Pancreatic cancer diagnosis and treatment nano probe with switching effect

Abstract

The invention discloses a pancreatic cancer diagnosis and treatment nano-probe with'switching 'effect, the nano-probe comprises a nano prodrug compound and a hydrophobic photosensitizer, the nano prodrug compound entraps the hydrophobic photosensitizer, the nano prodrug compound takes azide and maleimide modified low molecular weight heparin as a matrix, and the nano prodrug compound is prepared from azide and maleimide modified low molecular weight heparin as the matrix. A benzo-nitrogen five-membered ring hydrophobic anti-tumor drug and a targeting ligand are connected to the matrix. The nano prodrug compound has tumor metabolism regulation activity and can entrap a benzo-aza five-membered ring hydrophobic photosensitizer with an aggregation quenching effect, and the ROS response characteristic of the nano prodrug compound enables the probe to be specifically converted from a closed state to an open state in a tumor ROS environment, so that specific fluorescence imaging is realized; meanwhile, pancreatic cancer is treated in combination with photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pancreatic cancer diagnosis and treatment nanoprobe with a "switch" function. Background Art

[0002] Pancreatic cancer is currently the fourth leading cause of cancer death, with a median survival of less than 6 months, making it the tumor with the worst prognosis among all human cancers. The mortality rate of pancreatic cancer is extremely high, and the most important factor is the delayed diagnosis of the disease. Surgical resection is the only possible cure, but delayed diagnosis causes 80-85% of patients to be ineligible for surgical resection at the time of diagnosis. The widening prognostic gap between resectable and unresectable patients highlights the importance of improving prognosis through early diagnosis of pancreatic cancer.

[0003] Currently, the diagnostic methods for pancreatic cancer include abdominal ultrasound, computed tomography, magnetic resonance imaging, endoscopic ultrasound-guided puncture for cytological diagnosis, and measurement of blood cancer antigen 19-9 levels. However, the nonspecificity and low sensitivity of the diagnosis may result in the possibility of missing patients with early-stage disease. In order to improve the prognosis, a specific detection method that can diagnose pancreatic cancer at an early stage is needed. Another important factor is that pancreatic cancer is resistant to a variety of anti-tumor therapies. Even with the clinical first-line treatment of gemcitabine and its combination regimen or FOLFIRINOX regimen, the patient's survival period is only 5.91-11.1 months. For cases of advanced pancreatic cancer, non-pancreatic cancer-specific traditional therapies such as surgery, chemotherapy, and radiotherapy are only used to relieve the patient's symptoms, and there is still no clear cure. How to combine pancreatic cancer-specific diagnosis and treatment is of great significance to overcome the limitations of existing clinical therapies for pancreatic cancer.

[0004] Photodynamic therapy (PDT) and photodynamic diagnosis (PDD) are promising diagnostic and therapeutic methods. Both of them use the principle of light-excited photosensitizers and have natural dual-functional combined advantages. Studies have shown that the ROS generation of some photosensitizers is strongly correlated with fluorescence emission. The photosensitizers in the fluorescence quenching state are inactivated in the excited singlet state, and the number of photosensitizer molecules entering the excited triplet state is reduced, thereby reducing the generation of singlet oxygen and ROS. Due to their good fluorescence emission characteristics, they have the properties of fluorescent probes. When some photosensitizers are encapsulated in nanoparticles, they form H-aggregates, which will completely quench their fluorescence and reduce their extinction due to the aggregation-caused quenching (ACQ) effect. The photosensitizer in this state is in a relatively "off" state. Once the nanoparticles disaggregate or degrade, the photosensitizer is released and emits a strong fluorescence signal. Then, by controlling the conversion of the photosensitizer between H-aggregates and release, not only can the phototoxicity "switch" in PDT be achieved to reduce systemic side effects, but the fluorescence signal "switch" in PDD can also be achieved to improve the specificity of the diagnostic signal.

[0005] Precise localization of photosensitizers at the lesion site is the key to achieving early PDD and PDT for pancreatic cancer. Plectin-1 is highly expressed in the pancreatic cancer cell membrane, but only expressed at low levels in the cytoplasm and nucleus in normal human pancreatic ductal cells, fibroblasts and vascular endothelial cells. It is a specific marker for the localization of pancreatic cancer cell membrane. Pancreatic intraepithelial neoplasia (PanIN) III lesions are considered to be pre-invasive carcinoma in situ with invasive gene mutations. The detection of PanIN III lesions has a direct impact on clinical treatment decisions. Plectin-1 is expressed in less than 3.85% of PanIN I / II (chronic pancreatitis lesions), but as high as 60% in PanIN III. Therefore, Plectin-1 can not only be detected in early pancreatic cancer, but also distinguish between malignant pancreatic diseases and chronic pancreatitis.

[0006] Based on this, the present invention designs and constructs a specific diagnostic and therapeutic probe targeting pancreatic cancer to solve the current technical problems of non-pancreatic cancer-specific imaging of nanoprobes and insufficient penetration of pancreatic cancer tissue. Summary of the invention

[0007] One of the purposes of the present invention is to provide a nanoprobe, comprising a nanoprodrug complex and a hydrophobic photosensitizer, wherein the nanoprodrug complex encapsulates the hydrophobic photosensitizer, and the mass ratio of the hydrophobic photosensitizer to the nanoprodrug complex is 1:2-10;

[0008] The nano-prodrug complex is based on low molecular weight heparin modified with azide and maleimide, and a benzazapine five-membered ring hydrophobic anti-tumor drug and a targeting ligand are connected to the base.

[0009] The hydrophobic photosensitizer is selected from phthalocyanine, zinc phthalocyanine, lead phthalocyanine, silicon phthalocyanine, palladium phthalocyanine, nickel phthalocyanine, and copper phthalocyanine;

[0010] The benzazapine five-membered ring hydrophobic anti-tumor drug is selected from one of lonidamine, vemurafenib, selumetinib, indomethacin and 6-mercaptopurine.

[0011] Furthermore, the targeting ligand is the Plectin-1 targeting peptide Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys.

[0012] Furthermore, the molar ratio of the azide- and maleimide-modified low molecular weight heparin, the benzazapine five-membered ring hydrophobic anti-tumor drug and the targeting ligand in the nano-prodrug complex is 1:0.1-0.9:0.1-0.3.

[0013] In one embodiment of the present invention, the preparation method of the nano-prodrug complex is: connecting a hydroxyl-containing or hydroxyl-derivatized benzazapine five-membered ring hydrophobic anti-tumor drug to an alkynyl group through an esterification reaction; then coupling it with azide- or maleimide-modified low molecular weight heparin, and finally covalently binding it to a targeting ligand to obtain a nano-prodrug complex.

[0014] Furthermore, the specific steps of the method are as follows:

[0015] S1. Dissolve oxalyl chloride in dichloromethane, cool to 0°C, add 3-butyn-1-ol, and after the reaction of 3-butyn-1-ol is complete, add a hydroxyl-containing or hydroxyl-modified benzazazepine five-membered ring hydrophobic anti-tumor drug, and after the reaction is complete, separate and purify by column chromatography to obtain intermediate I; the molar ratio of 3-butyn-1-ol to oxalyl chloride is 1:1-2;

[0016] S2, dissolving low molecular weight heparin modified with azide or maleimide in formamide, adding a targeting ligand, reacting for 12 hours under inert gas protection, adding cold acetone to the system to precipitate the product after the reaction, re-dissolving the precipitate with water, centrifuging, placing the supernatant in a dialysis bag for dialysis or placing it in an ultrafiltration tube for ultrafiltration, and drying to obtain azide-modified low molecular weight heparin with targeting; wherein the dialysis is 1-2 days, and the ultrafiltration is 0.5-1 hour; the molar ratio of low molecular weight heparin modified with azide or maleimide to the targeting ligand is 1:0.1-0.3;

[0017] S3. Dissolve the targeted azide-modified low molecular weight heparin in formamide, add intermediate I, and under the protection of inert gas, add cuprous iodide or copper sulfate pentahydrate and a reducing agent to react for 2-24 hours; after the reaction, add cold acetone to precipitate the product, re-dissolve the precipitate with water, centrifuge, and place the supernatant in a dialysis bag for dialyzing or in an ultrafiltration tube for ultrafiltration for 1-2 days and ultrafiltration for 0.5-1 hour, and freeze-dry to obtain a nano-prodrug complex; the molar ratio of the targeted azide-modified low molecular weight heparin to the intermediate I is 1:0.1-0.9.

[0018] Preferably, the benzazapine five-membered ring hydrophobic tumor metabolism regulating drugs described in S1 include lonidamine, vemurafenib, selumetinib, indomethacin, and 6-mercaptopurine.

[0019] The preparation method of the nanoprobe described above is specifically as follows: dissolving the nanoprodrug complex with a certain amount of water; ultrasonically dissolving a certain amount of benzazapine five-membered ring hydrophobic photosensitizer with an appropriate amount of N-methylpyrrolidone; slowly dropping the benzazapine five-membered ring hydrophobic photosensitizer solution into the nanoprodrug complex solution under an ice bath environment, stirring for 0.5-2 hours, and ultrasonically treating the probe for 10-40 minutes under ice bath conditions to obtain the nanoprobe.

[0020] The second object of the present invention is to provide the use of the above nanoprobe in the preparation of tumor diagnosis and treatment products. Preferably, the tumor is pancreatic cancer.

[0021] In view of the technical problems of current nanoprobes for non-pancreatic cancer-specific imaging and insufficient penetration of pancreatic cancer tissue, the inventors, based on previous research (CN 111920959A), used low molecular weight heparin modified with azide and maleimide as a matrix, covalently linked benzazapine five-membered ring hydrophobic anti-tumor drugs and pancreatic cancer-specific targeting groups (Plectin-1 targeting peptides) to form a nanoprodrug complex. The benzazapine five-membered ring hydrophobic prodrug in its hydrophobic core can further encapsulate fluorescent probes, and because the fluorescent probe used also has the hydrophobic structure of the benzazapine five-membered ring structure, under stronger hydrophobic interactions and π-π interactions, the fluorescent probe and the hydrophobic prodrug are tightly stacked to construct a pancreatic cancer diagnosis and treatment nanoprobe. The pancreatic cancer targeting group is used to increase the pancreatic cancer localization of the probe, and the tightly stacked smaller nanosize is used to enhance the pancreatic tumor penetration ability. The oxalate structure in the complex efficiently decomposes and releases free drugs and fluorescent probes under the stimulation of pancreatic cancer ROS. Combined with the fluorescent probe with aggregation quenching effect, the pancreatic cancer specificity of the nanoprobe is achieved. It can greatly improve the pancreatic cancer-specific imaging of the probe, improve the diagnostic sensitivity, and achieve the combination of diagnosis and treatment.

[0022] The nano-prodrug complex used in the present invention has tumor metabolism regulating activity itself, and can encapsulate a benzazapine five-membered ring hydrophobic photosensitizer with aggregation quenching effect. Its ROS response characteristics can enable the probe to specifically switch from a "closed" to an "on" state in a tumor ROS environment, thereby achieving specific fluorescence imaging, and at the same time combined with photodynamic therapy to treat pancreatic cancer.

[0023] Beneficial effects:

[0024] (1) The nanoprobe of the present invention has a relatively small nanometer size. The nanoprodrug complex containing the benzazapine five-membered ring anti-tumor drug and the hydrophobic photosensitizer used have a similar benzazapine five-membered ring structure, and there is a strong hydrophobic interaction and π-π interaction between the two, which not only enables the nanoprodrug complex to have a good drug loading and encapsulation rate for the photosensitizer, but also compresses the nanometer size by loading the photosensitizer, which is beneficial to the penetration of the nanoprobe into pancreatic cancer and improves the diagnostic and therapeutic efficiency of the nanoprobe.

[0025] (2) The nanoprobe of the present invention has good pancreatic cancer targeting activity. The present invention uses the pancreatic cancer specific marker Plectin-1 as a target. In the early stage of pancreatic cancer, it is highly accumulated on the surface of pancreatic cancer cells and almost not distributed in non-cancerous cells, thus achieving efficient pancreatic cancer specific targeting of the nanoprobe.

[0026] (3) The nanoprobe of the present invention has a "switch" function. The ROS responsiveness of the nanoprodrug complex is combined with the aggregation quenching effect of the probe, so that the nanoprodrug complex is in a "closed" state of fluorescence quenching after loading the photosensitizer, and under the stimulation of tumor ROS, the photosensitizer is released and turned to the "open" state to emit fluorescence, which is combined with tumor targeting to further achieve pancreatic cancer-specific localization of the fluorescence signal.

[0027] (4) The nanoprobe of the present invention has the characteristics of integrated diagnosis and treatment. The nanoprodrug complex has a tumor metabolism regulation effect. The fluorescent probe prepared after loading the photosensitizer not only has a pancreatic cancer-specific fluorescence imaging effect, but also has a metabolic regulation and photodynamic combined treatment effect for pancreatic cancer, thus realizing the integrated diagnosis and treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are the results of investigating the “switch” function of the low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe in Test Example 8.

[0029] Figure 2 This is the uptake result of the low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe in pancreatic tumor cells in Test Example 9.

[0030] Figure 3This is the fluorescence localization imaging image of the low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe in tumor-bearing nude mice in Test Example 10.

[0031] Figure 4 This is the distribution result of the low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe in tumor-bearing nude mouse tissue in Test Example 10. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] In the following examples, the preparation process of the azidized and maleimidized low molecular weight heparin is as follows: 1 mmol of low molecular weight heparin is weighed and dissolved in 4 mL of formamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to react for 0.5 h, 2 mL of a formamide mixed solution of 2-azidoethanol and N-(3-aminopropyl)maleimide (the molar ratio of low molecular weight heparin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2-azidoethanol, and N-(3-aminopropyl)maleimide is 1:1.5:1.5:0.8:0.2) is added to react for 6 h, and 5 times the volume of cold acetone is added, stirred, and filtered to obtain a precipitate, thereby obtaining the azidized and maleimidized low molecular weight heparin.

[0036] Example 1

[0037] 1. Preparation of ROS-responsive low molecular weight heparin-selumetinib prodrug complex

[0038] 2 mmol oxalyl chloride was dissolved in 10 mL dichloromethane, cooled to 0°C, 2 mmol 3-butyn-1-ol was added, and the mixture was reacted at room temperature for 2 h. 2 mmol selumetinib was added, and the mixture was reacted at room temperature for 4 h. Intermediate I was obtained by separation and purification by column chromatography.

[0039] Weigh 1mmol of low molecular weight heparin modified with azide and maleimide and dissolve it in formamide, weigh 0.2mmol of Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys and add it to the system, and react for 12h under nitrogen protection. Add 5 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, and vacuum dry to obtain the targeted azide-modified low molecular weight heparin.

[0040] Weigh 1mmol of targeted azide-modified low molecular weight heparin and dissolve it in 10mL formamide, weigh 0.5mmol of intermediate I, add 0.1mmol of copper sulfate pentahydrate and 0.2mmol of ascorbic acid under nitrogen protection, and react for 12h after addition. Add 10 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, ultrafilter and vacuum dry to obtain the ROS-responsive low molecular weight heparin-selumetinib prodrug complex.

[0041] 2. Preparation of Nanoprobes

[0042] (1) Preparation of ROS-responsive low molecular weight heparin-selumetinib nanoprobes loaded with silicon phthalocyanine

[0043] Weigh 2 mg of silicon phthalocyanine and dissolve it in 4 ml of N-methylpyrrolidone. Dissolve 10 mg of ROS-responsive low molecular weight heparin-selumetinib prodrug complex in 2 mL of distilled water. Slowly add the silicon phthalocyanine solution dropwise into the ROS-responsive low molecular weight heparin-selumetinib prodrug complex aqueous solution, stir vigorously for 1 hour, and ultrasonicate in a water bath for 20 minutes to obtain the ROS-responsive low molecular weight heparin-selumetinib nanoprobe loaded with silicon phthalocyanine.

[0044] (2) Preparation of ROS-responsive low molecular weight heparin-selumetinib nanoprobes loaded with palladium phthalocyanine

[0045] Weigh 4 mg of palladium phthalocyanine and dissolve it in 6 ml of N-methylpyrrolidone. Dissolve 15 mg of ROS-responsive low molecular weight heparin-selumetinib prodrug complex in 3 mL of distilled water. Slowly add the palladium phthalocyanine solution dropwise into the aqueous solution of the ROS-responsive low molecular weight heparin-selumetinib prodrug complex, stir vigorously for 1.5 h, and ultrasonicate in a water bath for 40 min to obtain the ROS-responsive low molecular weight heparin-selumetinib nanoprobe loaded with palladium phthalocyanine.

[0046] (3) Preparation of ROS-responsive low molecular weight heparin-selumetinib nanoprobes loaded with lead phthalocyanine

[0047] Weigh 3 mg of lead phthalocyanine and dissolve it in 6 ml of N-methylpyrrolidone. Dissolve 12 mg of ROS-responsive low molecular weight heparin-selumetinib prodrug complex in 2 mL of distilled water. Slowly add the lead phthalocyanine solution dropwise into the aqueous solution of the ROS-responsive low molecular weight heparin-selumetinib prodrug complex, stir vigorously for 2 h, and ultrasonicate in a water bath for 20 min to obtain the ROS-responsive low molecular weight heparin-selumetinib nanoprobe loaded with lead phthalocyanine.

[0048] Example 2

[0049] 1. Preparation of ROS-responsive low molecular weight heparin-vemurafenib prodrug complex

[0050] Weigh vemurafenib and 2-hydroxyethylamine in an eggplant-shaped bottle, with a molar ratio of 2-hydroxyethylamine to vemurafenib of 2:1, dissolve in 2 mL of methanol, and stir at room temperature for 4 h under nitrogen protection. After the reaction is complete, hydroxy-modified vemurafenib is purified by column chromatography.

[0051] 1 mmol of oxalyl chloride was dissolved in 6 mL of dichloromethane, cooled to 0°C, 1 mmol of 3-butyn-1-ol was added, and the mixture was reacted at room temperature for 2 h. 1 mmol of hydroxy-modified vemurafenib was added, and the mixture was reacted at room temperature for 1 h. Intermediate I was obtained by separation and purification by column chromatography.

[0052] Weigh 1mmol of azide- and maleimide-modified low molecular weight heparin and dissolve it in formamide, weigh 0.2mmol of Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys and add it to the system, and react for 4 hours under nitrogen protection. Add 5 times the volume of cold acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, dialyze it in distilled water, and vacuum dry it to obtain the targeted azide-modified low molecular weight heparin.

[0053] Weigh 1 mmol of targeted azide-modified low molecular weight heparin and dissolve it in 10 mL of formamide, weigh 0.5 mmol of intermediate I, add 0.1 mmol of cuprous iodide under nitrogen protection, and react for 6 hours after addition. Add 10 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, ultrafilter and freeze-dry to obtain the ROS-responsive low molecular weight heparin-vemurafenib prodrug complex.

[0054] 2. Preparation of ROS-responsive low molecular weight heparin-vemurafenib nanoprobes loaded with copper phthalocyanine

[0055] 1.5 mg of copper phthalocyanine was weighed and dissolved in 2 ml of N-methylpyrrolidone. 12 mg of ROS-responsive low molecular weight heparin-vemurafenib prodrug complex was dissolved in 3 mL of distilled water. The copper phthalocyanine solution was slowly added dropwise to the ROS-responsive low molecular weight heparin-vemurafenib prodrug complex aqueous solution, stirred vigorously for 1 h, and then ultrasonicated in a water bath for 20 min to obtain the ROS-responsive low molecular weight heparin-vemurafenib nanoprobe loaded with copper phthalocyanine.

[0056] Example 3

[0057] 1. Preparation of ROS-responsive low molecular weight heparin-lonidamine prodrug complex

[0058] 1 mmol of lonidamine was dissolved in 5 mL of chloroform, cooled to 0°C, and 1 mmol of thionyl chloride was added. After the reaction was complete, 1 mmol of ethylene glycol was added and reacted at room temperature for 2 hours. The hydroxy-modified lonidamine was purified by column chromatography.

[0059] 1 mmol of oxalyl chloride was dissolved in 10 mL of dichloromethane, cooled to 0°C, 1 mmol of 3-butyn-1-ol was added, and the mixture was reacted at room temperature for 2 h. 1 mmol of hydroxy-modified lonidamine was added, and the mixture was reacted at room temperature for 2 h. Intermediate I was obtained by separation and purification by column chromatography.

[0060] Weigh 1mmol of azide- and maleimide-modified low molecular weight heparin and dissolve it in formamide, weigh 0.2mmol of Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys and add it to the system, and react for 12 hours under nitrogen protection. Add 5 times the volume of cold acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, dialyze it in distilled water, and vacuum dry it to obtain the targeted azide-modified low molecular weight heparin.

[0061] Weigh 1 mmol of targeted azide-modified low molecular weight heparin and dissolve it in 10 mL of formamide, weigh 0.5 mmol of intermediate I, add 0.1 mmol of cuprous iodide under nitrogen protection, and react for 12 hours after addition. Add 10 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, ultrafilter and freeze-dry to obtain the ROS-responsive low molecular weight heparin-lonidamine prodrug complex.

[0062] 2. Preparation of Nanoprobes

[0063] (1) Preparation of ROS-responsive low molecular weight heparin-lonidamine nanoprobes loaded with zinc phthalocyanine

[0064] Weigh 2 mg of zinc phthalocyanine and dissolve it in 4 ml of N-methylpyrrolidone. Dissolve 18 mg of ROS-responsive low molecular weight heparin-lonidamine prodrug complex in 3 mL of distilled water. Slowly add the zinc phthalocyanine solution dropwise into the aqueous solution of the ROS-responsive low molecular weight heparin-lonidamine prodrug complex, stir vigorously for 0.5 h, and then ultrasonicate in a water bath for 30 min. The ROS-responsive low molecular weight heparin-lonidamine nanoprobe loaded with zinc phthalocyanine is obtained.

[0065] (2) Preparation of ROS-responsive low molecular weight heparin-lonidamine nanoprobes loaded with nickel phthalocyanine

[0066] Weigh 3 mg of nickel phthalocyanine and dissolve it in 4 ml of N-methylpyrrolidone. Dissolve 10 mg of ROS-responsive low molecular weight heparin-lonidamine prodrug complex in 2 mL of distilled water. Slowly add the nickel phthalocyanine solution dropwise into the aqueous solution of the ROS-responsive low molecular weight heparin-lonidamine prodrug complex, stir vigorously for 1 hour, and then ultrasonicate in a water bath for 30 minutes. The ROS-responsive low molecular weight heparin-lonidamine nanoprobe loaded with nickel phthalocyanine is obtained.

[0067] Example 4

[0068] 1. Preparation of ROS-responsive low molecular weight heparin-indomethacin prodrug complex

[0069] 1 mmol of indomethacin was dissolved in 5 mL of chloroform, cooled to 0°C, and 1 mmol of thionyl chloride was added. After the reaction was complete, 1 mmol of ethylene glycol was added and reacted at room temperature for 2 hours. The hydroxy-modified indomethacin was purified by column chromatography.

[0070] 1 mmol of oxalyl chloride was dissolved in 10 mL of dichloromethane, cooled to 0°C, 1 mmol of 3-butyn-1-ol was added, and the mixture was reacted at room temperature for 2 h. 1 mmol of hydroxy-modified indomethacin was added, and the mixture was reacted at room temperature for 2 h. Intermediate I was obtained by separation and purification by column chromatography.

[0071] Weigh 1mmol of low molecular weight heparin modified with azide and maleimide and dissolve it in formamide, weigh 0.2mmol of Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys and add it to the system, and react for 5 hours under nitrogen protection. Add 5 times the volume of cold acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, dialyze it in distilled water, and vacuum dry it to obtain the targeted azide-modified low molecular weight heparin.

[0072] Weigh 1 mmol of targeted azide-modified low molecular weight heparin and dissolve it in 10 mL of formamide, weigh 0.5 mmol of intermediate I, add 0.1 mmol of cuprous iodide under nitrogen protection, and react for 5 hours after addition. Add 6 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, ultrafilter and freeze-dry to obtain the ROS-responsive low molecular weight heparin-indomethacin prodrug complex.

[0073] 2. Preparation of ROS-responsive low molecular weight heparin-indomethacin nanoprobes loaded with phthalocyanine

[0074] Weigh 3 mg of phthalocyanine and dissolve it in 5 ml of N-methylpyrrolidone. Dissolve 10 mg of ROS-responsive low molecular weight heparin-indomethacin prodrug complex in 2 mL of distilled water. Slowly add the phthalocyanine solution dropwise into the ROS-responsive low molecular weight heparin-indomethacin prodrug complex aqueous solution, stir vigorously for 2 h, and perform water bath ultrasound for 10 min to obtain the phthalocyanine-loaded ROS-responsive low molecular weight heparin-indomethacin nanoprobe.

[0075] Example 5

[0076] 1. Preparation of ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex

[0077] Weigh 6-mercaptopurine and 2-mercaptoethanol into an eggplant-shaped bottle, dissolve in 2 mL of methanol, the molar ratio of 6-mercaptopurine to 2-mercaptoethanol is 1:1, add 1 mL of H2O2 solution, react completely at room temperature, and purify by column chromatography to obtain hydroxyl-modified 6-mercaptopurine.

[0078] 1 mmol of oxalyl chloride was dissolved in 6 mL of dichloromethane, cooled to 0°C, 1 mmol of 3-butyn-1-ol was added, and the mixture was reacted at room temperature for 2 h. 1 mmol of hydroxy-modified vemurafenib was added, and the mixture was reacted at room temperature for 1 h. Intermediate I was obtained by separation and purification by column chromatography.

[0079] Weigh 1mmol of azide- and maleimide-modified low molecular weight heparin and dissolve it in formamide, weigh 0.2mmol of Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys and add it to the system, and react for 4 hours under nitrogen protection. Add 5 times the volume of cold acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, dialyze it in distilled water, and vacuum dry it to obtain the targeted azide-modified low molecular weight heparin.

[0080] Weigh 1 mmol of targeted azide-modified low molecular weight heparin and dissolve it in 10 mL of formamide, weigh 0.5 mmol of intermediate I, add 0.1 mmol of cuprous iodide under nitrogen protection, and react for 6 hours after addition. Add 10 times the volume of ice acetone to precipitate, centrifuge to obtain the precipitate, re-dissolve it with an appropriate amount of distilled water, ultrafilter and freeze-dry to obtain the ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex.

[0081] 2. Preparation of Nanoprobes

[0082] (1) Preparation of ROS-responsive low molecular weight heparin-6-mercaptopurine nanoprobes loaded with lead phthalocyanine

[0083] Weigh 3 mg of lead phthalocyanine and dissolve it in 5 ml of N-methylpyrrolidone. Dissolve 8 mg of ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex in 2 mL of distilled water. Slowly add the lead phthalocyanine solution dropwise into the ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex aqueous solution, stir vigorously for 1.5 h, and perform water bath ultrasound for 15 min to obtain the ROS-responsive low molecular weight heparin-6-mercaptopurine nanoprobe loaded with lead phthalocyanine.

[0084] (2) Preparation of ROS-responsive low molecular weight heparin-6-mercaptopurine nanoprobes loaded with zinc phthalocyanine

[0085] Weigh 2 mg of zinc phthalocyanine and dissolve it in 4 ml of N-methylpyrrolidone. Dissolve 8 mg of ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex in 2 mL of distilled water. Slowly add the zinc phthalocyanine solution dropwise into the ROS-responsive low molecular weight heparin-6-mercaptopurine prodrug complex aqueous solution, stir vigorously for 1.5 h, and perform water bath ultrasound for 15 min to obtain the ROS-responsive low molecular weight heparin-6-mercaptopurine nanoprobe loaded with zinc phthalocyanine.

[0086] Test Example 1

[0087] Characterization of ROS-responsive nanoprobes

[0088] 1. Particle size. The particle sizes of the prepared ROS-responsive nanoprodrug complex and nanoprobe solution were measured using a laser particle size measuring instrument.

[0089] 2. Degree of substitution. Accurately weigh 10 mg of the ROS-responsive prodrug complex, dissolve it in water and make it up to 10 mL; draw 1 mL, dilute it with methanol and make it up to 10 mL, break the emulsion by water bath ultrasound for 3 minutes, and use a UV spectrophotometer to measure its absorbance at the maximum absorption wavelength of the hydrophobic drug. Substituting it into the standard curve, the content of the hydrophobic drug can be calculated. The degree of substitution can be calculated using the following formula:

[0090]

[0091] In this formula, c is the content of the hydrophobic drug (g), calculated from the standard curve; m is the mass of the weighed ROS-responsive prodrug complex (g); M1 is the average molecular weight of the hydrophobic drug, and M2 is the unit average molecular weight of the low molecular weight heparin.

[0092] 3. Drug loading. Accurately weigh 10 mg of the ROS-responsive nanoprobe freeze-dried preparation, dissolve it in water and make up to 10 mL; draw 1 mL, dilute it with DMSO and make up to 10 mL, break the emulsion by water bath ultrasound for 3 minutes, and use an ultraviolet spectrophotometer to measure its absorbance at the maximum absorption wavelength of the photosensitizer. Substituting it into the standard curve, the content of the photosensitizer in the ROS-responsive nanoprobe can be calculated. The photosensitizer drug loading of the ROS-responsive nanoprobe can be calculated by the following formula:

[0093] Drug loading DL (%) = (mass of drug in nanoparticles / mass of nanoparticles) × 100%.

[0094] Table 1 Characterization of low molecular weight heparin-hydrophobic drug nanoprobes

[0095]

[0096] Group 1 is the ROS-responsive low molecular weight heparin-selumetinib nanoprobe loaded with silicon phthalocyanine; Group 2 is the ROS-responsive low molecular weight heparin-vemurafenib nanoprobe loaded with copper phthalocyanine; Group 3 is the ROS-responsive low molecular weight heparin-lonidamine nanoprobe loaded with zinc phthalocyanine; Group 4 is the ROS-responsive low molecular weight heparin-indomethacin nanoprobe loaded with phthalocyanine; Group 5 is the ROS-responsive low molecular weight heparin-6-mercaptopurine nanoprobe loaded with lead phthalocyanine.

[0097] From the results in Table 1, it can be seen that after the ROS-responsive nanoprodrug complex is loaded with a benzazapine five-membered ring hydrophobic photosensitizer to prepare a nanoprobe, the particle size is greatly reduced, and the smaller particle size helps to promote the nanoparticles to accumulate more in pancreatic cancer tissues through osmotic retention in the blood circulation. The degree of substitution of hydrophobic drugs is about 60%. Such a high degree of substitution can provide a large space for the subsequent encapsulation of photosensitizer drugs and has good drug loading potential. It can be seen from the drug loading that the drug loading of the photosensitizer is about 20%, which is greatly improved compared with the drug loading of ~5-10% reported in the literature.

[0098] Test Example 2

[0099] Stability of ROS-responsive nanoprobes

[0100] The nanoprobe was prepared into a 1 mg / mL aqueous solution and placed at room temperature. The particle size and polydispersity coefficient were measured at 0 h, 24 h, and 48 h to evaluate the placement stability of the ROS-responsive nanoprobe solution.

[0101] Table 2 Evaluation results of stability of ROS-responsive nanoprobes

[0102]

[0103]

[0104] The results in Table 2 show that the particle size and polydispersity coefficient of the ROS-responsive nanoprobes change little, indicating that the ROS-responsive nanoprobes have good stability.

[0105] Test Example 3

[0106] Detection of in vitro release behavior of ROS-responsive low molecular weight heparin-lonidamine prodrug complex by HPLC

[0107] Two portions of 2 mg ROS-responsive low molecular weight heparin-lonidamine prodrug complex were accurately weighed and dissolved in 2 mL 0.1 mM hydrogen peroxide and 2 mL 1 mM hydrogen peroxide, respectively, to obtain a 1 mg / mL ROS-responsive low molecular weight heparin-lonidamine prodrug complex solution. Oscillating at 37°C, 300 μL of the solution was taken at 2 h, 6 h, 12 h and 24 h, 2.7 mL of chromatographic methanol was added, and the nanoparticle structure was destroyed by ultrasound for 30 min. The treated solution was then added to an ultrafiltration tube (MWCO = 3000), centrifuged at 10000 rpm for 30 min, 0.5 mL of the filtrate was taken, filtered using a 0.22 μm organic filter membrane, and the lonidamine content in the solution was analyzed by HPLC to calculate the degradation fraction.

[0108] Table 5 ROS sensitivity study of low molecular weight heparin-lonidamine prodrug complex

[0109]

[0110] The degradation score of the low molecular weight heparin-lonidamine nanoprodrug complex in the 1 mM hydrogen peroxide group was significantly greater than that in the 0.1 mM hydrogen peroxide group, indicating that the complex is ROS-responsive and can be used to prepare ROS-responsive nanoprobes to improve the specific imaging and therapeutic effects of diagnostic and therapeutic probes at the tumor site.

[0111] Test Example 4

[0112] MTT assay to detect the therapeutic effect of ROS-responsive nanoprobe on pancreatic cancer cells

[0113] PANC-1 adherent cells in logarithmic growth phase and in good condition were seeded in 96-well plates at a density of 5000 cells / well and incubated at 37°C for 8 hours. The culture medium was aspirated and 100 μL of ROS-responsive nanoprobes and nanoprodrug complexes containing different concentrations were added and incubated at 37°C for 4 hours. The illumination group containing photosensitive probes was illuminated at 630 nm near-infrared light 500 mW / cm 2 After irradiation for 3 minutes, the cells were incubated at 37°C for 20 hours. 40 μL of tetramethyl blue azolate (2.5 mg / L) was added and the cells were incubated for 4 hours. The culture medium of each well was removed and 150 μL of dimethyl sulfoxide was added to each well. After shaking for 5 minutes, the absorbance (OD) at 570 nm was measured with an ELISA reader. N = 6. The cell survival rate was calculated according to the following formula:

[0114] Viability(%)=A sample / A control ×100%,

[0115] Among them: A sample is the absorbance value of the drug-added group, A control is the absorbance value of the blank control group.

[0116] The IC50 inhibition rate of ROS-responsive nanoprobes on PANC-1 cells was calculated based on the survival rate results. 50 .

[0117] Table 4 Evaluation results of the therapeutic effects of ROS-responsive nanoprobes and nanoprodrug complexes on pancreatic cancer cells

[0118]

[0119] The results showed that the ROS-responsive nanoprobe had a strong tumor inhibitory effect and had therapeutic effects on pancreatic cancer.

[0120] Test Example 5

[0121] Antitumor effect of ROS-responsive low molecular weight heparin-lonidamine / zinc phthalocyanine on pancreatic cancer

[0122] Pancreatic cancer BxPC-3 tumor-bearing mice were randomly divided into 4 groups, 6 mice in each group, and the grouping and dosage were as follows: (1) normal saline; (2) free lonidamine (0.75 mg / Kg) + zinc phthalocyanine (0.5 mg / Kg); (3) low molecular weight heparin-lonidamine (3.0 mg / Kg); (4) low molecular weight heparin-lonidamine encapsulated zinc phthalocyanine (3.5 mg / Kg). The drugs were injected into the tail vein on days 0, 3, and 6, and 500 mW / cm 2The tumor was irradiated with a 670nm laser for 5 minutes. From the first day of drug administration, the length and width of the tumor were measured every day with a vernier caliper, and the tumor volume was calculated according to the formula. On the 14th day after drug withdrawal, the animals were euthanized, the tumors were dissected out and weighed, and the tumor growth inhibition rate (IR) was calculated according to the formula to evaluate its tumor inhibition effect.

[0123]

[0124] Among them, IR represents tumor inhibition rate, W control refers to the average tumor weight of the control group, W test Refers to the average tumor weight of the drug-treated group.

[0125] The results showed that compared with the saline group, the low molecular weight heparin-lonidamine entrapped zinc phthalocyanine group could significantly inhibit tumor growth, with an inhibition rate of up to 65%, while the inhibition rates of free zinc phthalocyanine + lonidamine and low molecular weight heparin-lonidamine were 44% and 21%, respectively, which were significantly lower than the low molecular weight heparin-lonidamine entrapped zinc phthalocyanine group. This indicates that the low molecular weight heparin-lonidamine entrapped zinc phthalocyanine group has an extremely significant in vivo pancreatic cancer treatment effect.

[0126] Test Example 6

[0127] Antitumor effect of ROS-responsive low molecular weight heparin-selumetinib / silicon phthalocyanine on pancreatic cancer

[0128] Pancreatic cancer PANC-1 tumor-bearing mice were randomly divided into 4 groups, 6 mice / group, and the grouping and administration doses were as follows: (1) normal saline; (2) free selumetinib (1.0 mg / kg) + phthalocyanine silicon (1 mg / kg); (3) low molecular weight heparin-selumetinib (4.0 mg / kg); (4) low molecular weight heparin-selumetinib encapsulated with phthalocyanine silicon (5 mg / kg). 24 hours after administration, a 630 nm near-infrared laser with a power of 500 mW / cm 2 The tumor site was irradiated for 5 minutes. The drug was injected into the tail vein on days 0, 3, and 6, and 500 mW / cm 2 The tumor was irradiated with a 630nm laser for 5 minutes. From the first day of drug administration, the length and width of the tumor were measured every day with a vernier caliper, and the tumor volume was calculated according to the formula. On the 14th day after drug withdrawal, the animals were euthanized, the tumors were dissected out and weighed, and the tumor growth inhibition rate (IR) was calculated according to the formula to evaluate its tumor inhibition effect.

[0129]

[0130] Among them, IR represents tumor inhibition rate, W control refers to the average tumor weight of the control group, W test Refers to the average tumor weight of the drug-treated group.

[0131] The results showed that compared with the saline group, the low molecular weight heparin-selumetinib-encapsulated phthalocyanine silicon group could significantly inhibit tumor growth, with a tumor inhibition rate of up to 69%, while the tumor inhibition rates of free phthalocyanine silicon + selumetinib and low molecular weight heparin-selumetinib were 42% and 31%, respectively, which were significantly lower than the low molecular weight heparin-selumetinib-encapsulated phthalocyanine silicon group. This indicates that the low molecular weight heparin-selumetinib-encapsulated phthalocyanine silicon group has extremely significant in vivo pancreatic cancer treatment efficacy.

[0132] Test Example 7

[0133] Antitumor effect of ROS-responsive low molecular weight heparin-indomethacin / phthalocyanine on pancreatic cancer

[0134] MIAPaca-2 pancreatic cancer tumor-bearing mice were randomly divided into 4 groups, 6 mice / group, and the grouping and drug dosage were as follows: (1) normal saline; (2) free indomethacin (1.0 mg / Kg) + phthalocyanine (1.5 mg / Kg); (3) low molecular weight heparin-indomethacin (5.0 mg / Kg); (4) low molecular weight heparin-indomethacin encapsulated phthalocyanine (6.5 mg / Kg). 24 hours after drug administration, a 630 nm near-infrared laser with a power of 200 mW / cm 2 The tumor site was irradiated for 10 minutes. The drug was injected into the tail vein on days 0, 3, and 6, and 200 mW / cm 2 The tumor was irradiated with a 630nm laser for 10 minutes. From the first day of drug administration, the length and width of the tumor were measured every day with a vernier caliper, and the tumor volume was calculated according to the formula. On the 14th day after drug withdrawal, the animals were euthanized, the tumors were dissected out and weighed, and the tumor growth inhibition rate (IR) was calculated according to the formula to evaluate its tumor inhibition effect.

[0135]

[0136] Among them, IR represents tumor inhibition rate, W control refers to the average tumor weight of the control group, W test Refers to the average tumor weight of the drug-treated group.

[0137] The results showed that compared with the saline group, the low molecular weight heparin-indomethacin entrapped phthalocyanine group could significantly inhibit tumor growth, with an inhibition rate of up to 69%, while the inhibition rates of free phthalocyanine + indomethacin and low molecular weight heparin-indomethacin were 44% and 28%, respectively, which were significantly lower than low molecular weight heparin-indomethacin entrapped blood phthalocyanine. This indicates that the low molecular weight heparin-indomethacin entrapped phthalocyanine group has extremely significant in vivo anti-tumor therapeutic efficacy.

[0138] Test Example 8

[0139] The “switch” function of ROS-responsive low molecular weight heparin-lonidamine / zinc phthalocyanine

[0140] The "switch" function of ROS-responsive low molecular weight heparin-lonidamine / zinc phthalocyanine was investigated by small animal in vivo optical imaging. Two portions of low molecular weight heparin-lonidamine / zinc phthalocyanine freeze-dried powder of the same mass were weighed, and pure water or 1mM H2O2 aqueous solution was added to reconstitute them, and then observed and photographed by small animal in vivo optical imaging.

[0141] The results are as follows Figure 1 It can be seen that when reconstituted in water, the phthalocyanine zinc in the ROS-responsive low molecular weight heparin-lonidamine / phthalocyanine zinc nanoprobe is loaded inside the nanoprobe. Due to the aggregation of phthalocyanine zinc, the fluorescence quenching does not show the fluorescence signal (left picture), that is, the probe fluorescence signal is in the "off" state. When reconstituted in 1mM H2O2 aqueous solution, because the low molecular weight heparin-lonidamine can respond to ROS and degrade, and trigger the release of phthalocyanine zinc, it shows a strong red fluorescence signal (right picture), that is, the probe fluorescence signal is in the "on" state. The results show that the integrated diagnosis and treatment nanoprobe has a good signal "switch" function, which is conducive to its better pancreatic cancer-specific localization diagnosis.

[0142] Test Example 9

[0143] Specific recognition of pancreatic cancer cells by ROS-responsive low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe

[0144] Pancreatic cancer cells PANC-1 in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded in a 24-well plate at a density of 100 cells / well and placed in an incubator for 24 hours to adhere to the wall. The same dose of free photosensitizer zinc phthalocyanine (5 micrograms per milliliter) and low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe (22.8 micrograms per milliliter, containing 5 micrograms per milliliter of photosensitizer) were added, as well as blank culture medium, and placed in an incubator for 12 hours. The culture medium was removed, washed with PBS, the cells were trypsinized, centrifuged and resuspended with PBS, and the uptake of low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobes in pancreatic tumor cells was quantitatively analyzed by flow cytometry.

[0145] The results are as follows Figure 2 As shown, the fluorescence level of cells incubated with free photosensitizer phthalocyanine zinc was relatively low, and the uptake effect was poor; the fluorescence level of phthalocyanine zinc in the low molecular weight heparin-lonidamine / phthalocyanine zinc nanoprobe group reached 12.2 times that of the free phthalocyanine zinc group (P<0.001), indicating that the low molecular weight heparin-lonidamine / phthalocyanine zinc nanoprobe has a good specific recognition effect on pancreatic cancer cells, which helps to improve the pancreatic cancer specific localization of this nanoprobe.

[0146] Test Example 10

[0147] Localization imaging of pancreatic cancer by ROS-responsive low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe

[0148] The dynamic distribution of low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobes in vivo and their pancreatic cancer targeting ability were investigated using a small animal in vivo optical imager. The tumor volume was 300 mm 3 The low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe solution was injected into the tail vein of left and right PANC-1 tumor-bearing nude mice, where the dosage of zinc phthalocyanine was 1 mg / kg. Fluorescence imaging of the tumor-bearing nude mice was performed and photos were taken at 8 hours.

[0149] The results are as follows Figure 3 As shown in the figure, the fluorescence intensity of pancreatic tumor tissue in tumor-bearing nude mice is very strong, and obvious red fluorescence is observed in the tumor tissue, indicating that the low molecular weight heparin-lonidamine / zinc phthalocyanine nanoprobe has a good pancreatic cancer tissue targeting and positioning fluorescence imaging effect. Figure 4 As shown in the figure, the fluorescence intensity of pancreatic tumor tissue is significantly stronger than that of other tissues, which further illustrates the specificity of the probe's distribution and release in tumors. Combined with its therapeutic effect on pancreatic cancer, it can help to fully exert its integrated diagnosis and treatment function.

Claims

1. A nanoprobe, characterized in that: It comprises a nano-prodrug complex and a hydrophobic photosensitizer, wherein the nano-prodrug complex encapsulates the hydrophobic photosensitizer, and the mass ratio of the hydrophobic photosensitizer to the nano-prodrug complex is 1:2-10; The nano-prodrug complex is based on low molecular weight heparin modified with azide and maleimide, and a benzazapine five-membered ring hydrophobic anti-tumor drug and a targeting ligand are connected to the base. The hydrophobic photosensitizer is selected from phthalocyanine, zinc phthalocyanine, lead phthalocyanine, silicon phthalocyanine, palladium phthalocyanine, nickel phthalocyanine, and copper phthalocyanine; The benzazapine five-membered ring hydrophobic anti-tumor drug is selected from lonidamine, vemurafenib, selumetinib, indomethacin and 6-mercaptopurine.

2. The nanoprobe according to claim 1, characterized in that The targeting ligand is the Plectin-1 targeting peptide Lys-Thr-Leu-Leu-Pro-Thr-Pro-Cys.

3. The nanoprobe according to claim 1, characterized in that: The molar ratio of the low molecular weight heparin modified by azide and maleimide, the benzazapine five-membered ring hydrophobic anti-tumor drug and the targeting ligand in the nano-prodrug complex is 1:0.1-0.9:0.1-0.

3.

4. Use of the nanoprobe according to any one of claims 1 to 3 in the preparation of tumor diagnosis and treatment products.

5. The use according to claim 4, characterized in that: The tumor is pancreatic cancer.

Images