Fluorescent Polystyrene Nanospheres Research Articles

Facile Synthesis and Characterization of Fluorescent Polystyrene Nanospheres for Homogeneous Light-Initiated Chemiluminescence Immunoassay.

Homogeneous light-initiated chemiluminescence technology (LICA) is widely used in clinical diagnostics due to the advantages of high sensitivity, minimal reagent usage, and no need for washing. Luminescent microspheres receive singlet oxygen emitted by photosensitive microspheres to generate optical signals. Therefore,1O2-initiated luminescent nanospheres are crucial, but there are few reports on the preparation of 1O2-initiated luminescent nanospheres. Herein, monodisperse luminescent Eu/C-28@PS (Eps) nanospheres were prepared and optimized using chelate Eu (TTA)3phen and 4-(2-phenyl-5,6-dihydro1,4-oxathiin-3-yl)-N, N-ditetradecylbenzenamine (C-28) as probe dye via THF/water swelling-shrinking procedure. Various swelling parameters were studied to obtain the swelling conditions that produce the minimum particle size and narrow size distribution, which shows good results in uniform particle size distribution (~ 250nm, a PDI of 0.03), surface carboxylate content (1.18mmol/g), and BSA loading capability (129.8mg/g) in the case of 20mg total probe dosage and 2h of incubation at 40°C using 14% THF/water mixture as a co-solvent system. The composition of the entrapped probe has a gain effect on the 1O2-initiated fluorescent signal and the optimal ratio of Eu (TTA)3phen: C-28 (1:1) was obtained on a commercial analyzer using IgG and anti-human IgG as models in PBS buffer. These results indicate that monodisperse luminescent Eps nanospheres are suitable as light-initiated chemiluminescence sensors and have great application potential in early detection, screening tests, and prognostic evaluation of patients.

Primary and Secondary Plastic Particles Exhibit Limited Acute Toxicity but Chronic Effects on Daphnia magna.

Nanoplastics (NPs; <0.1 μm) are speculated to be a bigger ecological threat due to their predicted wider distribution, higher concentrations, and bioavailability. Primary NPs are manufactured to be that size, while secondary NPs originate from fragmentation of bigger debris. To date, the long-term impact of NPs in freshwater systems, particularly secondary NPs, is not well-understood. Thus, we employed a freshwater invertebrate, Daphnia magna, to investigate the chronic effects of model primary NPs, fluorescent polystyrene nanospheres (PS-NPs; 20 nm), and water leachate of weathered single-use plastics that contained micro- and nanosized particles. In experiment 1, parent Daphnia (F0) were exposed to 1 and 50 mg/L PS-NPs until the production of the neonates (F1) followed by a two-generation recovery. PS-NPs were mainly detected in the intestine and brood chamber in F0 and transferred to F1 and F2. PS-NPs significantly decreased the appendage curling and heartbeat rate in F0 and reduced reproduction in F2. In experiment 2, the plastic leachate also reduced the appendage curling rate but increased growth and reproduction. The results suggest that the acute toxicity of primary and secondary plastic particles is low even at high concentrations, but their chronic and sublethal effects should not be overlooked.

Essence of affinity and specificity of peanut agglutinin-immobilized fluorescent nanospheres with surface poly(N-vinylacetamide) chains for colorectal cancer

We have designed a novel colonoscopic imaging agent that is composed of submicron-sized fluorescent polystyrene nanospheres with two functional groups – peanut agglutinin (PNA) and poly(N-vinylaceamide) (PNVA) – on their surfaces. PNA is a targeting moiety that binds to β- d-galactosyl-(1-3)-N-acetyl- d-galactosamine (Gal-β(1-3)GalNAc), which is the terminal sugar of the Thomsen–Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells; it is anchored on the nanosphere surface via a poly(methacrylic) acid (PMAA) linker. PNVA is immobilized to enhance the specificity of PNA by reducing nonspecific interactions between the imaging agent and normal tissues. The essential nature of both functional groups was evaluated through in vivo experiments using PNA-free and PNVA-free nanospheres. The imaging agent recognized specifically tumors on the cecal mucosa of immune-deficient mice in which human colorectal cancer cells had been implanted; however, the recognition capability disappeared when PNA was replaced with wheat germ agglutinin, which has no affinity for Gal-β(1-3)GalNAc. PNA-free nanospheres with exclusively surface PNVA chains rarely adhered to the cecal mucosa of normal mice that did not undergo the cancer cell implantation. In contrast, there were strong nonspecific interactions between normal tissues and PNA-free nanospheres with exclusively surface PMAA chains. In vivo data proved that PNA and PNVA were essential for biorecognition for tumor tissues and a reduction of nonspecific interactions with normal tissues, respectively.

Water/Ionic Liquid Interfaces as Fluid Scaffolds for the Two-Dimensional Self-Assembly of Charged Nanospheres

Liquid-liquid interfaces formed between water and ionic liquids serve as fluid scaffolds to self-assemble anionic nanospheres two-dimensionally. When aqueous dispersions of anionic fluorescent polystyrene nanospheres (diameter ∼500 nm) are layered on ionic liquids, ordered monolayers are spontaneously formed at the interface. Fluorescent nanospheres are hexagonally packed in the interfacial monolayers, as observed by confocal laser scanning microscopy (CLSM). The adsorption and alignment of nanospheres at the interface are affected by the ionic strength and pH of the aqueous phase, indicating electrostatic interaction as the primary driving force for the self-assembly. CLSM observation of the water/ionic liquid interface reveals that the lower hemisphere of nanospheres is exposed to the ionic liquid phase, which effectively alleviates lateral electrostatic repulsion between charged nanospheres and promotes their close packing. The densely packed monolayer structure of nanospheres is stably immobilized on the surface of CLSM glass dishes simply by rinsing the ionic liquid layer with pure water, probably as a consequence of the gluing effect exerted by imidazolium cations. The fluidic nature of the water/ionic liquid interface facilitates the diffusion and ordering of nanospheres into a hexagonal lattice, and these features render the interface promising soft scaffolds to self-assemble anionic nanomaterials two-dimensionally.

Molecular recognition of DNA–protein complexes: A straightforward method combining scanning force and fluorescence microscopy

Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein–DNA complexes.

Abstract no.: 3 Vitreous– a barrier to non‐viral ocular gene therapy

Purpose Intravitreal injection of therapeutic DNA, complexed to non‐viral carriers like cationic liposomes, may be promising to treat many severe retinal eye diseases. However, after intravitreal injection such DNA/cationic liposome complexes, so called lipoplexes, which are typically some hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was both to elucidate whether vitreous is a barrier for the lipoplexes as well as to evaluate strategies to overcome this barrier.Methods Fluorescent polystyrene nanospheres and lipoplexes were mixed with vitreous and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy based technique. The stability of lipoplexes and naked pDNA in vitreous was studied by gel electrophoresis.Results We showed that polystyrene nanospheres, in our first experiments used as a model for the lipoplexes, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surface of the polystyrene nanospheres hydrophillic by attaching hydrophillic polyethyleneglycol (PEG) chains at their surface circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that ‘pegylated’ polystyrene nanospheres, as long as they are smaller than 500 nm in size, are indeed mobile in the vitreous. It was further demonstrated that lipoplexes severely aggregate in vitreous and strongly bind to biopolymers in the vitreous which immobilizes them completely. However, as observed for the polystyrene nanospheres, coating of the lipoplexes with PEG avoided both their aggregation in the vitreous as well as their binding to fibrillar structures.Conclusions Modifying the surface of lipoplexes with hydrophylic PEG chains prevents them from aggregation in vitreous. In this way lipoplexes are obtained which can freely move in vitreous, an absolute request to be able to reach the retina after intravitreal injection.

Method for trapping and manipulating nanoscale objects in solution

We present a device that allows a user to trap a single nanoscale object in solution at ambient temperature, and then to position the trapped object with nanoscale resolution. This anti-Brownian electrophoretic trap (ABEL trap) works by monitoring the Brownian motion of the particle (via fluorescence microscopy), and then applying a feedback voltage to the solution so that the electrophoretic drift exactly cancels the Brownian motion. The ABEL trap works on any object that can be imaged optically and that acquires a charge in water. The ABEL trap is noninvasive, is gentle enough to handle biological molecules, and can trap objects far smaller than can be trapped with laser tweezers. Our proof-of-principle device can trap fluorescent polystyrene nanospheres with diameters down to 20nm.

Nanoparticle-based in vivo investigation on blood-brain barrier permeability following ischemia and reperfusion.

The blood-brain barrier (BBB) represents a significant impediment to a large variety of central nervous system-active agents. In the current study, we applied fluorescent polystyrene nanospheres (20 nm) to study the BBB permeability following cerebral ischemia and reperfusion. A microdialysis probe was implanted in the cerebral cortex of an anesthetized rat injected with fluorescent polystyrene nanospheres. The circulating nanospheres extravasating to the brain extracellular fluids were collected by the probe. Fluorescence intensity in the microdialysates throughout the course of cerebral ischemia/reperfusion was measured. Cerebral ischemia and reperfusion induced transient accumulations of extracellular nanospheres in the brain. The accumulation of nanospheres may result from their extravasation from the blood vessels. The concurrent cerebral oxygen levels monitored using oxygen-dependent quenching of phosphorescence decreased following ischemia and returned to their original levels after reperfusion. In conclusion, we demonstrated that high temporal resolution measurements of BBB permeability in vivo can be obtained using fluorescence polystyrene nanospheres and that these data correlate with changes of cerebral oxygen concentration. This present investigation indicates that nanoparticles have potential clinical applications involving drug delivery and determination of therapeutic efficacy and on-site diagnosis.

Microfabricated silicon microneedles for nonviral cutaneous gene delivery.

The skin represents an accessible somatic tissue for therapeutic gene transfer. The superficial lipophilic layer of the skin, the stratum corneum, however, constitutes a major obstacle to the cutaneous delivery of charged macromolecules such as DNA. To determine whether silicon-based microneedles, microfabricated via a novel isotropic etching/BOSCH reaction process, could generate microchannels in the skin of sufficient dimensions to facilitate access of lipid : polycation : pDNA (LPD) nonviral gene therapy vectors. Scanning electron microscopy was used to visualize the microconduits created in heat-separated human epidermal sheets after application of the microneedles. Following confirmation of particle size and particle surface charge by photon correlation spectrocopy and microelectrophoresis, respectively, the diffusion of fluorescent polystyrene nanospheres and LPD complexes through heat-separated human epidermal sheets was determined in vitro using a Franz-type diffusion cell. In-vitro cell culture with quantification by flow cytometry was used to determine gene expression in human keratinocytes (HaCaT cells). The diffusion of 100 nm diameter fluorescent polystyrene nanospheres, used as a readily quantifiable predictive model for LPD complexes, through epidermal sheets was significantly enhanced following membrane treatment with microneedles. The delivery of LPD complexes either into or through the membrane microchannels was also demonstrated. In both cases considerable interaction between the particles and the epidermal sheet was observed. In-vitro cell culture was used to confirm that LPD complexes mediated efficient reporter gene expression in human keratinocytes in culture when formulated at the appropriate surface charge. These studies demonstrate the utility of silicon microneedles in cutaneous gene delivery. Further studies are required to elucidate fully the influence of the physicochemical characteristics of gene therapy vectors, e.g. particle diameter and surface charge, on their diffusion through microchannels and to quantify gene expression in vivo.

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules.

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.

Photon-burst analysis in two-photon fluorescence excitation flow cytometry.

We studied the use of a dramatically reduced testing zone in combination with two-photon excitation and photon-burst analysis in high-throughput rare-event detection simulation using a modified flow cytometer. Two-photon excitation measurements were performed with a mode-locked titanium:sapphire laser. Fluorescence emission was measured with a photon-counting avalanche photodiode. Measured signal was analysed offline by autocorrelation and burst detection methods. Test samples were composed of full blood and orange fluorescent polystyrene nanospheres mixed in full blood. Results show that two-photon fluorescence excitation and time-correlation analysis provide a good signal-to-noise ratio for rare-event particle detection in a turbid sample environment.

Further histological evidence of the gastrointestinal absorption of polystyrene nanospheres in the rat

Further histological evidence of the oral absorption of fluorescent polystyrene nanospheres is presented. Fluorescent polystyrene microspheres of diameter ranging from 50 nm to 3 μm were administered daily for 10 days by oral gavage (2.5% w/v; 12.5 mg kg −1) to female Sprague Dawley rats. All except the 3 μm non-ionic fluorescent polystyrene nanospheres and microspheres were concentrated in the serosal layer of Peyer's patches and could be seen thereafter traversing the mesentery lymph vessels. The migrating particles were subsequently found in the lymph nodes and liver tissues. No particles were found in the lung or heart. We have previously shown that uptake and translocation is size dependent, increasing with decreasing size. Special emphasis is placed on the fate of the smallest particles investigated — 50 nm non-ionic polystyrene microspheres — which were seen in kidney and were also present in the villi and GI crypts.