Lactoferrin nanometer material for sonodynamic regulation and control of permeability of blood-brain barrier and preparation method and application of lactoferrin nanometer material

Abstract

The invention belongs to the technical field of medical nano-materials, and particularly relates to a lactoferrin nano-material for sonodynamic regulation and control of blood-brain barrier permeability as well as a preparation method and application of the lactoferrin nano-material. The nanomaterial is nanoparticles LCe6 formed by self-assembly of lactoferrin and chlorin e6, can target brain microvascular endothelial cells, and can temporarily, reversibly and targeting open BBB by using the sonodynamic effect of the LCe6 in combination with transcranial ultrasonic stimulation, so that the accumulation of the nanoparticles LCe6 at a focus part is improved; in addition, the nanoparticle LCe6 also has a targeting property on brain tumor cells, the tumor cells can be accurately killed by utilizing the sonodynamic effect of the nanoparticle under secondary transcranial ultrasound, and the effect of sonodynamic treatment on brain glioma is exerted to the maximum extent; the nanoparticles LCe6 have excellent biocompatibility and low toxicity; according to the invention, the intracerebral delivery efficiency of the drug can be obviously improved, and a novel method for targeting open brain disease BBB is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical nanomaterials, and particularly relates to a lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability, a preparation method thereof, and an application thereof. Background Art

[0002] With the increasing incidence of various brain diseases, such as glioblastoma, Alzheimer's disease, Parkinson's disease, and depression, there is an urgent need to update the technology that can accurately deliver drugs to the brain parenchyma. In particular, for primary intracranial tumors - glioblastoma, which has the highest incidence rate, high-grade gliomas (grades III - IV) account for more than 50%, mainly glioblastoma multiform (GBM). Currently, the clinical treatment of GBM mainly relies on surgical resection, combined with comprehensive treatment methods such as radiotherapy and chemotherapy. Although relative progress has been made in clinical treatment, GBM has a poor prognosis, a high recurrence rate, and a poor cure rate. The median survival time of patients is still less than 16 months, and the 5-year survival rate is only 5.4%. The existence of the blood-brain barrier (BBB) severely limits the penetration and accumulation of drugs in the brain and tumor sites, preventing the drugs from reaching the effective concentration required for treatment, and is one of the main obstacles to the effective treatment of GBM. The BBB is an important physiological barrier between the peripheral blood and the brain parenchyma. In the cerebral microvascular system, the BBB is a special structure composed of cerebral microvascular endothelial cells, astrocyte end-feet, pericytes, tight junctions between endothelial cells, and the basement membrane. While protecting the brain, the BBB excludes more than 98% of drugs and all macromolecular therapeutic drugs from entering the brain parenchyma, resulting in the inability of drugs to enter the glioblastoma lesion site, greatly limiting the accumulation and efficacy of drugs at the tumor site, and leading to a poor prognosis, high recurrence rate, and high mortality rate in clinical glioblastoma patients. Therefore, it is necessary to study strategies for efficiently and accurately opening the BBB to improve the drug delivery efficiency and accumulation at the tumor site, thereby inhibiting tumor growth, prolonging the survival period, and achieving the efficient treatment of GBM.

[0003] Currently, active-targeted nano-drugs that utilize the transcytosis of vascular endothelial cells are limited by the receptor expression level of endothelial cells and the transcytosis efficiency, and cannot significantly improve the drug delivery efficiency in the brain. In addition, it has been reported that gold nanoparticles targeting the tight junctions between endothelial cells can open the BBB under transcranial light irradiation, improving the brain delivery efficiency and efficacy of paclitaxel. However, due to the weak penetration ability of light through the skull, this method is only suitable for the treatment of superficial GBM.

[0004] In recent years, the technology of delivering drugs to the brain using ultrasound has attracted attention. The combination of ultrasound and microbubbles has become a means of opening the blood-brain barrier (BBB) and promoting drug delivery in the brain with great clinical application potential. This technology achieves non-invasive, instantaneous, and reversible opening of the BBB through mechanical forces such as cavitation effects and acoustic radiation forces. After injecting microbubbles, ultrasound is precisely irradiated to the brain tumor site, causing the microbubbles in the blood to locally open the BBB, thereby improving the penetration of drugs into the brain parenchyma and exerting effects on tumor cells. However, the short circulating half-life, poor stability, and safety of microbubbles in the blood limit their development to a certain extent.

[0005] Sonodynamic therapy (SDT) is a new non-invasive treatment modality based on the interaction between sonosensitizers and ultrasound. It generates a large amount of reactive oxygen species (ROS) by transferring energy to sonosensitizers through ultrasound, which can kill tumor cells or bacteria, etc. Most organic small molecule sonosensitizers such as porphyrins or xanthenone compounds have poor water solubility, short blood circulation time, and are relatively unstable under physiological conditions. Chlorin e6 (Ce6), belonging to chlorophyll compounds, is widely used as a photosensitizer in photodynamic therapy (PDT) and also has sonodynamic activity. However, disadvantages such as poor water solubility and easy aggregation greatly limit the in vivo application of Ce6. At present, although some studies have shown that excessive endogenous ROS in the brain tissue can affect the normal function of the BBB, leading to an increase in BBB permeability, there is no method to precisely regulate the generation of ROS in endothelial cells through exogenous physical stimulation and further reversibly and safely regulate BBB permeability to open up a new path for the treatment of brain diseases. Summary of the Invention

[0006] Aiming at the above problems, the purpose of the present invention is to provide a lactoferrin nanomaterial for sonodynamically regulating blood-brain barrier permeability, its preparation method, and application.

[0007] The technical content of the present invention is as follows: The present invention provides a preparation method of a lactoferrin nanomaterial for sonodynamically regulating blood-brain barrier permeability, comprising the following steps: Dissolve lactoferrin and a reducing agent in a buffer solution and stir for a reduction reaction; After dissolving Chlorin e6, slowly drip it into the above lactoferrin solution and continue stirring; Perform dialysis in the above buffer solution using a dialysis bag and regularly change the buffer solution; After dialysis, filter and purify using an ultrafiltration tube, and filter through a filter membrane to obtain the final lactoferrin nanomaterial, namely lactoferrin-Chlorin e6 nanoparticles (LCe6); The molar ratio of the lactoferrin to the reducing agent used is 1:(50 - 500); The temperature of the stirred reduction reaction is 22~37°C; The reducing agent includes one or more of dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl); The molar ratio of lactoferrin to chlorin e6 is 1:(5~100); The buffer includes one or more of PBS, Tris, HEPES, and physiological saline; The solution in which chlorin e6 is dissolved includes one or more of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), and absolute ethanol; The pore size of the dialysis bag is 3500 Da~14000 Da; The pore size of the ultrafiltration tube is 30 KDa - 100 KDa.

[0008] The present invention also provides a lactoferrin nanomaterial prepared by the above method, which is lactoferrin-chlorin e6 nanoparticles (LCe6), has a spherical structure, and there is no aggregation between the nanoparticles.

[0009] The present invention also provides the application of the lactoferrin nanomaterial in the preparation of products for treating brain diseases; The product for treating brain diseases has the function of targeting brain microvessels; The product for treating brain diseases can transiently and reversibly open the BBB; The method for treating brain diseases is that after the lactoferrin nanomaterial is combined with ultrasound to transiently and reversibly open the BBB, it accumulates at the disease site, and a second ultrasound is induced to cause apoptosis of tumor cells, thereby achieving the effect of treating tumors; The brain diseases include brain tumors, Parkinson's disease, Alzheimer's disease, depression, encephalitis, stroke, multiple sclerosis, paralysis, Huntington's disease, epilepsy, thrombosis, embolism, cerebral infarction, brain infections caused by bacteria or viruses, etc.

[0010] The beneficial effects of the present invention are as follows: The lactoferrin nanomaterial of the present invention is a nanoparticle LCe6 formed by the self-assembly of lactoferrin and chlorin e6. It can target brain microvascular endothelial cells. Combining with transcranial ultrasound stimulation, it can transiently, reversibly and targetedly open the BBB by using the sonodynamic effect of LCe6, improving the accumulation of nanoparticle LCe6 at the lesion site. Moreover, the nanoparticle LCe6 also has targeting properties for brain tumor cells. By using the sonodynamic effect of the nanoparticle under secondary transcranial ultrasound, it can precisely kill tumor cells, maximizing the effect of sonodynamic therapy for glioblastoma. The nanoparticle LCe6 has excellent biocompatibility and low toxicity. The present invention can significantly improve the efficiency of drug delivery to the brain, providing a new method for targeted opening of the BBB in brain diseases. Brief Description of the Drawings

[0011] Figure 1 It is the transmission electron microscope and dynamic light scattering results of the LCe6 nanoparticles of the present invention; Figure 2 It is the ROS generation efficiency of the LCe6 nanoparticles of the present invention shown by the DPBF fluorescent indicator; Figure 3 It is the detection of the uptake of the LCe6 nanoparticles of the present invention in endothelial cells and the generation of ROS by LCe6 combined with ultrasound; Figure 4 It is a schematic diagram of the in vitro BBB cell model constructed and the growth curve of the TEER value during the growth process of the cell model, and the change curve of the TEER value of LCe6 combined with ultrasound to open the BBB; Figure 5 It is the fluorescence imaging and slice confocal images of the brain targeting of the LCe6 nanoparticles of the present invention, and the intracerebral penetration of Evans blue under ultrasound stimulation; Figure 6 It is the accumulation of drugs at the brain tumor site and the quantitative detection of drugs after the LCe6 nanoparticles of the present invention combined with ultrasound open the BBB; Figure 7 It is a schematic diagram of the LCe6 nanoparticles of the present invention combined with ultrasound to open the in vitro BBB model and induce the generation of ROS and apoptosis in tumor cells, the quantitative statistics of the generated ROS and the calcein / propidium iodide staining; Figure 8 It is a schematic diagram and treatment situation of the LCe6 nanoparticles of the present invention combined with ultrasound in the treatment of glioblastoma. Detailed Embodiments

[0012] The present invention will be further described in detail below through specific implementation cases and the description of the drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art fall within the scope defined by the appended claims of this application.

[0013] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0014] Example 1 Preparation of lactoferrin nanomaterial Dissolve lactoferrin (Lf, 16.98 mg, 0.223 μmol) and dithiothreitol (DTT, 6.46 mg, 41.9 μmol) in 8.4 mL of PBS (pH 7.0), and stir at 37 °C for 20 min for the reduction reaction; Subsequently, dissolve chlorin e6 (Ce6, 4.0 mg, 6.7 μmol) in 1.0 mL of DMSO, and slowly drop it into the lactoferrin solution, and stir at room temperature for 2 h. After the reaction is completed, dialyze in PBS (pH 7.4) using a dialysis bag with a pore size of 14 KDa for 24 h, and replace the PBS solution every 6 h.

[0015] Filter and purify using an ultrafiltration tube with a pore size of 30 KDa and filter through a 0.45 μm aqueous filter membrane to obtain the final lactoferrin nanomaterial, that is, lactoferrin-chlorin e6 nanoparticles (LCe6). Detect it with a transmission electron microscope and a Malvern particle size analyzer. The results are as Figure 1 shown in a and b. The nanoparticles of the prepared lactoferrin nanomaterial have a spherical structure, with a uniform and stable structure. The particle diameter is about 100 nm, and there is no aggregation among the nanoparticles.

[0016] Example 2 Preparation of lactoferrin nanomaterial Dissolve lactoferrin (Lf, 16.98 mg, 0.223 μmol) and dithiothreitol (DTT, 6.46 mg, 41.9 μmol) in 8.4 mL of PBS (pH 7.0), and stir at 30 °C for 25 min for the reduction reaction; Subsequently, dissolve chlorin e6 (Ce6, 5.32 mg, 8.92 μmol) in 1.0 mL of absolute ethanol, and slowly drop it into the lactoferrin solution, and stir at room temperature for 2 h. After the reaction is completed, dialyze in PBS (pH 7.4) using a dialysis bag with a pore size of 10 KDa for 24 h, and replace the PBS solution every 6 h.

[0017] Filter and purify using an ultrafiltration tube with a pore size of 50 KDa and filter through a 0.45 μm aqueous filter membrane to obtain the final lactoferrin nanomaterial, that is, lactoferrin-chlorin e6 nanoparticles (LCe6).

[0018] Example 3 Preparation of lactoferrin nanomaterial Dissolve lactoferrin (Lf, 16.98 mg, 0.223 μmol) and β-mercaptoethanol (BME) (DTT, 5.22 mg, 66.9 μmol) in 8.4 mL of PBS (pH 7.0), and stir at 37 °C for 25 min for the reduction reaction; Subsequently, dissolve chlorin e6 (Ce6, 3.33 mg, 5.58 μmol) in 1.0 mL of DMF, and slowly drip it into the lactoferrin solution, and stir at room temperature for 2 h. After the reaction is completed, dialyze in PBS (pH 7.4) using a dialysis bag with a pore size of 7500 Da for 24 h, and replace the PBS solution every 6 h.

[0019] Filter and purify using an ultrafiltration tube with a pore size of 100 KDa and filter through a 0.45 μm aqueous filter membrane to obtain the final lactoferrin nanomaterial, namely lactoferrin-chlorin e6 nanoparticles (LCe6).

[0020] Example 4 Preparation of lactoferrin nanomaterial Dissolve lactoferrin (Lf, 16.98 mg, 0.223 μmol) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl, 2.79 mg, 11.15 μmol) in 8.4 mL of PBS (pH 7.0), and stir at 22 °C for 25 min for the reduction reaction; Subsequently, dissolve chlorin e6 (Ce6, 6.65 mg, 11.15 μmol) in 1.0 mL of THF, and slowly drip it into the lactoferrin solution, and stir at room temperature for 2 h. After the reaction is completed, dialyze in PBS (pH 7.4) using a dialysis bag with a pore size of 10 KDa for 24 h, and replace the PBS solution every 6 h.

[0021] Filter and purify using an ultrafiltration tube with a pore size of 50 KDa and filter through a 0.45 μm aqueous filter membrane to obtain the final lactoferrin nanomaterial, namely lactoferrin-chlorin e6 nanoparticles (LCe6).

[0022] Comparative Example 1 Preparation of lactoferrin nanomaterial Dissolve lactoferrin (Lf, 16.98 mg, 0.223 μmol) and dithiothreitol (DTT, 6.46 mg, 41.9 μmol) in 8.4 mL of PBS (pH 7.0), and stir at 4 °C for 25 min for the reduction reaction; Subsequently, chlorin e6 (Ce6, 4.0 mg, 6.7 μmol) was dissolved in 1.0 mL of DMSO and slowly dropped into the lactoferrin solution, followed by stirring at room temperature for 2 h. After the reaction was completed, dialysis was performed in PBS (pH 7.4) using a dialysis bag with a pore size of 14 KDa for 24 h, and the PBS solution was changed every 6 h.

[0023] Filtration and purification were carried out using an ultrafiltration tube with a pore size of 30 KDa and filtration through a 0.45 μm aqueous filter membrane to obtain the final lactoferrin nanomaterial, namely lactoferrin-chlorin e6 nanoparticles (LCe6). Transmission electron microscopy and Malvern particle size analyzer were used for detection, and the results are as Figure 1 shown in c and d. The particles of the prepared lactoferrin nanomaterial were disordered, did not have a spherical structure, and were in a flocculent state. The particle diameters were distributed around 2, 20, and 300 nm, and the size was uneven. This indicates that during the preparation of LCe6 nanoparticles, too low temperature of the reduction reaction will affect its reaction activity, resulting in the failure to successfully synthesize uniform spherical nanoparticles.

[0024] Experimental Example 1 To evaluate the effect of LCe6 nanoparticles in generating ROS in response to ultrasound, the DPBF singlet oxygen indicator fluorescent probe was added to LCe6 nanoparticles. After applying ultrasonic stimulation, the absorbance of the mixed solution was detected using a UV spectrophotometer, and the results are as Figure 2 shown. The generation of a large amount of singlet oxygen reduced the UV absorption of DPBF, proving that LCe6 nanoparticles have a sonodynamic effect.

[0025] Experimental Example 2 To evaluate the effect that LCe6 nanoparticles can be significantly taken up by endothelial cells and target mouse brain microvessels a) After incubating LCe6 nanoparticles in the medium of mouse brain microvascular endothelial cells for 1 h, it was found by confocal microscopy observation and flow cytometry analysis that LCe6 was taken up by the cells, while small molecule Ce6 was not significantly taken up by the cells, and the results are as Figure 3 shown in a, b, and c.

[0026] b) After incubating LCe6 in the medium of mouse brain microvascular endothelial cells for 1 h, ultrasonic stimulation was applied (parameters: power 1.5 W / cm 2 , duty cycle 10%, frequency 1 MHz, 3 min), labeled with the DCFH-DA probe, and it was found by confocal microscopy observation and flow cytometry analysis that LCe6 could generate ROS under ultrasonic irradiation after being taken up by the cells. The results are shown in Figure 3 d, e, and f.

[0027] Experimental Example 3 To evaluate the ability of LCe6 combined with ultrasound to open the BBB, an in vitro BBB monolayer cell model was constructed. On the upper chamber of a 24-well transwell plate, 5×10 4 bEnd.3 cells per well were seeded. The transcellular electrical resistance value (TEER) was measured every 2 days using an epithelial volt-ohmmeter. After 8 days of culture, a plateau was reached, as shown in a and b of Figure 4 , indicating successful construction of the BBB monolayer cells. On the eighth day, LCe6 (50 μg / mL) was added and co-incubated for 1 hour. Ultrasound stimulation was applied to the lower chamber of the culture (parameters: power 1.5 W / cm 2 , duty cycle 10%, frequency 1 MHz, 3 min), as shown in Figure 4 c. After 1 h of ultrasound stimulation, the TEER decreased significantly, indicating that ultrasound stimulation could significantly reduce the TEER and temporarily open the BBB; after 6 - 12 h of stimulation, the TEER was still significantly lower than the initial value and showed a gradually increasing trend; after 24 h, the TEER returned to the initial value, indicating that the BBB had returned to normal. In other groups such as the PBS, PBS + ultrasound, and LCe6 groups, the TEER value of the in vitro BBB did not change, indicating that single application of ultrasound or LCe6 nanoparticles had no effect on the BBB. The above results showed that LCe6 combined with ultrasound could open the BBB transiently (within 24 h) and reversibly (open → closed).

[0028] Experimental Example 4 To evaluate the effect of LCe6 combined with ultrasound on opening the BBB in vivo, mice were fixed on a stereotaxic apparatus, the scalp was incised, and a drill was used to make a hole at a position 2.5 mm lateral and 0 mm anterior to the bregma. A glass electrode was used to inject 5×10 4 GL261-luc tumor cells into the cerebral striatum at a depth of 1.5 mm. After the injection, the needle was left in place for 5 min and the scalp was sutured. Six days after implantation, potassium D-luciferin (dose 15 mg / kg) was injected intraperitoneally for small animal in vivo bioluminescence imaging. The results were as shown in a of Figure 5 , and bioluminescence at the injection site demonstrated successful tumor implantation.

[0029] By injecting PBS, Ce6, and LCe6 (dose 5 mg / kg, Ce6 quantified) into the body via the tail vein, the fluorescence of Ce6 in the brain was shown by ex vivo fluorescence imaging 1 h later (the excitation wavelength of Ce6 was 640 nm and the emission wavelength was 680 nm), as well as the fluorescence statistical chart, specifically as shown in Figure 5as shown in a, b, and c. The results showed that at the same tumor size, the ex vivo fluorescence signal in the tumor site of the LCe6 group was significantly higher than that of the free Ce6 group and the PBS group. In addition, confocal images showed that there was no obvious Ce6 signal in the blood vessels of the control group and the small molecule Ce6 group, while the Ce6 signal in the LCe6 group was significantly accumulated in the blood vessels in a targeted manner, indicating that the LCe6 nanoparticles could specifically target the tumor site and specifically target the BBB by binding to the lactoferrin receptor.

[0030] By injecting LCe6 (at a dose of 5 mg / kg) into the body via the tail vein, ultrasonic stimulation was applied to the brain tumor area 1 hour later. Evans blue (2%, at a dose of 100 mg / kg) was injected via the tail vein at 1, 6, 12, and 24 h. The brain was taken 30 minutes after injection, and ex vivo fluorescence imaging of the whole brain and coronal sections was performed to show the penetration of Evans blue in the brain and fluorescence statistics, as well as the quantitative detection of Evans blue, specifically as Figure 5 shown in d, e, f, g, and h. It was found that when Evans blue was injected 1 h after applying ultrasonic stimulation in the LCe6 combined with ultrasound group, the ex vivo fluorescence signal was significantly enhanced compared with the LCe6 group, while there was no significant difference between the PBS group and the PBS combined with ultrasound group, and between the Ce6 group and the Ce6 combined with ultrasound group, indicating that LCe6 could target the microvessels of the tumor site, and the combined ultrasonic stimulation could open the BBB, enabling Evans blue to cross the BBB and enter the tumor site. As time extended, 6 h and 12 h after injecting Evans blue, the fluorescence signal and content gradually decreased, and the fluorescence signal and content of Evans blue returned to the initial level at 24 h, indicating that the BBB gradually closed after opening and was completely closed at 24 h. It was shown that LCe6 combined with ultrasound could significantly open the BBB, and the BBB could reversibly recover 24 h after opening.

[0031] Experimental Example 5 To evaluate that LCe6 could open the BBB under ultrasonic stimulation and improve the brain delivery efficiency of Ce6, LCe6 (at a dose of 5 mg / kg) was injected into the body via the tail vein, ultrasonic stimulation was applied to the brain tumor area 1 h later, and ex vivo fluorescence imaging of the brain was performed at 6, 12, 24, and 48 h respectively, and fluorescence signal statistics were carried out. The specific results were as Figure 6 shown in a and b, indicating that the LCe6 group could also cross the BBB through the receptor-mediated manner without ultrasound, and could open the BBB after applying ultrasound to significantly increase the accumulation of Ce6 at the brain tumor site, and the accumulation reached the highest at 24 h as time extended, and then Ce6 would gradually degrade and metabolize.

[0032] Experimental Example 6 To evaluate the induction of tumor cell apoptosis by LCe6 combined with ultrasound through the blood-brain barrier (BBB), a co-culture model of endothelial cells and tumor cells was constructed using a Transwell plate. After culturing bEnd.3 cells in the upper chamber for 7 days to form a monolayer cell model, LCe6 was added and then ultrasonic stimulation was applied 1 h later. The chamber and culture medium were transferred to a well plate containing GL261 cells, as specifically shown in Figure 7 a in the figure. Then, after culturing for 24 h, the second ultrasound was applied (parameters: power 3.0 W / cm 2 , duty cycle 20%, frequency 1 MHz, 5 min). After 4 h, DCFH-DA probe and calcein / propidium iodide were used to stain the tumor cells to verify the effect of LCe6 in inducing ROS and apoptosis in GL261 cells under ultrasonic stimulation, as specifically shown in Figure 7 b and c in the figure. The results showed that LCe6 combined with ultrasound could open the BBB in an in vitro BBB model, and LCe6 passed through the chamber and was taken up by GL261 cells in the lower chamber. After the second ultrasound was applied, ROS was generated in the tumor cells, further inducing tumor cell apoptosis. In the case of Ce6 combined with ultrasound, there was no significant tumor cell apoptosis signal, indicating that Ce6 combined with ultrasound could not pass through the BBB and enter the lower chamber. In addition, pure ultrasound had no obvious toxic effect on tumor cells.

[0033] Experimental Example 7 To evaluate the accumulation of nanomaterials at the tumor site after the BBB was opened by ultrasound combined with ultrasound, the second ultrasound was used to induce tumor cell apoptosis, and then the effect of treating tumors was achieved. After injecting the drug for 1 h, the first ultrasound stimulation was performed (parameters: power 1.5 W / cm 2 , duty cycle 10%, frequency 1 MHz, 3 min) to open the BBB and promote the entry of the drug into the brain. After 24 h, the second ultrasound stimulation was performed (parameters: power 3.0 W / cm 2 , duty cycle 20%, frequency 1 MHz, 5 min). The above steps were one treatment, and a total of 3 treatments were performed. The tumor treatment effect, body weight change, and survival period were observed using small animal in vivo imaging every 6 days, as shown in Figure 8 the figure. The results showed that the PBS group, PBS + ultrasound group, and Ce6 + ultrasound group had no obvious effect on the tumor sound field and survival period extension. The LCe6 + ultrasound group had the best treatment effect compared with other groups. The bioluminescence signal showed that the tumor did not increase significantly, and the median survival period was 45 days, with a significantly extended survival period. The LCe6 group could also inhibit tumor growth to a certain extent, indicating that LCe6 could accumulate in the brain tumor site to a certain extent without the effect of ultrasound opening the BBB and produce ROS under the action of the second sonodynamic therapy to exert an antitumor effect. However, the median survival period was 24 days, and there was no significant extension of the survival period compared with the PBS group.

Claims

1. A preparation method of a lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability, characterized in that, It includes the following steps: Dissolve lactoferrin and a reducing agent in a buffer solution and stir to carry out a reduction reaction; After dissolving chlorin e6, slowly drip it into the above lactoferrin solution and continue stirring; Carry out dialysis in the above buffer solution using a dialysis bag and regularly change the buffer solution; After dialysis, filter and purify using an ultrafiltration tube, and filter through a filter membrane to obtain the final lactoferrin nanomaterial, namely lactoferrin-chlorin e6 nanoparticles.

2. The preparation method of the lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability according to claim 1, wherein, The temperature of the reduction reaction is 22~37 °C.

3. The preparation method of the lactoferrin nanomaterial for sonic power regulation of blood-brain barrier permeability according to claim 1, wherein, The molar ratio of lactoferrin to the reducing agent used is 1:(50~500).

4. The preparation method of the lactoferrin nanomaterial for modulating the permeability of the blood-brain barrier by sonodynamic action according to claim 1, wherein, The reducing agent includes one or more of dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl).

5. The preparation method of the lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability according to claim 1, characterized in that, The molar ratio of lactoferrin to chlorin is 1:(5~100).

6. The preparation method of the lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability according to claim 1, wherein, The pore size of the dialysis bag is 3500 Da~14000 Da.

7. The preparation method of the lactoferrin nanomaterial for sonodynamic regulation of blood-brain barrier permeability according to claim 1, wherein The pore size of the ultrafiltration tube is 30 KDa - 100 KDa.

8. A lactoferrin nanomaterial obtained by the preparation method according to any one of claims 1-7, characterized in that, The nanomaterial is lactoferrin-chlorin e6 nanoparticles (LCe6) and has a spherical structure.

9. Use of the lactoferrin nanomaterial according to claim 8 in the preparation of a product for treating brain diseases, characterized in that, The product for treating brain diseases has the function of targeting brain microvessels; The product for treating brain diseases can transiently and reversibly open the blood-brain barrier.

10. The application according to claim 9, characterized in that, The method for treating brain diseases is that after transiently and reversibly opening the blood-brain barrier through the combination of the lactoferrin nanomaterial and ultrasound and accumulating at the disease site, a second ultrasound is induced to cause apoptosis of tumor cells, thereby achieving the effect of treating tumors.

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