A generic ⁸⁹Zr labeling method to quantify the in vivo pharmacokinetics of liposomal nanoparticles with positron emission tomography

Figures & data

Figure 1 ⁸⁹Zr labeling of an FA-decorated FA-DFO-liposome through a room-temperature ligand exchange reaction between the ⁸⁹Zr(8-HQ)₄ complex and the encapsulated DFO in the liposomal aqueous cavity.

Abbreviations: FA, folic acid; DFO, deferoxamine; 8-HQ, 8-hydroxyquinoline; PEG₂₀₀₀, (polyethylene glycol)-2000.

Figure 2 TEM images and particle size distributions of (A) DFO-liposome, (B) FA-DFO-liposome, and (C) PEG-DFO-liposome.

Abbreviations: TEM, transmission electron microscopy; DFO, deferoxamine; FA, folic acid; PEG, polyethylene glycol.

Table 1 Optimization of ⁸⁹Zr radiolabeling of liposomes

Entrya	Lipophilic ligandb	Liposome (300 µL)	Isolated RCYs
1c	8-HQ	DFO-liposome	98%±2% (n=3)
2	8-HQ	DFO-liposome	98%±2% (n=5)
3	8-HQ	FA-DFO-liposome	98%±1% (n=6)
4	8-HQ	PEG-DFO-liposome	97%±1% (n=3)
5c	8-HQ	Blank liposome	22%
6	N/A	DFO-liposome	81%±11% (n=3)
7	2-HQ	DFO-liposome	83%

Notes:

a ⁸⁹Zr (5.0 MBq) in HEPES buffer (100 µL, 1.0 M, pH 7.0).

b PBS (50 µL) for entry 6, or 2-HQ or 8-HQ (23 µg, 0.16 µmol) in HEPES buffer (50 µL, 10 mM, pH 7.0) for the rest of the experiments.

c One hundred and fifty microliters of the corresponding liposomes were used. Data presented as mean ± standard deviation.

Abbreviations: RCYs, radiochemical yields; 8-HQ, 8-hydroxyquinoline; DFO, deferoxamine; FA, folic acid; PEG, polyethylene glycol; N/A, not available; 2-HQ, 2-hydroxyquinoline; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PBS, phosphate-buffered saline.

Figure 3 (A and B) Sequential axial PET images at 6, 24, and 48 h and three-dimensional PET images at 24 h post-i.v. injection of two KB tumor xenograft-bearing (in the front-right flank indicated by white arrows) CD1 nude mice that received either the ⁸⁹Zr-FA-DFO-liposome or the ⁸⁹Zr-DFO-liposome. (C and D) Radioactivity accumulation in selected organs extracted from the corresponding ⁸⁹Zr-FA-DFO-liposome or ⁸⁹Zr-DFO-liposome PET scans.

Abbreviations: PET, positron emission tomography; i.v., intravenous; FA, folic acid; DFO, deferoxamine; PEG, polyethylene glycol; ID, injected dose.

Figure 4 (A) Ex vivo biodistribution of the ⁸⁹Zr-FA-DFO-liposome, ⁸⁹Zr-DFO-liposome, and ⁸⁹Zr-DFO in KB tumor xenograft-bearing CD1 nude mice (n=3) and ⁸⁹Zr-PEG-FA-DFO-liposome in healthy CD1 mice (n=3) at 48 h post-i.v. injection. (B) Tumor-to-organ ratio of ⁸⁹Zr-FA-DFO-liposome and ⁸⁹Zr-DFO-liposome at 48 h post-i.v. injection.

Abbreviations: FA, folic acid; DFO, deferoxamine; PEG, polyethylene glycol; i.v., intravenous; ID, injected dose.

Figure S1 HPLC chromatogram of crude reaction mixture of ⁸⁹Zr and 8-HQ. ⁸⁹Zr(8-HQ)₄ with retention time of 16.6 min detected by radioactivity detector. HPLC chromatography from UV detector (A); HPLC chromatography from radioactivity detector (B).

Abbreviations: HPLC, high-performance liquid chromatography; 8-HQ, 8-hydroxyquinoline; UV, ultraviolet; UV-A, ultraviolet absorption; ChA, chromatography; CPS, counts per second; Reg, region.

Figure S2 HPLC chromatogram of crude reaction mixture of ⁸⁹Zr and DFO. ⁸⁹Zr-DFO with retention time of 16.4 min detected by radioactivity detector. HPLC chromatography from UV detector (A); HPLC chromatography from radioactivity detector (B).

Abbreviations: HPLC, high-performance liquid chromatography; DFO, deferoxamine; UV, ultraviolet; UV-A, ultraviolet absorption; ChA, chromatography; CPS, counts per second.

Figure S3 RadioTLC of free ⁸⁹Zr, ⁸⁹Zr-DFO, and ⁸⁹Zr(8-HQ)₄.

Abbreviations: TLC, thin-layer chromatography; DFO, deferoxamine; 8-HQ, 8-hydroxyquinoline.

Figure S4 Size exclusion purification of ⁸⁹Zr-DFO-liposome eluted in the fraction from 0.4 to 2.2 mL.

Abbreviation: DFO, deferoxamine.

Figure S5 KB cell uptake of ⁸⁹Zr-FA-DFO-liposome and ⁸⁹Zr-DFO-liposome in vitro. The results are presented as % incubation dose per million cells.

Abbreviations: FA, folic acid; DFO, deferoxamine.

Table S1 Characterization of a thermosensitive DFO-liposome, an FA-decorated active targeting FA-DFO-liposome, and a PEG-DFO-liposome: mean size, PDI, and zeta potential

Samples	Size (nm)	PDI	Zeta potential (mV)
DFO-liposome	99.8±0.9	0.120	−22.2
FA-DFO-liposome	102.9±2.3	0.110	−20.1
PEG-DFO-liposome	101.2±3.1	0.126	−23.1

Note: Values are presented as the mean ± standard deviation (n=3).

Abbreviations: DFO, deferoxamine; FA, folic acid; PEG, polyethylene glycol; PDI, polydispersity index.

Table S2 Biodistribution of ⁸⁹Zr-FA-DFO-liposome, ⁸⁹Zr-DFO-liposome, and ⁸⁹Zr-DFO in KB tumor xenograft-bearing CD1 nude mice (n=3) at 48 h post-i.v. injection and ⁸⁹Zr-PEG-DFO-liposome in healthy CD1 mice (n=3) at 48 h post-i.v. injection expressed as % ID/g

Organ	^89Zr-FA-DFO-liposome (n=3)	^89Zr-DFO-liposome (n=3)	^89Zr-DFO (n=3)	^89Zr-PEG-DFO-liposome (n=3)
Blood	0.087±0.103	0.146±0.079	N/A	0.137±0.120
Liver	0.570±0.226	1.083±0.559	0.201±0.054	0.520±0.162
Heart	0.093±0.073	0.215±0.099	0.018±0.005	0.156±0.090
Kidney	3.926±2.898	1.321±0.117	2.054±0.275	4.872±2.161
Colon	0.105±0.065	0.526±0.700	0.204±0.154	0.309±0.206
Lung	0.203±0.142	0.328±0.112	0.209±0.231	0.142±0.067
Muscle	0.051±0.042	0.093±0.037	0.058±0.037	0.063±0.018
Spleen	0.377±0.227	1.639±0.141	0.050±0.006	0.445±0.262
Stomach	0.120±0.092	0.203±0.090	0.169±0.078	0.391±0.311
Bone	2.516±1.704	10.857±5.283	0.121±0.035	2.198±0.473
Small intestine	0.105±0.067	0.144±0.055	0.126±0.073	0.283±0.191
Tumor	0.588±0.518	1.533±0.552	0.069±0.019	N/A

Note: Data presented as mean ± standard deviation.

Abbreviations: FA, folic acid; DFO, deferoxamine; i.v., intravenous; PEG, polyethylene glycol; ID, injected dose; N/A, not available.