This study tested the hypothesis that controlled flow through microchannels can cause shear-induced intracellular loading of cells with molecules. The overall goal was to design a simple device to expose cells to fluid shear stress and thereby increase plasma membrane permeability. DU145 prostate cancer cells were exposed to fluid shear stress in the presence of fluorescent cell-impermeant molecules by using a cone-and-plate shearing device or high-velocity flow through microchannels. Using a syringe pump, cell suspensions were flowed through microchannels of 50 -300 μm diameter drilled through Mylar® sheets using an excimer laser. As quantified by flow cytometry, intracellular uptake and loss of viability correlated with the average shear stress. Optimal results were observed when exposing the cells to high shear stress for short durations in conical channels, which yielded uptake to over one third of cells while maintaining viability at approximately 80%. This method was capable of loading cells with molecules including calcein (0.62 kDa), large molecule weight dextrans (150 -2000 kDa), and bovine serum albumin (66 kDa). These results supported the hypothesis that shearinduced intracellular uptake could be generated by flow of cell suspensions through microchannels and further led to the design of a simple, inexpensive, and effective device to deliver molecules into cells. Such a device could benefit biological research and the biotechnology industry.
This study tested the hypothesis that ultrasound can target intracellular uptake of drugs into vascular endothelial cells (ECs) at low to intermediate energy and into smooth muscle cells (SMCs) at high energy. Ultrasound-enhanced delivery has been shown to enhance and target intracellular drug and gene delivery in the vasculature to treat cardiovascular disease, but quantitative studies of the delivery process are lacking. Viable ex vivo porcine carotid arteries were placed in a solution containing a model drug, TO-PRO ® -1, and Optison ® microbubbles. Arteries were exposed to ultrasound at 1.1 MHz and acoustic energies of 5.0, 66, or 630 J/cm 2 . Using confocal microscopy and fluorescent labeling of cells, the artery endothelium and media were imaged to determine the localization and to quantify intracellular uptake and cell death. At low to intermediate ultrasound energy, ultrasound was shown to target intracellular delivery into viable cells that represented 9 -24% of exposed ECs. These conditions also typically caused 7 -25% EC death. At high energy, intracellular delivery was targeted to SMCs, which was associated with denuding or death of proximal ECs. This work represents the first known in-depth study to evaluate intracellular uptake into cells in tissue. We conclude that significant intracellular uptake of molecules can be targeted into ECs and SMCs by ultrasound-enhanced delivery suggesting possible applications for treatment of cardivascular diseases and dysfunctions.
Noninvasive methods to measure and predict ultrasound effects on cells are needed to realize applications of ultrasound-mediated drug delivery to improve chemotherapy, gene therapy and targeted delivery. This study tested the hypothesis that (i) cellular bioeffects of ultrasound correlate with cavitation dose, (ii) broadband noise provides a measure of cavitation dose, and, thus, (iii) cellular bioeffects can be predicted by noninvasively measuring broadband noise. After exposing cell suspensions to ultrasound and measuring intracellular molecular uptake and loss of cell viability (bioeffects), a broad range of bioeffects were achieved by varying frequency, pressure, exposure time, cavitation nucleation site (Optison) concentration, and cell type. As a measure of cavitation activity, broadband noise measurements from acoustic spectra were collected during cell sonication and shown to be larger at elevated pressure and, after a high initial value, sharply decayed to a constant, background value at long exposure times. Combining these results, we found that broadband noise correlated well with molecular uptake and viability over the broad range of experimental conditions used (p-value <0.0001). This indicates that acoustic spectrum analysis provides a unifying parameter to correlate with bioeffects over a wide range of acoustic and experimental conditions. [Work supported by NIH, EKOS, DoEd GAANN Program.]
In this case study, we present an approach for employing modeling to help define the design space for a reaction with potential to generate an impurity that could impact the quality of an API. Our approach broadly consisted of (1) evaluating the reaction parameters that can affect the critical impurity level to develop appropriate assumptions for a mechanistic model, (2) developing and evaluating a mechanistic model to predict the formation of the critical impurity, (3) defining a design space based on the model output to reduce in practice the acceptable parameter space to a practical number of parameters, and (4) verifying the design space through experimental testing. This work resulted in a verified design space that can be practically employed and includes wide parameters ranges for manufacturing flexibility.
The majority of active pharmaceutical ingredients (APIs) and intermediates are isolated as solid products through crystallization, filtration, and drying. In some cases, filtration of APIs and intermediates exhibit long cycle times and may potentially become the bottleneck of the entire process train. Thus, early assessment of the cake properties is typically required to evaluate filtration performance prior to scale-up. This work presents two approaches to rapidly estimate the specific cake resistance through lab studies. Using the first approach, a first-order approximation of the specific cake resistance is estimated from data collected during a simple Buchner funnel filtration. The second approach provides a more accurate estimate from a more extensive filtration study incorporating dynamic pressure modulation (DPM, a single filtration with ascending pressures), improving the fidelity of filtration predictions. Results from several case studies demonstrate how a workflow combining these two approaches can be appropriately employed to assess the cake properties from laboratory filtrations for predictions of the pilot/manufacturing plant filtration performance.
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