Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

SLNs 1. 처리 <ol><li> SLNs의 합성 참고 : 하위 20 나노 SLNs 1을 제조 피트 방법 1,17,20을 사용합니다. <ol><li> 무균 기술, bioreagents 또는 세포 배양 등급 시약 및 살균 물질 (즉, 피펫, 주걱, 유리 병 등)를 사용합니다. </li><li> SLNs의 합성 (그림 2)에 대한 바이오 안전성 캐비닛을 사용합니다. </li><li> 형광 염료의 0.6 ㎎을 결합하고 바이알 (15 mL)에 Heneicosane (지질, 5 중량 / 중량 % 지질 농도) 0.10 g에서 공동 용융 (나일 레드 (NIR)을, NIR 농도는 약 0.3 ㎎ / ㎖ 임) 90 ° C 및 교반. </li><li> 0.11 g의 폴리 옥시 에틸렌 (10) 올레 일 에테르 (계면 활성제, 5.5 중량 / 중량 % 계면 활성제 농도)를 추가합니다. 90 ℃에서 교반하고, 생성 된 혼합물을 동시 - 용융. </li><li> 90 ° C의 혼합물, 열 멸균 물 1.79 g을 넣고, 투명 나노 에멀젼이 형성 될 때까지 저어. 참고 : 계속 교반 및 제어 냉각, SL에서NS 생성됩니다. 이러한 조건은 중유 에멀젼 유 중수 에멀젼의 반전을 일으킨다. 이러한 지질 방울에 NIR 파티션으로 친 유성 분자. </li><li> 대조군 샘플로서 기능하기 위해서는, 형광 색소를 첨가하지 않고 평행 나노 에멀젼을 제조. 90 ° C, 및 바이알에서 교반 (15 mL)을 공동으로 용융물 Heneicosane 0.10 g 및 0.11 g의 폴리 옥시 에틸렌 (10) 올레 일 에테르 결합. <ol><li> 90 ° C의 혼합물, 열 멸균 물 1.79 g을 넣고, 투명 나노 에멀젼이 형성 될 때까지 저어. </li></ol></li><li> 또한 멸균 0.2 μm의 필터를 사용하여 각각의 나노 에멀젼을 살균. </li></ol><img alt="그림 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> 그림 SLNs의 합성 2. 도식. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> </li><li> 입자 크기와 폴ydispersity <ol><li> 입자 크기 및 분산도 유리 큐벳을 사용 SLNs 1 및 입자 크기를 측정하기위한기구를 측정하기 위해 동적 광산란 (DLS)을 사용한다. </li><li> 시료의 측정에 앞서, 표준 제제와 측정을위한 제조사의 프로토콜은 다음 두 가지 알려진 표준 입자 크기를 측정한다. </li><li> 표준 용액 1.5 ㎖의 유리 큐벳을 채우고 샘플과 같은 측정 조건을 사용하여 표준 용액을 측정한다. 주 : 이전 단계는 각 표준에 대해 반복 하였다. </li><li> 제조 된 샘플을 측정합니다. 각각에 대해,하기 실험 파라미터들을 사용하여 100 초, 물의 굴절율 (1.33), 물의 점도의 실행 시간을 20 ° C (1.002 MPA · 초)에서. 주 :이 기술은 주파수가 나노 입자의 크기를 측정하기 위해 광을 시프트 이용한다. </li><li> 이상적 상대의 평균 입자 직경 및 분산도를보고CLE 크기 분포. </li></ol></li><li> 녹는 점과 녹는 잠열 <ol><li> SLNs (1)의 열적 거동을 조사하기 위해 자동 샘플러 및 액체 질소 냉각 소스를 구비 시차 주사 열량계 (DSC)를 사용한다. </li><li> 약 25 밀리그램의 질량과 40 μL의 알루미늄 팬에 AS-준비 SLNs를 피펫 및 기밀 DSC 스캔 중에 수분 손실을 최소화하기 위해 보편적 크림 퍼 프레스를 사용하여 팬을 밀봉. </li><li> 5 ~ 80 ° C에서의 각 나노 에멀젼을 측정한다. </li><li> 융점 계곡 융점 물질의 양으로 나눈 DSC 플롯에서 곡선 아래 면적의 적분을 나타내는 DSC 플롯에서 용융 잠열이 융해 잠열을 나타내는보고한다. </li></ol></li></ol> 섬유 아 세포와 수지상 세포 2. SLNs 상호 작용 <ol><li> 기본 인간 섬유 아세포의 문화 <ol><li> 문화 차 인간 Fi를broblasts 낮은 혈청 성장 보충 (LSGS) 키트 보충 (106 중) 인간 피부 섬유 아세포의 배양 액체 배지에서 제조업체의 지침에 따라. 80 % 합류에 도달시, 37 ℃, 5 % CO 2에서 배양 가습 분위기에서 세포를 유지한다. </li><li> MTT 및 이미징 웰 당 2 × 104 세포의 밀도로 분석 96 웰 플레이트에 시드 세포, 및 37 ° CO / N 종래 SLN 노광에서 평형을 허용 모두. </li><li> 지질 농도에 대하여, (도 4)에 표시하고, 각 웰 당 10 μL를 추가로 타임 제로에서, 완전 배지로 희석 SLNs 96 웰 플레이트에 웰 복제. </li><li> 인큐베이션 기간 동안, 37 ° C, 5 % CO 2에서 세포를 유지한다. </li></ol></li><li> MTT 독성 분석 <ol><li> SLNs으로 배양 24 시간 후에, 제조자의 프로토콜에 따라 MTT 분석을 이용하여 세포 생존력을 결정한다. &lt;/ 리&gt; <li> 한 번 세포를 세척하고 각 웰 (100 μL / 웰)에 새로운 배지를 추가합니다. 새로운 배지로 교체하기 전에 10 분 동안 70 % 메탄올 용액에 배양에 의해 제어 우물을 죽여. </li><li> 마이크로의 각 웰에 MTT 시약 (물론 당 10 μL)를 첨가하고, 37 ℃에서 O / N을 품어. </li><li> O / N 배양 한 후, 제조 업체의 프로토콜에 따라 제공되는 세제와 세포 내 formazin 결정을 (물론 당 100 μL) 용해. </li><li> RT에서 3 시간 배양 한 후, 마이크로 플레이트 리더를 사용하여 570 nm의 파장에서 측정 된 흡광도 값을 얻었다. 참고 : 3- (4,5- dimethylthiazolyl-2) -2,5- 디 페닐 테트라 졸륨 브로마이드 (V / W)은 1 % 미만이어야 사용 MTT 시약. </li></ol></li><li> 형광 현미경 <ol><li> SLN와 섬유 아세포의 투여 후 특정 시점에서, 멸균 인산 완충 식염수 (PBS)로 두 번 씻어 섬유 아세포를 차가운 70 % 메탄올로 10 분 동안 고정한다. </li><li> 10 분 후, 대역 통과 (BP) 50분의 690 nm의 필터 세트를 사용하여, 거꾸로 현미경을 사용하여 PBS 캡처 이미지와 메탄올을 대체합니다. </li></ol></li><li> 마우스 골수 수지상 세포의 문화 <ol><li> 유도 마우스 골수 유래의 수상 세포 (BMDC)는 앞서 설명한 23,24. 간략히, 재조합 쥐 과립구 - 대 식세포 콜로니 - 자극 인자 (GM-CSF, 300 U / ml) 및 재조합 뮤린 IL-4와 2 × 6 / 플레이트에서 100mm 배양 접시에서 C57BL / 10 골수 플레이트 단일 세포 현탁액 (B 세포 인자는, 200 U / ㎖의 자극). 3 일마다 미디어를 변경합니다. </li><li> 7 일째, 37 ° C에서 세포를 0.5 μg의 5.0 / ㎖ 지질의 최종 농도로 요리 근적외선 SLNs로드를 추가하고 유지, 노출, 원하는 길이의 5 % CO 2 인큐베이터. </li><li> 판의 철저한 세척 한 다음, 50 ㎖ 튜브에 수집, 판에있는 모든 미디어를 흡입하여 요리에서 세포를 수집 (느슨하게 광고를 꺼 내려4 mM의 에틸렌 디아민 테트라 아세트산 (EDTA)을 함유하는 인산염 완충 생리 식염수 5 ㎖ (PBS)와 내재 세포). 동일한 50 ㎖ 튜브에서 얻은 세포 현탁액을 수집한다. </li></ol></li><li> 유동 세포 계측법을 통해 SLNs 법인의 평가 <ol><li> 2.4.3 점에서 얻어진 세포 현탁액의 농도를 결정하고 12mm X 75mm의 둥근 바닥 FACS 튜브에 3 × 105 세포를 분산. </li><li> PBS 1 ㎖를 첨가 한 후 4 ℃의 온도에서, 400 XG에서 5 분 동안 튜브를 원심 분리하여 세포를 세척한다. </li><li> 상층 액을 제거하고 방지하는 CD11c-FITC 모노클로 날 항체 (0.25 μg의 / ㎖ 최종)를 함유하는 PBS / 2 mM의 EDTA / 0.1 % 소 혈청 알부민 100 ㎕ (BSA) (염색 완충액)으로 펠렛을 재현 탁. 어둠 속에서 얼음에 30 분 동안 품어. </li><li> PBS 1 ㎖로 세포를 씻어 준 후 4 ℃의 온도에서, 400 XG에서 5 분 동안 원심 분리 튜브. </li><li> 뜨는을 취소하고 각을 재현 탁염색 버퍼 400 μL로 펠렛. 셀은 유동 세포 계수기 (아마도 파란색과 빨간색 레이저가 장착)와 샘플 수집을위한 준비가되어 있습니다. </li><li> 분석 소프트웨어 유세포를 사용하는 CD11c + 세포 (FITC 양성)의 모집단에 게이팅 및 (를 PerCP-Cy5.5 또는 APC 채널에서 측정) SLNs 관련 형광의 세기를 비교하여 DC에 의한 SLNs 혼입 수준을 평가. </li></ol></li></ol>

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Trends in colloid and interface science XIV.</a> Progress in Colloid and Polymer Science. 115, 36-39 (2000).</li><li>Forgiarini, A., Esquena, J., Gonzalez, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+nano-emulsions+by+low-energy+emulsification+methods+at+constant+temperature.">Formation of nano-emulsions by low-energy emulsification methods at constant temperature.</a> Langmuir. 17 (7), 2076-2083 (2001).</li><li>Cabone, C., Tomasello, B., Ruozi, B., Renis, M., Puglisi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+optimization+of+PIT+solid+lipid+nanoparticles+via+statistical+factorial+design.">Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design.</a> Eur J Med Chem. 49, 110-117 (2012).</li><li>Raimondi, G., 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=Mammalian+Target+of+Rapamycin+Inhibition+and+Alloantigen-Specific+Regulatory+T+Cells+Synergize+To+Promote+Long-Term+Graft+Survival+in+Immunocompetent+Recipients.">Mammalian Target of Rapamycin Inhibition and Alloantigen-Specific Regulatory T Cells Synergize To Promote Long-Term Graft Survival in Immunocompetent Recipients.</a> J Immunol. 184, 624-636 (2010).</li><li>Jhunjhunwala, S., Raimondi, G., Thomson, A. W., Little, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+rapamycin+to+dendritic+cells+using+degradable+microparticles.">Delivery of rapamycin to dendritic cells using degradable microparticles.</a> J Control Release. 133 (13), 191-197 (2009).</li><li>Kapse-Mistry, S., Govender, T., Srivastava, R., Yergeri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodrug+delivery+in+reversing+multidrug+resistance+in+cancer+cells..">Nanodrug delivery in reversing multidrug resistance in cancer cells..</a> Front Pharmacol. 5 (159), 1-22 (2014).</li><li>Musacchio, T., Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+lipid-based+pharmaceutical+nanocarriers.">Recent developments in lipid-based pharmaceutical nanocarriers.</a> Front Biosci (Landmark Ed). 1 (16), 1388-1412 (2011).</li><li>Cerpnjak, K., Zvonar, A., Ga&#353;perlin, M., Vre&#269;er, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-based+systems+as+a+promising+approach+for+enhancing+the+bioavailability+of+poorly+water-soluble+drugs).">Lipid-based systems as a promising approach for enhancing the bioavailability of poorly water-soluble drugs).</a> Acta Pharm. 63 (4), 27-445 (2013).</li><li>Rodr&#236;guez-Gasc&#242;n, A., Pozo-Rodr&#236;guez, A., Solin&#236;s, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+nucleic+acid+vaccines:+use+of+self-amplifying+RNA+in+lipid+nanoparticles.">Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles.</a> Int J Nanomedicine. 9, 1833-1843 (2014).</li><li>Almeida, A. J., Souto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+lipid+nanoparticles+as+a+drug+delivery+system+for+peptides+and+proteins.">Solid lipid nanoparticles as a drug delivery system for peptides and proteins.</a> Adv Drug Deliv Rev. 59, 478-490 (2007).</li><li>Pardeshi, C., 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=Solid+lipid+based+nanocarriers:+an+overview.">Solid lipid based nanocarriers: an overview.</a> Acta Pharm. 62, 433-472 (2012).</li><li>Attama, A. A., Momoh, M. A., Builders, P. F., Sezer, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Nanoparticulate+Drug+Delivery+Systems:+A+Revolution+in+Dosage+Form+Design+and+Development.">Lipid Nanoparticulate Drug Delivery Systems: A Revolution in Dosage Form Design and Development.</a> Recent Advances in Novel Drug Carrier Systems. , 107-140 (2012).</li></ol>

The authors have nothing to disclose.

본 연구에서는 SLNs의 합성과 세포 내 표적 응용 프로그램에 대한 적용 가능성은 탐구했다. 이러한 생체 적합성 나노 입자는 약물 전달, 유전자 침묵, 그리고 백신 기술을 25 ~ 30 등의 여러 응용 프로그램에 대한 배송 차량 등의 약속을 보여 주었다. 초소형 SLNs가 용이 한 공정으로 합성하고, 주요 피부 세포 및 주요 면역 세포와의 상호 작용을 탐색 하였다. SLNs는 모델 치료화물을 역임 형광 ?...

나노 입자 기반 전달 비히클은 특히 셀룰러 시그널링 및 유전자 발현을 변경하는기구를 제공하고, 세포 내 표적 애플리케이션을위한 큰 약속을 보여 주었다. 이들 차량은 약물, 단백질 및 세포 반응에 영향과 표적 조직에서 원하는 효과를 달성하도록 설계된 핵산으로 로딩 될 수있다. nanocarriers 여러 유형의 지질, 폴리머, 실리콘, 및 자성 물질을 포함하여 치료 및 진단을 위해 유익 탐구되어왔다. 이러한 시스템으로 인해 치료 목표 조직에서 농도 및 독성의 감소를 증가 지역화 된 약물 전달에 대한 자신의 잠재력에 매력적이다. 고형분 지질 나노 입자 (SLNs)는 최근 유망한 약물 전달 비히클로 떠오르고있다 나노 입자 전달 시스템의 잘 연구 된 예이다. SLNs 용이 바이오 센싱 (1), (2) 화장품 및 t를 포함하여 다수의 애플리케이션 용으로 제형 화 될 수있다herapeutic 배달 3-7. 효용성은 향상된 생체 적합성 결과, 재 흡수성, 독성 지질 전적으로 구성되어 있다는 사실로부터 유래한다. 합성 과정에서, 친 유성 약물함으로써 비경 구 투여 용 약물 용해도 및 적합성을 증가 SLN 차량에 통합 될 수있다. SLN 차량은 또한 그 분해 및 클리어런스를 감소 및 치료 작용을 극대화 캡슐화 치료학을 안정하는 것을 돕는다. 이러한 차량으로 인해 체온 3,4,8,9에서 안정성에 특히 긴 연기, 제어 방출 제제에 적합하다. 중요한 지질 나노 입자의 약물의 캡슐화는 약물 분자의 극한 약동학 프로파일을 변경. 이것은 좁은 치료 지수를 가진 약물의 제어 방출을 허용함으로써 잠재적 이점을 제공한다. SLN-혼입 치료제의 방출 속도는 조정 지질 분해 속도 또는 약물의 확산 속도에 기초 할 수있다지질 매트릭스. SLNs는 종종 특정 표적 조직에 축적하도록 설계되었습니다. 예를 들어, 크기 (전형적으로는 10 nm 이상)는 종양 조직의 새는 혈관은 증착을 용이하게 순환의 유지를 증강시켜. 또한, 입자 투여 경로는 예컨대 림프절 10,11 특정 생리 학적 구조를 대상으로 잠재적으로 생체 분포를 변경하는 것으로 나타났다. 표적 조직에 침착, 적당한 세포 상호 작용을 달성하고 궁극적으로 나노 입자의 내재화에 의한 선택적으로 셀 (12)의 밖으로 이온 및 분자의 흐름을 제어하는 세포막의 능력에 도전. 세포 흡수를 용이하게하기 위해, 펩타이드, 작은 분자 및 모노클로 날 항체 (13, 14)을 포함하여 특정 리간드 nanocarriers을 수정하는 것이 가능하다. 수동 침투 및 나노 입자의 능동 수송을 모두 포함하는 여러 메커니즘세포막 이전 3,12,15을 설명 하였다. 일반적으로, 이것은 세포 - 나노 입자의 상호 작용은 이러한 세포 형태 또는 세포주기 단계 (12)와 같은 셀 - 특정 매개 변수 외에, 크기, 형상, 표면 전하 및 표면 화학을 포함하는 나노 입자의 물리 화학적 특성에 의해 영향을받는 것으로 입증되었다. 이전의 조사는 상전이 온도 (PIT)을 사용하는 방법 (17) (16) 및 국소 바이오 마커 검출 애플리케이션 1 서브 10 나노 SLNs의 합성을 증명했다. 이것은 온도가 서서히 변화시키면서 조성물은 일정하게 유지 2 부드러운 합성법이다. 가열 된 용액의 연속 교반,로는 나노 에멀젼의 RT 결과를 냉각시킨다. 보다 작은 입자 크기를 가진 1 SLNs의 합성 과정이 결과는 이전의 NaN 지질 합성을위한 다양한 방법을 이용하여보고oparticles 17 ~ 22. 얻어진 크기 규모는, 20nm 이하는, 증가 된 표면적과 상호 작용을위한 향상된 셀룰러 전위 세포 표적화 애플리케이션을위한 이점을 제공한다. 형광 염료 또는 치료를 제공하도록 설계 SLNs의 개략은,도 1에 도시된다. SLNs는 지질 내부 (예를 들면, 선형 알칸) 친 유성 화합물 (예를 들면, 염료 또는 치료제)의 결합을 허용하고, 계면 활성제의 외관의 구성 물에 둘러싸여 (예를 들어, 선형 비이 온성 계면 활성제). 본 연구에서는 SLNs는 형광 염료로드 된 입자 세포 상호 작용을 조사하기 위해 모델로 사용. 차 인간 피부 섬유 아세포 및 마우스 수지상 세포 독성 및 흡수 입자에 대하여 상호 작용을 특성화하기 위해 시간에 걸쳐 SLN 로딩 염료를 노출시켰다. 3- (4,5- 디메틸 티아 졸 -2- 일) -2,5- diphenylphenyltetrazolium 브로마이드 (MTT) 분석은 utili이었다적당한 투여 수준을 설정하기 위해 평탄화. 형광 현미경 및 유동 세포 계측법은 시험관 내에서 입자의 흡수를 검사하기 위해 사용하는 두 가지 방법이었다. <img alt="그림 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> 주요 성분을 표시 SLN의 그림 1. 도식. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

PIT 방법은 SLNs를 합성하는 데 사용하고, 상전이 온도 수조를 이용하여 측정 하였다. 샘플을 서서히 가열하여 맑은 용액이 등장 할 때까지 온화하게 교반한다. heneicosane 지질을 사용하여 제조 SLNs위한 상전이 온도는 45 ° C. 표 1은 입자 크기, 분산도, 융점 SLNs 위해 용융 잠열을 요약 한 것이다. 전술 한 공정 조건을 사용하여 합성 SLNs은 18.59 및 16.87 nm의 평균 입자 직경 및 5.83 및 4.47의 다 ...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

고체 지질 나노 입자 (SLNs) 세포 내 타겟팅을위한 응용 프로그램

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K 조회수.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

비디오: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

에 대한 바이러스 나노 입자<em&gt; 생체에</em&gt; 종양 이미징

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

연구

생체공학

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

재생 연구를위한 고조파 나노 입자

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

생산 및 일가 양자점 타겟팅

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

멀티 모달 분자 이미징 애플리케이션을위한 Biofunctionalized 프 러시안 블루 나노 입자

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

셀 라벨 및 초상 자성 산화철 나노 입자로 타겟팅

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

실크 나노 입자의 제조 및 약물 전달 응용 프로그램

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Falcarindiol 인간 중간 엽 줄기 세포에 의해 포함 된 새로운 지질 코팅 나노 입자의

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

암 세포 배양에서 수집된 매체에서 액체 크로마토그래피 질량 분광법에 의해 분석된 선택된 Kynurenines의 동시 정량화

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

미세 유체 혼합 플랫폼을 사용하여 유전자 전달을 위한 지질 나노 입자 제조 및 특성화

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...