The U/S system is composed of a software-modifi ed Vivid E9 (GE Healthcare Systems, Milwaukee, Wisconsin) permitting cus-tomized acoustic pulsing parameters. Location of the liver is initially confi rmed by ultrasound examination; and if required, a high- frequency linear array probe (12 L, GE Healthcare Systems) is used until profi ciency is established. For MB disruption a lower- frequency cardiac probe (3S, GE Healthcare Systems) is used. Once the injec-tion is initiated, we allow a 15–20 s period of low MI imaging (<0.4 MI) to facilitate the perfusion of the liver parenchyma with the MBs (Fig. 1 ). During this initial phase, one can identify the presence of the contrast effect. Once liver perfusion has been visually con-fi rmed, the MI is increased (≤1.3 MI) for 2 s before returning to low MI to allow vascular reperfusion. This sequence of alternating high and low MI is repeated for the duration of MB circulation, roughly 2 min in a rat. When the complete dose has been injected, a small bolus of saline is infused to ensure complete administration of dose by fl ushing the catheter tubing (200 μL in a 0.2 mm line). It is important that a slow rate of injection be used, roughly 30 s/mL to avoid MB disruption and allow adequate opportunity for cavitation and reperfusion during the serial-pulsed sequences. Additionally, a rapid infusion can result in acoustic signal attenuation. In this case, only a limited amount of superfi cial MBs will be disrupted reducing the delivery of pDNA to the hepatocytes.
Special attention must be given to probe positioning. With the rat placed in a supine position, the ultrasound probe should be placed just caudal to the xiphoid process at approximately a 45 ○ angle directing the beam cranially ( see Note 6 ). Through empiric studies, we determined that a multi-angle delivery is benefi cial.
Fig. 1 Upon initiation of microbubble injection via a tail vein of the rat, the con-trast effect will be apparent in approximately 20 s in the liver. This perfusion of vasculature is the point at which the sequence of high mechanical index acoustic energy should be initiated to cause MB cavitation and gene delivery. Without this clear visual confi rmation effi cacy may be compromised
That is, following each 2 s period of high MI, the probe should be moved slightly and the angle of insonation changed to provide U/S energy to each of the lobes of the liver over the course of the 2-min delivery session. This should include increasing and decreasing the angle as well as directing acoustic energy laterally.
Given the size of the rat, and using an unfocused ultrasound beam centered at 2 cm below the skin, a relatively large portion of the liver is captured in each two-dimensional slice of acoustic pulsing.
This approach is the reason that probe angulation and positioning is important in maximally interrogating all lobes of the liver (Fig. 2 ). If it is diffi cult to achieve suffi cient contrast effect within the liver parenchyma, improper probe placement is likely.
Note that while using a variable angulation of the probe is benefi cial, care must be given to limit off-target delivery of the agents resulting in decreased effi cacy. The liver focus can be main-tained by using direct visualization of liver perfusion. Additionally, minimal external mechanical pressure should be applied to the probe as excessive pressure may reduce liver perfusion.
For our studies, a variety of parameters were used to monitor animal health including a veterinary diagnostic blood analyzer (VetScan VS2, Abraxis Inc, Union City, California) that assesses 14 parameters of blood (albumin, alkaline phosphatase, alanine ami-notransferase, amylase, blood urea nitrogen, calcium, creatinine, globulin, glucose, potassium, sodium, phosphorus, total bilirubin, and total protein). No signifi cant deviation from baseline has been observed. Additionally, gross histological analysis of liver following treatment revealed no adverse morphological changes.
While numerous associated experimental components can and need to be addressed and controlled during the procedures to achieve an optimal outcome, our data support the concept that additional chemical binding of agents to the MB is not required.
Fig. 2 For early proof of concept studies pDNA coding for green fl uorescent protein was used to verify effi cacy of microbubble gene delivery to the liver of naïve rats. Additionally, demonstrated is the effect of single versus multiple planes of insonation during gene therapy. As can be seen above, a higher degree of reporter gene expression is achieved when directing U/S energy in multiple planes
Our results are specifi c for an agent that has low bioavailability (rapid sequestration/metabolism in systemic circulation) and poor native effi cacy.
As researchers undertake a program of microbubble gene therapy, they should bear in mind that while none of the steps or proce-dures stated above is complex, it is a magnifi cation of minor errors that can result in loss of experimental effi ciency. Seemingly trivial details become manifest and demand that an appropriate degree of attention is provided in a consistent manner. To this end by per-forming our studies with meticulous attention to detail, we achieved 93 % study effi cacy. Figure 3 is a representative data set that is a result of the materials and methods described above. These results are noteworthy due to the inherent variability associated with performance of in vivo systems.
4 Notes
1. To activate microbubbles, one must comply with the manufac-turer’s specifi cations included in the package inserts.
Alternatively, follow methods presented in literature if preparing a unique MB on the bench top.
Fig. 3 Peak HDL following treatment of either control or full microbubble gene therapy. The combination of co-injection of microbubble and ApoA-I pDNA with ultrasound acoustic energy (MB + U/S + pDNA) directed at the liver resulted in a signifi cant elevation of serum HDL (mg/dL). This HDL increase followed expres-sion of human mRNA, resulting in production of human ApoA-I protein in the rat within 12 h of treatment. This effect was not seen in any of the control cohorts.
Average HDL value (mg/dL) for each cohort is presented on the respective bar
2. Special attention need be addressed to needle gauge as excessive positive or negative pressure can burst MBs. Using improper gauge needles or drawing too rapidly can reduce the concen-tration of MBs thereby negatively effecting delivery.
3. Delivery regimes which rely on co-localization of gene/drug with MBs during ultrasound-directed cavitation in the tissue of interest may require simple mixing. If there is temporal separa-tion of the two solusepara-tions, the success of the delivery may be compromised. Mixing times will be dependent on bubble and drug properties. More complicated techniques in which agent is bound to or incorporated into the MB shell will require more elaborate procedures.
4. Whether bolus or a longer duration infusion, the proper rate of injection must be used to maximize drug delivery. Pushing too rapidly will allow a signifi cant percentage of drug/MB to pass through the target tissue without the benefi t of sonoporation.
Conversely, for longer injections, careful attention must be given to ensure proper agitation so that MBs do not fl oat out of solution.
5. Prior to imaging the abdomen, all hair should be removed, initially with clippers then with a standard facial razor to ensure adequate acoustic coupling of the transducer. It is very impor-tant to ensure as much fur as possible to prevent acoustic inter-ference. One technique is to shave as stated above, apply acoustic coupling gel to the area. This prevents the skin from drying out while the catheter is being placed.
6. Special attention must be given to probe positioning. With the rat placed in a supine position, the U/S probe should be placed just caudal to the xiphoid process at approximately a 45 ○ angle directing the beam cranially. Through numerous studies to identify the best probe mechanics, it was demonstrated that a multi-angle delivery is required. Using a 2D system treats only a single plain in each therapeutic pulse. To reach as much tar-get tissue as possible (e.g., liver) each pulse should be aimed in a slightly different direction.
The experimental protocol of this study conformed to the Guide for the Care and Use of Laboratory Animals and was approved by the IACUC at the General Electric Global Research (Niskayuna, NY), which is an AAALAC-, USDA-, and OLAW- accredited facility.
Acknowledgements
The authors would like to thank all members of the team who have made microbubble gene delivery a success in our lab. This includes physicist Kirk Wallace, ultrasound engineer David Mills, Chemists
Matthew Butts, Bruce Johnson, and Binil Kandapallil, biologists Mike Marino, Chris Morton, Jeannette Roberts and Andrew Torres as well as the leadership team who make it possible.
The project described was supported in part under NIH SBIR 1R44HL095238 “Development of Novel Tissue Directed Ultrasound Therapeutic Gene Delivery System” from DHHS, National Heart, Lung and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the offi cial views of DHHS, NIH, National Heart, Lung and Blood Institute as well as funds from GE Global Research, Niskayuna NY.
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