Ultra-pH-sensitive nanoprobe library with broad pH tunability and fluorescence emissions

Xinpeng Ma, Yiguang Wang, Tian Zhao, Yang Li, Lee-Chun Su, Zhaohui Wang, Gang Huang, Baran D Sumer, Jinming Gao, Xinpeng Ma, Yiguang Wang, Tian Zhao, Yang Li, Lee-Chun Su, Zhaohui Wang, Gang Huang, Baran D Sumer, Jinming Gao

Abstract

pH is an important physiological parameter that plays a critical role in cellular and tissue homeostasis. Conventional small molecular pH sensors (e.g., fluorescein, Lysosensor) are limited by broad pH response and restricted fluorescent emissions. Previously, we reported the development of ultra-pH-sensitive (UPS) nanoprobes with sharp pH response using fluorophores with small Stokes shifts (<40 nm). In this study, we expand the UPS design to a library of nanoprobes with operator-predetermined pH transitions and wide fluorescent emissions (400-820 nm). A copolymer strategy was employed to fine tune the hydrophobicity of the ionizable hydrophobic block, which led to a desired transition pH based on standard curves. Interestingly, matching the hydrophobicity of the monomers was critical to achieve a sharp pH transition. To overcome the fluorophore limitations, we introduced copolymers conjugated with fluorescence quenchers (FQs). In the micelle state, the FQs effectively suppressed the emission of fluorophores regardless of their Stokes shifts and further increased the fluorescence activation ratios. As a proof of concept, we generated a library of 10 nanoprobes each encoded with a unique fluorophore. The nanoprobes cover the entire physiologic range of pH (4-7.4) with 0.3 pH increments. Each nanoprobe maintained a sharp pH transition (on/off < 0.25 pH) and high fluorescence activation ratio (>50-fold between on and off states). The UPS library provides a useful toolkit to study pH regulation in many pathophysiological indications (e.g., cancer, lysosome catabolism) as well as establishing tumor-activatable systems for cancer imaging and drug delivery.

Figures

Figure 1
Figure 1
Schematic design of ultra-pH-sensitive (UPS) micellar nanoprobes. (a) In the unimer state (pH t), polymer dissociation resulted in fluorophore/quencher separation and strong fluorescence emission. In the micelle state (pH > pHt), fluorescence quenching dramatically suppresses the emission intensity of fluorophores. (b) A random copolymer strategy was used to achieve an operator-predetermined control of nanoprobe pHt by the ability to continuously fine tune the hydrophobicity of the PR segment.
Figure 2
Figure 2
Syntheses of dye- or fluorescence quencher (FQ)-conjugated PEO-b-P(R1-r-R2) copolymers. The hydrophobicity of the PR segment can be continuously controlled by varying the molar fractions of the two monomers (R1 or R2 = Et, ethyl; Pr, propyl; Bu, butyl; Pe, pentyl).
Figure 3
Figure 3
(a) Comparison of PDEA/PD5A molecular mixture vs P(DEA40-D5A40) copolymer strategies for control of pHt. (b) Normalized fluorescence intensity of P(DEAx-D5Ay) nanoprobes with different ratios of the two monomers as a function of pH. (c) Nanoprobe pHt is linearly correlated with the molar fraction of the DEA-MA monomer in the PR segment. Error bars were calculated from three repeating experiments (n = 3). Polymer concentrations were 0.1 mg/mL in these studies.
Figure 4
Figure 4
(a) Normalized fluorescence intensity as a function of pH for Cy5-conjugated P(DPAx-DBAy) nanoprobes. (b) Derivative fluorescence plot (dF/dpH, data from a) as a function of pH for P(DPAx-DBAy) vs P(DEA40-D5A40) nanoprobes. Use of methacrylate monomers with close hydrophobicity (i.e., DPA/DBA vs DEA/D5A) resulted in much sharper pH transitions. (c) Linear relationships of the nanoprobe pHt vs molar fraction of the less hydrophobic monomer for different copolymer compositions. These correlations serve as the standard curves for selecting the optimal copolymer composition to achieve an operator-predetermined pHt. (d) Representative library of UPS nanoprobes with 0.3 pH increment covering the physiologic range of pH 4–7.4. All nanoprobes were conjugated with the Cy5 dye. Polymer concentrations were at 0.1 mg/mL.
Figure 5
Figure 5
Introduction of FQ-conjugated PDPA copolymer significantly increased the fluorescence activation ratio of different PDPA-dye nanoprobes. Fluorescence intensity ratio at different pH to pH 7.4 (FpH/F7.4) was plotted for copolymer alone (a, c, and e) and with addition of FQ-conjugated copolymers (b, d, f). See main text for detailed description and Supporting Information Figure S19 for the structures of the dyes and FQs.
Figure 6
Figure 6
Exemplary UPS library consisting of 10 nanoprobes spanning a wide pH range (4–7.4) and large fluorescent emissions (400–820 nm). Each nanoprobe is encoded by its transition pH and fluorophore. Images of 4.4-AMCA and 4.7-MB were taken by a camera at an excitation light of 365 nm. Images of the rest of the nanoprobe solutions were taken on a Maestro Imaging system.

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