Recombinant III-type collagen with transdermal function as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of biological materials, and provides recombinant III-type collagen with a transdermal function and application thereof. The recombinant III-type collagen with the transdermal function comprises an oligopeptide segment with skin permeability, a flexible human III-type collagen segment located between the oligopeptide segment and a collagen functional structure segment, and a functional structure segment derived from human III-type collagen. According to the invention, an oligopeptide segment with a transdermal function is introduced into the molecular structure of the III-type collagen, so that the collagen keeps good biocompatibility and structural function, and meanwhile, the whole collagen obtains stable skin penetrating power, thereby expanding the application scene of the collagen in external application such as skin repair, aging resistance and skin care.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and particularly relates to recombinant type III collagen with transdermal function, its preparation method and application. Background Technology

[0002] Collagen is one of the most important structural proteins in human skin, especially abundant in the dermis, typically accounting for over 70% of the dermal dry weight. It plays a crucial role in maintaining skin tension, elasticity, and tissue mechanical stability. Simultaneously, collagen is a core component of the extracellular matrix (ECM), participating in various physiological processes such as cell adhesion, migration, and signal regulation. With the development of tissue engineering, medical dressings, and bio-cosmetic technologies, collagen-related materials are widely used in skin repair, anti-aging, wound care, and functional skin products. Among these, type III collagen, due to its excellent biocompatibility and tissue affinity, has become one of the most sought-after active ingredients in topical skin products.

[0003] However, in practical applications, the delivery efficiency of collagen, especially type III collagen, under topical conditions remains significantly limited. Collagen has a large molecular weight, a relatively rigid structure, and a stable spatial conformation, making it difficult to penetrate the skin's stratum corneum barrier via passive diffusion. Existing research indicates that the stratum corneum has strict limitations on the permeability of exogenous substances; the molecular weight threshold for freely penetrating the stratum corneum is generally considered to be approximately 500 Da. Collagen's functional structure far exceeds this range, and it lacks natural active transmembrane or translayer transport mechanisms. Therefore, traditional topical collagen mostly remains on the skin surface, struggling to penetrate the dermis to exert its biological functions. This significantly restricts the effectiveness of collagen in non-invasive skin products.

[0004] To improve the transdermal delivery of macromolecules, strategies utilizing short peptides with skin-permeable capabilities have been proposed in recent years. These short peptides, when applied topically, are those capable of participating in the skin barrier crossing process and promoting the entry of proteins or peptides into skin tissues. These transdermal peptides are typically small in molecular weight, possess strong hydrophilicity, and have specific amino acid compositions. They can promote the crossing of the skin barrier through various mechanisms. For example, short peptides such as TAT ​​peptide derived from HIV-1 and Penetratin have been reported to enhance the transdermal absorption of various drugs or proteins. These studies demonstrate that short peptides with skin permeability, as a versatile and modularly applicable transdermal enhancement tool, have broad application potential in the delivery of recombinant proteins. It should be noted that existing short peptides with skin permeability are mostly derived from viral proteins, cell membrane-penetrating domains, or artificially screened short peptides. Their sequences differ significantly from collagen itself in evolutionary origin, structural characteristics, and tissue compatibility. When fused with collagen, exogenous short peptides with skin permeability often struggle to achieve intrinsic coordination with the collagen structure at the sequence level, potentially introducing additional immune risks or structural instability. In contrast, if short peptides with skin permeability originate from the collagen sequence itself or its homologous structural regions, they are theoretically more likely to maintain consistency with the overall collagen structure in terms of molecular composition, spatial extensibility, and tissue environment adaptability, thereby endowing it with new delivery capabilities without significantly disrupting its biological properties.

[0005] However, directly fusing short peptides with skin permeability with functional proteins (such as collagen) still faces several challenges: First, short peptides with skin permeability differ significantly from large protein molecules in spatial conformation and structural characteristics, and direct splicing may cause local conformational interference, leading to reduced fusion expression efficiency, decreased stability, or loss of function. Second, existing technologies often use universal artificial linker sequences (such as Gly-Ser repeat sequences) as transitional structures, but these linker methods lack intrinsic matching with the natural structure of collagen, which may form conformational discontinuities or "structural breaks" in the fusion region, thereby affecting the overall structural integrity and functional performance of the fusion.

[0006] Furthermore, existing research has largely focused on enhancing collagen delivery capabilities by adding short peptide modules with skin permeability, while paying insufficient attention to whether a collagen sequence with transdermal capability can be obtained. In other words, current technology has not yet provided a solution that can achieve stable transdermal delivery capability at the overall molecular level by optimizing the sequence while maintaining the structural characteristics and biocompatibility of type III collagen.

[0007] Therefore, there is an urgent need to develop a recombinant type III collagen with stronger structural integration, more natural source, and more stable function, which not only solves the problem of transdermal delivery efficiency, but also maintains the correct conformation, solubility and activity of recombinant type III collagen in the expression system. Summary of the Invention

[0008] The purpose of this invention is to provide recombinant type III collagen with transdermal function, thereby addressing the problems mentioned in the background section.

[0009] The present invention is implemented as follows: a recombinant type III collagen with transdermal function includes a short peptide segment with skin permeability, a flexible human type III collagen segment located between the short peptide segment and the collagen functional structure segment, and a functional structure segment derived from human type III collagen.

[0010] The short peptide segment with skin permeability has the characteristics of low molecular weight, compact structure, and rich in hydrophilic and basic residues. It can assist recombinant type III collagen to penetrate the stratum corneum and enter the dermis through a variety of skin permeation mechanisms, including endocytosis, membrane perturbation, tight junction regulation or hair follicle permeation. Its amino acid sequence is as shown in SEQ ID NO:5, or a sequence with at least 80% identity with it.

[0011] The flexible human type III collagen segment can buffer the spatial structural tension between skin-permeable short peptides and collagen domains, while improving the overall stability and expression efficiency of recombinant type III collagen. It forms a transitional link between skin-permeable short peptides and functional structural segments of type III collagen, and has good hydrophilicity, spatial flexibility and conformational coordination. It can be stably expressed and maintain its function in different combinations of skin-permeable short peptides and collagen structural segments. Its amino acid sequence is selected from SEQ ID NO:10-13.

[0012] The functional structural segments derived from human type III collagen can provide biological support and collagen characteristics for recombinant type III collagen. Rich in multiple extracellular matrix recognition sites, they have the potential to promote dermal cell adhesion, growth, and signal activation, making them suitable for applications such as skin repair and anti-aging. Containing tripeptide repeat modules such as GPP, GPR, GEK, and GAG, they possess good hydrophilicity, spatial extensibility, and expression compatibility, enabling them to exist stably within recombinant type III collagen and maintain conformational stability and functional presentation in the dermal tissue environment. Their amino acid sequence is shown in SEQ ID NO:15. Rich in stable tripeptide repeat units and extracellular matrix (ECM) recognition sites, they exhibit good triple helix extensibility, contributing to the maintenance of the overall conformation of recombinant type III collagen and enhancing cell adhesion, migration, and tissue repair activity.

[0013] Another objective of this invention is to provide a nucleic acid molecule that encodes the aforementioned recombinant type III collagen with transdermal function.

[0014] Another objective of this invention is to provide a recombinant expression vector comprising the aforementioned nucleic acid molecules.

[0015] Another objective of this invention is to provide a host cell containing the aforementioned recombinant expression vector.

[0016] Another objective of this invention is to provide a method for preparing recombinant type III collagen with transdermal function, comprising the following steps:

[0017] (1) Recombinant expression vectors were obtained by recombining nucleic acid molecules into the vector pPICZαC;

[0018] (2) Transfect the recombinant expression vector into host cells to obtain recombinant cells;

[0019] (3) After culturing the recombinant cells, centrifuge and collect the supernatant;

[0020] (4) The recombinant type III collagen with transdermal function is obtained from the supernatant.

[0021] Another objective of this invention is to provide the use of recombinant type III collagen with transdermal function in the preparation of transdermal topical formulations.

[0022] Another objective of this invention is to provide the use of recombinant type III collagen with transdermal function in the preparation of skin repair or skin care biomaterial products.

[0023] This invention introduces short peptide segments with transdermal function into the molecular structure of type III collagen, enabling the collagen to maintain its good biocompatibility and structural function while achieving stable skin penetration. The molecular design integrates multiple functions, including transdermal delivery, biocompatibility, spatial conformational flexibility, and collagen helical structure stability. This significantly improves the delivery efficiency, expression stability, and structural controllability of collagen in topical skin applications while preserving its natural functions. Animal experiments have shown good skin absorption and tissue localization capabilities. Combined with its collagen functional modules, it can be used to develop topical protein products with repair, anti-inflammatory, and anti-aging functions, thereby expanding its application scenarios in skin repair, anti-aging, and skin care. Attached Figure Description

[0024] Figure 1This is a schematic diagram of the screening results of short peptides with transdermal delivery capability provided in Example 1 of the present invention;

[0025] Figure 2 This is a schematic diagram of the overall structure of the type III collagen construct with transdermal delivery capability provided in Embodiment 3 of the present invention;

[0026] Figure 3 This is a schematic diagram of the plasmid construction of the type III collagen construct with transdermal delivery capability provided in Embodiment 3 of the present invention;

[0027] Figure 4 This is a schematic diagram showing the transformation and colony growth of different type III collagen constructs provided in Example 3 of the present invention in the Pichia pastoris expression system;

[0028] Figure 5 This is a schematic diagram of the colony PCR identification results of different type III collagen constructs provided in Example 3 of the present invention;

[0029] Figure 6 The electrophoretic analysis results show the expression of the type III collagen construct provided in Example 3 of this invention at different induction time points;

[0030] Figure 7 This is a schematic diagram of the elution curve and main elution peaks of the type III collagen construct provided in Example 4 of the present invention after fermentation and chromatography purification;

[0031] Figure 8 The results of SDS-PAGE electrophoresis analysis of the purified products of different type III collagen constructs provided in Example 4 of this invention;

[0032] Figure 9 The transdermal distribution fluorescence microscopy results of the type III collagen construct provided in Example 5 of the present invention after FITC labeling in a rat skin model;

[0033] Figure 10 This is a schematic diagram of the cytotoxicity detection results of the type III collagen construct provided in Example 6 of the present invention;

[0034] Figure 11 The results of the transdermal accumulation and permeation rate analysis of the type III collagen construct provided in Example 7 of this invention in the Franz diffusion cell model. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1: Screening and validation of short peptide PTCPⅢ with transdermal delivery capability:

[0038] This invention is based on short peptide fragments derived from human type III collagen sequences. By constructing a candidate short peptide set, short peptides with potential transdermal delivery capabilities and skin permeability are screened and verified. The candidate short peptides are selected from flexible regions and non-highly structured segments in the natural sequence of type III collagen. Charged or hydrophilic amino acids are introduced into some sequences through point mutations to obtain multiple candidate short peptide sequences with a length of about 10-20 amino acid residues.

[0039] Under uniform screening conditions, candidate short peptides were contacted with skin-related model systems. By detecting the interaction response characteristics between different short peptides and the system, their potential transdermal delivery capabilities were compared and analyzed. During the screening process, short peptides with known transdermal functions were set as positive controls, and short peptides derived from type III collagen but obtained through random mutation or key site substitution were set as structural controls to exclude the influence of source factors on the results.

[0040] The control short peptides include: TAT peptide (as shown in SEQ ID NO:1), Penetratin peptide (as shown in SEQ ID NO:2), PKU12 peptide (as shown in SEQ ID NO:3), and SPACE™ peptide (as shown in SEQ ID NO:4), as well as three control short peptides derived from type III collagen sequences but with amino acid substitutions at key sites (as shown in SEQ ID NO:6-8). The response characteristics of each short peptide were compared under the same conditions.

[0041] The results are as follows Figure 1 As shown, significant differences were observed among the different short peptides. One candidate short peptide exhibited a stable and significantly reduced signal response during detection. Compared to the control group of short peptides known to have skin permeability and the collagen-derived mutant control group, the signal level of this short peptide was significantly reduced, and it showed good reproducibility in repeated experiments. Based on the characteristic performance of this short peptide in the screening system, it was identified as a skin-permeable short peptide with potential for transdermal delivery, and its amino acid sequence is shown in SEQ ID NO:5.

[0042] In contrast, the short peptide control group and the collagen-derived mutant control group, which are known to have skin permeability, did not show the same signal change trend as PTCPⅢ, indicating that the transdermal delivery potential of PTCPⅢ is closely related to its specific amino acid composition and sequence structure, rather than being determined solely by the collagen source itself. These results provide a reliable functional short peptide basis for the subsequent construction of recombinant type III collagen fusion sequences with transdermal delivery capability.

[0043] Example 2: Screening and validation of flexible linker peptides:

[0044] The present invention aims to screen out flexible linking peptides (linking peptides) from the Gly-XY region of human type III collagen that can serve as skin-permeable short peptides to collagen structural segments. The linking peptides should have the characteristics of natural origin, good hydrophilicity, strong conformational flexibility, and high expression stability, and be able to be compatible with different structural domains in the fusion expression construct, maintaining the stable output of overall conformation and function.

[0045] The original sequence used in the embodiments of the present invention (as shown in SEQ ID NO:35) is the GXY segment of the human type III collagen α1 chain, which is more than 1,000 amino acids in length. This region is a classic tripeptide repeat structure region with strong structural regularity, rich in proline and hydroxyproline, and has good biocompatibility and native conformation.

[0046] Using a sliding window algorithm, sequences such as SEQ ID NO:35 were progressively truncated in window lengths of 9-15 amino acid residues to obtain natural short peptide fragments of length 9-15, generating a total of 7916 candidate fragments. A candidate linker peptide database was constructed, and the flexible linking ability of all fragments was evaluated based on the following comprehensive scoring system:

[0047] First, an axial positioning score system was established. This score is based on the regular distribution characteristics of the Gly-XY repeating unit of the tripeptide structure. Different stability weights are given to the G, X and Y positions respectively. The G position (Glycine) is a key residue on the central axis of the triple helix and is given a high score. If there are large side chains or hydrophobic residues in the X / Y positions, the score will be reduced because it may cause conformational distortion.

[0048] Secondly, based on the physicochemical properties of amino acids themselves, a bonus item for flexible residues is set up to give positive points to residues with high flexibility and chain segment rotational freedom (such as Gly, Ser, Ala, Thr); at the same time, a penalty item for rigid / hydrophobic residues is set up to give negative points to amino acids such as Pro, Ile, Leu, Phe that are prone to structural rigidity or hydrophobic aggregation.

[0049] Furthermore, a repetitive fragment penalty factor is introduced to reduce the score of sequences with repetitive patterns such as "GPPGPP" and "GEKGEK" in order to reduce spatial arrangement repetition or spiral extension anomalies that may occur during fusion construction.

[0050] Finally, to improve the stability and functional compatibility of the construct expression, a conformational complexity correction factor is added, and negative weights are set for segments with potential structural kinking risks or spatial arrangement conflicts to avoid abnormal folding in the fusion region.

[0051] After standardizing the scores of all candidate sequences, they were sorted from high to low, and several representative linker peptide sequences were selected for subsequent validation.

[0052] After screening and normalization using the above scoring system, several representative high-scoring flexible linker peptides were obtained. Among them, the sequences shown in SEQ ID NO:10-13 are preferred fragments with high scores. As a comparison, the sequences shown in SEQ ID NO:9 are linker peptide sequences with lower scores and are retained in the construction system as negative controls to verify the key role of flexible linker peptides in fusion expression. In addition, the common artificial flexible linker peptide GGGGSGGGGS (as shown in SEQ ID NO:14) was selected as a standard linker peptide group reference to evaluate the superiority of the linker peptides screened in the embodiments of the present invention.

[0053] Example 3: Construction and expression verification of type III collagen construct:

[0054] In this embodiment of the invention, short peptides with skin permeability (as shown in SEQ ID NO:1-8) are selected as the N-terminal functional module, and linker peptides (as shown in SEQ ID NO:9-14) are used as intermediate modules to connect to the downstream collagen functional structural segment (as shown in SEQ ID NO:15), forming a complete fusion construct and a control group. Figure 2 As shown, each construct is named as follows:

[0055] Construct A: PTCPⅢ + sequence as shown in SEQ ID NO:10 + sequence as shown in SEQ ID NO:15 (collagen transdermal short peptide + flexible linker peptide + collagen functional structural segment);

[0056] Construct B: The sequence shown in SEQ ID NO:10 + the sequence shown in SEQ ID NO:15 (linker peptide + collagen functional segment);

[0057] Construct C: The sequence shown in SEQ ID NO:6 + the sequence shown in SEQ ID NO:10 + the sequence shown in SEQ ID NO:15 (control transdermal short peptide a + flexible linker peptide + collagen functional structural segment).

[0058] Construct D: TAT + sequence as shown in SEQ ID NO:10 + sequence as shown in SEQ ID NO:15 (transdermal short peptide + flexible linker peptide + collagen functional structural segment);

[0059] Construct E: Penetratin + sequence as shown in SEQ ID NO:10 + sequence as shown in SEQ ID NO:15 (transdermal short peptide + flexible linker peptide + collagen functional structural segment);

[0060] Construct F: PTCPⅢ+GGGGSGGGGS (as shown in SEQ ID NO:14) + sequence as shown in SEQ ID NO:15 (short collagen transdermal peptide + conventional linker peptide + collagen functional structural segment).

[0061] Constructor G: PTCPⅢ + sequence as shown in SEQ ID NO:15;

[0062] After codon optimization, the above-mentioned construct was chemically synthesized to obtain a full-length nucleic acid sequence, which was then cloned into the pPICZαC expression vector using seamless cloning to facilitate subsequent purification and identification. The recombinant vector map is shown below. Figure 3 As shown, to achieve efficient expression of the type III collagen construct, this embodiment of the invention uses the Pichia pastoris expression system and constructs a fusion expression vector using pPICZαC-A as the vector, following the procedure below:

[0063] First, the successfully constructed recombinant plasmid pPICZαC-A was transformed into TOP10 competent cells and then screened on LB plates containing Zeocin to obtain positive clones. The positive single clones were expanded by liquid LB culture and then high-purity plasmids were extracted on a large scale using the high-purity plasmid small-scale extraction kit from Tiangen Biotech (Beijing) Co., Ltd. to ensure the subsequent transformation efficiency and expression stability.

[0064] Pichia pastoris strain X33 was selected as the expression host. The expression plasmid was linearized using PmeI (50 μL enzyme digestion system, 37℃ for 2 hours). The reaction product was recovered by ethanol precipitation and resuspended in sterile water for later use. Logarithmic growth phase X33 competent cells were prepared and electroporated using a BTXECM399 electroporator (parameters: 1500V, 1mm gap electroporation cuvette) to introduce the linearized plasmid into the host.

[0065] Immediately after electroporation, 1 mL of pre-cooled 1M sorbitol buffer was added to the cells. After incubation at 30°C for 1 hour, the cells were evenly spread onto YPDS Zeocin selection plates (100 μg / mL). The plates were then incubated upside down at 30°C for approximately 72 hours. The colony growth of some constructs on the plates is shown below. Figure 4 As shown in the figure (where the left side is construct A and the right side is construct E), well-grown single clones were selected for colony PCR identification to confirm the correct insertion of the target gene. Some construct PCR identification results are shown below. Figure 5 As shown, the transformation and integration efficiency of each construct in Pichia pastoris is stable and the positive clone acquisition rate is high, indicating that the fusion sequence of the present invention does not have obvious toxicity or genetic instability problems in the host bacteria and has good engineering adaptability.

[0066] Positive clones were inoculated into 5 mL of BMGY pre-culture medium, and cultured with shaking at 28-30℃ for 24 hours. After centrifugation, the bacterial resuspended and transferred to 50 mL of BMMY culture system for small shake flask induction expression experiment. Methanol was added to the culture medium every 24 hours, with a final concentration of 0.5%, to maintain induction.

[0067] Induction time points (hours): 0, 24, 48, 72, and 96. At each of these time points, 1.0 mL of sample was taken from the expression culture system and transferred to a 1.5 mL sterile EP tube. The tubes were centrifuged at 12000g for 2-3 minutes at room temperature using a benchtop microcentrifuge. The supernatant was discarded, and the cell pellet was collected for subsequent expression detection. SDS-PAGE was used for preliminary analysis to compare the expression abundance at different induction times and determine the optimal expression period for the target protein. If necessary, grayscale scanning software was used for band quantification analysis to further evaluate expression efficiency. The protein expression results of construct A at different induction times are shown below. Figure 6 As shown, clones with good expression performance were finally selected for large-scale induced expression studies.

[0068] Example 4: Large-scale fermentation and protein purification:

[0069] To further obtain high-yield, high-purity type III collagen constructs, this invention, based on ShakeFlask expression validation, conducted high-density fermentation at a scale of 5L and established a simple and efficient downstream purification process:

[0070] Pichia pastoris X33 positive recombinant strains with good expression were selected, and after seed shake flask culture (BMGY medium), they were inoculated into 5L fermenters (Applikon glass fermenter system), and the following parameters were maintained using a fully automated control system:

[0071] Initial inoculation dose: 5-10% (v / v);

[0072] Culture medium composition: BSM high-salt fermentation medium for Pichia pastoris fermentation;

[0073] Temperature: 30℃;

[0074] pH: Maintained at 5.5-6.0, with automatic alkali replenishment adjustment;

[0075] Dissolved oxygen: Maintain at 20-30%, adjusted by stirring and aeration;

[0076] The fermentation process is divided into three phases: glycerol batch culture phase (biomass accumulation), glycerol feeding phase (increasing cell density), and methanol induction phase (target protein expression).

[0077] When the OD 600nm of the fermentation broth reaches about 160, the process is switched to the methanol induction stage. The methanol concentration is maintained at 0.5-1.0% by online feeding, and the induction is continued for 96 hours while monitoring protein expression.

[0078] After fermentation, the fermentation broth was centrifuged (8000 rpm, 4℃, 30 minutes), and the supernatant was collected. The crude extract was concentrated by ultrafiltration using a membrane with a molecular weight cutoff of 3 kDa, and the buffer was replaced simultaneously to transfer the sample into 25 mM phosphate buffer (pH 7.2) to prepare for subsequent purification.

[0079] The main purification step was performed using Smac MMC 40 chromatography media (Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and the procedure is as follows:

[0080] Sample loading: Protein concentrate is directly loaded onto a pre-equilibrated MMC chromatography column;

[0081] Elution: A linear gradient elution with NaCl was used, with 25 mM phosphate buffer as the eluent and the NaCl concentration gradient ranging from 0 to 500 mM.

[0082] Monitoring: The elution curve was monitored using both UV 280 nm and UV 240 nm, and the peaks corresponding to the target protein were collected.

[0083] The eluted sample was further purified by Superdex 100 molecular sieve chromatography to remove low molecular weight impurities and then replaced with PBS solution to obtain target proteins with uniform structure and higher purity.

[0084] The obtained construct proteins were used for functional validation experiments after analysis of the samples by SDS-PAGE electrophoresis and BCA method. The purification results of construct A are as follows: Figure 7 As shown, the electrophoresis results of the purified products of multiple constructs are as follows: Figure 8As shown (where lane M: 15-250 kDa protein marker; the remaining lanes are the constructs AG in sequence), after further purification by multi-mode chromatography and molecular sieve, each construct sample showed a single main band, with a significant reduction in impurities and no obvious degradation fragments, indicating that the constructs constructed in the embodiments of the present invention have good structural stability and integrity during expression and purification.

[0085] Example 5: Transdermal Experiment and Animal Validation:

[0086] To verify the skin permeability of the constructed transdermal short peptide-flexible linker peptide-collagen fusion construct, this invention employs FITC labeling technology to fluorescently label the fusion construct and performs a transdermal application experiment on the back of shaved rats. Its transdermal capacity is observed using a fluorescence microscope.

[0087] Dissolve the fusion construct to be labeled in 0.1M sodium carbonate buffer (pH 9.0), and control the protein concentration at ≥2mg / mL. To avoid pH changes caused by buffer degradation, it is recommended to prepare the buffer fresh and keep it away from light during use. Dissolve FITC in anhydrous DMSO to prepare a 1mg / mL stock solution. Prepare fresh before labeling and store in the dark. Slowly add 50μL of FITC solution to each 1mL of protein solution, preferably in portions (5μL each time), mixing gently while adding. Incubate the mixture at 4℃ in the dark for 8 hours. Then add NH4Cl to make the final concentration 50mM and continue incubating at 4℃ in the dark for 2 hours to terminate the reaction. Add 5% glycerol to stabilize the labeled protein. Finally, remove free FITC molecules by Sephadex G-25 gel filtration chromatography, collect the FITC-labeled protein solution and store it in the dark for later use.

[0088] Male SD rats aged 6-8 weeks, weighing 180-220g, were selected. After anesthesia, the hair on their backs was removed to ensure that the intact skin area was available for experimental use (all animal experiments were conducted in accordance with the "Regulations on the Management of Laboratory Animals" and the ethical standards for laboratory animals).

[0089] The labeled fusion construct solution was applied to the dorsal skin of shaved rats, repeated every 30 minutes for 2 hours. Animals were immediately sacrificed after the experiment, and the applied skin was rapidly embedded in OCT embedding medium and cryosectioned (thickness: 8-10 μm). The FITC signal distribution of the cryosections was observed under a fluorescence microscope to assess the skin penetration depth and efficiency of the fusion construct. Results are as follows: Figure 9 As shown, the observation results indicate that the signal distribution can penetrate deep into the dermis and diffuse continuously, confirming that the construct can effectively penetrate the skin barrier and reach the target tissue layer in a short time.

[0090] Example 6: Cytotoxicity, rat subcutaneous implantation compatibility, and guinea pig sensitization experiments:

[0091] To systematically evaluate the biosafety of the transdermal fusion collagen provided in the embodiments of the present invention, L929 cytotoxicity experiments, rat subcutaneous implantation compatibility experiments, and guinea pig skin sensitization experiments were conducted:

[0092] Lyophilized protein samples were dissolved in high-glucose DMEM medium at final concentrations of 0.1-2.0 mg / mL and used to treat L929 cells for 24 hours. Cell viability was then assessed using the CCK-8 assay. The results are as follows: Figure 10 As shown, within the concentration range of 0-200 μg / mL, after treating L929 cells with construct A containing a flexible linker peptide (as shown in SEQ ID NO:7) for 24 h, the cell viability was above 90%, with no significant difference compared to the blank control (P>0.05), and no obvious cytotoxicity was observed. Construct E containing a low-scoring linker peptide and construct G without a linker peptide showed a slight decrease in cell viability at high concentrations, but still remained above 80%, indicating that different linking methods all had good cell compatibility. Among them, the flexible linker peptide construct had the best safety, and the results showed that the cell viability of each concentration treatment group was above 90%, indicating that the fusion construct had no obvious toxicity to cells at the experimental concentration.

[0093] To assess the risk of skin sensitization, the guinea pig maximum dose method was used for sensitization experiments. A 10 mg / mL concentration of the fusion construct was applied to the back of guinea pigs to induce sensitization for 7 consecutive days. Local erythema and edema reactions were observed at 24 and 48 hours, and scored according to GB / T16886.10 standards. The scoring criteria in Table 1 were used to observe the sensitization at 24, 48, and 72 hours.

[0094] Table 1

[0095]

[0096] No sensitization reactions such as erythema and edema were observed on the back skin of guinea pigs in the transdermal fusion collagen group and the negative control (physiological saline) group provided in the embodiments of the present invention. The scores for erythema and edema were both 0, the sensitization rate was 0%, and the mean reaction value was 0.

[0097] Under the above experimental conditions, the fusion construct of the present invention showed no sensitizing effect on guinea pig skin, as shown in Table 2:

[0098] Table 2

[0099]

[0100] It can be seen that none of the animals in the experimental group showed a sensitization reaction of grade ≥1, and were therefore determined to be non-dermal sensitization.

[0101] Example 7: Transdermal kinetics and quantitative analysis experiment:

[0102] To systematically evaluate the skin penetration ability of the transdermal fusion collagen construct of the present invention, kinetic analysis was performed using FITC fluorescent labeling combined with the Franz diffusion cell model.

[0103] The FITC-labeled and purified fusion construct was dissolved in PBS buffer (pH 7.4) to a final concentration of 0.5 mg / mL. 1.0 mL was accurately pipetted into the donor cavity of the Franz diffusion cell and placed in contact with the pre-treated, hairless SD rat abdominal skin. PBS solution was added to the recipient cavity, and the temperature was maintained at a constant 37°C. Magnetic stirring was used to ensure uniform mass transfer. Recipient fluid was collected at 0, 1, 2, 4, 6, 8, 12, and 24 hours, and fluorescence intensity (excitation 488 nm, emission 520 nm) was measured. The protein transdermal accumulation and permeation rate were calculated. The results are shown in Table 3.

[0104] Table 3

[0105]

[0106] Using Excel 2019 software, a Q-time t plot was performed, and linear regression was conducted to obtain the kinetic fitting equation. The slope of this equation is the steady-state transmittance [µg / (h·cm)]. 2 [ ] indicates the transdermal absorption rate of the drug, and the cumulative permeation of the two protein groups versus time linear curves are shown below. Figure 11 As shown, the kinetic fitting equations and permeation parameters for the two groups are shown in Table 4 below:

[0107] Table 4

[0108]

[0109] Experimental results showed that, compared with collagen constructs without flexible linker peptides, constructs using preferred flexible linker peptides had significantly higher permeation rates and accumulation amounts, with peak values ​​occurring between 8 and 12 hours, indicating that they possess strong transdermal absorption capabilities.

[0110] In summary, the embodiments of the present invention introduce a screened functional short peptide segment with skin permeability into the structure of type III collagen, and combine it with a flexible segment derived from the natural sequence of type III collagen to construct a type III collagen fusion sequence that can penetrate the skin barrier and enter the skin tissue under topical application conditions. This fusion sequence takes into account both the biocompatibility and tissue functional characteristics of collagen in its overall structure, while endowing it with stable transdermal delivery capability.

[0111] Experimental results show that the functional short peptides screened in the embodiments of the present invention can effectively play a transdermal delivery role in the fusion sequence. Different constructs show clear differences in expression efficiency, structural stability and skin permeability. Among them, the recombinant type III collagen fusion sequence constructed by the preferred structural combination can achieve stable and efficient secretory expression in the Pichia pastoris system. After fermentation amplification and chromatography purification, a recombinant protein product with complete structure and stable performance is obtained.

[0112] Further verification through in vitro models and animal experiments showed that the above-mentioned recombinant type III collagen fusion sequence exhibited good transdermal delivery capability and biocompatibility in skin models, with no obvious cytotoxicity or sensitization reactions, indicating that it is suitable for non-invasive drug delivery and topical application scenarios.

[0113] Compared to traditional methods that rely solely on topical collagen or simply introduce exogenous short peptides with skin permeability, the recombinant type III collagen fusion sequence of this invention exhibits significant advantages in functional integration, expression stability, and transdermal delivery efficacy. It demonstrates excellent skin permeability, biocompatibility, and engineering adaptability in various in vitro and in vivo models, and can be widely applied in skin repair, skin care, and related biomaterials fields. This provides a new molecular-level solution for non-invasive collagen delivery, with promising application prospects and promotional value.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant type III collagen with transdermal function, characterized in that, It includes a short peptide segment with skin permeability, a flexible human type III collagen segment located between the short peptide segment and the collagen functional structure segment, and a functional structure segment derived from human type III collagen. The amino acid sequence of the short peptide segment with skin permeability is as shown in SEQ ID NO:5, or a sequence having at least 80% identity with it. The amino acid sequence of the flexible human type III collagen segment is selected from that shown in SEQ ID NO:10-13; The amino acid sequence of the functional structural segment derived from human type III collagen is shown in SEQ ID NO:

15.

2. The recombinant type III collagen with transdermal function according to claim 1, characterized in that, The amino acid sequence of the recombinant type III collagen is selected from that shown in SEQ ID NO:16-35.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant type III collagen with transdermal function as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the recombinant expression vector as described in claim 4.

6. The host cell according to claim 5, characterized in that, The host cells for the recombinant cells are selected from Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and mammalian cells.

7. A method for preparing recombinant type III collagen with transdermal function as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The nucleic acid molecule as described in claim 3 is recombined into the vector pPICZαC to obtain the recombinant expression vector as described in claim 4; (2) Transfect the recombinant expression vector into host cells to obtain the recombinant cells as described in claim 5; (3) After culturing the recombinant cells, centrifuge and collect the supernatant; (4) The recombinant type III collagen with transdermal function is obtained from the supernatant.

8. Use of a recombinant type III collagen with transdermal function as described in claim 1 or 2 in the preparation of a transdermal topical formulation.

9. The use of a recombinant type III collagen with transdermal function as described in claim 1 or 2 in the preparation of skin repair or skin care biomaterial products.

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