Elsevier

Biomaterials

Volume 33, Issue 33, November 2012, Pages 8714-8722
Biomaterials

Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles

https://doi.org/10.1016/j.biomaterials.2012.08.033Get rights and content

Abstract

Controlling the self-assembling of building blocks as nanoscale entities is a requisite for the generation of bio-inspired vehicles for nanomedicines. A wide spectrum of functional peptides has been incorporated to different types of nanoparticles for the delivery of conventional drugs and nucleic acids, enabling receptor-specific cell binding and internalization, endosomal escape, cytosolic trafficking, nuclear targeting and DNA condensation. However, the development of architectonic tags to induce the self-assembling of functionalized monomers has been essentially neglected. We have examined here the nanoscale architectonic capabilities of arginine-rich cationic peptides, that when displayed on His-tagged proteins, promote their self-assembling as monodisperse, protein-only nanoparticles. The scrutiny of the cross-molecular interactivity cooperatively conferred by poly-arginines and poly-histidines has identified regulatable electrostatic interactions between building blocks that can also be engineered to encapsulate cargo DNA. The combined use of cationic peptides and poly-histidine tags offers an unusually versatile approach for the tailored design and biofabrication of protein-based nano-therapeutics, beyond the more limited spectrum of possibilities so far offered by self-assembling amyloidogenic peptides.

Introduction

Viral capsid proteins self-assemble as complex, highly symmetric particles that act as natural cages for the cell-targeted delivery of their genomes. The tailored construction of virus-inspired complexes is a promising route to drug delivery [1], [2], [3], [4], [5], [6], [7]. Being devoid of any infectious material, “artificial viruses” [8] do not show the undesired biological side effects associated to administration of viruses in viral gene therapy [9]. In this context, virus-like particles (VLPs), microbial organelles, [10], multifunctional proteins [11] and a spectrum of diverse vesicular materials are under development as carriers for therapeutic nucleic acids or conventional drugs. Many functional peptides have been identified from nature or selected by directed molecular evolution as ligands for cell surface receptors, membrane-active peptides and nuclear localization signals [4], [5], [6], [12]. When conveniently pooled, these tag-mediated activities confer virus-like properties to the resulting multifunctional entities. In protein-only vehicles, all these domains can be covalently combined in single chain molecules that constitute the monomeric building blocks [11]. However, peptides enabling their holding proteins to organize as nanosized particles have so far been unidentified. The so-called self-assembling peptides, that might have been potentially promising for nanoparticle generation, are in general amyloidogenic protein segments that form fibers, membranes or hydrogels [13], [14]. When used in fusion proteins, these peptides induce protein aggregation [15], [16], being useless as tags for nanoparticle formation. Therefore, promoting the assembling of a selected protein as nanoparticles is so far excluded from rational engineering.

We have very recently described that a nine-arginine peptide (R9), when displayed on the surface of a recombinant, His-tagged EGFP, promotes the self-assembling of the whole fusion protein as regular nanoparticles of about 20 nm in diameter [17]. These constructs efficiently penetrate cultured mammalian cells by an endocytic pathway [18], cross the nuclear membrane, accumulate in the nucleus and allow the expression of a carried transgene [17]. The formation of these supramolecular complexes is completely distinguishable from unspecific protein aggregation [7], [19], [20]. Cationic peptides, including poly-arginines of different lengths, are well known by their membrane-crossing and DNA-condensation abilities, and widely used in gene therapy and more generally in drug delivery [5], [6], [12], [21]. However, if showing a general applicability, such a newly described architectonic ability would be specially promising for the easy engineering of protein nanoparticles formed by specific proteins with desired biological activities.

To explore the possibility of effectively controlling the assembly of protein nanoparticles, we have examined here the role of cationic peptides and poly-histidines as an architectonic tag pair. These agents, upon incorporated into monomeric building blocks, synergistically cooperate in promoting nanoparticle formation by balancing, in a regulatable way, protein–protein and protein–DNA interactions. The potential of the ‘nano-architectonic tag’ concept is discussed here in the context of the design of smart, protein-based particles by conventional genetic engineering.

Section snippets

Protein design and gene cloning

Several derivatives of R9–GFP–H6 containing decreasing numbers of arginine residues were constructed in house by site directed mutagenesis of the parental clone, by replacing these residues by glycines and alanines to keep the length of the peptide tag constant (Table 1). The new constructs R7–GFP–H6, R6–GFP–H6 and R3–GFP–H6 were efficiently produced in Escherichia coli Rosetta from the vector pET21b (Novagen 69744-3). Nine additional derivatives of GFP–H6 containing diverse amino terminal

Mapping the architectonic abilities of poly-arginines

While His-tagged GFP is exclusively found in a disassembled monomeric form (of around 5 nm), the addition of the cell-penetrating poly-arginine (R9) peptide at the amino terminus promoted the spontaneous organization of R9–GFP–H6 as building blocks of regularly sized nanoparticles of around 20 nm [17]. To map the architectonic properties of poly-arginines we constructed a series of arginine-based tags (Rn) with a decreasing number of arginine residues, to evaluate if they retained the ability to

Discussion

The construction of self-assembling protein-only nanoparticles from repetitive building blocks has been a rather neglected issue in nanomedicine, in contrast to the long run expertise accumulated in the fabrication of liposomes and polymeric particles with pre-defined nanoscale features [35], [36], [37], [38], [39]. Consequently, the current protein-based vehicles for drug delivery generated de novo include a catalog of rather amorphous entities [40] that are produced under no previous design.

Conclusion

The combination of a cationic peptide and a hexa-histidine tail fused to the amino and carboxy termini, respectively, of different proteins enable them to act as building blocks of self-assembling nanoparticles whose properties are regulatable by pH during particle formation. These vehicles are also able to condense and deliver expressible DNA into mammalian cells. The architectonic properties of the tag pair at the nanoscale are supported by electrostatic contacts, primarily driven by the

Acknowledgments

We appreciate the technical support of Fran Cortés from the Cell Culture Unit of Servei de Cultius Cel·lulars, Producció d’Anticossos i Citometria (SCAC), and from Servei de Microscòpia, both at the UAB, and the Protein Production Platform (PPP) of the CIBER de Bioingeniería, Biomateriales y Nanomedicina. We also acknowledge the financial support received for the design and production of artificial viruses for gene therapy to EV and AV from FISS (PS0900165), MINECO (ACI2009-0919), AGAUR (

References (81)

  • N.T. Huynh et al.

    Lipid nanocapsules: a new platform for nanomedicine

    Int J Pharm

    (2009)
  • C. Goldmann et al.

    Packaging of small molecules into VP1–virus–like particles of the human polyomavirus JC virus

    J Virol Methods

    (2000)
  • C.M. Malboeuf et al.

    Human papillomavirus-like particles mediate functional delivery of plasmid DNA to antigen presenting cells in vivo

    Vaccine

    (2007)
  • E. Rodriguez-Carmona et al.

    Nanostructured bacterial materials for innovative medicines

    Trends Microbiol

    (2010)
  • J.E. Straub et al.

    Principles governing oligomer formation in amyloidogenic peptides

    Curr Opin Struct Biol

    (2010)
  • E.R. Wright et al.

    Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences

    Adv Drug Deliv Rev

    (2002)
  • P.S. Satheshkumar et al.

    The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids

    J Mol Biol

    (2005)
  • D. Ejima et al.

    Arginine as an effective additive in gel permeation chromatography

    J Chromatogr A

    (2005)
  • M. Ferrari

    Frontiers in cancer nanomedicine: directing mass transport through biological barriers

    Trends Biotechnol

    (2010)
  • M. Uchida et al.

    Biological containers: protein cages as multifunctional nanoplatforms

    Adv Mater

    (2007)
  • E. Vazquez et al.

    Modular protein engineering in emerging cancer therapies

    Curr Pharm Des

    (2009)
  • E. Mastrobattista et al.

    Artificial viruses: a nanotechnological approach to gene delivery

    Nat Rev Drug Discov

    (2006)
  • M.L. Edelstein et al.

    Gene therapy clinical trials worldwide to 2007 – an update

    J Gene Med

    (2007)
  • J.L. Corchero et al.

    Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

    Microb Cell Fact

    (2011)
  • S. Koutsopoulos et al.

    Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold

    Proc Natl Acad Sci U S A

    (2009)
  • S. Zhang et al.

    Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane

    Proc Natl Acad Sci U S A

    (1993)
  • W. Wu et al.

    Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Microb Cell Fact

    (2011)
  • B. Zhou et al.

    Small surfactant-like peptides can drive soluble proteins into active aggregates

    Microb Cell Fact

    (2012)
  • E. Vazquez et al.

    Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide

    Nanomedicine (Lond)

    (2010)
  • E. Vazquez et al.

    Post-production protein stability: trouble beyond the cell factory

    Microb Cell Fact

    (2011)
  • M. Martinez-Alonso et al.

    Learning about protein solubility from bacterial inclusion bodies

    Microb Cell Fact

    (2009)
  • P. Kumar et al.

    Transvascular delivery of small interfering RNA to the central nervous system

    Nature

    (2007)
  • T.J. Dolinsky et al.

    PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations

    Nucleic Acids Res

    (2007)
  • L. Cavallo et al.

    POPS: a fast algorithm for solvent accessible surface areas at atomic and residue level

    Nucleic Acids Res

    (2003)
  • N. Eswar et al.

    Protein structure modeling with MODELLER

    Methods Mol Biol

    (2008)
  • L.A. Kelley et al.

    Protein structure prediction on the Web: a case study using the Phyre server

    Nat Protoc

    (2009)
  • N. Guex et al.

    SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling

    Electrophoresis

    (1997)
  • L. Willard et al.

    VADAR: a web server for quantitative evaluation of protein structure quality

    Nucleic Acids Res

    (2003)
  • C. Dominguez et al.

    HADDOCK: a protein–protein docking approach based on biochemical or biophysical information

    J Am Chem Soc

    (2003)
  • J.C. Phillips et al.

    Scalable molecular dynamics with NAMD

    J Comput Chem

    (2005)
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