Ultra-small size CeO2-Gd@BSA-cRGD targeted nanoparticles and green preparation method thereof

Abstract

The application provides an ultrasmall size CeO2-Gd@BSA-cRGD targeted nanoparticle and a green preparation method thereof, and belongs to the field of nanomedicine. The preparation method comprises the following steps: under stirring, a cerium salt solution is added into a BSA-cRGD solution prepared in step (1), then a gadolinium salt solution is added, after stirring, a mixed solution is obtained; under stirring, a sodium hydroxide solution is added into the mixed solution, the pH of the system is adjusted to 11-12, stirring reaction is carried out, dialysis purification is carried out, and the CeO2-Gd@BSA-cRGD targeted nanoparticle is obtained. The method is simple, easy to control, green, mild and suitable for large-scale production; the nanoparticle size is ultrasmall and the dispersity is good; the targeted tumor tissue permeability is better; the kidney metabolism can be carried out, and the side effects are reduced.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to an ultra-small CeO2-Gd@BSA-cRGD targeted nanoparticle and its green preparation method. Background Technology

[0002] Nanozymes are a new type of artificial enzyme that combines the properties of nanomaterials and natural enzymes. CeO2 nanozymes are rich in Ce. 3+ / Ce 4+ The redox pair of CeO2 nanozymes gives them various enzyme-mimicking activities, such as peroxidase, catalase, and glucose oxidase, making them a focus of scientific research in recent years. Currently, CeO2 nanozymes have been applied in various medical fields, including oncology, neurology, ophthalmology, cardiology, chronic inflammation, diabetes, and hepatology. However, the current synthesis of nano-CeO2 mainly relies on hydrothermal synthesis, which is subject to harsh reaction conditions, requires sophisticated equipment, uses organic solvents as surfactants, involves high reaction temperatures, and is cumbersome, significantly limiting its large-scale production. Patent CN 114261983 B discloses a layered, flattened spherical cerium oxide material, its preparation method, and its applications. This invention uses a cerium source, an alkali source, and a surfactant to prepare cerium oxide via hydrothermal synthesis at 100-200℃. The preparation process requires high reaction temperatures, and the introduction of organic solvents has adverse environmental impacts. Patent CN 115417443 B discloses nano-cerium oxide particles, their green synthesis process, and their applications. This invention uses plant extracts as a reducing agent and capping agent to reduce cerium trichloride to synthesize nanoscale particles. This process requires repeated heating and cooling for 60 minutes, followed by calcination in a muffle furnace at 280-400℃ for 2-6 hours. Although this preparation method is pollution-free, the synthesis process is cumbersome and requires significant investment in equipment and skilled operators.

[0003] Magnetic resonance imaging (MRI) has been widely used in disease diagnosis and medical research. Clinically, T1-weighted contrast agents are widely used because they shorten the longitudinal relaxation time of protons in surrounding water, thereby enhancing image brightness on T1-weighted images, which is beneficial for disease diagnosis and differential diagnosis. Currently, the main T1-weighted contrast agents used clinically are small molecules based on gadolinium chelates, which have no specificity for tumor tissue, rapid in vivo clearance, and nephrotoxicity.

[0004] Integrin α νβ3 is a transmembrane glycoprotein receptor heterodimer involved in regulating angiogenesis, tumor proliferation, invasion, and metastasis. It is selectively overexpressed in many malignant tumor cells (including TNBC) and tumor vascular endothelial cells, but is expressed at low levels or not at all in normal tissues. Therefore, integrin α... ν β3 is an important target for TNBC imaging and therapy. Cyclic arginine, glycine, and aspartic peptide (cRGD) are α-targets. ν β3-specific ligands can interact with α ν It binds to the β3 integrin receptor and targets tumor cells with high affinity.

[0005] Therefore, preparing cRGD-targeted, gadolinium-doped, ultra-small, and well-dispersed cerium oxide nanoparticles through a simple and environmentally friendly method for the diagnosis and treatment of TNBC has significant medical application value. Summary of the Invention

[0006] The purpose of this invention is to provide ultra-small CeO2-Gd@BSA-cRGD targeted nanoparticles and their green preparation method. The preparation method is simple and easy to control. The nanoparticles are ultra-small in size and have good dispersibility. The TEM particle size is 2.5-3.5 nm, the hydrated particle size is 4-7 nm, the targeting permeability to tumor tissue is better, and they can be metabolized by the kidneys, reducing side effects.

[0007] A green preparation method for ultra-small CeO2-Gd@BSA-cRGD targeted nanoparticles includes the following steps:

[0008] (1) Disperse bovine serum albumin (BSA) in distilled water and activate it with sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (sulfon-SMCC). Then add arginine-glycine-aspartic acid-D-phenylalanine-cysteine ​​(Arg-Gly-Asp-D-Phe-Cys, cRGDfc) and stir to obtain a solution of cRGD conjugated BSA (BSA-cRGD).

[0009] (2) Under stirring conditions, the cerium salt solution is added to the BSA-cRGD solution prepared in step (1), followed by the addition of the gadolinium salt solution. After stirring, a mixed solution is obtained.

[0010] (3) Under stirring conditions, sodium hydroxide solution was added to the mixed solution in step (2), the pH of the system was adjusted to 11-12, and the stirring reaction was carried out under alkaline-induced redox environment. After the stirring reaction was completed, the solution after the reaction was purified by dialysis to obtain gadolinium-doped cerium oxide nanoparticles (CeO2-Gd@BSA-cRGD targeted nanoparticles) encapsulated by BSA-cRGD.

[0011] Preferably, BSA is dispersed in distilled water, and the mass concentration of BSA is 2-4 mg / mL; the mass ratio of BSA, sulfonyl-SMCC and cRGDfc is (30-50):(10-20):(5-10).

[0012] Preferably, the amount of cerium salt used is 10-40 μmol, the atomic molar ratio of Ce to Gd is 1:(0.05-0.36), and the mass ratio of BSA to Ce is (12-13):1. By controlling the amount of raw materials, it is beneficial to prepare nanoparticles with ultra-small particle size.

[0013] Preferably, in step (1), the activation temperature is 4°C and the activation time is 1-4h; the stirring temperature is 4°C and the stirring time is 24-48h.

[0014] Preferably, in step (2), the cerium salt is CeCl3·6H2O or Ce(NO3)3·6H2O, and the gadolinium salt is GdCl3·6H2O or Gd(NO3)3·6H2O.

[0015] Preferably, in step (2), the stirring time is 5-10 min.

[0016] Preferably, in step (3), the stirring reaction temperature is 25-37℃ and the stirring time is 5-6h.

[0017] Preferably, in step (3), the molecular weight of the dialysis membrane is 10-14 kDa.

[0018] The ultra-small CeO2-Gd@BSA-cRGD targeted nanoparticles prepared by the green preparation method have cerium oxide nanoparticles as the core and Gd 3+ BSA is doped into the cerium oxide lattice and the surface of nano-cerium oxide is targeted by cRGD.

[0019] Preferably, the TEM particle size of the CeO2-Gd@BSA-cRGD targeted nanoparticles is 2.5-3.5 nm; the hydrated particle size of the CeO2-Gd@BSA-cRGD targeted nanoparticles is 4-7 nm.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention provides CeO2-Gd@BSA-cRGD nanoparticles and their green preparation method. Compared with existing technologies, these nanoparticles are assembled using bovine serum albumin as a template, with the pH of the system adjusted to 11-12, under alkaline conditions induced by redox environment. Furthermore, cRGD targeting molecules are modified on their surface, resulting in high enzyme activity and the ability to actively target integrin α. νβ3, thus more effectively enriched in TNBC tumor regions; moreover, the CeO2-Gd@BSA-cRGD nanoparticles prepared by this method are ultra-small in size and have good dispersibility, with a TEM particle size of 2.5-3.5 nm and a hydrated particle size of 4-7 nm, which improves the penetration into tumor tissues and can be metabolized by the kidneys, reducing side effects.

[0022] 2. The preparation method of the present invention is simple, the reaction conditions are easy to control, and the cost is low. The reaction process does not involve organic solvents, making it suitable for large-scale production and showing good prospects for clinical medical translational applications.

[0023] 3. The CeO2-Gd@BSA-cRGD nanoparticles prepared in this invention, by introducing gadolinium into cerium oxide nanoparticles, not only endow cerium oxide nanoparticles with MR imaging performance, enabling non-invasive, real-time, in vivo visualization of tumors, but also improve the catalytic activity and ability to generate reactive oxygen species of cerium oxide nanoparticles, further enhancing the anti-tumor performance of cerium oxide nanoparticles. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The image shows the XRD pattern of CeO2-Gd@BSA-cRGD nanoparticles in Example 3.

[0026] Figure 2 The hydration particle size distribution of CeO2-Gd@BSA-cRGD nanoparticles in Example 3 is shown.

[0027] Figure 3 This is a TEM image of the CeO2-Gd@BSA-cRGD nanoparticles in Example 3;

[0028] Figure 4 The UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-H2O2 (1.25-20mM) in Example 3 and the corresponding steady-state kinetic analysis diagrams of the Michaelis-Menten model are shown.

[0029] Figure 5UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-glucose (1.25-20 mM) and corresponding steady-state kinetic analysis using the Michaelis-Menten model;

[0030] Figure 6 The relaxation coefficient r1 of CeO2-Gd@BSA-cRGD nanoparticles in Example 3 is related to Gd. 3+ Concentration relationship graph;

[0031] Figure 7 The images shown are magnetic resonance T1WI images of CeO2-Gd@BSA-cRGD nanoparticle dispersions with different gadolinium ion concentrations in Example 3.

[0032] Figure 8 Figure 1. Detection results of CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD nanoparticles provided by the present invention for BEAS-2B cytotoxicity analysis.

[0033] Figure 9 Figure showing the results of CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD nanoparticles provided by this invention used in 4T1 cytotoxicity analysis.

[0034] Figure 10 The UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-H2O2 (10 mM) in Comparative Example 1 are shown.

[0035] Figure 11 The hydration particle size distribution of CeO2-Gd@BSA-cRGD nanoparticles in Comparative Example 2 is shown.

[0036] Figure 12 This is a SEM image of CeO2 nanoparticles in Comparative Example 3. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] A green preparation method for ultra-small CeO2-Gd@BSA-cRGD nanoparticles includes the following steps:

[0039] (1) Disperse BSA in distilled water under magnetic stirring. The mass concentration of BSA is 2-4 mg / mL. Add sulfonyl-SMCC for activation. The activation temperature is preferably 4℃ and the activation time is preferably 1-4 h. Then add cRGDfc and stir. The stirring temperature is 4℃ and the stirring time is preferably 24-48 h to obtain BSA-cRGD solution. The mass ratio of BSA, sulfonyl-SMCC and cRGDfc is (30-50):(10-20):(5-10).

[0040] (2) Add the cerium salt solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add the gadolinium salt solution. Stir for 5-10 minutes to obtain a mixed solution.

[0041] The cerium salt is preferably CeCl3·6H2O or Ce(NO3)3·6H2O, and the gadolinium salt is preferably GdCl3·6H2O or Gd(NO3)3·6H2O. The amount of cerium salt in the mixed solution is 10-40 μmol, the atomic molar ratio of Ce to Gd is 1:(0.05-0.36), and the mass ratio of BSA to Ce is (12-13):1.

[0042] (3) Add sodium hydroxide solution to the solution in step (2) under magnetic stirring, adjust the pH of the system to 10-11, the NaOH solution concentration is preferably 1M (mol / L), and stir continuously under alkaline-induced redox environment, the stirring temperature is preferably 25-37℃, the stirring time is preferably 5-6h, after the stirring reaction is completed, the solution after the reaction is purified by dialysis to obtain gadolinium-doped cerium oxide nanoparticles (CeO2-Gd@BSA-cRGD targeted nanoparticles) wrapped with BSA-cRGD, the dialysis membrane has a molecular weight of 10-14kDa, and the CeO2-Gd@BSA-cRGD targeted nanoparticles purified by dialysis are freeze-dried and stored.

[0043] The prepared CeO2-Gd@BSA-cRGD targeted nanoparticles, with cerium oxide nanoparticles as the core and Gd... 3+ Doped in cerium oxide lattice, with cRGD-targeted bovine serum albumin coated on the surface.

[0044] The present invention will be further illustrated below with reference to the embodiments, but the present invention is not limited to the embodiments described below.

[0045] Example 1

[0046] (1) Weigh 30mg BSA and add it to 10mL of distilled water under magnetic stirring. Add 10mg sulfonyl-SMCC to activate for 1h, then add 5mg cRGDfc and stir at 4℃ for 24h to obtain BSA-cRGD solution.

[0047] (2) Add (100mM, 168uL)Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add (100mM, 9uL)Gd(NO3)3·6H2O solution. Stir for 5 min to obtain a mixed solution.

[0048] (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6h after adjustment. After the reaction is completed, purify the solution by dialysis (dialysis membrane molecular weight 10-14kDa) to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles. After dialysis purification, CeO2-Gd@BSA-cRGD nanoparticles are freeze-dried and stored.

[0049] Example 2

[0050] (1) Weigh 30mg BSA and add it to 10mL of distilled water under magnetic stirring. Add 10mg sulfonyl-SMCC to activate for 1h, then add 5mg cRGDfc and stir at 4℃ for 24h to obtain BSA-cRGD solution.

[0051] (2) Add (100mM, 168uL)Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add (100mM, 25uL)Gd(NO3)3·6H2O solution. Stir for 8 min to obtain a mixed solution.

[0052] (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6h after adjustment. After the reaction is completed, purify the solution by dialysis (dialysis membrane molecular weight 10-14kDa) to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles. After dialysis purification, CeO2-Gd@BSA-cRGD nanoparticles are freeze-dried and stored.

[0053] Example 3

[0054] (1) Weigh 30mg BSA and add it to 10mL of distilled water under magnetic stirring. Add 10mg sulfonyl-SMCC to activate for 1h, then add 5mg cRGDfc and stir at 4℃ for 24h to obtain BSA-cRGD solution.

[0055] (2) Add (100mM, 168uL)Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add (100mM, 59uL)Gd(NO3)3·6H2O solution. Stir for 10 min to obtain a mixed solution.

[0056] (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6h after adjustment. After the reaction is completed, purify the solution by dialysis (dialysis membrane molecular weight 10-14kDa) to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles. After dialysis purification, CeO2-Gd@BSA-cRGD nanoparticles are freeze-dried and stored.

[0057] Figure 1 The XRD pattern of CeO2-Gd@BSA-cRGD nanoparticles in Example 3 confirms the formation of CeO2 crystals in the CeO2-Gd@BSA-cRGD nanoparticles, exhibiting three characteristic peaks consistent with the diffraction peaks of standard card PDF#43-1002, corresponding to the (111), (220), and (311) planes of the crystal structure, respectively. No diffraction peaks corresponding to Gd or Gd2O3 were observed in the XRD pattern, indicating that Gd... 3+ It is completely doped within the CeO2 lattice without altering the structure of CeO2.

[0058] Figure 2 The image shows a TEM image of the CeO2-Gd@BSA-cRGD nanoparticles from Example 3, indicating that the CeO2-Gd@BSA-cRGD nanoparticles have a uniform morphology, good dispersibility, and a particle size of 3.30 ± 0.13 nm. Figure 2 It is known that the preparation method of the present invention can synthesize ultra-small CeO2-Gd@BSA-cRGD nanoparticles. This small size makes it easier for the nanoparticles to enter cancer cells and can be metabolized and excreted by the kidneys, reducing the toxic side effects of the nanoparticles.

[0059] Figure 3 The image shows the hydrated particle size distribution of CeO2-Gd@BSA-cRGD nanoparticles in Example 3, with a hydrated particle size of approximately 5.03 ± 0.29 nm.

[0060] Figure 4 The UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-H2O2 (1.25-20 mM) in Example 3, along with the corresponding steady-state kinetic analysis using the Michaelis-Menten model, are shown. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) are 38.4 × 10⁻⁶.-8 Ms -1 And 1.88mM.

[0061] Figure 5 The UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-glucose (1.25-20 mM) and the corresponding steady-state kinetic analysis using the Michaelis-Menten model are shown. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) are 1.67 × 10⁻⁶. -6 Ms -1 And 0.7mM.

[0062] Figure 6 The relaxation coefficient r1 of CeO2-Gd@BSA-cRGD nanoparticles in Example 3 is related to Gd. 3+ The concentration relationship graph, obtained by linear fitting, yielded an r1 value of 3.047 ± 0.09 mM for CeO2-Gd@BSA-cRGD. -1 S -1 This indicates that the nanoparticles have a T1WI enhancement effect.

[0063] Figure 7 In Example 3, based on different Gd 3+ The magnetic resonance T1WI images of the CeO2-Gd@BSA-cRGD nanoparticle dispersion at a certain concentration indicate that the nanoparticles do indeed have a T1WI enhancement effect.

[0064] Figure 8 This study describes the in vitro MTT assay for BEAS-2B normal lung epithelial cell cytotoxicity. 8000 cells per well were seeded in 96-well plates and incubated for 12 h. Then, different concentrations (0, 25, 50, 100, 200, and 400 μg / mL) of unmodified cRGD-targeted CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD nanoparticle dispersions were added, and the cells were cultured for another 24 h. Figure 8 As shown, even at a concentration of 400 μg / mL, the relative cell viability still reached 88.96±8.71% and 89.88±5.25%, indicating that both CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD nanoparticles have good biocompatibility.

[0065] Figure 9This study used an in vitro MTT assay for 4T1 triple-negative breast cancer cytotoxicity. 8000 cells per well were seeded in 96-well plates and incubated for 12 hours. Then, different concentrations (0, 25, 50, 100, 200, and 400 μg / mL) of unmodified cRGD-targeted CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD nanoparticle dispersions were added, and the cells were cultured for another 24 hours. For the Blocking assay, free cRGD was pre-incubated for 3 hours, followed by the addition of different concentrations (0, 25, 50, 100, 200, and 400 μg / mL) of CeO2-Gd@BSA-cRGD nanoparticle dispersions, and the cells were cultured for another 24 hours. Figure 9 As shown, with increasing CeO2-Gd@BSA and CeO2-Gd@BSA-cRGD concentrations, the cell viability of 4T1 cells gradually decreased. This is attributed to the chemokinetic therapy and starvation therapy resulting from the interaction of endogenous H2O2 and glucose with the nanoparticles in 4T1 cells. At the same concentration, the cell viability of 4T1 cells in the CeO2-Gd@BSA-cRGD targeted group was significantly lower than that in the non-targeted CeO2-Gd@BSA group, mainly due to the greater uptake of CeO2-Gd@BSA-cRGD nanoparticles by 4T1 cells. The cell viability of the blocking group was increased compared to the CeO2-Gd@BSA-cRGD targeted group, further indicating that CeO2-Gd@BSA-cRGD actively targets α-cells. V β3 receptor.

[0066] Therefore, CeO2-Gd@BSA-cRGD targeted nanoparticles can be used as peroxidase and glucose oxidase in the treatment of triple-negative breast cancer cells; CeO2-Gd@BSA-cRGD nanoparticles can be used as multifunctional therapeutic nanoparticles to prepare agents for the detection and treatment of triple-negative breast cancer.

[0067] Compared to Example 3, if BSA is not added, the toxic side effects of CeO2-Gd@BSA-cRGD nanoparticles will increase; if Gd and cRGD are not added, the enzyme activity of CeO2-Gd@BSA-cRGD nanoparticles will decrease, and the uptake capacity of 4T1 cells will also decrease, resulting in a decrease in the killing ability of 4T1 cells.

[0068] Comparative Example 1

[0069] (1) Weigh 30mg BSA and add it to 10mL of distilled water under magnetic stirring. Add 10mg sulfonyl-SMCC to activate for 1h, then add 5mg cRGDfc and stir at 4℃ for 24h to obtain BSA-cRGD solution.

[0070] (2) Add (500mM, 168uL)Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add (100mM, 59uL)Gd(NO3)3·6H2O solution. Stir for 10 min to obtain a mixed solution.

[0071] (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6h after adjustment. After the reaction is completed, purify the solution by dialysis (dialysis membrane molecular weight 10-14kDa) to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles. After dialysis purification, CeO2-Gd@BSA-cRGD nanoparticles are freeze-dried and stored.

[0072] Figure 10 To compare the UV-Vis spectra of CeO2-Gd@BSA-cRGD nanoparticles and TMB-H2O2 (10mM) in Example 1, compared with Example 3, the UV-Vis absorption value of oxTMB at a wavelength of 652nm decreased.

[0073] Comparative Example 2

[0074] (1) Weigh 30 mg BSA and add it to 10 mL of distilled water under magnetic stirring. Add 10 mg sulfonyl-SMCC to activate for 1 h, then add 5 mg cRGDfc and stir at 4 °C for 24 h to obtain BSA-cRGD solution.

[0075] (2) Add (700mM, 168uL)Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add (100mM, 59uL)Gd(NO3)3·6H2O solution. The stirring time is preferably 10min to obtain a mixed solution.

[0076] (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6h after adjustment. After the reaction is completed, purify the solution by dialysis (dialysis membrane molecular weight 10-14kDa) to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles. After dialysis purification, CeO2-Gd@BSA-cRGD nanoparticles are freeze-dried and stored.

[0077] Figure 11 The image shows the hydrated particle size distribution of CeO2-Gd@BSA-cRGD nanoparticles in Comparative Example 2. The hydrated particle size is approximately 100.45 ± 10.85 nm, which is significantly larger than that in Example 3.

[0078] Comparative Example 3

[0079] (1) Weigh 40 mg Ce(NO3)3 and 30 mg hexamethylenetetramine and mix them with 50 mL of distilled water to obtain a clear solution;

[0080] (2) Add the clear solution to a polytetrafluoroethylene-lined reactor, heat to 180°C, and continue for 12 hours;

[0081] (3) Wash with distilled water, collect the precipitate by centrifugation, and calcine in a muffle furnace at 500°C for 2 hours to obtain CeO2 nanoparticles.

[0082] Figure 12 The image shows a SEM image of CeO2 nanoparticles in Comparative Example 3. Although the nanoparticles are small, they exhibit agglomeration, resulting in poor dispersibility.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A green method for the preparation of ultra-small size CeO2-Gd@BSA-cRGD targeted nanoparticles, characterized by, Includes the following steps: (1) Weigh 30 mg BSA and add it to 10 mL of distilled water under magnetic stirring. Add 10 mg of sulfonyl-SMCC to activate for 1 h, then add 5 mg of cRGDfc and stir at 4 °C for 24 h to obtain BSA-cRGD solution. (2) Add 100 mM, 168 uL Ce(NO3)3·6H2O solution to the BSA-cRGD solution prepared in step (1) under magnetic stirring, and then add 100 mM, 59 uL Gd(NO3)3·6H2O solution. Stir for 10 min to obtain a mixed solution. (3) Add 1M NaOH solution to the above mixed solution to adjust the pH value to 11-12, and stir continuously at 37℃ for 6 h after adjustment. After the reaction is completed, purify the solution by dialysis to obtain CeO2-Gd@BSA-cRGD targeted nanoparticles, wherein the molecular weight of the dialysis membrane is 10-14 kDa; The super-small size CeO2-Gd@BSA-cRGD targeted nanoparticles take nano cerium oxide as core, Gd 3+ Doped in cerium oxide crystal lattice, the surface of nano cerium oxide cRGD targeted BSA; The ultra-small CeO2-Gd@BSA-cRGD targeted nanoparticles have a TEM particle size of 3.30 ± 0.13 nm and a hydrated particle size of 5.03 ± 0.29 nm.

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