A method for judging and controlling the end point of ligand exchange on the surface of nano-iron oxide particles based on the kinetics feedback of relaxation rate

Abstract

The application belongs to the technical field of ligand exchange of nano iron oxide particles, and in particular to a method for judging the end point of ligand exchange on the surface of nano iron oxide particles and controlling the grading purification based on relaxation rate kinetics feedback, which can reflect the rearrangement kinetics process of the ligand exchange interface by detecting the relaxation rate, realize the objective quantitative control of the reaction end point, and improve the batch consistency of the ligand exchange; the temperature control slow-release pre-dispersion measure is adopted to avoid the particle agglomeration caused by sudden temperature change; in the purification, the cooperative control of the purification process and the exchange kinetics stage is realized, the recovery rate and purity are improved; by controlling the residual amount of ligand, the magnetic resonance performance is optimized while the stability is ensured, and the process complexity is reduced.

Description

Technical Field

[0001] This invention belongs to the field of nano-iron oxide particle surface engineering and ligand exchange technology, specifically involving a method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback. Background Technology

[0002] Currently, hydrophobic iron oxide nanoparticles prepared by high-temperature organic phase thermal decomposition typically have hydrophobic ligands such as oleic acid and oleylamine coated on their surface. These nanoparticles, with a particle size of 6–20 nm, exhibit uniform size, high crystallinity, and excellent magnetic properties, making them a crucial foundation for constructing magnetic resonance imaging (MRI) contrast agents, magnetic particle imaging (MPI) tracers, and magnetothermal therapy materials. However, the hydrophobic ligands coating these particles limit their dispersion to nonpolar organic solvents, preventing direct application in aqueous biomedical systems. Therefore, ligand exchange is necessary to convert them into hydrophilic systems.

[0003] In existing technologies, ligand exchange involves mixing hydrophobic particles with hydrophilic ligands (such as PEG derivatives) in an organic solvent and achieving ligand replacement through prolonged stirring. The determination of the endpoint of ligand exchange is based on a fixed reaction time or qualitative analysis such as infrared spectroscopy. The purification and separation of the reaction system is achieved by removing free ligands through high-speed centrifugation or constant magnetic field separation. The existing ligand exchange method has the following shortcomings: (1) The sudden temperature change leads to irreversible particle aggregation: After the high temperature discharge, the temperature drops rapidly to room temperature, causing the solubility of hydrophobic ligands to drop sharply and precipitate out. The hydrophobic attraction between particles is enhanced, and the aggregation rate is as high as 20% or more. Moreover, this aggregation is usually irreversible; (2) The reaction endpoint lacks objective quantitative criteria: The existing method relies on time or qualitative characterization, which cannot reflect the dynamic process of the ligands changing from physical adsorption to chemical anchoring, resulting in significant batch-to-batch differences; (3) The purification process lacks coupling control with the reaction stage: The constant intensity separation method does not consider the dynamic changes of particle surface (such as hydrophobic / hydrophilic ratio, surface charge, hydration layer) at different stages of ligand exchange, resulting in serious particle loss or incomplete purification; (4) The complete replacement strategy reduces material performance: Complete removal of hydrophobic ligands reduces relaxation performance and increases process complexity. Summary of the Invention

[0004] This invention aims to provide a method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback. This method avoids particle agglomeration caused by sudden temperature changes, improves batch-to-batch consistency of ligand exchange, achieves synergistic control of the purification process and exchange kinetic stages, and enables objective quantitative control of the reaction endpoint.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback is provided, including:

[0007] Temperature-controlled slow-release pre-dispersion: The hydrophobic iron oxide nanoparticle suspension is cooled at a certain rate and an aprotic polar solvent is injected;

[0008] Ligand adsorption: Adding hydrophilic ligands to the reaction system enhances adsorption with high shear, allowing the hydrophilic ligands to quickly contact the surface of hydrophobic iron oxide nanoparticles for initial adsorption;

[0009] ligand rearrangement: Incubation under low shear conditions allows the hydrophilic ligands adsorbed on the surface of iron oxide nanoparticles to rearrange and anchor.

[0010] Ligand equilibrium and endpoint determination: During the reaction phase of ligand reordering, the transverse relaxation rate r2 and longitudinal relaxation rate r1 of the reaction system are periodically detected, and the rate of change is calculated as Δ(r2 / r1) / Δt. When the rate of change is lower than the set rate of change threshold twice in a row, it is determined that the ligand reordering has reached a dynamic equilibrium state and the reaction is terminated.

[0011] Graded purification: In the different ligand exchange kinetic stages of ligand adsorption, ligand rearrangement, ligand equilibrium and endpoint determination, the reaction system is subjected to graded magnetic separation and synergistic purification treatment. The magnetic field strength of the magnetic separation is dynamically adjusted to dynamically match the ligand binding state.

[0012] Preferred options also include:

[0013] Hydrophobic ligand residual amount control: The residual amount of hydrophobic ligands is controlled by adjusting the ratio of the added hydrophilic ligands to the hydrophobic iron oxide nanoparticle suspension and the reaction time of the reaction system during ligand adsorption, ligand reordering and / or ligand equilibrium and endpoint determination.

[0014] Preferably, the temperature-controlled slow-release pre-dispersion step specifically involves: cooling the hydrophobic iron oxide nanoparticle suspension at a temperature of 80–120°C to 60–80°C at a rate of ≥20°C / min, and simultaneously introducing an aprotic polar solvent at a volume ratio of 0.5–1.0.

[0015] Preferably, the shear rate for high-shear enhanced adsorption of the reaction system is 1000–5000 s⁻¹. -1 .

[0016] Preferably, the low-shear condition in the weighted arrangement of the balancing weights is:

[0017] Low shear rate range: 10-200s -1 Preferred 50-100s -1The temperature of the reaction system is 50℃-65℃ under low shear.

[0018] Preferably, in the ligand equilibration step, the rate of change threshold is ≤0.02 min. -1 If the rate of change is lower than the rate of change threshold twice consecutively, it is determined that the balancing weights have reached a dynamic equilibrium state and the reaction is terminated.

[0019] Preferably, the stepwise magnetic separation and synergistic purification treatment of the reaction system includes:

[0020] In the ligand exchange kinetics stage of ligand adsorption, the purification conditions are: high gradient magnetic field 50-100 T / m, separation time 5-10 minutes;

[0021] In the ligand exchange kinetics stage of the ligand reordering, the purification conditions are: medium gradient magnetic field 20-30 T / m, combined with ultrafiltration, and separation time 10-15 minutes.

[0022] In the ligand exchange kinetics phase of ligand equilibrium, the purification conditions are: purification is performed using a pulsed magnetic field.

[0023] Preferably, the ultrafiltration conditions are: MWCO 50-100 kDa;

[0024] The pulsed magnetic field has 3-5 cycles, and one cycle of the pulsed magnetic field is 30s on and 30s off.

[0025] Preferably, the aprotic polar solvent is selected from one or more of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, or acetone.

[0026] Preferably, the hydrophilic ligand is Mal-PEG-NH2, HOOC-PEG-COOH, or Dopamine-PEG-OCH3.

[0027] According to statistics, this method shows the following improvements compared to traditional ligand exchange methods: 1. CV value comparison: traditional method batch CV value (>25%) vs. this invention method (<8%); 2. Aggregation rate comparison: traditional method (>30%) vs. this invention method (<2%); 3. Recovery rate and purity: recovery rate >92%, purity >99.5% (ICP-MS determination); 4. r2 relaxation rate comparison: traditional method (120~165 mM) -1 s -1 This method (180-220 mM) -1 s -1 ); 5. Comparison of process time: traditional method (more than 48 hours), this method (4-6 hours).

[0028] In this method, temperature-controlled slow release can be achieved by receiving the suspension in a constant-temperature oil bath / heating mantle, controlling the cooling rate with a programmed temperature-controlled cooling device (such as Julabo or Huber), and injecting an aprotic polar solvent via a syringe pump (such as Harvard Apparatus); high-shear adsorption uses a high-speed shear emulsifier (such as IKA Ultra-Turrax T25) or a high-pressure homogenizer (such as APVGaulin); low-shear rearrangement uses a magnetic stirrer (such as IKA C-MAG HS7) or a track shaker (such as IKA KS 4000); fractionation purification uses a high-gradient magnetic separator (such as Sepmag Q or Cestron); and endpoint detection uses a benchtop nuclear magnetic resonance relaxation analyzer (such as Bruker Minispec mq20).

[0029] The technical principle of this method is explained as follows: The transverse relaxation rate r2 of iron oxide nanoparticles is mainly affected by the thickness of the water molecule diffusion layer on the particle surface and the surface spin dynamics, while the longitudinal relaxation rate r1 is related to the rapid exchange of water molecules and the surface ligand density. In the initial stage of ligand exchange, hydrophilic ligands cover the particle surface through physical adsorption, while hydrophobic ligands have not yet completely dissociated. At this time, the particle surface is in a disordered mixed state, and the diffusion and exchange behavior of water molecules on the surface changes drastically, manifested as rapid fluctuations in the r2 / r1 ratio and a large value of Δ(r2 / r1) / Δt. As incubation progresses, hydrophilic ligands coordinate with the iron oxide surface through functional groups (such as amino, carboxyl, and catechol groups) or hydrogen bonding, forming a stable hydrophilic layer; at the same time, hydrophobic ligands remain or dissociate in a controlled manner, and the surface structure tends to become more ordered. When the ligand reordering and anchoring reach a dynamic equilibrium, the surface hydration layer thickness and ligand conformation tend to stabilize, and the diffusion and exchange rates of water molecules on the surface slow down. At this point, the rate of change of the r2 / r1 ratio, Δ(r2 / r1) / Δt, decreases significantly and approaches zero. Therefore, by setting a reasonable threshold for Δ(r2 / r1) / Δt (≤0.02 min...),... -1 This allows for an objective and quantitative determination of whether the ligand interface rearrangement has reached a dynamic equilibrium, thus avoiding overreaction or underreaction.

[0030] The core innovation of this invention lies in the discovery of a stable mapping relationship between the relaxation ratio change rate Δ(r2 / r1) / Δt and the evolution state of the ligand interface structure (physical adsorption → rearrangement → chemical anchoring). For the first time, this relaxation kinetic parameter has been transformed from a traditional offline performance characterization index into an online reaction endpoint feedback control signal, realizing adaptive termination control of the ligand exchange process.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: the method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback can reflect the rearrangement kinetics of the ligand exchange interface, realize objective quantitative control of the reaction endpoint, and improve batch-to-batch consistency of ligand exchange; the temperature-controlled slow-release pre-dispersion measure avoids particle agglomeration caused by sudden temperature changes; in purification, the purification process and the exchange kinetic stage are synergistically controlled, improving recovery rate and purity; by controlling the residual amount of ligands, the magnetic resonance performance is optimized while ensuring stability, and the process complexity is reduced. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling fractional purification based on relaxation rate kinetic feedback, the method comprising the following steps:

[0035] (1) Temperature-controlled slow-release pre-dispersion: Receive the suspension of iron oxide nanoparticles coated with thermally decomposed oleic acid (particle size 12±2nm, temperature 120℃, concentration 20 mg / mL), cool it to 80℃ at a cooling rate of 30℃ / min, and simultaneously inject N-methylpyrrolidone (volume ratio 1.0).

[0036] Since hydrophobic iron oxide nanoparticles prepared by thermal decomposition are usually dispersed in organic solvents, the main dispersion media are decalin or phenyl ether; toluene, chloroform, or dichloromethane (DCM) can also be used. In this embodiment, the suspension of thermally decomposed oleic acid-coated iron oxide nanoparticles is dispersed in decalin, with a particle size of 12±2 nm, a temperature of 120℃, and a concentration of 20 mg / mL (each mL of the thermally decomposed oleic acid-coated iron oxide nanoparticle suspension contains 20 mg of Fe).

[0037] In step (1), by cooling to 80°C at a cooling rate of 30°C / min, the precipitation of hydrophobic ligands can be suppressed, the aggregation driving force can be reduced, and the iron oxide nanoparticles can be prevented from agglomerating prematurely due to the slowing down of thermal motion during the cooling process.

[0038] Temperature-controlled slow release uses a constant temperature oil bath / heating mantle to receive the suspension, and the cooling rate is controlled by a programmable temperature cooling device (JulaboFP50). A nonprotic polar solvent is injected through a syringe pump (Harvard Apparatus).

[0039] N-methylpyrrolidone (NMP) is an aprotic polar solvent. Other alternative solvents include N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), acetonitrile, and acetone. The aprotic polar solvent is selected from one or more of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, or acetone.

[0040] When adding the aprotic polar solvent, it is introduced simultaneously at a volume ratio of 0.5–1.0, meaning the volume ratio of the aprotic polar solvent to the iron oxide nanoparticle suspension is 0.5–1.0. Introducing the aprotic polar solvent allows the hydrophobic iron oxide nanoparticles, stably dispersed in the oil phase (decahydronaphthalene), to gradually adapt to a more polar environment.

[0041] (2) Ligand adsorption: The solution obtained in the above steps was mixed with Mal-PEG-NH2 (molecular weight 3400 Da, concentration 50 mg / mL). The amount of Mal-PEG-NH2 solution added was ligand / Fe molar ratio 10:1. The reaction system was subjected to high shear-enhanced adsorption at a shear rate of 5000 s. -1 The residence time is 120 seconds, which allows the hydrophilic ligands to quickly come into contact with the surface of the hydrophobic iron oxide nanoparticles and undergo initial adsorption.

[0042] In this step, Mal-PEG-NH2 is the added hydrophilic ligand solution, the solute of which is a maleimide-polyethylene glycol-amino (Mal-PEG-NH2) compound, and the solvent is DMSO (dimethyl sulfoxide).

[0043] The amount of Mal-PEG-NH2 solution added is such that the ligand / Fe molar ratio is 10:1, meaning that the molar ratio of Mal-PEG-NH2 compound to Fe element in the iron oxide nanoparticle suspension in the ligand solution is 10:1.

[0044] The residence time is 120 seconds, which is the reaction time for high shear-enhanced adsorption.

[0045] In the ligand adsorption step, the high-shear equipment used is a high-speed shear emulsifier (IKA Ultra-Turrax T25 / T50), or a high-pressure homogenizer (APV Gaulin) or an ultrasonic cell disruptor (Sonics VCX800).

[0046] The purpose of this step is to rapidly perform ligand exchange, establishing a "non-equilibrium adsorption state" in the reaction system.

[0047] (3) Rearrangement of balancing components: Incubation was carried out under low shear conditions. The reaction system temperature was 65℃ during low shear incubation. A magnetic stirrer was used for the low shear equipment, and the low shear rate was 200 s. -1 The equipment parameters are: magnetic stirring speed 600 rpm. This allows the hydrophilic ligands adsorbed on the surface of iron oxide nanoparticles to rearrange and anchor.

[0048] Low shear conditions facilitate the rearrangement and anchoring of hydrophilic ligands on the surface of iron oxide nanoparticles, preventing the desorption of already adsorbed hydrophilic ligands. In other embodiments, the low shear device may also be a track shaker or a bottle rolling machine, with a low shear rate of 10-200 s⁻¹. -1 (Preferred time: 50-100 seconds) -1 Either is acceptable, with the following equipment parameters: magnetic stirring speed 200-600 rpm, or track shaking table speed 60-120 rpm.

[0049] (4) Ligand equilibrium: During the ligand rebalancing reaction phase, the transverse relaxation rate r2 and longitudinal relaxation rate r1 of the reaction system were periodically measured every 10 minutes, and the rate of change was calculated as Δ(r2 / r1) / Δt. The Δ(r2 / r1) / Δt measured at approximately 90 minutes was 0.018 min. -1 The rate of change meets the following condition: it is lower than the set rate of change threshold (≤0.02 min). -1 The reaction is terminated when the ligands are reorganized to a dynamic equilibrium.

[0050] In other embodiments, the rate of change may be lower than a set rate of change threshold (≤0.02 min) twice consecutively. -1 When the reaction reaches a dynamic equilibrium, the reaction is terminated to improve the accuracy of the reaction endpoint determination.

[0051] In this step, the transverse relaxation rate r2 and the longitudinal relaxation rate r1 are detected using a benchtop nuclear magnetic resonance relaxation analyzer (Bruker Minispec mq20 / mq60); in other embodiments, a portable NMR (Magritek Spinsolve) may also be used.

[0052] (5) Fractional purification: In different ligand exchange kinetic stages, including ligand adsorption, ligand rearrangement, ligand equilibrium, and endpoint determination, the reaction system is subjected to fractional magnetic separation and synergistic purification. The magnetic field strength of the magnetic separation is dynamically adjusted to match the ligand binding state. In the first stage (ligand adsorption), the magnetic field gradient is 100 T / m, the purification time is 10 min, and the flow rate is 2 mL / min, which is used to remove large particle aggregates. In the second stage (ligand rearrangement), the magnetic field gradient is 25 T / m, combined with ultrafiltration. The ultrafiltration conditions are MWCO 50 kDa to remove free ligands. In the third stage (ligand equilibrium and endpoint determination), the magnetic field gradient of the pulsed magnetic field is 25 T / m (30s / 30s, 5 cycles) to selectively separate equilibrium particles.

[0053] This fractional purification step is performed during the different ligand exchange kinetic stages of ligand adsorption, ligand reordering, ligand equilibration, and endpoint determination. The equipment used for fractional purification is a high-gradient magnetic separator (Sepmag Q) and an ultrafiltration system (Millipore Amicon). In other embodiments, a centrifuge (Beckman Optima) can also be used for assisted purification.

[0054] After each purification stage, the particle size distribution and zeta potential of the DLS particles were measured using a Malvern Zetasizer (DLS) nanoparticle size and zeta potential analyzer and a transmission electron microscope (TEM) (JEOL JEM-2100) to confirm the purification effect.

[0055] (6) Control of residual hydrophobic ligands: The residual amount of hydrophobic ligands is controlled by adjusting the ratio of the added hydrophilic ligands to the hydrophobic iron oxide nanoparticle suspension and the reaction time of the reaction system during the ligand adsorption, ligand reordering and / or ligand equilibrium and endpoint determination.

[0056] In this embodiment, the method for controlling the residual amount of hydrophobic ligands is performed during the preceding steps of ligand adsorption, ligand rebalancing to ligand equilibrium, and endpoint determination. In this embodiment, the ratio of the added hydrophilic ligand to the hydrophobic iron oxide nanoparticle suspension is 10:1 (ligand / Fe molar ratio in the Mal-PEG-NH2 solution), and the reaction time for ligand rebalancing to ligand equilibrium and endpoint determination is 90 minutes. Generally, the more ligand added and the longer the reaction time, the less residual hydrophobic ligands.

[0057] The purpose of residual control of hydrophobic ligands is to optimize magnetic resonance performance while ensuring product stability, thereby increasing the transverse relaxation rate r2 to 180–220 mM. -1 s -1 This improves magnetic resonance performance and reduces manufacturing complexity.

[0058] Residual oleic acid content was determined by TGA (TA Instruments Q500) to detect residual control results. The TGA instrument used for the determination was a TGA (TA Instruments Q500). The TGA determination method is as follows: 1. Sample preparation: Take 5-10 mg of purified sample and freeze-dry for 24 hours; 2. Instrument: TA Instruments Q500 (or Mettler Toledo TGA / DSC 1); 3. Test conditions: N2 atmosphere (flow rate 50 mL / min), heating rate 10℃ / min, temperature range 25-600℃; 4. Data processing: Weight loss in the 300-600℃ range is attributed to oleic acid decomposition, and the mass percentage is calculated.

[0059] The residual control detection results in this embodiment are as follows: TGA determination showed a residual oleic acid content of 0.5% w / w and a surface ligand density of 2.0 chains / nm. 2 .

[0060] Surface ligand density calculation formula: Surface ligand density = (TGA weight loss % x Avogadro constant) / (ligand molecular weight x BET specific surface area). Surface ligand density 2.0 chains / nm 2 This indicates that 2.0 PEG chains are grafted onto the surface of each square nanoparticle, or 2.0 chains / nm. 2 This indicates that high-density grafting can provide good colloidal stability and steric hindrance effect, preventing particle aggregation.

[0061] Final product test result: r2=215 mM -1 s -1 D_h=26 nm, PDI=0.10, recovery rate 93%.

[0062] The transverse relaxation rate r2 is 215mM -1 s -1The product meets the magnetic resonance performance requirements of this embodiment. D_h (Hydrodynamic Diameter) is the equivalent hydrodynamic diameter of the nanoparticles in solution, determined by dynamic light scattering (DLS). Its significance lies in reflecting the actual size of the particles in solution, including the surface ligand layer. In this embodiment, D_h = 26 nm, meeting the product performance requirements. PDI (Polydispersity Index) indicates the uniformity of particle size distribution, ranging from 0 to 1. Its significance is: PDI < 0.1 indicates high monodispersity, PDI < 0.2 indicates good monodispersity, and the target in this invention is: PDI < 0.15. Recovery rate refers to the total recovery rate of hydrophilic iron oxide nanoparticles from hydrophobic particles after ligand exchange and purification. The calculation formula is: Recovery rate = (Purified Fe mass / Initial Fe mass) x 100%; Fe mass is determined by ICP-MS or ICP-OES.

[0063] The final product test results show that all relevant indicators meet the requirements.

[0064] Example 2

[0065] A method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling fractional purification based on relaxation rate kinetic feedback, the method comprising the following steps:

[0066] (1) Temperature-controlled slow-release pre-dispersion: Receive the suspension of iron oxide nanoparticles coated with thermally decomposed oleic acid (particle size 12±2nm, temperature 100℃, concentration 12 mg / mL), cool it to 70℃ at a cooling rate of 25℃ / min, and simultaneously inject N-methylpyrrolidone (volume ratio 0.8 times).

[0067] (2) Ligand adsorption: The solution obtained in the above steps was mixed with HOOC-PEG-COOH (molecular weight 2000 Da). The amount of HOOC-PEG-COOH (molecular weight 2000 Da) solution added was 5:1 ligand / Fe molar ratio. The reaction system was subjected to high shear-enhanced adsorption at a shear rate of 3000 s. -1 The residence time is 60 seconds, which allows the hydrophilic ligands to quickly come into contact with the surface of the hydrophobic iron oxide nanoparticles and undergo initial adsorption.

[0068] In this step, HOOC-PEG-COOH is a hydrophilic ligand, which is added in solution form. The solvent is DMSO (dimethyl sulfoxide). The amount of HOOC-PEG-COOH added is 5:1 ligand / Fe molar ratio, that is, the molar ratio of HOOC-PEG-COOH compound to Fe element in the iron oxide nanoparticle suspension in the ligand solution is 5:1.

[0069] The residence time is 60 seconds, which is the reaction time for high shear-enhanced adsorption.

[0070] (3) Rearrangement of balancing components: Incubation was carried out under low shear conditions. The reaction system temperature was 65℃ during low shear incubation. A magnetic stirrer was used as the low shear equipment, and the low shear rate was 100 s. -1 This allows the hydrophilic ligands already adsorbed on the surface of iron oxide nanoparticles to rearrange and anchor.

[0071] (4) Ligand equilibrium and endpoint determination: During the ligand rebalancing reaction phase, the transverse relaxation rate r2 and longitudinal relaxation rate r1 of the reaction system were periodically measured every 8 minutes, and the rate of change was calculated as Δ(r2 / r1) / Δt. The Δ(r2 / r1) / Δt measured at approximately 120 minutes was 0.015 min. -1 The rate of change meets the following condition: it is lower than the set rate of change threshold (≤0.02 min). -1 The reaction is terminated when the ligands are reorganized to a dynamic equilibrium.

[0072] (5) Fractional purification: In different ligand exchange kinetic stages, including ligand adsorption, ligand rearrangement, ligand equilibrium, and endpoint determination, the reaction system is subjected to fractional magnetic separation and synergistic purification. The magnetic field strength of the magnetic separation is dynamically adjusted to match the ligand binding state. In the first stage (ligand adsorption), the magnetic field gradient is 80 T / m, the purification time is 7 min, and the flow rate is 1.5 mL / min, which is used to remove large particle agglomerates. In the second stage (ligand rearrangement), the magnetic field gradient is 25 T / m, combined with ultrafiltration. The ultrafiltration conditions are MWCO 75 kDa to remove free ligands. In the third stage (ligand equilibrium and endpoint determination), the magnetic field gradient of the pulsed magnetic field is 25 T / m (30s / 30s, 5 cycles) to selectively separate equilibrium particles.

[0073] This step-by-step purification process was performed during the different ligand exchange kinetics stages of ligand adsorption, ligand reordering, ligand equilibration, and endpoint determination. After each purification stage, the particle size distribution and zeta potential of the DLS (Malvern Zetasizer) nanoparticles and zeta potentials were measured using a JEOL JEM-2100 transmission electron microscope to confirm the purification effect.

[0074] (6) Control of residual hydrophobic ligands: The residual amount of hydrophobic ligands is controlled by adjusting the ratio of the added hydrophilic ligands to the hydrophobic iron oxide nanoparticle suspension and the reaction time of the reaction system during ligand adsorption, ligand reordering and / or ligand equilibrium and endpoint determination.

[0075] The method for controlling the residual amount of hydrophobic ligands is performed in the preceding steps of ligand adsorption and ligand rebalancing to ligand equilibrium and endpoint determination. In this embodiment, the ratio of the added hydrophilic ligand to the hydrophobic iron oxide nanoparticle suspension is 10:1 ligand / Fe molar ratio in the HOOC-PEG-COOH solution, and the reaction time for ligand rebalancing to ligand equilibrium and endpoint determination is 120 minutes.

[0076] The residual control detection results in this embodiment are as follows: TGA determination showed a residual oleic acid content of 1.2% w / w and a surface ligand density of 1.2 chains / nm. 2 .

[0077] Final product test result: r2=195 mM -1 s -1 The sample size was 28 nm, PDI was 0.12, and the recovery rate was 92%. No precipitation occurred after storage at 4℃ for 6 months. The final product test results showed that all relevant indicators met the requirements.

[0078] Example 3

[0079] A method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling fractional purification based on relaxation rate kinetic feedback, the method comprising the following steps:

[0080] (1) Temperature-controlled slow-release pre-dispersion: Receive the suspension of iron oxide nanoparticles coated with thermally decomposed oleic acid (particle size 8±1 nm, temperature 80℃, concentration 5 mg / mL), cool it to 60℃ at a cooling rate of 20℃ / min, and simultaneously inject dimethyl sulfoxide (0.5 times by volume).

[0081] (2) Ligand adsorption: The solution obtained in the above steps was mixed with Dopamine-PEG-OCH3 (molecular weight 2000 Da). The amount of Dopamine-PEG-OCH3 (molecular weight 2000 Da) added was 3:1 ligand / Fe molar ratio. The reaction system was subjected to high shear-enhanced adsorption at a shear rate of 1000 s. -1 The residence time is 30 seconds, which allows the hydrophilic ligands to quickly come into contact with the surface of the hydrophobic iron oxide nanoparticles and undergo initial adsorption.

[0082] In this step, Dopamine-PEG-OCH3 (molecular weight 2000 Da) is a hydrophilic ligand, added in solution form, with DMSO (dimethyl sulfoxide) as the solvent. The amount of Dopamine-PEG-OCH3 added is a ligand / Fe molar ratio of 3:1, meaning that the molar ratio of the Dopamine-PEG-OCH3 compound to the Fe element in the iron oxide nanoparticle suspension in the ligand solution is 3:1.

[0083] The residence time is 30 seconds, which is the reaction time for high shear-enhanced adsorption.

[0084] (3) Rearrangement of balancing components: Incubation was carried out under low shear conditions. The reaction system temperature was 60℃ during low shear incubation. A magnetic stirrer was used as the low shear equipment, and the low shear rate was 10 s. -1 This allows the hydrophilic ligands already adsorbed on the surface of iron oxide nanoparticles to rearrange and anchor.

[0085] (4) Ligand equilibrium and endpoint determination: During the ligand rebalancing reaction phase, the transverse relaxation rate r2 and longitudinal relaxation rate r1 of the reaction system were periodically measured every 5 minutes, and the rate of change was calculated as Δ(r2 / r1) / Δt. The Δ(r2 / r1) / Δt measured at approximately 60 minutes was 0.012 min. -1 The rate of change meets the following condition: it is lower than the set rate of change threshold (≤0.02 min). -1 The reaction is terminated when the ligands are reorganized to a dynamic equilibrium.

[0086] (5) Fractional purification: In different ligand exchange kinetic stages, including ligand adsorption, ligand rearrangement, ligand equilibrium, and endpoint determination, the reaction system is subjected to fractional magnetic separation and synergistic purification. The magnetic field strength of the magnetic separation is dynamically adjusted to match the ligand binding state. In the first stage (ligand adsorption), the magnetic field gradient is 50 T / m, the purification time is 5 min, and the flow rate is 0.5 mL / min, which is used to remove large particle agglomerates. In the second stage (ligand rearrangement), the magnetic field gradient is 20 T / m, combined with ultrafiltration. The ultrafiltration conditions are MWCO 100 kDa to remove free ligands. In the third stage (ligand equilibrium and endpoint determination), the magnetic field gradient of the pulsed magnetic field is 20 T / m (30 s / 30 s, 3 cycles) to selectively separate equilibrium particles.

[0087] The step of this graded purification is performed in different ligand exchange kinetic stages, namely ligand adsorption, ligand reordering, ligand equilibration, and endpoint determination. After each purification stage, the particle size distribution and zeta potential of the DLS (Malvern Zetasizer) nanoparticle size and zeta potential are sampled and detected by transmission electron microscopy (TEM) (JEOL JEM-2100) to confirm the purification effect.

[0088] (6) Control of residual hydrophobic ligands: The residual amount of hydrophobic ligands is controlled by adjusting the ratio of the added hydrophilic ligands to the hydrophobic iron oxide nanoparticle suspension and the reaction time of the reaction system during the ligand adsorption, ligand reordering and / or ligand equilibrium and endpoint determination.

[0089] The method for controlling the residual amount of hydrophobic ligands is performed in the preceding steps of ligand adsorption and ligand rebalancing to ligand equilibrium and endpoint determination. In this embodiment, the ratio of the added hydrophilic ligand to the hydrophobic iron oxide nanoparticle suspension is 3:1 ligand / Fe molar ratio in the Dopamine-PEG-OCH3 solution, and the reaction time for ligand rebalancing to ligand equilibrium and endpoint determination is 60 minutes.

[0090] The residual control detection results in this embodiment are as follows: TGA determination showed a residual oleic acid content of 2.0% w / w and a surface ligand density of 0.5 chains / nm. 2 .

[0091] Final product test result: r2=185 mM -1 s -1 The parameters were: D_h = 22 nm, PDI = 0.15, and recovery rate 90%. The final product test results showed that all relevant indicators met the requirements, making it particularly suitable for the monodispersity requirements of magnetic particle imaging (MPI).

[0092] In Examples 1-3 above, Example 1 involves adding a high molar ratio of hydrophilic ligands, and by controlling the reaction process, a product with low residual content is obtained; Example 2 involves adding a medium molar ratio of hydrophilic ligands, and by controlling the reaction process, a product with medium residual content is obtained; Example 3 involves adding a low molar ratio of hydrophilic ligands, and by controlling the reaction process, a product with high residual content is obtained. Different products can be used for different purposes.

[0093] Comparative Example

[0094] The traditional room temperature exchange method based on existing technology: After the hydrophobic iron oxide prepared by thermal decomposition is naturally cooled to room temperature (25°C), Mal-PEG-NH2 (ligand / Fe molar ratio 5:1) is added, and the mixture is stirred at 25°C for 24 hours.

[0095] Test results: Sudden temperature change caused particle agglomeration (>20%), requiring high-speed centrifugation (10000 rpm) for separation, with a yield of only 58% and an r² value of 165 mM. -1 s -1 (15% lower than in Example 2), and the r2 value of 5 batches was CV=28% (coefficient of variation) and PDI=0.25, which could not meet clinical requirements.

[0096] Based on the above embodiments, it can be seen that the method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback is a method based on kinetic feedback regulation and graded purification. In the temperature-controlled slow-release pre-dispersion step, the hydrophobic iron oxide nanoparticle suspension at a temperature of 80–120℃ is cooled to 60–80℃ at a rate of ≥20℃ / min, and an aprotic polar solvent (volume ratio 0.5–1.0) is introduced simultaneously, which achieves a stable transition of the system, can inhibit the precipitation of hydrophobic ligands, and reduce the driving force of aggregation.

[0097] In the high-shear-enhanced adsorption step, at a shear rate of 1000–5000 s⁻¹ -1 Under certain conditions, the hydrophilic ligands are brought into rapid contact with the particle surface to achieve 60–80% initial adsorption, thus establishing a “non-equilibrium adsorption state”.

[0098] In the low-shear rearrangement and endpoint feedback control steps, incubation is performed under low-shear conditions, and the transverse relaxation rate r2 and longitudinal relaxation rate r1 are periodically detected. The rate of change is calculated as Δ(r2 / r1) / Δt. When the change is below a set threshold (≤0.02 min), the result is recorded. -1 When the ligand interface rearrangement reaches a dynamic equilibrium, the reaction is terminated. This is determined by establishing the relaxation rate change rate. The mapping relationship between the evolution of ligand interface structure transforms relaxation behavior from a "characterization means" into a "feedback control signal," thereby achieving adaptive termination control of the reaction process.

[0099] In the staged magnetic separation and synergistic purification method, the magnetic field is dynamically adjusted according to the ligand exchange kinetics stage to dynamically match the separation intensity with the ligand binding state. Specifically, in the three kinetic stages of the ligand exchange reaction—adsorption, rearrangement, and equilibrium—the magnetic field separation parameters are dynamically adjusted according to the ligand exchange kinetics stage. The purification conditions during the adsorption stage are: a high gradient magnetic field of 50-100 T / m and a separation time of 5-10 minutes; the purification conditions during the rearrangement stage are: a medium gradient magnetic field of 20-30 T / m combined with ultrafiltration (MWCO 100 kDa) and a separation time of 10-15 minutes; and the purification conditions during the equilibrium stage are: a pulsed magnetic field (30s on / 30s off, 3-5 cycles). This achieves synergistic optimization of the separation process and reaction stages, improving the recovery rate and purity.

[0100] Among the residual ligand regulation measures adopted, the residual hydrophobic ligand content was controlled at 0.5–2.0% to improve colloidal stability and optimize relaxation performance.

[0101] Through the above measures, this method achieves objective quantitative control of the reaction endpoint, reducing CV from >25% to <8%; significantly reduces particle aggregation, decreasing the aggregation rate from >30% to <2%; achieves synergistic optimization of the separation process and reaction stage, with a recovery rate >92% and purity >99.5%; and improves magnetic resonance performance, increasing r² to 180–220 mM. -1 s -1 The process time has been significantly reduced from 48 hours to 4–6 hours.

[0102] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0103] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback, characterized in that, include: Temperature-controlled slow-release pre-dispersion: The hydrophobic iron oxide nanoparticle suspension is cooled at a certain rate and an aprotic polar solvent is injected; Ligand adsorption: Adding hydrophilic ligands to the reaction system enhances adsorption with high shear, allowing the hydrophilic ligands to quickly contact the surface of hydrophobic iron oxide nanoparticles for initial adsorption; ligand rearrangement: Incubation under low shear conditions allows the hydrophilic ligands adsorbed on the surface of iron oxide nanoparticles to rearrange and anchor. Ligand equilibrium and endpoint determination: During the reaction phase of ligand reordering, the transverse relaxation rate r2 and longitudinal relaxation rate r1 of the reaction system are periodically detected, and the rate of change is calculated as Δ(r2 / r1) / Δt. When the rate of change is lower than the set rate of change threshold, it is determined that the ligand reordering has reached a dynamic equilibrium state and the reaction is terminated. Graded purification: In the different ligand exchange kinetic stages of ligand adsorption, ligand rearrangement, ligand equilibrium and endpoint determination, the reaction system is subjected to graded magnetic separation and synergistic purification treatment. The magnetic field strength of the magnetic separation is dynamically adjusted to dynamically match the ligand binding state.

2. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that, Also includes: Hydrophobic ligand residual amount control: The residual amount of hydrophobic ligands is controlled by adjusting the ratio of the added hydrophilic ligands to the hydrophobic iron oxide nanoparticle suspension and the reaction time of the reaction system during ligand adsorption, ligand reordering and / or ligand equilibrium and endpoint determination.

3. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that, The specific steps of the temperature-controlled slow-release pre-dispersion are as follows: the hydrophobic iron oxide nanoparticle suspension at a temperature of 80–120℃ is cooled to 60–80℃ at a rate of ≥20℃ / min, and an aprotic polar solvent is introduced simultaneously at a volume ratio of 0.5–1.

0.

4. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that, The shear rate for high-shear enhanced adsorption of the reaction system is 1000–5000 s⁻¹. -1 .

5. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that, The low-shear condition in the weighted arrangement of the ballast is: Low shear rate range: 10-200s -1 Preferred 50-100s -1 The temperature of the reaction system is 50℃-65℃ under low shear.

6. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that: In the ligand equilibration step, the rate of change threshold is ≤0.02 min. -1 If the rate of change is lower than the rate of change threshold twice consecutively, it is determined that the balancing weights have reached a dynamic equilibrium state and the reaction is terminated.

7. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that, The stepwise magnetic separation and synergistic purification process of the reaction system includes: In the ligand exchange kinetics stage of ligand adsorption, the purification conditions are: high gradient magnetic field 50-100 T / m, separation time 5-10 minutes; In the ligand exchange kinetics stage of the ligand reordering, the purification conditions are: medium gradient magnetic field 20-30 T / m, combined with ultrafiltration, and separation time 10-15 minutes. In the ligand exchange kinetics phase of ligand equilibrium, the purification conditions are: purification is performed using a pulsed magnetic field.

8. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 7, characterized in that: The conditions for the ultrafiltration treatment are: MWCO 50-100 kDa; The pulsed magnetic field has 3-5 cycles, and one cycle of the pulsed magnetic field is 30s on and 30s off.

9. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that: The aprotic polar solvent is selected from one or more of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, or acetone.

10. The method for determining the endpoint of ligand exchange on the surface of nano-iron oxide particles and controlling graded purification based on relaxation rate kinetic feedback according to claim 1, characterized in that: The hydrophilic ligand is Mal-PEG-NH2, HOOC-PEG-COOH, or Dopamine-PEG-OCH3.