Positron emission tomography imaging of the stability of Cu-64 labeled dipalmitoyl and distearoyl lipids in liposomes

Abstract

Changes in lipid acyl chain length can result in desorption of lipid from the liposomal anchorage and interaction with blood components. PET studies of the stability of such lipids have not been performed previously although such studies can map the pharmacokinetics of unstable lipids non-invasively in vivo. The purpose of this study was to characterize the in vivo clearance of (64)Cu-labeled distearoyl- and dipalmitoyl lipid included within long circulating liposomes. Distearoyl and dipalmitoyl maleimide lipids (1mol%) in liposomes were labeled with a (64)Cu-incorporated bifunctional chelator (TETA-PDP) after the activation of pyridine disulfide to thiol by TCEP. Long circulating liposomes containing HSPC:DSPE-PEG2k-OMe:cholesterol: x (55:5:39:1), where x was (64)Cu-DSPE or (64)Cu-DPPE, or HSPC:DSPE-PEG2k-OMe:cholesterol:(64)Cu-DSPE:DPPC (54:5:39:1:1) were evaluated in serum (in vitro) and via intravenous injection to FVB mice. The time-activity curves for the blood, liver, and kidney were measured from PET images and the biodistribution was performed at 48h. In vitro assays showed that (64)Cu-DPPE transferred from liposomes to serum with a 7.9h half-life but (64)Cu-DSPE remained associated with the liposomes. The half clearance of radioactivity from the blood pool was 18 and 5h for (64)Cu-DSPE- and (64)Cu-DPPE liposome-injected mice, respectively. The clearance of radioactivity from the liver and kidney was significantly greater following the injection of (64)Cu-DPPE-labeled liposomes than (64)Cu-DSPE-labeled liposomes at 6, 18 and 28h. Forty eight hours after injection, the whole body radioactivity was 57 and 17% ID/cc for (64)Cu-DSPE and (64)Cu-DPPE, respectively. These findings suggest that the acyl chain length of the radiolabel should be considered for liposomal PET studies and that PET is an effective tool for evaluating the stability of nanoformulations in vivo.

Figure 1

a) liposomal labeling scheme using bifunctional chelator and (b) maleimide lipids exploited for ⁶⁴Cu-BFC conjugation

Figure 2

In vitro stability of radio labeled lipid in three liposomes (⁶⁴Cu-DSPE liposomes, ⁶⁴Cu-DPPE liposomes, ⁶⁴Cu-DSPE-1mol%DPPC liposomes). Liposomes were incubated for 48 hours with 50% serum (PBS). Radio TLCs were obtained from the highest radioactive liposomal fraction (a- and c-TLC) and serum fraction (b-TLC). Blue line, CPM (counts per minute) indicates radioactivity of ⁶⁴Cu-lipid detected by the gamma counter. Red line represents liposomal fluorescence (779 nm). Green line, absorbance, represents serum proteins measured at 280 nm.

Figure 3

Time dependent lipid transfer assay with ⁶⁴Cu-DPPE liposomes at 0, 5, 19, 41, and 48 hours. Blue line, CPM (counts per minute), indicates radioactivity of ⁶⁴Cu-lipid detected by the gamma counter. Red line represents liposomal fluorescence (779 nm). Green line, absorbance, represents serum proteins measured at 280 nm.

Figure 4

a) Small-animal PET images (projected image) acquired after 30 min scanning at (a) 0, (b) 6, and (c) 18 hours. Left: ⁶⁴Cu-DSPE liposomes, middle: ⁶⁴Cu-DPPE liposomes, right: ⁶⁴Cu-DSPE-1mol%DPPC liposomes.

Figure 5

Quantitative analysis (%ID/cc) of PET images with the time activity curve (TAC) of (a) blood pool and (b) liver and (c) whole body radioactivity. Three liposomes (⁶⁴Cu-DSPE liposomes, ⁶⁴Cu-DPPE liposomes, ⁶⁴Cu-DSPE-1mol%DPPC liposomes) were applied. (d) Comparison of stability of ⁶⁴Cu-DPPE liposomes from blood pool (in vivo) and serum incubation (in vitro) (curve fitted with one-phase exponential decay). Statistical analysis was performed by ANOVA, followed by Tukey’s multiple comparison test (error bars, mean ± STD; ***, P < 0.001)

Figure 6

Quantitative analysis (%ID/cc) of PET images of kidney. Statistical analysis was performed by ANOVA, followed by Tukey’s multiple comparison test (error bars, mean ± STD; *, P < 0.05; **, P < 0.01; ***, P < 0.001)