Engineered Nonviral Protein Cages Modified for MR Imaging

Diagnostic medical imaging utilizes magnetic resonance (MR) to provide anatomical, functional, and molecular information in a single scan. Nanoparticles are often labeled with Gd(III) complexes to amplify the MR signal of contrast agents (CAs) with large payloads and high proton relaxation efficiencies (relaxivity, r1). This study examined the MR performance of two structurally unique cages, AaLS-13 and OP, labeled with Gd(III). The cages have characteristics relevant for the development of theranostic platforms, including (i) well-defined structure, symmetry, and size; (ii) the amenability to extensive engineering; (iii) the adjustable loading of therapeutically relevant cargo molecules; (iv) high physical stability; and (v) facile manufacturing by microbial fermentation. The resulting conjugates showed significantly enhanced proton relaxivity (r1 = 11–18 mM–1 s–1 at 1.4 T) compared to the Gd(III) complex alone (r1 = 4 mM–1 s–1). Serum phantom images revealed 107% and 57% contrast enhancements for Gd(III)-labeled AaLS-13 and OP cages, respectively. Moreover, proton nuclear magnetic relaxation dispersion (1H NMRD) profiles showed maximum relaxivity values of 50 mM–1 s–1. Best-fit analyses of the 1H NMRD profiles attributed the high relaxivity of the Gd(III)-labeled cages to the slow molecular tumbling of the conjugates and restricted local motion of the conjugated Gd(III) complex.


General Methods
Unless otherwise indicated, all reactions were performed under a nitrogen atmosphere using oven-dried glassware. Anhydrous solvents were used in all reactions and obtained from a J.C.
Meyer solvent system (Laguna Beach, CA). Thin-layer chromatography (TLC) was performed on EMD 60 F254 silica gel plates. Standard grade 60 Å 230-400 mesh silica gel was used for normalphase column chromatography. Unless otherwise stated, all silica gel columns were flashed with air. 1 H and 13 C NMR spectra were obtained on a Bruker 500 MHz Avance III NMR spectrometer with DCH cryoprobe. ESI-MS was performed on a Bruker AmaZon-SL spectrometer.
Cyclen was purchased from Strem Chemical. Ethylenediaminetetraacetic acid (EDTA) was purchased from AppliChem GmbH. All other buffer components, salts, and reagents were purchased from Sigma Aldrich, Merck KGaA, Fisher Scientific, Acros Organics, or TCI and used without purification.
Analytical HPLC-MS was performed on an Agilent 1260 Infinity II HPLC system with an in-line Agilent 6120 Quad mass spectrometer. Semi-preparative HPLC was performed on an Agilent PrepStar 218 equipped with an Agilent 1260 Infinity diode array detector. HPLC purifications utilized deionized water (18.2 MΩ·cm) obtained from a Millipore Q-Guard System and HPLC grade MeCN, formic acid, and ammonium hydroxide (all obtained from Fisher Scientific).
All restriction enzymes, T4 polynucleotide kinase (PNK), Phusion ® High-Fidelity DNA polymerase, and T4 DNA ligase were obtained from New England BioLabs. Oligonucleotides were synthesized by Microsynth AG. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Fluorochem. Kanamycin sulfate was obtained from AppliChem GmbH. Ni-NTA agarose resin was obtained from Qiagen GmbH. Amicon Ⓡ Ultra centrifugal filters were purchased from Merck and PD Mini-/MidiTrap desalting columns from GE Healthcare. Millipore purification system was used to obtain Milli-Q water. All buffers were prepared using Milli-Q (MQ) water, pH adjusted for the temperature at which the buffer was used, and sterile-filtered (0.2 µm membrane filter).

Synthesis of benzyl acrylate
Benzyl acrylate was synthesized following literature procedure. 1

HR-MS Spectrum of Gd-C4-IA
Samples were prepared at 1 mg/mL in MQ H2O, and were analyzed using an Agilent 6230 Time of Flight (TOF) mass spectrometer with an electrospray ionization (ESI) source, attached to an Agilent 1200 series HPLC stack. Data was acquired on Agilent Mass Hunter Acquisition software and analyzed on Agilent Mass Hunter Qualitative Analysis software.

Cloning of OP variants
To provide specific handles for conjugation, one, two or three cysteine mutations were introduced per OP protein monomer, affording variants OP-1intC, OP-2intC, and OP-3intC, respectively. The residues targeted from mutation were Ser38, Arg66 and Arg103. The variant OP-1intC, which contains the S38C mutation, has been previously described. 4 As such, plasmid pET29b(+)_OPS38C was used as a basis for generation of the OP-2intC (S38C, R103C), and OP-3intC (S38C, R66C, R103C) variants. The genes for these variants were generated by "QuikChange" (Agilent) site-directed mutagenesis. The primers used for OP-2intC were:

Protein expression of AaLS-13 and OP cysteine mutants
AaLS-13 and OP cages were expressed in E. coli strain BL21-Gold (DE3) which was transformed with either pMG211-AaLS-13 or the appropriate pET29b(+)-OP. Cells were grown at 37 °C in selective LB medium until the OD600 reached ~0.6-0.8, at which point protein production was induced by adding IPTG to a final concentration of 0.1 mM. After culturing at 25 °C for 22 hours, cells were harvested by centrifugation at 5,000 g and 4 °C for 10 min. The cell pellet was stored at -20 °C until purification.

Cell lysis and Ni-NTA purification of AaLS-13
The cell pellet from a 400 mL culture was re-suspended in 20 mL lysis buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole) supplemented with lysozyme (0.1 mg/mL), DNase I (5 μg/mL), RNase A (5 μg/mL), and a protease inhibitor cocktail (Sigma). The lysate was incubated for 1 hour at room temperature. After lysis by sonication (using a 50% duty cycle and 80% amplitude setting on ice for 2 min, followed by cooling on ice for 2 min, repeated 5 times) and clearance by centrifugation at 9,500 g and 25 °C for 25 min, the supernatants were loaded S-38

Cell lysis and Ni-NTA purification of OP variants
Each cell pellet from 800 mL of culture was resuspended in 10 mL of lysis buffer (50 mM sodium

MS Spectra of Gd-Protein Conjugates
The stability of Gd(III)-labeled protein cages was evaluated by MS. DataAnalysis was used for data analysis and processing. All proteins were deconvoluted using MaxEntropy deconvolution. S-44 In, 159 Tb (chosen as internal standards for data interpolation and machine stability). Instrument performance is optimized daily through autotuning followed by verification via a performance report (passing manufacturer specifications).

UV-vis spectroscopy of Gd(III) complexes
Gd-C4-NH2 and Gd-C4-IA were dissolved in MQ H2O at 1 mM and the UV-vis spectra obtained to confirm the lack of absorption at 280 nm, the wavelength used to quantify protein concentration.
The absorption at 280 nm was measured for Gd-protein conjugates in 50 mM sodium phosphate

Relaxivity measurements at 7 T
A 60 µL aliquot of each sample from 1.4 T measurements was pipetted into flame sealed Pasteur pipettes. The pipette tips containing solution were scored, separated, and sealed with parafilm to make small capillaries containing solution. These capillaries were imaged using a Bruker

Phantom image measurements at 3 T
The Eppendorf tubes from experiment 2 were imaged using a Siemens 3 T Prisma MR imaging spectrometer. 1 relaxation times were measured using a dual gradient echo method (StaGE) with two different flip angles. 1 analysis was carried out using the image sequence analysis tool in Paravision 6.0 software (Bruker) to selected ROIs for each axial slice. S-60 2 -map pulse sequence, with static TR (5000 ms) and 32 fitted echoes in 11 ms intervals (11, 22, ..., 352 ms). Imaging parameters were as follows: field of view, 25 × 25 mm; matrix size, 256 × 256; number of axial slices, 4; slice thickness, 1.0 mm; and averages, 3. 1 and 2 analysis was carried out using the image sequence analysis tool in Paravision 6.0 software (Bruker) with monoexponential curve-fitting of image intensities of selected ROIs for each axial slice.

Equation for ΔR1
Percent change in relaxation rate is determined by Eq. S3. 6

Phantom image analysis
Determined contrast enhancement as percent change in relaxation rate (Eq. S3) with serum phantom image data from Trial 1 and Trial 2 T1 and T2 measurements (Figures S40-S43).