Self-carrier spherical polypeptide nanoparticles and preparation method and application thereof

Abstract

The present application provides a self-carrier spherical polypeptide nanoparticle and its preparation method and application. The self-carrier spherical polypeptide nanoparticle is a spherical nano-polypeptide with a 3D structure formed by self-assembly of multiple monomer molecules, wherein the monomer molecules include hydrophobic molecules and hydrophilic polypeptide molecules, wherein the hydrophobic molecules are on the inner side of the spherical nano-polypeptide and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptide. The hydrophilic polypeptide molecules are selected from at least one of polypeptide antigen molecules, targeting molecules and transmembrane peptide molecules. The self-carrier spherical polypeptide nanoparticle of the present application can be accumulated at the tumor site or targeted drug delivery to tumor cells or other cells to enhance the therapeutic effect of the drug.

Description

Technical Field

[0001] The present application relates to the technical field of drug preparation, and in particular to a self-carrier spherical polypeptide nanoparticle and a preparation method and application thereof. Background Art

[0002] In recent years, tumors have become one of the major public health issues that seriously threaten human health. Traditional single treatment methods such as surgery, chemotherapy and radiotherapy often fail to achieve ideal therapeutic effects, and tumor immunotherapy has gradually come into people's view. Tumor immunotherapy is a treatment method that activates the body's anti-tumor immune response to kill tumor cells in order to control tumor growth and eliminate tumors. At present, immunotherapy is mainly divided into the following categories: immune checkpoint inhibitor therapy, chimeric antigen receptor T cell immunotherapy, tumor vaccine therapy, oncolytic virus therapy and bispecific antibody therapy.

[0003] Tumor vaccines are an important part of tumor immunotherapy. The principle is to introduce various forms of tumor antigens (such as tumor antigen-related genes, tumor-related antigens or peptides, tumor cells, etc.) into the patient's body to break the tumor immunosuppression state and reactivate the body's humoral and cellular immune responses. Traditional nanopeptide particles as tumor vaccines are delivered by encapsulating peptide antigen molecules in nanocarriers, which makes it difficult to ensure the antigen loading. In addition, the targeting and delivery effect of drug delivery nanocarriers are difficult to meet ideal requirements. Summary of the invention

[0004] Based on this, it is necessary to provide a self-carrier spherical polypeptide nanoparticle to address the problems of low antigen loading capacity of traditional nanoparticles and poor targeting and delivery effect of drug delivery nanocarriers. The self-carrier spherical polypeptide nanoparticle is prepared by self-assembly by directly connecting hydrophobic molecules and hydrophilic polypeptide molecules to form amphiphilic monomer molecules, and using this as the base unit.

[0005] The specific technical solutions are as follows:

[0006] In the first aspect of the present application, a self-carrier spherical polypeptide nanoparticle is provided, which is a spherical nano-polypeptide with a 3D structure formed by self-assembly of multiple monomer molecules, wherein the monomer molecules comprise hydrophobic molecules and hydrophilic polypeptide molecules, wherein the hydrophobic molecules are on the inner side of the spherical nano-polypeptide, and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptide, and the hydrophilic polypeptide molecules comprise at least one of a polypeptide antigen molecule, a targeting molecule, and a transmembrane peptide molecule.

[0007] In one embodiment, the hydrophobic molecule is directly linked to the hydrophilic polypeptide molecule; alternatively, they are linked via an amide bond or a disulfide bond chemical bond.

[0008] In one embodiment, the hydrophobic molecule includes at least one of dioleoylphosphatidylethanolamine (phospholipid DOPE), monophosphoryl lipid A (MPLA), resiquimod (adjuvant R848) and imiquimod.

[0009] In one embodiment, the hydrophilic polypeptide molecule also includes an environmentally responsive molecule.

[0010] Optionally, the hydrophilic polypeptide molecule is selected from a polypeptide antigen molecule, a targeting molecule or a transmembrane peptide molecule.

[0011] Optionally, the hydrophilic polypeptide molecule is selected from environmental responsive molecules and polypeptide antigens, or is selected from environmental responsive molecules and targeting molecules.

[0012] In one embodiment, the polypeptide antigen molecule includes at least one of prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), mucin-1 (MUC-1), melanoma associated antigen (gp100), fibronectin associated with neovascularization related to tumor metastasis (ED-A), and anti-apoptosis protein (Survivin).

[0013] The targeting molecules include angiotensin I receptor polypeptide molecule AT1, integrin α v At least one of the β3 receptor molecule iRGD and the tumor angiogenesis targeting peptide NGR.

[0014] The cell-penetrating peptide molecule includes at least one of a transcriptional transactivator protein (TAT), a pH-responsive cell-penetrating peptide (TH), and a membrane-type amphipathic peptide MPG.

[0015] The environmental responsive molecules include at least one of matrix metalloproteinase responsive polypeptides, glutathione responsive molecules, enzymatically hydrolyzed α-lactalbumin polypeptides, and H2O2 responsive molecules.

[0016] In one embodiment, the interior of the self-carrier spherical polypeptide nanoparticles also contains one or more of antigen molecules, endosomal escape molecules, adjuvant molecules and hydrophilic chemotherapy drugs.

[0017] Optionally, the antigen molecule comprises at least one of PSA, PSMA and MUC-1 polypeptide;

[0018] Optionally, the endosomal escape molecule comprises at least one of a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide having an amino acid sequence as shown in SEQ ID NO.2.

[0019] Optionally, the adjuvant molecule comprises at least one of CpG ODN, QS21, IL-1, IL-2 and IFN-γ.

[0020] Optionally, the hydrophilic chemotherapy drug includes at least one of paclitaxel, doxorubicin and gemcitabine.

[0021] In one embodiment, the self-carrier spherical polypeptide nanoparticles include one or more types of monomer molecules.

[0022] The second aspect of the present application provides a method for preparing the self-carrier spherical polypeptide nanoparticles as described in any of the above items, comprising directly connecting a hydrophilic polypeptide molecule to a hydrophobic molecule to form the self-carrier spherical polypeptide nanoparticles.

[0023] In one embodiment, the method for preparing the self-carrier spherical polypeptide nanoparticles further comprises encapsulating one or more of antigen molecules, endosomal escape molecules, adjuvant molecules and hydrophilic chemotherapy drugs in the nanoparticles.

[0024] The third aspect of the present application provides the use of the above-mentioned self-carrier spherical polypeptide nanoparticles in the preparation of nanoformulations for disease treatment.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] The self-carrier spherical polypeptide nanoparticles provided in the present application are spherical nano-polypeptides with a 3D structure formed by self-assembly of multiple monomer molecules; the monomer molecules include hydrophobic molecules and hydrophilic polypeptide molecules; the hydrophobic molecules are on the inner side of the spherical nano-polypeptides, and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptides; the hydrophilic polypeptide molecules include at least one of polypeptide antigen molecules, targeting molecules and transmembrane peptide molecules.

[0027] Furthermore, the hydrophilic polypeptide molecules contain polypeptide antigens as self-carrier molecules to form spherical polypeptide nanoparticles, which can be used as polypeptide nanovaccines to achieve accurate antigen loading and improve the efficacy of vaccines. At the same time, after the nanoparticles are injected intravenously or subcutaneously into the body, they can be well accumulated at the tumor site due to the EPR effect and targeting, and act on immune cells in the tumor microenvironment. Moreover, they can be used as self-carrier nanoparticles with adjuvant function constructed by antigen peptides and hydrophobic adjuvant molecules, which can realize the co-delivery of antigen adjuvants to antigen-presenting cells, activate the body's antigen-specific immune response, and inhibit tumor growth.

[0028] Furthermore, hydrophilic polypeptide molecules can also replace any functional polypeptides. For example, spherical transmembrane peptide nanoparticles constructed by transmembrane peptide molecules can be used as drug nanodelivery carriers to greatly improve the delivery efficiency of the drugs they carry. Spherical polypeptide nanoparticles formed by targeting polypeptide molecules can be used as nanocarriers to load other drugs or mRNA for targeted delivery, which can greatly improve the targeted delivery efficiency of loaded mRNA or other drugs and achieve efficient targeted delivery.

[0029] Furthermore, the method for preparing the self-carrier spherical polypeptide nanoparticles of the present application is simple to operate and has high synthesis efficiency, and the synthesized polypeptide nanoparticles are stable and not easy to aggregate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the particle size distribution of nanoparticles of MPLA-MUC1 according to an embodiment of the present invention, wherein: Figure 1 A in the figure is the particle size diagram of different molar ratios of MPLA and MUC1. Figure 1 B in the figure is the particle size diagram when the molar ratio of MPLA to MUC1 is 1:1;

[0031] Figure 2 This is a particle size distribution diagram of the nanoparticles of DOPE-SS-Survivin according to an embodiment of the present invention;

[0032] Figure 3 This is a transmission electron microscopy image of DOPE-glutathione responsive molecule-gp100 nanoparticles according to an embodiment of the present invention;

[0033] Figure 4 This is a transmission electron microscope image of MPLA-MPG nanoparticles according to an embodiment of the present invention;

[0034] Figure 5 This is a study on the in vitro stability of imiquimod-iRGD nanoparticles according to an embodiment of the present invention;

[0035] Figure 6 This is an MPLA-PSMA nanoparticle according to an embodiment of the present invention, wherein: Figure 6 A in the figure shows the situation where MPLA-PSMA nanoparticles promote DC to take up antigens; Figure 6 B in the figure is a schematic diagram of the structure of MPLA-PSMA nanoparticles;

[0036] Figure 7 The cytokine secretion after the nanoparticles containing the cytokine IL-2 encapsulated by the adjuvant DOPE-AT1 polypeptide in one embodiment of the present invention were incubated with Treg cells;

[0037] Figure 8 This is the maturation promotion of BMDCs cells by R848-PSA nanoparticles in one embodiment of the present invention;

[0038] Fig. 9 This is a nanoformulation constructed by DOPE-MMP2-iRGD encapsulating paclitaxel PTX in one embodiment of the present invention, wherein Fig. 9 A in the figure is the effect of DOPE-MMP2-iRGD-encapsulated paclitaxel (PTX) nanoformulation on the tumor mass of tumor-bearing mice; Fig. 9B in the figure is the drug loading of DOPE-MMP2-iRGD nanoparticles;

[0039] Fig.10 This is the effect of imiquimod-ED-A spherical polypeptide nanovaccine on the survival of tumor-bearing mice in one embodiment of the present invention;

[0040] Fig.11 This is the effect of DOPE-SS-TH nanovaccine encapsulated with gemcitabine on the tumor volume of tumor-bearing mice according to one embodiment of the present invention. DETAILED DESCRIPTION

[0041] In order to facilitate understanding of the present application, the present application will be described in more detail below. However, it should be understood that the present application can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The optional range of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. The following examples are given for the purpose of illustrating various embodiments of the present application, and are not intended to limit the present application in any way. It will be understood by those skilled in the art that, as defined by the scope of the claims, variations and other uses thereof are included within the spirit of the present application. The materials, reagents, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial sources.

[0043] One embodiment of the present application provides a self-carrier spherical polypeptide nanoparticle, which is a spherical nano-polypeptide with a 3D structure formed by self-assembly of multiple monomer molecules; the monomer molecules include hydrophobic molecules and hydrophilic polypeptide molecules; the hydrophobic molecules are on the inner side of the spherical nano-polypeptide, and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptide; the hydrophilic polypeptide molecules include at least one of polypeptide antigen molecules, targeting molecules and transmembrane peptide molecules.

[0044] In one specific example, the hydrophobic molecule includes at least one of phospholipid DOPE, monophosphoryl lipid A, adjuvant R848 and imiquimod.

[0045] In one specific example, the hydrophobic molecule is directly connected to the hydrophilic polypeptide molecule, and optionally, the hydrophobic molecule is directly connected via an amide bond or a disulfide bond. After being taken up by DC cells, the self-carrier spherical polypeptide nanoparticles can act together as a whole nanocarrier, or the chemical bonds can be broken to act separately in individual forms.

[0046] Optionally, the hydrophilic polypeptide molecule is selected from a polypeptide antigen molecule or a targeting molecule or a membrane-penetrating peptide molecule.

[0047] In one specific example, the hydrophilic polypeptide molecule also includes an environmentally responsive molecule.

[0048] Optionally, the hydrophilic polypeptide molecule is selected from the group consisting of an environmental responsive molecule and a polypeptide antigen, or is selected from the group consisting of an environmental responsive molecule and a targeting molecule.

[0049] When there are multiple hydrophilic polypeptide molecules, the connection order will be designed differently according to different purposes. For example, spherical polypeptide nanoparticles that connect hydrophobic molecules-environmentally responsive molecules-polypeptide antigens in sequence, and spherical polypeptide nanoparticles that connect hydrophobic molecules-environmentally responsive molecules-targeting molecules in sequence. When the spherical polypeptide nanoparticles enter the body, the targeting molecules first target the tumor cells, and then MMP2 breaks, and the chemotherapy drugs are released to kill the tumor cells.

[0050] In one specific example, the environmental responsive molecule is selected from at least one of a matrix metalloproteinase responsive polypeptide, a glutathione responsive molecule, an enzymatically hydrolyzed α-lactalbumin polypeptide, and a H2O2 responsive molecule. The formed spherical polypeptide nanoparticles can be used as nanocarriers to load other drugs to achieve environmental responsive drug co-delivery, thereby improving the drug delivery efficiency in the body and achieving targeted drug delivery.

[0051] In one specific example, at least one of the polypeptide antigen molecules prostate-specific antigen, prostate-specific membrane antigen, mucin-1, melanoma-associated antigen gp100, fibronectin antigen polypeptide ED-A related to neovascularization associated with tumor metastasis, and anti-apoptosis protein Survivin forms a spherical polypeptide nanovaccine.

[0052] In one specific example, the targeting molecule includes at least one of AT1, iRGD, LHRH, NGR, RVG, and YIGSR, and the formed spherical polypeptide nanoparticles can be used as nanocarriers for targeted delivery of drugs or genes.

[0053] In one specific example, the cell-penetrating peptide molecules include at least one of TAT, TH, R9-TAT, K9, Pen and MPG, and the spherical polypeptide nanoparticles formed can efficiently deliver drugs to tumor cells or other cells, thereby enhancing the therapeutic effect of the drugs.

[0054] In one specific example, the interior of the self-carrier spherical polypeptide nanoparticles can also encapsulate one or more of antigen molecules, endosomal escape molecules, adjuvant molecules and hydrophilic chemotherapy drugs. Chemotherapy drugs are added to the self-carrier spherical polypeptide nanoparticles to obtain polypeptide nanoformulations encapsulating chemotherapy drugs, which can effectively inhibit tumor growth. The combination of self-carrier spherical polypeptide nanoparticles and chemotherapy drugs can achieve a synergistic therapeutic effect.

[0055] Optionally, the antigen molecule includes one or more of PSA, PSMA and MUC-1 polypeptide. Optionally, the endosomal escape molecule includes one or more of LLKKLLKKLLKK (SEQ ID NO.1) and LLHHLLHHLLHH (SEQ ID NO.2). Optionally, the adjuvant molecule includes one or more of CpG ODN, QS21, IL-1, IL-2 and IFN-γ. Optionally, the hydrophilic chemotherapy drug includes one or more of paclitaxel, doxorubicin and gemcitabine.

[0056] In one specific example, the self-supporting spherical polypeptide nanoparticles include one or more types of monomer molecules.

[0057] Optionally, the monomer molecule is selected from a hydrophobic molecule and a polypeptide antigen; optionally, the monomer molecule is selected from a hydrophobic molecule and a targeting molecule; optionally, the monomer molecule is selected from a hydrophobic molecule and a transmembrane peptide molecule; optionally, the monomer molecule is selected from a hydrophobic molecule and an environmental response molecule-polypeptide antigen; optionally, the monomer molecule is selected from a hydrophobic molecule and an environmental response molecule-targeting molecule.

[0058] In one specific example, the hydrophobic molecule is selected from monophosphoryl lipid A, the hydrophilic polypeptide molecule is selected from MUC-1 antigen, and the internal or encapsulated molecule is an antigen molecule that binds to the endosomal escape molecule (LLHHLLHHLLHHSTAPPAHGVTSAPDTRPAPGSTAPP, SEQ ID NO: 3).

[0059] In one specific example, the hydrophobic molecule is selected from DOPE, and the hydrophilic polypeptide molecule is selected from Survivin polypeptide antigen.

[0060] In one specific example, the hydrophobic molecule is selected from DOPE, and the hydrophilic polypeptide molecule is selected from H2O2 responsive molecules and gp100 antigens.

[0061] In one specific example, the hydrophobic molecule is selected from monophosphoryl lipid A, and the hydrophilic polypeptide molecule is selected from membrane amphiphilic peptide MPG.

[0062] In one specific example, the hydrophobic molecule is selected from imiquimod, and the hydrophilic polypeptide molecule is selected from iRGD transmembrane peptide.

[0063] In one specific example, the hydrophobic molecule is selected from monophosphoryl lipid A, and the hydrophilic polypeptide molecule is selected from PSMA antigen.

[0064] In one specific example, the hydrophobic molecule is selected from DOPE, the hydrophilic polypeptide molecule is selected from angiotensin I receptor polypeptide molecule AT1, and the cytokine IL-2 is encapsulated.

[0065] In one specific example, the hydrophobic molecule is selected from R848, and the hydrophilic polypeptide molecule is selected from PSA antigen polypeptide.

[0066] In one specific example, the hydrophobic molecule is selected from DOPE, the hydrophilic polypeptide molecule is selected from matrix metalloproteinase 2 MMP2 and iRGD transmembrane peptide, and the chemotherapy drug paclitaxel is encapsulated.

[0067] In one specific example, the hydrophobic molecule is selected from imiquimod, and the hydrophilic polypeptide molecule is selected from the fibronectin antigen ED-A antigen associated with neovascularization associated with tumor metastasis.

[0068] In one specific example, the hydrophobic molecule is selected from DOPE, the hydrophilic polypeptide molecule is selected from pH-responsive transmembrane peptide TH, and the chemotherapeutic drug gemcitabine is encapsulated.

[0069] It can be understood that, in other specific examples, the self-carrier spherical polypeptide nanoparticles are not limited to those listed above, and can also be other forms of self-carrier spherical polypeptide nanoparticles.

[0070] In one specific example, the particle size of the self-carrier spherical polypeptide nanoparticles can be 80 to 500 nm. Optionally, the particle size of the self-carrier spherical polypeptide nanoparticles can be 80 to 200 nm. Within this range, the self-carrier spherical polypeptide nanoparticles have a better therapeutic effect on the disease, and the specific particle size can be 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm.

[0071] Further, one embodiment of the present application provides a method for preparing the above-mentioned self-carrier spherical polypeptide nanoparticles, comprising directly connecting a hydrophilic polypeptide molecule to a hydrophobic molecule to form a self-carrier spherical polypeptide nanoparticle. The reaction conditions are stirring at 20°C to 30°C, and the reaction time is 12 hours to 16 hours. Specifically, the stirring temperature can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, and the reaction time can be 12 hours, 14 hours, 16 hours.

[0072] Furthermore, the self-carrier spherical polypeptide nanoparticles formed above can also encapsulate one or more of antigen molecules, endosomal escape molecules, adjuvant molecules and hydrophilic chemotherapy drugs. The reaction conditions are stirring at 20°C to 30°C, and the reaction time is 2 hours to 4 hours. Specifically, the stirring temperature can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, and the reaction time can be 2 hours, 3 hours, or 4 hours.

[0073] In one specific example, the hydrophobic molecule is directly connected to the hydrophilic polypeptide molecule through an amide bond or a disulfide bond. After being taken up by DC, the self-carrier spherical polypeptide nanoparticles can act together as a whole nanocarrier, or they can break the chemical bonds and act separately in individual forms.

[0074] In one specific example, the molar ratio of hydrophobic molecules to hydrophilic polypeptide molecules is (1:5) to (3:1), preferably, the molar ratio is (1:1) to (1:4). The self-supporting spherical polypeptide nanoparticles prepared within this range have better sphericity, uniform and stable particle size, and uniform dispersion. The specific molar ratio can be 1:1, 1:2, 1:3, or 1:4.

[0075] The self-carrier spherical polypeptide nanoparticles can be used as nanoformulations for treating diseases. Optionally, the disease can be a tumor or other diseases. Optionally, the nanoformulations for treating tumors include tumor vaccines and nanocarriers.

[0076] The self-carrier spherical polypeptide nanoparticles and the preparation method thereof of the present application are further described in detail below in conjunction with specific examples.

[0077] The polypeptides and their amino acid sequences involved in the following examples are as follows:

[0078] MUC1:APDTRPAPG, SEQ ID NO:4;

[0079] Survivin:LMLGEFLKL, SEQ ID NO:5;

[0080] gp100: YLEPGPVTA, SEQ ID NO: 6;

[0081] MPG:GALFLGFLGAAGSTMGA, SEQ ID NO:7;

[0082] iRGD: CDPGKDGRC, SEQ ID NO:8;

[0083] PSMA:ESWTKKSPSPEFSGM, SEQ ID NO:9;

[0084] AT1: GGGGDRVYIHPF, SEQ ID NO: 10;

[0085] PSA: VLSNDVCAQV, SEQ ID NO: 11;

[0086] MMP2: GILGVP, SEQ ID NO: 12;

[0087] TH: AGYLLGHINLHHLAHL, SEQ ID NO: 13.

[0088] It is understood that in other embodiments, the polypeptide and the amino acid sequence of the polypeptide used in the self-carrier spherical polypeptide nanoparticles are not limited thereto.

[0089] Example 1 Preparation of MPLA-MUC1 Nanoparticles

[0090] The molar ratio of hydrophobic molecules to hydrophilic polypeptide molecules in the self-supported spherical polypeptide nanoparticles of the present application is (1:4) to (3:1). Taking MPLA-MUC1 nanoparticles as an example, MPLA and MUC1 are prepared into self-supported spherical polypeptide nanoparticles at different molar ratios. Figure 1 As shown in A in the figure, when spherical polypeptide nanoparticles are prepared at a ratio greater than 1:1 and less than 1:4, nanoparticles with uniform particle size cannot be formed; when MPLA-MUC1 nanoparticles are prepared at a ratio of (1:1) to (1:4), the nanoparticle diameter is 50 to 500 nm, and the particle size is uniform and stable.

[0091] The specific steps for preparing MPLA-MUC1 nanoparticles using amide bonds are as follows: monophosphoryl lipid A (MPLA) and N,N'-carbonyldiimidazole are mixed in a molar ratio of 1:(1-3), reacted at room temperature for 2 hours to activate the hydroxyl group, and then the same molar amount of MUC1 polypeptide as monophosphoryl lipid A is added, mixed, and stirred at room temperature for 12 hours. Substances with larger molecular weights are collected by dialysis to obtain MPLA-MUC1 nanoparticles. The particle size and potential of the nanoparticles are measured by a Malvern particle size analyzer. Figure 1 B in the figure shows that its particle size is 159.85±4.05nm and its potential is -19.10±1.00.

[0092] Example 2 Preparation of Phospholipid DOPE-SS-Survivin Nanoparticles

[0093] The reduction-responsive anti-apoptotic protein Survivin nanoparticles were constructed using disulfide bonds. The specific steps are as follows: (1) The phospholipid DOPE and the cross-linking agent SPDP were mixed at a molar ratio of 1:1.5, reacted at room temperature for 4 to 8 hours, and then 3 times the molar volume of the reducing agent dithiothreitol DTT was added. The reaction continued for 2 hours, then dialyzed and freeze-dried. (2) The anti-apoptotic protein / peptide Survivin and the cross-linking agent SPDP were mixed at a molar ratio of 1:2, reacted at room temperature for 4 to 8 hours, then dialyzed and freeze-dried. (3) The freeze-dried mixture obtained in steps (1) and (2) was mixed at a molar ratio of 1:1.5, stirred and reacted at room temperature for 24 hours, and the DOPE-SS-Survivin nanoparticles were obtained after dialysis. Figure 2 As shown, the particle size of the nanoparticles is 85.23±5.34nm and the potential is -23.87±1.20.

[0094] Example 3 Preparation of phospholipid DOPE-glutathione responsive molecule-gp100 nanoparticles

[0095] The glutathione-responsive tumor vaccine, namely DOPE-glutathione-responsive molecule-gp100 nanoparticles, was prepared using disulfide bonds. The operating principle is as follows: the amino group of phospholipid DOPE is converted into a thiol group using the crosslinking agent SPDP and the reducing agent dithiothreitol DTT. The amino group of glutathione-responsive molecule-gp100 is then converted into pyridinedithiol using the crosslinking agent SPDP. The two freeze-dried powders are mixed at a molar ratio of 1:1.2, stirred at room temperature for 24 hours, and dialyzed to obtain DOPE-glutathione-responsive molecule-gp100 nanoparticles. Its morphology was observed under a transmission electron microscope. Figure 3 As shown, the nanoparticles are spherical particles with uniform size and uniform dispersion.

[0096] Example 4 Preparation of MPLA-MPG Nanoparticles

[0097] The hydrophobic adjuvant MPLA and the cell-penetrating peptide MPG were combined in a molar ratio of 1:1 using amide bonds to construct MPLA-MPG nanoparticles. The specific steps can be referred to Example 1. Figure 4 Transmission electron microscopy results showed that the nanoparticles were spherical, well dispersed, and had a particle size of about 200 nm.

[0098] Example 5 Preparation of hydrophobic adjuvant imiquimod-iRGD nanoparticles and study of their in vitro stability

[0099] Imiquimod and integrin α were bound by amide bond v Imiquimod-iRGD nanoparticles were prepared according to the molar ratio of 1:2 of β3 receptor molecule fragment (iRGD). After dialysis, the nanoparticles were placed in a PBS solution at 4°C, and the particle size was measured every 5 days to observe its stability. Figure 5 The results showed that the nanoparticles could exist stably for more than 30 days without obvious aggregation and precipitation.

[0100] The stability of the polypeptide nanoparticles prepared in other embodiments of the present application can be stable for more than 30 days, which will not be described in detail here.

[0101] Example 6 Preparation of hydrophobic adjuvant MPLA-PSMA nanoparticles and their promotion of antigen uptake by BMDCs

[0102] MPLA-PSMA nanoparticles were prepared by combining MPLA and PSMA in a molar ratio of 1:3 using an amide bond. In order to explore whether MPLA-PSMA nanoparticles can promote the uptake of antigens by BMDCs, a laser confocal microscope (Leica, TCSSP5) was used to observe the distribution of FITC-labeled MPLA-PSMA nanoparticles in BMDCs. The whole process was kept away from light, and free FITC-PSMA of the same concentration was used as a control. The results are shown in Figure 2. Figure 6 As shown in Figure A, MPLA-PSMA nanoparticles can significantly promote the uptake of PSMA by BMDCs. Figure 6 B in FIG. 1 is a schematic diagram of the structure of MPLA-PSMA nanoparticles.

[0103] The polypeptide nanoparticles prepared in other embodiments of the present application can significantly promote the uptake of antigens by BMDCs, which will not be described in detail here.

[0104] Example 7 Preparation of phospholipid DOPE-AT1 polypeptide-encapsulated cytokine IL-2 nanoformulation and cytokine secretion after incubation with Treg cells

[0105] Firstly, DOPE and angiotensin receptor I (AT1) targeting peptide were linked by disulfide bonds to construct reduction-responsive nano-delivery carrier DOPE-SS-AT1 nanoparticles. Then, a certain amount of cytokine IL-2 was added to the DOPE-SS-AT1 solution, which was shaken vigorously and then ultrafiltered using an ultrafiltration tube to obtain DOPE-AT1-IL-2NVs nano-preparation. Figure 7 The results showed that the nanoformulation could significantly increase the proliferation of suppressive T cells Treg in mice with myocardial infarction, promote the secretion of inhibitory cytokine IL-10, and promote myocardial remodeling.

[0106] The encapsulation rate of IL-2 was calculated to be 25.4±3.6% according to the formula: encapsulation rate = [(total IL-2 amount - free IL-2 amount) / total IL-2 amount] × 100%.

[0107] Example 8 Effect of adjuvant R848-prostate specific antigen PSA nanoparticles on maturation of BMDCs

[0108] The adjuvant molecule R848 and the prostate antigen PSA were reacted at a molar ratio of 1:1.5 using an amide bond to construct R848-PSA nanoparticles. Flow cytometry was used to detect the maturation of BMDCs by the nanoparticles. The results are as follows: Figure 8 As shown, the expression levels of co-stimulatory molecules CD40 and CD80 were significantly increased in cells treated with R848-PSA nanoparticles, which indicates that R848-PSA nanoparticles can better promote the maturation of BMDCs.

[0109] The polypeptide nanoparticles prepared in other embodiments of the present application can promote the maturation of BMDCs well, and will not be described in detail here.

[0110] Example 9 Preparation of phospholipid DOPE-MMP2-iRGD-encapsulated paclitaxel PTX nanoparticles and its effect on tumor mass in tumor-bearing mice

[0111] By chemically synthesizing DOPE-MMP2-iRGD nanoparticles (see Example 3 for details), adding the chemotherapy drug paclitaxel PTX into the reaction solution and shaking it vigorously, and then ultrafiltration, a spherical polypeptide nanoparticle with tumor targeting was obtained, which can efficiently deliver the chemotherapy drug PTX to the tumor site, significantly inhibit the growth of breast cancer in mice, and improve the therapeutic effect of tumor-bearing mice ( Fig. 9 A). The average tumor weight of mice treated with the DOPE-MMP2-iRGD-encapsulated PTX nanoformulation group was 0.496±0.13 g, which was significantly lower than that of the other three groups (PBS group 5.315±0.39 g, Free PTX group 2.07±0.13 g).

[0112] The DOPE-MMP2-iRGD nanoparticles prepared in this application contain chemotherapy drugs and enter the body. iRGD first targets tumor cells, and then MMP2 breaks and the chemotherapy drugs are released to kill tumor cells. Compared with directly connecting chemotherapy drugs to the outside, the chemotherapy drugs contained in this application can achieve a sustained and controlled release effect, and the drug loading is higher ( Fig. 9 In addition, the internal encapsulation method has simple steps, high synthesis efficiency and low economic cost.

[0113] Example 10 Preparation of adjuvant imiquimod-ED-A spherical polypeptide nanoparticles and its effect on the survival of tumor-bearing mice

[0114] The adjuvant imiquimod was covalently linked to the fibronectin antigen ED-A related to tumor metastasis-related angiogenesis using amide bonds, and tumor angiogenesis-targeted nanoparticles were constructed at a molar ratio of 1:2. When the nanoparticles entered the tumor tissue of tumor-bearing mice, they activated antigen-specific immune responses and significantly inhibited tumor angiogenesis, thereby achieving the effect of inhibiting tumor growth. A mouse melanoma model was prepared, and data were recorded from the day of administration and a mouse survival curve was drawn. The results are as follows Fig.10 As shown, imiquimod-ED-A nanoparticles can effectively inhibit tumor growth and prolong the survival of tumor-bearing mice.

[0115] Example 11 Preparation of DOPE-SS-TH nanoformulations encapsulating gemcitabine and its effect on tumor volume in tumor-bearing mice

[0116] Taking advantage of the pH-sensitive property of TH, pH-responsive spherical cell-penetrating peptide nanoparticles were prepared according to the molar ratio of DOPE:TH=1:1, and the chemotherapy drug gemcitabine was encapsulated to prepare a nanoformulation. A mouse pancreatic cancer tumor model was constructed to observe the effect of the nanovaccine on the tumor volume of tumor-bearing mice. The results are as follows Fig.11 As shown, compared with the other three groups, the DOPE-SS-TH nanovaccine loaded with gemcitabine significantly inhibited the growth of pancreatic cancer tumors and had good anti-tumor efficacy.

[0117] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A self-carrier spherical polypeptide nanoparticle, characterized in that: A spherical nano-polypeptide with a 3D structure formed by self-assembly of multiple monomer molecules; the monomer molecules are composed of hydrophobic molecules and hydrophilic polypeptide molecules, and the hydrophobic molecules are directly connected to the hydrophilic polypeptide molecules; the self-carrier spherical polypeptide nanoparticles are prepared by self-assembly by directly connecting the hydrophobic molecules and the hydrophilic polypeptide molecules to form amphiphilic monomer molecules, using the amphiphilic monomer molecules as primitives; the hydrophobic molecules are on the inner side of the spherical nano-polypeptide, and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptide; The self-carrier spherical polypeptide nanoparticles also contain one or more of an adjuvant molecule and a hydrophilic chemotherapeutic drug; The hydrophilic polypeptide molecule includes at least one of a polypeptide antigen molecule, a targeting molecule and a membrane-penetrating peptide molecule; The hydrophobic molecule is selected from at least one of phospholipid DOPE, monophosphoryl lipid A, adjuvant R848 and imiquimod; The polypeptide antigen molecule is selected from at least one of PSA, PSMA, MUC-1, gp100, ED-A and Survivin; The targeting molecule is selected from at least one of AT1, iRGD and NGR; The cell-penetrating peptide molecule is selected from at least one of TH and MPG; The adjuvant molecule is IL-2.

2. The self-carrier spherical polypeptide nanoparticle according to claim 1, characterized in that: The hydrophilic chemotherapy drugs include gemcitabine.

3. The self-carrier spherical polypeptide nanoparticle according to claim 1, characterized in that: Directly linked via amide or disulfide chemical bonds.

4. The self-carrier spherical polypeptide nanoparticle according to any one of claims 1 to 3, characterized in that: The monomer molecules in the self-carrier spherical polypeptide nanoparticles are of one or more types.

5. The self-carrier spherical polypeptide nanoparticle according to any one of claims 1 to 3, characterized in that: The hydrophilic polypeptide molecules also include environmental responsive molecules; the environmental responsive molecules are selected from at least one of matrix metalloproteinase responsive polypeptides, glutathione responsive molecules, enzymatically hydrolyzed α-lactalbumin polypeptides and H2O2 responsive molecules.

6. A self-carrier spherical polypeptide nanoparticle, characterized in that: A spherical nano-polypeptide with a 3D structure formed by self-assembly of multiple monomer molecules; the monomer molecules are composed of hydrophobic molecules and hydrophilic polypeptide molecules, and the hydrophobic molecules are directly connected to the hydrophilic polypeptide molecules; the self-carrier spherical polypeptide nanoparticles are prepared by self-assembly by directly connecting the hydrophobic molecules and the hydrophilic polypeptide molecules to form amphiphilic monomer molecules, using the amphiphilic monomer molecules as primitives; the hydrophobic molecules are on the inner side of the spherical nano-polypeptide, and the hydrophilic polypeptide molecules are on the outer side of the spherical nano-polypeptide; The interior of the self-carrier spherical polypeptide nanoparticles also contains paclitaxel; The hydrophilic polypeptide molecule includes at least one of a polypeptide antigen molecule, a targeting molecule and a membrane-penetrating peptide molecule; The hydrophobic molecule is selected from at least one of phospholipid DOPE, monophosphoryl lipid A, adjuvant R848 and imiquimod; The polypeptide antigen molecule is selected from at least one of PSA, PSMA, MUC-1, gp100, ED-A and Survivin; The targeting molecule is selected from at least one of AT1, iRGD and NGR; The cell-penetrating peptide molecule is selected from at least one of TH and MPG.

7. The self-carrier spherical polypeptide nanoparticle according to claim 6, characterized in that: The hydrophilic polypeptide molecules also include environmental responsive molecules; the environmental responsive molecules are selected from at least one of matrix metalloproteinase responsive polypeptides, glutathione responsive molecules, enzymatically hydrolyzed α-lactalbumin polypeptides and H2O2 responsive molecules.

8. The method for preparing the self-carrier spherical polypeptide nanoparticles according to any one of claims 1 to 4, characterized in that: include: Directly connecting a hydrophilic polypeptide molecule with a hydrophobic molecule to form a self-carrier spherical polypeptide nanoparticle, wherein the self-carrier spherical polypeptide nanoparticle is prepared by self-assembly by directly connecting the hydrophobic molecule with the hydrophilic polypeptide molecule to form an amphiphilic monomer molecule, using the amphiphilic monomer as a primitive; The nanoparticles are loaded with one or more of an adjuvant molecule and a hydrophilic chemotherapeutic drug.

9. Use of the self-carrier spherical polypeptide nanoparticles according to any one of claims 1 to 7 in the preparation of nanoformulations for treating diseases, wherein the disease is tumor.

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