CN113730613B

CN113730613B - Application of lutetium-labeled nano-carrier in preparation of medicine for treating neuroendocrine tumor

Info

Publication number: CN113730613B
Application number: CN202111056235.9A
Authority: CN
Inventors: 朱然; 施秀敏; 李庆; 王峰; 杨凯; 王广林; 张川; 梁茂林
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2022-03-29
Anticipated expiration: 2041-09-09
Also published as: CN113730613A

Abstract

The invention relates to the technical field of preparation of antitumor drugs, in particular to application of a lutetium-labeled nano-carrier in preparation of a drug for treating neuroendocrine tumors, and provides a preparation method of the lutetium-labeled nano-carrier. Experiment of the invention verifies¹⁷⁷Lu-SPN-GIP can realize the rapid ablation effect of photothermal therapy on neuroendocrine tumors, retain the DNA double-strand damage effect of nuclides on tumor cells, and simultaneously have the in-vivo tracing function of the nuclides.

Description

Application of lutetium-labeled nano-carrier in preparation of medicine for treating neuroendocrine tumor

Technical Field

The invention relates to the technical field of preparation of antitumor drugs, in particular to application of a lutetium-labeled nano-carrier in preparation of a drug for treating neuroendocrine tumors.

Background

Neuroendocrine tumors are a heterogeneous group of tumors originating from peptidergic neurons and neuroendocrine cells, capable of producing bioactive amines and polypeptide hormones. Can occur in various organs and tissues of the whole body, has benign tumors with inertia and slow growth, low-grade malignant tumors and high-metastatic and obvious malignant tumors, and is a general name of a large group of tumors.

Neuroendocrine tumors are classified into two major groups, non-functional (about 80%) and functional (about 20%) according to whether the tumor has a hormone-secreting function and the presence or absence of clinical symptoms caused by hormones. The non-functional gastrointestinal pancreatic neuroendocrine tumor is mainly manifested by non-specific digestive tract symptoms or tumor local occupation symptoms, such as progressive dysphagia, abdominal pain, abdominal distention, diarrhea, abdominal mass, jaundice or black stool; functional gastrointestinal-pancreatic neuroendocrine tumors are mainly characterized by related clinical symptoms caused by secretion of biologically active hormones from tumors, such as skin flushing, sweating, asthma, diarrhea, hypoglycemia, refractory digestive tract ulcer, diabetes and the like. The functional gastrointestinal and pancreatic neuroendocrine tumors are mainly pancreatic neuroendocrine tumors, including insulinoma, somatostatin tumor, glucagonoma, gastrinoma and the like.

The treatment means of the neuroendocrine tumor comprises endoscopic surgery, surgical treatment, radiation intervention treatment, radionuclide treatment, chemotherapy, biological treatment, molecular targeted treatment and the like, and the selection of the treatment means depends on the grading, stage and occurrence part of the tumor and whether the tumor has the function of secreting hormone.

Because of the promising basic clinical methods of radionuclide therapy and radionuclide diagnostics, the world is dealing with reactor nuclides¹⁷⁷The demand for Lu is increasing. As having a shorter halfStage of decay T_1/2Low energy beta emitters for =6.71 days,¹⁷⁷lu constitutes an excellent vehicle for the specific deposition of large amounts of energy in small volumes. These physical properties are used to a large extent in the form of radioimmuno-radionuclide therapy and peptide receptor radionuclide therapy in oncology, in particular for the therapy and diagnosis of tumors.

Therefore, how to prepare the lutetium-labeled nano-carrier according to the characteristics of the lutetium element and apply the lutetium-labeled nano-carrier to the preparation of the medicine for treating neuroendocrine tumors is a problem to be solved urgently by the technical staff in the field.

Disclosure of Invention

The invention aims to provide application of a lutetium-labeled nano-carrier in preparation of a medicine for treating neuroendocrine tumors, and the effect of inhibiting and ablating tumors can be remarkably achieved only through single administration treatment of 1.11 MBq.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of a lutetium-labeled nano-carrier in preparation of a medicine for treating neuroendocrine tumors.

Preferably, the neuroendocrine tumor treatment drug is for combined photothermal therapy and radionuclide therapy.

Preferably, the preparation method of the lutetium-labeled nano-carrier comprises the following steps:

(1) mixing glucose-dependent insulinotropic polypeptide and Mal-PEG₁₂Mixing DSPE and tri (2-carboxyethyl) phosphine, reacting, purifying and freeze-drying a reaction product, and dissolving the reaction product in tetrahydrofuran to obtain a solution A;

(2) adding dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅-mixing dimethyl sulfoxide solutions of DSPE to obtain a solution B;

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

(4) mixing the solution A, the solution B and the solution C, carrying out ultrasonic self-assembly in water, introducing nitrogen, passing through a column, carrying out ultrafiltration to obtain DOTA-SPN-GIP, and adding water to obtain a DOTA-SPN-GIP solution;

(5) the DOTA-SPN-GIP solution obtained in the step (4),¹⁷⁷LuCl₃Mixing HCl solution and sodium acetate solution, reacting, adding PBS or normal saline into the reaction product, and performing ultrafiltration to obtain the lutetium-labeled nano-carrier.

Preferably, in step (1), the glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The mass ratio of the DSPE to the tris (2-carboxyethyl) phosphine is 8-12: 1-3: 13-15;

before reaction, glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂Respectively adjusting the pH value of DSPE and tris (2-carboxyethyl) phosphine to 7.2-7.6, and then mixing for reaction;

the reaction is performed by shaking for 10-14 h at room temperature;

glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The volume ratio of the total mass of the DSPE and the tri (2-carboxyethyl) phosphine to the tetrahydrofuran is 1.6-3.6 g: 130-170L.

Preferably, DOTA-NHS is reacted with NH in dimethyl sulfoxide in step (2)₂-PEG₄₅The concentration of the DSPE in the dimethyl sulfoxide is 8-12 g/L;

the mixing is performed by oscillating for 1.5-2.5 h at room temperature;

dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅The volume ratio of dimethyl sulfoxide solution of DSPE is 3-5: 24-28.

Preferably, the mass volume ratio of the PCPDTBT to the tetrahydrofuran in the step (3) is 1-2 g: 1-2L.

Preferably, the water adding amount in the step (4) is 3-5 times of the total volume of the solution A, the solution B and the solution C; the ultrasonic frequency is more than or equal to 20 KHz; the self-assembly time is 12-18 min.

Preferably, in the step (4), the volume ratio of the solution A to the solution B to the solution C is 130-170: 25-35: 40-60; introducing nitrogen, passing through PD10 gel column, collecting intermediate product of specific section, and ultrafiltering;

when the dosage of the glucose-dependent insulinotropic polypeptide is 1mg, the intermediate product is 4-5 mL flowing out of a PD10 gel column;

when the dosage of the glucose-dependent insulinotropic polypeptide is 2Nmg, the intermediate product is NmL (3N + 1) - (3 + 2) from a PD10 gel column;

the volume ratio of DOTA-SPN-GIP to water is 3-7: 10.

Preferably, in the step (5), the¹⁷⁷LuCl₃The emission amount of the HCl solution is 148-185 MBq;

¹⁷⁷LuCl₃the volume ratio of the HCl solution, the DOTA-SPN-GIP solution and the sodium acetate solution is 3-5: 4-6: 0.5-1.5; the concentration of the sodium acetate solution is 0.15-0.35M;

the reaction is performed by shaking at room temperature for 25-35 min.

Preferably, in the step (5), the volume ratio of the reaction product to the added PBS or physiological saline is 1: 3-5.

Preferably, the purification in the step (1) is ultrafiltration, and the parameters are set to 4000-6000 rpm and 25-35 min, and the ultrafiltration is carried out for 5-8 times;

in the steps (4) and (5), the ultrafiltration parameters are set to 3000-4000 revolutions per minute, 4-6 minutes and ultrafiltration is carried out for 1-3 times.

Compared with the prior art, the invention has the following beneficial effects

1、¹⁷⁷Lu is a representative beta-emitting radiopharmaceutical for Molecular Radiotherapy (MRT) and is currently routinely used in europe for the treatment of NET (neuroendocrine tumor) patients. The therapy is generally carried out by injecting 7.4 GBq intravenously 2 or 4 times¹⁷⁷Lu marker drugs can significantly improve the progression-free survival period. In the combined treatment research of the invention, the effect of inhibiting the ablation tumor can be obviously achieved under a small dose through the single administration treatment of 1.11 MBq.

2、¹⁷⁷Lu-SPN-GIP can realize the rapid ablation effect of PTT on tumors, retain the DNA double-strand damage effect of nuclides on tumor cells, and simultaneously have the in-vivo tracing function of the nuclides. We have demonstrated from in vitro cell studies and in vivo treatments that combined photothermal and molecular radiotherapy can significantly synergistically kill CFPAC-1 tumor cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 shows different dosages¹⁷⁷The growth inhibitory effect of Lu-SPN-GIP in combination with photothermal therapy in CFPAC-1;

FIG. 2 is a fluorescent image of the survival of CFPAC-1 cells from different treatment groups;

FIG. 3 shows the survival rates of CFPAC-1 cells from different treatment groups;

FIG. 4 is a drawing showing¹⁷⁷Lu-SPN-GIP and¹⁷⁷LuCl₃retention effects;

FIG. 5 shows photothermal therapy and laser-only tumor temperature increase;

FIG. 6 shows the tumor volume changes in the different treatment groups within 21 days;

FIG. 7 shows NS group (Saline), SPN-GIP group (SPN),¹⁷⁷HE staining analysis of tumors in the LuCl3 group;

FIG. 8 shows a simple photothermal group (Laser only), an SPN-GIP plus photothermal group (SPN-GIP + Laser),¹⁷⁷Lu-SPN-GIP group (¹⁷⁷Lu-SPN-GIP) and¹⁷⁷Lu-SPN-GIP photothermal group (177 Lu-SPN-GIP + Laser) tumor HE staining analysis;

FIG. 9 shows the actual size of the tumors in each group after treatment;

FIG. 10 shows the weight change of each treatment group;

FIG. 11 is the blood glucose levels of each treatment group;

fig. 12 is liver function level for each treatment group;

FIG. 13 shows renal function levels in each treatment group;

FIG. 14 is an HE staining analysis of heart, liver, spleen, lung, kidney, pancreas, intestine for each treatment group;

FIG. 15 is CFPAC-1 cell passage¹⁷⁷Lu-SPN-GIP, photothermal therapy and gamma-H2 AX immunofluorescence imaging after combination therapy,blue represents cell nucleus, green represents DNA double-strand damage part, and scale represents 30 μm;

FIG. 16 is CFPAC-1 tumor trafficking¹⁷⁷Lu-SPN-GIP, photothermal therapy and gamma-H after combination therapy₂AX immunofluorescence imaging.

Detailed Description

In the present invention, the neuroendocrine tumor treating drug is used for a combination of photothermal therapy and radionuclide therapy.

The invention provides a preparation method of a lutetium-labeled nano carrier, which comprises the following steps:

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

(5) the DOTA-SPN-GIP solution obtained in the step (4),¹⁷⁷LuCl₃Mixing HCl solution and sodium acetate solution, reacting, adding PBS or normal saline into the reaction product, and ultrafiltering to obtain lutetium-labeled nano-carrier (¹⁷⁷Lu-SPN-GIP）。

In the present invention, in the step (1), the glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The mass ratio of the DSPE to the tris (2-carboxyethyl) phosphine is 8-12: 1-3: 13-15; preferably 9-11: 2: 14; further preferably 10: 2: 14;

in the present invention, the step (1) is to subject the glucose-dependent insulinotropic hormone to the reaction before the reactionPolypeptide, Mal-PEG₁₂Respectively adjusting the pH value of DSPE and tris (2-carboxyethyl) phosphine to 7.2-7.6, and then mixing for reaction; preferably adjusting the pH value to 7.3-7.5; more preferably, the pH is adjusted to 7.4;

in the invention, the reaction in the step (1) is performed by shaking at room temperature for 10-14 h; preferably 11-13 h; more preferably 12 h;

in the present invention, the glucose-dependent insulinotropic polypeptide, Mal-PEG, in the step (1)₁₂The volume ratio of the total mass of the DSPE and the tri (2-carboxyethyl) phosphine to the tetrahydrofuran is 1.6-3.6 g: 130-170L; preferably 2.0-3.2 g: 140-160L; further preferably 2.4-2.8 g: 145-155L; more preferably 2.6 g: 150L.

In the present invention, DOTA-NHS in said step (2) is reacted with NH in dimethyl sulfoxide₂-PEG₄₅The concentration of the DSPE in the dimethyl sulfoxide is 8-12 g/L; preferably 9-11 g/L; further preferably 10 g/L;

the mixing is performed by oscillating for 1.5-2.5 h at room temperature; preferably 1.7-2.3 h; further preferably 1.9-2.1 h; more preferably 2 h;

dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅The volume ratio of dimethyl sulfoxide solution of DSPE is 3-5: 24-28; preferably 4: 25-27; further preferably 4: 26.

In the invention, the mass-to-volume ratio of PCPDTBT to tetrahydrofuran in the step (3) is 1-2 g: 1-2L; preferably 1 g: 1L.

In the invention, the water adding amount in the step (4) is 3-5 times of the total volume of the solution A, the solution B and the solution C; preferably 4 times;

the ultrasonic frequency is more than or equal to 20 KHz; the self-assembly time is 12-18 min; preferably 13-17 min; further preferably 14-16 min; more preferably 15 min.

In the invention, in the step (4), the volume ratio of the solution A, the solution B and the solution C is 130-170: 25-35: 40-60; preferably 140-160: 27-33: 44-56; further preferably 145-155: 29-31: 48-52; more preferably 150: 30: 50;

introducing nitrogen, passing through PD10 gel column, collecting intermediate product of specific section, and ultrafiltering;

the volume ratio of DOTA-SPN-GIP to water is 3-7: 10; preferably 4-6: 10; further preferably 5: 10.

In the present invention, in the step (5), the¹⁷⁷LuCl₃The emission amount of the HCl solution is 148-185 MBq; preferably 158 to 175 MBq; more preferably 162 to 171 MBq; more preferably 165 MBq;

¹⁷⁷LuCl₃the volume ratio of the HCl solution, the DOTA-SPN-GIP solution and the sodium acetate solution is 3-5: 4-6: 0.5-1.5; preferably 4: 5: 0.7-1.3; further preferably 4: 5: 0.9-1.1; more preferably 4: 5: 1;

the concentration of the sodium acetate solution is 0.15-0.35M; preferably 0.20-0.30M; more preferably 0.25M;

the reaction is performed by shaking at room temperature for 25-35 min; preferably 27-33 min; further preferably 29-31 min; more preferably 30 min.

In the invention, in the step (5), the volume ratio of the reaction product to the added PBS or normal saline is 1: 3-5; preferably 1: 4.

In the invention, the purification in the step (1) is ultrafiltration, and the parameters are set to 4000-6000 r/min and 25-35 min, and the ultrafiltration is carried out for 5-8 times; preferably, the parameters are set to be 4500-5500 r/min, 27-33 min and 6-7 times of ultrafiltration; further preferably 4800-5200 r/min, 29-31 min, ultrafiltering 7 times; more preferably 5000 r/min, 30min, ultrafiltering for 7 times;

setting ultrafiltration parameters in the steps (4) and (5) to be 3000-4000 r/min, 4-6 min and carrying out ultrafiltration for 1-3 times; preferably setting parameters of 3300-3700 r/min, 5min, and carrying out ultrafiltration for 2 times; further preferably 3400-3600 r/min, 5min, ultra-filtering for 2 times; more preferably 3500 rpm/min, 5min, and 2 times of ultrafiltration.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Instruments and reagents for experimentation

Multifunctional enzyme mark instrument (Bio-Tek, USA)

LB2111 Multi Crystal Gamma Counter (BERTHOLD, USA)

Constant temperature oscillation mixer (Thermomixer, China)

High speed centrifuge (H1850 Hunan instrument centrifuge Co., Ltd.)

Ultra pure water system (Direct Q5, Merck Millipore USA)

Ultrasonic cleaning machine (KQ-100 KD, Kunshan ultrasonic apparatus Co., Ltd.)

GIP (Shanghai Jier Biochemical Co., Ltd., custom synthesis)

Mal-PEG₁₂DSPE (Santa, USA)

PCPDTBT (Sigma, USA)

TCEP (Amresco, USA)

Tetrahydrofuran (national drug group chemical reagent Co., Ltd.)

NH₂-PEG₄₅-DSPE (Aladdin, China)

DOTA-NHS (Macrocyclics, USA)

CCK8 kit (Donglian chemistry, Japan)

Self-prepared reagent

(1) 0.05M HCl 30% HCl 264. mu.L was diluted to a volume of 50 mL with ultrapure water.

(2) 0.25M sodium acetate (NaOAc) 1.025 g of anhydrous sodium acetate powder was weighed out and dissolved in 50 mL of ultrapure water and dissolved by sonication.

Example 1

A preparation method of a lutetium marked nano carrier comprises the following steps:

(1) before reaction, glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂-DSPE and tris (2-carboxyethyl) phosphine, respectively, adjusted to pH 7.2 and thenMixing the three, shaking at room temperature for 10h, ultrafiltering the reaction product (with parameters of 4000 rpm, 25min, and 5 times of ultrafiltration), lyophilizing, and dissolving in tetrahydrofuran to obtain solution A;

wherein the glucose-dependent insulinotropic polypeptide and Mal-PEG are₁₂-the mass ratio of DSPE to tris (2-carboxyethyl) phosphine is 8: 1: 13; glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The volume ratio of the sum of DSPE and tris (2-carboxyethyl) phosphine to tetrahydrofuran is 1.6 g: 130L;

(2) adding dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅Mixing dimethyl sulfoxide solutions of DSPE, and shaking for 1.5h at room temperature to obtain a solution B;

wherein DOTA-NHS is reacted with NH in dimethyl sulfoxide₂-PEG₄₅The concentration of DSPE in dimethyl sulfoxide is 8 g/L; dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅-volume ratio of dimethyl sulfoxide solution of DSPE 3: 24;

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

wherein the mass-volume ratio of PCPDTBT to tetrahydrofuran is 1 g: 2L

(4) Mixing the solution A, the solution B and the solution C, carrying out ultrasonic (ultrasonic frequency is more than or equal to 20 KHz) self-assembly for 12min in water, introducing nitrogen, passing through a PD10 gel column, connecting an intermediate product of a specific section, then carrying out ultrafiltration (ultrafiltration parameters are set to be 3000 r/min, 4min, and ultrafiltration is carried out for 1 time) to obtain DOTA-SPN-GIP, and adding water to obtain a DOTA-SPN-GIP solution;

wherein the water adding amount is 3-5 times of the total volume of the solution A, the solution B and the solution C; the volume ratio of the solution A to the solution B to the solution C is 130: 25: 40;

when the dosage of the glucose-dependent insulinotropic polypeptide is 2Nmg, the intermediate product is NmL (3N + 1) - (3 + 2) from a PD10 gel column; the volume ratio of DOTA-SPN-GIP to water is 3: 10;

(5) will be step (4)Obtained DOTA-SPN-GIP solution, 148 MBq¹⁷⁷LuCl₃Mixing HCl solution and 0.15M sodium acetate solution, oscillating at room temperature for 25min, adding PBS or normal saline into the reaction product, and ultrafiltering (setting ultrafiltration parameters are 3000 r/min, 4min, and ultrafiltering for 1 time) to obtain lutetium-labeled nano-carrier;

wherein the content of the first and second substances,¹⁷⁷LuCl₃the volume ratio of the HCl solution, the DOTA-SPN-GIP solution and the sodium acetate solution is 3: 4: 0.5; the volume ratio of the reaction product to the added PBS or physiological saline was 1: 3.

Example 2

(1) before reaction, glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂Respectively adjusting the pH value of DSPE and tris (2-carboxyethyl) phosphine to 7.6, mixing the three, oscillating at room temperature for 14h, ultrafiltering the reaction product (with the parameter set to 6000 r/min, 35min, and ultrafiltering for 8 times), freeze-drying, and dissolving in tetrahydrofuran to obtain solution A;

wherein the glucose-dependent insulinotropic polypeptide and Mal-PEG are₁₂-the mass ratio of DSPE to tris (2-carboxyethyl) phosphine is 12: 3: 15; glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The volume ratio of the sum of DSPE and tris (2-carboxyethyl) phosphine to tetrahydrofuran is 3.6 g: 170L;

(2) adding dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅Mixing dimethyl sulfoxide solutions of DSPE, and shaking for 2.5 hours at room temperature to obtain a solution B;

wherein DOTA-NHS is reacted with NH in dimethyl sulfoxide₂-PEG₄₅The concentration of DSPE in dimethyl sulfoxide is 12 g/L; dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅-volume ratio of dimethyl sulfoxide solution of DSPE 5: 28;

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

wherein the mass-volume ratio of PCPDTBT to tetrahydrofuran is 2 g: 1L

(4) Mixing the solution A, the solution B and the solution C, carrying out ultrasonic (ultrasonic frequency is more than or equal to 20 KHz) self-assembly for 18min in water, introducing nitrogen, passing through a PD10 gel column, connecting an intermediate product of a specific section, then carrying out ultrafiltration (ultrafiltration parameters are set to be 4000 revolutions per minute, 6min, and ultrafiltration is carried out for 1-3 times) to obtain DOTA-SPN-GIP, and adding water to obtain a DOTA-SPN-GIP solution;

wherein the water adding amount is 5 times of the total volume of the solution A, the solution B and the solution C; the volume ratio of the solution A to the solution B to the solution C is 170: 35: 60;

when the dosage of the glucose-dependent insulinotropic polypeptide is 1mg, the intermediate product is 5mL flowing out of a PD10 gel column;

when the dosage of the glucose-dependent insulinotropic polypeptide is 2Nmg, the intermediate product is NmL (3N + 1) - (3 + 2) from a PD10 gel column; the volume ratio of DOTA-SPN-GIP to water is 7: 10;

(5) the DOTA-SPN-GIP solution obtained in the step (4) and 185 MBq¹⁷⁷LuCl₃Mixing HCl solution and 0.35M sodium acetate solution, oscillating at room temperature for 35min, adding PBS or normal saline into the reaction product, and ultrafiltering (setting ultrafiltration parameters are 4000 r/min, 6min, and ultrafiltering for 3 times) to obtain lutetium-labeled nano-carrier;

wherein the content of the first and second substances,¹⁷⁷LuCl₃the volume ratio of the HCl solution, the DOTA-SPN-GIP solution and the sodium acetate solution is 5: 6: 1.5; the volume ratio of the reaction product to the added PBS or physiological saline was 1: 5.

Example 3

(1) before reaction, glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂Respectively adjusting the pH value of DSPE and tris (2-carboxyethyl) phosphine to 7.4, mixing the three, oscillating at room temperature for 10-14 h, ultrafiltering the reaction product (the parameters are set to 5000 r/min, 30min, and ultrafiltering for 7 times), freeze-drying, and dissolving in tetrahydrofuran to obtain a solution A;

wherein the glucose-dependent insulinotropic polypeptide and Mal-PEG are₁₂-the mass ratio of DSPE and tris (2-carboxyethyl) phosphine is 10: 2: 14; glucose-dependent insulinotropic polypeptide, Mal-PEG₁₂The volume ratio of the sum of DSPE and tris (2-carboxyethyl) phosphine to tetrahydrofuran is 2.6 g: 150L;

(2) adding dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅Mixing dimethyl sulfoxide solutions of DSPE, and shaking for 2 hours at room temperature to obtain a solution B;

wherein DOTA-NHS is reacted with NH in dimethyl sulfoxide₂-PEG₄₅The concentration of DSPE in dimethyl sulfoxide is 10 g/L; dimethyl sulfoxide solution of DOTA-NHS and NH₂-PEG₄₅-volume ratio of dimethyl sulfoxide solution of DSPE 4: 26;

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

wherein the mass-volume ratio of PCPDTBT to tetrahydrofuran is 1 g: 1L

(4) Mixing the solution A, the solution B and the solution C, carrying out ultrasonic (ultrasonic frequency is more than or equal to 20 KHz) self-assembly for 15min in water, introducing nitrogen, passing through a PD10 gel column, connecting an intermediate product of a specific section, then carrying out ultrafiltration (ultrafiltration parameters are all set to 3500 rpm, 5min, and 2 times of ultrafiltration) to obtain DOTA-SPN-GIP, and adding water to obtain a DOTA-SPN-GIP solution;

wherein the water adding amount is 4 times of the total volume of the solution A, the solution B and the solution C; the volume ratio of the solution A to the solution B to the solution C is 150: 30: 50;

when the dosage of the glucose-dependent insulinotropic polypeptide is 2Nmg, the intermediate product is NmL (3N + 1) - (3 + 2) from a PD10 gel column; the volume ratio of DOTA-SPN-GIP to water is 5: 10;

(5) the DOTA-SPN-GIP solution obtained in the step (4) and 165MBq¹⁷⁷LuCl₃Mixing HCl solution and 0.25M sodium acetate solution, oscillating at room temperature for 30min, adding PBS or normal saline into the reaction product, and ultrafiltering (setting of ultrafiltration parameters are 3500 rpm/min, 5min, and ultrafiltering for 2 times) to obtain lutetium-labeled nano-carrier;

wherein the content of the first and second substances,¹⁷⁷LuCl₃HCl solution, DOTA-SPNThe volume ratio of the GIP solution to the sodium acetate solution is 4: 5: 1; the volume ratio of the reaction product to the added PBS or physiological saline was 1: 4.

Example 4

1. Photothermal therapy in combination with nuclide therapy growth inhibition in CFPAC-1 cells (experimental with lutetium labeled nanocarriers obtained in example 3, the same applies below)

(1) Taking CFPAC-1 cells in logarithmic phase, digesting the CFPAC-1 cells with 0.25% pancreatin, adjusting the cell concentration, and spreading the cells on a 96-well plate; cell concentration of 10⁴mL, 5% CO at 37 ℃₂Incubate in incubator for 24 h.

(2) After the cells are attached to the wall, the medium is replaced, the medium with the specified radioactivity (0, 0.37, 3.7, 7.4, 11.1, 14.8, 18.5 MBq/mL) is added respectively for culturing for 24 h, and then the 808 laser is used for culturing at 1W/cm²Theoretical power heating for 10 minutes.

(3) After removing the medium, the medium was washed twice with PBS, the medium containing 10% CCK8 was replaced, incubated in an incubator for 1h, and then measured for OD (450 nm) with a microplate reader.

(4) Cell viability = (different dose groups plus light heat-blank)/(control group-blank) × 100%.

PTT (photothermal therapy) combination¹⁷⁷Lu-SPN-GIP Live-dead experiment

(1) Digesting CFPAC-1 cells in logarithmic growth phase with 0.25% pancreatin, adjusting cell concentration and spreading on a 96-well plate; cell concentration of 10⁴one/mL, 5% CO at 37 ℃₂Incubate in incubator for 24 h.

(2) After the cells are attached to the wall, the culture medium is replaced, and the normal culture medium, 50 mu g/mL SPN-GIP and 1.11 MBq/mL¹⁷⁷LuCl₃1.11 MBq/mL¹⁷⁷Lu-SPN-GIP, and divided into a non-treatment complete blank group (Control) and an SPN-GIP group according to different treatments,¹⁷⁷LuCl₃group, blank photothermal group, SPN-GIP photothermal treatment group,¹⁷⁷Lu-SPN-GIP group, and¹⁷⁷Lu-SPN-GIP combined photothermal treatment group.

(3) Using 808 laser with theoretical power of 1W/cm²And 5 minutes of photothermal treatment.

(4) mu.L each of PI/AM was added to 1.5 mL of the buffer solution, and the mixture was mixed with light and light.

(5) Each group of treated cells was washed twice with PBS and 100. mu.L of a PI/AM (1: 1000) -containing buffer was added to each well.

(6) And (4) incubating in the incubator for 20-30 minutes in the dark, taking out, and continuously photographing under a fluorescence microscope in the dark.

As a result: of different activity¹⁷⁷By using Lu-SPN-GIP in combination with photothermal therapy 24 hours later, it was observed that by combination therapy, cell survival was achieved when the radioactivity was 11.1 MBq/mL<10% effect, the growth inhibition effect is more efficient and rapid. We chose 1.11 MBq as the therapeutic dose in subsequent treatments. Meanwhile, the cell survival rate is verified by the combined photothermal effect of different materials and comparison¹⁷⁷Lu-SPN-GIP not only has the photothermal effect of SPN-GIP, but also has¹⁷⁷Radiation therapy effect of Lu nuclide. Combination therapy demonstrated excellent efficacy at the cellular level. The specific results are shown in FIGS. 1 to 3.

Example 5¹⁷⁷Evaluation of the Retention Effect of Lu-SPN-GIP in tumors

(1) A CFPAC-1 (human pancreatic cancer cell) animal model was constructed. Taking CFPAC-1 cells in logarithmic growth phase, digesting with 0.25% pancreatin, washing with PBS, resuspending, counting, and loading tumor at right hind leg of mouse at 2 × 10⁶ Volume 50. mu.L/mouse.

(2) Tumors with the diameter of about 4-5 mm are selected for treatment in groups 10 days after tumor loading, and 5 tumors are selected in each group.

(3) After adjustment of the marks¹⁷⁷Lu-SPN-GIP volume and activity 1.11 MBq/25. mu.L, likewise freed with sodium acetate¹⁷⁷LuCl₃The activity and volume were 1.11 MBq/25. mu.L.

(4) To give¹⁷⁷Lu-SPN-GIP group and¹⁷⁷LuCl₃images were acquired at 0, 24, 48, 4 d post-intratumoral injection on SPECT/CT.

(5) And reconstructing the collected data by using PMOD software and setting the same condition for analysis.

As a result:¹⁷⁷Lu-SPN-GIP and free¹⁷⁷LuCl₃The development is immediately collected on SPECT/CT after the intratumoral injection, the difference between the two is not seen at the time point of 0.5 h,¹⁷⁷the Lu-SPN-GIP group is still completely retained in the tumor at 24 h and 48 h, the in-tumor development is still clearly visible after 4 d, and the nuclide diffusion development is not seen outside the tumor or even in the whole body, but¹⁷⁷LuCl₃The group was found in 24 h not only in tumors but also in the whole body, and was completely metabolized in vivo by 48 h. Description of the invention¹⁷⁷Lu-SPN-GIP has excellent tumor retention effect, can exert the radiotherapy effect for a long time, and reduces the side effect of the whole body. The specific results are shown in FIG. 4.

Example 6 PTT Association¹⁷⁷Evaluation of the efficacy of Lu-SPN-GIP in a murine model of CFPAC-1 tumor bearing

(1) And selecting the established animal models to randomly group. Tumors with the diameter of about 4-5 mm are selected for treatment in groups 10 days after tumor loading, and 5 tumors are selected in each group. The experimental groups were NS group, SPN-GIP group,¹⁷⁷LuCl₃group, blank photothermal group, SPN-GIP photothermal group,¹⁷⁷Lu-SPN-GIP group and¹⁷⁷Lu-SPN-GIP photothermal group. The administration route is intratumoral injection.

(2) The body weight of the mice was measured before treatment and the length of the tumor was recorded.

(4) The photothermal treatment groups all use 808 lasers with the theoretical power of 1W/cm²The temperature is monitored by an imager in the heating process, and the temperature in the tumor is controlled to be 45-50 ℃ by adjusting the distance between a laser and the tumor.

(5) Body weight and tumor diameter were measured twice weekly after treatment to the end of the observation period, day 21. The tumor volume exceeds 1 cm in the treatment process³Or weight loss over 20% is the criteria for sacrifice.

(6) Tumor volume V = major diameter × minor diameter²÷2。

(7) By the end of the 21-day observation period, mice were euthanized to leave heart blood and serum was centrifuged, and the major tissue organs were dissected and stored in 4% paraformaldehyde fixing solution.

(8) Fixed tumor tissues are taken for HE staining and immunohistochemical analysis such as Ki67 and CD31 is carried out.

As a result: the evaluation of the therapeutic effect was divided into NS group, SPN-GIP group,¹⁷⁷LuCl₃group, simple photo-thermal group, SPN-GIP photo-thermal group,¹⁷⁷Lu-SPN-GIP group and¹⁷⁷Lu-SPN-GIP photothermal group. The administration route is intratumoral injection. The photothermal treatment group recorded the course of heating temperature control by photothermal only and photothermal treatment after injection of SPN drug, confirming that photothermal treatment was mainly effected by SPN, and the results are shown in fig. 5. The treatment effect is directly evaluated through the growth of the tumor volume and the tumor growth rate, the treatment effect of each group is evaluated through the range of tumor cells in the visual field in the specimen, and the specific result is shown in figures 5-9.

Example 7 PTT Association¹⁷⁷Toxic side effect evaluation of Lu-SPN-GIP in CFPAC-1 tumor bearing mouse model

1. Serological adverse reaction to blood sugar, liver and kidney functions and the like

(1) After observation for 21 days, the mice in each group are euthanized, blood is taken from the heart immediately, and the mice are placed for half an hour at normal temperature.

(2) Blood from each mouse was centrifuged at 1500 rpm for 10 minutes and the supernatant carefully aspirated and centrifuged at 12000 rpm at 4 ℃ for 15 minutes.

(3) And taking the upper layer serum and sampling the upper layer serum in a blood biochemical instrument to measure blood sugar and liver and kidney functions.

Pathological changes of each main tissue organ in mouse model with tumor

(1) Observed for 21 days, each group of mice was immediately dissected after euthanasia.

(2) The vital organs of heart, liver, spleen, lung, kidney, intestine and pancreas are carefully separated and completely soaked in 4% paraformaldehyde tissue fixing solution and stored in a refrigerator at 4 ℃.

(3) And performing HE staining analysis when the sample is placed until radioactivity can not be detected completely.

As a result: PTT federation¹⁷⁷Lu-SPN-GIP as a global monitor of the body weight change of CFPAC-1 tumor bearing mice during treatmentThe weight average of mice in each treatment group is not obviously reduced in the whole treatment process. As shown in fig. 10. In the process of further toxicity evaluation, the blood sugar level of each group of mice is detected to indicate that the pancreatic function is not affected, and the important tissue and organ HE staining observation of each group of liver function has no obvious pathological changes, thereby indicating that each treatment group has no obvious toxic or side effect. Specific results are shown in fig. 11-14 (evaluation of toxic and side effects of each treatment group).

Example 8¹⁷⁷DNA double-strand damage effect of Lu-SPN-GIP on tumor cells and tissues

(1) Collecting CFPAC-1 cells in logarithmic growth phase, digesting with pancreatin, and adjusting cell concentration to 5 × 10³The cells were then attached to each other in an incubator for 24 hours by placing each cell in a glass plate having a diameter of 15 mm.

(2) The control group was added with a normal medium,¹⁷⁷Lu-SPN-GIP group with 1.11 MBq¹⁷⁷Lu-SPN-GIP, 1.11 MBq for combination therapy¹⁷⁷Lu-SPN-GIP rear 808 laser 1W/cm²And 5 minutes.

(3) After 24 hours of treatment 4% paraformaldehyde was fixed for 30 minutes and washed three times with PBS for 5 minutes each.

(4) The cells were permeabilized with 0.2% triton (10. mu.L in 5mL PBS) for 15 minutes at room temperature and washed three times with PBS, 5 minutes each.

(5) Blocking was performed with 1% BSA (1% BSA: 0.1 g BSA in 10 mL PBS, 0.22 μm filter) for 1 hour.

（6）γ-H₂AX Primary antibody 1:250 dilution (6. mu.L in 1500. mu.L BSA, 200. mu.L per dish) was followed in the wet box overnight in a refrigerator at 4 ℃.

(7) After washing three times with PBS, a secondary antibody (1: 500) was added and incubated for 1 hour.

(8) After washing three times with PBS, Hoechst (1: 2000) was incubated for 15 minutes in the dark at room temperature.

(9) After washing with PBS for three times, add anti-quenching mounting solution, and keep out of the sun at room temperature.

(10) And (5) observing and photographing under a confocal microscope.

As a result:¹⁷⁷the killing effect of Lu on tumor is mainly achieved through betaThe result of the damage of the double strand of DNA of the tumor cells by the radiation. CFPAC-1 cells in gamma-H₂Adding into AX immunofluorescence¹⁷⁷Gamma-H can be observed in the Lu-SPN-GIP group₂AX was expressed positively and turned into clearly visible green fluorescence. The same was true in tumor tissue, albeit 21 days after the observation period¹⁷⁷Lu has completely decayed, but the effect of DNA double strand damage to tumor cells is still clearly visible. This may also be via¹⁷⁷An important factor in the long-lasting suppression of tumors after Lu-SPN-GIP treatment. Specific results thereof are shown in FIGS. 15 and 16 (DNA double strand damage effect).

In the invention develop¹⁷⁷Lu-labeled Semiconducting Polymer Nanoparticles (SPNs) for targeting glucose-dependent insulinotropic polypeptide receptors (GIPR) for combined isotope therapy and photothermal therapy (PTT) under in vivo imaging guidance. By in vivo tracking¹⁷⁷Lu-SPN-GIP and free¹⁷⁷LuCl₃The invention demonstrates the radioactivity of the label¹⁷⁷Lu-SPN-GIP has an excellent retention effect in a tumor model. By combining PTT and isotopic internal Radiation Therapy (RT) at 1W/cm²(ii) 808 nm laser for 5 minutes while receiving a 1.11 MBq dose¹⁷⁷Tumors were effectively eliminated in Lu-SPN-GIP mice. Has significant therapeutic advantages compared to monotherapy (PTT or RT only). In the PTT-only treatment group, local recurrence was observed in 3 mice after two weeks, and proliferation was rapid, and only¹⁷⁷Although the Lu-SPN-GIP radiation treatment group can observe that the proliferation of the tumor is obviously inhibited, the tumor ablation effect is not prominent. At the same time, biochemical and histological examination of blood showed that, at our therapeutic dose,¹⁷⁷Lu-SPN-GIP did not have any significant toxicity to the treated mice. The result of the invention presents a simple method for constructing the SPN-based multifunctional nano-treatment platform, and can provide combined treatment for refractory neuroendocrine tumors.

¹⁷⁷Lu-SPN-GIP can realize the rapid ablation of PTT on tumors, retain the DNA double-strand damage effect of nuclides on tumor cells, and simultaneously have the in-vivo tracing function of the nuclides. The invention proves that the combined PTT and RT can obviously and synergistically kill CFPAC-1 tumor cells from in vitro cell research and in vivo treatment. This treatment modality may lead to new considerations for cancer treatment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Nano carrier marked by lutetium¹⁷⁷The application of Lu-SPN-GIP in preparing the medicine for treating pancreatic cancer cells is characterized in that the preparation method of the lutetium-labeled nano carrier comprises the following steps:

(3) dissolving PCPDTBT in tetrahydrofuran to obtain a solution C;

2. The use according to claim 1,¹⁷⁷Lu-SPN-GIP is used in combination with a photothermal therapeutic agent.

3. The use according to claim 1,in the step (1), glucose-dependent insulinotropic polypeptide and Mal-PEG are added₁₂The mass ratio of the DSPE to the tris (2-carboxyethyl) phosphine is 8-12: 1-3: 13-15;

the reaction is performed by shaking for 10-14 h at room temperature;

4. The use according to claim 1, wherein said DOTA-NHS is reacted with NH in dimethyl sulfoxide in step (2)₂-PEG₄₅The concentration of the DSPE in the dimethyl sulfoxide is 8-12 g/L;

the mixing is performed by oscillating for 1.5-2.5 h at room temperature;

5. The use according to claim 1, wherein the mass-to-volume ratio of PCPDTBT to tetrahydrofuran in step (3) is 1-2 g: 1-2L.

6. The use of claim 1, wherein the amount of water added in the step (4) is 3-5 times of the total volume of the solution A, the solution B and the solution C; the ultrasonic frequency is more than or equal to 20 KHz; the self-assembly time is 12-18 min.

7. The use according to claim 1, wherein in the step (4), the volume ratio of the solution A, the solution B and the solution C is 130-170: 25-35: 40-60; introducing nitrogen, passing through PD10 gel column, collecting intermediate product of specific section, and ultrafiltering;

the volume ratio of DOTA-SPN-GIP to water is 3-7: 10.

8. Use according to claim 1, characterized in that, in step (5), said step (5) is carried out¹⁷⁷LuCl₃The emission amount of the HCl solution is 148-185 MBq;

the reaction is performed by shaking at room temperature for 25-35 min.

9. The use of claim 1 or 8, wherein in the step (5), the volume ratio of the reaction product to the added PBS or physiological saline is 1: 3-5;

the purification in the step (1) is ultrafiltration, the parameters are set to be 4000-6000 r/min and 25-35 min, and ultrafiltration is carried out for 5-8 times;