Modified ferritin, cross-linked ferritin and preparation method and application of modified ferritin and cross-linked ferritin
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI SHANGZEYONGZHI BIOTECHNOLOGY PARTNERSHIP (LLP)
- Filing Date
- 2024-09-27
- Publication Date
- 2026-05-05
AI Technical Summary
Existing dermal filling materials such as hyaluronic acid and collagen have limited support and are easy to degrade, resulting in short filling effect, requiring frequent injection, and are susceptible to environmental influences in the body to cause morphological changes and displacement.
Modified ferritin is prepared by genetic modification or chemical modification, and crosslinking ferritin is formed through crosslinking reactions, retaining its polymer structure, improving biocompatibility and anti-degradability.
Crosslinked ferritin has excellent support, anti-degradability and elasticity, can maintain the filling effect for a long time, reduce the injection frequency, and is highly stable in the body, making it difficult to shift or change in morphology.
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Abstract
Description
Modified ferritin, cross-linked ferritin, and preparation method and application thereof
[0001] This application claims priority to Chinese Patent Application No. 2023112753858, filed on September 28, 2023, and Chinese Patent Application No. 2024112962839, filed on September 14, 2024. The entire text of the above-mentioned Chinese patent application is incorporated herein by reference. Technical Field
[0002] The present invention belongs to the technical field of biomedical materials, and in particular relates to modified ferritin, cross-linked ferritin, and preparation methods and applications thereof. Background Art
[0003] The skin, the largest organ in the human body, plays a vital role in defending against external aggressions and maintaining a stable internal environment. With aging, facial tissue sagging and wrinkles develop. Subdermal fillers can effectively reduce facial sagging, replenish volume loss, reshape facial contours, and improve skin quality. Local injections of hyaluronic acid and / or collagen have become a common method due to their ease of use, minimal invasiveness, and excellent biocompatibility.
[0004] Hyaluronic acid, also known as hyaluronic acid, is one of the most commonly used dermal fillers in clinical practice due to its advantages of good tissue compatibility, ease of injection, immediate results, and minimal invasiveness. However, as hyaluronic acid is an acidic mucopolysaccharide, it cannot form a three-dimensional structure after cross-linking, resulting in limited support after filling and a tendency to experience Tyndall phenomenon, which affects the appearance of the filler. Hyaluronic acid is a high-molecular-weight polymer with hydroxyl and carboxyl groups in its chemical structure. It is easily hydrolyzed in the body upon interaction with water. Biological enzymes (especially hyaluronidase and human free radicals) have a strong degrading effect on hyaluronic acid. As a result, hyaluronic acid has a short retention time in the body, requiring repeated injections to achieve therapeutic effects. Skin cells quickly absorb the initial hyaluronic acid injection, and staying up late and drinking alcohol accelerate hyaluronic acid metabolism in the body. Areas of high activity also experience increased hyaluronic acid metabolism. Subdermal hyaluronic acid fillers typically last 6-8 months. Furthermore, impact can easily cause hyaluronic acid to shift and change its morphology. Therefore, hyaluronic acid as a subcutaneous filling material still has some defects and needs further product improvement.
[0005] Collagen is another common filler material, offering advantages such as good tissue compatibility, ease of injection, immediate results, and minimal invasiveness. The natural collagen found in human skin is type I collagen, whose amino acid sequence contains three uncommon amino acids: 4-hydroxyproline (9%), 3-hydroxyproline (0.1%), and 5-hydroxylysine (0.6%). Amino acid modifications allow for stable covalent bonds to form protocollagen molecules, which are then linked together into a right-handed triple helix. Each strand of the protocollagen molecule is also a left-handed helix, giving the natural collagen found in the skin strong support and tensile strength. Currently, the collagen used for subcutaneous fillers is mostly prokaryotically expressed collagen fragments, which are unable to form either a left-handed single helix or a right-handed triple helix. Consequently, they lack the strong support and tensile strength of collagen in the body and cannot fully function. Furthermore, because fillers are collagen fragments, they are easily hydrolyzed by collagenases in the body, resulting in a short retention time in the body.
[0006] Given the shortcomings of hyaluronic acid and collagen, such as limited support and easy degradation, there is an urgent need to develop new filling materials that are long-lasting, have better biomechanical support, and can be produced industrially on a large scale.
[0007] Ferritin is a naturally occurring hollow spherical protein in animals with an inner diameter of 8 nm and an outer diameter of 12 nm. It is composed of light and heavy chain subunits that self-assemble to form a 24-mer protein cage. Currently, ferritin is often used in tissue engineering or as a delivery vehicle for small molecule drugs, biologics, and nucleic acids. However, there are few reports on its use in the preparation of filler materials.
[0008] Summary of the Invention
[0009] To address the above-mentioned deficiencies in the prior art, the present invention provides modified ferritin, cross-linked ferritin, and preparation methods and applications. The prepared cross-linked ferritin has good biocompatibility and, in practical applications, exhibits excellent support, degradation resistance, and elasticity.
[0010] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:
[0011] The present invention provides a modified ferritin comprising a plurality of light chain subunits or heavy chain subunits of ferritin from natural sources, wherein the plurality of light chain subunits or the plurality of heavy chain subunits form a multimer, preferably a cage-shaped multimer.
[0012] In the present invention, "modified ferritin" refers to ferritin from non-natural sources, including, for example, artificial ferritin obtained by genetically modifying naturally derived ferritin. In the present invention, the amino acid sequence of the light chain subunit may be as shown in SEQ ID NO: 1, or may have at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, to the sequence shown in SEQ ID NO: 1.
[0013] In the present invention, the amino acid sequence of the heavy chain subunit may be as shown in SEQ ID NO: 2, 6 or 7, or have at least 80% identity with the sequence shown in SEQ ID NO: 2, 6 or 7, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.
[0014] In some embodiments, the modified ferritin comprises 8, 16, 24, or 48 light chain subunits or 8, 16, 24, or 48 heavy chain subunits.
[0015] In some embodiments, the natural source includes animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, more preferably a human.
[0016] In some preferred embodiments, the modified ferritin is composed of 24 light chain subunits forming a cage-shaped multimer, or is composed of 24 heavy chain subunits forming a cage-shaped multimer.
[0017] The present invention also provides a cross-linked ferritin; which is a cross-linked ferritin formed by cross-linking ferritin from a natural source or a variant thereof, and the cross-linked ferritin retains the multimeric structure of the ferritin from the natural source or the variant thereof; or a cross-linked ferritin formed by cross-linking the modified ferritin as described above, and the cross-linked ferritin retains the multimeric structure of the modified ferritin; wherein the variant has at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of the ferritin from the natural source.
[0018] Preferably, the cross-linked ferritin is in the form of a hydrogel or microsphere structure.
[0019] In the present invention, the swelling degree of the cross-linked ferritin may be lower than 1000%, preferably lower than 600%, and more preferably lower than 500%.
[0020] In the present invention, the elastic modulus of the cross-linked ferritin may be higher than 100 Pa, preferably higher than 600 Pa, and more preferably higher than 1000 Pa.
[0021] In the present invention, the viscosity modulus of the cross-linked ferritin may be higher than 10 Pa, preferably higher than 30 Pa, more preferably higher than 50 Pa, and even more preferably higher than 100 Pa.
[0022] In the present invention, the natural ferritin may be derived from animals, plants, fungi or bacteria, preferably animals; the animal is a vertebrate, preferably a mammal, and more preferably a human.
[0023] Preferably, the cross-linking is performed by a biological cross-linking reaction or a chemical cross-linking reaction.
[0024] In some embodiments, the ferritin is mouse heavy chain ferritin, preferably having an amino acid sequence having NCBI accession number NP_034369.1.
[0025] In some embodiments, the ferritin is mouse light chain ferritin, preferably having an amino acid sequence having NCBI accession number AAA37614.1.
[0026] In some embodiments, the ferritin is Drosophila melanogaster heavy chain ferritin, preferably, the amino acid sequence of which has NCBI accession number NP_001247366.1.
[0027] In some embodiments, the ferritin is Drosophila melanogaster light chain ferritin, preferably having an amino acid sequence with NCBI accession number NP_001263105.1.
[0028] In some embodiments, the ferritin is Caenorhabditis elegans ferritin, preferably having an amino acid sequence whose NCBI accession number is NP_491198.1.
[0029] In some embodiments, the ferritin is oyster ferritin, preferably having an amino acid sequence as shown in SEQ ID NO: 3.
[0030] In some embodiments, the ferritin is human mitochondrial ferritin, preferably having an amino acid sequence having NCBI accession number NC_000005.10.
[0031] In some embodiments, the ferritin is soybean ferritin, preferably, the amino acid sequence of which has an NCBI accession number of NP_001236534.2.
[0032] In some embodiments, the ferritin is Arabidopsis thaliana ferritin, preferably having an amino acid sequence whose NCBI accession number is NP_195780.1.
[0033] In some embodiments, the ferritin is beet chloroplast ferritin, preferably having an amino acid sequence with NCBI accession number XP_010676813.1.
[0034] In some embodiments, the ferritin is Mucor microsporum ferritin, preferably, the amino acid sequence of which has an NCBI accession number of XP_023465511.1.
[0035] In some embodiments, the ferritin is ferritin from Encephalitozoon cerevisiae, preferably having an amino acid sequence with NCBI accession number XP_003887486.1.
[0036] In some embodiments, the ferritin is a non-heme ferritin, preferably a non-heme ferritin of the Enterobacteriaceae family, and more preferably the NCBI accession number of its amino acid sequence is WP_000917208.1.
[0037] In some embodiments, the ferritin is Acinetobacter ferritin, preferably having an amino acid sequence having NCBI accession number WP_002117082.1.
[0038] In some embodiments, the ferritin is Campylobacter ferritin, preferably having an amino acid sequence having NCBI accession number WP_002786063.1.
[0039] In some embodiments, the ferritin is a Bacteroides ferritin, preferably having an amino acid sequence having NCBI accession number WP_010538613.1.
[0040] In some embodiments, the ferritin is Pseudomonas aeruginosa ferritin, preferably, its amino acid sequence is shown in SEQ ID NO:4.
[0041] In some embodiments, the ferritin is Pyrococcus aerobicus ferritin, preferably, the NCBI accession number of its amino acid sequence is NC_014804.1.
[0042] In some embodiments, the ferritin is Pyrococcus jacobense ferritin, and preferably the NCBI accession number of its amino acid sequence is NC_015680.1.
[0043] In some embodiments, the ferritin is a Deinococcus-Thermus ferritin, and preferably, the NCBI accession number of its amino acid sequence is WP_038069286.1.
[0044] In some embodiments, the ferritin is actinomycetin, preferably, the amino acid sequence of which has an NCBI accession number of NP_218358.1.
[0045] In some embodiments, the ferritin is a Proteobacteria ferritin, preferably, the NCBI accession number of its amino acid sequence is NP_417795.1.
[0046] In some embodiments, the ferritin is Escherichia coli ferritin, preferably, its amino acid sequence is shown in SEQ ID NO:5.
[0047] In some embodiments, the ferritin is an Archaeota ferritin, and preferably, the NCBI accession number of its amino acid sequence is WP_012309233.1.
[0048] In some embodiments, the ferritin is Pyrococcus furiosus ferritin, preferably having an amino acid sequence with NCBI accession number WP_011011871.1.
[0049] In some embodiments, the ferritin is a Methanosarcina ferritin, preferably having an amino acid sequence having NCBI accession number WP_048037772.1.
[0050] In some embodiments, the cross-linked ferritin further comprises hyaluronic acid and / or collagen.
[0051] Preferably, the volume ratio of the cross-linked ferritin to the hyaluronic acid is 1:5-5:1.
[0052] Preferably, the volume ratio of the cross-linked ferritin to the collagen is 1:5-5:1.
[0053] Preferably, the molar concentration ratio or mass ratio of the cross-linked ferritin to the hyaluronic acid is 1:5-5:1.
[0054] Preferably, the molar concentration ratio or mass ratio of the cross-linked ferritin to the collagen is 1:5-5:1.
[0055] The present invention also provides a method for preparing the cross-linked ferritin as described above, the method comprising the following steps:
[0056] The naturally derived ferritin or its variants, or the modified ferritin as described above, is made into cross-linked ferritin through a cross-linking reaction; the cross-linking reaction is a chemical cross-linking reaction or a biological cross-linking reaction.
[0057] In the present invention, when the cross-linking reaction is a chemical cross-linking reaction, the cross-linking reaction may include the following steps: mixing a neutral solution containing ferritin from a natural source or a variant thereof, or a neutral solution containing the modified ferritin as described above with a cross-linking agent, and performing a cross-linking reaction to obtain the product.
[0058] Wherein, after the cross-linking reaction, hyaluronic acid and / or collagen may be mixed into the ferritin hydrogel.
[0059] The cross-linking reaction may be followed by neutralization, washing and / or homogenization.
[0060] Among them, the cross-linking agent can be selected from one or more of 1,4-butanediol diglycidyl ether (BDDE), genipin (GP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), nordihydroguaiaretic acid (NDGA) and glutaraldehyde, preferably 1,4-butanediol diglycidyl ether (BDDE).
[0061] The volume percentage of the cross-linking agent relative to the neutral solution may be 3-30%, preferably 6%.
[0062] The cross-linking reaction may be carried out in a constant temperature water bath; the temperature of the cross-linking reaction may be 60-72° C., for example, 60° C.; and the time of the cross-linking reaction may be 20-150 min.
[0063] Wherein, the pH of the neutral cross-linked ferritin solution may be 8.
[0064] The concentration of the naturally derived ferritin or its variant, or the modified ferritin as described above may be 10-600 mg / mL, preferably 50-300 mg / mL, for example 100 mg / mL.
[0065] The neutralization may include pickling to neutrality; the pickling preferably includes leaching with an acidic agent; preferably, the acidic agent is selected from one or more of hydrochloric acid, sulfuric acid and acetic acid, such as acetic acid.
[0066] Wherein, the cleaning may include cleaning with a buffered saline solution; preferably, the buffered saline solution is selected from one or more of phosphate buffer, physiological saline, Tris-HCl buffer and HEPES, such as physiological saline; preferably, the concentration of the buffered saline solution is 10-100mM and the pH is 6-8.
[0067] The cleaning time may be 12-30 hours, for example 24 hours.
[0068] In the present invention, when the cross-linking reaction is a biological cross-linking reaction, the naturally derived ferritin can be cross-linked via disulfide bonds; preferably, the naturally derived ferritin is fused with a cysteine-containing polypeptide through genetic engineering to obtain cross-linked ferritin.
[0069] Preferably, the natural source includes animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, and more preferably a human.
[0070] In a specific embodiment, the bio-crosslinking reaction includes, for example, mixing ferritin modified with a cysteine-containing polypeptide and β-mercaptoethanol, magnetically stirring, and after a preliminary reaction, centrifuging to remove the supernatant, washing the protein precipitate with PBS, and then dissolving it in a DMSO solution (pH 3-8), and magnetically stirring until the reaction is complete.
[0071] The present invention also provides a use of a naturally derived ferritin or a variant thereof, or the modified ferritin as described above, in the preparation of a subcutaneous or tissue filler; wherein the variant has at least 80% identity, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, to the amino acid sequence of the naturally derived ferritin.
[0072] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0073] The reagents and raw materials used in the present invention are commercially available.
[0074] The positive progress effect of the present invention is:
[0075] This invention is the first to utilize cross-linked ferritin derived from naturally derived ferritin or its variants, or from modified ferritin, for subcutaneous and tissue filling. Compared to existing filler materials, the ferritin of this application, particularly the genetically modified ferritin, exhibits excellent biocompatibility and non-immunogenicity. The cross-linked ferritin retains its unique, intact multimeric structure (cage-like structure) and exhibits excellent elasticity, high support, low swelling, resistance to degradation, resistance to displacement, long in vivo retention, and injectability, resulting in superior subcutaneous and tissue filling results. BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG1 and FIG2 are the microscopic morphological characterization results of the hydrogels obtained in Examples 1-2 and Comparative Examples 1-4.
[0077] FIG3 shows the microscopic morphology of the hydrogels obtained in Example 1 and Examples 10-15 after dilution 100 times.
[0078] Figure 4 shows the characterization results of the anti-enzymatic ability of uncross-linked LFn, HFn, BSA, and Col at 25°C.
[0079] Figure 5 shows the characterization results of the enzymatic hydrolysis rates at 37°C of the hydrogels obtained in Examples 1-2 (LFn, HFn), commercial HA, commercial Col, and the hydrogels obtained in Examples 10-13 (LFn-HA, HFn-HA, LFn-Col, and HFn-Col).
[0080] FIG6 shows the swelling levels of the hydrogels obtained in Examples 1-2 and Comparative Examples 3-4 after immersion for 24 hours.
[0081] FIG7 shows the swelling degree of the hydrogels obtained in Examples 1-2 and Comparative Examples 3-4 after immersion for 24 hours.
[0082] FIG8 shows the elastic and viscous moduli of the hydrogels obtained in Examples 1-2 and Comparative Examples 3-4.
[0083] FIG9 and FIG10 show the gel point volume and displacement level of the hydrogels obtained in Example 1-2 and Comparative Examples 3-4 after subcutaneous injection.
[0084] FIG11 shows the gel point maintenance results of the hydrogels obtained in Examples 1-2 and Comparative Examples 3-4 after subcutaneous injection.
[0085] FIG12 shows the inflammatory factor detection results of the hydrogels obtained in Examples 1-2 and Comparative Examples 3-4 after subcutaneous injection. DETAILED DESCRIPTION
[0086] The embodiments of the technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. The reagents used in the examples are all commercially available conventional products unless otherwise specified.
[0087] definition
[0088] Ferritin is a common cage-shaped self-assembly protein that is widely present in animals, plants and microorganisms. Its main function is to convert toxic divalent iron into trivalent iron and store it in the internal cavity to regulate the iron metabolism balance of the organism.
[0089] The ferritin of the present invention refers to any ferritin capable of forming a ring or cage-like structure. It can be naturally derived ferritin, or a modified ferritin, such as a recombinantly expressed ferritin, or a variant thereof. It can be derived from prokaryotes, protozoa, fungi, plants, or animals, such as bacteria, fungi, insects, reptiles, birds, amphibians, fish, or mammals; for example, rodents, ruminants, non-human primates, or humans, such as mice, rats, guinea pigs, dogs, cats, horses, sheep, monkeys, gorillas, and humans. While the amino acid sequences of ferritins from different organisms, from bacteria to humans, vary greatly, their structures are similar and all can form a ring or cage-like protein shell structure.
[0090] In some embodiments or comparative examples, a neutral / alkaline crosslinking method was used to prepare the hydrogel: 6% by volume of the crosslinker BDDE was slowly added to the protein solution. Upon observation under light, the crosslinker slowly settled to the bottom or suspended in filaments, separating from the protein solution. The pipette tip was hovered over the separation point of the solution and gently pipetted slowly, observing under light until the separation and suspension completely disappeared, indicating that the solution was fully mixed. Crosslinking was then performed in a 60-72°C water bath for 20-150 minutes.
[0091] In some embodiments or comparative examples, a biocrosslinking method was also used to prepare the hydrogel: the gene sequence of the ferritin subunit was fused with the peptide chain through genetic engineering to obtain ferritin with a polypeptide connected to the outer surface; then, β-mercaptoethanol (β-ME) was added dropwise to the ferritin solution (1 mg / mL, solvent: 20 mM Tris-HCl) to a working concentration of 20 mmol / L, and magnetic stirring was applied for 10 minutes. The reaction solution was collected, centrifuged for 10 minutes, the supernatant was removed, and the protein precipitate was washed twice with PBS. The collected protein precipitate was dissolved in a 6% DMSO solution (pH 3-8) at a protein solution concentration of 2 mg / mL and magnetic stirring was applied until the reaction was complete. The polypeptide here can be any polypeptide chain containing cysteine.
[0092] The amino acid sequence of ferritin used in the examples or comparative examples of the present invention may be specifically as follows:
[0093] 1. Human light chain ferritin:
[0094] 2. Human heavy chain ferritin-1:
[0095] 3. Mouse heavy chain ferritin—NCBI Reference Sequence: NP_034369.1;
[0096] 4. Mouse light chain ferritin—GenBank:AAA37614.1;
[0097] 5. Drosophila melanogaster heavy chain ferritin—NCBI Reference Sequence: NP_001247366.1;
[0098] 6. Drosophila melanogaster light chain ferritin—NCBI Reference Sequence: NP_001263105.1
[0099] 7. Caenorhabditis elegans ferritin—NCBI Reference Sequence: NP_491198.1;
[0100] 8. Oyster ferritin (PDB:6LIJ):
[0101] 9. Human mitochondrial ferritin—NCBI Reference Sequence: NC_000005.10;
[0102] 10. Soybean ferritin—NCBI Reference Sequence: NP_001236534.2;
[0103] 11. Arabidopsis thaliana ferritin 1—NCBI Reference Sequence: NP_195780.1;
[0104] 12. Sugar beet chloroplast ferritin—NCBI Reference Sequence: XP_010676813.1;
[0105] 13. Mucor microsporidial ferritin—NCBI Reference Sequence: XP_023465511.1;
[0106] 14. Encephalitozoon ferritin—NCBI Reference Sequence: XP_003887486.1;
[0107] 15. Enterobacteriaceae non-heme iron protein—NCBI Reference Sequence: WP_000917208.1;
[0108] 16. Acinetobacter bacterial ferritin—NCBI Reference Sequence: WP_002117082.1;
[0109] 17. Campylobacter ferritin—NCBI Reference Sequence: WP_002786063.1;
[0110] 18. Bacteroides ferritin—NCBI Reference Sequence: WP_010538613.1;
[0111] 19. Pseudomonas aeruginosa ferritin (PDB: 6NLF):
[0112] 20. Pyrococcus aerophilus ferritin—NCBI Reference Sequence: NC_014804.1;
[0113] 21. Pyrococcus jacobinii ferritin—NCBI Reference Sequence: NC_015680.1;
[0114] 22. Ferritin from Deinococcus-Thermus—NCBI Reference Sequence: WP_038069286.1;
[0115] 23. Actinomycetin—NCBI Reference Sequence: NP_218358.1;
[0116] 24. Proteobacteria ferritin—NCBI Reference Sequence: NP_417795.1;
[0117] 25. Escherichia coli ferritin:
[0118] 26. Ferritin from Archaeota—NCBI Reference Sequence: WP_012309233.1;
[0119] 27. Pyrococcus furiosus ferritin—NCBI Reference Sequence: WP_011011871.1;
[0120] 28. Methanosarcina ferritin—NCBI Reference Sequence: WP_048037772.1;
[0121] 29. Human heavy chain ferritin-2:
[0122] 30. Human heavy chain ferritin-3:
[0123] Example 1
[0124] The ferritin used in this example is modified human ferritin (human light chain ferritin, LFn), which contains 24 light chain subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 1.
[0125] The specific preparation steps of human light chain ferritin (LFn) hydrogel are as follows:
[0126] Chemically cross-linked LFn protein gels were prepared by adding 6% 1,4-butanediol diglycidyl ether (BDDE) to a 200 mg / mL LFn aqueous solution at pH 8. The mixture was then placed in a thermostatic water bath at 60°C for 20-150 minutes to produce chemically cross-linked LFn protein gels. The cross-linked LFn protein gels were then neutralized with 0.05M acetic acid to a pH of 7, rinsed with physiological saline for 24 hours, homogenized, and potted to produce chemically cross-linked LFn hydrogels (neutral cross-linking).
[0127] Example 2
[0128] The ferritin used in this example is a modified human ferritin (human heavy chain ferritin, HFn), which contains 24 subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 2.
[0129] According to the same method as in Example 1, chemically cross-linked HFn hydrogel was prepared under neutral conditions (neutral cross-linking).
[0130] Example 3-Example 9
[0131] Place the aliquoted LFn protein solution into the sample chamber of a freeze dryer. After prefreezing, sublimation drying, and desorption drying, lyophilized LFn protein is obtained. Weigh 1 mL of lyophilized LFn protein according to the ratios shown in Table 1 and add 1 mL of BDDE (pH 8) aqueous solution to the solution. Mix thoroughly and place in a thermostatic water bath at 60°C for 20-150 minutes to produce different chemically cross-linked LFn protein gels.
[0132] Then the cross-linked LFn protein gel was neutralized to pH 7 by immersion in 0.05 M acetic acid, washed with physiological saline for 24 h, homogenized, and sealed.
[0133] Table 1
[0134] Example 10
[0135] The ferritin used in this example is modified human ferritin (human light chain ferritin, LFn), which contains 24 subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 1.
[0136] 50 mg of hyaluronic acid (HA, molecular weight: 200 kD) was added to a 200 mg / mL LFn solution at pH 8 and gradually stirred at room temperature to dissolve. BDDE was added to the LFn / HA mixture at a volume percentage of 6%. After uniform stirring, the mixture was placed in a 60°C constant temperature water bath for 1-1.5 hours to produce a gel of LFn-chemically cross-linked HA, hereinafter referred to as LFn-HA. The gel was then neutralized with 0.5% HAC to a pH of 7, washed with physiological saline for 15 hours, homogenized, and potted.
[0137] Example 11
[0138] The ferritin used in this example is modified human ferritin (human heavy chain ferritin, HFn), which contains 24 subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 2.
[0139] 50 mg of hyaluronic acid (HA, molecular weight: 200 kD) was added to a 200 mg / mL HFn solution at pH 8 and gradually stirred at room temperature to dissolve. BDDE was added to the HFn / HA mixture at a volume percentage of 6%. After uniform stirring, the mixture was placed in a 60°C constant temperature water bath for 1-1.5 hours to produce a HFn-crosslinked HA gel, hereinafter referred to as HFn-HA. The gel was then neutralized with 0.5% HAC to a pH of 7, rinsed with physiological saline for 15 hours, homogenized, and potted.
[0140] Example 12
[0141] The ferritin used in this example is modified human ferritin (human light chain ferritin, LFn), which contains 24 subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 1.
[0142] 20 mg of collagen (Col, dried porcine collagen) was added to a 200 mg / mL LFn solution at pH 8 and gradually dissolved in a 60°C water bath. BDDE was added to the LFn / Col solution at a volume percentage of 6%. After uniform stirring, the mixture was allowed to react in a 60°C water bath for 1.5-2 hours to produce a LFn-Col cross-linked gel, referred to as LFn-Col. The gel was then neutralized with 0.5% HAC to a pH of 7, washed with saline for 15 hours, homogenized, and potted.
[0143] Example 13
[0144] The ferritin used in this example is modified human ferritin (human heavy chain ferritin, HFn), which contains 24 subunits, and the amino acid sequence of each subunit is shown in SEQ ID NO: 2.
[0145] 20 mg of collagen (Col, dried porcine collagen) was added to a 200 mg / mL HFn solution at pH 8 and gradually dissolved in a 60°C water bath. BDDE was added to the HFn / Col solution at a volume percentage of 6%. After uniform stirring, the mixture was allowed to react in a 60°C water bath for 1.5-2 hours to produce a HFn-Col cross-linked gel, referred to as HFn-Col. The gel was then neutralized with 0.5% HAC to a pH of 7, rinsed with saline for 15 hours, homogenized, and potted.
[0146] Example 14
[0147] The ferritin used in this example is modified human ferritin (human heavy chain ferritin, HFn), which contains 8 heavy chain subunits (hereinafter referred to as HFn-8mer), and the amino acid sequence of each subunit is shown in SEQ ID NO:6.
[0148] According to the same method as in Example 1, a chemically cross-linked HFn-8mer hydrogel was prepared under neutral conditions.
[0149] Example 15
[0150] The ferritin used in this example is modified human ferritin (human heavy chain ferritin, HFn), which contains 16 heavy chain subunits (hereinafter referred to as HFn-16mer), and the amino acid sequence of each subunit is shown in SEQ ID NO:7.
[0151] According to the same method as in Example 1, a chemically cross-linked HFn-16mer hydrogel was prepared under neutral conditions.
[0152] Comparative Example 1
[0153] Referring to the method of Example 1, chemically cross-linked LFn hydrogel (alkaline cross-linking) was prepared under alkaline conditions of pH 12 (200 mg / mL LFn was dissolved in NaOH to obtain a premixed solution with a pH of 12).
[0154] Comparative Example 2
[0155] Referring to the method of Example 1, chemically cross-linked HFn hydrogel (alkaline cross-linking) was prepared under alkaline conditions of pH 12 (200 mg / mL HFn was dissolved in NaOH to obtain a premixed solution with a pH of 12).
[0156] Comparative Example 3
[0157] Dissolve 100 mg of HA in 1 mL of 75 mM NaOH and mix thoroughly to obtain a premix with a pH of 12. Add 6% BDDE to the premix at a volume percentage, mix thoroughly, and place in a thermostatic water bath at 45°C for 5 hours to produce a chemically cross-linked HA gel. The chemically cross-linked HA gel is then neutralized with 0.05 M acetic acid to a pH of 7, rinsed with physiological saline for 24 hours, homogenized, and potted to obtain a chemically cross-linked HA alkaline hydrogel.
[0158] Comparative Example 4
[0159] Dissolve 100 mg of Col in 1 mL of 0.25 M NaOH at 60°C and mix thoroughly to obtain a premix with a pH of 12. Add 6% BDDE by volume to the premix, mix thoroughly, and place in a thermostatic water bath at 60°C for 5-12 hours to produce a chemically cross-linked Col protein gel. The chemically cross-linked Col protein gel is then neutralized with 0.05 M acetic acid to a pH of 7, rinsed with physiological saline for 24 hours, homogenized, and potted to obtain a chemically cross-linked Col alkaline hydrogel.
[0160] Effect Example 1 Test Characterization Results
[0161] The hydrogels obtained in each embodiment and comparative example were characterized as follows:
[0162] 1. Micromorphology Measurement
[0163] (1) The microstructures of the hydrogels of Examples 1-2 and Comparative Examples 1-4 were observed using a field emission scanning electron microscope.
[0164] (2) The hydrogels of Examples 1 and 10-15 were diluted 100 times and observed using a field emission transmission electron microscope.
[0165] 2. Determination of anti-enzymatic ability
[0166] (1) Uncross-linked LFn, HFn, Col, and bovine serum albumin (BSA) were incubated with trypsin (trypsin) and collagenase (collagenase) at 25°C for 2 h and 24 h, respectively, and the anti-enzymatic effect was detected by SDS-PAGE.
[0167] (2) Hydrogels (LFn, HFn) and commercial HA fillers according to Examples 1-2 Commercial Col fillers The degradation rates of the hydrogels (LFn-HA, HFn-HA, LFn-Col, and HFn-Col) of Examples 10-13 were determined as follows:
[0168] Weigh a certain mass of sample and record it as m0. Place the sample in an EP tube and add 1 mL of different enzyme solutions (trypsin, collagenase) to each tube. After different time periods in a constant temperature oscillator at 37°C and 100 rpm, place it in a centrifuge at 8000 rpm for 5 minutes and then discard the supernatant. Dry the solution and record the mass after enzymatic hydrolysis, m. d The formula for calculating sample degradation rate is: D=(m0–m d ) / m0×100%.
[0169] 3. Thermal Stability Determination
[0170] The hydrogels of Examples 1, 2, and 14-15 were incubated in pure water at 37° C., 60° C., 80° C., and 100° C. for 30 min, respectively, and then centrifuged at 8000 rpm for 5 min to observe the precipitation of the samples.
[0171] 4. Swelling Determination
[0172] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, the mass of the dried material was weighed and recorded as m0. The material was immersed in 2 mL PBS at 37°C for 24 h. After reaching swelling equilibrium, the PBS was removed and the gel was placed on a 45° inclined plane for 1 min and then weighed and recorded as m S . Swelling degree calculation formula: S=(m S -m0) / m0×100%.
[0173] 5. Determination of elastic and viscous modulus
[0174] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, the viscoelasticity of the gels was measured using a Haake RS6000 (Thermo Fisher Scientific (China) Co., Ltd.) advanced rotational rheometer. The elasticity and viscosity of the hydrogels were dynamically measured at a test temperature of 25°C, a slit width of 1 mm, and a vibration frequency in the range of 0.1-100 Hz. The observation indicators were the elastic modulus (G') and the viscous modulus (G").
[0175] 6. Observation of Subcutaneous Retention Time and Retention Efficiency in Vivo
[0176] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, female nude mice, 8-10 weeks old, were subcutaneously injected with each hydrogel into two points in the middle of the back of the mice, 200 μL per point. The volume of the gel spots was measured every 7 days for 30 days. After 30 days, the mice were killed and the remaining gel spots were removed and weighed after dissection.
[0177] 7. Observation of filling position movement in vivo
[0178] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, female nude mice aged 8-10 weeks were subcutaneously injected with each hydrogel at two points in the middle of the back of the mice, with 200 μL at each point. The displacement distance of the gel points was measured every 7 days within 30 days.
[0179] 8. In vivo inflammatory response observation
[0180] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, Balb / c mice, 8-10 weeks old, were subcutaneously injected with each hydrogel at two points on the back of the mice, 200 μL at each point. The mice were killed 48 hours later, and multifactor detection was used to detect local tissue inflammatory factors. Mice that were not subcutaneously injected (Normal) were used as blank controls.
[0181] 9. Based on the clinician's injection experience score
[0182] The hydrogels of Examples 1 and 3-9 were evaluated for injectability by 5 dermatology clinicians. As a control.
[0183] The results are as follows:
[0184] In the following results, each hydrogel was prepared according to the previous method, and the actual application concentration was 100 mg / mL only during subcutaneous injection.
[0185] 1. Appearance and micromorphology characterization results
[0186] The cross-linked sample containing HFn is brown in color because it has ferroxidase activity and may participate in iron metabolism in the body as a filling material.
[0187] The microscopic morphology of the hydrogels of Examples 1-2 and Comparative Examples 1-4 is shown in Figures 1-2. The results show that after crosslinking under neutral conditions with LFn or HFn, they are all in a dense state, while crosslinking under alkaline conditions is the crosslinked product of the depolymerized monomers, with a porous structure.
[0188] The microscopic morphology of the hydrogels from Example 1 and Examples 10-15 after 100-fold dilution is shown in Figure 3. The results show that the wild-type LFn-24mer (Example 1) exhibits a complete cage-like structure; the HFn-8mer (Example 14) exhibits a half-bowl-like structure, not a cage-like structure; and the HFn-16mer (Example 15) exhibits a cage-like structure consisting of two half-bowls docked together. Furthermore, the LFn-HA, LFn-Col, HFn-HA, and HFn-Col hydrogels from Examples 10-13 all exhibit cage-like structures, demonstrating that further crosslinking of HA or Col with LFn or HFn does not affect the original cage structure.
[0189] 2. Characterization results of anti-enzymatic ability
[0190] The SDS-PAGE results of uncross-linked LFn, HFn, Col, and bovine serum albumin (BSA) are shown in Figure 4. The results indicate that collagenase can only specifically degrade collagen and has no significant degradation effect on uncross-linked LFn, HFn, and BSA. Under trypsin catalysis for 2-24 hours, uncross-linked LFn and HFn show little degradation, indicating that uncross-linked LFn and HFn are resistant to degradation by collagenase and trypsin.
[0191] Hydrogels (LFn, HFn) of Examples 1-2, commercial HA fillers Commercial Col fillers The enzymatic degradation rate results of the hydrogels (LFn-HA, HFn-HA, LFn-Col, and HFn-Col) of Examples 10-13 at 37 °C are shown in Figure 5. The results show that the degradation rates of LFn and HFn under the action of trypsin and collagenase are significantly lower; while commercial HA and commercial Col can be degraded by trypsin and collagenase almost within 2 h at 37 °C.
[0192] 3. Results of thermal stability measurement
[0193] It was observed that after the hydrogels of Examples 1, 2, and 14-15 were tested at 80 °C, significantly more protein precipitates were observed in the hydrogels of HFn-8mer (Example 14) and HFn-16mer (Example 15) after the test. It indicates that due to structural problems, the thermal stability of the hydrogels of HFn-8mer and HFn-16mer is relatively poor.
[0194] 4. Results of swelling level and swelling degree measurement
[0195] The swelling level results of the hydrogels of Examples 1-2 and Comparative Examples 3-4 are shown in Figure 6. The results show that different degrees of swelling occurred after washing. The higher the cross-linking degree, the lower the water absorption. Visually, HA showed a greater degree of swelling, Col showed a slight swelling, and LFn and HFn did not show a large degree of swelling, indicating that the cross-linking degrees of LFn and HFn are relatively high. The swelling level is LFn, HFn < Col < HA. If the swelling degree of the hydrogel is too high under physiological conditions, it will cause a decrease in the mechanical strength of the hydrogel, and may also affect the growth of surrounding tissues or cause abnormalities in body indicators.
[0196] The swelling degree results of the hydrogels of Examples 1-2 and Comparative Examples 3-4 are shown in Figure 7. The results results The results show that there is no significant difference in the swelling degrees of LFn (527.1%) and HFn (469.7%) compared with Col (608.7%), but they are significantly lower than HA (4756.2%), suggesting that the mechanical strengths of LFn and HFn are close to Col and higher than HA.
[0197] 5. Results of elastic and viscous modulus measurement
[0198] The elastic modulus and viscous modulus of the hydrogels of Examples l-2 and Comparative Examples 3-4 were detected, and the results are shown in Figure 8. G’ represents the elastic modulus, G” represents the viscous modulus, and G’>G”, indicating that the sample is in a liquid state. After the sample is stable, the elastic modulus of LFn is higher than 1000 Pa, that of Col is higher than 600 Pa, while those of HFn and HA are both lower than 200 Pa; the viscous modulus of LFn is higher than 100 Pa, that of Col is higher than 30 Pa, while those of HFn and HA are both lower than 20 Pa. Both the elasticity and viscosity are LFn>Col>HFn, HA, indicating that LFn has the best elasticity and viscosity, and HFn is close to HA.
[0199] 6. Observation results of in vivo subcutaneous retention time and filling position movement
[0200] For the hydrogels of Examples 1-2 and Comparative Examples 3-4, the in vivo epidermal degradation level (retention capacity / extension area) and displacement level are shown in Figure 9. The hydrogel was subcutaneously injected into two points in the middle back of mice, 200 μL per point, and the length, width, height and displacement were measured (as shown in Figure 10). Extension area = 4 / 3π(abc). The results show that the ferritin retention capacity in the middle back is higher than that of HA and Col. After 28 days, the remaining gel point volume LFn (1.12 cm 3 ) and HFn(0.95cm 3 ) is approximately the same, while HA and Col are degraded to 0.34 cm 3 and 0.47cm 3 The middle part of the back was relatively weak in motility. Within 28 days, the subcutaneous displacement of ferritin and HA (6.38 mm) was at a similar level, significantly lower than Col 9.30 mm. Among them, the displacement of LFn was relatively the smallest (3.98 mm) (as shown in Figure 10).
[0201] 7. In vivo retention efficiency observation results
[0202] Figure 11 shows the results of a subcutaneous hydrogel retention experiment in mice for the hydrogels of Examples 1-2 and Comparative Examples 3-4. The left figure shows the results after 30 days. The results show that LFn and HFn gel dots remained stably within one month, with no significant degradation. After 30 days, Col and HA were significantly degraded, with HA being the most significantly degraded. LFn and HFn showed better retention than HA and Col.
[0203] 8. In vivo inflammatory response observation results
[0204] Figure 12 shows the subcutaneous inflammatory factor assays for the hydrogels in Examples 1-2 and Comparative Examples 3-4. Serum was collected from Balb / c mice 48 hours after subcutaneous injection for inflammatory factors, and skin homogenates were taken from the gel points for inflammatory factors. Compared to the Normal group, subcutaneous injection of LFn and HFn did not increase the inflammatory factors IL-6 and TNF-α in serum, and LFn did not increase the inflammatory factors IL-6 and TNF-α in skin tissue, suggesting that LFn does not induce an inflammatory response. IL-6 in serum and skin tissue increased by 11.1-fold and 21.2-fold after HA injection.
[0205] 9. Based on the clinician's injection experience score results
[0206] The hydrogels of Examples 1 and 3-9 and Juvederm The injection experience score results are shown in Tables 2 and 3. The meaning of each score is:
[0207] "+" means: the injection can be pushed steadily under a certain force, the needle may occasionally be slightly blocked or stuck, and the material has a certain viscoelasticity.
[0208] “++” means: the injection is smooth, with basically no blockage or jamming, and the material’s viscoelasticity is acceptable.
[0209] “+++” means: easy to push without resistance, not easy to clog the needle, and the material has good viscoelasticity.
[0210] Table 2
[0211] Table 3
[0212] The results showed that in LFn hydrogel, as the protein concentration increased, the viscosity of the hydrogel became better; in LFn hydrogel, as the volume ratio of BDDE increased, the viscosity of the hydrogel became better.
Claims
1. A modified ferritin, characterized in that It comprises a plurality of light chain subunits or heavy chain subunits of ferritin from natural sources; wherein the plurality of light chain subunits or the plurality of heavy chain subunits form a multimer, preferably a cage-shaped multimer.
2. The modified ferritin according to claim 1, characterized in that The amino acid sequence of the light chain subunit is as shown in SEQ ID NO: 1 or has at least 80% identity with the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain subunit is as shown in SEQ ID NO: 2, 6 or 7 or has at least 80% identity with the sequence shown in SEQ ID NO: 2, 6 or 7; Preferably, the modified ferritin comprises 8, 16, 24 or 48 light chain subunits or 8, 16, 24 or 48 heavy chain subunits; and / or, the natural source comprises animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, more preferably a human; More preferably, the modified ferritin is a cage-shaped polymer composed of 24 light chain subunits, or a cage-shaped polymer composed of 24 heavy chain subunits.
3. A cross-linked ferritin, characterized in that It is a cross-linked ferritin formed by cross-linking of ferritin from a natural source or a variant thereof, and the cross-linked ferritin retains the polymer structure of the ferritin from a natural source or a variant thereof; or it is a cross-linked ferritin formed by cross-linking of the modified ferritin according to claim 1 or 2, and the cross-linked ferritin retains the polymer structure of the modified ferritin; wherein the variant has at least 80% identity with the amino acid sequence of the ferritin from a natural source; Preferably, the cross-linked ferritin is in the form of a hydrogel or microsphere structure.
4. The cross-linked ferritin according to claim 3, characterized in that The swelling degree of the cross-linked ferritin is less than 1000%, preferably less than 600%, more preferably less than 500%; and / or, the elastic modulus of the cross-linked ferritin is higher than 100 Pa, preferably higher than 600 Pa, more preferably higher than 1000 Pa; and / or, the viscosity modulus of the cross-linked ferritin is higher than 10 Pa, preferably higher than 30 Pa, more preferably higher than 50 Pa, and even more preferably higher than 100 Pa; And / or, the natural source includes animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, and more preferably a human; Preferably, the cross-linking is performed by a biological cross-linking reaction or a chemical cross-linking reaction.
5. The cross-linked ferritin according to claim 3, characterized in that The cross-linked ferritin further comprises hyaluronic acid and / or collagen; Preferably, the volume ratio of the cross-linked ferritin to the hyaluronic acid is 1:5-5:1; or, the volume ratio of the cross-linked ferritin to the collagen is 1:5-5:1; Or, the molar concentration ratio or mass ratio of the cross-linked ferritin to the hyaluronic acid is 1:5-5:1; or The molar concentration ratio or mass ratio of ligand to collagen is 1:5-5:
1.
6. A method for preparing cross-linked ferritin as claimed in any one of claims 3 to 5, characterized in that: The method comprises the following steps: The naturally derived ferritin or its variant, or the modified ferritin as claimed in claim 1 or 2, is made into cross-linked ferritin through a cross-linking reaction; the cross-linking reaction is a chemical cross-linking reaction or a biological cross-linking reaction.
7. The method according to claim 6, characterized in that When the cross-linking reaction is a chemical cross-linking reaction, the cross-linking reaction comprises the following steps: A neutral solution containing ferritin from natural sources or its variants, or a neutral solution containing the modified ferritin as claimed in claim 1 or 2, is mixed with a cross-linking agent to carry out a cross-linking reaction to obtain the product; And / or, after the cross-linking reaction, the step further includes mixing hyaluronic acid and / or collagen into the cross-linked ferritin; And / or, after the cross-linking reaction, neutralization, washing and / or homogenization are further included; When the cross-linking reaction is a biological cross-linking reaction, the natural ferritin is cross-linked via disulfide bonds; preferably, the natural ferritin is fused and expressed with a cysteine-containing polypeptide by genetic engineering to obtain cross-linked ferritin; Preferably, the natural source includes animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, and more preferably a human.
8. The method according to claim 7, characterized in that When the cross-linking reaction is a chemical cross-linking reaction, the method comprises one or more of the following conditions: ① The cross-linking agent is selected from one or more of 1,4-butanediol diglycidyl ether, genipin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, nordihydroguaiaretic acid and glutaraldehyde, preferably 1,4-butanediol diglycidyl ether; ② The volume percentage of the cross-linking agent relative to the neutral solution is 3-30%, preferably 6%; ③ The cross-linking reaction is carried out in a constant temperature water bath; the temperature of the cross-linking reaction is preferably 60-72°C, more preferably 60°C; the time of the cross-linking reaction is preferably 20-150min; ④ The pH of the neutral solution is 8; ⑤ The concentration of the naturally derived ferritin or its variant, or the modified ferritin as described in claim 1 or 2 is 10-600 mg / mL, preferably 50-300 mg / mL, for example 100 mg / mL.
9. The method according to claim 7 or 8, characterized in that The method includes one or more of the following conditions: ① The neutralization includes pickling to neutrality; the pickling preferably includes leaching with an acidic agent; preferably, the acidic agent is selected from one or more of hydrochloric acid, sulfuric acid and acetic acid, more preferably acetic acid; ② The cleaning includes cleaning with a buffered saline solution; preferably, the buffered saline solution is selected from one or more of phosphate buffer, saline, Tris-HCl buffer and HEPES; and or, the concentration of the buffered saline solution is 10-100 mM and the pH is 6-8; ③The cleaning time is 12-30h.
10. Use of ferritin from natural sources or its variants, or the modified ferritin as claimed in claim 1 or 2, in the preparation of a subcutaneous or tissue filler; wherein: The variant has at least 80% identity with the amino acid sequence of ferritin from natural sources; Preferably, the natural source includes animal, plant, fungal or bacterial sources, preferably animal sources; the animal is a vertebrate, preferably a mammal, and more preferably a human.