Bioconjug Chem. 2018 Apr 18;29(4):1060-1072. doi: 10.1021/acs.bioconjchem.7b00717. Epub 2018 Feb 23.
The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.
丝氨酸蛋白酶 Kallikrein 5（KLK5）的过表达和活性增加是炎症性皮肤病（如酒渣鼻）的特征。该酶的抑制剂（如 4-(2-氨基乙基)苯磺酰氟盐酸盐（AEBSF·HCl）或抗人重组 Kallikrein 5（抗-KLK5）抗体）在疾病治疗中的应用受到限制，因为它们的生物利用度低，因此将它们固定在药物递送剂中有助于使丝氨酸蛋白酶抑制剂在临床上有用。在这项工作中，我们合成了金纳米颗粒（GNP），其表面覆盖有羟基和羧基末端硫醇的混合物（GNP.OH/COOH），其羧基基团用于通过静电或共价方式（分别为 GNP.COOH AEBSF 和 GNP.AEBSF）进一步功能化纳米颗粒与丝氨酸蛋白酶抑制剂 AEBSF·HCl，或与抗-KLK5 抗体（GNP.antiKLK5）。合成并功能化的 GNP 具有高度的水溶性，并通过紫外-可见吸收光谱、透射电子显微镜（TEM）、动态光散射（DLS）和热重分析（TGA）进行了广泛的表征。GNP.OH/COOH 及其随后的功能化有效地抑制了 KLK5 的体外活性。使用共聚焦荧光显微镜证明了荧光团包被的 GNP.OH/COOH 在人角质形成细胞（HaCaT 细胞）中的内化。细胞活力测定表明，当游离 AEBSF 被掺入纳米颗粒中时，其细胞毒性显著降低，无论是离子形式（GNP.COOH AEBSF）还是最重要的共价形式（GNP.AEBSF）。功能化的纳米颗粒 GNP.AEBSF 和 GNP.antiKLK5 抑制了 HaCaT 细胞内 KLK5 的活性，并减少了 TLR-2 配体引发炎症条件下的 IL-8 分泌。这项研究表明，这些 GNP 作为一种新的细胞内递药策略，对于治疗酒渣鼻等皮肤疾病的小分子药物和抗体具有巨大的潜力。
