Name: a facile and efficient approach for the production of reversible disulfide cross-linked micelles
Uploaded: 2016-12-23T12:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

The authors thank Ms.Alisha Knudson for the editorial help. They would also like to acknowledge the financial support from the NIH/NCI (3R01CA115483, to K.S.L.), the DoD PRMRP Award (W81XWH-13-1-0490, to K.S.L.), the NIH/NCI (1R01CA199668, to Y.L.), and the NIH/NICHD (1R01HD086195, to Y.L.).

윤리 문 : 여성 무 흉선 누드 마우스 (민 / 뉴 주) 6-8 주령은 구입 후 AAALAC 지침에 따라 무균 상태에서 보관하고 실험하기 전에 적어도 4 일 동안 순응시켰다되었다. 모든 동물 실험은 기관의 지침에 따라 수행 캘리포니아 데이비스 대학의 동물 사용 및 관리 관리 자문위원회에 의해 승인 된 프로토콜 번호 07-13119 호 및 제 09-15584에 따라되었다. TD PEG 5K -Cys 1. 합성 4 -Ebes 8 -CA (8) <ol><li> 둥근 바닥 플라스크에, 얼음에 무수 디메틸 포름 아미드 (DMF) 및 냉기의 10 ~ 20 ml의 MEO-PEG 5K -NH 2 (2g, 0.4 밀리몰)을 녹인다. </li><li> 유리 비이커에 3 당량을 용해. 1- 히드 록시 -6- 클로로 - 벤조 트리아 졸, 벤조 트리아 졸 (HOBt), 3 당량의. N의 N &#39;이소 프로필 카보 다이이 미드 (DIC), 3 당량. 무수 D에서의 (FMOC) 2리스-OHMF (10-15 mL)을 첨가 하였다. 자기 볶음 접시에 15 ~ 20 분 동안 교반한다. </li><li> 메오 5K-PEG -NH 2. 얼음 욕을 제거하고 실온에서 밤새 반응 혼합물을 교반 함유하는 반응 플라스크에 혼합물을 추가한다. </li><li> 박막 크로마토 그래피 (TLC) 16 카이저 테스트 (17), 반응의 완료를 확인 (황색 무연 NH 2의 부재를 나타낸다). 반응 플라스크에 빙냉 에테르 약 200 ml에 첨가하여 중합체 생성물 1 (MEO 5K-PEG -Lys (NH-FMOC)를 2) 침전물. 원심 분리를 통해 침전 된 폴리머 (6 6,000 XG에 분, 4 ° C)를 분리합니다. <ol><li> TLC를 수행 실리카겔 코팅 TLC 플레이트 상에 시료를 발견. 디클로로 메탄 / 메탄올을 사용하여 (9 : 1)을 이동상으로서. TLC 판을 개발 한 후 UV 램프 아래 지점을 관찰한다. 하나는 핫 플레이트 아민 반점을 가시화 염색 시약을 닌히 드린 사용할 수있다. </li><li> 카이에 대한산이의 테스트는, 카이저의 시약을 포함하는 유리 튜브의 샘플을 조금 넣습니다. 5 분 동안 100 ℃에서 가열 한 상기 색상 변화 (용액의 색이 노란색 남아있는 경우, 다음 반응이 완료 될 때)을 찾는다. </li></ol></li><li> 무수 DMF (10-20 mL) 중의 생성물을 재용 해하고, 침전 및 원심 분리를한다 (단계 1.4 참조)를 반복한다. </li><li> 다음 반복 단계 1.5 및 빙냉 에테르 중합체 침전물을 세 번 세척 하였다. </li><li> 깨끗한 반응 플라스크에 폴리머 석출 한 전송 잔류 에테르를 제거 고진공 소스로 플라스크를 연결한다. </li><li> 준비하고 20 %의 약 20 ~ 30 ml에 추가 (v / v)의 DMF 4 메틸 피 1 중간을 중합체. 완전히 용해 될 때까지 저어. 3 시간 동안 반응을 실행합니다. </li><li> TLC 16 수행하고 황제의 시험 (청색 무료 NH 2의 존재를 확인) (17) (단계 1.4 참조) completio을 확인합니다반응 없음. 반응이 완료되면 제품 1 (1.4-1.6 단계)에 대해 언급 한 바와 같이, 에테르 침전를 진행합니다. </li><li> 고분자 제품 2 (MEO-PEG 5K -Lys (NH 2) 2) 진공에서 건조. </li><li> (1.1-1.7 단계) 제품 3을 생성하는 중간 2 상 (FMOC) 2리스-OH 결합 하나 더 라운드를 수행 (MEO-PEG 5K -Lys (리스 (NH-FMOC) 2) 2). (1.8-1.10 단계)를 FMOC 그룹 (4, MEO-PEG 5K -Lys (리스 (NH 2) 2) 2) 커플 (FMOC)리스 (BOC) (1.1-1.7 단계) -OH로 드 보호 3 세대 수지상 폴리 라이신 생성 (5, MEO-PEG 5K -Lys-리스 2 - ((FMOC)리스 (BOC)) 4) PEG 사슬의 한쪽 끝에서 네 BOC 및 FMOC 그룹이 종료되었습니다. </li><li> 반응 플라스크에 중간체 얻어진 중합체 5 옮긴다. 에서와 같이디클로로 메탄 (DCM)에서 1 (v / v)의 트리 플루오로 아세트산 (TFA) : eparate 반응 플라스크 1을 준비합니다. 폴리머 (5) 중간에 1 TFA / DCM (v / v)의 혼합물 : 1의 15 ~ 20 ML을 추가합니다. 중합체가 완전히 용해 될 때까지 혼합물을 교반한다. 추가로 3 시간 동안 교반한다. </li><li> TLC 16 카이저의 수행 및 시험 반응 종료를 확인하기 위해도 17의 (단계 1.4 참조) (청색 무연 NH (2)의 존재를 확인한다). 반응이 완료되면 점성 용액이 얻어 질 때까지 공기와 중합체 인 TFA / DCM 혼합물을 증발시켰다. 제품 1 (1.4-1.6 단계)에 대해 언급 한 바와 같이, 에테르 침전를 진행합니다. 진공에서 - (((FMOC)리스 (NH 2)) 4 -Lys-리스 2 MEO-PEG 5K) 고분자 제품 (6)을 건조. </li><li> 반응 플라스크에 6 중간 전사 중합체. 8 당량을 포함하는 무수 DMF의 약 40 ml를 사용합니다. N의 N의 -diisopropylethylami네브라스카 (DIEA)는 폴리머 (6) 중간을 용해한다. 유리 비이커에, 12 당량을 용해. 의 HOBt, 12 당량. DIC, 12 당량의. 무수 DMF의 20 ~ 25 ml의의의 (FMOC) 시스테인 (트리트) -OH. 10 ~ 15 분 동안 진탕 한 후 (6)을 함유하는 반응 플라스크에 반응 혼합물을 추가한다. 밤새 반응을 실행합니다. </li><li> TLC (16)와 카이저의 시험과 반응의 완료를 확인합니다 (단계 1.4 참조; 황색 무료 NH 2의 부재를 나타내는) 17. ((FMOC) 라이신 - - ((FMOC) Cys 또는 반응이 완료되면 제품 1 (단계 1.4-1.6)에 대해 언급 된 바와 같이, 제품 7 (MEO 5K-PEG -Lys-리스 2 분리, 에테르 침전 진행 (트리트))) 4. </li><li> 제품 8 얻기 위해, 단계 1.8에 설명 된대로, 7 FMOC 탈 보호를 수행합니다 (- (리스 (NH 2) - PEG 5K -Lys-리스 2 (시스테인 (NH 2) (트리트을))) 4). 커플 (FMOC) -PEG 2 -Suc-OH ( &quot;Ebes&quot;링커, 24 당량.)를의 HOBt에 대해 위에서 설명 된 절차를 사용하여 중간 고분자 / DIC 매개 커플 링에 중간 9 (MEO-PEG 5K -Lys-리스 2 -Lys 4 얻기 - (시스테인 (트리트을) ) 4 (Ebes (NH-FMOC)) 8). </li><li> 중간 10 얻을 수 FMOC 탈 보호 한 번 더 라운드 (1.6-1.8 단계) 수행 (MEO-PEG 5K -Lys-리스를 2 -Lys 4 - (시스테인 (트리트)) 4 (Ebes (NH 2)) 8). </li><li> 반응 플라스크에 폴리머 중간층 (10)을 전송하고 용해 무수 DMF (30-40 ml)로 추가한다. 또 다른 반응 플라스크에서 24 당량을 용해. 무수 DMF에 (이전에 게시 된 절차에 따라 제조) CAOSu (20 ~ 30 ㎖) 18. 48 당량을 추가합니다. 및 N, N- 디 이소 프로필 에틸 아민으로는 10 ~ 15 분 동안 교반 할 수 있습니다. 10을 함유하는 반응 플라스크에 콘텐츠를 전송하고, 반응 런 O하자vernight. </li><li> TLC 16 카이저의 테스트로 반응 종료를 확인하고 (단계 1.4 참조, 황색 무연 NH 2의 부재를 나타내는) 17. (Cys이고 (트리트)) 4 -Ebes - 반응이 완료되면 제품 1 (1.4-1.6 단계) 제품 (11)을 분리 (MEO 5K-PEG -Lys-리스 2 -Lys 4 바와 같이, 에테르 침전 진행 8 -CA 8). 탈 이온수를 투석하고, 백색 분말을 수득 샘플을 동결 건조. </li><li> 반응 플라스크에 중간 폴리머 (11)를 놓습니다. 준비하고 고분자 용액에 TFA / 1,2- 에탄 (EDT) / 트리 에틸 실란 (TIS)의 20 ㎖ / H 2 O (94 / 2.5 / 1 / 2.5, v / v)의 혼합물을 추가합니다. 완전히 용해 될 때까지 혼합물을 저어. 4 시간 동안 반응을 실행합니다. TLC (16)에 의해 반응 종료를 확인한다. </li><li> 흄 후드 아래에 공기를 불어 폴리머 TFA / EDT / TIS / H 2용액까지 O 혼합물의 점성이된다. 제품 1 (1.4-1.6 단계)에 대해 언급 한 바와 같이 최종 제품, 12 (PEG 5K -Cys 4 -Ebes 8 -CA 8)를 분리, 에테르 침전를 진행합니다. 아세토 니트릴에 용해하고, 백색 분말을 수득하여 동결 건조. </li></ol> PTX로드 미셀 2. 준비 <ol><li> 표준 증착 방법을 사용하여 PTX로드 PEG 5K -Cys 4 -Ebes 8 -CA 8 미셀을 준비합니다. <ol><li> 클로로포름 1 ㎖ (클로로포름)에서 PTX의 다른 양 (1-9 mg)을 함께 TD 20 mg을 녹이고. 균질 건조한 중합체 필름을 수득하는 회전 증발기를 사용하여 용매를 제거한다. 필요한 경우 약물로드 미셀의 형성을 허용하도록, 40 kHz에서 30 분 동안 초음파 처리 하였다 와류에 의한 인산 완충 용액 (PBS) 1 ㎖로 필름을 재구성한다. </li><li> 3 %의 (w / w) H 2 O 2 (1 당량 6 μl를 추가합니다. 호텔 fr하기TD를에 티올기를 산화) 티올기를 EE. 엘만 (Ellman) 테스트 (19)에 의해 지시 된 바와 같이 유리 티올 그룹의 레벨이 일정하게 낮은 값으로 유지되면 추가 특성에 대한 미셀 솔루션을 사용합니다. </li><li> 샘플을 살균 할 수있는 0.22 μm의 필터 솔루션을 필터링합니다. 9 번 아세토 니트릴을 첨가하고, 초음파 처리를 10 분간 수행하여 미셀의 약물 방출 후 HPLC 시스템 (20)의 미셀에로드 된 약물의 양을 분석한다. 이동상으로 아세토 니트릴 / 물 HPLC의 C18 컬럼을 사용합니다. </li><li> HPLC로 면적 값과 11 표준 약물 농도의 검량선에 따라 약물 로딩을 계산한다. 주 : 적재 효율이 초기 약물 함량 미셀에로드 약물의 비율로 정의된다. </li></ol></li></ol> 미셀 3 요소 특성 분석 <ol><li> 제 m의 크기 및 크기 분포를 측정동적 광산란 (DLS) 악기 (29)와 icelles. 실온에서 측정을 수행하고 ㎖ / 1 밀리그램의 미셀 농도를 유지한다. 주 : 입자 크기 분석을 수행 빈으로 PBS를 사용하고 실제 샘플 입자 크기를 기록한다. 샘플에 대한 세중의 측정을하고 측정 값을 평균. </li><li> 이전에 13,21 바와 같이, 이전과 소수성 형광 프로브로 피렌와 가교 후 PEG 5K -Cys 4 -Ebes 8 -CA (8)의 임계 미셀 농도 (CMC)을 측정하는 형광 스펙트럼을 사용합니다. 참고 : 일반적으로, 미셀 농도는 5 × 10-7 5 × 10 -4 M. 범위 </li></ol> 또는 환원제없이 SDS에서 미셀 4. 안정성 <ol><li> 소듐 도데 실 설페이트 스톡 용액 (SDS) 용액 (7.5 ㎎ / ㎖) 및 PBS에서 이황화 가교 미셀 (1.5 ㎎ / ㎖)을 준비한다. 다음, U를최종 SDS 농도로 된 혼합 용액을 상기 원액을 2.5 ㎖의 노래 밀리그램 / 및 미셀 농도가 1.0 ㎎ / ㎖이다. </li><li> 또는 10 mM의 글루타치온 (GSH)의 존재없이 소정의 시간 간격으로 미셀 용액 (단계 3.1에서 언급 한 바와 같이) 크기 및 크기 분포를 측정한다. </li></ol> 5. 용혈 분석 <ol><li> 로부터 수집 이전에 발행 절차 (11)와 누드 마우스에서 신선한 구연산 혈액을 사용하여 PTX-로드 가교 미셀 (PTX-된 DCM)에 따라 제조 PTX-로드, 비 가교 미셀 (PTX-NCMs)의 용혈성 잠재력을 평가 꼬리 베일. <ol><li> 2 %의 최종 농도로 PBS로 세포 펠렛을 다시 정지 후, 10 분간 1,000 XG에서 혈액 샘플 (1.0 mL)을 원심 분리하여 적혈구를 모아서 PBS로 세 번 세척하고. </li></ol></li><li> 서로 다른 농도 (0.2 및 1.0 적혈구 현탁액 200 μl를 혼합mg의 PTX-NCMs 및 PTX-된 DCM,의 / ㎖)을 각각 및 인큐베이터 통에 37 ° C에서 4 시간 동안 배양한다. </li><li> 5 분 동안 3,000 XG에서 미셀 적혈구 혼합물을 원심 분리기. 96 웰 플레이트의 모든 샘플의 상등액 100 ㎕를 옮긴다. 마이크로 플레이트 판독기를 사용하여 540 nm에서 상층 액의 헤모글로빈의 흡광도를 측정한다. 주 : 트리톤-100 (2 %) 및 PBS와 함께 배양 된 RBC를 각각 음성 및 양성 대조군으로 사용하여야한다. 적혈구의 퍼센트 용혈는 다음과 같은 공식으로 계산된다 적혈구 용혈 = (OD 샘플 - OD 대조군) / (OD 양성 대조군 - OD 대조군) × 100 %입니다. </li></ol>

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Y. Li and K.S.L. are the inventors of a pending patent on reversibly crosslinked micelle systems (US patent application 61/485,774). K.S.L. is the founding scientist of LamnoTherapeutics Inc., which plans to develop the nanotherapeutics described in the manuscript. The remaining authors declare no competing financial interests.

여러 가지 나노 입자는 약물 전달에 잠재적 인 사용을 위해 조사되었다. 리포좀 독소루비신과 파클리탁셀 (PTX)는 인간 혈청 알부민 나노 집계가 암 치료에 대한 FDA의 승인을 nanotherapeutics 중입니다 -loaded. 임상 적 효과가 있지만, 그러나, 이러한 nanotherapeutics 모두는 상대적으로 크기가 &quot;큰&quot;이며, 그들은 간과 폐에 축적되는 경향이있다. 상대적으로 작은 입자 크기와 높은 약물 적재 용량 고분...

나노 기술은 생물 의학 분야 (1)의 수를 도움이되고 빠른 신흥 분야이다. 나노 입자는 기존 치료제의 다른 유형 가능하지 않은 특성을 설계 및 튜닝을위한 기회를 제공한다. 나노 담체는 약물 순환 시간을 연장, 생분해에 대한 약물의 안정성을 향상시키는 약물 용해도 문제를 극복하고, 미세 조정 표적 약물 전달 및 공동 전달 조영제 1.2이 될 수있다. 나노 입자 기반 배달 시스템은 암 이미징 및 치료에 약속을 개최합니다. 종양 vasculatures는 거대 분자에 누설하고 향상된 침투성 및 보존 (EPR) 효과 3를 통해 종양 부위에 나노 입자를 순환의 우선 축적으로 이어질 수 있습니다. 적극적으로 항암 약물 캐리어로 추진되고있는 여러 가지 나노 캐리어 (예를 들어, 리포좀, 하이드로 겔 및 고분자 미셀) 중 고분자 미셀은 일을 통해 폭 넓은 인기를 얻고있다전자 지난 십 4,5. 고분자 미셀은 정맥 내 투여에 잠재적으로 그들의 unimers 해리 선도의 임계 미셀 농도 (CMC) 미만으로 희석 할 수 있고, 열역학 시스템이다. 가교 전략에 unimers 미셀 해리를 최소화하기 위해 사용되어왔다. 그러나, 과도하게 미셀 안정화시켜 전체 치료 효과를 감소 대상 부위에서 방출로부터 약물을 방지 할 수있다. 여러 가지 화학적 방법은 수 있도록 탐구 된 가교 산화 환원에 대한 응답으로 또는 환원 이황화 결합 6,7 및 pH의 절단 (8) 또는 가수 분해 가능한 에스테르 결합 9,10와 같은 외부 자극에 분해. 우리는 이전에 telodendrimers (TD)이라고 설계 및 수지상 콜산 (CA) 및 블록 선형 폴리에틸렌 글리콜 (PEG) 공중 합체로 이루어진 미셀 나노 입자의 합성을보고 11-15 </&gt; SUP. 이 TDs를은 (N = 분자량 킬로 달톤 (K)에서, y는 콜산의 수 (CA) 단위 =) PEG NK -CAy로 표시됩니다. 그들은 이러한 소수성 코어 파클리탁셀 (PTX)과 독소루비신 (DOX)로 캡슐화 약물에 작은 크기, 긴 수명, 높은 효율을 특징으로한다. 이러한 PEG, 리신, 및 CA 등 TD의 빌딩 블록은 생체 적합성 및 PEG 코로나의 존재는 망상 내피 시스템에 의해 미셀 나노 입자의 비특이적 흡수를 방지하는 &quot;은폐&quot;나노 입자의 특성을 부여 할 수있다. 티올 선형 덴드리머 용이 우리 표준의 TDS 수지상 올리고 라이신 백본에 시스테인을 도입함으로써 생성 될 수있다. 이 문서의 TDS 소수성 코어에 이황 가교 (도 1)을 도입함으로써 가역적 가교 미셀 약물 전달체의 제조를위한 손쉬운 프로토콜을 제공한다.

<table><tbody><tr><td>MeO-PEG&lt;sup&gt;5K&lt;/sup&gt;-NH&lt;sub&gt;2&lt;/sub&gt;</td><td>Rapp Polymere</td><td>125000-2</td><td></td></tr><tr><td>Fmoc-Lys(Fmoc)-OH</td><td>Aaptec</td><td>AFK107</td><td></td></tr><tr><td>Fmoc-Lys(Boc)-OH</td><td>Anaspec</td><td>AS-20132</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Aapptec</td><td>AAC105</td><td></td></tr><tr><td>Dimethylformamide</td><td>Fisher Scientific</td><td>BP1160-4</td><td></td></tr><tr><td>Ethyl ether</td><td>Fisher Scientific</td><td>E134-20</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>T6508</td><td>Corrosive, handle with care</td></tr><tr><td>4-methyl piperidine</td><td>Alfa-Aesar</td><td>L-02709</td><td></td></tr><tr><td>Ebes linker</td><td>Anaspec</td><td>AS-61924</td><td></td></tr><tr><td>Cholic acid</td><td>Sigma Aldrich</td><td>C1129</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>Sigma Aldrich</td><td>02390</td><td>Handle inside fume hood. Bleach gloves after usage.</td></tr><tr><td>Triisopropylsilane</td><td>Sigma Aldrich</td><td>233781</td><td></td></tr><tr><td>Chloroform (anhydrous)</td><td>Sigma Aldrich</td><td>288306</td><td></td></tr><tr><td>Hydrogen peroxide solution 30%</td><td>Aaron Industries</td><td>NA</td><td></td></tr><tr><td>HoBt-Cl</td><td>Aaptec</td><td>CXZ096</td><td></td></tr><tr><td>DIC</td><td>Sigma Aldrich</td><td>D125407</td><td></td></tr><tr><td>Female athymic nude mice (Nu/Nu strain), 6&amp;ndash;8 weeks age</td><td>Harlan (Livermore, CA)</td><td></td><td></td></tr></tbody></table>

a facile and efficient approach for the production of reversible disulfide cross-linked micelles

나노 의학은 생물 의학 응용 프로그램에 대한 규모 나노 미터 입자의 고유 한 특성을 이용합니다 치료의 새로운 형태이다. 치료 결과를 극대화하고, 약물과 관련된 부작용을 감소시키는 약물 전달을 향상 오늘날 나노 의학의 초석 일부이다. 특히 나노 입자는 암 치료의 다양한 응용 프로그램을 발견했다. 디자인, 응용 프로그램 및 종양 미세 환경에 따라 생산에 높은 수준의 유연성을 제공 나노 입자는 임상 적으로 신속한 번역 더 효과적 일 것으로 예상된다. 고분자 미셀 나노 캐리어는 약물 전달 응용 프로그램에 대한 인기있는 선택입니다. 이 글에서, 우리는 잘 정의 된 양친 선형 수지상 공중 합체 (telodendrimer, TD)의 자기 조립 (self-assembly)에 따라 약물로드, 이황화 가교 미셀을 합성하는 간단하고 효과적인 프로토콜을 설명합니다. TD는 폴리에틸렌 GL 구성된다친수성 세그먼트 용액 계 펩티드 화학 제를 사용하여 아민 종결 된 PEG에 코어 형성 용 소수성 잔기에 부착 단계적 같은 티올 콜산 클러스터와 ycol (PEG). 파클리탁셀 (PTX)과 같은 화학 요법 약물은 표준 용매 증발 법을 사용하여로드 될 수있다. 오 2 - 매개 산화는 이전에 TDS 농도에 무료 티올 그룹에서 내 미셀 이황화 가교를 형성하기 위해 사용 하였다. 그러나 반응이 느리고 대규모 생산에 가능하지 않았다. 최근는 H 2 O 2 - 매개 산화법 더 가능하고 효율적인 방식으로 탐구하였으며, 이는 96 배 빠른 속도로 이전에보고 된 방법보다이었다. 이 방법을 사용 PTX로드, 이황화 가교 나노 입자 50g을 성공적으로 좁은 입자 크기 분포와 높은 약물 적재 효율로 생산되고있다. 얻어진 미셀 용액의 안정성은 그러한 w 공동 배양 같은 방해 조건을 이용하여 분석또는 환원제없이 세제, 도데 실 황산나트륨, 제 i. 이들 비 - 가교 상대 비교할 때 약물로드 디설파이드 가교 미셀 적은 용혈 활성을 보여 주었다.

약물로드, 이황화 가교 미셀의 제조 및 특성 양친 매성 고분자 5K PEG -Cys -Ebes 4 8 8 -CA 암 약물 전달 디설파이드 가교 미셀 시스템을 형성 할 수있는 수지상 중합체. 구조적으로, 이는 분지 된 폴리 (라이신 시스테인 Ebes) 백본을 통해 선형 PEG 분자 (친수성 도메인 분자량 5K)의 일단에 연결된 콜릭 산 (...

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A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

가역 디설파이드 가교 미셀 제조를위한 손쉬운 효율적인 접근법

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

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Research

JoVE Journal

Biochemistry

8.8K 조회수.  University of California, Davis. To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

비디오: A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

구조 연구 생산 이황화 - 안정화 Transmembrane의 펩타이드 단지

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

연구

화학

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Photoredox 촉매를 사용하여 가시 광선 중재 분산 중합에 의해 웜 형상 미셀 손쉬운 합성

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

시스테인 잔류 물을 용매 접근성에 대한 분석<em&gt; 옥수수 rayado fino 바이러스</em에서 생산&gt; 바이러스 같은 입자<em&gt; Nicotiana benthamiana</emVLPs에 펩티드의&gt; 식물과 교차 연결

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

면역학 및 감염병학

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

구형 및 벌레 모양의 Micellar 나노 자기 조립, 분무, 결합 하 여 용 매 기반 구조 제어의 제조

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.

비 환 원 SDS-PAGE 분석 및 화학적 가교 결합으로 포유류 세포에서 이황화 물 조합에 의해 안정화 된 다이 성체 복합체를 배양에 검출

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a ...

발생학

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

동맥 경화 증의 영상에 대 한 펩 티 드 Amphiphile Micelles Monocyte 타겟팅의 합성

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

생체공학

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Aqueous One-step Crosslinking 및 Co-nanopolymerization을 이용한 Stimuli-responsive Nanogel 합성

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

리소리피드 함유 온도 민감성 리포좀의 미세유체 생산

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

생물의학용 알긴산나트륨 하이드로겔 마이크로스피어 개발

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

저자 스포트라이트: 생체적합성 알긴산나트륨 하이드로겔 마이크로스피어를 이용한 치료법 발전

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Microfluidic Electrospray 기술을 사용하여 다양한 금속 이온을 가진 가교 알긴산 나트륨 미세구 준비

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...