This Small Business Innovation Research (SBIR) Phase I project will develop nanoparticle imaging agents for diagnostic applications in clinical pathology. Currently, fluorescent imaging agents are used to image cells and tissue samples in both solid (biopsy) and liquid (blood sample) forms and to identify markers that indicate disease state or progression. These imaging agents have several limitations, including poor stability and reduced brightness, especially over time. Nanoparticle imaging agents can provide brighter images over longer periods of time, potentially enhancing diagnostic ability. Further, nanoparticle imaging agents may permit multiple markers to be imaged simultaneously reducing the amount of blood/tissue required for analysis. This proposal will examine the potential of fluorescent nanoparticle quantum dots for pathological diagnostic imaging by (i) showing that nanoparticles can be produced in sufficient volume to meet market need, (ii) demonstrating that nanoparticles can be used to identify standard markers in blood cells and (iii) comparing their performance and stability to products currently used in the clinic. This research and development will thus validate the potential of nanoparticle quantum dots in the clinical pathology market, and could lead to enhanced diagnostic tools for earlier disease detection with reduced blood/tissue sample requirements.
The broader impact/commercial potential of this project primarily impacts the pathology imaging market, which could have substantial benefits to society. Although diagnostic imaging agents are a $2.86B market, nanoparticles represent just $183M of that market. There is thus tremendous opportunity to enhance nanoparticle market share. Currently, few fluorescent nanoparticle agents are used in pathology because of a lack of stability. These research and development activities will evaluate the potential of nanoparticle imaging agents to improve diagnostic capabilities of disease. The proposed product, the Multidot, exhibits superior stability to current nanoparticle imaging agents potentially elevating a commercialization roadblock. If successful, the Multidot should provide a brighter signal, increasing disease detection capability, and also permit multiple markers to be imaged simultaneously. The latter not only reduces the amount of tissue required for imaging, but could enable additional diagnostics based on pairs or groupings of markers, enhancing personalized medicine approaches. Together, these benefits, will enhance clinical care by providing new types of diagnostic imaging agents with superior performance to those currently employed.
Pathology is a $48B/yr industry with $5B dedicated to molecular detection], of this fluorescent imaging comprises ~$500M. The pathology market for fluorescence imaging naturally splits into two segments based on the type of tissue analyzed: liquid (i.e.,blood) and solid (i.e., tissue) biopsies. Liquid biopsies are commonly analyzed via flow cytometry, primarily for diseases of the blood, including leukemias and human immunodeficiency virus (HIV). Because of the number of different cell types present in blood, several molecular markers are required to characterize its composition. In addition, as molecular medicine approaches (i.e., targeted treatment of a specific patient population based on the molecular expression profile associated with their pathology) increase, the ability to evaluate increased numbers of markers in the same cell is crucial. Fluorescent agents offer the potential for multiplexed imaging; that is the simultaneous imaging of multiple biomarkers. However, traditional fluorescent dyes used in flow cytometry have broad emission spectra that can overlap with each other and also cellular autofluorescence, limiting the ability to separate labeled cells into different populations. Further, discrimination between cells with low expression levels and background fluorescence can be challenging with any fluorophore. Increasing fluorophore brightness maximizes the signal to noise ratio, and therefore identification of target cells. Stable, bright fluorescent agents with narrow emission bandwidths would greatly improve multiplexed flow cytometry capabilities. Solid tissue samples are analyzed via a process known as immunohistochemistry (IHC). Specimens are stained with colored hematoxylin and eosin dyes that stain nuclei and the cytoplasm, respectively. Molecular analysis is typically performed for single markers only (1/per slide) using 3,3'-Diaminobenzidine (DAB) or horse radish peroxidase (HRP), both of which produce a colormetric (nonfluorescent) product. Current use of fluorescent reagents for molecular analysis in the clinic is primarily limited to fluorescence in situ hybridization (FISH) assays, which identify molecular (i.e., DNA, RNA) expression within a cell. FISH is used to identify patient sensitivity to molecular targeting drugs, and is thus an important pillar of personalized medicine, a rapidly growing field. For example, FISH testing for the Her-2 receptor is the gold standard for patient selection for treatment with Herceptin, a molecular therapy employed in breast cancer treatment. Currently, the FISH market is dominated by Abbott through their Vysis product lines, which utilize proprietary fluorophores (e.g., spectrum green, spectrum orange) for molecular detection. Molecular detection is proportional to the number of molecules expressed in the cell of interest, but also to the intensity of the fluorophores employed and the ability to distinguish their fluorescence from background. Brighter probes could increase the detection of low expressers, thus identifying additional patients that may benefit from molecular drug therapy. Thus, increased brightness and reduced overlap with autofluorescence channels are important features of fluorophores for FISH studies. There is an unmet need in the clinical pathology market for fluorescent diagnostic reagents that offer reliable, sensitive, and unequivocal diagnostic output for the analysis of multiple biomarkers. This finding was confirmed via 87 customer interviews performed through NSF the I-Corps program, which identified the greatest pain points in fluorescent clinical pathology imaging as (1) reagent stability under pathological processing and imaging conditions and (2) difficulty in identifying more than 4 molecular signals in the same cell as a result of spectral overlap between dyes. Core Quantum Technologies (CQT) proposed a new quantum dot reagent in the Phase I SBIR, the MultiDot, for use in in vitro pathological imaging that addresses the above concerns. The MultiDot consists of a patent-pending coating that permits multiple, organic QDs to be encapsulated within a polymer micelle, yielding a product stable in cell-friendly biological buffers. MultiDots are 4-13X brighter and 15X more stable to photo-oxidation than commercial QDs and organic dyes. In addition, MultiDots are manufactured using a proprietary high throughput manufacturing process that reduces unit cost, making MultiDots cost competitive with current pathology organic dye products. Our Phase I SBIR demonstrated that the electrospray high throughput manufacturing of the Multidots was able to be transferred to CQT from The Ohio State University. We were able to attach antibodies to the fluorescent reagent and label targeted cells with a greater than 99.7% positive signal, measurements were performed in both a research flow cytometer and in a clinical flow cytometer. Acceptance of this reagent in the clinical market after FDA approval will increase the sensitivity and specificity of disease diagnosis as well as advance personalized medicine, leading to earlier diagnosis of disease with more accuracy and the knowledge of which medications would work optimally in the therapy and remission of patient disease.
