UTMD stands for ultrasound targeted microbubble.Destruction. UTMD can be used to direct site specific delivery of bioactive molecules, including therapeutic genes to target organs accessible to ultrasound such as the heart and liver. In UTMD bioactive molecules such as negatively charged plasmid, DNA vectors encoding a gene of interest are added to the cation shells of lipid microbubble contrast agents, the vector bound microbubbles can be administered intravenously or directly to the left ventricle of the heart.
In larger animals, they can also be delivered by an trac coronary catheter with or without a dwell period. The subsequent delivery from the circulation to a target organ occurs optimally through acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the micro vasculature of the targeted region.
As a result of this sauna parion effect, the transfection efficiency into and across the endothelial cells is increased and transgene encoding vectors can be deposited into the surrounding tissue of interest. Plasma DNA remaining in the circulation is rapidly degraded by nucleases in the blood microbubble preparation. An aliquot of a prepared microbubble stock solution containing glucose phosphate buffered saline, diol glycerol three phosphatidylcholine and diol glycerol three.
Phosphatidyl ethanolamine is first incubated at 40 degrees Celsius for 15 minutes. The warmed microbubble solution is then transferred to a 1.5 milliliter micro tube containing glycerol and between one and two milligrams of purified plasma DNA encoding an expression construct for the gene of interest. Phosphate buffered saline is added to a final volume of 500 microliters.
The air in the micro tube is then replaced with Octa fluoro propane gas. The micro tube is then shaken vigorously in a dental amalgamator for 20 seconds. Equipment calibration prior to first use the one megahertz UTMD cavitation transducer needs to be calibrated to ensure proper mechanical index and pulse repetition.
A submerge one megahertz transducer is connected to a waveform generator through a power amplifier. The transducer is placed in a plastic container full of water aimed directly at a hydrophone, which has been connected to an oscilloscope via charge amplifier, waveform frequency amplitude, burst cycle, and power amplification can all be modified to obtain the proper duty cycle and mechanical index optimal to cavitate the microbubbles. In this setup, we have calibrated the system to a mechanical index equivalent to approximately 1.3 at one MEGAHERZ MICROBUBBLE delivery and UTMD prior to microbubble delivery and U-T-M-D-C 57 BL six mice are anesthetized with 100 milligrams per kilogram ketamine, and five milligrams per kilogram xylazine through IP injection.
Microbubble delivery is administered intravenously through a direct injection into the left ventricle of the heart or through tail vein catheterization, which will be shown later for the direct cardiac injection. A volume up to 100 microliters of the plasma DNA loaded microbubble solution is injected through a 30 gauge needle inserted at the anterior fourth intercostal space under ultrasonic visualization into the left ventricle of the heart. The microbubble solution bolus resulting from the left interventricular injection is visualized using visual sonic's high frequency ultrasound transducer placed in a stationary position on the thorax of the mouse in a long axis view.
Immediately following the injection, microbubble destruction is carried out for approximately five minutes, using a second smaller size low frequency, 1.0 megahertz transducer held directly over the desired organ targeting destruction to this region. The ultrasound is administered to the liver at a pulse repetition frequency of 1.0 megahertz with a mechanical index equivalent to approximately 1.3 to 1.5, and pulse repetition period of 100 milliseconds for every 20 cycles, or the pulse can be gated to the ECG of the mouse to a burst of three frames of ultrasound every four to six cardiac cycles. For the tail vein method of microbubble delivery, the mouse is anesthetized the same way a syringe containing the plasmid.
DNA bound microbubbles is connected to a 27 gauge needle tail vein catheter. The tail vein catheter is inserted into the distal third of either the right or left lateral veins that run along the tail of the mouse. The syringe containing the microbubbles is placed in an infusion pump that will automatically administer a uniform preset volume of solution over a preset period of time.
Bioluminescence analysis of UTMD, the effectiveness of the UTMD mediated plasma DNA delivery can be evaluated through a variety of methods depending upon the genes encoded in the construct. In particular, in vivo bioluminescence imaging is one method that allows you to monitor the presence and duration of gene expression. In mice transfected with the plasmid encoding a bioluminescence reporter gene such as luciferase.
The Enogen in vivo imaging system is used for bioluminescence. Imaging images are typically taken of all mice the first day after UTMD mediated transfection and is repeated every three to four days until bioluminescence gene expression is no longer visually detectable through the system. To prepare mice for bioluminescent imaging, mice first receive an IP injection of the luciferase reporter probe Lucifer.
The mice are then anesthetized. Approximately three minutes later, bio distribution of the Lucifer substrate is allowed to proceed for approximately 10 minutes before the animal or animals are placed in the Xeno Gen imaging chamber and a full body image scan is taken during the acquisition, the photons emitted from the fireplace. Luciferase de Lucifer photochemical reaction are determined by the system after each image is acquired.
Using the Xeno in vivo imaging system, the system software is used to measure and quantify the visible luminescent region of interest.
