Ultrasonically Assisted Preparation of Polysaccharide Microcontainers for Hydrophobic Drugs

Stable polysaccharide microcontainers are fabricated by ultrasonically assisted procedure. Ultrasound induces formation of permanent microcontainer shell due to interaction between chitosan and xanthan gum. The obtained system has a core-shell structure with high loading capacity for hydrophobic molecules. The permanent polymer shell thickness of 7-10 nm allows to maintain the microcontainer stability for more than 4 months. The microcontainers in a wide size range of 350-7500 nm were obtained by changing an overall emulsion viscosity. Uptake of the microcontainers by mouse melanoma M3 cells was studied by flow cytometry and confocal microcscopy. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3546

Progress in melanoma modeling in vitro

Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study melanoma new techniques, including spherical models, tumorospheres, and melanoma skin equivalents have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation, and are less expensive and time consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved. This article is protected by copyright. All rights reserved

РОЛЬ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА И АУТОФАГИИ В ПРОТИВООПУХОЛЕВОМ ОТВЕТЕ КЛЕТОЧНЫХ ЛИНИЙ МЕЛАНОМЫ НА ТАРГЕТНОЕ ИНГИБИРОВАНИЕ MEK и mTOR КИНАЗ

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma.Введение. Возникновение резистентности и дальнейшая опухолевая прогрессия остаются актуальной проблемой в лечении меланомы кожи. Процесс аутофагии и эпителиально-мезенхимальный переход (ЭМП) тесно связаны между собой и играют ключевую роль в опухолевой прогрессии. Таргетное коингибирование МЕК и mTOR киназ является потенциальной мишенью для терапии меланомы, нацеленной на блокирование ЭМП.Цель работы – изучение влияния ко-ингибирования МЕК и mTOR киназ на выживаемость, возможность формирования 3D-сфероидов и миграционные способности клеточных линий меланомы, а также взаимосвязь этих изменений с маркерами ЭМП и аутофагии.Материал и методы. Работа проведена на клеточных линиях меланомы Mel Z и Mel MTP, полученных от пациентов, проходивших лечение в НМИЦ онкологии им. Н.Н. Блохина. Оценку антипролиферативной активности биниметиниба и/или рапамицина исследовали МТТ-тестом. 3D-сфероиды получали на основе RGDпептидов, миграционную способность и инвазивность оценивали к камере Бойдена и базальном матриксе. Изменения экспрессии маркеров аутофагии и ЭМП исследованы иммуноцитохимически или иммуноблоттингом.Результаты. Рапамицин усиливал цитотоксичность биниметиниба как в 2D-, так и в 3D-культурах клеточных линий меланомы. При этом биниметиниб и рапамицин снижали инвазию, но не миграцию клеток меланомы in vitro. Эффективность комбинации связана со снижением маркеров ЭМП N-кадхерина и β-катенина и аутофагии в клетках меланомы – Беклин 1, р62/SQST M1 и LC3BII .Заключение. Инактивация аутофагии и ЭМП позволяет преодолевать резистентность к существующей терапии и может быть рассмотрена как перспективная мишень для терапии меланомы

Nouveaux modèles 3D in vitro à base de sphéroïdes multicellulaires tumoraux pour tester des substances anticancéreuses et des vecteurs de délivrance de médicaments

Multicellular tumor spheroids (MTS) are a promising tool in tumor biology. The aim of the Thesis was to develop a novel highly reproducible technique for MTS formation, and to demonstrate the availability of these spheroids as 3D in vitro model to test anticancer drugs and drug delivery vehicles. Cell self-assembly effect induced by an addition of cyclic RGD-peptides directly to monolayer cultures was studied for 16 cell lines of various origin. Cyclo-RGDfK peptide and its modification with triphenylphosphonium cation (TPP) were found to induce spheroid formation. The spheroids were used as a model to evaluate the cytotoxicity of antitumor drugs (doxorubicin, curcumin, temozolomide) and a number of nano- and micro- formulations (microcontainers, nano-emulsions and micelles).Les sphéroïdes multicellulaires tumoraux (SMT) constituent un outil prometteur dans le domaine de l’étude biologique des tumeurs. Le but de la thèse était de développer une technique de la formation de SMT et de démontrer la disponibilité de ces sphéroïdes comme modèle in vitro 3D pour tester l’efficacité de principes actifs anticancéreux ainsi que celle de formulations de délivrance de médicaments. L'effet d’auto-assemblage de cellules induit par une addition des peptides RGD cycliques a été étudié pour 16 lignées cellulaires de différentes origines. Le peptide cyclique RGDfK et sa modification avec le cation triphenylphosphonium (TPP) ont permis de mettre en évidence l’induction de formation de sphéroïdes. Les sphéroïdes ont été employés comme modèles pour évaluer la cytotoxicité de principes actifs antitumoraux (doxorubicine, curcumine, temozolomide) et un certain nombre de formulations nano- et micrométriques (microréservoirs, nano-émulsions et micelles)

Фотодинамическая терапия солидных опухолей in vitro и in vivo с применением комбинации рибофлавина и наноразмерных апконвертирующих фосфоров

Rationale: Riboflavin (vitamin B2) is one of the most promising agents for photodynamic therapy (PDT). However, its use is limited by the excitation in the ultraviolet (UV) and visible spectral ranges and, as a result, by a small penetration into biological tissue not exceeding a few millimeters. This problem could be solved by approaches ensuring excitation of riboflavin molecules within tumor tissues by infrared (IR) light. Upconversion nanoparticles (UCNPs) can be potentially considered as mediators able to effectively convert the exciting radiation of the near IR range, penetrating into biological tissue to a 3 cm depth, into the photoluminescence in the UV and visible spectral ranges.Aim: To evaluate the efficacy of UCNPs for IR-mediated riboflavin activation in the depth of tumor tissue during PDT. Materials and methods: The water-soluble riboflavin flavin mononucleotide (FMN, Pharmstandard-UfaVITA, Russia) was used as a photosensitizer in in vitro and in vivo experiments. The in vitro experiments were performed on human breast adenocarcinoma SK-BR-3, human glioblastoma U-87 MG, and rat glioma C6 cell lines. Lewis lung carcinoma (LLC) inoculated to hybrid BDF1 mice was used as a model to demonstrate the delivery of FMN to the tumor. UCNPs with a core/shell structure [NaYF4:Yb3+, Tm3+/NaYF4] were used for photoactivation of FMN in vivo. PDT based on FMN, UCNPs and laser radiation 975 nm (IR) was performed on mouse xenografts of human breast adenocarcinoma SKBR-3.Results: We were able to show that FMN could act as an effective in vitro photosensitizer for SK-BR-3, U-87 MG, and C6 cell lines. FMN IC50 values for glioma cells were ~30 μM, and for SK-BR-3 cell line ~50 μM (24 h incubation, irradiation 4.2 J/cm2). In the LLC model, the appropriate concentration of FMN (30 μM and above) can be achieved in the tumor as a result of systemic administration of FMN (at 2 and 24 hours after injection). The effect of PDT using near IR light for UCNP-mediated excitation of FMN was demonstrated in mouse xenografts SKBR-3, with the tumor growth inhibition of 90±5%.Conclusion: The study has demonstrated the possibility to use riboflavin (vitamin B2) as a photosensitizer for PDT. The photoexcitation of FMN via the anti-Stokes photoluminescence of UCNPs allows for implementation of the PDT technique with the near IR spectral range.Обоснование. Рибофлавин (витамин В2) считается одним из наиболее перспективных агентов для фотодинамической терапии. Однако его применение ограничено возбуждением в ультрафиолетовом (УФ) и синем диапазонах спектра и, как следствие, малой (не более нескольких миллиметров) глубиной проникновения в биоткань. Решением данной проблемы видится разработка подходов, обеспечивающих фотовозбуждение молекул рибофлавина под действием инфракрасного (ИК) света в глубине опухолевой ткани. В качестве посредника, способного эффективно преобразовывать возбуждающее излучение ближнего ИК диапазона, проникающее в биоткань на глубину до 3 см, в фотолюминесценцию УФ и видимого диапазона спектра, могут быть рассмотрены наноразмерные апконвертирующие фосфоры (НАФ).Цель – оценить эффективность использования НАФ для ИК-опосредованной активации рибофлавина в глубине опухолевой ткани при проведении фотодинамической терапии.Материал и методы. Водорастворимая форма рибофлавина – флавинмононуклеотид (ФМН) (Фармстандарт-УфаВИТА, Россия) – был использован в качестве фотосенсибилизатора в экспериментах in vitro и in vivo. Эксперименты in vitro выполнены на клеточных линиях аденокарциномы молочной железы человека SK-BR-3, глиобластомы человека U-87 MG и глиомы крысы C6. Карцинома легкого Льюис, перевитая мышам-гибридам BDF1, была использована в качестве модели для демонстрации доставки ФМН в опухолевую ткань. Для фотоактивации ФМН in vivo применялись НАФ со структурой «ядро/оболочка» [NaYF4:Yb3+, Tm3+/NaYF4]. Фотодинамическая терапия на основе ФМН, НАФ и лазерного излучения 975 нм проводилась на ксенографтах мыши SK-BR-3.Результаты. Показано, что ФМН может выступать в качестве эффективного фотосенсибилизатора in vitro в отношении клеточных линий SK-BR-3, U-87 MG и C6. Значения IC50 для клеток глиомы составляли ~30 мкМ ФМН, а для клеток карциномы молочной железы SK-BR-3 ~50 мкМ ФМН (24 ч инкубации, облучение 4,2 Дж/см2). С использованием модели карциномы легкого Льюис установлено, что соответствующая концентрация ФМН (30 мкМ и выше) может быть достигнута в опухоли в результате системного введения ФМН (через 2 и 24 часа после введения). На ксенографтах мыши SK-BR-3 продемонстрирован эффект фотодинамической терапии с использованием света ближнего ИК диапазона для НАФ-опосредованного возбуждения ФМН, торможение роста опухоли при этом составило 90±5%.Заключение. Продемонстрирована возможность применения рибофлавина (витамина В2) в качестве фотосенсибилизатора для фотодинамической терапии. Использование подхода, основанного на фотовозбуждении ФМН через антистоксовую фотолюминесценцию НАФ, позволяет реализовать метод фотодинамической терапии с применением света из ближнего ИК диапазона спектра

<i>In vitro и in vivo</i> photodynamic therapy of solid tumors with a combination of riboflavin and upconversion nanoparticles

Rationale: Riboflavin (vitamin B2) is one of the most promising agents for photodynamic therapy (PDT). However, its use is limited by the excitation in the ultraviolet (UV) and visible spectral ranges and, as a result, by a small penetration into biological tissue not exceeding a few millimeters. This problem could be solved by approaches ensuring excitation of riboflavin molecules within tumor tissues by infrared (IR) light. Upconversion nanoparticles (UCNPs) can be potentially considered as mediators able to effectively convert the exciting radiation of the near IR range, penetrating into biological tissue to a 3 cm depth, into the photoluminescence in the UV and visible spectral ranges.Aim: To evaluate the efficacy of UCNPs for IR-mediated riboflavin activation in the depth of tumor tissue during PDT. Materials and methods: The water-soluble riboflavin flavin mononucleotide (FMN, Pharmstandard-UfaVITA, Russia) was used as a photosensitizer in in vitro and in vivo experiments. The in vitro experiments were performed on human breast adenocarcinoma SK-BR-3, human glioblastoma U-87 MG, and rat glioma C6 cell lines. Lewis lung carcinoma (LLC) inoculated to hybrid BDF1 mice was used as a model to demonstrate the delivery of FMN to the tumor. UCNPs with a core/shell structure [NaYF4:Yb3+, Tm3+/NaYF4] were used for photoactivation of FMN in vivo. PDT based on FMN, UCNPs and laser radiation 975 nm (IR) was performed on mouse xenografts of human breast adenocarcinoma SKBR-3.Results: We were able to show that FMN could act as an effective in vitro photosensitizer for SK-BR-3, U-87 MG, and C6 cell lines. FMN IC50 values for glioma cells were ~30 μM, and for SK-BR-3 cell line ~50 μM (24 h incubation, irradiation 4.2 J/cm2). In the LLC model, the appropriate concentration of FMN (30 μM and above) can be achieved in the tumor as a result of systemic administration of FMN (at 2 and 24 hours after injection). The effect of PDT using near IR light for UCNP-mediated excitation of FMN was demonstrated in mouse xenografts SKBR-3, with the tumor growth inhibition of 90±5%.Conclusion: The study has demonstrated the possibility to use riboflavin (vitamin B2) as a photosensitizer for PDT. The photoexcitation of FMN via the anti-Stokes photoluminescence of UCNPs allows for implementation of the PDT technique with the near IR spectral range