Probe and use thereof in imaging and therapy

Abstract

The present invention relates to the technical field of imaging and therapeutic probes, and specifically relates to a probe and a use thereof in imaging and therapy. The present invention specifically provides a complex, comprising: 1) a protein shell, wherein the protein shell is a third structural domain of albumin or a variant thereof, or a third structural domain a-subunit of albumin or a variant thereof; 2) a small molecule binding to the protein shell; and 3) a functionalized fragment binding to the protein shell, wherein the functionalized fragment is a molecule capable of being applied to diagnosis, prevention or treatment of kidney-bladder clearance pathway diseases. Also provided is a use of the complex.

Description

A probe and its application in imaging and treatment

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311432730.4 and application date October 31, 2023, and claims its priority. The content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present application relates to the technical field of imaging probes, and in particular to a kidney metabolism probe based on albumin variants and its application. Background Art

[0004] Bladder cancer is one of the most common cancers worldwide. According to epidemiological statistics, approximately 550,000 new cases were reported in 2018, making it second only to prostate cancer in incidence among urological diseases. Bladder cancer is primarily caused by urothelial cell proliferation, with 75% of patients diagnosed with non-muscle-invasive bladder cancer (NMIBC). Currently, the mainstay of treatment for NMIBC is transurethral cystoscopic resection of the bladder tumor, followed by postoperative intravesical instillation of first-line drugs such as BCG or mitomycin C to reduce recurrence. However, approximately 60% of patients experience recurrence, and 25% experience disease progression. Cystoscopy is invasive and, due to its limitations, can easily lead to missed diagnoses. Despite recent regulatory approval of the anti-PD-1 (programmed cell death protein 1) drug pembrolizumab for the treatment of bladder cancer, its efficacy remains limited, leaving few effective salvage treatment options other than radical cystectomy. Therefore, the development of new treatments for high-risk NMIBC is urgently needed.

[0005] Imaging methods can provide early diagnosis of bladder cancer and non-invasive, accurate postoperative monitoring. In addition, the use of photosensitizers can be used for photodynamic therapy of bladder cancer under image guidance. In addition, radionuclide labeling can also achieve early imaging of bladder cancer (such as 64 Cu, 18 F, etc.) and treatment (therapeutic nuclides, such as 177 Lu et al.

[0006] Currently, near-infrared fluorescence imaging is increasingly being used in the diagnosis and treatment of tumors due to its favorable safety profile and good tissue penetration. Dyes used for near-infrared fluorescence imaging include inorganic nanomaterials (i.e., carbon nanotubes, quantum dots, and rare earth nanoparticles) and hydrophobic small-molecule organic dyes. Most inorganic nanomaterials with high quantum yields are often doped with heavy metals, resulting in low biocompatibility and potential long-term toxicity. Small-molecule organic dyes with high biosafety are more desirable. However, small organic molecules with near-infrared emission often have large conjugated surfaces and are primarily taken up by the epithelial reticular system and metabolized in the liver, gallbladder, and intestine, making them difficult to metabolize through the urinary system. An example of this is indocyanine green (ICG), a dye approved by the US Food and Drug Administration for clinical use. Furthermore, while cyanine / polymethine dyes, the most commonly used organic materials, offer advantages such as good biocompatibility and near-infrared tailing emission, their low free dye quantum yield and poor pharmacokinetics hinder their clinical translation.

[0007] Summary of the Invention

[0008] The present invention provides a complex formed by the covalent bonding of a protein shell and a cyanine dye molecule. The cyanine dye molecule serves as a core chromophore, covalently bonded to the protein shell. The protein shell can be functionalized through genetic engineering or chemical grafting. The complex has a small size and can therefore be used to treat diseases of major organs and tissues in the kidney-bladder metabolic pathway. Compared with currently used near-infrared fluorescent probes in clinical practice, it has strong photostability, high light energy conversion efficiency, high quantum yield, and significantly extended emission wavelength, making it suitable for deep tissue imaging and photodynamic therapy. It provides a promising treatment option, especially for high-risk NMIBC patients who are refractory to BCG therapy.

[0009] Therefore, the present application provides the following invention:

[0010] In a first aspect, the present application provides a complex comprising: 1) a protein shell, wherein the protein shell is the third domain of albumin or a variant thereof, or the α subunit of the third domain of albumin or a variant thereof, 2) a small molecule bound to the protein shell, and 3) a functionalized fragment connected to the protein shell, wherein the functionalized fragment is a molecule that can be used for the diagnosis, prevention or treatment of kidney-bladder clearance pathway diseases.

[0011] The albumin in the present invention can be mammalian serum albumin. In certain embodiments, the albumin is human serum albumin. In certain embodiments, the third domain is composed of amino acid residues 384-585 of human serum albumin. In certain embodiments, the third domain of human serum albumin has an amino acid sequence as shown in SEQ ID NO: 1, wherein the exemplary amino acid sequences of the a subunit and the b subunit are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In certain embodiments, the complex is formed by the third domain of albumin and a reactive group (e.g., Cl) on the dye molecule. - or Br - In certain embodiments, the complex comprises the third domain of albumin or its variant and the dye molecule in a molar ratio of 1:5 to 5:1 (eg, 1:1).

[0012] In the present invention, variants include fragments, fusion recombinant proteins, and repeat fragments, etc. In some embodiments, the small molecule is selected from one or more of a photosensitizer (eg, a dye molecule, such as a cyanine dye molecule), a nuclide, and a small molecule drug (eg, a small molecule chemotherapeutic drug).

[0013] In the present invention, small molecules refer to molecules with a molecular weight of no more than 500.

[0014] Albumin variants can be expressed in prokaryotic or eukaryotic cells to impart the biocompatibility of wild-type albumin. While organic dyes with high quantum yields of near-infrared emission often contain heavy metals, which can cause long-term cytotoxicity, the complexes of the present invention significantly reduce the toxicity of these drugs to the body.

[0015] The small molecules included in the complexes of the present invention can have reactive groups (e.g., Br, Cl) to allow them to attach to albumin. In some embodiments, the reactive groups of the small molecule react with linking groups (e.g., sulfhydryl, thiol, hydroxyl, carboxyl, or amino) of serum albumin to form a link between the small molecule and serum albumin. In some embodiments, the linking group of the serum albumin molecule is a sulfhydryl group. Thus, in some embodiments, the small molecule is covalently bound to the sulfhydryl group of serum albumin.

[0016] In some embodiments, the small molecule is a cyanine dye molecule.

[0017] In certain embodiments, the dye molecule has a backbone structure selected from any one of the following or its derivatives:

[0018] In certain embodiments, the dye molecule has a structure selected from any one of the following:

[0019] or a structure obtained by replacing Cl in the skeleton with F, Br or I in the above structure; or

[0020] Or the structure obtained by replacing Cl in the skeleton with F, Br or I in the above structure.

[0021] In certain embodiments, the complex comprises a cyanine dye molecule that is IR-780.

[0022] The functionalized fragments contained in the complex of the present invention can be small molecule chemical drugs that can be used for the diagnosis, prevention or treatment of kidney-bladder clearance pathway diseases, or functional peptides used in kidney-bladder clearance pathway diseases, which can be peptide chains or cyclic peptides, or therapeutic proteins for treating kidney or bladder diseases.

[0023] In certain embodiments, the third domain of albumin or its variant is combined with a functionalized fragment, or the third domain a subunit of albumin or its variant is combined with a functionalized fragment to form a fusion / recombinant protein to obtain a functionalized protein shell.

[0024] In certain embodiments, the functional fragment can be connected to the protein shell by a chemical, an amino acid or a peptide. In certain embodiments, the chemical is a small molecule coupling agent for chemical grafting (e.g., diphenylcyclooctyne-N-hydroxysuccinimide), which can also be replaced by any connector that effectively connects the protein shell and the functionalized fragment. In other embodiments, the protein shell can be prepared into a fusion protein with the functional peptide or with the therapeutic protein by genetic engineering editing (e.g., by eukaryotic expression or prokaryotic expression). For example, a eukaryotic / prokaryotic expression vector (e.g., Pichia pastoris, Escherichia coli, 293HT cells) can be used to express the fusion protein.

[0025] In certain embodiments, the functionalized fragment can be a short peptide PLZ4 or a cyclic peptide thereof targeting bladder cancer, wherein the amino acid sequence of the PLZ4 is QDGRMGF (SEQ ID NO: 10), and the amino acid sequence of the cyclic peptide (cPLZ4) is cQDGRMGFc (SEQ ID NO: 15), RGD or its fusion protein, prostate-specific membrane antibody (PSMA), and other short peptides and derivatives thereof targeting bladder cancer, or other small molecules.

[0026] In certain embodiments, the functionalized fragment is EphB4-EBD protein (abbreviated as EphB4 in some embodiments), a protein or other small molecule that has the effect of treating bladder cancer.

[0027] EphB4, also known as tyrosine kinase receptor B4, is a member of the Eph family of tyrosine kinase receptors. The signals induced by its interaction with its cognate ligand Ephrinb2 play a key role in vascular development. The structure of EphB4 consists of three parts: extracellular, transmembrane, and intracellular. The extracellular domain of EphB4 is located outside the cell and mainly includes three parts: (1) Ephrin binding domain (EBD): a globular domain that binds to the ephrinB2 ligand at the N-terminus, (2) Cys rich domain: a cysteine-rich domain, and (3) Fibronectin Type III like domain: two fibronectin domains. The intracellular domain of the EphB4 receptor is the tyrosine kinase active region, including: Tyr kinase domain (TK), sterile α motif (SAM) and PDZ domain, which can promote the phosphorylation of its own tyrosine residues to enhance enzyme activity, and then catalyze the phosphorylation of various substrate proteins in the cell, activate intracellular protein kinases, and thus transmit intracellular information to the extracellular space. The EphB4 receptor binds to the EphrinB2 ligand through its extracellular domain, thereby promoting phosphorylation of the intracellular tyrosine kinase active region, generating signal transduction, promoting endothelial cell adhesion and migration, and inhibiting the production of multiple growth factors and promoting angiogenesis in vivo. sEphB4 is a soluble extracellular domain of the EphB4 protein that binds to the EphrinB2 ligand, thereby blocking activation of the EphB4 and EphrinB2 signaling pathways and inhibiting tumor angiogenesis.

[0028] As mentioned above, the extracellular domain of the tyrosine kinase receptor B4 (EphB4) consists of three components: a globular domain (Ephrin binding domain, EBD) that binds to the ephrinB2 ligand, a cysteine-rich domain, and two fibronectin Type III-like domains. The EphB4-EBD protein can be attached to the protein shell, and the DIII-EphB4-EBD fusion protein can be expressed and purified through genetic engineering. The DIII-EphB4-EBD protein is a small molecule that can covalently bind to cyanine dye molecules and undergo drug metabolism through the renal clearance pathway. The DIII-EphB4-EBD protein can be used as an antagonist to block the signaling pathway between EphB4 and its ligand EphrinB2, thereby inhibiting tumor angiogenesis. At the same time, the covalent complex formed by the DIII-EphB4-EBD recombinant protein and the cyanine dye molecule can also be used for the imaging and treatment of diseases of major organs and tissues in the kidney-bladder metabolic pathway.

[0029] The EphB4 (EPH receptor B4) receptor protein described in the present invention can be an EphB4 receptor protein of human origin, wherein the EphB4-EBD protein can be the extracellular domain of EphB4, its subunits and variants, or the globular domain that binds to the ephrinB2 ligand, and its fragments, mutants, edited and modified products, and repeat fragments and other variants.

[0030] The amino acid sequence of an exemplary human EphB4 full-length protein is shown in SEQ ID NO:11.

[0031] The amino acid sequence of an exemplary EphB4-EBD protein is shown in SEQ ID NO:12.

[0032] In certain embodiments, in the complex of the present invention, the protein shell and the functionalized fragment form a fusion / recombinant protein. In certain embodiments, the functionalized fragment is selected from the group consisting of short peptide PLZ4, cyclic peptide cPLZ4, RGD, or EphB4-EBD protein.

[0033] In some embodiments, the fusion protein is (DIII) a -(RGD) b , wherein a and b are each independently selected from an integer ≥ 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0034] In some embodiments, the fusion / recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Furthermore, radionuclides can be labeled on the complexes of the present invention (e.g., on the protein shell or functionalized fragments) to achieve early imaging and treatment of diseases. The radionuclides can be imaging nuclides, such as Cu-64, F-18, etc., or therapeutic nuclides, such as Lu-177, Y-90, I-131, I-125, Ac-225, P-32, etc.

[0035] In one aspect, the present application also provides a method for preparing a functionalized protein shell, comprising the following steps:

[0036] Step S1: preparing solution A, which contains the third domain of albumin or a variant thereof, or the α subunit of the third domain of albumin or a variant thereof;

[0037] Step S2: After ultrafiltration and centrifugation of Solution A through an ultrafiltration tube, the filtrate is discarded, an alkaline solution (e.g., sodium bicarbonate solution) is added, and the mixture is vortex-mixed at room temperature to achieve an alkaline pH. The mixed solution is Solution B;

[0038] Step S3: Vortex mix the small molecule coupling agent for chemical grafting (e.g., diphenylcyclooctyne-N-hydroxysuccinimide) and solution B at room temperature, react at room temperature or under heating conditions, remove excess coupling agent by ultrafiltration and centrifugation through an ultrafiltration tube, and dissolve the product with phosphate buffer solution (PBS) to obtain compound 1.

[0039] Step S4: Vortex mix compound 1 and the raw material of the functional fragment (e.g., short peptide mother liquor) at room temperature, react overnight at room temperature or under heating conditions, remove excess functional fragments (e.g., short peptides) by ultrafiltration and centrifugation through an ultrafiltration tube, and finally dissolve the product with phosphate buffered saline (PBS) to obtain compound 2.

[0040] In certain embodiments, solution A in step S1 is the third domain of albumin or its variant, or the a subunit of the third domain of albumin or its variant dissolved in phosphate buffered saline (PBS) or pure water to prepare solutions of desired concentrations.

[0041] In certain embodiments, the ultrafiltration tube in step S2 is of 1 kDa-20 kDa model, more preferably 10 kDa; the centrifugal speed is 1000 g-12000 g, more preferably 4000 g; the centrifugal time is 10 min-120 min, more preferably 40 min; the centrifugal temperature is 0-60 ° C, more preferably 4 ° C; the pH range is 7.8-10, more preferably 8.3; the vortex mixing time is 5-60 s, more preferably 10 s.

[0042] In certain embodiments, the coupling agent in step S3 is prepared by dissolving in dimethyl sulfoxide (DMSO) solvent and stored at -20°C for future use; the molar ratio of the coupling agent to solution B is 0.01:1-1:10, more preferably 0.3:1; the vortex mixing time is 5-60s, more preferably 10s; the heating temperature range of the reaction is 20°C-50°C, more preferably 37°C; the reaction time range is 30min-180min, more preferably 60min; the ultrafiltration tube is a 1kDa-20kDa model, more preferably 10kDa; the centrifugal speed is 1000g-12000g, more preferably 4000g; the centrifugation time is 10min-120min, more preferably 40min; the centrifugation temperature is 0-60°C, more preferably 4°C.

[0043] The short peptide in step S4 can be any functional peptide used in kidney-bladder clearance pathway diseases. It can be a peptide chain or a cyclic peptide. The present invention uses bladder cancer as an example. The short peptide cPLZ4 (amino acid sequence: cQDGRMGFc) targeting bladder cancer is selected. The short peptide mother solution is dissolved with pure water or phosphate buffer (PBS) and stored at -20°C for standby use; the molar ratio of compound 1 to the short peptide is in the range of 0.5:1-1:1000, more preferably 1:100; the vortex mixing time is 5-60s, more preferably 10s; the heating temperature range is 20°C-50°C, more preferably 37°C; the overnight reaction time is 6-14h, more preferably 12h; the ultrafiltration tube is 1kDa-20kDa model, more preferably 10kDa; the centrifugation speed is 1000g-12000g, more preferably 4000g; the centrifugation time is 10min-120min, more preferably 40min; the centrifugation temperature is 0-60°C, more preferably 4°C.

[0044] The present invention also provides a method for preparing the composite, comprising the following steps:

[0045] S1: preparing solution A containing the functionalized protein shell, and preparing solution B containing small molecules (such as photosensitizers (such as dye molecules, such as cyanine dye molecules), nuclides, small molecule drugs (such as small molecule chemotherapy drugs)).

[0046] S2: Vortex-mixing solution A and solution B at room temperature to obtain the complex at room temperature or under heating conditions.

[0047] In certain embodiments, solution A in step S1 is a solution of the functionalized protein shell dissolved in phosphate buffer solution (PBS) or pure water to prepare a solution of different required concentrations; solution B is a small molecule mother solution prepared by dissolving in dimethyl sulfoxide (DMSO) solvent and stored at -20°C for future use.

[0048] In certain embodiments, the vortex mixing time in step S2 is 5-60s, more preferably 10s; the heating temperature range is 20°C-80°C, more preferably 60°C; the heating time is 5-120min, more preferably 10min; the molar ratio of solution A and solution B is 0.01:1-1:100, more preferably 1:1.

[0049] In one aspect, the present application provides use of the complex of the present invention as a fluorescent probe.

[0050] In one aspect, the present application provides a pharmaceutical composition comprising a complex of the present invention, optionally further comprising one or more pharmaceutical carriers or excipients. The pharmaceutical composition of the present invention can be administered by any suitable means, such as intravenous injection, intravesical administration, intratumoral injection, intraperitoneal injection, etc. The complex of the present invention can not only be used for near-infrared fluorescence imaging, but can also achieve tumor-killing effects through in vivo photodynamic therapy. Therefore, in certain embodiments, the pharmaceutical composition is a contrast agent, and in other embodiments, the pharmaceutical composition is a therapeutic agent.

[0051] The complex provided by the present invention has a small molecular weight, and a strong covalent bond can be formed between the third structural domain DIII of albumin and the small molecule, so it can be metabolized to the bladder through the renal clearance pathway. Therefore, it can be administered by intravenous injection to treat diseases of the main organs and tissues of the kidney-bladder metabolic pathway.

[0052] In another aspect, the present application provides a method for diagnosing, preventing, or treating a urinary system disease, comprising administering a complex of the present invention to a subject in need thereof. The disease includes, but is not limited to, urinary system tumors (e.g., renal cancer, Wilms' tumor, bladder rhabdomyosarcoma, renal pelvis / ureter cancer, bladder cancer, urethral cancer, epithelial urinary tract cancer, etc.), urinary system injuries (e.g., kidney damage), and the like. The disease may be a disease of a major organ or tissue in the kidney-bladder metabolic pathway. The method includes, but is not limited to, photodynamic therapy, imaging, surgical navigation, radiotherapy, and chemotherapy.

[0053] In another aspect, the present application provides the use of the complex of the present invention for preparing a medicament for diagnosing, preventing, or treating a urinary system disease. Such diseases include, but are not limited to, urinary system tumors (e.g., the tumors described above), urinary system injuries (e.g., kidney damage), and the like. Such diseases may occur in major organs and tissues in the kidney-bladder metabolic pathway.

[0054] In one aspect, the present application provides a kit comprising the complex of the present invention; optionally, the kit further comprises any one or more selected from the following: antibodies, primers, reagents for fixing and / or permeabilizing cells, or any combination thereof.

[0055] In one aspect, the present application provides a method for targeting cells, comprising contacting the complex as described above with the cells; optionally, after contact, irradiating the cells with a laser to obtain imaging of the cells.

[0056] In one aspect, the present application provides use of the aforementioned complex in preparing a kit for targeting cells or obtaining cell imaging.

[0057] In certain embodiments, the complex is targeted to the cell by binding to a cell surface molecule, cell surface protein, or cell surface receptor expressed on the cell.

[0058] In certain embodiments, the cells are located in the urinary system, such as the kidney, ureter, bladder, or urethra. In certain embodiments, the cells are tumor cells. The tumors may be tumors occurring in any part of the urinary system, including but not limited to renal cell carcinoma, Wilms' tumor, bladder rhabdomyosarcoma, renal pelvis / ureteral cancer, bladder cancer, urethral cancer, epithelial urinary tract cancer, and the like.

[0059] In certain embodiments, the cell is present in a tissue or a living organism.

[0060] In another aspect, the present application provides an imaging method, comprising using the aforementioned complex as an imaging agent.

[0061] In certain embodiments, the imaging is near infrared 1 (NIR-I) and / or near infrared 2 (NIR-II) fluorescence imaging.

[0062] In certain embodiments, the imaging method is fluorescence imaging of cells, tissues, or living organisms.

[0063] Definition of terms

[0064] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0065] As used herein, the term "wild-type human serum albumin" refers to biologically active, naturally occurring human serum albumin. The amino acid sequence of wild-type human serum albumin can be readily obtained from various public databases (e.g., the GenBank database). In certain embodiments, the GenBank database number for human serum albumin is AEE60908.1.

[0066] As used herein, the term "cysteine," also referred to as "Cys," is a common amino acid found in living organisms. Cysteine ​​is the only amino acid among the more than 20 amino acids that make up proteins that has a reducing group, a sulfhydryl group (-SH).

[0067] As used herein, the term "mercapto", also known as thiol or thiol, is a negative monovalent functional group consisting of a sulfur atom and a hydrogen atom connected together, and its chemical formula is -SH.

[0068] As used herein, the term "protein tertiary structure" refers to a three-dimensional structure in which the polypeptide chain of a protein is further coiled or folded into a regular pattern based on various secondary structures.

[0069] As used herein, the term "protein quaternary structure" refers to a protein composed of two or more polypeptide chains with independent tertiary structures, wherein the polypeptide chains are combined with each other through secondary bonds to form a spatial structure.

[0070] As used herein, the term "subunit" refers to each polypeptide chain with an independent tertiary structure in the quaternary structure of a protein.

[0071] As used herein, the term "domain" refers to the basic unit that constitutes the tertiary structure of a protein, which has a unique spatial conformation. Generally, different domains of a protein can be clearly distinguished in space. In certain embodiments, when a protein is composed of multiple polypeptide chains, the domain of the protein comprises multiple subunits.

[0072] Advantageous Effects of the Invention

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] 1. The complex of the present invention has a small molecular weight and size, allowing for intravenous administration to treat diseases of key organs and tissues involved in the kidney-bladder metabolic pathway. For example, compared with invasive intravesical instillation, intravenous administration avoids the pain associated with cystoscopy or urethral catheterization. However, cystoscopy is expensive, and intravenous administration can alleviate the patient's financial burden to a certain extent.

[0075] 2. Compared with the near-infrared fluorescent probes used clinically, the complex containing photosensitizers such as cyanine dyes of the present invention has strong photostability, high light energy conversion rate, high quantum yield, and significantly extended emission wavelength, and is suitable for deep tissue imaging and photodynamic therapy. Therefore, it can effectively carry out photodynamic therapy for diseases of the main organs and tissues of the kidney-bladder metabolic pathway. For high-risk NMIBC patients, not only do they face the economic pressure brought about by the extremely expensive treatment costs, but the efficacy of chemotherapy drugs (such as BCG, a first-line drug in clinical practice) is still limited. In addition to radical cystectomy, there are almost no other effective rescue treatment options. The present invention undoubtedly provides a good alternative treatment measure for high-risk NMIBC patients who are refractory to BCG.

[0076] 3. In the complex of the present invention, the small molecule can also be a nuclide or a small molecule chemotherapy drug, etc. Therefore, the complex of the present invention can be used in combination with multiple therapies such as radiotherapy, chemotherapy, and photodynamic therapy.

[0077] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG1 is a mass spectrum and electrophoresis diagram of the protein shell (DIII-cPLZ4) functionalized with the albumin third domain variant by chemical modification as described in Example 1 of the present invention.

[0079] FIG2 is the absorption and emission spectra of the complex Cyanine@cDIII-PLZ4 described in Example 1 of the present invention.

[0080] FIG3 is a fluorescence enhancement diagram of the cyanine dye described in Example 1 of the present invention after binding to the chemically modified functionalized albumin third domain variant.

[0081] FIG4 is an electrophoresis diagram of the fusion protein DIII-PLZ4 expressed by gene editing in Example 2 of the present invention ( FIG4A ) and fluorescence enhancement diagrams after binding to a cyanine dye ( FIG4B-C ).

[0082] FIG5 shows the photostability of the complex described in Example 2 of the present invention in phosphate buffered saline (PBS) and mouse urine, respectively.

[0083] FIG6 is a graph showing the pharmacokinetic comparison of the complex and free cyanine dye in Example 3 of the present invention in mice.

[0084] FIG7 is a graph showing the results of upright fluorescence microscopy and flow cytometry of the uptake of different drugs by different cells described in Example 4 of the present invention.

[0085] FIG8 shows the target binding site of the verification complex on bladder cancer cells described in Example 4 of the present invention.

[0086] FIG9 shows the killing effect of the complex described in Example 5 of the present invention on bladder tumor cells at different concentrations and light doses.

[0087] FIG10 is a graph showing the confocal microscopy and flow cytometry results of monitoring the photodynamically mediated generation of reactive oxygen species in bladder tumor cells by the complex described in Example 6 of the present invention.

[0088] FIG11 shows the construction of the orthotopic bladder cancer mouse model described in Example 7 of the present invention.

[0089] FIG12 shows the fluorescence enrichment of the complex described in Example 8 of the present invention in mice with orthotopic bladder cancer.

[0090] FIG13 shows the photodynamic therapy-mediated tumor killing and postoperative survival monitoring of the complex described in Example 9 of the present invention in mice bearing BALB / c orthotopic bladder cancer.

[0091] FIG14 shows the photodynamic therapy-mediated tumor killing and postoperative survival monitoring of the complex described in Example 10 of the present invention in C57BL / 6 mice with orthotopic bladder cancer.

[0092] FIG15 shows the toxic effects of the complex described in Example 11 of the present invention on healthy mice.

[0093] Figure 16 shows radionuclides. 64 Cu labeling and preliminary evaluation

[0094] Figure 17 shows radionuclides. 177 Lu labeling and preliminary evaluation

[0095] Figure 18 shows the effects of photodynamic therapy by intravenous injection (iv) and intravesical infusion (infusion) of Dili-fused tumor-targeting peptide (DIII-tar pep)

[0096] FIG19 is a diagram showing the yeast expression of EphB4-DIII fusion protein and its evaluation of binding to photosensitizers.

[0097] Figure 20 shows the expression of DIII-RGD and (DIII)m-(RGD)n and the evaluation of their binding to photosensitizers

[0098] Figure 21 shows the targeting of DIII-RGD and (DIII)m-(RGD)n expression to three different tumor cells

[0099] Figure 22 shows the evaluation of DIII-RGD and (DIII)m-(RGD)n in animals

[0100] Sequence information

[0101] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0102] Table 1: Description of sequences DETAILED DESCRIPTION

[0103] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0104] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE. CULTURE) (RI Freshney, ed. (1987)).

[0105] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0106] Example 1 Preparation of the complex Cyanine@DIII-cPLZ4

[0107] The complex of the present invention is formed by covalent bonding of the third domain of albumin and its variants and a dye molecule, wherein the core chromophore is a dye molecule, the covalently bonded portion outside the core chromophore is the albumin variant, and the protein shell portion of the probe can be functionalized by genetic engineering editing or chemical grafting.

[0108] This example provides a functionalized protein shell portion, DIII-cPLZ4, obtained by chemically modifying and linking the third domain of albumin with a cyclic PLZ4 (cPLZ4, amino acid sequence: cQDGRMGFc), a cyclic peptide that specifically targets the short peptide PLZ4. The chemical preparation method includes the following specific steps:

[0109] Solution A (85 μM, 100 μL) of the third domain of albumin was prepared. Solution A was ultrafiltered and centrifuged at 4°C through a 10 kDa ultrafiltration tube. The filtrate was discarded, and 0.1 M sodium bicarbonate solution was added and vortexed for 10 seconds at room temperature to adjust the pH to 8.3. This mixture was referred to as solution B. 2.565 μL of solution B was vortexed for 10 seconds with 10 mM diphenylcyclooctyne-N-hydroxysuccinimide (DBCO-NHS) at room temperature. The mixture was reacted at 37°C for 1 hour. The excess DBCO was removed by ultrafiltration and centrifugation at 4°C through a 10 kDa ultrafiltration tube. The volume of the dissolved product was made up to 100 μL with phosphate buffered saline (PBS) to obtain compound 1. Compound 1 and 3 μL of 8.55 mM cPLZ4 stock solution (amino acid sequence: cQDGRMGFc) were vortex-mixed at room temperature for 10 seconds, reacted overnight at 37°C for 12 hours, and ultrafiltration and centrifugation at 4°C through a 10 kDa ultrafiltration tube to remove excess cPLZ4. The product was dissolved in phosphate buffered saline (PBS) to a total volume of 100 μL to obtain the functionalized protein shell DIII-cPLZ4c, whose amino acid sequence is shown in SEQ ID NO: 13. Mass spectrometry (Figure 1A) and western blot electrophoresis (Figure 1B) indicated that the molecular weight of DIII-cPLZ4 was approximately 23 kDa.

[0110] This example also provides a method for preparing the complex Cyanine@DIII-cPLZ4:

[0111] The functionalized protein shell DIII-cPLZ4 and the cyanine dye IR-780 were mixed at a molar ratio of 1:1 and heated at 60°C for 10 minutes to obtain the compound Cyanine@DIII-cPLZ4.

[0112] Example 2 Fusion and recombinant expression of the complex Cyanine@DIII-PLZ4

[0113] The complex of the present invention is formed by covalently binding a fusion expression protein of the third structural domain DIII of albumin and a PLZ4 short peptide to a dye molecule, wherein the core chromophore is a dye molecule, the covalently bound portion outside the core chromophore is the albumin fusion protein, and the protein shell portion of the probe can be functionalized by genetic engineering editing or chemical grafting.

[0114] This embodiment provides a functionalized protein shell portion, DIII-PLZ4, which is formed by covalently binding a fusion recombinant protein of the third domain of albumin and a specific targeting short peptide PLZ4 to a dye molecule. The preparation method includes the following specific steps:

[0115] The Dili-PLZ4 fusion protein was expressed using a eukaryotic / prokaryotic expression vector (eg, Pichia pastoris, Escherichia coli, 293HT cells). Gel electrophoresis ( FIG. 4A ) confirmed that the molecular weight of Dili-PLZ4 was approximately 23 kDa.

[0116] This example also provides a method for preparing the dye@DIII-PLZ4 complex:

[0117] The functionalized protein shell DIII-PLZ4 and the dye IR-783 were mixed at a molar ratio of 1:1 and heated at 60°C for 10 minutes to obtain the compound dye@DIII-PLZ4.

[0118] Example 3 In vitro characterization of the complexes Cyanine@DIII-cPLZ4 and Cyanine@DIII-PLZ4

[0119] Absorption and emission spectra of the Cyanine@DIII-cPLZ4 complex prepared in Example 1 at varying gradient concentrations were measured, using phosphate buffered saline (PBS) as the base for zero adjustment. The results showed that the complex had absorption peaks (as shown in Figure 2A) and emission peaks (as shown in Figure 2B) in the near-infrared region I. Mixing the cyanine dye and DIII-PLZ4 significantly enhanced the fluorescence intensity (as shown in Figure 3A). Fluorescence imaging of the complex in the near-infrared region I (as shown in Figure 3B) and near-infrared region II (as shown in Figure 3C) also confirmed the significant fluorescence enhancement.

[0120] Figure 4 shows the electrophoresis diagram of the fusion protein DIII-PLZ4 expressed by gene editing (Figure 4A) and the fluorescence enhancement diagram after binding to cyanine dye (Figure 4B-C).

[0121] The 85 μM complex Cyanine@DIII-PLZ4 prepared in Example 2 was diluted 28.3-fold (3 μM) with phosphate buffered saline (PBS) and fresh mouse urine, respectively. The latter was incubated at 37°C for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h before near-infrared imaging in the first and second regions. The results showed that the fluorescence intensity of the complex was not significantly reduced in urine at physiological temperature (as shown in Figure 5A), and its fluorescence intensity did not change significantly after incubation for up to 6 h (as shown in Figure 5B).

[0122] Example 4 Comparison of the pharmacokinetic results of the complex Cyanine@DIII-PLZ4 and free cyanine dye in mice

[0123] 3 μM Cyanine@DIII-PLZ4 and 3 μM free cyanine dye prepared in Example 2 were injected into normal BALB / c mice undergoing full-body hair removal via tail vein injection. Near-infrared second-zone fluorescence imaging was performed on the front and side of the mice 30 minutes, 1 hour, 2 hours, and 3 hours later. The results showed that Cyanine@DIII-PLZ4 can be metabolized to the bladder through the renal clearance pathway, and fluorescence enrichment was observed in the bladder at 30 minutes, while the free cyanine dye was retained in the liver (Figure 6).

[0124] Example 5 Targeting ability of Cyanine@DIII-cPLZ4 to tumor cells

[0125] The targeting ability of Cyanine@DIII-PLZ4 to bladder cancer cells was observed using an upright fluorescence microscope. Human bladder cancer cell line T24 cells were plated onto 8-well cell culture slides, with 10,000 cells per well. After incubation for 24 hours, the culture medium was aspirated and the slides were gently washed 2-3 times with sterile phosphate-buffered saline (PBS). 100 μL of 4% paraformaldehyde was added to each well, and the slides were fixed on ice for 10-20 minutes, followed by repeated washing with phosphate-buffered saline (PBS). 100 μL of each of the three drug groups (Cyanine@DIII-PLZ4 diluted in serum-free medium, Cyanine@DIII diluted in serum-free medium, and serum-free medium) were added to each well. The slides were incubated for 10 minutes, washed repeatedly with PBS, and a mung bean-sized drop of DAPI reagent containing a fluorescence quencher was added to each well. The slides were sealed and observed under an upright fluorescence microscope. Compared to the other control groups, the Cyanine@DIII-PLZ4 group exhibited higher fluorescence targeting to T24 cells (see Figure 7a). Two human bladder cancer cell lines, T24 and 5637, and a normal urothelial cell line, SV-HUC-1, were plated in three 8-well cell culture slides, with 10,000 cells per well. All other steps were the same as above, and the cells were observed under an upright fluorescence microscope. The results showed that Cyanine@DIII-PLZ4 had high targeting activity against the two human bladder cancer cell lines, T24 and 5637, but low targeting activity against the normal urothelial cell line, SV-HUC-1 (see Figure 7B).

[0126] Flow cytometry was used to investigate the uptake of Cyanine@cDIII-PLZ4 by human bladder cancer cells. Two human bladder cancer cell lines were plated in six-well plates, with 200,000 cells per well. After incubation for 24 hours, the culture medium was aspirated and the cells were gently washed two to three times with phosphate-buffered saline (PBS). The cells were then replaced with serum-free medium containing Cyanine@DIII-PLZ4 and Cyanine@DIII (3 μM), respectively, and incubated at 37°C for 1, 2, 3, 4, 5, and 6 hours. Three replicates were set up at each time point, and a serum-free medium control was included as a control. After incubation, each well was washed three to five times with PBS to remove any unbound drug and aspirated. The adherent cells were then digested into single cells using trypsin, centrifuged at 1000 rpm for 3 minutes at room temperature, resuspended in serum-free medium, and immediately analyzed by flow cytometry. The results showed that compared with the control group, the two human bladder cancer cell lines had a higher uptake of the Cyanine@DIII-cPLZ4 drug (as shown in Figures 7C-D, GH), and as the incubation time increased, the tumor cells' uptake of the drug increased, and the uptake capacity after 6 hours could reach more than 90% (as shown in Figures 7E-F, GH).

[0127] Furthermore, the targeted binding site of Cyanine@DIII-cPLZ4 on bladder cancer cells was verified to be the αvβ3 integrin receptor. First, flow cytometry was used to examine the expression of the αvβ3 integrin receptor on the cell surfaces of two human bladder cancer cell lines, T24 and 5637, and a normal urothelial cell line, SV-HUC-1. Each cell line was plated in separate 6-well plates, with 200,000 cells per well, and incubated in an incubator for 24 hours. The adherent cells were then digested with trypsin to form single suspended cells, centrifuged at 1000 rpm for 3 minutes, resuspended in serum blocking solution (containing 0.5% phosphate buffered saline), incubated at room temperature for 20 minutes, and centrifuged at 1000 rpm for 3 minutes. A diluted primary antibody (1:100) was added, incubated at 37°C for 2 hours, and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, the cells were resuspended in PBS and washed for 5 minutes, and centrifuged at 1000 rpm for 3 minutes. The washing step was repeated three times. Diluted secondary antibody (1:100) was added dropwise, incubated at 37°C for 30 minutes, centrifuged at 1000 rpm for 3 minutes, the supernatant discarded, resuspended in PBS, washed for 5 minutes, and centrifuged at 1000 rpm for 3 minutes. This washing step was repeated three times. The results show that both human bladder cancer cells highly express αvβ3, while normal urothelial cells express it negatively (see Figure 8A). Next, T24 cells were plated in 8-well glass culture plates, with 10,000 cells per well. After incubation for 24 hours, anti-αvβ3 reagent was added, incubated at 37°C for 1 hour, and washed three times with PBS for 5 minutes. The cells were fixed with 4% paraformaldehyde on ice for 10-20 minutes and washed repeatedly with phosphate-buffered saline (PBS). 1001 μL of Cyanine@DIII-cPLZ4 diluted in serum-free medium at 1 μM was added to the cells and incubated for 10 minutes. After repeated washing with PBS, a mung bean-sized drop of DAPI reagent containing a fluorescence quencher was added to each well. The slides were sealed and observed under an upright fluorescence microscope. The results showed that the tumor-targeting ability of the group treated with anti-αvβ3 antibodies was significantly weakened (see Figure 8B), indicating that the αvβ3 integrin receptor is the binding site of the complex in targeting tumor cells.

[0128] Example 6 Killing effect of the complex on bladder tumor cells at different concentrations and light doses

[0129] MB-49 cell lines were plated in 96-well plates with 5,000 cells per well. After being placed in a cell culture incubator for 24 hours, the cells were washed twice with PBS. There were six groups for cell treatment, namely Cyanine+laser, Cyanine@DIII+laser, Cyanine@DIII-PLZ4+laser, Cyanine+dark, Cyanine@DIII+dark, and Cyanine@DIII-PLZ4+dark. Cyanine, Cyanine@DIII, and Cyanine@DIII-PLZ4 were diluted with serum-free culture medium to different concentrations of 0, 1.25, 2.5, 5, and 10 μM, respectively. After incubating the cells with different concentrations of drugs in the incubator for 4 hours, the drugs were repeatedly washed with PBS and replaced with serum-free culture medium. The culture dish of the laser group was placed under an 808 nm laser and irradiated for 8 minutes with a power of 0.3 w / cm 2 The non-laser treatment group was kept in the dark. After 12 hours in the incubator, the cells were incubated with CCK8 reagent diluted in serum-free medium for 30 minutes, and the results were analyzed using a microplate reader. The results showed that compared with the other control groups, the Cyanine@DIII-cPLZ4+laser group had a significant cell-killing effect, and the killing effect increased with increasing drug concentration. The non-laser treatment group showed almost no cell killing, indicating that the drug has a good biosafety (Figure 9A).

[0130] MB-49 cells were plated in a 96-well plate with 5,000 cells per well. After 24 hours in a cell culture incubator, the cells were washed twice with PBS. Cyanine@DIII-cPLZ4 was diluted to 5 μM in serum-free medium. After incubating the cells for 4 hours, the drug was repeatedly washed with PBS and replaced with serum-free medium. The culture dishes in the laser group were placed under 808 nm laser irradiation for 8 minutes, with light doses set at 0, 0.3, 0.5, and 1 w / cm 2 The cells were digested into single cells using trypsin, centrifuged, and the supernatant discarded. After staining with PI for 30 minutes, the cells were washed with PBS, resuspended, and centrifuged to discard the supernatant. This washing step was repeated several times before analysis by flow cytometry. The results showed that the cell-killing activity of Cyanine@DIII-PLZ4 increased with increasing light dose (Figure 9B).

[0131] MB-49 cells were incubated with 8.5 μM Cyanine, Cyanine@DIII-PLZ4 and PBS as controls for 4 h. The cells were treated as above and then irradiated with 808 nm, 0.3 w / cm 2The cells were irradiated with laser for 8 minutes and treated with Calcein AM / PI live-dead cell double staining kit. Under confocal microscopy, it was observed that Cyanine@DIII-PLZ4 drug had significant cell killing ability under laser irradiation (see Figure 9C).

[0132] Example 7 Cyanine@DIII-cPLZ4 on the Photodynamically Induced Reactive Oxygen Species Production in Bladder Tumor Cells

[0133] MB-49 cells were plated in confocal culture dishes with 300,000 cells per well. After 24 hours in a cell culture incubator, the cells were washed twice with PBS. Cyanine@DIII-cPLZ4, Cyanine, and PBS were diluted to 8.5 μM in serum-free medium. After incubating the cells for 4 hours, the drugs were repeatedly washed with PBS and replaced with serum-free medium. The culture dishes of the laser group were placed in an 808 nm, 0.3 w / cm 2 Laser irradiation was performed for 8 minutes. Excess medium was aspirated, and cells were incubated with 100 μL of 5 μM DCFH-DA dye diluted in serum-free medium for 15 minutes. The cells were washed three times with PBS for 5 minutes each, then replaced with serum-free medium and observed under a confocal microscope. The results show that the Cyanine@DIII-cPLZ4 group produced more reactive oxygen species under laser irradiation than the control group (see Figure 10A).

[0134] Flow cytometry also demonstrated similar results. MB-49 cells were plated in a 6-well plate, with 200,000 cells per well. Subsequent cell treatment, drug administration, and laser irradiation procedures were the same as described above. Under a standard microscope, the Cyanine@DIII-cPLZ4 group was observed to produce photodynamically induced reactive oxygen species (ROS) under laser irradiation, leading to swelling and rupture of the tumor cell membrane (see Figure 10C). The adherent cells were then digested with trypsin to single cells, centrifuged at 1000 rpm for 3 minutes, and washed twice with PBS for 5 minutes each. The cells were then incubated with 1 mL of 5μM DCFH-DA dye diluted in serum-free medium for 15 minutes at room temperature. The cells were centrifuged at 400g for 3 minutes, and the supernatant discarded. The cells were washed twice with PBS for 5 minutes each. The cells were resuspended in 1 mL of serum-free medium and analyzed by flow cytometry. The results show that the Cyanine@DIII-cPLZ4 group produced more ROS under laser irradiation than the control group (see Figure 10B).

[0135] Example 8 Construction of orthotopic bladder cancer model mice

[0136] The abdominal cavity of 6-8 week old BABL / c female mice was locally depilated in advance, and 100 μL of 7% chloral hydrate was injected into the abdominal cavity of each mouse. The bladder position and the surrounding skin were disinfected with alcohol and iodine tincture, a small incision was made at the position with surgical scissors, and the bladder was carefully exposed with sterilized tweezers. A 24G indwelling needle external catheter was slowly inserted into the mouse's urethra to the bladder, the mouse urine was slowly drained out, and PBS was injected into the bladder to flush and fill it. The indwelling needle was slowly inserted from the catheter to damage the epidermal mucosa of the bladder (as shown in Figure 11A). The PBS was removed, and 100 μL and 1×10 6 The cells were resuspended in 1 mL of MB-49 and the urethra was ligated for 1 hour. Subsequently, multimodal imaging, including physiological anatomy (see Figure 11B), hematoxylin and eosin staining (see Figures 11C-D), magnetic resonance imaging (see Figure 11E), and ultrasound imaging (see Figure 11F), confirmed the successful establishment of an orthotopic bladder cancer model. The hematoxylin and eosin staining results (see Figures 11C-D) demonstrate urothelial cell proliferation without damage to the middle and muscle layers, demonstrating the establishment of non-muscle invasive bladder cancer.

[0137] Example 9 Fluorescence enrichment and biodistribution of Cyanine@DIII-cPLZ4 in orthotopic bladder cancer tumor model mice.

[0138] The in situ bladder cancer mouse model described in Example 8 was constructed, and the mice were subjected to whole-body hair removal. The mice were injected with Cyanine@DIII-PLZ4 drug via the tail vein, and near-infrared two-zone fluorescence imaging was performed 10min, 30min, 1h, 2h, 3h, 4h, 5h, and 6h after drug injection. It can be seen from Figure 12A that the drug is metabolized to the bladder through the renal clearance pathway, and a clear fluorescence signal can be seen in the bladder tumor at 30min. The bladder was repeatedly rinsed with PBS to wash away the excess dye that was not bound to the bladder lining. The bladder tumor lining layer was still bound to the drug, indicating that the drug was well targeted to the bladder tumor (as shown in Figure 12E). After 3h, the bladder tumor was dissected down, and the retention of fluorescence signals in the bladder was observed (as shown in Figures 12B-D).

[0139] Example 10 Tumor killing effect of Cyanine@DIII-cPLZ4 on C57BL / 6 type orthotopic bladder cancer mice

[0140] Treatment strategy (see Figure 13A): On the first day, normal 7-8 week old C57BL / 6 female mice were subjected to intravesical mechanical injury and intravesical instillation of 100 μL, 2×10 6An orthotopic bladder cancer mouse model was constructed using the Luc-MB-49 mouse bladder cancer cell line, as shown in Figure 13A. On day 6, mice that had undergone local hair removal were intraperitoneally injected with 100 μL of 3 mg / mL D-Luciferin luciferin substrate. Bioluminescence imaging was performed 5 minutes later, and mice showing bioluminescence signals were selected for group treatment (see Figure 13B). MRI and ultrasound imaging were performed on the same day to further confirm the presence of tumor space in the bladder (see Figure 13C). Tumor-bearing mice were randomly divided into three groups (n=5 per group) for treatment: Cyanine@DIII-PLZ4+Laser group, PBS+Laser group, and BCG group. The first two groups of mice were injected with 100uL of 85uM Cyanine@DIII-PLZ4 and phosphate buffered saline (PBS) through the tail vein. After 2 hours, a 24G indwelling needle catheter was inserted into the mouse urethra to the bladder. Phosphate buffered saline (PBS) was instilled into the bladder through the catheter. The excess dye not bound to the bladder lining was repeatedly rinsed to wash away the excess dye, and the bladder was filled with PBS to keep it full. A 400uM optical fiber connected to an 808nm laser was inserted into the mouse bladder through the catheter. Under ultrasound imaging guidance, 0.3w / cm2 of light was irradiated to the mouse in situ bladder tumor. 2, 8 minutes. Mice in the BCG group received an intravesical instillation of 100 μL of Bacillus Calmette-Guérin (BCG) at 1 mg / mL diluted in sterile saline. Tumor growth in all three groups of mice began to be monitored using bioluminescence imaging, ultrasound, and MRI after the ninth day (i.e., the third day after treatment). On the ninth day, mice that had undergone local hair removal were intraperitoneally injected with 100 μL of 3 mg / mL D-Luciferin luciferin substrate. Bioluminescence imaging was performed 5 minutes later. Compared with pre-treatment, no bioluminescence signal was observed in the bladders of mice in the Cyanine@DIII-PLZ4 group, indicating tumor elimination. However, bioluminescence signals were still present in the BCG group and the PBS+Laser group, indicating that tumors were not significantly suppressed (see Figure 13B). Furthermore, the bioluminescence quantitative results on the ninth day (see Figure 13D) show that the bioluminescence signal in the Cyanine@DIII-PLZ4 group was significantly reduced compared to the PBS+Laser and BCG groups (P<0.01, P<0.05). On day 12, ultrasound and MRI imaging of the three groups of mice (see Figures 13C and 13E) revealed that the bladder tumor mass disappeared in the Cyanine@DIII-PLZ4 group, while it increased in the BCG and PBS+Laser groups. Bladders from the three groups were dissected and fixed with 4% paraformaldehyde. Paraffin sections were prepared and stained with HE. The results (see Figure 13F) revealed that the bladder tissue of the Cyanine@DIII-PLZ4 group showed no signs of tumor cell proliferation, while the bladder tissue of the BCG and PBS+Laser groups showed tumor cell proliferation with irregular borders.

[0141] Example 11 The tumor killing effect of Cyanine@DIII-cPLZ4 on BALB / c orthotopic bladder cancer in mice.

[0142] The treatment strategy is shown in Figure 14A: On the first day, normal 7-8 week old BALB / c female mice were subjected to intravesical mechanical injury and intravesically instilled with 100 μL of 2×10 6An orthotopic bladder cancer mouse model (Figure 14A) was constructed using the Luc-MB-49 mouse bladder cancer cell line. On day 6, mice that had undergone local hair removal were intraperitoneally injected with 100 μL of 3 mg / mL D-Luciferin luciferin substrate. Bioluminescence imaging was performed 5 minutes later, and mice displaying bioluminescence signals were selected for group treatment (Figure 14B). Ultrasound imaging was performed on the same day to further confirm tumor lesions within the bladder (Figure 14D). Tumor-bearing mice were randomly divided into three groups (n=5 per group) for treatment: Cyanine@DIII-PLZ4+Laser group, PBS+Laser group, and BCG group. Mice in the first two groups received a tail vein injection of 100 μL of 85 μM Cyanine@DIII-PLZ4 and phosphate buffered saline (PBS). Two hours later, a 24G indwelling catheter was inserted through the mouse urethra into the bladder. PBS was then perfused into the bladder through the catheter. Excess dye not bound to the bladder lining was repeatedly flushed away, and the bladder was then filled with PBS to maintain fullness. A 400 μM optical fiber connected to an 808 nm laser was inserted into the mouse bladder through a catheter, and 0.3 W / cm2 of light was irradiated to the mouse orthotopic bladder tumor under the guidance of ultrasound imaging. 2 , 8 min. Mice in the BCG group received intravesical instillations of 100 μL of 1 mg / mL Bacillus Calmette-Guérin (BCG) diluted in sterile saline. On days 9 and 13, mice that had undergone local hair removal were intraperitoneally injected with 100 μL of 3 mg / mL D-Luciferin luciferin substrate. Bioluminescence imaging was performed 5 minutes later. Compared to pre-treatment, the bioluminescence signal in the bladders of mice in the Cyanine@DIII-PLZ4 group gradually decreased and eventually disappeared. However, bioluminescence signal persisted in the BCG and PBS+Laser groups, and some mice died, indicating that tumors were not significantly suppressed (see Figure 14B). Furthermore, the bioluminescence quantification results on day 9 (see Figure 14C) show that the bioluminescence signal in the Cyanine@DIII-PLZ4 group was significantly reduced compared to the PBS+Laser and BCG groups (P < 0.05, P < 0.01). On day 13, ultrasound imaging of the bladders of the three groups of mice (see Figure 14D) revealed that the tumors in the Cyanine@DIII-PLZ4 group had been eliminated, while those in the PBS+Laser and BCG groups had significantly increased. Furthermore, on day 60, ultrasound and MRI imaging of the bladders of the surviving mice in the Cyanine@DIII-PLZ4 group revealed no tumors, indicating that photodynamic therapy with Cyanine@DIII-PLZ4 did not result in bladder cancer recurrence.

[0143] Example 12 Toxicity test of Cyanine@DIII-cPLZ4 in healthy mice.

[0144] On the first day, three healthy mice were injected with Cyanine@DIII-PLZ4 via the tail vein. Three healthy mice that received no treatment served as a control group. Urine, blood, and organ dissection were performed for toxicity testing (see Figure 15A). Twenty-four hours later, a 24G indwelling catheter was inserted through the mouse urethra into the bladder. Urine was collected several times into EP tubes, 150-250 μL per tube, and stored briefly at -80°C before routine urinalysis. Results showed that WBC, Sg, pH, and BLD values ​​in both the experimental and control groups were within normal ranges (see Figure 15J). Six mice were then blooded from the orbital vein using pre-prepared capillary tubes. 500-700 μL of blood was collected from each mouse and placed in a 1.5 mL centrifuge tube. The tubes were incubated at 37°C for 1 hour and then centrifuged at 3000 g for 15 minutes at 4°C. The supernatant was then transferred to a new centrifuge tube and temporarily stored at -80°C before being sent for serum biochemical analysis. The results showed that ALT, ASP, ALP, BUN, UA, and CR, indicators of liver and kidney function, showed no significant differences between the experimental and control groups and were all within normal ranges (see Figures 15E-I). On day 7, six mice were blooded from the orbital vein. 500-700 μL of blood was collected from each mouse and placed in an anticoagulant tube. The tubes were temporarily stored at 4°C and sent for routine blood analysis. The results showed that RBC, WBC, Lymph, Mon, and Gran levels showed no significant differences between the experimental and control groups and were all within normal ranges (see Figures 15C-D). Six mice were then dissected, and the heart, liver, spleen, lungs, kidneys, intestines, bladder and other organs were removed and placed in a centrifuge tube containing 4% paraformaldehyde fixative. The tubes were briefly stored at 4°C, and then paraffin sections were made and HE staining was performed. The results showed that the drug injection did not produce toxic effects on the various organs of normal mice (see Figure 15B).

[0145] Example 13 Radionuclides 64 Cu-labeling and preliminary PET evaluation.

[0146] Preparation of chelating agent: First, purchase p-SCN-Bn-DOTA (Cas: 127985-74-4, MedChemExpress). Dissolve p-SCN-Bn-DOTA in dimethyl sulfoxide (DMSO) to prepare a 20 nmol / μL solution 1. All other chemicals and reagents were from Sigma-Aldrich (Dorset, UK).

[0147] Prepare the protein / peptide to be labeled: Dissolve the lyophilized powder of protein DIII and DIII-PLZ4 successfully obtained in Example 2 in buffer ((HEPES, pH=8.9, 0.1 M)), take 50 μL for the following reaction, and mark it as solution 2.

[0148] Labeling reaction: Add solution 1 dropwise to solution 2 at a 40-fold molar ratio, mix gently, and react at 37°C. Incubate the mixture on a thermostatic shaker at 70 rpm for 2 hours, then at 2-8°C overnight to obtain solution 3. After incubation, transfer solution 3 to an ultrafiltration tube, dilute to 300 μL, and centrifuge again. Repeat this process three times, resuspending each step in 0.1 M ammonium acetate solution (pH 6) to remove excess p-SCN-Bn-DOTA, to obtain solution 4 (protein concentration ~2 mg / mL).

[0149] 64 Preparation of CuCl2 and 64 Cu labeling: performed on the CTI RDS cyclotron (112–11 meV) 64 Ni(p,n) 64 Cu reaction preparation 64 Cu. will be bombarded 64 Ni (10 mg) was dissolved from the gold-plated target using 100-150 μL of low-concentration hydrochloric acid. 64 Cu was purified by anion exchange column (Biorad AG1-X8 resin) 64 CuCl2. Excess 64 Ni is eluted in 9M HCl. Ni is eluted in 0.1M HCl. 64 Cu 2+ Previously, the pH was lowered with 6 M HCl. The target fraction in the eluate was diluted with an equal volume of 1 M ammonium acetate solution to a pH of 6. This solution contains approximately 0.5 M chloride and 0.5 M acetate.

[0150] The above-prepared 64 CuCl2 solution (37 MBq, 120 μL) was added to the coupling solution prepared above (solution 4, 240 μg, 120 μL) and incubated at room temperature for 20 min.

[0151] Radiochemical yield determination: Instant thin layer chromatography was performed using ITLC-SA (Varian) with a mobile phase of 0.1 M citrate buffer (pH 5); size exclusion high performance liquid chromatography was performed using a BioSep SEC-S-2000 column (Phenomenex, Macclesfield, UK) with a constant temperature isocratic mobile phase of 0.1 M phosphate buffer containing 50 mM EDTA, pH 7, at a flow rate of 1.0 mL / min.64 The retention time of Cu impurity is about 11 minutes.

[0152] Radiochemical purity analysis: Determine by high performance liquid chromatography (HPLC): Take 3 μL of radioactive sample into a 1 mL sample vial, determine the radioactivity, add PBS to dilute to 1 μCi / μL, and then place it into the sample tank to determine the radiochemical purity.

[0153] Verify labeling effect: Use a fluorescence spectrometer or other fluorescence imaging equipment to detect the labeled samples and confirm the labeling effect and fluorescence intensity.

[0154] Animal experiment: A humanized bladder cancer model (FVB mice, 6-8 weeks) was constructed as in Example 8. PET imaging was then performed on the tumor-bearing mice. Mice were anesthetized with isoflurane / O2 (2% v / v) before injection. 64 Cu-labeled protein (4.44-5.55 MBq / 120-150 μCi / mouse, 100 μL PBS). At the designated time points after injection, mice were scanned using an Inveon DPET scanner (Siemens Medical Solutions, Malvern, PA). PET images were reconstructed without correction for attenuation and scattering. Image analysis was performed using ASIPro VM™ software. As shown in the figure, the fluorescence signal of the mouse model with successful bladder cancer fully matched the radionuclide imaging signal, indicating that the PET images were reconstructed without correction for attenuation and scattering. 64 Cu-labeled Dili targeting protein was highly enriched in tumor areas ( FIG16 ).

[0155] Example 14 Radionuclide 177 Lu labeling and preliminary evaluation

[0156] First, p-SCN-Bn-DOTA (Cas: 127985-74-4, MedChemExpress) was purchased. Dissolve p-SCN-Bn-DOTA in dimethyl sulfoxide (DMSO) to prepare a 20 nmol / μL solution. All other chemicals and reagents were from Sigma-Aldrich (Dorset, UK). Chemicals of the highest available purity (containing the least metal ions) were used.

[0157] Prepare the protein / peptide to be labeled: Dissolve the lyophilized powder of protein DIII and DIII-PLZ4 successfully obtained in Example 2 in buffer (Na2CO3-NaHCO3, pH=9.5, 0.15M), take 1.5 mL and perform the following reaction, labeled as solution 2.

[0158] Labeling reaction: Add solution 1 dropwise to solution 2 at a 10-fold molar ratio. Mix gently and incubate at 37°C. Incubate the mixture on a thermostatic shaker at 70 rpm for 1 hour. After incubation, transfer the antibody to an ultrafiltration tube and add buffer solution 3 (NaOAc-Ac, pH 5.5, 0.5 M) to a volume of 300 μL. Centrifuge again. Repeat this process three times to remove excess p-SCN-Bn-DOTA, yielding solution 4.

[0159] 177 Lu labeling: Take a radioactivity of 115.4MBq (volume 12μL) [ 177 To the solution of Lu] LuCl3, 300 μL of the coupled DOTA-DIII (solution 4) was added, and the mixture was placed in a metal bath for reaction for 45 min at a set temperature of 45° C. Purification was performed using an albumin affinity column (see purification method in Example 1).

[0160] Radiochemical Yield Determination and Radiochemical Purity Analysis: Radiochemical yield was determined using instant thin layer chromatography (iTLC). Specifically, cut microfiber glass paper into strips 10 cm long and 1.5 cm wide. Mark a line 1.5 cm from the bottom to serve as the sample loading scale. Sodium citrate-citric acid system (concentration: 0.5 M, pH = 5.5) was used. After the coupling reaction, the DiIII variant was pipetted evenly. 2 μl of radioactive sample was pipetted onto the marked line on the microfiber glass paper strip. The spotted microfiber glass paper strip was placed into the sodium citrate developing system with the marked line facing downward, and the marked line should be above the liquid surface. Using a flashlight, observe the upward flow of the developing agent. When the developing system reaches 1 cm from the top, remove the microfiber strip, allow it to dry naturally, and then determine the radiochemical yield on Radio-iTLC.

[0161] Radiochemical purity: Determine by high performance liquid chromatography (HPLC): Take 3 μL of radioactive sample into a 1 mL sample vial, determine the radioactivity, add PBS to dilute to 1 μCi / μL, and then place it into the sample tank to determine the radiochemical purity.

[0162] Animal experiment: As in Example 13, after constructing the bladder cancer model, mice were injected and monitored. After the tumor size reached a certain level (about 7-10 days after inoculation), radionuclide imaging was performed. 1 μCi / μL of the labeled solution was injected into each mouse. 177 Lu-DIII-tar pep (200 μL PBS, pH 7.4). At different time points, the mice were anesthetized and killed, and the organs (including bladder tumors) were taken for radionuclide distribution measurement. As shown in Figure 17, 177 Lu-labeled Dili targeting peptide ( 177The enrichment of Lu-DIII-tar pep) in tumors was significantly higher than that in the control group 177 Lu-DIII. Neither treatment had a significant effect on mouse body weight during long-term monitoring (19-day testing period). Six mice per group.

[0163] Example 15 Photodynamic therapy effects of intravenous injection (iv) and intravesical infusion (infusion) of Cyanine@DIII fusion-expressed tumor targeting peptide (DIII-tar pep)

[0164] A T24 bladder cancer tumor-bearing mouse model was constructed according to Example 8. The DIII-targeting peptide (labeled as DIII-tar pep in FIG18 ) fused with the tumor-targeting peptide developed by the present invention (plz4 in this example) was incubated with a cyanine dye to form a complex (IR-783) as in Example 1. The complex was then administered via tail vein injection or ultrasound-guided bladder perfusion. Two hours after administration, an optical fiber was inserted into the bladder under ultrasound guidance, and the tumor site was irradiated with an 808 nm laser at 0.3 W / cm 2 Tumor growth was tracked and monitored using a small animal imaging system, ultrasound imaging, and MRI imaging. At the end of the experiment, the tumor was removed and photographed. As shown in Figure 18 , the Dili-fused tumor-targeting peptide complex developed by the present invention (labeled Dili-tar pep in Figure 18 ) significantly inhibited tumor growth and prolonged the survival of mice when administered either intravenously (IV) or via intravesical instillation.

[0165] Among them, G1: PBS control group; G2: BCG (bladder instillation) control group; G3: Cyanine@DIII group; G4: Cyanine@DIII-PLZ4 group; G5: PBS laser irradiation group; G6: Cyanine@DIII laser irradiation group; G7: Cyanine@DIII-PLZ4 (bladder instillation) laser irradiation group; G8: Cyanine@DIII-PLZ4 laser irradiation group. (Except for the marked bladder instillation method, the rest were administered by tail vein injection)

[0166] Example 16 Preliminary Application of DIII-EphB4-EBD Expression

[0167] DIII-EphB4-EBD was expressed and purified using the Pichia pastoris expression system.

[0168] (1). Construction of DIII-EphB4-EBD expression vector

[0169] First, the DIII-EphB4-EBD sequence (SEQ ID NO: 14 in Table 1) was cloned into the vector plasmid pPIC9K, and the constructed vector plasmid was transformed into the Pichia pastoris GS115 expression vector by electroporation. Positive transformants were screened on MD nutrient-deficient medium plates, and the positive transformants grown on the MD plates were collected, appropriately diluted with sterile water, and plated on YPD medium plates with an appropriate G418 concentration. High-copy positive transformants were screened to obtain the DIII-EphB4-EBD expression vector.

[0170] (2) Induction of DIII-EphB4-EBD expression

[0171] The high-copy DIII-EphB4-EBD expression strain obtained above was streaked onto MD plates and cultured in a constant temperature incubator at 30°C for 2-3 days. After a single clone grew, it was picked and activated in BMGY activation medium for 12-16 hours. The activated bacterial liquid was centrifuged, the culture supernatant was discarded, and the culture was transferred to expression medium BMMY for induced expression. 1% methanol was added every day for induction for 4-7 days.

[0172] (3) Purification, isolation and identification of DIII-EphB4-EBD protein

[0173] After induction, the supernatant of the BMMY-induced expression culture medium was collected and filtered through 0.8, 0.45, and 0.22 μm filter membranes, respectively. The purified products were then purified using an Albupure affinity purification column. The purified products were then identified by SDS gel electrophoresis and near-infrared imaging. As can be seen in Figure 19 (left), a highly pure purified product (in the red dotted box) was obtained in the final eluate. Furthermore, as can be seen in Figure 19 (right), after binding to the near-infrared fluorescent dye, different concentrations of DIII-EphB4-EBD (abbreviated as DIII-EphB4 in the figure), namely (Dye@DIII-EphB4-1 and Dye@DIII-EphB4-2), exhibited consistent fluorescence characteristics with the original DIII (Dye@DIII), indicating that the fusion strategy did not affect the fluorescence characteristics of DIII.

[0174] Example 17 Preliminary Application of DIII-RGD and (DIII)m-(RGD)n Expression

[0175] (1) Construction of recombinant DIII-RGD and (DIII)m-(RGD)n vectors

[0176] The sequence information of DIII-RGD and (DIII)m-(RGD)n is the DIII sequence in SEQ ID NO: 1 in Table 1 plus the RDG sequence (Arg-Gly-Asp). Various combinations of DIII and RDG are used. Here, m=2, n=3 and m=1, n=2 are used as examples for illustration. According to the characteristics of Escherichia coli, the recombinant encoding gene is artificially optimized to synthesize the target gene.

[0177] (2) Construction of recombinant DIII-RGD and (DIII)m-(RGD)n vectors

[0178] The target gene obtained in step (1) and the pET21b(+) plasmid double-digested with restriction endonucleases HindIII and NdeI (enzyme digestion conditions: 37°C, 4h) were subjected to agarose gel electrophoresis for identification. The target fragment was recovered using a commercial agarose gel recovery kit. The target fragment was ligated with the pET21b(+) vector plasmid using Gibson assembly solution. The ligation product was transformed into Escherichia coli DH5α competent cells, and positive single clones were selected for activation and colony PCR identification. An expression vector strain was obtained.

[0179] (3) Inducible expression of recombinant DIII-RGD and (DIII)m-(RGD)n expression vectors

[0180] Transfer the positive single clone of the recombinant E. coli expression strain BL21 (DE3) into 5-8 ml LB medium containing ampicillin resistance, activate with shaking at 37°C for 12-16 hours, transfer to 500 ml LB medium containing ampicillin resistance and culture at 37°C for 3-4 hours until the OD600 reaches 0.6-0.8. Add 0.1-5 mM IPTG, induce expression at 18-30°C for 8-20 hours, and harvest the bacteria.

[0181] (4) Isolation, purification and identification of recombinant DIII-RGD and (DIII)m-(RGD)n proteins

[0182] Centrifuge the recombinant DIII-RGD and (DIII)m-(RGD)n expressing bacteria at 3000-14000g for 10-20 minutes at 4°C. Discard the supernatant and collect the pellet. Resuspend the pellet with 10-20 mL of lysis buffer per 500 mL of culture medium. Sonicate and lyse the pellet. After sonication, centrifuge at 10000-15000g for 10-20 minutes at 4°C to collect the inclusion body pellet. Resuspend the pellet with 10-20 mL of wash buffer per 500 mL of culture medium. Centrifuge at 10000-15000g for 10-20 minutes at 4°C. Repeat 2-3 times to collect the inclusion body pellet. For every 500 mL of culture medium, 5-10 mL of inclusion body solubilization solution was added to dissolve the inclusion bodies. The cells were stirred at 4°C for 12-24 hours to completely dissolve the inclusion bodies. The cells were centrifuged at 10,000-15,000g for 10-20 minutes at 4°C, and the precipitate was discarded. The supernatant of the inclusion body solubilization solution was collected and 50-60 volumes of protein refolding solution were added. The cells were then incubated at 4°C for 1-12 hours to renature the inclusion bodies. The refolded target protein was purified using an Albupure affinity chromatography column to obtain the recombinant protein. The Western blotting results in Figure 20 (left) show that recombinant Dili-RGD and (Dli)m-(RGD)n were successfully expressed. After incubation with a fluorescent dye, the fluorescence intensity increased with increasing recombinant protein concentration, as shown in Figure 20 (right), indicating that the expressed protein is biologically active and retains the fluorescence enhancement properties consistent with Dili.

[0183] Example 18 Targeting of DIII-RGD and (DIII)m-(RGD)n to three different tumor cells

[0184] DIII-RGD, RGD-DIII-RGD, and RGD-DIII-RGD-DIII-RGD expressed, purified, and prepared according to Example 17 were complexed with a cyanine dye (IR-783 in this case) as described in Example 1, and their targeting of three cancer cells (U87MG, MDA-MB-231, and Hela) was observed using flow cytometry. The three bladder cancer cell lines were plated in several 6-well plates, with 200,000 cells per well. After incubation for 24 hours, the culture medium was aspirated and the cells were gently washed 2-3 times with PBS. The three cells were then replaced with serum-free medium (3 μM) containing dye@DIII, dye@DIII-RGD, dye@RGD-DIII-RGD, and dye@RGD-DIII-RGD-DIII-RGD, respectively. Each group was incubated in a 37°C incubator for 6 hours. Three replicates were set for each group, and a serum-free medium incubation group served as a control. After incubation, wash each well 3-5 times with PBS to wash off the unbound drug and absorb it dry. Add trypsin to each well to digest the adherent cells into single cells, centrifuge at 1000 rpm for 3 minutes at room temperature, resuspend in serum-free medium, and immediately send to flow cytometer for detection. The results show that dye@DIII alone has no targeting effect on tumor cells. Compared with the control group and dye@DIII group, dye@DIII-RGD, dye@RGD-DIII-RGD and dye@RGD-DIII-RGD-DIII-RGD drugs have strong targeting ability for the three types of tumor cells (as shown in Figure 21).

[0185] Example 19 Evaluation of DIII-RGD and (DIII)m-(RGD)n in Animals

[0186] The RGD-DIII-RGD-DIII-RGD, RGD-DIII-RGD, and DIII-RGD successfully expressed in Example 17 were incubated with a cyanine dye as described in Example 1 (the dye used was IR-783) to form complexes. These complexes were administered via tail vein injection, and their pharmacokinetic characteristics in mice were recorded at different time points using NIR-II fluorescence imaging. As shown in Figure 22, RGD-DIII-RGD-DIII-RGD, RGD-DIII-RGD, and DIII-RGD were primarily cleared to the bladder via the kidneys, and all were able to achieve long-term accumulation in the bladder.

[0187] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A complex comprising: 1) a protein shell, wherein the protein shell is the third domain of albumin or a variant thereof, or the a subunit of the third domain of albumin or a variant thereof, 2) a small molecule bound to the protein shell, and 3) a functionalized fragment connected to the protein shell, wherein the functionalized fragment is a molecule that can be used for the diagnosis, prevention or treatment of kidney-bladder clearance pathway diseases.

2. The complex of claim 1, wherein the albumin is mammalian serum albumin; Preferably, the albumin is human serum albumin.

3. The complex according to claim 1 or 2, wherein The small molecule is selected from one or more of a photosensitizer (eg, a dye molecule, such as a cyanine dye molecule), a nuclide, and a small molecule drug (eg, a small molecule chemotherapeutic drug).

4. The complex according to any one of claims 1 to 3, wherein the dye molecule has a backbone structure selected from any one of the following or a derivative thereof: Preferably, the cyanine dye molecule has any one of the following structures: or the above structures by replacing Cl in the skeleton with F, Br or I; or Or the structure obtained by replacing Cl in the skeleton with F, Br or I in the above structure.

5. The complex according to any one of claims 1 to 4, wherein the functionalized fragment is selected from: Small molecule drugs that can be used for the diagnosis, prevention or treatment of kidney-bladder clearance pathway diseases. Functional peptides, such as peptide chains or cyclic peptides, used in kidney-bladder clearance pathway diseases. therapeutic proteins for the treatment of kidney or bladder disease; Preferably, the functional fragment is linked to the protein coat via a chemical, an amino acid or a peptide; Preferably, the chemical is a small molecule coupling agent for chemical grafting (e.g., diphenylcyclooctyne-N-hydroxysuccinimide), which can also be replaced by any linker that effectively connects the protein shell and the functionalized fragment; Preferably, the protein coat is modified by genetic engineering (e.g., by eukaryotic expression or prokaryotic expression) to The functional peptide may be prepared into a fusion protein with the therapeutic protein; Preferably, the functionalized fragment is selected from short peptides and derivatives thereof, or other small molecules targeting bladder cancer, such as a short peptide PLZ4 targeting bladder cancer, whose amino acid sequence is QDGRMGF (SEQ ID NO: 10), or its cyclic peptide cQDGRMGFc (SEQ ID NO: 15), RGD or its fusion protein, prostate-specific membrane antibody (PSMA); Preferably, the functionalized fragment is a protein or other small molecule having a therapeutic effect on bladder cancer, such as EphB4-EBD protein; Preferably, the protein shell and the functionalized fragment form a fusion / recombinant protein; more preferably, the functionalized fragment is selected from the group consisting of short peptide PLZ4, cyclic peptide cPLZ4, RGD or EphB4-EBD protein.

6. The complex according to any one of claims 1 to 5, wherein the complex is labeled with a radionuclide (e.g., on a protein coat or a functionalized fragment); Preferably, the radionuclide is a nuclide used for imaging, such as 64 Cu, 18 F. 68 Ga; Preferably, the radionuclide is a therapeutic nuclide, such as 177 Lu, 90 Y. 131 I-, 125 I. 225 Ac, 32 P etc.

7. A method for preparing a functionalized protein shell contained in the complex according to any one of claims 1 to 6, comprising the steps of: Step S1: preparing solution A, which comprises the third domain of albumin or its variant according to claim 1 or 2, or the a subunit of the third domain of albumin or its variant; Step S2: After ultrafiltration and centrifugation of solution A through an ultrafiltration tube, the filtrate is discarded, an alkaline solution (such as sodium bicarbonate solution) is added, and then vortex mixed at room temperature to make the pH reach an alkaline condition, and the mixed solution is solution B; Step S3: Vortex mix the small molecule coupling agent for chemical grafting and solution B at room temperature, react at room temperature or under heating conditions, remove excess coupling agent by ultrafiltration and centrifugation through an ultrafiltration tube, and dissolve the product with phosphate buffer solution (PBS) to obtain compound 1. Step S4: Vortex mix compound 1 and the raw material of the functional fragment (e.g., short peptide mother liquor) at room temperature, react overnight at room temperature or under heating conditions, remove excess functional fragments (e.g., short peptides) by ultrafiltration and centrifugation using an ultrafiltration tube, and finally dissolve the product with phosphate buffered saline (PBS) to obtain compound 2.

8. A method for preparing the composite according to any one of claims 1 to 7, comprising the steps of: S1: preparing a solution A containing the functionalized protein shell, and preparing a solution B containing small molecules (such as photosensitizers (such as dye molecules, such as cyanine dye molecules), nuclides, small molecule drugs (such as small molecule chemotherapeutic drugs)), wherein the functionalized protein shell is prepared according to the method of claim 6; S2: Vortexing solution A and solution B at room temperature to obtain the composite at room temperature or under heating conditions.

9. Use of the complex according to any one of claims 1 to 8 as a fluorescent probe.

10. A pharmaceutical composition comprising the complex according to any one of claims 1 to 9, optionally further comprising one or more pharmaceutical carriers or excipients; Preferably, the pharmaceutical composition is administered by intravenous injection, intravesical administration, intratumoral injection or intraperitoneal injection; Preferably, the pharmaceutical composition is a contrast agent or a therapeutic agent.

11. A method for diagnosing, preventing or treating a urinary system disease, the method comprising administering the complex of any one of claims 1 to 6 to a subject in need thereof, the disease including but not limited to urinary system tumors and urinary system injuries; Preferably, the method is selected from photodynamic therapy, imaging, surgical navigation, radiotherapy, chemotherapy.

12. Use of the complex according to any one of claims 1 to 6 for preparing a medicament for diagnosing, preventing or treating a urinary system disease.

13. A kit comprising the complex according to any one of claims 1 to 6.

14. A method for targeting cells, the method comprising contacting the complex of any one of claims 1 to 6 with the cells; optionally, after contacting, irradiating the cells with a laser to obtain an image of the cells.

15. Use of the complex according to any one of claims 1 to 6 in the preparation of a kit for targeting cells or for obtaining imaging of cells.

16. An imaging method, comprising using the complex according to any one of claims 1 to 6 as an imaging developer.

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