作者要感谢毛里齐奥教授马西的富有成果的讨论和小姐基娅拉阿莱格雷蒂语言编辑。作者的研究是由阪乔瓦尼Ricercatori 2010部（Ministero della Salute的GR-2010- 2312573）的支持。

注:按照来文中使用的化学品。线性RGD购买，但是它可以通过标准Fmoc固相肽合成16,19来制备。溶剂是分析纯。透析需要与男女截止等于3500大使用膜。所合成的化合物的特征在于记录在400MHz分光计使用氯仿1 H NMR谱（CDCL 3）或氧化氘（D 2 O）作为溶剂，和化学位移被报告为每百万份δ值。此外，水凝胶使用KBr压片技术进行FT-IR分析和他们的物理特征包括使用倒置试管在37℃下评估凝胶化的研究。 1. 4- Azidobutanoyl合成氯化物1 <ol><li>在10ml二氯甲烷和0.5ml二甲基甲酰胺溶解500毫克的4- azidobutanoic酸（3.90毫摩尔）。 </li><li>冷却该溶液在0℃下，使用冰浴。 </li><li>添加505微升草酰氯（5.85毫摩尔）至5毫升二氯甲烷中，并缓慢加入滴加至反应体系中，同时搅拌。 </li><li>用冰浴于0℃1小时后，返回到室温。 </li><li>除去用旋转蒸发器在减压下除去溶剂。 </li><li>通过1 H-NMR谱表征所得产物，在CDCl 3中16的样品溶解。 </li></ol> 2. RGD的叠氮化物衍生物2的合成<ol><li>在1ml的1M NaOH中溶解50毫克RGD的（0.145毫摩尔）。 </li><li>在2ml四氢呋喃中溶解的1（0.16毫摩尔）24毫克。 </li><li>用冰浴的所有RGD溶液添加到溶液1中滴加在0℃。 </li><li>返回到室温并搅拌过夜。 </li><li>加入1ml的1M HCl。 </li><li>除去用旋转蒸发器在减压下除去溶剂。 </li><li>表征OBTained通过1 H-NMR光谱法的产品，将样品溶解在D 2 O中16。 </li></ol> 3. PAA炔修改3 <ol><li>溶解200毫克的35％w / w的在15毫升的蒸馏水中的PAA溶液（2.8毫摩尔）。 </li><li>加入15.4毫克盐酸炔丙基胺（0.20毫摩尔）的。 </li><li> 1体积/体积乙腈:14毫升1溶解42.8毫克1-羟基苯并三唑水合物的三唑（HOBt，0.28毫摩尔）通过加热蒸馏水溶液至50℃。 </li><li>添加所有的HOBt解决方案，以解决PAA在室温下。 </li><li> 53.6毫克ethyldimethylaminopropylcarbodiimide的（EDC，0.28毫摩尔）添加到反应混合物中。 </li><li>使用的1M HCl以调节pH至5.5，并过夜搅拌该反应体系在室温下。 </li><li>透析溶液。溶解11.2克氯化钠于2L蒸馏水，然后添加37％0.2毫升w / w的盐酸。利用透析膜与男女 3.5 kDa的的截止解决方案。 </li><li> PERFORM透析三天。每日用2L含有0.2毫升37％w / w的盐酸新制备的蒸馏水的改变透析溶液。 </li><li>储存在-80℃的最终解决方案。根据制造商的协议在冻干机冻干它。 </li><li>通过1 H-NMR谱表征官能化聚合物，在D 2 O中16中的样品溶解。 </li></ol> 4. PAA-RGD的合成聚合物4 <ol><li>溶解78毫克PAA的10毫升蒸馏水改性炔3（1.083毫摩尔）。 </li><li>在5毫升四氢呋喃溶解25毫克RGD叠氮化物2衍生物（0.0722毫摩尔）的。 </li><li>所有RGD溶液添加到聚合物溶液中。 </li><li>添加2.2毫克的碘化铜（0.0116毫摩尔）和2.2毫克抗坏血酸钠（0.0111毫摩尔）的。 </li><li>回流所得混合物过夜，在60℃，同时搅拌。 </li><li>冷却该混合物至25℃。 </li><li> DialyzË解决方案。溶解11.2克氯化钠于2L蒸馏水，然后添加37％0.2毫升w / w的盐酸。利用透析膜与男女 3.5 kDa的的截止解决方案。 </li><li>三天进行透析治疗。每日用2L含有0.2毫升37％w / w的盐酸新制备的蒸馏水的改变透析溶液。 </li><li>储存在-80℃的最终解决方案。根据制造商的协议在冻干机冻干它。 </li><li>通过1 H-NMR谱表征所得产物，在D 2 O中16中的样品溶解。 </li></ol> 5. RGD功能化合成水凝胶<ol><li>准备PBS。溶解645毫克的PBS盐在50毫升的蒸馏水。 </li><li>混合40毫克卡波姆和9毫升PBS（步骤5.1）的10毫克官能PAA 4，在室温下，直至完全溶解（30分钟）。 </li><li> 400毫克的PEG添加到该溶液中，并保持搅拌45分钟。 </li><li>停止搅拌并允许系统沉降30分钟。 </li><li>使用的1N NaOH以调节pH至7.4。 </li><li>到5ml所获得的混合物中，添加25毫克琼脂糖粉末。 </li><li>照射微波辐射系统在500瓦至沸腾，一时间通常为30秒和1分钟之间，并且电磁加热至80℃。 </li><li>离开暴露于室温下将该混合物直到其温度降低至50℃，加入5毫升的PBS（步骤5.1）的，以便获得在一个1的溶液:1体积比。 </li><li>准备12多孔板含钢瓶用直径1.1厘米</li><li>就拿从溶液500微升等分，并将它们放置到每个钢瓶。 </li><li>离开休息45分钟，直至系统完全凝胶化。 </li><li>除去使用不锈钢镊子以获得水凝胶的圆柱体。 </li></ol> 6.装载的治疗手段（药物或细胞） <ol><li>重复ST每股收益5.1-5.7。 </li><li>当混合物（已经在溶胶状态）达到37℃，加入5毫升含有所需药物溶液或细胞培养物的溶液中，以便在一个1以获得最终系统:1的体积比。 </li><li>重复步骤5.9-5.12以获得与凝胶内的物理包埋biocompounds聚合物网络。 </li></ol> 7.水凝胶表征<ol><li> FT-IR分析<ol><li>后形成凝胶，泡的原料合成的水凝胶中的一个在2.5ml蒸馏水中24小时。 </li><li>除去，其中水凝胶浸入水性介质和冷冻干燥液体N 2。 </li><li>根据KBr压片技术层压水凝胶样品。 <ol><li>添加铲满溴化钾到玛瑙研钵。取的水凝胶样品的少量并用溴化钾粉末混合（KBr压量，或者仅仅足以覆盖刮刀的尖端约0.1-2％）。 </li><li>研磨混合物直到粉末细而均匀。&lt;/ LI&gt; <li>使用KBr压片盒，以形成红外沉淀。按使用手动实验室按粉末:在压力容量等于5吨，然后进行3分钟，在10吨压力容量3分钟。 </li><li>释放压力，得到最终的沉淀为均质和透明的外观。将粒料进红外样本保持器，并运行的频谱16。 </li></ol></li></ol></li><li>凝胶研究<ol><li>填充2ml微量管900微升的PBS并平衡至37℃。 </li><li>添加100微升制备的聚合物溶液，以形成水凝胶，并在37℃下孵育。 </li><li>倒置该管，并且如果凝胶在1,2，5，10和20分钟流向观察。记录在其中凝胶不流动的凝胶化的时间。 </li></ol></li></ol>

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The authors state no conflict of interest and they have not received any payment in preparation of this manuscript.

The PAA post-polymerization modification with alkyne moieties and the RGD functionalization with the azide group guarantee the formation of a stable bond between the polymer and the peptide. Indeed, triazole serves as a rigid linking unit among the carbon atoms, attached to the 1,4 positions of the 1,2,3-triazole ring and it cannot be cleaved hydrolytically or otherwise. In addition, triazole is extremely difficult to oxidize and reduce, unlike other cyclic structures such as benzenoids and related aromatic heterocycles24. In this way, the chemical lability of PAA-RGD product is almost absent under the experimental conditions of hydrogel synthesis. The peptide is grafted to the 3D polymeric network due to the nature of its chemical bond and it is not physically entrapped (therefore, it could leave the system).
The alkyne PAA is prepared using propargylamine in an aqueous solution, in the presence of EDC and HOBt, to obtain the amine derivative. Experimental results indicate that the percentage of triple bond linking to the PAA is proportional to the amount of amine used. Referring to the NMR analysis, the polymer functionalization degree is between 90% and 100% when the molar ratio between the reagents PAA and propargylamine is 1:1 and is about 50% when the ratio is 1:0.5. Due to the fact that the formation of the gel network involves an esterification reaction, it is necessary to maintain some PAA carboxyl groups capable of reacting with hydroxyl groups of PEG and agarose. The modification of half PAA carboxyl groups is adequate for this, but hydrogels with improved physicochemical properties are obtained when the polymer functionalization degree is equal to 10% (molecular ratio PAA:propargylamine 1:0.1)25. The purification of the alkyne PAA is performed by dialysis, using a dialysis membrane with molecular weight cut-off (MWCO) of 3.5 kDa, allowing the removal of by-products and unreacted species. The product is finally isolated using lyophilization. 
RGD is characterized by the amino terminal group -NH2, which can be used as a coupling point to link the spacer-N3. The linkage between these compounds is obtained via an amide bond that is very stable and not too difficult to form16. In order to proceed in this way, 4-azidobutanoyl chloride is prepared as the linker of the spacer-N3 system. 
The alkyne moieties react with the RGD-azide derivative, through a click reaction, using copper iodide as a catalyst and sodium ascorbate as an antioxidant agent due to the possible oxidation of copper (Cu+ &#8594; Cu2+) in experimental atmospheric conditions. After screening different solvents and temperatures, the best results are performed by using a tetrahydrofuran/water mixture at 60 &#176;C16. According to the biological application, the post-synthesis retention of copper poses potential toxicity risks, especially for applications where the cells must remain viable. As a solution, dialysis is used to remove the copper catalyst and other by-products, obtaining a purified PAA-RGD compound suitable for hydrogel synthesis without any possible side effects. Click chemistry ensures a quantitative yield of the polymeric product grafting RGD. 
Due to the same reactivity of each PAA carboxyl group, the polymer functionalization is statistically evaluated. The exact number of modified carboxyl groups per PAA chain cannot be established and the reported functionalization degree is referred as the total number of carboxyl groups that have reacted with propargylamine. This estimation affects the subsequent assessment of the RGD functionalization and its effective amount loaded into each hydrogel. In addition, GPC analysis is not performed because in this work, the polymer is used and the protocol does not begin from monomers. The percentage of RGD functionalization is commonly verified with 1H-NMR and it is not possible to obtain reliable data from HPLC analysis. However, considering that each step of this work is replicated many times and the same experimental values and results are recorded, the illustrated results are reliable.
Another limitation is the use of copper as the catalyst as previously reported. The possible interaction of the copper with the PAA carboxyl groups during the click reaction and its subsequent presence as an entrapped molecule in the hydrogel network would reduce the biocompatibility of the system and would generate toxic effects in cell cultures. The three-day dialysis minimizes this potential limitation of the technique.
The functionalized PAA is used as a subunit in hydrogel formation, with carbomer, PEG and agarose. Carbomer and agarose are chosen because of their well-known biocompatibility in therapeutic approaches and tissue engineering. Carbomer also has anti-inflammatory properties while agarose is a suitable polymer for tissue repair strategies and provides a good environment for cell adhesion, spreading, proliferation, and protein drugs. Carbomer is a cross-linked poly-acrylic acid containing carboxyl groups (65%) that make it an ionizable molecule. The molecular weight is 106 Da. Its use is fundamental for gel synthesis due to its high number of reactive carboxyl groups. However, at the same time, it is very difficult to estimate a quantitative yield of any possible chemical functionalization because of its complex structure. For this reason, PAA is used due to its lower molecular weight (105 Da) and its linear structure. The combination of carbomer and PAA allows the design of gel networks with a RGD moiety that, otherwise, would not be viable. PEG is also introduced into the gel network due to its high biocompatibility, its ability to increase stem cell adhesion, and its high elasticity (being a small polymer) that can create elastic junctions between the carbomer and the agarose to form local interconnections through their own hydroxyl groups26. It also has a physical effect by increasing system viscosity and thus enhancing tight macromer cross-linking.
The use of the four polymers leads to a 3D network that is highly biocompatible, injectable and capable of maintaining its biochemical functionality with improved cell adhesion due to the presence of RGD. The latter is decisive in generating a suitable cell microenvironment within the hydrogel that otherwise would not give a satisfactory result27.
The hydrogel network can be consider as a "carbon skeleton" of macromolecules forming a stable structure comprised mostly of C-C bonds, where the cross-linking points are C-O bonds formed by esterification between carboxyl and hydroxyl groups. The microwave irradiation is a key point to ensure the agarose solubility in the aqueous reagent system and to avoid any further purification in the synthesis of sterile hydrogels via the sol/gel transition, because any initiators or monomers can be used and by-products are not formed. The microwave-assisted reaction also guarantees a high efficiency, and the functionalization degree of polymer chains with bioactive compounds or drugs generally does not affect the morphology, the structure, and the physical properties of the networks. These hydrogels are able to form in situ due to their ability to easily conform to any shape and be injected using minimally invasive surgical procedures. This is a significant advantage compared to the use of rigid and preformed scaffolds.
The synthesized networks can be used as an enhanced cell survival biomaterial, thanks to its high biocompatibility and the ability to create a hospitable cell microenvironment27. The improvement of this work is the ability to design a three-dimensional structure where the RGD is chemically linked through a stable and strong bond that is not cleavable in physiological conditions, thus avoiding RGD diffusion through the hydrogel pores to maintain its therapeutic effect at a high level for a long and sustained period of time. Alternative synthesis of chemical linkers, such as the ester or the hydrazone bonds, do not allow the same effect towards cells because they are cleavable, or pH/temperature sensitive under the cell microenvironment21,28,29. In addition, the tripeptide release is mostly driven by a pure diffusion mechanism that is very quick due to its small steric hindrance. The alternatives to modifications of the physical and mechanical parameters of the hydrogel network, for example reducing pores size, cannot be enough to maintain an adequate concentration of RGD within the matrix.
A possible additional functionalization is the use of fibronectin, which can optimize the hydrogel compatibility with the extracellular matrix in order to make viable living cells. One of the most important aspects is maintaining cell viability within hydrogel networks for days. In this way, cells can leave the matrix able to proliferate and differentiate in vivo. The first experimental results are published27. This highlights the comparison between RGD-functionalized hydrogels and non-functionalized hydrogels showing promising results. In particular, the RGD in the gel network improves stem cell survival and guarantees the immunomodulation of the pro-inflammatory and pro-regenerative effect at the lesion site in vivo. The presence of RGD linked to the hydrogel is a crucial point to achieve suitable therapeutic results. The RGD maintains its availability for cell interactions and it does not escape rapidly through hydrogel pores, maintaining its bioactive impact.

水凝胶是由亲水性的交联的聚合物，它们是天然或合成的，并且其特征在于，一个独特的三维结构形成的三维网络。这些设备是在药物递送，组织工程，基因的载体和智能传感器1,2的生物医学领域越来越有吸引力。事实上，它们的高水含量，以及它们的流变学和机械性能使之适合候选模拟软组织的微环境，使它们有效工具水溶性细胞因子或生长因子的递送。其中最有希望采用的是作为可注射生物材料携带细胞和生物活性化合物。水凝胶可以通过保持并在生理相关的方式精确地递送干细胞调节信号提高细胞存活和控制干细胞的命运，如在体外和体内实验3,4-观察。这样做的主要优点是有可能保持接种（原位 ）的区域内注射的细胞，减少细胞的叶面积和extravasates进入循环系统洪流，迁移遍布全身，失去靶目标5的量。三维水凝胶网络的稳定性是由于其交联位点，由聚合物链6之间的共价键或内聚力形成。 在此框架下，正交选择的化学品适用于聚合物链能够提高水凝胶演出7的多功能工具。事实上，用适当的化学基团的聚合物的改性可以帮助提供适当的化学，物理和机械性能，以提高细胞生存力以及它们在组织形成使用。以相同的方式，这些技术中以加载细胞或生长因子的凝胶基质中，使用了含RGD肽允许在细胞粘附和存活的改善。 RGD是由三肽精氨酸，甘氨酸和天冬氨酸，这是迄今为止的最有效和最经常使用的三肽，由于其处理多于一个细胞粘附受体的能力和对细胞锚定，行为和生存8,9-其生物学影响。在这项工作中，RGD官能水凝胶的合成进行了研究与设计网络的一个热情细胞微环境特征在于足够的生化特性的目的。 在水凝胶的合成使用微波辐射提供了一个简单的过程，以减少副反应，并在比常规热过程10更短的时间周期得到较高的反应速率和产率。此方法不需要纯化步骤和产量的无菌水凝胶由于聚合物之间的相互作用，并在反应系统11不存在有机溶剂。因此，它可以确保链接到聚合物网络，因为没有模RGD的高百分比ifications需要参与凝胶形成的聚合物的化学基团。羧基，由PAA与卡波姆和羟基，由PEG和琼脂糖，通过缩聚反应产生的水凝胶三维结构。所提到的聚合物可用于水凝胶在脊髓损伤的修复治疗12的合成。这些设备，如报告以前的作品13,14，显示出高生物相容性，以及类似于那些生活的许多组织和触变性质的机械和物理化学性质。此外，它们在原位保持局限性，在注射区域。 在这项工作中，PAA的羧基与炔部分修改（ 图1），和一个RGD叠氮化物化合物合成用的结构（CH 2）N利用该三肽末端基团-NH 2的反应与制备化学化合物- N 3（&lt;STRONG&gt;图2）。随后，将改性的PAA通过CuAAC点击反应15-17（ 图3）的RGD-叠氮化物衍生物进行反应。使用铜（I）催化剂的导致在这两个反应速率和区域选择性的重大改进。该CuAAC反应被广泛用于有机合成和在聚合物科学。它结合了高效率和高耐受性的官能团，并且它是通过使用有机溶剂的影响。高选择性，快速的反应时间和简单的纯化过程允许星形聚合物，嵌段共聚物或链接枝所需部分18的获得。此点击策略使得能够修改聚合物聚合后根据最后的生化应用定制的物理化学性质。所述CuAAC实验条件容易再现（反应不敏感，水，而可以最低限度发生铜氧化），和的性质形成三唑确保了产品的稳定性。使用铜金属可以被认为是一个临界点时，由于对细胞的潜在毒性作用，并在生物微环境，但透析用作纯化方法，以允许完全除去催化剂残留物的。最后，PAA改性RGD在水凝胶的合成（ 图4），将所得网络的物理化学性质使用进行了研究，以检查这些系统如细胞或药物载体的潜在功能。 <img alt="图1" src="/files/ftp_upload/54445/54445fig1.jpg" /> 图1:PAA改性炔合成炔基官能PAA的方案。 "N"表示与羧基与炔丙基反应的单体。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/54445/54445fig2.jpg" /> 图2:。RGD- 叠氮合成 RGD-叠氮衍生物的合成<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54445/54445fig3.jpg" /> 图3:点击反应 RGD-叠氮衍生物和炔PAA之间的点击反应方案。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54445/54445fig4.jpg" /> 图4:水凝胶SYNThesis。RGD功能化水凝胶合成过程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="67">
			<td height="67" style="height:67px;width:183px;">Poly(acrylic acid) solution average Mw ~ 100,000, 35 wt % in H2O</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:87px;">523925</td>
			<td style="width:145px;">CAS 9003-01-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) 2,000</td>
			<td>Sigma Aldrich</td>
			<td>84797</td>
			<td>CAS 25322-68-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbomeer 974P</td>
			<td>Fagron</td>
			<td>1387083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td>Invitrogen Corp.</td>
			<td style="width:87px;">16500-500</td>
			<td>UltraPure Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RGD peptide</td>
			<td style="width:108px;">abcam</td>
			<td>ab142698</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">4-azidobutanoic acid</td>
			<td style="width:108px;">Aurum Pharmatech</td>
			<td style="width:87px;">Z-2421</td>
			<td>&#160;CAS 54447-68-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxalyl chloride</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td>O8801</td>
			<td>CAS 79-37-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Propargylamine hydrochloride 95%</td>
			<td>Sigma Aldrich</td>
			<td>P50919</td>
			<td>CAS 15430-52-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Copper(I) iodide</td>
			<td>Sigma Aldrich</td>
			<td>3140</td>
			<td>CAS 7681-65-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>Y0000039</td>
			<td>CAS 134-03-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Dialysis Membrane</td>
			<td style="width:108px;">Spectrum Laboratories, Inc.</td>
			<td>132725</td>
			<td style="width:145px;">Spectra/Por 3 Dialysis Membrane&#160; Standard RC Tubing&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MWCO: 3,5 kD</td>
		</tr>
	</tbody>
</table>

the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

的PAA炔衍生物有效地从聚丙烯酸和炔丙胺合成，如在图1，其中n标签的羧基与胺反应的单体显示。该产物的身份通过1 H-NMR光谱法证实。 图5示出了具有三键改性的PAA的1 H-NMR谱。 <img alt="图5" src="/files/ftp_upload/54445/54445fig5.jpg" /> 图5:1 的PAA的H-NMR谱修改炔相关炔部分的信号被突出显示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 聚合物链的信号可以在范围2.75-1.50 ppm的观察;而在2.8的峰值<html> 0ppm的，有代表性的炔之H，并在4.20 ppm的峰，关系到-CH 2的2H，表征炔丙基部分。这证实了PAA已经被适当修改。炔官能化程度的评价已被下PAA峰积分的面积进行了（设置为3.00，根据每单体的氢的数目）和炔丙基结构部分，如在图5中示出。官能f的程度是计算公式如下: <img alt="方程" src="/files/ftp_upload/54445/54445eq1.jpg" /><img alt="方程" src="/files/ftp_upload/54445/54445eq2.jpg" />代表炔丙基残基的积分面积，炔的H区的总和（标记为<img alt="方程" src="/files/ftp_upload/54445/54445eq3.jpg" /> ）及-CH 2区域（表示为<img alt="方程" src="/files/ftp_upload/54445/54445eq4.jpg" /> ），而离子"SRC ="/文件/ ftp_upload / 54445 / 54445eq5.jpg"/&gt;指的是聚合物的信号的积分面积。官能度被计算为10％，它按水凝胶的合成，其中是考虑令人满意PAA具有穿过其剩余的羧基反应以形成三维网络。对于改性聚合物16得到定量产率。 以类似的方式， 图6示出了炔改性的PAA和RGD叠氮化物之间的CuAAC点击反应后的产物的1 H-NMR谱。在8.15 ppm的形成三唑的峰证实，发生在定量产率的反应和RGD强烈链接到PAA链。 图6示出所有的PAA链和RGD的特征信号。 <img alt="图6" src="/files/ftp_upload/54445/54445fig6.jpg" /> 图6:含RGD的 1 H-NMR谱 挂PAA。三唑的信号表示（标记为"A"）。执行通过CuAAC点击反应RGD聚合物功能化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> RGD官能水凝胶是通过四个聚合物（PAA，卡波姆，琼脂糖和PEG）通过微波辅助自由基聚合的化学交联来制备。加热到80℃导致较高的大分子的移动性，并因此提高了聚合物的羧基和羟基之间的短距离的互连。酯化反应将这些官能团之间发生并产生本地网络被称为"微凝胶"。 作为缩聚的进行，连续系统的粘度增加，WHILE相互作用的大分子活性位点之间的概率减小。然而，更近的官能团仍有效地进行交互，由于较慢的流动性。所得的物理化学条件的特征在于产生水凝胶的最终三维宏观微粒凝胶表面之间的"焊接"。酯化，氢键和羧化带来聚合物链具有统计学接近，因此形成一个稳定的非均相结构。由此产生的系统表现出的溶胶/凝胶行为，并过渡到凝胶状态5分钟之内。该时间间隔记录为凝胶时间。 含RGD官能水凝胶的化学性质进行了研究使用FT-IR分析。 图7示出了RGD叠氮化物化合物的红外光谱（绿线），无RGD官能（黑线）中合成的水凝胶之间的比较，以及用肽修饰（蓝线）的水凝胶。水凝胶规范TRA都特点是在3,600-3,200厘米宽信号-1范围，代表性的残余OH键的伸缩振动，并通过围绕2940厘米-1 CH拉伸的峰。该酯化羧基和羟基的聚合物组之间发生的验证是由峰约1600 -1 1400 -1给出，分别对应，对对称和非对称伸缩的 CO 2部分的。这些峰中的非官能化的水凝胶的频谱更为明显，而在RGD-凝胶光谱它们部分地被表示为酰胺频带I和II的信号覆盖。 <img alt="图7" src="/files/ftp_upload/54445/54445fig7.jpg" /> 图7:FT-IR光谱比较 RGD（绿线）的FT-IR光谱，水凝胶不RGD功能化（黑线）和RGD功能化水凝胶（蓝线）。该有关酰胺RGD指示信号。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> C的拉伸= O，标记为酰胺频带I（"酰胺I"，在图7中 ），提供于1650 -1的峰的三肽谱和它移位到约1670厘米-1的RGD-水凝胶样品在。的NH的弯曲，（ 图7中"酰胺Ⅱ"）与酰胺频带II，可以与信号记录周围1550厘米-1在RGD光谱，它也是将水凝胶样品中可识别的，在约1600厘米3 - 1。因为有在标准的水凝胶制剂没有酰胺组件，一个酰胺基性质的峰的存在表明PAA真的官能与含RGD和它能够形成与聚合物网络内肽位点的水凝胶。 水凝胶的FT-IR光谱也显示了琼脂糖的单糖单元和酯基之间的相关糖苷键的COC的伸缩振动的峰（900-1,000 -1范围内）。 以获得洞察三维结构和这些水凝胶的物理和机械性能，SEM分析，凝胶化，溶胀动力学和流变研究被执行，如在先前的工作13,20讨论。 SEM分析（ 图8）表明，水凝胶的特征是复杂的微观结构与包含小孔隙孔壁上一些较大的孔和一些纤维状的网络。此外，大多数的孔隙互连。缠结结构类似于以相同的方式，但没有RGD官能制备水凝胶的三维网络。这表明，RGD不改变聚合物网络。使用倒置试管试验中，水凝胶小号在5分钟内充分凝固，如在水凝胶样品中观察到的无RGD官能21。这个简短的凝胶时间强调其生物医学应用的适用性。 <img alt="图8" src="/files/ftp_upload/54445/54445fig8.jpg" /> 图8:。SEM分析 SEM图像显示了RGD功能化的凝胶样品（A）和无功能化（B）水凝胶的形态<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54445/54445fig8large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 溶胀平衡比表示吸收和保留大量的水的能力，这是水凝胶系统20,22的前端的功能之一。分析样品表现出快速膨胀动力学和他们到达的第一个小时内膨胀平衡。他们的膨胀ING均衡值Q被报道在我们以前的工作16，它是类似于无RGD通过水凝胶的分析而得到的值，在确认三肽集成与聚合物网络，并且不产生高妨碍凝胶化过程。 与流变研究，凝胶储能模量（G&#39;）被发现是大约一比损耗模量（G&#39;&#39;）更高量级，表示弹性，而不是粘性材料23和两个基本上独立于频率。 G&#39;和G&#39;&#39;类似的值记录没有修改肽16凝胶样本。这表明，RGD的聚合物网络内的存在不影响所述材料的流变性能，并保持特有特征争相注射系统用于生物医学应用。

Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

the-synthesis-rgd-functionalized-hydrogels-as-tool-for-therapeutic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.1K 次观看.  Politecnico di Milano. We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated. 

视频: The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

科研

生物工程

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...