Loading capacity was maximum at the lower ratio tested (1:1) for both Chi NPs and Chi-C48/80 NPs. B surface antigen loaded Chi-C48/80 NPs validated the adjuvanticity of the delivery system, demonstrating for the first time a successful association between a mast cell activator and chitosan nanoparticles as a vaccine adjuvant for hepatitis B computer virus, applied to a nasal vaccination strategy. of chitosan was suspended in 10 mL of a 1 M NaOH answer, and stirred for 3 h at 50 C. The combination was then filtered (0.45 m membrane, MerckMillipore, Darmstadt, Germany), and the resultant pellet washed with 20 mL of deionized water. The recovered chitosan was dissolved in 200 mL of 1% (and resuspended in acetate buffer, pH 5.7, 25 mM. Nanoparticles at a final concentration of 2.5 mg/mL were incubated with BSA, ovalbumin (OVA), or myoglobin in acetate buffer for 60 min at RT. Ratios from 7:1 to 1 1:1 (NP/protein) were tested for BSA, while OVA and myoglobin were incubated at a fixed excess weight ratio of 7:1. After incubation, particles were centrifuged at 12,000 for 20 min, and the supernatant was collected. The amount of protein loaded on nanoparticles was decided indirectly by measuring the concentration of non-bound protein in the nanoparticle supernatant using the BCA or Micro-BCA protein assay (Pierce, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturers instructions. Loading efficacy and loading capacity (LC) were determined by Equations (2) and (3), respectively. and the resultant pellet was washed 3 times with a mixture of methanol/water (70:30, for 10 min. Nasal and vaginal washes were collected on Day 42. Vaginal washes were collected by instilling 100 L of PBS into the vaginal cavity, and the lavage fluid was flushed in and out a few times before collection. Samples were centrifuged at 11,500 for 10 min, and supernatants were stored. Nasal lavage samples were collected from euthanized mice. The lower jaw of the mice was Desoximetasone cut way and the nasal lavage collected by instilling 200 L of sterile PBS posteriorly into the nasal cavity. Fluid exiting the nostrils was collected and spun at 11,500 at 4 C for 20 min. Collected and processed samples were stored until further analysis. 2.10.2. Determination of Serum IgG, Desoximetasone IgG1, IgG2c, and Secretory IgA Quantification of immunoglobulins was performed using a protocol optimized by our group [27,30]. The endpoint titers offered in the results represent the antilog of the last log2 dilution, for which the OD values were at least two-fold higher than that of the naive sample, equally diluted. The log 2 end-point titers were utilized for statistical analysis. 2.11. Statistical Analysis Statistical analysis was performed with GraphPad Prism v 5.03 (GraphPad Software Inc., La Jolla, CA, USA). Students t-test and ANOVA followed by Tukeys post-test were used for two samples or multiple comparisons, respectively. A p-value 0.05 was considered statistically significant (* p 0.05; ** p 0.01; *** p 0.001). 3. Results and Discussion 3.1. Purification of Chitosan Before use chitosan was submitted to a purification process to ensure the removal of any possible impurities. FTIR analysis was performed Desoximetasone before and after the purification process to confirm the preservation of structure and integrity of the commercial polymer. The spectra obtained were in agreement with previously published data [32,33]. FTIR spectrum of chitosan showed a broad band between 3500 and 3200 cm?1 (Determine 1) corresponding to the stretching vibration of OCH. The peak of NCH stretching from main amine groups was overlapped in the same region. The peak at 2869 cm?1 indicates CCH stretching vibrations. Peaks at 1650 and 1588 Itga10 cm?1 correspond to C=O stretch and NCH bending, respectively. The peak at 1419 cm?1 belongs to the NCC stretching and the bands at 1150 and 1025 cm?1 are characteristic of the CO.