Nano-selenium preparation for renal injury as well as preparation method and application of nano-selenium preparation

Abstract

The invention relates to a nano-selenium preparation for renal injury and a preparation method and application thereof, the nano-selenium preparation comprises a core and a shell, the core comprises selenium, the shell comprises distearoyl phosphatidyl ethanolamine-polyethylene glycol, and the shell wraps the surface of the core. The preparation method comprises the following steps: wrapping: respectively dissolving a raw material of the core and a raw material of the shell in a solvent to obtain a core material solution and a shell material solution; mixing the core material solution with the shell material solution, and wrapping the surface of the raw material of the core with the raw material of the shell to obtain a solution containing a nano-selenium preparation precursor; and preparing the nano-selenium preparation for renal injury: evaporating the solution containing the nano-selenium preparation precursor to dryness, and adding water to resuspend to obtain the nano-selenium preparation for renal injury. The nano-selenium preparation disclosed by the invention has a relatively good protection effect on AKI, has good kidney targeting property and biological safety, can effectively improve kidney functions, reduce pathological injury of kidney tissues and remarkably relieve mitochondrial injury and cell apoptosis, and has dose dependence.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nano-selenium preparation for treating kidney damage, and a preparation method and application thereof. Background Art

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, associated with significant morbidity and mortality. Epidemiological data show that AKI occurs in 20%-50% of hospitalized patients worldwide, with approximately 13.3 million new cases and approximately 1.7 million deaths annually.

[0003] AKI is of great clinical significance, but its pathophysiological mechanisms remain incompletely elucidated, involving multiple complex factors such as metabolic disorders, trace element imbalance, oxidative stress, and cell apoptosis. Studies have shown that selenium homeostasis is disrupted in patients with AKI, with serum selenium concentrations significantly decreased, and even continuous renal replacement therapy cannot restore serum selenium levels. Disturbances in selenium homeostasis can lead to decreased selenoprotein activity and increased production of reactive oxygen species (ROS), which can cause apoptosis or necrosis of renal tubular cells, further exacerbating acute kidney injury.

[0004] Current treatments for AKI are limited to symptomatic treatments such as volume management, hemodynamic support, and renal replacement therapy, lacking specific cures. Multiple studies have shown that selenium supplementation in mice or rats with AKI can improve renal damage and restore renal function. A retrospective cohort study showed that preoperative serum selenium levels can predict the incidence of AKI after adult cardiac surgery. Low serum selenium levels were significantly associated with a higher risk of AKI, suggesting that serum selenium may have a protective effect against AKI.

[0005] Selenium supplementation has numerous beneficial effects on various tissues. For patients with AKI, selenium supplementation not only does not burden renal excretion but also facilitates treatment and recovery. However, supplementing with inorganic selenium, such as sodium selenite and sodium selenate, is not only poorly absorbed but can also cause hair loss, nail deformation, and even poisoning. Direct ingestion of organic selenium, such as selenomethionine and selenocysteine, has a high absorption rate of 70%-90%, but may leave 20%-30% of inorganic selenium, posing risks with long-term intake.

[0006] Therefore, finding a preparation that can supplement serum selenium in AKI patients as a drug has important clinical application value in the treatment of AKI. Summary of the Invention

[0007] In response to the above-mentioned deficiencies in the prior art, the present invention proposes a nano-selenium preparation for kidney injury and a preparation method thereof, which can be used to prevent or treat acute kidney injury, and has the functions of improving renal function, reducing mitochondrial damage and inhibiting cell apoptosis.

[0008] The specific technical solutions of the present invention are as follows:

[0009] A nano-selenium preparation for kidney injury, characterized in that the nano-selenium preparation comprises a core and a shell, the core comprises selenium, the shell comprises distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and the shell is wrapped around the surface of the core.

[0010] In one embodiment, the raw material of the core includes organic selenium.

[0011] In one embodiment, the organic selenium comprises selenocystamine.

[0012] In one embodiment, the molecular weight of the DSPE-PEG is 1500-2500.

[0013] On the other hand, the present invention also provides a method for preparing the nano-selenium preparation, comprising the following steps:

[0014] Coating: dissolving the core material and the shell material in a solvent respectively to obtain a core material solution and a shell material solution; mixing the core material solution with the shell material solution to coat the surface of the core material with the shell material to obtain a solution containing a nano-selenium preparation precursor;

[0015] Preparation of a nano-selenium preparation for renal injury: evaporating a solution containing a precursor of the nano-selenium preparation to dryness, adding water and resuspending the solution to obtain a nano-selenium preparation for renal injury (Se@DSPE-PEG).

[0016] In one embodiment, the solvent comprises a volatile organic solvent.

[0017] In one embodiment, the volatile organic solvent comprises dichloromethane.

[0018] In one embodiment, the mass ratio of the raw material of the core to the raw material of the shell is 1:(3-5).

[0019] In one embodiment, the mixing method includes mixing under ultrasonic conditions, the evaporation method includes rotary evaporation, and the resuspending method includes resuspending under ultrasonic conditions.

[0020] In addition, the present invention also provides a drug, specifically a drug for preventing and / or treating kidney damage, comprising the nano-selenium preparation as described above, or the nano-selenium preparation obtained by the above preparation method. The drug may also include pharmaceutically acceptable excipients.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Good renal targeting and biosafety: The nano-selenium preparation of the present invention can be effectively accumulated in the damaged kidney and remain there after injection into the body, and no abnormal pathological changes were observed in the HE staining of major organs such as the heart, liver, spleen, lungs and kidneys.

[0023] (2) Protective effect on renal function: When nano-selenium preparations are injected into damaged kidneys, serum creatinine and urea nitrogen show a significant decrease, which can effectively protect renal function, and this protective effect has a dose effect.

[0024] (3) Effectively alleviate acute kidney injury: The nano-selenium preparation of the present invention can restore the expression of mitochondrial protein PGC-1α, mitochondrial outer membrane protein TOMM20, electron transport chain protein Cytb, mitochondrial dynamin OPA1 and mitochondrial fusion protein MFN2 in AKI mice, improve mitochondrial damage, and can also restore the expression of antioxidant enzyme GXP3, down-regulate the expression of apoptosis proteins Cleaved Caspase 3 and p53, and inhibit the production of acute kidney injury markers NGAL and KIM-1, thereby inhibiting cell apoptosis, and the recovery effect is dose-dependent.

[0025] In summary, the nano-selenium preparation of the present invention, obtained by combining selenium and distearoylphosphatidylethanolamine-polyethylene glycol, has good renal targeting and biosafety, has a protective effect on renal function, improves the antioxidant capacity of damaged kidneys by improving mitochondrial damage and inhibiting cell apoptosis, has a repair effect on acute kidney injury, and is dose-dependent. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preparation process of nano-selenium complex Se@DSPE-PEG2000;

[0027] Figure 2 Schematic diagram of the preparation and grouping of the acute kidney injury model;

[0028] Figure 3 This is the distribution diagram of the nano-selenium complex Se@DSPE-PEG2000 in the normal mice of the control group;

[0029] Figure 4 This is the distribution diagram of the nano-selenium complex Se@DSPE-PEG2000 in the AKI group mice;

[0030] Figure 5 HE staining of major organs of mice, including heart, liver, spleen, lung and kidney;

[0031] Figure 6 The nano-selenium complex Se@DSPE-PEG2000 has a protective effect on renal function in mice with acute kidney injury;

[0032] Figure 7 Nano-selenium complex Se@DSPE-PEG2000 improves mitochondrial damage in mice with acute kidney injury;

[0033] Figure 8 Nano-selenium complex Se@DSPE-PEG2000 improves cell apoptosis in mice with acute kidney injury;

[0034] Figure 9 Nano-selenium complex Se@DSPE-PEG2000 improves cell apoptosis in mice with acute kidney injury. DETAILED DESCRIPTION

[0035] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] Example 1

[0038] 1. A selenium-loaded nanocomposite and a preparation method thereof.

[0039] In this example, selenocystamine was encapsulated by DSPE-PEG2000 to form a selenium-loaded nanocomplex Se@DSPE-PEG2000, and the therapeutic effect on ischemia-reperfusion-induced AKI mice was observed by tail vein injection.

[0040] (1) Preparation of nano-selenium preparations

[0041] ① Weigh 1 mg of selenocystamine and dissolve it in 1 mL of dichloromethane;

[0042] ② Weigh 4 mg of DSPE-PEG2000 and dissolve it in 10 mL of dichloromethane;

[0043] ③ Mix the above two solutions under ultrasonic conditions and then evaporate to dryness using a rotary evaporator;

[0044] ④ The evaporated material was resuspended in water under ultrasonic conditions to obtain the nano-selenium complex Se@DSPE-PEG2000.

[0045] In this embodiment, the mass ratio of selenocystamine to DSPE-PEG2000 is 1:4.

[0046] (2) Preparation of acute kidney injury (AKI) model

[0047] This study used renal pedicle clamping to establish a bilateral renal ischemia-reperfusion injury model. Specifically, the abdominal cavity of mice was surgically exposed layer by layer, and both kidneys were located. The renal pedicles were carefully separated and clamped at 37°C for 30 minutes. The clamp was then released to restore renal blood flow, and the abdominal cavity was closed to produce AKI mice. Normal mice served as the control group.

[0048] Example 2

[0049] A high dose of fluorescent-labeled Se@DSPE-PEG2000 (2.0 mg / kg) was injected into normal mice and AKI mice through the tail vein, and the in vivo distribution of Se@DSPE-PEG2000 in normal control group mice and AKI group mice was observed using in vitro organ imaging.

[0050] Figure 3 This is the distribution diagram of Se@DSPE-PEG2000 in the normal mice of the control group. Figure 4 This is the distribution diagram of Se@DSPE-PEG2000 in the AKI group mice. Figure 5 HE staining images of the main organs such as heart, liver, spleen, lung and kidney of normal mice in the control group and mice in the AKI group.

[0051] As can be seen, in the control group of normal mice, Se@DSPE-PEG2000 was primarily distributed in the liver, reaching a peak at 10 minutes, while a small amount of Se@DSPE-PEG2000 was also present in the kidneys. At 3 hours, Se@DSPE-PEG2000 was still distributed in the liver, while distribution in the kidneys reached a peak. Subsequently, at 6 hours, considerable residual signal could still be detected in both the liver and kidneys, but metabolism was essentially complete at 12 and 24 hours, with almost no fluorescence signal detected. Throughout the observation process, the liver was the organ with the most Se@DSPE-PEG2000, reaching a peak signal retention at 10 minutes, while the kidneys reached a peak signal retention at 3 hours and still had a detectable fluorescence signal at 6 hours. No signal was detected in the heart, spleen, and lungs throughout the entire process.

[0052] In mice in the AKI group, Se@DSPE-PEG2000 was primarily distributed in the liver and kidneys at 10 minutes. Significant signal persistence persisted in these organs until 6 hours, while some residual signal was also detected at 12 and 24 hours. Throughout the observation period, the liver and kidneys reached peak signal persistence at 10 minutes and continued to show fluorescence signals at 12 and 24 hours. No signal was detected in the heart, spleen, or lungs.

[0053] The in vitro imaging results of the above two groups showed that the distribution of Se@DSPE-PEG2000 in the normal mice in the control group and the mice in the AKI group was relatively consistent, mainly distributed in the liver and kidneys. However, the signals of both organs in the mice in the AKI group reached a peak at 10 minutes, and the peak time was significantly earlier than that of the normal mice in the control group, and the resident signal was stronger than the peak signal of the normal mice in the control group. In addition, the signal residence time of the mice in the AKI group was significantly longer than that of the normal mice in the control group, and a certain amount of Se@DSPE-PEG2000 residue could still be detected at 24 hours. This shows that Se@DSPE-PEG2000 has good renal targeting to damaged kidneys, can be effectively enriched in damaged kidneys, and has the characteristics of continuous residence in the kidneys.

[0054] In addition, no abnormal pathological changes caused by Se@DSPE-PEG2000 were observed in the HE staining of major organs such as the heart, liver, spleen, lungs and kidneys of normal mice in the control group and AKI group injected with Se@DSPE-PEG2000, indicating that Se@DSPE-PEG2000 has good biosafety.

[0055] Example 3

[0056] Mice with AKI were randomly divided into four groups and injected with Se@DSPE-PEG2000 via the tail vein: surgery alone, surgery plus low-dose Se@DSPE-PEG2000 (0.5 mg / kg), surgery plus medium-dose Se@DSPE-PEG2000 (1.0 mg / kg), and surgery plus high-dose Se@DSPE-PEG2000 (2.0 mg / kg). A control group of normal mice received 0.2 mL of saline. Twenty-four hours later, euthanasia was performed and blood was collected. Serum was then extracted for renal function testing. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using an automated chemistry analyzer (AU480, Beckman Coulter). Serum creatinine and BUN levels are expressed in milligrams per deciliter (mg / dl). Figure 6 It can be seen that the concentrations of serum creatinine and urea nitrogen in the serum of mice in each group.

[0057] The results show that compared with the normal control group, the serum creatinine (Scr) and urea nitrogen (BUN) concentrations in the surgery-only group were significantly increased, indicating that the model was successful and that the mice had abnormal renal function. In contrast, the serum creatinine and BUN concentrations in the three surgery + Se@DSPE-PEG2000 groups were significantly decreased compared with the surgery-only group. Furthermore, the higher the dose of Se@DSPE-PEG2000 injected, the lower the serum creatinine and BUN concentrations. This suggests that tail vein injection of Se@DSPE-PEG2000 can effectively protect the renal function of AKI mice, and this protective effect is dose-dependent.

[0058] Example 4

[0059] AKI mice were randomly divided into four groups and injected with Se@DSPE-PEG2000 via the tail vein: surgery alone, surgery plus low-dose Se@DSPE-PEG2000 (0.5 mg / kg), surgery plus medium-dose Se@DSPE-PEG2000 (1.0 mg / kg), and surgery plus high-dose Se@DSPE-PEG2000 (2.0 mg / kg). Normal mice in the control group were injected with 0.2 mL of normal saline. After 24 hours, euthanasia procedures were performed and biological samples were collected to observe mitochondrial conditions. Figure 7 The expression of various mitochondrial proteins in each group of mice can be seen.

[0060] From the immunoblotting results, it can be seen that Se@DSPE-PEG2000 can restore the expression of mitochondrial gene protein PGC-1α, mitochondrial outer membrane protein TOMM20, electron transport chain protein Cytb, mitochondrial dynamic protein OPA1 and mitochondrial fusion protein MFN2 in AKI mice ( Figure 7 AF), and immunohistochemistry results also showed that Se@DSPE-PEG2000 could restore the expression of mitochondrial gene protein PGC-1α and mitochondrial dynamic protein OPA1 in AKI mice ( Figure 7 GJ), and these recovery effects are dose-dependent. The higher the dose of Se@DSPE-PEG2000 injected, the more obvious the improvement effect, indicating that the nano-selenium complex Se@DSPE-PEG2000 can effectively improve mitochondrial damage in mice with acute kidney injury.

[0061] Example 5

[0062] AKI mice were randomly divided into four groups and injected with Se@DSPE-PEG2000 via the tail vein: surgery alone, surgery plus low-dose Se@DSPE-PEG2000 (0.5 mg / kg), surgery plus medium-dose Se@DSPE-PEG2000 (1.0 mg / kg), and surgery plus high-dose Se@DSPE-PEG2000 (2.0 mg / kg). Normal mice in the control group were injected with 0.2 mL of normal saline. After 24 hours, euthanasia procedures were performed and biological samples were collected. Cell apoptosis was observed by immunoblotting and staining. Figure 8-9 It can be seen that the changes in various biological components that affect cell apoptosis in each group of mice.

[0063] Immunoblotting results show that Se@DSPE-PEG2000 can restore the expression of the antioxidant enzyme GXP3, downregulate the expression of apoptosis proteins Cleaved Caspase 3 and p53, and inhibit the production of NGAL, a marker of acute kidney injury. This effect is dose-dependent, with higher doses of Se@DSPE-PEG2000 injected resulting in more pronounced improvements. This suggests that the nano-selenium complex Se@DSPE-PEG2000 can enhance the antioxidant capacity of AKI mice, inhibit apoptosis, and alleviate acute kidney injury.

[0064] Glycogen staining results showed that Se@DSPE-PEG2000 reduced tubular necrosis and improved kidney damage in AKI mice. TUNEL staining results showed that Se@DSPE-PEG2000 inhibited the production of fragmented DNA in tubular cells and alleviated apoptosis in AKI mice. These protective effects were dose-dependent, with higher doses of Se@DSPE-PEG2000 being more pronounced.

[0065] These results are consistent with the above-mentioned immunoblotting results, indicating that the nano-selenium complex Se@DSPE-PEG2000 has the effect of improving the antioxidant capacity of mice with acute kidney injury, inhibiting cell apoptosis, and alleviating acute kidney injury.

[0066] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A nano-selenium preparation for kidney damage, characterized in that: The nano-selenium preparation comprises a core and a shell, wherein the core comprises selenium, the shell comprises distearoylphosphatidylethanolamine-polyethylene glycol, and the shell is wrapped on the surface of the core.

2. The nano-selenium preparation according to claim 1, characterized in that The raw material of the core includes organic selenium.

3. The nano-selenium preparation according to claim 2, characterized in that The organic selenium includes selenocystamine.

4. The method for preparing the nano-selenium preparation according to any one of claims 1 to 3, wherein: The following steps are involved: Coating: dissolving the core material and the shell material in a solvent respectively to obtain a core material solution and a shell material solution; mixing the core material solution with the shell material solution to coat the surface of the core material with the shell material to obtain a solution containing a nano-selenium preparation precursor; Preparation of nano-selenium preparation for kidney injury: evaporating a solution containing a precursor of the nano-selenium preparation to dryness, adding water and resuspending the solution to obtain the nano-selenium preparation for kidney injury.

5. The preparation method according to claim 4, characterized in that The solvent includes a volatile organic solvent.

6. The preparation method according to claim 5, characterized in that The volatile organic solvent includes dichloromethane.

7. The preparation method according to claim 6, characterized in that The mass ratio of the raw material of the core to the raw material of the shell is 1:(3-5).

8. The preparation method according to claim 4, characterized in that The mixing method includes mixing under ultrasonic conditions, the evaporation method includes rotary evaporation, and the resuspending method includes resuspending under ultrasonic conditions.

9. A drug, characterized in that The invention comprises a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient comprises the nano-selenium preparation according to any one of claims 1 to 3, or the nano-selenium preparation prepared by the preparation method according to any one of claims 4 to 8.

10. The drug according to claim 9, characterized in that The medicine is a medicine for preventing and / or treating kidney damage.

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