A composite nanoparticle and its use in the preparation of a drug for treating diabetes

Abstract

The present invention provides a composite nanoparticle, which is prepared by a method comprising the following steps: S1. Adding a potassium permanganate solution to a serum albumin solution, and carrying out a first reaction under stirring to obtain a first reaction product; S2. Subjecting the first reaction product to a first dialysis to obtain a first dialysis product; S3. Mixing the first dialysis product with a bilirubin solution, and carrying out a second reaction under oscillation to obtain a second reaction product; S4. Subjecting the second reaction product to a second dialysis to obtain a second dialysis product. The present invention also provides the use of the composite nanoparticle as described above in the preparation of a medicament for treating diabetes. Through the above technical solution, the present invention effectively improves the activity and success rate of stem cell transplantation by means of the composite nanoparticle, thereby achieving a better effect in treating diabetes.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular, to a composite nanoparticle and its use in the preparation of a drug for treating diabetes. Background Art

[0002] Diabetes, as a chronic non-communicable disease, has gradually become one of the major threats to human health. Traditional treatment methods are familiar to people and have certain curative effects, such as insulin injection treatment and chemical drug treatment. However, they also have drawbacks such as poor treatment effects, recurrence of the disease, and large side effects. Therefore, the development of new effective technologies or drugs is an urgent need in the field of diabetes treatment.

[0003] Stem cell technology is a research hotspot in the fields of tissue repair and disease treatment and is considered a new hope for the treatment of many refractory diseases, including diabetes treatment. For example, CN110628723B provides a recombinant stem cell that can overexpress Exendin 4 protein; using this recombinant stem cell, drugs for treating obesity, fatty liver, and type 2 diabetes can be prepared; this recombinant stem cell can significantly reduce blood glucose and lipid levels and improve insulin sensitivity. However, there are still many problems in stem cell treatment technology. Among them, problems such as the activity and apoptosis of transplanted cells are important reasons affecting the effect of stem cell transplantation. How to regulate and effectively improve the activity and success rate of cell transplantation is an urgent problem to be solved for the effect of stem cell transplantation in treating diabetes. Summary of the Invention

[0004] The purpose of the present invention is to further effectively improve the activity and success rate of cell transplantation for the preparation of a cell drug for treating diabetes.

[0005] To achieve the above purpose, the present invention provides a composite nanoparticle, which is prepared by a method including the following steps: S1. Add a potassium permanganate solution to a serum albumin solution and carry out a first reaction under stirring to obtain a first reaction product; S2. Carry out a first dialysis on the first reaction product to obtain a first dialysis product; S3. Mix the first dialysis product with a bilirubin solution and carry out a second reaction under oscillation to obtain a second reaction product; S4. Carry out a second dialysis on the second reaction product to obtain a second dialysis product.

[0006] The present invention also provides the use of the composite nanoparticle as described above in the preparation of a drug for treating diabetes.

[0007] Through the above technical solution, the present invention effectively improves the activity and success rate of stem cell transplantation through the composite nanoparticle, thereby achieving a better effect in treating diabetes.

[0008] Other features and advantages of the present invention will be described in detail in the following specific implementation section. Description of the Drawings

[0009] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0010] Figure 1 It is a graph of the particle size and Tyndall effect of the composite nanoparticles in Example 1.

[0011] Figure 2 It is a graph of the Zeta potential of the composite nanoparticles in Example 1.

[0012] Figure 3 It is a graph of the results of the catalytic hydrogen peroxide oxygen production performance of the composite nanoparticles in Example 1.

[0013] Figure 4 It is the result of the biocompatibility test of the composite nanoparticles in Example 1, specifically the MTT result of MIN6 cells and the composite nanoparticles in Example 1.

[0014] Figure 5 It is the result of the test on the protective effect of the composite nanoparticles in Example 1 on cranial bone stem cells, specifically the effect of the composite nanoparticles in Example 1 on improving the ATP activity of cranial bone stem cells under model conditions.

[0015] Figure 6 It is the result of the test on the protective effect of the composite nanoparticles in Example 1 on cranial bone stem cells, specifically reducing the proportion of senescent cells under model conditions. ** indicates P < 0.01, and the cells are magnified 200 times.

[0016] Figure 7 It is the result of the composite of the composite nanoparticles in Example 1 and cranial bone stem cells on improving the treatment effect of diabetic mice, specifically the effect of reducing the blood glucose of mice.

[0017] Figure 8 It is the result of the composite of the composite nanoparticles in Example 1 and cranial bone stem cells on improving the treatment effect of diabetic mice, specifically the effect of promoting the weight recovery of mice.

[0018] Figure 9 It is the result of the composite of the composite nanoparticles in Example 1 and cranial bone stem cells on improving the treatment effect of diabetic mice, specifically the effect of improving the survival rate of mice. ** indicates P < 0.01. Specific Implementation

[0019] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention.

[0020] The present invention provides a composite nanoparticle, which is prepared by a method comprising the following steps: S1. Add a potassium permanganate solution to a serum albumin solution, and carry out a first reaction under stirring to obtain a first reaction product; S2. Carry out a first dialysis on the first reaction product to obtain a first dialysis product; S3. Mix the first dialysis product with a bilirubin solution, and carry out a second reaction under oscillation to obtain a second reaction product; S4. Carry out a second dialysis on the second reaction product to obtain a second dialysis product.

[0021] The composite nanoparticle of the present invention has good biocompatibility, and has the functions of ROS scavenging, in-situ oxygen production, regulating oxidative stress and inflammatory response signaling pathways, and can protect the activity of cranial stem cells. Among them, the role of serum albumin may be a stabilizer; the role of potassium permanganate is to form manganese oxide, and manganese oxide may have the effect of improving the hypoxia tolerance of transplanted cells; the role of bilirubin may be antioxidant.

[0022] Preferably, the potassium permanganate solution is a fresh potassium permanganate solution; the concentration of the potassium permanganate solution is 1-20 mg / mL.

[0023] Preferably, the concentration of the serum albumin solution is 5-100 mg / mL.

[0024] Preferably, the serum albumin is bovine serum albumin; the serum albumin solution is a serum albumin solution obtained by dissolving the serum albumin in a phosphate buffer solution, and the concentration of the phosphate buffer solution is 0.01-0.1 M, and the pH value is 7.2-7.6.

[0025] Preferably, the conditions of the first reaction include: the time is 1-10 hours, the temperature is 5-40 °C, and the stirring speed is 10-200 revolutions per minute.

[0026] Preferably, the conditions of the second reaction include: the time is 1-10 hours, the temperature is 5-40 °C, and the oscillation frequency is 10-200 times per minute.

[0027] Preferably, the conditions of the first dialysis include: the dialysis membrane specification is 10-20 kDa, the dialysis time is 10-30 hours, and the temperature is 5-40 °C; the conditions of the second dialysis include: the dialysis membrane specification is 10-20 kDa, the dialysis time is 10-30 hours, and the temperature is 5-40 °C.

[0028] Preferably, the solvent of the bilirubin solution is phosphate buffer solution or physiological saline, the concentration of bilirubin in the bilirubin solution is 0.1 - 2 mg / mL, the concentration of the phosphate buffer solution is 0.01 - 0.1 M, and the pH value is 7.2 - 7.6.

[0029] Preferably, the particle size of the composite nanoparticles is 10 - 100 nm; the Zeta potential is -40 mV to 10 mV.

[0030] The present invention also provides the use of the composite nanoparticles as described above in the preparation of a drug for treating diabetes.

[0031] Preferably, the drug for treating diabetes further contains stem cells. Preferably, the stem cells are cranial bone stem cells or recombinant stem cells.

[0032] The present invention will be further described in detail below by way of examples. Unless otherwise specified, the raw materials used in the examples can be obtained through commercial channels.

[0033] Example 1: Preparation and detection of composite nanoparticles.

[0034] The composite nanoparticles were synthesized by the biomineralization method using bovine serum albumin as a template. Weigh 250 mg of bovine serum albumin and dissolve it in 10 mL of phosphate buffer solution (PBS), and then slowly add the freshly prepared potassium permanganate solution (7 mg / mL, 5 mL) to the albumin solution within 5 minutes. After adding, the solution immediately turns dark brown, and then stir it at room temperature for 4 h. After the reaction is completed, dialyze the obtained reaction solution in a dialysis bag (14 kDa) in deionized water for 24 h to remove unreacted monomer impurities, and then the MnO2 nanoparticles stabilized by albumin on the surface are obtained. The bilirubin was encapsulated by the incubation method. The aqueous solution of MnO2 nanoparticles and the bilirubin solution (the solvent is PBS, the concentration is 1 mg / mL) were incubated at 37 °C with constant shaking for 6 h. Using the special complexation effect between bilirubin and albumin, the bilirubin was encapsulated on the surface of the MnO2 nanozyme to form a dark brown solution. Dialyze the prepared sample solution in a dialysis bag for 12 h to remove organic solvents and free drugs, and then freeze-dry to obtain the composite nanoparticles.

[0035] The particle size and distribution of the composite nanoparticles were investigated by dynamic light scattering method, and the results are as Figure 1 shown; the surface charge of the composite nanoparticles was measured by a Zeta potential analyzer, and the results are as Figure 2 shown.

[0036] A portable dissolved oxygen meter was used to detect the situation of the composite nanoparticles catalyzing H2O2 to produce O2, and the results are as Figure 3 shown.

[0037] The composite nanoparticles were co-cultured with MIN6 cells, and the cell viability was detected by MTT to evaluate the biocompatibility. The results are as Figure 4 shown. The results indicate that the composite nanoparticles have good biocompatibility.

[0038] Example 2: Detection of the composite and protective effect of composite nanoparticles on cranial bone stem cells.

[0039] After chopping the cranial bone slices with a medical electric drill saw, transfer them to a 50 mL centrifuge tube, add 20 - 30 mL of normal saline, mix well, place it in a low-temperature shaker, and shake at 4°C - 10°C and a rotation speed of 100 - 200 revolutions per minute for 10 - 15 minutes. Filter through a 200-mesh sieve, centrifuge the filtrate at 1000 rpm for 6 minutes. After removing the supernatant, resuspend the precipitate with cell culture medium. Take a small amount of the suspension, count it using a fully automatic blood analyzer, and then inoculate it at a density of 2×10 5 / cm 2 into a 25 cm 2 plastic cell culture flask, place it in an incubator at 37°C with 5% CO2 for culture. After 48 hours, pour out the non-adherent cells, change to fresh culture medium, and then change the medium every 2 - 3 days. When the cells grow to 90% confluence, digest and passage them until the P3 - P5 generations to obtain fibroblast-like human cranial bone stem cells with a single morphology.

[0040] Add 10 mg / ml D-galactose to the cranial bone stem cells and culture for 48 hours to construct a cell senescence model. Then add 20 - 100 μM composite nanoparticles (calculated by the amount of Mn), co-culture for 48 hours, and then perform ATP activity detection and SA-β-Gal staining to detect the activity of cranial bone stem cells. The results are as Figure 5 and Figure 6 shown. The results indicate that the composite nanoparticles can increase the ATP activity of cranial bone stem cells under model conditions and inhibit cell senescence.

[0041] Example 3: Composite cranial bone stem cells with composite nanoparticles improve the treatment effect of diabetic mice.

[0042] Before modeling, fast BALB / c mice (20 - 25 g) for 12 - 16 h without water restriction. Weigh streptozotocin at 120 mg / kg, add an appropriate volume of citrate buffer to make a 2% streptozotocin solution, keep it in the dark on ice for use, and streptozotocin needs to be prepared and used immediately (injected within 30 minutes). For the mice fasted for 12 - 16 h, measure the fasting blood glucose and weigh them, and then inject streptozotocin intraperitoneally. Measure the blood glucose value and body weight of the modeled mice on the 1st day and the 3rd day respectively. If both blood glucose values are greater than 22.2 mmol / mL, it is confirmed that the diabetic modeling is successful and used for subsequent experiments.

[0043] The successfully modeled diabetic mice were randomly divided into: a model group, a cranial stem cell transplantation group (dose: 1×10 6 cells / mouse), and a composite nanoparticle and cranial stem cell complex transplantation group (dose: 5 nmol composite nanoparticles (calculated by the amount of Mn) + 1×10 6 cells / mouse). After anesthesia, the hair on the right kidney area of the test diabetic mice was shaved off and disinfected with 75% ethanol solution. Then, the outer skin and muscle layer were cut open. The right kidney was exposed with a cotton swab. After making a small incision on the renal capsule with a 1 mL injection needle, the self-made syringe was slowly inserted under the renal capsule. After confirming that all the complexes were transferred, the syringe tip was withdrawn, and the small incision was pressed with a cotton swab for several seconds to prevent extracellular overflow. Subsequently, the kidney was pushed back into the abdominal cavity, and the wound was sutured. The wound was smeared with erythromycin ointment, and 0.1 mL of penicillin was intramuscularly injected to prevent infection. The animal body weight was measured at different time points, the blood glucose concentration of the mice was measured, and the survival rate of the mice was counted. The results are as shown in Figure 7 、 Figure 8 and Figure 9 .

[0044] The results showed that the mice in the composite nanoparticle and cranial stem cell complex transplantation group had better body weight maintenance, lower blood glucose concentration, and higher survival rate, indicating better therapeutic effect on diabetes.

[0045] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0046] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0047] Furthermore, any combination can be made between different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A composite nanoparticle, characterized in that, The composite nanoparticles are prepared by a method comprising the following steps: S1. Add a potassium permanganate solution to a serum albumin solution, and carry out a first reaction under stirring to obtain a first reaction product; S2. Carry out a first dialysis on the first reaction product to obtain a first dialysis product; S3. Mix the first dialysis product with a bilirubin solution, and carry out a second reaction under oscillation to obtain a second reaction product; S4. Carry out a second dialysis on the second reaction product to obtain a second dialysis product; The potassium permanganate solution is a fresh potassium permanganate solution; The concentration of the potassium permanganate solution is 1 - 20 mg / mL; The concentration of the serum albumin solution is 5 - 100 mg / mL; The serum albumin is bovine serum albumin, and the serum albumin solution is a serum albumin solution obtained by dissolving the serum albumin in a phosphate buffer solution, the concentration of the phosphate buffer solution is 0.01 - 0.1 M, and the pH value is 7.2 - 7.6; The particle size of the composite nanoparticles is 10 - 100 nm; the Zeta potential is -40 mV to 10 mV.

2. The composite nanoparticles according to claim 1, wherein, The conditions of the first reaction include: the time is 1 - 10 hours, the temperature is 5 - 40 °C, and the stirring speed is 10 - 200 revolutions per minute; The conditions of the second reaction include: the time is 1 - 10 hours, the temperature is 5 - 40 °C, and the oscillation frequency is 10 - 200 times per minute.

3. The composite nanoparticles according to claim 1, wherein, The conditions of the first dialysis include: the dialysis membrane specification is 10 - 20 kDa, the dialysis time is 10 - 30 hours, and the temperature is 5 - 40 °C; the conditions of the second dialysis include: the dialysis membrane specification is 10 - 20 kDa, the dialysis time is 10 - 30 hours, and the temperature is 5 - 40 °C.

4. The composite nanoparticles according to claim 1, wherein, The solvent of the bilirubin solution is a phosphate buffer solution or physiological saline, the bilirubin concentration in the bilirubin solution is 0.1 - 2 mg / mL, the concentration of the phosphate buffer solution is 0.01 - 0.1 M, and the pH value is 7.2 - 7.

6.

5. Use of the composite nanoparticles according to claims 1 - 4 in the preparation of a medicament for treating diabetes.

6. The use according to claim 5, wherein, The medicament for treating diabetes further contains stem cells, and the stem cells are cranial stem cells or recombinant stem cells.

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