Schisandrin B-loaded oral liver-targeting nanoemulsion as well as preparation method and application thereof

Abstract

The invention provides a schizandrin B-loaded oral liver-targeting nano-emulsion as well as a preparation method and application thereof. The nano-emulsion is mainly prepared by taking a galactosylated protein conjugate synthesized by reaction of soybean lecithin, N-acetyl-D-galactosamine and bovine serum albumin and docosahexaenoic acid as carriers, the loaded drug is schisandrin B. The invention also provides an application of the nanoemulsion in the aspect of delivering a drug for promoting liver regeneration, especially liver regeneration after a liver cancer in-situ PHx operation. The SCHB nanoemulsion prepared by the invention takes SPC as a basic emulsifier, and has the characteristics of high drug loading capacity and simple preparation method. The surface-modified DHA is used for promoting intestinal epithelial permeation of the drug-loaded nano-emulsion, the Gal-BSA is used for improving the gastrointestinal stability of the nano-emulsion, accumulation of the drug in the liver is increased through the targeting effect, and the effect of SCHB for promoting liver regeneration is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of oral liver-targeted preparations, and specifically relates to an oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine and a preparation method thereof, and relates to the use of the nanoemulsion in promoting liver regeneration. Background Art

[0002] Primary liver cancer includes three pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and mixed HCC-ICC. HCC accounts for approximately 80% of all primary liver malignancies and is the third leading cause of cancer-related mortality worldwide. Surgical intervention for liver cancer, including partial hepatectomy (PHx) and liver transplantation, is the most critical way for patients to achieve long-term survival. The regenerative capacity of the residual liver after PHx is a key determinant of patient survival and postoperative prognosis. However, due to incomplete understanding of the mechanism of liver regeneration, there is currently no optimal drug in clinical practice that can significantly improve the regenerative capacity of the liver after PHx. Traditional Chinese medicine is safe and effective for the treatment and rehabilitation of postoperative patients. It can restore liver structure and function through multiple pathways, levels, and time stages. However, drugs often have problems such as poor target tissue distribution and low efficacy in actual clinical applications. Therefore, by constructing a delivery system with targeting ability, the concentration of drugs delivered to the lesion site can be increased, thereby effectively exerting the therapeutic effect. Therefore, it is urgent to discover and clinically promote new drugs that promote liver regeneration.

[0003] Schisandrin beta (SCHB) is one of the main lignan biophenol active ingredients extracted from Schisandra chinensis, which has the effect of promoting liver structural reconstruction and restoring liver function. Studies have shown that SCHB can activate key regulatory factors involved in hepatocyte proliferation in the PXR signaling pathway, leading to upregulation of PXR downstream proteins. This activation triggers the interaction between activated PXR and YAP, promoting hepatocyte proliferation and reconstruction of liver tissue structure. SCHB belongs to Schisandra chinensis lignans, which are mainly treated by oral administration. Previous studies have shown that it has poor water solubility and low oral bioavailability, resulting in low drug concentrations in target tissues. Therefore, it is crucial to develop oral drug nanoemulsions targeting the liver to ensure that the carrier is effectively absorbed and transported intact to the systemic circulation to promote accumulation in the target site.

[0004] In recent years, the strategy of combining oral drug delivery and targeted therapy has received increasing attention. Oral targeting is a non-invasive drug delivery strategy that uses nano-delivery systems to achieve systemic or remote targeting through oral routes. It can identify target tissues or target cells more accurately and efficiently, and has the advantages of convenient drug delivery and high compliance.

[0005] Oral targeting requires the surface functional design of the delivery system to overcome the obstruction and rejection of the gastrointestinal tract, increase the probability of the drug delivery system entering the systemic circulation, and ultimately complete its preset targeting function. Nanoemulsion, as a promising oral drug delivery system, has been used to treat a variety of diseases. Nanoemulsions can significantly improve the bioavailability and efficacy of drugs due to their easy absorption properties. Many proteins are nutritious, non-toxic, low-cost, and have good emulsification ability, and can be used as emulsifiers for nanoemulsions. However, existing nanoemulsions also have some problems, such as difficulty in breaking through biological barriers, etc.

[0006] Therefore, in addition to withstanding gastrointestinal digestion, oral nanodrug delivery systems are also required to be able to break through the biomembrane barrier, which also requires further research. Summary of the invention

[0007] Purpose of the invention: In view of the deficiencies of the prior art, the present invention provides an oral liver-targeted nanoemulsion loaded with schisandra chinensis alcohol B and a preparation method and application thereof.

[0008] Technical solution: In order to achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0009] An oral liver-targeted nanoemulsion for loading schisandra chinensis alcohol B. The nanoemulsion mainly uses soybean lecithin, N-acetyl-D-galactosamine, bovine serum albumin and docosahexaenoic acid as carriers, and the loaded drug is schisandra chinensis alcohol B. The galactosylated protein conjugate is mainly synthesized by using bovine serum albumin and N-acetyl-D-galactosamine through Maillard reaction.

[0010] As a specific embodiment, the schisandra alcohol ethyl includes schisandra alcohol ethyl or a derivative thereof.

[0011] As a specific implementation scheme, the mass ratio of bovine serum albumin to N-acetyl-D-galactosamine is 1:(1-4); preferably 1:3; the reaction conditions of the Maillard reaction are: reacting at pH 6-8, 55-65°C, and 75-85% humidity for 12-48 hours.

[0012] More specifically, the method for synthesizing the galactosylated protein conjugate comprises the following steps:

[0013] Mix and dissolve bovine serum albumin and N-acetyl-D-galactosamine, freeze-dry, place the freeze-dried powder in a saturated KBr sealed space, and react at pH 6-8, 55-65°C, and 75-85% humidity for 12-48 hours to obtain the product. Preferably, react at pH 8, 60°C, and 79% humidity for 24 hours.

[0014] The present invention also provides a method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis alcohol B, comprising the following steps:

[0015] (1) taking a galactosyl protein conjugate, adding deionized water to prepare a solution as the aqueous phase; dissolving soybean lecithin, docosahexaenoic acid and schisandrae alcohol ethyl in oil as the oil phase;

[0016] (2) Mixing the oil phase and the water phase to prepare the nanoemulsion.

[0017] As a specific embodiment, in step (1), the oil is selected from one or more of medium chain triglycerides, soybean oil, corn oil, peanut oil, and castor oil, preferably medium chain triglycerides.

[0018] As a specific implementation scheme, in step (2), the oil phase and the aqueous phase are mixed, wherein the mass ratio of galactosylated protein to soybean lecithin is (0.25-0.75):1, preferably 0.5:1, based on the mass of bovine serum albumin, and the content of docosahexaenoic acid in the oil phase is 10-80 μM, preferably 20 μM, and the mass ratio of docosahexaenoic acid, soybean lecithin and schisandrin B in the oil phase is (0.175-1.4):1:(1-2.56), preferably 0.35:1:1.28.

[0019] As a specific embodiment, in step (2), the method for preparing the nanoemulsion comprises: mixing the oil phase and the water phase and then sonicating to obtain a primary emulsion; and high-pressure homogenizing the primary emulsion to obtain the nanoemulsion.

[0020] Preferably, the ultrasonication is performed in an ice water bath at 60-70W for 2.5-3.5min (pulse 2s, pause 2s), and more preferably, the ultrasonication is performed in an ice water bath at 65W for 3min (pulse 2s, pause 2s); the high-pressure homogenization is performed 2-4 times at 5000-7000psi, and more preferably, the high-pressure homogenization is performed 3 times at 6000psi.

[0021] Finally, the present invention provides the use of the oral liver-targeted nanoemulsion loaded with schisandra chinensis alcohol B in the preparation of drugs for promoting liver regeneration.

[0022] Furthermore, the liver regeneration is liver regeneration after in situ PHx surgery for liver cancer.

[0023] The present invention first synthesizes a galactosylated protein (Gal-BSA) conjugate through a green and safe Maillard reaction. Oral liver targeting is different from conventional blood circulation targeting. Oral liver targeting requires not only the ability to break through the gastrointestinal barrier, but also the effective targeting of the lesion. Therefore, the present invention uses soybean lecithin (SPC) as the basis of the nanoemulsion, which not only has good biocompatibility, but also has a good drug encapsulation effect. The use of hydrophilic N-acetyl-D-galactosamine (Gal) can form an extended spatial network structure in the aqueous phase, produce steric hindrance, make up for the defect that the protein emulsion is easily affected by environmental factors in the gastrointestinal tract (pH, ionic strength and enzymatic hydrolysis, etc.), and is used to improve the stability of the delivery system. Therefore, bovine serum albumin (BSA) and Gal are grafted to synthesize a galactosylated conjugate (Gal-BSA) as an emulsifier to prepare a nanoemulsion that can withstand gastrointestinal digestion and improve the stability of the system in the gastrointestinal tract. In addition, Cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) as an absorption enhancer can improve the intestinal wall permeability of the nanoemulsion and increase the oral bioavailability of the drug. The present invention uses Gal-BSA for ligand modification, which can target the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes to improve the targeting effect of the nanoemulsion on the liver, thereby better exerting the therapeutic effect of the drug in promoting liver regeneration after PHx.

[0024] Secondly, the present invention adopts microfluidization high-pressure homogenization method to prepare nanoemulsion (SCHB@SPC / Gal-BSA / DHA) loaded with SCHB, which is used for oral administration to promote liver regeneration after 70% PHx in normal mice or liver regeneration after in situ PHx in liver cancer mice. The results show that DHA-modified SPC / galactosylated protein nanoemulsion can effectively encapsulate SCHB, improve small intestinal epithelial cell transport, and improve the oral bioavailability of SCHB. In addition, the nanoemulsion prolongs the residence time of SCHB in the gastrointestinal tract and realizes the targeted accumulation of drugs in the liver area, resulting in SCHB being able to play a higher role in promoting liver regeneration after PHx. Therefore, the SCHB@SPC / Gal-BSA / DHA nanoemulsion developed in this study has great potential in being used for oral targeted promotion of liver regeneration after PHx.

[0025] Beneficial effects: The SCHB nanoemulsion prepared by the present invention uses SPC as the basic emulsifier, has the characteristics of high drug loading and simple preparation method. Surface-modified DHA is used to promote the intestinal epithelial permeation of the drug-loaded nanoemulsion, Gal-BSA improves the gastrointestinal stability of the nanoemulsion, increases the accumulation of the drug in the liver, and finally targets the liver to promote liver regeneration, so that SCHB can promote liver regeneration after normal 70% PHx or in situ PHx of liver cancer. The @SPC / Gal-BSA / DHA nanoemulsion developed by the present invention can be used as an oral delivery platform for the liver regeneration drug SCHB. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The invention discloses the preparation and characterization of galactosylated protein (Gal-BSA) conjugates. A is the SDS-PAGE image of Gal-BSA under different reaction pH conditions, B is the grafting degree of Gal-BSA, and C is the browning degree of Gal-BSA; D is the SDS-PAGE image of Gal-BSA under different reaction times, E is the grafting degree of Gal-BSA, and F is the browning degree of Gal-BSA; G is the SDS-PAGE image of BSA and Gal at different ratios, H is the grafting degree of Gal-BSA, and I is the browning degree of Gal-BSA.

[0027] Figure 2 The preparation and characterization results of SCHB@SPC / Gal-BSA / DHA of the present invention. A is the encapsulation efficiency and drug loading changes of the preparation within 90 days; B is the changes in particle size and polydispersity coefficient of the preparation within 90 days; C is the potential change of the preparation within 90 days. DF is the TEM images of SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA.

[0028] Figure 3 The results of the study on the absorption-promoting mechanism of SCHB@SPC / Gal-BSA / DHA of the present invention are shown in Figure 1. A is the apparent permeability coefficient (Papp) of the preparation; B is the Caco-2 cell uptake of the preparation; C is the transmembrane electrical resistance (TEER) of the preparation; D is the ZO-1 immunofluorescence staining of the Caco-2 cell monolayer; and E is the fluorescence semi-quantification of ZO-1.

[0029] Figure 4 These are the results of the gastrointestinal stability study of SCHB@SPC / Gal-BSA / DHA of the present invention. A is the FRET of the preparation in artificial gastric juice; B is the FRET of the preparation in artificial intestinal juice; and C is the dynamic release of the preparation in artificial gastrointestinal juice.

[0030] Figure 5 The results of the in vitro permeability study of SCHB@SPC / Gal-BSA / DHA of the present invention are shown in Figure 1. A is the apparent permeability coefficient (Papp) of the preparation; B is the transmembrane electrical resistance (TEER) of the preparation; C is the TEM image of the preparation before and after it crosses the Caco-2 cell monolayer; D is the retention of DiR / SCHB@SPC / Gal-BSA / DHA in the intestine; EF is the fluorescence semi-quantitative of the DiR preparation in the stomach, small intestine and large intestine; H is the intestinal section of DiO / SCHB@SPC / Gal-BSA / DHA; I is the fluorescence semi-quantitative image of the intestinal section of the DiO preparation.

[0031] Figure 6 The results of the liver targeting study of DiR / SCHB@SPC / Gal-BSA / DHA of the present invention are shown in Figure 1. A is the tissue fluorescence distribution diagram of the DiR preparation; BF are the fluorescence semi-quantification of the DiR preparation in the heart, liver, spleen, lung and kidney tissues, respectively.

[0032] Figure 7 The pharmacokinetic study results of SCHB@SPC / Gal-BSA / DHA of the present invention are shown in Figure 1. A is the blood drug concentration curve of free SCHB and SCHB preparations changing with time; B is the pharmacokinetic parameters of free SCHB and SCHB preparations.

[0033] Figure 8 It is the result of the study on the targeting of SCHB@SPC / Gal-BSA / DHA to the liver after oral administration. AE are the drug distribution diagrams of free SCHB and SCHB preparation in liver, heart, spleen, lung and kidney tissues respectively.

[0034] Fig. 9 This is the research result of the present invention SCHB@SPC / Gal-BSA / DHA promoting liver regeneration after 70% PHx. A is a representative liver picture of normal mice on the 7th day after 70% PHx; B is a Ki-67 staining picture of the liver of normal mice after 70% PHx at different time points; C is a H&E staining picture of the liver of normal mice after 70% PHx at different time points.

[0035] Fig.10 These are the research results of the present invention on promoting liver regeneration after orthotopic PHx of liver cancer by SCHB@SPC / Gal-BSA / DHA. A is a representative liver image of a liver cancer mouse on the 7th day after orthotopic PHx; B is an ultrasound scan of liver thickness of a liver cancer mouse on the 7th day after orthotopic PHx; C is Ki-67 staining of the liver of a liver cancer mouse on the 7th day after orthotopic PHx; D is H&E staining of the liver of a liver cancer mouse on the 7th day after orthotopic PHx. DETAILED DESCRIPTION

[0036] The present invention can be better understood according to the following examples. However, the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the invention described in detail in the claims.

[0037] Example 1 Preparation and characterization of galactosylated protein (Gal-BSA) conjugate

[0038] Experimental method: Galactosylated protein (Gal-BSA) was prepared by green and safe Maillard reaction. First, bovine serum albumin (BSA) solution (15 mg / mL) was prepared by stirring in deionized water. Then N-acetyl-D-galactosamine (Gal) was added to the BSA solution, and the mass ratio of BSA to Gal was 1:1, 1:2, 1:3, and 1:4, respectively. After stirring at 25°C for 12 hours until completely dissolved, the pH values ​​were adjusted to 6, 7, and 8, respectively. After the mixed solution was freeze-dried, the freeze-dried powder was placed in a saturated KBr sealed container and placed at 60°C and 79% humidity for 12, 24, 36, and 48 hours. The reaction product was named Gal-BSA. In order to determine the synthesis of Gal-BSA, the synthesized Gal-BSA was first qualitatively analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The degree of grafting (DG) of Gal-BSA was quantitatively evaluated by the o-phthalaldehyde (OPA) method. In addition, the concentration of Gal-BSA and the production of byproducts (melanoidins) were monitored by measuring the absorbance at 294 and 420 nm during the reaction. Finally, the emulsification activity index (EAI) and emulsion stability index (ESI) were used to evaluate the emulsification ability of Gal-BSA.

[0039] Experimental results: SDS-PAGE of Gal-BSA ( Figure 1 A, D and G) show that BSA presents a distinct band in the range of 63-75 kDa. As the reaction proceeds, a smear band is detected in the molecular weight range of 75-180 kDa, indicating that the Gal-BSA conjugate is successfully formed. The OPA method is further used to quantitatively evaluate the grafting degree (DG) of Gal-BSA ( Figure 1 B, E and H), Gal-BSA has different grafting degrees under different feed ratios, reaction pH and reaction time. The concentration of Gal-BSA and the production of by-products (melanin) were monitored by measuring the absorbance at 294 and 420 nm during the reaction ( Figure 1 C, F and I), where Gal-BSA was generated faster in 24 h, had a higher Gal-BSA concentration, and had lower by-product generation. In addition, the reaction pH of BSA and Gal was optimized to 8, the reaction time was extended to 24 h, and the mass ratio was adjusted to 1:3, the emulsification performance of Gal-BSA was improved, and the stability was better.

[0040] Example 2 Preparation and Characterization of SCHB@SPC / Gal-BSA / DHA

[0041] Experimental method: Gal-BSA was prepared into a 5 mg / mL deionized water solution (prepared under the above optimal conditions, i.e., the reaction pH was 8, the reaction was carried out at 60°C and 79% humidity for 24 hours, and the mass ratio of BSA to Gal was 1:3) as the aqueous phase. SPC and DHA were dissolved in medium-chain triglycerides to obtain a mixed oil solution, and then SCHB was dissolved in the above mixed oil solution to prepare 31.25 mg / mL as the oil phase. Then, the oil phase and the aqueous phase were mixed at an oil-water volume ratio of 1:4. The mass ratio of SPC, Gal-BSA, DHA and SCHB in the entire solution system was 1:0.5:0.35:1.28. The solution was vortexed for 5 minutes, and then ultrasonicated in an ice water bath at 65W for 3 minutes (pulse 2s, pause 2s) to obtain a primary emulsion. Subsequently, microjet high-pressure homogenization was used for 3 times at 6000psi to obtain SCHB@SPC / Gal-BSA / DHA. SCHB@SPC and SCHB@SPC / Gal-BSA were prepared by the same method.

[0042] The particle size (PS), polydispersity index (PDI) and zeta potential (ZP) of the nanoemulsion loaded with SCHB were measured by nanoparticle size potentiometer. The drug encapsulation efficiency (LE) and drug loading (LC) of SCHB were determined by high performance liquid chromatography (HPLC) and UV detector. SCHB was detected using acetonitrile / water (65:35, v / v) as the mobile phase at λ=254nm. The LE (%) and LC (%) of SCHB were calculated by the following formula:

[0043]

[0044] Experimental results: Figure 2 As shown in AC, the changes in LE, LC, PS, PDI and ZP of SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA were monitored at 4°C for 90 days to evaluate the storage stability of the nanoemulsion. The results showed that LE was always above 80% within 90 days. In addition, the particle size of SCHB@SPC showed a rapid increase, accompanied by an increase in PDI from 0.1 to approximately 0.2. In contrast, the particle size of SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA increased more slowly and had a narrower size distribution. After storage at 4°C for 90 days, there was no significant change in the ZP of the three nanoemulsions. The morphology of the nanoemulsions was examined by transmission electron microscopy (TEM) ( Figure 2DF), the results show that SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA all present uniform spherical particles with excellent dispersion properties. Among them, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA show a hazy hydration layer on their surface, which may be related to the insertion of Gal-BSA.

[0045] Example 3 Study on the absorption promoting mechanism of SCHB@SPC / Gal-BSA / DHA

[0046] Experimental method: Caco-2 cells were cultured at 5×10 4 The cells were seeded at a density of 10 cells / well in the upper chamber of the transwell (pore size 0.4 μm) and cultured in MEM medium containing 20% ​​v / v fetal bovine serum (FBS) for 21 days. The Caco-2 cell monolayer resistance (TEER) was measured using a voltammeter. When the TEER value reached 500Ω·cm2, subsequent experiments were performed. When preparing the sample, SCHB@SPC / Gal-BSA / DHA was diluted with PBS (DHA contents were 10, 15, 20, 40, and 80 mM, respectively). Then, the culture medium was removed and equilibrated with preheated PBS at 37°C for 30 minutes. Then, the PBS upper chamber was replaced with 0.2 mL of sample, and the lower chamber was replaced with 0.6 mL of fresh PBS. After incubation for 2 hours, 0.5 mL of solution was taken out from the lower chamber, and the SCHB content was quantitatively analyzed by HPLC. The apparent permeability coefficient (Papp) of SCHB is calculated as follows:

[0047]

[0048] Where Q is the cumulative amount of SCHB delivered to the lower chamber (ng), A is the area of ​​the Caco-2 cell monolayer (cm 2 ), C0 is the initial SCHB concentration in the upper chamber (μg / mL), and t is the duration of the permeability experiment (s). In order to evaluate the uptake of DHA-containing nanoemulsions by Caco-2 cells, after incubation of SCHB@SPC / Gal-BSA / DHA (DHA content was 10, 15, 20, 40, and 80 mM, respectively) for 2 h, rinsing with PBS, RIPA cell lysis buffer was added to lyse the cells for 1 h, and finally the SCHB content was determined by HPLC. Finally, the TEER value was measured to monitor the effect of the SCHB preparation on the integrity of the Caco-2 monolayer. First, the initial TEER value of the Caco-2 cell monolayer was measured. Then, the upper chamber culture medium was replaced with 0.2 mL of SCHB preparation (DHA content was 10, 15, 20, 40, and 80 mM, respectively). After 2 h, the SCHB preparation was removed from the upper chamber and replaced with PBS, and the TEER value of the Caco-2 cell monolayer after different treatments was measured again.

[0049] In addition, Caco-2 cells were cultured on sterilized slides in 12-well plates until the cells reached about 70% fusion, and then cultured in antibiotic-free and serum-free medium for 24 h for subsequent treatment. 1 mL of SCHB@SPC / Gal-BSA / DHA (DHA content was 10, 15, 20, 40, and 80 mM, respectively) was added and incubated at 37°C (5% CO 2 ) for 2 hours. At the end of the incubation time, rinse with preheated PBS for 5 minutes × 3 times. Then perform immunofluorescence staining, the specific steps are as follows: a. Fixation: 4% paraformaldehyde, fix at room temperature for 20 minutes, rinse with preheated PBS for 5 minutes × 3 times; b. Permeabilization: Cover Caco-2 cells with 0.1% Triton X-100 prepared in 0.01M PBS for 10 minutes; c. Blocking: Incubate with 1% BSA, 22.52mg / mL glycine in PBST (PBS+0.1% Tween 20) at room temperature for 30 minutes, rinse with PBS for 5 minutes × 3 times; d. Primary antibody incubation: Use recombinant Anti-ZO1 tight junction protein antibody (antibody dilution 1:100), drop it on the slide to cover the cells (100μL per slide), incubate in a wet box at room temperature for 1 hour or at 4℃ overnight. e. Rewarming: Place the sample at room temperature for 15 minutes, remove the antibody working solution, and rinse with preheated PBS for 5 minutes × 3 times; f. Secondary antibody incubation: goat anti-rabbit IgG H&L (Alexa Fluor 488) (antibody dilution 1:1000) (500μL per slide), incubate at room temperature in the dark for 1h; g. Nuclear staining: 1μg / mL DAPI, incubate at room temperature in the dark for 10min; h. Sealing observation: PBS rinse 5min×3 times, drop a drop of anti-fluorescence quenching sealing liquid on the clean slide, carefully remove the slide and discard the water, turn it upside down on the slide, drop two drops of nail polish on the edge and seal the cover glass with nail polish (to prevent the sample from drying and moving under the microscope), dry at room temperature in the dark, and store at -20℃. Observe under a laser confocal microscope (Zeiss LSM 900), and it will emit green light after 488 excitation. Finally, use Image J software for fluorescence semi-quantification.

[0050] Results: The apparent permeability coefficient (Papp) values ​​of Caco-2 cell monolayers containing nanoemulsions with different DHA contents were evaluated. Figure 3 A) enhanced the permeability of SCHB across the Caco-2 cell monolayer. It is noteworthy that although increasing the DHA content did not affect the uptake of Caco-2 cells ( Figure 3 B), but it will reduce the transmembrane resistance of Caco-2 cell monolayer ( Figure 3C). The results showed that DHA and DHA modified nanoemulsion ( Figure 3 DE) may be through the interaction with Ca 2+ Formation of complexes to reduce surrounding Ca 2+ concentration, affecting the function of ZO-1 and instantaneously opening TJs. This interference can lead to the paracellular permeability of drug-encapsulated nanoemulsions through the small intestine. It is worth noting that this effect becomes more obvious with the increase of DHA content in the nanoemulsion. In summary, when the DHA content is 20mM, the paracellular permeability in the nanoemulsion can be effectively enhanced.

[0051] Example 4 Investigation of gastrointestinal stability of SCHB@SPC / Gal-BSA / DHA

[0052] Experimental methods: The gastrointestinal stability of SCHB@SPC / Gal-BSA / DHA in vitro was evaluated. Briefly, HIQ / NR@SPC, HIQ / NR@SPC / Gal-BSA and HIQ / NR@SPC / Gal-BSA / DHA were prepared using the FRET pair of HIQ and NR. The above preparations were then mixed with simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) in a 15 mL centrifuge tube. The mixture was then placed in a constant temperature oscillator (100 rpm / min) at 37 ° C for 0, 0.5, 1, 2, 4, 6, 8, and 12 h. The fluorescence intensity of the samples was measured by a fluorescence spectrophotometer.

[0053]

[0054] Where I HIQ and I NR Fluorescence intensity of HIQ and NR at 510 and 635 nm, respectively, with an excitation wavelength of 450 nm. To evaluate the release of SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA were placed in dialysis bags (MWCO, 3.5 kDa), dispersed in 20 mL SGF for the first 2 h, and then transferred to SIF. In a thermostatic oscillator (100 rpm / min, 37±0.5°C), 0.5 mL of culture medium was collected at predetermined time points and supplemented with the same amount of fresh culture medium. HPLC was used to analyze the release of SCHB.

[0055] Experimental results: Figure 4 As shown in A, in SGF, the FRET ratios of the three groups of nanoemulsions slowly decreased over time. Figure 4As shown in Figure B, in SIF, the FRET ratio of the HIQ / NR@SPC group decreased rapidly after 2 h, while the FRET ratio of the HIQ / NR@SPC / Gal-BSA and HIQ / NR@SPC / Gal-BSA / DHA groups did not change significantly, indicating that the Gal-BSA nanoemulsion was more stable. It is worth noting that the release behaviors of these nanoemulsions were different during the dynamic dialysis of SGF and SIF ( Figure 4 C). The interaction between bile salts and phospholipid molecules can promote the release of lipid drug molecules from SCHB@SPC, and the cumulative drug release reached 52.60% after 24 hours. In contrast, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA showed higher resistance to pancreatic lipase activity, and the cumulative release of both was significantly delayed and reduced, which was due to the hydrophilic barrier effect conferred by Gal-BSA, thereby preventing the binding of pancreatic lipase. Within 24 hours, the cumulative release rates of SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA were 37.32% and 33.33%, respectively. The results show that the nanoemulsion modified with Gal-BSA has good stability in SGF and SIF.

[0056] Example 5 In vitro permeability study of SCHB@SPC / Gal-BSA / DHA

[0057] Experimental method: Caco-2 cells were used as an in vitro gastrointestinal epithelial model. Briefly, Caco-2 cells were cultured at 5×10 4 The cells were seeded at a density of 10 cells / well in the upper chamber of a transwell (pore size 0.4 μm) and cultured in MEM medium containing 20% ​​v / v fetal bovine serum (FBS) for 21 days. The electrical resistance (TEER) of the Caco-2 cell monolayer was measured using a voltammeter. The TEER value reached 500 Ω·cm 2 , and conduct subsequent experiments. When preparing samples, Free SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA (100μg / mL SCHB) were diluted with PBS respectively. Then, the culture medium was removed and equilibrated with preheated PBS at 37°C for 30min. Then, the upper PBS chamber was replaced with 0.2mL sample, and the lower chamber was replaced with 0.6mL fresh PBS. After incubation for 2h, 0.5mL solution was taken out from the lower chamber, and the content of SCHB was quantitatively analyzed by HPLC. The apparent permeability coefficient (P app)The calculation formula is as described above. Finally, the TEER value was measured to monitor the effect of the SCHB preparation on the integrity of the Caco-2 monolayer. First, the initial TEER value of the Caco-2 cell monolayer was measured. Then, the upper chamber culture medium was replaced with 0.2mL SCHB preparation (100μg / mL SCHB). After 2h, the SCHB preparation was removed from the upper chamber and replaced with PBS, and the TEER value of the Caco-2 cell monolayer after different treatments was measured again. In order to evaluate the integrity of the nanoemulsion after penetrating the barrier, TEM images were obtained from the apical and basolateral sides of the Caco-2 cell monolayer after 2h of incubation with SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA. C57BL / 6 mice were fasted overnight and randomly divided into 4 groups (n=3). Nanoemulsions containing DiR / SCHB (Free DiR, DiR / SCHB@SPC, DiR / SCHB@SPC / Gal-BSA, DiR / SCHB@SPC / Gal-BSA / DHA) were prepared and mice were given DiR-nanoemulsions by gavage (2 mg / kg DiR; 100 mg / kg SCHB). The distribution of DiR formulations in the artificial gastrointestinal tract (GIT) was studied by imaging the intestinal segments of mice using an IVIS spectral system and a small animal anesthesia device at 0.5, 1, 4, 8, and 12 h after treatment.

[0058] In addition, DiO-loaded nanoemulsions were prepared to further study the distribution of @SPC / Gal-BSA / DHA in the GIT. Male C57BL / 6 mice were randomly divided into 4 groups (n=3) and orally administered with Free DiO, DiO / SCHB@SPC, DiO / SCHB@SPC / Gal-BSA, and DiO / SCHB@SPC / Gal-BSA / DHA (DiO 3 mg / kg, SCHB 100 mg / kg), respectively. The mice were killed at 0.5, 1, 4, 8, and 12 h after administration. The ileum was collected, opened, and gently rinsed with PBS. After fixation with 4% paraformaldehyde, paraffin sections were performed and then stained with DAPI. Finally, imaging was performed using a laser confocal microscope.

[0059] Experimental results: A Caco-2 cell monolayer model was established to simulate intestinal epithelial cells and their tight junctions. Papp was used as the main indicator for evaluating permeability to evaluate the transport of SCHB nanoemulsion through the Caco-2 cell monolayer ( Figure 5 A). Compared with Free SCHB, the nanoemulsion significantly improved the permeability of SCHB through intestinal epithelial cells, and the Papp value of each SCHB nanoemulsion formula was about 2 to 3 times that of Free SCHB. It is worth noting that the permeability of SCHB@SPC / Gal-BSA / DHA was the most significantly improved.

[0060] In addition, we evaluated the paracellular permeability of various SCHB nanoemulsions using TEER. Figure 5 As shown in B, all nanoemulsions can rapidly reduce the TEER value of the Caco-2 cell monolayer, among which the TEER value of the SCHB@SPC / Gal-BSA / DHA group was significantly reduced by 68.83% at 2 h. In contrast, the TEER value of the Free SCHB group did not change significantly. In addition, after the removal of each SCHB nanoemulsion, the TEER gradually recovered, indicating the recovery of TJs. To evaluate the integrity of the barrier after nanoemulsion penetration, TEM images were obtained from the apical and basolateral sides of the Caco-2 cell monolayer after 2 h of incubation with SCHB@SPC, SCHB@SPC / Gal-BSA, and SCHB@SPC / Gal-BSA / DHA ( Figure 5 C). The TEM image of SCHB@SPC on the basal side showed some non-round emulsion droplets, indicating that the nanoemulsion was unstable. Meanwhile, the TEM images of SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA still maintained a round emulsion morphology, indicating that these formulations had good stability after crossing the membrane.

[0061] DiR-labeled Free DiR, DiR / SCHB@SPC, DiR / SCHB@SPC / Gal-BSA, and DiR / SCHB@SPC / Gal-BSA / DHA formulations were orally administered to mice. Fluorescence signals of gastrointestinal organs were detected at different time points after the mice were anesthetized and killed. Figure 5 As shown in D, the overall residence time of the nanoemulsion in the stomach exceeds that of Free DiR. 4 h after administration, the fluorescence intensity in the small intestine of the FreeDiR group was significantly reduced. In contrast, the three nanoemulsion groups showed different degrees of fluorescence intensity retention in this intestinal segment. It is worth noting that the retention rate of the DiR / SCHB@SPC / Gal-BSA group and the DiR / SCHB@SPC / Gal-BSA / DHA group in the intestinal segment was significantly improved, which may be because they are more resistant to GIT enzyme degradation and are released more slowly in the intestinal fluid. Fluorescence quantitative evaluation showed that all nanoemulsions showed extensive fluorescence retention in the stomach, small intestine, and large intestine ( Figure 5 EG). In addition, the absorption of DiO-labeled formulations was evaluated ( Figure 5 H). At each time point, the small intestine was separated and the ileum was removed for subsequent sectioning. The absorption of DiO was then detected using laser confocal microscopy. It is worth noting that the signal of the free DiO group in the small intestinal sections was significantly weaker, indicating that a considerable portion of Free DiO failed to reach the intestinal microvilli. In contrast, DiO-labeled nanoemulsions were detected within the brush borders of the intestinal villi. At the same time, the fluorescence intensity of different intestinal segments was quantitatively evaluated ( Figure 5I), showing that all nanoemulsion groups showed prolonged retention time in the intestine. It is worth noting that the DiO / SCHB@SPC / Gal-BSA / DHA group had better fluorescence retention compared with other groups. In summary, @SPC / Gal-BSA / DHA has good intestinal absorption, which is beneficial to improve the bioavailability of drugs.

[0062] Example 6 Liver targeting ability of DiR / SCHB@SPC / Gal-BSA / DHA

[0063] Experimental methods: C57BL / 6 mice were fasted overnight and randomly divided into 4 groups (n=3). Nanoemulsions containing DiR / SCHB (Free DiR, DiR / SCHB@SPC, DiR / SCHB@SPC / Gal-BSA, DiR / SCHB@SPC / Gal-BSA / DHA) were prepared, and mice were orally administered DiR-nanoemulsions containing 2mg / kg DiR and 100mg / kg SCHB. After 0.5, 1, 4, 8, and 12h of treatment, the animals were imaged using an IVIS spectral system and a small animal anesthesia device. Next, the mice were anesthetized and killed, and the major organs, including the heart, liver, spleen, lungs, and kidneys, were separated for imaging. 4.5.5 The software detects the fluorescence intensity of the tissue.

[0064] Experimental results: Free DiR, DiR / SCHB@SPC, DiR / SCHB@SPC / Gal-BSA or DiR / SCHB@SPC / Gal-BSA / DHA were orally administered to C57BL / 6 mice, and their fluorescence intensity was monitored using an IVIS spectral system. To further elucidate the targeting ability of DiR nanoemulsions, organs and tissues were obtained for imaging 12 h after oral administration ( Figure 6 A). Semi-quantitative analysis of the distribution of DiR in various tissues ( Figure 6 BF). The fluorescence signal intensity of the Free DiR group was the smallest at different time points in different tissues and organs. The fluorescence signal of the DiR / SCHB@SPC / Gal-BSA / DHA group was the strongest at each time point. This enhancement may be attributed to the improvement of DHA-mediated absorption, while the modification of Gal-BSA further promoted the nanoemulsion to enter the portal vein and accumulate in the liver tissue.

[0065] Example 7 Pharmacokinetic study of SCHB@SPC / Gal-BSA / DHA

[0066] Experimental methods: Healthy male C57BL / 6 mice were randomly divided into 4 groups (n=6) and given Free SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA or SCHB@SPC / Gal-BSA / DHA by gavage at a dose of 100 mg / kg. The mice in each group were anesthetized by inhalation of isoflurane using a small animal anesthesia device, and blood was collected from the abdominal aorta of anesthetized mice at 0, 0.083, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after administration. The blood samples were then centrifuged at 1000×g for 10 min at 4°C, and plasma was collected and stored at -80°C for further analysis. Mouse plasma samples (30 μL) were mixed with 300 μL of methanol containing the internal standard (IS, berberine, 100 ng / mL) working solution and shaken for 3 min. Subsequently, the sample was centrifuged at 21 000 × g for 5 min, and 200 μL of the supernatant was transferred to a new tube and centrifuged at 21 000 × g for 5 min. 5 μL of the supernatant was taken for LC-MS / MS analysis. Finally, the pharmacokinetic software WinNonlin 8.3 was used to analyze the main pharmacokinetic parameters.

[0067] Experimental results: After oral administration of Free SCHB and various SCHB nanoemulsions, the drug concentration-time curves in the plasma of C57BL / 6 mice ( Figure 7 A). The concentration-time data were analyzed and the pharmacokinetic parameters ( Figure 7 B). It is worth noting that the SCHB area under the curve (AUC 0～∞ ) were significantly higher than those in the Free SCHB group. The relative bioavailabilities of SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA were 1.38, 1.46 and 1.50 times that of the Free SCHB group, respectively.

[0068] Example 8 Study on the targeting of SCHB@SPC / Gal-BSA / DHA to the liver after oral administration

[0069] Experimental methods: Male C57BL / 6 mice were used as experimental subjects and randomly divided into 4 groups (n=6). Free SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA were orally administered at a dose of 100 mg / kg, respectively. To determine the biodistribution of SCHB, mice were anesthetized and killed at 0.5, 1, 4 and 8 h after administration, and then the heart, liver, spleen, lung and kidney samples of the mice were collected. The tissue samples were rinsed with saline to remove blood, dried with filter paper and weighed. For analysis, about 50 mg of organs were weighed and homogenized with 1 mL of 80% methanol containing IS (100 ng / mg). The samples were then centrifuged at 21 000×g for 5 min, and 200 μL of the supernatant was transferred to a new tube and centrifuged at 21 000×g for 5 min. Finally, a volume of 5 μL of the supernatant was taken for LC-MS / MS analysis. The detailed parameters were the same as described previously.

[0070] Experimental results: This study comprehensively investigated the tissue distribution of SCHB in C57BL / 6 mice after oral nanoemulsion. The liver, heart, spleen, lung, and kidney were collected at 0.5, 1, 4, and 8 h after administration and homogenized to quantify the drug concentration in these tissues. After 0.5 h, SCHB@SPC / Gal-BSA / DHA rapidly accumulated in the liver ( Figure 8 A), indicating superior liver targeting ability. The maximum drug concentration of the SCHB@SPC / Gal-BSA / DHA group reached 1158.30 ng / mg, which was 7.64 times that of the free SCHB group. At the same time, the SCHB@SPC and SCHB@SPC / Gal-BSA groups reached the peak drug concentration in tissues other than the liver within 4 hours, and the difference was not statistically significant ( Figure 8 BE). The liver and kidney showed the highest SCHB concentrations due to increased angiogenesis and perfusion levels, and SCHB may be metabolized in the former and excreted through the latter.

[0071] Example 9 Study on SCHB@SPC / Gal-BSA / DHA promoting liver regeneration after 70% PHx

[0072] Experimental methods: Healthy male C57BL / 6 mice were randomly divided into Control, Model, Free SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA (n=7). For animal models, the 70% PHx model was established according to standard procedures. Except for the Control group, all other groups of mice were given 70% PHx. As described above, SCHB was suspended in 0.5% (w / v) sodium carboxymethylcellulose (CMC-Na) solution. The mice in the Control and Model groups were given 0.5% CMC-Na solution, and the other four groups of mice were given the corresponding dose of 100 mg / kg SCHB preparation. All mice were gavaged 5 minutes after PHx surgery, once a day until the end of the study. Then the mice were killed at the designated time (0, 1, 3, 5 and 7 days). After the treatment, liver tissues were collected and photographed, and the middle part of the right posterior lobe of the mouse liver was pathologically stained, including H&E staining and immunohistochemistry.

[0073] Experimental results: In order to evaluate the effect of SCHB nanoemulsion on liver regeneration, a C57BL / 6 mouse model with 70% PHx was established. Each group of mice took SCHB nanoemulsion 100 mg / kg orally once a day until the end of the experiment. At the same time, the control group and the model group took 0.5% CMC-Na orally every day. On the 7th day after PHx, the liver of the model group gradually became larger, because the liver has a certain regenerative ability, which can make up for the volume loss caused by liver lobe resection to a certain extent. However, the morphological images of the liver treated with the SCHB nanoemulsion group showed that the liver volume increased further after treatment, and the liver volume of mice treated with SCHB@SPC / Gal-BSA / DHA was close to the liver volume of the Control group ( Fig. 9 A). After PHx, the remaining mature hepatocytes initially undergo hypertrophy, enter the cell cycle, and proliferate. Therefore, hepatocyte proliferation was assessed by Ki-67 staining ( Fig. 9 B). After PHx, starting from day 0 (2 hours), the nanoemulsion group quickly started the cell cycle process and entered the proliferation phase, and maintained a high cell proliferation during the experimental observation time. The incidence of Ki-67 positive cells in the SCHB@SPC / Gal-BSA / DHA group was higher than that in other groups. H&E staining results ( Fig. 9 C) showed that the liver tissue cells of the model group mice were arranged in disorder and the nuclear division increased, suggesting that liver tissue edema may be caused by liver damage. The liver tissue cells treated with SCHB or SCHB nanoemulsion had a clear structure, neatly arranged cells, uniform size, and no nuclear division. The results showed that the hepatomegaly induced by SCHB and SCHB nanoemulsion was benign.

[0074] Example 10 Study on SCHB@SPC / Gal-BSA / DHA promoting liver regeneration after in situ PHx of liver cancer

[0075] Experimental methods: First, H22 animal model was established by orthotopic transplantation in male C57BL / 6 mice. Two weeks after H22 was orthotopically transplanted into the liver of normal mice, ultrasound examination was performed using a high-level color Doppler ultrasound instrument with a linear array probe (frequency L8-18i) to confirm the establishment of the orthotopic liver cancer mouse model. Then PHx was performed in orthotopic liver cancer in mice. Control and healthy PHx groups were established. Liver cancer PHx mice were randomly divided into Model, Free SCHB, SCHB@SPC, SCHB@SPC / Gal-BSA and SCHB@SPC / Gal-BSA / DHA (n=8), and 0.5% CMC-Na, Free SCHB or SCHB nanoemulsion were orally administered at a dose of 100 mg / kg once a day for 1 week. Tumor growth was monitored by ultrasound, and tumors with a diameter of about 0.5 cm were selected for surgical resection. At the end of treatment, probe ultrasound was used to monitor the thickness of the liver lobe. The mice were then anesthetized and killed, and the liver tissues were collected for photography. The liver tissues were fixed with 4% paraformaldehyde for 24 h, dehydrated, embedded, and sectioned. The sections were dewaxed to the aqueous phase before staining, and stained with H&E and Ki-67 immunohistochemistry to evaluate liver regeneration after in situ PHx of liver cancer.

[0076] Experimental results: To further study the potential of SCHB nanoemulsion to promote liver regeneration under pathological conditions, an orthotopic hepatocellular carcinoma mouse xenograft model was established, and tumor resection was performed to study the progress of liver regeneration under pathological conditions. The liver regeneration of the Model group mice was slower than that of the normal PHx group. After hepatocellular carcinoma resection, the thickness of the liver lobes of mice treated with Free SCHB and SCHB nanoemulsion increased to varying degrees, among which the thickness of the liver lobes of SCHB@SPC / Gal-BSA / DHA was the most obvious ( Fig.10 B). In addition, representative liver images obtained on day 7 confirmed liver regeneration and showed that the liver diameter after SCHB@SPC / Gal-BSA / DHA treatment reached the normal liver size ( Fig.10 A). These findings confirmed that Free SCHB and its nanoemulsion effectively promoted liver regeneration after orthotopic PHx surgery for liver cancer, and significant regenerative effects were observed in SCHB@SPC / Gal-BSA / DHA. Subsequently, Ki-67 immunohistochemical staining was used to further verify the effect of SCHB on liver regeneration after hepatectomy ( Fig.10 C). On day 7, the proliferation of cells in the normal liver PHx group and the HCC in situ PHx group was significant. H&E staining ( Fig.10 D) showed no liver damage or necrosis after treatment.

Claims

1. An oral liver-targeted nanoemulsion loaded with schisandra chinensis alcohol B, characterized in that: The nanoemulsion is mainly formed by using soybean lecithin, galactosyl protein conjugate and docosahexaenoic acid as carriers and loading schisandra alcohol B; the galactosyl protein conjugate is mainly synthesized by using bovine serum albumin and N-acetyl-D-galactosamine through Maillard reaction.

2. The oral liver-targeted nanoemulsion loaded with Schisandrin B according to claim 1, characterized in that: The mass ratio of bovine serum albumin to N-acetyl-D-galactosamine is 1:(1-4); the reaction conditions of the Maillard reaction are: reacting for 12-48 hours at pH 6-8, 55-65° C., and 75-85% humidity.

3. The method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine according to claim 1 or 2, characterized in that: The following steps are involved: (1) taking a galactosyl protein conjugate, adding deionized water to prepare a solution as the aqueous phase; dissolving soybean lecithin, docosahexaenoic acid and schisandrae alcohol ethyl in oil as the oil phase; (2) Mixing the oil phase and the water phase to prepare the nanoemulsion.

4. The method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine according to claim 3, characterized in that: In step (1), the oil is selected from one or more of medium chain triglycerides, soybean oil, corn oil, peanut oil, and castor oil.

5. The method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine according to claim 3, characterized in that: In step (2), the oil phase and the aqueous phase are mixed, wherein the mass ratio of galactosylated protein to soybean lecithin is (0.25-0.75):1, based on the mass of bovine serum albumin, and the content of docosahexaenoic acid in the oil phase is 10-80 μM, and the mass ratio of docosahexaenoic acid, soybean lecithin and schisandrin B in the oil phase is (0.175-1.4):1:(1-2.56).

6. The method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine according to claim 3, characterized in that: In step (2), the method for preparing the nanoemulsion comprises: mixing the oil phase and the water phase and then ultrasonicating to obtain a primary emulsion; and high-pressure homogenizing the primary emulsion to obtain the nanoemulsion.

7. The method for preparing the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine according to claim 6, characterized in that: The ultrasonic treatment was carried out in an ice water bath at 60-70W for 2.5-3.5min, with a pulse of 2s and a pause of 2s during the ultrasonic treatment. The high-pressure homogenization was carried out at 5000-7000psi for 2-4 times.

8. Use of the oral liver-targeted nanoemulsion loaded with schisandra chinensis acetylcholine as claimed in claim 1 or 2 in the preparation of a drug for promoting liver regeneration.

9. The use according to claim 8, characterized in that: The liver regeneration is liver regeneration after PHx surgery for liver cancer.

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