CN112807326B - Polypeptide nano-composite and preparation method and application thereof - Google Patents

Abstract

The invention discloses a polypeptide nano-composite and a preparation method and application thereof, wherein the polypeptide nano-composite has a general formula of [ peptide-S-Au ]] _m M is a positive integer, wherein peptide is (ALA-Hyp-Y-Hle-PM-) ^D R ^D C)‑PEG ₃ -MDMX; the polypeptide nano-composite is used for inhibiting the growth of tumors; further, the polypeptide nano-complex restores the anticancer functions of p53 and p73 by inducing the ubiquitination-dependent degradation of MDMX; the polypeptide nano-composite obtained by the invention can restore the anticancer functions of p53 and p73 by inducing the ubiquitination-dependent degradation of MDMX, and finally inhibit the growth of tumors; the polypeptide nano-composite prepared by the preparation method has the advantages of excellent solution stability and large drug loading capacity, and meanwhile, the degradation process of the polypeptide nano-composite has the advantages of high efficiency and similar catalysis, can realize intracellular continuous and stable protein degradation, and reduces the possibility of drug resistance of tumor cells.

Description

Polypeptide nano-composite and preparation method and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a polypeptide nano-composite as well as a preparation method and application thereof.

Background

Today, drug regulatory agencies around the world have approved formulations for approximately 400 human proteins and almost all can be classified as enzymes, transporters and receptors. Although human proteomics continues to identify a greater number of new therapeutic targets, over 85% of the 3000 disease-causing proteins are nonenzymatic proteins, transcription factors, scaffold proteins, and are considered "without patent potential" because they are unable to develop small molecule compounds that inhibit their biological function; even at 13% of druggable targets (400 out of 3000), traditional druggability strategies that occupy the active site often require high drug exposure doses in vivo, yet further increase the threat of off-target toxicity. To reverse this unfavorable situation, a great deal of work has been done to develop new means to modulate the concentration of the target protein. For this reason, translationally regulatable protein concentrations are present, by which intracellular disease-associated proteins can be down-regulated by antisense oligonucleotides, RNAi and the emerging CRISPR-Cas system. However, these nucleic acid-based tools depend largely on the half-life of the target protein. Therefore, a promising technology called PROTAC (proteolytic targeting chimera) was developed to fill the technical gap of targeted degradation of post-translational long-lived proteins.

ProTAC is a novel class of heterobifunctional molecules that bridge an intracellular target protein and an E3 ubiquitination ligase and cause ubiquitin-dependent degradation of the target protein in the proteasome. As a tool for basic research, PROTACs can achieve more direct, regulatable, and reversible target protein knockouts than nucleic acid-based tools. As a clinically applicable therapeutic approach, PROTACs can be attached to the surface of any target protein, rather than to its cognate active site, thereby allowing clearance of both established and "non-druggable" drug target proteins. Thus, protein degradation agents produced by PROTACs are receiving increasing attention, both in basic research and clinically. However, the number of E3 ligase and target proteins used to accomplish protein degradation is limited by the lack of specific small molecule ligands, and thus severely reduces the applicability and universality of PROTAC.

Due to the large interaction interface of the target protein, small molecule compounds are difficult to target, and once this challenge is fulfilled, polypeptide-derived protein degradation can not only break through the limitations of conventional drug development, focus more on binding site occupancy, but also expand the protein range involved in protein-protein interactions (PPI). MDMX, also known as MDM4, a traditional non-drug-forming PPI-associated protein, would be a beneficiary targeted by this emerging drug strategy.

MDMX is a binding protein of p 53-and p73-, and has a function of blocking its anticancer activity, and attenuated p53 and p73 cannot regulate cycle arrest and apoptosis after DNA damage, and directly results in tumor progression, poor prognosis and treatment resistance. In cancer cells, up-regulated MDMX inhibits the activity of p53 and p73, and has been the target of drug development for the treatment of retinoblastoma, pancreatic cancer, colorectal cancer, and breast cancer, but no MDMX antagonist has been put into clinical use.

Disclosure of Invention

The invention aims to provide a polypeptide nano-composite, a preparation method and an application thereof, wherein the polypeptide nano-composite can induce the ubiquitination-dependent degradation of MDMX so as to recover the anticancer functions of p53 and p 73.

The invention adopts the following technical scheme: a polypeptide nano-composite with the general formula of [ peptide-S-Au ]] _m M is a positive integer, wherein peptide is (ALA-Hyp-Y-Hle-PM-) ^D R ^D C)-PEG ₃ -MDMX。

Further, ALA-Hyp-Y-Hle-PM- ^D R ^D C is a fragment of Von Hippel Lindau factor.

A preparation method of a polypeptide nano-composite comprises the following steps:

step 1: the peptide is obtained by solid phase peptide synthesis according to FMOC chemical method, wherein the peptide is (ALA-Hyp-Y-Hle-PM- ^D R ^D C)-PEG ₃ -MDMX；

Step 2: mixing peptide and NH ₂ -PEG _n -SH is mixed to HAuCl after magnetic stirring ₄ Obtaining the polypeptide nano-composite in the solution.

An application of a polypeptide nano-composite, which is used for preparing a medicament for inhibiting the growth of tumors.

The invention has the beneficial effects that: the polypeptide nano-composite obtained by the invention can restore the anticancer functions of p53 and p73 by inducing the ubiquitination-dependent degradation of MDMX, and finally inhibit the growth of tumors; the polypeptide nano-composite prepared by the preparation method has the advantages of excellent solution stability and large drug loading capacity, and meanwhile, the degradation process of the polypeptide nano-composite has the advantages of high efficiency and similar catalysis, can realize intracellular continuous and stable protein degradation, and reduces the possibility of drug resistance of tumor cells.

Drawings

FIG. 1 is a schematic diagram of the synthesis and mechanism of action of the polypeptide nanocomposite of the present invention;

FIG. 2 is a schematic representation of the characterization of the physicochemical properties of the polypeptide nanocomplexes of the invention;

FIG. 3 is a schematic diagram of the in vivo mechanism of action of the polypeptide nanocomplexes of the present invention;

FIG. 4 is a graph showing the therapeutic effect of the polypeptide nanocomposite of the present invention on retinoblastoma in vivo;

FIG. 5 is a schematic diagram of the physicochemical characterization of the acid-sensitive molecule-modified polypeptide nanocomplex according to the present invention;

FIG. 6 is a graph showing the effect of PDX treatment of human pancreatic cancer in vivo by the polypeptide nanocomplexes of the present invention.

Detailed Description

The invention is described in detail below with reference to the drawings and the detailed description.

The invention discloses a polypeptide nano-composite, which has a general formula of [ peptide-S-Au ]] _m M is a positive integer, wherein peptide is (ALA-Hyp-Y-Hle-PM- ^D R ^D C)-PEG ₃ -MDMX，ALA-Hyp-Y-Hle-PM- ^D R ^D C is a fragment of Von Hippel Lindau factor.

The invention also discloses a preparation method of the polypeptide nano-composite, which comprises the following steps:

step 1: the peptide is obtained by solid phase peptide synthesis according to FMOC chemical method, wherein the peptide is (ALA-Hyp-Y-Hle-PM- ^D R ^D C)-PEG ₃ -MDMX；

Step 2: mixing peptide and NH ₂ -PEG _n -SH is mixed to HAuCl after magnetic stirring ₄ Obtaining the polypeptide nano-composite in the solution.

The invention also discloses application of the polypeptide nano-composite in preparing a medicament for inhibiting tumor growth, and the anti-cancer functions of p53 and p73 are recovered by inducing the ubiquitination-dependent degradation of MDMX.

The present invention contemplates polypeptide-derived PROTACs that bind to MDMX and the Von Hippel Lindau factor (VHL) E3 ligase listIn the interstices of the face, this polypeptide-derived PROTACs is called molecular glue MG, due to the bridging of MDMX and VHL like an adhesive. Furthermore, to overcome the pharmacological barrier of polypeptide-based PROTACs, [ Peptide-S-Au ] was obtained by self-assembly of the gold-polypeptide precursor Peptide-S-Au] _m Abbreviated as Nano-MG, as shown in fig. 1, which is a Nano-sized particle.

Design and Synthesis of Nano-MG

The MG consists of three parts: 1) MDMX binding groups (binding motif); 2) one composed of polyethylene glycol trimer (PEG) ₃ ) And 6-aminocaproic acid; 3) octapeptides consisting of hydroxyproline-and homoleucine-recognized by subunit VHL recruiting the target in the E3 ubiquitination ligase complex (fig. 2A).

Two additional dextrorotatory Cys and Arg residues were introduced at the C-terminus of MG for construction of nanoclusters. Notably, MG is easily synthesized by Solid Phase Polypeptide Synthesis (SPPS) according to FMOC chemistry (FMOC chemistry), with a yield of about 75% and a purity of > 95%, further improving its application potential, and nano-engineering of MG is performed by a "one-pot two-step" method under mild conditions.

Step 1: 1MG MG and 1MG NH ₂ -PEG _n -SH (MW 2000Da) was magnetically stirred for 5min and mixed to 5ml of 20mM HAuCl ₄ In solution.

Step 2: 5ml of 100mM HEPES (pH7.4) was added, and the solution became purple (FIG. 2B).

Characterization of Nano-MG

UV-Vis spectra confirmed the formation of Nano-MG, whose plasmon resonance (plasmon resonance) gave a characteristic UV absorbance (540 nm). Dynamic light scattering measurements indicated that the hydrodynamic size of the Nano-MG was 25nm (FIG. 2B). Fourier transform infrared spectroscopy (FTIR) in fig. 2C shows that the characteristic absorption peak of the free thiol group in the spectrum from MG shifts to the peak of the gold (I) -Thiolate (Thiolate) complex (complex) in the spectrum of Nano-MG, indicating that the gold ion has been bridged to MG by the sulfur atom in the sulfate, which is further characterized by X-ray photoelectron spectroscopy (XPS), where the Au (4f) peak position of Nano-MG is consistent with the result of conjugation of the Au (I) ion to alkanethiol in the Cys residue (fig. 2D).

These results indicate that the MG polypeptides have successfully assembled into supramolecular gold (I) -thiol-polypeptide nanocomplexes (Nano-MG). In addition, the morphology and elemental composition of Nano-MG were determined by High Resolution Transmission Electron Microscopy (HRTEM) images and X-ray spectroscopy (EDS). As shown in fig. 2E, HRTEM images and diffraction patterns (difractionatpatterren) show spherical microspheres, Nano-MG with single (single) good dispersion properties, good crystallinity. EDS analysis showed that the observed Nano-MG consists of Au, N, O and S (FIG. 2G), consistent with the composition of the polypeptide and gold. Elemental overlay (overlay chart) and TEM images show Au, N, O and S uniformly dispersed in the nanoparticles (fig. 2F), indicating good uniformity of Nano-MG. In summary, the above results demonstrate that Nano-MG was successfully constructed as a spherical supramolecular gold (I) -thiol-polypeptide nanocomposite.

Stability of Nano-MG

The three key characteristics of chemical stability, cell membrane permeability and controllable drug release are crucial to the drug effect of Nano-MG.

The chemical stability of Nano-MG was first examined by suspending it in PBS containing 20% FBS at pH 4.0, 6.0 and 7.4, respectively, and measuring the time-dependent particle size value (particle size) with DLS. FIG. 2H shows that the Nano-MG can remain monodisperse and unchanged in size throughout 24H, indicating that the Nano-MG can avoid the risk of undesirable aggregation (aggregation) and consequent uptake by the reticuloendothelial system.

To ascertain the cancer cell membrane penetrating ability of Nano-MG, Fluorescein Isothiocyanate (FITC) was bound to MG via the N-terminus and analyzed by flow cytometry after 6h incubation ^FITC MG and Nano- ^FITC Cellular uptake of MG. As shown in FIG. 2I, with ^FITC Internalized Nano- ^FITC MG levels were significantly increased. In addition, with 3mM amiloride (microcytosis inhibitor) and 2. mu.M cytochalasin D (actin inhibitor)) Pre-incubation to block Nano- ^FITC Internalization by MG also suggests actin-dependent microcytosis.

One key design of Nano-MG is that it increases the sensitivity of the cell to a reducing environment. Gold (I) -alkanethiol bonds release the loaded drug in response to high concentrations of Glutathione (GSH) in cancer cells, while remaining stable in the extracellular environment. It is expected that Nano-MGs release about 90% of MG (cargo) when incubated for 3h in a solution mimicking the intracellular environment at pH 6.0 with standard PBS, 5mM GSH (fig. 2J), whereas Nano-MGs generally maintain their integrity and release less than 10% when incubated for 12h in a solution mimicking the intracellular environment at pH7.4 with standard PBS, 5mM GSH (fig. 2J). Taken together, these data demonstrate that the supramolecular gold (I) -thiol-polypeptide nanocomplex is a stable and viable platform that can be used as a delivery-releasing polypeptide product (cargo) in cells.

Nano-MG facilitating MDMX degradation and subsequent functional restoration of p53 and p73

The ability of the synthesized Nano-MG to promote the degradation of MDMX in cells was first evaluated by immunoblotting (fig. 3A). The retinoblastoma cell line WERI-Rb-1 was selected for the initial assessment of Nano-MG, considering the presence of over-expressed MDMX and p53 and p73 in the wild-type state (wild-type status) in this cell line. Nano-MG down-regulated MDMX in vitro, stabilizing p53 and p73 (FIG. 3A). A downstream target p21 of p53, was also significantly down-regulated in response to Nano-MG (fig. 3A).

Notably, the potency (potency) of the Nano-MG stabilizing p53 and p73 is greater than that of MDMX-p53 inhibitors by the same method ^D PMI synthesized Nano- ^D PMI is more efficient. To further verify the degradation of MDMX at the protein level and to eliminate transcriptional interference, cells were pretreated with the ribosomal inhibitor CHX for 12 h. Treatment of WERI-Rb-1 cells with increasing concentrations of Nano-MG for 48h induced knock-out of MDMX levels, and 100nM was found to cause 95% of the highest MDMX degradation concentrations (FIG. 3B).

In addition, proteasome inhibitors MG132 and E2 ubiquitinase inhibitor PYR41 can inhibit the degradation of MDMX to some extent (figure)3B) It was shown that Nano-MG degrades MDMX in a ubiquitin-dependent manner. In addition, RNA sequencing analysis showed that differential changes in cell transcript levels were triggered after 24h treatment with Nano-MG compared to WERI-Rb-1 cells treated with PBS (FIG. 3C). 776 differentially expressed genes in response to Nano-MG were found, and Gene Set Enrichment Analysis (GSEA) showed consistent and reproducible enrichment of p53 regulated gene expression signatures (FIGS. 3D-F). Furthermore, in Nano-MG treated cells, the most prominent up-regulated pathways involved p73 signaling (fig. 3G), apoptosis (fig. 3H), cell cycle checkpoints (fig. 3I), and cell cycle mitosis (fig. 3J). As a result, the Nano-MG significantly inhibited the proliferation of WERI-Rb-1 in vitro (FIG. 3K), and induced its apoptosis (FIG. 3L) and cell cycle arrest. More importantly, compared with Nano- ^D PMI, Nano-MG, showed enhanced anti-cancer activity (FIGS. 3K and 3L). These results collectively demonstrate that Nano-MG can degrade MDMX and shows activity in vitro targeting the p53 and p73 pathways.

Nano-MG inhibition of retinoblastoma in xenograft mouse models

Retinoblastoma is the most common primary intraocular malignancy in children today, and its appearance can not only cause blindness, but also threaten the younger lives of patients. Thus, it is a suitable and meaningful model for testing for Nano-MG in the eye.

Will be 1 × 10 ⁵ The WERI-Rb-1 cells were implanted under the retina of a single eye of Balb/c nude mice to establish an orthotopic xenograft retinoblastoma mouse model (FIG. 4A). Retinoblastoma-bearing mice were randomly assigned into 3 groups (n-3/group): respectively injecting saline, Nano-MG and Nano- ^D PMI was treated as in fig. 4A. On day 11, it was found that Nano-MG treatment significantly hindered the progression of retinoblastoma compared to control (fig. 4B). Meanwhile, Nano-MG is more than Nano- ^D PMI was more therapeutically effective (fig. 4B). These results are further shown in H&Confirmed in E staining that the tumor area was significantly reduced after treatment with Nano-MG, compared to control and Nano- ^D The PMI treated formed a sharp contrast (fig. 4C).

In addition, these results were further supported by TUNEL immunofluorescence analysis of apoptosis, ki67 Immunohistochemical (IHC) detection of cell proliferation (fig. 4D). A significant decrease in MDMX levels was also found in tumors after Nano-MG treatment (FIG. 4D), suggesting a role in MDMX degradation. The recovery of p53 and p73 further validated the anticancer activity and mechanism of Nano-MG (fig. 4D). And using Nano-MG or Nano- ^D The PMI treatment did not cause significant changes in body weight and visceral pathological morphology, further indicating that the nanoparticles themselves are not acutely toxic. Taken together, these data indicate that Nano-MG is able to safely and effectively inhibit the development of retinoblastoma by degrading MDMX.

The Nano-MG can be excreted from the body after treatment

Removable nanoparticles have recently become a new class of engineered nanomedicines that can be targeted for drug delivery to a target site, while off-targeted nanoparticles and working nanocarriers can be rapidly cleared by renal excretion and/or the mononuclear macrophagy system (MPS). These nanoparticles can show great potential for attenuation of systemic toxicity by avoiding non-specific accumulation in healthy tissues or organs. These nanoparticles have an ultra-small size (< 6nm) and can be efficiently expelled from the body after systemic and/or local administration.

Nano-MG can be broken down into ultrafine nanoparticles of only about 5nm in size in response to intracellular reducing conditions (FIG. 5A), which is well documented in TEM images (FIG. 5B) after 5mM GSH treatment and DLS results. This ultra-micro size can confer permeability to the Nano-MG across the blood-ocular barrier, and following intravitreal injection, the kinetic distribution (disturbed kinetics) of the Nano-MG in mice is monitored by mass spectrometry (mass-spectroscopy), and inductively coupled plasma mass spectrometry (ICP-MS) is used to monitor tissue and organ permeability ¹⁹⁷ Au was measured and quantified. In the eye ¹⁹⁷ The change of Au with time is represented by ID%, the generated metabolic kinetics can exceed 70% after 8h ¹⁹⁷ Au was cleared from the eye and emptied after 2w (fig. 5C). In addition, after 2w injection, it was almost in the heart, liver, spleen, kidney, lung and brainCan not detect any ¹⁹⁷ Au (fig. 5C). Notably, liver and spleen are the major pairs ¹⁹⁷ Au is metabolized (fig. 5C), meaning that Nano-MG is expelled in an MPS-dependent manner. Taken together, these results indicate that Nano-MG is a nanoparticle that can be scavenged.

7. Nano-MG modified by imidazole further increases tumor specific accumulation

To prepare Tumor Microenvironment (TME) responsive nanoclusters, Polyacryloylmercaptoimidazole (PSI) was synthesized to package Nano-MG. For the synthesis of PSI, mercaptoimidazole was first reacted with N-succinimidyl 6-maleimidocaproate to produce N-succinimidyl 3-maleimidomercaptoimidazole (fig. 5D). Next, the activated imidazole was coupled to polyacrylamide (PAA, MW 20000Da) by reaction of the amino group of PAA with carboxyfluorescent diacetoxysuccinimide ester (fig. 5D). The product PSI can be coated on the Nano-MG exterior to yield Nano-MG @ PSI as evidenced by the increased hydrodynamic diameter (fig. 5E) and the decreased ZETA potential at pH7.4 (fig. 5F).

The dissociation constant (pKa) of imidazole is between 6 and 7, so on TME, PSI will be further protonated in pHe. As expected, the ZETA potential of the Nano-MG @ PSI can change from 28mV at pH7.4 to 50mV at pH 6.0. Nanoparticles with a strong positive charge can electrostatically attract a negative charge to the cancer cell membrane and subsequently initiate internalization of the cell. Nano-MG @ PSI enhanced internalization of cancer cells at pH 6.5 compared to physiological pH (FIG. 5G). This pH-responsive cellular internalization will confer Nano-MG @ PSI with the ability to enhance tumor accumulation.

For validation, organ and intratumor content were detected and quantified by ICP-MS ¹⁹⁷ And Au. The biology of the two nanoparticles is expressed as percent injected dose per gram of tissue or tumor (ID%/g), respectively, as shown in fig. 5H. Nano-MG @ PSI is expected to increase tumor accumulation 6h after injection. By further analysing these ¹⁹⁷ Au distribution, finding ratios of Nano-MG @ PSI in all tumors and normal organs were superior to Nano-MG (fig. 5I), indicating that imidazole modification further increased the tumor-specific accumulation of Nano-MG.

Nano-MG @ PSI induces the reversal of pancreatic cancer in PDX model (regression)

For decades, the mouse model of single cell (monocellula) tumor xenograft has been a standard tool for oncology research. However, more and more studies have demonstrated that single cell tumors and their actual tumors (actual tumors) differ greatly in major histological, genetic and microenvironmental features. Therefore, the PDX model will be directly xenografted from primary or metastatic tumors in patients to severely combined immunodeficient mice, with increasing popularity in therapeutic screening, biomarker discovery, and especially preclinical evaluation of drugs over the last decade.

To further investigate the therapeutic efficacy of Nano-MG @ PSI, the Nano-MG @ PSI was combined with Nano- ^D A comparative study was performed on the effects of PMI @ PSI on tumor growth, tumor weight, tumor apoptosis and MDMX, p53, p21 levels (fig. 6A).

When the tumor volume reaches 100 +/-25 mm ³ In time, rats were randomly divided into 3 groups: PBS (control), Nano-MG and Nano- ^D PMI. After 10 days of treatment, the tumor volume of the control mice increased more than 13 times, and 1.5MG/kg polypeptide equivalent (equivalent dose) of Nano-MG @ PSI and Nano- ^D PMI @ PSI inhibited tumor growth by 85.7% and 59.7%, respectively (fig. 6B). The isolated PDX tumor mass (fig. 6C) and morphology (image) (fig. 6D) support the superior anti-cancer activity of Nano-MG @ PSI. In addition, deoxyribonucleotide end-transferase mediated nick end labeling (TUNEL) analysis of residual tumors from different treatment groups showed that the DNA fragment was associated with PDS or Nano- ^D PMI @ PSI treatment significantly increased apoptosis compared to Nano-MG @ PSI treatment (FIG. 6E).

Immunohistochemical staining of tumor tissue sections showed significant down-regulation of MDMX (fig. 6F), p53 (fig. 6G) and p73 (fig. 6H) in Nano-MG @ PSI treated tumor tissues. In addition, Nano-MG @ PSI and Nano- ^D PMI @ PSI treated PDX mice had no statistical differences in body weight, blood biochemical indices (fig. 6I), visceral case sections (fig. 6J), indicating their safety. Taken together, these indicate that Nano-MG @ PSI enhances therapeutic efficacy in KRAS mutated pancreatic cancer by degrading MDMX and subsequently restoring p53, p73, while also maintaining highly beneficial biological safety.

Despite the significant advances in small molecule-derived PROTAC technology, polypeptide-derived degradants (degraders) have attracted considerable attention in the pharmaceutical arts because they expand the range of ligands for E3 ligase and target proteins with which targeted protein degradation can be accomplished. However, polypeptide preparations (therapeutics) are always compromised by their lack of cell permeability, resistance to proteolysis (proteolytic resistance), and accumulation at the site of interest (accumulation in the site of interest).

The invention expands the PROTAC technology, develops a polypeptide degrading agent targeting MDMX, and Nano-engineer the polypeptide degrading agent into a gold (I) -thiol-polypeptide Nano-complex named Nano-MG with potential drug candidate capability for malignant tumors over-expressing MDMX.

The invention concludes that: Nano-MG specifically induces MDMX ubiquitin-dependent degradation and subsequently restores p53 and p73 anti-cancer function. Nano-MG has high anti-retinoblastoma activity in vitro and, in vivo, exhibits highly advantageous removability by rapid expulsion from the organism following intravitreal injection. Furthermore, imidazole-modified Nano-MG showed tumor-specific accumulation after systemic injection and was able to effectively arrest tumor progression in PDX model of malignant pancreatic cancer with Kras G12D mutation. Milk exosome coating (coating) enabled oral dosing of Nano-MG and subsequently enabled significant inhibition of tumor growth in PDOX models of colon cancer. These results indicate that Nano-MG has the potential to be a candidate drug for targeting MDMX. Notably, the prior art has succeeded in targeting MDM2, a small molecule-based PROTACs that also has the ability to inhibit the p53, p73 homologous proteins of MDMX; polypeptide-based PROTACs that target MDMX or MDM2 display only limited cellular and biological functions. Therefore, the work fills the blank of MDMX degradation, and extends the PROTAC technology to the polypeptide field.

The invention is an application of successful combination of polypeptide and nanotechnology in constructing PROTAC molecules, and clearly shows the potential of polypeptide-derived nanometer degradation agents in targeting various PPIs.

In conclusion, the present invention not only verifies that MDMX degradation is a promising clinical strategy for anticancer therapy, but more importantly, provides a feasible approach to convert polypeptide-derived PROTACs into a potential drug candidate and is likely to stress the success of this class of drugs against a variety of disease "non-druggable" targets.

It is noted that Nano-MG has high loading efficiency because the loaded MG itself is one of the components. Nano-MG exhibits high activity against retinoblastoma in vitro and in vivo by degrading MDMX and subsequently restoring p53 and p73, while also exhibiting highly advantageous cleanability properties for rapid excretion in vivo. Furthermore, Nano-MG @ PSI can effectively inhibit tumor progression in a pancreatic cancer human tumor xenograft (PDX) model containing the Kras G12D mutation due to its charge reversal (charge reversal) properties in response to the Tumor Microenvironment (TME). The oral formulation Nano-MG @ M coated with milk exosomes was significantly effective in targeting and degrading MDMX in the human orthotopic transplantation tumor model (PDOX) of colon cancer. Thus, the present invention not only verifies that degrading MDMX is a promising clinical strategy for anti-tumor therapy, but more importantly, provides a viable means of converting polypeptide-derived PROTACs into potential drug candidates and potentially reassumes their discovery efforts in a variety of diseases.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims (4)

1. A polypeptide nano-composite is characterized in that the polypeptide nano-composite is represented by a general formula of [ peptide-S-Au ]] _m M is a positive integer, wherein peptide is (ALA-Hyp-Y-Hle-PM- ^D R ^D C)-PEG ₃ -MDMX；

Wherein the sequence of MDMX is ^D E ^D F ^D W ^D Y ^D V ^D E ^D F ^D E ^D K ^D L ^D L ^D R。

2. The polypeptide nanocomposite as claimed in claim 1, wherein the ALA-Hyp-Y-Hle-PM- ^D R ^D C is a fragment of the Von Hippel Lindau factor.

3. A method for preparing the polypeptide nanocomposite as claimed in claim 1 or 2, comprising the steps of:

step 1: peptide is obtained by solid phase peptide synthesis according to FMOC chemical method, wherein the peptide is (ALA-Hyp-Y-Hle-PM- ^D R ^D C)-PEG ₃ -MDMX；

Step 2: mixing peptide and NH ₂ -PEG _n -SH is mixed to HAuCl after magnetic stirring ₄ Obtaining the polypeptide nano-composite in the solution.

4. The use of a polypeptide nanocomplex according to any of claims 1 to 3, wherein said polypeptide nanocomplex is used for the preparation of a medicament for inhibiting the growth of a tumor.

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