이 작품은 암, 나노기술 우수 파일럿 부여, 대학 암 연구 기금과 국민 건강 연구소의 K - 12 경력 개발 수상 캐롤라이나 센터에 대한 골퍼에 의해 투자되었다.

1. PLGA - B - PEG 중합체의 합성 <ol><li> 터미널 카르복 그룹과 폴리 (D, L - lactide - 공동 glycolide) (PLGA)는 (PLGA - 카르복) 5mM의 농도에서 PLGA (같은 재료 섹션에서 언급된)에 대한 모든 용매에 녹아있다. PLGA는 부드러운 감동이 농도에서 해산 수 있습니다. </li><li> 보건국 (분자량 115.09) 및 EDC (분자량 191.7) 모두 25mM의 농도에서 PLGA 용액에 녹아 있습니다. (EDC와 NHS 모두 PLGA에 비해 5 배의 stoichiometric 초과에 추가됩니다.) PLGA - 카르복은 약 1 시간 동안 부드러운 감동과 PLGA - 카르복 솔루션 EDC와 NHS을 추가하여 PLGA - 보건국로 변환됩니다. </li><li> 반응 생성물 PLGA - 보건국은 세척 용액에 메탄올을 추가하여 밖으로 시켰던 것입니다. 메탄올의 약 10 배 볼륨 초과가 솔루션에 추가됩니다. 솔루션은 침전물 밖 PLGA - NHS를하고 (EDC와 NHS의 흔적을 제거합니다. 메탄올로 세척의이 절차는 적어도 세 번 반복 뜨는을 폐기 XG 2000에서 centrifuged입니다. </li><li> PLGA - 보건국 펠렛은 세척 솔루션의 모든 흔적을 제거하는 30 분 진공하에 건조. </li><li> PLGA - 보건국 펠렛 지금 PLGA를 해산하기 위해 처음 사용되었던 동일한 농도에서 같은 용매에 다시 녹아있다. heterobifunctional PEG (아민 - PEG - 카르복)는 다음 5mM의 농도에서 PLGA 솔루션에 추가됩니다 (1:1의 비율 stoichiometric). 혼합 솔루션은 지속적인 교반과 함께 24 시간 동안 incubated입니다. </li><li> 24 시간 후에, 반응 생성물 PLGA - B - PEG 블록 공중 합체는 초과 세척 용액에 메탄올을 추가하여 밖으로 시켰던 것입니다. 로 세 번 상기 세정 및 원심 분리 과정을 반복합니다. 이것은 모든 초과 unreacted PEG 제거됩니다. </li><li> PLGA - B - PEG 블록 공중 합체는 진공 하에서 건조됩니다. </li></ol> 2. PLGA - B - PEG의 nanoparticle 준비 표면에 PEG으로 덮여 PLGA 코어와 Nanoparticles 이러한 디블록 공중 합체와 함께 준비하실 수 있습니다. 다른 소수성 약물의 다양한 등 nanoparticles에서 캡슐 수 있습니다. 형광 화합물은 nanoparticles에 캡슐 될 수도 있고 아니면 PLGA에 복합 있으므로 이러한 nanoparticles가 형광 이미징에 사용될 수 있습니다. Nanoprecipitation 방법은 캡슐로 원하는화물 자연에서 높은 소수성 특히 때 nanoparticles를 만들기 위해 사용됩니다. <ol><li> PLGA - B - PEG 블록 공중 합체와 약물 /화물은 (캡슐 예정) PLGA를 녹이고있는 어떤 용매에 녹아 있습니다. PLGA는 acetonitrile, DCM, tetrahydrofuran, 아세톤 또는 에틸 아세테이트를 포함하여 여러 가지 일반적인 용매에 의해 용해 수 있습니다. 그것이 nanoparticle의 속성에 영향을 미친다 같은 용매의 선택은 중요합니다. 따라서, 용매 적절한는이 단계에서 사용해야합니다. </li><li> 고분자 / 약물 혼합물은 다음 주위 3 MG / ML의 최종 폴리머 농도를주는 감동의 물을 3-5 볼륨 dropwise 추가됩니다. (그림 2) </li><li> 교반은 nanoparticles 자기 조립에 의해 양식 수 있고 유기 용매의 흔적을 제거하는 감소 압력을 받고 2 시간 동안 계속됩니다. </li><li> 수확 및 정화 : nanoparticles 그 다음 씻은 Amicon 필터 (MWCO 20KDa)를 사용하여 10 분 2,700 X g에서 원심 분리에 의해 집중하고, PBS로 재구성됩니다. 이것은 모두 해제 속은 약물 /화물을 제거합니다. 기본 biophysical 같은 크기로 characterizations, 표면 요금 및 약물 로딩 효율 좋은 nanoparticles의 속성을 이해하기 위해 수행할 수 있습니다. </li></ol> 3. 저장 냉동 건조는 nanoparticles 11 저장하는 데 일반적으로 사용되는 방법입니다. 냉동 건조는 장기 안정성 12 nanoparticles의 물리적, 화학적 특성을 유지합니다. 동결 건조 프로세스가 입자에 스트레스 원인과 제제를 불안정하게 할 수 cryo - protectants (냉동 스트레스로부터 보호)와 lyo - protectants가 (건조 스트레스로부터 보호) 일반적으로 사용되고 있으므로. 이 protectants의 선택은 저장 시간이 13 원하는 길이에 의해 결정됩니다. <ol><li> 냉동 건조에서 그 TG 영하로 샘플의 총 응고가 있습니다. </li><li> 건조 단계에서 얼음이 승화에 의해 제거됩니다. 온도 및 압력이 효율적인 냉동 건조 과정을 달성하기 위해 최적화되어야합니다. </li></ol> 4. 대표 결과 : PLGA - B - PEG 디 - 블록 공중 합체의 특성 다른 기술은 폴리머의 성공적인 활용을 확인하는 데 사용할 수 있습니다. PLGA - B - PEG의 구성은 400 MHz의 상반기 핵 자기 공명 (NMR)를 사용하여 특징하실 수 있습니다. 형성된 제품의 분자량 (PLGA - B - PEG)는 겔 침투 크로마 토그래피 (GPC)에서 확인할 수 있습니다. PLGA - B - PEG 분자량 distributioN 곡선과 용출 시간은 PLGA와 PEG 혼자 다른되어야합니다. 조합에서는 이러한 기술은 형성된 제품 특징 및 활용 반응이 성공 여부를 결정한다. PLGA - B - PEG의 특성화는 nanoparticles 입자 크기 및 크​​기 분포는 동적 광 산란에 의해 측정할 수 있습니다. nanoprecipitation 과정에서 다른 매개 변수는 입자의 크기에 영향을줍니다. 처음 사용하는 고분자 (PLGA와 PEG 모두)의 분자 중량은 또한 입자 크기 분포에 영향을. 전환 전자 현미경 (TEM)은 또한 그림 3에서 볼 nanoparticles의 크기 분포와 구조를 확인하는 데 사용할 수 있습니다. 입자 크기 범위는 NM 범위에서 일반적이다. 고르지 크기 분포와 큰 입자 크기는 중 결합 반응 또는 nanoprecipitation 방법의 요구에 최적화 오류를 나타낼 수 있습니다. 또한, 표면 제타 전위는 ZetaPALS에 의해 측정할 수 있습니다. 약물 /화물 적재 효율 표준 HPLC과 계량 수 있습니다. 입자가 유기 용매와 HPLC에 녹아있는 아르 것은 약물 /화물 (그림 4)의 흡광도를 측정을 수행할 수 있습니다. nanoparticles의 고정 수량 30 슬라이드 - A - Lyzer 미니 투석 단위로 dialyzed 아르 알려진 어디 약물 릴리스 운동 연구는 할 수 있습니다. 고정 시간 간격으로, 투석 장치에있는 내용은 수집과 유기 용매의 동일한 볼륨 nanoparticles를 해산에 추가됩니다. HPLC는 약물 /화물 내용을 계량이 샘플에 이루어집니다. <img alt="그림 1" src="/files/ftp_upload/3398/3398fig1.jpg" /> 그림 1. EDC / NHS 화학 <img alt="그림 2" src="/files/ftp_upload/3398/3398fig2.jpg" /> 그림 2. 고분자 nanoparticles를 준비 Nanoprecipitation 방법은. PEG - PLGA 디블록와 입자에로드 될 수있는 약물이나화물을 포함 (acetonitrile 또는 DCM) 용매의 유기 솔루션은 H 2 O. 감동의 3-5 ML에 dropwise 추가됩니다 <img alt="그림 3" src="/files/ftp_upload/3398/3398fig3.jpg" /> 그림 3. nanopartices의 전송 전자 현미경은. PEG - PLGA의 TEM 이미지는 wortamin를 포함한 nanoparticles. Phosphotungstic 산은 대비 에이전트로 사용되었다. <img alt="그림 4" src="/files/ftp_upload/3398/3398fig4.jpg" /> 그림 4. PBS에서 투석 후 nanoparticles에서 Paclitaxel의 nanoparticle에서 약물의 관리 릴리스. 릴리스. 언급 당시 입자는 투석 카세트에서 제거되었고 acetonitrile에 solublized. 솔루션은 HPLC로 측정되었다. nanoparticles의 두 개의 서로 다른 많은가 비교되었다. <div id="myEventWatcherDiv"></div><div id="myEventWatcherDiv"></div>

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관심 없음 충돌 선언하지 않습니다.

디블록 공동 폴리머를 사용하여 nanoprecipitation 방법은 고분자 nanoparticles를 엔지니어링하는 간단하고 빠른 방법을 나타냅니다. 그 결과 nanoparticles는 저조한 수용성 화합물의 전달 활용할 수있는 소수성 핵심으로 구성되어 있습니다. 타겟팅 리간드로 가능성이 더 활용을위한 잔기를 제공하면서 표면 친수성 ​​계층은 우수한 수성 용해도 수 있습니다. liposomes 등 많은 nanopartic...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> EDC </td><td> 써모 과학 </td><td> 22,980 </td><td> 활용 시약 </td></tr><tr><td> 보건국 </td><td> 써모 과학 </td><td> 24,500 </td><td> 활용 시약 </td></tr><tr><td> 아민 - PEG - 카르복 </td><td> Laysan 바이오 주식 회사 </td><td> Nh2 - PEG - CM - 5000 </td><td> 폴리머는 (모든 PEG MW를 사용할 수 5,000 여기에 나열됩니다) </td></tr><tr><td> PLGA - carbxylate </td><td> Lactel </td><td> B6013 - 2 </td><td> 중합체 </td></tr><tr><td> Dichloromethane (DCM) </td><td> 시그마 - 올드 리치 </td><td> 34,856 </td><td> 용제 </td></tr><tr><td> Acetonitrile&gt; 99 % 순도 </td><td> 시그마 - 올드 리치 </td><td> 34,851 </td><td> 용제 </td></tr><tr><td> 메탄올&gt; 99 % 순도 </td><td> 시그마 - 올드 리치 </td><td> 34,860 </td><td> 빨래 </td></tr></tbody></table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

나노기술은 harnessing에게 규모 나노미터 (nanoparticles)입니다 입자의 고유한 속성을 포함 과학의 비교적 새로운 지점입니다. Nanoparticles들은 크기, 조성과 표면 화학은 신중하게 제어할 수있는 정확한 방식으로 설계하실 수 있습니다. 이것은 용해도, 확산 계수, biodistribution, 릴리스 특성 및 immunogenicity로서화물의 기본 속성 중 일부를 수정하는 전례없는 자유를 수 있습니다. 그들의 처음부터, nanoparticles는 약물 전달, 이미징 및 세포 생물학을 포함하여 1-4 과학 및 의학의 많은 분야에서 널리 사용되었습니다. 그러나, 그것은 완전히 인식 기술 장벽으로 인해 &quot;나노기술 연구소&quot;의 외부 활용되지 않았습니다. 이 문서에서는, 우리는 잠재적인 어플 리케이션의 광범있는 폴리머 기반의 nanoparticle 플랫폼을 종합하는 간단한 방법을 설명합니다. 첫 번째 단계는 소수성 친수성 ​​도메인과 도메인을 모두 가진 디블록 공동 고분자를 합성하는 것입니다. 모델 고분자와 같은 PLGA와 PEG를 사용하여, 우리는 EDC / NHS 화학 5 (그림 1)을 사용하여 결합 반응을 설명했다. 우리는 또한 폴리머 정화 과정을 논의합니다. 합성 디블록 공동 고분자는 소수성 - 친수성 ​​상호 작용을 통해 nanoprecipitation 과정에서 nanoparticles로 자기 조립하실 수 있습니다. 설명 폴리머 nanoparticle은 매우 다양한 있습니다. nanoparticle의 소수성 핵심은 약물 전달 experiments6에 대해 제대로 용해 약물을 휴대 이용하실 수 있습니다. 또한, nanoparticles는 DMSO와 같은 용매가 필요 wortmannin로 저조한 수용성 분자 생물 시약에 대한 독성 용매의 문제를 극복할 수 있습니다. 그러나, DMSO는 세포 독성하고 실험을 방해할 수 있습니다. 이러한 저조한​​ 수용성 약물과 시약이 효과적으로 최소한의 독성과 고분자 nanoparticles를 사용하여 전달할 수 있습니다. 폴리머 nanoparticles 또한 형광 염료와 함께로드되고 세포 연구에 대한 인신 매매 이용하실 수 있습니다. 마지막으로, 이러한 폴리머 nanoparticles는 표면 PEG 통해 리간드를 타겟팅 복합 수 있습니다. 이러한 타겟 nanoparticles는 70-10이나 세포에서 특정 epitopes 레이블을 이용하실 수 있습니다.

Watch this Scientific Journal Video about Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique at JoVE.com

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

이 문서 디블록 공동 폴리머를 사용하여 폴리머 기반 nanoparticles를 합성하는 nanoprecipitation 방법을 설명합니다. 우리는 디블록의 합성 공동 고분자, nanoprecipitation 기술, 그리고 잠재적인 애플 리케이션을 설명합니다.

Nanoprecipitation 기법을 통해 디블록 고분자 Nanoparticles의 제형

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

formulation-diblock-polymeric-nanoparticles-through-nanoprecipitation

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JoVE Journal

Bioengineering

37.1K 조회수.  University of North Carolina School of Medicine. 이 문서 디블록 공동 폴리머를 사용하여 폴리머 기반 nanoparticles를 합성하는 nanoprecipitation 방법을 설명합니다. 우리는 디블록의 합성 공동 고분자, nanoprecipitation 기술, 그리고 잠재적인 애플 리케이션을 설명합니다.

비디오: Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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 ...

연구

생체공학

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

미리 설계된 크기와 모양의 중합체 및 바이오 하이브리드 섬유의 미세 유체 제조

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

포토 리소그래피에 의해 고분자 미세 바늘 어레이 제작

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

바이오 메디컬 응용 프로그램에 대한 초 소수성 고분자 재료를 제조

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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

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

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

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

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

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

급속 하 고, 확장 가능한 어셈블리 및 플래시 Nanoprecipitation 통해 다양 한 합성 Nanocarriers에 생리 활성 단백질의 Immunostimulants 로드

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

단백질 키 니 아 제 C 델타 억제제 펩 티 드 배합 금 나노 입자를 사용 하 여

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

고분자 나노 입자에 소수 성 및 친수성 화합물의 캡슐화에 대 한 NanoPrecipitation 플래시

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

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

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

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

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

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