Development Of Novel Polymeric Microbubble With Matrix Metalloproteinase-2 Targeting Potential

Part One: Real-time Myocardial Contrast Echocardiography in Rat: Infusion vs. Bolus AdministrationAIM: To compare the feasibility of real-time myocardial contrast echocardiography (RTMCE) in rats with infusion and bolus administration of BR1.Methods: RTMCE was performed in 12 Sprague–Dawley rats using B-mode RTMCE following the BR1 infusion or bolus injection. The myocardium signal intensity (SI) was plotted against time and was fitted to exponential functions. The plateau SI (A) and rate of SI increase (β) for the infusion study and peak signal intensity (PSI) for the bolus study were obtained. 99mTc-Sestamibi and Evans blue were used to assess myocardial blood perfusion and to calculate the myocardium perfusion defect area ex vivo. Results: High-quality RTMCE images were successfully obtained using each method. At baseline, all LV segments showed even contrast distribution(Infusion: A*βLAD: 48.27±3.72dB, A*βLCx: 47.89±5.43dB, p>0.05; Bolus: PSI LAD: 48.27±3.72dB, PSI LCx: 47.89±5.43dB, p>0.05). Following left anterior descending coronary artery(LAD)ligation, significant perfusion defect was observed in LAD beds with a significantly decreased A*βand PSI values compared to LCx beds (Infusion: A*βLAD: 5.42±1.57dB, A*βLCx: 46.52±5.32dB, p<0.05; Bolus: PSI LAD: 2.11±0.67dB, PSI LCx: 20.68±0.72 dB, p<0.05), which was consistent with 99mTc-Sestamibi distribution findings. Myocardial perfusion defect areas assessed by both methods showed no differences and showed good correlation with Evans blue staining. ED frames were more favorable for imaging analysis.Conclusions: Both infusion and bolus administration of the contrast agent combined with RTMCE technique can provide a reliable and non-invasive approaches for myocardial perfusion assessment in rats, and the infusion method was more suitable for quantitative analysis of myocardial blood flow.Part Two: Development Research on Novel Polymeric Microbubble with Targeted AbilityAIM: To develop the novel polymeric microbubbles with targeted ability with polyethylene glycol (PEG) space arm. Method: Polyethylene glycol (PEG), carbohydrate, lipid, phospholipids, the polymer, the surface active agent and high fatty alcohol were used according to the different proportion to prepare microbubble membrane solution. During the solution sonication, the sulfur hexafluoride (SF6) gas were passed over simultaneously. The diameter, the density and the stability of microbubbles were tested to modify the microbubbles preparation condition. MMP2-targeted microbubbles (TMB2) were prepared by conjugating monoclonal MMP2 antibody to microbubble shell surface using PEG arm. Attachment of antibodies to the MB's surface was verified by flow cytometry and fluorescent microscopy studies.Results: The does and proportion of the microbubble member materials would affect the microbubbles features. An novel kind of polymeric microbubbles was generated with 3.36±1.62μm in size and 7×109/ml in concentration. The microbubbles can be kept for 5 days in room temperature and two months at 4°C. After incubating with monoclonal MMP2 antibody, fluorescent microscopy of the TMB2 revealed an even"green ring"around the microbubble surface. Anti-MMP2 mAb attachment to the TMB2 shell was also confirmed by flow cytometry.Conclusions: Novel polymeric microbubble has the high binding ability of monoclone antibody (mAb) on its shell via PEG arm. This application is ideal for both targeted imaging and/or targeted drug. Part Three: Targeted Ultrasound Contrast Imaging of Matrix Metalloproteinase-2 in Ischemia-Reperfusion Rat Model: ex vivo and in vivo StudiesAIM: Activation of matrix metalloproteinase-2 (MMP2) after myocardial ischemia-reperfusion (I/R) contributes to adverse left ventricular (LV) remodeling. We hypothesized that post I/R remodeling-associated MMP2 activation could be detected in vivo by using ultrasound with novel MMP2 targeted microbubble.Methods: MMP2-targeted microbubbles (TMB2) were prepared by conjugating monoclonal MMP2 antibody to microbubble shell surface using the polyethylene glycol (PEG) arm. Control MB (MBc) was also prepared. Attachment of antibodies to the MB's surface was verified by flow cytometry and fluorescent microscopy studies. In vitro studies, myocardium sections were incubated with MBc and TMB2, respectively. In vivo studies, real-time myocardial contrast echocardiography (MCE) was used to assess the myocardial perfusion and myocardial function. One week post I/R, triggered myocardial contrast echocardiography was performed to increase microvascular permeability. Subsequently, either MBc or TMB2 were intravenously administered followed by high-energy FLASH images. Myocardial remodeling and MMP2 activation post one week I/R were conformed by histological and immunofluorescence stainings.Results: In vitro studies, TMB2 significantly bound within the risk area (RA) of one week post-I/R myocardial sections, while rare binding was observed in the control area (CA). Neither TMB2 nor MBc bound to myocardial tissue in control rats. In vivo studies, high quality MCE short-axis images at the middle papillary muscle level were successfully achieved in all rats. At baseline, real-time MCE shows an even myocardial contrast perfusion of the left ventricular walls and the wall thickening fraction of the LV walls within the RA and the CA were not significantly different (62%±3% versus 63%±5%, p>0.01). After 30 min coronary ligation and reperfusion, gross histology revealed a decreased in wall thickness of the central MI region and a significant LV dilation. The significant perfusion decrease was observed in the RA with a decreased wall thickening fraction compared to that of the CA (25%±4% versus 65%±5%, p<0.01). One week post I/R, myocardial video intensity from the adhered TMB2 was significantly higher in the RA versus the CA compared to MBc (1.76±0.25 versus 1.02±0.19, p<0.01). There was no obvious retention of either MB species in control rats (TMB2: 1.02±0.14, MBc: 1.01±0.11, p>0.01).Conclusions: Novel polymer microbubble has the high binding ability of monoclone antibody (mAb) on its shell via a poly ethylene glycol (PEG) arm. Base on the temporary microvascular permeability increasing, MMP2-targeted contrast-enhanced ultrasound imaging may provide a novel and non-invasive approach to localize and study MMP2 activation in a post I/R remodeling rat model.