CN114452405A - Metal organic supermolecule with functions of inducing iron death and activating p53 - Google Patents

Metal organic supermolecule with functions of inducing iron death and activating p53 Download PDF

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CN114452405A
CN114452405A CN202210187516.6A CN202210187516A CN114452405A CN 114452405 A CN114452405 A CN 114452405A CN 202210187516 A CN202210187516 A CN 202210187516A CN 114452405 A CN114452405 A CN 114452405A
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成琨
罗永百
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First Affiliated Hospital of Medical College of Xian Jiaotong University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/242—Gold; Compounds thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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Abstract

The invention discloses a metal organic supermolecule with the functions of inducing iron death and activating p53, which is prepared from peptide-gold precursor polymer and CeO2The nano-particles are formed by self-assembly under the actions of metallophilic interaction and van der Waals force, the structure of the peptide-gold precursor polymer is shown as a formula (I), and the peptide-gold precursor polymer is formed by Au in chloroauric acid3+And spontaneous coordination formation between thiolated PMI polypeptides. The metal organic supermolecule can reactivate a p53 signal path in vitro, enhance iron death of lung cancer, has an anti-tumor effect in vivo, and has an effect in vivoThe safety is high.
Figure DDA0003524203560000011

Description

Metal organic supermolecule with functions of inducing iron death and activating p53
Technical Field
The invention belongs to the technical field of nano-drugs, relates to medical application of combination of metal and organic matters, and particularly relates to a metal organic supermolecule with functions of inducing iron death and activating p 53.
Background
Lung cancer is a leading cause of cancer-related death worldwide. Statistics from 185 countries or regions worldwide indicate that there may be 2,206,771 new cases and 1,796,144 deaths in 2020. Current treatment regimens in clinical settings are far from satisfactory. Due to their inherent limitations and the complex heterogeneity of cancer, conventional therapies are often accompanied by cancer recurrence and poor prognosis. Emerging immunotherapies also have several drawbacks, including narrow anti-cancer spectrum, low response rates, and potential toxicity due to autoimmunity. Therefore, there is an urgent need to develop innovative precise medical solutions. Other forms of non-apoptotic cell death, such as iron death, are induced to overcome drug resistance, indicating a new direction for cancer treatment. In addition, the combination with other therapies to increase the breadth and depth of response of iron death therapies may result in more effective anti-tumor effects. The natural anti-tumor effect of p53 and its enhanced susceptibility to iron death suggests that induction of iron death and p53 treatment may be an excellent combination.
Iron death is an iron-dependent programmed cell death caused by intracellular lipid peroxidation, and has different death characteristics compared to apoptosis, pyro-death, and autophagy. Given that iron death is dependent on lipid metabolism and oxidative stress, alterations in cellular energy metabolism can significantly affect the sensitivity of cells to iron death. On the other hand, metabolic reprogramming inevitably occurs during carcinogenesis, making cancer cells highly sensitive to iron death-inducing therapies. More importantly, induction of the iron death pathway by depletion of Xc (cystine/glutamate antiporter) or GPX4 (glutathione peroxidase) has been shown to be effective in killing drug-resistant cancer cells. New studies have also demonstrated an emerging role for iron death in cross-talk between tumor cells and immune cells. The above shows that targeted iron death is of great significance for anticancer therapy. However, cancer cells tend to impair iron death by increasing the expression of antioxidant enzymes or up-regulating prominin2 to promote iron transport. Abundant iron death targets and a regulation network provide available resources for iron death sensitization. Among them, p53, as a tumor suppressor, can enhance the sensitivity of cells to iron death both directly (transcription-dependent inhibition of SLC7a11 expression) and indirectly (by regulating amino acid metabolism, iron transport, PUFA (polyunsaturated fatty acid) metabolism, antioxidant defense, etc.).
The p53 protein is the most important tumor suppressor protein, and the signaling pathway is abnormal in most human tumors, wherein about 50% of the signaling pathway is caused by the mutational inactivation of the gene TP53 encoding the p53 protein. In a wild type TP53 tumor type, the expression level and the transcription function of the p53 protein are frequently negatively regulated by E3 ubiquitin ligase MDM2 and a homologous protein MDMX thereof, so that the cancer inhibition function of the protein is inhibited. Thus, the p53-MDM2/MDMX protein interaction is a rational and broad therapeutic target against TP53 wild-type tumors. Although a large number of small molecule drugs have been found to activate p53, such as Nutlins, imidazole WK2344, etc., high concentration administration and subsequent biotoxicity are inevitable due to the poor targeting and specificity of small molecules. Peptide drugs have the natural advantages of high affinity, good biological safety, etc., and are becoming competitive drugs for protein-protein interaction (PPI) modulators. At present, many studies report a great deal of effort in screening and modification of p53 activation peptides and achieve considerable therapeutic effects at the animal level. However, the search for higher affinity peptide fragments and overcoming their pharmacological barriers to promote their clinical transformation remains elusive.
Unlike traditional chemical molecules, supramolecular polymers are based on non-covalent interactions between molecules, such as metal coordination, hydrogen bonding, etc., and are gaining increasing attention as nanomedicines. However, the common supramolecules are often limited to a single functional component, inherent limitations and complex biological environments, resulting in undesirable therapeutic effects. Nano-platform based cascade engineering has been introduced ingeniously to optimize this cancer treatment, where metal organic supramolecules have been demonstrated to be an effective and rapidly developing strategy. It relies on metal-organic coordination interactions, based on abundant geometric structures and a large number of available resources, such as various metallic materials like gold, silver, iron, rare earth elements, etc., and organic modules like peptides, nucleic acids and small molecules. The vast number of combinatorial options offers greater possibilities to generate efficient cancer defense strategies that can either generate more therapeutic species at the tumor site or achieve stronger anti-tumor effects. Although many successful examples of metallo-organic supramolecules have been reported in tumor imaging, modulation of protein interactions, immunotherapy and combination therapies, there remains a great challenge in efficiently and simply synthesizing such complex nanosystems.
Various forms of supramolecular therapeutics have been widely used in cancer therapy to improve targeting specificity and therapeutic effect while reducing side effects on normal cells. Although all therapeutically approved monoclonal antibodies or fragments thereof are proteins directed against cell surface receptors or human secreted proteins, intracellular antigen targeting strategies remain to be translated into clinical applications. Although small molecule drugs convert some non-patent targets into patent targets, they are still limited by their size and lack of field of action. Peptide drugs between small molecule drugs and biological macromolecules, while meeting the requirements of moderate cell permeability and large drug target interfaces. Therefore, how to construct a supramolecular polymer capable of realizing the combination of the iron death induction therapy and the p53 activation therapy solves the clinical application problems of difficulty in cell membrane penetration and easiness in protein degradation of polypeptide, realizes the accumulation of the tumor suppressor protein p53, and becomes an extremely important topic in cancer treatment.
Disclosure of Invention
The invention aims to solve the clinical application problems that the polypeptide is difficult to penetrate cell membranes and easy to degrade.
In view of the above, the present invention addresses this need in the art by providing metallo-organic supramolecules with the ability to induce iron death and activate p 53. The metal organic supermolecule provided by the invention realizes polypeptide target protein-protein interaction; meanwhile, p53 is difficult to accumulate again by the polypeptide drug, and the dual anti-tumor effects of iron death and p53 activation are successfully realized.
In one aspect, the invention relates to a metallo-organic supramolecule composed of a peptide-gold precursor polymer and CeO2The nano-particles are formed by self-assembly under the actions of metallophilic interaction and van der Waals force, the structure of the peptide-gold precursor polymer is shown as a formula (I), and the peptide-gold precursor polymer is formed by Au in chloroauric acid3+And spontaneous coordination formation between thiolated PMI polypeptides.
Figure BDA0003524203540000041
Furthermore, in the metal organic supramolecules provided by the invention, the amino acid sequence of the PMI polypeptide is shown as SEQ ID No. 1; the SEQ ID No.1 is: TSFAEYWALLSP are provided.
Furthermore, in the metal organic supermolecule provided by the invention, the CeO2Before the nanoparticles are mixed with the peptide-gold precursor polymer, the CeO needs to be added2The nanoparticles were dissolved in 1mM hydrochloric acid; each 1mg of the CeO2The nanoparticles were dissolved in 58 μ L of the 1mM hydrochloric acid.
Further, in the metal organic supramolecules provided by the invention, the average particle size of the metal organic supramolecules is 31.74 nm.
Further, the present invention provides the metal-organic supramolecules, wherein the zeta potential of the metal-organic supramolecules in a PBS solution with pH7.4 is 19.9 mV.
On the other hand, the present invention is not particularly limited to the method for preparing the peptide-gold precursor polymer in the metal organic supramolecules provided by the present invention, and the method for preparing the peptide-gold precursor polymer in the metal organic supramolecules provided by the present invention includes:
2mg NH Per 2mg PMI2-PEGn-SH (MW 2000Da) and 4mL of deionized water were mixed, and 1mL of 10mM chloroauric acid solution was added, and stirred at 500rpm for 5 min.
In another aspect, the present invention provides a method for preparing metal organic supramolecules having the functions of inducing iron death and activating p53, comprising:
2mg PMI, 2mg NH2PEGn-SH (MW 2000Da) was mixed with 4mL of deionized water, and 1mL of 10mM chloroauric acid solution was added, and stirred at 500rpm for 5 min. During this process, the reaction system changed from a pale yellow turbid liquid to a purple red transparent solution with a marked tyndall effect. 0.002mM of nano CeO22.25mL of the solution was added to 2.25mL of 100mM HEPES, and then added to the peptide-gold precursor polymer solution for mild reduction. Nano CeO2The solution is 1mg of CeO2The nanoparticles were dissolved in 58 μ L of 1mM hydrochloric acid.
The invention constructs the method with p53 activation and cooperative iron death based on the cascade engineering of a nano platformMetal organic supramolecules (Nano-PMI @ CeO)2). Free radical generating module-CeO2The nanoparticle is used as a core, and the p 53-activated Peptide (PMI) -gold precursor polymer is reduced in situ and assembled on CeO2Surface as Nano-PMI @ CeO2The housing of (1). Nano-PMI @ CeO2Effectively up-regulate p53 levels in vitro and in vivo. Nano-PEG @ CeO as compared with an empty carrier2In contrast, Nano-PMI @ CeO2The better effect is achieved in the aspects of down-regulating GPX4 and promoting tumor cell apoptosis. More importantly, Nano-PMI @ CeO2The tumor progression of the lung cancer allograft model is significantly inhibited by p53 repair and induction of iron death, and exhibits good biosafety. Therefore, the invention further claims the application of the metal organic supramolecules provided by the invention in iron death induction medicines, p53 activation medicines and cancer treatment medicines, and further claims a cancer treatment medicine with iron death induction and p53 activation and a cancer treatment medicine, with the condition that the effective components of the medicine comprise the metal organic supramolecules provided by the invention.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the present invention provides a metal organic supramolecule having functions of inducing iron death and activating p53, which has dual functions of inducing iron death and activating p53, and shows excellent effects as a tumor therapeutic agent; the invention provides a metal organic supermolecule with functions of inducing iron death and activating p53, which shows good biological safety. The invention develops a tumor therapeutic agent with double functions of inducing iron death and activating p53, and proves a potential feasible treatment paradigm for sensitizing iron death through p53 activation, and the tumor therapeutic agent has the potential of clinical transformation. This also shows that metal organic supramolecules have great promise in transforming nanomedicine and treating human diseases.
Drawings
FIG. 1 shows Nano-PMI @ CeO2Schematic representation of the synthetic process and its targeting of iron droop at lung cancer sites induced by EPR effect and p53 pathway.
FIG. 2 shows Nano-PMI @ CeO2The characteristics of (a); a is Nano-PMI @ CeO2And an infrared spectrogram of PMI; b is Nano-PMI @ CeO2And Nano-PEG @ CeO2Ultraviolet-visible spectrum of (a); c is the measurement of Nano-PMI @ CeO by using a dynamic light scattering method2The hydrodynamic diameter of (a); d is Nano-PEG @ CeO2And Nano-PMI @ CeO2A TEM image of (a); e is the Nano-PMI @ CeO measured in PBS at pH7.42Surface charge (Zeta potential).
FIG. 3 shows Nano-PMI @ CeO2Activation of the p53 pathway, induction of iron death; a is the apoptosis of A549 cells detected by flow cytometry; b is the apoptosis rate of A549 cells under different treatments; c is 0.02mg/mLNano, 0.02mg/mL Nano-PMI @ CeO2、0.02mg/mL Nano-PEG@CeO2Western blot results for treated a549 cells COX2, Gpx4, p53, and SLC7a11 showed; d is the relative protein levels of COX2, GPX4, P53 and SLC7A11 calculated using Image J; the results are expressed as mean ± SE (n ═ 3), and P values were calculated by t test (, P)<0.001；**，P<0.01；*，P<0.001)。
FIG. 4 is Nano @ PMI @ CeO2Anti-tumor activity in vivo; a is a subcutaneous lung cancer transplantation model and a treatment process schematic diagram; b is tumor volume size change (n ═ 6); c is a photograph of the subcutaneous transplanted tumor at the end of the collection experiment; d is the average weight of the tumor (n ═ 6); e is tumor H&E, dyeing results; f is TUNEL staining; p values were calculated by t test (, P)<0.001；**，P<0.01；*，P<0.001)。
FIG. 5 shows Nano-PMI @ CeO2Mechanisms to induce tumor cell death in vivo; a is Nano-PMI @ CeO aiming at p53 pathway and inducing iron death2Schematic of the antitumor activity of (a); b is representative immunohistochemical staining of COX2, Gpx4 and P53 in tumor sections (scale bar: 100 μm); c is the IHC score analysis intratumoral p53, Gpx4 and COX2 protein levels; p values were determined by t test (, P)<0.001；**，p<0.01；*，p<0.05)。
FIG. 6 shows Nano-PMI @ CeO2Evaluating safety in vivo; a is a schematic diagram showing the tumor specificity of Nano-PMI @ CeO2 through an EPR effect; b is the influence of Nano-PMI @ CeO2, Nano-PEG @ CeO2 and Nano-PMI on weight change; c is the effect of the polypeptide on C57BL/6 mouse White Blood Cells (WBC); d is the shadow of the polypeptide on the blood platelet of C57BL/6 mouseSounding; e is the effect of the polypeptide on the Red Blood Cells (RBC) of the C57BL/6 mouse; effect of F polypeptide on C57BL/6 mouse hemoglobin; g histological HE staining images of heart, liver, spleen, lung and kidney of mice under different treatments (scale bar: 200 μm).
In the above figure, Nano-PMI @ CeO2The invention provides a metal organic supermolecule, Nano-PEG @ CeO, with the functions of inducing iron death and activating p532Is a metal organic supramolecule not loaded with PMI polypeptide; Nano-PMI is unbound CeO2The peptide-gold precursor polymer of (a); nano is Au3+Dissolved in HEPES.
Detailed Description
The following examples are given to illustrate the technical aspects of the present invention, but the present invention is not limited to the following examples.
In the examples below, all synthetic peptide sources are from CS bio co. All other chemicals used in this study were purchased from Sigma-Aldrich unless otherwise noted. All products were used without further purification.
In the examples described below, the human NSCLC cell line A549 and the mouse Lewis lung carcinoma cells (LLC) used were obtained from the cell bank of the Chinese academy of sciences (Shanghai, China), all maintained in DMEM medium supplemented with 10% FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin. Cell culture maintained at 5% CO2All cells were maintained at 37 ℃ in the gas atmosphere of (2).
Example 1
This example provides Nano-PMI @ CeO2、Nano-PEG@CeO2And a test for preparing Nano-PMI.
The peptides used in this example were all synthesized on the appropriate resins on a CS bio 336X automated peptide synthesizer using an optimized HBTU activation/DIEA in situ neutralization protocol developed by the HBTU/HOBT protocol of Fmoc chemical SPPS. Nano CeO used in this example2The solution is 1mg of CeO2The nanoparticles were dissolved in 58 μ L of 1mM hydrochloric acid.
(1)Nano-PMI@CeO2Preparation of
2mg PMI, 2mg NH2-PEGn-SH (MW 2000Da) was mixed with 4mL of deionized water1mL of 10mM chloroauric acid solution was added, and the mixture was stirred at 500rpm for 5 min. During this process, the reaction system changed from a pale yellow turbid liquid to a purple red transparent solution with a marked tyndall effect. 0.002mM of nano CeO22.25mL of the solution was added to 2.25mL of 100mM HEPES, and then added to the peptide-gold precursor polymer solution for mild reduction.
(2)Nano-PEG@CeO2Preparation of
2mg of NH2PEGn-SH (MW 2000Da) was mixed with 4mL of deionized water, and 1mL of 10mM chloroauric acid solution was added, and stirred at 500rpm for 5 min. In the process, the reaction system changed from a pale yellow turbid liquid to a purple red transparent solution. 0.002mM of nano CeO22.25mL of the solution was added to 2.25mL of 100mM HEPES, and then added to the peptide-gold precursor polymer solution for mild reduction.
(3) Preparation of Nano-PMI
2mg PMI, 2mg NH2PEGn-SH (MW 2000Da) was mixed with 8mL of deionized water, 2mL of 10mM chloroauric acid solution was added, and stirring was performed at 500rpm for 5 min. During this process, the stirring was terminated when the reaction system changed from a pale yellow turbid liquid to a purple red transparent solution.
Fourier Transform Infrared (FT-IR) spectroscopy (FIG. 2A) confirmed the disappearance of thiol groups in PMI-SH and Nano-PMI @ CeO2In the presence of Au-S. Nano-PMI @ CeO under a Transmission Electron Microscope (TEM)2And Nano-PEG @ CeO2All showed good dispersion and uniform size (fig. 2D). Measurement of nanoparticle size by Dynamic Light Scattering (DLS) shows Nano-PMI @ CeO2Has an average particle size of 31.74nm (FIG. 2C), and is in accordance with a TEM image. Zeta potential analysis shows that Nano-PMI @ CeO2The zeta potential in PBS solution (PH 7.4) was 19.9mV, indicating that the nanoparticles had good colloidal stability (fig. 2E). In summary, Nano-PMI @ CeO2Is based on CeO2Nanoparticles (metal part) and polypeptide PMI (organic part) are self-assembled to construct metal organic supramolecules.
Example 2
The embodiment provides Nano-PMI @ CeO2Reactivation of the p53 signaling pathway in vitro, and enhancement of iron death assays for lung cancer.
To confirm Nano-PMI @ CeO2Nanoparticles inhibit tumor growth potential in vitro, and the following experiments were performed with wild-type p53 and lung cancer cell line a549 overexpressing MDM2/MDMX as subjects. With Nano-PEG @ CeO2As a positive control, nanoparticles loaded with PEG-amine alone, referred to as Nano, served as a negative control. The level of apoptosis of the tumor cells was analyzed by flow apoptosis 48 hours after the intervention of the A549 cells in the different treatment groups. Apoptosis was measured by flow cytometry analysis by the FITC PE-7AAD apoptosis detection kit (BD, USA). A549 cells at 2X 105Cell/well density seeding was cultured in 6-well dishes for 24 hours, and then the cells were cultured with different drug groups at the indicated concentrations for 48 hours. Next, cells were harvested by brief centrifugation, washed twice with cold PBS and then resuspended at 1X 106Cells were resuspended in 1 × binding buffer at a concentration of individual cells/mL. 100 μ L of cell suspension (1X 10)5Individual cells) were transferred to 1.5mL culture tubes. Add 5. mu.L of E Annexin V and 5. mu.L of 7-AAD. Cells were vortexed gently and incubated at room temperature (25 ℃) protected from light for 15 minutes. Then 400. mu.L of 1 × Annexin V Binding Buffer was added to each tube. Analysis was performed by flow cytometry over 1 hour.
The test result shows that Nano-PMI @ CeO2Promote the apoptosis ratio of A549 cells to be remarkably increased (about 51 percent) which is far higher than that of Nano-PEG @ CeO2With the Nano group (FIG. 3A and FIG. 3B), Nano-PEG @ CeO2Also show antitumor activity. The above results indicate that Nano-PMI @ CeO2Tumor proliferation can be inhibited by inducing apoptosis.
The Nano-PMI @ CeO is proved2After having anti-cancer ability, the present example further explores its mechanism by Westernblot. Total protein was extracted from cells using RIPA lysis buffer containing protease inhibitors and equal amounts of protein lysate were separated by 10% SDS-PAGE, transferred to PVDF membrane and probed using primary and secondary antibodies.
The primary antibody is as follows: anti-p53(Santa Cruz Biotechnology, USA; 1:500), anti-GPX4(Santa Cruz, USA; 1:500), anti-SLC7A11(CST, USA; 1:1000), anti-COX2(Proteintech, USA; 1:500) and anti-GAPDH (Proteintech, USA; 1: 5000). ECL substrate (Millipore, MA, USA) was used for signal visualization. The band images were analyzed using ImageJ software (NIH) and normalized to GAPDH. Results of relative protein levels were analyzed using Image J.
Nano-PMI@CeO2(0.02mg/mL) with Nano-PEG @ CeO2(0.02mg/mL) intervened in A549 cells for 48h (FIG. 3C), compared to the control, with Nano-PEG @ CeO2The expression of SLC7A11 was significantly down-regulated in the group, suggesting that it may promote tumor cell iron death by inhibiting SLC7A 11. Furthermore, Nano-PMI @ CeO compared to the control group2The expression of p53 in (C) was significantly increased, indicating that Nano-PMI @ CeO2Accumulation of p53 in a549 cells was achieved by blocking the interaction between p53 and MDM2/MDMX (fig. 3C and 3D). Moreover, reactivation of p53 can significantly inhibit expression of SLC7a11 and GPX4, thereby further enhancing iron death of a549 cells.
Taken together, these results indicate that Nano-PMI @ CeO2Not only induces tumor cell death by promoting apoptotic pathways, but also sensitizes tumor cell iron death by reactivating the p53 signaling pathway.
Example 3
This example provides Nano-PMI @ CeO2And (3) testing the effect of inhibiting in vivo tumors.
All C57BL/6 mice were purchased from the laboratory animals center of Sichuan university of transportation. Animals were housed under standard pathogen-free conditions with standard food and typical light/dark cycles. All experimental procedures involving animals were performed according to the institutional guidelines and approved by the experimental animal center of the university of transport, west ampere. C57BL/6 mice (5-6 weeks old) were age matched for tumor inoculation. LLC cells (1X 10)6Individual cells/site) were implanted subcutaneously in the buttocks of C57BL/6 mice. When the tumor reaches 100mm3At the mean volume of (3), mice were randomly assigned to control group, Nano-PMI @ CeO2(2mg/Kg)、Nano-PMI(2mg/Kg)、Nano-PEG@CeO2(2mg/Kg) and control groups were divided into groups (6 mice each). Treatment was performed by intraperitoneal injection every other day. Small daily monitoringBody weight and condition of the mice. In addition, tumor length and width were measured daily with calipers and tumor volume was calculated using the following equation: v ═ length × width2)/2. And determining a humanoid endpoint according to the discomfort degree of the animal and the size of the tumor.
To evaluate Nano-PMI @ CeO2In vivo anti-tumor effect, this example constructed a mouse model of subcutaneous allograft tumor of lung cancer. LLC cells are injected subcutaneously to establish a C57BL/6 allogeneic lung cancer model. Mice were injected intraperitoneally with either the polypeptide drug or PBS (control group) once every other day for 6 times. Firstly, Lewis Lung Cancer (LLC) cells (1 x 10)6Mice) were inoculated subcutaneously into C57BL/6 mice, as shown in FIG. 4A, at tumors growing up to 100mm3At the left and right, respectively give Nano-PEG @ CeO by means of intraperitoneal injection2、Nano-PMI@CeO2And Nano-PMI treatment (2mg/Kg once every other day). And mice were monitored daily for subcutaneous graft tumor volume and body weight. Nano-PEG @ CeO as compared to PBS control2(2mg/kg) inhibited tumor growth by 51% at day 12 (FIG. 4B). Further, Nano-PMI @ CeO2(2mg/kg) has more remarkable cancer inhibition capacity, and the tumor inhibition rate is more than 74% (fig. 4B). At the same time, the gross tumor view (FIG. 4C) and the tumor weight (FIG. 4D) also demonstrated Nano-PMI @ CeO2The efficiency is highest compared to the other two groups.
Tissues were removed, formalin fixed, paraffin embedded (FFPE), sectioned and treated with hematoxylin-eosin (H) according to standard histopathology techniques&E) And (6) dyeing. All sections used for histological analysis were 4 μm thick. Immunohistochemistry used primary antibodies: anti-p53, anti-COX2(Abcam, USA; 1:200), anti-GPX4(Proteintech, USA; 1: 200). Images were taken by NIS Elements imaging software (Nikon) using a Nikon Eclipse Ni-U microscope and quantified by imagej (nih). The results of the tissue staining experiments are shown in fig. 4E and 4F. H of tumor tissue&The results of E staining (FIG. 4E) and TUNEL staining (FIG. 4F) also confirmed that Nano-PMI @ CeO2The antitumor effect of (1).
The results show that the metal organic supermolecule Nano-PMI @ CeO2Can be used as a promising anti-tumor p53 activator.
Example 4
This example provides Nano-PMI @ CeO2The tumor iron death verification test is activated through p53 reactivation in vivo.
Inorganic component Nano CeO in metal organic supermolecule Nano-PMI @ CeO22After in vivo release, SLC7A11 protein can be inhibited, further GPX4 is down-regulated to trigger iron death, and the organic functional peptide PMI can realize the reactivation of a p53 pathway of tumor cells in vivo through inhibiting the negative regulation of MDM2/MDMX, p53 can further inhibit SLC7A11 after activation, and further enhance the iron death related to GPX4 (figure 5A).
To further demonstrate Nano-PMI @ CeO2Specific regulatory mechanisms for in vivo antitumor, as found by IHC staining (FIG. 5B), Nano-PMI @ CeO2Can effectively promote the re-accumulation of p53 protein in tumor cells, and remarkably down-regulates GPX4, and synergistically induces the iron death process (figure 5B and figure 5C).
In conclusion, in vivo experiments prove that Nano-PMI @ CeO2The metal organic supermolecule provided by the invention can be passively accumulated at a tumor part through enhanced tumor permeability and in-vivo retention effect, promotes the reactivation of p53 and simultaneously coordinates the death of tumor cells by iron, and shows more efficient anti-tumor capability.
Example 5
This example provides Nano-PMI @ CeO2In vivo safety evaluation test.
Metallo-organic supramolecules generally enhance therapeutic performance by reducing the concentration of functional molecules in normal tissues and substantially increasing the concentration in tumors through Enhanced Permeability and Retention (EPR) effects (fig. 6A).
To evaluate Nano-PMI @ CeO2Safety in vivo, this example conducted a comprehensive toxicity study using C57BL/6 mice. Mice were given Nano-PMI @ CeO2Intraperitoneal injection is carried out once every other day for 12 days at a dose of 2mg/kg, and the weight change and the nutritional state of the mice are monitored. As shown in FIG. 6B, the four groups of mice gained weight gradually, Nano-PMI @ CeO2Although the weight gain rate of the group was slightly lower than that of the control group and the NanoPMI group at the later stage of the treatment, none of the groups hadThe difference was significant. Nano-PMI @ CeO2And Nano-PEG @ CeO2Safety of (b) was further confirmed by analysis of mouse peripheral blood lymphocytes (fig. 6C), platelets (fig. 6D), Red Blood Cells (RBCs) (fig. 6E), and hemoglobin (fig. 6F). In addition, histology H of liver and spleen, kidney, heart and lung&E staining supports the above findings and Nano-PMI @ CeO2Sufficiently safe and with significant therapeutic potential (FIG. 6G).
In conclusion, the present study shows that NanoPMI @ CeO2The medicine is used for treating the lung cancer, has effectiveness and safety, and provides preclinical evidence for clinical transformation of the lung cancer.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.

Claims (10)

1. A metallo-organic supramolecular comprising a peptide-gold precursor polymer and CeO2The nano-particles are formed by self-assembly under the actions of metallophilic interaction and van der Waals force, the structure of the peptide-gold precursor polymer is shown as a formula (I), and the peptide-gold precursor polymer is formed by Au in chloroauric acid3+And spontaneous coordination formation between thiolated PMI polypeptides.
Figure FDA0003524203530000011
2. The metalorganic supramolecular as claimed in claim 1, wherein said PMI polypeptide amino acid sequence is set forth in SEQ ID No. 1; the SEQ ID No.1 is: TSFAEYWALLSP are provided.
3. Metallo-organic supramolecules as claimed in claim 1, characterized in that said CeO2Nanoparticles and peptides-the CeO needs to be mixed with the gold precursor polymer before it is mixed2The nanoparticles were dissolved in 1mM hydrochloric acid; each 1mg of the CeO2The nanoparticles were dissolved in 58 μ L of the 1mM hydrochloric acid.
4. Metallo-organic supramolecules according to claim 1, characterized in that their average particle size is 31.74 nm.
5. Metallo-organic supramolecules according to claim 1, characterized in that the zeta potential of metallo-organic supramolecules is 19.9mV in PBS solution at pH 7.4.
6. Use of metallo-organic supramolecules as claimed in claim 1 in drugs inducing iron death.
7. Use of metallo-organic supramolecules as claimed in claim 1 in p 53-activated drugs.
8. Use of the metalorganic supramolecules as claimed in claim 1 in the preparation of medicaments for the treatment of cancer.
9. A therapeutic agent for cancer having both iron death induction and p53 activation, comprising the metallo-organic supramolecules in claim 1.
10. A cancer therapeutic agent comprising the metallo-organic supramolecules according to claim 1.
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