Kuo-Hsiang Chuang

Tumor-targeting prodrug-activating bacteria for cancer therapy

Increasing the specificity of chemotherapy may improve the efficacy of cancer treatment. Toward this aim, we developed a strain of bacteria to express enzymes for selective prodrug activation and non-invasive imaging in tumors. β-glucuronidase and the luxCDABE gene cluster were expressed in the DH5α strain of Escherichia coli to generate DH5α-lux/βG. These bacteria emitted light for imaging and hydrolyzed the glucuronide prodrug 9ACG to the topoisomerase I inhibitor 9-aminocamptothecin (9AC). By optical imaging, colony-forming units (CFUs) and staining for βG activity, we found that DH5α-lux/βG preferentially localized and replicated within CL1-5 human lung tumors in mice. The intensity of luminescence, CFU and βG activity increased with time, indicating bacterial replication occurred in tumors. In comparison with DH5α-lux/βG, 9AC or 9ACG treatment, combined systemic administration of DH5α-lux/βG followed by 9ACG prodrug treatment significantly (P<0.005) delayed the growth of CL1-5 tumors. Our results demonstrate that prodrug-activating bacteria may be useful for selective cancer chemotherapy.

An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β -glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (βG) on CT26 tumors. The fluorescent intensity in βG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA

Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alphaPEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.

Micro-PET Imaging of β-Glucuronidase Activity by the Hydrophobic Conversion of a Glucuronide Probe

PURPOSEnTo develop a new glucuronide probe for micro-positron emission topography (PET) that can depict beta-glucuronidase (betaG)-expressing tumors in vivo.nnnMATERIALS AND METHODSnAll animal experiments were preapproved by the Institutional Animal Care and Use Committee. A betaG-specific probe was generated by labeling phenolphthalein glucuronide (PTH-G) with iodine 131 ((131)I) or (124)I. To test the specificity of the probe in vitro, (124)I-PTH-G was added to CT26 and betaG-expressing CT26 (CT26/betaG) cells. Mice bearing CT26 and CT26/betaG tumors (n = 6) were injected with (124)I-PTH-G and subjected to micro-PET imaging. A betaG-specific inhibitor D-saccharic acid 1,4-lactone monohydrate was used in vitro and in vivo to ascertain the specificity of the glucuronide probes. Finally, the biodistributions of the probes were determined in selected organs after injection of (131)I-PTH-G to mice bearing CT26 and CT26/betaG tumors (n = 14). Differences in the radioactivity in CT26 and CT26/betaG tumors were analyzed with the Wilcoxon signed rank test.nnnRESULTSn(124)I-PTH-G was selectively converted to (124)I-PTH (phenolphthalein), which accumulated in CT26/betaG cells and tumors in vitro. The micro-PET images demonstrated enhanced activity in CT26/betaG tumors resulting from betaG-mediated conversion and trapping of the radioactive probes. Accumulation of radioactive signals was 3.6-, 3.4-, and 3.3-fold higher in the CT26/betaG tumors than in parental CT26 tumors at 1, 3, and 20 hours, respectively, after injection of the probe (for all the three time points, P < .05).nnnCONCLUSIONnHydrophilic-hydrophobic conversion of (124)I-PTH-G probe can aid in imaging of betaG-expressing tumors in vivo.

Engineering Chimeric Receptors To Investigate the Size- and Rigidity-Dependent Interaction of PEGylated Nanoparticles with Cells

Attachment of ligands to the surface of nanoparticles (NPs) is an attractive approach to target specific cells and increase intracellular delivery of nanocargos. To expedite investigation of targeted NPs, we engineered human cancer cells to express chimeric receptors that bind polyethylene glycol (PEG) and internalize stealth NPs in a fashion similar to ligand-targeted liposomes against epidermal growth factor receptor 1 or 2 (HER1 or HER2), which are validated targets for cancer therapy. Measurement of the rate of endocytosis and lysosomal accumulation of small (80-94 nm) or large (180-220 nm) flexible liposomes or more rigid lipid-coated mesoporous silica particles in human HT29 colon cancer and SKBR3 breast cancer cells that express chimeric receptors revealed that larger and more rigid NPs were internalized more slowly than smaller and more flexible NPs. An exception is when both the small and large liposomes underwent endocytosis via HER2. HER1 mediated faster and greater uptake of NPs into cells but retained NPs less well as compared to HER2. Lysosomal accumulation of NPs internalized via HER1 was unaffected by NP rigidity but was inversely related to NP size, whereas large rigid NPs internalized by HER2 displayed increased lysosomal accumulation. Our results provide insight into the effects of NP properties on receptor-mediated endocytosis and suggest that anti-PEG chimeric receptors may help accelerate investigation of targeted stealth NPs.

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39±0.5u2009nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100u2009μM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

High-Throughput Sorting of the Highest Producing Cell via a Transiently Protein-Anchored System

Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2u200a=u200a0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

Optimization of an anti-poly(ethylene glycol) (anti-PEG) cell-based capture system to quantify PEG and PEGylated molecules

Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL-1 levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL-1. All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.

Using anti-poly(ethylene glycol) bioparticles for the quantitation of PEGylated nanoparticles

Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify un-decomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEG-stabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01u2009nM, 0.1u2009nM, 15.63u2009ng/mL and 0.48u2009ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation.