Curcumin-loaded apotransferrin nanoparticles provide efficient cellular uptake and effectively inhibit HIV-1 replication in vitro

Abstract

Background: Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research.

Methodology/principal findings: In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI(50) = 15.6 µM) is twice more toxic than nano-curcumin (GI(50) = 32.5 µM), nano-curcumin (IC(50)<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC(50) = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis.

Conclusion: Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness.

Figure 1. Curcumin loading increases size of apotransferrin nanoparticles.

The preparations of curcumin-loaded apotransferrin nanoparticles (nano-curcumin; left) and apotransferrin nanoparticles without curcumin (nano-apotransferrin; right) were examined by A) TEM B) SEM and C) AFM as indicated.

Figure 2. Nanoparticle formulation increases curcumin uptake, which is inhibited by transferrin receptor blockade.

A) SUP-T1 cells were incubated for 1 h with curcumin formulations as indicated, then examined by confocal microscopy. (i) Cells without curcumin; (ii) 1 µM sol-curcumin; (iii) 1 µM nano-curcumin; or (iv) 1 µM nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). Each panel contains three images: fluorescence, bright field and merged. B) SUP-T1 cells (i) or stimulated PBMCs (ii) were incubated for 1 h with curcumin formulations, after which intrinsic fluorescence of intracellular curcumin was determined quantitatively by fluorometric analysis. Cells were treated with 5 µM sol-curcumin, 5 µM nano-curcumin, or 5 µM nano-curcumin in the presence of antibodies to the transferrin receptor (TrR-Ab; 100 ng/ml). All the values are normalized to that obtained from SUPT1 cells. Error bars indicate standard deviation (SD). ***, P≤0.001 compared to sol-curcumin; n.s.: non-significant.

Figure 3. Nanoparticle formulation exhibits increased cellular retention in SUP-T1 cells.

Cells were incubated with 1 µM (Panel A) and 10 µM (Panel B) sol-curcumin and nano-curcumin and examined by confocal microscopy at time points of 1, 2, 4 and 6 h. Each panel contains three images: fluorescence, bright field and merged.

Figure 4. Nanoparticle formulation exhibits increased cellular retention in stimulated PBMCs.

Cells were incubated with 1 µM (Panel A) and 10 µM (Panel B) sol-curcumin and nano-curcumin and examined by confocal microscopy at time points of 1, 2, 4 and 6 h. Each panel contains three images: fluorescence, bright field and merged.

Figure 5. Nanoparticle formulation decreases curcumin cytotoxicity.

SUPT1 cells (Panel A) or stimulated PBMCs (Panel B) were exposed to increasing concentrations (1, 5, 10, 25, 50 and 100 µM) of sol-curcumin, nano-curcumin, azidothymidine (AZT) or nano-apotransferrin (10, 50 and 100 µg) for 16 h, after which cell viability was determined by MTT assay. PBMCs were cultured in the presence of IL-2 (20 IU/ml). Cell viability in the absence of drug was defined as 0% cytotoxicity. Error bars indicate SD. ** P≤0.01, and ***, P≤0.001 compared to nano-curcumin. * indicates µg apotransferrin protein that carry equivalent molar concentration of the drug.

Figure 6. Nano-curcumin more effectively inhibits HIV-1 replication through a mechanism dependent on transferrin receptor.

A & C) SUPT1 cells (Panel A) or stimulated PBMCs (Panel C) were challenged for 2 h with HIV-1_93IN101 (1 mg p24/ml) in the presence of increasing concentrations (1, 2.5, 5, 10, 20 and 30 µM) of sol-curcumin, nano-curcumin, or nano-apotransferrin (10 and 50 µg). They were then incubated for a further 96 h, after which viral replication was measured by p24 antigen capture assay. *indicates µg apotransferrin protein that carry equivalent molar concentration of the drug. B & D) SUP-T1 cells or stimulated PBMCs were challenged for 2 h with HIV-1_93IN101 in the presence of 2.5 or 5.0 µM concentrations of sol-curcumin, nano-curcumin or nano-curcumin in the presence of transferrin receptor antibody (100 ng/ml). After 96 h incubation, viral replication was measured by p24 antigen capture assay. In both these experiments, viral replication in the absence of drug was defined as 0% inhibition; Azidothymidine (AZT) was employed as a positive control. Error bars indicate SD. ** P≤0.01, and ***, P≤0.001 compared to sol-curcumin; n.s.: non-significant.

Figure 7. Inhibition of HIV-1 replication by nano-curcumin is due to abolished viral cDNA synthesis and/or altered topology.

SUPT1 cells were challenged for 4 h with HIV-1_93IN101 in the presence of 5 µM of sol-curcumin, nano-curcumin or nano-apotransferrin. A) The expression of topoisomerase IIα was determined by (i) semi-quantitative and (ii) quantitative-PCR. B) Quantity of viral cDNA synthesized was shown by both (i) semi-quantitative and (ii) real-time PCR using gag-specific primers. Template from normal SUP-T1 cells was used as negative control. Azidothymidine (AZT) was employed as a positive control and 18S was used as an internal control in both experiments. Error bars indicate SD. ***, P≤0.001 compared to sol-curcumin.

Figure 8. Action of nano-curcumin against virus-induced inflammatory response.

A) SUPT1 cells were challenged for 4 h with HIV-1_93IN101 in the presence of 5 µM of sol-curcumin, nano-curcumin or nano-apotranferrin. The expression of topoisomerase IIβ, IL-1β, TNF-α and COX-2 was determined by semi-quantitative-PCR. Template from normal SUP-T1 cells was used as negative control. 18S was used as an internal control in both experiments. IL-1β (Panel B), COX-2 (Panel C) and TNF-α (Panel D) were estimated using commercial kits as described in the methods section. Error bars indicate SD. ***, P≤0.001 compared to nano-curcumin.