In silico model-based characterization of metabolic response to harsh sparging stress in fed-batch CHO cell cultures.

Mammalian cell culture platform has been successfully implemented for industrial biopharmaceutical production through the advancements in early stage process development including cell-line engineering, media design and process optimization. However, late stage developments such as scale-up, scale-down and large-scale cell cultivation still face many industrial challenges to acquire comparable process performance between different culture scales. One of them is the sparging strategy which significantly affects productivity, quality and comparability. Currently, it is mainly relying on the empirical records due to the lack of theoretical framework and scarcity of available literatures to elucidate intracellular metabolic features. Therefore, it is highly required to characterize the underlying mechanism of physiological changes and metabolic states upon the aeration stress. To this end, initially we cultivated antibody producing CHO cells under mild and harsh sparging conditions and observed that sparging stress leads to the decreased cell growth rate, viability and productivity. Subsequent in silicomodel-driven flux analysis suggested that sparging stress rewires amino acid metabolism towards the enriched H2O2 turnover rate by up-regulated fluxes of amino acid oxidases. Interestingly, many of these H2O2-generating reactions were closely connected with the production of NADH, NADPH and GSH which are typical reducing equivalents. Thus, we can hypothesize that increased amino acid uptake caused by sparging stress contributes to restore redox homeostasis against oxidative stress. The current model-driven systematic data analysis allows us to quickly define distinct metabolic feature under stress condition by using basic cell cultivation datasets.

Targeted vault nanoparticles engineered with an endosomolytic peptide deliver biomolecules to the cytoplasm.

Vault nanoparticles were engineered to enhance their escape from the endosomal compartment by fusing a membrane lytic peptide derived from adenovirus protein VI (pVI) to the N-terminus of the major vault protein to form pVI-vaults. We demonstrate that these pVI-vaults disrupt the endosomal membrane using three different experimental protocols including (1) enhancement of DNA transfection, (2) co-delivery of a cytosolic ribotoxin, and (3) direct visualization by fluorescence. Furthermore, direct targeting of vaults to specific cell surface epidermal growth factor receptors led to enhanced cellular uptake and efficient delivery of vaults to the cytoplasm. This process was monitored with fluorescent vaults, and morphological changes in the endosomal compartment were observed. By combining targeting and endosomal escape into a single recombinant vault, high levels of transfection efficiency were achieved using low numbers of vault particles. These results demonstrate that engineered vaults are effective, efficient, and nontoxic nanoparticles for targeted delivery of biomaterials to the cell cytoplasm.

Enhanced percolation and gene expression in tumor hypoxia by PEGylated polyplex micelles.

In regard to gene vectors for cancer gene therapy, their percolation into the tumor tissue should be essential for successful outcome. Here, we studied the tumor penetrability of nonviral vectors (polyplexes) after incubation with the multicellular tumor spheroid (MCTS) models and intratumoral (i.t.) injection into subcutaneous tumors. As a result, polyethylene glycolated (PEGylated), core-shell type polyplexes (polyplex micelles) showed facilitated percolation and improved transfection inside the tumor tissue, whereas conventional polyplexes from cationic polymers exhibited limited percolation and localized transfection. Furthermore, the transfection of hypoxia-responsive plasmid demonstrated that polyplex micelles allowed the transfection to the hypoxic region of the tumor tissue in both in vitro and in vivo experiments. To the best of our knowledge, our results demonstrated for the first time that polyplex micelles might show improved tumor penetrability over cationic polyplexes, thereby achieving transfection into the inside of the tumor tissue.

Targeting vault nanoparticles to specific cell surface receptors.

As a naturally occurring nanocapsule abundantly expressed in nearly all-eukaryotic cells, the barrel-shaped vault particle is perhaps an ideal structure to engineer for targeting to specific cell types. Recombinant vault particles self-assemble from 96 copies of the major vault protein (MVP), have dimensions of 72.5 x 41 nm, and have a hollow interior large enough to encapsulate hundreds of proteins. In this study, three different tags were engineered onto the C-terminus of MVP: an 11 amino acid epitope tag, a 33 amino acid IgG-binding peptide, and the 55 amino acid epidermal growth factor (EGF). These modified vaults were produced using a baculovirus expression system. Our studies demonstrate that recombinant vaults assembled from MVPs containing C-terminal peptide extensions display these tags at the top and bottom of the vault on the outside of the particle and can be used to specifically bind the modified vaults to epithelial cancer cells (A431) via the epidermal growth factor receptor (EGFR), either directly (EGF modified vaults) or as mediated by a monoclonal antibody (anti-EGFR) bound to recombinant vaults containing the IgG-binding peptide. The ability to target vaults to specific cells represents an essential advance toward using recombinant vaults as delivery vehicles.

Polyplex micelles from triblock copolymers composed of tandemly aligned segments with biocompatible, endosomal escaping, and DNA-condensing functions for systemic gene delivery to pancreatic tumor tissue.

PURPOSE: For systemic gene delivery to pancreatic tumor tissues, we prepared a three-layered polyplex micelle equipped with biocompatibility, efficient endosomal escape, and pDNA condensation functions from three components tandemly aligned; poly(ethylene glycol) (PEG), a poly(aspartamide) derivative with a 1,2-diaminoethane moiety (PAsp(DET)), and poly(L-lysine). MATERIALS AND METHODS: The size and in vitro transfection efficacy of the polyplex micelles were determined by dynamic light scattering (DLS) and luciferase assay, respectively. The systemic gene delivery with the polyplex micelles was evaluated from enhanced green fluorescence protein (EGFP) expression in the tumor tissues. RESULTS: The polyplex micelles were approximately 80 nm in size and had one order of magnitude higher in vitro transfection efficacy than that of a diblock copolymer as a control. With the aid of transforming growth factor (TGF)-beta type I receptor (TbetaR-1) inhibitor, which enhances accumulation of macromolecular drugs in tumor tissues, the polyplex micelle from the triblock copolymer showed significant EGFP expression in the pancreatic tumor (BxPC3) tissues, mainly in the stromal regions including the vascular endothelial cells and fibroblasts. CONCLUSION: The three-layered polyplex micelles were confirmed to be an effective gene delivery system to subcutaneously implanted pancreatic tumor tissues through systemic administration.

Transfection study using multicellular tumor spheroids for screening non-viral polymeric gene vectors with low cytotoxicity and high transfection efficiencies.

Polyplexes consisting of plasmid DNA and polycations have received much attention as promising vectors for gene transfer. For effective gene therapy, polycations with different polyamine structures in the side chain were developed to ensure their buffering capacity for endosomal escape, and their PEGylated block copolymers were developed to increase their stability and biocompatibility. The effects of the chemical structures of polycations and their PEGylation on transfection and cytotoxicity were elucidated by use of a three-dimensional multicellular tumor spheroid of human hepatoma HuH-7 cells. Various features of transfection with polyplex micelles, which have been hard to observe in conventional monolayer cultures, were revealed by the multicellular tumor spheroid (MCTS) model in terms of cytotoxicity and time-dependent behaviors of transfected gene expression under three-dimensional microenvironments. By using this system, the polyplex micelle from poly(ethylene glycol)-b-poly(N-substituted asparagine) copolymers having the N-(2-aminoethyl)-2-aminoethyl group in the side chain (PEG-b-P[Asp(DET)] polyplex micelle) was proved to achieve high transfection efficiencies as well as low cytotoxicity, both of which are critical properties for successful in vivo gene delivery.

Fed-batch cultures of Escherichia coli cells with oxygen-dependent nar promoter systems.

The recombinant proteins produced from Escherichia coli as a host cell need to be made at as low a cost as possible because of the end of the monopoly following expiry of the patent on early pharmaceutical proteins, and thus expanding applications to non-pharmaceutical large-scale products. We review in this article how the various promoters used in recombinant E. coli could affect its protein products, especially with emphasis on relatively new oxygen-dependent nar promoters for beta-galactosidase production. Several studies carried out in the authors' laboratory show that the nar promoter does not require any chemicals except 1% nitrate and oxygen for protein production. And according to recent work with the modified strains it is possible to produce the enzyme (beta-galactosidase) even without the nitrate ions at 45% of its total protein content when its cell density reached OD = 176.