Name: preparation and characterization of lipophilic doxorubicin pro-drug micelles
Uploaded: 2016-08-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

DOX-PA 1. 합성 <ol><li> 독소루비신의 390 밀리그램과 팔 미트 산 히드라 지드 243 mg의 무게를 측정하고, 둥근 바닥 플라스크에 전송할 수 있습니다. </li><li> 유리 주사기와 플라스크에 무수 메탄올 150 ㎖를 추가합니다. 피펫으로 트리 플루오로 아세트산 (TFA)의 39 μl를 추가합니다. 자기 교반기를 사용하여, 어두운 곳에서 실온에서 18 시간 동안 반응 혼합물을 교반 하였다. 비고 : 반응 물질의 양을 확장 할 수있는 아래 DOX-PA의 상이한 양을 얻었다. 반응물의 비율을 같은 비율로 유지되어야한다. 78 ㎎을 1,170 ㎎의 범위에서 DOX 수량을 사용하는 반응은 일반 화학 실험실에서 수행 될 수있다. </li><li> 실리카 겔 컬럼을 사용 DOX-PA 정제. 17 <ol><li> 로타리 증발기로 반응 혼합물 중의 용매를 제거한다. 혼합물의 부피를 약 20 mL로 감소 된 후 실리카겔 3g을 추가한다. 건조 분말을 얻었다 회전 증발을 계속 t 수 있도록그 실리카 겔에 흡착 제품. 건조 분말을 형성 한 후 추가로 30 분 동안 진공 상태에서 시료를 유지한다. </li><li> 용매로서 디클로로 메탄을 이용하여 컬럼에 실리카 겔 50g을 팩. 조심 컬럼에 흡착 생성물을 함유하는 실리카 겔 샘플을 추가한다. </li><li> 이에 서서히 극성 용매 (표 1)의 증가, 메탄올의 비율을 증가시키면서, 디클로로 메탄 및 메탄올의 혼합물로 컬럼을 용출시켰다. </li><li> 시험관에서의 용출액 분획 (25 ㎖ / 튜브)를 수집하고, 박층 크로마토 그래피 (T​​LC)에 의해 진행 상황을 모니터링한다. </li><li> 순수한 DOX-PA 포함하는 모든 분획을 합하고 건조 분말이 형성 될 때까지 회전 증발기를 사용하여 용매를 제거한다. 또한 진공 O의 /의 N에서 제품을 건조. </li></ol></li><li> TLC에 의해 DOX-PA의 분석. <ol><li> TLC 판의 4cm X 8cm 부분을 잘라. TLC 스포팅 모세관을 가진 판의 바닥에서 현장 샘플 용액 0.5 cm 충족 사용용매로 한올. </li><li> 디클로로 메탄 및 메탄올의 혼합물을 함유하는 현상 챔버로 TLC 판 위 (3/1, v / v)로. 용매의 깊이는 0.5 cm보다 그냥 작아야합니다. </li><li> 용매 전면 판의 상단에 도달 할 때 현상 실에서 플레이트를 제거합니다. 연필로 용매 앞의 위치를​​ 표시하고 판을 건조 할 수 있습니다. 샘플을 시각화하기 위해 포화 된 요오드 증기를 함유하는 염색 챔버로 TLC 플레이트를 놓는다. </li></ol></li><li> 1 H 핵 자기 공명 분광법 (1H-NMR)와 DOX-PA 분석. 18 <ol><li> 메틸 술폭 시드-D6 (DMSO) 1 ㎖에 DOX-PA 15 mg을 용해하고, NMR 튜브에 시료를 옮긴다. </li><li> 핵 자기 공명 악기의 자석에 NMR 튜브를 삽입합니다. 용매로서 DMSO를 선택 양성자 스펙트럼을 측정한다. 자석에서 NMR 튜브를 제거합니다. NMR의 결과 (18)를 분석합니다. </li></ol></li></ol> 이. 필름 분산 방법에 의해 DOX-PA 미셀 준비 <ol><li> DSPE-PEG (40 mg) 및 PA-DOX (4 mg)을 10 mL 유리 바이알에 2 ㎖의 메탄올로 용해. </li><li> 바이알의 박막이 형성 될 때까지 회전 증발기를 사용하여 진공하에 유기 용매를 제거한다. 대안 적으로, (예를 들어, 아르곤 또는 질소 가스) 막을 형성하고 상기 잔류 용매를 제거하고 진공 건조기에서 바이알을 유지하는 불활성 가스하에 유기 용매를 증발 : 참고. </li><li> 전송 유리 병에 둘 베코 인산염 완충 생리 식염수 (pH가 7.4, DPBS) 2 ㎖. </li><li> 미셀을 생성하는 RT에서 3 분 동안 초음파 욕조에 병을 놓습니다. 주 : 초음파 전력은 초음파 화장실의 다른 모델마다 다릅니다. 얇은 폴리머 / 약물 필름을 분산 할 수있는 충분한 초음파 전력을 생성 할 수있는 단위를 선택합니다. 이 프로토콜에서 사용되는 초음파 욕의 출력 전력은 110 (W)이다 </li><li> 단기 저장을 위해 4 ° C에서 미셀을 유지하고 -20 오래 ° C ~-term 저장. 참고 : 또한, 미셀은 또한 동결 건조 및 사용하기 전에 물을 재구성 할 수 있습니다. 일반적으로, 더 동결 방지제 또는 lyoprotectant이 제제 필요하지 않습니다. </li></ol> DOX-PA 미셀 3. 특성 <ol><li> 미셀의 약물 봉입 효율 DOX-PA 농도의 결정 <ol><li> 1 ㎍ / ㎖, 5 μg의 / ㎖, 20 ㎍ / ㎖, 50 ㎍ / ㎖, 100 μg의 / ㎖ : DOX-PA 다섯 가지 농도 DOX-PA 용액을 제조 DMSO에 이전 단계에서 합성 녹인다. 490 nm에서 UV-VIS 스펙트로 DOX-PA 용액의 흡광도를 측정한다. DOX-PA 약물 농도 및 490 nm에서 그들의 대응 흡수에 기초하여 표준 곡선을 생성한다. </li><li> DMSO 500 μL와 약물로드 미셀의 25 μl를 희석. 자외선 VIS 분광기로 490 nm에서 흡광도를 측정한다. 3.1.1에서 생성 된 표준 곡선과 약물 농도를 계산한다. </li><li> 다음 식을 사용하여 캡슐화 효율을 계산한다 : 약물의 포위 효율 (%) = (미셀의 약물의 양) / (첨가 약물의 양) × 100 % </li></ol></li><li> 동적 광 산란 입자 크기의 특성 (DLS) <ol><li> 1 ㎎ / ㎖의 최종 DSPE-PEG 농도 DPBS (PH 7.4)으로 희석 미셀. Z 축 평균 크기 및 분산도 지수 (PDI)를 획득하기 위해 입도 분석기 2 mL의 샘플을 분석한다. </li></ol></li><li> 생체 외 항암 활성 평가 참고 : 적절한 무균 기술을 사용하고 바이오 안전성 캐비닛 내부에 작동합니다. <ol><li> 세포 배양 플라스크에서 (예를 들어, T25)를 포함하는 DU-145 인간 전립선 암 세포를 세포 배양 배지를 제거하고 2 ㎖ DPBS (PH 7.4)에 세포를 헹군다. </li><li> 대기음 DPBS는 트립신 용액 (0.25 %) 1 mL를 넣고 세포를 분리하여 37 ℃에서 2 분간 배양한다. </li><li> 10m 추가세포 배양 배지 L (RPMI 1640 + 10 % 태아 소 혈청 + 1 % 항생제 - 안티 곰팡이), 세포의 대부분은 플라스크로부터 분리 될 때. 15 ㎖의 원심 분리기 튜브와 원심 분리기 5 분 1,000 XG에 세포에 세포를 전송합니다. </li><li> 세포 배양 배지 5 ㎖과 세포 펠렛을 다시 중지 및 혈구 세포 수를 카운팅하는 샘플을 제거한다. 50000 세포 / ml의 농도로 세포 배양 배지와 세포를 희석. 96 웰 세포 배양 플레이트에 희석하여 세포 현탁액을 추가 (100 μL / 웰). 세포 배양 인큐베이터에서 인큐베이션 셀 (37 ° C를 5 % CO 2) 18 시간 동안 세포 부착을 허용한다. </li><li> 각각 0.1 μM, 0.5 μM, 2 μM, 5 μM, 10 μM을 최종 약물 농도를 얻기 위하여 세포 배양 배지와 DOX의 디메틸 술폭 시드 (DMSO) 용액 DOX-PA DMSO 용액을 희석. 0.5 %에 위의 모든 샘플에서 최종 DMSO 농도를 유지합니다. 최종 약물 공동 얻었다 세포 배양 배지와 DOX-PA 미셀 희석0.1 μM의 ncentrations 각각 0.5 μM, 2 μM, 5 μM, 10 μM. 빈 세포 배양 배지에게 컨트롤을 사용합니다. </li><li> 배양기에서 96 웰 세포 배양 플레이트를 제거하고 (각 그룹에 대해 N = 4) 단계 3.3.5에서 제조 된 다른 치료제를 함유하는 배지 100 ㎕와 세포 배양액을 교체한다. (2 37 ° C, 5 % CO) 추가로 72 시간 동안 세포 배양기에서 세포를 인큐베이션. </li><li> 배지를 흡인 및 3- (4,5- 디메틸 티아 졸 -2- 일) 0.5 ㎎ / ㎖을 함유하는 배지 100 ㎕ -2,5- 디 페닐 테트라 졸륨 브로마이드 (MTT)를 추가한다. </li><li> 추가로 2 시간 동안 세포 배양기에서 세포를 인큐베이션. 조심스럽게 매체를 제거하고 포르 마잔 결정을 용해 DMSO 100 ㎕를 추가합니다. </li><li> 570 nm 파장과 670 nm의 참조 파장의 마이크로 플레이트 분광 광도계로 흡광도를 측정한다. </li><li> 다음 식을 사용하여 세포 생존율을 계산한다 : <strong&gt; 세포 생존율 (%) =는 (A 테스트 / A 컨트롤) × 100 % 주 : 변동 (ANOVA) 통계 시험의 분석을 사용하여 서로 다른 그룹의 세포 생존율을 비교한다. 약물 농도 데이터 대 세포 생존 능력에 기초하여, IC (50)를 계산한다. </li></ol></li></ol>

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(64), (2012).</li><li>Ulbrich, K., Etrych, T., Chytil, P., Jelinkova, M., Rihova, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPMA+copolymers+with+pH-controlled+release+of+doxorubicin:+in+vitro+cytotoxicity+and+in+vivo+antitumor+activity.">HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.</a> J Controlled Release. 87 (1-3), 33-47 (2003).</li><li>Patil, R., 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=Cellular+Delivery+of+Doxorubicin+via+pH-Controlled+Hydrazone+Linkage+Using+Multifunctional+Nano+Vehicle+Based+on+Poly(beta-L-Malic+Acid).">Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(beta-L-Malic Acid).</a> Int J Mol Sci. 13 (9), 11681-11693 (2012).</li><li>Hu, X., Liu, S., Huang, Y., Chen, X., Jing, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+block+copolymer-doxorubicin+conjugates+via+different+linkages:+preparation,+characterization,+and+in+vitro+evaluation.">Biodegradable block copolymer-doxorubicin conjugates via different linkages: preparation, characterization, and in vitro evaluation.</a> Biomacromolecules. 11 (8), 2094-2102 (2010).</li><li>Huynh, L., Neale, C., Pomes, R., Allen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+approaches+to+the+rational+design+of+nanoemulsions,+polymeric+micelles,+and+dendrimers+for+drug+delivery.">Computational approaches to the rational design of nanoemulsions, polymeric micelles, and dendrimers for drug delivery.</a> Nanomedicine. 8 (1), 20-36 (2012).</li><li>Shi, 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=A+drug-specific+nanocarrier+design+for+efficient+anticancer+therapy.">A drug-specific nanocarrier design for efficient anticancer therapy.</a> Nat Commun. 6, 7449 (2015).</li></ol>

The authors have nothing to disclose.

본 연구에서 우리는 미셀의 제조를위한 단순한, 신속한 필름 분산 법을 설명한다. 이 방법은, 양친 성 고분자의 자기 조립 특성을 이용한다 (예를 들어 DSPE-PEG)를 수성 환경에서 코어 - 쉘 구조의 미셀을 형성한다. 이 미셀의 제조 방법은 여러 가지 장점을 갖는다. 1. 일반적 리포좀, 나노 입자 및 nanoemulsions. 19 (2)의 제조에 사용되는 (예를 들면 압출 또는 균질화 같은) 복잡한 크기 환?...

화학 요법은 일반적으로 암의 다양한 형태를 치료하는 데 사용됩니다. 모든 경우 대부분은 화학 요법 약물은 더 많은 생명을 위협 조건, 오심, 설사 등의 관리가 작은 조건에 따라 다를 수 독성 부작용이있다. 대부분의 항암제는 독성이 있기 때문에 정상 조직에 대한이 약의 비 선택적 노출은 필연적으로 독성이 발생합니다. 따라서, 선택적으로 암세포에 약물을 전달할 수있는 치료 방법에 대한 중대한 필요성이있다. 항암제의 투여와 함께 또 다른 문제는 가난한 수용성이다. 일반적으로, 가용 화제는 이러한 난 용성 약물을 수립 할 필요가있다. 그러나, 디메틸 설폭 사이드와 같은 대부분의 가용 화제, (DMSO), 크레 모포 EL 및 폴리 솔 베이트 80 (트윈 80) 간 및 신장 독성, 용혈, 급성 과민 반응 및 말초 신경 병증의 원인이 될 수 있습니다. 1 따라서, 안전하고 생체 적합성 제제는 필요하다 가난한 사람들의 임상 사용LY 수용성 항암제. Nanocarriers은 상기 과제에 대한 약물 전달 시스템을 약속한다. 이 nanocarriers는 리포좀,이 나노 입자, 3 미셀, 4-7 고분자 - 약물 접합체, 8, 무기 물질을 포함한다. 9 몇 나노 의학 제품 (예를 들어, Doxil, 아브 락산 및 Genexol)는 암 환자를 치료하기 위해 규제 기관에 의해 승인되었습니다. (10) 고분자 미셀이 성공적으로 항암제의 전달에 사용 된 나노 크기의 약물 전달 담체를 약속한다. 4-7,11,12 전형적인 고분자 미셀은 자기 조립 공정을 통해 양친 성 중합체로부터 제조된다. 코어 - 쉘 구조 고분자 미셀은 친수성 ​​쉘과 소수성 코어를 포함한다. 친수성 쉘은 입체적으로 미셀을 안정화시키고 혈류에서의 순환을 연장 할 수 있습니다. 소수성 코어는 효과적으로 소수성 D를 캡슐화 할 수 있습니다양탄자. 때문에 미셀 (일반적으로 미만 200 ㎚) 및 장기 순환 속성의 작은 크기, 고분자 미셀이 향상 침투성 및 보존 (EPR) 효과 (수동 종양 타겟팅)을 대상으로 종양을 달성 할 것으로 추정된다. 봉입 안정성은 미셀의 능력을 표적 종양 중요하다. 최적 종양 타겟팅을 달성하기 위해, 미셀은 종양 부위에 도달하기 전에 최소 누설 약물이 아직 신속 암세포를 입력 한 후 약물을 방출한다. 제제의 안정성이 제품 개발의 실행 가능성뿐만 아니라 개발 된 제품의 저장 수명을 결정하기 때문에 또한 제제 안정성, 또한, 제품 개발을위한 필수 요건이다. 최근 많은 노력 전달 담체에 약물의 로딩을 개선하려고했다. 친 유성 전구 약물 접근 지질 나노 입자 에멀젼에 약물 로딩을 개선 탐색 된 전략이다. 13,14 접속사약물과 지질의 ugation 크게 자신의 친 유성을 개선하고 nanocarriers의 친 유성 성분의로드 및 보존을 향상시킬 수 있습니다. 여기서는 친 독소루비신 전구 약물로드 미셀을 제조하는 프로토콜을 기술한다. 우선, 친 유성 약물 독소루비신위한 합성 과정을 설명한다. 이어서, 필름 분산 법 마이셀을 생성하는 프로토콜이 도입된다. 이 방법을 성공적으로 이전 연구에서 사용되었다. 5 DSPE-PEG가 성공적 미셀 약물 전달을 위해 사용 되었기 때문에 미셀을 제조하기위한 담체 물질로서 선택 하였다. (15, 16)는 마지막으로, 미셀을 특성화하는 데 사용되는 여러 가지 시험 관내 분석을 서술 제형은 항암 활성을 평가 하였다.

<table><tbody><tr><td>DSPE-PEG2K</td><td>Cordenpharm</td><td>LP-R4-039</td><td>&gt;95%</td></tr><tr><td>Doxorubicin</td><td>LC Laboratories</td><td>D-4000</td><td>&gt;99%</td></tr><tr><td>Palmitic Acid Hydrazide</td><td>TCI AMERICA&amp;nbsp;&amp;nbsp;</td><td>P000425G</td><td>&gt;98.0%</td></tr><tr><td>Methanol</td><td>ACROS Organics</td><td>610981000</td><td>Anhydrous</td></tr><tr><td>Methylene chloride&amp;nbsp;</td><td>FISHER&amp;nbsp;</td><td>D151-4</td><td>99.90%</td></tr><tr><td>Methyl sulfoxide-d&lt;sub&gt;6&lt;/sub&gt;</td><td>ACROS Organics</td><td>AC320760075</td><td>NMR solvent</td></tr><tr><td>Trifluoroacetic Acid&amp;nbsp;</td><td>ACROS Organics</td><td>AC293811000</td><td>99.50%</td></tr><tr><td>Silica Gel</td><td>FISHER&amp;nbsp;</td><td>L-7446</td><td>230-400 mesh</td></tr><tr><td>Baker Flex TLC Plates</td><td>FISHER&amp;nbsp;</td><td>NC9990129</td><td></td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DU 145&amp;nbsp; Prostate Cancer Cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>MTT</td><td>ACROS Organics</td><td>158990050</td><td>98%</td></tr><tr><td>RPMI 1640 Medium</td><td>MEDIATECH INC&amp;nbsp;</td><td>10041CV</td><td></td></tr><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>15240062</td><td>100x stock solution</td></tr><tr><td>Fetal Bovine Serum</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>10437077</td><td></td></tr><tr><td>Nuclear Magnetic Resonance Spectroscopy</td><td>Varian, Inc</td><td></td><td>300 NMR&amp;nbsp;</td></tr><tr><td>B&amp;uuml;chi R-3 Rotavapor</td><td>Buchi</td><td>1103022V1&amp;nbsp;</td><td>Rotary evaporator</td></tr><tr><td>Ultrasonic Bath</td><td>BRANSON ULTRASONICS CORPORATION&amp;nbsp;</td><td>CPX952318R</td><td></td></tr><tr><td>UV-VIS spectrometer Biomate 3</td><td>Thermo Spectronic</td><td></td><td></td></tr><tr><td>Zetasizer Nano ZS90&amp;nbsp;</td><td>Malvern Instruments</td><td></td><td>Particle Size Analyer</td></tr><tr><td>Microplate Spectrophotometer&amp;nbsp;</td><td>Rio-Rad</td><td>Benchmark Plus&amp;nbsp;</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Napco</td><td>CO2 6000</td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>Nuaire</td><td></td><td></td></tr><tr><td>SigmaPlot&amp;nbsp;</td><td>Systat Software, Inc.</td><td></td><td>Analytical Software</td></tr><tr><td>96-Well Cell Culture Plate</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Trypsin&amp;nbsp; 0.25%</td><td>Corning Cellgro</td><td>25-053-CI</td><td></td></tr></tbody></table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

그림 1은 DOX-PA의 합성 방식을 보여줍니다. DOX-PA는 pH 민감성 히드라 존 결합을 통해 독소루비신 팔 미트 산의 결합에 의해 합성 하였다. 팔 미트 산 히드라 지드의 약간 과량은 반응의 완료를 용이하게하기 위해 사용되었다. 이 반응에있어서 매우 높은 효율이 독소루비신의 소량은 18 시간 반응 (도 2) 뒤에 남아 있었다. 수율은 약 88 %였다. 반응의 ?...

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

친 유성 독소루비신 전구 약물의 제조 및 특성화를위한 프로토콜 1,2- distearoyl- SN -glycero -3- phosphoethanolamine- N로드 - [아미노 (폴리에틸렌 글리콜) -2000 (PEG-DSPE) 미셀 설명한다.

친 유성 독소루비신 전구 약물 미셀의 제조 및 특성화

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K 조회수.  Hampton University.  친 유성 독소루비신 전구 약물의 제조 및 특성화를위한 프로토콜 1,2- distearoyl- SN -glycero -3- phosphoethanolamine- N로드 - [아미노 (폴리에틸렌 글리콜) -2000 (PEG-DSPE) 미셀 설명한다. 

비디오: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

가역 디설파이드 가교 미셀 제조를위한 손쉬운 효율적인 접근법

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

연구

생화학

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

암 연구학

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 ...

화학

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

탄소 나노 튜브에 의해 안정화 내부 자기 조립 된 지질 입자의 용이 한 제조

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

π-π 스태킹 상호 작용에 의해 안정화 된 블록 공중 합체 미셀 준비하는 양친 매성 공중 합체의 음이온 중합

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

표적 약물 전달을 위해 독소루비신으로 로드된 압타머-PEI-g-PEG 수정 금 나노 입자의 합성

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

액정 나노 입자 캐리어의 소수성화물 캡슐화의 대상 플라즈마 멤브레인 배달

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

생체공학

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

생리 활성 친수성 구형 단백질의 함정에 대한 나노 리포좀의 준비 및 특성화

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

리소리피드 함유 온도 민감성 리포좀의 미세유체 생산

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...