• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1209-1216
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• 2022

To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.

• 약학회지
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• 제40권6호
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• pp.705-712
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• 1996

We have previously shown that determination of glucose uptake using ${\alpha}$-methylglucose(${\alpha}$-MG) is very sensitive and rapid parameter for the assessment of loss of cellular fu nction in renal cell line($LLC-PK_1$). The present study was designed to elucidate the mechanism of inhibition of ${\alpha}$-MG uptake and the intracellular site of toxic action of cisplatin(CIS). $LLC-PK_1$ cells were exposed to various concentrations(5 ${\mu}$M-l00 ${\mu}$M) of CIS for 5 hrs or 24 hrs and ${\alpha}$-MG uptake was determined. Mitochondrial function was evaluated by measuring intracellular ATP content and MTT reduction. The activities of marker enzymes for the basolateral membrane(Na$^+$-K$^+$ ATPase) and brush border membrane (alkaline phosphatase: ALP) were also measured. CIS treatment significantly inhibited the ${\alpha}$-MG uptake in a time- and dose-dependent manner above 25 ${\mu}$M for 5 hrs. Intracellular ATP content and MTT reduction were affected by 24 hr-treatment of 50 ${\mu}$M CIS. The activities of Na$^+$-K$^+$ ATPase and ALP were significantly decreased at 10 ${\mu}$M and 5 ${\mu}$M of CIS for 24 hrs, respectively. The incubation with CIS for 5 hrs had no effects on the intracellular ATP content, MTT reduction and the activities of marker enzymes up to 100 ${\mu}$M. These results partly indicate that inhibition of ${\alpha}$-MG uptake by CIS may not be attributed to the disturbance of mitochondrial function or inhibition of the activity of Na$^+$-K$^+$ ATPase and can be resulted from direct effect of CIS on the Na$^+$/glucose cotransporter in brush border membrane. This study shows that additional mechanistic information, indicating the intracellular site of nephrotoxic action, can be gained by coupling the ${\alpha}$-MG uptake and ATP content or the activity of Na$^+$-K$^+$ ATPase.

• KSBB Journal
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• 제29권5호
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• pp.328-335
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• 2014

Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.

• Molecular & Cellular Toxicology
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• 제3권4호
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• pp.292-298
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• 2007

Zinc plays indispensable roles in metabolism, including cell growth, apoptosis, proliferation and differentiation. Kidneys are target organs for various regulators of mineral metabolism, and play a key role in zinc balance. To investigate the zinc uptake efficiency, we examined the zinc uptake and accumulation level in vivo and in vitro study. Plasma zinc concentration was peaked out at 1 hr after oral zinc administration. The renal zinc level was peaked out at 12 hr after oral zinc administration, and it was the highest in 40 mg/kg Zn-Asp administrated group in comparison with other groups. In addition, the m-RNA expression level of zinc transporter-1 (ZnT-1), zinc transporter-2 (ZnT-2) and high-affinity L-aspartate transporter (EAAT-3) in Zn-Asp administered group were increased compared with control groups and $ZnSO_4$ group. In order to investigate the intracellular zinc uptake mechanism, we performed the in vitro study by using human embryonic kidney cell line, HEK 293. Intracellular zinc level was peaked out at 3 hr after zinc treatment. In the same way, the mRNA expression level of ZnT-1 and EAAT-3 were increased compared with control group. This study showed that Zn-Asp is effective the zinc uptake into the kidney by increasing the zinc transporter expression.

• Journal of Pharmaceutical Investigation
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• 제38권3호
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• pp.163-169
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• 2008

Liposomes are spherical vesicles composed of lipid bilayer membranes. However, the conventional liposomes have been found to be plagued by rapid opsonization and taken up by the reticuloendothelial system (RES), resulting in shortened circulation time and limited intracellular uptake to target cell. In this study, polyethyleneglycol-cationic liposomes (PCL) containing cationic lipid and DSPE-mPEG were prepared by thin film cast-hydration method. The PEG liposomes had approximately $97.0{\pm}1.3\;nm$ of mean particle diameter and $-21.7{\pm}1.2\;mV$ of zeta potential value. PCL had $96.4{\pm}1.8\;nm$ of mean particle diameter and $-8.7{\pm}1.1\;mV$ of zeta potential value with a decrease of about 10 mV compared to the PEG liposomes. Loading of model drug, doxorubicin (DOX), in liposomes were carried out by using remote loading method and the loading efficiency of DOX in liposomes was about $95.0{\pm}1.9%$. Intracellular uptake and cytotoxicity of PCL were higher than that of PEG liposomes to murine B16F10 melanoma cells. In addition, anti-tumor activity of PCL was similar to that of PEG liposomes on growth of A549 human lung carcinoma in BALB/c mice. Consequently, PCL modified with cationic lipid may be applicable as anticancer drug carriers that can increase intracellular uptake and therapeutic efficacy.

• 대한의생명과학회지
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• 제11권3호
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• pp.301-305
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• 2005

MR spectroscopy of intracellularly located $^{133}Cs$ has been used to monitor the uptake of Gd-EOB-DTPA by the isolated rat liver. As shown by ${31}P$ spectroscopy, accumulation of $^{133}Cs$ ions in hepatocytes does not produce detectable effects on the metabolism. The hepatic internalization of Gd-EOB-DTPA was followed by the paramagnetic relaxation enhancement of the intracellular $^{133}Cs$ ions, and confirmed by parallel quantitations of Gd and Cs run by inductively coupled plasma analysis of liver samples and aliquots of perfusate. Two peaks are observed at -22.0 and -23.5 ppm, with respect to the line of the external reference arbitarily set to 0 ppm. Upon rinsing of the extracellular compartment with regular K-H free of CsCl, the high-field resonance disappears within 20min. The intracellular concentration was confirmed by ICP, which gives a $Cs^+$ content of $22.0\pm3.5mM$. The relaxation data significantly underestimate the Gd content, suggesting a potential compartmentation of $Cs^+$ and the contrast agent.

• Applied Biological Chemistry
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• 제43권3호
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• pp.163-168
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• 2000

토양 및 수계에 집적, 유입되어 있는 과다한 인산의 제거에 이용 가능한 미생물 자원을 확보하기 위하여 활성오니에서 분리된 Acinetobacter lwoffi PO8의 배양조건 및 외부환경조건에 따른 인산흡수 양상을 조사하였다. 배양액 내 초기 pH가 $7.5{\sim}8.5$ 범위에서 균생육과 인산흡수율이 가장 높았으며, 탄소원으로 glycerol 및 arabinose을 첨가하였을 경우 각각 약 93 및 91%의 높은 인산 흡수율을 보였다. 질소원 형태에 따른 인산흡수양상은 아미노태 보다 암모늄염이 효과적이었으며, 특히, $NH_4NO_3$,와 $(NH_4)_2SO_4$가 각각 95와 96%의 인산흡수율을 나타냈다. 금속이온 중에서 $Co^{2+}$ 첨가 시 균생육이 제해되었으나, 이외의 다른 금속이온은 균의 성장 및 인산흡수에 큰영향을 미치지 않았다. 아미노산 중 arginine, methionine 및 lysine 등은 아미노산을 첨가하지 않은 경우보다 $10{\sim}20%$ 더 높은 인산흡수율을 나타냈었으며, 이때 배양기간 중 glucose의 공급으로 배지 중 잔존 인산이 완전히 제거되었다. 또한 $^{32}P$를 사용하여 균의 인산 분포를 조사한 결과로 세포내 흡수된 인산은 대부분 세포의 원형질 내에 polyphosphate의 형태로 분포되어 있었다.

• Journal of Pharmaceutical Investigation
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• 제38권1호
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• pp.15-21
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• 2008

Liposomes as one of the efficient drug carriers have some shortcomings such as their short circulation time, fast clearance from human body by reticuloendothelial system (RES) and limited intracellular uptake to target cell. In this study, polyethylenglycol (PEG)-incorporated cationic liposomes were prepared by ionic complexation of positively charged liposomes with carboxylated polyethyleneglycol (mPEG-COOH). The cationic liposomes had approximately $98.6{\pm}1.0nm$ of mean particle diameter and $42.8{\pm}0.8mV$ of zeta potential value. The PEG-incorporated cationic liposomes had $110.1{\pm}1.2nm$ of mean particle diameter with an increase of about 10 nm compared to the cationic liposomes. Zeta potential value of them was $12.9{\pm}0.6mV$ indicating 30mV decrease of cationic charge compared to the cationic liposomes. The amount of PEG which was incorporated onto the cationic liposomes was assayed by using picrate assay method and the incorporation efficiency was $58.4{\pm}1.1%$. Loading efficiency of model drug, doxorubicin, into cationic liposomes or PEG-incorporated cationic liposomes was about $96.0{\pm}0.7%$. Results of intracellular uptake which were evaluated by flow cytometry analysis of doxorubicin loaded liposomes showed that intracellular uptake of PEG-incorporated cationic liposomes was higher than the cationic liposomes or DSPE-mPEG liposomes. In addition, cytotoxicity of PEG-incorporated cationic liposomes was comparable to cationic liposomes. Consequently, the PEG-incorporated cationic liposomes of which surface was incorporated with PEG by ionic complex may be applicable as anticancer drug carriers that can increase therapeutic efficacy.

• The Korean Journal of Physiology
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• 제30권1호
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• pp.63-76
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• 1996

The aim of present study was to characterize phosphate uptake and to investigate the mechanism for the insulin and insulin-like growth factor(IGF) stimulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells. Results were as follows : 1. The primary cultured proximal tubule cells had accumulated $6.68{\pm}0.70$ nmole phosphate/mg protein in the presence of 140 mM NaCl and $2.07{\pm}0.17$ nmole phosphate/mg protein in the presence of 140 mM KCl during a 60 minute uptake period. Raising the concentration of extracellular phosphate to 100 mM$(48.33{\pm}1.76\;pmole/mg\;protein/min)$ induced decrease in phosphate uptake compared with that in control cells maintained in 1 mM phosphate$(190.66{\pm}13.01\;pmole/mg\;protein/min)$. Optimal phosphate uptake was observed at pH 6.5 in the presence of 140 mM NaCl. Phosphate uptake at pH 7.2 and pH 7.9 decreased to $83.06{\pm}5.75%\;and\;74.61{\pm}3.29%$ of that of pH 6.5, respectively. 2. Phosphate uptake was inhibited by iodoacetic acid(IAA) or valinomycin treatment $(62.41{\pm}4.40%\;and\;12.80{\pm}1.64%\;of\;that\;of\;control,\;respectively)$. When IAA and valinomycin were added together, phosphate uptake was inhibited to $8.04{\pm}0.61%$ of that of control. Phosphate uptake by the primary proximal tubule cells was significantly reduced by ouabain treatment$(80.27{\pm}6.96%\;of\;that\;of\;control)$. Inhibition of protein and/or RNA synthesis by either cycloheximide or actinomycin D markedly attenuated phosphate uptake. 3. Extracellular CAMP and phorbol 12-myristate 13 acetate(PMA) decreased phosphate uptake in a dose-dependent manner in all experimental conditions. Treatment of cells with pertussis toxin or cholera toxin inhibited phosphate uptake. cAMP concentration between $10^{-6}\;M\;and\;10^{-4}\;M$ significantly inhibited phosphate uptake. Phosphate uptake was blocked to about 25% of that of control at 100 ng/ml PMA. 3-Isobutyl-1-methyl-xanthine(IBMX) inhibited phosphate uptake. However, in the presence of IBMX, the inhibitory effect of exogenous cAMP was not significantly potentiated. Forskolin decreased phosphate transport. Acetylsalicylic acid did not inhibit phosphate uptake. The 1,2-dioctanoyl-sn-glycorol(DAG) and 1-oleoyl-2-acetyl-sn- glycerol(OAG) showed a inhibitory effect. However, staurosporine had no effect on phosphate uptake. When PMA and staurosporine were treated together, inhibition of phosphate uptake was not observed. In conclusion, phosphate uptake is stimulated by high sodium and low phosphate and pH 6.5 in the culture medium. Membrane potential and intracellular energy levels are also an important factor fer phosphate transport. Insulin and IGF-I stimulate phosphate uptake through a mechanisms that involve do novo protein and/or RNA synthesis and decrease of intracellular cAMP level. Also protein kinase C(PKC) is may play a regulatory role in transducing the insulin and IGF-I signal for phosphate transport in primary cultured proximal tubule cells.

• Macromolecular Research
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• 제14권6호
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• pp.646-653
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• 2006

Highly hydrophilic, uniform, superparamagnetic and nontoxic maltotrionic acid (MA)-coated magnetite nano-particles (MAM) were prepared and characterized by TEM, DLS, XRD and VSM. MA was used to improve the biocompatibility, monodispersity and non-specific intracellular uptake of nanoparticles. Folic acid (FA) was subsequently conjugated to the MAM to preferentially target KB cells (cancer cells) that have folate receptors expressed on their surfaces and to facilitate nanoparticles in their transit across the cell membrane. Finally, fluorescence isothiocyanate (FITC) was added to the nanoparticles to visualize the nanoparticle internalization into KB cells. After the cells were cultured in a media containing the MAM and MAM-folate conjugate (FAMAM), the results of fluorescence and confocal microscopy showed that both types of nanoparticles were internalized into the cells. Nevertheless, the amount of FAMAM uptake was higher than that of MAM. This result indicated that nanoparticles modified with MA and FA could be used to facilitate the nanoparticle uptake to specific KB cells (cancer cells) for molecular imaging.