多价 DNA 功能化金纳米粒子转染胰岛。
Transfection of pancreatic islets using polyvalent DNA-functionalized gold nanoparticles.
机构信息
Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
出版信息
Surgery. 2010 Aug;148(2):335-45. doi: 10.1016/j.surg.2010.05.013.
BACKGROUND
Transplantation of pancreatic islets is an effective treatment for select patients with type 1 diabetes. Improved cellular therapy results may be realized by altering the gene expression profile of transplanted islets. Current viral and nonviral vectors used to introduce nucleic acids for gene regulation hold promise, but safety and efficacy shortcomings motivate the development of new transfection strategies. Polyvalent gold nanoparticles (AuNPs) densely functionalized with covalently immobilized DNA oligonucleotides (AuNP-DNA) are new single entity transfection and gene regulating agents (ie, not requiring lipids, polymers, or viral vectors for cell entry) able to enter cells with high efficiency and no evidence of toxicity. We hypothesize that AuNP-DNA conjugates can efficiently transfect pancreatic islets with no impact on viability or functionality, and can function to regulate targeted gene expression.
METHODS
AuNPs were surface-functionalized with control and antisense DNA oligonucleotides. Purified murine and human islets were exposed to AuNP-DNA conjugates for 24 hours. Islet AuNP-DNA uptake, cell viability, and functionality were measured. Furthermore, the ability of antisense AuNP-DNA conjugates to regulate gene expression was measured using murine islets expressing eGFP.
RESULTS
Collectively, fluorescent confocal microscopy, transmission electron microscopy, mass spectrometry, and flow cytometry revealed substantial penetration of the AuNP-DNA conjugates into the inner core of the islets and within islet cells. No change in cellular viability occurred and the insulin stimulation index was unchanged in treated versus untreated islets. Transplantation of AuNP-DNA treated islets cured diabetic nude mice. Functionally, antisense eGFP AuNP-DNA conjugates reduced eGFP expression in MIP-eGFP islets.
CONCLUSION
Polyvalent AuNP-DNA conjugates may represent the next generation of nucleic acid-based therapeutic agents for improving pancreatic islet engraftment, survival, and long-term function.
背景
胰岛移植是治疗 1 型糖尿病的有效方法。通过改变移植胰岛的基因表达谱，可以提高细胞治疗的效果。目前用于引入核酸进行基因调控的病毒和非病毒载体具有一定的前景，但安全性和有效性的不足促使新转染策略的发展。多价金纳米粒子（AuNP）通过共价固定的 DNA 寡核苷酸（AuNP-DNA）进行密集功能化，是新的单一实体转染和基因调控剂（即，不需要脂质、聚合物或病毒载体进入细胞），能够高效进入细胞，没有毒性的证据。我们假设 AuNP-DNA 缀合物可以有效地转染胰岛，而不会影响其活力或功能，并可以调节靶向基因表达。
方法
AuNP 表面用对照和反义 DNA 寡核苷酸功能化。纯化的鼠和人胰岛暴露于 AuNP-DNA 缀合物 24 小时。测量胰岛 AuNP-DNA 摄取、细胞活力和功能。此外，使用表达 eGFP 的鼠胰岛测量反义 AuNP-DNA 缀合物调节基因表达的能力。
结果
荧光共聚焦显微镜、透射电子显微镜、质谱和流式细胞术共同揭示了 AuNP-DNA 缀合物大量渗透到胰岛的核心内部和胰岛细胞内。细胞活力没有变化，处理组和未处理组的胰岛素刺激指数没有变化。AuNP-DNA 处理的胰岛移植治愈了糖尿病裸鼠。功能上，反义 eGFP AuNP-DNA 缀合物降低了 MIP-eGFP 胰岛中的 eGFP 表达。
结论
多价 AuNP-DNA 缀合物可能代表下一代基于核酸的治疗剂，可改善胰岛移植的植入、存活和长期功能。
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