Long COVID exhibits clinically distinct phenotypes at 3-6 months post-SARS-CoV-2 infection: results from the P4O2 consortium.

BACKGROUND: Four months after SARS-CoV-2 infection, 22%-50% of COVID-19 patients still experience complaints. Long COVID is a heterogeneous disease and finding subtypes could aid in optimising and developing treatment for the individual patient. METHODS: Data were collected from 95 patients in the P4O2 COVID-19 cohort at 3-6 months after infection. Unsupervised hierarchical clustering was performed on patient characteristics, characteristics from acute SARS-CoV-2 infection, long COVID symptom data, lung function and questionnaires describing the impact and severity of long COVID. To assess robustness, partitioning around medoids was used as alternative clustering. RESULTS: Three distinct clusters of patients with long COVID were revealed. Cluster 1 (44%) represented predominantly female patients (93%) with pre-existing asthma and suffered from a median of four symptom categories, including fatigue and respiratory and neurological symptoms. They showed a milder SARS-CoV-2 infection. Cluster 2 (38%) consisted of predominantly male patients (83%) with cardiovascular disease (CVD) and suffered from a median of three symptom categories, most commonly respiratory and neurological symptoms. This cluster also showed a significantly lower forced expiratory volume within 1 s and diffusion capacity of the lung for carbon monoxide. Cluster 3 (18%) was predominantly male (88%) with pre-existing CVD and diabetes. This cluster showed the mildest long COVID, and suffered from symptoms in a median of one symptom category. CONCLUSIONS: Long COVID patients can be clustered into three distinct phenotypes based on their clinical presentation and easily obtainable information. These clusters show distinction in patient characteristics, lung function, long COVID severity and acute SARS-CoV-2 infection severity. This clustering can help in selecting the most beneficial monitoring and/or treatment strategies for patients suffering from long COVID. Follow-up research is needed to reveal the underlying molecular mechanisms implicated in the different phenotypes and determine the efficacy of treatment.

Precision Medicine for More Oxygen (P4O2)-Study Design and First Results of the Long COVID-19 Extension.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic has led to the death of almost 7 million people, however, with a cumulative incidence of 0.76 billion, most people survive COVID-19. Several studies indicate that the acute phase of COVID-19 may be followed by persistent symptoms including fatigue, dyspnea, headache, musculoskeletal symptoms, and pulmonary functional-and radiological abnormalities. However, the impact of COVID-19 on long-term health outcomes remains to be elucidated. Aims: The Precision Medicine for more Oxygen (P4O2) consortium COVID-19 extension aims to identify long COVID patients that are at risk for developing chronic lung disease and furthermore, to identify treatable traits and innovative personalized therapeutic strategies for prevention and treatment. This study aims to describe the study design and first results of the P4O2 COVID-19 cohort. Methods: The P4O2 COVID-19 study is a prospective multicenter cohort study that includes nested personalized counseling intervention trial. Patients, aged 40-65 years, were recruited from outpatient post-COVID clinics from five hospitals in The Netherlands. During study visits at 3-6 and 12-18 months post-COVID-19, data from medical records, pulmonary function tests, chest computed tomography scans and biological samples were collected and questionnaires were administered. Furthermore, exposome data was collected at the patient's home and state-of-the-art imaging techniques as well as multi-omics analyses will be performed on collected data. Results: 95 long COVID patients were enrolled between May 2021 and September 2022. The current study showed persistence of clinical symptoms and signs of pulmonary function test/radiological abnormalities in post-COVID patients at 3-6 months post-COVID. The most commonly reported symptoms included respiratory symptoms (78.9%), neurological symptoms (68.4%) and fatigue (67.4%). Female sex and infection with the Delta, compared with the Beta, SARS-CoV-2 variant were significantly associated with more persisting symptom categories. Conclusions: The P4O2 COVID-19 study contributes to our understanding of the long-term health impacts of COVID-19. Furthermore, P4O2 COVID-19 can lead to the identification of different phenotypes of long COVID patients, for example those that are at risk for developing chronic lung disease. Understanding the mechanisms behind the different phenotypes and identifying these patients at an early stage can help to develop and optimize prevention and treatment strategies.

Ultrasonic Characterization of Ibidi µ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents.

Understanding and controlling the ultrasound contrast agent (UCA)'s response to an applied ultrasound pressure field are crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the UCA. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the UCA can be studied. The aim of our study was to determine the in situ ultrasound pressure amplitude in the ibidi µ -slide I Luer channel, an optically transparent chamber suitable for cell culture, including culture under flow, for all microchannel heights (200, 400, 600, and [Formula: see text]). First, the in situ pressure field in the 800- [Formula: see text] high channel was experimentally characterized using Brandaris 128 ultrahigh-speed camera recordings of microbubbles (MBs) and a subsequent iterative processing method, upon insonification at 2 MHz, 45° incident angle, and 50-kPa peak negative pressure (PNP). Control studies in another cell culture chamber, the CLINIcell, were compared with the obtained results. The pressure amplitude was -3.7 dB with respect to the pressure field without the ibidi µ -slide. Second, using finite-element analysis, we determined the in situ pressure amplitude in the ibidi with the 800- [Formula: see text] channel (33.1 kPa), which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, and [Formula: see text]) with either 35° or 45° incident angle, and at 1 and 2 MHz. The predicted in situ ultrasound pressure fields were between -8.7 and -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound in situ pressures demonstrate the acoustic compatibility of the ibidi µ -slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of UCAs for imaging and therapy.

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100-400 kPa, 100-1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.

Internalization of targeted microbubbles by endothelial cells and drug delivery by pores and tunnels.

Ultrasound insonification of microbubbles can locally enhance drug delivery by increasing the cell membrane permeability. To aid development of a safe and effective therapeutic microbubble, more insight into the microbubble-cell interaction is needed. In this in vitro study we aimed to investigate the initial 3D morphology of the endothelial cell membrane adjacent to individual microbubbles (n = 301), determine whether this morphology was affected upon binding and by the type of ligand on the microbubble, and study its influence on microbubble oscillation and the drug delivery outcome. High-resolution 3D confocal microscopy revealed that targeted microbubbles were internalized by endothelial cells, while this was not the case for non-targeted or IgG1-κ control microbubbles. The extent of internalization was ligand-dependent, since αvß3-targeted microbubbles were significantly more internalized than CD31-targeted microbubbles. Ultra-high-speed imaging (~17 Mfps) in combination with high-resolution confocal microscopy (n = 246) showed that microbubble internalization resulted in a damped microbubble oscillation upon ultrasound insonification (2 MHz, 200 kPa peak negative pressure, 10 cycles). Despite damped oscillation, the cell's susceptibility to sonoporation (as indicated by PI uptake) was increased for internalized microbubbles. Monitoring cell membrane integrity (n = 230) showed the formation of either a pore, for intracellular delivery, or a tunnel (i.e. transcellular perforation), for transcellular delivery. Internalized microbubbles caused fewer transcellular perforations and smaller pore areas than non-internalized microbubbles. In conclusion, studying microbubble-mediated drug delivery using a state-of-the-art imaging system revealed receptor-mediated microbubble internalization and its effect on microbubble oscillation and resulting membrane perforation by pores and tunnels.

Theranostic Microbubbles with Homogeneous Ligand Distribution for Higher Binding Efficacy.

Phospholipid-coated targeted microbubbles are used for ultrasound molecular imaging and locally enhanced drug delivery, with the binding efficacy being an important trait. The use of organic solvent in microbubble production makes the difference between a heterogeneous or homogeneous ligand distribution. This study demonstrates the effect of ligand distribution on the binding efficacy of phospholipid-coated ανß3-targeted microbubbles in vitro using a monolayer of human umbilical-vein endothelial cells and in vivo using chicken embryos. Microbubbles with a homogeneous ligand distribution had a higher binding efficacy than those with a heterogeneous ligand distribution both in vitro and in vivo. In vitro, 1.55× more microbubbles with a homogeneous ligand distribution bound under static conditions, while this was 1.49× more under flow with 1.25 dyn/cm2, 1.56× more under flow with 2.22 dyn/cm2, and 1.25× more in vivo. The in vitro dissociation rate of bound microbubbles with homogeneous ligand distribution was lower at low shear stresses (1-5 dyn/cm2). The internalized depth of bound microbubbles was influenced by microbubble size, not by ligand distribution. In conclusion, for optimal binding the use of organic solvent in targeted microbubble production is preferable over directly dispersing phospholipids in aqueous medium.

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms.

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0-12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.

Corrections to "Vibrational Responses of Bound and Nonbound Targeted Lipid-Coated Single Microbubbles".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 786, paragraph II-A, it should read "The coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG-40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG(2000), 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-biotin, 880129, Avanti Polar Lipids)."

The Impact of Lipid Handling and Phase Distribution on the Acoustic Behavior of Microbubbles.

Phospholipid-coated microbubbles are ultrasound contrast agents that can be employed for ultrasound molecular imaging and drug delivery. For safe and effective implementation, microbubbles must respond uniformly and predictably to ultrasound. Therefore, we investigated how lipid handling and phase distribution affected the variability in the acoustic behavior of microbubbles. Cholesterol was used to modify the lateral molecular packing of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based microbubbles. To assess the effect of lipid handling, microbubbles were produced by a direct method, i.e., lipids directly dispersed in an aqueous medium or indirect method, i.e., lipids first dissolved in an organic solvent. The lipid phase and ligand distribution in the microbubble coating were investigated using confocal microscopy, and the acoustic response was recorded with the Brandaris 128 ultra-high-speed camera. In microbubbles with 12 mol% cholesterol, the lipids were miscible and all in the same phase, which resulted in more buckle formation, lower shell elasticity and higher shell viscosity. Indirect DSPC microbubbles had a more uniform response to ultrasound than direct DSPC and indirect DSPC-cholesterol microbubbles. The difference in lipid handling between direct and indirect DSPC microbubbles significantly affected the acoustic behavior. Indirect DSPC microbubbles are the most promising candidate for ultrasound molecular imaging and drug delivery applications.

High-Resolution Imaging of Intracellular Calcium Fluctuations Caused by Oscillating Microbubbles.

Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbubble-cell interaction remains poorly understood. Because intracellular calcium (Cai2+) is a key cellular regulator, unraveling the Cai2+ fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Cai2+ fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Cai2+ uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cell-cell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 µm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Cai2+ fluctuations, providing new insight into the microbubble-cell interaction to aid clinical translation.

Opening of endothelial cell-cell contacts due to sonoporation.

Ultrasound insonification of microbubbles can locally increase vascular permeability to enhance drug delivery. To control and optimize the therapeutic potential, we need to better understand the underlying biological mechanisms of the drug delivery pathways. The aim of this in vitro study was to elucidate the microbubble-endothelial cell interaction using the Brandaris 128 ultra-high-speed camera (up to 25 Mfps) coupled to a custom-built Nikon confocal microscope, to visualize both microbubble oscillation and the cellular response. Sonoporation and opening of cell-cell contacts by single αVß3-targeted microbubbles (n = 152) was monitored up to 4 min after ultrasound insonification (2 MHz, 100-400 kPa, 10 cycles). Sonoporation occurred when microbubble excursion amplitudes exceeded 0.7 µm. Quantification of the influx of the fluorescent model drug propidium iodide upon sonoporation showed that the size of the created pore increased for larger microbubble excursion amplitudes. Microbubble-mediated opening of cell-cell contacts occurred as a cellular response upon sonoporation and did not correlate with the microbubble excursion amplitude itself. The initial integrity of the cell-cell contacts affected the susceptibly to drug delivery, since cell-cell contacts opened more often when cells were only partially attached to their neighbors (48%) than when fully attached (14%). The drug delivery outcomes were independent of nonlinear microbubble behavior, microbubble location, and cell size. In conclusion, by studying the microbubble-cell interaction at nanosecond and nanometer resolution the relationship between drug delivery pathways and their underlying mechanisms was further unraveled. These novel insights will aid the development of safe and efficient microbubble-mediated drug delivery.

Ligand Distribution and Lipid Phase Behavior in Phospholipid-Coated Microbubbles and Monolayers.

Phospholipid-coated targeted microbubbles are ultrasound contrast agents that can be used for molecular imaging and enhanced drug delivery. However, a better understanding is needed of their targeting capabilities and how they relate to microstructures in the microbubble coating. Here, we investigated the ligand distribution, lipid phase behavior, and their correlation in targeted microbubbles of clinically relevant sizes, coated with a ternary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), with PEG40-stearate and DSPE-PEG2000. To investigate the effect of lipid handling prior to microbubble production in DSPC-based microbubbles, the components were either dispersed in aqueous medium (direct method) or first dissolved and mixed in an organic solvent (indirect method). To determine the lipid-phase behavior of all components, experiments were conducted on monolayers at the air/water interface. In comparison to pure DSPC and DPPC, the ternary mixtures had an additional transition plateau around 10-12 mN/m. As confirmed by infrared reflection absorption spectroscopy (IRRAS), this plateau was due to a transition in the conformation of the PEGylated components (mushroom to brush). While the condensed phase domains had a different morphology in the ternary DPPC and DSPC monolayers on the Langmuir trough, the domain morphology was similar in the coating of both ternary DPPC and DSPC microbubbles (1.5-8 µm diameter). The ternary DPPC microbubbles had a homogenous ligand distribution and significantly less liquid condensed (LC) phase area in their coating than the DSPC-based microbubbles. For ternary DSPC microbubbles, the ligand distribution and LC phase area in the coating depended on the lipid handling. The direct method resulted in a heterogeneous ligand distribution, less LC phase area than the indirect method, and the ligand colocalizing with the liquid expanded (LE) phase area. The indirect method resulted in a homogenous ligand distribution with the largest LC phase area. In conclusion, lipid handling prior to microbubble production is of importance for a ternary mixture of DSPC, PEG40-stearate, and DSPE-PEG2000.

Combined Confocal Microscope and Brandaris 128 Ultra-High-Speed Camera.

Controlling microbubble-mediated drug delivery requires the underlying biological and physical mechanisms to be unraveled. To image both microbubble oscillation upon ultrasound insonification and the resulting cellular response, we developed an optical imaging system that can achieve the necessary nanosecond temporal and nanometer spatial resolutions. We coupled the Brandaris 128 ultra-high-speed camera (up to 25 million frames per second) to a custom-built Nikon A1R+ confocal microscope. The unique capabilities of this combined system are demonstrated with three experiments showing microbubble oscillation leading to either endothelial drug delivery, bacterial biofilm disruption, or structural changes in the microbubble coating. In conclusion, using this state-of-the-art optical imaging system, microbubble-mediated drug delivery can be studied with high temporal resolution to resolve microbubble oscillation and high spatial resolution and detector sensitivity to discern cellular response. Combining these two imaging technologies will substantially advance our knowledge on microbubble behavior and its role in drug delivery.

Acoustic Characterization of the CLINIcell for Ultrasound Contrast Agent Studies.

Ultrasound contrast agents consist of gas-filled coated microbubbles that oscillate upon ultrasound insonification. Their characteristic oscillatory response provides contrast enhancement for imaging and has the potential to locally enhance drug delivery. Since microbubble response depends on the local acoustic pressure, an ultrasound compatible chamber is needed to study their behavior and the underlying drug delivery pathways. In this study, we determined the amplitude of the acoustic pressure in the CLINIcell, an optically transparent chamber suitable for cell culture. The pressure field was characterized based on microbubble response recorded using the Brandaris 128 ultrahigh-speed camera and an iterative processing method. The results were compared to a control experiment performed in an OptiCell, which is conventionally used in microbubble studies. Microbubbles in the CLINIcell responded in a controlled manner, comparable to those in the OptiCell. For frequencies from 1 to 4 MHz, the mean pressure amplitude was -5.4 dB with respect to the externally applied field. The predictable ultrasound pressure demonstrates the potential of the CLINIcell as an optical, ultrasound, and cell culture compatible device to study microbubble oscillation behavior and ultrasound-mediated drug delivery.

Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery.

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3-D cell culture, perfusion flow, and membrane-free soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh-speed camera (17 million frames/s) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated drug delivery. When insonified at frequencies between 1 and 2 MHz, microbubbles in the OrganoPlate experienced larger oscillation amplitudes resulting from higher local pressures. Microbubbles responded similarly in both systems when insonified at frequencies between 2 and 4 MHz. Numerical simulations performed with a 3-D finite-element model of ultrasound propagation into the OrganoPlate and the OptiCell showed the same frequency-dependent behavior. The predictable and homogeneous pressure field in the OrganoPlate demonstrates its potential to develop an in vitro 3-D cell culture model, well suited to study ultrasound-mediated drug delivery.

Vibrational Responses of Bound and Nonbound Targeted Lipid-Coated Single Microbubbles.

One of the main challenges for ultrasound molecular imaging is acoustically distinguishing nonbound microbubbles from those bound to their molecular target. In this in vitro study, we compared two types of in-house produced targeted lipid-coated microbubbles, either consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C16:0 (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine, C18:0 (DSPC) as the main lipid, using the Brandaris 128 ultrahigh-speed camera to determine vibrational response differences between bound and nonbound biotinylated microbubbles. In contrast to previous studies that studied vibrational differences upon binding, we used a covalently bound model biomarker (i.e., streptavidin) rather than physisorption, to ensure binding of the biomarker to the membrane. The microbubbles were insonified at frequencies between 1 and 4 MHz at pressures of 50 and 150 kPa. This paper shows lower acoustic stability of bound microbubbles, of which DPPC-based microbubbles deflated most. For DPPC microbubbles with diameters between 2 and [Formula: see text] driven at 50 kPa, resonance frequencies of bound microbubbles were all higher than 1.8 MHz, whereas those of nonbound microbubbles were significantly lower. In addition, the relative radial excursions at resonance were also higher for bound DPPC microbubbles. These differences did not persist when the pressure was increased to 150 kPa, except for the acoustic stability which further decreased. No differences in resonance frequencies were observed between bound and nonbound DSPC microbubbles. Nonlinear responses in terms of emissions at the subharmonic and second harmonic frequencies were similar for bound and nonbound microbubbles at both pressures. In conclusion, we identified differences in vibrational responses of bound DPPC microbubbles with diameters between 2 and [Formula: see text] that distinguish them from nonbound ones.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.

Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.