A method for preparing a non-metallic organic magnetic resonance nano-diagnostic agent

Abstract

The invention discloses a preparation method for a non-metallic organic magnetic resonance nano-diagnostic agent, comprising the following steps: adding potassium persulfate and sodium p-styrene sulfonate to double distilled water and stirring; adding tert-butyl acrylate, ethylene glycol diacrylate and 2-methyl-2-acrylic acid-2,2,6,6-tetramethyl-4-piperidinyl ester piperidine, and resuspending by water bath ultrasound to obtain TBATP nanoparticles; adding sodium tungstate and hydrogen peroxide to react, centrifuging the obtained reaction solution to remove the supernatant, and washing the precipitate with water to obtain TBATM nanoparticles; adding olsalazine sodium to obtain TBATM@OS nano-diagnostic agent. The present invention can efficiently prepare a large number of TBATP nanoparticles at one time, load olsalazine sodium, and obtain TBATM@OS nano-diagnostic agent with magnetic resonance imaging function and effective treatment of enteritis. TEMPO contained in TBATM@OS has the ability of magnetic resonance imaging and scavenging reactive oxygen ROS; olsalazine sodium can be slowly released at the lesion site, realizing the precise treatment of inflammatory bowel disease visualized by magnetic resonance imaging, and providing new ideas for the diagnosis and treatment of clinical inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-diagnostic and therapeutic agents, and in particular to a method for preparing a non-metallic organic magnetic resonance nano-diagnostic and therapeutic agent. Background Art

[0002] The development of medical imaging visualization disease treatment technology is an important trend in the medical field. It integrates medical imaging and therapeutic drugs on the same nanoparticle to achieve real-time monitoring of the disease treatment process. Therefore, the development of new imaging visualization and treatment integrated nanoparticles is of great significance to improve the effect of clinical treatment. The diagnostic technology of medical imaging is becoming more and more abundant, among which magnetic resonance imaging (MRI) is one of the most widely used technologies. At present, clinical MRI requires the use of metal gadolinium-based contrast agents to enhance the signal intensity of specific areas. However, studies in recent years have shown that gadolinium-based contrast agents have the problem of chronic toxicity to kidney and brain tissue. Long-term or repeated use of metal-based contrast agents may cause tissue fibrosis for incurable nephrogenic diseases. In addition, some early approved iron-based contrast agents have been discontinued due to their possible neurotoxicity problems. Therefore, for the development of magnetic resonance imaging visualization disease treatment technology, it is very urgent and important to seek alternative, safer and more effective contrast agents. This will help to increase the scope of application of medical imaging in disease treatment and reduce potential risks to patient health.

[0003] Currently, the evaluation of inflammatory diseases of intestinal tissue mainly relies on visual inspection with endoscopes. However, the limitation of endoscopic surgery is that tissue sampling can only cover a limited part, which has a certain impact on the ability of clinicians to detect acute intestinal inflammatory lesions early. How to accurately evaluate the lesions of intestinal mucosal tissue and track the prognosis of patients after treatment remains a challenge. MRI, as an imaging technology without ionizing radiation and insensitive to tissue depth, can non-invasively obtain detailed three-dimensional anatomical images of various internal tissues of the human body. With the development of magnetic resonance imaging technology, the temporal and spatial resolution of magnetic resonance imaging has been continuously improved by increasing the magnetic field strength, applying multi-channel coils, using ultra-fast breath-hold scanning sequences and parallel acquisition technology. Compared with CT scans, magnetic resonance imaging is more sensitive for the early detection of inflammatory bowel disease. In addition, if MRI contrast agents are injected during magnetic resonance imaging, the diagnostic sensitivity and specificity of inflammatory bowel disease can be significantly improved. This combined application can more accurately evaluate the lesions of intestinal tissue, which helps clinicians better formulate treatment plans and track the efficacy and prognosis of patients.

[0004] In order to overcome the problems of nephrotoxicity and brain deposition of MRI metal-based contrast agents in clinical diagnosis, people have begun to pay attention to the use of all-organic non-metal contrast agents to obtain magnetic resonance images. Among them, organic nitrogen oxide free radical CAs, such as 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) and its derivatives, have attracted widespread interest among researchers because they can generate MRI signals through the relaxation mechanism of water protons in body tissues. However, the development of organic nitrogen oxide CAs still faces some challenges. First, they are rapidly reduced to MRI-inactive diamagnetic hydroxylamines in the body; second, organic nitrogen oxides themselves are not sufficiently water-dispersible; finally, they have relatively low relaxation rates because there is only one unpaired electron in each nitrogen oxide, while each Mn 2+ The ion has 5 unpaired electrons, Gd 3+ The ion has 7 unpaired electrons. To overcome these challenges, some research groups have adopted chemical coupling methods to connect nitrogen oxides to polymers such as lipids, cyclodextrins, polyacetylene, chitosan, polyurethane, etc., and developed organic nitrogen oxide contrast agents. These measures are aimed at improving the stability, water dispersibility and relaxivity of contrast agents to better meet the needs of clinical diagnosis. Suchada Rajca and his team successfully enhanced the relaxivity of organic nitrogen oxide free radicals using nitrogen oxide free radical polymerization technology. They developed a nitrogen oxide functionalized brush arm polymer (Rajca, A., Y. Wang, M. Boska, et al. (2012). "Organic Radical Contrast Agents for Magnetic Resonance Imaging." Journal of the American Chemical Society 134 (38): 15724-15727.), namely BASP-ORCA1. The polymer showed excellent MR imaging effects in mouse models. In addition, other researchers further enhanced the nitroxide relaxation rate of BASP-ORCA1 through a triple nitroxide loading strategy and created BASP-ORCA3. However, both BASP-ORCA1 and BASP-ORCA3 are MRI T2 contrast agents, which essentially produce dark images and have many defects in signal reading. Therefore, how to provide bright images, prolong the circulation time in the body, improve the dispersibility in water, in vivo stability, and enhance the sensitivity of MRI is of great significance for the clinical transformation of organic nitroxides. Research in these areas will promote the further application and development of organic nitroxides in the field of MRI imaging.

[0005] TEMPO can be used not only as an MRI contrast agent, but also as a scavenger with the ability to scavenge reactive oxygen species (ROS) for the treatment of ROS-induced inflammatory diseases. However, due to its poor water solubility, its further development in vivo application is limited. In order to improve the water solubility of hydrophobic drugs, the use of nanocarrier technology to deliver TEMPO can achieve high in vivo transport efficiency and improve its bioavailability. For example, in 2019, Professor Zhang Qixiong of Southwest University and his team (Rajca, A., Y. Wang, M. Boska, et al. (2012). "Organic Radical Contrast Agents for Magnetic Resonance Imaging." Journal of the American Chemical Society 134 (38): 15724-15727.) used ROS-reactive aromatic thione (ATK) to connect the anti-inflammatory drug budesonide (Bud) and the antioxidant TEMPO to prepare a self-assembled oxidatively degradable double prodrug Bud-ATK-Tem (B-ATK-T). This nanoparticle can release anti-inflammatory drugs in response to reactive oxygen species (ROS), achieving dual functions of anti-inflammatory and antioxidant, solving the problem of TEMPO delivery in vivo. This research opens up new possibilities for the application of TEMPO in disease treatment.

[0006] Inflammatory bowel disease lesions produce excessive ROS in macrophages due to excessive inflammatory factors. These ROS constantly stimulate the inflammatory site, which not only aggravates the inflammatory response, but also may expand the lesion area. Therefore, removing excessive ROS in the lesion site has become one of the effective strategies for treating inflammatory bowel disease. In addition, Olsalazine Sodium (OS), a first-line clinical drug for the treatment of inflammatory bowel disease, has an active ingredient of 5-aminosalicylic acid, which mainly acts on the inflamed mucosa, inhibits the synthesis of prostaglandins that cause inflammation, inhibits the formation of inflammatory mediators such as leukotrienes that enhance cell membrane permeability and cause edema. However, long-term use of small molecule drugs also has certain risks, which may lead to adverse reactions such as nausea, vomiting, and liver and kidney toxicity. The method of using nano drug carriers to target small molecule drugs is to use the unique microenvironment of intestinal inflammation to target the drug carrier to the inflammatory tissue and release the drug there to treat inflammation. This method can reduce the systemic toxic side effects caused by the drug and retain the drug at the site of inflammation as much as possible, thereby greatly improving the efficiency of drug treatment.

[0007] Therefore, the development of a non-metallic magnetic resonance imaging probe containing TEMPO and an organic combination of the two can monitor the tissue distribution and dynamic changes of the drug delivery system in the body in real time, dynamically and visually. Nitrogen oxide free radicals have the ability of magnetic resonance imaging and also have the ability to capture reactive oxygen. The diagnostic and therapeutic agent can effectively coordinate drug treatment by clearing reactive oxygen in the inflammatory lesion site, realizing the integrated diagnosis and treatment function of the drug, and will provide a precise and efficient new solution for the diagnosis and treatment of IBD. Summary of the invention

[0008] The purpose of the present invention is to overcome the problems of complex preparation process, low yield and high toxicity of existing non-metallic organic magnetic resonance contrast agents, and to provide a method for preparing a non-metallic organic magnetic resonance nano-therapeutic agent (TBATM@OS) with simple steps, high efficiency and low toxicity.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention adopts the emulsion polymerization method to synthesize TBATP nanoparticles by a one-step method from TEMPO precursor and tert-butyl acrylate, and after oxidation, loads the enteritis treatment drug olsalazine sodium (OS) to construct the TBATM@OS nanodiagnostic agent. Specifically, the steps are as follows:

[0011] A method for preparing a non-metallic organic magnetic resonance nano-diagnostic agent comprises the following steps:

[0012] (1) In a reaction vessel, add an initiator, potassium persulfate, and an emulsifier, sodium p-styrene sulfonate, into double distilled water and stir for 15 to 60 minutes under nitrogen flow;

[0013] (2) under nitrogen conditions, add tert-butyl acrylate, ethylene glycol diacrylate and 2-methyl-2-acrylate-2,2,6,6-tetramethyl-4-piperidinyl ester piperidine and stir thoroughly for 2 to 5 minutes;

[0014] (3) sealing the reaction vessel, heating in an oil bath, stirring the reaction for a certain period of time, and then cooling to room temperature;

[0015] (4) The reaction solution was diluted with pure water and then dispensed into centrifuge tubes. The supernatant was removed by centrifugation. Ethanol was added to the obtained precipitate and resuspended by ultrasonication in a water bath. The precipitate was washed repeatedly. The obtained product was TBATP nanoparticles.

[0016] (5) Add a small amount of ethanol to disperse the TBATP nanoparticles, then add sodium tungstate and hydrogen peroxide, and continue stirring until the reaction is complete;

[0017] (6) diluting the reaction solution obtained in step (5) with pure water and then placing it in centrifuge tubes, removing the supernatant by centrifugation, and repeatedly washing the precipitate with water to obtain the TBATM nanoparticles;

[0018] (7) Disperse TBATM nanoparticles in water, add olsalazine sodium, stir at room temperature, centrifuge to remove the supernatant, wash with water, and the resulting product is the TBATM@OS nano-diagnostic agent.

[0019] Preferably, in the above-mentioned preparation method, the mass ratio of potassium persulfate, sodium p-styrene sulfonate and double distilled water in step (1) is 1:2:2000.

[0020] Preferably, in the above-mentioned preparation method, the mass volume ratio of tert-butyl acrylate, ethylene glycol diacrylate and 2-methyl-2-acrylic acid-2,2,6,6-tetramethyl-4-piperidinyl ester piperidine in step (2) is 0.01 mL:0.5 mL:0.5 g.

[0021] Preferably, in the above preparation method, the temperature of the oil bath heating in step (3) is 70° C., the stirring speed is 600 rpm, and the stirring time is 3 hours.

[0022] Preferably, in the above preparation method, the mass volume ratio of the TBATP nanoparticles to ethanol in step (5) is 20 mg:1 mL; the mass volume ratio of sodium tungstate to hydrogen peroxide is 1 mg:1 mL; the stirring speed is 300 rpm; and the stirring time is 72 hours.

[0023] Preferably, in the above-mentioned preparation method, the mass ratio of the TBATM nanoparticles to olsalazine sodium in step (7) is 10:1, the stirring speed is 300 rpm, and the stirring time is 12 hours.

[0024] Compared with the prior art, the present invention has the following beneficial effects: the present invention prepares 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO) precursor small molecules into nanoparticles by a one-step emulsion polymerization method, and can efficiently prepare a large number of TBATP nanoparticles at one time. After the particles are oxidized, the drug olsalazine sodium (OS) for treating enteritis is loaded to obtain a TBATM@OS nanodiagnostic agent with magnetic resonance imaging (MRI) function and effective treatment of enteritis. The TEMPO contained in TBATM@OS has the ability of magnetic resonance imaging and scavenging reactive oxygen species (ROS); on the other hand, the loaded anti-inflammatory drug olsalazine sodium can be slowly released at the lesion site, realizing the precise treatment of inflammatory bowel disease visualized by magnetic resonance imaging, and providing new ideas for the diagnosis and treatment of clinical inflammatory bowel disease.

[0025] The present invention uses emulsion polymerization to prepare organic TEMPO precursor small molecules into nanoparticles. After TBATP oxidation, nanoparticles with magnetic resonance imaging function and free radical scavenging ability are obtained. After loading olsalazine sodium, TBATM@OS shows a better T relaxation rate than TBATM before drug loading. The longitudinal relaxation rates (r1) of TBATM and TBATM@OS in a 7T high-field magnetic resonance system are 0.369mM and 0.72mM, respectively. -1 S -1 、0.418mM -1 S -1 The scavenging rates of hydroxyl radicals, superoxide anions and the total antioxidant rates of TBATM@OS were 97.50%, 82.99% and 89.64%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a display diagram of TBATM@OS nano-diagnostic agents and raw materials;

[0027] Figure 2 This is the transmission electron microscopy image of TBATM@OS nano-diagnostic agent;

[0028] Figure 3 is the particle size distribution diagram of TBATM@OS nanoparticles;

[0029] Figure 4 This is the infrared absorption spectrum of TBATM@OS nano-diagnostic agent and its raw materials;

[0030] Figure 5 This is the ultraviolet absorption spectrum of TBATM@OS nano-diagnostic agent and its raw materials;

[0031] Figure 6 This is the ESR spectrum of TBATM@OS nanodiagnostic agent and its raw material nitroxide free radical;

[0032] Figure 7 is the scavenging ability of TBATM@OS on reactive oxygen species (ROS);

[0033] Figure 8 (A) is TBATM 1 H-weighted MRI images, where the concentrations of TEMPO were 0, 0.25, 0.50, 1, 1.5, 2, and 2.5 mg / mL;

[0034] Figure 8 (B) for TBATM@OS 1 H-weighted MRI images, where the concentrations of TEMPO were 0, 0.25, 0.50, 1, 1.5, 2, and 2.5 mg / mL;

[0035] Figure 8(C) is the relationship between the longitudinal relaxation rate (1 / T1) of TBATM and the TEMPO concentration;

[0036] Figure 8 (D) is the relationship between the longitudinal relaxation rate (1 / T1) of TBATM@OS and the TEMPO concentration;

[0037] Fig. 9 is the cell survival rate graph;

[0038] Fig.10 (A) is the graph of serum alanine aminotransferase levels in mice administered TBATM@OS and its raw materials by tail vein;

[0039] Fig.10 (B) is the graph of serum aspartate aminotransferase levels in mice dosed with TBATM@OS and its raw materials through tail vein;

[0040] Fig.10 (C) is a graph showing the serum alkaline phosphatase content in mice after tail vein administration of TBATM@OS and its raw materials;

[0041] Fig.10 (D) is the graph of serum urea content in mice after tail vein administration of TBATM@OS and its raw materials;

[0042] Fig.10 (E) is the serum creatinine content of mice administered TBATM@OS and its raw materials by tail vein;

[0043] Fig.10 (F) Graph showing the total protein content in serum of mice administered TBATM@OS and its raw materials through tail vein. DETAILED DESCRIPTION

[0044] Example 1 Synthesis and characterization of TEMPO nanoparticles TBATM@OS

[0045] The specific steps are as follows:

[0046] (1) Preparation and color change of TBATM@OS diagnostic agent: 10 mg potassium persulfate and 20 mg sodium p-styrene sulfonate were added to 20 mL double distilled water, stirred for 30 min under nitrogen, 1 mL tert-butyl acrylate, 20 μL ethylene glycol diacrylate and 1 g 2-methyl-2-acrylic acid-2,2,6,6-tetramethyl-4-piperidinyl ester piperidine were added, stirred for 2 to 5 min, the reaction vessel was sealed, and the reaction was continued under oil bath heating for 6 hours and then cooled to room temperature. The reaction solution was diluted with 20 mL double distilled water and divided into 2 mL centrifuge tubes, centrifuged at 4 ° C and the supernatant was removed. The resulting precipitate was added with 2 mL ethanol and resuspended by ultrasonication in an ice bath. The centrifugation and washing were repeated 3 times. The resulting product was TBATP nanoparticles with a yield of 93%. 1g TBATP nanoparticles were dispersed in 1mL ethanol, and then 14.8mg sodium tungstate and 2mL hydrogen peroxide were added. The mixture was stirred for 48h under closed conditions at room temperature. 1mL hydrogen peroxide was added every 12 hours, and the solution gradually changed from milky white to orange-yellow. After the reaction was completed, the mixture was divided into 2mL centrifuge tubes, centrifuged to remove the supernatant, and the resulting precipitate was added with 2mL double distilled water and resuspended in an ice bath ultrasonic bath. The centrifugation and washing were repeated twice. The resulting product was TBATM nanoparticles with a yield of 99%. 100mg TBATM was dispersed in 1mL ethanol, 10mg olsalazine sodium (OS) was added, stirred at room temperature for 12 hours, centrifuged to remove the supernatant, resuspended with 2mL double distilled water, loaded with a 14000 dialysis membrane, and dialyzed in 500mL double distilled water for 24h to obtain a yellow-green TBATM@OS nanodiagnostic agent.

[0047] (2) Morphology and size of TBATM@OS under transmission electron microscopy: Take 10 μL of sample aqueous solution and drip it on the front of the copper mesh. After it is naturally air-dried, use a transmission electron microscope to observe the microscopic morphology (morphology and size) of the nanoparticles under a voltage of 200 KV. Figure 2 As shown, under transmission electron microscopy, TBATM@OS is concentratedly distributed, relatively regular and round, with a uniform particle size of about 200 nm.

[0048] (3) DLS and Zeta Potentiel determination of TBATM@OS: 1 mL of sample aqueous solution was added to a particle size dish and a potential dish respectively, and the particle size, polydispersity coefficient and Zeta potential of the TBATM@OS nanodiagnostic agent were measured using a Malvern particle size analyzer. Figure 3 As shown in Table 1, the hydrated particle size of TBATM@OS is about 200 nm, which is consistent with the observed TEM results, and the particle size distribution is relatively concentrated, with good dispersibility; its Zeta potential is 6.72±0.33 mv.

[0049] Table 1. Dispersibility, particle size and Zeta potential of TBATM@OS nano-theranostics

[0050]

[0051] (3) Infrared absorption spectrum measurement: 2-3 mg of raw materials and TBATM@OS solid powder were placed in a microscope infrared instrument to perform infrared absorption spectrum analysis of the material. By comparing with monomer ATP, ATM, TBATP, and TBATM, the absorption peak measured in the TBATM@OS nanodiagnostic agent is almost consistent with the characteristic absorption peak of its raw material. Among them, ATP is at 1631 cm -1 The carbon-carbon double bond absorption peak (C=C) of ATM at 1625cm-1 is clearly visible, and the displacement absorption peak disappears in the nanopolymers TBATP, TBATM and TBATM@OS. The above evidence shows that: TBATM nanoparticles contain ATP monomers, and because OS does not have a special displacement photon group, the infrared absorption spectra of TBATM@OS nanodiagnostic agent and TBATM nanoparticles are almost the same. The infrared absorption spectra of TBATM@OS nanodiagnostic agent and its raw materials are shown in the figure below. Figure 4 shown.

[0052] (4) UV absorption spectrum measurement: 1.5 mL of sample solution was added to a quartz cuvette, and the absorption spectrum of each sample was measured using a UV spectrophotometer. By comparing with the absorption spectra of each raw material, the absorption spectrum of TBATM@OS showed an absorption peak of the free radical of the ATM monomer at 462 nm, and an absorption peak of olsalazine sodium (OS) at 365 nm, indicating that the TBATM@OS nanodiagnostic agent contains the target TEMPO and OS drug molecules, such as Figure 5 shown.

[0053] (5) Electron paramagnetic resonance (ESR) spectroscopy of nitroxide free radicals: 2 mg of sample was dissolved in 1 mL of deionized water, free radical scavenger DMPO was added, and then 10 μL of sample was taken for electron paramagnetic resonance spectroscopy (EPR) test. The results are as follows: Figure 6 As shown in the figure, the nitroxide free radical peak of small molecule ATM has 6 absorption peaks at 3450-3600nm, while the TBATP after polymerization has no absorption peak of nitroxide free radical. TBATM after oxidation with hydrogen peroxide has only one large absorption peak at 3450-3600nm. This is because the nitroxide free radical signal of TEMPO molecules on the nanoparticles is high, resulting in the peak not splitting. The ESR spectrum of TBATM@OS measured in the same way shows the same signal as that of TBATM, indicating that TBATM@OS contains TEMPO.

[0054] (6) Test of TBATM@OS's ability to scavenge reactive oxygen species (ROS): According to the instructions of the hydrogen peroxide fluorescence assay kit (near-infrared fluorescence), H2O2 working solution and H2O2 standard solution were prepared, and 0.5 mL of TBATM@OS sample solution and H2O2 standard solution with different concentration gradients were added to a 96-well plate. At the same time, blank wells were prepared. 50 μL of H2O2 working solution was added to each well, the well plate was sealed, and incubated at room temperature for 30 min; the fluorescence intensity was detected at Ex / Em=640 / 680nm (Cutoff=665nm) using a fluorescence spectrophotometer, and the scavenging efficiency of TBATM@OS for scavenging H2O2 was calculated. The absorbance value of each group was read at 550nm using a multifunctional microplate reader, and the scavenging efficiency of TBATM@OS for scavenging O2·- was calculated. According to the instructions of the hydroxyl radical test kit, the substrate application solution, 0.03% standard application solution, reagent three application solution and color developer were prepared; 0.2mL distilled water was added to the standard tube and the control tube respectively, and 0.4mL distilled water was added to the blank tube; 0.2mL 0.03% standard application solution was added to the standard tube, and then 0.2mL substrate application solution was added to the control tube and the measurement tube respectively; 0.2mL TBATM@OS sample solution with different concentration gradients was added to the measurement tube; in addition, 0.4mL reagent three application solution was added to the blank tube, standard tube, control tube and measurement tube, the EP tube was covered, and incubated at 37℃ for 1min; 2mL color developer was added to the blank tube, standard tube, control tube and measurement tube to terminate the reaction, mixed, and placed at 25℃ for 20min. The absorbance value of each group was read at 550nm using a multifunctional microplate reader, and the scavenging efficiency of TBATM@OS in scavenging ·OH was calculated. Prepare ABTS working solution and Trolox standard solution according to the instructions of the total antioxidant capacity test kit (ABTS method); add 200 μL of ABTS working solution to each test well in the 96-well plate; add 10 μL of distilled water to the blank control well; add 10 μL of Trolox standard solution of various concentrations to the standard curve test well; add 10 μL of various samples to the sample test well and mix gently. Incubate at 25°C for 5 minutes. Use a multifunctional microplate reader to read the absorbance value of each group at 734 nm and calculate the total antioxidant capacity of TBATM@OS. The scavenging ability of TBATM@OS for reactive oxygen species (ROS) is shown in Figure 2. Figure 7 shown.

[0055] (7) In vitro magnetic resonance imaging study: The TBATM@OS aqueous solution was diluted in a gradient manner and added to a 1 mL syringe tube prepared in advance. The piston was removed and the end was sealed with a sealing film (be careful not to have bubbles in the tube). The tube was scanned under an ultra-high field 7T magnetic resonance imaging instrument to characterize its T1-weighted imaging effect. Figure 8As shown in Figure 2, the longitudinal relaxation rates of TBATM and TBATM@OS are 0.369 mM -1 S -1 、0.418mM -1 S -1 .like Figure 8 The A and B mapping images in the figure show that the T1-weighted signal gradually increases with the increase of particle concentration, and because TBATM@OS loaded with the hydrophilic drug OS exhibits a higher relaxation rate than TBATM, it indicates that TBATM is an excellent carrier and TBATM@OS can be used as a T1 contrast agent for magnetic resonance imaging.

[0056] (8) In vitro biocompatibility study: The effect of TBATM@OS on the viability of RAW264.7 (macrophages) and NCM460 (normal human colon epithelial cells) was investigated by MTT colorimetry. The absorbance value A at 570 nm was detected by an ELISA instrument. The cell viability was calculated according to formula (2):

[0057]

[0058] like Fig. 9 As shown in the figure, as the sample concentration increases, the cell viability of RAW 264.7 and NCM460 remains at a high level. When the concentration of TBATM@OS is as high as 1000 μg / mL, the cell viability of RAW264.7 and NCM460 is still over 80% after 24 hours of co-incubation, indicating that TBATM@OS has good biocompatibility and low toxicity. Fig. 9 Cell viability chart of (A) RAW 264.7 (B) NCM460 cells incubated with different concentrations of TBATP, TBATM, and TBATM@OS for 24 hours.

[0059] (9) In vivo biocompatibility study: TBATM@OS was dispersed in sterile PBS to prepare a 2.5 mg / mL TBATM@OS solution, which was then administered to Balb / c mice with inflammatory bowel disease via the tail vein (100 μL; 2.5 mg / mL) once every 2 days for a total of 4 times. Fig.10 As shown, the evaluation of serum biochemical indexes showed that the liver and kidney function indicators of mice were normal, indicating that TBATM@OS has good biocompatibility and low toxicity.

Claims

1. A method for preparing a non-metallic organic magnetic resonance nano-diagnostic agent, Features The steps include: Add 10 mg of potassium persulfate and 20 mg of sodium p-styrene sulfonate to 20 mL of double distilled water, stir for 30 min under nitrogen, add 1 mL of tert-butyl acrylate, 20 μL of ethylene glycol diacrylate and 1 g of 2-methyl-2-acrylic acid-2,2,6,6-tetramethyl-4-piperidinyl ester, stir well for 2-5 min, seal the reaction vessel, continue stirring and react for 6 hours under oil bath heating, and then cool to room temperature; The reaction solution was diluted with 20 mL of double distilled water and then dispensed into 2 mL centrifuge tubes. The supernatant was removed by centrifugation at 4°C. The resulting precipitate was added with 2 mL of ethanol and resuspended by ultrasonication in an ice bath. The centrifugation and washing were repeated 3 times. The resulting product was TBATP nanoparticles. 1 g of TBATP nanoparticles was dispersed in 1 mL of ethanol, and then 14.8 mg of sodium tungstate and 2 mL of hydrogen peroxide were added. The mixture was stirred in a sealed condition at room temperature for 48 h, and 1 mL of hydrogen peroxide was added every 12 hours. The solution gradually changed from milky white to orange-yellow. After the reaction was completed, the mixture was divided into 2 mL centrifuge tubes, centrifuged to remove the supernatant, and the resulting precipitate was added with 2 mL of double distilled water and resuspended by ultrasonication in an ice bath. The centrifugation and washing were repeated twice, and the resulting product was TBATM nanoparticles. Disperse 100 mg TBATM in 1 mL of ethanol, add 10 mg of olsalazine sodium, stir at room temperature for 12 hours, centrifuge to remove the supernatant, resuspend with 2 mL of double distilled water, load with 14000 dialysis membrane, and dialyze in 500 mL of double distilled water for 24 hours to obtain the yellow-green TBATM@OS nanodiagnostic and therapeutic agent.

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