Dual Mode Nanoemulsions for 19F MRI and Fluorescence Detection

描述(申请人提供)：最近，卡内基梅隆大学(CMU)的科学家发明了一项引人注目的技术，可以通过磁共振成像(MRI)看到移植的细胞。用一种新型的全氟聚醚(PFPE)纳米乳液组合物体外标记感兴趣的细胞，然后将其引入动物或人类受试者体内；随后使用氟-19(19F)磁共振成像在体内监测细胞迁移。19F图像对标记细胞具有极高的选择性，在同一扫描过程中获得的共同配准的常规质子(1H)磁共振图像将标记细胞置于其精确的解剖环境中。CELSENSE公司最近开发和制造了一种商业版本的使能PFPE试剂，称为‘Cell Sense’(CS)。在进一步的发展中，CMU最近发表的研究已经证明了用于19F MRI和荧光检测的坚固的“双模式”纳米乳剂的配方。关键的创新之处在于，荧光染料在氟碳油中以少量存在，但在整个加工过程中与PFPE油以共价结合的方式保持在氟碳相中。这些新试剂稳定、无毒、可自我释放到多种细胞类型。~(19)F-核磁共振信号与标记细胞的荧光强度之间存在明显的线性关系。在这些结果的基础上，这一应用程序的目标是开发一种可商业扩展的Cell Sense荧光版本。目前的结果，包括生物测试，都是小规模的试点。为此，提出了向规模化发展和生产发展的必要措施。在这份修订后的申请中，这些扩大程序背后的技术挑战得到了非常详细的解决。PFPE油特殊的物理化学性质、高疏水性和显著的疏脂性，使得这些油的合成修饰和配方比传统的碳氢化合物乳化更加困难。在合成方面，本建议还涉及了PFPE共混、非共轭染料的去除和最终产品的纯化、纳米乳液制剂等问题。这种真正的双模制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，它将使研究人员能够在移植后几天和几周使用组织学或流式细胞术确定标记细胞的命运和表型。在临床领域，双模式试剂将用于确保使用定量荧光测量在等量的患者或供体来源的细胞中体外一致的CS标记。荧光CS将能够使用低成本、快速的多孔板读取器来验证输送到细胞的CS试剂的量。这一阶段的建议有三个具体目标：(1)综合。制定了大规模合成荧光共混型全氟聚乙烯酰胺(FBPA)分子的方案，足以生产&gt；1 L的纳米乳液；(2)配方。开发使用AIM 1中生产的FBPA生产双模纳米乳液的方案；(3)验证。评价双模纳米乳剂标记细胞的细胞标记效率、安全性和荧光与19F信号的线性验证。合成和配方工作(目标1，2)将在CMU进行，而生物测试(目标3)将在Celense，Inc.的实验室进行。在一年的时间框架内成功完成这一项目，将有助于我们过渡到在第二阶段项目中测试双模纳米乳剂在人体治疗细胞中的安全性和有效性，例如干细胞。 公共卫生相关性：目前，700多项涉及治疗性细胞(如淋巴细胞和干细胞)移植的临床试验正在进行中，以治疗癌症和心力衰竭等重大疾病。拟议的项目将通过为磁共振成像(MRI)和光学成像提供独特的试剂和工具，使人能够在移植到患者体内后跟踪细胞，从而帮助加快这些试验以及细胞疗法的常规临床应用。

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.