Butylphthalide nanoparticles, their preparation method, and applications
Butylphthalide nanoparticles were prepared by electrostatic adsorption on layered double hydroxides, which solved the problems of low water solubility and bioavailability of butylphthalide, achieved targeted activation of the PI3K-AKT signaling pathway, significantly improved renal ischemia-reperfusion injury, and provided a new method for the treatment of kidney diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF BENGBU MEDICAL COLLEGE
- Filing Date
- 2026-03-10
- Publication Date
- 2026-06-02
AI Technical Summary
In the prior art, the low water solubility and poor bioavailability of butylphthalide limit its efficacy in treating renal ischemia-reperfusion injury, and there is a lack of effective prevention and treatment methods.
Using layered double hydroxides as a carrier, butylphthalide nanoparticles were prepared by electrostatic adsorption, allowing them to be uniformly loaded between the layers of the layered double hydroxides. This resulted in butylphthalide nanoparticles with high drug loading and good stability, which targeted and activated the PI3K-AKT signaling pathway, inhibiting HK-2 cell apoptosis and oxidative stress.
Butylphthalide nanoparticles can significantly improve renal ischemia-reperfusion injury, target and activate the PI3K-AKT signaling pathway, inhibit cell apoptosis and oxidative stress, and restore mitochondrial function, providing a new strategy for the treatment of kidney diseases.
Smart Images
Figure CN122123989A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and in particular to a butylphthalide nanoparticle, its preparation method, and its application. Background Technology
[0002] Acute kidney injury (AKI) caused by ischemia-reperfusion (IR) is a common complication of kidney transplantation, cardiovascular and other surgical procedures. If left untreated, it can progress to renal failure, renal fibrosis, and chronic kidney disease. Its pathological process involves multiple mechanisms, including inflammatory responses, oxidative stress, mitochondrial dysfunction, and autophagy. Currently, clinical methods for the prevention and treatment of renal ischemia-reperfusion injury remain very limited. DL-3-n-butylphthalide (NBP) is a compound extracted from celery seeds with neuroprotective and anti-inflammatory activities. Recent studies have confirmed that NBP can not only alleviate renal ischemia-reperfusion injury and delay the progression of hypertensive nephropathy by inhibiting excessive inflammation and oxidative stress, but also improve diabetic nephropathy by reducing excessive fibrosis and podocyte apoptosis. However, the inherent low water solubility and poor bioavailability of NBP in treatment severely limit its efficacy and application in the treatment of kidney diseases. Summary of the Invention
[0003] Based on the technical problems existing in the background technology, the present invention proposes a butylphthalide nanoparticle, its preparation method and application. The butylphthalide nanoparticle of the present invention has high drug loading capacity and good stability, and can target and activate the PI3K-AKT signaling pathway, effectively inhibit HK-2 cell apoptosis and oxidative stress, restore mitochondrial function and improve renal ischemia-reperfusion injury.
[0004] This invention proposes a butylphthalide nanoparticle comprising: a layered double hydroxide and butylphthalide, wherein the butylphthalide is loaded on the layered double hydroxide.
[0005] Preferably, butylphthalide is uniformly loaded in the interlayer of the layered double hydroxide.
[0006] Preferably, the average particle size of the butylphthalide nanoparticles is 200-250 nm.
[0007] Preferably, the content of butylphthalide in the butylphthalide nanoparticles is 20-30 wt%.
[0008] Preferably, the layered double hydroxide has a hexagonal layered structure.
[0009] Preferably, the layered double hydroxide contains Mg and Al.
[0010] Preferably, in the layered double hydroxide, the molar ratio of Mg to Al is 2.8-3.2:1.
[0011] Preferably, the layered double hydroxide is prepared by hydrothermal coprecipitation.
[0012] Preferably, in the preparation process of the layered double hydroxide, the hydroxide, magnesium source, aluminum source and water are mixed, stirred and reacted, solid and liquid are separated, the solid is mixed with water and subjected to hydrothermal reaction to obtain the layered double hydroxide.
[0013] After the above hydrothermal reaction, the solid and liquid were separated, the solid was resuspended in water, and freeze-dried to obtain a layered double hydroxide.
[0014] Preferably, in the preparation of the layered double hydroxide, the hydroxide, magnesium source, aluminum source, and water are mixed and stirred in an inert gas atmosphere.
[0015] Preferably, during the preparation of the layered double hydroxide, the reaction is stirred at 50-70°C for 0.8-1.2 h.
[0016] Preferably, in the preparation of the layered double hydroxide, a hydrothermal reaction is carried out at 90-110°C for 14-18 hours.
[0017] Preferably, in the preparation of the layered double hydroxide, the hydroxide is a water-soluble hydroxide.
[0018] Preferably, in the preparation of the layered double hydroxide, the magnesium source is at least one of magnesium chloride and magnesium chloride hydrate.
[0019] Preferably, in the preparation of layered double hydroxides, the aluminum source is at least one of aluminum chloride and aluminum chloride hydrate.
[0020] Preferably, in the preparation process of the layered double hydroxide, the molar ratio of hydroxide, magnesium, and aluminum is 6.5-7:2.8-3.2:1.
[0021] The present invention also proposes a method for preparing the above-mentioned butylphthalide nanoparticles, comprising the following steps: mixing layered double hydroxide, butylphthalide and water, adsorbing, separating solid and liquid, drying, and obtaining butylphthalide nanoparticles.
[0022] Preferably, adsorption is carried out at 20-40℃ for 20-28 hours.
[0023] Preferably, shaking is maintained during the adsorption process.
[0024] Preferably, the weight ratio of layered double hydroxide to butylphthalide is 1:1.5-2.5.
[0025] Preferably, the drying method is freeze drying.
[0026] This invention also proposes the application of the above-mentioned butylphthalide nanoparticles in the preparation of drugs for treating acute renal ischemia-reperfusion injury.
[0027] The present invention also proposes a medicament for treating acute renal ischemia-reperfusion injury, comprising: the above-mentioned butylphthalide nanoparticles and pharmaceutically acceptable excipients.
[0028] The pharmaceutically acceptable excipients mentioned above can be sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, lubricants, diluents, etc.
[0029] The above-mentioned drugs can be injectable, and the diluent for the injectable drugs can be physiological saline, water for injection, etc. The dosage of the injectable drugs, calculated based on butylphthalide nanoparticles, can be 25 mg / kg / day.
[0030] This invention utilizes layered double hydroxide nanoparticles for electrostatic adsorption, allowing butylphthalide to be uniformly dispersed and loaded between the layers of the layered double hydroxide, successfully constructing butylphthalide nanoparticles with high drug loading and good stability, thus modifying the drug dosage form of butylphthalide.
[0031] This invention, through in vivo and in vitro experiments, preliminarily elucidates the specific mechanism of the butylphthalide nanoparticle drug delivery system in treating acute renal ischemia-reperfusion injury. Unexpectedly, it was discovered that butylphthalide nanoparticles can target and activate the PI3K-AKT signaling pathway, effectively inhibit HK-2 cell apoptosis and oxidative stress, restore mitochondrial function, and significantly improve renal function in rats, thus alleviating renal ischemia-reperfusion injury. Butylphthalide nanoparticles hold promise as a novel strategy for treating kidney diseases. Attached Figure Description
[0032] Figure 1 The images show transmission electron microscopy (TEM) images and elemental distribution diagrams of layered double hydroxides and butylphthalide nanoparticles, where A and C represent layered double hydroxides, and B and D represent butylphthalide nanoparticles.
[0033] Figure 2 The diagram shows the particle size distribution of layered double hydroxides and butylphthalide nanoparticles, where A represents layered double hydroxides and B represents butylphthalide nanoparticles.
[0034] Figure 3 The diagram shows the Zeta potentials of layered double hydroxides and butylphthalide nanoparticles, where A represents layered double hydroxides and B represents butylphthalide nanoparticles.
[0035] Figure 4 The images show X-ray diffraction patterns of layered double hydroxides, butylphthalide, and butylphthalide nanoparticles, where LDHs represents layered double hydroxides, NBP represents butylphthalide, and LDHs-NBP represents butylphthalide nanoparticles.
[0036] Figure 5 The images show the FTIR spectra of layered double hydroxides, butylphthalide, and butylphthalide nanoparticles, where LDHs represents layered double hydroxides, NBP represents butylphthalide, and LDHs-NBP represents butylphthalide nanoparticles.
[0037] Figure 6 The UV full-wavelength scan curve and standard curve for butylphthalide are shown.
[0038] Figure 7 The sustained-release curves of butylphthalide in butylphthalide nanoparticles under different pH environments are shown.
[0039] Figure 8 Images of liver and kidney function indicators and HE staining of various organs in rats in each group 7 days after intraperitoneal injection are shown. In the images, A represents liver function, B represents kidney function, and C represents HE staining.
[0040] Figure 9 HE staining results of kidneys in each group after establishing a renal ischemia-reperfusion injury model.
[0041] Figure 10 Volcano plot of differentially expressed transcriptomic genes between the I / R group and the Sham group, and between the I / R+LDHs-NBP group and the I / R group after constructing a renal ischemia-reperfusion injury model.
[0042] Figure 11 Heatmap of significantly differentially expressed genes in the I / R group, Sham group, and I / R+LDHs-NBP group after constructing a renal ischemia-reperfusion injury model.
[0043] Figure 12 KEGG enrichment analysis plots for the I / R group, Sham group, and I / R+LDHs-NBP group after constructing a renal ischemia-reperfusion injury model.
[0044] Figure 13 To establish a renal ischemia-reperfusion injury model, the immunohistochemical and protein expression levels of each group were obtained. In this study, A represents the immunohistochemical results of p-PI3K, p-AKT, and Nrf2 in rat kidneys, and B represents the protein expression levels in rat kidneys.
[0045] Figure 14 A shows the effect of different concentrations of layered double hydroxides on the proliferation activity of HK-2 cells.
[0046] Figure 14 B represents the effect of different concentrations of butylphthalide and butylphthalide nanoparticles on the proliferation activity of H / R model HK-2 cells.
[0047] Figure 15 The apoptosis rate of HK-2 cells in each group is shown.
[0048] Figure 16 The results show the mitochondrial membrane potential of HK-2 cells in each group.
[0049] Figure 17 The results show the ROS level and p-PI3K, p-AKT and Nrf2 protein expression levels of HK-2 cells in each group. AB represents the ROS level and CD represents the protein expression level. Detailed Implementation
[0050] The technical solution of the present invention will now be described in detail through specific embodiments.
[0051] Reagents, cells, and animal sources: Butylphthalide was purchased from CSPC Enbipu Pharmaceutical Co., Ltd. SDS, Tween 20 reagent, methanol, and tissue fixative were purchased from Sinopharm Chemical Reagents Group. DMEM, fetal bovine serum (Pronosei Corporation); Antibodies such as p-PI3K, PI3K, p-AKT, AKT, and Nrf2 (Affinity). CCK-8 kit, Hoechst 33342 reagent, Annexin V&PI apoptosis kit, BCA kit, ROS kit, Western Blot gel preparation kit, and JC-1 kit (Beyotime Biotechnology Co., Ltd.). The human renal cortical proximal tubule epithelial cell line HK-2 was purchased from Pronoss and cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2 saturated humidity. Sixty male SD rats, 180g±20g each (Jiangsu Qinglongshan Biotechnology Co., Ltd.), were housed at the Research Animal Center of Bengbu Medical University. The ambient temperature was 22-25℃ and the humidity was 55-60%.
[0052] Animal disposal methods must comply with animal ethics requirements.
[0053] Example 1 A method for preparing butylphthalide nanoparticles includes the following steps: Prepare 40 mL of an aqueous solution containing 272 mg (6.8 mmol) of sodium hydroxide. Under nitrogen purging, add 10 mL of a mixed aqueous solution (containing 241.4 mg (1 mol) of AlCl3·6H2O and 609.9 mg (3 mol) of MgCl2·6H2O). Stir the mixture in a water bath at 60 °C for 1 h, then centrifuge at 4000 rpm for 10 min. Resuspend the precipitate in 40 mL of ddH2O and centrifuge again. Resuspend the precipitate in 40 mL of ddH2O and transfer it to a reaction vessel. React the mixture hydrothermally at 100 °C for 16 h, then centrifuge at 12000 rpm for 1 h. Resuspend the precipitate in 2 mL of ddH2O and freeze-dry to obtain layered double hydroxides, denoted as LDHs. Store at -20 °C for later use. 10 mg of layered double hydroxide was added to 80 mL of an aqueous solution containing 20 mg of butylphthalide, and the mixture was adsorbed in a shaker at 37 °C for 24 h. Then, the mixture was centrifuged at 12000 rpm for 1 h, and the precipitate was freeze-dried to obtain butylphthalide nanoparticles, denoted as LDHs-NBP.
[0054] The layered double hydroxide LDHs and butylphthalide nanoparticles LDHs-NBP prepared in Example 1 were tested, and the results are as follows: Figure 1 As shown.
[0055] Figure 1 The images show transmission electron microscopy (TEM) images and elemental distribution diagrams of layered double hydroxides and butylphthalide nanoparticles, where A and C represent layered double hydroxides, and B and D represent butylphthalide nanoparticles.
[0056] Depend on Figure 1 It can be seen that the layered double hydroxide exhibits a typical hexagonal layered structure. Its morphology remains unchanged after loading with phthaloyl butadiene, and phthaloyl butadiene is uniformly dispersed between the layers. The molar ratio of Mg to Al in the layered double hydroxide is approximately 3:1, and the distribution is uniform.
[0057] Figure 2 The diagram shows the particle size distribution of layered double hydroxides and butylphthalide nanoparticles, where A represents layered double hydroxides and B represents butylphthalide nanoparticles.
[0058] Depend on Figure 2 It can be seen that the average particle size of the layered double hydroxide is 186.8±1.9 nm, which increases to 244.5±6.9 nm after loading with phthaloyl butyrate.
[0059] Figure 3 The diagram shows the Zeta potentials of layered double hydroxides and butylphthalide nanoparticles, where A represents layered double hydroxides and B represents butylphthalide nanoparticles.
[0060] Depend on Figure 3It can be seen that after loading butylphthalide with layered double hydroxides, the Zeta potential decreased from 40.8±0.98 mV to 24.3±1.1 mV, indicating that butylphthalide was successfully loaded through electrostatic interaction.
[0061] Figure 4 The images show X-ray diffraction patterns of layered double hydroxides, butylphthalide, and butylphthalide nanoparticles, where LDHs represents layered double hydroxides, NBP represents butylphthalide, and LDHs-NBP represents butylphthalide nanoparticles.
[0062] Depend on Figure 4 It can be seen that the butylphthalide nanoparticles retain the crystalline phase structure of the layered double hydroxide, indicating that the introduction of butylphthalide did not change the crystal stability.
[0063] Figure 5 The images show the FTIR spectra of layered double hydroxides, butylphthalide, and butylphthalide nanoparticles, where LDHs represents layered double hydroxides, NBP represents butylphthalide, and LDHs-NBP represents butylphthalide nanoparticles.
[0064] Depend on Figure 5 As can be seen, the FTIR spectrum of butylphthalide nanoparticles shows characteristic peaks of layered double hydroxides and butylphthalide, with no new peaks appearing, further confirming that butylphthalide is a physical loading.
[0065] Figure 6 The UV full-wavelength scan curve and standard curve for butylphthalide are shown.
[0066] The content of butylphthalide in the butylphthalide nanoparticles was determined to be approximately 25 wt% using the external standard method of ultraviolet spectrophotometer.
[0067] Figure 7 The sustained-release curves of butylphthalide in butylphthalide nanoparticles under different pH environments are shown.
[0068] Depend on Figure 7 It can be seen that butylphthalide nanoparticles have a better release effect at pH=6.4.
[0069] Example 2 The experiment was conducted using butylphthalide, the layered double hydroxide prepared in Example 1, and butylphthalide nanoparticles. The specific steps are as follows: Rats were acclimatized for 7 days and then divided into 6 groups, which were designated as LDHs group, NBP group, LDHs-NBP group, LDHs-NBP+LY294002 group, Sham group, and I / R group. The LDHs group received intraperitoneal injection of a saline suspension of lamellar double hydroxide, with a dosage of 20 mg / kg / day calculated as lamellar double hydroxide, for 7 consecutive days. The NBP group received intraperitoneal injection of butylphthalide saline solution, with a dosage of 5 mg / kg / day calculated as butylphthalide, for 7 consecutive days. The LDHs-NBP group received intraperitoneal injection of a saline suspension of butylphthalide nanoparticles, with a dosage of 25 mg / kg / day based on butylphthalide nanoparticles, for 7 consecutive days. The LDHs-NBP+LY294002 group received intraperitoneal injection of a saline suspension of butylphthalide nanoparticles at a dose of 25 mg / kg / day based on the butylphthalide nanoparticles, for 7 consecutive days. On day 1, the PI3K specific inhibitor LY294002 was administered intraperitoneally at a dose of 10 mg / kg. Sham group: Intraperitoneal injection of an equal volume of normal saline, continued for 7 days; In the I / R group, an equal volume of normal saline was injected intraperitoneally for 7 consecutive days.
[0070] Seven days after intraperitoneal injection, liver and kidney function were assessed in rats of the LDHs, NBP, LDHs-NBP, and Sham groups. Three rats from each group were euthanized, and the heart, liver, spleen, lungs, and kidneys of each group were stained with hematoxylin and eosin (HE). The results are as follows: Figure 8 As shown.
[0071] Figure 8 Images of liver and kidney function indicators and HE staining of various organs in rats in each group 7 days after intraperitoneal injection are shown. In the images, A represents liver function, B represents kidney function, and C represents HE staining.
[0072] Depend on Figure 8 It can be seen that compared with the Sham group, there were no significant differences in liver and kidney function indicators among the LDHs group, NBP group, and LDHs-NBP group. No significant pathological changes were observed in important organs such as the heart, liver, spleen, lungs, and kidneys, indicating that LDHs and LDHs-NBP have good biocompatibility.
[0073] After 7 days of continuous intraperitoneal injection of the reagent, a renal I / R model was established in rats in the I / R group. The specific steps were as follows: 6 mL / kg of 2.5% tribromoethanol was injected intraperitoneally, the abdomen was prepared and disinfected, the abdominal cavity was exposed by incision along the linea alba, blood was obtained by puncture of the inferior vena cava, the renal arteries were bluntly dissected, the right kidney was removed, and then reperfusion was performed for 24 h. After 7 days of continuous intraperitoneal injection of the reagent, rats in the Sham group underwent sham surgery. The specific steps were as follows: only the left kidney was freed without clamping the renal artery, and no reperfusion treatment was performed. Other procedures were the same as those in the I / R group. After 7 days of continuous intraperitoneal injection of the reagent, the left renal artery of the LDHs group, NBP group, LDHs-NBP group, and LDHs-NBP+LY294002 group was clamped with a non-invasive arterial clamp for 45 min; then each group was reperfused for 24 h (at this time, each group was recorded as: I / R+LDHs group, I / R+NBP group, I / R+LDHs-NBP group, and I / R+LDHs-NBP+LY294002 group, respectively). Then, the left kidneys of all rats were removed under anesthesia, and blood samples were collected for subsequent analysis. The rats were then euthanized, and the kidneys of each group were subjected to HE staining, transcriptome sequencing, immunohistochemical detection of p-PI3K, p-AKT, and Nrf2, and protein expression level detection. The results are as follows: Figure 9-13 .
[0074] Figure 9 HE staining results of kidneys in each group after establishing a renal ischemia-reperfusion injury model.
[0075] Figure 10 Volcano plot of differentially expressed transcriptomic genes between the I / R group and the Sham group, and between the I / R+LDHs-NBP group and the I / R group after constructing a renal ischemia-reperfusion injury model.
[0076] Figure 11 Heatmap of significantly differentially expressed genes in the I / R group, Sham group, and I / R+LDHs-NBP group after constructing a renal ischemia-reperfusion injury model.
[0077] Figure 12 KEGG enrichment analysis plots for the I / R group, Sham group, and I / R+LDHs-NBP group after constructing a renal ischemia-reperfusion injury model.
[0078] Figure 13 To establish a renal ischemia-reperfusion injury model, the immunohistochemical and protein expression levels of each group were obtained. In this study, A represents the immunohistochemical results of p-PI3K, p-AKT, and Nrf2 in rat kidneys, and B represents the protein expression levels in rat kidneys.
[0079] Depend on Figure 9 It can be seen that the kidneys in the I / R group showed typical damage, and both butylphthalide and butylphthalide nanoparticle treatment could significantly reduce pathological changes such as renal tubular dilation, brush border loss, interstitial edema and inflammatory cell infiltration. Among them, the protective effect of butylphthalide nanoparticles was more significant.
[0080] Depend on Figure 10-12 It can be seen that the differentially expressed genes between the I / R+LDHs-NBP group and the I / R group are significantly enriched in the PI3K-AKT signaling pathway.
[0081] Depend on Figure 13As can be seen from A, butylphthalide nanoparticles can significantly upregulate the expression of p-PI3K and p-AKT and promote Nrf2 nuclear translocation, with a stronger effect than NBP treatment alone.
[0082] Depend on Figure 13 B shows that AKT phosphorylation induced by butylphthalide nanoparticles was significantly reversed by LY294002, and Nrf2 expression was downregulated, confirming that its protective effect depends on the activation of the PI3K-AKT-Nrf2 pathway.
[0083] The PI3K-AKT signaling pathway plays an important role in regulating various pathophysiological processes such as metabolism, oxidative stress, and immune inflammation. Activation of this pathway can promote Nrf2 nuclear translocation. Nrf2 plays a crucial role in combating oxidative stress-related diseases. In the cell nucleus, Nrf2 binds to antioxidant response elements (AREs), thereby initiating the transcription of antioxidant genes, reducing ROS levels, and enhancing the cell's defense against oxidative stress.
[0084] Example 3 Butylphthalide, the layered double hydroxide prepared in Example 1, and butylphthalide nanoparticles were used to investigate their effects on the antioxidant stress capacity of HK-2 cells. The specific steps are as follows: 1. Prepare normal culture media containing different concentrations of layered double hydroxides (0, 2.5, 5, 10, 20, 40, and 80 μg / mL), and culture HK-2 cells for 24 h. Cell proliferation activity was measured using a CCK-8 assay kit. Each group was tested in at least three replicates. Results are as follows: Figure 14 As shown in Figure A.
[0085] Figure 14 A shows the effect of different concentrations of layered double hydroxides on the proliferation activity of HK-2 cells.
[0086] Depend on Figure 14 As can be seen from A, after treatment with different concentrations of layered double hydroxides for 24 h, there was no statistically significant difference in the proliferation activity of HK-2 cells, indicating that the cytotoxicity of layered double hydroxides to HK-2 cells was negligible.
[0087] 2. Culture HK-2 cells. When the HK-2 cells reach 70-80% confluence, wash them with PBS and then divide them into groups for modeling: the Control group was replaced with normal culture medium and cultured under normoxic conditions for 12 h. The H / R group was replaced with DMEM / F12 sugar-free medium and cultured under hypoxic conditions for 12 h, followed by reoxygenation for 4 h. The H / R+NBP group was replaced with DMEM / F12 sugar-free medium containing different concentrations of butylphthalide (2.5, 5, 10, and 20 μg / mL), and cultured under hypoxic conditions for 12 h, followed by reoxygenation for 4 h. The H / R+LDHs-NBP group was replaced with DMEM / F12 sugar-free medium containing different concentrations of butylphthalide nanoparticles (2.5, 5, 10, and 20 μg / mL), and cultured under hypoxic conditions for 12 h, followed by reoxygenation for 4 h.
[0088] Proliferation activity was determined using a CCK-8 assay kit, with each group undergoing at least three parallel experiments. Results are as follows: Figure 14 As shown in B.
[0089] Figure 14 B represents the effect of different concentrations of butylphthalide and butylphthalide nanoparticles on the proliferation activity of H / R model HK-2 cells.
[0090] Depend on Figure 14 B shows that the proliferation activity of HK-2 cells in the H / R group was significantly lower than that in the Control group, indicating that the model was successful; the proliferation activity of HK-2 cells in different concentrations of H / R+LDHs-NBP groups was higher than that in the H / R+NBP group; among the effective concentrations, 5 µg / mL butylphthalide nanoparticles had the best effect on promoting cell proliferation.
[0091] 3. Construct the Control group and H / R group according to the method in step 2; then take HK-2 cells from the H / R group and divide them into 5 groups, seed them into 6-well plates, and add no drug to 1 group. Continue to culture for 24 hours and it will still be the H / R group. The remaining four groups were added with a final concentration of 4 μg / mL layered double hydroxide, 1 μg / mL butylphthalide, 5 μg / mL butylphthalide nanoparticles, and (5 μg / mL butylphthalide nanoparticles and 20 μM LY294002), respectively. After each group was cultured for 24 h, they were named H / R+LDHs, H / R+NBP, H / R+LDHs-NBP, and H / R+LDHs-NBP+LY294002 groups, respectively. Then, following the instructions of the apoptosis kit, the apoptosis rate of HK-2 cells in each group was detected. The JC-1 experiment was performed on each group, and images were taken using a Zeiss fluorescence microscope. The results are as follows: Figure 15-16 As shown.
[0092] Figure 15 The results show the apoptosis rate of HK-2 cells in each group. Figure 16 The results show the mitochondrial membrane potential of HK-2 cells in each group.
[0093] Depend on Figure 15It can be seen that compared with the H / R+LDHs and H / R+NBP groups, butylphthalide nanoparticles LDHs-NBP can significantly improve cell apoptosis; the cell apoptosis rate of the H / R+LDHs-NBP+LY294002 group is significantly higher than that of the H / R+LDHs-NBP group, indicating that the effect of butylphthalide nanoparticles depends on the activation of the PI3K-AKT-Nrf2 pathway, which is consistent with the previous results.
[0094] Depend on Figure 16 It can be seen that: when the mitochondrial membrane potential is normal, JC-1 is in an aggregated state (showing red fluorescence); when the mitochondrial membrane potential decreases, JC-1 can depolymerize into monomers (showing green fluorescence), indicating that mitochondrial function is impaired; compared with the Control group, the HK-2 cells in the H / R group have damaged mitochondria and decreased membrane potential; the red / green fluorescence intensity ratio of the H / R+LDHs-NBP group is much higher than that of the H / R+LDHs, H / R+NBP, and H / R+LDHs-NBP+LY294002 groups; butylphthalide nanoparticles LDHs-NBP can significantly improve mitochondrial function and restore mitochondrial membrane potential; butylphthalide nanoparticles can improve mitochondrial function in HK-2 cells.
[0095] 4. The ROS level, p-PI3K, p-AKT, and Nrf2 protein expression levels of HK-2 cells in each group from step 3 were detected respectively. The results are as follows: Figure 17 As shown.
[0096] Figure 17 The results show the ROS level and p-PI3K, p-AKT and Nrf2 protein expression levels of HK-2 cells in each group. AB represents the ROS level and CD represents the protein expression level.
[0097] Depend on Figure 17 It can be seen that compared with the H / R+LDHs, H / R+NBP, and H / R+LDHs-NBP+LY294002 groups, butylphthalide nanoparticles LDHs-NBP are more effective in reducing ROS levels, and the protective effect can be reversed by the PI3K inhibitor LY294002. Butylphthalide nanoparticles LDHs-NBP can significantly activate the PI3K-AKT-Nrf2 signaling pathway, while LY24002 pretreatment inhibits the phosphorylation process of this pathway, resulting in a significant reduction in Nrf2 protein expression, suggesting that LY294002 can affect the nuclear translocation and expression of Nrf2 after activation. This indicates that butylphthalide nanoparticles LDHs-NBP may maintain redox balance by activating the PI3K-AKT-related Nrf2 pathway, thereby reducing excessive ROS accumulation and alleviating H / R-induced oxidative stress. Butylphthalide nanoparticles LDHs-NBP can activate the PI3K-AKT-Nrf2 pathway to inhibit oxidative stress.
[0098] The statistical processing was performed as follows: SPSS 28.0 software was used for statistical processing of the data, and the experimental results were analyzed using mean ± standard deviation (mean ± SD). Differences between groups were analyzed using t-tests and one-way ANOVA. P < 0.05 was considered statistically significant.
[0099] In summary, the butylphthalide nanoparticles (LDHs-NBP) of this invention can alleviate oxidative stress damage in both in vivo and in vitro treatments, while increasing the abundance of phosphorylated PI3K and AKT, promoting Nrf2 nuclear translocation and increasing its expression. Furthermore, this effect can be reversed by the PI3K inhibitor LY294002. This indicates that butylphthalide nanoparticles (LDHs-NBP) may exert a renal protective effect by targeting and regulating the PI3K-AKT-Nrf2 pathway, providing a new theoretical basis for related drug development and a new design concept for targeted renal therapy.
[0100] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A butylphthalide nanoparticle, characterized in that, include: Layered double hydroxide and butylphthalide, with butylphthalide loaded on the layered double hydroxide.
2. The butylphthalide nanoparticles according to claim 1, characterized in that, Butylphthalide is uniformly loaded in the interlayer of layered double hydroxides; preferably, the average particle size of butylphthalide nanoparticles is 200-250 nm; preferably, the content of butylphthalide in butylphthalide nanoparticles is 20-30 wt%.
3. The butylphthalide nanoparticles according to claim 1 or 2, characterized in that, The layered double hydroxide has a hexagonal layered structure; preferably, the layered double hydroxide contains Mg and Al; preferably, the molar ratio of Mg to Al in the layered double hydroxide is 2.8-3.2:
1.
4. The butylphthalide nanoparticles according to any one of claims 1-3, characterized in that, Layered double hydroxides are prepared by hydrothermal coprecipitation. Preferably, in the preparation process of layered double hydroxides, hydroxides, magnesium sources, aluminum sources and water are mixed, stirred and reacted, solid and liquid are separated, the solid is mixed with water and subjected to hydrothermal reaction to obtain layered double hydroxides.
5. The butylphthalide nanoparticles according to claim 4, characterized in that, In the preparation of layered double hydroxides, the hydroxide, magnesium source, aluminum source, and water are mixed and stirred in an inert gas atmosphere. Preferably, the reaction is carried out at 50-70°C for 0.8-1.2 hours. More preferably, the reaction is carried out at 90-110°C for 14-18 hours using hydrothermal reaction.
6. The butylphthalide nanoparticles according to claim 4, characterized in that, In the preparation of layered double hydroxides, the hydroxide is a water-soluble hydroxide; preferably, in the preparation of layered double hydroxides, the magnesium source is at least one of magnesium chloride and magnesium chloride hydrate; preferably, in the preparation of layered double hydroxides, the aluminum source is at least one of aluminum chloride and aluminum chloride hydrate; preferably, in the preparation of layered double hydroxides, the molar ratio of hydroxide, magnesium, and aluminum is 6.5-7:2.8-3.2:
1.
7. A method for preparing butylphthalide nanoparticles as described in any one of claims 1-6, characterized in that, The process includes the following steps: mixing layered double hydroxide, butylphthalide, and water, adsorbing, separating the solid and liquid phases, and drying to obtain butylphthalide nanoparticles.
8. The method for preparing butylphthalide nanoparticles according to claim 7, characterized in that, The adsorption is carried out at 20-40℃ for 20-28 hours; preferably, the adsorption is carried out by shaking; preferably, the weight ratio of layered double hydroxide to butylphthalide is 1:1.5-2.5; preferably, the drying is freeze drying.
9. The use of butylphthalide nanoparticles as described in any one of claims 1-6 in the preparation of a drug for treating acute renal ischemia-reperfusion injury.
10. A drug for treating acute renal ischemia-reperfusion injury, characterized in that, include: The butylphthalide nanoparticles according to any one of claims 1-6 and the pharmaceutically acceptable excipients.