이 작품은 부분적으로 교육 싱가포르 교육부 (CX에 RG 12분의 64)과 나노 의학의 NTU - 노스 웨스턴 대학에서 1 등급 학술 연구 기금에 의해 지원되었다.

1. 준비 <ol><li> 100 ㎖ 둥근 면도 왕수 (농질산 1 부 (HNO 3), 농축 염산 (HCL) 3 부) 및 DI 물로 씻어 플라스크를 이용하여 바닥 플라스크. 플라스크를 오토 클레이브에서 15 분 동안 100 ℃에서 열풍 오븐에서 건조한다. 싸서 사용할 때까지 무균 플라스크를 저장합니다. </li><li> 70 % 에탄올을 사용하는 휴대용 소형 압출기 세트 소독. </li><li> 회전 증발기를 켜고 각각 37 ° C 4 ° C의 온수 욕조와 냉각탑의 온도를 설정합니다. </li><li> 10 ml의 0.1 mm의 인산염 완충 생리 식염수 (PBS) (PH 7.4)에 칼 세인의 374 mg을 용해하여 원액을 칼 세인 60 밀리미터를 준비합니다. 1 M 수산화 나트륨 (NaOH) 용액을 사용하여 7.4의 pH를 조정한다. </li></ol> 리포좀 2. 합성 <ol><li> 지질을 제거 (1,2- Dipalmitoyl- SN -glycero -3- 포스 포 콜린 (DPPC), 1- 팔미 토일 -2- 하이드 록시 SN -glycero -3- phosphocho라인 (MPPC) 및 1,2- distearoyl- SN -glycero -3- phosphoethanol - 아민 N - [카르복시 (폴리에틸렌 글리콜) -2000 (암모늄 염) (PEG2000-DSPE))에서 냉동 (-20 ° C) 및 RT로 해동. </li><li> 15.9 mg의 DPPC, 1.3 mg의 MPPC, 2.8 mg의 DSPE-PEG2000 무게. 2 ml의 클로로포름을 함께 녹여. </li><li> 멸균 된 둥근 바닥 플라스크에 클로로포름 용액을 전송하고 얇은 건조한 지질 층을 형성하기 위해, 감압하에 회전 증발기를 사용하여 용매를 증발시켰다. </li><li> 단계 1.4에서 50 μL의 Au NPS를 (3.36 × 1016 입자 / ㎖)를 제조 칼 세인 1.95 mL의 60 mM의 함유를 30 분 동안 2 ㎖의 수용액 45 ° C에서 지질 층 수화물. </li><li> 45 ° C까지 가열 블록 예열. 필터 지지체와 200 nm의 폴리 카보네이트 멤브레인 필터를 놓고 미니 압출기 세트를 조립한다. DI 물 잠재적 인 누출을 확인합니다. </li><li> 단계 2.4 extru에서 리포좀 용액 1 ㎖와 주사기 중 하나를 입력합니다조립 미니 압출기의 타단 주사기 용액을 통과하여 샘플 드. 11 번 반복합니다. </li><li> 무료 금 NP에, 제조 업체의 프로토콜에 따라 용리액으로 PBS를 이용하여 지질과 칼 세인을 제거하기 위해 PD-10 탈염 칼럼을 통해 합성 된 리포좀을 실행합니다. </li><li> 4 ° C에서 멸균 튜브에 샘플을 보관하고 2 일 내에 사용합니다. </li></ol> 난방과 리포좀 3. 칼 세인 출시 <ol><li> 단계 2.2 DPPC, MPPC 및 DSPE-PEG2000의 몰 농도를 추가하여 원액의 지질 몰 농도를 계산합니다. 0.1 mM의 PBS 완충액 (pH 7.4)을 이용하여 5 mM의 지질 농도 리포솜 원액을 희석. 원심 분리 튜브 (2 ㎖)에 샘플을 이동. </li><li> 온수 욕에서 튜브를 배치하고 25 내지 70 ℃로 서서히 승온. 여기에 1 ℃ / min의 속도로 온도를 증가시킨다. </li><li> 다른 온도 점 (27, 32, 37, 39, 41 분액 (10 μL)을 모아서43, 45, 52, 57, 62, 67 및 70 ° C). </li><li> RT에서 10 분 동안 리포좀을 소화하고 칼 세인의 완전한 방출을 달성하기위한 리포좀 용액을 2 % 트리톤 X-2~100 mL의 분취 량의 10 μL를 추가한다. </li><li> 96 웰 마이크로 플레이트의 각 웰에 리포좀 용액 200 μl를 전송 및 형광 마이크로 플레이트 판독기를 사용하여 수집 된 샘플의 형광 강도를 측정한다. 칼 세인의 여기 및 방출 파장은 각각 480 및 515 나노 미터이다. </li><li> 100 % 방출로 트리톤 X-100 처리 된 샘플의 형광 강도를 고려하면, 상기 식을 이용하여 각 시점에서의 calcein 백분율을 계산 : <img alt="Formula1" src="/files/ftp_upload/53619/53619formula1.jpg" /> 피트 주어진 시점에서 용액의 형광 강도이다. F에서 I 및 F의 X는 각각 용액의 초기 및 최종 표준화 형광 강도이다. </li></ol> 4. 칼 세인 다시펄스 레이저와 리포좀에서 임대 <ol><li> 석영 큐벳에 100 ㎕의 리포좀 솔루션을 전송하고 큐벳 홀더에 넣습니다. 광원으로서 532 nm 파장에서 6 나노초의 펄스 지속 시간 : YAG 레이저를 사용하여 펄스의 Nd. 1 Hz에서 : 반복 속도 - 레이저 매개 변수를 다음과 같이 사용 레이저 에너지 밀도 : 1 엠제이 / cm 2; 빔 직경 : 0.5 mm. </li><li> 광 리포좀 용액을 통과하도록 큐벳으로 평행 레이저 광을 안내하고 다양한 펄스 후에 분취 액을 수집한다. </li><li> RT에서 10 분 동안 리포좀을 소화하고 칼 세인의 완전한 방출을 달성하기위한 리포좀 용액을 2 % 트리톤 X-2~100 mL의 분취 량의 10 μL를 추가한다. </li><li> 칼 세인을 감안하면 빛에 민감 레이저 실험 동안 탈색 될 수있는, 미리 측정 리포솜 릴리스 데이터를 정상화 칼 세인 용액에 펄스 레이저의 표백 효과. <ol><li> 특히, 칼 세인 용액 (60 mM)을 펄스 레이저 위스콘신에 노출펄스 수 (0, 25, 50 및 100)를 변화시키기위한 제 1 Hz에서의 주파수. 전과 레이저 노광 후, 각각 480 및 515 나노 미터의 여기 및 발광 파장 칼 세인의 형광 강도를 측정한다. </li><li> 표백의 양 (특정 펄스 수 후 칼 세인의 레이저 노광 / 형광 강도 전에 칼 세인의 형광 강도)를 계산한다. 인자 리포좀의 형광 강도를 곱하여 리포좀 샘플로부터 얻은 값을 정규화하기 위해이 비율을 사용한다. </li></ol></li><li> 각각 480 및 515 nm에서의 여기 및 방출 파장에서 형광 마이크로 플레이트 판독기를 사용하여 수집 된 샘플의 형광 강도를 측정한다. </li><li> 100 % 방출로 트리톤 X-100 처리 된 샘플의 형광 강도를 고려하면, 상기 식을 이용하여 각각의 펄스 수에서의 calcein 백분율을 계산 : <img alt="Formula1" src="/files/ftp_upload/53619/53619formula1.jpg" /> I 및 F의 X는 각각 용액의 초기 및 최종 표준화 형광 강도이다. </li></ol> 압력 자극 5. 측정 <ol><li> 현미경 슬라이드에 시료 100 ㎕를 놓고 샘플에 레이저의 초점을 설정합니다. </li><li> 용액에 바늘 수중 청음기 (1 mm 직경 450 네바다 / 아빠 감도)를 빠져. 주의 : 하이드로는 손상을 방지하기 위해, 레이저 광에 의해 조명 될 안된다. </li><li> 다양한 펄스 수 (0 ~ 100)과 펄스 에너지 (20-160 μJ / 펄스)과 펄스 레이저와 샘플을 조사. </li><li> 디지털 오실로스코프를 사용하여 압력 신호를 기록한다. </li></ol>

<ol>
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관심 없음 충돌이 선언되지 않습니다.

박막 수화 리포좀을 제조하는 통상적 인 방법이다. 유기 용매 (여기에서는 클로로포름) 제 플라스크에 지질 박막을 생성하기 위해 37 ℃에서 회전 증발기에서 지질을 용해하는 데 사용하고 제거 하였다. 이 지질 막을 60 mM의 칼 세인 5 nm의 금 NPS를 함유하는 수용액으로 수화 하였다. 수화 과정 동안, 온도는 50 ° C의 주위를 유지하고, 플라스크를 지속적 플라스크를 회전시켜 교반 하였다. 이 단계에?...

가능성은 극대화 특이 최소한의 부작용과, 공간 - temporal- 및 투여 량 조절 패션에 약물을 전달하는 매력적인 방법을 외부 자극이다 사용하여 약물 방출을 유발합니다. 외인성 자극 - 반응 시스템 (빛, 자기장, 초음파, 마이크로파 방사선)의 넓은 범위 중, 빛 트리거 플랫폼은 병원에서의 비 침습, 단순성 및 적응성에 의해, 매력적이다. 지난 10 년 1 광범위한 연구 그러나 이러한 근적외선 광 책임 금 플랫폼 기술의 다양성을 제공하고있다 (호주) nanocages 스마트 중합체 코팅, 약물 3 및 자기 조립 porphysome의 nanovesicles와 접합이 포토 - 불안정성 중합체 나노 입자 (NP에). (4) 이러한 기술 개발의 전임상 단계에서 정지하고, 개시하고 계속 과정에 관련된 파라미터들의 명확한 이해 및 최적화를 필요약물 방출 롤링. 이러한 시스템의 제조를위한 단순하고 용이하게 접근 방법 중 하나는 시장에서 널리 이용되고 광범위 전임상과 임상 실험에도 조사 된 둘 열 민감성 리포좀 5,6-과 금 NPS를 통합하는 것이다. 작은 동물이나 인간의 국소 전달을 위해 사용하는 경우 근적외선 활성화 금 나노 구조 (예를 들어, nanocages)에 비해 자신의 플라즈몬 파장 금 NP에의 깊은 조직 활성화의 한계에도 불구하고,이 시스템은 여전히 큰 약속을 보유하고있다. 7 빛 트리거 출시 리포좀과 금 NPS를 조합에서 일부 초기의 노력이 있습니다. 8-11 대부분이 재료의 참신에 집중하는 동안, 접근성 및 확장 성 문제를 해결해야합니다. 또한, 이러한 nanocarriers를 사용하여 분리 메커니즘에 대한 보고서는 여전히 제한되어 있습니다. 여기서의 제조 광 반응동시에 약물과 친수성의 Au NP에 탑재 리포좀이 설명되었다. 칼 세인이 캡슐화 효율 및 시스템의 방출 프로파일을 평가하기 위해 모델 화합물로서 사용된다. 또한,이 시스템에서의 Au NP에 의해 흡수되는 광은 국부 온도의 증가의 결과, 열의 형태로 주위의 미세 환경을 발산. 에어 마이크로 버블은 레이저 가열 중에 생성 된 리포좀 (도 1)의 기계적인 파괴의 원인이된다. 마이크로 버블 공동 현상의 메커니즘은 수중 청음기 측정에 의해 확인된다.

<table border="0" cellpadding="0" cellspacing="0" width="943">
	<tbody>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">850355P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC)</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">855675P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000)&#160;</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">880120P</td>
			<td dir="LTR" style="width:213px;">Powder, Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Gold Nanoparticles</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">752568-100mL</td>
			<td dir="LTR" style="width:213px;">5nm particles, stabilized at 0.1mM PBS</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Calcein</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">C0875-10g</td>
			<td dir="LTR" style="width:213px;">60mM, pH 7.4 - adjusted using NaOH</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:471px;">phosphate buffered saline (PBS)</td>
			<td dir="LTR" style="width:140px;">Sigma Aldrich</td>
			<td dir="LTR" style="width:119px;">P5493</td>
			<td dir="LTR" style="width:213px;">0.1 mM, pH 7.4</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Double distilled water</td>
			<td dir="LTR" style="width:140px;">Millipore Milli-DI water purification system</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;"></td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Triton X100&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Sigma, Life Sciences</td>
			<td dir="LTR" style="width:119px;">X-100</td>
			<td dir="LTR" style="width:213px;">To disrupt the liposomes to calculate total encapsulation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Rotavapor&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">R 210</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Heating bath</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">B 491</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Vacuum Controller&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">V-850</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Vacuum Pump</td>
			<td dir="LTR" style="width:140px;">Buchi (Switzerland)</td>
			<td dir="LTR" style="width:119px;">V-700</td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Recirculation bath with temperature controller</td>
			<td dir="LTR" style="width:140px;">Polyscience</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Used for Lipososme preparation</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">&#160;Mini-extruder assembly with heating block&#160;</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610000</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Syringes, 1000 uL</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610017</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Polycarbonate filter membrane, 200nm&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Whatmann</td>
			<td dir="LTR" style="width:119px;">800281</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Filter Support</td>
			<td dir="LTR" style="width:140px;">Avanti Polar Lipids (Alabama, US)</td>
			<td dir="LTR" style="width:119px;">610014</td>
			<td dir="LTR" style="width:213px;">Used for extrusion of liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">PD 10 Desalting coulumns, Sephadex G-25 medium</td>
			<td dir="LTR" style="width:140px;">GE Healthcare, Life sciences</td>
			<td dir="LTR" style="width:119px;">17-0851-01</td>
			<td dir="LTR" style="width:213px;">Used to purify the liposomes</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">Centrifuge&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Sigma Laboratory Centrifuges</td>
			<td dir="LTR" style="width:119px;">3K30</td>
			<td dir="LTR" style="width:213px;">Used to concentrate the liposomal solution&#160;</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:471px;">Rotor</td>
			<td dir="LTR" style="width:140px;">Sigma</td>
			<td dir="LTR" style="width:119px;">19777-H</td>
			<td dir="LTR" style="width:213px;">Used to concentrate the liposomal solution&#160;</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Zetasizer&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Nano ZS Malvern</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Used for the determination of liposome size and zetapotential</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:471px;">UV- Visible Spectrophotometer</td>
			<td dir="LTR" style="width:140px;">Shimadzu</td>
			<td dir="LTR" style="width:119px;">UV-2450</td>
			<td dir="LTR" style="width:213px;">Used to measure the absorbance of the samples</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Fluorescent Spectrofluorometer&#160;&#160;</td>
			<td dir="LTR" style="width:140px;">Molecular Devices</td>
			<td dir="LTR" style="width:119px;">SpectraMax M5</td>
			<td dir="LTR" style="width:213px;">Used to measure the fluorescence emission of the samples</td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:471px;">Nd:YAG Laser</td>
			<td dir="LTR" style="width:140px;">NewWave Research</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">532 nm; Maximum power: 17mJ; Width: 406 ns; Used for sample irradiation</td>
		</tr>
		<tr height="126">
			<td dir="LTR" height="126" style="height:126px;width:471px;">HNR Hydrophone</td>
			<td>ONDA</td>
			<td dir="LTR" style="width:119px;">HNR-1000</td>
			<td dir="LTR" style="width:213px;">1000 mm diameter and 450 nV/Pa sensitivity, Proper working frequency range: 0.25-10 MHz; Calibration: 50 mV/Bar; Used to measure the acoustic signals</td>
		</tr>
		<tr height="84">
			<td dir="LTR" height="84" style="height:84px;width:471px;">Digital Osciloscope</td>
			<td dir="LTR" style="width:140px;">LECORY - Wave Runner 64Xi-A</td>
			<td dir="LTR" style="width:119px;"></td>
			<td dir="LTR" style="width:213px;">Frequency: 600 MHz; Max sample rate : 10 Gs/s (at two channel); Used to record the measured acoustic signals</td>
		</tr>
	</tbody>
</table>

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.

10 : 4 또는 7.95 : 리포좀 86 몰비 DPPC, MPPC 및 DSPE-PEG2000과 종래의 박막 수화 기술을 사용하여 제조 된 0.65 :. 1.39 ㎎ / ㎖ (12)의 Au NP에의 크기는 광을 결정하는 것이 중요 다음 레이저 여기 실험 기간 동안의 변환 효율을 가열. 금 NP에의 작은 크기, 더 높은 13 따라서 5 nm의 금 NP에 공급 업체에서 가장 작은 샘플, 캡슐화를 위해 선택되었다. 열 변환 효율이다. 합성...

Watch this Scientific Journal Video about Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation at JoVE.com

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

이 프로토콜은 시판중인 물질과 광 반응 형 리포좀 집적 금 나노 입자에 대한 간단한 제조 방법을 설명하는데 이용된다. 또한, 펄스 레이저의 치료에 합성 리포좀 마이크로 버블 공동화 과정을 측정하는 방법을 보여준다.

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 ...

synthesis-gold-nanoparticle-integrated-photo-responsive-liposomes

Research

JoVE Journal

Bioengineering

8.1K 조회수.  Nanyang Technological University.  이 프로토콜은 시판중인 물질과 광 반응 형 리포좀 집적 금 나노 입자에 대한 간단한 제조 방법을 설명하는데 이용된다. 또한, 펄스 레이저의 치료에 합성 리포좀 마이크로 버블 공동화 과정을 측정하는 방법을 보여준다. 

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

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

높은 명령으로 Nanosheets에 Peptoid 폴리머 및 자기 조립의 고체 상 합성 Submonomer

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

연구

생체공학

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Monolayer의 합성, 조립, 그리고 특성화는 단백질 Monolayer의 전기에 대한 골드 Nanoparticle 필름을 보호

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

실버 씨앗 중재 성장 방법과 골드 Nanostar 합성

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

조합의 합성과 고분자 필름과 Nanoparticle 도서관에서 높은 처리량 단백질 출시

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

자기 공명 이미지 지침에 열성 리포좀에서 시스플라틴의 트리거 된 릴리스를위한 맞춤 설계 레이저 기반의 가열 장치

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

원격으로 업 컨버전 나노 중재 니 Photoactivation에 의해 세포내 신호 변환 하는 통합된 시스템

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

광 반응성 고분자 나노 캐리어로부터의 siRNA 방출의 시공간 조절을 통한 유전자 침묵 예측

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

기능성된 10 nm 폴리머-코팅 된 금 입자 피 대상에 대 한 및 약물 전달의 합성

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

준비, 정화, 및 지방산 포함 된 리의 사용

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

나노 입자와 그들의 항균 활동 및 세포 독성 속성의 평가 수정 다중 벽 탄소 나노튜브의 합성

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...