Preparation method of nanoparticles of controlled-release gas signal molecule H2S

Abstract

The invention discloses a preparation method of nano-particles of controlled-release gas signal molecule H2S, and relates to the technical field of nano-medicine, and the preparation method comprises the following steps: S1, preparing a bovine serum albumin solution with a preset volume and a preset concentration; s2, preparing a glutaraldehyde solution with a preset volume and a preset concentration; s3, under a preset temperature condition, adding GYY4137 with a preset mass into the solution in the step S1 which is continuously stirred; and S4, under a preset temperature condition, dropwise adding a preset volume of absolute ethyl alcohol into the continuously stirred solution obtained after the operation in the step S3 at a preset rate. According to the invention, a plurality of drug loading advantages of serum albumin are reasonably utilized, the nano-particles of the controlled-release gas signal molecule H2S with high drug loading capacity are synthesized, and a possibility is provided for research and development of a carrier material of an H2S donor.

Description

Technical Field

[0001] The invention relates to the technical field of nanomedicine, and in particular to a method for preparing nanoparticles of controlled-release gas signal molecule H2S. Background Art

[0002] H2S is a colorless, toxic gas with a rotten egg smell and is one of the most toxic substances in nature. However, in 1989, scientists detected endogenous H2S in rats and human brains and confirmed that it may have a preset physiological effect. Over the past 30 years, a large number of studies have confirmed that H2S has characteristics similar to nitric oxide (NO) and carbon monoxide (CO), and participates in signal transmission in multiple systems. Therefore, it is considered to be the third gas signal molecule in the body besides NO and CO.

[0003] Gaseous signaling molecules refer to gas molecules that can send out "signals" and cause signal transduction in biological cells, thereby producing biological effects. Endogenous H2S is mainly produced by sulfur-containing amino acid metabolism, and participates in many physiological functions such as brain development, blood pressure regulation, and inflammatory response by freely diffusing outside the cell. Under normal circumstances, the level of H2S in the body is produced by specific enzymes and maintained within a preset range. Various factors lead to the downregulation of the expression and / or activity of the enzyme, which will cause significant changes in the level of H2S in tissues and organs and induce the occurrence and development of diseases. Therefore, H2S has become a potential drug for the treatment of many diseases. However, H2S with a concentration of less than 300 μM is non-toxic and biologically active, and H2S has a short residence time in biological cells. Therefore, if it is administered in the way of conventional drugs, it usually has little effect. A common strategy is to deliver H2S donors to the affected area to control the release of H2S in situ to achieve a therapeutic effect. The main donors of exogenous H2S are sodium hydrosulfide (NaHS), sodium sulfide (Na2S) and sulfur-containing organic compounds. NaHS and Na2S will release a large amount of H2S in a very short time after contacting water; among sulfur-containing organic compounds, the new water-soluble H2S donor GYY4137 synthesized by Professor Moore in 2008 can continuously release H2S for a long time. A common disadvantage of these donors is the uncontrollable release of H2S. Therefore, the development of carrier materials for H2S donors has become a technical problem that needs to be solved urgently.

[0004] BSA nanoparticles are self-assembled from BSA. There are multiple drug loading sites inside the particles, which can be loaded with water-soluble drugs and fat-soluble hydrophobic drugs. They are widely used drug carriers in the field of controlled release therapy of cancer drugs. The degradation rate of BSA nanoparticles can be adjusted by their cross-linking degree, which provides a simple and feasible method for regulating the release rate of H2S.

[0005] Based on this, the present invention adopts desolvation technology to prepare BSA nanoparticles as carriers of H2S donor GYY4137. By embedding GYY4137 into the hydrophilic drug loading site of BSA, the possibility of GYY4137 contacting with water during the administration process is isolated, and the stable delivery of GYY4137 to the patient is achieved, thereby achieving the purpose of controllable release of H2S at the patient.

[0006] The above information disclosed in this Background section is only for enhancement of understanding of the background of the present disclosure and therefore it may contain information that does not form the prior art that is already known to one of ordinary skill in the art. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing nanoparticles of the controlled-release gas signal molecule H2S. The present invention rationally utilizes the many drug-loading advantages of serum albumin and synthesizes nanoparticles of the controlled-release gas signal molecule H2S with a high drug loading amount, which will provide a possibility for the research and development of carrier materials for H2S donors to solve the problems in the above-mentioned background technology.

[0008] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing nanoparticles of controlled-release gas signal molecule H2S, characterized in that it comprises the following steps: S1, preparing a BSA solution of a preset volume and a preset concentration; S2, preparing a glutaraldehyde solution of a preset volume and a preset concentration; S3, adding a preset mass of GYY4137 to the continuously stirred S1 solution under the preset temperature condition; S4, under a preset temperature condition, adding a preset volume of anhydrous ethanol dropwise at a preset rate to the solution obtained after the continuous stirring in step S3; S5, under a preset temperature condition, adding a preset volume of S2 solution to the solution obtained after the continuously stirred S4 step, and continuing stirring for a preset time; S6, centrifuging the solution obtained after the operation in step S5 at a preset temperature and a preset rate for a preset time to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol for a preset number of times under a preset temperature condition, and then washing the nanoparticles with a NaCl solution of a preset concentration and a preset pH value for a preset number of times; S8, dispersing the nanoparticles obtained after the operation of steps S1-S7 in a mannitol solution of a preset concentration and storing the solution under a preset temperature.

[0009] As a possible implementation manner, further, the solution solvent of step S1 is deionized water, the solution volume of step S1 is > 0 mL, and the solution concentration of step S1 is > 0 mg / mL.

[0010] As a possible implementation, further, in step S1, BSA can be replaced by any other serum albumin.

[0011] As a possible implementation manner, further, the solution solvent of step S2 is deionized water, the solution volume of step S2 is > 0 mL, and the solution concentration of step S2 is > 0%.

[0012] As a possible implementation manner, further, the condition temperature of step S3 is > 0 °C, and the mass of GYY4137 in step S3 is > 0 mg.

[0013] As a possible implementation manner, further, in step S3, the final concentration of GYY4137 in the BSA solution is 0 to 19.23 mg / mL.

[0014] As a possible implementation manner, further, the condition temperature of step S4 is > 0 °C, the volume of anhydrous ethanol in step S4 is > 0 mL, and the dropping rate of anhydrous ethanol in step S4 is > 0 mL / min.

[0015] As a possible implementation manner, further, the condition temperature of step S5 is > 0 °C, the volume of the solution in step S5 is > 0 μL, and the continuous stirring time in step S5 is 0 to 48 h.

[0016] As a possible implementation manner, further, the condition temperature of step S6 is > 0 °C, the centrifugation rate in step S6 is > 0 rpm, and the centrifugation time in step S6 is > 0 min.

[0017] As a possible implementation manner, further, the condition temperature of step S7 is > 0 °C, the number of times the nanoparticles are washed with anhydrous ethanol in step S7 is > 0 times, the concentration of the NaCl solution in step S7 is > 0 mM, the pH value of the NaCl solution in step S7 is 7.0-14.0, and the number of times the nanoparticles are washed with NaCl solution in step S7 is > 0 times.

[0018] As a possible implementation manner, further, the concentration of the mannitol solution in step S8 is > 0%, and the storage temperature in step S8 is > 0 °C.

[0019] In the above technical solution, the technical effects and advantages provided by the present invention are: The present invention rationally utilizes the many drug-carrying advantages of serum albumin and synthesizes nanoparticles of the controlled-release gas signal molecule H2S with a high drug-carrying amount, which will provide a possibility for the research and development of carrier materials for H2S donors.

[0020] The nanoparticles of the controlled-release gas signal molecule H2S obtained in the present invention are convenient to store and transport, have excellent comprehensive performance, have good clinical application prospects, and have important research value and clinical significance in the field of new nano-drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0022] Figure 1 The present invention is a flow chart of a method for preparing nanoparticles of the controlled-release gas signal molecule H2S.

[0023] Figure 2 The effect of cross-linking degree on the morphology (a), average particle size (b), polydispersity coefficient (c) and surface Zeta potential (d) of BSA nanoparticles loaded with GYY4137, where *p < 0.05, **p < 0.01.

[0024] Figure 3 This is the main element distribution diagram of BSA nanoparticles loaded with GYY4137 (cross-linking degree is 200%).

[0025] Figure 4 The effect of cross-linking degree on the release of H2S from GYY4137-loaded BSA nanoparticles in PBS environment (pH = 7.4).

[0026] Figure 5 Schematic diagram of the molecular mechanism of GYY4137 achieving controlled release of H2S through BSA nanoparticles. DETAILED DESCRIPTION

[0027] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in a variety of forms and should not be construed as limited to the examples set forth herein; rather, these example embodiments are provided so that the description of the present disclosure will be more comprehensive and complete, and the concept of the example embodiments will be fully conveyed to those skilled in the art.

[0028] The present invention provides Figures 1 to 5 A method for preparing nanoparticles of a controlled-release gas signal molecule H2S is shown, comprising the following steps: S1, preparing a bovine serum albumin solution of a preset volume and a preset concentration; S2, preparing a glutaraldehyde solution of a preset volume and a preset concentration; S3, adding a preset mass of GYY4137 to the continuously stirred S1 solution under the preset temperature condition; S4, under a preset temperature condition, adding a preset volume of anhydrous ethanol dropwise at a preset rate to the solution obtained after the continuous stirring in step S3; S5, under a preset temperature condition, adding a preset volume of S2 solution to the solution obtained after the continuously stirred S4 step, and continuing stirring for a preset time; S6, centrifuging the solution obtained after the operation in step S5 at a preset temperature and a preset rate for a preset time to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol for a preset number of times under a preset temperature condition, and then washing the nanoparticles with a NaCl solution of a preset concentration and a preset pH value for a preset number of times; S8, dispersing the nanoparticles obtained after the operation of steps S1-S7 in a mannitol solution of a preset concentration and storing the solution under a preset temperature.

[0029] Example 1: A method for preparing BSA nanoparticles loaded with GYY4137 having a cross-linking degree of 50%, characterized by comprising the following steps: S1, prepare 1 mL of 10 mg / mL BSA in deionized water; S2, prepare 1 mL of 8% glutaraldehyde solution in deionized water; S3, 1.0 mg of GYY4137 was added to the stirred S1 solution at 25 °C; S4, at 25 °C, 4 mL of anhydrous ethanol was added dropwise at a rate of 1 mL / min to the solution obtained after the continuous stirring in step S3; S5, at 25 °C, add 2.9 μL of S2 solution to the solution obtained after the S4 step operation with continuous stirring, and continue stirring for 24 h; S6, centrifuging the solution obtained after the operation in step S5 at 25 °C for 10 min at a rate of 25 °C to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol once at 25°C, and then washing the nanoparticles with a 10 mM NaCl solution with a pH value of 9.0 twice; S8, dispersing the nanoparticles obtained after the operation in step S7 in a 5% mannitol solution and storing them at 4 °C.

[0030] Example 2: A method for preparing BSA nanoparticles loaded with GYY4137 having a cross-linking degree of 100%, characterized by comprising the following steps: S1, prepare 1 mL of 10 mg / mL BSA in deionized water; S2, prepare 1 mL of 8% glutaraldehyde solution in deionized water; S3, 1.0 mg of GYY4137 was added to the stirred S1 solution at 25 °C; S4, at 25 °C, 4 mL of anhydrous ethanol was added dropwise at a rate of 1 mL / min to the solution obtained after the continuous stirring in step S3; S5, at 25 °C, add 5.9 μL of S2 solution to the solution obtained after the S4 step operation with continuous stirring, and continue stirring for 24 h; S6, centrifuging the solution obtained after the operation in step S5 at 25 °C for 10 min at a rate of 25 °C to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol once at 25°C, and then washing the nanoparticles with a 10 mM NaCl solution with a pH value of 9.0 twice; S8, dispersing the nanoparticles obtained after the operation in step S7 in a 5% mannitol solution and storing them at 4 °C.

[0031] Example 3: A method for preparing BSA nanoparticles loaded with GYY4137 having a cross-linking degree of 150%, characterized by comprising the following steps: S1, prepare 1 mL of 10 mg / mL BSA in deionized water; S2, prepare 1 mL of 8% glutaraldehyde solution in deionized water; S3, 1.0 mg of GYY4137 was added to the stirred S1 solution at 25 °C; S4, at 25 °C, 4 mL of anhydrous ethanol was added dropwise at a rate of 1 mL / min to the solution obtained after the continuous stirring in step S3; S5, at 25 °C, add 8.8 μL of S2 solution to the solution obtained after the S4 step operation with continuous stirring, and continue stirring for 24 h; S6, centrifuging the solution obtained after the operation in step S5 at 25 °C for 10 min at a rate of 25 °C to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol once at 25°C, and then washing the nanoparticles with a 10 mM NaCl solution with a pH value of 9.0 twice; S8, dispersing the nanoparticles obtained after the operation in step S7 in a 5% mannitol solution and storing them at 4 °C.

[0032] Example 4: A method for preparing BSA nanoparticles loaded with GYY4137 having a cross-linking degree of 200%, characterized by comprising the following steps: S1, prepare 1 mL of 10 mg / mL BSA in deionized water; S2, prepare 1 mL of 8% glutaraldehyde solution in deionized water; S3, 1.0 mg of GYY4137 was added to the stirred S1 solution at 25 °C; S4, at 25 °C, 4 mL of anhydrous ethanol was added dropwise at a rate of 1 mL / min to the solution obtained after the continuous stirring in step S3; S5, at 25 °C, add 11.8 μL of S2 solution to the solution obtained after the S4 step operation with continuous stirring, and continue stirring for 24 h; S6, centrifuging the solution obtained after the operation in step S5 at 25 °C for 10 min at a rate of 25 °C to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol once at 25°C, and then washing the nanoparticles with a 10 mM NaCl solution with a pH value of 9.0 twice; S8, dispersing the nanoparticles obtained after the operation in step S7 in a 5% mannitol solution and storing them at 4 °C.

[0033] Experimental Example 1: The nanoparticles obtained in Examples 1 to 4 were observed using a field emission scanning electron microscope and a Malvern nanoparticle size analyzer. It was found that the degree of crosslinking had no significant effect on the morphology, average particle size and polydispersity coefficient of the BSA nanoparticles loaded with GYY4137, while the absolute value of the Zeta potential on the surface of the nanoparticles increased with the increase of the degree of crosslinking ( Figure 1 ).

[0034] Experimental Example 2: The nanoparticles obtained in Example 4 were observed using a field emission transmission electron microscope. It can be seen that all elements are evenly distributed in the nanoparticles. The signals of C, N, O, and S elements are strong, mainly from bovine serum albumin. P element is a characteristic element of GYY4137. Although the signal is weak, these signals in the nanoparticle area (within the white dotted circle) are significantly stronger than those outside the nanoparticle area (outside the white dotted circle), which shows that GYY4137 has been successfully encapsulated in bovine serum albumin nanoparticles.

[0035] Experimental Example 3: By observing the H2S release behavior (release rate and cumulative release amount) of the nanoparticles obtained in Examples 1 to 4 under a physiological environment (PBS solution with pH = 7.4), it can be seen that the cross-linking degree only affects the release rate of H2S, but not the cumulative release amount of H2S. Therefore, the release rate of H2S can be adjusted by regulating the cross-linking degree of the BSA nanoparticles loaded with GYY4137 ( Figure 3 ).

[0036] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing nanoparticles of controlled-release gas signal molecule H2S, characterized in that: The following steps are involved: S1, preparing a bovine serum albumin (BSA) solution of a preset volume and a preset concentration; S2, preparing a glutaraldehyde solution of a preset volume and a preset concentration; S3, adding a preset mass of GYY4137 to the continuously stirred S1 solution under the preset temperature condition; S4, under a preset temperature condition, adding a preset volume of anhydrous ethanol dropwise at a preset rate to the solution obtained after the continuous stirring in step S3; S5, under a preset temperature condition, adding a preset volume of S2 solution to the solution obtained after the continuously stirred S4 step, and continuing stirring for a preset time; S6, centrifuging the solution obtained after the operation in step S5 at a preset temperature and a preset rate for a preset time to collect the nanoparticles; S7, washing the nanoparticles obtained after the operation in step S6 with anhydrous ethanol for a preset number of times under a preset temperature condition, and then washing the nanoparticles with a NaCl solution of a preset concentration and a preset pH value for a preset number of times; S8, dispersing the nanoparticles obtained after the operation of steps S1-S7 in a mannitol solution of a preset concentration and storing the solution under a preset temperature.

2. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The solution solvent of step S1 is deionized water, the solution volume of step S1 is > 0 mL, and the solution concentration of step S1 is > 0 mg / mL.

3. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: In step S1, BSA can be replaced by any serum albumin.

4. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The solution solvent of step S2 is deionized water, the solution volume of step S2 is > 0 mL, and the solution concentration of step S2 is > 0%.

5. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The condition temperature of step S3 is > 0 °C, and the mass of GYY4137 in step S3 is > 0 mg.

6. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The final concentration of GYY4137 in the BSA solution in step S3 is 0 to 19.23 mg / mL.

7. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The condition temperature of step S4 is > 0 °C, the volume of anhydrous ethanol in step S4 is > 0 mL, and the dropping rate of anhydrous ethanol in step S4 is > 0 mL / min.

8. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The condition temperature of step S5 is > 0 °C, the volume of the solution in step S5 is > 0 μL, and the continuous stirring time in step S5 is 0 to 48 h.

9. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The condition temperature of step S6 is > 0 °C, the centrifugation rate in step S6 is > 0 rpm, and the centrifugation time in step S6 is > 0 min.

10. The method for preparing nanoparticles of a controlled-release gas signal molecule H2S according to claim 1, characterized in that: The condition temperature of step S7 is > 0 °C, the number of times the nanoparticles are washed with anhydrous ethanol in step S7 is > 0 times, the concentration of the NaCl solution in step S7 is > 0 mM, the pH value of the NaCl solution in step S7 is 7.0-14.0, and the number of times the nanoparticles are washed with the NaCl solution in step S7 is > 0 times; The concentration of the mannitol solution in step S8 is > 0%, and the storage temperature in step S8 is > 0 °C.

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