The Parallel Plate Flow Chamber In Assessing Targeted Microbubbles Adhesion Under Shear Flow

ObjectiveThe general idea for using ultrasound contrast in molecular imaging is very straightforward. Targeted Microbubbles are administered in the circulation, it can used as tracers to detect blood flow, and also they can selectively bind to the molecular receptors on the diseased tissues and accumulated there. Excess circulating microbubbles are cleares from the bloodstream after a few minutes, and the target/normal tissue ratio of the ultrasound signal becomes enough for selective imaging of the vascular pathology and molecular changed. One strategy to prepare targeted microbubbles is by coupling ligands specific to target receptors onto the microbubble surface Via avidin-biotin bridge. Such site targeted microbubbles bind to specific receptors expressed on cell surfaces and are thereby retained in the tissue.Adhesion behavior of these targeted microbubbles has been studied in vivo, usually use the mouse cremasteric inflammation model. However, there are many other factor such as binding to leukocytes and Phagocytosed in vivo. And also a rigorous, complete characterization of microbubble binding and detachment kinetics is not possible in vivo because the local site density of the targeted molecule is unknown.The adhesion of leukocytes to endothelial cells has been extensively studied in vitro as well as in vivo. Parallel plate flow chamber systems have been used to study the adhesion of neutrophils, polystyrene beads and nanospheres to specific receptors on the target surface under static and flow conditions. Attachment of cells and beads depends on the site density of the adhesion molecules and on the fluid shear stress applied. In this study, a parallel plate flow chamber system was applied to gain insights into the adhesion of targeted microbubbles to immobilized substrates.This study was undertaken to establish a kind of biotinylated microbubbles. First, assessed the binding abilities of biotinylated microbubbles to streptavidin . And then, use the "avidin-biotin" bridge to attached a monoclonal antibodies against mouse IgG to this biotinylated microbubble , assecced whether it would bind to immobilized mouse IgG under controlled shear conditions. Finally, a targeted microbubble was designed by conjugating monoclonal antibodies against mouse P-selectin to the lipid shell of the microbubble via an "avidin-biotin" bridge. And the binding capability of microbubbles targeted to P-selectin was studied using the parallel plate flow chamber under physiologic flow conditions. Understanding the process of targeted microbubble binding and detachment may help in the assessment of successful targeted preparations and prediction the in vivo behavior and the using circumstance of targeted microbubbles.Methods1. Preparation of biotinylated microbubbles and used The Parallel plate flow chamber to assess biotinylated microbubbles targeted to streptavidin1.1. Preparation of biotinylated microbubbles and determine their biological properties.Biotinylated microbubbles(MBb) AND Control lipid microbubbles(MB) were prepared by sonication, the MB and MBb were used microscopy to observe and used coulter counter to counte. Using green fluorescent-labeled streptavidin to identify the biotin lipid on the microbubbles' surface. The ultrasound contrast effects of the MBb and MB were used the renal contrast -enhanced ultrasound(CEU) in rat to determine.1.2. Assessment of biotinylated microbubbles targeted to streptavidin with Parallel plate flow chamberThe binding abilities of MBb to streptavidin (5μg/ml, 50μg/ml) immobilized on a culture dish to different shear stresses (0.2-51.2dyn/cm~2 )in every 30 seconds were assessed in a parallel plate flow chamber. The Control object is the culture dish that lacks of streptavidin. And use MB to assesse the non-specific binding to 50μg/ml streptavidin substrates, all groups ues 3 samples according to the recommended protocol.2. The parallel plate flow chamber in assessing the microbubbles' binding ability targeted to mouse IgG2.1. monoclonal antibodies against mouse IgG was attached to biotinylated microbubbles via streptavidin-biotin coupling (MB-IgG), use microscopy and coulter counter to observe and counte. The ultrasound contrast effect of the MB-IgG was determined by using the hindlimb CEU in rat. And use the parallel plate flow chamber to evaluate the MB-IgG binding to mouse IgG.2.2. MB-IgG (5×10~6 /ml) were drawn through the flow chamber at a shear stress of 0.3 dyn/cm~2 for 6 min. Their adhesion to the target surface with different mouse IgG substrated (10ng/ml,100ng/ml and 1000ng/ml) was assessed. Control experiments were performed with plates with no mouse IgG and on mouse IgG coated plates blocked with excess goat anti mouse IgG monoclonal antibody prior to infusion of microbubbles. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area at 60-s intervals and a graph of microbubble accumulation with time was plotted.3. Binding capability of microbubbles targeted to P-selectin under physiologic flow conditions1. Targeted microbubble was designed by conjugating monoclonal antibodies against mouse P-selectin to the lipid shell of the microbubble via an "avidin-biotin" bridge. use microscopy and Coulter counter to observe and counte.2. The binding and retention of targeted microbubbles to P-selectin immobilized on a culture dish were assessed in a parallel-plate flow chamber. Targeted microbubbles drawn through the flow chamber coated with P-selectin (10-1000ng/ml) at a shear stress of 0.3 dyn/cm~2, and different shear stress(0.2-1.7dyn/cm~2) with 1000ng/ml P-selectin substrates. Control experiments on a plate that lack of P-selectin, or was blocked with excess monoclonal antibodies against P-selectin. Microbubble retention was tested by ramping up shear stress at 30-s intervals.Results1. Preparation of biotinylated microbubbles and used The Parallel plate flow chamber to assess biotinylated microbubbles targeted to streptavidin1.1. Preparation of biotinylated microbubbles and determination of their biological properties.1.1.1. Both MB and MBb were prepared successfully, the density of MB was about 1.5×10~9 /ml, the mean sizes was 2.83μm respectively; the density of MBb was about 1.3×10~9 /ml, and the mean sizes was 2.71μm. They can float to the top overnight and keep the same sizes and density after 30 days.1.1.2. Green fluorescent-labeled streptavidin shows that the streptavidin linked well to the surface of microbubbles, which observed with fluorescence microscopy.1.1.3. Both MB and MBb can achieve high quality contrast effects on rat renal CEU.1.2. Assessment of biotinylated microbubbles targeted to streptavidin with Parallel plate flow chamberBiotinylated microbubbles' binding ability increased at higher plate surface density of streptavidin while the control group results in minimal bind ability (F=170.777, P<0.01) . For a streptavidin substrate of 50μg/ml, half-maximal detachment was achieved at the shear stress of (6.4-12.8) dyn/cm~2.2. The parallel plate flow chamber in assessing the microbubbles' binding ability targeted to mouse IgG2.1. MB-IgG was prepared successfully via streptavidin-biotin couple, the density and the mean sizes remained the same to the MBb. It can contrast enhance rat's hindlimb very well with ultrasound.2.2. Keeping the shear stress and microbubble concentration constant at 0.3dyn/cm~2 and 5×10~6/ml, respectively, the microbubble accumulation over a 6-min interval at three mouse IgG site densities was assessed. Increased numbers of microbubbles accumulated at higher mouse IgG site density and the difference in the accumulation rates was found to be statistically significant (F=282.171, P<0.01) . Control experiments performed on culture dishes without any mouse IgG or on culture dishes coated with mouse IgG but blocked with excess goat anti murine IgG monoclonal antibody demonstrated minimal binding of microbubbles compared to that when mouse IgG was present on the culture dish.3. Binding capability of microbubbles targeted to P-selectin under physiologic flow conditions3.1. Both MBb and MBp were prepared successfully, the density of MBb was about 1.3×10~9 /ml, the mean sizes was about 2.71μm. The density of MBp was about 7.1×10~8 /ml, the mean sizes was 2.73μm.3.2. Keeping the shear stress and microbubble concentration constant at 0.3dyn/cm~2 and 5×10~6/ml, respectively, the microbubble binding over a 6-min interval at three mouse P-selectin site densities was assessed. Increased numbers of microbubbles binding at higher mouse P-selectin site density and the difference in the binding rates was found to be statistically significant (F=235.336, P<0.01) . Control experiments performed on culture dishes without any mouse P-selectin or on culture dishes coated with mouse P-delectin but blocked with excess anti mouse P-selectin monoclonal antibody demonstrated minimal binding of microbubbles compared to that when mouse P-selectin was present on the culture dish.3.3. Microbubble binding was also assessed at six different shear stresses keeping the mouse P-selectin substrates at 1000ng/ml. Linear regression analysis of the data sets was performed to assess the kinetics of microbubble binding on the target surface. Microbubbles' binding rate was found to be the greatest at a shear stress of 0.5dyn/cm2 as compared to the other shear stresses of 0.2, 0.3, 0.8, 1.2 and 1.7dyn/cm~2 (F=150.364,P<0.01) .3.4. Microbubble detachment was assessed by increasing the flow every 30 s and observing the number of microbubbles remaining bound. Better retention of microbubbles was found on the target surfaces with higher mouse P-selectin site densities (F=297.615, P<0.01) . For a mouse P-selectin substrates of 1000ng/ml, half-maximal detachment was achieved at a shear stress of～9.6dyn/cm~2.Conclusions1. Biotinylated microbubbles were prepared successful, it can coupled many kind of ligands via "avidin-biotin" bridge on to microbubbles' surface. 2. Parallel plate flow chamber experiments can use to assess biotinylated microbubbles' binding ability to streptavidin. The parallel plate flow chamber system can assessed targeted microbubbles' binding ability well, this system can be developed to perform flow studies to other kinds of targeted microbubbles.3. MB-IgG was prepared successfully via streptavidin-biotin couple, it can adhere to the mouse IgG firmly, using this method can provide many kinds of targeted microbubbles.4. Binding and retention of the targeted microbubbles to P-selectin is specific and effective under physiologic flow conditions.5. In vitro evaluation of binding capability of targeted microbubbles could be useful in predicting the effects of molecular ultrasound imaging and the using circumstance of microbubbles in vivo.