Exosome-coated Oxygen Nanobubble-laden Hydrogel Augments Intracellular Delivery of Exosomes for Enhanced Wound Healing

Characterization dat

Fluorescence Lifetime Cross Correlation Spectroscopy Resolves EGFR and Antagonist Interaction in Live Cells

Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their Kd value was determined to be 11 ± 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live HEK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 ± 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 ± 64 receptors/μm2 to 38 ± 7 receptors/μm2 upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein−protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR through live cell quantification and imaging

Hydrodynamic Size-Dependent Cellular Uptake of Aqueous QDs Probed by Fluorescence Correlation Spectroscopy

Aqueous quantum dots (QDs) directly synthesized with various thiol ligands have been investigated as imaging probes in living cells. However, the effect of the surface chemistry of these ligands on QDs’ cellular uptakes and their intracellular fate remains poorly understood. In this work, four CdTe QDs were directly synthesized under aqueous conditions using four different thiols as stabilizers and their interactions with cells were investigated. Fluorescence correlation spectroscopy (FCS), X-ray photoelectron spectroscopy (XPS), and zeta potential measurements on QDs primarily show that the surface structure of these QDs is highly dependent on the thiol ligands used in the preparation of QDs’ precursors, including its layer thicknesses, densities, and surface charges. Subsequently, FCS integrated with the maximum-entropy-method-based FCS (MEMFCS) was used to investigate the concentration distribution and dynamics of these QDs in living A-427 cells. Our findings indicate that QDs' surface characteristics affect cell membrane adsorption and subsequent internalization. More critically, we show that the cellular uptake of aqueous QDs is dependent on their hydrodynamic diameter and might have the potential to escape trapped environments to accumulate in the cytoplasm

Fluorescent Ag Clusters via a Protein-Directed Approach as a Hg(II) Ion Sensor

Proteins have proven to be particularly attractive as effective ligands in the synthesis of nano- and subnanoscaled materials because of their multiple chelating and functional groups imparting unique functionalities. However, protein-directed fluorescent metal cluster synthesis is still a challenge but a promising area of research. Here, we report on the synthesis of new water-soluble, stable, fluorescent Ag clusters via a facile, green method using denatured bovine serum albumin (dBSA) as a stabilizing agent. The dBSA with its 35 free cysteine residues could contribute to polyvalent interactions with the Ag clusters and serve as effective stabilizing agents for these clusters. The as-prepared Ag clusters showed high fluorescence emission at ∼637 nm and were stable even in 1 M NaCl. The fluorescent Ag clusters were then used in the detection of Hg2+ with high sensitivity and selectivity. The detection limit was 10 nM in the linear range from 10 nM to 5 μM

Quantitative Investigation of Compartmentalized Dynamics of ErbB2 Targeting Gold Nanorods in Live Cells by Single Molecule Spectroscopy

Understanding the diffusion dynamics and receptor uptake mechanism of nanoparticles in cancer cells is crucial to the rational design of multifunctional nanoprobes for targeting and delivery. In this report, for the first time, we quantify the localization and evaluate the diffusion times of Herceptin-conjugated gold nanorods (H-GNRs) in different cell organelles by fluorescence correlation spectroscopy (FCS) and examine the endocytic diffusion of H-GNRs in live ErbB2 overexpressing SK-BR-3 cells. First, by colocalizing H-GNRs in different cellular organelles depicted by the respective markers, we demonstrate that H-GNRs colocalize with the endosome and lysosome but not with the Golgi apparatus. Our study shows that Herceptin-conjugated GNRs have similar intracellular localization characteristics as Herceptin−ErbB2 complex, with a higher concentration found in the endosome (72 ± 20.6 nM) than lysosome (9.4 ± 4.2 nM) after internalization. The demonstrated approach and findings not only lay the foundations for a quantitative understanding of the fate of nanoparticle-based targeting but also provide new insights into the rational design of nanoparticle delivery systems for effective treatment

Periodic and Dynamic 3-D Gold Nanoparticle−DNA Network Structures for Surface-Enhanced Raman Spectroscopy-Based Quantification

The enhancement factor of gold nanoparticles linked by DNA in a three-dimensional (3-D) network structure was evaluated as 1.12 × 107 and shown to be greater than a two-dimensional (2-D) array by a factor of ∼10, possibly due to the dimensional expansion of resonance and periodicity of the so formed structures. Uniform and higher level of enhancement was possible from these DNA linked gold nanoparticle networks because of the matching of the resonant condition and the excitation wavelength (785 nm) to enable dynamic quantification of analytes by surface-enhanced Raman spectroscopy (SERS). The structure was first validated by obtaining a SERS measurement of 4-mercaptopyridine; then, rhodamine B was used as a test analyte to quantify the dye up to nanomolar concentrations using the proposed 3-D network structures dynamically in a liquid system. Since these constructs can be easily fabricated, we envision its application in in vitro quantification and intracellular studies in the near future

Multiplex Biosensor Using Gold Nanorods

Gold nanorods (GNRs) with different aspect ratios were fabricated through seed-mediated growth and surface activation by alkanethiols for the attachment of antibodies to yield gold nanorod molecular probes (GNrMPs). Multiplex sensing was demonstrated by the distinct response of the plasmon spectra of the GNrMPs to binding events of three targets (goat anti-human IgG1 Fab, rabbit anti-mouse IgG1 Fab, rabbit anti-sheep IgG (H+L)). Plasmonic sensors are highly specific and sensitive and can be used to monitor refractive index changes caused by molecular interactions in their immediate vicinity with potential to achieve single-particle biosensing. This technique can play a key role in developing novel optical biosensors for both in vivo and in vitro detection and single-receptor kinetics

Nuclear Targeting Dynamics of Gold Nanoclusters for Enhanced Therapy of HER2⁺ Breast Cancer

Recent advances in fluorescent metal nanoclusters have spurred tremendous interest in nanomedicine due to the ease of fabrication, excellent biocompatibility, and, more importantly, excellent wavelength-dependent tunability. Herein, we report our findings on fluorescent BSA-protected gold nanoclusters (AuNCs), ∼2 nm in size conjugated with Herceptin (AuNCs-Her), for specific targeting and nuclear localization in ErbB2 over-expressing breast cancer cells and tumor tissue as a novel fluorescent agent for simultaneous imaging and cancer therapy. More interestingly, we found that AuNCs-Her could escape the endolysosomal pathway and enter the nucleus of cancer cells to enhance the therapeutic efficacy of Herceptin. We elucidate the diffusion characteristics (diffusion time and number of diffusers) and concentration of the fluorescing clusters in the nucleus of live cells. Our findings also suggest that the nuclear localization effect of AuNCs-Her enhances the anticancer therapeutic efficacy of Herceptin as evidenced by the induction of DNA damage. This study not only discusses a new nanomaterial platform for nuclear delivery of drugs but also provides important insights on nuclear targeting for enhanced therapy

Dynamic Effect of β‑Lactam Antibiotic Inactivation Due to the Inter- and Intraspecies Interaction of Drug-Resistant Microbes

The presence of β-lactamase positive microorganisms imparts a pharmacological effect on a variety of organisms that can impact drug efficacy by influencing the function or composition of bacteria. Although studies to assess dynamic intra- and interspecies communication with bacterial communities exist, the efficacy of drug treatment and quantitative assessment of multiorganism response is not well understood due to the lack of technological advances that can be used to study coculture interactions in a dynamic format. In this study, we investigate how β-lactamase positive microorganisms can neutralize the effect of β-lactam antibiotics in a dynamic format at the inter- and intraspecies level using microbial bead technology. Three interactive models for the biological compartmentalization of organisms were demonstrated to evaluate the effect of β-lactam antibiotics on coculture systems. Our model at the intraspecies level attempts to mimic the biofilm matrix more closely as a community-level feature of microorganisms, which acknowledges the impact of nondrug-resistant species in shaping the dynamic response. In particular, the results of intraspecies studies are highly supportive of the biofilm mode of bacterial growth, which can provide structural support and protect the bacteria from an assault on host or environmental factors. Our findings also indicate that β-lactamase positive bacteria can neutralize the cytotoxic effect of β-lactam antibiotics at the interspecies level when cocultured with cancer cells. Results were validated using β-lactamase positive bacteria isolated from environmental niches, which can trigger phenotypical alteration of β-lactams when cocultured with other organisms. Our compartmentalization strategy acts as an independent ecosystem and provides a new avenue for multiscale studies to assess intra- and interspecies interactions

DataSheet1_Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors.docx

Three-dimensional (3D) tumor culture techniques are gaining popularity as in vitro models of tumoral tissue analogues. Despite the widespread interest, need, and present-day effort, most of the 3D tumor culturing methodologies have not gone beyond the inventors’ laboratories. This, in turn, limits their applicability and standardization. In this study, we introduce a straightforward and user-friendly approach based on standard 96-well plates with basic amenities for growing 3D tumors in a scaffold-free/scaffold-based format. Hanging drop preparation can be easily employed by flipping a universal 96-well plate. The droplets of the medium generated by the well-plate flip (WPF) method can be easily modified to address various mechanisms and processes in cell biology, including cancer. To demonstrate the applicability and practicality of the conceived approach, we utilized human colorectal carcinoma cells (HCT116) to first show the generation of large scaffold-free 3D tumor spheroids over 1.5 mm in diameter in single-well plates. As a proof-of-concept, we also demonstrate matrix-assisted tumor culture techniques in advancing the broader use of 3D culture systems. The conceptualized WPF approach can be adapted for a range of applications in both basic and applied biological/engineering research.</p