Name: delivery of therapeutic sirna to the cns using cationic and anionic liposomes
Uploaded: 2016-07-23T15:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

모든 마우스는 사육과 콜로라도 주립 대학에서 기관 애니멀 케어 및 사용위원회에 의해 승인 된 프로토콜에 따라 평가 및 실험 동물 관리 국제의 인증 협회의 인증을 받았으며 실험 동물 자원, 유지되었다. LSPCs 1. 준비 <ol><li> 1 DOTAP (1,2-dioleoyl -3- 트리메틸 프로판) : LSPCs을위한 콜레스테롤의 비율이 1을 사용합니다. 1 클로로포름 : 플라스크에 메탄올 용액을 4 nmol을 리포좀 제제의 경우, DOTAP 2 나노 몰과 (1) 10 ㎖에 콜레스테롤이 나노 몰을 혼합한다. </li><li> 흄 후드에서 N 2 가스를 이용하여 메탄올 용액 : 클로로포름 9 ㎖에 증발. 가스없이 밤새 흄 후드에서 용매의 마지막 1 mL로 증발시켰다. 플라스크의 바닥에 얇은 지질막을 관찰한다. </li><li> 55 ° C에 10 % 자당 용액의 열 10ml를. </li><li> / 천천히 분, 즉 1 ml에 얇은 지질 필름 상에 가열 된 10 % 자당 용액을 붓고 GE 동안ntly 플라스크를 소용돌이. 지질 분자는 겔 액상으로 유지하도록 55 ° C에서 지질과 10 %의 자당과 플라스크를 유지한다. 다중 층 소포 (MLV) 형성에 재수 결과. </li><li> 지질은 리포좀 크기를 조정하기 전에 적어도 한 시간 동안 55 ° C에서 재수 할 수 있습니다. </li><li> 1.0 μm의 필터 제조업체의 지침에 따라 압출기를 조립 또는 크기 1.0 μm의 주사기 필터를 사용합니다. </li><li> 11 번 압출기에 주사기 중 하나에 가열 된 리포좀 현탁액 1 ML을 추가하고, 두 주사기 사이의 정지를 전달합니다. 서스펜션은 (11)를 통과 한 후 시작 주사기보다 반대 주사기에 있는지 확인하십시오. </li><li> 리포좀 크기는 다시 0.45 μm의 다음으로, 0.2 ㎛의 필터는 큰 단일 층 소포 (LUVs)를 생성한다. 쉽게 크기 조정 가열 리포좀 현탁액을 유지합니다. </li><li> (4를 20 μM의 200 μL와 4 nmol을 리포좀 현탁액 (200 μM)의 20 μl를 품다nmol을 총)을 실온에서 10 분 동안 siRNA의 스톡 용액. </li><li> 500 μM 실온에서 10 분 동안의 siRNA / 리포좀 용액 (40 nmol을 총) 주식 RVG-9R 솔루션의 80 μl를 품어. LSPCs는 가능한 빨리 사용되어야하지만, 제조 후 수 시간이 사용될 수있다. 준비 후 몇 시간 동안 4 ° C에서 보관 LSPCs. </li></ol> PALETS 2. 준비 <ol><li> 40 : DSPE (1,2- 디스 테아-SN 글리세로 3 phosphoethanolamine) 중 5 몰비 : 콜레스테롤 : DSPE-PEG 또는 DOTAP : 콜레스테롤 : DSPE-PEG PALETS위한 55을 사용합니다. 1 클로로포름 : 플라스크에 메탄올 용액을 4 nmol을 리포좀의 제조를 위해, DSPE 또는 DOTAP 중 2.2 나노 몰, 콜레스테롤의 1.6 나노 몰 및 2 10 ㎖에 DSPE-PEG 0.2 나노 몰을 혼합한다. </li><li> 단계 1.1 이상 1.2에 설명 된대로 정확히 리포좀 현탁액을 준비합니다. 1X PBS 10 ㎖에 DSPE 리포좀을 재수 1 ㎖ / 분을 추가하여 75 ℃까지 가열 하였다. 지질가에서 75 ° C에서 재수 할 수 있도록 허용크기 전에 적어도 1 시간. </li><li> 크기는 리포좀 현탁액을 정확히 같은 단계 1.6-1.8에서 설명. </li><li> 피펫 DOTAP와 DSPE 리포좀 후 단일 사용 씩에 4 nmol을 (200 μM) 리포좀 현탁액의 각 20 μL은 크기되었습니다 및 벤치 탑 동결 건조기 (그림 2)를 사용하여 30 분 동안 동결 건조했다. </li><li> 20 μM DSPE PALETS 리포좀으로 캡슐화하는 siRNA를 실온에서 10 분 동안 프로타민 설페이트 26.6 μM 용액 1.4 μL (4 nmol을 총) siRNA의 스톡 용액 200 μl를 부화. </li><li> siRNA와의 4 nmol을 원액 200 μL로 PALETS DOTAP 리포좀을 재수 또는 siRNA의 / 프로타민 용액 201.4 μL와 DSPE PALETS 리포좀을 재수. RT에서 10 분 동안 리포좀 / siRNA의 용액을 인큐베이션. </li><li> 두 DOTAP 및 DSPE PALETS위한 리포좀 / siRNA의 용액에 60 mM의 1- 에틸 -3- (3- 디메틸 아미노 프로필) 카 보디이 미드 히드로 클로라이드 (EDC) 용액 10 μL를 추가한다. </li><li> 모두 DOTAP와 DSPE PALETS의 리포좀 / siRNA의 용액에 150 mM의 N 히드 록 (설포-NHS) 용액 10 μl를 추가합니다. </li><li> RT에서 2 시간 동안 siRNA의 / 리포좀 현탁액과 EDC 및 NHS 술포 부화. </li><li> 500 μM 실온에서 10 분 동안 솔루션을 가교의 siRNA / 리포좀과 (40 nmol을 총) 주식 RVG-9R 솔루션의 80 μl를 품어. 참고 : EDC / 설포-NHS가 PEG 지질에 공유 결합에 RVG-9R 수 있습니다. 준비 후 몇 시간 내에 PALETS를 사용합니다. 준비 후 몇 시간 동안 4 ° C에서 보관 PALETS. </li><li> PALETS의 siRNA를 캡슐화 효율을 확인하려면 : <ol><li> 이전과 프로타민 260 nm에서 설정된 분광 광도계 리포좀의 첨가 후에 siRNA의 농도를 측정한다. </li><li> 50 kDa의 원심 필터를 사용하여 리포좀 / 캡슐화 된 siRNA의 서스펜션에서 캡슐화 된 siRNA를 필터링합니다. 20 분 동안 1,000 XG에 원심 분리기. </li><li> 농도 측정봉입 여액 siRNA를 260 nm에서 분광 광도계를 사용하여 잔류 물로 캡슐화 된 siRNA의 농도. </li></ol></li></ol> 3. LSPCs 또는 PALETS와 마우스를 주입 <ol><li> 혈관을 팽창하기 위해 가열 램프에 5 ~ 10 분 동안 쥐를 노출. </li><li> 2-3 %의 이소 플루오 란 / 산소와 마우스를 마취하여 주입 전에 주입시 5-10 분 흐른다. 동물이 더 이상 보행과 호흡이 발가락 핀치를 수행하여 감속 때 마취를 확인하지 않습니다. 꼬리 정맥에 액세스하기 위해 후면 또는 측면에 마우스를 놓습니다. 마취 동안 건조를 방지하기 위해 마우스의 눈에 수의사 연고를 사용합니다. </li><li> / 지우기 70 % EtOH로 꼬리 뿌려 26 G 인슐린 주사기를 이용하여 마우스의 꼬리 정맥에 LSPCs 또는 PALETS 300 μL를 주입. 꼬리 정맥에 주입 말단 시작하고 정맥이 붕괴 또는 파열 경우 근위 이동합니다. </li><li> 이 흉골 드러 누움을 유지 때까지 깨끗한 새장에 마우스를 놓습니다. 모 두지 마십시오완전히 회복 될 때까지 다른 케이지 동료와 함께 사용합니다. 마우스는 5 ~ 15 분에 복구해야합니다. 이들은 가열 패드 또는 회복이 오래 걸리는 경우 체온을 유지하기위한 가열 램프 아래에 배치 될 수있다. 마우스는 과열에 대한 보호하기 위해 면밀히 모니터링해야한다. </li><li> 마취 기운이 떨어지기 보장하기 위해 주사 후 동물 몇 시간을 모니터하고 치료에는 부작용이 없습니다. LSPCs 또는 PALETS은 최소 24 시간 동안 순환 할 수 있습니다. </li></ol> 유동 세포 계측법을 통해 단백질 발현 4. 분석 <ol><li> 15 분 동안 20 % CO 2 유량 쥐를 안락사. </li><li> 가위를 사용하여 마우스의 피부와 두개골을 멀리 절단하여 뇌의 반 반구를 해부하다. 껍질 멀리 반 거리에 집게 나 가위의 끝 부분을 사용하여 두개골에서 뇌의 반구. </li><li> FACS 완충액 2.5 mL로하여 40 ㎛의 메쉬 셀 스트레이너를 통해 각 마우스에서 뇌 눌러 반 구체 (1X; 주사기의 플런저 페트리 접시에 PBS, 1 % 소 태아 혈청, 10 mM의 EDTA). </li><li> FACS 버퍼의 추가 2.5 ml의 셀 스트레이너와 페트리 접시를 씻어. 얼음에 15 ml의 원뿔 관에서 단일 세포 현탁액을 넣습니다. </li><li> 피펫 350 x g에서 5 분 동안 1.5 ml의 마이크로 원심 분리기 튜브에 5 ㎖ 단일 세포 현탁액 100 ㎕. FACS 완충액 1 ㎖에 재현 탁 상등액과 세포 펠렛을 버린다. 5 분 동안 350 XG 스핀. FACS 버퍼의 또 다른 1 ㎖로 반복합니다. 얼음에 세포 현탁액을 유지합니다. </li><li> (1) 100 ㎕의 세포를 품어 : FACS 버퍼에 0.5 ㎎ / ㎖ 쥐 항 - 마우스의 Fc 블록 (100) 희석을 얼음에 30 ~ 60 분. 펠렛 단계 4.5로 세포를 씻어. 얼음에 세포 현탁액을 유지합니다. </li><li> 30 ~ 60 분 동안 얼음에 FACS 버퍼에 7 % 마우스 혈청에서 마우스 PRP C에 대해 20 μg의 / ㎖ 형광 항체 100 ㎕의 세포를 품어. 펠렛 단계 4.5로 세포를 씻어. 세포 현탁액을 유지얼음 에다가요. </li><li> 펠릿 1 분간 적혈구 용해 완충액 1 ㎖ (1X PBS 155 mM의 NH 4 CL, 12 mM의 NaHCO3을 0.1 mM의 EDTA)으로 세포를 재 - 보류. 단계 4.5에서와 같이 펠릿은 FACS 완충액 1 ml의 세포를 재현 탁. 얼음에 세포 현탁액을 유지합니다. </li><li> 앞서 설명한 바와 같이 (10) 플로우 사이토 미터를 사용하여 C PRP 발현을 분석한다. 게이트는 총 세포 집단에서 세포를 살고 있습니다. 게이트 PRP C + 라이브 세포 집단에서 세포 MFI (평균 형광 강도)를 결정합니다. </li></ol>

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Biol. Chem. 276 (34), 32000-32007 (2001).</li><li>Li, S., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+transfer+via+intravenous+administration+of+cationic+lipid-protamine-DNA+(LPD)+complexes.">In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.</a> Gene Ther. 4 (9), 891-900 (1997).</li><li>B&#252;eler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+development+and+behaviour+of+mice+lacking+the+neuronal+cell-surface+PrP+protein.">Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.</a> Nature. 356 (6370), 577-582 (1992).</li><li>Dykxhoorn, D., Novina, C., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+the+messenger:+short+RNAs+that+silence+gene+expression.">Killing the messenger: short RNAs that silence gene expression.</a> Nat Rev Mol Cell Bio. 4 (6), 457-467 (2003).</li><li>Graham, A., Ray, M., Perry, E., Jaros, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+nicotinic+acetylcholine+receptor+subunit+expression+in+the+human+hippocampus.">Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus.</a> J Chem Neuroanat. 25 (2), 97-113 (2003).</li><li>Kretzschmar, H. A., Prusiner, S. B., Stowring, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+prion+proteins+are+synthesized+in+neurons.">Scrapie prion proteins are synthesized in neurons.</a> Am J Pathol. 122 (1), 1-5 (1986).</li><li>Moser, M., Colello, R., Pott, U., Oesch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+the+prion+protein+gene+in+glial+cells.">Developmental expression of the prion protein gene in glial cells.</a> Neuron. 14 (3), 509-517 (1995).</li><li>Cronier, S., Laude, H., Peyrin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+can+infect+primary+cultured+neurons+and+astrocytes+and+promote+neuronal+cell+death.">Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.</a> Proc Natl Acad Sci. 101 (33), 12271-12276 (2004).</li><li>Diedrich, J., Bendheim, P., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie-associated+prion+protein+accumulates+in+astrocytes+during+scrapie+infection.">Scrapie-associated prion protein accumulates in astrocytes during scrapie infection.</a> Proc Natl Acad Sci. 88 (2), 375-379 (1991).</li></ol>

The authors declare that they have no competing financial interests.

이 보고서는 효율적으로 CNS에의 siRNA를 전송이 표적 전달 시스템을 만들 수있는 프로토콜을 설명합니다. 중추 신경계에의 siRNA를 전달하는 이전 방법은 뇌, 대상의 siRNA의 정맥 주사, 또는 비 대상 리포좀의 siRNA 복합체의 정맥 주사에​​ 직접 siRNA를 /의 shRNA 벡터를 주입 포함되어 있습니다. 중추 신경계로의 siRNA / shRNA를 벡터의 주입은 목적 단백질의 발현 수준의 감소를 야기 않는다. 그러나, siRNA...

프리온은 중추 신경계에 영향을 미치는 심각한 신경 퇴행성 질환이다. 프리온 질병은 PRP 해상도라는 감염 체에 의해 정상 세포 프리온 단백질 PRP C의 미스 폴딩 기인한다. 이 질병은 인간 1-3에서 소 해면상 소에 뇌증, 스크래피 양에, cervids 만성 소모성 질환, 및 크로이츠 펠트 - 야콥병 등 종의 다양한 영향을 미친다. 프리온은 시냅스 손실 시작하고, 공포 화하기 위해 진행 신경 퇴행, 신경교 증, 신경 세포의 손실 및 플라크 침착을 일으킬. 결국, 4 개인 동물 /의 죽음의 결과로. 수십 년 동안, 연구자들은 늦추거나 프리온 질병의 진행을 중지하는 것을 의미 화합물을 조사 하였다. 그러나 연구진은 성공적인 치료 또는 효과적인 전신 배달 차량 중 하나를 발견하지 않았습니다. 내인성 PRP C 발현 프리온 질병 (5)의 발전에 필요한 </s&gt;입니다. 따라서, 감소 또는 제거 PRP C 표현이 지연 또는 질병의 경감 될 수 있습니다. 몇몇 그룹은 프리온 질환 PRP C 발현 수준의 역할을 조사하기 위해 쥐 뇌 조직에 직접 shRNA를 발현 PRP C의 감소 수준 또는 주입 lentivectors와 트랜스 제닉 마우스를 만들었다. 신경 PRP C의 양을 감소 발견 이들 연구자 프리온 질병의 병리학 시브를 중지 결과 및 6-9 동물의 수명을 연장. 우리는 마우스 신경 모세포종 세포 (10)의 PRP 해상도의 틈새에서 그 PRP C의 siRNA 처리 결과를보고했다. 이러한 연구는 치료의 사용은 작은 간섭 RNA (siRNA의) mRNA의 절단 같이 PRP C 발현 수준을 감소시키기에 충분히 프리온 질병의 진행을 지연시킬 수 있다는 것을 시사한다. 그러나 프리온 질환에 대한 조사 대부분의 치료는 실용적이지 않을 것입니다 방법으로 전달되었다임상있다. 따라서, siRNA의 치료는 CNS에 정맥 내 전달을 대상으로 전신 전달 시스템을 필요로한다. 연구자들은 유전자 치료제에 대한 전달 비히클로서 리포좀의 사용을 연구 하였다. 양이온 및 음이온 모두 지질 리포좀의 형성에 사용된다. 양이온 성 지질 및 DNA / RNA 사이의 전하의 차이를 효율적으로 포장 할 수 있기 때문에 양이온 성 지질 널리 음이온 지질 이상 사용된다. 양이온 성 지질의 또 다른 장점은 그들이 다른 지질 11-14보다 용이하게 세포막을 통과한다는 것이다. 그러나, 양이온 성 지질은 음이온 지질 (13, 14)보다 면역 원성 있습니다. 따라서 연구팀은 리포좀의 지질 음이온에 양이온을 사용하여 이동하기 시작했다. 유전자 치료 제품을 효율적으로 DNA / RNA 분자 15-19 집광 양전하 펩타이드 프로타민 설페이트를 사용하는 음이온 성 리포좀 내로 패키징 할 수있다. 음이온 LIPI 이후DS는 양이온 성 지질가 순환 시간을 증가 할 수 있으며, 더 많은 동물 모델 (13, 14)에서 허용 될 수보다 면역 원성 있습니다. 리포좀은 리포좀에 부착 된 펩티드를 대상으로 사용하여 특정 조직을 대상으로한다. 니코틴 성 아세틸 콜린 수용체에 결합하는 RVG-9R의 신경 펩타이드는 중추 신경계 17-20로의 siRNA와 리포좀을 대상으로 사용되어왔다. 이 보고서는 프로토콜은 세 가지의 siRNA 전달 비히클을 제조하고, 패키징하고 신경 세포 (도 1)에 siRNA를 전달하기 위해 설명. siRNA를 리포좀 - 펩티드 복합체 (LSPCs)의 siRNA를 리포좀으로 구성되는 펩티드 타겟팅 RVG-9R 정전 기적 리포솜의 외 표면에 부착. 펩티드 RVG-9R 공유 결합 지질 PEG 그룹에 결합하여 siRNA를 리포좀 내에 캡슐화 프로타민 구성되어 치료의 siRNA (PALETS)를 캡슐화 리포솜을 해결. 아래의 방법을 사용하고 LSPCs PALET를 생성S는 PRP C의 siRNA는 치료 또는 실질적으로 프리온 질병 병리학의 발병을 지연시키는 엄청난 약속을 보유하고 신경 세포의 90 %, 최대 PRP C의 발현을 감소시킨다.

<table><tbody><tr><td>DOTAP lipid</td><td>Avanti Lipids</td><td>890890</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Lipids</td><td>700000</td><td></td></tr><tr><td>DSPE</td><td>Avanti Lipids</td><td>850715</td><td></td></tr><tr><td>DSPE-PEG</td><td>Avanti Lipids</td><td>880125</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>AC268320010</td><td></td></tr><tr><td>Methanol</td><td>EMD Millipore</td><td>113351</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;sub&gt; Gas</td><td>AirGas</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>Extruder</td><td>Avanti Lipids</td><td>610023</td><td></td></tr><tr><td>1.0, 0.4, and 0.2 &amp;#956;m filters</td><td>Avanti Lipids</td><td>610010, 610007, 610006</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Protamine sulfate</td><td>Fisher Scientific</td><td>ICN10275205</td><td></td></tr><tr><td>EDC</td><td>Thermo Scientific</td><td>22980</td><td>Aliquoted for single use</td></tr><tr><td>Sulfo-NHS</td><td>Thermo Scientific</td><td>24510</td><td>Aliquoted for single use</td></tr><tr><td>40 &amp;#956;m Cell Strainer</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Rat anti-mouse CD16/CD32 Fc block</td><td>BD Pharmigen</td><td>553141</td><td></td></tr><tr><td>Anti-PrP antibody (PRC5)</td><td>Proprietary - PRC</td><td></td><td></td></tr></tbody></table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

음이온 PALETS 내의 siRNA의 캡슐화의 효율성을 증가시키기 위해, siRNA의 프로타민을 혼합 하였다. 1 (그림 3A) : 1 2 : siRNA를위한 최선의 프로타민의 농도를 결정하기 위해, siRNA를 1에서, 프로타민의 서로 다른 농도로 혼합 하였다. 프로타민을 사용하지 않고 음이온 성 리포좀에 60~65% siRNA의 캡슐화 효율이 있었다. 프로타민과 샘플 : 1 ~ 1.5 : 1에서 siRNA의 몰비 1 (133-266...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

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

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

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 ...

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.4K 조회수.  Colorado State University. The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases. 

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

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

SiRNA 암 세포에 배달에 대 한 Exosomes의 준비

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

연구

암 연구학

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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 ...

생체공학

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

설치류 중추 신경계에 약물을 직접 심실 배달

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

신경과학

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

생화학

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNA의 뇌의 특정 최저 대한 정위 주입하는 간단한 대안

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

체 외에서 의 효율적인 배달 양이온 Nanoliposomes의 세대 문자 메신저 RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

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 ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

중추 신경계에 비강 siRNA 전달 하는 동안 마우스의 배치에 대 한 위치 장치

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

면역학 및 감염병학

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

양이온 성 리포좀을 통해 특정의 siRNA 반복 전달하여 마우스 폐 Intersectin-1의 장기 침묵. 전자 현미경에 의한 넉다운 효과의 평가

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

생물학

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

쥐 중추 신경계에서 안티 센스 올리고 뉴클레오티드의 내 전달

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...