Preparation methods and applications of POCD nanoprobes

Abstract

Preparation method and application of POCD nanoprobes. These probes have MRI imaging capabilities and drug loading capabilities, and can promote drug release to achieve therapeutic effects. The methods include: (1) preparing Fe3O4 nanoparticles; (2) preparing Fe3O4@mPDA nanoparticles; (3) preparing LF-Fe3O4@mPDA nanoparticles; (4) preparing LF-RSV-Fe3O4@mPDA nanoparticles: RSV is dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles at a ratio of 5:1. The mixture is stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical detection and medical image processing, and particularly relates to a method for preparing a POCD nanoprobe, and the application of this nanoprobe in the treatment of postoperative cognitive impairment. Background Technology

[0002] Postoperative cognitive dysfunction (POCD) refers to acute or early-onset brain dysfunction that occurs after surgery in patients who did not have mental illness before the procedure. It is generally influenced by the surgery itself and is a postoperative complication. POCD has a high prevalence in the elderly population and is characterized by a range of negative effects, including impaired memory, decreased information processing ability, and reduced attention, significantly reducing patients' quality of life.

[0003] The exact mechanisms of POCD, including its etiology and pathophysiology, are not fully understood, but it is likely closely related to several factors, including anesthesia, surgical trauma, inflammation, and neurovascular changes. Anesthesia-related factors (such as the type and duration of anesthesia) and surgery-related factors (such as the invasiveness and duration of surgery) play important roles. Studies have shown that various factors can increase the risk of POCD, such as advanced age, pre-existing cognitive impairment, cardiovascular disease and complications such as diabetes, genetic factors, and perioperative influencing factors. While diagnostic criteria for POCD are constantly evolving, the commonly used method remains neuropsychological testing to assess cognitive function before and after surgery. However, this traditional method faces significant challenges in implementation. Firstly, in clinical practice, different scales must be selected based on different cognitive domains and lobes, and the variety of scales increases the difficulty for medical professionals. Secondly, the diversity of scales leads to longer interview times. Finally, it presents challenges for patients with lower levels of education, potentially resulting in inaccurate data. Furthermore, POCD treatment is primarily preventative, including optimizing perioperative care, reducing anesthesia exposure, effectively controlling pain, and providing effective postoperative cognitive stimulation training; currently, there is a lack of effective therapeutic drugs. Therefore, finding new treatment options for POCD patients is an important research objective of this study.

[0004] Blood-brain barrier (BBB) ​​dysfunction is a significant contributing factor to post-traumatic brain disease (POCD). Recent discoveries regarding the mechanisms of POCD have led to various findings, including inflammatory responses in the peripheral circulatory system and central nervous system induced by surgery, glial cell activation, and the impact of oxidative stress on neuronal integrity. The BBB is a natural protective barrier in the brain, and its highly selective permeability makes it a major obstacle to drug delivery. Neither macromolecular drugs nor most antitumor drugs can cross the BBB, hindering effective treatment of brain-related diseases. With the rapid development of research on brain drug delivery, nanoparticle-based drug delivery systems have shown great potential in treating brain diseases. Nanoparticles, including liposomes, micelles, inorganic nanoparticles, hybrid nanoparticles, and exosomes, have been applied in preclinical studies for treating brain diseases. Increasing research evidence suggests that BBB dysfunction is a crucial factor in POCD; however, the presence of the BBB also results in extremely low bioavailability and poor therapeutic effects for many macromolecular drugs.

[0005] Lactoferrin is a widely used targeting substance in brain drug delivery. A natural glycoprotein and member of the transferrin family, lactoferrin possesses antiviral, anti-inflammatory, antioxidant, anticancer, and immunostimulatory effects. Due to the overexpression of its receptor on many cell surfaces, lactoferrin exhibits significant targeting potential. It can bind to the highly expressed TF receptor (TFR) and LF intracellular receptor (LFR) on the surface of highly proliferating cancer cells and endothelial cells of the blood-brain barrier. LF can cross the blood-brain barrier and has been shown to be a good candidate for manufacturing nanocarriers. Resveratrol, a natural chemical extracted from plants, has anti-inflammatory effects, and its role in improving cognitive function is increasingly recognized, showing some improvement in postoperative cognitive impairment.

[0006] Photothermal therapy (PTT) has long been recognized as a valuable treatment method in the biomedical field; however, it also faces several critical challenges that urgently need to be addressed. One major issue is that overheating can cause thermal resistance in treated cells, potentially significantly reducing the therapeutic efficacy of PTT. Another problem is that uncontrolled heat can diffuse into surrounding healthy tissue, posing a potential threat. Therefore, optimizing PTT to address these issues is a key focus of current research. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a POCD nanoprobe, which has MRI imaging capability and drug loading capability, and can promote drug release, thereby achieving a therapeutic effect.

[0008] The technical solution of this invention is: a method for preparing POCD nanoprobes, comprising the following steps:

[0009] (1) Preparation of Fe3O4 nanoparticles;

[0010] (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in TMB solution and ultrasonically dispersed to form an emulsion-like liquid. P123, F127 and dopamine hydrochloride were dissolved in ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 was added to the above solution system, mixed and ultrasonically for 1 hour, and then equipped with a mechanical stirrer. After stirring evenly, ammonia water was quickly added to the reaction system and reacted for 4 hours. After centrifugation, Fe3O4@mPDA was collected and washed three times alternately with water and ethanol. The obtained product was freeze-dried and stored.

[0011] (3) Preparation of LF-Fe3O4@mPDA nanoparticles: Dissolve LF in PBS, add EDC:NHS to the LF solution, mix well and place in a 4℃ refrigerator for 4h of static activation, then add Fe3O4@mPDA to the activated LF solution, adjust the pH of the mixed solution to 8.5 with ammonia water, stir at room temperature for 12h, collect by centrifugation, wash with deionized water, freeze dry and store at -20℃ for later use;

[0012] (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles at a ratio of 5:1. The mixture was stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

[0013] This invention successfully constructed a novel nanoprobe LF-RSV-Fe3O4@mPDA, which possesses superior MRI imaging capabilities and photothermal effects, targeting performance and high biocompatibility, and drug-carrying capacity for experimental drug delivery. At the cellular level, the novel LF-RSV-Fe3O4@mPDA nanoprobe was demonstrated to have good cellular uptake capacity, enabling MRI imaging and reducing ROS generation at the cellular level.

[0014] It also provides applications for POCD nanoprobes, such as in the preparation of drugs for treating postoperative cognitive impairment. Attached Figure Description

[0015] Figure 1 This is a research technology roadmap for the present invention.

[0016] Figure 2 This is a schematic diagram of the synthesis of the POCD nanoprobe according to the present invention.

[0017] Figure 3 These are TEM images of the nanoprobe. (a) TEM image of Fe3O4, (b) TEM image of Fe3O4@mPDA.

[0018] Figure 4 The images show the infrared microscopy of Fe3O4@mPDA, RSV-Fe3O4@mPD, and LF-RSV-Fe3O4@mPDA.

[0019] Figure 5 Photothermal properties of LF-RSV-Fe3O4@mPDA. (a) Temperature-time curves after 10 minutes of irradiation with 808 lasers at different power densities. (b) Temperature-time curves at a power density of 1.0 W / cm². -2 Temperature-time curves after 10 minutes of 808 laser irradiation. (c) Real-time thermal image corresponding to Figure Ⅰa, real-time thermal image corresponding to Figure Ⅱb. (d) Photothermal stability of the nanoprobe.

[0020] Figure 6 Drug release from LF-RSV-Fe3O4@mPDA nanoprobes under NIR response.

[0021] Figure 7 MRI imaging performance of LF-RSV-Fe3O4@mPDA nanoprobes: (a) Relaxation fitting curves of LF-RSV-Fe3O4@mPDA nanoprobes: lateral relaxation image 1 / T2; (b) T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes at different concentrations.

[0022] Figure 8 The effect of different concentrations of H2O2 on the viability of SH-SY5Y cells was investigated, n=4.

[0023] ***p<0.001.

[0024] Figure 9 The effects of different concentrations of LF-RSV-Fe3O4@mPDA nanoprobes on the activity of SH-SY5Y and bEnd.3 cells.

[0025] Figure 10 T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes at different concentration gradients at the cellular level.

[0026] Figure 11 To evaluate the photothermal performance of nanoprobes at the cellular level, (a) the effect of different NIR irradiation times on cell viability. (b) cell viability treated with H2O2, free RSV, and nanoprobes.

[0027] (n=3).

[0028] Figure 12 The following are images of the cellular phagocytosis and targeting evaluation of the nanoprobes. (a) Microscopic images of cells after co-incubation with Fe3O4@mPDA and LF-Fe3O4@mPDA for 2 and 4 hours; blue fluorescence represents DAPI-labeled cell nuclei, and red fluorescence represents RhB-loaded nanoprobes; (b) Statistical graph of Rhodamine B fluorescence intensity analysis (n=3) using ImageJ software.

[0029] Figure 13 For ROS detection of nanoprobes: (a) control group; (b) fluorescence spectrum of LF-RSV-Fe3O4@mPDA group; (c) fluorescence spectrum of H2O2 group; (d) fluorescence spectrum of LF-RSV-Fe3O4@mPDA+H2O2 group; (e) statistical graph of average fluorescence intensity of each group (n=3, ***p<0.001).

[0030] Figure 14 An evaluation graph was constructed for the model. (a) Time spent in the new arm of the Y-maze mouse; (b) IL-6 level in mouse blood samples. Statistical results: *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 15 For the in vivo biocompatibility assessment of the nanoprobes. (a) Body weight change curves of mice 15 days after tail vein injection of nanoprobes; (b) Blood routine indicators of mice after tail vein injection of different nanoprobes (n=4); (c) Histological section analysis of major organs of mice after tail vein injection of different nanoprobes (HE, 200×). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] To make the description of this disclosure more detailed and complete, illustrative descriptions of embodiments and specific examples of the present invention are provided below; however, these are not the only forms of implementing or utilizing the specific examples of the present invention. The embodiments cover features of multiple specific examples and methods and steps for constructing and operating these specific examples, and their order. However, other specific examples may also be used to achieve the same or equivalent functions and order of steps.

[0034] like Figure 1 , 2 As shown, a method for preparing a POCD nanoprobe includes the following steps:

[0035] (1) Preparation of Fe3O4 nanoparticles;

[0036] (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in...

[0037] In TMB solution, ultrasonic dispersion is used to form an emulsion-like liquid. P123, F127 and dopamine hydrochloride are dissolved in ethanol solution and ultrasonically dissolved completely. TMB solution containing Fe3O4 is added to the above solution system, mixed and ultrasonically for 1 hour, and then equipped with a mechanical stirrer. After stirring evenly, ammonia water is quickly added to the reaction system and reacted for 4 hours. After centrifugation, Fe3O4@mPDA is collected, washed three times alternately with water and ethanol, and the obtained product is freeze-dried and stored.

[0038] (3) Preparation of LF-Fe3O4@mPDA nanoparticles: Dissolve LF in PBS, add EDC:NHS to the LF solution, mix well and place in a 4℃ refrigerator for 4h of static activation, then add Fe3O4@mPDA to the activated LF solution, adjust the pH of the mixed solution to 8.5 with ammonia water, stir at room temperature for 12h, collect by centrifugation, wash with deionized water, freeze dry and store at -20℃ for later use;

[0039] (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles at a ratio of 5:1. The mixture was stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

[0040] This invention successfully constructed a novel nanoprobe LF-RSV-Fe3O4@mPDA, which possesses superior MRI imaging capabilities and photothermal effects, targeting performance and high biocompatibility, and drug-carrying capacity for experimental drug delivery. At the cellular level, the novel LF-RSV-Fe3O4@mPDA nanoprobe was demonstrated to have good cellular uptake capacity, enabling MRI imaging and reducing ROS generation at the cellular level.

[0041] Preferably, in step (1), in the synthesis of the oleic acid iron complex, 10.8 g of ferric chloride and 36.5 g of sodium oleate are dissolved in a mixed solvent composed of ethanol, distilled water and n-hexane, heated to 70°C for 4 hours. After the reaction is completed, the upper organic layer containing the oleic acid iron complex is washed with distilled water in a separatory funnel. After washing, hexane is evaporated to remove the oleic acid iron complex. Then, 36 g of the oleic acid iron complex and 5.7 g of oleic acid are dissolved in 200 g of 1-octadecene. The reaction mixture is heated to 320°C and reacted for 30 min. The initially transparent solution turns into a turbid brownish-black solution. It is cooled to room temperature, and 500 ml of ethanol is added to the solution. After centrifugation to obtain oleic acid-stabilized iron nanoparticles, OA-stabilized Fe3O4 is collected.

[0042] Preferably, in step (2), the prepared Fe3O4 powder is redissolved in TMB solution and ultrasonically dispersed to facilitate the formation of an emulsion oily liquid of 1 mg / ml. 300 mg P123, 750 mg F127 and 1.5 g dopamine hydrochloride are dissolved in 100 ml of 40% ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 is added to the solution system, mixed and ultrasonically sonicated for 1 hour, and then equipped with a mechanical stirrer. After stirring evenly, 3.2 ml of 30% ammonia water is quickly added to the reaction system, and the reaction is carried out for 4 hours. After centrifugation, Fe3O4@mPDA is collected.

[0043] Preferably, in step (3), LF is dissolved in 10 mg / ml PBS, and EDC:NHS is added to the LF solution at a ratio of 4:3.

[0044] Preferably, the method further includes step (5): observing the microstructure of the mesopores using a transmission electron microscope and measuring the particle size of the nanoprobe; detecting the ultraviolet-visible absorption spectrum of the drug loaded on the nanoprobe using a spectrophotometer; studying the infrared spectral characteristics of the nanoprobe using Fourier transform infrared microscopy and analyzing the formation of chemical bond peaks; evaluating the photothermal efficiency and stability of the nanoprobe in vitro using an 808nm semiconductor laser and an infrared thermal imaging system; and finally, studying the MR imaging and relaxation performance of the nanoprobe using an MRI imaging system with a magnetic field strength of 1.5T.

[0045] Preferably, the method further includes step (6), testing the photothermal efficiency and stability of LF-RSV-Fe3O4@mPDA. First, nanoprobes of different concentrations are added to a cuvette, and then irradiated with 808nm NIR at different power densities. Simultaneously, an infrared thermal imaging system is used to monitor and record the temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution, with parameters set to record once every 10 seconds for a total duration of 10 minutes. Real-time thermal images of the nanoprobe solution are also recorded. This experiment uses power densities of 0.5, 1.0, 1.5, and 2.0 W cm⁻¹. -2 Irradiate 1 ml of nanoprobe solution with an 808 nm laser for 10 minutes; the Fe concentration is 50 μg / mL. -1 Based on temperature changes, a power rating of 1.0W cm was selected. -2 Nanoparticles of different concentrations were irradiated with an 808nm laser for 10 minutes. The Fe concentrations were 0, 25, 50, 75, 100, and 200 μg / mL. -1 ; Using 1.0W cm -2 The 808nm laser was switched on and off repeatedly every 5 minutes to record temperature changes; images and experimental data obtained from the infrared thermal imaging system were collected, and the data were fitted into a time-temperature curve.

[0046] Preferably, the method further includes step (7), evaluating the drug loading rate of the LF-RSV-Fe3O4@mPDA nanoprobe, stirring the probe and RSV at a ratio of 5:1, measuring the drug absorbance using a UV spectrophotometer and an ELISA reader, and obtaining the loading rate and encapsulation efficiency of the nanoprobe based on the fitted drug concentration standard curve; and conducting a drug release experiment, loading LF-RSV-Fe3O4@mPDA into two dialysis bags, each containing 1 mg / mL of the drug. -1 LF-RSV-Fe3O4@mPDA was used. 1 ml of the nanoprobe solution was filled into each dialysis bag, and two dialysis bags were then immersed in a pH 7.4 solution, for a total volume of 20 ml. The mixture was shaken at 100 rpm. At time points of 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 17 h, 24 h, 28 h, 36 h, and 48 h, 1 ml of dialysate was collected, and an equal volume of buffer was added to maintain the same total volume. Before each dialysate collection, 1 W cm... -2 The PBS buffer solution with a pH of 7.4 was irradiated with an NIR laser for 10 minutes. Finally, the concentration of RSV released was measured by an enzyme-linked immunosorbent assay (ELISA) reader based on the absorption wavelength of RSV. The amount of RSV released was calculated based on the dose-absorption curve of RSV.

[0047] Preferably, the method further includes step (8), evaluating the relaxation performance of the nanoprobe. First, the concentration of iron ions in LF-RSV-Fe3O4@mPDA is determined by ICP. LF-RSV-Fe3O4@mPDA is prepared into solutions with different concentrations of 0, 10, 25, 50, 75, and 100 μg / ml, placed in 1.5 ml centrifuge tubes, and placed on a centrifuge tube rack filled with water. The T2 relaxation time of the nanoprobe at different concentrations is obtained by scanning with an MRI magnetic resonance scanner. The corresponding relaxation rate fitting curve is plotted according to the unit conversion of the iron particle concentration measured by ICP, and analyzed to evaluate the MRI imaging performance of LF-RSV-Fe3O4@mPDA. The specific scanning parameters are as follows: T2 3D sequence, TR = 2000 ms, TE = 50 ms, FOV = 140 mm × 140 mm.

[0048] It also provides applications for POCD nanoprobes, such as in the preparation of drugs for treating postoperative cognitive impairment.

[0049] Preferably, it is used to target the blood-brain barrier.

[0050] A therapeutic nanoprobe, LF-RSV-Fe3O4@mPDA, was synthesized, possessing excellent targeting, drug-loading, and MRI imaging properties. First, LF targets the LF receptor on the blood-brain barrier. Second, leveraging the large specific surface area and excellent photothermal properties of its polydopamine mesoporous layer, precise RSV release and low-temperature photothermal therapy are achieved. Finally, the MRI imaging properties of Fe3O4 are utilized to image the blood-brain barrier and evaluate the treatment effect. In the synthesis of the nanoprobe, oleic acid-stabilized Fe3O4 was used as the core, coated with mesoporous dopamine as the drug-loading system, and LF was covalently linked to it via EDC / NHS, resulting in excellent targeting performance. However, many factors influenced the synthesis process; the control of the morphology and particle size of the mesoporous dopamine, as well as the aqueous phase conversion, all affected the final experimental results.

[0051] Figure 3 These are TEM images of the nanoprobe. (a) TEM image of Fe3O4, (b)

[0052] TEM image of Fe3O4@mPDA.

[0053] Figure 4 The images show the infrared microscopy of Fe3O4@mPDA, RSV-Fe3O4@mPD, and LF-RSV-Fe3O4@mPDA.

[0054] Figure 5Photothermal properties of LF-RSV-Fe3O4@mPDA. (a) Temperature-time curves after 10 minutes of irradiation with 808 lasers at different power densities. (b) Temperature-time curves at a power density of 1.0 W / cm². -2 Temperature-time curve after 10 minutes of 808 laser irradiation. (c) Real-time thermal image corresponding to Figure a.

[0055] Figure 2b shows the real-time thermal image (d) corresponding to the photothermal stability of the nanoprobe.

[0056] Figure 6 Drug release from LF-RSV-Fe3O4@mPDA nanoprobes under NIR response.

[0057] Figure 7 MRI imaging performance of LF-RSV-Fe3O4@mPDA nanoprobes: (a) Relaxation fitting curves of LF-RSV-Fe3O4@mPDA nanoprobes: lateral relaxation image 1 / T2; (b) T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes at different concentrations.

[0058] Figure 8 The effect of different concentrations of H2O2 on the viability of SH-SY5Y cells was investigated, n=4.

[0059] ***p<0.001.

[0060] Figure 9 The effects of different concentrations of LF-RSV-Fe3O4@mPDA nanoprobes on the activity of SH-SY5Y and bEnd.3 cells.

[0061] Figure 10 T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes at different concentration gradients at the cellular level.

[0062] Figure 11 To evaluate the photothermal performance of nanoprobes at the cellular level, (a) the effect of different NIR irradiation times on cell viability. (b) cell viability treated with H2O2, free RSV, and nanoprobes.

[0063] (n=3).

[0064] Figure 12 The following are images of the cellular phagocytosis and targeting evaluation of the nanoprobes. (a) Microscopic images of cells after co-incubation with Fe3O4@mPDA and LF-Fe3O4@mPDA for 2 and 4 hours; blue fluorescence represents DAPI-labeled cell nuclei, and red fluorescence represents RhB-loaded nanoprobes; (b) Statistical graph of Rhodamine B fluorescence intensity analysis (n=3) using ImageJ software.

[0065] Figure 13 For ROS detection of nanoprobes: (a) control group; (b) fluorescence spectrum of LF-RSV-Fe3O4@mPDA group; (c) fluorescence spectrum of H2O2 group; (d) fluorescence spectrum of LF-RSV-Fe3O4@mPDA+H2O2 group; (e) statistical graph of average fluorescence intensity of each group (n=3, ***p<0.001).

[0066] Figure 14 An evaluation graph was constructed for the model. (a) Time spent in the new arm of the Y-maze mouse; (b) IL-6 level in mouse blood samples. Statistical results: *p<0.05, **p<0.01, ***p<0.001.

[0067] Figure 15 For the in vivo biocompatibility assessment of the nanoprobes. (a) Body weight change curves of mice 15 days after tail vein injection of nanoprobes; (b) Blood routine indicators of mice after tail vein injection of different nanoprobes (n=4); (c) Histological section analysis of major organs of mice after tail vein injection of different nanoprobes (HE, 200×).

[0068] This study employed a soft-template method, which, compared to the hard-template method, is a synthesis method performed at the molecular level. The chemical reaction between the template agent and the carbon precursor in the soft template is a crucial factor in the synthesis process, which is relatively simple and less environmentally polluting. To further simplify the synthesis process, a dual soft-template method was used. Fe3O4 was redissolved in TMB and sonicated to form an emulsion, which was then added to a block copolymer (P123 and F127) solution with a certain concentration of ethanol as a solvent. After adding a certain proportion of dopamine hydrochloride and sonicating to homogenize, a certain amount of ammonia was added under mechanical stirring to provide alkaline conditions. During this synthesis process, we found that, consistent with existing literature reports, the filling parameters of the surfactant can be precisely controlled by finely adjusting the mass ratio of P123 to F127 in the reaction system. At the same time, the amount of TMB also has a significant impact, and the amount of dopamine hydrochloride is related to the thickness of the mesoporous dopamine. Temperature and the concentration of ethanol in the reaction system also have a significant impact on the morphology of the mesoporous dopamine. In the synthesis of mesoporous dopamine using the dual-template method, Guan BY et al. found that when the mass ratio of P123 to F127 reached 1:1 and 5:3, the morphology of the mesoporous dopamine transformed into non-porous, wrinkled nanosheets and nanodiscs. By continuously increasing the volume fraction of ethanol from 30% to 40%, and then to 45%, the resulting mesoporous dopamine structure evolved from a mixture of nanospheres and bowl-shaped nanoparticles to dendritic nanoparticles, and then to walnut-shaped nanoparticles. When the volume fraction of ethanol was further increased to 55%, non-porous particles with a wrinkled, sheet-like morphology were formed. Therefore, in this experiment, the mass ratio of each substance was continuously adjusted, and the temperature and reaction system were controlled during the synthesis process.

[0069] The particle size of carriers involved in brain drug delivery is generally below 200 nm, which facilitates endocytosis and smooth entry into the brain-to-brain barrier (BBB). Therefore, the nanoprobe constructed in this experiment, by controlling the amount of dopamine hydrochloride and the reaction time, showed a particle size of about 100 nm under transmission electron microscopy, which can better penetrate the BBB. However, the proportion of mesoporous morphology of the nanoprobe should be more precisely controlled to form a microporous structure, which may be more conducive to improving the drug loading rate.

[0070] Imaging examinations play a crucial role in disease diagnosis, treatment, and monitoring, offering advantages such as non-invasiveness, providing detailed structural and functional information, enabling early diagnosis, and monitoring disease progression. Non-invasive and radiation-free MRI has enormous development potential. To enhance contrast between tissues, exogenous contrast agents are introduced during examinations. Gadolinium is a common MRI contrast agent; however, it has a long residence time in the body, slow excretion, and potential nephrotoxicity, especially in patients with renal insufficiency. Studies have developed contrast agents based on iron nanoparticle pairs. Iron contrast agents are relatively safer and less damaging to the kidneys. Currently, five types of superparamagnetic iron oxide nanoparticles (SPION) have been designed and are undergoing clinical trials as MRI contrast agents, but they are only in use in a few countries.

[38] SPION's unique advantages make it a promising area for research in molecular imaging.

[0071] This study used Fe3O4 as an imaging material, which demonstrated good T2 imaging capability under MRI. The synthesized Fe3O4 nanoparticles had a particle size of approximately 12 nm. Transmission electron microscopy revealed that the synthesized Fe3O4@mPDA contained Fe3O4 particles encapsulated within a mesoporous structure. MRI results showed an r2 value of 5.704 mM. -1 s -1 This indicates that the LF-RSV-Fe3O4@mPDA nanoprobe is a good T2-weighted negative contrast agent.

[0072] This study investigated the in vitro photothermal conversion performance and photothermal stability of LF-RSV-Fe3O4@mPDA. Literature review indicated that LF-RSV-Fe3O4@mPDA can promote blood-brain barrier permeability and increase drug accumulation in the brain at 41-43℃, resulting in a better therapeutic effect on neurodegenerative diseases. In this experiment, a power density of 1.0 W / cm² was used. -2 808nm laser on 50μg mL -1The temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution reached 40.51℃ after irradiation for 10 minutes, indicating that the nanoprobe possesses strong photothermal conversion capabilities. Within one hour, the temperature rise and fall of the nanoprobe were similar under near-infrared light switching, demonstrating the good photothermal stability of LF-RSV-Fe3O4@mPDA. The combined results of in vitro photothermal testing and cellular-level photothermal performance evaluation validate that this nanoprobe exhibits excellent photothermal conversion performance and photothermal stability, making it a promising photothermal responsive material.

[0073] The blood-brain barrier (BBB) ​​is a highly selective, dynamic interface between the blood and the brain, protecting central neurons from damage. The BBB's presence makes it difficult for most central nervous system (CNS) drugs to reach the brain parenchyma, blocking the entry of almost all macromolecular drugs and nearly 98% of small molecule drugs, resulting in extremely low bioavailability for many drugs. Therefore, targeted therapy is extremely important in the CNS, and crossing the biological barrier is one of the most crucial steps in achieving CNS-targeted therapy. In recent years, nanoparticles have been widely studied in brain drug delivery due to their superior performance. Currently, among various CNS-targeting therapies, receptor-mediated targeting is the most extensively studied. Literature review reveals that LF, a glycoprotein belonging to the transferrin (TF) family, can bind to TF receptors (TFRs) and LF intracellular receptors (LFRs) highly expressed on the surface of highly proliferating cancer cells and BNS cells, thereby promoting their access to the cell nucleus. This advantage can be used to develop active targeted drug delivery systems that can easily cross the BBB, allowing drugs to reach the site of injury and achieve good therapeutic effects.

[0074] In this study, LF was covalently coupled using EDC / NSH. Microscopic infrared spectroscopy revealed characteristic peaks of amide bonds, confirming successful LF attachment to the nanoprobe surface. At the cellular level, co-incubating the targeted nanoprobe with untargeted probes and cells showed that the targeted nanoprobe was taken up by cells in greater quantities, demonstrating the good targeting performance of the synthesized nanoprobe. Previous studies have verified at the animal level that LF-attached nanoprobes reached more brain tissue than unattached nanoprobes, proving LF as a good targeting substance.

[0075] A growing body of research indicates that a high drug loading rate must be accompanied by good drug release capability to ensure effective drug delivery to the site of injury. Current research largely focuses on developing stimulus-responsive drug release systems that remain stable under extracellular conditions while responding to intracellular stimuli after internalization, including acidic pH environments, redox reactions, reactive oxygen species, and NIR photothermal stimulation, triggering rapid drug release.

[0076] In this study, we explored drug release under photothermal response. Using a pH of 7.4 in vitro to simulate the brain environment, we found that the nanoprobe could release drugs. Under NIR irradiation, the nano-drug-loaded system released more drug in the same amount of time, demonstrating the probe's photothermal responsive drug release capability. In previous research, our group also synthesized a black TiO2 nanoprobe targeting pancreatic tumors. This probe achieves MRI-guided photothermal chemotherapy by targeting the IGF1 receptor. Experimental results showed that this nanoprobe can promote the release of chemotherapeutic drugs and destroy tumors through photothermal effects and an acidic environment of pH 5, providing a new strategy and method for the treatment of pancreatic cancer.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing POCD nanoprobes, characterized in that: It includes the following steps: (1) Preparation of Fe3O4 nanoparticles; (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in TMB solution and ultrasonically dispersed to form an emulsion-like liquid. P123, F127 and dopamine hydrochloride were dissolved in ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 was added to the above solution system, mixed and ultrasonically for 1 hour, and then equipped with a mechanical stirrer. After stirring evenly, ammonia water was quickly added to the reaction system and reacted for 4 hours. After centrifugation, Fe3O4@mPDA was collected and washed three times alternately with water and ethanol. The obtained product was freeze-dried and stored. (3) Preparation of LF-Fe3O4@mPDA nanoparticles: Dissolve LF in PBS, add EDC:NHS to the LF solution, mix well and place in a 4℃ refrigerator for 4h of static activation, then add Fe3O4@mPDA to the activated LF solution, adjust the pH of the mixed solution to 8.5 with ammonia water, stir at room temperature for 12h, collect by centrifugation, wash with deionized water, freeze dry and store at -20℃ for later use; (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles at a ratio of 5:

1. The mixture was stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles. The RSV is resveratrol.

2. The method for preparing POCD nanoprobes according to claim 1, characterized in that: In step (1), during the synthesis of the oleic acid iron complex, 10.8 g of ferric chloride and 36.5 g of sodium oleate were dissolved in a mixed solvent consisting of ethanol, distilled water, and n-hexane. The mixture was heated to 70°C for 4 hours. After the reaction was completed, the upper organic layer containing the oleic acid iron complex was washed with distilled water using a separatory funnel. After washing, hexane was evaporated to remove the oleic acid iron complex. Then, 36 g of the oleic acid iron complex and 5.7 g of oleic acid were dissolved in 200 g of 1-octadecene. The reaction mixture was heated to 320°C and reacted for 30 min. The initially transparent solution turned into a turbid brownish-black solution. The solution was cooled to room temperature, and 500 ml of ethanol was added to the solution. After centrifugation, oleic acid-stabilized iron nanoparticles were obtained, and OA-stabilized Fe3O4 was collected.

3. The method for preparing POCD nanoprobes according to claim 2, characterized in that: In step (2), the prepared Fe3O4 powder is redissolved in TMB solution and ultrasonically dispersed to form an emulsion oily liquid of 1 mg / ml. 300 mg P123, 750 mg F127 and 1.5 g dopamine hydrochloride are dissolved in 100 ml of 40% ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 is added to the solution system, mixed and ultrasonically for 1 hour, and then equipped with a mechanical stirrer. After stirring evenly, 3.2 ml of 30% ammonia water is quickly added to the reaction system. The reaction is carried out for 4 hours, and Fe3O4@mPDA is collected after centrifugation.

4. The method for preparing POCD nanoprobes according to claim 3, characterized in that: In step (3), LF is dissolved in 10 mg / ml PBS, and EDC:NHS is added to the LF solution at a ratio of 4:

3.

5. The method for preparing POCD nanoprobes according to claim 4, characterized in that: The method also includes step (5): observing the microstructure of the mesopores using a transmission electron microscope and measuring the particle size of the nanoprobes; detecting the ultraviolet-visible absorption spectrum of the drug loaded on the nanoprobes using a spectrophotometer; studying the infrared spectral characteristics of the nanoprobes using Fourier transform infrared microscopy and analyzing the formation of chemical bond peaks; evaluating the photothermal efficiency and stability of the nanoprobes in vitro using an 808nm semiconductor laser and an infrared thermal imaging system; and finally, studying the MR imaging and relaxation properties of the nanoprobes using an MRI imaging system with a magnetic field strength of 1.5T.

6. The method for preparing POCD nanoprobes according to claim 5, characterized in that: The method also includes step (6), testing the photothermal efficiency and stability of LF-RSV-Fe3O4@mPDA. First, nanoprobes of different concentrations are added to cuvettes, and then irradiated with 808nm NIR at different power densities. Simultaneously, an infrared thermal imaging system is used to monitor and record the temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution, with parameters set to record every 10 seconds for a total duration of 10 minutes. Real-time thermal images of the nanoprobe solution are also recorded. This experiment uses power densities of 0.5, 1.0, 1.5, and 2.0 W / cm². -2 Irradiate 1 ml of nanoprobe solution with an 808 nm laser for 10 minutes; the Fe concentration is 50 μg / mL. -1 Based on temperature changes, a power rating of 1.0W cm was selected. -2 Nanoparticles of different concentrations were irradiated with an 808nm laser for 10 minutes. The Fe concentrations were 0, 25, 50, 75, 100, and 200 μg / mL. -1 ; Using 1.0W cm -2 The 808nm laser was switched on and off repeatedly every 5 minutes to record temperature changes; images and experimental data obtained from the infrared thermal imaging system were collected, and the data were fitted into a time-temperature curve.

7. The method for preparing POCD nanoprobes according to claim 6, characterized in that: The method also includes step (7), evaluating the drug loading rate of the LF-RSV-Fe3O4@mPDA nanoprobe. The probe and RSV are stirred at a ratio of 5:

1. The absorbance of the drug is measured using a UV spectrophotometer and an ELISA reader. Based on the fitted drug concentration standard curve, the loading rate and encapsulation efficiency of the nanoprobe are obtained. A drug release experiment is performed, in which LF-RSV-Fe3O4@mPDA is placed into two dialysis bags, each containing 1 mg / mL of the drug. -1 LF-RSV-Fe3O4@mPDA was used. 1 ml of the nanoprobe solution was filled into each dialysis bag, and two dialysis bags were then immersed in a pH 7.4 solution, for a total volume of 20 ml. The mixture was shaken at 100 rpm. At time points of 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 17 h, 24 h, 28 h, 36 h, and 48 h, 1 ml of dialysate was collected, and an equal volume of buffer was added to maintain the same total volume. Before each dialysate collection, 1 W cm... -2 The PBS buffer solution with a pH of 7.4 was irradiated with an NIR laser for 10 minutes. Finally, the concentration of RSV released was measured by an enzyme-linked immunosorbent assay (ELISA) reader based on the absorption wavelength of RSV. The amount of RSV released was calculated based on the dose-absorption curve of RSV.

8. The method for preparing POCD nanoprobes according to claim 7, characterized in that: The method also includes step (8), evaluating the relaxation performance of the nanoprobe. First, the concentration of iron ions in LF-RSV-Fe3O4@mPDA is determined by ICP. LF-RSV-Fe3O4@mPDA is prepared into solutions with different concentrations of 0, 10, 25, 50, 75, and 100 μg / ml and placed in 1.5 ml centrifuge tubes. The centrifuge tubes are placed on a centrifuge tube rack filled with water. The T2 relaxation time of the nanoprobe at different concentrations is obtained by scanning with an MRI magnetic resonance scanner. The corresponding relaxation rate fitting curve is plotted according to the unit conversion of the iron particle concentration measured by ICP and analyzed to evaluate the MRI imaging performance of LF-RSV-Fe3O4@mPDA. The specific scanning parameters are as follows: T2 3D sequence, TR = 2000 ms, TE = 50 ms, FOV = 140 mm × 140 mm.

9. The application of the POCD nanoprobe according to claim 1, characterized in that: Its application is in the preparation of drugs for treating postoperative cognitive impairment.

10. The application of the POCD nanoprobe according to claim 9, characterized in that: Its application is in targeting the blood-brain barrier.

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