Photodynamic therapy (PDT) is a clinically approved cancer therapy, based on a photochemical reaction between a light activatable molecule or photosensitizer, light, and molecular oxygen. When these three harmless components are present together, reactive oxygen species are formed. These can directly damage cells and/or vasculature, and induce inflammatory and immune responses. PDT is a two-stage procedure, which starts with photosensitizer administration followed by a locally directed light exposure, with the aim of confined tumor destruction. Since its regulatory approval, over 30 years ago, PDT has been the subject of numerous studies and has proven to be an effective form of cancer therapy. This review provides an overview of the clinical trials conducted over the last 10 years, illustrating how PDT is applied in the clinic today. Furthermore, examples from ongoing clinical trials and the most recent preclinical studies are presented, to show the directions, in which PDT is headed, in the near and distant future. Despite the clinical success reported, PDT is still currently underutilized in the clinic. We also discuss the factors that hamper the exploration of this effective therapy and what should be changed to render it a more effective and more widely available option for patients.

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.

A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm-2, with a fluence rate of 50 mW∙cm-2, and thereafter tumor growth was measured with a follow-up until 30 days. The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in clinical practice.

Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm−2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm−2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 95 and 50 50 J cm−2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm−2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm−2 is delivered in the first light fraction. Also, delivering 5 J cm−2 at 5 mW cm−2 95 J cm−2 at 50 mW cm−2 results in a reduction in visual skin damage from that obtained with 5 95 J cm−2 at 50 mW cm−2. A similar reduction in damage is observed if 5 45 J cm−2 are delivered at 50 mW cm−2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (μ_s′) and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g₂)/(1-g₁) and g₁ and g₂ the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of μ_s′(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of μ_s′(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.