The advancement of targeted drug delivery faces significant challenges for clinical translation, such as issues related to poor solubility, non-specific distribution, and limited bioavailability of cancer theranostics. Notably, promising developments in near-infrared (NIR) imaging, particularly those centered around Indocyanine Green (ICG), hold the potential for intraoperative tumor targeting. However, the field of medical imaging grapples with two persistent challenges: 1) non-targeted uptake and 2) incomplete elimination of imaging agents. In response to these issues, we focus on the creation of targeted NIR-I/II fluorescence agents possessing optimized physicochemical properties. These innovative compounds encompass zwitterionic organic nanocarriers, such as the noteworthy Harvard Dots (H-Dots). Importantly, these agents can be administered systemically, circumventing non-specific tissue uptake and achieving exclusive elimination through the urinary system. H-dots not only enable the precise determination of surgical margins through NIR image-guided procedures but also serve as effective carriers for targeted anticancer drug delivery. Their unique characteristics result in reduced uptake by the immune system, heightened selectivity for tumors, and enhanced tumor suppression compared to conventional free drugs. Consequently, these H-Dots represent a highly promising theranostic nanoplatform. This research paves the way for the creation of renal-clearable, tissue-specific NIR contrast agents, holding great promise for future advancements in image-guided cancer surgery and, ultimately, improved patient outcomes.
While ICG-based NIR imaging has shown great potential in intraoperative surgery, there are two fundamental and unsolved problems facing medical imaging: 1) nonspecific uptake of intravenously administered diagnostic and/or therapeutic agents by normal tissues and organs and 2) incomplete elimination of unbound targeted agents from the body. These problems make image-guided cancer surgery extremely difficult because the background signal is high, and therefore the TBR is low. Designing a targeted contrast agent that shows fast clearance from the background tissues and eventually from the body after complete targeting is the key to the success of image-guided interventions. “Structure-Inherent Targeting” is a strategy that combines tissue-specific targeting components and imaging domain into a single molecule for targeting and imaging specific tissues in real-time, where the compact structural design enables the unbound contrast agent to be easily cleared from the body after targeting.
Programed death ligand-1 (PD-L1) expression is currently the only predictive biomarker for cancer immunotherapy. Since PD-L1 expression in tumors is largely heterogeneous, in-vivo detection and quantification of PD-L1 in intact tumors is of major interest. Here we employ fluorescence lifetime (FLT) imaging for in-vivo detection and quantification of PD-L1 expression using an anti-PD-L1 antibody conjugated to IRDye800CW (αPDL1-800). We show that FLT imaging accurately identifies heterogeneous PD-L1 expression in tumors. Tumor areas of high PD-L1 levels were spatially correlated to significantly longer FLTs of αPDL1-800 and the distribution of PD-L1 in deep-seated (>1cm depth) tumors was achieved using FLT tomography.
Fluorescence imaging of cancers using continuous wave (CW) detection of receptor targeted probes offer poor sensitivity and specificity due to background autofluorescence and non-specific probe accumulation. Here we show that fluorescence lifetime (FLT) imaging can significantly improve tumor contrast using epidermal growth factor receptor (EGFR) and programed death ligand 1 (PD-L1) targeted probes in a preclinical model of human breast cancer. Our results suggest that these probes have significantly longer FLTs in tumors than in normal tissue and the FLT enhancement is receptor dependent. We also show potential for simultaneous quantification of EGFR and PD-L1 using in vivo FLT multiplexing.
Accurate mapping of gastrointestinal stromal tumors (GIST) during surgery is difficult, which contributes to the suboptimal diagnosis and recurrence of cancers. To overcome this limitation, we developed a near-infrared (NIR) fluorescent nanoprobe for real-time navigation of GIST using a targeted strategy against the CD117 ligand stem cell factor (SCF). A zwitterionic NIR fluorophore conjugated to SCF showed specific binding to a xenograft mouse model of CD117-positive GIST-T1 with minimal nonspecific tissue signals. This promising intraoperative imaging strategy could be further explored for early diagnosis and follow-up of GIST prognosis before and after surgical resection.
There remains a paucity of methodological tools to determine the biodistribution of vaccine antigens. In response to this, we established a near-infrared (NIR) imaging method using a NIR fluorophore, ZW800-1C, conjugated with different sizes of vaccine antigens that allows for real-time monitoring of the fate of delivered vaccines in vivo. The fluorescent signal observed using the system after a model vaccine injection in mice recapitulated the size-dependent transport of the vaccine into the secondary lymphoid tissue. This methodology can be broadly applied for optimization of formulations and safety evaluation of clinical vaccines.
Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology.
Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light.
Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature.
Results: A specific combination of wavelengths at low irradiances (250 to 400 mW / cm2 for 1064 nm and 55 to 65 mW / cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes.
Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.
Two fundamental and unsolved problems facing bioimaging and nanomedicine are nonspecific uptake of intravenously administered diagnostic and/or therapeutic agents by normal tissues and organs, and incomplete elimination of unbound targeted agents from the body. To solve these problems, we have synthesized a series of indocyanine near-infrared (NIR) fluorophores that varied systematically in net charge, conformational shape, hydrophilicity/lipophilicity, and charge distribution. Using 3D molecular modeling and optical fluorescence imaging, we have defined the relationship among the key independent variables that dictate biodistribution and tissue-specific targeting such as lung and sentinel lymph nodes (Nat Biotechnol. 2010), human prostate cancers (Nat Nanotechnol. 2010), and human melanomas (Nat Biotechnol. 2013). Recently, we have developed new pharmacophore design strategy “structure-inherent targeting,” where tissue- and/or organ-specific targeting is engineered directly into the non-resonant structure of a NIR fluorophore, thus creating the most compact possible optical contrast agent for bioimaging and nanomedicine (Angew Chem. 2015, Nat Med. 2015). The biodistribution and targeting of these compounds vary with dependence on their unique physicochemical descriptors and cellular receptors, which permit 1) selective binding to the target tissue/organ, 2) visualization of the target specifically and selectively, and 3) provide curing options such as image-guided surgery or photo dynamic therapy. Our study solves two fundamental problems associated with fluorescence image-guided surgery and lays the foundation for additional targeted agents with optimal optical and in vivo performance.
Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging.
Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance.
Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation
fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we
describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high
power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in
this article and the list of components are available online at www.frangionilab.org.
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