Biomedical subjects

T G Park

Publications and source records attributed to T G Park.

At least 19 recordsLinked to original sources

PubMed2006

Activated Notch1 interacts with p53 to inhibit its phosphorylation and transactivation.

We propose a biochemical mechanism for the negative role of Notch signaling on p53 transactivation function. Expression of the intracellular domain of human Notch1 (Notch1-IC) inhibits the expression of p53-responsive genes p21, mdm2, and bax in HCT116 p53(-/-) cells. Furthermore, Notch1-IC expression inhibits the phosphorylation of ectopically expressed p53 in HCT116 p53(-/-) cells as well as the phosphorylation of endogenous p53 in UV-treated HCT116 p53(+/+) cells. Transcriptional downregulation of p53-responsive genes by Notch1-IC was confirmed both by chromatin immunoprecipitation assay and Northern blot analysis. We found the intracellular interaction between Notch1-IC and p53 in HCT116 p53(+/+) cells and suggest that activated Notch1 interaction with p53 is an important cellular event for the inhibition of p53-dependent transactivation. The N-terminal fragment of Notch1-IC, which can interacts with p53, inhibits p53 phosphorylation and represses p53 transactivation. In addition, Notch signaling downregulated p53-dependent apoptosis induced by UV irradiation.

Apoptosis↗

PubMed2001

Microencapsulation of dissociable human growth hormone aggregates within poly(D,L-lactic-co-glycolic acid) microparticles for sustained release.

For the sustained release formulation of recombinant human growth hormone (rhGH), dissociable rhGH aggregates were microencapsulated within poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. rhGH aggregates were first produced by adding a small volume of aqueous rhGH solution into a partially water miscible organic solvent phase (ethyl acetate, EtAc) containing PLGA. These rhGH aggregates were then microencapsulated within PLGA polymer phase by extracting EtAc into an aqueous phase pre-saturated with EtAc. Release profiles of rhGH from these microparticles were greatly affected by changing the volume of incubation medium. The released rhGH species consisted of mostly monomeric form having a correct conformation. This study reveals that sustained rhGH release could be achieved by microencapsulating reversibly dissociable protein aggregates within biodegradable polymers.

Capsules↗

PubMed2001

Protein release microparticles based on the blend of poly(D,L-lactic-co-glycolic acid) and oligo-ethylene glycol grafted poly(L-lactide).

Bovine serum albumin (BSA), a model protein drug, was encapsulated with a microparticle based on the blend of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactide)-g-oligo(ethylene glycol) (PLLA-g-oligoEG). Effects of PLLA-g-oligoEG in the blend on degradation, characteristic properties, and release behavior of the microparticle were studied. Drug loading efficiency increased with increase in the graft frequency of oligoEG in the graft copolymer in the blend. The release of BSA was found to be more efficient for microparticles based on the blend than on the PLGA, which is due to the faster protein diffusion through the swollen phase of the hydrogel-like structure. The microparticles based on the blend showed a slower degradation and a lower pH shift compared to that of PLGA.

Calorimetry, Differential Scanning↗

PubMed2001

A new gene delivery formulation of polyethylenimine/DNA complexes coated with PEG conjugated fusogenic peptide.

A fusogenic peptide, KALA, was conjugated with poly(ethylene glycol) (PEG) for use as an endosome disruptive agent in the gene delivery formulation of polyethyleneimine (PEI). A maleimide terminated methoxy-PEG, a cysteine specific derivative, was reacted with KALA to produce a PEG-KALA conjugate. The conjugate was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and its hemolytic activity was determined relative to KALA. Positively charged PEG-KALA conjugate was coated onto the surface of negatively charged DNA/PEI complexes to form net positively charged PEG-KALA/DNA/PEI complexes. They were 200-400 nm in diameter with increasing amount of PEG-KALA, whereas DNA/PEI complexes coated with KALA aggregated to a great extent. This was because PEG chains surrounding the surface of the complexes suppressed the inter-particle interaction that was mediated by cationic KALA. Transfection efficiency progressively increased as the amount of PEG-KALA to be coated was increased, suggesting that fusogenic activity of KALA contributes to enhancing the level of gene expression.

Amino Acid Sequence↗

PubMed2001

Pegylation enhances protein stability during encapsulation in PLGA microspheres.

During encapsulation of proteins in biodegradable microspheres, a significant amount of the protein reportedly undergoes denaturation to form irreversible insoluble aggregates. Incomplete in vitro release of proteins from the microspheres is a common observation. An attempt was made to overcome this problem by pegylation of the protein to be encapsulated. Lysozyme, a model protein, was conjugated with methoxy polyethylene glycol (mPEG, MW 5000). The conjugate was characterized by SDS-PAGE, SE-HPLC, and MALDI-TOF mass spectroscopy. The pegylated lysozyme (Lys-mPEG) consisted of different isomers of mono-, di- and tri-pegylated with about 15% as native lysozyme. The specific activity of the protein was retained after pegylation (101.3+/-10.4%). The microsphere encapsulation process was simulated for pegylated and native lysozyme. Pegylated lysozyme exhibited much better stability than native lysozyme against exposure to organic solvent (dichloromethane), homogenization, and showed reduced adsorption onto the surface of blank PLGA microspheres. Release profiles of the two proteins from microspheres were very different. For native lysozyme, it was high initial release (about 50%) followed by a nearly no release (about 10% in 50 days). In contrast, Lys-mPEG conjugate showed a triphasic and near complete release over 83 days. This study shows that the pegylation of protein can provide substantial protection against the destabilization of protein during encapsulation.

Adsorption↗

PubMed2001

DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline).

A series of linear poly(ethylenimine) (L-PEI) containing varying amounts of cationic charge density in its backbone was produced by controlled hydrolysis of poly(2-ethyl-2-oxazoline) (PEtOz) for using as a nonviral DNA transfection agent. The effects of cationic charge density and molecular weight of the L-PEI on the cytotoxicity and transfection efficiency were studied. The efficiency of transfection was monitored by using a luciferase reporter gene system. Gel retardation assay and dynamic light scattering (DLS) showed that the condensation capacity of L-PEI was suitable for transfection. Highly compacted L-PEI/DNA complex ( approximately 150 nm) was obtained with a surface charge value of around +28.4 mV. Cell cytotoxicity was affected to a great extent by the hydrolysis percent of L-PEI as well as by the molecular weight. Transfection efficiency of luciferase plasmid DNA against NIH 3T3 fibroblast was largely dependent upon the hydrolysis percent (charge density) in the polymer backbone and the molecular weight of the L-PEI, but independent of the total amount of cationic charges used for DNA condensation. L-PEI with a hydrolysis percent of 88.0% exhibited comparable transfection efficiency to that of commonly used branched PEI.

3T3 Cells↗

PubMed2001

Degradation behaviors of biodegradable macroporous scaffolds prepared by gas foaming of effervescent salts.

Biodegradable polymeric scaffolds for tissue engineering were fabricated by a gas-foaming/salt-leaching method using a combination of two effervescent salts, ammonium bicarbonate and citric acid. Poly(D,L-lactic-co-glycolic acid) (PLGA) in a state of gel-like paste was first produced by precipitation of PLGA dissolved in chloroform into ethanol. The polymer slurry was mixed with sieved particles of ammonium bicarbonate, molded, and then immersed in an aqueous solution of citric acid to generate macroporous scaffolds. The scaffolds had relatively homogeneous pore structures throughout the matrix and showed an average pore size of 200 microm and over 90% porosity. By adjusting the concentration of citric acid in the aqueous medium, it was possible to control porosity as well as mechanical strength of the scaffolds. In vitro degradation studies of three different scaffolds having lactic/glycolic acid molar ratios of 75/25, 65/35, and 50/50 exhibited marked swelling behaviors at different critical time points. The swollen matrices had a hydrogel-like internal structure. It was found that massive water uptake into the degrading scaffolds induced matrix swelling, which facilitated the hydrolytic scission of PLGA chains with concomitant disintegration of the matrices.

Biocompatible Materials↗

PubMed2001

Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer.

Biodegradable polymeric micelles containing doxorubicin in the core region were prepared from a di-block copolymer composed of doxorubicin-conjugated poly(DL-lactic-co-glycolic acid) (PLGA) and polyethyleneglycol (PEG). The di-block copolymer of PLGA-PEG was first synthesized and the primary amino group of doxorubicin was then conjugated to the terminal hydroxyl group of PLGA, which had been pre-activated using p-nitrophenyl chloroformate. The resulting polymeric micelles in aqueous solution were characterized by measurement of size, drug loading, and critical micelle concentration. The micelles containing chemically-conjugated doxorubicin exhibited a more sustained release profile than PEG-PLGA micelles containing physically-entrapped doxorubicin. The cytotoxic activity of the micelles against HepG2 cells was greater than free doxorubicin, suggesting that the micelles containing conjugated doxorubicin were more effectively taken up cellularly, by an endocytosis mechanism rather than by passive diffusion. Confocal microscopic observation and flow cytometry analysis supported the enhanced cellular uptake of the micelles.

Antibiotics, Antineoplastic↗

PubMed2001

Protein-fatty acid complex for enhanced loading and stability within biodegradable nanoparticles.

Lysozyme was hydrophobically modified with a fatty acid, sodium oleate, via an ion-pairing mechanism. Ionic binding between an anionic carboxylic group of sodium oleate and basic amino groups in lysozyme was primarily utilized to form lysozyme-oleate complex. The complex formation was pH dependent. The lysozyme-oleate complex dissolved in an organic solvent exhibited much higher conformational stability at elevated temperature compared with free lysozyme in the same solvent. The complex was formulated into biodegradable nanoparticles by a spontaneous emulsion and solvent diffusion method. The resultant formulation showed near 100% encapsulation efficiency of lysozyme within nanoparticles with < 100 nm in diameter with a narrow size distribution. Lysozyme could be loaded into the nanoparticles up to 18.6% (w/w) with concomitantly increased particle sizes. This study demonstrates a new formulation method of biodegradable nanoparticles with highly efficient encapsulation of proteins, which are potentially useful for oral protein delivery including mucosal vaccination.

Biocompatible Materials↗

PubMed2001

The effect of charge increase on the specificity and activity of a short antimicrobial peptide.

By using short linear antimicrobial peptides as a model system, the effect of peptide charge on the specificity between Candida albicans (fungi) and Gram-positive bacteria was investigated. In a present study, we added and/or deleted lysine residue(s) at the C-terminal and/or N-terminal end(s) of an antimicrobial peptide (KKVVFKVKFK-NH(2)) and synthesized the peptides that had similar alpha helical structures in a lipid membrane mimic condition. The increase of peptide charge improved antifungal activity without the change of antibacterial activity. Structure-activity relationship study about the peptides revealed that the net positive charge must play an important role in the specificity between C. albicans and Gram-positive bacteria and the increase of the net positive charge without the moderate change of secondary structure could improve activity for C. albicans rather than Gram-positive bacteria.

Antifungal Agents↗

PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗

PubMed2000

Lysozyme microencapsulation within biodegradable PLGA microspheres: urea effect on protein release and stability.

Lysozyme was encapsulated within biodegradable poly(D, L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres.

Animals↗

PubMed2000

In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates.

Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] by an ester linkage and the doxorubicin-PLGA conjugate was formulated into nanoparticles. A carboxylic acid end group of PLGA was conjugated to a primary hydroxyl group of doxorubicin. The primary amine group of doxorubicin was protected during the conjugation process and then deprotected. The nanoparticles containing the conjugate exhibited sustained doxorubicin release profiles over a 1-month period, whereas those containing unconjugated free doxorubicin showed a rapid doxorubicin release within 5 days. Doxorubicin release patterns could be controlled by conjugating doxorubicin to PLGA having different molecular weights. The conjugated doxorubicin nanoparticles showed increased uptake within a HepG2 cell line, which was quantitated by a flow cytometry and visualized by confocal microscopy. The nanoparticles exhibited slightly lower IC(50) value against the HepG2 cell line compared to that of free doxorubicin. In vivo anti-tumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection. The conjugation approach of doxorubicin to PLGA was potentially useful for the formulation of nanoparticles that requires targeting for cancer cells as well as sustained release at the site.

Animals↗

PubMed2000

Hydrophobic ion pair formation between leuprolide and sodium oleate for sustained release from biodegradable polymeric microspheres.

Leuprolide acetate, an analogue of luteinizing hormone-releasing hormone (LH-RH), was hydrophobically ion paired with a long chain fatty acid, sodium oleate, in an aqueous solution. Solution behaviors of the complex formed between leuprolide and sodium oleate were investigated in terms of aqueous solubility, turbidity, particle size, and zeta potential as a function of molar ratio between the two species. It was found that with increasing the stoichiometric molar amounts of sodium oleate to leuprolide approached up to 2.5-3, the solution became gradually turbid with increasing particle sizes, indicating leuprolide precipitation as a result of hydrophobic ion pairing. On the other hand, beyond that critical molar ratio range, the solution turned into clear with much reduced particle size, indicative of micelle formation. The hydrophobically modified leuprolide-oleate complex was lyophilized and directly encapsulated within biodegradable poly(D, L-lactic-co-glycolic acid) (PLGA) microspheres via a single oil-in-water (O/W) emulsion method. Microsphere morphology, leuprolide release behavior, and polymer mass erosion profiles were examined in comparison to the PLGA microspheres prepared with free leuprolide.

Delayed-Action Preparations↗

PubMed2000

Effect of formulation and processing variables on the characteristics of microspheres for water-soluble drugs prepared by w/o/o double emulsion solvent diffusion method.

80% except for acetaminophen, due to its lower solubility in water and higher solubility in corn oil. The release profile of the drug was pH dependent. In acidic medium, the release rate was much slower, however, the drug was released quickly at pH 7.4. Tacrine showed unexpected release profiles, probably due to ionic interaction with polymer matrix and the shell structure and the highest release rate was obtained at pH 2.0. The prepared microspheres had a sponge-like inner structure with or without central hollow core and the surface was dense with no apparent pores.

Acetaminophen↗

PubMed2000

A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive.

Highly open porous biodegradable poly(L-lactic acid) ¿PLLA scaffolds for tissue regeneration were fabricated by using ammonium bicarbonate as an efficient gas foaming agent as well as a particulate porogen salt. A binary mixture of PLLA-solvent gel containing dispersed ammonium bicarbonate salt particles, which became a paste state, was cast in a mold and subsequently immersed in a hot water solution to permit the evolution of ammonia and carbon dioxide within the solidifying polymer matrix. This resulted in the expansion of pores within the polymer matrix to a great extent, leading to well interconnected macroporous scaffolds having mean pore diameters of around 300-400 microm, ideal for high-density cell seeding. Rat hepatocytes seeded into the scaffolds exhibited about 95% seeding efficiency and up to 40% viability at 1 day after the seeding. The novelty of this new method is that the PLLA paste containing ammonium bicarbonate salt particles can be easily handled and molded into any shape, allowing for fabricating a wide range of temporal tissue scaffolds requiring a specific shape and geometry.

Animals↗

PubMed1999

Microencapsulation of human growth hormone within biodegradable polyester microspheres: protein aggregation stability and incomplete release mechanism.

Recombinant human growth hormone (rhGH) was encapsulated within poly(D,L-lactic-co-glycolic acid) microspheres by a double emulsion solvent evaporation method. A mixture of methylene chloride and ethyl acetate in varying volume ratios was used for the microsphere preparation. Protein release profiles from three different microsphere formulations demonstrated initial burst effects ranging from 28.2% to 54.7% after a 1-day incubation and exhibited no further significant releases up to 19 days. This was because the encapsulated rhGH with the microspheres was largely aggregated in a noncovalent fashion during the formulation. Nonaggregated water soluble rhGH species within the microspheres are likely to be responsible for the rapid release upon incubation. The initially released rhGH in the incubation medium, however, was composed of mostly monomer species with a small amount of dimer as probed by size-exclusion chromatography. Circular dichroism spectra of the initially released rhGH in the medium revealed that the conformation of the released rhGH was correctly folded relative to that of native rhGH, with little variation in alpha-helix contents depending on the formulations. The "nonrelease" mechanism after the initial burst release was attributed to nonspontaneously dissociable noncovalent protein aggregation and surface adsorption of rhGH present within the microspheres.

Acetates↗

PubMed1999

Conjugation of drug to poly(D,L-lactic-co-glycolic acid) for controlled release from biodegradable microspheres.

Poly(d,l-lactic-co-glycolic acid) (PLGA) was chemically conjugated to a model drug, N-(9-fluorenylmethoxycarbonyl-N-tert-butoxycarbonyl-l-tryptophan (Fmoc-Trp(Boc)) via an ester linkage. Various coupling reaction conditions were tested to optimize the conjugation process between a hydroxyl terminal group of PLGA and a carboxylic acid group of Fmoc-Trp(Boc). Two different lactic/glycolic acid compositions of PLGA (50/50 and 75/25) were used for the conjugation. The Fmoc-Trp(Boc)-PLGA conjugates were formulated into microspheres by a solvent evaporation technique for controlled release of Fmoc-Trp(Boc) over an one month period. A linear constant release of Fmoc-Trp(Boc) and its water-soluble PLGA oligomer conjugates was observed over an extended period without any initial burst effect, while unconjugated Fmoc-Trp(Boc) encapsulated within microspheres exhibited a rapid release profile. In addition, Fmoc-Trp(Boc) release rate solely depended on the PLGA composition that affected polymer degradation rate. The release rate of Fmoc-Trp(Boc) conjugated with fast degrading 50/50 PLGA was more rapid than that conjugated with relatively slow degrading 75/25 PLGA. This study demonstrates that PLGA-drug conjugation approach is a new and novel strategy to control the drug release rate from PLGA microspheres by utilizing the chemical degradation rate of PLGA backbone.

Biocompatible Materials↗