Wonjung Kwak

Phage display selection of peptides that home to atherosclerotic plaques: IL-4 receptor as a candidate target in atherosclerosis

Imaging or drug delivery tools for atherosclerosis based on the plaque biology are still insufficient. Here, we attempted to identify peptides that selectively home to atherosclerotic plaques using phage display. A phage library containing random peptides was ex viv screened for binding to human atheroma tissues. After three to four rounds of selection, the DNA inserts of phage clones wer sequenced. A peptide sequence, CRKRLDRNC, was the most frequently occurring one. Intravenously injected phage displaying the CRKRLDRNC peptide was observed to home to atherosclerotic aortic tissues of low‐density lipoprotein receptor‐deficient (Ldlr−/–) mice at higher levels than to normal aortic tissues of wild‐type mice. Moreover, a fluorescein‐ or radioisotope‐conjugated synthetic CRKRLDRNC peptide, but not a control peptide, homed in vivo to atherosclerotic plaques in Ldlr−/– mice, while homing of the peptide to other organs such as brain was minimal. The homing peptide co‐localized with endothelial cells, macrophages and smooth muscle cells a mouse and human atherosclerotic plaques. Homology search revealed that the CRKRLDRNC peptide shares a motif of interleukin‐receptor (IL‐4) that is critical for binding to its receptor. The peptide indeed co‐localized with IL‐4 receptor (IL‐4R) at atherosclerotic plaques. Moreover, the peptide bound to cultured cells expressing IL‐4R on the cell surface and the binding was inhibited by the knock‐down of IL‐4R. These results show that the CRKRLDRNC peptide homes to atherosclerotic plaques through binding to IL‐4R as its target and may be a useful tool for selective drug delivery and molecular imaging of atherosclerosis.

In vivo imaging of tumor apoptosis using histone H1-targeting peptide

In vivo imaging of apoptosis could allow monitoring of tumor response to cancer treatments such as chemotherapy. Using phage display, we identified the CQRPPR peptide, named ApoPep-1(Apoptosis-targeting Peptide-1), that was able to home to apoptotic and necrotic cells in tumor tissue. ApoPep-1 also bound to apoptotic and necrotic cells in culture, while only little binding to live cells was observed. Its binding to apoptotic cells was not dependent on calcium ion and not competed by annexin V. The receptor for ApoPep-1 was identified to be histone H1 that was exposed on the surface of apoptotic cells. In necrotic cells, ApoPep-1 entered the cells and bound to histone H1 in the nucleus. The imaging signals produced during monitoring of tumor apoptosis in response to chemotherapy was enhanced by the homing of a fluorescent dye- or radioisotope-labeled ApoPep-1 to tumor treated with anti-cancer drugs, whereas its uptake of the liver and lung was minimal. These results suggest that ApoPep-1 holds great promise as a probe for in vivo imaging of apoptosis, while histone H1 is a unique molecular signature for this purpose.

Revival of TE2A; a better chelate for Cu(II) ions than TETA?

A highly effective synthetic route for TE2A was developed and the (64)Cu-labeled TE2A complexes showed higher kinetic inertness and faster clearance than most commonly used TETA analogs.

Detection of apoptosis in a rat model of focal cerebral ischemia using a homing peptide selected from in vivo phage display

Focal cerebral ischemia, known as stroke, is caused by a sudden interruption in the blood supply to the brain. We attempted to identify peptides that can home to ischemic stroke tissue and detect the apoptosis of cells. A phage library displaying random peptides was screened for homing peptides to ischemic stroke tissue in a rat transient middle cerebral artery (MCA) occlusion model. After three rounds of in vivo screening, a phage clone displaying the most frequently occurring CLEVSRKNC sequence was selected. The CLEVSRKNC-phage preferentially homed to ischemic stroke tissue after intravenous administration into the MCA occlusion rats. The fluorescein-labeled synthetic CLEVSRKNC peptide, but not a scrambled control peptide, homed to ischemic stroke tissue with a lack of homing to non-ischemic brain tissue. The CLEVSRKNC peptide co-localized with a portion of neuronal cells, rather than with astrocytes, undergoing apoptosis at the penumbra region of stroke lesions. In autoradiographic studies, the uptake of the (131)I-labeled CLEVSRKNC peptide into an ischemic lesion increased at the first day and peaked at the third day after the injury. These results demonstrate that the CLEVSRKNC peptide can home to ischemic stroke tissue, while detecting apoptotic neuronal cells, and suggest it has applications as a targeting moiety for molecular imaging and selective drug delivery to stroke tissue.

Vivid Tumor Imaging Utilizing Liposome-Carried Bimodal Radiotracer

By developing a new bimodal radioactive tracer that emits both luminescence and nuclear signals, a trimodal liposome for optical, nuclear, and magnetic resonance imaging is efficiently prepared. Fast clearance of the radiotracer from reticuloendothelial systems enables vivid tumor imaging with minimum background.

Combination Therapy and Noninvasive Imaging with a Dual Therapeutic Vector Expressing MDR1 Short Hairpin RNA and a Sodium Iodide Symporter

We investigated the feasibility of using combination gene therapy and noninvasive nuclear imaging after expression of the human sodium iodide symporter (hNIS) and inhibition of the multidrug resistance (MDR1) gene in colon cancer cells. Methods: HCT-15 cells were stably transfected with a dual expression vector, in which the hNIS gene, driven by a constitutive cytomegalovirus promoter, has been coupled to an MDR1 short hairpin RNA (shRNA) cassette. Cell lines stably expressing the hNIS gene and MDR1 shRNA (designated MN-61 and MN-62) were produced, and the expression of the NIS gene and MDR1 shRNA was examined by Western blotting, reverse transcription–polymerase chain reaction, and immunostaining. The functional activities of MDR1 shRNA were determined by paclitaxel uptake and sensitivity to doxorubicin. Functional NIS expression was confirmed by the uptake and efflux of 125I and the cytotoxicity of 131I. The effect of the combination of 131I and doxorubicin was determined by an in vitro clonogenic assay. In vivo NIS expression was examined by small-animal PET with 124I. Results: The shMDR-NIS–expressing cells showed a significant decrease in the expression of MDR1 messenger RNA and its translated product, P-glycoprotein. The inhibition of P-glycoprotein expression by shRNA enhanced the intracellular accumulation of paclitaxel, the cellular retention of which is mediated by P-glycoprotein, thereby increasing sensitivity to the anticancer drug. The shMDR-NIS–expressing cells showed a significant increase of 125I uptake, which was completely inhibited by KClO4. Although the iodide efflux rate was rapid, the cell survival rate was markedly reduced by 131I treatment. Interestingly, the combination of doxorubicin and a radioiodide (131I) displayed synergistic cytotoxicity that correlated with MDR1 inhibition and NIS expression in shMDR-NIS–expressing cells. Furthermore, in mice with shMDR-NIS–expressing tumor xenografts, small-animal PET with 124I clearly visualized shMDR1-NIS–expressing tumors. Conclusion: We developed a dual expression vector with the NIS gene and MDR1 shRNA. This study represents a promising first step in investigations of the potential use of a combination of the NIS gene and MDR1 shRNA as a new therapeutic strategy allowing RNA interference–based gene therapy, NIS-based radioiodine therapy, and in vivo monitoring based on NIS imaging.

Evaluation of kidney repair capacity using 99mTc-DMSA in ischemia/reperfusion injury models

Quantitative (99m)Tc-DMSA renal uptake was studied in different renal ischemia/reperfusion (I/R) mice models for the assessment of renal repair capacity. Mice models of nephrectomy, uni- and bi-lateral I/R together with sham-operated mice were established. At 1h, 1d, 4d, 1, 2 and 3 wk after I/R, (99m)Tc-DMSA (27.7 ± 1.3 MBq) was injected via tail vein and after 3h post-injection, the mice were scanned for 30 min with pinhole equipped gamma camera. Higher uptake of (99m)Tc-DMSA was measured in normal kidneys of uni-lateral I/R model and nephrectomized kidney I/R model at 3 wk post-surgery. Comparing the restoration capacities of the affected kidneys of nephrectomy, uni- and bi-lateral I/R models, higher repair capacity was observed in the nephrectomized model followed by bi-lateral then uni-lateral models. The normal kidney may retard the restoration of damaged kidney in uni-lateral I/R model. Moreover, 3 wk after Uni-I/R, the size of injured kidney was significantly smaller than non-ischemic contralateral and sham operated kidneys, while nephrectomy I/R kidneys were significantly enlarged compared to all others at 3 wk post-surgery. Very strong correlation between (99m)Tc-DMSA uptake and weight of dissected kidneys in I/R models was observed. Consistent with (99m)Tc-DMSA uptake results, all histological results indicate that kidney recovery after injury is correlated with the amount of intact tubules and kidney sizes. In summary, our study showed good potentials of (99m)Tc-DMSA scan as a promising non-invasive method for evaluation of kidney restoration after I/R injuries. Interestingly, mice with Bi-I/R injury showed faster repair capacity than those with uni-I/R.

Synthesis and evaluation of a radioiodinated bladder cancer specific peptide.

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine ((131/124)I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [(131/124)I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [(124)I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [(124)I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [(124)I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [(124)I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [(18)F]FDG. The experimental results suggest the potential of [(124)I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.