Dtx polypeptides that specifically target diphtheria toxin receptors and uses thereof

Abstract

The present application relates to the field of pharmacy, and particularly relates to a DTX polypeptide which is specifically targeted to diphtheria toxin receptor, crosses a biological membrane barrier, especially a blood-brain barrier, a modified complex of the DTX polypeptide, a drug delivery system, and application in diagnosis and treatment of brain diseases such as brain tumors. The present application uses computer-aided design to prepare a multifunctional targeting polypeptide molecule DTX which has high binding activity to diphtheria toxin receptor and the ability to cross a biological membrane barrier, and relates to preparation of DTX modified fluorescein and drugs, high molecular weight carrier materials and application thereof in construction of brain imaging and targeted therapy drug delivery systems. The biggest advantage of the DTX polypeptide is that it has double-stage targeting capability for the blood-brain barrier and brain tumors. The results of the present application show that the DTX can mediate drugs or nano drug delivery systems to cross a biological membrane barrier, actively seek targets, and have good application prospects in diagnosis and targeted treatment of intracerebral diseases such as intracerebral tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical science, specifically to a specific target for diphtheria toxin receptors and its application. Background Technology

[0002] Due to the presence of the blood-brain barrier (BBB), traditional drugs have difficulty penetrating the brain, making the treatment of brain diseases a major clinical challenge. Active targeted drug delivery systems are crucial in the treatment of brain diseases. The capillary endothelial cells that constitute the BBB contain various specific receptors, including low-density lipoprotein receptors, transferrin receptors, and diphtheria toxin receptors (DTR). DTR, also known as the pro-heparin-binding-epidermal growth factor (proHB-EGF) membrane-binding precursor, is a transmembrane glycoprotein expressed in smooth muscle cells, vascular endothelial cells, the BBB, and neurons. Furthermore, DTR expression is significantly upregulated in the BBB under conditions such as glioma. Moreover, glioma cells also highly express DTR, showing a positive correlation with the severity of the disease.

[0003] Utilizing the interaction between peptides, proteins, or antibodies and their corresponding ligands or antigens to mediate drug delivery to the brain is currently the main strategy for active targeted drug delivery. Among these, peptides are the most widely used due to their convenient synthesis, simple structure, high affinity, and low immunogenicity. Summary of the Invention

[0004] The purpose of this invention is to provide a targeting polypeptide molecule DTX that can target DTR and has the ability to cross biological membrane barriers, especially the blood-brain barrier, and its modified complexes and drug delivery systems for the diagnosis and treatment of brain diseases such as brain tumors, so as to achieve good tumor targeting effects.

[0005] This invention utilizes computer-aided design methods to construct drug-loaded complexes and nano-drug delivery systems using DTX and its derivatives. These systems can cross the brain border via receptor-mediated endocytosis, increasing drug delivery efficiency and enabling applications in the diagnosis and treatment of brain diseases such as brain tumors. This also provides a reference for the design of subsequent targeted peptides. The invention involves the design and preparation of DTX, and the modification of drug molecules or polymeric carrier materials with DTX to construct DTX complexes and DTX-modified drug delivery systems, thereby improving the targeted therapeutic effects on tumors.

[0006] In a first aspect, the present invention provides a DTX polypeptide that specifically targets diphtheria toxin receptors, wherein the DTX polypeptide has dual targeting capabilities against the blood-brain barrier and brain tumors.

[0007] Furthermore, the DTX polypeptide is a polypeptide composed of 13 consecutive amino acids: aspartic acid, histidine, threonine, lysine, valine, asparagine, serine, lysine, leucine, serine, leucine, phenylalanine, and phenylalanine. The polypeptide is an L-configuration polypeptide (i.e.,...). L D L H L T L K L V L N L S L K L L L S L L L F L F, SEQ ID NO.1). The amino acid configuration can be changed, or it can be a reverse D-configuration polypeptide of an L-configuration polypeptide (i.e.,...). D F D F D L D S D L D K D S D N D V D K D T D H D D, SEQ ID NO.2).

[0008] In a second aspect, the present invention provides a DTX-X complex obtained by covalently linking the DTX polypeptide as described above with imaging substance X, for imaging diagnosis and tracing of blood-brain barrier, peripheral solid tumors and brain tumors with high expression of diphtheria toxin receptor.

[0009] Furthermore, the imaging substance X in the composite is selected from fluorescent substances such as Fluorescein, near-infrared dyes such as Cy5, IR820, and DiR, magnetic resonance imaging agents such as Gd-DTPA, and radioactive imaging agents. 99m Tc-DTPA and other similar technologies can be used for imaging diagnosis and tracing of brain, brain tumors, or peripheral tumors.

[0010] This invention designs DTX peptides using computer-aided design methods, prepares DTX peptides using Boc solid-phase peptide synthesis, and, by introducing thiol groups, allows them to be functionalized with maleimide-based imaging substances (such as fluorescent substances Fluorescein, near-infrared dyes Cy5, IR820, DiR, magnetic resonance imaging agents Gd-DTPA, and radioactive imaging agents). 99m The complex is formed by the reaction of Tc-DTPA, etc.

[0011] In a third aspect, the present invention provides a DTX-Y complex obtained by covalently linking the DTX polypeptide as described above with an antitumor drug Y, for targeted therapy of peripheral tumors and brain tumors that highly express diphtheria toxin receptors.

[0012] Furthermore, the antitumor drug Y in the complex is selected from anthracycline drugs such as doxorubicin and epirubicin, taxane drugs such as paclitaxel, docetaxel and carbamate, camptothecin drugs such as camptothecin, hydroxycamptothecin and irinotecan, vinblastine drugs such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactone drugs such as triterpenoid, protein degradation targeting chimeras, p53 activating peptides, and other polypeptide drugs, and can be used for targeted therapy of brain tumors or peripheral tumors.

[0013] The DTX peptide-modified drugs designed in this invention include: forming pH-sensitive hydrazone bonds through maleimide hexamethylene hydrazine derivative reactions (involving drugs containing ketone or aldehyde groups such as doxorubicin and epirubicin); forming disulfide bonds through 3-(2-pyridinedimerol)propionic acid derivative reactions (involving drugs containing hydroxyl or amino groups such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, and vinorelbine); forming pH-sensitive borate esters through dopamine reactions with borate groups in drugs (involving drugs containing borate groups such as bortezomib); directly forming amide bonds through solid-phase synthesis (involving peptide drugs such as p53 activating peptides); or synthesizing DTX peptide-drug complexes such as protein degradation-targeting chimeras through linker bonds.

[0014] In a fourth aspect, the present invention provides a DTX-polyethylene glycol-Z complex obtained by covalently linking the DTX polypeptide as described above with a polyethylene glycol-Z complex for use in the preparation of a nano-drug delivery system.

[0015] Furthermore, in the composite, Z is phospholipid, polylactic acid (PLA), lactoglycolic acid copolymer (PLGA), or polycaprolactone (PCL).

[0016] Furthermore, DTX-polyethylene glycol-phospholipid complexes can be used in the preparation of liposome drug delivery systems, polymer micelle drug delivery systems, and polymer disk drug delivery systems; DTX-polyethylene glycol-polylactic acid complexes, DTX-polyethylene glycol-lactic-glycolic acid copolymer complexes, and DTX-polyethylene glycol-polycaprolactone complexes can be used in the preparation of polymer micelle drug delivery systems and nanoparticle drug delivery systems.

[0017] Furthermore, the liposome drug delivery system, polymer micelle drug delivery system, lipid disk drug delivery system, and nanoparticle drug delivery system can be used to encapsulate diagnostic drugs or tumor therapeutic drugs.

[0018] Furthermore, the diagnostic drugs are selected from fluorescent substances such as coumarin 6, rhodamine B, Cy7, IR820, DiR, DiD, and magnetic resonance imaging agent Gd-DTPA, which can be used for imaging diagnosis and tracing of brain, brain tumors, or peripheral tumors.

[0019] Furthermore, the tumor treatment drugs mentioned are selected from anthracycline drugs such as doxorubicin and epirubicin, taxane drugs such as paclitaxel, docetaxel and carbamate, camptothecin drugs such as camptothecin, hydroxycamptothecin and irinotecan, vinblastine drugs such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactone drugs such as triterpenoid, protein degradation targeting chimeras, p53 activating peptides and melitrizin, scorpion venom peptides and antimicrobial peptides, and other polypeptide drugs, which can be used for targeted therapy of brain tumors or peripheral tumors.

[0020] The DTX peptide designed in this invention, after introducing thiol groups, can be modified onto polymeric carrier materials such as polyethylene glycol-distearate phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic-glycolic acid copolymer (PEG-PLGA), and polyethylene glycol-polycaprolactone (PEG-PCL) containing maleimide functional groups. It can be used to construct nano-drug delivery systems such as liposomes, lipid disks, polymer micelles, and nanoparticles modified with DTX peptides.

[0021] The DTX peptide-modified nanoparticle drug delivery system designed in this invention can encapsulate doxorubicin, epirubicin, paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, bortezomib, carfilzomib, cyclophosphamide, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, mitoxantrone, mitomycin, bleomycin, irinotecan, cisplatin, oxaliplatin, p53 activating peptide, melione, scorpion venom peptide, bevacizumab, trastuzumab, etc.; it can also encapsulate fluorescent substances and magnetic resonance imaging agents, such as FAM, near-infrared dyes Cy5.5, IR820, DiR, DiD, Gd-DTPA, etc.

[0022] This invention provides the preparation and property evaluation of DTX peptides, as well as the material basis for the application of the modified drug complexes and nanodelivery systems in tumor diagnosis and treatment. Experimental results of this invention show that DTX peptides possess multifunctional targeting capabilities across the body's borders (BBB) ​​and targeting tumor cells, exhibiting superior tumor-targeting ability in animal models; the DTX-modified nanodelivery systems demonstrate good tumor-targeting performance and stronger anti-tumor effects.

[0023] To achieve the above objectives, the present invention adopts the following technical solution:

[0024] 1. Design, synthesis and characterization of DTX peptides

[0025] Using computer-aided design methods, the binding of diphtheria toxin to its receptor was simulated, and peptide fragments of diphtheria toxin with high affinity were extracted. DTX-Cys, incorporating cysteine, was prepared using a Boc protected solid-phase synthesis method.

[0026] 2. Evaluation of the in vitro and in vivo targeting ability of DTX peptides

[0027] The in vitro targeting of fluorescently labeled DTX peptides to brain capillary endothelial cells (rat primary capillary endothelial cells BCEC and bEND.3 cells), model tumor cells (U87 glioma cells), and a U87 tumor sphere model was investigated. The in vivo targeting of fluorescently labeled DTX peptides to brain tissue and tumor sites was investigated by intravenous injection of the peptides into normal mice, nude mice bearing U87 subcutaneous tumors, and nude mice bearing U87 orthotopic tumors.

[0028] 3. DTX peptide-DTPA-Gd and DTX peptide-DTPA- 99m Tc synthesis

[0029] The DTX peptide-DTPA was synthesized via a Michael addition reaction between maleimide and thiol groups, chelating Gd or 99m Tc yields DTX peptide-DTPA-Gd or DTX peptide-DTPA- 99m Tc.

[0030] 4. Preparation of DTX peptide-drug

[0031] The DTX peptide with cysteine ​​introduced reacts with the maleimide hexamethylene hydrazine derivative of the drug to form a peptide-drug complex containing a pH-sensitive hydrazone bond. The drugs involved include doxorubicin, epirubicin, and other drugs containing ketones or aldehydes.

[0032] The DTX peptide with cysteine ​​introduced reacts with the 3-(2-pyridinedimercapto)propionic acid derivative of the drug to form a peptide-drug complex containing disulfide bonds. The drugs involved include paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine, and other drugs containing hydroxyl or amino groups.

[0033] DTX peptides are modified with dopamine and then react with the borate groups of drugs to form peptide-drug complexes containing pH-sensitive borate esters. The drugs involved include bortezomib and other drugs containing borate groups.

[0034] DTX peptides are synthesized by direct condensation with peptide drugs via solid-phase synthesis, including peptide drugs such as p53 activating peptides.

[0035] 5. Construction and characterization of DTX peptide-modified nanodelivery systems

[0036] First, DTX peptide-modified polymers, including DTX peptide-PEG-DSPE, DTX peptide-PEG-PLA, DTX peptide-PEG-PLGA, and DTX peptide-PEG-PCL, were synthesized. The synthesis of these materials was achieved by reacting the free thiol groups of cysteine-containing DTX peptides with the maleimides contained in Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, and Mal-PEG-PCL. Specifically, Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, and Mal-PEG-PCL were dissolved in acetonitrile, rotary evaporated to form films, and then reacted with PBS (pH 8.0) containing thiol-containing AG to prepare the DTX peptide-modified polymers. 1 H-NMR characterization.

[0037] Then, DTX peptide-modified nanoparticle drug delivery systems were prepared. Certain amounts of DTX peptide-PEG-DSPE and mPEG-DSPE with phospholipids and cholesterol, or DTX peptide-PEG-DSPE and mPEG-DSPE, or DTX peptide-PEG-PLA and mPEG-PLA, or DTX peptide-PEG-PLGA and mPEG-PLGA, or DTX peptide-PEG-PCL and mPEG-PCL, along with a certain amount of drug, were used to prepare corresponding DTX peptide-modified liposomes, polymer micelles, polymer disks, and polymer nanoparticles using methods such as film-forming hydration. The particle size and particle size distribution of the nanoparticle drug delivery systems were characterized using a laser scattering particle size analyzer.

[0038] 6. In vivo targeting evaluation of DTX peptide-modified nanodelivery systems

[0039] The uptake of the DTX peptide-modified nanodelivery system was investigated in bEND.3 cells, U87 cells, U87 tumor spheres, and an in vitro BBB / U87 tumor sphere model.

[0040] The targeting ability of a DTX peptide-modified nanodelivery system loaded with DiR was investigated by intravenous injection of a nude mouse carrying a U87 subcutaneous xenograft tumor into the tail vein.

[0041] The distribution of a DTX peptide-modified nanodelivery system carrying DiR at different time points was investigated by intravenous injection of the system into the tail vein of a U87 orthotopic xenograft model mouse.

[0042] 7. Evaluation of the in vitro antitumor effects of DTX-modified peptide drugs

[0043] The MTT assay was used to investigate the in vitro growth inhibition of DTX peptide-modified drugs on U87 cells. The in vivo antitumor effect was evaluated by injecting DTX peptide-modified drugs into the tail vein of nude mice bearing U87 orthotopic xenograft tumors, with median survival and tumor cell apoptosis as indicators.

[0044] 8. Evaluation of the in vitro antitumor efficacy of DTX peptide-modified nanodelivery systems

[0045] The MTT assay was used to investigate the in vitro growth inhibition of U87 cells by a DTX peptide-modified nanodelivery system carrying tumor therapeutic drugs. The in vivo antitumor effect was evaluated by intravenous injection of the DTX peptide-modified nanodelivery system carrying tumor therapeutic drugs into nude mice with U87 orthotopic xenograft tumor model via the tail vein, using median survival of nude mice, tumor cell apoptosis, number of new blood vessels and mimic blood vessels as indicators.

[0046] The advantages of this invention are:

[0047] 1. This invention employs computer-aided design to prepare a multifunctional targeting polypeptide molecule, DTX, which exhibits high binding activity to the diphtheria toxin receptor and the ability to cross biological membrane barriers. It also relates to the preparation of DTX-modified fluorescein and drugs, polymeric carrier materials, and their application in the construction of brain imaging and targeted therapy drug delivery systems. Results show that the DTX and its modified model drugs can be specifically bound or taken up by positive cells expressing the diphtheria toxin receptor (such as human brain capillary endothelial cells and glioma cells), possessing the ability to cross biological membrane barrier constituent cells (such as blood-brain barrier endothelial cells) and target tumor cells. Nanoparticle drug delivery systems constructed from DTX-modified polymeric carrier materials (such as liposomes, polymer micelles, polymer disks, and nanoparticles) can more effectively deliver the encapsulated model drugs to brain tissue and cells expressing the diphtheria toxin receptor, significantly improving the therapeutic effect of brain diseases. The greatest advantage of the DTX polypeptide of this invention lies in its dual-level targeting capability against both the blood-brain barrier and brain tumors.

[0048] 2. The results of this invention show that DTX can mediate drug or nanodelivery systems to cross biological membrane barriers and actively seek targets, and has good application prospects in the diagnosis and targeted treatment of brain diseases such as brain tumors. Attached Figure Description

[0049] Figure 1 Simulated diagram of the diphtheria toxin-diphtheria toxin-receptor binding complex. Using computer-aided technology, the binding of diphtheria toxin to the diphtheria toxin receptor (PDB:1XDT) was simulated. The bound polypeptide fragment was extracted and named DTX, with the amino acid sequence DHTKVNSKLSLFF.

[0050] Figure 2 HPLC and ESI-MS chromatograms of DTX-Cys peptide. Figure A shows the HPLC chromatogram. Chromatographic method: Column (YMC, C18): 150 × 4.6 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution program: 28% B–53% B for 20 min; flow rate: 1 mL / min; column temperature: 40℃; detection: UV 214 nm. Its purity is 98.12%, which meets the requirements. Figure B shows the ESI-MS chromatogram. The molecular weight of DTX-Cys is 1638.79 Daltons, which is consistent with the theoretical molecular weight.

[0051] Figure 3 ESI-MS spectrum of DTX-Cy5. The measured molecular weight of DTX-Cy5 is 2243.8 Daltons, which is consistent with the theoretical molecular weight of 2244.59.

[0052] Figure 4 ESI-MS spectrum of DTX-DOX. The molecular weight of DTX-DOX is 2173.4 Daltons, which is consistent with the theoretical molecular weight.

[0053] Figure 5 .DTX-PEG-DSPE 1 H-NMR spectrum. The disappearance of the characteristic absorption peak of the maleimide (Mal) group in Mal-PEG-DSPE indicates the successful synthesis of DTX-PEG-DSPE.

[0054] Figure 6 Particle size distribution of liposomes. Figure A shows the hydrated particle size of blank liposomes loaded with doxorubicin (sLip / DOX). Figure B shows the particle size distribution of DTX-modified liposomes loaded with doxorubicin (DTX-sLip / DOX). The results show that the hydrated particle size of sLip / DOX is 92.56 nm, and the dispersion index is 0.136. The hydrated particle size of DTX-sLip / DOX is 97.76 nm, and the dispersion index is 0.186, indicating that the liposomes have suitable particle size and uniform particle size distribution.

[0055] Figure 7 Uptake of fluorescently labeled DTX peptide by brain capillary endothelial cells bEND.3. Figure A shows a representative laser confocal micrograph of uptake by bEND.3 cells and DTX-FAM. Blue represents DAPI-labeled cell nuclei, and green represents the fluorescence of fluorescein FAM. Scale bar: 20 μm. Figures B and C show the flow cytometry results and quantitative fluorescence statistical analysis of uptake by bEND.3 cells and DTX-FAM, respectively. ns: no statistical difference; ***p<0.001. The results suggest that bEND.3 cells can effectively uptake DTX peptide. Mean±SD, n=3.

[0056] Figure 8 Brain targeting ability of Cy5-labeled DTX peptide in normal mice. Figure A shows images of normal ICR mice 1 hour after tail vein injection of free Cy5 and Cy5-labeled DTX peptide. Figure B shows the semi-quantitative statistical analysis results of the corresponding fluorescence. The results show that DTX-Cy5 has strong fluorescence in the mouse brain, which is significantly different from that of free Cy5, indicating that the DTX peptide has good brain targeting ability. Mean ± SD, n = 3

[0057] Figure 9 Representative laser confocal micrographs of brain capillary endothelial cells (bEND.3) taking up fluorescently labeled DTX peptide-modified liposomes (DTX-sLip). Blue indicates DAPI-labeled cell nuclei, green indicates fluorescence of FITC, scale bar is 10 μm.

[0058] Figure 10 Uptake of fluorescently labeled DTX peptide by U87 glioma cells. Figure A shows a representative laser confocal microscopy image of U87 cells and DTX-FAM uptake. Blue represents DAPI-labeled cell nuclei, and green represents the fluorescence of fluorescein FAM. Scale bar is 20 μm. Figures B and C show the flow cytometry results and quantitative fluorescence statistical analysis of DTX-FAM uptake by U87 cells, respectively. ns: no statistical difference; ***p<0.001. The results suggest that U87 cells can effectively uptake DTX peptide. Mean±SD, n=3.

[0059] Figure 11 Laser confocal tomography (LCT) images of U87 tumor spheres after co-incubation with fluorescently labeled DTX peptide for 4 hours. The results indicate that the DTX peptide can be effectively taken up by U87 tumors and penetrates deep into the interior of the tumor spheres. Scale bar = 100 μm.

[0060] Figure 12Brain targeting of Cy5-labeled DTX peptide in U87 orthotopic tumor-bearing mice. The figure shows imaging images of various organs in U87 nude mice with orthotopic brain tumors 1 hour after tail vein injection of free Cy5 and Cy5-labeled DTX peptide. The results suggest that DTX peptide can effectively accumulate at the orthotopic tumor site and has good in vivo brain tumor targeting ability.

[0061] Figure 13 A representative laser confocal micrograph of U87 brain tumor cells taking up fluorescently labeled DTX peptide-modified liposomes (DTX-sLip). Blue represents DAPI-labeled cell nuclei, green represents the fluorescence of FITC, and the scale bar is 10 μm.

[0062] Figure 14 The in vitro antitumor efficacy of DTX-DOX on U87 cells. The figure shows the growth curves of U87 cells after 72 hours of treatment with different concentrations of the drug. The IC50 of DTX-modified doxorubicin (DTX-DOX) is also shown. 50 The concentration is 0.0134 μM, which is much lower than the IC50 of free DOX. 50 (1.3 μM). This suggests that DTX modification significantly enhances the inhibitory activity of the drug DOX on U87 cells. Mean ± SD, n = 3.

[0063] Figure 15 The in vitro antitumor efficacy of doxorubicin-loaded DTX-modified liposomes against U87 cells. The figure shows the growth curves of U87 cells after 72 hours of treatment with different drug concentrations. The IC50 values ​​are shown for both the blank doxorubicin-loaded liposomes (sLip / DOX) and the DTX-modified doxorubicin-loaded liposomes (DTX-sLip / DOX). 50 The values ​​were 119 and 93.32 nM, respectively. This suggests that DTX modification can enhance the inhibitory activity of doxorubicin liposomes on U87 cells. Mean ± SD, n = 3. Detailed Implementation

[0064] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0065] Example 1: Preparation of DTX, DTX-imaging agents, DTX-antitumor drugs, DTX-polymer complexes and their modified delivery systems

[0066] 1. Design and synthesis of DTX peptides

[0067] Using computer-aided design methods, the binding of diphtheria toxin to the diphtheria toxin receptor (PDB: 1XDT) was simulated. Figure 1 A polypeptide fragment with strong affinity for diphtheria toxin was extracted and named DTX, with the amino acid sequence DHTKVNSKLSLFF (SEQ ID NO.1).

[0068] DTX-Cys, incorporating cysteine, was prepared using a Boc-protected solid-phase synthesis method. The peptide was purified by preparative HPLC and lyophilized to obtain a pure peptide. The purity and molecular weight of the peptide were characterized by HPLC and ESI-MS, and the results are shown in the table below. Figure 2 Its purity is 98.12%, which meets the requirements. The molecular weight is 1638.79 Daltons, which matches the theoretical molecular weight.

[0069] 2. Preparation of DTX-FAM and DTX-Cy5

[0070] DTX-FAM or DTX-Cy5 was synthesized by the addition reaction of the thiol group of DTX-Cys with the maleimide of fluorescein. The peptide DTX was dissolved in a small amount of DMF with 1.5 times excess maleimide fluorescein (MAL-FAM) or maleimide Cy5 (MAL-Cy5), and 1% DIEA was added. The reaction was carried out for 2 h, monitored by high-performance liquid chromatography (HPLC). The reaction was stopped after the peptide reaction was complete, and the peptide was purified by preparative HPLC using an acetonitrile / water system (containing 0.1% TFA). The DTX was then freeze-dried to obtain FAM-labeled or Cy5-labeled DTX (DTX-FAM, DTX-Cy5). The peptides were characterized by ESI-MS mass spectrometry. Figure 3 ).

[0071] 3. Preparation of DTX-based antitumor drugs

[0072] An example of preparing a DTX-doxacin complex as a DTX-linked drug containing a ketone or aldehyde group was described. A certain amount of DTX-Cys peptide was dissolved in 3 mL of phosphate buffer (0.1 mM, pH 7.0), and 10 molar amounts of tris(2-carboxyethyl)phosphine (TCEP) were added. The mixture was stirred at 4 °C for 20 min. Four molar amounts of doxorubicin-6-maleimide hexazine derivative were added, and the mixture was reacted at room temperature in the dark for 1 h. The reaction solution was separated using a C18 preparative column (column: Waters X Bridge 19 × 300 mm; mobile phase: A - pure water, B - acetonitrile; elution method: 100% A ~ 100% B linear gradient). The corresponding fractions were collected and freeze-dried to obtain the DTX-doxacin complex. ESI-MS mass spectrometry characterization was performed. Figure 4 ).

[0073] The DTX-paclitaxel complex was used as an example of DTX linked to a drug via a disulfide bond. 200 mg of paclitaxel was dissolved in 10 mL of chloroform and cooled to 0–5 °C. 39.99 mg of DCC and 60.4 mg of 3-(2-pyridinedimerol)propionic acid were added sequentially. After the addition was complete, the mixture was allowed to react overnight at room temperature. The reaction solution was filtered and purified by column chromatography (CHCl3 / MeOH = 50:1–15:1, V / V elution) to obtain a paclitaxel 3-(2-pyridinedimerol)propionic acid derivative. The paclitaxel 3-(2-pyridinedimerol)propionic acid derivative was dissolved in 5 mL of DMF, and 1.5 molar amounts of DTX were dissolved in PBS / DMF. The pH of the solution was maintained at 4–5. The paclitaxel 3-(2-pyridinedimerol)propionic acid derivative was added dropwise to a thiol peptide solution and reacted at room temperature for 6 h. The peptide-paclitaxel complex was then prepared by preparative liquid chromatography and lyophilized.

[0074] An example of using a DTX-bortezomib complex as a DTX N-terminal modified drug. Following the synthesis of DTX, amino acids were sequentially added to the resin. After all amino acid residues of the peptide were added, the Boc protection at the N-terminus was removed with trifluoroacetic acid. A DMF solution containing 3 molar amounts of succinic anhydride and DIEA was added, and the reaction was carried out at room temperature for 30 min. After washing the resin, 5 molar amounts of trimethylchlorosilane were added to protect dopamine, and the reaction was carried out at room temperature for 1 h using HBTU / DIEA as a condensing agent. The resin was cleaved with HF, and the peptide-dopamine derivative was purified by preparative HPLC. The peptide-dopamine derivative was mixed with bortezomib at a molar ratio of 1:1 in a buffer solution at pH 7.4 to obtain the peptide-bortezomib complex.

[0075] An example of using the DTX-p53 activating peptide PMI complex as a DTX fusion peptide drug. It was prepared directly by solid-phase peptide synthesis. Specifically, after determining the DTX-PMI peptide sequence, amino acids were sequentially added using the same method as for DTX preparation. The DTX-PMI fusion peptide was then obtained after hydrofluoric acid cleavage and purification.

[0076] 4. DTX-DTPA-Gd and DTX-DTPA- 99m Preparation of Tc

[0077] Maleimide-DTPA was dissolved in DMF and mixed with a PBS solution of DTX-Cys peptide as described above. The mixture was then stirred and reacted to prepare a liquid-phase purification solution. After freeze-drying, pure DTX-DTPA was obtained. Gd or... 99m Tc yields either DTX-DTPA-Gd or DTX-DTPA- 99m Tc.

[0078] 5. Preparation of DTX-polymer complex

[0079] DTX peptide-modified polymers such as DTX peptide-PEG-DSPE, DTX peptide-PEG-PLA, DTX peptide-PEG-PLGA, and DTX peptide-PEG-PCL were synthesized. The synthesis of these materials was achieved by reacting the free thiol groups of cysteine-containing DTX peptides with the maleimides contained in Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, and Mal-PEG-PCL. 1 H-NMR characterization. Taking the preparation of DTX-PEG-DSPE as an example, DTX-Cys was dissolved in 0.1M PBS solution (pH 7.2), and Mal-PEG-DSPE was dissolved in DMF. The mixture was magnetically stirred and monitored by HPLC. The reaction was stopped after Mal-PEG-DSPE had completely reacted. Excess DTX-Cys and DMF were removed by dialyzing (molecular weight cutoff 3.5 kDa), and DTX-PEG-DSPE was obtained by freeze-drying. 1 H-NMR characterization revealed the disappearance of the characteristic absorption peak of MAL in MAL-PEG-DSPE, indicating successful synthesis of DTX-PEG-DSPE (see [link to H-NMR]). Figure 5 ).

[0080] 6. Construction and characterization of DTX peptide-modified nanodelivery systems

[0081] Prepared DTX peptide-modified nanoparticle drug delivery systems. Certain amounts of DTX peptide-PEG-DSPE and mPEG-DSPE with phospholipids and cholesterol, or DTX peptide-PEG-DSPE and mPEG-DSPE, or DTX peptide-PEG-PLA and mPEG-PLA, or DTX peptide-PEG-PLGA and mPEG-PLGA, or DTX peptide-PEG-PCL and mPEG-PCL, along with a certain amount of drug, were used to prepare corresponding DTX peptide-modified liposomes, polymer micelles, polymer disks, and polymer nanoparticles using methods such as film-forming hydration. The particle size and particle size distribution of the nanoparticle drug delivery systems were characterized using a laser scattering particle size analyzer.

[0082] Taking DTX-modified doxorubicin-loaded liposomes (DTX-sLip / DOX) as an example, the formulation of the PEG-liposome membrane material is HSPC / Chol / mPEG. 2000 -DSPE (52:43:5, mol / mol), the formulation of DTX-modified PEG liposome membrane material is HSPC / Chol / mPEG. 2000-DSPE / DTX-PEG-DSPE (52:43:3:2, mol / mol). The above membrane materials were weighed and dissolved in chloroform. The chloroform was removed by rotary evaporation under reduced pressure to obtain a homogeneous lipid membrane, which was then vacuum dried for 24 hours. A certain volume of 0.32M ammonium sulfate solution was added, and the mixture was shaken in a 60°C water bath for 2 hours to obtain a liposome suspension. In a 60°C water bath, the liposomes were sequentially extruded through 400, 200, and 100-pore nuclear pore membranes using a high-pressure homogenizer (if the liposome volume was less than 10 mL, a micro-extruder was used) to obtain blank liposomes. The blank liposomes were eluted with physiological saline and passed through a Sephadex G-50 gel column to replace the external aqueous phase. Doxorubicin physiological saline solution was added at a drug-lipid ratio of 1:10 (w / w), and the mixture was incubated in a 60°C water bath for 20 minutes. The mixture was then eluted with physiological saline and passed through a Sephadex G-50 gel column to remove free drug, yielding doxorubicin liposomes. Figure 6 The particle size distribution of the liposomes is shown. The results show that the hydrated particle size of DTX-sLip / DOX is 97.76 nm and the dispersion index is 0.186, indicating that the liposomes have suitable particle size and uniform dispersion.

[0083] Example 2: Evaluation of the brain-targeting ability of DTX and its modified nanocarriers

[0084] 1. Evaluation of the in vitro brain-targeting ability of DTX peptides

[0085] Diphtheria toxin receptor-positive brain capillary endothelial cells (bEND.3 cells) were evenly seeded on confocal culture dishes and incubated for 24 h. Cell confluence and morphology were observed under a microscope. A 5 μM fluorescently labeled DTX peptide (DTX-FAM) solution was prepared using DMEM containing 10% FBS. The culture medium was discarded, and the above peptide solution was added to each well of the cells. The cells were incubated at 37°C for 4 h, washed three times with PBS (pH 7.4), fixed with 4% paraformaldehyde for 10 min, stained with DAPI (4',6-diamidino-2-phenylindole), and observed under a confocal microscope. For quantitative analysis, cells were treated under the same conditions, digested with trypsin, and cell uptake was detected by flow cytometry. The results of DTX peptide uptake by bEND.3 cells are shown below. Figure 7 As shown in the figure, the results indicate that bEND.3 cells can effectively take up DTX peptides.

[0086] 2. In vivo brain-targeting ability of DTX peptides

[0087] A 100 μM solution of Cy5-labeled DTX peptide (DTX-Cy5) was prepared and injected intravenously into ICR mice. One hour later, the mice were anesthetized and perfused with physiological saline and 4% paraformaldehyde solution, respectively. The mice were then dissected, and brain tissue was collected. The distribution of the peptide molecules in the brain was observed using a small animal in vivo fluorescence imaging system. Figure 8 As shown, DTX-Cy5 exhibits strong fluorescence in the mouse brain, showing a significant difference from free Cy5, indicating that the DTX peptide has good in vivo brain targeting ability.

[0088] 3. Evaluation of the brain-targeting ability of DTX peptide-modified nanodelivery systems

[0089] Synthesis of functional material FITC-PEG using the specific reaction of amino groups with isothiocyanates 3400 -DSPE. The synthesis steps are roughly as follows: Weigh an appropriate amount of NH2-PEG. 3400 -DSPE and 1.2 equivalents of fluorescein isothiocyanate (FITC) were mixed and dissolved in DMF. Triethylamine was added, and the mixture was stirred at room temperature in the dark. The reaction was detected using ninhydrin. When the solution no longer turned blue, it indicated NH2-PEG. 3400 -DSPE reaction was complete. Excess FITC and DMF were removed by dialysis (molecular weight cutoff 3500), and NH2-PEG was obtained after freeze-drying. 3400 -DSPE. Further, fluorescently labeled DTX peptide-modified liposomes were prepared using a thin-film hydration method.

[0090] Diphtheria toxin receptor-positive brain capillary endothelial cells (bEND.3 cells) were evenly seeded on confocal culture dishes and incubated for 24 h. Cell confluence and morphology were observed under a microscope. The culture medium was discarded, and fluorescently labeled DTX peptide-modified liposomes were added to each well. The cells were incubated at 37°C for 4 h, washed three times with PBS (pH 7.4), fixed with 4% paraformaldehyde for 10 min, stained with DAPI, and observed under a confocal microscope. The uptake results of bEND.3 cells are shown below. Figure 9 As shown, DTX modification can significantly increase the uptake of liposomes in bEND.3 cells.

[0091] Example 3: Evaluation of the in vitro and in vivo targeting ability of DTX and its modified nanocarriers against brain tumors.

[0092] 1. Evaluation of the in vitro targeting ability of DTX peptides

[0093] Diphtheria toxin receptor-positive model tumor cells (U87 glioma cells) were evenly seeded on confocal culture dishes and incubated for 24 h. Cell confluence and morphology were observed under a microscope. A 5 μM fluorescently labeled DTX peptide (FAM-DTX) solution was prepared using DMEM containing 10% FBS. The culture medium was discarded, and the above peptide solution was added to each well of the cells. The cells were incubated at 37°C for 4 h, washed three times with PBS (pH 7.4), fixed with 4% paraformaldehyde for 10 min, stained with DAPI, and observed under a confocal microscope. For quantitative analysis, cells were treated under the same conditions, digested with trypsin, and cell uptake was detected by flow cytometry. The results of DTX peptide uptake by U87 cells are shown below. Figure 10 As shown in the figure, the results indicate that U87 cells can effectively take up DTX peptides.

[0094] 2. Evaluation of the permeability of DTX peptide tumor spheres

[0095] Prepare a 2% low molecular weight agarose solution using serum-free preparation, autoclave for 30 min, and while still hot, spread 150 μL into each well of a 48-well plate. Irradiate under UV light for another 30 min, then allow to cool and solidify. Digest U87 cells, seeding 4000 cells per well (400 μL per well). Shake the plate in the same direction to clump the cells. Incubate the 48-well plate in an incubator for 7 days to form U87 tumor spheres. Discard the culture medium from the tumor spheres and add DTX-FAM and free FAM solutions of equal fluorescence intensity. Incubate at 37°C for 4 h, then remove the tumor spheres, wash three times with PBS, fix with 4% paraformaldehyde for 30 min, place the tumor spheres in a confocal dish, and photograph using a laser confocal microscope. Figure 11 As shown, DTX peptides can be effectively taken up by U87 tumors and can penetrate deep into the tumor spheres.

[0096] 3. In vivo brain tumor targeting ability of DTX peptide

[0097] Nude mice weighing approximately 20g were anesthetized and fixed on a stereotaxic apparatus. 5μL of U87 cell suspension (6×10⁶ cells / mL) was drawn up using a microsyringe. 5 (Number of cells) were inoculated into the striatum of mice (0.6 mm anterior to the anterior fontanelle, 1.8 mm to the right, and 3 mm deep) to construct an orthotopic mouse model of glioma. Experiments began 7 days after tumor inoculation, with mice receiving DTX-Cy5 and free Cy5 solutions of the same fluorescence intensity via tail vein. One hour later, the mice were anesthetized, and the fluorescence distribution in the brain was observed using a small animal in vivo imaging system. The results are as follows: Figure 12 As shown, DTX peptides can effectively accumulate at the in situ tumor site and have good in vivo brain tumor targeting ability.

[0098] 4. In vivo targeting evaluation of DTX peptide-modified nanodelivery systems

[0099] U87 cells were evenly seeded on confocal culture dishes and incubated for 24 hours. Cell confluence and morphology were then observed under a microscope. The culture medium was discarded, and fluorescently labeled DTX peptide-modified liposomes (prepared as described in Example 2.3) were added to each well. The cells were incubated at 37°C for 4 hours, washed three times with PBS (pH 7.4), fixed with 4% paraformaldehyde for 10 minutes, stained with DAPI, and observed under a confocal microscope. The uptake results of U87 cells are shown below. Figure 13 As shown, DTX modification can significantly increase the uptake of liposomes in bEND.3 and U87 cells.

[0100] Example 4: Evaluation of the antitumor efficacy of DTX-antitumor drugs and DTX-modified nanodelivery systems

[0101] 1. Evaluation of the in vitro antitumor effects of DTX-modified peptide drugs

[0102] The inhibitory effect of DTX-modified peptides on U87 cells was evaluated using the MTT assay. U87 cells in logarithmic growth phase were digested and counted, and seeded at 5000 cells per well in 96-well plates, incubated for 24 h. Different concentrations of free doxorubicin (DOX), drug-drug complex (DTX-DOX), physical mixture (DTX+DOX), and free peptide (DTX) were added. Three replicates were set up for each concentration, with a negative control of cell culture medium without any sample added. The 96-well plates were incubated for another 72 h, then 20 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for another 4 h. The culture medium in the wells was aspirated, and 150 μL of DMSO was added to each well. The plates were then placed in an air shaker and shaken at low speed for 15 min to dissolve the blue-purple crystals at the bottom of the wells. The absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability of each well was calculated. The survival rate was plotted against the logarithm of drug concentration using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The result is as follows: Figure 14 As shown, the IC50 of DTX-modified doxorubicin (DTX-DOX) 50 The concentration is 0.0134 μM, which is much lower than the IC50 of free DOX. 50 (1.3 μm). This suggests that DTX modification can significantly enhance the inhibitory activity of the drug DOX on U87 cells.

[0103] 2. Evaluation of the in vitro antitumor efficacy of DTX peptide-modified nanodelivery systems

[0104] The MTT assay was used to evaluate the inhibitory effect of the DTX peptide-modified nanoparticle drug delivery system on U87 cells. U87 cells in the logarithmic growth phase were digested and counted, and seeded at 5000 cells per well in 96-well plates, incubated for 24 h. Different concentrations of DTX-modified doxorubicin-loaded liposomes (DTX-sLip / DOX) and doxorubicin-loaded blank liposomes (sLip / DOX) were added. Three replicates were set up for each concentration, and cell culture medium without any sample was reserved as a negative control. The 96-well plates were incubated for another 72 h, and 20 μL of 5 mg / mL MTT solution was added to each well. Incubation continued for another 4 h. The culture medium in the wells was aspirated, and 150 μL of DMSO was added to each well. The plates were then placed in an air shaker and shaken at low speed for 15 min to fully dissolve the blue-purple crystals at the bottom of the wells. The absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability of each well was calculated. The survival rate was plotted against the logarithm of drug concentration using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The result is as follows: Figure 15 As shown, the ICs for sLip / DOX and DTX-sLip / DOX are... 50 The values ​​were 119 and 93.32 nM, respectively. This suggests that DTX modification can enhance the inhibitory activity of doxorubicin liposomes on U87 cells.

[0105] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A DTX polypeptide that specifically targets diphtheria toxin receptors, characterized in that, The DTX polypeptide is a polypeptide composed of 13 consecutive amino acids: aspartic acid, histidine, threonine, lysine, valine, asparagine, serine, lysine, leucine, serine, leucine, phenylalanine, and phenylalanine. The polypeptide is an L-configuration polypeptide. L D L H L T L K L V L N L S L K L L L S L L L F L F, the amino acid sequence is shown in SEQ ID NO.

1.

2. A complex obtained by covalently linking the DTX polypeptide as described in claim 1 to an imaging material.

3. The complex according to claim 2, characterized in that, The imaging material is selected from fluorescent material Fluorescein, near-infrared dyes Cy5, IR820, or DiR, magnetic resonance imaging agent Gd-DTPA, and radioactive imaging agent. 99m Tc-DTPA.

4. The use of the DTX peptide as described in claim 1, or the complex as described in claim 2 or 3, in the preparation of imaging diagnostic and tracing reagents for brain tumors.

5. A DTX-Y complex obtained by covalently linking the DTX polypeptide as described in claim 1 to an antitumor drug Y, wherein the antitumor drug Y is selected from anthracycline drugs doxorubicin or epirubicin, taxane drugs paclitaxel, docetaxel or cabazitaxel, camptothecin drugs camptothecin, hydroxycamptothecin or irinotecan, vinblastine drugs vincristine or vinorelbine, proteasome inhibitors bortezomib or carfilzomib, and lactone drugs terbinolone.

6. The use of the DTX peptide as described in claim 1, or the DTX-Y complex as described in claim 5, in the preparation of targeted therapeutic drugs for brain tumors.

7. A DTX polypeptide as described in claim 1 is covalently linked to a polyethylene glycol-Z complex to obtain a DTX-polyethylene glycol-Z complex; wherein Z in the complex is selected from phospholipids, polylactic acid, lactoglycolic acid copolymer, and polycaprolactone.

8. The application of the DTX peptide as described in claim 1, or the DTX-polyethylene glycol-Z complex as described in claim 7, in the preparation of liposome drug delivery systems, polymer micelle drug delivery systems, polymer disk drug delivery systems, and nanoparticle drug delivery systems.

9. The application according to claim 8, characterized in that, The drug delivery system described herein carries diagnostic drugs or tumor treatment drugs.

10. The application according to claim 9, characterized in that, The diagnostic drugs are selected from fluorescent substances such as coumarin 6, rhodamine B, Cy7, IR820, DiR, and DiD, or magnetic resonance imaging agents such as Gd-DTPA.

11. The application according to claim 9, characterized in that, The tumor treatment drugs are selected from anthracycline drugs doxorubicin or epirubicin, taxane drugs paclitaxel, docetaxel or cabazitaxel, camptothecin drugs camptothecin, hydroxycamptothecin or irinotecan, vinblastine drugs vincristine or vinorelbine, proteasome inhibitors bortezomib or carfilzomib, and lactone drugs ternolactone.

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