Visualizing Ribbon-to-Ribbon Heterogeneity of Chemically Unzipped Wide Graphene Nanoribbons by Silver Nanowire-Based Tip-Enhanced Raman Scattering Microscopy.

Graphene nanoribbons (GNRs), a quasi-one-dimensional form of graphene, have gained tremendous attention due to their potential for next-generation nanoelectronic devices. The chemical unzipping of carbon nanotubes is one of the attractive fabrication methods to obtain single-layered GNRs (sGNRs) with simple and large-scale production.  The authors recently found that unzipping from double-walled carbon nanotubes (DWNTs), rather than single- or multi-walled, results in high-yield production of crystalline sGNRs. However, details of the resultant GNR structure, as well as the reaction mechanism, are not fully understood due to the necessity of nanoscale spectroscopy. In this regard, silver nanowire-based tip-enhanced Raman spectroscopy (TERS) is applied for single GNR analysis and investigated ribbon-to-ribbon heterogeneity in terms of defect density and edge structure generated through the unzipping process.  The authors found that sGNRs originated from the inner walls of DWNTs showed lower defect densities than those from the outer walls. Furthermore, TERS spectra of sGNRs exhibit a large variety in graphitic Raman parameters, indicating a large variation in edge structures. This work at the single GNR level reveals, for the first time, ribbon-to-ribbon heterogeneity that can never be observed by diffraction-limited techniques and provides deeper insights into unzipped GNR structure as well as the DWNT unzipping reaction mechanism.

Remote Excitation of Tip-Enhanced Photoluminescence with a Parallel AgNW Coupler.

Tip-enhanced photoluminescence (TEPL) microscopy allows for the correlation of scanning probe microscopic images and photoluminescent spectra at the nanoscale level in a similar way to tip-enhanced Raman scattering (TERS) microscopy. However, due to the higher cross-section of fluorescence compared to Raman scattering, the diffraction-limited background signal generated by far-field excitation is a limiting factor in the achievable spatial resolution of TEPL. Here, we demonstrate a way to overcome this drawback by using remote excitation TEPL (RE-TEPL). With this approach, the excitation and detection positions are spatially separated, minimizing the far-field contribution. Two probe designs are evaluated, both experimentally and via simulations. The first system consists of gold nanoparticles (AuNPs) through photoinduced deposition on a silver nanowire (AgNW), and the second system consists of two offset parallel AgNWs. This latter coupler system shows a higher coupling efficiency and is used to successfully demonstrate RE-TEPL spectral mapping on a MoSe2/WSe2 lateral heterostructure to reveal spatial heterogeneity at the heterojunction.

SERS Endoscopy for Monitoring Intracellular Drug Dynamics.

Understanding the dynamics and distribution of medicinal drugs in living cells is essential for the design and discovery of treatments. The tools available for revealing this information are, however, extremely limited. Here, we report the application of surface-enhanced Raman scattering (SERS) endoscopy, using plasmonic nanowires as SERS probes, to monitor the intracellular fate and dynamics of a common chemo-drug, doxorubicin, in A549 cancer cells. The unique spatio-temporal resolution of this technique reveals unprecedented information on the mode of action of doxorubicin: its localization in the nucleus, its complexation with medium components, and its intercalation with DNA as a function of time. Notably, we were able to discriminate these factors for the direct administration of doxorubicin or the use of a doxorubicin delivery system. The results reported here show that SERS endoscopy may have an important future role in medicinal chemistry for studying the dynamics and mechanism of action of drugs in cells.

Length-Controllable Gold-Coated Silver Nanowire Probes for High AFM-TERS Scattering Activity.

Tip-enhanced Raman scattering (TERS) microscopy is an advanced technique for investigation at the nanoscale that provides topographic and chemical information simultaneously. The TERS probe plays a crucial role in the microscopic performance. In the recent past, the development of silver nanowire (AgNW) based TERS probes solved the main tip fabrication issues, such as low mechanical strength and reproducibility. However, this fabrication method still suffers from low control of the protruded length of the AgNW. In this work, a simple water-air interface electrocutting method is proposed to achieve wide controllability of the length. This water cutting method was combined with a succedent Au coating on the AgNW surface, and the probe achieved an up to 100× higher enhancement factor (EF) and a 2× smaller spatial resolution compared to pristine AgNW. Thanks to this excellent EF, the water-cut Au-coated AgNW probes were found to possess high TERS activity even in the nongap mode, enabling broad applications.

Fluorescence Imaging of 3D Cell Models with Subcellular Resolution.

Over the past years, research has made impressive breakthroughs towards the development and implementation of 3D cell models for a wide range of applications, such as drug development and testing, organogenesis, cancer biology, and personalized medicine. Opposed to 2D cell monolayer culture systems, advanced 3D cell models better represent the in vivo physiology. However, for these models to deliver scientific insights, appropriate investigation techniques are required. Despite the potential of fluorescence microscopy to visualize these models with high spatial resolution, sample preparation and imaging assays are not straightforward. Here, we provide different protocols of sample preparation for fluorescence imaging, for both matrix-embedded and matrix-free models ( e.g ., organoids and spheroids, respectively). Additionally, we provide detailed guidelines for imaging 3D cell models via confocal multi-photon fluorescence microscopy. We show that using these protocols, images of 3D cell culture systems can be obtained with sub-cellular resolution. Graphical abstract.

All-Optical and One-Color Rewritable Chemical Patterning on Pristine Graphene under Water.

We report a facile all-optical method for spatially resolved and reversible chemical modification of a graphene monolayer. A tightly focused laser on graphene under water introduces an sp3-type chemical defect by photo-oxidation. The sp3-type defects can be reversibly restored to sp2 carbon centers by the same laser with higher intensity. The photoreduction occurs due to laser-induced local heating on the graphene. These optical methods combined with a laser direct writing technique allow photowriting and erasing of a well-defined chemical pattern on a graphene canvas with a spatial resolution of about 300 nm. The pattern is visualized by Raman mapping with the same excitation laser, enabling an optical read-out of the chemical information on the graphene. Here, we successfully demonstrate all-optical Write/Read-out/Erase of chemical functionalization patterns on graphene by simply adjusting the one-color laser intensity. The all-optical method enables flexible and efficient tailoring of physicochemical properties in nanoscale for future applications.

Gold-coated silver nanowires for long lifetime AFM-TERS probes.

Tip-enhanced Raman scattering (TERS) microscopy is an advanced technique for investigation at the nanoscale because of its excellent properties, such as its label-free functionality, non-invasiveness, and ability to simultaneously provide topographic and chemical information. The probe plays a crucial role in TERS technique performance. Widely used AFM-TERS probes fabricated with metal deposition suffer from relatively low reproductivity as well as limited mapping and storage lifetime. To solve the reproducibility issue, silver nanowire (AgNW)-based TERS probes were developed, which, thanks to the high homogeneity of the liquid-phase synthesis of AgNW, can achieve high TERS performance with excellent probe reproductivity, but still present short lifetime due to probe oxidation. In this work, a simple Au coating method is proposed to overcome the limited lifetime and improve the performance of the AgNW-based TERS probe. For the Au-coating, different [Au]/[Ag] molar ratios were investigated. The TERS performance was evaluated in terms of changes in the enhancement factor (EF) and signal-to-noise ratio through multiple mappings and the storage lifetime in air. The Au-coated AgNWs exhibited higher EF than pristine AgNWs and galvanically replaced AgNWs with no remarkable difference between the two molar ratios tested. However, for longer scanning time and multiple mappings, the probes obtained with low Au concentration showed much longer-term stability and maintained a high EF. Furthermore, the Au-coated AgNW probes were found to possess a longer storage lifetime in air, allowing for long and multiple TERS mappings with one single probe.

Versatile and Robust Method for Antibody Conjugation to Nanoparticles with High Targeting Efficiency.

The application of antibodies in nanomedicine is now standard practice in research since it represents an innovative approach to deliver chemotherapy agents selectively to tumors. The variety of targets or markers that are overexpressed in different types of cancers results in a high demand for antibody conjugated-nanoparticles, which are versatile and easily customizable. Considering up-scaling, the synthesis of antibody-conjugated nanoparticles should be simple and highly reproducible. Here, we developed a facile coating strategy to produce antibody-conjugated nanoparticles using 'click chemistry' and further evaluated their selectivity towards cancer cells expressing different markers. Our approach was consistently repeated for the conjugation of antibodies against CD44 and EGFR, which are prominent cancer cell markers. The functionalized particles presented excellent cell specificity towards CD44 and EGFR overexpressing cells, respectively. Our results indicated that the developed coating method is reproducible, versatile, and non-toxic, and can be used for particle functionalization with different antibodies. This grafting strategy can be applied to a wide range of nanoparticles and will contribute to the development of future targeted drug delivery systems.

Gold-Etched Silver Nanowire Endoscopy: Toward a Widely Accessible Platform for Surface-Enhanced Raman Scattering-Based Analysis in Living Cells.

Recently, our group introduced the use of silver nanowires (AgNWs) as novel non-invasive endoscopic probes for detecting intracellular Raman signals. This method, although innovative and promising, relies exclusively on the plasmonic waveguiding effect for signal enhancement. It, therefore, requires sophisticated operational tools and protocols, drastically limiting its applicability. Herein, an advanced strategy is offered to significantly enhance the performance of these endoscopic probes, making this approach widely accessible and versatile for cellular studies. By uniformly forming gold structures on the smooth AgNW surface via a galvanic replacement reaction, the density of the light coupling points along the whole probe surface is drastically increased, enabling high surface-enhanced Raman scattering (SERS) efficiency upon solely focusing the excitation light on the gold-etched AgNW. The applicability of these gold-etched AgNW probes for molecular sensing in cells is demonstrated by detecting site-specific and high-resolved SERS spectra of cell compartment-labeling dyes, namely, 4',6-diamidino-2-phenylindole in the nucleus and 3,3'-dioctadecyloxacarbocyanine on the membrane. The remarkable spectral sensitivity achieved provides essential structural information of the analytes, indicating the overall potential of the proposed approach for cellular studies of drug interactions with biomolecular items.

From 2D to 3D Cancer Cell Models-The Enigmas of Drug Delivery Research.

Over the past decades, research has made impressive breakthroughs towards drug delivery systems, resulting in a wide range of multifunctional engineered nanoparticles with biomedical applications such as cancer therapy. Despite these significant advances, well-designed nanoparticles rarely reach the clinical stage. Promising results obtained in standard 2D cell culture systems often turn into disappointing outcomes in in vivo models. Although the overall majority of in vitro nanoparticle research is still performed on 2D monolayer cultures, more and more researchers started acknowledging the importance of using 3D cell culture systems, as better models for mimicking the in vivo tumor physiology. In this review, we provide a comprehensive overview of the 3D cancer cell models currently available. We highlight their potential as a platform for drug delivery studies and pinpoint the challenges associated with their use. We discuss in which way each 3D model mimics the in vivo tumor physiology, how they can or have been used in nanomedicine research and to what extent the results obtained so far affect the progress of nanomedicine development. It is of note that the global scientific output associated with 3D models is limited, showing that the use of these systems in nanomedicine investigation is still highly challenging.

Correction: FRET-based intracellular investigation of nanoprodrugs toward highly efficient anticancer drug delivery.

Correction for 'FRET-based intracellular investigation of nanoprodrugs toward highly efficient anticancer drug delivery' by Farsai Taemaitree et al., Nanoscale, 2020, 12, 16710-16715, DOI: 10.1039/D0NR04910G.

FRET-based intracellular investigation of nanoprodrugs toward highly efficient anticancer drug delivery.

In order to overcome unpredictable side-effects and increased cytotoxicity of conventional carrier-based anticancer drug delivery systems, several systems that consist exclusively of the pure drug (or prodrug) have been proposed. The behavior and dynamics of these systems after entering cancer cells are, however, still unknown, hindering their progress towards in vivo and clinical applications. Here, we report a comprehensive in cellulo study of carrier-free SN-38 nanoprodrugs (NPDs), previously developed by our group. The work shows the intracellular uptake, localization, and degradation of the NPDs via FRET microscopy. Accordingly, new FRET-NPDs were chemically synthesized and characterized. Prodrug to drug conversion and therapeutic efficiency were also validated. Our work provides crucial information for the application of NPDs as drug delivery systems and demonstrates their outstanding potential as next-generation anticancer nanomedicines.

Two-Photon-Induced [2 + 2] Cycloaddition of Bis-thymines: A Biocompatible and Reversible Approach.

Despite having great value across a wide variety of scientific fields, two-photon polymerizations currently suffer from two significant problems: the need for photoinitiators, which generate toxic side products, and the irreversibility of the process. Hence, the design of a versatile approach that circumvents these issues represents a major scientific challenge. Herein, we report a two-photon absorption strategy where reversible [2 + 2] cycloaddition of bis-thymines was achieved without the need for any photoinitiator. The cycloaddition and cycloreversion reactions could be induced by simply changing the irradiation wavelength, and repeated writing and erasing cycles were performed. The simplicity, reversibility, and biocompatibility of this strategy open up a whole new toolbox for applications across a wide variety of scientific fields.

Controlled Fabrication of Optical Signal Input/Output Sites on Plasmonic Nanowires.

Silver nanowires have attracted considerable attention as subdiffraction limited diameter waveguides in a variety of applications including cell endoscopy and photonic integrated circuitry. Optical signal transport occurs by coupling light into propagating surface plasmons, which scatter back into light further along the wire. However, these interconversions only occur efficiently at wire ends, or at defects along the wire, which are not controlled during synthesis. Here, we overcome this limitation, demonstrating the visible laser light-induced fabrication of gold nanostructures at desired positions on silver nanowires, and their utility as efficient in/out coupling points for light. The gold nanostructures grow via plasmon-induced reduction of Au(III) and are shown to be excellent "hotspots" for surface-enhanced Raman scattering.

Polymeric Engineering of Nanoparticles for Highly Efficient Multifunctional Drug Delivery Systems.

Most targeting strategies of anticancer drug delivery systems (DDSs) rely on the surface functionalization of nanocarriers with specific ligands, which trigger the internalization in cancer cells via receptor-mediated endocytosis. The endocytosis implies the entrapment of DDSs in acidic vesicles (endosomes and lysosomes) and their eventual ejection by exocytosis. This process, intrinsic to eukaryotic cells, is one of the main drawbacks of DDSs because it reduces the drug bioavailability in the intracellular environment. The escape of DDSs from the acidic vesicles is, therefore, crucial to enhance the therapeutic performance at low drug dose. To this end, we developed a multifunctionalized DDS that combines high specificity towards cancer cells with endosomal escape capabilities. Doxorubicin-loaded mesoporous silica nanoparticles were functionalized with polyethylenimine, a polymer commonly used to induce endosomal rupture, and hyaluronic acid, which binds to CD44 receptors, overexpressed in cancer cells. We show irrefutable proof that the developed DDS can escape the endosomal pathway upon polymeric functionalization. Interestingly, the combination of the two polymers resulted in higher endosomal escape efficiency than the polyethylenimine coating alone. Hyaluronic acid additionally provides the system with cancer targeting capability and enzymatically controlled drug release. Thanks to this multifunctionality, the engineered DDS had cytotoxicity comparable to the pure drug whilst displaying high specificity towards cancer cells. The polymeric engineering here developed enhances the performance of DDS at low drug dose, holding great potential for anticancer therapeutic applications.

Tailored Ag-Cu-Mg multielemental nanoparticles for wide-spectrum antibacterial coating.

Bactericidal nanoparticle coatings are very promising for hindering the indirect transmission of pathogens through cross-contaminated surfaces. The challenge, limiting their employment in nosocomial environments, is the ability of tailoring the coating's physicochemical properties, namely, composition, cytotoxicity, bactericidal spectrum, adhesion to the substrate, and consequent nanoparticles release into the environment. We have engineered a new family of nanoparticle-based bactericidal coatings comprising Ag, Cu, and Mg and synthesized by a green gas-phase technique. These coatings present wide-spectrum bactericidal activity on both Gram-positive and Gram-negative reference strains and tunable physicochemical properties of relevance in view of their "on-field" deployment. The link between material and functional properties is rationalized based on a multidisciplinary and multitechnique approach. Our results pave the way for engineering biofunctional, fully tunable nanoparticle coatings, exploiting an arbitrarily wide number of elements in a straightforward, eco-friendly, high-throughput, one-step process.

Surface Density-of-States Engineering of Anatase TiO2 by Small Polyols for Enhanced Visible-Light Photocurrent Generation.

Enhancement of visible-light photocurrent generation by sol-gel anatase TiO2 films was achieved by binding small polyol molecules to the TiO2 surface. Binding ethylene glycol onto the surface, enhancement factors up to 2.8 were found in visible-light photocurrent generation experiments. Density functional theory calculations identified midgap energy states that emerge as a result of the binding of a range of polyols to the TiO2 surface. The presence and energy of the midgap state is predicted to depend sensitively on the structure of the polyol, correlating well with the photocurrent generation results. Together, these results suggest a new, facile, and cost-effective route to precise surface band gap engineering of TiO2 toward visible-light-induced photocatalysis and energy storage.