A method of preparing homogeneous amyloid fibrils
By preparing amyloid fibers with uniform particle size and high thermal stability, the problem of poor specificity of targeted drug binding has been solved, improving the efficacy and safety of drugs and expanding their application in the fields of biochemistry and pharmacy.
Patent Information
- Application Number
- CN202411652280.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2026-03-17
- Estimated Expiration
- 2044-11-19
AI Technical Summary
Existing targeted drugs have poor specificity when binding to different forms of amyloid aggregates, resulting in poor efficacy and potential safety threats, making it difficult to fully play a role in biomedical research.
Amyloid fiber samples with uniform particle size and high thermal stability were prepared through in vitro regulation and condition optimization. Amyloid fibers with high uniformity and stability were screened by multiple iterative incubation and ultrasonic treatment.
It improves the binding specificity of amyloid fibers, reduces the side effects caused by nonspecific binding, enhances drug efficacy, and expands its application prospects in the fields of biochemistry, pharmacy, and neuroscience.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fiber preparation technology, specifically relating to a method for preparing uniform amyloid fibers. Background Technology
[0002] Amyloid fibrils are highly ordered fibrous aggregates formed by the self-assembly of polypeptides or proteins, exhibiting enormous application potential in the biomedical field. They are not only pathological markers of various diseases but also key factors in disease development and progression, significantly impacting tissue and organ function. They hold broad application prospects in early disease diagnosis and intervention, targeted drug design and precise delivery, and disease model construction and mechanism analysis. For example, the U.S. Food and Drug Administration approved adunatumab (J. Sevigny, P. Chiao, T. Bussière, PHWeinreb, L. Williams, M. Maier, R. Dunstan, S. Salloway, T. Chen, Y. Ling, J. O'Gorman, F. Qian, M. Arastu, M. Li, S. Chollate, MS Brennan, O. Quintero-Monzon, RH Scannevin, HM Arnold, T. Engber, K. Rhodes, J. Ferrero, Y. Hang, A. Mikulskis, J. Grimm, C. Hock, RM Nitsch, A. Sandrock, Nature, 2016, 537, 50-56) and lencanemab (CHvan Dyck, CJ Swanson, P. Aisen, RJ Bateman, C. Chen, M. Gee, M. Kanekiyo, D. Li, L. Reyderman, S. Cohen, L. Froelich, S. Katayama, M. Sabbagh, B. Vellas, D. Watson, S. Dhadda, M. Irizarry, LD Kramer, T. Iwatsubo, N. Engl. J. Med., 2023, 388, 9-21), can selectively bind to amyloid plaque deposits in the brains of Alzheimer's disease patients, clearing the deposited protein from the brain by activating the immune system. Therefore, it has shown significant efficacy in early-stage Alzheimer's patients. Adunatumab has been shown to reduce amyloid plaque deposition in the brain and has shown a protective effect on cognitive function in some studies. Lencanemab, over an 18-month treatment period, slowed the rate of cognitive decline in early-stage Alzheimer's patients by 27%.
[0003] However, protein self-assembly is a complex and delicate dynamic equilibrium regulated by various environmental factors. This process produces a variety of different aggregates, which exhibit wide differences in morphology, structure, and function. These aggregates provide rich information in biomedical research but also present significant challenges; adunatumumab and lencanemumab face similar issues. Because they can target and recognize multiple different forms of aggregates, their binding specificity varies across different aggregate forms, resulting in suboptimal targeting of highly toxic amyloid aggregates. This variability in binding not only weakens the actual efficacy of the drug in vivo, hindering its full therapeutic effect, but also poses a potential threat to drug safety. Therefore, optimizing binding specificity and improving the precision of targeted binding to reduce side effects and enhance efficacy has become a critical issue that urgently needs to be addressed in the development and application of such targeted drugs. Through in vitro regulation and condition optimization, amyloid fiber samples with high homogeneity and stability were screened out. Based on these amyloid fibers, highly specific targeted drugs were designed, which can effectively avoid potential side effects caused by non-specific binding, effectively slow down the rate of cognitive decline, and provide patients with new treatment options.
[0004] Based on this, this application was developed. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing highly uniform amyloid fibers. This method, through in vitro regulation and condition optimization, screens and prepares amyloid fiber samples with high uniformity and stability. The prepared amyloid fibers exhibit characteristics such as uniform particle size and high thermal stability.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A method for preparing uniform amyloid fibers, comprising the following steps:
[0008] 1) Dissolve the lyophilized β-amyloid protein powder in sodium hydroxide solution, centrifuge at 10000–14000 rpm for 3–5 minutes at 4°C, and separate the supernatant; add an appropriate amount of phosphate buffer to the supernatant, transfer to a centrifugal filter, centrifuge at 3000–6000 rpm for 3–5 minutes at 4°C, and the filtrate is the protein monomer solution;
[0009] 2) Place the prepared protein monomer solution in a constant temperature shaker and incubate at 37±2℃ and 160–300 rpm for 3–5 days. Observe that the protein has aggregated to form primary amyloid fibers.
[0010] 3) Take the above primary amyloid fiber sample, sonicate it in a water bath for 3–5 minutes at room temperature, blow it evenly and mix it with the freshly prepared protein monomer solution, and then incubate it at 37±2℃ and 160–300 rpm for 3–5 days to obtain the second generation amyloid fiber sample.
[0011] 4) Pre-treat the second-generation amyloid fiber samples according to the method in step 3) above, and then prepare the third to twelfth generation amyloid fiber samples; wherein, for the third to sixth generations, each generation of amyloid fiber samples is incubated at 37±2℃ and 160–300rpm for 1–2 days with shaking, and for the seventh to twelfth generations, each generation of amyloid fiber samples is incubated at 37±2℃ and 160–300rpm for 4–8 hours with shaking; the twelfth generation fiber sample obtained by transmission electron microscopy is an amyloid fiber with uniform morphology, and the twelfth generation amyloid fiber sample is kept for later use.
[0012] 5) Using the prepared 12th generation amyloid fiber sample as a seed, sonicate in a water bath for 3–5 minutes at room temperature, blow it evenly and mix it with the freshly prepared protein monomer solution. Then, incubate it at 37±2℃ and 160–300 rpm for 2–4 days to obtain highly homogeneous amyloid fibers.
[0013] Specifically, in step 1), sodium hydroxide solution can be added to the lyophilized β-amyloid protein powder, and the final concentration after protein dissolution is 15–25 mg / mL.
[0014] More preferably, 50–100 mM sodium hydroxide solution can be added to 1 mg of lyophilized β-amyloid protein powder, and the final concentration of the protein after dissolution is 18–22 mg / mL.
[0015] Specifically, in step 1), an appropriate amount of 10–50 mM phosphate buffer can be added to the supernatant to make the final protein concentration 50–200 μM.
[0016] More preferably, in step 1), the phosphate buffer solution has a pH of 7.2–7.5 and contains 0.01–0.02% sodium azide.
[0017] Specifically, in step 3), after the primary amyloid fiber sample is blown evenly, it can be added to the freshly prepared protein monomer solution at a volume percentage of 2–10%.
[0018] Specifically, in step 5), after the 12th generation amyloid fiber sample is blown evenly, it can be added to the freshly prepared protein monomer solution at a volume percentage of 2–10%.
[0019] As a preferred embodiment, the above-mentioned method for preparing highly uniform amyloid fibers specifically includes the following steps:
[0020] 1) Add 50 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid protein powder, resulting in a final protein concentration of 20 mg / mL. Centrifuge at 13000 rpm for 3 minutes at 4°C, and separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide) to the supernatant to bring the final protein concentration to 100 μM. Transfer the solution to a centrifugal filter and centrifuge at 6000 rpm for 3 minutes at 4°C. The filtrate is the protein monomer solution.
[0021] 2) The prepared protein solution was transferred to a vial and placed in a constant temperature shaker. It was incubated at 37°C and 200 rpm for 4 days. The protein was observed to have aggregated to form primary amyloid fibers by transmission electron microscopy.
[0022] 3) Take the above primary amyloid fiber sample, sonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). Mix the two evenly and transfer them to a vial. Incubate the solution in a shaker at 37°C with shaking at 200 rpm. After 4 days, the second-generation amyloid fiber sample is obtained.
[0023] 4) Following the fiber pretreatment method described above, the second-generation fiber samples were subjected to water bath ultrasonic treatment to prepare third to twelfth-generation amyloid fiber samples. For the third to sixth generations, each sample was incubated at 37°C and 200 rpm for one day with shaking. For the seventh to twelfth generations, each sample was incubated at 37°C and 200 rpm for six hours with shaking. Transmission electron microscopy revealed that the twelfth-generation fiber sample was a uniformly morphological amyloid fiber, and this sample was retained for future use.
[0024] 5) Using the prepared 12th generation amyloid fiber sample as a seed, sonicate in a water bath for 5 minutes at room temperature, blow evenly, and add it to the freshly prepared protein monomer solution at a ratio of 2–10% (v / v). Incubate in a constant temperature shaker at 37℃ for 2–4 days to obtain highly uniform amyloid fibers.
[0025] The present invention provides a uniform amyloid fiber prepared by the above method.
[0026] This invention's method is applicable to the preparation of common amyloid fibers, such as α-synuclein fibers, β-amyloid fibers, and pancreatic islet amyloid polypeptide fibers. This paper uses the preparation of amyloid fibers from β-amyloid, a key protein in Alzheimer's disease, as an example to demonstrate the practicality and efficiency of this method. Compared with existing technologies, the beneficial effects of this invention are as follows:
[0027] 1. This invention prepares amyloid fiber samples with high homogeneity and stability through in vitro regulation and condition optimization. The prepared amyloid fibers exhibit uniform particle size and high thermal stability. These characteristics endow the amyloid fibers prepared by this method with the potential for further applications, greatly expanding their application scope in the fields of biochemistry and chemistry.
[0028] 2. This invention provides a novel method for preparing amyloid fibers, which enriches our understanding of the structure of amyloid fibrils and ensures their stability, thereby expanding the application prospects of amyloid proteins in biochemistry, pharmacy, neuroscience and other fields.
[0029] 3. This invention fibroses β-amyloid protein, a key protein in Alzheimer's disease, which is an effective and promising protein modification strategy to change its morphological characteristics. It is expected to develop a detection method for this type of pathological protein fiber, providing a new approach for the clinical diagnosis of the corresponding disease.
[0030] 4. This invention prepares an amyloid fiber with uniform particle size and high stability by iteratively incubating β-amyloid protein, a key protein in Alzheimer's disease, thereby improving the comprehensive utilization value of β-amyloid protein and showing great application potential in the fields of pharmacy and diagnostic medicine.
[0031] 5. This invention has low production costs, is simple to operate, and uses inexpensive and readily available raw materials. This invention modifies proteins to achieve high value, greatly enhancing the application value of amyloid fibers in the fields of pharmacy and diagnostic medicine. Attached Figure Description
[0032] Figure 1 The dynamic light scattering data are shown for the amyloid fibers prepared according to the present invention. It indicates that the amyloid fibers prepared by this method have a uniform particle size of 1101 nm.
[0033] Figure 2 The circular dichroism (CD) spectra of the amyloid fibers prepared according to this invention are shown in the left figure. The CD spectra of the amyloid fibers are obtained at 20℃ (the lowest temperature during the measurement process) and 90℃ (the highest temperature during the measurement process). The right figure depicts the concentration changes of species contributing to the secondary structure of the amyloid fibers under different temperature conditions. The melting point of the amyloid fibers was determined to be 72.2 ± 0.3℃, indicating that they possess high thermal stability.
[0034] Figure 3 The image shows the microstructural features of the amyloid fibers prepared according to the present invention, obtained by transmission electron microscopy. Detailed Implementation
[0035] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.
[0036] Example 1
[0037] A method for preparing highly uniform amyloid fibers, specifically comprising the following steps:
[0038] 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid protein to achieve a final protein concentration of 20 mg / mL. Centrifuge at 13000 rpm for 3 minutes at 4°C and separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to achieve a final protein concentration of 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 6000 rpm for 3 minutes at 4°C. The filtrate is the protein monomer solution.
[0039] 2) The prepared protein monomer solution was transferred to a vial and placed in a constant temperature shaker. It was incubated at 37°C and 200 rpm for 4 days. The protein was observed to have aggregated to form primary amyloid fibers by transmission electron microscopy.
[0040] 3) Take the above primary amyloid fiber sample, sonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37℃ with shaking at 200 rpm. After 4 days, the second generation amyloid fiber sample is obtained.
[0041] 4) Pretreatment of the second-generation amyloid fiber samples was performed according to the method in step 3) above, thereby preparing third to twelfth-generation amyloid fiber samples. For the third to sixth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 1 day. For the seventh to twelfth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 6 hours. Transmission electron microscopy revealed that the twelfth-generation fiber sample was a uniformly morphological amyloid fiber, and this sample was retained for future use.
[0042] 5) Using the prepared 12th generation amyloid fiber sample as a seed, sonicate in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). Mix well, and then incubate in a constant temperature shaker at 37℃ and 200 rpm for 2 days to prepare highly uniform amyloid fibers.
[0043] Figure 1 Dynamic light scattering data of the prepared amyloid fibers are presented. This indicates that the amyloid fibers prepared by this method have a uniform particle size of 1101 nm.
[0044] Figure 2 The circular dichroism spectral data of the prepared amyloid fibers are presented. The melting point of the amyloid fibers was determined to be 72.2 ± 0.3℃, indicating that they possess high thermal stability.
[0045] Figure 3 Transmission electron microscopy images of the prepared amyloid fibers are presented, showing the microstructural features of the fibers, with diameters in the nanometer scale and lengths extending to the micrometer scale.
[0046] Example 2
[0047] A method for preparing highly uniform amyloid fibers, specifically comprising the following steps:
[0048] 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid protein to achieve a final protein concentration of 20 mg / mL. Centrifuge at 13000 rpm for 3 minutes at 4°C and separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to achieve a final protein concentration of 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 6000 rpm for 3 minutes at 4°C. The filtrate is the protein monomer solution.
[0049] 2) The prepared protein monomer solution was transferred to a vial and placed in a constant temperature shaker. It was incubated at 37°C and 200 rpm for 4 days. The protein was observed to have aggregated to form primary amyloid fibers by transmission electron microscopy.
[0050] 3) Take the above primary amyloid fiber sample, sonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37℃ with shaking at 200 rpm. After 4 days, the second generation amyloid fiber sample is obtained.
[0051] 4) Process the second-generation amyloid fiber samples according to the method in step 3) above to prepare third to twelfth-generation amyloid fiber samples. For generations 3 to 6, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 1 day. For generations 7 to twelfth, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 6 hours. Transmission electron microscopy revealed that the twelfth-generation fiber sample was a uniformly morphological amyloid fiber, and this sample was retained for future use.
[0052] 5) Using the prepared 12th generation amyloid fiber sample as a seed, sonicate in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 2% (v / v). Mix well, and then incubate in a constant temperature shaker at 37℃ and 200 rpm for 4 days to prepare highly uniform amyloid fibers.
[0053] Example 3
[0054] A method for preparing highly uniform amyloid fibers, specifically comprising the following steps:
[0055] 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid protein to achieve a final protein concentration of 20 mg / mL. Centrifuge at 13000 rpm for 3 minutes at 4°C and separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to achieve a final protein concentration of 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 6000 rpm for 3 minutes at 4°C. The filtrate is the protein monomer solution.
[0056] 2) The prepared protein monomer solution was transferred to a vial and placed in a constant temperature shaker. It was incubated at 37°C and 200 rpm for 4 days. The protein was observed to have aggregated to form primary amyloid fibers by transmission electron microscopy.
[0057] 3) Take the above primary amyloid fiber sample, sonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37℃ with shaking at 200 rpm. After 4 days, the second generation amyloid fiber sample is obtained.
[0058] 4) Process the second-generation amyloid fiber samples according to the method in step 3) above to prepare third to twelfth-generation amyloid fiber samples. For generations 3 to 6, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 1 day. For generations 7 to twelfth, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm with shaking for 6 hours. Transmission electron microscopy revealed that the twelfth-generation fiber sample was a uniformly morphological amyloid fiber, and this sample was retained for future use.
[0059] 5) Using the prepared 12th generation amyloid fiber sample as a seed, sonicate in a water bath for 5 minutes at room temperature, blow it evenly, and then add it to the freshly prepared protein monomer solution at a ratio of 5% (v / v). Mix well, and then incubate in a constant temperature shaker at 37℃ and 200 rpm for 3 days to prepare highly uniform amyloid fibers.
[0060] In summary, the method of the present invention, through in vitro regulation and condition optimization, screens and prepares amyloid fiber samples with high uniformity and stability, and the prepared amyloid fibers have characteristics such as uniform particle size and high thermal stability.
Claims
1. A method of preparing homogeneous amyloid fibrils, characterized in that, The method comprises the following steps: 1) Dissolving the lyophilized β-amyloid powder in sodium hydroxide solution, centrifuging, separating the supernatant; adding an appropriate amount of phosphate buffer to the supernatant, centrifuging, and the filtrate is the protein monomer solution; 2) Placing the prepared protein monomer solution in a constant temperature shaker, incubating at 37±2℃ for 3-5 days, and observing that the protein has aggregated to form primary amyloid fibers; 3) Taking the primary amyloid fiber sample, ultrasonically treating in a water bath at room temperature, mixing with freshly prepared protein monomer solution, and then incubating at 37±2℃, and preparing the second generation amyloid fiber sample after 3-5 days; 4) Preparing the third to twelfth generation amyloid fiber samples according to the method of step 3) above; wherein the third to sixth generation amyloid fiber samples are incubated at 37±2℃ for 1-2 days, and the seventh to twelfth generation amyloid fiber samples are incubated at 37±2℃ for 4-8 hours; 5) Taking the prepared twelfth generation amyloid fiber sample as a seed, ultrasonically treating in a water bath, mixing with freshly prepared protein monomer solution, and then incubating at 37±2℃ for 2-4 days; Add sodium hydroxide solution to the lyophilized β-amyloid powder, and the final concentration of the dissolved protein is 15-25 mg / mL; In step 1), the pH of the phosphate buffer is 7.2-7.5, and it contains 0.01-0.02% sodium azide.
2. The method of claim 1, wherein the preparation of the homogeneous amyloid fibrils is characterized by, Add 50-100 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid powder, and the final concentration of the dissolved protein is 18-22 mg / mL.
3. The method of claim 1, wherein the preparation of the uniform amyloid fibrils is characterized by, In step 1), add an appropriate amount of 10-50 mM phosphate buffer to the supernatant to make the final protein concentration 50-200 μM.
4. The method of claim 1, wherein the preparation of the uniform amyloid fibrils is characterized by, In step 3), the primary amyloid fiber sample is mixed with freshly prepared protein monomer solution at a volume percentage of 2-10%.
5. The method of claim 1, wherein the preparation of the uniform amyloid fibrils is characterized by, In step 5), the twelfth generation amyloid fiber sample is mixed with freshly prepared protein monomer solution at a volume percentage of 2-10%.
6. The method of any one of claims 1 to 5 is used to prepare the uniform amyloid fibers.