这项工作得到了浙江树人大学研究项目 （2023R053 和 2023KJ237） 的支持。

实验中使用的血液来自体重 20-25 g、大约 7 周龄的 SPF 级 BALB/c 雌性小鼠。浙江树仁学院动物实验伦理委员会批准了所有动物护理和实验程序。
1. 溶液制备
<ol><li>称取 ALG 并在 50 °C 的水浴中搅拌，溶于超纯水中，得到 2% ALG 溶液。</li>
	<li>在超纯水中分别制备含有 5%（或摩尔质量浓度为 0.3 M）的 CaCl2、FeCl3、ZnSO4 和 CuSO4 溶液的质量分数。将每种溶液分别倒入收集皿中。</li>
</ol>2. 微流体电喷雾装置
<ol><li>取玻璃毛细管，将其放在喷灯火焰上软化，然后将其拉伸成各种直径 （50 μm-500 μm）。用玻璃切割机切掉精细部分，然后用优质碳化硅砂纸轻轻抛光喷嘴端口。清洁后，将毛细管的粗端连接到长管上，将管子的另一端连接到分配针和 5 mL 注射器（补充图 1）。 注意:玻璃管切口的锋利边缘很难插入软管;因此，在火焰边缘处，中断应呈圆角。</li>
	<li>将注射器连接到微流体注射泵上，并将毛细管的一端连接到支架上。将高压电源的红色高压端夹连接到注射器的分配针头上，将银色夹子放入收集液中。这种配置创建了一个简单的微流体电喷雾装置（图 1A）。 注意:将毛细管垂直于工作台放置。</li>
</ol>3. ALG 微球制备
<ol><li>将含有不同金属离子的单独收集皿直接放在毛细管下方。打开微流体注射泵并选择 Fast Forward 以使用 ALG 灌注管，然后设置适当的流速 （2 mL/h）。</li>
	<li>打开高压电源开关并转动旋钮以设置合适的电压 （5-7 kV）。</li>
	<li>向每个 90 mm 培养皿中加入 20 mL 溶液，以收集 ALG 微球以形成与不同金属离子交联的 ALGMS（图 1B）。</li>
	<li>ALG 在电场和重力的作用下，在几秒钟内在不同的离子收集溶液中形成微球。用消毒的移液管吸出微球，并将其转移到单独的 1.5 mL 离心管中，以获得 Zn2+、Ca2+、Cu2+和 Fe3+ -ALG 水凝胶微球（图 1C）。标记 Zn-ALGMS、Ca-ALGMS、Cu-ALGMS 和 Fe-ALGMS 管。 注:通过调整电压、针尖直径、流速和溶液浓度等参数，可以获得不同直径的微球。</li>
	<li>取少量制备好的微球，在 PBS 中尽可能均匀地分散，并将它们置于显微镜下进行观察。</li>
	<li>在同一批次的微球中随机选取 10 个观察场，将需要测量的图像导入 ImageJ 软件，将图像转换为合适的灰度模式，并使用阈值分割技术准确定义目标颗粒的面积并启动分析颗粒的功能，以便 ImageJ 能够识别和测量目标颗粒。获取微球的数据，然后将数据导入 Origin 软件进行分析和粒度映射。</li>
</ol>4. 抗菌性能测试
<ol><li>使用液体 Luria-Bertani （LB） 培养基制备 5 mL 初始细菌浊度为 0.2 mcF 的 金黄色葡萄球 菌（金黄色葡萄球菌）和 大肠杆菌 （大肠杆菌）悬浮液。</li>
	<li>将 10 mL 每种细菌溶液添加到 4 个 15 mL 无菌离心管中。然后，向每个试管中加入 0.5 mL 的 Zn-ALGMS、Ca-ALGMS、Cu-ALGMS 和 Fe-ALGMS ALG，并将它们置于 37 °C 和 200 rpm 的恒温振荡器中 12 小时。向对照组中加入等量的盐水。</li>
	<li>用无菌水将每种细菌溶液稀释至 105 倍。使用无菌玻璃珠将 200 μL 细菌溶液铺布在 LB 固体培养基上。</li>
	<li>将溶液快速均匀地铺在包衣板上，避免在同一区域来回涂抹。然后，将板置于 37 °C 的培养箱中 12 小时。观察不同群体的菌落。 注意:所有无菌程序都应在酒精灯附近进行。对微生物培养容器、接种器具和培养基进行消毒。</li>
</ol>5. 药物释放测试
<ol><li>为了评估不同微球的药物释放能力，使用牛血清白蛋白 （BSA） 作为负载模型药物。将 500 μL 的每个微球（Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS）添加到 1 mg/mL BSA 悬浮液中 24 小时。</li>
	<li>对于药物释放测定，将每个饱和样品加入 5.0 M 磷酸盐缓冲盐水 （PBS;pH 7.4） 中，然后在 37 °C 下搅拌 15 分钟，并以 80 rpm 的速度连续振荡。</li>
	<li>使用 100 μL 溶液，并在 1、3、6、12、24、36 和 48 小时用新鲜的培养基更换培养基。</li>
	<li>按照制造商的说明，使用 BCA 试剂盒在特定时间定量上清液蛋白浓度。经酶标定量测定释放的药物，得到相应的吸光度，并按标准曲线将实测值转换为实际药物浓度。使用以下公式计算药物释放速率: 药物释放百分比 = （特定时间点释放的药物浓度 / 初始药物浓度） x 100%</li>
	<li>定期从每个孔中取出等体积的 PBS，并用等体积的新鲜培养基替换。</li>
</ol>6. 溶血试验
<ol><li>将制备好的 Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS 放入 PBS 中，并在 37 °C 下孵育 24 小时以配制孵育溶液。</li>
	<li>通过眼静脉切开术从健康的 BALB/c 小鼠获得全血，收集在含有柠檬酸钠的抗凝管中。涡旋并以 1509 x g 离心 15 分钟以获得红细胞。</li>
	<li>用 PBS 冲洗红细胞 2 次至 3 次，并用 PBS 制备 10% 红细胞溶液。</li>
	<li>各种 ALGMS 的制备:实验组每个微球 500 μL，阳性对照组 1 mL 超纯水 （ddH2O），阴性对照组 1 mL PBS 溶液，每个与 20 μL 血细胞溶液混合。</li>
	<li>将样品在 37 °C 下孵育 4 小时，以 1509 x g 离心，15 分钟。将上清液放在一边，并在溶血后拍摄每组中的红细胞。</li>
	<li>取阳性对照组溶血率为100%，阴性对照组为0%。测量每组中上清液的吸光度值，并使用以下公式计算溶血速率: 溶血 （%） =（每组上清液的吸光度值 - 单独蒸馏水的吸光度值）/（阳性对照组的吸光度值 - 单独蒸馏水的吸光度值）x 100</li>
</ol>7. 细胞生物相容性试验
<ol><li>用 PBS 洗涤不同的 ALGMS 2 次，放入 5 mL 培养皿中 15 分钟，弃去上清液。</li>
	<li>在含有 10% FBS 的完全培养基 （DMEM） 中分别加入 0.2 mL 的不同 ALGMS，在 37 °C 下孵育 24 小时，并过滤溶液以获得渗滤液。</li>
	<li>在 24 孔板中将 NIH3T3 细胞培养至约 70% 的细胞密度，用微球浸出溶液处理 NIH3T3 细胞，并继续用完全培养基再培养 24 小时。</li>
	<li>取出细胞培养基并用 PBS 洗涤细胞。</li>
	<li>使用钙黄绿素-AM/PI 测定法评估细胞活力。取 2.5 μL 钙黄绿素-AM 溶液 （4 mM） 和 12.5 μL PI 溶液 （2 mM），将它们加入 5 mL 的 1x PBS 中并充分混合，以获得含有 2 μM 钙黄绿素-AM 和 5 μM PI 的工作溶液。</li>
	<li>将工作溶液以每孔 500 μL 的浓度添加到先前接种的细胞培养板中，在 37 °C 下避光孵育 15 分钟，并在倒置荧光显微镜下观察和拍照。为每个组设置三个仿行。</li>
	<li>根据以下公式计算细胞活力: 细胞活力 （%） = 活细胞数 / （活细胞数 + 死细胞数）</li>
</ol>

开发用于生物医学的海藻酸钠水凝胶微球

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不得披露任何利益冲突。

在该协议中，我们提出了一种基于微流体电喷雾技术制备 ALGMS 的方法。该方法操作简单，可产生大量圆度均匀、直径可控的微球。这种方法为研究人员提供了便利，可以促进水凝胶微球的研究和应用。此外，通过与不同的金属离子交联，ALGMS 的稳定性和生物活性得到提高。在抗菌实验中，Cu-ALGMS 和 Zn-ALGMS 表现出显著的细菌抑制作用。
水凝胶微球是具有出色可塑性的柔性微结...

药物递送系统是生物组织工程领域的研究热点，旨在提高药物递送效率和疗效，减少不良反应和副作用1.在这些系统中，水凝胶微球具有良好的生物相容性、可调的机械性能和功能性可塑性，是最常用的药物加载和递送载体之一2。它们可用于药物的缓释和受控释放，为药物提供良好的保护作用，避免或尽量减少药物在其他组织中的非特异性作用，并将药物靶向递送到特定的组织结构3。因此，水凝胶微球已成为一种新的高效药物递送系统，该领域的研究逐渐显现4.
水凝胶微球通常由可生物降解的材料合成，包括多糖、蛋白质和天然聚合物5。其中，ALG 是从海洋褐藻中提取的生物相容性、可生物降解的多糖6。其分子链包含游离羟基和羧基，可与大多数二价或多价阳离子交联，形成具有三维网络的水不溶性水凝胶结构5。ALG 形成的水凝胶微球可以在中性和碱性溶液中转化为带负电荷的聚电解质。负电荷之间的这种排斥导致微球膨胀，从而释放封装的活性成分或药物。这些特性导致人们认为 ALG 微球是广泛用于载药和控释的有前途的药物载体7。
制备水凝胶微球的方法多种多样。传统的 ALGMS 制备方法通常包括 sol-gel 法或 emulsion-sol 法。这些方法涉及沉淀、共沉淀和凝胶化反应等步骤，以获得目标微球8。近年来，随着微流控技术的不断发展，微流控电喷雾法逐渐成为一种高效、精密的微球制备方法9.该方法利用微流体技术，通过超细喷嘴电喷涂聚合物溶液，在随后的固化或交联过程中形成微米级的液滴和微球10。与传统方法相比，微流体电喷雾通过调整溶液流速、电压和细喷嘴尺寸等参数来精确控制微球粒径和形态11。它还能够高速连续制备微球，提高制备效率并保持温和的反应条件。此外，ALGMS 可以制备成具有多种功能，例如控释药物和负载催化剂，使其易于在各个领域应用。
在这里，我们提出了一种使用微流体电喷雾法制备 ALG 微球的方案。该过程包括将 ALG 溶液通过微流体装置并对其进行电喷雾。将所得液滴收集在含有不同金属离子（Ca2+、Cu2+、Zn2+ 和 Fe3+）的溶液中，以引发交联反应。该反应提高了微球的稳定性和粘附性，并赋予它们不同的功能。这种方法易于执行，并且合成的微球在其形态上表现出良好的尺寸均匀性。此外，我们还研究了它们的抗菌特性、缓慢的药物释放能力和生物相容性。该协议将有助于进一步的药物开发和生产。

<table><tbody><tr><td>120 mesh screen</td><td>Solarbio,China</td><td>YA0946</td><td></td></tr><tr><td>Alcohol burner</td><td>Solarbio,China</td><td>YA2320</td><td></td></tr><tr><td>BALB/c mice</td><td>Wukong Biotechnology,China</td><td></td><td></td></tr><tr><td>Bicinchoninic Acid Assay reagent</td><td>Meilunbio,China</td><td>MA0082</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Lablead,China</td><td>9048-46-8</td><td></td></tr><tr><td>CaCl2&amp;nbsp; powder</td><td>Aladdin,China</td><td>10043-52-4</td><td></td></tr><tr><td>Calcein-AM/PI</td><td>Biosharp,China</td><td>BL130A</td><td></td></tr><tr><td>Centrifuge tubes</td><td>Corning,America</td><td>430290</td><td></td></tr><tr><td>CuSO4&amp;nbsp; powder</td><td>Jnxinyuehuagong,China</td><td>7758-99-8</td><td></td></tr><tr><td>DMEM</td><td>Gibicol,China</td><td>C11995500BT</td><td></td></tr><tr><td>FeCl3&amp;nbsp; powder</td><td>Aladdin,China</td><td>7705-08-0</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HAKATA,China</td><td>HN-FBS</td><td></td></tr><tr><td>Glass tubes</td><td>Sartorius,Germany</td><td>CC0028</td><td></td></tr><tr><td>Light microscopy</td><td>Evidentscientific,Japan</td><td>BX53(LED)</td><td></td></tr><tr><td>Microfluidic syringe pump</td><td>Longerpump,England</td><td>LSP01-3A</td><td></td></tr><tr><td>NIH3T3</td><td>HyGyte,China</td><td>TCM-C752</td><td></td></tr><tr><td>Petri dish</td><td>Thermofisher,America</td><td>150464</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>Thermofisher,America</td><td>3002</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Thermofisher,America</td><td>Axia ChemiSEM</td><td></td></tr><tr><td>Sodium alginate powder&amp;nbsp;</td><td>Bjbalb,China</td><td>Y13095</td><td></td></tr><tr><td>ZnSO4 powder</td><td>Jnxinyuehuagong,China</td><td>7733-02-0</td><td></td></tr></tbody></table>

preparation of cross-linked sodium alginate microspheres with different metal ions using the microfluidic electrospray technology

微球是微米大小的颗粒，可以通过物理封装或吸附将药物加载并逐渐释放到聚合物表面和内部。在生物医学领域，水凝胶微球作为药物载体的应用已被广泛研究，因为它们能够减少给药频率、最大限度地减少副作用并提高患者依从性。海藻酸钠 （ALG） 是一种天然存在的线性多糖，具有三个主链糖苷键。聚合物的每个部分都存在两个辅助羟基，它们具有醇羟基部分的特性。合成的 ALG 单元可以与金属离子发生化学交联反应，形成聚合物堆栈的交联网络结构，最终形成水凝胶。水凝胶微球可以使用涉及 ALG 的离子交联特性的简单工艺制备。在这项研究中，我们使用微流体电沉积策略制备了基于 ALG 的水凝胶微球 （ALGMS）。由于精确控制了微流体电喷雾流，制备的水凝胶微球尺寸均匀且分散良好。采用微流控和高电场相结合的微流控电喷雾技术制备与不同金属离子交联的 ALGMS，并研究了其抗菌性能、缓慢药物释放能力和生物相容性。该技术有望应用于高级药物开发和生产。

与不同金属离子交联的 ALGMS 的表征 Ca-ALGMS、Cu-ALGMS、Zn-ALGMS 和 Fe-ALGMS 的光学形貌如图 2 所示，表现出良好的球形度、光滑的表面、均匀的粒径分布（补充图 2）和优异的单分散性。我们进一步使用扫描电子显微镜 （SEM） 和能量色散光谱 （EDS） 分析进行了显微表征。如图 3 所示，微球通常是球形的，具有明确的圆度。Zn-ALGMS...

作者聚焦:使用生物相容性海藻酸钠水凝胶微球推进治疗

该方案描述了使用用于药物载体设计的微流体装置制备与不同金属离子交联的海藻酸钠微球。还研究了这些微球的抗菌特性和缓慢的药物释放。

利用微流控电喷雾技术制备具有不同金属离子的交联海藻酸钠微球

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

preparation-cross-linked-sodium-alginate-microspheres-with-different

Research

JoVE Journal

Bioengineering

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

1.8K 次观看.  Zhejiang Shuren University. 氢微球的特点是相容性好、设计灵活性高、定制化程度高。它们在生物医学领域具有广泛的应用保护器，例如治疗药物递送和 TC 修复发现的构建。在这项研究中，我们使用简单的微流控装置制备了氢微球。最近，我们一直在尝试使用载有一些中医小分子的氢微球来促进所有愈合。在这项研究中，我们发现锌和铜离子交联海藻酸钠微球具有良好?...

视频: 作者聚焦:使用生物相容性海藻酸钠水凝胶微球推进治疗

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

的凹孔隙PDMS珠微泡制作

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

科研

化学

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

医学

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

生物工程

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

使用电喷雾技术合成载有黑籽油、海藻酸盐基的 pH 敏感磁珠

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned tissues due to convenience in the packaging of cells, improved cell retention, and minimal invasiveness. Moreover, the porous conformation of these microscale carriers could enhance the medium exchange and improve the level of nutrients and oxygen supplies. The present study illustrates the convenient fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (PLGA-HOPMs) by the facile microfluidic technology for cell delivery applications. The resultant monodispersed PLGA-HOPMs possessed particle sizes of ~400 &#181;m and open pores of ~50 &#181;m with interconnecting windows. Briefly, the emulsified oil droplets (PLGA solution in dichloromethane, DCM), wrapped with the 7.5% (w/v) gelatin aqueous phase, were introduced into the 1% (w/v) continuous flowing poly(vinyl alcohol) (PVA) aqueous solution through the coaxial nozzle in the customized microfluidic setup. Subsequently, the microspheres were subjected to solvent extraction and lyophilization procedures, resulting in the production of HOPMs. Notably, various formulations (concentrations of PLGA and porogen) and processing parameters (emulsifying power, needle gauge, and flow rate of dispersed phase) play crucial roles in the qualities and characteristics of the resulting PLGA HOPMs. Moreover, these architectures might potentially encapsulate various other biochemical cues, such as growth factors, for extended drug discovery and tissue regeneration applications.

Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

通过微流控技术 制造 高度开放的多孔微球（HOPM）

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned ...

利用微流控电喷雾技术制备具有不同金属离子的交联海藻酸钠微球

副本

摘要

探索更多视频

的凹孔隙PDMS珠微泡制作

生物医学应用海藻酸钠微球的一代

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

静电生长因子释放到微纤维支架

交变磁场敏感的混合明胶微凝胶药物控释

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

聚电解质复合物肝素结合域成骨生长因子交货

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

使用电喷雾技术合成载有黑籽油、海藻酸盐基的 pH 敏感磁珠

通过微流控技术制造高度开放的多孔微球（HOPM）

利用微流控电喷雾技术制备具有不同金属离子的交联海藻酸钠微球

副本

摘要

探索更多视频

的凹孔隙PDMS珠微泡制作

生物医学应用海藻酸钠微球的一代

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

静电生长因子释放到微纤维支架

交变磁场敏感的混合明胶微凝胶药物控释

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

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