Phonetic characteristics of German unstressed vowels: The case of opa vs Oper.

Kohler [Einführung in die Phonetik des Deutschen (Erich Schmidt Verlag, Berlin, Germany, 1995)] stated that German [ɐ] and [a] in unstressed syllables are merging. The present study tested this hypothesis. The contrast was found intact word-internally and word-finally. Neighborhood density enhanced its phonetic characteristics, but no effects of frequency and conditional probability were found.

Small anticancer drug release by light: Photochemical internalization of porphyrin-ß-cyclodextrin nanoparticles.

Aiming to engineer simple, neutral, strongly amphiphilic photoactive nanoparticles (NPs) to specifically target cancer cell lysosomes for drug transport and light-controlled release, new conjugates of ß-cyclodextrin with highly hydrophobic triphenylporphyrin bearing different alkyl chains, were synthesized. Although differently sized, all conjugates self-assemble into ~60 nm NPs in water and display similar photoactivity. The NPs target selectively the lysosomes of breast adenocarcinoma MCF-7 cells, embedding in vesicular membranes, as experiments with model liposomes indicate. Either empty or drug-loaded, the NPs lack dark toxicity for 48 h. They bind with differently structured anticancer drugs tamoxifen and gemcitabine as its N-adamantyl derivative. Red light irradiation of cells incubated with drug-loaded NPs results in major reduction of viability (>85 %) for 48 h displaying significant synergy of photo-chemotoxicity, as opposed to empty NPs, and to loaded non-irradiated NPs, in manifestation of photochemical internalization (PCI). Our approach expands the field of PCI into different small molecule chemotherapeutics.

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

Background: Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement. Methods: DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody. Results: VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models. Conclusions: VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Corrigendum: Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

[This corrects the article DOI: 10.3389/fimmu.2023.1278000.].

Sonochemical Internalization of Bleomycin Inhibits Adenocarcinoma Breast Tumor Development in an Orthotopic Rat Model.

One approach to reducing post-operative tumor recurrence and alleviate debilitating side effects of systemic chemotherapy, is work centered on the development of drug activation by focused and targeted externally applied physical energy thus providing site and temporal specificity. One such technique, light mediated photochemical internalization (PCI), has been shown to be a method to obtain enhanced chemotherapy efficacy for a wide variety of anti-cancer agents. A related technology, sonochemical internaization (SCI), is an extension of the PCI concept developed to overcome the limitations of poor light penetration in tissue. SCI utilizes ultrasonic energy, to activate sonosensitizers, co-administered with anti cancer agents. The purpose of the study reported here was to evaluate the inhibitory effects of SCI of bleomycin (BLM), both in vitro and in vivo, on the adenocarcinoma breast tumor rat cell line Mat B III. In vitro, the two aspects of sonication, sonoporation (SP) and sonochemical internalization (SCI) of BLM were examined. In vivo, BLM-SCI significantly inhibited tumor development, following Mat B III implantation, in an orthotopic breast tumor animal model using Fisher rats.

Inhibition of glioma development by doxorubicin-photochemical internalization generated macrophage vaccine: A survival study in rats.

BACKGROUND: The process known as immunogenic cell death (ICD) is characterized by dead and dying cancer cells exposing and releasing so-called damage associated molecular patterns (DAMPs). ICD has been shown to enhance the efficacy of antigen presenting cell (APC) immunotherapy. Both anthracycline drugs such as doxorubicin (DOX), and photodynamic therapy (PDT) have been shown to be inducers of ICD. It was therefore hypothesized that combined PDT and DOX i.e. photochemical internalization of DOX (DOX-PCI) would increase ICD compared to DOX acting as a single agent. MATERIALS AND METHODS: F98 glioma cells were treated with DOX-PCI in vitro and the ICD markers HMGB1, HSP70, and HSP90 were determined by ELISA assay. Peritoneal macrophages (Ma), obtained from Fisher rats acting as APCs, were co-incubated with dead F98 glioma cells killed via DOX or DOX-PCI treatment ex vivo. The pulsed Ma (Ma DOX or Ma DOX-PCI) were used to inoculate the animals either before (preventive) or after (curative) intra-cranially implantation of the glioma cells. RESULTS: F98 cells, treated with DOX-PCI in vitro, induced a significantly higher level of HGMB1, HSP70, and HSP90 than DOX acting alone. Ma DOX-PCI inoculated animals, in both preventive and curative protocols, had a pronounced survival benefit compared to either the non-treatment or MaDOX control groups. In the curative protocol, a second booster inoculation significantly improved survival, with 60% of the animals alive at day 60. CONCLUSION: Macrophages primed with DOX-PCI treated glioma cells appeared to be highly effective as APCs and, when injected into host animals, could delay and, in some cases, prevent tumor development.

Predictive biomarkers for 5-ALA-PDT can lead to personalized treatments and overcome tumor-specific resistances.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive, clinically approved therapy with numerous advantages over other mainstream cancer therapies. 5-aminolevulinic acid (5-ALA)-PDT is of particular interest, as it uses the photosensitiser PpIX, naturally produced in the heme pathway, following 5-ALA administration. Even though 5-ALA-PDT shows high specificity to cancers, differences in treatment outcomes call for predictive biomarkers to better stratify patients and to also diversify 5-ALA-PDT based on each cancer's phenotypic and genotypic individualities. AIMS: The present study seeks to highlight key biomarkers that may predict treatment outcome and simultaneously be exploited to overcome cancer-specific resistances to 5-ALA-PDT. METHODS AND RESULTS: We submitted two glioblastoma (T98G and U87) and three breast cancer (MCF7, MDA-MB-231, and T47D) cell lines to 5-ALA-PDT. Glioblastoma cells were the most resilient to 5-ALA-PDT, while intracellular production of 5-ALA-derived protoporphyrin IX (PpIX) could not account for the recorded PDT responses. We identified the levels of expression of ABCG2 transporters, ferrochelatase (FECH), and heme oxygenase (HO-1) as predictive biomarkers for 5-ALA-PDT. GPX4 and GSTP1 expression vs intracellular glutathione (GSH) levels also showed potential as PDT biomarkers. For T98G cells, inhibition of ABCG2, FECH, HO-1, and/or intracellular GSH depletion led to profound PDT enhancement. Inhibition of ABCG2 in U87 cells was the only synergistic adjuvant to 5-ALA-PDT, rendering the otherwise resistant cell line fully responsive to 5-ALA-PDT. ABCG2 or FECH inhibition significantly enhanced 5-ALA-PDT-induced MCF7 cytotoxicity, while for MDA-MB-231, ABCG2 inhibition and intracellular GSH depletion conferred profound synergies. FECH inhibition was the only synergism to ALA-PDT for the most susceptible among the cell lines, T47D cells. CONCLUSION: This study demonstrates the heterogeneity in the cellular response to 5-ALA-PDT and identifies biomarkers that may be used to predict treatment outcome. The study also provides preliminary findings on the potential of inhibiting specific molecular targets to overcome inherent resistances to 5-ALA-PDT.

RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer.

HER2 is a predictive biomarker for HER2-targeted therapeutics. For antibody-drug conjugates (ADCs; e.g., trastuzumab emtansine (T-DM1)), HER2 is utilized as a transport gate for cytotoxic agents into the cell. ADC biomarkers may therefore be more complex, also reflecting the intracellular drug transport. Here we report on a positive correlation between the early endosome marker RAB5A and T-DM1 sensitivity in five HER2-positive cell lines. Correlation between RAB5A expression and T-DM1 sensitivity is confirmed in breast cancer patients treated with trastuzumab emtansine/pertuzumab in the I-SPY2 trial (NCT01042379), but not in the trastuzumab/paclitaxel control arm. The clinical correlation is further verified in patients from the KAMILLA trial (NCT01702571). In conclusion, our results suggest RAB5A as a predictive biomarker for T-DM1 response and outline proteins involved in endocytic trafficking as predictive biomarkers for ADCs.

Squamous cell carcinoma of the ear treated by photochemical internalization.

A 77-year-old Caucasian male was diagnosed with squamous cell cancer of the right ear. The patient elected to take part in the first-in-man phase I TPCS2a based bleomycin photochemical internalization (PCI). On Day 0, The patient received the photosensitiser [Amphinex (TPCS2a)], by slow intravenous injection. Four days later, surface illumination based (PCI) was implemented 3 h after the slow infusion of Bleomycin. Four weeks following the infusion of the photosensitiser, the cancerous area turned into black rigid mass with clear demarcation from the macroscopically normal skin. The size of the treated area has been substantially reduced. Histopathologic assessment of the excised necrotic mass revealed no viable tumour and the excised margins (PCI-treated margins) were tumour-free. This case was a clear indication that PCI is a clinically relevant technique that has potential in the treatment of such cancers to avoid radical intervention.

Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements.

Metabolic modulation of macrophage activation has emerged as a promising strategy lately in immunotherapeutics. However, macrophages have a broad spectrum of functions and thus, understanding the exact metabolic changes that drive a particular immune response, is of major importance. In our previous work, we have reported a key role of nitric oxide (NO●) in two(2)-signal activated macrophages [M(2-signals)]. Further characterization using metabolic analysis in intact cells, showed that the basal and maximal respiration levels of M(2-signals) were comparable, with cells being unresponsive to the injections-inducd mitochondrial stress. Here, we show that excessive NO● secretion by the M(2-signals) macrophages, interferes with the oxygen (O2) consumption measurements on cells using the seahorse metabolic analyzer. This is attributed mainly to the consumption of ambient oxygen by NO● to form NO2- and/or NO3- but also to the reduction of O2 to superoxide anion (O2●-) by stray electrons from the electron transport chain, leading to the formation of peroxynitrite (ONOO-). We found that reactive species-donors in the absence of cells, produce comparable oxygen consumption rates (OCR) with M(2-signals) macrophages. Furthermore, inhibition of NO● production, partly recovered the respiration of activated macrophages, while external addition of NO● in non-activated macrophages downregulated their OCR levels. Our findings are crucial for the accurate metabolic characterization of cells, especially in cases where reactive nitrogen or oxygen species are produced in excess.

An improved in vitro photochemical internalization protocol for 3D spheroid cultures.

Photochemical internalization (PCI) is a modified form of photodynamic therapy (PDT) that enhances the efficacy of therapeutic agents in a site and temporal specific manner in both in vitro and in vivo publications. The purpose of the study reported here was to evaluate the benefits of a modified PCI protocol in a 3D rat glioma spheroid model. In the modified protocol, F98 glioma cells were incubated with photosensitizer (AlPcS2a) prior to spheroid generation, as opposed to post-spheroid formation photosensitizer exposure commonly used in conventional protocols. The efficacy of both bleomycin and doxorubicin PCI was evaluated using either the conventional or modified protocols. The formed spheroids were then exposed to light treatment from a diode laser, λ= 670 nm. Spheroid growth was monitored for a period of 14 days. The results of spheroid growth assays showed that there was no statistically significant difference in PCI efficacy between the conventional and modified protocols for both of the drugs tested. The direct PDT effect was significantly reduced using the modified protocol. Therefore, due to its several advantages, the modified protocol is recommended for evaluating the efficacy of PCI in tumor spheroid models.

Photosensitizer delivery by fibrin glue: potential for bypassing the blood-brain barrier.

Fibrin glue (FG) has potential as a delivery vehicle for photosensitizer directly to the resection cavity, so it may bypass the blood-brain barrier (BBB) and increase the concentration of successfully delivered photosensitizer. A specialized form of photodynamic therapy (PDT), photochemical internalization (PCI), which involves both photosensitizer and chemotherapeutic agent internalization, can locally inhibit the growth of cells. This will allow the reduction of recurrence of malignant gliomas around surgical resection. This study will look at the efficacy of FG loaded with drugs in mediating both PDT and PCI in inhibiting 3-dimensional tumor spheroid growth in vitro. Experiments were conducted on spheroids comprised of F98 glioma cells using photosensitizer AlPcS2a and chemotherapeutic drug bleomycin (BLM). At 2-, 24-, 48-, and 72-h increments, supernatant covering an FG layer within a well was collected and replaced by fresh medium, then added to spheroid-containing wells, which contained the respective chemicals for PDT and PCI. The wells were then exposed to light treatment from a diode laser, and after, spheroid growth was monitored for a period of 14 days. Significant spheroid growth inhibition was observed in both PDT and PCI modalities, but was far greater in PCI. Additionally, complete growth suppression was achieved via PCI at the highest radiant exposure. Achieving a slow photosensitizer release, significant F98 spheroid inhibition was observed in FG-mediated PDT and PCI. The present study showed BLM-PCI was the most efficacious of the two modalities.

Word class and spelling in English.

The spelling of an English word may reflect its part of speech, not just the sounds within it. In 2 preregistered experiments, we asked whether university students are sensitive to 1 effect of part of speech that has been observed by linguists: that content words (e.g., the noun inn) must be spelled with at least 3 letters, whereas function words (e.g., the preposition in) may have only 2 letters. Participants heard VC (vowel-consonant) and consonant-vowel-consonant (CVC; consonant-vowel-consonant) nonwords that were used as nouns (content words) or prepositions (function words). Participants either spelled the items on their own or chose between options with single and double final consonants (e.g., ib vs. ibb). Participants in the choice task favored final consonant doubling for VCs that were used as nouns. They usually chose single final consonants for VCs that were used as prepositions and for CVCs. Effects of word class were also found in the spelling production task. Final consonant doubling was less common in the production task than the choice task, reflecting participants' reluctance to produce this relatively uncommon spelling pattern. Our results support the view that spelling performance reflects the combined influences of multiple patterns, both phonological and nonphonological. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Enhanced gene transfection of macrophages by photochemical internalization: Potential for gene-directed enzyme prodrug therapy of gliomas.

BACKGROUND: Drawn by tumor synthesis of chemo-attractive factors, macrophages are frequently found in and around glioblastomas and play an important role both in augmenting as well as inhibiting tumor growth. Patient-derived macrophages have the potential, therefore, to act as targeted delivery vectors for a variety of anti-cancer treatments. Among these is ex vivo gene transfection and re-injection back into the patient of macrophages to target residual tumors. In this study, photochemical internalization (PCI) is investigated as a technique for the non-viral transfection of the cytosine deaminase (CD) prodrug activating gene into macrophages. The CD gene encodes an enzyme that converts the nontoxic antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU) - a potent chemotherapeutic agent. MATERIALS: PCI (photosensitizer + light treatment) mediated CD gene transfection of rat alveolar Ma cells was carried out in vitro. CD gene transfected NR8383 macrophages were co-cultured with F98 rat glioma cells in the presence or absence of 5-FC. Cell viability was assayed using the MTS colorimetric assay. RESULTS: Compared to the glioma cells, NR8383 demonstrated enhanced resistance to the toxic effects of 5-FU. PCI greatly increased the transfection efficiency of the CD gene in NR8383 cells. The viability of F98 cells was significantly inhibited by coculture with CD transfected NR8383 macrophages and 5-FC. CONCLUSION: Although gene insertion into macrophages has proven difficult, the results presented here show that non-viral transfection of the CD gene into these immune cells can be enhanced via PCI. CD transfected NR8383 cells could efficiently convert 5-FC to 5-FU and export the drug, producing a pronounced bystander toxic effect on adjacent non-transfected glioma cells. Compared to single treatment, repetitive PCI-induced transfection was more efficient at low CD plasmid concentration.

Drug-Loaded Lipid-Coated Hybrid Organic-Inorganic "Stealth" Nanoparticles for Cancer Therapy.

Hybrid porous nanoscale metal organic frameworks (nanoMOFs) made of iron trimesate are attracting increasing interest as drug carriers, due to their high drug loading capacity, biodegradability, and biocompatibility. NanoMOF surface modification to prevent clearance by the innate immune system remains still challenging in reason of their high porosity and biodegradable character. Herein, FDA-approved lipids and poly(ethylene glycol) (PEG)-lipid conjugates were used to engineer the surface of nanoMOFs by a rapid and convenient solvent-exchange deposition method. The resulting lipid-coated nanoMOFs were extensively characterized. For the first time, we show that nanoMOF surface modification with lipids affords a better control over drug release and their degradation in biological media. Moreover, when loaded with the anticancer drug Gem-MP (Gemcitabine-monophosphate), iron trimesate nanoMOFs acted as "Trojan horses" carrying the drug inside cancer cells to eradicate them. Most interestingly, the PEG-coated nanoMOFs escaped the capture by macrophages. In a nutshell, versatile PEG-based lipid shells control cell interactions and open perspectives for drug targeting.

Photodynamic Efficacy of Cercosporin in 3D Tumor Cell Cultures.

In the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco-PDT, in contrast to the other two cell lines (T47D and T98G). While 300 µm diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 µm) start to regrow following blue-light PDT and exhibit high viability. Cerco-PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (~590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco-PDT. U87 spheroids also experienced a respiratory collapse following cerco-PDT, but retained half their glycolytic activity.

Targeted Photodynamic Therapy of Human Head and Neck Squamous Cell Carcinoma with Anti-epidermal Growth Factor Receptor Antibody Cetuximab and Photosensitizer IR700DX in the Mouse Skin-fold Window Chamber Model.

Targeted photodynamic therapy (PDT) in head/neck cancer patients with a conjugate of the anti-epidermal growth factor receptor (EGFR) antibody, Cetuximab and a phthalocyanine photosensitizer IR700DX is under way, but the exact mechanisms of action are still not fully understood. In this study, the EGFR-overexpressing human head/neck OSC-19-luc2-cGFP tumor with transfected GFP gene was used in a skin-fold window chamber model in BALB/c nude mice. The uptake and localization of the conjugate in the tumor and its surrounding normal tissues were studied by an intravital confocal laser scanning microscopy with image analyses. The tumor was also irradiated with 690 nm laser light 24 h after conjugate administration. The vascular and tumor responses were examined by morphological evaluation and immunohistochemistry (IHC). The amount of conjugate in the tumor peaked at 24-48 h after injection. Image analyses of colocalization correlation parameters demonstrated a high fraction of the conjugate IR700DX colocalized in the GFP-expressing tumor cells. PDT-treated tumors showed extensive necrotic/apoptotic destruction with little vascular damage, while IHC showed no HIF-1α expression and decreased EGFR and Ki67 expression with activated caspase-3 overexpression, indicating a direct killing of tumor cells through both necrotic and apoptotic cell death.

Photochemically-Induced Release of Lysosomal Sequestered Sunitinib: Obstacles for Therapeutic Efficacy.

Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was found to accumulate in the membrane of endo/lysosomal compartments together with the photosensitizer disulfonated tetraphenylchlorin (TPCS2a). Furthermore, the treatment effect was potentiated by PCI in the human HT-29 and the mouse CT26.WT colon cancer cell lines. The cytotoxic outcome of sunitinib-PCI was, however, highly dependent on the treatment protocol. Thus, neoadjuvant PCI inhibited lysosomal accumulation of sunitinib. PCI also inhibited lysosomal sequestering of sunitinib in HT29/SR cells with acquired sunitinib resistance, but did not reverse the resistance. The mechanism of acquired sunitinib resistance in HT29/SR cells was therefore not related to lysosomal sequestering. Sunitinib-PCI was further evaluated on HT-29 xenografts in athymic mice, but was found to induce only a minor effect on tumor growth delay. In immunocompetent mice sunitinib-PCI enhanced areas of treatment-induced necrosis compared to the monotherapy groups. However, the tumor growth was not delayed, and decreased infiltration of CD3-positive T cells was indicated as a possible mechanism behind the failed overall response.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research.

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.

In-Vivo Optical Monitoring of the Efficacy of Epidermal Growth Factor Receptor Targeted Photodynamic Therapy: The Effect of Fluence Rate.

Targeted photodynamic therapy (PDT) has the potential to improve the therapeutic effect of PDT due to significantly better tumor responses and less normal tissue damage. Here we investigated if the efficacy of epidermal growth factor receptor (EGFR) targeted PDT using cetuximab-IRDye700DX is fluence rate dependent. Cell survival after treatment with different fluence rates was investigated in three cell lines. Singlet oxygen formation was investigated using the singlet oxygen quencher sodium azide and singlet oxygen sensor green (SOSG). The long-term response (to 90 days) of solid OSC-19-luc2-cGFP tumors in mice was determined after illumination with 20, 50, or 150 mW·cm-2. Reflectance and fluorescence spectroscopy were used to monitor therapy. Singlet oxygen was formed during illumination as shown by the increase in SOSG fluorescence and the decreased response in the presence of sodium azide. Significantly more cell death and more cures were observed after reducing the fluence rate from 150 mW·cm-2 to 20 mW·cm-2 both in-vitro and in-vivo. Photobleaching of IRDye700DX increased with lower fluence rates and correlated with efficacy. The response in EGFR targeted PDT is strongly dependent on fluence rate used. The effectiveness of targeted PDT is, like PDT, dependent on the generation of singlet oxygen and thus the availability of intracellular oxygen.