Animal Models in Eye Research: Focus on Corneal Pathologies.

The eye is a complex sensory organ that enables visual perception of the world. The dysfunction of any of these tissues can impair vision. Conduction studies on laboratory animals are essential to ensure the safety of therapeutic products directly applied or injected into the eye to treat ocular diseases before eventually proceeding to clinical trials. Among these tissues, the cornea has unique homeostatic and regenerative mechanisms for maintaining transparency and refraction of external light, which are essential for vision. However, being the outermost tissue of the eye and directly exposed to the external environment, the cornea is particularly susceptible to injury and diseases. This review highlights the evidence for selecting appropriate animals to better understand and treat corneal diseases, which rank as the fifth leading cause of blindness worldwide. The development of reliable and human-relevant animal models is, therefore, a valuable research tool for understanding and translating fundamental mechanistic findings, as well as for assessing therapeutic potential in humans. First, this review emphasizes the unique characteristics of animal models used in ocular research. Subsequently, it discusses current animal models associated with human corneal pathologies, their utility in understanding ocular disease mechanisms, and their role as translational models for patients.

Parametric Drug Release Optimization of Anti-Inflammatory Drugs by Gold Nanoparticles for Topically Applied Ocular Therapy.

Eye drops represent 90% of all currently used ophthalmic treatments. Only 0.02% of therapeutic molecules contained in eye drops reach the eye anterior chamber despite their high concentration. The tear film efficiently protects the cornea, reducing access to the target. Thereby, the increase in the drug bioavailability and efficiency must come from the mucoadhesion optimization of the drug delivery system. The gold nanoparticles, used as a drug delivery system in this study, already showcased ultrastable and mucoadhesive properties. The goal was to study the gold nanoparticles' ability to release two specific ophthalmic drugs, flurbiprofen and ketorolac. The parameters of interest were those involving the loading conditions, the gold nanoparticles properties, and the release experimental conditions. The drug release was measured using an in vitro model based on dialysis bags coupled with UV-visible spectroscopy. Gold nanoparticles showed an ability to release different molecules, whether hydrophobic or hydrophilic, in passive or active drug release environments. Based on these preliminary results, gold nanoparticles could represent a promising drug delivery system for ketorolac and flurbiprofen when topically applied through eye drops.

Biosensing Extracellular Vesicle Subpopulations in Neurodegenerative Disease Conditions.

Extracellular vesicles (EVs) are secreted nanoparticles that are involved in intercellular communication and that modulate a wide range of biological processes in normal and disease conditions. However, EVs are highly heterogeneous in terms of origin in the cell, size, and density. As a result, complex protocols are required to identify and characterize specific EV subpopulations, limiting biomedical applications, notably in diagnostics. Here, we show that combining quartz crystal microbalance with dissipation (QCM-D) and nanoplasmonic sensing (NPS) provides a facile method to track the viscoelastic properties of small EVs. We applied this multisensing strategy to analyze small EVs isolated by differential ultracentrifugation from knock-in mouse striatal cells expressing either a mutated allele or wild-type allele of huntingtin (Htt), the Huntington's disease gene. Our results validate the sensing strategy coupling QCM-D and NPS and suggest that the mass and viscoelastic dissipation of EVs can serve as potent biomarkers for sensing the intercellular changes associated with the neurodegenerative condition.

About the Influence of PEG Spacers on the Cytotoxicity of Titanate Nanotubes-Docetaxel Nanohybrids against a Prostate Cancer Cell Line.

The association between chemotherapeutic drugs and metal oxide nanoparticles has sparked a rapidly growing interest in cancer nanomedicine. The elaboration of new engineered docetaxel (DTX)-nanocarriers based on titanate nanotubes (TiONts) was reported. The idea was to maintain the drug inside cancer cells and avoid multidrug resistance mechanisms, which often limit drug efficacy by decreasing their intracellular concentrations in tumor cells. HS-PEGn-COOH (PEG: polyethylene glycol, n = 3000, 5000, 10,000) was conjugated, in an organic medium by covalent linkages, on TiONts surface. This study aimed to investigate the influence of different PEG derivatives chain lengths on the TiONts colloidal stability, on the PEGn density and conformation, as well as on the DTX biological activity in a prostate cancer model (human PC-3 prostate adenocarcinoma cells). In vitro tests highlighted significant cytotoxicities of the drug after loading DTX on PEGn-modified TiONts (TiONts-PEGn-DTX). Higher grafting densities for shorter PEGylated chains were most favorable on DTX cytotoxicity by promoting both colloidal stability in biological media and cells internalization. This promising strategy involves a better understanding of nanohybrid engineering, particularly on the PEGylated chain length influence, and can thus become a potent tool in nanomedicine to fight against cancer.

Design and Analytical Performances of a Diclofenac Biosensor for Water Resources Monitoring.

Because the broadly consumed pain killer diclofenac (DCF) is a recognized pollutant, monitoring of its concentration is routinely performed in surface waters. As a valuable alternative to chromatographic and immunochemical assays, we developed a piezoelectric immunosensor to quantify DCF, first in buffer (PBS) and then in river water samples. A sensing layer comprising DCF was built up on the surface of silica-coated quartz sensors using a robust coupling chemistry. Binding of a highly affine monoclonal anti-DCF antibody was monitored in real time by quartz crystal microbalance with dissipation (QCM-D) measurements from which were determined a dissociation constant KD of 0.24 nM and an acoustic antibody surface coverage of 1120 ng/cm2 at saturation. On the other hand, an optical antibody surface coverage of 260 ng/cm2 was determined by combined nanoplasmonic sensing measurement, giving a hydration percentage of 75% for the antibody monolayer. DCF assay was further set up following a competitive format for which binding of antibody to the sensing layer is inhibited by DCF in solution. The piezoelectric sensor response expressed as frequency shift ΔF was inversely related to the concentration of DCF with a dynamic range of 15-46 nM and a limit of detection (LoD) of 9.5 nM (2.8 µg/L) in PBS. This piezoelectric immunosensor was eventually applied to the assay of DCF in surface water samples taken at three different locations in the Seine and Marne rivers. The calculated concentration of DCF in these samples was in good agreement with official data published by the French center of water analysis eaufrance.

Isolating Nanoparticles from Complex Biological Media by Immunoprecipitation.

Polyethylene glycol (PEG) is considered the gold standard to prepare long circulating nanoparticles. The hydrophilic layer that sterically protects PEGylated nanomedicines also impedes their separation from biological media. In this study, we describe an immunoprecipitation method using AntiPEG antibodies cross-linked to magnetic beads to extract three types of radiolabeled PEGylated systems: polymeric nanoparticles, liposomes, and therapeutic proteins. The potential of the method is emphasized by isolating these systems after in vivo administration and ex vivo incubation in human biological fluids. Immunoprecipitation also allows a unique perspective on the size distribution of nanoparticles in the bloodstream after intravenous and intraperitoneal administrations. Further, we highlight the potential of the approach to inform on nanomaterial-associated drug in plasma as well as help characterize the protein corona. Altogether, we believe this method answers an unmet need in nanomedicine research and will contribute a fresh perspective on the interactions of nanomedicines with biological systems.

Titanate Nanotubes Engineered with Gold Nanoparticles and Docetaxel to Enhance Radiotherapy on Xenografted Prostate Tumors.

Nanohybrids based on titanate nanotubes (TiONts) were developed to fight prostate cancer by intratumoral (IT) injection, and particular attention was paid to their step-by-step synthesis. TiONts were synthesized by a hydrothermal process. To develop the customengineered nanohybrids, the surface of TiONts was coated beforehand with a siloxane (APTES), and coupled with both dithiolated diethylenetriaminepentaacetic acidmodified gold nanoparticles (Au@DTDTPA NPs) and a heterobifunctional polymer (PEG3000) to significantly improve suspension stability and biocompatibility of TiONts for targeted biomedical applications. The prefunctionalized surface of this scaffold had reactive sites to graft therapeutic agents, such as docetaxel (DTX). This novel combination, aimed at retaining the AuNPs inside the tumor via TiONts, was able to enhance the radiation effect. Nanohybrids have been extensively characterized and were detectable by SPECT/CT imaging through grafted Au@DTDTPA NPs, radiolabeled with 111In. In vitro results showed that TiONtsAuNPsPEG3000DTX had a substantial cytotoxic activity on human PC3 prostate adenocarcinoma cells, unlike initial nanohybrids without DTX (Au@DTDTPA NPs and TiONtsAuNPsPEG3000). Biodistribution studies demonstrated that these novel nanocarriers, consisting of AuNP- and DTXgrafted TiONts, were retained within the tumor for at least 20 days on mice PC3 xenografted tumors after IT injection, delaying tumor growth upon irradiation.

Core-Shell Gold/Silver Nanoparticles for Localized Surface Plasmon Resonance-Based Naked-Eye Toxin Biosensing.

The localized surface plasmon resonance (LSPR) phenomenon provides a versatile property for biodetection. Herein, this unique feature was employed to build a homogeneous optical biosensor to detect staphylococcal enterotoxin A (SEA) in solution down to very low levels by naked-eye readout. If the initial position of the LSPR band is located in the cyan region, even a small red shift (∼2-3 nm) induced by a refractive index change close to the surface of nanoparticles (NPs) could make the light absorption transit from cyan to green and become visually detectable via a concomitant change in the complementary colors. In this work, we aimed at synthesizing two types of NPs based on compositionally complex core-shell NPs-Ag shells on AuNPs (Au@AgNPs) and Ag inside gold nanoshells (Ag@AuNPs). By controlling the thickness of the shells and their surface chemistry with anti-SEA antibody (Ab), the LSPR band was tuned to near 495 and 520 nm for Ag@AuNPs and Au@AgNPs, respectively. The two particle systems were subsequently applied to spectroscopically and visually detect anti-SEA Ab-SEA interactions. Upon the addition of SEA, large red shifts of the LSPR band were observed spectroscopically and the limits of detection (LODs) were estimated to be 0.2 and 0.4 nM for Au@AgNPs and Ag@AuNPs, respectively. Although the two sets of NPs gave almost identical LODs, the Ag@AuNPs whose initial position of the LSPR band was tuned in the cyan to green region (∼500 nm) displayed a substantially more distinct color change from orange to red, as revealed by the naked eye. We foresee significant potential to this strategy in medical diagnostics and environmental monitoring, especially when basic laboratory infrastructure is sparse or nonexistent.

Silver-Based Plasmonic Nanoparticles for and Their Use in Biosensing.

The localized surface plasmon resonance (LSPR) property of metallic nanoparticles is widely exploited for chemical and biological sensing. Selective biosensing of molecules using functionalized nanoparticles has become a major research interdisciplinary area between chemistry, biology and material science. Noble metals, especially gold (Au) and silver (Ag) nanoparticles, exhibit unique and tunable plasmonic properties; the control over these metal nanostructures size and shape allows manipulating their LSPR and their response to the local environment. In this review, we will focus on Ag-based nanoparticles, a metal that has probably played the most important role in the development of the latest plasmonic applications, owing to its unique properties. We will first browse the methods for AgNPs synthesis allowing for controlled size, uniformity and shape. Ag-based biosensing is often performed with coated particles; therefore, in a second part, we will explore various coating strategies (organics, polymers, and inorganics) and their influence on coated-AgNPs properties. The third part will be devoted to the combination of gold and silver for plasmonic biosensing, in particular the use of mixed Ag and AuNPs, i.e., AgAu alloys or Ag-Au core@shell nanoparticles will be outlined. In the last part, selected examples of Ag and AgAu-based plasmonic biosensors will be presented.

Docetaxel-titanate nanotubes enhance radiosensitivity in an androgen-independent prostate cancer model.

Around 40% of high-risk prostate cancer patients who undergo radiotherapy (RT) will experience biochemical failure. Chemotherapy, such as docetaxel (DTX), can enhance the efficacy of RT. Multidrug resistance mechanisms often limit drug efficacy by decreasing intracellular concentrations of drugs in tumor cells. It is, therefore, of interest to develop nanocarriers of DTX to maintain the drug inside cancer cells and thus improve treatment efficacy. The purpose of this study was to investigate the use of titanate nanotubes (TiONts) to develop a TiONts-DTX nanocarrier and to evaluate its radiosensitizing in vivo efficacy in a prostate cancer model. In vitro cytotoxic activity of TiONts-DTX was evaluated using an MTS assay. The biodistribution of TiONts-DTX was analyzed in vivo by single-photon emission computed tomography. The benefit of TiONts-DTX associated with RT was evaluated in vivo. Eight groups with seven mice in each were used to evaluate the efficacy of the nanohybrid combined with RT: control with buffer IT injection ± RT, free DXL ± RT, TiONts ± RT and TiONts-DXL ± RT. Mouse behavior, health status and tumor volume were monitored twice a week until the tumor volume reached a maximum of 2,000 mm3. More than 70% of nanohybrids were localized inside the tumor 96 h after administration. Tumor growth was significantly slowed by TiONts-DTX associated with RT, compared with free DTX in the same conditions (P=0.013). These results suggest that TiONts-DTX improved RT efficacy and might enhance local control in high-risk localized prostate cancer.

Taxane-Grafted Metal-Oxide Nanoparticles as a New Theranostic Tool against Cancer: The Promising Example of Docetaxel-Functionalized Titanate Nanotubes on Prostate Tumors.

The combination of anticancer drugs and metal oxide nanoparticles is of great interest in cancer nanomedicine. Here, the development of a new nanohybrid, titanate nanotube-docetaxel (TiONts-DTX) is reported, the two parts of which are conjugated by covalent linkages. Unlike most nanoparticles currently being developed for biomedical purposes, TiONts present a needle-shaped morphology. The surface of TiONts is linked with 3-aminopropyl triethoxysilane and with a hetero-bifunctional polymer (polyethylene glycol) to create well-dispersed and biocompatible nanovectors. The prefunctionalized surface of this scaffold has valuable attachments to graft therapeutic agents (DTX in our case) as well as chelating agents (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to monitor the nanohybrids. To evaluate drug efficacy, in vitro tests have demonstrated that the association between TiONts and DTX shows cytotoxic activity against a hormone-refractory prostate cancer cell line (22Rv1) whereas TiONts without DTX do not. Finally, the first in vivo tests with intratumoral injections show that more than 70% of TiONts nanovectors are retained within the tumor for at least 7 d. Moreover, tumor growth in mice receiving TiONts-DTX is significantly slower than that in mice receiving free DTX. This nanohybrid can thus become a promising new tool in biomedicine to fight against prostate cancer.

Dispersion of titanate nanotubes for nanomedicine: comparison of PEI and PEG nanohybrids.

In the present study, we report the dispersion of titanate nanotubes (TiONts) via polymer grafting (PolyEthylene Glycol, PEG) or polymer adsorption (polyethylene imine, PEI) where different TiONts/polymer ratios have been investigated. The TiONts/PEI and TiONts/PEG nanohybrids were characterized by scanning and transmission electron microscopy as well as by zeta potential measurements in order to determine both their dispersion state and stability in water (at different pH for zetametry). The nature of the chemical bonds at the surface of these nanohybrids was investigated by Fourier-transformed infrared (FTIR) spectroscopy while the grafting densities of PEG on the nanotubes were quantified by thermogravimetric analyses (TGA). The nanohybrids reported here are promising tools for biotechnology applications due to their tubular morphology, their very good dispersion in water and the reactivity of their surface.