In vivo biodistribution and biocompatibility of liposomes

To determine the biodistribution of liposomes, mice were randomly divided into groups. Each group was consisted of 6 mice including 3 males and 3 females. Each group of mice were intravenously injected via tail with either PBS, free Dox, Free Erlo, Dox and Erlo loaded plain, Tf, TAT, QLPVM, Tf-TAT, and Tf-QLPVM at a dose 15.2 μmoles/ kg of body weight. At predetermined interval of 24 h, mice were sacrificed and various organs (brain, lungs, heart, liver, kidney, and spleen) were harvested and blood samples were collected. The harvested organs were washed with PBS and stored at −80°C until assayed. Group injected with PBS was considered as control. The various organs were homogenized, followed by extraction of drugs in acetonitrile: methanol (9:1). Then, the drugs extracted sample was centrifuged at 10,000 rpm for 15 min at 4°C. Then, the supernatant was evaporated using vacuum evaporator. The residual drugs exacted sample was further reconstituted in Methanol: PBS pH 5.5 (1:1) and vortexed. The unwanted proteins were separated from the reconstituted sample was again centrifuged at 4°C for 15 min at 10,000. The quantitative estimation of drugs was performed using HPLC. The Dox analysis was done as per described in Dox loading and the quantitative estimation of Erlo distribution was performed with some modifications as per Erlo loading. The mobile was comprised of 0.2 M potassium phosphate buffer pH 3.0: acetonitrile (52:48) at a room temperature with a flow rate 0.6 ml/min [30]. All the data was normalized and represented as the percent injected dose per gram of the tissue (%ID/g). To determine the qualitative distribution, mice in each group were intravenously injected lissamine-rhodamine labeled liposomes at a dose of 15.2 μmoles/ kg of body weight through tail vein. Mice were sacrificed 24h post-injection and the whole body as well as ex-vivo fluorescent images were taken by Kodak in vivo imaging system FX (Carestream Health Inc., Rochester, NY). The images were acquired by setting rhodamine channel.

Various organs including brain, heart, lungs, liver, spleen, and kidneys were histologically evaluated to determine biocompatibility of liposomes post injection. After sacrificing mice, the organs were harvested and fixed in 10% neutralized buffer formalin. Paraffin sectioning of organs were done and stained with hematoxylin and eosin (H&E) staining. The tissue slides were observed for histopathological evaluation.