Novel method for reducing drug-induced cell death

Abstract

The invention relates to a novel method for reducing drug-induced cell death, and belongs to the technical field of biological medicines. LP-Rh2 in the form of lipidosome is prepared by providing a skeleton structure by lipidosome and taking ginsenoside Rh2 as a functional component, and after LP-Rh2 and other drugs are jointly added into a cell culture system, the drug-induced cytotoxicity can be inhibited in various scenes, the cell viability is improved, and the drug-induced cytotoxicity can be inhibited. Therefore, the action mechanism of other candidate drugs with cytotoxicity can be better matched and researched in scientific research application, and potential safety risks possibly brought by drugs, especially vaccine adjuvants, can be reduced in drug application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a new method for reducing drug-induced cell death. BACKGROUND

[0002] An adjuvant is defined as a component added to a vaccine, which can enhance the immune response effect of the body to the antigen, and is a cross-discipline of immunology, chemistry and material science. At present, there are mainly 7 kinds of human adjuvants approved by the FDA of the United States, including liposomes, oil-in-water and special nanoparticle forms, and the traditional aluminum adjuvant is still more commonly used at present, and the research system thereof is incomplete. An excellent adjuvant should meet the requirements of low cost, high yield, uniformity, stability and safety, and the existing marketed adjuvants are limited in raw materials and technology and have advantages and disadvantages, and the development of new research ideas has far-reaching research significance for the development of vaccine preparations.

[0003] In recent years, many new substances with adjuvant function have been identified. For example, it is found in existing research that traditional statins for reducing cholesterol have good adjuvant effect, but they can induce significant death of antigen presenting cells (APC cells), and the APC cells are one of the key intermediate cells for the body to exert anti-infection immune response, which may produce unpredictable serious adverse reactions after actual use, greatly limiting the application of statin adjuvants in vaccines.

[0004] The liposome delivery system is a common delivery system widely used for medical, chemical, cosmetic and other purposes. In the present application, various liposomes are used as the basic framework to deliver the core component ginsenoside Rh2 in the present application to resist drug-induced cell death. SUMMARY

[0005] In order to solve the technical problems mentioned in the background art, the purpose of the present application is to provide a new method for reducing drug-induced cell death.

[0006] The purpose of the present application can be achieved by the following technical solutions: Based on the problem of statin adjuvant-induced death of APC cells, a new preparation, i.e. liposome-encapsulated ginsenoside Rh2 (Liposome-Rh2, hereinafter referred to as LP-Rh2), is developed through a large amount of high-throughput screening work. It can inhibit cell death and improve cell viability in various scenarios, so as to better cooperate with the mechanism of other candidate drugs with cytotoxicity in scientific research, and reduce the potential safety risks of drugs, especially vaccine adjuvants, in drug application. In the research, various liposome basic framework schemes are adopted, and the same effect of resisting drug-induced cell death can be achieved.

[0007] Take unsaturated phospholipid DOPC as an example, the LP-Rh2 is provided with DOPC (dioleoylphosphatidylcholine), CHOL (cholesterol) and DSPE-mPEG2000 (distearoylphosphatidyl ethanolamine-polyethylene glycol 2000) to form the skeleton structure of the liposome, and ginsenoside Rh2 is used as the functional component. The specific preparation method is as follows: Step S1, preparing the oil phase: DOPC, CHOL, DSPE-mPEG2000 and ginsenoside Rh2 are dissolved in ethanol respectively to form uniform dissolved solutions, and then the dissolved solutions are combined and stirred uniformly to prepare the oil phase.

[0008] Step S2, shearing emulsification: the oil phase is subjected to high-speed shearing at 6000-15000 rpm, and PBS buffer solution is rapidly injected, and the shearing emulsification is continuously performed for 5-10 min to form a uniform primary emulsion.

[0009] Step S3, purification homogenization: the primary emulsion is placed in a water bath and stirred at 70-100 rpm to distill ethanol until ethanol is precipitated, and then high-pressure homogenization treatment is performed, and finally a 0.22 μm filter is used for filtration to obtain the LP-Rh2 preparation.

[0010] Preferably, in the process of preparing the oil phase, the ethanol solution concentration of DOPC is 0.5 mg / mL, the ethanol solution concentration of CHOL is 0.25 mg / mL, the ethanol solution concentration of DSPE-mPEG2000 is 0.005 mg / mL, and the ethanol solution concentration of ginsenoside Rh2 is 0.1-3.0 mg / mL.

[0011] Preferably, the water bath temperature of the primary emulsion is 37-40°C.

[0012] Preferably, the high-pressure homogenization treatment is performed by step-by-step continuous treatment, and the total homogenization is 4 times, and the pressures are 10000 psi, 15000 psi, 20000 psi and 20000 psi respectively.

[0013] Further, the skeleton structure system can be HSPC (hydrogenated soybean lecithin), CHOL and DSPE-mPEG2000.

[0014] Further, the skeleton structure system can be DMPC (dimyristoylphosphatidylcholine), CHOL and DSPE-mPEG2000.

[0015] Further, the skeleton structure system can be DSPC (distearoylphosphatidylcholine), CHOL and DSPE-mPEG2000.

[0016] Further, the skeleton structure system can be DPPC (dipalmitoylphosphatidylcholine), DOPC, CHOL and DSPE-mPEG2000.

[0017] Advantages of the present application: Statins have been used in clinic for lowering cholesterol and other diseases for many years, and the safety data is sufficient. It is a promising and valuable new way to use "old drugs for new purposes" in vaccine adjuvants. However, in such studies, significant APC cytotoxicity has been found, and there is no related solution in the prior art, which will inevitably hinder its application in the adjuvant field. In the present application, the ginsenoside Rh2 is prepared into liposomes, which greatly relieves the induction of APC cell death. Mechanism studies show that it inhibits the apoptosis induced by statins, enhances cell activity, and enhances safety. In the cell death induced by the general apoptosis agent camptothecin, the present application LP-Rh2 also greatly relieves the induced apoptosis. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed for the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0019] Figure 1 The encapsulation structure characterization results of the LP-Rh2 preparation prepared for the present application embodiment 1; Figure 2 The test results of the LP-Rh2 preparation prepared for the present application embodiment 1 for reducing DC cell toxicity; Figure 3 The test results of the LP-Rh2 preparation prepared for the present application embodiment 1 for inhibiting statin-induced cell early apoptosis; Figure 4 The test results of the LP-Rh2 preparation prepared for the present application embodiment 1 for the influence on cell iron death, copper death and ammonia death; Figure 5 The test results of the LP-Rh2 preparation prepared for the present application embodiment 1 for the influence on cell autophagy and pyroptosis.

[0020] Figure 6 The characterization and anti-apoptosis test results of the LP-Rh2 preparation prepared for the present application embodiment 3 on cells; Figure 7 The characterization and anti-apoptosis test results of the LP-Rh2 preparation prepared for the present application embodiment 4 on cells; Figure 8 The anti-apoptosis test results of the LP-Rh2 preparation prepared for the present application embodiment 5 on cells; Figure 9The results of the anti-apoptotic test on cells are shown for the LP-Rh2 formulation prepared in Example 6 of this invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: LP-Rh2 was prepared using DOPC, CHOL, and DSPE-mPEG2000 as the liposome backbone structure. The specific implementation method is as follows: Step S1: Prepare the oil phase: Dissolve DOPC in ethanol to prepare a solution of 0.5 mg / mL, dissolve CHOL in ethanol to prepare a solution of 0.25 mg / mL, dissolve DSPE-mPEG2000 in ethanol to prepare a solution of 0.005 mg / mL, and dissolve ginsenoside Rh2 in ethanol to prepare a solution of 1.0 mg / mL; mix the solutions in equal volumes and stir well to prepare the oil phase.

[0023] Step S2, shearing emulsification: The oil phase is sheared at 10,000 rpm, and 10 times the volume of PBS buffer is rapidly injected. Shearing emulsification is continued for 8 minutes to form a uniform primary emulsion.

[0024] Step S3: Purification and Homogenization: The primary emulsion was placed in a water bath at 37°C and stirred at 70 rpm until no ethanol precipitation occurred. Then, it was transferred to a high-pressure homogenizer and homogenized continuously in stages for a total of 4 times at pressures of 10,000 psi, 15,000 psi, 20,000 psi, and 20,000 psi. After homogenization, the mixture was filtered through a 0.22 μm filter to obtain the LP-Rh2 formulation.

[0025] like Figure 1 The image shows the characterization results of the encapsulation structure of the LP-Rh2 formulation.

[0026] like Figure 2 The image shows the test results of the LP-Rh2 formulation prepared in Example 3 on reducing DC cell toxicity. Because statins are not soluble in water, the existing technology evaluation of its cytotoxicity scheme using DMSO dissolution system, in the actual test, the mortality rate of DC2.4 cells (a kind of APC cells) is as high as about 90%, therefore, the test uses the method of liposome delivery to wrap simvastatin (Simvastatin) and mevastatin (Mevastatin) into the corresponding liposome preparation form (LP-Simvastatin and LP-Mevastatin), and the liposome-encapsulated statins are added to DC2.4 cells for co-culture with LP-Rh2 at the same time, and the specific combination Figure 2 The data shows that LP-Rh2 can reverse statin-induced cell death under various conditions (with serum culture system and without serum culture system).

[0027] As Figure 3 shown, the test results of LP-Rh2 prepared in Example 3 to inhibit statin-induced cell early apoptosis. Specifically, LP-Simvastatin is used as a statin reagent, and JC-1 staining is used to detect cell status, and the combination of detection data shows that the addition of LP-Rh2 significantly increases the cell viability, and the mechanism of action is to inhibit drug-induced cell early apoptosis.

[0028] As Figure 4 and Figure 5 shown, in order to further explore other mechanisms of LP-Rh2 reversing cell death, we studied other cell death modes such as pyroptosis, autophagy, ferroptosis, cuproptosis and ammonium death, and the results showed that these death modes were not the main reason for LP-Rh2 reversing cell death and increasing cell viability.

[0029] Example 2, still using DOPC, CHOL and DSPE-mPEG2000 as the liposome skeleton structure, adjusting the loading amount of Rh2 for scaling test, the specific differences are shown in the following table: Rh2 content Emulsification parameter Rotavapor parameter Scaling test group 1 0.1 mg / mL 6000 rpm & 10 min 70 rpm & 37 °C Scaling test group 2 0.5 mg / mL 6000 rpm & 10 min 70 rpm & 37 °C Scaling test group 3 1.2 mg / mL 10000 rpm & 5 min 80 rpm & 37 °C Scaling test group 4 1.3 mg / mL 12000 rpm & 5 min 100 rpm & 37 °C Scaling test group 5 3.0 mg / mL 12000 rpm & 7 min 100 rpm & 40 °C Scaling test group 6 4.0 mg / mL 15000 rpm & 10 min 100 rpm & 40 °C Scaling test group 7 5.0 mg / mL 15000 rpm & 10 min 100 rpm & 40 °C In the scaling test as above, the addition of LP-Rh2 increases the cell viability, especially in the Rh2 content of 0.1-1.3 mg / mL, which shows a significant effect.

[0030] Example 3, using DMPC, CHOL and DSPE-mPEG2000 as the liposome skeleton structure, preparing LP-Rh2, the specific implementation method is the same, and the specific process difference is: DMPC: 1.0 mg / mL, CHOL: 0.25 mg / mL, DSPE-mPEG2000: 0.005 mg / mL; The water bath rotary evaporation conditions of the primary emulsion are 100 rpm & 60℃, and the rest of the implementation process is exactly the same as Example 1. As Figure 6Figure 2 shows the characterization of the LP-Rh2 formulation prepared in Example 3 and the results of the anti-apoptosis test on cells.

[0031] Example 4. LP-Rh2 was prepared with HSPC, CHOL and DSPE-mPEG2000 as the liposome skeleton structure, the specific implementation method was the same, and the specific process difference was: HSPC: 1.0 mg / mL, CHOL: 0.25 mg / mL, DSPE-mPEG2000: 0.005 mg / mL; the water bath rotary evaporation condition of the primary emulsion was 100 rpm & 60°C, and the rest of the implementation process was completely the same as Example 1. As shown in Figure 3, the characterization of the LP-Rh2 formulation prepared in Example 4 and the results of the anti-apoptosis test on cells. Figure 7

[0032] Example 5. LP-Rh2 was prepared with DSPC, CHOL and DSPE-mPEG2000 as the liposome skeleton structure, the specific implementation method was the same, and the specific process difference was: DSPC: 1.0 mg / mL, CHOL: 0.25 mg / mL, DSPE-mPEG2000: 0.005 mg / mL; the water bath rotary evaporation condition of the primary emulsion was 100 rpm & 60°C, and the rest of the implementation process was completely the same as Example 1. As shown in Figure 4, the anti-apoptosis test results of the LP-Rh2 formulation prepared in Example 5 on cells. Figure 8

[0033] Example 6. LP-Rh2 was prepared with DOPC, DPPC, CHOL and DSPE-mPEG2000 as the liposome skeleton structure, the specific implementation method was the same, and the specific process difference was: DOPC: 0.5 mg / mL, DPPC: 0.2 mg / mL, CHOL: 0.25 mg / mL, DSPE-mPEG2000: 0.005 mg / mL; the water bath rotary evaporation condition of the primary emulsion was 100 rpm & 60°C, and the rest of the implementation process was completely the same as Example 1. As shown in Figure 5, the anti-apoptosis test results of the LP-Rh2 formulation prepared in Example 6 on cells. Figure 9

[0034] During the test, camptothecin, which is generally recognized to induce cell apoptosis, was used for the experiment, and LP-Rh2 could significantly improve the cell survival rate in camptothecin, showing excellent effect of reducing drug-induced cell death.

[0035] ​​​In the description, references to "one embodiment," "an example," "certain examples" etc. mean that the particular feature, structure, material, or characteristic being described is included in at least one embodiment or example of the application. The appearances of an item in various examples or embodiments is not necessarily indicative of a frequency of occurrence in the various examples or embodiments. Moreover, descriptions of well-known methods associated are omitted so as not to obscure the disclosure.

[0036] The foregoing merely illustrates the principles of the application. Various modifications and adaptations will occur to those skilled in the art after consideration of the preceding description. All such modifications and adaptations employing the principles of the application are intended to be within the scope of the claims.

Claims

1. A novel method of reducing drug-induced cell death, characterized in that, The liposome provides a skeleton structure, and ginsenoside Rh2 is a functional component. When LP-Rh2 in the form of liposome is used together with other drugs, it can inhibit drug-induced cytotoxicity in various scenarios.

2. A novel method of reducing drug-induced cell death according to claim 1, characterized in that, The preparation method of LP-Rh2 is as follows: Step S1: Dissolve each liposome component and ginsenoside Rh2 in ethanol respectively, then mix the dissolved solutions to prepare an oil phase; Step S2: High-speed shear the oil phase and inject it into PBS buffer, continuously shear and emulsify to prepare a primary emulsion; Step S3: Place the primary emulsion in a water bath, stir and rotary evaporate until ethanol is precipitated, then perform high-pressure homogenization treatment, and finally filter with a 0.22 μm filter to obtain the LP-Rh2 preparation.

3. A novel method of reducing drug-induced cell death according to claim 1, wherein, The liposome skeleton structure system is DOPC, CHOL and DSPE-mPEG2000.

4. The novel method of reducing drug-induced cell death according to claim 1, wherein, The liposome skeleton structure system is HSPC, CHOL and DSPE-mPEG2000.

5. A novel method of reducing drug-induced cell death according to claim 1, wherein, The liposome skeleton structure system is DMPC, CHOL and DSPE-mPEG2000.

6. A novel method of reducing drug-induced cell death according to claim 1, wherein, The liposome skeleton structure system is DSPC, CHOL and DSPE-mPEG2000.

7. A novel method of reducing drug-induced cell death according to claim 1, wherein, The liposome skeleton structure system is DPPC, DOPC, CHOL and DSPE-mPEG2000.

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