이 작품은 전립선 암 연구의 마가렛 공주 병원 재단, 국립 과학 및 캐나다 건강 연구, 혁신의 캐나다 재단에 대한 캐나다 연구소의 공학 연구 협의회, 그리고 조이와 토비 타넨 바움 / 브라질 공 의자,에 의해 지원되었다 SUNY 연구 재단과 버팔로 대학에서 시작 지원을 부여합니다.

1. Pyropheophorbide 지질의 합성 <ol><li> 04 98.7 mg의 16시 lysophophatidylcholine (1 - 팔미 토일 -2 - 히드 록시 SN-글리세; (... 청 등, Bioconj 화학, 2002, 13 -392 앞서 설명한 바와 같이 퍼시 스피루리나 해조류로부터 제조) 200 밀리몰의 pyropheophorbide 겸용 지질 : EDC - -3 - 포스 포 콜린, 벤티 극지 지질 번호 855675), EDC의 76.3 mg을, 48.7 밀리그램의 DMAP (4 (디메틸 아미노) 5 ㎖ 아 밀렌 피리딘)은 1:1 : 2 : 2 파이의 비율로 클로로포름을 안정화 : DMPA. </li><li> 24 시간 동안 아르곤하에 실온에서 반응 혼합물을 교반 하였다. 40 ℃로 가열하면서 회전 증발기를 사용하여 감압 하에서 클로로포름을 증발 (파이로 SN-1 또는 SN-2 위치에 부착 됨) 파이 지질 이성질체의 혼합물을 생성한다. </li><li> 1mM의 염화칼슘, 50 mM 트리스 (pH가 13), 0.5 % 트리톤 X-100 및 5 ㎎ / ㎖의 농도에서 10 %의 메탄올을 Resuspend 파이 지질 이성질체 혼합물. 꿀벌의 독에서에서 포스 포 리파제 A2 (PLA2)를 추가0.1 ㎎ / ㎖의 농도와 SN-1 파이로 지질을 소화하고 순수한 SN-2 파이 지질 이성질체를 생산하기 위해 24 시간 동안 37 ° C에서 솔루션을 품어. </li><li> 추가 클로로포름 2 볼륨과 메탄올 추출물의 1.25 볼륨을 추가 한 다음 40 ℃에서 감압하에 회전 증발을 사용하여 용매를 제거 DCM 중의 1 % 메탄올로 분열 생성물을 재현 탁하고 순수한 이성질체 파이 지질위한 작은 디올 실리카 컬럼상에서 정제. </li></ol> 2. Porphysomes의 준비 <ol><li> pyropheophorbide 지질, 디스 테아-SN-글리세로 -3 - phosphoethanolamine-N-메 톡시 (폴리에틸렌 글리콜) (PEG-2000-PE) 및 클로로포름 콜레스테롤 별도로 녹여 각각 농도를 기록한다. </li><li> 65 몰 % 포르피린 지질, 5 몰 %의 PEG-2000-PE 및 30 몰 % 콜레스테롤을 결합하여 5 ㎎ / 튜브의 전체 지질 중량 12x75 mm 보로 실리케이트 시험관 porphysome 지질 필름을 준비한다. </li><li> 니트로 스트림하에 지질 필름 건조겐 가스 및이를 추가 1 시간 동안 진공하에 건조시킨. 미래의 수화 및 압출까지 아르곤하에 -20 ° C에서 지질 필름을 보관합니다. </li><li> 각 지질 필름 튜브에 1 ㎖의 인산염 완충 생리 식염수 (PBS, 150 mM의 NaCl을, 10 mM의 인산, 산도 7.4)를 추가합니다. 완전히 고정 될 때까지 액체 질소에 테스트 튜브를 동결하고, 샘플이 모두 녹을 때까지 65 ° C의 물을 욕조에 튜브를 해동. 소용돌이 30 초 총 (각 소용돌이로 교반 사이에 65 ° C의 물을 욕조에 5 초 다시 가열 10 초 각의 3 배)를위한 관. </li><li> 더 큰 집계 수화 용액에서 관찰 할 수있을 때까지 필요한시 5 사이클 이상 동결 융해를 반복합니다. 완료되면 얼음에 porphysome 서스펜션을 남겨주세요. </li><li> 수욕 순환 65 ° C에 대한 항온 큐레이터로 thermobarrel 압출기 연결 인치 설치이 누적 100 nm의 폴리 카보네이트 필터를 고압 압출기 (10 ml)에 조립 고압 동안 질소 탱크로 압출기 시스템을 연결확인 공급. </li><li> 9 인치 유리 전송 피펫을 사용하여 압출기 챔버에 porphysome 현탁액을 전송합니다. 200, 300 PSIG 사이에 독서가 초기 압출 압력이 관찰 될 때까지 질소 탱크 레귤레이터 다이얼을 돌려 압력을 설정합니다. 충분한 porphysome 유량이 달성되고, 압력 800 PSIG (5,440 kPa의)의 최대에 도달 할 때까지 서서히 압력 조정기의 밸브를 연다. </li><li> 제 압출 통과가 완료되면, 압력 조절 밸브를 닫고 압출기 상부에 압력 릴리프 밸브를 개방함으로써 압출기의 압력을 해제. 갓 압출 porphysome 용액 압출기 리필 및 최종 압출 된 용액은 균질 보장하기 위해 적어도 10 회 압출을 반복한다. </li><li> UV-VIS 분광계 메탄올 중 희석 된 샘플의 흡수를 측정하여 최종 porphysome 용액의 농도를 결정. 다음 식의 재치를 사용하여 농도를 계산H 97,000 M의 흡광 계수 -1 cm -1 pyropheophorbide 지질 410 nm에서. </li></ol><img alt="식 (1)" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li> 동적 광산란에 의해 맬버른 제타 ZS90을 사용 porphysomes의 최종 크기 분포를 측정한다. PBS에 porphysome 솔루션을 희석하고 평균 결과 15 실행 각각 세 가지 측정을 수행합니다. </li><li> 분광을 이용하여 (1 % 트리톤 X-100의 1 ㎜) 형광 담금질 porphysome의 방출 (PBS에 1 ㎜)과 unquenched porphysome을 비교하여 porphysome의 형광 담금질을 결정합니다. (Tirton X-100) 추가되기 전에 세제 후 스펙트럼을 가지고, 최대 형광을 읽고 신호를 정상화. 410 nm의 및 600 ~ 800 nm의 수에 발광 파장 범위로 여기를 설정합니다. </li><li> 나중에 사용할 때까지 4 ° C에서 아르곤 하에서 por​​physomes 솔루션을 유지하고, 알루미늄 F에 의해 빛으로부터 보호오일. </li></ol> 3. 동물 이종 이식 모델의 제조 <ol><li> 문화 KB의 RPMI-1640 세포 (10 % FBS)와, 37 ℃에서 5 % CO 2와 습기를 대기에있는 세포를 유지 </li><li> 표준 세포 배양 기술을 이용하여 수확 KB 세포. 주입 시간이 될 때까지 단기 저장을위한 얼음에 인산염 완충 생리 식염수 (PBS)에 세포 현탁액 (20,000 세포 / ㎖)를 유지한다. </li><li> 누드 마우스에 KB 이종 이식을 준비합니다. 표준식이 요법과 침구 (할렌 연구소 주어진; 다이어트 : 그들은 (NU (NCR) Foxn1의 뉴 6-7 주령 종류 번호의 CRL) : 20g 때 누드 마우스는 찰스 리버 연구소에서 구입 한 조사 (照射) LM-485, # 7912; 침구 : 옥수수 침구, ¼ &quot;, # 7097). 모든 동물 처리 도구 (주사기, 포셉, 동물 패드) 이전의 실험에 소독. 100 % 산소 캐리어 가스의 2 % 이소 플루 란의 V / V를 가진 마우스를 마취. 모든 동물 프로토콜은 기관 (대학 보건 느에 의해 승인twork이 경우) 동물 관리 및 사용위원회. </li><li> 마우스의 오른쪽 측면에 PBS의 피하에 현탁 종양 세포를 주입한다. 마우스는 종양이 4~5mm 2 ~ 3 ㎜의 두께의 직경에 도달하면 10 일 이후 사용할 준비가 된 것입니다. </li></ol> 4. 생체 광열 치료 <ol><li> 정맥 꼬리 정맥을 통해 (200 μL 볼륨에서 750 nmol의의 파이를 포함) porphysomes를 주입. porphysomes의 레이저 조사 24 시간 후 분사를 실시한다. </li><li> 이 섹션에서는 밀폐 된 레이저 안전 방에 모든 절차를 실시하고 있습니다. 레이저 조사시 레이저 안전 고글을 착용 할 것. </li><li> 레이저 조사 장치를 설정합니다. 스탠드에 클램프에 의한 레이저 섬유와 확산을 잡아. 낮은 수준의 레이저 파워를 설정하고 레이저의 전원을 켭니다. (DPSS 레이저, LaserGlow 테크놀로지스, 캐나다 토론토)는, AP를 사용하여 750 mW의 (1.18 W / cm 2)에 레이저 파워를 교정 직경 9mm의 조사 영역을 달성하기 위해, 레이저 섬유의 높이를 조정된 ower 다음 미터와는 동물을 준비하기 위해 레이저를 끄십시오. </li><li> 코 콘을 통해 100 % 산소 캐리어 가스의 2 % 이소 플루 란의 v / V를 사용하여 동물을 마취 없음) 이소 플루 란 인간의 건강에 위험 위험 분명 청소. 없습니다 동물이 동물의 뒷발에 포셉 또는 손가락으로 발가락 핀치를 수행하여 깊은 충분히 마취되어 있는지 확인합니다. 그들이이 절차에 응답하지 않는 경우 동물은 레이저 조사에 대한 준비가되어 있습니다. </li><li> 적외선 열 화상 카메라 (Mikroshot, LUMASENSE 기술)에 의해 종양의 온도를 모니터링합니다. 잘 집중하고 사전 PTT 이미지뿐만 아니라 PTT 조사가 시작되기 전에 일반 백색광 이미지를 가지고, 가까운 종양에 카메라를 놓습니다. </li><li> 다시 레이저를 켜고, 750 mW의에 가벼운 힘을 증가시킨다. 종양이 완전히 덮여 얻을 수있는 레이저 빔의 중앙에있는 종양을 놓고 조사를 시작 타이밍. 1 분의 레이저 치료에 대한 종양의 온도 이미지마다 5 초를 가져 가라. 1 분에 레이저를 끄십시오. 세인트영업 이익은 2 % 이소 플루 란 (isoflurane)의 가스 공급은 마취에서 동물이 회복 될 때까지 기다렸다가 다시 케이지에 넣어. </li><li> 대학 건강 네트워크의 승인을 동물의 프로토콜에 따라 12:12 시간 명암주기에 따라 normothermia 동물 룸에서 마우스를 유지합니다. 프레 놀핀 (0.05 ㎎ / kg, 상처로 인한 통증을 감소시키는, 레이저 조사는 다음의 주간 피하 하루에 두 번 주입한다. 이틀마다, 캘리퍼를 사용하여 종양 직경을 측정하고 사진을 가지고. 이용하여 종양 부피를 계산 다음 식 : V = π / 6 · · 긴 직경이고, B는 짧은 하나입니다 B 2. </li><li> 우리의 실험실에서 정의 된 엔드 포인트로 종양의 긴 직경이 10mm에 도달하면 CO 2 챔버를 넣어 동물을 제물로 바친다. CO2 처리 다음과 같은 자궁 경부 전위를 수행합니다. 생존 곡선을 그린다. </li></ol>

<ol>
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우리는 공개 아무것도 없어.

높은 약물 탑재량을 유지하면서도 약물 전달 기술의 발전으로, 다 기능성 나노 입자는 약물 전달체의 정확한 트래킹 넓은 조사 중이다. 포르피린로드 리포솜 나은 약동학 성질 및 포르피린 직접 투여보다 효율적인 전달을 위해 개발되었지만, 장애물 포르피린 제한 담지량 및 혈장 단백질 5,6 행 리포좀에서 포르피린 급속한 재분배 포함하여 존재한다. 자유 포르피린은 리포솜의 코어 또는...

Porphysomes는 복합 이미징 및 치료 1 할 수있다 우리는 최근에 개발 된 새로운 다기능 nanovesicles 있습니다. 이들은 자기 조립 포르피린 이중층 형성되고 큰 흡광 계수 독특한 구조 종속 형광 자기 소광 결과를 생성한다 (83, 이상 000/porphysome 입자) 포르피린의 극히 높은 밀도를 포함한다. Porphysomes 좋은 생체 내 약물 동력학 및 생체 분포 특성을 가지고 : 그들은 체계적으로 투여 후 12 시간의 혈중 반감기를 전시하고, 수동적으로 24 시간 주입 후 2에서 7.5 %의 ID / g의 이종 이식 종양에 축적. 자신의 고유 한 구조와 물리 화학적 특성이 복합 영상과 영상 유도 치료를위한 porphysome 좋은 후보를합니다. 우선, 포르피린을 함유 porphysomes 종양 축적 한시 종양의 형광 이미징을위한 본질적으로 적합하다.또한, 각 포르피린 따라서, porphysomes 쉽게 PET 영상 3 등 64Cu과 같은 방사성 동위 원소로 표지 할 수있는 킬레이트 방사성 동위 원소의 안정적 사이트가 있습니다. porphysomes 또한 고유의 광 음향 이미징 및 PTT 기능을 나타낼 수 있도록 porphysome 구조가 그대로있는 경우 또한, 흡수 된 빛 에너지는 레이저 조사의 노출, 열 소모된다. 그것은 porphysomes의 정맥 주사 후 24 시간, porphysome 축적 된 종양의 레이저 조사가 급격한 온도 증가와 강한 광열 종양 절제를 유도 것으로 나타났습니다. 이 porphysomes은 금 나노 입자 (금 나노 입자)의 높은 흡광 계수 효율적인 광열 강화 것을 보여 주었다 1. 한편, 금 나노 입자 등 다른 무기 광열 에이전트와 비교, porphysomes는 그들의 유기적 인 특성으로 바이오 안전성에서 뛰어난 장점을 보여줍니다. Porphysomes는 효소 분해하고 최소한의 급성 toxicit을 유도1,000 ㎎ / ㎏ 1의 높은 정맥 투여와 마우스의 Y. 또한, 리포좀과 유사한, porphysomes의 큰 수성 코어는 수동적으로 될 수 또는 적극적으로 치료 또는 영상 에이전트와로드. 광학 특성 및 porphysomes의 생체 적합성 바이오 포토 닉 이미징 및 치료를위한 유기 나노 입자의 복합 가능성을 보여줍니다. 본 논문에서는 pyropheophorbide-지질 복합체의 합성 방법, 제조 및 고압 압출을 사용 porphysomes의 특성화 방법을 소개한다. 생쥐의 생체 PTT는 종양 porphysome 활성화 PTT의 효율성을 입증뿐만 아니라 실시 피하 이종 이식 종양 모델을 사용하여 치료.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

우리는 최근에 본질적으로 다기능 nanovesicles로 porphysomes을 개발했다. 감광제는, α pyropheophorbide, 인지질에 접합 한 후 리포좀과 같은 구형 소포에 자기 조립. 때문에 포르피린 지질 이중층에 포르피린의 매우 높은 밀도, porphysomes 큰 멸종 계수, 구조에 의존 형광 자기 담금질, 우수한 광열 효과를 생성합니다. 우리의 제형, porphysomes 고압 압출을 사용하여 합성하고, 120 nm의 주위에, 평균 입자 크기를 표시. porphysomes의 스물 네 시간 포스트 정맥 주사, 종양의 국소 온도가 빠르게 750 mW의 (1.18 W / cm 2), 671 nm의 레이저 조사 1 분 노출에 따라 62 ° C, 30 ° C에서 증가했다. 종양의 완전한 열 절제에 이​​어, 형성 및 치유 2 주 안에, 대조군에서 종양 성장을 계속하면서 모두 정의 EN 도달 가피일반적으로 3 주 내에 차원 점. 이러한 데이터는 porphysomes가 유력한 광열 요법 (PTT) 에이전트로 사용할 수있는 방법을 보여줍니다.

Pyropheophorbide은 안정한 지질 단량체 (도 1A)으로 인지질에 접합 및 접합체의 고압 압출기를 사용하여 막 압출 법에 의해 porphysomes를 형성하는 자기 - 조립된다. 그것은 상대적으로 느린 유속 압출 제 3 사이클 동안 porphysome 지질 현탁액을 압출하는 것이 곤란하다. 더 돌출이 반복되는 바와 같이, 압출 된 용액의 유속은 점차 증가하고, 필요할 경우 압력은 다소 감소 될 수있다. Porphysome 크?...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

우리는 이와 같은 강력한 광열 치료 에이전트 기능, 구조에 의존 형광 자기 담금질 독특한 광열 속성이 porphysomes라는 새로운 내장 다기능 nanovesicles을 개발. 우리는 고압 압출을 사용하여 porphysomes을 공식화하고 이종 이식 종양 모델에서 자신의 광열 치료 효과를 조사 하였다.

한 분, Porphysomes를 사용하여 하위 한 와트 광열 종양 절제, 내장 다기능 Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

one-minute-sub-one-watt-photothermal-tumor-ablation-using-porphysomes

Research

JoVE Journal

Bioengineering

10.7K 조회수.  University of Toronto.  우리는 이와 같은 강력한 광열 치료 에이전트 기능, 구조에 의존 형광 자기 담금질 독특한 광열 속성이 porphysomes라는 새로운 내장 다기능 nanovesicles을 개발. 우리는 고압 압출을 사용하여 porphysomes을 공식화하고 이종 이식 종양 모델에서 자신의 광열 치료 효과를 조사 하였다. 

비디오: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

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

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

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

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

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

연구

생체공학

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

의 광 주파수 도메인 영상<em&gt; 예 생체</em&gt; 폐 절제술의 표본 : 조직 병리학 간 상관 관계에 하나에 하나의 이미지를 얻기

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Viral Nanoparticles for In vivo Tumor Imaging

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

Viral Nanoparticles for In vivo Tumor Imaging

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

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

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

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

종양의 크기 측정의 품질 보증을위한 조직 흉내 낸 재료를 사용하여 전임상 Multimodality 팬텀 건설

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

마이크로 웰 플레이트를 사용하여 크기 조절 종양 상체 많은 수의 생산

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

입자 추적 microrheology은을 사용하여 3D 종양 모델에서 세포 외 매트릭스 강성의 경도 측정

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

높은 강도의 실시간 모니터링의 초음파 (HIFU) 절제를 집중<em&gt; 체외</em&gt; 집중 초음파에 대한 조화 운동 이미징을 사용하여 개 간을 (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

에 내장 혈관 신생에 의하여 조직 공학<em&gt; 생체</em&gt; 조직 공학 상공 회의소

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

광열 각도 광산란와 신속하고 화학 무료 헤모글로빈 분석

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...