Multifaceted applications of nanomaterials in cell engineering and therapy

Nanomaterials with superior physiochemical properties have been rapidly developed and integrated in every aspect of cell engineering and therapy for translating their great promise to clinical success. Here we demonstrate the multifaceted roles played by innovatively-designed nanomaterials in addressing key challenges in cell engineering and therapy such as cell isolation from heterogeneous cell population, cell instruction in vitro to enable desired functionalities, and targeted cell delivery to therapeutic sites for prompting tissue repair. The emerging trends in this interdisciplinary and dynamic field are also highlighted, where the nanomaterial-engineered cells constitute the basis for establishing in vitro disease model; and nanomaterial-based in situ cell engineering are accomplished directly within the native tissue in vivo. We will witness the increasing importance of nanomaterials in revolutionizing the concept and toolset of cell engineering and therapy which will enrich our scientific understanding of diseases and ultimately fulfill the therapeutic demand in clinical medicine.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Utilization of PEI-modified Corynebacterium glutamicum biomass for the recovery of Pd(II) in hydrochloric solution

A new type of biosorbent was developed for binding anionic precious metals through cross-linking waste biomass Corynebacterium glutamicum with polyethylenimine (PEI). This biomass was evaluated for the removal and recovery of palladium and compared to commercial adsorbents, such as Amberjet 4200 Cl, Lewatit Monoplus TP 214, SPC-100, and SPS-200. The kinetic experiments revealed that the sorption equilibrium was reached with 30 min for the PEI-modified biomass. The maximum uptake of the biosorbent was 176.8 mg/g, which was calculated using the Langmuir model. The Pd(II) maximum uptake exhibited the following order: Amberjet 4200 Cl > Lewatit Monoplus TP 214 > PEI-modified biomass > SPC-100 > SPS-200. Acidified thiourea in 1.0 M HCl was used to desorb Pd(II) from all of the sorbents examined.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Chromatographic performance of a thin microporous bed of nitrocellulose

Chromatography along thin (125 μm) porous beds of nitrocellulose, layered on top of an polyester backing, shows good separation efficiency with plate heights of 10–20 μm. Flow is controlled by capillary forces and shows low rate variations between the individual disposable devices. Positively charged groups were introduced into the nitrocellulose and efficient separation of transferrin isoforms, differing by only 0.1 pI units, was found after a short migration distance (1 cm). The upper surface is not covered, which allows sample and reagents to be added, and the clear backing permits detection. The chromatography can easily be combined on-line with sensitive immunoassay detection down to the pM (10⁻¹² M) range. This microscaled combination device should have a wide range of applications in analytical biochemistry.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Polyethylenimine-cationized β-catenin protein transduction activates the Wnt canonical signaling pathway more effectively than cationic lipid-based transduction

The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by β-catenin protein transduction. Constitutively active β-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active β-catenin protein was added to HEK-293 cells, and induction of several Wnt/β-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active β-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active β-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.     

Dextran-g-PEI nanoparticles as a carrier for co-delivery of adriamycin and plasmid into osteosarcoma cells

Combination of chemotherapy and gene therapy of cancer has synergistic effects on overcoming drug resistance. Macromolecular materials such as dextran and PEI have been a potential module for chemotherapeutics and gene delivery. Herein, we hypothesize the combinational strategy of chemotherapy and gene therapy in a single dextran-PEI nanoplatform. The physicochemical properties, cytotoxicity, transfection efficiency were investigated in vitro. Ultra-violet spectrum and ¹H NMR revealed adriamycin and PEI were grafted to dextran chain. Agarose gel electrophoresis demonstrated that the migration of plasmid was completely retarded when the N/P ratio of complex was 4. The sizes of DEX-ADM-PEI/DNA nanoparticles decreased and the zeta potentials enhanced with the increasing N/P ratio. Transmission electron microscope indicated a round morphology of the nanoparticles. DEX-ADM-PEI conjugation has higher cytotoxicity, compared to free adriamycin, in MG-63 and Saos-2 osteosarcoma cells but DEX-PEI maintained over 65% cell viability at the concentration of 8 mg/mL. The transfection efficiency of DEX-ADM-PEI/pEGFP-N1 at N/P ratio of 4:1 both in MG-63 and Saos-2 cell were slightly low than that of PEI 25k. But our nanoplatform efficiently delivered both plasmid pEGFP-N1 and adriamycin into osteosarcoma cells. This study demonstrated that DEX-ADM-PEI efficiently and selectively delivered both plasmid pEGFP-N1 and adriamycin to osteosarcoma cells with low cytotoxicity.