Polypeptide type micromolecule glue and application thereof in preparation of cosmetics with skin anti-aging effect

Abstract

The invention discloses polypeptide type micromolecule glue and application thereof in preparation of cosmetics with a skin anti-aging effect. The polypeptide type small molecule glue comprises a hyaluronic acid binding peptide fragment and a proteoglycan binding peptide fragment. The polypeptide type small molecule glue provided by the invention is polypeptide with biological activity, the polypeptide can be combined with hyaluronic acid and proteoglycan after entering cells, the function of connecting the hyaluronic acid and the proteoglycan to protein 1 (HAPLN1) can be replaced, the content of collagen which is gradually reduced along with skin aging can be recovered to a certain extent, and the collagen type small molecule glue has the advantages of being good in biocompatibility, good in stability and good in stability. The composition may become a promising innovative strategy for preventing or repairing the aged skin.

Description

Technical Field

[0001] This application belongs to the field of synthetic biology, and in particular relates to amino acids, nucleotides, recombinant plasmids, transformants and targeted anti-aging agents of a polypeptide-type small molecule gel, and their application in the preparation of cosmetics with skin anti-aging effects. Background Technology

[0002] Hyaluronic acid and proteoglycan connexin 1 (HAPLN1) can bind to both hyaluronic acid and proteoglycans. Serum HAPLN1 levels decline with age, disrupting the integrity of the skin's extracellular matrix, which is primarily composed of type I collagen and hyaluronic acid. While levels of type I collagen and hyaluronic acid decrease in aging skin, they are significantly restored through HAPLN1 application. HAPLN1 also possesses anti-inflammatory and cell-damage-reducing effects. Therefore, HAPLN1 may be used to revitalize aging skin.

[0003] However, HAPLN1 has a relatively large molecular weight, approximately 40 kDa. If such a high-molecular-weight protein were used in skincare, it would be unable to penetrate the dermis, thus failing to exert its intended skincare benefits. Generally speaking, molecules larger than 3 kDa have significantly increased difficulty in transdermal absorption. Therefore, to enable molecules to penetrate the skin more easily and exert their skincare effects, their molecular weight should be controlled below 3 kDa.

[0004] Molecular glue acts like intermolecular adhesive, binding two different proteins together and bringing them close together spatially. Molecular glues form stable protein complexes through various mechanisms such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. Peptide-type molecular glues are biosynthesized peptides with molecular glue functions. Currently, there are no known cases of peptide-type molecular glues being used as ingredients in skincare products.

[0005] If a peptide-based molecular gel with a function similar to HAPLN1 and a molecular weight of less than 3 kDa is designed and synthesized, it could not only replace HAPLN1 in achieving the anti-aging effect of simultaneously binding hyaluronic acid and proteoglycans, but also be easily transdermal. Theoretically, this peptide-based molecular gel, which could replace HAPLN1, should consist of at least two parts: a peptide segment that binds hyaluronic acid and a peptide segment that binds proteoglycans.

[0006] The known peptide sequences that can bind hyaluronic acid are: CRRDDGAHWQFNALTVR; GAHWQFNALTVR; LKQKIKHVVKLKVVVKLRSQLVKRKQN; STMMSRSHKTRSHHV; GAHWQFNALTVRGGGS.

[0007] Fibulins are a family of seven members, of which fibulin-1 and fibulin-2 have been found to bind proteoglycans. Fibulin-1 binds to the CLDs of the proteoglycans Versican (Vcan) and Acan. Fibulin-2 binds to the CLDs of the proteoglycans Versican (Vcan), Acan, and brevican. The proteoglycan-binding sites on the fibulin-1 and fibulin-2 molecules are located in the central region of a calcium-binding EGF1-like repeat sequence. Based on domain analysis, the proteoglycan-binding peptides on the fibulin-1 molecule are LGESCINTVGSF; ICQNTLGSF; NTEGSYTCQ; CVNSPGSF; NTLGSYLCSCSVGF; VYGSYQCYC; PGSFQCSCPSSGY; TCFNIQGGF. The peptides that bind proteoglycans to fibulin-2 molecules are HCVNTLGSFHCY; QPGFLCQNT; NTVGSYTCQ; VCHNLPGSY; TCENTLGSY; IYGSYQCYC; TCHNIQGSF.

[0008] Theoretically, peptides with small molecular weights and strong binding affinity for proteoglycans include CVNSPGSF, VYGSYQCYC, and VCHNLPGSY. To increase the expression level of peptide-based molecular gels, tandem expression is necessary. However, after tandem expression, they still need to be cleaved into small peptides, as the active ingredient is the small peptide, which facilitates transdermal absorption. Peptides suitable for trypsin cleavage, which is relatively inexpensive, theoretically end in K or R. Therefore, the peptide-based molecular gel should end in K or R, while other parts should not contain K or R. The only hyaluronic acid-binding peptide sequence that meets these conditions is GHAHWQFNALTVR. The fused sequences of the two peptides are shown in SEQ ID NO. 1-3, all with molecular weights less than 3 kDa. Summary of the Invention

[0009] This application provides a polypeptide-type small molecule gel, which is a bioactive polypeptide whose various fragments have a synergistic effect that can significantly enhance anti-aging effects. This application also provides a targeted anti-aging agent and the application of the polypeptide-type small molecule gel and the targeted anti-aging agent in the preparation of cosmetics.

[0010] In a first aspect, embodiments of this application provide a polypeptide-type small molecule adhesive, wherein the polypeptide-type small molecule adhesive includes hyaluronic acid-binding peptide fragments and proteoglycan-binding peptide fragments.

[0011] According to one embodiment of this application, the polypeptide-type small molecule gel comprises an amino acid sequence as shown in any one of SEQ ID NO. 1-3.

[0012] According to an embodiment of one aspect of this application, the polypeptide-type small molecule gel comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence described in any one of SEQ ID NO. 1-3.

[0013] Secondly, embodiments of this application provide a nucleic acid molecule that encodes a polypeptide-type small molecule gel, as described in the first aspect.

[0014] According to one aspect of this application, a nucleic acid molecule comprises a nucleotide sequence shown in any one of SEQ ID NO. 4-6 and a tandem nucleic acid molecule formed thereon.

[0015] Thirdly, embodiments of this application provide a recombinant plasmid containing nucleic acid molecules or tandem nucleic acid molecules as described in the second aspect.

[0016] Fourthly, embodiments of this application provide a transformant, which includes a polypeptide-type small molecule gel (as described in the first aspect), a nucleic acid molecule (as described in the second aspect), or a recombinant plasmid (as described in the third aspect).

[0017] Fifthly, embodiments of this application provide a targeted anti-aging agent, including one obtained by cultivating a transformation system as described in the fourth aspect.

[0018] According to one embodiment of this application, the targeted anti-aging agent includes at least one of a transformant, a lysate, a fermentation broth, a fermentation broth extract, and a culture obtained by the transformant.

[0019] Sixthly, embodiments of this application provide the application of the polypeptide-type small molecule gel described in the first aspect or the targeted anti-aging agent described in the fifth aspect in the preparation of cosmetics with skin anti-aging effects.

[0020] The embodiments of this application have at least the following beneficial effects:

[0021] 1) The polypeptide-type small molecule gel of this application is composed of two parts: a hyaluronic acid-binding peptide fragment and a proteoglycan-binding peptide fragment. The selection of each peptide segment in both fragments enhances the anti-aging efficacy of the polypeptide-type small molecule gel. This polypeptide-type small molecule gel can not only replace the large HAPLN1 protein, but its molecular weight is also much smaller than that of HAPLN1 protein, resulting in a much higher transdermal penetration rate and significantly better anti-aging efficacy. Theoretically, this polypeptide-type small molecule gel has a stronger binding capacity to hyaluronic acid and proteoglycan, and exhibits better anti-aging effects than peptides with weaker binding capacity. Furthermore, selecting shorter polypeptide-type small molecule gel fragments and choosing longer fragments results in a smaller total molecular weight, making it easier to penetrate the skin and thus exert a better anti-aging effect. Moreover, the polypeptide-type small molecule gel of this application is expressed through tandem expression at several tens of times, which is equivalent to a several tens of times increase in yield. The optimized polypeptide-type small molecule gel exhibits significantly better anti-aging capabilities than other polypeptides.

[0022] 2) The polypeptide-type small molecule glue of this application is the only pure polypeptide-type molecular glue known to achieve skin anti-aging effects through molecular glue technology, setting a precedent for targeted anti-aging and precise aesthetics through a new molecular glue mechanism.

[0023] 3) The polypeptide-type small molecule gel of this application is a pure biological polypeptide. Pure biological polypeptides conform to the concept of green environmental protection, and no environmentally unfriendly chemical reagents are used in the synthesis process, ensuring high safety. The polypeptide-type small molecule gel of this application can be expressed in the transformant in one step, without the need for multiple cumbersome steps, resulting in lower cost. The polypeptide-type small molecule gel of this application directly targets and binds to hyaluronic acid fragments and proteoglycans, exhibiting significant anti-aging effects and can be used in anti-aging cosmetics. The anti-aging effect of the polypeptide-type small molecule gel of this application is significantly better than polypeptide molecules that simply inhibit aging proteins. The polypeptide-type small molecule gel of this application has a clear mechanism and well-defined targets, belonging to precision anti-aging. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows a comparison of the effects of Examples 1-3 and Comparative Examples 1-4 of this application on increasing the expression level of collagen I.

[0026] Figure 2 Transdermal permeability comparison charts of Examples 1-3 and Comparative Examples 1-4 of this application are shown.

[0027] Figure 3 The advantages and disadvantages of embodiments 1-3 of this application and comparative examples 1-4 are shown in the diagram. Detailed Implementation

[0028] To make the inventive objectives, technical solutions, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are merely illustrative and not intended to limit the scope of this application.

[0029] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an unspecified range.

[0030] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "several" in "one or more" means two or more. "Multiple" in "one or more" means two or more.

[0031] The above-described invention content is not intended to describe every disclosed embodiment or implementation method. The following description provides more specific examples of exemplary embodiments. Throughout this application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive. Research indicates that one of the main causes of skin aging is the alteration of the dermal structure. Many factors contribute to this structural change, such as a reduction in collagen I, which is insufficient to support the normal skin structure, leading to wrinkles, decreased elasticity, and other signs of aging. Increasing the expression level of collagen I is an important anti-aging method. HAPLN1 protein can link hyaluronic acid and proteoglycans; supplementing the skin with HAPLN1 protein can promote collagen I expression. Therefore, HAPLN1 protein has great potential for anti-aging. Unfortunately, HAPLN1 protein has a large molecular weight, making it difficult to penetrate the skin and exert its skincare effects. Small molecule peptides can replace the function of HAPLN1, namely, the function of linking hyaluronic acid and proteoglycans, and can, to some extent, restore the collagen content that gradually decreases with skin aging. Furthermore, the anti-aging mechanisms of many skincare ingredients, such as natural extracts, are unclear, requiring efficacy testing to determine their effectiveness. Peptide-based small molecule gels, however, can replace the function of HAPLN1—binding hyaluronic acid and proteoglycans—with a clear mechanism and well-defined targets, representing precise anti-aging. While chemically synthesized small molecule gels could theoretically also replace HAPLN1 in binding hyaluronic acid and proteoglycans to achieve anti-aging effects, they contradict the principles of green and environmentally friendly practices, and their safety is generally inferior to that of pure biological molecules. The synthesis of chemical small molecule gels often involves the use of environmentally unfriendly chemical reagents, increasing their risk. Furthermore, the synthesis of chemical small molecule gels may require multiple steps, making it cumbersome, complex, and costly. To date, no chemically synthesized small molecule gels for skin anti-aging have been discovered. This has prompted the development of small-molecule peptide-based small molecule gels to achieve targeted, precise, green, and safe anti-aging. Therefore, existing technologies urgently need improvement.

[0032] Based on this, this application develops a green, environmentally friendly, safe, low-cost, peptide-based, targeted and precise binding of hyaluronic acid and proteoglycans, small molecular weight, easy skin penetration, and peptide fragment sequence-optimized anti-aging technology and product.

[0033] A polypeptide-type small molecule gel

[0034] In a first aspect, embodiments of this application provide a polypeptide-type small molecule adhesive, wherein the polypeptide-type small molecule adhesive includes hyaluronic acid-binding peptide fragments and proteoglycan-binding peptide fragments.

[0035] In this embodiment, the polypeptide-type small molecule adhesive comprises two parts: a hyaluronic acid-binding peptide fragment and a proteoglycan-binding peptide fragment. This polypeptide-type small molecule adhesive can penetrate the skin and bind hyaluronic acid and proteoglycan through the hyaluronic acid-binding peptide fragment and the proteoglycan-binding peptide fragment, thereby achieving a skin-tightening effect.

[0036] In related technologies, the hyaluronic acid-binding peptide fragment and proteoglycan-binding peptide fragment in the polypeptide-type small molecule adhesive of this application are relatively short, with a small total molecular weight (only about 2 kDa), resulting in high skin penetration efficiency and stronger efficacy. Furthermore, the selection of the hyaluronic acid-binding peptide fragment and proteoglycan-binding peptide fragment affects the efficacy of the polypeptide-type small molecule adhesive. This application has carefully selected the peptide sequence, making this polypeptide-type small molecule adhesive highly effective at binding hyaluronic acid and proteoglycan, resulting in a very powerful anti-aging effect that is significantly superior to other designs.

[0037] There are multiple peptide options available for both hyaluronic acid-binding peptide fragments and proteoglycan-binding peptide fragments. This application selects fragments with smaller molecular weights, which is beneficial for transdermal absorption. Additionally, this application selects hyaluronic acid-binding peptide fragments ending in R and lacking K or R in other parts of the sequence, and proteoglycan-binding peptide fragments lacking K or R in the sequence. This design results in small peptide gels ending in R and lacking K or R in other parts. This design is suitable for multi-copy tandem expression, using low-cost trypsin to digest into several individual small peptide gels. Multi-copy tandem expression is beneficial for increasing expression levels. Fragments ending in R and lacking K or R in other parts are suitable for multi-copy tandem expression followed by digestion into several individual small peptide gels using low-cost trypsin.

[0038] In some alternative embodiments, the polypeptide-type small molecule gel includes, but is not limited to, the amino acid sequence shown in any one of SEQ ID NO. 1-3.

[0039] In some optional embodiments, the polypeptide-type small molecule gel comprises, but is not limited to, an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence described in any one of SEQ ID NO. 1-3.

[0040] Nucleic acid molecules

[0041] Secondly, embodiments of this application provide a nucleic acid molecule that encodes a polypeptide-type small molecule gel, as described in the first aspect.

[0042] In some alternative embodiments, the nucleic acid molecule includes, but is not limited to, the nucleotide sequence shown in any one of SEQ ID NO. 4-6 and its tandem nucleic acid molecule.

[0043] The nucleotide sequences shown in SEQ ID NO. 4-6 are derived by converting the amino acid sequences shown in SEQ ID NO. 1-3, respectively. The amino acid sequences shown in SEQ ID NO. 1-3 were first converted into nucleotide sequences using online amino acid-to-DNA conversion software (https: / / www.ebi.ac.uk / jdispatcher / st / emboss_backtranseq). Then, the nucleic acid sequences were optimized in Pichia pastoris using online gene sequence optimization software (https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html), avoiding the restriction enzyme sites at the multiple cloning site of the Pichia pastoris expression vector and the restriction enzyme sites used for linearization. The optimized nucleotide sequences were then tandemly converted approximately 20 times and synthesized into tandem nucleic acid molecules by Qingke Biotechnology Shanghai Branch.

[0044] In this application, the specific methods for avoiding restriction enzyme sites include: EcoR1, Not1, SnaB1, Avr11, and SacI when using the pPIC9K vector; EcoR1, Not1, Kpn1, Sac11, Xba1, Sal1, and SacI when using the pPICZαA vector; and EcoR1, Not1, Kpn1, Sac11, Xba1, and Avr II when using the pGAPZαA vector. Preferably, when using the pPIC9K, pPICZαA, or pGAPZαA vector, the restriction enzyme sites EcoR1, Not1, SacI, and Avr II must be avoided. Preferably, the vector is pGAPZαA.

[0045] Recombinant plasmid

[0046] Thirdly, embodiments of this application provide a recombinant plasmid containing nucleic acid molecules or tandem nucleic acid molecules as described in the second aspect.

[0047] In some embodiments, a method for preparing the above-mentioned recombinant plasmid is provided, comprising:

[0048] The second aspect involves ligating the tandem nucleic acid molecule with the expression vector after enzyme digestion to obtain the ligation product. The expression vector can be pPIC9K, pPICZαA, or pGAPZαA, preferably pGAPZαA.

[0049] The recombinant product was transformed into competent E. coli cells, and positive clones were screened out using a culture medium containing the corresponding antibiotics.

[0050] Positive clones of E. coli competent cells were cultured, and plasmids were extracted from them.

[0051] In some embodiments, the antibiotics include one or both of ampicillin and bleomycin.

[0052] In some embodiments, a ligase is used to ligate the gene fragment with the Pichia pastoris expression vector.

[0053] In some embodiments, when using the pPIC9K or pPICZαA expression vector, Sac1 linearization is used.

[0054] In some embodiments, when using the pGAPZαA vector, Avr II linearization is used.

[0055] Transformation

[0056] Fourthly, embodiments of this application provide a transformant, which includes a polypeptide-type small molecule gel (as described in the first aspect), a nucleic acid molecule (as described in the second aspect), or a recombinant plasmid (as described in the third aspect).

[0057] Targeted anti-aging agents

[0058] Fifthly, embodiments of this application provide a targeted anti-aging agent, including one obtained by cultivating a transformation system as described in the fourth aspect.

[0059] In some embodiments, the targeted anti-aging agent includes at least one of the following: a transformant, a lysate, a fermentation broth, a fermentation broth extract, and a culture obtained through the transformant.

[0060] Sixthly, embodiments of this application provide the application of the polypeptide-type small molecule gel described in the first aspect or the targeted anti-aging agent of the fifth aspect in the preparation of cosmetics with skin anti-aging effects. As an example, the polypeptide-type small molecule gel provided in the embodiments of this application is prepared into a polypeptide-type small molecule gel freeze-dried powder using freeze-drying technology. 0.01-5 parts by weight of the above-mentioned polypeptide-type small molecule gel freeze-dried powder are added to 100 parts of cosmetic liquid or emulsion to obtain a cosmetic with targeted anti-aging effects.

[0061] Example

[0062] The following embodiments describe the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on weight, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0063] The DMEM cell culture medium used in this application was purchased from Gibco's DMEM, high glucose, pyruvate medium, catalog number: 11995065.

[0064] The main components and concentrations of BMM medium are as follows: 100mM, pH 6.0, 1.34% potassium phosphate, 4×10-5% YNB, 0.5% biotin, and 0.5% methanol.

[0065] The main components and concentrations of BMGY medium are as follows: 2% (w / v) peptone; 1.34% (w / v) yeast nitrogen source; 1% (w / v) yeast extract; 1% (v / v) glycerol; 100 mM phosphate buffer (pH 6.0).

[0066] Example 1: A polypeptide-type small molecule gel and its preparation method

[0067] (1) Design the amino acid sequence of the polypeptide-type small molecule glue, wherein the amino acid sequence of the polypeptide-type small molecule glue is a sequence of hyaluronic acid-binding peptide fragment and proteoglycan-binding peptide fragment, as shown in SEQ ID NO.1.

[0068] (2) The amino acid sequence shown in SEQ ID NO.1 was converted into a nucleotide sequence using online amino acid-to-DNA conversion software (https: / / www.ebi.ac.uk / jdispatcher / st / emboss_backtranseq). The nucleic acid sequence was then optimized in Pichia pastoris using online gene sequence optimization software (https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html), avoiding the restriction enzyme sites EcoR1, Not1, Kpn1, Sac11, Xba1 at the multiple cloning site of the Pichia pastoris expression vector and the Avr II restriction enzyme site used for linearization. The nucleotide sequence shown in SEQ ID NO.4 was obtained.

[0069] (3) The nucleotide sequence shown in SEQ ID NO.4 is concatenated 20 times and named fzj1.

[0070] (3) The tandem nucleic acid molecule fzj1 sequence was sent to Qingke Biotechnology Shanghai Branch for synthesis. This nucleotide fragment was cloned into the pUC57 vector by Qingke Biotechnology Shanghai Branch and named pUC57-fzj1;

[0071] (4) pUC57-fzj1 and the vector pGAPZαA from Shanghai Qingke Biotechnology Co., Ltd. were digested with EcoRI and Not1. The digestion system is as follows:

[0072]

[0073] (5) The fzj1 fragment and the digested vector pGAPZαA were recovered using the Tiangen fragment recovery kit;

[0074] (6) The fzj1 fragment and the enzyme-digested vector pGAPZα were ligated together, and the ligation system was as follows:

[0075] (7) The ligation product was transformed into Escherichia coli Top10 competent cells (from Shanghai Qingke Biotechnology Co., Ltd.);

[0076] (8) Spread bleomycin on a plate containing 25 μg / mL. The culture medium in the plate is LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar.

[0077] (9) PCR detection of positive clones: the PCR products of positive clones can show specific bright bands during electrophoresis.

[0078] (10) The cloned product was cultured and the plasmid was extracted using a plasmid mini-prep kit to obtain the target plasmid p1-fzj1.

[0079] (11) All plasmids were linearized by Avr II restriction enzyme digestion. The enzyme digestion system is as follows:

[0080]

[0081] (12) The enzyme digestion product was run on 1% agarose agarose gel and a specific bright band was obtained. The band position was different from that of the non-enzyme digestion control, thus confirming that the plasmid was completely linearized.

[0082] (13) The linearized plasmid was electroporated into Pichia pastoris SMD1168H competent cells, cultured in a shaker at 220 rpm for 1.5 h, and collected by centrifugation at 6000 rpm for 3 min.

[0083] (14) The collected bacterial cells were screened through a plate containing 100 μg / mL bleomycin to identify Pichia pastoris k1-p1-fzj1 containing the fzj1 fragment;

[0084] (15) Transplant the engineered Pichia pastoris strain k1-p1-fzj1 into a 50 mL centrifuge tube containing 15 mL of BMGY medium with a breathable plug, and culture it in a shaker at 28 ° C and 220 rpm until OD600 = 4.

[0085] (16) Centrifuge at 6000 rpm for 3 min to collect bacterial cells;

[0086] (17) The bacterial cells were suspended in BMM medium and cultured on a shaker at 220 rpm for 4 days. The bacterial cells were then removed by centrifugation.

[0087] (18) A membrane filtration device was used to filter the supernatant using a 10 kDa standard membrane to obtain the retentate;

[0088] (19) A membrane filtration device was used to filter the retentate using a 100 kDa standard membrane to obtain the permeate;

[0089] (20) Trypsin digestion permeate;

[0090] (21) Use a 100 kDa standard membrane to filter the enzyme digestion solution to remove the enzyme, and recover the filtrate;

[0091] (22) Freeze-dry to obtain freeze-dried powder of polypeptide small molecule gel.

[0092] Example 2: A polypeptide-type small molecule gel and its preparation method

[0093] The difference between this embodiment and Embodiment 1 is that the amino acid sequence of the polypeptide-type small molecule gel is shown in SEQ ID NO.2. The nucleotide sequence encoding the polypeptide-type small molecule gel is shown in SEQ ID NO.5.

[0094] Example 3: A polypeptide-type small molecule gel and its preparation method

[0095] The difference between this embodiment and Example 1 is that the amino acid sequence of the polypeptide-type small molecule gel is shown in SEQ ID NO.3. The nucleotide sequence encoding the polypeptide-type small molecule gel is shown in SEQ ID NO.6.

[0096] Comparative Example 1: A macromolecular human HAPLN1 protein and its preparation method. This comparative example differs from Example 1 in that it directly expresses a macromolecular human HAPLN1 protein. The GenBank accession number for the human HAPLN1 protein is KAI2538140.1. Its amino acid and nucleotide sequences can be found in GenBank.

[0097] Comparative Example 2: A Long Peptide Molecular Gel and Its Preparation Method

[0098] The difference between this comparative example and Example 1 is that the selected hyaluronic acid-binding peptide and proteoglycan-binding peptide are longer polypeptides with much larger molecular weights than the small molecule gels used in the example, but smaller than the human HAPLN1 protein. Because the molecular weights are much larger than the small molecule gels, single-copy expression was used instead of tandem expression, resulting in a relatively lower expression level. The amino acid sequence of the fusion of the single-copy long peptide with the hyaluronic acid-binding peptide and the proteoglycan-binding peptide is shown in SEQ ID NO.7. The corresponding nucleotide sequence is shown in SEQ ID NO.8.

[0099] Comparative Example 3: A Non-Preferred Fragment Molecular Gel and Its Preparation Method

[0100] The difference between this comparative example and Example 1 is that the selected hyaluronic acid-binding peptide fragment and proteoglycan-binding peptide fragment are not preferred fragments; they are not peptide fragments with strong binding ability, but rather peptide fragments with weak binding ability. The amino acid sequence of the weakly binding fusion non-preferred hyaluronic acid-binding peptide fragment-non-preferred proteoglycan-binding peptide fragment is shown in SEQ ID NO. 9. The corresponding nucleotide sequence is shown in SEQ ID NO. 10.

[0101] Comparative Example 4: A simple anti-aging peptide and its preparation method

[0102] The difference between this comparative example and Example 1 is that it expresses a simple anti-aging peptide, not a molecular gel. The anti-aging peptide is a part of collagen peptides, and its amino acid sequence is shown in SEQ ID NO. 11. The corresponding nucleotide sequence is shown in SEQ ID NO. 12.

[0103] Test section

[0104] I. Collagen I expression level:

[0105] The testing steps include: 1. 3T3 cell resuscitation (our laboratory): cells are resuscitated in DMEM cell culture medium containing 10% FBS under culture conditions of 5% CO2 concentration, 37°C, and saturated humidity.

[0106] 2. Digest cells with 0.25% trypsin, and seed cells in 24-well plates at a rate of 1.6 × 10⁴ cells / well using DMEM containing 10% FBS as the culture medium, for a total volume of 0.5 ml. Incubate at 37°C for 24 h. 3. Remove the cell culture medium from the cell culture plates and, according to the experimental grouping system: negative control group (DMEM), model damage group (IL-1β + DMEM), and sample group (sample + IL-1β + DMEM), add 100 μL / well of fresh medium at a 100 μM sample concentration. Incubate at 37°C in a 5% CO₂ incubator for another 24 h.

[0107] 4. Extract RNA;

[0108] 5. qPCR was used to detect the expression level of collagen I;

[0109] Table 1: Expression level of collagen I

[0110]

[0111] Experimental results: As shown in Table 1, the expression levels of collagen I are plotted as bars. Figure 1 Table 1 shows the mean values ​​of three parallel experiments. The experimental results indicate that Examples 1-3 can significantly improve the expression level of collagen I, and are significantly better than Comparative Examples 1-4.

[0112] II. Transdermal Transmission Rate

[0113] 1. Select mice that have been fed for 1 week to prepare isolated mouse skin. Remove the hair on the back with a 6% sodium sulfide solution and euthanize the mice by removing the cervical vertebrae.

[0114] 2. Take a small piece of mouse skin, remove the subcutaneous fat, and fix it between the donor and recipient pools with the surface facing upwards.

[0115] 3. A diffusion cell was used, with physiological saline containing 0.5% sodium azide as the receiving solution. Transdermal absorption experiments were conducted at 32℃ and 600 r / min. The transdermal area was 2 cm², and the receiving solution volume was 15 mL.

[0116] 4. Prepare a 1% solution of the control (oligopeptide-1) and the sample using the acceptor solution.

[0117] 5. Take 1 mL of the prepared solution and add it to the supply tank. After 24 hours of transdermal treatment, take 1 mL of the receiving solution and make up the difference with fresh receiving solution.

[0118] 6. Determine the content of permeable substances in the receiving liquid and calculate the transmittance. The transmittance is the amount permeated divided by the original total amount.

[0119] Table 2: Transdermal Transfusion Rate

[0120]

[0121]

[0122] Experimental results: As shown in Table 2, the transdermal penetration rate (%) results are plotted as bars. Figure 2 Table 2 shows the mean values ​​of three parallel experiments. The experimental results indicate that Examples 1-3 are easily transdermal, comparable to the control, and significantly superior to Comparative Examples 1-4.

[0123] The above experimental results demonstrate that the polypeptide-type small molecule gel prepared in this application has no obvious toxicity, significantly improves the expression level of collagen I, and is easily transdermal. This application provides a polypeptide-type small molecule gel and its application in targeted anti-aging.

[0124] The polypeptide-type small molecule gel of this application provides a polypeptide that achieves anti-aging ability by replacing the HAPLN1 protein and binding hyaluronic acid and proteoglycans. It comprises two parts: a hyaluronic acid-binding peptide fragment and a proteoglycan-binding peptide fragment. Furthermore, through optimization of the sequences of the hyaluronic acid-binding peptide fragment and the proteoglycan-binding peptide fragment, it was found that the polypeptide-type small molecule gels of Examples 1-3 exhibit significantly enhanced anti-aging efficacy compared to other sequences. This is because the optimized hyaluronic acid-binding peptide fragment and the proteoglycan-binding peptide fragment have smaller molecular weights and stronger binding capacity to hyaluronic acid and proteoglycans.

[0125] The peptide-type small molecule adhesive of this application is the only known pure peptide-type small molecule adhesive that targets and binds hyaluronic acid and proteoglycans, setting a precedent for green, safe, targeted anti-aging, and precise aesthetics using peptide-type molecular adhesives.

[0126] It should also be noted that the exemplary embodiments mentioned in this application describe methods or systems based on a series of steps or apparatus. However, this application is not limited to the order of the above steps; that is, the steps can be performed in the order mentioned in the embodiments, or in a different order, or several steps can be performed simultaneously.

[0127] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A polypeptide-type small molecule gel, characterized in that, The polypeptide-type small molecule gel includes hyaluronic acid-binding peptide fragments and proteoglycan-binding peptide fragments.

2. The polypeptide-type small molecule gel according to claim 1, characterized in that, The polypeptide-type small molecule gel includes the amino acid sequence shown in any one of SEQ ID NO.1-3.

3. The polypeptide-type small molecule gel according to claim 2, characterized in that, The polypeptide-type small molecule gel contains an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence described in any one of SEQ ID NO. 1-3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide-type small molecule gel according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule comprises any one of the nucleotide sequences shown in SEQ ID NO. 4-6 and tandem nucleic acid molecules formed therefrom.

6. A recombinant plasmid, characterized in that, The recombinant plasmid contains the nucleic acid molecule as described in claim 4 or 5.

7. A transformant, characterized in that, The transformant comprises the polypeptide-type small molecule gel according to any one of claims 1-3, the nucleic acid molecule according to claim 4 or 5, or the recombinant plasmid according to claim 6.

8. A targeted anti-aging agent, characterized in that, This includes obtaining it through the transformation system described in claim 7.

9. The targeted anti-aging agent according to claim 8, characterized in that, The targeted anti-aging agent includes at least one of the following: a transformant, a lysate, a fermentation broth, a fermentation broth extract, and a culture obtained through the transformant.

10. The use of a polypeptide-type small molecule gel as described in any one of claims 1-3 or a targeted anti-aging agent as described in claim 8 or 9 in the preparation of cosmetics with skin anti-aging effects.

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