Dual Mode Nanoemulsions for 19F MRI and Fluorescence Detection

描述（由申请人提供）：最近，卡内基梅隆大学（CMU）的科学家发明了一种引人注目的技术，可以通过磁共振成像（MRI）使移植细胞可见。用新型全氟聚醚（PFPE）纳米乳液组合物离体标记感兴趣的细胞，然后将其引入动物或人受试者中;随后使用氟-19（19 F）MRI在体内监测细胞迁移。19 F图像对标记的细胞具有极高的选择性，并且在同一扫描会话中采集的共配准的常规质子（1H）MR图像将标记的细胞置于其精确的解剖背景中。最近，Celsense公司开发并生产了一种名为“Cell Sense”（CS）的商业版PFPE试剂。 在进一步的发展中，CMU最近发表的研究已经证明了用于19 F MRI和荧光检测的稳健的“双模式”纳米乳液的配方。关键的创新在于，荧光染料以低量存在于氟碳油中，而与PFPE油共价缀合的荧光染料在整个加工过程中保持在氟碳相中。这些新试剂稳定，无毒，可自行输送到各种细胞类型。观察到19 F NMR信号与标记细胞的荧光强度之间存在明显的线性相关性。在这些结果的基础上，本申请的目标是开发一种商业化可扩展的荧光版本的Cell Sense。目前的结果，包括生物测试，都是小规模的试验。因此，提出了进行更大规模开发和生产的必要步骤。这些规模扩大程序背后的技术挑战在本修订版申请中得到了非常详细的解决。PFPE油的特殊物理化学性质，高疏水性和显着的疏油性，使得这些油的合成改性和配制比传统的烃类乳化更困难。在合成中，如PFPE共轭混合，非共轭染料的去除和最终产品的纯化，纳米乳液的制备，也在本提案中得到解决。 这种真正的双模式试剂对于在临床前和临床研究中采用Cell Sense平台至关重要。在临床前研究中，它将允许研究人员在转移后数天和数周内使用19 F MRI后的组织学或流式细胞术积极识别标记细胞的命运和表型。在临床领域，双模式试剂将用于使用定量荧光测量确保在患者或供体来源的细胞的等分试样中离体CS标记一致。 荧光CS将能够使用低成本、快速多孔板读数器验证递送至细胞的CS试剂的量。第一阶段的提案有三个具体目标：（1）综合。开发用于大规模合成荧光共混PFPE酰胺（FBPA）分子的方案，足以产生>1 L的纳米乳液;（2）配制。制定使用目标1中生产的FBPA生产双模式纳米乳液的方案; (3)验证。评价细胞标记功效、安全性和用双模式纳米乳剂标记的细胞的荧光与19 F信号的线性验证。合成和配制工作（目标1、2）将在CMU进行，而生物学试验（目标3）将在Celsense，Inc.的实验室进行。在一年时间内成功完成该项目将有助于我们在II期项目中体外测试双模纳米乳剂在人类治疗细胞（如干细胞）中的安全性和有效性。 公共卫生相关性：目前，700多项涉及治疗性细胞移植的临床试验（例如，淋巴细胞和干细胞）正在进行中，以治疗主要疾病，如癌症和心力衰竭。拟议的项目将有助于加速这些试验，以及细胞疗法的常规临床使用，通过提供独特的磁共振成像（MRI）和光学成像试剂和工具，使人们能够在移植到患者体内后跟踪细胞。

DESCRIPTION (provided by applicant): Recently, scientists at Carnegie Mellon University (CMU) have invented a compelling technology that makes transplanted cells visible with magnetic resonance imaging (MRI). Cells of interest are labeled ex vivo with a novel perfluoropolyether (PFPE) nanoemulsion composition and then introduced into an animal or human subject; cell migration is subsequently monitored in vivo using fluorine-19 (19F) MRI. The 19F images are extremely selective for the labeled cells, and the co-registered conventional proton (1H) MR images acquired in the same scanning session places the labeled cells into their precise anatomical context. A commercial version of the enabling PFPE reagent, called 'Cell Sense' (CS), has recently been developed and manufactured by Celsense, Incorporated. In further developments, recently published research from CMU has demonstrated the formulation of robust, 'dual mode' nanoemulsions for 19F MRI and fluorescence detection. Key innovation lies in the fact that fluorescent dye is present in the fluorocarbon oil in low amount, yet covalently conjugated to PFPE oil remains in fluorocarbon phase throughout processing. These new reagents are stable, non-toxic and self-deliverable to a wide range of cell types. An apparent linear correlation between the 19F NMR signal and the fluorescence intensity of labeled cells is observed. Building on these results, the goal of this application is to develop a commercially-scalable fluorescent version of Cell Sense. The current results, including biological testing, are on small pilot scale. Therefore, necessary steps towards the development and production on a larger scale are proposed. The technical challenges underlying these scale up procedures are addressed in great detail in this revised application. Unusual physicochemical properties of PFPE oils, high hydrophobicity and significant lipophobicity, make the synthetic modifications and formulations of these oils more difficult than traditional emulsification of hydrocarbons. In the synthesis issues such as PFPE conjugate blending, unconjugated dye removal and purification of final product, nanoemulsion preparations, are also addressed in this proposal. This true dual mode agent is critical for the adoption of the Cell Sense platform in both preclinical and clinical studies. In preclinical studies, it will allow investigators to positively identify the fate and phenotype of labeled cells using histology or flow cytometry following 19F MRI, days and weeks after transfer. In the clinical domain, the dual mode agent will be used to ensure consistent CS labeling ex vivo in an aliquot of patient- or donor- derived cells using quantitative fluorescent measurements. Fluorescent CS will be able to validate the amount of CS reagent delivered to cells using a low cost, rapid multi-well plate reader. This Phase I proposal has three Specific Aims: (1) Synthesis. Develop protocols for the synthesis of fluorescent blended PFPE amide (FBPA) molecules on large-scale, sufficient to produce >1 L of nanoemulsion; (2) Formulation. Develop protocols for the production of dual mode nanoemulsions using the FBPA produced in Aim 1;.(3) Validation. Evaluate cell labeling efficacy, safety and linear validation of fluorescence versus 19F signal of cells labeled with dual mode nanoemulsions. The synthetic and formulation work (Aims 1,2) will be performed at CMU, while the biological testing (Aim 3) will be performed at the laboratories at Celsense, Inc. Successful completion of this project in a one-year time frame will facilitate our transition to testing safety and efficacy of dual mode nanoemulsions in human therapeutic cells, such as stem cells, in vitro in a Phase II project. PUBLIC HEALTH RELEVANCE: Currently, more than 700 clinical trials involving the transplantation of therapeutic cells (e.g., lymphocytes and stem cells) are underway to treat major diseases such as cancer and heart failure. The proposed project will help accelerate these trials, as well as the routine clinical use of cellular therapeutics, by providing unique reagents and tools for magnetic resonance imaging (MRI) and optical imaging that will enable one to follow cells after transplant into patients.