Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00
Description: Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

저자들은 보조금 번호 P20GM103541 및 부여 번호 P20GM10344615로 기관 개발 상 (IDeA)을 통한 재정 지원을 위해 국립 보건원 (NIH)의 국립 종합 의학 연구소에 감사를드립니다. 여기에있는 진술은 NIH의 견해를 반영하지 않습니다. 우리는 또한 첨단 기술 생명 과학 센터 (Bioscience CAT) 상 (12A00448)을 통해 재정 지원을 위해 델라웨어 생명 공학 연구소 (DBI)와 델라웨어 경제 개발 사무소 (DEDO)를 인정합니다.

1. siRNA 나노 캐리어의 제형 <ol><li> pH 6.0에서 20mM 4- (2- 하이드 록시 에틸) 피페 라진 -1- 에탄 설 폰산 (HEPES) 버퍼로 희석 된 동일한 부피의 siRNA 및 mPEG- b -P (APNBMA)의 개별 용액을 준비한다. <ol><li> 20 MM HEPES 솔루션에 32 μg / ML의 농도 siRNA를 추가합니다. 참고 : siRNA는 비 표적화 된 보편적 음성 대조군 시퀀스였다. 그러나, siRNA는 관심있는 모든 유전자를 표적으로하도록 설계 ?...

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tuning+of+siRNA+dose+response+by+combining+mixed+polymer+nanocarriers+with+simple+kinetic+modeling.">Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.</a> Acta Biomater. 50, 407-416 (2017).</li></ol>

이 방법에는 몇 가지 단계가 특히 중요합니다. nanocarriers을 수립 할 때, 구성 요소 추가 및 혼합 속도의 순서는 효능 (39)에 영향을 미치는 두 가지 중요한 매개 변수입니다. 이 프로토콜은 vortexing하는 동안 음이온 성 구성 요소 인 siRNA에 양이온 성 구성 요소 인 mPEG- b -P (APNBMA)가 첨가되도록 요구합니다. 총 배합량에 따라이 혼합 공정은 3-6 초가 소요됩니다. 나노 캐리어가 ?...

small interfering RNAs (siRNAs)는 사실상 모든 표적 유전자 1에 대해 매우 특이적이고 강력하며 맞춤형 인 촉매 RNAi 경로를 통해 전사 후 유전자 사일런 싱을 매개한다. 이러한 유망한 특성으로 인해 전이성 흑색 종 및 혈우병을 비롯한 수많은 질병의 치료를위한 인간 임상 시험에서 siRNA 치료제가 발전 할 수있었습니다 2 , 3 . 그러나 번역을 방해하는 중요한 배달 문제가 계속 발생합니다 4 . 특히, 운반체는 안정을 유지해야하며 세포 외 분해로부터 siRNA를 보호해야하며, 또한 세포질에 유핵 적재 물을 방출해야합니다 5 . 또한, 많은 RNAi 응용 프로그램은 siRNA 치료법 7 에서 부작용을 줄이고 변형 가능하게하는 공간 및 시간 6 에서 유전자 침묵을 조절하는 향상된 방법을 필요로합니다.약 발견 8 을위한 세포 microarrays에서 재생성 비계 9 에있는 세포 반응의 조음에 배열하는 신청에있는 dvances. 이러한 과제는 siRNA 나노 캐리어에서 결합 대 방출을보다 잘 제어 할 수있는 새로운 물질 및 방법의 필요성을 강조합니다. siRNA 방출을 제어하고 시공간 조절을 향상시키는 가장 유망한 전략 중 하나는 자극 반응 물질의 사용입니다 10 . 예를 들어, 생체 물질의 다양한 변화는 핵산 변형 산화 환원 전위 나 산도 또는 자장인가, 초음파 또는 광 (11)에 대응하여 결합 친 화성을 함께 설계 하였다. 이들 시스템 중 다수가 핵산 활성에 대한 개선 된 제어를 입증하지만, 트리거로서의 광의 사용은 순간적 시간 반응, 정확한 공간 분해 및 조율 용이성으로 인해 특히 유리하다 <sup class = &quot;xref&quot;&gt; 12. 또한, 유전자 발현 조절을위한 사진 민감 기술의 잠재력은 최첨단의 유도 성 프로모터 및 광 발생 조절 시스템에 의해 입증되었다. 그러나 이러한 시스템은 내인성 유전자를 규제하기위한 제한된 용량, 면역 원성과 같은 안전성 문제, 다중 부품 조립품의 운반이 어렵다는 것을 비롯하여 수많은 어려움을 겪고 있습니다 13 , 14 , 15 . 광 반응성 siRNA 나노 캐리어는 이러한 결점을 극복하고 유전자 발현을 공간적으로 조절하는보다 간단하고 견고한 접근법을 제공하는데 이상적으로 적합합니다 16 , 17 , 18 . 불행하게도, 결과 단백질의 녹다운 반응을 정확하게 예측하는 방법은 아직 파악하기 어렵습니다. 핵심 과제는 siRNA 방출의 정량적 평가가희귀 19 , 20 및이 평가가 수행 되더라도 siRNA / 단백질 회전율 역학의 분석에 결합되지 않았습니다. siRNA의 방출량과 지속성 / 수명은 생성 된 유전자 침묵 동역학의 중요한 결정 인자입니다. 따라서 그러한 정보가 부족하면 RNAi 21 의 용량 반응의 정확한 예측을 방해하는 주요 단절이 있습니다. 이러한 문제를 해결하기 nanocarriers의 적절한 구조 함수 관계의 형성을 촉진하고 더 바이오 물질 (22)을 설계 알리는 것이다. 또한, 이러한 접근법은보다 효과적인 siRNA 투약 프로토콜의 개발을 가능하게 할 것이다. 역동적 인 사일런스 반응을 이해하기 위해 여러 그룹이 RNAi 23 , 24 , 25 의 수학적 모델을 조사했습니다. 이 프레임 워크는유전자 발현의 siRNA 매개 된 변화에 대한 통찰력을 제공하고 속도 - 제한 단계 (26)를 식별하는 데 성공. 그러나, 이러한 모델은 제어 siRNA를 방출 할 수없는 상업적인 유전자 전달 시스템 (예를 들어, 리포 펙 타민 및 폴리에틸렌 이민 (PEI))에 적용되어, 상기 모델의 복잡성이 크게 그들의 유틸리티 (27)를 한정하고있다. 이러한 단점은 간결하고 사용하기 쉬운 예측 동역학 모델과 결합하여 정확하게 조정할 수있는 새로운 물질에 대한 충족되지 않은 요구를 강조합니다. 우리의 방법은 빛에 민감한 나노 캐리어 플랫폼과 결합 된 방법을 통합하여 자유로운 siRNA와 모델 RNAi 역학을 정량화함으로써 이러한 모든 문제를 해결합니다. 특히, 우리 플랫폼의 정확하게 제어 된 siRNA 릴리스 28 은 캡슐화 된 대 양성자를 정확하게 정량하기위한 두 가지 보완적인 방법으로 모니터링됩니다결합 된 siRNA. 이 분석에서 실험 데이터는 유전자 침묵 효율성 선험적 (29)을 예측하는 간단한 운동 모델로 입력됩니다. 마지막으로, nanocarriers의 온 / 오프 특성은 쉽게 세포 길이 규모에 공간 제어와 유전자 표현에 세포 패턴을 생성하는 데 활용됩니다. 따라서,이 방법은 세포 행동의 시공간 조절로부터 이익을 얻는 다양한 응용에서 유전자 침묵을 제어 및 예측하는 용이하게 적응 가능한 방법을 제공한다.

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			<td style="width:145px;">N/A</td>
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			<td height="21" style="height:21px;width:183px;">HEPES</td>
			<td style="width:157px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:183px;">sodium dodecyl sulfate&#160;</td>
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			<td height="21" style="height:21px;width:183px;">agarose</td>
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			<td style="width:145px;">BP160-100</td>
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			<td height="21" style="height:21px;width:183px;">ethidium bromide</td>
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			<td height="42" style="height:42px;width:183px;">siRNA labelled with Dy547</td>
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			<td style="width:195px;">fluorophore conjugated to 5&#8217; end of sense strand</td>
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			<td height="21" style="height:21px;width:183px;">microscope slide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">12-550-A3</td>
			<td style="width:195px;">pre-cleaned glass</td>
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			<td height="21" style="height:21px;width:183px;">LSM 780&#160;</td>
			<td style="width:157px;">Carl Zeiss</td>
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			<td height="21" style="height:21px;width:183px;">ZEN 2010</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">FCS analysis software</td>
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			<td height="21" style="height:21px;width:183px;">MATLAB</td>
			<td style="width:157px;">MathWorks</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">programming language</td>
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			<td height="21" style="height:21px;width:183px;">NIH/3T3 cells&#160;</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:145px;">ATCC CRL-1658</td>
			<td style="width:195px;"></td>
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			<td height="21" style="height:21px;width:183px;">DMEM</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">10-013-CV</td>
			<td style="width:195px;">growth media</td>
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			<td height="21" style="height:21px;width:183px;">fetal bovine serum</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">35-011-CV</td>
			<td style="width:195px;">heat-inactivated</td>
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			<td height="21" style="height:21px;width:183px;">penicillin-streptomycin</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">&#160;30-002-CI</td>
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			<td height="21" style="height:21px;width:183px;">6-well plates</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">08-772-1B</td>
			<td style="width:195px;"></td>
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			<td height="21" style="height:21px;width:183px;">Opti-MEM</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">11058021</td>
			<td style="width:195px;">transfection media</td>
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predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers

새로운 물질 및 방법은 유전자 활동의 정확한 조절을 필요로하는 광범위한 적용을위한 핵산의 결합 대 방출을보다 잘 제어하기 위해 필요합니다. 특히, 유전자 발현에 대한 시공간적 제어가 개선 된 새로운 자극 - 반응 물질은 약물 발견 및 재생 의학 기술에서 번역 가능한 플랫폼의 잠금을 해제합니다. 또한, 물질로부터의 핵산 방출을 제어하는 ​​능력이 향상되면 나노 캐리어 효능 을 선험적 으로 예측할 수있는 능률적 인 방법의 개발이 가능 해져 배달 차량의 신속한 스크리닝이 가능해진다. 여기, 우리는 유전자 침묵 효율을 예측하고 모듈 식 광 반응성 나노 캐리어 시스템을 통해 유전자 발현에 대한 시공간 제어를 달성하기위한 프로토콜을 제시한다. 작은 간섭 RNA (siRNA)는 mPEG- b -poly (5- (3- (아미노) 프로 폭시) -2- 니트로 벤질 메타 크릴 레이트) (mPEG- β -P (APNBMA)빛으로 조절되어 조정 가능한 ON / OFF siRNA 방출을 촉진 할 수있는 안정한 나노 캐리어입니다. 우리는 세포 내 환경을 모방 솔루션에서 siRNA 릴리스의 정확한 부량을위한 형광 상관 분광법과 겔 전기 영동을 사용하는 두 보완 assays을 설명합니다. 이 분석에서 얻은 정보는 다양한 광 자극 조건에 대한 동적 침묵 반응을 예측하기위한 간단한 RNA 간섭 (RNAi) 동역학 모델에 통합되었습니다. 이어서, 이러한 최적화 된 조사 조건은 시공간적으로 유전자 침묵을 제어하기위한 새로운 프로토콜의 개선을 가능하게했다. 이 방법은 cell-to-cell 분해능과 off-target 효과가없는 유전자 발현의 세포 패턴을 생성 할 수 있습니다. 종합하면, 우리의 접근 방식은 유전자 발현의 역동적 인 변화를 예측하고 시공간적으로 siRNA 활동을 정밀하게 제어 할 수있는 사용하기 쉬운 방법을 제공합니다. 이 assays 세트는 다양한 ot를 테스트하기 위해 쉽게 적용 할 수 있습니다그녀의 자극 반응 시스템은 생물 의학 연구 및 의학 분야의 수많은 어플리케이션과 관련된 핵심 과제를 해결합니다.

나노 캐리어의 제제화 후 siRNA 방출 분석을 수행하여 시험 관내 형질 감염 에 사용되는 조사 조건을 알려 주었다. 방출 된 siRNA의 백분율을 결정하기 위해 다양한 양의 빛을 가했다. 첫 번째 분석은 여전히 ​​폴리머와 복합체 / 결합 된 siRNA 분자에서 유리 siRNA 분자를 분리하기 위해 겔 전기 영동을 사용했습니다. 빛으로 치료되지 않은 나노 캐리어는 안정한 ...

Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

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

우리는 광 반응성 블록 공중 합체를 사용하는 새로운 방법을 제시한다.이 방법은 검출 불가능한 오프 - 타겟 효과없이 유전자 침묵을보다 효율적으로 공간적으로 제어 할 수있다. 또한 유전자 발현의 변화는 간단한 siRNA 방출 분석 및 간단한 운동 모델링을 사용하여 예측할 수 있습니다.

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 ...

The overall goal of these methods is to unable the prediction of small interfering RNA mediated gene silencing efficiencies and facilitate control over the resulting protein expression levels, in a spatial temporal manner. This method can elucidate structure function relationships and stimuli responsive delivery vehicles. Gaining better control over binding versus release, can unlock translatable platforms and drug discovery, as well as regenerative medicine technologies.The main advantages of this technique are the changes in gene expression can be controlled and predicted on the base as a simple, siRNA release assays and kinetic modeling. Though these methods employ novel photo-responsive polymers, this set of assays can be readily adapted to test the wide variety of other stimuli responsive systems. To begin, prepare a 32 micrograms per milliliter siRNA solution, by adding siRNA to a 20 micromolar HEPES solution at pH six.Then, prepare a polymer solution by adding dissolved mPEG block polyAPN BMA polymers to a 20 millimolar HEPES solution at pH six. To achieve a concentration of 220 micrograms per milliliter, this concentration will result in an N to P ratio of four, meaning there will be four times as many amine groups on the polymer as phosphate groups on siRNA. Next, add the polymer solution drop-wise to an equal volume of the siRNA solution, while gently mixing on a vortex machine.Continue to vortex for 30 seconds following polymer addition. Then, incubate the samples in the dark at room temperature for 30 minutes. Add the STS solution to each nanocarrier solution drop-wise while gently mixing on a vortex machine.Continue to vortex for 30 seconds following SDS edition. Centrifuge the samples at 3, 000 Gs for five seconds, then, incubate the samples in the dark at room temperature for 30 minutes. Calibrate and set a UV laser with a 365 nanometer filter to an intensity of 200 watts per square meter.Next, cut a hole into a rubber gasket, place the rubber gasket onto a washed and dried glass slide. Pipette the nanocarrier SDS solution onto the glass slide inside the hole of the rubber gasket. Load an excess of solution onto the glass slide, while avoiding contact with the rubber gasket.Place the second glass slide on top of the slide gasket. To avoid air bubble generation, place one end of the slide down first and then, slowly lower the other end. Attach binder clips to each side of the glass chamber to hold it close.Irradiate the samples for the desired length of time, using the UV laser with the 365 nanometer filter at an intensity of 200 watts per square meter. Remove the binder clips and open the chamber. Next, pipette 25 microliters of the irradiated nanocarrier SDS samples onto micro-centrifuge tubes.Incubate the solutions in the dark at room temperature for 30 minutes. Add five microliters of the loading buffer solution to 25 microliters of each nanocarrier SDS sample. Incubate the samples in the dark at room temperature for 10 minutes.Then, load 30 microliters of each nanocarrier SDS sample into a 2%agarose gel, pre-stained with ethidium bromide. Run the gel in the dark at 100 volts for 30 minutes. Image the gel, using a gel imaging system with ethidium bromide filters.Save the gel image files and proceed to bend intensity quantification as described in the text protocol. Remove the pieces of paper from a double sided adhesive spacer to expose the adhesive surface. Attach the spacer to a glass cover slip.Pipette the nanocarrier SDS solution onto the cover slip. Then, place a pre-washed glass slide on top of the cover slip with the hole from the adhesive spacer, centered around the sample. Push on the glass slide to ensure that the glass slide and cover slip are well attached and form a seal.Use a confocal microscope with a 40 times water immersive, aperchromal objective, having a numerical aperture of 1.2 for FCS measurements. Use the appropriate excitation laser channel to collect at least 30 measurements of 10 seconds each, per sample. Then, input the siRNA release data from FCS into a kinetic model to predict gene silencing responses, as detailed in the text protocol.To prepare in-vitro siRNA delivery, first add two milliliters of cell suspension to each well of the six well plate. Let the cells adhere and recover for 24 hours in the incubator. After washing the cells with phosphate buffered saline, prepare them for transvection by adding 1.5 milliliters of serum and antibiotic free transvection medium.Next, add 25 microliters of nanocarrier solution, containing 30 picomoles of siRNA tp each well. Gently pipette the medium up and down to mix. Place the cells in the incubator for three hours.Remove the transvection medium and wash each well with PBS. Then, add one milliliter of supplemented growth medium and place the cells in the incubator to recover for 30 minutes. To prepare the cells for treatment with a photo stimulus, remove the supplemented growth medium.Then, add one milliliter of transvection medium to each well. After calibrating the UV laser as described in the text protocol, place the cells on a hot plate set to 37 degree celsius. Remove the cover from the six well plate.Irradiate the cells from above the plate for the desired time, using the UV laser with a 365 nanometer filter at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium. Place the cells in incubator until further analysis.To prepare a photo mask that completely blocks 365 nanometer light and minimizes reflections, first cut, punch and/or machine the desired shape into the photo mask. Glue the photo mask to the bottom of the six well plate, with the pattern centered onto the well containing the cells and the anti-reflective side facing the plate. Set up two ring stands approximately 25 meters apart and detach a platform to each ring stand, so that the platforms are of equal height.Suspend the cell plate between the two stands by resting the plate on top of the platforms. Irradiate the cells from below the sample for the desired time, using a UV laser with a 365 nanometer filter, at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium.Then, place the cells in incubator to recover for at least 24 hours. Image the cells using fluorescence microscopy as described previously. Shown here, is an example of simple kinetic modeling of gene silencing dynamics.The model was run to predict the concentrations of mRNA, protein and siRNA, as a function of time following photo-induced siRNA release. The relative amounts of siRNA released following different dosages of light, were inputted as the initial siRNA concentration in the kinetic model. The resulting model predictions for the protein expression levels were an agreement with the experimentally determined expression levels obtained through western blotting.For visualization, GFP expressing cells were treated with GFP targeting siRNA and an anular photo mask was placed on the plate prior to irradiation to generate a circular pattern. Cells that were protected from the light exhibited fluorescence intensity that were indistinguishable from control samples not treated with siRNA and light. However, nearly all cells within the circular pattern exhibited no GFP expression, indicating efficient siRNA release and gene knockdown.Moreover, the use of light is a trigger enabled gene expression, to be controlled on cellular length scales. In summary, this method enables the rapid determination of irradiation conditions to spatio-temporally control siRNA release and predict gene silencing a priori. Employing this protocol will help elucidate the structure function relationships governing nanocarrier stability and efficacy.This technique may enable applications in regenerative medicine that requires spatio-temporal control over gene expression. These formulations also are well suited to the treatment of localized disorders such as skin cancer, as well as dermatological disorders.

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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 ...

연구

생체공학

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

Engineering a Bilayered Hydrogel to Control ASC Differentiation

ASC의 분화를 제어하는​​ Bilayered 히드로겔 꾸민

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

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

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 ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

사용<em&gt; 전의 VIVO</em&gt; 챈들러 루프 장치 수정 된 고분자 혈액 도관의 생체 적합성을 평가하기 위해

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

siRNA를 치료제의 전달을위한 다공성 실리콘 미세 입자

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Polymeric Microneedle Array Fabrication 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

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 ...

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

펄스 레이저인가 전압에 따라 금 나노 입자 통합 사진 응답 리포좀과 그들의 마이크로 버블 공동 현상의 측정의 합성

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

양이온과 음이온 리포좀을 사용 CNS에 치료의 siRNA 배달

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...