Mannitol이 家兎大動脈 切片의 收縮에 미치는 影響

손동열 漢陽大學校 1985 국내박사

뇌부종이 있는 뇌경색 환자들에서 만니톨로 인한 신부전증의 예측 인자

봉정빈 조선대학교 대학원 2016 국내석사

Background Renal failure is known as one of the mannitol’s most common side effects. It is very little known about cause, incidence, risk factors, and outcome of mannitol induced renal failure. The goal of this study is to determine predicting factors of mannitol induced renal failure. Methods We retrospectively reviewed the medical records of all ischemic stroke patients treated with mannitol due to brain edema from January 2008 to December 2010in Chosun university hospital. From among 125 patients, 25 patients received mannitol below 3 days were excluded and 100 patients were selected. A15% or 25% mannitol was administered intravenously by intermittent bolus and maximum dose was below 200g/day. Renal failure was defined as an increase in the creatine level of > 0.5 mg/dl if the base-line value is < 2 mg/dl or an increase > 1 mg/dl if the base-line value is > 2 mg/dl. Results The 14 patients(14%) were diagnosed as mannitol induced renal failure. Glucose level before use of mannitol and peak osmolarity during mannitol treatment were associated with renal failure in univariate analysis. In logistic regression analysis with suspected factors (P<0.1), independent predictive factors of mannitol induced renal failure were glucose level before use of mannitol, (1.014, 1.000-1.027,p=0.043) and peak osmolality (OR 1.041 95% CI 1.009-1.075, p=0.01). Conclusion Strict glucose control before mannitol treatment may be able to reduce the rate of renal failure occurrence. However, the recovery rate from renal failure was not confirmed in our study because of short follow up period.

Weissella cibaria 의 만니톨탈수소효소에 대한 생화학적 특성과 스타터 김치의 적용

서예슬 전남대학교 대학원 2014 국내석사

Mannitol is a naturally occurring six-carbon sugar alcohol. It is used as a bodying agent, humectant, anticaking agent and antioxidant and has a sweet cool taste. Because of these characteristics, mannitol is widely applied in the food industry. This study was performed to characterize the mannitol dehydrogenase (MDH) from Weissella cibaria to prepare for the preparation of starter kimchi containing mannitol. Also, physicochemical, microbiological and sensory characteristics of starter kimchi were investigated. Starter kimchi and regular kimchi were prepared in the presence of added 1% sucrose. Then, the kimchi were fermented at 15℃ for 3 d and stored at 4℃. The amount of mannitol and characteristics of kimchi were analyzed for 18 d with sensory evaluation. Mannitol producing ability in MRS broth and kimchi were measured using HPLC. Wei. cibaria was cultured in optimal conditions and then MDH was produced from Wei. cibaria. MDH was partially purified and biochemical properties were characterized. The amount of produced mannitol in MRS broth with 5% fructose was 2.27%. The mannitol concentration of starter kimchi and regular kimchi were 0.62% and 0.44%, respectively. These results suggest that Wei. cibaria could be used in mannitol-containing kimchi as a starter microorganism.

Candida magnoliae HH-01에 의한 과당으로부터 만니톨로의 생전환

백홍 韓國外國語大學校 大學院 2008 국내박사

The purpose of this thesis was to develop the optimal fermentation processes for the production of mannitol. For this purpose, isolation of producer strains, optimization of fermentation conditions such as carbon/nitrogen source, pH, temperature etc, and purification and characterization of key enzyme involved in mannitol biosynthesis were investigated. A novel microorganism which is able to produce mannitol when grown in the presence of fructose and glucose as a carbon source was isolated from the fermented product using various vegetables. The isolated strain was identified as Candida magnoliae based on the identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains. In this study, NADPH-dependent mannitol dehydrogenase from C. magnoliae HH-01 was purified to homogeneity by ion-exchange chromatography. The relative molecular masses of C. magnoliae mannitol dehydrogenase, as determined by sodium dedecyl sulfate-polyacrylamide gel electrophoresis was approximately 35 kDa. The enzyme catalyzed reduction of fructose into mannitol. The pH and temperature optima for fructose reduction was 7.0 and 35??, respectively. C. magnoliae mannitol dehydrogenase showed the highest activity with fructose as the substrate and used only NADPH. The enzyme activity was inhibited by CuCl2 and sulfhydryl compounds but not by EDTA. The mdh gene encodes mannitol dehydrogenase, an enzyme to convert fructose to mannitol. The oligonucleotides deduced from the peptide sequences and random primer were used to prepare the putative mdh gene from the chromosomal DNA of C. magnoliae by PCR. The gene encoding the enzyme was cloned and sequenced. The results demonstrated that a mdh gene consists of 852 bp and encodes 283 amino acids. Based on the cofactor binding site (GXXXGIG), structural stabilizing motif (DXXXNNAG), the active center (YXASK), catalysis enhancing site (PG), and H-bonding to carboxamide of nicotinamide ring (T), mannitol dehydrogenase has been placed with the NADPH-dependent short chain dehydrogenases/reductases (SDRs). The deduced amino acid sequences of C. magnoliae mannitol dehydrogenase showed a significant homology with SDRs from various sources, indicating that the C. magnoliae mannitol dehydrogenase is an NADPH-dependent tetrameric SDR. After fusion of six histidine codons to the 3' end of mdh gene and expression in E. coli, the active mannitol dehydrogenase from the culture broth of the recombinant E. coli was could be purified in a two-step procedure by affinity chromatography using a Ni+-NTA matrix column. For the study of the mdh gene expression, the putative promoter was isolated. GFP, a reporter protein, was expressed under the control of putative 158 bp fragment in C. magnoliae. To enhance the production of mannitol, cultivation conditions and medium composition were optimized. The most efficient substrate for mannitol production by C. magnoliae HH-01 was fructose. Glucose and sucrose also could also be converted into mannitol but with lower conversion yields. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration. The optimal pH and temperature for mannitol production were 4.5 - 5.0 and 33??, respectively. Under optimum conditions, a final mannitol production of 323 g․l-1 was obtained 333 g fructose․l-1 after 164 h, corresponding to an 1.97 g․l-1․h-1 and 97 % yield. 본 논문의 목적은 만니톨 생산을 위한 최적의 발효공정을 개발하는데 있다. 이 를 위해서 만니톨 생산균주의 분리 및 동정, 발효조건(탄소원, 질소원, pH, 온도) 의 최적화, 그리고 만니톨 생산에 관여하는 효소에 대한 정제 및 특성을 조사하 고자 하였다. 만니톨을 생산하는 새로운 미생물을 선별하기 위해서 다양한 장소에서 시료를 채취하였으며 이들을 과당과 포도당이 과량 함유된 선별배지에서 배양하였다. 그 결과 만니톨을 과량 생산하는 미생물을 선별하였으며 선별된 균주을 동정하기 위해서 26S rDNA의 D1/D2 domain의 염기서열을 근거로 하여 Candida magnoliae 임을 밝혀 내었다. 또한 선별한 균주와 C. magnoliae reference strain 의 탄소원 비교를 통해서 같은 종임을 밝혀 내었다. 본 연구에서 C. magnoliae로부터 NADPH-dependent mannitol dehydrogenase 을 이온교환 크로마토그래피을 이용하여 분리 정제를 하였다. Mannitol dehydrogenase의 분자량을 알아보기 위해서 SDS-PAGE을 수행하였으며 그 결 과 약 35 kDa의 크기를 가짐을 확인하였다. 이 효소는 과당에서 만니톨로 환원 시키는 반응을 촉매한다. 또한 과당 환원에 대한 최적 pH와 온도는 각각 7.0과 35℃였다. C. magnoliae에서 만니톨의 생산에 관여하는 효소의 기질과 조효소에 대한 특성을 알아보았으며 기질로 과당을 조효소로 NADPH을 사용하였을때 가 장 높은 활성을 보였다. 그리고 CuCl2와 sulfhydryl compounds에 의해 효소 활 성이 저해되었으나 EDTA는 전혀 영향을 주지 않았다. Mannitol dehydrogenase을 coding하는 mdh 유전자를 cloning하였다. Putative mdh 유전자를 얻기 위해서 C. magnoliae chromosomal DNA와 아미노산 서열 분석을 통해서 얻은 deduced oligonucleotides와 random primer을 사용하여 PCR 을 수행하였다. 그 결과 852 bp의 mannitol dehydrogenase gene을 얻었으며 유 전자 분석을 통해서 mannitol dehydrogenase가 283 개의 아미노산으로 구성되어 있음을 확인하였다. 또한 deduced amino acid 분석을 통해서 cofactor binding site (GXXXGIG), structural stabilizing motif (DXXXNNAG), the active center (YXASK), catalysis enhancing site (PG), 그리고 H-bonding to carboxamide of nicotinamide ring (T)을 확인하였으며 이와 같은 결과를 토대로 C. magnoliae mannitol dehydrogenase는 NADPH-dependent Short chain dehydrogenases /reductases(SDR)임을 확인하였다. C. magnoliae의 mannitol dehydrogenase는 다양한 다른 미생물의 SDR과 많은 유사성을 보였으며 NADPH-dependent tetrameric SDR임을 확인하였다. mdh 유전자의 3‘ 끝부분에 6개의 히스티딘 codon을 결합시킨 다음 대장균에서 발현하였으며 Ni+-NTA agarose에서 분리정 제를 수행하였다. mdh 유전자의 발현에 대한 연구를 위해서 putative promoter를 분리하였다. C. magnoliae에서 158bp의 putative promoter region에 의해서 GFP protein이 발현함을 확인하였다. 만니톨 생산의 최적화를 위해서 배양조건과 배양배지을 최적화 하였다. C. magnoliae에서 만니톨 생산에 최적의 탄소원은 과당이었다. 또한 포도당과 설탕 에서는 만니톨로 전환은 되지만 낮은 생산성을 보였다. Fed-batch fermentation 에서 포도당이 고갈 되었을때 과당에서 만니톨로의 전환이 감소하게 된다. 이때 다시 포도당을 첨가하면 과당에서 만니톨로의 전환이 다시 일어나게 된다. 이는 포도당이 만니톨의 생산에서 조효소인 NADPH의 regeneration에 중요한 역할을 담당하고 있다는 것을 내포하고 있다. 발효조건(pH, 온도등) 최적화를 수행하였 으며 pH 5.0, 33℃에서 배양시간 164시간동안 333 g l-1의 과당에서 323 g l-1 의 만니톨을 생산하며 1.97 g l-1 h-1의 productivity와 97 %의 yield로 만니톨을 생 산한다.

Therapeutic effects of intra-arterial MSC-Neurogenin1 transplantation in a rat model of chronic ischemic stroke

조다롱 아주대학교 2020 국내박사

Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN+ neurons, GFAP+ astrocytes, and Iba1+ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and Cleaved caspase-3. In our previous study, we showed that the transplantation of neural-induced mesenchymal stem cells by introducing Neurogenin1 (MSC/Ngn1) dramatically improved the stroke outcome compared to the parental MSCs in an acute stroke rat model. Here, we investigated the therapeutic effects of intra-arterial transplantation of MSC/Ngn1 cells in a chronic stroke model. Mannitol, hyperosmotic agent, used for opening the blood-brain barrier (BBB) to facilitate the infiltration of transplanted cells toward the infarcted area in a chronic stroke model. First, we identified that treatment of mannitol increased degradation of tight junction proteins, such as ZO-1 and claudin-5, and BBB permeability with measuring transendothelial electrical resistance (TEER) and diffusion of 150kDa FITC-dextran in vitro. Additionally, BBB permeability was evaluated with Evans blue dye extravasation into the brain parenchyma of chronic ischemic stroke model. Osmotic opening of BBB by intra-arterial infusion of mannitol was effective for 1 hour in both of normal and chronic stroke brain. Administering pre-treatment of mannitol has facilitated transmigration of MSC and MSC/Ngn1 through BBB in both in vitro trans-well migration assay and in vivo cell transplantation. MSCs or MSCs/Ngn1 were intra-arterial (IA) injected in the chronic stroke model after IA mannitol pre-treatment. The cells were labeled with superparamagnetic iron oxide (SPIO)-nanoparticles for magnetic resonance imaging (MRI) cell tracking. Significantly higher number of SPIO-labeled cells were in the mannitol-treated group compared with saline-treated group. MRI analysis revealed that mannitol increased SPIO-labeled MSCs and MSCs/Ngn1 delivery to ipsilateral hemisphere of chronic stroke rat model. To detect and quantify administered cells in the brain, human AluJ quantitative polymerase chain reaction (qPCR) analysis was performed. And we examined the rats with immunohistological analysis and behavioral tests. Transmigrated MSCs/Ngn1 were differentiated neuronal cells and mannitol-pretreated rats showed significantly promoted neurological functional recovery. These results indicated that mannitol facilitated the extravasation of stem cells and increased BBB permeability before intra-arterial transplantation has been shown to be even more effective in cell therapy of chronic stroke injury.

Characterization of the Mannitol, Exopolysaccharide Producing Lactic Acid Bacteria from Kimchi

강윤지 경상국립대학교 대학원 2023 국내석사

대한민국 경상남도 사천시의 사천용궁시장에서 파김치를 구매하여 만니톨과 EPS 생성능이 뛰어난 유산균을 분리하였다. 분리된 균주를 16S rRNA 유전자를 이용해 동정한 결과, 만니톨 생성 균주는 각각 Leuconostoc mesenteroides, Leuconostoc citreum로 동정되었으며, EPS 생성 균주는 각각 Weissella cibaria, Weissella confusa, Leuconostoc citreum로 동정되었다. 종균으로 사용하기 위해 다양한 온도, pH, 염 조건에서 생육 특성, 산 내성, 담즙산염 내성을 확인하였다. 만니톨 생성 균주를 이용하여 샤인머스켓 발효주스를 제조함에 있어 유산균주의 생육을 돕기 위해 yeast extract을 0.2% (w/v), tween 80을 0.1% (v/v) 첨가하였다. 발효 종균으로서는 L. mesenteroides SKP 88, L. citreum SKP 92 (10^7 CFU/ml)을 접종하였으며, 발효는 30℃, 48시간 동안 진행되었다. 발효 동안 pH는 감소함에 따라, TA는 증가되었으며, 생균수는 10배이상 증가하여 10^8 CFU/ml로 유지되었다. 또한 샤인머스켓 속 glucose와 fructose는 각각 에너지원으로, mannitol 생성으로 소비되어 감소하다 일정하게 유지되었으며 92 균주보다 88 균주가 만니톨 전환력이 더 우수하였다. EPS 생성 균주인 W. confusa SKP 173, W. cibaria SKP 182, L. citreum SKP 281이 생성한 EPS의 분석 결과 총당 함량은 62-65%, 총단백질 함량은 매우 소량으로 측정되었다. 173, 182의 EPS는 glucose로 이루어져 있으며, 281은 glucitol도 추가적으로 관찰되었다. 점도는 173이 생성하는 EPS가 전반적으로 가장 높았으며 세 균주의 EPS의 분자량은 2,000,000 Da 이상으로 측정되었다. 세 균주의 EPS는 α-(1,6) 및 α-(1,3) 글루코시드 결합을 하는 피라노스 당류로 관찰되어 결과적으로 EPS 생성 균주 모두 덱스트란으로 판단된다. EPS 생성 균주를 이용하여 요거트를 제조함에 있어 EPS 생성을 돕기 위해 sucrose를 5%(w/v) 첨가하였다. 발효 종균으로서는 EPS 생성 균주인 L. citreum SKP 281와 상업적 요거트 종균인 Streptococcus thermophilus, Lactobacillus bulgaricus (5 x 10^6 CFU/ml)을 접종하였으며, 37℃, 24시간 동안 발효, 10 ℃, 24시간 안정화 후 4℃, 24시간 보관하여 분석되었다. 발효 동안 pH는 감소함에 따라, TA는 증가되었으며, 생균수는 100배 이상 증가하여 10^8 CFU/ml로 유지되었다. 또한 EPS 생성 균주가 첨가되어 발효된 요거트에서는 EPS를 생성하여 유청분리를 감소시킴으로써 요거트의 품질을 향상시켰다. 따라서 만니톨, EPS 생성 유산균은 다양한 식품에 적용하여 건강기능성 식품으로 개발 가능하다고 예상되어진다.

Leuconostoc mesenteroides에 의한 Fructose에서 Mannitol로의 최적화 생물전환에 관한 연구

이구환 중부대학교 산업과학대학원 2003 국내석사

최근 전통적인 영양의 개념을 뛰어넘어 건강을 증진시키고 궁극적으로는 질병을 치료하는데 기여를 할 수 있는 기능성 식품에 대한 수요가 급격히 증가하고 있다. 기능성 식품 또는 식품첨가물 중에서 당알콜은 최근 그들의 탁월한 기능성이 알려지면서 특히 수요가 증가하고 있다. 당알코올 중에서도 만니톨은 (C_(6)H_(14)O_(6)) 식품, 화장품, 제약산업 등에 매우 광범위하게 이용되고 인체에 독성이 없어 미국 FDA에 의하여 GRAS(Generally Recognized As Safe)로 승인이 되어 이에 대한 수요량이 급격히 증가하는 추세이다. 본 연구는 Leuconostoc mesenetroides B-742를 이용하여 과당으로부터 만니톨 생산을 위한 최적 생물 전환공정을 개발하기 위한 것인데, 사용 되어진 염배지는 물 1리터에 3.0g KH_(2)PO_(4). 0.01g FeSO_(4) · H_(2)O, 0.01g MnSO_(4) · 4H_(2)O, 0.2 g MgSO_(4) · 7H_(2)O, 0.01 g NaCl, and 0.05 g CaCl_(2) 등을 사용하여 제조를 하였다. 최적공정 조건을 위하여 배양 pH, 배양 온도, 효모 추출물의 농도와 과당의 농도들이 선택이 되었고 측정 되어진 범위는 pH 4.5 to 7.5, 온도 22℃ to 34℃, 효묘 추출물의 농도 0.05 to 2.0% 와 과당의 농도는 5 to 350 g/L 의 범위였다. 연구 결과 최적 조건들은 pH 6.5, 배양온도 28℃, 효모 추출물의 농도 0.5%, 과당의 농도는 30 g/L 이었다. The production of functional foods providing health benefit is one of the fast growing fields in the food industry. Mannitol as GRAS (generally recognized as safe) is a functional food. Mannitol is about 70% as sweet as sucrose and slowly and incompletely absorbed from the intestine, suppling only about one-half energy value of glucose. Commecially, the mannitol is synthesized by catalytic or electrochemical reduction of glucose. However, as strong demand for natural products increased, biological techniques have been developed for mannitol production. The object of this study was to determine statistically optimum conditions for mannitol production by L. mesenteroides NRRL B-742. We optimized the processes' parameters such as pH, temperature, yeast extract concentration, and fructose concentration. The chosen ranges were 4.5 to 7.5 for pH, 22 to 34 for temperature, 0.05 to 2.0% for yeast extract and 5 to 350 g/l for fructose. The mineral medium consisted of 3.0 g KH_(2)PO_(4), 0.01 g FeSO_(4).H_(2)O, 0.01 g MnSO_(4).4H_(2)O, 0.2 g MgSO_(4).7H_(2)O, 0.01 g NaCl, and 0.05 g CaCl_(2) per 1 liter deionized water. The optimum values of pH, temperature, yeast extract, and fructose concentration were obtained at about pH 6.5, temperature 28℃, yeast extract 0.5%, and fructose 30 g/l. We hope that these findings are of particular importance for application of mannitol production industry.

Cytocidal effect of linear six-carbon sugar alcohols on Microcystis aeruginosa, a toxic cyanobacterium

서예린 중앙대학교 대학원 2021 국내석사

담수생태계에서 발생하는 남세균에 의한 녹조현상은 전세계적으로 수질오염을 유발하고 수생생물과 인간의 건강을 위협하고 있다. 특히 담수환경에 서식하는 유해 남세균의 한 종으로 알려진 Microcystis aeruginosa는 ‘마이크로시스틴 (Microcystin)’이라는 독성 물질을 생성하고 분비하는데, 마이크로시스틴이 인간과 동물의 체내로 들어가게 되면 간세포나 신경계에 치명적인 영향을 주는 것으로 알려져 있다. 따라서 M. aeruginosa의 생장과 번성을 제어하기 위한 방법에 대한 많은 연구가 진행되고 있다. 본 연구에서는 M. aeruginosa와 공생세균과의 상호작용과 이에 관여하는 대사물질을 연구하던 도중, M. aeruginosa의 생장을 조절할 것으로 예상되는 다양한 유기물질 중 만니톨이 M. aeruginosa에 대해 세포사멸 현상을 유도함을 관찰하여 이를 규명하기 위한 연구를 수행하였다. 만니톨을 포함한 선형 육탄당 알코올인 이디톨, 푸시톨, 갈락티톨, 소르비톨이 같은 현상을 보였으며, 이와 같은 현상은24시간 내에 25 μM 이상 농도 처리시 나타남을 관찰하였다. 반면 글리세롤, 에리스리톨, 이노시톨과 같은 다른 당 알코올은 M. aeruginosa에 영향을 미치지 않았다. 미분간섭 광학현미경 (DIC), 주사전자현미경 (SEM), 투과전자현미경 (TEM)과 같은 현미경 분석을 통해 손상된 세포에서의 세포내부물질의 유출과 함께 비정상적인 세포 형태, 주름진 세포벽과 세포막, 세포 외 소포 분비, 세포막의 손상 등이 유도됨을 확인하였다. 또한 유세포 분석을 통해 만니톨 처리시 M. aeruginosa의 형태적 변화를 유도하고 세포막 손상도가 증가함을 보여주었다. 뿐만 아니라 M. aeruginosa 배양액의 상층액에서 유출된 세포 내부 물질의 농도가 증가함을 확인하였다. LC-QTOF-MS 분석을 통해 M. aeruginosa가 만니톨을 세포 내로 흡수하지 못함을 확인하였고, 이를 통해 만니톨은 세포의 대사과정을 거쳐 작용하는 것이 아닌, 화학적 또는 물리적 손상에 의해 세포사멸효과를 나타낼 것으로 추측된다. 본 연구 결과를 통해 선형 육탄당 알코올이 유해 남세균인 M. aeruginosa의 빠른 세포사멸현상을 유도함을 규명하였다. 이를 통해 남세균의 증식을 억제하여 전세계적인 생태계 문제인 녹조현상을 제어하고 수질오염을 개선할 수 있는 연구 발전에 도움을 줄 수 있음을 예상한다. 하지만 정확한 작용기작 및 실제 환경에서의 적용가능성 탐색 등 추가 분석에 대한 필요성이 요구된다. Harmful cyanobacterial blooms (HCBs) are a global issue threatening water quality and human health. Microcystis aeruginosa, one of the dominant species causing HCBs, produces a toxic metabolite, microcystin, which can cause liver toxicity and dysfunction of the nervous system in humans. Many researchers have been studied to develop strategies that can control the growth of M. aeruginosa. While studying the interaction of M. aeruginosa and symbiotic bacteria via their metabolites, we found that linear six-carbon sugar alcohols including mannitol, iditol, fucitol, galactitol, and sorbitol caused rapid cell death of M. aeruginosa within 24 h at a concentration of >25 μM, however, other sugar alcohols such as glycerol, erythritol, and inositol did not affect the growth of M. aeruginosa. Microscopic observation showed that cytosol leaked out of the ruptured cell by mannitol. With FACS analysis, we confirmed that morphological modification and the loss of membrane integrity of M. aeruginosa cells were induced by mannitol. Released intracellular organic matter (IOM) and chlorophyll a in the supernatant were also increased. LC-QTOF-MS analysis revealed that M. aeruginosa could not uptake mannitol, which means that it does not involve in the cellular metabolic process. Transmission electron microscopy (TEM) images showed that mannitol induces the change of ultrastructure and intracellular organelles of M. aeruginosa compared to normal cells. Therefore, our results suggest that linear six-carbon sugar alcohols including mannitol can be an effective cytocidal substance to suppress the growth of M. aeruginosa.

가토의 근이완에 대한 Pancuronium과 Mannitol의 상호작용

전종헌 漢陽大學校 1989 국내석사

Development of poly(mannitol-co-PEI) gene transporter modified with rabies virus glycoprotein for brain targeted delivery of therapeutic siRNA

박태은 서울대학교 대학원 2015 국내박사

Alzheimer’s disease (AD) is the world’s most common dementing illness, but no defensive treatments are available currently. A key reason is that AD drug development has been focused on symptomatic management without addressing basic cause of the disease. Hence, genetic intervention to suppress AD causative genes could represent an alternative to standard pharmacological approach. With the advent in therapeutic approach of RNA interference (RNAi), down-regulation of AD problematic genes including BACE1 has been extensively studied using viral vectors. Although viral vectors offered potential advantages for AD RNAi therapeutics, their clinical applications are limited by safety issue associated with viral-mediated carcinogenesis and immunogenicity, which led to the finding of nanotechnology-based non-viral vectors. It is generally accepted that none of non-viral vectors is comparable to viral-vectors for delivery of genetic materials into host cells because non-viral vectors themselves are not equipped with modules for overcoming extracellular and intracellular barriers. Once delivered in body, therapeutic genes encounter extracellular barriers such as serum degradation, immune clearance, non-specific cell binding, and poor penetration of blood-brain barrier until they reach to target cells. After therapeutic genes are arrived in target cells, intracellular barriers inhibit RNAi activity which mainly includes cytotoxicity, poor endosomal escape and lysosomal degradation. The aim of the study is development of non-viral vector for AD RNAi therapeutics equipped with modules to conquer the biological barriers, which is divided into three parts; i) study 1: development of non-viral vector to overcome intracellular barriers, ii) study 2: surface functionalization of the non-viral vector to overcome extracellular barriers, and iii) study 3: verification of AD RNAi therapeutic potential of the developed non-viral vector in vivo. Study 1 mainly focused on an approach of controlling cellular uptake mechanism and consequent intracellular route of complexes to overcome the intracellular barriers. Since it became clear that uptake mechanism by which complexes are internalized determines their intracellular fates, caveolar endocytosis has emerged as an important endocytic target because it provides avoidance of lysosomal degradation. Caveolae vesicles are generally immobilized at the plasma membrane by the actin cytoskeleton, and internalized by certain stimulation via ligand-receptor interaction or osmotic stress. With a view to generating osmotically active gene carrier which facilitates caveolar endocytosis, degradable poly(mannitol-co-PEI) gene transporter (PMT) was generated by crosslinking low molecular weight PEI providing proton sponge effect and mannitol diacrylate as an osmolyte linker. PMT/DNA showed lower cytotoxicity compared to PEI/DNA complexes due to easily degradable ester groups and partially negatively charged mannitol backbone which can shield the remaining cationic charges of PEI. The proton sponge effect of PEI backbone partially contributed to gene delivery of PMT. More importantly, selective stimulation of caveolar endocytosis by mannitol backbone provided enhanced transfection efficiency via successful avoidance of lysosomal degradation of cargo. The action mechanism of PMT/DNA complexes on stimulation of caveolae-mediated endocytosis was associated with the activation of Src-kinase by mannitol part of PMT. In study 2, PMT was functionalized to overcome the extracellular barriers. Delivery of therapeutic siRNA to the brain is one of the biggest challenges for successful brain gene therapy because blood-brain barrier (BBB) permits selective entry of only few substances into the brain. In this study, ‘Trojan horse strategy’ was employed for ferrying PMT/siRNA complexes across the BBB. For this, PMT was conjugated with BBB-permeable rabies virus glycoprotein (RVG) via poly(ethylene glycol) (PEG) linker generating R-PEG-PMT. The PEG linker was expected to provide advantageous spatial arrangement of RVG peptide and stealth property on gene carrier. To determine the BBB penetration of complexes, in vitro BBB culture constructed by co-culture of bEnd.3 cells (mouse brain capillary endothelioma) and B23 cells (rat astrocytoma) on transwell system was utilized. The RVG ligand led to BBB penetration of PMT/siRNA complexes through receptor-mediated transcytosis via nicotinic acetylcholine receptors expressed on BBB. In mechanistic study, it was determined that improved BBB penetration of R-PEG-PMT/siRNA was achieved by stimulated receptor-mediated caveolar transcytosis. The enhanced accumulation in brain and biodistribution property of R-PEG-PMT/siRNA complexes was also demonstrated when systemically administrated. In study 3, RNAi therapeutic potential of R-PEG-PMT/siBACE1 complexes was examined in vivo using normal C57/BL6 mice. Intravenous administration of R-PEG-PMT/siBACE1 for three times within 2 weeks has shown brain-targeted suppression of BACE1 without significant systemic toxicity. Furthermore, decreased BACE1 led to inhibition of beta-amyloid poduction, a marker of AD pathogenesis in hippocampus and cortex parts. The overall results inferred that R-PEG-PMT is a promising tool for AD RNAi therapeutics. In aspect of overcoming intracellular barriers, i) degradable ester linkage of PMT reduced the cytotoxicity ii) PEI backbone of PMT has shown proton sponge effect, and iii) mannitol backbone provided the avoidance of lysosomal degradation via stimulation of caveolar endocytosis. In aspect of overcoming extracellular barriers, i) RVG offered neuronal cell targeting specificity, ii) stealth effect of PEG prevented the immune clearance in vivo, and iii) RVG directed the complexes across the BBB via receptor-mediated transcytosis and PMT facilitated trafficking of caveolae vesicle during transcytosis. The brain-targeted RNAi therapeutic effect of R-PEG-PMT/siBACE1 was demonstrated by suppression of BACE1 expression and beta-amyloid production. Consequently, R-PEG-PMT is a powerful gene carrier which can conquer the biological barriers itself, and possess the potential to be widely applicable in neurodegenerative diseases as a safe and efficient brain-targeted gene carrier.