Comparative Analysis of Tetra(2-naphthyl)tetracyano-porphyrazine and Its Iron Complex as Photosensitizers for Anticancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a rapidly developing modality of primary and adjuvant anticancer treatment. The main trends today are the search for new effective photodynamic agents and the creation of targeted delivery systems with the function of controlling the release of the agent in the tumor. Recently, the new group of cyanoarylporphyrazine dyes was reported, which combine the properties of photosensitizers and sensors of the local microenvironment. Such unique characteristics allow the release of the photosensitizer from the transport carrier to be assessed in real time in vivo. The aim of the present work was to compare the photophysical and photobiological properties of tetra(2-naphthyl)tetracyanoporphyrazine and its newly synthesized Fe(II) complex. We have shown that the chelation of the Fe(II) cation with the porphyrazine macrocycle leads to a decrease in molar extinction and an increase in the quantum yield of fluorescence and photostability. We demonstrate that the iron cation significantly affects the rate of dye accumulation in cells, the dark toxicity and photodynamic activity, and the direction of the changes depends on the particular cell line. However, in all the cases, the photodynamic index of a metal complex was higher than that of a metal-free base. In general, both of the compounds were found to be very promising for PDT, including for the use with transport delivery systems, and can be recommended for further in vivo studies.

Cyanoarylporphyrazines with High Viscosity Sensitivity: A Step towards Dosimetry-Assisted Photodynamic Cancer Treatment.

Despite the significant relevance of photodynamic therapy (PDT) as an efficient strategy for primary and adjuvant anticancer treatment, several challenges compromise its efficiency. In order to develop an "ideal photosensitizer" and the requirements applied to photosensitizers for PDT, there is still a need for new photodynamic agents with improved photophysical and photobiological properties. In this study, we performed a detailed characterization of two tetracyanotetra(aryl)porphyrazine dyes with 4-biphenyl (pz II) and 4-diethylaminophenyl (pz IV) groups in the periphery of the porphyrazine macrocycle. Photophysical properties, namely, fluorescence quantum yield and lifetime of both photosensitizers, demonstrate extremely high dependence on the viscosity of the environment, which enables them to be used as viscosity sensors. PzII and pz IV easily enter cancer cells and efficiently induce cell death under light irradiation. Using fluorescence lifetime imaging microscopy, we demonstrated the possibility of assessing local intracellular viscosity and visualizing viscosity changes driven by PDT treatment with the compounds. Thus, pz II and pz IV combine the features of potent photodynamic agents and viscosity sensors. These data suggest that the unique properties of the compounds provide a tool for PDT dosimetry and tailoring the PDT treatment regimen to the individual characteristics of each patient.

Targeting immunogenic cancer cell death by photodynamic therapy: past, present and future.

The past decade has witnessed major breakthroughs in cancer immunotherapy. This development has been largely motivated by cancer cell evasion of immunological control and consequent tumor resistance to conventional therapies. Immunogenic cell death (ICD) is considered one of the most promising ways to achieve total tumor cell elimination. It activates the T-cell adaptive immune response and results in the formation of long-term immunological memory. ICD can be triggered by many anticancer treatment modalities, including photodynamic therapy (PDT). In this review, we first discuss the role of PDT based on several classes of photosensitizers, including porphyrins and non-porphyrins, and critically evaluate their potential role in ICD induction. We emphasize the emerging trend of ICD induction by PDT in combination with nanotechnology, which represents third-generation photosensitizers and involves targeted induction of ICD by PDT. However, PDT also has some limitations, including the reduced efficiency of ICD induction in the hypoxic tumor microenvironment. Therefore, we critically evaluate strategies for overcoming this limitation, which is essential for increasing PDT efficiency. In the final part, we suggest several areas for future research for personalized cancer immunotherapy, including strategies based on oxygen-boosted PDT and nanoparticles. In conclusion, the insights from the last several years increasingly support the idea that PDT is a powerful strategy for inducing ICD in experimental cancer therapy. However, most studies have focused on mouse models, but it is necessary to validate this strategy in clinical settings, which will be a challenging research area in the future.

UCNP-based Photoluminescent Nanomedicines for Targeted Imaging and Theranostics of Cancer.

Theranostic approach is currently among the fastest growing trends in cancer treatment. It implies the creation of multifunctional agents for simultaneous precise diagnosis and targeted impact on tumor cells. A new type of theranostic complexes was created based on NaYF4: Yb,Tm upconversion nanoparticles coated with polyethylene glycol and functionalized with the HER2-specific recombinant targeted toxin DARPin-LoPE. The obtained agents bind to HER2-overexpressing human breast adenocarcinoma cells and demonstrate selective cytotoxicity against this type of cancer cells. Using fluorescent human breast adenocarcinoma xenograft models, the possibility of intravital visualization of the UCNP-based complexes biodistribution and accumulation in tumor was demonstrated.

Preclinical Study of Biofunctional Polymer-Coated Upconversion Nanoparticles.

Upconversion nanoparticles (UCNPs) are new-generation photoluminescent nanomaterials gaining considerable recognition in the life sciences due to their unique optical properties that allow high-contrast imaging in cells and tissues. Upconversion nanoparticle applications in optical diagnosis, bioassays, therapeutics, photodynamic therapy, drug delivery, and light-controlled release of drugs are promising, demanding a comprehensive systematic study of their pharmacological properties. We report on production of biofunctional UCNP-based nanocomplexes suitable for optical microscopy and imaging of HER2-positive cells and tumors, as well as on the comprehensive evaluation of their pharmacokinetics, pharmacodynamics, and toxicological properties using cells and laboratory animals. The nanocomplexes represent a UCNP core/shell structure of the NaYF4:Yb, Er, Tm/NaYF4 composition coated with an amphiphilic alternating copolymer of maleic anhydride with 1-octadecene (PMAO) and conjugated to the Designed Ankyrin Repeat Protein (DARPin 9_29) with high affinity to the HER2 receptor. We demonstrated the specific binding of UCNP-PMAO-DARPin to HER2-positive cancer cells in cultures and xenograft animal models allowing the tumor visualization for at least 24 h. An exhaustive study of the general and specific toxicity of UCNP-PMAO-DARPin including the evaluation of their allergenic, immunotoxic, and reprotoxic properties was carried out. The obtained experimental body of evidence leads to a conclusion that UCNP-PMAO and UCNP-PMAO-DARPin are functional, noncytotoxic, biocompatible, and safe for imaging applications in cells, small animals, and prospective clinical applications of image-guided surgery.

Photobiological properties of phthalocyanine photosensitizers Photosens, Holosens and Phthalosens: A comparative in vitro analysis.

Photobiological properties of phthalocyanine photosensitizers, namely, clinically approved Photosens and new compounds Holosens and Phthalosens were analyzed on transitional cell carcinoma of the urinary bladder (T24) and human hepatic adenocarcinoma (SK-HEP-1). Photosens is a sulfated aluminum phthalocyanine with the number of sulfo groups 3.4, which is characterized by a high degree of hydrophilicity, slow cellular uptake, localization in lysosomes and the lowest photodynamic activity. Holosens is an octacholine zinc phthalocyanine, a cationic compound with significant charge. Holosens more efficiently enters the cells; it is localized in Golgi apparatus in addition to lysosomes and exhibits a significant inhibitory effect on cell viability upon irradiation. The highest photodynamic activity was demostrated by Phthalosens. Phthalosens is a metal-free analog of Photosens with a number of sulfo groups 2.5, which determines its amphiphilicity. Phthalosens is characterized by the highest rate of cellular uptake through the outer cell membrane, localization in cell membrane as well as in lysosomes and Golgi apparatus, and the highest activity upon irradiation among the photosensitizers studied. In general, changes in the physicochemical properties of Holosens and Phthalosens ensured an increase in their efficiency in vitro compared to Photosens. The features of accumulation, intracellular distribution and their interrelation with photodynamic activity, revealed in this work, indicate the prospects of Phthalosens and Holosens for clinical practice.

Radioactive (90Y) upconversion nanoparticles conjugated with recombinant targeted toxin for synergistic nanotheranostics of cancer.

We report combined therapy using upconversion nanoparticles (UCNP) coupled to two therapeutic agents: beta-emitting radionuclide yttrium-90 (90Y) fractionally substituting yttrium in UCNP, and a fragment of the exotoxin A derived from Pseudomonas aeruginosa genetically fused with a targeting designed ankyrin repeat protein (DARPin) specific to HER2 receptors. The resultant hybrid complex UCNP-R-T was tested using human breast adenocarcinoma cells SK-BR-3 overexpressing HER2 receptors and immunodeficient mice, bearing HER2-positive xenograft tumors. The photophysical properties of UCNPs enabled background-free imaging of the UCNP-R-T distribution in cells and animals. Specific binding and uptake of UCNP complexes in SK-BR-3 cells was observed, with separate 90Y- and PE40-induced cytotoxic effects characterized by IC50 140 µg/mL (UCNP-R) and 5.2 µg/mL (UCNP-T), respectively. When both therapeutic agents were combined into UCNP-R-T, the synergetic effect increased markedly, ∼2200-fold, resulting in IC50 = 0.0024 µg/mL. The combined therapy with UCNP-R-T was demonstrated in vivo.

Effective delivery of porphyrazine photosensitizers to cancer cells by polymer brush nanocontainers.

Efficient drug delivery can be assigned to tasks that attract the most acute attention of researchers in the field of anticancer drug design. We have reported the first case of using amphiphilic polymer brushes as nanocontainers for photosensitizer delivery to cancer cells. Regular graft-copolymers of hydrophobic polyimides with hydrophilic polymethacrylic acid side chains were loaded with photosensitive dye tetra(4-fluorophenyl)tetracyanoporphyrazine (Pz) providing a sufficiently stable homogeneous fraction of fluorescent Pz-loaded nanoparticles with a size of 100-150 nm. Pz-loaded polymer brushes were substantially more efficient for Pz delivery into cells compared with other types of particles examined, Pz-polyethyleneglycol and Pz-methylcellulose. In vivo, an efficient Pz delivery to tumor can also be expected since the Pz-PB particle size is in the optimal range for passive targeting. Pz-PB showed pronounced photodynamic activity, while, that is important, in the absence of irradiation the PB carrier itself was significantly less toxic than the dye itself. Summing up, water-soluble polymer brushes with polyimide backbones and polymethacrylic acid side chains can be regarded as a novel type of nanocontainers providing efficient intracellular drug delivery for photodynamic therapy of cancers.

Dual use of porphyrazines as sensitizers and viscosity markers in photodynamic therapy.

Porphyrazines have recently emerged as a useful class of tetrapyrroles suitable for photodynamic therapy of cancer (PDT) with excellent uptake and retention properties in vivo. Here we demonstrate that the photophysical properties of cyano-aryl porphyrazine pz1 are strongly viscosity dependent, i.e. the fluorescence lifetime and the quantum yield of pz1 increase as a function of solution viscosity. We have calibrated pz1 as a red-emitting fluorescent 'molecular rotor' in a large range of viscosities from 80 to ca. 5500 cP, in solutions of various solvent compositions and temperatures. On the other hand, pz1 works as an efficient PDT sensitiser, i.e. it induces apoptosis and necrosis in cells upon irradiation with red light through formation of singlet oxygen. We demonstrate that PDT in cells using pz1 is accompanied by a significant viscosity increase by monitoring the fluorescence lifetime of the rotor. We suggest that this increase could be used as a completely new type of diagnostic and dosimetry tool in a PDT treatment.