Biomolecule-polynitrogen oxide conjugate and preparation method and application thereof

Abstract

The present invention provides a biomolecule-polynitrogen oxide conjugate and a preparation method and application thereof. The present invention discloses a method for preparing a biomolecule-polynitrogen oxide conjugate having a tumor hypoxia responsive function, wherein the conjugate is composed of a biomolecule and a polynitrogen oxide containing R(R')- + ‑O ‑ The structure of the nitrogen oxide monomer is polymerized by active free radicals. + ‑O ‑ The structure can be reduced under the CYP450 reductase that is highly expressed in the tumor area. The slightly acidic environment of the tumor leads to the cationization of the polymer, and the conjugate adheres to the negatively charged cell membrane, triggering endocytosis and transport, enhancing the permeability of the conjugate in tumor tissue and improving the efficacy. The conjugate not only retains the in vitro activity of the biomolecule well, but also responds to the hypoxic and acidic microenvironment of the tumor, greatly improving the half-life and biodistribution of the biomolecule in the body, and improving the anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to a biomolecule-polynitrogen oxide conjugate and a preparation method and application thereof. Background Art

[0002] Biomolecules such as proteins, peptides, antibodies, vaccines and nucleic acids play an important role in the prevention and treatment of diseases. However, biomolecules have complex structures, poor in vitro and in vivo stability, are difficult to penetrate cell membranes, are difficult to effectively enter deep into diseased tissues, have a single route of administration, and have poor compliance. Modifying biomolecules with polyethylene glycol (PEG) is an effective way to solve the above problems. However, the reaction yield of polyethylene glycol (PEG) modified biomolecules is low, the binding sites and coupling stoichiometry are difficult to control, the biological activity is severely reduced, there is a lack of targeting to the diseased site, and the tissue permeability is poor. The use of in situ growth technology to site-specifically couple polymers on biomolecules has the advantages of simple steps, mild reaction conditions, high retention of biological activity, and easy purification, which is particularly important for the improvement of biomolecules. In addition, coupling polymers that can respond to the characteristics of the tumor microenvironment (pH, enzymes, temperature, oxygen, etc.) will more accurately deliver biomolecules to deep parts of the tumor, thereby improving the utilization and therapeutic effect of the drug. Summary of the invention

[0003] The purpose of the present invention is to provide a novel biomolecule-polynitrogen oxide conjugate and a preparation method and application thereof.

[0004] The present invention is conceived as follows: protein drugs have been widely used in the treatment of tumors. However, some protein drugs have problems such as short half-life, poor stability, and poor tissue permeability. Protein-polymer conjugates prepared by coupling proteins and polymers can effectively improve the stability, pharmacokinetic and therapeutic effects of proteins. Tumor hypoxia is a major feature of the tumor microenvironment. Inspired by hypoxia prodrugs such as AQ4N and TPZ, the present invention provides a biomolecule-polynitrogen oxide conjugate that can be reduced by the reductase cytochrome P450 (CYP450) highly expressed in the tumor microenvironment.

[0005] To achieve the purpose of the present invention, in a first aspect, the present invention provides a biomolecule-polynitrogen oxide conjugate, wherein the conjugate is obtained by in situ polymerization of polynitrogen oxide from a biomolecule or by conjugating polynitrogen oxide with a biomolecule.

[0006] The biomolecules are selected from small molecule drugs, proteins, small peptides, antibodies, nucleotides, nucleic acid aptamers and other biomolecules.

[0007] The polynitrogen oxide is polymerized from a nitrogen oxide monomer, and the structure of the nitrogen oxide monomer is shown in Formula I:

[0008]

[0009] Wherein, R, R' and R" represent any group.

[0010] Nitrogen oxide monomers can be found in Xiang J, Shen Y, Zhang Y, et al. Multipotent Poly (Tertiary Amine-Oxide) Micelles for Efficient Cancer Drug Delivery. Advanced Science, 2022, 9(12): 2200173.

[0011] In the present invention, the protein includes but is not limited to insulin, blood factors, colony stimulating factors, growth hormone, interleukin, growth factors, therapeutic vaccines, calcitonin, tumor necrosis factor (TNF) and enzymes.

[0012] Preferably, the protein includes but is not limited to asparaginase, glutaminase, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, epidermal growth factor (EGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, coagulation factor, tumor necrosis factor, IFN, interleukin, gastrointestinal peptide, vasoactive intestinal peptide (VIP), intestinal caspase (CCK), gastrin, Secretin, erythropoietin, hormones, antidiuretic hormone, octreotide, pancreatic enzymes, superoxide dismutase, thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone, tissue-type plasminogen activator, interleukin-1, interleukin-15, receptor antagonist (IL-1RA), glucagon-like peptide-1 (GLP-1), leptin, growth hormone, granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), adenosine deaminase, uricase, asparaginase, human growth hormone, asparaginase, macrophage activation, chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vector, relaxin, cyclosporine, etc.

[0013] The nucleotides include, but are not limited to, natural or artificially synthesized, single-stranded or double-stranded polynucleotides or oligonucleotides.

[0014] Preferably, the nucleotides include antisense oligonucleotides, siRNA, anti-miR (target genes such as Bcl-2, V2R, EphA2, caveolin-1, TNF-α, MIF, GFPRAF-1, C-RAF, luciferase, vascular endothelial growth factor, SCV, FAS, INS2, Caspase-8 and HBsAg, etc.).

[0015] The nucleic acid aptamers include, but are not limited to, vascular endothelial growth factor (VEGF) aptamers, ricin aptamers, zucchini toxin aptamers, thrombin aptamers, activated plasma protein C aptamers, HIV-1 reverse transcriptase aptamers, HIV-1 integrase aptamers, protein kinase C aptamers, human neutrophil elastase aptamers, L-selectin aptamers, P-selectin aptamers, Yersinia protein tyrosine phosphatase aptamers, phospholipase A2 angiogenin aptamers, rhinovirus capsid protein aptamers, etc.

[0016] The other biological molecules include, but are not limited to, oxytocin, vasopressin, adrenocorticotropic hormone, prolactin, luteinizing hormone-releasing hormone, luteinizing hormone-releasing hormone, growth hormone-releasing factor, somatostatin, glucagon, gastrin, pentagastrin, tetragastrin, enkephalin, endorphin, angiotensin, renin, bradykinin, bacitracin, polymyxin, colistin, tyrocitracin, gramicidin and synthetic analogs, variants and pharmacologically active fragments, monoclonal antibodies and soluble vaccines, etc.

[0017] The small molecule drugs include but are not limited to doxorubicin, curcumin, SN38, CPT, tirapazamine, AQ4N, ibrutinib, sorafenib, afatinib, gefitinib, nilotinib, dasatinib, osimertinib, lenalidomide, palbociclib, palbociclib, pomalidomide, olaparib and other drugs and their derivatives.

[0018] The antibodies include, but are not limited to, monoclonal antibodies, single-chain antibodies, ankyrin repeat sequences, affibodies, and the like, and the like.

[0019] The polymer grown in situ from the drug gives the conjugate a special hypoxia responsive function. The "polynitrogen oxide" involved in the present invention includes homopolymers, heteropolymers, block polymers, copolymers, terpolymers and other polymers of "polynitrogen oxide" and their mixtures. Examples of precursor monomers oxidized to nitrogen oxides include but are not limited to dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, diisopropylaminoethyl methacrylate, and monomers containing monomers that can be oxidized to R(R')-N + -O - Monomers with a chemical structure and their derivatives are polymerized into polymers with a polynitrogen oxide structure through active free radical polymerization.

[0020] In a second aspect, the present invention provides a method for preparing the biomolecule-polynitrogen oxide conjugate. When the biomolecule is a protein, a small peptide or an antibody, the method for preparing the conjugate comprises:

[0021] In situ polymerization of polynitrogen oxides from biomolecules: first, molecules such as atom transfer radical initiators or chain transfer agents are grafted onto the surface of biomolecules, and then in situ copolymerization is carried out through atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization.

[0022] That is, biomolecule + atom transfer radical polymerization initiator or chain transfer agent → biomolecule initiator;

[0023] Biomolecule initiator + nitroxide monomer → biomolecule-polynitroxide conjugate;

[0024] or;

[0025] Conjugating poly(nitrogen oxide) to biomolecules: Nitrogen oxide monomers are first polymerized to poly(nitrogen oxide) from a functionalized atom transfer radical initiator or chain transfer reagent, and the resulting polymer is then conjugated to a biomolecule.

[0026] That is, functionalized atom transfer radical initiator or chain transfer agent + nitroxide monomer → polynitroxide;

[0027] Poly(N-oxide)+biomolecule→biomolecule-poly(N-oxide) conjugate.

[0028] Furthermore, the atom transfer radical initiator can be selected from any one of the following:

[0029]

[0030] Wherein, n is a positive integer from 1 to 10.

[0031] The chain transfer agent can be selected from any one of the following:

[0032]

[0033] Wherein, n is a positive integer of 1-10; and R is any alkane chain.

[0034] In a third aspect, the present invention provides an interferon-polynitrogen oxide conjugate (IFN-PODMA) prepared according to the method.

[0035] First, interferon containing LPETGGHHHHHH at the C-terminus was prepared by genetic engineering technology (prokaryotic expression), and then interferon-polynitrogen oxide conjugate (IFN-PODMA) was prepared by the above method.

[0036] In a fourth aspect, the present invention provides the use of the biomolecule-polynitrogen oxide conjugate (such as IFN-PODMA) in the preparation of anti-tumor drugs.

[0037] The invention utilizes site-specific coupling technology to connect an atom transfer radical polymerization (ATRP) initiator with the C-terminus of IFN through a Sortase A enzyme-mediated method to form IFN-Br, and polymerizes a nitrogen oxide monomer with CYP450 enzyme reduction characteristics under hypoxic conditions through the ATRP technology to form interferon-poly 2-(N-oxidized-N,N'-dimethylamino)-2-ethyl methacrylate (IFN-PODMA), which can be reduced by CYP450 enzymes highly expressed in tumor areas to form its precursor polymer interferon-poly 2-(N,N'-dimethylamino)-2-ethyl methacrylate (IFN-PDMA), which is protonated in the acidic microenvironment of the tumor and adheres to the negatively charged cell membrane, effectively improving the endocytic transport of IFN in the hypoxic area of ​​the tumor, enhancing protein permeability, drug metabolism kinetics and biodistribution, and improving the therapeutic effect.

[0038] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0039] The present invention provides a method for synthesizing a biomolecule-polynitrogen oxide conjugate (such as IFN-PODMA). Through the site-specific conjugation technology, the nitrogen oxide that can be reduced in the hypoxic environment of the tumor and under the condition of high expression of CYP450 enzymes is site-specifically conjugated at the C-terminus of IFN. The conjugate not only retains the in vitro biological activity well, improves the half-life, biodistribution and anti-tumor effect of IFN in the body, but also promotes the transcytosis of the conjugate and enhances the permeability of IFN in the tumor. Compared with the marketed gold standard drug PEGASYS, IFN-PODMA shows a more excellent anti-tumor effect, thereby laying a solid technical foundation for the clinical transformation of long-acting IFN. In addition, polynitrogen oxide can also be widely used in other proteins, small peptide drugs or small molecule drugs to improve their pharmacological characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of IFN-PODMA synthesis in a preferred embodiment of the present invention.

[0041] Figure 2 The SDS-PAGE gel images of IFN purified by nickel column affinity chromatography and IFN-PODMA before and after purification by atom transfer radical polymerization (ATRP) in a preferred embodiment of the present invention are shown.

[0042] Figure 3The NMR image of PODMA in deuterated water after enzymatic hydrolysis and IFN removal in a preferred embodiment of the present invention is shown in Figure 1. Wherein, a, b, c, d, and e are the chemical shifts of H in deuterated water (a: -CH 3 CH 2 -, b:-ONCH 3 ,c:-CH 2 CH 2 NO,d:-OCH 2 CH 2 ,e:-CCH 2 CH 3 ) ；

[0043] Figure 4 The molecular weights of IFN, IFN-Br and three molecular weights of IFN-PODMA analyzed by GPC in a preferred embodiment of the present invention.

[0044] Figure 5 The hydration radius of IFN, IFN-Br and three molecular weight IFN-PODMA analyzed by DLS in the preferred embodiment of the present invention.

[0045] Figure 6 The secondary structures of IFN, IFN-Br and IFN-PODMA of three molecular weights in the preferred embodiments of the present invention are shown.

[0046] Figure 7 In the preferred embodiment of the present invention, the MTT method was used to determine the anti-proliferative activity of IFN, PEGASYS, and IFN-PODMA with three molecular weights on Daudi B cells.

[0047] Figure 8 The pharmacokinetic data of IFN, PEGASYS, and IFN-PODMA of three molecular weights in the preferred embodiments of the present invention are shown.

[0048] Fig. 9 The cumulative distribution of IFN, PEGASYS and IFN-PODMA-2 in tumor area in the preferred embodiment of the present invention. *, ** and *** indicate significance at 5%, 1% and 0.1% level, respectively.

[0049] Fig.10 The figure shows the distribution of IFN, PEGASYS and IFN-PODMA-2 in the heart, liver, spleen, lung, kidney, gastrointestinal tract and skin in a preferred embodiment of the present invention.

[0050] Fig.11 The in vivo permeability of IFN, PEGASYS and IFN-PODMA-2 in the preferred embodiments of the present invention.

[0051] Fig.12The permeability of IFN, PEGASYS and IFN-PODMA-2 in 3D tumor cell spheroids under hypoxic conditions (1% oxygen) in a preferred embodiment of the present invention.

[0052] Fig.13 The chemical structure of IFN-PODMA is partially converted to IFN-PDMA in the presence of CYP450 reductase and NADPH under hypoxic conditions (1% oxygen) in a preferred embodiment of the present invention. Wherein, a, b, c, d, e are the chemical shifts of H on IFN-PODMA (a: -CH 3 CH 2 -, b:-ONCH 3 ,c:-CH 2 CH 2 NO,d:-OCH 2 CH 2 ,e:-CCH 2 CH 3 ) ； a′, b′, c′, d′, and e′ are the chemical shifts of H on IFN-PDMA (a: -CH 3 CH 2 -, b:-ONCH 3 ,c:-CH 2 CH 2 NO,d:-OCH 2 CH 2 ,e:-CCH 2 CH 3 ).

[0053] Fig.14 This is the change in the potential of IFN-PODMA under hypoxic conditions (1% oxygen) and in the presence of CYP450 reductase and NADPH in a preferred embodiment of the present invention.

[0054] Fig.15 This is the curve of tumor volume change during the treatment of mice in the preferred embodiment of the present invention. ** indicates significance at the 1% level.

[0055] Fig.16 This is a survival curve of mice during treatment in a preferred embodiment of the present invention. ** and *** indicate significance at the 1% and 0.1% levels, respectively.

[0056] Fig.17 This is a pathological tissue section of the tumor site after treatment in a preferred embodiment of the present invention.

[0057] Fig.18 These are pathological tissue sections of the heart, liver, lung, and kidney of mice after drug injection in a preferred embodiment of the present invention.

[0058] Fig.19 The figure shows the changes in the body weight of nude mice over time after drug injection in the preferred embodiment of the present invention. Among them, a represents the PBS group, b represents the IFN group, c represents the PEGASYS group, and d represents the IFN-PODMA group. There are 6-8 mice in each group, and the numbers represent the number of each mouse.

[0059] Fig. 20 The figure shows the changes in blood indicators of mice after drug injection in a preferred embodiment of the present invention.

[0060] Fig.21 The figure shows the changes of blood cell indexes in mice after drug injection in a preferred embodiment of the present invention.

[0061] Fig. 22 This is the hemolysis condition of the drug in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0062] The present invention provides a method for preparing a biomolecule-polynitrogen oxide having a tumor hypoxia responsive function, wherein the N + -O - The structure is reduced under CYP450 reductase, the slightly acidic environment of the tumor causes the polymer to be cationized, the conformation and function of the polymer chain change, and it adheres to the negatively charged cell membrane, triggers endocytosis and transport, increases the tumor tissue permeability of the conjugate, and improves the efficacy. The conjugate is polymerized by active free radicals. The biomolecule-polynitrogen oxide not only retains the in vitro biological activity well, but also responds to the tumor hypoxic environment, greatly improving the half-life, biodistribution and anti-tumor effect of the biomolecule in vivo.

[0063] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0064] The IFN involved in the following examples is IFN-α (NCBI accession number: AAA52715.1).

[0065] Example 1 Preparation method and application of interferon-polynitrogen oxide conjugate (IFN-PODMA)

[0066] 1. Preparation of IFN-PODMA

[0067] (1) A plasmid containing IFN-α was constructed by genetic engineering technology, and then the plasmid was transfected into Escherichia coli by molecular cloning method to express IFN-LPETGGH6, and the protein IFN-LPETGGH6 was extracted by nickel affinity chromatography.

[0068] (2) The protein IFN-LPETGGH6 is reacted with the compound AEBM (Formula II) under the guidance of Sortase A enzyme to recognize the LPETGG sequence, and the amino group of AEBM reacts with the terminal carboxyl group of IFN to obtain a complex (IFN-Br).

[0069]

[0070] (3) IFN-Br as a macromolecular initiator reacts with nitrogen oxide ODMA in the presence of a catalyst to undergo atom transfer radical polymerization (ATRP) to obtain interferon-polynitrogen oxide conjugate.

[0071] The reaction conditions of step (2) are: 100 mM LLPETGGHHHHHH, 50 mM sortase A and 2 mM ATRP initiator (AEBM) with 50 mM Tris·HCl, 150 mM NaCl, 10 mM CaCl 2 The mixture of 100 μg / ml and 100 μg / ml was incubated at room temperature (25 °C) overnight without stirring. Then, the reaction mixture was diluted and applied to HiTrap Capto Q anion column (GE Healthcare) on AKTA. 20 mM Tris·HCl, pH 7.4 was used as the equilibrium buffer, 20 mM Tris·HCl, 1 M NaCl, pH 7.4 was used as the eluent, and a continuous gradient elution was performed to remove the Sortase A enzyme, and the peak containing the desired product mixture (IFN-Br) was collected, and then the IFN-Br solution was buffer exchanged into 10 mM PBS (pH 7.4) through a desalting column and concentrated by ultrafiltration (3k-5k MWCO, Millipore).

[0072] The reaction conditions of step (3) are as follows: ATRP reaction of IFN-Br and ODMA is carried out in a PBS ice water bath. Briefly, 5 mL of IFN-Br (0.25 μmol) and ODMA (0.25 mmol for IFN-PODMA1; 0.375 mmol for IFN-PODMA2; 1 mmol for IFN-PODMA3) are added to a Schlenk tube, and the mixture is bubbled with nitrogen to remove oxygen in the tube. At the same time, 0.01 mmol CuCl, 0.03 mmol CuCl 2A catalyst solution in 2mL MilliQ water with 0.065mmol 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) was added to another Schlenk tube and deoxygenated with nitrogen bubbling. Afterwards, the deoxygenated catalyst solution was transferred to the first deoxygenated Schlenk tube via a cannula. The first Schlenk tube was then sealed, and the polymerization reaction was carried out in an ice-water bath for 2h, and then exposed to air to quench and terminate the reaction. IFN-PODMA was purified and separated using a desalting column and anion exchange chromatography to remove the catalyst and unreacted IFN-Br. Three molecular weights of interferon-polynitrogen oxides (IFN-PODMA1, IFN-PODMA2, IFN-PODMA 3) were obtained.

[0073] Schematic diagram of IFN-PODMA synthesis is shown in Figure 1 .

[0074] The SDS-PAGE gel images of IFN purified by nickel affinity chromatography and IFN-PODMA before and after purification by atom transfer radical polymerization (ATRP) are shown in Figure 2 .

[0075] 2. The chemical structures of monomeric nitrogen oxides and conjugates were characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC / MS).

[0076] The NMR image of PODMA in heavy water after enzymatic hydrolysis and removal of IFN is shown in Figure 3 .

[0077] 3. Nuclear magnetic resonance (NMR), Zeta potential and nanoparticle size analyzer (DLS) were used to characterize the chemical structure transformation and potential change of IFN-PODMA in the presence of CYP450 reductase highly expressed in the hypoxic area of ​​tumors.

[0078] 4. Analytical methods such as nanoparticle size analyzer (DLS), circular dichroism (CD) and gel permeation chromatography (GPC) were used to characterize the physicochemical properties of IFN-PODMA, such as the hydration radius, IFN secondary structure, and molecular weight of the conjugate.

[0079] The results of GPC analysis of the molecular weights of IFN, IFN-Br, and three molecular weights of IFN-PODMA are shown in Figure 4 The results of DLS analysis of the hydration radius of IFN, IFN-Br, and three molecular weight IFN-PODMA are shown in Figure 5 Their secondary structures can be seen in Figure 6 .

[0080] 5. The Daudi B cell line was used to test the in vitro biological activity of IFN, PEGASYS, and IFN-PODMA, that is, their ability to inhibit tumor cell proliferation in vitro.

[0081] C8161 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (v / v). 50 μL of approximately 10 4 50 μL of diluted samples (200, 400, 1000, 2000, 10000, 20000 pg / mL) were added to each well and incubated at 37°C for 96 h. The pure culture medium wells and pure cell wells were defined as background wells and control wells, and their survival rates were defined as 0% and 100%, respectively. The relative cell viability of the sample = (OD 样品 -OD 背景 ) / (OD 对照 -OD 背景 ) × 100%. Cell viability was calculated using a cell proliferation assay kit (Promega).

[0082] The antiproliferative activity of IFN, PEGASYS, and three molecular weight IFN-PODMA on Daudi B cells was determined by MTT assay. Figure 7 .

[0083] 6. Establish 3D tumor cell spheres (C8161 cell spheres) and test the in vitro permeability of IFN, PEGASYS, and IFN-PODMA under 1% oxygen concentration.

[0084] C8161 cells were plated at 10 4 The cells were seeded at a density of 100 cells / mL in a 96-well 3D cell sphere culture plate (primessurface) and cultured for 3 days to form 3D multicellular tumor spheres (3D MTS). IFN, PEGASYS or IFN-PODMA (1 μM) labeled with cy5 in 150 μL of fresh culture medium was added to the wells and incubated at 37°C for 4 hours under hypoxia (1% oxygen). All 3D MTS were imaged using a confocal microscope (Zeiss780).

[0085] The tumor regional cumulative distribution of IFN, PEGASYS, and IFN-PODMA-2 is shown in Fig. 9 .

[0086] The permeability of IFN, PEGASYS and IFN-PODMA-2 in 3D tumor cell spheroids under hypoxic conditions (1% oxygen) is shown in Fig.12 .

[0087] 7. Use the nude mouse model to test the pharmacokinetic properties of IFN, PEGASYS, and IFN-PODMA in vivo.

[0088] Female BALB / c nude mice (6 weeks old) were injected with IFN, PEGASYS or IFN-PODMA (1 mg / kg body weight) via the tail vein to evaluate the pharmacokinetics (n=3). Blood samples (20 μL) were collected from the tail vein of mice at designated time points (1 min, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h and 96 h). After centrifugation at 4500 g for 15 minutes, the concentrations of IFN, PEGASYS and IFN-PODMA in plasma were detected using human interferon-α2 enzyme-linked immunosorbent assay kit (PBL). Blood samples drawn before drug injection (0 h) were used as controls. Enzyme-linked immunosorbent assay data were quantified using the two-compartment model of DAS 3.0 software to generate pharmacokinetic parameters.

[0089] The pharmacokinetics of IFN, PEGASYS, and three molecular weight IFN-PODMA are shown in Figure 8 .

[0090] 8. Use the nude mouse model to test the in vivo permeability of IFN, PEGASYS, and IFN-PODMA.

[0091] Melanoma mice (150-200 mm 3 ) Cy5-labeled IFN, PEGASYS or IFN-PODMA were injected intravenously, and the IFN equivalent dose was 60 μg / mouse. Tumors were removed 4 hours later and rinsed 3 times with PBS. All tumors were embedded in OCT cryosection embedding medium (SAKURA Tissue-Tek) at -20°C to obtain 9 μm thick frozen sections. After fixing, the sections were stained with anti-CD31 antibody (primary antibody) and goat anti-mouse cy3-conjugated IgG (secondary antibody). Then, DAPI was used to stain the cell nuclei, and all sections were imaged with a confocal microscope (Zeiss780).

[0092] The distribution of IFN, PEGASYS and IFN-PODMA-2 in the heart, liver, spleen, lung, kidney, gastrointestinal tract and skin is shown in Fig.10 .

[0093] The in vivo permeability of IFN, PEGASYS, and IFN-PODMA-2 is shown in Fig.11 .

[0094] 9. Establish a nude mouse tumor model and test the anti-tumor effects of IFN, PEGASYS, and IFN-PODMA.

[0095] Each BALB / c nude mouse was subcutaneously implanted with 5.0×10 6 C8161 cells. About 7 days after tumor formation (about 50 mm in size) 3 ), the mice were randomly divided into 4 experimental groups (n = 6-8 in each group), and 1 mg / kg IFN or IFN equivalent PEGASYS, IFN-PODMA or PBS were intravenously injected weekly until all the mice in the control group (PBS, IFN and PEGASYS group) died. The tumor volume and body weight of the mice were measured every 3 or 4 days, and the calculation formula of tumor volume was ((width × width) × length) / 2. If the tumor volume of the mouse was 10 times larger than the initial volume or the weight loss exceeded 15% of the body weight, it was killed.

[0096] Under hypoxic conditions (1% oxygen) and in the presence of CYP450 reductase and NADPH, the chemical structure of IFN-PODMA is partially converted to IFN-PDMA ( Fig.13 ).

[0097] The shift in the potential of IFN-PODMA under hypoxic conditions (1% oxygen) and in the presence of CYP450 reductase and NADPH is shown in Fig.14 .

[0098] The change curve of tumor volume during the treatment of mice is shown in Fig.15 .

[0099] The survival curve of mice during treatment is shown in Fig.16 .

[0100] Pathological tissue sections of the tumor site after treatment Fig.17 .

[0101] Pathological tissue sections of the heart, liver, lung, and kidney of mice after drug injection Fig.18 .

[0102] The changes in nude mouse body weight over time after drug injection are shown in Fig.19 (a~d).

[0103] The changes in blood indicators of mice after drug injection are shown in Fig. 20 .

[0104] The changes in blood cell indicators after drug injection in mice are shown in Fig.21 .

[0105] Hemolysis of drugs is shown in Fig. 22 .

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A biomolecule-polynitrogen oxide conjugate, characterized in that: The conjugate is obtained by in situ polymerization of polynitrogen oxide from biomolecules or by conjugation of polynitrogen oxide with biomolecules; The biomolecules are selected from small molecule drugs, proteins, small peptides, antibodies, nucleotides, nucleic acid aptamers and other biomolecules; The polynitrogen oxide is polymerized from a nitrogen oxide monomer, and the structure of the nitrogen oxide monomer is shown in Formula I: Wherein, R, R', and R" represent groups, and the precursor monomer oxidized to nitrogen oxide is selected from dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, and diisopropylaminoethyl methacrylate.

2. The conjugate according to claim 1, characterized in that The protein is selected from the group consisting of insulin, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, tumor necrosis factors, and enzymes.

3. The conjugate according to claim 2, characterized in that The protein is selected from the group consisting of asparaginase, glutaminase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, epidermal growth factor, transforming growth factor, nerve growth factor, platelet-derived growth factor, bone morphogenetic protein, fibroblast growth factor, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, coagulation factors, IFN, interleukin, gastrointestinal peptides , vasoactive intestinal peptide, intestinal cholecystokinin, gastrin, secretin, erythropoietin, hormones, antidiuretic hormone, octreotide, pancreatic enzymes, superoxide dismutase, thyrotropin-releasing hormone, thyroid-stimulating hormone, tissue-type plasminogen activator, receptor antagonists, glucagon-like peptide-1, leptin, growth hormone, granulocyte-monocyte colony-stimulating factor, uricase, chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vector, relaxin, cyclosporine.

4. The conjugate according to claim 1, characterized in that The nucleotides are selected from natural or artificially synthesized, single-stranded or double-stranded polynucleotides or oligonucleotides.

5. The conjugate according to claim 1, characterized in that The nucleotide is selected from antisense oligonucleotide, siRNA, anti-miR.

6. The conjugate according to claim 1, characterized in that The nucleic acid aptamer is selected from vascular endothelial growth factor aptamer, ricin aptamer, zucchini toxin protein aptamer, thrombin aptamer, activated plasma protein C aptamer, HIV-1 reverse transcriptase aptamer, HIV-1 integrase aptamer, protein kinase C aptamer, human neutrophil elastase aptamer, L-selectin aptamer, P-selectin aptamer, Yersinia protein tyrosine phosphatase aptamer, phospholipase A2 angiogenin aptamer, and rhinovirus capsid protein aptamer.

7. The conjugate according to claim 1, characterized in that The small molecule drug is selected from doxorubicin, curcumin, camptothecin derivative SN38, camptothecin CPT, tirapazamine, banothramine hydrochloride AQ4N, ibrutinib, sorafenib, afatinib, gefitinib, nilotinib, dasatinib, osimertinib, lenalidomide, palbociclib, pomalidomide, olaparib and their derivatives.

8. The conjugate according to claim 1, characterized in that The antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, ankyrin repeats, an affibody and the like.

9. The method for preparing the biomolecule-polynitrogen oxide conjugate according to claim 1, characterized in that: In situ polymerization of polynitrogen oxides from biomolecules: firstly, an atom transfer radical initiator or a chain transfer agent is grafted onto the surface of the biomolecule, and then in situ copolymerization is carried out by atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization; That is, biomolecule + atom transfer radical polymerization initiator or chain transfer agent → biomolecule initiator; Biomolecule initiator + nitroxide monomer → biomolecule-polynitroxide conjugate; or; coupling the poly(nitrogen oxide) to a biomolecule by first polymerizing a nitroxide monomer to a poly(nitrogen oxide) from a functionalized atom transfer radical initiator or a chain transfer reagent, and then coupling the resulting polymer to a biomolecule; That is, functionalized atom transfer radical initiator or chain transfer agent + nitroxide monomer → polynitroxide; Poly(N-oxide)+biomolecule→biomolecule-poly(N-oxide) conjugate.

10. The method according to claim 9, characterized in that The atom transfer radical initiator is selected from any one of the following: Wherein, n is a positive integer from 1 to 10; The chain transfer agent is selected from any one of the following: Wherein, n is a positive integer of 1-10; and R is any alkane chain.

11. Use of the biomolecule-polynitrogen oxide conjugate according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

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