Proc. SPIE. 9723, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VIII
KEYWORDS: Infrared imaging, Near infrared, Proteins, Imaging systems, Magnetic resonance imaging, Luminescence, Inflammation, Flow cytometry, Colon, In vivo imaging, Fluorescent proteins, In vitro testing, Bacteria
Current treatment of inflammatory bowel disease (IBD) is largely symptomatic and consists of anti-inflammatory agents, immune-suppressives or antibiotics, whereby local luminal action is preferred to minimize systemic side-effects. Recently, anti-TNFα therapy has shown considerable success and is now being routinely used. Here we present a novel approach of using perfluorocarbon (PFC) nanoemulsion containing hydrogels (nanoemulgels) as imaging supported delivery systems for anti-TNF alpha probiotic delivery in IBD. To further facilitate image-guided therapy a food-grade lactic acid bacterium Lactococcus lactis capable of TNFα-binding was engineered to incorporate infrared fluorescent protein (IRFP). This modified bacteria was then incorporated into novel PFC nanoemulgels. The nanoemulgels presented here are designed to deliver locally anti-TNFα probiotic in the lower colon and rectum and provide dual imaging signature of gel delivery (MRI) across the rectum and lower colon and bacteria release (NIR). NIR imaging data in vitro demonstrates high IRFP expressing and TNFα-binding bacteria loading in the hydrogel and complete release in 3 hours. Stability tests indicate that gels remain stable for at least 14 days showing no significant change in droplet size, zeta potential and pH. Flow cytometry analyses demonstrate the NIRF expressing bacteria L. lactis binds TNFα in vitro upon release from the gels. Magnetic resonance and near-infrared imaging in vitro demonstrates homogeneity of hydrogels and the imaging capacity of the overall formulation.
In vitro studies were initiated to determine the suitability of murine and rat macrophages as delivery vehicles for gold
nanoshells in the treatment of gliomas. Visualization of macrophage accumulation in and around gliomas may be
accomplished using magnetic resonance imaging (MRI) and superparamagnetic iron oxide nanoparticles (SPIO). The
optimal loading of both murine and rat macrophages with SPIO was determined using inductively coupled plasma
atomic emission spectroscopy (ICP-AES). Higher concentrations of SPIO were observed in rat macrophages and the
optimal concentration in these cell lines was around 300 μg/ml. Higher concentrations resulted in significant cell
toxicity. SPIO were visualized in fixed rat brains subjected to high field MRI using T<sub>2</sub>*-weighted gradient echo pulse
sequences. Macrophages were found to be very sensitive to near infra-red (NIR) laser irradiation.
Specific absorption rate (SAR) heating using radiofrequency (RF) waves is affected by the RF frequency and amplitude,
and the conductivity of the tissue. Recently, conductive nanoparticles were demonstrated to induce hyperthermia in vitro
and in vivo upon irradiation with an external 13.56 MHz RF field. The addition of conductive nanoparticles was assumed
to increase the tissue conductivity and SAR. However, no quantitative studies have been performed that characterize the
conductivities of biocompatible colloids or tissues containing nanoparticles, and relate the conductivity to SAR.
The complex permittivities were measured for colloids containing single-wall carbon nanotubes (SWCNTs) in normal
saline with 0.32% w/v Pluronic F108 nonionic surfactant. The carbon concentrations of the colloids ranged from 0 to 88
mM. The permittivities were measured using a dielectric probe and RF network analyzer for RF frequencies from 200
MHz to 3 GHz. The nonionic surfactant was added to the colloids to minimize flocculation of the nanotubes during the
RF heating experiments. The results were compared with prior measurements of colloids containing 0.02% Pluronic
F108. The dielectric and conductivity of the 0.02% Pluronic colloids rose linearly with carbon concentration but the
0.32% Pluronic colloids varied from linearity.
Based on the permittivity results, selected colloid samples were placed inside a Bruker 7T/20 magnetic resonance (MR)
imaging (MRI) system and irradiated at 300 MHz using a high duty cycle RF pulse sequence. The temperature changes
were measured directly using fiber-optic thermometers and indirectly using MR thermometry and spectroscopy.
Temperature changes were consistent with the colloid conductivities.
Introduction: Failure to eradicate infiltrating glioma cells using conventional treatment
regimens results in tumor recurrence and is responsible for the dismal prognosis of
patients with glioblastoma multiforme (GBM). This is due to the fact that these migratory
cells are protected by the blood-brain barrier (BBB) which prevents the delivery of most
anti-cancer agents. We have evaluated the ability of photochemical internalization (PCI)
to selectively disrupt the BBB in rats. This will permit access of anti-cancer drugs to
effectively target the infiltrating tumor cells, and potentially improve the treatment
effectiveness for malignant gliomas.
Materials and Methods: PCI treatment, coupling a macromolecule therapy of
<i>Clostridium perfringens (Cl p)</i> epsilon prototoxin with AlPcS<sub>2a</sub>-PDT, was performed on
non-tumor bearing inbred Fisher rats. T1-weighted post-contrast magnetic resonance
imaging (MRI) scans were used to evaluate the extent of BBB disruption which can be
inferred from the volume contrast enhancement.
Results: The synergistic effect of PCI to disrupt the BBB was observed at a fluence level
of 1 J with an intraperitoneal injection of <i>Cl p</i> prototoxin. At the fluence level of 2.5J, the
extent of BBB opening induced by PCI was similar to the result of PDT suggesting no
synergistic effect evoked under these conditions.
Conclusion: PCI was found to be highly effective and efficient for inducing selective and
localized disruption of the BBB. The extent of BBB opening peaked on day 3 and the
BBB was completed restored by day 18 post treatment.