The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. 일단 회복 된 입자 준비 <ol><li> 2 시간 동안 95 ° C에서 30 % 과산화수소 용액에서 비 - 작용 화 된 다공성 실리콘 입자를 산화시킨다. 2 % 부드럽게 교반하면서 65 ℃에서 2 일 동안 이소 프로필 알콜 (3- 아미노 프로필) 트리에 톡시 실란 용액에 산화 된 입자를 Aminate. </li><li> 펠렛을 중단 간단한 초음파 처리를 이용하여, XG 18,800에서 30 분 동안 원심 분리 용액을 이소 프로필 알코올과 에탄올 중 세 번에 두 번 씻어 입자. 단계 1.3 및 1.4을 수행 할 때 에탄올 용액의 입자를 남겨주세요. </li><li> 10 ㎖ 동 중성자 원소 희석제 입자 현탁액 (예, 10 μL)의 공지 된 양을 첨가하고, 스톡 용액에 입자의 농도를 결정 입자 계수 분석기 입자를 카운트. </li><li> (3- 디메틸 아미노 프로필) - - N &#39;에틸 카보 다이이 미드 하이드로 클로라이드 (EDC, 0.1 nmol의) / N과 N-L 아르기닌 (0.1 nmol의)의 산 그룹을 활성화 </em&gt; 온화한 교반과 함께 RT에서 2 시간 동안 에탄올 20ml에 -hydroxysuccinimide (NHS, 0.1 nmol의). </li><li> 간단히 입자 원액을 초음파 처리하고 L 아르기닌 용액에 10 억 입자를 추가하고 부드러운 교반하면서 실온에서 18 시간 동안 반응을 둡니다. </li><li> N의 제 아스파라긴산 기 활성화 - (tert- 부 톡시 카르 보닐) -L- 아스파르트 산 (BOC-의 Asp-OH, 1 nmol의) 4 시간 동안 에탄올 20ml에 EDC (0.1 nmol의) / NHS (0.1 nmol의)과의 부드러운 교반하면서 4 ° C에서. </li><li> 10 ml의 에탄올에 50 mg의 폴리에틸렌 이민을 용해 및 BOC-의 ASP-OH 혼합물에 솔루션을 추가합니다. 반응이 부드러운 교반하면서 실온에서 24 시간 동안 진행하자. </li><li> 부드러운 교반하면서 6 시간 동안 4 ° C에서 EDC (0.1 nmol의) / NHS (0.1 nmol의)와 BOC-ASP를-OH / PEI 솔루션의 두 번째 아스파르트 산 그룹을 활성화합니다. </li><li> 일단 회복 된 입자를 얻으려면, 단계 1.8에서 BOC-ASP를-OH / PEI 솔루션으로 단계 1.5에서 입자 용액을 추가합니다. 반응이 완만 한 성으로 실온에서 18 시간 동안 할 수있게합니다<html> irring. </li><li> 펠렛을 중단 간단한 초음파 처리를 이용하여, XG 18,800에서 30 분 동안 용액을 원심 분리, 에탄올로 입자 용액을 세 번 세척 하였다. </li></ol> 2. 일단 회복 된 입자 특성 분석 <ol><li> 주사 전자 현미경 (SEM)을 사용하여 입자의 크기를 측정한다. <ol><li> 깨끗한 실리카 SEM 샘플 스텁에 입자 현탁액의 드롭 (에탄올에 10,000 개 / μL) 놓고 진공하에 실온에서 건조 할 수 있습니다. </li><li> 인 - 렌즈 검출기를 사용 3-5mm 작동 거리 8 kV의에서 SEM 이미지를 측정한다. </li></ol></li><li> 입자 분석기 시스템을 이용하여 입자의 제타 전위를 측정한다. <ol><li> 1 ㎖의 10 mM의 포스페이트 완충액 (pH 7.4)으로 입자 현탁액 10 μL 에탄올 (10,000 개 / μL)를 섞는다. </li><li> 절첩 모세관 세포에 샘플을 로딩하여 제조업체의 지시에 따른 제타 전위를 측정한다. </li></ol></li></ol>제작 &quot;&gt; 3. 일단 회복 된 입자에서 siRNA의로드 <ol><li> 진공 O / N에서 (PCPS 입자 제조 단계 1.9에서) 일단 회복 된 입자를 건조. </li><li> 건조 일단 회복 된 입자와 초음파 처리 살짝 siRNA를 핵산 분해 효소가없는 물 (20 μL) (4 μg의)를 추가합니다. siRNA의 비율로 다음과 같은 입자를 사용하여 2 × 105 개 / 0.2 μg의 siRNA를, 4 × 10 5 개 / 0.2 μg의 siRNA를, 6 × 10 5 개 / 0.2 μg의 siRNA를, 8 × 10 5 개 / 0.2 μg의 siRNA를, 10 × 10 5 개 / 0.2 μg의 siRNA를 12 × 105 개 / 0.2 μg의 siRNA의. </li><li> siRNA를가 입자에 결합 할 수 있도록 통에 4 ° C에서 3 시간 (1000 RPM)에 대해 품어. </li></ol> siRNA를 / PCPS 입자 비율 4. 최적화 <ol><li> siRNA의 비율에 다른 입자 (PCPS 입자에서 siRNA의로드 참조) PCPS / 제어 siRNA의 입자의 20 μL에 DNA 로딩 염료를 추가합니다. </li><li> 샘로드다 음과 DNA 겔 얼룩을 함유하는 2 % 아가로 스겔로. </li><li> 유동 완충액에서 20 분 동안 120 V의 정전압으로 전기 영동을 수행한다. </li><li> 이미지 획득 및 분석 소프트웨어 겔 분석. </li></ol> 일단 회복 된 입자에서의 siRNA의 5 출시 <ol><li> 소듐 도데 실 설페이트 (SDS, 2 %)에서 siRNA의 비율 (단계 3)와 상이한 입자 PCPS / 제어 siRNA를 입자의 20 μL를 혼합하고 실온에서 1 시간 동안 정치하자. </li><li> 샘플 DNA 로딩 염료를 추가합니다. </li><li> DNA 젤 얼룩을 포함하는 2 % 아가 로스 젤에로드 샘플. </li><li> DNA 전기 영동 장치 및 전원 공급 장치를 사용하여 유동 완충액에서 20 분 동안 120 V의 정전압으로 전기 영동을 수행한다. </li><li> 이미지 획득 및 분석 소프트웨어 겔 분석. </li></ol> 일단 회복 된 입자의 6 공 초점 현미경 <ol><li> PCPS / 형광 제어 siRNA의 입자 (10 × 10 5 개 / 0의 5 μl를 추가합니다.유리 커버 슬립 2 μg의 siRNA를 / 20 μL). </li><li> 공 초점 현미경으로 입자 층을 시각화. </li></ol> 의 Arg-PEI / 제어의 siRNA 나노 입자의 7 특성 <ol><li> 실리콘 물질을 저하시키고의 Arg-PEI / 제어 siRNA의 나노 입자를 형성하기 위해, 포스페이트 완충 염수 100 ㎕로 PCPS / siRNA의 입자 (10 × 106 PCPS 입자 / 2 μg의 siRNA를)를 첨가하고, 37 ℃에서 (1000 RPM) 흔들 이일. </li><li> 18,800 XG에서 30 분 동안 샘플을 원심 분리기 상층 액을 수집합니다. </li><li> 동적 광산란 (DLS)으로 형성의 Arg-PEI / siRNA의 나노 입자의 크기를 측정한다. <ol><li> 1 ml의 10 mM의 인산염 완충액 (pH 7.4)으로 뜨는 10 μl를 혼합하고 플라스틱 큐벳에 배치합니다. </li><li> 제조업체의 지시에 따라 입자 분석기 시스템을 이용하여 입자의 크기를 측정한다. </li></ol></li><li> 형성의 Arg / PEI-의 siRNA의 크기와 형태를 결정원자 힘 현미경으로 나노 입자. <ol><li> 실리콘 웨이퍼 상에 뜨는 위치의 10 μl를 가라. </li><li> 원자력 현미경 (AFM)으로 입자를 시각화. </li></ol></li></ol> 8. 세포 배양 <ol><li> 세포 배양 배지에서 배양 MDA-MB-231 인간 유방암 세포를 10 % 소 태아 혈청과 1 % 페니실린 - 스트렙토 마이신 5 % CO 2, 95 % 습도에서 37 ° C로 보충. </li></ol> 일단 회복 된 입자와 라이브 세포의 9 공 초점 현미경 <ol><li> 2 웰 배양 플레이트 세포는 24 시간 동안 / 웰 3 × 10 5 세포의 파종 밀도 슬라이드. </li><li> 세포에 형광 제어의 siRNA (10 × 10 5 개 / siRNA의 비율이 0.2 μg의 입자, 50 nM의 siRNA를)로드 일단 회복 된 입자를 추가합니다. </li><li> 입자 엑스포 다음 12 시간 동안 (실, 5 % CO 2, 95 %의 습도와 37 ° C로 제공됨) 공 초점 현미경으로 세포의 동영상 촬영확인합니다. </li></ol> 일단 회복 된 입자와 고정 세포의 10 공 초점 현미경 <ol><li> 2 웰 배양 플레이트 세포는 24 시간 동안 / 웰 3 × 10 5 세포의 파종 밀도 슬라이드. </li><li> 세포에 형광 제어의 siRNA (10 × 10 5 개 / siRNA의 비율이 0.2 μg의 입자, 50 nM의 siRNA를)로드 일단 회복 된 입자를 추가 1 일 7 일 10 일 동안 배양한다. </li><li> 인산염 완충 생리 식염수로 두 번 세포를 세척 한 다음 10 분 동안 4 % 파라 포름 알데히드 용액을 수정합니다. </li><li> 인산염 완충 생리 식염수 세포를 씻으십시오. </li><li> 10 분 동안 0.1 % 옥틸 페놀에 톡실 레이트와 세포를 Permeabilize 하시려면 다음 그들에게 인산염 완충 생리 식염수로 3 회 세척한다. </li><li> 부드럽게 교반하면서 RT에서 10 분 동안 인산 완충 생리 식염수에 소 혈청 (10 ㎎ / ㎖)에서 알부민 세포를 차단. </li><li> (1 μL / 40 μl를 형광 표지 팔로이 딘과 세포를 배양, 사상 액틴을 시각화하는 방법부드러운 교반하면서 실온에서 20 분 동안 솔루션을) 차단 한 후 인산염 완충 식염수로 세척한다. </li><li> 프레임에서 슬라이드를 제거하고 핵을 시각화하기 위해 4 &#39;, 6 diamidino -2- 페닐 인돌 (DAPI)와 antifade 시약을 추가합니다. </li><li> 상단에 커버 유리를 추가하고 공 초점 현미경으로 세포의 이미지를 가져 가라. </li></ol> PCPS / 형광 제어 siRNA의 입자와 세포의 11 유동 세포 계측법 <ol><li> 24 시간 동안 / 웰 3 × 10 5 세포의 파종 밀도에서 6 웰 플레이트에 플레이트 세포. </li><li> 세포에 형광 제어의 siRNA (10 × 10 5 개 / siRNA의 비율이 0.2 μg의 입자, 50 nM의 siRNA를)로드 일단 회복 된 입자를 추가하고 24 시간 동안 배양한다. </li><li> 인산염 완충 생리 식염수 세포를 씻고 셀 스크레이퍼로 긁어. </li><li> 분석에 앞서 2 % 우 태아 혈청 인산염 완충 생리 식염수에 세포를 보관합니다. 음성 대조군으로 치료 세포를 사용합니다. </li><li> 흐름 CY를 수행tometry. </li></ol> 일단 회복 된 입자 및 PCPS / 제어 siRNA의 입자와 세포의 12 세포 생존 <ol><li> 24 시간 동안 / 웰 3 × 10 3 세포의 세포 밀도로 96 웰 플레이트에 플레이트 세포. </li><li> PCPS 입자로 세포를 처리 (1.5 × 105 / 웰, 6 × 105 / 웰) 또는 PCPS / 제어 siRNA의 입자 (48) (10 × 10 5 개 / siRNA의 비가 0.2 μg의 입자, 10 nM 내지 100 nM의 siRNA의) 시간과 72 시간. 컨트롤 등의 인산염 완충 생리 식염수 (추가 된 입자와 같은 볼륨)로 처리 처리하지 않은 세포와 세포를 사용합니다. 세중 각 샘플을 분석. </li><li> 제조업체의 지시에 따라 세포 증식 분석을 수행한다. </li><li> 표준 편차 평균 ± 같은 데이터를 나타냅니다. </li></ol> PCPS / ATM 변이 된 siRNA의 입자와 세포의 13 서쪽 오점 <ol><li> 셀 밀도 2 × 10 5 세포 / w에게에서 6 웰 플레이트에 플레이트 세포24 시간 동안 엘. </li><li> PCPS / 제어의 siRNA (50 ㎚) 입자 또는 PCPS / ATM siRNA를 72 시간 동안 (50 ㎚) 입자와 세포를 품어. 대조군으로 치료 세포를 사용합니다. </li><li> 를 Lyse 프로테아제 저해제 칵테일로 보충 단백질 추출 시약을 이용하여 세포. </li><li> 14,000 XG에서 10 분 동안 세포 용 해물을 원심 분리기와 뜨는을 복구 할 수 있습니다. </li><li> 제조업체의 지시에 따라 단백질 정량 분석​​으로 단백질 농도를 결정한다. </li><li> (5 μL 2- 머 캅토 에탄올 / ㎖ 버퍼) 샘플 로딩 버퍼를 추가 샘플 및 99 ° C에서 6 분을 가열한다. </li><li> 유동 완충액에서 12 % SDS 폴리 아크릴 아미드 겔에서의 단백질 시료 (20 ㎍ / μL)을로드하고, 전기 영동 장치 및 전원 공급 장치를 사용하여 폴리 아크릴 아미드 겔 전기 영동 (1 시간, 120 V)를 수행한다. </li><li> 전기 영동 장치를 이용하여 니트로 셀룰로오스 막 (1 시간, 100 V) 내지 (20 % 메탄올로) 전송 버퍼에서 젤 옮기고전원 공급 장치. </li><li> 1 시간 동안 5 % 분유와 멤브레인을 차단합니다. </li><li> 1000 희석 O / N : 1 (단계 13.9)에서 차단 솔루션에 (토끼)에서 ATM 차 항체와 멤브레인을 품어. </li><li> 1 시간 동안 2500 희석 : 1에서 0.1 % 폴리에틸렌 글리콜 소르 비탄 모노 라우 레이트를 함유하는 인산염 완충 식염수로 멤브레인을 세척하고 (단계 13.9)에서 차단 용액에 이차 항체 (항 - 토끼)와 함께 배양한다. </li><li> 0.1 % 폴리에틸렌 글리콜 소르 비탄 모노 라우 레이트를 함유하는 인산염 완충 식염수로 세척하고 막을 이미지 획득 및 분석 소프트웨어를 사용하여 웨스턴 블롯 검출 시약과 단백질 밴드를 검출한다. </li><li> 부하 제어를 위해, 막 씻어 반복 (마우스에서 1 : 10,000 희석) β - 굴지 차 항체를 사용하여 13.9-13.12 단계 (항 - 마우스 1 : 4000 희석)와 이차 항체를. </li></ol>

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The authors declare that they have no competing financial interests.

이 프로토콜은 세포로의 siRNA의 형질 감염과 성공적으로 전달하기위한 방법을 설명한다. 특히,의 siRNA 전달 폴리 양이온 - 작용 PSI 입자로 구성된 다기능 플랫폼을 사용함으로써 달성된다. siRNA의 치료의 사용은 여러 종양 유전자가 높은 특이성으로 타겟팅 할 수있는 큰 잠재력 예, 암 치료를 갖는다. 따라서, siRNA의 치료 문제를 완화 할 수있는 siRNA를 전달 비히클을 개발할 필요가 존재한다. ...

작은 방해 RNA들 (siRNA가)는 유전자의 발현을 억제 할 수있는 이중 가닥 RNA 분자이다. 최근, siRNA는 임상 응용 1-5하는데있어서의 장래의 사용을 위해 치료 가능성을 보여 바이오 의약품의 차세대로서 개발되어왔다. 그러나, siRNA의 치료의 성공적인 구현 인해 클레아 불량 세포 내 흡수, 낮은 형질 감염 효율 및 엔도 / 5 리소좀에서 비효율적 릴리스 저하, 상당한 도전 남아있다. 이러한 장애의 대부분은 안전하고 효율적으로 환부 조직으로 siRNA를 전달을 제공 할 수있는 플랫폼의 개발에 의해 극복 될 수있다. 바이러스 캐리어에 비해 비 바이러스 플랫폼은 안전, 저가 및 원단의 용이성 등 여러 가지 이점을 제공한다. 이러한 폴리머 및 지질 등의 특정 성, 양이온 성 나노 입자에서, siRNA를 전달 3 유용 증명했다. 이전에, 우리는 원반형 닥터 개발전달 시스템 UG, 다단 벡터 (MSV)를 불린다. 이 플랫폼은 하나가 다른 차량으로부터 방출되는 연속 한 단계에 기초한다. 두번째 단계 차량 약물이나 조영제 -6,7- 로케이션 나노 입자 인 반면 제 1 스테이지 차량 생분해 다공성 실리콘 (PSI)에서 만든 미립자이다. 시 8 저하로 PSI 재료에 포함 된 나노 입자는, 서서히 방출된다. 실리콘 입자를 사용하는 이점은 형태 및 표면 특성을 용이하게 최적의 생체 내 분포 및 약물 방출을 달성하기 위해 맞추어 질 수 있다는 것이다. 최근에, 종양 조직에 대한 siRNA를 리포좀의 배달 MSV 플랫폼의 성공적인 사용은 난소 및 유방암 마우스 모델 9,도 10에 도시 하였다. 이 작품에서 우리는 MSV 플랫폼의 원칙에 따라 siRNA를위한 보편적 인 전달 시스템을 제작했다. 본 전달 시스템의 효능은 이전 우릴 입증되었다다른의 siRNA를 보내고 11 개의 분자. 시스템은 폴리에틸렌 이민 (PEI) 및 아르기닌 (의 Arg)로 이루어지는 그래프트 PSI, 폴리 양이온 기능화 된 다공성 실리콘 (PCPS) 담체이다. , 여기서 Arg 및 PSI는 PEI의 독성을 감소시키는 역할을 할 수있는 동안 이전 11 demonstarted로 PEI는 siRNA 로의 정전 기적 ​​상호 작용을 형성하는데 도움을 줄 수있다. PSI 미립자 siRNA를 보호 및 지속 방출을 가능하게하면서 또한, PEI의 존재는 세포 내 흡수 및 엔도 좀 탈출 어시스트 할 수있다. PSI 입자 서서히함으로써 뚜렷한 형태 및 좁은 크기 분포를 갖고 11의 Arg-PEI / siRNA의 나노 입자 (도 1)의 형성의 결과로, 생리적 조건 하에서 분해. PCPS / siRNA의 시스템의 안정성에 관한 자세한 내용은 쉔 등. (11)에 의해 연구를 참조하시기 바랍니다. 두번째 단계의 나노 입자가 초기 prese 나지 않으므로 PCPS 플랫폼은, 종래 MSV 상이1 단 캐리어 (11), (12), NT 퇴화 캐리어하지만 시간에 걸쳐 형성된다. PCPS 시스템의 siRNA의 적재 효율, 세포 독성 유전자 침묵 효율이 시험관 내에서 평가되었다. 형질 전환 효율은 DNA 복구에 관여하는 10 (ATM) 종양 유전자 돌연변이 실조증 확장증 대해 siRNA를 이용하여 측정 하였다. 이전에는, ATM의 억제는 유방암 모델 (10)에서 종양 성장을 감소시키는 것으로 밝혀졌다.

<table border="0" cellpadding="0" cellspacing="0" width="1812">
	<tbody>
		<tr height="55">
			<td height="55" style="height:55px;width:605px;">Name of the Material/Equipment</td>
			<td style="width:421px;">Company/Institution</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:653px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Polyethylenimine (PEI), branched</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408727</td>
			<td style="width:653px;">Average molecular weight ~25,000 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">L-Arginine</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A5006</td>
			<td style="width:653px;">Reagent grade, &#8805;98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Boc-Asp-OH</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408-468</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">(3-&#8203;Aminopropyl)&#8203;triethoxysilane</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">440140</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Hydrogen peroxide solution</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">216763</td>
			<td style="width:653px;">30&#160;wt. % in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Sulfuric acid</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">339741</td>
			<td style="width:653px;">100.00%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Isopropyl alcohol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">W292907</td>
			<td style="width:653px;">&#8805;99.7%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Ethanol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">459844</td>
			<td>&#8805;99.5%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>03449</td>
			<td>&#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-10G</td>
			<td>Blocking agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Ataxia-telangiectasia mutated siRNA&#160;</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Designed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Tris Acetate-EDTA buffer</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">T9650</td>
			<td style="width:653px;">For DNA agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Penicillin-Streptomycin</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fetal Bovine Serum</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>F2442</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">TWEEN 20</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:653px;">Polyethylene glycol sorbitan monolaurate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">2-Mercaptoethanol</td>
			<td style="width:421px;">Sigma Aldrich</td>
			<td>M6250</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium dodecyl sulfate</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>L3771</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate monobasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71496</td>
			<td style="width:653px;">For making phosphate buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate dibasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71640</td>
			<td style="width:653px;">For making phosphate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A4416</td>
			<td style="width:653px;">Secondary antibody (anti-mouse) for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">N-Hydroxysuccinimide (NHS)</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">130672</td>
			<td style="width:653px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td>
			<td style="width:421px;">Greiner Bio-One</td>
			<td style="width:133px;">655182</td>
			<td style="width:653px;">96-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">10X Tris-Glycine Liquid</td>
			<td style="width:421px;">Li-Cor&#160;</td>
			<td style="width:133px;">928-40010</td>
			<td style="width:653px;">Transfer buffer for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Paraformaldehyde solution 4% in PBS&#160;</td>
			<td style="width:421px;">Santa Cruz</td>
			<td>Sc-281692</td>
			<td style="width:653px;">Fixation of cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td>
			<td style="width:421px;">Promega</td>
			<td style="width:133px;">G5421</td>
			<td style="width:653px;">Proliferation assay</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Phosphate buffered saline</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP399-500</td>
			<td style="width:653px;">10 X Solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Corning cellgro Dulbecco&#39;s Modification of Eagle&#39;s (Mod.)</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">MT-15-017-CM</td>
			<td style="width:653px;">Cell culture media, 1X solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Triton X-100</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>AC21568-2500</td>
			<td style="width:653px;">Octyl phenol ethoxylate, permeabilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Cover glass</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>12-530C&#160;</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Methanol</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td>A412-1</td>
			<td style="width:653px;">For Western blot transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Plastic Cuvettes</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">14-377-010&#160;</td>
			<td style="width:653px;">For size measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Molecular BioProducts RNase AWAY Surface Decontaminant</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">14-754-34</td>
			<td style="width:653px;">Spray for removing RNAse contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Agarose</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP165-25</td>
			<td style="width:653px;">Low melting point, for running RNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td style="width:421px;">Invitrogen</td>
			<td>P36935</td>
			<td style="width:653px;">Antifade reagent with DAPI, nucelus detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">A12379</td>
			<td style="width:653px;">Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">S33102</td>
			<td style="width:653px;">Visualization of RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Negative Control siRNA</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1022076</td>
			<td style="width:653px;">Control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">AllStars Neg. siRNA AF 555</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1027294</td>
			<td style="width:653px;">Fluorescent control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Cell scraper</td>
			<td style="width:421px;">Celltreat</td>
			<td>229310</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite Multidishes and Microwell Plates</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>130184</td>
			<td style="width:653px;">6-well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce LDS Sample Loading Buffer (4X)&#160;</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>84788</td>
			<td style="width:653px;">Sample loading buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Pierce BCS Protein Assay Kit</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">23227</td>
			<td style="width:653px;">Protein quantification assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halt Protease Inhibitor Single-Use Cocktails (100X)</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">78430</td>
			<td style="width:653px;">Protease inhibitor cocktail, use at 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-PER Mammalian Protein Extraction Reagent</td>
			<td style="width:421px;">Thermo Scientific&#160;</td>
			<td style="width:133px;">78501</td>
			<td style="width:653px;">Protein extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sorvall Legend Micro 21R</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Beta Actin Antibody&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>MA1-91399</td>
			<td>&#946;-actin primary antibody (from mouse) for western blor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6X TriTrack DNA Loading Dye</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>R1161</td>
			<td style="width:653px;">DNA loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-Free Water&#160;</td>
			<td style="width:421px;">Life Technologies</td>
			<td>AM9938</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Fat Dry Milk</td>
			<td style="width:421px;">Lab Scientific</td>
			<td>M0841</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well BD Falcon culture slides&#160;</td>
			<td style="width:421px;">BD Biosciences</td>
			<td>354102</td>
			<td style="width:653px;">2-well culture slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Western blot detection reagent.&#160;</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td style="width:133px;">RPN2106</td>
			<td style="width:653px;">Western blot detection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BA Membranes</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td>10402096</td>
			<td style="width:653px;">Nitrocellulose membrane for Wester blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATM (D2E2) Rabbit mAb</td>
			<td style="width:421px;">Cell Signaling</td>
			<td>2873S</td>
			<td style="width:653px;">ATM primary antibody (from rabbit) for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:421px;">Cell Signaling&#160;</td>
			<td>7074</td>
			<td style="width:653px;">Secondary antibody (anti-rabbit) for Western blot&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Folded capillary cells</td>
			<td style="width:421px;">Malvern&#160;</td>
			<td style="width:133px;">DTS 1061</td>
			<td style="width:653px;">For zeta potentail measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">MDA-MB-231 cell line</td>
			<td style="width:421px;">ATCC</td>
			<td style="width:133px;">HTB-26</td>
			<td style="width:653px;">Mammary Gland/Breast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">12% Mini-PROTEAN TGX Gel</td>
			<td>Bio-rad</td>
			<td>456-1043&#160;</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biorad PowerPac HC</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td>Power supply for electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-rad</td>
			<td>161-0732</td>
			<td>Running buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wide Mini-Sub Cell GT Cell</td>
			<td>Bio-rad</td>
			<td>170-4405</td>
			<td>Electrophoresis equipment for DNA agarose gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra cell</td>
			<td>Bio-rad</td>
			<td>165-8000</td>
			<td>Electrophoresis equipment for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc XRS+ System with Image Lab Software</td>
			<td>Bio-rad</td>
			<td>170-8265</td>
			<td>Image acquisition and analysis software for gels and blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; (10cm) dia., 5x7mm diced Silicon Wafer</td>
			<td>Ted Pella&#160;</td>
			<td>16007</td>
			<td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td>Shaker&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoton II diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td>Isoton diluent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td>Particle counting analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Non-functionalized porous silicon particles&#160;</td>
			<td style="width:421px;">Microelectronics Research Center, University of Texas at Austin</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Dicoidal shape. 2.6 &#956;m (diameter) x 0.7 &#956;m (hight), provided in isopropyl alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Zetasizer Nano ZS</td>
			<td style="width:421px;">Malvern</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle analyzer system for size and zeta potential&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Scanning Electron Microscope</td>
			<td style="width:421px;">FEI</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Atomic Force Microscope</td>
			<td style="width:421px;">Bruker</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fluo ViewTM 1000 Confocal Microscope</td>
			<td style="width:421px;">Olympus</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Visualization of fixed and live cells</td>
		</tr>
	</tbody>
</table>

porous silicon microparticles for delivery of sirna therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

이 프로토콜은 안전하고 효율적인 siRNA를 형질 전환 용 비 바이러스 전달 시스템의 사용 방법에 대해 설명한다. SEM 결과 PCPS 입자는 원통형 형상이고 2.6 μm의 (도 2a)의 직경을 갖는 것을 알 수있다. 입자는 긍정적으로하여 음으로 대전 된 뉴클레오티드 바인딩 정전기를 가능하게 약 8.21의 제타 전위 (그림 2B)로 청구됩니다. 일단 회복 된 입자의 서로 다른 계층의 공 촛점 이...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

배달은 작은 간섭 RNA (siRNA의)의 치료 구현을위한 주요 과제로 남아. 이 프로토콜은 아르기닌 및 폴리에틸렌 이민 다공성 실리콘 그래프트 미립자로 구성된 다기능 및 생체 적합성의 siRNA 전달 플랫폼의 사용을 포함한다.

siRNA를 치료제의 전달을위한 다공성 실리콘 미세 입자

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.8K 조회수.  Houston Methodist Research Institute.  배달은 작은 간섭 RNA (siRNA의)의 치료 구현을위한 주요 과제로 남아. 이 프로토콜은 아르기닌 및 폴리에틸렌 이민 다공성 실리콘 그래프트 미립자로 구성된 다기능 및 생체 적합성의 siRNA 전달 플랫폼의 사용을 포함한다. 

비디오: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

소프트 리소그래피 작용화 및 Patterning 산화물없는 실리콘과 게르마늄

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

연구

생체공학

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

조직 공학 알긴산 미세 입자와 Thermogelling 폴리 (N- 이소 프로필) -graft - 콘드로이틴 황산 복합 재료의 합성

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

양이온과 음이온 리포좀을 사용 CNS에 치료의 siRNA 배달

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

신경 성장 인자의 사업자로 서 다공성 실리콘 필름을 디자인

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D 인쇄 다공성 셀 루 로스 나노 하이드로 겔 건설 기계

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

사이토 솔 릭 siRNA 전달을 위한 중성 충전, pH 반응 형 고분자 나노 입자의 제조

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

실리콘 나노 와이어 및 세포 간 전기 커플링의 조사를위한 광학 자극

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

실리콘 평면 피질 내 미세 전극의 표면 처리를위한 도구

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...