Nanoscale Advances

Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structure of these formulations is still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM) and the determination of LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well characterized pegylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size to Doxil, however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction implies that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilayers. Negative staining TEM shows 2-5 nm electron-dense spots in the liposoms interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply crosslink-stabilized supercoils. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the alternative possibility of hydrogen bonding between the mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of ionizable lipid in Comirnaty, ALC-0315, displaying free =O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These newly recognized structural features of mRNA-LNP may be important for the vaccines efficacy.

T-cell-dependent immunomodulation of carbohydrate antigens under benign conditions is the most promising approach for carbohydrate-based vaccine development. However, to achieve such adaptive immune responses, well-defined multifunctional nanocarriers loaded with immunogenic materials must be explored. Current efforts to use gold nanoparticles (AuNPs) as antigen carriers in vaccine development have conveniently introduced considerable diversity. Here, we show that the shape of AuNPs markedly influences carbohydrate-based antigen processing in murine dendritic cells (mDCs) and subsequent T-cell activation. In the study, CpG-adjuvant coated sphere-, rod-, and star-shaped AuNPs were conjugated to the tripodal Tn-glycopeptide antigen to study their DC uptake and the activation of T-cells in the DCs/T-cell co-culture assay. Our results showed that sphere- and star-shaped AuNPs displayed relatively weak receptor-mediated uptake but induced a high level of T helper-1 (Th1) biasing immune responses compared with rod-shaped AuNPs, showing that receptor-mediated uptake and cytokine secretion of nanostructures are two independent mechanisms. Significantly, the shapes of AuNPs and antigen/adjuvant conjugation synergistically work together to modulate the effective anti-Tn-glycopeptide immunoglobulin (IgG) antibody response after in vivo administration of the AuNPs. These results show that by varying the shape parameter, one can alter the immunomodulation, leading to the development of carbohydrate vaccines.

Acute respiratory distress syndrome (ARDS) involves death of lung epithelial cells. ARDS is a leading reason behind mortality in respiratory infections. Here we show a proof-of-concept that a Fullerene nanoformulation can be used for the regeneration of cells treated with apoptosis-inducing molecules, suggeting its potential for ARDS therapy.

Stealth properties of nanoparticles are essential for their proper functionalization in biological systems. To address limitations of polyethylene glycol (PEG), commonly used for this purpose, we explore the potential of polysarcosine (PSar) as stealth shell in peptide-functionalized dye-loaded polymeric NPs. To this end, polymeric NPs loaded with rhodamine dye with bulky hydrophobic counterion and bearing azide groups at their surface were grafted with PSar of different lengths ranging from 5 to 19 sarcosine units using strain-promoted cycloaddition. The obtained peptide-functionalized NPs showed remarkable colloidal stability in physiological media. The length of PSar showed a profound effect on stealth properties of NPs. The increase in the length of grafted PSar lead to decrease in the negative surface charge to nearly neutral values and decreased protein adsorption according to fluorescence correlation spectroscopy. The NPs with 19mer PSar showed minimal interactions with live cells and glass surfaces in a complex biological medium, in contrast to its shorter PSar analogues. These stealth NPs bearing HaloTag ligand enabled specific targeting of proteins at the cell surface. The obtained results show that a relatively short PSar peptide can be used for achieving stealth properties in polymeric NPs, allowing specific protein targeting with minimized non-specific interactions. The obtained PSar-functionalized polymeric NPs appear as a powerful platform for the fabrication of the next generation of nanomaterials for bioimaging and biosensing applications.

Alz-5 acts as a bifunctional chelator that can interact with various A{beta} aggregates and reduce their neurotoxicity. Single-cell ROS measurements provided by Pt-nanoelectrode technique revealed a significant antioxidant properties of Alz-5 in A{beta}42 - affected SH-SY5Y cell. AFM data obtained on A{beta}42 fibrils clearly indicate an anti-aggregating property of Alz-5. Youngs modulus mapping on living SH-SY5Y cells revealed an ability of Alz-5 to decrease cell rigidity in A{beta}42 - affected SH-SY5Y cells.

The impact on human health of the increasing use of silver nanoparticles (AgNPs) in medical devices remains understudied, even though AgNP-containing dressings are known to release silver in the bloodstream leading to accumulation and slow clearance in the liver. Cellular studies have shown the intracellular dissolution of AgNPs within endo-lysosomes followed by Ag(I) binding to biomolecular thiolate-containing molecules. However, the precise subcellular distribution of Ag(I) and the nature of the disrupted physiological pathways remained unknown. Novel imaging approaches enabled us to visualize the trafficking of AgNP-containing lysosomes towards a perinuclear location and a direct nuclear transfer of Ag(I) species with accumulation in the nucleoli. These Ag(I) species impaired nuclear receptor activity, disrupting critical mechanisms of liver physiology in very low dose exposure scenarios, thus justifying further research into defining a framework for the safe use of AgNPs.

The plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell lines including neurons (primary culture and tissue imaging). Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Multiple Sclerosis (MS) is a chronic, inflammatory disease of the central nervous system. Despite the pharmacological arsenal approved for MS, there are treatment-reluctant patients for whom cell therapy appears as the only therapeutic alternative. Myeloid-derived suppressor cells (MDSCs) are immature cells of the innate immune response able to immunosuppress T lymphocytes and to promote oligodendroglial differentiation in experimental autoimmune encephalomyelitis (EAE), a preclinical model for MS. Culture devices need to be designed so that MDSCs maintain a state of immaturity and immunosuppressive function similar to that exerted in the donor organism. Graphene oxide (GO) has been described as a biocompatible material with the capacity to biologically modulate different cell types, including immune cells. In the present work, we show how MDSCs isolated from immune organs of EAE mice maintain an immature phenotype and highly immunosuppressive activity on T lymphocytes after being cultured on 2D reduced GO films (rGO200) compared to those grown on glass. This activity is depleted when MDSCs are exposed to slightly rougher and more oxidized GO substrates (rGO90). The greater reduction in cell size of cells exposed to rGO90 compared to rGO200 is associated with the activation of apoptosis processes. Taken together, the exposure of MDSCs to GO substrates with different redox state and roughness appears as a good strategy to control MDSC activity in vitro. This versatility of GO nanomaterials and the impact of their physico-chemical properties in immunomodulation open the door to its possible selective therapeutic use for pathologies where MDSCs need to be enhanced or inhibited.

Localized hyperthermia is a promising approach to cancer therapy. However, its clinical potential is limited by heterogeneous heat distribution within tumors, and advanced methods to measure temperature at the sub-micron level are therefore required. To address this challenge, luminescent nanothermometers, such as lanthanide-doped nanocrystals (Ln-NC) operating in the near-infrared (NIR), have been investigated for accurate spatiotemporal thermal monitoring. In this study, the synthesis of neodymium-doped, sodium yttrium fluoride nanocrystals (Nd-NCs) was optimized to achieve high photoluminescence (PL) intensity by adjusting the dopant concentration and by shelling with inert layers. Standard curves for luminescence-based temperature readout were developed using ratiometric analysis of the temperature-dependent PL spectra in the 850-920 nm biological window, showing excellent linearity and high thermal sensitivity. A silica shell was added to the particles and shown to confer excellent aqueous stability and biocompatibility in A549 lung cancer cells. Finally, luminescent thermal readout was demonstrated in vitro in A549 cells by spectrally resolving the diffraction-limited luminescence spots at a single-particle scale over a clinically relevant temperature range from 20-50 {degrees}C. The application of the developed nanothermometer as preclinical tools for NP-HT characterization could provide crucial information on the therapeutic temperature achieved in and around the tumor area. This could be key to optimizing NP properties and therapeutic parameters, for the development of viable hyperthermal cancer treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/579538v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b06abdorg.highwire.dtl.DTLVardef@12fa23corg.highwire.dtl.DTLVardef@4439c1org.highwire.dtl.DTLVardef@12bcedd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM) as a non-Abbe limited technique gives access to the cellular ultra-structure yet results in grey-scale images and averts unambiguous (co-)localization of biomolecules. Multimodal nanoparticle-based immunolabels for correlative cathodoluminescence electron microscopy (CCLEM) and energy-dispersive X-ray spectromicroscopy (EDX-SM) are presented. The single-particle STEM-cathodoluminescence (CL) and characteristic X-ray emissivity of sub-20 nm lanthanide-doped nanoparticles were exploited as unique spectral fingerprints for precise localization and label identification. To maximize the nanoparticle brightness, lanthanides were incorporated in a low-phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles were then functionalized with either folic (terbium-doped) or caffeic acid (europium-doped). Their potential for immunolabeling was successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations showed single-particle CL emission along with a distinctive energy-dispersive X-ray signal, with the latter enabling colour-based localization of receptors within swift imaging times well below 2 mins per {micro}m2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to color immunolabelling based on electron spectromicroscopy. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/585848v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1f7c2d3org.highwire.dtl.DTLVardef@117be1eorg.highwire.dtl.DTLVardef@1c2bab2org.highwire.dtl.DTLVardef@16f3677_HPS_FORMAT_FIGEXP M_FIG Small (sub-20 nm) lanthanide-doped nanoparticles were successfully utilized in electron microscopy to label biological structures and contextualize them in the cells ultrastructure. Leveraging unique energy-dispersive X-ray signatures, the nanoparticles location and doping-identity was easily and fast retrieved, demonstrating the methods potential to (co)-localize labels while supplying a holistic impression of the underlying processes, as entire cells could be mapped. C_FIG

1Self-assembled monolayers (SAMs) have been widely utilized as a way of tailoring surface chemistry through the adsorption of organic molecules to different materials. SAMs are easy to prepare and offer a wide variety of organic molecules that afford additional or improved properties to the coated material. Spatial control of SAM placement has been achieved over many length-scales, even at the nanoscale. However, nanopatterned SAMs are usually prepared through serial processes utilizing atomic scanning probes or soft-lithography utilizing elastomeric masters. These techniques are expensive or not repeatable. Here we present the use of nanospheres for the creation of nanopatterned Au:Cu films which spatially control the grafting of a thermoresponsive SAM made from poly(N-isopropyl acrylamide) (PNIPAM). Chemical characterization validates the presence of PNIPAM and environmental atomic force microscopy showed its response to temperature which was evidenced by a change in stiffness. Our approach represents an affordable large area methodology for repeatable spatial control of SAMs at the nanoscale.

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, either directly in the case of lung exposure, or after the particles have crossed the epithelial barriers in case of oral or dermal exposure. It is thus important to study the potential effects of micro and nanoplastics on macrophages. Most studies have used an experimental scheme in which the cells of interest are exposed to a single dose of plastics, and where the readout of the studied parameters is made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. true-to-life polyethylene terephthalate nanoparticles and to a biodegradable nanoplastic, i.e. polycaprolactone, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser extent than under the acute exposure mode, indicating cell adaptation. However, polyethylene terephthalate nanoparticles induced oxidative stress and a pro-inflammatory response, while polycaprolactone nanoparticles induced a depression of macrophages functions, indicating harmful effects even in the repeated exposure scheme.

Therapeutic delivery of anti-cancer drugs is a major goal of modern medicine. In particular, microrobots (MRs) have recently been studied for their ability to navigate difficult-to-reach regions in the human body to deliver therapeutics for microscopically localized interventions. However, the control of individual and swarms of MRs to precisely target localized cellular regions remains a significant challenge, preventing their applications as delivery systems in cancer research. In this study, magnetic MRs were used to target cancer cells and create localized magnetic oscillations which resulted in magnetolysis of cancer cells. The magnetic MRs were selectively steered towards Hepatocarcinoma cells (HepG2 cells) using our custom-built magnetic controller under a rotating magnetic field at different frequencies. After internalization of the microrobots by cancer cells, magnetic oscillation of varying dosages was applied to disrupt the internal structure of cancer cells which leads to subsequent cell death.

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Spectral fingerprinting has emerged as a powerful tool, adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Through the use of Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages as compared to M2 and naive phenotypes. NIR fluorescence data also indicate that distinctive intra-endosomal environments of these cell types give rise to significant differences in many optical features such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of > 95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications such as cell phenotyping or mapping intra-endosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT6 give rise to more improved model accuracy (> 87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/583608v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11e2c60org.highwire.dtl.DTLVardef@1f3553forg.highwire.dtl.DTLVardef@61dcb9org.highwire.dtl.DTLVardef@e216b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oncotheranostic nanoagents are powerful artificial tools that significantly outperform traditional antitumor therapies. Among the wide variety of nanoagents, polymer nanoparticles have proven to be one of the most successful candidates for translation into clinical practice. Here we developed an oncotheranostic platform that is based on poly(lactic-co-glycolic acid) nanoparticles and possesses red/green light dual-activated diagnostic/therapeutic properties for targeted photodynamic therapy of HER2-positive aggressive breast cancer. PLGA nanoparticles were loaded with a red light-activated dye Nile Blue with pronounced solvatochromic properties for diagnostic applications and with a green light-activated photodynamic sensitizer Rose Bengal for photodynamic therapy. Targeted delivery was ensured by the non-covalent decoration of nanoparticles with anti-HER2 antibodies, which can be readily adapted for large-scale biotechnological production. In vitro and in vivo studies proved the effectiveness of red light-mediated HER2-specific imaging and green light-induced cytotoxicity of anti-HER2 PLGA. Interestingly, these particles fluoresced only after cellular internalization, which minimizes background signals inside the organism, thus facilitating real-time diagnostics. The particles allowed efficient and selective visualization of the HER2-positive primary tumor node and metastases spread and led to complete remission in BALB/c Nu/Nu mice with the HER2-positive xenografts after a single session of photodynamic therapy.

Ovarian cancer immunotherapy remains a challenge based on the "cold" tumor microenvironment. Herein we present a rational design to create immunogenic nanoparticles as a multi-agent platform that promotes immune response in a mouse model of ovarian cancer. The hybrid lipid-silica nanosystem is capable of co-loading four types of cargo molecules including a model antigen, nucleic acid-based adjuvant Cytosine-p-linked to Guanine (CpG, TLR3/9 agonist), lipid-based adjuvant (MPLA, TLR4 agonist) integrated into the lipid coat, and optionally a small molecule drug, such as the chemotherapeutic agent oxaliplatin, a well-established treatment for ovarian cancer. The optimization of the nanoplatform in terms of lipid composition, functionalized silica dendritic core formation, and final charge, as well as their compatibility with the complex loading profile highlights an opportunity for enhanced survival of mice with advanced ovarian cancer compared to monotherapy. Furthermore, intraperitoneal administration led to preferential accumulation within tumor-burdened tissues with selective accumulation in myeloid cells. High myeloid cell cytotoxicity negated the benefits of oxaliplatin. The inclusion of CpG in the nanoparticle formulation enhanced the survival of mice with ovarian cancer. To interpret these outcomes and guide future design, we also developed a mathematical model of nanoparticle-driven immune activation, which quantified treatment efficacy and identified key parameters governing tumor response. The presented hybrid nanoparticle i tunable, enabling delivery of alternative molecules therefore, thereby highlighting a promising platform for the treatment of peritoneal cancers. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/657862v1_ufig1.gif" ALT="Figure 1000"> View larger version (26K): org.highwire.dtl.DTLVardef@1434599org.highwire.dtl.DTLVardef@18e79e6org.highwire.dtl.DTLVardef@e5285corg.highwire.dtl.DTLVardef@bcc604_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite advances in diagnosis and management, cardiovascular diseases (CVDs) remain the leading cause of death in the US. Atherosclerosis is the most common form of CVD and the vulnerability of atherosclerotic plaques to rupture is a primary determinant for risk of catastrophic ischemic events. Current imaging of atherosclerotic disease focuses on assessing plaque size and the degree of luminal stenosis, which are not good predictors of plaque stability. Functional methods to identify biomarkers of inflammation in plaques could facilitate assessment of plaque instability to allow early intervention. In this study, we validate the use of a purpose-built, magnetic resonance imaging (MRI)-compatible positron emission tomography (PET) insert for multimodal, molecular imaging of vulnerable plaques in mice. We illustrate the application of PET to screen for inflamed regions to guide the application of MRI. Molecular MRI visualizes regions of vascular inflammation and is coupled with anatomical MRI to generate detailed maps of the inflammatory marker within the context of an individual vessel. As a testbed for this imaging methodology, we developed a multimodal, iron oxide nanoparticle (NP) targeting vascular cell adhesion molecule-1 (VCAM-1) for simultaneous PET/MRI of vascular inflammation performed on a mouse carotid ligation model. In vitro cell studies confirmed that the NPs are not cytotoxic to liver cells. In vivo simultaneous PET/MRI imaging identified regions of inflammation. Three-dimensional rendering of the MRI data facilitated high-resolution visualization of patterns of inflammation along the injured vessel. Histology validated the co-localization of the NPs with VCAM-1 expression at sites of induced inflammation. The results of this work validate the utility of the simultaneous PET/MR insert as a research tool for small animals and lays groundwork to further advance the potential clinical utility of integrated imaging systems.

AO_SCPLOWBSTRACTC_SCPLOWWe have developed Fullerene-C60 nanoformulations containing discrete sized nanoparticles by dispersing concentration range of Fullerene. Small sized particles are cytotoxic while larger ones are cell proliferative. The cell proliferative property is used for tissue repair in cellular and animal wound models.

Withdrawn reasonThe author has withdrawn the manuscript because additional results with complementary techniques are expected to complement the already gathered data and thus, having a more complete overview of the impact of several conditions of interest on the S100A9 fibrillation processes. Therefore, the author do not wish this work to be cited as reference for the project. If you have any questions, please contact with the author.