ex vivo fluorescence microscopy of murine brain tissue sections

<meta charset="utf8"></head><body>マウス神経膠芽腫における画像誘導治療送達のための酸化鉄ナノ粒子

Glioblastoma multiforme (GBM) is the highest grade of astrocytoma for which there is virtually no cure. Approximately 15,000 people are diagnosed with glioblastoma annually, which has a dismal median survival of about 15 months and a 5-year survival rate of 5%1. In the past decades, there has been marginal improvement in prognosis despite multiple efforts to advance therapeutic options. The current standard of care for GBM includes maximal surgical resection, when feasible, followed by radiotherapy and chemotherapy2. Temozolomide (TMZ), the chemotherapy of choice, was the latest therapy for glioblastoma discovered to show notable clinical efficacy; however, at least 50% of GBM tumors show TMZ resistance3. In spite of this rigorous therapeutic regimen, there is still a significant clinical need for improved glioblastoma therapy.
The development of therapeutics for GBM and other brain-related diseases is significantly hampered by the selective nature of the blood-brain barrier (BBB). The BBB is a physiological barrier comprised of endothelial cells, pericytes, and astrocyte feet-ends, which creates the semi-permeable membrane between the circulatory system and the brain, restricting the free passage of molecules and cells into the brain4. While protective in normal physiology and critical for brain homeostasis, the BBB prevents many therapeutics from reaching the brain, complicating the treatment of GBM. Efforts to enhance the delivery of therapeutics to GBM have led to the development of nanoparticle-based delivery vehicles, focused ultrasound drug delivery enhancement, and receptor-mediated drug delivery5,6.
Nanoparticles have emerged as a promising medium for developing therapeutics for a myriad of diseases, including cancers. The application of nanoparticles for imaging and therapeutic purposes in GBM has been attempted using various nanoparticle constructs7,8. With the focus on delivering drugs to GBM in conjunction with in vivo imaging of the delivery, the proposed approach utilizes magnetic nanoparticles (MN) consisting of an iron oxide core and covered by dextran for stability. The magnetic properties of these nanoparticles afford for their detection by magnetic resonance (MR) imaging, while simple conjugation chemistry to the aminated dextran coating allows for conjugation of therapeutic moieties such as RNA molecules, additional targeting moieties, or imaging moieties (such as Cy5.5 near-infrared optical dye)9,10. In addition to the imaging capabilities, the nano platform is able to extend the half-life of RNA therapeutics by protecting the oligonucleotide from endogenous nucleases, improving therapeutic delivery. Here, the application of this nano platform for in vivo delivery of therapeutic oligonucleotides (termed MN-anti-miR10b) to GBM, monitored by in vivo imaging, is presented. Previously, the ability of this nano platform to accumulate was demonstrated in GBM cells in vitro, causing significant loss of viability of tumor cells11. Prior to performing therapeutic in vivo studies, it is necessary to demonstrate in vivo delivery of this nano platform to GBM tumors in animal models. To achieve this, orthotopic GBM animal models were produced, and intravenous administration of the construct was performed followed by in vivo imaging. Outlined here are the protocols of these studies showing accumulation in the tumor region confirmed by in vivo imaging and ex vivo microscopy.

<meta charset="utf8"></head><body>著者スポットライト:膠芽腫治療のための酸化鉄ナノ粒子による革新的ながん治療

神経膠芽腫多形動物モデルにおけるオリゴヌクレオチドペイロードのナノ粒子送達

マウス脳組織切片の Ex Vivo 蛍光顕微鏡法

ex-vivo-fluorescence-microscopy-of-murine-brain-tissue-sections

Research

JoVE Journal

Cancer Research

Ex Vivo Fluorescence Microscopy of Murine Brain Tissue Sections

237 ビュー. まず、最適な切断温度で保存された瞬間凍結ブレインを摂氏80度の貯蔵庫から取り出し、クライオスタットチャンバー内に置きます。サンプルをチャンバー温度まで温めます。片刃のカミソリを使用して、注射部位で脳を冠状に切断します。最適な切断温度を少量使用して、サンプルを試料ディスクにマウントします。チャンバー内に冷や?...

ビデオ: マウス脳組織切片の Ex Vivo 蛍光顕微鏡法

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...