Hydrogel containing liver microsome and preparation method thereof

Abstract

The invention belongs to the field of biological manufacturing and tissue engineering, and particularly relates to hydrogel containing liver microsomes and a preparation method of the hydrogel. According to the hydrogel, an in-vitro liver metabolism module with long-term stability and high physiological correlation is constructed by integrating the metabolic function of liver microsomes and a bionic 3D matrix, and the hydrogel is used for drug metabolism evaluation and toxicity dynamic monitoring. Through the synergistic effect of the photosensitive matrix and the temperature response material, efficient loading of liver microsomes and long-acting maintenance of enzyme activity are achieved, the problems that in a traditional static culture system, the metabolic rate is unstable, the enzyme activity is prone to inactivation and the like are solved, and a more reliable in-vitro prediction tool is provided for drug research and development.

Description

Background Art

[0001] Hepatic metabolism is generally considered a key determinant in drug development. To understand drug biotransformation, a variety of in vitro metabolic studies have been established and applied. However, these traditional methods have inherent flaws, leading to inconsistent results between in vitro and in vivo experiments—primarily because these static culture systems fail to mimic the dynamic transport processes in the liver. During drug development, in vitro studies often take precedence over in vivo experiments (particularly human trials). In the early stages of drug discovery and development, in vitro data can not only be used to rapidly screen compounds, but also to elucidate the mechanism of drug action and provide a basis for subsequent research and decision making [1.Kim J, Hayward RC. Mimicking dynamic in vivoenvironments with stimuli-responsive materials for cell culture. TrendsBiotechnol 2012;30:426e39.2.Brandon EF, Raap CD, Meijerman I, Beijnen JH,Schellens JH. An update on in vitro testmethods in human hepatic drugbiotransformation research: pros and cons. Toxicol Appl Pharmacol 2003;189:233e46.].

[0002] The liver, a crucial metabolic organ in the human body, undertakes key functions in drug metabolism and clearance. Drug metabolites may be more active or toxic than the parent drug, directly impacting drug efficacy and safety in humans. Therefore, hepatic biotransformation is a key factor in determining the overall disposition characteristics of a drug, necessitating the development of reliable in vitro metabolic systems to accurately predict human metabolic behavior. Currently, a variety of in vitro metabolic systems have been developed to study liver extraction processes, including hepatocytes, microsomes, cytosol, S-9 fractions, and liver slices derived from humans or experimental animals [3. Meng Q. Three-dimensional culture of hepatocytes for prediction of drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 2010;6:733e46. 4. Lee MY. Three-dimensional cellular microarray for high-throughput toxicology assays. Proc Natl Acad Sci USA, 2008;105:59e63]. These models can be used to determine kinetic parameters such as the maximum reaction rate (Vmax) and Michaelis constant (Km) in vitro, and the in vivo intrinsic clearance can be calculated based on the intrinsic clearance (CLint=Vmax / Km) under linear conditions using enzyme content scaling [Ma B, Zhang G, Qin J, Lin B. Characterization of drugmetabolites and cytotoxicity assay simultaneouslyusing an integrated microfluidic device. Lab Chip 2009;9:232e8.].

[0003] Currently, in vitro liver metabolism research mainly relies on hepatocytes, microsomes, or recombinant enzyme systems, but these models have significant limitations: primary hepatocytes are scarce and easily inactivated during culture; microsomes have a short incubation time, making it difficult to simulate long-term metabolic processes; and static culture systems cannot reproduce the dynamic microenvironment of hepatic sinusoids. In addition, in traditional methods, microsomes often exist in a free form, resulting in rapid attenuation of enzyme activity due to uneven spatial distribution or an unsuitable microenvironment. Although studies have attempted to encapsulate microsomes in hydrogels to prolong activity, their mechanical properties and biocompatibility are still insufficient to support the long-term stable expression of complex metabolic functions. Therefore, there is an urgent need to develop a new hydrogel system that combines dynamic responsiveness, biomimetic matrix properties, and efficient enzyme loading capacity. Summary of the Invention

[0004] To address technical issues in existing in vitro liver metabolism models, such as short-lived liver microsome activity, uneven spatial distribution of metabolic enzymes, and inadequate simulation of dynamic metabolic function, the present invention provides a liver microsome-containing hydrogel, its preparation method, and its application. Through innovative material combinations and structural design, this hydrogel achieves long-term, stable expression of liver microsomal metabolic function, providing a more reliable in vitro evaluation platform for drug metabolism research.

[0005] To address the above-mentioned technical problems, the present invention provides a hydrogel containing liver microsomes and a method for its preparation. The hydrogel comprises two hydrogel systems with different crosslinking characteristics: a photocrosslinking system and a temperature-sensitive crosslinking system. This approach improves the internal and external unevenness of photocrosslinked hydrogel systems and the difficulty in controlling gel viscosity in temperature-sensitive systems. During preparation, the basic hydrogel form is first constructed using a temperature-sensitive gel, and then photocrosslinking is performed using a 3D printer to create hydrogel systems of varying shapes. The photocrosslinking system comprises two acrylic polymer systems: methacryloylated sodium alginate (MA-Alg) and polyethylene glycol diacrylate (PEG-DA). The crosslinking of MA-Alg achieves initial shaping, while the photocrosslinking of PEG-DA further strengthens the structure and prevents printing collapse. Together, these two systems enable a three-dimensional dynamic model of liver microsomes, enabling an in vitro drug metabolism evaluation system that closely resembles real-world scenarios.

[0006] The hydrogel system of this invention possesses the following key features: First, MA-Alg is used as a photosensitive matrix with a methacrylation degree of 60-80%, ensuring efficient photocrosslinking. Second, a temperature-responsive network is constructed using a poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer (PLGA-PEG-PLGA), which, together with PEG-DA, forms a dual-curing mechanism, resulting in the hydrogel possessing both excellent mechanical properties (storage modulus 500-2500 Pa) and controllable sol-gel transition characteristics (28-32°C). Third, the addition of decellularized liver extracellular matrix (dECM) retains ≥70% of native collagen IV, laminin, and growth factors, providing a biomimetic microenvironment for liver microsomes. Specifically, freeze-dried liver microsomes with a particle size of ≤0.9 μm are uniformly dispersed in the hydrogel, retaining ≥85% of enzyme activity. Furthermore, a gradient loading method is employed to avoid activity loss caused by localized high concentrations.

[0007] The preparation method of this invention includes the following key steps: first, MA-Alg is dissolved in pre-chilled PBS, followed by the sequential addition of PLGA-PEG-PLGA, PEG-DA, dECM, and a photoinitiator to form a homogenous precursor solution; then, the gel is formed by 3D printing combined with light irradiation (2-5 minutes); and finally, liver microsomes are loaded using a gradient method to ensure uniform distribution within the gel. This method is simple to operate, has good reproducibility, and is suitable for large-scale production.

[0008] This hydrogel demonstrates significant advantages in its applications: when used as a dynamic metabolic module in a microfluidic chip, it maintains over 75% CYP450 enzyme activity and maintains excellent structural integrity (weight loss ≤15%) for seven days. Compared to traditional free microsomal systems, its metabolic stability is more than two-fold improved, enabling more accurate prediction of drug interaction risks at clinical doses. Furthermore, this hydrogel can serve as a spacer in a "liver microsome-liver matrix" co-culture system, enabling simultaneous evaluation of drug metabolism and toxicity, providing a valuable in vitro screening tool for innovative drug development.

[0009] Furthermore, the hydrogel liver microsome system includes a photosensitive matrix: methacrylated sodium alginate; a polymer network: a polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid triblock copolymer bioactive scaffold, a polyethylene glycol diacrylate biomimetic matrix, a decellularized liver extracellular matrix, a photoinitiator (LAP or Irgacure 2959), and a load: liver microsomes, which contain CYP3A4, CYP2C9, CYP2D6, and CYP2C19 metabolic enzyme systems.

[0010] Furthermore, in the hydrogel, the molecular weight of methacrylated sodium alginate is 80-100 kDa; the molecular weight of the poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer is 4000-6000 Da, of which the molecular weight of PEG is 1500-3000 Da; the ratio of poly(lactic-co-glycolic acid) is 75:25, and the terminal segment is an ester. The molecular weight of polyethylene glycol diacrylate is 600-700 Da. Furthermore, in the hydrogel, the weight ratio of methacrylated sodium alginate, polylactic acid glycolic acid-polyethylene glycol-polylactic acid glycolic acid triblock copolymer and polyethylene glycol diacrylate (PEG-DA, 0.08-0.5 wt%) is 1:(0.2-0.3):(0.2-0.3).

[0011] Furthermore, in the hydrogel, the concentration of methacrylated sodium alginate is 0.1%-0.5%; the polymer network: the concentration of polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid triblock copolymer is 0.02%-0.15%, the concentration of polyethylene glycol diacrylate is 0.02%-0.15%, the concentration of decellularized liver extracellular matrix (dECM) is 0.01%-0.03%, the concentration of photoinitiator (LAP or Irgacure 2959) is 0.01%-0.03%, and the concentration of liver microsomes is 0.1 mg / ml-1 mg / ml. DETAILED DESCRIPTION

[0012] MA-Alg used in the examples has a Mw of 80-100 kDa and was purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. PEG-DA Mw: 600-700 Da, purchased from Shanghai Chuangsai Technology Co., Ltd.; PLGA-PEG-PLGA Mw: 4500 Da, purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.; dECM was purchased from Beijing Dikang Pharmaceutical Investment Management Co., Ltd.

[0013] Example 1: Preparation of moderate metabolic hydrogel 1. Material Preparation: Weigh 2g of MA-Alg and dissolve it in 4°C pre-chilled PBS to a concentration of 0.2%. Add 1g of PLGA-PEG-PLGA and 1g of PEG-DA and vortex to mix thoroughly. Add 0.7g of dECM and 0.2g of LAP in sequence, protected from light, and stir at 4°C for 0.5 hours. 2. 3D printing: Inject the mixture into the bioprinter and irradiate with UV light for 3 minutes at 4°C to print a 0.1 cm × 0.1 cm × 0.1 cm block of gel. 3. Microsome loading: Freeze-dried human liver microsomes (0.5 mg / mL) were gradiently mixed into the gel precursor and stirred at 4°C for 30 minutes.

[0014] Hydrogel Viscosity Measurement: The stirred mixture obtained in step 1 above was placed on a flat plate rheometer (20 mm diameter fixture) for measurement. The storage modulus (G') and loss modulus (G'') of the hydrogel during photogelation were plotted over time. The gel point is defined as the intersection where G' exceeds G''. The time sweep test parameters were as follows: 0.01% strain, 1 rad / s angular frequency, 0.2 mm gap between fixture and platform, and 900 s photoirradiation time. Results: G' (37°C): 2000 Pa.

[0015] Example 2: Preparation of highly active hydrogel 1. Material Preparation: Weigh 5g of MA-Alg and dissolve it in 4°C pre-chilled PBS to a concentration of 0.5%. Add 1.5g of PLGA-PEG-PLGA and 1.5g of PEG-DA and vortex to mix thoroughly. Add 1g of dECM and 0.3g of LAP in sequence, in the dark, at 4°C and stir for 0.5 hours. 2. 3D printing: Inject the mixture into a bioprinter and irradiate with UV light for 3 minutes at 4°C to print a 0.1 cm × 0.3 cm × 0.1 cm block of gel. 3. Microsome loading: Freeze-dried human liver microsomes (1 mg / mL) were gradiently mixed into the gel precursor and stirred at 4°C for 30 minutes.

[0016] Example 3: Preparation of basal metabolic hydrogel 1. Material Preparation: Weigh 1g of MA-Alg and dissolve it in 4°C pre-chilled PBS to a concentration of 0.1%. Add 0.2g of PLGA-PEG-PLGA and 0.2g of PEG-DA and vortex to mix thoroughly. Add 0.4g of dECM and 0.1g of LAP in sequence, in the dark, at 4°C and stir for 0.5 hours. 2. 3D printing: Inject the mixture into a bioprinter and irradiate with UV light for 3 minutes at 4°C to print a 0.1 cm × 0.3 cm × 0.1 cm block of gel. 3. Microsome loading: Freeze-dried human liver microsomes (0.1 mg / mL) were gradiently mixed into the gel precursor and stirred at 4°C for 30 minutes.

[0017] Example 4: Experimental steps of the microfluidic chip liver microsome hydrogel system (taking midazolam metabolism as an example) (1) Hydrogel preparation. MA-Alg, PLGA-PEG-PLGA, and PEG-DA were dissolved in 4°C pre-cooled PBS. The photoinitiator LAP and freeze-dried human liver microsomes were added at concentrations similar to those in Example 1. After vortex mixing, the mixture was allowed to stand in the dark for 30 minutes to form a homogeneous precursor solution. The mixture was injected into a bioprinter and irradiated with UV light for 3 minutes at 4°C to print a 0.1 cm × 0.5 cm × 0.1 cm strip of gel. (2) Microfluidic chip loading. The gel was placed in the main channel of the PDMS microfluidic chip and irradiated with UV light for 60 seconds to solidify the hydrogel to form an embedded liver microsome hydrogel column (5 mm in length). The side channel was perfused with 37°C preheated hepatocyte culture medium (containing 10% FBS) and equilibrated for 2 hours. (3) Midazolam metabolism experiment. PBS buffer containing midazolam (10 μM) and NADPH regeneration system (1 mM) was pumped in at a flow rate of 0.5 μL / min and incubated at 37°C in the dark. The effluent was collected from the outlet at 0, 1, 2, 4, 6, and 8 hours, and an equal volume of acetonitrile was added to terminate the reaction. The supernatant was collected by centrifugation at 12,000 rpm for 10 minutes. (4) Metabolite detection. UPLC-MS / MS was used to analyze the amount of 1-hydroxymidazolam produced. Chromatographic conditions: C 18 The column was 2.1 × 50 mm, 1.7 μm, and the mobile phase consisted of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL / min. Mass spectrometry conditions included positive ESI mode and multiple reaction monitoring (MRM) of midazolam (m / z 326→291) and 1-hydroxymidazolam (m / z 342→297). The metabolic rate (pmol / min / mg protein) and intrinsic clearance (CL) were calculated and compared with those in a static incubation system (96-well plate).

[0018] Note: ΔC: Change in metabolite concentration (pmol / mL, calculated by standard curve).

[0019] Δt: incubation time (min). W: protein amount (mg), protein concentration of microsomes / cell lysate in the incubation system (mg / mL).

[0020] Dilution factor: The dilution ratio of the sample during pretreatment (1 if undiluted).

[0021] Note: CL: intrinsic clearance (μL / min / mg); V1: metabolic rate (pmol / min / mg); V2: incubation volume (ml); W: protein amount (mg); C: initial substrate concentration (μmol / L); Application Verification The hydrogel from Example 1 was placed in a microfluidic chip, and midazolam (10 μM) and the NADPH regeneration system were perfused at a flow rate of 1 μL / min. HPLC-MS / MS analysis showed that after 7 days, the amount of 1-hydroxymidazolam generated remained at 78% of the initial value, which was significantly higher than that of the free microsome group (35%). Comparative Example 1: Single cross-linked hydrogel When only MA-Alg was used (without PLGA-PEG-PLGA and PEG-DA), the gel formation time was extended to 8 minutes, and the enzyme activity retention rate after microsome loading was reduced to 69.21%. Comparative Example 2: No dECM System After removal of dECM, the enzyme activity retention after microsome loading dropped to 68.75%, indicating that dECM plays a key role in enzyme stability. Comparative Example 3: No MA-Alg system After removing MA-Alg, the gel formation time was extended to 12 min, and the enzyme activity retention rate after microsome loading decreased to 65.39%. Comparative Example 4: Free Liver Microsomes Group Without using a gel system or dECM, a conventional incubation system was used. The incubation system (200 μL PBS buffer containing 0.5 mg / mL liver microsomes, 1-10 μM midazolam, and 1 mM NADPH regeneration system) was first prepared. After a 5-minute preincubation at 37°C, the reaction was initiated by the addition of NADPH. At time points of 0, 5, 10, 20, and 30 minutes, 50 μL of the reaction solution was aspirated and terminated with 150 μL acetonitrile (containing an internal standard). After centrifugation, the supernatant was collected for LC-MS / MS analysis. Metabolite production was used to calculate the metabolic rate (pmol / min / mg protein) and intrinsic clearance (CL = metabolic rate / substrate concentration × 1000). Negative controls without NADPH and a positive control for testosterone were also included to verify system activity. Results showed that enzyme activity retention decreased to 35.51% after microsomal loading.

[0022] Experimental data

Claims

1. A hydrogel containing liver microsomes, characterized in that: It is composed of the following components: photosensitive matrix: sodium methacrylated alginate; polymer network: poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer, polyethylene glycol diacrylate biomimetic matrix, decellularized liver extracellular matrix (dECM), photoinitiator (LAP or Irgacure2959); load: liver microsomes, which contain CYP3A4, CYP2C9, CYP2D6, and CYP2C19 metabolic enzyme systems.

2. The hydrogel according to claim 1, wherein The molecular weight of the methacrylated sodium alginate is 80-100 kDa; the molecular weight of the polylactic-co-glycolic acid-polyethylene glycol-polylactic-co-glycolic acid triblock copolymer is in the range of 4000-6000 Da, wherein the molecular weight of PEG is 1500-3000 Da; the ratio of polylactic-co-glycolic acid is 75:25, and the terminal segment is an ester end; the molecular weight of polyethylene glycol diacrylate is 600-700 Da.

3. The hydrogel according to claim 1, wherein The weight ratio of the methacrylated sodium alginate, the polylactic acid glycolic acid-polyethylene glycol-polylactic acid glycolic acid triblock copolymer and the polyethylene glycol diacrylate is 1:(0.2-0.3):(0.2-0.3).

4. The hydrogel according to claim 1, wherein The concentration of methacrylated sodium alginate is 0.1%-0.5%; the polymer network: the concentration of poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer is 0.02%-0.15%, the concentration of polyethylene glycol diacrylate is 0.02%-0.15%, the concentration of decellularized liver extracellular matrix (dECM) is 0.04%-0.1%, the concentration of photoinitiator (LAP or Irgacure 2959) is 0.01%-0.03%, and the concentration of liver microsomes is 0.1-1 mg / ml.

5. The hydrogel according to claim 1, wherein The methacrylation degree of the methacrylated sodium alginate is 60-80%.

6. A method for preparing the hydrogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: a. Sterilize methacrylated sodium alginate with acetone and dissolve in 4°C pre-cooled PBS to form a clear solution. b. Add poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) triblock copolymer and vortex mix; c. Add polyethylene glycol diacrylate, dECM and photoinitiator and stir for 0.5 hours in the dark; d. Place it in a 3D printer and use the photocrosslinking mode to irradiate it with UV-visible light while printing the hydrogel shape, which is in the form of a block with a size of 0.001 cm 3 - 0.20 cm 3 e. Add the freeze-dried liver microsomes to the mixture in step d according to the final concentration; f. Stir at 300-500 rpm for 30 minutes under sterile conditions at 4°C to complete the loading of the liver microsomes in the hydrogel.

7. The method according to claim 6, characterized in that In step d, the liver microsomes were loaded using a gradient loading method: the gel precursor was first pre-mixed at a concentration of 0.1 mg / mL, and then gradient mixing and adsorption were performed to the target concentration.

8. Use of the hydrogel according to any one of claims 1 to 5 in in vitro drug metabolism evaluation, characterized in that: Used to construct a liver microsome-liver matrix co-culture system as a dynamic metabolic module in a microfluidic chip, for a high-throughput in vitro evaluation platform of liver metabolism.

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