Preparation method of a cathepsin E-responsive preparation and its application in pancreatic cancer-specific photodiagnosis and treatment

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a cathepsin E-responsive preparation and its application in pancreatic cancer-specific photodiagnosis and treatment. The method for preparing the cathepsin E-responsive preparation of the present invention is to modify the carboxyl group on the outside of gold nanoparticles and couple a polypeptide connected to a small molecule fluorescent dye to finally obtain a cathepsin E-responsive preparation. The cathepsin E-responsive preparation has uniform particle size, good dispersibility, and a negative surface Zeta potential. The synthesis steps are simple, the operability is strong, and the preparation has good biosafety. The cathepsin E-responsive preparation provided by the present invention has good stability, can form aggregates at the tumor site, and can realize dual-modal imaging and photothermal treatment of the tumor in combination with AIE signals. In addition, the present invention has universal applicability and can be promoted and applied to other drugs according to different therapeutic purposes, with high commercial value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a cathepsin E-responsive preparation and its application in pancreatic cancer-specific photodiagnosis and treatment. Background Art

[0002] Pancreatic cancer theranostics remain a significant challenge due to delayed detection and limited effectiveness of systemic treatments. Notably, phototheranostics, which converts absorbed light into various forms of energy or signals, offers numerous opportunities. Among phototherapeutic agents, gold nanoparticles (AuNPs) have shown great potential for cancer photoacoustic (PA) imaging / photothermal therapy (PTT), attributed to their excellent biocompatibility, multifunctional modification, and photothermal conversion. However, the biological and optical properties of AuNPs are largely dependent on their size, with large AuNPs exhibiting excellent light absorption but poor penetration in pancreatic tumors with a dense extracellular matrix, while small AuNPs exhibit strong tumor penetration but negligible near-infrared absorption. To reconcile this discrepancy between AuNP size and absorption, small AuNPs have been found to self-assemble into large aggregates at tumor sites by integrating pH-sensitive bonds, photoresponsive molecules, or block copolymers. However, the complex physiological environment and limited light penetration may lead to the aggregation of AuNPs at non-tumor sites. Therefore, there is an urgent need to design a formulation that can achieve the “small to large” conversion of AuNPs in the tumor microenvironment, thereby precisely improving the photothermal efficiency and reducing non-target thermal damage.

[0003] In addition, another issue worthy of attention is the specific non-invasive imaging diagnosis of pancreatic cancer. Currently, the diagnosis of pancreatic cancer relies on traditional imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and endoscopic ultrasound (EUS). Despite this, the specificity and sensitivity of these imaging modalities still need to be improved, and non-real-time invasive biopsy is usually required to assist diagnosis. Therefore, high-sensitivity, non-invasive fluorescence imaging has attracted widespread attention in tumor diagnosis. In particular, aggregation-induced emission (AIE) molecules have recently emerged, which exhibit very weak emission in solution but strong signals when aggregated, with wide Stokes shifts, long-term cell tracking, and significant photostability. Therefore, AIE molecules have become an excellent choice for co-delivery with therapeutic agents to achieve precise cancer treatment and diagnosis. However, weak tumor specificity seriously hinders the biomedical application of these AIE molecules. Therefore, the development of AIE-based tumor-specific imaging probes is particularly important for accurate diagnosis and dynamic monitoring of pancreatic cancer.

[0004] Notably, molecular imaging probes targeting specific cancer biomarkers can help detect tumor progression quickly and accurately without relying on traditional invasive pathological analysis. Cathepsin E (CTSE) is a highly specific tumor-associated aspartate proteolytic enzyme that is upregulated in pancreatic cancer and has been shown to be positively correlated with disease progression. This highlights the great potential of CTSE-activated imaging probes for pancreatic cancer visualization and treatment response assessment. Therefore, exploring CTSE-specific triggering of AuNP and AIE aggregation has important prospects for improving pancreatic cancer-specific therapeutic diagnosis. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to propose a preparation method of a cathepsin E-responsive preparation and its pancreatic cancer-specific phototherapy application. By integrating AuNP, AIE (Quinoline-malononitrile-COOH) and CTSE-triggered responsive peptide (AGFSLPAGC), a cathepsin E-responsive preparation (AuHQ) was prepared. This responsive preparation can accurately achieve dual self-assembly of AuNP and AIE in pancreatic tumors, promoting fluorescence and photoacoustic imaging-guided PTT for targeted dual-modal pancreatic cancer treatment and diagnosis.

[0006] Based on the above objectives, the present invention provides a method for preparing a cathepsin E-responsive preparation, characterized in that it comprises the following steps:

[0007] S1: The response peptide was grafted onto quinoline-malononitrile by solid-phase synthesis to obtain Quinoline-malononitrile-AGFSLPAGC;

[0008] S2: Preparation of AuNPs by thermal reduction method;

[0009] S3: Through SH-PEG 5000 -COOH was modified onto the surface of AuNP to obtain AuC;

[0010] S4: esterified AuC is obtained by reacting 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and AuC;

[0011] S5: reacting the esterified AuC with N-(2-aminoethyl)maleimide via an amide condensation reaction to obtain AuM;

[0012] S6: AuM and quinoline-malononitrile-AGFSLPAGC were reacted by a click reaction to obtain AuHQ, which is a cathepsin E-responsive preparation;

[0013] The response peptide can be specifically recognized by cathepsin.

[0014] Preferably, the specific preparation steps of Quinoline-malononitrile-AGFSLPAGC in step S1 are as follows:

[0015] (1) Under an argon atmosphere, 2-methylquinoline and iodomethane were dissolved in anhydrous acetonitrile in sequence, reacted at 80°C for 20-24 hours, cooled to room temperature, filtered, washed, and dried to obtain a crude product;

[0016] (2) In an ice-water bath, the crude product, malononitrile, anhydrous ethanol, and sodium ethoxide were mixed and reacted under an argon atmosphere for 4 h. The resulting mixture was added to ice water, the pH was adjusted to 7-8, filtered, dried, and column chromatography was performed to obtain quinoline-malononitrile;

[0017] (3) Under nitrogen atmosphere, quinoline-malononitrile and 4-hydroxybenzaldehyde were dissolved in acetonitrile containing piperidine, then refluxed at 80°C for 24 h, cooled to room temperature, evaporated under reduced pressure, and column chromatography was performed to obtain quinoline-malononitrile-OH;

[0018] (4) Under an argon atmosphere, quinoline-malononitrile-OH and ethyl bromoacetate were dissolved in acetonitrile, potassium carbonate was added, and the mixture was refluxed for 12 h to obtain a crude product; the crude product and NaOH were then dissolved in TNF / water, the pH was adjusted to 5-6, and the mixture was filtered and recrystallized to obtain quinoline-malononitrile-COOH;

[0019] (5) The resin was mixed with a 20% piperidine solution, and after reacting for 40 minutes, a small amount of the resin was taken for ninhydrin detection, which was blue. Subsequently, the rink-amide-MBHA-resin resin, Fmoc-AA-OH, HBTU, HOBT and DIPEA were mixed, reacted in DMF at room temperature for 1 hour, washed, and a small amount of the resin was taken for ninhydrin detection, which was colorless. After reacting with a 20% piperidine solution for 40 minutes, a small amount of the resin was taken for ninhydrin detection, which was blue or reddish brown. Subsequently, the rink-amide-MBHA-resin resin, Quinoline-malononitrile-COOH, HBTU, HOBT and DIPEA were mixed, reacted for 1 hour, washed and subjected to ninhydrin detection, which was colorless. Finally, the mixture was treated with a lysis buffer at room temperature for 2 hours, concentrated, centrifuged, and purified by HPLC to obtain Quinoline-malononitrile-AGFSLPAGC.

[0020] Preferably, the washing in step (1) is performed using acetonitrile.

[0021] Preferably, in step (2), the pH is adjusted to 7-8 using 1M hydrochloric acid.

[0022] Preferably, the lysis solution in step (4) is a mixture of TFA, TIS, H2O, and EDT in a mass ratio of 94:1:2.5:2.5.

[0023] Preferably, in step (4), acetic acid is used to adjust the pH to 5-6.

[0024] Preferably, the volume ratio of TNF / water in step (4) is 7:3.

[0025] Preferably, the order of adding Fmoc-AA-OH in step (5) is Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH.

[0026] Preferably, in step (5), the equivalent ratio of rink-amide-MBHA-resin, Fmoc-AA-OH, HBTU, HOBT and DIPEA is 1:3:2.5:3:6.

[0027] Preferably, in step (5), the equivalent ratio of rink-amide-MBHA-resin, Quinoline-malononitrile-COOH, HBTU, HOBT and DIPEA is 1:3:2.5:3:6.

[0028] Preferably, the preparation steps of AuNP in step S2 are as follows:

[0029] Dissolve tetrachloroauric acid trihydrate and sodium citrate dihydrate in deionized water to obtain a tetrachloroauric acid solution and a sodium citrate solution, respectively. Mix the tetrachloroauric acid solution with deionized water, then add the sodium citrate solution, reflux, cool, add the tetrachloroauric acid solution and the sodium citrate solution again, repeat three times, and cool to room temperature to obtain a solution of AuNPs.

[0030] Preferably, the mass ratio of tetrachloroauric acid trihydrate to sodium citrate dihydrate is 1:2-6.

[0031] Preferably, the volume ratio of the tetrachloroauric acid solution to the sodium citrate solution is 1:5-10.

[0032] Preferably, the tetrachloroauric acid solution and the sodium citrate solution are each divided into four equal parts and added in sequence.

[0033] Preferably, the cooling temperature is 85°C and the reaction time is 0.5-1h

[0034] Preferably, the specific preparation steps of the AuC in step S3 are as follows:

[0035] SH-PEG 5000 -COOH was added to the AuNP solution, stirred for 24 h, and washed to obtain precipitated AuC;

[0036] Preferably, the specific preparation steps of the esterified AuC in step S4 are as follows:

[0037] The precipitated AuC was resuspended, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, and the mixture was stirred at 20-25° C. for 0.5-1 h. The mixture was centrifuged to obtain a precipitate, which was the esterified AuC.

[0038] Preferably, the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1-5:1.

[0039] Preferably, the specific preparation steps of the AuM in step S5 are as follows:

[0040] The precipitate in step S4 was resuspended, N-(2-aminoethyl)maleimide hydrochloride and triethylamine were added, stirred and mixed evenly, and centrifuged to obtain precipitated AuM.

[0041] Preferably, the molar mass ratio of the N-(2-aminoethyl)maleimide hydrochloride to triethylamine is 1:1.

[0042] Preferably, the specific preparation steps of AuHQ in step S6 are as follows:

[0043] The precipitated AuM was resuspended, and Quinoline-malononitrile-AGFSLPAGC was added, stirred at 20-25°C for 24 hours, centrifuged, and resuspended to obtain the final material AuHQ, which is the cathepsin E-responsive preparation.

[0044] Preferably, the organic solvent used for resuspension in steps S3, S4, and S5 is dimethyl sulfoxide.

[0045] Preferably, the cathepsin E-responsive preparation has a particle size of 20-50 nm.

[0046] Furthermore, the present invention also provides the application of a cathepsin E-responsive preparation, which is mainly used in the field of pancreatic cancer-specific photodiagnosis and treatment.

[0047] Beneficial effects of the present invention:

[0048] (1) The preparation method of the cathepsin E-responsive preparation of the present invention has mild synthesis conditions and high stability under physiological conditions. In tumor tissue, the cathepsin E-responsive preparation can lead to the self-assembly of AuNPs and AIE under CTSE-responsive stimulation, which can achieve strong dual-modal imaging and photothermal therapy effects of tumors.

[0049] (2) The preparation method of the cathepsin E-responsive preparation of the present invention can be extended to other coupled preparations, has good universality and scalability, and has good conversion value.

[0050] (3) The preparation method of the cathepsin E-responsive preparation of the present invention can be used in conjunction with a variety of treatment methods according to different needs to achieve synergistic tumor treatment and thus achieve the best tumor treatment effect.

[0051] (4) The preparation method of the cathepsin E-responsive preparation of the present invention has the characteristics of simple synthesis process, low cost, mild requirements and stable product. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description:

[0053] Figure 1 Schematic diagram of the process for preparing AuHQ in Example 1 of the present invention;

[0054] Figure 2 This is the LC-MS spectrum of the purified Quinoline-malononitrile-AGFSLPAGC prepared in Example 1 of the present invention;

[0055] Figure 3 This is a transmission electron micrograph of AuHQ prepared in Example 1 of the present invention;

[0056] Figure 4 This is the particle size distribution diagram of AuHQ prepared in Example 1 of the present invention;

[0057] Figure 5 This is the ESI-MS spectrum of Quinoline-malononitrile-AGFSLPAGC prepared in Example 1 of the present invention before and after enzyme digestion;

[0058] Figure 6 The fluorescence emission spectra of AuHQ prepared in Example 1 of the present invention before and after incubation with CTSE for different times;

[0059] Figure 7 The UV-visible absorption spectra of AuHQ prepared in Example 1 of the present invention before and after incubation with CTSE for different times;

[0060] Figure 8 Transmission electron microscopy images of AuHQ prepared in Example 1 of the present invention before and after incubation with CTSE for 24 hours;

[0061] Figure 9 This is the particle size distribution diagram of AuHQ prepared in Example 1 of the present invention after incubation with CTSE for 24 hours;

[0062] Figure 10 The AuHQ prepared in Example 1 of the present invention is subjected to 808nm laser (1W / cm 2 ) Temperature rise curves and infrared thermal images before and after incubation with CTSE under irradiation;

[0063] Figure 11 This is a temperature rise and fall curve of AuHQ prepared in Example 1 of the present invention after incubation with CTSE for 24 hours;

[0064] Figure 12 The photoacoustic signal changes of AuHQ prepared in Example 1 of the present invention before and after incubation with CTSE for different times;

[0065] Figure 13 Flow cytometry analysis of each preparation group in the pancreatic cancer cell apoptosis experiment;

[0066] Figure 14 This is a diagram showing the distribution of AuHQ prepared in Example 1 of the present invention in tumor-bearing mice;

[0067] Figure 15 This is a diagram showing the photoacoustic signal changes of AuHQ prepared in Example 1 of the present invention in tumor-bearing mice;

[0068] Figure 16 The graph shows the relative tumor volume of Panc02 tumor-bearing mice treated with different therapies. DETAILED DESCRIPTION

[0069] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0070] Example 1: A method for preparing a cathepsin E-responsive preparation, the specific preparation steps are as follows:

[0071] (1) Under argon atmosphere, 2-methylquinoline (1.43 g, 10 mmol, 1.38 mL) and iodomethane (4.71 g, 30 mmol, 2.49 mL) were dissolved in anhydrous acetonitrile (30 mL) in sequence, refluxed at 80°C for 20-24 h, cooled to room temperature, filtered, and the crude product was washed with acetonitrile and dried to obtain a crude product (2.552 g, 8.8 mmol);

[0072] (2) The crude product was dissolved in anhydrous ethanol in an ice-water bath, and malononitrile (1.47 g, 22 mmol) and sodium ethoxide (3.74 g, 55 mmol) were added. After reacting for 4 h in an argon atmosphere in the dark, the mixture was poured into 200 mL of ice water and the pH was adjusted to 7-8 with 1 M hydrochloric acid. The mixture was filtered, dried, and purified by column chromatography to obtain quinoline-malononitrile (800 mg, 3.4 mmol).

[0073] (3) Under nitrogen atmosphere, quinoline-malononitrile and 4-hydroxybenzaldehyde (1.0381 g, 8.5 mmol) were dissolved in acetonitrile (20 mL) containing piperidine (1 mL), refluxed at 80°C for 24 h, cooled to room temperature, and the solvent was evaporated under reduced pressure. After column chromatography, quinoline-malononitrile-OH (638 mg, 1.914 mmol) was obtained.

[0074] (4) Quinoline-malononitrile-OH and ethyl bromoacetate (319 mg, 1.914 mmol) were dissolved in acetonitrile (10 mL) at room temperature, potassium carbonate (456.2 mg, 3.254 mmol) was added, and the mixture was refluxed under an argon atmosphere for 12 h to obtain a crude product. The crude product (779.4 mg, 1.818 mmol) and NaOH (79.4 mg, 1.976 mmol) were dissolved in TNF / water (v / v = 7:3, 10 mL). The mixture was stirred at room temperature for 9 h, and the pH was adjusted to 5-6 with acetic acid. The mixture was filtered and recrystallized to obtain Quinoline-malononitrile-COOH (662.49 mg, 1.545 mmol).

[0075] (5) 2.1834 g (1 mmol) of resin was placed in a solid phase synthesis tube, and a small amount of DMF was added to swell for 1 hour. The mixture was reacted with 20% piperidine solution (10 mL) for 40 minutes. A small amount of resin was then taken for ninhydrin detection, which was blue. Then, rink-amide-MBHA-resin resin, Fmoc-AA-OH, HBTU, HOBT and DIPEA were mixed in an equivalent ratio of 1:3:2.5:3:6 and reacted in DMF at room temperature for 1 hour. The mixture was washed alternately with DMF and DCM 6 times. A small amount of resin was taken for ninhydrin detection, which was colorless. The mixture was then reacted with 20% piperidine solution for 40 minutes to remove the Fmoc protecting group on the amino group. A small amount of resin was tested with ninhydrin, resulting in a blue or reddish-brown color. Rin-amide-MBHA-resin resin, Quinoline-malononitrile-COOH, HBTU, HOBT, and DIPEA were then mixed in an equivalent ratio of 1:3:2.5:3:6. After reacting for 1 hour, the mixture was washed and tested with ninhydrin, resulting in a colorless product. Finally, the mixture was treated with 10 mL of lysis buffer (TFA / TIS / H2O / EDT = 94:1:2.5:2.5) at room temperature for 2 hours. After concentration, the mixture was centrifuged with icy ether to obtain a crude product, which was then purified by HPLC to obtain Quinoline-malononitrile-AGFSLPAGC.

[0076] (6) Synthesis of AuNPs: 80 mg of tetrachloroauric acid trihydrate was dissolved in 3.2 mL of deionized water to obtain a tetrachloroauric acid solution, and 320 mg of sodium citrate dihydrate was dissolved in 32 mL of deionized water to obtain a sodium citrate solution;

[0077] (7) Mix 0.8 mL of tetrachloroauric acid solution with 200 mL of deionized water, boil, then add 8 mL of sodium citrate solution and reflux for 30 min. After cooling to 85 °C, add 0.8 mL of tetrachloroauric acid solution and 8 mL of sodium citrate solution into the reaction system and react for 30 min. Repeat three times. After the reaction is completed, turn off the heating and continue stirring. After cooling to room temperature, collect the AuNP solution.

[0078] (8) Synthesis of AuC: 20 mg SH-PEG 5000 -COOH was added to 100 mL of AuNP solution, stirred for 24 h, then centrifuged at 13000 rpm for 16 min and washed twice with water to obtain precipitated AuC;

[0079] (9) Synthesis of AuM: The precipitated AuC was resuspended in 2 mL of dimethyl sulfoxide, 4 mg of EDC and 2.4 mg of NHS were added, and the mixture was stirred at room temperature for 1 h. Subsequently, the mixture was centrifuged at 13,000 rpm for 16 min. The precipitate was resuspended in 1 mL of dimethyl sulfoxide, 1.84 mg of N-(2-aminoethyl)maleimide hydrochloride and 3.6 μL of triethylamine were added, and the mixture was stirred for 24 h. The mixture was centrifuged at 13,000 rpm for 16 min to obtain the precipitated AuM.

[0080] (10) The precipitated AuM was resuspended in 1 mL of dimethyl sulfoxide, and then 2 mg of quinoline-malononitrile-AGFSLPAGC was added. The mixture was stirred at room temperature for 24 h and centrifuged at 13,000 rpm for 16 min. The resulting precipitate was resuspended in 1 mL of deionized water to obtain the nanomaterial AuHQ, which was a cathepsin E-responsive preparation.

[0081] Example 2: Studying the changes of Quinoline-malononitrile-AGFSLPAGC before and after enzyme digestion, comprising the following steps:

[0082] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The quinoline-malononitrile-AGFSLPAGC in Example 1 was resuspended in an acidic PBS solution;

[0083] (2) After 24 hours, freeze-dry and use ESI-MS to detect the spectrum before and after enzyme digestion. The detection results are as follows Figure 5 shown.

[0084] Example 3: Using a microplate reader to detect the fluorescence emission spectrum of the cathepsin E-responsive preparation obtained in Example 1, comprising the following steps:

[0085] (1) CTSE was added to a sodium acetate buffer solution (pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution.

[0086] (2) The fluorescence emission spectra of the AuHQ solution at 0 h, 1 h, 4 h, 8 h, 12 h, and 24 h were recorded using an enzyme marker in the wavelength range of 550-750 nm. The test results are as follows: Figure 6 shown.

[0087] Example 4: This example uses an ultraviolet spectrophotometer to detect the ultraviolet-visible absorption spectrum of nanomaterials, including the following steps:

[0088] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution;

[0089] (2) The UV-visible absorption spectra of the AuHQ solution at 0 h, 3 h, 6 h, 12 h, and 24 h were recorded using a UV spectrophotometer in the wavelength range of 400-1000 nm. The test results are as follows: Figure 7 shown.

[0090] Example 5: Using a transmission electron microscope to detect the changes in the morphology of AuHQ before and after incubation with CTSE, the method comprises the following steps:

[0091] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution;

[0092] (2) The morphology of AuHQ before and after incubation with CTSE enzyme for 24 h was examined using transmission electron microscopy. Figure 8 shown.

[0093] Example 6: Using DLS to detect the change in the particle size of AuHQ after incubation with CTSE, comprising the following steps:

[0094] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution;

[0095] (2) After incubation for 24 h, the particle size of AuHQ was detected by DLS. The test results are as follows: Figure 9 shown.

[0096] Example 7: This example uses an infrared thermal imager to record the temperature change of nanomaterials, including the following steps:

[0097] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution;

[0098] (2) Using 808nm laser (1W / cm 2 ) for 10 min, and the temperature of the AuHQ solution at 0 h, 3 h, 6 h, 12 h, and 24 h was recorded using an infrared thermal imager. The test results are as follows Figure 10shown.

[0099] Example 8: Using an infrared thermal imager to record the temperature rise and fall of a nanomaterial to calculate the photothermal conversion efficiency, comprising the following steps:

[0100] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution to a final concentration of 200 μg / mL;

[0101] (2) The temperature of the AuHQ solution was recorded for 24 h using an infrared thermal imager and the temperature was measured using an 808 nm laser (1 W / cm 2 ) irradiate for 10 minutes, then turn off the laser and stop recording when the temperature drops to the starting temperature. The test results are as follows Figure 11 shown.

[0102] Example 9: Use The LAZR small animal multimodal imaging system was used to detect the photoacoustic signal changes of AuHQ before and after incubation with CTSE for different times, including the following steps:

[0103] (1) CTSE was added to a buffer solution (sodium acetate buffer solution / DMSO = 98:2, pH 4) and incubated at 37°C for 0.5 h to stimulate its activity. The nanomaterial AuHQ in Example 1 was resuspended in an acidic PBS solution;

[0104] (2)Use The LAZR small animal multimodal imaging system recorded the photoacoustic signal values ​​of the AuHQ solution at 0 h, 3 h, 6 h, 12 h, and 24 h. The detection results are as follows Figure 12 shown.

[0105] Example 10: Using the Annexin V-FITC / PI apoptosis kit to detect the killing effect of each preparation group on pancreatic cancer cells, comprising the following steps:

[0106] (1) Panc02 cells were seeded into 12-well plates and grown in a constant temperature incubator for 12 h;

[0107] (2) Discard the old culture medium, add 1 mL of culture medium to each of the Control and Laser groups; add 1 mL of culture medium containing AuNPs (200 μg / mL) to each of the AuNP and AuNP+L groups; and add 1 mL of culture medium containing AuHQ (200 μg / mL) to each of the AuHQ and AuHQ+L groups. Continue culturing for 24 h.

[0108] (3) Laser group, AuNP+L group, and AuHQ+L group were irradiated with light for 5 min (1 W / cm 2 ), and continue to culture for 2 h;

[0109] (4) Trypsin digestion and collection of cells were performed, and the cells were stained using the Annexin V-FITC / PI apoptosis kit and detected by flow cytometry. The test results were as follows: Figure 13 shown.

[0110] Example 11: Detecting the biodistribution of nanomaterials using a small animal in vivo imaging system, comprising the following steps:

[0111] (1) Take 5 million Panc02 cells in the logarithmic growth phase and inoculate them subcutaneously on the back of 5-6 week-old C57BL / 6 male mice.

[0112] (2) When the tumor volume reaches 100 mm 3 Around 3 d, 100 μL of 4 mg / mL AuHQ saline solution was injected into tumor-bearing mice via the tail vein, and then the mice were subjected to fluorescence imaging at 0, 1, 2, 4, 6, 8, 12, 24, and 48 h;

[0113] (3) After 48 hours, the mice were killed, the tumors and major organs were removed and imaged, and the test results were as follows: Figure 14 shown.

[0114] Example 12: Use The LAZR small animal multimodal imaging system was used to detect the changes in the photoacoustic signal of AuHQ in tumor-bearing mice before and after different time periods, including the following steps:

[0115] (1) Take 5 million Panc02 cells in the logarithmic growth phase and inoculate them subcutaneously on the back of 5-6 week-old C57BL / 6 male mice.

[0116] (2) When the tumor volume reaches 200 mm 3 Around 24 hours after the onset of scintigraphy, 100 μL of 4 mg / mL AuHQ saline solution was injected into the tumor-bearing mice through the tail vein. Then, photoacoustic imaging was performed on the mice at 0, 3, 6, 12, 24, and 48 hours. The detection results were as follows: Figure 15 shown.

[0117] Example 13: Studying the in vivo anti-tumor effect of the nanomaterial AuHQ obtained in Example 1, comprising the following steps:

[0118] (1) Take 5 million Panc02 cells in the logarithmic growth phase and inoculate them subcutaneously on the back of 5-6 week-old C57BL / 6 male mice.

[0119] (2) When the tumor volume reaches 100 mm3 Around 24 hours after the injection, Panc02 tumor-bearing mice were randomly divided into 6 groups: (1) Control group, (2) AuNP group, (3) AuHQ group, (4) Laser group, (5) AuNP+L group, and (6) AuHQ+L group. Then, 100 μL of the corresponding preparation was injected into the tail vein of mice in different groups. 24 hours after the injection, the tumor site of mice in groups (4), (5), and (6) was irradiated with 808 laser for 5 minutes (1 W / cm 2 );

[0120] (3) Tumor size was measured every 3 days for a total of 8 times. The tumor volume (V) was calculated as follows:

[0121]

[0122] Among them, W and L are the shortest and longest diameters of the tumor, respectively. The test results are as follows: Figure 16 shown.

[0123] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0124] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a cathepsin E-responsive preparation, characterized in that: The following steps are involved: S1: The response peptide was linked to quinoline-malononitrile by solid-phase synthesis to obtain Quinoline-malononitrile-AGFSLPAGC; S2: Preparation of AuNPs by thermal reduction method; S3: Through SH-PEG 5000 -COOH was modified onto the surface of AuNP to obtain AuC; S4: esterified AuC is obtained by reacting 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and AuC; S5: reacting the esterified AuC with N-(2-aminoethyl)maleimide via an amide condensation reaction to obtain AuM; S6: AuM and Quinoline-malononitrile-AGFSLPAGC were reacted via a click reaction to obtain the final material AuHQ, which is the cathepsin E-responsive preparation; The response peptide is specifically recognized by cathepsins.

2. The method for preparing a cathepsin E-responsive preparation according to claim 1, wherein: The specific preparation steps of Quinoline-malononitrile-AGFSLPAGC in step S1 are as follows: (1) Under an argon atmosphere, 2-methylquinoline and iodomethane were dissolved in anhydrous acetonitrile in sequence, reacted at 80 °C for 20-24 h, cooled to room temperature, filtered, washed, and dried to obtain a crude product; (2) In an ice-water bath, the crude product, malononitrile, anhydrous ethanol, and sodium ethoxide were mixed and reacted under an argon atmosphere for 4 h. The resulting mixture was added to ice water, the pH was adjusted to 7-8, filtered, dried, and column chromatography was performed to obtain quinoline-malononitrile; (3) Under nitrogen atmosphere, quinoline-malononitrile and 4-hydroxybenzaldehyde were dissolved in acetonitrile containing piperidine, then refluxed at 80 °C for 24 h, cooled to room temperature, evaporated under reduced pressure, and column chromatography was performed to obtain quinoline-malononitrile-OH. (4) Under an argon atmosphere, quinoline-malononitrile-OH and ethyl bromoacetate were dissolved in acetonitrile, potassium carbonate was added, and the mixture was refluxed for 12 h to obtain a crude product. The crude product and NaOH were then dissolved in TNF / water, the pH was adjusted to 5-6, and the mixture was filtered and recrystallized to obtain quinoline-malononitrile-COOH. (5) The resin was mixed with 20% piperidine solution, and after reacting for 40 minutes, a small amount of resin was taken for ninhydrin detection, which was blue. Subsequently, rink-amide-MBHA-resin resin, Fmoc-AA-OH, HBTU, HOBT and DIPEA were mixed, and reacted in DMF at room temperature for 1 hour, washed, and a small amount of resin was taken for ninhydrin detection, which was colorless. After reacting with 20% piperidine solution for 40 minutes, a small amount of resin was taken for ninhydrin detection, which was blue or reddish brown. Subsequently, rink-amide-MBHA-resin resin, Quinoline-malononitrile-COOH, HBTU, HOBT and DIPEA were mixed, and after reacting for 1 hour, washed and ninhydrin detection, which was colorless. Finally, it was treated with cleavage solution at room temperature for 2 hours, concentrated, centrifuged, and purified by HPLC to obtain Quinoline-malononitrile-AGFSLPAGC.

3. The method for preparing a cathepsin E-responsive preparation according to claim 1, wherein: The preparation steps for preparing AuNPs described in step S2 are as follows: Tetrachloroauric acid trihydrate and sodium citrate dihydrate were dissolved in deionized water respectively to obtain tetrachloroauric acid solution and sodium citrate solution. The tetrachloroauric acid solution was mixed with deionized water, and then sodium citrate solution was added to reflux. The temperature was lowered, and the tetrachloroauric acid solution and sodium citrate solution were added again. The process was repeated three times, and the AuNP solution was obtained after cooling to room temperature.

4. The method for preparing a cathepsin E-responsive preparation according to claim 1, wherein: The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide in step S4 is 1-5:

1.

5. The method for preparing the cathepsin E responsive preparation according to claim 2, characterized in that: The order of adding Fmoc-AA-OH in step (5) is Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH.

6. The method for preparing a cathepsin E-responsive preparation according to claim 2, wherein: The lysis solution in step (5) is a mixture of TFA, TIS, H2O, and EDT in a mass ratio of 94:1:2.5:2.

5.

7. The method for preparing a cathepsin E-responsive preparation according to claim 2, wherein: The equivalent ratio of rink-amide-MBHA-resin, Fmoc-AA-OH, HBTU, HOBT and DIPEA in step (5) is 1:3:2.5:3:

6.

8. The method for preparing a cathepsin E-responsive preparation according to claim 2, wherein: The equivalent ratio of rink-amide-MBHA-resin, Quinoline-malononitrile-COOH, HBTU, HOBT and DIPEA in step (5) is 1:3:2.5:3:

6.

9. The method for preparing a cathepsin E-responsive preparation according to claim 1, wherein: The particle size of the AuHQ is 20-50 nm.

10. A use of a cathepsin E responsive preparation, characterized in that: Use of the cathepsin E-responsive preparation prepared by the method for preparing a cathepsin E-responsive preparation according to any one of claims 1 to 9 in the preparation of a pancreatic cancer-specific photodiagnostic and therapeutic reagent.

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