Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.
Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the
near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica) /shell (gold) ratio. In addition to
spectral tunability, GNS are inert and bioconjugatable making them potential labels for in vivo imaging and therapy of
tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow band
imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells
in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating
phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the
optimum wavelengths for ex-vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected
with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and
retention (EPR) effect. Ex-vivo NBI of tumor xenografts demonstrated tumor specific heterogeneous distribution of GNS
with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as
contrast agents to visualize tumors using NBI.
Given their tunable optical properties and high optical absorption and scattering cross sections, gold nanoshells (GNS) have been explored for a number of in vitro and in vivo imaging contrast and cancer therapy agents. While it has been shown that GNSs preferentially accumulate at the tumor site, little is known about the accumulation kinetics within the tumor. We demonstrate accumulation kinetics of GNSs in bulk tumors and histology slides using two-photon induced photoluminescence (TPIP) imaging. We found that GNSs had a heterogeneous distribution with higher accumulation at the tumor cortex. In addition, GNSs were observed in unique patterns surrounding the perivascular region. These results demonstrate that direct luminescence based imaging of metal nanoparticles provides high resolution and molecular specific multiplexed images.
Noninvasive imaging of epidermal growth factor (EGF) receptor (EGFR) expression can provide valuable molecular
information that could aid diagnostic and therapeutic decisions, particularly with targeted cancer therapies utilizing anti-EGFR antibodies. In this study we report on the development and validation of a nanoprobe for in-vivo imaging and
discrimination of EGFR-overexpressing tumors from surrounding normal tissues that also expresses EGFR. Near-infrared
quantum dots (QDs) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes.
These nanoprobes demonstrated excellent in-vitro and in-vivo binding affinity. In-vivo imaging demonstrated three
distinct phases of tumor influx (~3min), clearance (~60min) and accumulation (1-6hrs) of EGF-QD nanoprobes. Both
QD and EGF-QD demonstrated non-specific rapid tumor influx and clearance followed by an apparent dynamic
equilibrium at ~60min. Subsequently (1-6hrs), while QD concentration gradually decreased in tumors, EGF-QDs
progressively accumulated in tumors. At 24hrs, tumor fluorescence decreased to near baseline levels for both QD and
EGF-QD. Ex vivo whole-organ, tissue-homogenate fluorescence, confocal microscopy and immunofluorescence staining
confirmed tumor-specific accumulation of EGF-QD nanoprobes at an early time-point (4hrs). The favorable
pharmacokinetics, the ability to discriminate EGFR-overexpressing tumors from surrounding normal tissues using low
concentration (10-pmol) of EGF-QD nanoprobe underscores the clinical relevance of this probe to evaluate therapeutic
intervention.
Despite convincing evidence for hyperthermic radiosensitization, the invasive means of achieving and monitoring
hyperthermia and the lack of good thermal dosimetry have hindered its use in routine clinical practice. A non-invasive
method to generate and monitor hyperthermia would provide renewed enthusiasm for such treatments. Near-infrared
absorbing gold nanoshells have been shown to accumulate preferentially in tumors via the enhanced permeability and
retention effect and have been used for thermal ablation of tumors. We evaluated the use of these nanoshells to generate
hyperthermia to evaluate the anti-tumor effects of combining gold nanoshell mediated hyperthermia with radiotherapy.
Laser settings were optimized for hyperthermia in a mouse xenograft model to achieve a temperature rise of 40- 41°C in
the tumor periphery and 37-38°C (ΔT=4-5°C) deeper within the tumors. The ΔT measurements were verified using both
thermocouple and magnetic resonance thermal imaging (MRTI) temperature measurements. Tumor re-growth delay was
estimated by measuring tumor size after treatment with radiation (10Gy single dose), hyperthermia (15 minutes at 40°C),
and hyperthermia followed by radiation and control. Significant difference (p <0.05) in the tumor volume doubling time
was observed between the radiation group (13 days) and combination treatment group (25 days). The
immunofluorescence staining for the hypoxic, proliferating cells and the vasculature corroborated our hypothesis that the
radiosensitization is in part mediated by increased initial perfusion and subsequent collapse of vasculature that leads to
acute inflammatory response in the tumor. The increased vascular perfusion immediately after gold nanoshell mediated
hyperthermia is confirmed by dynamic contrast enhanced magnetic resonance imaging.
KEYWORDS: Skin, Cryogenics, Injuries, Blood vessels, Temperature metrology, Ear, Laser irradiation, Laser therapeutics, In vivo imaging, In vitro testing
Cutaneous laser treatment in dark skin patients is challenging due to significant light absorption by the melanin at the basal layer of epidermis, which can result in irreversible nonspecific thermal injury to the epidermis. Cryogen spray cooling (CSC) with R-134a (boiling point –26.2°C at 1 atm), which is currently used during cutaneous laser treatment, has shown poor efficacy in protecting dark human skin. We investigated the potential of CSC with R-404a (boiling point –46.5°C at 1 atm), which has a lower boiling point than R-134a, for improved therapeutic outcome in dark human skin at three levels: in vitro (epoxy resin skin phantom), ex vivo (normal dark human skin sample), and in vivo (skin of the rabbit external ear). The skin phantom was used to acquire the surface and internal temperature profiles in response to CSC with R-134a or R-404a at various spurt durations, based upon which CSC-induced heat removal from the skin phantom was estimated using an algorithm that solved a one-dimensional inverse heat conduction problem. CSC with R-404a increased the temperature reductions within the phantom and subsequently the amount of heat removal from the phantom in comparison to that with R-134a. Normal ex vivo Fitzpatrick types V-VI human skin samples were used to investigate the thermal response of dark human skin epidermis to CSC (R-134a or R-404a) at various spurt durations in conjunction with 595-nm pulsed dye laser irradiation at various radiant exposures. Cryogen R-404a increased the threshold radiant exposures for irreversible thermal injury to the epidermis in dark pigmentation skin. No obvious CSC-induced morphological changes to human skin was observed when sprayed with R404-a spurts using durations up to 300 ms. In vivo rabbit ear vasculature was used as a model of cutaneous anomalies to assess the influences of CSC (with R-134a or R-404a) on the photothermolysis of dermal blood vessels. CSC (R-134a or R-404a) with the spurt durations of 100 to 300 ms increased the most superficial depth of ...
While initially confined to the epidermis, squamous cell carcinoma can eventually penetrate into the underlying tissue if not diagnosed early and treated. The noninvasive early detection of the carcinoma is important to achieve a complete treatment of the disease. Of the various non-invasive optical techniques, the synchronous fluorescence (SF) technique is considered to provide a simplified spectral profile with more sharp spectral signatures of the endogenous fluorophores in complex systems. The potential use of the SF technique in the characterization of the sequential tissue transformation in 7,12-dimethylbenz(a)anthracene–12-O-tetradecanoylphorbol-13-acetate (DMBA-TPA)-induced mouse skin tumor model in conjunction with simple statistical analysis is explored. The SF spectra show distinct differences during the earlier weeks of the tumor-induction period. Intensity ratio variables are calculated and used in three discriminant analyses. All the discriminant analyses show better classification results with accuracy greater than 80%. From the observed differences in the spectral characteristics and the ratio variables that resulted in better classification between groups, it is concluded that tryptophan, collagen, and NADH are the key fluorophores that undergo changes during tissue transformation process and hence they can be targeted as tumor markers to diagnose normal from abnormal tissues using the SF technique.
The use of autofluorescence technique in the characterization of the sequential tissue transformation process in 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate (DMBA & TPA) induced two-stage mouse skin carcinogenesis model in conjunction with a suitable statistical method is being explored. The fluorescence excitation emission matrix (EEM) from experimental group (n=40; DMBA/TPA application), control group (n=6; acetone application) and the blank group (n=6; no application of DMBA/TPA or acetone) were measured every week using Fluoromax3 spectrofluorometer coupled with a waveguide fiber optic bundle (JY Horiba, NJ). The EEM was recorded for 19 excitation wavelengths from 280 to 460 nm at 10 nm intervals and the fluorescence emission was scanned from 300 to 750 nm. During the tissue transformation the epithelial tissues underwent biochemical and structural changes that are manifested in the tissue fluorescence. To correlate the tissue morphology with the observed fluorescence differences in the fluorescence emission, animals were sacrificed and the tissue biopsies were subjected to histopathological evaluation. The fluorescence emission corresponding to different fluorophores was extracted from the EEM, and the spectral data were used in multivariate statistical algorithm for the earliest diagnosis of the onset of tissue transformation. The intrinsic fluorescence from tryptophan, NADH and prophyrins showed distinct differences in the spectral signature during the tissue transformation, due to the altered metabolic activities of the cells. The statistical analysis of the spectral data corresponding to each excitation wavelength showed better classification accuracy at 280, 320, 350 and 405 nm excitations, corresponding to tryptophan, collagen, NADH and porphyrins with the classification accuracy of 74.3, 68.1, 64.6 and 74.7 % respectively. The variations in the spectral signature and the results of the statistical analysis suggest that porphyrins, tryptophan and NADH can be targeted as potential tumor markers in the early detection of the tissue transformation process.
Indocyanine Green (ICG) is clinically used as a fluorescent dye for imaging purposes. Its rapid circulation kinetics and minimal toxicity has prompted investigation into ICG's utility as a photosentitizer for therapeutic applications. Traditionally, optically mediated tumor therapy has focused on photodynamic therapy, which employs a photochemical mechanism resulting from the absorption of low intensity CW laser light by localized photosensitizers such as Photofrin II, Benzoporphyrin Derivative (BPD), ICG. Treatment of cutaneous vascular malformations such as port-wine stains, on the other hand, is based on a photothermal mechanism resulting from the absorption of high intensity pulsed laser light by hemoglobin. In this study, we compared the effectiveness of combining photochemical and photothermal mechanisms during application of ICG in conjunction with laser irradiation with the intention that the combined approach may lead to a reduction in the threshold dose of pulsed laser light required to treat hypervascular malformations. The blood vessels in rabbit ears were used as an in vivo model for targeted vasculature. Irradiation of the ears with IR light (λ=785 nm, Δτ = 3 min, Io = 120 mW) was used to elicit photochemical damage, while photothermal damage was brought about using pulses from a ruby laser (λ=694 nm, τ = 3 ms) with different fluences. For the combined modality, photochemical damage was induced first and followed by photothermal irradiation. This modality was compared with photothermal irradiation alone. The effectiveness of each irradiation scheme was assessed using histopathological analysis. We present preliminary data that suggests that pretreatment with photodynamic therapy before photothermal coagulation results in more severe vascular damage with lower photothermal fluence levels. The results of this study provide the foundation work for further exploration of the therapeutic potentials of photochemical and photothermal effects during application of ICG in conjunction with laser irradiation.
KEYWORDS: Photodynamic therapy, Tumors, Tissues, Luminescence, Mathematical modeling, In vivo imaging, Diffusion, Data modeling, Laser therapeutics, Cancer
In the recent years (delta) -aminolevulinic acid ((delta) -ALA) a precursor for the endogenous production of protoporphyrin IX (PPIX) has gained importance in the Photodynamic Therapy (PDT) of superficial and early-stage cancers. Though (delta) -ALA is present naturally in the cells, systemic administration of exogenous (delta) -ALA leads to the production of intracellular endogenous PP IX in both the tumor and the normal cells, but with varying concentration. However, the PPIX is accumulated more in the tumor tissues as the fast growing tumor cells take up the administered (delta) -ALA more than the normal cells. As the therapeutic efficacy of PDT is dependent on the post (delta) -ALA incubation time, at which the tumor to normal ratio of the PPIX concentration is high, the concentration of the PPIX in the normal and the tumor site were estimated using fluorescence spectroscopy. However, the estimation of the PPIX concentration during/after PDT is mandatory, as the PDT dosimetry is dependent on the sensitizer concentration at the target of interest. The observed variation in the concentration of PPIX in the tumor site with respect to the unexposed normal surrounding tissues, may be attributed to the diffusion of PPIX from the surrounding normal tissues to the tumor site, across the concentration gradient. Based on this a mathematical model has been proposed, to estimate the rate parameter for the diffusion of PPIX from the surrounding normal tissues in to the tumor tissue (Km), due to photobleaching during PDT at two different fluence. The Km value at two different fluences, 57.6 and 36 J/cm2 are estimated as 5.444+/- 1.186 and 3.221+/- 0.957, respectively. Further, the rate parameter for the cleavage and efflux of (delta) -ALA (K1), and the rate parameter for the evasion of the PPIX from the tumor tissues during PDT (Kt), were also estimated by fitting the experimental data to the developed mathematical model. The estimated parameters will be utilized to estimate the exact concentration of PPIX in the tumor tissues for a better PDT efficacy.
Native fluorescence spectra of tissues of both normal and cervical carcinoma were measured at 320 nm excitation. The spectra were normalized with respect to maximum peak intensity and average fluorescence spectra were computed for normal and cancerous cervical tissues. The average fluorescence spectrum of normal cervix is found to be significantly different from that of cancer. Both normal and cancerous cervical tissues show a distinct primary emission peak around 380 nm which may be attributed to the structural proteins, collagen and elastin. In addition, the average spectrum of normal cervix shows a less intense peak around 440 nm, which is red shifted to 460nm and more intense in the case of cancerous cervical tissues, when compared to normal. A ratio parameter I380/I460 is introduced and a critical value of 1.9 for this ratio parameter is found to classify cervical cancerous tissues from normal with a sensitivity and specificity of 100% and 85.7% respectively. Also the slope value analysis between 420 to 440nm & 440 to 460nm of the normal and cancerous samples gives 100% accuracy in the classification of normal from the cancerous samples. Further, discriminant analysis together with Principal component analysis (PCA) was performed to determine the statistical significance and to confirm the above two analysis. The potentiality of the ratio parameter and slope values analysis is improved by the support of multivariate statistical analysis.
Photo-oxidation was studied during PDT in a model system using NADPH as the target substrate and Photofrin II as the photosensitizer. The efficiency of NADPH oxidation per photon absorbed by photosensitizer was determined as a function of substrate concentration. The kinetics of changes in absorbance was measured to assay oxidation of NADPH. The efficiency of oxidation increased with increasing NADPH concentration and approached a stable value of 0.0048. Using typical values for the quantum efficiency of the Photofrin triplet state generation and the efficiency of singlet oxygen production we obtained a value of the efficiency of interaction between singlet oxygen and NADPH. Efficiency of diffusion of singlet oxygen was modeled with molecular diffusion theory using typical values for the singlet oxygen lifetime and the diffusion coefficient in aqueous solutions.
A new photosensitizer, Trithia Sapphyrin was synthesized with 3 sulfurs and 2 nitrogen inside the macrocycle. The relative effectiveness is influenced by molecular properties, which control the penetration and distribution of the sensitizer in the system prior to the photon capture. To be precise, the effectiveness of the sensitizer depends on (1) the ability to partition from the bathing medium to a region of the cell membrane where it is exposed to a low polarity environment, (2) ability to absorb light in such a low polarity environment, (3) its triplet quantum yield in this environment. In view of these factors, the partition coefficient for different concentrations, ranging from 1 to 50 (mu) g/ml of trithia sapphyrin in the Octanol/Saline system was determined. The partition coefficient for 5 (mu) g/ml is observed to be the highest, which indicated the higher partitioning of trithia sapphyrin towards the octanol phase i.e. towards the membrane. The uptake of sensitizer was analyzed in 0.05% hematocrit for different periods of incubation ranging from 5 minutes to 5 hour, in order to find (1) binding of sensitizer to the cell membrane, (2) partition of the sensitizer molecule between the aqueous phase and lipid phase and (3) diffusion of the pigment molecule into the intracellular aqueous phase. With these observations, Photohemolysis studies were carried out for different pre and post incubation time as a function of light dose and sensitizer concentration.
Recently, laser pretreatment of dental enamel has emerged as a new technique in the field of orthodontics. However, the changes in the morphology of the enamel surface is very much dependent on the wavelength of laser, emission mode of the laser, energy density, exposure time and the nature of the substance absorbing the energy. Based on these, we made a comparative in vitro study on laser etching with acid etching with reference to their bond strength. Studies were conducted on 90 freshly extracted, non carious, human maxillary or mandibular anteriors and premolars. Out of 90, 60 were randomly selected for laser irradiation. The other 30 were used for conventional acid pretreatment. The group of 60 were subjected to Nd-YAG laser exposure (1060 nm, 10 Hz) at differetn fluences. The remaining 30 were acid pretreated with 30% orthophosphoric acid. Suitable Begg's brackets were selected and bound to the pretreated surface and the bond strength were tested using Instron testing machine. The bond strength achieved through acid pretreatment is found to be appreciably greater than the laser pretreated tooth. Though the bond strength achieved through the acid pretreated tooth is found to be significantly greater than the laser pretreated specimens, the laser pretreatement is found to be successful enough to produce a clinically acceptable bond strength of > 0.60 Kb/mm. Examination of the laser pre-treated tooth under SEM showed globule formation which may produce the mechanical interface required for the retention of the resin material.
Native fluorescence characteristics of blood plasma was studied in the visible region, during the regenerating phase of the rat liver tissue. Animals were subjected to partial hepatectomy (PH). Blood samples were collected after several intervals of post PH time and also from control animals. Native fluorescence spectra of blood plasma were measured at 405 nm excitation. In addition to the primary emission peak around 440 nm, the fluorescence spectra of experimental group of animals showed distinct secondary emission peak around 620 nm, which is found to be absent in the case of control animals. This may be attributed to the presence of endogenous porphyrins. The fluorescence intensity at 620 nm was found to be maximum at about 16 hrs of post PH time and it decreased thereafter with increasing post PH time. The spectral differences between controls and experimental animals were found to be minimal at 240 hrs of post PH time.
Many applications of native fluorescence spectroscopy of intrinsic biomolecules such as Try, Tyr, Phe, NADH and FAD are reported on both the characterization and the discrimination of malignant tissues from the normal. In the field of diagnostic oncology, extensive studies have been made to distinguish the normal from malignant condition in breast, cervix, colon and bronchus. From the studies made by Alfano and co-workers, it was found that the emission at 340 and 440 nm under UV excitation have shown statistically significant difference between normal and malignant tissues. As tissues are highly complex in nature, it is worth to known whether the changes arise from cells or from other extracellular tissue components, so as to enable us to have better understanding on the transformation mechanism of normal into malignant and to go for an improved approach in the effective optical diagnosis. In this context, the present study addresses the question of whether there are differences in the native cellular fluorescence characteristics between normal and malignant epithelial cells from human larynx. With this aim, the UV fluorescence emission spectra in the wavelength region of excitation between 270 - 310 nm and the excitation spectra for 340 nm emission were measured and analyzed. In order to quantify the altered fluorescence signal between the normal and malignant cells, different ratio parameters were introduced.
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