Skip to main content

Encapsulation of Apoptotic Proteins in Lipid Nanoparticles to Induce Death of Cancer Cells


According to previous reports, cationic nanoparticles (cNPs) consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a cationic lipid, without any cargos, induce generation of reactive oxygen species (ROS) and toxicity in cells. We investigated the effect of DOTAP-based cNPs containing encapsulated human pro-apoptotic proteins (P-cNPs), tBid, Bax, or cytochrome c (Cyt c) on the death of HepG2, cancer cells. Upon the treatment to cells, cNPs containing Cyt c-cargo were the most effective in inducing cell death, followed by tBid- and Bax-cNPs. Among three P-cNPs, Cyt c-cNPs also induced the highest levels of ROS production and caspase-3 activity in the cells under the treatment with the same DOTAP concentration. The incorporation of dioleoylglycerol (DOG), a neutral phospholipid, in cNPs at the expense of DOTAP resulted in an increased amount of the encapsulated protein and consequently enhanced cell death in a DOG concentration-dependent manner. Thus, the present cNP formulation with protein cargo may elicit apoptotic effects in cancer cells and serve as a rational background for the preparation of cationic lipid-based NP formulation for cancer treatment.

This is a preview of subscription content, access via your institution.













glyceraldehyde 3-phosphate dehydrogenase


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


reactive oxygen species


  1. 1.

    Wang, Y., L. Miao, A. Satterlee, and L. Huang (2015) Delivery of oligonucleotides with lipid nanoparticles. Adv. Drug Deliv. Rev. 87: 68–80.

    Article  Google Scholar 

  2. 2.

    Kim, J. M., E. Shin, S. M. Ryou, J. H. Yeom, and K. Lee (2013) Gene delivery platforms. Biotechnol. Bioprocess Eng. 18: 637–647.

    CAS  Article  Google Scholar 

  3. 3.

    Liu, D., J. Hu, W. Qiao, Z. Li, S. Zhan, and L. Cheng (2005) Synthesis and characterization of a series of carbamate-linked cationic lipids for gene delivery. Lipids. 40: 839–848.

    CAS  Article  Google Scholar 

  4. 4.

    Podesta, J. E. and K. Kostarelos (2009) Engineering cationic liposome siRNA complexes for in vitro and in vivo delivery. Methods Enzymol. 464: 343–354.

    CAS  Article  Google Scholar 

  5. 5.

    Elmore, S. (2007) Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35: 495–516.

    CAS  Article  Google Scholar 

  6. 6.

    Kluck, R. M., E. Bossy-Wetzel, D. R. Green, and D. D. Newmeyer (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 275: 1132–1136.

    CAS  Article  Google Scholar 

  7. 7.

    Zhang, L., J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein (2000) Role of BAX in the apoptotic response to anticancer agents. Science. 290: 989–992.

    CAS  Article  Google Scholar 

  8. 8.

    Saxena, M., Y. Delgado, R. K. Sharma, S. Sharma, S. L. P. L. Guzmán, A. D. Tinoco, and K. Griebenow (2018) Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate. PLoS One. 13: e0195542.

    Google Scholar 

  9. 9.

    Méndez, J., M. Morales Cruz, Y. Delgado, C. M. Figueroa, E. A. Orellano, M. Morales, A. Monteagudo, and K. Griebenow (2014) Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells. Mol. Pharm. 11: 102–111.

    Article  Google Scholar 

  10. 10.

    Kim, S. K., M. B. Foote, and L. Huang (2012) The targeted intracellular delivery of cytochrome-C protein to tumors using lipid-apolipoprotein nanoparticles. Biomaterials. 33: 3959–3966.

    CAS  Article  Google Scholar 

  11. 11.

    Cho, E. Y., C.-H. Yun, and T. Ahn (2012) Effects of phospholipids on the functional regulation of tBID in membranes. Mol. Cell. Biochem. 363: 395–408.

    CAS  Article  Google Scholar 

  12. 12.

    Zhai, D., Q. Miao, X. Xin, and F. Yang (2001) Leakage and aggregation of phospholipid vesicles induced by the BH3-only Bcl-2 family member, BID. Eur. J. Biochem. 268: 48–55.

    CAS  Article  Google Scholar 

  13. 13.

    Szoka Jr, F. and D. Papahadjopoulos (1978) Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. USA. 75: 4194–4198.

    CAS  Article  Google Scholar 

  14. 14.

    Dooley, G. W. and C. K. Payne (2011) Cellular binding of nanoparticles in the presence of serum proteins. Chem. Commun. 47: 466–468.

    Article  Google Scholar 

  15. 15.

    Yang, Y., J. Cao, and Y. Shi (2004) Identification and characterization of a gene encoding human LPGAT1, an endoplasmic reticulum-associated lysophosphatidylglycerol acyltransferase. J. Biol. Chem. 279: 55866–55874.

    CAS  Article  Google Scholar 

  16. 16.

    Bhagwat, S. V., C. Vijayasarathy, H. Raza, J. Mullick, and N. G. Avadhani (1998) Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain. Biochem. Pharmacol. 56: 831–839.

    CAS  Article  Google Scholar 

  17. 17.

    Stern, I. and B. Shapiro (1953) A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood. J. Clin. Pathol. 6: 158–160.

    CAS  Article  Google Scholar 

  18. 18.

    Yun, C. H., C. S. Bae and T. Ahn (2016) Cargo-free nanoparticles containing cationic lipids induce reactive oxygen species and cell death in HepG2 cells. Biol. Pharm. Bull. 39: 1338–1346.

    CAS  Article  Google Scholar 

  19. 19.

    Fröhlich, E. (2012) The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomedicine. 7: 5577–5591.

    Article  Google Scholar 

  20. 20.

    Simon, H.-U., A. Haj-Yehia, and F. Levi-Schaffer (2000) Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 5: 415–418.

    CAS  Article  Google Scholar 

  21. 21.

    Porter, A. G. and R. U. Jänicke (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6: 99–104.

    CAS  Article  Google Scholar 

  22. 22.

    Ahn, T., Y. T. Chi, and C. H. Yun (2009) Effect of nonlamellar-prone lipids on protein encapsulation in liposome. Macromol. Res. 17: 956–962.

    CAS  Article  Google Scholar 

  23. 23.

    Battistuzzi, G., M. Borsari, D. Dallari, I. Lancellotti, and M. Sola (1996) Anion binding to mitochondrial cytochrome c studied through electrochemistry. Effects of the neutralization of surface charges on the redox potential. Eur. J. Biochem. 241: 208–214.

    CAS  Article  Google Scholar 

  24. 24.

    Bae, C. S. and T. Ahn (2018) Diacylglycerol in cationic nanoparticles stimulates oxidative stress-mediated death of cancer cells. Lipids. 53: 1059–1067.

    CAS  Article  Google Scholar 

  25. 25.

    Cowell, C. F., H. Döppler, I. K. Yan, A. Hausser, Y. Umezawa and P. Storz (2009) Mitochondrial diacylglycerol initiates protein-kinase D1-mediated ROS signaling. J. Cell Sci. 122: 919–928.

    CAS  Article  Google Scholar 

  26. 26.

    Redza-Dutordoir, M., and D. A. Averill-Bates (2016) Activation of apoptosis signaling pathways by reactive oxygen species. Biochim. Biophys. Acta. 1863: 2977–2992.

    CAS  Article  Google Scholar 

  27. 27.

    Buytaert, E., G. Callewaert, J. R. Vandenheede, and P. Agostinis (2006) Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum. Autophagy. 2: 238–240.

    CAS  Article  Google Scholar 

  28. 28.

    Wrobel, I. and D. Collins (1995) Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta. 1235: 296–304.

    Article  Google Scholar 

  29. 29.

    Basáñez, G., M. B. Ruiz-Argüello, A. Alonso, F. M. Goñi, G. Karlsson, and K. Edwards (1997) Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion. Biophys. J. 72: 2630–2637.

    Article  Google Scholar 

  30. 30.

    Mclntosh, T. J. (1996) Hydration properties of lamellar and non-lamellar phases of phosphatidylcholine and phosphatidyl-ethanolamine. Chem. Phys. Lipids. 81: 117–131.

    Article  Google Scholar 

Download references


This study was financially supported by Chonnam National University (Grant number: 2018-3390, C.-S. Bae) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03034829).

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

Author information



Corresponding author

Correspondence to Taeho Ahn.

Additional information

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bae, CS., Lee, CM. & Ahn, T. Encapsulation of Apoptotic Proteins in Lipid Nanoparticles to Induce Death of Cancer Cells. Biotechnol Bioproc E 25, 264–271 (2020).

Download citation


  • apoptosis
  • cell death
  • cytochrome c
  • nanoparticle
  • reactive oxygen species