Self-assembled nanoparticles of glycosylated AABB-type phthalocyanines for selective photodynamic therapy.

Photodynamic therapy represents a very attractive therapeutic tool considered to be effective, minimally invasive and minimally toxic. However, conventional photodynamic therapy actually has two main constraints: the limited penetration depth of visible light needed for its activation, and the lack of selectivity. Considering this, this work reports the synthesis and evaluation of a novel nanoconjugate for imaging and selective photodynamic therapy against HER2-positive breast cancer, a particularly aggressive form of the disease. It was demonstrated that upon 975 nm near infrared light exposure, the red emission of the NaYF4:Yb,Er up-conversion nanoparticles (UCNPs) can be used for optical imaging and simultaneously represent the source for the excitation of a covalently bound zinc tetracarboxyphenoxy phthalocyanine (ZnPc), a photosensitizer that in turn transfers energy to ground state molecular oxygen to produce cytotoxic singlet oxygen. The specificity of our nanoconjugates was achieved by immunoconjugation with Trastuzumab (Tras), a specific monoclonal antibody for selective detection and treatment of HER2-overexpressing malignant breast cancer cells. Selective tracking of SKBR-3 HER2-positive cells was verified by confocal microscopy analysis, and the photodynamic therapy effect was considerably improved when Trastuzumab was incorporated into the nanoconjugate, the UCNPs-ZnPc-Tras being practically inert in the absence of infrared light exposure but reducing the HER2-positive cell viability up to 21% upon 5 min of the irradiation. This theranostic nanoconjugate represents a valuable alternative for HER2-positive breast cancer imaging and selective photodynamic therapy.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.ImagesFigure 3Figure 4

To evaluate the feasibility, efficacy, and selectivity of photodynamic therapy (PDT) using targeted delivery of verteporfin to choroidal neovascularization (CNV) in the rat laser-injury model of CNV. We performed PDT in rat eyes on experimental CNV and normal retina and choroid using verteporfin conjugates. A targeted verteporfin conjugate was made by conjugating verteporfin (after isolation from its liposomal formulation) to a modified polyvinyl alcohol (PVA) polymer (verteporfin-PVA) followed by linkage to the peptide ATWLPPR known to bind the receptor for vascular endothelial growth factor, VEGFR2. The verteporfin-PVA conjugate served as a control. We performed fluorescent fundus angiography to determine the optimal timing of light application for PDT using the conjugates. Closure of CNV was assessed angiographically and graded in a masked standardized fashion. We used standardized histological grading to compare the effects on normal retina and choroid. The verteporfin-PVA conjugation ratio was on average 28:1. The conjugate retained typical emission/excitation spectra and photosensitizing activity and was as efficient as an equivalent amount of verteporfin. Peak intensity of targeted verteporfin in CNV was detected angiographically at 1 hour after intravenous injection. Photodynamic therapy using targeted verteporfin (3 or 4.5 mg/m2) with light application 1 hour after drug injection showed angiographic closure of all treated CNV (17/17) 1 day after treatment. Photodynamic therapy using verteporfin-PVA at the same drug dose achieved closure in 18 of 20 CNV. Histological examination after PDT of normal retina and choroid using targeted verteporfin and irradiation at 1 hour showed minimal effect on retinal pigment epithelium and no injury to photoreceptors, whereas PDT using verteporfin-PVA resulted in retinal pigment epithelium necrosis and mild damage to photoreceptors. Verteporfin bound to the targeting peptide, ATWLPPR, retained its spectral and photosensitizing properties. Angiography demonstrated localization of the targeted verteporfin 1 hour after injection. Photodynamic therapy using targeted verteporfin and the control conjugate were more effective in causing CNV closure than standard liposomal verteporfin. The targeted verteporfin resulted in more selective treatment than the control conjugate or standard verteporfin. These results suggest that targeted PDT strategies based on selective expression of receptors on CNV vasculature may improve current therapy. Targeted PDT for CNV is feasible and may offer a qualitative improvement in current treatments for patients with age-related macular degeneration. This study provides the basis for further preclinical studies of targeted PDT strategies and subsequent clinical trials.

Photodynamic therapy (PDT) is now a well-recognized modality for the treatment of cancer. While PDT has developed progressively over the last century, great advances have been observed in the field in recent years. The concept of dual selectivity of PDT agents is now widely accepted due to the relative specificity and selectivity of PDT along with the absence of harmful side effects often encountered with chemotherapy or radiotherapy. Traditionally, porphyrin-based photosensitizers have dominated the PDT field but these first generation photosensitizers have several disadvantages, with poor light absorption and cutaneous photosensitivity being the predominant side effects. As a result, the requirement for new photosensitizers, including second generation porphyrins and porphyrin derivatives as well as third generation photosensitizers has arisen, with the aim of alleviating the problems encountered with first generation porphyrins and improving the efficacy of PDT. The investigation of nonporphyrin photosensitizers for the development of novel PDT agents has been considerably less extensive than porphyrin-based compounds; however, structural modification of nonporphyrin photosensitizers has allowed for manipulation of the photochemotherapeutic properties. The aim of this review is to provide an insight into PDT photosensitizers clinically approved for application in oncology, as well as those which show significant potential in ongoing preclinical studies.

Clinical photodynamic therapy (PDT) schedules are based on the assumption that optimum drug–light intervals are times at which there is a maximum differential between photosensitiser retention in the tumour and surrounding normal tissue. However, vascular-mediated effects contribute to tumour destruction by PDT; therefore, plasma sensitiser levels and endothelial cell drug exposure could also be important determinants of PDT response. The purpose of this study was to investigate the influence of tumour, tissue and plasma concentrations of the photosensitiser Foscan® (meta-tetrahydroxyphenylchlorin, mTHPC) on PDT response. Groups of BalbC nude mice, bearing human mesothelioma xenografts (H-MESO1) were injected (i.v.) with a single dose of 14C-labelled mTHPC, or with two doses, separated by 72 h. Drug levels in plasma, tumour and normal tissues were measured at 5 min to 120 h after drug administration. The PDT tumour and skin responses were evaluated by illuminating separate groups mice at intervals of 5 min to 120 h after injection of Foscan (nonlabelled). Drug levels in both tumour and skin increased during the first 24 h after a single injection, and remained almost constant for at least 120 h. The second injection produced a further, rapid increase in mTHPC levels in tumours and skin, with steady state being maintained from 20 min to 120 h. By contrast, PDT response of both tumours and skin were maximal for illumination at 1–3 h after drug, with very little response when illumination was given 48–120 h after drug. There was no significant correlation between tumour or skin drug level and PDT response. There was, however, a significant correlation between plasma drug levels and tumour or skin response, excluding an initial distribution time of 20 min. These studies demonstrate a pronounced disassociation between tumour drug levels and optimum drug–light intervals for PDT response with Foscan. We suggest that the PDT effect, in both tumours and normal tissues, is largely mediated via vascular damage and that the selectivity of PDT is not based on differential tumour drug uptake.

Photodynamic therapy (PDT) following surgical tumor resection is leading to improved local tumor control and might be useful for selected intrathoracic malignancies. However, optimal tumor selectivity of PDT is mandatory to avoid injury of adjacent normal tissues. (1) PDT was applied on human tumor xenografts (malignant mesothelioma, squamous cell carcinoma of the neck, adenocarcinoma of the colon). M-tetrahydroxyphenylchlorin (mTHPC) and polyethylene glycol-derived mTHPC (MD-mTHPC) were administered i.p. The tumor and normal tissue of the hind leg were irradiated with 652 nm laser-light. Drug and light doses and drug-light intervals were varied. The extent of necrosis was assessed histologically. (2) Intrathoracic PDT was performed in minipigs with drug-light doses optimized in nude mice. After administration of the sensitizers i.v., intrathoracic structures were irradiated and analyzed histologically. The tumor selectivity of PDT increased in the xenograft model by: (1) choosing an appropriate drug light interval; (2) decreasing the drug dose while increasing the light dose; and (3) applying MD-mTHPC instead of mTHPC. In the minipig model, the extent of injury of intrathoracic structures was equally related to modulation of treatment conditions. The modification of chlorins and the modulation of the drug-light conditions improved the tissue selectivity of PDT. Nevertheless, further methodological optimizations are prerequisites for clinical use of PDT, especially for intraoperative application in thoracic surgery.

Photodynamic therapy (PDT) is a clinically approved non-invasive and curative procedure for different oncological and non-oncological applications. PDT is still under development due to several limitations which lead to partially successful photodynamic response. The crucial steps in PDT procedure are binding of the photosensitizer to outer cell membrane, its penetration and subcellular localization which envisage the target sites of reactive oxygen species generated during irradiation. Since the surrounding normal cells are also exposed to the photosensitizer and the ambient daylight can be harmful for healthy tissues after therapeutic light application, the challenging task in PDT research is to optimize the procedure in a way to reach tumor cell selectivity. The present study outlines the influence of a light exposure pre-treatment (prior therapeutic light) with specific wavelengths (365 nm and 635 nm) on the uptake, the localization and further re-localization of galactose-substituted Zn(II) phthalocyanines into MDA-MB-231 breast cancer cells. The in vitro photodynamic effect towards tumor cells was studied in comparison to the normal cell line Balb/c 3T3 (clone 31) after pre-irradiation with UV light (365 nm) and red LED (635 nm). The results suggest that the galactose functional groups of Zn(II) phthalocyanine and the harmless UV light at 365 nm favor the selective PDT response.

Molecular beacons are FRET-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets, thus are useful tools for in vivo cancer imaging. Photodynamic therapy (PDT) is a cell-killing process by light activation of a photosensitizer (PS) in the presence of oxygen. The key cytotoxic agent is singlet oxygen ((1)O(2)). By combining these two principles (FRET and PDT), we have introduced a concept of photodynamic molecular beacons (PMB) for controlling the PS's ability to generate (1)O(2) and, ultimately, for controlling its PDT activity. The PMB comprises a disease-specific linker, a PS, and a (1)O(2) quencher, so that the PS's photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease. Here, we report the full implementation of this concept by synthesizing a matrix metalloproteinase-7 (MMP7)-triggered PMB and achieving not only MMP7-triggered production of (1)O(2) in solution but also MMP7-mediated photodynamic cytotoxicity in cancer cells. Preliminary in vivo studies also reveal the MMP7-activated PDT efficacy of this PMB. This study validates the core principle of the PMB concept that selective PDT-induced cell death can be achieved by exerting precise control of the PS's ability to produce (1)O(2) by responding to specific cancer-associated biomarkers. Thus, PDT selectivity will no longer depend solely on how selectively the PS can be delivered to cancer cells. Rather, it will depend on how selective a biomarker is to cancer cells, and how selective the interaction of PMB is to this biomarker.

Glioblastoma (GBM), the most aggressive and lethal primary brain tumor, poses a significant therapeutic challenge due to its highly invasive nature and resistance to conventional therapies, including surgery, chemotherapy, and radiotherapy. Despite advances in standard treatments, patient survival remains limited, requiring the exploration of innovative strategies. Photodynamic therapy (PDT) has emerged as a promising approach, leveraging light-sensitive photosensitizers (PSs), molecular oxygen, and specific light wavelengths to generate reactive oxygen species (ROS) that selectively induce tumor cell death. Originally developed for skin cancer, PDT has evolved to target more complex malignancies, including GBM. The refinement of second- and third-generation PS, coupled with advancements in nanotechnology, has significantly improved PDT's selectivity, bioavailability, and therapeutic efficacy. Moreover, the combination of PDT with chemotherapy, targeted therapy, and immunotherapy, among other therapeutic modalities, has shown potential in enhancing therapeutic outcomes. This review provides a comprehensive analysis of the preclinical and clinical applications of PDT in GBM, detailing its mechanisms of action, the evolution of PS, and novel combinatory strategies that optimize treatment efficacy. However, several challenges remain, including overcoming GBM-associated hypoxia, enhancing PS delivery across the blood-brain barrier, and mitigating tumor resistance mechanisms. The integration of PDT with molecular and genetic insight, alongside cutting-edge nanotechnology-based delivery systems, may revolutionize GBM treatment, offering new prospects for improved patient survival and quality of life.

Recent advances in strategies for overcoming hypoxia in photodynamic therapy of cancer.

Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice.

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.

SummaryIn some malignant tumor, especially for pancreatic tumor, poor solid-tumor penetration of nanotherapeutics impedes their treatment efficacy. Herein, we develop a polymer-peptide conjugate with the deep tissue penetration ability, which undergoes a cascade process under ultrasound (US), including (1) the singlet oxygen 1O2 is generated by P18, (2) the thioketal bond is cleaved by the 1O2, (3) the departure of PEG chains leads to the in situ self-assembly, and (4) the resultant self-assembled PK nanoparticles show considerable cellular internalization. Owing to the synergistic effect of US on increasing the membrane permeability, the endocytosis and lysosome escape of PK nanoparticles are further enhanced effectively, resulting in the improved therapeutic efficacy. Thanks to the high tissue-penetrating depth and spatial precision of US, PTPK presents enhanced tumor inhibition in an orthotopic pancreatic tumor model. Therefore, the US-activated cascade effect offers a novel perspective for precision medicine and disease theranostics.

Development of targeted nanoparticles as carriers to deliver photosensitizers to cancer cells is highly beneficial for ensuring the expected therapeutic outcome of photodynamic therapy. Herein, polyacrylic acid (PAA) coated superparamagnetic iron oxide nanoparticles (SPIONs), conjugated with endothelial growth factor receptor (EGFR) targeting Cetuximab (Cet) were loaded with a BODIPY‐based (BOD‐Se‐I) photosensitizer (Cet‐PAA@SPION/BOD‐Se‐I) to achieve enhanced and selective photodynamic therapy on colon cancer cells. In vitro studies showed that Cet‐PAA@SPION/BOD‐Se‐I improved phototoxicity dramatically compared to free BOD‐Se‐I on the HT29 cell line due to high uptake of the photosensitizer via endothelial growth factor receptor. Most importantly, the developed nano‐agent completely eliminated the phototoxicity of BOD‐Se‐I on the healthy L929 cell line.