Preparation method of frozen microneedle patch for preventing skin photoaging

Abstract

The invention belongs to the technical field of skin photoaging, and particularly relates to a preparation method of a frozen microneedle patch for preventing skin photoaging, and the preparation method comprises the following steps: S1, placing venous blood in a blood collection tube for centrifugation; s2, centrifuging, and then taking out platelet-rich fibrin gel on the middle layer; s3, injecting the platelet-rich fibrin gel into a microneedle mold, filling a needle cavity with the platelet-rich fibrin gel, and standing; s4, after the microneedle mold is precooled, freezing forming is conducted, demolding is conducted after forming, and the frozen microneedle patch is obtained.The fibrin rich in platelets is combined with the frozen microneedle technology, the action mechanism of the skin photoaging phenotype is improved, efficient loading and long-acting release of growth factors are achieved, and a new direction is provided for regenerative medicine of photoaging.

Description

Technical Field

[0001] This invention belongs to the field of skin photoaging technology, specifically relating to a method for preparing a cryomicroneedle patch for preventing skin photoaging. Background Technology

[0002] While sun protection is the most basic measure to prevent photoaging of the skin, existing sunscreen products still have many shortcomings. First, most sunscreens are insufficiently protective against the UVA1 band (370-400 nm), and UVA1 is a significant factor causing photoaging, photoimmunosuppression, and pigmentation. Second, some organic sunscreens are sensitizing and irritating to humans, and may affect the endocrine system after transdermal absorption. Although nano-sized inorganic sunscreens improve skin feel, the risk of transdermal absorption raises safety concerns, requiring caution when used on infants, pregnant women, and areas with damaged skin. Furthermore, the actual use of sunscreen products often suffers from insufficient application, uneven application, and failure to reapply in time, significantly reducing their protective effect. Topical anti-photoaging agents face the dual challenges of skin permeability and stability. The stratum corneum, as a natural barrier, limits the transdermal absorption of large molecules and hydrophilic substances. Many active ingredients, such as vitamin C, polyphenolic compounds, and growth factors, have large molecular weights or strong hydrophilicity, making it difficult for them to effectively penetrate the skin to reach the site of action. Even with delivery systems such as nanocarriers and liposomes, the penetration efficiency still needs to be improved.

[0003] Stability is another major issue. Many natural active ingredients are sensitive to environmental factors such as light, heat, and oxygen, and are easily degraded and inactivated. For example, although anthocyanins have strong antioxidant activity, their poor photostability limits their application in sunscreen products. Vitamin C is easily oxidized, requiring special packaging and formulation design. Furthermore, the metabolic fate of active ingredients in the complex skin microenvironment, the duration of their effect, and potential local irritation also need to be considered. Interactions between different ingredients in the formulation can affect their individual stability and activity.

[0004] While physical therapy techniques such as radiofrequency, laser, and photodynamic therapy can produce immediate results, they also have significant limitations. First, these treatments typically require specialized equipment and highly trained operators, resulting in high costs and limiting accessibility. Second, treatment effectiveness is highly dependent on the operator's experience and skill level; improper operation can lead to complications such as burns, scarring, and pigmentation abnormalities. Third, most physical therapies require multiple sessions to achieve satisfactory results, increasing the patient's time and financial burden. The durability of treatment effects is also an issue; without daily protection and maintenance, the improved skin condition may gradually regress. Significant individual differences exist; the same treatment parameters may produce different effects on different individuals, and standardized treatment protocols are lacking. Furthermore, some physical therapies may cause discomfort, such as the heat sensation from radiofrequency treatment and photosensitivity reactions after photodynamic therapy, affecting patient compliance. Their impact on deeper skin structures is limited, primarily improving the appearance of the superficial layers, with relatively weak effects on collagen remodeling in the deeper dermis. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a method for preparing a cryomicroneedle patch for preventing skin photoaging. This method combines platelet-rich fibrin with cryomicroneedle technology to achieve a mechanism of action that improves the skin photoaging phenotype and realizes the purpose of efficient loading and long-lasting release of growth factors.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] The present invention aims to provide a method for preparing a cryomicroneedle patch for preventing photoaging of the skin, comprising the following steps:

[0008] S1. Place the venous blood in a blood collection tube and centrifuge it.

[0009] S2. After centrifugation, remove the middle layer of platelet-rich fibrin gel.

[0010] S3. Inject platelet-rich fibrin gel into the microneedle mold and fill the needle cavity, then let it stand.

[0011] S4. After pre-cooling the microneedle mold, it is then frozen and molded. After molding, it is demolded to obtain a frozen microneedle patch.

[0012] Furthermore, the collected venous blood was centrifuged within 2 minutes.

[0013] Furthermore, the venous blood was centrifuged at 3000 rpm for 15 minutes at room temperature.

[0014] Further, after centrifugation, the middle layer of platelet-rich fibrin gel was removed and processed under sterile conditions.

[0015] Furthermore, the microneedle mold is a 10x10 array mold with a needle height of 120 μm and a needle tip angle of 19°.

[0016] Furthermore, after injecting platelet-rich fibrin gel into the microneedle mold, it is subjected to vibration to remove bubbles or low-speed centrifugation. After the platelet-rich fibrin gel fills the needle cavity, it is left to stand at room temperature for 2-3 minutes.

[0017] Furthermore, the microneedle mold filled with platelet-rich fibrin gel was pre-cooled at -20°C for 2-3 hours and then frozen at -80°C for 8 hours to form the shape.

[0018] Platelet-Rich Fibrin (PRF) is rich in various bioactive factors, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1). These growth factors work synergistically to significantly promote angiogenesis, cell proliferation, collagen synthesis, and inflammation resolution, thereby accelerating tissue repair and regeneration. In skin repair, PRF has been widely used in facial rejuvenation procedures, such as PRF fillers and PRF facelifts, effectively improving skin texture, increasing skin thickness, and elasticity. Regarding anti-aging mechanisms, the growth factors in PRF can activate signaling pathways such as Wnt / β-catenin and PI3K / Akt in fibroblasts, upregulating the expression of type I collagen and elastin while inhibiting the activity of MMPs, thus reversing collagen degradation caused by photoaging. Furthermore, PRF possesses natural biocompatibility and is non-immunogenic, avoiding the rejection reactions that may occur with allogeneic tissue transplantation, making it an ideal carrier for skin repair. Using PRF in transdermal drug delivery systems not only enables deep delivery of growth factors through its microneedle technology, but also provides a continuous microenvironment for the skin through its scaffold structure, promoting tissue regeneration.

[0019] The core principle of cryo-microneedling technology is to rapidly freeze a microneedle mold in a cryogenic medium such as liquid nitrogen to form a microneedle array, which is then applied to the skin surface. During application, the microneedle tips contact the skin, and the instantaneous freezing effect creates a freezing channel at the interface between the needle tip and the epidermis, thereby achieving deep drug delivery and enhanced penetration without damaging the epidermis. Compared with traditional physical microneedles (such as roller microneedles and microneedles), cryo-microneedling has significant technological advantages. First, the freezing effect instantly freezes the interface between the microneedle and the skin, forming a tight cryo-seal that effectively prevents liquid leakage and microbial infection, greatly reducing the risk of infection. Second, cryo-microneedles do not require the addition of adhesives or water-soluble carriers during preparation, avoiding the biocompatibility problems that may arise from the high molecular weight polymers commonly used in traditional microneedles. Furthermore, cryo-microneedles have high mechanical strength, enabling them to overcome the resistance of the stratum corneum and penetrate into the dermis to release drugs or growth factors. In terms of drug delivery, cryo-microneedling technology is particularly suitable for encapsulating thermally unstable biomolecules such as proteins, peptides, and growth factors. Because the freezing process is fast, biological macromolecules are less likely to denature or become inactive, thus maintaining their biological activity.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0021] 1. This invention is the first to combine platelet-rich fibrin (PRF) with cryomicroneedle technology to develop a novel transdermal drug delivery system for skin photoaging repair. Through theoretical analysis and experimental verification, the mechanism by which the PRF microneedle delivery system improves the skin photoaging phenotype by regulating the p53 / p21 pathway and TGF-β1 expression, and influencing neutrophil (LY6G+) infiltration, is explored, providing a new direction for regenerative medicine in photoaging.

[0022] 2. This invention designs a microneedle mold with a specific taper and height, achieving efficient loading and long-lasting release of growth factors. Simultaneously, by controlling the freezing rate and temperature gradient, the mechanical strength and drug loading capacity of the microneedles are optimized, ensuring they have sufficient rigidity to penetrate the skin barrier without breaking.

[0023] 3. The present invention provides a simple and efficient in vivo drug delivery and evaluation system: a patch-type PRF cryomicroneedle drug delivery method was developed, and the comprehensive effect was evaluated by combining macroscopic skin indicators (erythema, wrinkles, etc.) and specific molecular indicators (p53, p21, TGF-β1, LY6G).

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the preparation process of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention.

[0026] Figure 2 This is a schematic diagram of the experimental process of the present invention.

[0027] Figure 3 This is a physical image of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention.

[0028] Figure 4 This is a side view of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention.

[0029] Figure 5 This is an enlarged photograph of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention.

[0030] Figure 6 This is a schematic diagram of the needle tip angle of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention.

[0031] Figure 7 This is a schematic diagram of a cryo-microneedle patch based on platelet-rich fibrin according to the present invention. All dimensions in the figure are in mm.

[0032] Figure 8 The trend graphs show the detection of p53, p21, TGF-β1, and LY6G in the blank group, control group, PRP microneedle group, and PRF microneedle group in the experimental examples of this invention.

[0033] Figure 9 The images show the macroscopic changes in the skin on the backs of nude mice in each experimental group during the fourth week of the experiment in this invention. From top to bottom, they represent the blank group, control group, PRP microneedle group, and PRF microneedle group.

[0034] Figure 10 The images show the macroscopic changes in the skin on the backs of nude mice in each experimental group at week eight in the experimental examples of this invention. From top to bottom, they represent the blank group, control group, PRP microneedle group, and PRF microneedle group.

[0035] Figure 11 This is a diagram showing the stress experiment results of the PRF microneedle assembly in this invention.

[0036] Figure 12 This is a diagram showing the stress experiment results of the PRP microneedle assembly in this invention. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0038] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0039] A method for preparing PRF cryo-microneedles includes the following steps:

[0040] A) Collect 10 ml of the subject's own venous blood and inject it directly into a dedicated PRF blood collection tube (such as a vacuum blood collection tube without anticoagulants). This blood collection tube is specially treated, with no anticoagulant on its inner wall, ensuring that the blood can immediately begin the coagulation cascade reaction after collection. The blood collection process should be performed under medical operating procedures to ensure the safety and sterility of the blood collection. The collected blood should be stored at room temperature and centrifuged within 2 minutes after collection to ensure that the blood is in its optimal physiological state at centrifugation. The time interval between collecting venous blood in step A and starting centrifugation in step B is ≤120 s. In practical applications, the donor and recipient are the same individual to avoid the risk of biological contamination and achieve the effect of immediate use by the user.

[0041] B) The collected blood was centrifuged at 3000 rpm for 15 minutes at room temperature (20-25°C). This parameter was chosen based on the following scientific basis: the relative centrifugal force (RCF) is calculated as RCF = 1.118 × r × (N / 1000)², where r is the rotor radius (in cm) and N is the rotational speed (in rpm). Assuming a centrifuge tube radius of approximately 8 cm, 3000 rpm corresponds to a relative centrifugal force of approximately 800 g. This level of centrifugal force is higher than the 400 g of traditional L-PRF, but lower than high-speed centrifugation (above 1000 g), minimizing mechanical damage to cells and growth factors while ensuring sufficient solid-liquid separation. The 15-minute centrifugation time is an optimized parameter; under the same relative centrifugal force, extending the centrifugation time improves fibrin aggregation and membrane mechanical strength. A 15-minute centrifugation time achieves complete stratification of blood cells without causing excessive cell rupture or excessive release of growth factors.

[0042] C) After centrifugation, the resulting product consists of three layers: an upper layer of serum (similar to a pale yellow liquid), a middle layer of PRF gel (a red and yellow aggregate), and a lower layer of concentrated red blood cells (dark red). Carefully remove the middle layer of PRF gel using sterile medical forceps. This operation must be performed in a sterile environment to prevent contamination. At this stage, the PRF should appear as a cohesive gel block with a certain degree of firmness. If the PRF appears as scattered fragments or is too soft and loose, the centrifugation parameters may need adjustment. Generally, high-quality PRF should have sufficient structural strength to be picked up by forceps without crumbling. The collected PRF should be immediately transferred to a sterile container; the process should be rapid to avoid prolonged exposure to air, which could lead to drying and loss of bioactivity. The PRF gel is taken from the intermediate gel layer between the plasma and red blood cell layers, retaining the adjacent gel areas. PRF is defined as a three-dimensional fibrin network obtained from autologous whole blood through a single centrifugation process. It is rich in platelets and leukocytes and can release various growth factors, making it an important representative of second-generation platelet concentrate systems. Unlike PRP systems, which require anticoagulants and exogenous activators, PRF emphasizes "additive-free, on-site preparation, and natural coagulation to form a fibrin scaffold," making it less risky and more acceptable for clinical use. PRF sustainably releases multiple growth factors and cytokines, including TGF-β, PDGF, VEGF, EGF, and bFGF, and maintains release kinetics within a certain time window. This is considered one of the important mechanisms by which it promotes angiogenesis, collagen deposition, epithelialization, and tissue remodeling.

[0043] D) Inject the PRF gel into a microneedle mold prepared by 3D printing technology and fill the needle cavity; let it stand at room temperature for 2-3 minutes to allow it to fully expand and form a stable gel structure. This process allows the fibrin in the PRF to further polymerize, increasing the mechanical strength of the gel. The microneedle mold is a 10x10 array mold with a needle height of 120 μm and a needle tip angle of 19°. After injecting the PRF gel, vibrate to remove bubbles or perform short-term low-speed centrifugation to improve the needle cavity filling rate.

[0044] E) After filling the microneedle mold, pre-cool it in a -20°C freezer for 2-3 hours, then transfer it to a -80°C freezer for final freezing for 8 hours to shape it; the low temperature environment of -80°C can effectively inhibit the growth and metabolic activities of microorganisms, while minimizing the diffusion of biomolecules (especially growth factors and cytokines) in PRF.

[0045] F) Demold to obtain PRF cryogenic microneedles.

[0046] Test case

[0047] In vivo experiments: A skin photoaging model was established by irradiating the back skin of nude mice with a combination of UVA and UVB.

[0048] Materials: Platelet-rich fibrin (PRF) was prepared from fresh venous blood of healthy volunteers, and in vivo experiments were conducted using BALB / c male nude mice.

[0049] Reagents and drugs: including phosphate-buffered saline (PBS), p53 antibody, p21 antibody, TGF-β1 antibody, LY6G antibody, etc.

[0050] Instruments and equipment include centrifuges, liquid nitrogen tanks, optical microscopes, scanning electron microscopes (SEM), universal testing machines, ELISA readers, quantitative PCR instruments, tissue sectioners, etc.

[0051] The specific protocol was as follows: Except for the normal control group (no irradiation, no intervention), all other groups received irradiation starting from week 1, with an initial dose of 100 mJ / cm² (one minimum erythema dose, 1 MED), increasing by 100 mJ / cm² weekly until reaching 400 mJ / cm², at which point the dose was maintained for a total of 8 weeks. The simple irradiation control group received continuous irradiation for 8 weeks; the PRF cryomicroneedling group and the platelet-rich plasma (PRP) cryomicroneedling positive control group received microneedling intervention immediately after each irradiation starting from week 4, continuing until the experimental endpoint at week 8. Macroscopic changes in the skin were observed regularly, and samples were collected at the experimental endpoint for histological, molecular biological, and immunohistochemical analysis, focusing on detecting the expression levels of skin aging-related proteins such as p53, p21, and TGF-β1, as well as indicators such as LY6G+ neutrophil infiltration rate.

[0052] Nude mice were irradiated with a combination of UVA and UVB on their backs. The initial dose was 100 mJ / cm² (1 MED) in the first week, increased weekly by 100 mJ / cm², reaching 400 mJ / cm² and maintaining the dose thereafter. From week 4 onwards, mice were divided into groups: a normal control group received no irradiation or intervention, while the irradiation-only control group received continuous irradiation. The PRF cryo-microneedling group, PRP cryo-microneedling group, and blank (water) cryo-microneedling group were treated immediately after irradiation and intervened for 8 weeks. Immunofluorescence staining was performed at 200× field of view, with random counting in 5 fields. Positive expression rate = (number of positive cells / total number of cells) × 100%. Data are expressed as mean ± standard deviation (x ± s). Compared with the irradiation-only control group, *P < 0.05, **P < 0.01, ***P < 0.001. The experimental results are shown in Table 1 below.

[0053] Table 1

[0054]

[0055] Observe macroscopic changes in the skin and evaluate the indicators as shown in Table 2 below.

[0056] Table 2

[0057]

[0058] The results of macroscopic skin changes in the fourth week are shown in Table 3 below.

[0059] Table 3

[0060]

[0061] Verification of model effectiveness: The scores of all photoaging indicators in the control group were significantly higher than those in the blank group (erythema: t=13.86, P<0.001; wrinkles: t=18.52, P<0.001; telangiectasia: t=17.01, P<0.001; color uniformity: t=16.23, P<0.001), and... Figure 9 The typical photoaging features shown in the second image from the left (dense deep wrinkles, numerous fused erythema, and clear red blood vessels) are consistent, confirming the successful 4-week ultraviolet modeling.

[0062] Comparison of PRP and PRF microneedling repair effects:

[0063] The scores of all indicators in the PRP microneedling group were significantly lower than those in the control group (P < 0.01), but the group still maintained a moderate level of photoaging, suggesting that PRP microneedling has a certain repair effect, but the effect is limited.

[0064] The scores of all indicators in the PRF microneedling group were significantly lower than those in the PRP microneedling group (erythema: t=3.46, P=0.004; wrinkles: t=3.82, P=0.002; capillary dilation: t=4.17, P=0.001; color uniformity: t=3.19, P=0.006). Among them, the scores of capillary dilation and color uniformity were close to the level of the blank group, while the scores of erythema and wrinkles were significantly reduced. This suggests that the photoaging repair effect of PRF microneedling is significantly better than that of PRP microneedling, and it can more effectively improve vascular damage and pigmentation abnormalities.

[0065] The results of macroscopic changes in the skin at week 8 are shown in Table 4 below.

[0066] Table 4

[0067]

[0068] Verification of the effectiveness of long-term photoaging: After 8 weeks of ultraviolet irradiation, the control group showed severe levels of all photoaging indicators, which were significantly higher than those of the blank group (erythema: t=19.05, P<0.001; wrinkles: t=23.09, P<0.001; capillary dilation: t=16.83, P<0.001; color uniformity: t=18.52, P<0.001). The results showed extensive fusion of erythema, dense and deep wrinkles, reticular dilation of capillaries, and severe uneven pigmentation, confirming the success of long-term photoaging modeling and a significant increase in the degree of damage compared to 4 weeks.

[0069] Comparison of long-term repair effects of PRP and PRF microneedling:

[0070] The scores of all indicators in the PRP microneedling group were significantly lower than those in the control group (P < 0.001), and further improved compared with 4 weeks, suggesting that the repair effect of PRP has a certain degree of persistence, but mild photoaging manifestations still remain (erythema grade 1.33, wrinkle grade 1.25).

[0071] The scores of all indicators in the PRF microneedling group were significantly lower than those in the PRP microneedling group (erythema: t=4.33, P<0.001; wrinkles: t=4.58, P<0.001; telangiectasia: t=3.71, P=0.002; color uniformity: t=3.46, P=0.004), and the scores of all indicators were close to those of the blank group. Among them, the telangiectasia score was only 0.08, and the skin wrinkles basically returned to their natural state. This suggests that the long-term repair effect of PRF microneedling is more significant and stable, and can effectively reverse the structural damage and functional abnormalities caused by long-term photoaging.

[0072] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0073] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for preparing a cryomicroneedle patch for preventing skin photoaging, characterized in that, Includes the following steps: S1. Place the venous blood in a blood collection tube and centrifuge it. S2. After centrifugation, remove the middle layer of platelet-rich fibrin gel. S3. Inject platelet-rich fibrin gel into the microneedle mold and fill the needle cavity, then let it stand. S4. After pre-cooling the microneedle mold, it is then frozen and molded. After molding, it is demolded to obtain a frozen microneedle patch.

2. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: The collected venous blood was centrifuged within 2 minutes.

3. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: Venous blood was centrifuged at 3000 rpm for 15 minutes at room temperature.

4. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: After centrifugation, the middle layer of platelet-rich fibrin gel was removed and processed under aseptic conditions.

5. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: The microneedle mold is a 10x10 array mold with a needle height of 120 μm and a needle tip angle of 19°.

6. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: After injecting platelet-rich fibrin gel into the microneedle mold, the mold is vibrated to remove bubbles or centrifuged at low speed. Once the platelet-rich fibrin gel fills the needle cavity, it is left to stand at room temperature for 2-3 minutes.

7. The method for preparing a cryomicroneedle patch for preventing skin photoaging as described in claim 1, characterized in that: After pre-cooling the microneedle mold filled with platelet-rich fibrin gel at -20°C for 2-3 hours, it was frozen at -80°C for 8 hours to form the shape.

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