THPE014 - Poster Exhibition
Gold nanoparticles to improve drug delivery to the central nervous system: targeting HIV reservoirs in the brain
C. Garrido1, N. Dahl1, C.A. Simpson2, J. Bresee2, D. Feldheim2, C. Melander3, D. Margolis1
1University of North Carolina - Chapel Hill, Medicine, Chapel Hill, United States, 2University of Colorado, Boulder, United States, 3North Carolina State University, Raleigh, United States
Background: The entry of ART to the central nervous system may be suboptimal due to the blood-brain-barrier, allowing the creation of a viral reservoir. To improve treatment delivery into the brain, we have developed gold nanoparticles, which are inorganic crystals that can be covalently conjugated to 2-1000 drug molecules. In addition, they can be co-fabricated with targeting molecules, allowing selective delivery to specific anatomic sanctuaries.
Methods: Gold nanoparticles were initially conjugated to TAMRA to allow visualization. The ability to enter into cells was assessed by 24-hour incubation with human lymphocytes, macrophages, astrocytes and brain microendothelial cells (HBMECs). Penetration was observed by confocal microscopy using CD4, CD14, anti-GFAP and b-catenin respectively, along with DAPI. Absorption of nanoparticles by PBMCs was also evaluated by flow cytometry. Nanoparticle's ability to cross the BBB was analyzed in vitro using transwell plates seeded with HBMECs, and in vivo by intravenous tail injection in mice. Fluorescence and gold content measured by ICP-MS were used to evaluate systemic nanoparticle distribution. Finally, gold nanoparticles were conjugated to a raltegravir derivative, and antiviral activity was assessed.
Results: Confocal images revealed the presence of TAMRA fluorescence in all cell types. FACS analysis of PBMCs after 6-24 hour incubation showed up to 15%CD4+T-cells with TAMRA
[Percentage of TAMRA+CD4+T-cells]
|10000 nM TAMRA||12%||15%|
|5000 nM TAMRA||9%||15%|
|1000 nM TAMRA||10%||7.5%|
|100 nM TAMRA||5%||5.3%|
|10 nM TAMRA||3.5%||4.2%|
|0 nM TAMRA||1.8%||3%|
. Mouse brain gold content reached up to 869 ppb/gram of tissue, and was also observed by microscopy. Nanoparticles conjugated to a Raltegravir derivative reduced viral replication to 25.24-38.57% at day 5.
[% of HIV replication]
Conclusions: Gold nanoparticles are able to enter into lymphocytes, macrophages, astrocytes and HBMECs, as well as crossing the blood-brain-barrier both in vitro and in vivo. Antiretroviral drugs can be conjugated to the nanoparticle and exert antiviral activity. Ongoing experiments include antiviral evaluation of the nanoparticles in the transwell system. Next steps include simultaneous conjugation of different molecules to the nanoparticles and further in vivo assays.
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