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In-depth understanding of the biophysicochemical interactions at the nano-bio interface is important for basic cell biology and applications in nanomedicine and nanobiosensors. Here, the extracellular surface potential and topography changes of live cell membranes interacting with polymeric nanomaterials using a scanning ion conductance microscopy-based potential imaging technique are investigated. Two structurally similar amphiphilic conjugated polymer nanoparticles (CPNs) containing different functional groups (i.e., primary amine versus guanidine) are used to study incubation time and functional group-dependent extracellular surface potential and topographic changes. Transmembrane pores, which induce significant changes in potential, only appear transiently in the live cell membranes during the initial interactions. The cells are able to self-repair the damaged membrane and become resilient to prolonged CPN exposure. This study provides an important observation on how the cells interact with and respond to extracellular polymeric nanomaterials at the early stage. This study also demonstrates that extracellular surface potential imaging can provide a new insight to help understand the complicated interactions at the nano-bio interface and the following cellular responses.

Core-shell conjugated polymer nanoparticles (CPNs) were fabricated by complexing a semi-flexible, primary amine-containing conjugated polymer (CP) with hyaluronic acid (HA). Flexibility introduced in the rigid rod conjugated backbone allows backbone reorganization to increase π-π interaction under ionic complexation, resulting in core-shell nanoparticles with a hydrophobic CP core wrapped with a HA shell. The core-shell nanoparticles exhibited no cellular toxicity and high cancer cell specificity with minimal binding to normal cells.

Understanding the cellular entry pathways of synthetic biomaterials is highly important to improve overall labeling and delivery efficiency. Herein, cellular entry mechanisms of conjugated polymer nanoparticles (CPNs) are presented. CPNs are intrinsic fluorescent materials used for various biological applications. While CPNs cause no toxicity, decreased CPN uptake is observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME is further confirmed by high co-localization with caveolin-1 proteins found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.

We developed a new synthetic approach to high molecular weight poly(p-phenylenebutadiynylene) s (PPBs) by increasing backbone flexibility. The introduction of a small amount of flexible units along the backbone improved both the physical and photophysical properties of the polymers. These materials were successfully fabricated into conjugated polymer nanoparticles (CPNs) and used for fluorescent live cell imaging for the first time.

Biodegradable conjugated polymer nanoparticles (CPNs) were prepared for high mitochondrial targeting in live cancer cells. The degradable CPNs are nontoxic and specifically localized to the mitochondria of live tumor cells through macropinocytosis followed by intracellular degradation and trafficking.

Understanding and controlling aggregation structures of conjugated polymers (CPs) in aqueous solutions is critical to improving the physical and photophysical properties of CPs for biological applications. Here, we present spectroscopic evidence, including nuclear magnetic resonance (NMR) spectroscopic results, that different organic acid treatment induces different aggregation structures and photophysical properties of CPs in water. Conjugated polymer nanoparticles (CPNs) were fabricated by treating a non-aqueous soluble, primary amine-containing poly(phenylene ethynylene) (PPE-NH(2)) with organic acids followed by dialysis. CPNs formed by acetic acid (AA) treatment (CPN-AAs) exhibit characteristics of loose aggregation with minimal π-π stacking, while CPNs formed by tartaric acid (TA) treatment (CPN-TAs) exhibit a high degree of π-π stacking among PPE-NH(2) chains. The controlled aggregation for a specific application was demonstrated by comparing the fluorescence quenching abilities of the CPN-AAs and the CPN-TAs. A doubled Stern-Volmer constant was obtained from the densely packed CPN-TAs compared to that of the loosely aggregated CPN-AAs.

Understanding cellular interactions and entry pathways of synthetic biomaterials are highly important to improve overall labeling and delivery efficiency. Conjugated polymer nanoparticles (CPNs) are emerging, fluorescent materials that have been used for cancer cell labeling and small interfering RNA (siRNA) delivery. In this contribution, detailed biophysical properties of CPNs including entry mechanisms and subcellular localization were studied using fluorescent-based techniques. While CPNs cause no toxicity, decreased CPN uptake was observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME was further confirmed by high co-localization with caveolin-1 proteins, which are found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.

Background Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

Aminosilylated substrate was treated with aziridines in order to prepare hyperbranched polymers on solid supports such as silicon wafer and fused silica. It is observed that the primary amine on the substrate is good enough to initiate the ring-opening polymerization of aziridine. Measuring the thickness of the film and the absolute density of the primary amine functionality shows that a very highly branched poly(ethyleneimine) is formed upon the reaction. The surface density of the primary amine functional group (-NH(2)) on the very top surface increased dramatically (from 3.5 amines/nm(2) to 66 amines/nm(2)). A protected aziridine, benzyl 1-aziridinecarboxylate, was employed for the stepwise growth of the film. Two step-processes, chain growth and deprotection, were successful in growing the molecular layer, and a linear chain was formed without branching. Copyright 2000 Academic Press.

Fabrication, characterization, and application of poly(phenylene ethynylene) (PPE)/silica composite particles are described. PPE is a class of conjugated polymers, which has been used for various sensory materials. However, its hydrophobic nature makes its application difficult in the aqueous phase, especially for biological substance detection. In this report, we utilized non-aqueous soluble PPE, 15 nm of colloidal silica particles, and aminosilane to fabricate a biosensory platform. The resulting composite showed high aqueous compatibility, large surface area, high quantum efficiency, and versatile chemical modification including oligonucleotide coupling. By monitoring the fluorescence quenching of PPE, we could detect a quencher-labeled target oligonucleotide specifically. Stern-Volmer (SV) analysis showed different accessibility of fluorophores (PPE) to a quencher labeled target oligonucleotide. The accessibility of fluorophores and SV constant are determined to be 0.54 and 4.2 x 10(7)M(-1), respectively, from a modified SV plot. This method will broaden the capability of conjugated polymers for the sensitive detection of biological substances.

The subcellular localizations and toxicity of conjugated polymer nanoparticles (CPNs) are dependent on the chemical structure of the side chain and backbone structures. Primary amine-containing CPNs exhibit high Golgi localization with no toxicity. Incorporation of short ethylene oxide and tertiary amine side chains contributes to decreased Golgi localization and increased toxicity, respectively.

Ionic attractions were applied to the construction of non-linear optical (NLO) chromophoric monolayers. In order to fully utilize such an interaction, stilbene-type NLO chromophores having a long alkyl chain and an anionic group at one end of the chain were designed; of this family of chromophores, sodium 11-[4-(trans-4′-pyridylstyryl)oxy] undecan-1-yl sulfate (3) was synthesized. By allowing a few minutes, this anionic chromophore self-assembles successfully on a cationically charged surface, which is prepared by treating clean fused silica with 3-aminopropyltriethoxysilane and then with iodomethane. The pyridine moiety of the self-assembled chromophore can be methylated to augment the molecular hyperpolarizability, β. The characteristics of the monolayer were examined viacontact angle measurements, UV–VIS spectroscopy, grazing-angle FTIR spectroscopy, and NLO property measurements.