이 작품은 건강 연구의 캐나다 학회에 의해 지원 하 고, 걸 레-42546 및 걸 레 119514 부여 운영 합니다.

1. 준비의 아미노산 솔루션 <ol><li>준비 및 레이블 두 50 mL 원뿔 원심 분리기 튜브 각 아미노산에 대 한 (하나 둘 다를 위해 각 &#34; 낮은 &#34; 및 &#34; 높은 &#34; 농도).</li>
	<li>순화 된 물 (18.2 ㏁ 25 ° C에서)를 포함 하는 큰 2 L 플라스 크를 준비.</li>
	<li>원하는 농도 도달 (그램)에서 각 아미노산의 양을 계산 하 고 주걱을 사용 하 여 그들의 각각 50 mL 원심 분리기 튜브로 아미노산의 적절 한 금액을 무게. 
	: 참고는 &#34; 높은 &#34; 2의 농도 부정 청구 아미노산, PBS 물 대신 사용 됩니다. 우리는 그들의 낮은 물 가용성 및 낮은 pH를 유지 하기 위해 물 수 대신 PBS를 사용 하 여 그들의 농도 증가 하지 않을 수 있습니다. 또한, 농도 계산 각 아미노산 솔루션에 대 한 40 mL의 최종 볼륨을 사용 하 여 얻은 했다. 모든 아미노산 농도 표 3에 설명 되어 있습니다. 오염을 피하기 위하여 아미노산 사이 주걱을 씻어 해야 합니다. 물 린스, 70% 에탄올으로 닦아 다음 것이 좋습니다.</li>
	<li>순화 된 물 (PBS)의 각 50 mL에 추가 40 mL 혈 청 학적인 피 펫을 사용 하 여 튜브. 튜브와 소용돌이 닫습니다 또는 해산 때까지 적극적으로 악수. 물 목욕 쥡니다 (실내 온도, 130 W, 40 kHz) 용 해도 프로세스에 도움을 사용할 수 있습니다. 
	참고: 다음 아미노산 솔루션은 빛에 민감한 및 알루미늄 호 일로 덮어야 한다: 트립토판, 페닐알라닌, 티로신 (이루어져 방향족 고리 구조) 시스테인 (반응-SH 그룹).</li>
</ol> 2. SAP-AA 솔루션의 준비 <ol><li>준비 20 mL 섬광 튜브 자기 조립에 대 한 펩 티 드. 주어진된 자기 조립 펩 티 드에 대 한 준비 아미노산 솔루션 (각 조합에서에서 이루어집니다 별도 유리병) 당 한 유리병을 준비.</li>
	<li>각 유리병의 바닥에 약 1 ± 0.2 mg 펩 티 드의 무게 (와 함께 한 가독성 또는 그이 하 0.1 mg까지), 높은-성능 분석 균형을 사용 하 여. 무게 후 뚜껑과 뚜껑에 펩 티 드의 정확한 무게를 기록</li>
	<li>펩 티 드, 펩 티 드 (0.1 mg/mL 16 아미노산의 길이가 긴 펩 티 드 또는 0.2 mg/mL에 대 한 자기 조립의 원하는 농도 도달 하기 위하여 포함 된 각 유리병에 적절 한 양의 아미노산 솔루션 (섹션 1에서 준비 하는) 플라스틱 8 아미노산의 길이가 짧은 펩 티 드).</li>
	<li>는 물 목욕 sonicator (130 W, 40 kHz) 실내 온도에, 튜브 내에서 솔루션 물 목욕에 완전히 몰입 하 보장에서 10 분 Sonicate.</li>
</ol> 3. DMSO 약물 또는 약물 에탄올 재고 솔루션의 준비 <ol><li>결합 (이 경우에는 100 %DMSO curcumin)에서 약물의 1 mg와 두 개의 재고 솔루션을 만드는 100% 에탄올과 다른 1mg. 
	참고: 우리는 DMSO curcumin와 에탄올 curcumin 주식 되었고 5 mg/mL 2.5 mg/mL, 각각, 각 용 매;에 있는 다양 한 가용성 때문에 DMSO와 400 µ L 에탄올의 200 µ L을 추가 그러나, 그것은 재고의 집중 관심의 소수 성 약물에 따라 조정 되어야 한다 주의 하는 것이 중요입니다. 약물 용 해도 등 효과적인 생물학 농도 요인이이 가치 결정에 중요 하다. 또한, 그 재고 것입니다 희석 약 및 50-fold DMSO와 에탄올 정립에서 각각, SAP AA 솔루션 (섹션 4 참조)와 함께 하는 마음에 계속. 공식 요구의 수에 따라 재고의 더 큰 볼륨을 준비를 선호 수 있습니다 &#8211;이 경우에, 약 1 mg 이상 사용 될 것 이라고. 주식-20 ° C에 저장 될 수 있다 얼음에 소용돌이 사용 하기 전에 해 동.</li>
	<li>소용돌이 15 리 바이 알 약을 완전히 분해 하는 s.</li>
</ol> 4. 약물 제형의 준비 <ol><li>준비, 1.5 mL microcentrifuge 튜브 각 배합에 대 한. 펩 티 드, 아미노산 (고 농도), 자체 조립 하는 의도를 레이블 튜브 반드시과 공동 용 매.</li>
	<li>마약 DMSO 주식, 또는 적절 한 microcentrifuge 튜브 20 µ L 마약 에탄올 재고 추가 10 µ L.</li>
	<li>추가 990 µ L의 SAP AA 산 성 솔루션을 적절 한 분류 마약 DMSO 주식과 마약 에탄올 주식 포함을 980 µ L을 포함 하는 microcentrifuge 튜브. 이 1 %DMSO 또는 2% 에탄올 1 mL 약물 제형 생산. 
	참고: 모든 curcumin 공식의 최종 농도 프로토콜에 따라 0.5 mg/mL 이었다. 다시 말하지만,이 때 달라 집니다 다른 소수 성 화합물을 사용 하 여 또는 다른 재고 농도와 시작 (단계 3.1 참조)</li> <li>30에 대 한 적극적으로 소용돌이 s 30 분 동안 휴식을 공식 허용</li></ol>
 5. 가용성 테스트 <ol><li>휴식 기간, 소용돌이 적극적으로 한 번 다시 30 후 미</li>
	<li>1 분에 대 한 14,220 x g에서 공식 원심</li>
	<li>강수량 microcentrifuge 관의 하단 (시각화) 하 여 분석.</li>
</ol>

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저자는 공개 없다.

수립 절차에서 다양 한 중요 한 단계 및 문제 해결에 고려해 야 할 점이 있다. 첫째, 우리는 다양 한 구성 요소와 농도 일으로 프로토콜을 통해 여러 개의 소용돌이 단계 모든 농도 균일 하 고 올바른 인지 확인 합니다. 고 농도, 소수 성 아미노산 솔루션의 일부 수 있습니다 여전히 하지 완전히 용 해 vortexing, 후 고이 경우에, 그들은 과정에서 도움을 손으로 적극적으로 흔들릴 수 있습니다. 마찬가?...

얼 간 재생 의학1,2,,34에서 3D 공중 발판으로 광범위 하 게 연구 하는 생체 재료의 클래스는. 그러나 최근에, 그들은 있다 되었습니다 악용 치료제 때문에 그들의 독특한 생물 속성5,6,,78의 납품을 위한 차량으로. 얼 간 자연스럽 게 안정 nanostructures9, 마약 캡슐화 하 고 보호 하는 수단 제공으로 조립. 얼 간은 amphipathic, 소수 성 및 친수성 아미노산 반복, 운전의 특정 패턴의 구성 그들의 자기 조립을9,10 , 소수 성 및 친수성 사이 매체 역할을 수 있도록 환경입니다. 따라서, 소수 성 약물-임상 배달에 있는 매우 낮은 생체 이용률과 신체에 흡수 수성 환경11,12 -가용성의 부족으로 인해 얼 간은 배달 약속 차량입니다. 또한, 그들의 순서 패턴 또한 그 얼 간 합리적으로 설계 하 고 수 하 어떤 주어진 약물 호환성 최대화 또는 복합 (즉, 기능 그룹에 따라) 추가 가용성 엔지니어링을 의미 합니다.
얼 간 효과적인 마약 배달 차량 많은 생체 외에서 그리고 vivo에서 설정13,14,,1516으로 적용 되어 있다. 그들은 또한 중대 한 안전 및 생체 적합성 표시. 그러나, SAP 약물 준비의 낮은 osmolarity, 때문에 그들은 임상 설정13에서 정 맥 주사에 대 한 사용할 수 없습니다. 이 구속을 고려 하면 우리 최근 얼 간 독성 공동의 사용을 감소 하 고 배합 osmolarity 증가 아미노산 솔루션과 결합 하는 전략을 개발 했다 따라서, 임상 관련성. 우리 얼 간의 빌딩 블록으로 아미노산을 사용 하기로 이미 임상 허용, 고 얼 간와 함께, 그들은 증가 소수 성 약물의 용 해도17,18필수 SAP의 양을 줄이는.
우리는 자세히 조사 SAP AA 조합 소수 성 약물의 용 해도 및 후속 배달을 위한 일반화 된 플랫폼으로 여러 단계의 심사 파이프라인을 만들고 Src 반응 억제제, PP2 모델 소수 성 화합물으로 서 그것을 적용 하 여. 이 과정에서 우리는 정립의 구성 요소를 변경-궁극적으로 테스트 6 다른 얼 간, 2 다양 한 농도 (낮은, 높은, 낮은에 따라 기존 임상 응용 프로그램에 높은 농도에서 모두 20 아미노산의 효과 검사 농도 했다 2 배, 3 배, 또는 물에서 각 아미노산의 최대 용 해도 기반으로 하는 임상 농도 5 배), 그리고 2 다른 공동 용 매-및 추가 분석을 위해 PP2 solubilized 선택한 조합. 이 약물 배합 vivo 모델 intratracheal 및 정 맥 행정을 사용 하 여 세포 배양에 약 배달 차량으로 효과가 입증 했다. 마찬가지로, 우리의 작업 solubilizing 여러 SAP AA 조합의 다양성에 감동을; 수성 환경에서 구조적으로 다른 소수 성 화합물 특히,는 약 rottlerin 및 curcumin18. 이 원고는 SAP AA 배합 방법 및 우리의 심사 파이프라인에서 기본 단계의 예로 curcumin 가용성의 분석을 설명합니다. 이 프로토콜 어떤 주어진된 소수 성 화합물을 분해 최적의 SAP AA 조합에 대 한 화면으로 간단 하 고 재현성을 제공 합니다.

<table style="border-collapse:collapse;width:1034pt" width="1379">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-I</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20" style="width:510pt" width="680">Sequence: AEAKAEAKAEAKAEAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAKAKAEAEAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-IV</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAEAEAKAKAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EFK8-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: FEFEFKFK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">A6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AAAAAAKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">P6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: PPPPPPPKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Alanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7469-100G</td>
			<td class="xl20">L-Alanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Isoleucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">I7403-100G</td>
			<td class="xl20">L-Isoleucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Leucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">L8912-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Leucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Methionine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">M5308-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Methionine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Proline</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5607-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Proline</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Valine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">V0513-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Valine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Phenylalanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5482-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Phenylalanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tryptophan</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8941-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Tryptophan</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tyrosine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8566-100G</td>
			<td class="xl20">L-Tyrosine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glycine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8790-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glycine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Asparagine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A4159-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Asparagine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8540-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Serine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Serine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Threonine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8441-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Threonine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Histidine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">H6034-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Histidine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Lysine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl17" style="width:101pt" width="135">L5501-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Lysine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Arginine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A8094-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Arginine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Aspartic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Aspartic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8415-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Cysteine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">C7352-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Cysteine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Dimethyl Sulfoxide</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">D4540-500ML</td>
			<td class="xl22">DMSO</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Ethanol</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">277649-100ML</td>
			<td class="xl22">Anhydrous</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Curcumin</td>
			<td class="xl21" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl22">08511-10MG</td>
			<td class="xl22">Hydrophobic drug, curcumin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Rottlerin</td>
			<td class="xl21" style="width:106pt" width="141">EMD Millipore</td>
			<td class="xl22">557370-10MG</td>
			<td class="xl22">Hydrophobic drug, rottlerin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">PP2</td>
			<td class="xl21" style="width:106pt" width="141">Enzo&#160;</td>
			<td class="xl22">BML-EI297-0001</td>
			<td class="xl21" style="width:510pt" width="680">Hydrophobic drug, PP2</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Scintillation Vials</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl21" style="width:101pt" width="135">2650-66022-081</td>
			<td class="xl22">Borosilicate Glass, with Screw Cap, 20 mL. Vials for weighing peptide.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Falcon 50 mL Conical Centrifugation Tubes</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl23" style="width:101pt" width="135">352070</td>
			<td class="xl22">Polypropylene, Sterile, 50 mL. For amino acid solutions.</td>
		</tr>
	</tbody>
</table>

solubility of hydrophobic compounds in aqueous solution using combinations of self-assembling peptide and amino acid

자가 조립 펩 티 드 (얼 간)는 임상 응용 프로그램; 소수 성 치료제의 배달에 대 한 유망한 차량 그들의 amphipathic 속성의 수성 환경에서 소수 성 화합물을 분해 하도록 수 있습니다. 그러나, 자가 조립 펩 티 드 솔루션은 가난한 혈액 호환성 (예를 들어, 낮은 osmolarity), 정 맥 행정을 통해 그들의 임상 응용 프로그램을 방해. 우리는 최근이 아미노산 솔루션 (SAP-AA) 약물 용 해도 향상 및 배합 osmolarity 임상 사용에 대 한 요구 사항에 도달 증가 얼 간 결합 소수 성 약물 전달에 대 한 일반화 된 플랫폼을 개발 했습니다. 이 공식 전략 3 구조적으로 다른 소수 성 화합물-PP2, rottlerin, 및 curcumin-그 다양성을 설명 하기 위하여의 맥락에서 철저 하 게 테스트 되었습니다. 또한, 우리 6 다른 얼 간, 낮은 농도에서 20 자연스럽 게 기존 아미노산 및 두 개의 서로 다른 공동 용 디 메 틸 sulfoxide (DMSO) 및 에탄올을 분석 하 여 배합 구성 요소 변경의 효과 검사 합니다. 특정된 소수 성 약물 및 공식화 억제제, PP2, 치료 기능에 대 한 구성 요소를 최적화에 효과가 입증 하는 우리의 전략은 체 외에서 그리고 vivo에서모두 관찰 되었다. 이 원고는 소수 성 화합물 및 기능적 연구에이 공식의 사용 가능성을 향한 첫 걸음으로 용 해도의 분석에 대 한 SAP-AA 조합을 사용 하 여 우리의 일반적인된 배합 방법을 설명 합니다. 우리는 소수 성 화합물, curcumin의 정립에 대 한 대표적인 용 해도 결과 포함 하 고 어떻게 우리의 방법론 미래 생물학 연구 및 질병 모델에 대 한 플랫폼 역할을 토론.

소수 성 약물, curcumin에 대 한 우리 공식 증거의 원칙으로 하나의 SAP, EAK16-II와 함께에서 낮은 농도에서 아미노산을 자연스럽 게 존재 하는 모든 20를 사용 하 여 제작. 우리는 또한 공식 공동 용 매로 DMSO와 에탄올을 사용 하 여 테스트. 총에서이 각각 서로 다른 구성 요소를 포함 하는 40 curcumin 공식 생산. Src 억제제, PP2를 사용 하 여 우리의 이전 연구에 우리 SAP (6 총)와 아미?...

Watch this Scientific Journal Video about Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid at JoVE.com

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

자기 조립 펩타이드와 아미노산의 조합을 사용 하 여 수 용액에서 소수 성 화합물의 용 해도

이 프로토콜 자체 조립 펩타이드와 아미노산 솔루션의 조합을 사용 하는 수성 환경에서 소수 성 화합물을 녹 이기의 임상 적용 방법을 설명 합니다. 우리의 방법은 소수 치료 학, 임상 설정으로 안전 하 고 효율적 수단 가용성 및 배달 방법의 부족의 주요 한계를 해결 합니다.

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

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16.6K 조회수.  University Health Network. 이 프로토콜 자체 조립 펩타이드와 아미노산 솔루션의 조합을 사용 하는 수성 환경에서 소수 성 화합물을 녹 이기의 임상 적용 방법을 설명 합니다. 우리의 방법은 소수 치료 학, 임상 설정으로 안전 하 고 효율적 수단 가용성 및 배달 방법의 부족의 주요 한계를 해결 합니다.

비디오: Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

를 사용하여 비 비만 당뇨병 마우스의 위 비우는의 평가 [<sup&gt; 13</sup&gt; C]-octanoic 산성 호흡 시험

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

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Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

실험 경추 척수 손상의 신경 전구체 세포 및 자기 조립 펩티드의 시너지 사용

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

멀티 플루오르 담즙 산 및 사용<em&gt; 생체</em&gt; 자기 공명 영상은 측정하는 담즙 산 전송

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Zebrafish 배아를 이용한 화학 화합물의 독성에 대한 신속한 평가

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

이식 품질 평가를 위해 화학 생검 사용

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

메타볼로믹스 및 리피도믹스 분석을 위한 뇌종양의 현장 샘플링 및 추출

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

생체 내 적용을 위한 나린제닌 용액의 제조

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

배양된 뼈 세포와 분리된 뼈축의 아미노산 소비 평가

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...