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PURPOSE: Radiation therapy (RT) plays an important role in the treatment of glioblastoma multiforme (GBM). However, inherent intrinsic resistance of tumors to radiation, coupled with the need to consider the tolerance of normal tissues and the potential effects on neurocognitive function, impose constraints on the amount of RT that can be safely delivered. A strategy for augmenting the effectiveness of RT involves the utilization of radiation sensitizers (RS). Directly implanting RS-loaded fibrin glue (FG) into the tumor resection cavity would by-pass the blood brain barrier, potentially enhancing the impact of RT on tumor recurrence. This study investigated the ability of FG to incorporate and release, in non-degraded form, the radiation sensitizers 5-Fluorouracil (5FU) and Motexafin gadolinium (MGd). METHODS: FG layers were created in a 24-well plate by combining thrombin, fibrinogen, and 5FU or MGd. Supernatants from these layers were collected at various intervals and added to F98 glioma spheroid cultures in 96-well plates. Radiation was applied either before or after RS application as single or fractionated dosages. Spheroid growth was monitored for 14 days. RESULTS: Combined treatment of FG-released 5FU and RT significantly inhibited spheroid growth compared to RS or RT as a single treatment. As a free drug, MGd demonstrated its efficacy in reducing spheroid volume, but had diminished potency as a released RS. Fractionated radiation was more effective than single dose radiation. CONCLUSION: Non-degraded RS was released from the FG for up to 72 h. FG-released 5FU greatly increased the efficacy of radiation therapy.

The purpose of this study was to investigate the effect of photothermal treatment (PTT) with gold nanoshell (ANS) using a macrophage-mediated delivery system in a head and neck squamous cell carcinoma (HNSCC) cell line. To achieve this, ANS-loaded rat macrophages (ANS-MAs) were prepared via the coculture method with ANS. The human HNSCC (FaDu cell) and macrophage (rat macrophage; NR8383 cell) hybrid spheroid models were generated by the centrifugation method to determine the possibility of using ANS-MAs as a cancer therapy. These ANS-MAs were set into the tumor and macrophage hybrid spheroid model to measure PTT efficacy. Kinetic analysis of the spheroid growth pattern revealed that this PTT process caused a decreasing pattern in the volume of the hybrid model containing ANS-MAs (p < 0.001). Comparison with empty macrophages showed harmony between ANS and laser irradiation for the generation of PTT. An annexin V/dead cell marker assay indicated that the PTT-treated hybrid model induced increasing apoptosis and dead cells. Further studies on the toxicity of ANS-MAs are needed to reveal whether it can be considered biocompatible. In summary, the ANS was prepared with a macrophage as the delivery method and protective carrier. The ANS was successfully localized to the macrophages, and their photoabsorption property was stationary. This strategy showed significant growth inhibition of the tumor and macrophage spheroid model under NIR laser irradiation. In vivo toxicology results suggest that ANS-MA is a promising candidate for a biocompatible strategy to overcome the limitations of fabricated nanomaterials. This ANS-MA delivery and PTT strategy may potentially lead to improvements in the quality of life of patients with HNSCC by providing a biocompatible, minimally invasive modality for cancer treatment.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.

The purpose of this study was to investigate the effect of photothermal treatment (PTT) with gold nanoshell (ANS) using a macrophage-mediated delivery system in a head and neck squamous cell carcinoma (HNSCC) cell line. To achieve this, ANS-loaded rat macrophages (ANS-MAs) were prepared via the coculture method with ANS. The human HNSCC (FaDu cell) and macrophage (rat macrophage; NR8383 cell) hybrid spheroid models were generated by the centrifugation method to determine the possibility of using ANS-MAs as a cancer therapy. These ANS-MAs were set into the tumor and macrophage hybrid spheroid model to measure PTT efficacy. Kinetic analysis of the spheroid growth pattern revealed that this PTT process caused a decreasing pattern in the volume of the hybrid model containing ANS-MAs (p < 0.001). Comparison with empty macrophages showed harmony between ANS and laser irradiation for the generation of PTT. An annexin V/dead cell marker assay indicated that the PTT-treated hybrid model induced increasing apoptosis and dead cells. Further studies on the toxicity of ANS-MAs are needed to reveal whether it can be considered biocompatible. In summary, the ANS was prepared with a macrophage as the delivery method and protective carrier. The ANS was successfully localized to the macrophages, and their photoabsorption property was stationary. This strategy showed significant growth inhibition of the tumor and macrophage spheroid model under NIR laser irradiation. In vivo toxicology results suggest that ANS-MA is a promising candidate for a biocompatible strategy to overcome the limitations of fabricated nanomaterials. This ANS-MA delivery and PTT strategy may potentially lead to improvements in the quality of life of patients with HNSCC by providing a biocompatible, minimally invasive modality for cancer treatment.

Gold based nanoparticles with strong near infra-red (NIR) absorption are ideally suited for photothermal therapy (PTT) of brain tumors. The goal of PTT is to induce rapid heating in tumor tissues while minimizing thermal diffusion to normal brain. PTT efficacy is sensitively dependent on both nanoparticle concentration and distribution in tumor tissues. Nanoparticle delivery via passive approaches such as the enhanced permeability and retention (EPR) effect is unlikely to achieve sufficient nanoparticle concentrations throughout tumor volumes required for effective PTT. A simple approach for improving tumor biodsitribution of nanoparticles is the use of cellular delivery vehicles. Specifically, this review focuses on the use of monocytes/macrophages (Mo/Ma) as gold nanoparticle delivery vectors for PTT of brain tumors. Although the efficacy of this delivery approach has been demonstrated in both in vitro and animal PTT studies, its clinical potential for the treatment of brain tumors remains uncertain.

Chemotherapeutic agents, either in the form of systemically injected free drug or encapsulated in nanoparticles transport vehicles, must overcome three main obstacles prior to reaching and interacting with their intended target inside tumor cells. Drugs must leave the circulation, overcome the tissue-tumor barrier and penetrate the cell's plasma membrane. Since, many agents enter the cell by endocytosis, they must avoid entrapment and degradation by the intracellular endolysosome complex. Ultrasound has demonstrated potential to enhance the efficacy of chemotherapy by reducing these barriers. The purpose of this review is to highlight the potential of ultrasound in combination with sonosensitizers to enhance the efficacy of chemotherapy by optimizing the anticancer agent's intracellular ability to engage and interact with its target.

Nonviral gene transfection overcomes some of the disadvantages of viral vectors, such as undesired immune responses, safety concerns, issues relating to bulk production, payload capacity, and quality control, but generally have low transfection efficiency. Here we describe the effects of a modified form of photodynamic therapy (PDT), i.e., photochemical internalization (PCI) to: (1) greatly increase nonviral cytosine deaminase gene (CD) transfection into tumor cells, significantly increasing the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and (2) enhance the toxic efficacy of the locally produced 5-FU to induce cell death on both transfected and non-transfected bystander cells.

Light-based treatment modalities such as photothermal therapy (PTT) or photochemical internalization (PCI) have been well documented both experimentally and clinically to enhance the efficacy of chemotherapy. The main purpose of this study was to examine the cytotoxic effects of silica-gold nanoshell (AuNS)-loaded macrophage-mediated (MaNS) PTT and bleomycin BLM-PCI on monolayers of squamous cell carcinoma cells. The two modalities were applied separately and in simultaneous combination. Two different wavelengths of light were employed simultaneously, one to activate a highly efficient PCI photosensitizer, AlPcS2a (670 nm) and the other for the MaNS-mediated PTT (810 nm), to evaluate the combined effects of these modalities. The results clearly demonstrated that macrophages could ingest sufficient numbers of silica-gold nanoshells for efficient near infrared (NIR) activated PTT. A significant synergistic effect of simultaneously applied combined PTT and PCI, compared to each modality applied separately, was achieved. Light-driven therapies have the advantage of site specificity, non-invasive and non-toxic application, require inexpensive equipment and can be given as repetitive treatment protocols.

BackgroundMacrophage (Ma) vectorization of chemotherapeutic drugs has the advantage for cancer therapy in that it can actively target and maintain an elevated concentration of drugs at the tumor site, preventing their spread into healthy tissue. A potential drawback is the inability to deliver a sufficient number of drug-loaded Ma into the tumor, thus limiting the amount of active drug delivered. This study examined the ability of photochemical internalization (PCI) to enhance the efficacy of released drug by Ma transport.MethodsTumor spheroids consisting of either F98 rat glioma cells or F98 cells combined with a subpopulation of empty or doxorubicin (DOX)-loaded mouse Ma (RAW264.7) were used as in vitro tumor models. PCI was performed with the photosensitizer AlPcS2a and laser irradiation at 670 nm.ResultsRAW264.7 Ma pulsed with DOX released the majority of the incorporated DOX within two hours of incubation. PCI significantly increased the toxicity of DOX either as pure drug or derived from monolayers of DOX-loaded Ma. Significant growth inhibition of hybrid spheroids was also observed with PCI even at subpopulations of DOX-loaded Ma as low as 11% of the total initial hybrid spheroid cell number.ConclusionResults show that RAW264.7 Ma, pulsed with DOX, could effectively incorporate and release DOX. PCI significantly increased the ability of both free and Ma-released DOX to inhibit the growth of tumor spheroids in vitro. The growth of F98 + DOX loaded Ma hybrid spheroids were synergistically reduced by PCI, compared to either photodynamic therapy or released DOX acting alone.

Immunotherapy of brain tumors involves the stimulation of an antitumor immune response. This type of therapy can be targeted specifically to tumor cells thus sparing surrounding normal brain. Due to the presence of the blood-brain barrier, the brain is relatively isolated from the systemic circulation and, as such, the initiation of significant immune responses is more limited than other types of cancers. The purpose of this study was to show that the efficacy of tumor primed antigen presenting macrophage (MaF98) vaccines can be increased by: (1) photodynamic therapy (PDT) of the priming tumor cells and (2) intracranial injection of allogeneic glioma cells directly into the tumor site. Experiments were conducted in an in vivo brain tumor development model using Fischer rats and F98 (syngeneic) and BT4C (allogeneic) glioma cells. The results showed that immunization with Ma (acting as antigen-presenting cells), primed with PDT-treated tumor cells (MaF98), significantly slowed but did not prevent the growth of F98-induced tumors in the brain. Complete suppression of tumor development was obtained via MaF98 inoculation combined with direct intracranial injection of allogeneic glioma cells. No deleterious effects were noted in any of the animals during the 14-day observation period.

Macrophages (Ma) loaded with gold based nanoparticles, which convert near infrared light to heat, have been studied as targeted transport vectors for photothermal therapy (PTT) of tumors. The purpose of the experiments reported here was to compare the efficacy of gold-silica nanoshells (AuNS) and gold nanorods (AuNR) in macrophage mediated PTT. PTT efficacy was evaluated in hybrid glioma spheroids consisting of human glioma cells and either AuNS or AuNR loaded Ma, designated MaNS and MaNR respectivly. Spheroids were irradiated for 10 min. with light from an 810 nm diode laser at irradiances ranging from 0 to 28 W/cm2. PTT efficacy was determined from spheroid growth over a 14-day period. The uptake by Ma of pegylated AuNR (3.9 ± 0.9 %) was twice that of pegylated AuNS, (7.9 ± 0.7 %). Hybrid spheroids consisting of a 5:1 ratio of glioma cells to loaded Ma exhibited significant growth inhibition with MaNS when subjected to irradiances of 7 W/cm2 or greater. In contrast, no significant growth inhibition was observed for the MaNR hybrid spheroids at this 5:1 ratio, even at the highest irradiance investigated (28 W/cm2). Although AuNR were taken up by Ma in larger numbers then AuNS, MaNS were shown to have greater PTT efficacy compared to MaNR for equivalent numbers of loaded Ma.

Cells infiltrating into normal brain from malignant brain tumors are protected by the blood brain barrier (BBB) which prevents the delivery and limits the effects of anti-tumor agents. We have evaluated the ability of photochemical internalization (PCI) to limit the effects of an agent known to broadly open the BBB to a target region of the brain. The PCI-based relocation and activation of macromolecules into the cell cytosol has the advantage of minimal side effects since the effect is localized to the area exposed to light, allowing the access of chemotherapeutic agents only to these regions. Non tumor bearing inbred Fisher rats were treated with photosesitizer, and a nontoxic intraperitoneal dose of Clostridium perfringens epsilon prototoxin (ETXp) followed by light exposure. Post-contrast T1 MRI scans were used to monitor the degree BBB disruption. F98 tumor cells were implanted into the brains of other animals that were subsequently treated 24 h later with ETXp-PCI BBB opening followed by the i.p. administration of bleomycin (BLM). PCI delivery of ETXp at low fluence levels demonstrated significant MRI enhancement. No effect on the BBB was observed if photosesitizer and light was given in the absence ETXp. The survival of animals implanted with F98 tumor cells was significantly extended following ETXp-PCI BBB opening and BLM therapy compared to controls. PCI delivered ETXp was effective in opening the BBB in a limited region of the brain. ETXp-PCI mediated BBB opening clearly increased the efficacy of BLM therapy.

Cells infiltrating into normal brain from malignant brain tumors are protected by the blood brain barrier (BBB) which prevents the delivery and limits the effects of anti-tumor agents. We have evaluated the ability of photochemical internalization (PCI) to limit the effects of an agent known to broadly open the BBB to a target region of the brain. The PCI-based relocation and activation of macromolecules into the cell cytosol has the advantage of minimal side effects since the effect is localized to the area exposed to light, allowing the access of chemotherapeutic agents only to these regions. Non tumor bearing inbred Fisher rats were treated with photosesitizer, and a nontoxic intraperitoneal dose of Clostridium perfringens epsilon prototoxin (ETXp) followed by light exposure. Post-contrast T1 MRI scans were used to monitor the degree BBB disruption. F98 tumor cells were implanted into the brains of other animals that were subsequently treated 24 h later with ETXp-PCI BBB opening followed by the i.p. administration of bleomycin (BLM). PCI delivery of ETXp at low fluence levels demonstrated significant MRI enhancement. No effect on the BBB was observed if photosesitizer and light was given in the absence ETXp. The survival of animals implanted with F98 tumor cells was significantly extended following ETXp-PCI BBB opening and BLM therapy compared to controls. PCI delivered ETXp was effective in opening the BBB in a limited region of the brain. ETXp-PCI mediated BBB opening clearly increased the efficacy of BLM therapy.

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 degrees C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 degrees C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40-46 degrees C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT.

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 degrees C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 degrees C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40-46 degrees C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT.

The effects of photodynamic therapy (PDT) in human glioma spheroids incubated in 5-aminolevulinic acid (ALA), or ALA esters, are investigated. Spheroid survival and growth are monitored following PDT at representative drug concentrations, light doses, and dose rates. The primary finding of this study is that the response of human glioma spheroids to PDT with lipophilic ester derivatives, such as benzyl-ALA and hexyl-ALA, is equivalent to that observed with ALA, however, this equivalency is obtained for ester concentrations 10-20 times lower than the parent compound. The enhanced efficiency of the esters is likely due to their increased membrane penetrance. Potential clinical advantages of using lipophilic esters in PDT of gliomas are discussed.

The effects of photodynamic therapy (PDT) in human glioma spheroids incubated in 5-aminolevulinic acid (ALA), or ALA esters, are investigated. Spheroid survival and growth are monitored following PDT at representative drug concentrations, light doses, and dose rates. The primary finding of this study is that the response of human glioma spheroids to PDT with lipophilic ester derivatives, such as benzyl-ALA and hexyl-ALA, is equivalent to that observed with ALA, however, this equivalency is obtained for ester concentrations 10-20 times lower than the parent compound. The enhanced efficiency of the esters is likely due to their increased membrane penetrance. Potential clinical advantages of using lipophilic esters in PDT of gliomas are discussed.

The translation of photodynamic therapy (PDT) to the clinical setting has primarily been limited to easily accessible and/or superficial diseases, for which traditional light delivery can be performed noninvasively. Cerenkov radiation, as generated from medically relevant radionuclides, has been suggested as a means to deliver light to deeper tissues noninvasively to overcome this depth limitation. This article investigates the utility of Cerenkov radiation, as generated from the radionuclide yttrium-90, for activating the PDT process using clinically approved aminolevulinic acid at 1.0 mm and also the more efficient porphyrin-based photosensitizer mesotetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS2a) at 1.2 µm. Experiments were conducted with monolayer cultured glioma and breast tumor cell lines. Although aminolevulinic acid proved to be ineffective for generating a therapeutic effect at all but the highest activity levels, TPPS2a produced at least a 20% therapeutic effect at activities ranging from 6 to 60 µCi/well for the C6 glioma cell line. Importantly, these results demonstrate for the first time, to our knowledge, that Cerenkov radiation generated from a radionuclide can be used to activate PDT using clinically relevant photosensitizers. These results therefore provide evidence that it may be possible to generate a phototherapeutic effect in vivo using Cerenkov radiation and clinically relevant photosensitizers.

Moderate hyperthermia (MHT) has been shown to enhance the effects of chemotherapeutic agents in a wide variety of cancers. The purpose of this study was to investigate the combined effects of commonly used chemotherapeutic agents with MHT induced by near-infrared (NIR) activation of gold nanoshell (AuNS)-loaded macrophages (Ma). AuNS-loaded murine Ma combined with human FaDu squamous cells, in hybrid monolayers, were subjected to three cytotoxic drugs (doxorubicin, bleomycin, cisplatin) with or without NIR laser irradiation. For all three drugs, efficacy was increased by NIR activation of AuNS-loaded Ma. The results of this in vitro study provide proof-of-concept for the use of AuNS-loaded Ma for photothermal enhancement of the effects of chemotherapy on squamous cell carcinoma.

Activation of sonosensitizers via focused ultrasound (FUS), i.e., sonodynamic therapy has been proposed as an extension to light-activated photodynamic therapy for the treatment of brain as well as other tumors. The use of FUS, as opposed to light, allows treatment to tumor sites buried deep within tissues as well as through the intact skull. We have examined ultrasonic activation of sonosensitizers together with the anticancer agent bleomycin (BLM), i.e., sonochemical internalization (SCI). SCI is a technique that utilizes FUS for the enhanced delivery of endo-lysosomal trapped macromolecules into the cell cytoplasm in a similar manner to light-based photochemical internalization. The released agent can, therefore, exert its full biological activity, in contrast to being degraded by lysosomal hydrolases. Our results indicate that, compared to drug or FUS treatment alone, FUS activation of the sonosensitizer AlPcS2a together with BLM significantly inhibits the ability of treated glioma cells to grow as three-dimensional tumor spheroids in vitro.

In this paper we propose using Array Waveguide Gratings (AWGs), working in the visible range, in order to implement the technique of Wavelength-Division-(de)Multiplexing for multi-point stimulation of deep-brain neurons. We've developed a CMOS compatible fabrication process and fabricated two sets of AWGs, working in the red and blue wavelengths. Experimental data demonstrating the functionality of these AWGs is presented.

The translation of photodynamic therapy (PDT) to the clinic has mostly been limited to superficial diseases where traditional light delivery is noninvasive. To overcome this limitation, a variety of mechanisms have been suggested to noninvasively deliver light to deep tissues. This work explores the minimum amount of light required by these methods to produce a meaningful PDT effect in the in vitro setting under representative low fluence and wavelength conditions. This threshold was found to be around 192 mJ/cm(2) using the clinically approved photosensitizer aminolevulinic acid and 12 mJ/cm(2) for the more efficient, second generation photosensitizer TPPS2a.

Gold-based nanoparticles have been used in a number of therapeutic and diagnostic applications. The purpose of this study was to investigate the efficacy of gold-silica nanoshells (AuNS) in photothermal therapy (PTT) of rat gliomas. Rat alveolar macrophages (Ma) were used as nanoparticle delivery vectors. Uptake of AuNS (bare and PEGylated) was investigated in Ma. AuNS were incubated with Ma for 24 h. Phase contrast microscopy was used to visualize the distribution of loaded Ma in three-dimensional glioma spheroids. PTT efficacy was evaluated for both empty (Ma) and AuNS-loaded Ma (Ma(NS)) in both monolayers and spheroids consisting of C6 rat glioma cells and Ma. Monolayers/spheroids were irradiated for 5 min with light from an 810-nm diode laser at irradiances ranging from 7 to 28 W cm(-2). Monolayer survival was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay while PTT efficacy in spheroids was determined from growth kinetics and live/dead fluorescence microscopy. PTT efficacy was investigated in vivo using a Sprague-Dawley rat glioma model. Five rats received direct intracranial injection of a mixture of 10(4) C6 glioma cells and, 2 days later, an equal number of Ma(NS). Three rats received laser treatment (810 nm; 10 min; 1 W) while the remaining two served as controls (no laser treatment). The uptake ratio of bare to PEGylated AuNS by Ma was 4:1. A significant photothermal effect was observed in vitro, albeit at relatively high radiant exposures (2.1-4.2 kJ cm(-2)). PTT proved effective in vivo in preventing or delaying tumor development in the PTT-treated animals.

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.

Background and Objective: Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field optical imaging technology currently being developed to investigate the feasibility of quantitative non-invasive evaluation of burn wound severity in a rat model. Our objective is to determine the potential of SFDI for mapping quantitative changes in spatially resolved tissue oxygen saturation and water concentration may be indicative of burn wound severity, healing, and further complications. In this portion of the investigation, we focus on the development of a rat burn model and the acute response of tissue to burn wounds. Study Design/Materials and Methods: A controlled burn protocol involving a heated brass comb was applied to 6 rats. Imaging was acquired at 17 evenly spaced wavelengths in the near-infrared from 650 to 970 nm. Over the course of the 3 hour post-burn period, we were able to map quantitative changes in spatially resolved chromophores. Burn severities were verified post-experiment using standard H and E histology and optical microscopy. Results/Conclusion: In total, we were able to induce 12 superficial-partial thickness burns, 8 deep-partial thickness burns, and 4 full thickness burns in our rat models. While several tissue chromophores were tracked, we found that changes in oxygen saturation and water concentration to be sensitive indicators of burn severity. Future work will include additional longitudinal studies over a period of days in order to investigate which parameters are correlated to tissue healing. © 2013 SPIE.

Acquired subglottic stenosis is a narrowing of the airway caused by prolonged endotracheal intubation. Currently, there are no non-invasive means to diagnose the disease. A previous study by this same group introduced optical coherence tomography (OCT) as a means of monitoring the progression of stenosis. The aim of the current study was to qualitatively and quantitatively analyze OCT images obtained from a subglottic stenosis model of the rabbit airway. 15 rabbits were used throughout the study, and a MEMs based OCT probe was utilized. The OCT images obtained were analyzed using a free software program, 3D Slicer. The region of scarred tissue was grown out and measured quantitatively. This study demonstrated the feasibility of using a program to quantify the progression of scarring in OCT images, in addition to qualitatively correlating between histology, endoscopic, and OCT images. Future works may include utilization of a long-range probe and use of a pressure necrosis model to better emulate the actual onset of neonatal subglottic stenosis. © 2013 SPIE.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT)-induced disruption of the blood-brain barrier (BBB) has been investigated as a technique for the delivery of therapeutic agents to selective regions of the brain. The purpose of this study was to determine the effects of PDT on the migration of systemically administered exogenous macrophages (Ma) loaded with iron oxide nanoparticles in non-tumor bearing rats. MATERIALS AND METHODS: A control group consisting of three Sprague-Dawley rats was injected with iron oxide-loaded rat alveolar Ma via jugular vein catheter while two animals were subjected to intracranial injection of iron oxide-loaded Ma. PDT-treated animals were injected with photosensitizer (AlPcS2a ; 1 mg/kg i.p.) followed by light irradiation (wavelength = 670 nm; light dose = 2.5 J) 48 hours later. Light irradiation was performed through the skull. Prior to light irradiation, iron oxide-loaded Ma were administered to each animal. Animals in all groups were imaged in a 7 Tesla (T) magnetic resonance (MR) imager to determine the extent of PDT-induced edema and to evaluate for the presence of iron oxide nanoparticles. Animals were sacrificed 7 days post-Ma administration and their brains analyzed for the presence of iron oxide using Perls staining. RESULTS: Significant uptake of iron oxide nanoparticles by rat alveolar Ma was observed thus providing the rationale for their use as delivery vectors. Histopathological analyses failed to find evidence of iron oxide in normal rat brain. Accumulations of iron oxide-loaded Ma were observed in both MR images and histological sections of non-tumor bearing rat brain following PDT-induced disruption of the BBB. CONCLUSIONS: MR imaging was shown to be useful for localizing iron-oxide loaded Ma in rat brains. Exogenous Ma are incapable of traversing the normal BBB and therefore, the use of Ma as delivery vehicles into the brain requires selective disruption of the BBB.

One common component of otolaryngological surgeries is the reshaping of cartilage. Previous studies have demonstrated the efficient achievement of this procedure through electromechanical reshaping (EMR), a technique that involves the direct application of voltage to cartilage that is mechanically deformed in a jig. Two main parameters, voltage and application time, may be regulated to achieve varying degrees of shape change. Although prior research has correlated these EMR parameters with degree of shape change, it remains necessary to correlate the same parameters with the degree of change in the mechanical properties of tissue. Once this is accomplished, an ideal balance may be determined, in which shape change is maximized while intrinsic tissue damage is minimized This study satisfies this need by providing comprehensive data on the pre- and post-EMR stiffness of both septal and auricular cartilage over a range of voltages (2-8V) with constant application time (2 min for septal, 3 min for auricular). EMR was applied using flat platinum electrodes to one of two 15 mm X 5 mm samples obtained from the same cartilage specimen, while the second sample was maintained as a control. Following a 15 min re-hydration period, the Young's modulus of the tissue was calculated for both the control and experimental sample from data obtained through a uniaxial tension test. A general reduction in stiffness was observed beginning at 3V, with the magnitude of reduction increasing at 6V. © 2010 Copyright SPIE - The International Society for Optical Engineering.

An emerging clinical treatment option for articular cartilage injury includes bone marrow stimulation techniques, such as microfracture, which has grown increasingly popular among athletes. During the microfracture procedure, the surgeon penetrates the subchondral bone with an awl and creates "microholes" deep enough to ensure bleeding from the bone marrow. This procedure triggers a spontaneous repair response that results in the formation of fibrocartilaginous repair tissue. This preliminary study aimed to evaluate the potential use of femtosecond lasers and Erbium:YAG lasers as alternatives to microfracture surgery of the knee by assessing the effects of ablation on bovine femoral condyles. Bovine femoral condyles were obtained and 8mm cube blocks were extracted. The specimen were ablated with various laser dosimetry parameters and observed using a high power dissecting microscope to examine the effects of the lasers. Further imaging with conventional histology (hematoxylin and eosin staining) was done to provide more accurate information. Preliminary results show some carbonization but demonstrate little thermal damage to surrounding tissues. The femtosecond laser offers a more precise and efficient ablation than the Erbium:YAG laser, but both are demonstrated to be possible alternatives to the surgical-skill dependent microfracture procedure. © 2010 Copyright SPIE - The International Society for Optical Engineering.

Electromechanical reshaping (EMR) of cartilage is a promising noninvasive technique with potential for broad application in reconstructive surgery. EMR involves applying direct current electrical fields to localized stress regions and initiating a series of oxidation-reduction reactions, thus effecting a shape change. Previous EMR studies have focused on macroscopic structural measurements of the shape change effect or monitoring of electrical current flow. Only limited investigation of structural changes in the tissue at the histologic level have been performed, and not in real time. This study is the first to use optical coherence tomography (OCT) to examine structural changes in cartilage during EMR. Two platinum needle electrodes were inserted into fixed rectangular rabbit nasal septal cartilage specimens. The spectral domain OCT probe was then positioned above the section of cartilage in which the anode needle was inserted. A constant voltage of 6V was applied for 3 minutes, and images were obtained (8 frames/second). OCT was also performed in specimens undergoing dehydration under ambient conditions and during pH changes produced by the addition of HCl, as both processes accompany EMR. The OCT data identified distinct findings among the three conditions, suggesting that EMR causes a much greater degree of reshaping on a molecular level than dehydration or a change in pH alone. OCT provides a means to gauge structural changes in the tissue matrix during EMR. The application of OCT to image the EMR process will add to our understanding of the mechanisms of action involved and potentially facilitate optimization of this process. © 2010 Copyright SPIE - The International Society for Optical Engineering.

Cryopreservation of cartilage has been investigated for decades and is currently an established protocol. However, the reliability and applicability of cartilage cryopreservation for the use in electromechanical reshaping (EMR) has not been studied exclusively. A system to cryopreserve large numbers of tissue specimens provides a steady source of cartilage of similar quality for experimentation at later dates. This will reduce error that may arise from different cartilage stock, and has the potential to maximize efficiency under time constraints. Our study utilizes a unique methodology to cryopreserve septal cartilage for use in EMR studies. Rabbit septal cartilage specimens were harvested and standardized to 20 x 8 x 1 mm, and placed in one of three solutions (normal saline, PBS, 10% DMSO in PBS) for four hours in a cold storage room at 4 degrees Celsius. Then, each cartilage specimen was vacuumed and sealed in an anti-frost plastic bag and stored in a freezer at -80 degrees Celsius for 1 to 3 weeks duration. EMR was performed using 2 and 6 volts for 2 minutes application time. Bend angle measurements of the cryopreserved cartilage specimens were compared to bend angles of fresh cartilage which underwent EMR using the same parameters. Results demonstrate that normal saline, phosphate buffered saline (PBS), and PBS with DMSO were effective in cryopreservation, and indicated no significant differences in bend angle measurements when compared to no cryopreservation. Our methodology to cryopreserve cartilage specimens provides a successful approach for use in conducting large-scale EMR studies. © 2010 Copyright SPIE - The International Society for Optical Engineering.

During laser therapy of port wine stain (PWS) birthmarks, regions of persistent perfusion may exist. We hypothesize that such regions, which are not readily visible, exist even during laser surgery performed by highly experienced clinicians. The objective of this study was to use objective feedback to assess the acute vascular response to laser therapy. We have developed a clinic-friendly laser speckle imaging (LSI) instrument to provide the clinician with real-time images of blood flow during laser therapy. We acquired images from patients undergoing laser therapy of PWS birthmarks at Scripps Clinic and Beckman Laser Institute and Medical Clinic. We extracted blood flow maps from the acquired imaging data. Collectively, we have observed two regimes of patient response to therapy: 1) an immediate increase in perfusion within minutes after laser therapy; and 2) an overall decrease in blood perfusion approximately one hour after laser therapy, with distinct regions of persistent perfusion apparent in the majority of post-treatment blood-flow images. A comparison of blood flow in PWS and adjacent normal skin demonstrated that PWS blood flow can be greater than or sometimes equivalent to that of normal skin. Regions of persistent perfusion frequently exist immediately after laser therapy of PWS birthmarks. Existence of these regions may be correlated to the need for multiple treatment sessions to improve substantially PWS skin appearance. With the use of intraoperative LSI, immediate retreatment of these regions may improve the outcome of each session. © 2009 SPIE.

Electromechanical reshaping (EMR) of cartilage is a novel technique that has significant potential for use in facial reconstructive surgery. EMR achieves permanent shape change by initiating electrochemical redox reactions in the vicinity of stress concentrations, thereby altering mechanical properties of tissue matrix. This study reports the use of a six electrode needle-based geometric configuration to reshape cartilage. Rectangular samples (24 × 12 × 1 mm) of rabbit nasal septal cartilages were bent at a right angle in a precision-machined reshaping jig. Two parallel arrays of three platinum needle electrodes were each inserted into cartilage along the bend at 3 mm from the bend line. One array served as an anode and the other as cathode. Constant voltage at 1, 2, 4, 6, and 8 volts was applied to the arrays for 2 minutes. The specimens were then removed from the jig and rehydrated for 15 minutes in phosphate buffered saline. Following rehydration, bend angles and thicknesses were measured. Bend angle increased with increasing voltage and application time. No statistically significant bending was observed below 6 volts for 2 minutes application time. Maximum bend angle of 33 ± 8 degrees or reshaping degree of 33% was observed at 8 volts applied for 2 minutes. Current flow was small (< 0.1 A) for each case. Sample thickness was 0.9 ± 0.2 mm. ANOVA analysis showed that cartilage thickness had no significant impact on the extent of reshaping at given voltage and application time. The six needle electrode geometric configuration conforms to the voltage- and time-dependent trends predicted by previous EMR studies. In the future, the reshaping properties of other geometric configurations will be explored. © 2009 SPIE.

Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and has been used to image the human larynx during surgical endoscopy. The design of a long GRIN lens based probe capable of capturing images of the human larynx by use of swept-source OCT during a typical office-based laryngoscopy examination is presented. In vivo OCT imaging of the human larynx is demonstrated with 40 fame/second. Dynamic vibration of the vocal folds is recorded to provide not only high-resolution cross-sectional tissue structures but also vibration parameters, such as the vibration frequency and magnitude of the vocal cord, which provide important information for clinical diagnosis and treatment, as well as in fundamental research of the voice. Office-based OCT is a promising imaging modality to study the larynx. © 2009 SPIE.

Current methods for analysis of sebum excretion have limitations, such as irreproducible results in repeatable measurements due to the point measurement method, user-dependent artifact due to contact measurement or qualitative evaluation of the image, and long measurement time. A UV-induced fluorescent digital imaging system was developed to acquire facial images so that the distribution of sebum excretion on the face could be analyzed. The imaging system consisted of a constant UV-A light source, digital color camera, and head-positioning device. We describe the system characterization for acquisition of a fluorescent facial image and the image analysis method. The imaging modality provided uniform light distribution on the facial mannequin model and presented a discernible color fluorescent image. Valuable parameters of sebum excretion were obtained after image analysis. The imaging system, which provides a non-contact method, was proven to be a useful tool to evaluate sebum excretion and to characterize the pattern of sebum excretion. When compared to conventional "Wood's lamp" and "Sebutape" methods that provide similar parameters for sebum excretion, the method described herein is simpler and more reliable to evaluate the dynamics of sebum excretion in nearly real-time.

Neurologic complications have been reported with spinal transforaminal injections. Causes include intraneural injection, plus embolization occlusion of the radicular artery with subsequent spinal cord infarction. 1 Optical coherence tomography (OCT) is a non-invasive imaging modality, which is used to image tissue microstructure with very high resolution (less than 20 microns) in real-time. With a view toward needle tip OCT visualization of the spinal neuroforamen, we conducted animal studies to explore OCT imaging of paraspinal neurovascular structures. With institutional animal care committee approval, we performed ex-vivo and in situ OCT studies in a euthanized dog, pig, and rabbit. Image data was gathered on spinal nerve roots, dura, and brachial plexus. Two systems were used: frequency domain OCT imaging system developed at California Institute of Technology, and time domain Imalux NIRIS system with a 2.7 mm diameter probe. In a euthanized pig, excised dura was punctured with a 17-gauge Tuohy needle. FDOCT dural images of the puncture showed a subsurface cone-shaped defect. In a rabbit in situ study, puncture of the dura with a 26-gauge needle is imaged as a discontinuity. FDOCT imaging of both small artery and large arteries will be presented, along with H&E and OCT images of the brachial plexus.

The effects of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) and gamma radiation, and PDT and hyperthermia (HT) are investigated in human glioma spheroids. In the case of ionizing radiation and PDT, the degree of interaction between the two modalities depends in a complex manner on a number of factors, including light fluence, fluence rate and radiation dose. It is shown that, under certain conditions, PDT and gamma radiation interact synergistically to produce enhanced cell kill. The degree of interaction appears to be independent of both sequence and time intervals investigated. TUNEL assays show that low fluence-rate ALA-PDT is a very efficient inducer of apoptosis, whereas neither high-fluence rate PDT nor ionizing radiation results in significant apoptosis in this system. Synergistic interactions between HT and PDT are observed when the two modalities are given concurrently. The degree of synergism increases with increasing temperature and light fluence. It is shown that apoptosis is the primary mode of cell death following combined HT and low-fluence rate ALA-PDT.

Although many results on the near infrared spectroscopy and imaging of the human brain have been already published, the signal-to-noise ratio (SNR) of the instrumentation used in these studies has never been systematically optimized for the anatomy of the human head. Assuming a typical anatomical structure of the adult human head, we have studied the SNR of the frequency-domain instrument as a function of the modulation frequency and the source-detector distance. The study was performed both experimentally (using a pulsed laser system) and numerically (using the diffusion approximation and Monte Carlo simulations). For the specified geometry we have found that changes caused by non-homogeneities in the phase and in the ratio of the modulation amplitude to the mean intensity significantly increase with the modulation frequency and exhibit peaks between 400 and 1100 MHz, depending on the source-detector distance. Assuming the shot noise, we have found that the corresponding SNR peaks between 400 and 600 MHz. Increase in the source-detector distance results in the SNR increase only for distances below 30 mm. At larger source-detector distances the higher noise decreases the SNR. Our results show that increasing modulation frequency only to 300 MHz offers a very significant improvement in the functional near-infrared spectro-imaging of the adult human brain compared to the 100 MHz instrument.

Near-infrared wavelengths are absorbed less by epidermal melanin mainly located at the basal layer of epidermis (dermo-epidermal junction), and penetrate deeper into human skin dermis and blood than visible wavelengths. Therefore, laser irradiation using near-infrared wavelength may improve the therapeutic outcome of cutaneous hyper-vascular malformations in moderately to heavily pigmented skin patients and those with large-sized blood vessels or blood vessels extending deeply into the skin. A mathematical model composed of a Monte Carlo algorithm to estimate the distribution of absorbed light followed by numerical solution of a bio-heat diffusion equation was utilized to investigate the thermal response of human skin to near-infrared laser irradiation, and compared it with that to visible laser irradiation. Additionally, the effect of skin surface cooling on epidermal protection was theoretically investigated. Simulation results indicated that 940 nm wavelength is superior to 810 and 1064 nm in terms of the ratio of light absorption by targeted blood vessel to the absorption by the basal layer of epidermis, and is more efficient than 595 nm wavelength for the treatment of patients with large-sized blood vessels and moderately to heavily pigmented skin. Dermal blood content has a considerable effect on the laser-induced peak temperature at the basal layer of epidermis, while the effect of blood vessel size is minimum.

The response of human glioma spheroids to repetitive 5-aminolevulinic acid-mediated photodynamic therapy (PDT) was investigated. In all cases, light fluences were kept below toxic thresholds to simulate conditions typically found at 1-2 cm depths in brain adjacent to tumor. Significant inhibition of spheroid growth was observed following multiple PDT treatments at sub-threshold light fluences. The effect appears to be insensitive to the treatment intervals investigated (weekly or bi-monthly). In all cases, suppression of growth was observed for the duration of treatment. Low fluence rates (< or = 5 mW cm(-2)) appear to be more effective than high fluence rates (25 mW cm(-2)). No evidence of PDT resistance was found in this investigation.

The response of human glioma spheroids to repetitive 5-aminolevulinic acid-mediated photodynamic therapy (PDT) was investigated. In all cases, light fluences were kept below toxic thresholds to simulate conditions typically found at 1-2 cm depths in brain adjacent to tumor. Significant inhibition of spheroid growth was observed following multiple PDT treatments at sub-threshold light fluences. The effect appears to be insensitive to the treatment intervals investigated (weekly or bi-monthly). In all cases, suppression of growth was observed for the duration of treatment. Low fluence rates (< or = 5 mW cm(-2)) appear to be more effective than high fluence rates (25 mW cm(-2)). No evidence of PDT resistance was found in this investigation.

The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm(-2) and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm(-2)) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.

The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm(-2) and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm(-2)) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.