Advertisement

Cellular and Molecular Bioengineering

, Volume 10, Issue 5, pp 387–403 | Cite as

Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm

  • Zachary I. Imam
  • Laura E. Kenyon
  • Grant Ashby
  • Fatema Nagib
  • Morgan Mendicino
  • Chi Zhao
  • Avinash K. Gadok
  • Jeanne C. Stachowiak
Article

Abstract

Introduction

From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers.

Objectives

Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface.

Methods

We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry.

Results

Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least four-fold in comparison to homogenous liposomes.

Conclusions

Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

Keywords

DOTAP Fusion Transmembrane delivery Membrane Biophysics Biomaterials 

Abbreviations

DPPC

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

DOPC

1,2 Dioleoyl-sn-glycero-3-phosphocholine

DOTAP

1,2 Dioleoyl–3-trimethylammonium-propane

PEG2000-DPPE

1,2 Dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]

Texas Red-DPPE

Texas Red-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine

Oregon Green-DPPE

Oregon Green-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine

mol%

Molar fraction

GUV

Giant unilamellar vesicle

SUV

Small unilamellar vesicle

Rhodamine B-dextran

Rhodamine B isothiocyanate-dextran average molecular weight of 10,000

TRITC-dextran

Tetramethylrhodamine isothiocyanate-dextran average molecular weight of 20,000

Notes

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation Division of Materials Research (DMR 1352487 to Stachowiak) and also National Institute of General Medical Science (Grant No. GM112065). We thank the BME Community of Undergraduate Research Scholars for Cancer (BME CUReS Cancer) an NSF sponsored Research Experience for Undergraduates (REU) at The University of Texas at Austin for enabling Grant Ashby to work in the Stachowiak Laboratory at UT Austin. We thank the laboratories of Professors Aaron Baker and Janet Zoldan for assistance with lentiviral transfection.

CONFLICT OF INTEREST

All authors, including Z. I. Imam, L. E Kenyon, G. Ashby, F. Nagib, M. Mendicino, C. Zhao, A. K. Gadok, and J. C. Stachowiak, declare that they have no conflict of interest.

ETHICAL APPROVAL

No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

Supplementary material

12195_2017_489_MOESM1_ESM.docx (2.5 mb)
Supplementary material 1 (DOCX 2517 kb).

References

  1. 1.
    Alvarez-Erviti, L., Y. Q. Seow, H. F. Yin, C. Betts, S. Lakhal, and M. J. A. Wood. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29:341–345, 2011.CrossRefGoogle Scholar
  2. 2.
    Angelova, M. I., and D. S. Dimitrov. Liposome Electroformation. Faraday Discuss. 81:303–311, 1986.CrossRefGoogle Scholar
  3. 3.
    Baker, A. H., A. Kritz, L. M. Work, S. A. Nicklin, and A. Nicklin. Cell-selective viral gene delivery vectors for the vasculature. Exp. Physiol. 90:27–31, 2005.CrossRefGoogle Scholar
  4. 4.
    Batrakova, E. V., and M. S. Kim. Using exosomes, naturally-equipped nanocarriers, for drug delivery. J. Controll. Release 219:396–405, 2015.CrossRefGoogle Scholar
  5. 5.
    Blosser, M. C., J. B. Starr, C. W. Turtle, J. Ashcraft, and S. L. Keller. Minimal effect of lipid charge on membrane miscibility phase behavior in three ternary systems. Biophys. J. 104:2629–2638, 2013.CrossRefGoogle Scholar
  6. 6.
    Charras, G. T., M. Coughlin, T. J. Mitchison, and L. Mahadevan. Life and times of a cellular bleb. Biophys. J. 94:1836–1853, 2008.CrossRefGoogle Scholar
  7. 7.
    Charras, G. T., C. K. Hu, M. Coughlin, and T. J. Mitchison. Reassembly of contractile actin cortex in cell blebs. J. Cell Biol. 175:477–490, 2006.CrossRefGoogle Scholar
  8. 8.
    Chazal, N., and D. Gerlier. Virus entry, assembly, budding, and membrane rafts. Microbiol. Mol. Biol. Rev. 67:226–237, 2003.CrossRefGoogle Scholar
  9. 9.
    Choi, K. S., H. Aizaki, and M. M. C. Lai. Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release. J. Virol. 79:9862–9871, 2005.CrossRefGoogle Scholar
  10. 10.
    Chollet, P., M. C. Favrot, A. Hurbin, and J. L. Coll. Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. J. Gene Med. 4:84–91, 2002.CrossRefGoogle Scholar
  11. 11.
    Ciani, L., A. Casini, C. Gabbiani, S. Ristori, L. Messori, and G. Martini. DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery studied by circular dichroism and other biophysical techniques. Biophys. Chem. 127:213–220, 2007.CrossRefGoogle Scholar
  12. 12.
    Ciani, L., S. Ristori, L. Calamai, and G. Martini. DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra. Biochim. Biophys. Acta Biomembr. 1664:70–79, 2004.CrossRefGoogle Scholar
  13. 13.
    Egleton, R. D., and T. P. Davis. Bioavailability and transport of peptides and peptide drugs into the brain. Peptides 18:1431–1439, 1997.CrossRefGoogle Scholar
  14. 14.
    Filion, M. C., and N. C. Phillips. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim. Biophys. Acta Biomembr. 1329:345–356, 1997.CrossRefGoogle Scholar
  15. 15.
    Friedl, P., and K. Wolf. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat. Rev. Cancer 3:362–374, 2003.CrossRefGoogle Scholar
  16. 16.
    Futami, J., M. Kitazoe, T. Maeda, E. Nukui, M. Sakaguchi, J. Kosaka, M. Miyazaki, M. Kosaka, H. Tada, M. Seno, Y. Sasaki, N. H. Huh, M. Namba, and H. Yamada. Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization. J. Biosci. Bioeng. 99:95–103, 2005.CrossRefGoogle Scholar
  17. 17.
    Gibbs, J. B. Mechanism-based target identification and drug discovery in cancer research. Science 287:1969–1973, 2000.CrossRefGoogle Scholar
  18. 18.
    Gordon, V. D., M. Deserno, C. M. J. Andrew, S. U. Egelhaaf, and W. C. K. Poon. Adhesion promotes phase separation in mixed-lipid membranes. EPL 84:48003, 2008.CrossRefGoogle Scholar
  19. 19.
    Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, and M. L. Dustin. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227, 1999.CrossRefGoogle Scholar
  20. 20.
    Gunawan, R. C., and D. T. Auguste. Immunoliposomes that target endothelium in vitro are dependent on lipid raft formation. Mol. Pharm. 7:1569–1575, 2010.CrossRefGoogle Scholar
  21. 21.
    Gunawan, R. C., and D. T. Auguste. The role of antibody synergy and membrane fluidity in the vascular targeting of immunoliposomes. Biomaterials 31:900–907, 2010.CrossRefGoogle Scholar
  22. 22.
    Hac, A. E., H. M. Seeger, M. Fidorra, and T. Heimburg. Diffusion in two-component lipid membranes—a fluorescence correlation spectroscopy and Monte Carlo simulation study. Biophys. J. 88:317–333, 2005.CrossRefGoogle Scholar
  23. 23.
    Heberle, F. A., and G. W. Feigenson. Phase Separation in Lipid Membranes. Cold Spring Harb. Perspect. Biol. 3:a004630, 2011.CrossRefGoogle Scholar
  24. 24.
    Hood, J. L., M. J. Scott, and S. A. Wickline. Maximizing exosome colloidal stability following electroporation. Anal. Biochem. 448:41–49, 2014.CrossRefGoogle Scholar
  25. 25.
    Imam, Z. I., L. E. Kenyon, A. Carrillo, I. Espinoza, F. Nagib, and J. C. Stachowiak. Steric pressure among membrane-bound polymers opposes lipid phase separation. Langmuir 32:3774–3784, 2016.CrossRefGoogle Scholar
  26. 26.
    Immordino, M. L., F. Dosio, and L. Cattel. Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int. J. Nanomed. 1:297–315, 2006.CrossRefGoogle Scholar
  27. 27.
    Kim, S. K., M. B. Foote, and L. Huang. The targeted intracellular delivery of cytochrome C protein to tumors using lipid-apolipoprotein nanoparticles. Biomaterials 33:3959–3966, 2012.CrossRefGoogle Scholar
  28. 28.
    King, J. E., E. A. Eugenin, C. M. Buckner, and J. W. Berman. HIV tat and neurotoxicity. Microbes Infect. 8:1347–1357, 2006.CrossRefGoogle Scholar
  29. 29.
    Lechardeur, D., K. J. Sohn, M. Haardt, P. B. Joshi, M. Monck, R. W. Graham, B. Beatty, J. Squire, H. O’Brodovich, and G. L. Lukacs. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6:482–497, 1999.CrossRefGoogle Scholar
  30. 30.
    Lee, H., J. H. Jeong, and T. G. Park. PEG grafted polylysine with fusogenic peptide for gene delivery: high transfection efficiency with low cytotoxicity. J. Controll. Release 79:283–291, 2002.CrossRefGoogle Scholar
  31. 31.
    Li, W., A. Asokan, Z. Wu, T. Van Dyke, N. DiPrimio, J. S. Johnson, L. Govindaswamy, M. Agbandje-McKenna, S. Leichtle, D. E. Redmond, T. J. McCown, K. B. Petermann, N. E. Sharpless, and R. J. Samulski. Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Mol. Ther. 16:1252–1260, 2008.CrossRefGoogle Scholar
  32. 32.
    Li, S., and N. Malmstadt. Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter 9:4969–4976, 2013.CrossRefGoogle Scholar
  33. 33.
    Mae, M., and U. Langel. Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery. Curr. Opin. Pharmacol. 6:509–514, 2006.CrossRefGoogle Scholar
  34. 34.
    Mansourian, M., A. Badiee, S. A. Jalali, S. Shariat, M. Yazdani, M. Amin, and M. R. Jaafari. Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN. Immunol. Lett. 162:87–93, 2014.CrossRefGoogle Scholar
  35. 35.
    Mills, J. C., N. L. Stone, and R. N. Pittman. Extranuclear apoptosis: the role of the cytoplasm in the execution phase. J. Cell Biol. 146:703–707, 1999.CrossRefGoogle Scholar
  36. 36.
    Moghimi, S. M., P. Symonds, J. C. Murray, A. C. Hunter, G. Debska, and A. Szewczyk. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther. 11:990–995, 2005.CrossRefGoogle Scholar
  37. 37.
    Momin, N., S. Lee, A. K. Gadok, D. J. Busch, G. D. Bachand, C. C. Hayden, J. C. Stachowiak, and D. Y. Sasaki. Designing lipids for selective partitioning into liquid ordered membrane domains. Soft Matter 11:3241–3250, 2015.CrossRefGoogle Scholar
  38. 38.
    Morille, M., C. Passirani, A. Vonarbourg, A. Clavreul, and J. P. Benoit. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials 29:3477–3496, 2008.CrossRefGoogle Scholar
  39. 39.
    Murthy, N., J. Campbell, N. Fausto, A. S. Hoffman, and P. S. Stayton. Design and synthesis of pH-responsive polymeric carriers that target uptake and enhance the intracellular delivery of oligonucleotides. J. Controll. Release 89:365–374, 2003.CrossRefGoogle Scholar
  40. 40.
    Paluch, E., C. Sykes, J. Prost, and M. Bornens. Dynamic modes of the cortical actomyosin gel during cell locomotion and division. Trends Cell Biol. 16:5–10, 2006.CrossRefGoogle Scholar
  41. 41.
    Parker, J. N., G. Y. Gillespie, C. E. Love, S. Randall, R. J. Whitley, and J. M. Markert. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc. Natl. Acad. Sci. USA 97:2208–2213, 2000.CrossRefGoogle Scholar
  42. 42.
    Pecheur, E. I., and D. Hoekstra. Peptide-induced fusion of liposomes. Methods Mol. Biol. 199:31–48, 2002.Google Scholar
  43. 43.
    Pires, P., S. Simoes, S. Nir, R. Gaspar, N. Duzgunes, and M. C. P. de Lima. Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. Biochim. Biophys. Acta Biomembr. 1418:71–84, 1999.CrossRefGoogle Scholar
  44. 44.
    Rawle, R. J., B. van Lengerich, M. Chung, P. M. Bendix, and S. G. Boxer. Vesicle fusion observed by content transfer across a tethered lipid bilayer. Biophys. J. 101:L37–L39, 2011.CrossRefGoogle Scholar
  45. 45.
    Regelin, A. E., S. Fankhaenel, L. Gurtesch, C. Prinz, G. von Kiedrowski, and U. Massing. Biophysical and lipofection studies of DOTAP analogs. Biochim. Biophys. Acta Biomembr. 1464:151–164, 2000.CrossRefGoogle Scholar
  46. 46.
    Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. L. Yang, J. Kopinja, M. D. Zhang, M. T. McManus, F. B. Gertler, M. L. Scott, and L. Van Parijs. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33:401–406, 2003.CrossRefGoogle Scholar
  47. 47.
    Sezgin, E., H. J. Kaiser, T. Baumgart, P. Schwille, K. Simons, and I. Levental. Elucidating membrane structure and protein behavior using giant plasma membrane vesicles. Nat Protoc. 7:1042–1051, 2012.CrossRefGoogle Scholar
  48. 48.
    Skaug, M. J., M. L. Longo, and R. Faller. the impact of texas red on lipid bilayer properties. J. Phys. Chem. B 115:8500–8505, 2011.CrossRefGoogle Scholar
  49. 49.
    Sollner, T., S. W. Whitehart, M. Brunner, H. Erdjumentbromage, S. Geromanos, P. Tempst, and J. E. Rothman. Snap receptors implicated in vesicle targeting and fusion. Nature 362:318–324, 1993.CrossRefGoogle Scholar
  50. 50.
    Tu, C. Y., L. Santo, Y. Mishima, N. Raje, Z. Smilansky, and J. Zoldan. Monitoring protein synthesis in single live cancer cells. Integr. Biol. 8:645–653, 2016.CrossRefGoogle Scholar
  51. 51.
    van Dommelen, S. M., P. Vader, S. Lakhal, S. A. A. Kooijmans, W. W. van Solinge, M. J. A. Wood, and R. M. Schiffelers. Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J. Controll. Release 161:635–644, 2012.CrossRefGoogle Scholar
  52. 52.
    Veatch, S. L., and S. L. Keller. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 85:3074–3083, 2003.CrossRefGoogle Scholar
  53. 53.
    Veatch, S. L., and S. L. Keller. Seeing spots: complex phase behavior in simple membranes. Biochim. Biophys. Acta Mol. Cell Res. 1746:172–185, 2005.CrossRefGoogle Scholar
  54. 54.
    White, S. J., S. A. Nicklin, H. Buning, M. J. Brosnan, K. Leike, E. D. Papadakis, M. Hallek, and A. H. Baker. Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors. Circulation 109:513–519, 2004.CrossRefGoogle Scholar
  55. 55.
    Wieber, A., T. Selzer, and J. Kreuter. Physico-chemical characterisation of cationic DOTAP liposomes as drug delivery system for a hydrophilic decapeptide before and after freeze-drying. Eur. J. Pharm. Biopharm. 80:358–367, 2012.CrossRefGoogle Scholar
  56. 56.
    Xu, Y. H., S. W. Hui, P. Frederik, and F. C. Szoka. Physicochemical characterization and purification of cationic lipoplexes. Biophys. J. 77:341–353, 1999.CrossRefGoogle Scholar
  57. 57.
    Yamazaki, Y., M. Nango, M. Matsuura, Y. Hasegawa, M. Hasegawa, and N. Oku. Polycation liposomes, a novel nonviral gene transfer system, constructed from cetylated polyethylenimine. Gene Ther. 7:1148–1155, 2000.CrossRefGoogle Scholar
  58. 58.
    Yang, S. T., E. Zaitseva, L. V. Chernomordik, and K. Melikov. Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid. Biophys. J. 99:2525–2533, 2010.CrossRefGoogle Scholar
  59. 59.
    Young, L. S., P. F. Searle, D. Onion, and V. Mautner. Viral gene therapy strategies: from basic science to clinical application. J. Pathol. 208:299–318, 2006.CrossRefGoogle Scholar
  60. 60.
    Zhao, J., J. Wu, and S. L. Veatch. Adhesion stabilizes robust lipid heterogeneity in supercritical membranes at physiological temperature. Biophys. J. 104:825–834, 2013.CrossRefGoogle Scholar
  61. 61.
    Zhu, S. J., D. S. P. Lansakara-P, X. R. Li, and Z. R. Cui. Lysosomal delivery of a lipophilic gemcitabine prodrug using novel acid-sensitive micelles improved its antitumor activity. Bioconjugate Chem. 23:966–980, 2012.CrossRefGoogle Scholar
  62. 62.
    Zhu, S. J., M. M. Niu, H. O’Mary, and Z. R. Cui. Targeting of tumor-associated macrophages made possible by peg-sheddable, mannose-modified nanoparticles. Mol. Pharm. 10:3525–3530, 2013.CrossRefGoogle Scholar
  63. 63.
    Zhu, S. J., P. Wonganan, D. S. P. Lansakara-P, H. L. O’Mary, Y. Li, and Z. R. Cui. The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression. Biomaterials 34:2327–2339, 2013.CrossRefGoogle Scholar
  64. 64.
    Zimmerberg, J., and M. M. Kozlov. How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7:9–19, 2006.CrossRefGoogle Scholar
  65. 65.
    Zuidam, N. J., and Y. Barenholz. Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim. Biophys. Acta Biomembr. 1329:211–222, 1997.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  • Zachary I. Imam
    • 1
  • Laura E. Kenyon
    • 1
  • Grant Ashby
    • 1
  • Fatema Nagib
    • 1
  • Morgan Mendicino
    • 1
  • Chi Zhao
    • 1
  • Avinash K. Gadok
    • 1
  • Jeanne C. Stachowiak
    • 1
  1. 1.Department of Biomedical EngineeringThe University of Texas at AustinAustinUSA

Personalised recommendations