Nasal application of low-dose mB29a-PLGA containing particles reduces severity of arthritis.

<p><b>A and B.</b> Effect of mB29a-nanoparticles on nasally induced suppression of PG-induced arthritis in BALB/c mice. Mice received 30 µg of mB29a peptide i.n. dissolved in PBS or encapsulated in PLGA or PLGA-TMC nanoparticles prior to arthritis induction. Arthritis scores of mB29a-PBS (black squares), PLGA (black circles) or PLGA-TMC (black triangles) treated mice as assessed by swelling and redness of the paws. Data are shown as the mean arthritis scores ± SEM. of n = 3 mice per group. Statistically significant: **, <i>P</i><0.01.</p

Nanoparticle mediated enhanced antigen presentation capacity of BMDCs <i>in vitro</i>.

<p>BMDC were incubated in the presence of sOVA-FITC or OVA-FITC encapsulated into PLGA, PLGA-TMC or TMC-TPP nanoparticles at different concentrations. External FITC signaling was silenced by trypan blue. <b>A.</b> The ΔMFI of OVA-FITC was assessed by subtraction of FITC signaling at 4°C from 37°C. <b>B.</b> OVA-FITC uptake by BMDC shown as the net percentage of OVA-FITC positive cells. <b>C.</b> CFSE-labeled CD4⁺ T-cells were incubated with BMDC stimulated with sOVA or OVA encapsulated in nanoparticles. Gray filled histograms; unstimulated CD4⁺ T-cells, Black overlays; CD4⁺ T-cell division patterns at different OVA concentrations after 72 hours. <b>D–F.</b> Cytokine concentrations of IL-2, IFN-γ and IL-10 (ng/ml) were determined in culture supernatants, after 72 h of culture. Data are representative for 3 independent experiments; mean ± SEM. Statistically significant: *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Onset of disease and maximum arthritis scores.

<p>Hsp70-mB29a peptide loaded PLGA, PLGA-TMC nanoparticles or PBS control (10 µg) were given i.n. on day −7, −5 and −3 and arthritis was induced by PG/DDA immunization on day 0 and 21. Arthritis symptoms were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026684#s4" target="_blank">materials and methods</a>. Day of onset and maximum arthritis scores were depicted as mean ± SEM. of n = 3 mice per group of one experiment.</p

Enhanced OVA-specific CD4⁺ T-cell proliferation, after nanoparticle administration.

<p><b>A and D.</b> OVA-specific CFSE labeled CD4⁺ T-cells were transferred to BALB/c recipient mice one day prior to vaccination. Mice received a single i.n. application of 30 µg of sOVA or OVA encapsulated into PLGA, PLGA-TMC or TMC-TPP nanoparticles. For induction of a non-mucosal response, mice received a single i.m. immunization in the hind limbs. At 72 h post OVA administration, <i>in vivo</i> T-cell division was addressed in spleen, nose-draining NALT and CLN as well as the thigh-draining ILN. Data are representative for at least 3 i.n. and 2 i.m. independent transfer studies. <b>B, C and E.</b> Total mRNA was purified from single cell suspensions from NALT, CLN, and ILN. Relative mRNA expression to HPRT of Foxp3 was determined 72 h post OVA application. Cells isolated from NALT were pooled per group. LN data are representative for at least 3 to 5 mice per group; mean ± SEM. Statistically significant: *, <i>P</i><0.05.</p