这项工作得到加拿大卫生研究所的支持, MOP-42546 和 MOP-119514 的运作赠款。

1. 氨基酸溶液的制备 <ol> <li> 为每种氨基酸准备和标记两个50毫升圆锥离心管 (分别用于两个和 #34; 低和 #34; 和 #34; 高 #34; 浓度). </li>
	<li> 准备一个大2升瓶, 含纯净水 (18.2 米和 #937; #183; cm 在25和 #176; C). </li>
	<li> 计算每种氨基酸 (克) 达到所需浓度, 并使用刮刀将适当数量的氨基酸称量到各自的50毫升离心管中. 
	注: 对于 #34; 高 #34; 两种带负电荷的氨基酸的浓度, 用 PBS 代替水。由于水的溶解度低, 我们不能增加其浓度, 而使用 PBS 代替水有助于维持低 pH 值。此外, 每种氨基酸溶液的浓度计算都是通过40毫升的最终体积得到的。所有氨基酸浓度在 表 3 中概述。一定要清洗在氨基酸之间的刮刀, 以避免污染。我们建议用水冲洗, 然后用70% 乙醇擦拭. </li>
	<li> 使用血清吸管将40毫升纯净水 (或 PBS) 加入每50毫升的试管中。帽管和涡旋或剧烈摇动直至溶解。水浴超声 (室温, 130 W, 40 赫) 也可用于协助溶解度过程. 
	注: 以下氨基酸溶液对光敏感, 应覆盖铝箔: 色氨酸、苯丙氨酸和酪氨酸 (包括芳香环状结构) 和半胱氨酸 (活性 SH 组). </li>
</ol> 2。准备 SAP-AA 解决方案 <ol> <li> 为自肽准备20毫升闪烁小瓶。对于一个给定的自肽, 准备一瓶每个准备的氨基酸溶液 (每个组合将在一个单独的小瓶). </li>
	<li> 使用高性能的分析平衡 (可读性降低到0.1 毫克或更低), 重约1和 #177; 0.2 毫克的肽到每个小瓶的底部。盖后称量, 并记录的确切重量的肽的上限. </li>
	<li> 将适当数量的氨基酸溶液 (在1节中制备) 放入每一个含有肽的小瓶中, 以便达到所需的自肽浓度 (0.1 毫克/毫升, 用于长肽, 长度为16氨基酸, 或0.2 毫克/毫升短肽, 长度为8氨基酸). </li>
	<li> 几种在室温下为10分钟的水浴 sonicator (130 W, 40 赫), 确保瓶子内的溶液完全浸入水浴中. </li>
</ol> 3。药物-二甲基亚砜或药物乙醇库存溶液的制备 <ol> <li> 将1毫克的药物 (在本例中为姜黄素) 与100% 的二甲基亚砜结合, 另1毫克与100% 乙醇一起创建两个股票解决方案. 
	注: 我们增加了200和 #181; l 二甲基亚砜和400和 #181; l 乙醇, 分别为5毫克/毫升和2.5 毫克/毫升, 由于每个溶剂中的溶解度不同, 而使其成为两甲基苯甲酸二甲砜-姜黄素和乙醇-姜黄素的股票;然而, 重要的是要注意, 股票的浓度应调整取决于疏水性药物的利益。诸如药物溶解度和有效生物浓度等因素在确定这一价值时很重要。同时, 请记住, 当与 SAP-AA 解决方案结合使用时, 该股票将分别被稀释100倍和50倍的二甲基亚砜和乙醇制剂 (见第4节)。根据所需配方的数量和 #8211, 最好准备大量的存货; 在这种情况下, 将使用超过1毫克的药物。库存可储存在-20 和 #176;使用前解冻冰和涡旋. </li>
	<li> 漩涡瓶十五年代完全溶解药物. </li>
</ol> 4。药物制剂的制备 <ol> <li> 为每个配方准备清晰的、1.5 毫升的离心管。一定要用预定的自肽、氨基酸 (和浓度) 和溶剂标签管. </li>
	<li> 添加10和 #181; 药物-二甲基亚砜库存, 或20和 #181; l 药物乙醇库存到适当的离心管. </li>
	<li> 添加990和 #181; SAP-AA 酸解决方案的适当标记离心管含有药物-二甲基亚砜的股票, 和980和 #181; 我对那些含有药物乙醇的股票。这产生1毫升药物配方与1% 二甲基亚砜或2% 乙醇. 
	注: 根据该协议, 所有姜黄素制剂的最终浓度为0.5 毫克/毫升。同样, 当使用其他疏水化合物和/或开始与不同的存货集中 (参见步骤 3.1) </li> <li> 漩涡强烈三十年代和允许公式化休息为 30 min. </li> </ol> 时, 这将有所不同。
 5. 溶解度测试 <ol> <li> 在休息期过后, 漩涡又一次剧烈地三十年代. </li>
	<li> 离心 14220 x g 的配方1分钟. </li>
	<li> 分析离心管底部的降水 (通过可视化). </li>
</ol>

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作者没有什么可透露的。

在制定过程中, 有许多关键步骤和问题需要考虑。首先, 当我们使用不同的组件和浓度时, 整个协议中的多个涡旋步骤确保所有浓度都是一致和正确的。一些高浓度的疏水性氨基酸溶液在涡流后仍可能无法完全溶解, 在这种情况下, 它们可以用手使劲摇动, 以协助过程。同样, sap-AA 解决方案必须经过步骤2.4 中概述的超声步骤, 因为结构调整结构自然倾向于聚合, 而超声将帮助分割 sap 集群, 从而产生更统一的解决方案。其次, 对于给定的疏水性药物, SAP-AA 制剂中的库存和最终浓度应取决于该化合物在生物环境中的有效浓度。该议定书应相应地加以修改, 以反映这种积极的集中。此外, 药物的装载能力也是一个重要的考虑因素;这可能是每个药物都有一个独特的负载能力使用这种策略, 每个 SAP-AA 组合可能支持不同数量的药物的基础上的兼容性。这表明了筛选的重要性, 找到最佳的 SAP-AA 组合为给定的化合物。
使用我们的技术对其他人有许多好处;更具体地说, 在传统的方法中, 单独使用结构化结构来封装和潜在的化合物的交付是非常重要的。如前所述, 氨基酸已被临床接受, 并加入这些解决方案的 SAP 配方增加渗透减少溶血活性, 这样, 静脉注射在临床情况是可能的。我们还发现, 它们大大增加疏水性复合溶解度的情况下, 单独的结构, 只有 sap 是不够的溶解度17,18。多种不同组合的 sap 和氨基酸参与允许扩展到高通量的方法筛选疏水性药物溶解度。可对溶解度数据进行详细分析, 揭示趋势;我们发现, 配方成分 (SAP 或氨基酸) 的分类结果显示, 每种疏水药物都有一个独特的模式。例如, 正电荷的氨基酸改善了姜黄素的溶解度 (图 1), 而我们先前的研究表明, 带负电荷的氨基酸对 PP218是更好的。这些趋势有助于确定特定成分对类似化学结构的药物溶解的适用性。此外, 我们的溶解度屏幕的简单性是一个优点和局限性;虽然它是容易执行, 有更多的技术和准确的方法来实验评估化合物的溶解度在解决方案 (如, 光谱学或色谱方法)。然而, 该协议中概述的筛选策略允许快速而高效地选择 SAP-AA 组合, 从而导致最高的药物溶解度, 因此, 是进一步分析的最高潜在生物活性。由于有许多配方的不同组合的自肽, 氨基酸, 氨基酸浓度, 和溶剂, (480 总在我们以前的手稿18), 这是一个必要的步骤, 缩小到寻找最佳特定药物的成分。在发现可溶性药物制剂后, 可通过更多的技术方法对其溶解度进行评估, 并在功能分析中进一步验证, 以评估生物活性和安全性。这些功能性的分析应该是量身定做的药物的预期用途, 在我们的手稿优化 PP2 配方18。扩大我们的平台, 在其他疏水性化合物将揭示更多的趋势和机制, 以提高溶解度, 并协助工程新的 sap 临床配方的特定疏水性化合物。
我们的方法的未来在于潜在的药物输送管道, 以及它的自动化能力。有许多步骤, 涉及重量粉和配药液体, 这是主要的 time-limiting 因素在制定过程中。尽管在实验室设置中执行过程可能看起来很冗长, 但这些步骤可以使用自动设备轻松地执行。同样, 该方法有很大的潜力, 以扩大到商业化生产, 使用自动秤和分配器, 以同时测试的溶解度, 许多疏水性药物。这将大大加快制定和筛选过程, 同时提高准确性和减少人为错误。因此, 我们的药物配方方法组成的 SAP-AA 组合是一个通用平台的溶解度和交付疏水性化合物, 并将大大受益于高通量技术。

sap 是一类生物材料广泛研究的3D 支架在再生医学1,2,3,4。然而, 最近, 由于其独特的生物学特性5、6、7、8, 它们被作为治疗药物的载体而被利用。sap 自然组装成稳定的纳米结构9, 从而提供了一种药物的封装和保护手段。sap 是亲, 由一个特定的疏水性和亲水性氨基酸重复的模式, 驱动他们的自组装9,10 , 并允许他们作为一个介乎疏水性和亲水性环境.因此, 对于疏水性药物的临床交付--由于在水环境中由于缺乏溶解度而在人体内具有极低的生物利用度和吸收率11,12 -sap 是希望作为交付车辆.此外, 他们的序列模式还意味着, sap 可以合理设计和工程, 以最大限度地与任何特定药物或化合物 (即, 基于功能基团) 的兼容性, 并进一步协助溶解度。
在许多体外和体内设置13、14、15、16中, sap 已被应用为有效的药物运载工具。它们也显示出极大的安全性和生物相容性。然而, 由于低渗透的 SAP 药物制剂, 它们不能用于静脉注射, 在临床设置13。考虑到这种限制, 我们最近制定了一项战略, 将 sap 与氨基酸解决方案结合起来, 以减少毒性 co-solvents 的使用, 并增加处方渗透, 从而提高临床相关性。我们选择使用氨基酸, 因为它们是结构化结构的基石, 已经被临床接受, 并与 sap 结合, 它们增加了疏水性药物的溶解度, 同时减少了需要的17,18的数量。
我们已经审查了 SAP-AA 组合作为一个通用平台的疏水性药物溶解度和后续交付通过创建一个多步骤筛选管道, 并将其应用于 Src 抑制剂, PP2, 作为一个模型疏水性化合物。在这个过程中, 我们研究了改变配方的成分-最终测试6种不同的结构, 所有20种氨基酸在2不同浓度 (低和高; 低根据现有临床应用的浓度, 和高浓度是 2x, 3x, 或5x 的临床浓度的基础上, 每个氨基酸在水中的最大溶解度), 和2不同的 co-solvents-和选择的组合, 溶解 PP2 进一步分析。这种药物制剂在细胞培养中被证明是一种有效的药物载体, 同时也在使用气管和静脉的管理的体内模型。同样, 我们的工作涉及的多功能性的 SAP-AA 组合在磷多, 结构不同的疏水性化合物在水环境中;具体来说, 药物 rottlerin 和姜黄素18。本文概述了液-AA 的配方方法和姜黄素溶解度分析为例, 在我们的筛选管道的主要步骤。该协议提供了一个简单的, 可重现的方式来筛选最佳的 SAP-AA 组合, 它可以溶解任何给定的疏水性化合物。

<table><tbody><tr><td>EAK16-I</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: AEAKAEAKAEAKAEAK, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>EAK16-II</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: AEAEAKAKAEAEAKAK, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>EAK16-IV</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: AEAEAEAEAKAKAKAK, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>EFK8-II</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: FEFEFKFK, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>A6KE</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: AAAAAAKE, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>P6KE</td><td>CanPeptide Inc.</td><td>Custom peptide</td><td>Sequence: PPPPPPPKE, N-terminus acetylation and C-terminus amidation, &gt;95% pure by HPLC</td></tr><tr><td>Alanine</td><td>Sigma-Aldrich</td><td>A7469-100G</td><td>L-Alanine</td></tr><tr><td>Isoleucine</td><td>Sigma-Aldrich</td><td>I7403-100G</td><td>L-Isoleucine</td></tr><tr><td>Leucine</td><td>Sigma-Aldrich</td><td>L8912-100G</td><td>L-Leucine</td></tr><tr><td>Methionine</td><td>Sigma-Aldrich</td><td>M5308-100G</td><td>L-Methionine</td></tr><tr><td>Proline</td><td>Sigma-Aldrich</td><td>P5607-100G</td><td>L-Proline</td></tr><tr><td>Valine</td><td>Sigma-Aldrich</td><td>V0513-100G</td><td>L-Valine</td></tr><tr><td>Phenylalanine</td><td>Sigma-Aldrich</td><td>P5482-100G</td><td>L-Phenylalanine</td></tr><tr><td>Tryptophan</td><td>Sigma-Aldrich</td><td>T8941-100G</td><td>L-Tryptophan</td></tr><tr><td>Tyrosine</td><td>Sigma-Aldrich</td><td>T8566-100G</td><td>L-Tyrosine</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8790-100G</td><td>L-Glycine</td></tr><tr><td>Asparagine</td><td>Sigma-Aldrich</td><td>A4159-100G</td><td>L-Asparagine</td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G8540-100G</td><td>L-Glutamine</td></tr><tr><td>Serine</td><td>Sigma-Aldrich</td><td>A7219-100G</td><td>L-Serine</td></tr><tr><td>Threonine</td><td>Sigma-Aldrich</td><td>T8441-100G</td><td>L-Threonine</td></tr><tr><td>Histidine</td><td>Sigma-Aldrich</td><td>H6034-100G</td><td>L-Histidine</td></tr><tr><td>Lysine</td><td>Sigma-Aldrich</td><td>L5501-100G</td><td>L-Lysine</td></tr><tr><td>Arginine</td><td>Sigma-Aldrich</td><td>A8094-100G</td><td>L-Arginine</td></tr><tr><td>Aspartic Acid</td><td>Sigma-Aldrich</td><td>A7219-100G</td><td>L-Aspartic Acid</td></tr><tr><td>Glutamic Acid</td><td>Sigma-Aldrich</td><td>G8415-100G</td><td>L-Glutamic Acid</td></tr><tr><td>Cysteine</td><td>Sigma-Aldrich</td><td>C7352-100G</td><td>L-Cysteine</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>D4540-500ML</td><td>DMSO</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>277649-100ML</td><td>Anhydrous</td></tr><tr><td>Curcumin</td><td>Sigma-Aldrich</td><td>08511-10MG</td><td>Hydrophobic drug, curcumin</td></tr><tr><td>Rottlerin</td><td>EMD Millipore</td><td>557370-10MG</td><td>Hydrophobic drug, rottlerin</td></tr><tr><td>PP2</td><td>Enzo&amp;nbsp;</td><td>BML-EI297-0001</td><td>Hydrophobic drug, PP2</td></tr><tr><td>Scintillation Vials</td><td>VWR</td><td>2650-66022-081</td><td>Borosilicate Glass, with Screw Cap, 20 mL. Vials for weighing peptide.</td></tr><tr><td>Falcon 50 mL Conical Centrifugation Tubes</td><td>VWR</td><td>352070</td><td>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

自组装多肽 (sap) 是有前途的车辆提供疏水疗法的临床应用;它们的亲特性使它们能够在人体的水环境中溶解疏水化合物。然而, 自肽解决方案有较差的血液相容性 (例如, 低渗透), 阻碍了他们的临床应用通过静脉注射管理。我们最近开发了一个通用的疏水药物输送平台, 它将 sap 与氨基酸溶液 (液-AA) 结合起来, 提高药物的溶解度, 增加配方渗透, 达到临床使用的要求。在三种不同的疏水性化合物--PP2、rottlerin 和姜黄素--的背景下, 对这种配方进行了彻底的测试, 以证明其通用性。此外, 我们研究了改变配方成分的影响, 分析了6种不同的结构, 20 种天然氨基酸在低和高浓度, 和两个不同的 co-solvents 二甲基亚砜和乙醇。我们的策略被证明是有效的, 以优化一个给定的疏水性药物的成分, 和治疗功能的配方抑制剂, PP2, 被观察到的体外和在体内。这篇手稿概述了我们的通用配方方法使用 SAP-AA 组合疏水性化合物, 并分析溶解度作为第一步, 以潜在使用这些配方在更多的功能性研究。我们包括有代表性的溶解度结果的配方疏水性化合物, 姜黄素, 并讨论如何作为一个平台, 未来的生物研究和疾病模型的方法。

对于疏水性药物, 姜黄素, 我们生产的配方使用所有20种天然存在的氨基酸在低浓度, 结合只有一个 SAP, EAK16-II, 作为一个原理。我们还测试了使用二甲基亚砜和乙醇作为 co-solvents 的配方。总的, 这产生了40姜黄素配方, 每个含有不同的成分。重要的是要注意, 在我们以前的研究使用 Src 抑制剂, PP2, 我们包括更多的选择的 SAP (共 6) 和氨基酸浓度 (临床, 以及较高的浓度), 这产生了共480种不同的配方。在选择 EAK16-II 作为本研究的 SAP 时, 这项工作的趋势被考虑在内。各种组件的浓度包括在表 1、表 2和表 3中作为参考。所有疏水性药物制剂通过可视化的方法进行药物溶解度筛选, 如果溶液完全清除离心后的任何沉淀物, 则被认为是可溶性的 (图 1)。如果药物沉淀到底部的管, 这被认为是不, 并没有经过进一步的测试。此外, 溶解度由两个不同的个体来检验;如果这些结果不能重现, 配方也不被认为是真正的可溶性。
在本研究中测试的40配方中, 7 配方成功溶解了姜黄素 (表 4)。按组件分组配方确定了两个主要趋势: 乙醇似乎是溶解姜黄素的较好溶剂, 而正电荷的氨基酸赖氨酸 (K) 和精氨酸 (R) 也似乎是溶解姜黄素的最佳组分 (表4). 有趣的是注意到含有 R 和 K 的配方的颜色变化, 这表明姜黄素是溶解在碱性条件 (图 1)。通过各种成分的性质对配方进行分组来进行观察是很有帮助的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56158/56158fig1.jpg"> 
图 1: 降水分析的例子.对于这些含有肽 EAK16-II、乙醇和带电氨基酸的姜黄素配方, 在离心后的离心管中可以清楚地看到沉淀。含有赖氨酸 (K)、精氨酸 (R) 或天门冬氨酸 (D) 的配方溶解姜黄素 (no 沉淀), 而含有组氨酸 (H) 或谷氨酸 (E) 的制剂不 (沉淀, 用红色圆圈)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56158/56158fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>药物</td>
			<td>配方浓度 (毫克/毫升)</td>
		</tr>
		<tr>
			<td>PP2</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>姜黄素</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>Rottlerin</td>
			<td>0.02</td>
		</tr>
	</tbody>
</table>
表 1:制剂中使用的药物浓度.药物制剂浓度各不相同, 各有不同的生物活性浓度, 也有不同的负荷能力。
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>自组装肽</td>
			<td>属性</td>
			<td>配方浓度 (毫克/毫升)</td>
		</tr>
		<tr>
			<td>EAK16-I</td>
			<td>家庭, 长</td>
			<td>0。1</td>
		</tr>
		<tr>
			<td>EAK16-II</td>
			<td>家庭, 长</td>
			<td>0。1</td>
		</tr>
		<tr>
			<td>EAK16-IV</td>
			<td>家庭, 长</td>
			<td>0。1</td>
		</tr>
		<tr>
			<td>EFK8-II</td>
			<td>修改的讲, 短</td>
			<td>0。2</td>
		</tr>
		<tr>
			<td>A6KE</td>
			<td>表面活性剂样, 短</td>
			<td>0。2</td>
		</tr>
		<tr>
			<td>P6KE</td>
			<td>表面活性剂样, 短</td>
			<td>0。2</td>
		</tr>
	</tbody>
</table>
表 2: 配方中使用的自肽的浓度.随着氨基酸的增加, 只有小浓度的自肽是必需的 (0.1-0.2 毫克/毫升)。较短的肽是双倍的浓度比较长的肽, 因为他们有一半的序列长度 (8 氨基酸与16氨基酸)。
<img alt="Table 3" src="/files/ftp_upload/56158/56158table3.jpg"> 
表 3: 配方中使用的氨基酸溶液的浓度.根据现有的临床应用, 选择低浓度的氨基酸。高浓度是 2x, 3x, 或5x 的临床浓度和在水中的每个氨基酸的最大溶解度。此图已从帕切科et al.中修改18
<img alt="Table 4" src="/files/ftp_upload/56158/56158table4.jpg"> 
表 4: 姜黄素具有代表性的溶解度结果摘要的 SAP-AA 组合, 有效溶解姜黄素筛选后的溶解度。此图已从帕切科et al.中修改18

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

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

本议定书描述了一种临床适用的方法, 溶解疏水化合物在水环境中使用自肽和氨基酸溶液的组合。我们的方法解决疏水疗法的一个主要局限, 缺乏安全, 有效的手段溶解和交付方法入临床设置。

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

solubility-hydrophobic-compounds-aqueous-solution-using-combinations

Universidad San Sebastian

Research

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JoVE Journal

JoVE Core

Organic Chemistry

Medicine

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

16.7K 次观看.  University Health Network. 本议定书描述了一种临床适用的方法, 溶解疏水化合物在水环境中使用自肽和氨基酸溶液的组合。我们的方法解决疏水疗法的一个主要局限, 缺乏安全, 有效的手段溶解和交付方法入临床设置。

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

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

科研

化学

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

生物工程

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

自组装多胺基多肽 Amphiphiles (PPAs) 及相关生物材料的简易合成协议

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

12-Dithiolane 改性自组装肽的合成与表征

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

<meta charset="utf8"></head><body> 肽水凝胶:ECF-5 的环境响应性凝胶化方法

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

<meta charset="utf8"></head><body>作者聚焦:改进自组装纤维和肽水凝胶的生产以获得卓越的生物相容性

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

机械稳定的自组装肽水凝胶的制备

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

生物化学

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

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此视频中的章节

生物分子有序结构由短肽的自组装形成

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

自组装多胺基多肽 Amphiphiles (PPAs) 及相关生物材料的简易合成协议

12-Dithiolane 改性自组装肽的合成与表征

信息承载Peptoids及其序列定向动态共价自组装的合成

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

机械稳定的自组装肽水凝胶的制备

机械稳定的自组装肽水凝胶的制备

机械稳定的自组装肽水凝胶的制备

一种三肽稳定的油酸纳米乳液