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

1. DOX-PA的合成<ol><li>称量390毫克的阿霉素和243毫克棕榈酸酰肼，并转移到一个圆底烧瓶中。 </li><li>添加150毫升无水甲醇中，用玻璃注射器烧瓶中。添加39微升三氟乙酸（TFA）的用移液管。用磁力搅拌器，搅拌在RT在黑暗中18小时，将反应混合物。 注意:反应材料的量可被放大或缩小，以获得不同量的DOX的功率放大器。反应物的比例应在相同的比例被维持。使用DOX量在78毫克至1,170毫克的范围内的反应可在常规的化学实验室中进行。 </li><li>用硅胶柱DOX-PA的纯化。17 <ol><li>用旋转蒸发器除去在反应混合物中的溶剂。加入3克硅胶的混合物的体积减少到大约20之后ml。继续旋转蒸发，得到干的粉末，并允许将T他吸附产品走上硅胶。保持在真空下将样品另外的30分钟，形成了干燥粉末后。 </li><li>包50克硅胶成使用二氯甲烷作为溶剂的柱。小心加入含吸附水的产品到柱硅胶样本。 </li><li>洗脱用二氯甲烷和甲醇的混合物的塔，同时逐渐增加甲醇的比例，从而增加溶剂极性（ 表1）。 </li><li>收集在试管洗脱液级分（25毫升/管）并监测通过薄层色谱（TLC）的进展。 </li><li>结合含有纯DOX-PA的所有级分，并用旋转蒸发器，直至形成干粉除去溶剂。进一步干燥真空O / N下的产物。 </li></ol></li><li>分析DOX-PA通过TLC的。 <ol><li>切TLC板的4cm的×8cm的部分。从在用TLC点样毛细管板的底部斑点样品溶液0.5厘米使用满足hanol作为溶剂。 </li><li>放置TLC板到含有二氯甲烷和甲醇的混合物中的显影腔室（3/1，体积/体积）。的溶剂中的深度应只是比0.5厘米以下。 </li><li>从显影腔移除所述板当溶剂前沿到达板的顶部。标记溶剂前沿用铅笔的位置，并允许所述板干燥。放置TLC板到含有以显现样品饱和碘蒸气染色室中。 </li></ol></li><li> 1 H-核磁共振光谱（1H-NMR）DOX-PA分析。18 <ol><li>溶解15mg的DOX的PA在1ml甲基亚砜-D6（DMSO）中的和样品转移到一个NMR管中。 </li><li>将NMR管进入核磁共振仪器的磁铁。测量质子光谱，选择DMSO作为溶剂。从磁体取出NMR管。分析核磁共振结果18。 </li></ol></li></ol> 2。 DOX-PA胶束由薄膜分散法制备<ol><li>溶解DSPE-PEG（40毫克）和DOX-PA（4毫克）在10毫升玻璃小瓶2ml甲醇。 </li><li>使用旋转蒸发，直到在小瓶的薄膜形成真空下除去有机溶剂。 注意:可替换地，蒸发的有机溶剂在惰性气体下（ 例如 ，氩气或氮气），以形成薄膜，并保持小瓶在真空干燥器以进一步除去残留的溶剂。 </li><li>转印2毫升Dulbecco氏磷酸盐缓冲盐水（pH值7.4，DPBS）的玻璃小瓶中。 </li><li>放置在小瓶在超声浴中3分钟在室温下，以产生胶束。 注:超声波浴的不同型号之间的超声波功率变化。选择能产生足够的超声功率驱散薄的聚合物/药物薄膜的单位。在这个协议中使用超声波浴的输出功率为110 W. </li><li>保持胶束在4℃下短期储存和-20℃下长期-term存储。 注意:可替换地，微胶粒还可以是冷冻干燥并在使用前用水重构。通常情况下，需要对这一提法没有冷冻保护剂或冻干保护剂。 </li></ol> 3. DOX-PA胶束的表征<ol><li> DOX-PA浓度的胶束和药物包封率的测定 <ol><li> 1微克/毫升，5微克/毫升，20微克/毫升，50微克/毫升和100微克/毫升:溶解DOX-PA在DMSO中前面的步骤，制备了五种不同浓度的DOX-PA的溶液合成。测量DOX的PA解决方案的吸收与在490nm处的UV-VIS光谱仪。基于所述DOX-PA的药物浓度和在490nm处的相应吸收率的标准曲线。 </li><li>稀释25微升载药胶束的500微升DMSO的。测量在490nm处用UV-VIS分光计的吸收。计算药物浓度与3.1.1中产生的标准曲线。 </li><li>使用以下等式计算包封效率: 药物包封率（％）=（在胶束药物量）/（加入的药物量）×100％ </li></ol></li><li> 粒径用动态光散射表征（DLS） <ol><li>稀释用DPBS（pH 7.4）中的胶束为1毫克/毫升的最终的DSPE-PEG浓度。分析2ml样品用粒度分析仪得到的Z均尺寸和多分散指数（PDI）。 </li></ol></li><li> 体外抗癌活性的评价 注意:使用适当的无菌技术和生物安全柜的内部运作。 <ol><li>从细胞培养烧瓶中取出细胞培养基（ 例如，T25）含有DU-145人前列腺癌细胞并用2ml的DPBS（pH7.4）中冲洗细胞。 </li><li>抽吸DPBS，加入1毫升胰蛋白酶溶液（0.25％），并在37℃孵育2分钟以分离细胞。 </li><li>添加10米细胞培养基的L（RPMI 1640 + 10％胎牛血清+ 1％抗菌 - 抗霉菌），当大多数细胞从烧瓶脱落。转移细胞到15毫升离心管中并离心将细胞以1000 xg离心5分钟。 </li><li>重悬用5ml细胞培养基的细胞沉淀并除去的样​​品用于与血球计数细胞数。稀释用细胞培养基将细胞以50,000细胞/ ml的密度。添加稀释的细胞悬液到96孔细胞培养板（100μl/孔）。孵育在细胞培养孵化器中的细胞（37℃，5％CO 2）18 ​​小时，以允许细胞附着。 </li><li>稀DOX二甲基亚砜（DMSO）溶液和DOX-PA DMSO溶液用细胞培养基分别以获得0.1微米的最终药物浓度，0.5μM，2微米，5微米和10微米。保持最终DMSO浓度以上所有的样品中至0.5％。稀DOX-PA胶束用细胞培养基以得到最终的药物共0.1μMncentrations，分别为0.5微米，2微米，5微米和10微米。使用空白细胞培养液控制。 </li><li>从培养箱中取出96孔细胞培养板，并用100微升培养基含有在步骤3.3.5制备（n = 4时为每个组）不同处理剂更换细胞培养基。孵育在细胞培养孵化器中的细胞（37℃，5％CO 2）的额外72小时。 </li><li>吸出培养基并加入100μl的培养基中含有0.5mg / ml的3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑鎓溴化物（MTT）。 </li><li>孵育额外的2小时的细胞培养孵化细胞。小心取出介质，并添加100微升DMSO来溶解甲晶体。 </li><li>在570纳米的波长和670nm的参考波长测定用微孔板分光光度计吸光度。 </li><li>使用下列公式计算细胞存活率: <strong&gt;的细胞活力（％）=（A 测试 / 控制 ）×100％ 注意:使用方差分析（ANOVA）统计检验的单向分析比较不同组之间的细胞生存力。基于细胞生存力相对于药物浓度的数据计算IC 50。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项工作中，我们描述了微胶粒的制备的简单，快速的膜分散法。这种方法利用一个两亲性聚合物的自组装特性（ 如 DSPE-PEG）以形成在含水环境中的核-壳结构的胶束。此胶束制备方法具有几个优点。 1.它涉及一个简单配制方法，它避免了使用脂质体，纳米颗粒和纳米乳剂。19 2的制备常用复杂小型化步骤（如挤出或均化）。它具有良好的重复性。一旦该制剂进行了优化，并建立?...

化疗通常用于治疗各种形式的癌症。大多数，如果不是全部，化疗药物具有毒性副作用可以从管理次要条件，如恶心和腹泻而变化，更危及生命的状况。因为大多数抗癌药物的毒性，这些药物非选择性暴露于正常组织不可避免地会导致毒性。因此，为治疗性的方法，可以选择性地递送药物进入肿瘤细胞非常需要。与抗肿瘤药物的管理的另一个挑战是他们的水溶性差。一般，需要增溶剂来配制这些难溶性药物。然而，大多数的增溶剂，如二甲基亚砜（DMSO），聚氧乙烯蓖麻油，和聚山梨醇酯80（吐温80）可能引起肝，肾毒性，溶血，急性过敏反应和周围神经病变。1因此，需要对安全性和生物相容性制剂临床使用的差LY水溶性抗癌药物。纳米载体是有希望的药物递送系统用于解决上述挑战。这些纳米载体包括脂质体， 纳米粒子，3胶束，4-7聚合物药物结合物，8和无机材料。9一些纳米产品（ 如 DOXIL，Abraxane的，和Genexol）已通过监管机构来治疗癌症病人。 10 聚合物胶束是有希望的纳米级药物递送载体，其中已被成功地用于抗癌药物的输送。通过自组装过程从两亲聚合物制备4-7,11,12典型聚合物胶束。芯 - 壳结构的聚合物胶束包括亲水​​壳和疏水核心。亲水壳可以在空间上稳定胶束并延长其在血液中循环。疏水核可以有效地封装疏水ð地毯。因为胶束（通常小于200nm）和长循环特性的小尺寸的，聚合物胶束被认为达到肿瘤通过增强的渗透性和保留（EPR）效果（被动靶向肿瘤）定位。 载药量稳定对肿瘤靶向胶束的能力至关重要。以达到最佳的肿瘤定位，微胶粒应具有最小的药物泄漏在到达肿瘤部位之前，尚未进入癌细胞后迅速释放药物。此外，制剂稳定性也是产品开发的一项基本要求，因为制剂稳定性决定产品开发的可行性，以及开发的产品的保质期。最近，许多努力已经取得了改善的药物装载到递送载体。所述亲脂性前药的方法是已被探索，以改善药物加载到脂质纳米颗粒和乳剂的策略。13,14的连词用药物脂质ugation可以显著改善他们的亲脂性，提高装载和固定在纳米载体的亲脂成分。 在这里，我们描述了准备亲脂阿霉素前药胶束的协议。首先，对于阿霉素亲脂性前药的合成方法进行说明。然后，被引入用于产生与一个薄膜分散法胶束的协议。这种方法已在我们以前的研究已成功地用于5 DSPE-PEG，被选定为，因为它已被成功地用于胶束药物递送制备胶束载体材料15,16最后，我们描述了用于表征胶束几个体外测定制剂，并评价抗癌活性。

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			<td style="width:119px;">158990050</td>
			<td>98%</td>
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			<td style="width:191px;">Varian, Inc</td>
			<td style="width:119px;"></td>
			<td>300 NMR&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
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			<td style="width:119px;">CO2 6000</td>
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			<td height="21" style="height:21px;width:216px;">Biological Safety Cabinet</td>
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			<td style="width:191px;">Becton Dickinson</td>
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			<td height="21" style="height:21px;width:216px;">Trypsin&#160; 0.25%</td>
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			<td style="width:119px;">25-053-CI</td>
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preparation and characterization of lipophilic doxorubicin pro-drug micelles

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

图1示出DOX的PA的合成方案。 DOX-PA用棕榈酸的偶联通过pH敏感腙键阿霉素合成。稍微过量的棕榈酸酰肼的使用，以促进该反应的完成。此反应方法具有非常高的效率，仅阿霉素的少量残留的18小时的反应（ 图2）之后。产率是大约88％。在反应结束时，DOX-PA用硅胶柱纯化，干燥，得到纯的红色固体产物。 DOX-PA用薄层色谱法，这表明对纯化的DOX的PA（ <...

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

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

一个亲脂性的阿霉素前体药物的制备和表征协议加载1,2- distearoyl- SN -glycero -3- phosphoethanolamine- N - [氨基（聚乙二醇）-2000]（DSPE-PEG）胶束描述。

制备和表征亲脂阿霉素前药胶束

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

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K 次观看.  Hampton University. 一个亲脂性的阿霉素前体药物的制备和表征协议加载1,2- distearoyl- SN -glycero -3- phosphoethanolamine- N - [氨基（聚乙二醇）-2000]（DSPE-PEG）胶束描述。 

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

Construction and Characterization of a Novel Vocal Fold Bioreactor

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

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

一种新型声带生物反应器的构建与鉴定

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

科研

生物工程

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

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

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

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

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

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

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

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

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

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

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

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

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

含有宏观结构的光学组织幻影的制备与表征

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

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

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

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

含脂肪酸脂质体的制备、纯化及应用

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

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

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

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

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

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

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

Growth and Characterization of Irradiated Organoids from Mammary Glands

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

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

乳腺辐照有机体的生长与表征

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

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

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

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

生物活性亲水球状蛋白的纳米脂质体制备与表征

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

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

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

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

聚焦 Ion 光束平版印刷到蚀刻纳米架构到微电极

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

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

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

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

功能性与极化胆囊肿的生成与定量特征

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