2.7. In-Vivo Nanoparticle Biodistribution

Male Swiss albino mice (8 weeks old, weighing 25–30 g), were purchased from Theodor Bilharz Research Institute (TBRI) Cairo, Egypt. The mice were initially divided into two groups (healthy controls and fibrotic mice). Chronic liver damage was induced by intra-peritoneal (IP) injection of 10% CCl₄ in olive oil (2.5 µL/g body weight) twice a week for one month [29]. The second group served as the healthy controls and received the same volume of olive oil through IP injections. To confirm the establishment of fibrosis after 4 weeks of CCl₄ injections, one mouse was sacrificed by cervical dislocation and liver tissue samples were obtained, for histopathological investigations. Briefly, livers were washed in tap water and then in serial dilutions of methyl, ethyl and absolute ethyl alcohol to achieve dehydration of the tissue. Specimens were cleared in xylene and embedded in paraffin at 56 °C in a hot air oven for 24 h. Paraffin tissue blocks were prepared for sectioning at 4-μm thicknesses by sledge microtome. The obtained tissue sections were collected on glass slides, de-paraffinized, stained with hematoxylin and eosin (H&E) for examination under the light microscope [30]. To determine whether chitosan collagen binding affects the NP in-vivo biodistribution and whether the excess deposition of collagen in fibrotic livers would facilitate NP accumulation in fibrotic livers Collagenase-loaded chitosan nanoparticles were prepared as detailed in earlier work from our group [25]. The healthy and fibrotic groups were divided each into two groups; the first group received collagenase-NPs intravenously (IV) for one week followed by fluorescent CS-NPs, LP-NPs and HP-NPs, while the second group received PBS instead of collagenase-NPs followed by fluorescent CS-NPs, LP-NPs and HP-NPs. A total of 0.8 mg NPs per mouse was administered intravenously through the tail vein. The total NPs amount was divided over 3 doses given at 2-h intervals [2]. One hour after the last dose, the mice were sacrificed by cervical dislocation and liver tissue samples were harvested. Livers were homogenized in PBS to yield a homogenate with a final concentration of 0.25 g/mL [2]. The concentration of NPs in the livers was then determined by fluorometry using CS-NPs, LP-NPs and HP-NPs calibration curves constructed in liver homogenates. Additionally, to determine whether CS-NPs distribution to other organs changed as a function of fibrosis and excessive collagen deposition, healthy and fibrotic animals were divided into two groups. The first group received intravenous fluorescent CS-NPs (0.8 mg over 3 doses), whereas the second group received the same volume of PBS. One hour after the last dose, the mice were sacrificed by cervical dislocation, and livers, spleens, kidneys, brains and lungs were harvested and homogenized in PBS to yield a homogenate with a final tissue concentration of 0.25 g/mL. The fluorescent intensities of organ homogenates obtained from CS-NP-receiving mice were normalized to fluorescent intensities of organ homogenates form PBS-receiving mice. Figure S1 provides a schematic representation of the in-vivo study Animal care and all experimental procedures were conducted according to the ethical guidelines of the Research Ethics Committee of Faculty of Pharmacy, German University in Cairo (GUC), Project ID: 2019-03-TC-SMH-MK (approved on 16 February 2019).