Convergent evolution of skin surface microarchitecture and increased skin hydrophobicity in semi-aquatic anole lizards.

Animals that habitually cross the boundary between water and land face specific challenges with respect to locomotion, respiration, insulation, fouling and waterproofing. Many semi-aquatic invertebrates and plants have developed complex surface microstructures with water-repellent properties to overcome these problems, but equivalent adaptations of the skin have not been reported for vertebrates that encounter similar environmental challenges. Here, we document the first evidence of evolutionary convergence of hydrophobic structured skin in a group of semi-aquatic tetrapods. We show that the skin surface of semi-aquatic species of Anolis lizards is characterized by a more elaborate microstructural architecture (i.e. longer spines and spinules) and a lower wettability relative to closely related terrestrial species. In addition, phylogenetic comparative models reveal repeated independent evolution of enhanced skin hydrophobicity associated with the transition to a semi-aquatic lifestyle, providing evidence of adaptation. Our findings invite a new and exciting line of inquiry into the ecological significance, evolutionary origin and developmental basis of hydrophobic skin surfaces in semi-aquatic lizards, which is essential for understanding why and how the observed skin adaptations evolved in some and not other semi-aquatic tetrapod lineages.

Magnetic layer-by-layer coated particles for efficient MRI of dendritic cells and mesenchymal stem cells.

AIM: Cell detection by MRI requires high doses of contrast agent for generating image contrast. Therefore, there is a constant need to develop improved systems that further increase sensitivity, and which could be used in clinical settings. In this study, we devised layer-by-layer particles and tested their potential for cell labeling. MATERIALS & METHODS: The advantages of layer-by-layer technology were exploited to obtain magnetic particles of controllable size, surface chemistry and magnetic payload. RESULTS: Flexibility in size and surface charge enabled efficient intracellular delivery of magnetic particles in mesenchymal stem cells and dendritic cells. Owing to the high magnetic payload of the particles, high MRI contrast was generated, even for very low cell numbers. Subcutaneous injection of the particles and subsequent uptake by dendritic cells enabled clear visualization of dendritic cells homing towards nearby lymph nodes in mice. CONCLUSION: The magnetic particles offer several possibilities as efficient cellular MRI contrast agents for direct in vitro or in vivo cell labeling.

Tailoring layer-by-layer capsules for biomedical applications.

Polymeric capsules have attracted great interest as versatile carrier systems in the area of medicine and pharmaceutics. These capsules are made by stepwise layer-by-layer adsorption of polymers onto a template core, which can be removed to produce hollow capsules. The cavity of these capsules can host various cargo molecules while the capsules' wall can be functionalized towards desired properties by embedding specific moieties into the multilayers. Tuning of the capsules' properties influences their interaction with cells and tissues and paves the way towards the development of stimuli-responsive capsules releasing their payload at a target site. In this review, we describe the generation of tailored layer-by-layer capsules and focus hereby on numerous potential applications of this multifunctional delivery platform in biomedical settings. We review the current status in the field and discuss the opportunities, as well as the hurdles, to be overcome to successfully transfer this technology to therapeutic and diagnostic applications.

Polyelectrolyte LbL microcapsules versus PLGA microparticles for immunization with a protein antigen.

The transition from organism-based traditional vaccines to the use of safer subunit vaccines has implemented the use of adjuvants to enhance immunogenicity. This study compares the potential of two types of polymeric microparticles as delivery systems for the model antigen ovalbumin. The delivery systems encompassed polyelectrolyte microcapsules, assembled via Layer-by-Layer technology, and PLGA microparticles fabricated by spray-drying. Mice were immunized subcutaneously either by a single injection or by two injections separated by four weeks with an equivalent dose of the OVA-loaded particles. Both particulate formulations mediated high, long-term IgG(1) responses whereas the IgG(2c) titers remained low. Additionally, Th1 and Th2 phenotype immune responses against OVA were assessed by quantifying the production of cytokines in CD4+ T-cells derived from the spleens of immunized mice at 6 months after the first injection. Immunization with particulate formulations led to significantly increased IL-2, IL-4, IL-10 and IFN-γ production by splenic CD4+ T-cells compared to control animals. LbL microcapsules and PLGA microparticles generated strong immune responses in vivo, characterized by a mixed Th1/Th2 type response with predominance of Th2 immunity. Both particulate formulations elicited a comparable type of immune response and appear to be promising for antigen delivery.

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.

Particulate vaccines: on the quest for optimal delivery and immune response.

Subunit vaccines offer a safer alternative to traditional organism-based vaccines, but their immunogenicity is impaired. This hurdle might be overcome by the use of micro- and nanodelivery systems carrying the antigen(s). This review discusses the rationale for the use of particulate vaccines and provides an overview of antigen-delivery vehicles currently under investigation. It further highlights the cellular uptake, antigen processing and the presentation by antigen-presenting cells because these processes are partially governed by particle characteristics and eventually determine the immunological outcome. Finally, we address the attractive concept of concomitant delivery of antigens and immunopotentiators. The condensed knowledge could be an asset for rationally designing antigen-delivery vehicles to obtain safe and efficacious vaccines.

Encapsulation performance of layer-by-layer microcapsules for proteins.

This study reports on the encapsulation efficiency of proteins in dextran sulfate/poly-L-arginine-based microcapsules, fabricated via layer-by-layer assembly (LbL). For this purpose, radiolabeled proteins are entrapped in CaCO(3) microparticles, followed by LbL coating of the CaCO(3) cores and subsequent dissolving of the CaCO(3) using EDTA. To allow to improve protein encapsulation in LbL microcapsules, we studied all steps in the preparation of the microcapsules where loss of protein load might occur. The encapsulation efficiency of proteins in LbL microcapsules turns out to be strongly dependent on both the charge and molecular weight of the protein as well as on the number of polyelectrolyte bilayers the microcapsules consist of.

Lyophilization of protein-loaded polyelectrolyte microcapsules.

PURPOSE: To evaluate if lyophilization can be used to obtain a dry formulation of polyelectrolyte microcapsules, which have emerged as a new class of microparticles for the encapsulation and delivery of biomacromolecules. METHODS: Microcapsules composed of dextran sulfate and poly-L-arginine were obtained by coating CaCO(3) microparticles by means of the layer-by-layer technique. Microcapsules were lyophilized using different stabilizers; intactness was checked by CLSM and SEM. Horseradish peroxidase was encapsulated as model enzyme and retained activity after freeze-drying was determined using a fluorescence assay. Ovalbumin was encapsulated as model antigen; immunogenicity after lyophilization was evaluated in vitro by a T-cell proliferation assay and in vivo by measuring the antibody titer in mice. RESULTS: The results clearly demonstrate the necessity of using polyols in the formulation to prevent rupture of the microcapsules and to preserve the activity of encapsulated enzymes. Lyophilized microcapsules appeared as a promising adjuvant for antigen delivery, as both in vitro as in vivo assays showed higher immune activation compared to free antigen. CONCLUSIONS: Lyophilization is a promising strategy towards improved stability of protein-loaded microcapsules.