Magnetic nanoformulation of azidothymidine 5'-triphosphate for targeted delivery across the blood-brain barrier

Abstract

Despite significant advances in highly active antiretroviral therapy (HAART), the prevalence of neuroAIDS remains high. This is mainly attributed to inability of antiretroviral therapy (ART) to cross the blood-brain barrier (BBB), thus resulting in insufficient drug concentration within the brain. Therefore, development of an active drug targeting system is an attractive strategy to increase the efficacy and delivery of ART to the brain. We report herein development of magnetic azidothymidine 5'-triphosphate (AZTTP) liposomal nanoformulation and its ability to transmigrate across an in vitro BBB model by application of an external magnetic field. We hypothesize that this magnetically guided nanoformulation can transverse the BBB by direct transport or via monocyte-mediated transport. Magnetic AZTTP liposomes were prepared using a mixture of phosphatidyl choline and cholesterol. The average size of prepared liposomes was about 150 nm with maximum drug and magnetite loading efficiency of 54.5% and 45.3%, respectively. Further, magnetic AZTTP liposomes were checked for transmigration across an in vitro BBB model using direct or monocyte-mediated transport by application of an external magnetic field. The results show that apparent permeability of magnetic AZTTP liposomes was 3-fold higher than free AZTTP. Also, the magnetic AZTTP liposomes were efficiently taken up by monocytes and these magnetic monocytes showed enhanced transendothelial migration compared to normal/non-magnetic monocytes in presence of an external magnetic field. Thus, we anticipate that the developed magnetic nanoformulation can be used for targeting active nucleotide analog reverse transcriptase inhibitors to the brain by application of an external magnetic force and thereby eliminate the brain HIV reservoir and help to treat neuroAIDS.

Keywords: AZTTP; HIV-1 infectivity; blood–brain barrier; magnetic liposomes; transendothelial migration.

Figure 1

TEM micrograph of magnetic liposomes. The average size of the liposomes is ∼150 nm.

Figure 2

Magnetic AZTTP liposomes inhibit HIV-1 p24 production. PBMCs (1 × 10⁶ cells/mL) obtained from normal subjects were infected with native HIV-1 IIIB (NIH AIDS Research and Reference Reagent Program Cat# 398) at a concentration of 10^3.0 TCID₅₀/mL cells for 3 hours and washed 3 times with Hank’s balanced salt solution (GIBCO-BRL, Grand Island, NY) before being returned to culture with and without free AZTTP or magnetic AZTTP liposomes (1–100 nM) for 7 and 14 days. The culture supernatants were quantitated for HIV-1 p24 antigen using a p24 ELISA kit (ZeptoMetrix Corporation, Buffalo, NY). The data represent the average of 3 independent experiments and are expressed as ng/mL. Statistical analysis was done using one way ANOVA with Bonferroni adjustment. ^#Comparison between MP-AZTTP liposome and free AZTTP group. Abbreviations: AZTTP, azidothymidine 5’-triphosphate; MP-AZTTP, magnetic nanoparticles bound AZTTP.

Figure 3

Transmigration of MP-AZTTP liposomes across the blood–brain barrier (BBB) model. Apparent permeability coefficients (Papp) of MP-AZTTP transport across the BBB model as free and in magnetic liposomes. The data represents the mean ± SE of 3 independent experiments and is expressed as cm/min. Statistical analysis was performed using unpaired Student’s t-test. Abbreviations: AZTTP, azidothymidine 5’-triphosphate; MP-AZTTP, magnetic nanoparticles bound AZTTP.

Figure 4

Uptake of magnetic liposomes by human monocytes. Monocytes were co-cultured with rDHPE-magnetic liposomes for 2 and 4 hours and their intracellular localization was assessed by fluorescence microscopy (Zeiss, Germany). Engulfed magnetic liposomes can be visualized as red fluorescence located within the cell cytoplasm.

Figure 5

Transmigration of monocytes across the in vitro blood–brain barrier (BBB). Magnetic liposome loaded monocytes were added in the upper chamber of the BBB model with or without a magnet placed underneath for the duration of experiment. At 2 and 4 hours after plating, migrated monocytes were counted in the lower chamber. Results are expressed as mean ± SE of three independent experiments. Statistical significance was determined using unpaired Student’s t-test.