Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of k-carrageenan hydrogels for the delivery of stem cells obt ained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation met hod and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v k-carrageenan solution at a cell density of 5  10 6 cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that k-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mec hanical analysis demonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that k-carrageenan exhibits properties t hat enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.

Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Numerical prediction of nanoparticle formation in flames

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2012

Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients.

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

Behavior of endogenous tumor-associated macrophages assessed in vivo using a functionalized nanoparticle.

Tumor-associated macrophages (TAMs) invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma). AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an "M2" macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

Oxidative stress and inflammation response after nanoparticle exposure : differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-a and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-a concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure. [Authors]

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

Nanoparticle reactivity toward dithiothreitol

Hazard determination of nanoparticles (NP) is challenging and should be based on a predictive and pragmatic tier approach. The biological effects of NP appear to be related not only to surface/size but also to their ability to generate free radicals/oxidants. We propose that the measurement of this property by acellular assays could be helpful for NP toxicity screening. This study investigated the effect of dispersing conditions on the oxidative capacity of a small selection of carbonaceous NP toward dithiothreitol (DTT). Increasing surfactant concentration decreased the DTT reactivity on these studied particles. Afterward, a panel of NP were studied under constant conditions. Classification of the NP panel based on their DTT reactivity was found to be dependent on the metric used (mass or surface) but not on the surfactant type. The DTT assay may be useful for an initial evaluation of the hazardousness of manufactured or unintentionally produced NP. However, the predictive potential of such a test towards biological effects still needs to be evaluated. [Authors]

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung: a dialog between aerosol science and biology

The introduction of engineered nanostructured materials into a rapidly increasing number of industrial and consumer products will result in enhanced exposure to engineered nanoparticles. Workplace exposure has been identified as the most likely source of uncontrolled inhalation of engineered aerosolized nanoparticles, but release of engineered nanoparticles may occur at any stage of the lifecycle of (consumer) products. The dynamic development of nanomaterials with possibly unknown toxicological effects poses a challenge for the assessment of nanoparticle induced toxicity and safety.In this consensus document from a workshop on in-vitro cell systems for nanoparticle toxicity testing11Workshop on 'In-Vitro Exposure Studies for Toxicity Testing of Engineered Nanoparticles' sponsored by the Association for Aerosol Research (GAeF), 5-6 September 2009, Karlsruhe, Germany. an overview is given of the main issues concerning exposure to airborne nanoparticles, lung physiology, biological mechanisms of (adverse) action, in-vitro cell exposure systems, realistic tissue doses, risk assessment and social aspects of nanotechnology. The workshop participants recognized the large potential of in-vitro cell exposure systems for reliable, high-throughput screening of nanoparticle toxicity. For the investigation of lung toxicity, a strong preference was expressed for air-liquid interface (ALI) cell exposure systems (rather than submerged cell exposure systems) as they more closely resemble in-vivo conditions in the lungs and they allow for unaltered and dosimetrically accurate delivery of aerosolized nanoparticles to the cells. An important aspect, which is frequently overlooked, is the comparison of typically used in-vitro dose levels with realistic in-vivo nanoparticle doses in the lung. If we consider average ambient urban exposure and occupational exposure at 5mg/m3 (maximum level allowed by Occupational Safety and Health Administration (OSHA)) as the boundaries of human exposure, the corresponding upper-limit range of nanoparticle flux delivered to the lung tissue is 3×10-5-5×10-3μg/h/cm2 of lung tissue and 2-300particles/h/(epithelial) cell. This range can be easily matched and even exceeded by almost all currently available cell exposure systems.The consensus statement includes a set of recommendations for conducting in-vitro cell exposure studies with pulmonary cell systems and identifies urgent needs for future development. As these issues are crucial for the introduction of safe nanomaterials into the marketplace and the living environment, they deserve more attention and more interaction between biologists and aerosol scientists. The members of the workshop believe that further advances in in-vitro cell exposure studies would be greatly facilitated by a more active role of the aerosol scientists. The technical know-how for developing and running ALI in-vitro exposure systems is available in the aerosol community and at the same time biologists/toxicologists are required for proper assessment of the biological impact of nanoparticles.

Nanoparticle usage and protection measures in the manufacturing industry : a representative survey

Addressing the risks of nanoparticles requires knowledge about release into the environment and occupational exposure. However, such information currently is not systematically collected; therefore, this risk assessment lacks quantitative data. The goal was to evaluate the current level of nanoparticle usage in Swiss industry as well as health, safety, and environmental measures, and the number of potentially exposed workers. A representative, stratified mail survey was conducted among 1626 clients of the Swiss National Accident Insurance Fund (SUVA), which insures 80,000 manufacturing firms, representing 84% of all Swiss manufacturing companies (947 companies answered the survey for a 58.3% response rate). The extrapolation to all Swiss manufacturing companies results in 1309 workers (95% confidence interval [CI]: 1073 to 1545) potentially exposed to nanoparticles in 586 companies (95% CI: 145 to 1027). This corresponds to 0.08% of workers (95% CI: 0.06% to 0.09%) and to 0.6% of companies (95% CI: 0.2% to 1.1%). The industrial chemistry sector showed the highest percentage of companies using nanoparticles (21.2%). Other important sectors also reported nanoparticles. Personal protection equipment was the predominant protection strategy. Only a few applied specific environmental protection measures. This is the first nationwide representative study on nanoparticle use in the manufacturing sector. The information gained can be used for quantitative risk assessment. It can also help policymakers design strategies to support companies developing a safer use of nanomaterial. Notingthe current low use of nanoparticles, there is still time to proactively introduce protective methods. If the predicted "nano-revolution" comes true, now is the time to take action. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of occupational and Environmental Hygiene for the following free supplemental resource: a pdf file containing a detailed description of the approach to statistical analyses, English translation of the questionnaire, additional information for Figure 1, and additional information for the SUVA-code.] [Authors]

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .