Progress and Challenges of Water-soluble NIR-II Organic Fluorophores for Fluorescence Imaging In vivo

The small-molecule fluorophores for the second near-infrared(NIR-II,1000–1700 nm)window have attracted increasing attention in basic scientific research and preclinical practice owing to their deep-photo penetration,minimal physiological toxicity and simplicity of chemical modification.However,most of the reported small-molecule NIR-II fluorophores suffered from poor water solubility,which can easily cause organ toxicity.In addition,the aggregation caused by their poor water solubility in the aqueous solution would also result in weak fluorescence of these NIR-II fluorophores.Thus,it is highly desirable and valuable to develop water-soluble small-molecule NIR-II fluorophores with excellent photophysical properties for high-contrast in vivo imaging.In this review,we summarize the recent research advances in water-soluble small-molecule NIR-II fluorophores and highlight the representative bioimaging applications.Moreover,the potential challenges and perspectives of water-soluble small-molecule NIR-II fluorophores are discussed as well.We anticipate this review can help researchers to grab the latest information of water-soluble small-molecule fluorophores for NIR-II imaging,sequentially boosting their further development.

Recent developments of fluorescence imaging technology in the second near-infrared window

Background:Fluorescence bio-imaging in the second near-infrared window(NIR-II FL,1000-1700nm)has great potential in clinical theranostics,which is of great importance providing precise locations of lesions and molecular dynamic actions simultaneously in a single nanoprobe.Methods:T here has been an upsurge of multidisciplinary research focusing on developing functional types of inorganic and organic nanoprobes that can be used for NIR-II FL with the high spatiotemporal resolution,deep tissue penetration,and negligible auto-fluorescence.Results:In this mini-review,we summarize recent progress in inorganic/organic NIR-II FL nanoprobes.We introduce the design and properties of inorganic and organic nanoprobes,in the order of single-walled carbon nanotubes,quantum dots,rare-earth-doped nanoparticles,metal nanoclusters and organic fluorophores,expect to realize precise diagnosis and efficient image-guided therapy.Conclusion:Meanwhile,to elucidate the problems and perspectives,we aim to offer diverse biological applications of inorganic/organic NIR-II FL nanoprobes and accelerate the clinical transformation progress.

Near-infrared-II deep tissue fluorescence microscopy and application

Fluorescence imaging has become an essential tool in biomedical research.However,non-invasive deep-tissue threedimensional optical in vivo imaging with the high spatiotemporal resolution is challenging due to the interaction between photons and tissues.Beam shaping has been used to tailor microscopy techniques to enhance microscope performance.The nearinfrared window(NIR)between 700 and 1,700 nm,generally emphasized as the NIR-II(1,000–1,700 nm)window,has been developed into a promising bio-optical solution chosen as the lower interaction effect in this spectrum,showing potential in basic biological research and clinical application.In this review,we summarize the existing methods to increase penetration depth and extensively describe biological microscopy techniques,NIR-II spectral windows,and fluorophores.Strategies to improve bioimaging performance and NIR-II imaging applications are introduced.Based on the current research achievements,we elucidate the main challenges and provide some recommendations and prospects for deep tissue penetration fluorescence for future biomedical applications.

NIR-Ⅱ Fluorophores:From Synthesis to Biological Applications

Fluorescence imaging is a useful tool in the field of biomedical applications.However,its imaging capacity is limited by the depth of tissue that can be penetrated when using visible light(400-700 nm)or the first near-infrared window(NIR-Ⅰ,700-900 nm).To overcome the problem,fluorescence imaging in the second near-infrared window(NIR-Ⅱ,1000-1700 nm)has been developed to reduce photon scattering,auto-absorption and tissue autofluorescence to achieve high spatiotemporal resolution and deep imaging penetration.The key to NIR-Ⅱimaging is obtaining and analyzing highly selective information from functional fluorophores that emit in the 1000-1700 nm range.With the rapid development of multidisciplinary research,various types of NIR-Ⅱfluorophores have been produced and used in non-invasive,real-time NIR-Ⅱbiomedical applications.This review summarizes some of the most prevalent NIR-Ⅱfluorophores and their synthesis,such as organic fluorophores(OFs),single-walled carbon nanotubes(SWCNTs),quantum dots(QDs),and rare-earth nanoparticles(RENPs).On this basis,we describe the applications of these fluorophores in biomedical fields,including bioimaging,biosensing,phototherapy and surgical navigation.Additionally,major challenges and prospects of NIR-Ⅱbiomedical application will be further explored.

Promoting near-infrared II fluorescence efficiency by blocking long-range energy migration

Generally,long wavelength absorbed near-infrared II(NIR-II)dyes have a low fluorescence efficiency in aggregate states for aggregate-caused quenching effect,simultaneously enhancing efficiency and extending absorption is a challenging issue for NIR-II dyes.Here,three benzo[1,2-c:4,5-c’]bis[1,2,5]thiadiazole(BBT)derivatives(TPA-BBT,FT-BBT,and BTBT-BBT)are used to clarify fluorescence quenching mechanisms.When the BBT derivatives are doped into a small molecule matrix,they show quite different fluorescence behaviors.Structuredistorted TPA-BBT displays fluorescence quenching originating from short-range exchange interaction,while FT-BBT and BTBT-BBT with a co-planar-conjugated backbone exhibit concentration-dependent quenching processes,namely changing from long-range dipole-dipole interaction to exchange interaction,which could be majorly ascribed to large spectral overlap between absorption and emission.By precisely tuning doping concentration,both FT-BBT and BTBT-BBT nanoparticles(NPs)present the optimal NIR-II fluorescence brightness at∼2.5 wt%doping concentration.The doped NPs have good biocompatibility and could be served as fluorescence contrast agents for vascular imaging with a high resolution under 980-nm laser excitation.Those paradigms evidence that molecular doping can promote fluorescence efficiency of long wavelength-absorbed NIR-II fluorophores via suppressing long-range energy migration.

Small Molecular NIR-II Fluorophores for Cancer Phototheranostics

Phototheranostics integrates deep-tissue imaging with phototherapy(containing photothermal therapy and photodynamic therapy),holding great promise in early diagnosis and precision treatment of cancers.Recently,second near-infrared(NIR-II)fluorescence imaging exhibits the merits of high accuracy and specificity,as well as real-time detection.Among the NIR-II fluorophores,organic small molecular fluorophores have shown superior properties in the biocompatibility,variable structure,and tunable emission wavelength than the inorganic NIR-II materials.What’s more,some small molecular fluorophores also display excellent cytotoxicity when illuminated with the NIR laser.This review summarizes the progress of small molecular NIR-II fluorophores with different central cores for cancer phototheranostics in the past few years,focusing on the molecular structures and phototheranostic performances.Furthermore,challenges and prospects of future development toward clinical translation are discussed.

Recent advances on small-molecule fluorophores with emission beyond 1000 nm for better molecular imaging in vivo

In the second near-infrared channel(NIR-II, 1000–1700 nm), organic and inorganic fluorophores are designed with superior chemical/optical properties to provide real-time information with deeper penetration depth and higher resolution owing to the innate lower light scattering and absorption of the NIR-II imaging than conventional optical imaging. Among them, the small-molecule based fluorophores have been highlighted due to their desirable biocompatibility and favorable pharmacokinetics. In this review, we introduced the latest research progress of the rational design of small-molecule NIR-II fluorophores and their impressively biological applications including the NIR-II signal imaging,multimodal imaging and theranostic.

Repurposing organic semiconducting nanomaterials to accelerate clinical translation of NIR-II fluorescence imaging

Optical imaging possesses important implications for early disease diagnosis,timely disease treatment,and basic medical as well as biological research.Compared with the traditionary near-infrared(NIR-I)window(650-950 nm)optical imaging,the emerging second near-infrared(NIR-II)window optical imaging technology owns the great superiorities of non-invasiveness,nonionizing radiation,and real-time dynamic imaging with the low biological interference,can significantly improve the tissue penetration depth and detection sensitivity,thus expecting to achieve accurate and precise diagnosis of major diseases.Inspired by the conspicuous superiorities,an increasing number of versatile NIR-II fluorophores have been legitimately designed and engineered for precisely deep-tissue mapping-mediated theranostics of life-threatening diseases.Organic semiconducting nanomaterials(OSNs)are derived from organic conjugated molecules withπ-electron delocalized skeletons,which show greatly preponderant prospects in the biomedicine field due to the excellent photoelectric property,tunable energy bands,and fine biocompatibility.In this review,the superiorities of NIR-II fluorescence imaging using OSNs for brilliant visualization various of diseases,including tongue cancer,ovarian cancer,osteosarcoma,bacteria or pathogens infection,kidney dysfunction,rheumatoid arthritis,liver injury,and cerebrovascular function,are emphatically summarized.Finally,the reasonable prospects and persistent efforts for repurposing OSNs to facilitate the clinical translation of NIR-II fluorescence phototheranostics are outlined.

Zn-doping enhances the photoluminescence and stability of PbS quantum dots for in vivo high-resolution imaging in the NIR-II window

Lead sulfide(PbS)quantum dots(QDs)are important near infrared(NIR)luminescent materials with tunable and strong emission covering a broad NIR region.However,their optical properties are quite sensitive to air,water,and high temperature due to the surface oxidation,thus limiting their applications in optoelectronic devices and biological imaging.Herein,a cation-doping strategy is presented to make a series of high-quality Zn-doped PbS QDs with strong emission covering whole second near-infrared window(NIR-II,1,000-1,700 nm).First-principle calculations confirmed that Zn dopants formed dopant states and decreased the recombination energy gap of host PbS.Notably,the Zn dopants significantly improved the quantum yield,photoluminescence lifetime and thermal stability of PbS QDs.Moreover,the PEGylated Zn-doped PbS QDs emitting in the NIR-llb window(1,500-1,700 nm)realized the noninvasive imaging of cerebral vascular of mouse with high resolution,being able to distinguish blood capillary.This material not only provides a new tool for deep tissue fluorescence imaging,but is also promising for the development of other NIR related devices.

NIR-II light activated photodynamic therapy with proteincapped gold nanoclusters

The use of near-infrared （NIR） light for photodynamic therapy （PDT） is a promising strategy to circumvent the limitations of current PDT, in which visible light with limited tissue penetration depth is usually used. In the present stud~ alkyl thiolated gold nanoclusters （AuNCs） were co-modified with human serum albumin （HSA） and catalase （CAT）, and then employed as a multifunctional, optical, theranostic nano-agent. In the AuNC@HSA/CAT system, the AuNCs were able to produce singlet oxygen under excitation by a 1,064-nm laser, which locates in the second NIR window （NIR-II）, and featured much lower tissue absorption and scattering, enabling NIR-II-triggered PDT. The HSA coating greatly improved the physiological stability of the nanoparticles, which showed efficient tumor retention after intravenous injection, as revealed by detecting the AuNC fluorescence. Moreover, the presence of CAT in the nanoparticles triggered decomposition of tumor endogenous H202 to generate oxygen, thereby enhancing the efficacy of PDT by relieving tumor hypoxia. Compared with conventional PDT using visible light, NIR-II-triggered PDT exhibits remarkably increased tissue penetration. Thus, we developed a new type of photosensitizing nano-agent that simultaneously enables in vivo fluorescence imaging, tumor hypoxia relief, and NIR-II light-induced in vivo PDT in the treatment of cancer.

Tracking the in vivo spatio-temporal patterns of neovascularization via NIR-II fluorescence imaging

The in vivo spatio-temporal patterns of neovascularization are still poorly understood because it is limited to multi-scale techniques from the cellular level to living animal level.Owing to deep tissue-penetration and zero autofluorescence background,the second near-infrared(NIR-II,1,000–1,700 nm)fluorescence imaging recently shows promise in breaking through this dilemma by dynamically tracking the pathophysiological process of neovascularization in vivo.Here,NIR-II fluorescence imaging was recruited for monitoring blood vessels in order to visualize the vascular injury and quantitively assess neovascularization in mouse models of acute skeleton muscle contusion and hindlimb ischemia.The temporal analysis of real-time NIR-II fluorescence intensity demonstrated that the blood flow perfusion of ischemia area was able to rapidly restore to 96%of pre-ischemic state within one week.Moreover,the spatial analysis revealed that the lower and outer quadrants of ischemia area in the mouse model of hindlimb ischemia always had relatively high blood flow perfusion compared with other quadrants during three weeks post-ischemia,and even exceeded pre-ischemic quantity at 21 days post-ischemia.In conclusion,this in vivo imaging technique has significant potential utility for studying the spatio-temporal patterns of neovascularization in vivo.

NIR-II emissive dye based polymer nanoparticle targeting EGFR for oral cancer theranostics

Oral cancer is a common malignant tumor of the head and neck,and surgery combined with radiotherapy and chemotherapy is the primary treatment modality.However,a positive resection margin that may lead to recurrence after surgery has always been a critical issue to address.Furthermore,radiotherapy and chemotherapy also have shortcomings such as resistance to chemotherapy and radiation,lack of targeting,and severe side effects.Therefore,exploring new methods of tumor surgical navigation and tumor treatment is of great significance for oral cancer.Although,the emerging near-infrared II(NIR-II,1,000–1,700 nm)region fluorescent imaging has revolutionized surgical navigation,a high tumor-targeting fluorescent probe remains lacking.Furthermore,while emerging photothermal therapy(PTT)can overcome chemoradiotherapy’s shortcomings and achieve precise treatment of tumors,its clinical application is still limited by the lack of high photothermal conversion efficiency,high photothermal stability,and highly penetrating materials.Herein,a NIR-II dye SQ890 is developed for tumor imaging and PTT of oral cancer.By assembling into nanoparticles(NPs)and being modified with epithelial growth factor receptor(EGFR)-targeting peptides GE11,SQ890 NPs-Pep can specifically accumulate in tumor sites via active targeting,and realize photoacoustic/NIR-II fluorescence dual-modality imaging-guided PTT of oral cancer.

In vivo and in situ real-time fluorescence imaging of peripheral nerves in the NIR-II window

The peripheral nervous system(PNS)is essential for performing and maintaining various motor and sensory functions.Abnormalities can lead to a series of peripheral neurological conditions,such as paraesthesia,pain,or spasms,which are debilitating and lowering the quality of life.Thecurrent guidelines for diagnosis rely predominantly on clinical symptoms resulting from PNS dysfunction,which occur already at an advancedstage.There are currently no effective methods that visually reflect the extent of peripheral neuropathy.In our study,we present a novel in vivoand in situ real-time imaging of peripheral nerves based on the second near-infrared window(NIR-II)fluoresce nee.In NIR-II system,lead sulfidequa ntum dots(PbS Qds)with NIR-II fluoresce nee specifically bound to motor neuro rvspecific protein agrin,acting as image con trast.In micemodel,peripheral nerves were visible as soon as after 2 h post injection.We provide evidenee for the efficacy of this approach,which allows todirectly dem on strate peripheral nerves,their structure,and pote ntial damagesites and degree.Furthermore,our products were of goodbiocompatibility,while the n eural fluoresce nee signal was solid,bright and stable for 4 h in vivo.Thus,overall,our results suggest that NIR-II isan effective new method for direct imaging of peripheral nerves in vivo,opening new horizons on early,improved and more precise,targeteddiag no sis.A resulti ng more rapid installatio n of perso nalized therapy facilitates a better prognosis of clinical peripheral neuropathy.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration

Owing to the prevalence of rotator cuff(RC)injuries and suboptimal healing outcome,rapid and functional regeneration of the tendon-bone interface(TBI)after RC repair continues to be a major clinical challenge.Given the essential role of the RC in shoulder movement,the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair.Here,we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology.In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI.The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model.In particular,we adopted nondestructive,consecutive,and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo.Furthermore,the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo.In summary,this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration,demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Sensitively detecting antigen of SARS-CoV-2 by NIR-Ⅱ fluorescent nanoparticles

Early detection of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection is an efficient way to prevent the spread of coronavirus disease 2019(COVID-19).Detecting SARS-CoV-2 antigen can be rapid and convenient,but it is still challenging to develop highly sensitive methods for effective diagnosis.Herein,a lateral flow assay(LFA)based on fluorescent nanoparticles emitting in the second near-infrared(NIR-II)window is developed for sensitive detection of SARS-CoV-2 antigen.Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence,such NIR-II based LFA allows enhanced signal-to-background ratio,and the limit of detection is down to 0.01 ng·mL^(−1)of SARS-CoV-2 antigen.In the clinical swab sample tests,the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test.The clinical samples with low antigen concentrations(~0.015–~0.068 ng·mL^(−1))can be successfully detected by the NIR-II LFA,but fail for the colloidal gold LFA.The NIR-II LFA can provide a promising platform for highly sensitive,rapid,and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection.