In Press
Hailey Monaco, Shinya Yokomizo, Haksoo Choi, and Satoshi Kashiwagi. In Press. “Quickly evolving near-infrared photoimmunotherapy provides multifaceted approach to modern cancer treatment.” VIEW, Pp. 20200110. Publisher's VersionAbstract
Abstract Among modalities of cancer immunotherapy, near-infrared photoimmunotherapy (NIR-PIT) has reached significant preclinical and clinical stages and quickly evolved over the last 5 years. NIR-PIT uses deep-penetrable NIR light to induce physicochemical changes in the antibody–photosensitizer conjugate (APC), leading to resultant necrosis and immunogenic cell death (ICD) of the cancer cells. Alternatively, other types of photomedicine use photosensitizers to convert absorbed light energy either into reactive oxygen species for photodynamic therapy (PDT) or into heat for photothermal therapy (PTT). ICD is a unique and relevant outcome of NIR-PIT because it induces long-lasting antitumor host immunity, which overcomes the immunosuppressive network of cancer. Due to its high specificity and durable antitumor effects, NIR-PIT is now considered a promising cancer therapy, and optimized NIR-PIT is readily expanding its applicability to many different types of cancer. Along with the traditional method of NIR-PIT, new avenues in its realm of treatment are currently being explored, such as the targeting of other immunosuppressive elements, delivery of NIR light through a catheter, real-time imaging for tumor detection, and the use of tumor-seeking small molecules for improved efficacy and safety. In addition, its effect on hyperpermeability has opened a door for a wide array of combination therapies with other modalities. This review summarizes the recent findings in clinical and preclinical studies of NIR-induced photomedicine and its future significance in the field of cancer research.
Chengeng Yang, Haoran Wang, Shinya Yokomizo, Morgan Hickey, Hyejin Chang, Homan Kang, Takeshi Fukuda, Mi Young Song, Sang Yeul Lee, Jin Woo Park, Kai Bao, and Haksoo Choi. 6/14/2021. “ZW800-PEG: A Renal clearable zwitterionic near‐infrared fluorophores for potential clinical translation.” Angew Chem Int Ed Engl, 60, 25, Pp. 13847-13852.Abstract
Near-infrared (NIR) fluorescence imaging has advanced medical imaging and image-guided interventions during the past three decades. Despite tremendous advances in imaging devices, surprisingly only a few dyes are currently available in the clinic. Previous fluorophores, ZW800-1A and ZW800-1C, significantly improved the poor performance of the FDA-approved indocyanine green. However, ZW800-1A is not stable in serum and ZW800-1C induces severe stacking in aqueous media. To solve such dilemmas, ZW800-PEG was designed by introducing a flexible yet stable thiol PEG linker. ZW800-PEG shows high solubility in both aqueous and organic solvents, thus improving renal clearance with minimal binding to serum proteins during systemic circulation. The sulfide group on the meso position of the heptamethine core improves serum stability and physicochemical properties including the maximum emission wavelength shift to 800 nm, enabling the use of ZW800-PEG for image-guided interventions and augmenting photothermal therapy.
H. Lee, Y.H. An, T.K. Kim, J. Ryu, G. K. Park, M.J. Park, J. Ko, H. Kim, H. Choi, Nathaniel S. H., and T.H. Park. 2021. “Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin‐Penetrating Property of bFGF Using 30Kc19α‐Based Fusion Protein.” Advanced Biology, Pp. 2000176.
Hye Hyeon Han, Homan Kang, Seong-Jong Kim, Rahul Pal, Anand T. N. Kumar, Haksoo Choi, and Sei Kwang Hahn. 2021. “Fluorescent nanodiamond – hyaluronate conjugates for target-specific molecular imaging.” RSC Adv., 11, Pp. 23073-23081. Publisher's VersionAbstract
Despite wide investigation on molecular imaging contrast agents, there are still strong unmet medical needs to enhance their signal-to background ratio, brightness, photostability, and biocompatibility with multimodal imaging capability. Here, we assessed the feasibility of fluorescent nanodiamonds (FNDs) as carbon based photostable and biocompatible materials for molecular imaging applications. Because FNDs have negatively charged nitrogen vacancy (NV) centers, they can emit bright red light. FNDs were conjugated to hyaluronate (HA) for target-specific molecular imaging. HA is a biocompatible, biodegradable, and linear polysaccharide with abundant HA receptors in the liver, enabling liver targeted molecular imaging. In vitro cell viability tests revealed the biocompatibility of HA–FND conjugates and the competitive cellular uptake test confirmed their target-specific intracellular delivery to HepG2 cells with HA receptors. In addition, in vivo fluorescence lifetime (FLT) assessment revealed the imaging capability of FNDs and HA–FND conjugates. After that, we could confirm the statistically significant liver-targeted delivery of HA–FND conjugates by in vivo imaging system (IVIS) analysis and ex vivo biodistribution tests in various organs. The renal clearance test and histological analysis corroborated the in vivo biocompatibility and safety of HA–FND conjugates. All these results demonstrated the feasibility of HA–FND conjugates for further molecular imaging applications.
Soon Hee Kim, Jin Seon Kwon, Jae Gu Cho, Kate G. Park, Tae Hyeon Lim, Moon Suk Kim, Haksoo Choi, Chan Hum Park, and Sang Jin Lee. 2021. “Non-invasive in vivo monitoring of transplanted stem cells in 3D-bioprinted constructs using near-infrared fluorescent imaging.” Bioengineering & Translational Medicine, 6, 2, Pp. e10216. Publisher's VersionAbstract
Abstract Cell-based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue-engineered scaffolds to the process of tissue regeneration remain poorly understood. Near-infrared (NIR) fluorescence imaging systems have great potential to non-invasively monitor the transplanted cell-based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127-labeled MSCs (NIR-MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR-MSCs-loaded bioink showed excellent printability. In addition, NIR-MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non-invasively monitor the NIR-MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR-based imaging system for non-invasive cell monitoring in vivo could play an active role in validating the cell fate in cell-based tissue engineering applications.
Y. J. Suh, T. H. Lim, H. S. Choi, M. S. Kim, S. J. Lee, S. H. Kim, and C. H. Park. 2020. “3D Printing and NIR Fluorescence Imaging Techniques for the Fabrication of Implants.” Materials, 13.Abstract
Three-dimensional (3D) printing technology holds great potential to fabricate complex constructs in the field of regenerative medicine. Researchers in the surgical fields have used 3D printing techniques and their associated biomaterials for education, training, consultation, organ transplantation, plastic surgery, surgical planning, dentures, and more. In addition, the universal utilization of 3D printing techniques enables researchers to exploit different types of hardware and software in, for example, the surgical fields. To realize the 3D-printed structures to implant them in the body and tissue regeneration, it is important to understand 3D printing technology and its enabling technologies. This paper concisely reviews 3D printing techniques in terms of hardware, software, and materials with a focus on surgery. In addition, it reviews bioprinting technology and a non-invasive monitoring method using near-infrared (NIR) fluorescence, with special attention to the 3D-bioprinted tissue constructs. NIR fluorescence imaging applied to 3D printing technology can play a significant role in monitoring the therapeutic efficacy of 3D structures for clinical implants. Consequently, these techniques can provide individually customized products and improve the treatment outcome of surgeries.
H. G. Moon, S. J. Kim, M. K. Lee, H. Kang, H. S. Choi, A. Harijith, J. Ren, V. Natarajan, J. W. Christman, S. J. Ackerman, and G. Y. Park. 2020. “Colony-stimulating factor 1 and its receptor are new potential therapeutic targets for allergic asthma.” Allergy, 75, Pp. 357-369.Abstract
BACKGROUND: A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. OBJECTIVE: To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. METHODS: We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. RESULTS: Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. CONCLUSION: The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.
G. Jones, S. K. Goswami, H. Kang, H. S. Choi, and J. Kim. 2020. “Combating iron overload: a case for deferoxamine-based nanochelators.” Nanomedicine, 15, 13, Pp. 1341-1356.Abstract
While iron is a nutrient metal, iron overload can result in multiple organ failures. Iron chelators, such as deferoxamine, are commonly used to ameliorate iron overload conditions. However, their uses are limited due to poor pharmacokinetics and adverse effects. Many novel chelator formulations have been developed to overcome these drawbacks. In this review, we have discussed various nanochelators, including linear and branched polymers, dendrimers, polyrotaxane, micelles, nanogels, polymeric nanoparticles and liposomes. Although these research efforts have mainly been focused on nanochelators with longer half-lives, prolonged residence of polymers in the body could raise potential safety issues. We also discussed recent advances in nanochelation technologies, including mechanism-based, long-acting nanochelators.
X.-H. Pham, E Hahm, T.H Kim, H.-M. Kim, S.H. Lee, S.C. Lee, H. Kang, H.-Y. Lee, D.H. Jeong, H. S. Choi, and B.-H. Jun. 2020. “Enzyme-amplified SERS immunoassay with Ag-Au bimetallic SERS hot spots.” Nano Res, 13, 12, Pp. 3338-3346.Abstract
Surface-enhanced Raman scattering (SERS) enables rapid detection of single molecules with high specificity. However, quantitative and sensitive SERS analysis has been a challenge due to the lack of reliable SERS-active materials. In this study, we developed a quantitative SERS-based immunoassay using enzyme-guided Ag growth on Raman labeling compound (RLC)-immobilized gold nanoparticle (Au NP)-assembled silica NPs (SiO2@Au-RLC@Ag). The enzyme amplified Ag+ to Ag, forming an Ag hot spot on the reduction as well as Ag growth on the RLC-immobilized Au NP-assembled silica NPs (SiO2@Au-RLC), which resulted in a significant increase in SERS signal. In the presence of target antigens such as IgG or PSA, Ab1-Antigen-Ab2 immune complex with alkaline phosphatase triggered an enzyme-catalyzed reaction to convert 2-phospho-L-ascorbic acid (2-phospho-L-AA) to ascorbic acid (AA). As produced AA reduced Ag+ surface of SiO2@Au-RLC, which enhanced the SERS signal of SiO2@Au-RLC@Ag in a solution with a target antigen concentration. The plasmonic immunoassay for IgG detection showed a high linearity of SERS intensity in the range of 0.6 ng/mL to 9.0 ng/mL with a detection limit (LOD) of 0.09 ng/mL, while an LOD of 0.006 ng/mL was obtained for PSA. The results indicate that the sensitivity of our novel SERS-based immunoassay is higher than that of conventional enzyme-based colorimetric immunoassays.
H. I. Kilian, H. Kang, N. Nyayapathi, T. Fukuda, E. Adluru, H. Zhang, B. Quinn, J. Xia, H. S. Choi, and J. F. Lovell. 2020. “Facile formulation of a long-wavelength cyanine for optical imaging in the second near-infrared window.” Biomater Sci, 8, Pp. 4199-4205.Abstract
The second near-infrared window (NIR-II) beyond 1000 nm has attracted attention for optical contrast imaging in small animals. We sought to assess whether commercially available NIR-II dyes could be easily formulated for this purpose. 13 hydrophobic NIR-II dyes were purchased and screened by formulating them in simple solubilizing agents with established use in humans: propylene glycol, Cremaphor EL, Kolliphor HS15 (HS15), Tween 80, and cyclodextrin. Based on the absorption at 1064 nm (matching the Nd:YAG laser output commonly used in photoacoustic imaging), three of the dyes were further assessed at varying dye and surfactant concentrations. Of these, benzo indole butyl diphenylaminocyclopentene heptamethine (BIBDAH) tetrafluoroborate in HS15 generally showed the most favorable NIR-II character. 1 mg mL−1 BIBDAH in 25% HS15 exhibited a single absorption peak at 1030 nm with a calculated intensity greater than 100, which was relatively stable for weeks in storage. Following intravenous administration to mice, determination of BIBDAH pharmacokinetics was possible by absorption measurements of sampled plasma, revealing a circulating half-life of about one hour. Most of the dye was taken up by the liver. BIBDAH was used in vitro and in vivo as a photoacoustic contrast imaging agent and its accumulation could be detected in subcutaneous tumors in mice. BIBDAH was used for fluorescence imaging of blood vessels in mice, including in the brain (through intact skull), and dye clearance from blood to the liver was visualized. Taken together, this study confirms that accessible, strongly-absorbing dye can readily be formulated for injection by simply dissolving them in biocompatible surfactants and used for high-contrast preclinical optical imaging in the second NIR window.
J. H. Lee, S. Y. Jung, G. K. Park, K. Bao, H. Hyun, G. El Fakhri, and H. S. Choi. 2020. “Fluorometric imaging for early diagnosis and prognosis of rheumatoid arthritis.” Adv Sci, 7, Pp. 1902267.Abstract
Early diagnosis and monitoring of disease progress are of significant importance in the effective treatment of rheumatoid arthritis (RA), because the continuing inflammation can lead to irreversible joint damage and systemic complications. However, applying imaging modalities for the prognosis of RA remains challenging, because no tissue-specific guidelines are available to monitor the progressive course of RA. In this study, fluorometric imaging of RA is reported using bioengineered targeted agents of the blood vessel, bone, and cartilage in combination with the customized optical fluorescence imaging system. Separate but simultaneous tissue-specific images of synovitis, cartilage destruction, and bone resorption are obtained from a mouse model of RA, which allows quantification of the prognosis of diseases at each stage. Thus, the fluorometric imaging of RA by using tissue-specific contrast agents plays a key role in the systemic treatment of RA by monitoring structural damage and disease progression.
W. Katagiri, G. Lee, A. Tanushi, K. Tsukada, H. S. Choi, and S. Kashiwagi. 2020. “High-throughput single-cell live imaging of photobiomodulation with multispectral near-infrared lasers in cultured T cells.” J Biomed Opt, 25, Pp. 1-18.Abstract
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.
M. M. N. Mannan, M. A. Kamran, S. Kang, H. S. Choi, and M. Y. Jeong. 2020. “A Hybrid Speller Design Using Eye Tracking and SSVEP Brain-Computer Interface.” Sensors, 20, Pp. 891.Abstract
Steady-state visual evoked potentials (SSVEPs) have been extensively utilized to develop brain-computer interfaces (BCIs) due to the advantages of robustness, large number of commands, high classification accuracies, and information transfer rates (ITRs). However, the use of several simultaneous flickering stimuli often causes high levels of user discomfort, tiredness, annoyingness, and fatigue. Here we propose to design a stimuli-responsive hybrid speller by using electroencephalography (EEG) and video-based eye-tracking to increase user comfortability levels when presented with large numbers of simultaneously flickering stimuli. Interestingly, a canonical correlation analysis (CCA)-based framework was useful to identify target frequency with a 1 s duration of flickering signal. Our proposed BCI-speller uses only six frequencies to classify forty-eight targets, thus achieve greatly increased ITR, whereas basic SSVEP BCI-spellers use an equal number of frequencies to the number of targets. Using this speller, we obtained an average classification accuracy of 90.35 +/- 3.597% with an average ITR of 184.06 +/- 12.761 bits per minute in a cued-spelling task and an ITR of 190.73 +/- 17.849 bits per minute in a free-spelling task. Consequently, our proposed speller is superior to the other spellers in terms of targets classified, classification accuracy, and ITR, while producing less fatigue, annoyingness, tiredness and discomfort. Together, our proposed hybrid eye tracking and SSVEP BCI-based system will ultimately enable a truly high-speed communication channel.
H. J. Ju, M. Park, J. H. Park, G. R. Shin, H. S. Choi, M. W. Suh, and M. S. Kim. 2020. “In vivo imaging of click-crosslinked hydrogel depots following intratympanic injection.” Materials, 13, 14, Pp. 3070.Abstract
In this study, we developed injectable intratympanic hyaluronic acid (HA) depots for the treatment of hearing loss. We prepared an injectable click-crosslinking formulation by modifying HA with tetrazine (HA-TET) and trans-cyclooctene (HA-TCO), which crosslinked to form an HA depot (Cx-HA). Preparation of the click-crosslinking HA formulation was facile, and Cx-HA depot formation was reproducible. Additionally, the Cx-HA hydrogel was significantly stiffer than HA hydrogel. To monitor the degradation pattern of hydrogels, we mixed a zwitterionic near-infrared (NIR) fluorophore (e.g., ZW800-1C) in the click-crosslinking HA formulation. Then, HA-TET and HA-TCO solutions containing ZW800-1C were loaded separately into the compartments of a dual-barrel syringe for intratympanic injection. The Cx-HA depots formed quickly, and an extended residence time in the tympanic cavity was confirmed by performing NIR fluorescence imaging. We have successfully prepared an injectable click-crosslinking HA formulation that has promise as an intratympanic drug depot.
H. Kim, H. S. Choi, J. B. Eom, and Y. Choi. 2020. “Mini-platform for off-on near-infrared fluorescence imaging using peptide-targeting ligands.” Bioconjug Chem, 31, Pp. 721-728.Abstract
Here, we propose a zwitterionic near-infrared (NIR) fluorophore-tryptophan (Trp) conjugate with a cleavable linker as a minimal-sized versatile platform (MP) for the preparation of peptide ligand-based off-on type molecular probes. The zwitterionic NIR fluorophore in MP undergoes fluorescence quenching via a photoinduced electron transfer mechanism when in close proximity to tryptophan, and nonspecific binding with serum proteins is minimized by the zwitterionicity of the fluorophore. The linker can be cleaved inside cancer cells in response to tumor-associated stimuli. As a proof-of-concept experiment, ATTO655 was covalently linked with Trp via a diarginine linker to form an MP. A cyclic peptide consisting of Arg-Gly-Asp-d-Phe-Lys (cRGD) was used as a cancer-targeting ligand and was conjugated to the MP to form cRGD-MP. The NIR fluorescence of cRGD-MP could be selectively turned on inside the target cancer cells, thereby enabling specific fluorescence imaging of integrin alphavbeta3-overexpressing cancer cells in vitro and in vivo.
H. S. Choi and H. K. Kim. 2020. “Multispectral image-guided surgery in patients.” Nat Biomed Eng, 4, Pp. 245-246.
Y. Ji, C. Jones, Y. Baek, G. K. Park, S. Kashiwagi, and H. S. Choi. 2020. “Near-infrared fluorescence imaging in immunotherapy.” Adv Drug Deliv Rev, 167, Pp. 121-134.Abstract
Near-infrared (NIR) light possesses many suitable optophysical properties for medical imaging including low autofluorescence, deep tissue penetration, and minimal light scattering, which together allow for high-resolution imaging of biological tissue. NIR imaging has proven to be a noninvasive and effective real-time imaging methodology that provides a high signal-to-background ratio compared to other potential optical imaging modalities. In response to this, the use of NIR imaging has been extensively explored in the field of immunotherapy. To date, NIR fluorescence imaging has successfully offered reliable monitoring of the localization, dynamics, and function of immune responses, which are vital in assessing not only the efficacy but also the safety of treatments to design immunotherapies optimally. This review aims to provide an overview of the current research on NIR imaging of the immune response. We expect that the use of NIR imaging will expand further in response to the recent success in cancer immunotherapy. We will also offer our insights on how this technology will meet rapidly growing expectations in the future.
S. H. Kim, J. H. Park, J. S. Kwon, J. G. Cho, K. G. Park, C. H. Park, J. J. Yoo, A. Atala, H. S. Choi, M. S. Kim, and S. J. Lee. 2020. “NIR fluorescence for monitoring in vivo scaffold degradation along with stem cell tracking in bone tissue engineering.” Biomaterials, 258, Pp. 120267.Abstract
Stem cell-based tissue engineering has the potential to use as an alternative for autologous tissue grafts; however, the contribution of the scaffold degradation along with the transplanted stem cells to in vivo tissue regeneration remains poorly understood. Near-infrared (NIR) fluorescence imaging has great potential to monitor implants while avoiding autofluorescence from the adjacent host tissue. To utilize NIR imaging for in vivo monitoring of scaffold degradation and cell tracking, we synthesized 800-nm emitting NIR-conjugated PCL-ran-PLLA-ran-PGA (ZW-PCLG) copolymers with three different degradation rates and labeled 700-nm emitting lipophilic pentamethine (CTNF127) on the human placental stem cells (CT-PSCs). The 3D bioprinted hybrid constructs containing the CT-PSC-laden hydrogel together with the ZW-PCLG scaffolds demonstrate that NIR fluorescent imaging enables tracking of in vivo scaffold degradation and stem cell fate for bone regeneration in a rat calvarial bone defect model. This NIR-based monitoring system can be effectively utilized to study cell-based tissue engineering applications.
Peixia Wang, Shili Liu, Mingxiang Hu, Hongwei Zhang, Demin Duan, Jiuyang He, Juanji Hong, Ruitao Lv, Haksoo Choi, Xiyun Yan, and Minmin Liang. 2020. “Peroxidase-Like Nanozymes Induce a Novel Form of Cell Death and Inhibit Tumor Growth In Vivo.” Advanced Functional Materials.
G. K. Park, J. H. Lee, E. Soriano, M. Choi, K. Bao, W. Katagiri, D. Y. Kim, J. H. Paik, S. H. Yun, J. V. Frangioni, T. E. Clancy, S. Kashiwagi, M. Henary, and H. S. Choi. 2020. “Rapid and selective targeting of heterogeneous pancreatic neuroendocrine tumors.” iScience, 23, Pp. 101006.Abstract
Design of tissue-specific contrast agents to delineate tumors from background tissues is a major unmet clinical need for ultimate surgical interventions. Bioconjugation of fluorophore(s) to a ligand has been mainly used to target overexpressed receptors on tumors. However, the size of the final targeted ligand can be large, >20 kDa, and cannot readily cross the microvasculature to meet the specific tissue, resulting in low targetability with a high background. Here, we report a small and hydrophilic phenoxazine with high targetability and retention to pancreatic neuroendocrine tumor. This bioengineered fluorophore permits sensitive detection of ultrasmall (<0.5 mm) ectopic tumors within a few seconds after a single bolus injection, highlighting every tumor in the pancreas from the surrounding healthy tissues with reasonable half-life. The knowledge-based approach and validation used to develop structure-inherent tumor-targeted fluorophores have a tremendous potential to improve treatment outcome by providing definite tumor margins for image-guided surgery.