CN106729778B - Molecular image nanoparticle probe and preparation and application thereof - Google Patents

Abstract

The invention belongs to the field of nuclear medicine molecular imaging probes, and particularly relates to a molecular imaging nanoparticle probe and preparation and application thereof. The molecular imaging nano-particle probe is characterized in that the basic nano-particles are rare earth nano-particles. Compared with the known nuclear medicine molecular imaging probe, the molecular imaging nanoparticle probe has the size of less than 10nm, and is surface modified with a biological reaction active group or a molecular recognition group; the preparation method is simple, the separation and purification are convenient and quick, the F18 adsorption capacity is strong, the probe can be used as a radioactive probe for molecular image detection, diagnosis and tracing, the imaging sensitivity is high, and the specificity is strong.

Description

A molecular imaging nanoparticle probe and its preparation and application

technical field

The invention belongs to the field of nuclear medicine molecular imaging probes, in particular to a molecular imaging nanoparticle probe and its preparation and application.

Background technique

Molecular imaging technology has been more and more widely used in biomedical research and clinical diagnosis due to its sensitivity and non-invasiveness. Especially, positron emission tomography (PET) technology has high in vivo imaging sensitivity and has been widely used. in clinical diagnosis. The probes currently used in clinical PET imaging are mainly F18-labeled glucose analogs based on tissue/cell glucose metabolism: F18-FDG. However, F18-FDG has no molecular specificity and can only be used for functional imaging based on glucose metabolism, but not for the detection and tracking of specific molecular events such as proteins or drugs. At present, in the preparation of nuclide probes, the main method is to connect the positron nuclide F18 (half-life 109 minutes) to the probe molecule by covalent bond, which has many reaction steps, harsh conditions, long time, low yield, separation and purification. Difficulties and other problems severely limit the development and application of F18-labeled probes. Therefore, it is very important to develop F18-labeled molecular imaging probes with specificity, high sensitivity, easy labeling, high yield and easy separation.

SUMMARY OF THE INVENTION

In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide a molecular imaging nanoparticle probe and its preparation and application.

In order to achieve the above purpose and other related purposes, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a molecular imaging nanoparticle probe, the basic nanoparticles of which are rare earth nanoparticles.

Preferably, the rare earth nanoparticles are nanoparticles doped with one or more rare earth elements.

Preferably, the rare earth element is selected from lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc) or yttrium (Y).

Preferably, the rare earth nanoparticles are fluoride particles, oxide particles, composite oxide particles, hydroxide particles, sulfide particles, carbonate compound particles, phosphoric acid compound particles, doped with one or more rare earth elements. Titanic acid compound particles, boric acid compound particles, vanadic acid compound particles, tungstic acid compound particles, complex cationic compound or complex anion compound particles.

Preferably, the rare earth nanoparticles are fluoride particles doped with one or more rare earth elements.

Preferably, the rare earth nanoparticles are selected from REF ₃ , MREF ₄ or REOF, wherein RE refers to a trivalent rare earth element, and M is selected from the alkali metal elements Li, Na or K.

Further preferably, the rare earth nanoparticles are NaGdF ₄ nanoparticles.

Preferably, the surface of the basic nanoparticle is modified with a molecular recognition group, so that it can acquire the ability to actively target the object to be tracked. The molecular recognition group modified on the surface of the basic nanoparticle and the molecular recognition group modified on the surface of the object to be tracked form a molecular recognition pair.

Preferably, the molecular recognition pair is selected from the group consisting of antibody-antigen, ligand-receptor, protein-substrate, protein-inhibitor, protein/polypeptide-protein/polypeptide, aptamer-protein, complementary oligonucleotides Pairs, bioorthogonal reactive groups and clathrates/complexes.

In some embodiments of the present invention, the molecular recognition pair is tetrazine and trans-cyclooctene. That is, the surface of the basic nanoparticle is modified with the molecular recognition group tetrazine, thereby endowing the molecular imaging nanoparticle probe with the ability to actively target the target to be tracked modified with trans-cyclooctene.

Preferably, the molecular imaging nanoparticle probes are labeled with positron nuclides so that they can be used for PET imaging. In some embodiments of the present invention, the positron nuclide is listed as F18. That is, the positron nuclide F18 is labeled on the base nanoparticles to form a molecular imaging nanoparticle probe that can be used for PET imaging.

Preferably, the particle size range of the molecular imaging nanoparticle probe is 1-10 nm. In some embodiments of the present invention, the particle size of the molecular imaging nanoparticle probe is ≤4 nm.

A second aspect of the present invention provides a method for preparing the aforementioned molecular imaging nanoparticle probe, comprising the steps of:

(1) preparing basic particles; (2) modifying the basic nanoparticles obtained in step (1) with molecular recognition groups; (3) continuing to carry out positronization of the basic nanoparticles modified with molecular recognition groups obtained in step (2) nuclide labeling.

In some embodiments of the present invention, it is listed that the basic particles are rare earth nanoparticles-NaGdF4 nanoparticles, in step ( ₁ ), the basic particles _- NaGdF4 nanoparticles are prepared by a solvothermal method, which specifically includes steps: 1) Rare earth element gadolinium ion salt, oleic acid and octadecene were heated under vacuum to form a transparent solution. After cooling to room temperature, methanol solution containing NaOH and NH ₄ F was added, the reaction was stirred at room temperature, the methanol was removed by heating, washed with ethanol and centrifuged, Collect the precipitate and disperse it in cyclohexane to obtain oil phase rare earth NaGdF ₄ nanoparticles; 2) add the oil phase rare earth NaGdF ₄ nanoparticles obtained in step (1), add dichloromethane and trichloroperoxybenzoic acid, and stir to react , L-cysteine was added, the reaction was continued to be stirred, the obtained solution was centrifuged with ethanol to collect the precipitate, and the precipitate was washed and then vacuum-dried to obtain aqueous rare earth NaGdF ₄ nanoparticles.

In step 1), the rare earth element gadolinium ion salt can be the chloride of rare earth gadolinium, for example, gadolinium chloride hexahydrate: molecular formula is H12Cl3GdO6; molecular weight is 371.7936; CAS number is 13450-84-5; gadolinium chloride: molecular formula is Cl3Gd ; molecular weight is 263.61; CAS number is 10138-52-0.

Correspondingly, in step (2), the method of click chemistry (Click chemistry) is used to react the aqueous rare earth _NaGdF nanoparticles obtained in step (1) with the substance shown in formula I to carry out molecular recognition group-tetrazine modification;

Correspondingly, in the step (3), the F18 anion solution is added to the basic nanoparticle solution modified with the molecular recognition group obtained in the step (2), and incubated to obtain the positron nuclide F18 labeling.

The third aspect of the present invention provides the use of the aforementioned molecular imaging nanoparticle probe in molecular imaging detection, diagnosis and tracing in vitro and in vivo.

Specifically, the aforementioned molecular imaging nanoparticle probes can be used for biological detection, diagnosis and tracking, including detection, diagnosis and tracking of molecules, viruses, bacteria, cells and materials in vitro, and molecules, viruses, bacteria, cells, Organs and tissues, and molecules (including small molecular compounds, high molecular polymers and biological macromolecules such as polypeptides, proteins and nucleic acids, etc.), drug preparations, nanoparticles, biological materials, etc. Tracking, or using the aforementioned molecular imaging nanoparticle probe to label the above substances in vitro for in vivo detection, diagnosis and tracking.

In some embodiments of the present invention, the preferred biological assays are tumor diagnosis and imaging of drug and cell tissue distribution in animals, wherein the animals are live animals.

Preferably, the aforementioned molecular imaging nanoparticle probes can be used to prepare PET contrast agents.

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

Compared with the known nuclear medicine molecular imaging probes, the molecular imaging nanoparticle probe of the present invention has a size of less than 10 nm, and the surface is modified with biologically reactive groups or molecular recognition groups; the preparation method is simple, the separation and purification are convenient and rapid, and it has very good performance. The strong adsorption capacity of F18 can be used as a radioactive probe for molecular imaging detection, diagnosis and tracing, with high imaging sensitivity and specificity.

Description of drawings

Figure 1: TEM image of ultra-small rare earth nanoparticles NaGdF4 in oil phase.

Figure 2: TEM image of ultra-small rare earth nanoparticles NaGdF4 in aqueous phase.

Figure 3: Characterization of Tz modification of NaGdF4.

Figure 4: Stability determination of NaGdF4-Tz adsorbed F18 in different solutions.

Figure 5: Distribution and metabolism of F18-adsorbed NaGdF4-Tz nanoparticles in mice.

Figure 6: PET tracing and tumor pretargeting imaging of Tz-NaGdF4 nanoparticles for tumor pretargeting distribution of TCO-SNPs.

Figure 7: Hydrogen spectrum of Ad-PEG.

Figure 8: Hydrogen spectrum of 6-ots-β-CD.

Figure 9: Hydrogen spectrum of CD-PEI.

Detailed ways

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention. In the following examples, the test methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by various manufacturers.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques are well described in the existing literature, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolfe, eds. .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999 et al.

Example 1 Preparation of oily and aqueous ultra _- small NaGdF4 nanoparticles

1 mmol of gadolinium chloride hexahydrate was heated to 140 °C under vacuum with 4 ml of oleic acid and 15 ml of octadecene until a clear solution was formed, and after cooling to room temperature, methanol containing 2.5 mmol of sodium hydroxide and 4 mmol of NH ₄ F was added dropwise. The solution was 10 mL, stirred overnight for 15 h, the mixture was slowly heated to 70 °C, methanol was removed, cooled to room temperature, centrifuged twice with ethanol at 4500 rpm, and finally dispersed in 5 mL of cyclohexane. The TEM image of the obtained oil phase rare earth NaGdF4 nanoparticles is shown in Fig. ₁ .

20mg of oil phase rare earth NaGdF4 nanoparticles _were dispersed in 4ml of cyclohexane, 2ml of dichloromethane and 5mg of 3-chloroperoxybenzoic acid were added, mixed and stirred at 40°C for 3h, after cooling to room temperature, 0.02g of L-cysteine was added. The mixed solution was stirred at room temperature for 5 hours, and the aqueous phase rare earth nanoparticles were collected by washing with ethanol for several times and vacuum drying. The TEM image of the obtained aqueous rare earth NaGdF4 nanoparticles is shown in Fig. ₂ .

Example 2 _Tetrazine (Tz) functional modification of ultra-small NaGdF4 nanoparticles

The surface of the cysteine-modified nanoparticles has amino groups. _The aqueous NaGdF4 nanoparticles are dispersed in mes buffer with pH=7.4, and a certain amount of DMF solution in which 2 mg of Tetrazine-PEG5-NHS Ester is dissolved is added to react. 3h, dialyzed for three days to remove unreacted Tetrazine-PEG5-NHS Ester, and collected by lyophilization. Some of the freeze-dried samples were dissolved in DMSO for NMR characterization. The NMR results are shown in Figure 3. There are both Tz-PEG ₅ -NHS peaks and nanoparticle peaks in the NMR peaks, indicating that Tz has been successfully attached to the nanoparticles.

In this example, Tetrazine-PEG5-NHS Ester: the molecular formula is C27H36N9O10; the molecular weight is 604.41, and the structural formula is:

Example 3 F18 labeling and stability testing of ultra-small Tz- _NaGdF nanoparticles

Dissolve Tz-NaGdF ₄ in physiological saline, add positron nuclide F18 ^- aqueous solution, incubate at room temperature for 10 min, and remove free F18 ^- by dialysis. After placing the F18-adsorbed nanoparticles in pure water, normal saline, PBS, and serum for a period of time, the radiation dose of the solution in the tube was measured, and then dialyzed to determine the efficiency of F18 adsorption by the nanoparticles in the solution. Observe whether the adsorption efficiency changes after a period of time. The results are shown in Figure 4. The nanoparticle adsorption of F18 in pure water, normal saline, and serum can be basically stable within 4 hours.

Example 4F18 _- labeled ultra-small Tz-NaGdF4 nanoparticles in vivo distribution and clearing PET imaging

We injected the F18-labeled nanoparticles in Example 3 into mice through the tail vein before PET scanning, and scanned at 0 h, 2 h, and 4 h respectively. The scanning results are shown in Figure 5 . Observing the clearance of Tz-NaGdF ₄ nanoparticles in the organs of mice, we can conclude from Figure 5 that the nanoparticles in the organs can be basically cleared after 2 hours of blood circulation of Tz-NaGdF ₄ nanoparticles.

Example 5F18 _- labeled ultra-small Tz-NaGdF4 nanoparticles for tumor pretargeting distribution of TCO-modified self-assembled drug carriers (TCO-SNPs) PET tracking imaging and tumor pretargeting imaging

Preparation of adamantane-modified polyethylene glycol Ad-PEG: Weigh 47 mg of adamantane hydrochloride with a molecular weight of 187.74 g/mol and dissolve it in 10 ml of CH ₂ Cl ₂ , add a stirring bar, and stir at an appropriate rotation speed to dissolve 1-Ad in dichloromethane. Subsequently, 26 mg of triethylamine and 50 mg of mPEG-NHS were added successively, stirred at room temperature for 2 hours, removed dichloromethane by a rotary evaporator, and dissolved in water. The liquid was placed in a dialysis bag with a molecular weight of 3500 for dialysis for three days, and placed in a freeze dryer. Lyophilized overnight.

The structure of the product was characterized, and the characterization data are shown in Figure 7:

Characterization data are as follows: ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 3.42-3.54 (440H, OCH ₂ ), 1.13-1.18 (15H, protons on Ad).

Preparation of 6-O-(p-toluenesulfonic acid)-β-cyclodextrin: 20g β-CD (17.6mmol) was added to 400mL 0.4mol/L NaOH solution in a 500ml three-necked flask, and it was completely dissolved and violently Stir until colorless (pre-ice water bath 0-5°C). Under nitrogen protection, slowly add 15 ml of acetonitrile solution dissolved in 6.723 g of p-toluenesulfonyl chloride (35.2 mmol) with a constant pressure funnel within 90.0 min, and then react in an ice-water bath for 5 h in the reactor, and discard the unreacted TsCl by suction filtration. , take the filtrate. Acidify with 1 mol/L HCl to pH 2-3 to form a white solid. The filtrate was allowed to stand at 4 degrees Celsius for 12 h overnight, filtered with suction, the white solid was recrystallized twice with water, and dried in vacuo to obtain a white powder.

The structure of the product was characterized, and the characterization data are shown in Figure 8:

The structural characterization data of the product are as follows: ¹ H NMR (400MHz, DMSO), δ 3.18-3.78 (C-2, -3, -4, -5); 4.79 (C-1); 5.74 (C-2, -3OH).

Preparation of β-cyclodextrin-modified polyethyleneimine CD-PEI: 100mg PEI (molecular weight 10KD) was dissolved in 100ml DMSO, and 6-OTs-β-CD prepared in Example 4 was added to the solution, The reaction was carried out at 70°C for 3 days, dialyzed with a dialysis bag with a molecular weight of 10KD for 6 days, filtered to remove unreacted 6-OTs-β-CD, and the samples were collected by lyophilization.

The structure of the product was characterized, and the characterization data are shown in Figure 9:

The structural characterization data for the product are as follows: ¹ H NMR (400 MHz, DMSO) δ 4.92 (C ₁ H on cyclodextrin), 3.27-3.66 (C _2-6 H on cyclodextrin), 2.3-3.0 (on PEI) OCH2 ₎ .

Preparation of adamantane-modified PAMAM Ad-PAMAM: The PAMAM used in this example is a first-generation polyamide dendrimer with a 1,4-diaminobutane core and amine ends. The PAMAM is available from Dendritic Nanotechnologies, Inc (Mount pleasant, MI).

A solution of PAMAM (20% wt, 100 mg, 0.069 mmol) in methanol was added to the round bottom flask. The methanol was evaporated in vacuo and the viscous solid was redissolved in 10 ml of dry THF. 1-adamantane isocyanate (244.6 mg, 1.38 mmol) in 10 ml of THF was added directly to the PAMAM solution. After the reaction mixture was stirred at room temperature for 2 hours, the solvent was removed in vacuo. Diethyl ether (100 ml) was added to the reaction residue to produce a white precipitate, which was collected by filtration. The white precipitate (100 ml×3) was washed with ether and dried to obtain Ad-PAMAM as a white solid. According to characterization data analysis, there are 8 Ads attached to each PAMAM.

We attached the TCO component to the long chain of adamantane-modified PEG (Ad-PEG), the same as cyclodextrin-modified PEI (CD-PEI), adamantane-modified PAMAM (Ad-PAMAM) through the interaction between Ad and CD TCO-SNPs are self-assembled by the inclusion complexation between them to form nanoparticle TCO-SNPs with a particle size of about 150 nm. The specific method includes steps: Ad-PEG-TCO (10mg/ml): 52.8ul; Ad-PAMAM: (2.1mg/ml) 18.86ul; /ml): 15ul vortex for 10s and let stand for 20min.

After injection into mice via tail vein, the TCO-SNPs could accumulate at tumor sites through the tumor EPR effect. 24 hours later, F18-labeled ultra-small Tz-NaGdF ₄ nanoparticles were injected through the tail vein, and PET scanning was performed 2 hours later. The white dotted circle in the figure is the subcutaneous tumor location. The obtained PET image is shown in Fig. 6, the liver, lung, spleen and other organs basically have no radioactive signal, while the tumor shows a very high radioactive signal. The results showed that the ultra-small rare earth nanoparticles bound to the TCO components remained at the tumor site, while the unbound nanoparticles were rapidly excreted, enabling in vivo tracking of TCO-SNPs and high tumor/tissue contrast imaging. Our imaging method is based on the tumor EPR effect. Currently, 18F-FDG contrast agent is mostly used in clinical practice, which is based on the high glucose metabolism of tumors. In contrast, our probe is universal and eliminates the possibility of false positives.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form or substance. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, the Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those skilled in the art, without departing from the spirit and scope of the present invention, can utilize the above-disclosed technical content to make some changes, modifications and equivalent changes of evolution, all belong to the present invention. Equivalent embodiments; at the same time, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solutions of the present invention.

Claims (10)

1. A method for preparing a molecular imaging nanoparticle probe, wherein the basic nanoparticle is a rare earth nanoparticle, the molecular imaging nanoparticle probe is labeled with a positron nuclide, and the surface of the basic nanoparticle is modified with a molecular recognition group , so that it can obtain the ability to actively target the object to be tracked; the molecular recognition group modified on the surface of the basic nanoparticle and the molecular recognition group modified on the surface of the object to be tracked form a molecular recognition pair, and the molecular recognition pair is four oxazine and trans-cyclooctene, the method comprises the steps: (1) Preparation of basic particles; (2) modifying the basic nanoparticles obtained in step (1) with molecular recognition groups; (3) Continue to carry out positron nuclide labeling on the basic nanoparticles modified with molecular recognition groups obtained in step (2). 2 . The method for preparing a molecular imaging nanoparticle probe according to claim 1 , wherein the rare earth nanoparticles are nanoparticles doped with one or more rare earth elements. 3 . 3 . The method for preparing a molecular imaging nanoparticle probe according to claim 2 , wherein the rare earth nanoparticles are fluoride particles, oxide particles, composite oxide particles doped with one or more rare earth elements. 4 . particles, hydroxide particles, sulfide particles, carbonate compound particles, phosphoric acid compound particles, titanic acid compound particles, boric acid compound particles, vanadic acid compound particles, tungstic acid compound particles, complex cationic compound or complex anionic compound particles. 4 . The method for preparing a molecular imaging nanoparticle probe according to claim 2 , wherein the rare earth nanoparticles are fluoride particles doped with one or more rare earth elements. 5 . 5. The method for preparing a molecular imaging nanoparticle probe according to claim 2, wherein the rare earth nanoparticles are selected from REF ₃ , MREF ₄ or REOF, wherein RE refers to a trivalent rare earth element, and M is selected from From the alkali metal elements Li, Na or K. 6 . The method for preparing a molecular imaging nanoparticle probe according to claim 2 , wherein the rare earth nanoparticles are NaGdF4 nanoparticles. ₇ . 7 . The method for preparing a molecular imaging nanoparticle probe according to claim 1 , wherein the particle size range of the molecular imaging nanoparticle probe is 1-10 nm. 8 . 8. The method according to claim 1, wherein when the basic particles are rare earth nanoparticles _- NaGdF4 nanoparticles, in step ( ₁ ), the basic particles NaGdF4 nanoparticles are prepared by a solvothermal method, specifically Including steps: 1) heating rare earth element gadolinium ion salt, oleic acid and octadecene under vacuum to form a transparent solution, after cooling to room temperature, adding methanol solution containing NaOH and NH ₄ F, stirring and reacting at room temperature, heating to remove methanol , wash and centrifuge with ethanol, collect the precipitate, and disperse it in cyclohexane to obtain oil phase rare earth NaGdF ₄ nanoparticles; 2) add the oil phase rare earth NaGdF ₄ nanoparticles obtained in step (1), add dichloromethane and triclosan oxybenzoic acid, stirring the reaction, adding L-cysteine, continuing the stirring reaction, using the obtained solution to collect the precipitate with ethanol centrifugation, washing the precipitate and vacuum drying to obtain the aqueous phase rare earth NaGdF ₄ nanoparticles. 9. method according to claim 8, is characterized in that, adopts the method of click chemistry to react the obtained water phase rare earth NaGdF of step ( ₁ ) nano-particles and the substance shown in formula I, carry out molecular recognition group-tetrazine retouch. 10. The method according to claim 8, wherein in step (3), the F18 anion solution is added to the basic nanoparticle solution modified with molecular recognition groups obtained in step (2), and incubated to obtain a positive Electron nuclide F18 labeling.

Images