Porous material with endotoxin adsorption function and preparation method thereof
By using porous hydrogel materials to absorb endotoxins using hydrophobic and electrostatic interactions, the problems of limited adsorption efficiency and capacity in the prior art are solved, and efficient and stable endotoxin removal effect is achieved.
Patent Information
- Application Number
- CN202510036003.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art has problems with limited adsorption efficiency and capacity in removing endotoxins, and the microcarriers modified with charged functional groups are poor in stability and are easily inactivated by temperature and pH.
Porous hydrogel materials are used to produce hydrophobic and electrostatic interactions with endotoxins through the structure of the material molecules themselves, and combined with the porous structure to increase the contact area, achieving efficient adsorption of endotoxins.
This material has excellent endotoxin adsorption ability, can efficiently and quickly adsorb endotoxins in liquids, and has good stability and is not easily affected by temperature and pH.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedical materials, and in particular relates to a preparation and use method of a porous hydrogel material with endotoxin adsorption function. Background Art
[0002] Endotoxin is a heterogeneous polymer structure, consisting of three parts: lipid A region, core polysaccharide region and O-antigen region. Hydrophobic lipid A is the active center and toxicity center of endotoxin. It is composed of a hydrophilic skeleton and a hydrophobic fatty chain, and contains C14 and C12 acyl chains and negative phosphate groups. Endotoxin molecules show a net negative charge in the physiological environment. The presence of lipid A makes endotoxin molecules have the properties of causing biological effects, forming macromolecules and strong heat resistance. Endotoxins are released into the environment during bacterial cell death, lysis, growth and proliferation, and are ubiquitous and potentially threatening pyrogens. Endotoxins show high toxicity in animals. Even if a small amount of endotoxin enters the animal body, it will induce systemic inflammatory response syndrome, multiple organ failure syndrome and sepsis, thereby endangering the life safety of the animal. When endotoxins are present in large quantities, they will cause septic shock through cytokine signals, leading to intravascular coagulation and even death. Various diseases caused by endotoxins seriously endanger human health. Therefore, the pharmacopoeias and regulations of various countries have strict endotoxin threshold limits for parenteral drugs and implantable medical devices that come into direct contact with human blood and myelin.
[0003] The main methods for removing endotoxins include chemical decomposition by strong acids, strong bases or strong oxidants, as well as ultrafiltration, extraction, affinity adsorption, etc. Ultrafiltration is one of the membrane separation technologies driven by pressure. It is often used to remove endotoxins from salts, small molecule therapies or water, but it is not suitable for most separation schemes. Extraction is an early method used to remove endotoxins from proteins. This method provides a high endotoxin removal efficiency for highly contaminated samples, but may result in product loss or residues in the target component. Affinity adsorption is a more ideal method for removing endotoxins. It uses the interaction between the structure of endotoxins themselves (a large number of negative phosphates, carbon-carbon double bonds and alkyl hydrophobic chains) and the adsorbent (electrostatic interactions, π-π interactions and hydrophobic interactions) to achieve the removal of endotoxins.
[0004] Commercially available adsorption products are usually based on the principle of cationic affinity adsorption. The single action principle limits their efficiency and capacity for endotoxin adsorption. Products are often in the form of microsphere fillers, and the uniformity of filler filling will greatly affect the adsorption and separation effect. In addition, the charged functional groups modified on the surface of the microcarriers have poor stability and are easily detached or inactivated by temperature and pH. Summary of the invention
[0005] The present invention introduces a method for preparing a porous material with endotoxin adsorption function. The adsorbent generates hydrophobic and electrostatic interactions between the material molecules and endotoxins. The porous structure of the adsorbent increases the contact area with the endotoxins, and the combination of the hydrophobic and electrostatic interactions makes the material have excellent adsorption capacity. The material is easy to use and can efficiently adsorb endotoxins in liquid materials.
[0006] The present invention introduces a method for preparing a porous hydrogel material with endotoxin adsorption function. The material with a large number of amino groups and hydrophobic molecular chains is used to produce hydrophobic and electrostatic interactions with endotoxins, and the endotoxins are adsorbed efficiently and quickly, thereby achieving the effect of removing endotoxins from liquid materials.
[0007] A method for preparing a porous material having the function of adsorbing endotoxins comprises the following steps:
[0008] (1) dissolving methacryloyl chitosan, methacryloyl polylysine, polyether F127 diacrylate and a photoinitiator in an acetic acid solution to obtain a hydrogel precursor solution;
[0009] (2) using a dispersed phase to fully disperse the prepared hydrogel precursor solution;
[0010] (3) photocuring the obtained fully dispersed system;
[0011] (4) The photocured hydrogel is freeze-dried, then swollen and perforated under negative pressure, and then soaked in an alkaline solution (such as NaOH solution). Finally, the sample is washed and freeze-dried to obtain a porous hydrogel material.
[0012] Furthermore, in step (2), a porous sieve membrane connector is used to connect the syringes containing the dispersed phase and the gel precursor solution respectively, and the two syringes are injected alternately, which is repeated multiple times to mix the two phases into a foam state.
[0013] More specifically, a porous material having endotoxin adsorption function comprises the following steps:
[0014] (1) preparing a hydrogel precursor solution: adding methacrylated chitosan, methacrylated polylysine, polyether F127 diacrylate and a photoinitiator to an acetic acid solution for dissolution;
[0015] (2) Add the prepared hydrogel precursor solution (continuous phase) into a syringe, and add the dispersed phase into another syringe in a certain proportion; Figure 1 As shown;
[0016] (3) Connect the two syringes mentioned above to the two sides of the porous sieve membrane connector respectively, inject the dispersed phase into the continuous phase, and then inject the continuous phase into the dispersed phase. Repeat the above operation n times and irradiate with a light source of fixed wavelength for curing.
[0017] (4) freeze-drying the hydrogel prepared in step (3), slicing it, swelling it with negative pressure, soaking it in an alkaline solution, washing it, and freeze-drying it to obtain a porous hydrogel material.
[0018] Furthermore, in step (1), the mass percentage concentration of the acetic acid (water) solution is 1 to 5%, and more preferably 2%.
[0019] Furthermore, in step (1), in order to speed up the dissolution rate, the materials can be dissolved in a certain order, for example, the photoinitiator can be first dissolved in an acetic acid solution to prepare a photoinitiator solution, and then the methacrylated chitosan and methacrylated polylysine are dissolved in the photoinitiator solution, and stirred at room temperature until completely dissolved. Then, polyether F127 diacrylate is dissolved in the above solution.
[0020] Furthermore, in step (1), the molecular weight of methacryloyl chitosan is 50-300 kDa, and the degree of substitution is 30-50%. The molecular weight of methacryloyl polylysine is 3-10 kDa, and the degree of substitution is 20-30%. The molecular weight of polyether F127 diacrylate is 15 kDa, and the degree of substitution is ≥90%. Among them, methacryloyl chitosan with a molecular weight of 100 kDa and a degree of substitution of 40% and methacryloyl polylysine with a molecular weight of 4 kDa and a degree of substitution of 25% are preferred.
[0021] Further, in step (1), the mass ratio of methacryloyl chitosan, methacryloyl polylysine, polyether F127 diacrylate, photoinitiator and acetic acid solution is 1-3: 2-6: 0.5-2: 0.1-1: 100. As further preferred, in step (1), the mass ratio of methacryloyl chitosan, methacryloyl polylysine, polyether F127 diacrylate, photoinitiator and 2% acetic acid solution is 1-2: 2-5: 0.5-2: 0.1-1: 100.
[0022] Furthermore, in the hydrogel precursor solution, the mass fraction of the methacrylylated chitosan is 1% to 2%, and the mass fraction of the methacrylylated polylysine is 2% to 5%. They not only have good biocompatibility, but also can participate in photopolymerization reaction after methacrylylation modification to form a stable hydrogel structure.
[0023] Furthermore, in step (1), in the hydrogel precursor solution, the mass fraction of the polyether F127 diacrylate is 0.5% to 2%. The material acts as a pore-forming stabilizer and a cross-linking agent, so that the continuous phase and the dispersed phase form a more stable foam in the subsequent process.
[0024] Furthermore, the photoinitiator in step (1) is preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and its mass fraction in the hydrogel precursor solution is 0.1% to 1%, preferably 0.2 to 0.3%, and more preferably 0.25%.
[0025] Furthermore, in step (2), the dispersed phase is an inert gas, such as one or more of argon, nitrogen and helium.
[0026] Furthermore, in step (2), the volume ratio of the dispersed phase to the continuous phase is 1:1 to 1:5, preferably 1:2 to 1:4; and more preferably 1:3.
[0027] Furthermore, the pore size of the porous sieve membrane in the porous sieve membrane connector can be selected to be 0.05-50 um.
[0028] Furthermore, the above operation is repeated n times as described in step (3), where n>2, and the two phases in the syringe are mixed into a foam state. In this step, the inert gas is evenly dispersed in the precursor solution (continuous phase) by using a porous sieve membrane, which helps to form a pore structure during the polymerization process and improve the adsorption efficiency.
[0029] Furthermore, in step (3), the wavelength of the light curing source is 350-450nm, and the curing time is 15s-3min; as a further preference, the fixed wavelength of the light source is preferably 405nm, and the curing time is 15s-3min, preferably 1min.
[0030] Furthermore, in step (4), the freeze-drying process is to first freeze the hydrogel at -90 to -70°C for 2 to 5 hours, and then transfer it to a freeze dryer for freeze drying. Furthermore, in step (4), the freeze-drying process is to first freeze the hydrogel for 3 hours, and then transfer it to a freeze dryer for freeze drying. It is preferred to freeze at -80°C or lower. Ice crystals will be smaller when the temperature drops rapidly, which will have less impact on the pore structure of the hydrogel block itself, and the pores of the hydrogel obtained after freeze drying will be more uniform and connected.
[0031] Furthermore, in step (4), the hydrogel swells and passes through the negative pressure hole, and the specific operation is: soak the hydrogel in water for 4-8 hours. After it is completely swollen, place it in an environment of -0.05 to -0.08 MPa for 2 to 3 minutes, and then take it out to a normal environment. Repeat the above operation 3 to 10 times until the hydrogel is uniform and transparent.
[0032] Furthermore, in step (4), the base includes one or more of sodium hydroxide and potassium hydroxide.
[0033] Further, taking sodium hydroxide solution as an example, in step (4), the hydrogel is immersed in NaOH solution, and the specific washing operation is: the hydrogel after swelling negative pressure through hole is immersed in 0.1-0.3 mol / L NaOH solution for 1-3 hours, and then washed with ultrapure water with endotoxin <0.001EU / mL until the conductivity of the washing liquid is less than 50uS / cm. Further, the hydrogel is immersed in 0.15 mol / L NaOH solution for 3 hours, and then washed with ultrapure water (endotoxin <0.001EU / mL) until the conductivity of the washing liquid is less than 50uS / cm. NaOH plays a role in secondary physical cross-linking and endotoxin removal.
[0034] The porous hydrogel material obtained by the present invention can be soaked in a high salt solution when in use to further improve the adsorption activity of the hydrogel. For example, before use, the porous hydrogel material can be placed in a salt solution of 10-30mM (such as 20mM (mmol / L)) PB and 0.5-2mol / L (such as 1M) NaCl, which is 5 times the volume of the hydrogel, and soaked for about 0.5-2h (such as 1h), and then washed with ultrapure water (endotoxin <0.001EU / mL) until the conductivity of the washing liquid is less than 50uS / cm. The above operation plays a role in cleaning and activating the porous hydrogel material, which can further improve the adsorption efficiency of endotoxin.
[0035] At the same time, after use, the hydrogel of the present invention can be regenerated and reused by soaking in the high-salt solution, so as to remove endotoxins by filtering when using different materials.
[0036] The present invention uses chitosan and polylysine, which not only have good biocompatibility, but also can utilize a large number of amino groups and hydrophobic regions on the molecules to produce hydrophobic effects and electrostatic interactions with endotoxins, thereby adsorbing endotoxins to achieve the effect of removing endotoxins from liquid materials. After methacrylation modification, free radicals can be generated by a photoinitiator, so that the double bonds on the modified chitosan and polylysine undergo free radical polymerization reactions, thereby solidifying the hydrogel and forming a stable hydrogel structure. Using polyether F127 diacrylate as a pore-forming stabilizer and cross-linking agent can make the continuous phase and the dispersed phase form a more stable foam during step (3). A large number of pore structures can greatly improve the efficiency of endotoxin adsorption. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 Schematic diagram of the preparation process of porous hydrogel using porous sieve membrane in the present invention.
[0038] Figure 2This is the SEM image of the porous hydrogel material prepared in Example 1.
[0039] Figure 3 This is the SEM image of the porous hydrogel material prepared in Example 2.
[0040] Figure 4 The endotoxin adsorption amount of the porous hydrogel materials prepared in Examples 1 and 2 of the present invention when adsorbing endotoxin (the results are represented by a bar graph). DETAILED DESCRIPTION
[0041] In order to make the present invention easier to understand, the present invention is further described in detail with reference to the following drawings and embodiments:
[0042] Example 1: A method for preparing a porous material with endotoxin adsorption function, comprising the following steps:
[0043] Prepare hydrogel precursor solution: first dissolve 0.01g phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in 4ml 2% acetic acid solution to prepare a photoinitiator solution. Then dissolve 0.08g methacryloyl chitosan (100kDa, degree of substitution 40%) and 0.2g methacryloyl polylysine (4kDa, degree of substitution 25%) in the photoinitiator solution, and stir at room temperature until completely dissolved. Dissolve 0.04g polyether F127 diacrylate (15kDa, degree of substitution ≥90%) in the above solution and refrigerate at 4°C to dissolve.
[0044] Transfer the hydrogel precursor solution into a 5 ml syringe;
[0045] Another 5 ml syringe collects 2 ml of argon;
[0046] Connect two syringes to the two sides of the porous sieve membrane connector respectively, inject the dispersed phase into the continuous phase, and then inject the continuous phase into the dispersed phase. Repeat the above operation 6 times to produce relatively uniform foam.
[0047] Irradiate with a light source with a wavelength of 405 nm for 1 min, during which the syringe is rotated to ensure uniform irradiation.
[0048] The porous sieve membrane connector was removed, the hydrogel was completely pushed out, the hydrogel was frozen at -80°C for 3 hours, and then transferred to a freeze dryer for freeze drying. The freeze-dried porous hydrogel was cut into porous hydrogel sheets with a thickness of about 2 mm.
[0049] Place the porous hydrogel sheet in a container filled with ultrapure water, soak for 6 hours, place the container in a -0.05MPa environment for 2 minutes, and take it out to a normal environment. Repeat the above operation 5 times until the hydrogel is uniform and transparent.
[0050] The hydrogel was immersed in a 0.15 mol / L NaOH solution for 3 h, and then washed with ultrapure water (endotoxin < 0.001 EU / mL) until the conductivity of the washing solution was less than 50 uS / cm.
[0051] The hydrogel was placed in a -80°C environment and frozen for 3 h, and then transferred to a freeze dryer for freeze drying.
[0052] Example 2: A method for preparing a porous material with endotoxin adsorption function, comprising the following steps:
[0053] Prepare a hydrogel precursor solution, the solution is the same as that in Example 1;
[0054] Transfer the hydrogel precursor solution into a 5 ml syringe;
[0055] Another 5 ml syringe collects 2 ml of argon;
[0056] Connect two syringes to the two sides of the porous sieve membrane connector respectively, inject the dispersed phase into the continuous phase, and then inject the continuous phase into the dispersed phase. Repeat the above operation 6 times to produce relatively uniform foam.
[0057] Irradiate with a light source with a wavelength of 405 nm for 1 min, during which the syringe is rotated to ensure uniform irradiation.
[0058] The porous sieve membrane connector was removed, the hydrogel was completely pushed out, the hydrogel was frozen at -80°C for 3 hours, and then transferred to a freeze dryer for freeze drying. The freeze-dried porous hydrogel was cut into porous hydrogel sheets with a thickness of about 2 mm.
[0059] The hydrogel was immersed in a 0.15 mol / L NaOH solution for 3 h, and then washed with ultrapure water (endotoxin < 0.001 EU / mL) until the conductivity of the washing solution was less than 50 uS / cm.
[0060] The hydrogel was placed in a -80°C environment and frozen for 3 h, then transferred to a freeze dryer for freeze drying;
[0061] The SEM images of the porous hydrogel materials prepared in Example 1 and Example 2 are shown in FIG. Figure 2 and Figure 3 ,Depend on Figure 2 It can be seen that the porous hydrogel material prepared in Example 1 has better internal pore connectivity.
[0062] Example 3: Adsorption capacity of porous hydrogel materials for endotoxin
[0063] The porous hydrogel prepared in Examples 1 and 2 (about 0.5 g) was placed in 10 mL of a salt solution of 20 mM PB (phosphate buffer) and 1 M NaCl and soaked for about 1 hour, and then washed with ultrapure water (endotoxin < 0.001 EU / mL) until the conductivity of the washing solution was less than 50 uS / cm. The porous hydrogel was then placed in a pyrogen-free shaking bottle, and 5 mL of endotoxin working standard was added to prepare a 100 EU / ml solution, which was fully shaken and allowed to stand for 5 minutes before the supernatant was collected. The endotoxin content of the supernatant was determined using a kit. The results are shown in FIG. Figure 4 As shown by Figure 4 It can be seen that the amount of endotoxin adsorbed by the porous hydrogel material prepared in Example 1 is greater than that of the material prepared in Example 2. Since the negative pressure perforation operation was not performed in Example 2, the pore penetration of the prepared material was poor, which affected its endotoxin adsorption capacity.
Claims
1. A method for preparing a porous material having the function of adsorbing endotoxin, characterized in that: The steps include: (1) dissolving methacryloyl chitosan, methacryloyl polylysine, polyether F127 diacrylate and a photoinitiator in an acetic acid solution to obtain a hydrogel precursor solution; (2) using a dispersed phase to fully disperse the prepared hydrogel precursor solution; (3) photocuring the obtained fully dispersed system; (4) freeze-drying the hydrogel obtained by photocuring, swelling it under negative pressure, and then soaking it in an alkaline solution. Finally, the sample is washed and freeze-dried to obtain a porous hydrogel material.
2. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: The molecular weight of the methacryloyl chitosan is 50-300 kDa, and the degree of substitution is 30-50%; the molecular weight of the methacryloyl polylysine is 3-10 kDa, and the degree of substitution is 20-30%; the molecular weight of polyether F127 diacrylate is 15 kDa, and the degree of substitution is ≥90%; the alkali is one of sodium hydroxide and potassium hydroxide, or a mixture of the two.
3. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: The mass ratio of methacryloyl chitosan, methacryloyl polylysine, polyether F127 diacrylate, photoinitiator and acetic acid solution is 1-3:2-6:0.5-2:0.1-1:
100.
4. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: In step (2), a porous sieve membrane connector is used to connect the syringes containing the dispersed phase and the gel precursor solution respectively, and the two syringes are injected alternately, which is repeated multiple times to mix the two phases into a foam state.
5. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 4, characterized in that: The pore size of the porous sieve membrane in the porous sieve membrane connector is 0.05-50 microns; the number of repetitions n>2; the gas is an inert gas, and the volume ratio of the dispersed phase to the continuous phase gel precursor solution is 1:1-5.
6. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: The wavelength of the light curing light source is 350-450nm, the curing time is 15s-3min, and the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.
7. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: In step (4), the freeze-drying process is to first freeze the hydrogel at -90°C to -70°C for 2 to 5 hours, and then transfer it into a freeze dryer for freeze drying.
8. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: In step (4), the specific operation of swelling negative pressure through hole is: immersing the freeze-dried hydrogel in water, placing it in an environment of -0.05MPa to -0.08MPa for 2 to 3 minutes after it is completely swollen, and then taking it out to a normal environment; repeating the above operation 3 to 10 times until the hydrogel is uniform and transparent.
9. The method for preparing a porous material having the function of adsorbing endotoxin according to claim 1, characterized in that: In step (4), the alkaline solution is a NaOH solution, and the specific operation of soaking in the NaOH solution is: soaking the hydrogel after swelling with negative pressure through-holes in a 0.1-0.3 mol / L NaOH solution for 1-3 hours, and then washing with ultrapure water with endotoxin <0.001EU / mL until the conductivity of the washing liquid is less than 50uS / cm.
10. A porous material having the function of adsorbing endotoxin, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 9.