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.

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.

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.

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.

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.