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Biomedical subjects

K J Hwang

Publications and source records attributed to K J Hwang.

At least 19 recordsLinked to original sources
PubMed1995

Studies using fluorescent tetrahydrochrysene estrogens for in situ visualization of the estrogen receptor in living cells.

We have analyzed four fluorescent nonsteroidal estrogens for their potential to serve as vital cytological stains to visualize the estrogen receptor (ER) in a model receptor expression system. The novel estrogen fluorophores are based on the rigidified stilbene-like structure of 5,6,11,12-tetrahydrochrysene (THC), and they embody electron-donor (hydroxyl) and electron-acceptor groups (nitrile, amide, ester, or ketone) that afford efficient, long wavelength, and environment-sensitive fluorescence. These probes bind with high affinity to human ER (relative binding affinity, 22-85 vs. estradiol, 100), and they stimulate the transcriptional activity of this receptor. The strong fluorescence of the estrogenic THCs permits visualization, using conventional epifluorescence microscopy, of ER in transfected Cos-7 cells that express elevated levels of receptor. Cell staining by the donor-acceptor THCs characteristically displays a nonuniform pattern of nuclear fluorescence that can be fully inhibited by nonfluorescent estrogens such as estradiol or diethylstilbestrol. Additionally, this staining appears to be specific for ER, since it coincides with the distribution of receptor as determined by indirect immunofluorescence analysis using an ER-specific monoclonal antibody. Using these probes, we have analyzed the intracellular distribution of ER mutants containing a variety of deletions. Evidence is presented to show that removal of amino-terminal sequences within the ER polypeptide results in an altered pattern of intranuclear distribution with preferential accumulation of receptor protein within the nucleolus. These THC fluorophores therefore represent excellent probes for cytological studies involving ER expressed in cultured cells and represent an important advance toward the goal of exploiting fluorescence technology to analyze the expression and distribution of ER within tissue samples.

Animals↗
PubMed1994

Identification of Borrelia burgdorferi isolated in Korea using outer surface protein A (OspA) serotyping system.

Two characteristic strains (935T, 934U) of B. burgdorferi isolated from Ixodes persulcatus and a wild rodent (Apodemus agrarius) in Korea were selected and analyzed by an immunoblot method using the monoclonal antibodies directed to different epitopes of outer surface protein A (OspA). The reactive pattern of strain 934U with these monoclonal antibodies was identical to that of strains belonging to B. afzelii and that of strain 935T was different from other isolates. Monoclonal antibody (5TEE3) which is specific to strain 935T did not react with any other Western and Japanese isolates. So, it was suggested that there exist at least two groups of B. burgdorferi in Korea. One could be classified as B. afzelii and the other is a divergent group from three known species of B. burgdorferi sensu stricto, B. garinii and B. afzelii.

Animals↗
PubMed1992

Donor-acceptor tetrahydrochrysenes, inherently fluorescent, high-affinity ligands for the estrogen receptor: binding and fluorescence characteristics and fluorometric assay of receptor.

We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-11 positions, is required for the highest relative binding affinity (RBA), and the trans-5,11-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NO2) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (phi f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (approximately 10(-9) M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal-dextran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when bound to ER, or when bound to non-receptor proteins. It is also the first demonstration of ER assay by fluorescence under equilibrium conditions.

Animals↗
PubMed1992

A sensitive fluorometric assay for reducing sugars.

A simple and rapid fluorometric assay for reducing sugars that is sensitive to the nanomolar range has been developed. The assay involves the derivatization of a given sugar with hydrazine at pH 3 to form a hydrazone, which is reacted with fluorescamine following adjustment of pH to first 9.4 and then 7.4. The amount of sugar in a sample is quantitated by measuring the fluorescence intensity at an excitation wavelength of 400 nm and an emission wavelength of 490 nm. The assay is precise and reproducible, as indicated by intra- and inter-run variations of at most 3% and 4%, respectively. In addition to reducing sugars, the assay can also be used to measure aliphatic and aromatic aldehydes, but not acetone. Compared with an existing fluorometric sugar assay, the assay reported here does not require chromatographic separation of the fluorescent derivative from unreacted fluorescamine. The assay can, however, be potentially adapted for postcolumn detection of aldehydes, reducing sugars, and hydrazones in HPLC.

Aldehydes↗
PubMed1989

Targeting small unilamellar liposomes to hepatic parenchymal cells by dose effect.

A major research goal of liposome pharmacology is the selective delivery of drugs to target cell populations while minimizing extraction by phagocytic macrophages and blood monocytes of the reticuloendothelial system. The liver is an ideal organ for studying targeting strategies using a variety of liposomes, inasmuch as its discontinuous capillaries have fenestrae through which liposomes less than 0.2 microns in diameter may escape into the extravascular space. In a previous kinetic study, we proposed that the hepatic uptake of small unilamellar vesicles (SUV) in mice was compatible with a model of uptake involving dual, parallel pathways. One is a saturable, phagocytic pathway of uptake mediated by Kupffer cells, the other is a nonsaturable, pinocytotic pathway of uptake mediated by parenchymal cells, favoring the latter pathway at high liposomal dose (Beaumier et al., 1983). In the present study, we demonstrated by the techniques of liver cells fractionation that the uptake of either the bovine brain sphingomyelin/cholesterol (2:1; mole/mole) SUV or distearoyl phosphatidylcholine/cholesterol (2:1; mole/mole) SUV by hepatic parenchymal cells was enhanced markedly by increasing the amount of injected dose of SUV. As high as 85 to 90% of the total liver dose can be attributed to the uptake of SUV by the hepatic parenchymal cells alone, when the injected dose reaches at or above 7.5 to 10 micrograms of lipid per g b.wt. The dose effect on the uptake of liposomes by hepatocytes appears to be a general phenomenon of neutral SUV. Our data suggested that blockade by dose permits a feasible approach to target SUV to hepatic parenchymal cells.

Animals↗
PubMed1988

Preparation of liposomes entrapping a high specific activity of 111In3+-bound inulin.

Targeting liposomes to specific tissues or cells require the unequivocal determination of the uptake of liposomes at the cellular level. The present report describes the preparation of liposomes entrapping a high specific activity of 111In3+-bound inulin, and the potential applications of a multiple labeling technique for characterizing the extent of uptake of liposomes by tissues or different cells in a given tissue in vivo. The labeling method involves the application of the technique of acetylacetone-mediated, ionophoric loading of 111In3+ into liposomes entrapping an inulin derivative to which a strong chelating agent, diethylenetriamine-pentaacetic acid (DTPA), is bound. Subsequent ionophoric removal of the weakly bound 111In3+ by incubating the previously 111In3+-loaded liposomes with 10 mM nitrilotriacetic acid and 100 microM tropolone at room temperature for 20 min results in the preparation of liposomes entrapping 111In3+-DTPA-inulin. Our method of preparation yields net efficiencies of converting 63-78% of the externally added 111In3+ to liposome-entrapped 111In3+-DTPA-inulin.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Effect of lipid composition on insulin-mediated fusion of small unilamellar liposomes: a kinetic study.

Liposome-entrapped insulin could be used to prolong the hypoglycemic action of insulin. Also, the conjugation of insulin to the surface of liposomes allows the potential application of using insulin as a transporting molecule to deliver liposome-entrapped drugs to insulin-receptor rich tissues. The success of these two approaches of drug delivery depends on how insulin may interact with liposomes. The present study describes the application of the principle of kinetics to investigate the effect of insulin on the stability of various preparations of liposomal drug carriers. The technique of fluorescence resonance energy transfer was employed to study the destabilization of liposomal formulations through the process of insulin-mediated fusion of liposomes. The kinetics of insulin-mediated fusion appeared to be compatible with a model whereby the initial rate of fusion is governed by the mechanism of fusion of two small unilamellar, unfused liposomal particles. The rate constants of insulin-mediated fusion of various liposomal formulations were estimated from the initial rate of fusion, using the model of two-particle fusion. Arrhenius analysis of the rate constants of fusion at different temperatures suggests that the mechanism of insulin-mediated fusion of small unilamellar vesicles is not governed merely by the energy and frequency of collision between liposomal particles. Other factors, such as the binding of insulin with the surface of liposomes and the temperature effect on the dynamics of the liposomal membrane, as well as conformation of insulin, could potentially be important.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Synthesis of diethylenetriaminepentaacetic acid conjugated inulin and utility for cellular uptake of liposomes.

The synthesis, binding of radioactive cations, liposomal encapsulation, and biodistribution of the oxidized-inulin reaction product with ethylenediamine and diethylenetriaminepentaacetic acid (4) are described. The four-step synthesis of the inulin derivative proceeded in a good overall yield of 72%. The complex of the inulin derivative with either 67Ga3+ or 111In3+ was stable in vivo and did not readily distribute into tissues, being excreted primarily in urine after intravenous administration to mice. The liposome-entrapped inulin derivative can be loaded with radioactive heavy metal cations by mobile ionophores in high radiochemical yields of 80-91%. Following the intravenous administration of the liposomal encapsulation of the indium-111-labeled inulin derivative, the entrapped compound had a biodistribution characteristic of liposomes and allowed an estimation of the extent of the intracellular uptake of liposomes. The ability of the inulin derivative to chelate many different types of metals will allow the use of this probe for studying subtle differences in tissue distribution resulting from different drug targeting or delivery protocols in the same animal by multiple labeling techniques. Moreover, the chelate-conjugated inulin permits studies of the applications of drug delivery systems in primates or human subjects by noninvasive techniques such as gamma-scintigraphic or nuclear magnetic resonance imaging methods.

Animals↗
PubMed1987

Uptake of small liposomes by non-reticuloendothelial tissues.

The distribution of liposomes within the intravascular space and the extent to which they escape into extravascular space strongly impact on the application of lipid vesicles as a carrier for pharmacologically active agents. The present study investigates how intact small unilamellar vesicles (SUV) may be taken up by different tissues after intravenous injection into mice, using various types of SUV with different entrapped markers, lipid composition, size, doses of liposomal lipids and stability in the blood. Our focus was specifically on sphingomyelin (or distearoyl phosphatidylcholine)/cholesterol (2:1, mol/mol) SUV, which are known to be stable in the blood circulation. Our results indicated that, in addition to the reticuloendothelial tissues, intact SUV were taken up in several other parts of the body, including intestine, skin, carcass and legs. It appears that the accumulation of SUV in the intestine and the skin increases with time post-injection. Furthermore, from the kinetic data, the process of uptake of SUV by the skin and intestine is compatible with a non-saturable pathway, which follows first-order kinetics. This suggests that the cells involved in the uptake of SUV in the intestine and skin are not phagocytic cells, which are normally saturable.

Animals↗
PubMed1987

Mechanism of ionophoric transport of indium-111 cations through a lipid bilayer membrane.

The use of mobile ionophores to facilitate the transport of 111In through a lipid bilayer membrane has broad applications in liposome technology and cell labeling. However, the mechanism of such ionophore-mediated transport of 111In through a lipid bilayer membrane is not completely clear. The present report describes the correlations of the behaviors of ionophoric loading of 111In into liposomes with the lipophilicity and the indium-binding affinity of three ionophores, namely, 8-hydroxyquinoline, acetylacetone, and tropolone. Our results suggest that the mechanism of the ionophoric transport of 111In through a lipid bilayer membrane involves the rapid exchange of 111In cations among the ionophores in both the aqueous solution and the lipid bilayer. Furthermore, the effectiveness of an ionophore in facilitating the transport of 111In from the external aqueous compartment to the entrapped nitrilotriacetic acid depends not only on the lipophilicity of the [111In]ionophore complex, but also on the lipophilicity of the free ionophore itself and the competition of 111In between nitrilotriacetic acid inside the inner aqueous compartment of the liposome and the ionophore imbedded in the lipid bilayer membrane of the liposome.

Animals↗
PubMed1986

Peptide-carrier interaction: induction of liposome fusion and aggregation by insulin.

As the roles of peptidic agents in therapy expand, the need for gaining the knowledge for formulating peptides and/or polypeptides becomes increasingly urgent. In an attempt to study various approaches to formulating peptidic agents for therapeutic applications, we investigated the interactions between drug carriers and peptides, using liposomes and insulin as a model. The fusion and aggregation properties of dipalmitoylphosphatidylcholine (DPPC) small, unilamellar liposomes, on the binding of insulin was studied by the techniques of resonance energy transfer of fluorescent labeled lipids, electron microscopy, and right-angle scattering. Within 1 h of adding insulin to DPPC liposomes at 25 degrees C, the average size of the liposomes increased from 239 to 361 A in diameter. There was no further increase in the size of the liposomes after the fused liposomes reached this size. However, the aggregation of the fused liposomes continued to increase for several hours after the insulin-induced fusion stopped. Our results suggest that insulin induces the aggregation of newly fused liposomes, when the temperature is below the gel----liquid crystalline phase-transition temperature (Tc) of the liposomes. The aggregation of fused liposomes is markedly affected by not only the zinc content of insulin but also the pH and ionic strength of the solution. The results of the present study demonstrate that an amphyphilic molecule, such as insulin, could induce the fusion of liposomes via hydrophobic interaction and facilitate liposome aggregation via hydrophilic interaction. Thus, when entrapping insulin by small, unilamellar liposomes, care should be taken to avoid fusion and aggregation.

Chemistry, Pharmaceutical↗
PubMed1986

Application of anion-exchange resin to remove lipophilic chelates from liposomes.

Lipophilic chelates such as 8-hydroxyquinoline, acetylacetone, and tropolone are useful to load high levels of radioactive cations into the inner aqueous compartments of liposomes for investigating the fate of liposomes by the technique of gamma imaging or gamma-ray perturbed angular correlation measurements. However, if lipophilic chelates are not completely removed from liposomes the very same lipophilic chelates can also cause leakage of the entrapped cations from liposomes. Thus, it is essential to make sure that all the lipophilic chelates are removed from liposomes after the loading process. The results of the present study show that more than 99.85% of acetylacetone in liposomal suspension can be removed by a minicolumn of AG1-X8 (phosphate form) anion exchange resin. Virtually all the 8-hydroxyquinoline and tropolone in liposomal suspension are adsorbed tightly to the resin. The procedure is rapid, and the dilution of liposomes is minimal. For experiments involving high levels of gamma-emitting radionuclides, the cleaning up process of removing lipophilic chelates from liposomes can be conveniently operated behind a lead glass.

Chelating Agents↗
PubMed1983

Effects of liposome size on the degradation of bovine brain sphingomyelin/cholesterol liposomes in the mouse liver.

The relative rates of degradation of the outer lipid bilayer of large multilamellar and small unilamellar bovine brain sphingomyelin/cholesterol (2:1; mol/mol) liposomes in the livers of Balb/c mice were compared. The rate of the release of entrapped In-111 ions from the aqueous reservoir of small unilamellar liposomes or from the outermost aqueous compartment of multilamellar liposomes was used to monitor the rate of degradation of the exterior lipid bilayer surface of these liposomes. The technique of gamma-ray perturbed angular correlation and a method for loading In-111 ions into the outermost aqueous compartment of liposomes were used in this investigation. It was found that in the liver the exterior lipid bilayer of large multilamellar liposomes was degraded more rapidly than the bilayer of small unilamellar liposomes in vivo. In contrast to the situation for small unilamellar liposomes, the degradative process for large multilamellar liposomes in the liver was not maintained under ischemic conditions. Our results suggest that multiple pathways operate in the degradation of liposomes in the liver. The rate of degradation of liposomes in the liver may depend on accessibility of liposomes to degradative sites.

Animals↗
PubMed1983

Effect of liposome dose on the elimination of small unilamellar sphingomyelin/cholesterol vesicles from the circulation.

The effect of the liposome dose of bovine brain sphingomyelin/cholesterol (2/1; mol/mol) small unilamellar vesicles (mean diameter 187 42A) on the rate of elimination of the vesicles from the circulation of mice was investigated. The results of the study indicated that the relative rate of elimination of the vesicles from the blood depended on the amount of intravenously administered liposomal lipid. The distribution of the liposomes in vivo was followed by monitoring entrapped In-111. In all tissues examined, the uptake of the liposomes was a dose-dependent process. An examination of the dose dependency of the distribution of the administered liposomes in the blood and liver at 23 hours post-injection, and of the kinetics of the elimination of these vesicles from the blood, suggests a hepatic uptake process involving two parallel pathways. One pathway is apparently a capacity-limited Michaelis-Menten process; the other pathway is a linear, non-saturable process. These pathways operate in parallel but respectively dominate at the low end and the high end of the dose range examined.

Animals↗
PubMed1982

Volume of distribution and transcapillary passage of small unilamellar vesicles.

This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/1; M/M) small unilamellar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapillary passage of the SUV. The complex of nitrilotriacetic acid with In-111 or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that initially the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours.

Animals↗
PubMed1982

Encapsulation, with high efficiency, of radioactive metal ions in liposomes.

The encapsulation of radioactive metalic cations, such as 111In3+ or 67Ga3+, in the internal aqueous compartment of liposomes can be achieved with an efficiency of about 90%. The efficient loading of a high specific activity of cations into liposomes involves the transport of 111In3+ or 67Ga3+ through the lipid bilayer to an encapsulated strong chelate, such as nitrilotriacetic acid, by 8-hydroxyquinoline, in conjunction with an efficient anion-exchange resin technique for the removal of the external cations. The efficiency of loading cations to liposomes is affected markedly by the concentration of 8-hydroxyquinoline-metal, and the presence of the chelating agents in the loading incubation mixture. However, the loading efficiency is not affected by the pH of the internal aqueous compartment of liposomes over a range of pH 5-9, the concentration of the liposomes, the method of liposomal preparation, the lamellar structure of the liposomes, and the composition of liposomes. Furthermore, the loading procedures do not appear to affect the size and the permeability of liposomes. There is a good agreement in the tissue distributions of the liposomes prepared by the present loading methods and those by the conventional method of encapsulation by sonication. Liposomes entrapping high specific activity of 67Ga3+ or 111In3+ will be useful for future studies of the in vivo kinetics of liposomes by the combined techniques of scintigraphic imaging and the gamma-ray perturbed angular correlation.

Animals↗