Representative Publications

    J1. Fabrication, Characterization, and Evaluation in Drug Release Properties of Magnetoactive Poly(ethylene oxide)-Poly(l-lactide) Electrospun Membranes. Savva I, Odysseos AD. Evaggelou L, Marinica O, Vasile E, Vekas L, Sarigiannis Y,  Krasia –Christoforou T. Biomacromolecules 2013.Dec9:14(12):4436-46

    The fabrication of electrospun magnetoactive fibrous nanocomposite membranes based on the water-soluble and biocompatible poly(ethylene oxide) (PEO), the biocompatible and biodegradable poly(L-lactide) (PLLA) and preformed oleic acid-coated magnetite nanoparticles (OA.Fe3O4) is reported. Visualization of the membranes by electron microscopy techniques reveals the presence of continuous fibers of approximately 2 μm in diameter, with the magnetic nanoparticles being evenly distributed within the fibers, retaining at the same time their nanosized diameters (≈ 5 nm). Thermal gravimetric analysis measurements suggest that the magnetic nanoparticles embedded within the polymer fibers affect favorably the thermal stability of the membranes. Moreover, assessment of their magnetic characteristics by vibrating sample magnetometry discloses tunable superparamagnetic behavior at ambient temperature. For the first time, the biocompatibility and biodegradability of PEO/PLLA and the tunable magnetic activity of the OA.Fe3O4 are combined in the same drug delivery system, with N-acetyl-p-aminophenol (acetaminophen) as a proof-of-concept pharmaceutical. Furthermore, their heating ability under alternating current (AC) magnetic field conditions is evaluated using frequency of 110 kHz and corresponding magnetic field strength of 25 mT (19.9 kA/m). Consequently, these magnetoactive fibrous nanocomposites exhibit promising characteristics for future exploitation in magnetothermally triggered drug delivery.

    J2. The role of constitutive behaviour interaction with the host tissue on the state of stress and growth of solid tumors. Voutouris C, Mpekris F, Michael S, Papageorgis P, Odysseos AD, Stylianopoulos T. PlosOne2014. Aug11: 9(8):1-9.

    Mechanical forces play a crucial role in tumor patho-physiology. Compression of cancer cells inhibits their proliferation rate, induces apoptosis and enhances their invasive and metastatic potential. Additionally, compression of intratumor blood vessels reduces the supply of oxygen, nutrients and drugs, affecting tumor progression and treatment. Despite the great importance of the mechanical microenvironment to the pathology of cancer, there are limited studies for the constitutive modeling and the mechanical properties of tumors and on how these parameters affect tumor growth. Also, the contribution of the host tissue to the growth and state of stress of the tumor remains unclear. To this end, we performed unconfined compression experiments in two tumor types and found that the experimental stress-strain response is better fitted to an exponential constitutive equation compared to the widely used neo-Hookean and Blatz-Ko models. Subsequently, we incorporated the constitutive equations along with the corresponding values of the mechanical properties - calculated by the fit - to a biomechanical model of tumor growth. Interestingly, we found that the evolution of stress and the growth rate of the tumor are independent from the selection of the constitutive equation, but depend strongly on the mechanical interactions with the surrounding host tissue. Particularly, model predictions - in agreement with experimental studies - suggest that the stiffness of solid tumors should exceed a critical value compared with that of the surrounding tissue in order to be able to displace the tissue and grow in size. With the use of the model, we estimated this critical value to be on the order of 1.5. Our results suggest that the direct effect of solid stress on tumor growth involves not only the inhibitory effect of stress on cancer cell proliferation and the induction of apoptosis, but also the resistance of the surrounding tissue to tumor expansion.

    J3. Stylianopoulos T, Kokonou M, Michael S, Tryfonos A, Rebholz C, Odysseos AD, Doumanidis C. Tensile mechanical properties and hydraulic permeabilities of electrospun cellulose acetate fiber meshes. J Biomed Mater Res B Appl Biomater. 2012 Nov;100(8):2222-30. doi: 10.1002/jbm.b.32791. Epub 2012 Aug 9.

    The mechanical properties and hydraulic permeabilities of biomaterial scaffolds play a crucial role in their efficacy as tissue engineering platforms, separation processors, and drug delivery vehicles. In this study, electrospun cellulose acetate fiber meshes of random orientations were created using four different concentrations, 10.0, 12.5, 15.0, and 17.5 wt % in acetone or ethyl acetate. The tensile mechanical properties and the hydraulic permeabilities of these meshes were measured, and a multiscale model was employed to predict their mechanical behavior. Experimentally, the elastic modulus ranged from 3.5 to 12.4 MPa depending on the polymer concentration and the solvent. Model predictions agreed well with the experimental measurements when a fitted single-fiber modulus of 123.3 MPa was used. The model also predicted that changes in fiber alignment may result in a 3.6-fold increase in the elastic modulus for moderately aligned meshes and a 8.5-fold increase for highly align meshes. Hydraulic permeabilities ranged from 1.4 x 10(-12) to 8.9 x 10(-12) m(2) depending on polymer concentration but not the choice of solvent. In conclusion, polymer concentration, fiber alignment, and solvent have significant impact on the mechanical and fluid transport properties of electrospun cellulose acetate fiber meshes

    J4. Khodr Issa, Panayiota Stylianou, Ana-Belen D’Avilla-Ibanez, Sofia Iliopouloa, Jean-Michel Siaugue, Andreas, Andreas Evdokiou, Triantafyllos Stylianopoulos, Costas Pitris and Andreani D. Odysseosa   Spatiotemporal Distribution of Silica-Coated, Dye-Doped Machemite Nanoparticles in Normal Brain Tissue and Orthotopic Glioblastoma Models (Under publication. Nanomedicine)

    Therapeutic responses of Glioblastoma Multiforme to small molecule targeted therapies remain insufficient mainly due to inadequate uptake by the invasive tumor peripheral zone.  We introduce end-functionalized, pegylated silica-coated, bimodal nanoparticles capable to preferentially accumulate in these regions. Spatial or temporal distribution of either nanoparticle systems across diverse regions of orthotopic glioblastoma tumors and their correlation with blood vessel characteristics, has been quantified by the nanoparticle fluorescence intensity per tissue area, which is analogous to nanoparticle concentration, both outside the tumor and inside as a function of distance from the tumor rim, applying  image processing algorithms. Anionic and smaller size nanoparticles have followed a more favorable distribution pattern within the highly vascularized tumor boundaries. Their correlation with tumor vessel characteristics has been verified by numerical modelling. These findings introduce a promising solution for delivery of therapeutics within the invasive tumor zone, prevent glioblastoma recurrence and reduce morbidity.

    J5. Patent: EP 1517412121403 and USPO 9,890,187: Prototype systems of theranostic biomarkers for in vivo molecular management of cancer (Odysseos AD, Pitris C, Keramidas A).

    Representative Projects

    P1. Management of Resistance to Tyrosine Kinase Inhibitors with Advanced Nanosystems. FP7-PEOPLE-IAPP: NANORESISTANCE 286125: Nov 2011-October 2015.  € 1,400.000. Principal Investigator: A. Odysseos.

    NANORESISTANCE introduces for the first time (i) receptor -independent targeting of Epidermal Growth Factor Receptor-kinase activity, (ii) nuclear delivery of anti-Epidermal Growth Factor Receptor therapy with novel grafting techniques and (iii) the deciphering of resistance and lack of responsiveness to anti-EGFR therapies in the preclinical setting with mathematical models of interstitial biodistribution. This work defines an unprecedented integrated approach for the comprehensive management of failure to anti-EGFR therapy and treatment monitoring. This partnership will play a structuring role by allowing researchers to acquire key skills equally relevant to the public and private sectors including cutting edge nanobiotechnology techniques for fabrication of nanotheranostic conjugates for targeted nuclear drug delivery and imaging, pioneering approaches for intracellular targeting with carbon nanotubes (CNT), innovative mathematical models and assessment of biodistribution, state-of-the-art Surface Plasmon Resonance for assessing drug-target interactions, emerging technologies for in vivo protein-protein and theranostic compound-protein interaction with Bimolecular Fluorescence Complementation Assays (BIFCs).These parallel approaches provide a promising innovative solution in the multifaceted challenge of the overall resistance to anti-EGFR therapies. This will be achieved with the development of multimodal CNT-based nanoplatforms carrying the fluorescent conjugates of EGFR inhibitors intracellularly independently of EGFR extracellular recognition. This system will further deliver anti-EGFR and fluorescent attributes to the nucleus. The partnership offers and a well-structured scheme of complementary skills highly inspired by the entrepreneurial spirit of academicians and research commitment of the industrial partners securing significant impact on their employability in their sector.

    P2. Mechanistic Approaches in Cellular and Biological Barriers- Sub-project: The Blood-Brain Tumour Barrier  FP7-PEOPLE-ITN: PATHCHOOSER: October 2013-September 2017, € 3,178.803 (Lead of Training, A. Odysseos)

    Nanomedicine offers capability to significantly change the course of treatment for lifethreatening diseases. Many of the most significant current therapeutic targets, to be viable in practice, require the efficient crossing of at least one biological barrier. However, the efficient and controlled crossing of the undamaged barrier is difficult. The range of small molecules that can successfully do so (via diffusive or other non-specific processes) is limited in size and physiochemical properties, greatly restricting the therapeutic strategies that may be applied. In practice, after several decades of limited success, there is a broad consensus that new multidisciplinary, multi-sectoral strategies are required. Key needs include detailed design and understanding of the bionano-interafce, re-assessment of in vitro models used to assess transport across barriers, and building regulatory considerations into the design phase of nanocarriers. The overarching premises of the PathChooser ITN are that (i) significant advances can only be made by a more detailed mechanistic understanding of key fundamental endocytotic, transcytotic, and other cellular processes, especially biological barrier crossing; (ii) elucidating the Mode of Action / mechanism of successful delivery systems (beyond current level) will ensure more rapid regulatory and general acceptance of such medicines. Paramount in this is the design and characterization of the in situ interface between the carrier system and the uptake and signalling machinery. (iii) inter-disciplinary knowledge from a range of scientific disciplines is required to launch a genuine attack on the therapeutic challenge. The PathChooser ITN program of research and training will equip the next generation of translational scientists with the tools to develop therapies for a range of currently intractable (e.g. hidden in the brain) and economically unviable diseases (e.g. orphan diseases affecting a limited population).

    P3. Multipotent Theranostic Metal-Based Scaffolds for Molecular Targeting of Colorectal Cancer (Cyprus Research Promotion Foundation; 2012-2015) June. 2012- May. 2014. € 178.803. Project Coordinator: C. Pitris ; Partner: A. Odysseos

    This project brings together expertise in medical optics and optoelectronics, magnetics, coordinate Lanthanide chemistry, bio-organic synthesis and molecular oncology, aiming at introducing a break-through solution for the overall management of colorectal cancer and potentially other malignancies expressing the epidermal growth factor family of receptors (EGFR). The solutions is based on prototype metal-based functionalized compounds engaging cutting-edge synthetic and complexation approaches. Initially, a panel  of kinetically stable prototype lanthanides complexes with potent (a) optical (e.g near infrared spectroscopic) and (b) magnetic /relaxation properties will be designed and synthesized as building blocks for heterometallic arrays and subsequently the lanthanide complexes will be functionalized to a series of biologically active bimodal theranostic agents with anti-EGFR recognizing and inhibiting properties.  Functionalization of the probes with the recognizing and therapeutic ligands (i.e. anilinoquinazolines) will increase the molecular size, thus optimizing proton relaxivity and magnetic efficacy. Diagnostic specificity will be assessed by ERB-B1 recognition and internalization of the compounds on (a) cell monolayers and (b) 3-dimensional tissue phantoms of cell lines differentially expressing ERB-B1 under inverted NIR microscope. Highly sensitive and efficacious compounds will be considered for further development as theranostic agents for early detection and therapy of CRC in subsequent pre-clinical models with the application of multimodal Molecular Imaging. This approach enables the quantitative imaging of defined cancer biomarkers in a non-invasive manner, aiding in lesion detection, patient stratification, new drug development and validation, dose optimization and treatment monitoring.

    P4.   Nanofiber Electrospinning and Rapid Prototyping for Intestinal Tissue Engineering (Cyprus Research Promotion Foundation; 2009-2011). €137.688   Partner and Deputy Coordinator, A. Odysseos

    Adaptation of Nanofiber Electrospinning for Rapid Prototyping: A mesoscale-resolution electrospinning RP system based on a 3-axis CNC machine was set up and configured. A collector for layered deposition based on spraying of graphite microparticles in sacrificial intermediate layers was tested, as well as a polystyrene foil mask on the target to patern electrospun areas The innovative idea of structuring of the electrospun fiber membrane surface to conform to the epithelial geometry of intestinal tissue by patterned electrochemical roughening of the metal foil target, onto which the fibers are deposited during electrospinning, was introduced. For software integration, an electrospinning process-structure database was assembled by parametric studies for cellulose acetate , and is to be combined with imaging software (STL format) for tissue geometry encoding .

    Tissue model design 1 was based on surgical collection of 37 tissue specimens at the Nicosia General Hospital, with tissues already imaged by optical microscopy, SEM and 3D laser scanning, white-light profilometry and X-ray microtomography, with SEM proving the most appropriate technique. Fabrication of tissue scaffolds was performed at the HO with SEM parametric studies for electrospinning of fiber membranes made of CA (also with PANI), PEO, PLLA and PEO/PLLA mixtures. Functional analysis was realized with dynamic mechanical analysis (DMA) and rheological analysis of a great variety of CA nanofiber membranes.  SEM analysis was performed on the cell-cultured scaffolds for subsequent intestinal tissue processing and engineering. Tissue phantom have been developed with intestinal cells incorporated in a collagen mixture. The partners introduced the family of vitamin E derivatives (tocopherols-tocotrienols) as a new super-family of growth and differentiation factors in the normal intestinal cell.

    P5. Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells. COST CM1106 (2012-2016).

    This COST Action aims to unite researchers with expertise in rational drug design and the medicinal chemistry of synthetic and natural compounds with biomedical investigators dedicated to the understanding the mechanisms governing drug resistance in cancer stem cells. Cancer stem cells (CSC) are a subpopulation of cells within tumors that exhibit enhanced tumor-initiating attributes and are a major contributing factor to current cancer therapy failure. The CSC phenotypic state comprises distinct molecular and functional differences that underpin resistance to current treatments and the unique ability spread and to seed new tumors throughout the body. This insight necessitates an entirely new approach to cancer drug development to effectively target tumor CSCs. Through exchange of information, experience and expertise, researcher mobility and fostering new collaboration between chemistry and biology groups, the Action endeavours to develop new, effective methods for identifying novel compounds and drug candidates that target drug-resistant cancer stem cells.

    Representative Publications

    J1. T. Nakano, Y. Okaie, S. Kobayashi, T. Koujin, C.-H. Chan, Y.-H. Hsu, T. Obuchi, T. Hara, Y. Hiraoka, and T. Haraguchi, “Performance evaluation of leader-follower-based mobile molecular communication networks for target detection applications,” IEEE Transactions on Communications, vol. 65, no. 2, pp. 636-676, 2017.

    This paper proposes a leader–follower-based model of mobile molecular communication networks for target detection applications. The proposed model divides the application functionalities of molecular communication networks into two types of mobile bio-nanomachine: leader and follower bio-nanomachines. Leader bio-nanomachines distribute in the environment to detect a target and create an attractant gradient around the target. Follower bio-nanomachines move according to the attractant gradient established by leader bio-nanomachines; they approach the target and perform necessary functionalities, such as releasing drug molecules. This paper develops mathematical expressions for the proposed model, describes wet laboratory experiments designed to estimate model parameters, and performs biologically realistic computer simulation experiments to evaluate the performance of the proposed model.

    J2. T. Nakano, S. Kobayashi, T. Suda, Y. Okaie, Y. Hiraoka, and T. Haraguchi, “Externally Controllable Molecular Communication,” IEEE Journal on Selected Areas in Communications, vol. 32, no. 12, pp. 2417-2431, 2014.

    An open research issue in molecular communication is to establish interfaces to interconnect the molecular communication environment (e.g., inside the human body) and its external environment (e.g., outside the human body). Such interfaces allow conventional devices in the external environment to control the location and timing of molecular communication processes in the molecular communication environment and expand the capability of molecular communication. This paper first describes an architecture of externally controllable molecular communication and introduces two types of interfaces for biological nanomachines; bio-nanomachine to bio-nanomachine interfaces (BNIs) for bio-nanomachines to interact with other biological nanomachines, and inmessaging and outmessaging interfaces (IMIs and OMIs) for bio-nanomachines to interact with devices in the external environment. This paper then describes a proof-of- concept design and wet laboratory implementation of the IMI and OMI, using biological cells. It further demonstrates how an architecture of externally controllable molecular communication with BNIs and IMIs/OMIs may apply to pattern formation, a promising nanomedical application of molecular communication.

    J3. T. Nakano, T. Suda, Y. Okaie, M. Moore, A. V. Vasilakos, “Molecular Communication among Biological Nanomachines: A Layred Architecture and research issues, ” IEEE
    Transactions on Nanobioscience, vol. 13, no. 3, pp. 169-197, 2014.

    Establishing a layered architecture of molecular communication helps organize various research issues and design concerns into layers that are relatively independent of each other, and thus accelerates research in each layer and facilitates the design and development of applications of molecular communication. This paper describes the layered architecture of molecular communication to identify research issues that molecular communication faces at each layer of the architecture. Specifically, this paper applies a layered architecture approach, traditionally used in communication networks, to molecular communication, decomposes complex molecular communication functionality into a set of manageable layers, identifies basic functionalities of each layer, and develops a descriptive model consisting of key components of the layer for each layer.

    J4. T. Nakano, A. Eckford, T. Haraguchi, Molecular Communication, Cambridge University Press, 2013.

    The book, molecular communication, is a comprehensive guide to research on molecular communication. It starts by describing biological nanomachines, the basics of biological molecular communication and the microorganisms that use it. It then proceeds to engineered molecular communication and the molecular communication paradigm, with mathematical models of various types of molecular communication and a description of the information and communication theory of molecular communication. Finally, it presents the practical aspects of designing molecular communication systems including a review of the key applications.

    J5. T. Nakano, M. Moore, F. Wei, A. V. Vasilakos, J. Shuai, “Molecular Communication and Networking: Opportunities and Challenges”, IEEE Transactions on Nanobioscience, vol. 11, no. 2, pp. 135-148, 2012.

    This paper presents the state-of-the-art in the area of molecular communication by discussing its architecture, features, applications, design, engineering, and physical modeling. It also discusses challenges and opportunities in developing networking mechanisms and communication protocols to create a network from a large number of bio-nanomachines for future applications.

    Representative Projects

    P1. Molecular communication – standardization and medical applications. JSPS KAKENHI JP 17H00733. April 2017 – March 2022. JPY43,290,000. Principal Investigator: T. Nakano.

    This project (currently underway) aims to design and develop medical applications of molecular communication, and to standardize a set of protocols, models, tools to investigate medical applications of molecular communication. It focuses on drug delivery by bio-nanomachines in blood vessels and interactions between bio-nanomachines and cancer cells. Mathematical modelling, computer simulations and web laboratory experiments are to be conducted.

    P2. Inbody bionano-sensor networks: JSPS KAKENHI JP16K12416. JPY3,380,000. April 2016 – March 2018.

    This project (currently underway) explores epidemic-based mechanisms to quickly disseminate information among mobile bio-nanomachines. It first develops a model of bio-nanomachines based on the observation of how biological cells migrate in the environment. It then uses the model to simulate collective dynamics and evaluate the performance of the proposed information dissemination method. It also derives performance bounds analytically. Further, it shows how the proposed method apply to target detection and tracking applications.

    P3. Molecular communication – systems engineering. JSPS KAKENHI JP25240011. April. 2013 – March 2017. JPY 36,270,000. Principal Investigator: T. Nakano.

    This project investigated systems engineering methods for molecular communication. One of the research challenges in molecular communication is to develop interfaces between bio-nanomachines (a-c) and between a bio-nanomachine and an external device. This project extended the previously proposed architecture with these interfaces, performed proof-of-concept experiments, and conducted numerical experiments to demonstrate the potential of the extended architecture.

    P4. Biologically compatible communication systems. JSPS KAKENHI JP22680006. April. 2010 – March 2013. JPY 15,860,000. Principal Investigator: T. Nakano.

    This project investigated a biologically compatible communication system. In particular, this project focused on the design and engineering of networking mechanisms based on epithelial cells, gap junction channels and calcium signalling. It designed signal amplification mechanisms, identified through computer simulations conditions that signals are amplified and propagated, and validated the design through wet laboratory experiments.

    P5. Molecular communication – a new ICT paradigm. SCOPE. JPY16,126,000. August 2010 – March 2013. Principal Investigator: T. Nakano.

    This project investigated theoretically physical layer characteristics of molecular communication. It developed the mathematical and simulation models of diffusion-based molecular communication (A), confined diffusion-based molecular communication (B), reaction-diffusion-based molecular communication (C) and  diffusion-with-drift-based molecular communication (D), and identified their physical layer characteristics such as distance and speed of communication. This project also chose calcium signalling as an instance of molecular communication and investigated the physical layer characteristics.

    Representative Publications

    J1. M. Donohoe, S. Balasubramaniam, B. Jennings, J. M. Jornet, “Nanodevice Arrays for Peripheral Nerve Fascicle Activation Using Ultrasound Energy-harvesting”, to appear in IEEE Transactions on Nanotechnology, 2017.

     We propose the use of wireless, energy-harvesting, implanted nanodevice arrays with electrodes for selective stimulation of peripheral nerves in the human body. We calculate the input ultrasound energy and harvested power for single fixed-size nanowire-based nanodevices at different tissue depths and compare these with the current and voltage levels required for peripheral neural stimulation. We model the dimensioning of arrays of nanodevices, embedded in biocompatible tissue patches, to meet these neural stimulation requirements. Selectivity of activation of particular nerve bundles requires that the output voltage and current of the array can be varied to increase or decrease penetration into the neural tissue. This variation can be achieved by changing the energised area of the array and/or by decreasing the incident ultrasound power. However, the array must be implanted horizontally relative to the incident ultrasound as any tilting of the nanodevices will reduce the harvested energy. The proposed approach provides a long-term implant solution for nerve stimulation that allows the patient greater freedom of movement than with embedded tethered electrodes.

    J2. B. D. Unluturk, S. Balasubramaniam, I. F Akyildiz, “The Impact of Social Behavior on the Attenuation and Delay of Bacterial Nanonetworks”, IEEE Transactions on Nanobioscience, vol. 15, no. 8, 2016.

    Molecular communication (MC) is a new paradigm for developing communication systems that exchanges information through the transmission and reception of molecules. One proposed model for MC is using bacteria to carry information encoded into DNA plasmids, and this is termed bacterial nanonetworks. However, a limiting factor in the models that have been studied so far is the environment considered only in ideal conditions with a single population. This is far from realistic in natural environments, where bacteria coexist in multiple populations of same and different species, resulting in a very complex social community. This complex community has social interactions that include cooperation, cheating, as well as competition. In this paper, the effects of these social interactions on the information delivery in bacterial nanonetworks are studied in terms of delay, attenuation and data rate. The numerical results show that the cooperative behavior of bacteria improves the performance of delay and attenuation leading to a higher data rate, and this performance can be degraded once their behavior switches towards cheating. The competitive social behavior shows that the performance can degrade delay as well as attenuation leading to slower data rates, as the population with the encoded DNA plasmids are prevented from reaching the receiver. The analysis of social interactions between the bacteria will pave the way for efficient design of bacterial nanonetworks enabling applications such as intrabody sensing, drug delivery, and environmental control against pollution and biological hazards.

    J3. S. A. Wirdatmadja, M. T. Barros, Y. Koucheryavy, J. M. Jornet, S. Balasubramaniam, “Wireless Optogenetic Nanonetworks for Brain Stimulation: Device Model and Charging Protocols”, IEEE Transactions on Nanobioscience, vol. 16, no. 8, 2017

    In recent years, numerous research efforts have been dedicated toward developing efficient implantable devices for brain stimulation. However, there are limitations and challenges with the current technologies. They include neuron population stimulation instead of single neuron level, the size, the biocompatibility, and the device lifetime reliability in the patient’s brain. We have recently proposed the concept of wireless optogenetic nanonetworking devices (WiOptND) that could address the problem of long term deployment, and at the same time target single neuron stimulation utilizing ultrasonic as amode for energy harvesting. In addition, a number of charging protocols are also proposed, in order to minimize the quantity of energy required for charging, while ensuring minimum number of neural spike misfirings. These protocols include the simple charge and fire, which requires the full knowledge of the raster plots of neuron firing patterns, and the predictive sliding detection window, and its variant Markov-chain based time-delay patterns,whichminimizes the need for full knowledge of neural spiking patterns as well as number of ultrasound charging frequencies. Simulation results exhibit a drop for the stimulation ratio of _ 25% and more stable trend in its efficiency ratio (standard deviation of _0.5%) for the Markov-chain based time-delay patterns protocol compared with the baseline change and fire. The results show the feasibility of utilizing WiOptND for long-term implants in the brain, and a new direction toward precise stimulation of neurons in the cortical microcolumn of the brain cortex.

    J4 A. Giaretta, S. Balasubramaniam, M. Conti, “Security Vulnerabilities and Countermeasures for Target Localization in Bio-NanoThings Communication Networks”, IEEE Transactions on Information Forensics and Security, vol. 11, no. 4, 2016.

    The emergence of molecular communication has provided an avenue for developing biological nanonetworks. Synthetic biology is a platform that enables reprogramming cells, which we refer to as Bio-NanoThings, that can be assembled to create nanonetworks. In this paper, we focus on specific Bio-NanoThings, i.e, bacteria, where engineering their ability to emit or sense molecules can result in functionalities, such as cooperative target localization. Although this opens opportunities, e.g., for novel healthcare applications of the future, this can also lead to new problems, such as a new form of bioterrorism. In this paper, we investigate the disruptions that malicious Bio-NanoThings (M-BNTs) can create for molecular nanonetworks. In particular, we introduce two types of attacks: blackhole and sentry attacks. In blackhole attack M-BNTs emit attractant chemicals to draw-in the legitimate Bio-NanoThings (L-BNTs) from searching for their target, while in the sentry attack, the M-BNTs emit repellents to disperse the L-BNTs from reaching their target. We also present a countermeasure that L-BNTs can take to be resilient to the attacks, where we consider two forms of decision processes that includes Bayes' rule as well as a simple threshold approach. We run a thorough set of simulations to assess the effectiveness of the proposed attacks as well as the proposed countermeasure. Our results show that the attacks can significantly hinder the regular behavior of Bio-NanoThings, while the countermeasures are effective for protecting against such attacks.

    J5. M. Barros, S. Balasubramaniam, B. Jennings, “Comparative End-to-end Analysis of Ca2+ Signaling-based Molecular Communication in Biological Tissues”, IEEE Transactions on Communications, vol. 63, no. 12, 2015.

    Calcium (Ca2+)-signaling-based molecular communication is a short-range communication process that diffuses and propagates ions between the cells of a tissue. The communication process is initiated via stimulation and amplification of the production of Ca2+ ions within a cell; these ions then diffuse through a physical connection between cells called a gap junction. Ca2+ signaling can be found in different classes of cell. In excitable cells, initiation of the Ca2+-signaling process is accompanied by an electrical component; for nonexcitable cell types, the electrical component is absent; while hybrid cells exhibit both behaviors. This paper provides a comparison and analysis of the communication behavior in tissues comprised three specific cell types that utilize Ca2+ signaling: epithelium cells (nonexcitable), smooth muscle cells (excitable), and astrocytes (hybrid). The analysis focuses on spatiotemporal Ca2+ concentration dynamics and how they are influenced by the intracellular signaling process, the molecular diffusion delay, the gain and capacity of the communication channel, as well as intracellular signaling interference. This analysis of the communication behavior in the context of tissues provides insights useful for, inter alia, the design of nanomachines that are situated within tissues and that use analysis of the communication channel to infer tissue health.

    J6. I. F Akyildiz, M. Pierobon, S. Balasubramaniam, Y. Koucheryavy, “Internet of Bio-Nano Things”, IEEE Communications Magazine, vol. 53, no. 3, March 2015.

    The Internet of Things (IoT) has become an important research topic in the last decade, where things refer to interconnected machines and objects with embedded computing capabilities employed to extend the Internet to many application domains. While research and development continue for general IoT devices, there are many application domains where very tiny, concealable, and non-intrusive Things are needed. The properties of recently studied nanomaterials, such as graphene, have inspired the concept of Internet of NanoThings (IoNT), based on the interconnection of nanoscale devices. Despite being an enabler for many applications, the artificial nature of IoNT devices can be detrimental where the deployment of NanoThings could result in unwanted effects on health or pollution. The novel paradigm of the Internet of Bio-Nano Things (IoBNT) is introduced in this paper by stemming from synthetic biology and nanotechnology tools that allow the engineering of biological embedded computing devices. Based on biological cells, and their functionalities in the biochemical domain, Bio-NanoThings promise to enable applications such as intra-body sensing and actuation networks, and environmental control of toxic agents and pollution. The IoBNT stands as a paradigm-shifting concept for communication and network engineering, where novel challenges are faced to develop efficient and safe techniques for the exchange of information, interaction, and networking within the biochemical domain, while enabling an interface to the electrical domain of the Internet.

    Representative Projects

    P1. Theoretical and Experimental Development of Protocols for Molecular Communications. Academy of Finland Research Fellow. Sept. 2014-Aug. 2019, € 835. 000. Principal Investigator: S. Balasubramaniam

    P2. Science Foundation Ireland Future Research Centre for Future Networks and Communications. Jan. 2015-Sept. 2021. € 150.000. Funded Investigator: S. Balasubramaniam

    P3. Nano Communication in Microfluidic Devices. Tampere University of Technology, Strategic Application. Jan. 2014-Dec. 2014. € 100.000. Principal Investigator: S. Balasubramaniam

    P4. A Biologically Inspired Framework supporting Network Management for the Future Internet. Science Foundation Ireland. Oct. 2009- Sept. 2013. € €362.291. Principal Investigator: S. Balasubramaniam

    P5. Application of Control Theory in Molecular Communications for the Treatment of Alzheimer's Disease. Government of Ireland Postdoctoral fellowship, Irish Research Council. Oct 2016 - Oct 2018. €91.790. Principal Investigator: M. T. Barros

    Representative Publications

    J1. Y. Chahibi, I. F. Akyildiz, and I. Balasingham. Propagation Modeling and Analysis of Molecular Motors in Molecular Communication. IEEE Transactions on NanoBioscience, 2016;15(8):917 – 927.

     Molecular motor networks (MMNs) are networks constructed from molecular motors to enable nanomachines to perform coordinated tasks of sensing, computing, and actuation at the nano- and micro- scales. Living cells are naturally enabled with this same mechanism to establish point-to-point communication between different locations inside the cell. Similar to a railway system, the cytoplasm contains an intricate infrastructure of tracks, named microtubules, interconnecting different internal components of the cell. Motor proteins, such as kinesin and dynein, are able to travel along these tracks directionally, carrying with them large molecules that would otherwise be unreliably transported across the cytoplasm using free diffusion. Molecular communication has been previously proposed for the design and study of MMNs. However, the topological aspects of MMNs, including the effects of branches, have been ignored in the existing studies. In this paper, a physical end-to-end model for MMNs is developed, considering the location of the transmitter node, the network topology, and the receiver nodes. The end-to-end gain and group delay are considered as the performance measures, and analytical expressions for them are derived. The analytical model is validated by Monte-Carlo simulations and the performance of MMNs is analyzed numerically. It is shown that, depending on their nature and position, MMN nodes create impedance effects that are critical for the overall performance. This model could be applied to assist the design of artificial MMNs and to study cargo transport in neurofilaments to elucidate brain diseases related to microtubule jamming.

    J2. M. Veletic, P. A. Floor, Y. Chahibi, and I. Balasingham. On the Upper Bound of the Information Capacity in Neuronal Synapses. IEEE Transactions on Communications, 2016;64(12):5025—5036

    Neuronal communication is a biological phenomenon of the central nervous system that influences the activity of all intra-body nano-networks. The implicit biocompatibility and dimensional similarity of neurons with miniature devices make their interaction a promising communication paradigm for nano-networks. To understand the information transfer in neuronal networks, there is a need to characterize the noise sources and unreliability associated with different components of the functional apposition between two cells-the synapse. In this paper, we introduce analogies between the optical communication system and the neuronal communication system to apply results from optical Poisson channels in deriving theoretical upper bounds on the information capacity of both the bipartite and tripartite synapses. The latter refer to the anatomical and functional integration of two communicating neurons and surrounding glia cells. The efficacy of information transfer is analyzed under different synaptic setups with progressive complexity, and is shown to depend on the peak rate of the communicated spiking sequence and neurotransmitter (spontaneous) release, neurotransmitter propagation, and neurotransmitter binding. The results provided serve as a progressive step in the evaluation of the performance of neuronal nano-networks and the development of new artificial nano-networks.

    J3. M. Veletic, P. A. Floor, Z. Babic, and I. Balasingham. Peer-to-Peer Communication in Neuronal Nano-Network. IEEE Transactions on Communications, 2016;64(3):1153–1164.

     Serving as peers in the central nervous system, neurons make use of two communication paradigms, electrochemical, and molecular. Owing to their effective coordination of all the voluntary and involuntary actions of the body, an intriguing neuronal communication nominates as a potential paradigm for nano-networking. In this paper, we propose an alternative representation of the neuron-to-neuron communication process, which should offer a complementary insight into the electrochemical signals propagation. To this end, we apply communication-engineering tools and abstractions, represent information about chemical and ionic behavior with signals, and observe biological systems as input-output systems characterized by a frequency response. In particular, we inspect the neuron-to-neuron communication through the concepts of electrochemical communication, which we refer to as the intra-neuronal communication due to the pulse transmission within the cell, and molecular synaptic transmission, which we refer to as the inter-neuronal communication due to particle transmission between the cells. The inter-neuronal communication is explored by means of the transmitter, the channel, and the receiver, aiming to characterize the spiking propagation between neurons. Reported numerical results illustrate the contribution of each stage along the neuronal communication pathway, and should be useful for the design of a new communication technique for nano-networks and intrabody communications.

    J4. F. Mesiti, P. A. Floor, and I. Balasingham. Astrocyte to Neuron Communication Channels with Applications. IEEE Transactions on Molecular, Biological and Multi-Scale Communications, 2015;1(2):164 – 175.

    Bioinspired communication techniques are emerging with increasing interest in parallel with recent advancements of nanotechnology. Particular interest is observed in the development of neuronal interfaces for human-machine communication and nanoscale neuronal devices. We propose a novel description of the communication pathways existing in the neuronal circuits, based on the abstract dynamics between different components of the neuronal communication. In the analysis, a critical role is played by glia cells, such as the astrocytes, which support and actively modulate the neuronal activity of adjacent neurons, as shown in experiments conducted the last decades. For this reason, the concept of tripartite synapse, where two neurons are interfaced with the astrocyte, is central in our abstraction. First, we define the layers of the bidirectional neuron-astrocyte communication and describe mathematically the relations connecting different quantities, i.e., intracellular molecular concentrations and currents produced on the cellular membrane. Second, the astrocytic Ca2+ signaling is investigated for the design of a neuronal communication interface based on the propagation of calcium waves through the astrocytic network. The proposed analysis provides an organized framework for an alternative description of the synaptic communication as well as for aiding the development of artificial biomimetic devices and prostheses.

    J5. F. Mesiti and I. Balasingham. Nanomachine-to-Neuron Communication Interfaces for Neuronal Stimulation at Nanoscale. IEEE Journal on Selected Areas in Communications - Special Issue on Emerging Technologies in Communications, 2013;31(12):695 – 705.

     The recent advancements in nanotechnology have been instrumental in initiating research and development of intelligent nanomachines, in a variety of different application domains including healthcare. The stimulation of the cerebral cortex to assist the treatment of brain diseases have been investigated with growing interest in the past, where nanotechnology offers a dramatic breakthrough. In this paper, we discuss the feasibility of a nanomachine-to-neuron interface to design a nanoscale stimulator device called synaptic nanomachine (SnM), compatible with the neuronal communication paradigm. An equivalent neuron-nanomachine model (EqNN) is proposed to describe the behavior of neurons excited by a network of SnMs. Sample populations of neurons are simulated under different stimulation scenarios. The assessment of the existing correlation between SnM stimulus and response, as well as between neurons and clusters of neurons, has been performed using statistical methods. The obtained results reveal that a controlled nanoscale stimulation induces apparently an oscillatory behavior in the neuronal activity and localized synchronization between neurons. Both effects are expected to have the basis of important cognitive and behavioral functions such as learning and brain plasticity.

    Representative Projects

    P1. Wireless In-body Sensor and Actuator Networks (WINNOW), Research Council of Norway, 01.05.2017 – 30.04.2021, budget NOK 16 million (€1.75 million). Project Manager/PI: Dr. Balasingham 

    The project studies synthetic cardiomyocytes encapsulated in nanoscale wireless implants using calcium signals for information transfer and develops truly the future leadless pacemaker that can be programmed remotely and last for more than 10 years without battery replacement.

    P2. Wireless In-Body Environment (WiBEC), H2020- MARIE Skodowska-CURIE ACTIONS (MSCA-ITN-2015), 01.01.2016-31.12.2019, budget €3.957 million. Coordinator: Dr. Balasingham

    The project studies intra-body sensor networks for future leadless pacemaker and monitoring bleeding and cancer in gastrointestinal track using a combination of human body and RF technologies.

    P3. Medical Sensing, Localization, and Communications using Ultra Wideband Technology (MELODY II), Research Council of Norway, 01.01.2013 - 31.12.2017, budget NOK 14.7 million (€ 1.6 million). Project Manager/PI: Dr. Balasingham

    The project designed a wireless in-body communication system for future wireless HD video capsule endoscopy and demonstrated a working prototype in animal studies. It is envisioned the results can facilitate population ide screening application. The project has produced 14 papers, filed 1 patent application, and 2 PhDs and 2 Postdocs.

    P4. Medical Sensing, Localization, and Communications using Ultra Wideband Technology (MELODY I), Research Council of Norway, 01.09.2008 - 31.12.2012, budget NOK 36 million (€ 4 million). Project Manager/PI: Dr. Balasingham

    The project designed and developed ultra wideband RF technology and demonstrated 1) non-contact sensing of central blood pressure and heart rate, 2) 2D heart imaging, and 3) localizing and tracking wireless capsule endoscopy. The published 100 papers, filed 3 patents, and produced 7 PhDs and 4 Postdocs.

    Energy + Data

    External Control Unit

    Transponder including blood pressure sensor

    P5. Invivo Ultrasonic Transponder System for Biomedical Applications (ULTRASPONDER) European Union 7th Framework Program, STREP, 01.09.2008 -31.08.2011, budget € 4.5 million. WP Leader: Dr. Balasingham  

    The project developed a ultrasound based wireless communication and power transfer for implants and demonstrated the results for heart implants in animals.

    Representative Publications

    J1. Stab J., Zlatev I., Raudszus B., Meister S., Pietrzik C. U., Langer K., von Briesen H. and Wagner S.: “Flurbiprofen-loaded Nanoparticles Can Cross a Primary Porcine In vitro Blood-brain Barrier Model to Reduce Amyloid-ß42 Burden.” J Nanomedine Biotherapeutic Discov 6:140. doi:10.4172/2155-983X.1000140

    Elevated amyloid-β42 (Aβ42) in the brain is expected to cause Alzheimer’s Disease (AD). Reducing Aβ42 is therefore a cornerstone in causal drug development. Nevertheless, many promising substances failed in clinical trials, because reaching the target organ in vivo is difficult. The brain is protected by the Blood-Brain Barrier (BBB) that shields off most molecules to maintain the brain homeostasis. Brain-targeted nanoparticles are one successful tool to bypass this problem: by acting as Trojan horses they carry embedded drugs across the BBB for brain disorder treatment.

    J2. Linz U., Hupert M., Santiago-Schübel B., Wien S., Stab J., Wagner S.: “ Transport of treosulfan and temozolomide accross an in-vitro blood-brain barrier model” Anti-Cancer Drugs 26, 728-36, 2015 (

    In vitro, treosulfan (TREO) has shown high effectiveness against malignant gliomas. However, a first clinical trial for newly diagnosed glioblastoma did not show any positive effect. Even though dosing and timing might have been the reasons for this failure, it might also be that TREO does not reach the brain in sufficient amount. Surprisingly, there are no published data on TREO uptake into the brain of patients, despite extensive research on this compound. An in-vitro blood–brain barrier (BBB) model consisting of primary porcine brain capillary endothelial cells was used to determine the transport of TREO across the cell monolayer. Temozolomide (TMZ), the most widely used cytotoxic drug for malignant gliomas, served as a reference. An HPLC-ESI-MS/MS procedure was developed to detect TREO and TMZ in cell culture medium. Parallel to the experimental approach, the permeability of TREO and the reference substance across the in-vitro BBB was estimated on the basis of their physicochemical properties. The detection limit was 30 nmol/l for TREO and 10 nmol/l for TMZ. Drug transport was measured in two directions: influx, apical-to-basolateral (A-to-B), and efflux, basolateral-to-apical (B-to-A). For TREO, the A-to-B permeability was lower (1.6%) than the B-to-A permeability (3.0%). This was in contrast to TMZ, which had higher A-to-B (13.1%) than B-to-A (7.2%) permeability values. The in-vitro BBB model applied simulated the human BBB properly for TMZ. It is, therefore, reasonable to assume that the values for TREO are also meaningful. Considering the lack of noninvasive, significant alternative methods to study transport across the BBB, the porcine brain capillary endothelial cell model was efficient to collect first data for TREO that explain the disappointing clinical results for this drug against cerebral tumors.

    J3. Neubauer J. C., Sébastien I., Germann A.,  Müller S. C., Meyerhans A., von Briesen H., Zimmermann H.: “Towards standardized automated immunomonitoring: an automated ELISpot assay for safe and parallelized functionality analysis of immune cellsCytotechnology. 2017 Feb;69(1):57-73. doi: 10.1007/s10616-016-0037-4

    The ELISpot assay is used for the detection of T cell responses in clinical trials and vaccine evaluations. Standardization and reproducibility are necessary to compare the results worldwide, inter- and intra-assay variability being critical factors. To assure operator safety as well as high-quality experiment performance, the ELISpot assay was implemented on an automated liquid handling platform, a Tecan Freedom EVO. After validation of the liquid handling, automated loading of plates with cells and reagents was investigated. With step by step implementation of the manual procedure and liquid dispensing optimization on the robot platform, a fully automated ELISpot assay was accomplished with plates remaining in the system from the plate blocking step to spot development. The mean delta difference amounted to a maximum of 6%, and the mean dispersion was smaller than in the manual assay. Taken together, we achieved with this system not only a lower personnel attendance but also higher throughput and a more precise and parallelized analysis. This platform has the potential to guarantee validated, safe, fast, reproducible and cost-efficient immunological and toxicological assays in the future.

    J4. Neubauer, J.C., Beier, A.F., Stracke, F., Zimmermann, H. (2015) “Vitrification in pluripotent stem cell banking: Requirements & technical solutions for large-scale biobanks.” Chapter 24 in: 2nd Edition of the book Vitrification in Assisted Reproduction: From Basic Science to Clinical Application (Liebermann, J. and Tucker, M.J., eds.). CRC press, 203-224.

    J5. M. Oeri, W. Bost, S. Tretbar, M. Fournelle (2016) Calibrated Linear Array-Driven photoacoustic/Ultrasound tomography. Ultrasound Med Biol. 42(11), 2697-2707.

    The anisotropic resolution of linear arrays, tools that are widely used in diagnostics, can be overcome by compounding approaches. We investigated the ability of a recently developed calibration and a novel algorithm to determine the actual radial transducer array distance and its misalignment (tilt) with respect to the center of rotation in a 2-D and 3-D tomographic setup. By increasing the time-of-flight accuracy, we force in-phase summation during the reconstruction. Our setup is composed of a linear transducer and a rotation and translation axis enabling multidimensional imaging in ultrasound and photoacoustic mode. Our approach is validated on phantoms and young mice ex vivo. The results indicate that application of the proposed analytical calibration algorithms prevents image artifacts. The spatial resolution achieved was 160 and 250 μm in photoacoustic mode of 2-D and 3-D tomography, respectively

    Representative Projects

    P1. EBiSC – European Bank of Pluripotent Stem Cells (Innovative Medicine Initiative IMI 115582-project, 2013-2018, Principal Investigators: H. Zimmermann / J. Neubauer. Project volume: € 36.50.000

    P2. DropTech - Hanging Drop based automated and parallelized cell technology platform for production and testing (EU FP7 601865-project, 2014-2017). Project Coordinator: H. Zimmermann / J. Neubauer. Project volume: € 5.250.000

    P3. Hyperlab-High yield and performance stem cell lab (EU FP7 22301–project, 2009-2012). Project Coordinator: H. Zimmermann / J. Neubauer. Project volume: € 3.950.000

    P4. NanoBrain- Alzheimer drugs incorporated in nanoparticles for specific transport over the blood-brain barrier (ERA-Net NEURON –project, 2010-2013, 01EW1010)

    P5. HISENTS – High level Integrated Sensor for Nano Toxicity Screening (EU H2020, 2016-2019)

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