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

Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages

International audienceTo improve the biological properties of calcium phosphate (CaP) bone substitute, new chemical compositions are under development. In vivo such materials are subject to degradation that could lead to particles release and inflammatory reactions detrimental to the bone healing process. This study aimed at investigating the interactions between a murine macrophage cell line (RAW 264.7) and substituted hydroxyapatite particles presenting promising biological properties. Micron size particles of stoichiometric and substituted hydroxyapatites (CO 3 substitution for PO 4 and OH; SiO 4 substitution for PO 4; CO 3 and SiO 4 co-substitution) were obtained by aqueous precipitation followed by spray drying. Cells, incubated with four doses of particles ranging from 15 to 120 µg/mL, revealed no significant LDH release or ROS production, indicating no apparent cytotoxicity and no oxidative stress. TNF-α production was independent of the chemistry of the particles; however the particles elicited a significant dose-dependent pro-inflammatory response. As micron size particles of these hydroxyapatites could be at the origin of inflammation, attention must be paid to the degradation 2 behavior of substituted hydroxyapatite bone substitute in order to limit, in vivo, the generation of particulate debris

Impact of Airborne Particle Size, Acoustic Airflow and Breathing Pattern on Delivery of Nebulized Antibiotic into the Maxillary Sinuses Using a Realistic Human Nasal Replica

International audiencePurpose:Improvement of clinical outcome in patients with sinuses disorders involves targeting delivery of nebulized drug into the maxillary sinuses. We investigated the impact of nebulization conditions (with and without 100 Hz acoustic airflow), particle size (9.9 μm, 2.8 μm, 550 nm and 230 nm) and breathing pattern (nasal vs. no nasal breathing) on enhancement of aerosol delivery into the sinuses using a realistic nasal replica developed by our team.Methods:After segmentation of the airways by means of high-resolution computed tomography scans, a well-characterized nasal replica was created using a rapid prototyping technology. A total of 168 intrasinus aerosol depositions were performed with changes of aerosol particle size and breathing patterns under different nebulization conditions using gentamicin as a marker.Results:The results demonstrate that the fraction of aerosol deposited in the maxillary sinuses is enhanced by use of submicrometric aerosols, e.g. 8.155 ± 1.476 mg/L of gentamicin in the left maxillary sinus for the 2.8 μm particles vs. 2.056 ± 0.0474 for the 550 nm particles. Utilization of 100-Hz acoustic airflow nebulization also produced a 2- to 3-fold increase in drug deposition in the maxillary sinuses (e.g. 8.155 ± 1.476 vs. 3.990 ± 1.690 for the 2.8 μm particles).Conclusions:Our study clearly shows that optimum deposition was achieved using submicrometric particles and 100-Hz acoustic airflow nebulization with no nasal breathing. It is hoped that our new respiratory nasal replica will greatly facilitate the development of more effective delivery systems in the future

Assessing sinus aerosol deposition: benefits of SPECT-CT imaging

International audiencePurpose: Aerosol inhalation therapy is one of the methods to treat rhinosinusitis. However the topical drug delivery to the posterior nose and paranasal sinuses shows only limited efficiency. A precise sinusal targeting remains a main challenge for aerosol treatment of sinus disorders. This paper proposes a comparative study of the nasal deposition patterns of micron and submicron particles using planar gamma-scintigraphy imaging vs. a new 3-dimensional (3D) imaging approach based on SPECT-CT measurements. Methods: Radiolabelled nebulizations have been performed on a plastinated model of human nasal cast coupled with a respiratory pump. First, the benefits provided by SPECT-CT imaging were compared with 2D gamma-scintigraphy and radioactive quantification of maxillary sinus lavage as reference for the sonic 2.8 μm aerosol sinusal deposition. Then, the impact on nasal deposition of various airborne particle sizes was assessed. Results: The 2D methodology overestimates aerosol deposition in the maxillary sinuses by a factor 9 whereas the 3D methodology is in agreement with the maxillary sinus lavage reference methodology. Then with the SPECT-CT approach we highlighted that the higher particle size was mainly deposited in the central nasal cavity contrary to the submicron aerosol particles (33.8 ± 0.6% of total deposition for the 2.8 μm particles vs. 1 ± 0.3% for the 230 nm particles). Conclusion: Benefits of SPECT/CT for the assessment of radiolabelled aerosol deposition in rhinology are clearly demonstrated. This 3D methodology should be preferentially used for scintigraphic imaging of sinusal deposition in Human

Testicular biodistribution of silica-gold nanoparticles after intramuscular injection in mice

International audienceWith the continuing development of nanomaterials, the assessment of their potential impact on human health, and especially human reproductive toxicity, is a major issue. The testicular biodistribution of nanoparticles remains poorly studied. This study investigated whether gold-silica nanoparticlescould be detected in mouse testes after intramuscular injection, with a particular focus on their ability to cross the blood– testis barrier. To that purpose, well-characterized 70-nm gold core–silica shell nanoparticles were used to ensure sensitive detection using high-resolution techniques. Testes were collected at different time points corresponding to spermatogenesis stages in mice. Transmission electronmicroscopy and confocal microscopy were used for nanoparticle detection, and nanoparticle quantification was performed by atomic emission spectroscopy. All these techniques showed that no particles were able to reach the testes. Results accorded with the normal histological appearance of testes even at 45 days post sacrifice.High-resolution techniques did not detect 70-nm silica-gold nanoparticles in mouse testes after intramuscular injection.These results are reassuring about the safety of nanoparticles with regard to male human reproduction, especially in the context of nanomedicine

Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms

International audienceBackground: The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments. Methods: Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production]. Results and conclusion: Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49±11%) than for amine 1-µm beads (18±5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446±7 and 139±12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H2O2 production

Quantitative cellular uptake of double fluorescent core-shelled model submicronic particles

International audienceThe relationship between particles' physicochemical parameters, their uptake by cells and their degree of biological toxicity represent a crucial issue, especially for the development of new technologies such as fabrication of micro- and nanoparticles in the promising field of drug delivery systems. This work was aimed at developing a proof-of-concept for a novel model of double fluorescence submicronic particles that could be spotted inside phagolysosomes. Fluorescein isothiocyanate (FITC) particles were synthesized and then conjugated with a fluorescent pHrodo™ probe, red fluorescence of which increases in acidic conditions such as within lysosomes. After validation in acellular conditions by spectral analysis with confocal microscopy and dynamic light scattering, quantification of phagocytosis was conducted on a macrophage cell line in vitro. The biological impact of pHrodo functionalization (cytotoxicity, inflammatory response, and oxidative stress) was also investigated. Results validate the proof-of-concept of double fluorescent particles (FITC + pHrodo), allowing detection of entirely engulfed pHrodo particles (green and red labeling). Moreover incorporation of pHrodo had no major effects on cytotoxicity compared to particles without pHrodo, making them a powerful tool for micro- and nanotechnologies

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro

International audienceBackground: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. Methods: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Results and conclusion: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed

Development of innovative pH sensor to evaluate phagocytosis of nanoparticles

International audienceInhaled nanoparticles (NP) exhibit variable toxicity levels which mainly depend on their physicochemical characteristics (size, morphology, crystallinity, chemical surface composition...). Biological effects monitoring thanks to usual tests (ROS, TNFα, LDH) are performed on alveolar macrophages collected from the respiratory system. In this frame, evaluation of NP uptake by macrophages appears as complementary data useful for NP toxicity assessment. The aim of this work deals with the development of pH sensible NP allowing the quantification of NP phagocytosed by macrophages, specially the step of fusion between phagosomes and lysosomes