Functionalized Single-walled Carbon Nanotubes for Topical Skin Delivery of Tyrosinase

이민정 서울대학교 대학원 2023 국내석사

Tyrosinase is an essential enzyme that mediates melanin synthesis in melanocytes to protect skin from ultraviolet (UV) induced damage. If melanocytes stop producing functional tyrosinases, skin diseases such as albinism and vitiligo can occur. One promising way to treat these skin diseases is the delivery of tyrosinase directly to the skin to produce synthetic melanin. Since protein-based drugs are sensitive to environmental change, topical delivery is an appropriate method for tyrosinase delivery. However, the large size of tyrosinase (31.5 kDa) makes it hard to penetrate the skin barrier. Therefore, a functionalized single-walled carbon nanotube (SWNT) was introduced as a nanocarrier of tyrosinase and a reverse electrodialysis (RED) battery was also utilized to accelerate the transport of the tyrosinase–SWNT complex (TYR–SWNT) by electrostatic repulsion. The characteristic properties and dimensions of the TYR–SWNTs were analyzed to confirm that it is suitable for topical skin delivery while maintaining the tyrosinase activity. The penetration efficiency of TYR–SWNT was observed in ex vivo porcine skin and in vivo mouse skin using confocal Raman microscopy. It was demonstrated that with the assistance of SWNT and RED, tyrosinase could be effectively delivered to the skin, which was sufficient to synthesize melanin with endogenous L-tyrosine. This study presents a carbon nanocarrier-based topical protein delivery platform that can be utilized for various medical applications. 티로시나아제는 피부 표피 기저막에 위치하고 있는 멜라닌세포에서 멜라닌 합성을 촉진하는 산화 효소이다. 티로시나아제가 피부 내에서 생성되지 못하거나 오작동하게 되면 백반증이나 알비노 등의 피부질환이 발생하며, 자외선으로 인한 손상으로부터 피부를 보호할 수 없게 된다. 티로시나아제를 직접 피부에 전달하는 것이 이러한 피부 질환을 치료하기 위한 하나의 방안이 될 수 있다. 티로시아나제를 피부에 전달하기 위한 방법으로 국소 부위 전달 방식이 주목받고 있는데, 국소 부위 전달 방법은 전달 과정에서 환경의 변화가 적기 때문에 단백질 약물의 분해나 변성을 방지할 수 있다는 장점이 있다. 그러나 티로시아나제는 31.5 kDa의 큰 분자량을 가지고 있기 때문에 피부 장벽을 투과하기 어렵다. 따라서, 본 연구에서는 기능화된 단일벽 탄소 나노 튜브(Single-walled carbon nanotube, SWNT)를 나노전달체로써 사용하여 티로시나아제의 피부 투과 능력을 향상시키려 하였다. 또한 역전기 투석을 기반으로 한 배터리를 이용하여 정전기적 반발력을 유도해 티로시네나아제와 SWNT의 복합체(Tyrosinase immobilized SWNT, TYR–SWNT)의 전달 효율을 높이고자 하였다. 카복시메틸셀룰로스, 아자이드 기능기, 지질로 기능화된 세 가지 종류의 SWNT 후보군을 제작하여 피부 투과 능력을 공초점 라만 현미경을 통해 분석하였다. 소수성 성질을 갖는 지질로 기능화된 SWNT (Lipid–SWNT)가 피부를 투과하기 가장 적합함을 확인하였으며, Lipid–SWNT를 티로시나아제의 전달체로 선정하여 TYR–SWNT를 제작하였다. TYR–SWNT가 피부에 전달하기 적합한 전하 및 크기를 가지고 있음을 확인하였으나, SWNT에 고정화된 티로시나아제는 모노페놀 산화 효소 초기 활성에 지연 현상이 있는 것을 관찰했다. TYR–SWNT는 생체 외 돼지 피부와 생체 내 알비노 쥐 피부에 전달되었으며, 두 경우 모두 멜라닌을 합성할 수 있는 깊이인 200 μm 이상까지 투과된 반면, 다른 장기에는 전달되지 않는 것을 공초점 라만 현미경을 통해 확인하였다. 본 연구를 통해 개발된 단일벽 탄소나노뷰트 기반의 티로시나아제 피부 국소 전달 시스템은 향후 다양한 단백질 단백질 약물 전달에 널리 활용될 수 있을 것으로 기대된다.

Development of a dissolving microneedle-based enhanced micro-channeling system for skincare

공성대 Graduate School, Yonsei University 2023 국내석사

Topical liquid formulations, dissolving microneedles (DMNs), and microscale needles made of biodegradable materials have gained widespread use for the transdermal delivery of active compounds in skincare. However, skin barriers hinder the transdermal delivery of active compounds through topical liquid formulation application, and the effectiveness of DMNs in skincare is limited by low encapsulation capacity and incomplete insertion. This study employed a dissolvable micro-channeling system (DMCS) for the topical application of serum to enhance serum delivery through micro-channels embedded with DMNs. The efficacy of a DMCS for enhancing transdermal serum delivery was investigated by evaluating the intensity of allophycocyanin (APC) loaded with serum in porcine skin after topical-serum-only and combinatorial serum application. The results revealed that the APC intensity was significantly higher in the skin layer at a depth of 120-270 μm upon combinatorial serum application compared to serum-only application. Moreover, the clinical assessment of skin hydration, depigmentation, wrinkle improvement, elasticity, dermal density, skin pores, and skin soothing showed significantly improved efficacy with combinatorial serum application through DMCS, without any safety issues, compared to serum-only application. These findings demonstrate that combinatorial serum application via DMCS can potentially enhance skincare treatments with optimal transdermal delivery of active compounds.

Application of microemulsion for enhancing topical skin absorption of 20(S)-protopanaxadiol and oral absorption of rebamipide

김기택 서울대학교 대학원 2017 국내박사

20(S)-protopanaxadiol (20S-PPD) is an aglycosylated metabolite of ginsenosides such as compound K and ginsenoside Rb1, and possesses a potent skin anti-aging activity. However, due to its low aqueous solubility and large molecular size, a suitable formulation strategy is required in order to enhance skin deposition of 20S-PPD by improving its solubility and skin permeability. Rebamipide (RBP) is a potent anti-ulcer and anti-oxidative agent, which belongs to biopharmaceutics classification system (BCS) class IV with a poor oral bioavailability of less than 10%. Thus, enhancing the systemic exposure of RBP may increase its pharmacological activities after oral administration. The objective of the study was to develop microemulsion (ME)-based systems for the topical skin delivery of 20S-PPD and for the oral delivery of RBP. 20S-PPD-loaded ME and ME-based hydrogel (MEH) formulations were prepared and evaluated in terms of their particle size distribution, morphology, maximum loading capacity, viscosity, and pH value. Then, the in vitro and in vivo deposition or permeation profiles of 20S-PPD in the selected MEH formulation were studied using hairless mouse skin model and artificial skin model, Strat-M® membrane. A Carbopol-based MEH system of 20S-PPD was successfully prepared with a mean droplet size of 110 nm and polydispersity index of 0.436. The formulation was stable at least for 56 days, and its viscosity was high enough for its topical skin application. It significantly enhanced the in vitro and in vivo skin deposition of 20S-PPD with no influence on its systemic absorption in hairless mice. Notably, it was found that the rank of the tested formulaions in the order of decreasing deposition of 20S-PPD in in vitro Strat-M® membrane and in vitro/in vivo hairless mouse skin was same. For RBP-loaded ME, characterization study (i.e. maximum loading capacity, particle size distribution, and morphology), in vitro drug release study, in vivo pharmacokinetic study, and intestinal toxicity study were performed. Capmul MCM EP and Solutol HS15-based ME system of RBP had spherical nano-sized droplets with polydispersity index of 0.265 and neutral zeta potential. Moreover, the prepared ME significantly enhanced the dissolution and oral bioavailability of RBP with no discernible intestinal toxicity. Taken together, the ME-based systems developed in this study could serve as a potentially effective topical skin and oral delivery system for enhancing the absorption of poorly soluble compounds including 20S-PPD and RBP.

Liposome Formulation of Coenzyme Q10 for Topical Delivery

Rengarajan Baskaran 영남대학교 대학원 2009 국내석사

연구배경: 코엔자임 Q10은 자외선에 의한 광노화를 줄이는 산화방지제이다.기존의 제품은 국소적으로 적용되는 리포솜 성분으로 피부 감염이나 노화의 치료에 사용된다. 또한 이 성분은 산화방지제를 포함하는데 이는 감염된 조직에서 자유 라디칼을 제거하며 자유 라디칼로부터 손상 방지를 도운다. 연구목적: 연구의 목적은 코엔자임 Q10의 리포좀 제형을 통해 피부 축적을 높임으로써 국소적 용도로 적용하는데 있다. 실험방법: 리포좀 제형은 얇은 지방 필름 기술을 통해 만들었으며, 크기 감소를 위해 초음파 bath에서 초음파 처리 하였다. 리포좀의 입자 크기는 전기영동 광산란 광도계로 측정하였다 (ELS-8000,OTSUKA Electronics Co.Ltd.,일본). 봉입율은 Degli et al에 나와있는 분배 방법에 의해 측정되었다. 또한, 헥세인과 리포솜 현탁액(1:1 v/v)을 섞은 후, 30초간 vortex 한 후 원심분리 하였다. 리포좀 현탁액 하층의 코엔자임 Q10은 고성능 액체 크로마토그래피를 이용해 분석하였다. 피부투과 동물실험은 hairless mice의 정돈된 피부와 2.27 cm2 확산 면적의 Franz diffusion cell을 사용하였다. 각 샘플의 은 미리 결정된 간격에 의해 시행되었고, 코엔자임 Q10의 피부투과 정량은 고성능 액체크로마토그래피로 측정되었다. 피부투과 시험 마지막에, 피부를 깨끗이 잘게 잘라서 분쇄한 후, 피부 내의 코엔자임 Q10의 함량을 분석하였다. 모든 실험은 암실에서 진행되었고, 샘플은 광분해로부터 보호되었다. 결론: 6개의 지방 조성중에서 0.75 비율의 CH/PL 이 안정성 측면에서 이상적이었으며, 모든 리포좀 제형이 대조군에 비해서 피부 잔류량이 많았다. 이는 리포좀 제형을 이용한 코엔자임 Q10이 효과적인 국소 제제로 개발될 수 있는 가능성을 나타낸다. Background: Coenzyme Q10 (CoQ) is a naturally occurring, fat-soluble compound, with characteristics common to vitamins and, like vitamins, is essential for the optimal functioning of all organisms. The physicochemical properties such as high hydrophobicity, high molecular weight, and photolytic nature make CoQ difficult to develop a novel delivery system. However, liposomes, one of promising novel delivery systems, are biocompatible and better in incorporating lipophilic drugs and it has been investigated as effective drug carrier. Purpose: The primary objective of the present study was to evaluate the penetration/retention of topically applied liposome formulations containing CoQ. The formulated liposomes were also investigated for physicochemical properties such as size, polydispersity index (PDI), entrapment efficiency, size evolution, and storage stability was also investigated. Methods: Liposome formulations of different phospholipon and cholesterol compositions were prepared by lipid thin film technique followed by size reduction. The formulated liposomes were also investigated for physicochemical properties such as size, polydispersity index (PDI), entrapment efficiency, size evolution, and storage stability. In vitro skin permeation/accumulation study was performed using freshly removed skin of male hairless mice skin and Franz diffusion cell with 2.27 cm2 diffusion area. All the experiments were carried out in dark condition and the samples were protected from light degradation. Conclusion: In conclusion, among the six lipid compositions, 0.75 molar ratio of CH to PL was optimal in terms of stability, and all the liposomal formulations showed high skin retention compared to the control. This finding suggests the possibility of developing an effective topical system of CoQ utilizing liposomes as carrier.

Microemulsion-based hydrogel formulation of itraconazole for topical delivery

이은아 서울대학교 대학원 2007 국내석사

마이크로에멀젼 제제를 이용한 가공인삼 성분의 피부흡수 촉진 연구

노가야 서울대학교 대학원 2011 국내석사