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

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

<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

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

FT-IR spectral analysis.

<p>The FT-IR analysis of Pure Lactoferrin proteins (A), Nano Lactoferrin (Lacto nano) (B), AZT molecule (C) and AZT loaded lactoferrin nanoparticle (AZT-Lactonano) (D). It reveals that in AZT-Lactonano, AZT is physically entrapped/adsorbed with the lactoferrin nanoparticles; it didn’t take part in any sort covalent interaction.</p

Histopathological analysis of tissues.

<p>Rats were orally administered with sol AZT and AZT-lactonano (10mg/kg body weight), after completion of 24hr time point, organs were removed and processed for cryo-sectioning followed by Hematoxylin and Eosin (H&E) staining. It revealed that no toxicity was found in above indicated organs, when AZT was delivered via nanoparticle form as compared to its sol form. Lesion or any abnormalities present was denoted by arrow. Scale bar is equal to 100μm.</p

pH sensitivity and time dependent drug release profile of AZT-Lactonano.

<p>(A) 600μg of drug was incubated in the buffers of different pH, the release of AZT was maximum at pH-5. This is followed by pH-6 and pH-4. The release was between 1–10% with the remaining fluids. SGF: Simulated Gastric Fluid, SIF: Simulated Intestinal Fluid. (B) Cumulative percentage release profile of AZT-Lactonano at pH 5.0 and pH 7.4. Each data points were taken in triplicate and presented as Mean ± SD. Value of significance, **P < 0.005, *P < 0.05.</p