Freddy T. Nguyen, MD, PhD

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

Publication

Molecular Recognition and In Vivo Detection of Temozolomide and 5-Aminoimidazole-4-carboxamide for Glioblastoma Using Near-Infrared Fluorescent Carbon Nanotube Sensors

There is a pressing need for sensors and assays to monitor chemotherapeutic activity within the human body in real time to optimize drug dosimetry parameters such as timing, quantity, and frequency in an effort to maximize efficacy while minimizing deleterious cytotoxicity. Herein, we develop near-infrared fluorescent nanosensors based on single walled carbon nanotubes for the chemotherapeutic Temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide using Corona Phase Molecular Recognition as a synthetic molecular recognition technique. The resulting nanoparticle sensors are able to monitor drug activity in real-time even under in vivo conditions. Sensors can be engineered to be biocompatible by encapsulation in poly(ethylene glycol) diacrylate hydrogels. Selective detection of TMZ was demonstrated using U-87 MG human glioblastoma cells and SKH-1E mice with detection limits below 30 μM. As sensor implants, we show that such systems can provide spatiotemporal therapeutic information in vivo, as a valuable tool for pharmacokinetic evaluation. Sensor implants are also evaluated using intact porcine brain tissue implanted 2.1 cm below the cranium and monitored using a recently developed Wavelength-Induced Frequency Filtering technique. Additionally, we show that by taking the measurement of spatial and temporal analyte concentrations within each hydrogel implant, the direction of therapeutic flux can be resolved. In all, these types of sensors enable the real time detection of chemotherapeutic concentration, flux, directional transport, and metabolic activity, providing crucial information regarding therapeutic effectiveness.

Publication

Emerging technologies in cancer detection

Exciting, modern technologies for cancer detection are under development in academic and industrial laboratories worldwide. This chapter provides a synopsis of technologies reaching greater importance as they advance toward clinical practice. These methods include significant advances in current methods as well as fundamentally new platforms. We place a special emphasis on point-of-care technologies for use in clinical settings as well as novel methods for use as at-home measurements and wearable devices. We also provide a synopsis on the involvement of artificial intelligence-based data analytics such as machine learning algorithms in both existing and developing assessments.

Publication

A wavelength-induced frequency filtering method for fluorescent nanosensors in vivo

Fluorescent nanosensors hold the potential to revolutionize life sciences and medicine. However, their adaptation and translation into the in vivo environment is fundamentally hampered by unfavourable tissue scattering and intrinsic autofluorescence. Here we develop wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption peak of a nanosensor, allowing the emission signal to be separated from the autofluorescence background, increasing the desired signal relative to noise, and internally referencing it to protect against artefacts. Using highly scattering phantom tissues, an SKH1-E mouse model and other complex tissue types, we show that WIFF improves the nanosensor signal-to-noise ratio across the visible and near-infrared spectra up to 52-fold. This improvement enables the ability to track fluorescent carbon nanotube sensor responses to riboflavin, ascorbic acid, hydrogen peroxide and a chemotherapeutic drug metabolite for depths up to 5.5 ± 0.1 cm when excited at 730 nm and emitting between 1,100 and 1,300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, nanosensors aided by WIFF detect the chemotherapeutic activity of temozolomide transcranially at 2.4 ± 0.1 cm through the porcine brain without the use of fibre optic or cranial window insertion. The ability of nanosensors to monitor previously inaccessible in vivo environments will be important for life-sciences research, therapeutics and medical diagnostics.

Temporal Imaging of Live Cells by High-Speed Confocal Raman Microscopy
Publication

Temporal Imaging of Live Cells by High-Speed Confocal Raman Microscopy

Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.

Transcutaneous Measurement of Essential Vitamins Using Near-Infrared Fluorescent Single-Walled Carbon Nanotube Sensors
Publication

Transcutaneous Measurement of Essential Vitamins Using Near-Infrared Fluorescent Single-Walled Carbon Nanotube Sensors

Vitamins such as riboflavin and ascorbic acid are frequently utilized in a range of biomedical applications as drug delivery targets, fluidic tracers, and pharmaceutical excipients. Sensing these biochemicals in the human body has the potential to significantly advance medical research and clinical applications. In this work, a nanosensor platform consisting of single-walled carbon nanotubes (SWCNTs) with nanoparticle corona phases engineered to allow for the selective molecular recognition of ascorbic acid and riboflavin, is developed. The study provides a methodological framework for the implementation of colloidal SWCNT nanosensors in an intraperitoneal SKH1-E murine model by addressing complications arising from tissue absorption and scattering, mechanical perturbations, as well as sensor diffusion and interactions with the biological environment. Nanosensors are encapsulated in a polyethylene glycol diacrylate hydrogel and a diffusion model is utilized to validate analyte transport and sensor responses to local concentrations at the boundary. Results are found to be reproducible and stable after exposure to 10% mouse serum even after three days of in vivo implantation. A geometrical encoding scheme is used to reference sensor pairs, correcting for in vivo optical and mechanical artifacts, resulting in an order of magnitude improvement of p-value from 0.084 to 0.003 during analyte sensing.

Publication

Implantable Nanosensors for Human Steroid Hormone Sensing In Vivo Using a Self-Templating Corona Phase Molecular Recognition

Dynamic measurements of steroid hormones in vivo are critical, but steroid sensing is currently limited by the availability of specific molecular recognition elements due to the chemical similarity of these hormones. In this work, a new, self‐templating synthetic approach is applied using corona phase molecular recognition (CoPhMoRe) targeting the steroid family of molecules to produce near infrared fluorescent, implantable sensors. A key limitation of CoPhMoRe has been its reliance on library generation for sensor screening. This problem is addressed with a self‐templating strategy of polymer design, using the examples of progesterone and cortisol sensing based on a styrene and acrylic acid copolymer library augmented with an acrylated steroid. The pendant steroid attached to the corona backbone is shown to self‐template the phase, providing a unique CoPhMoRE design strategy with high efficacy. The resulting sensors exhibit excellent stability and reversibility upon repeated analyte cycling. It is shown that molecular recognition using such constructs is viable even in vivo after sensor implantation into a murine model by employing a poly (ethylene glycol) diacrylate (PEGDA) hydrogel and porous cellulose interface to limit nonspecific absorption. The results demonstrate that CoPhMoRe templating is sufficiently robust to enable a new class of continuous, in vivo biosensors.

Publication

A Fiber Optic Interface Coupled to Nanosensors: Applications to Protein Aggregation and Organic Molecule Quantification

Fluorescent nanosensors hold promise to address analytical challenges in the biopharmaceutical industry. The monitoring of therapeutic protein critical quality attributes such as aggregation is a longstanding challenge requiring low detection limits and multiplexing of different product parameters. However, general approaches for interfacing nanosensors to the biopharmaceutical process remain minimally explored to date. Herein, we design and fabricate a integrated fiber optic nanosensor element, measuring sensitivity, response time, and stability for applications to the rapid process monitoring. The fiber optic-nanosensor interface, or optode, consists of label-free nIR fluorescent single-walled carbon nanotube transducers embedded within a protective yet porous hydrogel attached to the end of the fiber waveguide. The optode platform is shown to be capable of differentiating the aggregation status of human immunoglobulin G, reporting the relative fraction of monomers and dimer aggregates with sizes 5.6 and 9.6 nm, respectively, in under 5 min of analysis time. We introduce a lab-on-fiber design with potential for at-line monitoring with integration of 3D-printed miniaturized sensor tips having high mechanical flexibility. A parallel measurement of fluctuations in laser excitation allows for intensity normalization and significantly lower noise level (3.7-times improved) when using lower quality lasers, improving the cost effectiveness of the platform. As an application, we demonstrate the capability of the fully-integrated lab-on-fiber system to rapid monitoring of various bioanalytes including serotonin, norepinephrine, adrenaline, and hydrogen peroxide, in addition to proteins and their aggregation states. These results in total constitute an effective form factor for nanosensor based transducers for applications in industrial process monitoring.

Publication

DNA-SWCNT Biosensors Allow Real-Time Monitoring of Therapeutic Responses in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic cancer with limited treatment options. There is an urgent need for tools that monitor therapeutic responses in real time. Drugs such as gemcitabine and irinotecan elicit their therapeutic effect in cancer cells by producing hydrogen peroxide (HO). In this study, specific DNA-wrapped single-walled carbon nanotubes (SWCNT), which precisely monitor HO, were used to determine the therapeutic response of PDAC cells and tumors . Drug therapeutic efficacy was evaluated by monitoring HO differences using reversible alteration of Raman G-bands from the nanotubes. Implantation of the DNA-SWCNT probe inside the PDAC tumor resulted in approximately 50% reduction of Raman G-band intensity when treated with gemcitabine versus the pretreated tumor; the Raman G-band intensity reversed to its pretreatment level upon treatment withdrawal. In summary, using highly specific and sensitive DNA-SWCNT nanosensors, which can determine dynamic alteration of hydrogen peroxide in tumor, can evaluate the effectiveness of chemotherapeutics. SIGNIFICANCE: A novel biosensor is used to detect intratumoral hydrogen peroxide, allowing real-time monitoring of responses to chemotherapeutic drugs.

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 leadership experience in community building in healthcare innovation, research, medical, and physician-scientist communities.

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