Controlled release and targeting of polypeptide-deposited liposomes by enzymatic degradation

Article metrics


We prepared biobased nanocapsules with enzymatic degradability, which were generated by the layer-by-layer deposition of enzymes and polypeptide over the liposomal surface. Here, we demonstrate two different systems based on the enzymatic degradation of polymer layers. First, the deposition of trypsin and polyarginine (PArg), which is cleavable by trypsin, was carried out over a negatively charged liposome. The enzymatic cleavage of PArg resulted in exposure of the lipid membrane, which facilitated release of the cargo. Next, we attempted to degrade the outer polymer layer of the multilayered capsule wall to display the inner polymer layer by enzymatic degradation. This approach enabled the accumulation and targeting of the nanocapsules through the affinity between the displayed polymer layer and the target hydroxyapatite (HAp). The polymer wall was constructed with an inner layer consisting of poly-L-glutamic acid (PGlu) and an outer layer consisting of trypsin and PArg onto the liposome. The degradation of the outer PArg by trypsin allowed the surface to display the inner PGlu, which has bone-targeting ability. In addition, the polymer wall was constructed from an inner layer of PArg and an outer layer of pepsin and PGlu. The degradation of the outer PGlu by pepsin led to inner PArg on the surface to achieve cell-penetrating activity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Musyanovych A, Landfester K. Polymer micro and nanocapsules as biological carriers with multifunctional properties. Macromol Biosci. 2014;14:458–77.

  2. 2.

    van Dongen SFM, de Hoog HPM, Peters R, Nallani M, Nolte RJM, van Hest JCM. Biohybrid polymer capsules. Chem Rev. 2009;109:6212–74.

  3. 3.

    Ringsdorf H, Schlarb B, Venzmer J. Molecular Architecture and function of polymeric oriented systems—models for the study of organization, surface recognition, and dynamics of biomembranes. Angew Chem-Int Ed 1988;27:113–58.

  4. 4.

    Ruysschaert T, Germain M, Gomes J, Fournier D, Sukhorukov GB, Meier W, et al. Liposome-based nanocapsules. IEEE Trans Nanobiosci. 2004;3:49–55.

  5. 5.

    Bronich TK, Solomatin SV, Yaroslavov AA, Eisenberg A, Kabanov VA, Kabanov AV. Steric stabilization of negatively charged liposomes by cationic graft copolymer. Langmuir. 2000;16:4877–81.

  6. 6.

    Takeuchi H, Matsui Y, Yamamoto H, Kawashima Y. Mucoadhesive properties of carbopol or chitosan-coated liposomes and their effectiveness in the oral administration of calcitonin to rats. J Control Release. 2003;86:235–42.

  7. 7.

    Takeuchi H, Yamamoto H, Niwa T, Hino T, Kawashima Y. Enteral absorption of insulin in rats from mucoadhesive chitosan-coated liposomes. Pharm Res. 1996;13:896–901.

  8. 8.

    Thongborisute J, Tsuruta A, Kawabata Y, Takeuchi H. The effect of particle structure of chitosan-coated liposomes and type of chitosan on oral delivery of calcitonin. J Drug Target. 2006;14:147–54.

  9. 9.

    Maruyama K. Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Adv Drug Deliv Rev. 2011;63:161–9.

  10. 10.

    Anderson VC, Thompson DH. Triggered release of hydrophilic agents from plasmalogen liposomes using visible-light or acid. Biochim Biophys Acta. 1992;1109:33–42.

  11. 11.

    Frankel DA, Lamparski H, Liman U, Obrien DF. Photoinduced destablization of bilayer vesicles. JACS. 1989;111:9262–3.

  12. 12.

    Kono K. Thermosensitive polymer-modified liposomes. Adv Drug Deliv Rev. 2001;53:307–19.

  13. 13.

    Maeda T, Fujimoto K. A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating. Colloids Surf B-Biointerfaces 2006;49:15–21.

  14. 14.

    Decher G. Fuzzy nanoassemblies: Toward layered polymeric multicomposites. Science. 1997;277:1232–7.

  15. 15.

    Caruso F. Hollow capsule processing through colloidal templating and self-assembly. Chem-a Eur J 2000;6:413–9.

  16. 16.

    Caruso F, Caruso RA, Mohwald H. Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating. Science. 1998;282:1111–4.

  17. 17.

    Antipov AA, Sukhorukov GB, Donath E, Mohwald H. Sustained release properties of polyelectrolyte multilayer capsules. J Phys Chem B. 2001;105:2281–4.

  18. 18.

    Diaspro A, Silvano D, Krol S, Cavalleri O, Gliozzi A. Single living cell encapsulation in nano-organized polyelectrolyte shells. Langmuir. 2002;18:5047–50.

  19. 19.

    Fujimoto K, Toyoda T, Fukui Y. Preparation of bionanocapsules by the layer-by-layer deposition of polypeptides onto a liposome. Macromolecules. 2007;40:5122–8.

  20. 20.

    Fukui Y. Preparation of liponanocapsules via construction of bio-derived capsule wall on a liposomal template. Kobunshi Ronbunshu. 2017;74:396–409.

  21. 21.

    Fukui Y, Fujimoto K. The Preparation of sugar polymer-coated nanocapsules by the Layer-by-Layer deposition on the liposome. Langmuir. 2009;25:10020–5.

  22. 22.

    Hu XR, Feeney MJ, McIntosh E, Mullahoo J, Jia F, Xu QB, et al. Triggered release of encapsulated cargo from photoresponsive polyelectrolyte nanocomplexes. Acs Appl Mater Interfaces 2016;8:23517–22.

  23. 23.

    Itoh Y, Matsusaki M, Kida T, Akashi M. Enzyme-responsive release of encapsulated proteins from biodegradable hollow capsules. Biomacromolecules 2006;7:2715–8.

  24. 24.

    Wang D, Miller SC, Kopeckova P, Kopecek J. Bone-targeting macromolecular therapeutics. Adv Drug Deliv Rev. 2005;57:1049–76.

  25. 25.

    Fujisawa R, Wada Y, Nodasaka Y, Kuboki Y. Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals. Biochim Et Biophys Acta-Protein Struct Mol Enzymol 1996;1292:53–60.

  26. 26.

    Yamamoto S, Fukui Y, Kaihara S, Fujimoto K. Preparation and assembly of poly(arginine)-coated liposomes to create a free-sanding bioscaffold. Langmuir. 2011;27:9576–82.

  27. 27.

    Chiu K, Agoubi LL, Lee I, Limpar MT, Lowe JW, Goh SL. Effects of polymer molecular weight on the size, activity, and stability of PEG-functionalized trypsin. Biomacromolecules 2010;11:3688–92.

  28. 28.

    Durchschlag H, Zipper P Calculation of hydrodynamic parameters of biopolymers from scattering data using whole body approaches. In: Jaenicke R, Durchschlag H, editors. Analytical ultracentrifugation Iv. Progress in colloid and polymer science. Regensburg: Steinkopff Darmstadt; 1997. p. 43–57.

  29. 29.

    Ndou TT, Vonwandruszka R. Pyrene fluorescence in premicellar solutions - the effects of solvents and temperature. J Lumin. 1990;46:33–8.

  30. 30.

    Drin G, Cottin S, Blanc E, Rees AR, Temsamani J. Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem. 2003;278:31192–201.

  31. 31.

    Sakai N, Matile S. Anion-mediated transfer of polyarginine across liquid and bilayer membranes. JACS. 2003;125:14348–56.

  32. 32.

    Vives E, Schmidt J, Pelegrin A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochim Et Biophys Acta-Rev Cancer 2008;1786:126–38.

Download references

Author information

Correspondence to Keiji Fujimoto.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark