Implementation of P22 Viral Capsids As Intravascular Magnetic

Jun 12, 2013 - Bumb , A.; Brechbiel , M. W.; Choyke , P. Acta Radiol. 2010, 51, 751– 767. [Crossref], [PubMed], [CAS]. 23. Macromolecular and ...
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Implementation of P22 Viral Capsids As Intravascular Magnetic Resonance T1 Contrast Conjugates via Site-Selective Attachment of Gd(III)-Chelating Agents Junseon Min,† Hoesu Jung,† Hyun-Hee Shin,† Gyunggoo Cho,‡ HyungJoon Cho,*,† and Sebyung Kang*,† †

School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Korea, ‡ Korea Basic Science Institute, Ochang, 363-883, Korea S Supporting Information *

ABSTRACT: P22 viral capsids and ferritin protein cages are utilized as templating macromolecules to conjugate Gd(III)chelating agent complexes, and we systematically investigates the effects of the macromolecules’ size and the conjugation positions of Gd(III)-chelating agents on the magnetic resonance (MR) relaxivities and the resulting image contrasts. The relaxivity values of the Gd(III)-chelating agent-conjugated P22 viral capsids (outer diameter: 64 nm) are dramatically increased as compared to both free Gd(III)-chelating agents and Gd(III)-chelating agent-conjugated ferritins (outer diameter: 12 nm), suggesting that the large sized P22 viral capsids exhibit a much slower tumbling rate, which results in a faster T1 relaxation rate. Gd(III)-chelating agents are attached to either the interior or exterior surface of P22 viral capsids and the conjugation positions of Gd(III)-chelating agents, however, do not have a significant effect on the relaxivity values of the macromolecular conjugates. The contrast enhancement of Gd(III)-chelating agent-conjugated P22 viral capsids is confirmed by in vitro phantom imaging at a short repetition times (TR) and the potential usage of Gd(III)-chelating agent-conjugated P22 viral capsids for in vivo MR imaging is validated by visualizing a mouse’s intravascular system, including the carotid, mammary arteries, the jugular vein, and the superficial vessels of the head at an isotropic resolution of 250 μm.

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INTRODUCTION Magnetic resonance imaging (MRI) is one of the most versatile in vivo imaging techniques that provide highly resolved anatomical and functional information in a noninvasive manner. In addition, contrast agent enhanced MRI benefits from increased vessel/tissue contrast from the background and both positive (T1-weighted, brightening) and negative (T2weighted, darkening) contrast agents are being actively explored for in vivo experiments.1−8 Paramagnetic gadolinium ion (Gd(III)), which is frequently used as a positive contrast agent, enhances the image contrast with increased signal intensity from T1-weighted image acquisition due to the greatly reduced spin−lattice relaxation times produced by the interaction between the proton and unpaired electron spins of the contrast agent.6 However, the free form of Gd(III) is well-known to be toxic, so it is invariably complexed with poly(aminocarboxylate) compound chelating agents such as, tetraazacyclododecane tetraacetic acid (DOTA) and diethylenetriamine pentaacetic acid (DTPA) for in vivo applications.8 These chelated Gd(III)-based contrasting agents, however, exhibit rapid clearance, difficult functionalization, and relatively low relaxivity values (2−4 mM−1 s−1 at 60 MHz, 37−40 °C), and thus have limited applications for imaging experiments with high temporal resolution where accumulation of multiple scans cannot be envisaged.9−12 On the other hand, high-resolution © 2013 American Chemical Society

MR angiography (MRA) of circulatory blood vessels provide critical information regarding vascular diseases and physiology, but require relatively long-term blood pool T1 contrast agent with high relaxivity values.13−16 One promising way to improve both the circulation time and relaxivity value for high resolution/contrast MRA image acquisition is to conjugate Gd(III)-chelating agent complexes to macromolecular templates,17−21 thus preventing rapid clearance and decreasing the molecular tumbling rates of the contrast agents. Macromolecular conjugates have been widely studied as MRI contrast agents using polymers, modified proteins, dendrimers, liposomes, carbohydrates, and inorganic nanoparticles as templating macromolecules.18,22−26 In particular, viral capsids have been widely used as templating macromolecules because viral capsids have a well-defined monodisperse and multivalent structure that has a fairly large size that can decelerate the tumbling rate of the conjugates in a solution.19,20,27,28 P22 viral capsids have approximately twice the diameter of other viral capsids that are commonly used, which may allow for a much slower tumbling rate and a large accumulation of Gd(III)chelating agent complexes within them.29 The Salmonella Received: April 2, 2013 Revised: June 11, 2013 Published: June 12, 2013 2332

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Figure 1. Site-selective conjugations of either Gd(III)−DOTA or Gd(III)−DTPA complexes (red dots) to the interior surfaces of small-sized hPf_Fn (outer diameter: 12 nm) (A), and large-sized P22 K118C WB capsids (outer diameter: 64 nm) (B), or to the exterior surfaces of large-sized P22 S133C WB capsids (C), via thiol-maleimide Michael-type addition. to remove cell debris and the resulting supernatant was heated at 65 °C for 10 min. The further purification of thrombin cleavage peptide inserted Pf_Fn was achieved by using a 10 × 300 mm superose 6 size exclusion column. hPf_Fn protein cages were obtained by incubating thrombin cleavage peptide inserted Pf_Fn with 10 units/mL of thrombin at 37 °C overnight followed by SEC fractionation. The purified hPf_Fn was further characterized by UV/vis spectroscopy and TEM. Conjugations of Gd(III)−DOTA or Gd(III)−DTPA to the P22 WB Capsids and hPf_Fn Protein Cages. Complexes of Gd(III) and either maleimido-monoamide-DOTA (DOTA-mal) or maleimidomonoamide-DTPA (DTPA-mal) (Macrocyclics, Inc.) were formed with 1:1 molar ratio prior to conjugation. Subsequently, protein cages (P22 K118C or S133C WB capsids and hPf_Fn) were incubated with 5 mol equiv of Gd(III)−DOTA-mal or Gd(III)−DTPA-mal at room temperature with vigorous shaking overnight. Unreacted Gd(III)− DOTA-mal or Gd(III)−DTPA-mal chelating complexes were removed by extensive dialysis. The extent of the conjugation was determined by subunit mass measurements using electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS; Xevo G2 TOF, Waters). Mass Spectrometry. The subunit masses of untreated and chemically modified P22 viral capsids and hPf_Fn protein cages were analyzed using an ESI-TOF MS (Xevo G2 TOF, Waters) interfaced to a Waters UPLC and an autosampler. Samples were loaded onto the MassPREPTM Micro desalting column (Waters) and eluted with a gradient of 5−95% (v/v) acetonitrile containing 0.1% formic acid with a flow rate of 300 μL/min.37 ESI generally produces a series of multiply charged ions, and the charges are generally distributed as a continuous series with a Gaussian intensity distribution; the molecular masses of each species can be determined from the charges and the observed mass-to-charge (m/z) ratio values. Mass spectra were acquired in the range of m/z 500−3000 and processed using MaxEnt 1 and MaxEnt 3 from MassLynx version 4.1 to obtain the average mass from multiple charge state distributions. For clarity, only deconvoluted masses are presented. Determination of Protein Concentration. Protein concentration was determined by Bradford protein assay. Bradford dye reagent was prepared by diluting one part dye reagent concentrate with four parts water. Ten dilutions of a protein standard (bovine serum albumin (BSA)) were prepared to establish the standard curve. The linear range of this BSA assay was approximately 0.2 mg/mL to 0.8 mg/mL. One microliter of each standard and sample solution was mixed with the 200 μL of diluted dye reagent, and each mixed solution was put into separate plate wells. After 1 min incubation at room temperature, absorbance at 595 nm of each solution was measured. Time Domain-Nuclear Magnetic Resonance (TD-NMR). TDNMR experiments were carried out on a Minispec MQ60 NMR Analyzer (Bruker), operating at a resonance frequency of 60 MHz (1.4T). The temperature was maintained at 40 °C. Data were acquired using the Minispec software (Bruker). In the measurement, the T1 saturation recovery (SR) and T2 Carr Purcell Meiboom Gill (CPMG) NMR pulse sequence were used. The 90° and 180° pulse length were

typhimurium bacteriophage P22 assembles from 415 copies of the 46.6 kDa coat protein with the aid of approximately 300 copies of the 33.6 kDa scaffolding protein to form an icosahedral P22 procapsid.30,31 The P22 procapsid has a diameter of 58 nm and undergoes a structural transformation initiated by DNA packaging to form the infectious 64 nm diameter mature capsid.30,31 The P22 capsid transformation, from procapsid to mature capsid, can be mimicked in vitro by gentle heating (65 °C for 10 min).32 Extended heating (75 °C for 15 min) induces the selective release of subunits from the twelve 5-fold icosahedral vertices (pentons) to produce another capsid form, affectionately known as the “wiffle-ball”, which has a 10 nm hole at each of the twelve 5-fold vertices (Figure 1B, C).32,33 Wiffle-ball (WB) capsids are identical in structure to the mature capsids except for the absence of the subunits at the 5-fold vertices.32,34 The large 10 nm diameter pores of the WB at the 12 vertices ensure free molecular exchange between the interior and exterior environments of the capsid and could potentially be used as portals for entry of large molecular species to modify the interior of the capsid.

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EXPERIMENTAL SECTION

Preparation and Purification of P22 K118C and S133C WB Capsids. The point mutations (K118C and S133C) were conducted by using established polymerase chain reaction (PCR) protocols using pET-3a based plasmids encoding genes for scaffolding and coat proteins as templates.33,35 The amplified DNAs were transformed into a CaCl2-treated competent Escherichia coli strain BL21(DE) and selected for ampicillin resistance. Mutant procapsids were overexpressed in E. coli and purified by ultracentrifugation using 35% sucrose cushion. The empty procapsid shells were prepared by repeated extraction of scaffolding protein with phosphate buffer (50 mM phosphate, 100 mM NaCl, pH 6.5) with 0.5 M GuHCl at 4 °C. Purified empty procapsids were heated at 75 °C for 15 min to obtain WB capsids. The WB capsids were further purified by 10−35% sucrose gradient centrifugation and subsequently dialyzed against Tris buffer (50 mM Tris, 100 mM NaCl, pH 7.0) to successfully produce conjugated capsids. Each individual capsid form was verified using native agarose gels and transmission electron microscopy (TEM). Preparation and Purification of hPf-Fn. The sequence GGLVPRGSGAS was inserted into the 146 position of Pf_Fn with point mutation (S133C) by an established PCR protocol using pET30b based plasmids for constructing the full-length Pf_Fn S133C with thrombin cleavage peptide.36 The amplified DNAs were transformed into the competent E. coli strain BL21 (DE). The thrombin cleavage peptide inserted Pf_Fn was overexpressed and purified as described previously.36 Briefly, cells were harvested by centrifugation and subsequently resuspended in lysis buffer (50 mM phosphate, 100 mM NaCl, pH 6.5). The suspension was treated with lysozyme (50 μg/mL) and incubated for 30 min on ice. The solution was centrifuged 2333

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Figure 2. Characterization of Gd(III)-chelating agent-conjugated protein cages. Molecular mass measurements of dissociated subunits of (A) P22 K118C WB capsids and (B) hPf_Fn: untreated (bottom spectra), Gd(III)−DTPA-conjugated (middle spectra), and Gd(III)−DOTA-conjugated ones (top spectra). The calculated and observed molecular masses are indicated. (C) Native agarose gel analyses of untreated P22 K118C WB capsids and Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids. (D) Transmission electron micrographic images of Gd(III)− DOTA (top) or Gd(III)−DTPA (bottom) conjugated P22 K118C WB capsids stained with 2% uranyl acetate. 18.24 and 36.26 μs respectively, and the between 90° and 180° pulses was 2.00 ms. For each run, 15 scans were collected with a recycle delay of 10.00 s. The gain was set to 50 dB for hPf_Fn and 47 dB for P22 WB. In Vitro Phantom Imaging. Representative phantom was prepared with tubes of Gd−DOTA (43 μM), Gd−DTPA(43 μM), P22, and tap water. The T1-weighted MR images of the phantom were obtained using RAREVTR sequence using a volume coil with 4.7T MRI scanner (Bruker Biospec). The imaging parameters were as follows: The imaging parameters were as follows: TR = 70, 140, 210, 300, 400, 600, 800, 1000, 2000, 3000, 6000, and 9000 ms, TE = 8 ms, and flip angle =90°. For clarity, three short TRs (140, 400, 1000 ms) were chosen and presented in the main text for the demonstration of maximal signal enhancement of Gd(III)−DTPA conjugated P22 K118C WB capsids due to reduced T1 relaxivity. The relaxivities of P22 WB capsids conjugated with Gd(III)-DOTA or Gd(III)-DTPA were estimated by RAREVTR at three different concentrations (43, 215, and 430 μM) In Vivo Imaging of a Mouse’s Vascular System. 3D-FLASH images of BALB/C nude (orient-bio) were acquired using a volume coil with 4.7T MRI scanner (Bruker Biospec) before and 100 min after the injections of Gd−DTPA-loaded conjugated viral capsids (total injected dose of Gd: 25 μM Gd(III)/kg). The images were reconstructed using maximum intensity projection (MIP) protocol with Bruker Paravision software (PV4). The imaging parameters were as follows: flip angle = 60°, TR = 30 ms, TE = 2.3 ms, field of view = 16 × 16 × 32 mm3, matrix = 64 × 64 × 128, NEX = 1.

diameter pores (Figure 1)32,34 with a single site mutation (K118C), were prepared as has been previously described.33 Residue 118C of the P22 K118C WB capsid is known to be exposed to the interior surface of P22 WB capsids, and it has been used as a site for the attachment of small molecules.38 It was thought that twelve 10-nm pores might allow for efficient conjugation of Gd(III)-chelating agent complexes to the interior surface (K118C) of the capsids, as well as the rapid water exchange that is essential for efficient MR relaxation pathways.39 As a smaller protein cage for purposes of comparison, we used Pf_Fn, which does not have large holes as P22 WB capsids do. Gd(III)-chelating agent complexes were too large to freely diffuse into the interior cavity of wild-type Pf_Fn and we could not conjugate Gd(III)-chelating agent complexes to the interior surface of wild-type Pf_Fn. To generate holes that would be large enough for Gd(III)-chelating agent complexes to pass through and allow for rapid water accessibility, we genetically introduced thrombin cleave peptide into the flexible loop region of Pf_Fn, which is located at a 4fold axis of symmetry exposed on the surface of protein cage, as has been described previously.37 Recently, we demonstrated that thrombin effectively cleaves the inserted peptide, generating 1.5-nm diameter holes at 4-fold axes, without disrupting the cage architecture.36 In addition, we substituted serine residue at position 133 with cysteine (S133C), which is situated in the interior surface of Pf_Fn in order to attach the Gd(III)-chelating agent complexes securely inside.37 To construct Gd(III)-chelating agent complexes/protein cage conjugates, either DOTA-mal or DTPA-mal was first incubated with an excess amount of Gd(III) to saturate the Gd(III)-chelating agent complexes. Subsequently, the P22 K118C WB capsids and Pf_Fn with holes (hPf_Fn) were

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RESULTS AND DISCUSSION To systematically investigate the effect of the templating macromolecule size on T1 relaxivity, we chose two different protein cages: P22 viral capsids (outer diameter: 64 nm) and ferritin (outer diameter: 12 nm) that was isolated from hyperthermophile Pyrococcus furiosus (Pf_Fn). P22 WB capsids, which consist of 360 identical subunits having twelve 10-nm2334

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Figure 3. Measurements of T1 SR time (time range, 0−20 000 ms) of P22 K118C WB (A), hPf_Fn (B), and P22 S133C WB (C) capsids conjugated with Gd(III)−DTPA (bottom graphs) and Gd(III)−DOTA (top graphs) complexes using the TD-NMR, at 1.4T, 40 °C. The inverse values of the T1 relaxation time (1/T1) of each construct were plotted against the Gd(III) ion concentration (insets).

treated with either Gd(III)−DOTA-mal or Gd(III)−DTPAmal complexes for attachment. The extent of modification per cage of either the P22 K118C WB capsids or hPf_Fn was determined by ESI-TOF MS, following the dissociation of the capsids (Figure 2A,B).40 The molecular masses of P22 K118C WB capsid subunits that were treated with either Gd(III)−DOTA-mal or Gd(III)−DTPAmal complexes were determined to be 47278.0 and 47267.0 Da, respectively, which are in an excellent agreement with the corresponding calculated values of 47278.9 and 47267.9 Da (Figure 2A). The molecular masses of the hPf_Fn subunits that were treated with either Gd(III)−DOTA-mal or Gd(III)− DTPA-mal complexes were also measured as 18432.0 and 18421.0 Da, respectively, which are in a great agreement with the corresponding calculated values of 18434.4 and 18423.4 Da (Figure 2B). These data suggest that each subunit of the P22 K118C WB capsids and hPf_Fn was modified with only one Gd(III)-chelating complex without any side reaction. As a result, each P22 K118C WB and hPf_Fn possesses 360 and 24 Gd(III)-chelating complexes per cage, respectively (Figure 2A,B). To confirm the Gd-content of Gd(III)−DTPAconjugated P22 K118C WB capsids independently, inductively coupled plasma-mass spectrometry (ICP-MS) measurement was performed and the Gd-content was determined to be 0.83 Gd(III) per subunit, which is slightly lower than the value determined by MS probably due to slight sample loss during ICP-MS sample preparation. To avoid overestimation of relaxivity values of Gd(III)-chelating agent conjugated P22 K118C, we used the Gd-content determined by MS hereafter. The biophysical properties of the Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids and hPf_Fn were individually characterized. Native agarose gel is sensitive to changes in the charge, shape, and size of viral

capsids. The P22 K118C WB capsids and Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids migrated to the same position, suggesting that Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids maintain their shell integrity and shape even after conjugation (Figure 2C). TEM images of negatively stained Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids confirmed their intact cage architecture with a uniform size distribution regardless of the modification of the interior surface of the capsid (Figure 2D). The shell integrity of Gd(III)−DOTA- or Gd(III)−DTPAconjugated hPf_Fn was examined using size exclusion chromatography (SEC) and TEM (Supporting Figure S1). With SEC, the Gd(III)−DOTA- or Gd(III)−DTPA-conjugated hPf_Fn eluted at the same position as untreated hPf_Fn suggesting that the interior modification with Gd(III)-chelate agent complexes does not significantly alter the cage architecture or overall size of hPf_Fn. TEM images of the negatively stained Gd(III)−DOTA- or Gd(III)−DTPA-conjugated hPf_Fn also confirmed their intactness and uniform size (Figure S1). To examine the size effect of templating macromolecules on the MR relaxivity, we measured the T1 and T2 relaxation times of the Gd(III)-chelating agent-conjugated hPf_Fn and the P22 K118C WB capsids using TD-NMR (Bruker Minispec MQ60 TD-NMR). The T1 saturation recovery (SR) time method was utilized (saturation recovery time range, 0−20 000 ms) to measure the T1 relaxation times of the Gd(III)-chelating agentconjugated P22 K118C WB capsids and hPf_Fn using 60 MHz at 40 °C. As the Gd(III) concentration increased, the T1 relaxation times became shorter (Figure 3A,B and Supporting Table S1), suggesting that a myriad of Gd(III) complexes accelerated the recovery of the net magnetization.41 To obtain 2335

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the relaxivity values (ionic r1) of the Gd(III)-chelating agent conjugated P22 K118C WB capsids and hPf_Fn, the inverse values of the T1 relaxation time (1/T1) of each construct were plotted against the Gd(III) concentration ([Gd(III)]), and the slopes of each plot were determined (Figure 3, insets). It was found that the values of 1/T1 increased linearly as the [Gd(III)] increased (Figure 3 and Figure S2). The relaxivity values of the Gd(III)-chelating agentconjugated P22 K118C WB capsids and hPf_Fn were calculated respectively using the following equation on the basis of the measured T1 relaxation time values: Relaxivity (Ionic r1) =

⎛ 1 ⎜ − ⎝ T1,conjugated

conjugated P22 K118C WB capsids and conventional Gd(III)− DOTA agent (Dotarem) at 37 °C (Figure S4 and Table S4), and we did not observe significant difference between them, suggesting that all the relaxivity values we obtained are clinically relevant. Although P22 WB capsids and hPf_Fn have holes that are large enough for water to pass through rapidly,33,36 the interior cavities of these cages may not be accessible due to spatial constraints. To test this possibility, we prepared P22 WB capsids having cysteines on their exterior surface (S133C)34 using a previously described method,33 conjugated Gd(III)chelating agents (Gd(III)−DOTA-mal and Gd(III)−DTPAmal) in the same way, and compared with the relaxivity of the P22 K118C WB capsids conjugated with Gd(III)-chelating agents. Mass spectrometric analysis indicated that each subunit of the P22 S133C WB capsids was conjugated with only one Gd(III)-chelating agent (Figure S5A) per subunit same as the P22 K118C WB capsids, and TEM images confirmed their shell integrity after conjugation (Figure S5B). The relaxivity values of the Gd(III)−DOTA- or Gd(III)− DTPA-conjugated P22 S133C WB capsids were also measured under the same condition as those of the P22 K118C WB capsids using TD-NMR. They showed similar relaxation patterns to those of the P22 K118C WB capsids (Figure 3C) and exhibited relaxivity values of 26.2 mM−1 s−1 and 53.8 mM−1 s−1 (Table 1), respectively, which are similar or slightly lower than those of P22 K118C WB capsids (35.8 mM−1 s−1 and 57.0 mM−1 s−1, respectively). These results suggest that the conjugated Gd(III) within the cavity makes sufficient contact with the surrounding water due to its large holes and that the conjugation position is not a critical issue for P22 WB capsids. When we consider one capsid as a contrast agent unit, the P22 K118C WB capsids conjugated with Gd(III)−DTPA exhibited a relaxivity value as high as 20,503 mM−1 s−1 (Table 1). By contrast, the Gd(III)−DTPA-conjugated hPf_Fn exhibited a relaxivity value of 590 mM−1 s−1 (Table 1), 30 times lower than those of the P22 WB capsids, a result that is probably due to the combination of its relatively small size and low Gd(III) holding capacity (24 hPf_Fn vs 360 P22 WB capsid subunits). Recently, remarkable amounts of payload of Gd(III) (approximately 1900 Gd(III) ions) inside each P22 WB capsid have been achieved to maximize the total relaxivity through the branched oligomerization of p-SCN-Bn−DTPA−Gd(III) and 2-azido-1-azidomethyl-ethylamine (DAA) via stepwise click reactions.29 While they obtained a relaxivity value as high as 41 300 mM−1 s−1 per capsid, which is almost twice higher than the current study, the ionic relaxivity of them was 21.7 mM−1 s−1, which is almost half the value of the current study, probably due to the different configuration (branched network and linker length) and density of Gd(III) ions and different experimental conditions (0.65T, 25 °C).29 Together with these data, we could conclude that the relaxivity of the conjugated Gd(III) highly depends upon the size of the templating macromolecules, but not on the positions conjugated, at least in the case of P22 WB capsids. To evaluate the possible usage of Gd-(III)-chelating agentconjugated P22 viral capsids for in vivo MR imaging, we first conducted in vitro phantom imaging with Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids, which exhibited the highest relaxivity values (Table 1) according to TD-NMR, and untreated P22 K118C WB capsids and water as controls as shown in Figure 4. The T1-weighted MR images of the phantom were obtained by a volume coil with a 4.7T MRI

1 ⎞ ⎟ T1,cage ⎠

[Gd(III)]

where T1,conjugated is the T1 relaxation time of the Gd(III)chelating agent-conjugated P22 K118C WB capsids and hPf_Fn, T1,cage is the T1 relaxation time of the untreated P22 K118C WB capsids and hPf_Fn, and [Gd(III)] is the Gd(III) concentration of the samples. Although the Gd(III)-chelating agent-conjugated hPf_Fn exhibited significantly high relaxivity values (19.9 mM−1s−1 with DOTA and 24.6 mM−1s−1 with DTPA) as compared to the free Gd(III)-chelating agents (4−5 mM−1s−1),42 the Gd(III)-chelating agent-conjugated P22 WB capsids showed much higher relaxivity values (35.8 mM−1s−1 with DOTA and 57.0 mM−1s−1 with DTPA), which were approximately twice as large as those of the hPf_Fn conjugates (Table 1). These data suggest that the large-sized P22 WB Table 1. Relaxivity Values of Gd(III)-Chelating AgentConjugated Capsids at 60 MHz (40 °C) capsids P22 K118C WB hPf_Fn P22 S133C WB

conjugated Gd(III)chelating agents

T1 relaxivity (mM−1 s−1)

relaxivity/capsid (mM−1 s−1)

Gd(III)−DOTA Gd(III)−DTPA Gd(III)−DOTA Gd(III)−DTPA Gd(III)−DOTA Gd(III)−DTPA

35.8 57.0 19.9 24.6 26.2 53.8

12880.9 20502.6 476.4 589.9 9423.9 19354.8

capsids exhibit a much slower tumbling rate as compared to both free Gd(III)-chelating agent complexes and the smallsized hPf_Fn in solution resulting in a faster T1 relaxation rate.43 The construction of viral capsid-based MRI contrast composites has been explored under the diverse experimental conditions and most of studies exhibited the relaxivity values of 10−30 mM−1 s−1 depending on the experimental conditions (magnetic field and temperature), types of viral capsids, and Gd(III)-chelating agent complexes.42 We obtained similar or higher ralaxivity values with hPf_Fn and P22 K118 WB. It is known that the optimal ratio of the longitudinal and transverse relaxivities (r1/r2) for T1 contrast agent ranges from 0.5 to 0.9.29 The T2 relaxation times of the Gd(III)-chelating agentconjugated protein cages were measured by TD-NMR (Figure S3 and Table S2) and the measured r1/r2 values of Gd(III)chelating agent-conjugated protein cages exhibited the highest and lowest ratios of 0.78 and 0.55, respectively, for magnetic fields 1.4T at 40 °C representing that these complexes have the potential to be optimal T1 contrast agents (Table S3). To obtain clinically relevant relaxivity values of Gd(III)-chelating agent-conjugated P22 K118C WB capsids, we attempted to measure T1 and T2 relaxation times of Gd(III)-chelating agent2336

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conjugated P22 K118C WB capsids were 7.9 mM−1 s−1 and 13.7 mM−1 s−1, respectively, at 4.7 T (Figure S6). Considering the relaxivities for commercial Dotarem (Gd(III)−DOTA, 2.8 mM−1 s−1) and Magnevist (Gd(III)−DTPA, 3.2 mM−1 s−1), significant enhancements in T1 relaxivity were maintained at high field magnet (4.7T, Figure S6), as observed at low-field magnet (1.4T, Table 1). The P22 K118C WB capsids have an outer diameter of 64 nm, and the injection of Gd(III)−DTPA-conjugated P22 K118C WB capsids into the blood vessels may allow them to stay in the bloodstream with long enough circulation time to obtain high resolution MR vascular images, which might be useful in the detection of blood vessel-related diseases, such as arteriosclerosis and stroke.15 To investigate this hypothesis, the Gd(III)−DTPA-conjugated P22 K118C WB capsids were administrated to a BALB/C nude mouse. The Gd(III)− DTPA-conjugated P22 K118C WB capsids were chosen because of their highest relaxivity. The Institutional Animal Care and Use Committee of the Korea Basic Science Institute approved the in vivo animal experiments conducted in this study. 3D-FLASH images of the BALB/C nude mouse were acquired using a volume coil with 4.7T MRI scanner both before and 100 min after the injections of the Gd(III)−DTPAconjugated P22 K118C WB capsids (25 μM Gd(III)/kg). The images were reconstructed using the MIP protocol with Bruker Paravision software (PV4). The imaging parameters were flip angle = 60°, TR = 30 ms, TE = 2.3 ms, field of view = 16 × 16 × 32 mm3, matrix = 64 × 64 × 128, and NEX = 1. Axial, sagittal, and coronal MIP images from the mouse’s head to the top of its thorax, respectively, are shown in Figure 5. Following the injection of the Gd(III)−DTPA-conjugated P22 viral capsids, clear enhancement of the blood vessels can be seen in the images, including the carotid, mammary arteries, the jugular vein, and the superficial vessels of the head at an

Figure 4. T1-weighted in vitro phantom images of P22 K118C WB capsids conjugated with Gd(III)−DOTA (A), Gd(III)−DTPA (B), or water (C), and untreated P22 K118C WB capsids (D) at 4.7T, obtained using a RAREVTR sequence at three TRs (140, 400, 1000 ms).

scanner (Bruker BioSpec) using a RAREVTR sequence. The imaging parameters were as follows: TR = 70, 140, 210, 300, 400, 600, 800, 1000, 2000, 3000, 6000, and 9000 ms; TE = 8 ms; and flip angle = 90°. For clarity, only three TRs (140, 400, 1000 ms) were chosen and presented for the demonstration of signal enhancement at short TRs due to accelerated T1 relaxivity (Figure 4). At a short TR, the MR signal intensity of the Gd(III)−DTPA-conjugated P22 K118C WB capsids was the greates,t which was in agreement with the measured shortest T1 relaxation time (Table 1). The MR signal intensities of both untreated P22 K118C WB capsids and water were almost identical (Figure 4), suggesting that viral capsids themselves do not significantly have effect on the T1 relaxivity or contrast enhancement results from Gd(III)-chelating agent complexes conjugated to P22 K118C WB capsids. Measured relaxivities for the Gd(III)−DOTA- and Gd(III)−DTPA-

Figure 5. Axial pre- and postinjection (100 min after injection) 3D-FLASH images of BALB/C nude mouse (A-1 and B-1, respectively), along with the mean signal intensities their respective vascular regions (marked by a red-circle). MIPs of preinjection images are shown along the sagittal (A-2) and coronal (A-3) directions. The corresponding postinjection (100 min) images are shown along the sagittal (B-2) and coronal (B-3) directions as well for direct comparison. The identified vessels are labeled in B-2 and B-3. Three-dimensional rotation movies are provided as supplememtary data to provide clear visualization (SI movies). 2337

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isotropic resolution of 250 μm due to the reduced intravascular T1 relaxation (Figure 5). The monodisperse and multivalent structure of P22 viral capsids allows us to manipulate them genetically and chemically in a controlled manner and their large size allows us to observe mouse vasculature in vivo. In addition to the positions in which Gd(III)-chelating agents are attached, P22 viral capsids have an exterior surface for the presentation of various types of targeting ligands and dye molecules, as well as additional addressable sites on the interior surface for the attachment of molecular cargoes, such as drugs. Combined with additional targeting and drug encapsulation capabilities, P22 viral capsidbased MRI contrast conjugates with further biodistribution, blood half-life, and in vivo toxicity studies may provide new opportunities for developing new theranostic nanoplatforms.

ACKNOWLEDGMENTS This work was supported by Advanced Research Center (No. 2012-0008996, No. 2012-0008999) of MEST through NRF of Korea and the Collaborative Research Program for Convergence Technology (Seed-11-6) of the Korea (KRCF).

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CONCLUSIONS In this study, we constructed Gd(III)-chelating agent conjugated P22 viral capsids using genetic and chemical modifications and demonstrated significant enhancements in relaxivity. Contrast enhancements in vitro and in vivo were confirmed by in vitro phantom imaging and in vivo vascular imaging of a mouse. The effect of the templating macromolecule size was investigated by comparing the relaxivities of Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB capsids and hPf_Fn. TD-NMR measurements demonstrated that large P22 K118C WB conjugated with Gd(III)chelating agents has higher relaxivity values than those of small hPf_Fn, probably due to the decrease in the rates of molecular tumbling. The relaxivity values of Gd(III)−DOTA- or Gd(III)−DTPA-conjugated P22 K118C WB and P22 S133C WB, which conjugate Gd(III)-chelating agents on the interior and exterior surfaces of the capsids, respectively, were similar. This implies that the conjugation positions do not significantly influence the relaxivity values of the Gd(III)-chelating agentP22 WB capsid conjugates. In vitro phantom imaging of the Gd(III)-chelating agent-conjugated P22 WB capsids was conducted and the Gd(III)−DTPA-conjugated P22 K118C WB exhibited the brightest image in a short TR due to a reduced T1 value. Using the Gd(III)−DTPA-conjugated P22 K118C WB, we demonstrated the potential use of P22 viral capsids−Gd(III) conjugates as in vivo MRI contrast agents by imaging the blood vessels of a mouse including the carotid, mammary arteries, the jugular vein and, the superficial vessels of the head. ASSOCIATED CONTENT

S Supporting Information *

Detailed data for Gd(III)-chelating agent conjugated hPf_Fn, T2 relaxation times of Gd(III)-chelating agent conjugated protein cages, tables of T1 and T2 relaxation values, and supporting movies are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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*Tel: +82-52-217-2523. Fax: +82-52-217-2509. E-mail: [email protected] (S.K.); [email protected] (H.C.). Notes

The authors declare no competing financial interest. 2338

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