Umbilical cord stem cell and application thereof in preparation of anti-aging drugs and medical beauty products

Abstract

The invention discloses engineered umbilical cord stem cells and application thereof in preparation of anti-aging drugs and medical beauty products. A fusion polypeptide TAT-SIRT1-SOD (SEQ ID NO: 1) and an anti-p16INK4a monoclonal antibody (mAb-p16) are designed to synergistically modify umbilical cord stem cells, and a 3D dynamic culture technology is combined to construct an engineered cell system with an efficient anti-aging function. The fusion polypeptide mediates transmembrane through TAT cell-penetrating peptide, activates SIRT1 and removes mitochondrial ROS; the mAb-p16 is specifically combined with p16INK4a on the surface of the senescent cell, and the senescent cell is eliminated through an ADCC effect. Experiments show that the engineered umbilical cord stem cell can increase the senescent cell clearance rate by 86.1%, increase the SOD activity by 300%, significantly inhibit secretion of inflammatory factors IL-6, increase the skin collagen density by 148% and reduce the wrinkle depth by 67% in an animal model. The invention provides a novel and efficient technical scheme for anti-aging medicines and medical beauty products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and regenerative medicine, and specifically relates to umbilical cord stem cells and their application in the preparation of anti-aging drugs and medical beauty products. Background Art

[0002] Umbilical cord stem cells (UC-MSCs), with their high proliferation capacity, low immunogenicity, and multidirectional differentiation potential, have shown great potential in the anti-aging field and have become a core raw material for related research and product development. However, current anti-aging application technologies for UC-MSCs still have significant limitations, hindering further development in this field.

[0003] In terms of stem cell culture in vitro, traditional UC-MSCs will show obvious signs of aging when they are subcultured to P5 or above. Studies have shown that as the number of subcultures increases, the cell proliferation rate slows down, the activity of β-galactosidase increases significantly, and its secretome function also decreases, which greatly reduces the efficacy of the transplanted cells in the body. For example, in patent CN117797174A, cell aging is delayed only by adding common activating drugs such as growth factors. This method cannot fundamentally solve the problems of oxidative stress and accumulated DNA damage faced by cells during the culture process, and it is difficult to maintain the long-term stable activity and function of the cells.

[0004] While research has attempted improvements in exosome delivery, such as patent CN119033668A, which encapsulates UC-MSC exosomes within PLGA nanoparticles, improving exosome stability to some extent, the key issue of exosome targeting remains unresolved. Furthermore, this technology fails to address the need to eliminate the aging microenvironment, making it difficult for exosomes to precisely target the target area when exerting their anti-aging effects, reducing their overall effectiveness.

[0005] At the antibody application level, the current application scenarios of related technologies are relatively single. Although patent CN119074916A combines UC-MSCs with monoclonal antibodies (anti-PAFAH1B3) for use in the anti-cancer field, it does not involve anti-aging scenarios; patent CN115073605B uses anti-COX-2 monoclonal antibodies to treat photoaging, but does not achieve synergistic effects with stem cells, and cannot fully exert the combined advantages of the two in anti-aging. It can be seen that the existing technology has many problems that need to be solved in the anti-aging application of umbilical cord stem cells, and it is urgent to develop new technical solutions to break through these bottlenecks. Summary of the Invention

[0006] The purpose of the present invention is to provide a modified umbilical cord stem cell that significantly enhances its anti-aging and medical aesthetics efficacy by introducing specific fusion polypeptides and monoclonal antibodies, thereby providing a new technical solution for the preparation of highly effective anti-aging drugs and medical aesthetics products.

[0007] Therefore, on the one hand, the present invention discloses a fusion polypeptide TAT-SIRT1-SOD, which is delivered across the membrane by TAT transmembrane peptide (YGRKKRRQRRR), connecting the SIRT1 activation domain (CRARLSSHLQE) and the SOD mimetic enzyme (DLGVTKIEREE), and maintaining the functional domain independence through a flexible connecting peptide (GGGGS). It can penetrate the cell membrane to activate SIRT1 deacetylase and clear mitochondrial ROS, reducing DNA damage. The amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown in SEQ ID NO: 1.

[0008] The present invention also discloses an anti-p16INK4a monoclonal antibody mAb-p16, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody mAb-p16 is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; the monoclonal antibody mAb-p16 specifically binds to the amino acid sequence at positions 89 to 108 of the human p16INK4a protein, wherein the amino acid sequence at positions 89 to 108 of the human p16INK4a protein is SWTRWWCCTGPGRGWTCAM.

[0009] The present invention also discloses a method for preparing engineered umbilical cord stem cells, which comprises the following steps:

[0010] (1) Isolation and culture of umbilical cord mesenchymal stem cells: UC-MSCs were isolated from the umbilical cord Wharton's jelly using collagenase digestion. Flow cytometry analysis showed that the positive rates of CD73, CD90, and CD105 were >95%.

[0011] (2) The monoclonal antibody mAb-p16 was coupled to the microcarrier surface through the PNIPAM thermosensitive material to form a monoclonal antibody mAb-p16 modified thermosensitive microcarrier, wherein the molar ratio of the monoclonal antibody mAb-p16 to the PNIPAM was 1:5; thereby achieving the anchoring of the monoclonal antibody mAb-p16 on the cell surface;

[0012] (3) culturing stem cells in a fusion polypeptide TAT-SIRT1-SOD enhanced medium, wherein the amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown in SEQ ID NO: 1; thereby achieving intracellular delivery of the fusion polypeptide TAT-SIRT1-SOD;

[0013] (4) 3D dynamic culture was performed in a rotating bioreactor with a working volume of 50 mL, a density of 5 g / L of thermosensitive microcarriers modified with monoclonal antibody mAb-p16, a rotation speed of 25 rpm, a temperature of 37°C, and a 5% CO2 infusion rate of 50 mL / min. During the culture, the cells were first statically inoculated for 2 hours, and the rotational culture was started after the attachment rate was greater than 80%. 1 / 2 volume of the fusion polypeptide TAT-SIRT1-SOD enhanced culture medium was replaced every 3 days.

[0014] Preferably, the fusion polypeptide TAT-SIRT1-SOD enhanced culture medium of the present invention is a basal culture medium containing the fusion polypeptide TAT-SIRT1-SOD at a final concentration of 20 μM, wherein the basal culture medium is DMEM / F12+10% FBS.

[0015] The present invention also discloses an anti-aging pharmaceutical preparation, which comprises the engineered umbilical cord stem cells or exosomes thereof prepared according to the method, and a pharmaceutically acceptable carrier.

[0016] Preferably, the preparation of the present invention is an injection, wherein the injection comprises the following components: 1×10 7 engineered umbilical cord stem cells, 0.5 mg / mL monoclonal antibody mAb-p16, 20 μM fusion peptide TAT-SIRT1-SOD and normal saline.

[0017] Preferably, the preparation of the present invention is an exosome anti-wrinkle essence for medical aesthetics, and the exosome anti-wrinkle essence for medical aesthetics comprises the following components:

[0018] (1) Exosomes extracted from engineered umbilical cord stem cells prepared according to the method of claim 3, wherein the exosome concentration is 1×10 10 particles / mL;

[0019] (2) HA-PLA nanocarriers, which self-assembled with exosomes at a mass ratio of 1:5 at 4°C to form a stable structure with an average particle size of 152 ± 8 nm and a potential of -25 ± 3 mV;

[0020] (3) 1 mg / mL monoclonal antibody mAb-p16;

[0021] (4) 5% trehalose;

[0022] (5) 0.5% phenoxyethanol;

[0023] (6) Deionized water is added to 100%.

[0024] The present invention also discloses an engineered umbilical cord stem cell freezing solution, which contains 90% fetal bovine serum, 10% DMSO and 5 μg / mL monoclonal antibody mAb-p16.

[0025] The engineered umbilical cord stem cells prepared by the present invention through antibody-peptide synergistic modification combined with 3D culture exhibit significant multi-dimensional and synergistic beneficial effects. At the cellular level, the TAT-SIRT1-SOD peptide can penetrate the cell membrane to activate SIRT1 deacetylase and clear mitochondrial ROS, reducing DNA damage and effectively delaying cell aging. The mAb-p16C antibody specifically recognizes p16INK4a on the surface of senescent cells and efficiently eliminates senescent cells through the ADCC effect. These two antibodies work synergistically with the low-shear microenvironment provided by 3D culture to increase the clearance rate of senescent cells by 73.3%, increase cell proliferation capacity by 138.5% compared to conventional culture, and increase exosome production by 3.2 times. In terms of application effect, the prepared anti-aging injection preparation has been preclinically verified to significantly reduce the levels of IL-6 and MDA in the blood of aging mice, increase SOD activity, and reduce the proportion of senescent cells in liver tissue. Clinical testing of the exosome anti-wrinkle essence has shown that it can increase the skin elasticity of subjects by 47.6% and reduce the melanin index by 60%. Furthermore, the optimized cryopreservation system increased cell viability by 13.6% and functional retention to over 95%. As summarized in this review, engineered umbilical cord stem cells and their exosomes can be developed into various forms, including injectable formulations and skincare products, offering both safe and effective solutions for aging-related diseases and medical aesthetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 SDS-PAGE detection results of anti-p16INK4a monoclonal antibody (mAb-p16).

[0027] Figure 2 Western blot results of different cells. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Example 1: Synthesis of fusion polypeptide TAT-SIRT1-SOD

[0031] 1. Fusion polypeptide structure design: The amino acid sequence of TAT-SIRT1-SOD (SEQ ID NO: 1) is: YGRKKRRQRRR-GGGGS-CRARLSSHLQE-GGGGS-DLGVTKIEREE; wherein:

[0032] (1) TAT transmembrane peptide (positions 1-11): YGRKKRRQRRR, mediates transmembrane delivery;

[0033] (2) Connector peptide (positions 12-16): flexible linker GGGGS, maintaining the independence of functional domains;

[0034] (3) SIRT1 activation domain (positions 17-27): CRARLSSHLQE;

[0035] (4) Connector peptide (positions 28-32): GGGGS;

[0036] (5) SOD mimetic enzyme (positions 33-43): DLGVTKIEREE, mimicking the active center structure of Cu / Zn-SOD.

[0037] 2. Synthesis of fusion polypeptide: TAT-SIRT1-SOD (SEQ ID NO: 1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The purity was >98% as determined by HPLC.

[0038] 3. Mechanism of action: TAT-SIRT1-SOD penetrates the cell membrane and activates SIRT1 deacetylase, while simultaneously clearing mitochondrial ROS and reducing DNA damage.

[0039] Example 2: Preparation and testing of anti-p16INK4a monoclonal antibody (mAb-p16)

[0040] 1. Antibody Design and Preparation

[0041] 1.1 Antigen design and synthesis

[0042] Based on the amino acid sequence (SWTRWWCCTGPGRGWTCAM) of amino acids 89-108 of the human p16INK4a protein (UniProtKB: R9S252), the hydrophilicity index and surface accessibility of this region were analyzed using Antigenic Prediction software and confirmed as a dominant antigenic epitope. This region was synthesized using the Fmoc solid-phase synthesis method (Shanghai Sangon Biotech Co., Ltd.), with a purity of >98% as determined by HPLC.

[0043] 1.2 Phage display screening

[0044] The storage capacity is 2.5×10 10The humanized scFv phage display library of CFU was screened in three rounds using a biopanning strategy:

[0045] Round 1: Maxisorp plates were coated with 10 μg / mL antigen, blocked with 1% BSA, and then the library was added. The plates were incubated at 4°C for 2 hours, eluted with 0.1 M Glycine-HCl (pH 2.2), and the eluate was infected with ER2738 bacteria, with an enrichment factor of 5.2.

[0046] Round 2: 5 μg / mL antigen coating, competitive elution conditions remain unchanged, and the enrichment factor increases to 12.8;

[0047] Round 3: 1 μg / mL antigen coating, 100-fold excess free antigen was added for competitive elution, and the enrichment factor reached 47.3.

[0048] After screening, positive clones were initially screened by ELISA, and the clones with the highest affinity were selected for sequencing to obtain the heavy and light chain variable region sequences. After humanization optimization, the affinity was tested using Biacore T200. The antigen was immobilized on a CM5 chip (coupling density 1000RU), and the antibody concentration gradient was 0.3125-20nM. The flow rate was 30μL / min at 37°C, and the KD value was measured to be 1.2×10 -9 M, compared with commercial antibody (ab270058) (KD = 2.3 × 10 -8 M) increased by 19.2 times.

[0049] 1.3 Antibody expression and purification

[0050] The humanized optimized heavy chain variable region (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 3) were cloned into the pFUSE-CHIg-hG1 and pFUSE2-CLIg-hk vectors, respectively. After verification by Xho I / EcoR I double enzyme digestion, the heavy chain and light chain expression cassettes were connected through the IRES sequence to construct a bicistronic vector and transfected into CHO-S cells (electroporation parameters: 150V, 10ms, 3 pulses). Dual resistance screening was performed using 800μg / mL G418 and 200μg / mL Zeocin, and monoclonal cell lines were obtained by limiting dilution. Fed-batch culture was carried out in serum-free medium CD-CHO, and the cell density reached 12-15×10 on day 14. 6cells / mL, and the antibody yield was 350-420 mg / L. The purification process was as follows: MabSelect SuRe ProteinA column (column volume 10 mL, flow rate 5 mL / min, eluent 0.1 M Glycine-HCl pH 3.0) → SP Sepharose FF cation exchange column (equilibration solution 20 mM PB pH 6.0, eluent 20 mM PB + 1 M NaCl pH 6.0) → Q Sepharose FF anion exchange column (equilibration solution 20 mM Tris-HCl pH 8.0, eluent 20 mM Tris-HCl + 1 M NaCl pH 8.0). The final antibody purity was >95% (SDS-PAGE, e.g. Figure 1 ), endotoxin <0.1EU / μg.

[0051] 2. Monoclonal Antibody Testing

[0052] 2.1 Antigen binding specificity verification

[0053] Western blot: Take H2O2-induced senescent fibroblasts (SA-β-gal positive rate > 80%) and young fibroblast lysates (20 μg each loading), the primary antibody is mAb-p16 or commercial antibody (ab270058) diluted 1:1000, and the secondary antibody is HRP-labeled goat anti-human IgG or goat anti-rabbit IgG (1:5000). The results showed that mAb-p16 only detected a 16kDa specific band (gray value 1256±112) in senescent cells, and there was no signal in young cells; the commercial antibody (ab270058) had a band gray value of 873±95 in senescent cells, and non-specific bands appeared in young cells (gray value 321±45). Figure 2 shown.

[0054] 2.2 ADCC assay

[0055] The LDH release method was used, and the effector cells were human peripheral blood NK cells (isolated from healthy volunteers, flow cytometry sorted CD56 + The target cells were senescent fibroblasts (E:T = 20:1). After incubation for 4 hours with different antibody concentrations, the supernatant was centrifuged and assayed for LDH activity. The results showed that 1 μg / mL mAb-p16 induced 35±4% cytotoxicity, while the commercial antibody (ab270058) induced 18±3%. At 10 μg / mL, mAb-p16 reached 68±5%, a 61.9% increase compared to (ab270058) (42±4%). At 50 μg / mL, mAb-p16 cytotoxicity reached a plateau (72±6%), while the cytotoxicity of the control IgG1 was less than 5%.

[0056] 2.3 Cell apoptosis induction experiment

[0057] Senescent fibroblasts were treated with 10 μg / mL of the antibody for 24 hours, followed by Annexin V / PI double staining and flow cytometry analysis. The apoptosis rate in the mAb-p16 group was 42±6% (early apoptosis 28±5%, late apoptosis 14±3%), compared to 28±5% in the commercial antibody (ab270058) group. Caspase-3 / 7 activity assays revealed a 65.6% increase in fluorescence intensity (5.3±0.8) in the mAb-p16 group compared to 3.2±0.6 in the ab270058 group (substrate: Z-DEVD-R110, incubation at 37°C for 1 hour, excitation / emission wavelengths: 485 / 530 nm).

[0058] 2.4 Tissue Penetration Assessment

[0059] In a 3D skin model (comprising keratinocytes and dermal fibroblasts, cultured for 14 days to form a complete epidermal-dermal structure), 10 μg / mL of fluorescently labeled antibody (AlexaFluor 488) was added and incubated for 24 hours. Immunofluorescence analysis of frozen sections revealed that mAb-p16 had an epidermal penetration rate of 85±7% and a dermal penetration depth of 120±15 μm. Compared to a commercial antibody (ab270058), the epidermal penetration rate was 62±8% and the dermal penetration depth was 75±12 μm. This represents a 37% (epidermal) and 60% (dermal) improvement in the penetration ability of mAb-p16 compared to existing antibodies.

[0060] 3. Experimental Summary

[0061] In this example, the anti-p16INK4a monoclonal antibody mAb-p16 was successfully prepared through precise epitope design, high-affinity phage display screening, and efficient expression in CHO cells. Compared with the existing antibody (ab270058), mAb-p16 demonstrated significant advantages in key indicators such as KD value, antigen binding specificity, ADCC activity, and tissue penetration. In particular, mAb-p16 demonstrated a 19.2-fold increase in affinity, a 60-90% enhancement in cytotoxicity, and no nonspecific binding to young cells. This antibody specifically recognizes p16INK4a on the surface of senescent cells and efficiently eliminates senescent cells through ADCC, providing a novel targeting molecule for the development of anti-aging drugs and medical aesthetics products.

[0062] Example 3: Preparation of engineered umbilical cord stem cells

[0063] 1. Isolation and Primary Culture of UC-MSCs

[0064] 1.1 Umbilical cord tissue processing: The umbilical cord of a healthy, voluntary, full-term pregnant woman delivered by cesarean section (in accordance with ethical requirements) was placed in ice-bathed PBS containing 100 U / mL penicillin and 100 μg / mL streptomycin for transportation within 2 hours after delivery. The surface blood vessels of the umbilical cord were removed with a scalpel in a biosafety cabinet, and Wharton's jelly was cut into 1-2 mm 3 The tissue blocks were washed three times with PBS containing double antibodies, each time for 5 minutes.

[0065] 1.2 Enzyme digestion and cell separation, using a two-step digestion method:

[0066] (1) Step 1: Add 0.1% collagenase II and digest at 37°C for 60 min (120 rpm), with pipetting every 15 min.

[0067] (2) Step 2: Add 0.25% trypsin-EDTA and continue digestion at 37°C for 15 minutes. Add DMEM / F12 medium containing 10% fetal bovine serum to terminate the digestion.

[0068] (3) The digestion solution was filtered through a 100 μm cell sieve and centrifuged at 1200 rpm for 8 minutes. The supernatant was discarded and treated with red blood cell lysis buffer for 5 minutes. After centrifugation again, the cells were resuspended in culture medium.

[0069] 1.3 Primary culture and identification

[0070] (1) Culture conditions: inoculation density 1×10 6 cells / cm 2 The culture medium was DMEM / F12+10% FBS+1% double antibody, cultured at 37°C, 5% CO2, and the medium was changed half every 3 days;

[0071] (2) Cell identification: When passaged to P3, surface markers were detected by flow cytometry. The positive rates of CD73>95%, CD90>95%, CD105>90%, and CD34, CD45, and HLA-DR were all <2%;

[0072] 2. Antibody-peptide collaborative modification process

[0073] 2.1 Preparation of antibody-functionalized microcarriers

[0074] (1) Synthesis of PNIPAM thermosensitive material: PNIPAM (molecular weight 30 kDa) was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization and terminally modified with N-hydroxysuccinimide (NHS) active ester;

[0075] (2) Conjugation of mAb-p16 with PNIPAM: The reaction was carried out in PBS (pH 7.4) at a molar ratio of 1:5 for 2 h at room temperature. Sulfo-SMCC was used as a cross-linker (antibody to cross-linker molar ratio of 1:10). Free PNIPAM was removed by passing through a PD-10 column.

[0076] (3) Preparation of microcarrier coating: The amino-dextran microcarriers were incubated with the antibody-PNIPAM conjugate at a ratio of 1:10 (w / w) at 37°C for 4 h, washed three times with PBS, and the protein loading of the microcarriers was determined by the BCA method to be (23.5±2.1) μg / mg microcarrier.

[0077] 2.2 Preparation of Fusion Peptide TAT-SIRT1-SOD Enhanced Culture Medium

[0078] (1) TAT-SIRT1-SOD2 preparation: Dissolve the TAT-SIRT1-SOD2 prepared in Example 1 in sterile PBS to a 10 mM stock solution and store at -20°C;

[0079] (2) Culture medium addition: Dilute the mother solution to 20 μM before use and add it to the basal culture medium (DMEM / F12 + 10% FBS). Replace the culture medium every 48 hours and add fresh polypeptide at the same time.

[0080] 2.3 Verification of modification efficiency

[0081] (1) Flow cytometry: FITC-labeled mAb-p16 showed that 91 ± 3% of UC-MSCs surface bound to the antibody;

[0082] (2) Peptide internalization: TAT-SIRT1-SOD2 was labeled with AlexaFluor 555, and confocal microscopy showed that peptide fluorescence was present in 85±4% of cells, and 78±5% was localized in mitochondria.

[0083] 3.3D dynamic culture process

[0084] 3.1 Rotating bioreactor setup

[0085] (1) Reactor parameters: Synthecon RCCS-2 rotary bioreactor with a working volume of 50 mL and a microcarrier density of 5 g / L (approximately 1×10 6 cells / mL), rotation speed 25 rpm, temperature 37°C, 5% CO2 infusion rate 50 mL / min;

[0086] (2) Culture process: First, statically inoculate the cells for 2 hours, and start rotational culture after the attachment rate is greater than 80%. Replace 1 / 2 volume of fusion polypeptide TAT-SIRT1-SOD enhanced culture medium (basal culture medium containing a final concentration of 20 μM fusion polypeptide TAT-SIRT1-SOD, the basal culture medium is DMEM / F12 + 10% FBS) every 3 days.

[0087] 3.2 Cell growth and exosome detection

[0088] (1) Cell proliferation: After 7 days of culture, the cell density was determined by MTT assay and was (1.8±0.2)×10 6 cells / mL, compared with 2D culture (0.56±0.07)×10 6 cells / mL increased by 3.2 times;

[0089] (2) Exosome isolation: The culture supernatant was centrifuged at 300 × g for 10 minutes and 2000 × g for 20 minutes to remove cell debris, and then ultracentrifuged at 100,000 × g for 70 minutes, and the exosomes were resuspended in PBS;

[0090] (3) Exosome characterization (NTA assay): Exosome concentration in 3D culture group (5.8±0.6)×10 11 particles / mL, compared with 2D group (1.8±0.3)×10 11 Particles / mL increased by 3.2 times, and the particle size distribution of 100-150nm accounted for 72%;

[0091] 4. Cell Cryopreservation and Thawing

[0092] 4.1 Preparation of freezing solution (by volume): 90% FBS + 10% DMSO + 5 μg / mL mAb-p16, sterilize by filtration through a 0.22 μm filter membrane, and pre-cool at 4°C.

[0093] 4.2 Cryopreservation and thawing steps

[0094] (1) Cryopreservation: The 3D cultured microcarrier-cell complex was digested with 0.05% trypsin, centrifuged at 1000 rpm for 5 minutes, and resuspended in cryopreservation solution to (5-10) × 10 6 cells / mL, dispensed into cryovials, stored in a programmed cooling box (-80°C overnight) and then transferred to liquid nitrogen for storage;

[0095] (2) Recovery: Remove the cryovial from liquid nitrogen, thaw quickly in a 37°C water bath, dilute with 10-fold volume of culture medium containing 10% FBS, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in culture medium.

[0096] 4.3 Resuscitation effect detection

[0097] (1) Cell viability: Trypan blue staining showed that the cell viability after recovery was (92±3)%, which was 13.6% higher than that of the traditional cryopreservation medium (containing 10% DMSO + 90% FBS) (81±4);

[0098] (2) Functional retention: SIRT1 activity in revived cells was (2.1±0.2) nmol / min / mg protein, which was not significantly different from that before cryopreservation (2.3±0.3) nmol / min / mg protein.

[0099] (3) Antibody binding rate: Flow cytometry showed that the mAb-p16 binding rate was still (88±4)% after recovery.

[0100] 5. Experimental results and summary

[0101] 5.1 Key data comparison is shown in Table 1.

[0102] Table 1 Summary and comparison of experimental results

[0103] Detection indicators 2D culture group 3D culture group Improvement ratio Cell density (cells / mL) <![CDATA[0.56±0.07×10 6 ]]> <![CDATA[1.8±0.2×10 6 ]]> 3.2 times Exosome concentration (particles / mL) <![CDATA[1.8±0.3×10 11 ]]> <![CDATA[5.8±0.6×10 11 ]]> 3.2 times Cryopreservation and resuscitation survival rate 81±4％ 92±3％ 13.6％ <![CDATA[p16 + Senescent cell clearance rate]]> 45±5％ 78±6％ 73.3％

[0104] 5.2 Analysis of Technical Advantages

[0105] (1) Antibody-peptide synergistic modification: mAb-p16 is anchored to the stem cell surface through a PNIPAM thermosensitive coating, and the TAT-SIRT1-SOD2 peptide enhances the intracellular antioxidant capacity. The two synergistically increase the clearance rate of senescent cells by 73.3%;

[0106] (2) 3D dynamic culture: The rotating bioreactor provides a low shear force microenvironment, promoting cell-microcarrier adhesion (adhesion rate > 90%). The exosome production is significantly improved compared with 2D culture, and the expression of anti-aging related miRNAs (such as miR-21 and miR-146a) in exosomes increases by 2.5-3.0 times;

[0107] (3) Optimization of the freezing system: Adding mAb-p16 to the freezing solution can reduce ice crystal damage, maintain the antibody binding activity on the cell surface, and the cell function retention rate after recovery is over 95%.

[0108] Example 4: Verification of the anti-aging properties of engineered stem cells

[0109] 1. Experimental Design and Grouping

[0110] 1. Cell preparation: Prepare according to the method of Example 3.

[0111] 2. Grouping (n=6 replicates / group):

[0112] Group A: conventional UC-MSCs (unmodified), 2D cultured in DMEM / F12 + 10% FBS;

[0113] Group B: cells from group A + 20 μM TAT-SIRT1-SOD2 peptide, with daily medium changes;

[0114] Group C: cells from group A were seeded on mAb-p16C functionalized microcarriers (loading amount 23.5 ± 2.1 μg / mg) and cultured at 37°C;

[0115] Group D: treatment of group B+group C+culture in 3D rotating bioreactor (rotation speed 25 rpm, microcarrier density 5 g / L).

[0116] 3. Culture conditions: 37°C, 5% CO2 for 72 hours. Groups B and D were supplemented with 20 μM peptide every 24 hours. Groups A and C were cultured in DMEM / F12 medium containing 10% FBS.

[0117] 2. Testing indicators and methods

[0118] 1. β-Galactosidase (SA-β-gal) staining: Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and incubated in staining solution (containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg / mL X-gal) at 37°C for 16 h (in the absence of CO2). Five fields of view were randomly selected under an optical microscope (200×), and the percentage of SA-β-gal-positive cells was counted.

[0119] 2. SOD activity assay: Cell lysate (containing 1 mM PMSF) was ultrasonically disrupted in an ice bath (power 300 W, 3 × 10 s), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. The reaction system consisted of 50 μL sample + 250 μL working solution, incubated at 37°C for 20 min. The absorbance at 450 nm was measured, and the activity (U / mg protein) was calculated according to the standard curve.

[0120] 3. IL-6 secretion detection: Follow the instructions of the commercial ELISA detection kit.

[0121] 4. Cell proliferation rate determination: Follow the instructions of the CCK-8 detection kit.

[0122] 3. Experimental results are shown in Table 2.

[0123] 1. Senescent Cell Clearance Efficiency: The proportion of SA-β-gal-positive cells in Group D was only 5.3±0.9%, an 86.1% decrease compared to Group A, demonstrating that antibody-peptide synergistic modification combined with 3D culture can effectively eliminate senescent cells. Groups B (peptide treatment) and C (antibody treatment) showed reductions of 49.0% and 40.6%, respectively, confirming that both treatments independently can reduce senescent cells, but combined treatment exhibited a significant synergistic effect (p<0.01).

[0124] 2. Enhanced Antioxidant Capacity: Group D achieved SOD activity of 102.6±7.2 U / mg, a 300% increase compared to Group A, the highest among all groups. Group B saw a 148% increase in SOD activity due to direct supplementation of the SOD mimetic enzyme with the TAT-SIRT1-SOD2 peptide and activation of SIRT1. Group C saw a 21.4% increase in SOD activity due to the removal of senescent cells and the reduction of oxidative stressors. However, combined treatment exponentially enhanced antioxidant capacity.

[0125] 3. Inflammatory cytokine inhibition: IL-6 secretion in Group D was 45.3±4.2 pg / mL, a 78.5% decrease compared to Group A. In Group C, targeted elimination of senescent cells overexpressing p16INK4a using mAb-p16C reduced IL-6 by 53.1%. In Group B, peptides were used to inhibit mitochondrial ROS production, indirectly reducing inflammatory signaling and resulting in a 35.8% decrease in IL-6. This combined treatment further enhanced the anti-inflammatory effect.

[0126] 4. Cell Proliferation: The proliferation rate in Group D was OD450 = 1.24 ± 0.08, a 138.5% increase compared to Group A. The microcarrier adherent environment provided by 3D culture (a 63.5% increase in the proliferation rate in Group C) and the proliferation-promoting effect of the peptide (a 50.0% increase in Group B) synergistically promoted cell division. Flow cytometry showed that the proportion of cells in the S phase in Group D reached 32.7 ± 3.1%, significantly higher than the 14.5 ± 2.3% in Group A.

[0127] Table 2 Summary of experimental results

[0128]

[0129] *Compared with group A, p < 0.01 (One-way ANOVA, Tukey's post hoc test);

[0130] **Sample size: n = 3 independent experiments, 6 replicates per group, data expressed as Mean ± SD.

[0131] 4. Experimental Summary

[0132] This experiment confirmed that antibody-peptide synergistic modification combined with 3D culture can significantly improve the anti-aging properties of UC-MSCs from multiple dimensions. Among them, the TAT-SIRT1-SOD2 peptide delays cell senescence through the dual pathways of mitochondrial-targeted antioxidant and SIRT1 activation, the mAb-p16C antibody specifically eliminates senescent cells, and the 3D culture optimizes the cell microenvironment. The three form a synergistic anti-aging network of "prevention-elimination-proliferation". Experimental data show that group D (combined treatment group) performs best in four key indicators: aging markers (SA-β-gal), antioxidant enzyme activity (SOD), inflammatory factors (IL-6), and proliferation capacity, and is significantly better than single component treatment. Based on this, this engineered stem cell system has great application value and can be developed into injectable cell preparations to target the elimination of senescent cells and rebuild the tissue microenvironment; its exosome extracts with high SOD activity can be used in antioxidant formulas for skin care products; at the same time, its strong aging resistance also makes it suitable for cell cryopreservation, extending the activity retention time of stem cells after cryopreservation.

[0133] Example 5: Anti-skin aging effect in animal models

[0134] 1. Experimental Design and Methods

[0135] 1. Animal Model Construction

[0136] (1) Experimental animals: 6-week-old female C57BL / 6 mice, acclimated for 7 days.

[0137] (2) Photoaging induction:

[0138] Ultraviolet irradiation equipment: UVA (320-400nm, intensity 8mW / cm 2 )+UVB(280-320nm, intensity 0.3mW / cm 2 )Dual light source UV box.

[0139] Irradiation protocol: Irradiation was performed 5 days a week for 30 minutes per day for 8 weeks. The mice were shaved on the back before irradiation and were covered with eye protection during irradiation.

[0140] Successful modeling criteria: Skin histological examination showed disordered collagen fibers, broken elastic fibers, and wrinkle depth ≥100 μm.

[0141] 2. Grouping and processing

[0142] (1) Group setting (n=10 / group):

[0143] Control group: saline gel (gel matrix is ​​0.5% hyaluronic acid);

[0144] Group A: conventional UC-MSCs exosome gel (exosome concentration 1×10 10particles / g gel)

[0145] Group D: Engineered UC-MSCs (antibody-peptide synergistic modification + 3D culture) exosome gel (exosome concentration 1×10 10 particles / g gel)

[0146] (2) Administration: 0.1 g of gel was injected subcutaneously on the back once a week for 4 consecutive weeks.

[0147] 3. Detection indicators and methods

[0148] (1) Determination of skin water content: Before testing, mice were fasted but not watered for 12 hours. Three points were selected on the non-injected area of ​​the back for measurement, and the average value was taken.

[0149] (3) Collagen density detection (Masson staining): Dorsal skin tissue (5 mm × 5 mm) was obtained and fixed with 4% paraformaldehyde for 24 hours. The tissue was then embedded in paraffin and sectioned at 4 μm. The tissue was then stained with hematoxylin for 5 minutes, then with ponceau acid fuchsin for 10 minutes, then differentiated with phosphomolybdic acid solution for 5 minutes, and finally stained with aniline blue for 10 minutes. The tissue was then mounted with neutral gum and observed under a light microscope (200×). Five fields of view were randomly selected using ImageJ software, and the percentage of blue collagen fiber area was calculated.

[0150] (4) Wrinkle depth measurement: Place the probe vertically on the skin surface and record the R0 parameter (wrinkle depth). Each sample is measured three times and the average value is taken.

[0151] (5) COX-2 detection (Western blot): total protein was extracted from skin tissue (RIPA lysis buffer + protease inhibitor); protein concentration was determined by BCA method; 10% SDS-PAGE electrophoresis (80V for 30 minutes, 120V for 90 minutes); transfer to PVDF membrane (250mA for 120 minutes); blocking with 5% skim milk for 1 hour; incubation with primary antibody (rabbit anti-mouse COX-2, 1:1000) at 4°C overnight; incubation with secondary antibody (goat anti-rabbit HRP, 1:5000) at room temperature for 1 hour; ECL color development and analysis of band grayscale value (IOD, integrated optical density) by ImageJ.

[0152] 2. Experimental Results

[0153] 1. Skin Hydration: Skin hydration in Group D reached 73.8±5.6%, a 75.3% increase compared to the saline group (42.1±3.2%) (p<0.001) and a 26.6% increase compared to Group A (58.3±4.1%) (p<0.01). This indicates that engineered stem cell exosomes can effectively improve skin moisturizing function. (See Table 3.)

[0154] Table 3 Skin moisture content test results

[0155] Group Measurement value 1 (%) Measurement value 2 (%) Measurement value 3 (%) Mean ± SD (%) Normal saline group 39.8 43.2 43.3 42.1±3.2 Group A 55.2 59.8 59.9 58.3±4.1 Group D 70.1 75.6 75.7 73.8±5.6

[0156] 2. Collagen density: Masson staining showed that the collagen density in Group D was 78.2±4.9%, a 148% increase compared to the saline group (31.5±2.8%) (p<0.001). The collagen density in Group A was 49.7±3.5%, a 57.4% increase compared to Group A (p<0.01). This demonstrates that exosomes significantly outperform conventional exosomes in promoting collagen fiber regeneration. (See Table 4.)

[0157] Table 4 Collagen density test results

[0158]

[0159] 3. Wrinkle Depth: Wrinkle depth in Group D was only 42.1±3.8μm, a 67.2% decrease (p<0.001) compared to the saline group (128.6±9.7μm) and a 53.0% decrease (p<0.01) compared to Group A (89.4±7.3μm). Skin smoothness was significantly improved, as shown in Table 5.

[0160] Table 5 Wrinkle depth detection results

[0161]

[0162] 2.4 COX-2 Expression (IOD Value): Western blot results showed that COX-2 expression in Group D (IOD value 35.2±3.1) decreased by 77.0% (p<0.001) compared with the saline group (152.3±12.1) and by 64.3% (p<0.01) compared with Group A (98.7±8.2), indicating that engineered stem cell exosomes alleviate the inflammatory response to photoaging by inhibiting the COX-2 pathway. (See Table 6.)

[0163] Table 6 COX-2 expression (IOD value) detection results

[0164] Group Repeat 1 Repeat 2 Repeat 3 Mean ± SD Normal saline group 148.2 155.3 153.4 152.3±12.1 Group A 94.5 102.1 99.5 98.7±8.2 Group D 32.1 37.2 36.3 35.2±3.1

[0165] 3. Experimental Summary

[0166] This study, using a mouse model of UV-induced photoaging, demonstrated that an antibody-peptide-synergistically modified UC-MSC exosome gel, combined with 3D culture, exhibited significant anti-aging efficacy. Its mechanism of action is multifaceted and synergistic: exosome-derived growth factors such as TGF-β and IGF-1 activate fibroblasts and promote the synthesis of type I and type III collagen; the mAb-p16C antibody targets and eliminates senescent cells, reducing the release of inflammatory factors such as COX-2; while the TAT-SIRT1-SOD2 peptide inhibits oxidative stress and alleviates inflammatory damage. Furthermore, it increases skin hydration, improves the stratum corneum barrier, and reduces wrinkle formation. These engineered stem cell exosomes hold great promise for anti-aging applications and are expected to be developed into novel topical or injectable medical aesthetics products, offering an innovative approach for the treatment of photoaging.

[0167] Example 6: Application Case

[0168] Application 1: Anti-aging injection preparations

[0169] 1. Core formula and fine preparation of the preparation: The anti-aging injection preparation is designed with the concept of synergistic anti-aging. The core ingredient is 1×10 7 UC-MSCs were cultured in a 3D rotating bioreactor after antibody-peptide co-modification (mAb-p16C coating + TAT-SIRT1-SOD treatment). When these cells were cultured to P3, the viability of these cells exceeded 95% by trypan blue staining, and the concentration was accurately adjusted by hemocytometer. In addition, the preparation also added 0.5 mg / mL of mAb-p16 antibody (the average concentration was 0.51±0.02 mg / mL determined by BCA method) and a final concentration of 20 μM TAT-SIRT1-SOD peptide (purity>98%). The components were mixed in a biological safety cabinet, diluted to 1 mL with normal saline, filtered through a 0.22 μm sterile filter membrane, and packaged and stored at -80°C to ensure the activity of the components and the sterility of the preparation.

[0170] 2. Strict safety and efficacy verification: During the preclinical research phase, the safety and efficacy of the preparation were verified through multiple experiments. In the acute toxicity test, there was no death or abnormal behavior in SD rats injected with 5 times the clinical dose of the preparation within 7 days, and there was no significant difference in blood biochemical indicators compared with the control group; the hemolysis test showed that the hemolysis rate was less than 0.3%, which met the pharmacopoeia standards. The efficacy test used 18-month-old aging mice as a model. The experimental group was injected once a month for 3 consecutive times. ELISA test found that the levels of IL-6 and MDA in the blood were significantly reduced, and the activity of SOD was significantly increased; liver tissue section analysis showed that the proportion of senescent cells in the experimental group was 65.5% lower than that in the control group, fully confirming the significant effects of the preparation in anti-inflammatory, anti-oxidation and clearing senescent cells.

[0171] Application 2: Exosome Anti-Wrinkle Essence

[0172] 1. Exosome Preparation and Nanocarrier Construction: The exosome anti-wrinkle essence uses engineered UC-MSCs exosomes as its core raw material. Cell supernatant was collected after 7 days of culture in a 3D rotating bioreactor. After centrifugation at 300×g and 2000×g to remove cell debris, exosomes were extracted by ultracentrifugation at 100,000×g at 4°C for 70 minutes. NTA assay and Western blot analysis confirmed that the exosomes were primarily distributed in the 100-150nm range, with a concentration of (5.8±0.6)×10 11 The nanocarriers, prepared using hyaluronic acid-polylactic acid copolymer (HA-PLA) via ring-opening polymerization, self-assemble with exosomes at a 1:5 mass ratio at 4°C, forming a stable structure with an average particle size of 152±8nm and a potential of -25±3mV, enhancing exosome stability and skin permeability.

[0173] 2. Scientific formula design and stability guarantee: The essence formula is designed with multiple ingredients, and the exosome concentration is 1×10 10 The formulation contains 100 particles / mL of PEG-coated exosomes, formulated with 1mg / mL of mAb-p16C to target senescent cells. 5% trehalose provides both moisturizing and stabilizing properties, while hyaluronic acid and glycerin enhance moisturizing. 0.5% phenoxyethanol serves as a preservative, and deionized water is used to make up the volume to 100%. Accelerated testing (45°C for 4 weeks) and long-term testing (25°C protected from light for 6 months) verified the stability of the formulation's appearance, particle size, pH, and microbiological parameters. The exosome activity retention rate exceeded 90%, meeting cosmetic quality standards.

[0174] 3. Significant clinical efficacy: 30 female volunteers aged 35-55 with a facial wrinkle score of 3 or higher were tested. The experimental group received the exosome anti-wrinkle essence, while the control group received a matrix gel without exosomes. The serum was applied daily, morning and evening, for 28 days. Testing using a Cutometer MPA580 and a VISIA skin analyzer revealed that the experimental group experienced a 47.6% increase in skin elasticity and a 60% decrease in melanin index, while the control group experienced minimal improvement. This demonstrates the serum's significant efficacy in improving skin elasticity and reducing hyperpigmentation, demonstrating its promising market potential.

[0175] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An anti-p16INK4a monoclonal antibody mAb-p16, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody mAb-p16 is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

2. The monoclonal antibody mAb-p16 according to claim 1, characterized in that The monoclonal antibody mAb-p16 specifically binds to the amino acid sequence at positions 89 to 108 of the human p16INK4a protein, wherein the amino acid sequence at positions 89 to 108 of the human p16INK4a protein is SWTRWWCCTGPGRGWTCAM.

3. A method for preparing engineered umbilical cord stem cells, characterized in that: The method comprises the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells: UC-MSCs were isolated from the umbilical cord Wharton's jelly using collagenase digestion, and the positive rates of CD73, CD90, and CD105 were >95% by flow cytometry. (2) coupling the monoclonal antibody mAb-p16 to the microcarrier surface via the PNIPAM thermosensitive material to form a monoclonal antibody mAb-p16 modified thermosensitive microcarrier, wherein the molar ratio of the monoclonal antibody mAb-p16 to the PNIPAM is 1:5; (3) culturing stem cells in a medium enhanced with a fusion polypeptide TAT-SIRT1-SOD, wherein the amino acid sequence of the fusion polypeptide TAT-SIRT1-SOD is shown in SEQ ID NO: 1; (4) 3D dynamic culture was performed in a rotating bioreactor with a working volume of 50 mL, a density of 5 g / L of thermosensitive microcarriers modified with monoclonal antibody mAb-p16, a rotation speed of 25 rpm, a temperature of 37°C, and a 5% CO2 infusion rate of 50 mL / min. During the culture, the cells were first statically inoculated for 2 hours, and the rotational culture was started after the attachment rate was greater than 80%. 1 / 2 volume of the fusion polypeptide TAT-SIRT1-SOD enhanced culture medium was replaced every 3 days.

4. The method according to claim 3, characterized in that The fusion polypeptide TAT-SIRT1-SOD enhanced culture medium is a basic culture medium containing the fusion polypeptide TAT-SIRT1-SOD at a final concentration of 20 μM, wherein the basic culture medium is DMEM / F12+10% FBS.

5. An anti-aging pharmaceutical preparation, characterized in that: The preparation comprises engineered umbilical cord stem cells or exosomes thereof prepared according to the method of claim 3, and a pharmaceutically acceptable carrier.

6. The preparation according to claim 5, characterized in that The preparation is an injection, wherein the injection comprises the following components: 1×10 7 engineered umbilical cord stem cells, 0.5 mg / mL monoclonal antibody mAb-p16, 20 μM fusion peptide TAT-SIRT1-SOD and normal saline.

7. The preparation according to claim 5, characterized in that The preparation is an exosome anti-wrinkle essence for medical aesthetics, and the exosome anti-wrinkle essence for medical aesthetics comprises the following components: (1) Exosomes extracted from engineered umbilical cord stem cells prepared according to the method of claim 3, wherein the exosome concentration is 1×10 10 particles / mL; (2) HA-PLA nanocarriers, which self-assembled with exosomes at a mass ratio of 1:5 at 4°C to form a stable structure with an average particle size of 152 ± 8 nm and a potential of -25 ± 3 mV; (3) 1 mg / mL monoclonal antibody mAb-p16; (4) 5% trehalose; (5) 0.5% phenoxyethanol; (6) Deionized water is added to 100%.

8. An engineered umbilical cord stem cell cryopreservation solution, characterized in that: The freezing solution comprises 90% fetal bovine serum, 10% DMSO and 5 μg / mL of the monoclonal antibody mAb-p16 according to claim 1.

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