Synovial fluid pH sensor for early detection of prosthetic hip infections.

We describe an implantable sensor developed to measure synovial fluid pH for noninvasive early detection and monitoring of hip infections using standard-of-care plain radiography. The sensor was made of a pH responsive polyacrylic acid-based hydrogel, which expands at high pH and contracts at low pH. A radiodense tantalum bead and a tungsten wire were embedded in the two ends of the hydrogel in order to monitor the change in length of the hydrogel sensor in response to pH via plain radiography. The effective pKa of the hydrogel-based pH sensor was 5.6 with a sensitivity of 3 mm/pH unit between pH 4 and 8. The sensor showed a linear response and reversibility in the physiologically relevant pH range of pH 6.5 and 7.5 in both buffer and bovine synovial fluid solutions with a 30-minute time constant. The sensor was attached to an explanted prosthetic hip and the pH response determined from the X-ray images by measuring the length between the tantalum bead and the radiopaque wire. Therefore, the developed sensor would enable noninvasive detection and studying of implant hip infection using plain radiography.

Contrast agents for x-ray luminescence computed tomography.

Imaging probes are an important consideration for any type of contrast agent-based imaging method. X-ray luminescence imaging (XLI) and x-ray luminescence computed tomography (XLCT) are both contrast agent-based imaging methods that employ x-ray excitable scintillating imaging probes that emit light to be measured for optical imaging. In this work, we compared the performance of several select imaging probes, both commercial and self-synthesized, for application in XLI/XLCT imaging. Commercially available cadmium telluride quantum dots (CdTe QDs) and europium-doped gadolinium oxysulfide (GOS:Eu) microphosphor as well as synthesized NaGdF4 nanophosphors doped with either europium or terbium were compared through their x-ray luminescence emission spectra, luminescence intensity, and also by performing XLCT scans using phantoms embedded with each of the imaging probes. Each imaging probe displayed a unique emission spectrum that was ideal for deep-tissue optical imaging. In terms of luminescence intensity, due to the large particle size, GOS:Eu had the brightest emission, followed by NaGdF4:Tb, NaGdF4:Eu, and finally the CdTe QDs. Lastly, XLCT scans showed that each imaging probe could be reconstructed with good shape and location accuracy.

Upconversion Spectral Rulers for Transcutaneous Displacement Measurements.

We describe a method to measure micron to millimeter displacement through tissue using an upconversion spectral ruler. Measuring stiffness (displacement under load) in muscles, bones, ligaments, and tendons is important for studying and monitoring healing of injuries. Optical displacement measurements are useful because they are sensitive and noninvasive. Optical measurements through tissue must use spectral rather than imaging approaches because optical scattering in the tissue blurs the image with a point spread function typically around the depth of the tissue. Additionally, the optical measurement should have low background and minimal intensity dependence. Previously, we demonstrated a spectral encoder using either X-ray luminescence or fluorescence, but the X-ray luminescence required an expensive X-ray source and used ionizing radiation, while the fluorescence sensor suffered from interference from autofluorescence. Here, we used upconversion, which can be provided with a simple fiber-coupled spectrometer with essentially autofluorescence-free signals. The upconversion phosphors provide a low background signal, and the use of closely spaced spectral peaks minimizes spectral distortion from the tissue. The small displacement noise level (precision) through tissue was 2 µm when using a microscope-coupled spectrometer to collect light. We also showed proof of principle for measuring strain on a tendon mimic. The approach provides a simple method to study biomechanics using implantable sensors.

An implanted pH sensor read using radiography.

A biomedical sensor was developed to measure local pH near orthopedic implants to detect and study implant-associated infection. The sensor is read using plain radiography, a technique which is noninvasive, inexpensive, ubiquitously available in medical facilities, and routinely used in diagnosis and follow-up. The sensor comprises a radiopaque tungsten indicator pin embedded within a chemically responsive hydrogel that exhibits a pH-dependent swelling. A stainless steel well holds this hydrogel and attaches to an orthopedic plate. The local pH may be determined from the extent of hydrogel swelling by radiographically measuring the indicator position relative to the well. We calibrated the sensor in a series of standard pH buffers and tested it during bacterial growth in culture. The sensor was robust: its response was negligibly affected by changes in temperature, ionic strength within the normal physiological range, or long-term incubation with reactive oxygen species generated from hydrogen peroxide and copper. Pooled data from several sensors fabricated at different times and tested in different conditions had a root-mean-square deviation from a pH electrode reading of 0.24 pH units. Radiographic measurements were also performed in cadaveric tissue with the sensor attached to an orthopedic plate fixed to a tibia. Pin position readings varied by 100 µm between observers surveying the same radiographs, corresponding to 0.065 pH units precision in the range pH 4-8. The sensor was designed to augment standard radiographs of tissue, bony anatomy, and hardware by also indicating local chemical concentrations.

One-Pot Hydrothermal Synthesis of TbIII13(GeO4)6O7(OH) and K2TbIVGe2O7: Preparation of a Stable Terbium(4+) Complex.

Two terbium germanates have been synthesized via high-temperature and high-pressure hydrothermal synthesis with 20 M KOH as a mineralizer using Tb4O7 as a starting material. Tb13(GeO4)6O7(OH) crystallizes in trigonal space group R3̅, is built up of isolated GeO4 units, and contains a complex arrangement of terbium oxide polyhedra. K2TbGe2O7 is a terbium(4+) pyrogermanate that is isostructural with K2ZrGe2O7 and displays a rare stable Tb4+ oxidation state in the solid state.

Synthesis of brightly PEGylated luminescent magnetic upconversion nanophosphors for deep tissue and dual MRI imaging.

A method is developed to fabricate monodispersed biocompatible Yb/Er or Yb/Tm doped ß-NaGdF4 upconversion phosphors using polyelectrolytes to prevent irreversible particle aggregation during conversion of the precursor, Gd2 O(CO3 )2.H2 O:Yb/Er or Yb/Tm, to ß-NaGdF4 :Yb/Er or Yb/Tm. The polyelectrolyte on the outer surface of nanophosphors also provided an amine tag for PEGylation. This method is also employed to fabricate PEGylated magnetic upconversion phosphors with Fe3 O4 as the core and ß-NaGdF4 as a shell. These magnetic upconversion nanophosphors have relatively high saturation magnetization (7.0 emu g(-1) ) and magnetic susceptibility (1.7 × 10(-2) emu g(-1) Oe(-1) ), providing them with large magnetophoretic mobilities. The magnetic properties for separation and controlled release in flow, their optical properties for cell labeling, deep tissue imaging, and their T1 - and T2 -weighted magnetic resonance imaging (MRI) relaxivities are studied. The magnetic upconversion phosphors display both strong magnetophoresis, dual MRI imaging (r1 = 2.9 mM(-1) s(-1) , r2 = 204 mM(-1) s(-1) ), and bright luminescence under 1 cm chicken breast tissue.

Multifunctional yolk-in-shell nanoparticles for pH-triggered drug release and imaging.

Multifunctional nanoparticles are synthesized for both pH-triggered drug release and imaging with radioluminescence, upconversion luminescent, and magnetic resonance imaging (MRI). The particles have a yolk-in-shell morphology, with a radioluminescent core, an upconverting shell, and a hollow region between the core and shell for loading drugs. They are synthesized by controlled encapsulation of a radioluminescent nanophosphor yolk in a silica shell, partial etching of the yolk in acid, and encapsulation of the silica with an upconverting luminescent shell. Metroxantrone, a chemotherapy drug, was loaded into the hollow space between X-ray phosphor yolk and up-conversion phosphor shell through pores in the shell. To encapsulate the drug and control the release rate, the nanoparticles are coated with pH-responsive biocompatible polyelectrolyte layers of charged hyaluronic acid sodium salt and chitosan. The nanophosphors display bright luminescence under X-ray, blue light (480 nm), and near infrared light (980 nm). They also served as T1 and T2 MRI contrast agents with relaxivities of 3.5 mM(-1) s(-1) (r1 ) and 64 mM(-1) s(-1) (r2 ). These multifunctional nanocapsules have applications in controlled drug delivery and multimodal imaging.

Nanotechnologies for noninvasive measurement of drug release.

A wide variety of chemotherapy and radiotherapy agents are available for treating cancer, but a critical challenge is to deliver these agents locally to cancer cells and tumors while minimizing side effects from systemic delivery. Nanomedicine uses nanoparticles with diameters in the range of ∼1-100 nm to encapsulate drugs and target them to tumors. The nanoparticle enhances local drug delivery efficiency to the tumors via entrapment in leaky tumor vasculature, molecular targeting to cells expressing cancer biomarkers, and/or magnetic targeting. In addition, the localization can be enhanced using triggered release in tumors via chemical, thermal, or optical signals. In order to optimize these nanoparticle drug delivery strategies, it is important to be able to image where the nanoparticles distribute and how rapidly they release their drug payloads. This Review aims to evaluate the current state of nanotechnology platforms for cancer theranostics (therapeutic and diagnostic particles) that are capable of noninvasive measurement of release kinetics.

Detecting De-gelation through Tissue Using Magnetically Modulated Optical Nanoprobes (MagMOONs).

Alginate gels are widely used for drug delivery and implanted devices. The rate at which these gels break down is important for controlling drug release. Since the de-gelation may be different in vivo, monitoring this process in situ is essential. However, it is challenging to monitor the gel through tissue due to optical scattering and tissue autofluorescence. Herein we describe a method to detect through tissue the chemically-induced changes in viscosity and de-gelation process of alginate gels using magnetically modulated optical nanoprobes (MagMOONs). The MagMOONs are fluorescent magnetic microspheres coated with a thin layer of opaque metal on one hemisphere. The metal layer prevents excitation and emission light from passing through one side of the MagMOONs, which creates orientation-dependent fluorescence intensity. The magnetic particles also align in an external magnetic field and give blinking signals when they rotate to follow an external modulated magnetic field. The blinking signals from these MagMOONs are distinguished from background autofluorescence and can be tracked on a single particle level in the absence of tissue, or for an ensemble average of particles blinking through tissue. When these MagMOONs are dispersed in calcium alginate gel, they become sensors for detecting gel degradation upon addition of either ammonium ion or alginate lyase. Our results show MagMOONs start blinking approximately 10 minutes after 2 mg/mL alginate lyase addition and this blinking is clearly detected even through up to 4 mm chicken breast. This approach can potentially be employed to detect bacterial biofilm formation on medical implants by sensing specific proteases that either activate a related function or regulate biofilm formation. It can also be applied to other biosensors and drug delivery systems based on enzyme-catalyzed breakdown of gel components.

Spatial Resolution for X-ray Excited Luminescence Chemical Imaging (XELCI).

Measuring chemical concentrations at the surface of implanted medical devices is important for elucidating the local biochemical environment, especially during implant infection. Although chemical indicator dyes enable chemical measurements in vitro, they are usually ineffective when measuring through tissue because the background obscures the dye signal and scattering dramatically reduces the spatial resolution. X-ray excited luminescent chemical imaging (XELCI) is a recent imaging modality which overcomes these limitations using a focused X-ray beam to excite a small spot of red light on scintillator-coated medical implants with well-defined location (because X-rays are minimally scattered) and low background. A spectrochemical indicator film placed over the scintillator layer, e.g., a polymer film containing pH-indicator dyes, absorbs some of the luminescence according to the local chemical environment, and this absorption is then detected by measuring the light intensity/spectrum passing through the tissue. A focused X-ray beam is used to scan point-by-point with a spatial resolution mainly limited by the X-ray beam width with minimum increase from X-ray absorption and scattering in the tissue. X-ray resolution, implant surface specificity, and chemical sensitivity are the three key features of XELCI. Here, we study spatial resolution using optically absorptive targets. For imaging a series of lines, the 20-80% knife-edge resolution was ∼285 (±15) µm with no tissue and 475 ± 18 and 520 ± 34 µm, respectively, through 5 and 10 mm thick tissue. Thus, doubling the tissue depth did not appreciably change the spatial resolution recorded through the tissue. This shows the promise of XELCI for submillimeter chemical imaging through tissue.

Oxygenation imaging in deep tissue with X-Ray luminescence computed tomography (XLCT).

Oxygenation concentration of tissue is an important factor in culturing stem cells and in studying the therapy response of cancer cells. The hypoxia bone marrow is the site to harbor cancer cells. Thus, direct high-resolution measurements of molecular 𝑂2 would provide powerful means of monitoring cultured stem cells and therapied cancer cells. We proposed an imaging approach to measure oxygenation concentration in deep tissues, based on the XLCT, with combined strengths of high chemical sensitivity and high spatial resolution. We have developed different biosensing films for oxygenation measurements and tested these films with X-ray luminescent experiments. We have also performed phantom experiments with multiple targets to validate the XLCT imaging system with measurements at two channels.

LITE-1 mediates behavioral responses to X-rays in Caenorhabditis elegans.

Rapid sensory detection of X-ray stimulation has been documented across a wide variety of species, but few studies have explored the underlying molecular mechanisms. Here we report the discovery of an acute behavioral avoidance response in wild type Caenorhabditis elegans to X-ray stimulation. The endogenous C. elegans UV-photoreceptor protein LITE-1 was found to mediate the locomotory avoidance response. Transgenic expression of LITE-1 in C. elegans muscle cells resulted in paralysis and egg ejection responses to X-ray stimulation, demonstrating that ectopic expression of LITE-1 can confer X-ray sensitivity to otherwise X-ray insensitive cells. This work represents the first demonstration of rapid X-ray based genetically targeted (X-genetic) manipulation of cellular electrical activity in intact behaving animals. Our findings suggest that LITE-1 has strong potential for use in this minimally invasive form of neuromodulation to transduce transcranial X-ray signals for precise manipulation of neural activity in mammals, bypassing the need for invasive surgical implants to deliver stimulation.

Lignocellulosic-Based Nanoparticles with Photoluminescent Properties for Bioimaging.

Natural and renewable resources from plants or animals are an important source of biomaterials due to their biocompatibility and high availability. Lignin is a biopolymer present in the biomass of plants, where it is intertwined and cross-linked with other polymers and macromolecules in the cell walls, generating a lignocellulosic material with potential applications. We have prepared lignocellulosic-based nanoparticles with an average size of 156 nm that exhibit a high photoluminescence signal when excited at 500 nm with emission in the near-infrared (NIR) region at 800 nm. The advantage of these lignocellulosic-based nanoparticles is their natural luminescent properties and their origin from rose biomass waste, which eliminates the need for encapsulation or functionalization of imaging agents. Moreover, the in vitro cell growth inhibition (IC50) of lignocellulosic-based nanoparticles is about 3 mg/mL, and no in vivo toxicity was registered up to 57 mg/kg, which suggests that they are suitable for bioimaging applications. In addition, these nanoparticles can circulate in the blood and are excreted in urine. The combined high luminescence signal in NIR, small size, low in vitro toxicity, low in vivo toxicity, and blood circulation support the potential of lignin-based nanoparticles as a novel bioimaging agent.

Surface-enhanced raman scattering detection of pH with silica-encapsulated 4-mercaptobenzoic acid-functionalized silver nanoparticles.

Sensors based upon surface-enhanced Raman spectroscopy (SERS) are attractive because they have narrow, vibrationally specific spectral peaks that can be excited using red and near-infrared light which avoids photobleaching, penetrates tissue, and reduces autofluorescence. Several groups have fabricated pH nanosensors by functionalizing silver or gold nanoparticle surfaces with an acidic molecule and measuring the ratio of protonated to deprotonated Raman bands. However, a limitation of these sensors is that macromolecules in biological systems can adsorb onto the nanoparticle surface and interfere with measurements. To overcome this interference, we encapsulated pH SERS sensors in a 30 nm thick silica layer with small pores which prevented bovine serum albumin (BSA) molecules from interacting with the pH-indicating 4-mercaptobenzoic acid (4-MBA) on the silver surfaces but preserved the pH-sensitivity. Encapsulation also improved colloidal stability and sensor reliability. The noise level corresponded to less than 0.1 pH units from pH 3 to 6. The silica-encapsulated functionalized silver nanoparticles (Ag-MBA@SiO(2)) were taken up by J774A.1 macrophage cells and measured a decrease in local pH during endocytosis. This strategy could be extended for detecting other small molecules in situ.

Advances in functional X-ray imaging techniques and contrast agents.

X-rays have been used for non-invasive high-resolution imaging of thick biological specimens since their discovery in 1895. They are widely used for structural imaging of bone, metal implants, and cavities in soft tissue. Recently, a number of new contrast methodologies have emerged which are expanding X-ray's biomedical applications to functional as well as structural imaging. These techniques are promising to dramatically improve our ability to study in situ biochemistry and disease pathology. In this review, we discuss how X-ray absorption, X-ray fluorescence, and X-ray excited optical luminescence can be used for physiological, elemental, and molecular imaging of vasculature, tumors, pharmaceutical distribution, and the surface of implants. Imaging of endogenous elements, exogenous labels, and analytes detected with optical indicators will be discussed.

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Super-Fast Three-Dimensional Focused X-ray Luminescence Computed Tomography with a Gated Photon Counter.

X-ray luminescence computed tomography (XLCT) is a hybrid molecular imaging modality combining the merits of both x-ray imaging (high spatial resolution) and optical imaging (high sensitivity to tracer nanophosphors). Narrow x-ray beam based XLCT imaging has shown promise for high spatial resolution imaging, but the slow acquisition speed limits its applications for in vivo imaging. We introduced a continuous scanning scheme to replace the selective excitation scheme to improve imaging speed in a previous study. Under the continuous scanning scheme, the main factor that limits the scanning speed is the data acquisition time at each interval position. In this work, we have used a gated photon counter (SR400, Stanford Research Systems) to replace the high-speed oscilloscope (MDO3104, Tektronix) to acquire measurement data. The gated photon counter only counts the photon peaks in each measurement interval, while the oscilloscope records the entire waveform including both background noise data and photon peak data. The photon counter records much less data without losing any relevant information, which makes it ideal for super-fast three-dimensional (3D) imaging. We have built prototype XLCT imaging systems of both types and performed both single target and multiple target phantom experiments in 3D. The results have verified the feasibility of our proposed photon counter based system and good 3D imaging capabilities of XLCT within a reasonable time, yielding a 14 times faster scanning time compared with the oscilloscope based XLCT system. Now, the total scan time is reduced to 27 seconds per transverse section.

pH variation in medical implant biofilms: Causes, measurements, and its implications for antibiotic resistance.

The advent of implanted medical devices has greatly improved the quality of life and increased longevity. However, infection remains a significant risk because bacteria can colonize device surfaces and form biofilms that are resistant to antibiotics and the host's immune system. Several factors contribute to this resistance, including heterogeneous biochemical and pH microenvironments that can affect bacterial growth and interfere with antibiotic biochemistry; dormant regions in the biofilm with low oxygen, pH, and metabolites; slow bacterial growth and division; and poor antibody penetration through the biofilm, which may also be regions with poor acid product clearance. Measuring pH in biofilms is thus key to understanding their biochemistry and offers potential routes to detect and treat latent infections. This review covers the causes of biofilm pH changes and simulations, general findings of metabolite-dependent pH gradients, methods for measuring pH in biofilms, effects of pH on biofilms, and pH-targeted antimicrobial-based approaches.

Polyphenol effects on CuO-nanoparticle-mediated DNA damage, reactive oxygen species generation, and fibroblast cell death.

The ability of ten polyphenolic antioxidants to prevent CuO nanoparticle (NPCuO) and H2O2-mediated DNA damage and cytotoxicity was investigated. Five of the polyphenols (MEPCA, PREGA, MEGA, ECG, and EGCG) prevent NPCuO/H2O2-mediated DNA damage (IC50 values of 7.5-800 µM), three have no effect (PCA, VA, and EC), and two (GA and EGC) result in increased DNA damage. Most polyphenols had similar antioxidant/prooxidant activity in the presence of NPCuO or free copper ions. Electron paramagnetic resonance (EPR) spectroscopy of reactive oxygen species (ROS) generated by NPCuO/H2O2 in the presence of representative polyphenols correlate with results of DNA damage studies: in the presence of NPCuO/H2O2, MEPCA prevents ROS formation, VA has no effect on ROS levels, and EGC increases ROS levels. EPR results with CuO nanoparticles washed to remove dissolved copper in solution (wCuO) in the presence of H2O2/ascorbate suggest that MEPCA prevents ROS formation on the nanoparticle surface in addition to preventing ROS formation from dissolved copper. In mouse fibroblast (L929) cells, combining NPCuO with H2O2 results in significantly greater cytotoxicity than observed for either component alone. After 3 h incubation with MEPCA or MEGA, the viability loss in L929 cells induced by NPCuO/H2O2 challenge was significantly rescued at physiologically relevant polyphenol levels (1 µM). These studies show that polyphenols can protect DNA and inhibit cytotoxicity generated by NPCuO under oxidative stress conditions.

High-resolution chemical imaging through tissue with an X-ray scintillator sensor.

We describe a novel method for high-resolution chemical imaging on a surface embedded in tissue. The sensor surface consists of an X-ray scintillator film coated in a thin film loaded with chemical indicator dye. A narrow scanning X-ray beam is used to excite luminescence from X-ray scintillators located within the beam. This luminescence passes through the indicator film, and the spectrum is analyzed to measure chemical concentrations at that location. A pH sensor is demonstrated with a dynamic range between pH 6-9 and noise level of 0.05 pH units using methyl-red dyed pH paper. The location of the interface between two types of scintillator films is obtained with 0.30 mm spatial resolution even though the images are highly blurred by 10 mm of chicken breast. This work has important applications for detecting pH changes on surfaces of implanted medical devices.