A polynucleotide complex anti-aging microneedle patch and a preparation method thereof

Abstract

This invention belongs to the field of microneedle formulation technology and discloses a polynucleotide composite anti-aging microneedle patch and its preparation method. The microneedle patch includes a needle body and a backing, and contains the following raw materials in the indicated mass fractions: polyvinyl alcohol 4-7%, waterborne polyurethane 1-3%, sodium hyaluronate 1-2%, polynucleotide 0.1-2%, glycerin 0.5-1%, trehalose 0-2%, and the balance being water. Through microneedle-mediated physical penetration, PN directly reaches the target point in the dermis, which is significantly superior to topical application. The PVA-PU composite matrix endows the microneedles with excellent mechanical toughness, with a single needle breaking force greater than 0.1N, making it less prone to breakage during puncture and maintaining high needle tip integrity upon demolding.

Description

Technical Field

[0001] This invention relates to the field of microneedle formulation technology, and in particular to a polynucleotide composite anti-aging microneedle patch and its preparation method. Background Technology

[0002] Microneedling is an innovative technique that penetrates tissue in a minimally invasive and painless manner. It not only avoids the additional scarring that can result from traditional methods but also precisely delivers medication to the wound site for targeted treatment. Microneedles with their unique structures show great promise for controlled wound healing. However, several technical challenges remain to be addressed in the field of microneedle-mediated wound healing.

[0003] Skin aging, especially photoaging and natural aging, is characterized by a decrease in the proliferation capacity of dermal fibroblasts, a reduction in the synthesis of type I and type IV collagen, and disordered degradation of the extracellular matrix (ECM), ultimately leading to skin laxity, deeper wrinkles, and dermal atrophy.

[0004] Currently, mainstream anti-aging methods targeting dermal regeneration have significant limitations: Low delivery efficiency of topical preparations: Conventional topical polynucleotide (PN) preparations such as lotions and serums are limited by the barrier function of the stratum corneum. PN with larger molecular weights cannot effectively penetrate into the dermis and can only play a role in epidermal moisturization. They are difficult to achieve deep collagen regeneration and have weak anti-aging effects.

[0005] Injection therapy is highly invasive: Clinically, PN is injected directly into the dermis through water-light injection. Although the effect is certain, it is an invasive procedure with risks of pain, bruising, and infection. In addition, it is highly dependent on professional physicians, and patients have poor compliance, which cannot meet the needs of long-term home maintenance.

[0006] Existing microneedles have insufficient regenerative capacity: Commercially available soluble microneedles are mostly loaded with ingredients such as hyaluronic acid, peptides or vitamins, lacking core ingredients with medical-grade dermal repair activity. They cannot activate fibroblasts from the root to promote the massive regeneration of collagen, and their anti-aging depth and effect are limited.

[0007] Therefore, developing a deep dermal regeneration technology that can both break through the stratum corneum barrier and achieve efficient delivery of macromolecular active substances, while also being non-invasive, safe, and suitable for home use, is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a polynucleotide composite anti-aging microneedle patch and its preparation method, thereby solving the above-mentioned problems existing in the prior art.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a polynucleotide composite anti-aging microneedle patch, comprising a needle body and a backing; The polynucleotide composite anti-aging microneedle patch contains the following raw materials in the indicated mass fractions: 4-7% polyvinyl alcohol, 1-3% waterborne polyurethane, 1-2% sodium hyaluronate, 0.1-2% polynucleotide, 0.5-1% glycerin, 0-2% trehalose, and the remainder is water.

[0010] Preferably, the molecular weight of the polynucleotide is 500~1500 bp.

[0011] Preferably, the polyvinyl alcohol is PVA1788.

[0012] This invention also provides a method for preparing a polynucleotide composite anti-aging microneedle patch, comprising the following steps: Polyvinyl alcohol, waterborne polyurethane, glycerin, sodium hyaluronate, and water are mixed to obtain a matrix solution. After cooling the matrix solution, polynucleotides and trehalose are added and stirred at low speed to obtain a microneedle solution. The microneedle solution is vacuum degassed and poured into a microneedle mold, and then centrifuged. After centrifugation, it is cured, then demolded and sterilized to obtain a polynucleotide composite anti-aging microneedle patch.

[0013] Preferably, the temperature of the matrix solution after cooling is 2~6℃.

[0014] Preferably, the speed of the low-speed stirring is 100~300 rpm.

[0015] Preferably, the vacuum degree of the vacuum degassing is 0.08~0.1MPa, and the time is 20~40min.

[0016] Preferably, the centrifugation speed is 3000~5000 rpm and the time is 10~20 min.

[0017] Preferably, the curing temperature is 4~10℃, the vacuum degree is -0.07~-0.09MPa, and the time is 16~24h.

[0018] In this invention, the polynucleotide composite anti-aging microneedle patch has the following characteristics: (1) It is the first to combine medical-grade PN with soluble microneedle technology. Through the nucleic acid biological effect of PN, it regulates fibroblast function at the gene transcription level and realizes deep collagen regeneration in the dermis, which is different from traditional microneedles that only provide physical support or nutritional supply. (2) It transforms clinical injection-grade PN into a soluble microneedle patch, filling the dosage form gap of potent dermal regeneration products for home use, and realizing non-invasive and efficient transdermal delivery of macromolecular active substances. (3) It adopts a low-temperature drug loading and mild mixing process, which effectively avoids the degradation of the long chain structure of PN by temperature and shear force during the preparation process, and ensures the efficient performance of its in vivo biological activity. (4) Through the formulation design (polyvinyl alcohol-waterborne polyurethane composite system and hyaluronic acid compound), the microneedles have sufficient mechanical strength to puncture the skin. At the same time, the needle tip is rich in PN, which dissolves and releases the drug quickly after piercing the dermis, realizing the combination of rapid effect and sustained effect. (5) The laboratory-available polyvinyl alcohol + waterborne polyurethane compound system completely replaces the traditional polyvinyl alcohol skeleton. The low concentration of sodium hyaluronate avoids the problem of dissolution and clumping. The waterborne polyurethane flexible toughening modified polyvinyl alcohol matrix solves the defects of high dry brittleness and easy breakage of pure polyvinyl alcohol microneedles, and takes into account both puncture hardness and molding integrity.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) High-efficiency delivery: Through microneedle-mediated physical penetration, PN directly reaches the target point in the dermis, which is significantly better than topical application.

[0020] (2) Safe and non-invasive: The microneedles only penetrate the stratum corneum and do not touch the pain nerves, so as to achieve painless drug delivery; the material is completely soluble and there is no risk of needle residue, so it can be used safely at home.

[0021] (3) Excellent performance: The PVA-PU composite matrix gives the microneedles excellent mechanical toughness, with a single needle breaking force greater than 0.1N, making them less prone to breakage during puncture and ensuring high integrity of the needle tip after demolding.

[0022] (4) Simple preparation and controllable cost: The process conditions are mild, the whole process is an aqueous system, no organic solvents are required, no high temperature and high pressure equipment is required, and conventional laboratory equipment can be used to complete the production; the raw materials are all bulk medical grade polymer materials, which are widely available and have stable prices, significantly reducing the cost of raw material procurement and production threshold, and making it easy to scale up production and promote industrialization.

[0023] (5) Strong process adaptability: Differentiated drying schemes are set up, low temperature vacuum protects the activity of PN-containing samples, and the blank control group can use higher temperatures to accelerate drying, adapting to different laboratory equipment conditions.

[0024] (6) Good economic efficiency: The process steps are simple, the energy consumption is low, there are no special environmental requirements, and it has good economies of scale and industrial transformation potential. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0026] Figure 1 This is a process flow diagram for the preparation of the PN composite anti-aging microneedle patch of the present invention; Figure 2 The images show the morphology of the PN composite anti-aging microneedle patch prepared in Example 1; where (a) is a physical image of the microneedle patch, (b) is a top view of the microneedle array under an optical microscope, and (c) is a magnified image of the microneedles. Figure 3 The mechanical property test curve of the PN composite anti-aging microneedle patch prepared in Example 1 is shown. Figure 4 The image shows the cell compatibility evaluation results of the PN composite anti-aging microneedle patch prepared in Example 1; where (a) is a quantitative bar chart of the viability of fibroblasts in different treatments detected by CCK-8 method, and (b) is a Calcein-AM live cell fluorescence staining image of the blank microneedle group and the PN composite microneedle group. Figure 5 The images show the effect of the PN composite anti-aging microneedle patch prepared in Example 1 on the healing and repair of mouse skin wounds; where (a) is a representative photo of the mouse back wound before treatment, and (b) is a representative photo of the mouse back wound healing after treatment. Detailed Implementation

[0027] This invention provides a polynucleotide composite anti-aging microneedle patch, comprising a needle body and a backing.

[0028] In this invention, the polynucleotide composite anti-aging microneedle patch contains the following raw materials in the following mass fractions: 4-7% polyvinyl alcohol, 1-3% waterborne polyurethane (PU), 1-2% sodium hyaluronate (HA), 0.1-2% polynucleotide (PN), 0.5-1% glycerol, 0-2% trehalose, and the balance being water.

[0029] In this invention, the needle body and the backing are integrally formed from the same polymer matrix, and the active ingredient PN is mainly concentrated in the needle body, while the PN content in the backing is relatively low.

[0030] In this invention, the mass fraction of the polyvinyl alcohol is preferably 4-6%, and more preferably 5-6%.

[0031] In this invention, the mass fraction of the waterborne polyurethane is preferably 1.5 to 2.5%, and more preferably 2 to 2.5%.

[0032] In this invention, the mass fraction of sodium hyaluronate is preferably 1.2-1.8%, and more preferably 1.3-1.6%.

[0033] In this invention, the mass fraction of the polynucleotide is preferably 0.5-1.5%, more preferably 0.8-1%.

[0034] In this invention, the mass fraction of glycerol is preferably 0.6-0.9%, more preferably 0.7-0.8%.

[0035] In this invention, the mass fraction of trehalose is preferably 0.5-1.5%, and more preferably 1-1.5%.

[0036] In this invention, the molecular weight of the polynucleotide is preferably 500-1500 bp, and more preferably 800-1200 bp.

[0037] In this invention, the polyvinyl alcohol is preferably PVA1788, sourced from Sinopharm Chemical Reagent Co., Ltd.; degree of alcoholysis 87-89%; analytical grade.

[0038] In this invention, the aqueous polyurethane is sourced from Shanghai McLean Biochemical Technology Co., Ltd.; W650804, analytical grade.

[0039] In this invention, the sodium hyaluronate is sourced from Bloomage Biotechnology Co., Ltd.; its molecular weight is 100,000 to 1,500,000 Daltons; it is injectable grade sodium hyaluronate.

[0040] In this invention, the polynucleotide is sourced from Saimeng Biotechnology Co., Ltd.; long-chain salmon protamine DNA sodium (PN-M); medical injection grade.

[0041] In this invention, the glycerin is sourced from Sinopharm Chemical Reagent Co., Ltd.; it is pharmaceutical grade excipient.

[0042] In this invention, the trehalose is sourced from Shanghai Yuanye Biotechnology Co., Ltd.; pharmaceutical grade; S11052, purity ≥98%.

[0043] This invention also provides a method for preparing a polynucleotide composite anti-aging microneedle patch, the preparation process of which is shown in the schematic diagram below. Figure 1 As shown, it includes the following steps: Polyvinyl alcohol, waterborne polyurethane, glycerin, sodium hyaluronate, and water are mixed to obtain a matrix solution. After cooling the matrix solution, polynucleotides and trehalose are added and stirred at low speed to obtain a microneedle solution. The microneedle solution is vacuum degassed and poured into a microneedle mold, and then centrifuged. After centrifugation, it is cured, then demolded and sterilized to obtain a polynucleotide composite anti-aging microneedle patch.

[0044] In this invention, the preferred method for mixing polyvinyl alcohol, waterborne polyurethane, glycerin, sodium hyaluronate, and water is as follows: heat water to 80-90°C, add polyvinyl alcohol, and stir at a constant temperature of 400-500 rpm for 1.5-2 hours until completely dissolved; cool to room temperature, add glycerin and waterborne polyurethane, and stir for 30 minutes to mix evenly; then add sodium hyaluronate in portions and stir until completely dissolved.

[0045] In this invention, the temperature of the matrix solution after cooling is preferably 2~6℃, and more preferably 3~5℃.

[0046] In this invention, the speed of the low-speed stirring is preferably 100~300 rpm, and more preferably 150~250 rpm.

[0047] In this invention, the vacuum degree of the vacuum degassing is preferably 0.08~0.1MPa, more preferably 0.09~0.1MPa, and the time is preferably 20~40min, more preferably 25~35min.

[0048] In this invention, the centrifugation speed is preferably 3000~5000 rpm, more preferably 3200~4500 rpm, and the centrifugation time is preferably 10~20 min, more preferably 15~18 min.

[0049] In this invention, the curing temperature is preferably 4~10℃, more preferably 4~6℃, the vacuum degree is preferably -0.07~-0.09MPa, more preferably -0.08~-0.09MPa, and the time is preferably 16~24h, more preferably 18~22h.

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] A PN composite anti-aging microneedle patch comprises the following raw materials by mass fraction: PVA1788 5%, PU 2%, HA 1.5%, PN (1000bp) 1%, glycerol 0.8%, trehalose 1%, deionized water 88.7%.

[0053] Accurately weigh the following 10g microneedle solution: PVA1788: 0.500g, PU: 0.200g, HA: 0.150g, PN (1000bp): 0.100g, glycerol: 0.080g, trehalose: 0.100g, deionized water: 8.870g.

[0054] The preparation method of PN composite anti-aging microneedle patch includes the following steps: (1) Take 8.870g of deionized water and heat it to 85℃. Add 0.500g of PVA1788 and stir for 2 hours to dissolve. Cool to room temperature and add 0.080g of glycerin and 0.200g of PU and stir for 30 minutes. Sprinkle 0.150g of sodium hyaluronate powder on the liquid surface in 3 batches while stirring continuously. After the first batch of powder has fully swollen and dispersed, add the next batch of powder. Stir until the system is completely clear and free of fish-eye lumps to obtain the matrix solution.

[0055] (2) Cool the matrix solution to 4°C, add 0.100g PN and 0.100g trehalose, stir at 200rpm for 20min to mix well, use low speed and gentle stirring to avoid generating bubbles and mechanical shearing force to destroy the nucleic acid structure of PN, so as to fully preserve its biological activity and obtain microneedle solution; (3) Degas the microneedle solution under vacuum of 0.09 MPa for 30 min to remove dissolved microbubbles from the system; (4) After degassing, the mixture was poured into a polydimethylsiloxane (PDMS) microneedle mold (needle height 800 μm), and then the mold was placed in a centrifuge and centrifuged at 4000 rpm for 15 min. (5) After centrifugation, the product is dried under vacuum at 4℃ and -0.09MPa for 20 hours to solidify. (6) Demolding and sterilization to obtain the finished PN composite anti-aging microneedle patch.

[0056] Example 2

[0057] Unlike Example 1, the mass fractions in this example are: PVA1788 7%, PU 1%, HA 1%, PN (1000bp) 0.5%, glycerol 0.5%, trehalose 0.5%, and deionized water 89.5%.

[0058] The preparation process is the same as in Example 1.

[0059] Example 3

[0060] Unlike Example 1, the mass fractions in this example are: PVA1788 4%, PU 3%, HA 2%, PN (1000bp) 2%, glycerol 1%, trehalose 2%, and water 86%.

[0061] The preparation process is the same as in Example 1.

[0062] Comparative Example 1

[0063] Unlike Example 1, in this comparative example, PN was removed from the raw material and water was added to make up to 100%; in step (5), drying was performed at 40°C under vacuum for 10 hours. This was recorded as the blank microneedle group.

[0064] Comparative Example 2

[0065] Unlike Example 1, instead of preparing microneedles, a 1% aqueous solution of PN (1000bp) was used as a topical application and applied directly to the skin surface. This group was designated as the PN topical application group.

[0066] Comparative Example 3

[0067] Unlike Example 1, PN was replaced with an equal mass fraction of a conventional anti-aging ingredient (vitamin C), while the remaining components and preparation process remained unchanged. This was designated as the conventional microneedle group.

[0068] Performance testing and effect verification

[0069] 1. Characterization of microneedle morphology

[0070] The morphology of the PN composite anti-aging microneedle patch prepared in Example 1 was observed using an optical microscope. The results are as follows: Figure 2 As shown, the microneedle patch is intact, the needles are evenly arranged, and each microneedle is conical in shape. The needles are intact, without breaks or bubbles, and the needle height is about 800 μm, indicating that the preparation method of the present invention has good formability.

[0071] 2. Microneedle Mechanical Property Testing

[0072] The microneedles prepared in Example 1 were subjected to compression tests using a universal tensile testing machine, and the force-displacement curves were recorded. The results are as follows: Figure 3 As shown, the breaking force of a single needle is greater than 0.1N, which is significantly higher than the minimum force required to penetrate the stratum corneum of human skin (approximately 0.045N), proving that it has good puncture performance.

[0073] 3. Cell compatibility evaluation

[0074] The effects of microneedle extract on the survival of human skin fibroblasts were investigated using the CCK-8 assay and Calcein-AM live cell fluorescence staining method, and the biosafety of the PN composite anti-aging microneedle patch of the present invention was evaluated. The microneedle extract was prepared as follows: Duchenne modified Eagle medium (DMEM, high-glucose) with 10% fetal bovine serum was used as the extraction medium; blank microneedle patches from Comparative Example 1 and PN composite anti-aging microneedle patches from Example 1 were soaked at a ratio of sample mass:extraction medium = 0.1 g: 1 mL, and allowed to stand at 37°C for 24 h. The supernatant was separated and used as the blank microneedle extract and the PN composite microneedle extract, respectively. Human skin fibroblasts were divided into three groups: (a) control group (normal culture, without microneedles); (b) blank microneedle group (with blank microneedle extract); (c) PN composite microneedle group (with PN composite microneedle extract). After co-culturing for 24 hours, CCK-8 quantification and Calcein-AM live-cell fluorescence staining were performed, and the results are as follows: Figure 4 As shown.

[0075] Depend on Figure 4 The CCK-8 quantitative results in (a) showed that the cell viability of both the blank microneedle group and the PN composite microneedle group remained above 90%, with no significant difference compared with the control group (P>0.05), indicating that neither the blank microneedle matrix nor the PN composite microneedle extract had a significant inhibitory effect on fibroblast proliferation.

[0076] Depend on Figure 4 The Calcein-AM live cell fluorescence staining results in (b) show that the PN composite microneedle group has abundant green live cell fluorescence signals, and the cells are evenly distributed, plump, and spread normally. There is no significant difference compared with the control group, and no large number of cell apoptosis, detachment, or damage are observed.

[0077] The above results indicate that the PN composite anti-aging microneedle patch of the present invention has good cell compatibility, no significant cytotoxicity, and good biosafety, making it suitable for transdermal delivery to the skin.

[0078] 4. Evaluation of the efficacy of internal wound healing

[0079] A full-thickness skin resection wound model was established on the mouse back to evaluate the in vivo healing-promoting effect of the PN composite anti-aging microneedle patch of the present invention. After disinfecting the mouse back skin, full-thickness skin resection wounds with a diameter of 6 mm were made symmetrically on both sides of the midline of the back using a punch. The PN composite anti-aging microneedle patch prepared in Example 1 was then applied to the wound site, and gentle pressure was applied to allow the microneedles to penetrate the skin. The patch was changed every 2 days for 10 consecutive days. Representative photographs of the mouse back wounds were taken before treatment (day 0) and after treatment (day 10). The results are as follows: Figure 5 As shown.

[0080] Depend on Figure 5 As shown in (a), before treatment, the mice had circular full-thickness skin excision wounds with consistent diameter on their backs, with clear wound surfaces, regular boundaries, and consistent wound conditions.

[0081] Depend on Figure 5 As shown in Figure (b), after 10 days of continuous treatment with PN composite anti-aging microneedle patches, the wound area on the back of the mice was significantly reduced, the wound surface was basically closed, and only a small area of ​​incomplete epithelialization remained, indicating a good healing effect. During the treatment, the mice were in good mental condition, had normal eating and activity levels, and no infection or adverse reactions were observed.

[0082] The above results indicate that the PN composite anti-aging microneedle patch of the present invention can accurately deliver the PN active ingredients to the wound tissue, effectively promoting the healing and tissue repair of full-thickness skin excision wounds.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polynucleotide composite anti-aging microneedle patch, characterized in that, Includes the needle body and backing; The polynucleotide composite anti-aging microneedle patch contains the following raw materials in the indicated mass fractions: 4-7% polyvinyl alcohol, 1-3% waterborne polyurethane, 1-2% sodium hyaluronate, 0.1-2% polynucleotide, 0.5-1% glycerin, 0-2% trehalose, and the remainder is water.

2. The polynucleotide composite anti-aging microneedle patch according to claim 1, characterized in that, The molecular weight of the polynucleotide is 500~1500bp.

3. The polynucleotide composite anti-aging microneedle patch according to claim 1, characterized in that, The polyvinyl alcohol is PVA1788.

4. A method for preparing a polynucleotide composite anti-aging microneedle patch according to any one of claims 1 to 3, characterized in that, Includes the following steps: Polyvinyl alcohol, aqueous polyurethane, glycerol, sodium hyaluronate, and water were mixed to obtain a matrix solution. After cooling the matrix solution, polynucleotides and trehalose were added, and the mixture was stirred at low speed to obtain a microneedle solution. The microneedle solution was degassed under vacuum and then poured into a microneedle mold, followed by centrifugation. After centrifugation, the material is solidified, then demolded and sterilized to obtain a polynucleotide composite anti-aging microneedle patch.

5. The method for preparing a polynucleotide composite anti-aging microneedle patch according to claim 4, characterized in that, The temperature of the matrix solution after cooling is 2~6℃.

6. The method for preparing a polynucleotide composite anti-aging microneedle patch according to claim 4, characterized in that, The speed of the low-speed stirring is 100~300 rpm.

7. The method for preparing a polynucleotide composite anti-aging microneedle patch according to claim 4, characterized in that, The vacuum degassing process is performed at a vacuum level of 0.08~0.1MPa for a time of 20~40min.

8. The method for preparing a polynucleotide composite anti-aging microneedle patch according to claim 4, characterized in that, The centrifugation speed is 3000~5000 rpm, and the time is 10~20 min.

9. The method for preparing a polynucleotide composite anti-aging microneedle patch according to claim 4, characterized in that, The curing temperature is 4~10℃, the vacuum degree is -0.07~-0.09MPa, and the time is 16~24h.

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