技术领域Technical Field
本发明属于生物材料和皮肤科学领域，涉及一种基于聚多巴胺(PDA)纳米粒子的促毛发再生材料，尤其涉及一种促毛发再生新型ROS基微针贴片的应用。The invention belongs to the field of biomaterials and skin science, and relates to a hair regeneration promoting material based on polydopamine (PDA) nanoparticles, and in particular to the application of a novel ROS-based microneedle patch for promoting hair regeneration.
背景技术Background technique
脱发，或称为秃发，是一种普遍的皮肤问题，对患者的心理健康和社交地位产生显著影响。脱发可以由多种因素引起，包括遗传、激素变化、年龄增长、疾病及环境因素等。在目前的治疗方法中，药物疗法和外科手术是最常见的两种方式。然而，这些方法常伴随着限制性的效果、副作用和较低的患者依从性。Hair loss, or baldness, is a common skin problem that has a significant impact on patients' mental health and social status. Hair loss can be caused by a variety of factors, including genetics, hormonal changes, aging, disease, and environmental factors. Among the current treatments, drug therapy and surgery are the two most common methods. However, these methods are often accompanied by restrictive effects, side effects, and low patient compliance.
传统的药物治疗，如米诺地尔(Minoxidil)和非那雄胺(Finasteride)，虽然在临床上被证实可以促进毛发再生，但需要持续使用以维持效果，并可能引起皮肤刺激、性功能障碍等副作用。此外，这些药物主要通过非特异性方式促进毛囊进入生长期，其机制并未直接针对毛囊干细胞(HFSCs)，从而限制了其疗效。在外科手术方面，如毛发移植手术，虽然能够提供即时效果，但手术成本高、恢复时间长，且存在一定的风险。这些方法无法解决患者自然毛囊功能衰退的根本问题。Traditional drug treatments, such as minoxidil and finasteride, have been clinically proven to promote hair regeneration, but they require continuous use to maintain the effect and may cause side effects such as skin irritation and sexual dysfunction. In addition, these drugs mainly promote the growth phase of hair follicles in a non-specific manner, and their mechanism does not directly target hair follicle stem cells (HFSCs), thus limiting their efficacy. In terms of surgical procedures, such as hair transplantation, although they can provide immediate results, the operation is costly, the recovery time is long, and there are certain risks. These methods cannot solve the fundamental problem of the decline of the patient's natural hair follicle function.
此外，毛囊是毛发再生的关键组织结构，其生长、休止和退化周期对毛发的健康生长至关重要。HFSCs在这一周期中扮演着核心角色，但在某些生理或病理条件下，HFSCs的激活不足或增殖受抑，导致毛囊无法正常进入生长期，从而引起脱发。因此，研究和开发一种能够直接针对毛囊干细胞并有效激活它们的新型治疗方法，是当前脱发治疗领域的一个重大挑战和发展趋势。In addition, hair follicles are key tissue structures for hair regeneration, and their growth, rest, and regression cycles are essential for healthy hair growth. HFSCs play a central role in this cycle, but under certain physiological or pathological conditions, HFSCs are insufficiently activated or their proliferation is inhibited, resulting in the inability of hair follicles to enter the growth phase normally, thus causing hair loss. Therefore, researching and developing a new treatment method that can directly target hair follicle stem cells and effectively activate them is a major challenge and development trend in the current field of hair loss treatment.
当前脱发治疗方法，如药物治疗和外科手术，虽然在一定程度上能够促进毛发再生，但存在多方面的局限性和问题。首先，现有药物治疗如米诺地尔和非那雄胺虽然FDA批准，但需持续使用以维持效果，且可能引起多种副作用，包括性功能障碍、增加前列腺/乳腺癌风险等。此外，这些药物通常无法直接作用于HFSCs，因此效果有限。外科手术方法则成本高昂，且不适用于所有患者。Current hair loss treatments, such as medication and surgery, can promote hair regeneration to a certain extent, but they have many limitations and problems. First, although existing drug treatments such as minoxidil and finasteride are approved by the FDA, they require continuous use to maintain the effect and may cause a variety of side effects, including sexual dysfunction and increased risk of prostate/breast cancer. In addition, these drugs usually cannot act directly on HFSCs, so their effects are limited. Surgical methods are costly and not suitable for all patients.
综上所述，现有的脱发治疗方法存在效果有限、副作用明显、患者依从性差等多种不足。因此，有必要开发一种新的、更安全、更有效的治疗试剂和治疗方法，以满足日益增长的患者需求，改善他们的生活质量。In summary, existing hair loss treatment methods have many shortcomings, such as limited effects, obvious side effects, and poor patient compliance. Therefore, it is necessary to develop a new, safer, and more effective therapeutic agent and treatment method to meet the growing needs of patients and improve their quality of life.
发明内容Summary of the invention
本发明要解决的技术问题是提供一种包括聚多巴胺纳米粒子的毛发再生材料的应用，包括在制备毛发再生相关药剂中的应用。本发明旨在开发一种直接激活HFSCs的治疗方法，减少副作用并提高患者依从性，提供一种便捷、无需专业操作的治疗方式，克服现有脱发治疗方法的限制，提供一种更有效、安全且用户友好的毛发再生方法。The technical problem to be solved by the present invention is to provide an application of a hair regeneration material including polydopamine nanoparticles, including application in the preparation of hair regeneration-related agents. The present invention aims to develop a treatment method for directly activating HFSCs, reduce side effects and improve patient compliance, provide a convenient treatment method that does not require professional operation, overcome the limitations of existing hair loss treatment methods, and provide a more effective, safe and user-friendly hair regeneration method.
本发明提供了所述的一种毛发再生材料在制备毛发再生药物或者试剂中的应用。本发明所述的毛发再生材料包括聚多巴胺纳米粒子。其中，PDA颗粒可被细胞胞吞，通过调节PDA纳米粒子的氧化还原状态，可以实现胞内精确控制ROS的生成和释放，并在动力学上维持其有效浓度水平，从而提高治疗的安全性和有效性。本发明采用的水凝胶和可溶型微针作为PDA纳米粒子的传递载体，能够确保ROS在目标部位的持续和有效释放，同时最小化对周围健康组织的影响。此外，微针的局部使用可通过周围干细胞激活和ROS扩散实现大面积毛发再生效果。The present invention provides the use of a hair regeneration material in the preparation of a hair regeneration drug or reagent. The hair regeneration material of the present invention includes polydopamine nanoparticles. Among them, PDA particles can be endocytosed by cells. By adjusting the redox state of PDA nanoparticles, the generation and release of ROS can be precisely controlled within the cell, and its effective concentration level can be maintained kinetically, thereby improving the safety and effectiveness of treatment. The hydrogel and soluble microneedles used in the present invention as delivery carriers of PDA nanoparticles can ensure the continuous and effective release of ROS at the target site while minimizing the impact on surrounding healthy tissues. In addition, the local use of microneedles can achieve large-area hair regeneration effects through the activation of surrounding stem cells and the diffusion of ROS.
优选地，所述的毛发再生材料也可以用于制备可溶型微针，或者制成ROS供体材料负载水凝胶的形式。所述的毛发再生药物或者试剂作用于毛囊干细胞，是具有下列中的一种或者几种功能的药物或者试剂：Preferably, the hair regeneration material can also be used to prepare soluble microneedles, or made into a ROS donor material loaded hydrogel. The hair regeneration drug or reagent acts on hair follicle stem cells and has one or more of the following functions:
刺激毛囊干细胞的增殖活化，Stimulate the proliferation and activation of hair follicle stem cells.
激活Wnt/β-连环蛋白信号通路，和/或Activation of the Wnt/β-catenin signaling pathway, and/or
促进毛囊进入生长期并触发毛发再生。Promotes hair follicles to enter the growth phase and triggers hair regrowth.
所述的毛发再生材料可以通过下述制备方法获得，所述的方法包括：The hair regeneration material can be obtained by the following preparation method, which comprises:
搅拌质量体积比为0.5-5mg/ml的多巴胺溶液并调节pH值范围至9-10，在含有氧气的水中反应12小时以上合成PDA纳米粒子，得到初始状态的PDA NPs；和/或Stirring a dopamine solution with a mass volume ratio of 0.5-5 mg/ml and adjusting the pH value to 9-10, reacting in water containing oxygen for more than 12 hours to synthesize PDA nanoparticles to obtain PDA NPs in an initial state; and/or
将PDA以1-2mg/mL浓度分散在含氧气的水中，26-37℃孵育10天以上，每天换水1-2次，获得几乎不产生ROS的氧化态PDA NPs(OXI)；和/或Dispersing PDA at a concentration of 1-2 mg/mL in oxygenated water, incubating at 26-37°C for more than 10 days, changing the water 1-2 times a day, to obtain oxidized PDA NPs (OXI) that generate almost no ROS; and/or
通过添加0-500mM还原剂，不包括0，调节PDA纳米粒子的氧化还原状态，控制ROS的生成，生成ROS的还原态PDA NPs(RED)。The redox state of PDA nanoparticles was adjusted by adding 0-500 mM reducing agent, excluding 0, and the generation of ROS was controlled to generate ROS-reduced PDA NPs (RED).
优选地，搅拌的速度范围是300-600转/分钟，更优地，搅拌的速度范围是400-500转/分钟。使用强力搅拌的同时调节pH值，可以使多巴胺溶液迅速混匀，快速准确调节其酸碱度。多巴胺溶液的pH值范围优选是9.4-9.6，更优是9.53-9.56。调节多巴胺溶液的pH值优选使用质量体积比为25-35％的氨水，更优的是28-30％的氨水。使用质量体积比为28-30％的氨水调节多巴胺溶液的pH值。Preferably, the stirring speed range is 300-600 rpm, more preferably, the stirring speed range is 400-500 rpm. Using strong stirring while adjusting the pH value can quickly mix the dopamine solution and quickly and accurately adjust its pH. The pH value range of the dopamine solution is preferably 9.4-9.6, and more preferably 9.53-9.56. To adjust the pH value of the dopamine solution, it is preferred to use ammonia water with a mass volume ratio of 25-35%, and more preferably 28-30% ammonia water. Use ammonia water with a mass volume ratio of 28-30% to adjust the pH value of the dopamine solution.
优选地，所述的含有氧气的水是饱和氧气的水溶液。溶解在水中的空气中的分子态氧称为溶解氧，水中的溶解氧的含量与空气中氧的分压、水的温度都有密切关系；在自然情况下，空气中的含氧量变动不大，故水温是主要的因素，水温愈低，水中溶解氧的含量愈高；溶解于水中的分子态氧称为溶解氧，通常记作DO，用每升水里氧气的毫克数表示。水温保持在25摄氏度情况下，通常饱和溶解氧是28.3毫升每升。本发明的多巴胺在含有氧气的水中反应时间通常不少于5天，优选地，不少于8天，更优地，在含有氧气的水中反应时间是10-15天。Preferably, the water containing oxygen is an aqueous solution saturated with oxygen. Molecular oxygen in the air dissolved in water is called dissolved oxygen. The content of dissolved oxygen in water is closely related to the partial pressure of oxygen in the air and the temperature of the water. Under natural conditions, the oxygen content in the air does not change much, so the water temperature is the main factor. The lower the water temperature, the higher the content of dissolved oxygen in the water. Molecular oxygen dissolved in water is called dissolved oxygen, usually denoted as DO, expressed in milligrams of oxygen per liter of water. When the water temperature is maintained at 25 degrees Celsius, the saturated dissolved oxygen is usually 28.3 milliliters per liter. The reaction time of dopamine in water containing oxygen of the present invention is usually not less than 5 days, preferably not less than 8 days, and more preferably, the reaction time in water containing oxygen is 10-15 days.
优选地，所述的还原剂是抗坏血酸(或二硫苏糖醇)，还原剂的浓度是10-300mM，更优的，还原剂的浓度是50-300mM。Preferably, the reducing agent is ascorbic acid (or dithiothreitol), and the concentration of the reducing agent is 10-300 mM, more preferably, the concentration of the reducing agent is 50-300 mM.
优选地，所述的多巴胺溶液通过以下步骤获得：将多巴胺盐酸盐溶解于乙醇水溶液，乙醇水溶液中乙醇的含量为50％-95％体积比；多巴胺溶液的质量体积比为0.8-3mg/ml。更优地，在20-30℃下，按照每500毫克的盐酸多巴胺完全溶解于含100毫升去离子水和40-55毫升乙醇的溶液中的比例，制备多巴胺溶液。Preferably, the dopamine solution is obtained by the following steps: dissolving dopamine hydrochloride in an ethanol aqueous solution, wherein the ethanol content in the ethanol aqueous solution is 50%-95% by volume; the mass volume ratio of the dopamine solution is 0.8-3 mg/ml. More preferably, the dopamine solution is prepared at 20-30° C. in a ratio of 500 mg of dopamine hydrochloride completely dissolved in a solution containing 100 ml of deionized water and 40-55 ml of ethanol.
本发明还提供了一种ROS供体材料负载可溶型微针，所述的ROS供体材料负载可溶型微针含有上述毛发再生材料。在本发明的一个优选例中，可以根据《Role ofpolydopamine’s redox-activity on its pro-oxidant,radical-scavenging,andantimicrobial activities》中的方法制备上述毛发再生材料。本发明的ROS供体材料负载可溶型微针，可以制成微针贴剂，以非侵入性方式促进毛发再生。The present invention also provides a ROS donor material loaded soluble microneedle, wherein the ROS donor material loaded soluble microneedle contains the above-mentioned hair regeneration material. In a preferred embodiment of the present invention, the above-mentioned hair regeneration material can be prepared according to the method in "Role of polydopamine's redox-activity on its pro-oxidant, radical-scavenging, and antimicrobial activities". The ROS donor material loaded soluble microneedle of the present invention can be made into a microneedle patch to promote hair regeneration in a non-invasive manner.
所述的ROS供体材料负载可溶型微针可以通过以下方法制备：使用带有圆锥形孔的PDMS模具和透明质酸制备可溶型微针，负载毛发再生材料中的PDA纳米粒子。具体的，所述的ROS供体材料负载可溶型微针的制备方法，可以包括以下步骤：The ROS donor material loaded soluble microneedle can be prepared by the following method: using a PDMS mold with conical holes and hyaluronic acid to prepare a soluble microneedle, and loading the PDA nanoparticles in the hair regeneration material. Specifically, the preparation method of the ROS donor material loaded soluble microneedle can include the following steps:
(1)使用PDMS模具制备微针，模具中带有圆锥形孔；每个微针长度不小于1000微米，基部直径不大于600微米，微针间中心距为600-2000微米；；(1) preparing microneedles using a PDMS mold with a conical hole in the mold; each microneedle is not less than 1000 μm in length, the base diameter is not more than 600 μm, and the center distance between microneedles is 600-2000 μm;
(2)在模具上沉积透明质酸，并在底部使用真空制造负压，随后在干燥过夜，使溶剂蒸发并形成可溶型微针尖；OXI/RED被分散在HA溶液中，以制造负载ROS供体材料的微针；和/或(2) depositing hyaluronic acid on the mold and applying vacuum to the bottom to create negative pressure, followed by drying overnight to evaporate the solvent and form soluble microneedle tips; OXI/RED is dispersed in the HA solution to make microneedles loaded with ROS donor material; and/or
在模具上沉积透明质酸，将模具放置在离心管中离心20-30分钟，转速高于4000-5000rpm，随后干燥过夜，OXI/RED被分散在HA溶液中，以制造负载ROS供体材料的微针；Depositing hyaluronic acid on the mold, placing the mold in a centrifuge tube and centrifuging for 20-30 minutes at a speed higher than 4000-5000 rpm, followed by drying overnight, OXI/RED was dispersed in the HA solution to fabricate microneedles loaded with ROS donor materials;
(3)在HA微针背面沉积聚乙烯醇和聚乙烯吡咯烷酮，作为基底贴片，随后真空处理；在室温下干燥12小时以上，将微针贴剂从模具中分离；和/或(3) depositing polyvinyl alcohol and polyvinyl pyrrolidone on the back of the HA microneedles as a base patch, followed by vacuum treatment; drying at room temperature for more than 12 hours, and separating the microneedle patch from the mold; and/or
在HA微针背面沉积聚乙烯醇和聚乙烯吡咯烷酮，作为基底贴片，随后离心处理；在室温下干燥12小时以上，将微针贴剂从模具中分离。Polyvinyl alcohol and polyvinyl pyrrolidone were deposited on the back of the HA microneedles as a base patch, followed by centrifugation; the microneedle patch was separated from the mold after drying at room temperature for more than 12 hours.
本发明还提供了一种ROS供体材料负载水凝胶，其制备方法包括以下步骤：The present invention also provides a ROS donor material loaded hydrogel, the preparation method of which comprises the following steps:
获取分子量范围是5-30kDa的四臂-PEG-SC、四臂-PEG-SS和四臂-PEG-NH2；Four-arm-PEG-SC, four-arm-PEG-SS, and four-arm-PEG-NH2 with a molecular weight range of 5-30 kDa were obtained;
四臂-PEG-NH2溶于浓度为0.1-0.5M、pH 6-7的磷酸盐缓冲溶液中，配制成质量百分比为8％-15％浓度作为凝胶前体A；Four-arm-PEG-NH2 was dissolved in a phosphate buffer solution with a concentration of 0.1-0.5 M and a pH of 6-7 and prepared into a concentration of 8%-15% by mass as gel precursor A;
四臂-PEG-SS和四臂-PEG-SC分别溶于磷酸盐缓冲溶液中，分别配制成质量百分比为3％-7％浓度作为凝胶前体B；Four-arm-PEG-SS and four-arm-PEG-SC were dissolved in phosphate buffer solution and prepared into a concentration of 3% to 7% by mass as gel precursor B;
将OXI/RED以3-10mg/ml的浓度分散于A溶液中；Disperse OXI/RED in solution A at a concentration of 3-10 mg/ml;
将A和B溶液混合，获得负载ROS供体材料的水凝胶。The A and B solutions were mixed to obtain a hydrogel loaded with ROS donor material.
优选的，四臂-PEG-SC、四臂-PEG-SS和四臂-PEG-NH2的分子量是5-10kDa。Preferably, the molecular weight of four-arm-PEG-SC, four-arm-PEG-SS and four-arm-PEG-NH2 is 5-10kDa .
优选的，磷酸盐缓冲溶液的浓度为0.2-0.3M，pH 6.4-6.8，四臂-PEG-NH2溶于磷酸盐缓冲溶液中，以10wt％的浓度作为凝胶前体A。Preferably, the concentration of the phosphate buffer solution is 0.2-0.3 M, pH 6.4-6.8, and four-arm-PEG-NH2 is dissolved in the phosphate buffer solution at a concentration of 10 wt % as the gel precursor A.
优选的，四臂-PEG-SS和四臂-PEG-SC分别溶于磷酸盐缓冲溶液中，以7wt％和3wt％的浓度作为凝胶前体B。Preferably, four-arm-PEG-SS and four-arm-PEG-SC are dissolved in phosphate buffer solution at concentrations of 7 wt % and 3 wt %, respectively, as gel precursor B.
优选的，OXI/RED以5-7mg/ml的浓度分散于A溶液中。Preferably, OXI/RED is dispersed in solution A at a concentration of 5-7 mg/ml.
本发明的ROS供体材料负载水凝胶施用于皮肤下层，可以在皮肤下层逐渐释放PDA纳米粒子，确保ROS的持续和有效传递。The ROS donor material loaded hydrogel of the present invention is applied to the subcutaneous layer, and can gradually release the PDA nanoparticles in the subcutaneous layer, thereby ensuring the continuous and effective transfer of ROS.
PDMS(polydimethylsiloxane，聚二甲基硅氧烷)在室温下是一种无色、粘稠的液体，被称为硅油，是软光刻工艺中常用的聚合物。PDMS (polydimethylsiloxane) is a colorless, viscous liquid at room temperature, known as silicone oil, and is a polymer commonly used in soft lithography.
四臂PEG-SC是一种多功能交联PEG试剂，可和氨基反应，是一种四臂PEG-NHS酯试剂。PEG-SS(琥珀酰琥珀酸酯)是PEG-NHS试剂的一种。四臂PEG-SS是一种多臂PEG衍生物,在连接到一个季戊四醇核的四臂的每个末端具有琥珀酰亚胺NHS酯基。四臂PEG胺是一种多臂PEG衍生物,四臂的每个末端都有胺基连接到一个季戊四醇芯。反应性伯胺或NH2基团可与活化羧酸(如NHS酯)快速反应,形成稳定的酰胺键。Four-arm PEG-SC is a versatile cross-linking PEG reagent that reacts with amino groups and is a four-arm PEG-NHS ester reagent. PEG-SS (succinylsuccinate) is a type of PEG-NHS reagent. Four-arm PEG-SS is a multi-arm PEG derivative with succinimidyl NHS ester groups at each end of four arms attached to a pentaerythritol core. Four-arm PEG amine is a multi-arm PEG derivative with amine groups at each end of four arms attached to a pentaerythritol core. Reactive primary amines orNH2 groups react rapidly with activated carboxylic acids such as NHS esters to form stable amide bonds.
本发明中，OXI是指氧化态PDA NPs，即PDA NPs(聚多巴胺纳米粒子)在饱和氧气的水中孵育后获得的几乎不生成ROS的氧化态PDA NPs。RED是指还原态PDA NPs，是氧化PDANPs与还原剂孵育反应后获得的能够生成ROS的还原态PDA NPs。In the present invention, OXI refers to oxidized PDA NPs, that is, oxidized PDA NPs (polydopamine nanoparticles) obtained by incubating in water saturated with oxygen and generating almost no ROS. RED refers to reduced PDA NPs, which are reduced PDA NPs that can generate ROS after oxidized PDA NPs are incubated with a reducing agent.
本发明相比现有技术具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1.胞内递送ROS和精确控制ROS释放：PDA颗粒可被细胞胞吞，通过调节PDA纳米粒子的氧化还原状态，可以实现胞内精确控制ROS的生成和释放，并在动力学上维持其有效浓度水平，从而提高治疗的安全性和有效性。这一点在现有技术中往往难以实现。1. Intracellular delivery of ROS and precise control of ROS release: PDA particles can be endocytosed by cells. By adjusting the redox state of PDA nanoparticles, the generation and release of ROS can be precisely controlled intracellularly and their effective concentration level can be maintained kinetically, thereby improving the safety and effectiveness of treatment. This is often difficult to achieve in existing technologies.
2.直接激活毛囊干细胞：本发明利用PDA纳米粒子产生的ROS直接作用于HFSCs，促进其活化和增殖。这与现有药物治疗方法不同，后者通常无法针对性激活HFSCs。2. Direct activation of hair follicle stem cells: The present invention uses ROS generated by PDA nanoparticles to directly act on HFSCs, promoting their activation and proliferation. This is different from existing drug treatments, which are usually unable to specifically activate HFSCs.
3.减少副作用和提高患者依从性：相比于传统药物和外科手术，本发明提供的治疗方法副作用较小，易于患者接受和使用。特别是通过微针贴剂的设计，患者可以方便地自行应用，无需复杂的操作或长期药物治疗。3. Reduce side effects and improve patient compliance: Compared with traditional drugs and surgical procedures, the treatment method provided by the present invention has fewer side effects and is easy for patients to accept and use. In particular, through the design of microneedle patches, patients can easily apply them by themselves without complicated operations or long-term drug treatment.
4.创新的传递系统：本发明采用的水凝胶和可溶型微针作为PDA纳米粒子的传递载体，能够确保ROS在目标部位的持续和有效释放，同时最小化对周围健康组织的影响。此外，微针的局部使用可通过周围干细胞激活和ROS扩散实现大面积毛发再生效果。伴随ROS的扩散效应和干细胞激活会影响周围干细胞功能，该微针贴片的使用效果会由局部到全皮层生发。4. Innovative delivery system: The hydrogel and soluble microneedles used in the present invention as delivery carriers for PDA nanoparticles can ensure the continuous and effective release of ROS at the target site while minimizing the impact on surrounding healthy tissues. In addition, the local use of microneedles can achieve large-area hair regeneration effects through surrounding stem cell activation and ROS diffusion. With the diffusion effect of ROS and stem cell activation affecting the function of surrounding stem cells, the effect of the use of the microneedle patch will range from local to full-thickness hair growth.
5.广泛的应用潜力：除了促进毛发再生，由于ROS在许多生物学过程中的重要作用，本发明的技术也可应用于其他需要精确控制ROS水平的医疗和研究领域。5. Wide application potential: In addition to promoting hair regeneration, due to the important role of ROS in many biological processes, the technology of the present invention can also be applied to other medical and research fields that require precise control of ROS levels.
6.非侵入性和低创伤性：相比于外科植发等治疗方式，本发明提供的方法非侵入性、低创伤性，适用于更广泛的患者群体，尤其是对手术有顾虑的患者。6. Non-invasive and low-traumatic: Compared with surgical hair transplantation and other treatment methods, the method provided by the present invention is non-invasive, low-traumatic, and suitable for a wider range of patient groups, especially those who are concerned about surgery.
综上所述，本发明在毛发再生治疗领域提供了一种新的、有效的解决方案，具有较高的安全性和患者依从性，同时开辟了ROS在医学应用中的新途径。In summary, the present invention provides a new and effective solution in the field of hair regeneration treatment, which has high safety and patient compliance, and opens up a new way for ROS in medical applications.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是ROS供体材料基础形貌和体外ROS释放性能表征图。FIG1 is a diagram showing the basic morphology of the ROS donor material and the characterization of its in vitro ROS release performance.
其中，图1(A)是OXI和RED的扫描电子显微镜照片，由图片可知OXI与RED呈现粒径均一的球形，尺寸分布在400nm左右。图1(B)是RED中酚基团含量与VC浓度关系图，酚基团随VC用量增加而增加，并在300mM VC附近趋于饱和。图1(C)是RED的H2O2总释放量与VC浓度关系图，H2O2总释放量随VC用量增加而增加，并在300mM VC附近达到最高，约220μM。Among them, Figure 1 (A) is a scanning electron microscope photo of OXI and RED. It can be seen from the picture that OXI and RED are spherical with uniform particle size, and the size distribution is about 400nm. Figure 1 (B) is a graph showing the relationship between the phenol group content in RED and the VC concentration. The phenol group increases with the increase of VC dosage and tends to be saturated near 300mM VC. Figure 1 (C) is a graph showing the relationship between the total H2 O2 release of RED and the VC concentration. The total H2 O2 release increases with the increase of VC dosage and reaches the highest level near 300mM VC, about 220μM.
图2是ROS供体材料调控细胞内氧化还原状态表征图。FIG. 2 is a characterization diagram of the regulation of intracellular redox state by ROS donor materials.
其中，图2(A)是OXI和RED的细胞相容性检测图，RED材料能够提高L929细胞活力。图2(B)是激光共聚焦显微镜拍摄的经OXI和RED处理后的L929细胞骨架染色照片。图2(C)是由流式细胞仪检测的L929细胞吞噬OXI-CFDA和RED-CFDA后细胞内荧光强度分布直方图。图2(D)是图2(C)的峰面积定量统计对比，OXI-CFDA和RED-CFDA被细胞吞噬的数量没有显著差异。图2(E-G)使用Hyperion/Hyperion-c、iNap/iNapc和RoGFP探针转染的Hela细胞评估材料对细胞内氧化还原状态的调控，RED可以显著上调细胞内H2O2水平、下调NADPH水平和GSH水平。Among them, Figure 2 (A) is a cell compatibility test diagram of OXI and RED, and the RED material can improve the viability of L929 cells. Figure 2 (B) is a photo of L929 cytoskeleton staining after OXI and RED treatment taken by laser confocal microscopy. Figure 2 (C) is a histogram of intracellular fluorescence intensity distribution after L929 cells engulfed OXI-CFDA and RED-CFDA detected by flow cytometry. Figure 2 (D) is a quantitative statistical comparison of the peak area of Figure 2 (C). There is no significant difference in the number of OXI-CFDA and RED-CFDA engulfed by cells. Figure 2 (EG) Hela cells transfected with Hyperion/Hyperion-c, iNap/iNapc and RoGFP probes were used to evaluate the regulation of the material on the intracellular redox state. RED can significantly upregulate theintracellularH2O2 level and downregulate the NADPH level and GSH level.
图3是ROS供体材料负载水凝胶及其诱导体内毛发生长的表征图。FIG. 3 is a characterization diagram of ROS donor material loaded hydrogel and its induction of hair growth in vivo.
其中，图3(A)是ROS供体水凝胶体外成胶实物照片。图3(B)是ROS供体水凝胶的扫描电镜照片和实物照片，ROS供体纳米颗粒在水凝胶中均匀分布。图3(C)是ROS供体水凝胶体外降解率统计图，水凝胶在8天内基本降解完毕。图3(D)是从第0天到第20天使用不同水凝胶时毛发再生的宏观和局部放大图，RED组的毛发生长速率显著高于Blank和OXI组。图3(E)是注射20天后皮肤的H&E染色和高倍放大局部特征，RED组毛囊数量分布密集，并且毛囊形状饱满，体积较大，RED促进了毛囊生长和激活。Among them, Figure 3 (A) is a photo of the in vitro gelation of the ROS donor hydrogel. Figure 3 (B) is a scanning electron microscope photo and a photo of the ROS donor hydrogel, and the ROS donor nanoparticles are evenly distributed in the hydrogel. Figure 3 (C) is a statistical chart of the in vitro degradation rate of the ROS donor hydrogel, and the hydrogel is basically degraded within 8 days. Figure 3 (D) is a macroscopic and local magnified picture of hair regeneration when using different hydrogels from day 0 to day 20. The hair growth rate of the RED group is significantly higher than that of the Blank and OXI groups. Figure 3 (E) is the H&E staining and high-magnification local features of the skin 20 days after injection. The number of hair follicles in the RED group is densely distributed, and the hair follicles are full in shape and large in size. RED promotes the growth and activation of hair follicles.
图4是ROS供体材料负载水凝胶体内H2O2释放的评估。FIG. 4 is an evaluation of H2 O2 release in vivo from ROS donor material loaded hydrogels.
其中，图4(A)是显示将负载ROS供体材料体内注射到HyPerion小鼠的背部示意图。图4(B-C)是ROS供体材料对HyPerion小鼠皮肤组织中H2O2水平影响的定性和定量分析。3小时H2O2化学计量依赖的Ratio500/430升高证实细胞内H2O2释放，显著影响小鼠皮下局部氧化还原微环境。表明负载PDA纳米颗粒的水凝胶能够在皮下缓释ROS，确保ROS的持续和有效胞内递送。Among them, Figure 4 (A) is a schematic diagram showing the in vivo injection of ROS donor materials loaded into the back of HyPerion mice. Figure 4 (BC) is a qualitative and quantitative analysis of the effect of ROS donor materials on H2 O2 levels in the skin tissue of HyPerion mice. The increase in Ratio500/430, which is dependent on the stoichiometry of H2 O2 , at 3 hours confirmed the release of intracellular H2 O2 , which significantly affected the local redox microenvironment of the subcutaneous area of mice. It shows that the hydrogel loaded with PDA nanoparticles can release ROS subcutaneously, ensuring the continuous and effective intracellular delivery of ROS.
图5是ROS供体材料负载微针贴片及其诱导体内毛发生长的表征图。FIG. 5 is a characterization diagram of a ROS donor material loaded microneedle patch and its induction of hair growth in vivo.
其中，图5(A)是ROS供体材料负载微针贴片实物照片。图5(B)是ROS供体材料负载微针贴片扫描电镜照片，ROS供体纳米颗粒在微针针尖中分布均匀。图5(C)是从第9天到第15天使用不同微针贴片时毛发再生的宏观照片，RED组的毛发生长速率显著高于Blank和OXI组。图5(D)是各组单根毛发的扫描电镜照片，RED组的新生毛发直径显著高于其他组别。图5(E)是新生毛发的直径统计图。Among them, Figure 5(A) is a real photo of the microneedle patch loaded with ROS donor material. Figure 5(B) is a scanning electron micrograph of the microneedle patch loaded with ROS donor material, and the ROS donor nanoparticles are evenly distributed in the microneedle tip. Figure 5(C) is a macroscopic photo of hair regeneration when different microneedle patches are used from day 9 to day 15. The hair growth rate of the RED group is significantly higher than that of the Blank and OXI groups. Figure 5(D) is a scanning electron micrograph of a single hair in each group. The diameter of the new hair in the RED group is significantly higher than that in other groups. Figure 5(E) is a statistical graph of the diameter of the new hair.
图6是ROS供体材料通过ROS促进毛发生长的原理示意图。FIG. 6 is a schematic diagram showing the principle of ROS donor materials promoting hair growth via ROS.
ROS供体材料能够精确控制ROS释放和胞内递送：通过调节PDA纳米粒子的氧化还原状态，可以精确控制ROS的生成、释放和胞内递送，从而提高治疗的安全性和有效性。ROS donor materials enable precise control of ROS release and intracellular delivery: By regulating the redox state of PDA nanoparticles, the generation, release, and intracellular delivery of ROS can be precisely controlled, thereby improving the safety and efficacy of treatment.
图7是ROS供体材料在体内和体外的作用以及促进毛发生长的效果展示图。FIG. 7 is a diagram showing the effects of ROS donor materials in vivo and in vitro and their effects on promoting hair growth.
ROS供体材料制作成贴片或水凝胶，其中包裹的PDA纳米颗粒是局部作用于皮肤(即注射或者贴片作用区域)，其首先可被注射区域细胞内吞，实现胞内精准可控递送ROS，激活注射区毛囊干细胞。之后，伴随ROS的扩散效应和干细胞激活会影响周围干细胞功能，该微针贴片的使用效果会由局部到全皮层生发。图中显示，该微针贴片能够显著促进毛发生长，通过在预设位置使用该微针贴片能够精确控制促进毛发生长的区域和毛发生长速率。The ROS donor material is made into a patch or hydrogel, in which the encapsulated PDA nanoparticles act locally on the skin (i.e., the injection or patch action area). They can first be internalized by the cells in the injection area to achieve precise and controllable intracellular delivery of ROS and activate the hair follicle stem cells in the injection area. Afterwards, the diffusion effect of ROS and stem cell activation will affect the function of surrounding stem cells, and the effect of the use of the microneedle patch will range from local to full-thickness dermal growth. The figure shows that the microneedle patch can significantly promote hair growth. By using the microneedle patch in a preset position, the area where hair growth is promoted and the hair growth rate can be precisely controlled.
具体实施方式Detailed ways
一方面，本发明开发了一种直接激活毛囊干细胞的治疗方法：现有治疗手段往往无法有效针对毛囊干细胞，而本发明提供的方法能够直接作用于HFSCs，激活其增殖和活化，从而更高效地促进毛发再生。On the one hand, the present invention has developed a treatment method for directly activating hair follicle stem cells: existing treatment methods are often unable to effectively target hair follicle stem cells, while the method provided by the present invention can directly act on HFSCs, activating their proliferation and activation, thereby promoting hair regeneration more efficiently.
第二方面，本发明能够减少副作用并提高患者依从性：传统药物治疗常伴随多种潜在副作用，且需持续使用以维持效果。本发明提出的基于聚多巴胺纳米粒子的材料生物学调节疗法(MBMT)旨在提供一种安全、可控的治疗方式，通过精确控制ROS的释放量来最小化副作用，同时简化治疗流程，提高患者的治疗依从性。Secondly, the present invention can reduce side effects and improve patient compliance: Traditional drug treatments are often accompanied by a variety of potential side effects and require continuous use to maintain the effect. The material biological modulation therapy (MBMT) based on polydopamine nanoparticles proposed in the present invention aims to provide a safe and controllable treatment method, which minimizes side effects by precisely controlling the release of ROS, simplifies the treatment process, and improves patient compliance.
第三方面，本发明提供了一种便捷、无需专业操作的治疗方式。现有的外科手术方法成本高昂且需要专业操作，本发明则通过微针贴剂的设计，使得患者或非专业人员能够方便地自我应用，大幅降低治疗的复杂性和成本。Thirdly, the present invention provides a convenient treatment method that does not require professional operation. Existing surgical methods are expensive and require professional operation. The present invention uses the design of microneedle patches to enable patients or non-professionals to easily apply them themselves, greatly reducing the complexity and cost of treatment.
通过这些创新解决方案，本发明意在克服现有脱发治疗方法的限制，提供一种更有效、安全且用户友好的毛发再生方法。Through these innovative solutions, the present invention aims to overcome the limitations of existing hair loss treatment methods and provide a more effective, safe and user-friendly hair regrowth method.
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.
实施例1.ROS供体材料的合成Example 1. Synthesis of ROS donor materials
(1)在室温下，将500毫克的盐酸多巴胺(98％纯度)完全溶解于含100毫升去离子水和40毫升乙醇的溶液中。(1) At room temperature, 500 mg of dopamine hydrochloride (98% purity) was completely dissolved in a solution containing 100 ml of deionized water and 40 ml of ethanol.
(2)在强力搅拌(400转/分钟)下，迅速使用28-30％的氨水溶液将混合物的pH值调节至9.55，溶液颜色由淡黄色变为黑色。(2) Under strong stirring (400 rpm), the pH value of the mixture was quickly adjusted to 9.55 using 28-30% aqueous ammonia solution, and the color of the solution changed from light yellow to black.
(3)反应10小时后，通过离心分离目标的ROS供体材料——聚多巴胺纳米粒子(PDANPs)，并用去离子水洗涤三次，随后进行冷冻干燥。(3) After 10 h of reaction, the target ROS donor material, polydopamine nanoparticles (PDANPs), was separated by centrifugation, washed three times with deionized water, and then freeze-dried.
(4)使用场发射电子显微镜(FE-SEM,S4800 Hitachi)观察ROS供体材料的形态，并用动态激光光散射谱仪(ALV/CGS-5022F)检测纳米粒子的大小。(4) The morphology of the ROS donor material was observed using a field emission electron microscope (FE-SEM, S4800 Hitachi), and the size of the nanoparticles was detected using a dynamic laser light scattering spectrometer (ALV/CGS-5022F).
结果显示，OXI与RED呈现粒径均一的球形，尺寸分布在400nm左右(图1A)。The results showed that OXI and RED were spherical with uniform particle size, and the size distribution was around 400 nm ( Figure 1A ).
实施例2.ROS供体材料的可控ROS生成Example 2. Controllable ROS Generation of ROS Donor Materials
(1)将制备好的PDA NPs在饱和氧气的水中孵育12天，每12小时更换一次水。(1) The prepared PDA NPs were incubated in oxygen-saturated water for 12 days, and the water was replaced every 12 hours.
(2)经过离心和用去离子水洗涤后，得到未生成ROS的氧化PDA NPs(OXI)，作为阳性对照组。(2) After centrifugation and washing with deionized water, oxidized PDA NPs (OXI) without ROS generation were obtained and used as the positive control group.
(3)随后，将OXI在10-300mM的抗坏血酸溶液中孵育1小时。再经过离心和用无氧水洗涤后，得到能够生成ROS的还原PDA NPs(RED)。(3) Subsequently, OXI was incubated in a 10-300 mM ascorbic acid solution for 1 hour. After centrifugation and washing with oxygen-free water, reduced PDA NPs (RED) capable of generating ROS were obtained.
结果显示，酚基团随VC用量增加而增加，并在300mM VC附近趋于饱和。H2O2总释放量随VC用量增加而增加，并在300mM VC附近达到最高，约220μM(参见图1B和C)。The results showed that the phenol groups increased with the increase of VC dosage and tended to be saturated near 300 mM VC. The total amount of H2 O2 released increased with the increase of VC dosage and reached the highest level of about 220 μM near 300 mM VC (see Figure 1B and C).
实施例3.ROS生成和生物相容性评估Example 3. ROS generation and biocompatibility assessment
(1)设立五个组用于检测ROS生成：OXI,OXI-10mM VC,OXI-20mM VC,OXI-50mM VC,OXI-300mM VC。(1) Five groups were set up to detect ROS generation: OXI, OXI-10mM VC, OXI-20mM VC, OXI-50mM VC, and OXI-300mM VC.
(2)将样品在37℃的离心管中与PB孵育一定时间(每个管中加入1mg的ROS供体材料和1ml的PBS)，然后取一部分溶液稀释并使用过氧化氢测定试剂盒(AmpliteTMFluorimetric Hydrogen Peroxide Assay Kit,AAT Bioquest)测试H2O2生成。(2) The samples were incubated with PB in a centrifuge tube at 37°C for a certain period of time (1 mg of ROS donor material and 1 ml of PBS were added to each tube), and then a portion of the solution was diluted and tested for H2 O2 generation using a hydrogen peroxide assay kit (AmpliteTM Fluorimetric Hydrogen Peroxide Assay Kit, AAT Bioquest).
(3)使用Folin-Ciocalteu比色法确定经还原剂处理的RED中的儿茶酚含量。(3) The catechol content in RED treated with reducing agents was determined using the Folin-Ciocalteu colorimetric method.
(4)用小鼠成纤维细胞系L929细胞进行体外生物相容性评估。细胞在含有10％胎牛血清的DMEM培养基中培养，于37℃、5％CO2的湿润环境中培养。使用MTT测试细胞增殖，通过FITC-鬼笔环肽和DAPI共染色检查细胞骨架组装情况。(4) In vitro biocompatibility assessment was performed using mouse fibroblast cell line L929 cells. The cells were cultured in DMEM medium containing 10% fetal bovine serum in a humidified environment at 37°C and 5% CO2. Cell proliferation was tested using MTT, and cytoskeleton assembly was examined by co-staining with FITC-phalloidin and DAPI.
结果显示，RED材料能够提高L929细胞活力(图2A和B)。The results showed that RED materials could enhance the viability of L929 cells (Figure 2A and B).
实施例4.检测ROS供体材料被细胞内化情况Example 4. Detection of the internalization of ROS donor materials by cells
(1)小鼠成纤维细胞系L929细胞与1mg OXI-CFDA和1mg RED-CFDA在微酸性介质(pH＝6.5)中孵育4小时。(1) Mouse fibroblast cell line L929 cells were incubated with 1 mg OXI-CFDA and 1 mg RED-CFDA in a slightly acidic medium (pH = 6.5) for 4 hours.
(2)孵育后，使用DAPI和罗丹明-鬼笔环肽共染色细胞核和骨架。流式细胞仪(Beckman coulter,United States)分析细胞内CFDA荧光，并由激光共聚焦显微镜(Nikon,Japan)记录。(2) After incubation, DAPI and rhodamine-phalloidin were used to co-stain the nucleus and skeleton. Intracellular CFDA fluorescence was analyzed by flow cytometry (Beckman coulter, United States) and recorded by laser confocal microscopy (Nikon, Japan).
(3)使用透射电子显微镜(TEM,JEOL JSM-2100)进一步检测材料的内吞作用。(3) Transmission electron microscopy (TEM, JEOL JSM-2100) was used to further detect the endocytosis of the material.
结果显示，OXI-CFDA和RED-CFDA被细胞吞噬的数量没有显著差异(图2C)。使用Hyperion/Hyperion-c、iNap/iNapc和RoGFP探针转染的Hela细胞评估材料对细胞内氧化还原状态的调控，RED可以显著上调细胞内H2O2水平、下调NADPH水平和GSH水平(图2D)。The results showed that there was no significant difference in the amount of OXI-CFDA and RED-CFDA engulfed by cells (Figure 2C). Hela cells transfected with Hyperion/Hyperion-c, iNap/iNapc and RoGFP probes were used to evaluate the regulation of the material on the intracellular redox state. RED could significantly upregulate theintracellularH2O2 level and downregulate the NADPH level and GSH level (Figure 2D).
实施例5.通过ROS供体材料调控细胞内氧化还原状态的评估Example 5. Evaluation of the regulation of intracellular redox state by ROS donor materials
(1)为产生稳定的细胞系，通过共转染两种逆转录病毒包装载体(pMD2.G和psPAX2)和一个感应器质粒在HEK293T细胞中转染Hela细胞。(1) To generate stable cell lines, HeLa cells were transfected by co-transfection of two retroviral packaging vectors (pMD2.G and psPAX2) and a sensor plasmid in HEK293T cells.
(2)Hela细胞接种于96孔板中，每孔密度为6000个细胞。孵育24小时后，原培养基被替换为含RED(0.25mg/mL,0.5mg/mL)、OXI(0.25mg/mL,0.5mg/mL)或H2O2(50μM,100μM)的新培养基，并孵育24小时。(2) Hela cells were seeded in 96-well plates at a density of 6000 cells per well. After 24 hours of incubation, the original medium was replaced with new medium containing RED (0.25 mg/mL, 0.5 mg/mL), OXI (0.25 mg/mL, 0.5 mg/mL) or H2 O2 (50 μM, 100 μM) and incubated for 24 hours.
(3)随后，细胞被洗涤两次，并在HBSS缓冲液(pH 7.4)中含有100mM HEPES和25mM葡萄糖在37℃下进行测量。使用双激发比率由BioTek Synergy Neo 2多模式微孔板阅读器(BioTek,Synergy Neo2,US)获取，使用485BP 20nm或420BP 27nm激发，和532BP 40nm发射带通滤波器。(3) Subsequently, cells were washed twice and measured in HBSS buffer (pH 7.4) containing 100 mM HEPES and 25 mM glucose at 37° C. Dual excitation ratios were acquired by a BioTek Synergy Neo 2 multi-mode microplate reader (BioTek, Synergy Neo2, US), using 485BP 20 nm or 420BP 27 nm excitation, and 532BP 40 nm emission bandpass filters.
(4)通过减去未表达感应器的细胞样品的强度来校正背景荧光值。使用Pitstop 2(20μM)和Genistein(50μM)确定内吞途径的详细作用，并通过ROS探针-DCFH-DA检测。值得注意的是，由于CFDA的干扰荧光，因此在此背景下使用DCFH-DA探针进行细胞内ROS检测。(4) The background fluorescence value was corrected by subtracting the intensity of the cell sample that did not express the sensor. Pitstop 2 (20 μM) and Genistein (50 μM) were used to determine the detailed role of the endocytic pathway and detected by the ROS probe-DCFH-DA. It is worth noting that due to the interference fluorescence of CFDA, the DCFH-DA probe was used for intracellular ROS detection in this background.
实施例6.ROS供体材料负载水凝胶的制备Example 6. Preparation of ROS donor material loaded hydrogel
(1)从SINOPEG,China购买的四臂-PEG-SC(分子量＝10kDa)、四臂-PEG-SS(分子量＝10kDa)和四臂-PEG-NH2(分子量＝10kDa)。(1) Four-arm-PEG-SC (molecular weight = 10 kDa), four-arm-PEG-SS (molecular weight = 10 kDa), and four-arm-PEG-NH2 (molecular weight = 10 kDa) purchased from SINOPEG, China.
(2)四臂-PEG-NH2溶于磷酸盐缓冲溶液(0.2M,pH 6.8)中，以10wt％的浓度作为凝胶前体A，而四臂-PEG-SS和四臂-PEG-SC分别溶于磷酸盐缓冲溶液中，以7wt％和3wt％的浓度作为凝胶前体B。(2) Four-arm-PEG-NH2 was dissolved in phosphate buffer solution (0.2 M, pH 6.8) at a concentration of 10 wt % as gel precursor A, while four-arm-PEG-SS and four-arm-PEG-SC were dissolved in phosphate buffer solution at concentrations of 7 wt % and 3 wt %, respectively, as gel precursor B.
(3)将OXI/RED以5mg/ml的浓度分散于A溶液中。(3) Disperse OXI/RED in solution A at a concentration of 5 mg/ml.
(4)将A和B溶液混合，获得负载ROS供体材料的水凝胶。(4) Mixing solutions A and B to obtain a hydrogel loaded with ROS donor material.
(5)通过将水凝胶浸入37℃的PBS中10天来测试水凝胶的可降解性。(5) The degradability of the hydrogels was tested by immersing them in PBS at 37 °C for 10 days.
由ROS供体水凝胶的扫描电镜照片和实物照片显示，水凝胶成胶时间为2min(图3A),ROS供体纳米颗粒在水凝胶中均匀分布(图3B)，水凝胶在8天内基本降解完毕(图3C)，从第0天到第20天使用不同水凝胶时毛发再生的宏观和局部放大图，RED组的毛发生长速率显著高于Blank和OXI组(图3D)。注射20天后皮肤的H&E染色和高倍放大局部特征，RED组毛囊数量分布密集，并且毛囊形状饱满，体积较大，RED促进了毛囊生长和激活(图3E)。Scanning electron microscopy and physical photos of the ROS donor hydrogel showed that the gelation time of the hydrogel was 2 minutes (Figure 3A), the ROS donor nanoparticles were evenly distributed in the hydrogel (Figure 3B), and the hydrogel was basically degraded within 8 days (Figure 3C). From the macroscopic and local magnified images of hair regeneration using different hydrogels from day 0 to day 20, the hair growth rate of the RED group was significantly higher than that of the Blank and OXI groups (Figure 3D). H&E staining and high-magnification local features of the skin 20 days after injection showed that the number of hair follicles in the RED group was densely distributed, and the hair follicles were full in shape and large in size. RED promoted the growth and activation of hair follicles (Figure 3E).
实施例7.体内H2O2释放的评估Example 7. Evaluation of H2 O2 Release in Vivo
(1)使用同型合子转基因Hyperion小鼠进行实验。(1) Experiments were performed using homozygous transgenic Hyperion mice.
(2)使用脱毛膏去除背部毛发。随后，将动物随机分为8组(n＝6)，以评估ROS供体材料应用后的体内H2O2释放情况。(2) Remove the back hair using depilatory cream. Subsequently, the animals were randomly divided into 8 groups (n=6) to evaluate the in vivo H2 O2 release after application of ROS donor materials.
(3)负载PDA纳米粒子的水凝胶被皮下注射，随后使用IVIS Lumina Series III活体成像系统(PerkinElmer,Inc.)在特定时间点进行拍摄。(3) The hydrogel loaded with PDA nanoparticles was injected subcutaneously and then imaged at specific time points using an IVIS Lumina Series III in vivo imaging system (PerkinElmer, Inc.).
结果显示，负载PDA纳米颗粒的水凝胶能够在皮下缓释ROS，确保ROS的持续和有效胞内递送(图4)。The results showed that the hydrogel loaded with PDA nanoparticles was able to slowly release ROS subcutaneously, ensuring sustained and effective intracellular delivery of ROS ( Figure 4 ).
实施例8.ROS供体材料负载可溶型微针的制备Example 8. Preparation of soluble microneedles loaded with ROS donor materials
(1)使用PDMS模具制备微针，模具中带有圆锥形孔。每个微针长1500微米，基部直径500微米，微针间中心距为1000微米。(1) Microneedles were prepared using a PDMS mold with a conical hole in the mold. Each microneedle was 1500 μm long, 500 μm in diameter at the base, and 1000 μm between the centers of the microneedles.
(2)在模具上沉积14wt％的10kDa透明质酸(HA)和6wt％的800kDa透明质酸(HA)，并在底部使用真空制造负压，随后在室温下干燥过夜，使溶剂蒸发并形成可溶型微针尖。注意，OXI/RED被分散在HA溶液中，以制造负载ROS供体材料的微针。(2) 14 wt% of 10 kDa hyaluronic acid (HA) and 6 wt% of 800 kDa hyaluronic acid (HA) were deposited on the mold, and a vacuum was used to create negative pressure at the bottom, followed by drying at room temperature overnight to evaporate the solvent and form a soluble microneedle tip. Note that OXI/RED was dispersed in the HA solution to make microneedles loaded with ROS donor material.
(3)在HA微针背面沉积15wt％的聚乙烯醇(PVA)和5wt％的聚乙烯吡咯烷酮(PVP)，作为基底贴片，随后真空处理。在室温下干燥12小时后，小心地将微针贴剂从模具中分离。(3) 15 wt% polyvinyl alcohol (PVA) and 5 wt% polyvinyl pyrrolidone (PVP) were deposited on the back of the HA microneedles as a base patch, followed by vacuum treatment. After drying at room temperature for 12 hours, the microneedle patch was carefully separated from the mold.
(4)微针贴剂的制备和储存在无菌条件下进行。使用FE-SEM获得微针的形态。ROS供体材料负载微针贴片扫描电镜照片表明，ROS供体纳米颗粒在微针针尖中分布均匀(图5A和B)。(4) The preparation and storage of the microneedle patch were carried out under sterile conditions. The morphology of the microneedles was obtained using FE-SEM. The scanning electron microscopy images of the ROS donor material loaded microneedle patch showed that the ROS donor nanoparticles were evenly distributed in the microneedle tip (Figure 5A and B).
实施例9.体内毛发生长的评估Example 9. Assessment of in vivo hair growth
(1)使用六周龄雄性C57BL/6小鼠进行体内研究，并允许它们在新环境中适应一周。(1) Six-week-old male C57BL/6 mice were used for in vivo studies and were allowed to acclimate to the novel environment for one week.
(2)使用脱毛膏去除背部毛发以观察粉红色皮肤。随后，将动物随机分组(n＝6)，以测试ROS供体材料应用后的毛发再生情况。(2) The back hair was removed using a depilatory cream to observe the pink skin. Subsequently, the animals were randomly divided into groups (n=6) to test the hair regeneration after the application of ROS donor materials.
(3)负载PDA纳米粒子的水凝胶被皮下注射等待自然生物降解，而微针则手动应用于皮肤2分钟，6小时后移除。(3) The hydrogel loaded with PDA nanoparticles was injected subcutaneously and allowed to biodegrade naturally, while the microneedles were manually applied to the skin for 2 min and removed after 6 h.
实验结果显示，从第9天到第15天使用不同微针贴片时毛发再生的宏观照片，RED组的毛发生长速率显著高于Blank和OXI组。各组单根毛发的扫描电镜照片显示，RED组的新生毛发直径显著高于其他组别(图5C-E)。这表明，PDA纳米粒子能够有效促进毛发的生长和再生，证明了该方法的有效性和潜在的临床应用价值。The experimental results showed that the macroscopic photos of hair regeneration when using different microneedle patches from day 9 to day 15 showed that the hair growth rate in the RED group was significantly higher than that in the Blank and OXI groups. Scanning electron microscopy photos of single hairs in each group showed that the diameter of new hair in the RED group was significantly higher than that in other groups (Figure 5C-E). This shows that PDA nanoparticles can effectively promote hair growth and regeneration, proving the effectiveness and potential clinical application value of this method.
综上所述，本发明利用PDA纳米粒子的独特氧化还原特性和ROS释放能力，结合高效的传递系统，为毛发再生提供了一种新颖有效的治疗方法。In summary, the present invention utilizes the unique redox properties and ROS release ability of PDA nanoparticles, combined with an efficient delivery system, to provide a novel and effective treatment method for hair regeneration.
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化和修改。It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.
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| CN202410182678.XACN118236394A (en) | 2024-02-19 | 2024-02-19 | Application of a hair regeneration promoting material based on polydopamine nanoparticles |
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