Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads.

Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362 nt with the limit of detection down to 0.3 fm using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.

Supramolecular Heterodimer Peptides Assembly for Nanoparticles Functionalization.

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VHHs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VHHs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.

Long-Range Energy Transfer between Dye-Loaded Nanoparticles: Observation and Amplified Detection of Nucleic Acids.

Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics, and biosensing, but its operating range is fundamentally limited by the Förster radius of ≈5 nm. In this work, FRET between fluorescent organic nanoparticles (NPs) is studied in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distances of 15 and 20 nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this long-distance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding the cancer marker survivin, bringing together donor and acceptor NPs at ≈15 nm distance. In this nanoprobe, a single-molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based biosensing.

Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining.

The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers.

Study of surfactant cross-linking by click chemistry on a model water/oil interface.

In this study, we explored how chemical reactions of amphiphile compounds can be characterized and followed-up on model interfaces. A custom-made surfactant containing three alkyne sites was first adsorbed and characterized at a water/oil interface. These amphiphiles then underwent interfacial crosslinking by click chemistry upon the addition of a second reactive agent. The monolayer properties and dilatational elasticity, were compared before and after the polymerization. Using bulk phase exchange, the composition of the aqueous bulk phase was finely controlled and washed to specifically measure the interfacial effects of the entities adsorbed and trapped at the interface. In this study, we aim to emphasize an original experimental approach to follow complex phenomena occurring on model interfaces, and also show the potential of this method to characterize multifactorial processes.

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway.

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here, we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles, and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

Engineering Novel 3D Models to Recreate High-Grade Osteosarcoma and its Immune and Extracellular Matrix Microenvironment.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

In Vivo Imaging Evaluation of Fluorescence Intensity at Tail Emission of Near-Infrared-I (NIR-I) Fluorophores in a Porcine Model.

Over the last decade fluorescence-guided surgery has been primarily focused on the NIR-I window. However, the NIR-I window has constraints, such as limited penetration and scattering. Consequently, exploring the performance of NIR-I dyes at longer wavelengths (i.e., the NIR-II window) is crucial to expanding its application. Two fluorophores were used in three pigs to identify the mean fluorescence intensity (MFI) using two commercially available NIR-I and NIR-II cameras. The near-infrared coating of equipment (NICE) was used to identify endoluminal surgical catheters and indocyanine green (ICG) for common bile duct (CBD) characterization. The NIR-II window evaluation showed an MFI of 0.4 arbitrary units (a.u.) ± 0.106 a.u. in small bowel NICE-coated catheters and an MFI of 0.09 a.u. ± 0.039 a.u. in gastric ones. In CBD characterization, the ICG MFI was 0.12 a.u. ± 0.027 a.u., 0.18 a.u. ± 0.100 a.u., and 0.22 a.u. ± 0.041 a.u. at 5, 35, and 65 min, respectively. This in vivo imaging evaluation of NIR-I dyes confirms its application in the NIR-II domain. To the best of our knowledge, this is the first study assessing the MIF of NICE in the NIR-II window using a commercially available system. Further comparative trials are necessary to determine the superiority of NIR-II imaging systems.

Anionic amphiphilic calixarenes for peptide assembly and delivery.

Shape-persistent macrocycles enable superior control on molecular self-assembly, allowing the preparation of well-defined nanostructures with new functions. Here, we report on anionic amphiphilic calixarenes of conic shape and their self-assembly behavior in aqueous media for application in intracellular delivery of peptides. Newly synthesized calixarenes bearing four phosphonate groups and two or four long alkyl chains were found to form micelles of âˆ¼ 10 nm diameter, in contrast to an analogue with short alkyl chains. These amphiphilic calixarenes are able to complex model (oligo-lysine) and biologically relevant (HIV-1 nucleocapsid peptide) cationic peptides into small nanoparticles (20-40 nm). By contrast, a control anionic calixarene with short alkyl chains fails to form small nanoparticles with peptides, highlighting the importance of micellar assembly of amphiphilic calixarenes for peptide complexation. Cellular studies reveal that anionic amphiphilic calixarenes exhibit low cytotoxicity and enable internalization of fluorescently labelled peptides into live cells. These findings suggest anionic amphiphilic macrocycles as promising building blocks for the preparation of peptide delivery vehicles.

Dynamic covalent chemistry in live cells for organelle targeting and enhanced photodynamic action.

Organelle-specific targeting enables increasing the therapeutic index of drugs and localizing probes for better visualization of cellular processes. Current targeting strategies require conjugation of a molecule of interest with organelle-targeting ligands. Here, we propose a concept of dynamic covalent targeting of organelles where the molecule is conjugated with its ligand directly inside live cells through a dynamic covalent bond. For this purpose, we prepared a series of organelle-targeting ligands with a hydrazide residue for reacting with dyes and drugs bearing a ketone group. We show that dynamic hydrazone bond can be formed between these hydrazide ligands and a ketone-functionalized Nile Red dye (NRK) in situ in model lipid membranes or nanoemulsion droplets. Fluorescence imaging in live cells reveals that the targeting hydrazide ligands can induce preferential localization of NRK dye and an anti-cancer drug doxorubicin in plasma membranes, mitochondria and lipid droplets. Thus, with help of the dynamic covalent targeting, it becomes possible to direct a given bioactive molecule to any desired organelle inside the cell without its initial functionalization by the targeting ligand. Localizing the same NRK dye in different organelles by the hydrazide ligands is found to affect drastically its photodynamic activity, with the most pronounced phototoxic effects in mitochondria and plasma membranes. The capacity of this approach to tune biological activity of molecules can improve efficacy of drugs and help to understand better their intracellular mechanisms.

Amplified Fluorescence in Situ Hybridization by Small and Bright Dye-Loaded Polymeric Nanoparticles.

Detection and imaging of RNA at the single-cell level is of utmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex, and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dye-loaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin, and polyA tails), using a simple 1 h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green-, red-, and far-red-emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semiquantitative, correlating with RT-qPCR. In comparison with the commercial locked nucleic acid (LNA)-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only three steps vs ∼20 steps together with a much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.

Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe.

Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.

Confronting molecular rotors and self-quenched dimers as fluorogenic BODIPY systems to probe biotin receptors in cancer cells.

Probing receptors at the cell surface to monitor their expression level can be performed with fluorogenic dyes. Biotin receptors (BRs) are particularly interesting as they are overexpressed in cancer cells. Herein, to image BRs, we adapted and systematically compared two fluorogenic systems based on BODIPYs: a molecular rotor and a self-quenched dimer that light up in response to high viscosity and low polarity of the membrane, respectively. The fluorogenic dimer proved to be more efficient than the rotor and allowed BRs to be imaged in cancer cells, which can effectively be discriminated from non-cancer cells.

Intraoperative ureter identification with a novel fluorescent catheter.

Iatrogenic ureteral injuries (IUI) occur in 0.5-1.3% of cases during abdominal surgery. If not recognized intraoperatively, IUI increase morbidity/mortality. A universally accepted method to prevent IUI is lacking. Near-infrared fluorescent imaging (NIRF), penetrating deeper than normal light within the tissue, might be useful, therefore ureter visualization combining NIRF with special dyes (i.e. IRDye 800BK) is promising. Aim of this work is to evaluate the detection of ureters using stents coated with a novel biocompatible fluorescent material (NICE: near-infrared coating of equipment), during laparoscopy. female pigs underwent placement of NICE-coated stents (NS). NIRF was performed, and fluorescence intensity (FI) was computed. Successively, 0.15 mg/kg of IRDye 800BK was administered intravenously, and FI was computed at different timepoints. Ureter visualization using NS only was further assessed in a human cadaver. Both methods allowed in vivo ureter visualization, with equal FI. However, NS were constantly visible whereas IRDye 800BK allowed visualization exclusively during the ureteral peristaltic phases. In the human cadaver, NS provided excellent ureter visualization in its natural anatomical position. NS provided continuous ureteral visualization with similar FI as the IRDye 800BK, which exclusively allowed intermittent visualization, dependent on ureteral peristalsis. NS might prove useful to visualize ureters intraoperatively, potentially preventing IUI.

Enzyme-free amplified detection of cellular microRNA by light-harvesting fluorescent nanoparticle probes.

Detection of cellular microRNA biomarkers is an emerging powerful tool in cancer diagnostics. Currently, it requires multistep tedious protocols based on molecular amplification of the RNA target, e.g. RT-qPCR. Here, we developed a one-step enzyme-free method for microRNA detection in cellular extracts based on light-harvesting nanoparticle (nanoantenna) biosensors. They amplify the fluorescence signal by effective Förster resonance energy transfer (FRET) from ultrabright dye-loaded polymeric nanoparticle to a single acceptor and thus convert recognition of one microRNA copy (through nucleic acid strand displacement) into a response of >400 dyes. The developed nanoprobes of 17-19 nm diameter for four microRNAs (miR-21, let-7f, miR-222 and miR-30a) exhibit outstanding brightness (up to 3.8 × 107 M-1cm-1) and ratiometric sequence-specific response to microRNA with the limit of detection (LOD) down to 1.3 pM (21 amol), equivalent to 24 RT-qPCR cycles. They enable quantitative detection of the four microRNAs in RNA extracts from five cancerous cell lines (human breast cancer - T47D and MCF7, head and neck cancer - CAL33 and glioblastoma - LNZ308 and U373) and two non-cancerous ones (Hek293 and MCF10A), in agreement with RT-qPCR. The results confirmed that let-7f and especially miR-21 are systematically overexpressed in all studied cancerous cell lines. These nanoparticle biosensors are compatible with low-cost portable fluorometers and small detection volumes (11 amol LOD), opening a route to rapid point-of-care cancer diagnostics.

Fluorogenic Squaraine Dendrimers for Background-Free Imaging of Integrin Receptors in Cancer Cells.

To overcome the limited brightness of existing fluorogenic molecular probes for biomolecular targets, we introduce a concept of fluorogenic dendrimer probe, which undergoes polarity-dependent switching due to intramolecular aggregation-caused quenching of its fluorophores. Based on a rational design of dendrimers with four and eight squaraine dyes, we found that octamer bearing dyes through a sufficiently long PEG(8) linker displays >400-fold fluorescence enhancement from water to non-polar dioxane. High extinction coefficient (≈2,300,000 m-1 cm-1 ) resulted from eight squaraine dyes and quantum yield (≈25 %) make this octamer the brightest environment-sensitive fluorogenic molecule reported to date. Its conjugate with cyclic RGD used at low concentration (3 nm) enables integrin-specific fluorescence imaging of cancer cells with high signal-to-background ratio. The developed dendrimer probe is a "golden middle" between molecular probes and nanoparticles, combining small size, turn-on response and high brightness, important for bioimaging.

Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress.

Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.

Simultaneous multipurpose fluorescence imaging with IRDye® 800BK during laparoscopic surgery.

BACKGROUND: IRDye® 800BK is a fluorophore, currently undergoing clinical translation, which has both biliary and renal clearance. To date, there is no description of a fluorophore, which can be simultaneously used for non-invasive, near-infrared fluorescence-based (NIRF) visualization of different structures and perfusion evaluation. The purpose of this study was to evaluate IRDye® 800BK for the simultaneous assessment of bowel perfusion, lymphography, ureter and bile duct delineation. METHODS: Six pigs received a 0.15 mg/kg dye as a single bolus intravenous injection (IV). With the FLER (fluorescence-based enhanced reality) software, fluorescence intensity (FI) of 5 regions of interest (ROI) in an ischemic bowel loop was measured along with the time to reach the FI peak, and capillary lactate was measured from the same ROI, followed by the assessment of the ureters and bile ducts for a maximal duration of 180 min after dye administration. In 3 animals, the procedure was initiated via gastroscopic injection of a 0.6 mg (1 mg/mL) dye in the gastric submucosa followed by lymphography in a NIRF setting. RESULTS: Excellent visualization of the ureters and bowel perfusion was obtained under NIRF imaging. Additionally, the bile duct and gastric lymph ducts and nodes were visualized. A positive correlation was found between the time to peak FI in the ischemic bowel loop and the corresponding capillary lactate levels (rho 0.59, p < 0.001). CONCLUSION: In this study, we successfully demonstrated the simultaneous multipurpose IRDye® 800BK applicability during laparoscopic surgery. This fluorophore has the potential to become a powerful and versatile image-guided surgery tool.

Preoperative endoscopic marking of the gastrointestinal tract using fluorescence imaging: submucosal indocyanine green tattooing versus a novel fluorescent over-the-scope clip in a survival experimental study.

BACKGROUND: Intraoperative localization of endoluminal lesions is can be difficult during laparoscopy. Preoperative endoscopic marking is therefore necessary. Current methods include submucosal tattooing using visible dyes, which in case of transmural injection can impair surgical dissection. Tattooing using indocyanine green (ICG) coupled to intraoperative near-infrared (NIR) laparoscopy has been described. ICG is only visible under NIR-light, therefore, it doesn't impair the surgical workflow under white light even if there is spillage. However, ICG tattoos have rapid diffusion and short longevity. We propose fluorescent over-the-scope clips (FOSC), using a novel biocompatible fluorescent paint, as durable lesion marking. METHODS: In six pigs, gastric and colonic endoscopic tattoos using 0.05 mg/mL of ICG and markings using the fluorescent OSC were performed (T0). Simultaneously, NIR laparoscopy was executed. Follow-up laparoscopies were conducted at postoperative day (POD) 4-6 (T1) and POD 11-12 (T2). During laparoscopy, fluorescence intensity was assessed. In one human cadaver, FOSC was used to mark a site on the stomach and on the sigmoid colon, respectively. Intraoperative detection during NIR laparoscopy was assessed. RESULTS: Gastric and colonic ICG tattooing and OSC markings were easily visible using NIR laparoscopy on T0. All FOSC were visible at T1 and T2 in both stomach and colon, whereas the ICG tattooing at T1 was only visible in the stomach of 2 animals and in the colon of 3 animals. At T2, tattoos were not visible in any animal. FOSC were still visible in both stomach and colon of the human cadaver at 10 days. CONCLUSION: Endoscopic marking using FOSC can be an efficient and durable alternative to standard methods.

Near-infrared fluorescent coatings of medical devices for image-guided surgery.

Rapidly expanding field of image-guided surgery needs new materials for near-infrared imaging with deep tissue penetration. Here, we introduce near-infrared coating of equipment (NICE) for image-guided surgery based on a series of lipophilic cyanine-7.5 dyes with bulky hydrophobic counterions and a biocompatible polymer, poly(methyl methacrylate). The NICE material exhibits superior brightness (15-20-fold higher) and photostability compared to fluorescent coatings based on commonly used indocyanine green (ICG). It can be deposited on different surfaces and devices, such as steel and gold fiducials, silicone and PVC catheters, polymeric surgical sutures and gauzes. Such coated medical devices show excellent stability in air and buffer for ≥150 days. Accelerated ageing revealed their shelf-life of ≥3 years. They are also stable in serum-containing media, whereas ICG-based coating shows rapid dye leakage. NICE is compatible with standard sterilization protocols based on ethylene oxide and vapor. Moreover, our coating material is biocompatible, where cultured cells spread effectively without signs of cytotoxicity. Ex vivo studies suggest that NICE on fiducials can be visualized as deep as 0.5 cm, and NICE on catheters enables their visualization inside ureters and esophagus. Finally, NICE on different medical devices has been validated for image-guided surgery in porcine and human cadaver models. Thus, the developed NIR coating material emerges as a powerful tool for a variety of medical applications.