PLGA (poly (lactic-co-glycolic acid))-based paricalcitol nano preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a PLGA-based paricalcitol nano preparation as well as a preparation method and application thereof, the technical key points are as follows: the nano preparation provided by the invention is mainly composed of active vitamin D paricalcitol (pari) and a high polymer material PLGA, the particle size of the nano preparation is 114.0 nm, and the surface charge is-34.4 mV. The preparation method comprises the following steps: under a specific mass ratio, loading paricalcitol on PLGA by using a two-phase method to form a paricalcitol nano preparation with good stability, and removing a solvent through dialysis to obtain a water-soluble substance of PLGA-paricalcitol (PLGA-pari). The paricalcitol nanoparticles can be enriched in kidneys, show a good renal tubular epithelial cell protection effect, and can be used as a treatment preparation for various acute kidney diseases.

Description

Technical Field

[0001] The patent of this invention relates to the field of biomedicine technology, and specifically, to a PLGA-based paricalcitol nanoformulation and its preparation method and application. Background Art

[0002] Acute kidney injury (AKI) can be caused by a variety of factors, such as sepsis, nephrotoxic drugs, cardiovascular surgery, etc. The incidence of AKI in hospitalized patients is as high as 10-15%, and about 41.2% of patients do not recover their renal function at discharge and progress to chronic kidney disease. The incidence of acute kidney injury in ICU patients is as high as 60%, and the mortality rate is over 50%. Therefore, the prevention and treatment of AKI is a clinical scientific issue that needs to be solved urgently.

[0003] At present, the treatment of various types of AKI is mainly symptomatic supportive therapy, such as fluid resuscitation, anti-infection, and blood purification, and there is still a lack of renal protection treatment methods targeting the pathogenesis. More and more evidence shows that AKI caused by different causes has a common pathophysiological process, including inflammation, oxidative stress, ischemia-reperfusion injury, cell regeneration arrest and fibrosis. Renal tubular epithelial cells are important intrinsic cells of the kidney. Their main physiological function is reabsorption. Maintaining this physiological function requires a large amount of aerobic oxidation to provide energy. Therefore, renal tubular epithelial cells are easily damaged by stress factors such as hypoxia and cytotoxic drugs. After being damaged by the above factors, renal tubular epithelial cells can undergo apoptosis, iron necrosis, necroptosis and other cell death, and the regeneration ability of renal tubular epithelial cells after death is extremely weak. In the past, renal tubular epithelial cells were considered to be the main victims of AKI. However, recent studies have shown that damaged renal tubular epithelial cells themselves also release inflammatory factors and profibrotic factors, which also play an important role in promoting the progression of AKI. Therefore, alleviating or preventing renal tubular epithelial cell damage is expected to become a new approach to prevent and treat AKI.

[0004] Vitamin D receptor (VDR) is a nuclear transcription factor with a wide range of biological effects. Active vitamin D is its natural ligand and agonist. After the two bind, they enter the cell nucleus and participate in the transcriptional regulation of downstream target genes. Studies have found that VDR expression defects exist in renal tissues of AKI caused by various etiologies, and high-dose active vitamin D analog paricalcitol can stimulate VDR expression and activate the VD / VDR signaling pathway. In sepsis AKI, high-dose paricalcitol can reduce the expression of renal miR-155 and reduce the apoptosis of renal tubular epithelial cells, thereby delaying the progression of sepsis AKI; VDR also inhibits the reprogramming of renal tubular epithelial cell glucose metabolism by activating p-AMPK and downregulating the aerobic oxidation rate-limiting enzyme p-PDHA1 / PDHA1, thereby improving sepsis-induced oxygen consumption rate decrease and increased lactate accumulation, thereby delaying the progression of sepsis AKI. In cisplatin-induced AKI, it was found that paricalcitol can upregulate the target gene GPX4, protect the kidney by inhibiting ferroptosis of renal tubular epithelial cells; it can also inhibit NF-κB activation and downregulate the NLRP3 / Caspase-1 / GSDMD renal tubular epithelial cell pyroptosis pathway. In ischemia-reperfusion AKI, it was found that paricalcitol can upregulate the antioxidant enzyme GPX3, reduce oxidative stress and apoptosis of renal tubular epithelial cells; it can also downregulate the target gene ATF4 to regulate endoplasmic reticulum stress of renal tubular epithelial cells.

[0005] The traditional physiological function of active vitamin D is to regulate calcium and phosphorus metabolism, so low-dose (0.25ug / d) active vitamin D is often used clinically to prevent and treat hypocalcemia and secondary hyperparathyroidism. Only a larger dose of active vitamin D can activate the VDR signaling pathway and exert a renal protective effect, but when using paricalcitol in large doses, it is necessary to be vigilant about its systemic side effects. Therefore, the active vitamin D currently used in clinical practice is not suitable for directly increasing the dose to prevent and treat AKI, but needs to be modified in dosage form to have both effective local drug concentration and low systemic toxicity and side effects.

[0006] In recent years, nanotechnology has shown great advantages in targeted drug delivery and can effectively reduce the toxic and side effects of drugs on non-target organs. Nano drugs can enter renal tubular epithelial cells to exert their efficacy through two pathways: passive targeting or active targeting. Many classic drugs for treating kidney diseases, such as prednisolone, tripterygium wilfordii, and rapamycin, have been transformed into nano drugs targeting the kidneys. The research results show good efficacy and lower incidence of side effects. PLGA is a commonly used nano drug delivery system with good biocompatibility and biodegradability. PLGA nano carriers can encapsulate a variety of drugs, such as chemotherapeutic drugs, genes, proteins, etc., and effectively deliver drugs to tumors or other lesions through different targeting strategies, such as passive targeting, active targeting, and magnetic targeting. Therefore, PLGA can be used to nano-modify paricalcitol, so that it has a renal tubular epithelial cell targeting effect, activates the renal VDR signaling pathway, and protects the kidneys, while reducing non-target organ toxic and side effects.

[0007] To this end, the present application provides a preparation method and application of a PLGA-based paricalcitol nanoformulation to solve the problem that the current treatment of various types of AKI is mainly symptomatic supportive therapy, such as fluid resuscitation, anti-infection, and blood purification, and there is still a lack of renal protection treatment methods targeting the pathogenesis. Summary of the invention

[0008] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology, and to provide a PLGA-based paricalcitol nanoformulation and a preparation method and application thereof; the PLGA@pari nanoparticles in the scheme of the present invention have excellent renal tubular epithelial cell targeting ability, and have a stronger renal tubular epithelial cell protective effect than the same dose of paricalcitol, and the PLGA@pari nanoparticles can be used in the preparation and application of a potential and highly effective drug for the prevention and treatment of acute kidney injury.

[0009] The above-mentioned purpose of the present invention is achieved like this:

[0010] The present invention provides a PLGA-based paricalcitol nano preparation, which consists of PLGA and paricalcitol, wherein paricalcitol is loaded on PLGA, and the particle size of the nano preparation is 114.0 nm and the surface charge is -34.4 mV.

[0011] The present invention also provides a method for preparing a PLGA-based paricalcitol nanoformulation, comprising the following steps:

[0012] S1, dissolving PLGA in DMSO to obtain PLGA-DMSO solution (5 μg / μL);

[0013] S2, dissolving paricalcitol in DMSO to obtain paricalcitol-DMSO solution (0.5 μg / μL);

[0014] S3, mixing the PLGA-DMSO solution and the paricalcitol-DMSO solution to obtain a paricalcitol-PLGA-DMSO mixed solution;

[0015] S4. Dissolve PVA in double distilled water in another container to obtain a 1% PVA solution;

[0016] S5, placing a 1% PVA solution 5 times the amount of the paricalcitol-PLGA-DMSO mixed solution in an ultrasonic water bath, adding the paricalcitol-PLGA-DMSO mixed solution dropwise, and continuing the ultrasonic water bath for 5 minutes after the addition is complete to obtain an emulsion;

[0017] S6. The emulsion was transferred into a dialysis bag and dialyzed in double distilled water for 14 hours to obtain the final product, PLGA@paricalcitol (PLGA@pari) nanoformulation.

[0018] Furthermore, the mass ratio of PLGA to paricalcitol in step S3 is 50:1; and the volume ratio of the paricalcitol-PLGA-DMSO mixed solution to 1% PVA in step S5 is 1:5.

[0019] Furthermore, the water bath ultrasound power in step S5 is 300 W, the water bath ultrasound temperature is 37° C., and the water bath ultrasound time is 5 minutes.

[0020] Furthermore, the molecular weight cut-off of the dialysis bag in step S6 is 2.5 kDa, and the dialysis time is 14 hours.

[0021] The present invention also provides the use of the above-mentioned PLGA-based paricalcitol nanoformulation or the nanoformulation prepared by the preparation method in the preparation of drugs for preventing and treating acute kidney injury caused by sepsis (mainly supplementing renal tubular epithelial cell protective agents on the basis of current treatment).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The scheme design of the present invention provides a nano-preparation that is easy to prepare. By utilizing the mature PLGA carrier, the specific accumulation of paricalcitol in renal tubular epithelial cells is achieved. In the scheme of the present invention, paricalcitol is loaded on PLGA by a two-phase method to prepare PLGA@pari nanoparticles with a particle size of 114.0 nm and a negative surface charge. The PLGA@pari nanoparticles have the ability to enrich renal tubular epithelial cells and can achieve a higher concentration of paricalcitol in the local kidney than in the whole body, thereby preventing and treating acute kidney injury while reducing its systemic side effects.

[0024] 2. The scheme of the present invention is expected to solve the limitations of the current treatment methods for acute kidney injury and the clinical problem that the use of active vitamin D protozoa is not effective in treating acute kidney injury. At the same time, it can improve the blood half-life, tumor targeting effect and penetration ability of the drug. The scheme of the present invention provides new theoretical support for the development of new anti-cancer drugs and related clinical detection and treatment, and has important scientific significance, practical value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The actual picture, particle size and surface charge of PLGA@pari nanoparticles;

[0026] Figure 2 Transmission electron microscopy image of PLGA@pari nanoparticles;

[0027] Figure 3 The biodistribution and blood half-life of PLGA@Ce6 nanoparticles in mice;

[0028] Figure 4 The biosafety of PLGA@pari nanoparticles;

[0029] Figure 5 The protective effect of PLGA@pari nanoparticles on renal tubules;

[0030] Figure 6 The efficacy of PLGA@pari nanoparticles in preventing and treating acute kidney injury. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0033] With reference to the drawings, the following are preferred embodiments of the present invention.

[0034] In the following examples, unless otherwise specified, the raw materials and instruments used are commercially available.

[0035] Embodiment 1:

[0036] (1) Preparation of PLGA@pari nanoparticles: 10 μg of paricalcitol was dissolved in 20 μL of DMSO, and 500 μg of PLGA was dissolved in 100 μL of DMSO, and the two were mixed to prepare paricalcitol-PLGA-DMSO; in another flask, 1 g of PVA was dissolved in 100 mL of double distilled water to prepare 1% PVA. The EP tube was placed in an ultrasonic water bath, 600 μL of 1% PVA solution was added first, and then 120 μL of paricalcitol-PLGA-DMSO mixed solution was added dropwise with a pipette. After the addition was completed, the ultrasonic water bath was continued for 5 minutes to obtain a PLGA@pari nanoemulsion. Finally, the prepared PLGA@pari nanoemulsion was dialyzed in a 2.5 kDa dialysis bag for 14 hours (the dialysis external fluid was double distilled water) to obtain a PLGA@pari nanoformulation as a uniformly dispersed solution (such as Figure 1 ).

[0037] (2) Characterization of PLGA@pari nanoformulations: Figure 1 B in Figure 1 As shown in C, the PLGA@pari nanoparticles prepared in this example were subjected to particle size measurement and zeta potential analysis. The results showed that the particle size of the PLGA@pari nanoparticles was 114.0 nm and the surface charge was -34.4 mv. Figure 2 As shown, the PLGA@pari nanoparticles prepared in this example were subjected to transmission electron microscopy image analysis, and the results showed that the PLGA@pari nanoparticles were uniformly dispersed spherical.

[0038] Embodiment 2:

[0039] A PLGA@Ce6 nanoformulation similar to the PLGA@pari nanoformulation prepared in Example 1 was prepared and characterized, and its biodistribution and blood half-life were determined by semi-quantitative means of detecting fluorescence intensity.

[0040] 100 μL of Ce6 and PLGA@Ce6 at a concentration of 5 mg / kg was intraperitoneally injected, and in vivo imaging of the animals was performed 18 hours later. Figure 3 A in Figure 3 The in vivo fluorescence image of B in Figure 2 shows that PLGA@Ce6 is enriched in kidney tissue. The results of tissue fluorescence quantitative analysis show that the fluorescence intensity of the PLGA@Ce6 group is significantly higher than that of the Ce6 group, indicating that the PLGA-loaded paricalcitol nanoformulation has the ability to enrich kidney tissue.

[0041] 100 μL of Ce6 and PLGA@Ce6 at a concentration of 5 mg / kg were intraperitoneally injected, and blood samples were collected at different time points for fluorescence intensity measurement. Figure 3 C in Figure 3As shown in D, PLGA@Ce6 still has a relatively stable fluorescence intensity at 24 hours, while Ce6 has tended to disappear at 12 hours, indicating that the paricalcitol nanoformulation carried by PLGA is beneficial to prolong the blood circulation time.

[0042] Embodiment 3:

[0043] The PLGA@pari nanoformulation prepared in Example 1 was used to test its biosafety.

[0044] Zebrafish were treated with PBS, pari, PLGA, and PLGA@pari, and the survival status, heart rate, body length, and hatching rate of zebrafish were detected. Figure 4 Therefore, the results showed that the survival status of zebrafish under various treatments was good, with average heart rates of 148.8, 152.8, 155, and 152 beats / min, average body lengths of 3.106, 3.24, 3.176, and 3.4 mm, and 72h hatching rates of 90%, 90%, 100%, and 100%, respectively. This shows that pari, PLGA, and PLGA@pari all have good biosafety.

[0045] Embodiment 4:

[0046] The PLGA@pari nanoformulation prepared in Example 1 was used to detect its protective effect on renal tubular epithelial cells.

[0047] Flow cytometry was used to detect the effects of PBS, pari, and PLGA@pari on HK-2 cell apoptosis induced by lipopolysaccharide. ROS fluorescence staining and semi-quantitative methods were used to detect the production of ROS induced by PBS, pari, and PLGA@pari. Figure 5 As shown in Figures A and 5B, after LPS treatment, the apoptosis rate of HK-2 cells was 37.57%, while the apoptosis rates of HK-2 cells treated with pari and PLGA@pari nanoparticles after LPS treatment were reduced to 25.10% and 8.72%, respectively. Figure 5 As shown in C and D, Figure 4 Figure C shows that after LPS treatment, the positive detection rate of ROS in the visual field of HK-2 cells was 5.11%, while the detection rates of ROS in the visual field of HK-2 cells treated with pari and PLGA@pari nanoparticles were reduced to 3.13% and 0.92%, respectively, after LPS treatment, indicating that PLGA@pari nanoparticles have a good protective effect on renal tubular epithelial cells.

[0048] Embodiment 5:

[0049] The PLGA@pari nanoformulation prepared in Example 1 was used for the prevention and treatment of sepsis-related acute kidney injury in vivo.

[0050] Intraperitoneal injection of pari or PLGA@pari nanoformulation (200 ng / kg in pari) was used to prevent and treat sepsis-related acute kidney injury. Figure 6 As shown in A and B in Figure 3, the PLGA@pari nanoparticle group can effectively inhibit the progression of sepsis-related acute kidney injury, and creatinine and urea nitrogen decreased by 25% compared with the pari group. In addition, based on the mechanism that paricalcitol exerts its therapeutic effect by reducing glomerular epithelial cell apoptosis, we investigated the pathological changes of renal tubules in different groups. The results are shown in Figure 3. Figure 6 As shown in the CI in the figure, the renal tubular injury score of the PLGA@pari nanoparticle group was significantly improved compared with that of the paricalcitol group. This indicates that the PLGA@pari nanoparticle preparation we constructed can effectively protect renal tubular epithelial cells and prevent and treat sepsis-related acute kidney injury.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A PLGA-based paricalcitol nanoformulation, characterized in that: The preparation is composed of PLGA and paricalcitol, wherein paricalcitol is loaded on PLGA, and the particle size of the nano preparation is 114.0 nm and the surface charge is -34.4 mV.

2. The method for preparing a PLGA-based paricalcitol nanoformulation according to claim 1, characterized in that: The following steps are involved: S1, dissolving PLGA in DMSO to obtain a PLGA-DMSO solution; S2, dissolving paricalcitol in DMSO to obtain a paricalcitol-DMSO solution; S3, mixing the PLGA-DMSO solution and the paricalcitol-DMSO solution to obtain a paricalcitol-PLGA-DMSO mixed solution; S4. Dissolve PVA in double distilled water in another container to obtain a 1% PVA solution; S5, placing a 1% PVA solution 5 times the amount of the paricalcitol-PLGA-DMSO mixed solution in an ultrasonic water bath, adding the paricalcitol-PLGA-DMSO mixed solution dropwise, and continuing the ultrasonic water bath for 5 minutes after the addition is complete to obtain an emulsion; S6. The emulsion was transferred into a dialysis bag and dialyzed in double distilled water for 14 hours to obtain the final product, PLGA@paricalcitol (PLGA@pari) nanoformulation.

3. The method for preparing a PLGA-based paricalcitol nanoformulation according to claim 2, characterized in that: The mass ratio of PLGA to paricalcitol in step S3 is 50:1; the volume ratio of the paricalcitol-PLGA-DMSO mixed solution to 1% PVA in step S5 is 1:

5.

4. The method for preparing a PLGA-based paricalcitol nanoformulation according to claim 3, characterized in that: The water bath ultrasonic power in step S5 is 300 W, the water bath ultrasonic temperature is 37° C., and the water bath ultrasonic time is 5 minutes.

5. The method for preparing a PLGA-based paricalcitol nanoformulation according to claim 4, characterized in that: The molecular weight cut-off of the dialysis bag in step S6 is 2.5 kDa, and the dialysis time is 14 hours.

6. The PLGA-based paricalcitol nanoformulation according to claim 1 or the preparation method according to any one of claims 2 to 5, characterized in that: The nano preparation is used in preparing a drug for preventing and treating acute kidney injury caused by sepsis.

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