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In this report, we developed novel chlorin/arylaminoquinazoline conjugates for targeted photodynamic therapy of cancer. The synthesized photosensitizers consisted of chlorin- e 6 metallocomplexes (Zn, In, or Pd) conjugated with arylaminoquinazoline ligands with high affinity for epidermal growth factor receptors (EGFR). Additionally, the selectivity and antitumor properties of the conjugates were investigated in the EGFR-expressing A431 human tumor cell line in vitro . Among the tested molecules, the In-containing conjugate effectively inhibited tumor cell proliferation at nanomolar concentrations, a rare property for conventional photosensitizers. In in vivo experiments, the conjugates rapidly accumulated at the tumor site in nude mice bearing A431 xenograft tumors. Subsequent distribution analysis among different tissues was carried out using fluorescence imaging and elemental analysis. Finally, we demonstrated that the most promising In-containing conjugate was capable of inhibiting xenograft tumor growth in mice through combinational therapy. This therapeutic approach, combined with the conjugate's confirmed safety profile, highlights its potential for effective and safe cancer treatment.

The extracellular matrix (ECM) and its primary chemical components, including collagen, play a pivotal role in carcinogenesis and tumor progression. The ECM actively regulates cell proliferation, migration, and, importantly, resistance to various adverse factors. It is widely recognized as a key factor in modifying the resistance of tumor cells to various treatment modalities and cytotoxic compounds. However, the role of the ECM in tumor cell adaptation to nutritional deficiencies and hypoxic conditions remains significantly less studied. Since it is generally accepted that tumor cells resistance increases when cultured in a three-dimensional matrix, we sought to experimentally test the universality of this statement. In this work, we analyzed the responses of tumor cells with varying origins and proliferative activities, including human bladder carcinoma, epidermoid carcinoma, and ovarian carcinoma, to deprivation of serum, glucose and oxygen. We compared cell resistance to suboptimal conditions when cultured in a monolayer on tissue culture (TC)-treated polystyrene, on collagen-coated surfaces, or within a three-dimensional hydrogel composed of collagen type I. All three cell lines were stably transfected with fluorescent protein genes. To register the cell growth dynamics, we used a fluorescence-based technique that allows long-term quantitative observations without disrupting the hydrogel. The analyzed cell lines demonstrated different patterns of relative sensitivity to suboptimal conditions. We revealed that the direction and intensity of the collagen matrix effect depend on the cell type. Slowly proliferating ovarian carcinoma cells showed no noticeable changes in their behavior when cultured in a gel compared to a monolayer. In the case of bladder carcinoma, we registered predominantly resistance-stimulating effect of the collagen matrix, but it was significant only under serum deprivation. The most pronounced effect of collagen was registered for epidermoid carcinoma. Importantly, this effect was ambivalent: gel-embedded cells demonstrated significantly enhanced resistance to serum deprivation, but, at the same time, they were more responsive to glucose starvation and hypoxic conditions. We attribute the registered phenomenon to the individual characteristics of tumor cells with different origins and metabolic activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Modern radiotherapy utilizes a broad range of sources of ionizing radiation, both low-dose-rate (LDR) and high-dose-rate (HDR). However, the mechanisms underlying specific dose-rate effects remain unclear, especially for corpuscular radiation. To address this issue, we have irradiated human epidermoid carcinoma A431 cells under LDR and HDR regimes. Reducing the dose rate has lower lethality at equal doses with HDR irradiation. The half-lethal dose after HDR irradiation was three times less than after LDR irradiation. The study of mechanisms showed that under HDR irradiation, the radiation-induced halt of mitosis with the accompanying emergence of giant cells was recorded. No such changes were recorded after LDR irradiation. The level of DNA damage is significantly greater after HDR irradiation, which may be the main reason for the different mechanisms of action of HDR and LDR irradiations. Comparing the mechanisms of cell response to LDR and HDR irradiations may shed light on the mechanisms of tumor cell response to ionizing radiation and answer the question of whether different dose rates within the same dose range can cause different clinical effects.

In this report, we present a novel prodrug strategy that can significantly improve the efficiency and selectivity of combined therapy for bladder cancer. Our approach involved the synthesis of a conjugate based on a chlorin-e 6 photosensitizer and a derivative of the tyrosine kinase inhibitor cabozantinib, linked by a β-glucuronidase-responsive linker. Upon activation by β-glucuronidase, which is overproduced in various tumors and localized in lysosomes, this conjugate released both therapeutic modules within targeted cells. This activation was accompanied by the recovery of its fluorescence and the generation of reactive oxygen species. Investigation of photodynamic and dark toxicity in vitro revealed that the novel conjugate had an excellent safety profile and was able to inhibit tumor cells proliferation at submicromolar concentrations. Additionally, combined therapy effects were also observed in 3D models of tumor growth, demonstrating synergistic suppression through the activation of both photodynamic and targeted therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax . We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae ). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Research in the past decade on immunogenic cell death (ICD) has shown that the immunogenicity of dying tumor cells is crucial for effective anticancer therapy. ICD induction leads to the emission of specific damage-associated molecular patterns (DAMPs), which act as danger signals and as adjuvants to activate specific anti-tumor immune responses, leading to the elimination of tumor cells and the formation of long-term immunological memory. ICD can be triggered by many anticancer treatment modalities, including photodynamic therapy (PDT). However, due to the variety of photosensitizers used and the lack of a universally adopted PDT protocol, there is a need to develop novel PDT with a proven ICD capability. In the present study, we characterized the abilities of two photoactive dyes to induce ICD in experimental glioma in vitro and in vivo. One dye was from the tetracyanotetra(aryl)porphyrazine group with 9-phenanthrenyl ( pz I ), and the other was from the 4-(4-fluorobenzyoxy)phenyl ( pz III ) group in the aryl frame of the macrocycle. We showed that after the photosensitizers penetrated into murine glioma GL261 cells, they localized predominantly in the Golgi apparatus and partially in the endoplasmic reticulum, providing efficient phototoxic activity against glioma GL261 cells upon light irradiation at a dose of 20 J/cm 2 (λex 630 nm; 20 mW/cm 2 ). We demonstrated that pz I -PDT and pz III -PDT can act as efficient ICD inducers when applied to glioma GL261 cells, facilitating the release of two crucial DAMPs (ATP and HMGB1). Moreover, glioma GL261 cells stimulated with pz I -PDT or pz III -PDT provided strong protection against tumor growth in a prophylactic subcutaneous glioma vaccination model. Finally, we showed that dendritic cell (DC) vaccines pulsed with the lysates of glioma GL261 cells pre-treated with pz-I -PDT or pz-III -PDT could act as effective inducers of adaptive anti-tumor immunity in an intracranial orthotopic glioma mouse model.

Photodynamic therapy is known as an effective primary and adjuvant anticancer treatment. Compounds with improved properties or additional modalities are still needed to create an 'ideal' photosensitizer. In this article, we review cyanoarylporphyrazine dyes for photodynamic (anticancer) therapy that we have synthesised to date. The review provides information on the chemistry of cyanoarylporphyrazines, photophysical properties, cellular uptake features and the use of various carriers for selective delivery of cyanoarylporphyrazines to the tumour. The potential of cyanoarylporphyrazines as photodynamic anti-tumour agents also has been evaluated. The most interesting feature of cyanoarylporphyrazines is the dependence of the fluorescence quantum yield and excited state lifetime on the viscosity of the medium, which makes it possible to use them as viscosity sensors in photodynamic therapy. In the future, we expect that the unique combination of photosensitizer and viscosity sensor properties of cyanoarylporphyrazines will provide a tool for dosimetry and tailoring treatment regimens in photodynamic therapy to the individual characteristics of each patient., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8-3.6 kPa) did support the epithelial-like SKBR-3 cells' viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM.

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 (Pyr 4 CN 4 Pz). Similarly to its structural unit, Pyr 4 CN 4 Pz 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, Pyr 4 CN 4 Pz 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.

Today's research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression.

Development of combined schemes for the treatment of oncological diseases is a promising strategy to improve the effectiveness of antitumor therapy. This paper shows the fundamental possibility of multiplying the antitumor effect by combining targeted and photodynamic therapy. It was demonstrated that sequential treatment of HER-2 positive breast cancer cells with the targeted toxin DARPin-LoPE and the photoactive compound photodithazine leads to a synergistic enhancement of their effect. In the future, this approach is intended to achieve the maximum therapeutic effect while minimizing the risks of negative side effects., (© 2022. Pleiades Publishing, Ltd.)

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.

The use of 3D in vitro tumor models has become a common trend in cancer biology studies as well as drug screening and preclinical testing of drug candidates. The transition from 2D to 3D matrix-based cell cultures requires modification of methods for assessing tumor growth. We propose the method for assessing the growth of tumor cells in a collagen hydrogel using macro-scale registration and quantification of the gel epi-fluorescence. The technique does not require gel destruction, can be used for real-time observation of fast (in seconds) cellular responses and demonstrates high agreement with cell counting approaches or measuring total DNA content. The potency of the method was proven in experiments aimed at testing cytotoxic activity of chemotherapeutic drug (cisplatin) and recombinant targeted toxin (DARPin-LoPE) against two different tumor cell lines genetically labelled with fluorescent proteins. Moreover, using fluorescent proteins with sensor properties allows registration of dynamic changes in cells' metabolism, which was shown for the case of sensor of caspase 3 activity.

Glioma is the most common brain tumor, for which no significant improvement in life expectancy and quality of life is yet possible. The creation of stable fluorescent glioma cell lines is a promising tool for in-depth studies of the molecular mechanisms of glioma initialization and pathogenesis, as well as for the development of new anti-cancer strategies. Herein, a new fluorescent glioma GL261-kat cell line stably expressing a far-red fluorescent protein (TurboFP635; Katushka) was generated and characterized, and then validated in a mouse orthotopic glioma model. By using epi-fluorescence imaging, we detect the fluorescent glioma GL261-kat cells in mice starting from day 14 after the inoculation of glioma cells, and the fluorescence signal intensity increases as the glioma progresses. Tumor growth is confirmed by magnetic resonance imaging and histology. A gradual development of neurological deficit and behavioral alterations in mice is observed during glioma progression. In conclusion, our results demonstrate the significance and feasibility of using the novel glioma GL261-kat cell line as a model of glioma biology, which can be used to study the initialization of glioma and monitor its growth by lifetime non-invasive tracking of glioma cells, with the prospect of monitoring the response to anti-cancer therapy.

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. Pz II 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.

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.

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/cm 2 . 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Photodynamic therapy (PDT) is widely used in cancer treatment; however, several challenges compromise its efficiency. We propose a synergistic action between PDT and ferroptotic cell death. PDT acts as a source of reactive oxygen species for the Fenton reaction, which may reinforce ferroptosis induction and increase PDT efficacy in anticancer therapy., Competing Interests: Declaration of Interests No interests are declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Creation of various photoluminescent nanomaterials has significantly expanded the arsenal of approaches used in modern biomedicine. Their unique photophysical properties can significantly improve the sensitivity and specificity of diagnostic methods, increase therapy effectiveness, and make a theranostic approach to treatment possible through the application of nanoparticle conjugates with functional macromolecules. The most widely used nanomaterials to date are semiconductor quantum dots; gold nanoclusters; carbon dots; nanodiamonds; semiconductor porous silicon; and up-conversion nanoparticles. This paper considers the promising groups of photoluminescent nanomaterials that can be used in medical biotechnology: in particular, for devising agents for optical diagnostic methods, sensorics, and various types of therapy., (Copyright ® 2021 National Research University Higher School of Economics.)

The immunogenicity of dying cancer cells determines the efficacy of anti-cancer therapy. Photodynamic therapy (PDT) can induce immunogenic cell death (ICD), which is characterized by the emission of damage-associated molecular patterns (DAMPs) from dying cells. This emission can trigger effective anti-tumor immunity. Only a few photosensitizers are known to induce ICD and, therefore, there is a need for development of new photosensitizers that can induce ICD. The purpose of this work was to analyze whether photosensitizers developed in-house from porphyrazines (pz I and pz III) can induce ICD in vitro and in vivo when used in PDT. We indetified the optimal concentrations of the photosensitizers and found that, at a light dose of 20 J/cm 2 (λ ex 615-635 nm), both pz I and pz III efficiently induced cell death in cancer cells. We demonstrate that pz I localized predominantly in the Golgi apparatus and lysosomes while pz III in the endoplasmic reticulum and lysosomes. The cell death induced by pz I-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) but not by ferrostatin-1 and DFO (ferroptosis inhibitors) or by necrostatin-1 s (necroptosis inhibitor). By contrast, the cell death induced by pz III-PDT was inhibited by z-VAD-fmk and by the necroptosis inhibitor, necrostatin-1 s. Cancer cells induced by pz I-PDT or pz III-PDT released HMGB1 and ATP and were engulfed by bone marrow-derived dendritic cells, which then matured and became activated in vitro. We demonstrate that cancer cells, after induction of cell death by pz I-PDT or pz III-PDT, are protective when used in the mouse model of prophylactic tumor vaccination. By vaccinating immunodeficient mice, we prove the role of the adaptive immune system in protecting against tumours. All together, we have shown that two novel porphyrazines developed in-house are potent ICD inducers that could be effectively applied in PDT of cancer.

In the natural fluidic environment of a biological system, nanoparticles swiftly adsorb plasma proteins on their surface forming a "protein corona", which profoundly and often adversely affects their residence in the systemic circulation in vivo and their interaction with cells in vitro. It has been recognized that preformation of a protein corona under controlled conditions ameliorates the protein corona effects, including colloidal stability in serum solutions. We report on the investigation of the stabilizing effects of a denatured bovine serum albumin (dBSA) protein corona formed on the surface of upconversion nanoparticles (UCNPs). UCNPs were chosen as a nanoparticle model due to their unique photoluminescent properties suitable for background-free biological imaging and sensing. UCNP surface was modified with nitrosonium tetrafluoroborate (NOBF 4 ) to render it hydrophilic. UCNP-NOBF 4 nanoparticles were incubated in dBSA solution to form a dBSA corona followed up by lyophilization. As produced dBSA-UCNP-NOBF 4 demonstrated high photoluminescence brightness, sustained colloidal stability after long-term storage and the reduced level of serum protein surface adsorption. These results show promise of dBSA-based nanoparticle pretreatment to improve the amiability to biological environments towards theranostic applications.

Upconversion nanoparticles (UCNPs) are a promising nanoplatform for bioreagent formation for in vivo imaging, which emit UV and blue light under the action of near-infrared radiation, providing deep tissue penetration and maintaining a high signal-to-noise ratio. In the case of solid tumor visualization, the UCNP surface functionalization is required to ensure a long circulation time, biocompatibility, and non-toxicity. The effective UCNP accumulation in the solid tumors is determined by the disturbed architecture of the vascular network and lymphatic drainage. This work demonstrates an approach to the UCNP biofunctionalization with endogenous polysialic acid for in vivo bioreagent formation. Bioreagents possess a low level of nonspecific protein adsorption and macrophage uptake, which allow the prolongation of the circulation time in the bloodstream up to 3 h. This leads to an intense photoluminescent signal in the tumor.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.

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 NaYF 4 : 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.

Photodynamic therapy (PDT) is a clinically approved procedure for targeting tumor cells. Though several different photosensitizers have been developed, there is still much demand for novel photosensitizers with improved properties. In this study we aim to characterize the accumulation, localization and dark cytotoxicity of the novel photosensitizers developed in-house derivatives of porphyrazines (pz I-IV) in primary murine neuronal cells, as well as to identify the concentrations at which pz still effectively induces death in glioma cells yet is nontoxic to nontransformed cells. The study shows that incubation of primary neuronal and glioma cells with pz I-IV leads to their accumulation in both types of cells, but their rates of internalization, subcellular localization and dark toxicity differ significantly. Pz II was the most promising photosensitizer. It efficiently killed glioma cells while remaining nontoxic to primary neuronal cells. This opens up the possibility of evaluating pz II for experimental PDT for glioma., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of the study was to compare the relevance of ovarian adenocarcinoma spheroids with that of a monolayer culture for assessing photodynamic effect of the tetrakis(4-benzyloxyphenyl)tetracyanoporphyrazine photosensitizer., Materials and Methods: The work was performed on SKOV-3 human ovary adenocarcinoma cells grown in vitro in a monolayer culture and in the form of tumor spheroids obtained using culture plates with ultra-low attachment. We determined the photoinduced toxicity of porphyrazine on a monolayer culture using the MTT assay; the effect on the spheroids was tested by assessing the dynamics of their growth. Cellular uptake of porphyrazine was analyzed by confocal microscopy., Results: Porphyrazine has a pronounced photodynamic effect on SKOV-3 cells. When exposed to light at a dose of 20 J/cm 2 , the IC 50 value 24 h after exposure was 2.3 μM for SKOV-3 monolayer culture. For the spheroids, the effect manifested after a latency period: significant growth retardation of the treated spheroids appeared no sooner than 5 and 9 days after exposure. Notably, no decrease in the initial size of the treated spheroids was observed under any of the photodynamic regimes. The penetration depth of porphyrazine into spheroids was 50-100 μm during 24 h incubation., Conclusion: The limited penetration of the photosensitizer into the body of spheroids and its predominant accumulation in the surface layers can be one of the key factors behind the significant differences in the photodynamic response between the surface and deep layers of a spheroid. For cells located close to the spheroid surface, the photodynamic effect is comparable to that for a monolayer culture, while in deeper layers, the cells remain viable and support/maintain the growth of the spheroid even under intense photo-exposure. The fact that the in vitro distribution is similar to the inhomogeneous accumulation of photosensitizers in tumors in vivo allows us to consider spheroids more relevant than a monolayer culture for studying photodynamic anti-tumor effects., Competing Interests: Conflict of interest. The authors declare no conflict of interest.

A new water-soluble conjugate, consisting of a chlorin- e 6 photosensitizer part, a 4-arylaminoquinazoline moiety with affinity to epidermal growth factor receptors, and a hydrophilic β-d-maltose fragment, was synthesized starting from methylpheophorbide- a in seven steps. The prepared conjugate exhibited low levels of dark cytotoxicity and pronounced photoinduced cytotoxicity at submicromolar concentrations in vitro, with an IC 50 (dark)/IC 50 (light) ratio of ∼368 and a singlet oxygen quantum yield of about 20%. In tumor-bearing Balb/c nude mice, conjugate 1 preferentially accumulates in the tumor tissue. Irradiation of the nude mice bearing A431 xenograft tumors after intravenous administration of the prepared conjugate with a relatively low light dose (50 J/cm 2 ) produced an excellent therapeutic effect with profound tumor regression and low systemic toxicity.

Background: Anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). Therefore, when developing new treatment strategies, it is extremely important to choose methods that induce ICD and thereby activate anti-tumor immune response leading to the most effective destruction of tumor cells. The aim of this work was to analyze whether the clinically widely used photosensitizers, photosens (PS) and photodithazine (PD), can induce ICD when used in photodynamic therapy (PDT)., Methods: Cell death in murine glioma GL261 or fibrosarcoma MCA205 cells was induced by PS- or PD-PDT and cell death was analyzed by MTT or flow cytometry. Intracellular distribution of PS and PD was studied by using the laser scanning microscope. Calreticulin exposure and HMGB1 and ATP release were detected by flow cytometry, ELISA and luminescence assay, respectively. Immunogenicity in vitro was analyzed by co-culturing of dying cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and maturation (CD11c + CD86 + , CD11c + CD40 + ) of BMDCs and production of IL-6 in the supernatant were measured. In vivo immunogenicity was analyzed in mouse tumor prophylactic vaccination model., Results: We determined the optimal concentrations of the photosensitizers and found that at a light dose of 20 J/cm 2 (λex 615-635 nm) both PS and PD efficiently induced cell death in glioma GL261 and fibrosarcoma MCA205 cells. We demonstrate that PS localized predominantly in the lysosomes and that the cell death induced by PS-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) and by ferrostatin-1 and DFO (ferroptosis inhibitors), but not by the necroptosis inhibitor necrostatin-1 s. By contrast, PD accumulated in the endoplasmic reticulum and Golgi apparatus, and the cell death induced by PD-PDT was inhibited only by z-VAD-fmk. Dying cancer cells induced by PS-PDT or PD-PDT emit calreticulin, HMGB1 and ATP and they were efficiently engulfed by BMDCs, which then matured, became activated and produced IL-6. Using dying cancer cells induced by PS-PDT or PD-PDT, we demonstrate the efficient vaccination potential of ICD in vivo., Conclusions: Altogether, these results identify PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy.

Low-level laser therapy (LLLT) is widely used in clinical practice for treatment of various pathologies. It is assumed that LLLT impact on microcirculation is among the mechanisms underlying its therapeutic effect. The microcirculation disorder is observed in the pathogenesis of any inflammatory process and is significantly influenced by red blood cells (RBCs). On this point, studying the RBCs morphology under the influence of LLLT on alterated organism is of scientific interest and practical importance. The aim of the present study was to analyze the LLLT effect on morphokinetic parameters of RBCs in hyperadrenalinemia. The LLLT effect was analyzed on rats intraperitoneally injected with adrenaline hydrochloride solution (0.1 mg/kg). As the comparison groups, the effects of LLLT, adrenaline, or saline injection as well as the parameters of intact animals were studied. LLLT was applied on the occipital region of rats for 10 min. The light irradiation with pulse frequency 415 Hz at 890 nm wavelength and average power density in the plane of the output window at 193 μW/cm 2 was used. The dynamics of morphological characteristics of RBCs was studied by phase interference microscopy; the RBC electrophoretic mobility was tested by microelectrophoresis technique; photometric analyses of the RBCs amount, hemoglobin content, and osmotic fragility were performed. The adrenaline injection resulted in a significant increase in the amount of RBC pathological forms and a decrease in discocytes and normocytes by more than 50%. An increase in the optical density of RBC phase portraits, a decline in osmotic resistance, and electronegativity of RBC membranes and a reduction of their number in peripheral blood were also registered. The revealed effects persisted for 1 week after the adrenaline administration. LLLT did not significantly impact on the RBC parameters 1 h after adrenaline injection. However, a day later, LLLT reduced the severity of the adrenaline effect on RBSs, which was manifested in a decreased amount of the pathological forms of RBCs, restored RBC phase portraits, higher electrophoretic mobility and osmotic resistance, and RBSs amount in peripheral blood restored up to the level of intact animals. We suppose that the mechanism of LLLT action is realized both at cellular level through the laser radiation effect on RBC membranes, and at systemic level through the activation of stress-realizing systems of the organism with subsequent limitation of inflammatory response.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Background: Hydrogen peroxide (H 2 O 2 ) is thought to be one of the key components involved in the responses of tumor cells to chemotherapy. The aim of this study was to reveal the pathways and the phases of cisplatin-induced cell death that are characterized by changes of H 2 O 2 level., Methods: The genetically encoded cytosolic fluorescent sensor HyPer2 was used for flow cytometric analysis of the cisplatin-induced changes in H 2 O 2 level in HeLa Kyoto cells. Using a vital dye and the apoptotic markers PE Annexin V or TMRE the pathways and stages of cell death were investigated simultaneously with HyPer2 response. The H 2 O 2 level was studied separately in viable and early apoptotic cells after 12, 18, 24 h's incubation with cisplatin at several concentrations with or without the scavenger of reactive oxygen species NAC., Results: Cisplatin causes dose- and time-dependent increase of H 2 O 2 level in TMRE-positive and PE Annexin V-negative cancer cells. The scavenging of ROS by NAC decreased the H 2 O 2 level and restored cell viability., Conclusion: Н 2 О 2 generation begins in cells that have already lost mitochondrial membrane potential but have not yet externalized phosphatidylserine. Prevention of apoptosis by NAC confirmed the role of H 2 O 2 in apoptosis induction., General Significance: This is the first time that the sensor HyPer2 has been used in parallel with apoptotic markers and vital dye to demonstrate the role of H 2 O 2 in different stages and types of tumor cell death under chemotherapeutic action., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Delivery of particle-based theranostic agents via their transportation on the surfaces of red blood cells, commonly referred to as RBC-hitchhiking, has historically been developed as a promising strategy for increasing the extremely poor blood circulation lifetime, primarily, of the large-sized sub-micron agents. Here, we show for the first time that RBC-hitchhiking can be extremely efficient for nanoparticle delivery and tumor treatment even in those cases when no circulation prolongation is observed. Specifically, we demonstrate that RBC-hitchhiking of certain small 100 nm particles, unlike that of the conventional sub-micron ones, can boost the delivery of non-targeted particles to lungs up to a record high value of 120-fold (and up to 40% of the injected dose). To achieve this remarkable result, we screened sub-200 nm nanoparticles of different sizes, polymer coatings and ζ-potentials and identified particles with the optimal RBC adsorption/desorption behavior. Furthermore, we demonstrated that such RBC-mediated rerouting of particles to lungs can be used to fight pulmonary metastases of aggressive melanoma B16-F1. Our findings could change the general paradigm of drug delivery for cancer treatment with RBC-hitchhiking. It is not the blood circulation lifetime that is the key factor for nanoparticle efficiency, but rather the complexation of nanoparticles with the RBC. The demonstrated technology could become a valuable tool for development of new strategies based on small nanoparticles for the treatment of aggressive and small-cell types of cancer as well as other lung diseases.

Malignant tumors are characterized by structural and molecular peculiarities providing a possibility to directionally deliver antitumor drugs with minimal impact on healthy tissues and reduced side effects. Newly formed blood vessels in malignant lesions exhibit chaotic growth, disordered structure, irregular shape and diameter, protrusions, and blind ends, resulting in immature vasculature; the newly formed lymphatic vessels also have aberrant structure. Structural features of the tumor vasculature determine relatively easy penetration of large molecules as well as nanometer-sized particles through a blood⁻tissue barrier and their accumulation in a tumor tissue. Also, malignant cells have altered molecular profile due to significant changes in tumor cell metabolism at every level from the genome to metabolome. Recently, the tumor interaction with cells of immune system becomes the focus of particular attention, that among others findings resulted in extensive study of cells with preferential tropism to tumor. In this review we summarize the information on the diversity of currently existing approaches to targeted drug delivery to tumor, including (i) passive targeting based on the specific features of tumor vasculature, (ii) active targeting which implies a specific binding of the antitumor agent with its molecular target, and (iii) cell-mediated tumor targeting.

Carcinomas overexpressing EGFR family receptors are of high clinical importance, because the receptors have prognostic value and are used as molecular targets for anticancer therapy. Insufficient drug efficacy necessitates further in-depth research of the receptor biology and improvement in preclinical stages of drug evaluation. Here, we review the currently used advanced 3D in vitro models of tumors, including tumor spheroids, models in natural and synthetic matrices, tumor organoids and microfluidic-based models, as a potent tool for studying EGFR biology and targeted drug development. We are especially focused on factors that affect the biology of tumor cells, causing modification in the expression and basic phosphorylation of the receptors, crosstalk with other signaling pathways and switch between downstream cascades, resulting ultimately in the resistance to antitumor agents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We report combined therapy using upconversion nanoparticles (UCNP) coupled to two therapeutic agents: beta-emitting radionuclide yttrium-90 ( 90 Y) 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 90 Y- and PE40-induced cytotoxic effects characterized by IC 50 140 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 IC 50 = 0.0024 μg/mL. The combined therapy with UCNP-R-T was demonstrated in vivo., Competing Interests: The authors declare no conflict of interest.

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.

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 IC 50dark /IC 50light ratio of 11-18. In tumor-bearing mice, the conjugate preferentially accumulates in the tumor tissue., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

An interest to H 2 O 2 accumulation under photodynamic treatment can be explained by its participation in intracellular signal cascades. It is important not only to detect H 2 O 2 generation, but also to trace the dynamics of its intracellular content. In the present study the dynamics of cellular H 2 O 2 content under photodynamic treatment was analyzed using genetically encoded reversible H 2 O 2 -sensitive sensor HyPer. Real-time detecting of H 2 O 2 production after photodynamic treatment was performed using the protein sensor and individual features of action of different photosensitizers were revealed. Photodynamic treatment with a number of chlorin and phthalocyanine photosensitizers was found to induce secondary production of H 2 O 2 in the cells. Three types of dynamic responses were registered: monotonous increase of H 2 O 2 level during the entire observation time in the presence of Fotoditazin and Holosens; transient short-term accumulation in the presence of Radachlorin and Phthalosens; and relatively low-level stable increase in the presence of Photosens. The listed photosensitizers differ significantly in intracellular localization and physicochemical properties, which can determine the differences in the response of H 2 O 2 after the photodynamic treatment. In general, it has been shown that the rapid transient H 2 O 2 response is typical for hydrophobic compounds localized in membrane cell structures, whereas in the presence of more hydrophilic dyes a prolonged monotonous H 2 O 2 accumulation occurs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: A promising strategy for cancer diagnosis and therapy is the development of an agent for multimodal imaging and treatment. In the present paper we report on two novel multifunctional agents prepared on the porphyrazine pigment platform using a gadolinium (III) cation chelated by red-fluorescent tetrapyrrole macrocycles (GdPz1 and GdPz2)., Methods: Spectral and magnetic properties of the compounds were analyzed. Monitoring of GdPz1 and GdPz2 accumulation in the murine colon carcinoma CT26 was performed in vivo using fluorescence imaging and MRI. The photobleaching of GdPz1 or GdPz2 and tumor growth rate after photodynamic therapy (PDT) were assessed., Results: GdPz1 and GdPz2 demonstrated the selective accumulation in tumor that was indicated by higher fluorescence intensity in the tumor area in comparison with the normal tissues. The results of MRI in vivo showed that GdPz1 or GdPz2 provided significant contrast enhancement of the tumor in T1 MR images. PDT with GdPz2 resulted in ~20% decrease in fluorescence intensity of the compound and the inhibition of tumor growth., Conclusions: We assessed the efficiency of two innovative Gd(III) cation-porphyrazine chelates as bimodal MR and fluorescent probes and photosensitizers for PDT and showed their potentials for tumor diagnostics and treatment., General Significance: Water-soluble structures simple in preparation and administration into the body represent special interest for theranostics of tumors. Novel porphyrazine macrocycles chelating a central gadolinium cation demonstrated a good prospect as effective multimodal agents, representing a new approach to MRI and fluorescence imaging guided PDT., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The necessary precondition for efficient boron neutron capture therapy (BNCT) is control over the content of isotope 10 B 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 e ₆-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 e ₆-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., Competing Interests: The authors declare no conflict of interest.

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of targeted toxins based on non-immunoglobulin targeting molecules appears to be one of the most advanced approaches in the targeted therapy of malignant tumors with a high expression of the HER2 receptor. Earlier, we showed that the targeted toxin DARPin-PE40 consisting of the HER2-specific non-immunoglobulin polypeptide (the targeting module) and a fragment of Pseudomonas exotoxin A (the toxic module) exhibits an antitumor effect in vivo against the HER2-positive adenocarcinoma xenograft. In this work, an in-depth analysis of the effect of DARPin-PE40 on the growth dynamics of experimental xenograft tumors was carried out. DARPin-PE40 was shown to inhibit tumor growth at a dose of 25 and 50 μg/animal and to cause tumor node reduction at a dose of 80 μg/animal, followed by growth resumption at the end of therapy. An evaluation of the tumor growth dynamics revealed statistically significant differences in tumor volume in mice in the experimental groups compared to the control group. The results testify to the potential of using the created targeted toxin as an agent for the targeted therapy of HER2-overexpressing tumors.

Tumor response to therapeutic treatment is largely determined by its heterogeneity and the presence of intercellular junctions, hindering the penetration of large molecules deep into the three-dimensional structure of the tumor. In that context, 3D in vitro tumor models such as cancer cell spheroids are becoming increasingly popular. We obtained spheroids of human breast adenocarcinoma SKBR-3 overexpressing the HER2 cancer marker. The toxicity of HER2-targeted immunotoxin 4D5scFv-PE40 against spheroids was shown to be several orders of magnitude lower compared to a monolayer cell culture. The significant difference in the severity of the immunotoxin effect can be explained by the fact that it ineffectively penetrates the spheroid and predominantly influences the cells of the outer layers. The resulting tumor spheroid model can be used in development of drugs for targeted therapy as well as to study ways to improve the efficiency of anticancer agents by targeting cell-cell contacts.

Recombinant immunotoxins are extremely promising agents for the targeted therapy of tumors with a certain molecular profile. In this work, we studied the properties of a new recombinant HER2-specific immunotoxin composed of the scFv antibody and a fragment of Pseudomonas exotoxin A (4D5scFv-PE40). High affinity of the immunotoxin for the HER2 tumor marker, its selective cytotoxicity against HER2-overexpressing cells, and its storage stability were demonstrated. The 50% inhibitory concentration (IC50) of the 4D5scFv-PE40 immunotoxin for HER2-overexpressing cancer cells was 2.5-3 orders of magnitude lower compared to that for CHO cells not expressing this tumor marker and was 2.5-3 orders of magnitude lower than IC50 of free PE40 for HER2-overexpressing cancer cells. These findings provide a basis for expecting in the long run high therapeutic index values of the 4D5scFv-PE40 immunotoxin for its use in vivo.

Targeted delivery of semiconductor quantum dots (Q Ds) to tumors overexpressing HER2 cancer marker has been. demonstrated on immunocompromised mice bearing human breast cancer xenografts. To obtain targeted QDs complexes we applied the approach based on the use of protein adaptor system, RNAase barnase and its inhibitor barstar. Specific binding to target cancer marker was achieved through bivalent fusion protein containing two fragments of4D5scFv recombinant antibody and a fragment of barnase. QDs were conjugated to barstar, and final assembly of targeted complexes was obtained through non-covalent specific interaction of barstar, attached to QD, and barnase, that is part of the recombinant targeting protein. The efficient delivery of QDs to HER2-expressing tumor demonstrates the possibilities and prospects of the approach for targeted delivery of nanoparticles to cancer cells in vivo as the way to improve the efficiency of diagnosis and promote development of therapies based on the use of nanoparticles.