Near-infrared enzyme-activated fluorescent phospholipids for molecular imaging / Theresa M. Mawn.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Mawn, Theresa M.

Contributor:

Delikatny, E. James, advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Bioengineering.

Bioengineering--Penn dissertations.

Local Subjects:

Penn dissertations--Bioengineering.

Bioengineering--Penn dissertations.

Physical Description:

xv, 150 pages : illustrations (some color) ; 29 cm

Production:

2009.

Summary:

With the aim of being able to detect disregulated choline lipid metabolism in cancer and elucidate the pathways involved in the malignant transformation, our group is designing and synthesizing a series of phospholipase-specific NIR molecular beacons. The focus of my dissertation has been to characterizing these NIR enzyme-activated fluorescent phospholipids and to assess their potential as suitable in vivo phospholipase molecular beacons. Several key criteria were necessary to be determined a successful in vivo phospholipase probe: (1) sensitivity and specificity to phospholipase activity, (2) adequate signal amplification, (3) high enzyme-substrate affinity allowing for sufficient in vivo activation, (4) ability to overcome biological delivery barriers, and (5) in vivo pharmacokinetics that allows for sufficient tumor absorption and activation.

These criteria have been explored here for the first enzyme-activated fluorescent phospholipid to be synthesized by our group, Pyro-PL-BHQ. Pyro-PL-BHQ is highly specific to phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine (PC). Incubation of Pyro-PL-BHQ with PC-PLC in solution demonstrated a 150-fold amplification. Enzyme kinetics determined an apparent Km of 1.4--1.9 muM and Vmax of 160.7 +/- 29.2 nmol/min/mg. The PC-PLC inhibitor, tricyclodecan-9-yl xanthogenate (D609), inhibited probe activation with an IC50 of 34 +/- 8 muM. Pyro-PL-BHQ was internalized by DU145 human prostate cells, and subsequently activated. Tumor-bearing mice injected with Pyro-PL-BHQ, followed by in vivo NIR imaging, resulted in a 4-fold tumor-specific increase in radiance over background. Tumor probe activation was inhibited with administration of D609. As Pyro-PL-BHQ has satisfied the above prerequisites, we present Pyro-PL-BHQ as the first NIR phospholipase-activated molecular beacon.

Preliminary characterization has been performed on the second enzyme-activated phospholipid synthesized, Pyro-C12-PL-BHQ. This substrate displays high specificity for secreted phospholipase A2 type IB (sPLA2 IB). Enzyme kinetics determined an apparent Km of 2 muM. Tumor-bearing mice injected with Pyro-C12-PL-BHQ presented activation specifically in the gut, an expected result given that sPLA2 IB is primarily a digestive enzyme.

These results demonstrate the feasibility of designing a phospholipase-specific molecular imaging probe capable of directly, sensitively and quantitatively measuring phospholipase activity in vivo, an important contribution to the study of lipid metabolism as it pertains to cancer.

Notes:

Adviser: E. James Delikatny.

Thesis (Ph.D. in Bioengineering) -- University of Pennsylvania, 2009.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 3363698.