Photobiomodulation of avian embryos by red laser.

The current research focuses on the effect of variable doses of red laser on the chick embryonic development. He-Ne laser of 632-nm wavelength was used as an irradiation source in the first 48 h post-laying of chicken eggs. We have used five different doses: 2, 1, 0.3, 0.2, and 0.1 mJ/cm2 that needed a time range for about 400-20 s. Those irradiated embryos were left for additional 11 days for incubation in normal conditions, where they are blindly studied after the 11th day. Light microscopy was used in this study to investigate the histological and pathological features of the different experimental groups compared to the control one. However, electron microcopy was utilized to trace the apoptotic distribution in the developmental embryos. Minor abnormalities that are dependent on the laser dose have been shown in the irradiated embryos when compared to the sham group, where the highest laser dose showed about 12% embryonic development anomalies when related to the other irradiated groups. Irradiated embryos were found to express more INF-γ and IL-2 as circulating cytokines relative to the unexposed group, where the levels of IL-2 were highly significantly increased by all laser doses (0.3 mJ/cm2 light dose recipient group showed significant increase only when compared to the control group). IFN-γ levels were significantly increased as well by light doses above 0.2 mJ/cm2. This IFN-γ increase trend seemed to be laser dose-dependent. Simultaneously, these combined results propose the ability of high laser doses in inducing incurable changes in the embryonic development and consequently such alterations can have potential therapeutic applications through what is known as photobiomodulation.

MicroRNAs and exosomes: key players in HIV pathogenesis.

OBJECTIVES: HIV infection is well known to cause impairment of the human immune system, and until recently was a leading cause of death. It has been shown that T lymphocytes are the main targets of HIV. The virus inactivates T lymphocytes by interfering with a wide range of cellular and molecular targets, leading to suppression of the immune system. The objective of this review is to investigate to what extent microRNAs (miRNAs) are involved in HIV pathogenesis. METHODS: The scientific literature (Pubmed and Google scholar) for the period 1988-2019 was searched. RESULTS: Mounting evidence has revealed that miRNAs are involved in viral replication and immune response, whether by direct targeting of viral transcripts or through indirect modulation of virus-related host pathways. In addition, exosomes have been found to act as nanoscale carriers involved in HIV pathogenesis. These nanovehicles target their cargos (i.e. DNA, RNA, viral proteins and miRNAs) leading to alteration of the behaviour of recipient cells. CONCLUSIONS: miRNAs and exosomes are important players in HIV pathogenesis. Additionally, there are potential diagnostic applications of miRNAs as biomarkers in HIV infection.

WITHDRAWN: Effect of He-Ne laser irradiation on embryonic development in chicken eggs.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). These articles are retracted at the request of the authors. The joint Editors-in-Chief agree with this decision.

Randomized Controlled Trial Comparing the Effects of Far-Infrared Emitting Ceramic Fabric Shirts and Control Polyester Shirts on Transcutaneous PO2.

Our aim was to confirm earlier studies showing tcPO2 to be higher under clothing made with polyethylene terephalate (PET) fabric containing ceramic particles (CEL) compared to standard PET fabric. In previous studies PET garments were donned first to avoid possible persistent effects from ceramic particles. This study randomized donning sequence to avoid bias. METHODS: Subjects were randomized to don either PET shirts first (PETF n=73) or CEL first (CELF n=80), switching garments after 90 minutes. Skin temperature (ST), arterial oxygen saturation (O2sat), and tcPO2 were measured every 30 minutes. RESULTS: Baseline ST and O2 sat were nearly identical in the two groups. Baseline tcPO2 was modestly higher in the CELF group than with PETF: 66.4 ± 18.9 vs. 63.9 ± 18.8 mmHg (n.s). Independent of donning sequence, tcPO2 measurements 90 minutes after wearing CEL were 6.7% higher than after 90 minutes wearing PET (p<0.0003). Sequence analysis found tcPO2 in PETF subjects to gradually rise before and after switching garments, but tcPO2 fell immediately after switching garments in CELF subjects. PETF baseline O2sat of 98.1 ± 1.3 increased insignificantly after 90 minutes, and then increased further to 98.6 ± 0.8 after wearing CEL ninety minutes (p=0.0001). CELF baseline O2sat of 97.9 ± 1.7 increased to 98.5 ± 1.1 90 minutes after donning CEL (p=0.0002) and fell to 98.3 ± 1.0 ninety minutes after switching to PET (p=0.0033). CONCLUSIONS: The ability of ceramic-embedded fabric to induce higher tcPO2 measurements is not due to sequence bias.

Zinc phthalocyanines attached to gold nanorods for simultaneous hyperthermic and photodynamic therapies against melanoma in vitro.

Studies indicate that hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of phthalocyanines on the surface of gold nanorods (well-characterized devices for hyperthermia generation) for the elimination of melanoma, one of the most important skin cancers due to its high lethality. Following the synthesis of nanorods through a seed-mediated method, the efficacy of photodynamic therapy (PDT) and hyperthermia was assessed separately. We chose to coat the nanorods with two tetracarboxylated zinc phthalocyanines - with or without methyl-glucamine groups. After the coating process, the phthalocyanines formed ionic complexes with the cetyltrimethylammonium bromide (CTAB) that was previously covering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by transmission electron microscopy (TEM), and their singlet oxygen and hydroxyl radical generation yields were assessed. Furthermore, they were tested in vitro with melanotic B16F10 and amelanotic B16G4F melanoma cells. The cells with nanoparticles were irradiated with laser (at 635nm), and the cell viability was assessed. The results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached on the nanorods surface, and the combination of PDT and hyperthermia was able to eliminate over 90% of melanoma cells. This is a novel study because two tetracarboxylated phthalocyanines were used and because the same wavelength was irradiated to activate both the nanorods and the photosensitizers.

Photobiomodulation of breast and cervical cancer stem cells using low-intensity laser irradiation.

Breast and cervical cancers are dangerous threats with regard to the health of women. The two malignancies have reached the highest record in terms of cancer-related deaths among women worldwide. Despite the use of novel strategies with the aim to treat and cure advanced stages of cancer, post-therapeutic relapse believed to be caused by cancer stem cells is one of the challenges encountered during tumor therapy. Therefore, further attention should be paid to cancer stem cells when developing novel anti-tumor therapeutic approaches. Low-intensity laser irradiation is a form of phototherapy making use of visible light in the wavelength range of 630-905 nm. Low-intensity laser irradiation has shown remarkable results in a wide range of medical applications due to its biphasic dose and wavelength effect at a cellular level. Overall, this article focuses on the cellular responses of healthy and cancer cells after treatment with low-intensity laser irradiation alone or in combination with a photosensitizer as photodynamic therapy and the influence that various wavelengths and fluencies could have on the therapeutic outcome. Attention will be paid to the biomodulative effect of low-intensity laser irradiation on cancer stem cells.

Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells.

There is an unmet clinical need for novel wound healing strategies to treat full thickness skin defects, especially in diabetic patients. We hypothesized that a scaffold could perform dual roles of a biomechanical support and a favorable biochemical environment for stem cells. Human umbilical cord perivascular cells (HUCPVCs) have been recently reported as a type of mesenchymal stem cell that can accelerate early wound healing in skin defects. However, there are only a limited number of studies that have incorporated these cells into natural scaffolds for dermal tissue engineering. The aim of the present study was to promote angiogenesis and accelerate wound healing by using HUCPVCs and decellularized dermal matrix (DDM) in a rat model of diabetic wounds. The DDM scaffolds were prepared from harvested human skin samples and histological, ultrastructural, molecular and mechanical assessments were carried out. In comparison with the control (without any treatment) and DDM alone group, full thickness excisional wounds treated with HUCPVCs-loaded DDM scaffolds demonstrated an accelerated wound closure rate, faster re-epithelization, more granulation tissue formation and decreased collagen deposition. Furthermore, immunofluorescence analysis showed that the VEGFR-2 expression and vascular density in the HUCPVCs-loaded DDM scaffold treated group were also significantly higher than the other groups at 7days post implantation. Since the rates of angiogenesis, re-epithelization and formation of granulation tissue are directly correlated with full thickness wound healing in patients, the proposed HUCPVCs-loaded DDM scaffolds may fulfil a role neglected by current treatment strategies. This pre-clinical proof-of-concept study warrants further clinical evaluation. STATEMENT OF SIGNIFICANCE: The aim of the present study was to design a novel tissue-engineered system to promote angiogenesis, re-epithelization and granulation of skin tissue using human umbilical cord perivascular stem cells and decellularized dermal matrix natural scaffolds in rat diabetic wound models. The authors of this research article have been working on stem cells and tissue engineering scaffolds for years. According to our knowledge, there is a lack of an efficient system for the treatment of skin defects using tissue engineering strategy. Since the rates of angiogenesis, re-epithelization and granulation tissue are directly correlated with full thickness wound healing, the proposed HUCPVCs-loaded DDM scaffolds perfectly fills the niche neglected by current treatment strategies. This pre-clinical study demonstrates the proof-of-concept that necessitates clinical evaluations.

The application of antimicrobial photodynamic therapy (aPDT) in dentistry: a critical review.

In recent years there have been an increasing number of in vitro and in vivo studies that show positive results regarding antimicrobial photodynamic therapy (aPDT) used in dentistry. These include applications in periodontics, endodontics, and mucosal infections caused by bacteria present as biofilms. Antimicrobial photodynamic therapy is a therapy based on the combination of a non-toxic photosensitizer (PS) and appropriate wavelength visible light, which in the presence of oxygen is activated to produce reactive oxygen species (ROS). ROS induce a series of photochemical and biological events that cause irreversible damage leading to the death of microorganisms. Many light-absorbing dyes have been mentioned as potential PS for aPDT and different wavelengths have been tested. However, there is no consensus on a standard protocol yet. Thus, the goal of this review was to summarize the results of research on aPDT in dentistry using the PubMed database focusing on recent studies of the effectiveness aPDT in decreasing microorganisms and microbial biofilms, and also to describe aPDT effects, mechanisms of action and applications.

Real-time evaluation of two light delivery systems for photodynamic disinfection of Candida albicans biofilm in curved root canals.

Antimicrobial photodynamic therapy (APDT) combined with endodontic treatment has been recognized as an alternative approach to complement conventional root canal disinfection methods on bacterial biofilms. We developed an in  vitro model of bioluminescent Candida albicans biofilm inside curved dental root canals and investigated the microbial reduction produced when different light delivery methods are employed. Each light delivery method was evaluated in respect to the light distribution provided inside curved root canals. After conventional endodontic preparation, teeth were sterilized before canals were contaminated by a bioluminescent strain of C. albicans (CEC789). Methylene blue (90 µM) was introduced into the canals and then irradiated (λ = 660 nm, P = 100 mW, beam diameter = 2 mm) with laser tip either in contact with pulp chamber or within the canal using an optical diffuser fiber. Light distribution was evaluated by CCD camera, and microbial reduction was monitored through bioluminescence imaging. Our findings demonstrated that the bioluminescent C. albicans biofilm model had good reproducibility and uniformity. Light distribution in dental tissue was markedly dependent on the light delivery system, and this strategy was directly related to microbial destruction. Both light delivery systems performed significant fungal inactivation. However, when irradiation was performed with optical diffuser fiber, microbial burden reduction was nearly 100 times more effective. Bioluminescence is an interesting real-time analysis to endodontic C. albicans biofilm inactivation. APDT showed to be an effective way to inactivate C. albicans biofilms. Diffuser fibers provided optimized light distribution inside curved root canals and significantly increased APDT efficiency.

Photodynamic therapy plus regulatory T-cell depletion produces immunity against a mouse tumour that expresses a self-antigen.

BACKGROUND: Photodynamic therapy (PDT) can lead to development of antigen-specific immune response and PDT-mediated immunity can be potentiated by T regulatory cell (Treg) depletion. We investigated whether the combination of PDT with cyclophosphamide (CY) could foster immunity against wild-type tumours expressing self-antigen (gp70). METHODS: Mice with CT26 tumours were treated with PDT alone or in combination with low-dose CY. T regulatory cell numbers and transforming growth factor-ß (TGF-ß) levels were measured at several time points after treatment. Mice cured by PDT+CY were rechallenged with CT26 and monitored for long-term survival. RESULTS: Photodynamic therapy+CY led to complete tumour regression and long-term survival in 90% of treated mice while the absolute numbers of Treg decreased after PDT+CY and the TGF-ß levels were reduced to a level comparable to naïve mice. Sixty-five percent of the mice treated with PDT+CY that survived over 90 days tumour free rejected the rechallenge with the same tumour when a second dose of CY was administered before rechallenge but not without. CONCLUSION: Administration of CY before PDT led to depletion of Treg and potentiated PDT-mediated immunity, leading to long-term survival and development of memory immunity that was only uncovered by second Treg depletion.

Decacationic [70]Fullerene Approach for Efficient Photokilling of Infectious Bacteria and Cancer Cells.

Photodynamic inactivation of pathogenic bacteria and cancer cells by novel water-soluble decacationic fullerene monoadducts, C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2], were investigated. In the presence of a high number of electron-donating iodide anions as parts of quaternary ammonium salts in the arm region, we found that C70[>M(C3N6+C3)2] produced more highly reactive HO• radical than C60[>M(C3N6+C3)2], in addition to singlet oxygen (1O2). This finding offers an explanation of the preferential killing of Gram-positive and Gram-negative bacteria by C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2], respectively. The hypothesis is that 1O2 can diffuse more easily into porous cell walls of Gram-positive bacteria to reach sensitive sites, while the less permeable Gram-negative bacterial cell wall needs the more reactive HO• to cause real damage.

Topical antimicrobials for burn wound infections.

Throughout most of history, serious burns occupying a large percentage of body surface area were an almost certain death sentence because of subsequent infection. A number of factors such as disruption of the skin barrier, ready availability of bacterial nutrients in the burn milieu, destruction of the vascular supply to the burned skin, and systemic disturbances lead to immunosuppression combined together to make burns particularly susceptible to infection. In the 20th century the introduction of antibiotic and antifungal drugs, the use of topical antimicrobials that could be applied to burns, and widespread adoption of early excision and grafting all helped to dramatically increase survival. However the relentless increase in microbial resistance to antibiotics and other antimicrobials has led to a renewed search for alternative approaches to prevent and combat burn infections. This review will cover patented strategies that have been issued or filed with regard to new topical agents, preparations, and methods of combating burn infections. Animal models that are used in preclinical studies are discussed. Various silver preparations (nanocrystalline and slow release) are the mainstay of many approaches but antimicrobial peptides, topical photodynamic therapy, chitosan preparations, new iodine delivery formulations, phage therapy and natural products such as honey and essential oils have all been tested. This active area of research will continue to provide new topical antimicrobials for burns that will battle against growing multidrug resistance.

Ultraviolet C inactivation of dermatophytes: implications for treatment of onychomycosis.

BACKGROUND: Onychomycosis responds to systemic antifungals and sometimes to topical lacquers, but alternative treatments are desirable. Topical application of germicidal ultraviolet (UV) C radiation may be an acceptable and effective therapy for infected nails. OBJECTIVES: To test the ability of UVC to inactivate dermatophyte suspensions in vitro and to sterilize a novel ex vivo model of nail infection. METHODS: Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum and Microsporum canis suspensions were irradiated with UVC (254 nm) at a radiant exposure of 120 mJ cm(-2) and surviving colony-forming units quantified. T. rubrum infecting porcine hoof slices and human toenail clippings was irradiated with UVC at radiant exposures of 36-864 J cm(-2). RESULTS: In vitro studies showed that 3-5 logs of cell inactivation in dermatophyte suspensions were produced with 120 mJ cm(-2) UVC irradiation. Depending on factors such as the thickness and infectious burden of the ex vivo cultures, the radiant exposure of UVC needed for complete sterilization was usually in the order of tens to hundreds of J cm(-2). Resistance of T. rubrum to UVC irradiation did not increase after five cycles of subtotal inactivation in vitro. CONCLUSIONS: UVC irradiation may be a less invasive treatment option for onychomycosis, when the appropriate consideration is given to safety.

A green fluorescent protein-expressing murine tumour but not its wild-type counterpart is cured by photodynamic therapy.

The ideal cancer treatment should both destroy the primary tumour and at the same time educate the immune system to recognise the tumour as foreign so that distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the i.v. administration of photosensitisers followed by illumination of the tumour with red light producing reactive oxygen species that eventually cause vascular shutdown and tumour cell death by apoptosis and necrosis. Anti-tumour immunity is stimulated after PDT due to the acute inflammatory response, generation of tumour-specific antigens, and induction of heat-shock proteins. Green fluorescent protein (GFP) is used as an optical reporter to noninvasively image the progression of mouse tumours, and in addition, may act as a foreign (jellyfish) antigen. We asked whether GFP-expressing tumours could be used to monitor the response of tumour-bearing mice to PDT, and whether the tumour response differed when a nonimmunogenic tumour cell line was transduced with GFP. We injected RIF-1 or RIF-1 EGFP (stably transduced with a retroviral vector) cells in the leg of C3H/HeN mice and both the cells and tumour grew equally well. We used PDT with benzoporphyrin derivative and a short drug-light interval. There were complete cures and 100% mouse survival of RIF-1 EGFP while RIF-1 wild-type tumours all recurred. Cured mice were resistant to rechallenge with RIF-1 EGFP cells and a rechallenge with wild-type RIF-1 cells grew significantly slower. There was also slower RIF-1 EGFP rechallenge growth but no rejection when RIF-1 EGFP tumours were surgically removed. There was a low rate of PDT cure of tumours when RIF-1 cells were transduced with an empty retroviral vector. The presence of antibodies against EGFP in mouse serum suggests EGFP can act as a foreign antigen and PDT can then stimulate a long-term memory immune response.

Macrophage-targeted photodynamic therapy: scavenger receptor expression and activation state.

Macrophage-targeted photodynamic therapy (PDT) may have applications in the selective killing of cells involved in atherosclerosis, inflammation and tumor. We have previously shown that a conjugate between the photosensitizer chlorin(e6) (ce6) and maleylated bovine serum albumin (BSA-mal) gives highly selective targeting to macrophages. In this report we examine the effect of macrophage activation and scavenger receptor class A (SRA) expression on this targeting in two murine macrophage tumor cell lines (RAW264.7 and P388D1) and a control murine mammary sarcoma cell line (EMT-6). Cells were pretreated with interferon gamma (IFNgamma) and/or lipopolysaccharide (LPS) followed byBSA-ce6-mal addition, and SRA expression, tumor necrosis factor alpha (TNFalpha) release, conjugate uptake and PDT killing were measured. Both macrophage cell lines expressed SRA and took up conjugate specifically in an SRA-dependent manner, but differences were observed in their response to activation. RAW264.7 expressed increasingly more SRA and took up increasingly more BSA-ce6-mal in response to IFNgamma, LPS, and IFNgamma+LPS, respectively. The PDT killing did not follow the same pattern as the uptake of the photosensitizer. The increase in uptake in the IFNgamma treated cells did not lead to an increase in PDT killing, while stimulation with LPS or IFNgamma + LPS resulted in a significant protection against PDT, despite a significant increase in photosensitizer uptake. P388D1 was responsive to neither IFNgamma, nor to LPS, or to IFNgamma +LPS with respect to SRA expression, conjugate uptake, and PDT killing. These data may have implications for the use of PDT to target physiologically undesirable macrophage subtypes implicated in disease, and on how manipulation of the activation status of the macrophage will influence the PDT effect.

Photodynamic therapy targeted to pathogens.

Photodynamic therapy (PDT) employs a non-toxic dye termed a photosensitizer (PS) together with low intensity visible light, which, in the presence of oxygen, produce cytotoxic species. PS can be targeted to its destination cell or tissue and, in addition, the irradiation can be spatially confined to the lesion giving PDT the advantage of dual selectivity. This promising approach can be used for various applications including microbial inactivation and the treatment of infections. Resistance to PDT has not been shown and multiantibiotic-resistant strains are as easily killed as naive strains. It is known that Gram (+) bacteria are more sensitive to PDT as compared to Gram (-) species. However, the use of cationic PS or agents that increase the permeability of the outer membrane allows for the effective killing of Gram (-) organisms. Some PS have an innate positive charge, but our approach is to link PS to a cationic molecular vehicle such as poly-L-lysine. This modification dramatically increases PS binding to and penetrating through the negatively charged bacterial permeability barrier. Due to focused light delivery the use of PDT is possible only for localized infections. Nonetheless numerous diseases can be treated. Selectivity of the PS for microbes over host cells, accurate delivery of the PS into the infected area, and PDT dose adjustment help minimize side effects and give PDT an advantage over conventional therapy. There are only a few reports about the use of antimicrobial PDT in animal models and clinical trials. We have used genetically modified bioluminescent bacteria to follow the effect of PDT in infected wounds, burns, and soft tissue infections in mice. Not only were bacteria infecting wounds, burns, and abscesses killed, but mice were saved from death due to sepsis and wound healing was improved.

Macrophage-targeted photodynamic therapy.

Photodynamic therapy (PDT) uses light activatable molecules that after illumination produce reactive oxygen species and unwanted tissue destruction. PDT has dual selectivity due to control of light delivery and to some extent selective photosensitizer (PS) accumulation in tumors or other diseased tissue, additional targeted selectivity of PS for disease is necessary. The delivery of drugs to selected lesions can be enhanced by the preparation of targeted macromolecular conjugates that employ cell type specific targeting by ligand-receptor recognition. Macrophages and monocytes express a scavenger-receptor that is a high-capacity route for delivering molecules into endocytic compartments in a cell-type specific manner. We have shown that by attaching PS to scavenger-receptor ligands it is possible to get three logs of selective cell killing in macrophages while leaving non-macrophage cells unharmed. The capability to selectively kill macrophages has applications in treating cancer and in the detection and therapy of vulnerable atherosclerotic plaque and possibly for autoimmune disease and some infections.

Pegylation of charged polymer-photosensitiser conjugates: effects on photodynamic efficacy.

Conjugates between photosensitisers (PS) and charged polymeric carriers are under investigation for photodynamic therapy of cancer and may allow targeting to certain cell types or compartments in tumours. Covalent attachment of polyethylene glycol to macromolecules (pegylation) may alter their pharmacokinetics, cell type targeting, and photophysical properties. Macrophages may take up large amounts of aggregated PS, thus lessening the selectivity for cancer cells in tumours. We investigated the effect of pegylation on the uptake and phototoxicity of poly-L-lysine chlorin(e6) conjugates with either cationic or anionic charges in two cell lines, human ovarian cancer cells and mouse macrophages. The cationic conjugate after pegylation became less aggregated, consumed less oxygen and had reduced cellular uptake. However, the phototoxicity corrected for cellular uptake increased three- to five-fold. In contrast, the anionic succinylated conjugate on pegylation became more aggregated, consumed similar amounts of oxygen, and had higher cellular uptake. The anionic conjugate showed the highest relative phototoxicity towards both the cell lines (compared to the other three conjugates) and it decreased most towards the macrophages after pegylation. Pegylation reduced the amount of oxygen consumed per chlorin(e6) molecule when photosensitised cells were illuminated. These in vitro studies suggest that pegylation alters the phototoxicity of PS conjugates depending on the effect produced on the aggregation state.

Pegylation of a chlorin(e6) polymer conjugate increases tumor targeting of photosensitizer.

Photodynamic therapy is emerging as a viable modality for the treatment of many cancers. A limiting factor in its use against intracavity tumors such as disseminated ovarian cancer is insufficient selectivity of the photosensitizer for tumor compared with normal tissue. We report on an approach to improve tumor targeting by exploiting differences between cell types and by chemical modification of a photosensitizer conjugate. Attachment of polyethylene glycol (pegylation) to a polyacetylated conjugate between poly-l-lysine and chlorin(e6) increased the relative phototoxicity in vitro toward an ovarian cancer cell line (OVCAR-5) while reducing it toward a macrophage cell line (J774), compared with the nonpegylated conjugate. Surprisingly, the increased phototoxicity of the pegylated conjugate correlated with reduced oxygen consumption. Pegylation also reduced the tendency of the conjugate to aggregate and reduced the consumption of oxygen when the conjugates were illuminated in solution in serum containing medium, suggesting a switch in photochemical mechanism from type II (singlet oxygen) to type I (radicals or electron transfer). Pegylation led to more mitochondrial localization as shown by confocal fluorescence microscopy in OVCAR-5 cells, and, on illumination, produced a switch in cell death mechanism toward apoptosis not seen with J774 cells. Conjugates were injected i.p. into nude mice bearing i.p. OVCAR-5 tumors, and the pegylated conjugate gave higher amounts of photosensitizer in tumor and higher tumor:normal tissue ratios and increased the depth to which the chlorin(e6) penetrated into the peritoneal wall. Taken together, these results suggest that pegylation of a polymer-photosensitizer conjugate improves tumor-targeting and may increase the efficacy of photodynamic therapy for ovarian cancer.

Epidermal growth factor receptor-targeted immunophotodiagnosis and photoimmunotherapy of oral precancer in vivo.

Immunophotodiagnosis uses a fluorescence-labeled monoclonal antibody (MAb) that recognizes a tumor-associated antigen to image the fluorescence emitted from the fluorophore-bound MAb that has localized in the tissue. It may be used to diagnose malignant or precancerous lesions, to delineate the margins for tumor resection, or as a feedback mechanism to assess response to treatment. In oral precancer, the epidermal growth factor receptor (EGFR) is overexpressed and could be used as a marker for early detection or as a target for therapy. The goal of this study was to test an anti-EGFR MAb (C225) coupled to either the near-infrared fluorescent dye N,N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid for detection or a photochemically active dye (chlorin(e6)) for therapy of early premalignancy in the hamster cheek pouch carcinogenesis model. Fluorescence levels in the carcinogen-treated tissue correlated with the histological stage of the lesions when the C225-N,N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid conjugate was used but did not do so with the irrelevant conjugates. Discrete areas of clinically normal mucosa with high fluorescence (hot spots) were subsequently shown by histology to contain dysplastic areas. The best contrast between normal and carcinogen-treated cheek pouches was found at 4-8 days after injection. To test the potential of immunophotodiagnosis as a feedback modality for therapeutic intervention, experiments were conducted with the same MAb conjugated to chlorin(e6) followed by illumination to reduce expression of the EGFR by a photodynamic effect. Subsequent immunophotodiagnosis showed that this treatment led to a significant reduction in fluorescence in the carcinogen-treated cheek pouch compared with nonilluminated areas. This difference between illuminated and dark areas was not seen in the normal cheek pouch. Taken together, the results demonstrate the potential for development of immunophotodiagnosis as a diagnostic tool and as a method of monitoring response to therapy and that the EGFR may be an appropriate target in head and neck cancer.