The feasibility of multi-wavelength photoplethysmography for the real-time sensing of absorptive and scattering agents in pulsatile blood is discussed. The use of pulsatile signals extracted from trans-illumination of an accessible section of tissue allows us to calculate the concentration of the optically extinctive species in the pulsatile blood. This technology, initially used for pulse oximetry and dye densitometry, can be applied to monitor in vivo concentration and clearance of various absorptive species. Recently, our prototype has been used monitor the concentration of therapeutic gold nanoparticles, antimalarial quinine, and the antifungal agent amphotericin B. The assessment of the optical properties, device specifications, and signal quality for each compound are presented. We observe that this technology can be used to monitor numerous extinctive drug and nano-materials that present features in the 350-1100 nm range. The rationale for using this technology in a clinical setting would be to improve outcomes by real-time pharmacological feedback and/or control at point of care in addition to the elimination of invasive blood draws for collection of data.
KEYWORDS: Blood, In vivo imaging, Nanoparticles, Absorption, Signal attenuation, Therapeutic agents, Oxygen, LabVIEW, Signal detection, Photoplethysmography
A novel multi-wavelength photoplethysmograph (PPG), previously utilized to quantify optically absorptive circulating gold nanoparticles, has demonstrated the potential to enhance therapeutic treatment predictability as pharmacokinetic metrics are provided throughout the intravenous delivery and clearance phase of amphotericin b (injected in the lipid form Abelcet®) in real-time. This report demonstrates how the PPG could be used to assess the real-time bioavailability of intravenously delivered optically-absorbing therapeutic agents. The drug currently under investigation is antifungal amphotericin b (absorption peak ~355 nm). We describe how the algorithm has been adapted to quantify the concentration of amphotericin b in the pulsatile, circulating blood based on its extinction at three wavelengths (355, 660 and 940 nm) corresponding to the peaks of amphotericin b and wavelengths for oxygen saturation measurements, respectively. We show an example of the system collecting data representing the baseline, injection, and the clearance phases. The PPG device showed a measurement range of concentrations between 0.0987 mg/mL to 0.025mg/ml in blood. An examination of the data obtained suggests that the system is well suited to sense the concentration of amphotericin b at a therapeutic dose (≈5 mg/kg/day).
More than a decade into the development of gold nanoparticles for cancer therapies, with multiple clinical trials underway, ongoing pre-clinical research continues towards better understanding in vivo interactions with the goal of treatment optimization through improved best practices. In an effort to collect information for healthcare providers, enabling informed decisions in a relevant time frame, instrumentation for real-time plasma concentration (multi-wavelength pulse photometry) and protocols for rapid elemental analysis (energy dispersive X-Ray fluorescence) of biopsied tumor tissue have been developed in a murine model. An initial analysis, designed to demonstrate the robust nature and utility of the techniques, revealed that area under the bioavailability curve (AUC) alone does not currently inform tumor accumulation with a high degree of accuracy (R2=0.32), This finding suggests that the control of additional experimental and physiological variables may yield more predictable tumor accumulation. Subject core temperature are blood pressure were monitored, but did not demonstrate clear trends. An effort to modulate AUC has produced an adjuvant therapy which is employed to enhance circulation parameters, including the AUC, of nanorods and gold nanoshells. Preliminary studies demonstrated a greater than 300% increase in average AUC through the use of a reticuloendothelial blockade agent versus control groups. Given a better understanding of the relative importance of the physiological factors which impact rates of tumor accumulation, a proposed set of experimental best practices is presented.
A novel multi-wavelength photoplethysmograph (PPG), previously utilized to quantify optically absorptive circulating gold nanoparticles, has demonstrated the potential to enhance therapeutic treatment predictability as pharmacokinetic metrics are provided throughout the intravenous delivery phase of quinine in real-time. This report demonstrates how the PPG could be used to assess the real-time bioavailability of other types of intravenously delivered optically-absorbing nanoparticles and drugs. The drug currently under investigation is anti-malarial quinine (absorption peak ~350 nm). We describe how the algorithm has been adapted to quantify the concentration of quinine in the pulsatile, circulating blood based on its extinction at three wavelengths (340, 660 and 940 nm). We show an example of the system collecting data representing the baseline, injection, and the clearance phases. An examination of the raw signal suggests that the system is well suited to sense the concentration of quinine in the therapeutic range (10mg/kg).
Researchers employ increasingly complex sub-micron particles for oncological applications to deliver bioactive
therapeutic or imaging compounds to known and unknown in vivo tumor targets. These particles are often
manufactured using a vast array of compounds and techniques resulting in a complex architecture, which can be
quantified ex vivo by conventional metrology and chemical assays. In practice however, experimental homogeneity
using nanoparticles can be difficult to achieve. While several imaging techniques have been previously shown to
follow the accumulation of nanoparticles into tumor targets, a more rapid sensor that provides a quantifiable estimate
of dose delivery and short-term systemic response could increase the clinical efficacy and greatly reduce the
variability of these treatments. We have developed an optical device, the pulse photometer, that when placed on an
accessible location will estimate the vascular concentration of near-infrared extinguishing nanoparticles in murine
subjects. Using a technique called multi-wavelength photoplethysmography, the same technique used in pulse
oximetry, our pulse photometer requires no baseline for each estimate allowing it to be taken on and off of the
subject several times during experiments employing long circulating nanoparticles. We present a formal study of
our prototype instrument in which circulation half-life and nanoparticle concentration of gold nanorods is
determined in murine subjects with the aid of light anesthesia. In this study, we show good agreement between
vascular nanorod concentrations (given in optical density) as determined by our device and with UV-VIS
spectrophotometry using low volume blood samples.
Researchers employ increasingly complex sub-micron particles for oncological applications to deliver bioactive
therapeutic or imaging compounds to known and unknown in vivo tumor targets. In practice, experimental homogeneity
using nanoparticles can be difficult to achieve. While several imaging techniques have been previously shown to follow
the accumulation of nanoparticles into tumor targets, a more rapid sensor that provides a quantifiable estimate of dose
delivery and short-term systemic response could increase the clinical efficacy and greatly reduce the variability of these
treatments. We have developed a pulse photometer that when placed on an optically accessible location will estimate the
concentration of near-infrared absorbing nanoparticles. The goal is to monitor the accuracy of the delivered dose and the
effective circulation time of nanoparticles immediately after intravenous delivery but prior to therapeutic intervention.
We present initial tests of our prototype using murine models to assess its ability to quantify circulation half-life and
nanoparticle concentration. Four mice were injected with nanoparticles and circulation half-life estimates ranged from 3-
43 minutes. UV-Vis spectrophotometry was used to independently verify these measurements using 5μL blood samples.
Linear models relating the two methods produced R2 values of 0.91, 0.99, 0.88, and 0.24.
Laser induced thermal therapy is used in conjunction with gold coated silica core nanoshells and magneticresonance
temperature imaging (MRTI). The nanoshells are embedded in phantom or in vivo tumors and
heat preferentially compared to surrounding tissue when the laser is applied. The tissues thermal response
is varied by either the laser power or the nanoshell concentration. In this way precise control of the heating
can be achieved. This results in the ability to quantitatively monitor therapeutic temperature changes that
occur in a spatiotemporally controlled way. This provides an unprecedented means proscribing and
monitoring a treatment in real time and the ability to make precise corrections when necessary.
A novel thermal therapy delivery technique using low power near infrared irradiation delivered to a distribution of gold-silica nanoshell particles under MR-guidance has been recently introduced. This research expands upon the previous research by using MR temperature imaging as a tool to investigate the spatiotemporal temperature distribution associated with accumulations of nanoshells after an intravenous injection of nanoshells into tumor bearing mice. Tumors were inoculated and grown subcutaneously in mice and intravenously injected nanoshells were allowed to accumulate passively through the associated leaky vasculature. MRI was used in the planning and post-therapy evaluation of treated sites while realtime MR temperature imaging (MRTI) monitored the distribution of temperature within tissue during the procedure. MRTI was demonstrated to be an excellent tool for determining the extent of thermal energy
delivered to the treatment region and could be useful for evaluating the efficiency of nanoshell uptake into a target tissue. This preliminary data demonstrates the feasibility of using MR-guidance for the control of in vivo nanoshell-based photo-thermal therapy. Furthermore, this feasibility study validates previous research that nanoshells will passively accumulate in tumor target tissues at clinically relevant concentrations.
Gold nanoshells are a new class of nanoparticles with tunable optical absorption that can be placed in the near infrared. Gold nanoshells consist of a spherical silica core surrounded by a thin gold shell. The ratio of the sizes of the core diameter to the shell thickness as well as the total size of the nanoshell determines the optical absorption properties. Previous experiments have shown that these nanoparticles are stable at >325°C for durations typical of laser tissue welding. We have investigated the use of gold nanoshells as exogenous NIR absorbers to facilitate ex vivo laser tissue soldering. For ex vivo testing, gold nanoshells with peak absorption at approximately 820 nm were suspended in an albumin solder formulation and applied to muscle strips, followed by irradiation of the tissue at 821 nm. Mechanical testing of nanoshell-solder welds in muscle revealed successful fusion of tissues with tensile strengths of the weld site equal to the native tissue. The use of thermally stable nanoshells as an exogenous absorber allows the usage of light sources that are minimally absorbed by tissue components, thereby minimizing damage to surrounding tissue and producing welds sufficient for wound closure.
This report focuses on the treatment parameters leading to successful nanoshell-assisted photo-thermal therapy (NAPT). NAPT takes advantage of the strong near infrared (NIR) absorption of gold-silica nanoshells, a new class of nanoparticles with tunable optical absorptivities that are capable of passive extravasation from the abnormal tumor vasculature due to their nanoscale size. Under controlled conditions nanoshells accumulate in tumors with superior efficiency compared to surrounding tissues. For this treatment: (1) tumors were inoculated in immune-competent mice by subcutaneous injection, (2) polyethylene glycol coated nanoshells (≈150 nm diameter) with peak optical absorption in the NIR were intravenously injected and allowed to circulate for 6 - 48 hours, and (3) tumors were then extracorporeally illuminated with a collimated diode laser (808 nm, 2-6 W/cm2, 2-4 min). Nanoshell accumulations were quantitatively assessed in tumors and surrounding tissues using neutron activation analysis for gold. In order to assess temperature elevation, laser therapies were monitored in real-time using a mid-infrared thermal sensor. NAPT resulted in complete tumor regression in >90% of the subjects. This simple, non-invasive procedure shows great promise as a technique for selective photo-thermal tumor treatment.
Low molecular weight molecules are typically very difficult to detect directly in solution using commercially available SPR (surface plasmon resonance) instruments. This is because the mass change on binding is not sufficient to cause a detectable change in refractive index on binding to surface- bound receptors (e.g., antibodies). Some receptors, however, undergo extensive changes in tertiary structure upon binding ligands. Here we present data suggesting conformational changes in surface-bound receptors such as periplasmic binding proteins and calcium-binding proteins can be detected by SPR. This SPR response can be used to monitor specific binding of carbohydrates and calcium even though the molecular weight of these analytes would be difficult to detect using traditional SPR methods. Therefore this approach has potential applications for developing optical biosensors for such small molecules.
Glucose monitoring is of critical importance in the life of Type I and many Type II diabetics. This research furthers work toward a minimally invasive implantable glucose sensor based on fluorescence detection. Current experimental models use heterogeneous fluorescence resonance energy transfer (FRET) systems for sensing; ideally, the response of one fluorophore bound to a large polysaccharide is enhanced greatly in the presence of glucose while the other fluorophore bound to a glucose sensitive protein is diminished or unaffected. Many fluorophores are affected by environmental factors such as pH and temperature. FRET experiments using two fluorophores, tetramethylrodamine isothiocyanate (TRITC) and fluoroscein isothiocyanate (FITC), are performed evaluating the effects of fluctuations over the range of pH 4-8 and temperature 25-45 degree(s)C for various concentrations of glucose in a flow cell. TRITC is bound to the lectin Concanavalin A (Con A), and FITC is bound to dextran molecules of varying sizes.
Progress towards a painless and hygienic glucose monitoring procedure for diabetics continues as the growth of diabetes mellitus reaches epidemic proportions in the American population. Utilizing an implantable fluorescence based glucose assay, the minimally invasive approach presented here has previously shown promise towards this goal in terms of glucose specificity and quantification for in vitro environments. However, in realistic physiological circumstances the depth of the implant can vary and optical properties of skin can change due to normal physiological conditions. Additionally, naturally occurring auto-fluorescence can obscure the sensor signal. An important concern under these conditions is that variations of fluorescent intensity due to these or other causes might be mistaken for glucose concentration fluctuations. New data shows that fluorescence-based glucose assays can be probed and interpreted in terms of glucose concentrations through pig skin at depths of up to 700 mm when immobilized in a bio-compatible polymer. When a combination of two fluorophores are employed as demonstrated here, reasonable changes in skin thickness and the confounding effects of the variations inherent in skin can be overcome for this glucose sensing application.
A painless monitoring procedure for diabetics has proven to be an elusive goal. While completely noninvasive measurements are the desired technique, minimally invasive procedures using implanted fluorescence sensor chemistry offer significant advantages in specificity over current noninvasive approaches. The goal was to evaluate the potential for transdermal glucose sensing using intensity measurements from implanted microspheres. A fiber-optic probe and spectrometer were custom-built for collection of in vivo data. Comparisons with commercial fluorometers show the constructed device is adequate for this project.
The detection of sever brain trauma remains difficult when employing traditional methods in part due to the pathophysiological complexity of the condition. Current brain trauma detection includes schemes that require bulky, expensive equipment to deduce regional cerebral blood flow. These methods are difficult to use in conjunction with patients requiring ongoing intensive care and constant monitoring. Our previous studies have shown that surface- enhanced Raman spectroscopy (SERS) with silver colloids has the ability to measure physiological concentrations of in vivo brain analytes linked to brain trauma using short scan times. More recently, after implementing a damage model for ischemia in rats, an ex vivo analysis of brain microdialysis samples shows a correlation between SERS spectral features and the occurrence and location of known localized ischaemia. A near real-time measurement system could provide relevant clinical information in anticipation of surgical or pharmaceutical interventions for severely head injured patients.
Traditionally methods for the detection of excitatory amino acids, which have been linked to secondary injury following head trauma, can be excessively time consuming clinically. A near real-time measurement system could provide clinical information in anticipation of pharmaceutical intervention for head injured patients. Our studies have shown that surface-enhanced Raman spectroscopy (SERS) with silver colloids has the ability to measure physiological concentrations of in vitro excitatory amino acids using short scan times. Employing a damage model for ischemia, preliminary ex vivo rat extracellular grain fluid analysis shows an intriguing correlation between SERS spectral features and expected Glutamate concentration fluctuations following head injuries.
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