## 2 Materials and Methods  
### Manufacturing of Gondotropin-Releasing Hormone (GnRH)-Loaded Chitosan Nanoparticles  
Chitosan (chitosan extra pure; with a degree of deacetylation >85%; molecular weight = 300-350 KD; Alpha Chemika, Mubia, India) and sodium tripolyphosphate (TPP; Thermo Fisher GmbH, Kandel, Germany) were used in the fabrication of a nano-carrier polymer following the steps of the ionic gelation method [10; 11; 12]. Briefly, chitosan (0.1%, wt/vol) was vigorously stirred in an aqueous acidic solution (1%, wt/vol) to obtain polymeric chitosan cations. An aqueous solution of TPP (0.1 g/dL) was also prepared. Afterward, the chitosan-TPP nanoparticles were prepared by slowly dropping the TPP solution into a chitosan solution (chitosan to TPP weight ratio was 2:1) under constant magnetic stirring (800 rpm) for 2 h at room temperature. The pH of the chitosan-TPP solution was adjusted to 5.5 and then stored in the refrigerator before exposure to any analysis or application. GnRH-loaded chitosan nanoparticles (GnRH-ChNPs) were prepared by dripping-wise the hormone (GnRH; Receptal(r), MSD, Intervet International GmbH, Unterschleissheim, Germany) solution to the fabricated ChNPs in a ratio of 1:1. The mixture was adjusted to a pH of 6.5 and after that stirred gently at 800 rpm for 60 min at room temperature and incubated overnight to allow the hormonal adsorption on the surface of the nanoparticles. In Vitro Assessment of the Physicochemical Properties and Release Patterns of GnRH-Loaded Nanoparticles  
Particle size, zeta potential, and size distribution (polydispersity index, PdI) of the free ChNPs and GnRH-ChNPs were measured using a Zetasizer instrument (Malvern Instruments, Malvern, UK), which is based on dynamic light scattering (DLS) techniques. The samples were analyzed in triplicate and the average value (\(\pm\)SD) of each parameter was calculated. The functional groups of chitosan (Ch), ChNPs, and GnRH-ChNPs were identified by Fourier transform infrared spectrophotometer (FTIR, a Perkins Elmer 1600, USA) in the range from 4000 to 400/cm using potassium bromide discs (KBr) (5 mg of particles, 100 mg KBr pellets). Morphological characteristics of the free ChNPs and GnRH-ChNPs were examined using transmission electron microscope (TEM; JEOL JEM-1400, 120 kV, Peabody, MA, USA). Freshly fabricated ChNPs and GnRH-ChNPs solutions were diluted with deionized water with adjusted pH close to neutral. A one-drop sample was placed on a carbon-coated film 300 mesh copper grid and was left for 10 min until air-dried. The sample was stained with 1 M uranyl acetate solution for 1.5 min at 7 \({}^{\circ}\)C and any excess uranyl acetate was removed with filter paper before viewing on the TEM at magnification \(\times\) 20,000. The hormonal loading efficiency (LE%) was determined by the separation of the nanoparticles from the aqueous medium by centrifugation at 1200\(\times\) g at 4 \({}^{\circ}\)C for 20 min. The amount of free hormone in the supernatant of GnRH-ChNPs solution was measured using UV spectrophotometry at a wavelength of 280 nm (Optizen Pop, Mecasys Co. Ltd., Daejeon, Korea) and using the supernatant of ChNPs solution as a blank [13]. Afterward, LE% was calculated as ([initial GnRH concentration - free GnRH concentration]/initial GnRH concentration \(\times\) 100). The assessment of release patterns was carried out for 48 h to plot the kinetic release profile of GnRH from ChNPs according to the method described by [PERSON] and co-workers [13]. In brief, a pre-weighed sample of GnRH-ChNPs was divided into three aliquots and dissolved in equal volumes of phosphate buffer solution (PBS; pH 7.4). The samples were then incubated at 37 \({}^{\circ}\)C in a shaker adjusted at 600 rpm for 48 h. At allocated time intervals (0.5, 1, 2, 4, 6, 10, 12, 24, 48 h), the samples were removed and centrifuged at 14,000 rpm and 4 \({}^{\circ}\)C for 20 min. The concentration of free GnRH released into the supernatant was evaluated by spectrophotometry at the 280 nm wavelength (Optizen pop, Mecasys Co. Ltd. Daejeon, Korea).