Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.
1. Microbubble Production
2. Nanoparticle Fabrication
3. Composite Delivery Vehicle Fabrication (Protocol Adapted from VisEn Chemistry Notes)
4 .Tumor Model
All animal experiments were in compliance with an animal protocol approved by the University of Virginia Animal Care and Use Committee.
5. In Vivo Ultrasound Application
6. Tumor Perfusion Quantification
7. Nanoparticle Biodistribution in Tumor
8. Tumor Growth Rate
9. Tumor Processing and Analysis
10. Representative Results
1. Nanoparticle Fabrication (2.0)
2. Targeted Drug Delivery (5.4.1)
3. Tumor Ablation (5.4.3)
Figure 1. Characterization of PLAGA nanoparticles bearing BSA. (A) SEM image of properly fabricated nanoparticles. (B) SEM image of improperly fabricated nanoparticles.
Figure 2. (A) Fluorescence-mediated tomography (FMT) images of ultrasound treated (top) and control treated (bottom) subcutaneous gliomas immediately following treatment with a MNCAs, where nanoparticles (NPs) are bearing 680 fluorescence signals are superimposed on grayscale planar excitation light image.
Figure 3. Representative fold change in tumor growth following microbubble insonation with the five-burst extended pulsing protocol.
Figure 4. B-Mode (A,C) and contrast-enhanced ultrasonography (B,D) images of a subcutaneous C6 glioma tumor in a mouse. In the initial pretreatment image (A) the boundary of mostly hypoechoic tumor has been traced in blue. Time average enhancement after intravenous injection of a contrast agent is shown in (B) pretreatment. Posttreatment, before contrast injection, tumor is again mostly hypoechoic (D). After contrast injection, there is significantly less enhancement in the tumor.
Critical Steps
Cannulation of mouse tail vein:
Intravenous injection into the mouse tail vein can be a challenging procedure. However, a tail vein catheter can greatly improve the likelihood of a successful injection. To make the catheter, repeatedly bend back and forth a 25 gauge needle until it breaks from the hub. Insert the blunt end into the end of PE 20 tubing and seal the connection with silicon glue. To prepare the catheter for cannulation, attach a syringe loaded with 1% heparinized saline to the cather and infuse liquid into the dead space of the catheter. Position an anesthetized mouse on its side so the lateral tail vein is in view. Dilate the tail with warm water (105° – 110°F). Insert the needle into the vein. If successful blood will usually enter the catheter. Verify that the needle is in the vein by clearing the vein with a small amount of saline. Secure the catheter in place with tape before attaching the syringe to the infusion pump.
Nanoparticle Resuspension:
Nanoparticles may aggregate following lyophilization. Resuspend particles in PBS, repeatedly vortex and sonicate briefly (10 sec) in a sonic water bath. It is critical to properly resuspend the lyophilized sample. Characterize the suspension with SEM microscopy and light scattering techniques following lyophilization and resuspension.
Possible Modifications:
In terms of adjusting the nanoparticle fabrication protocol, BSA serves as a surrogate drug and can be interchanged for a multitude of therapeutic agents. Depending on the solubility of the therapeutic agent, nanoparticles can be fabricated as an oil-in-water emulsion or as an oil-in-water-in-oil emulsion. The loading efficiency and release of the therapeutic agent from PLAGA NPs, may also be adjusted considerably if desired. To increase the loading efficiency, increase the wt/wt loading of drug in polymer carriers via the optimization of NP fabrication parameters. To tailor the release rate of the therapeutic agent, vary the hydrolysis rate constant via changes in molecular weight and the lactic/glycolic ratio of PLAGA. By talioring both the loading efficiency and release rate of therapeutic agent, in and from PLAGA NPs, the desired local concentration can be delivered to tissue. In terms of adjusting the delivery protocol, the most important factors are the degree of ultrasonic MNCA destruction and microvascular permeabilization. It has been reported that albumin shelled microbubbles have a half-life of 1.3 ± 0.69 minutes (mean ± SD) (Optison 2008). We hypothesize that continuous agent infusion and prolonged ultrasound application will increase the extent of microvessel permeabilization. By increasing treatment time we aim to transiently heighten permeabilization of the microvasculature.
The non-thermal mechanical ablation protocol may be adjusted by changing MB concentration or the ultrasound peak pressure. It is expected that increasing MB concentration and/or ultrasound peak pressure will increase the degree of damage to the tumor vasculature.
Applications:
We are developing techniques to lower the acoustic power required by transcranial high-intensity focused ultrasound (HIFU) to reach the desired therapeutic effect.
In part, due to complications associated with treatment through the skull and importance of surrounding tissue, thermal tumor tissue ablation with high intensity focused ultrasound (HIFU) has not yet achieved widespread use as a therapeutic option for brain cancer. Preliminary conclusions from the first three patients of a clinical train have indicated deep brain HIFU treatment at sub-ablative focal temperatures results in cranial heating (McDannold 2009). The key to success of HIFU as a clinical treatment for transcranial treatment of brain tumors is the capacity to localize the delivery of acoustic energy to a well-delineated region. The ability to deliver acoustic energy is complicated by bone or air interfaces, such as the skull and previous surgical resections, which generate phase and power aberrations as the ultrasound energy is attenuated along the propagation path (Tanter 1998). These aberrations often contribute to pre-focal heating (McDonnald 2009) and cavitations in healthy tissues.
Over the past several years, the use of microbubbles to lower the acoustic power for targeted, reversible BBB disruption and tumor ablation has attracted much research interest (Hynynen 2001, Sheikov 2004, McDannold 2006a; 2006b, Meairs 2005). McDannold et al. (2006a) have demonstrated that the intravascular injection of microbubbles at the time of HIFU treatment resulted in a 91% reduction in the time-averaged acoustic power threshold for mechanical damage compared to controls in which no microbubbles were present, showing the potential for generating lesions with reduced temperature elevation. Reducing the thermal threshold for lesion formation in turn lowers the probability of heat accumulation in off target tissue or bone. Furthermore, it has been shown that the intravenous administration of microbubbles at the time of ultrasound treatment results in transient, localized, BBB opening with powers approximately two orders of magnitudes lower than those required for lesion formation.
It is the goal of this work to develop ultrasound-microbubbles techniques to both lower the acoustic power required by transcranial HIFU and control the degree of microvascular permeabilization. The two specific therapeutic applications of this work in the brain are targeted drug delivery and non-thermal ablation. The relative levels of increased permeability and permanent ablation can be controlled by adjusting acoustic power levels depending on whether the emphasis is on improved drug delivery, permanent microvascular ablation, or a combination of drug delivery and permanent microvascular ablation. This potential ability to control how the microvessels respond creates an opportunity to develop different permutations of our treatment strategy for specific transcranial therapeutic applications. We believe this approach has the potential to drastically transform how brain tumors are treated.
The authors have nothing to disclose.
Supported by NIH R01 HL74082, the Hartwell Foundation, and the Focused Ultrasound Surgery Foundation.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
ApoptTag kit | Intergen Co. | S7110 | ||
un-capped 85:15 poly(lactic-co-glycolic acid) (PLAGA) | Lakeshore Biomaterials | Custom | ||
Vivo Tag 680 | VisEn Medical | 10120 | Used to Tag BSA | |
Poly(vinyl alcohol) | Sigma-Aldrich | 363136 | ||
MicroTip Sonicator | Misonix | S-4000 | ||
Sequoia | Simons Medical | P.O.A | Equipped with CPS | |
FreeZone 2.5 | Labconco | 7670020 | Equipped with Nitrogen Trap | |
Methylene chloride (CH2Cl2) | Fisher Scientific | D37-500 | ||
FMT 250 | VisEn Medical | P.O.A | ||
F-12K Nutrient Mixture | Gibco | 21127-022 | ||
polyethyleneglycol-40 stearate | Sigma Chemical | 9004-99-3 | ||
distearoyl phosphatidylcholine | Avanti Polar Lipids | 770365 | ||
Multisizer Coulter Counter | Beckman Coulter | P.O.A | ||
Waveform Generator | Tektronix, Inc. | AFG-310 | ||
water-based ultrasound gel | Parker Laboratories | Aquasonic 100 | ||
Infusion pump | Harvard Apparatus | Harvard Apparatus PHD 2000 | ||
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) | Pierce Biotechnology | 25952-53-8 | ||
N-hydroxysulfosuccinimide (Sulfo-NHS) | Pierce Biotechnology | 106627-54-7 | ||
Succinic anhydride | Sigma Aldrich | 603902 | ||
Power Amplifier | Electronic Navigation Industries | ENI 3100LA | ||
Needle Thermocouple Probe | Omega | HYP1-30-1/2-T-G-60-SMPW-M | ||
BioGel (P100, medium) | Bio-Rad | 150-4170 | ||
.75’’ diameter 1 MHz unfocused transducer | Panametrics | A314S |