Research Fellow @ Massachusetts Institute of Technology, Transfusion Medicine Fellow @ Dartmouth-Hitchcock Medical Center

Publication

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.

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.

Publication

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.

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.

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.

Magnetomotive optical coherence microscopy for cell dynamics and biomechanics

Magnetomotive microscopy techniques are introduced to investigate cell dynamics and biomechanics. These techniques are based on magnetomotive transducers present in cells and optical coherence imaging techniques. In this study, magnetomotive transducers include magnetic nanoparticles (MNPs) and fluorescently labeled magnetic microspheres, while the optical coherence imaging techniques include integrated optical coherence (OCM)and multiphoton (MPM) microscopy,and diffraction phase microscopy (DPM). Samples used in this study are murine macrophage cells in culture that were incubated with magnetomotive transducers. MPMis used to visualize multifunctional microspheres based on their fluorescence, while magnetomotive OCM detects sinusoidal displacements of the sample induced by a magnetic field. DPM is used to image single cells at a lower frequency magnetic excitation, and with its Fourier transform light scattering (FTLS) analysis, oscillation amplitude is obtained, indicating the relative biomechanical properties of macrophage cells. These magnetomotive microscopy method shave potential to be used to image and measure cell dynamics and biomechanical properties. The ability to measure and understand biomechanical properties of cells and their microenvironments, especially for tumor cells, is of great importance and may provide insight for diagnostic and subsequently therapeutic interventions.

Publication

Targeted multifunctional multimodal protein-shell microspheres as cancer imaging contrast agents

PURPOSE: In this study, protein-shell microspheres filled with a suspension of iron oxide nanoparticles in oil are demonstrated as multimodal contrast agents in magnetic resonance imaging (MRI), magnetomotive optical coherence tomography (MM-OCT), and ultrasound imaging. The development, characterization, and use of multifunctional multimodal microspheres are described for targeted contrast and therapeutic applications.PROCEDURES: A preclinical rat model was used to demonstrate the feasibility of the multimodal multifunctional microspheres as contrast agents in ultrasound, MM-OCT and MRI. Microspheres were functionalized with the RGD peptide ligand, which is targeted to α(v)β₃ integrin receptors that are over-expressed in tumors and atherosclerotic lesions.RESULTS: These microspheres, which contain iron oxide nanoparticles in their cores, can be modulated externally using a magnetic field to create dynamic contrast in MM-OCT. With the presence of iron oxide nanoparticles, these agents also show significant negative T2 contrast in MRI. Using ultrasound B-mode imaging at a frequency of 30 MHz, a marked enhancement of scatter intensity from in vivo rat mammary tumor tissue was observed for these targeted protein microspheres.CONCLUSIONS: Preliminary results demonstrate multimodal contrast-enhanced imaging of these functionalized microsphere agents with MRI, MM-OCT, ultrasound imaging, and fluorescence microscopy, including in vivo tracking of the dynamics of these microspheres in real-time using a high-frequency ultrasound imaging system. These targeted oil-filled protein microspheres with the capacity for high drug-delivery loads offer the potential for local delivery of lipophilic drugs under image guidance.

Publication

Fourier Transform Light Scattering (FTLS) of Cells and Tissues

Fourier transform light scattering (FTLS) has been recently developed as a novel, ultrasensitive method for studying light scattering from inhomogeneous and dynamic structures. FTLS relies on quantifying the optical phase and amplitude associated with a coherent image field and propagating it numerically to the scattering plane. In this paper, we review the principle and applications of FTLS to static and dynamic light scattering from biological tissues and live cells. Compared with other existing light scattering techniques, FTLS has significant benefits of high sensitivity, speed, and angular resolution. We anticipate that FTLS will set the basis for disease diagnosis based on intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.

Publication

Congressionally Directed Medical Research Programs – Breast Cancer Research Program

Freddy Nguyen, an M.D./Ph.D. student in Professor Stephen Boppart’s Biophotonics Imaging Laboratory, was awarded an FY07 BCRP
Predoctoral Traineeship Award to optimize the use of an innovative imaging technology, magnetomotive optical coherence tomography (MM-OCT), which can provide real-time microscopic analysis of tumor
cells. Specifically, Mr. Nguyen’s project is to develop and optimize protein microspheres as a multimodal contrast agent to be used in conjunction with MM-OCT.
Mr. Nguyen has focused on encapsulating iron oxide nanoparticles and fluorescent dyes into the inner cores of modified protein microspheres capable of specifically targeting tumor neovessels, which are the blood vessels that tumors form to support their rapid growth. Tumor neovessel specificity was achieved by coating the microspheres with an arginine-glycine-asparatate (RGD) peptide, which binds to the αvβ3 integrin receptor on the surface of tumor neovessel endothelial cells. Preliminary studies confirmed that the microspheres preferentially bind to the tumor cells because they overexpress αvβ3 integrins in vitro. The microspheres accumulated in the neoves- sels at the tumor sites when injected into tumor-bearing rats. Mr. Nguyen plans to further pur- sue the cancer-specific targeting of the protein microspheres as a potential diagnostic contrast agent as well as a therapeutic agent in the treatment of breast cancer.

Physician-scientist with extensive experience developing and translating nanotechnologies and biomedical optical technologies from the bench to clinic in areas of genetics, oncology, and cardiovascular diseases. Extensive experience in community building in healthcare innovation, research, medical, and physician-scientist communities through various leadership roles.

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