Single Layer Graphene Biointerface: Studying Neuronal Network Development and Monitoring Cell Behavior over Time

The objective of my Ph.D. thesis is the investigation of the role of Single Layer Graphene (SLG) as a biointerface for its possible future exploitation in various biomedical applications; in particular for the development of biosensors, substrates for regenerative medicine, interfacing platforms for better recording of electrophysiological activity of neuronal networks, among others. This Ph.D. project is multidisciplinary involving both the material transfer and characterization part from one side and the biological part from another side. The material part offers an in-depth explanation of SLG synthesis, transfer, characterization and functionalization while the biological section sheds light on the studies performed for investigation of the behavior of different types of cell lines on SLG substrates. For better understanding of the sequence of the performed work, I have divided this thesis into separate chapters. In the beginning and end of every chapter, I added an introduction and conclusions related to it. Chapter 1 acts as a general introduction to graphene and graphene-related materials where a detailed explanation on the evolution of those materials as a cell interface is provided leading to the introduction of SLG in the end of this chapter along with its production process. Chapter 2 is oriented on the surface characterization of SLG substrates; in this chapter, I described the SLG transfer method, creation of the micrometric ablated geometric patterns on the transferred substrates using excimer laser micromachining, a technique developed in our lab, then further functionalization of the substrates and finally all the techniques employed for their physicochemical characterization. Chapter 3 is dedicated to the biological part of the project; i.e. studying the behavior of different cell lines on the SLG substrates. In this chapter, I have described and explained the interest of using the selected cell lines and the experiments that were performed on them. Chapter 4 has been devoted to a complete and separate project that I performed in collaboration with the Neuroscience and Brain Technologies department. The main focus of the project was the functionalization of the commercial multi-electrode arrays (MEAs) with SLG and studying the neuronal network activity on them throughout the complete network development. Although the main focus of my Ph.D. project was studying SLG biointerface, I have also been involved in side projects, among which, studying the neuronal-like response of mouse neuroblastoma (N2a) living cells to nanoporous patterns of thin supported anodic alumina which I have described in Appendix A, and studying the surface potential of graphene by polyelectrolyte coating which I have presented in Appendix B. To summarize, this thesis reports an original investigation, since, to the best of our knowledge, there is no report yet about the study of the effect of SLG functionalized MEA on the neuronal network activity throughout the complete network maturation. Furthermore, proliferation curves of different cell lines on SLG versus control substrates have been presented; in addition to physicochemical characterization of ablated and functionalized SLG substrates as means of possible explanation of a certain cellular behavior on graphene

A new Strategy to Improve Drug Delivery to the Maxillary Sinuses: The Frequency Sweep Acoustic Airflow

International audiencePurpose Enhancement of intra-nasal sinus drug deposition involves nebulization of a drug superimposed by the acoustic airflow characterized by a specific frequency. We investigated the impact of fixed frequency versus frequency sweep acoustic airflow on the improvement of aerosolized drug penetration into the maxillary sinuses.Methods Fixed frequency and frequency sweep acoustic airflow were generated using a prototype of variable frequency nebulizing system, and their effect on the intra-nasal sinus aerosol deposition in a ‘nasal replica’, a transparent, water-resistant, non-porous resin replica of the human plastinated cast created using a stereolithography technique, was tested. Sodium fluoride and gentamicin were chosen as markers. In addition to this, the effect of sweep cycle and intensity variation was also studied. Results Studies performed using fixed frequency acoustic airflow showed that each of the maxillary sinuses of the ‘nasal replica’ required specific frequency for the optimal intra-nasal sinus aerosol deposition; depending on the ostia of the left and right maxillary sinuses which are different. Intra-nasal sinus drug deposition experiments under the effect of the frequency sweep acoustic airflow showed an optimal aerosol deposition into both maxillary sinus of the ‘nasal replica’. Studies on the effect of the duration of the sweep cycle showed that the shorter the cycle the better the deposition.Conclusion Our study demonstrates the benefit of frequency sweep acoustic airflow on the drug deposition into maxillary sinuses of the ‘nasal replica’ characterized by ostia of different geometry. However, the delivery rates of the ‘nasal replica’ cannot be directly applied to real human chronic rhinosinusitis condition; further in vivo studies have to be conducted

Impact of acoustic airflow on intrasinus drug deposition: New insights into the vibrating mode and the optimal acoustic frequency to enhance the delivery of nebulized antibiotic

International audienceAim: We investigated the impact of vibrating acoustic airflow, the high frequency (f ≥ 100 Hz) and the low frequency (f ≤ 45 Hz) sound waves, on the enhancement of intrasinus drug deposition.Methods: 81mKr-gas ventilation study was performed in a plastinated human cast with and without the addition of vibrating acoustic airflow. Similarly, intrasinus drug deposition in a nasal replica using gentamicin as a marker was studied with and without the superposition of different modes of acoustic airflow.Results: Ventilation experiments demonstrate that no sinus ventilation was observed without acoustic airflow although sinus ventilation occurred whatever the modes of acoustic airflow applied. Intrasinus drug deposition experiments showed that the high frequency acoustic airflow led to 4-fold increase in gentamicin deposition into the left maxillary sinus and to 2-fold deposition increase into the right maxillary sinus. Besides, the low frequency acoustic airflow demonstrated a significant increase of 4-fold and 2-fold in the right and left maxillary sinuses respectively.Conclusion: We demonstrated the benefit of different modes of vibrating acoustic airflow for maxillary sinus ventilation and intrasinus drug deposition. The degree of gentamicin deposition varies as a function of frequency of the vibrating acoustic airflow and the geometry of the ostia

Step-by-step surface potential tuning of patterned graphene by polyelectrolyte coating

The fine control of the interfacial properties of functionalized graphene is a key point for its applications, especially in biosensing devices. We have here used an in-house developed technique to fabricate microsized patterned graphene via laser ablation and then we have functionalized the interface with poly-D-lysine, a biocompatible polyelectrolyte normally used as a promoter for cell adhesion. Scanning Kelvin probe microscopy shows that a surface potential contrast appears at the patterned regions, with ablated regions of silicon substrate exhibiting higher surface potential than the surrounding background, whereas both levels have negative values. By subsequent coating with the poly-D-lysine it is possible to change stepwise the surface potential levels, while keeping the contrast at the patterned regions constant, up to neutralizing the initial negative values. With further dipping in a polyelectrolyte solution of opposite sign, such as polystyrene sulfonate, it is then possible to decrease the surface potential shifting it again back to negative values. The starting substrate chosen for graphene transfer allows deciding the sign of the surface potential contrast between two adjacent regions of the pattern

Ex vivo detection and quantification of gold nanoparticles in human seminal and follicular fluids

International audienceIncreasing consumption of engineered nanoparticles and occupational exposure to novel, ultrafine airborne particles during the last decades has coincided with deterioration of sperm parameters and delayed fecundity. In order to prevent possible adverse health effects and ensure a sustainable growth for the nanoparticle industry, the ability to investigate the nanosized, mineralogical load of human reproductive systems is becoming a real clinical need. Toward this goal, the current study proposes two methods for the detection and quantification of engineered nanoparticles in human follicular and seminal fluid, developed with the use of well-defined 60 nm Au particles. Despite the complexity of these biological fluids, simple physical and chemical treatments allow for the precise quantification of more than 50 and 70% wt of the spiked Au nanoparticles at low μg ml−1 levels in follicular and seminal fluids, respectively. The use of electron microscopy for the detailed observation of the detected analytes is also enabled. The proposed method is applied on a small patient cohort in order to demonstrate its clinical applicability by exploring the differences in the metal and particulate content between patients with normal and low sperm count