Lynda J.M. Juffermans

Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

Ultrasound and microbubble-induced intra- and intercellular bioeffects in primary endothelial cells.

Recent developments in the field of ultrasound (US) contrast agents have demonstrated that these encapsulated microbubbles can not only be used for diagnostic imaging but may also be employed as therapeutic carriers for localized, targeted drug or gene delivery. The exact mechanisms behind increased uptake of therapeutic compounds by US-exposed microbubbles are still not fully understood. Therefore, we studied the effects of stably oscillating SonoVue microbubbles on relevant parameters of cellular and intercellular permeability, i.e., reactive oxygen species (ROS) homeostasis, calcium permeability, F-actin cytoskeleton, monolayer integrity and cell viability using live-cell fluorescence microscopy. US was applied at 1-MHz, 0.1MPa peak-negative pressure, 0.2% duty cycle and 20Hz pulse repetition frequency to primary endothelial cells. We demonstrated increased membrane permeability for calcium ions, with an important role for H(2)O(2). Catalase, an extracellular H(2)O(2) scavenger, significantly blocked the influx of calcium ions. Further changes in ROS homeostasis involved an increase in intracellular H(2)O(2) levels, protein nitrosylation and a decrease in total endogenous glutathione levels. In addition, an increase in the number of F-actin stress fibers and F-actin cytoskeletal rearrangement were observed. Furthermore, US-exposed microbubbles significantly affected endothelial monolayer integrity, but importantly, disrupted cell-cell interactions were restored within 30min. Finally, cell viability was not affected. In conclusion, these data provide more insight in the interactions between US, microbubbles and endothelial cells, which is important for understanding the mechanisms behind US and microbubble-enhanced uptake of drugs or genes.

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

Ultrasound and microbubble-targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.)

Intravital microscopy of localized stem cell delivery using microbubbles and acoustic radiation force

The use of stem cells for the repair of damaged cardiac tissue after a myocardial infarction holds great promise. However, a common finding in experimental studies is the low number of cells delivered at the area at risk. To improve the delivery, we are currently investigating a novel delivery platform in which stem cells are conjugated with targeted microbubbles, creating echogenic complexes dubbed StemBells. These StemBells vibrate in response to incoming ultrasound waves making them susceptible to acoustic radiation force. The acoustic force can then be employed to propel circulating StemBells from the centerline of the vessel to the wall, facilitating localized stem cell delivery. In this study, we investigate the feasibility of manipulating StemBells acoustically in vivo after injection using a chicken embryo model. Bare stem cells or unsaturated stem cells (<5 bubbles/cell) do not respond to ultrasound application (1 MHz, peak negative acoustical pressure P_ = 200 kPa, 10% duty cycle). However, stem cells which are fully saturated with targeted microbubbles (>30 bubbles/cell) can be propelled toward and arrested at the vessel wall. The mean translational velocities measured are 61 and 177 μm/s for P‐ = 200 and 450 kPa, respectively. This technique therefore offers potential for enhanced and well‐controlled stem cell delivery for improved cardiac repair after a myocardial infarction. Biotechnol. Bioeng. 2015;112: 220–227.

Secondary Bjerknes Forces Deform Targeted Microbubbles

Secondary Bjerknes forces can rupture the binding of targeted microbubbles. We have shown before that this effect can be used to quantify the adhesion strength between bubble and target surface [1]. At lower pressures however, microbubbles were observed to snap back to their original position within 100 µs after ultrasound application. In this study the mechanism of this restoring force was investigated in more detail using simultaneous top and side view high speed imaging [2]. Moreover, some results on the process of microbubble detachment (peeling versus uniform rupture) are presented.

Therapeutic Application of Adipose Derived Stem Cells in Acute Myocardial Infarction: Lessons from Animal Models

The majority of patients survive an acute myocardial infarction (AMI). Their outcome is negatively influenced by post-AMI events, such as loss of viable cardiomyocytes due to a post-AMI inflammatory response, eventually resulting in heart failure and/or death. Recent pre-clinical animal studies indicate that mesenchymal stem cells derived from adipose tissue (ASC) are new promising candidates that may facilitate cardiovascular regeneration in the infarcted myocardium. In this review we have compared all animal studies in which ASC were used as a therapy post-AMI and have focused on aspects that might be important for future successful clinical application of ASC.

ULTRASOUND AND MICROBUBBLE-TARGETED DELIVERY OF SMALL INTERFERING RNA INTO PRIMARY ENDOTHELIAL CELLS IS MORE EFFECTIVE THAN DELIVERY OF PLASMID DNA

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.

Endogenous C1-inhibitor production and expression in the heart after acute myocardial infarction.

BACKGROUND Complement activation contributes significantly to inflammation-related damage in the heart after acute myocardial infarction. Knowledge on factors that regulate postinfraction complement activation is incomplete however. In this study, we investigated whether endogenous C1-inhibitor, a well-known inhibitor of complement activation, is expressed in the heart after acute myocardial infarction. MATERIALS AND METHODS C1-inhibitor and complement activation products C3d and C4d were analyzed immunohistochemically in the hearts of patients who died at different time intervals after acute myocardial infarction (n=28) and of control patients (n=8). To determine putative local C1-inhibitor production, cardiac transcript levels of the C1-inhibitor-encoding gene serping1 were determined in rats after induction of acute myocardial infarction (microarray). Additionally, C1-inhibitor expression was analyzed (fluorescence microscopy) in human endothelial cells and rat cardiomyoblasts in vitro. RESULTS C1-inhibitor was found predominantly in and on jeopardized cardiomyocytes in necrotic infarct cores between 12h and 5days old. C1-inhibitor protein expression coincided in time and colocalized with C3d and C4d. In the rat heart, serping1 transcript levels were increased from 2h up until 7days after acute myocardial infarction. Both endothelial cells and cardiomyoblasts showed increased intracellular expression of C1-inhibitor in response to ischemia in vitro (n=4). CONCLUSIONS These observations suggest that endogenous C1-inhibitor is likely involved in the regulation of complement activity in the myocardium following acute myocardial infarction. Observations in rat and in vitro suggest that C1-inhibitor is produced locally in the heart after acute myocardial infarction.

Wistar rats from different suppliers have a different response in an acute myocardial infarction model.

The Wistar rat is a commonly used strain for experimental animal models. Recently it was shown that results vary between studies using Wistar rats of different suppliers. Therefore we studied whether Wistar rats obtained from Harlan Laboratories (Ha, n=24) and Charles River (CR, n=22) had a different outcome in an acute myocardial infarction (AMI) model. AMI was induced in both Ha and CR Wistar rats by one operator. This resulted in a significantly higher survival rate for Ha (79.2±10.2%) compared with CR rats (54.2±10.2%, p<0.05). Furthermore, CR rats had lost significantly more weight after 7 days (-5.9±3.1%) compared with Ha rats (-0.8±1.7%; p<0.001), indicating a worse health status of the CR rats. Paradoxically, the induced infarct was smaller in CR rats (7.3±3.6% of the heart) compared with Ha rats (12.1±4.7%, p<0.05). This indicates that CR rats were less sensitive for the cardiomyocyte damage subsequent to AMI induction, but remarkably showed more clinical side effects indicating that Wistar rats from two suppliers had a different response within the same AMI model.