Fabrication and characterization of UV-emitting nanoparticles as novel radiation sensitizers targeting hypoxic tumor cells.

Radiation therapy is one of the primary therapeutic techniques for treating cancer, administered to nearly two-thirds of all cancer patients. Although largely effective in killing cancer cells, radiation therapy, like other forms of cancer treatment, has difficulty dealing with hypoxic regions within solid tumors. The incomplete killing of cancer cells can lead to recurrence and relapse. The research presented here is investigating the enhancement of the efficacy of radiation therapy by using scintillating nanoparticles that emit UV photons. UV photons, with wavelengths between 230 nm and 280 nm, are able to inactivate cells due to their direct interaction with DNA, causing a variety of forms of damage. UV-emitting nanoparticles will enhance the treatment in two ways: first by generating UV photons in the immediate vicinity of cancer cells, leading to direct and oxygen-independent DNA damage, and second by down-converting the applied higher energy X-rays into softer X-rays and particles that are more efficiently absorbed in the targeted tumor region. The end result will be nanoparticles with a higher efficacy in the treatment of hypoxic cells in the tumor, filling an important, unmet clinical need. Our preliminary experiments show an increase in cell death using scintillating LuPO4:Pr nanoparticles over that achieved by the primary radiation alone. This work describes the fabrication of the nanoparticles, their physical characterization, and the spectroscopic characterization of the UV emission. The work also presents in vitro results that demonstrate an enhanced efficacy of cell killing with x-rays and a low unspecific toxicity of the nanoparticles.

Compact dual-mode diffuse optical system for blood perfusion monitoring in a porcine model of microvascular tissue flaps.

In reconstructive surgery, the ability to detect blood flow interruptions to grafted tissue represents a critical step in preventing postsurgical complications. We have developed and pilot tested a compact, fiber-based device that combines two complimentary modalities-diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy-to quantitatively monitor blood perfusion. We present a proof-of-concept study on an in vivo porcine model (n=8). With a controllable arterial blood flow supply, occlusion studies (n=4) were performed on surgically isolated free flaps while the device simultaneously monitored blood flow through the supplying artery as well as flap perfusion from three orientations: the distal side of the flap and two transdermal channels. Further studies featuring long-term monitoring, arterial failure simulations, and venous failure simulations were performed on flaps that had undergone an anastomosis procedure (n=4). Additionally, benchtop verification of the DCS system was performed on liquid flow phantoms. Data revealed relationships between diffuse optical measures and state of occlusion as well as the ability to detect arterial and venous compromise. The compact construction of the device, along with its noninvasive and quantitative nature, would make this technology suitable for clinical translation.

A capillary-mimicking optical tissue phantom for diffuse correlation spectroscopy.

Optical tissue phantoms are necessary for instrument benchmarking and providing a consistent baseline for experiments in various fields of tissue spectroscopy, including diffuse correlation spectroscopy (DCS). To provide the most useful comparisons, a phantom would ideally mimic tissue as closely as possible, including the geometry of static and dynamic scatterers. A branching design that keeps the capillary cross section constant ensures that the same flow velocity is found throughout the phantom while allowing for single input and output fittings to feed all of the capillaries simultaneously. The direction of each capillary is randomized every few millimeters by randomly allocating 2 by 2 "twisting" squares within each layer. These squares swap the locations of four adjacent artificial capillaries either clockwise or counterclockwise. Numerical simulations were used to verify the random walk-like behavior of the capillary paths resulting from this pattern. This is a step toward replicating the randomly varying directionality of actual capillaries. This design was verified by taking DCS measurements at different flow rates of Intralipid through the phantom, demonstrating the effect of the flow rate on the characteristic decay time of the autocorrelation.

Novel diffuse optics system for continuous tissue viability monitoring - extended recovery in vivo testing in a porcine flap model.

In reconstructive surgery, tissue perfusion/vessel patency is critical to the success of microvascular free tissue flaps. Early detection of flap failure secondary to compromise of vascular perfusion would significantly increase the chances of flap salvage. We have developed a compact, clinically-compatible monitoring system to enable automated, minimally-invasive, continuous, and quantitative assessment of flap viability/perfusion. We tested the system's continuous monitoring capability during extended non-recovery surgery using an in vivo porcine free flap model. Initial results indicated that the system could assess flap viability/perfusion in a quantitative and continuous manner. With proven performance, the compact form constructed with cost-effective components would make this system suitable for clinical translation.

In vivo preclinical verification of a multimodal diffuse reflectance and correlation spectroscopy system for sensing tissue perfusion.

In reconstructive surgery, impeded blood flow in microvascular free flaps due to a compromise in arterial or venous patency secondary to blood clots or vessel spasms can rapidly result in flap failures. Thus, the ability to detect changes in microvascular free flaps is critical. In this paper, we report progress on in vivo pre-clinical testing of a compact, multimodal, fiber-based diffuse correlation and reflectance spectroscopy system designed to quantitatively monitor tissue perfusion in a porcine model's surgically-grafted free flap. We also describe the device's sensitivity to incremental blood flow changes and discuss the prospects for continuous perfusion monitoring in future clinical translational studies.

Design verification of a compact system for detecting tissue perfusion using bimodal diffuse optical technologies.

It is essential to monitor tissue perfusion during and after reconstructive surgery, as restricted blood flow can result in graft failures. Current clinical procedures are insufficient to monitor tissue perfusion, as they are intermittent and often subjective. To address this unmet clinical need, a compact, low-cost, multimodal diffuse correlation spectroscopy and diffuse reflectance spectroscopy system was developed. We verified system performance via tissue phantoms and experimental protocols for rigorous bench testing. Quantitative data analysis methods were employed and tested to enable the extraction of tissue perfusion parameters. This design verification study assures data integrity in future in vivo studies.

Instrument to detect syncope and the onset of shock.

Currently the diagnosis of hemorrhagic shock is essentially clinical, relying on the expertise of nurses and doctors. One of the first measurable physiological changes that marks the onset of hemorrhagic shock is a decrease in capillary blood flow. Diffuse correlation spectroscopy (DCS) quantifies this decrease. DCS collects and analyzes multiply scattered, coherent, near infrared light to assess relative blood flow. This work presents a preliminary study using a DCS instrument with human subjects undergoing a lower body negative pressure (LBNP) protocol. This work builds on previous successful DCS instrumentation development and we believe it represents progress toward understanding how DCS can be used in a clinical setting.

A disposable, flexible skin patch for clinical optical perfusion monitoring at multiple depths.

Stable, relative localization of source and detection fibers is necessary for clinical implementation of quantitative optical perfusion monitoring methods such as diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy (DRS). A flexible and compact device design is presented as a platform for simultaneous monitoring of perfusion at a range of depths, enabled by precise location of optical fibers in a robust and secure adhesive patch. We will discuss preliminary data collected on human subjects in a lower body negative pressure model for hypovolemic shock. These data indicate that this method facilitates simple and stable simultaneous monitoring of perfusion at multiple depths and within multiple physiological compartments.

A scalable correlator for multichannel diffuse correlation spectroscopy.

Diffuse correlation spectroscopy (DCS) is a technique which enables powerful and robust non-invasive optical studies of tissue micro-circulation and vascular blood flow. The technique amounts to autocorrelation analysis of coherent photons after their migration through moving scatterers and subsequent collection by single-mode optical fibers. A primary cost driver of DCS instruments are the commercial hardware-based correlators, limiting the proliferation of multi-channel instruments for validation of perfusion analysis as a clinical diagnostic metric. We present the development of a low-cost scalable correlator enabled by microchip-based time-tagging, and a software-based multi-tau data analysis method. We will discuss the capabilities of the instrument as well as the implementation and validation of 2- and 8-channel systems built for live animal and pre-clinical settings.

A compact instrument to measure perfusion of vasculature in transplanted maxillofacial free flaps.

The vascularization and resulting perfusion of transferred tissues are critical to the success of grafts in buried free flap transplantations. To enable long-term clinical monitoring of grafted tissue perfusion during neovascularization and endothelialization, we are developing an implantable instrument for the continuous monitoring of perfusion using diffuse correlation spectroscopy (DCS), and augmented with diffuse reflectance spectroscopy (DRS). This work discusses instrument construction, integration, and preliminary results using a porcine graft model.

Advances in CMOS Solid-state Photomultipliers for Scintillation Detector Applications.

Solid-state photomultipliers (SSPMs) are a compact, lightweight, potentially low-cost alternative to a photomultiplier tube for a variety of scintillation detector applications, including digital-dosimeter and medical-imaging applications. Manufacturing SSPMs with a commercial CMOS process provides the ability for rapid prototyping, and facilitates production to reduce the cost. RMD designs CMOS SSPM devices that are fabricated by commercial foundries. This work describes the characterization and performance of these devices for scintillation detector applications. This work also describes the terms contributing to device noise in terms of the excess noise of the SSPM, the binomial statistics governing the number of pixels triggered by a scintillation event, and the background, or thermal, count rate. The fluctuations associated with these terms limit the resolution of the signal pulse amplitude. We explore the use of pixel-level signal conditioning, and characterize the performance of a prototype SSPM device that preserves the digital nature of the signal. In addition, we explore designs of position-sensitive SSPM detectors for medical imaging applications, and characterize their performance.

Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements.

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound H93G myoglobin (Mb) mutants to study the influence of the proximal ligand on the CO rebinding kinetics. In H93G mutants, where the proximal linkage with the protein is eliminated and the heme can bind exogenous ligands (e.g., imidazole, 4-bromoimidazole, pyridine, or dibromopyridine), we observe significant effects on the CO rebinding kinetics in the 10 ns to 10 ms time window. Resonance Raman spectra of the various H93G Mb complexes are also presented to aid in the interpretation of the kinetic results. For CO-bound H93G(dibromopyridine), we observe a rapid large-amplitude geminate phase with a fundamental CO rebinding rate that is approximately 45 times faster than for wild-type MbCO at 293 K. The absence of an iron proximal ligand vibrational mode in the 10 ns photoproduct Raman spectrum of CO-bound H93G(dibromopyridine) supports the hypothesis that proximal ligation has a significant influence on the kinetics of diatomic ligand binding to the heme.

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.