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.

The capacity of photodynamic therapy (PDT) to induce localized cell death and tissue damage suggests that when applied to tumors it could create a local depot of tumor-associated antigens, which would be available for uptake and presentation to the immune system, potentially leading to improved tumor control. Dendritic cells (DCs) are the most potent cells for antigen uptake, presentation, and stimulation of the immune system. However, it is unclear whether DCs would retain their viability and functional capacity for the requisite trafficking to draining lymph nodes when adoptively transferred in close temporal and anatomic proximity to the site of PDT-induced cytotoxicity. We conducted studies of combined PDT and adoptive DC therapy, "immunophototherapy," in a female, Fisher 344 rat orthotopic mammary tumor model. Using 5-aminolevulinic acid as a pro-drug, we demonstrated kinetically favorable biologic conversion to the photosensitive protoporphyrin IX, appropriate trafficking of syngeneic bone marrow-derived DCs injected into PDT-treated tumors within 15 min of completion of therapy, and improved survival over either modality alone. These data indicate that DCs rapidly administered into the site of PDT retain their viability and functional status, supporting the further evaluation of immunophototherapy strategies.

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 spectroscopic properties, ultrafast kinetics and utilization of a photochromic molecule as a bi-stable fluorescing sensor of polarity in live cells are described. This molecule is a photochromic fulgimide, 2,3-dialkylidenesuccinimide, which emits fluorescence that can be switched optically on and off. The fluorescence intensity is a function of the polarity of the molecular environment, namely it fluoresces strongly when the molecule is in its polar isomeric structure form. We demonstrate that this molecule enters live cells without inducing damage, it binds primarily to internal membranous organelles (mitochondria) and its fluorescence can be switched optically "on" and "off" repeatedly while inside the living cell. A possible use as a bi-stable, on/off sensor is discussed.

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 fluence rate are investigated in human glioma spheroids incubated in 5-aminolevulinic acid (ALA). It is shown that the response of glioma spheroids to ALA-mediated PDT depends strongly on the rate at which the light dose is delivered. At low doses, lower fluence rates are more effective. For example, at a dose of 50 J/cm2, near total spheroid kill is observed at fluence rates of as low as 10 mW/cm2. Below 10 mW/cm2, however, treatments are not as effective. The fluence rate effect is not as pronounced at higher doses where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.

An in vivo model has been developed for the study of photodynamic therapy (PDT) and anti-angiogenic treatments. Significant damage to the vasculature of the chick chorioallantoic membrane (CAM) was observed immediately following PDT with 5-aminolevulinic acid (ALA). Multicell human glioma spheroids were placed on the CAM at day five of embryonic development, however, neovascularization was not observed.

Background

Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image.

Objective

To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time.

Methods

Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days.

Results

The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts.

Conclusions

The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.

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 structural origin of scattering contrast from single cells is examined by using a combined optical coherence and multiphoton microscope based on a 12 fs Ti:sapphire source and a 0.95 NA objective. High-resolution coherence-gated scattering images from single cells are coregistered and compared with two-photon-excited fluorescence images. Scattering contrast is observed from mitochondria, plasma membrane, actin filaments, and the boundary between cytoplasm and nucleus. There is little contribution to scattering from regions inside the nuclear core. These results confirm that light scattering signals from specific subcellular structures can be visualized by using coherent reflectance geometry.