Novel method to label solid lipid nanoparticles with 64cu for positron emission tomography imaging

Abstract

Solid lipid nanoparticles (SLNs) are submicrometer (1-1000 nm) colloidal carriers developed in the past decade as an alternative system to traditional carriers (emulsions, liposomes, and polymeric nanoparticles) for intravenous applications. Because of their potential as drug carriers, there is much interest in understanding the in vivo biodistribution of SLNs following intravenous (i.v.) injection. Positron emission tomography (PET) is an attractive method for investigating biodistribution but requires a radiolabeled compound. In this work, we describe a method to radiolabel SLN for in vivo PET studies. A copper specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), conjugated with a synthetic lipid, was incorporated into the SLN. Following incubation with (64)CuCl(2) for 1 h at 25 °C in 0.1 M NH(4)OAc buffer (pH 5.5), the SLNs (∼150 nm) were successfully radiolabeled with (64)Cu (66.5% radiolabeling yield), exhibiting >95% radiolabeled particles following purification. The (64)Cu-SLNs were delivered intravenously to mice and imaged with PET at 0.5, 3, 20, and 48 h post injection. Gamma counting was utilized post imaging to confirm organ distributions. Tissue radioactivity (% injected dose/gram, %ID/g), obtained by quantitative analysis of the images, suggests that the (64)Cu-SLNs are circulating in the bloodstream after 3 h (blood half-life ∼1.4 h), but are almost entirely cleared by 48 h. PET and gamma counting demonstrate that approximately 5-7%ID/g (64)Cu-SLNs remain in the liver at 48 h post injection. Stability assays confirm that copper remains associated with the SLN over the 48 h time period and that the biodistribution patterns observed are not from free, dissociated copper. Our results indicate that SLNs can be radiolabeled with (64)Cu, and their biodistribution can be quantitatively evaluated by in vivo PET imaging and ex vivo gamma counting.

Fig. 1. Schematic of SLN-BAT

The lipid-PEG-BAT (left) molecule is incorporated into the SLN such that the stearic acid lipid tail is embedded in the stearic acid lipid core, while the PEG is associated with the lecithin monolayer, and the BAT chelator remains accessible from outside the SLN (right). We anticipate that van der Walls forces would cause the lipid portion of the lipid-PEG-BAT molecule to associate with the lipid core of the SLN and there be incorporated inside. We also anticipate that van der Walls forces would cause the hydrophilic PEG portion of the lipid-PEG-BAT to be associated with the hydrophilic headgroups of the lecithin molecule, leaving the BAT accessible on the surface of the SLN. By testing the ability for the SLNs to radiolabel ⁶⁴Cu, we validate the incorporation of lipid-PEG-BAT into the SLN and the accessibility of the BAT groups on the surface.

Fig. 2. Size characterization of SLNs

(a) Average Hydrodynamic diameter (145.2 ± 0.07 nm) of the optical SLNs, measured using dynamic light scattering (DLS). (b) Transmission electron microscopy (TEM) images of the 10-fold (left) and 20-fold (right) diluted SLN suspensions showing the mean dehydrated SLN diameter = 89 nm; scale bar = 100 nm.

Fig. 3. Radiolabeling yield (%) and purification of SLNs

(a) Elution profiles of ⁶⁴Cu-SLN after incubation with ⁶⁴CuCl₂ (1 hr) and EDTA (25 min) at 25°C and separation through a size-exclusion column. (b) Following purification, radio TLC of ⁶⁴Cu-SLN on a silica plate was developed with methanol/ammonium acetate (10%) (50:50, v/v) and recorded by a radio-TLC Imaging Scanner. Results show >95% radiolabeled nanoparticles.

Fig. 4. Stability of SLNs

Stability of radiolabeled solid lipid nanoparticles (SLNs) over 48 hr in serum (pH 7.4), saline (pH 5.5), saline (pH 4), and saline (pH 2). Bars represent mean ± standard deviation (n = 3/group).

Fig. 5. Radio TLC of SLNs incubated with serum

Cu-EDTA (a) and SLNs incubated with serum at 24 (b) and 48 hr (c) were developed on a silica plate with methanol/ammonium acetate (10%) (50:50, v/v) and recorded by a radio-TLC Imaging Scanner. The absence of free ⁶⁴Cu-EDTA in b) and c) confirm that the ⁶⁴Cu-SLNs were stable in plasma after 24 and 48 hr.

Fig. 6

Radio TLC of supernatant resulting from centrifugation filtration of SLNs incubated with saline (pH 2) and saline (pH 4) at 3 and 20 hr. Samples were developed on a silica plate was developed with methanol/ammonium acetate (10%) (50:50, v/v) and recorded by a radio-TLC Imaging Scanner. The TLC plots confirm that the loss of radiolabel observed (Figure 4) for ⁶⁴Cu-SLNs incubated with saline (pH 2) and saline (pH 4) were a result of ⁶⁴Cu-EDTA dissociating from the particle in these extreme conditions.

Fig. 7. Biodistribution with gamma counting

Biodistribution of ⁶⁴Cu-SLNs in mice at 52–55 hr post injection. Error bars represent mean ± standard deviation (n = 3). Liver activity (%ID/g) is significantly higher (p value < 0.01) than all other organs.

Fig. 8. Preliminary biodistribution with PET

Coronal view of a microPET image acquired at (a) 0.5-hr, (b) 3-hr, (c) 20-hr, and (d) 48-hr post i.v. injection of ⁶⁴Cu-SLNs. Radioactive signal is present in C = carotid, H = heart, L = liver, S = spleen, and I = intestines. A relative scale (the brightest spot is maximum) was applied for the images; e) Liver biodistribution obtained using ROI analysis on the microPET images. Error bars represent mean ± standard deviation (n = 3).

Fig. 9

Time activity curve (TAC) of blood after bolus i.v. injection of ⁶⁴Cu-SLNs in mice. TACs were obtained with region-of-interest (ROI) analysis using ASIPro software and expressed as the percentage of injected dose per cubic centimeter (%ID/cc). Error bars represent mean ± standard deviation (n = 3). The monoexponential curve fit to the data was as follows: y = 8.4363x^−0.593.