Multi-modal detection of colon malignancy by NIR-tagged recognition polymers and ultrasound contrast agents.

To increase colonoscopy capability to discriminate benign from malignant polyps, we suggest combining two imaging approaches based on targeted polymeric platforms. Water-soluble cationized polyacrylamide (CPAA) was tagged with the near infrared (NIR) dye IR-783-S-Ph-COOH to form Flu-CPAA. The recognition peptide VRPMPLQ (reported to bind specifically to CRC tissues) was then conjugated with the Flu-CPAA to form Flu-CPAA-Pep which was then incorporated into echogenic microbubbles (MBs) made of polylactic acid (PLA) that are highly responsive to ultrasound. The ultimate design includes intravenous administration combined with local ultrasound and intra-colon inspection at the NIR range. In this proof of principle study PLA MBs were prepared by the double emulsion technique and loaded with several types of Flu-CPAA-Pep polymers. After insonation the submicron PLA fragments (SPF)-containing Flu-CPAA-Pep were examined in vitro for their ability to attach to colon cancer cells and in vivo (DMH induced rat model) for their ability to attach to colon malignant tissues and compared to the specific attachment of the free Flu-CPAA-Pep. The generation of SPF-containing Flu-CPAA-Pep resulted in a tissue attachment similar to that of the free, unloaded Flu-CPAA-Pep. The addition of VRPMPLQ to the polymeric backbone of the Flu-CPAA reduced cytotoxicity and improved the specific binding.

Detection of a long non-coding RNA (CCAT1) in living cells and human adenocarcinoma of colon tissues using FIT-PNA molecular beacons.

Although the function and mechanism of action of long non-coding RNAs (lncRNA) is still not completely known, studies have shown their potential role in the control of gene expression and regulation, in cellular proliferation and invasiveness at the transcriptional level via multiple mechanisms. Recently, colon cancer associated transcript 1 (CCAT1) lncRNA was found to be expressed in colorectal cancer (CRC) tumors but not in normal tissue. This study aimed to study the ability of a CCAT1-specific peptide nucleic acid (PNA) based molecular beacons (TO-PNA-MB) to serve as a diagnostic probe for in vitro, ex vivo, and in situ (human colon biopsies) detection of CRC. The data showed enhanced fluorescence upon in vitro hybridization to RNA extracted from CCAT1 expressing cells (HT-29, SW-480) compared to control cells (SK-Mel-2). Uptake of TO-PNA-MBs into cells was achieved by covalently attaching cell penetrating peptides (CPPs) to the TO-PNA-MB probes. In situ hybridization of selected TO-PNA-MB in human CRC specimens was shown to detect CCAT1 expression in all (4/4) subjects with pre-cancerous adenomas, and in all (8/8) patients with invasive adenocarcinoma (penetrating the bowel wall) tumors. The results showed that CCAT1 TO-PNA-MB is a powerful diagnostic tool for the specific identification of CRC, suggesting that with the aid of an appropriate pharmaceutical vehicle, real time in vivo imaging is feasible. TO-PNA-MB may enable identifying occult metastatic disease during surgery, or differentiating in real time in vivo imaging, between benign and malignant lesions.