Uptake Of Model Drug Research Articles

Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface: Towards a pre-clinical model of periodontal diseases and personalized periodontal tissue engineering

While periodontal (PD) disease is among principal causes of tooth loss worldwide, regulation of concomitant soft and mineralized PD tissues, and PD pathogenesis have not been completely clarified yet. Besides, relevant pre-clinical models and in vitro platforms have limitations in simulating human physiology. Here, we have harnessed three-dimensional bioprinting (3DBP) technology for developing a multi-cellular microtissue model resembling PD ligament-alveolar bone (PDL-AB) biointerface for the first time. 3DBP parameters were optimized; the physical, chemical, rheological, mechanical, and thermal properties of the constructs were assessed. Constructs containing gelatin methacryloyl (Gel-MA) and hydroxyapatite-magnetic iron oxide nanoparticles showed higher level of compressive strength when compared with that of Gel-MA constructs. Bioprinted self-supporting microtissue was cultured under flow in a microfluidic platform for >10 days without significant loss of shape fidelity. Confocal microscopy analysis indicated that encapsulated cells were homogenously distributed inside the matrix and preserved their viability for >7 days under microfluidic conditions. Immunofluorescence analysis showed the cohesion of stromal cell surface marker-1+ human PDL fibroblasts containing PDL layer with the osteocalcin+ human osteoblasts containing mineralized layer in time, demonstrating some permeability of the printed constructs to cell migration. Preliminary tetracycline interaction study indicated the uptake of model drug by the cells inside the 3D-microtissue. Also, the non-toxic levels of tetracycline were determined for the encapsulated cells. Thus, the effects of tetracyclines on PDL-AB have clinical significance for treating PD diseases. This 3D-bioprinted multi-cellular periodontal/osteoblastic microtissue model has potential as an in vitro platform for studying processes of the human PDL.

Ultrasound Imaging of Microbubble Activity during Sonoporation Pulse Sequences

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied on many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells and Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation or the use of sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side, and the remainder of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high-frame-rate and high-resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1–1 MPa, 20–1000 cycles and 10,000 to 10,000,000 microbubbles/mL), there is strong and non-linear acoustic attenuation, as well as bubble destruction, gas diffusion and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was found that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.

Investigating the role of lipid transfer in sonoporation

The permeabilisation of cell membranes following exposure to microbubbles and ultrasound has considerable potential for therapeutic delivery. Recent studies have demonstrated that transfer of material takes place between phospholipid-coated microbubbles and cell membranes. The aim of this study was to investigate the impact of this transfer on membrane properties and cell permeability. Microbubbles were prepared with matched concentrations and size distributions from a series of formulations utilising phospholipids with differing molecular characteristics. Quantitative fluorescence microscopy techniques were used to quantify changes in the molecular packing, viscosity, and permeability to model drugs of cancer cells under mild ultrasound exposure (15 s of 1 MHz centre frequency, 125 kPa peak negative pressure, 1 kHz pulse repetition frequency, and 10% duty cycle). The molecular “packing” of cancer cell membranes was found to be significantly affected by the transfer of both phospholipids and other microbubble coating constituents. This was particularly pronounced in the case of microbubbles containing a specific class of lipids, which promoted a ~4-fold increase in model drug uptake following ultrasound exposure. Similar results were observed in an investigation of permeability in an in vitro blood brain barrier model suggesting that microbubble composition may play a key role in the efficacy of ultrasound mediated delivery.

Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates.To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally.Photobleaching rates up to 5.0 10−3 s−1 were measured when applying power densities up to 0.2 W.cm−2. By applying the three-compartment model, the model drug uptake rate of 6.0 10−3 s−1 was measured independent of the applied laser power.The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization.

Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles.

Site-specific perforation of the plasma membrane can be achieved through ultrasound-triggered cavitation of a single microbubble positioned adjacent to the cell. However, for this perforation approach (sonoporation), the recovery manoeuvres invoked by the cell are unknown. Here, we report new findings on how membrane blebbing can be a recovery manoeuvre that may take place in sonoporation episodes whose pores are of micrometres in diameter. Each sonoporation site was created using a protocol involving single-shot ultrasound exposure (frequency: 1 MHz; pulse length: 30 cycles; peak negative pressure: 0.45 MPa) which triggered inertial cavitation of a single targeted microbubble (diameter: 1-5 µm). Over this process, live confocal microscopy was conducted in situ to monitor membrane dynamics, model drug uptake kinetics and cytoplasmic calcium ion (Ca(2+)) distribution. Results show that blebbing would occur at a recovering sonoporation site after its resealing, and it may emerge elsewhere along the membrane periphery. The bleb size was correlated with the pre-exposure microbubble diameter, and 99% of blebbing cases at sonoporation sites were inflicted by microbubbles larger than 1.5 µm diameter (analysed over 124 sonoporation episodes). Blebs were not observed at irreversible sonoporation sites or when sonoporation site repair was inhibited via extracellular Ca(2+) chelation. Functionally, the bleb volume was found to serve as a buffer compartment to accommodate the cytoplasmic Ca(2+) excess brought about by Ca(2+) influx during sonoporation. These findings suggest that membrane blebbing would help sonoporated cells restore homeostasis.

Duration of ultrasound-mediated enhanced plasma membrane permeability

Ultrasound (US) induced cavitation can be used to enhance the intracellular delivery of drugs by transiently increasing the cell membrane permeability. The duration of this increased permeability, termed temporal window, has not been fully elucidated. In this study, the temporal window was investigated systematically using an endothelial- and two breast cancer cell lines. Model drug uptake was measured as a function of time after sonication, in the presence of SonoVue™ microbubbles, in HUVEC, MDA-MB-468 and 4T1 cells. In addition, US pressure amplitude was varied in MDA-MB-468 cells to investigate its effect on the temporal window. Cell membrane permeability of HUVEC and MDA-MB-468 cells returned to control level within 1–2h post-sonication, while 4T1 cells needed over 3h. US pressure affected the number of cells with increased membrane permeability, as well as the temporal window in MDA-MB-468 cells. This study shows that the duration of increased membrane permeability differed between the cell lines and US pressures used here. However, all were consistently in the order of 1–3h after sonication.

Cellular uptake of coumarin-6 under microfluidic conditions into HCE-T cells from nanoscale formulations.

In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.

Anionic cellulose beads for drug encapsulation and release

Cellulose beads were prepared from water- based solvent and oxidised by modified Anelli's reac- tion at 20-80 � C for 2-48 h (Fig. 1). The maximum amount of anionic groups (AGs) was 1:85 mmol g � 1 . The distribution of AGs was verified by absorption of cationic dyes and imaging with confocal fluorescent microscopy. Structural changes were studied spectro- scopically and with electron microscopy. Oxidation of the beads drastically increased the swelling capacity of air-dried beads. Uptake of model drug was more than doubled in never-dried beads. This is due to the changes in pore size distribution, mainly opening and widening of the closed pores and narrow cavities. Release profiles of the drug were studied at physio- logical pH of 7.4 and showed a controlled release rate independently of the amount of the drug encapsulated and amount of AGs.

Tracking of Cell Nuclei for Assessment of In Vitro Uptake Kinetics in Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation. Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells. Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93% of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision. Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.

Quantitative Evaluation of Ultrasound-Mediated Cellular Uptake of a Fluorescent Model Drug

This study aims to quantitatively analyze cellular uptake following local ultrasound (US)-mediated cell permeabilization. A 2μM cell-impermeable dye Sytox Green was co-injected with 3 × 10(7) microbubbles in the presence of C6 rat glioblastoma cell monolayer in total volume of 10ml. A 5.8-mm diameter mono-element US transducer was positioned at a distance of 8mm to the Opticell® membrane. Acoustical pressure of pulsed US was varied from 0.62MPa peak-to-peak (p-p) to 1.25MPa p-p. Large field of view (FOV = 15 × 15mm) 22 × 22 mosaic acquisitions were done under epifluorescence Leica DMR microscope and analyzed in Metamorph software to evaluate cell density as well as model drug uptake percentage. The size of acoustical field of the transducer closely matches the spatial pattern of the model drug internalized into the cells by US. Maximum of uptake percentage (42 ± 15%) was found at 0.88MPa p-p. Spatial aspect of US-mediated model drug uptake has been quantitatively evaluated on adherent cells using robust 2D-mapping approach.

Real-Time Assessment of Ultrasound-Mediated Drug Delivery Using Fibered Confocal Fluorescence Microscopy

Transport across the plasma membrane is a critical step of drug delivery for weakly permeable compounds with intracellular mode of action. The purpose of this study is to demonstrate real-time monitoring of ultrasound (US)-mediated cell-impermeable model drug uptake with fibered confocal fluorescence microscopy (FCFM). An in vitro setup was designed to combine a mono-element US transducer, a cell chamber with a monolayer of tumor cells together with SonoVue microbubbles, and a FCFM system. The cell-impermeable intercalating dye, SYTOX Green, was used to monitor US-mediated uptake. The majority of the cell population showed fluorescence signal enhancement 10s after US onset. The mean rate constant k of signal enhancement was calculated to be 0.23 ± 0.04min(-1). Feasibility of real-time monitoring of US-mediated intracellular delivery by FCFM has been demonstrated. The method allowed quantitative assessment of model drug uptake, holding great promise for further local drug delivery studies.