Ncr nu nu athymic female mice

5×10⁶ OVCAR-8-RFP cells suspended in PBS were injected intraperitoneally into NCr nu/nu athymic female mice (N=5) aged 10 to 12 weeks (Taconic, Rensselaer, NY). Tumor burden was monitored on a weekly basis using a Xenogen IVIS^® Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA). Mice were also given weekly vaginal lavages using 200 μL of sterile PBS throughout the study to collect cells from the local microenvironment of the reproductive organs. Cells sourced from lavages were counted and spun down to remove PBS as it has been shown to suppress MALDI-TOF ionization.^{21 (link)} This initial centrifugation step also removes mucous and mucous-associated proteins, such as mucin. Cells are then normalized to 10,000 cells/μL using DI water and stored at −80°C following collection. Upon addition of DI water, cells undergo osmotic stress and lyse, which removes the need for a wash step as all remaining proteins will be in solution. This lysing step prior to analysis also allows for proteins or other similarly sized molecules to be detected via MALDI-TOF MS. After two months of tumor progression, all animals were humanely sacrificed followed by collection of tumors and reproductive organs.

All animals were treated in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and the established Animal Care and Use Committees at the Ohio State University (OSU, protocol #2009A0196-R2) the University of Illinois at Chicago (UIC, protocol #16–035). In vivo studies conducted at OSU (maximum tolerated dose, bioavailability) utilized ICR male mice 6 weeks in age and around 20 grams in weight (Harlan Laboratories), and studies at UIC (hollow fiber assay, intraperitoneal xenografts) utilized NCr nu/nu athymic female mice 6–8 weeks in age (Taconic). Mice used in all in vivo assays were housed in a temperature- and light-controlled environment under 12:12 hour light:dark cycles and provided food and water ad libitum.

All animals were treated in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and the established Institutional Animal Use and Care protocol at the University of Illinois, Chicago. Xenograft studies utilized NCr nu/nu athymic female mice 6-8 weeks in age (Taconic). Mice were housed in a temperature and light-controlled environment (12 hours light and 12 hours dark) and provided food and water ad libitum. For xenograft experiments, OVCAR8-RFP cells (5 x 10⁶) were injected intraperitoneally (IP) per mouse and tumor growth was monitored using Xenogen IVIS® Spectrum In Vivo Imaging System (PerkinElmer) as previously described (20 ). Once all the mice formed tumors (~4 weeks), the mice were separated into 2 treatment groups and dosed once every two days with 0.5 mg/kg of verticillin A encapsulated nanoparticles (eNP-VA) and empty nanoparticles (eNP) for a total of 12 days. Mice were IVIS imaged twice weekly (535 nm excitation, and 620 nm emission, Exposure time: 2 seconds, F stop: 2). Living Image 4.0 software was used to quantify the average abdominal radiant efficiency and normalization was performed using Day 0 radiant efficiency. At week 7, all animals were sacrificed and tumors were collected for histological analysis.