Super-small nanodots with dual targeting and efficient penetration of BBB and preparation thereof

Abstract

The application discloses an anti-Alzheimer's disease (AD) ultra-small nanodot with high BBB penetration and double targeting, and a preparation method and application thereof, and belongs to the field of nanomaterial preparation and biomedical application. The ultra-small nanodot is obtained by a self-assembly method according to a certain ratio of protein molecules, a five-membered peptide, ferrous ions (Fe 2+ ), and flavonoid drugs. The obtained ultra-small nanodot has the abilities of double targeting, high BBB penetration and multi-target point treatment of AD. Meanwhile, the preparation method is simple, raw materials are easy to obtain, the biological safety is good, the stability is high, the treatment level is various, and the ultra-small nanodot can be rapidly metabolized in a living body, and has the potential to be a safe and efficient multi-target point anti-AD therapeutic agent.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and biomedical applications, specifically relating to an ultra-small nanodot, its preparation method and application. This ultra-small nanodot can promote efficient drug penetration of the BBB through the ultra-small size effect combined with receptor-mediated transport effect, and improve the efficacy of anti-AD through multiple targets (including degradation of Aβ protein fibers, inhibition of Aβ aggregation and scavenging of ROS). Background Technology

[0002] Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disease characterized by a continuous decline in cognitive and memory functions, along with various neuropsychiatric symptoms and behavioral disorders. Surveys indicate that in 2015, 47 million people worldwide suffered from Alzheimer's disease, and this number is projected to reach 75 million by 2030 and 131 million by 2050. The increase in AD cases is particularly pronounced in low- and middle-income countries, placing immense social and economic pressure on them. Therefore, developing effective treatments and interventions for AD has become a priority in 21st-century healthcare research.

[0003] As a neurological disease influenced by multiple factors, Alzheimer's disease (AD) has seen the development of a series of drugs targeting different pathological mechanisms, such as Aβ, microglia, and Tau, in recent years, based on research into its etiology. However, the clinical trial results of these drugs have not been optimistic, largely because most free drugs cannot effectively reach the brain to exert their intended effects. First, the brain border (BBB) ​​is a major barrier to drug delivery in brain tumors and neurodegenerative diseases (such as Alzheimer's and Parkinson's). Over 98% of small molecule drugs and almost 100% of protein, peptide, and gene therapy drugs cannot cross the BBB to enter the brain and thus cannot achieve their intended therapeutic effects. Second, once drugs enter the brain, they cannot accurately target diseased cells; transport through simple diffusion pathways significantly reduces their therapeutic efficacy against diseased cells and can also affect healthy tissues and cells. More importantly, AD is related to multiple pathological mechanisms, and single drugs, due to their inherent limitations such as easy degradation and poor stability, are difficult to treat effectively. Therefore, developing nanotherapeutic agents with high BBB penetration and multi-target therapy for AD is of great significance for the clinical treatment of AD. Summary of the Invention

[0004] Given the current difficulties in effectively delivering drugs into the brain and the need for developing drugs to treat Alzheimer's disease (AD), this invention aims to provide an ultra-small nanodot with secondary targeting and efficient BBB penetration capability for anti-AD, as well as its preparation method. In this ultra-small nanodot, a pentapeptide can specifically bind to Aβ through hydrophobic interactions, and protein molecules can promote the targeted penetration of nanoparticles into the BBB via receptor-mediated mechanisms. 2+ - The flavonoid complex exhibits multi-target anti-AD therapeutic effects. More importantly, the ultra-small size effect of nanodots can further enhance the BBB penetration efficiency of the drug, while simultaneously enabling the rapid metabolism of nanomedicines in vivo. Therefore, it holds promise as a safe, efficient, and multi-target anti-AD therapeutic agent.

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

[0006] A highly efficient BBB-penetrating, dual-targeting ultra-small nanodot, composed of protein molecules, pentapeptides, and ferrous ions (Fe2+). 2+ Flavonoid drugs are self-assembled; they are monodisperse spherical particles with a size of 2.5~6.5 nm, a potential of -15 mV~-22 mV, and a drug loading rate of 50%~60%.

[0007] Furthermore, the protein molecule is lactoferrin (Lf) with brain-targeting BBB function; the pentapeptide is any one of KLVFF, LPFFD, and LVFFA that has targeting affinity for Aβ protein aggregates; and the flavonoid drug is any one of quercetin (Que), silymarin (SLB), and rutin.

[0008] The method for preparing the ultra-small nanodots includes the following steps:

[0009] 1) Dissolve the pentapeptide fully in ultrapure water to obtain a pentapeptide solution;

[0010] 2) Dissolve the protein thoroughly in ultrapure water to obtain a protein solution;

[0011] 3) Dissolve the ferrous ion precursor in deionized water to obtain a ferrous solution;

[0012] 4) Dissolve the flavonoid drug thoroughly in an organic solvent to obtain a drug solution;

[0013] 5) Mix the obtained pentapeptide solution and protein solution evenly at a certain speed, add an appropriate amount of PBS solution and ferrous solution, react for a period of time, then add anhydrous ethanol and Tris solution to adjust the pH of the reaction solution, and finally add the drug solution to react.

[0014] 6) After centrifuging the solution after the reaction in step 5), the supernatant was taken and purified by ultrafiltration to obtain the ultra-small nanodots.

[0015] Furthermore, the ferrous ion precursor mentioned in step 3) is FeSO4.

[0016] Further, the organic solvent mentioned in step 4) is selected from any one of dimethyl sulfoxide, anhydrous acetone and anhydrous ethanol.

[0017] Further, the amounts of the pentapeptide solution, protein solution, ferrous solution, and drug solution used in step 5) are determined according to the proportions of pentapeptide, protein, ferrous iron, and ferrous sulfate. 2+ The mass ratio of the drug to flavonoids is 1:1.25~2.5:0.45~0.7:0.2~0.4, with a preferred ratio of 1:2.5:0.56:0.378.

[0018] In this invention, the ultra-small size of the nanodots is formed by the co-assembly of pentapeptides and protein molecules at a mass ratio of 1:1.25~2.5. During the assembly process, both act as dispersants and compete with flavonoids for Fe. 2+ The coordination sites are used to regulate particle size.

[0019] Furthermore, in step 5), the first reaction time is 3-5 minutes; the pH range is adjusted to 7-10, preferably 9; and the reaction time after adding the drug solution is 15-30 minutes.

[0020] Furthermore, the high-speed centrifugation speed in step 6) is 8000~12000 rpm, preferably 10000 rpm.

[0021] This invention provides an ultra-small nanodot with efficient BBB penetration and dual targeting capabilities. The pentapeptide composing this ultra-small nanodot specifically binds to Aβ via hydrophobic interactions, enabling the nanoparticles to target Aβ aggregates. The protein molecules composing this ultra-small nanodot can target BBB surface receptors, thereby enhancing its BBB-crossing efficiency. Furthermore, it can synergistically and efficiently penetrate the BBB through size effects, achieving effective drug accumulation in the brain. Using flavonoid drugs as assembly units, it can... 2+ The chelating coordination and synergistic effect of the nanostructure enhances the stability, water solubility, and antioxidant properties of flavonoid drugs, thereby enabling them to degrade Aβ protein fibers, inhibit Aβ aggregation, and scavenge ROS. This allows for the single-drug, multi-target, multi-pathway inhibition of AD-related pathogenic factors. More importantly, due to their ultra-small size, they can be rapidly metabolized in vivo, avoiding the toxic side effects of prolonged drug accumulation in normal tissues and organs during actual drug use. Therefore, the ultra-small nanodots obtained in this invention can be used to prepare anti-Alzheimer's disease drugs.

[0022] The significant advantages of this invention are:

[0023] (1) The ultra-small nanodots prepared in this invention can simultaneously target Aβ and BBB through receptor-mediated transport effect, size effect and drug treatment, and can efficiently penetrate BBB to achieve efficient drug accumulation in brain lesion area and achieve significant anti-AD effect.

[0024] (2) The ultra-small nanodots prepared by the present invention have small size, good dispersibility, high stability, high drug loading rate, and high BBB penetration. They can also be rapidly metabolized in vivo, avoiding the toxic side effects of drugs accumulating in the body for a long time on normal tissues and organs. Attached Figure Description

[0025] Figure 1 The KLVFF-Lf-Fe prepared in Example 1 2+ -Particle size diagram (a) and potential diagram (b) of Que NDs.

[0026] Figure 2 The KLVFF-Lf-Fe prepared in Example 1 2+ -Que NDs (a) and KLVFF-BSA-Fe 2+ - TEM image of Que NDs(b).

[0027] Figure 3 The KLVFF-Lf-Fe prepared in Example 1 2+ -Particle size variation of Que NDs over seven days in PBS (a) and 1640 culture medium (b).

[0028] Figure 4 The KLVFF-Lf-Fe prepared in Example 1 2+ - Comparison of ROS scavenging capabilities between Que NDs (2, 4, 6, 8, 10 μg / mL) and Que (2, 4, 6, 8, 10 μg / mL).

[0029] Figure 5 The KLVFF-Lf-Fe prepared in Example 1 2+ - A biocompatibility characterization diagram of Que NDs.

[0030] Figure 6 For application of Aβ fiber and KLVFF-Lf-Fe in Example 1 2+ - Degradation curves of Que NDs (0, 10, 20, 30, 40 μg / mL) at different time points (a) and degradation rate data (b) after co-incubation for 48 hours.

[0031] Figure 7 For application of Aβ fiber and KLVFF-Lf-Fe in Example 1 2+ -CLSM plot of Que NDs after 48 hours of co-incubation, where a is Aβ fiber (20 μM) and b is Aβ fiber (20 μM) + 10 μg / mL KLVFF-Lf-Fe 2+ -Que NDs, c is Aβ fiber (20 μM) + 20 μg / mL KLVFF-Lf-Fe 2+ -Que NDs.

[0032] Figure 8 To apply the Aβ monomer reservoir solution and KLVFF-Lf-Fe in Example 1 2+ -Growth curves (a) and inhibition rate data (b) of Que NDs (0, 10, 20, 30, 40 μg / mL) co-incubated at different time points.

[0033] Figure 9 To apply the Aβ monomer reservoir solution and KLVFF-Lf-Fe in Example 1 2+ -CLSM plot of Que NDs after 48 hours of co-incubation, where a is Aβ monomer (20 μM) and b is Aβ monomer (20 μM) + 10 μg / mL KLVFF-Lf-Fe 2+ -Que NDs, c is Aβ monomer (20 μM) + 20 μg / mL KLVFF-Lf-Fe 2+ -Que NDs.

[0034] Figure 10 To apply the Aβ monomer storage solution in Example 1 to KLVFF-Lf-Fe 2+ -Que NDs、Lf-Fe 2+ - Comparison of inhibition rates of Aβ fibrosis after co-incubation with QueNDs for 48 hours.

[0035] Figure 11 For application example 1, RhB, KLVFF-BSA-Fe 2+ -Que NDs (20 μg / mL, RhB labeled), KLVFF-Lf-Fe 2+ - Comparison of penetration rates of Que NDs (20 μg / mL, RhB labeled) against the BBB model.

[0036] Figure 12 To apply the BBB model in Example 1 to the lower chamber PC12 cells to RhB, KLVFF-BSA-Fe 2+-Que NDs (20 μg / mL, RhB labeled), KLVFF-Lf-Fe 2+ - Confocal plot of Que NDs (20 μg / mL, RhB labeled) uptake.

[0037] Figure 13 To apply KLVFF-Lf-Fe at different time points (1, 6, 12, 24 h) in Example 1 2+ Confocal plot of the metabolism of Que NDs (20 μg / mL, RhB labeled) in zebrafish. Detailed Implementation

[0038] An ultra-small nanodot with high efficiency in penetrating the BBB and dual targeting is prepared by the following steps:

[0039] 1) Dissolve the pentapeptide fully in ultrapure water to obtain a pentapeptide solution;

[0040] 2) Dissolve the protein thoroughly in ultrapure water to obtain a protein solution;

[0041] 3) Dissolve FeSO4 in deionized water to obtain a ferrous solution;

[0042] 4) Dissolve the flavonoids thoroughly in dimethyl sulfoxide, anhydrous acetone or anhydrous ethanol to obtain a drug solution;

[0043] 5) Mix the obtained pentapeptide solution and protein solution evenly at a certain speed, add an appropriate amount of PBS buffer solution and ferrous solution, react for 3-5 minutes, then add anhydrous ethanol and Tris solution to adjust the pH of the reaction solution to 7-10, and finally add the drug solution and react for 15-30 minutes; the amounts of pentapeptide solution, protein solution, ferrous solution and drug solution used should be based on the proportions of pentapeptide, protein, ferrous iron, and ferrous iron. 2+ The mass ratio of flavonoids to flavonoids is 1:1.25~2.5:0.45~0.7:0.2~0.4 for conversion.

[0044] 6) After centrifuging the solution after the reaction in step 5) at high speed of 8000~12000 rpm, the supernatant was taken and purified by ultrafiltration to obtain the ultra-small nanodots.

[0045] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0046] Example 1

[0047] 1) Weigh 4.0 mg of KLVFF and dissolve it in 1.0 mL of ultrapure water to prepare a 4.0 mg / mL KLVFF solution.

[0048] 2) Weigh 10.0 mg of Lf and dissolve it in 1.0 mL of ultrapure water to prepare a 10.0 mg / mL Lf solution.

[0049] 3) Weigh 0.0056 g of FeSO4·7H2O and dissolve it in 100 μL of HCl (0.1 M) solution, then add 900 μL of ultrapure water to prepare a 0.02 M FeSO4·7H2O solution. 2+ Solution.

[0050] 4) Weigh 0.0121 g of quercetin (Que) and dissolve it in 2.0 mL of anhydrous ethanol to prepare a 0.02 M Que solution.

[0051] 5) Add 200 μL of KLVFF solution and 200 μL of Lf solution to the reaction flask, then add 200 μL of PBS buffer solution (pH=7.4) and 80 μL of Fe under stirring at 1000 rpm. 2+ The solution was reacted for 3-5 minutes, then 100 μL of anhydrous ethanol and 200 μL of 0.1 M Tris solution were added to bring the pH to 9.0. Next, 50 μL of Que solution was added, and the reaction was continued for 15 minutes. The reaction solution was then centrifuged twice at 10000 rpm. The supernatant was transferred to an ultrafiltration tube and washed three times with water at 4500 rpm to obtain ultra-small nanodots KLVFF-Lf-Fe. 2+ -Que NDs (denoted as KLFQ NDs).

[0052] 1. The obtained KLVFF-Lf-Fe 2+ -Que NDs were added to pure water, and the drug content of Que was used for quantification to prepare a solution with a concentration of 10 μg / mL. The particle size and potential were measured, and the results are as follows: Figure 1 As shown.

[0053] Depend on Figure 1 It can be seen that KLVFF-Lf-Fe 2+ The particle size of the -Que NDs is 4.45±2.0 nm (a) and the potential is -17.5 mV (b).

[0054] 2. Add 10 μg / mL KLVFF-Lf-Fe 2+ -Que NDs and KLVFF-BSA-Fe 2+ -Que NDs solution was added dropwise to a copper grid, and after drying, it was characterized by electron microscopy. The results are as follows: Figure 2 As shown. Replace Lf with bovine serum albumin (BSA) and synthesize KLVFF-BSA-Fe according to the steps above. 2+-Que NDs (denoted as KBFQ NDs) are used as a reference.

[0055] Depend on Figure 2 It can be seen that KLVFF-Lf-Fe 2+ -Que NDs (a) and KLVFF-BSA-Fe 2+ -Que NDs(b) have a particle size of 4~7 nm, are uniform in size, and are solid spherical nanodots with good dispersibility.

[0056] 3. The KLVFF-Lf-Fe obtained from the reaction 2+ -Que NDs were added to PBS and 1640 culture medium, respectively, and sealed for storage. Samples were taken daily for one week to measure particle size changes. The results are as follows: Figure 3 As shown.

[0057] Depend on Figure 3 It is evident that the particle size of the nanoparticles did not change significantly within seven days in PBS and 1640 culture medium, indicating that KLVFF-Lf-Fe 2+ -Que NDs exhibit good dispersibility and stability.

[0058] 4. In order to investigate KLVFF-Lf-Fe 2+ The ROS scavenging ability of -Que NDs was assessed using the DPPH method to measure the ROS scavenging rate. The specific procedure was as follows: First, a 1.0 mM DPPH ethanol solution was prepared. 100 µL of this solution was then added with different concentrations of KLVFF-Lf-Fe. 2+ -Que NDs, then add anhydrous ethanol to bring the volume to 1.0 mL, so that the final DPPH concentration is 0.1 mM, KLVFF-Lf-Fe 2+ The concentrations of Que NDs were prepared sequentially at 2, 4, 6, 8, and 10 μg / mL. Que reaction solutions with concentrations of 2, 4, 6, 8, and 10 μg / mL were prepared using the same method. The reaction was carried out under light-protected conditions for half an hour, and the UV absorbance values ​​of each group were measured. The results were then analyzed. Figure 4 As shown.

[0059] Depend on Figure 4 It can be seen that at concentrations of 6, 8, and 10 μg / mL, KLVFF-Lf-Fe 2+ -Que NDs have a stronger ability to scavenge ROS than Que, indicating that at a certain concentration, KLVFF-Lf-Fe 2+ -Que NDs have a higher ROS removal capability than Que.

[0060] 5. To investigate KLVFF-Lf-Fe 2+ -Que NDs' biocompatibility, KLVFF-Lf-Fe2+ -Que NDs were co-incubated with mouse pheochromocytoma cells (PC12 cells) to determine KLVFF-Lf-Fe 2+ - The toxicity of Que NDs. The specific operation is as follows:

[0061] Once the PC12 cells have grown to approximately 90% confluence, perform cell passage. This involves removing the old culture medium from the culture flask, washing the cells three times with 2.0 mL of PBS solution, then digesting the cells with 1.0 mL of trypsin for about 1 minute to remove the trypsin. Next, add 2.0 mL of RPMI-1640 medium to stop the digestion and gently pipette the cells to suspend them in the cell culture medium. Take 1 / 4 of the cell culture medium and aliquot it into new culture flasks. Incubate the flasks at 37°C in a 5% CO2 incubator for further culture.

[0062] Take a portion of the PC12 cell stock solution and dilute it with RPMI-1640 culture medium to a density of 10. 5 cells / mL, and 10 cells / well 4 Cells were seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Afterward, the old culture medium was removed from the plates, and KLVFF-Lf-Fe was added at concentrations of 10, 20, 40, 60, and 80 μg / mL, respectively. 2+ -Que NDs culture medium was used, with four replicate wells for each concentration. After 6 hours of incubation, the culture medium was aspirated and the sample was washed twice with PBS. 100 μL of fresh culture medium was added, and the sample was incubated for another 18 hours. Then, 10 μL of 5.0 mg / mL MTT was added to each well, and the sample was incubated for 4–6 hours. The culture medium was carefully removed, and 150 μL of DMSO was added. The sample was then incubated in a shaker at 37°C and 150 rpm for 15 minutes. The absorbance of each solution at 490 nm was measured using a microplate reader, and the viability was calculated to evaluate KLVFF-Lf-Fe. 2+ -The biocompatibility of Que NDs, the results are as follows Figure 5 As shown.

[0063] Depend on Figure 5 It can be seen that KLVFF-Lf-Fe 2+ -Que NDs at a concentration of 80 μg / mL also showed a cell viability of over 80%, indicating that KLVFF-Lf-Fe 2+ -Que NDs have good biocompatibility.

[0064] Application Example 1

[0065] a) The Aβ protein was pretreated by dissolving the Aβ monomer protein in the highly polar solvent hexafluoroisopropanol (HFIP), stirring at 4°C and 450 rpm for 2 hours, freeze-drying, and then redissolving it in PBS solution with pH=7.4 to obtain an Aβ monomer stock solution with a concentration of 230 μM.

[0066] b) Dilute the Aβ monomer stock solution prepared in step a) to 20 μM with PBS solution at pH=7.4, and then add it to a 96-well plate at 100 μL per well. Incubate in a constant temperature shaker at 37°C and 150 rpm for 48 hours to allow fibers to grow.

[0067] 1. At 0, 6, 12, 24, 30, 36, and 48 hours after fiber formation, the KLVFF-Lf-Fe prepared in Example 1 was subjected to [further treatment / processing]. 2+ -Que NDs were added to the fibers obtained in step b), respectively, so that the KLVFF-Lf-Fe mixture at each time point was concentrated. 2+ -QueNDs concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL were added, and the mixture was incubated in a constant-temperature shaker at 37°C and 150 rpm. At the last time point, the particles were transferred into a 96-well plate, and 100 μL of 0.1 mM ThT solution was added to each well for 15 min of further incubation. The fluorescence intensity of each well was detected using a multi-functional fluorescence detector (excitation wavelength set to 450 nm, emission wavelength set to 490 nm). Aβ fiber degradation curves were plotted at different time points, and the degradation curves were used to analyze the degradation of different concentrations of KLVFF-Lf-Fe after 48 hours of fiber formation. 2+ -Que NDs calculated the degradation rate of Aβ fibers, and the results are as follows: Figure 6 As shown.

[0068] Depend on Figure 6 As observed, ThT-stained Aβ fibers exhibited high fluorescence intensity that did not change significantly over time. Meanwhile, KLVFF-Lf-Fe... 2+ With the addition of -Que NDs, the fluorescence intensity gradually decreased and showed a time- and concentration-dependent effect (a); 48 hours after fiber formation, the higher the particle concentration, the greater the degradation rate. 2+ - When the concentration of Que NDs is 40 μg / mL, the degradation rate of Aβ fibers can be as high as 85% (b).

[0069] 2. Different concentrations of KLVFF-Lf-Fe 2+ -Que NDs were mixed with the Aβ fiber solution obtained in step b), and the volume was adjusted to 1.0 mL with PBS buffer to ensure the KLVFF-Lf-Fe content in the mixture was within acceptable limits.2+ The concentrations of -Que NDs were 0, 10, and 20 μg / mL, and the concentration of Aβ was 20 μM. The mixtures were then transferred to 48-well plates and incubated in a constant-temperature shaker for 48 h. Afterward, 100 μL of 0.1 mM ThT solution was added to each well, and incubation continued for 15 min. The mixtures were then removed, centrifuged, washed twice with water (3000 rpm), and redispersed in 500 μL of ultrapure water. The solutions were then evenly spread on the surface of a confocal microscope, and the morphology and fluorescence intensity of each sample were observed using a confocal microscope (excitation wavelength set to 488 nm). The results are as follows: Figure 7 As shown.

[0070] Depend on Figure 7 As can be seen, Aβ fibers are mostly large-area network structures, exhibiting strong green fluorescence after ThT staining, while KLVFF-Lf-Fe... 2+ The fluorescence intensity of the -Que NDs-treated group was significantly reduced, and the large fiber structures were depolymerized into dispersed small patches, which directly demonstrated that KLVFF-Lf-Fe 2+ -Que NDs can effectively degrade Aβ fibers.

[0071] 3. The different concentrations of KLVFF-Lf-Fe prepared in Example 1 were used... 2+ -Que NDs were mixed with the Aβ monomer stock solution prepared in step a) and stored at 4°C. The KLVFF-Lf-Fe in the mixture... 2+ The concentrations of Que in the Que NDs were 0, 10, 20, 30, and 40 μg / mL, and the concentration of Aβ protein was 20 μM. At 0, 6, 12, 24, 30, 36, and 48 h after mixing, 100 μL of each solution was added to a 96-well plate and incubated at 37℃ and 150 rpm for 48 h. Then, 100 μL of 0.1 mM ThT solution was added to each well, and incubation was continued for 15 min. The fluorescence intensity of each well was detected using a multi-functional fluorescence detector (excitation wavelength 450 nm, emission wavelength 490 nm). Curves of Aβ protein fibrillation inhibition at different time points were plotted, and the concentrations of KLVFF-Lf-Fe after 48 hours of co-incubation were calculated using the growth curves. 2+ -QueNDs' inhibition rate of Aβ, the results are as follows Figure 8 As shown.

[0072] Depend on Figure 8It is evident that Aβ protein continuously aggregates and grows to form various Aβ oligomers, which further fibrillate to form Aβ fibrils and amyloid plaques, resulting in high fluorescence intensity after ThT staining. However, the addition of particles inhibits the Aβ aggregation and growth process, slows the growth curve, and gradually reduces the maximum fluorescence intensity, exhibiting a time- and particle concentration-dependent trend. This suggests that KLVFF-Lf-Fe 2+ -Que NDs exhibit a significant inhibitory effect on Aβ fibrillation (a); when the particle concentration is 40 μg / mL, the inhibition rate of Aβ can reach 68% after co-incubation for 48 hours (b).

[0073] 4. Different concentrations of KLVFF-Lf-Fe 2+ -Que NDs were mixed with the Aβ monomer stock solution obtained in step a), and the volume was adjusted to 1.0 mL with PBS buffer. The mixture contained KLVFF-Lf-Fe 2+ The concentrations of -Que NDs were 0, 10, and 20 μg / mL, and the concentration of Aβ protein was 20 μM. Each mixture was transferred to a 48-well plate and incubated for 48 h in a constant-temperature shaker. Then, 100 μL of 0.1 mM ThT solution was added to each well, and incubation continued for 15 min. The mixtures were then removed, centrifuged, washed twice with water (3000 rpm), and redispersed in 500 μL of ultrapure water. The solutions were then evenly spread on the surface of a confocal microscope, and the morphology and fluorescence intensity of each sample were observed using a confocal microscope (excitation wavelength set to 488 nm). The results are as follows: Figure 9 As shown.

[0074] Depend on Figure 9 As can be seen, Aβ protein aggregates and grows into large-area Aβ fibrillary plaques, which exhibit obvious green fluorescence after ThT staining, KLVFF-Lf-Fe 2+ -Que NDs can inhibit the fibrillation process of Aβ monomers, preventing Aβ monomers from forming large-sized fiber structures. The green fluorescence is also significantly weakened, further demonstrating the effectiveness of KLVFF-Lf-Fe. 2+ -Que NDs have a strong inhibitory effect on Aβ fibrosis.

[0075] 5. To investigate KLVFF-Lf-Fe 2+ -Que NDs' targeting ability for Aβ, using Lf-Fe assembled without KLVFF 2+-Que NDs were used as a control group. Specifically, the two types of particles were mixed separately with the Aβ monomer stock solution prepared in step a), resulting in mixtures with Que concentrations of 0, 10, and 20 μg / mL, and Aβ protein concentrations of 20 μM. The mixtures were then incubated at 37°C and 150 rpm for 48 hours in a constant-temperature shaker. Afterward, 100 μL of 0.1 mM ThT was added to each well, and incubation continued for 15 min. The fluorescence intensity of each well was detected using a multi-functional fluorescence detector (excitation wavelength set to 450 nm, emission wavelength set to 490 nm). The results are as follows: Figure 10 As shown.

[0076] Depend on Figure 10 It is evident that, at the same drug concentration, KLVFF-Lf-Fe 2+ -Que NDs are more effective than Lf-Fe in inhibiting Aβ fibrosis. 2+ The -Que NDs treatment group showed an increase of approximately 12%, which is because the strong binding of KLVFF to Aβ can promote the inhibitory effect of particles, strongly demonstrating the targeting effect of KLVFF on Aβ.

[0077] 6. To investigate KLVFF-Lf-Fe 2+ -Que NDs penetrated the BBB, a BBB model was constructed using bEnd.3 cells, and KLVFF-BSA-Fe was synthesized using bovine serum albumin (BSA). 2+ -Que NDs (denoted as KBFQ NDs) were used as the control group. The specific procedure was as follows: First, bEnd.3 cells (1×10⁻⁶) were... 5 (Number of cells / well) were seeded into the upper chambers of a Transwell plate and incubated for 8-12 days. During this period, transendothelial cell resistance (TEER) in the BBB model was measured periodically using a Millicell ERS (Millipore, USA) cytometer. A TEER greater than 200 Ω·cm was considered a threshold. 2 BBB modeling was considered successful at this point. Then, 1.6 mL of PC12 cell suspension was added to the lower chamber of the Transwell plate, and after culturing for another 24 h, it was ready for subsequent experiments. RhB-labeled KLVFF-Lf-Fe... 2+ -Que NDs with KLVFF-BSA-Fe 2+Que NDs were dispersed in fresh DMEM medium, and 600 μL of each was added to the upper chamber of a Transwell plate (Que concentration in the particles was 20 μg / mL). After culturing for 12 h, the culture medium in the upper and lower chambers of each group was collected, and the fluorescence intensity of RhB in the culture medium was detected using a multi-functional fluorescence detector (excitation wavelength 550 nm, emission wavelength 590 nm). The BBB penetration rate of each group of particles was calculated, and the results are as follows: Figure 11 As shown.

[0078] Depend on Figure 11 It is evident that RhB alone only achieved a cell penetration rate of 8.4%, indicating that the BBB can prevent small molecules from entering the brain. Meanwhile, 20 μg / mL KLVFF-Lf-Fe... 2+ -Que NDs had a penetration rate of 54.8%, and at the same concentration, the non-targeting KLVFF-BSA-Fe 2+ -Que NDs also had a penetration rate of 40.7%, indicating that the prepared nanodots can effectively improve the penetration ability of the BBB by their own size effect, and the Lf-mediated receptor transport effect can further promote the transport of particles to the brain.

[0079] Meanwhile, the fluorescence intensity in the lower chamber PC12 cells of each treatment group was observed using confocal microscopy (excitation wavelength set to 543 nm). The results are as follows: Figure 12 As shown.

[0080] Depend on Figure 12 As can be seen, compared with the weak fluorescence after RhB treatment alone, KLVFF-BSA-Fe 2+ -Que NDs showed significantly enhanced red fluorescence after co-incubation with cells, indicating that the nanoparticles can efficiently penetrate the BBB due to their ultra-small size. KLVFF-Lf-Fe 2+ The fluorescence intensity of -Que NDs was further improved, indicating that Lf can efficiently promote particle crossing of the BBB through receptor transport combined with the penetration effect of the ultra-small size of nanodots.

[0081] 7. 3 dpf zebrafish fry were reared in RhB-tagged KLVFF-Lf-Fe 2+ Zebrafish larvae were immersed in an aqueous solution of Que NDs (Que concentration in the particles was 20 μg / mL) for two days, and then transferred to clean water for soaking. The fluorescence of the kidneys and livers of the zebrafish larvae was observed using a confocal fluorescence microscope after soaking for different times (1, 6, 12, 24 h) (excitation wavelength set to 543 nm). The results are as follows: Figure 13 As shown.

[0082] Depend on Figure 13 It is evident that after zebrafish were transferred to fresh water, the fluorescence intensity in the liver and gastrointestinal tract gradually decreased with prolonged metabolic time. After 24 hours of metabolism, the fluorescence in the liver almost completely disappeared, indicating that KLVFF-Lf-Fe... 2+ -Que NDs can be rapidly metabolized out of the body due to their ultra-small size, preventing drug residues from accumulating in the body and causing side effects. This further validates the efficacy of KLVFF-Lf-Fe in in vivo experiments. 2+ - The safety of Que NDs as anti-AD drugs during long-term continuous use in actual treatment.

[0083] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An ultra-small nanodot with efficient BBB penetration and dual targeting capabilities, characterized in that, The ultra-small nanodots are self-assembled from protein molecules, pentapeptides, ferrous ions, and flavonoids; they are monodisperse spherical particles with a size of 2.5~6.5 nm, a potential of -15 mV~-22 mV, and a drug loading rate of 50%~60%; wherein the mass ratio of pentapeptides, protein molecules, ferrous ions to flavonoids is 1:1.25~2.5:0.45~0.7:0.2~0.

4. The protein molecule is lactoferrin, which has brain-targeting BBB function; the pentapeptide is any one of KLVFF, LPFFD, and LVFFA, which has targeting affinity for Aβ protein aggregates; the flavonoid drug is any one of quercetin, silymarin, and rutin.

2. A method for preparing ultra-small nanodots as described in claim 1, characterized in that, Includes the following steps: 1) Based on the solubility characteristics of pentapeptides, protein molecules, ferrous ions precursors and flavonoids, they are dissolved in suitable solvents to obtain pentapeptide solutions, protein solutions, ferrous ions solutions and drug solutions. 2) Mix the pentapeptide solution, protein solution and ferrous solution obtained in step 1) in proportion, react for a period of time, then adjust the pH and add the drug solution to continue the reaction; 3) After centrifuging the solution after the reaction in step 2), the supernatant was purified by ultrafiltration to obtain the ultra-small nanodots.

3. The method for preparing ultra-small nanodots according to claim 2, characterized in that, The precursor of the ferrous ions mentioned in step 1) is FeSO4.

4. The method for preparing ultra-small nanodots according to claim 2, characterized in that, In step 2), the first reaction time is 3-5 minutes; the pH range is adjusted to 7-10; and the reaction time after adding the drug solution is 15-30 minutes.

5. The method for preparing ultra-small nanodots according to claim 2, characterized in that, The high-speed centrifugation speed mentioned in step 3) is 8000~12000 rpm.

6. The application of the ultra-small nanodots as described in claim 1 in the preparation of anti-Alzheimer's disease drugs.

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