C O M M U N I C A T I O N S
the activity of bCD-PLL remained nearly unaffected throughout
the remaining 4 day incubation. We hypothesize that, like poly-
(
ethylene glycol) (PEG),16 the attached polylysine in bCD-PLL
may produce a steric exclusion space that prevents the proteolysis
of bCD by proteases such as cathepsins and matrix metallopro-
teinases (MMPs) that are overexpressed in highly invasive cancer
cell lines, such as MDA-MB-231.17
In summary, we have developed a novel cancer therapeutic
enzyme labeled with multimodal imaging reporters demonstrating
high relaxivity, low cytotoxicity, improved enzymatic specificity
to prodrug, efficient cell uptake, and high enzymatic stability in
serum as well as in breast cancer cell culture. This conjugate is the
first prototype for noninvasive image-guided enzyme/prodrug cancer
therapy. In vivo studies of this conjugate in tumor xenograft models
are currently underway.
Figure 1. (A) In vitro cytotoxicity of bCD, PLL, and bCD-PLL in a human
breast cancer cell line, MDA-MB-231, acquired from an MTT assay at a
dose of 4.3 µM that will be used for in vivo studies. Standard deviations
are shown with error bars (n ) 4); // indicates statistical significance at
levels of p < 0.01 for the PLL or bCD versus control. (B) Internalization
of bCD-PLL within MDA-MB-231 cells after treatment with bCD-PLL
(
100 ng/mL) for 30 min. Rhodamine fluorescence is shown in red, and
nuclei counterstained with DAPI are displayed in blue.
Acknowledgment. This work was supported by NIH P50
CA103175 (JHU ICMIC Program). We thank Dr. Barry Stoddard
of the Fred Hutchinson Cancer Research Center for providing the
pET-bCD expression vector. We thank Dr. Michal Neeman, Dr.
Venu Raman, and Dr. Peter Senter for helpful discussions. We thank
Dr. Maria Mikhaylova for her assistance with the DLS and ICP-
AES studies. We gratefully acknowledge the support of Dr.
Jonathan S. Lewin.
Supporting Information Available: Synthesis and characterization
of the bCD-PLL conjugate, kinetic studies, in vitro cytotoxicity and
cell uptake studies, enzymatic stability studies in serum, and cell culture.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. (A) Relative cell viability of MDA-MB-231 cells after incubation
with therapeutic enzyme (0.13 µM) for 0-72 h following additional 5 day
treatment with prodrug 5FC (1.5 mg/mL). (B) Dynamic enzymatic activity
of bCD and bCD-PLL in fresh mouse serum at 37 °C. Enzymatic activity
was measured in 0.5 mM cytosine, Tris-HCl (50 mM), pH 7.5, 25 °C.
Standard deviations are shown with error bars (n ) 4).
mise its enzymatic activity. In fact, compared to bCD, bCD-PLL
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