Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H⁺/K⁺ ionophore to equalize the intra- and extracellular pH‚ showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565–630 nm) to that from the UCNP red emission channel (640–680 nm), produced a sigmoidal pH response curve with an apparent pK_a for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator–modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis.

Graphical abstract

上转换纳米粒子（UCNP）应该特别适合细胞内部的测量，因为它们可以使用荧光显微镜近乎实时地成像到亚微米尺寸，并且它们克服了常见的问题，例如光漂白、自发荧光和深层组织穿透在细胞成像应用中遇到。在本研究中，研究了用 pH 敏感染料 (pHAb) 修饰的 UCNP 的性能。染料（发射波长 580 nm）附着在 UCNP 上的聚乙烯亚胺 (PEI) 涂层中，并在 980 nm 激发下通过 540 nm UCNP 发射进行激发。不同 pH 值下的 UC 共振能量转移效率范围为 25% 至 30%，从这些结果预测福斯特距离为 2.56 nm。人神经母细胞瘤 SH-SY5Y 细胞，用尼日利亚菌素 H ⁺ /K ⁺离子载体平衡以平衡细胞内和细胞外 pH，显示 UCNP-pHAb 缀合物颗粒的吸收，并取从 pHAb 发射通道收集的强度比 (565 –630 nm）与来自 UCNP 红色发射通道（640–680 nm）的波长相比，产生了 UCNP-pHAb 的表观_pKa约为 5.1 的 S 形 pH 响应曲线。 UCNP-pHAb 与 LysoBrite 染料（一种溶酶体标记物）共定位。研究了分别干扰网格蛋白介导的内吞作用和小凹介导的内吞作用的药物抑制剂，例如氯丙嗪（CPZ）和制霉菌素（NYS），以阐明纳米颗粒摄取到细胞中的机制。这项初步研究表明，pH 指示剂修饰的 UCNP（例如 UCNP-pHAb）可以报告 SH-SY5Y 细胞中的 pH，并且纳米颗粒通过网格蛋白介导的内吞作用掺入细胞中。

 图形概要