Detection of enzyme activity is one of the major goals of molecular imaging to aid in the assessment oftumor aggressiveness, tumor phenotyping to select the appropriate molecular therapeutic, and to monitortherapeutic response. Ultrasound, the most versatile and most commonly used imaging modality that boastshigh spatial and temporal resolution as well as high sensitivity to microbubbles, has been limited to thedetection of intravascular targets. This project aims to bring to ultrasound the ability to detect enzymes. Wehave successfully produced emulsions and nanobubbles with particles less than 100nm that are capable ofexiting the vascular space. We propose to use two populations of particles where each carries one of thepairs of an adhesion molecule pair, such as biotin-avidin, whose ability to interact is blocked until exposed tothe enzyme of interest. The enzyme activates the interaction to produce an aggregate of particles thatbecomes trapped in the tumor. Because ultrasound backscatter is related to the scatterer's radius raised tothe 6th power, signal increases dramatically allowing aggregate detection. To avoid the use of biotion-avidinbecause of the known allergic effects, we selected complimentary DMA strands as the adhesion pairbecause they rapidly form a tight double helix when exposed to each other. To prevent plasma degradationand to block interaction, each DMA strand is cyclized with a peptide that can be cleaved by the enzyme ofinterest. To accomplish this we propose 5 aims that proceed with increasing complexity. We will first useliquid fluorocarbon emulsions and then proceed to the more fragile nanobubbles: 1) We will first prove invitrousing DNA strands cyclized by a disulfide bond that are opened by TCEP, a reducing agent, that whenthe strands open aggregation occurs and ultrasound signal increases. 2) We will then cyclize the DNA with apeptide linker cleavable by thrombin and as in Aim 1, prove that signal increases in vitro in the presence ofthrombin and then in vivo using an acute thrombus that provides an intravascular target. 3) We will repeatAim 1, but the peptide linker will be replaced by a peptide cleavable by matrix metalloproteinase (MMP). Wewill then prove signal increase in vivo using mice with an MMP+ or MMP- tumor. 4) We will then repeat Aims1-3 using nanobubbles after they have been optimized to maximize signal difference between their nonaggregatedand aggregated state. 5) For aims 1 to 4 the DNA strands were attached to the particles usingbiotin/avidin linkers for convenience. In parallel with Aims 1-4 we will work to attach the DNA strands directlyto the shell to increase the probability of translating the agent to the clinic.
Showing the most recent 10 out of 35 publications
