Article pubs.acs.org/Biomac

Phage Lambda Capsids as Tunable Display Nanoparticles Jenny R. Chang,† Eun-Ho Song,‡ Eri Nakatani-Webster,† Lucas Monkkonen,† Daniel M. Ratner,‡ and Carlos E. Catalano*,† †

Department of Medicinal Chemistry, School of Pharmacy, University of Washington H-172, Health Sciences Building, Box 357610, Seattle, Washington 98195-7610, United States ‡ Department of Bioengineering, University of Washington, William H. Foege Building, Room N210D, Box 355061, Seattle, Washington 98195-5061, United States S Supporting Information *

ABSTRACT: Nanoparticle technologies provide a powerful tool for the development of reagents for use in both therapeutic and diagnostic, or “theragnostic” biomedical applications. Two broad classes of particles are under development, viral and synthetic systems, each with their respective strengths and limitations. Here we adapt the phage lambda system to construct modular “designer” nanoparticles that blend these two approaches. We have constructed a variety of modified “decoration” proteins that allow site-specific modification of the shell with both protein and nonproteinaceous ligands including small molecules, carbohydrates, and synthetic display ligands. We show that the chimeric proteins can be used to simultaneously decorate the shell in a tunable surface density to afford particles that are physically homogeneous and that can be manufactured to display a variety of ligands in a defined composition. These designer nanoparticles set the stage for development of lambda as a theragnostic nanoparticle system.

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INTRODUCTION Therapeutic and diagnostic nanoparticles fall into two broad categories, viral and nonviral, each with their respective strengths and weaknesses. A major strength of the nonviral systems is that they are modular and “tunable”, allowing the display of both biological and synthetic polymers in the particle.1 Incorporation of carbohydrates into synthetic nanoparticles, for instance, has attracted the attention of the delivery community due to their potential as targeting ligands capable of engaging cell-associated carbohydrate binding proteins for receptor-mediated uptake.2 This carbohydrate-based targeting strategy appears particularly well suited for nanoparticle-based delivery systems. There is growing evidence that displaying carbohydrate moieties on a particulate platform has distinct advantages, most notably they may mimic the high-density glycan display found on pathogens such as viruses and bacteria.3,4 Unfortunately, formulation of these carbohydratebased nanoparticles has largely been centered on complex polymer architectures that require specialized synthetic and polymer chemistry expertise. Viral-based systems similarly hold promise because they provide an ideal platform, which if appropriately optimized, can be modified to present a high density of ligands in a homogeneous and defined symmetric pattern. This type of display can increase the avidity of binding to target biomolecules, and viral capsids have been modified to display a number and variety of synthetic and biological ligands by covalent modification of the capsid surface.5−8 In general, this relies on cloning and expressing protein fusion constructs in the context of an infectious virus in vivo, or “shot-gun” chemical © 2014 American Chemical Society

modification of the isolated capsids in vitro. More recently, Prevelige, Douglas, and co-workers introduced specific cysteine residues and hexa-histidine (H6) tags into the capsid protein of bacteriophage P22 and demonstrated that mutant capsids isolated from Escherichia coli cells can be selectively modified with cysteine-directed reagents and are recognized by anti-H6 antibodies, respectively.9,10 Notwithstanding, these approaches require optimization for each new particle design, and control of display density can be difficult, especially if decoration with multiple ligands is desired. An ideal “theragnostic” reagent would blend the strengths of each of the above systems to afford particles that are (i) chemically and physically homogeneous, (ii) modular to allow the display of multiple ligands, (iii) well-defined with respect to the number and density of the displayed ligands, (iv) structurally defined with respect to the symmetry of the presented ligands, and (v) easily modified to allow unrestricted incorporation of a variety of biological and synthetic display ligands as required for specific theragnostic requirements.11,12 Bacteriophages provide ideal systems with which to develop theragnostic platforms due to their size, relative ease of production, and facile biochemical manipulation. Specifically, bacteriophage lambda has been used in phage display applications,13,14 as a gene delivery particle,15−17 and as a vaccine presentation platform.13,18 More recently, we have demonstrated that the lambda capsid can be chemically Received: August 8, 2014 Revised: October 2, 2014 Published: October 15, 2014 4410

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Figure 1. (a) Bacteriophage lambda capsid assembly in vivo. The procapsid shell is assembled from 415 copies of the major capsid protein (gpE, blue) and 12 copies of the portal protein (gpB, green). The scaffolding protein (gpNu3, orange) copolymerizes with the major capsid protein to chaperone shell assembly. The viral protease (gpC, gray oval) is autoproteolytic, degrading itself and the scaffolding protein with the resulting products exiting the shell interior. The terminase enzyme (cyan) packages phage DNA into the mature procapsid; this triggers an expansion transition to the immature capsid. While a monomer in solution, the decoration protein (gpD, magenta triangles) adds to the expanded shell as trimer spikes assembled at the 140 3-fold axes of the icosahedral shell (420 copies total) to form the mature capsid. (b) Expansion and decoration of the lambda procapsid in vitro. Urea is used to trigger procapsid expansion. Subsequently, 420 copies of gpD add to the expanded shell.

modified with polyethylene glycol (PEG), primarily at a single residue on the major capsid protein.8 PEGylation attenuates nonspecific capsid internalization into transferrin receptorexpressing HeLa cells, which is partially restored by additional conjugation of the PEG with transferrin. Here, we further develop the lambda system utilizing the capsid decoration protein to design reagents that blend the homogeneity and symmetry of viral nanoparticles with the modularity and tunability of the synthetic systems. Lambda capsid assembly in vivo affords a procapsid shell into which viral DNA is packaged (Figure 1a).19,20 Genome packaging triggers a remarkable procapsid expansion transition to a thinner, angularized capsid shell.21,22 The lambda capsid decoration protein, gpD, is expressed as a monomer in the phage-infected cell but assembles as trimer spikes at the 140 3fold icosahedral axes of the expanded shell surface (420 copies per shell; Figures 1, 2a).23,24 Upon packaging the full-length genome, finishing proteins and a viral tail are appended to afford an infectious lambda particle.19,25 Infectious lambda phages have been used as display platforms generating polypeptide libraries through peptide fusions with either gpD26 or the major tail protein (gpV).27 GpD is ideally suited to this purpose because it is symmetrically displayed at high density on the capsid surface. A number of studies have shown that gpD may be modified at either the N- or Cterminus to present peptides at the capsid surface for phage display applications.28 More recently, a “genetically tunable” lambda display system was developed, which allows the expression of plasmid borne gpD fusion proteins and a D− lambda lysogen to produce infectious phage that display the desired tag.28 While these studies show promise for the design of useful lambda nanoparticles, current applications that utilize gpD have been limited to proteinaceous fusion constructs expressed within E. coli cells in the context of an infectious lambda virus. Recent studies have utilized modified decoration proteins from bacteriophages P2229 and T430 to decorate viral capsids in vitro, and they have demonstrated that appended ligands are accessible on the capsid surface. Our in vitro system provides the opportunity to similarly decorate the lambda capsid with modified gpD constructs under defined reaction conditions. We previously described the construction of a plasmid that

Figure 2. (a) CryoEM reconstruction of gpD trimer spikes assembled at the icosahedral 3-fold axes of a lambda capsid shell. The major capsid proteins have been removed for clarity. The structural data was kindly provided by Dr. Gabriel Lander. (b) Crystal structure of a gpD trimer (PDB #1C5E) shown from a side elevation as it appears on the capsid shell surface. The gpD trimer is shown in cartoon representation with each subunit colored a different shade of blue/ green and with Serine 42 depicted as red spheres in each subunit. The N-terminal 14 residues of each monomer are disordered in the crystal structure and are not visible in the high-resolution structure; however, their location on the capsid surface is revealed by symmetric densities emanating from the trimer spikes as shown in panel a. (c) The gpD variants constructed in this study.

expresses all of the lambda structural capsid proteins and have demonstrated that functional procapsids may be purified in high yield.31 gpD is also expressed from this plasmid, and the 4411

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protein may similarly be purified in high yield.31 We have further shown that lambda procapsids can be expanded with urea in vitro and that purified gpD efficiently adds to the intact, expanded shells to afford fully decorated and biologically functional capsids (Figure 1b).22 Here we describe a protocol that harnesses the lambda system to construct hybrid nanoparticles that can be modified to simultaneously display multiple biological and synthetic ligands. This system provides a modular, tunable, and easily customized platform for the construction of “designer” nanoparticles as theragnostic reagents.

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reagents (Monkonnen and Catalano, unpublished). Therefore, gpD(S42C) purifications utilized buffers containing 1 mM dithiothreitol (DTT) in place of β-ME. H6-gpD-GFP was expressed as described above with modification. Briefly, 20 mM glucose was added to the growth media and the cell pellet was resuspended in Buffer H (20 mM Tris, pH 8, 150 mM NaCl, 0.1 mM EDTA, 7 mM β-ME, 25 mM imidazole). The cells were disrupted either by sonication or by French Press, and the clarified lysate was loaded onto a HisTrap FF (5 mL) column equilibrated with Buffer H. Bound protein was eluted with a 10-column volume gradient to 500 mM imidazole and the H6-gpD-GFP-containing fractions (bright green in color) were dialyzed overnight against Buffer H (minus imidazole). The protein was concentrated using an Amicon Ultra-0.5 filter and stored at 4 °C until use. Alternatively, the sample was dialyzed against Buffer H containing 20% glycerol for long-term storage at −20 °C. The primary sequence of H6-gpD-GFP was confirmed by mass spectrometry, which provided ∼78% sequence coverage (data not shown). Conjugation and Purification of gpD(S42C::PEG). Methoxypolyethylene glycol maleimide, PEG average Mn 5000 (MeO-PEG-Mal), was purchased from Sigma-Aldrich. To a solution of gpD(S42C) (0.86 mg, 75 μM) in phosphate buffered saline (PBS) buffer was added a solution of the MeO-PEG-Mal (0.375 mg, 1 equiv) in PBS buffer (pH 6.6, 50 mM, 1 mL). The reaction mixture was placed on a rocking platform at room temperature (R.T.) for 24 h and then buffer exchanged with Buffer B using an Amicon Ultra-0.5 filter to remove unreacted MeO-PEG. The reaction mixture was applied to a Superose 6 10/300 GL column (GE Healthcare) equilibrated and developed with Buffer B. The gpD(S42C::PEG)-containing fractions were pooled, concentrated in an Amicon Ultra-15 filter, and stored at 4 °C until further use. Synthesis of 6′-Maleimidohexanamido-Polyethylene Glycol Mannoside (Med. Chem. Lett. 2007, 17, 5379−5383). All commercially available chemicals were used without additional purification unless otherwise noted. 1H and 13C NMR were obtained at 500 and 125 MHz on a Bruker AV-500 NMR. Mass spectra (MS) were recorded on Bruker Esquire liquid chromatograph-ion trap mass spectrometer. Chemical shifts are reported in parts per million downfield relative to tetramethylsilane (TMS, 0.00 ppm) and coupling constant are reported in Hertz (Hz). The following abbreviations are used for the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; and br = broad.

EXPERIMENTAL SECTION

Materials and Methods. Construction of pT7Cap Dam7am43. The plasmid pT7capDam7am43 expresses the lambda major capsid protein (gpE), the scaffolding protein (gpNu3), the portal protein (gpB) and the capsid protease (gpC), which spontaneously assemble functional procapsids that can be purified in high yield. This plasmid was constructed through modification of pT7cap to obviate the expression of gpD;31 codons 7 (UUU) and 43 (UCC) were mutated to amber stop codons (UAG) using the QuikChange II site-directed mutagenesis kit (Agilent) according to the manufacturers instructions. Construction of Variant gpD Expression Plasmids. A summary of the proteins described in this work is presented in Figure 2c, each of which was expressed from plasmids constructed as follows. First, plasmids that express wild-type gpD protein, without and with a Nterminal 6-histidine tag (gpD-WT and H6-gpD-WT, respectively) were constructed by polymerase chain reaction (PCR) amplification of the D gene using genomic λ DNA as the template. The primers are described in Table S1 and the plasmids in Table S2 (Supporting Information). The expected PCR products were purified using the Wizard SV Gel and PCR Clean-up System (Promega), digested with NdeI and BamHI, and cloned into similarly digested pET21a (Novagen) to afford plasmids pD and pH6-gpD. Both plasmids served as templates for mutagenesis of serine 42 to cysteine via the QuikChange II site-directed mutagenesis kit (Agilent) to afford plasmids that expressed mutated gpD proteins (gpD(S42C) and H6gpD(S42C), respectively; Figure 2c). To generate gpD proteins containing N- and C-terminal linkers terminating with a unique cysteine residue (cys-gpD-H6 and H6-gpD-cys, respectively; Figure 2c), the D gene was amplified by PCR using genomic λ DNA as a template and the primers presented in Table S1. The plasmids were constructed in the same manner as those described above. To construct the plasmid expressing the N-terminal 6-histidine-tagged gpD-GFP (H6-gpD-GFP; Figure 2c), the D gene with a linker region encoded following its 3′-end was inserted in-frame into the vector pRSET_EmGFP (Invitrogen) between the restriction sites NdeI and NcoI. The PCR primers are described in Table S1. Multiple cloning sites were added to the 5′- and 3′-end of the D gene separately to generate plasmids that will express proteins of choice fused to gpD via linkers at the N- and C-termini, respectively. The plasmids were constructed in a similar manner as those described above. The PCR primers are described in Table S1. Purification of the Modified gpD Proteins. Expression and purification of all gpD protein constructs (except H6-gpD-GFP) was performed as described previously,32 with the following modifications. The gpD-containing cell lysis supernatant was loaded onto a HiTrap Q HP (5 mL) column equilibrated with Buffer B (20 mM Tris, pH 8, 20 mM NaCl, 0.1 mM EDTA, 7 mM β-ME). Bound protein was eluted in a 10-column volume gradient to 1 M NaCl and the gpD-containing fractions were dialyzed overnight against Buffer B. The dialyzed protein was concentrated in Amicon Ultra-15 filters and applied to a Superose 6 10/300 GL (GE Healthcare) column equilibrated and developed with Buffer B. The purified proteins were stored in 20% glycerol at −20 °C unless otherwise specified. We note that purification of gpD(S42C) in β-ME containing buffers results in adduction of the reducing agent to the cysteine residue which precludes chemical modification of the residue with maleimide

To a solution of 2-(2-(2-(amido)ethoxy-ethoxy)ethyl-O-α-D-mannoside 1 (20 mg, 6.4 × 10−2 mmol) in MeOH (1 mL) was added a solution of 6-maleimidohexanoic acid N-hydroxysuccinimide ester 2 (24 mg, 7.7 × 10−2 mmol) in MeOH (0.5 mL). The reaction mixture was stirred at R.T. for 2 h. The product 3 (28 mg, 87%) was obtained after removal of solvent under reduced pressure followed by purification through silica column chromatography. The identity of product was confirmed by mass spectrometry and by 1H and 13C NMR (Figure S1). 1H NMR (500 MHz, CDCl3) δ 8.03 (br, 1H), 6.83 (s, 1H), 4.83 (d, J = 1.4 Hz, 1H), 3.90−3.84 (m, 3H), 3.75−3.68 (m, 7H), 3.65−3.64 (m, 2H), 3.61−3.60 (m, 1H), 3.56 (dd, J = 5.5, 5.5 Hz, 2H), 3.52 (dd, J = 7.1, 7.1, 2H), 3.38 (dd, J = 5.6, 6.3, 2H), 2.22 (dd, J = 7.4, 7.4, 2H), 1.68−1.58 (m, 4H), 1.36−1.30 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 174.73, 171.21, 134.14, 133.81, 100.35, 4412

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73.24, 71.23, 71.10, 70.81, 70.61, 70.22, 69.91, 69.27, 67.21, 38.97, 37.01, 35.40, 27.89, 25.92, 25.07; MS-ESI (m/z): [M + H]+ calcd. for C 22 H37 N 2 O 11 , 505.23; found, 505.3, [M + Na]+ calcd. for C22H36N2NaO11, 527.22; found, 527.3. Conjugation and Purification of gpD(S42C::sMannose). To a solution of gpD(S42C) (2.84 mg, 248 μM) in PBS buffer (1 mL) was added a solution of the maleimide-activated mannose 3 (0.098 mg, 0.9 equiv) in PBS buffer (pH 6.6, 50 mM, 1 mL). The reaction mixture was gently shaken at R.T. for 24 h and then loaded onto a HiTrap Con A (1 mL) column equilibrated with 20 mM Tris buffer, pH 7.4 (4 °C), containing 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2. The protein was eluted over a 0−100% gradient to Buffer X (20 mM Tris buffer, pH 6.4 at 4 °C, containing 0.2 M methyl-α-D-mannopyranoside and 0.5 M NaCl). The gpD(S42C::sMannose)-containing fractions were pooled and dialyzed against 50 mM Tris buffer, pH 8, containing 150 mM NaCl, concentrated using an Amicon Ultra-0.5 filter and stored at 4 °C until further use. The expected product was confirmed by matrixassisted laser desorption/ionization (MALDI) mass spectrometry (data not shown). Procapsid Expansion and Decoration. Procapsids were isolated from E. coli BL21[DE3](pT7capDam7am43) cells and purified as described previously31 except that anion exchange chromatography employed a HiTrap Q HP (5 mL) column in place of a 50 mL DEAESepharose column. Expanded capsid shells were prepared and decorated with the gpD constructs and the reaction products were fractionated on a 1.2% agarose gel as previously described.22 The gpD surface density for each of the decorated particles was determined using a gel assay as previously described.33,34 Briefly and unless otherwise stated, unincorporated decoration protein was removed from the reaction mixture by buffer exchange using an Amicon Ultra0.5 Filter (Millipore). The samples were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining of the proteins. The intensities of the stained gpD and gpE bands were quantified by video densiometry, and the raw data were normalized for molecular weight and corrected for mass staining efficiency based on a standard concentration curve for each construct (not shown). The gpD:gpE ratios (i.e., surface density) are reported relative to that observed for gpD-WT (100%, Table S3). Agglutination Assay. Decorated capsids (1 μM gpD equivalent) were added to 10 mM HEPES buffered saline solution containing 150 mM NaCl, 1 mM CaCl2 and 1 mM MgCl2. Concanavalin A (Con A, 1 μM) in the 10 mM HEPES buffered saline solution was then added to initiate the reaction and agglutination was detected by monitoring the increase in absorbance (350 nm) at 1 min intervals. α-D-mannose was added to a final concentration of 5 mM after a total incubation time of 20 min to competitively displace the sMannose.

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RESULTS AND DISCUSSION Decoration of Lambda Capsids with gpD-Green Fluorescent Protein in Vitro. The ability to decorate lambda capsids with modified gpD constructs provides an attractive approach to develop “designer” nanoparticles of defined composition and symmetric presentation. Toward this end, we first constructed a plasmid that expresses a 6 histidinetagged protein composed of an N-terminal gpD domain fused to a C-terminal green fluorescent protein domain (H6-gpDGFP; Figure 2c). This protein is analogous to the previously described gpD-EYFP fusion protein used to decorate phage particles in vivo.33 H6-gpD-GFP can be purified to homogeneity and in high yield (Figure S2). We next used an agarose gel assay22 to demonstrate that H6-gpD-GFP can be added to the expanded lambda capsid under defined conditions in vitro. Because of the fluorescent nature of GFP, the decorated capsids are visualized two ways: by Coomassie blue staining for total protein content (Figure 3a) and by fluorescence imaging for the presence of GFP (Figure S3a). As previously demonstrated,

Figure 3. Tunable decoration of expanded capsids with H6-gpD-GFP and gpD(S42C::PEG) constructs. (a) Expanded capsids were incubated with gpD-WT and H6-gpD-GFP as indicated and the reaction products were analyzed by agarose gel stained with Coomassie blue. The position of the undecorated capsid is denoted with an arrowhead at the left of the gel. The diffuse bands that appear in the middle of the gel represent free H6-gpD-GFP protein that has not bound to the capsid. (b) Unincorporated H6-gpD-GFP was removed from the reaction mixtures by buffer exchange and the particles were analyzed by denaturing polyacrylamide gel stained with 4413

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yield of infectious virus, as observed for gpD-EYFP fusion constructs.33 Second, the current phage display systems generally require construction of modified lambda genomes and induction of lambda lysogens in vivo to afford the modified particle. A recent approach describes the use of a λ Dam lysogen (amber mutation in the D gene) combined with the expression of modified gpD from an introduced plasmid in vivo. This system requires plasmid and lysogen induction and affords decorated phage particles. These approaches are laborious and ultimately controlled by the whims of the E. coli cell, which does not allow facile manipulation of the capsid decoration density. We reasoned that the in vitro gpD-decoration procedure described herein could be adapted for the display of any peptide/protein on the capsid surface and at defined surface densities. Toward this end, we constructed a vector that possesses a multicloning site immediately downstream from the D gene (gpD-MCS) and an analogous vector that allows facile generation of N-terminal tagged fusion proteins (MCS-gpD, Figure 2c). These vectors allow for rapid and efficient expression of gpD-fusion proteins that can be used to decorate the lambda capsid in vitro with control of surface display density as described for H6-gpD-GFP above. A third major limitation of current lambda display platforms is that the systems are limited to peptide and protein tags. Teschke and co-workers recently demonstrated that a cysteine residue introduced into the Dec decoration protein could be modified with a fluorescent tag and that the labeled protein adds to the P22 capsid shell in vitro.29 We reasoned that gpD could be similarly modified, and we next constructed plasmids that express gpD containing a unique cysteine residue appended to either the N-terminus (cys-gpD-H6) or the Cterminus (H6-gpD-cys) of the protein (Figure 2c). This allows either of the purified proteins to be site-specifically modified by a variety of nonproteinaceous ligands using simple thiol-based chemistry.35 This is discussed further below. A final limitation of lambda display systems centers on the locations of the N- and C-termini of gpD bound to the capsid surface. gpD is a monomer in solution but assembles as a trimeric spike to the 140 3-fold axes of the icosahedral shell (Figure 2a,b). The N-terminus of each subunit directly interacts with the capsid shell to provide stabilizing contacts required for shell integrity, while the C-terminus exits the gpD trimer spike proximate to the shell surface.24 Appending bulky ligands to either terminus could hinder gpD trimer assembly at the capsid surface and as a consequence interfere with stabilization of the capsid and subsequently phage viability. Indeed, our observation that capsid decoration with H6-gpD-GFP is incomplete in vitro (Table S3) and the observation that infectious phage decorated with gpD-EYFP or gpD-GFP are less viable than wild-type in vivo28,33,36 highlight this concern. The crystal structure of the gpD trimer spike reveals that Ser42 is positioned at the apex of the spike in all three subunits (Figure 2b). We reasoned that modification of this residue would place the desired tag projecting away from the capsid surface and into solution for optimal display, and with minimal insult to both gpD spike assembly and shell integrity. We therefore constructed plasmids (with and without N-terminal histidine tags) that express gpD in which Ser42 has been changed to Cys42 (H6-gpD(S42C) and gpD(S42C), respectively; Figure 2c). Importantly, because the native protein does not contain any cysteine residues, this construct provides a unique site for modification using thiol-based chemistry.

Figure 3. continued Coomassie blue. (c) Expanded capsids were incubated with gpD-WT and gpD(S42C::PEG) as indicated and the reaction products were analyzed by agarose gel stained with Coomassie blue. The position of the undecorated capsid is denoted with an arrowhead at the left of the gel. Note that capsids decorated with 100% gpD(S42C::PEG) migrate “backwards” toward the cathode, suggesting an effective positive charge on the particle. (d) Unincorporated gpD(S42C::PEG) was removed from the reaction mixtures by buffer exchange and the particles were analyzed by denaturing polyacrylamide gel stained with Coomassie blue. Quantitation of the gel as described in Materials and Methods affords the surface density presented in Table S3.

capsids decorated with gpD-WT migrate as a distinct, higher mobility band in the agarose gel. In the presence of increasing amounts of H6-gpD-GFP, a progressively slower migrating species appears, consistent with decoration of the capsids with increasing GFP density. At maximal densities, two distinct particles are apparent in the gel, indicating that two populations are present. Exactly what these two species represent is unclear, but we presume that the upper and lower bands are partially versus completely decorated capsid shells, respectively. Quantitation of the relative fluorescence:Coomassie staining of the two bands is consistent with this suggestion (Figure S3b). Within this context, we note that the analogous “Dec” decoration protein binds selectivity to some (but not all) of the quasi 3-fold axes, but none of the strict 3-fold sites on the phage P22 capsid shell.29,34 A similar differential affinity may exist with the lambda capsid, which while not perceptible with wild-type gpD may result in incomplete trimer spike assembly with the bulky gpD-GFP construct. This is discussed further below. To confirm that the addition of H6-gpD-GFP to expanded capsids afforded stably decorated particles and to determine the surface density, the capsids were separated from unreacted material using a buffer exchange protocol, and the protein content of the decorated shells was quantified as described in Materials and Methods. The data present in Figure 3b demonstrates that both gpD-WT and H6-gpD-GFP stably add to the capsid shell. Importantly, the surface density of each can be tuned by adjusting the ratio of the proteins included in the reaction mixture; as the relative ratio of H6-gpD-GFP is increased in the reaction mixture, the density of the modified protein on the decorated particles increases to ultimately yield a relative surface density of ∼49%. Particle decoration does not appear to be linear, suggesting that gpD-WT outcompetes H6gpD-GFP during addition to the capsid surface (data not shown). The GFP domain (28.7 kDa) adds significant bulk to the small gpD polypeptide (11.4 kDa), which at high densities likely interferes with trimer spike assembly at the capsid surface and consequently results in incomplete shell decoration, as discussed above. Construction of gpD-Vectors for Diverse Phage Display Applications. GpD has been used in a variety of lambda phage display applications.13 To date, the gpD fusion constructs have appended peptides to either the N-terminus or C-terminus of the protein and decorated phages have been assembled in vivo from the induction of the modified lambda lysogens. While useful, this approach has several limitations. First, in vivo display restricts potential ligands to those that do not interfere with the development of viable viral particles or with the robustness of the E. coli cell. Unfortunately, it is quite likely that many desired ligands will indeed adversely affect the 4414

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Assembly of PEGylated Lambda Capsids of Defined Surface Density. A major thrust of this work is to construct tunable lambda-based nanoparticles for use as theragnostic agents. A complication faced by all nanoparticles is the induction of an immune response that results in rapid clearance of the reagent from circulation.37,38 The serum half-life of infectious phages in mammalian organisms depends on their surface properties and sequestering these immunogenic features increases their serum stability.38 PEGylation of biologics has profound effects on the pharmacokinetic profiles of a variety of proteins39 and it has been demonstrated that PEGylation of infectious phages significantly increases their circulation time in mice.40 Furthermore, we8 and others12 have demonstrated that PEGylation of viral capsids attenuates nonspecific uptake by eukaryotic cells and that specif ic uptake can be restored, at least in part, by secondary conjugation with targeting ligands. These studies employed shotgun chemical modification of the capsid, which limits the extent by which the system can be tuned. Therefore, we developed a novel approach to PEGylate the lambda capsid in a specific and tunable manner. The gpD(S42C) construct was chemically modified with methoxypolyethylene glycol maleimide (PEG average Mn 5000) using maleimide chemistry to afford gpD(S42C::PEG). This is a semisynthetic decoration protein that contains a single PEG5000 moiety site-specifically appended at Cys42 (Materials and Methods). The protein was purified to near homogeneity (Figure S2) and then used to decorate lambda capsids in vitro. As anticipated, gpD-WT adds to expanded capsids to afford a unique band of altered mobility in the agarose gel compared to undecorated capsids (Figure 3c). Increasing the relative fraction of gpD(S42C::PEG) in the reaction mixture results in gradual retardation of capsid migration in the gel consistent with progressive decoration with PEGylated gpD. To confirm this presumption, the protein content of the decorated particles was quantified as described in the Materials and Methods section, which demonstrates that both gpD-WT and gpD(S42C::PEG) stably add to the capsid shell (Figure 3d, Table S3). Two features in the PEG decoration study are noteworthy; first, the surface density can be tuned by adjusting the ratio of unmodified:PEGylated gpD in the reaction mixture. Second, migration of the PEGylated capsid shell when fully decorated is “backwards”, toward the anode (Figure 3c); this indicates an overall positive charge on the particle. We interpret this observation as an indication that gpD(S42C::PEG) bound to the capsid surface efficiently screens the surface properties of the proteinaceous shell, an indication that immunogenic epitopes can similarly be sequestered. Decoration of Lambda Capsids with Synthetic Glycoproteins. Mannose-containing glycoproteins provide cell-targeting epitopes that are recognized by cellular C-type lectin receptors. These receptors are employed by macrophages and other immune cells to identify pathogen-associated carbohydrate ligands.41 As described above, the formulation of nanoparticle delivery systems displaying functional carbohydrate ligands presently requires specialized synthetic and polymer chemistry expertise. Few strategies exist that support glyconanoparticle formulation by nonspecialized laboratories. Therefore, to further develop the chemical diversity possible in the lambda system, we next synthesized 6′-maleimidohexanamido-polyethylene glycol mannoside (sMannose) as described in the Materials and Methods section. GpD(S42C) was chemically modified with sMannose to afford the gpD(S42C::sMannose) neoglycoprotein, which was purified to

homogeneity (Figure S2). Like H6-gpD-GFP and gpD(S42C::PEG), increasing the fraction of gpD(S42C::sMannose) included in the capsid decoration reaction mixture results in a progressive retardation of capsid migration in the agarose gel, consistent with progressive decoration by the gpD neoglycoprotein (Figure 4a). This presumption was confirmed by analysis of their protein content as described in the Materials and Methods section (Figure 4b, Table S3). The Lambda Capsid Can Be Decorated with Multiple Tags in a Defined Composition. As demonstrated above, modified gpD constructs can be used to decorate the lambda capsid with a large protein (GFP), a synthetic polymer (PEG), and with a synthetic mannose neoglycoprotein (sMannose). To demonstrate that the capsid shell can be decorated with multiple ligands simultaneously, lambda capsids were incubated with gpD-WT, gpD(S42C::sMannose), and H6-gpD-GFP alone or in combination. The reaction mixture was analyzed by agarose gel electrophoresis, which clearly demonstrates that the proteins, alone and in combination, add to the capsid shell to afford decorated particles with a unique mobility in the gel (Figure 4c). Analysis of the isolated, decorated shells by SDSPAGE demonstrates that (i) each construct efficiently adds to the shell and that (ii) all three proteins can add to the capsid when simultaneously present in the reaction mixture (Figure 4d). Functional Display of Mannose on the Nanoparticle Surface. Because gpD-WT and the gpD(S42C::sMannose) neoglycoprotein migrate closely in SDS-PAGE, we utilized an agglutination assay to confirm the presence of gpD(S42C::sMannose) on the multipartite ligand decorated capsid surface. The presence of mannose is detected by agglutination of the nanoparticles with Concanavalin (Con A), a mannosebinding plant lectin. As expected, neither gpD-WT nor gpD(S42C) decorated capsids exhibit a positive agglutination response, while H6-gpD-GFP decorated capsids exhibit a weak (nonspecific) agglutination response (Figure 4e). In contrast, capsids decorated with gpD(S42C::sMannose), either fully or partially, show a strong response indicating (i) mannose is present on the capsid surface and (ii) it is efficiently displayed for interaction with Con A. Importantly, this demonstrates that the mannose residues are presented in a bioactive fashion, as demonstrated by specific recognition by Con A, a soluble surrogate for cellular C-type lectin receptors. These results set the stage for utilizing the biofunctional capsids for receptormediated delivery in vitro and in vivo. Structural Characterization of the Decorated Lambda Capsids. We have demonstrated that a variety of display tags of known composition can be site-specifically attached to the gpD decoration protein. We have further shown that these constructs efficiently add to the capsid shell, alone or in combination, in a tunable manner to afford particles of defined chemical composition. It is important, however, to confirm that the decorated capsids retain structural integrity. We therefore utilized electron microscopy to examine the decorated shells. Capsids decorated with gpD-WT reveal an intact icosahedral shell with thin walls and significant angular facets (Figures 5a, S4); this morphology is identical to capsids observed in an infectious lambda particle.21,24 The gpD(S42C) decorated capsids show an identical morphology, consistent with the conservative mutation in the protein. Capsids decorated with H6-gpD-GFP, gpD(S42C::PEG), or gpD(S42C::sMannose) also possess the thin wall, angularized phenotype. Interestingly, capsids decorated with H6-gpD-GFP have extra density on the 4415

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Figure 4. continued incubated with gpD-WT and gpD(S42C::sMannose) as indicated, and the reaction products were analyzed by agarose gel stained with Coomassie blue. The position of the expanded capsid is denoted with an arrowhead at the left of the gel. (b) Unincorporated gpD(S42C::sMannose) was removed from the reaction mixtures by buffer exchange, and the particles were analyzed by denaturing polyacrylamide gel stained with Coomassie blue. Quantitation of the gel as described in the Materials and Methods section affords the surface density presented in Table S3. (c) Expanded capsids were decorated with the indicated gpD constructs, and the reaction products analyzed by agarose gel stained with Coomassie blue. The position of the undecorated capsid is denoted with an arrowhead at the left of the gel. (d) Unincorporated gpD proteins were removed from the reaction mixtures by buffer exchange and the particles were analyzed by denaturing polyacrylamide gel stained with Coomassie blue. Quantitation of the gel as described in the Materials and Methods section affords the surface density presented in Table S3. (e) Capsids decorated with the indicated gpD constructs were used in the agglutination assay using Concanavalin A (Con A), a mannose-specific lectin (Materials and Methods). ■, gpD(S42C::sMannose); ⧫, 40:40:20 WT:sMannose:GFP; ▲, H6-gpD-GFP; ●, gpD-WT; □, gpD(S42C). The data demonstrate that mannose residues are presented in a bioactive fashion.

exterior of the shell, which we presume reflects the presence of the large GFP domain appended to the capsid surface (Figures 5a and S4). Decorated capsids isolated from reaction mixtures containing gpD-WT, gpD(S42C::sMannose), and H6-gpD-GFP in a 40:40:20 ratio also possess extra staining density at the capsid shell, though not as prominent as those fully decorated with H6-gpD-GFP. This is consistent with the lower surface density of H6-gpD-GFP on these particles. Functional Characterization of the Decorated Lambda Capsids. Procapsids serve as receptacles for the viral genome, and gpD is required to provide structural integrity to the DNA-filled shell (Figure 1a).22,42 In the absence of gpD, the capsid cannot withstand the intense pressures generated by the packaged genome (>25 atm) and the shell fractures, rendering the DNA accessible to DNase digestion.42 Here, we utilize an in vitro DNA packaging assay to determine if capsids decorated with modified gpD constructs retain biological activity and are structurally robust. The genome packaging reaction was performed by published procedure31,43 using capsids decorated with either wild-type gpD or the modified gpD constructs. The gpD(S42C) and gpD(S42C::sMannose) capsids are as efficient as wild-type gpD in packaging DNA (Figure 5b). Although slightly attenuated, capsids fully decorated with gpD(S42C::PEG) retain significant DNA packaging activity. In contrast, genome packaging into H6-gpD-GFP decorated capsids is seriously compromised when the surface density of the protein exceeds 50% (Figure 5c). This is consistent with the observation that infectious lambda viruses decorated with gpD-EYFP or gpD-GFP are less stable compared to wild-type virus in vivo.28,33 We presume that this reflects incomplete and/or inefficient decoration of the shell by the bulky gpD-GFP construct, as described above, and a concomitant inability to impart the requisite structural integrity required to package the full-length genome.

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CONCLUSIONS We have developed a hybrid nanoparticle assembly system that harnesses bacteriophage lambda as a homogeneous surface

Figure 4. Tunable decoration of expanded capsids with gpD(S42C::sMannose) and H6-gpD-GFP. (a) Expanded capsids were 4416

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gpD trimer spikes that assemble at the 140 3-fold icosahedral axes, each with the potential to present one or more display tags. The decoration protein can be specifically conjugated with protein and/or synthetic moieties in specific ratios to enhance cellular targeting and uptake of the particle, to avoid immune surveillance, and/or to improve the pharmacokinetic profile of the particles. Recent studies have demonstrated that phage nanoparticles can deliver reporter genes to eukaryotic cells and that the genes can be efficiently expressed in vivo.30,44 The observation that the decorated lambda shell is competent to package DNA sets the stage for developing the lambda system as a gene delivery nanoparticle. Alternatively, the nanoparticles may be optimized to enhance immune response to the capsid as a defined antigenic particle that presents both antigen and adjuvant in the same particle. In summary, these engineered shells can be tailored in specific ways to afford “designer” nanoparticles with defined surface characteristics for both diagnostic and therapeutic applications.

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ASSOCIATED CONTENT

S Supporting Information *

The oligonucleotide primers used to construct the expression vectors and the names of the vectors are presented in Tables S1 and S2, respectively. Quantitation of capsid decoration efficiency is presented in Table S3. NMR and MS data for 6′-maleimidohexanamido-polyethylene glycol mannoside is presented in Figure S1. SDS-PAGE showing purity of the gpD constructs and a fluorescent gel showing decoration of capsids with gpD-GFP are shown in Figures S2 and S3, respectively. An expanded collage of EM images showing capsids decorated with each of the gpD constructs is presented in Figure S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Department of Medicinal Chemistry, University of Washington, H-172 Health Science Building, Box 357610, Seattle, WA 98195; Tel: (206) 685-2468; Fax: (206) 685-3252; E-mail: [email protected].

Figure 5. Structural and functional characterization of the decorated capsids. (a) Negatively stained EM images of capsids decorated with gpD-WT (gpD), gpD(S42C), gpD(S42C::PEG) (PEG), H6-gpD-GFP (GFP), gpD(S42C::sMannose) (sMannose), and with a mixture of WT, sMannose, and GFP in a 40:40:20 ratio (40/40/20). A collage of images is presented in the Supporting Information (Figure S4). (b) Genome packaging activity of the decorated capsids. Capsids decorated with modified gpD constructs, as indicated, were used in the in vitro genome packaging assay (Materials and Methods). Packaging activity relative to gpD-WT is indicated. Each rectangular bar represents the average of three independent experiments; standard deviations are indicated with error bars. (c) Capsids were decorated with gpD-WT and H6-gpD-GFP as indicated and used in the in vitro genome packaging assay. Each rectangular bar represents the average of three independent experiments; standard deviations are indicated with error bars.

Author Contributions

C.E.C., D.M.R., and J.R.C. conceived and designed the experiments; E.-H.S. synthesized 6′-Maleimidohexanamidopolyethylene glycol mannoside and optimized the conjugation reactions with gpD(S42C); L.M. conducted the MALDI and MS protein sequencing experiments; J.R.C. performed the experiments and analyzed the data; the electron micrographs were taken by E.N.-W.; C.E.C., D.M.R., and J.R.C. cowrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. Gabriel Lander for providing the gpD cryoEM reconstruction data presented in this study. We would also like to thank Dr. Ross Lawrence and Dale Whittington of the University of Washington Mass Spectrometry Center and Dr. Miklós Guttman for their technical assistance with mass spectrometry. We are further indebted to Shannon Kruse Lambert for the EM images of the gpD(S42C::PEG) particles. The electron microscopy was conducted at the University of Washington NanoTech User

platform onto which multiple ligands can be displayed in a tunable manner. The protocols developed here allow for simultaneous display of both proteinaceous and nonproteinaceous ligands in a modular fashion. The system further allows display ligands to be appended at multiple points on the decoration protein to minimize interference with gpD assembly at the capsid surface and to optimize presentation of the ligand on the nanoparticle. The shell is symmetrically decorated with 4417

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Facility, a member of the NSF National Nanotechnology Infrastructure Network (NNIN). This work was funded by the Washington State Life Sciences Discovery Fund Grant #2496490 (C.E.C. and D.M.R.) and by the National Science Foundation Grant #MCB 1158107 (C.E.C.).

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