TW202340344A - Modified membrane and the manufacturing method thereof - Google Patents

Abstract

The present application provides a modified membrane comprising a hydrophobic porous membrane; and a polymer coating, wherein the polymer is Poloxamer. In addition, a manufacturing method for the membrane as the present application is provided.

Description

Modified film and preparation method thereof

The present application relates to a modified membrane, especially a hydrophobic porous membrane modified with Poloxamer, for hemodialysis/filtration.

Blood purification is the process of draining blood outside the body through different filters and adsorbers, using diffusion, convection, adsorption, ultrafiltration, filtration and other methods to remove the original pathogenic factors in the human body to achieve the effect of relieving symptoms. As a The scope of supportive therapy has expanded from the early stage of kidney disease to auxiliary treatment for severe acute pancreatitis, systemic inflammatory syndrome (SIRS), artificial liver support therapy, acute respiratory failure, etc., and has become a multi-organ support An important member of Multiple Organ Support Therapy (MOST).

Hemodialysis and peritoneal dialysis are the two main methods of treating uremic patients, with hemodialysis (HD) (commonly known as kidney dialysis) being the main treatment method. Blood purification methods also include hemofiltration (HF) and hemodiafiltration (HDF). As the core component of the artificial kidney system, hemodialysis membrane is used to treat uremia and renal failure.

The principle of hemodialysis is that the two sides of the dialysis membrane are the patient's blood and dialysate. According to the Gibbs-Donman membrane equilibrium principle, urea, creatinine and excess salt in the blood enter the dialysate through the dialysis membrane, while sugars, proteins, The blood cells and the like remain on the blood side and flow back into the patient's body, thus achieving the effect of purifying the blood. Therefore, the dialysis membrane is the core component in the hemodialysis process and plays a key role in the therapeutic effect of hemodialysis.

In addition, using hemofiltration or hemodiafiltration, some inflammatory factors with larger molecular weights such as IL-1, IL-6, TNF-α, etc. can be removed, effectively removing excess inflammatory factors from the circulatory system. Cytokines rebalance the body's homeostasis mechanism and restore normal physiological immune mechanisms, allowing patients with severe sepsis to avoid organ damage caused by some inflammatory substances. Currently, there is also technology to coat the surface of the dialysis filtration membrane with adsorbent materials or adsorbed proteins, or add an external adsorber during the dialysis/filtration process to use different ionic interactions to adsorb positively charged cytokines and negatively charged endotoxins. Further strengthen the adsorption of pathogenic cytokines in plasma to achieve the effect of purifying blood. However, microvesicles (MVs) are of great importance in immunity and septic shock, but so far, few studies have attempted to remove MVs.

Currently commonly used dialysis/filtration membranes are mainly divided into cellulose-based membranes and synthetic polymer membranes based on material composition. Cellulose-based membrane materials include regenerated cellulose and modified cellulose, while synthetic polymer membrane materials include bisphenol-based membranes. Polystyrene (PSF), polyetherstyrene (PES), polyacrylonitrile (PAN), polyamide (PA), polymethylmethacrylate (PMMA) and ethylene-vinyl alcohol copolymer (EVOH), etc.

Synthetic polymer materials have the advantages of wide variety, easy processing, and easy modification to obtain required properties. Therefore, they have gradually occupied the market as the second generation of dialysis/filtration membranes, including polyethylene, polyether ether, polyacrylonitrile, and polyethylene. Methyl methacrylate, etc., can be regarded as petroleum-based synthetic polymer materials according to their sources. Among them, the polyurethane hollow fiber dialysis membrane invented by Fresenius in Germany in 1985 is the most representative, occupying most of the global dialysis market, and is currently the mainstream dialysis/filtration membrane material.

However, polymer materials are usually hydrophobic, resulting in poor biocompatibility and blood compatibility. At the same time, there are problems with protein adsorption, deposition and contamination, resulting in membrane fouling, higher filtration resistance, and clinical adsorption saturation. Disadvantages: Patients often experience adverse reactions during dialysis/filtration use, which can easily cause coagulation and various oxidative stress reactions during application. Therefore, there is still a need to develop dialysis/filtration membranes that are resistant to protein contamination and have hemocompatibility.

In view of the problems existing in the prior art, this application provides a modified film, including: a hydrophobic porous film; and a polymer coating, the polymer is represented by the following compound of formula I: Formula I.

In one embodiment, preferably, a:b is 1:0.2 to 1:20. Preferably, the molecular weight of the compound of formula I is greater than 1000 Da. Preferably, the density of the compound of formula I on the hydrophobic porous membrane is greater than 0.2 mg/cm ² . More preferably, a:b is 1:8 to 1:10, the molecular weight of the compound of formula I is greater than 2500 Da, and the density on the hydrophobic porous membrane is greater than 0.5 mg/cm ² .

In one embodiment, the hydrophobic porous membrane is selected from the group consisting of polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), poly(ethylene/tetrafluoroethylene) (ETFE), poly(ethylene/chlorotrifluoroethylene) Ethylene) (ECTFE), perfluoropolyether (PFPE), polymethylmethacrylate (PMMA), polyethylene, polypropylene, polyacrylonitrile (PAN), ethylene-vinyl alcohol copolymer (EVOH), polyvinyl alcohol , polyether styrene (PSF) and polyether styrene (PES).

In one embodiment, more preferably, the hydrophobic porous membrane is polyvinylidene fluoride (PVDF), a:b is 1:8 to 1:10, and the molecular weight of the compound of formula I is 2800 Da.

In addition, this application further provides a method for preparing the film of this application, which includes the following steps: Provide a hydrophobic porous membrane; Immersing the hydrophobic porous membrane in a Poloxamer solution; and Rinse the hydrophobic porous membrane.

In one embodiment, the concentration of the Poloxamer solution is 10 mg/mL – 500 mg/mL, preferably 50 mg/mL – 400 mg/mL.

In one embodiment, the hydrophobic porous membrane is made by the following steps: Dissolve polyvinylidene fluoride (PVDF) and polyvinylpyrrolidone (PVP) in N,N-dimethylacetamide to form a casting solution; Spread the casting solution on a flat surface with a uniform thickness; and Precipitate in a water coagulation bath to form the hydrophobic porous membrane.

In one embodiment, preferably, the concentration of polyvinylidene fluoride (PVDF) is 15% - 20%, the concentration of polyvinylpyrrolidone (PVP) is 5%, and the casting solution is spread at a thickness of 300 μm. on this plane.

The compound of formula I is a Poloxamer, which is a copolymer with hydrophilic ends at both ends and hydrophobicity in the middle. Therefore, its hydrophobic affinity (hydrophobic interaction) can be used to fix the molecules on the surface of the hydrophobic porous membrane, making the hydrophobic The surface of the porous membrane is more hydrophilic. The modified membrane can reduce the amount of protein adsorbed and prolong the coagulation reaction time, thus improving blood compatibility.

The advantages and features of the invention, as well as the methods for achieving them, will be more readily understood when described in more detail with reference to exemplary embodiments and the accompanying drawings. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make this disclosure more thorough and complete and fully convey the scope of the invention to those of ordinary skill in the art, and the invention is only within the scope of the appended claims. defined. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be understood that terms such as those defined in commonly used dictionaries are to be understood to have meanings consistent with the context of the relevant art and are not to be overly idealistic or overly formal unless expressly defined herein. understand the meaning. As used throughout this specification, range values are intended to be shorthand representations of each and every value within the range, and any number within the range may be selected as the endpoint of the range.

Poloxamer (compound of formula I) is a polyoxyethylene polyoxypropylene ether block copolymer with the following structure: Formula I

The polyethylene oxide at both ends of the Poloxamer is hydrophilic, and the Polypropylene oxide in the middle is hydrophobic. Therefore, through a simple coating process, a hydrophobic porous membrane (such as Driven by the hydrophobic interaction between PVDF) and polyoxypropylene, the hydrophilic polyoxyethylene is anchored to the surface of the hydrophobic porous membrane (such as PVDF).

Common hydrophobic porous membranes used for hemodialysis/filtration include polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), poly(ethylene/tetrafluoroethylene) (ETFE), poly(ethylene/chlorotrifluoroethylene) ) (ECTFE), perfluoropolyether (PFPE), polymethylmethacrylate (PMMA), polyethylene, polypropylene, polyacrylonitrile (PAN), ethylene-vinyl alcohol copolymer (EVOH), polyvinyl alcohol, Polyether styrene (PSF) and polyether styrene (PES), etc. The following examples take polyvinylidene fluoride (PVDF) as an example, but are not limited thereto.

Example 1 - Preparation of Polyvinylidene Fluoride (PVDF) Film

This experiment used 15% - 20% PVDF (MW: 534 kDa, Sigma) and 5% PVP (MW: 58 kDa, Alfa Aesar) dissolved in DMAc (Sigma) at 70 °C overnight to form a casting solution (Casting solution). Next, the casting solution was evenly spread on the glass plane at a thickness of 300 µm using a casting knife at room temperature. Afterwards, it is precipitated in a water coagulation bath to form a PVDF film of the solid.

Example 2 - Preparation of Poloxamer modified PVDF film

This experiment uses three types of Poloxamers for modification, namely L31 (MW: 1100 Da, hydrophilic segment ratio 10%), L35 (MW: 1900 Da, hydrophilic segment ratio 50%) and L81 (MW: 2800 Da, hydrophilic segment ratio 10%) (Sigma). First, the PVDF film prepared in Example 1 was immersed in poloxamer (L31, L35 and L81) solutions with concentrations of 50 mg/mL, 100 mg/mL, 200 mg/mL and 400 mg/mL respectively. 1 hour. Then, the PVDF films were rinsed twice with PBS to remove the weakly adsorbed poloxamer.

The amount of poloxamer adsorbed to the PVDF film was determined by the weight increase per unit membrane area (mg/cm ² ), and the average value was presented from at least 4 repeated experiments. The results are shown in Figure 1. As The poloxamer concentration increased from 50 mg/mL to 400 mg/mL. The coating density on the PVDF film using L31 and L35 can both reach more than 0.2 mg/cm ² , and L81 can even reach at least 0.5 mg/cm ² . However, only the coating density of L81 on the PVDF film can be gradually increased. This is because poloxamer L81 has a higher molecular weight and more hydrophobic domains than L31 and L35, which can produce stronger van der Waal interactions with the surface of the PVDF film. force and hydrophobicity. Therefore, L31, L35 and L81 can all be coated on PVDF, especially L81 has the best effect. Therefore, the PVDF film modified with poloxamer L81 was selected for subsequent tests in this case.

Example 3 - Blood purification test of poloxamer-modified PVDF membrane

Blood samples were obtained from therapeutic phlebotomy of polycythemia patients at National Taiwan University Hospital, and platelet-poor plasma (PPP) was prepared by centrifugation at 2500<i>g for 12 min with Institutional Review Board approval, and the supernatant was collected and centrifuged again to further reduce platelets. Pooled PPP was obtained from three patients, and 4- and 60-fold PPP dilutions were prepared in PBS for plasma filtration and protein adsorption assays, respectively.

In this example, a plasma protein adsorption test was performed to study the antifouling ability of the poloxamer-modified PVDF film. The film prepared in Example 2 was seeded in a 12-well plate, moistened with 1 ml of ethanol, washed with 1 ml of PBS, and incubated with 1 ml of 60-fold diluted plasma at 37°C for 24 hours. Afterwards, the suspension in each well was collected and protein analysis was performed by BCA protein assay (Thermo Fisher Scientific). The protein adsorption amount was calculated by the following formula:

In the above formula, C _o is the original plasma protein concentration, C _f is the plasma protein concentration after adsorption, V is the plasma volume, and A is the membrane area. Each independent adsorption test was performed in 4 replicates and 6 independent experiments were performed. The results of the test are shown in Figure 2.

Figure 2 shows that the protein adsorption result on the unmodified PVDF film (control group) is 72.23±6.83 µg/cm ² , while the protein adsorption result after poloxamer concentration is 50 mg/mL, 100 mg/mL, 200 mg /mL and 400 mg/mL modified PVDF films, the protein adsorption results were significantly reduced to 48.22±6.32 µg/cm ² , 41.63±2.30 µg/cm ² , 35.40±4.49 µg/cm ² and 11.70±7.15 µg respectively. /cm ² . This is consistent with previous research on reducing protein adsorption on PVDF membranes by reducing hydrophobicity, and also further ensures the successful hydrophilic modification of the hydrophobic PVDF membrane surface.

Additionally, coagulation assays were studied in recalcified plasma with a 20 mM Ca ²⁺ concentration. Poloxamer-modified PVDF films were incubated with 0.5 ml of recalcified plasma in 24-well plates, and clotting time was determined as the onset of absorbance transition by ELISA at 660 nm in kinetic mode at 37 °C. Research below. Each independent clotting time test was performed in 4 replicates and 6 independent tests were performed. The results of the test are shown in Figure 3.

Figure 3 shows that the coagulation time of the unmodified PVDF film (control group) is only 14.94±0.71 minutes, while the coagulation time of the PVDF film modified with different concentrations of poloxamer can be effectively extended to 17.56 minutes respectively. ±0.5 minutes, 20.26±0.91 minutes, 22.87±1.29 minutes and 28.91±1.60 minutes. Therefore, the hydration layer formed by the hydrophilic domains in the poloxamer reduces the attachment of coagulation proteins to the surface of the PVDF film and reduces their activation.

Furthermore, in order to test whether the poloxamer-modified membrane affects its filtration ability, poloxamer-modified membranes with concentrations of 100 mg/mL and 400 mg/mL were selected for further research, and the area was 17.35 cm The membranes of ² are assembled into a cross-flow filtration system. First, PBS was flowed into the filtration system through a peristaltic pump (DG300N, DGS, China) for 30 minutes to generate a stable flow, and then 250 mL of 4 times diluted plasma was filtered through a suction pump (A-3S, Eyla, Japan). to create a pressure gradient of approximately 20 mmHg. The permeate was collected into 15 ml centrifuge tubes after 10 minutes to analyze the removal of microvesicles and cytokines.

Microvesicles were characterized and quantified by flow cytometry (FACS Calibur). A low flow rate was chosen, the collection time for each interval was fixed at 3 min, a PBS blank value was used to clarify background interference, and 1um size standard beads (Thermo Fisher Scientific) were used to define the exact position of the microbubbles, forward scatter (FSC) and side scatter. Scattering (SSC) was set to logarithmic mode and the background threshold was adjusted to zero. Filtered plasma (5 µL) was diluted with 500 µL PBS (filtered with a 0.22um filter) and used for flow cytometric analysis. Each independent filtration test was performed in 3 replicates and 4 independent experiments were performed. The results of microbubble removal are shown in Figure 4.

Figure 4 shows that in the group of PVDF membranes modified with poloxamer, the filtration performance of removing microbubbles from plasma decreased slightly, and when modified with a higher concentration of poloxamer (400 mg/mL) Lower microbubble removal performance was observed on the PVDF films, but no significant difference was found between the unmodified and modified films.

Furthermore, this example further analyzes the effectiveness of the poloxamer-modified PVDF film in removing cytokines. Levels of the cytokines IL-1β, IL-6, and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the manufacturer's instructions. Each independent experiment was performed in triplicate. Three independent experiments were performed. The results of IL-1β, IL-6, and IL-8 removal are shown in Figures 5 to 7, respectively.

Figures 5 to 7 show the filtration performance of poloxamer-modified PVDF membranes in removing IL-1β, IL-6, and IL-8 from plasma compared to unmodified PVDF membranes, in terms of PVDF The amount of poloxamer coated on the film surface increases accordingly. Especially in this study, the PVDF film modified with 100 mg/mL poloxamer significantly showed the best removal rate for IL-1β and IL-6, while the removal rate of IL-8 could be increased by 44%. , but no significance was found, and the PVDF membrane modified with 400 mg/mL poloxamer did not further improve the cytokine removal rate.

In summary, the test results show that this application uses a simple modification method without complicated chemical reactions, which not only reduces the adsorption of plasma proteins by the hydrophobic film, but also improves the blood compatibility of the hydrophobic film. More importantly, the Poloxamer-modified hydrophobic film did not lose its performance in removing microvesicles and cytokines and even showed more effective results.

without

Figure 1 shows the coating density analysis results of poloxamer-modified PVDF film.

Figure 2 shows the analysis results of the protein adsorption test.

Figure 3 shows the analysis results of the coagulation test.

Figure 4 shows the analysis results of the microbubble removal test.

Figure 5 shows the analytical results of the IL-1β removal assay.

Figure 6 shows the analytical results of the IL-6 removal assay.

Figure 7 shows the analytical results of the IL-8 removal assay.

Claims (10)

A modified film, including: a hydrophobic porous membrane; and a polymer coating, the polymer is represented by the following compound of formula I: Formula I. Such as the film of claim 1, wherein a:b is 1:0.2 to 1:20. The film of claim 1, wherein the compound of formula I has a molecular weight greater than 1000 Da. The film of claim 1, wherein the density of the compound of formula I on the hydrophobic porous film is greater than 0.2 mg/cm ² . The film of claim 1, wherein the hydrophobic porous membrane is selected from polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), poly(ethylene/tetrafluoroethylene) (ETFE), poly(ethylene/chlorine) Trifluoroethylene) (ECTFE), perfluoropolyether (PFPE), polymethyl methacrylate (PMMA), polyethylene, polypropylene, polyacrylonitrile (PAN), ethylene-vinyl alcohol copolymer (EVOH), poly A group consisting of vinyl alcohol, polystyrene (PSF) and polyetherstyrene (PES). Such as the film of claim 1, wherein the hydrophobic porous film is polyvinylidene fluoride (PVDF), a:b is 1:8 to 1:10, and the molecular weight of the compound of formula I is 2800 Da. A method for preparing the film of claim 1, comprising the following steps: Provide a hydrophobic porous membrane; Immersing the hydrophobic porous membrane in a Poloxamer solution; and Rinse the hydrophobic porous membrane. For example, the preparation method of claim 7, wherein the concentration of the Poloxamer solution is 10 mg/mL – 500 mg/mL. According to the preparation method of claim 7, the hydrophobic porous membrane is made by the following steps: Dissolve polyvinylidene fluoride (PVDF) and polyvinylpyrrolidone (PVP) in N,N-dimethylacetamide to form a casting solution; Spread the casting solution on a flat surface with a uniform thickness; and Precipitate in a water coagulation bath to form the hydrophobic porous membrane. The preparation method of claim 9, wherein the concentration of polyvinylidene fluoride (PVDF) is 15% - 20%, the concentration of polyvinylpyrrolidone (PVP) is 5%, and the casting solution is spread on the plane with a thickness of 300 µm superior.