2017 Fall Meeting
BIO- AND NANO MATERIALSU
Engineering surfaces to control and elucidate cellular response
A growing number of studies has been exploring the role of micro and nanostructured surfaces on the precise mechanisms that guide cellular and bacterial response. Adhering cells including bacteria sense and respond to the substrate’s physicochemical properties as well as surface mechanics and cell deformations at the micro- and nanoscale.
This symposium will cover recent advances on the mechanisms that control how mammalian cells and bacteria respond to micro- and nano-engineered surfaces. A particular emphasis will be given to the effects of the topographical (e.g. micro and nanostructures) and chemical (e.g. surface chemistry, surface functionalization, chemical gradients) environment, both at the micro and nanoscale, on the events that ultimately dictate cell fate, including cell migration, cell deformation and shape, biomechanical alterations, development of adhesion structures (e.g. focal adhesions, lamellopodia and filopodia), bacterial protrusions, proteins and gene expression, among others. The contribution of the physical/mechanical environment (e.g. substrate’s stiffness, stiffness gradients) to these events will also be considered. The symposium will consider the creation of novel ultra-small features that recently have been attracting much attention, such as nanometric clusters of adhesion molecules and cellular protrusions that have been referred to as nanopodia. Finally, the effects of micro- and nano-engineered surfaces under in vivo conditions will be distinctively addressed. A particular emphasis will be given to micro and nanofabrication technologies that allow to fabricate structures that mimic the natural architecture of the extracellular matrix (ECM) and biological tissues, highlighting emerging approaches that have permitted to achieve an increasingly more sophisticated ability to manufacture three-dimensional substrates to investigate cell response. These technologies will encompass both “bottom-up” and “top-down” strategies that permit to engineer and fine tune the physicochemical properties of substrates. In addition, the most advanced experimental techniques for both the characterization of surfaces (e.g. multimodal imaging) and cell response (e.g. super-resolution microscopy) will be considered.
Hot topics to be covered by the symposium:
- Cell- and bacteria-surface interactions;
- Micro- and nano-engineered substrates to control cell fate;
- Biomechanical and biochemical changes in response to micro and nanostructured surfaces;
- Cellular and bacterial adhesion on micro and nanostructured surfaces;
- Cell motility, shape and deformation;
- Antibacterial surfaces;
- Chemical and physical gradients;
- Micro and Nano-fabrication;
- Characterization techniques of surfaces and cellular response;
List of invited speakers (confirmed):
- Anna Lagunas [Institute for Bioengineering of Catalonia]
- Cornelia Lee-Thedieck [Karlsruhe Institute of Technology]
- Geetha Manivasagam [VIT University, India]
- Diego Mantovani [Laval University]
- Antonio Nanci [Université de Montréal]
- Ketul Popat [Colorado State University]
- Thomas J. Webster [Northeastern University]
Tentative list of scientific committee members:
- Fabio Variola [University of Ottawa]
- Krasimir Vasilev [University of South Australia]
- Elena Martinez [Institute for Bioengineering of Catalonia]
- Aldo Boccaccini [University of Erlangen-Nuremberg]
- Alessandro Lauria [ETH]
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Session 1 - Cell-surface interactions : -
Authors : A. Nanci1,2
Affiliations : 1Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dental Medicine, and 2Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada.
Resume : An attractive opportunity to improve the performance of medically-relevant metals is to alter their surface on the nanoscale. Our group introduced an oxidative method for nanocavitating the surface of Ti and its alloys by exposure to mixtures of H2SO4 and H2O2 . This generates a thin amorphous layer of TiO2 on the surface consisting of an intricate network of nanometric pores. The physicochemical characteristics of this mesoporous surface selectively influence the growth and activity of various cells in vitro, including stem cells, and promote osteogenic activity both in vitro and in vivo. These surfaces also simultaneously hamper the adhesion and/or retention of bacteria, in addition to affecting the integrity of a yeast variety. In this presentation, I will review these results and present recent structural and molecular data showing that nanocavitated titanium surfaces favor the formation and maturation of focal adhesions, as well as the extension of abundant filopodia that emit unique ultra-small lateral protrusion. Altogether, these are likely to have a major influence on the relationship of cells with surfaces, and on the activation of cell signaling pathways. We believe that the ability to nanocavitate thin mesoporous surface layers on materials of medical interest embodies a conceptual advance that allows a technology shift away from the inherent limitations of traditional adlayers. Supported by CIHR, FRQ_RSBO, NSERC and NIH.
Authors : Alejandra Rodriguez-Contreras, Dainelys Guadarrama, Antonio Nanci
Affiliations : Département de Biochimie et de Médecine Moléculaire, Laboratory for the study of calcified tissues & biomaterials, Faculté de médecine dentaire, Université de Montreal
Resume : Surface morphology influences material interactions between microorganisms, cells and extracellular matrix. Especially, biocompatible nano-structured surfaces control the cell behaviour and tissue integration processes of medical devices and implants. Stainless steel (SS) is one of the predominant materials used for the manufacture of medical devices, hospital equipment and instruments, surgical tools, and implants 1. For some of these applications, biocompatibility is essential, for others, an antimicrobial effect is required. Finding a simple way to attain these divergent objectives is clearly a daunting challenge. We have previously produced mesoporous Ti surfaces that interact selectively with different types of cells, and that simultaneously exhibit antimicrobial capacity 2-7. In this work, we have also found a way to similarly nanocavitate the surface of both stainless steel SS304 and SS316 with the purpose of controlling eukaryotic and prokaryotic cell behavior through surface topography. Thus, we show how high range anodization conditions using a mixture of H2SO4/H2O2 as an atypical electrolyte can efficiently nanocavitate the surface of SS to create a topography with advantageous biomedical characteristics. The characterization of the new nanocavitated SS surfaces revels an intricate 3D network of nanopores, forming a thin mesoporous layer of crystalline oxide. While deposited layers, unquestionably have some qualities, we submit that such thicker adlayers also have weaknesses and inherent limitations 8. Distinctively, the intricate 3D network of mesopores we obtained, is achieved within the bulk of the material. We also propose the combination of nanocorrosion/transpassivation/repassivation mechanisms for the understanding of its creation. In order to better understand its influence and effect on body cells, in vitro studies with fibroblasts, osteoblast and smooth muscular cells were performed. Nanocavitated surfaces have discerning impacts on cellular activity; in particular, adhesion and growth are promoted for osteogenic and smooth muscle cells, but not for fibroblasts. The fact that the created surface can selectively modulate cell activity, stimulate cell attachment, promote the formation of filopodia, and accelerate the maturation of focal adhesions is a major advance for the biocompatibility of SS alloys. In addition, the ability of our mesoporous surface to influence membrane fluidity, resulting in the formation of nanoscale lateral membrane protrusions, represents a novel cellular response that can be achieved by nanotopography. Bacterial essays were performed with both a gram positive and a gram negative bacterial strains. The treated surface exceptionally inhibits bacterial activity, which effect is relevant for preventing cross-infections without the use of synthetic active principles such as antibiotics, thus avoiding side effects in the body preventing drug-resistant infections. To our knowledge, this is the first time that surface treatment of SS can simultaneously achieve all these effects, and the obtained nanocavitated surface is actually the key for such successful and exciting results. Our strategy provides a simple, commercially attractive way to improve the material biocompatibility and control the adhesion of microorganisms, making nanostructured SS broadly useful in hospital environments, in manufacturing medical devices, as well as offering possibilities for non-medical applications. Thus, our work further highlights the fact that while sophisticated surface modification methodologies are undoubtedly powerful, there may be alternative approaches to achieve equivalent if not better outcomes.
Authors : Donata Kuczyńska, Agata Sotniczuk, Piotr Kwaśniak, Halina Garbacz
Affiliations : Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland
Resume : The aim of this study was to characterize the titanium grade 2 surface after laser modification, designed for biomedical applications. For this purpose, Ti grade 2 sheets were functionalized using the DLIL (Direct Laser Interference Lithography) technique in order to obtain surface with periodic/multimodal patterns. Method presented in this study was developed in order to locally functionalize the surface of prefabricated elements with original roughness. Fabrication of multimodal topography can be used to take advantage of favourable micro- and nano-roughness and to mimic the bone hierarchical surface structure after the remodelling process. Furthermore, appropriately designed texture of the surface may by beneficial for osteogenic passes of the stem cells. The hierarchical topography with roughness in a range from nano- to micrometers was characterized in terms of shape, roughness, chemical composition, microstructure and mechanical properties. In order to obtain all information, numerous research methods have been used: scanning electron microscopy, energy dispersive spectroscopy, optical profilometry, atomic force microscopy, micro hardness measurements and X-ray photoelectron spectroscopy. In order to estimate the potential use in biomedical applications, corrosion measurements in solution simulating the effect of body fluids were performed. Developed methodology can be used as an effective tool for manufacturing controlled surface structures improving the bone–implants interactions.
Session 2 - Cell-surface interactions : -
Authors : Cristina Plamadeala1, Habed Habibzadeh1, Sabrina V. Kirner3, Agnes Weth2, Jörg Krüger3, Jörn Bonse3, Armin Sebastian Guntner4, Achim Walter Hassel4, Werner Baumgartner2, Johannes Heitz1
Affiliations : 1. Institute of Applied Physics, Johannes Kepler University Linz, Linz, Austria; 2. Institute of Biomedical Mechatronics, Johannes Kepler University Linz, Linz, Austria; 3. Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany; 4. Christian Doppler Laboratory for Combinatorial Oxide Chemistry (at ICTAS), Johannes Kepler University Linz, Linz, Austria
Resume : The low density and high resistance to corrosion of Ti-6Al-4V alloy led to its wide use in biomechanical applications, such as implants and prosthesis. It is well known that not only surface topography, but also surface chemistry is an important factor that regulates cell adhesion on Ti-6Al-4V alloys. Therefore, in our study, the Ti-6Al-4V samples were processed with a femtosecond laser and then electrochemically treated. In a further step, fibroblasts were seeded on the samples. After 2 weeks in culture medium, a difference in cell morphology and cell number was observed. Contact angle measurements revealed that the laser-induced surface roughness and the electrochemical treatment together rendered the titanium alloy superhydrophilic. Moreover, biofilms were cultivated on the Ti samples, and a significant reduction of bacteria adhesion and growth was observed. In view of these observations, we consider that laser structuring together with chemical treatment make a powerful combination for controlling cellular and bacterial adhesions on Ti-6Al-4V alloys, which is of great significance in certain biomedical applications.
Authors : K. Brassat, J. Bürger, D. Kool, J. K. N. Lindner
Affiliations : Nanostructuring, Nanoanalysis and Photonic Materials (NNP), Dept. of Physics, Paderborn University, Paderborn, Germany; Center for Optoelectronics and Photonics Paderborn (CeOPP), Paderborn, Germany
Resume : The use of topographically and chemically patterned surfaces offers new possibilities to tailor the immobilization sites for biological entities. We present micro- and nanopatterning techniques, which allow for a highly adjustable surface modification: we use nanosphere lithography for the creation of regular surface patterns with motive sizes of few hundred nanometers to microns and block copolymer lithography for nanopore formation with pore diameters of sub-20 nm. Both techniques are based on self-assembly processes and are thus suitable for large-area surface patterning, exhibiting both, topographical and chemical surface design. We show patterned material thin films with regular arrays of free dot-like substrate areas, called antidot patterns. They exhibit chemical contrast by choosing combinations of substrate and thin film materials. The topography can be tailored in antidot size, film thickness and array pitch. The combination of nanosphere and block copolymer lithography allows for the creation of more advanced biomimetic hierarchical nanopore arrangements. Similar hierarchical surface patterns can be found on diatom frustules in nature. We have previously shown that these structured substrates can be used as templates for site-selective bio unit deposition such as protein micelles or DNA origami. In this contribution we present a detailed characterization of surfaces by correlating contact angle measurements, SEM, AFM and analytical cross-section TEM.
Authors : Geetha Manivasagam
Affiliations : Professor and Director,Centre for Biomaterials, Cellular and Molecular Theranostics (CBCMT) VIT University,Vellore, TN,India
Resume : Development of materials for dental and orthopedic applications has to an extent reached a saturation and most of the current research is focused on engineering the surfaces to prevent their failure. Failure on surfaces include corrosion, wear , bacterial infections and poor osseointegration. The success of an implant is dictated by the formation of long lasting integration with the surrounding bone. Several surface modification techniques have evolved to induce ossteointegration and improve the service period of an implant. Though several studies have demonstrated that the factors such as roughness, surface topography, surface energy, wettability and surface chemistry and microstructure plays a major on inducing osseointegration, till date there is no clear picture on the required surface factors that will lead to required bone bonding. Past research have well documented that the surface etching and blasting have improved osseointegration on Ti dental implants and coatings of hydroxyapatite on Ti orthopedic implants, the failure of these surface modified implants is still reported. A clear understanding of bone functioning and the molecular path ways that induce cell formation will throw more light on development of apt surfaces for osseointegration. Tailoring the surfaces at nano level has proven to induce superior bone bonding when compared to micron features and thus in this talk the research work carried out by our group on the development of novel surfaces on Titanium alloys using Anodization, EPD, SMAT and plasma treatment to enhance osseointegration will be presented. Research work carried out on understanding the pathways also will be discussed.
Session 3 - Cell-surface interactions : -
Authors : Kevin Bartlett (1), Sanli Movafaghi (1), Arun K. Kota (1,2) and Ketul C. Popat (1,2)
Affiliations : 1. Department of Mechanical Engineering, Colorado State University, Fort Collins CO 2. School of Biomedical Engineering, Colorado State University, Fort Collins CO
Resume : Many materials used for implants have excellent biocompatibility with different tissues in the body. However, in contact with the blood, occurrence of the incidents such as platelet/leukocyte adhesion and activation leads to further thrombosis and sometimes failure of these implants. It is well known that the platelet functionality can be modulated by the surface chemistry and the texture. Very few studies have investigated the effect of superhydrophobicity on hemocompatibility. Superhemophobic surfaces are extremely repellent to water (contact angle for water is greater than 150˚) and water droplets easily roll off from the surfaces. In this study, we have developed surfaces with different superhemophobic coatings. The surfaces were characterized using contact angle goniometry and X-ray photoelectron spectroscopy (XPS), and platelet adhesion and activation was investigated using fluorescence microscopy and scanning electron microscopy (SEM). The results indicate significantly better hemocompatibility of superhydrophobic surfaces as compared to unmodified surfaces.
Authors : Verónica Hortigüela (1), Enara Larrañaga (1), Francesco Cutrale (2), Anna Seriola (3), María García-Díaz (1), Anna Lagunas (4,1), Jordi Andilla (5), Pablo Loza-Alvarez (5), Josep Samitier (1,4,6), Samuel Ojosnegros (2), Elena Martínez (1,4,6)
Affiliations : (1) Institute for Bioengineering of Catalonia (IBEC), c/Baldiri Reixac 10-12, 08903 Barcelona, Spain; (2) University of Southern California, Translational Imaging Center, Molecular and Computational Biology, 105 Childs Way, Los Angeles, CA 90089, USA; (3) Center of Regenerative Medicine in Barcelona, Barcelona Biomedical Research Park, c/Dr. Aiguader 88, 08003 Barcelona, Spain; (4) Centro de Investigación Biomédica en Red (CIBER), Av. Monforte de Lemos 3-5, Pabellon 11, Planta 0, 28029 Madrid; (5) ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels (Barcelona), Spain; (6) Department of Engineering: Electronics, University of Barcelona (UB), c/Martí i Franquès 1-11, 08028 Barcelona, Spain.
Resume : Herein we present a nanostructured surface able to produce multivalent effects of surface-bound ephrinB1 ligands on the dynamics of oligomerization of EphB2 receptors. We create ephrin B1 nanopatterns of regular size (< 30 nm in diameter) by using self-assembled diblock copolymers. We then use an enhanced version of the Number and Brigthness technique, which can discriminate with molecular sensitivity the oligomeric state of diffusive species, to quantitatively track the EphB2 receptor oligomerization process in real time. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signalling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. This feature can benefit applications such as the design of new bioactive materials and drug-delivery systems.
Authors : Jordi Comelles1, Núria Berlanga1, Elena Martínez1, 2, 3
Affiliations : 1 Biomimetic Systems for Cell Engineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain 2 Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Zaragoza, Spain 3 Department of Engineering: Electronics, University of Barcelona, Barcelona, Spain
Resume : The physical properties of the extracellular matrix (ECM) play a key role in the regulation of cell processes that determine their fate and function. For example, it is well documented that cells can sense the stiffness of their environment, and change their differentiation lineage accordingly . In a similar way, cells exposed to topographical features can also change their differentiation profile . Other important cell functions, such as migration, are also affected by stiffness  and topography . Although the mechanisms by which cells sense stiffness have been extensively studied [5,6], much less is known about how cells sense the topography of their surroundings. The current view suggests that cells have conserved pathways for both stiffness and topography sensing . However, most of the works done on topographical patterns are carried out on stiff substrates, thus both cues are not disentangled. Here, we developed topographically modified substrates at the micrometer scale (2-10 µm) within a range of biologically relevant stiffness (from 6 to 160 kPa) and studied the response of 3T3 fibroblasts to topographical patterns of different stiffness. We used a method combining polyacrylamide chemical crosslinking and replica molding to obtain microstructured hydrogels of different stiffness. We then characterized the effect of the hydrogel swelling on the dimensions and shape of the microstructures for different rigidities by phase contrast and atomic force microscopies. We used 2, 5 and 10 µm wide lines to study the cell response to topography as a function of the stiffness. We found that cells aligned along the microstructured grooves, showing that their response to the topographical features is independent of the stiffness of the pattern in the range of stiffness studied. Interestingly, we observed that the cellular localization of Yes-Associated protein (YAP), a transcription factor involved in mechanosensing , was not altered by the presence of the topographical features, although it responded to the change in stiffness. Altogether, we present a platform that allows the study of topography sensing disentangled from the effects of the substrate stiffness. References  A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, Cell 126, 677 (2006).  M. J. Dalby, N. Gadegaard, R. Tare, A. Andar, M. O. Riehle, P. Herzyk, C. D. W. Wilkinson, and R. O. C. Oreffo, Nat. Mater. 6, 997 (2007).  C. M. Lo, H. B. Wang, M. Dembo, and Y. L. Wang, Biophys. J. 79, 144 (2000).  J. Comelles, D. Caballero, R. Voituriez, V. Hortigüela, V. Wollrab, A. L. Godeau, J. Samitier, E. Martínez, and D. Riveline, Biophys. J. 107, 1513 (2014).  A. Elosegui-Artola, E. Bazellières, M. D. Allen, I. Andreu, R. Oria, R. Sunyer, J. J. Gomm, J. F. Marshall, J. L. Jones, X. Trepat, and P. Roca-Cusachs, Nat. Mater. 13, 631 (2014).  S. Dupont, L. Morsut, M. Aragona, E. Enzo, S. Giulitti, M. Cordenonsi, F. Zanconato, J. Le Digabel, M. Forcato, S. Bicciato, N. Elvassore, and S. Piccolo, Nature 474, 179 (2011).  I. A. Janson and A. J. Putnam, J. Biomed. Mater. Res. - Part A 103, 1246 (2015).
Session 4 - Cell-surface interactions : -
Authors : Antonio Sechi, Alexander Töpel*, Andrij Pich (*presenting author)
Affiliations : DWI Leibniz Institute for Interactive Materials e.V. RWTH Aachen University
Resume : Aqueous nanogels are crosslinked colloidal polymer networks swollen in water. Nanogels exhibit unique properties like reversible deformability, surface activity, and stimuli-responsiveness and can display sensitivity to temperature, pH, light or ionic strength. In addition, nanogels can be used as building blocks for the design of well-ordered nanostructured soft materials of different dimensions and complexity.  By controlled self-assembly of nanogels in solution, on interfaces or surfaces defined architectures like colloidosomes, fibers, networks, arrays or films can be obtained. This contribution will focus on use of nanogels as building blocks for the decoration of biointerfaces. Different approaches for the attachment of nanogels to different surfaces by adsorption, use of anchoring peptides or formation of covalent bonds were developed. Recently we developed a new technique that allows printing nanogels on solid substrates.  Highly functional and stimuli-responsive nanogel arrays on glass surface were successfully fabricated by printing process using wrinkled PDMS templates. Using low-temperature plasma treatment, nanogels were chemically grafted onto glass supports thus leading to highly stable nanogel layers in cell culture media. Liquid cell AFM investigations showed that surface-grafted nanogel retained their swelling behaviour in aqueous media and that extracellular matrix protein coating did not alter both their stability and topography. We demonstrated that surface-grafted nanogels could serve as novel substrates for the analysis of cell adhesion and migration. Nanogels influenced size, speed and dynamics of focal adhesions as well as cell motility forcing cells to move along highly directional trajectories. Moreover, modulation of nanogel state or spacing served as an effective tool for regulation of cell motility. We suggest that nanogel arrays deposited on solid surfaces could be used to provide a precise and tuneable system to understand and control cell migration. Additionally, such nanogel arrays will contribute to the development of implantable systems aimed at supporting and enhancing cell migration during, for instance, wound healing and tissue regeneration.  A. Melle, A. Balaceanu, M. Kather, Y. Wu, E. Gau, W. Sun, X. Shi, A. Pich, J. Mater. Chem. 2016, DOI: 10.1039/C6TB01196A.  S. Hiltl, M. Schürings, A. Balaceanu, V. Mayorga, C. Liedel, A. Pich, A. Böker, Soft Matter 2011, 7, 8231-8238.  A. S. Sechi, S. Ullmann, J. M. G. Freitas, R. P. Takehara, P. Wünnemann, R. Schröder, M. Zenke, A. Böker, G. Aydin, S. Rütten, Andrij Pich, Adv Mater. Interf. 2016, DOI 10.1002/admi.201600455 .
Authors : Shutao Wang
Affiliations : Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, China
Resume : Circulating tumor cells (CTCs) have become an emerging “biomarker” for monitoring cancer metastasis and prognosis. Although there are existing technologies available for isolating/counting CTCs, the most common of which using immunomagnetic beads, they are limited by their low capture efficiencies and low specificities. By introducing a three-dimensional (3D) nanostructured substrate – specifically, a silicon-nanowire (SiNW) array coated with anti-EpCAM – we can capture CTCs with much higher efficiency and specificity. The conventional methods of isolating CTCs depend on biomolecular recognitions, such as antigen-antibody interaction. Unlikely, we here proposed that nanoscaled local topographic interactions besides biomolecular recognitions inspired by natural immuno-recognizing system. This cooperative effect of physical and chemical issues between CTCs and substrate leads to increased binding of CTCs, which significantly enhance capture efficiency. Recently, we have developed a 3D cell capture/release system triggered by enzyme, electrical potential and temperature as well as magnetic field, which is effective and of “free damage" to capture and release cancer cells. In addition, immune cells have also been employed as living template for greatly improving the limitation of traditional immunomagnetic beads. Furthermore, the potential pollution from biochip waste can be successfully disposed by employing self-cleaning substrates. Therefore, these bio-inspired interfaces open up a light from cell-based disease diagnostics to subsequent safety treatment of biomedical waste. 1. X. Liu, S. T. Wang*, Chem. Soc. Rev. 2014, 43, 2385-2401. 2. C. Huang, G.Yang, Q. Ha, J. Meng, S. T. Wang*, Adv. Mater. 2015, 27, 310-313. 3. Y. Li, Q. Lu, H. Liu, J. Wang, P. Zhang, H. Liang, L. Jiang, S. T. Wang*, Adv. Mater. 2015, 27, 6848-6854.
Authors : Xiangzhong Chen, Marcus Hoop, Fajer Mushtaq, Bradley J. Nelson, Salvador Pané
Affiliations : Multi-Scale Robotics Lab (MSRL), Institute of Robotics and Intelligent Systems (IRIS), ETH Zurich, Tannenstrasse 3, CH-8092 Zurich, Switzerland
Resume : Apart from mechanical and topographic cues, electrical cues can also regulate growth of cells. Current studies investigating the influence of electrical cues have mostly been shown on conductive material surface. However, conductive materials have limited application as scaffold because they require electric wires. Therefore, it is necessary to develop a scaffold with the ability of wirelessly generating localized electric output to induce tissue regeneration. Piezoelectric materials can generate transient surface charge variations when subject to mechanical deformation such as vibration induced by ultrasonic waves. This unique feature makes these materials ideal candidates for cell stimulation. In this presentation, we will show recent studies of cell growth behaviors on piezoelectric materials conducted in our lab. Neuron-like cells and bone-like cells are selected as models to study the applicability of piezoelectric materials. Piezoelectric materials, such as PVDF and PLLA, are made into either thin films or 3D porous scaffolds to support cell growth. We find that pure piezoelectric stimulation with ultrasound on neurite generation in neuron-like cells is comparable to stimulation induced by neuronal growth factor (NGF). The use of ultrasound in combination with piezoelectric polymers is advantageous since focused power can be transmitted deep into biological tissues, which holds great promise for the development of non-invasive tissue regenerative devices.
Authors : Young-Jin Kim, Dong-Gil Kim
Affiliations : Department of Biomedical Engineering, Catholic University of Daegu, Gyeongsan 38430, Republic of Korea
Resume : The cataract refers to the opacification of the crystalline lens or the lens capsule. Osmotic changes, protein aggregation, and slow down of the metabolic processes alter the proper transmission of light through the lens. The extraction of the opacified crystalline lens combined with intraocular lens (IOL) implantation is the first therapy for cataracts. Postoperative ocular inflammation and posterior capsule opacification (PCO) are the main concerns of the IOL implantation. Hydrophobic acrylic lenses have excellent properties such as remarkable optical transparency and low incidence of PCO. However, these hydrophobic acrylic lenses have higher surgically induced inflammation. Therefore, we prepared new highly biocompatible IOLs by introducing biomimetic materials such as 2-methyacryloyloxyethyl phosphorylcholine (MPC) for reducing postoperative ocular inflammation and PCO. We first fabricated hydrophobic acrylic IOLs containing different amount of MPC via radical polymerization at 50 °C under nitrogen. The resulting IOLs were transparent and exhibited smooth morphology. ATR-FTIR and XPS measurements confirmed the introduction of MPC to IOLs. Among the resulting samples, 20 wt% of MPC-introduced IOL (MPC20) showed significantly enhanced biocompatibility, meaning that the adsorption of proteins and the proliferation of epithelial cells on MPC20 were effectively suppressed. In addition, the pro-inflammatory cytokine expression was also substantially restrained on MPC20. Based on these results, the hydrophobic acrylic IOLs containing biomimetic material may contribute to the development of a new generation of IOL for reducing postoperative ocular inflammation and PCO.
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Session 5 - Cell-surface interactions : -
Authors : Cornelia Lee-Thedieck
Affiliations : Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces
Resume : Blood is replenished with billions of fresh cells every day throughout the entire life span. The source of these cells are the so-called hematopoietic (= blood forming) stem cells (HSCs). Their ability to reconstitute the entire blood system makes them the key to the cure of many hematological diseases. Upon transplantation from a healthy donor, they are able to replace the diseased hematopoietic system of the patient. However, this treatment is restricted by the limited availability of HSCs. To overcome that limitation, controlling HSC behavior in terms of proliferation or differentiation in vitro, is an important goal of nowadays HSC research. In vivo HSCs are controlled by a highly specialized microenvironment – the niche – within the bone marrow. In this niche HSCs are supported by mutual cell-cell as well as cell-matrix interactions. While it is clear that biological and/or chemical parameters play an important role in this interplay, surprisingly little attention was paid to physical signals that are transmitted by the niche microenvironment. In the last years, we found that these physical signals include matrix stiffness, nanostructure as well as the three-dimensional architecture. In reductionist approaches, in which we studied only one parameter at a time, we could show that all of these parameters impact HSC behavior. In order to achieve the goal of a synthetic stem cell niche to guide HSC behavior, the complexity of the natural HSC niche, which combines a variety of different signals, has to be taken into account. For this purpose, we increased the complexity of our systems to study the synergistic effects of different biological and/or physical signals. With these experiments we hope to get one step closer towards a synthetic stem cell niche that is as simple as possible but as complex as necessary to instruct HSCs.
Authors : Insung S. Choi
Affiliations : Center for Cell-Encapsulation, Department of Chemistry, KAIST, Daejeon 34141, Korea
Resume : Topography, the physical characteristics of an environment, is one of the most prominent stimuli neurons can encounter in the body. Many aspects of neurons and neuronal behavior are affected by the size, shape, and pattern of the physical features of the environment. A recent increase in the use of nanometric topographies, due to improved fabrication techniques, has resulted in new findings on neuronal behavior and development. Factors such as neuron adhesion, neurite alignment, and even the rate of neurite formation have all been highlighted through nanotopographies as complex phenomena that are driven by intricate intracellular mechanisms. The translation of physical cues is a biologically complex process thought to begin with recognition by membrane receptors as well as physical, cell-to-surface interactions, but the internal biological pathways that follow are still unclear. In this respect, nanotopography would be a more suitable platform on which to study receptor interfaces than microtopography because of the subcellular topographical features that are relevant in scale to the receptor activity. Ultimately, the characterization of this unknown network of pathways will unveil many aspects of the behavior and intracellular processes of neurons, and play an important role in the manipulation of neuronal development for applications in neural circuits, neuroregenerative medicine and prostheses, and much more. . K. Kang, Y.-S. Park, M. Park, M. J. Jang, S.-M. Kim, J. Lee, J. Y. Choi, D. H. Jung, Y.-T. Chang, M.-H. Yoon, J. S. Lee, Y. Nam, I. S. Choi, Nano Lett. 2016, 16, 675-680. . K. Kang, S. Y. Yoon, S.-E. Choi, M.-H. Kim, M. Park, Y. Nam, J. S. Lee, I. S. Choi, Angew. Chem. Int. Ed. 2014, 53, 6075-6079. . K. Kang, S.-E. Choi, H.-S. Jang, W. K. Cho, Y. Nam, I. S. Choi, J. S. Lee, Angew. Chem. Int. Ed. 2012, 51, 2855-2858. . W. K. Cho, K. Kang, G. Kang, M. J. Jang, Y. Nam, I. S. Choi, Angew. Chem. Int. Ed. 2010, 49, 10114-10118. . M.-H. Kim, M. Park, K. Kang, I. S. Choi, Biomater. Sci. 2014, 2, 148-155. (Minireview)
Session 6 - Cell-surface interactions : -
Authors : Tonazzini I 1,2, Masciullo C 2, Van Woerden GM 3, Elgersma Y 3, Beltram F 2 and Cecchini M 2
Affiliations : 1 Fondazione Umberto Veronesi, Milano, Italy 2 NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Pisa, Italy 3 Department of Neuroscience, Erasmus Medical Center, Rotterdam, the Netherlands
Resume : In the brain, cells are exposed to extracellular stimuli determined by the micro/nano-environment. Here Focal Adhesions (FAs) act as sensors, contributing to the final neuronal pathfinding. Nano-engineered substrates can induce specific topographical stimuli to cells, resembling in vitro several features of the in vivo extracellular matrix cues, and consequently tune and study the processes that regulate neuronal sensing (e.g. neurite growth). Although the dynamics of neuronal extracellular sensing is emerging as crucial for connectivity, little is known about these processes in pathological conditions. Ubiquitin E3A ligase (UBE3A) has crucial functions in the brain, being its unbalance the most prevalent genetic origin for autisms. Changes in its expression levels indeed lead to neurodevelopmental disorders (lack to Angelman Syndrome, increase to 15dup-autism). Here, we study the contact guidance and morpho-functional features (e.g. axonal development) of Wild-Type, UBE3A-deficient and -overexpressing neurons by exploiting nano/micro-grooved substrates (GRs) with the aim to compare their capability to follow directional stimuli. We further analyze the effect of pharmacological treatments acting on cytoskeleton contractility for restoring correct contact guidance. We finally analyze the molecular processes regulating FA pathway by western blot and by EGFP-paxillin transfection and live-cell TIRF microscopy. Thanks to GRs, we show that UBE3A-deficient neurons have impaired contact guidance, which is linked to impaired FA development and pathway activation. This impaired sensing leads in particular to defective axonal polarization, which can be restored by the modulation of the actin contractility.
Authors : Ignasi Casanellas (1,2), Anna Lagunas (3,1,*), Iro Tsintzou (1), Yolanda Vida (4,5), Daniel Collado (4,5), Ezequiel Pérez-Inestrosa (4,5), Josep Samitier (1,3,2)
Affiliations : (1) Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; (2) Department of Engineering Electronics, University of Barcelona (UB), Barcelona, Spain; (3) Networking Biomedical Research Center (CIBER), Madrid, Spain; (4) Instituto de Investigación Biomédica de Málaga (IBIMA), Department of Organic Chemistry, Universidad de Málaga (UMA), Málaga, Spain; (5) Andalusian Centre for Nanomedicine and Biotechnology-BIONAND, Málaga, Spain
Resume : Cartilage is an avascular tissue in which intercellular communication is maintained mainly by passive diffusion or through gap junction (GJ) communication. The formation of a GJ connectivity network among cells is imperative for the proper development of the new cartilage tissue. We previously showed that nanopatterns based on arginine-glycine-aspartate (RGD) dendrimers with uneven local densities, allow to control cell-substrate adherence and influence stem cell differentiation towards cartilage (1,2). We here demonstrate that nanopatterned growth substrates facilitate the formation of a GJ connectivity network among human stem cells undergoing chondrogenesis. Results show that a medium concentration of RGD dendrimers, leading to intermediate cell-substrate adherence, promotes chondrogenic differentiation and the formation of an extended GJ network. This can be observed through the immunostaining quantification and 3D mapping of connexin 43 (Cx43) expression. (1) Lagunas, A., Castaño, A. G., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, E., Gorostiza, P., Claros, S., Andrades, A., Samitier, J. Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nano Research. 2014, 7(3): 399-409 (2) Lagunas, A., Tsintzou, I., Vida, Y., Collado, D., Pérez-Inestrosa, E., Rodriguez Pereira, C., Magalhaes, J., Andrades, A., Samitier, J. Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment. Nano Research. 2016, 1-13
Authors : Thomas J. Webster
Affiliations : 1st Past President, U.S. Society For Biomaterials, Department of Chemical Engineering, Northeastern University, Boston, MA 02115
Resume : There is an acute shortage of organs due to disease, trauma, congenital defects, and most importantly, age related maladies. The synthetic materials used in tissue engineering applications today are typically composed of millimeter or micron sized particles and/or fiber dimensions. Although human cells are on the micron scale, their individual components, e.g. proteins, are composed of nanometer features. By modifying only the nanofeatures on material surfaces without changing surface chemistry, it is possible to increase tissue growth of any human tissue by controlling the endogenous adsorption of adhesive proteins onto the material surface. In addition, our group has shown that these same nanofeatures and nano-modifications can reduce bacterial growth without using antibiotics, which may further accelerate the growth of antibiotic resistant microbes. Inflammation can also be decreased through the use of nanomaterials. Finally, nanomedicine has been shown to stimulate the growth and differentiation of stem cells, which may someday be used to treat incurable disorders, such as neural damage. This strategy also accelerates FDA approval and commercialization efforts since new chemistries are not proposed, rather chemistries already approved by the FDA with altered nanoscale features. This invited talk will highlight some of the advancements and emphasize current nanomaterials approved by the FDA for human implantation.
Session 7 - Cell-surface interactions : -
Authors : D. Altamura1, S.G. Pastore1 , M.G. Raucci2, I. Fasolino2, D. Mele3, F. Intranuovo4, A. Terzi1, E. Altamura4, F. Mavelli4, F. Scattarella5, D. Siliqi1, L. Ambrosio2, C. Giannini1
Affiliations : 1Institute of Crystallography (IC), National Research Council, Bari 70126, Italy 2Institute of Polymers, Composites, and Biomaterials (IPCB), National Research Council, Naples, Italy 3Department of Geology, University of Bari, 70126 Bari, Italy 4Chemistry Department , University of Bari, Via E. Orabona 4, I–70125 Bari, Italy 5Istituto Officina dei Materiali, National Research Council (IOM-TASC-CNR), Area Science Park – Basovizza, Bld MM SS 14, Trieste, 34149, Italy
Resume : Gelatin is a good candidate to mimic the chemical composition of natural collagen in tissue engineered scaffolds. Here, a porous scaffold made of bovine gelatin has been treated with a biomimetic solution and a proper peptide mimicking BMP-2 growth factor to obtain a system biocompatible, biomineralized, and therefore suitable for use in bone defect regeneration. The influence on the scaffold architecture of chemical and biological factors added during the fabrication process has been evaluated, as well as hMSCs differentiation and colonization of the scaffold. X-ray microtomography was employed to obtain direct images of the 3D distribution of mass density through absorption contrast to evaluate possible microscale structure/morphology variations in terms of porosity and interconnectivity of the scaffold due to the addition of the chemical and biological factors. Confocal microscopy was employed to obtain direct images of the 3D distribution of osteocalcin content through fluorescent probe (Cy5) labelling, representing a marker for osteogenic differentiation. Table top scanning X-ray microdiffraction has been used to provide information at the atomic scale, about the crystalline moiety of the sample resulting from the mineralization process. Extended sample areas with mm2 size were mapped by scanning SAXS with hundred micrometer spatial resolution, so as to unambiguously detect hydroxyapatite nanocrystals and obtain their distribution on large scales. D. Altamura et al. ACS Appl. Mater. Interfaces 2016, 8, 8728?8736. Con il contributo del Ministero degli Affari Esteri e della Cooperazione Internazionale, Direzione Generale per la Promozione del Sistema Paese
Authors : Valentina Dinca1#, Valentina Mitran2, Simona Brajnicov1,3, Anca Bonciu1,4, Laurentiu Rusen1, Maria Dinescu1 and Anisoara Cimpean2
Affiliations : 1Lasers, National Institute for Lasers, Plasma and Radiation Physics, Bucharest, Romania 2 University of Bucharest - Department of Biochemistry and Molecular Biology, Bucharest, Romania 3University of Craiova, Romania 4 University of Bucharest -Faculty of Physics,Bucharest, Romania
Resume : Graphene have received increasing attention in the last years for biomedical applications for bone regeneration research. Nevertheless, by combining its specific characteristics with those of natural bioactive protein, enhanced biointerfaces can be obtained. In this work, we explore the feasibility of using the graphene sericin composites as potential coatings for testing in vitro osteoblast behavior. Matrix Assisted Pulsed Laser Evaporation technique was used for obtaining uniform coatings, with variable roughness depending on the deposition parameters and target composition. Characterization and evaluation of the coated substrates were carried out using different techniques (Fourier transform infrared spectroscopy, contact angle measurements, atomic force microscopy). The cellular model used to evaluate the in vitro biocompatibility of sericin, graphene and sericin-graphene composite films was represented by MC3T3-E1 pre-osteoblasts. The cell culture-based studies (phase contrast microscopy, LDH and MTT assays) have proved biocompatibility and the osteoblast behavior was correlated with both chemical composition and roughness. Our results give indications on the use of graphene based composites for tissue engineering applications. Acknowledgement: The authors acknowledge financial support from Ministerul Educatiei nNationale and CNCS-UEFISCDI via project PCCA 213 and PN-II-RU-TE-2014-4-2434 (TE 24).
Authors : V. Dinca1, L. Rusen1, M. Icriverzi2,3, V. Malheiro4, L. E. Sima2, M. Uta2, N. Nichita-Branza2, A. Roseanu2, E. C. Sirigim4, P. Hoffmann4 and M. Dinescu1
Affiliations : 1National Institute for Lasers, Plasma and radiation Physics, Bucharest, Romania 2Institute of Biochemistry of the Romanian Academy IBAR, Bucharest, Romania 3University of Bucharest, faculty of Biology, Bucharest, Romania 4EMPA, Switzerland
Resume : One of the major challenge in biomaterials research nowadays to guarantee the long term success of an implant is the design of implantable systems which have controlled characteristics and for which the cell and tissue response can be predicted and tailored. Within this context, the design and processing of instructive surfaces with synergic mechanical, chemical or topographical properties capable of directing cell behaviour represent the main strategy. In this work, we propose laser processing of micro-structured polycarbonate with convex and concave topographies to be used for evaluating the role of materials surface topography on mammalian cell lines (macrophages, modified hepotocites HepaRGDsRed cells) responses. KrF Excimer laser ablation was used with half-tone masks to produce convex and concave topographies with controlled surface dimensional parameters. All the laser processed surfaces or replicated surfaces were analysed by Atomic Force Microscopy and Scanning Electron Microscopy. Contact angle measurements revealed a direct correlation with the material type and morphology of the sample. Moreover, PDMS replica starting from laser processed surfaces were used for in vitro studies as models for silicone implants or for hepatocite like cells differentiation. Mesenchymal stem cells in convex and concave topographies were compared to planar surfaces showed different behaviour from alignment to stretching depending on the topography and material stiffness while the macrophage shape and size was different in concave and convex PDMS replicated surfaces, but no correlation was found with the cell polarization state. We have further used replicated microstructured gradients of polydimethylsiloxane (PDMS) that allow three-dimensional manipulation in vitro, to monitor HepaRGDsRed differentiation in real time. The regulated expression of the DsRed reporter proved a valuable tool not only for rapid screening of novel cell growth substrates favoring cell differentiation, but also, to enrich the hepatocyte-like cell population by fluorescence-activated cell sorting to investigate liver-specific processes in vitro.
Authors : A. R. Gillett, S. D. Hodgson, G. C. Smith
Affiliations : Thornton Science Park, University of Chester, Chester, UK
Resume : Surfaces which prevent bacterial fouling through their physical structure represent a key area of research for food and medical technology. Correlating bacterial adhesion with the physicochemical properties of the surface has seen limited success. However, the ratio between the Liftshitz-Van der Waals and the electron donor component (ɣLW /ɣ-) has been found to be a good predictor of bacterial adhesion to unmodified biomedical polymers1. In this study, advancing and receding contact angles of water, formamide and diiodomethane were used to calculate the physicochemical properties by the Liftshitz-Van der Waals acid-base approach on laser surface engineered polyethylene terephthalate films. Changes in surface characteristics were evaluated through surface roughness and XPS. Morphological changes were observed by SEM and Light microscopes. Escherichia coli attachment was monitored using SEM and enumerated by total viable counts. Preliminary results suggest using advancing contact angle to calculate the surface energy components yields no relationship between E.coli attachment and the ɣLW /ɣ- ratio and also the total interaction energy ΔEtot. However when using the receding contact angle, the adhesion of E.coli reduces as the ratio between ɣLW/ɣ- decreases. This is significant because receding contact angles have previously been related to the adhesion properties of textured surfaces2. Chemical analysis of the engineered surfaces indicate that changes to the ɣLW, ɣ- components and the wettability of the laser engineered PET, are as a result of surface morphology changes rather than a modification of the chemical structure. Relating the ɣLW/ɣ- ratio to bacterial adhesion could provide a reliable method for predicting the antibiofouling capabilities of textured surfaces. 1. Moreira, J.M., Simões, M., Melo, L.F. & Mergulhão, F.J. Escherichia coli adhesion to surfaces–a thermodynamic assessment. Colloid and Polymer Science 293, 177-185 (2015). 2. Samuel, B., Zhao, H. & Law, K.-Y. Study of Wetting and Adhesion Interactions between Water and Various Polymer and Superhydrophobic Surfaces. The Journal of Physical Chemistry C 115, 14852-14861 (2011).
Session 8 - Nanoparticles in biological systems : -
Authors : NGUYEN TK THANH
Affiliations : Biophysics Group, Department of Physics and Astronomy, University College London
Resume : In this presentation I will present the most recent results of our group on synthesis and functionalisation of nanoparticles (Au nanorods, Au nanoworms, high magnetic moment iron oxide nanoparticles, as well as novel structure of magnetic core@shell) for biomedical applications such as photo- and magnetic induced hyperthermia cancer treatment. The interaction of nanoparticles and cells/biological systems are still poorly understood, it would be useful for the community to assess these systems and find a better way to have more systematic approach in studying these interactions. Fig 1. Fine tuning Au nanorods and their optical properties. Ref: 1. Pallares, R. M., Bosman, M., Thanh, N.T.K. *, and Su, X. (2016) Plasmonic multi-logic gate platform based on sequence-specific binding of estrogen receptors and gold nanorods. Nanoscale. 8: 19919–20126. Front cover. 2. Thanh, N. T. K. (2016) Preface of Theme issue ”Multifunctional nanostructures for diagnosis and therapy of diseases’. Interface Focus, 6: 20160077. 3. Baber, R., Mazzei, L., Thanh, N. T. K., Gavriilidis, A. (2016) Synthesis of silver nanoparticles using microfluidic impinging jet reactors. Journal of Flow Chemistry. 6: 268-278. 4. Pallares, R. M., Lim, S. H., Thanh, N.T.K.*, and Su, X. (2016) Growth of Anisotropic Gold Nanoparticles in Photoresponsive Fluid and Application to UV Exposure Sensing and Erythema Prediction. Nanomedicine. 11: 2845-2860 5. Mameli, V., Musinu, A., Ardu, A., Ennas, G. , Peddis, D. , Niznansky, D., Sangregorio, C., Innocenti, C., Thanh, N. T. K.* and Cannas, C. (2016) Studying the exclusive effect of Zn-substitution on the magnetic and hyperthermic properties of cobalt ferrite nanoparticles. Nanoscale. 8, 10124-10137. 6. Monteforte, M., Kobayashi, S., Tung, L. D., Higashimine, K., Mott, D. M., Maenosono, S., Thanh, N. T. K., and Robinson, I. K. (2016) Quantitative Two Dimensional Strain Mapping of Small Core-Shell FePt@Fe3O4 Nanoparticles. New Journal of Physics. 18: 033016 7. Hervault, A., Lim, M., Boyer, C., Dunn, A., Mott, D., Maenosono, S. and Thanh, N. T. K.* (2016) Doxorubicin loaded dual pH- and thermo-responsive magnetic nanocarrier for combined magnetic hyperthermia and targeted controlled drug delivery applications. Nanoscale. 8: 12152-12161 8. Hachani, R., Lowdell, M., Birchall, M., Hervault, A., Merts, D., Begin-Colin, S., Thanh, N.T.K.* Polyol synthesis, functionalisation, and biocompatibility studies of superparamagnetic iron oxide nanoparticles for potential MRI contrast agents. (2016) Nanoscale. 8, 3278-3287. 9. Pallares, R. M., Su, X., Lim, S. H., Thanh, N. T. K* (2016) Fine-Tuning Gold Nanorods Dimensions and Plasmonic Properties Using the Hofmeister Salt Effects. Journal of Material Chemistry C. 4: 53-61 10. Blanco-Andujar, C., Southern, P., Ortega, D., Nesbitt, S.A., Pankhurst, Q.A., and Thanh, N.T.K. (2016) Real-time tracking of delayed-onset cellular apoptosis induced by intracellular magnetic hyperthermia. Nanomedicine. 11: 121-136.
Authors : I. Ojea-Jiménez, R. Capomaccio, D. Mehn, I. Osório, G. Ceccone, F. Rossi, L. Calzolai, D. Gilliland
Affiliations : European Commission, Joint Research Centre, Directorate F – Health, Consumers and Reference Materials, Consumer Products Safety Unit (F.2), Via E. Fermi, 2749, 21027 Ispra (VA) Italy (for all the authors)
Resume : Functionalized nanoparticles are attractive candidates for use in diagnostic and therapeutic applications as they can be made with polyvalent surface chemistry which can be tailored to effectively bind biological targeting vectors. In practice, achieving the correct balance of targeting potential and stealth characteristic in the final NP conjugate is critical to the pharmacokinetics, biodistribution and toxicity. Researchers are now beginning to capitalize on this great potential but a number of major challenges remain undisclosed. A major limitation in developing the proposed multifunctional vectors is to ensure that they not only have the flexibility to accommodate multiple binding chemistries but to verify that each surface component is present in the appropriate concentration to fulfill its function. Unfortunately, it seems that there is currently an incomplete understanding and connection between the physico-chemical properties of a nanomaterial and its real functionalization state, and as a result it remains unclear how to optimally synthesize and chemically modify nanomaterials for biological applications. In this work, we make use of simple NP models (i.e. gold nanoparticles) to investigate first the effect of coating density of a poly(ethylene oxide) ligand on nonspecific protein adsorption and quantify the ratios of ligand molecules per particle. Then, we monitor the grafting density of covalently bound proteins per nanoparticle by controlling the ratio of 2 differently functionalized PEO-ligands on the surface of the particles. Strong evidence on functionalization is supported by a robust set of complementary characterization techniques (CLS, DLS, AF4, TEM, Z-potential, SDS-PAGE, CD, AUC and XPS) after systematic modification of the surface characteristics via fine-tuning of the reaction conditions. By understanding the influence of key parameters such as the density of particles, grafting density and arrangement of ligands as well as conformation of biomolecules, this study establishes principles for the rational design of multi-functionalized NPs with controlled biomolecule adsorption.
Authors : Kinga Matuła a); Łukasz Richter a); Adrian Silesian b); Natalia Derebecka b); Joanna Wesoły b); Mikołaj Grzeszkowiak c); Barbara Peplińska c); Stefan Jurga c); Elżbieta Wyroba d); Jan Paczesny a); Robert Hołyst a)
Affiliations : a) Institute of Physical Chemistry PAS, 44/52 Kasprzaka, 01-224 Warsaw, Poland b) Faculty of Biology, Adam Mickiewicz University, 89 Umultowska, 61-614, Poznań, Poland c) Nanobiomedical Centre, Adam Mickiewicz University, 85 Umultowska, 61-614, Poznań, Poland d) Nencki Institute of Experimental Biology PAS, 3 Pasteur Street, 02-093 Warsaw, Poland
Resume : Bacterial evolution under antibiotic pressure is currently the topic of great importance, as only recently strains resistant to all known available drugs emerged. Nanoparticles are still believed to be promising remedy. Here we unravel a physical mode of action of nanoparticles. We show that bacteria Escherichia coli, exposed to sharp ZnO nanorods, evolve in 72-hour experiment by developing mechanical resistance. The pressure exerted at the bacterium is from 0,1 to 100 MPa. Microscopic studies (TEM, SEM with EDS, cryo-SEM, confocal microscopy) revealed that resistant bacteria change their shape from rod-like to spherical and develop thicker and more dense cell wall. Gram-negative E. coli bacteria with thicker cell wall falsified Gram test. We identified around 25 point mutations in genome and changes in gene expression profile of bacteria exposed to nanorods. Our research demonstrates that evolution of bacteria under external physical stress occurs in short time scales comparable to acquisition of resistance to antibiotics.
Authors : C. C. Mardare1,2, Z. Gajarska3, K. Zelenka2,3, N. Müller4, A. W. Hassel1,2,3
Affiliations : 1 Christian Doppler Laboratory for Combinatorial Oxide Chemistry (COMBOX) at Institute for Chemical Technology of Inorganic Materials, Johannes Kepler University Linz, Altenberger Str. 69, 4040, Linz, Austria; 2 CEST Competence Center for Electrochemical Surface Technology, Viktor Kaplan Str. 2, 2700 Wiener Neustadt, Austria 3 Institute for Chemical Technology of Inorganic Materials, Johannes Kepler University Linz, Altenberger Str. 69, 4040, Linz, Austria; 4 Institute of Organic Chemistry, Johannes Kepler University Linz, Altenberger Str. 69, 4040, Linz, Austria;
Resume : The increase in the occurrence of nosocomial infections together with the development of antibiotics resistant microorganisms represent a problem which annually leads to hundreds of thousands avoidable deaths and billions of euros spent on health care only in Europe. One solution to decrease the spread and proliferation of these microorganisms is to develop bactericidal materials, especially for the use as inanimate surfaces in health care facilities. Our current work has been focused on the synthesis, characterization, antibacterial testing and mechanistic proof of powders from the mixed Mo-W system. Powders with discrete ratio of Mo/W ranging from pure MoO3 to pure WO3 with a step of 5 to 10 wt.% increase of W were synthesized by spray drying method, followed by calcination. The morphological characterization revealed the formation of spherical hollow particles and the crystalline phases change as a function of composition. Antibacterial tests performed on a model gram-negative bacterium (E. coli) related the composition variation and the phases present to the antibacterial activity, simultaneously providing a detailed screening of the activity vs. composition transition. Furthermore, the mechanism of action has been proposed relating media acidification and the inability of bacteria to live at low pH values . These powders show adequate features for being implemented in hybrid inorganic-polymer systems.  C. C. Mardare, A. W. Hassel, ACS Comb. Sci. 16 (2014) 631
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