CN105727319A - Preparation and application of fluorescent-nuclear magnetic resonance bifuntional nano particles - Google Patents

Abstract

The invention relates to a fluorescent-nuclear magnetic resonance double-functional nanoparticle and its application in biomedical imaging. Using organic polyacids and gadolinium salts as raw materials, gadolinium-containing carbon quantum dots are synthesized in one step by hydrothermal method. Due to the fluorescent properties of carbon quantum dots, after doping with gadolinium, they can have two functions of fluorescence and nuclear magnetic resonance imaging at the same time, and then modified by tumor-targeting groups to prepare tumor-targeting nanoparticles, which can realize tumor-targeting fluorescence. Duplex imaging with MRI. The nanoparticle synthesized by the invention is simple, has high quantum yield and relaxation rate, and has tumor targeting property, and is expected to be applied in the field of biomedical imaging.

Description

Preparation and Application of a Fluorescence-NMR Bifunctional Nanoparticle

technical field

The invention relates to the field of biomedical imaging, in particular to the preparation and application of a fluorescence-nuclear magnetic resonance dual-function nanoparticle.

Background technique

Molecular probes can identify targets of interest through special signals emitted by themselves, such as light, electricity, and magnetism. Therefore, molecular imaging of tumors is highly dependent on molecular probes. In nuclear magnetic resonance (MR) imaging, Gd(III) can shorten the longitudinal relaxation time of surrounding water protons through paramagnetic substances, that is, increase the T1 relaxation rate (1/T1), and increase the T1WI signal (Coordin.Chem.Rev.2006, 250, 1562-79). As a T1WI contrast agent, Gd(III) is currently mainly used in the form of small molecule coordination chelation or nanostructure. Small molecule contrast agents have low relaxation rate and fast clearance, which are not conducive to MR molecular imaging. The nanostructure of Gd(III) is mainly composed of inorganic nanoparticles (such as Gd ₂ O ₃ , Gd ₂ O(CO ₃ ) ₂ ), which has a high relaxation rate, but has disadvantages such as large particle size, poor water solubility, and high toxicity. , and due to factors such as particle size and biocompatibility, it is often taken up in large quantities by the reticuloendothelial system in the body, and it is difficult to reach tumor tissues (Adv. Funct. Mater. 2008, 18, 766-76). Carbon quantum dots (carbondots, CDs) are a new type of fluorescent nanomaterials, which have the characteristics of small particle size, low toxicity, good biocompatibility, and good water solubility, making them widely used in the biological field, including biosensing and biological imaging (Theranostics, 2012, 2, 295-301; Angew. Chem. Int. Edit., 2010, 49(38): 6726-44). Carbon quantum dots have passive targeting. Studies by Huang et al. found that they can be enriched in tumor tissue through permeability enhancement and retention effects, and the clearance rate is fast, and only a small amount is phagocytosed by the reticuloendothelial system (ACSnano, 2013, 7( 7): 5684-5693). Compared with inorganic nanoparticles, Gd(III) contrast agents with high quantum yield carbon quantum dots are more suitable for carrier imaging. The preparation methods of gadolinium-containing carbon quantum dots that have been discovered so far are complicated, and the quantum yield and relaxation rate are very low, which is far from the actual application requirements (J.Mat.Chem., 2012, 22(44): 23327-30). Therefore, it is of great significance to synthesize nanomaterials with tumor targeting, high quantum yield and high relaxation rate through simple methods and use them in vivo.

Contents of the invention

Aiming at the existing problems such as low quantum yield and relaxation rate of gadolinium-containing carbon quantum dots and complicated preparation methods, the present invention provides a method for preparing nanomaterials with high quantum yield and high relaxation rate in one step, and then The tumor-targeting fluorescence-nuclear magnetic resonance double-functional nanoparticle is prepared after being modified by the tumor-targeting group, and its application in the fields of living imaging and the like.

The method is simple and easy to implement, and the obtained nanomaterial has high quantum yield and relaxation rate, is easily soluble in water, and is a good fluorescence-magnetic resonance dual-function imaging material.

The present invention is achieved through the following technical solutions:

The fluorescence-nuclear magnetic resonance dual-functional nanoparticles provided by the present invention are prepared by the following steps:

(1) dissolving the gadolinium salt in water, adding an aqueous polybasic acid solution, and the molar ratio of the polybasic acid to the gadolinium salt is 0.1-100:1;

(2) Add polyamine aqueous solution to the mixture in step (1), the molar ratio of polyamine to gadolinium salt is 0-500:1;

(3) Put the mixture in an autoclave, heat it to 110-300°C, and keep it for 0.5-24 hours;

(4) After cooling, filter the insolubles and collect the filtrate;

(5) Purify the filtrate obtained in step (4) by using one or more methods of gel chromatography, dialysis or ultrafiltration, and dry it after concentrating by rotary evaporation to obtain carbon quantum dots containing gadolinium.

The gadolinium salt described in step (1) is any one of gadolinium chloride, gadolinium sulfate, gadolinium acetate or gadolinium nitrate, or a mixture of any several in any proportion.

The polybasic acid described in the step (1) is any one of citric acid, tartaric acid, malic acid, succinic acid, maleic acid or fumaric acid, or any mixture of any several in any proportion.

The polyamine in step (2) is any one of urea, biuret, ethylenediamine or ethylenediamine polymer, or a mixture of any several in any proportion.

The drying method described in step (5) is vacuum drying or freeze drying.

The quantum yield of the prepared nanometer material can reach up to 75.5%, the relaxation rate R1 can reach 17.95mM ^-1 ·S ^-1 , and R2 can reach 19.77mM ^-1 ·S ^-1 .

The nanoparticles can be applied to medicines, medical imaging materials, and magnetic resonance-fluorescence dual-mode imaging probes; the nanoparticles are modified with tumor-targeting groups to prepare tumor-targeting nanoparticles, which can realize tumor-targeting fluorescence Duplex imaging with MRI.

Description of drawings

Fig. 1 is the fluorescence-nuclear magnetic resonance bifunctional nanoparticle TEM photo that the present invention makes and particle size distribution figure;

Fig. 2 is the fluorescent spectrogram of fluorescence-nuclear magnetic resonance bifunctional nanoparticles prepared by the present invention;

Fig. 3 is the red tile spectrogram of the fluorescence-nuclear magnetic resonance bifunctional nanoparticle that the present invention makes;

Fig. 4 is the in vivo T1WI imaging diagram of the nanomaterial prepared by the present invention to healthy mice;

Fig. 5 is a cell imaging diagram of fluorescence-nuclear magnetic resonance bifunctional nanoparticles prepared in the present invention.

detailed description

Example 1

Weigh 16 mg GdCl ₃ and dissolve it in 5 mL of water, and take another 1.05 g of citric acid and dissolve it in 20 mL of water, mix the two, place in a 50 mL polytetrafluoroethylene-lined autoclave, seal it and put it in a muffle furnace. The temperature was raised from room temperature to 200°C in 1 hour and maintained at this temperature for 8 hours. After naturally cooling to room temperature, the liquid was taken out and filtered through a 0.22 μm polyethersulfone filter membrane to obtain a filtrate, which was concentrated to 4 mL by rotary evaporation. The concentrate was separated with Sephadex G-25 gel, the fluorescent part was collected, concentrated by rotary evaporation again, and then freeze-dried to obtain brown-black solid nanoparticles.

Example 2

CLT-1 peptide labeling: adding dicyclohexylcarbodiimide to Gd-CDs to activate the carboxyl groups on the surface of nanoparticles, followed by adding N-hydroxyl-activated carboxyl sulfosuccinimide to the reaction system, and then carboxyl groups were combined with 1- Coupling of the amino group on azido-3-aminopropane forms a stable amide bond to give Gd-CDs-N ₃ . After grafting, a part of the carboxyl group on the surface of Gd-CDs-N ₃ was converted into an azide group, which reacted with the short peptide CLT1 modified by the alkyne group at the end, and the CLT-1 peptide was modified by click chemistry to achieve tumor targeting.

Example 3

Characterization of properties of gadolinium-containing carbon quantum dots:

1. Particle size distribution

As shown in Figure 1, the prepared nanoparticles were observed with a JEM-2000 transmission electron microscope (TEM) (acceleration voltage 75kV, magnification 80000 times), and the calculation and statistics showed that the particle size of the nanomaterials was mainly between 3.5 and 6.5nm.

2. Fluorescence spectrum and quantum yield

As shown in Figure 2, fully dissolve the nanomaterial with deionized water, take 2mL and place it in a quartz vessel, and use a fluorescence spectrophotometer to measure the fluorescence emission spectra under different wavelengths of excitation light, the excitation wavelengths are 300, 320, 340, 360, 380, 400, 420nm.

Choose quinine sulfate solution as reference sample, dissolve quinine sulfate in the sulfuric acid solution of 0.1mol/L, prepare 3 parallel reference sample solutions of quinine sulfate (to ensure that the absorbance value of its solution at 360nm is not higher than 0.05, and the fluorescence emission peak does not exceed the detection limit of the instrument). Excited at a wavelength of 360nm with a slit width of 5nm, the fluorescence emission curve was measured to ensure that the maximum value of fluorescence did not overflow, and the peak area of the fluorescence emission curve was calculated. Then measure the UV absorption value of the quinine sulfate reference sample at a wavelength of 360nm, and the samples are respectively prepared into 3 parallel sample solutions (the parallel sample solutions are the aqueous solutions of the above-mentioned nanomaterials, ensuring that the absorbance value of the solution at 320nm is not higher than 0.05, and the fluorescence emission peak value does not exceed the detection limit of the instrument), take the excitation wavelength of 320nm, measure the ultraviolet absorption value of the three sample liquids at 320nm (guarantee that the ultraviolet absorption value is not greater than 0.05), select the gap width of 5nm to measure the fluorescence emission curve, and then calculate the peak area of the fluorescence emission curve. Plug the calculated peak areas and measured UV absorbance values into the following equation:

In the formula, Be the quantum yield QY, A is the ultraviolet absorption value, F is the integrated area of the fluorescence emission curve, n is the refractive index of the solvent, the subscript S is a reference, and X is the sample to be measured, It is measured that the nanometer material produced by the method of the present invention has a fluorescence quantum yield of up to 75.5%.

3. Relaxation rate

Weigh about 5 mg of the nanomaterial prepared in the example, weigh it accurately, and then dissolve it in 250 μl of concentrated nitric acid, and use a volumetric flask to make up to 10 ml after complete ablation. The Gd element in the sample was quantified by ICP-atomic absorption spectrometry with reference to the standard solution of Gd element. The result is that the gadolinium content of the nanomaterial prepared in Example 1 is 49 μmol/g. , the sample was prepared as a Gd molar concentration of 0.4mmol/L mother liquor. Then the mother liquor was diluted to four concentrations of 0.2, 0.1, 0.05, and 0.025mmol/L respectively, with a total volume of 5mL, and placed in a 5mL flat-bottomed centrifuge tube. The T1 relaxation time was measured by the IR sequence of the nuclear magnetic resonance scanner: the TW value was 5500ms; the T2 time was measured by the CPMG sequence: the TW value was 5500ms. Calculate r1, r2 values. The nanometer material produced by the method of the present invention has a relaxation rate R1 up to 17.95mM ^-1 ·S ^-1 , and R2 up to 19.77mM ^-1 ·S ^-1 .

4. Infrared Spectroscopy Characterization

Figure 3 shows that 3435cm ^-1 is the stretching vibration absorption peak of OH, and 2111cm _- ¹ is the stretching vibration peak of N3. There is no such characteristic peak before grafting N3, but there are obvious characteristic peaks after grafting. After reacting with alkynyl CLT1, The characteristic peak decreases, indicating that the covalent combination of N3 and alkynyl leads to a small number of N3, 1720cm ^-1 and 1612cm ^-1 are the stretching vibration absorption peaks of C=O, and 1207-1035cm ^-1 is the stretching vibration absorption peak of CO , the peak before grafting CLT1 was narrower, but the peak became wider after grafting. The results of infrared spectroscopy showed that CLT1 had been grafted onto the surface of nanoparticles, and the CO absorption peak was broadened, which was related to the short-chain ether structure at the end of CLT1, which also indicated that CLT1 had been grafted onto the surface of nanoparticles.

Example 4

In vivo T1WI MRI

The nanoparticle produced by the method of the present invention is used to perform enhanced scanning on the tumor-bearing mice, and the in vivo situation of the nanoparticle is observed. Magnetic resonance parameters: Mini-Rat low-field nuclear magnetic resonance analyzer, mouse coil; SE sequence T1WI coronal scan: TR300ms, TE19.2ms, 100×100mm, slicewidth3mm, slicegap2mm.

Mice were gas anesthetized by ventilation with isoflurane mixed with oxygen. After the mice were anesthetized, they were placed on a polytetrafluoroethylene mouse bed, and T1WI images were obtained by plain scanning. The experimental mice were injected with the tumor-targeting fluorescence-nuclear magnetic resonance dual-functional nanoparticles produced by the method of the present invention through the tail vein of the mice, and the dose was 0.1 mmol/kg body weight. The time point data of 2min and 20min (Fig. 4) were collected respectively, and the enhancement was observed. It can be seen that the enhancement of the tumor site in the tumor-bearing mice injected with Gd-CDs-CLT1 was significantly higher than that in the tumor-bearing mice injected with Gd-CDs, showing a stronger expression. Good tumor targeting ability.

Example 5

cell imaging

Dissolve 40 mg of Gd-CDs-CLT1 nanoparticles in 2 mL of cell culture medium and mix well. Tumor cells were digested with trypsin to form a single-cell suspension, which was seeded into a culture dish with a diameter of 1 cm at a density of 1×10 ⁴ (Cells/ml), and 1 mL of culture solution mixed with carbon dots was added to each culture dish. Place in an incubator and incubate for 24h (temperature is 37°C, CO ₂ is 5%). The cultured cells were eluted twice with 0.1M PBS to completely wash away the culture medium and dead cells. The slides were placed on the stage of a fluorescence microscope, and the cell morphology was observed using 405nm and 488nm excitation, respectively. It was confirmed by tumor cell imaging (Figure 5) that Gd-CDs-CLT1 nanoparticles were used as fluorescent probes to label cells. Compared with the control (without nanoparticles) cells, the cells containing nanoparticles emitted obvious blue light when excited at 405nm, and at 488nm Excitation emits distinct green light, demonstrating that the nanoparticles can be used to label tumor cells in vitro.

Claims (8)

1. the preparation of a fluorescence-nuclear magnetic resonance bifunctional nanoparticle, is characterized in that: the preparation of described nanoparticle is made up of following steps: (1) dissolving the gadolinium salt in water, adding an aqueous polybasic acid solution, and the molar ratio of the polybasic acid to the gadolinium salt is 0.1-100:1; (2) Add polyamine aqueous solution to the mixture in step (1), the molar ratio of polyamine to gadolinium salt is 0-500:1; (3) Put the mixture in an autoclave, heat it to 110-300°C, and keep it for 0.5-24 hours; (4) After cooling, filter the insolubles and collect the filtrate; (5) Purify the filtrate obtained in step (4) by using one or more methods of gel chromatography, dialysis or ultrafiltration, and dry it after concentrating by rotary evaporation to obtain carbon quantum dots containing gadolinium. 2. preparation method as claimed in claim 1, is characterized in that: the gadolinium salt described in step (1) is any one in gadolinium chloride, gadolinium sulfate, gadolinium acetate or gadolinium nitrate, or any several mixture of proportions. 3. preparation method as claimed in claim 1, is characterized in that: the polybasic acid described in step (1) is any in citric acid, tartaric acid, malic acid, succinic acid, maleic acid or fumaric acid, Or any mixture of several in any proportion. 4. preparation method as claimed in claim 1 is characterized in that: the polyamine described in step (2) is any one in urea, biuret, ethylenediamine or ethylenediamine polymer, or any several Mixtures in any proportion. 5. The preparation method according to claim 1, characterized in that: the drying method in step (5) is vacuum drying or freeze drying. 6. The nanomaterial prepared by the method according to any one of claims 1-5, characterized in that the quantum yield of the nanomaterial can reach up to 75.5%, and the relaxation rate R1 can reach 17.95mM ^-1 ·S ^-1 , R2 can reach 19.77mM ^-1 ·S ^-1 . 7. The application of the fluorescence-nuclear magnetic resonance dual-functional nanoparticles according to claim 1, characterized in that: the nanoparticles can be applied to medicines, medical imaging materials and magnetic resonance-fluorescence dual-mode imaging probes . 8. The application of nanoparticles as claimed in claim 7, characterized in that: the nanoparticles are modified by tumor targeting groups to obtain tumor targeting nanoparticles, which can realize dual imaging of tumor targeting fluorescence and nuclear magnetic resonance imaging. functional imaging.