Towards the development of brain-penetrating gold nanoparticle-transactivator of transcription (TAT) peptide conjugates

Objectives: Multifunctional gold nanoparticles (AuNPs) are highly attractive drug delivery vehicles due to their small size, flexibility, and biocompatibility. However, surface modifications are essential if they are to cross the blood-brain barrier. The i-colloid AuNPs used in this work were produced by femtosecond laser ablation with surfactant-free surface. The conjugation of functional ligands to the AuNPs surface can be tunable between 0 and 100%. Transactivator of transcription (TAT) peptide derived from HIV has been used to deliver a wide range of cargoes across lipophilic barriers and proven to improve brain penetration of AuNPs. ^1,2 Thus, we synthesized AuNPs quantitatively conjugated with TAT and then labeled with Cu-64. The cellular uptakes and biodistribution of normal mice are reported.

Methods: Chelator-free Cu-64 labeling: Sodium ascorbate was dissolved in borate buffer (pH 8.6). ⁶⁴CuCl₂ aqueous solution was diluted with ascorbate solution and added to AuNPs. The mixture was allowed to react at room temperature for 1 hour before washed by centrifugation. Cellular uptake: PC3 cells were cultured in 6-well plates and ⁶⁴Cu-AuNPs were added and then incubated overnight. The media were removed and the cells were washed with PBS. The PBS solution was removed and the cells were extracted by Trypsin. The radioactivities of the cells and of each removed solution were measured. The percentage of radioactivity of cells over the total radioactivity was calculated. Biodistribution: Female CD-1 mice were anesthetized and 15-25 μCi of radiotracer was injected via tail vein. Mice were sacrificed at different time points (n=3 at each time point). The brain and selected organs were rapidly removed. Tissue samples were weighed and radioactivities were counted. The radioactivity in each organ calculated as the percent-injected dose per gram of tissue. Results: Chelator-free Cu-64 labeling: Two kinds of i-colloid AuNPs (size 10 nm) were used for the experiments. One is PEGylated only (1500 molecules of PEG5K per particle) as control and another is AuNP conjugated with 500 molecules of PEG(5K) and molecules of 1000 TAT on each particle surface. The chelator-free Cu-64 labeling method gave excellent yields, 40.1% and 38.5% for AuNP-PEG and AuNP-TAT respectively. Cellular uptake: The uptake of cells was 0.03% and 7.4% for AuNP-PEG and AuNP-TAT respectively. Biodistribution: The brain uptake are between 0.02-0.06 %ID/gram for all animals at different time points. Conclusions: We have successfully synthesized TAT-modified AuNPs and ⁶⁴Cu-labeled using chelator-free method with excellent yields, and demonstrated they have proven improved cell penetration of none-modified AuNPs. However, smaller sized nanoparticles may still be required to cross the blood-brain barrier. Acknowledgements: We thank Jenelle Stauff, Janna Arteaga and Phillip Sherman for assisting with animal studies. We acknowledge financial support from the program of China Scholarship Council (No. 201607060008). References: (1) Morshed, et al., Cell-penetrating peptide-modified gold nanoparticles for the delivery of doxorubicin to brain metastatic breast cancer. Mol. Pharmaceutics, 2016, 1843-54. (2) Cheng et al., Blood-brain barrier permeable gold nanoparticles: An efficient delivery platform for enhanced malignant glioma therapy and imaging. Small, 2014, 5137-50.