destroying the ferritin shell to release Fe 2+ , catalyzing lipid peroxidation through the Fenton reaction; simultaneously, the traditional Chinese medicine ingredient gambogic acid (GNA) inhibits the GCH1 pathway, weakening the cellular antioxidant defense system and synergistically enhancing lipid peroxidation accumulation, ultimately triggering ferroptosis. These three factors work synergistically in time and space, overcoming the limitations of the tumor microenvironment and achieving a multi-mechanism anti-tumor effect involving targeted accumulation, photodynamic oxidative damage, and ferroptosis metabolic remodeling, offering broad application prospects in the diagnosis and treatment of tumors.

[0024] A third technical solution of the present invention provides a multifunctional nano-delivery system based on targeted ferritin for use in the preparation of a drug for treating non-small cell lung cancer. The drug for treating non-small cell lung cancer is combined with near-infrared light to activate PDT, synergistically inducing ferroptosis.

[0025] The administration method of the drug for treating non-small cell lung cancer is intravenous injection.

[0026] Furthermore, experimental verification shows that the combination of the drug for treating non-small cell lung cancer and near-infrared light has the following technical effects: 1) Targeting: The ferritin-targeting peptide HKN15 specifically binds to tumor cell ferritin, increasing tumor accumulation rate; 2) Synergistic treatment: ROS generated by PDT and GNA-induced antioxidant defense collapse synergistically trigger ferroptosis; 3) Safety: Hemolysis rate <0.1%, no significant toxicity to major organs; 4) Tumor inhibition effect: In vivo experiments showed that the tumor volume inhibition rate reached 78.5% (the control group was 0%).

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has precise targeting: the ferritin targeting peptide HKN15 specifically binds to ferritin overexpressed in tumor cells, significantly increasing the accumulation rate of the drug in the tumor site and reducing toxic side effects on normal tissues; (2) The present invention can achieve synergistic treatment through multiple mechanisms: the reactive oxygen species (ROS) generated by photodynamic therapy (PDT) and the collapse of the antioxidant defense system induced by gambogic acid (GNA) form a spatiotemporal synergistic effect, breaking through the limitations of the tumor microenvironment on single-modality treatment, enhancing lipid peroxidation accumulation and triggering ferroptosis; (3) The present invention has high safety and stability: the hemolysis rate of the carrier-free self-assembled nanosystem is less than 0.1%, and in vivo experiments show no pathological damage to major organs. At the same time, the nanomaterial remains stable in the physiological environment, solving the problems of high toxicity and low encapsulation efficiency of traditional nanocarriers; (4) The preparation process of the present invention is simpler: the core-shell structure is formed by self-assembly through hydrophilic and hydrophobic interactions, without the need for complex carrier materials. The process is simple and the encapsulation efficiency reaches 88.6%, reducing production costs.

[0028] In summary, the present invention provides a highly effective and safe comprehensive treatment for non-small cell lung cancer through targeted delivery, synergistic therapy, and a low-toxic design. Compared to existing technologies, this patent utilizes subassembly nanoparticles that can carry the traditional Chinese medicine monomer gambogic acid, the photosensitizer Ce6, and the ferritin-targeting peptide HKN15. These nanoparticles exhibit excellent in vivo stability and targeting, while also generating ROS through photodynamic therapy and binding to ferritin for synergistic tumor killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Characterization of Ce6-PEG-HKN15-GNA (CHG) nanomaterials prepared in Example 1; the symbols in the figure represent: Figure 1 A: DLS, Figure 1 B:TEM, Figure 1 C: Zeta potential, Figure 1 D: UV-visible spectrum; Figure 2Singlet oxygen production from CHG nanomaterials prepared in Example 1 1 Determination of O2 capacity; the numbers in the figure indicate: Figure 2 A: SOSG fluorescence intensity-time curve of CHG nanomaterials under 633 nm laser irradiation; Figure 2 B: Control curve of SOSG fluorescence intensity of CHG nanomaterials under no illumination conditions; Figure 2 C: SOSG fluorescence intensity curve of CHG nanomaterials under laser irradiation after adding L-ascorbic acid; Figure 3 This is the biosafety assessment of the CHG nanomaterial prepared in Example 1; the numbers in the figure indicate: Figure 3 AB: hemolysis test results; Figure 3 C: HE staining image; Figure 4 The in vivo biodistribution and targeting performance of the CHG nanomaterial prepared in Example 1; the numbers in the figure indicate: Figure 4 AB: Whole-body fluorescence imaging of tumor-bearing mice; Figure 4 CD: quantitative analysis of fluorescence intensity in the tumor area; Figure 5 The in vivo tumor inhibition effect of the CHG nanomaterial prepared in Example 1; the numbers in the figure indicate: Figure 5 AC: Actual tumor image (A), tumor volume growth curve (B) and detached tumor weight statistics (C). DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, all raw materials in the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0032] Example 1: Preparation of a multifunctional nanodelivery system based on targeting ferritin (1) Materials and reagents: Polyethylene glycol 2000 (PEG-2000, Aladdin), HKN15 peptide (Shanghai Jiepeptide Biotechnology Co., Ltd.), dihydrochlorin e6 (Ce6, Aladdin), neogambogic acid (GNA, MCE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Aladdin), N-hydroxysuccinimide (NHS, Aladdin), N,N-dimethylformamide (DMF, Aladdin, anhydrous grade), and dialysis bag (Viskase).

[0033] The amino acid sequence of the ferritin targeting peptide HKN15 is HKNKGKKNGKHNGWK, and the purity is ≥98%.

[0034] (2) Preparation steps: ① Activation of PEG-2000 carboxyl groups: Weigh 100 mg of PEG-2000 and dissolve it in 20 ml of deionized water. Add EDC and NHS at a molar ratio of 1.2:1 (based on the carboxyl content of PEG-2000) (if PEG-2000 contains 0.05 mmol of carboxyl groups, add 0.06 mmol of EDC and 0.05 mmol of NHS) to activate the carboxyl groups. Adjust the pH of the reaction system to 5.5 with 0.1 M hydrochloric acid or sodium hydroxide. Stir continuously at 300 rpm at room temperature for 30 minutes. ② Preparation of PEG-HKN15 conjugate: Add 20 mg of HKN15 peptide to the above reaction system and stir at 300 rpm under nitrogen for 24 hours at room temperature. After the reaction is complete, place the mixture in a dialysis bag and dialyze in deionized water for 48 hours to remove unreacted products (deionized water was replaced every 8 hours). Then, freeze-dry (-50°C, 0.01 MPa, 24 hours) to obtain the PEG-HKN15 conjugate. ③ Preparation of CH complex: 10 mg of Ce6 was dissolved in 30 ml of anhydrous DMF and sonicated at 300 W power for 5 minutes in the dark to completely dissolve it. 25 mL of the above-mentioned non-freeze-dried PEG-HKN15 solution (corresponding to 20 mg of PEG-HKN15) was taken and added dropwise to the Ce6 DMF solution. The mixture was sonicated at 300 W power for 5 minutes in the dark to form a uniform mixture. Under nitrogen protection, the mixture was magnetically stirred at 800 rpm for 24 hours at room temperature in the dark for self-assembly reaction. After the reaction was completed, the mixture was placed in a dialysis bag and dialyzed with pure water for 36 hours (the water was changed every 12 hours) to remove the organic solvent. The liquid in the dialysis bag was collected and freeze-dried (-50°C, 0.01 MPa, 24 hours) to obtain a dark green powdery CH complex. ④ Preparation of CHG nanoparticles: 10 mg of CH complex and 1 mg of GNA were dispersed in 5 mL of anhydrous ethanol at a mass ratio of 10:1. After complete dissolution, the mixture was magnetically stirred at 500 rpm in a dark environment. At the same time, 5 mL of deionized water was added dropwise at a rate of 1 mL / min. The solution was observed to change from clear to turbid. The mixture was placed at 40-45°C and a vacuum degree of 0.09 MPa and rotary evaporated to a volume of less than 1 mL. 10 mL of deionized water was added and rotary evaporated again until the ethanol was completely removed. The resulting solution was freeze-dried (-50°C, 0.01 MPa, 24 h) to finally obtain yellow-green powdered CHG nanoparticles.

[0035] Example 2: Nanomaterial Characterization (1) Morphology analysis: An appropriate amount of CHG nanoparticles prepared in Example 1 was dispersed in deionized water to a concentration of 0.5 mg / mL and ultrasonically dispersed for 10 minutes to make them uniformly dispersed. 10 μL of the dispersion was added dropwise to the surface of the copper mesh. After natural drying, it was negatively stained with 2% phosphotungstic acid solution for 30 seconds. The excess dye was absorbed by filter paper and dried at room temperature. The surface was observed by transmission electron microscopy (TEM, model such as JEM-2100) at an accelerating voltage of 120 kV. Figure 1 As shown in Figure B, CHG nanoparticles have a regular spherical structure without obvious aggregation, and the average particle size is 155.3±3.2 nm.

[0036] (2) Particle size distribution: Take the above 0.5 mg / mL CHG nanoparticle dispersion and measure it using a dynamic light scattering instrument (such as Zetasizer Nano ZS) with the temperature set at 25°C, the equilibrium time at 2 minutes, and the detection angle at 90°. Figure 1 A results showed that the hydrated particle size was 156.4 nm and the polydispersity index (PDI) was 0.172, indicating that the particle size distribution was uniform; Figure 1 As shown in C, the zeta potential is -5.49 mV; (3) UV-visible spectral analysis: Take CHG nanoparticle dispersion, CH, Ce6 and GNA respectively, and scan them in the wavelength range of 200-800 nm using a UV-visible spectrophotometer (such as UV-2600).

[0037] like Figure 1 As shown in Figure D, the results show that CHG nanoparticles have characteristic absorption peaks at 400 nm and 670 nm, which are consistent with the characteristic peak positions of free Ce6, confirming that Ce6 is effectively loaded in the nanoparticles.

[0038] In summary, from Figure 1It can be seen that the prepared prodrug is spherical with a particle size of about 156 nm and contains Ce6 and GNA components, indicating that the CHG prodrug was successfully prepared.

[0039] Example 3: Singlet oxygen 1 Determination of O2 capacity Materials: Singlet oxygen sensing green (SOSG), PBS buffer (pH 7.4), CHG nanoparticles prepared in Example 2, L-ascorbic acid, and a 633 nm laser (power density 100 mW / cm²).

[0040] Instrument: Fluorescence spectrometer (monitoring fluorescence intensity at 525 nm).

[0041] (1) Prepare a PBS solution (pH 7.4) containing 5 μM singlet oxygen sensor green (SOSG) as a singlet oxygen detection system; (2) Experimental groups: Control group: Take an appropriate amount of CHG nanoparticles and add SOSG-PBS solution to make the equivalent concentration of Ce6 in the system reach 10 μg / mL, without laser irradiation; Illumination group: The same amount of CHG nanoparticles as the control group was taken, SOSG-PBS solution (Ce6 equivalent concentration 10 μg / mL) was added, and the irradiation was carried out under 633 nm laser (power density 100 mW / cm 2 ) continuous irradiation to simulate photodynamic triggering conditions.

[0042] Inhibition group: In addition to the light-exposed group, 1 mM L-ascorbic acid (singlet oxygen scavenger) was added, and the other conditions were the same as those of the light-exposed group.

[0043] (3) Use a fluorescence spectrometer to continuously monitor the changes in the fluorescence intensity of SOSG at 525 nm and record the fluorescence signals at different time points (0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min) within 0-7 minutes of laser irradiation.

[0044] Figure 2 A shows that under continuous 633 nm laser irradiation, the fluorescence intensity of SOSG in the CHG group increased significantly over time, indicating that it efficiently generated the fluorescence required for photodynamic therapy. 1 O2. Figure 2 B shows that the fluorescence intensity of SOSG in the CHG group without illumination did not change significantly, verifying that light excitation was the trigger 1 Necessary conditions for the generation of O2. Figure 2 C shows that after adding 1 mmol / L L-ascorbic acid, the SOSG fluorescence enhancement effect of the CHG+Laser group was inhibited, confirming the reverse. 1The specificity of O2 generation eliminated interference from other oxidative products. After 7 minutes of laser irradiation, the fluorescence intensity of the illuminated group increased 8.3-fold, significantly higher than that of the control and inhibition groups, demonstrating that CHG has photodynamic activity.

[0045] Example 4: In vivo biodistribution (1) Animal model: 6-week-old BALB / c nude mice were subcutaneously inoculated with HCC1833 tumor cells and the tumor volume reached 150±20 mm. 3 Experiments were conducted; (2) Group administration: Tumor-bearing nude mice were randomly divided into two groups and injected with CHG preparation or free Ce6 solution at an equivalent Ce6 dose of 2 mg / kg via tail vein; (3) In vivo imaging monitoring: Using a small animal imaging system (excitation 640 nm / emission 680 nm), in vivo fluorescence images of mice were collected at different time points (such as 0 h, 2 h, 4 h, 6 h, 8 h, etc.), and the changes in fluorescence intensity at the tumor site were analyzed to determine the time when the fluorescence intensity at the tumor site in the CHG group reached its peak (the peak was reached at 8 h, which was significantly higher than that in the free Ce6 group).

[0046] (4) Tissue distribution detection: Mice were killed 36 hours after administration, and organs (heart, liver, spleen, lung, kidney and tumor tissue) were taken for analysis. The accumulation of Ce6 in each tissue was detected. The analysis found that the accumulation of Ce6 in the liver and kidney tissues of the CHG group basically disappeared after 12 hours, but it still existed in the tumor tissue after 36 hours, confirming its tumor retention. Blood compatibility test ( Figure 3 Experimental Procedures: CHG solutions were prepared at concentrations of 1, 10, 50, and 100 μg / mL, with PBS and deionized water as controls. Fresh mouse red blood cells were incubated with the solutions, and the hemolysis rate was measured. Results: Within the CHG concentration range of 1-100 μg / mL, the hemolysis rate remained below 0.1%, well below the biosafety threshold of 5%, demonstrating good hemocompatibility and meeting the requirements for intravenous administration.

[0047] Histopathological examination ( Figure 3 C) Experimental procedures: Multiple control groups (Con group, Con + Laser group, Ce6 group, Ce6 + Laser group, CHG group, CHG + Laser group, and GNA group) were set up. After corresponding treatments, heart, liver, spleen, lung, and kidney tissues of mice were obtained, and pathological sections were prepared and stained with HE to observe tissue morphology and structure. Figure 3 C shows that the heart, liver, spleen, lung, and kidney tissue structures of mice in the CHG treatment groups (CHG group and CHG+Laser group) were intact, without abnormal pathological changes such as inflammatory infiltration or necrosis, confirming its safety in vivo and no obvious toxic side effects on major organs after intravenous injection.

[0048] Example 5: In vivo tumor inhibition experiment Tumor-bearing mouse model: BALB / c nude mice were subcutaneously inoculated with HCC1833 tumor cells to establish a tumor-bearing mouse model. The mice were raised until the tumor volume stabilized (approximately 150±20mm 3 ), and conduct subsequent treatment experiments.

[0049] Grouping and administration: The tumor-bearing mice were randomly divided into control group (NC group, NC+Laser group), free Ce6 group (Ce6 group, Ce6+Laser group), CHG group (CHG group, CHG+Laser group), and GNA group.

[0050] Treatment plan: For the group requiring laser treatment, CHG (0.3 mg / kg) or free Ce6 solution or GNA solution was injected into the tail vein once every 48 hours, combined with 633 nm laser irradiation of the tumor site; the CHG group was given the drug once every 48 hours, and the intervention continued for 14 days.

[0051] In vivo and tissue fluorescence imaging Figure 4 As shown, in vivo imaging ( Figure 4 A, B) Using a small animal imaging system (excitation 640 nm, emission 680 nm), the tumor fluorescence signal of the CHG group reached its peak 3 hours after administration, and the signal was significantly higher than that of the free Ce6 group within 24 hours, indicating that CHG can be rapidly enriched in the tumor and maintain effective retention. Figure 4 C, D) Tumor tissue fluorescence was measured at different time points. The fluorescence signal in the CHG group remained high after 24 hours, while the fluorescence in the free Ce6 group rapidly decayed. Combined with the enhanced permeability and retention (EPR) effect and the active targeting of the targeting peptide HKN15 to tumor cells, CHG demonstrated that it could prolong tumor accumulation to 24 hours, significantly surpassing the rapid metabolic clearance of free Ce6, laying the foundation for sustained photodynamic therapy.

[0052] Evaluation of tumor inhibition effect Figure 5 As shown, the tumor appearance and volume ( Figure 5 A, B) show that after 14 days of treatment, the tumor volume in the CHG+Laser group was significantly smaller than that in the other groups, with a volume inhibition rate of 78.5%. The tumor volume in the control group (NC group, NC+Laser group) and the single-drug group (Ce6 group, GNA group) increased significantly. Although the Ce6+Laser group had some inhibition, the effect was weaker than that in the CHG+Laser group. Figure 5C): Tumor weight in the CHG+Laser group was significantly lower than in the other groups (P<0.01), further confirming its potent tumor-suppressing effect. The CHG and GNA groups showed no significant tumor-suppressing effect, indicating that photodynamic therapy is key to CHG's efficacy. After treatment, heart, liver, spleen, lung, and kidney tissues were harvested for pathological examination. The CHG+Laser group showed intact organ tissue structure, with no inflammatory infiltration, necrosis, or other pathological lesions. This confirms the safety of the treatment regimen and the lack of significant toxic side effects on the major organs of tumor-bearing mice.

[0053] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A nano-delivery system targeting ferritin, characterized in that: The nanodelivery system is denoted as Ce6-PEG-HKN15-GNA; The components of the nano-delivery system include: photosensitizer Ce6, ferritin targeting peptide HKN15, polyethylene glycol PEG, and traditional Chinese medicine active ingredient gambogic acid GNA; the amino acid sequence of the ferritin targeting peptide HKN15 is HKNKGKKNGKHNGWK; The nanodelivery system forms a core-shell structure through self-assembly, wherein the photosensitizer Ce6 is the hydrophobic core and the PEG-HKN15 complex is the hydrophilic shell. The PEG-HKN15 complex is formed by coupling polyethylene glycol PEG and the ferritin targeting peptide HKN15 through EDC / NHS, and GNA is loaded on the surface of the core-shell structure.

2. The method for preparing a nano-delivery system targeting ferritin according to claim 1, comprising the following steps: S1: Polyethylene glycol (PEG) was coupled to the ferritin targeting peptide HKN15 via EDC / NHS to form a PEG-HKN15 complex; S2: The photosensitizer Ce6 and the PEG-HKN15 complex are self-assembled through hydrophobic interaction to form a Ce6-PEG-HKN15 complex, namely, a CH complex; S3: The new gambogic acid GNA was loaded onto the surface of the CH complex by solvent replacement method to obtain the multifunctional nanodelivery system Ce6-PEG-HKN15-GNA.

3. The method for preparing a nano-delivery system targeting ferritin according to claim 2, characterized in that: In step S1, the dosage of polyethylene glycol PEG and ferritin targeting peptide HKN15 is (80-120) mg: (10-30) mg; In step S2, the dosage of the photosensitizer Ce6 and the PEG-HKN15 complex is (5-20) mg: (10-30) mg; In step S3, the dosage of the neogambogic acid GNA and the CH complex is (1-3) mg: (5-20) mg.

4. The method for preparing a nano-delivery system targeting ferritin according to claim 3, characterized in that: In step S1, the specific operation of the EDC / NHS coupling is: S1.

1. Activate the carboxyl groups of polyethylene glycol (PEG): Dissolve PEG in deionized water, add a mixture of EDC and NHS, adjust the pH, and stir at room temperature. S1.

2. Peptide coupling: Add ferritin-targeting peptide HKN15 and stir at room temperature under inert gas protection; S1.

3. Purification: After stirring, the mixed reaction solution was placed in a dialysis bag and dialyzed against deionized water at room temperature. After dialysis, the mixture was freeze-dried to obtain a white flocculent PEG-HKN15 complex.

5. The method for preparing a nano-delivery system targeting ferritin according to claim 4, characterized in that: In step S1.1, the molar ratio of EDC to NHS is (1-1.5):1; the molar amount of NHS to the molar amount of carboxyl groups in PEG is 1:1; the pH is adjusted to 5-6, and the stirring is carried out at room temperature for 30-120 minutes; The stirring time at room temperature in step S1.2 is 24-30 hours; the dialysis time is 48 hours, and the water is changed every 8 hours.

6. The method for preparing a nano-delivery system targeting ferritin according to claim 5, characterized in that: The specific operation of the hydrophobic interaction self-assembly in step S2 is: S2.

1. Dissolution of organic phase: Dissolve photosensitizer Ce6 in anhydrous DMF in the presence of light and ultrasound; S2.

2. Complex assembly: add PEG-HKN15 complex solution dropwise and sonicate in the dark to form a homogeneous mixture; S2.

3. Self-assembly reaction: introduce inert gas to remove oxygen and stir at room temperature in the dark; S2.

4. Purification: The stirred solution was placed in a dialysis bag, dialyzed with pure water, and freeze-dried to obtain a dark green powder of Ce6-PEG-HKN15 complex, namely, CH complex.

7. The method for preparing a nano-delivery system targeting ferritin according to claim 6, characterized in that: In step S2.3, the stirring speed is 800-1200 rpm at room temperature in the dark for 24-48 h; The dialysis duration in step S2.4 was 36 h, with water changes every 8 h.

8. The method for preparing a nano-delivery system targeting ferritin according to claim 7, characterized in that: The specific operation of the solvent replacement method described in S3 is: S3.1, organic phase dispersion: dissolve the CH complex and neogamonic acid GNA in anhydrous ethanol; S3.2, Solvent replacement: Add deionized water dropwise, stirring in the dark, until the solution changes from clear to turbid; S3.

3. Remove solvent: Rotary evaporate to a volume < 1 mL. Add deionized water and continue rotary evaporation until the ethanol is completely removed. S3.

4. Solidification: Freeze-dry the multifunctional nanodelivery system Ce6-PEG-HKN15-GNA into yellow-green powder.

9. The method for preparing a nano-delivery system targeting ferritin according to claim 8, characterized in that: In step S3.2, the volume ratio of deionized water to anhydrous ethanol in step S3.1 is (0.8-1.2):1; the stirring speed is 500-1000 rpm; The temperature of the rotary evaporation in step S3.3 is 40-45° C.; the volume of the added deionized water is 10-15 mL.

10. Use of the ferritin-targeted nano-delivery system according to claim 1 in the preparation of a drug for treating non-small cell lung cancer.

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