Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

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

Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120

Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region

Improved Safety, Bioavailability and Pharmacokinetics of Zidovudine through Lactoferrin Nanoparticles during Oral Administration in Rats

<div><p>Zidovudine (AZT) is one of the most referred antiretroviral drug. In spite of its higher bioavailability (50–75%) the most important reason of its cessation are bone marrow suppression, anemia, neutropenia and various organs related toxicities. This study aims at the improvement of oral delivery of AZT through its encapsulation in lactoferrin nanoparticles (AZT-lactonano). The nanoparticles (NPs) are of 50–60 nm in size and exhibit 67% encapsulation of the AZT. They are stable in simulated gastric and intestinal fluids. Anti-HIV-1 activity of AZT remains unaltered in nanoformulation in acute infection. The bioavailability and tissue distribution of AZT is higher in blood followed by liver and kidney. AZT-lactonano causes the improvement of pharmacokinetic profile as compared to soluble AZT; a more than 4 fold increase in AUC and AUMC in male and female rats. The serum C_max for AZT-lactonano was increased by 30%. Similarly there was nearly 2-fold increase in T_max and t_1/2. Our in vitro study confirms that, the endosomal pH is ideal for drug release from NPs and shows constant release from up to 96h. Bone marrow micronucleus assay show that nanoformulation exhibits approximately 2fold lower toxicity than soluble form. Histopathological and biochemical analysis further confirms that less or no significant organ toxicities when nanoparticles were used. AZT-lactonano has shown its higher efficacy, low organs related toxicities, improved pharmacokinetics parameter while keeping the antiviral activity intact. Thus, the nanoformulation are safe for the target specific drug delivery.</p></div

<i>In vitro</i> stability of nanoparticles.

<p>All the data (n = 3) were presented as mean ± standard deviation.</p><p>^<b>#</b> Drug present in the particles was estimated by using HPLC, in milligrams. The amount of drug present initially at zero hour at 4°C and room temperature are considered as 100% drug present.</p><p><b>*</b> Room temperature: The temperature used here was an average equal to 23°C.</p><p><i>In vitro</i> stability of nanoparticles.</p

Biochemical safety analysis profile.

<p>Safety analysis was done using biochemical kits after oral administration of nano and soluble AZT (10mg/kg) in both male and female rats. Liver damage was estimated by Bilirubin and AST level whereas Kidney toxicity was checked by Urea and creatinine level. AZT-lactonano showed no toxicity to both liver and kidneys on the other hand it exhibited minimal urea levels when compared to the soluble AZT.</p

Size of Lactoferrin nanoparticles increases after loading of AZT.

<p>AZT containing Lactoferrin nanoparticles were prepared using sol-oil chemistry as described in methods section. The size of nanoparticles was assessed by Field emission scanning electron microscopy (top panel) and Atomic force microscopy (bottom panel). Lactonano: Lactoferrin nanoparticle without any drug loading into it, AZT Lacto nano: Lactoferrin nanoparticle loaded with AZT.</p

Bone marrow toxicity profile.

<p><b>(A)</b> It shows the frequency of polychromatic erythrocyte (PCE) in bone marrow cells after oral administration of sol AZT and AZT-lactonano at 4, 8 and 16h. Data were presented as Mean ± SD. Value of significance, **P < 0.005, *P < 0.05. <b>(B)</b> Bone marrow cells (after 8h of treatment) showing the presence of enucleated Normochromatic erythrocyte (NCE) and nucleated polychromatic erythrocyte (PCE) cells. One PCE holds a micronucleus (MN); indicated by arrow. This images was captured at 100x under oil immersion objective.</p

Tissue distribution of AZT.

<p>Single dose of sol AZT and equivalent weight of AZT-lactonano (10mg/kg body weight) was orally administered to Wistar rats. After completion of indicated time points, rats were sacrificed under proper anesthesia. AZT was extracted and estimated in blood, liver, kidney, heart, spleen, bone marrow, lungs, brain, oesophagus, stomach, small intestine and large intestine. Male-Nano and Female-Nano denotes the AZT concentration (delivered via AZT-lactonano) present in Male rats (n- = 3) and female rats (n = 3) respectively. Same nomenclature has been followed for Male-Sol and Female-Sol. Differences between groups were assessed by ANOVA. Data were presented as Mean ± SD. Value of significance, **P < 0.005, *P < 0.05.</p