Weaned piglet immunoregulation nano-particles with liver targeting property and preparation method of weaned piglet immunoregulation nano-particles

Abstract

The invention belongs to the technical field of animal immunopotentiators, and particularly relates to weaned piglet immunoregulation nanoparticles with liver targeting and a preparation method thereof.The preparation method comprises the following steps that S1, galactomannan is added into deionized water, butyl glyceride is added after stirring and dissolving, the pH is adjusted, a reaction is conducted under shielding gas, and a mixture is obtained; after the reaction is finished, dialyzing, purifying and freeze-drying to obtain butyl glyceride modified galactomannan; s2, dissolving the butyl glyceride modified galactomannan prepared in S1 into a buffer solution, stirring until the butyl glyceride modified galactomannan is dissolved, dropwise adding a silibinin ethanol solution into the buffer solution, and performing ultrasonic reaction to obtain silibinin-galactomannan self-assembled nanoparticles; and S3, adding a cross-linking agent into the silibinin-galactomannan self-assembled nanoparticles, carrying out a cross-linking reaction, then carrying out dialysis purification, and carrying out freeze drying so as to obtain the immunoregulation nanoparticles. According to the invention, silibinin is used as an immunomodulator, and based on the natural recognition characteristics of galactomannan and mannose receptors on the surfaces of weaned pig liver kuhn cells and dendritic cells, the nano packaging efficiency and stability of galactomannan to silibinin are enhanced by using the hydrophobic characteristic of butyl glyceride; the invention constructs a novel immunomodulator with weaned piglet liver targeting.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal immune enhancers, and particularly relates to a liver-targeted immune-regulating nanoparticle for weaned piglets and a preparation method thereof. Background Art

[0002] Intensive farming improves the feeding efficiency of piglets while also causing the livers of piglets to be prone to immune stress. Currently, existing technologies such as Chinese Patent CN118000302A provide an immune enhancer for piglet drinking water, its preparation method and application. By including a traditional Chinese medicine composition, a compound amino acid additive, a vitamin additive, β-glucan, and sodium selenite in the immune enhancer, it can significantly increase the contents of immunoglobulin G, immunoglobulin M, immunoglobulin A, and the conversion rate of T lymphocytes, and improve the immunity of piglets.

[0003] Although various current studies have shown that a variety of natural active products exhibit anti-inflammatory potential under in vitro conditions, their bioavailability and liver targeting are low under in vivo conditions. At the same time, due to the hydrophobicity of most natural active products (such as flavonoids, terpenoids, polyphenolic compounds), it is difficult to dissolve in the aqueous environment of the gastrointestinal tract, affecting their absorption and diffusion. After oral administration, natural products need to be absorbed through the intestine and enter the liver for metabolism. During this process, they may be rapidly degraded by intestinal microorganisms and metabolic enzymes in the intestine and liver or converted into inactive forms, resulting in a significant reduction in the active ingredients entering the systemic circulation. Moreover, due to the unstable molecular structure of some natural products, they are easily enzymolyzed or oxidized in the blood or tissues, have a short half-life, and it is difficult to maintain an effective blood drug concentration. Importantly, traditional dosage forms of natural active products (such as monomeric compounds, crude extracts) lack a targeted delivery system, cannot protect the active ingredients from gastric acid damage or regulate the release rate, resulting in insufficient absorption. In summary, due to defects such as physicochemical property limitations, significant first-pass effects, intestinal absorption barriers, rapid in vivo metabolism, or poor liver targeting, the efficiency of natural active products with anti-inflammatory potential is very low in practical applications, and often higher doses or repeated use are required to achieve the desired effect, resulting in an increase in breeding costs.

[0004] Therefore, there is an urgent need to develop an immune enhancer with good liver targeting and excellent therapeutic effects. Summary of the Invention

[0005] In view of the technical problems of low targeting and poor treatment effect existing in the existing immune enhancement reagents for piglets, the present invention provides a liver-targeted immune regulatory nanoparticle for weaned piglets and a preparation method thereof. By using silybin with good immune regulation effect as an immune regulator, based on the natural recognition characteristics of galactomannan and mannose receptors on the surface of Kupffer cells and dendritic cells in the liver of weaned piglets, and using the hydrophobic property of butyl glycerol ester to enhance the nano-encapsulation efficiency and stability of galactomannan for silybin, a novel immune regulator with liver targeting for weaned piglets is constructed.

[0006] The present invention provides a preparation method of a liver-targeted immune regulatory nanoparticle for weaned piglets, comprising the following steps:

[0007] S1. Add galactomannan to deionized water, stir to dissolve, then add butyl glycerol ester, adjust the pH, react under a protective gas, and after the reaction, dialyze and purify and then freeze-dry to obtain butyl glycerol ester-modified galactomannan;

[0008] S2. Dissolve the butyl glycerol ester-modified galactomannan prepared in S1 in a buffer solution, stir until dissolved, then dropwise add an ethanol solution of silybin, and carry out ultrasonic reaction to obtain silybin-galactomannan self-assembled nanoparticles;

[0009] S3. Add a cross-linking agent to the silybin-galactomannan self-assembled nanoparticles, carry out cross-linking reaction, then dialyze and purify and freeze-dry to obtain immune regulatory nanoparticles.

[0010] Further, in the step S1, the mass ratio of galactomannan to butyl glycerol ester is 25:1 to 30:1, the reaction pH is 8.5 to 9.5, the reaction temperature is 55 to 65 °C, and the reaction time is 12 to 24 h.

[0011] Further, in the step S1, the dialysis cut-off molecular weight is 3.5 kDa, and the dialysis time is 48 to 72 h.

[0012] Further, in the step S2, the concentration of the ethanol solution of silybin is 50 to 60 mg / mL, and the concentration of the dissolved butyl glycerol ester-modified galactomannan is 2 to 3 mg / mL.

[0013] Further, in the step S2, the ultrasonic reaction power is 200 w, the reaction time is 10 to 15 min, and the reaction environment is an ice bath.

[0014] Further, in the step S2, the particle size of the silybin-galactomannan nanoparticles is 200.82 ± 21.87 nm, and the Zeta potential is -47.9 ± 5.16 mV.

[0015] Further, in step S3, the crosslinking agent is a 0.5% w / v genipin solution, and the volume ratio of the silybin-galactomannan self-assembled nanoparticle solution to the genipin solution is 1000:1 to 500:1.

[0016] The present invention also provides a weaned piglet immune-regulating nanoparticle with liver targeting, comprising an immunomodulator, a targeting molecule, and a crosslinking agent. The immunomodulator is silybin, the targeting molecule is butyl glycerol ester-modified galactomannan, and the crosslinking agent is genipin.

[0017] The beneficial effects of the present invention are as follows:

[0018] The present invention selects silybin as the immunomodulator, modifies galactomannan with butyl glycerol ester, utilizes the hydrophobic property of butyl glycerol ester to enhance the encapsulation efficiency of galactomannan for silybin, and improves the stability of the nanoparticles by adding a crosslinking agent, ensuring that the drug molecules in the nanoparticles will not be rapidly enzymatically hydrolyzed or oxidized in piglets, and prolonging the action time of the drug molecules. At the same time, due to the specific targeting of galactomannan to the mannose receptors on Kupffer cells and dendritic cells in the piglet liver, it can ensure that the nanoparticles prepared by the present invention can aggregate in the inflammatory area of the piglet liver in vivo, improving the utilization efficiency of the drug molecules. Description of the Drawings

[0019] Figure 1 It is the experimental result graph of Experiment 4 in the experimental examples of the present invention;

[0020] Figure 2 It is the experimental result graph of Experiment 5 in the experimental examples of the present invention;

[0021] Figure 3 It is the experimental result graph of Experiment 6 in the experimental examples of the present invention;

[0022] Figure 4 It is the TEM graph of the silybin-galactomannan nanoparticles prepared by the present invention;

[0023] Figure 5 It is the statistical graph of the experimental results of Experiment 8 in the experimental examples of the present invention. Detailed Embodiments

[0024] The specific embodiments of the present invention will be described in detail below with reference to the specific drawings. It should be noted that the technical features described in the following embodiments or the combinations of technical features should not be considered in isolation, and they can be combined with each other to achieve better technical effects.

[0025] Embodiment

[0026] This embodiment provides a preparation method for a weaned piglet immune-regulating nanoparticle with liver targeting, comprising the following steps:

[0027] S1. Add galactomannan (2% w / v) to deionized water at 60 °C and stir until completely dissolved. Add galactomannan in a certain proportion, and the mass ratio of galactomannan to butyl glyceride is 25:1 to 30:1. Use NaOH solution to adjust the pH of the solution to 8.5 - 9.5. React for 12 - 24 hours under nitrogen protection at a reaction temperature of 55 - 65 °C. Dialyze the reaction solution for 48 - 72 hours with a cut-off molecular weight of 3.5 kDa to remove unreacted reagents, and freeze-dry to obtain butyl glyceride-modified galactomannan.

[0028] S2. Dissolve silybin in ethanol to prepare a silybin ethanol solution with a concentration of 50 - 60 mg / mL. Then dissolve butyl glyceride-modified galactomannan in PBS solution (pH 7.4) to prepare a butyl glyceride-modified galactomannan solution with a concentration of 2 - 3 mg / mL, and stir magnetically until transparent. Dropwise add the silybin ethanol solution to the butyl glyceride-modified galactomannan solution, set the ultrasonic power at 200 W, and treat it in an ice bath for 10 - 15 minutes to obtain silybin-galactomannan self-assembled nanoparticles.

[0029] S3. Add genipin solution to the silybin-galactomannan self-assembled nanoparticle solution, with a concentration of 0.5% w / v, and the volume ratio of the two is 1000:1 to 500:1. The cross-linking time is 2 hours, the cross-linking reaction temperature is 25 °C. After dialysis purification and freeze-drying, silybin-galactomannan nanoparticles are obtained, and the nanoparticles are as shown in the appendix Figure 4 as follows.

[0030] The experimental parameters and encapsulation efficiency test of the examples and comparative examples of the present invention are shown in Table 1 below:

[0031] Table 1 Test results of experimental parameters and encapsulation efficiency of examples and comparative examples

[0032]

[0033]

[0034] As can be seen from the above examples and comparative examples, the present invention can obtain silybin-galactomannan nanoparticles with a higher encapsulation efficiency by controlling the three factors of the mass ratio of galactomannan to butyl glyceride, reaction pH, and silybin solution concentration, thereby improving the utilization efficiency of silybin.

[0035] Experimental Example

[0036] Experimental method: Thirty-six healthy weaned piglets at 21 days of age were randomly divided into 6 groups, namely the normal control group, the immune stress group, the immune stress-silibinin control group, the immune stress-nanoparticle test group I, the immune stress-nanoparticle test group II, and the immune stress-nanoparticle test group III, with 6 replicates in each group and 1 piglet in each replicate. The feeding period of the piglets was from 21 to 35 days of age. The normal control group and the immune stress group were intragastrically administered normal saline once a day. The immune stress-silibinin control group was intragastrically administered a silibinin solution at a dose of 100 mg / kg once a day. The immune stress-nanoparticle test groups I, II, and III were intragastrically administered a silibinin-galactomannan nanoparticle dispersion at doses of 20, 50, and 100 mg / kg once a day (calculated based on the effective content of silibinin in the nanoparticles). On the 30th, 32nd, and 34th days of age, the normal control group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with an Escherichia coli lipopolysaccharide (LPS) solution at a dose of 10 mg / kg. On the 35th day of age, blood samples of the piglets were collected, and the growth performance of the piglets after immune stress was statistically analyzed. The piglets in the immune stress-nanoparticle test group with the best growth performance after immune stress and other control groups were euthanized, and tissue samples were collected for biochemical index determination.

[0037] Experiment 1: Effects of Silibinin-Galactomannan Nanoparticles on the Growth Performance of Immunologically Stressed Weaned Piglets

[0038] Experimental results: As shown in Table 2, compared with the normal control (N-CON) group, LPS stimulation significantly decreased the average daily gain (ADG) and average daily feed intake (ADFI) of the immune stress control (LPS-CON) group while increasing the feed conversion ratio (FCR, P<0.05). Intragastric administration of 100 mg / kg of the silibinin prototype (LPS-SAL) or 20 mg / kg (LPS-SALN-L), 50 mg / kg (LPS-SALN-M), and 100 mg / kg (LPS-SALN-H) of silibinin-galactomannan nanoparticles could effectively increase the ADG and ADFI of the piglets within 5 days after LPS challenge (P<0.05) and decrease the FCR during this period (P<0.05), which had a positive effect on improving the growth performance of immunologically stressed piglets.

[0039] Table 2 Effects of Silibinin-Galactomannan Nanoparticles on the Growth Performance of Immunologically Stressed Weaned Piglets

[0040]

[0041] Note: * indicates a significant difference between the N-CON group and the LPS-CON group (P<0.05); # indicates a significant difference compared with the LPS-CON group (P<0.05).

[0042] Experiment 2: Effects of silybin-galactomannan nanoparticles on plasma transaminase activities in immunologically stressed weaned piglets

[0043] Experimental results: As shown in Table 3 below, compared with the N-CON group, LPS stimulation led to a significant increase in the activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in piglets (P<0.05, Table 2). Oral administration of silybin or silybin-galactomannan nanoparticles at different levels could significantly reduce the activities of plasma ALT and AST in immunologically stressed piglets (P<0.05), and the plasma ALT activity in the LPS-SALN-M group and LPS-SALN-H group was significantly lower than that in the LPS-SAL group (P<0.05).

[0044] Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to detect liver cell apoptosis. Compared with normal piglets, the apoptosis rate of liver cells in immunologically stressed piglets increased significantly (P<0.05). On the contrary, oral administration of silybin or silybin-galactomannan nanoparticles at different levels could significantly reduce the apoptosis rate of liver cells in immunologically stressed piglets (P<0.05), and the apoptosis rate of liver cells in the LPS-SALN-M group and LPS-SALN-H group was significantly lower than that in the LPS-SAL group (P<0.05).

[0045] Table 3 Effects of silybin-galactomannan nanoparticles on plasma transaminase activities and liver cell apoptosis levels in immunologically stressed weaned piglets

[0046]

[0047] Note: * Indicates a significant difference between the N-CON group and the LPS-CON group (P<0.05); # Indicates a significant difference compared with the LPS-CON group (P<0.05); & Indicates a significant difference compared with the LPS-SAL group (P<0.05).

[0048] Experiment 3: Effects of silybin-galactomannan nanoparticles on plasma cytokine contents in immunologically stressed weaned piglets

[0049] Experimental results: As shown in Table 4 below, LPS stimulation induced an increase in the contents of plasma tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in piglets, which were significantly higher than those in the N-CON group (P<0.05). Administration of silybin or silybin-galactomannan nanoparticles at different levels could significantly reduce the content of TNF-α in the plasma of immunostressed piglets (P<0.05), and the medium and high doses of silybin-galactomannan nanoparticles could also inhibit the increase in the content of plasma IL-1β. In addition, the content of plasma interleukin 6 (IL-6) in the LPS-SALN-M group of piglets was significantly lower than that in the LPS-CON group (P<0.05).

[0050] Table 4 Effects of silybin-galactomannan nanoparticles on the contents of plasma cytokines in immunostressed weaned piglets (pg / mL)

[0051]

[0052] Note: * indicates a significant difference between the N-CON group and the LPS-CON group (P<0.05); # indicates a significant difference compared with the LPS-CON group (P<0.05).

[0053] Experiment 4: Effects of silybin-galactomannan nanoparticles on the liver tissue morphology of immunostressed weaned piglets

[0054] Experimental results: As shown in the appendix Figure 1 The histological results showed that the liver tissue structure of the N-CON group was normal, the hepatic parenchyma had a uniform density, and no obvious inflammatory cell infiltration was observed. In the LPS-CON group, some hepatocytes showed vacuolization and nuclear shrinkage, accompanied by congestion and inflammatory cell infiltration. Administration of silybin or silybin-galactomannan nanoparticles at different levels could effectively improve the liver morphology of immunostressed piglets, reduce the occurrence of hepatocyte swelling or vacuolization, and the LPS-SALN-H group had the most obvious effect, with neatly arranged hepatocytes, relatively dense cytoplasm, and no obvious difference in tissue morphology compared with the N-CON group.

[0055] Experiment 5: Effects of silybin-galactomannan nanoparticles on the ultrastructure of the liver of immunostressed weaned piglets

[0056] Experimental results: As shown in the appendix Figure 2 Compared with the N-CON group, there were obvious differences in the liver ultrastructure of the LPS-CON group, specifically manifested as swelling of the endoplasmic reticulum and mitochondrial structures. Administration of silybin or silybin-galactomannan nanoparticles at different levels could alleviate the abnormal changes in the liver ultrastructure of immunostressed piglets to varying degrees, and the LPS-SALN-H group had the best effect.

[0057] Experiment 6: Effects of Silybin-galactomannan Nanoparticles on the Expression of Nuclear Factor kappaB (NF-κB) p65 Protein in the Liver Nucleus of Immunologically Stressed Weaned Piglets

[0058] Experimental results: As shown in the appendix Figure 3 , compared with the N-CON group, the expression of NF-κB p65 protein in the liver nucleus of piglets in the LPS-CON group increased significantly (P<0.05, Figure 4 ). Nanoparticles of silybin-galactomannan at different levels could all inhibit the increase in the expression of NF-κB p65 protein in the liver nucleus of immunologically stressed piglets (P<0.05). Although intragastric administration of silybin could reduce the expression of NF-κB p65 protein in the liver nucleus of immunologically stressed piglets to a certain extent, there was no significant difference compared with the LPS-CON group (P>0.05). * in the figure indicates a significant difference between the N-CON group and the LPS-CON group (P<0.05); # indicates a significant difference compared with the LPS-CON group (P<0.05).

[0059] Experiment 7: Effects of Silybin-galactomannan Nanoparticles on the Expression Levels of Liver Inflammation-related Genes in Immunologically Stressed Weaned Piglets

[0060] Experimental results: As shown in Table 5, LPS stimulation induced an increase in the transcription of toll-like receptor 4 (TLR4), TNF-α, IL-1β, and IL-6 mRNA in the livers of piglets (P<0.05). Intragastric administration of silybin had a significant effect on inhibiting the expression of TNF-α in the livers of immunologically stressed piglets (P<0.05). Compared with the LPS-CON group, nanoparticles of silybin-galactomannan at different doses could all down-regulate the expression of TLR4 and TNF-α mRNA in the livers of immunologically stressed piglets (P<0.05), and the LPS-SALN-M group and the LPS-SALN-H group also had an obvious inhibitory effect on the expression of IL-1β and IL-6 (P<0.05). In addition, the LPS-SALN-M group significantly reduced the expression level of interleukin 18 (IL-18) in the livers of immunologically stressed piglets (P<0.05).

[0061] Table 5 Effects of Silybin-galactomannan Nanoparticles on the Expression Levels of Liver Inflammation-related Genes in Immunologically Stressed Weaned Piglets

[0062]

[0063] Note: * indicates a significant difference between the N-CON group and the LPS-CON group (P<0.05); # indicates a significant difference compared with the LPS-CON group (P<0.05)

[0064] Experiment 8: Specific Liver Targeting Experiment of Silybin-Galactomannan Nanoparticles

[0065] Experimental results: As shown in the appendix Figure 5 It was found that the study on the distribution of silybin in piglets after intragastric administration of the silybin prototype group (SAL) and the silybin-galactomannan nanoparticle group (SALN-H) showed that the nanoparticles loaded with galactomannan increased the content of silybin in the liver, reaching a peak at 6 h, which was about 7.04 times that of the prototype group. This was due to the fact that silybin-galactomannan nanoparticles had appropriate particle size, negative charge characteristics and the specificity to target and recognize Kupffer cells and dendritic cells in the liver. Controlling the particle size could endow the polysaccharide nanocarrier with the advantages of high permeability, low clearance rate and liver targeting. After absorption, the polysaccharide carrier rapidly entered the liver through the portal vein. Those with a size less than 100 nm could directly pass through the fenestrae of hepatic sinusoidal endothelial cells (~150 nm) and then be taken up by hepatocytes. If the particle size was too large, it was not easy to pass through the portal vein quickly to the liver, reducing the absorption efficiency of the nanoparticles. Only those with appropriate particle size and negative charge were easily captured by Kupffer cells, and then the loaded substances were released intracellularly. Importantly, galactomannan was a natural ligand of the surface receptors of Kupffer cells and dendritic cells, so it greatly improved the liver-targeted delivery characteristics.

[0066] Although several embodiments of the present invention have been given in this article, those skilled in the art should understand that the embodiments in this article can be changed without departing from the spirit of the present invention. The above embodiments are only exemplary and should not be used as the limitation of the scope of the rights of the present invention.

Claims

1. A method for preparing liver-targeted immunomodulatory nanoparticles for weaned piglets, characterized in that: The method comprises the following steps: S1, adding galactomannan to deionized water, stirring and dissolving, adding butyl glyceride, adjusting the pH and reacting under protective gas, dialyzing and purifying after the reaction, and then freeze-drying to obtain butyl glyceride-modified galactomannan; S2, dissolving the butyl glyceride-modified galactomannan prepared in S1 in a buffer solution, stirring until dissolved, and then adding silybin ethanol solution dropwise thereto, and performing ultrasonic reaction to obtain silybin-galactomannan self-assembled nanoparticles; S3. Add a cross-linking agent to the silybin-galactomannan self-assembled nanoparticles, perform dialysis purification after the cross-linking reaction, and freeze-dry to obtain the immunomodulatory nanoparticles.

2. The method for preparing the liver-targeted immunomodulatory nanoparticles for weaned piglets according to claim 1, characterized in that: In step S1, the mass ratio of galactomannan to butyl glyceride is 25:1-30:1, the reaction pH is 8.5-9.5, the reaction temperature is 55-65° C., and the reaction time is 12-24 h.

3. The method for preparing a liver-targeted weaned piglet immunomodulatory nanoparticle according to claim 2, characterized in that: In step S1, the dialysis cut-off molecular weight is 3.5 kDa, and the dialysis time is 48 to 72 hours.

4. The method for preparing the liver-targeted immunomodulatory nanoparticles for weaned piglets according to claim 3, characterized in that: In step S2, the concentration of the silybin ethanol solution is 50-60 mg / mL, and the concentration of the butyl glyceride-modified galactomannan after dissolution is 2-3 mg / mL.

5. The method for preparing the liver-targeted immunomodulatory nanoparticles for weaned piglets according to claim 4, characterized in that: In step S2, the ultrasonic reaction power is 200W, the reaction time is 10-15min, and the reaction environment is an ice bath.

6. The method for preparing the liver-targeted immunomodulatory nanoparticles for weaned piglets according to claim 1, characterized in that: In step S2, the particle size of silybin-galactomannan nanoparticles is 200.82±21.87 nm and the Zeta potential is -47.9±5.16 mV.

7. The method for preparing the liver-targeted immunomodulatory nanoparticles for weaned piglets according to claim 1, characterized in that: In step S3, the cross-linking agent is a 0.5% w / v genipin solution, and the volume ratio of the silybin-galactomannan self-assembled nanoparticle solution to the genipin solution is 1000:1-500:

1.

8. A liver-targeted weaned piglet immunomodulatory nanoparticle, characterized in that: The invention comprises an immunomodulator, a targeting molecule and a cross-linking agent, wherein the immunomodulator is silybin, the targeting molecule is galactomannan modified with butyl glyceride, and the cross-linking agent is genipin.

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