Solid Drug Nanoparticle Dispersions for Improved delivery of Efavirenz to Macrophages.

NJ Liptrott, P Martin, M Giardiello, T McDonald, S Rannard, A Owen. University of Liverpool, Liverpool, UK

Background: Nanomedicine approaches to drug formulation offer a number of potential benefits in the treatment of HIV such as improving bioavailability and/or targeting to cell types capable of sustaining viral replication[1, 2]. A reduction in pill burden and the associated production costs would have a positive impact, particularly in resource-limited settings. Macrophages are particularly relevant in HIV infection as they are a cellular reservoir for viral replication [3]. Since nanoparticles are within the size range phagocytosed by macrophages, an emulsion-templated freeze-drying approach was used to create efavirenz (EFV) solid drug nanoparticle (SDN) dispersions (70% w/w EFV in solid state) and their uptake into and impact upon monocyte derived macrophages (MDM) was assessed.

Methods: Anonymised, healthy volunteer blood was obtained from the National Blood Service. MDM were differentiated from healthy volunteer CD14+ monocytes and incubated (300,000 cells, N=4) with 10µM (0.3µCi) of either a nanoparticle dispersion or aqueous solution of EFV to investigate uptake. EFV concentrations within the cell pellet and media were then measured by liquid scintillation counting and used to calculate a cellular accumulation ratio (CAR; [intracellular]/[extracellular])[4, 5]. Second, the route of nanoparticle uptake was explored by assessing the impact of co-incubation with inhibitors of endocytosis (dynasore, prevents clathrin coated pit formation via inhibition of dynamin, 100µM; indomethacin, caveloae inhibitor, 100µM; cytochalasin, inhibitor of actin polymerisation and phagocytosis, 5µM) on CAR. Finally, the impact of nanoparticles and aqueous solution of EFV on phagocytic potential and activation (measured by cytokine release) was conducted as part of a putative safety assessment with cytochalasin included as a positive control. Following incubation for 24h, determination of phagocytic capacity of MDM was achieved using pHrodo reagent. Activation of MDM was assessed by incubating MDM in the presence of either lipopolysaccharide (LPS, known agonist of the CD14/TLR4/MD-2 complex on macrophages, 1µg/mL), aqueous solution or nanoformulated EFV (10µM) for 24h. Cell culture supernatants were then harvested for cytokine analysis using a Bioplex 200 analyser with recommended gating settings.

Results: The accumulation of nanoformulated EFV was greater than aqueous EFV (2.2-fold, P=0.01). Accumulation of nanoparticles in MDM treated with dynasore (32%, P=0.047) and indomethacin (37%, P=0.048) was significantly lower than in controls. A trend to lower accumulation with cytochalasin (31%, P=0.056) was also observed. MDM treated with cytochalasin B (phagocytosis inhibitor) showed 71% less uptake of pHrodo particles compared to untreated cells (P=0.048). Neither aqueous nor nanoformulated EFV had a significant impact on MDM phagocytic capacity. LPS activated MDM and significantly upregulated secretion of IL-1β (32 fold, P=0.029), IL-6 (1360 fold, P=0.029), IL-8 (158 fold, P=0.021) and TNFα (458 fold, P=0.029). Neither aqueous nor nanoformulated EFV had a significant impact on cytokine secretion from MDM.

Conclusions: These data indicate that EFV solid drug nanoparticles accumulate in MDM to a greater extent than dissolved EFV via an endocytic mechanism. Reassuringly, the uptake of these particles did not significantly impact on macrophage function. An integrated analysis of pharmacology and safety is required to fully understand the potential for the application of nanotechnologies to improve antiretroviral drug deployment.

1. Daum N, Tscheka C, Neumeyer A, Schneider M. Novel approaches for drug delivery systems in nanomedicine: effects of particle design and shape. Wiley interdisciplinary reviews Nanomedicine and nanobiotechnology 2012; 4:52-65.

2. Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress. Journal of controlled release : official journal of the Controlled Release Society 2012; 161:175-87.

3. Koppensteiner H, Banning C, Schneider C, Hohenberg H, Schindler M. Macrophage internal HIV-1 is protected from neutralizing antibodies. Journal of virology 2012; 86:2826-36.

4. Kwan WS, Janneh O, Hartkoorn R, et al. Intracellular \\'boosting\\' of darunavir using known transport inhibitors in primary PBMC. British journal of clinical pharmacology 2009; 68:375-80.

5. Janneh O, Jones E, Chandler B, Owen A, Khoo SH. Inhibition of P-glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes. The Journal of antimicrobial chemotherapy 2007; 60:987-93.