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

Purpose

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.

Procedures

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.

Results

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.

Conclusions

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.     

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality

BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.     

Evaluation of the Temporal Window for Drug Delivery Following Ultrasound-Mediated Membrane Permeability Enhancement

Purpose  

Ultrasound-induced cavitation facilitates cellular uptake of drugs via increased membrane permeability. Here, the purpose was to evaluate the duration of enhanced membrane permeability following ultrasound treatment in cell culture.     

Ultrasound-Mediated Targeted Drug Delivery Generated by Multifocal Beam Patterns: An In vitro Study

Ultrasound-mediated targeted drug delivery has been a subject for a dedicated research activity for several decades. Nevertheless, in vitro studies in this field of research are characterized by their inconsistencies. To improve the repeatability of such experiments, a novel approach of multifocal spot generation was investigated. A multifocal pattern of 16 spots was utilized using an iterative Gerchberg–Saxton algorithm. The pattern was applied to insonate a 96-well Petri dish plate using a clinically available planar-phased array transducer with approximately 1000 elements with a central frequency of 0.55 MHz. The pattern was acoustically characterized and applied to a monolayer of human breast cancer cell line in the 96-well plate. With the help of ultrasonic contrast agents, the intracellular drug uptake was increased by an average factor of 3.5 compared with the control group.     

Targeted Gene Transfection from Microbubbles into Vascular Smooth Muscle Cells Using Focused,Ultrasound-Mediated Delivery

We investigated a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0 to 950 kPa) and pulse durations (0 to 100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA or ultrasound alone. Microbubbles in combination with ultrasound (500-kPa, 1-MHz, 50-cycle bursts at a pulse repetition frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 ± 27%) and without (19 ± 8%). Maximal gene transfection (∼1% of cells) occurred using 50-cycle, 1-MHz pulses at 300 kPa, which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. (E-mail: jh7fj@virginia.edu)     

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

Purpose

This study aims to quantitatively analyze cellular uptake following local ultrasound (US)-mediated cell permeabilization.

Procedures

A 2 μM cell-impermeable dye Sytox Green was co-injected with 3?×?10⁷ microbubbles in the presence of C6 rat glioblastoma cell monolayer in total volume of 10 ml. A 5.8-mm diameter mono-element US transducer was positioned at a distance of 8 mm to the Opticell® membrane. Acoustical pressure of pulsed US was varied from 0.62 MPa peak-to-peak (p-p) to 1.25 MPa p-p. Large field of view (FOV?=?15?×?15 mm) 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.

Results

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 0.88 MPa p-p.

Conclusions

Spatial aspect of US-mediated model drug uptake has been quantitatively evaluated on adherent cells using robust 2D-mapping approach.     

Transcranial Photoacoustic Detection of Blood-Brain Barrier Disruption Following Focused Ultrasound-Mediated Nanoparticle Delivery

Molecular Imaging and Biology - Blood-brain barrier disruption (BBBD) is of interest for treating neurodegenerative diseases and tumors by enhancing drug delivery. Focused ultrasound (FUS) is a...     

Ultrasound-Mediated Delivery of Chemotherapy into the Transgenic Adenocarcinoma of the Mouse Prostate Model

Ultrasound (US) in combination with microbubbles (MB) has had promising results in improving delivery of chemotherapeutic agents. However, most studies are done in immunodeficient mice with xenografted tumors. We used two phenotypes of the spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model to evaluate if US + MB could enhance the therapeutic efficacy of cabazitaxel (Cab). Cab was either injected intravenously as free drug or encapsulated into nanoparticles. In both cases, Cab transiently reduced tumor and prostate volume in the TRAMP model. No additional therapeutic efficacy was observed combining Cab with US + MB, except for one tumor. Additionally, histology grading and immunostaining of Ki67 did not reveal differences between treatment groups. Mass spectrometry revealed that nanoparticle encapsulation of Cab increased the circulation time and enhanced the accumulation in liver and spleen compared with free Cab. The therapeutic results in this spontaneous, clinically relevant tumor model differ from the improved therapeutic response observed in xenografts combining US + MB and chemotherapy.     

Comparison of Acoustofluidic and Static Systems for Ultrasound-Mediated Molecular Delivery to T Lymphocytes

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0–30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate–dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 μg/mL vs. 94 μg/mL) and shorter treatment time (～2–3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.     

超声显微镜技术评定粥样硬化组织声学特性的研究

尸检获取人主动脉壁正常及粥样硬化斑块处标本，１００个区域，２０６块。利用超声显微镜技术对正常内膜组（Ｎ），脂肪沉积组（Ｆ），纤维化组（ＦＳ）及钙化组（ＣＡ）进行超声显微镜成像，同时测量各组的声学指标－超声传导速度（Ｖｓ）值。结果显示：超声显微镜能清晰显示主动脉壁正常及病理改变时各层次结构，定量测定Ｖｓ值时，Ｆ组较Ｎ组显著减低（Ｐ＜０．００１），ＦＳ组及ＣＡ组较Ｎ组显著增高。提示：组织的病理成份改变对声学参数产生明显影响     

Endothelial Cells,First Target of Drug Delivery Using Microbubble-Assisted Ultrasound

Microbubble-assisted ultrasound has emerged as a promising method for local drug delivery. Microbubbles are intravenously injected and locally activated by ultrasound, thus increasing the permeability of vascular endothelium for facilitating extravasation and drug uptake into the treated tissue. Thereby, endothelial cells are the first target of the effects of ultrasound-driven microbubbles. In this review, the in vitro and in vivo bioeffects of this method on endothelial cells are described and discussed, including aspects on the permeabilization of biologic barriers (endothelial cell plasma membranes and endothelial barriers), the restoration of their integrity, the molecular and cellular mechanisms involved in both these processes, and the resulting intracellular and intercellular consequences. Finally, the influence of the acoustic settings, microbubble parameters, treatment schedules and flow parameters on these bioeffects are also reviewed.     

Assessment of Malaria In Vitro Drug Combination Screening and Mixed-Strain Infections Using the Malaria Sybr Green I-Based Fluorescence Assay

Several drug development strategies, including optimization of new antimalarial drug combinations, have been used to counter malaria drug resistance. We evaluated the malaria Sybr green I-based fluorescence (MSF) assay for its use in in vitro drug combination sensitivity assays. Drug combinations of previously published synergistic (atovaquone and proguanil), indifferent (chloroquine and azithromycin), and antagonistic (chloroquine and atovaquone) antimalarial drug interactions were tested against Plasmodium falciparum strains D6 and W2 using the MSF assay. Fifty percent inhibitory concentrations (IC₅₀s) were calculated for individual drugs and in fixed ratio combinations relative to their individual IC₅₀s. Subsequent isobologram analysis and fractional inhibitory concentration determinations demonstrated the expected drug interaction pattern for each combination tested. Furthermore, we explored the ability of the MSF assay to examine mixed parasite population dynamics, which are commonly seen in malaria patient isolates. Specifically, the capacity of the MSF assay to discern between single and mixed parasite populations was determined. To simulate mixed infections in vitro, fixed ratios of D6 and W2 strains were cocultured with antimalarial drugs and IC₅₀s were determined using the MSF assay. Dichotomous concentration curves indicated that the sensitive and resistant parasites composing the genetically heterogeneous population were detectable. Biphasic analysis was performed to obtain subpopulation IC₅₀s for comparison to those obtained for the individual malaria strains alone. In conclusion, the MSF assay allows for reliable antimalarial drug combination screening and provides an important method to discern between homogenous and heterogeneous parasite populations.Malaria is a severe global health problem that is compounded by the emergence of drug-resistant parasites. The emergence of these multidrug-resistant Plasmodium species, particularly P. falciparum, has made decisions regarding malaria chemoprophylaxis and treatment more complicated. Furthermore, it is predicted that increased incidences of clinical infections and subsequent deaths are likely as the rapid spread of resistant parasites occurs (15, 23, 35, 39). Several drug development strategies have been used to counter malaria drug resistance, including optimization of new antimalarial drug combinations. Several groups have examined drug combinations in laboratory settings that may have promising efficacies in clinical settings (5, 7, 30). Artemisinin-based combination therapies, one of the most successful therapeutic combinations, are currently used in areas where malaria is endemic (39). However, it is predicted that even with an aggressive prophylactic and treatment campaign, resistance to these drugs will certainly emerge (8). In fact, resistance to these combinations has already been observed. Wongsrichanalai and others reported the decreasing efficacy of the artesunate-mefloquine combination on the Cambodian-Thai border (41). Thus, there is a need to discover novel combinations between existing antimalarials and/or new chemical entities that can be used in the treatment of severe malaria.In addition to resistance to antimalarial combination therapies, there is also a concern with Plasmodium mixed infections. A mixed infection is defined as an infection with more than one type of species or genotype of Plasmodium (24). Although highly understudied, the implications of a mixed infection are profound. Mixed infections can cause a relapse as a result of emergence of the resistant subpopulation of parasites after the sensitive subpopulation has been eradicated by drug therapy. The existence of a resistant population may be a result of both divergent evolution, where parasites have acquired resistance mechanisms, and/or two cohabitating parasites when the individual is infected (16, 24). This phenomenon has been observed in areas of malaria endemicity in Africa and Southeast Asia, where the mixed-infection prevalence is as high as 30% (24). However, there has been conflicting evidence as to the true frequency of Plasmodium mixed infections (27, 34). Furthermore, this problem is confounded by the inability to properly identify and differentiate Plasmodium mixed infections.The [³H]hypoxanthine incorporation assay has been used as the gold standard in P. falciparum drug susceptibility testing (11). Despite the assay''s reliability and accuracy, it is very expensive, involves multiple processing steps, and requires special handling and waste disposal procedures. In contrast, dye-based technologies, such as those using 4′,6-diaminino-2-phenylindole, Pico green, YOYO-1, and Sybr green, have been shown to have comparable results to radioactive assays (2, 10, 17, 19, 36, 38). Many of these assays use DNA dye intercalation, which accurately measures parasite growth. Use of these assays has increased because they are relatively simple and inexpensive to run compared to their radioactive and enzyme-linked immunosorbent assay-based counterparts.While several in vitro drug sensitivity assays have been used to analyze antimalarial drug interactions, the ability of the malaria Sybr green I-based fluorescence (MSF) assay for this purpose has not been fully characterized. The [³H]hypoxanthine incorporation assay has been shown to detect differences in susceptibility patterns as well as being able to identify drug interactions (11, 30). However, radioactivity usage makes it costly and difficult to routinely use in research and clinical settings, particularly in a resource-limited environment. The MSF assay utilizes the Sybr green I dye (Invitrogen, San Diego, CA), which is relatively inexpensive and has been shown to reliably measure P. falciparum in vitro drug sensitivities. As the prevalence of Plasmodium mixed infections increases, there is a need for an assay that can reliably identify and differentiate drug-sensitive subpopulations in a particular infection. In this study, we examined the capability of the MSF assay to determine drug interactions and discern between single and mixed P. falciparum populations.     

Normative Values for Corneal Nerve Morphology Assessed Using Corneal Confocal Microscopy: A Multinational Normative Data Set

OBJECTIVECorneal confocal microscopy is a novel diagnostic technique for the detection of nerve damage and repair in a range of peripheral neuropathies, in particular diabetic neuropathy. Normative reference values are required to enable clinical translation and wider use of this technique. We have therefore undertaken a multicenter collaboration to provide worldwide age-adjusted normative values of corneal nerve fiber parameters.RESULTSThere was a significant linear age-dependent decrease in CNFD (−0.164 no./mm² per year for men, P < 0.01, and −0.161 no./mm² per year for women, P < 0.01). There was no change with age in CNBD (0.192 no./mm² per year for men, P = 0.26, and −0.050 no./mm² per year for women, P = 0.78). CNFL decreased in men (−0.045 mm/mm² per year, P = 0.07) and women (−0.060 mm/mm² per year, P = 0.02). CNFT increased with age in men (0.044 per year, P < 0.01) and women (0.046 per year, P < 0.01). Height, weight, and BMI did not influence the 5th percentile normative values for any corneal nerve parameter.CONCLUSIONSThis study provides robust worldwide normative reference values for corneal nerve parameters to be used in research and clinical practice in the study of diabetic and other peripheral neuropathies.     

基于角膜共聚焦显微镜的神经纤维人工智能分析方法

糖尿病周围神经病变(diabetic peripheral neuropathy,DPN)是糖尿病最常见的慢性并发症之一.基于临床症状体征以及电生理检查的传统DPN诊断方法主要用于检测大神经纤维病变,而DPN最早出现损伤的部位是小神经纤维.角膜共聚焦显微镜(corneal confocal microscopy,CCM...     

New Scores for the Assessment of Mitral Stenosis Using Real-Time Three-Dimensional Echocardiography

Nonsurgical management of patients with symptomatic mitral valve stenosis has been established as the therapeutic modality of choice for two decades. Catheter-based balloon dilation of the stenotic valvular area has been shown, at least, as effective as surgical interventions. Unfavorable results of catheter-based interventions are largely due to unfavorable morphology of the valve apparatus, particularly leaflets calcification and subvalvular apparatus involvement. A mitral valve score has been proposed in Boston, MA, about two decades ago, based on morphologic assessment of mitral valve apparatus by two-dimensional (2D) echocardiography to predict successful balloon dilation of the mitral valve. Several other scores have been developed in the following years in order to more successfully predict balloon dilatation outcome. However, all those scores were based on 2D echocardiography, which is limited by ability to distinguish calcification and subvalvular involvement. The introduction of new matrix-based ultrasound probe has allowed 3D echocardiography (3DE) to provide more detailed morphologic analysis of mitral valve apparatus including calcification and subvalvular involvement. Recently, a new 3DE scoring system has been proposed by our group, which represents an important leap into refinement of the use of echocardiography guiding mitral valve interventions.     

Quantitative Assessment of Bladder Neck Compliance by Using Transvaginal Real-Time Elastography of Women

To assess the feasibility of using ultrasound real-time elastography (RTE) to measure bladder neck compliance, we performed real-time elastography measurements by manually applying repetitive compression with the transducer on the scan position of the bladder neck. Instant elastography index (EI) and mean EI of anterior and posterior lips of the bladder neck were calculated. The EI values of anterior and posterior lips of the bladder neck were analyzed in relation to age, body surface area, body mass index, detrusor wall thickness and length, width and thickness of the bladder neck in healthy women. The intra-observer and inter-observer repeatability of measurements in different parts of the bladder neck were assessed using intra-class correlation coefficients with 95% confidence intervals and Bland–Altman analysis. There were no statistically significant differences between elastography measurements made by the same or two different observers in each area measured. There was no significant difference between anterior and posterior lip thickness of the bladder neck. The distribution of the elastography measurements indicated that the anterior lip of the bladder neck was slightly harder than the posterior lip. On the whole, from the results of the study, it was clear that EIs of the bladder neck were related to age in healthy women. Stepwise multiple regression analysis results revealed that age was the only independent factor modulating compliance of the bladder neck in healthy women. It is possible to provide a reproducible semi-quantification of real-time elastography in bladder neck compliance.     

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.     

A Comparison Study of Real-Time Ultrasound Elastography and Electron Microscopy for the Assessment of Liver Damage Induced by Brain Death

The aim of this study was to investigate the specificity and sensitivity of real-time ultrasound elastography (RTE) in the evaluation of liver damage induced by brain death and the correlation with ultrastructural changes in liver tissue. Eleven RTE parameters before brain death and at 0, 3, 6 and 9 h after brain death in 12 miniature pigs were collected and analyzed, and the correlation of these parameters with electron microscopy results was explored. Six of the RTE parameters, namely, mean relative strain value within the region of interest, standard deviation of the relative strain value within the region of interest, area of low strain within the region of interest, complexity of low strain area within the region of interest, skewness and correlation, significantly differed among the time periods. Categorical data were analyzed using the χ²-test. Spearman's correlation analysis was used for evaluating correlations between RTE parameters and electron microscopy results, and the correlation coefficients (r) were calculated. Electron microscopy results revealed that liver damage gradually increased after brain death, with significant differences between 0 and 9 h (χ²?=?14.143, p value?=?0.027). In addition, the six aforementioned RTE parameters significantly correlated with electron microscopy results, with the mean relative strain value within the region of interest being the strongest (r?=?–0.59, p value < 0.001) correlated parameter. RTE could provide preliminary assessment of liver damage induced by brain death, and correlates to ultrastructural changes in liver tissue.     

Ultrasound-Mediated Microbubble Enhancement of Radiation Therapy Studied Using Three-Dimensional High-Frequency Power Doppler Ultrasound

Tumor responses to high-dose (>8 Gy) radiation therapy are tightly connected to endothelial cell death. In the study described here, we investigated whether ultrasound-activated microbubbles can locally enhance tumor response to radiation treatments of 2 and 8 Gy by mechanically perturbing the endothelial lining of tumors. We evaluated vascular changes resulting from combined microbubble and radiation treatments using high-frequency 3-D power Doppler ultrasound in a breast cancer xenograft model. We compared treatment effects and monitored vasculature damage 3 hours, 24 hours and 7 days after treatment delivery. Mice treated with 2 Gy radiation and ultrasound-activated microbubbles exhibited a decrease in vascular index to 48 ± 10% at 24 hours, whereas vascular indices of mice treated with 2 Gy radiation alone or microbubbles alone were relatively unchanged at 95 ± 14% and 78 ± 14%, respectively. These results suggest that ultrasound-activated microbubbles enhance the effects of 2 Gy radiation through a synergistic mechanism, resulting in alterations of tumor blood flow. This novel therapy may potentiate lower radiation doses to preferentially target endothelial cells, thus reducing effects on neighboring normal tissue and increasing the efficacy of cancer treatments.