A method for preparing a kidney-clearable optical diagnostic probe-cell conjugate and its application.

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a renal-clearable optical diagnostic probe-cell conjugate and its application. The invention involves fusing a renal-clearable optical diagnostic probe with a maleimide-modified polyethylene glycol chain, followed by incubation with Traut's-pretreated cells to obtain the renal-clearable optical diagnostic probe-cell conjugate. This method is simple, time-saving, and efficient, applicable to various renal-clearable optical diagnostic probes and cell types. Furthermore, this method has minimal impact on cell viability, disease targeting ability, and ability to cross physiological barriers. In addition, the renal-clearable optical diagnostic probe-cell conjugate constructed using this method can release renal-clearable fragments in the disease microenvironment, thereby enabling early diagnosis of various diseases through urine analysis, exhibiting high sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a kidney-clearable optical diagnostic probe-cell conjugate and its application. Background Technology

[0002] Currently, the diagnosis of deep tissue diseases in clinical practice mainly relies on imaging examinations (ultrasound, CT, X-ray, MRI, etc.) and histopathological biopsies. Imaging examinations, based on the physical morphological changes of diseased tissue, are difficult to detect effectively in the early stages of disease; tissue biopsies, due to their highly invasive nature and high risks, have their clinical application strictly limited. Renal-clearable optical diagnostic probes are a class of highly promising diagnostic reagents. With proper design, these probes can react with disease biomarkers, releasing fragments that are excreted through the kidneys and eventually accumulate in the urine. However, similar to most small molecule drugs, renal-clearable optical diagnostic probes rely on passive transport through the bloodstream and lack active targeting to the lesion site. This limits their application scope in disease diagnosis, as well as their diagnostic specificity and sensitivity.

[0003] As the basic structural units of mammals, cells can sense disease information and respond accordingly in complex physiological environments. For example, when a blood vessel is damaged, platelets are rapidly recruited to the wound to seal it; when the body is infected, neutrophils can quickly respond to chemokines released at the site of infection and migrate, adhere, and infiltrate along the chemokine gradient. In participating in these response processes, cells also produce informational substances such as cfDNA, cytokines, and extracellular vesicles. These informational substances are of great value for disease diagnosis and are an important basis for the diagnosis of blood biochemical indicators. However, in the early stages of disease, these substances are often difficult to detect due to factors such as low concentration, easy degradation, and difficulty in separation, thus greatly limiting the early diagnosis of diseases.

[0004] In summary, there is an urgent clinical need for novel, non-invasive early diagnostic methods. Summary of the Invention

[0005] To address the shortcomings of existing technologies and overcome the low sensitivity and poor specificity of current diagnostic methods, this invention provides a method for preparing a renal-clearable optical diagnostic probe-cell conjugate. This method uses cells as transport carriers for the probe, actively migrating it to the disease site. After the probe-cell conjugate reacts with disease markers, it releases a fragment that can be cleared by the kidneys and accumulated in urine. Compared to naturally produced informational substances by cells, the renal-clearable optical diagnostic probe-cell conjugate of this invention releases a fragment that rapidly accumulates in urine, is not diluted or interfered with by blood substances, and is easy to detect, showing promise for applications in thrombotic diseases and chronic osteomyelitis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a renal-clearable optical diagnostic probe-cell conjugate, specifically comprising: fusing a renal-clearable optical diagnostic probe with maleimide-modified polyethylene glycol, and then incubating it with Traut's pretreated cells to obtain the renal-clearable optical diagnostic probe-cell conjugate; wherein the renal-clearable optical diagnostic probe is a disease marker-responsive polypeptide linked to a fluorescent group and a fluorescence quenching group, and the disease marker-responsive polypeptide can release a fragment excreted into the urine through the kidney after reacting with a disease marker; wherein the cells include platelets, neutrophils, erythrocytes, macrophages, monocytes, and mesenchymal stem cells.

[0007] Preferably, the disease markers include thrombin, Staphylococcus aureus SspA (SSPA) protease, matrix metalloproteinase MMP-2, matrix metalloproteinase MMP-9, collagenase, β-galactosidase, caspase-8, caspase-3, cathepsin K, cathepsin L, cathepsin B, cathepsin S, Staphylococcus aureus transpeptidase sorting enzyme A (SrtA), gamma-glutamyl transferase, leukocyte esterase, angiotensin-converting enzyme, and renin.

[0008] More preferably, the disease biomarker responsive peptides include thrombin-responsive peptides KGFPRGFGA, GFPRGFPAGGC, KALNNGSGFPRGRAKC, LTPRGWRLGGC, DPRSFL, GFPRGFPAGG, GF-Pip-RSGGGGKC, GFPRGFPAGG, GF-Pip-RSGGGGKC, GFPRGFPA, GDARSWG, GFPRGFGAGG, GFPRGLPAGG, GFPRVFPAGG, GFPRGF, GFPRGFPAGAGG, GFPRGFPAGGK, GFPRGFPAGGK, GGLVPRGSGAS, KGGPRSGGGC, KGALVPRGSAGK; Staphylococcus aureus SspA protease-responsive peptide KGFLEGT; and renin-responsive peptides DRVYIHPFHLVIH, DRVYIHPFHLLYHN. DRVYIHPFHLLYYS; LasB responsive peptide AFK; cathepsin responsive peptides HPGGPQ, GHPGGPQG; serine protease responsive peptide AGPR; Caspase-3 responsive peptide ZDEVD; angiotensin-converting enzyme responsive peptides FRK, LFK; SrtA protease responsive peptides QALPETGEE, LPETG; MMP protease responsive peptides PLGLW, APPGFSPFR, LR, GGRK, VHPK, SQNYPIVQ, PLGLG, PLGL, HLI, FRKP, FVR, AAA, KPLGLAR, AGFSLPA, AGFSLAKR, APA; GramB responsive peptide IEDT; cathepsin K responsive peptides HPGGPQ, KPLGL.

[0009] In the above polypeptide sequence, A represents alanine, C represents cysteine, D represents aspartic acid, E represents glutamic acid, F represents phenylalanine (including D and L configurations), G represents glycine, H represents histidine, I represents isoleucine, K represents lysine, L represents leucine, M represents methionine, N represents asparagine, P represents proline, Q represents glutamine, R represents arginine, S represents serine, T represents threonine, V represents valine, W represents tryptophan, Y represents tyrosine, and pip represents the Piperidine group.

[0010] Preferably, the fluorescent group includes cyanine, indocyanine, and rhodamine dye groups, with indocyanine green (ICG) being the most preferred; the fluorescence quenching group includes IRDye-QC, BHQ-0, BHQ-1, BHQ-2, and BHQ-3.

[0011] Preferably, the cells are derived from mammals, including rodents, rabbits, pigs, or primates. More preferably, they are cells from mice, rabbits, pigs, or humans.

[0012] Preferably, in the maleimide-modified polyethylene glycol, the molecular weight of the polyethylene glycol ranges from 200 to 7000 Da. For example, the molecular weight of the polyethylene glycol is 500, 1000, 2000, 3000, or 5000 Da.

[0013] Preferably, the renal-clearable optical diagnostic probe is fused with maleimide-modified polyethylene glycol via an amide condensation-mediated method involving NHS-activated esters and amino groups: the renal-clearable optical diagnostic probe is dissolved in an organic solvent, then maleimide-modified polyethylene glycol and N,N-diisopropylethylamine are added, and the mixture is reacted at room temperature for 3-8 hours under inert gas protection, followed by dialysis and drying to obtain the final product.

[0014] More preferably, the maleimide-modified polyethylene glycol is selected from succinimide ester-polyethylene glycol 1000-maleimide (NHS-PEG1000-Mal; CAS No.: 756525-92-5).

[0015] Preferably, the method for incubating the probe with Traut's pretreated cells is as follows: 10 8 Orders of magnitude of cells were resuspended in PBS and incubated with Traut's reagent at room temperature for 20-60 minutes. After incubation, excess Traut's reagent was removed, and the cells were washed. The cells were then incubated with a probe fused with 20-30 μM of polyethylene glycol at 30-40°C in the dark for 0.5-3 hours. After incubation, excess probe was removed. Renal-clearable optical diagnostic probes were then attached to the cell surface using an addition reaction between maleimide and thiol groups.

[0016] More preferably, the concentration of the Traut's reagent for pretreatment is 0.025-0.2 mg / mL, such as 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL or 0.2 mg / mL; the Traut's reagent is dissolved in PBS, and the solvent pH is 7.0-8.5 during pretreatment.

[0017] Preferably, the fluorescent group and the fluorescence quencher group are covalently linked to the disease biomarker response peptide by amide condensation, click chemistry, or Michael addition.

[0018] More preferably, a fluorescent group is attached to the disease biomarker response peptide using an amide condensation method: the disease biomarker response peptide, the fluorescent group, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine are dissolved in an anhydrous organic solvent, reacted at room temperature for 5-10 hours, and then dialyzed and dried to obtain the final product.

[0019] Furthermore, the molar ratio of the responsive peptide to the fluorescent group is 1:2.

[0020] More preferably, a click chemistry method is used to attach a fluorescence quencher group to the disease biomarker response peptide: the response peptide-fluorescent group complex and the fluorescence quencher group are dissolved in an organic solvent-water mixture, and then CuSO4·5H2O and sodium ascorbate are added. After reacting at room temperature for 5-8 hours under inert gas protection, the product is obtained by dialysis and drying.

[0021] Furthermore, the molar ratio of the responsive peptide-fluorescent group complex to the fluorescence quencher group is 1:1.

[0022] Furthermore, in the organic solvent-water mixed solvent, the volume ratio of organic solvent to water is 1-3:1.

[0023] Preferably, the cells are obtained using differential centrifugation, density gradient centrifugation, or flow cytometry.

[0024] Preferably, the disease biomarker response peptide is prepared using a standard solid-phase synthesis method, with a resin to amino acid molar ratio of 1:(1-2.5).

[0025] The second aspect of the present invention also provides a kidney-clearable optical diagnostic probe-cell conjugate prepared by the preparation method described in the first aspect.

[0026] The third aspect of the present invention also provides the application of the renal-clearable optical diagnostic probe-cell conjugate described in the second aspect in the diagnosis of diseases, including thrombosis, osteomyelitis, aneurysm, atherosclerosis, bone cancer, and bloodstream infection.

[0027] The kidney-clearable optical diagnostic probe constructed by the method of this invention exhibits good responsiveness and specificity to specific disease biomarkers. Figures 1-4 Further, a renal-clearable optical diagnostic probe-cell conjugate was constructed, and its cell morphology and probe distribution were analyzed by scanning electron microscopy (SEM). Figure 5 ), confocal microscope ( Figure 6 ) The observation was completed. This conjugate can be effectively enriched at the disease site ( Figures 7-8 ), and has high biosafety ( Figure 9 At the same time, its specific renally clearable fragments can be detected in urine. Figures 10-12 ).

[0028] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses a method for preparing a renal-clearable optical diagnostic probe-cell conjugate. The method involves fusing a renal-clearable optical diagnostic probe with a maleimide-modified polyethylene glycol chain, followed by incubation with Traut's-pretreated cells to obtain the renal-clearable optical diagnostic probe-cell conjugate. This method is simple, time-saving, and efficient, applicable to various renal-clearable optical diagnostic probes and cell types. Furthermore, this method has minimal impact on cell viability, disease targeting ability, and ability to cross physiological barriers. In addition, the renal-clearable optical diagnostic probe-cell conjugate constructed using this method can release renal-clearable fragments in the disease microenvironment, enabling early diagnosis of various diseases through urine analysis, exhibiting high sensitivity and specificity.

[0029] Specifically, the present invention has the following advantages: (1) This invention couples a kidney-clearable optical diagnostic probe to cells, enabling cells to produce fragments that can be cleared by the kidneys in disease environments, thereby achieving non-invasive and highly sensitive diagnosis of diseases through urine analysis.

[0030] (2) This invention utilizes the rapid coupling reaction between thiol and maleimide to efficiently modify the renal clearable optical diagnostic probe onto the cell surface within 1.5 h. The operation is simple, time-saving and efficient.

[0031] (3) The method of the present invention can flexibly select different cells as carriers according to different disease types to achieve non-invasive diagnosis of a variety of diseases and has good application flexibility. Attached Figure Description

[0032] Figure 1 The figure shows the enzymatic hydrolysis rate of thrombin by the renal-clearable thrombin probe in Example 1, indicating that the renal-clearable thrombin probe can be rapidly hydrolyzed by thrombin.

[0033] Figure 2 The specific response of the renal-clearable thrombin probe in Example 1 to thrombin demonstrates the specific recognition between the renal-clearable thrombin probe and thrombin.

[0034] Figure 2 In the diagram, the horizontal axis from left to right represents: Blank, Reactive Oxygen Species (ROS) including hydrogen peroxide (H2O2), and peroxynitrosoanion (ONOO). - ), hypochlorite (OCl) - Other enzymes include caspase-3, caspase-8, cathepsin D, gamma-glutamyl transferase (GGT), and metal ions including ferrous ions (Fe). 2+ ), copper ions (Cu) 2+ ), magnesium ions (Mg 2+ ), potassium ions (K) + ) and thrombin.

[0035] Figure 3 The figure shows the enzymatic cleavage rate of the renal-clearable SSPA probe against the SSPA protease in Example 2, indicating that the renal-clearable SSPA probe can be rapidly hydrolyzed by the SSPA protease.

[0036] Figure 4 The rapid response of the renal-clearable SSPA probe to SSPA protease in Example 2 demonstrates the specific recognition between the renal-clearable SSPA probe and SSPA protease.

[0037] Figure 5The scanning electron microscopy results for the renal-clearable thrombin probe-platelet conjugate and the renal-clearable SSPA probe-neutrophil conjugate in Examples 1 and 2 show that the cell membrane remains intact after being connected to the renal-clearable optical diagnostic probe. The scale bar is 1 μm.

[0038] Figure 6 The confocal scanning results of the renal-clearable thrombin probe-platelet conjugate and the renal-clearable SSPA probe-neutrophil conjugate in Examples 1 and 2 show the uniform distribution of the renal-clearable optical diagnostic probes on the cell membrane surface, with a scale bar of 1 μm or 5 μm.

[0039] Figure 7 The image shows the enrichment of the renal-clearable thrombin probe-platelet conjugate from Example 1 at the thrombus site in mice, demonstrating the targeting of the renal-clearable thrombin probe-platelet conjugate to the thrombus site.

[0040] Figure 8 The image shows the enrichment of the renal-clearable SSPA probe-neutrophil conjugate in the osteomyelitis site of rats in Example 2, demonstrating the targeting of the renal-clearable SSPA probe-neutrophil conjugate for osteomyelitis.

[0041] Figure 9 Following tail vein injection of the renal-clearable thrombin probe-platelet conjugate of Example 1 or the renal-clearable SSPA probe-neutrophil conjugate of Example 2, changes in serum C-reactive protein, TNF-α, and interleukin-6 levels in rats, as well as HE-stained sections of major organs, showed that the renal-clearable thrombin probe-platelet conjugate and the renal-clearable SSPA probe-neutrophil conjugate have good biocompatibility.

[0042] Figure 10 After intravenous injection of the renal-clearable thrombin probe-platelet conjugate of Example 1 into rats with thrombotic diseases, a renal-clearable fragment with near-infrared fluorescence was detected in the urine, indicating that the renal-clearable thrombin probe-platelet conjugate has a good diagnostic effect on thrombotic diseases.

[0043] Figure 11 After intravenous injection of the renal-clearable thrombin probe-platelet conjugate of Example 1 into mice with diffuse thrombotic disease, a renal-clearable fragment with near-infrared fluorescence was detected in the urine, indicating that the renal-clearable thrombin probe-platelet conjugate has a good diagnostic effect on diffuse thrombotic disease.

[0044] Figure 11In the figure, the three groups on the horizontal axis are the healthy group, the lipopolysaccharide model group (LPS), and the heparin + LPS intervention group.

[0045] Figure 12 After intravenous injection of the renal-clearable SSPA probe-neutrophil conjugate of Example 2 into rats with osteomyelitis, a renal-clearable fragment with near-infrared fluorescence was detected in the urine, indicating that the renal-clearable SSPA probe-neutrophil conjugate has a good diagnostic effect on osteomyelitis.

[0046] Figure 12 In the figure, the horizontal axis represents the post-inoculation time, which is 3 days, 5 days, 7 days, 10 days, 14 days, and 21 days respectively. The groups are, in order, healthy group, sham operation group, E. coli group, and Staphylococcus aureus group. Detailed Implementation

[0047] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0048] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0049] Example 1: Preparation method of renal-clearable thrombin probe-platelet conjugate The method includes the following steps: (1) Synthesis of thrombin-responsive peptide (peptide sequence: KGFPRGFGA) using standard solid-phase synthesis: 1.4 g of Rink amide resin (0.88 mmol / g) was weighed into the peptide synthesis reactor, and 15 mL of N,N-dimethylformamide (DMF) solvent was added. After swelling for 40 minutes, the first amino acid was added and reacted for 3 hours under continuous air blowing into the suspension. After the reaction, the Fmoc group was deprotected using 20% ​​piperidine / DMF solution (3 × 20 mL, 10 minutes each time). After thorough washing with DMF (3 × 20 mL, 3 minutes each time), subsequent amino acids were added. After the reaction, the mixture was thoroughly washed with isopropanol and n-hexane in sequence (washed 3 times with each solvent, 20 mL each time, 3 minutes each time). The resin was then eluted three times with trifluoroacetic acid / dichloromethane (1:99, v:v; 3 × 20 mL, 5 minutes each time) (3 × 20 mL), and the filtrate was collected. Finally, the filtrate was concentrated under vacuum to a colorless oily substance, and ether was added (oily substance: ether = 1:100, v:v) to precipitate thrombin-responsive peptides. The precipitate was then separated by centrifugation to obtain the polypeptide product.

[0050] (2) Synthesis of ZWCY, a near-infrared fluorescent group derived from cyanine: The synthesis procedure was performed according to the article by Huang et al. (doi.org / 10.1039 / C6SC05059J). Under nitrogen protection, tert-butyl (4-hydroxyphenylethyl) carbamate (500 mg, 2.1 mmol) was dissolved in 10 mL of DMF solvent, and potassium carbonate (1.45 g, 10.5 mmol) was added. The mixture was stirred at room temperature for 0.5 hours. Then, N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (CAS No.: 63857-00-1, 569 mg, 0.7 mmol) was added, and the reaction mixture was stirred at 60 °C for 2 hours. The reaction solution was then cooled to room temperature and concentrated. Finally, 20 mL of a mixture of trifluoroacetic acid and water (volume ratio 1:1) was added, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by analytical HPLC. After the reaction was complete, the resulting mixture was concentrated under reduced pressure at 55°C and purified by preparative HPLC (mobile phase: methanol-water, containing 0.1% formic acid, elution gradient of 20-70%) to obtain pure product ZWCY (400 mg, yield 62%). 1¹H NMR (400 MHz, DMSO-d⁶) data are as follows: δ 7.96 (s, 2H), 7.83 (s, 1H), 7.67 (s, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 6.24 (d, J = 14.0 Hz, 2H), 4.19 (s, 4H), 3.55 (s, 4H), 3.10 (s, 20H), 2.89 (s, 2H), 2.83 (s, 2H), 2.75 (s, 4H), 2.14 (s, 4H). 1.93 (s, 2H), 1.29 (s, 12H). (3) Synthetic renal-clearable thrombin probe The fluorescent group ZWCY was linked to the thrombin-responsive peptide using an amide condensation method: The thrombin-responsive peptide (15 mg, 0.0075 mmol), the fluorescent group ZWCY (9.4 mg, 0.015 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU; 11.4 mg, 0.03 mmol), 1-hydroxybenzotriazole (HOBT; 4.1 mg, 0.03 mmol), and N,N-diisopropylethylamine (3.8 mg, 0.3 mmol) were dissolved in 20 mL of anhydrous DMF and reacted at room temperature for 8 hours with stirring. The resulting reaction solution was then dialyzed against ultrapure water for 12 hours (MWCO 1 kDa), and then freeze-dried to obtain the thrombin-responsive peptide-ZWCY complex (17.0 mg, yield 80%).

[0051] A click chemistry method was used to attach a fluorescent quencher group, IRDye-QC Azide (Qiyue Biotechnology, Cat. No.: Q-0127542), to a thrombin-responsive peptide: The thrombin-responsive peptide-ZWCY complex (17 mg, 0.0056 mmol) and IRDye-QC Azide (5.3 mg, 0.0056 mmol) were dissolved in 5 mL of a DMSO / H2O (2:1 v / v) mixture. Then, CuSO4·5H2O (0.7 mg, 0.0028 mmol) and sodium ascorbate (0.5 mg, 0.0028 mmol) were added, and the reaction was carried out under argon protection with stirring at room temperature for 6 hours. After the reaction, the reaction solution was dialyzed against ultrapure water for 12 hours (MWCO 2 kDa) and then freeze-dried to obtain the final product, a renal-clearable thrombin probe (20.0 mg, yield 89%).

[0052] (4) Fusion of renal-clearable thrombin probe with polyethylene glycol chain: The above-mentioned 20.0 mg purified renal-clearable thrombin probe was dissolved in 2 mL of DMF solvent, and then NHS-PEG1000-Mal (25 mg, purchased from PonsureBiological) and N,N-diisopropylethylamine (3 µL) were added. After stirring at room temperature for 5 hours under nitrogen protection, the resulting reaction solution was dialyzed in ultrapure water for 12 hours (MWCO 3 kDa), and then freeze-dried to obtain renal-clearable thrombin probe-polyethylene glycol-Mal (30.0 mg, yield 57%).

[0053] (5) Obtain anticoagulated whole blood from the veins of rats or mice (whole blood is anticoagulated with 4% sodium citrate, and the volume ratio of whole blood to anticoagulant is 9:1). Platelets are separated by differential centrifugation: first, centrifuge at 100g for 20 minutes at room temperature, take out the upper platelet-rich plasma, and then centrifuge the platelet-rich plasma at 800g for 20 minutes at room temperature to separate the platelets.

[0054] (6) Platelets (2×10) 8 Platelets were resuspended in 400 μL PBS (pH 7.8), then mixed with an equal volume of 0.1 mg / mL Traut's reagent (2-iminothione; dissolved in pH 7.8 PBS) and incubated at room temperature for 30 minutes. Excess Traut's reagent was then removed by centrifugation (800 g, 10 min), and the platelets were washed twice with Tyrode's buffer (pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.16 mM NaH2PO4, 3 mM NaHCO3, 5.5 mM MMglucose). The washed platelets (2 × 10⁻⁶) were then... 8 (A sample of platelet-renal thrombin probe) was incubated with 1 mL of a renal-clearable thrombin probe-polyethylene glycol-mal (25 μM, dissolved in PBS, pH 7.4) at 37°C in the dark for 1 hour. After incubation, excess probe was removed by centrifugation (800g, 10 min), and the platelet-renal thrombin probe was washed twice with Tyrode's buffer. The final renal-clearable thrombin probe-platelet conjugate was prepared and stored in Tyrode's buffer at 4°C in the dark.

[0055] Example 2: Preparation of a renal-clearable SSPA probe-neutrophil conjugate The preparation method includes the following steps: (1) Synthesis of SSPA-responsive peptide (peptide sequence: KGFLEGT) using standard solid-phase synthesis: 1.4 g of Rink amide resin (0.88 mmol / g) was weighed into the peptide synthesis reactor, and 15 mL of DMF solvent was added. After swelling for 40 minutes, the first amino acid was added to the suspension under continuous air blowing. After 3 hours of reaction, the Fmoc group was deprotected using 20% ​​piperidine / DMF solution (3 × 20 mL, 10 minutes each time). After thorough washing with DMF (3 × 20 mL, 3 minutes each time), subsequent amino acids were added. After the reaction was completed, the reaction mixture was washed thoroughly with isopropanol and n-hexane in portions (3 times with each solvent, 20 mL each time, 3 minutes each time). The resin was then eluted three times with trifluoroacetic acid / dichloromethane (1:99, v:v; 3 × 20 mL, 5 minutes each time), and the filtrate was collected. Finally, the filtrate was concentrated under vacuum to a colorless oily substance, and diethyl ether was added to precipitate the SSPA-responsive peptide (oily substance: diethyl ether = 1:100, v:v). The precipitate was then separated by centrifugation to obtain the polypeptide product.

[0056] (2) Synthetic renal-clearable SSPA probe The fluorescent group ZWCY was linked to the SSPA-responsive peptide using an amide condensation method: the SSPA-responsive peptide (15 mg, 0.0075 mmol), the fluorescent group ZWCY (9.4 mg, 0.015 mmol), HATU (11.4 mg, 0.03 mmol), HOBT (4.1 mg, 0.03 mmol), and N,N-diisopropylethylamine (3.8 mg, 0.3 mmol) were dissolved in 7 mL of anhydrous DMF and reacted at room temperature with stirring for 8 hours. The resulting reaction solution was dialyzed against ultrapure water for 12 hours and then freeze-dried to obtain the SSPA-responsive peptide-ZWCY complex (19.0 mg, yield 85%).

[0057] A click chemistry method was used to attach the fluorescence quencher group IRDye-QC Azide to the SSPA-responsive peptide: The SSPA-responsive peptide-ZWCY complex (19 mg, 0.005 mmol) and IRDye-QC Azide (5.3 mg, 0.0056 mmol) were dissolved in 5 mL of DMSO / H2O (2:1 v / v), and then CuSO4·5H2O (0.7 mg, 0.0028 mmol) and sodium ascorbate (0.5 mg, 0.0028 mmol) were added. The reaction was carried out under argon protection with stirring at room temperature for 6 hours. After the reaction was completed, the reaction solution was dialyzed against ultrapure water for 12 hours (MWCO 1 kDa) and then freeze-dried to obtain the final product, a renal-clearable SSPA probe (15.0 mg, yield 78%).

[0058] (3) Fusion of renal-clearable SSPA probe with polyethylene glycol chain: The purified renal-clearable SSPA probe (15.0 mg) was dissolved in 2 mL of DMF solvent, and then NHS-PEG1000-Mal (25 mg, purchased from PonsureBiological) and N,N-diisopropylethylamine (3 µL) were added. After stirring at room temperature for 5 hours under nitrogen protection, the resulting reaction solution was dialyzed in ultrapure water for 12 hours (MWCO 2 kDa), and then freeze-dried to obtain renal-clearable SSPA probe-polyethylene glycol-Mal (20.0 mg, yield 40%).

[0059] (4) Neutrophil isolation For peripheral blood neutrophils, the Ficoll-Paque method was used for separation: 2.5 mL of Histopaque 1119 solution and 2.5 mL of Histopaque 1077 solution were sequentially stacked in a 15 mL centrifuge tube to construct a discontinuous density gradient. Then, 4 mL of fresh anticoagulated whole blood was gently added to the top layer of the gradient. This three-layer gradient system was centrifuged at 400 g for 45 minutes. After centrifugation, the upper layer containing the mononuclear cell ring was discarded, and the second ring-shaped cell layer was collected. The resulting cells were then washed three times with PBS. Afterward, erythrocyte lysis buffer (Beyotime, Cat. No.: C3702) was added to the washed cells. After lysis, the cell suspension was centrifuged at 500 g for 10 minutes and washed twice with HBSS buffer.

[0060] For bone marrow-derived neutrophils, Percoll density gradient centrifugation was used for separation: After euthanizing mice or rats, the bones were soaked in 75% ethanol for 10 min, and then the femur and tibia were quickly separated. After carefully removing the attached muscle and fascia, the bone marrow was immersed in RPMI 1640 medium (the medium covered the bone to prevent cell drying and inactivation) for later use. Then, the bone marrow was washed out from the bone marrow with phosphate-buffered saline (PBS), centrifuged at 200g for 3 min, and resuspended in PBS. The resulting single-cell suspension was then gently added to a 55%, 65%, and 78% Percoll discontinuous density gradient separation system (prepared in PBS), centrifuged at 1000g for 30 min, and mature neutrophils at the 65% and 78% Percoll gradient interface were collected and washed three times with pre-cooled PBS to obtain the final neutrophils.

[0061] (5) Neutrophils (1×10 8The neutrophils were resuspended in 400 μL PBS (pH 7.5) and then incubated with an equal volume of 0.05 mg / mL Traut's reagent (2-iminothione; dissolved in pH 7.5 PBS) at room temperature for 30 minutes. Excess Traut's reagent was then removed by centrifugation (500 g, 10 min), and the cells were washed twice with PBS buffer. Next, the washed neutrophils were incubated with 25 μM renal-clearable SSPA probe-polyethylene glycol-mal (dissolved in PBS, pH 7.5) at 37°C in the dark for 40 minutes. After incubation, excess probe was removed by centrifugation (500 g, 10 min), and the cells were washed twice with PBS buffer. The renal-clearable SSPA probe-neutrophil conjugate was then prepared and stored in PBS for later use.

[0062] Experimental example: like Figure 1 As shown, to investigate whether the renal-clearable thrombin probe in Example 1 could be rapidly hydrolyzed by thrombin, different concentrations of the renal-clearable thrombin probe (10, 30, 50, 70, 100, 200, 300, 400 μM; dissolved in PBS buffer) were reacted with 0.1 U / mL thrombin at 37°C, and the maximum reaction rate of the enzymatic reaction was determined using a fluorescence spectrometer. The results indicate that the renal-clearable thrombin probe in Example 1 is an effective substrate for thrombin and can be rapidly hydrolyzed by thrombin.

[0063] like Figure 2 As shown, to investigate the specificity of the renal-clearable thrombin probe in Example 1 to thrombin, different types of interfering reagents (90 µM reactive oxygen species, 90 µM metal ions, or 0.1 U / mL of other types of proteases; dissolved in PBS buffer) were reacted with 25 μM of the renal-clearable thrombin probe at 37 °C for 1 h, and the fluorescence intensity of the reaction solution was measured using a fluorescence spectrometer. The results indicate that the renal-clearable thrombin probe in Example 1 can only be hydrolyzed by thrombin and has high specificity.

[0064] like Figure 3 As shown, to investigate whether the renal-clearable SSPA probe in Example 2 could be rapidly hydrolyzed by the SSPA protease, different concentrations of the renal-clearable SSPA probe (10, 30, 50, 80, 100, 200, 300, 400, 500 μM; dissolved in PBS buffer) were reacted with 0.1 U / mL of SSPA protease at 37°C, and the maximum reaction rate was determined using a fluorescence spectrometer. The results indicate that the renal-clearable SSPA probe in Example 2 is an effective substrate for the SSPA protease and can be rapidly hydrolyzed by it.

[0065] like Figure 4 As shown, to investigate the specificity of the renal-clearable SSPA probe in Example 2 for SSPA protease, different types of interfering reagents (90 µM reactive oxygen species, 90 µM metal ions, or 0.1 U / mL of other types of proteases; dissolved in PBS buffer) were reacted with 25 μM of the renal-clearable SSPA probe at 37 °C for 1 h, and the fluorescence intensity of the reaction solution was measured using a fluorescence spectrometer. The results indicate that the renal-clearable SSPA probe in Example 2 can only be hydrolyzed by SSPA protease and has high specificity.

[0066] like Figure 5 As shown, to investigate whether the cell morphology of the cells in Examples 1 and 2 would change after being coupled with the renal-clearable optical diagnostic probe, the renal-clearable thrombin probe-platelet conjugate and the renal-clearable SSPA probe-neutrophil conjugate were fixed in 2.5% glutaraldehyde (pentanediol dissolved in PBS) for 24 hours, respectively. They were then dehydrated using a gradient of alcohols (50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol, 10 minutes per gradient), followed by gold sputtering and observation using a scanning electron microscope. The results showed that the cell membranes of platelets and neutrophils maintained their intact morphology after being coupled with the renal-clearable optical diagnostic probe.

[0067] like Figure 6 As shown, to investigate the distribution of the renal-clearable optical diagnostic probes on the cell surface in Examples 1 and 2, the distribution of the renal-clearable thrombin probe-platelet conjugate and the renal-clearable SSPA probe-neutrophil conjugate was fixed with mounting medium and observed using a confocal scanning microscope. The results showed that the renal-clearable optical diagnostic probes were uniformly distributed on the cell surface.

[0068] like Figure 7 As shown, to investigate the targeting ability of the renal-clearable thrombin probe-platelet conjugate in Example 1 to the thrombus site, the purified thrombin probe-platelet conjugate was labeled with a near-infrared fluorescent group (ZWCY) and then intravenously injected into a mouse model of deep vein thrombosis (the deep vein thrombosis model was constructed using the inferior vena cava 90% stenosis method, and the injection dose of the thrombin probe-platelet conjugate was 1×10⁻⁶. 8 (Thrombin probe-platelet conjugate per kg mouse body weight). Twenty-four hours after injection, the deep vein thrombus was removed, and the near-infrared fluorescence intensity of the thrombus was measured using an in vivo imaging system. The results indicate that the renal-clearable thrombin probe-platelet conjugate in Example 1 has excellent targeting specificity to the thrombus site.

[0069] like Figure 8 As shown, to investigate the targeting ability of the renal-clearable SSPA probe-neutrophil conjugate in Example 2 to the site of infection, the purified SSPA probe-neutrophil conjugate was labeled with a near-infrared fluorescent group (ZWCY) and then intravenously injected (injection dose of 1×10⁻⁶). 7 (1 × 10⁻⁶ cells / kg rat body weight) was injected into a rat model of osteomyelitis (the osteomyelitis model was established by injecting 1 × 10⁻⁶ cells / kg rat body weight into the right tibia of the rat). 6 (Constructed from Staphylococcus aureus ATCC25923). Twenty-four hours after injection of the SSPA probe-neutrophil conjugate, both tibias of the rats were removed, and the near-infrared fluorescence intensity of the tibias was measured using an in vivo imaging system. The results showed that the kidney-clearable SSPA probe-neutrophil conjugate in Example 2 exhibited excellent targeting of the infection site.

[0070] like Figure 9 As shown, to investigate the biosafety of the renal-clearable optical diagnostic probe-cell conjugates in Examples 1 and 2, the renal-clearable optical diagnostic probe-cell conjugate (thrombin probe-platelet conjugate) was injected into healthy rats at a dose of 3.5 × 10⁻⁶. 7 The dose of SSPA probe-neutrophil conjugate was 1 × 10⁻⁶ / kg rat body weight. 7 (Number of rats / kg body weight). At 2, 12, and 24 hours after injection, 500 μL of blood was collected from the orbital venous plexus of rats, and serum was harvested by centrifugation at 3000 rpm for 15 minutes to measure the levels of C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Twenty-four hours after injection, major organs were removed for pathological section analysis. The results showed that intravenous injection of the renal-clearable optical diagnostic probe-cell conjugate did not cause changes in serum CRP, IL-6, and TNF-α levels in rats, and had no effect on major organs. This indicates that the renal-clearable optical diagnostic probe-cell conjugate in Examples 1 and 2 has good biocompatibility.

[0071] like Figure 10 As shown, to investigate the diagnostic ability of the renal-clearable thrombin probe-platelet conjugate in Example 1 for thrombotic diseases, the thrombin probe-platelet conjugate was intravenously injected into a rat model of deep vein thrombosis (dose: 3.5 × 10⁻⁶). 7(Thrombin probe-platelet conjugate per kg rat body weight), and urine samples were collected from rats 2 hours after injection for analysis. The results showed that after injection of the thrombin probe-platelet conjugate, renally clearable fragments (with near-infrared signals) appeared in the urine of rats with deep vein thrombosis. The concentration of renally clearable fragments increased over time, indicating that the renally clearable thrombin probe-platelet conjugate in Example 1 has good diagnostic ability for deep vein thrombosis.

[0072] like Figure 11 As shown, to investigate the diagnostic ability of the renal-clearable thrombin probe-platelet conjugate in Example 1 for thrombotic diseases, the thrombin probe-platelet conjugate was administered intravenously (dose 1 × 10⁻⁶). 8 (6 mg / kg mouse body weight) cells were injected into a mouse model of diffuse thrombosis (the model was constructed by intratracheal infusion of lipopolysaccharide (LPS) at a dose of 6 mg / kg mouse body weight). Urine samples were collected from the mice within 1 hour after injection of the cell conjugate for analysis. The results showed that after injection of the thrombin probe-platelet conjugate, renally clearable fragments (with near-infrared signals) appeared in the urine of mice with diffuse thrombosis, and the renally clearable fragments were reduced in the treatment group, indicating that the renally clearable thrombin probe-platelet conjugate in Example 1 has good diagnostic capabilities for diffuse thrombosis.

[0073] like Figure 12 As shown, to investigate the diagnostic capability of the renal-clearable SSPA probe-neutrophil conjugate in Example 2 for Staphylococcus aureus infection, the SSPA probe-neutrophil conjugate was administered via tail vein injection (dose: 1 × 10⁻⁶). 7 One SSPA probe-neutrophil conjugate / kg rat body weight was injected into rats with Staphylococcus aureus-induced osteomyelitis (the osteomyelitis model was established by injecting 1×10⁻⁶ SSPA probe-neutrophil conjugate / kg rat body weight into the right tibia of the rat). 6 The sample was constructed from Staphylococcus aureus ATCC25923, and urine samples from rats were subsequently collected for analysis. The results showed that after injection of the SSPA probe-neutrophil conjugate, renally clearable fragments (with near-infrared signals) appeared in the urine of Staphylococcus aureus-infected rats. Furthermore, these renally clearable fragments only appeared in Staphylococcus aureus infections, indicating that the renally clearable SSPA probe-neutrophil conjugate in Example 2 has good diagnostic capabilities for Staphylococcus aureus infections.

[0074] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing a kidney-clearable optical diagnostic probe-cell conjugate, characterized in that, A renal-clearable optical diagnostic probe was fused with maleimide-modified polyethylene glycol and then incubated with Traut's pretreated cells to obtain a renal-clearable optical diagnostic probe-cell conjugate. The renal-clearable optical diagnostic probe is a disease biomarker-responsive peptide linked with a fluorescent group and a fluorescence quencher group. After reacting with a disease biomarker, the disease biomarker-responsive peptide can release a fragment excreted into the urine through the kidneys. The cells include platelets, neutrophils, erythrocytes, macrophages, monocytes, and mesenchymal stem cells.

2. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, The disease markers include thrombin, Staphylococcus aureus SspA protease, matrix metalloproteinase MMP-2, matrix metalloproteinase MMP-9, collagenase, β-galactosidase, caspase-8, caspase-3, cathepsin K, cathepsin L, cathepsin B, cathepsin S, Staphylococcus aureus transpeptidase sorting enzyme A, gamma-glutamyl transferase, leukocyte esterase, angiotensin-converting enzyme, and renin.

3. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 2, characterized in that, The disease biomarker responsive peptides include thrombin-responsive peptides KGFPRGFGA, GFPRGFPAGGC, KALNNGSGFPRGRAKC, LTPRGWRLGGC, DPRSFL, GFPRGFPAGG, GF-Pip-RSGGGGKC, GFPRGFPAGG, GF-Pip-RSGGGGKC, GFPRGFPA, GDARSWG, GFPRGFGAGG, GFPRGLPAGG, GFPRVFPAGG, GFPRGF, GFPRGFPAGAGG, GFPRGFPAGGK, GFPRGFPAGGK, GGLVPRGSGAS, KGGPRSGGGC, KGALVPRGSAGK; Staphylococcus aureus SspA protease-responsive peptide KGFLEGT; and renin-responsive peptides DRVYIHPFHLVIH, DRVYIHPFHLLYHN. DRVYIHPFHLLYYS; LasB responsive peptide AFK; cathepsin responsive peptides HPGGPQ, GHPGGPQG; serine protease responsive peptide AGPR; Caspase-3 responsive peptide ZDEVD; angiotensin-converting enzyme responsive peptides FRK, LFK; SrtA protease responsive peptides QALPETGEE, LPETG; MMP protease responsive peptides PLGLW, APPGFSPFR, LR, GGRK, VHPK, SQNYPIVQ, PLGLG, PLGL, HLI, FRKP, FVR, AAA, KPLGLAR, AGFSLPA, AGFSLAKR, APA; GramB responsive peptide IEDT; cathepsin K responsive peptides HPGGPQ, KPLGL.

4. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, The fluorescent groups include anthocyanin, indocyanine, and rhodamine dye groups; the fluorescence quenching groups include IRDye-QC, BHQ-0, BHQ-1, BHQ-2, and BHQ-3.

5. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, In the maleimide-modified polyethylene glycol, the molecular weight of the polyethylene glycol ranges from 200 to 7000 Da.

6. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, A kidney-clearable optical diagnostic probe was fused with maleimide-modified polyethylene glycol via an NHS-activated ester-amino amide condensation-mediated method: The kidney-clearable optical diagnostic probe was dissolved in an organic solvent, and then maleimide-modified polyethylene glycol and N,N-diisopropylethylamine were added. After reacting at room temperature for 3-8 hours under inert gas protection, the product was obtained by dialysis and drying.

7. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, The method for incubating the probe with Traut's pretreated cells is as follows: 10 8 Orders of magnitude of cells are resuspended in PBS and incubated with Traut's reagent at room temperature for 20-60 minutes. After incubation, excess Traut's reagent is removed, and the cells are washed. The cells are then incubated with 20-30 μM of probe fused with polyethylene glycol at 30-40°C in the dark for 0.5-3 hours. After incubation, excess probe is removed to obtain the final product.

8. The method for preparing a kidney-clearable optical diagnostic probe-cell conjugate according to claim 1, characterized in that, The cells are derived from mammals, including rodents, rabbits, pigs, or primates.

9. A kidney-clearable optical diagnostic probe-cell conjugate prepared by the preparation method according to any one of claims 1-8.

10. The application of the renal-clearable optical diagnostic probe-cell conjugate of claim 9 in disease diagnosis, characterized in that, The types of diseases mentioned include thrombosis, osteomyelitis, aneurysm, atherosclerosis, bone cancer, and bloodstream infection.

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