The assembly of plasmonic nanoparticles with precise spatial and orientational order may lead to structures with new electromagnetic properties at optical frequencies. The directed self-assembly method presented controls the interparticle-spacing and symmetry of the resulting nanometer-sized elements in solution. The self-assembly of three-dimensional (3D), icosahedral plasmonic nanosclusters (NCs) with resonances at visible wavelengths is demonstrated experimentally. The ideal NCs consist of twelve gold (Au) nanospheres (NSs) attached to thiol groups at predefined locations on the surface of a genetically engineered cowpea mosaic virus with icosahedral symmetry. In situ dynamic light scattering (DLS) measurements confirm the NSs assembly on the virus. Transmission electron micrographs (TEM) demonstrate the ability of the self-assembly method to control the nanoscopic symmetry of the bound NSs, which reflects the icosahedral symmetry of the virus. Both, TEM and DLS show that the NCs comprise of a distribution of capsids mostly covered (i.e., 6–12 NS/capsid) with NSs. 3D finite-element simulations of aqueous suspensions of NCs reproduce the experimentalbulkabsorbance measurements and major features of the spectra. Simulations results show that the fully assembled NCs give rise to a 10-fold surface-averaged enhancement of the local electromagnetic field.
Since more than 30 % of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, we used Ag nanoparticles (NPs) to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there was also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 had less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag+. In contrast to the more intense acute pulmonary effects of C20, C110 induced mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag+release leading to a sub-chronic injury response. Interestingly, the released metallic Ag gets incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.
Polyethylene glycol (PEG)ylated Raman-active gold nanoparticles (PEG-R-AuNPs) consist of an interchangeable Raman organic molecule layer held onto a gold nanocore by a silica shell. PEG-R-AuNPs have been shown preclinically to increase the sensitivity and specificity of Raman spectroscopy, with picomolar sensitivity and multiplexing capabilities. Although clinical trials are being designed to use functionalized PEG-R-AuNPs in various applications (e.g., to target dysplastic bowel lesions during colonoscopy), the effects of these nanoparticles on human cells remain unknown. The occurrence and mechanisms underlying any potential cytotoxicity induced by these nanoparticles (0–1000 PEG-R-AuNPs/cell) are investigated in immortalized human HeLa and HepG2 cell lines at several time points (0–48 h) after exposure. Using fluorometric assays, cell viability (MTT), reactive oxygen species (ROS) generation (dichlorofluoresceindiacetate), protein oxidation (protein carbonyl content), and total cellular antioxidant concentrations the concentrations (metmyoblobin-induced oxidation of ABTS) are assessed. Analysis of lipid oxidation using an enzyme immunoassay (8-isoprostane concentrations), gene expression of antioxidant enzymes using quantitative reverse transcription polymerase chain reactions, and the intracellular location of PEG-R-AuNPs using transmission electron microscopy is also undertaken. PEG-R-AuNPs cause no cytotoxicity in either HeLa or HepG2 cells in the acute setting as ROS generation is balanced by antioxidant enzyme upregulation. Following prolonged exposures (48 h) at relatively high concentrations (1000 PEG-R-AuNPs/cell), nanoparticles are found within vesicles inside cells. Under these conditions, a minimal amount of cytotoxicity is seen in both cell lines owing to increases in cellular oxidative stress, most likely due to ROS overwhelming the antioxidant defenses. Evidence of oxidative stress-induced damage includes increased lipid and protein oxidation. Although further in vivo toxicity studies are necessary, these initial encouraging results show that PEG-R-AuNPs cause minimal toxicity in human cells in the acute setting, which bodes well for potential future applications of these nanoparticles in living subjects.
Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Raman’s limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with64Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (μCi) (3.7 megabecquerel (MBq)) of64Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g?1) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g?1; 24 h = 8.27% ID g?1) than IR-injected mice (5 h = 0.09% ID g?1; 24 h = 0.08% ID g?1). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g?1; 24 h = 0.42% ID g?1) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.
Clinical impact of biotechnology has been constrained by the limitations oftraditional hypodermic injection of biopharmaceuticals. Microneedle patches have beenproposed as a minimally invasive alternative. In this study, we assess the translation ofa dissolving microneedle patch designed for simple, painless self-administration ofbiopharmacetucials that generates no sharp biohazardous waste. To study pharmacokineticsand safety of this approach, human growth hormone (hGH) was encapsulated in 600 μmlong dissolving microneedles composed of carboxymethylcellulose and trehalose using anaqueous, moderate-temperature process that maintained complete hGH activity afterencapsulation and retained most activity after storage for up to 15 months at roomtemperature and humidity. After manual insertion into the skin of hairless rats, hGHpharmacokinetics were similar to conventional subcutaneous injection. After patch removal,the microneedles had almost completely dissolved, leaving behind only blunt stubs. Thedissolving microneedle patch was well tolerated, causing only slight, transient erythema.This study suggests that a dissolving microneedle patch can deliver hGH and otherbiopharmaceuticals in a manner suitable for self-administration without sharp biohazardouswaste.
This paper advances the design of stimuli-responsive materials based on colloidal particles dispersed in liquid crystals (LCs). Specifically, we report that thin films of colloid-in-liquid crystal (CLC) gels can undergo easily visualized ordering transitions in response to reversible and irreversible (enzymatic) biomolecular interactions occurring at aqueous interfaces of the gels. In particular, we demonstrate that LC ordering transitions can propagate across the entire thickness of the gels. We observe, however, that confinement of the LC to small domains with lateral sizes of ~10 μm does change the nature of the anchoring transitions, as compared to films of pure LC, due to the effects of confinement on the elastic energy stored in the LC. The effects of confinement are also observed to cause the response of individual domains of the LC within the CLC gel to vary significantly from one another, indicating that manipulation of LC domain size and shape can provide the basis of a general and facile method to tune the response of these LC-basedphysical gels to interfacial phenomena. Overall, the results presented in this paper establish that CLC gels offer a promising approach to the preparation of self-supporting, LC-based stimuli-responsive materials.
Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid-based therapeutics. A facile surface-mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface-mediated delivery, and the control of surface topography. Uniform disc-like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ~ 818 nm and thickness of ~ 195 nm. The microwell array-mediated delivery of lipoplexes containing FAM-oligodeoxynucleotides is ~ 18.6 and ~ 10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non-small cell lung cancer cells) and a suspension cell line (KG-1a acute myelogenous leukemia cells), respectively. MicroRNA-29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA-29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine.
Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structuresin vivoand suitable local cellular microenvironments for functional tissue engineering.
Nucleic acid detection with label-free biosensors circumvents costly fluorophore functionalization steps associated with conventional assays by utilizing transducers of impressive ultimate detection limits. Despite this technological prowess, molecular recognition at a surface limits the biosensors’ sensitivity, specificity, and reusability. It is therefore imperative to integrate novel molecular approaches with existing label-free transducers to overcome those limitations. Here, we demonstrate this concept by integrating a DNA strand displacement circuit with a micron-scale whispering gallery mode (WGM) microsphere biosensor. The integrated biosensor exhibits at least 25-fold improved nucleic acid sensitivity, and sets a new record for label-free microcavity biosensors by detecting 80 pM (32 fmol) of a 22nt oligomer; this improvement results from the catalytic behavior of the circuit. Furthermore, the integrated sensor exhibits extremely high specificity; single nucleotide variants yield 40- to 100-fold lower signal. Finally, the same physical sensor was demonstrated to alternatingly detect 2 different nucleic acid sequences through 5 cycles of detection, showcasing both its reusability and its versatility.
Engineered nanomaterials (ENMs) continue to attract significant attentions because they have novel physicochemical properties that can improve the functions of products that will benefit human lives. However, the physicochemical properties that make ENMs attractive could interact with biological systems and induce cascades of events that cause toxicological effects. Recently, there are more studies suggesting inflammasome activation may play an important role in ENMs-induced biological responses. Inflammasomes are a family of multi-protein complexes and are increasingly recognized as major mediators of host immune system. Among these, NLRP3 inflammasome is the most studied one that could directly interact with ENMs to generate inflammatory responses. In this review, we aim to link the ENM physicochemical properties to NLRP3 inflammasome activation. The understanding of the mechanisms of ENMs-NLRP3 inflammasome interaction will provide us strategies for safer nanomaterial design and therapy.
To assure a responsible and sustainable growth of nanotechnology, the environmental health and safety (EHS) aspect of engineered nanomaterials and nano-related products needs to be addressed at a rate commensurate with the expansion of nanotechnology. Zebrafish has been demonstrated as a correlativein vivovertebrate model for such task, and the current advances of using zebrafish for nano EHS studies are summarized here. In addition to morphological and histopathological observations, the accessibility of gene manipulation would greatly empower such a model for detailed mechanistic studies of any nanoparticles of interest. The potential for establishing high-throughput screening platforms to facilitate the nano EHS studies is highlighted, and a discussion is presented on how toxicogenomics approaches represent a future direction to guide the identification of toxicity pathways.
Inorganic nanostructures have been used extensively to package nucleic acids into forms useful for therapeutic applications. Here we report that the two products of transcription, RNA and inorganic pyrophosphate, can self-assemble to form composite microsponge structures composed of nanocrystalline magnesium pyrophosphate sheets (Mg2P2O7·3.5H2O) with RNA adsorbed to their surfaces. The microsponge particles contain high loadings of RNA (15–21 wt.%) that are protected from degradation and can be obtained through a rolling circle mechanism as large concatemers capable of mediating RNAi. The morphology of the RNAi microsponges is influenced by the time-course of the transcription reaction and interactions between RNA and the inorganic phase. Previous work demonstrated that polycations can be used to condense RNAi microsponges into nanoparticles capable of efficient transfection with low toxicity. Our new findings suggest that the formation of these nanoparticles is mediated by the gradual dissolution of magnesium pyrophosphate that occurs in the presence of polycations. The simple one-pot approach for assembling RNAi microsponges along with their unique properties could make them useful for RNA-based therapeutics.
Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2O2is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2O2alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.