Utilization of algal sugars and glycerol for enhanced cephalosporin C production by Acremonium chrysogenum M35.

In our previous study, glycerol was utilized as an additional carbon source for the production of cephalosporin C (CPC) by Acremonium chrysogenum M35. In this study, algal sugars extracted from the third-generation biomass were utilized in the CPC production for the first time. The CPC production improved about twofold when using the algal sugars as the carbon source. The complex medium including algal sugars and glycerol was utilized, and 7·3 g l-1 CPC production was achieved in a 250-ml shaking flask. To determine the important variables for the CPC production, Plackett-Burman design was carried out and 6·18 g l-1 of CPC was estimated under the numerically optimized conditions. Under the optimized conditions, the CPC production was performed in a 5-l scale bioreactor, affording CPC production at a rate of 7·1 g l-1 . Moreover, 6·7 g l-1 CPC was produced using crude glycerol as the substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: Microalgae are the biomass containing various components, such as carbohydrates, lipids, and amino acids. In this study, carbon sources contained in microalgae were obtained by acid extraction, and cephalosporin C (CPC), a ß-lactam antibiotic intermediate, was produced by using Acremonium chrysogenum M35. In addition, the increase of CPC production was not distinct for A. chrysogenum M35 with algal sugars as the only carbon source; therefore, glycerol was added, increasing the CPC production. Thus, cheap residues such as algal sugars form microalgal and glycerol form biodiesel process could be used as the alternative sources for the production of various products.

TerplexDNA gene carrier system targeting artery wall cells.

The development of non-viral gene carrier systems becomes more urgent and important due to the major biosafety considerations involved with application of viral vector systems for clinical gene therapy. We recently developed a novel non-viral gene carrier system, termed TerplexDNA, which showed high gene transfer efficiency when compared to the lipofectamine gene delivery system both in HepG2 and A7R5 cell lines in vitro. In present studies, we demonstrated that the TerplexDNA gene carrier system specifically delivered the reporter genes (LacZ and Luciferase) and therapeutic gene (hrVEGF(165) cDNA) into bovine aortic artery wall cells (endothelial cells and smooth muscle cells) by receptor mediated endocytosis. We found that the transfection efficiency to these primary artery wall cells, when mediated by the TerplexDNA system, was dose-dependent, saturable and was significantly inhibited by excess free LDL. The transfection efficiency of the TerplexDNA gene carrier system was approximately 60-fold higher than that of the lipofectamine gene carrier system. The TerplexDNA gene carrier system is a useful and promising tool for artery wall gene transfer.

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium.

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.

Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment.

Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.

Biodegradable polyester, poly[alpha-(4-aminobutyl)-L-glycolic acid], as a non-toxic gene carrier.

PURPOSE: The aim of this study was to develop a non-toxic polymeric gene carrier. For this purpose, biodegradable cationic polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) was synthesized. PAGA was designed to have ester linkage because polyesters usually show biodegradability. METHODS: Degradation of PAGA in an aqueous solution was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PAGA/DNA complexes were characterized by gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS). The transfection was measured by using the beta-galactosidase reporter gene. RESULTS: PAGA was degraded in aqueous solution very quickly and the final degradation product was a monomer (L-oxylysine). Formation of self-assembling biodegradable complexes between PAGA and DNA at a charge ratio 1:1 (+/-) was confirmed by gel band shift assay and AFM. In these studies, controlled release of DNA from the complexes could be seen. The complexes showed about 2-fold higher transfection efficiency than DNA complexes of poly-L-lysine (PLL), a structural analogue of PAGA, which is the most commonly used poly-cation for gene delivery. The polymer did not show cytotoxicity, possibly because of its degradability and the biocompatibility of the monomer. CONCLUSIONS: The use of the biodegradable poly-cation, PAGA, as a DNA condensing agent will be useful in safe gene delivery.

Repression of GAD autoantigen expression in pancreas beta-Cells by delivery of antisense plasmid/PEG-g-PLL complex.

It was previously reported that silencing of the expression of glutamic acid decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet beta-cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can prevent autoimmune destruction of pancreatic beta-cells. To repress GAD expression in islet beta-cells, we delivered an antisense GAD mRNA expression plasmid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid: PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in beta-cells and expression was dependent on glucose level. The repression of GAD after transfection of pRIP-AS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression.