title: Design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain.
abstract: Hepatocytes and acinar cells exhibit high-efficiency, fiber-dependent internalization of adenovirus; however, viral capsids have unpredictable immunological effects and are challenging to develop into targeted drug carriers. To exploit this internalization pathway and minimize the use of viral proteins, we developed a simple gene product that self assembles nanoparticles decorated with the knob domain of adenovirus serotype 5 fiber protein. The most significant advantages of this platform include: (i) compatibility with genetic engineering; (ii) no bioconjugate chemistry is required to link fusion proteins to the nanoparticle surface; and (iii) it can direct the reversible assembly of large nanoparticles, which are monodisperse, multivalent, and biodegradable. These particles are predominantly composed from diblock copolymers of elastin-like polypeptide (ELP). ELPs have unique phase transition behavior, whereby they self-assemble above a transition temperature that is simple to control. The diblock ELP described contains two motifs with distinct transition temperatures, which assemble nanoparticles at physiological temperatures. Analysis by non-denaturing-PAGE demonstrated that the purified knob-ELP formed trimers or dimers, which is a property of the native knob/fiber protein. Dynamic light scattering indicated that the diblock copolymer, with or without knob, is able to self assemble into nanoparticles ~40 nm in diameter. To examine the functionality of knob-ELP, their uptake was assessed in a hepatocyte cell-line that expresses the receptor for adenovirus serotype 5 fiber and knob, the coxsackievirus and adenovirus receptor (CAR). Both plain ELP and knob-ELP were bound to the outside of hepatocytes; however, the knob-ELP fusion protein exhibits more internalization and localization to lysosomes of hepatocytes. These findings suggest that functional fusion proteins may only minimally influence the assembly temperature and diameter of ELP nanoparticles. These results are a proof-of-principal that large fusion proteins (>10 kDa) can be assembled by diblock ELPs without the need for bioconjugate chemistry, which greatly simplifies the design and evaluation of targeted drug carriers.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, United States.
authors.forename: Guoyong
authors.initials: G
authors.lastname: Sun
authors.forename: Pang-Yu
authors.initials: PY
authors.lastname: Hsueh
authors.forename: Siti M
authors.initials: SM
authors.lastname: Janib
authors.forename: Sarah
authors.initials: S
authors.lastname: Hamm-Alvarez
authors.forename: J Andrew
authors.initials: JA
authors.lastname: MacKay
mesh_terms: D000260:Adenoviruses, Human / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics; D018929:Cell Culture Techniques; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D006224:Cricetinae; D003412:Cricetulus; D015195:Drug Design; D004549:Elastin / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D006367:HeLa Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D053758:Nanoparticles / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011991:Receptors, Virus / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D044366:Transition Temperature
chemical_list: C568771:CLMP protein, human; C568772:CLMP protein, mouse; D036022:Capsid Proteins; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D010455:Peptides; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C018315:hexon capsid protein, Adenovirus; D004549:Elastin