Research Fellow @ Massachusetts Institute of Technology, Resident Physician @ Mount Sinai Hospital

Publication

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.

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.

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.

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.

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

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.

RGD coated protein microspheres as a dual fluorescent and magnetomotive contrast agent for targeted cancer imaging with magnetomotive optical coherence tomography

Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications ranging from ophthalmology to oncology. OCT is analogous to ultrasound technology but with micron by using light waves instead of sound waves providing detailed morphological or structural information at the cellular level about the tissue specimen. Magneto-motive OCT (MM-OCT) is a recently developed modality of OCT in which a magnetic field is modulated on and off during imaging. With the development of this modality, exogeneous contrast agents are becoming more important to target markers that are expressed prior to morphological changes that structural OCT can only detect. Modified protein microspheres consisting of an oil core and a hydrophilic BSA protein shell are being presented as a multi-modal contrast agent vehicle. The protein microspheres are encapsulated with iron oxide in the oil core to provide the magnetic signal contrast and a near infrared dye to provide a fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Under MM-OCT, these agents have been detected above various levels of background tissue scattering demonstrating that these agents can provide added contrast to OCT through the magnetic signal. These agents were incubated with various cell lines with differing levels of alpha(v)beta(3) integrin receptor expression that were quantified using western blotting and fluorescent antibody immunohistochemical staining. The normal control cell line used was the CRL-4010. The breast cancer cell lines studied included CRL-2314, SK-BR-3, MCF-7, and 13762 MAT B III cells. These studies address the binding specificity and sensitivity of the RGD functionalized protein microspheres to the alpha(v)beta(3) integrin receptors. In addition, a quantitative analysis is being performed to correlate the relative levels of bound microspheres to the cells, measured through MM-OCT measurements and through their fluorescence signals of the microspheres, and the cell’s alpha(v)beta(3) integrin receptor expression derived from the western blot experiments. Preliminary results indicate that these agents have a higher affinity to the cancer cells over the normal epithelial cells and are also internalized by the cells and could have to potential to become localized targeted drug delivery vehicles. In an NMU carcinogen induced rat animal model, the targeted protein microspheres were injected in-vivo. These preliminary results, using a multi-spectral dark box imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor. These microspheres are being presented as a novel contrast agent to a novel high resolution imaging modality targeted at cancer.

Magnetic protein microspheres as dynamic contrast agents for magnetomotive optical coherence tomography

Optical coherence tomography (OCT) is an emerging biomedical imaging modality that has been developed over the last 15 years. More recently, OCT has been used for the intraoperative imaging of tumor margins in breast cancer and axillary lymph nodes providing a real time in-vivo assessment of the tissue morphology. Traditional OCT images are limited by only being able to observe morphological structures. As diagnostic medicine continues to push for earlier detection, one must develop functional imaging modalities that would detect molecular information in-vivo allowing a real-time microscopic analysis of the tissue specimen. A novel modality of OCT called magnetomotive-OCT (MMOCT) has been developed by our group, employing an induced modulated magnetic field with a magnetic contrast agent to create the added contrast to structural OCT images. Modified protein microspheres with a BSA protein shell functionalized with RGD peptide sequences for targeting and an oil core have been designed and synthesized. Magnetic nanoparticles (Fe3O4) and Nile Red dye have been encapsulated into its oil core. These microspheres have previously been demonstrated to target cancer cells by functionalizing them with a layer of RGD peptides and could be functionalized with monoclonal antibodies. Preliminary results show that these magnetic microspheres, which are 2.0- 5.0 microns in size, are readily detectable under MM-OCT when embedded in a 5% agarose gel, in a 3-D scaffold of macrophage cells previously incubated with the microspheres, and when injected in-vivo into a tumor from an NMUcarcinogen rat animal model for breast cancer.

Publication

Multimodal biomedical imaging with asymmetric single-walled carbon nanotube/iron oxide nanoparticle complexes

Magnetic iron oxide nanoparticles and near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWNT) form heterostructured complexes that can be utilized as multimodal bioimaging agents. Fe catalyst-grown SWNT were individually dispersed in aqueous solution via encapsulation by oligonucleotides with the sequence d(GT)15, and enriched using a 0.5 T magnetic array. The resulting nanotube complexes show distinct NIR fluorescence, Raman scattering, and visible/NIR absorbance features, corresponding to the various nanotube species. AFM and cryo-TEM images show DNA-encapsulated complexes composed of a approximately 3 nm particle attached to a carbon nanotube on one end. X-ray diffraction (XRD) and superconducting quantum interference device (SQUID) measurements reveal that the nanoparticles are primarily Fe2O3 and superparamagnetic. The Fe2O3 particle-enriched nanotube solution has a magnetic particle content of approximately 35 wt %, a magnetization saturation of approximately 56 emu/g, and a magnetic relaxation time scale ratio (T1/T2) of approximately 12. These complexes have a longer spin-spin relaxation time (T2 approximately 164 ms) than typical ferromagnetic particles due to the smaller size of their magnetic component while still retaining SWNT optical signatures. Macrophage cells that engulf the DNA-wrapped complexes were imaged using magnetic resonance imaging (MRI) and NIR mapping, demonstrating that these multifunctional nanostructures could potentially be useful in multimodal biomedical imaging.

Research Profiles

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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.

Email: freddytn@mit.edu

Arnold O. Beckman Postdoctoral Fellow
Institute for Medical Engineering and Science

Research Fellow, MIT Innovation Initiative
Former Co-Director, MIT Hacking Medicine
Regional Director – Europe, MIT Hacking Medicine
Co-Director, MIT COVID-19 Challenge
Co-Director, MIT Hacking Racism Challenge

Massachusetts Institute of Technology
77 Massachusetts Avenue
Cambridge, MA 02139

Email: freddy.nguyen@mountsinai.org

Resident Physician, PGY-3,
Department of Pathology, Molecular and Cell-Based Medicine

Icahn School of Medicine at Mount Sinai
Mount Sinai Hospital
One Gustave L. Levy Place, Box 1194
New York, NY 10029