Retroductal 纳米微粒注射液对小鼠颌下腺的治疗
Summary
局部药物向颌下腺的传递对了解涎腺生物学和新疗法的发展有很浓厚的兴趣。我们提出了一个更新和详细的 retroductal 注射协议, 旨在提高交付准确性和实验再现性。本文提出的应用是高分子纳米粒子的传递。
Abstract
涎腺治疗的两个共同目标是在自身免疫或辐射损伤后组织功能障碍的预防和治疗。通过局部向涎腺提供生物活性化合物, 可以安全地实现更大的组织浓度, 而不是系统性的管理。此外, 离体超腺堆积物的靶组织效应可以显著降低。在这方面, retroductal 注射液是一种广泛应用的方法, 以调查涎腺生物学和病理生理学。Retroductal 对生长因子、原代细胞、腺病毒载体和小分子药物的管理在损伤的设置中支持腺体功能。我们以前已经证明了 retroductally 注射纳米粒子-siRNA 策略在辐照后维持腺体功能的有效性。在这里, 一个高效和可重复的方法管理纳米材料的小鼠颌下腺通过沃顿的管道是详细 (图 1)。我们描述了进入口腔, 并概述了必要的步骤 cannulate 沃顿的管道, 并进一步的意见, 作为质量检查整个过程。
Introduction
涎腺功能障碍有许多病因, 包括 Sjögren 综合征, 自身免疫介导的功能性分泌组织丧失, 以及辐射诱发的 hyposalivation (RIH), 共同 sequella 头颈肿瘤放疗1。由于任一条件而导致的唾液功能丧失会诱发个人口腔和全身感染、蛀牙、消化和吞咽功能障碍、言语障碍和主要抑郁症1,2,3。因此, 生活质量显著受损, 干预措施仅限于缓解症状, 而不是治疗4。为了研究新的治疗方法在体内, 直接对涎腺进行生物活性化合物是有好处的。
Retroductal 注射液是一种很有价值的方法, 可以直接向唾液腺提供生物活性化合物, 并检测疾病、损伤或正常组织稳态下的疗效。三个主要唾液腺是腮腺 (PG), 颌下腺 (SMG) 和舌下 (SLG), 所有这些都空入口腔通过排泄导管。该小鼠的解剖允许通过插管的沃顿商学院的管道直接进入, 位于舌下的口底部5。插管后, 溶解药物可以直接管理到 SMG。在 retroductal 分娩后, 附加腺的扩散受周围组织胶囊的限制, 该囊调节与周围结构的材料交换6。smg 和它的导管是相似的结构在人, 并且经常被访问在 SMG 手术期间和 sialoendoscopy7。在人类和小鼠, PG 同样可以通过 Stensen 的导管在颊粘膜8。
在 RIH 的小鼠模型中, SMG retroductal 注射液用于提供治疗方法, 包括生长因子、原代细胞、腺病毒载体、细胞因子和抗氧化化合物, 以调节细胞对损伤的反应, 并减少由此产生的组织损伤5,9,10,11,12,13,14,15,16。retroductal 注射液的临床成功最显著的是对腺病毒载体的管理, 以直接表达的水道 (水通道蛋白 1;AQP1) 治疗头颈癌放疗后的患者17。
以前, 我们已经开发并显示了 retroductally 注射聚合物纳米 siRNA 系统的功效, 以保护唾液腺功能从 RIH11,18,19,20。作为我们过去工作的延伸, 在这里, 我们展示了我们的协议 retroductal SMG 注射使用一个荧光标记纳米粒子 (NP) 能够加载和交付否则不太可溶性药物21,22,23。
我们已经合成了 NP 从两嵌段共聚物组成的聚 (苯乙烯-alt-马来酸酐)-b 聚 (苯乙烯) (PSMA) 通过可逆添加链碎片 (筏) 聚合, 如前所述21。通过溶剂交换, 这些聚合物自发地自组装成胶束 NP 结构, 具有疏水性内部和亲水性外部的21。NPs 被标记与得克萨斯红色荧光允许验证 NP 交付入腺体, 不用牺牲动物。活体动物成像和 SMG 免疫组化显示在注射后1小时和1天。
此更新和可重现的插管协议应使其他人能够实现 retroductal 注射。我们预计, 这种精制技术将成为体内研究和治疗发展的关键24,25。
Protocol
Representative Results
Discussion
Retroductal 注射液对涎腺的局部药物传递至关重要。该技术在筛选治疗剂的条件, 包括干燥综合征和 RIH9,10,28的应用。通过 retroductal 注射液直接向 SMG 提供药物, 在降低靶向效应 (包括免疫激活11) 的潜在效果方面比系统性管理具有重要优势。在不积累周围组织的情况下, 最大限度地提高当地药物的输送能力, 也能?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该出版物中报告的研究得到国家牙科和颅面研究研究所 (NIDCR) 和国立卫生研究院全国癌症研究所 (R56 DE025098、UG3 DE027695 和 F30 CA206296) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这项工作也得到了 NSF DMR 1206219 和 IADR 口腔护理奖 (2016) 创新的支持。
我们要感谢詹 Gavrity 在 IVIS 实验中的帮助。我们要感谢凯伦. 宾利在执行 EM 方面的投入和协助。我们要感谢裴翁对 IHC 的协助。我们要感谢马修·格尔斯在图准备方面的协助。我们要感谢伊莲 Smolock 博士和艾米莉. 吴对这篇手稿的批判性阅读。
Materials
| Pilocarpine hydrochloride | Sigma Aldrich | P6503 | Pilocarpine |
| Student Vannas Spring Scissors | Fine Science Tools | 91500-9 | Spring Scissors for Tracheostomy |
| Sterile Saline Solution | Medline | RDI30296H | Saline |
| Dumont #7 Forceps | Fine Science Tools | 11274-20 | Curved Forceps |
| Dumont #5 Forceps | Fine Science Tools | 11251-10 | Straight Forceps |
| Standard Pattern Forceps | Fine Science Tools | 11000-12 | Blunt Forceps |
| Fine Scissors- Tungsten Carbide | Fine Science Tools | 14568-09 | Dissection Scissors |
| Microhematocrit Heparinized Capillary Tubes | Fisher Scientific | 22362566 | Capillary tubes |
| Lubricant Eye Ointment | Refresh | N/A | Refresh Lacri-Lube |
| Goat polyclonal anti-Nkcc1 | Santa Cruz Biotech | SC-21545 | Nkcc1 Antibody |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | DAPI |
| GraphPad Prism | GraphPad | ver6.0 | Statistical Software |
| Cotton tipped applicator | Medline | MDS202000 | Applicator for eye ointment |
| 0.5cc Insulin Syringe, 29G x 1/2" | BD | 7629 | Syringe for intraperitoneal injection |
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