Protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate and application thereof

Abstract

The invention discloses a protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate and application of the protein / polysaccharide composite dialysis membrane, a lanthanum carbonate loaded protein / polysaccharide composite aggregate is used for modifying a porous filter membrane, and the dialysis membrane with a lanthanum carbonate loaded protein / polysaccharide self-assembled membrane as a compact skin layer and the porous filter membrane as a supporting layer is formed. By integrating a membrane separation mechanism of hemodialysis and an adsorption principle of blood perfusion, the skin layer of the dialysis membrane has molecular sieve separation and chemical adsorption functions, efficient removal of molecular uremic toxins (phosphorus, creatine liver, bilirubin, p-cresol sulfate and indol sulfate) with different sizes is synergistically realized, macromolecular proteins in blood are effectively intercepted by pore size screening, and the dialysis membrane has the advantages of simple structure, low cost and high efficiency. According to the present invention, by using the adsorption effect of the functional group on the surface of the membrane, the high removal rate on the protein-bound urotoxin is achieved, the high removal effect on phosphorus is particularly achieved, the blood compatibility is considered, the large-scale preparation is easily achieved, and the new idea is provided for the development of the high-performance blood purification material.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a dialysis membrane with a cortex made of a supported protein / polysaccharide composite membrane material having molecular sieving and chemical adsorption functions. Background Art

[0002] Currently, the number of uremia patients globally is approximately 3 million, and it is expected to increase significantly to 5.44 million by 2030. The mortality rate associated with uremia is as high as 80%, and hemodialysis is the main treatment method for this disease. Hyperphosphatemia is a common complication among dialysis patients and has been found to be related to an increased mortality rate in uremia. Traditional hemodialysis membranes mainly rely on molecular sieving to remove uremic toxins, and their efficacy in removing low-molecular-weight uremic toxins (0.5 kDa, such as urea and creatinine) has been proven. However, their ability to remove phosphate, medium-molecular-weight toxins (0.5 - 60 kDa, such as β2-microglobulin), and protein-bound toxins (such as indoxyl sulfate and p-cresyl sulfate) is still limited. In addition, most hemodialysis membranes can cause an immune response when encountering blood, resulting in blood incompatibility. Therefore, it is necessary to develop a new type of hemodialysis membrane that has the ability to efficiently remove small-molecular-weight uremic toxins phosphorus, medium-molecular-weight uremic toxins, and protein-bound toxins, and at the same time retain macromolecular serum proteins.

[0003] The cortical layer of the hemodialysis membrane is in direct contact with the blood. Studying its role as a solute separation barrier is crucial for the separation performance of the dialysis membrane. To optimize the clearance of uremic toxins and achieve an efficacy comparable to that of the endogenous kidney, surface modification of the polymer membrane can optimize its dialysis performance for uremic toxins. For example, grafting heparin, vitamin E, etc. onto the surface of the polymer membrane can improve the biocompatibility of the membrane. However, there are problems such as complex experiments and harsh conditions in the surface grafting method of the polymer membrane, and it cannot effectively remove medium-molecular-weight uremic toxins. Removing uremic toxins by adsorption using hemoperfusion technology is simple and easy to operate, and it also shows effective removal ability even at low initial concentrations. Based on this, an organic / inorganic hybrid matrix membrane constructed by incorporating inorganic particles into an organic porous polymer matrix can enhance the adsorption performance of the membrane. This design can effectively reduce the spatial separation between the solute and the binding sites, thereby improving the overall efficiency of membrane separation and reducing the mass transfer resistance. The selectivity of the membrane is affected by its inorganic composition, while the polymer membrane matrix provides support, stability, and flow control. To successfully separate the target substance, it is necessary to screen inorganic fillers with high selectivity and high adsorption capacity, and the compatibility and stability when the inorganic filler binds to the polymer matrix also need to be considered. For example, lanthanum-based adsorbents have a high affinity for phosphorus, lower toxicity compared to other metals, and relatively low cost, and are widely used for the removal of phosphorus in the blood. Incorporating the phosphorus removal adsorbent into the membrane by methods such as blending, coating, and in-situ growth can endow the membrane with high phosphorus adsorption performance. However, it should be noted that although nanoparticles show significant advantages in phosphorus absorption kinetics, their large-scale application still faces the technical bottleneck of the uniform dispersion of nanoparticles; more critically, the traditional physical blending modification strategy can only limitedly improve the adsorption capacity of the membrane material for the target molecule, and these two challenges severely restrict its practical efficacy in the field of blood purification. In addition, as a blood purification membrane, it is challenging to achieve a delicate balance between removing small / medium-molecular-weight toxic substances and preserving large-molecular-weight beneficial proteins to achieve the desired results.

[0004] In recent years, the research on the construction mechanism and performance regulation of protein-based separation membranes has been continuously deepened. The core scientific issues focus on how to construct a functional separation layer with an ultrathin structure, low defect density, and high porosity through controllable interface engineering. Research shows that the co-impregnation-filtration strategy or directed self-assembly technology based on two-dimensional heterogeneous interfaces such as solid / liquid and liquid / liquid can achieve the controllable preparation of protein nanofilms. It is worth noting that disulfide bonds, as the key chemical bonds maintaining the stability of the protein tertiary structure, directly affect the mechanical strength and durability of the membrane. When external physical stimuli (such as thermal stress, UV irradiation, interfacial tension perturbation) or chemical environment changes (such as urea / guanidine hydrochloride denaturants, pH shift) cause disulfide bond cleavage, it will trigger an irreversible protein unfolding process. During this process, the exposure of intramolecular hydrophobic groups will trigger strong hydrophobic interactions, which become the main driving force for protein molecular rearrangement and membrane structure formation. For example, lysozyme and bovine serum albumin, due to their unique β-sheet domain and disulfide bond network, can not only be used as model proteins for amyloid fibril research but also show unique advantages in the development of biomedical membrane materials, and their easy availability of raw materials further enhances the potential for engineering applications. CN108854599A uses a dialysis membrane obtained by modifying a polymer membrane with lysozyme amyloid aggregates, which has a high clearance rate for small and medium-sized urinary toxins. However, in practical applications, this membrane only separates urinary toxins of different molecular sizes by pore size and has a low clearance rate for protein-bound urinary toxins (the clearance rate of indoxyl sulfate is 33.1%). In view of this problem, based on the advantages of the adhesion and film-forming properties of protein amyloid aggregates, and by loading inorganic nanoparticles with excellent adsorption performance for urinary toxins, a composite adsorption filtration membrane material with both molecular sieving and chemical adsorption functions is designed. By integrating the membrane separation mechanism of hemodialysis and the adsorption principle of hemoperfusion, it is expected to synergistically achieve the efficient clearance of urinary toxins. Summary of the Invention

[0005] The object of the present invention is to provide a protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate to solve the problems such as complex production process and poor ability to remove urinary toxins of existing dialysis membranes.

[0006] For the above object, the dialysis membrane provided by the present invention is to modify a porous filter membrane with an amyloid-like protein / polysaccharide composite aggregate in-situ loaded with lanthanum carbonate nanoparticles, forming a dialysis membrane with a protein / polysaccharide self-assembly membrane loaded with lanthanum carbonate as the dense skin layer and the porous filter membrane as the support layer.

[0007] Furthermore, preferably, the above-mentioned protein is bovine serum albumin or lysozyme.

[0008] Furthermore, preferably, the above-mentioned polysaccharide is sodium alginate or sodium carboxymethyl cellulose.

[0009] Further, the porous filter membrane is an ultrafiltration membrane or a hollow fiber membrane in a dialyzer assembly.

[0010] Further, preferably, the membrane thickness of the ultrafiltration membrane is 12 - 240 μm, and the pore size is 50 - 200 nm; the dialyzer assembly is a PVDF ultrafiltration membrane assembly, and the pore size of the hollow fiber membrane is 50 - 200 nm.

[0011] The preparation method of the protein / polysaccharide composite dialysis membrane based on in-situ loading of lanthanum carbonate in the present invention is as follows: adjust the aqueous solution of 30 - 100 mmol / L tris(2-carboxyethyl)phosphine to a pH value of 6.0 - 9.0 with NaOH, then mix it with a 2 - 50 mg / mL protein aqueous solution and a 0.5 - 12.5 mg / mL polysaccharide aqueous solution in equal volume. After incubating at room temperature for 6 - 24 hours, add LaCl3 and Na2CO3 and stir for 0.5 - 2 hours to obtain a solution containing amyloid-like protein / polysaccharide composite aggregates loaded with lanthanum carbonate nanoparticles. Then immerse the porous filter membrane in the solution for 2 - 12 hours to form a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate at the solid-liquid interface, that is, a dialysis membrane with a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate as the dense skin layer and the porous filter membrane as the support layer is obtained.

[0012] In the above preparation method, preferably, the mass ratio of the protein to the polysaccharide is 2:1 - 15:1, the mass ratio of the protein to LaCl3 is 50:1 - 500:1, and the mass ratio of LaCl3 to Na2CO3 is 1.1:1 - 2:1.

[0013] In the above preparation method, more preferably, the mass ratio of the protein to the polysaccharide is 4:1 - 10:1, and the mass ratio of the protein to LaCl3 is 200 - 300:1.

[0014] The present invention also provides the application of the protein / polysaccharide composite dialysis membrane based on in-situ loading of lanthanum carbonate in removing uremic toxins, and the uremic toxins are phosphorus, creatinine, p-cresol phosphate, indoxyl sulfate, bilirubin, and β2-microglobulin.

[0015] The present invention further provides the application of the protein / polysaccharide composite dialysis membrane based on in-situ loading of lanthanum carbonate in the adsorption of mixed antibiotics, and the mixed antibiotics are enrofloxacin, norfloxacin, roxithromycin, oxytetracycline, erythromycin, sulfamethoxazole, and sulfadiazine.

[0016] The present invention uses tris(2-carboxyethyl)phosphine hydrochloride, a disulfide bond reducing agent, to induce phase transition of proteins while adding polysaccharides to obtain a solution containing amyloid-like protein / polysaccharide composite aggregates. After in-situ loading of lanthanum carbonate nanoparticles onto the amyloid-like protein / polysaccharide composite aggregates in this solution, a porous membrane is immersed in the solution for modification to form a dialysis membrane with a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate as the dense skin layer and the porous membrane as the support layer. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The present invention directly in-situ loads lanthanum carbonate nanoparticles onto amyloid-like protein / polysaccharide composite aggregates to prepare a composite adsorption and filtration dialysis membrane skin layer with both molecular sieving and chemical adsorption functions. The thickness of the membrane can be controlled according to the film formation time, and the membrane thickness increases from 60 nm to 90 nm as the film formation time increases.

[0018] 2. The dialysis membrane of the present invention can be used as a blood purification membrane. It has a good scavenging effect on uremic toxins of different molecular sizes by the chemical adsorption of functional groups on the membrane surface and lanthanum carbonate, especially showing a high scavenging effect on phosphorus. The maximum adsorption amount of phosphorus reaches 1235.1 mg / g, and it also has a good scavenging efficiency for protein-bound uremic toxins. The maximum adsorption amounts of p-cresol sulfate, indoxyl sulfate, and bilirubin are 260 mg / g, 730.2 mg / g, and 768.4 mg / g respectively, which are much higher than the scavenging effects of traditional adsorbents for blood perfusion.

[0019] 3. The dialysis membrane of the present invention has a good retention effect on macromolecular proteins by pore size sieving. The retention rate of serum albumin is ≥98%. After 4 hours of dialysis, it has a good scavenging effect on phosphorus, p-cresol sulfate, indoxyl sulfate, bilirubin, and β2-microglobulin, and the scavenging rates are 98%, 85%, 61%, 73%, and 26% respectively. The scavenging rate of protein-bound uremic toxins is higher than that of existing dialysis membrane materials.

[0020] 4. The dialysis membrane of the present invention can effectively adsorb antibiotics, including enrofloxacin, norfloxacin, roxithromycin, oxytetracycline, erythromycin, sulfamethoxazole, and sulfadiazine.

[0021] 5. The skin layer of the dialysis membrane of the present invention is easy to be prepared on a large scale, with the characteristics of mild reaction conditions, simple preparation process, low cost, low energy consumption, and environmental protection, avoiding the cumbersome steps and environmental pollution problems in the surface modification process of traditional blood purification polymer membranes. Compared with traditional blood purification membranes, this dialysis membrane synergistically realizes the efficient scavenging of uremic toxins of different sizes (phosphate, creatinine, bilirubin, p-cresol sulfate, indoxyl sulfate) by integrating the membrane separation mechanism of hemodialysis and the adsorption principle of hemoperfusion, while taking into account blood compatibility, providing new ideas for the development of high-performance blood purification materials. Description of the Drawings

[0022] Figure 1 is the thickness of the self - assembled film of bovine serum albumin / sodium alginate loaded with lanthanum carbonate at different times on the long film.

[0023] Figure 2 is the scanning electron micrograph of the surface of the polyamide ultrafiltration membrane (left) and the self - assembled film of bovine serum albumin / sodium alginate loaded with lanthanum carbonate (right) in Example 1.

[0024] Figure 3 is the atomic force micrograph of the self - assembled film of bovine serum albumin / sodium alginate loaded with lanthanum carbonate in Example 1.

[0025] Figure 4 is the transmission electron micrograph plan view of bovine serum albumin / sodium alginate loaded with lanthanum carbonate in Example 1.

[0026] Figure 5 is the phosphorus adsorption amount of the dialysis membrane PTB / SA / LC in Example 1 and the dialysis membrane PTB / SA in Comparative Example 1.

[0027] Figure 6 is the phosphorus adsorption rate of the dialysis membrane PTB / SA / LC in Example 1 at different pH values.

[0028] Figure 7 is the adsorption amount of indoxyl sulfate, p - cresol sulfate and bilirubin by the dialysis membrane PTB / SA / LC in Example 1.

[0029] Figure 8 is the clearance rate of uremic toxins by the dialysis membrane PA - PTB / SA / LC in Example 1.

[0030] Figure 9 is the adsorption amount of antibiotics by the dialysis membrane PTB / SA / LC in Example 1.

[0031] Figure 10 is the adhesion of platelets to the dialysis membrane PTB / SA / LC in Example 1.

[0032] Figure 11 is the cytotoxicity of the dialysis membrane PTB / SA / LC to cells in Example 1.

[0033] Figure 12 is the clearance rate of uremic toxins by the hollow fiber membrane in the PTB / SA / LC self - assembled film - modified dialyzer in Example 3. Detailed implementation manners

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0035] Example 1

[0036] Adjust the pH value of 50 mL of 50 mmol / L tris(2-carboxyethyl)phosphine aqueous solution to 8.0 with NaOH, and then mix it evenly with 50 mL of 5 mg / mL bovine serum albumin aqueous solution and 50 mL of 1.25 mg / mL sodium alginate aqueous solution. After incubating at room temperature for 12 hours, a homogeneous solution containing amyloid-like bovine serum albumin / sodium alginate composite aggregates is obtained. Then, add 1.02 mg of LaCl3 and 0.66 mL of 1 mg / mL Na2CO3 aqueous solution to the obtained solution, and stir for 30 minutes to obtain a solution containing amyloid-like bovine serum albumin / sodium alginate composite aggregates loaded with lanthanum carbonate nanoparticles. The solution is homogeneous and stable. After immersing a polyamide ultrafiltration membrane in this solution for 6 hours, a dialysis membrane (named PA-PTB / SA / LC) is formed with a bovine serum albumin / sodium alginate self-assembled membrane loaded with lanthanum carbonate nanoparticles (named PTB / SA / LC) as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0037] In this example, an imaging ellipsometer was used to measure the membrane thickness of the bovine serum albumin / sodium alginate self-assembled membrane loaded with lanthanum carbonate formed on the surface of the polyamide ultrafiltration membrane during the immersion process. The results showed that the membrane thickness increased from 60 nm to 90 nm as the immersion time extended (as Figure 1 ). The self-assembled membrane was characterized by scanning electron microscopy. The results showed that after the bovine serum albumin / sodium alginate self-assembled membrane loaded with lanthanum carbonate was formed on the polyamide ultrafiltration membrane, the pores on the surface of the polyamide ultrafiltration membrane were completely covered, and the surface presented a relatively uniform, dense and smooth thin film (as Figure 2 ). The self-assembled membrane is a composite aggregate of amyloid-like proteins formed by the phase transition of bovine serum albumin and sodium alginate. The surface of the self-assembled membrane presents uniformly distributed nanoscale spherical aggregates (as Figure 3 ). Through high-resolution transmission electron microscopy analysis of the dialysis membrane, the lanthanum carbonate in the bovine serum albumin / sodium alginate self-assembled membrane loaded with lanthanum carbonate presented a rod-shaped nanoparticle morphology. The bright field image showed uniform lattice fringes, and the measured interplanar spacing was d = 0.505 nm (as Figure 4 ), verifying that the sample was lanthanum carbonate.

[0038] Comparative Example 1

[0039] Adjust the pH value of 50 mL of 50 mmol / L tris(2-carboxyethyl)phosphine aqueous solution to 5.0 with NaOH, and then mix it evenly with 50 mL of 5 mg / mL bovine serum albumin aqueous solution. After immersing a polyamide ultrafiltration membrane in this solution for 6 hours, a dialysis membrane (named PA-PTB) is formed with a bovine serum albumin self-assembled membrane (named PTB) as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0040] Comparative Example 2

[0041] Adjust the pH value of 50 mL of 50 mmol / L tris(2 - carboxyethyl)phosphine aqueous solution to 8.0 with NaOH, then mix it evenly with 50 mL of 5 mg / mL bovine serum albumin aqueous solution and 50 mL of 1.25 mg / mL sodium alginate aqueous solution. After incubating at room temperature for 12 hours, a homogeneous solution containing amyloid - like bovine serum albumin / sodium alginate composite aggregates is obtained. Then, immerse the polyamide ultrafiltration membrane in this solution for 6 hours to form a dialysis membrane (named PA - PTB / SA) with the bovine serum albumin / sodium alginate self - assembled membrane (named PTB / SA) as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0042] Comparative Example 3

[0043] Adjust the pH value of 50 mL of 50 mmol / L tris(2 - carboxyethyl)phosphine aqueous solution to 8.0 with NaOH, then mix it evenly with 50 mL of 5 mg / mL bovine serum albumin aqueous solution and 50 mL of 1.25 mg / mL sodium alginate aqueous solution. After incubating at room temperature for 12 hours, a homogeneous solution containing amyloid - like bovine serum albumin / sodium alginate composite aggregates is obtained. Then, add 2 mg of lanthanum carbonate nanoparticles (particle size of 30 nm) to the obtained solution and stir for 30 minutes to obtain a solution containing amyloid - like bovine serum albumin / sodium alginate composite aggregates loaded with lanthanum carbonate nanoparticles. Immerse the polyamide ultrafiltration membrane in this solution for 6 hours to form a dialysis membrane (named PA - PTB / SA / LCP) with the bovine serum albumin / sodium alginate self - assembled membrane loaded with lanthanum carbonate nanoparticles (named PTB / SA / LCP) as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0044] Example 2

[0045] In this example, replace the 1.25 mg / mL sodium alginate aqueous solution in Example 1 with an equal - volume 1.25 mg / mL sodium carboxymethylcellulose aqueous solution, and the other steps are the same as in Example 1 to form a dialysis membrane with the bovine serum albumin / sodium carboxymethylcellulose self - assembled membrane loaded with lanthanum carbonate nanoparticles as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0046] Example 3

[0047] Adjust 50 mL of 25 mmol / L tris(2-carboxyethyl)phosphine aqueous solution to pH 7.0 with NaOH, then mix it evenly with 50 mL of 20 mg / mL lysozyme aqueous solution and 50 mL of 2 mg / mL sodium alginate aqueous solution. After incubating at room temperature for 12 hours, a homogeneous solution containing amyloid-like lysozyme / sodium alginate composite aggregates is obtained. Then, add 2 mg of LaCl3 and 1.5 mL of 1 mg / mL Na2CO3 aqueous solution to the obtained solution, and stir for 30 minutes to obtain a solution containing amyloid-like lysozyme / sodium alginate composite aggregates loaded with lanthanum carbonate nanoparticles. The solution is homogeneous and stable. After soaking a polyamide ultrafiltration membrane in this solution for 6 hours, a dialysis membrane is formed with a lysozyme / sodium alginate self-assembled membrane loaded with lanthanum carbonate nanoparticles as the dense skin layer and the polyamide ultrafiltration membrane as the support layer.

[0048] Example 4

[0049] In this example, the solution containing amyloid-like bovine serum albumin / sodium alginate composite aggregates loaded with lanthanum carbonate nanoparticles was circulated in a commercial dialyzer through a dialysis pump for 12 hours. Other steps were the same as in Example 1, and a dialysis membrane with a bovine serum albumin / sodium alginate self-assembled membrane loaded with lanthanum carbonate nanoparticles as the skin layer and a hollow fiber membrane as the support layer was obtained.

[0050] Example 5

[0051] Application of the dialysis membrane PA-PTB / SA / LC of Example 1

[0052] 1. Adsorption of uremic toxins by the dialysis membrane PA-PTB / SA / LC

[0053] Place the dialysis membrane PA-PTB / SA / LC in 10 mL of phosphorus solution (dissolve 219.35 mg of potassium dihydrogen phosphate in 50 mL of ultrapure water to prepare a 1 g / L phosphate solution, and dilute the low-concentration phosphorus solution with ultrapure water). After standing for 12 hours, use an ultraviolet spectrophotometer to measure the residual phosphorus concentration in the solution after adsorption. And make a comparison of the adsorption performance with the dialysis membranes of Comparative Examples 1-3. The factors affecting the adsorption performance of the dialysis membrane include: pH, phosphorus concentration, and adsorption time. The experimental results show that as the initial concentration of the phosphorus solution (5-500 mg / L) increases, the adsorption rate of PTB / SA / LC to phosphorus is higher than that of the dialysis membrane PTB / SA without loaded lanthanum carbonate in Comparative Example 2. When the concentration is lower than 200 mg / L, the adsorption rate > 60%. As the phosphorus concentration increases, the adsorption rate gradually decreases. Compared with the dialysis membrane PTB / SA without loaded lanthanum carbonate, after loading lanthanum carbonate, the adsorption performance of the dialysis membrane PTB / SA / LC is significantly improved, and the maximum adsorption capacity reaches 1235.1 mg / g (as Figure 5) This is because the introduction of rare earth elements increases the density and binding energy of surface active sites. However, for the dialysis membrane PA-PTB formed only with bovine serum albumin in Comparative Example 1, the phosphorus adsorption capacity is less than 50 mg / g, indicating that the introduction of polysaccharides increases the phosphorus binding sites. Compared with the in-situ loading of lanthanum carbonate in Example 1, for the dialysis membrane PA-PTB / SA / LCP obtained only by physical blending in Comparative Example 3, compared with PTB / SA without loaded lanthanum carbonate, the phosphorus adsorption capacity only increased slightly (452.4 mg / g). This is because particle sedimentation easily occurs during the static film-forming stage, resulting in uneven distribution of lanthanum carbonate in the vertical direction of the membrane layer. The in-situ loading process, through coordination-induced molecular-level assembly, anchors lanthanum carbonate in a monodispersed form to the protein / polysaccharide network, forming a functional layer with uniform thickness, effectively solving the phase separation problem of the traditional blending method, and thus effectively improving the adsorption interface efficiency. Therefore, the above results show that the combination of protein and polysaccharide helps to increase the phosphorus adsorption capacity, and the phosphorus adsorption capacity is significantly improved after in-situ loading of lanthanum carbonate.

[0054] In addition, the results show that when the dialysis membrane PA-PTB / SA / LC is placed in 50 mL of 25 mg / L phosphorus solution and left standing for 1 hour, the phosphorus adsorption basically reaches saturation, and the adsorption rate is 80%, indicating that this dialysis membrane can adsorb phosphorus relatively quickly in a short time.

[0055] As Figure 6 shown, in the pH range of 2 - 10, the phosphorus adsorption rate of the dialysis membrane PA-PTB / SA / LC always remains above 80%, and reaches a peak of 93% at pH = 6. This broad-spectrum pH adaptability stems from the dynamic matching between the charged groups on the dialysis membrane surface and the phosphate ion form: amino groups can capture phosphate ions through electrostatic attraction, while lanthanum carbonate and phosphate ions can form a coordination complex. The dual mechanisms cooperate to ensure continuous and efficient adsorption in a wide pH range. Based on this, it can be used for blood purification, and the blood pH is 7.35 - 7.45.

[0056] The dialysis membrane PA-PTB / SA / LC was placed in 10 mL of different concentrations of uremic toxins (p-cresol sulfate, indoxyl sulfate, bilirubin) solutions respectively. After standing for 12 hours, the residual concentration of uremic toxins in the solution after adsorption was measured with an ultraviolet spectrophotometer. When the adsorption equilibrium was reached, the maximum adsorption capacities of the dialysis membrane PA-PTB / SA / LC for indoxyl sulfate, p-cresol sulfate, and bilirubin were 260 mg / g, 730.2 mg / g, and 768.4 mg / g respectively (as Figure 7 ). Compared with traditional blood purification membranes, the adsorption capacity increased by more than 10 times.

[0057] 2. Dialysis of uremic toxins by the dialysis membrane PA-PTB / SA / LC

[0058] The simulated solution is an aqueous solution containing 1 mg / mL bovine serum albumin, 1 mg / mL lysozyme, 1 mg / mL phosphorus, 40 mg / mL p-cresol sulfate, 40 mg / mL indoxyl sulfate, and 1 mg / mL bilirubin. The flow rates of both the simulated solution and the dialysis solution (water) are 10 mL / min, and dialysis is carried out for 4 hours using the dialysis membrane PA-PTB / SA / LC. The clearance rates of the dialysis membrane for phosphorus, p-cresol sulfate, indoxyl sulfate, bilirubin, and β2-microglobulin are tested. At the same time, comparative experiments are conducted with the dialysis membranes of Comparative Examples 1 to 3, and the results are as Figure 8 shown. The results show that the dialysis membrane PA-PTB / SA / LC can retain macromolecular bovine serum albumin and has good clearance effects on phosphorus, p-cresol sulfate, indoxyl sulfate, bilirubin, and β2-microglobulin, and the clearance rates are 98%, 85%, 61%, 73%, and 26% in sequence. The dialysis membrane of Comparative Example 1 can hardly retain bovine serum albumin, and the dialysis membranes of Comparative Example 2 and Comparative Example 3 can retain bovine serum albumin, indicating that the introduction of polysaccharides can make the pores of the protein membrane smaller, but the clearance rates of phosphorus, p-cresol sulfate, indoxyl sulfate, bilirubin, and β2-microglobulin are all reduced by 40% - 60%.

[0059] 3. Adsorption of the dialysis membrane PA-PTB / SA / LC to antibiotics

[0060] The dialysis membrane PA-PTB / SA / LC is respectively placed in aqueous solutions of enrofloxacin (ENR), norfloxacin (NOR), roxithromycin (ROX), oxytetracycline (TER), erythromycin (ERY), sulfamethoxazole (SMX), and sulfadiazine (SDZ) with different concentrations (50 - 500 mg / mL). After standing at room temperature for 12 hours, the permeability of the solution is tested by ultraviolet-visible absorption spectroscopy. The experimental results show that this dialysis membrane has significant adsorption capacities for enrofloxacin, norfloxacin, roxithromycin, oxytetracycline, erythromycin, sulfamethoxazole, and sulfadiazine, and their maximum adsorption amounts are 139, 134, 119, 117, 107, 95, and 92 mg / g respectively (as Figure 9 ).

[0061] 4. Adhesion of the PTB / SA / LC self-assembled membrane to platelets

[0062] Centrifuge fresh blood at 1000 rpm for 10 minutes to obtain plasma. Then, add 40 mL of centrifuged plasma containing platelets to silicon wafers coated with PTB / SA / LC self-assembled membranes (prepared in the same way as in Example 1, only replacing the polyamide ultrafiltration membrane with silicon wafers) and silicon wafers without the membrane for incubation. After incubating at 37 °C for 2 hours, rinse the silicon wafers three times with PBS buffer. Finally, fix with 1.0% glutaraldehyde by mass at room temperature for 8 hours, and then wash with PBS buffer. Subsequently, dehydrate the samples with a series of ethanol aqueous solutions with different concentration gradients (50% - 100%). Use a scanning electron microscope to observe the adhesion performance of blood cells on the PTB / SA / LC self-assembled membrane (as Figure 10 ). The experimental results show that the number of platelets adhered to the surface of the PTB / SA / LC self-assembled membrane decreases, and most of them maintain a discoid resting morphology, indicating that the dialysis membrane of the present invention has a good anti-platelet adhesion effect.

[0063] 5. Cytotoxicity of the dialysis membrane PA-PTB / SA / LC to cells

[0064] Cut the dialysis membrane PA-PTB / SA / LC into strips and immerse them in 20 mL of DMEM medium, and incubate at 37 °C for 72 hours to obtain an extract. Add 100 μL of cultured I929 cells and 1 mL of the extract to a 96-well plate, mix well, and then place them in an incubator for 24, 48, and 72 hours. After sucking out the solution in the incubated 96-well plate, add 2 mL of cck-8 solution, incubate at 37 °C for 1 hour, and then measure with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cytotoxicity (as Figure 11 ). The experimental results show that after incubating the cells for 24, 48, and 72 hours, the dialysis membrane PA-PTB / SA / LC does not have a great impact on the cell viability, and the cell viability is above 90%, higher than the ISO safety threshold (≥70%), indicating that the dialysis membrane is almost non-toxic and can significantly improve the cell compatibility of the dialysis membrane.

[0065] Example 6

[0066] Dialysis application of the dialyzer in Example 4 for uremic toxins

[0067] The simulation solution is an aqueous solution containing 1 mg / mL bovine serum albumin, 1 mg / mL lysozyme, 1 mg / mL phosphorus, 40 mg / mL p-cresol sulfate, 40 mg / mL indoxyl sulfate, and 1 mg / mL bilirubin. The flow rates of the simulation solution and the dialysis solution (water) are both 10 mL / min, and dialysis is carried out with the dialyzer for 4 hours. Test the clearance rates of the modified hollow fiber membrane in the dialyzer for phosphorus, p-cresol sulfate, indoxyl sulfate, bilirubin, and β2-microglobulin, as Figure 12As shown, after 4 hours of dialysis, the dialysis rates of the dialyzer for phosphorus, lysozyme, creatinine, p-cresol sulfate, indoxyl sulfate, and bilirubin were 91%, 18%, 56%, 42%, 61%, and 78%, respectively. In addition, the dialyzer was also able to retain bovine serum albumin with a retention rate of 98%.

[0068] As can be seen from the above, the protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate of the present invention can effectively reduce the non-specific adsorption of platelets and bacteria, and in vitro hemolysis and MTT cell activity tests prove that the dialysis membrane has good blood compatibility.

Claims

1. A protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate, characterized in that: The dialysis membrane is prepared by modifying a porous filter membrane with an amyloid-like protein / polysaccharide composite aggregate in-situ loaded with lanthanum carbonate nanoparticles, and forming a dialysis membrane with a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate as the dense skin layer and the porous filter membrane as the support layer.

2. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 1, wherein: The protein is bovine serum albumin or lysozyme, and the polysaccharide is sodium alginate or sodium carboxymethyl cellulose.

3. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 1 or 2, characterized in that: The preparation method of the dialysis membrane is as follows: Adjust the aqueous solution of 30 - 100 mmol / L tris(2-carboxyethyl)phosphine to a pH value of 6.0 - 9.0 with NaOH, then mix it with an equal volume of a 2 - 50 mg / mL protein aqueous solution and a 0.5 - 12.5 mg / mL polysaccharide aqueous solution, incubate at room temperature for 6 - 24 hours, then add LaCl3 and Na2CO3, and stir for 0.5 - 2 hours to obtain a solution containing an amyloid-like protein / polysaccharide composite aggregate loaded with lanthanum carbonate nanoparticles. Then, immerse the porous filter membrane in the solution for 2 - 12 hours to form a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate at the solid-liquid interface, that is, a dialysis membrane with a protein / polysaccharide self-assembled membrane loaded with lanthanum carbonate as the dense skin layer and the porous filter membrane as the support layer is obtained.

4. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 3, characterized in that: The mass ratio of the protein to the polysaccharide is 2:1 - 15:1, the mass ratio of the protein to LaCl3 is 50:1 - 500:1, and the mass ratio of LaCl3 to Na2CO3 is 1.1:1 - 2:

1.

5. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 3, characterized in that: The mass ratio of the protein to the polysaccharide is 4:1 - 10:1, and the mass ratio of the protein to LaCl3 is 200 - 300:

1.

6. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 1, wherein: The porous filter membrane is an ultrafiltration membrane or a hollow fiber membrane in a dialyzer assembly.

7. The protein / polysaccharide composite dialysis membrane based on in-situ loaded lanthanum carbonate according to claim 6, wherein: The membrane thickness of the ultrafiltration membrane is 12 - 240 μm, and the pore size is 50 - 200 nm; the dialyzer assembly is a PVDF ultrafiltration membrane assembly, and the membrane pore size of the hollow fiber membrane is 50 - 200 nm.

8. Application of the protein / polysaccharide composite dialysis membrane based on in-situ loading of lanthanum carbonate as claimed in claim 1 in removing uremic toxins, wherein the uremic toxins are phosphorus, creatinine, p-cresol phosphate, indoxyl sulfate, bilirubin, and β2-microglobulin.

9. Application of the protein / polysaccharide composite dialysis membrane based on in-situ loading of lanthanum carbonate as claimed in claim 1 in adsorbing a mixture of antibiotics, wherein the mixture of antibiotics is enrofloxacin, norfloxacin, roxithromycin, oxytetracycline, erythromycin, sulfamethoxazole, and sulfadiazine.

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