In vivo detection of drug-induced apoptosis in tumors using Raman spectroscopy

We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.

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Characterization of magnetic nanoparticle-seeded microspheres for magnetomotive and multimodal imaging

Magnetic iron-oxide nanoparticles have been developed as contrast agents in magnetic resonance imaging (MRI) and as therapeutic agents in magnetic hyperthermia. They have also recently been demonstrated as contrast and elastography agents in magnetomotive optical coherence tomography and elastography (MM-OCT and MM-OCE, respectively). Protein-shell microspheres containing suspensions of these magnetic nanoparticles in lipid cores, and with functionalized outer shells for specific targeting, have also been demonstrated as efficient contrast agents for imaging modalities such as MM-OCT and MRI, and can be easily modified for other modalities such as ultrasound, fluorescence, and luminescence imaging. In addition to multimodal contrast-enhanced imaging, these microspheres could serve as drug carriers for targeted delivery under image guidance. Although the preparation and surface modifications of protein microspheres containing iron oxide nanoparticles has been previously described and feasibility studies conducted, many questions regarding their production and properties remain. Since the use of multifunctional microspheres could have high clinical relevance, here we report a detailed characterization of their properties and behavior in different environments to highlight their versatility. The work presented here is an effort for the development and optimization of nanoparticle-based microspheres as multi-modal contrast agents that can bridge imaging modalities on different size scales.

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Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy

Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA) was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation.

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Optical coherence tomography and targeted multi-modal protein microspheres for cancer imaging

The field of biomedical optics has grown quickly over the last two decades as various technological advances have helped increase the acquisition speeds and the sensitivity limits of the technology. During this time, optical coherence tomography (OCT) has been explored for a wide number of clinical applications ranging from cardiology to oncology to primary care. In this thesis, I describe the design and construction of an intraoperative clinical OCT system that can be used to image and classify breast cancer tumor margins as normal, close, or positive. I also demonstrate that normal lymph nodes can be distinguished from reactive or metastatic lymph nodes by looking at the difference in scattering intensity between the cortex and the capsule of the node. Despite the advances of OCT in the detection and diagnosis of breast cancer, this technology is still limited by its field of view and can only provide structural information about the tissue. Structural OCT would benefit from added contrast via sub-cellular or biochemical components via the use of contrast agents and functional OCT modalities. As with most other optical imaging techniques, there is a trade off between the imaging field of view and the high-resolution microscopic imaging. In this thesis, I demonstrate for the first time that MM-OCT can be used as a complimentary technique to wide field imaging modalities, such as magnetic resonance imaging (MRI) or fluorescence imaging, using targeted multi-modal protein microspheres. By using a single contrast agent to bridge the wide field and microscopic imaging modalities, a wide field imaging technique can be used to initially localize the contrast agent at the site of interest to guide the location of the MM-OCT imaging to provide a microscopic view. In addition to multi-modal contrast, the microspheres were functionalized with RGD peptides that can target various cancer cell lines. The cancer cells readily endocytosed bound protein microspheres, revealing the possibility that these protein microspheres could also be used as therapeutic agents. These investigations furthered the utility of the OCT technology for cancer imaging and diagnosis.

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Low-coherence interferometry and optical coherence tomography for image-guided surgical treatment of solid tumors

A method of forming an image of tissue. The method includes beginning an invasive procedure on a patient exposing tissue. The method then includes acquiring OCT data from the exposed tissue and converting the OCT data into at least one image. The method also includes ending the invasive procedure after the converting of the data.

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Challenges and opportunities for reinvigorating the physician-scientist pipeline

Physician-scientists, with in-depth training in both medicine and research, are uniquely poised to address pressing challenges at the forefront of biomedicine. In recent years, a number of organizations have outlined obstacles to maintaining the pipeline of physician-scientists, classifying them as an endangered species. As in-training and early-career physician-scientists across the spectrum of the pipeline, we share here our perspective on the current challenges and available opportunities that might aid our generation in becoming independent physician-scientists. These challenges revolve around the difficulties in recruitment and retention of trainees, the length of training and lack of support at key training transition points, and the rapidly and independently changing worlds of medical and scientific training. In an era of health care reform and an environment of increasingly sparse NIH funding, these challenges are likely to become more pronounced and complex. As stakeholders, we need to coalesce behind core strategic points and regularly assess the impact and progress of our efforts with appropriate metrics. Here, we expand on the challenges that we foresee and offer potential opportunities to ensure a more sustainable physician-scientist workforce.

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High resolution live cell Raman imaging using subcellular organelle-targeting SERS-sensitive gold nanoparticles with highly narrow intra-nanogap

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 μW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules.

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Measuring uptake dynamics of multiple identifiable carbon nanotube species via high-speed confocal Raman imaging of live cells

Carbon nanotube uptake was measured via high-speed confocal Raman imaging in live cells. Spatial and temporal tracking of two cell-intrinsic and nine nanotube-derived Raman bands was conducted simultaneously in RAW 264.7 macrophages. Movies resolved single (n, m) species, defects, and aggregation states of nanotubes transiently as well as the cell position, denoted by lipid and protein signals. This work portends the real-time molecular imaging of live cells and tissues using Raman spectroscopy, affording multiplexing and complete photostability.

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Toward a better understanding of the retention of physician-scientists in the career pipeline

We question the implications of the study by Jeffe and Andriole,1 who assembled a novel database from disparate sources to investigate the role of Medical Scientist Training Program (MSTP) funding for MD/PhD student training. MSTPs (i.e., MSTP programs) were stratified by duration of MSTP funding to their respective institutions. As reported in Table 2, recent MSTPs were more similar in student prematriculation characteristics to non-MSTPs than they were to long-standing MSTPs. Because the authors did not compare all MSTPs with all non-MSTPs, their concluding recommendation that future studies take into account the MSTP funding status of MD/PhD trainees should be evaluated with the duration of MSTP funding to the institution in mind.

The authors found that female and minority students were more likely to graduate from long-standing MSTPs than from non-MSTPs. However, the analysis did not normalize the ethnic and gender diversity of the MD/PhD cohort to the overall medical student cohort at each respective school. Thus, it is unclear whether the increased diversity is due to MSTP funding or certain institution-specific factors. Intra-institutional normalization could also have been performed on other variables (e.g., MCAT score and the undergraduate institution’s Carnegie Classification).

Another potential confounder of the analysis is the research milieu in which the long-standing MSTPs function, that is, the home medical school. For example, as a crude analysis, out of the 43 medical schools with MSTPs in 2010–2011, 36 (84%) were concurrently among the top 43 medical school recipients of NIH funding in 2010.2 We maintain that the institutional environment plays a more integral role in the development of physician–scientists than does the funding mechanism. Institutions giving higher priority to research are more likely to have invested in the proper infrastructure and resources to support MD/PhD students and to fully fund them. With the authors’ finding of increased MD/PhD graduate debt linked to increased likelihood of pursuing a nonuniversity clinical practice, further investigation is warranted regarding the role of institutional trainee support, level of financial support, and sources of funding beyond MSTP support alone.

Based on Table 4, there was no significant difference between long-standing MSTP, recent MSTP, and non-MSTP graduates regarding pursuing a career outside that of full-time faculty/research scientist. This suggests that obtaining both the MD and PhD degrees, regardless of MSTP funding, is in itself sufficient for this outcome. However, a comparison of the students’ research career intentions at the time of matriculation—from the AAMC Matriculating Student Questionnaire (MSQ)—with their intentions at the time of graduation would have been a better measurement of the influence of MSTP funding on the persistence of career intentions. The fact remains that no studies have shown the predictive value of career intentions on actual outcomes.3,4

Incorporating information from the MSQ and implementing postgraduation longitudinal studies would provide a better understanding of the impact of factors such as training environment and funding support on the retention of physician–scientists in the career pipeline.

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Targeted multi-modal protein microspheres for cancer imaging

Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications from ophthalmology to oncology. OCT is analogous to ultrasound but with micron-scale resolution by using light waves instead of sound waves to provide detailed structural information at the cellular level. The development of contrast agents has been critical to the development of OCT and its functional modalities such as magneto-motive OCT (MM-OCT). MM-OCT is a modality of OCT in which a small external magnetic field is modulated on and off during imaging, providing an added dimension of contrast from the magnetic particle responses. Protein microspheres consisting of a hydrophobic oil core and a hydrophilic BSA protein shell provide the vehicle for a multi-modal contrast agent. The microspheres encapsulate iron oxide nanoparticles in the oil core, providing magnetic signal contrast, and dyes such as Nile Red and DiR iodide, providing fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Using dynamic light scattering, we found the size distribution of the microspheres to be between 1-5 µm. Under SEM and TEM, we were able to visualize the various layers and coatings, such as silica and RGD peptides, of the microsphere. The microspheres were optimized to maximize the magnetic contrast under MM-OCT and MRI, and the fluorescent contrast under a dark box fluorescence imaging system, and fluorescence microscopy. These studies validated the use of MM-OCT as a method for quantifying the relative amount of iron oxide and the relative number of microspheres in the samples. To address the binding specificity and sensitivity of the RGD coated microspheres to the integrin receptors, the microspheres were incubated with cell lines of varying expression levels of the alpha(v)beta(3) integrin receptor and visualized under fluorescence microscopy. The cell lines used in this study included a normal epithelial cell line: hTERT-HME1, and several human breast cancer cell lines: HCC38, SK-BR-3, MCF-7, ZR-75-1, MDA-MB-231, and MDA-MB-435S. These results were externally validated by quantification of the receptors using indirect immunohistochemical staining and flow cytometry. Preliminary results, using the multi-spectral dark box fluorescence imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor and to lymph nodes. This is highly suggestive of the microsphere’s selective binding to the vasculature. By combining the benefits of these various imaging modalities in a single agent, it becomes possible to use a wide-field imaging method such as MRI or small animal fluorescence imaging to initially locate the agents in-vivo, to use MM-OCT to provide micron scale resolution structural images in-vivo, and to use fluorescence microcopy to confirm the localization of these particles ex-vivo.

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