PDRN-sodium hyaluronate cross-linked double-drug-loading microneedle patch as well as preparation method and application of PDRN-sodium hyaluronate cross-linked double-drug-loading microneedle patch

Abstract

The invention relates to a PDRN-sodium hyaluronate cross-linked double-drug-loading microneedle patch as well as a preparation method and application of the PDRN-sodium hyaluronate cross-linked double-drug-loading microneedle patch. The double-drug-loading microneedle patch is composed of a needle point drug-loading layer and a cross-linked substrate drug-loading layer, the needle point medicine carrying layer is prepared from L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, glycine, sodium hyaluronate and trehalose; the cross-linked substrate medicine carrying layer is prepared from a PDRN-sulfhydrylated HA copolymer, minoxidil, L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, glycine and trehalose. The amino acid anti-hair loss and hair growth microneedle system is innovatively developed, a double-drug-loading layered design is adopted, the needle point drug-loading layer is formed by wrapping quick-release amino acid with low-molecular-weight hyaluronic acid (HA), the cross-linked substrate drug-loading layer is formed by wrapping slow-release amino acid, PDRN and minoxidil with high-molecular-weight hyaluronic acid, and instant and long-acting synergy is achieved.

Description

Technical Field

[0001] The present invention relates to a PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch and a preparation method and application thereof, belonging to the technical field of hair loss prevention and regeneration. Background Art

[0002] As an important appendage of the human skin, hair growth directly impacts personal image and mental health. Keratin, the primary component of hair, is a fibrous protein formed by a complex spatial structure of multiple amino acids. Its unique chemical composition and molecular structure directly determine the mechanical strength, elasticity, and biological functions of hair. Modern research indicates that healthy hair growth depends on the homeostasis of the hair follicle microenvironment and a continuous supply of nutrients. Deficiency or metabolic abnormalities of specific amino acids are key factors contributing to hair loss.

[0003] In the amino acid composition of hair keratin, cysteine ​​is a key structural amino acid, with a content as high as 10-17%. The sulfhydryl group (-SH) in the cysteine ​​molecule forms a stable disulfide bond (-SS-) through oxidation, forming a three-dimensional network structure of keratin, providing mechanical support for hair. At the same time, cysteine ​​also has significant antioxidant activity, which can effectively remove reactive oxygen free radicals (ROS) around the hair follicles and protect hair follicle stem cells from oxidative damage. Clinical data show that the cysteine ​​content in the hair shaft of patients with hair loss is generally 15-20% lower than that of normal people, which directly leads to a decrease in hair strength and easy breakage.

[0004] Glutamate accounts for 12-16% of hair keratin and is a core substance in hair follicle energy metabolism. As a key player in the tricarboxylic acid cycle, glutamate participates in ATP synthesis through conversion to α-ketoglutarate, providing energy support for rapidly dividing hair matrix cells. Studies have confirmed that glutamate metabolic activity is significantly higher during the anagen phase of hair follicles than during the catagen and telogen phases. Furthermore, the carboxyl group of glutamate can form hydrogen bonds with water molecules, maintaining proper hydration of keratin and ensuring hair flexibility and gloss.

[0005] Arginine (6-8%) plays multiple roles in regulating hair growth. On the one hand, arginine produces NO through the nitric oxide synthase (NOS) pathway, causing vasodilation around the hair follicles, increasing blood perfusion and nutrient delivery. On the other hand, arginine can upregulate the expression of vascular endothelial growth factor (VEGF) and promote the proliferation of dermal papilla cells. Animal experiments have shown that topical application of arginine can extend the growth phase of hair follicles in mice by 30% and increase hair density by 25%. Other essential amino acids also play irreplaceable roles: serine (7-9%) participates in the formation of the α-helical structure of keratin; lysine stabilizes keratin structure through ε-amino cross-linking and promotes the absorption of trace elements such as iron and zinc; proline is an important raw material for collagen synthesis and can reduce fibrotic changes around the hair follicles in patients with androgenic alopecia; and glycine activates hair follicle stem cells by regulating the Wnt / β-catenin signaling pathway, promoting hair follicle regeneration.

[0006] However, traditional anti-hair loss and hair growth products face numerous challenges: oral amino acid supplements have low bioavailability (<10%) and systemic distribution makes it difficult to target hair follicles; topical preparations are limited by the stratum corneum barrier, with a transdermal absorption rate of less than 5%. Single active ingredients, such as minoxidil, are only 30-40% effective for androgenic alopecia (even lower at 20-30% in women), are virtually ineffective against types such as cicatricial alopecia and alopecia areata, and fail to improve structural changes such as follicular fibrosis, limiting their efficacy. Within 3-6 months after discontinuation of the drug, the rate of new hair loss is as high as 80%, requiring lifelong maintenance treatment due to impaired transdermal absorption and high drug dependency. Furthermore, it only improves blood supply but cannot repair damaged keratin (lack of structural amino acids such as cysteine), inhibit DHT (requiring combination with a 5α-reductase inhibitor), or combat oxidative stress in hair follicles. It is difficult to comprehensively address the multiple pathological mechanisms involved in hair loss.

[0007] For example, Chinese patent document CN116570549A discloses a hyaluronic acid microneedle loaded with minoxidil, its preparation method and application. However, this patent only targets a single hair loss mechanism (vasodilation) and cannot synergistically solve multiple problems such as hair follicle atrophy and oxidative stress. Conventional hair growth fluids have low transdermal efficiency (<5%) and are easily degraded by sebum. In view of the current single function: existing microneedles are mostly loaded with a single hair growth component (such as minoxidil or PDRN), lacking multi-target synergy. Release is uncontrollable: non-cross-linked microneedles release drugs suddenly (>90% release within 24 hours), and cannot maintain long-term treatment. Insufficient penetration: traditional smear-type amino acids have technical defects such as difficulty penetrating the stratum corneum to reach the hair follicles. In response to the shortcomings of the existing technology, the present invention provides a method for preparing a PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch. The present invention provides a dual-drug-loaded microneedle system that quickly releases an amino acid combination through the needle tip to instantly improve the hair follicle microenvironment. The PDRN-sodium hyaluronate cross-linked network enables sustained release of amino acid combination, PDRN and minoxidil from the substrate, which activates dermal papilla cells for a long time

[0008] In view of this, starting from a soluble microneedle array, developing a product that can break through the skin barrier, deliver anti-hair loss and hair growth ingredients directly to the hair follicle area, and at the same time improve the skin environment will provide a breakthrough solution to the problem of hair loss. Summary of the Invention

[0009] To address the shortcomings of the existing technology, the present invention provides a PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch, its preparation method, and its application. The present invention rapidly releases an amino acid combination from the needle tip, instantly improving the hair follicle microenvironment. Simultaneously, the PDRN-sodium hyaluronate cross-linked network enables sustained release of the amino acid combination, PDRN, and minoxidil from the base, resulting in long-lasting activation of dermal papilla cells. This patch has promising application prospects and economic value in preventing hair loss and regeneration.

[0010] The technical solutions of the present invention are as follows:

[0011] A PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch, consisting of a needle tip drug-loaded layer and a cross-linked base drug-loaded layer;

[0012] The needle tip drug-carrying layer comprises the following components in parts by weight: 1 to 5 parts of L-cysteine, 1 to 5 parts of L-glutamine, 1 to 3 parts of L-arginine, 1 to 3 parts of serine, 0.1 to 1.5 parts of L-lysine hydrochloride, 0.1 to 1.5 parts of L-proline, 0.1 to 1.5 parts of glycine, 20 to 25 parts of sodium hyaluronate, and 1 to 5 parts of trehalose;

[0013] The cross-linked base drug-carrying layer includes the following components in parts by mass: 20 to 50 parts of PDRN-thiolated HA copolymer, 1 to 10 parts of minoxidil, 2 to 10 parts of L-cysteine, 2 to 10 parts of L-glutamine, 3 to 8 parts of L-arginine, 3 to 8 parts of serine, 1 to 5 parts of L-lysine hydrochloride, 1 to 5 parts of L-proline, 1 to 5 parts of glycine, and 5 to 15 parts of trehalose.

[0014] According to the present invention, the needle tip drug-carrying layer preferably comprises the following components in parts by mass: 2.25 parts of L-cysteine, 2.25 parts of L-glutamine, 1.5 parts of L-arginine, 1.5 parts of serine, 0.75 parts of L-lysine hydrochloride, 0.75 parts of L-proline, 0.75 parts of glycine, 22.5 parts of sodium hyaluronate, and 2.5 parts of trehalose;

[0015] The cross-linked substrate drug-carrying layer includes the following components in parts by mass: 40 parts of PDRN-thiolated HA copolymer, 2 parts of minoxidil, 6.75 parts of L-cysteine, 6.75 parts of L-glutamine, 4.5 parts of L-arginine, 4.5 parts of serine, 2.25 parts of L-lysine hydrochloride, 2.25 parts of L-proline, 2.25 parts of glycine, and 6 parts of trehalose.

[0016] Preferably according to the present invention, the molecular weight of the sodium hyaluronate is 100 to 300 KDa.

[0017] Preferably, according to the present invention, the PDRN-thiolated HA copolymer is prepared according to the following method:

[0018] (1) Under nitrogen protection, sodium hyaluronate and iminothiolane were added to PBS buffer, stirred and mixed evenly, and then stirred and reacted at 20-30° C. for 2-6 hours to obtain a thiolated hyaluronic acid solution;

[0019] (2) After adjusting the pH of the thiolated hyaluronic acid solution to 6.0-7.0, PDRN and a cross-linking agent were added, and cross-linked at 4°C for 2-4 hours to obtain a PDRN-thiolated HA gel;

[0020] (3) After homogenizing the PDRN-thiolated HA gel for 5 to 30 minutes, ethanol was added. After the final volume of ethanol reached 60 to 80%, the supernatant was removed and ethanol was continued to be added. After the final volume of ethanol reached 80 to 90%, the supernatant was removed and the lower layer solid-liquid mixture was obtained; after filtration and freeze-drying, the PDRN-thiolated HA copolymer was obtained.

[0021] Further preferably, in step (1), the molecular weight of the sodium hyaluronate is 1000-2000 KDa; and the PBS buffer is a PBS buffer containing 1 mM EDTA and a pH of 8.0-8.5.

[0022] Further preferably, in step (1), the mass-to-volume ratio of the sodium hyaluronate and the PBS buffer is (5-15):450, unit: g / mL; the mass-to-volume ratio of the iminothiolane and the PBS buffer is (1-2):450, unit: g / mL.

[0023] Further preferably, in step (2), the mass-to-volume ratio of the PDRN and sodium hyaluronate is 1:(5-15); the cross-linking agent is a composition of EDC and NHS, the molar ratio of EDC and NHS is 1:1.5, and the added amount is 90-95% of the mass of sodium hyaluronate.

[0024] The preparation method of the above-mentioned PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch comprises the following steps:

[0025] 1) L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, and glycine were dissolved in PBS buffer according to the ratio and stirred to obtain an amino acid solution; sodium hyaluronate and trehalose were added to the amino acid solution according to the ratio, centrifuged at 2500-3500 rpm for 10-20 minutes, and then poured into the needle tip of the PDMS mold to obtain a needle tip drug loading layer;

[0026] 2) According to the ratio, PDRN-thiolated HA copolymer, minoxidil, L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, glycine, and trehalose were dissolved in PBS buffer, stirred and mixed evenly, and poured into the base of the PDMS mold after centrifugation at 2500-3500 rpm for 10-20 min. The mixture was vacuum dried for 20-30 h, the microneedle array was removed, pressurized at 0.1 MPa for 5-15 s, and dried again at 20-30°C for 10-15 h to obtain a PDRN-sodium hyaluronate cross-linked dual-drug microneedle patch.

[0027] According to the preferred embodiment of the present invention, in step 1), the PBS buffer is a PBS buffer with a pH of 5.0.

[0028] According to the preferred embodiment of the present invention, in step 2), the PBS buffer solution contains 1-5 mM Zn 2+ , PBS buffer with pH = 5.5-6.5.

[0029] The above-mentioned PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch can be used to prepare hair loss prevention and regeneration products.

[0030] The technical features and beneficial effects of the present invention are as follows:

[0031] 1. The present invention innovatively develops an amino acid anti-hair loss microneedle system. The provided PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch adopts a dual-drug-loaded layered design. The needle tip drug-loaded layer is a low molecular weight hyaluronic acid (HA) wrapped with rapid-release amino acids, and the cross-linked base drug-loaded layer is a high molecular weight hyaluronic acid wrapped with sustained-release amino acids, PDRN and minoxidil, achieving immediate and long-term synergy. In addition, the present invention breaks through the skin barrier through a soluble microneedle array, and delivers a carefully proportioned amino acid combination directly to the hair follicle area. At the same time, it combines the cell repair function of polydeoxyribonucleotides (PDRN) and the slow release and strong hydration of sodium hyaluronate to improve the skin environment. It realizes multi-target, multi-mechanism synergistic treatment to achieve the effect of hair loss prevention and regeneration.

[0032] 2. The needle tip drug-carrying layer of the present invention is pH responsive and can dissolve quickly in the weakly acidic environment of the scalp (pH = 5.5-6.0). The HA and PDRN in the cross-linked base drug-carrying layer form a protective network through EDC / NHS cross-linking, which can avoid the destruction of minoxidil activity in a neutral environment and achieve a sustained release effect. In addition, L-cysteine ​​in the cross-linked base drug-carrying layer can bind to the HA-SH disulfide bond, and L-arginine can bind to Zn 2+ Complex, L-glutamine in Zn 2+ Zn is formed under the conditions 2+ -Glutamine-HA ternary complex. These three binding modes make the amino acids in the cross-linked base drug-carrying layer more tightly bound to the PDRN-thiolated HA gel, and the sustained-release ability is stronger.

[0033] 3. The preparation method of the PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch provided by the present invention has few steps, simple operation, uses green and safe raw materials, has no adverse toxic side effects on the human body, and has high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Evaluation of the in vitro release performance of the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch of Example 1. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with specific drawings and examples. The examples described below are only preferred embodiments of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention. In the following examples, the experimental methods used are conventional methods unless otherwise specified. The reagents used in the examples are all commercially available products.

[0036] Example 1

[0037] A PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch, consisting of a needle tip drug-loaded layer and a cross-linked base drug-loaded layer;

[0038] The needle tip drug-carrying layer includes the following components by mass: L-cysteine ​​2.25g, L-glutamine 2.25g, L-arginine 1.5g, serine 1.5g, L-lysine hydrochloride 0.75g, L-proline 0.75g, glycine 0.75g, sodium hyaluronate 22.5g, and trehalose 2.5g;

[0039] The cross-linked base drug-carrying layer includes the following components in parts by mass: 40 g of PDRN-thiolated HA copolymer, 2 g of minoxidil, 6.75 g of L-cysteine, 6.75 g of L-glutamine, 4.5 g of L-arginine, 4.5 g of serine, 2.25 g of L-lysine hydrochloride, 2.25 g of L-proline, 2.25 g of glycine, and 6 g of trehalose.

[0040] The preparation method of the above-mentioned PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch comprises the following steps:

[0041] (1) Preparation of needle tip drug loading layer

[0042] L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, and glycine were dissolved in 150 mL of PBS buffer (pH 5.0) according to the ratio and stirred to obtain an amino acid solution. Sodium hyaluronate and trehalose were added to the amino acid solution according to the ratio, and the mixture was centrifuged at 3000 rpm for 15 minutes before being poured into the needle tip of the PDMS mold to obtain a needle tip drug-loading layer.

[0043] The PDMS mold includes a needle tip and a base, with a total depth of 600 μm, a depth of the needle tip of 150 μm, and a depth of the base of 450 μm.

[0044] (2) Preparation of PDRN-thiolated HA copolymer

[0045] Under nitrogen protection, 9 g of sodium hyaluronate and 1.5 g of iminothiolane were added to 450 mL of PBS buffer containing 1 mM EDTA, pH = 8.0, stirred and mixed thoroughly, and then stirred and reacted at 25°C for 4 h to obtain a thiolated hyaluronic acid solution;

[0046] After adjusting the pH of the thiolated hyaluronic acid solution to 6.5 using sodium dihydrogen phosphate, 1 g of PDRN, 4.31 g of EDC, and 3.88 g of NHS were added and cross-linked at 4°C for 4 h to obtain PDRN-thiolated HA gel;

[0047] After homogenizing the PDRN-thiolated HA gel for 10 minutes, ethanol was added. After the final volume of ethanol reached 60-80%, the supernatant was removed. Ethanol was continued to be added. After the final volume of ethanol reached 80-90%, the supernatant was removed to obtain a lower layer solid-liquid mixture. After suction filtration and freeze-drying, a PDRN-thiolated HA copolymer was obtained.

[0048] (3) Preparation of PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patches

[0049] According to the ratio, PDRN-thiolated HA copolymer, minoxidil, L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, glycine, and trehalose were dissolved in 450 mL of 3 mM Zn 2+ The mixture was stirred and evenly mixed in PBS buffer with pH = 6, centrifuged at 3000 rpm for 15 min, and then poured into the base of the PDMS mold. It was vacuum dried for 24 h, and the microneedle array was taken out and turned upside down on a wet PVA film. It was further bonded by applying pressure at 0.1 MPa for 10 s, and dried again at 25 ° C for 12 h to obtain a PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch.

[0050] Example 2

[0051] A PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch, consisting of a needle tip drug-loaded layer and a cross-linked base drug-loaded layer;

[0052] The needle tip drug-carrying layer includes the following components by mass: L-cysteine ​​3g, L-glutamine 3g, L-arginine 1g, serine 1g, L-lysine hydrochloride 0.5g, L-proline 0.5g, glycine 0.5g, sodium hyaluronate 20g, and trehalose 3g;

[0053] The cross-linked base drug-carrying layer includes the following components in parts by mass: 30 g of PDRN-thiolated HA copolymer, 5 g of minoxidil, 5 g of L-cysteine, 5 g of L-glutamine, 3 g of L-arginine, 3 g of serine, 1.5 g of L-lysine hydrochloride, 1.5 g of L-proline, 1.5 g of glycine, and 10 g of trehalose.

[0054] The preparation method is the same as that of Example 1.

[0055] Example 3

[0056] A PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch, consisting of a needle tip drug-loaded layer and a cross-linked base drug-loaded layer;

[0057] The needle tip drug-carrying layer includes the following components by mass: L-cysteine ​​1.5g, L-glutamine 1.5g, L-arginine 2g, serine 2g, L-lysine hydrochloride 1.0g, L-proline 1.0g, glycine 1.0g, sodium hyaluronate 25g, and trehalose 1g;

[0058] The cross-linked base drug-carrying layer includes the following components in parts by mass: 50 g of PDRN-thiolated HA copolymer, 1 g of minoxidil, 8 g of L-cysteine, 8 g of L-glutamine, 6 g of L-arginine, 6 g of serine, 3 g of L-lysine hydrochloride, 3 g of L-proline, 3 g of glycine, and 5 g of trehalose.

[0059] The preparation method is the same as that of Example 1.

[0060] Test example

[0061] 1. The in vitro release performance of the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch prepared in Example 1 was evaluated as follows:

[0062] The PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch (1 cm 2 , containing 50 to 100 needles) is fixed to the donor chamber of the Franz diffusion cell and gently pressed (50 g / cm 2 , 10s) to ensure that the needle tip penetrates the semipermeable membrane (simulating skin puncture). Set the temperature to 32±0.5℃ (simulating body surface temperature) and the stirring speed to 500rpm to ensure the uniformity of the receiving medium; the sampling time points are 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h, covering the rapid release and sustained release stages; 0.5mL is sampled each time (rehydration is performed in time) to maintain a constant volume in the receiving chamber. HPLC was used to detect the cumulative release rate of amino acids and minoxidil, and an ultra-micro spectrophotometer was used to detect the cumulative release rate of PDRN. Results Table 1 and Figure 1 shown.

[0063] Table 1

[0064]

[0065]

[0066] From Table 1 and Figure 1It can be seen that the minoxidil and PDRN in the PDRN-sodium hyaluronate cross-linked dual-drug microneedle patch prepared in Example 1 of the present invention are released at 80-85% within 72 hours, while the rapid release of amino acids is achieved to replenish pores and hair nutrition, and the drug delivery is more durable. Conventional minoxidil spray directly contacts the skin through spraying and has a fast release rate. According to literature reports (such as JPharmSci.2018), the skin penetration rate of 5% minoxidil solution in 2 hours is >90%.

[0067] 2. Hair experiment

[0068] The patients were divided into two groups: microneedle group and spray group. The drug used in the microneedle group was the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch prepared in Example 1, and the drug used in the spray group was a common spray containing minoxidil on the market.

[0069] 6-8 week old C57BL / 6 mice were selected and their backs were depilated (electric shaving + depilatory cream) to enter the synchronized resting phase. Before grouping, it was confirmed that the skin was free of damage (screening criteria). The experiments and measurements were performed according to Table 2 below.

[0070] Table 2

[0071]

[0072] The comparison of key experimental results is as follows:

[0073] (1) Hair regeneration efficiency The results are shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] As shown in Table 3, the sustained-release effect and deep-dose delivery of the PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch prepared in Example 1 of the present invention significantly accelerated the entry of hair follicles into the growth phase (Anagen). Common minoxidil-containing sprays on the market have a low transdermal penetration rate (<5%) and require frequent administration to achieve partial results.

[0078] (2) The expression results of hair follicle molecular markers are shown in Table 4.

[0079] Table 4

[0080] markers Changes in the microneedle group Spray group changes VEGF The expression level increased by 3.2 times The expression level increased by 1.5 times β-catenin The positive cell rate increased by 70% The positive cell rate increased by 30% Ki67 (proliferation) Dermal papilla cells increased by 60% Dermal papilla cells increased by 25%

[0081] As shown in Table 4, the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch prepared in Example 1 of the present invention simultaneously delivers minoxidil (vasodilation) and PDRN (activation of the Wnt pathway), producing a synergistic effect. Common minoxidil-containing sprays on the market rely solely on the single vascular effect of minoxidil, requiring frequent administration to achieve partial results.

[0082] (3) Skin irritation results are shown in Table 5.

[0083] Table 5

[0084] Observation Microneedle kit Spray Group Erythema / edema None (0 / 10) Mild (4 / 10) Epidermal thickening No significant changes Thickening of the stratum corneum Inflammatory factors No increase in TNF-α TNF-α increased 2.1 times

[0085] As shown in Table 5, the raw materials used in the PDRN-sodium hyaluronate cross-linked dual-drug-loaded microneedle patch prepared in Example 1 of the present invention are green and safe, have no adverse side effects on the human body, and have high biosafety. However, the common minoxidil-containing sprays on the market contain solvents such as propylene glycol, which may cause irritation.

[0086] In summary, the present invention has innovatively developed a kind of amino acid anti-hair growth microneedle system, the double-drug-loaded microneedle patch of the PDRN-sodium hyaluronate cross-linked provided adopts double-drug-loaded layered design, and the needle tip drug-loaded layer is low molecular weight hyaluronic acid (HA) wrapped quick-release amino acid, and the cross-linked substrate drug-loaded layer is high molecular weight hyaluronic acid wrapped slow-release amino acid, PDRN and minoxidil, realizing immediate and long-term synergy. In addition, the present invention breaks through the skin barrier by soluble microneedle array, and the amino acid combination of careful proportion is directly delivered to the hair follicle area, while combining the cell repair function of polydeoxyribonucleotide (PDRN) and the slow release of sodium hyaluronate and the characteristics such as strong hydration to improve skin environment, realize multi-target, multi-mechanism collaborative treatment, achieve the effect of anti-hair loss regeneration. And the raw materials used are green and safe, have no adverse side effects to human body, and have high biosafety.

[0087] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch, characterized in that: It consists of a needle tip drug-carrying layer and a cross-linked base drug-carrying layer; The needle tip drug-carrying layer comprises the following components in parts by weight: 1 to 5 parts of L-cysteine, 1 to 5 parts of L-glutamine, 1 to 3 parts of L-arginine, 1 to 3 parts of serine, 0.1 to 1.5 parts of L-lysine hydrochloride, 0.1 to 1.5 parts of L-proline, 0.1 to 1.5 parts of glycine, 20 to 25 parts of sodium hyaluronate, and 1 to 5 parts of trehalose; The cross-linked base drug-carrying layer includes the following components in parts by mass: 20 to 50 parts of PDRN-thiolated HA copolymer, 1 to 10 parts of minoxidil, 2 to 10 parts of L-cysteine, 2 to 10 parts of L-glutamine, 3 to 8 parts of L-arginine, 3 to 8 parts of serine, 1 to 5 parts of L-lysine hydrochloride, 1 to 5 parts of L-proline, 1 to 5 parts of glycine, and 5 to 15 parts of trehalose.

2. The PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch as claimed in claim 1, characterized in that The needle tip drug-carrying layer comprises the following components by weight: 2.25 parts of L-cysteine, 2.25 parts of L-glutamine, 1.5 parts of L-arginine, 1.5 parts of serine, 0.75 parts of L-lysine hydrochloride, 0.75 parts of L-proline, 0.75 parts of glycine, 22.5 parts of sodium hyaluronate, and 2.5 parts of trehalose; The cross-linked substrate drug-carrying layer includes the following components in parts by mass: 40 parts of PDRN-thiolated HA copolymer, 2 parts of minoxidil, 6.75 parts of L-cysteine, 6.75 parts of L-glutamine, 4.5 parts of L-arginine, 4.5 parts of serine, 2.25 parts of L-lysine hydrochloride, 2.25 parts of L-proline, 2.25 parts of glycine, and 6 parts of trehalose.

3. The PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch as claimed in claim 1, characterized in that The molecular weight of the sodium hyaluronate is 100-300 KDa.

4. The PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch as claimed in claim 1, characterized in that The PDRN-thiolated HA copolymer was prepared as follows: (1) Under nitrogen protection, sodium hyaluronate and iminothiolane were added to PBS buffer, stirred and mixed evenly, and then stirred and reacted at 20-30° C. for 2-6 hours to obtain a thiolated hyaluronic acid solution; (2) After adjusting the pH of the thiolated hyaluronic acid solution to 6.0-7.0, PDRN and a cross-linking agent were added, and cross-linked at 4°C for 2-4 hours to obtain a PDRN-thiolated HA gel; (3) After homogenizing the PDRN-thiolated HA gel for 5 to 30 minutes, ethanol was added. After the final volume of ethanol reached 60 to 80%, the supernatant was removed and ethanol was continued to be added. After the final volume of ethanol reached 80 to 90%, the supernatant was removed and the lower layer solid-liquid mixture was obtained; after filtration and freeze-drying, the PDRN-thiolated HA copolymer was obtained.

5. The PDRN-sodium hyaluronate cross-linked dual-drug microneedle patch as claimed in claim 4, characterized in that In step (1), the molecular weight of the sodium hyaluronate is 1000-2000 KDa; the PBS buffer is a PBS buffer containing 1 mM EDTA and a pH of 8.0-8.5; the mass-to-volume ratio of the sodium hyaluronate to the PBS buffer is (5-15):450, unit: g / mL; the mass-to-volume ratio of the iminothiolane to the PBS buffer is (1-2):450, unit: g / mL.

6. A PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch as claimed in claim 4, characterized in that In step (2), the mass volume ratio of the PDRN and sodium hyaluronate is 1:(5-15); the cross-linking agent is a composition of EDC and NHS, the molar ratio of EDC and NHS is 1:1.5, and the added amount is 90-95% of the mass of sodium hyaluronate.

7. The method for preparing the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch according to any one of claims 1 to 6, characterized in that: The steps are as follows: 1) L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, and glycine were dissolved in PBS buffer according to the ratio and stirred to obtain an amino acid solution; sodium hyaluronate and trehalose were added to the amino acid solution according to the ratio, centrifuged at 2500-3500 rpm for 10-20 minutes, and then poured into the needle tip of the PDMS mold to obtain a needle tip drug loading layer; 2) According to the ratio, PDRN-thiolated HA copolymer, minoxidil, L-cysteine, L-glutamine, L-arginine, serine, L-lysine hydrochloride, L-proline, glycine, and trehalose were dissolved in PBS buffer, stirred and mixed evenly, and poured into the base of the PDMS mold after centrifugation at 2500-3500 rpm for 10-20 min. The mixture was vacuum dried for 20-30 h, the microneedle array was removed, pressurized at 0.1 MPa for 5-15 s, and dried again at 20-30°C for 10-15 h to obtain a PDRN-sodium hyaluronate cross-linked dual-drug microneedle patch.

8. The preparation method according to claim 7, wherein In step 1), the PBS buffer is a PBS buffer with a pH of 5.0; in step 2), the PBS buffer is a PBS buffer containing 1-5 mM Zn 2+ , PBS buffer with pH = 5.5-6.

5.

9. The use of the PDRN-sodium hyaluronate cross-linked dual-drug loaded microneedle patch according to any one of claims 1 to 6, characterized in that: Used to prepare hair loss prevention and regeneration products.

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