CN104162175B - Functionalized dendrimer-based SPECT-CT bimodal imaging contrast agent and preparation method thereof - Google Patents

Abstract

The invention relates to a functionalized dendrimer-based SPECT-CT dual-mode imaging contrast agent and a preparation method thereof, using pegylated fifth-generation polyamide-amine dendrimer as a polymer carrier materials, by covalently grafting diethylenetriaminepentaacetic acid on the surface of dendrimers, wrapping gold nanoparticles by in-situ synthesis, and acetylating or hydroxylating the remaining amino groups on the surface to mark ^99m Tc, namely have to. The invention has good SPECT/CT imaging effect, realizes SPECT/CT imaging of different tissue parts in mice, lays a good foundation for the development of multifunctional tissue-specific contrast agents, and has broad application prospects.

Description

Functionalized dendrimer-based SPECT-CT dual-modality imaging contrast agent and its Preparation

technical field

The invention belongs to the field of contrast agents, in particular to a functional dendrimer-based SPECT-CT dual-mode imaging contrast agent and a preparation method thereof.

Background technique

Nuclear medicine imaging uses radionuclide-labeled chemical substances as imaging agents or radiopharmaceuticals to be introduced into living organisms to display the physiological and biochemical processes in the body. By receiving the radiation emitted by radionuclides by nuclear medicine SPECT or PET imaging equipment, it is possible to clearly observe the functional information of the contrast agent participating in various physiological, biochemical and even metabolic aspects of the organism, and obtain qualitative, quantitative and localized clinical disease diagnosis and treatment results. The most prominent advantage of radionuclide imaging in nuclear medicine is its functional imaging. Due to its unique functional imaging characteristics, compared with morphological imaging methods (X-ray radiography, B-ultrasound, CT, MRI), it can reflect visceral Changes in blood flow in organs or tissues, changes in receptor density and activity, and changes in metabolism and function have become one of the most advanced technologies for clinical diagnosis and treatment of diseases. However, nuclear medicine imaging has the disadvantages of low spatial resolution and the inability to clearly locate the anatomical position of the image. With the continuous advancement of technology, the appearance of PET/CT and SPECT/CT successfully solved this problem. Through SPECT/CT examination, the functional metabolic information of SPECT and the anatomical diagnostic information of CT can be obtained at the same time in the body, and the image fusion and diagnostic efficiency double (Wang Rongfu, Li Xianfeng, Wang Qiang. The latest application progress of SPECT/CT[J]. CT theory and Applied Research, 2012,21(3):577-582.). Therefore, compared with SPECT or CT alone, SPECT/CT has more clinical significance in diagnosing diseases and evaluating prognosis.

Although SPECT/CT dual-modal imaging equipment has been greatly promoted clinically, the research and development of corresponding SPECT/CT dual-modal imaging contrast agents has not received enough attention. Although a variety of CT or SPECT imaging contrast agents can complete a variety of corresponding contrast imaging, the use of two contrast agents not only causes inconvenience to clinical operations, but also increases the toxic and side effects of contrast agents on patients. It is difficult to coordinate between them, and there are differences in the body. Therefore, a multifunctional contrast agent system that can complete SPECT/CT dual-mode imaging contrast diagnosis will greatly improve the sensitivity and accuracy of diagnosis, and reduce the pain and potential side effects caused to patients. will have great application value.

Polyamidoamine (PAMAM) dendrimers are macromolecules with a highly branched structure, which are formed through repeated reactions of branched unit structures. PAMAM is highly monodisperse and highly branched, and possesses a controllable and regular structure. It not only has numerous modifiable amino groups on the surface, but also has a unique cavity structure inside. Because PAMAM can be used as a multifunctional carrier material, modify functional groups on the surface, and wrap drugs or inorganic nanoparticles with internal cavities to achieve multifunctional integration and form multifunctional nanoprobes to meet the requirements of clinical applications. For example (Wen S, LiK, Cai H, et al.Multifunctional dendrimer-entrapped gold nanoparticles for dualmode CT/MR imaging applications[J].Biomaterials,2013,34(5):1570-1580.) using modified gadolinium ion chelate The fifth-generation PAMAM dendrimers of the mixture prepared gold nanoparticles by in situ reduction of templates, and chelated gadolinium ions to prepare CT/MR dual-modal imaging contrast agents, which obtained better dual-modal imaging in vivo and in vitro Effect. Therefore, the use of dendrimers as carriers to prepare SPECT/CT dual-modal contrast agents will meet the needs of clinical applications. At the same time, according to the modifiability of dendrimer surfaces, different surface modifications can be performed to achieve different in vivo tissue and organ imaging. become possible.

A search of domestic and foreign literature and patents on SPECT/CT dual-modal contrast agents shows that: at present, no preparation and application of SPECT/CT contrast agents based on dendrimer-encapsulated gold nanoparticles chelating ^99m Tc have been found. reports.

Contents of the invention

The technical problem to be solved by the present invention is to provide a functionalized dendrimer-based SPECT-CT dual-mode imaging contrast agent and its preparation method. The contrast agent has good SPECT/CT imaging effect and is a multifunctional contrast agent. A good foundation has been laid for the development of agents.

A functionalized dendrimer-based SPECT-CT dual-mode imaging contrast agent of the present invention uses pegylated fifth-generation polyamide-amine dendrimers as polymer carrier materials, through co- Valence grafting connects diethylenetriaminepentaacetic acid to the surface of dendrimers, wraps gold nanoparticles by in-situ synthesis, acetylates or hydroxylates the remaining amino groups on the surface, and marks ^99m Tc to obtain the product.

A preparation method of a functionalized dendrimer-based SPECT-CT dual-mode imaging contrast agent of the present invention, comprising:

(1) Dissolve G5.NH ₂ (purchased from Dendritech) in water, and add an aqueous solution of diethylenetriaminepentaacetic acid cyclic anhydride cDTPAA (purchased from Sigma Aldrich), stir and react at room temperature for 8-12h to obtain G5-DTPA , then add mPEG-COOH activated by EDC (purchased from Sigma Aldrich) (purchased from Shanghai Yanyi Biotechnology Co., Ltd.), and stir for 1-3d to obtain a functionalized dendrimer G5-DTPA-mPEG solution;

(2) Add chloroauric acid solution to the G5-DTPA-mPEG solution and stir for 20-40min, then add sodium borohydride solution, and stir for 1-4h;

(3) Acetylation: add triethylamine and stir for 20-40min, then add acetic anhydride, stir for 12-24h;

Or hydroxylation: add glycidol, stir and react for 12-24h; dialyze the reaction solution, and finally freeze-dry the aqueous solution of the product to obtain Au-Ac DENPs or Au-Gly DENPs;

(4) Dissolve the above-mentioned Au-Ac DENPs or Au-Gly DENPs in phosphate buffer, add SnCl ₂ , then add sterile radioactive persphenate solution and mix, and separate by column chromatography to obtain the chelated nucleus ^99m Tc-Au-Ac DENPs or ^99m Tc-Au-Gly DENPs of functionalized dendrimers wrapped gold nanoparticles by prime ^99m Tc.

In the step (1), the molar ratio of G5.NH ₂ and cDTPAA is 1:5-10; the molar ratio of mPEG-COOH and EDC is 1:8-15, and the activation time is 2-4 hours; G5-DTPA and The molar ratio of mPEG-COOH is 1:15-25.

The molecular weight of mPEG-COOH in the step (1) is 5000.

In the step (1)-(4), the reaction can also be carried out in PBS buffer solution, dimethyl sulfoxide, dimethylformamide, polar solvents such as dimethylacetamide except that it can be carried out in pure water reaction.

In the step (2), the molar ratio of G5-DTPA-mPEG to chloroauric acid is 1:150-250; the molar ratio of G5-DTPA-mPEG to sodium borohydride is 1:750-1250.

In the step (3), the molar ratio of G5-DTPA-mPEG and triethylamine is 1:400-800; the molar ratio of G5-DTPA-mPEG and acetic anhydride is 1:350-750; G5-DTPA-mPEG and The molar ratio of glycidol is 1:500-1000.

The specific process of the dialysis in the step (3) is: use a dialysis bag to dialyze in a PBS buffer solution with a pH of 7.4, and then dialyze in distilled water. The dialysis bag is a cellulose dialysis membrane with a molecular weight cut-off of 8000-14000.

Au-Ac DENPs and _SnCl in the described step (4) The mass ratio is 1:0.1-0.5; The ratio of Au-Ac DENPs and radioactive persphenate is 1mg: 1850-3700mBq; Au-Gly DENPs and SnCl The mass ratio of ₂ is 1:0.1-0.5; the ratio of Au-Gly DENPs and radioactive permethanate is 1mg:1850-3700mBq.

The pH of the phosphate buffer in the step (4) is 7.2-7.4.

The specific process of column chromatography separation in the step (4) is: using PD-10 desalting chromatographic column to purify the target product to remove free ^99m Tc and unreacted substances.

In the present invention, the ^99m Tc chelating agent cDTPAA is grafted onto the surface of the fifth-generation polyamide-amine dendrimer whose terminal is an amino group, and cDTPAA is added dropwise to the dendrimer solution under rapid stirring, To ensure the uniformity of dendrimer grafted DTPA.

In the present invention, 5-20 times the amount of EDC is used to activate the carboxyl group of mPEG-COOH to enhance the activity of reacting with the dendrimer, and the activated mPEG-COOH is also added dropwise to the dendrimer solution to Ensure the uniformity of dendrimer grafted PEG. At the same time, the addition of PEG can increase the circulation time of the material in the body, and increase the amount of subsequent wrapped nano-gold to achieve a more sensitive CT contrast effect.

In the present invention, after the chloroauric acid solution is added, stir for 20-40min to fully mix the chloroauric acid molecules with the dendrimer, and utilize the affinity of AuCl ₄ ^- and the amino group of the dendrimer to make it enter the inner cavity of the dendrimer , and then rapidly reduced by a strong reducing agent NaBH ₄ to obtain gold nanoparticles wrapped in dendrimers, which well prevents the aggregation of gold nanoparticles.

In the present invention, acetylation and hydroxylation are used to modify the remaining amino groups on the surface of the dendrimer to reduce its surface potential, improve the biocompatibility of the material, and endow it with different surface groups to achieve different functions in the body. Tissue imaging performance.

Use ¹ H NMR (Nuclear Magnetic Resonance Popper), UV-Vis (Ultraviolet Visible Spectroscopy), TEM (Transmission Electron Microscopy), MTT Test (Cell Viability Analysis), and in vivo SPECT/CT imaging to characterize the dual-modality obtained by the present invention Dendrimers with imaging function, the results are as follows:

(1) ¹ H NMR test results

¹ H NMR test results show that the SPECT/CT dual-modal contrast agent particles Au-AcDENPs and Au-Gly DENPs prepared in the present invention both have the characteristic absorption peak of PEG at 3.5 ppm, see FIG. 2 . At the same time, the characteristic peak of G5 appears between 2.1-3.3ppm, and the characteristic absorption of acetyl group appears at 1.8ppm after acetylation modification.

(2) UV-Vis test results

The UV-Vis test results show that the surface plasmon resonance (SPR) peaks of the SPECT/CT dual-modal contrast agent particles Au-AcDENPs and Au-Gly DENPs prepared in the present invention are located at 530 and 510 nm, respectively, see FIG. 3 . This indicates that gold nanoparticles are prepared in the present invention, and different surface modifications have different SPR peak positions.

(3) TEM test results

The TEM test results show the size and size distribution of the two gold nanoparticles, see Figure 4. The two gold nanoparticles are similar in size, with average diameters of 3.3nm and 3.2nm, respectively, and their size distribution is narrow, with good dispersion.

(4) MTT test results

SK-OV-3 cells were used to study the cytocompatibility of Au-Ac DENPs and Au-Gly DENPs. After Au-Ac DENPs and Au-Gly DENPs with different Au concentrations (0, 2.5, 5, 10, 20, 40, 80 μM) were co-cultured with KB cells for 24 hours, the viability of the cells was detected by the MTT method, see Figure 5. It can be seen from the figure that compared with the control SK-OV-3 cells treated with PBS, Au-AcDENPs and Au-GlyDENPs complexes still have no effect on cell growth when the Au concentration is as high as 80 μM, showing good Cytocompatibility.

(5) micro-SPECT/CT imaging of ^99m Tc-Au-Ac DENPs mice

Inject 200 μL of ^99m Tc-Au-Ac DENPs ([ ^99m Tc]=370MBq/mL, [Au]=0.08M) tail vein into a mouse weighing 22g, and scan and detect it with a micro-SPECT/CT small animal imager The obtained SPECT/CT pictures (Fig. 6, Fig. 8). It can be seen from the figure that the acetylated surface-treated ^99m Tc-Au-Ac DENPs material can realize specific imaging of the lungs of mice, and its brightness is significantly higher than that of other major organs such as the liver, and the liver, kidney, The signal of spleen and bladder increased, and the imaging time could last at least 2 hours, and the metabolic signal gradually weakened (Fig. 6). It is proved that the ^99m Tc-Au-Ac DENPs synthesized by this method has better SPECT/CT dual-modal imaging effect.

(6) In vivo micro-CT imaging of ^99m Tc-Au-Gly DENPs mice

Inject 200 μL of ^99m Tc-Au-Gly DENPs ([ ^99m Tc]=370MBq/mL, [Au]=0.08M) tail vein into mice weighing 22g, and scan and detect them by micro-SPECT/CT small animal imager CT images of mice were obtained (Fig. 7, Fig. 9). The ^99m Tc-Au-Gly DENPs material with hydroxylation surface treatment can stay in the blood for a long time, and complete blood pool imaging such as the heart, kidney, abdominal aorta, etc., as time goes on, the material can be metabolized into the spleen, bladder and Liver (Fig. 7). It is proved that the ^99m Tc-Au-Gly DENPs synthesized by this method has better SPECT/CT dual-modal imaging effect. The in vivo imaging results of the two materials show that the synthesized contrast agents with different surface modifications have significantly different metabolic distribution behaviors in vivo, and can perform different tissue-organ-specific imaging functions.

The functionalized dendritic macromolecular material prepared by the invention, in which the gold nanoparticle is wrapped with chelate, has a narrow size distribution of the gold nanoparticle and good dispersibility. It has good cytocompatibility in vitro, and in vivo experiments also show its good SPECT/CT dual-modality imaging performance.

Using polyamide-amine dendrimers with a large number of amino groups on the surface, a large number of cavities inside and a precise and controllable structure as a template and stabilizer, a functional dendrimer-based SPECT/CT dual-modal imaging contrast was prepared. agent, the present invention has involved five basic principles:

(1) Make full use of a large number of modifiable amino groups on the surface of polyamide-amine dendrimers, graft ^99m Tc chelating agent DTPA and mPEG-COOH, not only modify the SPECT contrast agent ^99m Tc chelating agent DTPA, but also improve the Blood circulation time and biocompatibility of contrast media.

(2) Make full use of the internal cavity of polyamide-amine dendrimers to wrap stable gold nanoparticles. _The rapid reduction of metal ions by the strong reducing agent NaBH4 produces stable gold nanoparticles inside the dendrimers.

(3) Gold nanoparticles have good attenuation ability to X-rays. In vivo CT imaging tests in mice demonstrate that the dendrimer-coated gold nanoparticle contrast agent can be used as a promising CT contrast agent.

(4) ^99m Tc has a good contrast effect on SPECT imaging. The SPECT test results show that the material has better SPECT imaging contrast. It shows that the dendrimer- ^99m Tc imaging contrast agent can be used as a promising SPECT contrast agent.

(5) Use a large number of modifiable amino groups on the surface of polyamide-amine dendrimers for different surface modification and functionalization to achieve different in vivo imaging characteristics and complete different in vivo imaging functions to facilitate clinical specific tissue and organ inspection .

The key factor for preparing SPECT/CT dual-mode contrast agent is to find a suitable carrier platform to load different contrast elements to form a multifunctional contrast agent complex. PAMAM is a kind of macromolecule with highly branched structure, which is a dendritic macromolecule formed by stepwise and repeated reactions of branched unit structures. It is highly monodisperse and highly branched with a controlled and regular structure. It not only has numerous modifiable amino groups on the surface, but also has a unique cavity structure inside. Because PAMAM can be used as a multifunctional carrier material, modify functional groups on the surface, and wrap drugs or inorganic nanoparticles with internal cavities, realize multifunctional integration, form multifunctional nanoprobes, and meet the requirements of clinical applications. Taking advantage of the excellent monodispersity of dendrimers, the modifiability of a large number of functional groups on the surface, the drug loading of the internal cavity structure and the good biocompatibility after modification, the dendrimers can be used to Modification, assembly and biological functionalization of inorganic nanoparticles, as well as the grafting of various small molecule contrast agents, prepare excellent multifunctional molecular imaging contrast agents for SPECT/CT dual-modal imaging, in order to expect to be applied to the diagnosis and treatment of diseases. Early diagnosis.

The present invention uses the specific structure and properties of dendrimers to graft ^99m Tc chelating agent DTPA on the surface of dendrimers to chelate ^99m Tc to realize SPECT imaging, and then graft EDC-activated mPEG-COOH on dendrimers. Molecular surface to increase its blood circulation time and improve biocompatibility, and finally wrap nano gold particles inside the dendrimer for CT imaging, and acetylate or hydroxylate the remaining amino groups on the surface of the dendrimer to reduce the surface positive charge , in order to reduce its toxicity, and prepare an excellent SPECT/CT dual-modal contrast agent to meet different imaging requirements.

Beneficial effect

(1) The SPECT/CT dual-mode imaging contrast agent of the present invention has a good SPECT/CT imaging effect, laying a good foundation for the development of multifunctional contrast agents;

(2) The preparation process of the present invention is simple, the experimental conditions are normal temperature and pressure, easy to operate, the preparation procedures adopted can be used to prepare other functional dendrimers, and SPECT and CT contrast agents are used for SPECT/CT dual Modal imaging, with good use value;

(3) The dual-modal contrast agent with different surface groups prepared by the present invention has different in vivo characteristics, can complete different tissue and organ imaging, has good clinical application value, and lays a solid foundation for different surface modifications to achieve different tissue and organ imaging a good foundation;

(4) The preparation process adopted in the present invention can be used to prepare targeted SPECT and CT contrast agents for SPECT/CT targeted dual-modal imaging, and has good use value.

Description of drawings

Fig. 1 is a schematic diagram of the reaction equation of the present invention.

Fig. 2 is the ¹ H NMR spectrum of Au-Ac DENPs (a) and Au-Gly DENPs (b) prepared in the present invention.

Fig. 3 is the ultraviolet absorption spectrum of Au-Ac DENPs (a) and Au-Gly DENPs (b) prepared in the present invention.

Fig. 4 is the TEM pictures (a, c) and particle size distribution histograms (b, d) of Au-Ac DENPs (a, b) and Au-Gly DENPs (c, d) prepared in the present invention.

Figure 5 is the MTT analysis of the viability of SK-OV-3 cells treated with Au-Ac DENPs and Au-Gly DENPs prepared in the present invention at different Au concentrations.

Figure 6 shows 200 μL of ^99m Tc-Au-Ac DENPs ([ ^99m Tc ]=370MBq/mL) injected into the tail vein of mice, and the lungs, liver, kidneys, and spleens of mice detected by micro-SPECT/CT scanning And the SPECT/CT pictures of the bladder (as indicated by the arrow), from top to bottom are 30min (a), 60min (b), and 120min (c) after injection.

Figure 7 shows the mouse heart, ^{inferior vena} cava, liver, SPECT/CT images of kidney, spleen and bladder (as indicated by arrows), from top to bottom are 30min (a), 60min (b), 120min (c) after injection.

Figure 8 shows the mouse ^lungs , heart, liver, kidney and bladder (such as The CT images indicated by arrows) are before injection (a), 30min (b), 60min (c) and 120min (d) after injection.

Figure 9 shows the mouse heart, liver, kidney and bladder (as indicated by the arrows) detected by micro-CT scanning after 200 μL of ^99m Tc-Au-Gly DENPs ([Au]=0.08M) was injected into the tail vein of the mouse. ) CT images, in order before injection (a), 30min (b), 60min (c) and 120min (d) after injection.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

As shown in Figure 1, ^99m Tc-Au-Ac DENPs were prepared.

(1) Dissolve 20.0 mg of fifth-generation polyamidoamine dendrimer (G5.NH ₂ , purchased from Dendritech Company) in 15 mL of water, and add 2 mL of diethylenetriaminepentaacetic acid cyclic anhydride (cDTPAA, purchased from Sigma Aldrich) aqueous solution (1.1mg/mL), stirred at room temperature for 8h to obtain G5-DTPA; at the same time, added 5mL 1-ethyl-(3-di Methylaminopropyl) carbodiimide (EDC, purchased from Sigma Aldrich) in aqueous solution (7.69mg/mL) and stirred for 2h, then mPEG-COOH (purchased from Shanghai Yanyi Biotechnology Co., Ltd.) after EDC activation Add to the G5-DTPA solution, stir and react for 1-3d to obtain a functionalized dendrimer (G5-DTPA-mPEG) solution;

(2) Add 2.1mL chloroauric acid solution (30mg/mL) to the G5-DTPA-mPEG solution obtained in (1) and stir for 30min, the solution turns light yellow, then add 2.8mL sodium borohydride aqueous solution (10mg/mL ), the solution instantly turns dark red, and the reaction is stirred for 1-4h. Then, 78 μL of triethylamine was added and stirred for 30 min, and finally 42 μL of acetic anhydride was added, and stirred for 15 h. Finally, the reaction product was dialyzed in phosphate buffer solution 2L×3 and distilled water 2L×3 successively for 3 days with a cellulose dialysis membrane (MWCO=14000), and finally the purified product was freeze-dried to obtain Au-Ac DENPs;

(3) Dissolve 0.2 mg of Au-Ac DENPs prepared in step (2) in 0.25 mL of phosphate buffer (PBS, pH=7.2-7.4), add 0.05 mg of SnCl ₂ , and then add 1.5 mL of sterile radioactive Permethanate (radioactive ^99m Tc concentration is 370MBq/mL) solution, mixed and reacted rapidly for 30min, purified and separated by PD-10 desalting chromatographic column, to obtain functionalized dendrimers with chelated nuclide ^99m Tc wrapped gold nanoparticles ( ^99m Tc-Au-Ac DENPs);

During the synthesis process, the surface modification of the dendrimer was characterized by nuclear magnetic resonance (NMR), as shown in Figure 2a. The integral calculation of each peak showed that 17.6 mPEG molecules were modified on the surface of the dendrimer carrier. The ultraviolet absorption characterization of the obtained product Au-Ac DENPs is shown in Figure 3a. The surface plasmon resonance (SPR) peak of the gold nanoparticles is at 530nm, which proves the formation of gold nanoparticles. The TEM pictures (Figures 4a and 4b) show that the gold nanoparticles The particle diameter is about 3.3nm, the size distribution is narrow, and it has good dispersion. The content of Au in the product was further measured by inductively coupled plasma emission spectrometer (ICP), and the results showed that the average gold loading capacity of each dendrimer was 198.0 gold atoms. The radioactive purity of ^99m Tc-Au-Ac DENPs is as high as 95% as tested by fast thin-layer chromatography, and can be stable for more than 6 hours in normal saline and plasma. These test results show that the designed and synthesized functionalized dendrimers ^99m Tc-Au-Ac DENPs have been successfully prepared.

Example 2

As shown in Figure 1, ^99m Tc-Au-Gly DENPs were prepared.

(1) Dissolve 20.0mg G5.NH ₂ in 15mL water, and add 2mL cDTPAA aqueous solution (1.1mg/mL) dropwise, stir and react at room temperature for 8h to obtain G5-DTPA; at the same time, in 10mL mPEG-COOH aqueous solution (7.69mg/mL) was added to 5mL EDC aqueous solution (7.69mg/mL) and stirred for 2h, then the mPEG-COOH activated by EDC was added to the G5-DTPA solution, and stirred for 1-3d to obtain the functionalized dendrimer Molecular (G5-DTPA-mPEG) solution;

(2) Add 2.1mL chloroauric acid solution (30mg/mL) to the G5-DTPA-mPEG solution obtained in (1) and stir for 30min, the solution turns light yellow, then add 2.8mL sodium borohydride aqueous solution (10mg/mL ), the solution instantly turns dark red, and the reaction is stirred for 1-4h. Then 84 μL glycidol was added and the reaction was stirred for 24 h. Finally, the reaction product was dialyzed in phosphate buffer solution 2L×3 and distilled water 2L×3 successively for 3 days with a cellulose dialysis membrane (MWCO=14000), and finally the purified product was freeze-dried to obtain Au-Gly DENPs;

(3) Dissolve 0.2 mg of Au-Gly DENPs prepared in step (2) in 0.25 mL of PBS, add 0.05 mg of SnCl ₂ , and then add 1.5 mL of sterile radioactive persinate (radioactive ^99m Tc concentration is 370 MBq/mL ) solution and rapidly mixed and reacted for 30 minutes, and then purified and separated by PD-10 desalting chromatographic column to obtain the functionalized dendrimer ( ^99m Tc-Au-Gly DENPs) of the chelated nuclide ^99m Tc-coated gold nanoparticles;

During the synthesis process, the surface modification of the dendrimer was characterized by NMR, as shown in Figure 2b. The integral calculation of each peak shows that 17.6 mPEG molecules are modified on the surface of the dendrimer carrier. The ultraviolet absorption characterization of the obtained product Au-Gly DENPs is shown in Figure 3b. The surface plasmon resonance (SPR) peak of the gold nanoparticles is at 510nm, which proves the formation of gold nanoparticles. The TEM pictures (Figures 4c and 4d) show that the gold nanoparticles The particle diameter is about 3.2nm, the size distribution is narrow, and it has good dispersion. The content of Au in the product was further measured by inductively coupled plasma emission spectrometer (ICP), and the results showed that the average gold loading capacity of each dendrimer was 198.0 gold atoms. The radioactive purity of ^99m Tc-Au-Gly DENPs is as high as 95% as tested by fast thin-layer chromatography, and can be stable for more than 6 hours in normal saline and plasma. These test results show that the designed and synthesized functionalized dendrimers ^99m Tc-Au-Gly DENPs have been successfully prepared.

Example 3

MTT assay was used to study the cytotoxicity of the prepared Au-Ac DENPs and Au-Gly DENPs.

Collect the logarithmic phase human ovarian cancer cell line (SK-OV-3 cells), add it to a 96-well cell culture plate, add 200 μL of cell-containing RPMI1640 medium to each well to make the cell density to 8000/well; then place in a cell culture incubator (5% CO ₂ , 37°C) for 24 hours, discard the medium and add 180 μL of fresh medium, and then add 20 μL of PBS buffer containing different concentrations of Au-Ac DENPs and Au-Gly DENPs (the final concentrations of gold were respectively 0, 2.5, 5, 10, 20, 40, 80 μM) to verify the effect of the material on the growth of SK-OV-3 cells. All test groups were set with 5 wells as a parallel group; after incubation in the incubator for 24 hours, 20 μL of MTT solution (5 mg/mL) was added to each well, and after 4 hours of incubation, the culture solution in the wells was carefully sucked, and the Add 200 μL DMSO, place on a shaker and shake in the dark for 15 minutes, and then measure the absorbance of the MTT formazan solution in each well at 570 nm in an enzyme-linked immunosorbent detector. The analysis results showed the toxic effect of Au-Ac DENPs and Au-Gly DENPs on cells by cell viability. The viability of the treated cells was detected by MTT method, see Figure 5. It can be seen from the figure that, compared with untreated SK-OV-3 cells, Au-AcDENPs and Au-Gly DENPs are not toxic to cells when the gold concentration is as high as 80 μM, showing good biocompatibility.

Example 4

200 μL of ^99m Tc-Au-Ac DENPs ([ ^99m Tc]=370MBq/mL, [Au]=0.08M) obtained in Example 1 or 3 was intravenously injected into mice with a body weight of 22g, and the It was detected by CT scanning to obtain a control CT picture (Fig. 8), and 30 minutes, 60 minutes, and 120 minutes after administration, the CT pictures of mouse bladder and kidney were obtained by SPECT/CT scanning respectively. The SPECT/CT pictures obtained by scanning and detecting with the micro-SPECT/CT small animal imager (Fig. 6, Fig. 8). It can be seen from the figure that the acetylated surface-treated ^99m Tc-Au-Ac DENPs material can realize specific imaging of the lungs of mice, and its brightness is significantly higher than that of other major organs such as the liver, and the liver, kidney, The signal of spleen and bladder increased, and the imaging time could last at least 2 hours, and the metabolic signal gradually weakened (Fig. 6). It is proved that the ^99m Tc-Au-Ac DENPs synthesized by this method has better SPECT/CT dual-modal imaging effect.

Example 5

200 μL of ^99m Tc-Au-Gly DENPs ([ ^99m Tc]=370MBq/mL, [Au]=0.08M) obtained in Example 1 or 3 was intravenously injected into the body of a mouse with a body weight of 22g. It was detected by CT scanning to obtain a control CT picture (Fig. 9), and 30 minutes, 60 minutes, and 120 minutes after administration, the CT pictures of mouse bladder and kidney were obtained by SPECT/CT scanning respectively. The SPECT/CT pictures obtained by scanning and detecting with the micro-SPECT/CT small animal imager (Fig. 7, Fig. 9). It can be seen from the figure that the hydroxylated surface-treated ^99m Tc-Au-Gly DENPs material can stay in the blood for a long time to complete blood pool imaging such as the heart, kidney, and abdominal aorta. With the extension of time, the material can Metabolism enters the spleen, bladder and liver (Fig. 7). It is proved that the ^99m Tc-Au-Gly DENPs synthesized by this method has better SPECT/CT dual-modal imaging effect. The in vivo imaging results of the two materials show that the synthesized contrast agents with different surface modifications have significantly different metabolic distribution behaviors in vivo, and can perform different tissue-organ-specific imaging functions.

Claims (10)

1. a kind of functionalization the spect-ct bimodal image-forming contrast medium based on dendrimer it is characterised in that: with poly- second 5th PAMAM dendrimer of diolation modification as polymer carrier, by covalence graft by diethyl three Triamine pentaacetic acid is connected to dendrimer surface, wraps up gold nano grain by the method for fabricated in situ, and by surface residual ammonia Base carries out acetylation or hydroxylating, labelling^99mTc, obtains final product；The method of wherein fabricated in situ is: using sodium borohydride to chlorine gold Acid solution is reduced.

2. the preparation method of the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization, comprising:

(1) by the 5th PAMAM dendrimer g5.nh₂It is dissolved in water, and add diethylenetriamine pentaacetic acid cyclic acid anhydride The aqueous solution of cdtpaa, is stirred at room temperature reaction 8-12h and obtains g5-dtpa, add the mpeg-cooh after edc activation, stir Mix reaction 1-3d, obtain functional dendritic macromole g5-dtpa-mpeg solution；

(2) add chlorauric acid solution stirring 20-40min in g5-dtpa-mpeg solution, add sodium borohydride solution, stirring Reaction 1-4h；

(3) add triethylamine stirring 20-40min, be subsequently added acetic anhydride, stirring reaction 12-24h or addition (+)-2,3-Epoxy-1-propanol, Stirring reaction 12-24h；Reactant liquor is dialysed, finally the aqueous solution lyophilization of product is obtained au-ac denps or au-gly denps；

(4) above-mentioned au-ac denps or au-gly denps is dissolved in phosphate buffer, adds sncl₂, then add again Enter aseptic radioactivity peracid saline solution and mix, column chromatography for separation, obtain final product chelating nucleic^99mTc wraps up the work(of golden nanometer particle Dendrimer can be changed^99mTc-au-ac denps or^99mtc-au-gly denps.

3. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: g5.nh in described step (1)₂Mol ratio with cdtpaa is 1:5-10；Mpeg-cooh and The mol ratio of edc is 1:8-15, and soak time is 2-4 hour；The mol ratio of g5-dtpa and mpeg-cooh is 1:15-25.

4. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: the molecular weight of the mpeg-cooh in described step (1) be 5000.

5. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: in described step (2) mol ratio of g5-dtpa-mpeg and gold chloride be 1:150-250； The mol ratio of g5-dtpa-mpeg and sodium borohydride is 1:750-1250.

6. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: in described step (3) mol ratio of g5-dtpa-mpeg and triethylamine be 1:400-800； The mol ratio of g5-dtpa-mpeg and acetic anhydride is 1:350-750；The mol ratio of g5-dtpa-mpeg and (+)-2,3-Epoxy-1-propanol is 1: 500-1000.

7. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: the dialysis in described step (3) specifically comprises the processes of: is first 7.4 in ph using bag filter Dialyse in pbs buffer, then dialyse in distilled water, bag filter is cellulose dialysis film, molecular cut off is 8000-14000.

8. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: au-ac denps and sncl in described step (4)₂Mass ratio be 1:0.1-0.5； The ratio of au-ac denps and radioactivity persalt is 1mg:1850-3700mbq；Au-gly denps and sncl₂Quality Than for 1:0.1-0.5；The ratio of au-gly denps and radioactivity persalt is 1mg:1850-3700mbq.

9. the spect-ct bimodal image-forming contrast medium based on dendrimer of a kind of functionalization according to claim 2 Preparation method it is characterised in that: the ph=7.2-7.4 of the phosphate buffer in described step (4).

10. the spect-ct bimodal imaging contrast based on dendrimer of a kind of functionalization according to claim 2 The preparation method of agent it is characterised in that: column chromatography for separation in described step (4) specifically comprises the processes of: using pd-10 desalination chromatograph Column purification target product, removes free^99mTc and unreacting substance.