• Title/Summary/Keyword: Cell-based assay

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Downregulatory Effect of Extracts from Mistletoe (Viscum album) and Pueraria Root (Pueraria radix) on Cellular NF-κB Activation and heir Antioxidant Activity (겨우살이(Viscum album)와 칡뿌리(Pueraria radix) 추출물의 NF-κB활성 억제 및 항산화 효과)

  • Song, Hee-Sun;Park, Yeon-Hee;Kim, Seung-Kyoon;Moon, Won-Kuk;Kim, Dong-Woo;Moon, Ki-Young
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.10
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    • pp.1594-1600
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    • 2004
  • Effects of mistletoe (Viscum album) extract and pueraria (Pueraria radix) extract on cellular NF-$textsc{k}$B activity were evaluated in human malignant keratinocytes (SCC-13) to elucidate the possible correlation of NF-$textsc{k}$B with antioxidant activity. The antioxidant activities of these natural extracts were examined in four different evaluation methods, i.e., lipid peroxidation value (POV) evaluation test, I,l-diphenyl-2-picrylhydrazyl radical (DPPH), nitric oxide (NO) scavenging test, and reducing power assay. Pueraria extract (0.5 mg) and mistletoe extract (5 mg) downregulated the cellular NF-$textsc{k}$B activation up to 35% and 10% compared to the control, respectively, although their effects were lower than the known NF-$textsc{k}$B downregulator, vitamin C (8.8 mg, 53%) in a cell-based NF-$textsc{k}$B activity assay system. In the POV test, relative antioxidant activities of mistletoe extract (86%) and pueraria extract (75%) were significantly higher than the known antioxidant, vitamin C (48%) at the same concentration (10 mg) and the degree of activity increased in a dose-dependent manner. Pueraria extract showed more potential radical scavenging activities than those of mistletoe extract evaluated in both DPPH and NO test. Especially, the NO radical scavenging activity of pueraria extract ($SC_{50}$/, 88 $\mu$g) was comparable to that of vitamin C ($SC_{50}$/, 77 $\mu$g). Even pueraria extract possessed a much less reducing power compared to vitamin C, it also revealed higher reducing power than that of mistletoe extract. These results indicate that mistletoe extract and pueraria extract may serve as an useful natural antioxidant agents, and led to suggest the hypothesis that compounds having an antioxidant activity, i.e., radical scavenging activity or reducing power may be correlated with the downregulation of NF-$textsc{k}$B activation in human keratinocytes.

Urine endothelin-1 as a Biomarker for Urinary Tract Infections in Children (Urine endothelin-1의 소아 요로 감염 환아에서의 진단적 유용성)

  • Kee, Hyung Min;Yi, Dae Yong;Yun, Ki Wook;Lim, In Seok;Ha, Tae-Seon
    • Childhood Kidney Diseases
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    • v.18 no.1
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    • pp.36-41
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    • 2014
  • Purpose: Urinary tract infections (UTIs) are the most common source of bacterial infections in infants and young children. Accurate diagnosis and treatment is important because of their association with renal scarring, which can lead to complications. Urine endothelin-1 (ET-1) is the major renal isoform produced and released by renal mesangial cells in response to glomerular injury. This study aimed to investigate whether urinary levels of ET-1 can be used as a biomarker for UTI diagnosis. Method: We conducted a prospective study using medical records of 70 patients below the age of 18 years, who visited Chung-Ang University Hospital from July 2012 to July 2013. We classified the patients into the UTI and control groups based on urine culture studies. The UTI group was further divided into upper and lower UTI groups using 99m-Technetium dimercaptosuccinic acid scintigraphy. Urine ET-1 was measured using enzyme linked immunosorbent assay with 0.3 mL urine. Results: The UTI and control groups were comprised of 45 and 25 patients, respectively. Mean urine ET-1 levels were significantly higher in the UTI group than in the control group ($1.41{\pm}0.35$ pg/mL vs. $0.33{\pm}0.07$ pg/mL, P =0.04). There was no significance difference in the quantitative value between the upper and lower UTI groups (P =0.552). There was no correlation between urine ET-1 and serum C-reactive protein (Pearson correlation [R]=0.24), urine ET-1 and serum white blood cell count (R=0.19). Conclusion: Our study suggests that urine ET-1 can be used for early diagnosis of UTI in children.

Improving effect of psoriasis dermatitis by yakuchinone A in the TNF-α stimulated HaCaT cells (TNF-α 자극에 활성화된 HaCaT 세포주에서 Yakuchinone-A에 의한 건선 피부염 개선 효과)

  • Kim, Min Young;Hwang, Hyung Seo
    • Journal of Applied Biological Chemistry
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    • v.63 no.1
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    • pp.95-101
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    • 2020
  • Psoriasis is an autoimmune skin disease that is accompanied by hyper proliferation of the epidermis, erythema of various sizes, and ulceration. However, the mechanism of the development of psoriasis dermatitis is unclear. Recently, it is known that the inflammatory cytokines and Th17 cells as well as chemokine (CC motif) ligand 20 (CCL20) are involved in the process of keratinocytes hyper-differentiation, which is common in psoriasis dermatitis. Therefore, we studied the effects of yakuchinone-A, an active ingredient of Alpinia oxyphylla Miquel known for its anti-inflammatory activity, to improve psoriasis dermatitis. First, cytotoxicity of yakuchinone-A was observed in cell counting kit-8 assay and not observed in 10 ㎍/mL concentration on the human keratinocyte HaCaT cells. Yakuchinone-A in the presence of tumor necrosis factor-alpha (TNF-α) on HaCaT cells inhibited mRNA expression of IL-6, IL-8, and TNF-α by up to 61.4±7.5, 23.6±1.5, 46.0±4.8%. CCL20, a chemokine that attracts immune cells such Th17 cells to the inflammation location, was also significantly suppressed by yakuchinone-A. In addition, IκB and STAT3 phosphorylation involved in the CCL20 expression was inhibited by yakuchinone-A in a concentration-dependent manner up to the level of 79.1±5.0, 80.8±2.3%. Furthermore, yakuchinone-A downregulated CCL20 mRNA expression level on IL-17A-activated HaCaT cells as a concentration-dependent manner. Based on these results, yakuchinone-A is expected to be developed as a new material for improving psoriasis dermatitis in the future.

LC-MS/MS analysis and anti-inflammatory effects of crude extract from Coptidis Rhizoma (황련 추출물의 LC-MS/MS 분석 및 항염증 효과)

  • Min-Jung, Kim;Ye-Jin, Yang;Kwang-Youn, Kim;Hun Hwan, Kim;Jae Dong, Son;Ju-Hye, Yang;Dong bin, Lee;Woo Hyun, Kim;Hu-Jang, Lee;Seon Been, Bak;Kwang-Il, Park
    • Herbal Formula Science
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    • v.31 no.1
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    • pp.1-10
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    • 2023
  • Objectives : The main aim of this study was to examine the LC-MS/MS used to identify phenolic compounds of CRE(Coptidis Rhizoma 70% EtOH Extract). Also, we investigated antioxidative activities and Anti-inflammatory activities. Methods : LC-MS/MS Analysis HPLC and LC-MS/MS were performed on a 1260 series HPLC system (Agilent Technologies, Inc., California, USA) and 3200 QTrap tandem mass system (Sciex LLC) operated in positive ion mode (spray voltage set at -4.5 kV). The solvent used was DW and Acetonitrile containing 0.1% formic acid, a gradient system was used at a flow rate of 0.5 mL/min for analysis, and a Prontosil C18 column (length, 250 mm; inner diameter, 4.6 mm; particle size, 5 ㎛; Phenomenex Co., Ltd., California, USA, Biochoff Chromatography) was used. The solvent conditions used in the mobile phases were 0-10 min at 10-15% B, 10-20 min at 20% B, 20-30 min at 25%, 30-40 min at 40%, 40-50 min at 70%, 50-60 min at 95%, and 60-70 min at 95%. The analysis was performed at a wavelength of 284 nm and a temperature of 35℃. The cell viability was measured using a 3-(4,5-dimethyethiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. We examined the effects of CRE on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in a RAW 264.7 cells Results : The chemical analysis CRE by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that Rosmarinic acid, Ferrulic acid, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid as phenolic components. DPPH radical scavenging activity was the inhibitory activity of CRE showed at 200 ㎍/mL a statistically significant level. MTT assay demonstrated that the CRE did not have a cytotoxic effect in RAW 264.7 and LPS-induced RAW264.7 cells. Also, CRE reduced NO production in RAW 264.7 cells stimulated with LPS. Conclusions : Based on these findings, The chemical analysis 4 major components CRE such as Rosmarinic acid, Ferrulic acid, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid. Moreover, we confirmed that CRE has effects antioxidant and anti-inflammatory. The results demonstrate that CRE can be used as an antioxidant and a powerful chemopreventive ingredient against inflammatory diseases.

Neuronal Cell Protective Effect of Dendropanax morbifera Extract against High Glucose-Induced Oxidative Stress (High Glucose로 유도된 산화 스트레스에 대한 황칠나무 잎 추출물의 뇌신경세포 보호 효과)

  • Kim, Jong Min;Park, Seon Kyeong;Guo, Tian Jiao;Kang, Jin Yong;Ha, Jeong Su;Lee, Du Sang;Kwon, O-Jun;Lee, Uk;Heo, Ho Jin
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.45 no.7
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    • pp.938-947
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    • 2016
  • Antioxidant activities and neuroprotective effects of ethyl acetate fraction from Dendropanax morbifera (EFDM) against high glucose-induced oxidative stress and neurotoxicity were investigated to confirm their physiological activities. An 80% ethanolic extract of D. morbifera showed the highest contents of total phenolic compounds as well as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities. The extract was fractionated using several solvents, and the ethyl acetate fraction showed the highest activities in ferric reducing/antioxidant power and malondialdehyde inhibitory assays. To evaluate the neuroprotective effect based on antioxidant activities, cell viability was assessed using PC12 and MC-IXC cells in $H_2O_2$- and high glucose-induced cytotoxic assays, respectively. EFDM evidently showed neuroprotective effects in all cells (neuron-like PC12 cells and human brain-originated neuroblastoma MC-IXC cells). Inhibitory effect of the extract on acetylcholinesterase (AChE) as an acetylcholine-hydrolyzing enzyme was performed to examine the effect on cognitive function. EFDM presented an AChE inhibitory effect. Finally, high-performance liquid chromatography analysis showed that the major phenolic compound of EFDM is probably a rutin.

An Investigation of Glyceollin I's Inhibitory Effect on The Mammalian Adenylyl (글리세올린 I의 아데니닐 고리화 효소 활성 억제 효능과 결합 부위 비교 분석)

  • Kim, Dong-Chan;Kim, Nam Doo;Kim, Sung In;Jang, Chul-Soo;Kweon, Chang Oh;Kim, Byung Weon;Ryu, Jae-Ki;Kim, Hyun-Kyung;Lee, Suk Jun;Lee, Seungho;Kim, Dongjin
    • Journal of Life Science
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    • v.23 no.5
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    • pp.609-615
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    • 2013
  • Glyceollin I has gained attention as a useful therapy for various dermatological diseases. However, the binding property of glyceollin I to the mammalian adenylyl cyclase (hereafter mAC), a critical target enzyme for the down-regulation of skin melanogenesis, has not been fully explored. To clarify the action mechanism between glyceollin I and mAC, we first investigated the molecular docking property of glyceollin I to mAC and compared with that of SQ22,536, a well-known mAC inhibitor, to mAC. Glyceollin I showed superiority by forming three hydrogen bonds with Asp 1018, Trp 1020, and Asn 1025, which exist in the catalytic site of mAC. However, SQ22,536 formed only two hydrogen bonds with Asp 1018 and Asn 1025. Secondly, we confirmed that glyceollin I effectively inhibits the formation of forskolin-induced cAMP and the phosphorylation of PKA from a cell-based assay. Long term treatment with glyceollin I had little effect on the cell viability. The findings of the present study also suggest that glyceollin I may be extended to be used as an effective inhibitor of hyperpigmentation.

Photoprotection and Anti-inflammatory Effects of Chinese Medical Plants (약용식물추출물의 광보호 효과와 항염증 효과 연구)

  • Jin-Hwa, Kim;Sung-Min, Park;Gwan-Sub, Sim;Bum-Chun , Lee;Hyeong-Bae, Pyo
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.2
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    • pp.227-233
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    • 2004
  • Chronic exposure to solar radiation, particularly ultraviolet (UV) light, causes a variety of adverse reactions on human skin, such as sunburn, photoaging and photocarcinogenesis. Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental facts play critical roles in cellular damage. And, repeated-UV irradiation activated the expression of the matrix metalloproteinase (MMP) and induced skin irritation. Therefore, the development of effective and safe photoprotectants that can reduce and improve the skin damage has been required. The purpose of this study was to investigate the photo-protective effect of several chinese medical plants (Juniperus chinensis) on the UV -induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. UVA induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. Expression of prostaglandin E$_2$ (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay(EIA) using PGE$_2$ monoclonal antibody. In the human skin we tested anti-irritation effect on the SLS-induced damage skin after appling the extract containing emulsion. We found that Juniperus chinensis extract had potent radical scavenging effect by 98% at 100$\mu\textrm{g}$/mL. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97% at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79% at 25$\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. In the test of proinflammatory cytokines of human keratinocytes Juniperus chinensis extract decreased expression of interleukin 6 about 30%. The amount of PGE$_2$ by HaCaT keratinocytes was significantly increased at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB (p < 0.05). At the concentrations of 3.2-25$\mu\textrm{g}$/mL of this extract, the production of PGE$_2$ by HaCaT keratinocytes (24 h after 10mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p < 0.05). In SLS-induced skin irritation model in vivo, we found to reduce skin erythema and improve barrier recovery after appling Juniperus chinensis extract containing emulsion when compared to irritated non-treated and placebo-treated skin. Our results suggest that Juniperus chinensis extract can be effectively used for the prevention of UV and SLS-induced adverse skin reactions and applied as anti-aging and anti-irritation cosmetics.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Effects of Fermented Soybean Paste Chungkukjang on the Immunoreactivity in Ovariectomized Mice (청국장이 난소적출 마우스의 면역 기능에 미치는 영향)

  • Park, Hyunjin;Yoon, Leena;Kim, Hyun-Sook
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.42 no.12
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    • pp.1930-1939
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    • 2013
  • It has been reported that Chungkukjang, one of Korean traditional fermented soybean products, may improve hypertension, diabetes, and hyperlipidemia. In this study, we sought to investigate the immunoenhancing effects of Chungkukjang in ovariectomized mice. For the first period, female SLC ddy mice were either sham-operated (Sham; n=27) or ovariectomized (OVX; n=27). As a basal diet, ovariectomized mice were fed low-calcium diet for faster induction of osteoporosis for six weeks, and those in the Sham group were fed AIN-76 diet. For the second period, half of the OVX group (n=9) and the Sham group (n=9) were fed a Chungkukjang-based diet (CKJ); whereas the other half (OVX; n=9/ Sham; n=9) were fed a casein-based diet (CSI) for 8 weeks. After a second period, we collected the blood via heart puncture and measured the splenocytes proliferation, T lymphocyte subsets by flowcytometry, and levels of serum cytokines (IL-2, IL-4, IL-6, IL-10, IFN-${\gamma}$ and TNF-${\alpha}$) by ELISA assay. The OVX+CKJ group showed higher splenocytes proliferation, higher ratio of CD4/CD8, and lower levels of IL-6 and TNF-${\alpha}$ cytokines compared to the OVX+CSI group. The Sham+CKJ group showed cytokine productions, such as higher levels of IL-10 and IFN-${\gamma}$, and lower levels of IL-6 and TNF-${\alpha}$ compared to the Sham+CSI group. The result of this study suggests that Chungkukjang may lower the proinflammatory cytokine levels in both the OVX and Sham groups. In addition, Chungkukjang could make a balance of T cell subset proliferations and enhance the splenocyte proliferations in the OVX group.

Development of High Intensity Focused Ultrasound (HIFU) Mediated AuNP-liposomal Nanomedicine and Evaluation with PET Imaging

  • Ji Yoon Kim;Un Chul Shin;Ji Yong Park;Ran Ji Yoo;Soeku Bae;Tae Hyeon Choi;Kyuwan Kim;Young Chan Ann;Jin Sil Kim;Yu Jin Shin;Hokyu Lee;Yong Jin Lee;Kyo Chul Lee;Suhng Wook Kim;Yun-Sang Lee
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.9 no.1
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    • pp.9-16
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    • 2023
  • Liposomes as drug delivery system have proved useful carrier for various disease, including cancer. In addition, perfluorocarbon cored microbubbles are utilized in conjunction with high-intensity focused-ultrasound (HIFU) to enable simultaneous diagnosis and treatment. However, microbubbles generally exhibit lower drug loading efficiency, so the need for the development of a novel liposome-based drug delivery material that can efficiently load and deliver drugs to targeted areas via HIFU. This study aims to develop a liposome-based drug delivery material by introducing a substance that can burst liposomes using ultrasound energy and confirm the ability to target tumors using PET imaging. Liposomes (Lipo-DOX, Lipo-DOX-Au, Lipo-DOX-Au-RGD) were synthesized with gold nanoparticles using an avidin-biotin bond, and doxorubicin was mounted inside by pH gradient method. The size distribution was measured by DLS, and encapsulation efficiency of doxorubicin was analyzed by UV-vis spectrometer. The target specificity and cytotoxicity of liposomes were assessed in vitro by glioblastoma U87mg cells to HIFU treatment and analyzed using CCK-8 assay, and fluorescence microscopy at 6-hour intervals for up to 24 hours. For the in vivo study, U87mg model mouse were injected intravenously with 1.48 MBq of 64Cu-labeled Lipo-DOX-Au and Lipo-DOX-Au-RGD, and PET images were taken at 0, 2, 4, 8, and 24 hours. As a result, the size of liposomes was 108.3 ± 5.0 nm at Lipo-DOX-Au and 94.1 ± 12.2 nm at Lipo-DOX-Au-RGD, and it was observed that doxorubicin was mounted inside the liposome up to 52%. After 6 hours of HIFU treatment, the viability of U87mg cells treated with Lipo-DOX-Au decreased by around 20% compared to Lipo-DOX, and Lipo-DOX-Au-RGD had a higher uptake rate than Lipo-DOX. In vivo study using PET images, it was confirmed that 64Cu-Lipo-DOX-Au-RGD was taken up into the tumor immediately after injection and maintained for up to 4 hours. In this study, drugs released from liposomes-gold nanoparticles via ultrasound and RGD targeting were confirmed by non-invasive imaging. In cell-level experiments, HIFU treatment of gold nanoparticle-coupled liposomes significantly decreased tumor survival, while RGD-liposomes exhibited high tumor targeting and rapid release in vivo imaging. It is expected that the combination of these models with ultrasound is served as an effective drug delivery material with therapeutic outcomes.