Russian Journal of BiotherapyRussian Journal of BiotherapyРоссийский биотерапевтический журнал1726-97841726-9792Publishing House ABV Press119110.17650/1726-9784-2019-19-1-71-80ORIGINAL REPORTSОРИГИНАЛЬНЫЕ СТАТЬИCYTOTOXICITY AND HEMOCOMPATIBILITY OF DOXORUBICIN-LOADED PLGA NANOPARTICLESЦИТОТОКСИЧНОСТЬ И ГЕМОСОВМЕСТИМОСТЬ PLGA НАНОЧАСТИЦ, НАГРУЖЕННЫХ ДОКСОРУБИЦИНОМhttps://orcid.org/0000-0002-9771-3992MalinovskayaYu. A.МалиновскаяЮ. А.
Russian Federation

Юлия Александровна Малиновская

j.malinowskaya@gmail.comhttps://orcid.org/0000-0001-8119-8247KovalenkoE. I.КоваленкоЕ. И.
Russian Federation
https://orcid.org/0000-0002-0398-4645KovshovaT. S.КовшоваТ. С.
Russian Federation
https://orcid.org/0000-0002-2814-9064OsipovaN. S.ОсиповаН. С.
Russian Federation
https://orcid.org/0000-0002-1230-4680MaksimenkoO. O.МаксименкоО. О.
Russian Federation
https://orcid.org/0000-0002-5744-7060BalabanyanV. Yu.БалабаньянВ. Ю.
Russian Federation
RazzhivinaV. A.РазживинаВ. А.
Russian Federation
GrechikhinaM. V.ГречихинаМ. В.
Russian Federation
https://orcid.org/0000-0002-8996-2905BoikoA. A.БойкоА. А.
Russian Federation
https://orcid.org/0000-0003-1113-6715GelperinaS. E.ГельперинаС. Э.
Russian Federation
Drug Technology LLCООО «Технология лекарств»M. V. Lomonosov Moscow State UniversityМосковский государственный университет им. М.В. ЛомоносоваD. Mendeleev University of Chemical Technology of RussiaРоссийский химико-технологический университет им. Д.И. МенделееваShemyakin and Ovchinnikov Institute of Bioorganic ChemistryФГБУН «Институт биоорганической химии им. акад. М.М. Шемякина и Ю.А. Овчинникова РАН»2203202019171802203202022032020https://bioterapevt.abvpress.ru/jour/article/view/1191

Introduction. The use of polymeric biodegradable nanoparticles (NP) as drug delivery systems is a promising approach to overcome histohematomatic barriers. Thus, poloxamer 188-coated poly (lactide-co-glycolide) (PLGA) NP are able to overcome blood-brain barrier and to deliver therapeutic agents, in particular doxorubicin, into intracranial tumour upon intravenous administration. It is important to evaluate NP interaction with blood components in preclinical studies.

The objective of the study was to investigate cytotoxicity and hemocompatibility of doxorubicin-loaded PLGA NP (Dox-PLGA NP), to essess NP uptake by glioblastoma cells.

Materials and methods. The influence of NP on coagulation cascade was evaluated by prothrombin time measuring before and after plasma incubation with NP. To assess NP thrombogenicity the platelet activation level was determined by flow cytometry. The NP hemolytic activity (released hemoglobin concentration) was measured spectrophotometrically. NP cytotoxicity was determined by MTS assay. NP uptake by human glioblastoma cells was evaluated by flow cytometry.

Results. Dox-PLGA NP did not influence blood coagulation time and thrombocyte activity at concentrations up to 100 mcg/mL: PT values were 12–15 s for all tested samples, and P-selectin expression level did not exceed 15 %. All samples were not hemolytic after 3 h of incubation. Cytotoxicity of doxorubicin released from PLGA NP on glioma U87MG cells was comparable to that of free doxorubicin. As shown by flow cytometry Dox-PLGA NP were efficiently internalized into the cells.

Conclusion. The study of hemocompatibility confirmed the safety of Dox-PLGA NP: NP did not influence blood coagulation system and did not induce hemolysis. NP were efficiently internalized into the human glioblastoma cells and produced considerable antitumor effect in vitro.

Введение. Использование полимерных биодеградирумых наночастиц (НЧ) в качестве средств доставки лекарственных веществ является перспективным способом преодоления гистогематических барьеров. Так, НЧ из сополимера молочной и гликолевой кислот (PLGA), модифицированные полоксамером 188, способны преодолевать гематоэнцефалический барьер и доставлять лекарственные вещества, в частности доксорубицин, в интракраниальную опухоль при внутривенном введении. На этапе доклинических исследований было важно оценить возможное токсическое действие НЧ на компоненты крови.

Цель исследования – оценка цитотоксичности и гемосовместимости PLGA НЧ, нагруженных доксорубицином (Dox-PLGA НЧ), изучение кинетики захвата наночастиц клетками глиобластомы человека.

Материалы и методы. Для изучения влияния НЧ на свертывающую систему крови определяли протромбиновое время до и после инкубации плазмы с НЧ. Уровень активации тромбоцитов определяли на проточном цитофлуориметре по уровню экспрессии Р-селектина. Гемолитическую активность НЧ определяли спектрофотометрически по концентрации высвободившегося гемоглобина. Цитотоксичность НЧ оценивали с помощью MTS-теста. Захват НЧ клетками изучали с помощью проточного цитофлуориметра.

Результаты. Dox-PLGA НЧ не оказывали влияния на время коагуляции плазмы крови и активность тромбоцитов в диапазоне концентраций 0,1–100 мкг/мл: показатель протромбинового времени составил 12–15 с для всех тестируемых образцов и уровень экспрессии Р-селектина не превысил 15 %. НЧ не вызывали гемолиз после 3 ч инкубации с образцами крови. Цитотоксический эффект Dox-PLGA НЧ на клетки глиобластомы U87MG был сравним с действием субстанции доксорубицина. Результаты проточной цитометрии показали, что НЧ активно захватываются клетками.

Заключение. Проведенное исследование подтвердило гемосовместимость Dox-PLGA НЧ: НЧ не оказывали влияния на свертывающую систему крови и не вызывали гемолиз в исследуемом диапазоне концентраций. НЧ активно захватывались клетками глиобластомы и оказывали выраженный цитотоксический эффект.

 

PLGA nanoparticlesbiocompatibilityhemocompatibilitycytotoxicityinternalizationPLGA наночастицыбиосовместимостьгемосовместимостьцитотоксичностьинтернализацияThis study was supported by the Federal target program “Pharma 2020” (Contract No 13411.1008799.13.144).Исследование проведено при поддержке Федеральной целевой программы «Фарма 2020» (Госконтракт № 13411.1008799.13.144).Afanasieva D.A., Baryshnikova M.A., Shcherbakov A.I. et al. The development of anticancer liposomal vaccine model. Immunologiya = Immunology 2014;35(6):317–21 (In Russ.).Weissig V., Pettinger T.K., Murdock N. Nanopharmaceuticals (part 1): products on the market. Int J Nanomedicine 2014;9:4357–73. DOI: 10.2147/IJN.S46900.Pathak Y., Thassu D. Drug delivery nanoparticles formulation and characterization. Boca Raton, USA, CRC Press, 2009, 416 р.Wang A.Z., Langer R., Farokhzad O.C. Nanoparticle Delivery of Cancer Drugs. Annu Rev Med 2012;63(1):185–98. DOI: 10.1146/annurev-med040210-162544.Makadia H.K., Siegel S.J. Poly lacticco-glycolic acid(PLGA) as biodegradable controlled drug delivery carrier. Polymers (Basel) 2011;3(3):1377–97. DOI: 10.3390/polym3031377.Lü J.M., Wang X., Marin-Muller C. et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 2009;9(4):325–41. DOI: 10.1586/erm.09.15.Mundargi R.C., Babu V.R., Rangaswamy V. et al. Nano/micro technologies for delivering macromolecular therapeutics using poly(D, L-lactide-co-glycolide) and its derivatives. J Control Release 2008;125(3):193–209. DOI: 10.1016/j.jconrel.2007.09.013.Shive M.S., Anderson J.M. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 1997;28(1):5–24. DOI: 10.1016/S0169-409X(97)00048-3.Szymonowicz M., Rybak Z., Witkiewicz W. et al. In vitro hemocompatibility studies of (poly-(L-lactide) and poly(L-lactide-co-glycolide) as materials for bioresorbable stents manufacture. Acta Bioeng Biomech 2014;16(4):131–9. DOI: 10.5277/ABB-00055-2014-03.Landes C.A., Ballon A., Roth C. Maxillary and mandibular osteosyntheses with PLGA and P(L/DL)LA implants: a 5-year inpatient biocompatibility and degradation experience. Plast Reconstr Surg 2006;117(7):2347–60. DOI: 10.1097/01. prs.0000218787.49887.73.Mau L.P., Cheng C.W., Hsieh P.Y., Jones A.A. Biological complication in guided bone regeneration with a polylactic acid membrane: a case report. Implant Dent 2012;21(3):171–4. DOI: 10.1097/ID.0b013e31824eece1.Maiborodin I.V., Kuznetsova I.V., Beregovoy E.A. et al. Absence of complete resorption of polylactide material in the organism. Novosti khirurgii = Surgery News 2014;22(1):26–32. (In Russ.).Майбородин И.В., Кузнецова И.В., Береговой Е.А и др. Отсутствие полной резорбции полилактидного материала в организме. Новости хирургии 2014;22(1):26–32.Gentile P., Chiono V., Carmagnola I., Hatton P.V. An overview of poly(lactic-co-glycolic)acid(PLGA)-based biomaterials for bone tissue engineering. Int J Mol Sci 2014;15(3):3640–59. DOI: 10.3390/ijms15033640.Pérez-Herrero E., Fernández-Medarde A. Advanced targeted therapies in cancer: drug nanocarriers, the future of chemotherapy. Eur J Pharm Biopharm 2015;93:52–79. DOI: 16.1016/j.ejpb.2015.03.018.Tariq M., Alam M.A., Singh A.T. et al. Biodegradable polymeric nanoparticles for oral delivery of epirubicin: in vitro, ex vivo, and in vivo investigations. Colloids Surf B Biointerfaces 2015;128:448–56. DOI: 10.1016/j.colsurfb.2015.02.043.Derakhshandeh K., Soheili M., Dadashzadeh S., Saghiri R. Preparation and in vitro characterization of 9-nitrocamptothecin-loaded long circulating nanoparticles for delivery in cancer patients. Int J Nanomedicine 2010;5:463–71. DOI: 10.2147/IJN.S11586.Derakhshandeh K., Hosseinalizadeh A., Nikmohammadi M. The effects of PLGA microparticles on intestinal absorption of p-glycoprotein substrate using the everted rat intestinal sac model. Arch Pharm Res 2011;34(11):1989–97. DOI: 10.1007/s12272-011-1120-1.Sobot D., Mura S., Couvreur P. Nanoparticles: Blood Components interaction. Encyclopedia of polymeric nanomaterials, 2015, 1352 p. DOI: 10.1007/978-3-642-29648-2_227.Fornaguera C., Caldero G., Mitjans M. et al. Interactions of PLGA nanoparticles with blood components: protein adsorption, coagulation, activation of the complement system and hemolysis studies. Nanoscale 2015;7(14):6045–58. DOI: 10.1039/c5nr00733j.Kim D., El-Shall H., Dennis D., Morey T. Interaction of PLGA nanoparticles with human blood constituents. Colloids Surf B Biointerfaces 2005;40(2):83–91. DOI: 10.1016/j.colsurfb.2004.05.007.Fornaguera C., Solans C. Methods for the in vitro characterization of nanomedicines – biological component interaction. J Pers Med 2017;7(1):2. DOI: 10.3390/jpm7010002.Rahimian S., Fransen M.F., Kleinovink J.W. et al. Particulate systems based on poly(lactic-co-glycolic)acid(pLGA) for immunotherapy of cancer. Curr Pharm Des 2015;21(29):4201–16. DOI: 10.2174/1381612821666150901100247.Desai N. Challenges in development of nanoparticle-based therapeutics. AAPS J 2012;14(2):282–95. DOI: 10.1208/s12248-012-9339-4.Gelperina S., Maksimenko O., Khalansky A. et al. Drug delivery to the brain using surfactant-coated poly(lactide-coglycolide) nanoparticles: influence of the formulation parameters. Eur J Pharm Biopharm 2010;74(2):157–63. DOI: 10.1016/j.ejpb.2009.09.003.Wohlfart S., Khalansky A.S., Gelperina S. et al. Efficient chemotherapy of rat glioblastoma using doxorubicin-loaded PLGA nanoparticles with different stabilizers. PloS one 2011;6(5):e19121. DOI: 10.1371/journal.pone.0019121.Rejman J., Oberle V., Zuhorn I.S., Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochem J 2004;377(1):159–69. DOI: 10.1042/BJ20031253.Hillaireau H., Couvreur P. Nanocarriers’ entry into the cell: relevance to drug delivery. Cell Mol Life Sci 2009;66(17):2873–96. DOI: 10.1007/s00018-009-0053-z.Fehrenbacher N., Jäättelä M. Lysosomes as targets for cancer therapy. Cancer Res 2005;65(8):2993–5. DOI: 10.1158/0008-5472. CAN-05-0476.Ilinskaya A.N., Dobrovolskaia M.A. Nanoparticles and the blood coagulation system. Part II: safety concerns. Nanomedicine 2013;8(6):969–81. DOI: 10.2217/nnm.13.49.Gunness P., Aleksa K., Kosuge K. et al. Comparison of the novel HK-2 human renal proximal tubular cell line with the standard LLC-PK1 cell line in studying drug-induced nephrotoxicity. Can J Physiol Pharmacol 2010;88(4):448-55. DOI: 10.1139/y10-023.Ramirez T., Strigun A., Verlohner A. et al. Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells. Arch Toxicol 2018;92(2):893–906. DOI: 10.1007/s00204-017-2079-6.Wolff J.E., Trilling T., Mölenkamp G. et al. Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 1999;125(8–9):481–6. DOI: 10.1007/s004320050305.Clark M.J., Homer N., O’Connor B.D. et al. U87MG decoded: the genomic sequence of a cytogenetically aberrant human cancer cell line. PLoS Genet 2010;6(1):e1000832. DOI: 10.1371/journal.pgen.1000832.Chittasupho C., Xie S.X., Baoum A. et al. ICAM-1 targeting of doxorubicin-loaded PLGA nanoparticles to lung epithelial cells. Eur J Pharm 2009;37(2):141–50. DOI: 10.1016/j.ejps.2009.02.008.Lundqvist M., Stigler J., Elia G. et al. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A 2008;105(38):14265–70. DOI: 10.1073/pnas.0805135105.Aggarwal P., Hall J.B., McLeland C.B. et al. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev 2009;61(6):428–37. DOI: 10.1016/j.addr.2009.03.009.Oslakovic C., Cedervall T., Linse S., Dahlbäck B. Polystyrene nanoparticles affecting blood coagulation. Nanomedicine 2012;8(6):981–6. DOI: 10.1016/j.nano.2011.12.001.Neun B.W., Dobrovolskaia M.A. Method for in vitro analysis of nanoparticle thrombogenic properties. Methods Mol Biol 2011;697:225–35. DOI: 10.1007/978-1-60327-198-1_24.Li X., Radomski A., Corrigan O.I. et al. Platelet compatibility of PLGA, chitosan and PLGA-chitosan nanoparticles. Nanomedicine (Lond) 2009;4(7): 735–46. DOI: 10.2217/nnm.09.65.Jackson S.P. The growing complexity of platelet aggregation. Blood 2007;109(12):5087–95. DOI: 10.1182/blood-2006-12-027698.Cenni E., Granchi D., Avnet S. et al. Biocompatibility of poly(D,L-lactideco-glycolide) nanoparticles conjugated with alendronate. Biomaterials 2008;29(10):1400–11. DOI: 10.1016/ j.biomaterials.2007.12.022.Merten M., Thiagarajan P. P-selectin expression on platelets determines size and stability of platelet aggregates. Circulation 2000;102(16):1931–6. DOI: 10.1161/01.CIR.102.16.1931.Bosi S., Feruglio L., da Ros T. et al. Hemolytic effects of water-soluble fullerene derivatives. J Med Chem 2004;47(27):6711–5. DOI: 10.1021/jm0497489.Bannunah A.M., Vllasaliu D., Lord J., Stolnik S. Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge. Mol Pharm 2014;11(12):4363–73. DOI: 10.1021/mp500439c.Kumar R., Kulkarni A., Nabulsi J. et al. Facile synthesis of PEGylated PLGA nanoparticles encapsulating doxorubicin and its in vitro evaluation as potent drug delivery vehicle. Drug Deliv Transl Res 2013; 3(4):299–308. DOI: 10.1007/s13346-012-0124-9.