A mineralized albumin-based 1 H / 19 F magnetic resonance contrast agent and its preparation method and application

Abstract

The present invention belongs to the technical field of biopharmaceutical materials, and specifically relates to a method for preparing a mineralized albumin-based 1 H / 19 F magnetic resonance contrast agent and its preparation method and application. The present invention utilizes a disulfide bond reducing agent to destroy the disulfide bond of albumin to deform it and fully expose the metal binding group, thereby efficiently combining albumin with Gd. The gadolinium content in the obtained Gd-albumin is high, and the yield of Gd-albumin is greatly improved by optimizing the preparation process. It shows a better T1 relaxation rate and good T2 relaxation performance than commercial gadolinium-based contrast agents (Gd-DTPA), and can effectively achieve T1-T2 dual-modality imaging; F@Gd-albumin is prepared by further coating perfluorocarbon on the basis of Gd-albumin, which can achieve excellent 1 H / 19 F multi-modal magnetic resonance imaging capabilities provide new methods for accurate imaging diagnosis of cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical materials. More specifically, it relates to a method based on mineralized albumin. 1 H / 19 F magnetic resonance contrast agent and its preparation method and application. Background Art

[0002] Among modern clinical medical imaging technologies, magnetic resonance imaging (MRI) is one of the most powerful diagnostic tools. It has extremely high tissue penetration and does not require the use of ionizing radiation or radioactive agents, which makes it a huge advantage in clinical diagnostic practice and biomedical research. Magnetic resonance imaging is widely used in the field of cancer diagnosis due to its high spatial resolution and tomography capabilities of soft tissues. In order to further enhance the MRI signal and improve imaging resolution, magnetic resonance contrast agents are often used to reduce the longitudinal (T1) or transverse (T2) relaxation time of water protons. These contrast agents affect both T1 and T2 relaxation times, but the degree of influence varies. The type of contrast agent can be determined by comparing the relaxation rates ri (ri=1 / (C·Ti)) of different materials, where i=1 or 2. Generally speaking, when the contrast agent's r2 / r1 is less than 3, it means that its T1 contrast effect is better than T2, and it is suitable for T1 contrast agents; when r2 / r1 is between 3 and 10, it means that the contrast agent has T1 and T2 contrast effects and can be used as T1-T2 dual-mode contrast agents; and when r2 / r1 is greater than 10, it means that its T2 contrast effect is better than T1, and it is suitable for T2 contrast agents. These judgment criteria are of great guiding significance for clinical selection of appropriate contrast agents and optimization of imaging effects.

[0003] T1-weighted imaging provides higher tissue resolution, while T2-weighted imaging is more sensitive in disease detection. However, it is usually difficult to simultaneously obtain two types of images of the same tissue area using a single T1 or T2 contrast agent. In addition, due to the influence of factors such as fat, hemorrhage, thrombus, air, calcification, and metal deposition, single-mode T1 or T2-weighted magnetic resonance imaging may produce artifacts, resulting in ambiguous diagnostic results, which seriously affects the accurate diagnosis of diseased tissues. The combination of T1 and T2-weighted MRI can make up for each other's shortcomings, obtain more comprehensive and accurate information, and show great potential in clinical diagnosis. Combining the advantages of the two imaging modes, contrast agents with T1-T2 dual-mode MRI contrast capabilities are of great significance in improving the accuracy of disease diagnosis and have attracted widespread attention and attention.

[0004] Gadolinium-based contrast agents are one of the most widely used MRI contrast agents. However, the current gadolinium-based contrast agents are mainly T1 contrast agents. Therefore, the development of a safe and efficient new gadolinium-based T1-T2 dual-mode contrast agent to adapt to clinical diagnosis has broad application prospects. At present, Gd-based contrast agents are mainly divided into gadolinium complexes and inorganic gadolinium-based nanoparticles. However, traditional gadolinium-based contrast agents are only T1 contrast agents and can only improve the T1-weighted imaging effect. To achieve T1-T2 dual-modality imaging on the same magnetic resonance imaging instrument, it is necessary to develop corresponding gadolinium-based contrast agents. Gadolinium-albumin contrast agents prepared by the existing technology level are also rarely used in T1-T2 dual-modality imaging. For example, Chinese patent application CN115317628A discloses a gadolinium-based magnetic resonance contrast agent with a particle size of less than 10 nm. The contrast agent can achieve T1-weighted imaging, but does not focus on T2-weighted imaging. Further research and development and technological breakthroughs are still needed to obtain gadolinium-albumin contrast agents with excellent T1-T2 dual-modality imaging capabilities.

[0005] on the other hand, 19 F MRI is also an important development direction in the field of magnetic resonance imaging. 19 F has 100% natural abundance and is the only stable fluorine isotope. 1 H has the same spin quantum number and similar gyromagnetic ratio, and has higher NMR sensitivity. 19 F has almost no endogenous background signal and has a wide chemical shift range, which makes 19 F MRI images can obtain high signal-to-noise ratio results, and the imaging signal is linearly related to the contrast agent content and magnetic field strength, which can achieve accurate positioning and quantitative analysis of imaging. 19 F MRI probes have been explored, including perfluorocarbons, fluorinated polymers, fluorinated ionic liquids, and inorganic fluorides. Among them, perfluorocarbon emulsions are the most typical 19 One of the F MRI contrast agents, it has multiple equivalent fluorine atoms and can produce strong 19 However, existing perfluorocarbon probes have defects such as poor stability and uneven size, which limits their application in 19 Therefore, the development of new types of F MRI with better stability and uniform size is necessary. 19 F MRI probe is of great significance and is expected to promote 19F MRI technology is more widely used in clinical and research (Yoon JK, Burgess D J. Interfacial properties as stability predictors of lecithin-stabilized perfluorocarbon emulsions [J]. Pharmaceutical development and technology, 1996, 1 ​​(4): 333-341.). 19 F MRI probes are often difficult to prepare and difficult to mass produce, and their safety remains to be verified.

[0006] 1 H MRI has high resolution and high sensitivity but is susceptible to endogenous signal interference. 19 F MRI can hardly detect endogenous signals in the human body. Therefore, combined with T1-T2 dual-mode 1 H MRI and 19 F MRI can provide more accurate and comprehensive image information, providing clinicians with better diagnostic evidence. At the same time, combining these two technologies on one instrument can simultaneously collect three layers of different magnetic resonance information, achieve precise matching of spatiotemporal imaging, and thus achieve accurate monitoring of tumor growth and treatment process. However, since molecules with high F content are often both hydrophobic and oleophobic, they are not compatible with F MRI. 1 The interaction of water molecules required for H MRI is difficult to achieve, which makes 1 H / 19 The development of high-field multimodal MRI contrast agents is quite challenging. Therefore, it is necessary to develop a dual-mode MRI agent that can simultaneously detect T1 and T2 images. 1 H MRI and 19 Ultra-high-field contrast agents capable of F MRI are of great significance for achieving accurate diagnosis of diseases such as cancer, and are also one of the important challenges faced in current research. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defect that the traditional gadolinium-based contrast agent can only improve the T1-weighted imaging ability. The primary purpose of the present invention is to provide a nanoparticle Gd-albumin based on mineralized albumin. The obtained nanoparticle Gd-albumin shows a better r1 relaxation rate than the commercial gadolinium-based contrast agent (Gd-DTPA), and good r2 relaxation performance, so that 3<r2 / r1<10, has the contrast effect of both T1 and T2 modes, and can effectively realize T1-T2 dual-modality imaging.

[0008] Another object of the present invention is to provide a method for preparing the F@Gd-albumin based on mineralized albumin.

[0009] Another object of the present invention is to provide F@Gd-albumin prepared by the preparation method, wherein the obtained F@Gd-albumin has good biosafety and stability, and shows a better r1 relaxation rate than the commercial gadolinium-based contrast agent (Gd-DTPA), and good r2 relaxation performance, such that 3<r2 / r1<10, and has the same T1-T2 dual-modality imaging capability as Gd-albumin, and can also achieve 1 H / 19 FMultimodal imaging.

[0010] Another object of the present invention is to provide applications of the nanoparticle Gd-albumin or F@Gd-albumin.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention protects a nanoparticle Gd-albumin based on mineralized albumin, wherein the nanoparticle Gd-albumin is prepared by the following method, and the preparation method comprises the following steps:

[0013] S1. Disperse albumin in water, add a disulfide bond reducing agent, add a gadolinium salt solution under stirring, adjust the pH to 11-12, continue stirring until the reaction is complete, centrifuge, and take the supernatant;

[0014] S2. Collect the supernatant obtained in step S1, perform gradient dialysis with an arginine solution having a pH of 7 to 8, and then perform dialysis with deionized water, collect the supernatant after dialysis, and perform freeze-drying to obtain Gd-albumin.

[0015] Since the relaxation rate, imaging ability and other effects of contrast agents are closely related to the structure and properties of contrast agents, the relaxation rate, imaging ability and other effects of nanoparticles can be changed by surface modification, shape adjustment and size control. Traditional gadolinium-based contrast agents and even gadolinium albumin contrast agents are only T1 contrast agents, which can only improve T1 weighted imaging ability. The present invention uses a disulfide bond reducing agent to destroy the disulfide bonds of albumin to deform it and fully expose the metal binding group, thereby enhancing its binding ability to metal ions, thereby efficiently combining albumin with Gd. The gadolinium content in the obtained Gd-albumin is high, and the yield of Gd-albumin is greatly improved by optimizing the preparation process. It shows a better T1 relaxation rate than commercial gadolinium-based contrast agents (Gd-DTPA) and good T2 relaxation performance, making 3<r2 / r1<10, with T1 and T2 contrast effects, and can effectively achieve T1-T2 dual-modality imaging.

[0016] Preferably, the gadolinium salt is selected from one or more of gadolinium chloride, gadolinium sulfate and gadolinium nitrate.

[0017] Preferably, the disulfide bond reducing agent is selected from one or more of reduced glutathione, mercaptoethanol, dithiothreitol, mercaptoformamide, and mercaptopropionic acid.

[0018] More preferably, the disulfide bond reducing agent is reduced glutathione.

[0019] Preferably, the albumin is human serum albumin or bovine serum albumin, more preferably bovine serum albumin.

[0020] Preferably, the molar ratio of the albumin, the disulfide bond reducing agent and the gadolinium salt is 1:(17-20):(15-20).

[0021] Furthermore, the particle size of the nanoparticle Gd-albumin is 30-50 nm.

[0022] Furthermore, the content of Gd in the nanoparticle Gd-albumin is 6-10 wt%.

[0023] Furthermore, the gadolinium salt is added slowly by injection, and the injection pump parameter is 1-1.5 mL / min.

[0024] Preferably, in step S1, the stirring speed is 400-800 rpm.

[0025] Preferably, the time for the reaction to be complete is 12 to 14 hours.

[0026] Preferably, in step S2, the method of collecting the supernatant after the reaction is completed is centrifugation, and the parameters of the centrifugation are: 12000-13000 rpm, 25-35 min.

[0027] The present invention also protects a method for preparing F@Gd-albumin based on mineralized albumin, comprising the following steps: dispersing the nanoparticle Gd-albumin in water, adding perfluorocarbon and ethanol, using ultrasound to fully wrap the Gd-albumin with the loaded perfluorocarbon, filtering (for the purpose of removing large-sized particles), and taking the filtrate as the F@Gd-albumin based on mineralized albumin;

[0028] Among them, the mass volume ratio of Gd-albumin to perfluorocarbon is 1: (0.4~2) mg / μL.

[0029] In view of the problem that artifacts in single-mode magnetic resonance imaging affect the diagnostic results, and the defects of existing perfluorocarbon liposome probes such as poor stability and uneven size, their application in 19 F MRI has been further applied, while other types of 19 F MRI probes are often difficult to prepare and difficult to mass produce. Their safety also needs to be verified. In addition, molecules with high F content are both hydrophobic and oleophobic. 1 H MRI needs to interact with water molecules. 1 H MRI and 19 F MRI has the problem of being difficult to combine. The present invention uses albumin as a biological template to synthesize the biomimetic material Gd-albumin, and then coats perfluorocarbon through hydrophobic interaction to obtain F@Gd-albumin. The obtained F@Gd-albumin has good biological safety and stability, achieving 1 H / 19 F multimodal MRI applications, and showed better T1 relaxation rate than commercial gadolinium-based contrast agents (Gd-DTPA), as well as good T2 relaxation performance, making 3<r2 / r1<10, with the same T1-T2 dual-modality imaging capability as Gd-albumin, and can also achieve 1 H / 19 FMultimodal imaging.

[0030] Preferably, the perfluorocarbon is selected from one or more of perfluoro-15-crown-5-ether, perfluorooctyl bromide, perfluorotributylamine, and perfluoropolyether.

[0031] Preferably, the volume ratio of ethanol to water is 1:(2-4).

[0032] During the gradient dialysis process, the dialysis solution was gradually changed from arginine solution (pH 7-8) to deionized water to prevent protein product precipitation.

[0033] Furthermore, the method of fully dispersing is to use a cell disruptor for dispersion, and the operating parameters of the cell disruptor are: ultrasonic interval of 1 to 4 s, and ultrasonic time of 3 to 10 min.

[0034] Furthermore, the filtration is performed using a 220 nm filter.

[0035] The present invention also protects a F@Gd-albumin based on mineralized albumin, which is prepared by the above preparation method.

[0036] Furthermore, the content of Gd in the F@Gd-albumin is 2-4 wt%.

[0037] Furthermore, the loading amount of perfluorocarbon in the agent F@Gd-albumin is 30-75 wt%, preferably 60-70 wt%.

[0038] The present invention also protects the use of the nanoparticle Gd-albumin or the F@Gd-albumin in preparing a contrast agent.

[0039] Furthermore, the nanoparticle Gd-albumin or the F@Gd-albumin is used in the preparation of a T1-T2 magnetic resonance contrast agent.

[0040] Furthermore, the F@Gd-albumin is prepared 1 H / 19 F Application of multimodal magnetic resonance contrast agents.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention utilizes a disulfide bond reducing agent to destroy the disulfide bond of albumin to deform it and fully expose the metal binding group, thereby efficiently combining albumin with Gd. The content of gadolinium in the obtained Gd-albumin is high, and the yield of Gd-albumin is greatly improved by optimizing the preparation process. The obtained Gd-albumin shows a better r1 relaxation rate and good r2 relaxation performance than the commercial gadolinium-based contrast agent (Gd-DTPA), and can effectively realize T1-T2 dual-modality imaging. F@Gd-albumin is prepared by further coating perfluorocarbon on the basis of Gd-albumin, which can achieve excellent 1 H / 19 F multi-modal magnetic resonance imaging capabilities provide new methods for accurate imaging diagnosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the particle size distribution diagram of Gd-BSA prepared in Example 1.

[0044] Figure 2 This is the particle size distribution diagram of PFCE@Gd-BSA prepared in Example 4.

[0045] Figure 3 Statistical graph of PFCE drug loading and encapsulation efficiency in PFCE@Gd-BSA at different feed ratios.

[0046] Figure 4 This is a statistical diagram of the particle size stability of PFCE@Gd-BSA under different feed ratios.

[0047] Figure 5This is a statistical graph of the particle size stability of PFCE@Gd-BSA prepared in Example 4 in different solvents at different temperatures.

[0048] Figure 6 This is the transmission electron microscope image of PFCE@Gd-BSA prepared in Example 4 and its local magnified image.

[0049] Figure 7 Statistical graph of the yield data of Gd-BSA prepared in Example 4 and Comparative Examples 5-7.

[0050] Figure 8 Statistical graph of the Gd element content of Gd-BSA prepared in Example 1 and PFCE@Gd-BSA prepared in Example 4.

[0051] Fig. 9 This is a statistical graph showing the effect of the presence or absence of a disulfide bond reducing agent on the Gd element content in Gd-BSA.

[0052] Fig.10 This is the nuclear magnetic resonance fluorine spectrum of PFCE@Gd-BSA prepared in Example 4.

[0053] Fig.11 T1-weighted (A) and T2-weighted MRI images (B) of Gd-DTPA, Gd-BSA and PFCE@Gd-BSA; statistical graphs of the relationship between the longitudinal relaxation rate (1 / T1) (C) and transverse relaxation rate (1 / T2) (D) of Gd-DTPA, Gd-BSA and PFCE@Gd-BSA and Gd concentration.

[0054] Fig.12 A is a schematic diagram of sample placement; B is PFCE@Gd-BSA 19 F is MRI image; C is PFCE@Gd-BSA 19 F Statistical graph of the relationship between MRI signal-to-noise ratio and PFCE concentration.

[0055] Fig.13 The data statistics of cell survival rate after incubation of HUVEC cells with different concentrations of Gd-BSA, PFCE@Gd-BSA for 24 h.

[0056] Fig.14 A in the figure is the tumor site after intratumoral injection of PFCE@Gd-BSA 19 F is the magnetic resonance image; B is the tumor site after intratumoral injection of PFCE@Gd-BSA 19 Statistical diagram of the signal-to-noise ratio of F magnetic resonance signals.

[0057] Fig.15T1-weighted MRI images (A) and T2-weighted MRI images (B) of Gd-BSA prepared with and without disulfide bond reducing agents; as well as their longitudinal relaxation rate (1 / T1) (C) and transverse relaxation rate (1 / T2) (D) with Gd 3+ Concentration relationship data. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0059] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0060] Example 1 Preparation method of Gd-BSA

[0061] The specific steps are as follows:

[0062] S1. Disperse 1 g of bovine serum albumin in 20 mL of deionized water, add 80 mg of reduced glutathione, and stir at 200 rpm for 15 min. Then, under stirring at 600 rpm, add 5 mL of 20 mg / mL gadolinium chloride hexahydrate solution dropwise at a speed of 1 mL / min using a syringe pump to obtain a mixed solution.

[0063] S2. The pH value of the mixed solution obtained in step S1 was adjusted to 12, and the mixture was stirred at 600 rpm for 14 h until the reaction was complete. Meanwhile, the pH value was checked every three hours and an appropriate amount of NaOH solution was added to maintain the pH value unchanged.

[0064] S3. Centrifuge the solution obtained after the reaction at 13,000 rpm for 30 min and collect the supernatant. Use 0.1M arginine solution (pH 7) to perform gradient dialysis on the supernatant (the specification of the dialysis bag used is 14,000 Da). The concentration of arginine is halved each time. After 3 dialysis, the dialysis solution is replaced with deionized water, and then dialyzed twice with deionized water. The dialyzed purified supernatant is freeze-dried to obtain Gd-BSA.

[0065] Example 2 Preparation method of Gd-BSA

[0066] The difference from Example 1 is that the reaction time in step S2 is 12 h, and the other steps are the same as Example 1.

[0067] Example 3 Preparation method of Gd-BSA

[0068] The difference from Example 1 is that the disulfide bond reducing agent in step S1 is mercaptoethanol, and the other steps are the same as Example 1.

[0069] Example 4 Preparation method of PFCE@Gd-BSA

[0070] Take 20 mg of Gd-BSA obtained according to the method of Example 1 and dissolve it in 8 mL of deionized water, then add 10 μL of PFCE (perfluoro-15-crown-5-ethyl ether) and 2 mL of anhydrous ethanol to the solution. After slight shaking, use a cell disruptor (ultrasonic interval of 3 s, amplitude of 20%, ultrasonic time of 5 min) to ultrasonically mix the solution in an ice bath. Finally, filter with a 220 nm filter head, and take the filtrate to obtain the target magnetic resonance contrast agent PFCE@Gd-BSA.

[0071] Example 5 Preparation method of PFCE@Gd-BSA

[0072] The difference from Example 4 is that 30 μL of PFCE was weighed.

[0073] For other steps and parameters, refer to Example 3.

[0074] Example 6 Preparation of PFCE@Gd-BSA

[0075] The difference from Example 4 is that Gd-BSA prepared according to the method of Example 2 is weighed, and the other steps are the same as Example 4.

[0076] Example 7 Preparation of PFCE@Gd-BSA

[0077] The difference from Example 4 is that Gd-BSA prepared according to the method of Example 3 is weighed, and the other steps are the same as Example 4.

[0078] Comparative Example 1 Preparation method of PFCE@Gd-BSA

[0079] The difference from Example 4 is that 60 μL of PFCE was weighed.

[0080] For other steps and parameters, refer to Example 4.

[0081] Comparative Example 2 Preparation method of PFCE@Gd-BSA

[0082] The difference from Example 4 is that 100 μL of PFCE was weighed.

[0083] The other steps and parameters are the same as those in Example 4.

[0084] Comparative Example 3 Preparation method of PFCE@Gd-BSA

[0085] The difference from Example 4 is that 150 μL of PFCE was weighed.

[0086] The other steps and parameters are the same as those in Example 4.

[0087] Comparative Example 4 Preparation of Gd-BSA and PFCE@Gd-BSA

[0088] Preparation of PFCE@Gd-BSA: The difference from Example 4 is that Gd-BSA is different, and the other steps are the same as Example 4.

[0089] Preparation of Gd-BSA: The difference from Example 1 is that in step S1, reduced glutathione is not added, and the other steps are the same as Example 1.

[0090] Comparative Example 5 Preparation method of Gd-BSA and PFCE@Gd-BSA

[0091] Preparation of PFCE@Gd-BSA: The difference from Example 4 is that Gd-BSA is different, and the other steps are the same as Example 3.

[0092] Preparation of Gd-BSA: The difference from Example 1 is that in step S3, ultrapure water is directly used to dialyze the supernatant.

[0093] Comparative Example 6 Preparation of Gd-BSA and PFCE@Gd-BSA

[0094] Preparation of PFCE@Gd-BSA: The difference from Example 4 is that Gd-BSA is different, and the other steps are the same as Example 4.

[0095] Preparation of Gd-BSA: The difference from Example 1 is that the pH is not adjusted to be constant in step S2.

[0096] Comparative Example 7 Preparation Method of Gd-BSA and PFCE@Gd-BSA

[0097] Preparation of PFCE@Gd-BSA: The difference from Example 4 is that Gd-BSA is different, and the other steps are the same as Example 4.

[0098] Preparation of Gd-BSA: The difference from Example 1 is that in step S2, the pH is not adjusted to be constant; in step S3, the supernatant is directly dialyzed using deionized water.

[0099] Experimental Example 1 DLS / Zeta Potentiel Determination

[0100] 1. Experimental methods

[0101] The particle size distribution of Gd-BSA prepared in Example 1, the particle size (Average Size), polydispersity index (PDI) and Zeta potential (Zeta Potential) of PFCE@Gd-BSA obtained with different feed ratios (i.e., Examples 4-5 and Comparative Examples 1-3) were detected using a Malvern particle size analyzer. Specifically, the sample to be tested was weighed and dispersed in 2 mL of deionized water at room temperature and then measured using a Malvern particle size analyzer.

[0102] 2. Experimental results

[0103] Table 1 Dispersibility, particle size and Zeta potential of PFCE@Gd-BSA at different feed ratios

[0104]

[0105] Depend on Figure 1 It can be seen that the particle size distribution range of the Gd-BSA prepared by the present invention is 30-50 nm, and the size is uniform and the distribution is even.

[0106] The particle size distribution results of Examples 2-3 are substantially the same as those of Example 1 and will not be described in detail herein.

[0107] Based on Gd-BSA, PFCE@Gd-BSA magnetic resonance contrast agents with different feed ratios were further prepared. The results are shown in Table 1. Compared with Comparative Examples 1 to 3, the PdI of PFCE@Gd-BSA obtained in Examples 4 to 5 is smaller, indicating that the obtained magnetic resonance contrast agents are uniform in size and evenly distributed. Particle size distribution of magnetic resonance contrast agents obtained in Example 4 ( Figure 2 ) once again verified that the size of the magnetic resonance contrast agent obtained under the feed ratio of the embodiment is more uniform and evenly distributed.

[0108] The particle size distribution results of Examples 6 and 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0109] Experimental Example 2 Determination of encapsulation efficiency and drug loading

[0110] 1. Experimental methods

[0111] The formula for determining the encapsulation efficiency and loading amount of the sample to be tested is as follows:

[0112] Encapsulation efficiency (%) = M P / M×100%

[0113] Drug loading (%) = M P / (M B +M P )×100%

[0114] Where M represents the total mass of PFCE input, MP represents the mass of PFCE loaded in PFCE@Gd-BSA, M B represents the mass of Gd-BSA in PFCE@Gd-BSA.

[0115] Gd-BSA mass (M B ) was obtained according to the following method: the absorbance values ​​A at 260 nm and 280 nm of the samples to be tested (PFCE@Gd-BSA prepared in Examples 4 to 5 and Comparative Examples 1 to 3 (wherein Example 4 is Group 2, Example 5 is Group 1, and Comparative Examples 1 to 3 are Groups 3 to 5, respectively)) were directly detected on a UV spectrophotometer. 260 , A 280 . BSA concentration in sample = 1.45 × A 280 -0.74×A 260 (mg / mL), and then the mass of Gd-BSA can be calculated.

[0116] PFCE mass (M P ) was obtained by the following method: PFCE@Gd-BSA prepared in Examples 4-5 and Comparative Examples 1-3 was dispersed in a mixed solution consisting of 10 vol% D2O and 90 vol% H2O, and 10 mM sodium trifluoroacetate (TFANa) was added as an internal reference. Then it was placed in a nuclear magnetic resonance tube and detected by a nuclear magnetic resonance spectrometer to obtain 19 F NMR spectrum. The calculation formula of PFCE concentration is as follows:

[0117]

[0118] In the formula, C PFCE is the molar concentration of PFCE, C TFANa is the molar concentration of TFANa, m is the integral value of PFCE (in 19 In the calculation of F NMR spectra, the integral value of TFANa is fixed to 1), n ​​is the number of equivalent fluorine atoms (n TFANa =3, n PFCE =20).

[0119] 2. Experimental results

[0120] The results are as follows Figure 3 As shown in the figure, as the PFCE feed increases, the drug loading gradually increases and the encapsulation efficiency gradually decreases. After group 2 (ie, Example 4), the PFCE@Gd-BSA loading capacity of PFCE reaches 60% to 80%. If the PFCE feed is further increased, the PFCE drug loading capacity increases very little.

[0121] Experimental Example 3 In vitro stability study

[0122] 1. Experimental methods

[0123] The PFCE@Gd-BSA contrast agents obtained in Examples 4-5 and Comparative Examples 1-3 were dispersed in 2 mL of different solutions (PBS solution, PBS solution containing 10 vol% FBS (fetal bovine serum)), respectively, and incubated at different temperatures. 1 mL of the solution was taken out at different time points, and the particle size change was measured using a Malvern particle size analyzer to evaluate the particle size stability.

[0124] 2. Experimental results

[0125] Some studies have used albumin to prepare perfluorocarbon emulsions, but their stability still needs to be improved. The stability of nanoparticles is a prerequisite for their imaging applications. The method of the present invention obtains PFCE@Gd-BSA magnetic resonance contrast agents with uniform size, uniform distribution and excellent stability. The stability results are as follows Figure 4 As shown, embodiments 4 to 5 (corresponding to Figure 4 PFCE@Gd-BSA of Group 1~2 in Example 4, Example 5 is Group 1) showed excellent stability, and the particle size remained basically unchanged after being placed at 4°C for 21 days, while Comparative Examples 1~3 (corresponding to Figure 4 The particle size of PFCE@Gd-BSA in groups 3~5) increased significantly after 5 days of storage, especially the PFCE@Gd-BSA in comparative examples 2~3.

[0126] Taking the PFCE@Gd-BSA obtained in Example 4 as an example, the stability of PFCE@Gd-BSA under different storage conditions was further determined. The results are as follows: Figure 5 As shown, in different solutions, the average particle size of PFCE@Gd-BSA obtained in Example 4 remained stable; in a 4°C aqueous solution, PFCE@Gd-BSA could even maintain a stable particle size for 3 weeks, showing good stability.

[0127] Experimental Example 4 Transmission electron microscopy observation of the morphology and particle size of PFCE@Gd-BSA

[0128] 1. Experimental methods

[0129] 10 μL of the sample solution obtained in Example 4 was dripped onto the carbon support film and naturally air-dried at room temperature. The morphology, particle size and dispersion of the nanoparticles were observed using a transmission electron microscope at 200 KV.

[0130] 2. Experimental results

[0131] The results are as follows Figure 6As shown in the figure, the prepared PFCE@Gd-BSA is spherical with a particle size of about 150 nm. The results of transmission electron microscopy and dynamic light scattering (DLS) are different because the latter obtains the particle size of the nanoparticles in a hydrated state, and the solvent effect makes the nanoparticles show a larger hydrated particle size.

[0132] In addition, the experimental results of Examples 6 and 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0133] Experimental Example 5 Gd-BSA yield analysis and optimization

[0134] 1. Experimental methods

[0135] After the Gd-BSA prepared in Example 1 and Comparative Examples 5 to 7 were completely freeze-dried, the mass of the freeze-dried powder was weighed and the yield was calculated.

[0136] 2. Experimental results

[0137] Since albumin is very sensitive to the pH and salt concentration of the solution, drastic changes in pH and salt concentration during the reaction and dialysis may cause the protein to deform and precipitate. Therefore, by controlling the pH constant during the reaction and gentle gradient dialysis, the precipitation of protein products during the dialysis process can be reduced, which effectively improves the yield of Gd-BSA. Under the same reaction conditions, the yield of Gd-BSA in the experimental group with constant reaction pH and gradient dialysis can reach 56%, which is much higher than the yield of Gd-BSA in other groups (such as Figure 7 as shown).

[0138] In addition, the experimental results of Examples 2-3 are basically the same as those of Example 1, and will not be repeated here.

[0139] Experimental Example 6 Elemental Analysis

[0140] 1. Experimental methods

[0141] Taking the Gd-BSA obtained in Example 1 and the PFCE@Gd-BSA prepared in Example 4 as representative test objects, a certain concentration of Gd-BSA and PFCE@Gd-BSA solution was taken, and an appropriate amount of concentrated nitric acid was added respectively, and the mixture was slowly heated to 130°C in an oil bath for nitration for 48 h until the solution became colorless, transparent and without precipitation. The nitration solution was diluted with deionized water and filtered, and the final volume was 10 mL. The content of Gd in PFCE@Gd-BSA was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-OES). The content of Gd in the Gd-BSA obtained in Comparative Example 4 was determined according to the above method.

[0142] 2. Experimental results

[0143] like Figure 8As shown in the figure, the content of Gd in Gd-BSA and PFCE@Gd-BSA was 6.14wt% and 2.05wt% respectively determined by ICP-OES (excluding PFCE, the Gd content in PFCE@Gd-BSA was 6.2wt%). The results show that through biomimetic mineralization, Gd ions are successfully combined with BSA, and the obtained Gd-BSA is used to further encapsulate PFCE. The Gd element has a strong ability to bind to BSA, and the Gd content is basically not lost. The obtained PFCE@Gd-BSA can be used as a Gd-containing contrast agent for T1-weighted magnetic resonance imaging.

[0144] Fig. 9 The results show that during the preparation of PFCE@Gd-BSA magnetic resonance contrast agent, the disulfide bond of BSA is cut by adding a disulfide bond reducing agent, which fully exposes the metal binding group of BSA, thereby enhancing its binding ability to gadolinium ions and significantly increasing the gadolinium content in Gd-BSA. Under the same reaction conditions, the gadolinium content of Gd-BSA (Comparative Example 4) without the addition of a disulfide bond reducing agent (glutathione) is only 1.2wt%, while the gadolinium content of Gd-BSA with the addition of glutathione during the preparation process is as high as 6.14wt%.

[0145] In addition, the experimental results of Examples 2-3 are substantially the same as those of Example 1, and the experimental results of Examples 6-7 are substantially the same as those of Example 4, which will not be described in detail here.

[0146] Experimental Example 7 Nuclear Magnetic Resonance Spectrum and PFCE Content Analysis

[0147] 1. Experimental methods

[0148] The PFCE@Gd-BSA prepared in Example 4 was used as a representative test object. The PFCE@Gd-BSA solution prepared in Example 4 was mixed with a mixed solution consisting of 10 vol% D2O and 90 vol% H2O, and 10 mM sodium trifluoroacetate (TFANa) was added as an internal reference. Then, the mixture was loaded into an NMR tube, and the PFCE@Gd-BSA was measured using a 400 MHz NMR spectrum. 19 F NMR spectrum. In addition, 19 F NMR spectroscopy can also be used to calculate the concentration of PFCE, and the calculation formula is as follows:

[0149]

[0150] In the formula, C PFCE is the molar concentration of PFCE, C TFANa is the molar concentration of TFANa, m is the integral value of PFCE (in 19 In the calculation of F NMR spectra, the integral value of TFANa is fixed to 1), n ​​is the number of equivalent fluorine atoms (nTFANa =3, n PFCE =20).

[0151] 2. Experimental results

[0152] The results are as follows Fig.10 As shown in the figure, in addition to the signal peak of sodium trifluoroacetate at -75.32 ppm, PFCE@Gd-BSA has an obvious signal peak belonging to PFCE at -91.92 ppm. After analysis, the T2 relaxation time of PFCE@Gd-BSA is 601.3 ms and the T1 relaxation time is 879.3 ms. The results show that PFCE is successfully encapsulated in the nanoparticles. At the same time, the T2 relaxation time of PFCE@Gd-BSA is long, which is very suitable for 19 F MRI.

[0153] In addition, the experimental results of Examples 5 to 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0154] Experimental Example 8 In vitro 1 H MRI studies

[0155] 1. Experimental methods

[0156] Taking the Gd-BSA obtained in Example 1 and the PFCE@Gd-BSA prepared in Example 4 as representative test objects, the Gd-BSA, PFCE@Gd-BSA and Gd-DTPA (commercial T1 contrast agent) solutions were gradiently diluted, added to a 24-well plate, and scanned under a high-field 3T magnetic resonance imaging apparatus to characterize their weighted imaging effects.

[0157] 2. Experimental results

[0158] The results are as follows Fig.11 As shown in Figure 2, with the increase of particle concentration, the T1-weighted signal gradually increased, and the longitudinal relaxation rates of Gd-BSA and PFCE@Gd-BSA were 10.34 mM, respectively. -1 s -1 , 10.78 mM -1 s -1 , which is about 3 times that of Gd-DTPA (commercial T1 contrast agent). PFCE@Gd-BSA loaded with perfluoro-15-crown-5-ether showed a similar relaxation rate to Gd-BSA, indicating that coating PFCE does not affect the T1-weighted imaging ability of PFCE@Gd-BSA, and PFCE@Gd-BSA can be used as a T1 contrast agent for magnetic resonance imaging. In addition, Gd-BSA and PFCE@Gd-BSA also showed excellent T2-weighted imaging effects, with transverse relaxation rates of 34.06 mM and 24.77 mM, respectively. -1 s -1、33.06 mM -1 s -1 , which is about 9 times that of Gd-DTPA, making 3<r2 / r1<10. It has the contrast effect of both T1 and T2 modes and can effectively realize T1-T2 dual-modality imaging.

[0159] In addition, the experimental results of Examples 5 to 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0160] Experimental Example 9 In vitro 19 F MRI studies

[0161] 1. Experimental methods

[0162] Taking the PFCE@Gd-BSA obtained in Example 4 as a representative test object, the PFCE@Gd-BSA solution was diluted in a gradient manner and added to a 1 mL syringe prepared in advance. The piston was removed and the end was sealed with a sealing film. The ultra-high field 9.6 T nuclear magnetic resonance imaging instrument was used to detect the concentration of the PFCE@Gd-BSA solution. 19 F MRI FLASH sequence for detection and characterization 19 F magnetic resonance imaging effects and quantitative analysis of signal-to-noise ratio changes.

[0163] 2. Experimental results

[0164] The results are as follows Fig.12 As shown in Figure A, the middle circle is PBS solution, and the surrounding circles contain PFCE. The concentrations of PFCE are 2, 4, 6, 8, 12, and 24 mM, respectively. As the concentration of PFCE in the PFCE@Gd-BSA solution increases, the image becomes brighter (e.g. Fig.12 B), 19 The F MRI signal and signal-to-noise ratio (SNR) are constantly increasing, and the lowest imaging concentration is 6 mM. The results show that PFCE@Gd-BSA magnetic resonance contrast agent has good 19 F MRI imaging capabilities.

[0165] In addition, the experimental results of Examples 5 to 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0166] Experimental Example 10 In vitro biocompatibility study

[0167] 1. Experimental methods

[0168] The Gd-BSA and PFCE@Gd-BSA obtained in Example 4 were used as representative test objects, and the effects of Gd-BSA and PFCE@Gd-BSA on the viability of HUVEC (human umbilical vein endothelial cells) were investigated by MTT colorimetry. The absorbance value A at 570 nm was detected by an ELISA instrument. The cell viability was calculated according to the following formula:

[0169]

[0170] 2. Experimental results

[0171] The results are as follows Fig.13 As shown in the figure, as the sample concentration increases, the cell viability of HUVEC remains at a high level. When the concentration of Gd-BSA and PFCE@Gd-BSA is as high as 600 μg / mL, the cell viability of HUVEC is still over 90% after 24 h of co-incubation, indicating that both Gd-BSA and PFCE@Gd-BSA have good biocompatibility.

[0172] In addition, the experimental results of Examples 5 to 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0173] Experimental Example 11 In vivo 19 F MRI studies

[0174] 1. Experimental methods

[0175] Taking the PFCE@Gd-BSA obtained in Example 4 as a representative test object, 100 μL of PFCE@Gd-BSA magnetic resonance contrast agent (PFCE: 20 mM) was injected into tumor-bearing mice by intratumoral injection. After the injection, the mice were anesthetized and imaged using an ultra-high field 9.6 T magnetic resonance imaging device. 19 F MRI.

[0176] 2. Experimental results

[0177] The results are as follows Fig.14 As shown in the figure, after intratumoral injection of PFCE@Gd-BSA magnetic resonance contrast agent, obvious 19 F magnetic resonance signal, the signal-to-noise ratio is close to 20. This shows that PFCE@Gd-BSA magnetic resonance contrast agent can achieve good tumor 19 F Magnetic resonance imaging.

[0178] In addition, the experimental results of Examples 5 to 7 are substantially the same as those of Example 4 and will not be described in detail herein.

[0179] Experimental Example 12 Effect of Adding a Disulfide Bond Reducing Agent on the In vitro 1 Effects of H MRI

[0180] 1. Experimental methods

[0181] Taking the Gd-BSA obtained in Example 1 and the Gd-BSA obtained in Comparative Example 4 as representative test objects, the Gd-BSA solutions obtained in Example 1 and the Gd-BSA solutions obtained in Comparative Example 4 were gradiently diluted, added to a 48-well plate, and scanned under a high-field 3T nuclear magnetic resonance imaging apparatus to characterize their T1 and T2 weighted imaging effects.

[0182] 2. Experimental results

[0183] The results are as follows Fig.15 As shown in the figure, with the increase of particle concentration, the T1-weighted signal gradually increases. The longitudinal relaxation rates (r1) of Gd-BSA obtained in Example 1 and Gd-BSA obtained in Comparative Example 4 are 10.34 mM, respectively. -1 s -1 , 7.14 mM -1 s -1 ; The transverse relaxation rate (r2) is 34.06 mM -1 s -1 、13.14 mM -1 s -1 ; r2 / r1 are 3.79 mM -1 s -1 and 1.84 mM -1 s -1 This indicates that the addition of the disulfide bond reducing agent effectively enhances the binding of gadolinium ions to albumin and improves the relaxivity of Gd-BSA, making 3<r2 / r1<10, with contrast effects in both T1 and T2 modes, and can effectively achieve T1-T2 dual-mode magnetic resonance imaging.

[0184] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A nanoparticle Gd-albumin based on mineralized albumin, characterized in that: The nanoparticle Gd-albumin is prepared by the following preparation method, which comprises the following steps: S1. Disperse albumin in water, add a disulfide bond reducing agent, add a gadolinium salt solution under stirring, adjust the pH to 11-12, continue stirring until the reaction is complete, centrifuge, and take the supernatant; S2, collecting the supernatant obtained in step S1, performing gradient dialysis with an arginine solution having a pH of 7 to 8, and then dialyzing with water, collecting the dialyzed supernatant and performing freeze-drying to obtain Gd-albumin; The albumin is human serum albumin or bovine serum albumin; The molar ratio of the albumin, the disulfide bond reducing agent and the gadolinium salt is 1:(17-20):(15-20).

2. The nanoparticle Gd-albumin according to claim 1, characterized in that: The gadolinium salt is selected from one or more of gadolinium chloride, gadolinium sulfate and gadolinium nitrate.

3. The nanoparticle Gd-albumin according to claim 1, characterized in that: The disulfide bond reducing agent is selected from one or more of reduced glutathione, mercaptoethanol, dithiothreitol, mercaptoformamide, and mercaptopropionic acid.

4. The nanoparticle Gd-albumin according to claim 1, characterized in that: The particle size of the nanoparticle Gd-albumin is 30-50 nm.

5. A method for preparing F@Gd-albumin based on mineralized albumin, characterized in that: The method comprises the following steps: dispersing the nanoparticle Gd-albumin according to any one of claims 1 to 4 in water, adding perfluorocarbon and ethanol, using ultrasound to fully wrap the loaded perfluorocarbon in Gd-albumin, filtering, and taking the filtrate as F@Gd-albumin; The mass volume ratio of Gd-albumin to perfluorocarbon is 1: (0.4-2) mg / μL.

6. The preparation method according to claim 5, characterized in that: The perfluorocarbon is selected from one or more of perfluoro-15-crown-5-ether, perfluorooctyl bromide, perfluorotributylamine, and perfluoropolyether.

7. A F@Gd-albumin based on mineralized albumin, characterized in that: Prepared by the preparation method of claim 5 or 6.

8. Use of the nanoparticle Gd-albumin according to any one of claims 1 to 4 or the F@Gd-albumin according to claim 7 in the preparation of a contrast agent.

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