Enhanced optical imaging and fluorescent labeling for visualizing drug molecules within living organisms

Abstract

The visualization of drugs in living systems has become key techniques in modern therapeutics. Recent advancements in optical imaging technologies and molecular design strategies have revolutionized drug visualization. At the subcellular level, super-resolution microscopy has allowed exploration of the molecular landscape within individual cells and the cellular response to drugs. Moving beyond subcellular imaging, researchers have integrated multiple modes, like optical near-infrared II imaging, to study the complex spatiotemporal interactions between drugs and their surroundings. By combining these visualization approaches, researchers gain supplementary information on physiological parameters, metabolic activity, and tissue composition, leading to a comprehensive understanding of drug behavior. This review focuses on cutting-edge technologies in drug visualization, particularly fluorescence imaging, and the main types of fluorescent molecules used. Additionally, we discuss current challenges and prospects in targeted drug research, emphasizing the importance of multidisciplinary cooperation in advancing drug visualization. With the integration of advanced imaging technology and molecular design, drug visualization has the potential to redefine our understanding of pharmacology, enabling the analysis of drug micro-dynamics in subcellular environments from new perspectives and deepening pharmacological research to the levels of the cell and organelles.

Keywords: Drug visualization; Fluorophore labeling; Optical imaging; Therapeutics.

Graphical abstract

Figure 1

Principles underlying imaging-based technologies for drug visualization and screening.

Figure 2

Application of NIR-II fluorescence microscopy. (A) Schematic illustration of the NIR-II fluorescence microscopic imaging system. Reprinted with the permission from Ref. . Copyright © 2020 The Authors, some rights reserved. (B) NIR-II fluorescence microscopy images of the cerebrovasculature through a thinned-skull window. (C) Distributions of 3D fluorescence intensity in brain blood vessels before and after induction of prothrombic ischemic stroke. Reprinted with the permission from Ref. . Copyright © 2021 Wiley-VCH GmbH. (D) Schematic diagram of the NIR-II endoscopy system. (E) Simultaneous white light and fluorescence images of representative tumors. Reprinted with the permission from Ref. . Copyright © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 3

Advanced image quantification tools. (A) Procedure for fluorescence-based GFP-WIPI1 image acquisition and analysis using CellProfiler. Reprinted with the permission from Ref. . Copyright © 2023 The Authors, some rights reserved. (B) M-value calculations indicate mitochondria-lysosome contact events in ATG13KO cells (1,2) with or without treatment with the mitochondrial damaging agent. Reprinted with the permission from Ref. . Copyright © 2020 Elsevier Ltd. (C) Determination of the L/W value to evaluate changes to mitochondrial morphology in response to a drug. (D) Mitochondrial morphology of distribution parameters of L/W. (E) Use of the L/W value to evaluate mitochondrial morphological responses to CCCP treatment. Reprinted with the permission from Ref. . Copyright © 2020 Springer-Verlag GmbH Germany.

Figure 4

Recommend fluorophores from the visible window to near-infrared (NIR) Ⅱ window.

Figure 5

The main elements of directly fluorescently labeled drug technology for drug visualization.

Figure 6

Direct fluorescent labeling for the visualizing of small molecule drugs. (A) The structure of a novel multifunctional prostate cancer-targeting fluorescent inhibitor. (B) In vivo targeting studies of novel multifunctional prostate-specific membrane antigen (PSMA) inhibitors. Reprinted with the permission from Ref. . Copyright © 2019 American Chemical Society. (C) Structured illumination microscopy-captured distribution of Cy5-labeled dextran. (D) Different angle images of the localization of Cy5-dextran to mitochondria. Reprinted with the permission from Ref. . Copyright © 2022 Elsevier B.V. (E) The dynamics process of photoactivated Pt₂L escaping from the autolysosomes to the nucleus. Reprinted with the permission from Ref. . Copyright © 2020 Wiley-VCH GmbH.

Figure 7

The types of natural label-free fluorescent derivatives for drug visualization.

Figure 8

Specific fluorophores targeting organelles. (A) Schematic diagram of the responsiveness of magnoflorine to mitochondrial ClO^‒. (B) Effect of MF on ferroptosis and the morphological distribution of mitochondria. (C) Quantitative analysis of mitochondrial morphology. Reproduced with permission. Data as shown as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, all compared with untreated cells. Reprinted with the permission from Ref. . Copyright © 2022 Elsevier B.V. (D) Schematic representation of mitoFluo accumulation and uncoupling activity. Reprinted with the permission from Ref. . Copyright© 2014 Royal Society of Chemistry.

Figure 9

De novo-designed near-infrared probes for imaging therapeutics. (A) Schematic representation of organelle-specific photodynamic cancer therapy targeted to lipid droplets or mitochondria. (B) NIR AIEgens exhibit deeper tissue penetration than Mito-Tracker. Reprinted with the permission from Ref. . Copyright © 2018 American Chemical Society. (C) Design of NIR-II probes for noninvasive in situ detection of acute kidney injury (AKI). (D) 3D-MSOT images of control and AKI model mice at 5 h post-injection of the probe. Reprinted with the permission from Ref. . Copyright © 2023 American Chemical Society. (E) LET-1052 as a pH/viscosity-activatable molecule for acidic tumor microenvironment turn-on photothermal therapy. Reprinted with the permission from Ref. . Copyright © 2022 Wiley-VCH GmbH. (F) Chemical structure and surface electrostatic potential of TPy-Chpt and TTPy-Chpt. (G) Comparison images of tumors after PDT treatment. (H) Images of PC-3 tumor-bearing nude mice intratumorally injected with TPy-Chpt or TTPy-Chpt at different times. Reprinted with the permission from Ref. . Copyright © 2022 American Chemical Society.

Figure 10

Schematic representation of the proposed design strategy for drug visualization.