CN114366822A

CN114366822A - Active targeting multi-modal molecular imaging probe and preparation method and application thereof

Info

Publication number: CN114366822A
Application number: CN202011103960.2A
Authority: CN
Inventors: 吴爱国; 王胤杰; 李娟�; 曹奕
Original assignee: Ningbo Institute of Material Technology and Engineering of CAS; Cixi Institute of Biomedical Engineering CNITECH of CAS
Current assignee: Ningbo Institute of Material Technology and Engineering of CAS; Cixi Institute of Biomedical Engineering CNITECH of CAS
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2022-04-19

Abstract

The application discloses an active targeting multi-modal molecular imaging probe, a preparation method and an application thereof, wherein the active targeting multi-modal molecular imaging probe comprises an active targeting group, a nano-carrier and a multi-modal imaging signal molecule; the active targeting group is coupled with the nano-carrier through the active targeting group; the multimode imaging signal molecule is coupled with the outside of the nano carrier or carried in the nano carrier. The active targeting multi-modal molecular imaging probe provided by the application can simultaneously perform multiple imaging including fluorescence, isotope imaging and magnetic resonance imaging, and enhances the imaging effect through the complementary matching of multiple imaging. And has the characteristics of good targeting property, low dosage and high sensitivity, and simultaneously has good in vivo circulation stability and high biocompatibility, and reduces the toxic and side effects on normal tissues and organs.

Description

Active targeting multi-modal molecular imaging probe and preparation method and application thereof

Technical Field

The application relates to an active targeting multi-modal molecular imaging probe, a preparation method and application thereof, belonging to the field of biomedicine.

Background

Molecular imaging is a science that uses imaging means to display specific molecules at the tissue level, cell level and subcellular level, reflects the change of molecular level in the living body state, and qualitatively and quantitatively studies the biological behavior in the aspect of imaging. The molecular imaging technology is a new technology combining medical imaging technology with molecular biology, chemistry, physics, radiation medicine, nuclear medicine and computer science. It integrates genetic information, biochemistry and new imaging probes, detects by precise imaging technology, and then achieves the purpose of displaying the biological process of living tissue on molecular and cell level by a series of image post-processing technologies.

Cancer has now become the second leading cause of death after cardiovascular and cerebrovascular diseases, seriously threatening human health. Early diagnosis and treatment are one of the important means for diagnosing and treating cancer. At present, in the early diagnosis of cancer, molecular imaging is mainly relied on to scan the affected part of a patient. The common molecular imaging diagnosis comprises nuclide imaging (such as PET), but the method lacks targeting to the tumor, has insufficient measurement precision, has low concentration of nuclide diagnostic drugs which can be enriched at the tumor part, cannot be obviously imaged and observed, can be scanned when the side length of the tumor tissue exceeds 5mm, can be transferred to a plurality of malignant tumors at the moment, and causes fatal consequences, so that the effect of enriching the drugs by the high permeability and retention effect of the simple solid tumor is not obvious.

The targeting transportation is a mode of coupling targeting molecules on the surface of a nano carrier, specifically enriching imaging signal molecules at a tumor part through the specific combination of a ligand and a receptor and enhancing the imaging specificity. The targeted transportation is combined with the isotope imaging signal molecules, so that the isotope imaging signal molecules can be efficiently enriched at the tumor part, and the purpose of reducing the using dose of tumors with the same size or enhancing the enrichment of tumors with small volume so as to improve the imaging sensitivity is achieved. However, a single imaging mode is limited by the inherent limitations of imaging technical means during imaging, and the defect that the imaging cannot be improved is often existed, and due to the fact that the multi-modal probe can simultaneously perform multiple imaging, the signals obtained by different imaging modes are complementary, and the molecular imaging with higher precision and high accuracy can be realized.

Disclosure of Invention

According to one aspect of the application, an active targeting multi-modal molecular imaging probe is provided, the probe targets a neuropeptide Y receptor to over-express tumor cells and tissues, has the performance of multi-modal imaging, and can realize high specificity and high sensitivity imaging on tumors.

The active targeting multi-modal molecular imaging probe comprises an active targeting group, a nano-carrier and a multi-modal imaging signal molecule;

the active targeting group is coupled with the nano-carrier through the active targeting group;

the multimode imaging signal molecule is coupled with the outside of the nano carrier or carried in the nano carrier.

Alternatively, the active targeting group is derived from a ligand for the neuropeptide Y receptor;

the ligand for the neuropeptide Y receptor is a targeting ligand polypeptide.

Optionally, the targeting ligand polypeptide comprises a peptide chain selected from at least one of the following: NPY (28-36), [ Arg6, Pro34] pNPY, [ Phe6, Pro34] pNPY, [ Asn6, Pro34] pNPY, [ Cys6, Pro34] pNPY, [ Phe6, Pro34] pNPY, [ D-His 34, Pro34] NPY, [ Phe 34, Pro34] pNPY, [ Pro34, Nle 34, Bpa 34, Leu34] NPY (28-36), [ Pro34, Nal 34, Leu34] Y (28-36), [ Pro34, Nle 34, Nal 34, Leu34] NPY (28-36), [ D-Arg 34] -NP, [ D-34 ] -NPY, [ D-Arg 34, D-34 ] -NPY, [ Pro34] NPY, [ Pro34, Nal 34] Nap 34, Leu34] NPY (28-Pro 34), [ Pro-Ala 34], pNPY-34, pNPY (Pro-34, pNPY-P-34, pNPY-34, NPY-34, pNPY-34, NPY-P-34, NPY-36-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-34, NP, [ Pro30, Tyr32, Pro34] NPY (25-36), [ Pro30, Tyr32] NPY (25-36), [ Pro30, Trp32] NPY (25-36), [ Asn28, Pro30, Trp32] NPY (25-36), [ Pro30, Tyr31, Trp32] NPY (25-36), NPY (25-36).

Optionally, the nano-carrier is a polymer material, and the polymer material is connected with the active targeting group through an amide bond.

Optionally, the high molecular material is selected from at least one of polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL), polyethylene glycol-polyethyleneimine (PEG-PEI), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), distearoylphosphatidylethanolamine-polylactic acid (DSPE-PLA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), Distearoylphosphatidylethanolamine (DSPE), polyethylene glycol (PEG), polyethylene oxide-polypropylene oxide-polyethylene oxide (PEG-PPG-PEG).

Optionally, the multimode imaging signal molecule comprises three molecules of a nuclide signal molecule, a fluorescent signal molecule and a magnetic resonance imaging signal molecule.

Optionally, the nuclide signal molecule is a radionuclide-containing molecule¹¹C、⁶⁴Cu、⁶⁷Cu、¹²⁴I、¹²⁵I、¹³¹I、¹¹¹In、²¹³Bi、⁶⁸Ga、¹⁸F、⁹⁹mTc、⁹⁰Y、¹⁷⁷Lu、²¹¹At least one medical drug of At;

the fluorescence signal molecule is selected from any one of Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Propidium Iodide (PI), anthocyanin (CY5), chlorophyll protein (PerCP), Texas red (ECD) and heptamethine cyanine (IR 780);

the magnetic resonance imaging signal molecule is selected from any one of gadolinium-based substances, iron-based substances and manganese-based substances.

Preferably, the gadolinium-based substance is any one of gadodiamide, gadoxetic acid disodium, gadopentetate dimelamine, a mixture of gadodiamide and sodium carpodiamide, gadoterate dimelamine, gadoteridol, gadolinium trichloride and the like;

the iron-based substance being Fe₃O₄Superparamagnetic iron oxide particles, and the like;

the manganese-based substance is Mn-DPDP or Mn₃O₄And the like.

Alternatively, the internal mounting method includes any one of a chemical bonding method, a dialysis method, an emulsification method, and a solvent evaporation method.

Optionally, the mass of the active targeting group accounts for 0.1-60 wt% of the total mass of the nano-carrier;

the particle size of the nano-carrier is 5-200 nm;

preferably, the particle size of the nano-carrier is 10-150 nm;

the carrying rate of the multimode imaging signal molecules is 10-80 wt%;

preferably, the carrying rate of the multimode imaging signal molecules is 30-80 wt%;

further preferably, the carrying rate of the multimode imaging signal molecule is 50-80 wt%.

Specifically, the lower limit of the mass of the active targeting group in the total mass of the nano-carrier can be independently selected from 0.1 wt%, 1 wt%, 5 wt%, 7 wt% and 10 wt%; the upper limit of the mass of the active targeting group in the total mass of the nano-carrier can be independently selected from 20 wt%, 30 wt%, 40 wt%, 50 wt% and 60 wt%.

Specifically, the lower limit of the carrying rate of the multimode imaging signal molecules can be independently selected from 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%; the upper limit of the carrying rate of the multimode imaging signal molecule may be independently selected from 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%.

In another aspect of the present application, a method for preparing the active targeting multi-modal molecular imaging probe is provided, which at least includes the following steps:

and reacting the active targeting group source, the nano-carrier and the multimode imaging signal molecular source to obtain the active targeting multimode molecular imaging probe.

Optionally, the method comprises the steps of:

s100, reacting a high polymer material source with an active targeting group source I to obtain an active targeting group/nano-carrier compound;

s200, reacting the active targeting group/nano-carrier compound with a fluorescence signal molecule source II to obtain an active targeting group/fluorescence signal molecule/nano-carrier compound;

s300, reacting the active targeting group/fluorescent signal molecule/nano carrier compound with a nuclide signal molecule source and a magnetic resonance imaging signal molecule source III to obtain the active targeting multi-modal molecular imaging probe.

Optionally, in step S100, the molar ratio of the polymer material source to the active targeting group source is 1: 1-10: 1

Specifically, in the molar ratio of the polymer material source to the active targeting group source, the lower limit of the ratio of the polymer material source can be independently selected from 1, 2, 3, 4 and 5; the upper limit of the proportion of the source of polymeric material may be independently selected from 6, 7, 8, 9, 10.

Optionally, in step S200, the molar ratio of the active targeting group/nanocarrier complex to the source of fluorescent signal molecules is 1: 1-1: 10.

specifically, in the molar ratio of the active targeting group/nano-carrier compound to the fluorescent signal molecule source, the lower limit of the ratio of the fluorescent signal molecule source can be independently selected from 1, 2, 3, 4 and 5; the upper limit of the ratio of the fluorescent signal molecule source may be independently selected from 6, 7, 8, 9, 10.

Alternatively, in step S300,

the molar ratio of the nano-carrier containing the fluorescence signal molecules to the nuclide signal molecule source is 1: 100-1: 200.

specifically, in the molar ratio of the nano-carrier containing the fluorescent signal molecules to the nuclide signal molecule source, the lower limit of the ratio of the nuclide signal molecule source can be independently selected from 100, 110, 120, 130 and 140; the upper limit of the ratio of the nuclide signal molecular source may be independently selected from 150, 160, 170, 180, 200.

The molar ratio of the active targeting group/fluorescent signal molecule/nano-carrier compound to the magnetic resonance imaging signal molecule source is 1: 50-1: 100.

specifically, in the molar ratio of the active targeting group/fluorescent signal molecule/nano-carrier compound to the magnetic resonance imaging signal molecule source, the lower limit of the ratio of the magnetic resonance imaging signal molecule source can be independently selected from 50, 55, 60, 65 and 70; the upper limit of the ratio of the magnetic resonance imaging signal molecule source can be independently selected from 80, 85, 90, 95 and 100.

Specifically, the polymer material source refers to a polymer material containing at least one amino group at the end of the main chain, preferably the end of the main chain of the polymer material contains an amino group and a carboxyl group;

the fluorescence signal molecule source is any one of carboxylated Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Propidium Iodide (PI), anthocyanin (CY5), chlorophyll protein (PerCP), Texas red (ECD) and heptamethine cyanine (IR 780);

the nuclide signal molecule source is a solution of a radioactive isotope and a chelating agent;

the magnetic resonance imaging signal molecule source refers to the aqueous solution of various magnetic resonance contrast agents.

In another aspect of the present application, there is provided an active-targeting multi-modal molecular imaging probe of any one of the above mentioned items or an active-targeting multi-modal molecular imaging probe prepared by any one of the above mentioned methods, for use in preparing an imaging agent for diagnosing a disease, wherein the disease includes at least one of breast cancer, ovarian cancer, renal cancer, gastric cancer, and brain cancer.

The beneficial effects that this application can produce include:

1. the active targeting multi-modal molecular imaging probe provided by the application can simultaneously perform multiple imaging including fluorescence, isotope imaging and magnetic resonance imaging, and enhances the imaging effect through the complementary matching of multiple imaging.

2. The active targeting multi-modal molecular imaging probe provided by the application has good targeting property.

3. The active targeting multi-modal molecular imaging probe provided by the application has the characteristics of low dosage and high sensitivity.

4. The active targeting multi-modal molecular imaging probe provided by the application has good in vivo circulation stability and high biocompatibility, and reduces toxic and side effects on normal tissues and organs.

Drawings

Fig. 1 is a particle size distribution diagram of an active-targeting multi-modal molecular imaging probe according to example 1 of the present application;

fig. 2 is a fluorescence imaging diagram of the active-targeted multi-modal molecular imaging probe according to example 1 of the present application.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise specified, the raw materials and the like in the examples of the present application were purchased from commercial sources,

the particle size distribution of the probe in the application adopts a dynamic light scattering particle size analyzer (model number is Nano-ZS, Malvern, England);

the molecular weight of the probe in the application adopts a time-of-flight mass spectrometer (model LC-Q-TOF 4600, AB Sciex, USA);

fluorescence detection in small animals using a small animal fluorescence imager (type IVIS Lumina III, PerkinElmer, USA);

detecting radionuclide accumulation condition by using isotope imager (type is Discovery PET/CT 690Elite, GE, USA);

the intensity of the imaging signal was measured using a nuclear magnetic resonance imager (model Ingenia 1.5T, Philips, the Netherlands).

Example 1

NPY-PEG-FITC Loading⁶⁸Synthesis of probes (P1) for Ga-DOTATATE and gadopentetate meglumine

The synthesis method comprises the following steps:

(1) COOH-PEG-NH with a molar ratio of 1:1:1.5 at room temperature₂NHS and EDC, adding 1 mol ratio of targeting ligand polypeptide containing pNPY after 2 hours of activation, stirring at room temperature for 48 hours, dialyzing with dialysis bag with molecular weight of 3000 for 48 hours, freeze-drying and storing to obtain pNPY-PEG-NH₂。

(2) At room temperature, pNPY-PEG-NH with the molar ratio of 1:1:1.5₂NHS and EDC, stirring for 48 hours at room temperature, dialyzing for 48 hours by a dialysis bag with the molecular weight of 5000, and freeze-drying and storing to obtain pNPY-PEG-FITC.

(3) Taking 5 g of pNPY-PEG-FITC to dissolve in 2 ml of acetone,⁶⁸2 ml of Ga-DOTATATE solution (wherein⁶⁸Ga concentration is 100 mmol/l), 2 ml of gadopentetate dimeglumine solution (wherein the gadopentetate dimeglumine concentration is 50 mmol/l) are mixed uniformly.

(4) Dropwise adding the mixed solution obtained in the step (3) into 2 ml of deionized water, stirring for five minutes, and rotationally evaporating at 37 ℃ to remove acetone;

(5) and (5) passing the solution obtained in the step (4) through a 0.22-micron hydrophilic filter membrane to obtain a product, namely the active targeting multi-modal molecular imaging probe.

Analysis of the molecular weight of the product using a time-of-flight mass spectrometer confirmed that the resulting compound was the target compound.

The product was analyzed by using a dynamic light scattering particle size analyzer, and the result is shown in fig. 1, which shows that the particle size distribution of the active targeting multi-modal molecular imaging probe is concentrated near 100 nm, the distribution index is 0.178, and the particle size of the nanoprobe is uniform.

Using a small animal fluorescence imager, the results are shown in fig. 2, and a fluorescence signal at the tumor in the mouse can be observed.

With an isotope imager, the accumulation of gallium elements in breast cancer tumor cells can be observed.

Using a magnetic resonance imager, T1 weighted imaging signal enhancement can be observed.

Example 2

NPY-PLGA-FITC Loading⁶⁸Synthesis of probes for Ga-DOTATATE and gadoxetic acid disodium (P2)

The synthesis method comprises the following steps:

the synthesis procedure is as in example 1, except that in step (2) the solution of gadopentetate dimeglumine is replaced by a solution of gadoxetic acid disodium.

And analyzing the product by using a dynamic light scattering particle size analyzer to obtain the distribution index of the active targeting multi-modal molecular imaging probe, which is 0.162.

Using a small animal fluorescence imager, a fluorescent signal can be observed at the tumor in the mouse.

The accumulation of gallium elements in tumor cells can be observed using an isotope imager.

Example 3

NPY-DSPE-FITC Loading⁶⁸Synthesis of Ga-DOTATATE and gadolinium diamine Probe (P3)

The synthesis method comprises the following steps:

the synthesis procedure is the same as in example 1, except that the solution of gadopentetate dimeglumine in step (2) is replaced by a solution of gadodiamide.

And analyzing the product by using a dynamic light scattering particle size analyzer to obtain the distribution index of the active targeting multi-modal molecular imaging probe, which is 0.193.

Example 4 Synthesis of probes P4 to P315

In this example, compounds P4 to P315 were synthesized by the same procedure as in example 1; see table 1 for the remaining conditions.

TABLE 1

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. An active targeting multi-modal molecular imaging probe is characterized in that the active targeting multi-modal molecular imaging probe comprises an active targeting group, a nano-carrier and a multi-modal imaging signal molecule;

the active targeting group is coupled with the nano-carrier;

2. The active-targeting multi-modal molecular imaging probe of claim 1, wherein the active-targeting group is derived from a ligand of a neuropeptide Y receptor;

the ligand of the neuropeptide Y receptor is a targeting ligand polypeptide;

preferably, the targeting ligand polypeptide comprises a peptide chain selected from at least one of the following: NPY (28-36), [ Arg6, Pro34] pNPY, [ Phe6, Pro34] pNPY, [ Asn6, Pro34] pNPY, [ Cys6, Pro34] pNPY, [ Phe6, Pro34] pNPY, [ D-His 34, Pro34] NPY, [ Phe 34, Pro34] pNPY, [ Pro34, Nle 34, Bpa 34, Leu34] NPY (28-36), [ Pro34, Nal 34, Leu34] Y (28-36), [ Pro34, Nle 34, Nal 34, Leu34] NPY (28-36), [ D-Arg 34] -NP, [ D-34 ] -NPY, [ D-Arg 34, D-34 ] -NPY, [ Pro34] NPY, [ Pro34, Nal 34] Nap 34, Leu34] NPY (28-Pro 34), [ Pro-Ala 34], pNPY-34, pNPY (Pro-34, pNPY-P-34, pNPY-34, NPY-34, pNPY-34, NPY-P-34, NPY-36-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-P-34, NPY-34, NP, [ Pro30, Tyr32, Pro34] NPY (25-36), [ Pro30, Tyr32] NPY (25-36), [ Pro30, Trp32] NPY (25-36), [ Asn28, Pro30, Trp32] NPY (25-36), [ Pro30, Tyr31, Trp32] NPY (25-36), NPY (25-36).

3. The active targeting multi-modal molecular imaging probe of claim 1, wherein the nano-carrier is a polymer material, and the polymer material is connected with the active targeting group through an amide bond;

preferably, the polymer material is selected from at least one of polyethylene glycol-polylactic acid-glycolic acid copolymer, polyethylene glycol-polycaprolactone, polyethylene glycol-polyethyleneimine, polyethylene glycol-distearoylphosphatidylethanolamine, distearoylphosphatidylethanolamine-polylactic acid, polylactic acid-glycolic acid copolymer, distearoylphosphatidylethanolamine, polyethylene glycol, polyethylene oxide-polypropylene oxide-polyethylene oxide.

4. The active targeting multi-modal molecular imaging probe of claim 1, wherein the multi-modal imaging signal molecules comprise a nuclear species signal molecule, a fluorescent signal molecule, and a magnetic resonance imaging signal molecule;

preferably, the nuclide signal molecule comprises a radionuclide¹¹C、⁶⁴Cu、⁶⁷Cu、¹²⁴I、¹²⁵I、¹³¹I、¹¹¹In、²¹³Bi、⁶⁸Ga、¹⁸F、⁹⁹mTc、⁹⁰Y、¹⁷⁷Lu、²¹¹At least one medical drug of At;

preferably, the fluorescent signal molecule is selected from any one of fluorescein isothiocyanate, phycoerythrin, propidium iodide, anthocyanin, chlorophyll protein, texas red and heptamethine cyanine;

preferably, the magnetic resonance imaging signal molecule is selected from any one of gadolinium-based substances, iron-based substances and manganese-based substances.

5. The active-targeting multi-modal molecular imaging probe of claim 1, wherein the internal loading method comprises any one of chemical bonding, dialysis, emulsification, and solvent evaporation.

6. The active targeting multi-modal molecular imaging probe of claim 1, wherein the active targeting group accounts for 0.1-60 wt% of the total mass of the nanocarrier;

the particle size of the nano carrier is 5-200 nm;

the carrying rate of the multimode imaging signal molecules is 10-80 wt%.

7. The method for preparing an active targeting multi-modal molecular imaging probe of any one of claims 1 to 6, comprising at least the following steps:

8. The method for preparing according to claim 7, characterized in that it comprises the following steps:

s200, reacting the active targeting group/nano-carrier compound with a fluorescent signal molecule source II to obtain an active targeting group/fluorescent signal molecule/nano-carrier compound;

9. The preparation method according to claim 8, wherein in step S100, the molar ratio of the polymer material source to the active targeting group source is 1: 1-10: 1;

preferably, in step S200, the molar ratio of the active targeting group/nanocarrier complex to the source of fluorescent signaling molecules is 1: 1-1: 10;

preferably, in step S300, the step of,

the molar ratio of the active targeting group/nanocarrier complex to the radionuclide signal molecule source, on a molar basis of radionuclide, is 1: 100-1: 200 of a carrier;

10. use of the active-targeted multi-modal molecular imaging probe of any one of claims 1 to 6 or the active-targeted multi-modal molecular imaging probe prepared by the method of any one of claims 7 to 9 for the preparation of an imaging agent for diagnosing a disease comprising at least one of breast cancer, ovarian cancer, renal cancer, gastric cancer, brain cancer.