이 작품은 건강 / 국립 암 연구소 (R01CA129822 및 R01CA140995)의 국립 연구소에서 LKM-K에 교부금에 의해 투자되었다. 박사 메디나 Kauwe 지속적인 지원을 위해 C. 레이, M. M-Kauwe 및 D. Revetto을 감사드립니다.

1. 생체 조직 배달 <ol><li> 6-8 주 이전 NU / NU 마우스 베어링 피하 양측 측면 이종 이식 종양에 대한 주입 당 HerDox의 0.004 ㎎ / ㎏ 용량의 0.2 ML를 동일시하는 멸균 식염수로 충분히 HerDox 섞는다. </li><li> 부드럽게 거품을 피하고, 29G 바늘로 장착 3 / 10 cc의 인슐린 주사기에 HerDox 혼합물을 그립니다. </li><li> 0.5 L / 분, isoflurane을 농도 : 3~4% (또는 이하) 마취 가스 청소 시스템 (산소 유량을 갖춘 유도 챔버에 짧은 isoflurane을 노출에 의해 유도된다. </li><li> 마취 마우스 (주입 당 0.2 ML)의 꼬리 정맥에 전체 혼합물을 주입한다. IV 주입은 또한 제지, unanesthetized 마우스를 수행 할 수 있습니다. </li><li> 하루에 한 번, 여섯 개의 연속 일 같은 마우스 주사를 반복합니다. </li></ol> 2. 생체 내 형광 이미징 종양 HerDox 형광의 축적 할 수멀티 영상을 사용하여 주입의 마지막 날 (7 일)에 의해 탐지 가능한 수. 아래 절차에서는 사용자 정의 매크로 조명 및 탐지 시스템 (그림 1) 8의 사용을 수반. <ol><li> 생체 광학 영상에서 다중 모드를 켭니다. </li><li> doxorubicin의 형광 검출에 적합 방출 대역 통과 필터 (590 nm의 ± 30 nm의)를 선택합니다. </li><li> 아르곤 - 크립톤 레이저를 켜고 레이저 광 경로에 여기 대역 통과 필터 (488 nm의 ± 10 nm의)를 배치합니다. </li><li> 마취 시스템을 켠 후 마취 챔버 (산소 유량에 마우스를 올려 : 0.5 L / 분, isoflurane을 농도 : 3-4% (또는 이하) 가스 청소 시스템을 갖추고 있습니다. </li><li> 마취 챔버에서 마우스를 마취입니다 생체 광학 영상에서 다중의 이미징 챔버에 마우스를 전송합니다. </li><li> 마우스의 코를 통해 노즈콘를 배치하고 지속적인 마취를 관리하는 흐름을 엽니 다영상 획득시 SIA (산소 유량 : 0.5 L / 분, isoflurane을 농도 : 2~3% (또는 이하). </li><li> 15 초 노출 시간을 사용하여 형광 이미지를 획득. </li><li> 배경 정정 또는 대비 조정 등의 이미지 분석 및 처리를 수행합니다. </li></ol> 3. 형광 이미징 생체 HerDox 형광 종양 특정 기관에 군데 할 수 있습니다 (간을 포함, 신장, 비장, 심장과 골격 근육) HerDox의 마지막 (7 일) 주사 후 24 시간에서 안락사 생쥐에서 수확. <ol><li> 생체 광학 영상에서 다중 모드를 켭니다. </li><li> 독소루비신 형광 검출을위한 방출 대역 통과 필터 (580 nm의 ± 20 nm의)를 선택합니다. </li><li> 아르곤 - 크립톤 레이저를 켜고 레이저 광 경로에 여기 대역 통과 필터 (488 nm의 ± 10 nm의)를 배치합니다. </li><li> 장소 종양과 특정 장기 이미징 챔버 오에 페트리 접시에 배치F 생체 광학 영상에서 다중. </li><li> 15 초 노출 시간을 사용하여 조직의 형광 이미지를 획득. 초기 형광 이미지 획득의 예는 그림 2a에 표시됩니다. 배경 (그림 2B)로 제공되는, 페트리 접시 빈을 사용하여 동일한 작업을 반복합니다. </li><li> 배경 정정 또는 대비 조정 등의 이미지 분석 및 처리를 수행합니다. 수정 된 이미지의 예는 그림 2a에서 그림 2B의 뺄셈의 결과, 그림 2C에 표시됩니다. </li></ol> 4. 종양의 현장 공 촛점 이미징 현장 공 촛점 이미징 세포 수준에서 탐지 및 HerDox 종양 축적 분석 할 수 있습니다. <ol><li> 라이카 SPE 공 촛점 현미경을 켭니다. </li><li> 독소루비신에 대한 독소루비신 및 배출 파장 (560-620 nm의)에 여기에 대한 488 nm의 레이저 광을 선택형광 검출. </li><li> 대물 렌즈 40X 또는 63X 목적 및 드롭 침수 오일을 선택합니다. </li><li> 절차 2에서 설명한대로 이전 HerDox 및 모의 치료를받은 안락사 생쥐에서 신선한 종양을 추출합니다. </li><li> 얼음에 페트리 접시에 장소 종양 조직의 저하를 방지하고 공 촛점 이미징을위한 델타 T 챔버에 종양을 전송합니다. </li><li> 연속 초점 깊이 (: 1 ㎛, 두께 : 20 μm의 스텝 크기)에서 종양의 공 촛점 이미지를 획득. Z-축을 따라 순차적으로 획득 한 이미지의 예는 그림 3의 왼쪽 패널에 표시됩니다. </li><li> 최대 강도 이미지의 Z - 프로젝션을 수행합니다. Z-스택 이미지의 최대 강도 투사는 그림 3, 오른쪽 패널에 표시됩니다. </li><li> . 최대 강도의 형광 강도 Z 의미 계산 -보기의 전체 필드에 이미지의 형광 강도를 평균 투사 이미지는 계산 하였다D ImageJ에 사용. </li></ol> 5. 비례 스펙트럼 이미징 및 분석 비례 스펙트럼 이미징 및 분석 독스 형광자가 형광 사이의 차별을 할 수 있습니다. <ol><li> 형광 공 촛점 현미경 스캐닝 레이저의 전원을 켭니다. </li><li> 단계 10 나노 미터의 크기, 라이카 SPE 공 촛점 현미경을 사용하여 488 nm의 빛을 여기에, 510-650 ㎚의 스펙트럼 범위 내에서 지정된 깊이에서 HerDox 처리 및 치료 종양의 15 이미지를 획득. </li><li> 독소루비신의 100 μM 솔루션을 준비합니다. </li><li> 독스 형광 (: 510-650 nm의 스텝 크기 : 10 nm의 스펙트럼 범위)의 순수한 스펙트럼 서명을 얻기 위해 100 μM의 독소루비신 솔루션의 스펙트럼 이미징을 수행합니다. 그래프가 그림 4에 표시된대로 분광 이미징 및 형광 스펙트럼 결과에서 이미지 수집의 전형적인 결과를 꾸몄다. </li><li> 이미지 C에서자가 형광 스펙트럼 서명을 취득우베 (스펙트럼 범위 : 510-650 nm의 스텝 크기 : 10 nm의) 치료 종양의 스펙트럼 영상으로 얻었다. 그래프가 그림 5에 표시된대로 분광 이미징 및 형광 스펙트럼 결과에서 이미지 수집의 전형적인 결과를 꾸몄다. </li><li> 우리는 9 개발 프로그램을 사용하여 4 개의 기준 스펙트럼 서명 (순수자가 형광, 0.1.doxorubin +0.9.자가 형광, 0.2. 독소루비신 +0.8.자가 형광, 0.3.doxorubin +0.7.자가 형광) 생성합니다. 4 개의 기준 스펙트럼 특성을 보여주는 전형적인 곡선은 그림 6에 나와 있습니다. </li><li> 우리가 이전 9 개발 한 프로그램을 사용하여 유클리드 거리 측정을 통해 참조 스펙트럼 서명에 의해 정의 된 이미지의 스펙트럼 분류를 수행합니다. </li><li> 비례 스펙트럼 이미징 및 분석 비교를 위해 우리가 7을 개발 스펙트럼 unmixing 프로그램 (ImageJ에 플러그 - 인), 10를 사용하여 해당 이미지의 선형 스펙트럼 unmixing을 수행합니다. 전자선형 스펙트럼 unmixing하여자가 형광에서 HerDox 형광을 분리 xample는 그림 7에 나와 있습니다. </li></ol>

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저자 다니엘 Farkas의는 스펙트럼 분자 영상의 회장이다. 나머지 저자는 더 경쟁 이익이 없습니다.

DOX 형광 종양이 피하 때 멀티 영상을 이용하여 생체 내에서 탐지 가능한 할 수 있습니다. 그러나 HerDox의 치료 적 용량 (0.004 ㎎ / ㎏)의 단일 복용 후 아래의 감지 임계 값입니다. 반면, 7 매일 주사 (7 일 1x/day) 후, 입자의 종양의 축적과 보존 Dox가 형광의 시각화를 가능하게하기에 충분합니다. 독스 또는 클린 기술이 사용되는 생체 내 이미징을위한 다른 형광 물질?...

종양 표적 항암 화학 요법의 것이 전달되는 치료보다 오히려 비 종양 조직에 배포하는 것보다 의도 한 대상에 축적 할 수 있기 때문에 타겟이 불분명 한 약물에 비해 낮은 복용량에서 암 세포를 제거하는 잠재력을 가지고있다. 후자의 상황이 약물의 효능을 희석하기 때문에 과다 복용 효과가 필요 하듯이, 종양 타겟팅 표준 비 표적 치료를 통해 모두 치료 및 안전 이점이있다. 자기 조립 나노 입자에 캡슐화 화학 요법을 표적으로하는 약물이 화학적으로 공유 결합 대상 분자에 연결되어있는 약물과는 대조적으로 변경되지 않은 상태를 유지할 수 있습니다. 이러한 결합은 약물과 표적 분자 모두의 활동을 변경할 가능성이 있기 때문에, 비공유 어셈블리 약물 효능을 유지 할 수 있습니다. 우리는 이전에 소설은 세 가지 구성 요소, 자기 조립 복잡한 HerDox는 HER2 + 종양을 대상으로하는 것으로 나타났습니다 <em&gt; 생체 및 심장 1 등, 정상 조직을 살려주는 동안 종양의 성장 절제를 이끌어. HerDox이 수용체 결합 세포 침투 단백질, HerPBK10 및 화학 요법 에이전트 사이의 비공유 상호 작용을 통해 형성되고, 작은 핵산 링커를 통해 독소루비신 (독스). endosomal 막 침투가 아데노 바이러스 파생 penton 기본 캡시드 단백질 4-6의 결합을 통해 수행되는 동안 HerPBK10는, 인간 표피 성장 인자 수용체 (HER) 바인딩 2-4 수용체 매개 세포 내 이입을 트리거합니다. 단백질의 양전하 도메인 핵산에게 DNA-협재 독스가 타겟 배달을 위해 운송 할 수있는을 통해 바인딩 4, 5를 할 수 있습니다. 전자 성, 윤달, 그리고 아마도 단백질 올리고머의 상호 작용은 서로 다른 온도 1에 혈액 및 확장 보관하는 안정 라운드 10-20 nm의 입자로 자기 조립을 용이하게합니다. 우선 그녀에게 대상HER2가 상승 할 때 2 + 종양 세포는 강화 된 리간드 친화력에 의해 촉진된다. 우리의 이전 연구는 비 종양 조직에 종양과 비교 HerDox 금리 우대 체계적 축적 배달 생체 내 7에있는 종양 세포에 독스 1 및 침투 불분명하는 것으로 나타났습니다. 우리가 관찰 한 종양 세포 기입 한 후 HerDox 릴리스는 독스, 핵 1에 독스 축적을 허용. 종양의 축적은 상대적으로 낮은 HER2 발현 종양이 비교적 높은 HER2 레벨 1에 비해 적은 HerDox 축적으로, 수용체 수준과 상관 관계가 나타납니다. 또한, 효과적인 세포 사멸 농도는 다른 세포 표면에게 HER2 레벨 1을 표현하는 종양 세포 라인에서 HER2 디스플레이와 역 상관 관계를 나타낸다. 종양 살해 10 배 낮은 복용량 이상에서 발생으로 HerDox의 압축을 풉니에 비해 불분명 독스를 통해 치료 및 안전 이점을 전시geted 약물과 타겟이 불분명 독스 1 대조적으로, 심장 (심장 초음파 검사 및 조직 학적 염색에서 감지) 또는 간 (얼룩 TUNEL에 의해 감지) 조직에는 감지 악영향을 얻을 수 없습니다. 바이러스 캡시드 단백질로부터 유도에도 불구하고, HerPBK10는 치료 수준이 전혀 검출 면역 원성을 전시하지 않습니다. 전체 아데노 바이러스에 기존의 항체가 HerPBK10을 인식 할 수있는 반면, 그들은 2 바인딩 셀을 방지 할 수 없습니다. 시간이 지남에 따라 측정 된 종양의 부피는 표적 치료제의 치료 효과를 평가하는 표준 방법이며, HerDox의 치료 효능을 평가하기 위해 고용되었다. 생체 내 및 생체 형광 강도 이미징에이 방법을 보완하면 더 나은 효율성 7을 대상으로 평가하기 위해 우리를 허용했다. 우리는 특히 그 HerDox 전혀 확인하지 않도록 Dox가 형광 스펙트럼 분석을 절제 종양의 현장 공 촛점 이미징 통합했습니다T는 생체 내 종양에 축적하지만, 세포질 및 핵 7에 종양 세포와 전달 독스에 침투. 스펙트럼 분석은 또한 우리가자가 형광 7에서 독스 형광을 구별 할 수있었습니다. 여기에서 우리는 조직 배달 후 생체 내에서 HerDox을 평가 자세하게 우리의 접근 방식을 보여, 그리고 가장 중요한, 평가하기위한 멀티 이미징 방법과 분석을 통해 대상.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

HER2 + 종양 표적 나노 입자, HerDox는 HER2 + 암 동물 모델에서 종양 특혜 축적과 종양 성장 절제를 전시하고 있습니다. HerDox가 작은 핵산 링커를 통해, 화학 요법 제, 독소루비신과 종양 표적 세포 침투 단백질의 비공유 자기 조립에 의해 형성된다. 전자 성, 층간 및 올리고머 상호 작용의 조합은 라운드 10-20 nm의 입자로 자기 조립을 용이하게합니다. HerDox 서로 다른 온도에서 혈액뿐만 아니라, 확장 된 스토리지 안정성을 전시한다. 심장과 간 (타겟이 불분명 한 독소루비신에 의한 표시 손상을 받아야 함) 등의 비 종양 조직에 탐지되지 않는 부작용을 가진 종양 세포 죽음 종양 베어링 마우스 결과에 HerDox의 전신 배달. HER2 고도 세포 발현에 비해 따라서 높은 HER2 수준을 표시하는 종양이 HerDox의 큰 축적을 전시, 인간 표피 성장 인자 수용체를 발현하는 세포를 표적으로 용이in vitro와 in vivo에서 모두 낮은 수준을 노래. 현장 공 초점 및 스펙트럼 분석과 결합 된 형광 강도 영상은 우리가 대상으로 생체 내 종양 조직 출산 후 HerDox의 종양 세포의 침투에 확인 할 수있다. 조직 배달 후 멀티 영상을 통해 표적 종양을 평가하는 여기에 우리가 세부 사항 우리의 방법을.

그림 1은 형광 강도, 스펙트럼, 수명 2 - 광자, 내 생명 공 초점 및 생물 발광 영상 등 여러 양식에 따라 이미지 수집의 목적을 위해 만들어진 생체 광학 영상 프로토 타입에서 보여줍니다. 또한,이 시스템에 통합 냉각 고감도 카메라와 고성능 레이저 라인은 특히 독소루비신 형광의 생체 검출을위한 상용 광학 이미징 시스템 11에 비해 높은 반면 형광 이미지...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

이 문서 상세 종양 표적 나노 입자, HerDox의 광학 영상 분석을위한 절차에 대해 설명합니다. 특히, 표적 종양 침투를 평가 종양 검출을위한 멀티 이미징 장치에 대한 자세한 사용은 여기에 설명되어 있습니다.

표적으로 바이러스 성 단백질의 분석은 HER2 + 종양에 전달 나노 입자

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

Research

JoVE Journal

Bioengineering

10.9K 조회수.  University of Southern California. 이 문서 상세 종양 표적 나노 입자, HerDox의 광학 영상 분석을위한 절차에 대해 설명합니다. 특히, 표적 종양 침투를 평가 종양 검출을위한 멀티 이미징 장치에 대한 자세한 사용은 여기에 설명되어 있습니다.

비디오: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

초음파 타겟 Microbubble 파괴 기술 소개

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

연구

생체공학

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

타겟 안정 동위 원소 희석 액체 크로마 토그래피 - 질량 분광법 분석을위한 세포 지질 추출

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

표피 성장 인자를 사용하여 인간의 췌장 암 세포의 유전자 전달 및 Transfection은 다행 기반 엔지니어링된 Nanovectors를 수용체 타겟

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

에 대한 바이러스 나노 입자<em&gt; 생체에</em&gt; 종양 이미징

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

고분자 유전자 배달에 대한 평가는 Nanoparticle 추적 분석 및 높은 처리량 유동 세포 계측법에 의해 나노 입자

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

재생 연구를위한 고조파 나노 입자

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

액체에서의 펄스 레이저 어블 레이션에 의한 금속 나노 입자의 제조 : 나노 입자의 항균성 연구를위한 도구

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

상호 빛 전자 현미경 검사 법 (클레 멘 타인) 추적 하 고 바이러스 성 단백질을 이미징 관련 Cryo 움직일 셀 구조

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

단백질 키 니 아 제 C 델타 억제제 펩 티 드 배합 금 나노 입자를 사용 하 여

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...