Ferrocene cross-linked membrane cracked polyamino acid nanoparticles for cancer treatment as well as preparation method and application of ferrocene cross-linked membrane cracked polyamino acid nanoparticles

Abstract

The invention provides a ferrocene cross-linked membrane cracked polyamino acid composite material. The ferrocene cross-linked membrane cracked polyamino acid composite material is prepared from ferrocene dicarboxaldehyde and membrane cracked polyamino acid, the membrane cracking polyamino acid comprises triblock polyamino acid of polyethylene glycol monomethyl ether, polylysine and polyphenylalanine. According to the invention, ferrocene cross-linked membrane cleavage polyamino acid nanoparticles with an apoptosis and ferroptosis synergistic killing mechanism are designed, mitochondria can be efficiently targeted and damaged, oxidative stress is amplified through membrane cleavage polyamino acid-ferrocene cascade to activate apoptosis, active oxygen depletion glutathione in tumor cells is improved to activate ferroptosis, and the effect of killing apoptosis and ferroptosis is enhanced. Meanwhile, the ferrocene cross-linked structure can effectively shield surface cations of the membrane cleavage polyamino acid, so that the biological safety of the traditional membrane cleavage polyamino acid is greatly improved, and the problems that a membrane cleavage polyamino acid material is poor in tumor killing effect and high in toxicity are solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of film-splitting polyamino acid material preparation, and relates to a ferrocene cross-linked film-splitting polyamino acid composite material and a preparation method and application thereof, in particular to a ferrocene cross-linked film-splitting polyamino acid nanoparticle for cancer treatment and a preparation method and application thereof. BACKGROUND

[0002] The tumor treatment field has long relied on traditional anti-tumor materials (such as chemotherapy drugs, targeted antibodies, radioactive particles, etc.), but such materials generally have poor selectivity, significant toxic side effects, and are prone to cause drug resistance. In recent years, film-splitting polyamino acids have been widely concerned as a new treatment strategy due to their unique physical and chemical action mechanisms. Unlike traditional drugs, film-splitting polyamino acids achieve killing by directly destroying tumor cell membrane structures, for example, by combining cationic polymers with negatively charged tumor cell membranes to form pores through electrostatic interaction, resulting in increased cell membrane permeability, content leakage, and cell lysis death. Such mechanisms do not rely on tumor-specific receptors or metabolic pathways and can avoid drug resistance caused by gene mutations or abnormal signal pathways, and are particularly suitable for solid tumors with high heterogeneity.

[0003] However, existing film-splitting polyamino acids still face two major bottlenecks: first, the single mechanism of action, existing technologies mostly rely on a single membrane disruption mechanism, while tumor cells can quickly restore homeostasis through membrane repair mechanisms (such as lipid metabolism reprogramming and membrane protein recombination), resulting in reduced killing efficiency. In addition, a single mechanism cannot cope with the heterogeneity of different cell subpopulations in the tumor microenvironment, for example, the killing effect on dormant tumor cells with dense membrane structures is limited; second, the lack of biological safety, traditional film-splitting polyamino acids (such as polyethylene imine and polylysine derivatives) are not degradable or have toxic degradation products, which need to be limited to systemic exposure by local injection (such as intratumoral administration), greatly limiting the clinical application scenarios. For example, poly-cationic materials are prone to binding with blood proteins, causing coagulation risks, and residual polymer fragments may accumulate in the liver and kidneys, causing long-term toxicity.

[0004] Therefore, how to find a more suitable film-splitting polyamino acid material to solve the above technical problems of film-splitting polyamino acid materials has become one of the focuses of many forward-looking first-line researchers in the industry. SUMMARY

[0005] Therefore, the present application aims to provide a ferrocene cross-linked membrane lysing polyamino acid composite material, a preparation method and application thereof, in particular, a ferrocene cross-linked membrane lysing polyamino acid nanoparticle for cancer treatment.

[0006] The present application provides a ferrocene cross-linked membrane lysing polyamino acid composite material, which is obtained by preparing ferrocene dimethylaldehyde and membrane lysing polyamino acid.

[0007] The membrane lysing polyamino acid comprises a triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine.

[0008] Preferably, the composite material has a cross-linked network structure formed by the membrane lysing polyamino acid and ferrocene.

[0009] The formation is specifically cross-linking by forming an imine bond between the aldehyde group in the ferrocene dimethylaldehyde and the amino group at the end of the polylysine segment.

[0010] The cross-linked structure is specifically a pH-sensitive dynamic cross-linked network structure.

[0011] The pH sensitivity is specifically that the imine bond is broken, releasing ferrocene molecules and exposing free amino groups of polylysine, when the pH value is 4.5-5.5.

[0012] Preferably, in the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the polyethylene glycol monomethyl ether has a polymerization degree of 100-120.

[0013] In the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the polylysine has a polymerization degree of 65-75.

[0014] In the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the polyphenylalanine has a polymerization degree of 8-12.

[0015] The molar ratio of ferrocene to membrane lysing polyamino acid is (4.5-5.5):1.

[0016] Preferably, the ferrocene cross-linked membrane lysing polyamino acid composite material comprises ferrocene cross-linked membrane lysing polyamino acid nanoparticles.

[0017] The ferrocene cross-linked membrane lysing polyamino acid nanoparticle has a particle size of 80-150 nm;

[0018] The ferrocene cross-linked membrane lysing polyamino acid nanoparticle has a micellar structure.

[0019] The ferrocene cross-linked membrane lysing polyamino acid nanoparticle comprises an outer layer formed by polyethylene glycol monomethyl ether segments, an intermediate layer formed by a ferrocene cross-linked polylysine network, and an inner core formed by polyphenylalanine segments.

[0020] The present application provides a preparation method of a ferrocene cross-linked membrane lysing polyamino acid composite material, comprising the following steps:

[0021] 1) mixing a triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine and an organic solvent, and then mixing with water to obtain a membrane lysing polyamino acid nanoparticle solution;

[0022] 2) mixing the membrane lysing polyamino acid nanoparticle solution obtained in the above step and a 1,1'-ferrocene dimethyl formaldehyde organic solution to react, to obtain a ferrocene cross-linked membrane lysing polyamino acid nanoparticle.

[0023] Preferably, the number average molecular weight of the triblock polyamino acid is 14000-17000.

[0024] In step 1), the mixing mode comprises adding the solution of the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine and the organic solvent to water for stirring and mixing.

[0025] The stirring and mixing time is 25-35 min.

[0026] Preferably, the organic solvent comprises dimethyl sulfoxide.

[0027] The mass concentration of the membrane lysing polyamino acid nanoparticle solution is 0.5-1.0 mg / mL.

[0028] The mass ratio of the membrane lysing polyamino acid nanoparticle to 1,1'-ferrocene dimethyl formaldehyde is 100:(25-35).

[0029] Preferably, the solvent in the 1,1'-ferrocene dimethyl formaldehyde organic solution comprises dimethyl sulfoxide.

[0030] In step 2), the mixing mode comprises adding the 1,1'-ferrocene dimethyl formaldehyde organic solution to the membrane lysing polyamino acid nanoparticle solution for stirring and mixing.

[0031] The reaction time is 3.5-4.5 hours.

[0032] Preferably, the reaction is followed by a step of purifying the reaction solution by dialysis.

[0033] The dialysis bag used for the dialysis has a molecular weight cut-off of 8000 Da.

[0034] The purification time is 3 days.

[0035] The application also provides the use of the ferrocene crosslinked membrane lysing polyamino acid composite material in the preparation of an antitumor drug.

[0036] The application provides a ferrocene crosslinked membrane lysing polyamino acid composite material, which is obtained after preparation of ferrocene dimethyl formaldehyde and membrane lysing polyamino acid; the membrane lysing polyamino acid comprises a triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine. Compared with the prior art, the ferrocene crosslinked membrane lysing polyamino acid nanoparticle with an apoptosis and ferroptosis synergistic killing mechanism is specially designed for cancer treatment. The nanoparticle can efficiently target and damage mitochondria compared with traditional membrane lysing polyamino acid antitumor materials, activate apoptosis through a membrane lysing polyamino acid-ferrocene cascade amplification oxidative stress, improve glutathione depletion in active oxygen in tumor cells, and activate ferroptosis, so that the purpose of efficiently killing tumor cells is achieved. Meanwhile, the ferrocene crosslinked structure can effectively shield the surface cations of the membrane lysing polyamino acid, greatly improving the biological safety of the traditional membrane lysing polyamino acid, and solving the problems of poor tumor killing effect and high toxicity of the membrane lysing polyamino acid material.

[0037] The application provides a unique structural design of the ferrocene crosslinked membrane lysing polyamino acid nanoparticle, which is constructed through the following steps: molecular structure customization: a triblock polyamino acid main chain is sequentially connected by a polyethylene glycol monomethyl ether (mPEG) segment (hydrophilic shell), a polylysine (PLL) segment (cationic membrane damage unit), and a polyphenylalanine (PPhe) segment (hydrophobic core) to form an amphiphilic self-assembled nanoparticle; ferrocene dynamic crosslinking: a Schiff base reaction is used to form an imine bond (-C=N-) between a dialdehyde ferrocene (Fc(CHO)2) and an amino group at the end of the polylysine segment, so as to construct a pH-sensitive dynamic crosslinking network and endow the nanoparticle with tumor acid microenvironment responsiveness; tumor microenvironment triggered dissociation: lysosome acid response: after the nanoparticle is endocytosed by a tumor cell, it enters a lysosome (pH 4.5-5.5), the imine bond is broken under acidic conditions, ferrocene molecules are released, and free amino groups of the polylysine are exposed; proton sponge effect escape: the free amino groups are protonated under low pH, the osmotic pressure of the lysosome is increased, the lysosome membrane is ruptured, and the nanoparticle is released into the cytoplasm.

[0038] The ferrocene cross-linked film polyamino acid composite provided by the application can realize the synergistic activation of double lethal pathways. After the nanoparticles escape into the cytoplasm, the synergistic killing of apoptosis-iron death is realized through the following mechanisms: (1) mitochondrial membrane targeted damage: targeted positioning: the polyphenylalanine hydrophobic segment is combined with the mitochondrial membrane phospholipid (such as cardiolipin) through hydrophobic-hydrophobic interaction, realizing mitochondrial specific targeting; membrane damage effect: the cationic groups of polylysine insert into the mitochondrial membrane, destroy the membrane potential and form pores, leading to mitochondrial swelling, disintegration of the ridge structure and leakage of contents (such as cytochrome c and hydrogen peroxide). (2) Activation of the apoptosis pathway: cytochrome c release: the outflowing cytochrome c is combined with the Apaf-1 protein in the cytoplasm, activates the caspase-9 / caspase-3 cascade reaction, and starts the classical apoptosis program; ROS signal amplification: mitochondrial damage further produces reactive oxygen species (ROS), activates the p53 pathway, inhibits the expression of anti-apoptotic protein Bcl-2, and enhances the sensitivity to apoptosis. (3) Activation of the ferroptosis pathway: Fenton reaction catalysis: the released ferrocene (Fe 2+ ) occurs Fenton reaction (Fe 2+ + H2O2→ Fe 3+ + ·OH + OH-) with hydrogen peroxide (H2O2), generating a large amount of hydroxyl radicals (·OH); lipid peroxidation storm: ·OH attacks polyunsaturated fatty acids (PUFA) in the cell membrane and mitochondrial membrane, triggering the lipid peroxidation chain reaction; antioxidant system collapse: hydroxyl radicals synchronously consume glutathione (GSH), inhibit glutathione peroxidase 4 (GPX4) activity, and block the lipid peroxide repair pathway. The application can also realize the self-amplification effect of oxidative stress cascade. The core innovation of the application is to form a positive feedback loop through the interaction of double lethal pathways: apoptosis promotes ferroptosis: the mitochondrial membrane permeability increases during the apoptosis process, releasing more H2O2 and iron ions to provide substrates for the Fenton reaction; ferroptosis strengthens apoptosis: lipid peroxide accumulation leads to further loss of mitochondrial membrane integrity, exacerbating cytochrome c leakage; self-amplification killing network: the above interaction forms a cascade cycle of “mitochondrial damage→ROS / iron release→double pathway activation→secondary mitochondrial damage”, which significantly improves the removal efficiency of drug-resistant tumor cells.

[0039] The ferrocene cross-linked membrane split polyamino acid nanoparticle for cancer treatment has excellent anti-tumor synergy, and compared with single mechanism therapy, the double-pathway synergy effect has the following advantages: overcoming drug resistance: apoptosis-resistant tumor cells (such as Bcl-2 high expression type) can be cleared through the ferroptosis pathway; ferroptosis-inhibited tumor cells (such as GPX4 overexpression type) can be killed through the apoptosis pathway; enhancing the killing efficiency: the joint attack of double-pathway on the metabolic network of tumor cells (energy metabolism blockage + oxidative reduction homeostasis destruction) leads to the "synthetic lethal" effect; the cascade self-amplification mechanism enables low-dose administration to achieve high killing effect (EC 50 3-5 times lower than single mechanism system); high biological safety: tumor-specific activation: imine bond is only broken in acidic lysosomes, avoiding non-specific release in normal tissues. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The particle size distribution and morphology diagram of the mPEG-b-PLL-b-PPA nanoparticle (PNPs) prepared in the application.

[0041] Figure 2 The particle size distribution and morphology diagram of the mPEG-b-PLL-b-PPA / Fc nanoparticle (FNPs) prepared in the application.

[0042] Figure 3 The FT-IR spectrum of the nanoparticle PNPs and FNPs prepared in the application.

[0043] Figure 4 The Zeta potential of the nanoparticle PNPs and FNPs prepared in the application.

[0044] Figure 5 The XPS spectrum (Fe2p) of the nanoparticle FNPs prepared in the application.

[0045] Figure 6 The drug release kinetics curve of the FNPs prepared in the application at different pH values.

[0046] Figure 7 The cytotoxicity of the PNPs and FNPs prepared in the application to 4T1;

[0047] Figure 8 The tumor inhibition curve of the FNPs prepared in the application;

[0048] Figure 9 The immunofluorescence section of the tumor tissue of the mouse after treatment with the FNPs prepared in the application (Tunel, Bcl-2, Cleaved-caspase3 and Cyt c are apoptosis indicators, and GPX4 is an iron death indicator). DETAILED DESCRIPTION

[0049] For further understanding of the present application, the preferred embodiments of the present application are described below in conjunction with the examples, but it should be understood that the description is only for further illustrating the features and advantages of the present application and is not a limitation on the patent claims of the present application.

[0050] All raw materials of the present application are not particularly limited in source, and can be purchased on the market or prepared according to the conventional methods well known to those skilled in the art.

[0051] All raw materials of the present application are not particularly limited in purity, and the present application preferably adopts the purity requirements conventional in the field of preparation of medical pure or film lysing polyamino acid materials used for cancer treatment.

[0052] All raw materials of the present application are of conventional grades and abbreviations in the art, and each grade and abbreviation is clear and explicit in the field of its relevant use, and those skilled in the art can purchase or prepare them by conventional methods according to the grade, abbreviation and corresponding use.

[0053] The present application provides a ferrocene cross-linked film lysing polyamino acid composite material, which is obtained after preparation of ferrocene dimethylaldehyde and film lysing polyamino acid.

[0054] The film lysing polyamino acid includes a triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine. It can be mPEG (polyethylene glycol monomethyl ether)-b-PLL (polylysine)-b-PPhe (polyphenylalanine).

[0055] In the present application, the composite material preferably has a cross-linked network structure formed by the film lysing polyamino acid and ferrocene.

[0056] In the present application, the formation is specifically preferably the formation of an imine bond by the aldehyde group in ferrocene dimethylaldehyde and the amino group at the end of the polylysine segment.

[0057] In the present application, the cross-linked structure is specifically preferably a pH-sensitive dynamic cross-linked network structure.

[0058] In the present application, the pH sensitivity is specifically that the imine bond is broken, releasing ferrocene molecules and exposing free amino groups of polylysine, when the pH value is preferably 4.5-5.5, more preferably 4.7-5.3, and more preferably 4.9-5.1.

[0059] In the present application, the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid, the polymerization degree of the polyethylene glycol monomethyl ether is preferably 100-120, more preferably 103-118, more preferably 105-115, more preferably 107-113. Specifically, the polymerization degree is preferably n=113. The number average molecular weight Mn is 5000.

[0060] In the present application, the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid, the polymerization degree of the polylysine is preferably 65-75, more preferably 67-73, more preferably 69-71.

[0061] In the present application, the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid, the polymerization degree of the polyphenylalanine is preferably 8-12, more preferably 8.5-11.5, more preferably 9-11, more preferably 9.5-10.5.

[0062] In the present application, the molar ratio of the ferrocene to the film cleavage polyamino acid is preferably (4.5-5.5):1, more preferably (4.7-5.3):1, more preferably (4.9-5.1):1. Specifically, it can be 5:1.

[0063] In the present application, the ferrocene cross-linked film cleavage polyamino acid composite material preferably includes ferrocene cross-linked film cleavage polyamino acid nanoparticles. Specifically, the ferrocene cross-linked film cleavage polyamino acid composite material is a spherical nanoparticle.

[0064] In the present application, the particle size of the ferrocene cross-linked film cleavage polyamino acid nanoparticle is preferably 80-150 nm, more preferably 90-140 nm, more preferably 100-130 nm, more preferably 110-120 nm, and specifically can be 114.83±3.72 nm.

[0065] In the present application, the ferrocene cross-linked film cleavage polyamino acid nanoparticle preferably has a micellar structure.

[0066] In the present application, the ferrocene cross-linked film cleavage polyamino acid nanoparticle preferably includes an outer layer formed by a polyethylene glycol monomethyl ether segment, a middle layer formed by a ferrocene cross-linked polylysine network, and an inner core formed by a polyphenylalanine segment.

[0067] The present application provides a preparation method of a ferrocene cross-linked film cleavage polyamino acid composite material, comprising the following steps:

[0068] 1) mixing the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid and the organic solvent, and then mixing with water to obtain a film cleavage polyamino acid nanoparticle solution;

[0069] 2) mixing the film lysing polyamino acid nanoparticle solution obtained in the above step and the 1,1'-ferrocene dimethyl formaldehyde organic solution, and then reacting to obtain ferrocene cross-linked film lysing polyamino acid nanoparticles.

[0070] The film lysing polyamino acid nanoparticle solution is obtained by mixing the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid and the organic solvent, and then mixing with water again.

[0071] In the present application, the number average molecular weight of the triblock polyamino acid is preferably 14000-17000, more preferably 14500-16500, and more preferably 15000-16000. Specifically, it can be 15444±935.

[0072] In the present application, the mixing method in step 1) preferably includes dropwise adding the solution obtained by mixing the polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid and the organic solvent to water and stirring to mix.

[0073] In the present application, the stirring time is preferably 25-35 min, more preferably 27-33 min, and more preferably 29-31 min, and specifically can be 30 min.

[0074] In the present application, the organic solvent preferably includes dimethyl sulfoxide.

[0075] The film lysing polyamino acid nanoparticle solution obtained in the above step and the 1,1'-ferrocene dimethyl formaldehyde organic solution are mixed and then reacted to obtain ferrocene cross-linked film lysing polyamino acid nanoparticles.

[0076] In the present application, the mass concentration of the film lysing polyamino acid nanoparticle solution is preferably 0.5-1.0 mg / mL, more preferably 0.6-0.9 mg / mL, and more preferably 0.7-0.8 mg / mL.

[0077] In the present application, the mass ratio of the film lysing polyamino acid nanoparticles to 1,1'-ferrocene dimethyl formaldehyde is preferably 100:(25-35), more preferably 100:(27-33), and more preferably 100:(29-31).

[0078] In the present application, the solvent in the 1,1'-ferrocene dimethyl formaldehyde organic solution preferably includes dimethyl sulfoxide.

[0079] In the present application, the mixing method in step 2) preferably includes dropwise adding the 1,1'-ferrocene dimethyl formaldehyde organic solution to the film lysing polyamino acid nanoparticle solution and stirring to mix.

[0080] In the present application, the reaction time is preferably 3.5-4.5 hours, more preferably 3.7-4.3 hours, more preferably 3.9-4.1 hours, and specifically can be 4 hours. Specifically, the reaction is a room temperature reaction.

[0081] In the present application, after the reaction, a step of purifying the reaction solution by dialysis is also preferably included.

[0082] In the present application, the molecular weight cut-off of the dialysis bag used for dialysis is preferably 8000 Da.

[0083] In the present application, the purification time is preferably 3 days.

[0084] The present application breaks through the single path limitation of traditional membrane lysis technology through deep coupling of material chemical design and biological mechanism, provides an innovative strategy with high efficiency and safety for solid tumor treatment, can realize apoptosis-ferroptosis dual mechanism killing of tumor cells by targeting mitochondrial damage and cascade amplification of intracellular oxidative stress compared to traditional membrane lysis polyamino acid, and at the same time, the ferrocene crosslinking structure shields a large number of surface positive charges of the membrane lysis polyamino acid, improves the biological safety, and solves the problems of poor effect caused by single killing mechanism of the membrane lysis polyamino acid and high toxicity caused by high positive charge.

[0085] The present application provides the application of the ferrocene crosslinked membrane lysis polyamino acid composite material in the preparation of an antitumor drug.

[0086] The present application is a complete and detailed overall technical solution, which better ensures the composition and structure of the ferrocene crosslinked membrane lysis polyamino acid nanoparticle, further reduces the toxicity of the ferrocene crosslinked membrane lysis polyamino acid composite material, and improves the killing effect on tumor cells, and the ferrocene crosslinked membrane lysis polyamino acid nanoparticle for cancer treatment, the preparation method and the application can specifically include the following contents.

[0087] The present application is based on the unique structure design of the ferrocene crosslinked membrane lysis polyamino acid nanoparticle, which is constructed by the following steps:

[0088] The triblock polyamino acid main chain is sequentially connected by a polyethylene glycol monomethyl ether (mPEG) segment (hydrophilic shell), a polylysine (PLL) segment (cationic membrane destruction unit) and a polyphenylalanine (PPhe) segment (hydrophobic core) to form an amphiphilic self-assembled nanoparticle;

[0089] Ferrocene dynamic crosslinking: By using dialdehyde ferrocene (Fc(CHO)2) to form imine bonds (-C=N-) with the amino groups at the end of polylysine segments through Schiff base reaction, a pH-sensitive dynamic crosslinking network is constructed, endowing nanoparticles with responsiveness to the acidic tumor microenvironment.

[0090] The present invention provides a ferrocene cross-linked membrane-lytic polyamino acid nanoparticle for cancer treatment, its preparation method, and its application. The present invention specifically designs ferrocene cross-linked membrane-lytic polyamino acid nanoparticles with a synergistic killing mechanism of apoptosis and ferroptosis for cancer treatment. Compared with traditional membrane-lytic polyamino acid antitumor materials, these nanoparticles can efficiently target and damage mitochondria. Through the amplification of oxidative stress and activation of apoptosis via the membrane-lytic polyamino acid-ferrocene cascade, they increase the depletion of reactive oxygen species in tumor cells, glutathione-activated ferroptosis, thereby achieving the goal of highly efficient killing of tumor cells. Simultaneously, the ferrocene cross-linked structure can effectively shield the surface cations of the membrane-lytic polyamino acid, greatly improving the biosafety of traditional membrane-lytic polyamino acids and solving the problems of poor tumor-killing effect and high toxicity of membrane-lytic polyamino acid materials.

[0091] This invention provides a unique structural design for ferrocene cross-linked membrane-disrupting polyamino acid nanoparticles, constructed through the following steps: Molecular structure customization: Triblock polyamino acid backbone: composed of polyethylene glycol monomethyl ether (mPEG) segments (hydrophilic shell), polylysine (PLL) segments (cationic membrane disruption unit), and polyphenylalanine (PPhe) segments (hydrophobic core), forming amphiphilic self-assembled nanoparticles; Ferrocene dynamic cross-linking: utilizing dialdehyde ferrocene (Fc(CHO)2) and the amino group at the end of the polylysine segment to form an imine bond (-C=N-), constructing a pH-sensitive dynamic cross-linking network, endowing the nanoparticles with responsiveness to the tumor acidic microenvironment; Tumor microenvironment-triggered dissociation: Lysosomal acid response: after being internalized by tumor cells, the nanoparticles enter the lysosome (pH... (4.5–5.5) The imine bond breaks under acidic conditions, releasing ferrocene molecules and exposing the free amino group of polylysine; proton sponge effect escape: the free amino group is protonated at low pH, causing an increase in lysosomal osmotic pressure, which leads to lysosomal membrane rupture and release of nanoparticles into the cytoplasm.

[0092] The ferrocene cross-linked membrane cleavage polyamino acid composite material provided by this invention can achieve dual lethal pathway synergistic activation. After the nanoparticles escape into the cytoplasm, they achieve apoptosis-ferroptosis synergistic killing through the following mechanisms: (1) Mitochondrial membrane targeted destruction: Targeted localization: The hydrophobic segments of polyphenylalanine bind to mitochondrial membrane phospholipids (such as cardiolipin) through hydrophobic-hydrophobic interactions, achieving mitochondrial-specific targeting; Membrane damage effect: The cationic groups of polylysine insert into the mitochondrial membrane, destroying the membrane potential and forming pores, leading to mitochondrial swelling, cristae disintegration and leakage of contents (such as cytochrome c, hydrogen peroxide). (2) Apoptosis pathway activation: Cytochrome c release: The effluxed cytochrome c binds to Apaf-1 protein in the cytoplasm, activating the caspase-9 / caspase-3 cascade reaction and initiating the classical apoptosis program; ROS signal amplification: Mitochondrial damage further generates reactive oxygen species (ROS), activating the p53 pathway, inhibiting the expression of the anti-apoptotic protein Bcl-2, and enhancing apoptosis sensitivity. (3) Ferroptosis pathway activation: Fenton reaction catalysis: The released ferrocene (Fe 2+ ) undergoes the Fenton reaction with hydrogen peroxide (H2O2) (Fe 2+ +H₂O₂→Fe 3+ The reaction of hydroxyl radicals (·OH) generates a large number of hydroxyl radicals (·OH); lipid peroxidation storm: ·OH attacks polyunsaturated fatty acids (PUFAs) in the cell membrane and mitochondrial membrane, triggering a chain reaction of lipid peroxidation; antioxidant system collapse: hydroxyl radicals simultaneously consume glutathione (GSH), inhibit glutathione peroxidase 4 (GPX4) activity, and block the lipid peroxidation repair pathway. This invention can also achieve a self-amplified effect of oxidative stress cascade. The core innovation of this invention lies in the positive feedback loop formed by the interaction of two lethal pathways: apoptosis promotes ferroptosis: during apoptosis, mitochondrial membrane permeability continuously increases, releasing more H2O2 and iron ions, providing substrates for the Fenton reaction; ferroptosis enhances apoptosis: the accumulation of lipid peroxides leads to further loss of mitochondrial membrane integrity, exacerbating cytochrome c leakage; self-amplified killing network: the above interactions form a cascade loop of "mitochondrial damage → ROS / iron release → dual pathway activation → secondary mitochondrial damage", significantly improving the clearance efficiency of drug-resistant tumor cells.

[0093] The ferrocene cross-linked membrane lysed polyamino acid nanoparticles for cancer treatment provided by this invention exhibit excellent anti-tumor properties through a synergistic mechanism. Compared to single-mechanism therapies, the dual-pathway synergistic effect of this invention has the following significant advantages: overcoming drug resistance: apoptosis-resistant tumor cells (such as those overexpressing Bcl-2) can be cleared through the ferroptosis pathway; ferroptosis-inhibited tumor cells (such as those overexpressing GPX4) can be killed through the apoptosis pathway; enhanced killing efficiency: the combined attack of the dual pathways on the tumor cell metabolic network (energy metabolism blockade + redox homeostasis disruption) leads to a "synthetic lethality" effect; the cascade self-amplification mechanism enables high killing effects with low-dose administration (EC). 50 (3-5 times lower than single-mechanism systems); High biosafety: Tumor-specific activation: Imine bonds break only in acidic lysosomes, avoiding non-specific release in normal tissues.

[0094] To further illustrate the present invention, the following detailed description of a ferrocene crosslinked membrane pyrolysis polyamino acid composite material, its preparation method, and its application are provided in conjunction with embodiments. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are given only to further illustrate the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0095] Example 1

[0096] A ferrocene cross-linked membrane cleavage polyamino acid nanoparticle with a synergistic killing mechanism of apoptosis and ferroptosis.

[0097] mPEG 113 -b-PLL 70 -b-PPA 10 (PLP) synthesis

[0098] First, prepare the NCAs of two amino acids. In a dry, anhydrous three-necked round-bottom flask, add 200 mL of anhydrous tetrahydrofuran (THF). Dissolve L-lysine (20 g) and triphosgene (12 g) in the THF under nitrogen protection, stirring in an oil bath at 55 °C. When the turbid solution becomes clear (approximately 2 hours), increase the nitrogen flow rate. Remove as much THF as possible, concentrate the reaction solution, and when approximately 50 mL of THF remains, pour the concentrated reaction solution into 1500 mL of pre-cooled n-hexane for precipitation, stirring rapidly during precipitation. Then, allow it to stand at -20 °C for 4 hours. After standing, the solvent was removed by filtration through a Buchner funnel. The resulting white solid product was dissolved in 250 mL of cold ethyl acetate and washed four times with 60 mL of ice water. Then, it was placed in an Erlenmeyer flask with an appropriate amount of anhydrous magnesium sulfate and dried overnight at -20°C. The next day, the anhydrous magnesium sulfate was removed by suction filtration through a G4 sintered glass funnel. The resulting liquid product was then subjected to reduced pressure to remove ethyl acetate. Finally, the final product was obtained by recrystallization.

[0099] Subsequently, the ring-opening polymerization of NCA was initiated using mPEG-NH2. Specifically, 1 g of mPEG-NH2 was added to 250 mL of dry toluene, and the mixture was heated in an oil bath at 125 °C for azeotropic dehydration for 2-3 hours. The toluene was then dried using a vacuum pump connected to a cold trap. 4.7 g of L-Lys NCA dissolved in 40 mL of anhydrous N,N-dimethylformamide (DMF) was added, and the mixture was stirred at room temperature for 3 days, with nitrogen purging three times daily. Then, 0.5 g of L-Phe NCA dissolved in 40 mL of anhydrous DMF was added, and the mixture was stirred at room temperature for another 3 days, with nitrogen purging three times daily. After the reaction was complete, the mixture was settled using ice-cold diethyl ether with rapid stirring during the settling process. The mixture was then filtered through a Buchner funnel, dried under vacuum, and weighed to obtain a white solid product. Then, deprotection was performed. The product was dissolved in 10 times its mass volume of trifluoroacetic acid (TFA), and then 3 times its mass volume of hydrobromic acid / acetic acid solution was added. The mixture was stirred at room temperature for 1 hour, precipitated again with ice-cold ether, filtered through a Buchner funnel, and the solid was dissolved in an appropriate amount of DMF and placed in an 8000 Da dialysis bag. After dialyzing in deionized water for 3 days, the mixture was lyophilized to obtain mPEG. 113 -b-PLL 70 -b-PPA 10 (PLP).

[0100] The reaction formula is shown below:

[0101]

[0102] Synthesis of Ferrocene Crosslinked Membrane-Cracked Polyamino Acid Nanoparticles (FNPs)

[0103] First, PLP (50 mg) was dissolved in 10 mL of dimethyl sulfoxide (DMSO). After the solid was completely dissolved, it was slowly added dropwise to 40 mL of deionized water and stirred for 30 minutes to obtain a solution of mPEG-b-PLL-b-PPA nanoparticles (PNPs).

[0104] Subsequently, mPEG-b-PLL-b-PPA / Fc nanoparticles (FNPs) were prepared by reacting PNPs solution with 1,1'-ferrocene dicarboxaldehyde. The specific steps are as follows:

[0105] 1,1'-ferrocene dicarboxaldehyde (Fc) (13.7 mg, 0.064 mmol) was dissolved in 5 mL of DMSO. After complete dissolution, the solution was slowly added dropwise to 50 mL of PNPs solution. The reaction was stirred for 4 hours. The reaction solution was then purified with deionized water for 3 days in an 8000 Da dialysis bag to obtain a concentration of 600 μg / mL. -1 FNPs solution.

[0106] The products prepared in the embodiments of the present invention were characterized and tested.

[0107] See Figure 1 , Figure 1 The image shows the particle size distribution and morphology of the mPEG-b-PLL-b-PPA nanoparticles (PNPs) prepared in this invention.

[0108] See Figure 2 , Figure 2 The particle size distribution and morphology of the mPEG-b-PLL-b-PPA / Fc nanoparticles (FNPs) prepared in this invention are shown in the figure.

[0109] See Figure 3 , Figure 3 The FT-IR spectra of the PNPs and FNPs nanoparticles prepared in this invention are shown.

[0110] See Figure 4 , Figure 4 The zeta potentials of the PNPs and FNPs nanoparticles prepared in this invention.

[0111] See Figure 5 , Figure 5 XPS pattern (Fe2p) of the nanoparticles FNPs prepared in this invention.

[0112] Pharmaceutical experiments were conducted on the mPEG-b-PLL-b-PPA / Fc nanoparticles (FNPs) prepared in the embodiments of the present invention.

[0113] See Figure 6 , Figure 6 Drug release kinetic curves of FNPs prepared in this invention at different pH values.

[0114] See Figure 7 , Figure 7 The PNPs and FNPs prepared for this invention exhibit cytotoxicity against 4T1.

[0115] See Figure 8 , Figure 8 The tumor inhibition curves of the FNPs prepared in this invention are shown.

[0116] See Figure 9 , Figure 9 Immunofluorescence sections of mouse tumor tissue after FNPs treatment prepared for this invention (Tunel, Bcl-2, Cleaved-caspase 3 and Cyt c are apoptosis markers, and GPX4 is a ferroptosis marker).

[0117] The foregoing provides a detailed description of the ferrocene cross-linked membrane pyrolysis polyamino acid nanoparticles for cancer treatment, their preparation method, and applications. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these embodiments are merely to aid in understanding the method and core ideas of the invention, including the best mode, and to enable any person skilled in the art to practice the invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the wording of the claims, or if they include equivalent structural elements that are not substantially different from the wording of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A ferrocene cross-linked membrane pyrolysis polyamino acid composite material, characterized in that, The ferrocene crosslinked membrane pyrolysis polyamino acid composite material is prepared by ferrocene diformaldehyde and membrane pyrolysis polyamino acid; The membrane-lytic polyamino acid comprises a triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine.

2. The composite material according to claim 1, characterized in that, The composite material has a cross-linked network structure formed by membrane-lytic polyamino acids and ferrocene; Specifically, the formation involves the cross-linking of aldehyde groups in ferrocene diformaldehyde with amino groups at the ends of polylysine segments to form imine bonds. The cross-linking structure is specifically a pH-sensitive dynamic cross-linking network structure; Specifically, the pH sensitivity means that at a pH value of 4.5 to 5.5, the imine bond breaks, releasing ferrocene molecules and exposing the free amino group of polylysine.

3. The composite material according to claim 1, characterized in that, In the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the degree of polymerization of polyethylene glycol monomethyl ether is 100-120. In the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the degree of polymerization of polylysine is 65-75. In the triblock polyamino acid of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine, the degree of polymerization of polyphenylalanine is 8-12. The molar ratio of ferrocene to membrane-degradable polyamino acid is (4.5–5.5):

1.

4. The composite material according to claim 1, characterized in that, The ferrocene cross-linked membrane pyrolysis polyamino acid composite material includes ferrocene cross-linked membrane pyrolysis polyamino acid nanoparticles. The particle size of the ferrocene crosslinked membrane pyrolyzed polyamino acid nanoparticles is 80–150 nm. The ferrocene crosslinked membrane cleaves polyamino acid nanoparticles with a micelle structure. The ferrocene cross-linked membrane cleaved polyamino acid nanoparticles comprise an outer layer formed by polyethylene glycol monomethyl ether segments, an intermediate layer formed by a ferrocene cross-linked polylysine network, and a core formed by polyphenylalanine segments.

5. A method for preparing a ferrocene crosslinked membrane pyrolysis polyamino acid composite material, characterized in that, Includes the following steps: 1) After mixing polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid with an organic solvent, and then mixing it with water again, a membrane lysis polyamino acid nanoparticle solution was obtained. 2) The membrane lysis polyamino acid nanoparticle solution obtained in the above steps is mixed with 1,1'-ferrocene diformaldehyde organic solution and reacted to obtain ferrocene crosslinked membrane lysis polyamino acid nanoparticles.

6. The composite material according to claim 5, characterized in that, The number-average molecular weight of the triblock polyamino acid is 14,000 to 17,000. In step 1), the method of mixing again includes adding the solution of polyethylene glycol monomethyl ether-polylysine-polyphenylalanine triblock polyamino acid and organic solvent dropwise to water and stirring to mix. The mixing time is 25 to 35 minutes.

7. The composite material according to claim 5, characterized in that, The organic solvent includes dimethyl sulfoxide; The mass concentration of the membrane lysis polyamino acid nanoparticle solution is 0.5–1.0 mg / mL; The mass ratio of the membrane-disrupting polyamino acid nanoparticles to 1,1'-ferrocene dicarboxaldehyde is 100:(25-35).

8. The composite material according to claim 5, characterized in that, The solvent in the 1,1'-ferrocene diformaldehyde organic solution includes dimethyl sulfoxide; In step 2), the mixing method includes adding 1,1'-ferrocene diformaldehyde organic solution dropwise to the membrane lysis polyamino acid nanoparticle solution and stirring to mix. The reaction time is 3.5 to 4.5 hours.

9. The composite material according to claim 5, characterized in that, The reaction also includes a step of purifying the reaction solution by dialysis. The molecular weight cutoff of the dialysis bag used for dialysis is 8000 Da; The purification process takes 3 days.

10. The application of the ferrocene cross-linked membrane pyrolysis polyamino acid composite material according to any one of claims 1 to 4 or the ferrocene cross-linked membrane pyrolysis polyamino acid composite material prepared by the preparation method according to any one of claims 5 to 9 in the preparation of antitumor drugs.

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