Lysosome-targeting chimeric nanoparticles based on low-density lipoprotein receptor, their preparation method and application

Abstract

This invention relates to the field of biomaterials technology, specifically to lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors, their preparation method, and their applications. The nanoparticles of this invention can effectively degrade PD-L1 in lysosomes via LDLR-mediated endocytosis to regulate the immunosuppressive checkpoint signaling pathway. Simultaneously, they possess excellent optical properties; under irradiation with 660 nm wavelength light, PpIX generates singlet oxygen. 1 O2 activates photoimmunotherapy for tumors, promotes dendritic cell (DC) maturation, and induces immunogenic cell death in tumor cells. The nanoparticles of this invention effectively enhance anti-tumor immune responses by inducing immunogenic cell death in tumor cells through photoimmunotherapy and by degrading PD-L1 and reprogramming the immunosuppressive checkpoint PD-1 / PD-L1 signaling pathway.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors, their preparation methods, and their applications. Background Technology

[0002] Immunity is an important physiological function acquired during biological evolution, used to defend against the invasion of pathogenic microorganisms. The immune system has multiple functions, including eliminating senescent cells, maintaining homeostasis, monitoring cell mutations, and preventing tumor development through the action of immune cells and molecules.

[0003] Cancer immunotherapy is an emerging therapeutic strategy aimed at combating malignant tumors by activating the body's innate immune system, particularly cytotoxic T lymphocytes. Among various immunotherapy strategies, immune checkpoint blockade (ICB) has achieved significant success. Clinically, this is typically achieved using immune checkpoint inhibitors (antibodies or small molecule inhibitors), which block these immune checkpoints by occupying binding pockets or active sites. However, these small molecule- or antibody-based checkpoint inhibitors often suffer from low tumor targeting efficiency, insufficient biocompatibility, and a high incidence of immune-related adverse events (irAEs).

[0004] Lysosome-targeted chimeric (LYTACs) technology is a targeted extracellular protein degradation technology. LYTACs degrade cell membrane or extracellular proteins via the lysosomal pathway, overcoming the limitation of proteasome-targeted chimeric (PROTACs) technology, which can only process intracellular proteins. Compared to immune checkpoint inhibitors, the strategy of using LYTACs to degrade immune checkpoint proteins and thus block related pathways has advantages such as higher tumor targeting efficiency, lower toxicity, and persistence. However, LYTACs rely on lysosomal transport receptors (such as CI-M6PR or ASGPR) to mediate endocytosis. If the tumor cell receptor expression level is low, the degradation efficiency decreases significantly.

[0005] High circulating cholesterol levels are considered a high-risk factor for increased cancer incidence and recurrence rates. Low-density lipoprotein (LDL) is the primary carrier of cholesterol, entering cells via endocytosis mediated by the LDL receptor (LDLR) and further transported to lysosomes. Studies have shown that LDLR expression and LDL uptake are increased in various cancer cell lines, including breast cancer, lung cancer, pancreatic cancer, leukemia, prostate cancer, colon cancer, and liver cancer. LDLR is overexpressed in multiple cancer cell lines, making it an ideal target for targeted cancer therapy.

[0006] Therefore, utilizing LDLR as a lysosomal targeting receptor to prepare novel LYTACs for the efficient degradation of cancer cell membrane proteins, thereby improving the therapeutic efficiency of drugs and reducing toxic side effects, has important clinical application prospects. Summary of the Invention

[0007] This invention provides lysosomal targeting chimeric nanoparticles based on low-density lipoprotein receptors, their preparation method, and their applications, in order to solve the problems of insufficient tumor-specific targeting and uncontrollable degradation efficiency of existing LYTAC nanoparticles.

[0008] According to one aspect of the present invention, lysosome-targeting chimeric nanoparticles based on low-density lipoprotein receptors are provided. The lysosome-targeting chimeric nanoparticles are co-assembled from an amphiphilic chimeric peptide targeting low-density lipoprotein and an amphiphilic chimeric peptide of a programmed death receptor ligand. Each of the two amphiphilic chimeric peptides has a hydrophilic polypeptide body and a hydrophobic drug body. The hydrophilic polypeptide body comprises a receptor-targeting polypeptide sequence, a polypeptide linker sequence, and an enzyme-responsive polypeptide sequence. The hydrophobic drug body is a photosensitizer. The receptor-targeting polypeptide sequence and the hydrophobic drug body are linked by the polypeptide linker sequence and the enzyme-responsive polypeptide sequence.

[0009] Furthermore, the two target receptor polypeptide sequences are polypeptides targeting LDLR and PD-L1, respectively.

[0010] Furthermore, the two target receptor polypeptide sequences are RLTRKRGLK and CVRARTR, respectively.

[0011] Furthermore, the polypeptide linker sequence is GSGS.

[0012] Furthermore, the enzyme-responsive polypeptide is a polypeptide that can be cleaved by cathepsin B.

[0013] Furthermore, the enzyme-responsive polypeptide is FK.

[0014] Furthermore, the photosensitizer is protoporphyrin.

[0015] Furthermore, the amphiphilic chimeric peptides are PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR, respectively.

[0016] Furthermore, the lysosome-targeting chimeric nanoparticles have a particle size of 120-150 nm.

[0017] The present invention also provides a method for preparing the lysosomal targeting chimeric nanoparticles based on low-density lipoprotein receptors as described above, characterized by comprising the following steps:

[0018] Step 1: Using amino acids and photosensitizers as raw materials, amphiphilic chimeric peptides PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR were synthesized by solid-phase synthesis.

[0019] Step 2: Dissolve the two amphiphilic chimeric peptides separately in dimethyl sulfoxide to prepare 5mM CP. LDLR and CP PD-L1 Stock solution;

[0020] Step 3, mix the two stock solutions with (CP) LDLR :CP PD-L1 The mixture was prepared by mixing the ingredients in a ratio of 5:1 (5:1) until homogeneous.

[0021] Step 4: Under ultrasonic conditions, the mixture obtained in step 3 is slowly dripped into ultrapure water to form nanoparticles through co-assembly.

[0022] The present invention also provides the application of the lysosome-targeting chimeric nanoparticles based on low-density lipoprotein receptor as described above in the preparation of antitumor drugs.

[0023] The lysosome-targeting chimeric nanoparticles based on low-density lipoprotein receptors of the present invention have tumor-specific targeting properties and are a highly efficient and safe nano-LYTAC. The polypeptide co-assembled nanoparticle materials are non-toxic, have good biocompatibility, and have a wide range of applications.

[0024] The lysosomal-targeting chimeric nanoparticles based on the low-density lipoprotein receptor (LDLR) of this invention are nanoparticles co-assembled from amphiphilic chimeric peptides, primarily prepared via a solid-phase polypeptide synthesis strategy, without involving the introduction of non-natural amino acids or complex synthesis. The LYTAC nanoparticles with dual-targeting function provided by this invention can effectively utilize the endocytosis of LDLR to degrade PD-L1 via the lysosomal pathway, blocking the immunosuppressive checkpoint PD-1 / PD-L1 signaling pathway and effectively enhancing the anti-tumor immune response. Simultaneously, in combination with PpIX-mediated photodynamic therapy, it induces immunogenic cell death in tumor cells through photoimmunotherapy, further enhancing the anti-tumor immune response. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0026] Figure 1a This is a structural diagram of the amphiphilic chimeric peptide provided in Example 1 of the present invention;

[0027] Figure 1b This is a structural diagram of the amphiphilic chimeric peptide provided in Example 2 of the present invention;

[0028] Figure 2a The mass spectrum of the amphiphilic chimeric peptide provided in Example 1 of this invention;

[0029] Figure 2b The mass spectrum of the amphiphilic chimeric peptide provided in Example 2 of this invention;

[0030] Figure 3 This is a particle size distribution diagram of the nanoparticles provided in Example 3 of the present invention;

[0031] Figure 4 This is a TEM image of the nanoparticles provided in Example 3 of the present invention;

[0032] Figure 5 The ultraviolet-visible absorption spectrum of the nanoparticles provided in Example 3 of this invention;

[0033] Figure 6 The fluorescence spectrum of the nanoparticles provided in Example 3 of this invention;

[0034] Figure 7 The high-performance liquid chromatogram of nanoparticles provided in Example 3 of the present invention;

[0035] Figure 8 This is a quantitative diagram of reactive oxygen species generation in nanoparticles provided in Example 3 of the present invention;

[0036] Figure 9 This is a quantitative diagram of cellular uptake of nanoparticles provided in Example 3 of the present invention;

[0037] Figure 10 This is a quantitative PD-L1 degradation diagram of the nanoparticles provided in Example 3 of the present invention;

[0038] Figure 11 This is a quantitative diagram of intracellular reactive oxygen species generation of nanoparticles provided in Example 3 of the present invention;

[0039] Figure 12 This is a cell activity test diagram of the nanoparticles provided in Example 3 of the present invention;

[0040] Figure 13 This is a quantitative map of HMGB1 in cells of nanoparticles provided in Example 3 of the present invention;

[0041] Figure 14 This is a flow cytometry analysis diagram showing the effect of co-culturing nanoparticles with 4T1 cells on the maturation of dendritic cells, as provided in Example 3 of the present invention. Detailed Implementation

[0042] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] It should be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of a descriptive feature, integral, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or sets.

[0044] To keep the drawings concise, only the parts relevant to the invention are shown schematically in each figure, and they do not represent the actual structure of the product. Furthermore, for ease of understanding, in some figures, only one of components with the same structure or function is shown schematically, or only one is labeled. In this document, "one" can mean not only "only one" but also "more than one".

[0045] It should also be further understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0046] In the embodiments shown in the accompanying drawings, the directional indications (such as up, down, left, right, front, and back) used to explain the structure and movement of the various components of the invention are relative rather than absolute. These descriptions are appropriate when these components are in the positions shown in the drawings. If the descriptions of the positions of these components change, these directional indications also change accordingly.

[0047] Furthermore, in the description of this application, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without any creative effort.

[0049] Please refer to Figure 1, and in conjunction with Figure 2 and Figure 3As shown, the present invention discloses a lysosomal-targeting chimeric nanoparticle based on a low-density lipoprotein receptor, which is co-assembled from an amphiphilic chimeric peptide targeting low-density lipoprotein and an amphiphilic chimeric peptide of a programmed death receptor ligand. Each of the two amphiphilic chimeric peptides has a hydrophilic polypeptide body and a hydrophobic drug body. The hydrophilic polypeptide body contains a receptor-targeting polypeptide sequence, a polypeptide linker sequence, and an enzyme-responsive polypeptide sequence, and the hydrophobic drug body is a photosensitizer. The receptor-targeting polypeptide sequence and the hydrophobic drug body are linked by the polypeptide linker sequence and the enzyme-responsive polypeptide sequence.

[0050] Among them, the two target receptor polypeptide sequences are polypeptides targeting LDLR and PD-L1, respectively. Furthermore, the two target receptor polypeptide sequences are RLTRKRGLK and CVRARTR, respectively. The two polypeptides achieve efficient targeting and treatment of tumor sites through specific recognition of LDLR and PD-L1.

[0051] The polypeptide linker sequence is GSGS, and the enzyme-responsive polypeptide is a polypeptide that can be cleaved by cathepsin B.

[0052] Among them, the enzyme-responsive peptide is FK. The ε-amino group of lysine (K) is easily recognized and cleaved by lysosomal cathepsins, thus achieving the separation of the peptide from the photosensitizer.

[0053] The photosensitizer is protoporphyrin (PpIX), which acts as an activating group in photodynamic therapy (PDT) and generates reactive oxygen species (ROS) under specific wavelengths of light. This induces immunogenic death in tumor cells and synergistically enhances the anti-tumor effect.

[0054] The amphiphilic chimeric peptides of the present invention are PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR, respectively.

[0055] Preferably, the lysosome-targeting chimeric nanoparticles have a particle size of 120-150 nm.

[0056] The lysosome-targeting chimeric nanoparticles of this invention, possessing dual-targeting functions, are applied to tumor immune checkpoint blockade combined with photodynamic therapy. The lysosome-targeting chimeric nanoparticles degrade PD-L1 via lysosomes, thereby blocking the PD-1 / PD-L1 tumor immunosuppressive pathway and activating the anti-tumor immune response mediated by effector T cells (Teffs). After entering the lysosome, the LYTAC nanoparticles are cleaved by CatB, which is highly expressed in the lysosome, releasing PpIX. Under irradiation with 660 nm wavelength light, PpIX generates singlet oxygen. 1 O2 activates tumor photoimmunotherapy, promotes DC maturation, and induces immunogenic cell death in tumor cells.

[0057] The nanoparticles of this invention have tumor-specific targeting properties and are a highly efficient and safe nano-LYTAC. The polypeptide co-assembled nanoparticle material is non-toxic and has good biocompatibility.

[0058] This invention also provides a method for preparing lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors, comprising the following steps:

[0059] Step 1: Using amino acids and photosensitizers as raw materials, amphiphilic chimeric peptides PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR were synthesized by solid-phase synthesis.

[0060] Step 2: Dissolve the two amphiphilic chimeric peptides separately in dimethyl sulfoxide to prepare 5mM CP. LDLR and CP PD-L1 Stock solution;

[0061] Step 3, mix the two stock solutions according to (CP) LDLR :CP PD-L1 The mixture was prepared by mixing the ingredients in a ratio of 5:1 (5:1) until homogeneous.

[0062] Step 4: Under ultrasonic conditions, the mixture obtained in step 3 is slowly dripped into ultrapure water to form nanoparticles through co-assembly.

[0063] To further verify the technical effects of the present invention, specific embodiments are described in detail below:

[0064] The raw materials in the following examples were sourced from the following sources: 2-Chlorotrityl Chloride Resin resin (resin substitution degree 1.365 mmol / g), phenylalanine with an amino group protected by N-fluorene-9-methoxycarbonyl (Fmoc-Phe-OH), lysine with an amino group and a carboxyl group on its side group protected by N-fluorene-9-methoxycarbonyl and benzyloxycarbonyl, respectively (Fmoc-Lys(Boc)-OH), glycine with an amino group protected by N-fluorene-9-methoxycarbonyl (Fmoc-Gly-OH), serine with an amino group and a carboxyl group on its side group protected by N-fluorene-9-methoxycarbonyl and tert-butyl, respectively (Fmoc-Ser(tBu)-OH), cysteine ​​with an amino group and a carboxyl group on its side group protected by N-fluorene-9-methoxycarbonyl and triphenylmethyl, respectively (Fmoc-Cys(Trt)-OH), and valine with an amino group protected by N-fluorene-9-methoxycarbonyl. Arginine (Fmoc-Val-OH), arginine (Fmoc-Arg(Pbf)-OH) with its amino group and side group carboxyl group protected by N-fluorene-9-methoxycarbonyl and 2,2,4,6,7-pentamethyldihydrobenzofuran-3-sulfonyl groups, alanine (Fmoc-Ala-OH) with its amino group protected by N-fluorene-9-methoxycarbonyl, threonine (Fmoc-Thr(tBu)-OH) with its amino group and side group carboxyl group protected by N-fluorene-9-methoxycarbonyl and tert-butyl groups, leucine (Fmoc-Leu-OH) with its amino group protected by N-fluorene-9-methoxycarbonyl, benzotriazole-N,N,N',N'-tetramethylureafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) were all purchased from Shanghai Jier Biochemical Co., Ltd.

[0065] Among them, protoporphyrin (PpIX) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0066] Ninhydrin, trifluoroacetic acid (TFA), and N,N-diisopropylethylamine (DIEA) were all purchased from Aladdin.

[0067] Dichloromethane (DCM), triisopropylsilane (TIS), N,N-dimethylformamide (DMF), methanol (MeOH), piperidine, and diethyl ether were all purchased from Sinopharm Group.

[0068] Example 1

[0069] This embodiment provides an amphiphilic chimeric peptide CP. PD-L1 Its structural formula is: PpIX-FK-GSGS-CVRARTR-OH, and the specific synthesis steps are as follows:

[0070] Step 1: Weigh a certain amount of resin and place it in the polypeptide solid-phase synthesis column. Wash it three times with DMF, drain the solvent, and then swell it with an appropriate amount of DMF for 1 hour before draining the solvent.

[0071] Step 2: Add 4 molar equivalents of Fmoc-Arg(Pbf)-OH and an appropriate amount of 10 molar equivalents of DIEA DMF solution to the synthesis column, and stir slowly for 3 hours.

[0072] Step 3: Take a small amount of resin from Step 2 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0073] Step 4: Remove the Fmoc protecting group from Fmoc-Arg(Pbf)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0074] Step 5: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Thr(tBu)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0075] Step 6: Take a small amount of resin from Step 5 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0076] Step 7: Remove the Fmoc protecting group from Fmoc-Thr(tBu)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0077] Step 8: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Arg(Pbf)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0078] Step 9: Take a small amount of resin from Step 8 and place it in a methanol solution of ninhydrin (0.01 mg / mL). Heat to boiling for 3-5 minutes and test the color. If no color change occurs, it indicates that the amino acid coupling is successful.

[0079] Step 10: Remove the Fmoc protecting group from Fmoc-Arg(Pbf)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0080] Step 11) Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Ala-OH, 3.6 molar equivalents of HBTU, HOBT and 7.5 molar equivalents of DIEA to the synthesis column and stir slowly for 2 hours.

[0081] Step 12) Take a small amount of resin from step 11) and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0082] Step 13: Remove the Fmoc protecting group from Fmoc-Ala-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0083] Step 14: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Arg(Pbf)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0084] Step 15: Take a small amount of resin from Step 14 and place it in a methanol solution of ninhydrin (0.01 mg / mL). Heat to boiling for 3-5 minutes and test the color. If no color change occurs, it indicates that the amino acid coupling is successful.

[0085] Step 16: Remove the Fmoc protecting group from Fmoc-Arg(Pbf)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0086] Step 17: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Val-OH, 3.6 molar equivalents of HBTU, HOBT and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0087] Step 18: Take a small amount of resin from Step 17 and place it in a methanol solution of ninhydrin (0.01 mg / mL). Heat to boiling for 3-5 minutes and test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0088] Step 19: Remove the Fmoc protecting group from Fmoc-Val-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0089] Step 20: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Cys(Trt)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0090] Step 21: Take a small amount of resin from step 20 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0091] Step 22: Remove the Fmoc protecting group from Fmoc-Cys(Trt)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0092] Step 23: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Ser(tBu)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0093] Step 24: Take a small amount of resin from step 23 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0094] Step 24: Remove the Fmoc protecting group from Fmoc-Ser(tBu)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0095] Step 25: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Gly-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0096] Step 26: Take a small amount of resin from step 25 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0097] Step 27: Remove the Fmoc protecting group from Fmoc-Gly-OH. Add a 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0098] Step 28: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Ser(tBu)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0099] Step 29: Take a small amount of resin from step 28 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0100] Step 30: Remove the Fmoc protecting group from Fmoc-Ser(tBu)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0101] Step 31: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Gly-OH, 3.6 molar equivalents of HBTU, HOBT and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0102] Step 32: Take a small amount of resin from step 31 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0103] Step 33: Remove the Fmoc protecting group from Fmoc-Gly-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0104] Step 34: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Lys(Boc)-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0105] Step 35: Take a small amount of resin from step 34 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0106] Step 36: Remove the Fmoc protecting group from Fmoc-Lys(Boc)-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0107] Step 37: Add an appropriate amount of DMF solution containing 3 molar equivalents of Fmoc-Phe-OH, 3.6 molar equivalents of HBTU, HOBT, and 7.5 molar equivalents of DIEA to the synthesis column, and stir slowly for 2 hours.

[0108] Step 38: Take a small amount of resin from step 37 and put it into a methanol solution of ninhydrin (0.01 mg / mL). Heat and boil for 3-5 minutes to test the color. If there is no color change, it indicates that the amino acid coupling is successful.

[0109] Step 39: Remove the Fmoc protecting group from Fmoc-Phe-OH. Add 30% Piperidine / DMF (V / V) deprotection solution to the solid-phase synthesis column, stir for 20 min, wash three times with DMF, and drain the solvent.

[0110] Step 40: Add an appropriate amount of DMF solution containing 3 molar equivalents of PpIX, 3.6 molar equivalents of HBTU, HOBT, and 9 molar equivalents of DIEA to the synthesis column, and stir slowly for 24 hours.

[0111] Step 41: Wash the resin with DMF, MeOH and DCM respectively, and dry it under vacuum at room temperature. The dried resin is ready for use.

[0112] Step 42, cleavage of peptides. A certain volume of a mixed liquid of TFA / H2O / TIS (V / V / V=95% / 2.5% / 2.5%) was added to the dried resin synthesis column obtained in Step 41. The mixture was stirred at room temperature for 2 hours. The cleaved liquid was collected and concentrated by rotary evaporation to a viscous liquid. Then, it was added dropwise to cold diethyl ether to precipitate the peptide. After centrifugation, the supernatant was removed. The peptide powder was then dried under vacuum at room temperature for 12 hours and stored at low temperature for later use.

[0113] For this embodiment CP PD-L1 The polypeptide sequence was detected. Figure 1a This is a structural diagram of the amphiphilic chimeric peptide obtained in this embodiment; Figure 2a This is the mass spectrometry of the amphiphilic chimeric peptide obtained in this embodiment. The results show that the actual molecular weight of the chimeric peptide is consistent with the theoretical molecular weight, CP PD-L1 The structure of the polypeptide is PpIX-FK-GSGS-CVRARTR-OH.

[0114] Example 2

[0115] This embodiment provides a novel amphiphilic chimeric peptide CP. LDLRIts structural formula is: PpIX-FK-GSGS-RLTRKRGLK-OH. The preparation method of this embodiment is the same as that of Example 1, except that the amino acids used are different. The amino acids used in this embodiment are Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, and Fmoc-Thr(tBu)-OH.

[0116] Figure 1b This is a structural diagram of the amphiphilic chimeric peptide obtained in this embodiment; Figure 2b This is the mass spectrometry of the amphiphilic chimeric peptide obtained in this embodiment. The results show that the actual molecular weight of the chimeric peptide is consistent with the theoretical molecular weight, CP LDLR The structure of the polypeptide is PpIX-FK-GSGS-RLTRKRGLK-OH.

[0117] Example 3

[0118] This embodiment provides a lysosomal-targeting chimeric nanoparticle based on a low-density lipoprotein receptor for immunotherapy of mouse 4T1 breast cancer cells. The nanoparticle preparation method includes the following steps:

[0119] (1) Using amino acids and photosensitizers as raw materials, an amphiphilic chimeric peptide CP was synthesized by solid-phase synthesis. LDLR and CP PD-L1 .

[0120] (2) The two amphiphilic chimeric peptides were dissolved in dimethyl sulfoxide to prepare 5 mM CP solutions. LDLR and CP PD-L1 Stock solution;

[0121] (3) Mix the two stock solutions with (CP) LDLR :CP PD-L1 The mixture was prepared by mixing the ingredients in a ratio of 5:1 (5:1) until homogeneous.

[0122] (4) Under ultrasonic conditions, the mixture obtained in step (2) is slowly dripped into ultrapure water to form nanoparticles through co-assembly, named N. LP。

[0123] (5) Using the same method, prepare only CP-containing LDLR Nanoparticles (N L ) and only containing CP PD-L1 Nanoparticles (N P () as a control material.

[0124] To examine the physical characteristics of the nanoparticles, experiments were conducted on the three embodiments described above. All statistical calculations were performed using OriginPro 2026 software, and all data are presented as mean ± standard deviation. Statistical analysis of the experimental data employed one-way ANOVA supplemented by Tukey's test, or a two-tailed Student's t-test. For all statistical tests, a p-value less than 0.05 was considered statistically significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0125] First, the prepared N L N P N LP Through dynamic light scattering (DLS) analysis, N L N P N LP The hydrated particle sizes were 120 nm, 140 nm, and 140 nm, respectively. The experimental results are as follows: Figure 3 As shown, the nanoparticles exhibit good dispersibility. Furthermore, the morphology of the nanoparticles was characterized by TEM. Figure 4 It can be seen that nanoparticles N L N P N LP The particle sizes were 120 nm, 165 nm, and 121 nm, respectively, indicating small particle size and uniform distribution. These results suggest that CP... LDLR and CP PD-L1 It exhibits excellent co-assembly properties, enabling it to co-assemble into nanoparticles with similar morphology and uniform size through hydrophilic-hydrophobic interactions.

[0126] To examine the optical properties of the nanoparticles, a UV-Vis spectrophotometer and fluorescence spectroscopy were used to detect them. The results are as follows: Figure 5 As shown. Figure 5 It can be seen from N L N P N LP The UV-Vis absorption spectrum exhibits characteristic absorption peaks at 505, 540, 575, and 625 nm. Figure 6 Indicates N L N P N LP The fluorescence emission spectrum further revealed characteristic emission peaks at 625 nm and 680 nm, which were identical to the characteristic absorption peaks of porphyrin molecules. The results indicate that N L N P N LP Nanoparticles all possess excellent optical properties.

[0127] To examine the specific activation effect of cancer biomarkers (CatB) on nanoparticles, in N L or N P After incubation with CatB, high-performance liquid chromatography (HPLC) analysis was performed. Please refer to the attached image for the results. Figure 7 As shown. N L and N P Elution peaks appeared at 14.5 min and 16.0 min, respectively, corresponding to CP. LDLR and CP PD-L1 Chimeric peptides. Conversely, N L and N P No such peaks were detected after incubation with CatB, further confirming that CatB is effective against N. L and N P The specific activation effect of nanoparticles. Furthermore, transmission electron microscopy images and dynamic light scattering results both confirm the dissociation of nanoparticles (…). Figure 3 , Figure 4 The above results indicate that N L N P N LP It can specifically dissociate in the presence of CatB, which is attributed to its CatB-responsive FK sequence.

[0128] To test PpIX, N L N P N LP The photodynamic activity of singlet oxygen was investigated. The singlet oxygen fluorescent probe 1,3-diphenylisobenzofuran (DPBF) was used to detect singlet oxygen. 1 O2 generation. Under 660nm light irradiation, the power intensity is 30mW / cm². 2 At 2°C, the UV-Vis absorption intensity of DPBF at 410 nm gradually decreases over time. For example... Figure 8 As shown, after 5 minutes of 660nm light irradiation, N L N P N LP The UV-Vis absorption intensity ratio decreased to 5% of the initial intensity. The results indicate that N L N P N LP Nanoparticles exhibit good ability to generate singlet oxygen in aqueous solutions. 1 The ability of O2.

[0129] To test the cellular uptake capacity and PD-L1 degradation efficiency of nanoparticles, nanoparticles with different proportions were constructed. LP (CP LDLR :CP PD-L1 Co-culture with cells using ratios of 0:1, 1:10, 1:5, 1:1, 5:1, 10:1, 1:0. N was studied using confocal fluorescence imaging.LP Nanoparticle cellular uptake capacity. For example... Figure 9 As shown, based on the average fluorescence intensity analysis of the nanoparticles, CP was found in the nanoparticles. LDLR The higher the proportion, the stronger the cellular uptake capacity, and the better the CP. LDLR :CP PD-L1 At a ratio of 5:1, the endocytosis efficiency reached 84.7% of that at 1:0. Next, the mean immunofluorescence intensity of PD-L1 was analyzed; the results can be found in [link to analysis]. Figure 10 As shown, that is, CP LDLR :CP PD-L1 At a 5:1 ratio, the green fluorescence of PD-L1 protein in 4T1 cells decreased the most compared to the control group, with a PD-L1 degradation efficiency of 69.6%. Based on the combined analysis of cellular uptake capacity and degradation efficiency, the preparation of N... PL The ratio of nanoparticles is 5:1 (CP) LDLR :CP PD-L1 The results showed that the nanoparticles had excellent cellular uptake efficiency and high PD-L1 degradation capacity.

[0130] To examine the generation of intracellular reactive oxygen species (ROS) after co-culturing with nanoparticles, the generation of intracellular ROS was detected using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) ROS fluorescent probe. After being taken up by 4T1 cells, H2DCFDA was hydrolyzed by intracellular esterases and oxidized by ROS to the fluorescent 2′,7′-dichlorofluorescein (DCF). 4T1 cells were then co-cultured with nanoparticles... L N P N LP The cells were incubated for a total of 12 hours. Afterward, they were incubated with the H2DCFDA probe for 30 minutes. Following this, the cells were either exposed to 660 nm light (30 mW / cm²) or not. 2 (6 min). Fluorescence images of the cells were captured using a confocal laser scanning microscope. Based on the confocal images and quantitative analysis, compared to unirradiated cells, N... L N P N LP The average fluorescence intensity of DCF in incubated cells increased by approximately 5-fold. Figure 11 The results show that: N L N P N LP It exhibits excellent ROS generation capability under 660nm light irradiation.

[0131] To test the in vitro antitumor activity of the nanoparticles, 4T1 cells were inoculated with 0.1, 0.5, 1, 2.5, 5, 10, and 20 μM N2, respectively. L N P N LPAfter 12 hours of incubation, cells that had not undergone any treatment served as the control group, with cells receiving either no 660nm light irradiation (30mW / cm²). 2 After treatment (6 min), continue culturing for 12 h, and detect using an MTS kit. Figure 12 As shown, N L N P N LP All three drugs exhibited varying degrees of concentration-dependent cytotoxic effects in 4T1 cells, with N showing the highest concentration-dependent cytotoxicity in the absence of 660nm light irradiation. L N P N LP The cell viability of the incubated 4T1 cells was all above 80%, indicating negligible cytotoxicity, suggesting that the nanoparticles themselves possess low cytotoxicity. Under 660 nm light irradiation, N... L N P N LP The cytotoxicity of incubated cells increased in a concentration-dependent manner. At a concentration of 20 μM, after incubation with N... LP Cell viability decreased to approximately 30% after incubation and light irradiation. The results indicate that under 660 nm light irradiation, N... LP It has excellent photodynamic therapy capabilities.

[0132] To examine the ability of nanoparticles to induce immunogenic cell death (ICD) and dendritic cell (DC) maturation in 4T1 cells, HMGB1 expression was detected using immunofluorescence imaging. High-mobility group box 1 (HMGB1) is an important biomarker for assessing ICD. Quantitative analysis using confocal imaging showed that, compared to unirradiated cells, HMGB1 expression was significantly higher. L N P N LP The mean fluorescence intensity of anti-HMGB1 antibody decreased by 61.7%, 64.3%, and 66.9% in cells incubated and exposed to light, respectively, indicating that HMGB1 was effectively released from the cell nucleus into the cell supernatant. Figure 13 Next, the maturation status of dendritic cells (DCs) was assessed by flow cytometry. Bone marrow-derived dendritic cells (BMDCs) were co-incubated with supernatants of 4T1 cells after different treatments. After 12 hours of incubation, they were then compared with N... L N P N LP After being treated and co-incubated with the supernatant of 4T1 cells exposed to light, mature DCs (CD80) + CD86 + The proportions of cells treated with light were 45.7%, 49.3%, and 47.8%, respectively, significantly higher than those of cells not treated with light. Figure 14 As shown. The results indicate that: N LPMediated therapy can induce the maturation of tumor ICDs and DCs, thereby increasing tumor immunogenicity and enhancing anti-tumor immune responses.

[0133] The nanoparticles of this invention can effectively degrade PD-L1 in lysosomes via LDLR-mediated endocytosis to regulate the immunosuppressive checkpoint signaling pathway. Simultaneously, they possess excellent optical properties; under irradiation with light at a wavelength of 660 nm, PpIX generates singlet oxygen. 1 O2 activates tumor photoimmunotherapy, promotes dendritic cell (DC) maturation, and induces immunogenic cell death in tumor cells. The nanoparticles of this invention effectively enhance anti-tumor immune responses by inducing ICD in tumor cells through photoimmunotherapy and by degrading PD-L1 and reprogramming the immunosuppressive checkpoint PD-1 / PD-L1 signaling pathway.

[0134] Finally, the method described in this application is merely a preferred embodiment and is not intended to limit the scope of protection of this invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. Lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors, characterized in that, The lysosome-targeting chimeric nanoparticles are co-assembled from an amphiphilic chimeric peptide targeting low-density lipoprotein and an amphiphilic chimeric peptide targeting a programmed death receptor ligand. Each of the two amphiphilic chimeric peptides has a hydrophilic polypeptide body and a hydrophobic drug body. The hydrophilic polypeptide body contains a target receptor polypeptide sequence, a polypeptide linker sequence, and an enzyme-responsive polypeptide sequence. The hydrophobic drug body is a photosensitizer. The target receptor polypeptide sequence and the hydrophobic drug body are linked by the polypeptide linker sequence and the enzyme-responsive polypeptide sequence.

2. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The two target receptor polypeptide sequences are polypeptides targeting LDLR and PD-L1, respectively.

3. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The two targeted receptor polypeptide sequences are RLTRKRGLK and CVRARTR, respectively.

4. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The polypeptide linker sequence is GSGS.

5. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The enzyme-responsive polypeptide is a polypeptide FK that can be cleaved by cathepsin B.

6. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The photosensitizer is protoporphyrin.

7. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The amphiphilic chimeric peptides are PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR, respectively.

8. The lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, The lysosome-targeted chimeric nanoparticles have a particle size of 120-150 nm.

9. A method for preparing lysosomal-targeting chimeric nanoparticles based on low-density lipoprotein receptors according to claim 1, characterized in that, Includes the following steps: Step 1: Using amino acids and photosensitizers as raw materials, amphiphilic chimeric peptides PpIX-FK-GSGS-RLTRKRGLK and PpIX-FK-GSGS-CVRARTR were synthesized by solid-phase synthesis. Step 2, two amphiphilic chimeric peptides were dissolved in dimethyl sulfoxide, respectively, to prepare 5 mM CP LDLR and CP PD-L1 stock solution; Step 3, mix the two stock solutions with (CP) LDLR :CP PD-L1 The mixture was prepared by mixing the ingredients in a ratio of 5:1 (5:1) until homogeneous. Step 4: Under ultrasonic conditions, the mixture obtained in step 3 is slowly dripped into ultrapure water to form nanoparticles through co-assembly.

10. The application of the lysosome-targeting chimeric nanoparticle based on low-density lipoprotein receptor according to claim 1 in the preparation of antitumor drugs.

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