We have evaluated the key properties of the polyethylenimine (PEI)-polyethylene glycol (PEG)-TAT peptide polyplex nanoparticles including their behavior in cells and compared them with the transfection efficacy (TE) using 11 different cell lines. We found statistically significant positive correlation between TE and the share of 50-75 nm fraction in the whole mixture of nanoparticles estimated with atomic force microscopy. Variations in PEG/PEI and N/P ratios (PEI nitrogen to DNA phosphate ratio) enabled us to find their optimal combinations, which resulted in up to 100% TE for several cell lines. Surfaces of the TE dependence of both PEG/PEI and N/P turned out to be similar in appearance for all investigated cell lines, while maximum TEs were different. We investigated subcellular transport kinetics and unpacking of the polyplex nanoparticles labeled with quantum dots (plasmid DNA) and AlexaFluor647 (block-copolymer part) using Förster Resonance Energy Transfer approach. The results demonstrated clear and statistically significant positive correlation of TE with the cellular uptake rate of the nanoparticles and negative correlation with the rate constant of their unpacking within endo/lysosomal compartments in the living cells.
We have synthesized and investigated properties of new PEI-PEG-based polyplexes containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (targeted polyplexes), and control polyplexes without this ligand peptide (non-targeted polyplexes). The targeted polyplexes demonstrated receptor-mediated transfection of Cloudman S91 (clone M-3) murine melanoma cells that was more efficient than with the non-targeted ones. Transfection with the targeted polyplexes was inhibited by chlorpromazine, an inhibitor of the clathrin-mediated endocytosis pathway, and, to a lesser extent, by filipin III or nystatin, inhibitors of the lipid-raft endocytosis pathway, whereas transfection with the non-targeted polyplexes was inhibited mainly by nystatin or filipin III. The targeted polyplexes caused significantly higher in vivo transfection of melanoma tumor cells after intratumoral administration compared to the non-targeted control. The targeted polyplexes carrying the HSVtk gene, after ganciclovir administration, more efficiently inhibited melanoma tumor growth and prolonged the lifespan of DBA/2 tumor-bearing mice compared to the non-targeted ones. Packed targeted polyplexes appeared and accumulated in the melanoma cells six hours earlier than the non-targeted ones. The targeted polyplexes enter into the nuclei of the melanoma cells more rapidly than the non-targeted control, and this difference may also be attributed to processes of receptor-mediated endocytosis. We believe that these data may be useful for the optimization of polyplex systems.
The manufacturer (developer) has to prepare a specification for each newly developed biomedical cell product (BCP) that has passed the stage of preclinical studies. The specification is included into the registration dossier when applying for marketing authorisation of a BCP. In accordance with the Order of the Ministry of Health of the Russian Federation No. 14n of 19 January 2017 «On approval of the specification format for a biomedical cell product» the specification should contain information about authenticity of the cell line used in the BCP, namely: morphological characteristics, expression of specific markers, expression of specific genes, expression of specific proteins, as well as markers of cell line stability. At present Russia has no practical experience in BCP quality evaluation. The aim of the study was to substantiate methodological approaches to authentication of cell lines used in BCPs as illustrated by quality evaluation of the DF-2 model cell line using test methods that allow for characterisation of the morphological, genetic, immunophenotypic, and cytogenetic profile of the cell line. Materials and methods: the study analysed the DF-2 cell line — human dermal fibroblasts (mesenchymal stem cells) obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg). The following analytical test methods were used in the study: morphological analysis; flow cytometry for immunophenotyping of the DF-2 model cell line; short tandem repeats for creating an allelic profile of the model cell line; cytogenetic analysis — differential DAPI staining of metaphase chromosomes. Results: the paper summarises methodological approaches to identification testing of medicines containing living human cells (BCP analogues) currently used in international practice, and presents the results of authentication of the model cell line. Conclusions: methods used for BCP identification testing should ensure unambiguous authentication of the cell line according to its specification. The study performed helped to work out the procedure of authentication of a model cell line.
Метод проточной цитометрии -наиболее информативный метод идентификации и количественного определения поверхностных маркеров клеток. Проточная цитометрия дает возможность проводить под-счет клеток, а также характеризацию их типов и подтипов путем мечения клеток моноклональными анти-телами, конъюгированными с флуорохромом. В настоящее время производителями продуктов на основе клеток человека накоплен значительный опыт применения проточной цитометрии, разработано боль-шое количество методик, подлежащих валидации и включению в спецификацию на клеточный продукт. В обзоре авторами рассмотрен опыт применения метода проточной цитометрии для оценки качества клеточных линий человека, используемых, в частности, для создания препаратов с целью применения в клеточной терапии. Учитывая обязательное наличие клеточного компонента в составе биомедицинских клеточных продуктов (БМКП), метод проточной цитометрии будет являться обязательным при подтверж-дении подлинности в ходе экспертизы качества БМКП в Российской Федерации. Про-филактика, диагностика, лечение 2018; 18(1): 16-24. DOI: 10.30895/2221 18(1): 16-24. DOI: 10.30895/ -996Х-2018-24 * Контактное лицо: Трусов Георгий Александрович; trusov@expmed.ru Flow cytometry is the most common method of identification and quantitation of cell surface markers. Flow cytometry can be used for cell counting and characterization of cell types and subtypes by labeling cells with fluorochrome-conjugated monoclonal antibodies. Manufacturers of human cell-based medicinal products have accumulated significant experience in flow cytometry and developed a large number of procedures that can be validated and included into cell products specifications. The present review summarises the experience gained with the use of flow cytometry for characterization of human cell lines used to develop cell therapy products. Since all biomedical cell products (BMCPs) have a cellular component, it will be necessary to use the flow cytometry method for identification testing of BMCPs during evaluation of their quality.
ФГБУ «Центр стратегического планирования и управления медико-биологическими рисками здоровью» Федерального медико-биологического агентства (ФГБУ «ЦСП» ФМБА России), г. МоскваОсновной задачей представленной аналитической работы является описание и характеристика выявленных в ходе клинических исследований эпигенетических маркеров, ассоциированных с воздействием некоторых поллютантов на организм человека, а именно твердых частиц с разным аэродинамическим диаметром (PM 0,1 , PM 2,5 , PM 10 , PM 2,5-10 ), к которым сорбируются различные химические соединения. Целью проведенной работы было сформировать обобщенный перечень наиболее информативных биомаркеров, обнаруживаемых у человека. Практическая значимость созданного перечня из более чем полутора сотен эпигенетических сигнатур воздействия поллютантов на организм человека заключается в возможности разработки на его основе методологического подхода, который можно будет использовать для ранней диагностики заболеваний у людей, подвергшихся токсическому воздействию различных загрязнителей окружающей среды, в частности твердых частиц. Кроме того, данная работа может стать предпосылкой для разработки принципиально новых систем очистки воздуха.
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