Shi Jianquan, Hu Jin, Yuan Yeshuang, Zhang Bo, Guo Wenting, Wu Yuanhao, Jiang Lingjuan
Department of Intensive Care Unit, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Department of Medical Research Center, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China.
ACS Omega. 2021 Mar 12;6(11):7931-7940. doi: 10.1021/acsomega.1c00532. eCollection 2021 Mar 23.
Therapeutic proteins such as enzymes, hormones, and cytokines suffer from poor stability, inefficient cellular penetration, and rapid clearance from circulation. Conjugation with polymers (such as poly(ethylene glycol)) and fusion with long-acting proteins (such as albumin and Fc fragments) have been utilized to partially address the delivery issues, but these strategies require the introduction of new macromolecular substances, resulting in potential immunogenicity and toxicity. Herein, we report an easy strategy to increase the intracellular delivery efficiency and stability of proteins by combining of sortase-mediated protein cyclization and cell-penetrating peptide (CPP)-mediated intracellular delivery. We, for the first time, genetically constructed a green fluorescence protein (GFP) fused with a CPP, a transacting activator of transcription (TAT) peptide, at its C-terminus for intracellular internalization, and two sortase recognition sequences, pentaglycine and LPETG, at its N- and C-termini for cyclization. Notably, he cyclized GFP-TAT (cGFP-TAT) not only highly retained the photophysical properties of the protein but also significantly improved the stability compared with the native linear GFP (lGFP) and linear TAT peptide-fused GFP (lGFP-TAT).Moreover, cGFP-TAT showed better cellular internalization ability compared with lGFP. In C26 tumor-inoculated mice, cGFP-TAT exhibited enhanced tumor retention, with increases of 7.79- and 6.52-fold relative to lGFP and lGFP-TAT in tumor retention 3 h after intratumor administration. This proof-of-concept study has provided an easy strategy to increase the stability, intracellular delivery efficiency, and tumor retention of GFP, which would be applicable to numerous therapeutic proteins and peptides for clinical practice.
诸如酶、激素和细胞因子等治疗性蛋白质存在稳定性差、细胞穿透效率低以及从循环中快速清除的问题。与聚合物（如聚乙二醇）缀合以及与长效蛋白质（如白蛋白和Fc片段）融合已被用于部分解决递送问题，但这些策略需要引入新的大分子物质，从而导致潜在的免疫原性和毒性。在此，我们报告了一种简单的策略，即通过将分选酶介导的蛋白质环化与细胞穿透肽（CPP）介导的细胞内递送相结合，来提高蛋白质的细胞内递送效率和稳定性。我们首次通过基因工程构建了一种绿色荧光蛋白（GFP），其C末端融合了一种CPP，即转录反式激活因子（TAT）肽，用于细胞内内化，其N末端和C末端有两个分选酶识别序列，即五甘氨酸和LPETG，用于环化。值得注意的是，环化的GFP-TAT（cGFP-TAT）不仅高度保留了蛋白质的光物理性质，而且与天然线性GFP（lGFP）和线性TAT肽融合的GFP（lGFP-TAT）相比，稳定性显著提高。此外，与lGFP相比，cGFP-TAT表现出更好的细胞内化能力。在接种C26肿瘤的小鼠中，瘤内给药3小时后，cGFP-TAT在肿瘤中的保留增强，相对于lGFP和lGFP-TAT，肿瘤保留增加了7.79倍和6.52倍。这项概念验证研究提供了一种简单的策略来提高GFP的稳定性、细胞内递送效率和肿瘤保留率，这将适用于众多用于临床实践的治疗性蛋白质和肽。
