저자는 미국 육군 의학 연구 및 Materiel 명령을 감사드립니다 (권한 부여 # W81XWH-10-1-0806)와 재정 지원을위한 국립 보건원 (권한 부여 # U19 AI091031-01 및 권한 부여 # 1R01GM090280)는 것이다. BN은 화학 및 생물 공학 Balloun 교수를 인정하고 NLBP은 학제간 공학 윌킨슨의 교수로 인정한다. 우리는 nanoparticle의 작용화 실험을 수행하는 그녀의 도움 줄리아 벨라 감사드립니다.

1. 높은 처리량 탄수화물 합성 <ol><li> 이전 dimannoside의 자동화 합성, 적절한 보호 설탕 기증자, 일반적으로 trichloroacetimidate 및 수용체 주로 알케닐 fluorous 알코올에이 벤치 위에 합성됩니다. </li><li> 프로그램 dimannoside의 자동 합성을 위해 쓰여집니다. 기본적인 자동화된 절차의 도식 표현은 그림 2에 표시됩니다. 프로그램에서, 그것은 발기인의 추가하기 전에 기증자와 수용체의 혼합물은 최소 30 분 동안 흔들 것을 보장합니다. </li><li> 합성 기증자, 수용체 및 trimethylsilyltrifluoromethanesulfonate의 솔루션은 dichloromethane으로 만들어집니다. 톨루엔과 dichloromethane는 글리코 실화 반응을 위해 가장 자주 사용됩니다. </li><li> 또한, 80 % 메탄올 및 100 %의 메탄올에 임시 보호 그룹 deprotection위한 시약의 솔루션을 준비합니다. </li><li> 프로그램의 시작하기 전에되도록 그 relat방에있는 필자 습도는 자동화 챔버 30 % 이하이다. 높은 습도는 글리코 실화 반응에 대한 해로운 것입니다. </li><li> 프로그램이 시작되면 로봇 팔이 순차적으로 반응 유리병에 기증자와 수용체의 솔루션을 전송합니다. 그런 다음 혼합물은 30 분 동안 흔들 수 있습니다. </li><li> 로봇 팔이 혼합물로 trimethylsilyltrifluoromethanesulfonate의 0.2-0.3 등가물를 전송 다음으로, 일반적으로 상온에서 -20와 같은 낮은 온도 ° C는 획득 수 있지만. 반응 혼합물은 30 분 동안 흔들 수 있습니다. </li><li> 30 분 후 반응이 멈춘 작은 나누어지는는 반응 진행을 모니터링하기 위해 제거됩니다. 완료되지 않은 경우, 반응은 계속 수 있으며 궁극적으로 필요한 시간을 수정할 수 있습니다. </li><li> 반응이 완료되면 반응 혼합물은 정화를 위해 C 8 F 17-수정된 실리카 겔을 포함 fluorous 고체 상 추출 (FSPE) 카트리지로 전송됩니다. </li><li> 장바구니산등성이 먼저 비 fluorous 분수를 떼어 버리려고 80 % 메탄올 - 물 혼합 (8 ML)로 씻어 있습니다. </li><li> 그러면 카트리지는 원하는 fluorous 태그가 추가된 제품을 얻기 위해 100 % 메탄올로 씻어 있습니다. 추가적인 정화가 필요한 경우 컴퓨터가 정지하고 반응 생성물 (들)은 추가적인 방법으로 정화를 위해 제거할 수 있습니다. </li><li> 정화 사이클 후 로봇 팔이 반응 유리병에 나트륨 methoxide을 dispenses. 반응은 2 시간 동안 흔들 수 있습니다. 완료되지 않은 경우, 반응은 다시 긴 기간 동안 계속 수 있으며 궁극적으로 필요한 프로그램의 시간은 수정할 수 있습니다. </li><li> 반응의 완료 후, 제품은 FSPE에 의해 정화되어 후 잔류 수분을 제거하는 증발 다음에 무수 톨루엔에 용해에 노출. </li><li> 원하는 체인 길이 대상 분자에 대해 얻은 것입 때까지 그 다음주기가 (6 단계 13 일부터) 반복됩니다. </li><li> 자동화에서 얻은 보호되는 제품은 일입니다도중에 더 정화와 완전히 같은 핵 자기 공명 (NMR) 분광법과 같은 기술이 특징. 그것은 보통 폭발성 수소 가스와 팔라듐을 포함하기 때문에 최종 목표 분자의 완전한 deprotection은 (모든 남아있는 보호 그룹의 삭제) 후 원칙적으로 자동화 플랫폼 밖 완료됩니다. 최종 deprotection 단계는 자동화 플랫폼 밖 벤치 위에 실시되었다. 첫 번째 단계는 카르복실산로 제작 알데히드의 산화 다음 fluorous 태그에 이중 결합의 온존 분해했습니다. 제품은 칼럼 크로마 토그래피에 의해 정화되었다. 마지막 단계는 팔라듐 - 촉매의 첨가에 의한 벤질 에테르 그룹 deprotection했습니다. 제품은 순수하게 최종 제품을 얻기 위해 팔라듐 없애 celite 패드 통과되었다. </li></ol> 2. 높은 처리량 Nanoparticle 표면 작용화 <ol><li> 높은 처리량 고분자 합성과 nanoparticle의 제조는 동일한 PR에 따라 수행됩니다otocol과 로보트가 피터슨 외 19에 기술된 설정합니다. 입자 제조에 사용되는 공중 합체 시스템이 sebacic 산성 (SA)과 1,6 - 비스 (파라 carboxyphenoxy) 헥산 (CPH) 및 1,8 - 비스 (를 기준으로합니다 패러 carboxyphenoxy) -3,6-dioxaoctane (CPTEG) 및 CPH. 활용 로봇 증착 장치의 개략도 표현은 그림 1B에 표시됩니다. </li><li> nanoparticle 제조 따라 nanoparticle 라이브러리와 튜브를 포함하는 홀더는 선형 액추에이터 무대 reattached 있습니다. </li><li> polyanhydride 입자의 표면에 탄수화물의 첨부 파일이 들어 연속 2 반응으로 구성된 아민 - 카르복실산 커플링 반응 20 수행됩니다. </li><li> 첫 번째 반응 들어, 첫 번째 프로그램 주사기 펌프에 주사기를 1 - 에​​틸 -3 - (3 - 디메틸) 10 등가물 (eq.) (입자 표면의 평균 어금니 카르복실산 농도의 등가물)으로 가득합니다-CArbodiimide 하이드로 클로라이드 (EDC)와 10 EQ. 수용액에서의 에틸렌의 두 번째 프로그램 주사기 펌프의 주사기는 12 EQ와 함께로드되는 동안. 수용액에서 N-hydroxysuccinimide (보건국)의. </li><li> LabVIEW 프로그램을 사용하면, 시약의 현탁액은 nanoparticle 라이브러리 *로 입금됩니다. </li><li> 다음 각 예제는 sonicated됩니다 (40 Hz에서 30들)과 튜브 홀더는 로봇 플랫폼에서 분리됩니다. </li><li> Nanoparticle의 현탁액은 4 ° C.에 일정한 회전 구 H에 **에 대한 incubated있다 </li><li> 반응 시간이 완료되면 튜브는 centrifuged (5 분 12,000 XG)와 두 세척 단계를 수행할 수있는 로봇 스테이션에 반환됩니다. </li><li> 두 번째 주사기 펌프의 주사기는 차가운 물로 채워진 상태에서 세척 들어, 주사기는 비어와 최초의 프로그램 가능한 주사기 펌프에 로드된 남아 있습니다. 각 튜브의 표면에 뜨는는 빈 주사기와 보조 펌프의 예금 찬물에​​ 철회된다. </li><li> 나노의 균질화2.6 단계에서 설명한대로 입자 현탁액이 수행됩니다. 튜브 그때 centrifuged됩니다 (5 분 12,000 XG)와 단계 2.9에 설명된 두 번째 세척 단계가 수행됩니다. </li><li> 두 번째 반응의 경우, 두 증착 단계가 사용됩니다. 최초의 증착 단계에서, 12 EQ. EDC 중 한 펌프 및 12 개의 EQ와 함께로드됩니다. 의 보건국은 보조 펌프와 함께로드됩니다. </li><li> 두 번째 증착 단계는 10 EQ를 포함합니다. 첫 번째와 두 번째 펌프 (즉, 갈락토 오스, 유당 또는 디 - mannose) *** 10 EQ와 삼분의 일 펌프에 대한 구체적인 saccharide니다. 의 glycolic 산 (제어로 사용 ****). </li><li> 4 ° C.에서 단계 2.6에 설명된 지속적인 회전 구 H 위해 incubated로 Nanoparticle의 현탁액이 균질하고 있습니다 </li><li> 반응 시간이 완료되면 단계 2.8, 2.9 및 2.10에 설명한 바와 같이, 세탁 단계가 수행됩니다. </li><li> 작용 nanoparticle 라이브러리 후 적어도 2 시간 동안 건조 진공 챔버에 배치됩니다. </li><li> 작용 nanoparticles 그런 다음 문자 위치X 선 광전자 분광법 각각 표면 조성과 saccharide의 농도를 결정하는 높은 처리량 페놀 - 황산 분석하여 ized. 전자 현미경 및 동적 광 산란을 스캐닝하는 것은 입자 크기, 크기 분포 및 표면 요금을 결정하기 위해 활용됩니다. </li></ol> 주 : * 증착 볼륨이 각 튜브에 포함된 nanoparticles의 질량에 따라 다릅니다. 첫 번째와 두 번째 반응을위한 ** 반응 시간은 최종 saccharide 농도를 조정하도록 변경할 수 있습니다. *** 각 saccharide이 원하는 그룹에 따라 테스트 튜브로 입금됩니다. deprotected saccharides 이미 nanoparticle의 표면에 대한 부착을 허용 covalently 연결이 분자를 가지고 있기 때문에 **** 탄수화물의 첨부 파일이 연구에 사용된 구체적인 반응의 경우, glycolic 산은 링커 컨트롤로 사용됩니다. 3. 대표 결과 ful그림 2에 표시된 요 보호 dimannoside은 자동화 플랫폼을 사용하여 합성되었다. 합성 화합물은 용매로 CDCl 3을 사용 VXR 400 MHz의 분광계에 1 H NMR에 의해 특징되었습니다. NMR 스펙트럼은 그림 3에 표시됩니다. polyanhydride의 높은 처리량 nanoparticle의 제조 및 작용화을 이용하는 것은 본 설명한 nanoparticles, dimannose, 락토 오스 및 갈락토 오스의 첨부 파일이 성공적으로 10, 11 실시되었습니다. 이 설정을 사용하여 최적의 반응 조건 (즉, 반응 온도 및 시간) 원하는 nanoparticle의 작용화 조직 형태를 달성하는 확인되었다. 반응이 4 시에 실시되었을 때 ° C ~ 대신 상온의, nanoparticle의 집합의 감소가 SEM에서 관찰되었다 (데이터가 표시되지 않음). 디 - mannose이나 자중 CPH의 nanoparticles : 표 1은 작용 50:50 CPTEG의 특성을 대표하는 결과를 보여줍니다4에서 합성 유당, ° C. 데이터 작용화로 인해 평균 nanoparticle 직경의 작은 증가를 나타냅니다. 비 작용 nanoparticles는 약 부정적인 제타 잠재력을 가지고 있지만. -20 MV는 작용 입자 nanoparticle 표면의 성공 작용화 보여주 긍정 제타 잠재적인 가치를 보여주었다. 락토 오스 및 디-mannose는 모두 중립 설탕되지만 에틸렌 무료로 아민 그룹 saccharides를 연결 활용 링커가 긍정적인 제타 전위의 책임 수도 디아민. 반응 시간은 nanoparticles의 최종 형태와 달성 설탕 첨부의 학위를 모두 영향을 미칠 수있는 또 다른 변수입니다. 반응 시간을 조정하여, nanoparticles 표면에 부착된 최종 설탕 농도는 그림 4A와 같이 제어할 수 있습니다. 예상했던대로 50:50 CPTEG의 표면에 dimannose의 농도 : CPH는 nanoparticles가 함께 증가반응의 전체 시간과 18 시간 후 최대에 도달했습니다. 24 시간 총 반응 시간과 기능화 Nanoparticles은 마우스 뼈 - 골수 파생 돌기 세포 (DC가)에 CLRs을 타겟팅하는 능력을 평가하는 데 사용되었다. 유동세포계측법 두 CL 수용체 (즉, CIRE (CD209, DC-로그인) 및 mannose 수용체 (CD206)) 이외의 작용과 자극 후, 그리고 유당 및 DI-mannose 작용 nanoparticles (그림 4B)의 표현을 평가하는 데 사용되었다. 세포가 락토 오스 및 디-mannose 작용 nanoparticles 모두 함께 자극했을 때 효과적인 타겟팅을 나타내는 것입니다 두 수용체의 높은 표현은, 얻은 것입니다. 그러나, 디-mannose-작용 입자 공부했다 수용체이 리간드의 특이성을 나타내는 표현의 높은 수준을 보여주었다. <table border="1"><tbody><tr><td> Nanoparticle 유형 </td><td> 평균 입자 직경 (NM) </td><td> 번가분노의 입자 ζ-잠재적인 (MV) </td></tr><tr><td> 비 작용 </td><td> 162 ± 43 </td><td> -20 ± 0.6 </td></tr><tr><td> 락토 오스 </td><td> 235 ± 34 </td><td> 26 ± 2.4 </td></tr><tr><td> 디 - mannose </td><td> 243 ± 32 </td><td> 30 ± 4.2 </td></tr></tbody></table> 표 1. Nanoparticle 특성화. 비 작용과 작용이 유사 탄성 광 산란 및 제타 잠재력 측정을 특징으로했다. 입자 크기 데이터는 평균 값이 ± 세 독립적인 실험에서 수집한 동적 광 산란 데이터의 표준 편차 (SD)를 나타냅니다. 제타 잠재적인 데이터는 평균 값이 ± SD 세 독립적인 신호를 나타냅니다. CPH nanoparticle의 표면 :​​ 제타 전위의 기호의 변화는 설탕 50:50 CPTEG에 효율적으로 복합했습니다 보여줍니다. <img alt="그림 1" src<html> = &quot;/ files/ftp_upload/3967/3967fig1.jpg&quot;/&gt; 그림 1. 탄수화물 polyanhydride의 nanoparticles의 작용화 및 설명 높은 처리량 방식으로 설계할 수있는 작용 nanoparticle 라이브러리의 예제를 통해 추구하는 접근법 (A) 그래픽 표현. (B) 자동 증착 장치의 도식 표현는 (i) 세 NE 1,000 펌프로 구성되어 입자 작용화, 활용, (ii) 본 로봇 무대는 두 액츄에이터 (Zaber)에 의해 통합 : X 방향의 움직임을위한 하나의 다른 두 인접한 랙에은 (튜브 및 cuvettes에 적합한) 세 액츄에이터, 각 방향에 대해 하나 (X, Y 및 Z)으로 구성된과 (3) 두 번째 로봇 무대, Y 방향의 운동입니다. 펌프 다섯 액츄에이터의 총 시리즈에 연결되어 있습니다. 발전소 펌프는 LabVIEW 소프트웨어를 사용하여 컴퓨터에 의해 운영됩니다. 이 그림은 규모에 없습니다.arge.jpg &quot;대상 =&quot;_blank &quot;&gt; 큰 그림을 보려면 여기를 클릭하십시오. <img alt="그림 2" src="/files/ftp_upload/3967/3967fig2.jpg" /> 그림 2. 자동의 그래픽 표현은 예로 mannose를 사용하여 탄수화물의 합성을 반복. <img alt="그림 3" src="/files/ftp_upload/3967/3967fig3.jpg" /> 그림 3. 보호 dimannoside의 1 H NMR. <img alt="그림 4" src="/files/ftp_upload/3967/3967fig4.jpg" /> 그림 4. () saccharide의 nanoparticle의 표면 농도에 대한 반응 시간의 영향. 표시된 데이터에 50:50 CPTEG : CPH의 nanoparticles는 서로 다른 반응 시간에 dimannose으로 기능화되었으며 반응은 4 시에 진행되었습니다 ° C. 두 개의 독립적인 작용화 실험의 평균 및 표준 오류가 표시됩니다. (B) 유당과 디 - mannose 작용 nanoparticles취득 표현에 비해 CPH nanoparticles : 뼈 작용 50:50 CPTEG로 자극 후이 두 마커의 향상된 표현에 의해 증명으로 골수 파생 돌기 세포에 효과적으로 대상 DC-기호 (CIRE, CD209)와 mannose 수용체 (CD206) 비 작용 입자로.

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NLBP는 cofounder이며 탄수화물 회사 LuCella Biosciences 주식 회사의 지분을 보유하고

면역 세포에 직접 nanoparticle의 상호 작용에 에이전트를 대상으로 같은 탄수화물의 약효는 이전 10, 11을 입증되었습니다. 우리 실험실에서 이전 연구는이를 더욱 T 세포 활성화 10, 11 중요할 수도 면역 세포의 활성을 향상 polyanhydride의 nanoparticles에 연결된 특정 설탕은 항원 제시 세포 (APCs)에 다른 CLRs를 타겟팅할 수있는 것으로 나타났습니다. 그러나 몇 가지 매개 변수 - 같은 polyanhydr...

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 동력 XYZ 스테이지 : 배 T-LSM050A, 축 당 50 밀리미터 여행 </td><td> Zaber 기술 </td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000 단일 주사기 펌프 </td><td> 새로운 시대 펌프 시스템 </td><td> NE-1000 </td></tr><tr><td> Pyrex * 비스타 * 무테 재사용 유리 문화 튜브 </td><td> 코닝 </td><td> 07-250-125 </td></tr><tr><td> ASW 1000 </td><td> Chemspeed 기술 </td><td></td></tr><tr><td> LabVIEW </td><td> 내쇼날 인 스트 루먼트 </td><td> 776671-35 </td></tr><tr><td> SGE 가스 타이트 주사기, Luer Loc 데이 </td><td> 시그마 알드리치 </td><td> 509,507 </td></tr><tr><td> XL-2000 Sonicator </td><td> Qsonica </td><td> Q55 </td></tr><tr><td> 미니 튜브 회전 </td><td> 피셔 과학 </td><td> 05-450-127 </td></tr></tbody></table>

high-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles

이러한 자료 디자인, 나노 테크놀로지, 화학, 면역학 등의 분야를 포함 Transdisciplinary 방법은 이성적 효능 백신 캐리어를 디자인하는 데 활용해야합니다. Nanoparticle 기반 플랫폼은 백신에게 immunogenicity 1을 향상시킬 수있는 백신 항원의 지속성을 연장하실 수 있습니다. 여러 생분해성 폴리머는 백신 배달 차량 1로 연구되어, 특히 polyanhydride 입자는 안정적인 단백질 항원의 지속적인 출시를 제공하고 항원 제시 세포를 활성화하고 면역 반응에게 2-12를 조절하는 능력을 증명하고있다. 이러한 백신 운반선의 분자 설계 폴리머 속성의 합리적인 선택뿐만 아니라 적절한 타겟팅 대리인의 법인을 통합해야합니다. 리간드와 작용 입자를 대상으로 높은 처리량을 자동으로 지어낸 다양한 R을 공부하는 능력을 향상시킬 수있는 강력한 도구입니다속성의 엔지와 재현성 백신 전달 장치의 디자인으로 이어질 것입니다. 면역 세포의 특정 수용체에 의해 인식되는 수있는 리간드를 타겟팅 이외는 10,11,13 C 형 렉틴 수용체 (CLRs가)에 탄수화물이 존재 인식 패턴 인식 수용체 (PRRs)입니다 변조 및 재단사 면역 반응을 보여줘왔다 병원균의 표면. CLRs 통해 면역 세포의 자극은 항원과 더욱 T 세포 활성화 14,15위한 후속 프레 젠 테이션의 향상된 내면화을 허용합니다. 따라서 탄수화물 분자는 면역 반응의 연구에 중요한 역할을하지만, 이러한 biomolecules의 사용은 종종 구조적으로 잘 정의되고 순수한 탄수화물의 가용성의 부족을 앓고. 솔루션 위상 반응을 반복을 바탕으로 자동화 플랫폼은 크게 낮은 B를 사용하여 이러한 synthetically 도전 분자의 신속하고 제어 합성을 활성화할 수 있습니다전통적인 고체 상 16,17 방법보다 블록 수량 uilding. 여기에 우리는 이러한 중간 정화를위한 fluorous 고체 상 추출과 mannose 기반 타겟팅 리간드로서 oligosaccharides의 자동화 솔루션 상 합성을위한 프로토콜을보고합니다. 탄수화물 기반 타겟팅 요원이되기 위해 자동화된 방법을 개발 후, 우리는 이전에 10 설명된대로 LabVIEW에서 운영하는 자동화된 로봇 세트를 고용 polyanhydride nanoparticles의 표면에 그들의 부착을위한 방법을 설명합니다. 탄수화물과 표면 작용화는 CLRs에게 10,11을 대상으로하고 발굴 다중 파라메 트릭 시스템과 관련된 복잡 아주 가치 (그림 1A)이 될에게 제조 방법의 처리량 증가에 효험을 보이지 않고있다.

Watch this Scientific Journal Video about High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles at JoVE.com

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

이 문서에서는 높은 처리량 방법은 항원 제시 세포의 특정 수용체​​를 타겟에 더 이상 사용할 수 polyanhydride의 nanoparticles의 표면에 oligosaccharides과 첨부 파일의 합성을 위해 제공됩니다.

Polyanhydride의 Nanoparticles의 높은 처리량 탄수화물의 합성과 작용화

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

high-throughput-synthesis-carbohydrates-functionalization

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Bioengineering

11.3K 조회수.  Iowa State University. 이 문서에서는 높은 처리량 방법은 항원 제시 세포의 특정 수용체​​를 타겟에 더 이상 사용할 수 polyanhydride의 nanoparticles의 표면에 oligosaccharides과 첨부 파일의 합성을 위해 제공됩니다.

비디오: High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Biomaterials 높은 처리량 발견을위한 폴리머 Microarrays

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

연구

생체공학

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 ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Polyanhydride의 Nanoparticles에 의해 유도된 Murine Macrophages 폐포 수확 및 평가 세포 활성화

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

높은 처리량 마이크로 캡슐 단일 셀 및 다중 셀

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

고분자 유전자 배달에 대한 평가는 Nanoparticle 추적 분석 및 높은 처리량 유동 세포 계측법에 의해 나노 입자

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

동맥 경화의 빠른 검색을위한 산화철 나노 입자의 전자 레인지 중심의 합성

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

의료 기기에 대한 박테리아의 높은 처리량 식별 구충제 폴리머

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

간염 E 바이러스 나노 화학 활용 방법을 사용 하 여의 표면 기능화

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...