Thymoquinone-loaded mesenchymal stem cell-derived exosome as an efficient nano-system against breast cancer cells

Document Type : Original Article

Authors

1 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

4 Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Objective(s): Exosomes became the subject of extensive research in drug delivery approach due to their potential applicability as therapeutic tools for cancer therapy. Thymoquinone (Tq) is an anti-cancer agent due to its great anti-proliferative effect. However, poor solubility and weak bioavailability restrict its therapeutic applications. In this study, exosomes secreted from human adipocyte-derived mesenchymal stem cells (AdMSCs) were isolated and the efficacy of a novel encapsulation method for loading of Tq was investigated. Finally, the cytotoxic effect of Tq incorporated exosomes against cancer cells was evaluated.
Materials and Methods: Exosomes secreted from AdMSCs were isolated via ultracentrifugation and characterized by electron microscopy and western blotting. Then, through a novel encapsulation approach, Tq was loaded into exosomes by the combination of three methods including incubation, freeze-thawing, and surfactant treatment. Then, the encapsulation efficiency, in vitro cellular uptake, and cytotoxicity of Tq incorporated exosomes (Tq@EXOs) in MCF7 and L929 cells were estimated. 
Results: Tq loading into exosomes through our novel method caused a significant improvement in encapsulation efficiency of about 60%. The fluorescent microscopy and flow cytometry outcomes indicated the efficient uptake of Tq@EXOs-FITC by cells throughout 4 hr. Furthermore, MTT results displayed the ability of Tq@EXOs in effectively decreasing the cell viability of MCF7 without causing any obvious cytotoxicity on L929 as normal cells.
Conclusion: The results suggest that our approach provides effective loading of Tq into exosomes which offer a valuable and safe platform for drug delivery to cancer cells thus having a great potential for clinical studies.  

Keywords


1.    Pullan JE, Confeld MI, Osborn JK, Kim J, Sarkar K, Mallik S. Exosomes as drug carriers for cancer therapy. Mol Pharm 2019; 5:1789-1798.
2.    Xia X, Wang Y, Huang Y, Zhang H, Lu H, Zheng JC. Exosomal miRNAs in central nervous system diseases: biomarkers, pathological mediators, protective factors and therapeutic agents. Prog Neurobiol 2019; 183:101694-101748.
3.    Théry C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, et al. Molecular characterization of dendritic cell-derived exosomes. selective accumulation of the heat shock protein hsc73. J Cell Biol 1999; 3:599-610.
4.    Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS. Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood 2007; 9:3234-3244.
5.    Clayton A, Turkes A, Dewitt S, Steadman R, Mason MD, Hallett MB. Adhesion and signaling by B cell-derived exosomes: the role of integrins. FASEB J 2004; 9:977-979.
6.    Nolte-’t Hoen EN, Buschow SI, Anderton SM, Stoorvogel W, Wauben MH. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1. Blood 2009; 9:1977-1981.
7.    Nikfarjam S, Rezaie J, Zolbanin NM, Jafari R. Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine. J Trans Med 2020; 1:449-469.
8.    Jia Y, Zhu Y, Qiu S, Xu J, Chai Y. Exosomes secreted by endothelial progenitor cells accelerate bone regeneration during distraction osteogenesis by stimulating angiogenesis. Stem Cell Res Ther 2019; 1:12-24.
9.    Skogberg G, Lundberg V, Berglund M, Gudmundsdottir J, Telemo E, Lindgren S, et al. Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens. Immunol Cell Biol 2015; 8:727-734.
10.    Osaki M, Okada F. Exosomes and their role in cancer progression. Yonago Acta Med 2019; 2:182-190.
11.    Das CK, Jena BC, Banerjee I. Exosome as a novel shuttle for delivery of therapeutics across biological barriers. Mol Pharm 2019; 1:24-40.
12.    He C, Zheng S, Luo Y, Wang B. Exosome theranostics: biology and translational medicine. Theranostics 2018; 1:237-255.
13.    Soltani F, Parhiz H, Mokhtarzadeh A, Ramezani M. Synthetic and biological vesicular nano-carriers designed for gene delivery. Curr Pharm Des 2015; 42:6214-6235.
14.    Lee ST, Im W, Ban JJ, Lee M, Jung KH, Lee SK, et al. Exosome-based delivery of miR-124 in a huntington’s disease model. J Mov Disord 2017; 1:45-52.
15.    Gilligan KE, Dwyer RM. Engineering exosomes for cancer therapy. Int J Mol Sci 2017; :6:1122-1133.
16.    Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 2014; 35:2383-2390.
17.    Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et al. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine 2016; 12:655-664.
18.    Osterman CJ, Lynch JC, Leaf P, Gonda A, Ferguson Bennit HR, Griffiths D, et al. Curcumin modulates pancreatic adenocarcinoma cell-derived exosomal function. PloS One 2015; 10:132845-132861.
19.    Vakhshiteh F, Atyabi F, Ostad SN. Mesenchymal stem cell exosomes: a two-edged sword in cancer therapy. Int J Nanomed 2019; 14:2847-2859.
20.    Imran M, Rauf A, Khan IA, Shahbaz M, Qaisrani TB, Fatmawati S, et al. Thymoquinone: a novel strategy to combat cancer: a review. Biomed Pharmacother 2018; 106:390-402.
21.    Asaduzzaman Khan M, Tania M, Fu S, Fu J. Thymoquinone, as an anticancer molecule: from basic research to clinical investigation. Oncotarget 2017; 8:51907-51919.
22.    Yimer EM, Tuem KB, Karim A, Ur-Rehman N, Anwar F. Nigella sativa L. (black cumin): a promising natural remedy for wide range of illnesses. Evidence Based Complement Altern Med 2019; :1528635-1528650.
23.    Patra JK, Das G, Fraceto LF, Campos EVR, Rodriguez-Torres MDP, Acosta-Torres LS, et al. Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnology 2018; 16:71-71.
24.    Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol 2015; 33:941-951.
25.    Azimifar MA, Salmasi Z, Doosti A, Babaei N, Hashemi M. Evaluation of the efficiency of modified PAMAM dendrimer with low molecular weight protamine peptide to deliver IL-12 plasmid into stem cells as cancer therapy vehicles. Biotechnol Prog 2021; 37:3175-3203.
26.    Mohammadi M, Alibolandi M, Abnous K, Salmasi Z, Jaafari MR, Ramezani M. Fabrication of hybrid scaffold based on hydroxyapatite-biodegradable nanofibers incorporated with liposomal formulation of BMP-2 peptide for bone tissue engineering. Nanomedicine 2018; 14:1987-1997.
27.    Salmasi Z, Hashemi M. Mesenchymal stem cells engineered by modified polyethylenimine polymer for targeted cancer gene therapy, in vitro and in vivo. Biotechnol Prog 2020; 36:3025-3054.
28. Jafari F, Emami SA, Javadi B, Salmasi Z, Tayarani-Najjaran M,  Tayarani-Najara Z. Inhibitory effect of saffron, crocin, crocetin, and safranal against adipocyte differentiation in human adipose-derived stem cells. J Ethnopharmacol 2022; 294:115340.
29. Adelipour M, Allameh AA, Sheikhi AK, Ranjbaran M, Naghashpour M, Nazeri Z, et al. Role of the mesenchymal stem cells derived from adipose tissue in changing the rate of breast cancer cell proliferation and autophagy, in vitro and in vivo. Iran J Basic Med Sci 2021;24:98-107.
30. Ebrahimian M, Shahgordi S, Yazdian-Robati R, Etemad L,  Hashemi M, Salmasi Z. Targeted delivery of galbanic acid to colon cancer cells by PLGA nanoparticles incorporated into human mesenchymal stem cells. J Phytomed 2022; 12:295-308.
31. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 2006; 3:1-29.
32. Jabarpour M, Siavashi V, Asadian S, Babaei H, Jafari SM, Nassiri SM. Hyperbilirubinemia-induced pro-angiogenic activity of infantile endothelial progenitor cells. Microvasc Res 2018; 118:49-56.
33. Mahdipour E, Salmasi Z, Sabeti N. Potential of stem cell-derived exosomes to regenerate β islets through Pdx-1 dependent mechanism in a rat model of type 1 diabetes. J Cell Physiol 2019; 234:20310-20321.
34.    Nooshabadi VT, Khanmohammadi M, Shafei S, Banafshe HR, Malekshahi ZV, Ebrahimi-Barough S, et al. Impact of atorvastatin loaded exosome as an anti-glioblastoma carrier to induce apoptosis of U87 cancer cells in 3D culture model. Biochem Biophys Rep 2020; 23:100792-100801.
35. Neamat Karimi , Fatemeh Soghra Karami Tehrani. Expression of SR-B1 receptor in breast cancer cell lines, MDAMB-468 and MCF-7: Effect on cell proliferation and apoptosis. Iran J Basic Med Sci 2021; 24:1069-1077.
36.    Salmasi Z, Mokhtarzadeh A, Hashemi M, Ebrahimian M, Farzad SA, Parhiz H, et al. Effective and safe in vivo gene delivery based on polyglutamic acid complexes with heterocyclic amine modified-polyethylenimine. Colloids Surf B Biointerfaces 2018; 172:790-796.
37.    Ebrahimian M, Hashemi M, Maleki M, Abnous K, Hashemitabar G, Ramezani M, et al. Induction of a balanced Th1/Th2 immune responses by co-delivery of PLGA/ovalbumin nanospheres and CpG ODNs/PEI-SWCNT nanoparticles as TLR9 agonist in BALB/c mice. Int J Pharmaceutics 2016; 515:708-720.
38.    Bunggulawa EJ, Wang W, Yin T, Wang N, Durkan C, Wang Y, et al. Recent advancements in the use of exosomes as drug delivery systems. J Nanobiotechnology 2018; 16:81-94.
39.    Muralikumar M, Manoj Jain S, Ganesan H, Duttaroy AK, Pathak S, Banerjee A. Current understanding of the mesenchymal stem cell-derived exosomes in cancer and aging. Biotechnol Rep 2021; 31:658-671.
40.    Hood JL, Scott MJ, Wickline SA. Maximizing exosome colloidal stability following electroporation. Anal Biochem 2014; 448:41-49.
41.    Oskouie MN, Aghili Moghaddam NS, Butler AE. Therapeutic use of curcumin-encapsulated and curcumin-primed exosomes. J Cell Physiol 2019; 234:8182-8191.
42.    Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 2017; 38:754–763.
43.    Fuhrmann G, Serio A, Mazo M, Nair R, Stevens MM. Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins. J Control Release 2015; 205:35-44.
44.    Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 2017; 38:754-763.
45.    Haney MJ, Klyachko NL, Zhao Y, Gupta R, Plotnikova EG, He Z, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release 2015; 207:18-30.
46.    Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacologica Sinica 2017; 38:754-763.
47.    Kooijmans SA, Vader P, Van Dommelen SM, Van Solinge WW, Schiffelers RM. Exosome mimetics: a novel class of drug delivery systems. Int J Nanomedicine 2012; 7:1525-1541.
48.    Simons M, Raposo G. Exosomes--vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009; 21:575-581.
49.    Xu M, Yang Q, Sun X, Wang Y. Recent advancements in the loading and modification of therapeutic exosomes. Front Bioeng Biotechnol 2020; 8:1173-1185.
50.    Salarpour S, Forootanfar H, Pournamdari M, Ahmadi-Zeidabadi M, Esmaeeli M, Pardakhty A. Paclitaxel incorporated exosomes derived from glioblastoma cells: comparative study of two loading techniques. DARU J Pharm Sci 2019; 27:533-539.
51.    Qu M, Lin Q, Huang L, Fu Y, Wang L, He S, et al. Dopamine-loaded blood exosomes targeted to brain for better treatment of Parkinson’s disease. J Control Release 2018; 287:156-166.
52.    Carobolante G, Mantaj J, Ferrari E. Cow milk and intestinal epithelial cell-derived extracellular vesicles as systems for enhancing oral drug delivery. Pharmaceutics 2020; 12:226-237.
53.    Zou X, Yuan M, Zhang T, Wei H, Xu S, Jiang N, et al. Extracellular vesicles expressing a single-chain variable fragment of an HIV-1 specific antibody selectively target Env(+) tissues. Theranostics 2019; 9:5657-5671.
54.    Bagheri E, Abnous K, Farzad SA, Taghdisi SM, Ramezani M, Alibolandi M. Targeted doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting against colorectal cancer. Life Sci 2020; 261:118369-118379.
55.    Smyth TJ, Redzic JS, Graner MW, Anchordoquy TJ. Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro. Biochim Biophys Acta 2014; 1838:2954-2965.
56.    Greco KA, Franzen CA, Foreman KE, Flanigan RC, Kuo PC, Gupta GN. PLK-1 silencing in bladder cancer by siRNA delivered with exosomes. Urology 2016; 91:241-247.