Article
Coating nanoparticles with plant-produced transferrinhydrophobin fusion protein enhances their uptake in cancer cells Lauri Johannes Reuter, Mohammad Ali Shahbazi, Ermei M. Mäkilä, Jarno J. Salonen, Reza Saberianfar, Rima Menassa, Hélder A. Santos, Jussi Joonas Joensuu, and Anneli Ritala Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
Coating nanoparticles with plant-produced transferrin-hydrophobin fusion protein enhances their uptake in cancer cells AUTHORS Lauri J. Reuter1, Mohammad-Ali Shahbazi2,3, Ermei M. Mäkilä4, Jarno J. Salonen4, Reza Saberianfar5, Rima Menassa5, Hélder A. Santos2,6, Jussi J. Joensuu1, Anneli Ritala1* AFFILIATIONS 1
VTT Technical Research Centre of Finland Ltd., Finland
2
Division of Pharmaceutical Chemistry and Technology, Drug Research Program, Faculty of
Pharmacy, University of Helsinki, Finland 3
Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark
4
Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku,
Finland 5
Agriculture and Agri Food Canada, London, Ontario, Canada
6 Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Finland
ACS Paragon Plus Environment
1
Bioconjugate Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 30
ABSTRACT
Encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality: The hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for functionalizing hydrophobin coatings with transferrin as a targeting ligand.
KEYWORDS hydrophobin, hydrophobin fusion protein, porous silicon nanoparticle, tobacco BY-2 cells, drug targeting
ACS Paragon Plus Environment
2
Page 3 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
INTRODUCTION Drug delivery is one of the biggest challenges in modern medicine, especially for cytotoxic drugs used in the treatment of cancer due to their severe off-target effects1 and poor bioavailability. Thus, new formulations are needed to enhance the delivery to specific target cells. Encapsulating drugs in nanoparticles is a potential solution for both increasing their solubility and controlling their delivery and release, thus diminishing the unwanted side effects. However, nanoparticles need to be stable, degradable, non-immunogenic and reach specifically their targets2. In this regard, porous silicon (PSi) nanoparticles have been used extensively as carriers in oral3 and intravenous drug delivery4 for different biochemical applications 5,6. Fungal hydrophobins (HFB) are small, globular proteins with extraordinary surface active properties due to their unique amphipathic structure: One other end of the molecule is hydrophilic, while the surface of the other end forms a hydrophobic patch soluble in water, but form multimers at high concentrations
9,10
7,8
. HFBs are highly
. The multimerization occurs
presumably to reach an energetically favorable state where the hydrophobic patches are facing each other and surrounding water molecules displaced. When in contact with a hydrophilic hydrophobic interphase, such as a silicon surface in aqueous solution, HFBs self-assemble into a monolayer
10,11
(Figure 1). The self-assembly of Trichoderma reesei HFBI and HFBII has been
successfully utilized to formulate nanoparticles from poorly water-soluble drug compounds 12,13. When applied on PSi nanoparticles, the HFB coating improved the solubility and biocompatibility of the particles, while allowing the controlled release of the payload
14
. The
coated particles were stable in simulated gastrointestinal fluids and the oral administration to rats increased the transit time from stomach to intestine
15
. The HFB coating also influenced the
biodistribution of the PSi nanoparticles when administered intravenously to rats
16
. A major
ACS Paragon Plus Environment
3
Bioconjugate Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 30
problem in parenteral administration is the adsorption of plasma proteins around the nanoparticles as a corona, causing aggregation and loss of activity
17,18
. However, HFB-coated
PSi nanoparticles and polystyrene nanoparticles recruited significantly less plasma proteins than naked particles 16,19. When linked to other proteins, HFBs convey some of their properties to the respective fusion partner. Several HFBI fusion proteins have been expressed in plants
20–25
. Recently, we have
shown that also HFBII and HFBIV are potential candidates as fusion partners and HFBII are structurally similar
7,9
26
. While HFBI
, the amino acid sequence and the hydropathy profile of
HFBIV are distinctly different, possibly also influencing its properties
27
. Nevertheless, HFBIV
appears to bind to both polar and hydrophobic surfaces, similarly to HFBI and HFBII 27. The HFB fusion technology has been applied for purification of fusion proteins using surfactant based aqueous two-phase separation (ATPS) in fungal 28,29, insect 30, plant 21 and plant cell based production platforms
31
. A fusion protein combining HFBI and a dual cellulose
binding domain has also been used for adding functionality to HFB-coated nanoparticles. The fusion protein enabled formulation of the nanoparticles, similar as non-fused HFB, but also bound the nanoparticle to cellulose nanofibrils within cellulose hydrogel allowing improved formulation
12
. In this study, we took one step further and investigated whether a HFB fusion
protein could be used for active targeting of nanoparticles utilizing transferrin receptor-mediated endocytosis. Transferrin (Tf) is an 80 kDa glycoprotein with 19 intramolecular disulphide bridges. Challenges in production of the complex molecule in bacterial systems
32
and risks
involved in purification from human plasma have encouraged search of alternative production platforms, such as plants and yeasts. Human Tf was first expressed in tobacco 32 and is currently produced commercially in rice under trade name Optiferrin 33. Plant-derived recombinant Tf has
ACS Paragon Plus Environment
4
Page 5 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
been shown to be structurally and functionally similar to native human protein although it appears not to be glycosylated 32–34. The main function of Tf is iron sequestration and transport in serum
1,33
. When free Tf (apo
form) binds ferric iron, its conformation changes (holo form) and the affinity to Tf receptor increases. The Tf/Tf-receptor complex is taken-up by the cells through endocytosis and dissociation of iron occurs at low pH in the endosomal compartments. Subsequently, the protein is recycled back to the bloodstream 1,33. The Tf receptor is ubiquitously expressed on normal cell types, but is upregulated on various tumors 1. This, combined with the capacity of Tf to cross the blood-brain barrier, has made it an interesting molecule for drug targeting via direct conjugation to the active molecule 32,35,36, or to nanocarriers 1,37,38. The aims of this study were to engineer a fusion protein (Figure 1) that exhibits the functional characteristics of both HFB and Tf, to produce the protein in plant cell culture and to test whether it can be used to facilitate targeting and uptake of nanoparticles in cancer cells. Such a fusion protein could advance the development of affordable nanoparticle drugs for cancer therapy by offering a tool for simple one-step formulation of targeted nanoparticles from poorly water-soluble pharmaceutical compounds 13.
ACS Paragon Plus Environment
5
Bioconjugate Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 30
Figure 1. Schematic presentation of a HFB-Tf fusion protein and its adsorption on the surface of a PSi nanoparticle. In aqueous solutions the HFBs assemble as monolayers on hydrophobic surfaces presumably to reach an energetically favorable state, where surrounding water molecules are displaced from between the hydrophobic patch of the HFB and the nanoparticle surface.
RESULTS AND DISCUSSION Construct screening in Nicotiana benthamiana We have recently reported that in addition to T. reesei HFBI, also HFBII and HFBIV are potential candidates for expression of fusion proteins in plants
26
. To find a suitable fusion
strategy, we built six fusion constructs where human Tf was connected by a linker either N- or C-terminally to T. reesei hydrophobins HFBI, HFBII or HFBIV (Figure 2A). The constructs and a non-fused Tf were transiently expressed in N. benthamiana and accumulation levels were determined by immunoblot analysis (Figure 2B and C). All fusion constructs resulted in lower accumulation in comparison to non-fused Tf (43±19% of TSP, mean±SD, n=4). There was no statistically significant difference (p
