Design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain

• Published in: Journal of controlled release Vol. 155; no. 2; pp. 218 - 226
• Main Authors: Sun, Guoyong, Hsueh, Pang-Yu, Janib, Siti M., Hamm-Alvarez, Sarah, Andrew MacKay, J.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Kidlington Elsevier B.V 30.10.2011; Elsevier
• Subjects: Acinar cells; Adenovirus; Adenoviruses, Human - chemistry; Animals; Biodegradability; Biological and medical sciences; Blotting, Western; capsid; Capsid Proteins - chemistry; Capsid Proteins - genetics; Capsids; Cell Culture Techniques; Cellular uptake; CHO Cells; composite polymers; Controlled release; Copolymers; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Coxsackievirus; Cricetinae; Cricetulus; drug carriers; Drug delivery; Drug Design; Drug development; Elastin - chemistry; Elastin - genetics; Elastin like polypeptides; Electrophoresis, Polyacrylamide Gel; Endocytosis; Escherichia coli - genetics; Fibers; Fusion protein; General pharmacology; genes; Genetic Engineering; HeLa Cells; Hepatocyte; Hepatocytes; Hepatocytes - metabolism; Humans; Knob; Light scattering; Lysosomes; Medical sciences; Mice; Microscopy, Fluorescence; Molecular Weight; Nanoparticles; Nanoparticles - chemistry; Peptides - chemistry; Peptides - genetics; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Phase transition; polypeptides; Protein Binding; Receptors, Virus - biosynthesis; Receptors, Virus - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Self; Serotypes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; temperature; Temperature effects; Transition Temperature; viral proteins; Nanoparticles; Cellular uptake; Elastin like polypeptides; Hepatocyte; Knob; Adenoviridae; Pharmaceutical technology; Nanoparticle; Control release polymer; Elastin; In vitro; Uptake; Aminoacid polymer; Protein; Virus; Design; Polypeptide; Internalization; Microparticle; Genetic engineering
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/j.jconrel.2011.06.010

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. [Display omitted]