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 border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">DSPE-PEG2K</td>
			<td style="width:191px;">Cordenpharm</td>
			<td style="width:119px;">LP-R4-039</td>
			<td style="width:167px;">&#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxorubicin</td>
			<td style="width:191px;">LC Laboratories</td>
			<td style="width:119px;">D-4000</td>
			<td>&#62;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Palmitic Acid Hydrazide</td>
			<td style="width:191px;">TCI AMERICA&#160;&#160;</td>
			<td style="width:119px;">P000425G</td>
			<td>&#62;98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">610981000</td>
			<td>Anhydrous</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">D151-4</td>
			<td>99.90%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl sulfoxide-d6</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC320760075</td>
			<td>NMR solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic Acid&#160;</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC293811000</td>
			<td>99.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silica Gel</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">L-7446</td>
			<td>230-400 mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BAKER FLEX TLC PLATES&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">NC9990129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DU 145&#160; Prostate Cancer Cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:119px;">HTB-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">158990050</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium</td>
			<td style="width:191px;">MEDIATECH INC&#160;</td>
			<td style="width:119px;">10041CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic-Antimycotic&#160;</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">15240062</td>
			<td>100x stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">10437077</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nuclear Magnetic Resonance Spectroscopy</td>
			<td style="width:191px;">Varian, Inc</td>
			<td style="width:119px;"></td>
			<td>300 NMR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chi R-3 Rotavapor</td>
			<td style="width:191px;">Buchi</td>
			<td style="width:119px;">1103022V1&#160;</td>
			<td>Rotary evaporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic Bath</td>
			<td style="width:191px;">BRANSON ULTRASONICS CORPORATION&#160;</td>
			<td style="width:119px;">CPX952318R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-VIS spectrometer Biomate 3</td>
			<td style="width:191px;">Thermo Spectronic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zetasizer Nano ZS90&#160;</td>
			<td style="width:191px;">Malvern Instruments</td>
			<td style="width:119px;"></td>
			<td>Particle Size Analyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
			<td style="width:191px;">Rio-Rad</td>
			<td style="width:119px;">Benchmark Plus&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:191px;">Napco</td>
			<td style="width:119px;">CO2 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biological Safety Cabinet</td>
			<td style="width:191px;">Nuaire</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigmaPlot&#160;</td>
			<td style="width:191px;">Systat Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>Analytical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-Well Cell Culture Plate</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin&#160; 0.25%</td>
			<td style="width:191px;">Corning Cellgro</td>
			<td style="width:119px;">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

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

소설 보컬 폴드 생물 반응기의 구조 및 특성

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

신경 성장 인자 인 캡슐 레이션을위한 새로운 HDL 모방 나노 입자의 제조 및 특성 규명

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

동맥 경화 증의 영상에 대 한 펩 티 드 Amphiphile Micelles Monocyte 타겟팅의 합성

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

제조 및 Macrostructure를 포함 하는 광학 조직 팬텀의 특성화

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

준비, 정화, 및 지방산 포함 된 리의 사용

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

나노 제형에도 Limulus Amoebocyte Lysate (LAL) 분석 실험을 사용 하 여

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

유 방 땀 샘에서 조사 된 유기 체의 성장과 특성화

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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 ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

마이크로 전극에 에칭 나노 아키텍처에 초점을 맞춘 이온 빔 리소그래피

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

기능성 및 편광 담즙 상피 낭종의 생성 및 정량적 특성화

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...