Gray Mikaela A, de Janon Alejandro, Seeler Michelle, Heller William T, Panoskaltsis Nicki, Mantalaris Athanasios, Champion Julie A
Chemical and Biomolecular Engineering, Georgia Institute of Technology Atlanta, Georgia 30332-0002, United States.
Biomedical Systems Engineering Laboratory, Georgia Institute of Technology, Atlanta, Georgia 30332-0002, United States.
ACS Omega. 2025 Jan 14;10(3):2628-2639. doi: 10.1021/acsomega.4c07666. eCollection 2025 Jan 28.
Proteins can perform ideal therapeutic functions. However, their large size and significant surface hydrophilicity and charge prohibit them from reaching intracellular targets. These chemical features also render them poorly encapsulated by nanoparticles used for intracellular delivery. In this work, a novel combination of protein vesicles and hydrophobic ion pairing (HIP) was used to load protein cargo and achieve cytosolic delivery to overcome the limitations of previous protein vesicle properties. Protein vesicles are thermally self-assembling nanoparticles made from elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly, physiological stability, and small size, the ELP sequence was modified to include histidine and tyrosine residues. HIP was used to load and release protein cargo requiring endosomal escape for cytosolic function. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and a significant reduction in viability in both a traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. By examining the uptake of positively and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cytosolic cargo delivery and how HIP loading influences protein vesicle self-assembly and disassembly using microscopy, small-angle X-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins as therapeutics for various diseases and extend protein vesicles to deliver other biomacromolecules, as the strategy developed here resulted in the first cytosolic protein cargo delivery using protein vesicles.
蛋白质可以发挥理想的治疗功能。然而，它们的大尺寸以及显著的表面亲水性和电荷特性阻碍了它们到达细胞内靶点。这些化学特性还使得它们难以被用于细胞内递送的纳米颗粒有效包裹。在这项工作中，一种蛋白质囊泡与疏水离子对（HIP）的新型组合被用于装载蛋白质货物并实现胞质递送，以克服先前蛋白质囊泡特性的局限性。蛋白质囊泡是由与富含精氨酸的亮氨酸拉链融合的弹性蛋白样多肽（ELP）以及与富含谷氨酸的亮氨酸拉链融合的球状蛋白质制成的热自组装纳米颗粒。为了赋予刺激响应性解组装、生理稳定性和小尺寸特性，ELP序列被修饰以包含组氨酸和酪氨酸残基。HIP被用于装载和释放需要从内体逃逸以发挥胞质功能的蛋白质货物。HIP囊泡能够递送细胞色素c（一种具有胞质活性的蛋白质），并且在传统的二维（2D）人类癌细胞系培养以及急性髓系白血病的仿生三维（3D）类器官模型中均能显著降低细胞活力。通过检测由HIP装载的带正电和负电的荧光蛋白质货物的摄取情况，这项工作揭示了HIP对于胞质货物递送的必要性，以及HIP装载如何通过显微镜、小角X射线散射和纳米颗粒跟踪分析影响蛋白质囊泡的自组装和解组装。HIP蛋白质囊泡有潜力拓宽细胞内蛋白质作为各种疾病治疗药物的应用范围，并将蛋白质囊泡扩展用于递送其他生物大分子，因为此处开发的策略首次实现了使用蛋白质囊泡进行胞质蛋白质货物递送。
