Pavlo Khort

Purpose: Photodynamic therapy (PDT) for carcinomas is an alternative treatment method for this pathology. PDT has a number of advantages, such as targeted therapy, weak side effects and high anticancer efficiency. Polymer branched nanocarriers in a combination with gold nanoparticles (AuNPs) can potentially reduce the side effects of hydrophobic photosensitizers (PS) and improve their pharmacokinetic properties. The aim of the study was to investigate the effect of anionic star-like copolymers of the dextran-graft-(polyacrylamide-co-polyacrylic acid) with a dextran core of 20 kDa and 500 kDa on the combined AuNPs and Temoporfin PDT of squamous cell carcinoma. Patients and Methods: AuNPs were synthesized in situ in a matrix of dextran-graft-(polyacrylamide-co-polyacrylic acid) hybrid polymers with a dextran core of 20 kDa (D20-PAAan) or 500 kDa (D500-PAAan) with grafted Polyacrylamide (PAA) chains. The size and morphology of nanosystems were characterized by DLS and scanning electron microscopy. Cytotoxicity and phototoxicity studies were performed on the Hep-2, KB and MAEC cell lines in vitro. The cytotoxicity of the individual components of the nanosystems, cell inactivation under PDT conditions following irradiation with 660 nm light (15 J/cm²), intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨ_m), and overall cytomorphological changes were assessed. Results: The optimal concentrations of gold (as AuNPs) 4.3 μg/mL, polymer 5.0 μg/mL and Temoporfin 0.625 μg/mL were determined, at which the maximum plasmon enhancement is achieved with the competing effect of Förster resonance energy transfer. The cytotoxicity of D20-PAA (100 μg/mL) was 80%, 65%, and 45% against KB, Hep-2 and MAEC cells respectively. The cytotoxicity of D500-PAA at the same concentration did not exceed 40%. ROS levels were increased 1.8-fold in Hep-2 cells following incubation with the ternary nanosystems and subsequent irradiation, regardless of the carrier polymer. ΔΨ_m was increased 1.4-fold in Hep-2 cells treated with D20-PAAan/AuNPs/Tem and 2.1-fold in cells treated with D500-PAAan/AuNPs/Tem followed by 660 nm light irradiation. In KB and MAEC cells, these changes were less pronounced. Conclusion: D20-PAAan based systems showed a stronger influence on cells by briefly changing the intracellular ROS levels and ΔΨ_m. Nanosystems based on D20-PAAan should be more promising for PDT applications.

Photodynamic therapy (PDT) is a non-invasive anticancer treatment that uses special photosensitizer molecules (PS) to generate singlet oxygen and other reactive oxygen species (ROS) in a tissue under excitation with red or infrared light. Though the method has been known for decades, it has become more popular recently with the development of new efficient organic dyes and LED light sources. Here we introduce a ternary nanocomposite: water-soluble star-like polymer/gold nanoparticles (AuNP)/temoporfin PS, which can be considered as a third-generation PDT system. AuNPs were synthesized in situ inside the polymer molecules, and the latter were then loaded with PS molecules in an aqueous solution. The applied method of synthesis allows precise control of the size and architecture of polymer nanoparticles as well as the concentration of the components. Dynamic light scattering confirmed the formation of isolated particles (120 nm diameter) with AuNPs and PS molecules incorporated inside the polymer shell. Absorption and photoluminescence spectroscopies revealed optimal concentrations of the components that can simultaneously reduce the side effects of dark toxicity and enhance singlet oxygen generation to increase cancer cell mortality. Here, we report on the optical properties of the system and detailed mechanisms of the observed enhancement of the phototherapeutic effect. Combinations of organic dyes with gold nanoparticles allow significant enhancement of the effect of ROS generation due to surface plasmonic resonance in the latter, while the application of a biocompatible star-like polymer vehicle with a dextran core and anionic polyacrylamide arms allows better local integration of the components and targeted delivery of the PS molecules to cancer cells. In this study, we demonstrate, as proof of concept, a successful application of the developed PDT system for in vitro treatment of triple-negative breast cancer cells under irradiation with a low-power LED lamp (660 nm). We consider the developed nanocomposite to be a promising PDT system for application to other types of cancer.

Introduction: Cancer chemotherapy faces two major challenges – high toxicity of active substances and tumor resistance to drugs. Low toxic nanocarriers in combination with anticancer agents can significantly increase the effectiveness of therapy. Modern advances in nanotechnology make it easy to create materials with the necessary physical and chemical properties. Methods: Two hybrid nanosystems of dextran-polyacrylamide/ zinc oxide nanoparticles (D-PAA/ZnO NPs) were synthesized in aqueous solution with zinc sulphate (D-PAA/ZnO NPs (SO₄^2-)) and zinc acetate (D-PAA/ZnO NPs (-OAc)). The light absorption, fluorescence, dynamic light scattering and transmission electron microscopy for nanocomposite characterization were used. MTT, neutral red uptake and scratch assays were selected as fibroblasts cytotoxicity assays. Cytotoxicity was tested in vitro for normal fibroblasts, MAEC, prostate (LNCaP, PC-3, DU-145) and breast (MDA-MB-231, MCF-7) cancer cells lines. Immunocytochemical methods were used for detection of Ki-67, p53, Bcl-2, Bax, e-cadherin, N-cadherin and CD44 expression. Acridine orange was used to detect morphological changes in cells. Results: The radius of ZnO NPs (SO₄^2-) was 1.5 nm and ZnO NPs (-OAc) was 2 nm. The nanosystems were low-toxic to fibroblasts, MAEC. Cells in the last stages of apoptosis with the formation of apoptotic bodies were detected for all investigated cancer cell lines. Proapoptotic proteins expression in cancer cells indicates an apoptotic death. Increased expression of E-cadherin and N-cadherin was registered for cancer cells line LNCaP, PC-3, DU-145 and MCF-7 after 48 h incubation with D-PAA/ZnO NPs (SO₄^2-). Conclusion: The nanosystems were low-toxic to fibroblasts, MAEC. The D-PAA/ZnO NPs nanosystem synthesized using zinc sulphate demonstrates high cytotoxicity due to destruction of various types of cancer cells in vitro and potentially increases adhesion between cells. Thus, our findings indicate the selective cytotoxicity of D-PAA/ZnO NPs against cancer cells and can be potentially used for cancer treatment.

The thermoresponsive Zinc TetraPhenylPorphyrin photosensitizer/Dextran poly (N-isopropylacrylamide) graft copolymer/Au Nanoparticles (ZnTPP/D-g-PNIPAM/AuNPs) triple hybrid nanosystem was synthesized in aqueous solution as a nanodrug for potential use in thermally driven and controlled photodynamic therapy applications. The aqueous solution of the nanosystem has demonstrated excellent stability in terms of aggregation and sedimentation several days after preparation. Optimal concentrations of the components of hybrid nanosystem providing the lowest level of aggregation and the highest plasmonic enhancement of electronic processes in the photosensitizer molecules have been determined. It has been revealed that the shrinking of D-g-PNIPAM macromolecule during a thermally induced phase transition leads to the release of both ZnTPP molecules and Au NPs from the ZnTPP/D-g-PNIPAM/AuNPs macromolecule and the strengthening of plasmonic enhancement of the electronic processes in ZnTPP molecules bound with the polymer macromolecule. The 2.7-fold enhancement of singlet oxygen photogeneration under resonant with surface plasmon resonance has been observed for ZnTPP/D-g-PNIPAM/AuNPs proving the plasmon nature of such effect. The data obtained in vitro on wild strains of Staphylococcus aureus have proved the high potential of such nanosystem for rapid photodynamic inactivation of microorganisms particular in wounds or ulcers on the body surface.

A zinc tetraphenylporphyrin photosensitizer/dextran graft polyacrylamide anionic copolymer/Au nanoparticles (ZnTPP/D- g -PAAan/Au NPs) triple hybrid nanosystem has been proposed as a nanodrug for potential photodynamic therapy applications.