Fluorescent Tracking Research Articles

Spatiotemporal Tracking of Near‐Infrared Fluorescent Single‐Walled Carbon Nanotubes inC. ElegansNematodes Confined in a Microfluidics Platform

AbstractCaenorhabditis elegans (C. elegans) nematodes are a powerful model organism for diverse biological and biomedical studies, benefiting from their genetic similarities to humans, small size, and transparency. However, fluorescence imaging ofC. eleganscan be challenging due to the strong autofluorescence in the visible range, which obscures the signal of common fluorescent proteins or dyes. Single‐walled carbon nanotubes (SWCNTs) fluoresce in the near‐infrared (NIR) range, where there is no autofluorescence background. Herein, a platform is developed for in vivo NIR imaging ofC. elegansgastrointestinal tract using biocompatible SWCNTs functionalized by single‐stranded DNA or phospholipid‐polyethylene glycol (PEG). The SWCNTs serve as fluorescent tracking probes within the worm gut, following internalization along with food intake. A microfluidic confinement device is employed to ensure an anesthetics‐free feeding environment, allowing spatiotemporal control over the SWCNTs intake imaging. Furthermore, improvements in spectral colocalization, real‐time detection of intracorporeal SWCNT dynamics, and digestive trajectory tracking are demonstrated. Owing to the unique optical properties of SWCNTs and the confinement of the worms in the microfluidics system, the proposed platform facilitates advanced in vivo imaging ofC. elegansin both the visible and NIR regions, opening numerous avenues for advancing research ofC. elegansand other microscopic model organisms.

Precise Synergistic Photothermal Therapy Guided by Accurate Temperature‐Dependent NIR‐II Fluorescence Imaging

AbstractPhotothermal therapy (PTT) is considered a promising treatment strategy for solid tumors. However, local hyperthermia (over 45°C) during PTT can cause severe side effects in neighboring healthy tissues. PTT with accurate temperature feedback is a compelling strategy to ablate tumors and reduce side effects, but it still faces challenges. Here, a new kind of phototheranostic nanoparticle, namely 17‐RF@Ag2Se is developed, enabling in vivo NIR‐II fluorescence tracking, PTT and fluorescence nanothermometry as well as synergistic heat‐shock protein (HSP) inhibition. Precise PTT with high spatiotemporal resolution is achieved with the help of the designed NIR‐II fluorescence imaging‐photothermal therapy linkage apparatus. Upon intravenous injection, 17‐RF@Ag2Se is specifically accumulated in tumors targeted by the overexpressed integrin αvβ3, which is monitored by NIR‐II fluorescence imaging of Ag2Se QDs. Further, the release of HSP inhibitor, tanespimycin (17‐AAG), enhances the thermosensitivity of tumor cells. Subsequently, the internal temperature of the tumor is precisely monitored and adjusted during PTT via the temperature‐dependent NIR‐II fluorescence feedback of Ag2Se QDs and the linkage apparatus calibration, thereby achieving efficient and safe tumor PTT. Also, the results present a new method for accurate temperature monitoring and control in vivo, which can be applied to other biomedical studies.

Polyvalent anion-induced J-aggregation and its use in ratiometric fluorescence tracking of hypotonic in Arabidopsis

J-aggregate dyes have been widely used in biomedicine, chemical engineering, etc., due to their structure and photophysical properties. J-aggregation theory describes the nearest-neighbor molecules' transition dipole moments in a sliding stacking arrangement. However, we found that the intervention of polyvalent anions can induce the J-aggregation of dyes via the intermolecular van der Waals and electrostatic interactions, which is an essential way once overlooked. Experimental results and theoretical calculations revealed that the more negatively charged or large size of anions within a specific range, the more efficiently the dye will form J-aggregates. As an application, the content of polyvalent anion salts in a salt-stressed Arabidopsis model was evaluated based on the ratiometric fluorescence emission resulting from the polyvalent anion-induced dye J-aggregation.

Alpha particle spectrometry in fluorescent nuclear track detectors with an automatic 3D track reanalysis algorithm

The main obstacle to applying aluminum oxide doped with carbon and magnesium (Al2O3:C,Mg)-based fluorescent nuclear track detectors (FNTDs) in radionuclide therapy with alpha emitters is that it has not been known how to achieve an accurate and in situ dose assessment for in vitro cellular experiments. In this study, we developed an automatic 3D track reanalysis algorithm (ATRA) to analyze the alpha energy spectra in FNTDs. The algorithm provides an excellent track identification rate and an excellent track linking rate (0.961) without significant variations in various track densities. The algorithm also achieves a reliable track detection rate exceeding 0.927 for track densities at the level of in situ measurement, and it successfully measures the alpha energy spectra for obtaining the absorbed dose. We also confirmed that the track overlap clearly interferes with accurate track reconstruction for track densities beyond 1.5 × 106 tracks cm−2. We provided a correction equation that can be used to obtain the actual alpha tracks for high track densities; the equation may also correct estimations of absorbed doses due to alpha particles.

Fluorescent imaging and smartphone detection of ClO- using sensors based on trifluoromethylquinoline-substituted pyranonitrile

In the field of novel fluorescent sensors, achieving rapid and sensitive tracking of target substances has become an urgent imperative. Applying this technology to the detection of biomarkers for biological diseases holds profound practical significance. Hypochlorous acid (HClO) is a significant reactive oxygen species (ROS) within biological systems. Aberrant metabolism of HClO can result in disruptions of physiological functions and oxidative stress, consequently influencing the development of various diseases. In this context, our study has successfully developed a novel fluorescent probe named W-4a, which employs trifluoromethyl quinoline as a substitute for traditional pyranonitrile compounds, enabling the rapid (<40 s) and highly sensitive (UV limit of detection: 1.62 μM; fluorescence limit of detection: 0.14 μM) detection of ClO-, during the detection process, ClO- effectively activated the fluorescent probe and showed a distinct red fluorescent signal at 687 nm. Furthermore, the W-4a probe exhibits excellent biocompatibility and excels in both exogenous and endogenous ClO- detection imaging within cells, Co-localization experiments indicate that probe W-4a effectively targets the Golgi apparatus. Additionally, in conjunction with the W-4a probe, we have devised a portable smart detection method for hypochlorous acid in environmental settings, which holds the potential to serve as a flexible and potent tool for monitoring environmental toxicity.

A Pyr-loaded polymer microparticle for effectively controlling Solenopsis invicta (Hymenoptera: Formicidae) in the nest

Human interference and incorrect use of pesticides are easy to induce red imported fire ant (RIFA) escape and migrate from a nest, resulting in ineffective control of RIFA. In order to avoid RIFA alert, we designed an amphiphilic PSI-mPEG-Boc-DAH loaded Pyr to make the microparticles with effective controlled release. The investigation showed that the quantity of Pyr released by Pyr@PSI-mPEG-Boc-DAH under acidic environment was only 36.40 ± 1.90% at 48 h, whereas the release rate of original Pyr was 75.23 ± 5.71%. And the RIFA mortality rate of 1 ppm Pyr in Pyr@PSI-mPEG-Boc-DAH microparticles at 48 h was only 7.78%, which was significantly lower than that of the Pyr (47.78%). Futhermore, the death rate increased sharply after 48 h, and reached 95.84% within a week after using Pyr@PSI-mPEG-Boc-DAH microparticles. Moreover, PSI-mPEG-Boc-DAH carriers could be absorbed and even transported to crop of the RIFA for subsequent trophallaxis by using fluorescence tracking. In the field experiment, the reduction rate of Pyr@PSI-mPEG-Boc-DAH treatment was achieved 99.89% after 7 d. Pyr@PSI-mPEG-Boc-DAH didn’t cause RIFA to be alarmed within 48 h and could kill nearly all of ants in the nest after 7 d, which showed a very good control effect in the field experiment. This work provided a new idea and guidance for the effective control RIFA and the development of sustainable agriculture.

Using Functionalized Liposomes to Harvest Extracellular Vesicles of Similar Characteristics in Dermal Interstitial Fluid.

Extracellular vesicles (EVs) are used by living cells for the purpose of biological information trafficking from parental-to-recipient cells and vice versa. This back-and-forth communication is enabled by two distinct kinds of biomolecules that constitute the cargo of an EV: proteins and nucleic acids. The proteomic-cum-genetic information is mediated by the physiological state of a cell (healthy or otherwise) as much as modulated by the biogenesis pathway of the EV. Therefore, in mirroring the huge diversities of human communications, the proteins and nucleic acids involved in cell communications possess seemingly near limitless diversities, and it is this characteristic that makes EVs so highly heterogeneous. Currently, there is no simple and reliable tool for the selective capture of heterogeneous EVs and the delivery of their undamaged cargo for research in extracellular protein mapping and spatial proteomics studies. Our work is a preliminary attempt to address this issue. We demonstrated our approach by using antibody functionalized liposomes to capture EVs from tumor and healthy cell-lines. To characterize their performance, we presented fluorescence and nanoparticle tracking analysis (NTA) results, TEM images, and Western blotting analysis for EV proteins. We also extracted dermal interstitial fluid (ISF) from healthy individuals and used our functionalized synthetic vesicle (FSV) method to capture EVs from their proteins. We constructed three proteomic sets [EV vs ISF, (FSV+EV) vs ISF, and (FSV+EV) vs EV] from the EV proteins and the free proteins harvested from ISF and compared their differentially expressed proteins (DEPs). The performance of our proposed method is assessed via an analysis of 1095 proteins, together with volcano plots, heatmap, GO annotation, and enriched KEGG pathways and organelle localization results of 213 DEPs.

Satellite-based solar-induced fluorescence tracks seasonal and elevational patterns of photosynthesis in California’s Sierra Nevada mountains

Robust carbon monitoring systems are needed for land managers to assess and mitigate the changing effects of ecosystem stress on western United States forests, where most aboveground carbon is stored in mountainous areas. Atmospheric carbon uptake via gross primary productivity (GPP) is an important indicator of ecosystem function and is particularly relevant to carbon monitoring systems. However, limited ground-based observations in remote areas with complex topography represent a significant challenge for tracking regional-scale GPP. Satellite observations can help bridge these monitoring gaps, but the accuracy of remote sensing methods for inferring GPP is still limited in montane evergreen needleleaf biomes, where (a) photosynthetic activity is largely decoupled from canopy structure and chlorophyll content, and (b) strong heterogeneity in phenology and atmospheric conditions is difficult to resolve in space and time. Using monthly solar-induced chlorophyll fluorescence (SIF) sampled at ∼4 km from the TROPOspheric Monitoring Instrument (TROPOMI), we show that high-resolution satellite-observed SIF followed ecological expectations of seasonal and elevational patterns of GPP across a 3000 m elevation gradient in the Sierra Nevada mountains of California. After accounting for the effects of high reflected radiance in TROPOMI SIF due to snow cover, the seasonal and elevational patterns of SIF were well correlated with GPP estimates from a machine-learning model (FLUXCOM) and a land surface model (CLM5.0-SP), outperforming other spectral vegetation indices. Differences in the seasonality of TROPOMI SIF and GPP estimates were likely attributed to misrepresentation of moisture limitation and winter photosynthetic activity in FLUXCOM and CLM5.0 respectively, as indicated by discrepancies with GPP derived from eddy covariance observations in the southern Sierra Nevada. These results suggest that satellite-observed SIF can serve as a useful diagnostic and constraint to improve upon estimates of GPP toward multiscale carbon monitoring systems in montane, evergreen conifer biomes at regional scales.

Exosomal miR-331-3p derived from chemoresistant osteosarcoma cells induces chemoresistance through autophagy

BackgroundOsteosarcoma is a common malignant bone tumor, and chemotherapy can effectively improve the prognosis. MicroRNA-331 (MiR-331) is associated with poor cancer outcomes. However, the role of miR-331 in osteosarcoma remains to be explored.MethodsDrug-resistant osteosarcoma cells were cultured, and their exosomes were purified. The secretion and uptake of exosomes by drug-resistant osteosarcoma and osteosarcoma cells were confirmed using a fluorescence tracking assay and Transwell experiments. The effects of drug-resistant exosomes on cell proliferation were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. siRNA-Drosha and neutral sphingomyelinase inhibitor GW4869 were used to determine the transfer of miRNAs. qRT-PCR and western blotting were used to detect the role of autophagy in the regulation of drug-resistant cell-derived exosomal miR-331-3p.ResultsExosomal miR-331-3p levels in drug-resistant cells were higher than in exosomes from osteosarcoma cells. The exosomes secreted by the drug-resistant osteosarcoma cells could be absorbed by osteosarcoma cells, leading to acquired drug resistance in previously non-resistance cells. Inhibition of miRNAs resulted in reduced transmission of drug resistance transmission by exosomes. Exosomes from drug-resistant osteosarcoma cells transfected with siRNA-Drosha or treated by GW4869 could not enhance the proliferation of MG63 and HOS cells. Finally, miR-331-3p in the exosomes secreted by drug-resistant osteosarcoma cells could induce autophagy of osteosarcoma cells, allowing them to acquire drug resistance. The inhibition of miR-331-3p decreased drug resistance of osteosarcoma cells.ConclusionExosomes secreted from chemoresistant osteosarcoma cells promote drug resistance through miR-331-3p and autophagy. Inhibition of miR-331-3p could be used to alleviate drug resistance in osteosarcoma.

New Rhodamine-based sensor for high-sensitivity fluorescence tracking of Cys and simultaneously colorimetric detection of H2S

Sulfhydryl-containing compounds including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) are involved in many physiological processes. The development of single-molecule optical sensor for the distinguish detection of these bio-thiols is a critical and challenging effort. In this work, we designed a one-step synthesis of the Rhodamine-based sensor FR for specific fluorescent response of Cys and simultaneously colorimetric detection of H2S, in which the aldehyde and fluorine groups act as response sites. Sensor FR displays significant fluorescence enhancement at 565 nm toward Cys with high selectivity and low detection limits (49 nM) due to the low background fluorescent signal of the spirocyclic closed-state in Rhodamine structure. Meantime, after treatment of H2S, the color of the sensor changes significantly from colorless to blue-purple, which can be used as a visual colorimetric method to detect H2S. These response mechanisms were systematically characterized by 1H NMR and Mass spectrometry. Finally, sensor FR could be used to monitor exogenous and endogenous of intracellular Cys changes.

Visualising the adsorption behaviour of sodium dodecyl sulfate corrosion inhibitor on the Mg alloy surface by a novel fluorescence labeling strategy

Corrosion inhibitors, have the characteristics of environmental friendly and high efficient, are widely used to inhibit the corrosion of alloys; however, their corrosion inhibition mechanism and adsorption behavior is still unclear. Herein, a cationic dye (1,8-naphthalenimide, NP) is synthesized for fluorescent visualisation of the sodium dodecyl sulfate (SDS) adsorption behavior on the α-Mg and β-Li phases. In-situ observation showed that obvious fluorescence adsorption preferentially appeared on the α-Mg phase via physicochemical adsorption. Moreover, stronger fluorescence intensity was observed on the local corrosion sites due to the precipitation of Mg-SDS complex. Micro-scale in situ observation under electrochemical control is applied to explain the inhibition mechanism from the perspective of corrosion kinetics. The inhibition efficiency is evidently improved (>90%) after the addition of SDS, and the optimum value reaches 95.1% at the SDS concentration of 0.04 M. Surface characterizations are conducted to validate the adsorption behaviour of SDS on the Mg alloy surface. The inhibition mechanism of SDS is proposed by experimental and theoretical calculations. This work advances the development and application of specific fluorescence tracking to elucidate the adsorption behaviour of corrosion inhibitors at the micro-level and provides a new idea for the development of efficient corrosion inhibitors.

THU572 Structure, Organisation And Mechanistic Insight Of Cytochrome P450 17A1

Abstract Disclosure: L.L. Martin: None. P. O'Leary: None. S. Arumugam: None. S. Bell: None. R.J. Rodgers: None. Introduction: Cytochrome P45017A1 (CYP17) is a heme-containing, membrane bound enzyme situated at a key branch within steroidogenic pathways. CYP17 is a dual function enzyme that initially catalyses 17α-hydroxylation of pregnenolone and progesterone to 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. CYP17 can further metabolize these steroids via 17,20-lyase reactions to produce androgen/estrogen precursors. Activity of CYP17 requires electrons delivered by cytochrome P450 oxido-reductase (CPR) and and accessory protein cytochrome b5 (cyt b5) for the 17,20-lyase reaction. Previously, we showed that (i) CYP17 could homodimerise and heterodimerise with CPR and cyt b5 in cells using FRET [1] and computer models [2] and (ii) protein-protein interaction of cyt b5 with CYP17 slowed the rate of electron transfer to CYP17 [3] by electrochemistry. The current study focuses on sub-cellular organisation of CYP17 and mechanistic insight gained via analysis of the structural and molecular interactions of CYP17 with its partner proteins associated with 17α-hydroxylase and 17,20-lyase reactions. Methods: Single molecule fluorescence microscopy (SMFM) was used to observe fluorescently tagged CYP17 in live HEK 293 cells. Molecular Dynamics (MD) simulations were done for CYP17 in membranes, in the presence and absence of substrates, while interacting with CPR and cyt b5. Results: Halo-tagged CYP17 was imaged in real-time within the endoplasmic reticulum, providing kinetic data and sub-cellular localisation dynamics of individual molecules.MD simulations, within a membrane environment, allowed the dynamics of full-length models of CYP17 to be simulated: (i) in the absence of substrate, (ii) with a hydroxylase substrate (pregnenolone), and (iii) with a lyase substrate (17α-OH pregnenolone). Intriguingly, the protein-protein interaction of cyt b5 decreased the movement and flexibility within the active site of CYP17, however, regions not necessarily obvious for haem-substrate rearrangement were also altered. Single molecule fluorescence revealed CYP17 molecules moved dynamically along the endoplasmic reticulum. Conclusions: MD analyses provided structural insight on the mechanism underlying the allosteric coupling between the cyt b5 to the CYP17 interface and specifically to the active site. Single molecule fluorescence tracking indicated that CYP17 actively moves dynamically in the endoplasmic reticulum. 1. Praporski, S., et al., Organization of Cyt P450 Enzymes Involved in Sex Steroid Synthesis. J. Biol. Chem., 2009. 284 33224-33232. 2. Holien, J.K., et al., A Homodimer Model Can Resolve the Conundrum as to How CPR and Cyt b5 Compete for the Same Binding Site on Cyt P450c17. Curr Protein Pept Sci, 2017. 18 515-521. 3. Simonov, A.N., et al., Mechanistic Scrutiny Identifies a Kinetic Role for Cyte b5 Regulation of Human Cyt P450c17. PLoS ONE, 2015. 10 e0141252. Presentation Date: Thursday, June 15, 2023

Metal-Organic Framework-Based Insecticide and dsRNA Codelivery System for Insecticide Resistance Management.

Targeted silencing of resistance-associated genes by specific double-stranded RNA (dsRNA) is an attractive strategy for overcoming insecticide resistance in insect pests. However, silencing target genes of insect pests by feeding on dsRNA transported via plants remains challenging. Herein, a codelivery system of insecticide and dsRNA is designed by encapsulating imidacloprid and dsNlCYP6ER1 within zeolitic imidazolate framework-8 (ZIF-8) nanoparticles to improve the susceptibility of Nilaparvata lugens (Stål) to imidacloprid. With an average particle size of 195 nm and a positive surface charge, the derived imidacloprid/dsNlCYP6ER1@ZIF-8 demonstrates good monodispersity. Survival curve results showed that the survival rates of N. lugens treated with imidacloprid and imidacloprid@ZIF-8 were 82 and 62%, respectively, whereas, in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group, the survival rate of N. lugens is only 8%. Pot experiments demonstrate that the survival rate in the imidacloprid/dsNlCYP6ER1@ZIF-8 treatment group was much lower than that in the imidacloprid treatment group, decreasing from 54 to 24%. The identification of NlCYP6ER1 expression and the fluorescence tracking of ZIF-8 demonstrate that ZIF-8 can codeliver dsRNA and insecticide to insects via rice. Safety evaluation results showed that the dsNlCYP6ER1@ZIF-8 nanoparticle had desirable biocompatibility and biosafety to silkworm. This dsRNA and insecticide codelivery system may be extended to additional insecticides with potential resistance problems in the future, greatly enhancing the development of pest resistance management.

Multiphase jet penetration in gas-solid systems

Thermochemical conversion of biomass in fixed/fluidized bed reactors requires efficient feedstock penetration and dispersion through reliable feed injection systems. Pneumatic biomass injection produces a gas-solid jet in the reactor that requires proper momentum to disperse the feedstock. Assessing multiphase jet penetration in fluidized beds is challenging because of system's complexity, opaqueness and lack of a universal measurement technique adapted for tracking the associated phenomena. In this work, three measurement techniques (fluorescent particle tracking, pitot tubes and Electric Capacitance Tomography (ECVT)) were employed and compared to assess the penetration of air-biomass jets into fixed/fluidized beds. Results from all techniques indicated that the jet penetration increased with solids mass flux, and jets with similar momentum provide greater penetration in fluidized beds compared to fixed beds. Tridimensional jet propagation and biomass concentration across the jet length were obtained for different operating conditions providing a useful database for CFD and other mechanistic models validation.

Bio-based Carbon dots Loaded with 5-Fu: A Multifunctional drug Delivery System.

In the present work, a simple and efficient stirring method was used to successfully synthesize a novel multifunctional carbon dots-drug delivery system AMP-CDs@5-Fu in the form of intertwined filaments. The results showed that AMP-CDs@5-Fu had the highest final release in the medium mimicking the physiological environment of the human small intestine compared to that of 5-Fu and that the drug release behaviors followed a zero-grade drug release within the first 3h. The results also showed that AMP-CDs@5-Fu could be used to reduce the toxicity of 5-Fu while significantly improving the anticancer ability. In vitro hemolysis and anticancer assays showed that AMP-CDs@5-Fu could significantly improve the anticancer ability while decreasing the toxicity of 5-Fu, and the hemolysis rate of AMP-CDs@5-Fu was significantly lower than that of 5-Fu; their IC50 against 4T1 cancer cells were 201.63 ± 8.94µg 5-Fu/mL and 241.24 ± 11.05µg 5- Fu/mL. In addition, AMP-CDs@5-Fu allowed clear cell imaging. Therefore, AMP-CDs@5-Fu is expected to improve the bioavailability of 5-Fu as a novel oral agent with fluorescent properties and very promising as a novel fluorescence tracking drug loading system, which is expected to be used in the field of anticancer targeted therapy and fluorescence tracking to monitor the distribution of drugs.

In vivo fluorescent labeling and tracking of extracellular matrix.

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.

Nano-invasomes for simultaneous topical delivery of buprenorphine and bupivacaine for dermal analgesia.

Opioid and local anaesthetic receptors are abundantly concentrated in different layers of the skin. Therefore, simultaneous targeting of these receptors can produce more potent dermal anaesthesia. Herein, we developed lipid-based nanovesicles for the co-delivery of buprenorphine and bupivacaine to efficiently target skin-concentrated pain receptors. Invasomes incorporating two drugs were prepared by ethanol injection method. Subsequently, the size, zeta potential, encapsulation efficiency, morphology, and in-vitro drug release of vesicles were characterized. Ex-vivo penetration features of vesicles were then investigated by the franz diffusion cell on the full-thickness human skin. Wherein, it was demonstrated that invasomes penetrated the skin deeper and delivered bupivacaine more effectively than buprenorphine to the target site. The superiority of invasome penetration was further evidenced by the results of ex-vivo fluorescent dye tracking. Estimation of in-vivo pain responses by the tail-flick test revealed that compared with the liposomal group, the group receiving invasomal formulation and drug-free invasomal formulation (only containing menthol) displayed increased analgesia in the initial times of 5 and 10 min. Also, no signs of oedema or erythema were observed in the Daze test in any of the rats receiving the invasome formulation. Finally, ex-vivo and in-vivo assays demonstrated efficiency in delivering both drugs into deeper layers of skin and exposing them to the located pain receptors, which improves the time of onset and the analgesic effects. Hence, this formulation appears to be a promising candidate for tremendous development in the clinical setting.

Multiplexed trackable intratumor microdosing of the investigational STING agonist TAK-676 alone and in combination in the native tumor microenvironment of patients with head and neck cancer: A phase 0 trial.

2579 Background: TAK-676 is a novel synthetic STING agonist developed to induce an inflammatory state through release of interferon (IFN), production of proinflammatory chemokines and cytokines, and activation of innate and adaptive immune cells in the tumor microenvironment (TME). To gain early mechanistic insights on the effects of TAK-676 in the native TME alone and in combination with standard-of-care chemotherapies, we completed a Phase 0 intratumoral microdosing trial in head and neck squamous cell carcinoma patients using the Comparative In Vivo Oncology (CIVO) platform. CIVO enables direct evaluation of biomarker response in trackable, distinct regions of drug exposure upon resection. These studies were designed to identify rational combinations that could be evaluated in future clinical trials with therapeutic intent. Methods: This IRB-approved study was conducted at four centers within the United States. Fifteen subjects provided Informed Consent and enrolled in the trial. Microdoses (≤1/100th systemic dose) of TAK-676, carboplatin, 5-FU, paclitaxel, and combinations thereof, were simultaneously injected via CIVO to eligible patients presenting with a surface-accessible primary or metastatic lesion ≥ 2 cm and planned surgical resection of the target lesion at 24 (n=8) or 72–96 (n=7) hours. Injection sites were identified via co-injection of fluorescent tracking markers (CIVO GLO). Multiplexed ISH, IHC, and spatial molecular profiling via GeoMx and CosMx were used to resolve molecular responses at each injection site. Patients were monitored for adverse events (AEs) for 28 days following injection. Results: TAK-676 exposure was associated with increased IFN signaling in all samples analyzed at the 24-hour cohort. This was accompanied by increased expression of pro-inflammatory cytokines and chemokines, “M1-like” macrophages, NK cells, and CD8+ T cells. Spatial profiling with GeoMx and CosMx revealed an IFN signature and elevated pro-inflammatory markers across multiple cell types throughout the TME. In addition, exposure to TAK-676 plus chemotherapy resulted in localized enhancement of apoptosis vs. chemotherapy alone across multiple tumors. The responses observed at 24 hours were transient and not detected in the 72–96-hour cohort. No AEs related to injection procedure or contents were reported. Conclusions: Intratumor microdosing via CIVO of TAK-676 alone and in combination with chemotherapy was shown to promote an early pro-inflammatory response, providing evidence of an on-target mechanism of action. The absence of effect at the late timepoints could be due to pharmacokinetics of drug exposure or compensatory signaling within the TME. In this Phase 0 clinical trial we safely demonstrated precise, mechanistic effects of TAK-676 on the native TME of intact human tumors. Clinical trial information: NCT04541108 .

Cancer Therapeutic siRNA Delivery and Imaging by Nitrogen- and Neodymium-Doped Graphene Quantum Dots.

While small interfering RNA (siRNA) technology has become a powerful tool that can enable cancer-specific gene therapy, its translation to the clinic is still hampered by the inability of the genes alone to cell transfection, poor siRNA stability in blood, and the lack of delivery tracking capabilities. Recently, graphene quantum dots (GQDs) have emerged as a novel platform allowing targeted drug delivery and fluorescence image tracking in visible and near-infrared regions. These capabilities can aid in overcoming primary obstacles to siRNA therapeutics. Here, for the first time, we utilize biocompatible nitrogen- and neodymium-doped graphene quantum dots (NGQDs and Nd-NGQDs, respectively) for the delivery of Kirsten rat sarcoma virus (KRAS) and epidermal growth factor receptor (EGFR) siRNA effective against a variety of cancer types. GQDs loaded with siRNA noncovalently facilitate successful siRNA transfection into HeLa cells, confirmed by confocal fluorescence microscopy at biocompatible GQD concentrations of 375 μg/mL. While the GQD platform provides visible fluorescence tracking, Nd doping enables deeper-tissue near-infrared fluorescence imaging suitable for both in vitro and in vivo applications. The therapeutic efficacy of the GQD/siRNA complex is verified by successful protein knockdown in HeLa cells at nanomolar siEGFR and siKRAS concentrations. A range of GQD/siRNA loading ratios and payloads are tested to ultimately provide substantial inhibition of protein expression down to 31-45%, comparable with conventional Lipofectamine-mediated delivery. This demonstrates the promising potential of GQDs for the nontoxic delivery of siRNA and genes in general, complemented by multiwavelength image tracking.

Engineered Cytokine-Primed Extracellular Vesicles with High PD-L1 Expression Ameliorate Type 1 Diabetes.

Type 1 diabetes (T1D), which is a chronic autoimmune disease, results from the destruction of insulin-producing β cells targeted by autoreactive T cells. The recent discovery that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) function as therapeutic tools for autoimmune conditions has attracted substantial attention. However, the in vivo distribution and therapeutic effects of MSC-EVs potentiated by pro-inflammatory cytokines in the context of T1D have yet to be established. Here, it is reported that hexyl 5-aminolevulinate hydrochloride (HAL)-loaded engineered cytokine-primed MSC-EVs (H@TI-EVs) with high expression of immune checkpoint molecule programmed death-legend 1 (PD-L1) exert excellent inflammatory targeting and immunosuppressive effects for T1D imaging and therapy. The accumulated H@TI-EVs in injured pancreas not only enabled the fluorescence imaging and tracking of TI-EVs through the intermediate product protoporphyrin (PpIX) generated by HAL, but also promoted the proliferative and anti-apoptotic effects of islet β cells. Further analysis revealed that H@TI-EVs exhibited an impressive ability to reduce CD4+ T cell density and activation through the PD-L1/PD-1 axis, and induced M1-to-M2 macrophage transition to reshape the immune microenvironment, exhibiting high therapeutic efficiency in mice with T1D. This work identifies a novel strategy for the imaging and treatment of T1D with great potential for clinical application.