Preparation method of diagnosis and treatment integrated preparation based on PET-CT (positron emission tomography-computed tomography) and application of diagnosis and treatment integrated preparation in tumor treatment

Abstract

The invention provides a preparation method of a diagnosis and treatment integrated preparation based on PET-CT (positron emission tomography-computed tomography) and application of the diagnosis and treatment integrated preparation in tumor treatment, and relates to the technical field of biomedical materials. According to the technical scheme, a PET-CT contrast agent and an optical treatment drug are connected through a connecting unit, one end of the connecting unit comprises a functional group used for being connected with glucose, and the other end of the connecting unit comprises a functional group used for being connected with a photosensitive anti-cancer drug. The invention aims to develop a PET-CT (positron emission tomography-computed tomography)-based diagnosis and treatment integrated preparation for diagnosing and treating cancers. 18F-FDG is a common contrast agent for PET-CT, an optical therapeutic drug is introduced into an 18F-FDG analogue molecule by using a connecting unit, the feasibility of the 18F-FDG analogue molecule for cancer diagnosis / treatment is discussed, and the 18F-FDG analogue has important practical prospects and social values in the field of preparation of cancer diagnosis and treatment integrated drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a PET-CT contrast agent. 18 F-FDG analogues were used as the basis and modified to introduce tumor optical therapy drugs to prepare integrated diagnosis and treatment nano-preparations. The application research of this nano-preparation in tumor photothermal / photodynamic therapy while being used as a PET-CT contrast agent for imaging was investigated. Background Art

[0002] Cancer is one of the most serious diseases that threaten human health and cause death. One of the main reasons why cancer treatment is difficult and has a high mortality rate is that it is difficult to diagnose the disease in the early stages. When the disease is discovered, it is mostly in the middle and late stages, so it is difficult to cure it completely. In addition, due to the complex environment of cancer and its middle and late stages, the current commonly used clinical treatment methods are less efficient. Usually, before cancer treatment, diagnostic methods such as imaging are needed to understand the phenotype and heterogeneity of cancer cells, and then determine the treatment plan.

[0003] At present, the commonly used cancer diagnosis methods in clinic include: computed tomography (CT), magnetic resonance imaging (MRI), ultrasonic imaging diagnosis technology and positron emission tomography combined with computed tomography (PET-CT). Among them, PET-CT can obtain tomographic images of all directions of the whole body in one imaging, which has the characteristics of sensitivity, accuracy, specificity and precise positioning. It can understand the overall condition of the whole body at a glance, and achieve the purpose of early detection of lesions and diagnosis of diseases. Phototherapy (mainly including photodynamic therapy and photothermal therapy) is a new cancer treatment method developed in recent years. It has attracted widespread attention due to its convenient operation, non-invasiveness, local selectivity, low drug resistance and low side effects. The working principle of phototherapy is based on the photochemical reaction of phototherapy drugs (photosensitizers and photothermal preparations). After intravenous injection, phototherapy drugs will be preferentially absorbed and enriched in the lesion tissue. The lesion site is directly irradiated with a light source of a specific wavelength to excite the photosensitizer and undergo a photochemical reaction, thereby generating reactive oxygen or local high temperature to induce tumor cell apoptosis and (or) death.

[0004] However, since diagnostic agents and anticancer drugs are two independent entities with differences in selectivity and dispersion, the flow routes of anticancer drugs and diagnostic agents in the body are not the same, resulting in the therapeutic effect not meeting expectations. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing a PET-CT integrated diagnosis and treatment preparation and its application in tumor treatment, and to solve the problems raised in the above background technology through the following technical solutions.

[0006] The first object of the present invention is to provide a method for preparing a PET-CT integrated diagnosis and treatment preparation, comprising: connecting a PET-CT contrast agent and a phototherapy drug through a connecting unit, wherein one end of the connecting unit comprises a functional group for connecting to glucose and the other end comprises a functional group for connecting to the surface of a nanoparticle, specifically comprising the following steps:

[0007] S1. Determine PET-CT contrast agents and phototherapy drugs;

[0008] S2, preparing a connection unit;

[0009] S3, introducing the linking unit into the phototherapy drug to obtain an intermediate linker;

[0010] S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been obtained as integrated diagnostic and therapeutic preparations.

[0011] Preferably, the functional group at one end of the linking unit for connecting with glucose is selected from one or more of phenylboronic acid and phenylboronic acid analogs.

[0012] Preferably, the functional group at the other end of the linking unit for connecting to the surface of the nanoparticle is selected from one or more of a thiol, dopamine, a dopamine analogue, and a functional group containing a group that reacts directly with a phototherapeutic drug, and the functional group containing a group that reacts directly with a phototherapeutic drug is selected from one or more of a functional group containing an amino group, a functional group containing a hydroxyl group, a functional group containing a carboxyl group, a functional group containing a halogen atom, a functional group containing a carbonyl group, a functional group containing a nitro group, a functional group containing a sulfonic acid group, and a functional group containing a thiol group.

[0013] Preferably, the linking unit is selected from 3-aminophenylboronic acid (PBA) or lipoic acid-phenylboronic acid LA-PBA.

[0014] Preferably, the phototherapeutic drug is photosensitivity and has the function of producing substances that induce apoptosis and / or death of cancer cells under irradiation with light of a specific wavelength. The phototherapeutic drug is selected from one or more of protoporphyrin, hematoporphyrin, dihydrochlorin, porphyrin derivatives, photosensitizers that can produce reactive oxygen species by light induction, and gold nanorods, copper sulfide nanoparticles, indocyanine green, and photothermal preparations that can produce heat by light induction.

[0015] The second object of the present invention is to provide an integrated diagnosis and treatment preparation prepared by the above-mentioned preparation method.

[0016] The third object of the present invention is to provide the application of the integrated diagnosis and treatment preparation, based on the expanded application of the PET-CT integrated diagnosis and treatment preparation in the biomedical field.

[0017] Preferably, the application of the integrated diagnosis and treatment preparation is based on the PET-CT integrated diagnosis and treatment preparation for the manufacture of tumor treatment drugs.

[0018] Preferably, the application of the integrated diagnosis and treatment preparation is based on the application of the integrated diagnosis and treatment preparation of PET-CT in fluorescence imaging and photoacoustic imaging.

[0019] Preferably, the application of the integrated diagnosis and treatment preparation is based on the PET-CT integrated diagnosis and treatment preparation for the manufacture of disease diagnosis preparations.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention relates to an integrated diagnosis and treatment preparation based on PET-CT. This application combines the originally independent cancer diagnostic preparation with anticancer drugs, aiming to develop a preparation that can be used for both cancer diagnosis and treatment (integrated diagnosis and treatment), overcoming the problem that traditional diagnostic preparations and anticancer drugs are independent individuals and have unsatisfactory treatment effects due to differences in selectivity and dispersion. This preparation can overcome the above problems and effectively improve the efficiency of cancer treatment. As a new type of integrated anticancer diagnosis and treatment preparation, it has good application prospects. This preparation is universal for the diagnosis / treatment of tumors. If the diagnosis result shows no canceration, there is no need to start the treatment mode and it has no effect on the patient; if the diagnosis result confirms cancer, laser irradiation can be directly used to accurately treat the lesion.

[0022] (2) The present invention relates to a method for preparing a PET-CT integrated diagnosis and treatment preparation. The preparation method modifies the existing clinical diagnostic preparation and introduces optical therapeutic drugs. While diagnosing cancer, light-induced cancer treatment is performed to improve the efficiency of cancer treatment.

[0023] (3) The present invention relates to an application of a PET-CT-based integrated diagnosis and treatment preparation, which has applications in many fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objectives and advantages of the present invention will become more apparent from a reading of the detailed description of non-limiting embodiments made with reference to the following accompanying drawings.

[0025] Figure 1 This is a schematic diagram of the preparation of GNRs-LA-PBA-(Ac-FDG) in Example 1 of the present application;

[0026] Figure 2 Schematic diagram of the results of Experimental Example 1 of the present application, including LA, PBA, Ac-FDG and GNRs-LA-PBA-(Ac-FDG) of Example 1 1 Schematic diagram of H NMR spectrum (A), and Ce6, Ac-FDG and Ce6-PBA-(Ac-FDG) of Example 91 Schematic diagram of H NMR spectrum (B);

[0027] Figure 3 Schematic diagram of the ultraviolet absorption spectra of GNRs and GNRs-LA-PBA-(Ac-FDG) of the present application;

[0028] Figure 4 A schematic diagram of the dynamic light scattering (DLS) spectrum of the GNRs of the present application (A) and a schematic diagram of the dynamic light scattering (DLS) spectrum of GNRs-LA-PBA-(Ac-FDG) (B);

[0029] Figure 5 Schematic diagram of the transmission electron microscope (TEM) photograph of the GNRs of the present application (A) and the schematic diagram of the transmission electron microscope (TEM) photograph of GNRs-LA-PBA-(Ac-FDG) (B);

[0030] Figure 6 Schematic diagram of the present application shows the photothermal conversion curve of the integrated diagnostic and therapeutic preparation GNRs-LA-PBA-(Ac-FDG) under constant power (A) and constant concentration (B) conditions;

[0031] Figure 7 The change of ultraviolet absorption intensity of benzofuran after 650nm laser irradiation of Ce6-PBA-(Ac-FDG) of the present application;

[0032] Figure 8 This is a schematic diagram of the results of Experimental Example 6 of the present application, including the phosphorylation of ATP and GNRs-LA-PBA-(Ac-FDG) with ATP in Example 1. 31 Schematic diagram of P NMR spectrum (A), and the phosphorylation of ATP and Ce6-PBA-(Ac-FDG) with ATP in Example 9 13 Schematic diagram of C NMR spectrum (B);

[0033] Fig. 9 Schematic diagram of the dark toxicity (A) and phototoxicity (B) of the integrated diagnosis and treatment preparation GNRs-LA-PBA-(Ac-FDG) on HeLa cells in Example 1 of the present application;

[0034] Fig.10 Schematic diagram of the dark toxicity (A) and phototoxicity (B) of the integrated diagnostic and therapeutic preparation Ce6-PBA-(Ac-FDG) of Example 9 of the present application on HeLa cells. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods and drawings.

[0036] The term "and / or" as used herein includes any and all combinations of one or more of the related listed items. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further treatment, and the instruments used in the examples are commercially available. All technical and scientific terms used in this application have the same meaning as those commonly understood by technicians in the technical field of the present invention. The terms used in the specification of the present invention in this application are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0037] The present application provides a PET-CT diagnosis and treatment integrated preparation and preparation method, specifically including:

[0038] S1. Determine PET-CT contrast agents and phototherapy drugs

[0039] PET-CT is a commonly used diagnostic method in clinical practice. It can obtain tomographic images of the whole body in all directions in one imaging. It has the characteristics of sensitivity, accuracy, specificity and precise positioning. It can clearly understand the overall condition of the whole body and achieve the purpose of early detection of lesions and diagnosis of diseases. Among them, 18-fluorodeoxyglucose ( 18 F-FDG) is the main contrast agent and is commonly used in positron emission tomography (PET) medical imaging devices. 18 The fluorine in the F-FDG molecule is a positron-emitting radioisotope. 18 F, patients taking 18 After F-FDG, the PET scanner can build a reflection 18 Doctors use these images to diagnose health conditions. 18 F-FDG is radioactive and needs to be tested under specific experimental conditions. 18 The feasibility of this strategy was explored using the analogs of F-FDG, 2-(acetylamino)-2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-pyranose.

[0040] Phototherapy mainly includes photodynamic therapy and photothermal therapy, and its working principle is based on the photochemical reaction of phototherapy drugs (photosensitizers and photothermal preparations), which in turn produces reactive oxygen or local high temperature to induce apoptosis and (or) death of tumor cells. This application selects easily modified phototherapy drugs, including but not limited to protoporphyrin, hematoporphyrin, dihydrochlorin, and other porphyrin derivatives that can induce the generation of photosensitizers with reactive oxygen, as well as gold nanorods, copper sulfide nanoparticles, indocyanine green and other photothermal preparations as photothermal therapy drugs.

[0041] S2. Preparation of connection unit

[0042] According to the structural characteristics of contrast agents and phototherapy drugs, one or two or more reagents are selected to form a linking unit for combining contrast agents and phototherapy drugs. One end of the linking unit contains a functional group for connecting to glucose, and the other end contains a functional group for connecting to the surface of nanoparticles. Specifically, the functional group of the linking unit for connecting to the surface of the nanoparticle is selected from one or more of thiol, dopamine, dopamine analogs, and functional groups containing groups that react directly with phototherapy drugs, and the functional group containing groups that react directly with phototherapy drugs is selected from one or more of amino-containing functional groups, hydroxyl-containing functional groups, carboxyl-containing functional groups, halogen-containing functional groups, carbonyl-containing functional groups, nitro-containing functional groups, sulfonic acid-containing functional groups, and thiol-containing functional groups.

[0043] The linking unit is selected from 3-aminophenylboronic acid (PBA) or lipoic acid-phenylboronic acid LA-PBA.

[0044] S3. Introducing the linking unit into the phototherapy drug to obtain an intermediate linker

[0045] The connecting unit is connected to the phototherapy drug through a chemical reaction to obtain an intermediate connecting body. The specific operation is as follows:

[0046] When the linking unit is 3-aminophenylboronic acid (PBA), its amino group is used to combine with the phototherapy drug. The phototherapy drug is dissolved in DMSO, and then NHS, EDC, PBA and TEA are added in sequence, mixed, stirred at room temperature in the dark for 24h-48h under a nitrogen atmosphere to obtain an intermediate linker, and then the intermediate linker solution is slowly dripped into a centrifuge tube containing ice water by the suspension drop method, centrifuged at 10000rpm for 30min, the supernatant is taken out, and vacuum dried to obtain an intermediate linker.

[0047] When the linking unit is lipoic acid-phenylboronic acid LA-PBA, the phototherapeutic drug and LA-PBA are mixed in a ratio of 1:1-10, stirred at room temperature in the dark for 12h-48h, dialyzed for 12h-72h to remove coupling agents NHS, EDC, TEA and unreacted LA and PBA, and freeze-dried to obtain an intermediate linker.

[0048] S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0049] Phenylboronic acid is combined with the hydroxyl groups on the 3rd and 4th carbon atoms in the diagnostic agent and its similar molecules, and an intermediate connector connected with a phototherapeutic drug is introduced into the diagnostic agent molecule to obtain an integrated diagnostic and therapeutic agent.

[0050] Add contrast agent to weakly alkaline deionized water 18 F-FDG analogues 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) or 2-deoxy-2-fluoro-D-pyranose (F-FDG), then add the intermediate linker, stir at room temperature in the dark for 48h-72h, and dialyze for 24h to remove the unreacted contrast agent. 18 The F-FDG analogue is freeze-dried to obtain the final product, which is an integrated diagnostic and therapeutic preparation.

[0051] In some embodiments, the invention includes the expanded application of the above-mentioned PET-CT integrated diagnosis and treatment preparation in the biomedical field, including the application in the field of manufacturing tumor treatment drugs, the application in fluorescence imaging and photoacoustic imaging, and the use of the PET-CT integrated diagnosis and treatment preparation in the manufacture of disease diagnosis preparations.

[0052] Example 1

[0053] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0054] The integrated diagnosis and treatment preparation provided in this embodiment is GNRs-LA-PBA-(Ac-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically gold nanorods (GNRs), and the PET-CT contrast agent is a contrast agent 18 F-FDG analog 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG).

[0055] like Figure 1 As shown, the preparation method specifically includes:

[0056] S1. Determine PET-CT contrast agents and phototherapy drugs

[0057] First, prepare a gold seed solution: weigh 0.3553 g of hexadecyltrimethylammonium bromide (CTAB) and dissolve it in 9.75 mL of pure water (0.1 mol / L), then add 0.25 mL of 0.01 mol / L chloroauric acid solution, and quickly add 0.6 mL of freshly prepared 0.01 mol / L sodium borohydride solution under vigorous stirring. The color of the solution changes from yellow to tea brown, indicating that gold seeds are formed. After stirring for 2 minutes, let it stand at room temperature for 2h-4h to obtain a gold seed solution.

[0058] Secondly, preparation of the growth solution: weigh 0.328 g of CTAB and dissolve it in 9 mL of pure water (0.1 mol / L), then add 0.5 mL of 0.01 mol / L chloroauric acid solution, 0.1 mL of 0.01 mol / L silver nitrate solution and 0.2 mL of 1 mol / L HCl solution in sequence, mix well and add 0.08 mL of 0.1 mol / L ascorbic acid solution, the color of the solution changes from dark yellow to colorless.

[0059] Next, prepare photothermal gold nanorods (GNRs): after the growth solution is stirred for 2 minutes, 1 mL of gold seed solution is quickly added, and then the resulting solution is allowed to stand at room temperature overnight. The resulting solution is centrifuged for 30 minutes (12000 rpm), the supernatant is discarded and dispersed in pure water again to obtain a gold nanorod solution.

[0060] S2. Preparation of connection unit

[0061] Thioctanoic acid (LA) (1.21 g; 5.87 mmol) was completely dissolved in 35 mL of MeOH, and NHS (1.95 g; 10.14 mmol) and EDC (1.167 g; 10.14 mmol) were added in sequence, stirred for 30 min (550 rpm), and then PBA (0.964 g, 7.04 mmol) and TEA (3 mL; 21.12 mmol) were added. The reaction mixture was stirred at room temperature in a nitrogen atmosphere in the dark for 24 h to obtain the product, i.e., the connecting unit thioctic acid-phenylboronic acid LA-PBA.

[0062] S3. Introducing the linking unit into the phototherapy drug to obtain an intermediate linker

[0063] Add 100 mL of GNRs prepared in step S1 to a round-bottom flask, and then add the LA-PBA solution prepared in step S2 to a volumetric flask. After stirring at room temperature in the dark for 24 hours, transfer the liquid to a dialysis membrane and dialyze in ultrapure water for 24 hours to remove coupling agents NHS, EDC, TEA and unreacted LA and PBA. Then, freeze-dry the dialyzed sample to obtain the intermediate connector GNRs-LA-PBA.

[0064] S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0065] Take a 250ml volumetric flask and add 150ml of ultrapure water, then take about 2ml of 1mol / L NaOH solution and adjust the pH of 150ml of ultrapure water to about 8, so that the solution is weakly alkaline, and then add the contrast agent 18F-FDG analogue 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) (10 mmol), then add GNRs-LA-PBA (10 mmol) obtained in step S3 and stir at room temperature in the dark for 48 hours. The liquid is transferred to a dialysis membrane and dialyzed in ultrapure water for 24 hours to remove unreacted Ac-FDG, and then the dialyzed sample is freeze-dried to obtain the GNRs-LA-PBA-(Ac-FDG) product.

[0066] Example 2

[0067] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0068] The integrated diagnosis and treatment preparation provided in this embodiment is GNRs-LA-PBA-(F-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically gold nanorods (GNRs), and the PET-CT contrast agent is a contrast agent 18 F-FDG analogue 2-deoxy-2-fluoro-D-glucopyranose (F-FDG).

[0069] The preparation method of the preparation is basically the same as that of Example 1, except that the contrast agent selected in step S4 is 18 The F-FDG analog is 2-deoxy-2-fluoro-D-glucopyranose (F-FDG), and the obtained product is GNRs-LA-PBA-(F-FDG).

[0070] Example 3

[0071] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0072] The integrated diagnosis and treatment preparation provided in this embodiment is CuSNPs-LA-PBA-(Ac-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically copper sulfide nanoparticles (CuSNps), and the PET-CT contrast agent is a contrast agent 18 F-FDG analog 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG).

[0073] Step S2 in the preparation method is the same as that in Example 1, except that:

[0074] S1. Determine PET-CT contrast agents and phototherapy drugs

[0075] 0.1 mL of sodium sulfide solution (1 M) was added to 100 mL of aqueous solution containing copper chloride (0.01345 g, 0.1 mmol) and sodium citrate (0.02 g, 0.068 mmol) and stirred at room temperature. The light blue copper chloride solution immediately turned dark brown after the addition of sodium sulfide. After 5 min, the reaction mixture was heated to 90 ° C and stirred for 15 min until a dark green solution was obtained. The mixture was transferred to ice water. Citric acid-coated photothermal copper sulfide nanoparticles (CuSNPs) were obtained and stored in a refrigerator for later use.

[0076] S3. Introducing the linking unit into the phototherapy drug to obtain an intermediate linker

[0077] 100 mL of the photothermal agent CuSNPs prepared in step S1 was added to a round-bottom flask, and then the LA-PBA solution prepared in step S2 was added to a volumetric flask. After stirring at room temperature in the dark for 24 h, the liquid was transferred to a dialysis membrane and dialyzed in ultrapure water for 24 h to remove the coupling agents NHS, EDC, TEA and unreacted LA and PBA, and then the dialyzed sample was freeze-dried to obtain the intermediate connector CuSNPs-LA-PBA.

[0078] S4. Connecting the intermediate connector to the contrast agent 18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0079] Take a 250ml volumetric flask and add 150ml of ultrapure water, then take about 2ml of 1mol / L NaOH solution and adjust the pH of 150ml of ultrapure water to about 8, so that the solution is weakly alkaline, and then add the contrast agent 18 F-FDG analogue 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) (10 mmol), then add CuSNPs-LA-PBA (10 mmol) obtained by the reaction in step S3 and stir at room temperature in the dark for 48 hours. The liquid is transferred to a dialysis membrane and dialyzed in ultrapure water for 24 hours to remove unreacted Ac-FDG, and then the dialyzed sample is freeze-dried to obtain the CuSNPs-LA-PBA-(Ac-FDG) product.

[0080] Example 4

[0081] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0082] The integrated diagnosis and treatment preparation provided in this embodiment is CuSNPs-LA-PBA-(F-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically copper sulfide nanoparticles (CuSNps), and the PET-CT contrast agent is a contrast agent 18F-FDG analogue 2-deoxy-2-fluoro-D-glucopyranose (F-FDG).

[0083] The preparation method is basically the same as that of Example 3, except that the contrast agent selected in step S4 is 18 The F-FDG analog is 2-deoxy-2-fluoro-D-glucopyranose (F-FDG), and the obtained product is CuSNPs-LA-PBA-(F-FDG) product.

[0084] Example 5

[0085] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0086] The integrated diagnosis and treatment preparation provided in this embodiment is ICG-PBA-(Ac-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically indocyanine green (ICG), and the PET-CT contrast agent is a contrast agent 18 F-FDG analog 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG).

[0087] The preparation method specifically comprises:

[0088] S1. Determine PET-CT contrast agents and phototherapy drugs

[0089] Carboxylated indocyanine green (ICG-COOH) was selected as the photothermal agent.

[0090] S2. Preparation of connection unit

[0091] 3-Aminophenylboronic acid (PBA) was selected as the linking unit.

[0092] S3. Introducing the linking unit into the phototherapy drug to obtain an intermediate linker

[0093] ICG-COOH (1 g; 1.37 mmol) was completely dissolved in DMSO (35 mL), and NHS (237 mg; 2.1 mmol), EDC (433 mg; 2.1 mmol), PBA (364 mg, 2.1 mmol) and TEA (4.45 mL; 31.99 mmol) were added in sequence. The reaction mixture was stirred at room temperature in a nitrogen atmosphere and protected from light for 550 r. After 24 h, the product ICG-PBA was obtained. Then, the solution after the reaction was slowly dripped into a centrifuge tube containing ice water by the suspension drop method. All centrifuge tubes were centrifuged at 35000 r for 30 min. This was repeated 3-4 times until the supernatant was basically clear. The supernatant was taken out and dried in a vacuum drying oven to finally obtain the intermediate connector ICG-PBA.

[0094] S4. Connecting the intermediate linker and the contrast agent18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0095] Take a 250ml volumetric flask and add 150ml of ultrapure water, then take about 2ml of 1mol / L NaOH solution and adjust the pH of 150ml of ultrapure water to about 8, so that the solution is weakly alkaline, and then add the contrast agent 18 F-FDG analogue 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) (10 mmol), then add ICG-PBA (10 mmol) obtained in step S1 and stir at room temperature in the dark for 48 hours. Transfer the liquid to a dialysis membrane and dialyze it in ultrapure water for 24 hours to remove unreacted Ac-FDG, and then freeze-dry the dialyzed sample to obtain ICG-PBA-(Ac-FDG).

[0096] Example 6

[0097] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0098] The integrated diagnosis and treatment preparation provided in this embodiment is ICG-PBA-(F-FDG), wherein the optical therapeutic drug is a photothermal preparation, specifically ICG, and the PET-CT contrast agent is a contrast agent 18 F-FDG analogue 2-deoxy-2-fluoro-D-glucopyranose (F-FDG).

[0099] The preparation method is basically the same as that of Example 5, except that the contrast agent selected in step S4 is 18 The F-FDG analog is 2-deoxy-2-fluoro-D-glucopyranose (F-FDG), and the resulting product is ICG-PBA-(F-FDG).

[0100] Example 7

[0101] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0102] The integrated diagnosis and treatment preparation provided in this embodiment is PP-PBA-(Ac-FDG), in which the optical therapeutic drug is a photosensitizer, specifically protoporphyrin (PP), and the PET-CT contrast agent is a contrast agent 18 F-FDG analog 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG).

[0103] The preparation method specifically comprises:

[0104] S1. Determine PET-CT contrast agents and phototherapy drugs

[0105] Protoporphyrin (PP) was selected as the photosensitizer.

[0106] S2. Preparation of connection unit

[0107] 3-Aminophenylboronic acid (PBA) was selected as the linking unit.

[0108] S3. Introducing the connecting unit into the photothermal preparation to obtain an intermediate connecting body

[0109] PP (1 g; 1.77 mmol) was completely dissolved in DMSO (35 mL), NHS (1.48 g; 12.80 mmol) and EDC (2.46 g; 12.80 mmol) were first added, and stirred for 550 r, 30 min, and then PBA (1.85 g, 10.67 mmol) and TEA (4.45 mL; 31.99 mmol) were added. The reaction mixture was stirred at room temperature in a nitrogen atmosphere and away from light for 550 r, and the product PP-PBA was obtained after 24 h. Then, the solution after the reaction was slowly dripped into a centrifuge tube containing ice water by the suspension drop method, and all the centrifuge tubes were centrifuged at 35000 r for 30 min in a centrifuge, and the reaction was repeated 3-4 times until the supernatant was basically clear, and the supernatant was taken out and dried in a vacuum drying oven to obtain the product, i.e., the intermediate connector PP-PBA.

[0110] S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0111] Take a 250ml volumetric flask and add 150ml of ultrapure water, then take about 2ml of 1mol / L NaOH solution and adjust the pH of 150ml of ultrapure water to about 8, so that the solution is weakly alkaline, and then add the contrast agent 18 F-FDG analogue 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) (4 mmol), then add PP-PBA (2 mmol) obtained by the reaction in step 1) and stir at room temperature in the dark for 48 hours. The liquid is transferred to a dialysis membrane and dialyzed in ultrapure water for 24 hours to remove unreacted Ac-FDG, and then the dialyzed sample is freeze-dried to obtain PP-PBA-(Ac-FDG).

[0112] Example 8

[0113] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0114] The integrated diagnosis and treatment preparation provided in this embodiment is PP-PBA-(Ac-FDG), in which the optical therapeutic drug is a photosensitizer, specifically protoporphyrin (PP), and the PET-CT contrast agent is a contrast agent 18 F-FDG analogue 2-deoxy-2-fluoro-D-glucopyranose (F-FDG).

[0115] The preparation method is basically the same as that of Example 7, except that the contrast agent selected in step S4 is 18 The F-FDG analog is 2-deoxy-2-fluoro-D-glucopyranose (F-FDG), and the resulting product is PP-PBA-(F-FDG).

[0116] Example 9

[0117] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0118] The integrated diagnosis and treatment preparation provided in this embodiment is Ce6-PBA-(Ac-FDG), in which the optical therapeutic drug is a photosensitizer, specifically chlorin (Ce6), and the PET-CT contrast agent is a contrast agent 18 F-FDG analog 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG).

[0119] The preparation method specifically comprises:

[0120] S1. Determine PET-CT contrast agents and phototherapy drugs

[0121] Chlorin (Ce6) was selected as the photosensitizer.

[0122] S2. Preparation of connection unit

[0123] 3-Aminophenylboronic acid (PBA) was selected as the linking unit.

[0124] S3. Introducing the connecting unit into the photothermal preparation to obtain an intermediate connecting body

[0125] Ce6 (0.4 g; 0.67 mmol) was completely dissolved in DMSO (20 mL), and NHS (0.622 g; 12.80 mmol) and EDC (0.371 g; 12.80 mmol) were first added, and stirred for 550 r, 30 min, and then PBA (0.37 g, 2.68 mmol) and TEA (0.45 mL; 3.22 mmol) were added. The reaction mixture was stirred at room temperature in a nitrogen atmosphere and protected from light for 550 r. After 24 h, the product Ce6-PBA was obtained. The liquid was transferred to a dialysis membrane and dialyzed in ultrapure water for 24 h to remove the solvent DMSO and the remaining NHS, EDC, and TEA after the reaction, and then the dialyzed sample was freeze-dried to obtain the intermediate connector Ce6-PBA.

[0126] S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been developed into integrated diagnostic and therapeutic preparations

[0127] Take a 250ml volumetric flask and add 150ml of ultrapure water, then take about 2ml of 1mol / L NaOH solution and adjust the pH of 150ml of ultrapure water to about 8, so that the solution is weakly alkaline, and then add the contrast agent 18 F-FDG analogue 2-(acetylamino)-2-deoxy-D-glucose (Ac-FDG) (6mmol), then add Ce6-PBA (2mmol) obtained in step 1) and stir at room temperature in the dark for 48h. Transfer the liquid to a dialysis membrane and dialyze in ultrapure water for 24h to remove unreacted Ac-FDG, and then freeze-dry the dialyzed sample to obtain Ce6-PBA-(Ac-FDG).

[0128] Example 10

[0129] This embodiment provides a PET-CT-based integrated diagnosis and treatment preparation and a preparation method.

[0130] The integrated diagnosis and treatment preparation provided in this embodiment is Ce6-PBA-(F-FDG), in which the optical therapeutic drug is a photosensitizer, specifically chlorin (Ce6), and the PET-CT contrast agent is a contrast agent 18 F-FDG analogue 2-deoxy-2-fluoro-D-glucopyranose (F-FDG).

[0131] The preparation method is basically the same as that of Example 9, except that the contrast agent selected in step S4 is 18 The F-FDG analog is 2-deoxy-2-fluoro-D-glucopyranose (F-FDG), and the obtained product is Ce6-PBA-(F-FDG).

[0132] The present application utilizes an intermediate connector to connect optical therapeutic drugs with PET-CT commonly used contrast agent analogs to form an integrated diagnosis and treatment preparation. The system has application potential in the field of tumor diagnosis and treatment. In order to further verify the technical solution of the present invention and the beneficial effects achieved, Example 1 is verified. The synthetic schematic diagram of Example 1 is shown in FIG. Figure 1 shown.

[0133] Test Example 1

[0134] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 was used as a sample. 1 H NMR characterization, the results are as follows Figure 2 (A) As shown; Ce6-PBA-(Ac-FDG) prepared in Example 9 was used as a sample 1 H NMR characterization, the results are as follows Figure 2 (B) shown.

[0135] Test Example 2

[0136] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 was selected as a sample, and an absorption spectrum experiment was performed on it. The results are as follows: Figure 3 As shown, the characteristic absorption peak of gold nanorods between 800nm ​​and 900nm can be clearly seen from the ultraviolet absorption spectrum of GNRs-LA-PBA-(Ac-FDG), further confirming that the integrated diagnosis and treatment preparation GNRs-LA-PBA-(Ac-FDG) was successfully prepared.

[0137] Test Example 3

[0138] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 was selected as a sample, and its hydration diameter was characterized. The results are as follows: Figure 4 As shown, Figure 4 The dynamic light scattering (DLS) spectra of GNRs (A) and GNRs-LA-PBA-(Ac-FDG) (B). As shown in the figure, GNRs and GNRs-LA-PBA-(Ac-FDG) show double peaks, proving that they have a rod-like structure. Compared with GNRs, the size of GNRs-LA-PBA-(Ac-FDG) has increased, proving the introduction of contrast agent analogs.

[0139] Test Example 4

[0140] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 was selected as a sample, and its morphological characteristics were characterized. The results are as follows: Figure 5 As shown, Figure 5 Transmission electron microscopy images of GNRs (A) and GNRs-LA-PBA-(Ac-FDG) (B). As shown in the figure, GNRs-LA-PBA-(Ac-FDG) has a rod-like structure and is evenly dispersed.

[0141] Test Example 5

[0142] The GNRs-LA-PBA-(Ac-FDG) sample prepared in Example 1 and the Ce6-PBA-(Ac-FDG) sample prepared in Example 9 were selected to characterize their photothermal and photodynamic properties, respectively. The results are as follows: Figure 6 and Figure 7 As shown, Figure 6 This is the photothermal conversion curve of GNRs-LA-PBA-(Ac-FDG). Under the irradiation of 808nm laser, GNRs-LA-PBA-(Ac-FDG) exhibits good photothermal conversion properties. With the increase of nanoparticle concentration (A) or laser intensity (B), the solution temperature increases faster. Figure 7The ultraviolet absorption spectrum of Ce6-PBA-(Ac-FDG) aqueous solution irradiated by 605nm laser to generate active oxygen bleaching benzofuran. Therefore, GNRs-LA-PBA-(Ac-FDG) and Ce6-PBA-(Ac-FDG) can be used for photothermal therapy and photodynamic therapy of tumors, respectively.

[0143] Test Example 6

[0144] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 and the Ce6-PBA-(Ac-FDG) prepared in Example 9 were selected as samples, and an in vitro phosphorylation experiment was performed on them using adenosine triphosphate (ATP). The results were as follows: Figure 8 (A) and Figure 8 (B) shown. Figure 8 (A) After the introduction of Ac-FDG into the photothermal agent, the glucose phosphorylation process was simulated in vitro 31 The PNMR results show that the modified Ac-FDG can still undergo the phosphorylation process, indicating that the modified glucose contrast agent can still undergo the phosphorylation process and does not affect the tumor diagnosis process. Figure 8 (B) After the introduction of photosensitizer into Ac-FDG, the glucose phosphorylation process was simulated in vitro 13 C NMR results show that the modified Ac-FDG can still undergo phosphorylation, indicating that the modified glucose contrast agent can still undergo phosphorylation and does not affect the tumor diagnosis process.

[0145] Test Example 7

[0146] The GNRs-LA-PBA-(Ac-FDG) prepared in Example 1 and the Ce6-PBA-(Ac-FDG) prepared in Example 9 were selected as samples and subjected to in vitro cell experiments. The results were as follows: Fig. 9 and Fig.10 shown. Fig. 9 The cell experiment results of GNRs-LA-PBA-(Ac-FDG) show that after the prepared integrated diagnosis and treatment preparation was co-cultured with Hela cells, GNRs-LA-PBA-(Ac-FDG) showed good biocompatibility under conditions without laser irradiation. After 808nm laser irradiation, GNRs-LA-PBA-(Ac-FDG) showed obvious phototoxicity, so it is expected to be used in tumor photothermal therapy. Fig.10The cell experiment results of Ce6-PBA-(Ac-FDG) show that after the prepared integrated diagnosis and treatment preparation was co-cultured with Hela cells, Ce6-PBA-(Ac-FDG) showed good biocompatibility under conditions without laser irradiation. After 650nm laser irradiation, Ce6-PBA-(Ac-FDG) showed obvious phototoxicity, so it is expected to be used in tumor photodynamic therapy.

[0147] The above description is a detailed description, basic principles and beneficial effects of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Without departing from the spirit or basic features of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work based on the embodiments in the present invention belong to the scope of protection of the present invention, and all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Although this specification is described in accordance with the implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should take the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing a PET-CT integrated diagnosis and treatment preparation, characterized in that: include: The PET-CT contrast agent is connected to the phototherapy drug via a connecting unit, wherein one end of the connecting unit comprises a functional group for connecting to glucose and the other end comprises a functional group for connecting to the surface of the nanoparticle, specifically comprising the following steps: S1. Determine PET-CT contrast agents and phototherapy drugs; S2, preparing a connection unit; S3, introducing the linking unit into the phototherapy drug to obtain an intermediate linker; S4. Connecting the intermediate linker and the contrast agent 18 F-FDG analogs have been obtained as integrated diagnostic and therapeutic preparations.

2. The preparation method according to claim 1, characterized in that: The functional group at one end of the linking unit for connecting with glucose is selected from one or more of phenylboronic acid and phenylboronic acid analogs.

3. The preparation method according to claim 2, characterized in that: The functional group at the other end of the connecting unit for connecting to the surface of the nanoparticle is selected from one or more of a thiol group, dopamine, a dopamine analogue, and a functional group containing a group that directly reacts with a phototherapeutic drug, and the functional group containing a group that directly reacts with a phototherapeutic drug is selected from one or more of a functional group containing an amino group, a functional group containing a hydroxyl group, a functional group containing a carboxyl group, a functional group containing a halogen atom, a functional group containing a carbonyl group, a functional group containing a nitro group, a functional group containing a sulfonic acid group, and a functional group containing a thiol group.

4. The preparation method according to claim 2 or 3, characterized in that: The linking unit is selected from 3-aminophenylboronic acid (PBA) or lipoic acid-phenylboronic acid LA-PBA.

5. The preparation method according to claim 1, characterized in that: The phototherapy drug is photosensitivity and has the function of producing substances that induce apoptosis and / or death of cancer cells under irradiation with light of a specific wavelength. The phototherapy drug is selected from one or more of protoporphyrin, hematoporphyrin, dihydrochlorin, porphyrin derivatives, photosensitizers that can generate reactive oxygen species by light induction, and gold nanorods, copper sulfide nanoparticles, indocyanine green, and photothermal preparations that can generate heat by light induction.

6. A diagnosis and treatment integrated preparation, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 5.

7. A use of the integrated diagnosis and treatment preparation as claimed in claim 6, characterized in that: The expanded application of the PET-CT integrated diagnosis and treatment preparation in the biomedical field.

8. The use according to claim 7, characterized in that: The PET-CT-based integrated diagnosis and treatment preparation is used to manufacture drugs for treating tumors.

9. The use according to claim 7, characterized in that: The application of the PET-CT integrated diagnosis and treatment preparation in fluorescence imaging and photoacoustic imaging.

10. The use according to claim 7, characterized in that: The PET-CT-based integrated diagnosis and treatment preparation is used to manufacture disease diagnosis preparations.

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