Hyaluronan-Stabilized Redox-Sensitive Nanoassembly for Chemo-Gene Therapy and Dual T1/T2 MR Imaging in Drug-Resistant Breast Cancer Cells.

Tailoring combinatorial therapies along with real-time monitoring strategies has been the major focus of overcoming multidrug resistance in cancer. However, attempting to develop a multifunctional nanoplatform in a single construct leads to compromising therapeutic outcomes. Herein, we developed a simple, theranostic nanoassembly containing a hyaluronic acid-stabilized redox-sensitive (HART) polyethylenimine polyplex composed of a doxorubicin (DOX) intercalated Bcl-2 shRNA encoded plasmid along with a green-synthesized hausmannite (Mn3O4) and hematite (Fe3O4) nanoparticle (GMF). The highly stable HART nanoassembly has enhanced CD44-mediated intracellular uptake along with hyaluronidase (hylase) and redox-responsive drug-gene release. With Bcl-2 gene silencing induced by the successful delivery of HART in multidrug-resistant MCF7 breast cancer cells, the synergistic cytotoxic effect of Bcl-2 silencing and DOX was achieved. In addition, the HART nanoassembly containing GMF exhibited excellent dual MRI contrast (T1/T2) by reducing artifact signals. Overall, the HART nanoassembly with its enhanced theranostic properties has the potential to improve the therapeutic efficacy in future preclinical and clinical trials.

Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue. MATERIALS AND METHODS: The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue. RESULTS: The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody. CONCLUSION: The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

Genetic Programming by Nitric Oxide-Sensing Gene Switch System in Tumor-Targeting Bacteria.

Recent progress in synthetic biology has enabled bacteria to respond to specific disease signals to perform diagnostic and/or therapeutic tasks. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) colonization of tumors results in increases in nitric oxide (NO) levels, suggesting that NO may act as a candidate inducer of tumor-specific gene expression. The present study describes a NO-sensing gene switch system for triggering tumor-specific gene expression in an attenuated strain of S. Typhimurium. The genetic circuit was designed to sense NO via NorR, thus initiating the expression of FimE DNA recombinase. This was found to lead sequentially to the unidirectional inversion of a promoter region (fimS), which induced the expression of target genes. Target gene expression in bacteria transformed with the NO-sensing switch system was triggered in the presence of a chemical source of NO, diethylenetriamine/nitric oxide (DETA/NO) in vitro. In vivo results revealed that the gene expression is tumor-targeted, and specific to NO generated by inducible nitric oxide synthase (iNOS) after S. Typhimurium colonization. These results showed that NO was a promising inducer to finely tune the expression of target genes carried by tumor-targeting bacteria.

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy.

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein-engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Imaging Calreticulin for Early Detection of Immunogenic Cell Death During Anticancer Treatment.

Surface-exposed calreticulin (ecto-CRT) is a well-known "eat-me" signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or 18F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion: The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment.