이 작품은 국립 경력 재단 (RV에 CBET-1336073), ACS 석유 연구 기금 (RV에 52345-DN17), 미국 심장 협회 (JGJ에 BGIA), 국립 과학 재단 (National Science Foundation) (경력에 의해 지원되었다 CBET-1055942에 JGJ), 건강 / 국립 심장, 폐 및 혈액 연구소 JGJ에 (1R21HL110330), 루이와 복숭아 오웬과 텍사스 아동 병원의 국립 연구소.

정렬 폴리실록산 LCEs 1. 합성 <ol><li> 무수 0.6 ml의 반응성 메소 겐 (4- 메 톡시 페닐 4- (3- 부 테닐 옥시) 벤조 에이트)의 166.23 ㎎, 폴리 (hydromethylsiloxane) 40 ㎎, 가교제 및 12.8 mg의 (1,4- 디 (10 undecenyloxybenzene) 30 수확기 작은 유리 병에 톨루엔 (직경 약 13 ㎜, 길이 100 ㎜) 교반 막대가 충전. 용해 25 분 동안 35 ℃에서 용액을 교반 하였다. </li><li> 별도의 바이 얼에서, 1 중량 %의 디클로로 (1,5- 시클로 옥타 디엔) 디클로로 메탄 -platinum (II) 촉매 용액을 제조 하였다. , 피펫을 통해 단계 1.1 시약에 촉매 용액 30 μl를 추가 혼합 교반 및 주문 제작 (3cm X 2cm X 1cm) 직사각형 폴리 테트라 플루오로 에틸렌 (PTFE) 금형에 솔루션을 붓는다. 처음 15 분 동안 거품을 제거하기 위해 주기적으로 진탕하면서 30 분 동안 60 ° C로 가열 한 오븐에서 유리 슬라이드와 장소 느슨하게 몰드 커버. </li><li> 가열 오븐 쿠에서 곰팡이를 제거작은 용기에 액체 질소를 주입하고 2 초 동안 액체 질소 PTFE 몰드의 바닥과 접촉하여 액체 질소 L. <ol><li> 혼합물을 냉각되면, 조심스럽게 금속 주걱을 사용하여 금형에서 엘라스토머를 제거하고 PTFE 시트의 상단에 배치합니다. 면도날을 사용 LCE의 가장자리 트리밍 세 개의 동일한 크기 (약. 2.7 cm 길이 및 0.5 cm 폭)로 그 길이를 따라 LCE 잘라. </li></ol></li><li> 수평 막대의 한쪽 끝에 각 조각을 끊고 LCE의 다른 쪽 끝 10 클립 (4.4 g)을 연결합니다. 테이프를 사용 장소에 LCE를 잡고 10 분 단위로 한 번에 클립 하나를 추가합니다. 길이와 균일 성 변화를 지적, 실온에서 7 일간 LCE을 끊습니다. 눈물이나 휴식 어떤 샘플을 폐기하십시오. 샘플을 제거하고 주변에 저장합니다. </li></ol> 전기 응답 폴리실록산의 LCE의 나노 복합 재료의 2. 준비 <ol><li> 를 통해 분산 된 카본 블랙 LCE 나노 복합 재료를 준비하려면위의 1.4 - 샘플의 대부분은, 첫 번째 반복 1.1 단계를 반복합니다. 반응성 메소 젠, 가교제, 실록산을 포함하는 반응 액에 4.38 mg의 카본 블랙 나노 입자를 추가한다. 5 클립 대신로드 10의 총을 사용합니다. </li><li> LCE 표면에 추가적인 카본 블랙의 나노 입자를 추가하기 위해서, 톨루엔, 카본 블랙 나노 w / v 용액 1 %를 준비한다. 20 분 동안 초음파 처리는 나노 입자를 분산 한 후 배양 접시에 부어 분산액. 6 시간 동안 나노 입자 분산 단계 2.1에서 LCEs을 담가. </li><li> 6 시간 후, 배양 접시에서 솔루션을 철회 피펫을 사용하고 엘라스토머는 공기에 건조 할 수 있습니다. 부드럽게 테이프 또는 면봉 표면에 과잉의 탄소 입자를 청소. </li></ol> 가역 에폭시 기반 LCEs 3. 준비 <ol><li> 4,4&#39;- diglycidyloxybiphenyl 31, 101 mg의 박산의 246.15 ㎎, 헥사 산의 71.6 ㎎, 및 카복시 종료 polydi 76 mg의 혼합주문 제작 (3cm X 2cm X 1cm) 직사각형 PT​​FE 금형에 메틸 실록산. 180 ℃에서 핫 플레이트 상에 배치하여 샘플을 가열한다. <ol><li> (1,5,7-triazabicyclo [4.4.0] 데크 5 엔) 촉매의 11.48 mg을 추가하고 180 ℃로 예열 금속 핀셋을 사용하여 교반한다. 혼합물을 약 20 분 후, 겔이 형성 될 때까지 계속 반응시켜, 반응에 의해 생성 된 기포를 제거하기 위해 주기적으로 교반한다. </li></ol></li><li> 핫 플레이트에서 PTFE 보트를 제거하고 실온으로 냉각 할 수 있습니다. PTFE 금형에서 엘라스토머를 분리하기 위해 면도날을 사용합니다. </li><li> 180 ° C에서 고분자 언론에 두 PTFE 시트를 놓습니다. PTFE 시트 사이의 단계 3.2에서 엘라스토머를 배치하고, 0.3의 두께로 샘플을 압축 - 0.5 mm. 4 시간 동안 180 ℃에서 가열을 계속합니다. </li><li> RT에 샘플과 시원한를 제거합니다. 직사각형 조각 (약 2.5 cm 길이 0.5 cm 폭)에 샘플을 잘라. 가열 오븐 내부에 폴리이 미드 테이프를 사용하여 한 끝에서 샘플을 끊습니다. 12 파를 부착샘플의 자유 단 perclips (8.88 g). 16 시간 - 165 ° CO / N, 또는에 대한 12로 가열 오븐의 온도를 설정합니다. </li><li> 가열 오븐에서 엘라스토머를 제거 길이의 변화를주의. 다음 잔류 응력을 제거 RT로 다시 냉각 핫 플레이트에 80 ° C에 샘플을 가열. </li></ol> LCEs 4. 시험 및 특성 <ol><li> 카메라와 120 ° C에 열판 및 이미징에 샘플을 가열하여 가역적 인 변형을 측정합니다. 실온으로 다시 냉각 한 후 RT에서 초기 시료 길이 120 ° C까지 가열 한 후 시료의 길이 및 길이를 참고. LCEs은​​ 약 30 % 수축 및 냉각에 초기 길이로 반환해야합니다. 도 1a 및도 1b에 도시 된 예시적인 이미지를 참조. </li><li> 가열 / CO에서 150 ° C 0 ° C에서 각각 LCE 스캔으로부터 작은 조각을 절단하여 시차 주사 열량계 (DSC)에 의한 상 전이 온도 유리 전이 분석10 ° C의 속도를 oling / 32, 33 민. </li><li> X 선 회절 측정에 의해 액정 배향의 정도를 정량화. 2 차원 촬상 기능을 X 선 회절 장치에 넣어 샘플. 33은도 2에 도시 된 예 회절 이미지를 참조. 참고 회절상 이방성이어야 LCE (33)의 배향을 반영. 폴리실록산 LCEs은​​ 네마 에폭시 계 LCEs는 스 멕틱 상을 나타내고있다. </li><li> LCE 길이 변화를 측정 강성 및 동적 기계적 분석 (DMA)을 사용하여 폭. 기록 길이 LCEs 및 LCE 나노 복합체에 대한 전위의 함수로서 온도의 함수로서 강도 변화. <ol><li> 열 - 기계적 측정을 위해 수동으로 X 0.3 cm 2cm의 크기로 샘플을 잘라 면도날을 사용하여 조심스럽게 텐션 클램프 사이에 고정합니다. 느슨한 부분을 제거하기 위해 1 백만의 힘을 적용합니다. <ol><li> 열 30 ° C 형 FOLLO에서 샘플을 평형5 ° C / min으로 가열 및 냉각 사이클에 의해 결혼. 120 ° C에 30 ° C에서 열 샘플. 온도의 변화를 측정하는 동안 DMA 기록 샘플의 길이 및 폭의 변화를 생성한다. LCE 샘플의 열 기계적 측정도 3a를 참조하십시오. </li></ol></li><li> 전기 측정을 위해 직접 X 0.3 cm 2cm의 크기로 LCE 나노 복합 샘플을 잘라내어은 에폭시를 사용 LCE 나노 복합체의 양단 동선 접착제. 장력을 이용하여 LCE 나노 복합 1 백만 긴장 클램프 고정합니다. <ol><li> 범위의 전압에서 구리 와이어를 통해 전위를 적용 0 - 30 초 - 60 V, 60 Hz의 주파수 및 0.1 초에서부터 / 오프 펄스 구간에. </li><li> 전위에 응답하여 기록 모양으로 변경됩니다. 느슨 함을 제거하기 위해 1 백만의 고정 된 힘을 적용합니다. 클램프의 위치 변화는 샘플의 변화 모양에 대응한다. Figu보기LCE 나노 복합 시료의 전기 측정 3B 재. </li></ol></li></ol></li></ol> LCE 나노 복합 재료의 전기 자극을 통해 5 활성 세포 배양 <ol><li> 30 초 동안 산소 플라즈마에서 LCE 나노 복합 재료의 일면을 취급합니다. 스핀은 톨루엔 폴리스티렌의 용액 300 μl를 캐스팅 (승 / 1 % v)의 플라즈마 세정 표면의 상단에 1 분 동안 3,300 rpm에서. 톨루엔을 제거하고, 30 초간 산소 플라즈마를 이용 LCE 나노 복합체의 폴리스티렌 코팅 표면을 처리하기 위해 12 시간 동안 진공하에 건조 엘라스토머. </li><li> 표면을 소독하기 위해 30 분 동안 70 %의 에탄올 용액에 LCE 나노 복합 재료를 놓습니다. <ol><li> 인산염 완충 식염수 LCE 나노 복합체를 세척하고 폴리스티렌 코팅 된면이 위로 향하게하여 건조 페트리 접시에 LCE를 전송합니다. 래트 꼬리 콜라겐 타입 I 용액 (0.02 N 아세트산 중 50 ㎍ / ㎖) 5 ㎖에 침지 코팅 LCE의 전체 표면. LCE 없음을 품어적어도 30 분 동안 37 ° C에서 anocomposite 5 % CO 2. </li></ol></li><li> 신생아 쥐 심실의 심근 세포를 분리 이전 11보고 높은 혈청 도금 미디어에서 일시 중지합니다. <ol><li> / cm 2 600,000 세포 - LCE 기판 위에 세포 플레이트 100,000의 밀도로서 전술. 전후 24 시간 후, 저 혈청 유지 매체 (DMEM, M199 18.5 %, 5 % HS 1 % FBS와 항생제)에 세포를 옮긴다. 심근 4 일 동안 LCE의 표면에 부착 및 증식 할 수 있습니다. </li></ol></li><li> 디자인은 3 차원 프린터를 사용하여 제조사의 프로토콜을 사용하여도 4에 도시 된 용기의 개략도를 사용하여 사용자 용기를 제작. 참고 : 3D는 선박을 인쇄하는 것은 60mm X 40mm X 20mm와 50mm X 30mm X 15mm의 내부 차원의 외형 치수를 가진 직사각형 컨테이너입니다. 두 측면에서, 도전성 카본 봉을 삽입하는 데 사용 5mm 구멍 두 가지가있다. 골짜기컨테이너의 상단에 구멍 주위 최대 ES는 양쪽 끝에 자리에 LCE를 개최 사각형 플라스틱 조각 용기에서 (52.5 mm X 12mm X 4mm의 크기)를 배치 할 수 있습니다. 구멍 사이의 거리가 용기의 한쪽 3mm이고,도 4에 도시 된 바와 같이, 노치 구멍 주위에 위치된다. 이는 상술 LCE 기판의 크기와 호환되도록 설계된다. 재료 보충와 같이 전도성 탄소 막대는 상용 공급 업체를 통해 얻을 수있다. <ol><li> 선박에서 구멍을 통해 탄소 막대를 삽입하고 의료용 실리콘 접착제를 사용하여 장소에서 개최합니다. 접착 O의 /의 N을 치료. </li></ol></li><li> 세포 배양 유지 보수 매체와 전기 공급 장치에 연결 병렬 전도성 카본 봉으로 가득 사용자 정의 3D 인쇄 된 용기 심근으로 전송 LCE 나노 복합 재료. 탄소 막대에 걸쳐 LCE를 놓고 전기적 접촉을 보장하기 위해 한 끝에 수정. <ol><li> 하나 또는 양쪽 끝에서 장소에 LCE를 유지하지만, LCE 샘플을 통해 느슨하게이를 배치하는 3-D 용기의 홈을 통해 사각형 플라스틱 조각을 삽입합니다. 전기적으로 24 시간의 총 온 / 오프 시간 5 초와 40 V AC 전위의인가를 통해 LCE을 자극한다. </li></ol></li><li> 앞서 설명한 바와 같이 11 칼 세인 AM을 사용하여 살아있는 세포의 막 얼룩이. </li><li> 핵 염색을 위해, 거꾸로 형광 현미경 이미징 전에 설치 매체 DAPI이 함유로 세포를 다룹니다. 살아있는 세포의 개수를 세어 가장 적합 함수를 사용하여 셀의 배향 각을 결정하기를 ImageJ에 사용한다. </li></ol> 직접 난방을 사용 LCEs 6. 활성 세포 배양 <ol><li> 블랙 나노 입자를 첨가 탄소없이 순수한 LCE를 사용하여 위의 5.3 - 반복 5.1 단계를 반복합니다. 이 절차는 종래 공보에 상세하게 설명한다. (11) </li><li> 페트리 D에 심근과 LCE로 이동세포 배양 배지 및 유지 0.5 &quot;X 2&quot;캡톤 저항 히터 틱. 최소 24 시간 동안과 5 초 간격으로 열 떨어져 12 W. 사이클의 가열 힘으로 저항 히터를 돌려 LCE에 공급 열. </li></ol>

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The authors have nothing to disclose.

In order to produce monodomain LCEs, the LCEs need to be uniaxially loaded during crosslinking. This is challenging in practice because the LCE is loaded when it is only partially crosslinked, and therefore is not mechanically robust and can easily break or tear. The procedure described above (steps 1.1 - 1.4) can produce monodomain LCEs consistently. One critical step is the removal of the LCE from the PTFE mold for loading at the appropriate time. If the LCE is removed too quickly, it will easily break or tear. On the...

빠른 가역 및 프로그램 모양 변화를 나타낼 수있다 재료 새로운 응용 프로그램 1-9의 번호 바람직하다. 모양 응답 스텐트는 상처 치유 및 치료 7 지원할 수 있습니다. 인공 로봇 탐사 또는 유해하거나 인간 (10)에 대한 안전하지 환경에서 작업을 수행하는데 도움이 될 수 있습니다. 모양 응답 탄성 중합체 세포 활성 환경에서 배양하는 활성 세포 배양에 사용하기에 바람직하다. 11-14 다른 응용 프로그램 포장, 감지 및 약물 전달을 포함한다. 액정 엘라스토머 (LCE)는 액정이 15 ~ 20 주문과 고분자 네트워크입니다. LCEs은​​ 메소 겐으로 알려진 액정 분자 유연한 고분자 네트워크를 결합하여 제조된다. LCEs의 응답은 메소 겐의 순서에 영향을 고분자 네트워크 균주 및 자극 액정 순서의 결합으로부터 유도되는 것 유전자속도의 네트워크 균주, 반대의 경우도 마찬가지. 외부 부하가없는 상태에서 크고 가역적 형상 변경을 달성하기 위해, 메소 겐은 LCE 단일 방향으로 정렬되어야한다. LCEs 작업에 일반적인 실제 문제는 LCEs을 모노 도메인 생성된다. 또 다른 과제는 직접 가열 이외의 자극에 반응하여 형상 변화를 발생한다. 이것은 LCE 네트워크를 21-28로 나노 입자 또는 염료의 추가를 통해 수행 할 수 있습니다. 여기, 우리는 모노 도메인 LCEs 및 LCE 나노 복합 재료의 제조를 보여줍니다. 먼저, 제 Kupfer 등에 의해보고 된 두 단계 방법을 사용하여 모노 도메인 LCEs의 제조를 보여준다. (29)이 여전히 어려울 수 샘플 사이의 균일 한 배향 및 일관성 모노 도메인 LCEs 제조되지만 달성하는 가장 인기있는 잘 알려진 방법 . 우리는 샘플링에 자세한 포함 용이 표준 실험실 장비를 사용하여 구현 될 수있는 방법을 보여취급 및 준비. 다음으로, 도전성 카본 블랙이 도전성 나노 입자, 전기적 응답 LCEs 생산 LCEs에 첨가 될 수있는 방법을 보여준다. 우리는 그 다음 에폭시 계 LCEs의 합성 및 정렬을 보여줍니다. 이들 물질은 교환 네트워크 결합을 나타내고 고온으로 가열하고 균일 한 하중을인가하여 배향 할 수있다. 모든 LCEs는 육안 샘플 영상, X 선 회절 측정 및 동적 기계적 분석을 특징으로한다. 마지막으로, 우리는 활성 세포 배양을위한 형상 반응 기판과 같은 LCEs의 하나의 잠재적 인 응용 프로그램을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="859">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:379px;">4-methoxyphenyl 4-(3-butenyloxy)benzoate</td>
			<td style="width:167px;">TCI America</td>
			<td style="width:145px;">M2106</td>
			<td style="width:168px;">Reactive mesogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">poly(methylhydrosiloxane)</td>
			<td style="width:167px;">Gelest</td>
			<td style="width:145px;">HMS-993</td>
			<td>Reactive polysiloxane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-di(10-undecenyloxybenzene)</td>
			<td style="width:167px;">N/A</td>
			<td style="width:145px;">N/A</td>
			<td>see: Ali, S. A., Al-Muallem, H. A., Rahman, S. U. &#38; Saeed, M. T. Bis-isoxazolidines: A new class of corrosion inhibitors of mild steel in acidic media. Corrosion Science 50 (11), 3070&#8211;3077, doi:10.1016/j.corsci.2008.08.011 (2008)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(dichloro(1,5-cyclooctadiene)-platinum(II)&#160;</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">244937</td>
			<td>Pt catalyst</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PTFE mold</td>
			<td style="width:167px;">N/A</td>
			<td>N/A</td>
			<td>fabricated at Rice machine shop</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carbon black nanoparticles</td>
			<td style="width:167px;">Cabot</td>
			<td>VULCAN&#174;&#160;XC72R</td>
			<td>used in the synthesis of LCE nanocomposites</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">polystyrene</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td>331651</td>
			<td>linear polystyrene&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">4,4&#39;-diglycidyloxybiphenyl</td>
			<td style="width:167px;">N/A</td>
			<td style="width:145px;">N/A</td>
			<td>see:&#160; Giamberjni, M., Amendola, E. &#38; Carfagna, C. Liquid Crystalline Epoxy Thermosets. Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals 266 (1), 9&#8211;22, doi:10.1080/10587259508033628 (1995).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">sebacic acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">283258</td>
			<td>C8 linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">hexadecanedioic acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">177504</td>
			<td>C16 linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carboxydecyl-terminated polydimethylsiloxane</td>
			<td style="width:167px;">Gelest</td>
			<td style="width:145px;">DMS-B12</td>
			<td>Siloxane linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">1,5,7-triazabicyclo[4.4.0] dec-5-ene</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">345571</td>
			<td>catalyst for reversible LCEs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carbon rods</td>
			<td style="width:167px;">Ladd Research&#160;</td>
			<td style="width:145px;">30250</td>
			<td>used in cell culture experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">medical grade silicone adhesive</td>
			<td style="width:167px;">Silbione</td>
			<td style="width:145px;">MED ADH 4100 RTV</td>
			<td>used to adhere carbon rods to vessel</td>
		</tr>
	</tbody>
</table>

preparation of monodomain liquid crystal elastomers and liquid crystal elastomer nanocomposites

LCEs 완전히 가역적 인 형태 변화와 의학의 잠재적 인 응용 프로그램, 조직 공학, 인공 근육, 그리고 부드러운 로봇 모양 응답 물질이다. 여기, 우리는 그들의 형상 응답의 특성, 기계적 특성 및 미세 구조와 함께 형상 응답 액정 엘라스토머 (LCEs) 및 LCE 나노 복합 재료의 제조를 보여줍니다. LCEs 두 종류 - 폴리실록산 계, 에폭시 계는 - 합성 정렬 한 특징이다. 폴리실록산 계 LCEs는 모노 도메인 LCEs 결과적으로 두 가교 단계, 하중하에 초 통해 제조된다. 폴리실록산 LCE 나노 복합 재료는 LCE의 대부분에 걸쳐 및 LCE 표면에 모두 전도성 카본 블랙 나노 입자의 첨가를 통해 제조된다. 에폭시 계 LCEs은​​ 가역적 에스테르 화 반응을 통해 제조된다. 에폭시 계 LCEs는 (160 ° C) 상승 t에서 단축 부하의 응용 프로그램을 통해 정렬emperatures. 정렬 LCEs 및 LCE 나노 복합체는 영상의 2 차원 X 선 회절 측정, 시차 주사 열량 및 동적 기계적 분석의 조합을 사용하여 가역 변형, 기계적 강도 및 액정 정렬에 대한 특징이다. LCEs 및 LCE 나노 복합 재료는 제어 가능하게 세포 배양 배지에 균주를 생성하는 열 및 / 또는 전위 자극, 그리고 우리는 사용자 정의 만든 장치를 이용하여 세포 배양을위한 형상 반응 기판으로 LCEs의 적용을 설명 할 수있다.

모노 도메인 LCEs 인해 액정 순서와 네트워크 형태의 커플 링 모양 응답입니다. 난방 LCEs 주 배향 방향을 따라 고분자 네트워크의 수축을 생성 액정 오더 파라미터의 감소 결과. 도 1a 및도 1b에 도시 된 바와 같이이 용이 열판 LCE 배치하여 가시화된다. 수축 RT 샘플의 길이를 따라 LCE 계약에서와 등방성 전이 온도 이상으로 가열에서 최대이다. 약간의...

Watch this Scientific Journal Video about Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites at JoVE.com

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

모노 도메인 액정 탄성체 및 액정 엘라스토머 나노 복합 재료의 제조

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

preparation-monodomain-liquid-crystal-elastomers-liquid-crystal

Research

JoVE Journal

Bioengineering

12.5K 조회수.  Rice University. We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

비디오: Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

향상된 마이크로 및 Nanoparticle 조작에 대한 Plasmonic 및 광자 크리스탈 Nanostructures 활용

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

연구

생체공학

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 ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

시투에서 액체에 있는 생물학 어셈블리의 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

액상 사출 성형을위한 3D 인쇄 금형을 사용하여 의료 기기의 신속하고 저렴한 프로토 타이핑

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

생물 액체 배지에서 확장 성, 금속 높은 가로 세로 비율 나노 복합 재료의 생성

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

제어 약물 릴리스에 대한 자기장 응답 하이브리드 젤라틴 마이크로 겔을 교류

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

모듈형 DNA 장치 조립 로봇 액체 처리 자동화

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

액정 나노 입자 캐리어의 소수성화물 캡슐화의 대상 플라즈마 멤브레인 배달

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...