그랜트 지원 NHLBI HL080532, NHLBI HL073449, NCRR RR16453, 그리고 그랜트 -에 도움이 수상 (RVS)를 AHA 국가를 포함하고 있습니다. 특별 감사는 비디오 제작과 함께 자신의 지원 거리 코스 설계 및 컨설팅 (DCDC) 그룹 dcdcgroup.org,하고 DCDC를 설립 교육 부여 번호 P336C050047 미국 이민국에 연장됩니다.

1. Microbubble 주식 준비 <ol><li> 10 PBS의 MLS는 1g의 포도당 200 MG 1,2 - dipalmitoyl - SN - glycero - 3 - phosphatidylcholine 50 MG 1,2 - dipalmitoyl - SN - glycero - 3 - phosphatidylethanolamine를 섞는다. </li><li> 피펫은 5 분마다 혼합, 끓는 물 목욕 20~30분의 혼합물을 가열. </li><li> 솔루션은 최대 6 개월까지 4 ° C에 저장할 수 있습니다. </li></ol> 2. Microbubble 준비 <ol><li> 40 준비 microbubble 재고 솔루션과 부화 ° 15 분 C의 250 μl를 가져가라. </li><li> 미리 예열 microbubble 솔루션은 다음 글리세롤의 50 μl를 포함하는 1.5 ML의 microtube로 전송됩니다. </li><li> 표현이 관심의 유전자 (4mg/ml의 최적 농도로 Qiagen 내독소 무료 MegaPrep 키트, Qiagen, 제르, MD에 의해이 예제에서 정화)에 대한 건설 인코딩 플라스미드 DNA를 정화 1-2 MG. 2.4) 인산 버퍼 호수 500 μl의 최종 볼륨에 추가됩니다. 내독소 무료 Qiagen의 maxipreps은 불임을 보장하기 위해서뿐만 아니라 멸균 PBS로 사용됩니다. </li><li> microtube에있는 공기는 다음 Octafluoropropane 가스로 대체됩니다. </li><li> microtube 그 다음 20 초 동안 치과 amalgamator에 적극적으로 흥분 상태입니다. </li><li> microbubbles에 바인딩되지 않은 subnatant 포함된 잔류 DNA와 버퍼는 다음 조심스럽게 제거하고 microbubble 계층은 무소속의 DNA를 제거하기 위해 멸균 PBS로 세 번 씻어, 각 씻어주기 사이에 얼음에 위치합니다. 우리는 일반적으로 30-40%의 결합 효율을 얻을 수 있습니다. 모든 시약은 살균하고주의 오염을 최소화하기 위해 만들어졌다. </li><li> 플라스미드 DNA - 바운드 microbubbles 그런 다음 사용 때까지 서로 다른 두 시간 동안 얼음에 게재됩니다. </li><li> 혼합 및 PBS 세척 후 microtube에서 제거 subnatant는 사용 260nm의 파장에서이 솔루션의 광학 밀도를 측정하여 언바운드 DNA의 농도와 마찬가지로 알려진 초기 농도에 따라 바운드 금액을 결정하는 데 사용할 수 있습니다 분광 광도계. </li></ol> 3. 장비 보정 <ol><li> 처음 사용하기 전에, 1 MHz의 캐비테이션 변환기는 적절한 기계 색인 및 펄스 반복 수 있도록 조정해야합니다. submergible 1 MHz 이상, 13mm, 산만 변환기는 전력 증폭기를 통해 20 MHz의 기능 / 임의 파형 발생기에 연결됩니다. </li><li> 변환기는 무료 증폭기를 통해 500 MHz의 오실로 스코프에 연결되었습니다 수중 처음기에서 직접 겨냥한 물로 가득 찬 플라스틱 용기에 배치됩니다. </li><li> 파형, 주파수, 진폭, 버스트주기 및 전원 증폭은 모든 적절한 듀티 사이클 및 microbubbles을 cavitate하기 위해 최적의 기계적 인덱스를 얻기 위해 수정할 수 있습니다. 이 특정 실험을 위해, 우리는 1 MHz 이상에서 ~ 1.3에 상응하는 기계 색인 시스템을 보정합니다. </li></ol> 4. Microbubble 배송 &amp; UTMD <ol><li> 이전 microbubble 전달 및 UTMD에, C57BL / 6 마​​우스는 IP 분사를 통해 마취제와 5mg/kg xylazine을 100mg/kg로 anesthetized되었습니다. </li><li> Microbubble 배달은 심장의 좌심실로 또는 꼬리 정맥 catheterization 통해 직접 분사를 통해 정맥 투여했다. 직접 심장 주사, 최대 플라스미드 DNA - 로드된 microbubble 솔루션을 100 μl에 볼륨이 심장의 좌심실에 초음파 시각화 아래 앞쪽에 4 번째 늑간 공간에서 삽입 30 게이지 바늘을 통해 주입합니다. </li><li> 왼쪽 interventricular 주입으로 인한 microbubble 솔루션 볼러스은 Vevo 2100 이미징 시스템 비주얼 Sonics &#39;VisualSonics 사용하여 긴 축에보기에서 마우스의 흉부에 고정 위치에 배치 38 MHz의 고주파 초음파 변환기&#39;를 사용하여 시각입니다. 모든 주사기와 바늘이 주사 가능한 가장 멸균 환경을 할려고, 멸균 수 있습니다. </li><li> 즉시 주사 다음, microbubble 파괴는 두 번째, 작은 크기의 낮은 주파수 1.0 MHz의 변환기를 사용하여 ~ 5 분이 지역에 파괴를 대상으로, 원하는 기관을 통해 직접적인 개최를 위해 수행됩니다. 이 예제에서는 초음파는 기계 약 1.3-1.5 색인에 상응하고, 매 20 사이클 100 MS의 펄스 반복 기간, 1.0 MHz의의 펄스 반복 주파수에서 간장을 실시했다. 또는 펄스 초음파 3 프레임마다 4-6 심장 사이클의 버스트에 (이 실험에 표시되지 않음) 마우스의 ECG에 문이 수 있습니다. 우리는 초음파의 파열 사이의 거품과 함께 리필에 모세관 침대를 허용 프로토콜과 transfection의 효율성을 구하십시오. </li></ol> 5. 대체 납품 방법 우리는 절차의 복잡으로 인해 interventricular 주사를 강조하기 위해 선택하지만, 이러한 microbubbles의 장기간 주입에 꼬리 정맥으로 많은 경우에jection이 선호하는 방법입니다. microbubble 배달의 꼬리 - 정맥 방법은 마우스 동일한 방법 anesthetized 수 있습니다. 플라스미드 DNA - 바운드 microbubbles를 포함하는 주사기는 27 게이지 바늘 / 꼬리 정맥 카테터에 연결됩니다. 꼬리 정맥 카테터는 그 마우스의 꼬리를 따라하거나 오른쪽 또는 왼쪽 측면 정맥의 말초 세번째에 삽입됩니다. microbubbles를 포함하는 주사기는 자동으로 시간이 미리 설정된 기간 동안 솔루션의 유니폼을 미리 볼륨을 관리 주입 펌프에 배치됩니다. 우리는 일반적으로 3ml/hour의 속도로 200 300μl를 달이다. 동물을 사용 모든 동물은 관련 국가 및 / 또는 현지 동물 복지 기관에 의해 정의된 좋은 동물 관행에 따라 처리되었고, 모든 동물의 작업은 (하와이 기관 애니멀 케어 및 사용위원회의 대학 승인 번호 07-100 해당위원회에 의해 승인되었다 -3). 적절한 마취 (케타민을 / zylazine)을 사용하고 진통제 (Bupivicaine 및 Buprenorphine)이 필요하지만, 사용할 수 있었어요. 6. 대표 결과 : UTMD - 중재 플라스미드 DNA 전달의 효율성은 구성 등로 인코딩된 유전자에 따라 다양한 방법을 통해 평가하며 이에 제한되지 않음 수 있으며 생체내 이미징, B - 여자 예 생체내의 얼룩 및 / 또는 루시페라제 immunohistology. 특히, 생체내 bioluminescence 이미징의 하나가 플라스미드 인코딩 발광 생물 리포터 유전자 (루시페라제)와 transfected 생쥐에서 순차적으로 유전자 발현의 존재와 시간을 모니터할 수 있습니다. VIVO 이미징 시스템 (IVIS)에서 Xenogen는 (칼리퍼 생명 과학, 홉킨튼, MA) 발광 생물 이미징에 사용됩니다. 이미지는 일반적으로 모든 마우스 UTMD 중재 transfection 후 첫날 찍은되고 발광 생물 유전자 발현이 더 이상 시스템 (그림 1)를 통해 시각적으로 감지할 수 없습니다 때까지 모든 3-4일 반복됩​​니다. 발광 생물 이미징을 위해 마우스를 준비하려면, 마우스 먼저 프로브 D - luciferin (칼리퍼 생명 과학)와 그때 anesthetized 아르 ~ 3 분 후 루시페라제 기자의 IP 주사를 받게됩니다. D - luciferin 기판의 Biodistribution는 동물 IVIS 이미징 챔버에 배치되고 몸 전체에 이미지 검색이 촬영되기 전에 ~ 10 분 동안 진행하는 허용됩니다. 인수 동안 반딧불 루시페라제 / D - luciferin 광화학 반응에서 방출 광자는 측정됩니다. 그림 1은 UTMD 다음 간을 유사한 IVIS 발광 생물 이미징을 설명하고, 그림 2는 안티 루시페라제 차 항체 (시그마 - 알드리치)과 AlexFluor - 568 복합 이차 항체 (Invitrogen)를 사용하여 transfected 간을 epifluorescence (100X) 이미지입니다 . 그것은 UTMD 중재 간 transfection은 내피 세포뿐만 아니라 영향을 것을보고 분명하지만 hepatocyes뿐. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="그림 1" /> 그림 1 심장의 Xenogen / IVIS 영상은 치료 쥐를 UTMD (A) 대조군 마우스 :.. 플라스미드 + PBS 심장 감독 UTMD 다음, (B) 트리 트먼트 마우스 : 플라스미드 + 심장 다음 Microbubbles가 UTMD 감독, 그리고 (C) 치료 마우스 : 간 다음 플라스미드 이상 Microbubbles는 UTMD 감독. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="그림 2" /> 그림없이 UTMD 2. 간 UTMD의 Immunohistochemistry (빨간색으로 반 루시페라제.) (A) 플라스미드 및 UTMD와 (B) 플라스미드. 공촛점 이미지 (100X), 핵은 DAPI 얼룩 푸른 있습니다.

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관심 없음 충돌 선언하지 않습니다.

UTMD은 유전자 전달에 대한 새로운 접근 방식을 나타냅니다. 플랫폼 기술로 그것은 조직 특이성의 높은 수준을 원하는 것입니다 bioactive 분자의 무수한를 제공하기 위해, 많은 잠재적인 유전자 치료 전략의 결합 수 있습니다. 기술의 주요 생물 학적 한계는 transfection의 낮은 효율성이다. 또 다른 중요한 고려 사항은 현저하게 뼈 또는 공기를 개입 감소 수있는 초음파 검사로 대상 기관의 접근이다. ?...

<table border="1"><tbody><tr><th> 시약 / 장비 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 1,2 - dipalmitoyl - SN - glycero - 3 - phosphatidylcholine </td><td> 시그마 - 알드리치 </td><td> P - 5911 </td><td> microbubble 지질 껍질 성분 </td></tr><tr><td> 1,2 - dipalmitoyl - SN - glycero - 3 - phosphatidylethanolamine </td><td> 시그마 - 알드리치 </td><td> P - 3275 </td><td> microbubble 지질 껍질 성분 </td></tr><tr><td> 포도당 </td><td> 시그마 - 알드리치 </td><td> G5400 </td><td> microbubbles을 안정 생각 </td></tr><tr><td> 생리 인산염 버퍼 </td><td> 시그마 - 알드리치 </td><td> P5368 </td><td></td></tr><tr><td> 글리세린 </td><td> 시그마 - 알드리치 </td><td> G5516 </td><td> coalescing에서 microbubbles을 방지하기 위해 믿고 </td></tr><tr><td> Octafluoropropane 가스 </td><td> AirGas </td><td> N / A </td><td> 임상 응용에 사용되는 불활성 가스 </td></tr><tr><td> VialMix 치과 amalgamator </td><td> 브리스톨 - 마이어스 Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz 이상, 13mm, 산만 변환기 </td><td> 올림푸스 </td><td> A303S - SU </td><td></td></tr><tr><td> 20 MHz의 기능 / 임의 파형 발생기 </td><td> 애질런트 </td><td> 33220A </td><td></td></tr><tr><td> 전력 증폭기 </td><td> 크론 - 하이트 (주) </td><td> 모델 7500 </td><td></td></tr><tr><td> 수중 처음기 </td><td> 브루엘 및 Kjaer </td><td> 유형 1803 </td><td></td></tr><tr><td> 충전 앰프 </td><td> 브루엘 및 Kjaer </td><td> 유형 2634 </td><td></td></tr><tr><td> 500 MHz 이상 오실로 스코프 </td><td> 르크로이의 </td><td> 9354L </td><td></td></tr><tr><td> 34 MHz의 변환기와 VisualSonics &#39;Vevo 2100 이미징 시스템 </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G 반장님 꼬리 정맥 카테터 </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> 지니 플러스 주입 펌프 </td><td> 켄트 과학 </td><td> 요정 </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

이러한 관심의 유전자를 인코딩 부정 청구 플라스미드 DNA를 벡터로 UTMD, bioactive 분자에서, 지질 microbubble 대비 요원 7-9의 양이온 포탄에 추가됩니다. 생쥐에서 이러한 벡터 운반 microbubbles는 심장의 좌심실 자에게하거나 직접 관리하실 수 있습니다. 큰 동물에 그들은 또한 카테터를 통해 주입 intracoronary 수 있습니다. 순환에서 대상 기관에 대한 후속 배달 microbubbles의 공진 주파수에서 음향 캐비테이션에 의해 발생합니다. 그것은 기계적 에너지 또는 대상 지역 10 microvasculature의 내피 세포 사이의 과도 기공 형성에 microbubble 파괴 결과에 의해 생성되는 가능성이 보인다. 이 sonoporation 효과의 결과로로와 내피 세포에 걸쳐 transfection 효율이 향상되며, transgene 인코딩 벡터는 주변 조직으로 입금됩니다. 순환에 남아 플라스미드 DNA는 신속하게 추가로 이외의 sonicated 조직에 전달 가능성을 감소하고 매우 구체적인 목표 - 장기 transfection로 연결 혈액에 nucleases로 저하됩니다.

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

초음파 타겟 Microbubble의 파괴 (UTMD)가 같은 마음으로 간 초음파 검사에 액세스할 수 기관을 대상으로, 치료 유전자를 포함 bioactive 분자의 특정 사이트 게재를 직접하는 데 사용할 수 있습니다<sup&gt; 1-6</sup&gt;.

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 ...

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.4K 조회수.  University of Hawaii. 초음파 타겟 Microbubble의 파괴 (UTMD)가 같은 마음으로 간 초음파 검사에 액세스할 수 기관을 대상으로, 치료 유전자를 포함 bioactive 분자의 특정 사이트 게재를 직접하는 데 사용할 수 있습니다 1-6.

비디오: Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

명암 초음파 마약 베어링 Nanoparticle 배달 및 Microvascular 절제를 통해 마우스에서 Gliomas의 치료 타겟

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

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 ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

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. ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

높은 가위 균질화 및 시리얼 농도로 농축, 지질 기반 산소 마이크로 버블 에멀젼의 제조

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

미세 유체 기술은 셀 변형능을 검사 할

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMS 샘플 소개를위한 미세 유체 칩

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

펄스 레이저인가 전압에 따라 금 나노 입자 통합 사진 응답 리포좀과 그들의 마이크로 버블 공동 현상의 측정의 합성

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

뇌 이미징 응용 프로그램에서 CMUT 기술에 기반 하는 새로운 초음파 프로브의 성능을 평가 하기 위한 실험 프로토콜

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 보완 방법을 사용 하 여 태 반 대상 약물 전달의 안전과 효율성의 종합 평가

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

세포가 없는 세균 추출물, 리포좀 및 에멀젼 전달을 사용하여 합성 세포를 생산하는 단백질 을 준비합니다.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...