CN105816887B - A kind of CT contrast agent and its preparation method and application based on Pt nano particle@BSA biomimetic materials - Google Patents

Abstract

The invention discloses a CT contrast agent based on nano-Pt@BSA bionic material and its preparation method and application. The contrast agent is composed of bovine serum albumin and nano-platinum particles. Packing, the particle diameter of the contrast agent is 1-10nm. Compared with traditional iodine-based small-molecule contrast agents in clinical applications, CT contrast agents based on nano-Pt@BSA biomimetic materials have the following characteristics: higher X-ray absorption coefficient; the bovine serum albumin-coated nano-platinum particles have good Stability, aqueous solution dispersibility, biocompatibility and longer blood residence time; easy surface functional modification; the reaction of bionic nanomaterials synthesized with protein as a template is carried out in aqueous solution, which is in line with the idea of green synthesis and can Reduce environmental pollution to a certain extent.

Description

A CT contrast agent based on nano-Pt@BSA biomimetic material and its preparation method and application

technical field

The invention belongs to the technical field of contrast agents, and in particular relates to a CT contrast agent based on a nano-Pt@BSA bionic material and its preparation method and application.

Background technique

Early diagnosis and treatment of malignant tumors is a research hotspot and difficulty in the field of biomedicine today. Molecular imaging integrates molecular biochemistry, data processing nanotechnology, image processing and other technologies, and uses imaging methods to conduct qualitative and quantitative research on biological processes in vivo at the cellular and molecular levels, and directly displays cells or molecules through images. Physiological and pathological processes at the same level, so as to non-invasively explore the early biological changes, metabolic level, genetic abnormalities, etc. of the disease, and then realize the early diagnosis of the disease, the screening of active drugs in vivo, and the direct, rapid and even real-time evaluation of the treatment effect. Because of its high specificity, high sensitivity and image resolution, it can truly provide qualitative, positioning and quantitative data for clinical diagnosis in the future. Among them, computerized tomography (CT), as an advanced non-destructive detection technology, is one of the most commonly used clinical detection methods at present. Compared with other detection technologies, it has strong penetrating power, high resolution and fast detection speed. , low price, intuitive test results, no need to contact with the tested items and many other advantages. Therefore, since it was invented in the 1970s, CT has become an important tool in medical imaging, and its application is becoming more and more extensive.

Currently, CT contrast agents widely used clinically are small iodine-containing molecules, such as iopromide and iohexol. However, due to its small molecular weight, it can be rapidly metabolized in the human kidney, resulting in a relatively short blood circulation time in vivo, which greatly limits its application in specific targeted imaging techniques and angiography. Although better and newer iodine contrast agents with low toxicity and permeability have been developed, their low X-ray absorption coefficient and incapable of targeted modification have not been really overcome. Other limitations include: (1) toxicity: nephrotoxicity in some cases, renal failure, anaphylactic shock; (2) high osmolarity and viscosity can also cause some adverse toxic side effects; (3) low iodine The molecular weight makes it have a low X-ray absorption number, which limits the improvement of its sensitivity.

In recent years, CT contrast agents based on nanomaterials have attracted extensive attention from researchers. Since 2004, most of the researches based on inorganic nanoparticles as CT imaging agents are on gold, silver, platinum, bismuth, and gadolinium nanoparticles or their complexes. Among them, platinum nanoparticles are not only stable, but also chemically inert, and have good properties in self-assembly, quantum size effects, and catalytic and biological applications. Platinum nanoparticles are used in medical diagnosis and X-ray CT imaging agents, which are summarized as follows: (1) Compared with conventional CT contrast agents, they have a higher X-ray absorption coefficient; (2) are easy to surface functional modification, such as The antibody can be modified to target the tumor; (3) it is non-toxic and has good biocompatibility. Based on the above, the present invention draws lessons from the principle of biomineralization, uses bovine serum albumin (BSA) as a biological template, and sodium borohydride as a reducing agent, and obtains a core-shell biomimetic nano-platinum composite material through a one-pot biomimetic synthesis method (Pt@BSA). The applied biological template bovine serum albumin (BSA) is a globular protein that exists in large quantities in the serum of living organisms and is widely used in nanobiotechnology, such as polymerase chain reaction (PCR), molecular imaging, drug delivery , Molecular self-assembly and assisted synthesis of metal nanoparticles. The study found that the bovine serum albumin-bound quantum dots showed good stability, which can effectively prevent the occurrence of flocculation, increase the quantum yield and reduce cytotoxicity.

Contents of the invention

The present invention aims to provide a CT contrast agent based on nano Pt@BSA biomimetic material and its preparation method and application.

Technical scheme of the present invention is:

A CT contrast agent based on nano-Pt@BSA bionic material, which is composed of bovine serum albumin (BSA) and nano-platinum particles, characterized in that the nano-platinum particles are wrapped by bovine serum albumin (BSA), and the contrast agent The particle diameter is 1-10nm.

The preparation method of the above-mentioned CT contrast agent based on the nano-Pt@BSA biomimetic material comprises the following steps:

(1) Prepare bovine serum albumin (BSA) into a solution, add chloroplatinic acid (H ₂ PtCl ₆ 6H ₂ O) solution, and stir for 5-30 minutes;

(2) Prepare sodium borohydride (NaBH ₄ ) into a solution and place it in an ice bath;

(3) Add sodium borohydride (NaBH ₄ ) solution into bovine serum albumin (BSA) and chloroplatinic acid solution, and stir for 0.5-6h;

(4) dialysis and freeze-drying treatment to obtain solid powder of nano-platinum particles wrapped by bovine serum albumin.

The molar ratio of the chloroplatinic acid (H ₂ PtCl ₆ .6H ₂ O) to bovine serum albumin (BSA) is 25:1-200:1;

The molar ratio of platinum in the sodium borohydride (NaBH ₄ ) and chloroplatinic acid (H ₂ PtCl ₆ ·6H ₂ O) is 2:1-6:1.

The bovine serum albumin-wrapped nano-platinum particles prepared by the present invention have good stability, aqueous solution dispersibility and biocompatibility, show good CT imaging effects, and have excellent in vivo vascular imaging and tumor CT contrast effects in vitro ( Such as lung cancer cell imaging), therefore, has potential application prospects of CT contrast.

The Pt@BSA nanoparticle synthesized by the present invention is just a platinum nanoparticle assembly wrapped by BSA, which has a higher X-ray absorption coefficient, good biocompatibility, and is easy to modify and connect a large number of targets compared with traditional clinical iodine agents. To the group and other characteristics. The exterior of the nanoparticles is wrapped by BSA molecules, which makes the material have good biocompatibility, and based on the rich functional groups (such as amino, carboxyl, hydroxyl, etc.) of the BSA molecule itself, it can also serve as a multifunctional interface for targeting specific modification of the molecule. In addition, the synthesis of nanomaterials by green biomimetic methods has attracted extensive attention and has great potential application prospects. Using proteins as templates to synthesize nanomaterials has many advantages, such as good biological safety and stability, and is suitable for biological imaging research. In conclusion, the method of the present invention is simple and feasible, and using bovine serum albumin as a template for the synthesis of platinum nanomaterials has some obvious advantages. First of all, the synthesis of the material is carried out in aqueous solution, and the process does not use organic solvents, which can reduce environmental pollution to a certain extent. Secondly, as a macromolecule with good biocompatibility, bovine serum albumin protein has good biocompatibility and high biosafety, and is easy to modify the surface, so that it can be used as a template to synthesize nanostructured materials. The field of biomedical imaging has a wide range of potential applications.

Compared with traditional iodine-based small-molecule contrast agents (such as iopromide and iohexol) in clinical use, CT contrast agents based on nano-Pt@BSA biomimetic materials have the following characteristics:

1. Higher X-ray absorption coefficient. A high X-ray attenuation coefficient means higher contrast enhancement at lower dose levels.

2. The bovine serum albumin-coated nano-platinum particles have good stability, aqueous solution dispersibility, biocompatibility and long blood residence time. For intravenous administration, any X-ray contrast agent must have high aqueous dispersibility. The contrast agent has excellent chemical properties and pharmacokinetic properties after intravenous injection, and has excellent in vivo angiography and tumor CT contrast effects in vitro (such as lung cancer cell imaging).

3. Easy surface functional modification. The outside of the contrast agent is wrapped by BSA molecules, which makes the material have good biocompatibility, and based on the rich functional groups (such as amino, carboxyl, hydroxyl, etc.) Specific modifications to molecules for targeted imaging of tumors in vitro and in vivo.

4. The reaction of biomimetic nanomaterials synthesized with protein as a template is carried out in aqueous solution, which is in line with the idea of green synthesis and can reduce environmental pollution to a certain extent.

Description of drawings

In Fig. 1, the transmission electron microscope (a) and (c), the particle size distribution diagram (b) and the ultraviolet absorption diagram (d) of the nano-Pt@BSA biomimetic material.

Figure 2 shows the cytotoxicity test results of the nano-Pt@BSA biomimetic material.

In Fig. 3, the CT image (a) and the corresponding CT value (b) of the nano-Pt@BSA biomimetic material and iopromide, in which 1 is the nano-Pt@BSA biomimetic material and 2 is iopromide.

Detailed ways

The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings to describe the technical solution of the present invention in detail.

Example:

(1) Experimental reagents

99% lyophilized bovine serum albumin (BSA, molecular weight ~66.4kDa), dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetra Triazolium bromide (MTT) was purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and 1640 cell culture medium were purchased from Shanghai Baihuishen Biotechnology Co., Ltd. Chloroplatinic acid hexahydrate (H ₂ PtCl ₆ ·6H ₂ O), sodium borohydride (NaBH ₄ ), absolute ethanol (CH ₃ CH ₂ OH) and other commercially available chemical reagents were purchased from Shanghai Chemical Reagent Company. A549 cells were provided by the Shanghai Cell Institute of the Chinese Academy of Sciences. The water used in the experiment was deionized water (18.2 MΩ).

(2) Preparation of nano-Pt@BSA biomimetic materials

30 mL of BSA solution with a concentration of 26 mg/mL was prepared by using freeze-dried bovine serum albumin and deionized water, and 30 mL of chloroplatinic acid solution with a concentration of 19.3 mM was quickly added under continuous magnetic stirring. After continuing to stir for 10 min, slowly add 0.112 g of NaBH ₄ (dissolved in 2 mL of ice water), and stir vigorously for 30 min. Then the obtained product was dialyzed for 8h, and finally the final product was dried in a freeze dryer. An appropriate amount of the prepared biomimetic nano-platinum material (Pt@BSA) coated with bovine serum albumin (Pt@BSA) was dissolved in PBS to make a solution, and the platinum content was precisely determined by ICP, and stored in a refrigerator at 4 °C until use.

Figure 1 shows the transmission electron microscope, particle size distribution diagram and ultraviolet absorption diagram of the nano-Pt@BSA biomimetic material. It can be seen from Figure 1 that the average particle size of the nano-Pt@BSA biomimetic material prepared by the present invention is about 2nm, and it can be seen that its surface is wrapped by BSA through the light and shade difference under a high-power lens. In the ultraviolet absorption spectrum, the absorption peak around 280nm is the characteristic absorption of BSA, which further proves that the platinum nanoparticles are wrapped by BSA.

(3) Biosafety study of nano-Pt@BSA biomimetic materials

MTT analysis was used to study the biocompatibility of biomimetic gold nanomaterials. First, 8000 A549 cells were inoculated in each well of two 96-well cell culture plates, and incubated overnight at 37°C, 5% CO2, when the cells were in the logarithmic phase of good growth. Then the old medium was discarded, and 100 μL of complete medium containing different concentrations of platinum (concentration gradient: 0, 0.01, 0.05, 0.1, 0.3, 0.6, 1.2, 2.5 mM) was re-added to each well, and the cells were routinely cultured for 24 hours and 48 hours. At the same time, 4 duplicate wells and a blank control group were set up. After terminating the culture, discard the supernatant, add 100 μL 1mg/mL MTT solution to each well, incubate at 37°C for 4 hours, then discard the supernatant, add 100 μL DMSO to each well, shake at 37°C for 10 minutes, and use an enzyme-linked immunosorbent assay instrument The absorbance value (OD value) of each hole at 490nm was measured, and the relationship diagram between the cell survival rate and the incubation concentration was obtained after data processing.

Figure 2 shows the survival rate of A549 cells incubated with different concentrations of Pt@BSA for 24 hours and 48 hours respectively, and the untreated cells were used as a comparison. The invention uses the MTT method to detect the activity of A549 cells after interacting with the biomimetic nano-platinum material, so as to evaluate the biosafety of the biomimetic nano-platinum material. The results showed that the biomimetic nano-platinum material had little effect on the survival rate of A549 cells, and the cell survival rate remained at about 80% even at the maximum concentration of 2.5mM. Interestingly, however, we found that the survival rate of cells increased when cells were incubated with the material for 48 hours compared with 24 hours, which may be because BSA provides a good interface for cell growth. MTT results show that the biomimetic nano-platinum material involved in the present invention has low toxicity to A549 cells, good biocompatibility, and has potential application value in biomedical imaging. Experiments also show that the nano-Pt@BSA biomimetic material involved in the present invention has good dispersibility in water, 1640 medium and PBS buffer solution (pH 7.4).

(4) Study on CT imaging performance of nano-Pt@BSA biomimetic material

Disperse Pt@BSA in PBS solution, and prepare PBS solutions with different concentration gradients of biomimetic nano-platinum materials. 1.5mLEP tube for use.

Gemstone CT (Discovery CT750HD, Milwaukee, USA) from GE Company was used to scan, and the CT imaging effects of PBS solutions at different concentrations were tested. Select FOV = 25.0cm, layer thickness = 0.625mm, scan mode, tube voltage 120kVp, mA fixed setting, select the same layer on the scanned 6 groups of images, select the region of interest of the same size, and measure the interest The CT value of the area. Images were processed using the GSI Viewer browser. Figure 3 is a comparison of CT contrast imaging between the nano-Pt@BSA bionic material and iopromide. It can be seen from the figure that the CT imaging effect of the nano-Pt@BSA bionic material involved in the present invention is much higher than that of the commonly used clinical iodine agent iopromide. When the molar concentration is 12mM, the imaging effect of the nano-Pt@BSA biomimetic material is close to three times that of the iodine agent iopromide. Moreover, the prepared nano-Pt@BSA biomimetic material has excellent in vitro tumor CT contrast effects (such as lung cancer cell imaging) and in vivo vascular imaging effects as a CT contrast agent, and has potential application prospects in the field of biomedical imaging.

Claims (3)

1. A method for preparing a CT contrast agent based on a nano-Pt@BSA biomimetic material, the CT contrast agent based on a nano-Pt@BSA biomimetic material is composed of bovine serum albumin (BSA) and nano-platinum particles, and the nano-platinum particles Wrapped by bovine serum albumin (BSA), the particle diameter of the contrast agent is 1-10nm, characterized in that the preparation method comprises the following steps: (1) Prepare bovine serum albumin (BSA) into a solution, add chloroplatinic acid (H ₂ PtCl ₆ 6H ₂ O) solution, and stir for 5-30 minutes; (2) Prepare sodium borohydride (NaBH ₄ ) into a solution and place it in an ice bath; (3) Add sodium borohydride (NaBH ₄ ) solution into bovine serum albumin (BSA) and chloroplatinic acid solution, and stir for 0.5-6h; (4) dialysis and freeze-drying treatment to obtain solid powder of nano-platinum particles wrapped by bovine serum albumin. 2. the preparation method of the CT contrast agent based on nanometer Pt@BSA biomimetic material according to claim 1, the molar ratio of described chloroplatinic acid (H ₂ PtCl ₆ 6H ₂ O) and bovine serum albumin (BSA) is 25:1-200:1; 3. The preparation method of the CT contrast agent based on nano-Pt@BSA biomimetic material according to claim 1, the platinum in the sodium borohydride (NaBH ₄ ) and chloroplatinic acid (H ₂ PtCl ₆ 6H ₂ O) The molar ratio is 2:1-6:1.