HL671795, HL048743 및 HL108630 :이 작품은 NIH 보조금에 의해 지원되었다.

SDF-1α 글리 칸 나노 입자의 1. 준비 생체 내 전달의 목적으로 인해, 제조에 사용 된 모든 용기, 피펫 팁을 소독. <ol><li> 초순수에서 다음 원액을 준비 : 1 % 덱스 트란 황산; 1 M의 NaOH (PES 막으로 멸균 여과); 0.1 % (0.8 연속적 0.22 μm의 필터를 통해 필터 및 그 후에 NaOH로 pH가 5.5로 조정), 0.2 % 빙초산 키토산; 0.1 M ZnSO 4; 15 % 만니톨; 0.92 ㎎ / ㎖ SDF-1α (80 ° C에서 분취 액에 저장되고, 4 ℃에서 보관 일단 해동 C). </li><li> 0.22 μm의 필터 멤브레인을 통해 재고 솔루션을 소독. 리 물러의 amebocyte 용 해물 (LAL) 젤 응고 분석에 준비된 용액에 독소 수준을 평가합니다. 수준이 &lt;0.06 EU / ㎖ 있는지 확인합니다. </li><li> 자기 교반 막대를 함유하는 1.5 mL 유리 바이알에 0.18 ml의 초순수를 추가한다. 800 rpm으로 교반 속도를 설정합니다. 0.1 ml의 1 %를 추가덱스 트란 설페이트 및 2 분 동안 교반한다. 0.08 mg을 SDF-1α (0.92 Mg를 0.087 ㎖ / ml의 SDF-1α 용액)를 첨가하고, 20 분 동안 교반한다. </li><li> 0.33 ml의 0.1 % 키토산 적가하고 추가 5 분 동안 교반한다. 변경 볶음 최대 속도 (1 분 이상) 0.1 ML의 주사기로 천천히 0.1 ml의 0.1 M ZnSO 4를 추가합니다. </li><li> 800 rpm으로 교반 속도를 반환하고, 30 분 동안 교반한다. 0.4 ml의 15 % 만니톨을 추가하고 5 분 동안 교반한다. 1.5ml의 미세 원심 분리 튜브에 반응 혼합물을 전송. 10 분 동안 4 ° C에서 16,000 XG에 원심 분리기. 주 : 만니톨의 존재는 원심 분리 후 입자의 부유를 용이하게한다. 펠릿의 소형화에 따라 만니톨의 농도는 0 %에서 5 %로 변화 될 수있다. 원심 분리 후, 각 입자의 완전 재현 탁하여 최종 현탁액 응집체를 피하기 위해 중요하다. </li><li> 기음 뜨는 천천히 액체의 마지막 한 방울을 제거하기 위해 피펫을 사용합니다. 0.2 ml의 5 % 만니톨을 추가합니다. 0.5 ml의 펠렛을 일시 중단, 29 G의 옛 성은DLE 주사기. 1 ml의 5 % 만니톨을 추가합니다. 15 분 동안 16,000 XG에 원심 분리기. </li><li> 단계를 반복 1.6. </li><li> 뜨는을 기음. 0.2 ml의 5 % 만니톨 펠렛을 재현 탁. 4 ° C에서 입자 현탁액을 저장합니다. 주 : 입자 현탁액도 저장 -80 ° C에서 동결 또는 동결 건조 될 수있다. 서스펜션 포함 5 % 만니톨 동결 및 해동 후 또는 동결 건조 및 수분 보충 후 입자의 응집을 방지하기 위해 필수적이다. 만니톨 보존 후의 현탁액을 원심 분리에 의해 제거 될 수있다. </li></ol> 입자 크기와 제타 전위 2. 측정 입경 및 제타 전위를 소재리스트에 표시된 입자 분석기로 각각 동적 광산란 및 전기 영동 광 산란에 의해 분석된다. <ol><li> 축적 시간 : 다음 입자 크기 측정을위한 파라미터를 설정 (70); 반복 시간 : 4; 온도 : 25 ° C;희석제 : 물; 강도 조절 : 자동. </li><li> 물 (10 배 희석)와 SDF-1α-DS-CS 샘플을 희석. 이러한 에펜 도르프 베트로 일회용 UV 큐벳에 시료 100 ㎕를 넣습니다. 셀 홀더에 큐벳을 삽입합니다. &quot;최적&quot;(~ 10,000 CPS)에 도달하기 위해 강도 조정 기다립니다. 데이터 수집을 시작합니다. </li><li> 측정이 완료되면, 직경 (㎚) 및 다 분산도 지수 cumulants 결과를 기록한다. 4 반복 판독 각각으로부터 얻어진 결과를 평균치와 표준 편차를 계산한다. </li><li> 로드 제타 전위 측정 플로우 셀에 10 배 희석 한 시료의 입자 500 μL. 누적 시간 : 다음 측정을위한 매개 변수를 설정합니다 (10); 반복 시간 : 5; 온도 : 25 ° C; 희석제 : 물; 강도 조절 : 자동. 제타 전위 (MV)의 결과를 기록한다. (5) 판독 반복의 각각으로부터 얻어진 결과를 평균 및 표준을 계산 deviatioN. </li></ol> 입자의 SDF-1α의 3 정량화 <ol><li> 1.3 램 믈리 샘플 버퍼에 0.01, 0.02, 0.03, 0.04, 0.05 ㎎ / ㎖의 농도 자유 양식 SDF-1α를 희석. 40 μL의 1.3 배 램 믈리 샘플 버퍼 6 μL SDF-1α-DS-CS 샘플을 희석. (나눈다. 작은 분취 량으로 원액을 0.25 M 트리스 -HCl, pH가 7.5, 8 % SDS, 40 % 글리세롤, 0.05 ㎎ / 브로 모 페놀 블루,는 ml 8 % 2- 머 캅토 에탄올을 함유 4X 램 믈리 스톡 완충액을 준비하고 -20 ℃로 유지 단일 사용하기 위해 C를 °.) </li><li> 10 분 동안 100 ℃에서 샘플을 가열한다. 소용돌이 입자를 완전히 해리시키기 위해 10 분 동안 가열 시간 배 샘플 (최대 속도로 10 초 동안 각각). 2 분 동안 RT에 샘플을 냉각. 0.5 분 10,000 XG에 원심 분리기는 응축수를 가져올 수 있습니다. 소용돌이는 다시 잘 섞는다. 참고 :이 시점에서, 시료 용액이 눈에 보이는 침전물 존재와 명확해야한다. </li><li> 로드 10# 181; L 샘플 / 웰에 4-20 %의 SDS 젤. 20 분 동안 200 V의 전기를 실행합니다. 코마시 블루 단백질 얼룩 젤을 얼룩. </li><li> 분자 이미 저와 농도계 분석 소프트웨어를 사용하여 SDF-1α의 단백질 밴드 밀도를 검사합니다. 무료 SDF-1α 표준 구성된 표준 곡선에 대한 입자 SDF-1α의 양을 계산합니다. </li></ol> 생체 외 자료 분석 4. <ol><li> 1 칼슘과 마그네슘 (D-PBS)없이 둘 베코 인산염 완충 생리 식염수와 SDF-1α 글리 칸 입자를 혼합 : 1의 비율 (v / v)로. </li><li> 1.5 ml의 튜브에 0.05 ㎖의 분취 량으로 입자 현탁액을 나눕니다. 튜브의 바닥에 샘플을로드합니다. 기포를 도입 또는 액체의 표면을 방해하지 않도록. 파라 필름으로 튜브의 상단을 밀봉합니다. 주 : 이때, 액체는 튜브 믹서 후속 상하 회전시 하단에 남아있을 것이다. </li><li> 37에서 튜브를 회전# 176; C 회전 마이크로 튜브 믹서에. 지정된 시간에 튜브에서 분취 액을 제거하고 즉시 4 ° C에서 10 분 동안 16,000 XG에서 원심 분리. </li><li> 상층 액과 펠렛을 분리, 0.05 ml의 D-PBS로 펠렛을 재구성. -20 ° C에서 샘플을 저장합니다. 모든 샘플이 수집 된 후 전술 한 바와 같이, SDS 겔상에서 상등액과 펠릿을 조사. </li></ol> 5. 마이그레이션 분석 이 분석은 SDF-1α의 화성 활동을 측정합니다. 세포 표면의 수용체 (CXCR4)와 SDF-1α의 상호 작용은 SDF-1α의 그라데이션으로 세포의 이동을 야기한다. 이 분석에서, 세포는 낮은 잘으로하고 SDF-1α 솔루션 (낮은 우물에서 반투과성 막에 의해 분리) 상부 우물에로드됩니다. <ol><li> SDF-1α 희석 (유리 또는 입자 - 결합 된 형태) -3- FO에서 이주 완충액 (0.5 % 소 혈청 알부민을 함유하는 RPMI-1640 배지)100, 33, 11, 3.7, 1.2, 0.41, 0.14, 및 0.05 ng를 / ㎖의 최종 농도로 용액을 희석 LD. </li><li> 24 웰 플레이트에 웰에 희석 된 SDF-1α 솔루션 또는 마이그레이션 버퍼 만 (대조군)의 0.6 ML을 추가합니다. 잘 (입력 셀 제어)에 0.57 ml의 마이그레이션 버퍼를 추가합니다. 30 분 동안 37 ℃에서 배양한다. 이러한 낮은 잘 위에없이 Transwell (기공 크기 5 μm의 직경 6.5 mm)로 투과 세포 배양 삽입을 놓습니다. 트랜스 웰에 0.1 ml의 된 Jurkat 세포 (5 × 10 5)를 삽입로드. 잘 입력 세포 제어에 직접 0.03 ml의 세포를로드합니다. 5 % CO 2 인큐베이터에서 2 시간 동안 37 ℃에서 플레이트를 인큐베이션. </li><li> 트랜스 웰 인서트를 제거합니다. 4 ml의 폴리스티렌 튜브에 낮은 우물로 마이그레이션 한 세포를 전송합니다. 유량 계측기와 마이그레이션 된 세포를 계산합니다. </li><li> negat에 숫자의 감산 입력 후 세포 수 (X 웰 100/30 입력 셀 컨트롤 세포 수)의 백분율로 계산 이주컨트롤 (에만 이동 완충액을 함유하는 웰에 마이그레이션 셀) 필자. </li></ol>

<ol>
	<li>Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+polyelectrolyte+complexes+of+chitosan+and+dextran+sulfate+towards+versatile+nanocarriers+of+bioactive+molecules.">Colloidal polyelectrolyte complexes of chitosan and dextran sulfate towards versatile nanocarriers of bioactive molecules.</a> Eur J Pharm Biopharm. 78 (1), 10-18 (2011).</li><li>Morachis, J. M., Mahmoud, E. A., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+strategies+for+therapeutic+delivery+by+using+polymeric+nanoparticles.">Physical and chemical strategies for therapeutic delivery by using polymeric nanoparticles.</a> Pharmacol Rev. 64 (3), 505-519 (2012).</li><li>Yue, Z. 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=Surface+charge+affects+cellular+uptake+and+intracellular+trafficking+of+chitosan-based+nanoparticles.">Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles.</a> Biomacromolecules. 12 (7), 2440-2446 (2011).</li><li>Ghadge, S. K., Muhlstedt, S., Ozcelik, C., Bader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDF-1alpha+as+a+therapeutic+stem+cell+homing+factor+in+myocardial+infarction.">SDF-1alpha as a therapeutic stem cell homing factor in myocardial infarction.</a> Pharmacol Ther. 129 (1), 97-108 (2011).</li><li>Sharma, M., Afrin, F., Satija, N., Tripathi, R. P., Gangenahalli, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-derived+factor-1/CXCR4+signaling:+indispensable+role+in+homing+and+engraftment+of+hematopoietic+stem+cells+in+bone+marrow.">Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow.</a> Stem Cells Dev. 20 (6), 933-946 (2011).</li><li>Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+stromal+cell-derived+factor-1alpha-heparin+complex.">Characterization of the stromal cell-derived factor-1alpha-heparin complex.</a> J Biol Chem. 276 (11), 8288-8296 (2001).</li><li>Amara, A., 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=Stromal+cell-derived+factor-1alpha+associates+with+heparan+sulfates+through+the+first+beta-strand+of+the+chemokine.">Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine.</a> J Biol Chem. 274 (34), 23916-23925 (1999).</li><li>Sadir, R., Imberty, A., Baleux, F., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate/heparin+oligosaccharides+protect+stromal+cell-derived+factor-1+(SDF-1)/CXCL12+against+proteolysis+induced+by+CD26/dipeptidyl+peptidase+IV.">Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV.</a> J Biol Chem. 279 (42), 43854-43860 (1074).</li><li>Yin, T., 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=SDF-1alpha+in+glycan+nanoparticles+exhibits+full+activity+and+reduces+pulmonary+hypertension+in+rats.">SDF-1alpha in glycan nanoparticles exhibits full activity and reduces pulmonary hypertension in rats.</a> Biomacromolecules. 14 (11), 4009-4020 (2013).</li><li>Huang, M., Vitharana, S. N., Peek, L. J., Coop, T., Berkland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+complexes+stabilize+and+controllably+release+vascular+endothelial+growth+factor.">Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor.</a> Biomacromolecules. 8 (5), 1607-1614 (2007).</li><li>McCall, R. L., Sirianni, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA+nanoparticles+formed+by+single-+or+double-emulsion+with+vitamin+E-TPGS.">PLGA nanoparticles formed by single- or double-emulsion with vitamin E-TPGS.</a> J Vis Exp. (82), 51015 (2013).</li><li>Carrillo-Conde, B. R., Roychoudhury, R., Chavez-Santoscoy, A. V., Narasimhan, B., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+synthesis+of+carbohydrates+and+functionalization+of+polyanhydride+nanoparticles.">High-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles.</a> J Vis Exp. (65), 3967 (2012).</li><li>Xu, J., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+gene+delivery+and+transfection+in+human+pancreatic+cancer+cells+using+epidermal+growth+factor+receptor-targeted+gelatin+nanoparticles.">Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles.</a> J Vis Exp. (59), e3612 (2012).</li><li>Lauten, E. 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=Nanoglycan+complex+formulation+extends+VEGF+retention+time+in+the+lung.">Nanoglycan complex formulation extends VEGF retention time in the lung.</a> Biomacromolecules. 11 (7), 1863-1872 (2010).</li><li>Schatz, C., Domard, A., Viton, C., Pichot, C., Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+and+efficient+formation+of+colloids+of+biopolymer-based+polyelectrolyte+complexes.">Versatile and efficient formation of colloids of biopolymer-based polyelectrolyte complexes.</a> Biomacromolecules. 5 (5), 1882-1892 (2004).</li></ol>

The authors declare that they have no competing financial interests.

전술 한 바와 같이, DS-CS 나노 입자 폴리 음이온 (DS)과 폴리 양이온 (CS) 분자간 전하 중성화를 통해 형성된다. 전하 상호 작용은 분자 충돌시 쉽게 발생하기 때문에, 혼합 중에 중합체 용액 및 교반 속도의 농도는 생성 된 입자의 크기가 중요하다. 일반적인 경향은 더 DS 및 CS 솔루션 (15) 및 작은 입자의 높은 교반 속도 결과를 희석하고 있다는 것이다. SDF-1α 글리 칸의 ?...

덱스 트란 황산 (DS)과 키토산 (CS)는 (DS)에 여러 대체 음으로 하전 된 황산 그룹과 폴리 사카 라이드, 또는 양전하 아민 그룹입니다 (CS를 탈 아세틸). 수용액에 혼합 할 때, 두 가지 다당류는 정전 기적 상호 작용을 통해 고분자 전해질 복합체를 형성한다. 얻어진 착물 수용액 (침전물)로부터 상분리한다 큰 응집체 또는 수분 산성 (콜로이드)의 소형 입자를 형성 할 수있다. 이러한 결과에 기여하는 구체적인 조건은 광범위하게 연구되어 왔으며, 최근의 리뷰에서 요약 한 상세히 도시되어있다. 이러한 조건 중에서, 수분 산성 입자를 제조하기위한 두 가지 기본적인 요구 반대로 대전 된 중합체는 1)이 크게 상이한 몰 질량을 가져야이고; 2) 비 화학량 론적 비율로 혼합 될 수있다. 이러한 조건에 의해 생성 된 전하 전하 중성 복합체 중합체 세그먼트를 허용중화는 분리 및 입자의 코어를 형성하고, 여분의 중합체는 외부 쉘을 형성하기 위해 하나한다. 이 프로토콜에 기재된 당쇄 입자는 폐 전달을 위해 의도되고, 음으로 하전 된 순으로 설계되고, 나노 사이즈의. 음극 표면 전하 입자 2,3의 세포 흡수의 가능성을 감소시킨다. 나노 사이즈의 입자는 원위기도 통로를 통해 용이하게한다. 이 목표를 달성하기 위해, 본 제조에 사용되는 DS의 양은 CS 초과 (중량비 3 : 1); 고 분자량은 DS (중량 평균 MW 500,000)와 저분자 CS (MW kDa의 범위 50-190, 75~85% 탈 아세틸)이 사용된다. SDF-1α는 화성 활동을 통해 원점 복귀 기능을 발휘 줄기 세포 호밍 ​​요인이다. SDF-1α는 원점과 골수 조혈 줄기 세포의 유지에 중요한 역할을하고 proge의 모집부상 수리 4,5의 말초 조직에 nitor 세포. SDF-1α는 단백질이 프로테아제 (CD26 / DPPIV) 불활로부터 보호, 헤파린 / 헤파 란 설페이트, 형태 다이머에 결합하고, 세포 표면 수용체를 통해 표적 세포와 상호 작용할 수 있도록 그 단백질 서열에 헤파린 결합 부위를 갖는다 6-8. DS는 헤파린 / 헤파 란 황산과 유사한 구조적 특성이있다; 따라서, DS에 대한 SDF-1α의 결합은 천연 고분자 리간드의 그것과 유사하다. 다음 프로토콜에서 우리는 SDF-1α-DS-CS 나노 입자의 제조 방법을 설명합니다. 절차는 이전에 9를 연구 된 제제 중 하나를 나타냅니다. 프로토콜은 원래 VEGF-DS-CS 나노 입자 (10)의 조사에서 적합하다. 소규모 제조 용이 동일한 스톡 용액 및 제조 조건으로 확장 할 수있는 기술된다. 제조 후, 입자 b를 특징Y는 크기, 제타 전위, SDF-1α 통합의 정도, 체외 방출 시간 및 통합 SDF-1α의 활동을 조사.

<table border="0" cellpadding="0" cellspacing="0" width="489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Name</td>
			<td style="width:136px;">Company</td>
			<td style="width:99px;">Catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulfate</td>
			<td>Fisher</td>
			<td>BP1585-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, low molecular weight&#160;</td>
			<td>Sigma</td>
			<td>448869</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>204986</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure water&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>10977-023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>Prepared according to reference 8.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, PES membrane 0.22 um.&#160;&#160;&#160;</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic Micro Stirring Bars (2 x 7 mm)</td>
			<td>Fisher&#160;</td>
			<td>14-513-63</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass vial Kit; SUN-SRi</td>
			<td>Fisher&#160;</td>
			<td>14-823-182</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Delsa Nano C Particle Analyzer&#160;</td>
			<td>Backman Coulter</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf UVette Cuvets</td>
			<td>Eppendorf</td>
			<td>952010069</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4&#8211;20% Mini-PROTEAN TGX Gel</td>
			<td>Bio-Rad</td>
			<td>456-1096</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GelCode Blue Safe Protein Stain</td>
			<td>Fisher&#160;</td>
			<td>PI-24592</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Imager VersaDoc MP 4000 System</td>
			<td>BioRad</td>
			<td>170-8640</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Transwell Permeable Supports</td>
			<td>Corning</td>
			<td>3421</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accuri C6 Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>D8537</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyrogent plus kit</td>
			<td>Fisher</td>
			<td>NC9753738</td>
		</tr>
	</tbody>
</table>

preparation and characterization of sdf-1&#945;-chitosan-dextran sulfate nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

제조 된 SDF-1α-DS-CS 입자의 크기 및 제타 전위는 입자 분석기로 측정된다. 1 사이즈의 측정 분석을 보여준다. 네 개의 반복 된 측정에 의한 결과로부터 cumulants, SDF-1α-DS-CS 입자의 유체 역학적 평균 직경은 661 ± 8.2 (㎚)이고 분산도는 0.23 ± 0.02이다. 제타 전위 측정의 결과는도 2에 도시되어있다. 다섯 반복 측정에서, SDF-1α-DS-CS 입자의 제타 전위는 -24.8 ± 0.5 MV이?...

Watch this Scientific Journal Video about Preparation and Characterization of SDF-1Î±-Chitosan-Dextran Sulfate Nanoparticles at JoVE.com

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α-키토산 덱스 트란 황산 나노 입자의 제조 및 특성

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

preparation-characterization-sdf-1-chitosan-dextran-sulfate

Research

JoVE Journal

Chemistry

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

12.3K 조회수.  Brigham and Women's Hospital and Harvard Medical School. The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

비디오: Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

전자 레인지를 이용한 산 - 촉매 반응을 통해 실리카 나노 입자의 제조

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

연구

화학

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

준비 및 개인의 특성과 다중 약물은 물리적으로 고분자 미셀을 포획로드

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

합성, 특성 및 하이브리드 금 / CD 및 금 /은 ZnS 코어 / 쉘 나노 입자의 작용 화

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

비스 - iminoguanidinium의 리간드 선택적 결정화에 의한 황산염 분리

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

준비 및<em&gt; 체외</em&gt; 자기 공명 영상에 대한 덴드리머 기반 대조 대리인의 특성

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

준비 및 C60/Graphene 하이브리드 Nanostructures의 특성화

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

Ligand 중재 Nucleation 및 팔라듐 금속 나노 입자의 성장

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

단일 금속 나노 입자의 고분해능 물리적 특성화

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

합성 및 암화 금 나노 입자의 특성화

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...