AMPK-mediated autophagy pathway activation promotes ΔFosB degradation to improve levodopa-induced dyskinesia.

BACKGROUND: Parkinson's disease patients on chronic levodopa often suffer from motor complications, which tend to reduce their quality of life. Levodopa-induced dyskinesia (LID) is one of the most prevalent motor complications, often characterized by abnormal involuntary movements, and the pathogenesis of LID is still unclear but recent studies have suggested the involvement of autophagy. METHODS: The onset of LID was mimicked by chronic levodopa treatment in a unilateral 6-hydroxydopamine (6-OHDA) -lesion rat model. Overexpression of ΔFosB in HEK293 cells to mimic the state of ΔFosB accumulation. The modulation of the AMP-activated protein kinase (AMPK)-mediated autophagy pathway using by metformin, AICAR (an AMPK activator), Compound C (an AMPK inhibitor) and chloroquine (an autophagy pathway inhibitor). The severity of LID was assessed by axial, limb, and orofacial (ALO) abnormal involuntary movements (AIMs) score and in vivo electrophysiology. The activity of AMPK pathway as well as autophagy markers and FosB-ΔFosB levels were detected by western blotting. RT-qPCR was performed to detect the transcription level of FosB-ΔFosB. The mechanism of autophagy dysfunction was further explored by immunofluorescence and transmission electron microscopy. RESULTS: In vivo experiments demonstrated that chronic levodopa treatment reduced AMPK phosphorylation, impaired autophagosome-lysosomal fusion and caused FosB-ΔFosB accumulation in the striatum of PD rats. Long-term metformin intervention improved ALO AIMs scores as well as reduced the mean power of high gamma (hγ) oscillations and the proportion of striatal projection neurons unstable in response to dopamine for LID rats. Moreover, the intervention of metformin promoted AMPK phosphorylation, ameliorated the impairment of autophagosome-lysosomal fusion, thus, promoting FosB-ΔFosB degradation to attenuate its accumulation in the striatum of LID rats. However, the aforementioned roles of metformin were reversed by Compound C and chloroquine. The results of in vitro studies demonstrated the ability of metformin and AICAR to attenuate ΔFosB levels by promoting its degradation, while Compound C and chloroquine could block this effect. CONCLUSIONS: In conclusion, our results suggest that long-term metformin treatment could promote ΔFosB degradation and thus attenuate the development of LID through activating the AMPK-mediated autophagy pathway. Overall, our results support the AMPK-mediated autophagy pathway as a novel therapeutic target for LID and also indicate that metformin is a promising therapeutic candidate for LID.

Analysis and experimental validation of necroptosis-related molecular classification, immune signature and feature genes in Alzheimer's disease.

Necroptosis, a programmed cell death pathway, has been demonstrated to be activated in Alzheimer's disease (AD). However, the precise role of necroptosis and its correlation with immune cell infiltration in AD remains unclear. In this study, we conducted non-negative matrix factorization clustering analysis to identify three subtypes of AD based on necroptosis-relevant genes. Notably, these subtypes exhibited varying necroptosis scores, clinical characteristics and immune infiltration signatures. Cluster B, characterized by high necroptosis scores, showed higher immune cell infiltration and was associated with a more severe pathology, potentially representing a high-risk subgroup. To identify potential biomarkers for AD within cluster B, we employed two machine learning algorithms: the least absolute shrinkage and selection operator regression and Random Forest. Subsequently, we identified eight feature genes (CARTPT, KLHL35, NRN1, NT5DC3, PCYOX1L, RHOQ, SLC6A12, and SLC38A2) that were utilized to develop a diagnosis model with remarkable predictive capacity for AD. Moreover, we conducted validation using bulk RNA-seq, single-nucleus RNA-seq, and in vivo experiments to confirm the expression of these feature genes. In summary, our study identified a novel necroptosis-related subtype of AD and eight diagnostic biomarkers, explored the roles of necroptosis in AD progression and shed new light for the clinical diagnosis and treatment of this disease.

Activation of mGlu2/3 receptors in the striatum alleviates L-DOPA-induced dyskinesia and inhibits abnormal postsynaptic molecular expression.

Group II metabotropic glutamate receptors (mGlu2/3 receptors) have been regarded as promising candidates for the treatment of L-DOPA-induced dyskinesia (LID); however, confirmation is still lacking. As the hub of the basal ganglia circuit, the striatum plays a critical role in action control. Supersensitive responsiveness of glutamatergic corticostriatal input may be the key mechanism for the development of LID. In this study, we first examined the potency of LY354740 (12 mg/kg, i.p.) in modulating glutamate and dopamine release in lesioned striatum of stable LID rats. Then, we injected LY354740 (20nmoL or 40nmoL in 4 µL of sterile 0.9 % saline) directly into the lesioned striatum to verify its ability to reduce or attenuate L-DOPA-induced abnormal involuntary movements. In experiment conducted in established LID rats, after continuous injection for 4 days, we found that LY354740 significantly reduced the expression of dyskinesia. In another experiment conducted in parkinsonism rat models, we found that LY354740 attenuated the development of LID with an inverted-U dose-response curve. The role of LY354740 in modulating striatal expressions of LID-related molecular changes was also assessed after these behavioral experiments. We found that LY354740 significantly inhibited abnormal expressions of p-Fyn/p-NMDA/p-ERK1/2/p-HistoneH3/ΔFosB, which is in line with its ability to alleviate abnormal involuntary movements in both LID expression and induction phase. Our study indicates that activation of striatal mGlu2/3 receptors can attenuate the development of dyskinesia in parkinsonism rats and provide some functional improvements in LID rats by inhibiting LID-related molecular changes.

Identification of metabolism-related subtypes and feature genes in Alzheimer's disease.

BACKGROUND: Owing to the heterogeneity of Alzheimer's disease (AD), its pathogenic mechanisms are yet to be fully elucidated. Evidence suggests an important role of metabolism in the pathophysiology of AD. Herein, we identified the metabolism-related AD subtypes and feature genes. METHODS: The AD datasets were obtained from the Gene Expression Omnibus database and the metabolism-relevant genes were downloaded from a previously published compilation. Consensus clustering was performed to identify the AD subclasses. The clinical characteristics, correlations with metabolic signatures, and immune infiltration of the AD subclasses were evaluated. Feature genes were screened using weighted correlation network analysis (WGCNA) and processed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Furthermore, three machine-learning algorithms were used to narrow down the selection of the feature genes. Finally, we identified the diagnostic value and expression of the feature genes using the AD dataset and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: Three AD subclasses were identified, namely Metabolism Correlated (MC) A (MCA), MCB, and MCC subclasses. MCA contained signatures associated with high AD progression and may represent a high-risk subclass compared with the other two subclasses. MCA exhibited a high expression of genes related to glycolysis, fructose, and galactose metabolism, whereas genes associated with the citrate cycle and pyruvate metabolism were downregulated and associated with high immune infiltration. Conversely, MCB was associated with citrate cycle genes and exhibited elevated expression of immune checkpoint genes. Using WGCNA, 101 metabolic genes were identified to exhibit the strongest association with poor AD progression. Finally, the application of machine-learning algorithms enabled us to successfully identify eight feature genes, which were employed to develop a nomogram model that could bring distinct clinical benefits for patients with AD. As indicated by the AD datasets and qRT-PCR analysis, these genes were intimately associated with AD progression. CONCLUSION: Metabolic dysfunction is associated with AD. Hypothetical molecular subclasses of AD based on metabolic genes may provide new insights for developing individualized therapy for AD. The feature genes highly correlated with AD progression included GFAP, CYB5R3, DARS, KIAA0513, EZR, KCNC1, COLEC12, and TST.

Shortwave-infrared-light-emitting probes for the in vivo tracking of cancer vaccines and the elicited immune responses.

Tracking and imaging immune cells in vivo non-invasively would offer insights into the immune responses induced by vaccination. Here we report a cancer vaccine consisting of polymer-coated NaErF4/NaYF4 core-shell down-conversion nanoparticles emitting luminescence in the near-infrared spectral window IIb (1,500-1,700 nm in wavelength) and with surface-conjugated antigen (ovalbumin) and electrostatically complexed adjuvant (class-B cytosine-phosphate-guanine). Whole-body wide-field imaging of the subcutaneously injected vaccine in tumour-bearing mice revealed rapid migration of the nanoparticles to lymph nodes through lymphatic vessels, with two doses of the vaccine leading to the complete eradication of pre-existing tumours and to the prophylactic inhibition of tumour growth. The abundance of antigen-specific CD8+ T lymphocytes in the tumour microenvironment correlated with vaccine efficacy, as we show via continuous-wave imaging and lifetime imaging of two intravenously injected near-infrared-emitting probes (CD8+-T-cell-targeted NaYbF4/NaYF4 nanoparticles and H-2Kb/ovalbumin257-264 tetramer/PbS/CdS quantum dots) excited at different wavelengths, and by volumetrically visualizing the three nanoparticles via light-sheet microscopy with structured illumination. Nanoparticle-based vaccines and imaging probes emitting infrared light may facilitate the design and optimization of immunotherapies.

Common mechanisms underlying axonal transport deficits in neurodegenerative diseases: a mini review.

Many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are characterized by the accumulation of pathogenic proteins and abnormal localization of organelles. These pathological features may be related to axonal transport deficits in neurons, which lead to failures in pathological protein targeting to specific sites for degradation and organelle transportation to designated areas needed for normal physiological functioning. Axonal transport deficits are most likely early pathological events in such diseases and gradually lead to the loss of axonal integrity and other degenerative changes. In this review, we investigated reports of mechanisms underlying the development of axonal transport deficits in a variety of common neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease to provide new ideas for therapeutic targets that may be used early in the disease process. The mechanisms can be summarized as follows: (1) motor protein changes including expression levels and post-translational modification alteration; (2) changes in microtubules including reducing stability and disrupting tracks; (3) changes in cargoes including diminished binding to motor proteins. Future studies should determine which axonal transport defects are disease-specific and whether they are suitable therapeutic targets in neurodegenerative diseases.

Dissecting the molecular features of bovine-arrested eight-cell embryos using single-cell multi-omics sequencing†.

The regulation of mammalian early-embryonic development is a complex, coordinated process that involves widespread transcriptomic and epigenetic remodeling. The main cause of developmental failure in preimplantation embryos after in vitro fertilization is the irreversible arrested-at-cleavage stage. To deepen our understanding of this embryonic block, we profiled a single-cell multi-omics map of copy number variations (CNVs), the transcriptome, the DNA methylome, and the chromatin state of bovine eight-cell embryos with a two-cell fate that either arrested or developed into blastocysts. To do this, we sequenced a biopsied blastomere and tracked the developmental potential of the remaining cells. Aneuploid embryos inferred by CNVs from DNA- and RNA-library data tended to lose their developmental potency. Analysis of distinct genomic regions of DNA methylation and chromatin accessibility revealed that enrichment of gene function and signaling pathways, such as the MAPK signaling pathway, was altered in arrested euploid eight-cell embryos compared with blastocyst-developed euploid eight-cell embryos. Moreover, the RNA expression and chromatin accessibility of embryonic genome activation-associated genes were lower in arrested euploid embryos than in blastocyst-developed embryos. Taken together, our results indicate that the developmental block of eight-cell embryos can be caused by multiple molecular layers, including CNVs, abnormality of DNA methylation and chromatin accessibility, and insufficient expression of embryonic genome activation-associated genes. Our integrated and comprehensive data set provides a valuable resource to further dissect the exact mechanisms underlying the arrest of bovine eight-cell embryos in vitro.

Phosphorylcholine-conjugated gold-molecular clusters improve signal for Lymph Node NIR-II fluorescence imaging in preclinical cancer models.

Sentinel lymph node imaging and biopsy is important to clinical assessment of cancer metastasis, and novel non-radioactive lymphographic tracers have been actively pursued over the years. Here, we develop gold molecular clusters (Au25) functionalized by phosphorylcholine (PC) ligands for NIR-II (1000-3000 nm) fluorescence imaging of draining lymph nodes in 4T1 murine breast cancer and CT26 colon cancer tumor mouse models. The Au-phosphorylcholine (Au-PC) probes exhibit 'super-stealth' behavior with little interactions with serum proteins, cells and tissues in vivo, which differs from the indocyanine green (ICG) dye. Subcutaneous injection of Au-PC allows lymph node mapping by NIR-II fluorescence imaging at an optimal time of ~ 0.5 - 1 hour postinjection followed by rapid renal clearance. Preclinical NIR-II fluorescence LN imaging with Au-PC affords high signal to background ratios and high safety and biocompatibility, promising for future clinical translation.

In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors.

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is to extend the excitation and emission wavelengths to the second near-infrared window (NIR-II) at >1,000 nm, also called the short-wavelength infrared window. Here we show biocompatible core-shell lead sulfide/cadmium sulfide quantum dots emitting at ~1,880 nm and superconducting nanowire single-photon detectors for single-photon detection up to 2,000 nm, enabling a one-photon excitation fluorescence imaging window in the 1,700-2,000 nm (NIR-IIc) range with 1,650 nm excitation-the longest one-photon excitation and emission for in vivo mouse imaging so far. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~1,100 µm through an intact mouse head, and enabled non-invasive cellular-resolution imaging in the inguinal lymph nodes of mice without any surgery. We achieve in vivo molecular imaging of high endothelial venules with diameters as small as ~6.6 µm, as well as CD169 + macrophages and CD3 + T cells in the lymph nodes, opening the possibility of non-invasive intravital imaging of immune trafficking in lymph nodes at the single-cell/vessel-level longitudinally.

High-precision tumor resection down to few-cell level guided by NIR-IIb molecular fluorescence imaging.

In vivo fluorescence/luminescence imaging in the near-infrared-IIb (NIR-IIb, 1,500 to 1,700 nm) window under <1,000 nm excitation can afford subcentimeter imaging depth without any tissue autofluorescence, promising high-precision intraoperative navigation in the clinic. Here, we developed a compact imager for concurrent visible photographic and NIR-II (1,000 to 3,000 nm) fluorescence imaging for preclinical image-guided surgery. Biocompatible erbium-based rare-earth nanoparticles (ErNPs) with bright down-conversion luminescence in the NIR-IIb window were conjugated to TRC105 antibody for molecular imaging of CD105 angiogenesis markers in 4T1 murine breast tumors. Under a ∼940 ± 38 nm light-emitting diode (LED) excitation, NIR-IIb imaging of 1,500- to 1,700-nm emission afforded noninvasive tumor­to­normal tissue (T/NT) signal ratios of ∼40 before surgery and an ultrahigh intraoperative tumor-to-muscle (T/M) ratio of ∼300, resolving tumor margin unambiguously without interfering background signal from surrounding healthy tissues. High-resolution imaging resolved small numbers of residual cancer cells during surgery, allowing thorough and nonexcessive tumor removal at the few-cell level. NIR-IIb molecular imaging afforded 10-times-higher and 100-times-higher T/NT and T/M ratios, respectively, than imaging with IRDye800CW-TRC105 in the ∼900- to 1,300-nm range. The vastly improved resolution of tumor margin and diminished background open a paradigm of molecular imaging-guided surgery.

Ultrafast isolated molecule imaging without crystallization.

Crystallography is the standard for determining the atomic structure of molecules. Unfortunately, many interesting molecules, including an extensive array of biological macromolecules, do not form crystals. While ultrashort and intense X-ray pulses from free-electron lasers are promising for imaging single isolated molecules with the so-called "diffraction before destruction" technique, nanocrystals are still needed for producing sufficient scattering signal for structure retrieval as implemented in serial femtosecond crystallography. Here, we show that a femtosecond laser pulse train may be used to align an ensemble of isolated molecules to a high level transiently, such that the diffraction pattern from the highly aligned molecules resembles that of a single molecule, allowing one to retrieve its atomic structure with a coherent diffraction imaging technique. In our experiment with CO2 molecules, a high degree of alignment is maintained for about 100 fs, and a precisely timed ultrashort relativistic electron beam from a table-top instrument is used to record the diffraction pattern within that duration. The diffraction pattern is further used to reconstruct the distribution of CO2 molecules with atomic resolution. Our results mark a significant step toward imaging noncrystallized molecules with atomic resolution and open opportunities in the study and control of dynamics in the molecular frame that provide information inaccessible with randomly oriented molecules.

Carbon Nanotubes-Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis.

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

Bright NIR-II Photoluminescence in Rod-Shaped Icosahedral Gold Nanoclusters.

Fluorophores with high quantum yields, extended maximum emission wavelengths, and long photoluminescence (PL) lifetimes are still lacking for sensing and imaging applications in the second near-infrared window (NIR-II). In this work, a series of rod-shaped icosahedral nanoclusters with bright NIR-II PL, quantum yields up to ≈8%, and a peak emission wavelength of 1520 nm are reported. It is found that the bright NIR-II emission arises from a previously ignored state with near-zero oscillator strength in the ground-state geometry and the central Au atom in the nanoclusters suppresses the non-radiative transitions and enhances the overall PL efficiency. In addition, a framework is developed to analyze and relate the underlying transitions for the absorptions and the NIR-II emissions in the Au nanoclusters based on the experimentally defined absorption coefficient. Overall, this work not only shows good performance of the rod-shaped icosahedral series of Au nanoclusters as NIR-II fluorophores, but also unravels the fundamental electronic transitions and atomic-level structure-property relations for the enhancement of the NIR-II PL in gold nanoclusters. The framework developed here also provides a simple method to analyze the underlying electronic transitions in metal nanoclusters.

In vivo NIR-II structured-illumination light-sheet microscopy.

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

Deep learning for in vivo near-infrared imaging.

Detecting fluorescence in the second near-infrared window (NIR-II) up to ∼1,700 nm has emerged as a novel in vivo imaging modality with high spatial and temporal resolution through millimeter tissue depths. Imaging in the NIR-IIb window (1,500-1,700 nm) is the most effective one-photon approach to suppressing light scattering and maximizing imaging penetration depth, but relies on nanoparticle probes such as PbS/CdS containing toxic elements. On the other hand, imaging the NIR-I (700-1,000 nm) or NIR-IIa window (1,000-1,300 nm) can be done using biocompatible small-molecule fluorescent probes including US Food and Drug Administration-approved dyes such as indocyanine green (ICG), but has a caveat of suboptimal imaging quality due to light scattering. It is highly desired to achieve the performance of NIR-IIb imaging using molecular probes approved for human use. Here, we trained artificial neural networks to transform a fluorescence image in the shorter-wavelength NIR window of 900-1,300 nm (NIR-I/IIa) to an image resembling an NIR-IIb image. With deep-learning translation, in vivo lymph node imaging with ICG achieved an unprecedented signal-to-background ratio of >100. Using preclinical fluorophores such as IRDye-800, translation of ∼900-nm NIR molecular imaging of PD-L1 or EGFR greatly enhanced tumor-to-normal tissue ratio up to ∼20 from ∼5 and improved tumor margin localization. Further, deep learning greatly improved in vivo noninvasive NIR-II light-sheet microscopy (LSM) in resolution and signal/background. NIR imaging equipped with deep learning could facilitate basic biomedical research and empower clinical diagnostics and imaging-guided surgery in the clinic.

Cross-Link-Functionalized Nanoparticles for Rapid Excretion in Nanotheranostic Applications.

Most NIR-IIb fluorophores are nanoparticle-based probes with long retention (≈1 month or longer) in the body. Here, we applied a novel cross-linked coating to functionalize core/shell lead sulfide/cadmium sulfide quantum dots (PbS/CdS QDs) emitting at ≈1600 nm. The coating was comprised of an amphiphilic polymer followed by three crosslinked amphiphilic polymeric layers (P3 coating), imparting high biocompatibility and >90 % excretion of QDs within 2 weeks of intravenous administration. The P3 -QDs were conjugated to an engineered anti-CD8 diabody (Cys-diabody) for in vivo molecular imaging of CD8+ cytotoxic T lymphocytes (CTLs) in response to anti-PD-L1 therapy. Two-plex molecular imaging in combination with down-conversion Er nanoparticles (ErNPs) was performed for real-time in vivo monitoring of PD-L1 positive tumor cells and CTLs with cellular resolution by non-invasive NIR-IIb light sheet microscopy. Imaging of angiogenesis in the tumor microenvironment and of lymph nodes deep in the body with a signal-to-background ratio of up to ≈170 was also achieved using P3 -QDs.

Breaking 50 Femtosecond Resolution Barrier in MeV Ultrafast Electron Diffraction with a Double Bend Achromat Compressor.

We propose and demonstrate a novel scheme to produce ultrashort and ultrastable MeV electron beam. In this scheme, the electron beam produced in a photocathode radio frequency (rf) gun first expands under its own Coulomb force with which a positive energy chirp is imprinted in the beam longitudinal phase space. The beam is then sent through a double bend achromat with positive longitudinal dispersion where electrons at the bunch tail with lower energies follow shorter paths and thus catch up with the bunch head, leading to longitudinal bunch compression. We show that with optimized parameter sets, the whole beam path from the electron source to the compression point can be made isochronous such that the time of flight for the electron beam is immune to the fluctuations of rf amplitude. With a laser-driven THz deflector, the bunch length and arrival time jitter for a 20 fC beam after bunch compression are measured to be about 29 fs (FWHM) and 22 fs (FWHM), respectively. Such an ultrashort and ultrastable electron beam allows us to achieve 50 femtosecond (FWHM) resolution in MeV ultrafast electron diffraction where lattice oscillation at 2.6 THz corresponding to Bismuth A_{1g} mode is clearly observed without correcting both the short-term timing jitter and long-term timing drift. Furthermore, oscillating weak diffuse scattering signal related to phonon coupling and decay is also clearly resolved thanks to the improved temporal resolution and increased electron flux. We expect that this technique will have a strong impact in emerging ultrashort electron beam based facilities and applications.

Cross-Link-Functionalized Nanoparticles for Rapid Excretion in Nanotheranostic Applications.

Most NIR-IIb fluorophores are nanoparticle-based probes with long retention ( ≈ 1 month or longer) in the body. Here, we applied a novel cross-linked coating to functionalize core/shell lead sulfide/cadmium sulfide quantum dots (PbS/CdS QDs) emitting at ≈ 1600 nm. The coating was comprised of an amphiphilic polymer followed by three crosslinked amphiphilic polymeric layers (P3 coating), imparting high biocompatibility and > 90% excretion of QDs within 2 weeks of intravenous administration. The P3-QDs were conjugated to an engineered anti-CD8 diabody (Cys-diabody) for in vivo molecular imaging of CD8 + cytotoxic T lymphocytes (CTLs) in response to anti-PD-L1 therapy. Two-plex molecular imaging in combination with down-conversion Er nanoparticles (ErNPs) was performed for real-time in vivo monitoring of PD-L1 positive tumor cells and CTLs with cellular resolution by non-invasive NIR-IIb light sheet microscopy. Imaging of angiogenesis in the tumor microenvironment and of lymph nodes deep in the body with a signal-to-background ratio of up to ≈ 170 was also achieved using P3-QDs.

A theranostic agent for cancer therapy and imaging in the second near-infrared window.

Theranostic nanoparticles are integrated systems useful for simultaneous diagnosis and imaging guided delivery of therapeutic drugs, with wide ranging potential applications in the clinic. Here we developed a theranostic nanoparticle (~ 24 nm size by dynamic light scattering) p-FE-PTX-FA based on polymeric micelle encapsulating an organic dye (FE) fluorescing in the 1,000-1,700 nm second near-infrared (NIR-II) window and an anti-cancer drug paclitaxel. Folic acid (FA) was conjugated to the nanoparticles to afford specific binding to molecular folate receptors on murine breast cancer 4T1 tumor cells. In vivo, the nanoparticles accumulated in 4T1 tumor through both passive and active targeting effect. Under an 808 nm laser excitation, fluorescence detection above 1,300 nm afforded a large Stokes shift, allowing targeted molecular imaging tumor with high signal to background ratios, reaching a high tumor to normal tissue signal ratio (T/NT) of (20.0 ± 2.3). Further, 4T1 tumors on mice were completed eradicated by paclitaxel released from p-FE-PTA-FA within 20 days of the first injection. Pharmacokinetics and histology studies indicated p-FE-PTX-FA had no obvious toxic side effects to major organs. This represented the first NIR-II theranostic agent developed.

In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles.

The near-infrared-IIb (NIR-IIb) (1,500-1,700 nm) window is ideal for deep-tissue optical imaging in mammals, but lacks bright and biocompatible probes. Here, we developed biocompatible cubic-phase (α-phase) erbium-based rare-earth nanoparticles (ErNPs) exhibiting bright downconversion luminescence at ~1,600 nm for dynamic imaging of cancer immunotherapy in mice. We used ErNPs functionalized with cross-linked hydrophilic polymer layers attached to anti-PD-L1 (programmed cell death-1 ligand-1) antibody for molecular imaging of PD-L1 in a mouse model of colon cancer and achieved tumor-to-normal tissue signal ratios of ~40. The long luminescence lifetime of ErNPs (~4.6 ms) enabled simultaneous imaging of ErNPs and lead sulfide quantum dots emitting in the same ~1,600 nm window. In vivo NIR-IIb molecular imaging of PD-L1 and CD8 revealed cytotoxic T lymphocytes in the tumor microenvironment in response to immunotherapy, and altered CD8 signals in tumor and spleen due to immune activation. The cross-linked functionalization layer facilitated 90% ErNP excretion within 2 weeks without detectable toxicity in mice.