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CN-121986109-A - Modified ferritin, cross-linked ferritin, preparation method and application thereof

CN121986109ACN 121986109 ACN121986109 ACN 121986109ACN-121986109-A

Abstract

Modified ferritin, cross-linked ferritin, and preparation method and application thereof are provided. The modified ferritin comprises a plurality of naturally derived ferritin light chain subunits or heavy chain subunits, wherein the plurality of light chain subunits or the plurality of heavy chain subunits form a multimer, preferably a cage multimer. The cross-linked ferritin is ferritin of natural origin or a variant thereof, or is cross-linked ferritin formed by cross-linking of modified ferritin. The cross-linked ferritin still maintains a unique cage-shaped structure of ferritin, has the characteristics of good elasticity, high support degree, small swelling degree, degradation resistance and the like, and can realize better subcutaneous and tissue filling effects.

Inventors

  • SU YONG

Assignees

  • 上海尚泽涌致生物科技合伙企业(有限合伙)

Dates

Publication Date
20260505
Application Date
20240927
Priority Date
20230928

Claims (10)

  1. A modified ferritin, characterized in that it comprises a plurality of light chain subunits or heavy chain subunits of ferritin of natural origin, wherein the plurality of light chain subunits or the plurality of heavy chain subunits form a multimer, preferably a cage-like multimer.
  2. The modified ferritin according to claim 1, wherein the amino acid sequence of the light chain subunit is shown in SEQ ID No.1 or has at least 80% identity to the sequence shown in SEQ ID No.1 and the amino acid sequence of the heavy chain subunit is shown in SEQ ID No.2, 6 or 7 or has at least 80% identity to 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 an animal, plant, fungal or bacterial source, preferably an animal source; More preferably, the modified ferritin consists of a cage polymer of 24 light chain subunits or a cage polymer of 24 heavy chain subunits.
  3. A crosslinked ferritin, characterized in that it is a crosslinked ferritin formed by crosslinking a naturally derived ferritin or a variant thereof, said crosslinked ferritin retaining the multimeric structure of said naturally derived ferritin or a variant thereof, or a crosslinked ferritin formed by crosslinking a modified ferritin according to claim 1 or2, said crosslinked ferritin retaining the multimeric structure of said modified ferritin, wherein said variant has at least 80% identity to the amino acid sequence of a naturally derived ferritin; Preferably, the cross-linked ferritin is in a hydrogel or microsphere structure.
  4. A crosslinked ferritin according to claim 3 wherein the crosslinked ferritin has a swelling degree of less than 1000%, preferably less than 600%, more preferably less than 500%; And/or the elastic modulus of the crosslinked ferritin is higher than 100Pa, preferably higher than 600Pa, more preferably higher than 1000Pa; and/or the viscosity modulus of the crosslinked ferritin is higher than 10Pa, preferably higher than 30Pa, more preferably higher than 50Pa, still more preferably higher than 100Pa; and/or the natural source comprises animal, plant, fungal or bacterial sources, preferably animal sources, the animal being a vertebrate, preferably a mammal, more preferably a human; Preferably, the crosslinking is performed by a biological crosslinking reaction or a chemical crosslinking reaction.
  5. A cross-linked ferritin according to claim 3 wherein said 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 the mass ratio of the crosslinked ferritin to the hyaluronic acid is 1:5-5:1, or the molar concentration ratio or the mass ratio of the crosslinked ferritin to the collagen is 1:5-5:1.
  6. A method for preparing a cross-linked ferritin as claimed in any one of claims 3 to 5, said method comprising the steps of: Preparing crosslinked ferritin from naturally derived ferritin or a variant thereof or comprising a modified ferritin according to claim 1 or 2 by a crosslinking reaction, said crosslinking reaction being a chemical crosslinking reaction or a biological crosslinking reaction.
  7. The method of claim 6, wherein when the crosslinking reaction is a chemical crosslinking reaction, the crosslinking reaction comprises the steps of: Mixing a neutral solution containing naturally-derived ferritin or a variant thereof, or a neutral solution containing modified ferritin according to claim 1 or2 with a crosslinking agent, and performing a crosslinking reaction; And/or, after the crosslinking reaction, mixing hyaluronic acid and/or collagen into the crosslinked ferritin; And/or, after the crosslinking reaction, neutralization, washing and/or homogenization are further included; When the crosslinking reaction is a biological crosslinking reaction, the naturally-derived ferritin is crosslinked through disulfide bonds, and preferably, the naturally-derived ferritin and the polypeptide containing cysteine are fused and expressed through genetic engineering to obtain crosslinked ferritin; Preferably, the natural source comprises animal, plant, fungal or bacterial sources, preferably animal sources, the animal being a vertebrate, preferably a mammal, more preferably a human.
  8. The method of claim 7, wherein when the crosslinking reaction is a chemical crosslinking 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 under the condition of constant temperature water bath, the temperature of the cross-linking reaction is preferably 60-72 ℃, more preferably 60 ℃, and the time of the cross-linking reaction is preferably 20-150min; ④ The neutral solution has a pH of 8; ⑤ The concentration of the naturally derived ferritin or variant thereof, or the modified ferritin according to claim 1 or2, is 10-600mg/mL, preferably 50-300mg/mL, e.g. 100mg/mL.
  9. The method of claim 7 or 8, wherein the method comprises one or more of the following conditions: ① The neutralization comprises pickling to neutrality, preferably pickling with an acidic reagent, preferably one or more of hydrochloric acid, sulfuric acid and acetic acid, more preferably acetic acid; ② The washing comprises washing with a buffer salt solution, preferably one or more selected from phosphate buffer, physiological saline, tris-HCl buffer and HEPES, and/or the concentration of the buffer salt solution is 10-100mM, and the pH is 6-8; ③ The cleaning time is 12-30h.
  10. Use of a naturally derived ferritin or a variant thereof, or a modified ferritin according to claim 1 or 2 in the preparation of a subcutaneous or tissue filler, wherein said variant has at least 80% identity to the amino acid sequence of a naturally derived ferritin; Preferably, the natural source comprises animal, plant, fungal or bacterial sources, preferably animal sources, the animal being a vertebrate, preferably a mammal, more preferably a human.

Description

Modified ferritin, cross-linked ferritin, preparation method and application thereof The present application claims priority from chinese patent application 2023112753858, whose application date is 2023, 9, 28 and priority from chinese patent application 2024112962839, whose application date is 2024, 9, 14. The present application incorporates the entirety of the above-mentioned chinese patent application. Technical Field The invention belongs to the technical field of biomedical materials, and particularly relates to modified ferritin, cross-linked ferritin, a preparation method and application thereof. Background The skin is the largest organ of the human body and plays an important role in resisting external invasion and maintaining the stability of the internal environment. With age, skin aging can occur as facial tissues relax and wrinkles develop. By subcutaneous filling, facial laxity sagging, capacity deficiency supplementing, facial contour remodeling, skin quality improvement, etc. can be well reduced. Local injection of hyaluronic acid and/or collagen has the advantages of simple operation, small damage, good biocompatibility and the like, and has become a common method. Hyaluronic acid is also called hyaluronic acid, has the advantages of good tissue compatibility, easy injection, immediate effect after filling injection and minimally invasive effect, and is one of the most commonly used dermal fillers in clinic. Because hyaluronic acid is acidic mucopolysaccharide, a three-dimensional space structure cannot be formed after crosslinking, the support degree is limited after filling, and the tyndall phenomenon is easy to occur, so that the appearance after filling is influenced. Hyaluronic acid is a high molecular polymer, the chemical structure of the hyaluronic acid is hydroxyl and carboxyl, the hyaluronic acid is easy to hydrolyze in vivo after the hyaluronic acid interacts with water, and biological enzymes (especially hyaluronidase and human free radicals) have strong degradation effect on hyaluronic acid, so that the hyaluronic acid has short residence time in vivo and can achieve the curative effect by repeated injection. Meanwhile, skin cells can quickly absorb hyaluronic acid after primary injection, and the metabolism of hyaluronic acid in vivo can be accelerated after staying up and drinking. The rate of hyaluronic acid metabolism is also increased in areas where activity is frequent. The hyaluronic acid is filled subcutaneously and generally maintained for a period of 6-8 months. Meanwhile, the displacement and the shape change of hyaluronic acid are easy to be caused by collision. Thus, hyaluronic acid has some defects as a subcutaneous filling material, and further products are required to be perfected. Collagen is another common filling material, and has the advantages of good tissue compatibility, easy injection, instant effect after filling injection and minimally invasive effect. The natural collagen existing in human skin is type I collagen, the amino acid sequence of the collagen contains 3 unusual amino acids, namely 4-hydroxyproline (9%), 3-hydroxyproline (0.1%) and 5-hydroxylysine (0.6%), stable covalent bond connection can be formed through amino acid modification, a procollagen molecule (protocollagen) of a right-hand triple helix is further formed, each strand of procollagen molecule is a left-hand helix, and therefore the natural collagen existing in the skin has strong supporting force and tensile strength (TENSILESTRENGTH). The subcutaneous filling collagen used in the current market is mostly collagen fragments expressed by pronucleus, can not form a left-handed spiral single chain or a right-handed triple spiral, so that the collagen lacks strong supporting force and tensile strength in vivo and cannot play the real functions. Meanwhile, as the filler is collagen fragments, the filler is easy to hydrolyze by collagenase in the body, and the in-vivo residence time is short. In view of the disadvantages of hyaluronic acid and collagen, such as limited supporting force and easy degradation, development of a novel filling material which is long-acting, has better biomechanical supporting degree and can be produced in a large-scale industrialized manner is needed. Ferritin is a hollow spherical protein naturally occurring in animals, has an inner diameter of 8nm and an outer diameter of 12nm, and is a 24-mer protein cage structure formed by self-assembly of light chain subunits and heavy chain subunits. In the current research, ferritin is often used for tissue engineering or as a delivery carrier of small molecule drugs, biological agents, nucleic acids and the like, and related researches on the ferritin in preparation of filling materials are rarely reported. Disclosure of Invention In order to solve the defects in the prior art, the invention provides modified ferritin, cross-linked ferritin, a preparation method and application thereof, and the prepared cross-linked