Probiotic-palladium hydride biological hydrogen storage system as well as preparation method and application thereof

Abstract

The invention discloses a probiotic-palladium hydride biological hydrogen storage system as well as a preparation method and application thereof. The preparation method comprises the following steps: co-incubating probiotics and bivalent palladium salt, adding or not adding organic sodium salt to continue incubation, introducing hydrogen into the obtained incubation liquid, and reacting to obtain the probiotic-palladium hydride biological hydrogen storage system. The technical problem that due to the special properties of small molecular size, non-polarity, low solubility, easy diffusion and the like of hydrogen, targeted transportation and controllable release of hydrogen are difficult to achieve through a traditional hydrogen treatment means at present is solved, synthesis of palladium nano-enzyme on the surfaces of probiotics and loading of hydrogen are achieved through a one-step method, the palladium nano-enzyme serves as a hydrogen carrier, and the preparation method is simple and convenient. Under the combined action of the probiotics, directional transportation of the hydrogen is realized, so that the functions of resisting inflammation, resisting oxidative stress and regulating intestinal flora of the hydrogen are realized in intestinal tracts.

Description

Technical Field

[0001] The present invention relates to the technical field of biological hydrogen storage, and in particular to a probiotics-palladium hydride biological hydrogen storage system and a preparation method and application thereof. Background Art

[0002] In recent years, the application of hydrogen in the biomedical field has attracted widespread attention due to its unique biological effects. A large number of studies have shown that molecular hydrogen has the ability to selectively remove cytotoxic oxygen free radicals (such as hydroxyl radicals ·OH) without affecting the characteristics of physiological reactive oxygen species (ROS) involved in normal cell signal transduction, thus showing good anti-inflammatory, anti-oxidative stress and intestinal flora regulation effects. These characteristics make hydrogen have great potential in biomedical applications. However, due to the special properties of hydrogen such as small molecular size, non-polarity, low solubility and easy diffusion, traditional hydrogen therapy methods are difficult to achieve precise targeted transport and controllable release of hydrogen. Therefore, the development of a stable hydrogen storage system is of great significance for the biomedical application of hydrogen.

[0003] At present, researchers have tried to use carrier materials such as mesoporous silica to load hydrogen-releasing prodrug ammonia borane, but the connection between ammonia borane and the carrier is mainly achieved through hydrogen bonding, which may lead to a sudden release of the drug during delivery, thereby affecting the therapeutic effect. In addition, the high hydrogen storage capacity of palladium (Pd) can also be used to prepare PdH nanoparticles to transport and release active hydrogen. Nanoscale palladium not only has excellent hydrogen storage capacity, but also has high enzyme-like activity, which can remove OH from the lesion site. However, although palladium hydride can stably store hydrogen and release it continuously, it is difficult to accumulate in the target site, which will reduce its therapeutic effect to a certain extent. In order to solve the accumulation and targeting problems of nanozymes, some researchers have tried to combine nanozymes with biologically active bacteria. For example, iron nanozymes are first prepared and then connected to the surface of Bifidobacterium longum by covalent modification to enhance the colonization and accumulation of nanozymes in the intestine. However, this method not only increases the production cost and process complexity, but also may introduce potential biosafety issues due to the use of linkers, thereby affecting the stability and therapeutic effect of probiotics.

[0004] Therefore, developing a more efficient, simple and safe hydrogen loading and transportation system to overcome the limitations of existing technologies has become an urgent need in current research. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a probiotic-palladium hydride biological hydrogen storage system and its preparation method and application. Under the action of the probiotics themselves, a probiotic-palladium hydride biological hydrogen storage system with good enzyme-like activity is synthesized in one step, which not only realizes the loading and targeted transport of hydrogen, but also protects bacteria against the complex gastrointestinal environment and the inflammatory environment of enteritis.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for preparing a probiotic-palladium hydride biological hydrogen storage system, comprising the following steps:

[0008] The probiotics are co-incubated with a divalent palladium salt, an organic sodium salt is added for further incubation, hydrogen is introduced into the obtained incubation solution A, and the probiotics-palladium hydride biological hydrogen storage system is obtained by reaction; the molar ratio of the divalent palladium salt to the organic sodium salt is (1-5):(1-3); or

[0009] The probiotics are co-incubated with a divalent palladium salt, and hydrogen is introduced into the obtained incubation solution B to react to obtain the probiotics-palladium hydride biological hydrogen storage system.

[0010] The present invention solves the technical problem that due to the special properties of hydrogen such as small molecular size, non-polarity, low solubility and easy diffusion, traditional hydrogen treatment methods are difficult to achieve targeted transport and controllable release of hydrogen. The synthesis of palladium nanoenzymes (palladium nanoparticles) on the surface of probiotics and the loading of hydrogen are achieved through a one-step method, and a probiotic-palladium hydride biological hydrogen storage system is prepared with a hydrogen sustained-release function. On the one hand, hydrogen loading and targeted transport can be achieved, and on the other hand, palladium has antioxidant enzyme activity and can be used as a nano armor to protect bacteria from complex gastrointestinal environments and the inflammatory environment of enteritis.

[0011] Furthermore, the probiotics are selected from one or more of Escherichia coli Nissle1917 (EcN), Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium longum, Lactobacillus bulgaricus and Lactobacillus rhamnosus.

[0012] Furthermore, the probiotics are co-incubated with the divalent palladium salt for 30-50 minutes.

[0013] Furthermore, the organic sodium salt is one or more of sodium formate (HCOONa), sodium lactate and sodium acetate.

[0014] Furthermore, the organic sodium salt is added and the incubation time is continued for 7-9 hours.

[0015] Furthermore, the concentration of the organic sodium salt in the incubation solution A is 0.5-3 mM.

[0016] Furthermore, the concentration of the divalent palladium salt in the incubation solution B is 1-5 mM.

[0017] Furthermore, the divalent palladium salt is selected from one or more of sodium chloropalladate (Na2PdCl4), palladium chloride, palladium acetate, palladium iodide and palladium carbonate.

[0018] Furthermore, the time for introducing hydrogen is 5-10 min.

[0019] Furthermore, a liquid bacterial agent of probiotics is co-incubated with a divalent palladium salt, and the absorbance value (OD 600 ) is 0.8-1.

[0020] Furthermore, the liquid bacterial agent of the probiotics is washed multiple times with physiological saline and resuspended.

[0021] The present invention protects the probiotics-palladium hydride biological hydrogen storage system prepared by the above method.

[0022] The present invention also protects the use of the probiotic-palladium hydride biological hydrogen storage system in the preparation of drugs for preventing or treating radiation intestinal damage, anti-inflammatory drugs, anti-oxidative stress drugs or drugs for regulating intestinal flora.

[0023] Beneficial effects of the present invention:

[0024] The present invention solves the current complex process of synthesizing the nanozyme and then connecting it to the surface of the probiotics through a linker, as well as the current relatively single mode of action. The loading of palladium nanozymes on the surface of the probiotics can be achieved in one step, and hydrogen loading can be achieved on the basis of the probiotics loaded with nanozymes. The palladium nanozyme serves as a hydrogen carrier to achieve directional transport of hydrogen, so as to achieve the anti-inflammatory, anti-oxidative stress and intestinal flora regulation functions of hydrogen in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the process for preparing EcN@PdH NPs using the sodium formate method.

[0026] Figure 2 Schematic diagram of the process for preparing EcN@PdH NPs by direct hydrogen introduction.

[0027] Figure 3 TEM images of Escherichia coli Nissle1917 (EcN) and EcN@PdH NPs prepared in Example 1.

[0028] Figure 4 This is the EDX palladium element mapping result of EcN@PdH NPs prepared in Example 1.

[0029] Figure 5 This is a graph showing the hydrogen loading test results of the EcN@PdH NPs prepared in Example 1 and the EcN@Pd NPs prepared in Comparative Example 2.

[0030] Figure 6 The graph shows the test results of the antioxidant enzyme activity (SOD-like activity, scavenging ·OH) of Escherichia coli Nissle1917 (EcN), EcN@Pd NPs prepared in Comparative Example 2, and EcN@PdH NPs prepared in Example 1.

[0031] Figure 7 The stability test results of Escherichia coli Nissle1917 (EcN) and EcN@PdH NPs prepared in Example 1 in gastrointestinal fluid; wherein, (a) is a data graph of the bacterial activity of EcN and EcN@PdH NPs, and (b) is a data graph of the superoxide anion scavenging ability of EcN@PdH NPs after treatment with PBS, SGF, and SIF, respectively.

[0032] Figure 8 The figures are the protection test results of Escherichia coli Nissle1917 (EcN) and EcN@PdH NPs prepared in Example 2 against radiation intestinal damage; wherein, (a) is a graph showing the changes in mouse body weight, (b) is a data graph showing spleen index, and (c) is a graph showing blood white blood cell count. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0034] The present invention provides a method for preparing a probiotic-palladium hydride biological hydrogen storage system, comprising the following steps:

[0035] The probiotics are co-incubated with a divalent palladium salt, an organic sodium salt is added and the incubation is continued, hydrogen is introduced into the obtained incubation solution A, and the probiotics-palladium hydride biological hydrogen storage system is obtained by reaction; the molar ratio of the divalent palladium salt to the organic sodium salt is (1-5):(1-3).

[0036] The preparation method co-incubates probiotics with divalent palladium ions, then adds organic sodium salt as an electron donor, and under the action of the probiotics' own enzymes, the adsorbed divalent palladium ions are reduced to zero-valent palladium nanoparticles, and then hydrogen is passed through the system to achieve hydrogen loading.

[0037] The present invention also provides a method for preparing a probiotic-palladium hydride biological hydrogen storage system, comprising the following steps:

[0038] The probiotics are co-incubated with a divalent palladium salt, and hydrogen is introduced into the obtained incubation solution B to react to obtain the probiotics-palladium hydride biological hydrogen storage system.

[0039] The preparation method optimizes the method of using organic sodium salt to prepare a probiotic-palladium hydride biological hydrogen storage system with a sustained-release function. Hydrogen is used as an electron donor to form a palladium nanozyme load on the surface of the probiotic and at the same time the palladium nanozyme adsorbs hydrogen. The probiotic-palladium hydride biological hydrogen storage system with a hydrogen sustained-release function is synthesized in one step. The synthesis time of this method is greatly shortened.

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0042] Example 1

[0043] A method for preparing an Escherichia coli-palladium hydride biological hydrogen storage system (EcN@PdH NPs) comprises the following steps:

[0044] (1) E. coli Nissle1917 was cultured overnight and the volume was adjusted to OD 600 1, 1 mL Nissle1917 was washed twice with saline and resuspended, and the centrifugation parameters were set to 5000 rpm, 5 min;

[0045] (2) Take the resuspended Nissle1917, add 0.6 mL of physiological saline and 0.3 mL of Na2PdCl4 aqueous solution (10 mM), and incubate at 37°C in a bacterial shaker for 30 min. Add 0.1 mL of HCOONa aqueous solution (10 mM) to the system and incubate at 37°C in a bacterial shaker for 8 h. Then, introduce hydrogen for 5 min, wash twice with physiological saline and resuspend, and set the centrifugation parameters to 5000 rpm for 5 min to obtain EcN@PdH NPs.

[0046] Figure 1 Schematic diagram of the process for preparing EcN@PdH NPs using the sodium formate method.

[0047] Example 2

[0048] A method for preparing an Escherichia coli-palladium hydride biological hydrogen storage system (EcN@PdH NPs) comprises the following steps:

[0049] (1) E. coli Nissle1917 was cultured overnight and the volume was adjusted to OD 600 1, 1 mL Nissle1917 was washed twice with saline and resuspended, and the centrifugation parameters were set to 5000 rpm, 5 min;

[0050] (2) Take the resuspended Nissle1917, add 0.7 mL of saline and 0.3 mL of Na2PdCl4 aqueous solution (10 mM), and incubate at 37 °C in a bacterial shaker for 30 min. Then, introduce hydrogen for 5 min, wash twice with saline and resuspend. The centrifugation parameters were set to 5000 rpm for 5 min to obtain EcN@PdH NPs.

[0051] Figure 2 Schematic diagram of the process for preparing EcN@PdH NPs by direct hydrogen introduction.

[0052] Comparative Example 1

[0053] A method for preparing an Escherichia coli-palladium biological hydrogen storage system comprises the following steps:

[0054] (1) E. coli Nissle1917 was cultured overnight and the volume was adjusted to OD 600 1, 1 mL Nissle1917 was washed twice with saline and resuspended, and the centrifugation parameters were set to 5000 rpm, 5 min;

[0055] (2) Take the resuspended Nissle1917, add 0.6 mL of physiological saline and 0.3 mL of Na2PdCl4 (3 mM), and incubate at 37°C in a bacterial shaker for 30 min. Add 0.1 mL of HCOONa (10 mM) to the system and incubate at 37°C in a bacterial shaker for 8 h. Then, hydrogen gas is introduced for 5 min. No synthesis of biohydride palladium is observed, as indicated by a significant change in the color of the solution to black.

[0056] Comparative Example 2

[0057] A method for preparing an Escherichia coli-palladium biological hydrogen storage system (EcN@Pd NPs) comprises the following steps:

[0058] (1) E. coli Nissle1917 was cultured overnight and the volume was adjusted to OD 600 1, 1 mL Nissle1917 was washed twice with saline and resuspended, and the centrifugation parameters were set to 5000 rpm, 5 min;

[0059] (2) Take the resuspended Nissle1917, add 0.6 mL of normal saline and 0.3 mL of Na2PdCl4 (10 mM), and incubate at 37°C in a bacterial shaker for 30 min. Add 0.1 mL of HCOONa (10 mM) to the system and incubate at 37°C in a bacterial shaker for 8 h. Wash twice with normal saline and resuspend. Set the centrifugation parameters to 5000 rpm for 5 min to obtain EcN@Pd NPs.

[0060] Test Example 1

[0061] The morphology of Escherichia coli Nissle1917 (EcN) and the EcN@PdH NPs prepared in Example 1 were characterized by transmission electron microscopy (TEM). Figure 3 As shown, the TEM image shows that a large number of nano-sized granular substances appear on the surface of bacteria.

[0062] Energy dispersive X-ray spectrometer (EDX) was used to test the EcN@PdH NPs prepared in Example 1. The palladium element mapping results are as follows: Figure 4 As shown, these nanoparticles contain a large amount of palladium, further proving that palladium is successfully loaded on the surface of probiotics.

[0063] The methylene blue experiment was used to detect the hydrogen loading. The detection method was as follows: the release of reduced hydrogen by EcN@Pd NPs and EcN@PdH NPs in saline was measured using a methylene blue (MB) probe. Blue MB can be quickly reduced to colorless dihydromethylene blue (MBH2) by hydrogen, so the decrease in the absorbance of MB at 664nm (MB's characteristic peak) is linearly correlated with the hydrogen content. The test results are shown in Figure 2. Figure 5 As shown, the characteristic absorption peak of EcN@PdH NPs at 664 nm is reduced compared with that of EcN@Pd NPs, demonstrating the successful loading of hydrogen.

[0064] The characterization results of EcN@PdH NPs prepared in Example 2 are the same as those in Example 1.

[0065] Test Example 2

[0066] The antioxidant enzyme activities (SOD-like activity, scavenging ·OH) of Escherichia coli Nissle1917 (EcN), EcN@Pd NPs prepared in Comparative Example 2, and EcN@PdH NPs prepared in Example 1 were tested.

[0067] The method for testing the activity of SOD-like substances is as follows: xanthine oxidase is used to catalyze the reaction of hypoxanthine to generate O2 ·- BMPO was used as a free radical scavenger, and electron spin resonance (ESR) spectrometer was used to detect the O2 ·- The test results are as follows: Figure 6 As shown in the figure, the control group (blank control group with deionized water added) produced a characteristic four-line peak with a signal intensity of 1:1:1:1, which is consistent with the signal of BMPO / ·OOH adduct, indicating that the reaction system successfully generated a large amount of superoxide anions. With the introduction of EcN@PdH NPs, O2 ·- The signal intensity becomes lower, indicating that EcN@PdHNPs have a certain ability to scavenge O2 ·- role.

[0068] The method for testing the removal of ·OH is to use the classic Fenton reaction to generate ·OH, use DMPO as a free radical scavenger, and use an ESR spectrometer to detect the changes in the ·OH content after treatment with EcN, EcN@Pd NPs, and EcN@PdH NPs. The test results are shown in Figure 2. Figure 5 As shown in the figure, in the control group, the Fenton reaction produced a characteristic four-line peak with a signal intensity of 1:2:2:1, which is consistent with the signal of DMPO / ·OH adduct, indicating that the reaction system successfully generated a large number of hydroxyl radicals. With the introduction of EcN@PdH NPs, the signal intensity became lower, indicating that EcN@PdH NPs have a certain effect of scavenging ·OH.

[0069] The above results show that the introduction of palladium effectively improves the antioxidant enzyme activity of probiotics. On this basis, hydrogen adsorption further improves the free radical scavenging activity of the probiotic-palladium hydride biological hydrogen storage system.

[0070] Test Example 3

[0071] The stimulating environment of gastrointestinal fluid not only easily affects the bacterial activity and thus the therapeutic effect, but also may affect the activity of the enzyme due to its pH change. Therefore, the stability of Escherichia coli Nissle1917 (EcN) and EcN@PdH NPs prepared in Example 1 in gastrointestinal fluid was tested, including the effect on bacterial activity and the effect on the bacterial active oxygen scavenging ability.

[0072] The test method for the effect on bacterial activity is as follows: EcN and EcN@PdH NPs were incubated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) for a period of time, respectively, washed with physiological saline, and then plated in bacterial solid culture medium. After incubation at 37°C for 12 hours, the number of colonies formed was counted.

[0073] The detection method for the bacterial active oxygen scavenging ability is as follows: EcN and EcN@PdH NPs are incubated in simulated gastric fluid and simulated intestinal fluid for a period of time, respectively, washed with physiological saline, and then detected with ESR.

[0074] Test results such as Figure 7 As shown, from Figure 7 As can be seen in (a), the formation of palladium hydride can significantly protect bacteria from the stimulation of simulated gastric juice; Figure 7 As can be seen in (b), after being treated with simulated gastric fluid and simulated intestinal fluid, EcN@PdH NPs can also exhibit good superoxide anion scavenging ability.

[0075] Test Example 4

[0076] The protective effect of EcN@PdH NPs prepared in Example 2 on radiation-induced intestinal damage was investigated at the animal level. The test method was as follows:

[0077] Twenty-four 6-8 week old C57BL / 6 mice were randomly divided into four groups, namely, Control group (blank control group), PBS+10Gy group, EcN+10Gy group, and EcN@PdH NPs+10Gy group. They were gavaged with PBS, PBS, EcN, and EcN@PdHNPs for 7 consecutive days. The latter three groups were given 10 Gy abdominal irradiation on the sixth day. The mice were sacrificed and samples were collected on the tenth day.

[0078] Test results such as Figure 8 As shown, from Figure 8 As can be seen in (a), EcN@PdH NPs treatment can improve the weight loss caused by irradiation. Figure 8 As can be seen in (b), EcN@PdH NPs treatment can improve the decrease in spleen index caused by irradiation. Figure 8As can be seen in (c), after irradiation, the white blood cells in the PBS+10Gy group were significantly reduced compared with the Control group; compared with the PBS+10Gy group, EcN@PdH NPs treatment can improve radiation-induced blood system damage.

[0079] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art should understand that other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. 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 claims of the present invention.

Claims

1. A method for preparing a probiotic-palladium hydride biological hydrogen storage system, characterized in that: The following steps are involved: The probiotics are co-incubated with a divalent palladium salt, an organic sodium salt is added for further incubation, hydrogen is introduced into the obtained incubation solution A, and the probiotics-palladium hydride biological hydrogen storage system is obtained by reaction; the molar ratio of the divalent palladium salt to the organic sodium salt is (1-5):(1-3); or The probiotics are co-incubated with a divalent palladium salt, and hydrogen is introduced into the obtained incubation solution B to react to obtain the probiotics-palladium hydride biological hydrogen storage system.

2. The preparation method according to claim 1, characterized in that: The probiotics are selected from one or more of Escherichia coli Nissle 1917, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium longum, Lactobacillus bulgaricus and Lactobacillus rhamnosus.

3. The preparation method according to claim 1, characterized in that: The probiotics and the divalent palladium salt are co-incubated for 30-50 minutes.

4. The preparation method according to claim 1, characterized in that: The organic sodium salt is added and the incubation time is continued for 7-9 hours.

5. The preparation method according to claim 1, characterized in that: The concentration of the organic sodium salt in the incubation solution A is 0.5-3 mM.

6. The preparation method according to claim 1, characterized in that: The concentration of the divalent palladium salt in the incubation solution B is 1-5 mM.

7. The preparation method according to claim 1, characterized in that: The time for introducing hydrogen is 5-10 minutes.

8. The preparation method according to claim 1, characterized in that: A liquid bacterial agent of probiotics is co-incubated with a divalent palladium salt, and the absorbance value of the liquid bacterial agent of probiotics at a wavelength of 600nm is 0.8-1.

9. A probiotics-palladium hydride biological hydrogen storage system prepared by the method according to any one of claims 1 to 8.

10. Use of the probiotic-palladium hydride biological hydrogen storage system according to claim 9 in the preparation of drugs for preventing or treating radiation-induced intestinal damage, anti-inflammatory drugs, anti-oxidative stress drugs or drugs for regulating intestinal flora.

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