Albumin-immobilized cellulose and composition for treating alzheimer's disease comprising same

Abstract

The present invention relates to: an albumin-immobilized cellulose; and a composition for treating Alzheimer's disease, the composition comprising the albumin-immobilized cellulose. More specifically, the present invention relates to an albumin (human serum albumin, HSA)-immobilized cellulose having excellent amyloid beta (Aβ) adsorption capacity, and a composition for treating Alzheimer's disease by using an in vitro amyloid beta (Aβ) trap, wherein the composition comprises the albumin-immobilized cellulose.

Description

Albumin-fixed cellulose and composition for treating Alzheimer's disease comprising the same

[0001] The present invention relates to albumin-fixed cellulose and a composition for treating Alzheimer's disease comprising the same, and more particularly, to cellulose fixed with albumin (human serum albumin, HSA) having excellent amyloid beta (Aβ) adsorption capacity and a composition for treating Alzheimer's disease by in vitro amyloid beta (Aβ) trapping comprising the same.

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by impaired neurocognitive function, mental retardation, and developmental delays. This disease is characterized by neuronal loss, neurodegeneration, the formation and accumulation of amyloid beta (Aβ) plaques, and neurofibrillary tangles mediated by the tau protein. Aβ plays a crucial role in the onset and progression of AD, making it a crucial factor in the etiology of AD. Aβ accumulation in extracellular brain regions is a hallmark of the pathological condition. To date, only well-established hypotheses and concepts regarding the etiology of AD and potential drug targets are known. While the accumulation of Aβ aggregates and fibrils of tau protein is a key driver of AD's pathophysiology, these aggregations can also be caused by age-related dementias other than AD. However, pathological changes associated with AD occur long before the onset of actual signs and symptoms of the disease. Aβ peptides, containing 40 and 42 amino acid residues, are the major component of plaques formed in AD by cleavage of the transmembrane amyloid precursor protein. Aβ peptides circulate in the blood and cerebrospinal fluid (CSF).

[0003] The World Health Organization has declared Alzheimer's disease a "global health priority" because there is no permanent cure. Details about the chronological and biological progression of Alzheimer's disease, as well as the underlying therapeutic rationale, remain lacking. The interaction of Aβ oligomers with neurons and glial cells leads to a variety of pathophysiological features. These interactions include mitochondrial dysfunction, activation of glycogen synthase kinase, which induces calcium metabolism dysregulation, phosphorylation of tau protein, induction of inflammatory pathways that support oxidative stress, stimulation of neuronal cell death, and apoptosis. Therapies adopted to treat Alzheimer's disease-related pathology, including immunotherapy and pharmacological therapies, have failed in preclinical and clinical trials due to low bioavailability, difficulty crossing the blood-brain barrier (BBB), and short half-lives. Therefore, there is an urgent need to develop novel disease-modifying approaches that can prevent or slow the progression of this life-threatening neurological disease. Based on well-explained hypotheses and concepts, currently adopted treatments only aim to slow the progression of Alzheimer's disease or modify the disease.

[0004] Many therapeutic strategies among systemic approaches aim to develop AD treatments through peripheral clearance of Aβ. Recently, albumin-based plasma exchange therapy has demonstrated clinical benefit in patients with mild to moderate AD. Other approaches to lower plasma Aβ levels include adsorption approaches using Aβ-binding molecules, such as soluble low-density lipoprotein receptor-related protein (LRP) 1, proteins, and anti-Aβ antibodies. Researchers continue to focus on HSA to shift the equilibrium between CSF and plasma Aβ levels, given that the progression of AD is sensitive to plasma HSA levels. The Aβ balance between peripheral and central compartments is primarily regulated by transmembrane receptors. Specifically, these are receptors for LRP and advanced glycation end products (RAGE), which are present in the brain endothelium. LRP receptors transport Aβ from the CSF into the peripheral circulation, whereas RAGE receptors transport peripheral Aβ into the brain.

[0005] Under these technical backgrounds, the inventors of the present application confirmed the possibility of treating Alzheimer's disease by in vitro amyloid beta (Aβ) trapping using cellulose immobilized with albumin (human serum albumin, HSA) having excellent amyloid beta (Aβ) adsorption capacity, and completed the present invention.

[0006] The purpose of the present invention is to provide a composition for treating Alzheimer's disease.

[0007] An object of the present invention is to provide a medical device coated with the above composition.

[0008] In order to achieve the above-described purpose, the present invention relates to a composition for treating Alzheimer's disease by trapping amyloid beta (Aβ) in vitro, comprising a substrate on which human serum albumin (HSA) is fixed.

[0009] The present invention relates to a medical device coated with the above composition.

[0010] According to the present invention, utilizing HSA as an Aβ sequestering ligand is expected to offer several advantages, including easy integration and adoption in clinical settings, given its excellent biocompatibility, tolerability, and safety. Furthermore, HSA has already been utilized in the medical field to coat stents and tubes due to its remarkable properties. It has demonstrated antithrombotic properties and corrosion resistance, primarily due to its electrostatic and hydrophilic properties. Furthermore, it readily adheres to surfaces, mitigates endothelial cell death, provides antioxidant protection, and effectively inhibits platelet activation and aggregation. Overall, these properties highlight the potential of HSA as a valuable biomaterial for the development of medical devices specifically designed for the ex vivo treatment of Alzheimer's disease.

[0011] Figure 1. (A) Schematic representation of the functionalization and derivatization reactions of CA membrane, (B) optical side images of (a) clean CA surface, (b) CA-APTS, (c) CA-APTS-GA, and (d) CA-APTS-GA-HSA.

[0012] Figure 2a. Raman spectra of untreated and functionalized CA films: 10 scans recorded using a 1064 nm laser line. Figure 2b. FTIR spectra of HSA, untreated, and functionalized CA films. 32 measurements are recorded. Established observations that are not influenced by environmental experimental parameters (e.g., CO2 and water vapor). Figure 2c. XPS spectra of untreated, functionalized, and derivatized CA films. The recorded peaks (285, 532, 99, and 400 eV) correspond to carbon (C1s), oxygen (O1s), Si2p, and N1s, respectively. Figure 2d. TGA of untreated, functionalized, and derivatized CA films obtained in the temperature range from room temperature to 600°C and at a temperature ramp of 10°C / min.

[0013] Figure 3. SEM images of the surfaces of (A1-A3) pristine CA, (B1-B3) functionalized and derivatized CA-APTS, (C1-C3) CA-APTS-GA, and (D1-D3) CA-APTS-GA-HSA; images captured using a voltage of 5.0 kV, magnification levels of 1, 4.98, and 25 kx, and visualized distances of 50, 10, and 2 μm.

[0014] Figure 4. (A) Esterase-like activity of 5 μM, (B) 10 μM, and (C) 15 μM control HSA solutions. (D) Immobilized HSA in response to various PNPA concentrations (100, 350, and 700 μM). (E) Reaction rates observed in the control HSA solution and immobilized HSA are shown. Experiments were performed in triplicate.

[0015] Figure 5. (A) Expression vector encoding GFP-Aβ fusion protein, pQE80-GFPAβ. (B) SDS-PAGE analysis of expressed GFP-Aβ fusion protein: lane 1, before induction; lane 2, lysate collected 20 h after induction; lane 3, lysate collected 24 h after induction; lane 4, soluble fraction; lane 5, insoluble fraction; lane 6, non-adsorbed eluate from affinity chromatography; lane 7, eluate from the wash step; lane 8, purified GFP-Aβ. (C) SDS-PAGE results of GFP-Aβ purified via anion exchange chromatography; lanes 1–9: fractions eluted from the anion exchanger.

[0016] Figure 6. ITC-based analysis of the interactions between (A) HSA and GFP-Aβ, (B) HSA and Aβ-GFP, and (C) HSA and GFP.

[0017] Figure 7. (A, a) Particle size analysis of GFP-Aβ after 0 h and (A, b) 24 h of incubation; the 0 h sample exhibits a multimodal distribution, while the 24 h sample exhibits a single distribution. The particle size after 24 h is larger than that of the 0 h sample. The insert shows the increased turbidity of the GFP-Aβ solution. (B) TEM images of GFP-Aβ after 0 h and (C) 24 h of incubation; the 0 h sample exhibits a monomeric distribution of GFP-Aβ, whereas the 24 h sample exhibits fibers due to aggregation. (D) TEM analysis of freshly prepared GFP-Aβ. (E) Examination of the sample stored at -80°C after 45 days confirms the formation of fibers.

[0018] Figure 8. (A) Binding of GFP-Aβ to HSA at 1, 5, 10, 20, and 30 μM; 10 μM GFP-Aβ solution was treated as the ligand after immobilization for 1 h in amine-coupled 96-well plates, showing that 1 μM HSA was the optimal ligand sequestration concentration. (B) Concentration-dependent GFP-Aβ sequestration potential of HSA immobilized at 1 μM in amine-coupled plates and (C) GFP-Aβ sequestration potential of CA surface-immobilized HSA; data represent the means of three replicates and the standard error of each mean.

[0019] Unless otherwise defined, all 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 pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0020] Amyloid beta (Aβ) is a peptide that plays a key role in the development of Alzheimer's disease (AD). In patients with Alzheimer's disease, amyloid beta plaques accumulate in the brain. These plaques form when amyloid beta proteins aggregate and accumulate within and around neurons. These deposits disrupt communication between brain cells, leading to neuronal damage and cell death.

[0021] The medical significance of removing amyloid-beta from peripheral blood lies in its potential to mitigate the progression of Alzheimer's disease. Because Aβ plays a central role in AD pathology, lowering Aβ levels in the body could slow disease progression. Immobilizing human serum albumin (HSA) on the surface of cellulose acetate (CA) to remove amyloid-beta provides a platform for effectively capturing and removing Aβ from the blood. This could have therapeutic potential to help manage the progression of Alzheimer's disease by reducing the burden of Aβ in the brain.

[0022] Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurocognitive dysfunction and mental developmental delay. It is believed to be caused by the accumulation of amyloid beta (Aβ) in plaque form. Targeting Aβ has been considered a promising approach for the treatment of AD. We designed an in vitro Aβ blocker by covalently conjugating human serum albumin (HSA), a natural Aβ binder, to the surface of cellulose acetate (CA) membranes. HSA immobilized on CA membranes at a concentration of 3.06 ± 0.22 μg / mm2 was found to be functionally active, as evidenced by its esterase-like activity, which converts p-nitrophenyl acetate to p-nitrophenol. As a model ligand, a recombinantly produced green fluorescent protein-Aβ (GFP-Aβ) fusion protein exhibited the properties of native Aβ. These characteristics include a propensity to form aggregates or fibrils and an affinity for HSA with a dissociation constant (KD) of 0.91 μM. HSA on CA membranes was shown to sequester GFP-Aβ in a concentration-dependent manner in the 1–10 μM range. Furthermore, it exhibited greater binding affinity than HSA immobilized on commercial amine-conjugated plates. These results suggest that covalent immobilization of HSA on the CA surface could serve as a potential platform for sequestering Aβ to alleviate AD.

[0023] Based on this, the present invention relates to a composition for treating Alzheimer's disease by trapping amyloid beta (Aβ) in vitro, comprising a substrate on which human serum albumin (HSA) is fixed.

[0024] Human serum albumin, as described above, is the most abundant plasma protein derived from human plasma, accounting for 60% by weight of the total plasma protein content. HSA is composed of a single non-glycosylated polypeptide chain of 585 amino acids with a molecular weight of 66,500 Da.

[0025] Due to its relatively small molecular weight and net negative charge at physiological pH, human serum albumin accounts for 85% of the osmotic effect of normal plasma. Therefore, HSA is the primary regulator of plasma volume. A secondary role of human serum albumin is to bind small molecules produced by catabolism (e.g., fatty acids and bilirubin). Albumin represents the primary transport vehicle for these key metabolites, which are poorly soluble at physiological pH.

[0026] Human serum albumin provides abundant plasma protein, and approximately 90% of Aβ is expected to be bound to the protein. Therefore, human serum albumin serves as one of the most important physiological transport proteins for numerous endogenous and exogenous compounds.

[0027] Direct interaction between human serum albumin and Aβ helps to inhibit the initiation and progression of AD by reducing Aβ aggregation. The progression of AD is associated with a decrease in HSA-Aβ complex formation, suggesting a malfunction in human serum albumin-mediated Aβ transport. The biomolecular driving force behind this Aβ equilibrium shift remains unclear. Nevertheless, clinical improvement in patients with AD has been observed when CSF-Aβ levels were lowered using Grifol 5% and 20% HSA solutions (Albutein®). HSA concentrations are significantly reduced in the CSF because they cannot freely cross the tight junctions of the BBB.

[0028] The limitations of pharmacological approaches and the encouraging results of the Grifols Albutaine® solution led the authors to develop a novel HSA-based Aβ removal strategy. In this context, HSA was immobilized on a CA membrane, serving as a scavenging platform to capture Aβ. The CA membrane was chosen due to its enhanced biocompatibility with proteins. Furthermore, the immobilization process produced fewer toxic residues. In vitro assay characteristics and concentration-dependent Aβ removal potential suggest that CA membrane-immobilized HSA could be used as an in vitro therapeutic agent to improve AD.

[0029] Given its excellent biocompatibility, tolerability, and safety, utilizing human serum albumin as an Aβ sequestering ligand is expected to offer several advantages, including easy integration and adoption in clinical settings. Furthermore, human serum albumin has already been utilized in the medical field to coat stents and tubes due to its remarkable properties. It has demonstrated antithrombotic properties and corrosion resistance, primarily due to its electrostatic and hydrophilic properties. Furthermore, it readily adheres to surfaces, mitigates endothelial cell death, provides antioxidant protection, and effectively inhibits platelet activation and aggregation. Overall, these properties highlight the potential of human serum albumin as a valuable biomaterial for the development of medical devices specifically targeted for the ex vivo treatment of Alzheimer's disease.

[0030] The substrate may be, for example, a cellulose resin or a cellulose membrane. Any cellulose resin or cellulose membrane suitable for fixing human serum albumin, non-toxic, and biocompatible may be used without limitation. However, for example, the substrate may be a cellulose acetate (CA) resin or a cellulose acetate (CA) membrane.

[0031] Human serum albumin is fixed to the above-described substrate. Human serum albumin may be linked to the cellulose resin or cellulose membrane, for example, a cellulose acetate (CA) resin or cellulose acetate (CA) membrane, for example, through covalent bonding or affinity formation, but is not limited thereto.

[0032] Specifically, human serum albumin (HSA) can be fixed to the surface of the cellulose acetate (CA) membrane through a covalent bond. It can be characterized in that human serum albumin (HSA) is linked to the cellulose acetate (CA) membrane by including glutaraldehyde (GA) as a linker and 3-aminopropyltriethoxysilane (APTS) as a spacer, thereby forming a CA-APTS-GA-HSA bond.

[0033] The above cellulose acetate (CA) membrane can be functionalized for fixation. The cellulose acetate (CA) membrane can be partially hydrolyzed by treating it with NaOH, then treated with aminopropyl triethoxysilane (APTS) to generate a CA-APTS surface, then treated with glutaraldehyde (GA) to attach a linker moiety to APTS to generate a CA-APTS-GA surface, and then HSA can be fixed to form CA-APTS-GA-HSA.

[0034] Specifically, it starts with partial hydrolysis of a cellulose acetate (CA) membrane using NaOH, which allows the hydroxyl groups to react with sodium ions, forming covalent CA-O- bonds. The CA membrane is then treated with an aminopropyl triethoxysilane (APTS) solution, which forms CA-O-Si-(CH2)3-NH2 bonds, where the ethoxy groups of APTS are covalently bonded to the CA membrane. Glutaraldehyde (GA) is then applied to attach the linker moiety, forming CA-O-Si-(CH2)3-N=CH-CHO bonds. The aldehyde group of GA forms a covalent bond with the amino group of APTS. HSA is then immobilized on the surface, forming CA-O-Si-(CH2)3-NH-CO-HSA bonds, which involve covalent bonds between the aldehyde groups of HSA and GA.

[0035] Protein immobilization is achieved by using GA as a linker molecule to connect HSA molecules and APTS as a spacer to stably link GA molecules to the CA surface. Otherwise, protein immobilization is difficult due to the steric hindrance of the hydroxyl (OH) groups present on the CA surface. Functionalization and derivatization of CA membranes containing OH groups are problematic because these groups can react with other OH and amino groups on the APTS backbone. It is said that the use of a weakly basic catalyst in the temperature range of 80–100°C can promote the synthetic reaction between OH and amino groups by synergistically activating the reactivity of the amino groups. However, the use of a strong basic catalyst in the reaction results in the formation of ether derivatives with low amino reactivity. In contrast, the ethoxy group of APTS undergoes hydrolysis and represents an intermediate step in the chemical reaction.

[0036] The GFP-Aβ fusion protein was used as a model ligand to evaluate the Aβ adsorption capacity of the above HSA-immobilized substrate. GFP-Aβ is a protein in which GFP, a fluorescent protein, and Aβ, a ligand of HSA, are genetically fused, and the adsorption pattern of Aβ can be easily observed through fluorescence. GFP-Aβ had an affinity (K) of 1.10 μM for HSA in the ITC (isothermal thermal calorimetry) analysis. D ) and still possessed the original fibril or aggregation forming properties of Aβ, so it was evaluated as a useful ligand for evaluating the Aβ adsorption capacity of albumin-coated cellulose membranes.

[0037] HSA was covalently immobilized on CA membranes using APTS and GA under mild alkaline conditions. Membrane derivatization and functionalization were confirmed by analytical techniques (Raman spectroscopy, FTIR, SEM, XPS, and TGA). The esterase-like activity of HSA verified its structural integrity. The kit-based quantification of the measured immobilized HSA was 3.06 ± 0.22 μg / mm². Furthermore, the GFP-Aβ fusion protein served as a model ligand by retaining the intrinsic properties of Aβ, including fiber formation and particle size increase, upon storage. Analysis using ITC showed that the K between GFP-Aβ and HSA D was found to be 0.91 μM. HSA immobilized at 1 μM on amine-binding plates showed optimal binding to GFP-Aβ in a concentration-dependent manner. HSA immobilized on CA membranes (except for the initial 1 nM concentration) showed concentration-dependent GFP-Aβ sequestration.

[0038] Nearly 90% of Aβ in the systemic circulation binds to HSA, which regulates Aβ oligomerization in the brain by regulating Aβ levels in the CSF and blood. Implementation of an albumin-based plasma exchange strategy results in Aβ mobilization and stabilization of cognitive function, memory, and language abilities in AD patients.

[0039] In an experiment to determine the optimal HSA concentration for effectively sequestering Aβ, the HSA concentration that exhibits optimal binding ability to GFP-Aβ was evaluated, and the human serum albumin may be included at a concentration of 1 μM or more. The human serum albumin may be specifically included at a concentration of 1 to 30 μM. Specifically, it was confirmed that 1 μM HSA was the optimal ligand sequestration concentration.

[0040] The development of Alzheimer's disease treatments has been a mixed bag, with numerous successes and failures. While several therapeutic options have shown promising results in treating AD, they often induce severe neurological and immunogenic reactions. Leveraging HSA's natural ability to bind Aβ as an ex vivo Aβ-trapping platform could be an attractive, biocompatible, and tunable in vitro option for improving AD.

[0041] Based on this, the composition according to the present invention can be characterized by external absorption of amyloid beta (Aβ) in the blood of an Alzheimer's disease patient. External AD treatment is possible through fixation of HSA to the CA membrane.

[0042] Utilizing CA as a matrix aligns with sustainable and environmentally sound practices and offers advantages in terms of reduced environmental impact compared to alternative materials used in the reported technology. By demonstrating excellent Aβ binding capacity, confirming structural integrity through esterase-like activity, and demonstrating concentration-dependent GFP-Aβ sequestration, this method is expected to establish itself as a safe and highly biocompatible in vitro AD treatment platform.

[0043] "Treatment" refers to any sign of success in the treatment or amelioration of an impairment, pathology, or condition, including any subjective or objective parameter, such as alleviation; remission; making symptoms or impairments more tolerable to the patient; reducing the pathology or condition; slowing the rate of regression or decline; creating a less debilitating final point of regression; or improving the physical or mental well-being of the patient. Treatment or amelioration of symptoms may be based on objective or subjective parameters, including physical examination, neuropsychiatric testing, and / or psychiatric evaluation.

[0044] An "effective amount" is generally an amount sufficient to reduce the severity or frequency of a symptom, eliminate a symptom, its underlying cause, prevent the onset of a symptom or its underlying cause, or ameliorate or correct impairment resulting from or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to correct a disease state or symptom, particularly a condition or symptom associated with a disease state, or otherwise prevent, impede, delay, or reverse the progression of a disease state or any other undesirable symptom associated with the disease in any way.

[0045] The composition according to the present invention may be a pharmaceutical composition. "Effective amount" or "therapeutically effective amount" refers to the amount necessary (with respect to the dosage, duration, and method of administration) to achieve the desired therapeutic result. An effective amount is at least the minimum amount of an active agent necessary to provide therapeutic benefit to a subject, and is less than a toxic amount.

[0046] The extent to which amyloid beta (Aβ) is adsorbed from the blood of an Alzheimer's disease patient in vitro in a pharmaceutical composition can be determined by other factors known to those skilled in the art. Depending on the severity of the symptoms / disease, the degree of the patient's symptoms / disease, needs, age, responsiveness, etc., the dosage may be adjusted based on the professional judgment of the administration supervisor, taking into account comprehensive factors. The concentration range presented in the present invention is merely an example and is not intended to limit the embodiments of the claimed composition to this range. A single treatment or multiple divided treatments at lower concentrations or amounts may also be administered.

[0047] The present invention also relates to a medical device coated with the composition. The device may be a tube.

[0048] Specifically, it may be a blood purification CA tube having its inner lining coated with HSA-immobilized CA or packed with HSA-immobilized CA beads. Such a device may be introduced as part of an extracorporeal blood circulation device or blood purification device for removing Aβ from the blood of AD patients.

[0049] Example

[0050] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0051] Manufacturing example

[0052] 1. Materials and Methods

[0053] 1.1 Materials: Cellulose acetate (CA, MW 50000) and N,N-dimethylformamide (DMF) were all purchased as high-purity grades from Daejung Chemical, Korea. Aminopropyl triethoxysilane (APTS, purity 99%), para-nitrophenyl acetate (PNPA, purity ≥98%), and HSA (purity ≥98%) were purchased from Sigma-Aldrich, Korea. 50% glutaraldehyde (GA) solution was procured from TCI, Japan, and high-purity sodium hydroxide was procured from Deoksan, Korea.

[0054] 1.2 Derivatization, functionalization and HSA immobilization synthesis of CA membranes:

[0055] CA membranes were synthesized via a solution casting technique. Powdered CA was dried overnight at 100°C. CA powder (12% by weight) was dissolved in DMF (88% by weight) at 60°C for 3 h with constant stirring at 600 rpm until completely dissolved. The homogeneous dope solution was stored at room temperature (RT) overnight to remove air bubbles and then cast onto a flat glass plate using a doctor blade. The cast membrane and the glass plate were immersed in a deionized water bath (2.0 L of deionized water). After 1 h, the molded membrane was peeled from the glass plate and removed from the deionized water bath. Finally, the obtained membrane was washed three times with deionized water to remove residual DMF solvent and then dried overnight at RT.

[0056] The membrane was vacuum-dried at 80°C for 8 h and then treated in a weakly basic alkaline environment at 37°C for 4 h to partially hydrolyze the membrane (30 mL, 0.1 N NaOH). The hydrolyzed membrane was then placed in 20 mL of a 20% ultrapure APTS solution, and 2 mL of a 0.1 N NaOH solution was added, and the membrane was treated at 37°C for 24 h to generate a CA-APTS surface. The membrane was thoroughly washed several times with sufficient ultrapure water to remove any traces of unreacted APTS. The membrane was then placed in 20 mL of a 50% GA solution at 40°C for 4 h, and 0.1 mL of a 0.1 N NaOH solution was added to attach the linker moiety to APTS, generating a CA-APTS-GA surface. Finally, the membranes were immersed in 40 mL of each HSA solution (typically 1%) at 37°C for 6 h to immobilize HSA on the pre-activated surface, generating a CA-APTS-GA-HSA adhesion surface. After the immobilization step was completed, the membranes were thoroughly washed with ultrapure water and stored in ultrapure water at 4°C to prevent microbial growth and contamination. The synthetic scheme for CA surface functionalization and derivatization is illustrated in Figure 1(A).

[0057] 1.3 Analytical Characterization of Derivatized, Functionalized, and HSA-Immobilized CA Membranes:

[0058] The prepared membrane is 600-4000 cm -1The functionalization and derivatization steps were investigated by Fourier transform infrared spectroscopy (ATR-FTIR) using an FT-4100 FT-IR spectrophotometer (JASCO, Japan). The recorded spectra are the average of consecutive measurements after removing the band noise of the atmosphere, the effects of atmospheric carbon dioxide, and water vapor. Raman spectroscopy was performed using a Raman-DXR Raman microscope from Thermo Scientific using a 1064 nm laser line. A total of 10 scans were performed to obtain the Raman spectra. To evaluate the surface morphology of the CA membranes after each functionalization and derivatization step, scanning electron microscopy (SEM) TESCAN (VEGA Ⅱ LSU, Czech Republic) was used at a voltage of 5.0 kV, magnifications of 1.00, 4.98, and 25.0 k×, and viewing distances of 50, 10, and 2 μm. RT~600 using TA Instruments (Japan TMA Q400) with the temperature raised by 10 degrees while maintaining nitrogen flow at 50 mL / min. o The thermal stabilities of the pristine and functionalized CA membranes were evaluated by thermogravimetric analysis (TGA) in the C range. Additionally, the pristine and derivatized membranes were analyzed by X-ray photoelectron spectroscopy (XPS), and the elemental quantification achieved by APTS coating and HSA immobilization on the CA surface was evaluated by an XPS-K ALPHA (Thermo Scientific) spectrophotometer. Surface analysis of the pristine and activated membranes was performed using a scanning electron microscope (SEM) equipped with a TESCAN (VEGA II LSU, Czech Republic) instrument.

[0059] 1.4 Quantification of fixed HSA molecules per unit area:

[0060] Quantification of immobilized HSA per unit area on the CA membrane surface was performed using the Thermo Scientific® Pierce Coomassie Plus (Bradford) Protein Assay Kit (Product No. 23236). A calibration curve was constructed for HSA solutions ranging from 0 to 2000 μg / mL. CA membranes functionalized with APTS, GA, and HSA of defined dimensions (18 mm length, 16 mm width) were tested. Experiments were performed in triplicate for each membrane. HSA-immobilized CA membranes, clean CA membranes (control), and naked HSA solutions were incubated with 1500 μL of the Bradford solution provided in the kit and incubated at RT for 10 min. The absorbance was then recorded at a wavelength of 595 nm using a Harvard Bioscience™ BioDrop DUO UV / Vis spectrophotometer (Sweden). The amount of fixed HSA per unit area (mm2) of the CA surface was determined with reference to the constructed calibration curve.

[0061] 1.5 Esterase-like activity of immobilized HSA:

[0062] To assess the functional preservation of HSA molecules after immobilization on CA membranes, the esterase-like activity of immobilized HSA molecules was performed using PNPA as a substrate. Various concentrations of HSA (5, 10, and 15 μM) were prepared in phosphate-buffered saline (PBS, pH 7.4) and incubated with increasing PNPA concentrations of 100, 350, and 700 μM. Similarly, HSA immobilized on CA membranes was prepared by placing the functionalized CA membranes in 20 mL of 1%, 2.5%, and 5% PNPA in the presence of 2 mL of 0.1 N NaOH solution. The absorbance of the released product, p-nitrophenol, was recorded at 400 nm using a Harvard Bioscience™ BioDrop DUO UV / Vis spectrophotometer (Sweden) at 2, 7, and 14 min of incubation using a molar extinction coefficient of 17700 M-1 cm-1. The reaction rate (V, μM / s) was calculated by determining the time-dependent conversion of substrate. In enzyme kinetics, the initial reaction rate is determined by monitoring the change in optical density (OD) over a short time interval. This rate can be obtained by dividing the change in OD by the time interval. The rate is then converted to concentration per time by dividing by the product of the extinction coefficient and the path length to normalize the measurements.

[0063] 1.6 Expression, isolation, and characterization of GFP-Aβ fusion proteins (GFP-Aβ, Aβ-GFP)

[0064] pET21-AβM1-42-GFP was synthesized by Bionics (Korea). The codons were optimized for expression in Escherichia coli. This plasmid DNA served as a template for PCR amplification of the Aβ gene (126 bp). To express and isolate Aβ-GFP fusions, expression vectors were generated by infinite cloning

[0032] . Two types of pQE80 expression vectors (pQE80-AβM1-42-sfGFP and pQE80-sfGFP-Aβ1-42) containing Aβ1-42 and super-spliced ​​GFP (sfGFP) were constructed by infinite cloning. These expression vectors encode Aβ1-42 and sfGFP proteins with a flexible linker, containing (G4S)3 repeats in the linker sequence between Aβ and sfGFP. A sixfold His tag was added to the N- or C-terminus of sfGFP for purification. The pQE80 expression vector was transformed into Escherichia coli BL21(DE3) cells. Cells were cultured in a culture medium at 37°C until the culture reached an OD600 of 0.5–0.7, then induced with 1 mM IPTG and cultured at 37°C for 20 h. Cells were harvested (8,000 × G, 20 min) and resuspended in Tris lysis buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4, and 0.1 mg / mL lysozyme). The cell lysate was homogenized by sonication at 50% amplitude for 30 cycles (30 s on, 30 s off) and centrifuged at 12,000 rpm for 20 min at 4°C. Sixfold His-tagged sfGFP-Aβ1-42 was purified using Ni-NTA resin (Thermo Fisher, USA) according to the manufacturer's instructions. Secondary purification was performed using FPLC equipped with a HiTrap Q HP anion-exchange chromatography column (Cytiva, Korea). The eluted sfGFP-Aβ1-42 was buffer-exchanged into phosphate-buffered saline (PBS, 10 mM 154 mM NaCl) using a PD-10 desalting column (Cytiva, Korea).Proteins were analyzed on 5–15% SDS-PAGE gels and stained with Coomassie blue.

[0065] 1.7 Isothermal titration calorimetry (ITC) analysis of the interaction between GFP-Aβ and Aβ-GFP and HSA

[0066] The interactions of GFP-Aβ, Aβ-GFP, and HSA, and the formation of Aβ aggregates after incubation for a certain period of time were evaluated by ITC, differential light scattering (DLS), and transmission electron microscopy (TEM). The thermal behavior of the interaction of HSA and GFP-Aβ was studied at 25°C in 10 mM PBS (containing 154 mM NaCl, pH 7.4) using an Auto-iTC200 microcalorimeter at the Korea Basic Science Institute. To perform the analysis, the sample cell and syringe of the calorimeter were filled with the respective molar concentrations of HSA (40 μL of 100 μM) and GFP-Aβ solutions (200 μL of 10 μM). During the titration, GFP-Aβ was injected in small amounts into the HSA-loaded sample cell. The heat exchange generated by the interaction of the two samples was measured by the ITC calorimeter. The ITC system was operated by allowing an appropriate time interval between sample injections to reach equilibrium. Data were obtained in the form of heat exchange versus time and fitted to a set of binding site models, and the results were presented in the form of heat exchange curves obtained as a function of the molar concentrations of HSA, GFP-Aβ, and Aβ-GFP. All ITC experiments were performed under constant temperature and pressure and analyzed using MicroCal Origin™ software. Samples were analyzed in three replicates and the average values ​​were reported to ensure accuracy between results.

[0067] 1.8 Analysis of the self-aggregation tendency of GFP-Aβ

[0068] The formation of aggregates due to self-interactions of GFP-Aβ molecules, which confirms the formation of Aβ plaques and subsequent tangle formation, was assessed by DLS analysis using an Anton Paar (Litesizer500) instrument and TEM. GFP-Aβ solutions prepared at a concentration of 10 μM were stirred at RT (600 rpm) for 24 h, and then the samples were analyzed by DLS, and particle size and the associated intensity distribution were assessed at both 0 and 24 h after incubation. The results were plotted as the % of intensity distribution versus particle size (nm) over the relevant time range. The aggregation of GFP-Aβ was further cross-checked by TEM using an HT-7800 (Hitachi High-Technologies) at magnifications of 20, 50, 100, and 200 kJ and an operating voltage of 100 kV. These stored GFP-Aβ samples were also examined by TEM and compared with freshly prepared samples to determine the presence of aggregation.

[0069] 1.9 Binding of HSA and GFP-Aβ fusion

[0070] The GFP-Aβ binding assay was performed in a double-check manner. In the first assay protocol, the binding ability of HSA to GFP-fused Aβ was assessed in a concentration-dependent manner by immobilizing an optimized HSA concentration (1 μM) on G-bioscience 96-well amine-conjugated plates (Cat. # 786-756), whose surfaces were pre-activated with maleic acid. In the first step, various concentrations of HSA (1 μM, 5 μM, 10 μM, 20 μM, and 30 μM) were immobilized to determine the HSA concentration that could sufficiently bind to GFP-Aβ, and the plates were incubated with 10 μM GFP-Aβ for 1 h. The assay was then performed according to the manufacturer's instructions with minor modifications. Briefly, the wells of the well-coated amine-conjugated plates were washed three times with ultrapure water. Control and treated samples (200 μL each, HSA, GFP, GFP-Aβ) were prepared with binding buffer (PBS, pH 7.4, Cat. # P5493) and incubated overnight at 37°C with periodic shaking for effective binding. The samples were then removed, and 300 μL of blocking buffer (Cat. # 11112589001) was added to each well for 60 min to block unreacted sites. The wells were then washed three times with 300 μL of phosphate-buffered saline-Tween (PBST, 0.05% Tween 20, pH 7.4) washing buffer. 200 μL of 1 nM–10 μM GFP-Aβ was added to the HSA-bearing wells, incubated for 1 h, and then washed four times with PBS buffer. Finally, absorbance was recorded using a microplate reader for GFP at excitation and emission wavelengths of 485 nm and 520 nm, respectively. HSA binding capacity was determined by subtracting the absorbance of the control wells without HSA.

[0071] For binding of immobilized HSA to GFP-Aβ on CA membrane-based plates, CA membranes were cut into circular discs with the same size as the bottom of a 96-well plate. These discs were vacuum-dried and immobilized with a 1% HSA solution. The derivatized and functionalized CA membrane discs were stored in 20 mL of each HSA solution and then stored in cold ultrapure water at 4°C. Before placing the HSA-immobilized discs, the discs were dried at 45°C for 12 h. The discs were then placed in triplicate on a 96-well coated black plate, and the binding procedure and calculations (average) were performed as described above. The intrinsic fluorescence of the CA membrane was determined by measuring the absorbance of the naked CA discs and subtracted from the final calculations.

[0072] Example

[0073] Example 1. HSA-immobilized CA membrane

[0074] Polymeric membranes derived from biorenewable resources have been primarily used in various biomedical applications. The suitability of the membrane surface depends on the availability of specific functional groups that support the biomechanical properties of the resulting potential product. Various approaches have been employed to introduce various functionalities into the membranes to make them suitable, tunable, and modifiable for their intended biological applications. Membrane-based immobilization of biologically active molecules can yield promising biomedical and environmental outcomes. Among various biopolymers, cellulose and its derivatives (e.g., ethyl cellulose and CA) have attracted considerable attention due to their enhanced biocompatibility and reduced post-use impact. Membranes synthesized from cellulose release glucose and acetyl groups as a result of degradation. Therefore, the presence of proton-donating moieties leads to a decrease in pH due to the formation of acetic acid. The degradation of cellulose membranes is entirely dependent on the pH level of the environment. This can be optimally controlled by adjusting the degree of acetylation of the cellulose derivatives used.

[0075] Protein immobilization is achieved by using GA as a linker molecule to connect HSA molecules and APTS as a spacer to stably link GA molecules to the CA surface. Otherwise, protein immobilization is difficult due to the steric hindrance of the hydroxyl (OH) groups present on the CA surface. Functionalization and derivatization of CA membranes containing OH groups are problematic because these groups can react with other OH and amino groups on the APTS backbone. It is said that the use of a weakly basic catalyst in the temperature range of 80–100°C can promote the synthetic reaction between OH and amino groups by synergistically activating the reactivity of the amino groups. However, the use of a strong basic catalyst in the reaction results in the formation of ether derivatives with low amino reactivity. In contrast, the ethoxy group of APTS undergoes hydrolysis and represents an intermediate step in the chemical reaction.

[0076] Optical side images of the CA membrane before, after, and during the derivatization process are shown in Figure 1(B). The CA membrane remains white during initial hydrolysis. Subsequent derivatization with APTS reveals a slight yellowish tinge. Upon GA linkage, the membrane turns reddish-brown and fluoresces when analyzed using an analytical instrument. Therefore, the sample is analyzed using the 1064 nm Raman laser line. HSA immobilization results in a slightly enhanced color, as observed for GA linked to the CA surface.

[0077] Example 2. Analytical characterization of synthesized and functionalized CA membranes.

[0078] Using various analytical techniques, we confirmed the stepwise linking strategy and the final immobilization of HSA molecules on the clean CA membrane surface. The Raman spectroscopy results are shown in Figure 2a. The Raman spectrum of the clean CA membrane shows a characteristic peak at 659 cm -1 The small peak observed at 978 cm is attributed to the C-OH group, while -1 The absorbance recorded at is due to the CO function. Similarly, the recorded absorbance values ​​(1382, 1435 and 1736 cm -1 ) are attributed to acetyl, C=O and CH groups, respectively. Finally, 2934 cm -1 A characteristic peak can be observed in , which shows the CH stretching of the glycosidic bond present in the chemical skeleton of CA.

[0079] For the CA-APTS Raman spectrum, the peaks at 300, 1485, and 1575 cm in the pristine CA film -1 An increase in intensity is observed as the bands appear, indicating that Si-O-Si and NH2 groups are bonded after the APTS molecule is attached under weak basic conditions. Also, the bands at 1100 and 900-1200 cm -1The bands of represent the COC functionality and Si-O-cellulose interaction, respectively. The glycosidic CH stretch becomes less intense and shifts to lower wavenumbers due to the attachment of the spacer molecule. In the case of GA and HSA attachment, which yields CA-APTS-GA and CA-APTS-GA-HSA, the peaks are intensified, indicating the binding of the two molecules. The characteristic peaks of CN in GA are located at 1400-1700 cm -1 This is clearly evident in the region. The CH stretch of the glycosidic bond broadens and shifts to lower wavenumbers as a result of the GA linkage, and becomes sharper and more intense after the HSA molecule is attached. Similarly, membranes analyzed under the 633 nm laser line were evaluated as reported in the literature, but the observation was unreliable due to the fluorescence of the GA molecules.

[0080] The FTIR (Fourier Transform Infrared) spectrum of HSA shows the presence of pristine, functionalized, and derivatized CA films (Fig. 2b). The FTIR spectrum of HSA (immobilized component) shows the peaks at 1631, 1670, 3070, and 3300 cm, respectively. -1 It showed characteristic bands representing amide-I, amide-II, amide-B, and amide-A of the HSA backbone. In contrast to the original CA, the derivatized and functionalized membranes showed significant changes in the FTIR spectra. Partial hydrolysis of the original CA increased the number and size of surface OH groups. However, the bands of the acetyl groups decreased and the intensity of the bands specific for C=O decreased significantly. The untreated CA membrane and the derivatized CA membrane showed characteristic bands representing amide-I, amide-II, amide-B, and amide-A in the FTIR spectra at 800–1200 cm -1 It has various peaks in the range of 950 cm -1 The distinct peaks observed are considered to be due to the presence of CO bonds. The functional bonds at each step can be observed with different recorded intensities in the FTIR spectra of all functionalized and derivatized CA films.

[0081] APTS-based functionalization shifts the original CA band due to the influence of Si moieties. The intensity of the peak observed at 1250 cm-1 due to the ether groups decreases after functionalization due to hydrolysis of the acetyl groups or polymer chains. The decrease in the band intensity of this particular peak indicates partial hydrolysis of the CA film. The functionalization of APTS shifts the intensity of the CA band at 1145 cm-1 due to Si-O-Si atoms. -1 Indicates the bands transformed with APTS. Overall, APTS functionalization results in an overall decrease in band intensity. This is thought to be due to cleavage rather than the formation of multiple binding sites due to steric hindrance caused by the high molecular weight of APTS. In the HSA immobilization process, several amino and carboxyl groups show the same reactivity tendency under the given reaction conditions. The functional link between HSA and GA is at 1650 cm -1 In -C=N-. This linkage also exists between GA and APTS. However, as can be seen from the band intensity, the linkage between HSA and GA is stronger. The functional linkage based on the carboxyl group of GA and HSA is at about 1320 cm due to the established -CO-OO- bond. -1 The bands appearing in the FTIR results are supported by the findings of Raman spectroscopy.

[0082] Further evidence of derivatization and functionalization is provided by X-ray photoelectron spectroscopy (XPS) survey scans, as shown in Figure 2c, which illustrate the chemical composition of CA before and after the functionalization strategy. The XPS scan of the untreated CA membrane shows two inconspicuous peaks at 285 eV and 532 eV, which correspond to C1 and O1, respectively. The CA XPS survey spectrum did not observe any solvent residue peaks, demonstrating the efficient synthesis of the CA membrane. The CA-APTS XPS scan shows the appearance of a small new peak at 99 eV, which is attributed to Si 2p, demonstrating APTS anchoring and silane coupling on the CA surface. The same peak is also observed in the CA-APTS-GA and CA-APTS-GA-HSA XPS scans, with a significant decrease in intensity after the binding of HSA molecules. In addition, a small peak is observed at 400 eV, which corresponds to N1. The intensity remains the same after the binding of GA, but is significantly enhanced after the immobilization of HSA molecules. Then, the Si peak decreases due to the joint-like attenuation of HSA molecules on the pre-activated CA surface. The original CA film contains 43.3% carbon (mass concentration: 36.4%) and 56.2% oxygen (mass concentration: 62.9%).

[0083] CA-APTS and CA-APTS-GA contain 70.6% carbon (mass concentration, 63.8%), 26.3% oxygen (mass concentration, 31.7%), 1.2% silicon (mass concentration, 2.6%), and 1.8% nitrogen (mass concentration, 1.9%). CA-APTS-GA-HSA has the contents of 67.71% carbon (mass concentration, 61.4%), 26.46% oxygen (mass concentration, 26.4%), 2.85% silicon (mass concentration, 2.4%), and 9.4% nitrogen (mass concentration, 9.9%). The XPS results support those of FTIR and Raman spectroscopy, clearly confirming the functionalization and immobilization of HSA molecules on the CA surface.

[0084] Thermal analysis of products is essential for commercial and industrial applications. Therefore, this was implemented for both untreated and functionalized CA membranes. As shown in Figure 2d, thermal analysis evaluates and compares temperature-dependent membrane degradation. The untreated CA membrane exhibits two typical stages of degradation and degradation with increasing temperature. The first stage is due to the loss of bound water molecules, and the second stage is due to the dissociation and degradation of polymer chains. The thermal stability of CA-APTS-GA differs significantly from other functionalized CA membranes due to the strong interchain bonds established between the chains. The same derivatized membrane also exhibits a slightly different thermal response to the loss of water molecules immediately after the initiation of thermal profiling, with subsequent changes observed at 240°C and 320°C. The residue formation in the CA-APTS membrane is significantly longer. This is believed to be due to the presence of SiO2 moieties and demonstrates the occurrence of silane coupling on the CA membrane surface.

[0085] Thermal properties are stable after coupling and attachment of HSA molecules. The HSA molecules lose mass due to the evaporation of water molecules during the second thermal shift, which occurs at 260 °C. All CA membranes exhibit a mass loss of up to 15% until the temperature reaches 200 °C (except for CA-APTS-GA, which shows a mass loss of approximately 40%). As the temperature increases, a degradation shift of the CA membrane occurs due to the degradation of the cellulose moiety. The untreated CA membrane exhibited the lowest observed residual mass, demonstrating that the required structural integrity and biomedical compatibility are maintained after partial hydrolysis of the membrane.

[0086] The surface morphology of untreated and functionalized CA membranes was evaluated by scanning electron microscopy (SEM). The results are shown in Figure 3. The untreated CA membrane exhibited a clean and smooth surface morphology with no noticeable residual polymer formation (Figures 3A1-A3). This initial result serves as a baseline for understanding the subsequent changes induced by surface modification. Exposure of the CA membrane to sequential treatments with APTS and GA resulted in distinct topographical changes. The APTS treatment resulted in a softer and more brittle membrane texture (Figures 3B1-B3).

[0087] A significant increase in surface roughness was observed. This is attributed to the polymer tightening effect resulting from the cross-linking of GA molecules and APTS (Figs. 3C1-C3). These cross-linking-induced changes become more evident with increasing magnification, and the appearance of small, porous spherical structures contributing to the overall roughness of the membrane becomes apparent. The increased networking observed in the CA-APTS-GA membrane is thought to be due to dimerization reactions with the alkyl groups of multiple molecules. The smoothness and brittleness of the membrane returned to normal after subsequent GA and HSA treatments (Figs. 3D1-D3), demonstrating a crucial step in the functionalization process. The networking and irregularities caused by the previous APTS and GA treatments were significantly alleviated upon exposure to HSA, resulting in a more uniform and smoother surface, and the covalent bonding of HSA molecules to the pre-activated CA surface emphasized the layering. Ultimately, SEM analysis revealed complex, step-by-step changes in surface morphology occurring at various stages of membrane functionalization.

[0088] Example 3. Quantification of fixed HSA per unit area

[0089] The amount of immobilized HSA per unit area of ​​the CA membrane is determined by the Bradford protein assay. Typically, the amount of immobilized protein is estimated by subtracting the amount of void and wash fractions, but this strategy is not consistently feasible. This assay kit allows for direct and indirect protein estimation. In acidic media, Coomassie blue dye binds to proteins, which is observed as a shift in the absorbance maximum from 465 nm to 595 nm and a change in color from brown to blue. The measured absorbance increases at 595 nm, indicating that the protein sample is in a liquid or solid state, allowing for a direct estimation. Conversely, the absorbance decreases at 465 nm, indicating that unbound dye has been removed from the solution in the presence of the protein sample. 288 nm 2 The average amount of HSA immobilized on the CA membrane surface was 3.06 ± 0.22 μg / mm 2 It was found to be 881 μg, corresponding to the surface density of .

[0090] Example 4. Esterase-like activity of immobilized HSA

[0091] The effect of immobilization on the intrinsic enzymatic function of HSA was assessed by measuring its esterase-like activity. The esterase-like activity of serum albumin is of great pharmacological and biological importance. Serum albumin was monitored by the absorbance of p-nitrophenol released at 400 nm upon hydrolysis of p-nitrophenyl acetate (PNPA). When interacting with other molecules, HSA exhibited significant three-dimensional flexibility and allosteric modulation. The esterase-like activity of HSA is similar to that of the acetylcholinesterase family of proteins, which possess a triple catalytic complex consisting of a histidine nucleophile, a serine nucleophyll, and an acidic group. This group cooperates to cleave a CO bond in the substrate. The non-canonical catalytic triad, consisting of a lysine nucleophyll, a histidine nucleophyll, and a histidine-stabilizing carbonyl group from the backbone, is exhibited by HSA site-I and is considered to be the origin of HSA's catalytic activity. The experimental results of the analysis are shown in Figure 4, demonstrating that naked HSA solutions exhibit concentration-dependent esterase activity as the substrate molecule concentration increases. As the concentration of PNPA increased during incubation, immobilized HSA also exhibited the same pattern of activity, demonstrating preservation of function after immobilization.

[0092] Example 5. Expression, isolation, and characterization of GFP-Aβ fusion protein.

[0093] High yields of Aβ are required for in vitro and in vivo studies of Aβ, but obtaining large quantities from neural tissue is difficult. Furthermore, isolated Aβ peptides can rapidly aggregate, making Aβ extracted from neural tissue unsuitable for biological activity. Advances in genetic engineering tools have facilitated the mass production of Aβ. However, efficient expression of Aβ is not possible using existing Escherichia coli expression systems due to the poor solubility and significant toxicity potential of Aβ. These limitations can be overcome by using a fusion tag as a soluble and non-toxic partner for Aβ. We recombinantly produced GFP-fused Aβ (GFP-Aβ) in E. coli using the expression vector pQE80-GFPAβ (Fig. 5A).

[0094] A 15-amino acid long linker sequence was introduced to link Aβ and sfGFP, generating a GFP-Aβ fusion construct. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that GFP-Aβ was strongly expressed at 0 h after induction with an expected molecular weight of 33 kDa (Fig. 5(B), lane 2). Using GFP as a solubility tag, GFP-Aβ was found mostly in the soluble fraction (Fig. 5(B), lane 4 vs. lane 5). Affinity chromatography and anion exchange chromatography using Ni-NTA agarose in a gravity column purified GFP-Aβ with a purity of 95% and a yield of 51 mg / L per culture (Fig. 5(C)). An Aβ-GFP fusion protein, in which Aβ is fused to the N-terminus of GFP, was produced and purified similarly to GFP-Aβ. GFP-Aβ, in which the C-terminus of Aβ is not sterically hindered, can bind to HSA, whereas Aβ-GFP cannot, indicating that the C-terminal region of Aβ contributes more significantly to the HSA interaction. The region spanning residues (K28-M35) located near the C-terminus of Aβ42 has the highest binding affinity and is the major interaction site with HSA. The importance of the C-terminal region of Aβ42 in the HSA interaction has also been demonstrated by NMR studies and MD simulations.

[0095] Example 6. Quantitative binding strength of fusion proteins to HSA

[0096] Intermolecular binding processes always involve some type of heat exchange due to the interaction between the two moieties. Calculating and measuring the amount of heat absorbed or released can provide valuable information for characterizing the binding and interaction. Isothermal titration calorimetry (ITC) is one of the thermodynamic detection techniques readily available for efficient thermal analysis of intermolecular interactions. Unlike other spectroscopic methods, ITC is a robust and robust technique. Furthermore, ITC can analyze turbid, colored, and opaque solutions. ITC accuracy ranges from medium to high affinity, with the most accurate calculations typically occurring in the nanomolar to millimolar range, mimicking biological interaction analysis. A single ITC experiment can determine heat exchange, stoichiometry, and interaction-related binding constants. The heat exchanged is equivalent to the change in binding enthalpy (ΔH0) due to the conformational change occurring during binding and can be used to indirectly calculate the Gibbs free energy. The thermal behavior of expressed fusion proteins (i.e., GFP-Aβ and Aβ-GFP) interacting with HSA is shown in Figure 6. Unlike Aβ-GFP, GFP-Aβ exhibited an endothermic interaction with HSA with a dissociation constant (KD) of 0.91 μM, indicating a moderate affinity between HSA and the ligand. HSA and wild-type GFP did not show a reasonable binding pattern. Based on ITC data, Aβ-GFP exhibited a very low binding affinity for HSA, so GFP-Aβ was selected as the ligand for further binding studies.

[0097] Example 7. Self-aggregation tendency of GFP-Aβ

[0098] Dynamic light scattering analysis of samples before and after aggregation is shown in Figure 7(A). The multiple intensity distribution peaks of various sizes in the nanometer scale observed at 0 h (i.e., before aggregation) were converted to a single distribution peak after 24 h of incubation at 37°C. After 24 h of treatment, the size of the aggregates increased (~600 nm) compared to the original size of 3–5 nm observed in the sample before aggregation. Aggregation after 24 h of incubation was also confirmed in transmission electron microscopy (TEM) images taken at various magnifications (Figures 7(B) and (C)). Visual observations revealed that GFP-Aβ self-aggregated during storage. The self-aggregation of the stored GFP-Aβ sample was also examined by TEM and compared with that of the freshly prepared sample (Figures 7(D) and (E)). TEM images indicate that GFP-Aβ self-associates to form fibrils. Therefore, fresh GFP-Aβ samples were prepared for experimental purposes to confirm the monomeric state of the fused form of Aβ.

[0099] Example 8. Concentration-dependent binding of GFP-Aβ to HSA or CA-APTS-GA-HSA

[0100] Nearly 90% of Aβ in the systemic circulation binds to HSA, which regulates Aβ oligomerization in the brain by regulating Aβ levels in the CSF and blood. Implementation of an albumin-based plasma exchange strategy results in Aβ mobilization and stabilization of cognitive status, memory, and language abilities in AD patients. The stoichiometry and affinity of the HSA-Aβ interaction are not yet fully elucidated. Various kinetic outcomes have been reported for the interaction of HSA and Aβ. It is important to determine whether HSA binds to monomeric or oligomeric Aβ. Furthermore, experimental efforts are needed to identify the optimal HSA concentration for effective Aβ sequestration. Therefore, it is necessary to determine the binding capacity of HSA to GFP-Aβ. Among various concentrations of HSA, 1 μM was found to be the optimal concentration for binding to the 10 μM test concentration of GFP-Aβ, as shown in Figure 8(A). Therefore, we evaluated the concentration-dependent sequestration potential of HSA by gradually increasing the concentration of GFP-Aβ after immobilizing HSA on amine-conjugated 96-well plates using this optimal concentration. Figure 8(B) and (C) show the GFP-Aβ blocking potential of plate-immobilized 1 μM HSA and CA membrane-immobilized HSA, respectively. Both plate-immobilized HSA and CA membrane-immobilized HSA exhibit concentration-dependent GFP-Aβ sequestration, except for 1 nM GFP-Aβ binding with CA membrane-immobilized HSA. CA membrane-immobilized HSA shows the best GFP-Aβ sequestration potential compared to HSA immobilized at 1 μM on amine-conjugated pre-activation-coated well plates. Silane conjugation and GA-based protein immobilization appear to be excellent strategies for functional protein immobilization.

[0101] Before the nucleation process of Aβ monomers, a 1 μM concentration of HSA can delay the onset of the disease by prolonging the elongation of Aβ fibrils. A uniform monomer distribution was observed whenever freshly prepared GFP-Aβ samples were used, as evidenced by DLS and TEM data. Therefore, 1 μM of HSA is sufficient for ligand binding and removal.

[0102] The development of Alzheimer's disease treatments has been a mixed bag, with numerous successes and failures. While several therapeutic options have shown promising results in treating AD, they often induce severe neurological and immunogenic reactions. Leveraging HSA's natural ability to bind Aβ as an ex vivo Aβ-trapping platform could be an attractive, biocompatible, and tunable in vitro option for improving AD.

[0103] As described above, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby.

[0104] According to the present invention, utilizing HSA as an Aβ sequestering ligand is expected to offer several advantages, including easy integration and adoption in clinical settings, given its excellent biocompatibility, tolerability, and safety. Furthermore, HSA has already been utilized in the medical field to coat stents and tubes due to its remarkable properties. It has demonstrated antithrombotic properties and corrosion resistance, primarily due to its electrostatic and hydrophilic properties. Furthermore, it readily adheres to surfaces, mitigates endothelial cell death, provides antioxidant protection, and effectively inhibits platelet activation and aggregation. Overall, these properties highlight the potential of HSA as a valuable biomaterial for the development of medical devices specifically designed for the ex vivo treatment of Alzheimer's disease.

Claims

1. A composition for treating Alzheimer's disease by trapping amyloid beta (Aβ) in vitro, comprising a substrate on which human serum albumin (HSA) is fixed.

2. A composition according to claim 1, characterized in that the substrate is a cellulose acetate (CA) film.

3. A composition characterized in that human serum albumin (HSA) is fixed to the surface of the cellulose acetate (CA) membrane through covalent bonding in the first paragraph.

4. A composition characterized in that, in the third paragraph, human serum albumin (HSA) is linked to the cellulose acetate (CA) film by including glutaraldehyde (GA) as a linker and 3-aminopropyltriethoxysilane (APTS) as a spacer, thereby forming a CA-APTS-GA-HSA bond.

5. A composition according to claim 1, characterized in that the human serum albumin is contained at a concentration of 1 to 30 μM.

6. A composition characterized in that, in the first paragraph, amyloid beta (Aβ) in the blood of an Alzheimer's disease patient is adsorbed externally.

7. A medical device coated with a composition according to any one of claims 1 to 6.

8. In the 7th paragraph, the device is a tube device.

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