Rational Design of NIR-II Ratiometric Fluorescence Probes for Accurate Bioimaging and Biosensing In Vivo.

Intravital fluorescence imaging in the second near-infrared window (NIR-II, 900-1700 nm) 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.

Multichannel-optical imaging for in vivo evaluating the safety and therapeutic efficacy of stem cells in tumor model in terms of cell tropism, proliferation and NF-κB activity.

Stem cell-based cancer treatment has garnered significant attention, yet its safety and efficacy remain incompletely understood. The nuclear factor-kappa B (NF-κB) pathway, a critical signaling mechanism involved in tumor growth, angiogenesis, and invasion, serves as an essential metric for evaluating the behavior of stem cells in tumor models. Herein, we report the development of a triple-channel imaging system capable of simultaneously monitoring the tropism of stem cells towards tumors, assessing tumor proliferation, and quantifying tumor NF-κB activity. In this system, we generated a CRISPR-Cas9 gene-edited human glioblastoma cell line, GE-U87-MG, which provided a reliable readout of the proliferation and NF-κB activity of tumors by EF1α-RFLuc- and NF-κB-GLuc-based bioluminescent imaging, respectively. Additionally, near infrared-II emitting Tat-PEG-AgAuSe quantum dots were developed for tracking of stem cell tropism towards tumor. In a representative case involving human mesenchymal stem cells (hMSCs), multichannel imaging revealed no discernible effect of hMSCs on the proliferation and NF-κB activity of GE-U87-MG tumors. Moreover, hMSCs engineered to overexpress the necrosis factor-related apoptosis-inducing ligand were able to inhibit NF-κB activity and growth of GE-U87-MG in vivo. Taken together, our imaging system represents a powerful and feasible approach to evaluating the safety and therapeutic efficacy of stem cells in tumor models.

Multiscale NIR-II Imaging-Guided Brain-Targeted Drug Delivery Using Engineered Cell Membrane Nanoformulation for Alzheimer's Disease Therapy.

Effective drug delivery in the central nervous system (CNS) needs to have long blood-circulation half-lives, to pass through the blood-brain barrier (BBB), and subsequently to be taken up by target cells. Herein, a traceable CNS delivery nanoformulation (RVG-NV-NPs) is developed by encapsulating bexarotene (Bex) and AgAuSe quantum dots (QDs) within Lamp2b-RVG-overexpressed neural stem cell (NSC) membranes. The high-fidelity near-infrared-II imaging by AgAuSe QDs offers a possibility of in vivo monitoring the multiscale delivery process of the nanoformulation from the whole-body to the single-cell scale. It was revealed the synergy of acetylcholine receptor-targeting of RVG and the natural brain-homing and low immunogenicity of NSC membranes prolong the blood circulation, facilitate BBB crossing and nerve cell targeting of RVG-NV-NPs. Thus, in Alzheimer's disease (AD) mice, the intravenous delivery of as low as 0.5% of oral dose Bex showed highly effective up-regulation of the apolipoprotein E expression, resulting rapid alleviation of ∼40% ß-amyloid (Aß) level in the brain interstitial fluid after a single dose administration. The pathological progression of Aß in AD mice is completely suppressed during a 1 month treatment, thus effectively protecting neurons from Aß-induced apoptosis and maintaining the cognitive abilities of AD mice.

An oral ratiometric NIR-II fluorescent probe for reliable monitoring of gastrointestinal diseases in vivo.

Early monitoring of gastrointestinal diseases via orally delivered NIR-II ratiometric fluorescent probes represents a promising noninvasive diagnostic modality, but is challenging due to the limitation of harsh digestive environment. Here, we report a single-component NIR-II ratiometric molecular nanoprobe (LC-1250 NP) to monitor gastrointestinal disease with high specificity to its biomarker H2O2 via oral administration. LC-1250 NP displays stable fluorescence in the channel of 1250 long-pass (F1250LP) before and after the gastrointestinal disease detection as the reference, while it presents significantly enhanced fluorescence signal in the response channel of 1150 nm short-pass (F1150SP) in diseased gastrointestinal environment due to the intramolecular cyclization of LC-1250 molecules activated by H2O2. The fluorescence ratio (F1150SP/F1250LP) increases linearly with the concentration of H2O2 with a low detection limit of 20 nM. Therefore, when delivered orally, LC-1250 NP can accurately map the diseased areas and surmount the false-positive interference from biological heterogeneity by NIR-II ratiometric fluorescence imaging, providing sensitive and reliable evaluation for the progress of gastroenteritis.

Single-particle fluorescence tracking combined with TrackMate assay reveals highly heterogeneous and discontinuous lysosomal transport in freely orientated axons.

Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including the forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases.

Recent advances of engineered and artificial drug delivery system towards solid tumor based on immune cells.

Precise drug delivery in cancer treatment is a long-standing concern of modern medicine. Compared with traditional molecular medicines and nano-medicines, emerging cell-based biomimetic delivery strategies display numerous merits, including successive biological functions, innate biocompatibility and superior security since they originate from living organisms, providing a very promising approach. Among them, immune cells receive increasing attention because of their inherent ability in tumor resistance, pathogen elimination, and other significant physiological functions. Herein, we investigated the recent advances on immune cell-based high efficient delivery and therapeutic strategies in solid tumor treatment, mainly focus on T cells, natural killer cells and macrophages, which have been used as drug cargos directly or provided membrane/exosomes as nanoscale drug delivery systems. We also discuss the further potential applications and perspective of this innovative strategy, as well as the predictable challenges in forward exploration in this emerging area.

Linear-branched poly(ß-amino esters)/DNA nano-polyplexes for effective gene transfection and neural stem cell differentiation.

Controllable regulation of stem cell differentiation is a critical concern in stem cell-based regenerative medicine. In particular, there are still great challenges in controlling the directional differentiation of neural stem cells (NSCs) into neurons. Herein, we developed a novel linear-branched poly(ß-amino esters) (S4-TMPTA-BDA-DT, STBD) through a two-step reaction. The synthesized linear-branched polymers possess multiple positively charged amine terminus and degradable intermolecular ester bonds, thus endowing them with excellent properties such as high gene load, efficient gene delivery, and effective gene release and transcription in cells. In the mCherry transfection test, a high transfection efficiency of approximately 70% was achieved in primary NSCs after a single transfection. Moreover, STBD also showed high biocompatibility to NSCs without disturbing their viability and neural differentiation. With the high gene delivery property, STBD is capable of delivering siRNA (shSOX9) expression plasmid into NSCs to significantly interfere with the expression of SOX9, thus enhancing the neuronal differentiation and maturation of NSCs. The STBD/DNA nano-polyplex represents a powerful non-viral approach of gene delivery for manipulating the differentiation of stem cells, showing broad application prospects in NSC-based regenerative therapy for treating neurodegenerative diseases.

Variable metal resistance of P. putida CZ1 biofilms in different environments suggests its remediation application scope.

Pseudomonas putida is potentially used in the bioremediation of heavy metals (HMs). Its response to different HMs in different environments is still not fully understood. This study investigated resistance against 12 kinds of metals by P. putida CZ1 planktonic cells and its biofilm in LB and mineral medium (MM). P. putida CZ1 biofilms have high resistance and accumulation capacity for Cu2+, Zn2+, Pb2+, Fe3+, Mn2+, Al3+ and Ni2+, but less resistance to Co2+, Cd2+, Cr2O72-, Ag+ and Hg2+. Biofilms were 2-8 times more resistant to Cu2+ and Zn2+ than planktonic cells. There was a strong correlation between the P content and the accumulation of Cu2+, Zn2+, Fe3+, Mn2+, Pb2+, Ni2+and Al3+ respectively. Confocal laser scanning microscopy (CLSM) combined with live/dead staining study found that cells in the biofilms can keep viable after 36 h under MIC of Cu2+ or Zn2+ both in LB and MM. When the metal concentration increased, cells can be killed gradually. For Cu2+, Zn2+, Fe3+, Mn2+, Pb2+ and Ni2+, higher resistance was found in MM (2-4 times higher) than in LB and higher accumulation of these metals were also found in MM. P. putida CZ1 biofilm cultured in MM with citric acid as carbon source had stronger resistance and accumulation ability to Cu2+, Zn2+, Pb2+, Fe3+, Mn2+, and Ni2+. This suggested that P. putida CZ1 had greater remediation potential for these metals in organic acid rich environments.

Whole-Body Fluorescence Imaging in the Near-Infrared Window.

Fluorescence imaging is one of the most widely used in vivo imaging methods for both fundamental research and clinical practice. Due to the reduced photon scattering, absorption, and autofluorescence in tissues, the emerging near-infrared (NIR) imaging (650-1700 nm) can afford deep tissue imaging with high spatiotemporal resolution and in vivo report the anatomical structures as well as the physiological activities in a whole-body level. Here, we give a brief introduction to fluorescence imaging in the first NIR (NIR-I, 650-950 nm) and second NIR (NIR-II, 1000-1700 nm) windows, summarize the recently developed NIR fluorophores and their applications in whole-body vascular system imaging, precision cancer theranostics, and regenerative medicine. Finally, the clinical applications and future prospects of in vivo NIR fluorescence imaging are also discussed.

A Nanoformulation-Mediated Multifunctional Stem Cell Therapy with Improved Beta-Amyloid Clearance and Neural Regeneration for Alzheimer's Disease.

Alzheimer's disease (AD) is a common dementia that is currently incurable. The existing treatments can only moderately relieve the symptoms of AD to slow down its progress. How to achieve effective neural regeneration to ameliorate cognitive impairments is a major challenge for current AD treatment. Here, the therapeutic potential of a nanoformulation-mediated neural stem cell (NSC) therapy capable of simultaneous Aß clearance and neural regeneration is investigated in a murine model. Genetically engineered NSCs capable of stably and continuously expressing neprilysin (NEP) are developed to enhance Aß degradation and NSC survival in the brain. A PBAE-PLGA-Ag2 S-RA-siSOX9 (PPAR-siSOX9) nanoformulation with high gene/drug deliverability is synthesized to overcome AD microenvironment-associated adverse effects and to promote neuronal differentiation of the NEP-expressing NSCs. For achieving accurate stereotactic transplantation, Ag2 S quantum-dot-based fluorescence imaging is used to guide NSC transplantation in real time. This strategy shows numerous benefits, including efficient and long-lasting Aß degradation, improved neural regeneration, and accurate cell transplantation. It is shown that a single administration of this therapy achieves long-term efficacy (6 months) with respect to memory reversal and improvement of learning deficits.

A Targeted Activatable NIR-IIb Nanoprobe for Highly Sensitive Detection of Ischemic Stroke in a Photothrombotic Stroke Model.

Ischemic stroke is a devastating disease resulting in high morbidity and mortality. To date, its early diagnosis still faces challenges. Herein, an efficient detection strategy is proposed, in which a targeted activatable NIR-IIb nanoprobe (V&C/PbS@Ag2 Se) is constructed for in vivo highly sensitive detection of early ischemic stroke in a photothrombotic stroke model. At first, the fluorescence of V&C/PbS@Ag2 Se displays an "off" state due to the competitive absorption of excitation irradiation between Cy7.5 fluorophores and PbS@Ag2 Se quantum dots (QDs). Upon intravenous injection, the V&C/PbS@Ag2 Se quickly accumulates in the lesion regions based on VCAM1 binding peptide target to the inflamed vascular endothelium of ischemic stroke. Later, the nanoprobes can be rapidly activated via Cy7.5 oxidation by peroxynitrite (ONOO- ), the prodromal biomarker of ischemic stroke, instantly illuminating the lesion regions. Such a targeted activatable strategy offers a favorable approach for in vivo early real-time assessment of ischemic stroke, which can be expanded to other diseases as a general mothed for in vivo precise diagnosis.

Rapid Unperturbed-Tissue Analysis for Intraoperative Cancer Diagnosis Using an Enzyme-Activated NIR-II Nanoprobe.

Accurate intraoperative tissue identification is critical to tumor surgery. However, conventional methods are labor- and time-intensive, which greatly delay the intraoperative decision-making. Herein, a matrix metalloproteinase (MMP)14-activated NIR-II nanoprobe (A&MMP@Ag2 S-AF7P) is presented for rapid unperturbed-tissue analysis for ex vivo and in vivo neuroblastoma diagnosis. A&MMP@Ag2 S-AF7P displays negligible fluorescence in normal tissues but is activated quickly by inhibiting the fluorescence resonance energy transfer (FRET) between Ag2 S QDs and A1094 mediated by MMP14 overexpressed in neuroblastoma; meanwhile, the exposure of the membrane penetrating peptide R9 (TAT-peptide) results in efficient internalization of nanoprobes in the cancer cells, providing superior tumor-to-normal (T/N) tissue ratio. Instant illumination of the lesion and well-defined tumor margins make the nanoprobes a suitable rapid diagnostic reagent for cancer surgical or tissue biopsy procedures.

Advanced Fluorescence Imaging Technology in the Near-Infrared-II Window for Biomedical Applications.

Fluorescence imaging has become a fundamental tool for biomedical applications; nevertheless, its intravital imaging capacity in the conventional wavelength range (400-950 nm) has been restricted by its extremely limited tissue penetration. To tackle this challenge, a novel imaging approach using the fluorescence in the second near-infrared window (NIR-II, 1000-1700 nm) has been developed in the past decade to achieve deep penetration and high-fidelity imaging, and thus significant biomedical applications have begun to emerge. In this Perspective, we first examine recent discoveries and challenges in the development of novel NIR-II fluorophores and compatible imaging apparatuses. Subsequently, the recent advances in bioimaging, biosensing, and therapy using such a cutting-edge imaging technique are highlighted. Finally, based on the achievement in the representative studies, we elucidate the main concerns regarding this imaging technique and give some advice and prospects for the development of NIR-II imaging for future biomedical applications.

Recent Progress of Hybrid Optical Probes for Neural Membrane Potential Imaging.

The neural membrane potential of nerve cells is the basis of neural activity production, which controls advanced brain activities such as memory, emotion, and learning. In the past decades, optical voltage indicator has emerged as a promising tool to decode neural activities with high-fidelity and excellent spatiotemporal resolution. In particular, the hybrid optical probes can combine the advantageous photophysical properties of different components such as voltage-sensitive molecules, highly fluorescent fluorophores, membrane-targeting tags, and optogenetic materials, thus showing numerous advantages in improving the photoluminescence intensity, voltage sensitivity, photostability, and cell specificity of probes. In this review, the current state-of-the-art hybrid probes are highlighted, that are designed by using fluorescent proteins, organic dyes, and fluorescent nanoprobes as the fluorophores, respectively. Then, the design strategies, voltage-sensing mechanisms and the in vitro and in vivo neural activity imaging applications of the hybrid probes are summarized. Finally, based on the current achievements of voltage imaging studies, the challenges and prospects for design and application of hybrid optical probes in the future are presented.

Advanced Near-Infrared Light for Monitoring and Modulating the Spatiotemporal Dynamics of Cell Functions in Living Systems.

Light-based technique, including optical imaging and photoregulation, has become one of the most important tools for both fundamental research and clinical practice, such as cell signal sensing, cancer diagnosis, tissue engineering, drug delivery, visual regulation, neuromodulation, and disease treatment. In particular, low energy near-infrared (NIR, 700-1700 nm) light possesses lower phototoxicity and higher tissue penetration depth in living systems as compared with ultraviolet/visible light, making it a promising tool for in vivo applications. Currently, the NIR light-based imaging and photoregulation strategies have offered a possibility to real-time sense and/or modulate specific cellular events in deep tissues with subcellular accuracy. Herein, the recent progress with respect to NIR light for monitoring and modulating the spatiotemporal dynamics of cell functions in living systems are summarized. In particular, the applications of NIR light-based techniques in cancer theranostics, regenerative medicine, and neuroscience research are systematically introduced and discussed. In addition, the challenges and prospects for NIR light-based cell sensing and regulating techniques are comprehensively discussed.

Tumor Microenvironment-Activated NIR-II Nanotheranostic System for Precise Diagnosis and Treatment of Peritoneal Metastasis.

Activatable theranostic systems show potential for improved tumor diagnosis and therapy owing to high detection specificities, effective ablation, and minimal side-effects. Herein, a tumor microenvironment (TME)-activated NIR-II nanotheranostic system (FEAD1) for precise diagnosis and treatment of peritoneal metastases is presented. FEAD1 was fabricated by self-assembling the peptide Fmoc-His, mercaptopropionic-functionalized Ag2 S quantum dots (MPA-Ag2 S QDs), the chemodrug doxorubicin (DOX), and NIR absorber A1094 into nanoparticles. We show that in healthy tissue, FEAD1 exists in an NIR-II fluorescence "off" state, because of Ag2 S QDs-A1094 interactions, while DOX remains in stealth mode. Upon delivery of FEAD1 to the tumor, the acidic TME triggers its disassembly through breakage of the Fmoc-His metal coordination and DOX hydrophobic interactions. Release of A1094 switches on Ag2 S fluorescence, illuminating the tumor, accompanied by burst release of DOX within the tumor tissue, thereby achieving precise tumor theranostics. This TME-activated theranostic strategy holds great promise for future clinical applications.

An Activatable NIR-II Nanoprobe for In†Vivo Early Real-Time Diagnosis of Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the most dangerous acute diseases resulting in high morbidity and mortality. Current methods remain limited with respect to early diagnosis and real-time feedback on the pathological process. Herein, a targeted activatable fluorescent nanoprobe (V&A@Ag2 S) in the second near-infrared window (NIR-II) is presented for in vivo optical imaging of TBI. Initially, the fluorescence of V&A@Ag2 S is turned off owing to energy transfer from Ag2 S to the A1094 chromophore. Upon intravenous injection, V&A@Ag2 S quickly accumulates in the inflamed vascular endothelium of TBI based on VCAM1-mediated endocytosis, after which the nanoprobe achieves rapid recovery of the NIR-II fluorescence of Ag2 S quantum dots (QDs) owing to the bleaching of A1094 by the prodromal biomarker of TBI, peroxynitrite (ONOO- ). The nanoprobe offers high specificity, rapid response, and high sensitivity toward ONOO- , providing a convenient approach for in vivo early real-time assessment of TBI.

NIR-II Fluorescent Self-Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis.

Fluorescence-guided cytoreductive surgery is one of the most promising approaches for facile elimination of tumors in situ, thereby improving prognosis. Reported herein is a simple strategy to construct a novel chainlike NIR-II nanoprobe (APP-Ag2 S-RGD) by self-assembly of an amphiphilic peptide (APP) into a nanochain with subsequent chemical crosslinking of NIR-II Ag2 S QDs and the tumor-targeting RGD peptide. This probe exhibits higher capability for cancer cell detection compared with that of RGD-functionalized Ag2 S QDs (Ag2 S-RGD) at the same concentration. Upon intraperitoneal injection, superior tumor-to-normal tissue signal ratio is achieved and non-vascularized tiny tumor metastatic foci as small as about 0.2 mm in diameter could be facilely eliminated under NIR-II fluorescent imaging guidance. These results clearly indicate the potential of this probe for fluorescence-guided tumor staging, preoperative diagnosis, and intraoperative navigation.

Programmable Chemotherapy and Immunotherapy against Breast Cancer Guided by Multiplexed Fluorescence Imaging in the Second Near-Infrared Window.

Combined chemotherapy and immunotherapy have demonstrated great potential in cancer treatment. However, it is difficult to provide clear information of the pharmacokinetics and pharmacodynamics of chemodrugs and transplanted immune cells in vivo by traditional approaches, resulting in inadequate therapy. Here, a multiplexed intravital imaging strategy by using fluorescence in the second near-infrared window (NIR-II) is first developed to visualize the two events of chemotherapy and immunotherapy in vivo, so that a combinational administration is programed to improve the therapeutical effects against a mouse model of human breast cancer. In detail, Ag2 Se quantum dots (QDs) (λEm = 1350 nm) loaded with stromal-cell-derived factor-1α (SDF-1α) and chemodrug doxorubicin (DOX) are first administrated to deliver the SDF-1α and DOX to the tumor site. After their arrival, monitored by Ag2 Se QD fluorescence, natural killer (NK)-92 cells labeled with Ag2 S QDs (λEm = 1050 nm) are intravenously injected so that the cells are recruited to the tumor by the chemotaxis of SDF-1α, which is visualized by Ag2 S QD fluorescence. Such an imaging approach allows simultaneous evaluation of the behaviors of individual injections in vivo, and facilitates optimized administration regimens, resulting in enhanced tumor inhibition.