背景

白癜风是一种以皮肤黑素细胞破坏为核心的自身免疫性疾病，目前的治疗方法存在疗效不稳定、复发率高及副作用明显等局限性。酪氨酸酶（tyrosinase，TYR）作为黑色素合成的限速酶，其活性降低与白癜风的发病密切相关。mRNA疗法通过递送功能性基因实现瞬时蛋白表达，具有高效性与安全性，但其应用受到递送系统效率和稳定性的限制。纳米脂质体（Lipid nanoparticles，LNP）作为成熟的递送载体，能够增强mRNA的稳定性和靶向性，而微针技术则为局部精准给药提供了新思路。本研究旨在通过优化酪氨酸酶mRNA序列、构建LNP递送系统以及建立白癜风动物模型，探索mRNA疗法在白癜风治疗中的可行性。

目的

1.    筛选高表达、高活性的TYR-mRNA序列；

2.    构建并评价负载TYR-mRNA的LNP载体系统；

3.    验证TYR-mRNA-LNP载体系统的生物活性及安全性；

4.    建立稳定可靠的白癜风小鼠模型；

5.    评估微针技术递送TYR-mRNA-LNP的治疗潜力。

方法

1.    mRNA序列优化与筛选：设计三条TYR-mRNA序列（TYR-mRNA-1/2/3），通过体外转录合成，并转染HEK-293T细胞，检测Flag标签表达量及TYR活性。

2.    LNP载体构建：采用微流控技术封装TYR-mRNA-2，分析其粒径、Zeta电位和包封率，通过透射电镜观察LNP形态。

3.    生物活性与安全性评价：以原代人黑素细胞（HEM细胞）为模型，检测不同浓度的LNP对细胞活力、氧化应激指标（ROS、MDA、SOD、GPx）及黑色素含量的影响。

4.    白癜风模型构建：在C57BL/6J Nifdc小鼠身上外用40%莫诺苯宗乳膏持续3周，通过白癜风小鼠模型的色素脱失指数（Vitiligo mouse model depigmentation index，VMMDI）、组织病理学和免疫组化方法验证模型的有效性。

5.    治疗可行性验证：通过空心微针将TYR-mRNA-LNP载体系统递送至白癜风小鼠的脱色区域，观察复色进程，并评价皮肤局部及系统的安全性。

结果

1.    TYR-mRNA-2被确定为最优酪氨酸酶mRNA序列：三条候选序列（TYR-mRNA-1/2/3）中，TYR-mRNA-1体外转录产量最高（2004.4 ng/μL，产量100.2 μg），但其蛋白表达稳定性不足（Western Blot显示Flag标签表达显著低于TYR-mRNA-2，P<0.05）；TYR-mRNA-3虽转染后酪氨酸酶活性最高（0.650±0.055 U/10⁵ cells），但合成成本过高（产量与浓度最低）。功能验证显示，TYR-mRNA-2在酶活性（0.740±0.039 U/10⁵ cells）与TYR-mRNA-1无差异（P>0.05）的前提下，兼具高表达稳定性与适中的合成效率（产量与浓度仅次于TYR-mRNA-1）。综合评估表明，TYR-mRNA-2在规模化生产成本（较TYR-mRNA-3降低35%）、蛋白表达效率（较TYR-mRNA-1提升18%）及功能活性（较TYR-mRNA-3提升13.8%）间实现最优平衡，满足递送系统对序列稳定性和功能可控性的核心需求。

2.    成功构建适用于皮肤递送的酪氨酸酶mRNA-LNP载体系统：通过微流控技术制备的LNP粒径均一（动态光散射直径125.2±3.2 nm，PDI=0.102±0.03），近中性表面电荷（Zeta电位-2.1332±0.7 mV）可有效降低皮肤角质层的静电排斥，增强局部滞留；透射电镜证实其完整球形结构（无聚集或缺陷），与光散射数据高度一致（粒径分布误差<5%）。mRNA包封效率达88.03±0.07%，表明制备工艺能高效保护核酸免于降解。该系统的物理特性（粒径<150 nm、PDI<0.15）符合FDA对纳米药物递送载体的标准，其高包封效率与稳定结构为后续体内外功能研究奠定了技术基础。

3.    TYR-mRNA-LNP的生物学效应呈现浓度依赖性阈值：1 μg/mL剂量可安全上调黑素合成功能，酪氨酸酶活性（较对照组提升28.6%，P<0.05）与黑色素含量（提升22.3%，P<0.05）显著增加，且未引发细胞毒性（活力>95%）；而≥2 μg/mL时，尽管酶活性仍高于对照组（P<0.05），但细胞活力下降（2 μg/mL组第48小时降至78.4%，P<0.05），伴随ROS水平（升高1.8倍）及脂质过氧化标志物（MDA含量增加2.1倍）的剂量依赖性积累（P均<0.05）。机制研究表明，≥2 μg/mL剂量可显著抑制SOD（下降37.2%）与GPx（下降41.5%）活力（P<0.05），破坏氧化还原稳态。上述结果明确界定1 μg/mL为功能-安全性平衡阈值，为后续剂量优化提供直接依据。

4.    外用40%莫诺苯宗乳膏诱导的白癜风小鼠模型具有高可靠性：连续外用3周后，90.48%的小鼠（n=21）呈现稳定脱色表型（VMMDI评分11.38±0.75），表皮基底层黑素颗粒密度下降62.3%（P<0.01），酪氨酸酶免疫组化信号强度较对照组降低34.7%（P<0.05），与临床白癜风患者病理特征高度吻合（毛囊黑素细胞损失>70%）。模型表型可持续超过观察周期（>8周），且未引发系统性毒性（肝转氨酶ALT/AST、肌酐等血生化指标均在生理范围内）。组织学检测显示，造模过程仅伴随短暂表皮屏障损伤（早期鳞屑评分1.2±0.3）及真皮浅层噬色素细胞浸润（密度<5个/HPF），证实该模型兼具表型稳定性（成功率>90%）、病理可验证性（酪氨酸酶活性下降与临床一致）及操作安全性（无不可逆损伤），适用于治疗药物的临床前评价。

5.    微针递送TYR-mRNA-LNP促进白癜风小鼠色素再生：10 μg剂量组在介入第4天即显现毛囊外根鞘黑素颗粒沉积量增加，第7天复色面积扩展达45%（P<0.01），且表皮基底层黑素细胞密度同步上升；5 μg组的疗效稍弱（第7天复色面积增幅30%），但两组间差异随时间推移缩小。免疫组化的结果显示酪氨酸酶活性在毛囊区域优先激活，与HE染色揭示的黑素再生时空规律一致。安全性方面，微针递送未引发真皮炎症（炎细胞浸润计数<3个/HPF）或系统毒性（血常规及肝肾功能指标波动<10%）。值得注意的是，对照组迟发性微弱复色（第7天面积增幅<5%）提示需在临床研究中排除自然病程干扰，进一步验证治疗的特异性。

结论

1.    通过体外转录与功能验证，TYR-mRNA-2在合成效率、蛋白表达稳定性与TYR活性间达到较优的平衡，被确定为后续研究的核心序列。

2.    基于微流控技术构建的TYR-mRNA-LNP系统具有均一粒径、高包封率及近中性表面电荷，其理化性质符合纳米药物递送标准。

3.    在H₂O₂氧化应激条件下，1μg/mL TYR-mRNA-LNP可显著提升黑素细胞酪氨酸酶活性与黑色素合成，但2-4μg/mL TYR-mRNA-LNP会引发细胞活力下降及氧化损伤，提示需严格控制TYR-mRNA-LNP的给药浓度。

4.    用莫诺苯宗诱导的白癜风小鼠模型能成功模拟白癜风的临床病理特征，且无系统毒性，为治疗效果的评价提供标准化平台。

5.    微针递送的治疗潜力：微针递送酪氨酸酶mRNA-LNP可促进局部复色，显著增加毛囊黑素颗粒密度，且未引发局部皮肤的炎症反应或系统毒性，证实其局部靶向性与安全性。

Background

Vitiligo is an autoimmune skin disorder characterized by the destruction of melanocytes. Current treatment options are limited by inconsistent efficacy, high relapse rates, and significant side effects. Tyrosinase (TYR), the rate-limiting enzyme in melanin biosynthesis, plays a critical role in vitiligo pathogenesis due to its reduced enzymatic activity. mRNA therapy enables transient protein expression through functional gene delivery and offers high efficacy and safety. However, its clinical application is constrained by the efficiency and stability of the delivery system. Lipid nanoparticles (LNPs), as a well-established delivery platform, can improve mRNA stability and targeting. Meanwhile, microneedle technology provides a novel approach for localized and precise administration. This study aims to evaluate the feasibility of mRNA therapy for vitiligo treatment by optimizing TYR-mRNA sequences, constructing an LNP-based delivery system, and developing a vitiligo animal model.

Objectives

1.    To identify TYR mRNA sequences with high expression and enzymatic activity.

2.    To construct and evaluate an LNP delivery system loaded with TYR-mRNA.

3.    To validate the bioactivity and safety of the TYR-mRNA-LNP system.

4.    To establish a stable and reliable mouse model of vitiligo.

5.    To assess the therapeutic potential of microneedle-mediated delivery of TYR-mRNA-LNP.

Methods

1.    mRNA sequence optimization and selection: Three TYR-mRNA sequences (TYR-mRNA-1/2/3) were designed, synthesized via in vitro transcription, and transfected into HEK-293T cells. Flag-tag expression and TYR activity were analyzed.

2.    LNP formulation: TYR-mRNA-2 was encapsulated using microfluidic technology. Particle size, Zeta potential, and encapsulation efficiency were measured, and LNP morphology was examined by transmission electron microscopy.

3.    Bioactivity and safety evaluation: Primary human epidermal melanocytes (HEM cells) were treated with different concentrations of LNPs to assess cell viability, oxidative stress markers (ROS, MDA, SOD, GPx), and melanin content.

4.    Vitiligo model establishment: C57BL/6J Nifdc mice were topically treated with 40% monobenzone cream for three weeks. The effectiveness of the model was validated using the vitiligo mouse model depigmentation index (VMMDI), histopathology, and immunohistochemistry.

5.    Therapeutic feasibility evaluation: TYR-mRNA-LNP were delivered to depigmented skin areas of vitiligo mice using hollow microneedles. Repigmentation progress and local/systemic safety were assessed.

Results

1.    TYR-mRNA-2 was identified as the optimal tyrosinase mRNA sequence. Among the three candidate sequences (TYR-mRNA-1/2/3), TYR-mRNA-1 exhibited the highest in vitro transcription yield (2004.4 ng/μL, 100.2 μg total), but demonstrated insufficient protein expression stability (Western blot showed significantly lower Flag tag expression than TYR-mRNA-2, P < 0.05). TYR-mRNA-3 showed the highest enzymatic activity post-transfection (0.650 ± 0.055 U/10⁵ cells) but incurred the highest synthesis cost due to its lowest yield and concentration. TYR-mRNA-2 displayed enzymatic activity comparable to TYR-mRNA-1 (0.740 ± 0.039 U/10⁵ cells, P > 0.05), along with superior expression stability and moderate synthesis efficiency (yield and concentration second only to TYR-mRNA-1). Comprehensive evaluation revealed that TYR-mRNA-2 achieved an optimal balance across production cost (35% lower than TYR-mRNA-3), protein expression efficiency (18% higher than TYR-mRNA-1), and functional activity (13.8% higher than TYR-mRNA-3), making it the most suitable sequence for stable, functional, and scalable delivery.

2.    A tyrosinase mRNA-LNP delivery system suitable for cutaneous administration was successfully constructed. LNPs prepared via microfluidic technology exhibited uniform particle size (125.2 ± 3.2 nm, PDI = 0.102 ± 0.03), near-neutral surface charge (Zeta potential: -2.1332 ± 0.7 mV), which facilitates enhanced skin retention by reducing electrostatic repulsion from the stratum corneum. Transmission electron microscopy confirmed a complete spherical morphology without aggregation or defects, in close agreement with light scattering data (size distribution error <5%). The encapsulation efficiency reached 88.03 ± 0.07%, indicating robust protection of mRNA from degradation. The physical parameters (size <150 nm, PDI <0.15) meet FDA standards for nanoparticle-based drug delivery systems, laying a solid technical foundation for further in vitro and in vivo functional studies.

3.    The biological effects of TYR-mRNA-LNPs exhibited a concentration-dependent threshold. At 1 μg/mL, TYR-mRNA-LNPs significantly increased tyrosinase activity (28.6% higher than control, P < 0.05) and melanin content (22.3% increase, P < 0.05) without cytotoxicity (cell viability >95%). However, at ≥2 μg/mL, while tyrosinase activity remained elevated (P < 0.05), cell viability declined (78.4% at 48 h for 2 μg/mL group, P < 0.05), accompanied by dose-dependent increases in ROS (1.8-fold) and MDA (2.1-fold) levels (P < 0.05). Mechanistically, ≥2 μg/mL doses significantly suppressed antioxidant enzymes SOD (37.2%) and GPx (41.5%) (P < 0.05), disrupting redox homeostasis. These findings identify 1 μg/mL as the optimal threshold balancing efficacy and safety, providing a rational basis for subsequent dose optimization.

4.    Topical 40% monobenzone-induced vitiligo mouse model demonstrated high reliability. After three weeks of topical application, 90.48% of mice (n=21) developed a stable depigmentation phenotype (VMMDI score: 11.38 ± 0.75). Melanin granule density in the basal epidermis decreased by 62.3% (P < 0.01), and tyrosinase immunohistochemical signal was reduced by 34.7% (P < 0.05) compared to controls, closely resembling the histopathological features of clinical vitiligo (including >70% loss of follicular melanocytes). The phenotype persisted beyond 8 weeks without systemic toxicity (ALT/AST, creatinine, and other biochemical indices remained within physiological range). Histological examination revealed only transient epidermal barrier disruption (early desquamation score: 1.2 ± 0.3) and mild dermal melanophage infiltration (<5 cells/HPF), confirming the model's high reproducibility, clinical relevance, and procedural safety, thus providing a standardized platform for therapeutic evaluation.

5.    Microneedle-mediated delivery of TYR-mRNA-LNPs promoted repigmentation in vitiligo mice. In the 10 μg group, increased melanin granule deposition in the follicular outer root sheath was observed by day 4, with repigmentation expanding to 45% of the depigmented area by day 7 (P < 0.01), accompanied by elevated basal epidermal melanocyte density. The 5 μg group showed slightly weaker effects (30% repigmentation on day 7), though the intergroup difference narrowed over time. Immunohistochemistry revealed preferential activation of tyrosinase in hair follicles, aligning with HE staining findings of temporospatial melanocyte regeneration. Importantly, no inflammatory response (dermal inflammatory cells <3/HPF) or systemic toxicity (variations in hematology and liver/kidney function <10%) was observed. Notably, the control group displayed minimal spontaneous repigmentation (<5% area on day 7), highlighting the need to account for natural disease course in clinical translation to confirm treatment specificity.

Conclusion

1.    TYR-mRNA-2 was identified as the optimal sequence based on in vitro transcription and functional assays, achieving the best balance between synthesis efficiency, protein expression stability, and tyrosinase activity.

2.    The TYR-mRNA-LNP system developed using microfluidic technology demonstrated uniform size, high encapsulation efficiency, and near-neutral surface charge, meeting regulatory standards for nanoparticle delivery.

3.    Under oxidative stress, TYR-mRNA-LNP at 1 μg/mL significantly enhanced melanocyte tyrosinase activity and melanin synthesis without cytotoxicity, whereas doses of 2–4 μg/mL induced decreased viability and oxidative damage, indicating the necessity for strict dosage control of TYR-mRNA-LNP.

4.    The monobenzone-induced vitiligo mouse model effectively simulated clinical and pathological manifestations of vitiligo without systemic toxicity, providing a reliable preclinical platform for therapeutic evaluation.

5.    Microneedle-mediated delivery of TYR-mRNA-LNPs effectively promoted localized repigmentation and increased follicular melanin granule density without triggering cutaneous inflammation or systemic side effects, confirming its targeted delivery potential and safety profile.