Innovative drug delivery and translational research by emerging leaders in the field.

The field of drug delivery continues to be at the forefront of innovation, driven by the relentless pursuit of more effec- tive, targeted, and safer therapies. Young scientists are playing a pivotal role in this progress, bringing fresh per- spectives, and a deep commitment to addressing the com- plex challenges of drug delivery. With this special issue in Drug Delivery and Translational Research on "Young Sci- entists in Drug Delivery," we are delighted to showcase a selection of 20 exceptional articles led by emerging leaders in the field, including late-stage PhD students, postdoctoral researchers, and early-career principal investigators

Invasin-functionalized liposome nanocarriers improve the intracellular delivery of anti-infective drugs

Intracellular infections caused by invasive pathogens continue to prove difficult to combat, due in part to the commonly poor membrane permeability of anti-infective drugs. The aim of this study was to improve the intracellular delivery of one such poorly permeable (but broad-spectrum) anti-infective, gentamicin. Gentamicin was encapsulated into liposomal nanocarriers which were then surface functionalized with InvA497, a bacteria-derived invasion protein. Treatment of HEp-2 cells infected with the enteroinvasive bacteria Yersinia pseudotuberculosis or Salmonella enterica with gentamicincontaining, InvA497-functionalized liposomes resulted in a significantly greater reduction in infection load than treatment with non-functionalized liposomes, indicating that such a bacteriomimetic nanocarrier was not only able to promote successful cellular uptake of gentamicin but was also able to mediate anti-infective drug delivery to both cell cytoplasm and intracellular compartments. The developed InvA497-functionalized liposomal nanocarrier therefore holds great promise as a strategy for improving the therapy of intracellular infections

Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections.

The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa. Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas-infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells

Mechanism and determinants of nanoparticle penetration through human skin

The ability of nanoparticles to penetrate the stratum corneum was the focus of several studies. Yet, there are controversial issues available for particle penetration due to different experimental setups. Meanwhile, there is little known about the mechanism and determinants of their penetration. In this paper the penetration of four model gold nanoparticles of diameter 6 and 15 nm, differing in surface polarity and the nature of the vehicle, through human skin was studied using multiphoton microscopy. This is in an attempt to profoundly investigate the parameters governing particle penetration through human skin. Our results imply that nanoparticles at this size range permeate the stratum corneum in a similar manner to drug molecules, mainly through the intercellular pathways. However, due to their particulate nature, permeation is also dependent on the complex microstructure of the stratum corneum with its tortuous aqueous and lipidic channels, as shown from our experiments performed using skin of different grades of barrier integrity. The vehicle (toluene-versus-water) had a minimal effect on skin penetration of gold nanoparticles. Other considerations in setting up a penetration experiment for nanoparticles were also studied. The results obtained are important for designing a new transdermal carrier and for a basic understanding of skin–nanoparticle interaction

Interaction of nanoscale particles with the skin barrier

Skin penetration of nanoparticles was the focus of several recent studies. This is of major importance in basic research for potential future applications, e.g. designing topical and transdermal delivery systems, as well as for health risk analysis. Yet, there is a controversy among researchers on the status of their skin penetration due to different experimental setups. Meanwhile, there is little known about the mechanism and determinants of nanoparticle penetration. The main thesis objective was hence to study the penetration of model gold nanoparticles of different physicochemical and formulation parameters through human skin of different degrees of barrier integrity. Multiphoton microscopy was used for nanoparticle detection. Imaging parameters were determined in terms of resolution and depth profiling of gold nanoparticles in skin. A semiquantitative approach based on pixel analysis of gold nanoparticles was developed to compare nanoparticle localization in different skin locations under different conditions. Based on penetration experiments, determinants that favor or limit particle penetration were determined as well as the barrier to penetration (intercellular lipids). Finally nanoparticle penetration was successfully enhanced using a chemical enhancement approach. Results obtained are important to enhance our understanding of nanoparticle interaction with the skin barrier. Future studies are required to reduce the gap between research and applications.Die Penetration von Nanopartikeln ist Gegenstand der aktuellen Forschung. Diese Frage ist von großer Bedeutung für die Anwendung im Bereich der Nanomedizin als auch für die Abschätzung des Risikopotenzials bei Kontakt mit solchen Systemen. Bis dato sind allerdings keine eindeutigen Aussagen möglich. Das Ziel dieser Arbeit war daher die Untersuchung des Penetrationsverhaltens anhand von kolloidalem Gold (AuNP). Dieses Modellsystem erlaubt die Untersuchung der Penetration in Abhängigkeit von verschiedenen physikochemischen Eigenschaften der Partikel (oberflächenmodifiziert), als auch von Formulierungseigenschaften (Vehikel). Die AuNP erlauben eine Visualisierung mittels Multiphotonen Mikroskopie. Daher wurden die Auflösung und die optischen Parameter für AuNP in Haut bestimmt. Des Weitern wurde ein Pixel-basiertes Verfahren ermittelt, das eine semiquantitative Analyse der penetrierten Objekten ermöglicht. Dies erlaubt eine Abschätzung der Partikelpenetration. Penetrationsexperimente erlaubten die Parameter, die die Penetration beeinflussen, hinsichtlich Größe und Oberflächenpolarität einzuschränken. Außerdem konnte gezeigt werden, dass auch die Penetration von Nanopartikeln mit Hilfe von Penetrationsverbesserern gesteigert werden kann. Die Ergebnisse dieser Arbeit sind wichtige Bausteine für das Verständnis der Interaktion von Nanopartikeln mit der Hautbarriere. Zukünftige Studien sind dennoch nötig, um die Lücke zwischen Forschung und möglicher Anwendung zu schließen

RELATING CELLULAR ASSOCIATION WITH LIPOSOME CYTOTOXICITY IN HUMAN ENDOTHELIAL CELLS

INTRODUCTION Interactions with the endothelium play a key role in the behaviour of intravenously administered nanoparticle drug carriers[1]. Hence, quantifying cellular association (membrane adhesion and cell internalization) of liposomes with endothelial cells is an effective screening method of biocompatibility and success of new drug carriers. Current methods are inaccurate as concentration does not necessarily equate to local cellular association. The focus of this experiment is to quantify the cellular association between liposomes and two types of human endothelial cells and compare the associations with cells’ cytotoxic response. Cellular association of liposomes as well as cell viability were quantified on cellular level at different concentrations of liposomes. METHODS Two different types of cells, Human Umbilical Vein Endothelial cell, which is a common cell type used in vitro studies, and Human MicroVascular Cell, which is more accurate representation of in vivo, were used[2]. HUVEC and HMVEC were cultured and passaged onto chamber slides using standard cell culture techniques. The confluent cells were exposed to fluorescent liposomes with hydrodynamic diameter of 90.4 nm at concentrations ranging from 0.08nM to 8nM for 24 hours, membrane stained with CellMask Deep Red and fixed with paraformaldehyde, following same protocols for both types of cells. Cell viability on exposure to the same concentration range of liposomes was determined using Vialight assay using manufacturer protocols. Z-stacks of the treated cells were obtained using Olympus Fluoview FV1000 confocal microscope. Region of interest, limited by cell membranes, was set using the membrane stain channel using ImageJ. The region of interest was superimposed onto the fluorescent liposome channel to determine exclusively the fluorescence of cell adhered and cell internalized liposomes RESULTS Compared to HUVECs, higher cellular association of liposomes was observed for HMVC as shown in Figure 1.While cellular association of liposomes increased with concentration, cell viability was in the range of 85 to nearly 100% for the concentration range of 0.08-4 nM with no significant difference. Only at 8 nM, cell viability decreased significantly to approximately 62 %. DISCUSSION AND CONCLUSIONS Liposome cellular association provide insight into the cytotoxicity and the endothelial cytotoxicity of the liposomes at low concentration of 8nM raises cautions on documented innocuous properties of liposomes. Cytotoxicity and cellular association upon comparison showed exponential relationship. Because the cytotoxicity and cellular association relationship is exponential, slight over-administration can cause severe toxicity. 8nM is lower than concentration of current intravenous liposome-based drug doxorubicin[3]. High toxicity and exponential relationship raise caution on the importance of proper safe dosage

(E)-N′-(4-Chloro­benzyl­idene)-3,4,5-trimethoxy­benzohydrazide

The title compound, C17H17ClN2O4, was synthesized from 3,4,5-trimethoxy­benzohydrazide and 4-chloro­benzaldehyde. In the crystal structure, packing is stabilized by intramolecular C—H⋯O and inter­molecular N—H⋯O and C—H⋯O hydrogen-bonding inter­actions

RELATING QUANTUM DOT ASSOCIATION WITH HUMAN ENDOTHELIAL CELLS WITH THEIR CYTOTOXIC EFFECTS

INTRODUCTION Advances in the field of nanotechnology have enabled researchers to pursue biomedical applications of nanoparticles. Quantum dots are commonly used fluorescent probes because they are brighter and less prone to photobleaching than other fluorophores [1]. However, despite the advantages, potential for toxicity must be acknowledged. Quantum dots are commonly made with toxic metal elements, which can cause oxidative stress [2]. Cadmium ions have been shown to disrupt mitochondria activity, leading to cell death [2]. Quantum dots have been shown to attach to the cell membrane as well as be internalized through endocytic mechanisms [3]. In this study, we aim to quantify quantum dot association and compare results from cytotoxicity assays for identical conditions, relating cellular association with cytotoxicity. METHODS Human Umbilical Vein Endothelial Cells (HUVECs) and Human Micro-vascular Endothelial Cells (HMVECs) were cultured in static conditions in 8-well chamber slides then exposed to amino-PEG quantum dots at a concentration of 0.2nM to 200nM. After exposure for 24 hours, the cells were washed, fixed, and stained. Z-stacks were obtained using an Olympus Fluoview FV1000 confocal microscope. Images were analyzed using ImageJ software to quantify mean fluorescence intensity within the defined region of interest, selected from the boundaries of stained cell membranes. Statistical analysis using one-way Analysis of Variance (ANOVA) and post-hoc Tukey HSD test was performed. Finally, Vialight assay was used to test cell viability after exposure to quantum dots under the same experimental conditions used for association experiments. RESULTS Exposure to different concentrations of quantum dots results in significant changes in the observed fluorescence intensity per area. Non-linear dependence of cellular association of quantum dots on exposure concentration was observed. A representative example of mean fluorescence intensity of quantum dots associated with HUVECs is shown in Figure 1.A significant decrease in the viability of HUVECs was observed on exposure to quantum dots (30-50% cell viability relative to 100% for non-exposed cells). However, no significant difference in cell viability was observed between 0.2nM to 200nM concentrations. DISCUSSION AND CONCLUSIONS Nanoparticle association studies play a vital role in predicting cell viability in nanoparticle cytotoxicity studies. The non-linear trend observed suggests that for the range of concentrations examined, cellular association does not increase linearly with exposure concentration, and that cytotoxicity can be related to association, rather than just to exposure concentration. This experiment provides an approach to advance future studies relating cellular association to cytotoxicity

(E)-N′-(2-Hydroxy­benzyl­idene)-3,4,5-trimethoxy­benzohydrazide

The title compound, C17H18N2O5, was synthesized from 3,4,5-trimethoxy­benzohydrazide and 2-hydroxy­benzaldehyde. The dihedral angle between the planes of the two benzene rings is 29.9 (2)°. The crystal structure involves intra­molecular O—H⋯N, and inter­molecular N—H⋯O and C—H⋯O hydrogen bonds

Perspectives on utilization of edible coatings and nano-laminate coatings for extension of postharvest storage of fruits and vegetables

It is known that in developing countries, a large quantity of fruit and vegetable losses results at postharvest and processing stages due to poor or scarce storage technology and mishandling during harvest. The use of new and innovative technologies for reducing postharvest losses is a requirement that has not been fully covered. The use of edible coatings (mainly based on biopolymers) as a postharvest technique for agricultural commodities has offered biodegradable alternatives in order to solve problems (e.g., microbiological growth) during produce storage. However, biopolymer-based coatings can present some disadvantages such as: poor mechanical properties (e.g., lipids) or poor water vapor barrier properties (e.g., polysaccharides), thus requiring the development of new alternatives to solve these drawbacks. Recently, nanotechnology has emerged as a promising tool in the food processing industry, providing new insights about postharvest technologies on produce storage. Nanotechnological approaches can contribute through the design of functional packing materials with lower amounts of bioactive ingredients, better gas and mechanical properties and with reduced impact on the sensorial qualities of the fruits and vegetables. This work reviews some of the main factors involved in postharvest losses and new technologies for extension of postharvest storage of fruits and vegetables, focused on perspective uses of edible coatings and nano-laminate coatings.María L. Flores-López thanks Mexican Science and Technology Council (CONACYT, Mexico) for PhD fellowship support (CONACYT Grant Number: 215499/310847). Miguel A. Cerqueira (SFRH/BPD/72753/2010) is recipient of a fellowship from the Fundação para a Ciência e Tecnologia (FCT, POPH-QREN and FSE Portugal). The authors also thank the FCT Strategic Project of UID/ BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project ‘‘BioInd Biotechnology and Bioengineering for improved Industrial and AgroFood processes,’’ REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP, CE Brazil (CI10080-00055.01.00/13)