[68Ga]Ga-THP-tetrazine for bioorthogonal click radiolabelling: pretargeted PET imaging of liposomal nanomedicines

Pretargeted PET imaging using bioorthogonal chemistry is a leading strategy for the tracking of long-circulating agents such as antibodies and nanoparticle-drug delivery systems with short-lived isotopes. Here, we report the synthesis, characterisation and in vitro/vivo evaluation of a new 68Ga-based radiotracer [68Ga]Ga-THP-Tetrazine ([68Ga]Ga-THP-Tz) for bioorthogonal click radiochemistry and in vivo labelling of agents with slow pharmacokinetics. THP-tetrazine (THP-Tz) can be radiolabelled to give [68/67Ga]Ga-THP-Tz at room temperature in less than 15 minutes with excellent radiochemical stability in vitro and in vivo. [68Ga]Ga-THP-Tz was tested in vitro and in vivo for pretargeted imaging of stealth PEGylated liposomes, chosen as a leading clinically-approved platform of nanoparticle-based drug delivery, and for their known long-circulating properties. To achieve this, PEGylated liposomes were functionalised with a synthesised transcyclooctene (TCO) modified phospholipid. Radiolabelling of TCO-PEG-liposomes with [68/67Ga]Ga-THP-Tz was demonstrated in vitro in human serum, and in vivo using both healthy mice and in a syngeneic cancer murine model (WEHI-164 fibrosarcoma). Interestingly in vivo data revealed that [68Ga]Ga-THP-Tz was able to in vivo radiolabel liposomes present in the liver and spleen, and not those in the blood pool or in the tumour. Overall, these results demonstrate the potential of [68Ga]Ga-THP-Tz for pretargeted imaging/therapy but also some unexpected limitations of this system.


Section 3. Characterisation of TCO-PEG-liposomes 1. Nanoparticle Tracking Analysis
The concentration and the hydrodynamic of synthesised TCO-PEG-liposomes were measured by NTA using NanoSight LM10 and NTA software v3.2 (Malvern Panalytical).The stock sample was diluted to achieve ~100 particles/viewing frame.Measurements were made in triplicates for 60 s with a 488 nm laser, for up to three serial dilutions of the sample.

Section 5. 68 Ga purification
All radioactive work was performed manually with all necessary radioprotection measures in place. 68Ga was manually eluted from 68 Ge/ 68 Ga generator with 0.1 N HCl.The peak radioactivity containing 1 mL elution was used for radiolabelling after buffering with 3.4 M Sodium acetate/1 M Sodium carbonate to pH 6. Buffered 68 Ga was used for all radiolabelling and i.v.injections after removal of colloids using saline pre-rinsed centrifugal filter MW cutoff 50 kDa.
The colloid formation during neutralisation of generator eluate competes with the radiolabelling of the chelator and provides an impure final reaction mixture requiring a need for purification of the radiolabelling mixture post labelling.To minimise the presence of colloids, post buffering of generator eluate to pH 6, buffered Ga was purified using a centrifugal spin filter which removed any colloids formed.This was verified using radioTLC as seen in figure S6.Section 8. PET/SPECT imaging reconstruction method PET/CT images were reconstructed using Tera-Tomo 3D reconstruction (400-600 keV energy window, 1-3 coincidence mode, 4 iterations and subsets) at a voxel size of (0.4 × 0.4 × 0.4) mm3 and corrected for attenuation, scatter, and decay.The data were binned into 17 frames (1 × 1, 10 × 3, 5 × 5 and 1 × 4 min) for dynamic analysis.SPECT images were reconstructed using HiSPECT standard method.
14 Section 9.In vivo pretargeted imaging at t = 4 hr time point

Section 10. In vivo biodistribution data
The tables below show the biodistribution data obtained from the in vivo experiments performed on this study.

Figure S2 .
Figure S2.Purification of THP-tetrazine: (A) HPLC chromatogram of the semi-prep HPLC method used for isolation of THP-tetrazine from the reaction mixture.Fraction collected between t r =29 min-33 min contained the THP-tetrazine; (B) Mass spectra of the THP-tetrazine crude reaction mixture showing the THP-tetrazine as the major product alongside unreacted Methyl-tetrazine amine and hydrolysed THP; and (C) Mass spectra of the THP-tetrazine fraction collected after semi-prep HPLC showing the pure isolated THP-tetrazine.

Figure S3 .
Figure S3.(A) Schematic representation of the synthesis of TCO-PL; (B) Characterisation of TCO-PL.NMR spectra of TCO-PL: The synthesised TCO-PL is characterised by the presence of characteristic peaks of TCO incorporated in the phospholipid: δ 2.5 -CH; 5.5 =CH in cyclooctene.The integration of the peak's accounts for 283 out of 293 protons.The integration is not quantitative due to the relative high intensity of protons from ethylene peak of the PEG2000 chain which decrease the resolution of other peaks.

Figure S4 .
Figure S4.The mass spectrum of the TCO-PL (A) and DSPE-PEG2000 amine (B): The envelope nature observed in both spectra is due to the polydispersity of the PEG2000 chain which is characterised by a difference of 44 between each peak within the envelope.The TCO-PL is characterised by the higher m/z=2700-3700 compared to the lower m/z for DSPE-PEG2000 amine m/z=2400-3200.

( 5
μL) of TCO-PL-liposomes or PEGylated liposomes were deposited on the carbon grids in Vitrobot Mark IV (FEI).The excess liquid was removed by blotting with filter paper (Agar Scientific); Parameters: blotting time = 2 s, wait time = 30 s, and blotting force = 2.The grids were instantly frozen in liquid ethane (−188 °C) and maintained in liquid N 2 (−196 °C) in a grid box and transferred into a cryo-transfer holder.CryoEM of these samples was recorded on TECNAI 12 G2 (FEI) system interfaced with a TemCam-F216 camera and operated using Temmenu v4 software (Tietz Video & Image Processing Systems GmbH, Germany).Parameters used to capture images are as follows: electron acceleration = 120 kV, magnification = 52,000×, acquisition time = 1 s, and spot size = 5.Section 4 Purification methods The following HPLC/FPLC methods were used: Method 1. Analytical HPLC method for LC/MS for characterisation of THP-tetrazine Solvent A= Water (0.1% Trifluoroacetic acid v/v), Solvent B= Acetonitrile (0.1% Trifluoroacetic acid v/v) Method 2. Semi-prep HPLC method for purification of THP-tetrazine Solvent A= Acetonitrile (0.1% Trifluoroacetic acid v/v), Solvent B= Water (0.1% Trifluoroacetic acid v/v) Method 3. Analytical radioHPLC method for characterisation of [ 68 Ga]Ga-THP-tetrazine Solvent A= Water (0.1% Trifluoroacetic acid v/v), Solvent B= Acetonitrile (0.1% Trifluoroacetic acid v/v) Method 4. Serum stability HPLC method Solvent A = phosphate buffered saline Method 5. Size exclusion purification method A G-25 size exclusion column pre-equilibrated with 15 column volumes of 0.9% filtered saline was used.The test sample for purification is applied to the column in 500 µL volume, followed by 750 µL fractions which are collected in separate vials.Liposomes elute in the first 750 µL fraction as seen in figure below.

Figure S5 .
Figure S5.Procedure for purification of liposomes using size exclusion chromatography column.Created with BioRender.com.

Figure S6 .
Figure S6.Purification of 68 Ga: The colloids generated after neutralisation of gallium generator eluate are removed and the different gallium samples pre and post purification are characterised using ITLC (A) Generator eluate; (B) Neutralised 68 Ga pre-purification and (C) Neutralised 68 Ga post-purification

Figure S8 .
Figure S8.HPLC SEC control serum stability run: (A) RadioHPLC chromatogram showing the elution of serum bound 68 Ga; (B) UV chromatogram (280 nm) of proteins present in human serum.

Figure S8 .
Figure S8.Pretargeting test in vivo group at t = 4 h(n=4): (A) Schematic of pretargeting test group; (B) PET image of animal administered with TCO-PL-liposomes at t= 0 h followed by administration of [ 68 Ga]Ga-THP-tetrazine at t = 3 h and PET scan at t = 4 h; (C) Image quantification performed on the PET images acquired at t = 4 h.