Gold standard assessment of immunogenic cell death induced by photodynamic therapy: From in vitro to tumor mouse models and anti-cancer vaccination strategies.

The discovery of the concept of immunogenic cell death (ICD) is a cornerstone in the development of novel anti-cancer immunotherapeutic approaches. Induction of the ICD pathway by specific anti-cancer therapeutic regimens can eliminate cancer cells by directly killing them during therapy and by activation of strong and specific anti-cancer immunity, leading to a long-lasting immunological memory that prevents cancer recurrence. ICD encompasses different forms of regulated cell death and can be triggered by many anti-cancer treatment modalities, including photodynamic therapy (PDT). PDT is a multistep procedure involving the accumulation of a light-sensitive dye known as a photosensitizer (PS) in tumor cells, followed by its activation by irradiation with a light of an appropriate wavelength. In the presence of molecular oxygen, the irradiated PS leads to the generation of cytotoxic reactive oxygen species, which can lead to ICD induction in the cancer cells. Here, we first describe in vitro methods to help optimize the PDT procedure for a specific PS. We also provide a collection of protocols and techniques for assessing ICD in vitro, including analysis of the emission of damage associated molecular patterns (DAMPs), efferocytosis, and the maturation and activation state of antigen presenting cells. Next, we describe in detail protocols for diverse tumor mouse models for assessing and characterizing ICD in vivo, such as murine tumor vaccination models. Finally, as an immunotherapeutic vaccine, we suggest using either PDT-induced dead cancer cells, preferably undergoing ICD, or dendritic cells loaded with lysates of PDT-induced cancer cells in a syngeneic orthotopic glioma model. Overall, this methodological article provides a quantitative, comprehensive set of validated tools that can be successfully used, with some adaptations, to identify, optimize and validate novel PSs in vitro and in vivo for the efficient induction of ICD during photodynamic treatment.

Current Principles, Challenges, and New Metrics in pH-Responsive Drug Delivery Systems for Systemic Cancer Therapy.

The paradigm of drug delivery via particulate formulations is one of the leading ideas that enable overcoming limitations of traditional chemotherapeutic agents. The trend toward more complex multifunctional drug carriers is well-traced in the literature. Nowadays, the prospectiveness of stimuli-responsive systems capable of controlled cargo release in the lesion nidus is widely accepted. Both endogenous and exogenous stimuli are employed for this purpose; however, endogenous pH is the most common trigger. Unfortunately, scientists encounter multiple challenges on the way to the implementation of this idea related to the vehicles' accumulation in off-target tissues, their immunogenicity, the complexity of drug delivery to intracellular targets, and finally, the difficulties in the fabrication of carriers matching all imposed requirements. Here, we discuss fundamental strategies for pH-responsive drug delivery, as well as limitations related to such carriers' application, and reveal the main problems, weaknesses, and reasons for poor clinical results. Moreover, we attempted to formulate the profiles of an "ideal" drug carrier in the frame of different strategies drawing on the example of metal-comprising materials and considered recently published studies through the lens of these profiles. We believe that this approach will facilitate the formulation of the main challenges facing researchers and the identification of the most promising trends in technology development.

Novel Rigidochromic and Anti-Kasha Dual Emission Fluorophores Based on D-π-A Dyads as the Promising Materials for Potential Applications Ranging from Optoelectronics and Optical Sensing to Biophotonics and Medicine.

Today we see an increasing demand for new fluorescent materials exhibiting various sensory abilities due to their broad applicability ranging from the construction of flexible devices to bioimaging. In this paper, we report on the new fluorescent pigments AntTCNE, PyrTCNE, and PerTCNE which consist of 3-5 fused aromatic rings substituted with tricyanoethylene fragments forming D-π-A diad. Our studies reveal that all three compounds exhibit pronounced rigidochromic properties, i.e., strong sensitivity of their fluorescence to the viscosity of the local environment. We also demonstrate that our new pigments belong to a very rare type of organic fluorophores which do not obey the well-known empirical Kasha'rule stating that photoluminescence transition always occurs from the lowest excited state of an emitting molecule. This rare spectral feature of our pigments is accompanied by an even rarer capability of spectrally and temporally well-resolved anti-Kasha dual emission (DE) from both higher and lowest electronic states in non-polar solvents. We show that among three new pigments, PerTCNE has significant potential as the medium-bandgap non-fullerene electron acceptor. Such materials are now highly demanded for indoor low-power electronics and portable devices for the Internet-of-Things. Additionally, we demonstrate that PyrTCNE has been successfully used as a structural unit in template assembling of the new cyanoarylporphyrazine framework with 4 D-π-A dyads framing this macrocycle (Pyr4CN4Pz). Similarly to its structural unit, Pyr4CN4Pz is also the anti-Kasha fluorophore, exhibiting intensive DE in viscous non-polar medium and polymer films, which strongly depends on the polarity of the local environment. Moreover, our studies showed high photodynamic activity of this new tetrapyrrole macrocycle which is combined with its unique sensory capacities (strong sensitivity of its fluorescent properties to the local environmental stimuli such as viscosity and polarity. Thus, Pyr4CN4Pz can be considered the first unique photosensitizer that potentially enables the real-time combination of photodynamic therapy and double-sensory approaches which is very important for modern biomedicine.

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.

Poly(methacrylic Acid)-Cellulose Brushes as Anticancer Porphyrazine Carrier.

The prospective strategy for treatment of cancer is based on the application of nano-sized macromolecular carriers, which are able penetrate inside and can be accumulated within tumor tissue. In this work graft copolymers of cellulose and poly(methacrylic acid) has been prepared and tested as a nanocontainers for the delivery of drug to tumor. For this purpose, two derivatives of porphyrazine suitable for photodynamic cancer therapy were loaded into prepared polymer brush. Fluorescence imaging was applied for monitoring of accumulation of porphyrazine in the CT26 murine colon carcinoma. The selective accumulation of cellulose brush loaded with porphyrazine in tumor was demonstrated by fluorescence intensity contrast between the tumor area and normal tissues. The tumor growth rate after photodynamic therapy were assessed and inhibition of its growth was revealed.

The localization of the photosensitizer determines the dynamics of the secondary production of hydrogen peroxide in cell cytoplasm and mitochondria.

Photodynamic therapy (PDT) is based on the production of the cytotoxic reactive oxygen species (ROS) by light irradiation of a photosensitizer dye in the presence of molecular oxygen. Along with photochemical ROS production, it becomes evident that PDT induces massive secondary production of ROS which is registered long after the irradiation is completed. We created cell lines of human epidermoid carcinoma with the cytoplasmic and mitochondrial localization of protein sensor HyPer sensitive to hydrogen peroxide to compare its concentration in two cellular compartments. The lag-period between irradiation and accumulation of hydrogen peroxide in cells was registered; its duration was dose-dependent and increased up to 80 min when lowering the exposition dose from 50 to 15 J/cm2. We have shown that localization of the photosensitizer determines the spatiotemporal pattern of the cell response to PDT: secondary hydrogen peroxide accumulation in cell cytoplasm induced by photodynamic treatment with lysosome-localized phtalocyianine Photosens occurs several minutes prior to that in mitochondria; on the contrary, membranotropic arylcyanoporphyrazine dye leads to massive mitochondrial hydrogen peroxide production followed by its cytoplasmic accumulation. We hypothesize that photosensitizers with various physicochemical properties and intracellular localization can trigger different patterns not only of primary but also secondary ROS production leading to different cell fate outcomes.

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.

Synthesis and biological evaluation of new water-soluble photoactive chlorin conjugate for targeted delivery.

A new water-soluble conjugate, consisting of a chlorin-based photosensitizing part, and a 4-arylaminoquinazoline moiety with high potential affinity to an epidermal growth factor receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), suitable for photodynamic therapy (PDT), was synthesized starting from methylpheophorbide-a in seven steps. An increased accumulation of this compound in A431 cells with high level of EGFR expression, in comparison with CHO and HeLa cells with low EGFR expression was observed. The prepared conjugate exhibits dark and photoinduced cytotoxicity at micromolar concentrations with IC50dark/IC50light ratio of 11-18. In tumor-bearing mice, the conjugate preferentially accumulates in the tumor tissue.

Liposomal Form of Tetra(Aryl)Tetracyanoporphyrazine: Physical Properties and Photodynamic Activity In Vitro.

Tetra(aryl)tetracyanoporphyrazines are the promising group of dyes for photodynamic therapy of tumors with unique combination of photosensitizer properties and sensitivity of fluorescence parameters to the environment viscosity. However, in vivo application of such hydrophobic photosensitizers requires using of drug carriers ensuring efficient delivery to the tumor site. The present study is focused on obtaining liposomes loaded with tetrakis(4-benzyloxyphenyl)tetracyanoporphyrazine and examining their properties depending on lipid composition. An efficient loading of the dye and a high long-term stability were proved for the liposomes composed of phosphatidylcholine with cholesterol and phosphatidylglycerol. This can be explained by the presence of negatively charged lipids in the bilayer and, as a consequence, a high value of the surface potential. A high rate of cellular uptake and a strong photoinduced toxicity give the prerequisites for the further use of the liposomal form of the photosensitizer for photodynamic therapy of tumors.

Pharmacokinetics of Chlorin e6-Cobalt Bis(Dicarbollide) Conjugate in Balb/c Mice with Engrafted Carcinoma.

The necessary precondition for efficient boron neutron capture therapy (BNCT) is control over the content of isotope 10B in the tumor and normal tissues. In the case of boron-containing porphyrins, the fluorescent part of molecule can be used for quantitative assessment of the boron content. Study Objective: We performed a study of the biodistribution of the chlorin e6-Cobalt bis(dicarbollide) conjugate in carcinoma-bearing Balb/c mice using ex vivo fluorescence imaging, and developed a mathematical model describing boron accumulation and release based on the obtained experimental data. Materials and Methods: The study was performed on Balb/c tumor-bearing mice (CT-26 tumor model). A solution of the chlorin e6-Cobalt bis(dicarbollide) conjugate (CCDC) was injected into the blood at a dose of 10 mg/kg of the animal's weight. Analysis of the fluorescence signal intensity was performed at several time points by spectrofluorimetry in blood and by laser scanning microscopy in muscle, liver, and tumor tissues. The boron content in the same samples was determined by mass spectroscopy with inductively coupled plasma. Results: Analysis of a linear approximation between the fluorescence intensity and boron content in the tissues demonstrated a satisfactory value of approximation reliability with a Spearman's rank correlation coefficient of r = 0.938, p < 0.01. The dynamics of the boron concentration change in various organs, calculated on the basis of the fluorescence intensity, enabled the development of a model describing the accumulation of the studied compound and its distribution in tissues. The obtained results reveal a high level of correspondence between the model and experimental data.

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.

Simultaneous in vivo imaging of diffuse optical reflectance, optoacoustic pressure and ultrasonic scattering.

We present reflection-mode bioimaging system providing complementary optical, photoacsoutic and acoustic measurements by acoustic detector after each laser pulse. While the photons absorbed within the sample provide optoacoustic (OA) signals, the photons absorbed by the external electrode of a detector provide the measurable diffuse reflectance (DR) from the sample and the probing ultrasonic (US) pulse. To demonstrate the in vivo capabilities of the system we present the results of complementary DR/OA/US imaging of a mouse tumor, head of a newborn rat, and the back of a newborn rat with 3.5mm/50µm/35µm lateral resolution. Trimodal approach allows visualization of mechanical structures in healthy and pathological tissues along with peculiarities of blood supply. The system may be used for diagnostics of diseases accompanied by the defects of vascularization as well as for assessing the mechanisms of vascular changes when monitoring response to therapy.

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.