Mun Hwan Choi

Amphiphilic PHA-mPEG copolymeric nanocontainers for drug delivery: preparation, characterization and in vitro evaluation.

Amphiphilic biodegradable core-shell nanoparticles were prepared by emulsification-solvent evaporation technique from diblock copolymers which were synthesized by chemical coupling of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) to monomethoxy poly(ethylene glycol) (mPEG) through transesterification reaction. The nanoparticles were found to be assembled in aqueous solution into an outer hydrophilic shell of mPEG connected to the interior hydrophobic polyhydroxyalkanoate (PHA) copolymer core, which was identified by a comparative analysis of enzymatic degradation of the mPEG-coupled and non-coupled PHA nanoparticles. Morphological examination under atomic force microscope showed the formation of smooth spherically shaped nanoparticles. The average particle sizes and zeta potentials of amphiphilic nanoparticles were in the range of 112-162 nm and -18 to -27 mV, respectively. A hydrophobic drug thymoquinone was encapsulated in the nanoparticles and its release kinetics was studied. The in vitro cytotoxicity evaluation of the nanoparticles on prenatal rat neuronal hippocampal and fibroblast cells revealed that biocompatibility of the amphiphilic nanoparticles was generally independent of the ratio of comonomer units in the PHA block. In conclusion, the amphiphilic nanoparticles contained the hydrophobic PHA segments buried in the core and could thus be used as safe carriers for the controlled release of variety of hydrophobic drugs.

Metabolic relationship between polyhydroxyalkanoic acid and rhamnolipid synthesis in Pseudomonas aeruginosa: Comparative 13C NMR analysis of the products in wild-type and mutants

Polyhydroxyalkanoic acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty acids were also investigated using octanoic-1-¹³C acid. ¹³C NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-¹³C being converted from the octanoic acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-¹³C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-¹³C via fatty acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depended on the type of carbon sources. The rhlB mutant could be exploited as an efficient producer of the important biosurfactant HAAs (e.g., ~700 mg/L HAAs was obtained when grown on 60 mM octanoic acid).

Amorphous amphiphilic P(3HV-co-4HB)-b-mPEG block copolymer synthesized from bacterial copolyester via melt transesterification: Nanoparticle preparation, cisplatin-loading for cancer therapy and in vitro evaluation

Cisplatin is a chemotherapeutic agent used against a variety of tumors. We determined the efficacy and bioavailability of cisplatin in the form of cisplatin-loaded self-assembled amphiphilic copolymer nanoparticles (NPs). Non-crystallizing bacterial copolyester was employed as hydrophobic segment to increase drug loading efficiency. Novel amorphous amphiphilic block copolymer P(3HV-co-4HB)-b-mPEG was synthesized from bacterial copolyester poly(3-hydroxyvalerate-co-4-hydroxybutyrate) coupled via transesterification reaction using bis(2-ethylhexanoate) tin catalyst to monomethoxypoly(ethylene glycol). The product was characterized, and core-shell particles with nanometer size range were prepared by emulsification-solvent evaporation method. Transmission electron microscopy (TEM) examination revealed that the NPs took the shape of spheres with inner concealed core of hydrophobic P(3HV-co-4HB) polymer and the outer shell formed by hydrophilic mPEG segment. The in vitro release profile of cisplatin from the core hydrophobic domain showed a sustained release of the drug. TEM and confocal microscopy examination revealed clearly the internalization of cisplatin-loaded NPs into the tumor cells. MTT assay, flow cytometry, western blot and confocal microscopy revealed a suppression effect by the NPs on tumor cell growth, and enhancement of apoptotic process of the tumor cells compared to free drug treated cells. The amorphous polymeric NPs could be effective vehicles for the sustained delivery of toxic anticancer drugs.

Local Sequence Dependence of Polyhydroxyalkanoic Acid Degradation in Hydrogenophaga pseudoflava

The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions inHydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, 13C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. 13C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and “native” PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.

Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2-Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain-Length

Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

Shifting of the Distribution of Aromatic Monomer-Units in Polyhydroxyalkanoic Acid to Longer Units by Salicylic Acid in Pseudomonas fluorescens BM07 Grown With Mixtures of Fructose and 11-Phenoxyundecanoic Acid

Medium‐chain‐length‐polyhydroxyalkanoic acids (MCL‐PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer‐units whose precursors are supplied via β‐oxidation degradation of MCL fatty acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl‐group transfer from 3‐hydroxyacyl‐ACPs derived from acetyl‐CoA to coenzyme A. It was found that salicylic acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer‐units derived from a MCL fatty acid co‐added as carbon source (e.g., 11‐phenoxyundecanoic acid (11‐POU)) to longer monomer‐units. Both SA and acrylic acid were found to induce high accumulations of 3‐ketohexanoic acid in BM07 wild‐type cells grown with n‐hexanoic acid as well as to inhibit the formation of acetyl‐CoA from acetoacetyl‐CoA by BM07 cell extract, suggesting that 3‐ketoacyl‐CoA thiolase is their common β‐oxidation target. The structural motif of acrylic acid present in the molecular structure of SA may self‐explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild‐type and ΔphaG mutant cells grown on the mixtures of fructose and 11‐POU revealed that both PhaG and β‐oxidation inhibitor may play a critical role in the synthesis of PHA with longer side‐chain ω‐functional substitutions. Biotechnol. Bioeng. 2009;102: 1209–1221.

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k(1)), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k(1) value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k(1) value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k(1) values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with ω-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid β-oxidation inhibition.

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07.

Lipid Emulsion Inhibits Apoptosis Induced by a Toxic Dose of Verapamil via the Delta-Opioid Receptor in H9c2 Rat Cardiomyoblasts

The goals of this study were to investigate the effects of lipid emulsion (LE) on apoptosis induced by a toxic dose of verapamil in H9c2 cells and to elucidate the associated cellular mechanism. The effects of LE alone and combined with an inhibitor on the decreases in cell counts and viability induced by verapamil and diltiazem were examined using the MTT assay. The effects of verapamil alone, combined LE and verapamil treatment, and combined inhibitor, LE and verapamil treatment on cleaved caspase-3, caspase-8 and Bax expression, were examined using Western blotting. The effects of verapamil alone and combined with LE on the number of TUNEL-positive H9c2 cells were also examined. LE attenuated the decreases in cell counts and viability induced by verapamil and diltiazem. However, the magnitude of the LE-mediated attenuation of decreased cell viability was enhanced by verapamil compared with diltiazem treatment. Naloxone, naltrindole hydrochloride, LY294002 and MK-2206 inhibited the LE-mediated attenuation of increased cleaved caspase-3 and caspase-8 expression induced by verapamil. LE attenuated the increase in the number of TUNEL-positive cell induced by verapamil. These results suggest that LE attenuates apoptosis induced by verapamil via activation of the delta-opioid receptor, phosphoinositide 3-kinase and Akt.

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens BM07 enhancing the production of polyhydroxyalkanoic acid but deficient in cold-induced exobiopolymer production

Pseudomonas fluorescens BM07 is known to produce cold-induced exobiopolymer, which is mainly composed of water-insoluble hydrophobic polypeptides (up to 85%) and saccharides (8%), by decreasing the culture temperature down to as low as 10 degrees C. We screened for transposon insertion mutants of P. fluorescens BM07 that were unable to produce the exobiopolymer. Among the eight mutants that showed the deficiency of exobiopolymer and O-lipopolysaccharide, one mutant BM07-59 that had the highest polyhydroxyalkanoates (PHA) production was selected. The transposon inserted gene in BM07-59 was identified as galU. The disruption of the gene galU coded for the putative product, UDP-glucose pyrophosphorylase (GalU), resulted in 1.5-fold more accumulation of PHA compared with the wild-type strain from 70 mM fructose or galactose at 30 degrees C. Electrophoretic analysis of lipopolysaccharide showed that the mutant lacked the O-antigen lipopolysaccharide bands. The glycosyl composition of the lipopolysaccharide produced by the mutant strain was significantly different from that of the wild-type strain. We suggest that the deletion of galU could be a way to shift carbon flux efficiently from exobiopolymer toward PHA in P. fluorescens BM07.