biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2024-24-2-188-199biopreparat-579Research ArticleТЕМА НОМЕРА: ВЫСОКОТЕХНОЛОГИЧНЫЕ ЛЕКАРСТВЕННЫЕ ПРЕПАРАТЫISSUE TOPIC: ADVANCED THERAPY MEDICINAL PRODUCTSКинетика накопления в клетках флуоресцентно-меченного олигонуклеотида при использовании адресных фолатсодержащих катионных липосомTime course of fluorescent-labelled oligonucleotide accumulation in cells with the use of folate receptor-targeted cationic liposomeshttps://orcid.org/0000-0003-3727-4905ШмендельЕ. В.ShmendelE. V.

Шмендель Елена Васильевна, канд. хим. наук

просп. Вернадского, д. 86, Москва, 119571

Elena V. Shmendel, Cand. Sci. (Chem.)

86 Vernadsky Ave, Moscow 119571

elena_shmendel@mail.ruhttps://orcid.org/0000-0002-8381-1890МарковО. В.MarkovO. V.

Марков Олег Владимирович, канд. биол. наук

проспект Академика Лаврентьева, д. 8, г. Новосибирск, 630090

Oleg V. Markov, Cand. Sci. (Biol.)

8 Academician Lavrentyev Ave, Novosibirsk 630090

markov_oleg@list.ruhttps://orcid.org/0000-0003-4044-1049ЗенковаМ. А.ZenkovaM. A.

Зенкова Марина Аркадьевна, д-р. биол. наук, проф., член-корр. РАН

проспект Академика Лаврентьева, д. 8, г. Новосибирск, 630090

Marina A. Zenkova, Dr. Sci. (Biol.), Professor, Corr. Member of RAS

8 Academician Lavrentyev Ave, Novosibirsk 630090

marzen@niboch.nsc.ruhttps://orcid.org/0000-0002-5372-1325МасловМ. А.MaslovM. A.

Маслов Михаил Александрович, д-р хим. наук, доц.

просп. Вернадского, д. 86, Москва, 119571

Mikhail A. Maslov, Dr. Sci. (Chem.), Assoc. Prof.

86 Vernadsky Ave, Moscow 119571

mamaslov@mail.ruФедеральное государственное бюджетное образовательное учреждение высшего образования «МИРЭА — Российский технологический университет», Институт тонких химических технологий имени М.В. ЛомоносоваРоссияMIREA — Russian Technological University, Lomonosov Institute of Fine Chemical TechnologiesRussian FederationФедеральное государственное бюджетное учреждение науки Институт химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of SciencesRussian Federation202403062024242Высокотехнологичные лекарственные препараты188199Copyright © Шмендель Е.В., Марков О.В., Зенкова М.А., Маслов М.А., 20242024Шмендель Е.В., Марков О.В., Зенкова М.А., Маслов М.А.Shmendel E.V., Markov O.V., Zenkova M.A., Maslov M.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/579

ВВЕДЕНИЕ. Использование катионных липосом является перспективным подходом для доставки терапевтических нуклеиновых кислот (НК) в клетки-мишени, так как позволяет защитить НК от деградации внеклеточными нуклеазами. Однако для обеспечения селективной доставки НК к месту терапевтического действия данный подход нуждается в совершенствовании, в том числе за счет модификации поверхности липосом адресными лигандами.ЦЕЛЬ. Проведение сравнительного исследования кинетики накопления в клетках флуоресцентно-меченного олигонуклеотида (модельного препарата нуклеиновых кислот) с помощью адресных фолатсодержащих катионных липосом и обычных липосом.МАТЕРИАЛЫ И МЕТОДЫ. Адресные фолатсодержащие катионные липосомы (F) и обычные липосомы (L) готовили с использованием следующих компонентов: поликатионный амфифил 2X3, цвиттер-ионный липид DOPE, фолатный липоконъюгат F12. Физикохимические характеристики липосом изучали с помощью методов динамического светорассеяния и трансмиссионной электронной микроскопии. Комплексы липосом с флуоресцентно-меченным олигонуклеотидом (FITC-ODN) формировали с использованием различных соотношений компонентов (N/P). Кинетика накопления комплексов на клетках рака шейки матки человека (линия КВ-3-1) и почки эмбриона человека (линия НЕК 293) была изучена с помощью методов проточной цитометрии, флуоресцентной и конфокальной микроскопии.РЕЗУЛЬТАТЫ. Полученные липосомы L и F представляли собой сферические частицы диаметром от 75 до 100 нм. Сформированы комплексы катионных липосом (L и F) с FITC-ODN при оптимальном соотношении N/P, равном 2/1, при котором наблюдается образование однородных (индекс полидисперсности меньше 0,200) частиц с размером 112,4–125,1 нм. Фолатсодержащие липосомы на 25% эффективнее доставляли FITC-ODN в клетки КВ-3-1 через 90, 120 и 240 мин после начала трансфекции по сравнению с обычными липосомами, не содержащими адресного лиганда. Согласно данным флуоресцентной и конфокальной микроскопии после начала трансфекции клеток наблюдалась совместная колокализация флуоресцентных сигналов липосом и олигонуклеотида и далее происходило накопление олигонуклеотида в цитоплазме.ВЫВОДЫ. Показана эффективная доставка флуоресцентно-меченного олигонуклеотида в цитоплазму опухолевых клеток с помощью катионных липосом. Использование адресных фолатсодержащих липосом позволяет увеличить количество трансфицированных клеток и эффективность доставки олигонуклеотида по сравнению с обычными липосомами, не содержащими адресного лиганда. Полученные результаты могут быть использованы для дальнейшей разработки препаратов направленного действия на основе терапевтических нуклеиновых кислот и липосом.

INTRODUCTION. The use of cationic liposomes is a promising approach to the delivery of therapeutic nucleic acids to target cells because liposomes can protect nucleic acids from degradation by extracellular nucleases. However, to ensure selective delivery to the site of action, this approach needs modification, including liposome surface functionalisation with targeting ligands.AIM. This study aimed to compare the time courses of the accumulation of a fluorescent-labelled oligonucleotide (FITC-ODN), which simulated a nucleic acid-based medicinal product, in cells with the use of folate receptor-targeted (F) and conventional (L) cationic liposomes.MATERIALS AND METHODS. F- and L-liposomes were prepared using the polycationic amphiphile 2X3, the zwitterionic helper lipid DOPE, and the folate lipoconjugate F12. Physicochemical characterisation of the liposomes was performed using dynamic light scattering and transmission electron microscopy. Liposome–FITC-ODN complexes were formed at various nitrogen to phosphate (N/P) charge ratios. Flow cytometry, fluorescence microscopy, and confocal microscopy methods were used to study the accumulation of liposome–FITC-ODN complexes in human cervical carcinoma (KB-3-1) and human embryonic kidney (HEK 293) cells.RESULTS. The prepared F- and L-liposomes were spherical particles with a diameter of 75–100 nm. The authors selected the optimal N/P ratio of 2/1 to obtain complexes of F- and L-liposomes with the FITC-ODN. This N/P ratio yielded homogeneous liposome–FITC-ODN complexes having a polydispersity index below 0.200 and a size of 112.4–125.1 nm. F-liposomes were 25% more efficient than L-liposomes in FITC-ODN delivery to KB-3-1 cells at 90, 120, and 240 minutes after transfection. In the first few minutes of cell transfection, fluorescence and confocal microscopy data on the distribution of liposome–FITC-ODN complexes showed that cationic liposome fluorescence signals colocalised with FITC-ODN signals. Later, FITC-ODN accumulation in the cytoplasm was observed.CONCLUSIONS. Cationic liposomes demonstrated efficient FITC-ODN delivery into the cytoplasm of cancer cells. F-liposomes enhanced the percentage of transfected cells and improved FITC-ODN delivery compared with L-liposomes. The results obtained can be used in the further development of targeted medicinal products based on therapeutic nucleic acids and liposomes.

терапевтическая нуклеиновая кислотаантисмысловой олигонуклеотидфлуоресцентномеченный олигонуклеотидлипосомафолатадресная доставкаадресные фолатсодержащие катионные липосомыкинетика накопленияопухолевые клеткиtherapeutic nucleic acidsantisense oligonucleotidefluorescent-labelled oligonucleotideliposomefolatetargeted deliveryfolate receptor-targeted cationic liposomesaccumulation time coursecancer cellsИсследование выполнено при финансовой поддержке Российского научного фонда в рамках проекта № 23-73-10168. Работа Маркова О.В. и Зенковой М.А. поддержана в рамках бюджетного финансирования Российской Федерации, проект № 121031300044-5.The study reported in this publication was funded by the Russian Science Foundation, Project No. 23-73-10168. O.V. Markov and M.A. Zenkova received public funding from the government of the Russian Federation, Project No. 121031300044-5.ReferencesShchaslyvyi AY, Antonenko SV, Tesliuk MG, Telegeev GD. 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The authors declare that there are no conflicts of interest present.