Difunctional albumin nanoparticles, preparation method, pharmaceutical composition and application
By preparing albumin nanoparticles, modifying their surface with chelated gadolinium ions and encapsulating boron-containing compounds, the challenge of monitoring boron drug distribution in boron neutron capture therapy was solved, thereby improving the precision and safety of treatment.
Patent Information
- Application Number
- CN202511023567.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies make it difficult to achieve real-time monitoring of the distribution and enrichment of boron drugs in the body during boron neutron capture therapy, and nanoparticles simply loaded with gadolinium contrast agents cannot meet the needs of integrated treatment and monitoring.
Using albumin as a carrier, the surface is modified with a macrocyclic ligand chelating agent that chelates gadolinium ions, and a hydrophobic boron-10 compound is encapsulated inside to prepare bifunctional albumin nanoparticles, which are then combined with magnetic resonance imaging and boron neutron capture therapy.
It enables real-time distribution and enrichment monitoring of boron drugs in vivo, improving the precision of treatment, and has good drug loading capacity and tissue penetration, as well as excellent biocompatibility and safety.
Smart Images
Figure CN121015873A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to nanomedicines, and more particularly to a bifunctional albumin nanoparticle, its preparation method, pharmaceutical composition, and its application. Background Technology
[0002] Boron neutron capture therapy (BNCT) is a precise radiotherapy based on a unique nuclear reaction principle. Its core mechanism involves the capture of boron-10 (… 10 B) Specific drugs are delivered to the tumor site. When the tumor tissue is exposed to a neutron beam of appropriate energy, boron atoms capture neutrons and undergo nuclear fission, releasing highly lethal alpha particles and lithium ions at extremely close range. This energy release is strictly limited to boron-containing tumor cells, giving it excellent tumor selectivity and low systemic toxicity, making it an important research direction for treating refractory tumors.
[0003] However, the success of BNCT treatment hinges heavily on precise control of boron-containing drugs within the body, particularly in tumor tissue, and their temporal and spatial variations. Current clinical practice primarily relies on positron emission tomography (PET) to monitor boron distribution, but PET technology suffers from significant drawbacks, including high cost and the inability to provide real-time dynamic imaging. Magnetic resonance imaging (MRI), a widely used and important non-invasive diagnostic technique, possesses superior soft tissue resolution, clearly revealing internal structures and playing a crucial role in precise tumor localization, staging assessment, and dynamic monitoring of treatment efficacy. However, while nanoparticles simply loaded with gadolinium (Gd) contrast agent offer good MRI imaging capabilities, they are insufficient to meet the integrated treatment and monitoring requirements of BNCT. Summary of the Invention
[0004] Purpose of the invention: The purpose of this invention is to provide a bifunctional albumin nanoparticle with magnetic resonance imaging capability that can be used for boron neutron capture therapy. The second purpose is to provide the preparation method, pharmaceutical composition and application of the nanoparticle.
[0005] Technical solution: The bifunctional albumin nanoparticles of the present invention use albumin as a carrier, are modified with macrocyclic ligand chelating agents that chelate gadolinium ions on the surface, and are encapsulated with hydrophobic boron-10 compounds inside.
[0006] Preferably, the albumin is human serum albumin.
[0007] Preferably, the macrocyclic ligand chelating agent is a carboxyl-containing macrocyclic ligand chelating agent.
[0008] Preferably, the hydrophobic boron-containing compound is a boron-10 cholesterol derivative, Chol. 10 B.
[0009] The preparation method of the bifunctional albumin nanoparticle comprises the following steps:
[0010] (1) coupling a macrocyclic ligand chelator to albumin;
[0011] (2) mixing the albumin coupled with the macrocyclic ligand chelator with a gadolinium salt solution, removing free gadolinium ions after reaction to obtain albumin with the macrocyclic ligand chelator modified on the surface and chelated with gadolinium ions;
[0012] (3) adding a hydrophobic boron-10-containing compound solution to the albumin obtained in the foregoing step after heat treatment, uniformly mixing, treating in an ice water bath, and concentrating by ultrafiltration to obtain the bifunctional albumin nanoparticle.
[0013] Preferably, step 1 is coupling an N-hydroxysuccinimide activated ester of the macrocyclic ligand chelator containing a carboxyl group to albumin.
[0014] Preferably, step 2 uses a gadolinium chloride solution, wherein the molar amount of gadolinium chloride is 5-10 times that of the macrocyclic ligand chelator coupled to albumin.
[0015] Preferably, the heat treatment temperature in step 3 is 50-60°C, the time is 10-20 min, and 0.8-1.2 mmol of the hydrophobic boron-10-containing compound is added per gram of albumin in step 1.
[0016] The pharmaceutical composition comprises the bifunctional albumin nanoparticle of claim 1 as an active ingredient and a pharmaceutically acceptable excipient.
[0017] The bifunctional albumin nanoparticle or the pharmaceutical composition is used in the preparation of an antitumor drug.
[0018] Preferably, the application is used in the preparation of an antitumor drug for magnetic resonance imaging and boron neutron capture therapy.
[0019] Compared with the prior art, the bifunctional albumin nanoparticle has the following advantages: 1. The bifunctional albumin nanoparticle has good T1-weighted magnetic resonance imaging capability, can be used to track the distribution and enrichment of the boron-containing drug in the body in real time, realize the synergistic application with boron neutron capture therapy, and help to evaluate the treatment window and improve the accuracy of treatment; 2. The prepared nanoparticle has uniform particle size, stable zeta potential, good drug loading capacity and tissue penetration, and is beneficial to targeted enrichment in tumor tissue; 3. The bifunctional albumin nanoparticle preferably uses human serum albumin as a nanoparticle carrier, has excellent biocompatibility and good in vivo stability, is high in safety, and is beneficial to clinical transformation. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1Figure for particle size characterization of bifunctional albumin nanoparticle;
[0021] Figure 2 Figure for surface potential characterization of bifunctional albumin nanoparticle;
[0022] Figure 3 Figure for transmission electron microscope observation of bifunctional albumin nanoparticle;
[0023] Figure 4 Figure for Gd content detection of bifunctional albumin nanoparticle; 3+
[0024] Figure 5 Figure for Chol B encapsulation rate detection of bifunctional albumin nanoparticle; 10
[0025] Figure 6 Figure for biocompatibility determination of bifunctional albumin nanoparticle;
[0026] Figure 7 Figure for in vitro MRI performance determination of bifunctional albumin nanoparticle;
[0027] Figure 8 Figure for in vivo MRI performance determination of bifunctional albumin nanoparticle;
[0028] Figure 9 Figure for boron neutron capture therapy performance evaluation of bifunctional albumin nanoparticle. DETAILED DESCRIPTION
[0029] The technical solutions of the present application are described below.
[0030] Example 1: Preparation and characterization of bifunctional albumin nanoparticle
[0031] 1. Preparation of bifunctional albumin nanoparticle
[0032] (1) Take 10 mg of human serum albumin (HSA) and dissolve it in 5 mL of 0.1 M phosphate buffer at pH 8.5, add 875 μg of DOTA-NHS activated ester (CAS No.: 170908-81-3), react at room temperature in the dark for 24 h, and then treat by dialysis and ultrafiltration to obtain DOTA-conjugated albumin (DOTA-HSA);
[0033] (2) Take DOTA-HSA and dissolve it in 0.1 M ammonium acetate buffer at pH 5.5, add GdCl3 solution, and react at room temperature for 24 h, and then treat by dialysis and ultrafiltration to obtain bifunctional albumin nanoparticle (Gd-DOTA-HSA); 3 + at 37℃, 150 rpm for 8 hours. After the reaction, the mixture was loaded into a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed in ultrapure water for 24 hours to remove unchelated free gadolinium ions. After dialysis, the product was concentrated by centrifugation to obtain gadolinium ion-loaded DOTA-HSA;
[0034] (3) Take 100 μL of the above prepared gadolinium ion-loaded DOTA-HSA, heat treat at 55℃ for 15 min, then add 1 mL of 10 mM Chol 10 B-ethanol solution, immediately after mixing, ice water bath, then use 100 kD ultrafiltration centrifuge tube to concentrate and purify at 3000 rpm for 15 min, to obtain stable bifunctional albumin nanoparticles (Gd-nab-chol 10 B);
[0035] wherein Chol 10 B) was synthesized according to the method described in Zhu JY, Zeng X, Qin SY, et al. Acidity-responsive gene delivery for "superfast" nuclear translocation and transfection with high efficiency. Biomaterials. 2016; 83: 79-92. 10 B) was synthesized after reaction.
[0036] 2. Characterization of bifunctional albumin nanoparticles
[0037] (1) Particle size and zeta potential of bifunctional albumin nanoparticles
[0038] The bifunctional albumin nanoparticles prepared in Example 1 were characterized by dynamic light scattering (DLS), with an average particle size of 186.84 ± 6.3 nm and a PDI of 0.162, indicating a uniform particle size distribution. The zeta potential measurement result was -21.4 ± 1.2 mV, showing good colloidal stability.
[0039] (2) Morphological characterization of bifunctional albumin nanoparticles
[0040] The morphology of the bifunctional albumin nanoparticles prepared in Example 1 was characterized using transmission electron microscopy (TEM). The nanoparticle sample was diluted with ultrapure water to approximately 0.1 mg / mL, and 10 μL was dropped onto a 200-mesh copper mesh supported by a carbon film. The mixture was allowed to stand for 2 min to promote particle adsorption. Excess liquid was then gently blotted away with filter paper, and the sample was negatively stained with 1% phosphotungstic acid. After standing for 30 s, the sample was blotted dry again. After the sample was allowed to air dry at room temperature, it was observed and images were captured using a transmission electron microscope to obtain the morphology information of the nanoparticles.
[0041] The results are as follows Figure 3 As shown, the bifunctional albumin nanoparticles are spherical, well-dispersed, and have a complete morphology.
[0042] (3) Gd in bifunctional albumin nanoparticles 3 +Content
[0043] Gd in the bifunctional albumin nanoparticles prepared in Example 1 was determined using inductively coupled plasma mass spectrometry (ICP-MS). 3+ The content of.
[0044] 500 μL of bifunctional albumin nanoparticles were analyzed by Beijing Laixi Testing Technology Co., Ltd. The digestion method was as follows: a mixed solution of 67-69% concentrated nitric acid and 30% hydrogen peroxide (3:1 volume ratio) was added, and microwave digestion was performed at a maximum temperature of 180℃ for 30 minutes until complete digestion. After cooling to room temperature, the solution was diluted to 50 mL with ultrapure water and filtered through a 0.22 μm filter membrane. 159 Tb was used as an internal standard to correct for matrix effects in ICP-MS analysis.
[0045] Test results as follows Figure 4 As shown, Gd in bifunctional albumin nanoparticles 3 The gadolinium concentration was 9.68 mM, indicating high labeling efficiency and sufficient gadolinium loading capacity for MRI imaging.
[0046] (4) Chloroprotein nanoparticles with bifunctional albumin 10 B Encapsulation rate
[0047] Take 20 μL of the Gd prepared in Example 1 3+ Bifunctional albumin nanoparticles with a concentration of 9.68 mM were added to 380 μL of pure acetonitrile for demulsification, and then filtered through a 0.22 μm microporous membrane.
[0048] Encapsulation efficiency was determined by high-performance liquid chromatography (HPLC). An Agilent Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm) was used. The mobile phase was 80% acetonitrile-water solution, the flow rate was 1 mL / min, and the detection wavelength was 210 nm. The standard curve was calculated as follows: Y = 12861419X – 9896088 (R²). 2 =0.9965) Calculate Chol 10 B is the concentration, where Y is the peak area and X is the drug concentration.
[0049] Encapsulation efficiency (EE%) is calculated using the following formula:
[0050] EE%=(C×V×D×Vtotal) / Minitial×100%
[0051] Where C is the measured Chol 10 B is the concentration (μM), V is the detection volume (0.4 mL), D is the dilution factor (20), Vtotal is the total volume of nanoparticles (8 mL), and Minimum is the initial dosage (10 μmol). High-performance liquid chromatography (HPLC) is as follows: Figure 6 HPLC analysis showed that the encapsulation efficiency of the bifunctional albumin nanoparticles with Chol10B was 56.8%.
[0052] Example 2: Performance Testing of Bifunctional Albumin Nanoparticles
[0053] 1. Biocompatibility of bifunctional albumin nanoparticles
[0054] The toxicity of bifunctional albumin nanoparticles to 4T1 cells was evaluated using the CCK-8 assay.
[0055] In a 96-well plate, 5×10⁻⁶ mm per well. 3 4 T1 cells were seeded at a density of 1 / 4, and the culture medium was discarded after the cells adhered; the medium was then replaced with Gd. 3 +Introduce complete culture medium containing bifunctional albumin nanoparticles at final concentrations of 0.1, 0.5, 1, 5, and 9.68 mM, and continue culturing for 24 h;
[0056] After culturing, remove the cells, discard the culture medium, add 100 μL of complete culture medium containing 10% CCK-8 reagent to each well, incubate in an incubator for 1 h, and measure the absorbance at 450 nm using a microplate reader;
[0057] The results are as follows Figure 6 As shown, in the range of 0.1-9.68 mM Gd 3+Within the specified concentration range, cell survival rates were >90%, indicating that the bifunctional albumin nanoparticles have no significant cytotoxicity and good biocompatibility.
[0058] 2. In vitro nuclear magnetic resonance imaging performance of bifunctional albumin nanoparticles
[0059] The bifunctional albumin nanoparticles (Gd-nab-chol) prepared in Example 1 were used. 10 B) Prepared with deionized water using Gd 3+ Solutions with concentration gradients of 0.01, 0.03, and 0.05 mM were analyzed, and T1-weighted images were acquired using a Bruker BioSpec 7T MRI system.
[0060] The results are as follows Figure 7 As shown, the signal strength varies with Gd 3+ The concentration gradually increased with increasing concentration. This indicates that Gd-nab-chol 10 The development effect of B is similar to that of Gd 3+ It is positively correlated with concentration and has good T1 enhancement ability.
[0061] 3. In vivo properties of bifunctional albumin nanoparticles
[0062] Eighteen female BALB / c mice aged 6–8 weeks and weighing 18–22g were selected and housed in a specific pathogen-free (SPF) environment.
[0063] All mice were subcutaneously inoculated with 4T1 breast cancer cells in the right axilla to establish a tumor model, and the tumor volume was allowed to reach 80–100 mm. 3 Then, they were randomly divided into 3 groups of 6 each:
[0064] The first group was the KB group, which received an equal volume of PBS via tail vein injection as a control; the second group was the KB+BNCT group, which received a single boron neutron capture therapy (BNCT) irradiation 24 hours after tail vein injection of PBS; the third group received Gd-nab-chol. 10 In the B+BNCT group, the bifunctional albumin nanoparticles prepared in Example 1 were injected via tail vein (dose was 0.1 mmol / kg, with Gd...). 3+ (Calculation), also receiving a single BNCT irradiation 24 hours after injection,
[0065] All BNCT treatment groups received a flux of 2 × 10⁻⁶ neutrons in the neutron irradiation device. 10 n / cm 2 The patient was irradiated with a thermal neutron beam of / s for 150 minutes, and the irradiation area was the whole body.
[0066] All mice were imaged using a Bruker BioSpec 7T MRI system to acquire T1 -weighted images at 2, 6, 24 and 48 h after injection of Gd-Nab-chol10B; in addition, tumor volume changes were continuously monitored after administration to evaluate the therapeutic effect.
[0067] The MRI results showed that the T1 signal in the tumor region was continuously enhanced at 2 h, 6 h, 24 h and 48 h after injection, indicating that it had good tumor enrichment ability and imaging persistence. Figure 8 The tumor volume statistical results showed that the dual-functional albumin nanoparticle combined with BNCT treatment group achieved significant results in tumor inhibition rate. Figure 9 The tumor volume statistical results showed that the dual-functional albumin nanoparticle combined with BNCT treatment group achieved significant results in tumor inhibition rate.
Claims
1. A bifunctional albumin nanoparticle, characterized in that, The nanoparticles use albumin as a carrier, are surface-modified with macrocyclic ligand chelating agents that chelate gadolinium ions, and encapsulate hydrophobic boron-10 compounds inside.
2. The bifunctional albumin nanoparticles according to claim 1, characterized in that, The albumin in question is human serum albumin.
3. The bifunctional albumin nanoparticles according to claim 1, characterized in that, The macrocyclic ligand chelating agent is a carboxyl-containing macrocyclic ligand chelating agent.
4. The bifunctional albumin nanoparticles according to claim 1, characterized in that, The hydrophobic boron-containing compound is a boron-10 cholesterol derivative, Chol. 10 B.
5. A method for preparing bifunctional albumin nanoparticles according to claim 1, characterized in that the step... include: (1) Couple macrocyclic ligand chelating agents to albumin; (2) The albumin with the macrocyclic ligand chelating agent coupled with the gadolinium salt solution was mixed and the free gadolinium ions were removed after the reaction to obtain albumin with the macrocyclic ligand chelating agent with the surface modified with gadolinium ions. (3) After the albumin obtained above is heated, a hydrophobic boron-10 compound solution is added, mixed evenly, treated in an ice-water bath, and then concentrated by ultrafiltration to obtain bifunctional albumin nanoparticles.
6. The preparation method according to claim 5, characterized in that, Step 1 involves coupling the N-hydroxysuccinimide activated ester of a carboxyl-containing macrocyclic ligand chelating agent to albumin.
7. The preparation method according to claim 5, characterized in that, In step 3, the heating temperature is 50-60℃ and the time is 10-20 minutes.
8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the bifunctional albumin nanoparticles of claim 1 as the active ingredient, and pharmaceutically acceptable excipients.
9. The use of the bifunctional albumin nanoparticle of claim 1, or the pharmaceutical composition of claim 7, in the preparation of an antitumor drug.
10. The application according to claim 9, characterized in that, The application is in the preparation of anti-tumor drugs that combine magnetic resonance imaging with boron neutron capture therapy.