Anti-quenching NIR-II molecular fluorophores for in vivo high-contrast imaging and pH sensing

Noninvasive fluorescence (FL) imaging and high-performance photocatalytic therapy (PCT) are opposing optical properties that are difficult to combine in a single material system. Herein, we report a facile approach to introducing oxygen-related defects in carbon dots (CDs) via post-oxidation with 2-iodoxybenzoic acid (IBX), in which some nitrogen atoms were substituted by oxygen atoms. Unpaired electrons in these oxygen-related defects rearranged the electronic structure of the oxidized carbon dots (ox-CDs), resulting in an emerging near-infrared (NIR) absorption band. These defects not only contributed to enhanced NIR bandgap emission but also acted as trappers for photoexcited electrons to promote efficient charge separation on the surface, leading to abundant photo-generated holes on the ox-CDs surface under visible-light irradiation. Under white LED torch irradiation, the photo-generated holes oxidized hydroxide to hydroxyl radicals in the acidification of the aqueous solution. In contrast, no hydroxyl radicals were detected in the ox-CDs aqueous solution under 730nm laser irradiation, indicating noninvasive NIR FL imaging potential. Utilizing the Janus optical properties of the ox-CDs, the in vivo NIR FL imaging of sentinel lymph nodes around tumors and efficient photothermal enhanced tumor PCT were demonstrated. This article is protected by copyright. All rights reserved.

Near-infrared (NIR) fluorescence imaging in the wavelength of 1000-1400 nm (2nd-NIR optical window) is expected to offer better spatiotemporal deep-tissue imaging due to the lower autofluorescence and scattering than that in the 1st-NIR optical window (700-1000 nm). During the past decade considerable progress has been made in the development of NIR fluorescent materials that emit over 1000 nm for non-invasive fluorescence imaging. Although single-walled carbon nanotube (SWNT), rare-earth metal ion doped nanomaterials and silver sulfide (Ag2S) quantum dots (QDs) have been reported as fluorescent probes in the 2nd-NIR optical window, their fluorescence brightness is relatively low, with quantum yields (QY) less than 6 %. To achieve deep-tissue imaging with high spatiotemporal resolution, we have developed fluorescent lead sulfide (PbS) QDs with high quantum yields (QY >10 %) in the 2nd-NIR region. We present the synthesis of 2nd-NIR emitting PbS QDs and their applications to the non-invasive fluorescence imaging of lymph nodes, cerebral blood vessels, breast tumors, and phagocytic cells in mice.

Near-infrared (NIR) fluorescence imaging in the wavelength of 1000–1400 nm (2nd-NIR optical window) is expected to offer better spatiotemporal deep-tissue imaging due to the lower autofluorescence and scattering than that in the 1st-NIR optical window (700–1000 nm). During the past decade considerable progress has been made in the development of NIR fluorescent materials that emit over 1000 nm for non-invasive fluorescence imaging. Although single-walled carbon nanotube (SWNT), rare-earth metal ion doped nanomaterials and silver sulfide (Ag2S) quantum dots (QDs) have been reported as fluorescent probes in the 2nd-NIR optical window, their fluorescence brightness is relatively low, with quantum yields (QY) less than 6%. To achieve deep-tissue imaging with high spatiotemporal resolution, we have developed fluorescent lead sulfide (PbS) QDs with high quantum yields (QY >10%) in the 2nd-NIR region. This paper summarize the synthesis of 2nd-NIR emitting PbS QDs and their applications to the non-invasive fluorescence imaging of lymph nodes, cerebral blood vessels, breast tumors, and phagocytic cells in mice.

Noninvasive Real-Time Fluorescence Imaging of the Lymphatic uptake of BSA–IRDye 680 Conjugate Administered Subcutaneously in Mice

Organic fluorophores have been widely used for biological imaging in the visible and the first near-infrared windows. However, their application in the second near-infrared window (NIR-II, 1000-1700 nm) is still limited mainly due to low fluorescence quantum yields (QYs). Here, we explore molecular engineering on the donor unit to develop high performance NIR-II fluorophores. The fluorophores are constructed by a shielding unit-donor(s)-acceptor-donor(s)-shielding unit structure. Thiophene is introduced as the second donor connected to the shielding unit, which can increase the conjugation length and red-shift the fluorescence emission. Alkyl thiophene is employed as the first donor connected to the acceptor unit. The bulky and hydrophobic alkyl thiophene donor affords larger distortion of the conjugated backbone and fewer interactions with water molecules compared to other donor units studied before. The molecular fluorophore IR-FTAP with octyl thiophene as the first donor and thiophene as the second donor exhibits fluorescence emission peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the highest for molecular NIR-II fluorophore reported so far. Superior temporal and spatial resolutions have been demonstrated with IR-FTAP fluorophore for NIR-II imaging of the blood vessels of a mouse hindlimb.

The fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) has emerged as a new method for in vivo imaging and attracted considerable attention in the past decade. Owing to the suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in NIR-II window can afford deep tissue penetration depth with high clarity. Inorganic nanoparticle-based fluorescent probes in the NIR-II window have greatly prospered the field into a development stage because of their superior traits, including adjustable emission covering the whole NIR-II window and abundant surface functional groups that facilitate chemical modification and bioconjugation, etc. In this Feature, we introduce the unique imaging performance of the NIR-II optical window and highlight the latest development of noninvasive biological fluorescent imaging in NIR-II window using inorganic nanoparticle-based probes. A perspective on the challenge and future direction of inorganic nanoparticle-based NIR-II probes is also discussed.

One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery.

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Noninvasive fluorescence imaging (NFI) is a powerful tool to study physiology and pathophysiology in animal disease models. NFI has been successfully applied in a number of animal disease models including cancer, arthritis, and stroke. Furthermore, several applications in humans have been described. NFI is widely available in research laboratories because it has a number of advantages: It uses non-ionizing radiation and requires comparably simple, inexpensive instrumentation, and easy to handle. Fluorochromes can be detected with high sensitivity, and image acquisition time is relatively short. Furthermore, a plethora of fluorescent imaging agents is available including unspecific, target-specific, and activatable imaging probes. With these probes, biological processes such as inflammation, cell death or enzyme activity, and many others can be visualized in living animals. This review offers an overview of current approaches in NFI of stroke pathophysiology in animal models of cerebral ischemia. First, the instrumentation and the different types of imaging agents for NFI are described. Second, a short introduction to animal models of stroke is provided. Third, examples for NFI in animal models of stroke are given. Finally, the use of NFI in human stroke is critically discussed.

Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.

Fluorescence imaging in the second near-infrared window (NIR-II) enables deep-tissue visualization with high spatial-temporal resolution. Developing molecular fluorophores with high brightness and stability in aqueous media is therefore critical. However, most NIR-II excited fluorophores suffer from pronounced nonradiative decay and fluorescence quenching in water. Here, we propose a unimolecular micellization strategy to construct high-brightness NIR-II fluorophores that self-assemble into stable unimolecular micelles (UIMs) in aqueous solution. The designed star-shaped amphiphilic molecule IR-FCT8CP carries long alkyl chains that collapse into a compact hydrophobic core upon micellization, effectively shielding the fluorophore from water-induced quenching and restricting intermolecular interactions. The resulting IR-FCT8CP UIMs exhibit absorption and emission maxima at 979 and 1181nm, respectively, with a quantum yield of 0.05% and a molar absorption coefficient of 1.67×104 M-1·cm-1 in aqueous solution, yielding higher brightness than IR-FCDP and IR-FCTP UIMs. The IR-FCT8CP UIMs enable dynamic in vivo vascular imaging under 1064nm excitation using a 1500nm long-pass filter, clearly resolving vascular networks with a high signal-to-background ratio. This unimolecular micellization strategy offers a general design concept for developing stable, high-brightness NIR-II molecular fluorophores for efficient bioimaging in physiological environments.

Introduction: Tumor dimensionality creates a dynamic three dimensional (3D) architecture that is influenced by the associated microenvironment, including stromal cells and the extracellular matrix. These paracrine interactions impact therapeutic efficacy and can alter drug response in vivo , yet most current in vitro models do not accurately recapitulate the dimensionality or the stromal microenvironment of human tumors. In vitro models that are more recapitulative of the human tumor microenvironment have broad applicability in evaluation of signaling pathways driving cancer progression, therapeutic efficacy, and mechanisms involved in therapeutic resistance and tumor recurrence. There is a great need to adapt traditional analytical methods, developed for two dimensional cell culture, for use in 3D tissue models. Herein, a novel perfusion bioreactor system is used to support the multi-week growth and development of 3D breast carcinoma tissue surrogates (measuring 1.0 cm in maximum dimension) consisting of breast carcinoma epithelial cell lines and cancer associated fibroblasts (CAF) in a supportive extracellular matrix. Further, non-invasive imaging techniques, commonly employed to evaluate in vivo animal model systems, were used to measure growth of the surrogates overtime. Methods: 3D breast carcinoma surrogates were generated by incorporating MDA-MB-231 cells (tagged with GFP and luciferase) or MCF-7 cells (tagged with GFP and luciferase), with or without CAF, into an extracellular matrix. Surrogates were cultured in a perfusion bioreactor system for up to 3 weeks. Cell growth was measured on histologic sections of surrogates by counting the number of nucleated cells per surrogate cross-sectional area (cell density). Growth and viability were also determined in the same surrogates over time by using non-invasive fluorescence and luminescence imaging (IVIS 100 system). Results: The use of a flow perfusion bioreactor system resulted in a marked increase in the cell density of surrogates compared to non-perfused surrogates (perfused: 93.3 nucleated cells/area vs. non-perfused: 32.1 nucleated cells/area) at 21 days culture. Fluorescence and luminescence imaging of surrogates, containing increasing concentrations of breast cancer epithelial cells, were imaged at day 0 to confirm a correlation between signal intensity and cell number using each imaging modality (GFP: R2=0.97, p<0.01, Luciferase: R2=0.99, p=0.053 ). Next, fluorescence imaging of the same 3D breast carcinoma surrogates (containing breast carcinoma cells and CAF) overtime was completed at days 0, 7, and 14 of culture and showed an increase in signal (7.9 fold higher signal at day 14 compared to day 0) indicating growth throughout culture. Similar results were seen with imaging of the luciferase signal where the signal was 28.2 fold higher at day 14 compared to day 0. Conclusions: The presence of perfusion allows the growth and development of a recapitulative breast carcinoma surrogate with a size similar to human breast carcinomas at the time of detection and with an appropriate tumor microenvironment. Non-invasive imaging methods have successfully been adapted to evaluate growth of the breast carcinoma surrogates throughout multi-week culture. Future Directions: The use of these perfused breast carcinoma surrogates and imaging modalities in the evaluation of established and candidate cancer therapeutics will be assessed. Citation Format: Kayla F. Goliwas, Jillian R. Richter, Lauren E. Marshall, Joel L. Berry, Andra R. Frost. Evaluation of in vitro three dimensional breast cancer surrogates using histologic morphology and non-invasive imaging to monitor growth and viability throughout culture. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A05.

Intravital fluorescence imaging in the second near-infrared window (NIR-II, 900-1700nm) has emerged as a promising method for non-invasive diagnostics in complex biological systems due to its advantages of less background interference, high tissue penetration depth, high imaging contrast, and sensitivity. However, traditional NIR-II fluorescence imaging, which is characterized by the "always on" or "turn on" mode, lacks the ability of quantitative detection, leading to low reproducibility and reliability during bio-detection. In contrast, NIR-II ratiometric fluorescence imaging can realize quantitative and reliable analysis and detection in vivo by providing reference signals for fluorescence correction, generating new opportunities and prospects during in vivo bioimaging and biosensing. In this review, the current design strategies and sensing mechanisms of NIR-II ratiometric fluorescence probes for bioimaging and biosensing applications are systematically summarized. Further, current challenges, future perspectives and opportunities for designing NIR-II ratiometric fluorescence probes are also discussed. It is hoped that this review can provide effective guidance for the design of NIR-II ratiometric fluorescence probes and promote its adoption in reliable biological imaging and sensing in vivo.

Advances in Improving Healthcare with Aggregation-Induced Emission.

MoS2 nanoflowers surface decorated with CuS nanorods and carbon dots for fluorescent and ultrasound imaging in cancer therapy.