Vicente Nuñez

Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications.

This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.

Erythrocyte-derived nano-probes functionalized with antibodies for targeted near infrared fluorescence imaging of cancer cells.

Constructs derived from mammalian cells are emerging as a new generation of nano-scale platforms for clinical imaging applications. Herein, we report successful engineering of hybrid nano-structures composed of erythrocyte-derived membranes doped with FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG), and surface-functionalized with antibodies to achieve molecular targeting. We demonstrate that these constructs can be used for targeted imaging of cancer cells in vitro. These erythrocyte-derived optical nano-probes may provide a potential platform for clinical translation, and enable molecular imaging of cancer biomarkers.

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

ABSTRACT Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples.

Microfluidic space-domain time-resolved emission spectroscopy of terbium(III) and europium(III) chelates with pyridine-2,6-dicarboxylate.

This article describes the utilization of laminar microflows for time-resolved emission measurements with steady-state excitation and detection. Passing a laminar flow through a short illuminated section of a microchannel provided a means for pulsed-like photoexcitation of the moieties carried by the fluid. Imaging the microchannel flows carrying thus photoexcited chelates of lanthanide ions allowed us to extract their excited-state lifetimes from the spatial distribution of the changes in the emission intensity. The lifetime values obtained using this space-domain approach agreed well with the lifetimes from time-domain measurements. This validated space-domain microfluidic approach reveals a means for miniaturization of time-resolved emission spectroscopy.

Microfabrication of three-dimensional filters for liposome extrusion

Liposomes play a relevant role in the biomedical field of drug delivery. The ability of these lipid vesicles to encapsulate and transport a variety of bioactive molecules has fostered their use in several therapeutic applications, from cancer treatments to the administration of drugs with antiviral activities. Size and uniformity are key parameters to take into consideration when preparing liposomes; these factors greatly influence their effectiveness in both in vitro and in vivo experiments. A popular technique employed to achieve the optimal liposome dimension (around 100 nm in diameter) and uniform size distribution is repetitive extrusion through a polycarbonate filter. We investigated two femtosecond laser direct writing techniques for the fabrication of three-dimensional filters within a microfluidics chip for liposomes extrusion. The miniaturization of the extrusion process in a microfluidic system is the first step toward a complete solution for lab-on-a-chip preparation of liposomes from vesicles self-assembly to optical characterization.

High-resolution 3D printing for drug delivery

Liposomes are spherical particles comprising single or multiple concentric lipid bilayers commonly referred to as lamellae.1 Liposomes are used for drug delivery to specific sites in the body, and are usually fabricated by hydrating phospholipid films. Following hydration, liposomes can be loaded with reagents of interest, surface functionalized for targeted drug delivery, and tailored in size for more efficient delivery. The effectiveness of nanotherapeutic delivery strongly depends on particle size.2 The immune system can rapidly remove colloidal drug delivery particles by activating the complement system and by cells of the mononuclear phagocyte system. To evade such immune responses, nanoscale particles are required. Furthermore, the leading strategy for delivery to tumor sites is through the enhanced permeability and retention effect. That is, the vascular network surrounding a tumor site is known to be highly leaky because of enhanced requirements for oxygen and nutrients by the tumor. As a result, gaps between the thin layer of cells lining the interior surface of blood vessels can reach a couple of micrometers in size. Thus, nanoparticles are able to preferentially accumulate at tumor sites. An automated nanoparticle preparation platform— encompassing synthesis, functionalization, loading, and characterization in a total analytical system—would benefit drug delivery by liposomal nanocarriers.3 Miniaturizing such a device would indeed also improve performance in several ways from faster response times to reduced reagent consumption. Furthermore, such a device would expedite and stimulate the study of several aspects of liposome preparation and activity in both academic and industrial settings where fast prototyping is required. We have investigated the use of femtosecond laser direct writing for just such a device. In particular, we considered the advantages and disadvantages of two-photon polymerization (TPP) and femtosecond laser irradiation followed by chemical etching (FLICE).4, 5 Figure 1. Reproduction of Michelangelo’s David fabricated by two-photon polymerization (TPP).

Effects of ICG concentration on the optical properties of erythrocyte- derived nano-vectors

Erythrocyte-based nanoparticle platforms can offer long circulation times not offered by traditional drug delivery methods. We have developed a novel erythrocyte-based nanoparticle doped with indocyanine green (ICG), the only FDA-approved near-infrared chromophore. Here, we report on the absorption and fluorescence emission characteristics of these nanoparticles fabricated using ICG concentrations in the range of 161-323 μM. These nanoparticles may serve as biocompatible optical materials for various clinical imaging and phototherapeutic applications.

Erythrocyte-derived optical nano-vesicles as theranostic agents

We have engineered nano-vesicles, derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETS) since in response to NIR photo-excitation they can generate heat or emit fluorescent light. Using biochemical methods based on reduction amination, we have functionalized the surface of NET with antibodies to target specific biomolecules. We present results that demonstrate the effectiveness of NETs in targeted imaging of cancer cells that over-express the human epidermal growth factor receptor-2 (HER2).

Dynamic staining of bacteria at a single-cell level

Bacterial infectious diseases remain one of the major health hazards nation- and worldwide. The expedience of detection and identification of bacterial pathogens determines how early the diagnosis is, and hence, what the treatment and the outcome of the illness would be. As we have previously reported, the dynamics of fluorescence staining provides venues for the development of expedient assays for detection and identification of bacterial species[1]. We measured the kinetics of bacterial staining with cyanine and thioflavin dyes and investigated their photophysical properties. We demonstrated that the pseudo first-order kinetic constants of the fluorescence staining processes have species specificity without contrition dependence. Combining the dynamics of staining with real-time fluorescence microscopy we characterized the fluorescence staining process at the single-cell level with improved sensitivity and contrast.