Characterization of nSSL before and after GC loading.

<p>Values shown in each category are the average±SD for at least 10 formulations (13 empty nSSL, 27 nSSL-BMS, and 17 nSSL-MPS formulations). No significant differences were observed when comparing size according to intensity, number, or volume. No significant differences were observed when comparing nSSL-MPS and nSSL-BMS. PdI: an indication of size distribution variance between different batches prepared. A low PdI (<0.2) indicates that the sample is monodispersed.% drug encapsulated  = 100×([drug]/[lipid]_{after Dowex anion exchanger})/([drug]/[lipid]_{after dialysis}).</p

The effect of nSSL-BMS treatment at late stages of ECM, followed by artemisone.

<p>PbAus-infected C57Bl/6 mice (n = 10 per group, non-treated; n = 6 per group, nSSL-BMS) were administered 5% dextrose or 20 mg/kg nSSL-BMS (arrows) every other day, starting on day 5p.i., followed by 2×10 mg/kg/d artemisone after the cerebral phase, on days 11–15 p.i. (double arrows). No effect on the development of parasitemia was observed following administration of 5% dextrose. Administration of nSSL-BMS significantly reduced clinical score when started on day 5 p.i. or day 6 p.i. (p<0.001, p<0.05 vs. no treatment, respectively) and increased survival (p<0.01 and p<0.001 vs. non-treated), creating a time-window for antiplasmodial treatment and cure. *Day 15 p.i. nSSL-BMS: n = 6 (days 5,7,9 p.i. and days 6,8 p.i.).</p

Survival rates after early treatment with 10 mg/kg free or nSSL-encapsulated MPS (upper graph) or BMS (lower graph).

<p>Representative results for ICR mice infected with PbA are presented. Arrows denote treatment administration. ECM prevention is reflected in longer survival times, due to the development of severe anemic malaria, and as a result creation of a survival time-window for anti-plasmodial administration. Significant differences in survival were seen between non-treated and nSSL-MPS-treated groups (p = 0.01) and between non-treated mice and mice administered free or nSSL-BMS (p<0.01).</p

Scoring of clinical signs of ECM.

<p>Scoring of clinical signs of ECM.</p

Hemorrhages post-PbAus infection and following free BMS or nSSL-BMS administration.

<p>H&E staining: representative pictures depicting hemorrhage size, day 9 p.i. (magnification ×20). No hemorrhages were observed in the brains of healthy mice. PbAus-infected C57Bl/6 mice were administered placebo (5% dextrose, n = 22), 20 mg/kg free BMS (n = 17) or nSSL-BMS (n = 18) on days 3, 5, 7, and 9 p.i. Survival rates on day 13p.i., at the end of the cerebral phase, were 0 in the placebo group and 12% in the free BMS group, vs. 88% in the nSSL-BMS group.</p

Structures of the glucocorticoid prodrugs methylprednisolone hemisuccinate (MPS, left) and β-methasone hemisuccinate (BMS, right).

<p>Both compounds are amphipathic weak acids suitable for loading into nSSL.</p

Levels of BMS originating from nSSL-BMS or free BMS in tissues of naive healthy mice and mice with ECM.

<p>Mice at initial clinical stages of ECM were injected (on day 6 p.i.) with either free or nSSL-BMS. Healthy mice were injected with nSSL-BMS. *n.d, not done.</p

Effect of sequential steroid-artemisone treatment on the development of infection.

<p>PbAus-infected C57Bl/6 mice (n = 10 per group) were treated with 10 mg/kg free or nSSL-BMS on days 3, 5, 7, and 9 post-inoculation. Although parasitemias were not affected (p>0.05, all groups), treatment led to reduced clinical scores (p<0.05, non-treated vs. free BMS; p<0.001, vs. nSSL-BMS, days 1–12 p.i.), an effect more pronounced in liposome-treated mice (p<0.05, free vs. nSSL). Administration of 2×20 mg/kg/d artemisone on days 11–15p.i. led to cure. *One mouse (of the total 17 administered artemisone) relapsed and died of ECM. Arrows represent free or nSSL-BMS injections; double arrows represent artemisone injections.</p

Effect of steroid treatment on clinical score during the ECM-susceptible phase of infection.

<p>PbAus-infected C57Bl/6 mice (n = 10, non-treated group, empty nSSL group, 5 mg/kg and 10 mg/kg nSSL-BMS groups; n = 13, 20 mg/kg nSSL-BMS group) were administered empty nSSL or nSSL-BMS on days 3, 5, 7, and 9 post-inoculation. A clear dose-response was seen, which translated to improved clinical score. Clinical scores of mice treated with 10 mg/kg or 20 mg/kg nSSL-BMS were significantly lower than those of control mice. *p = 0.8, non-treated vs. empty nSSL; p = 0.08, non-treated vs. 5 mg/kg nSSL-BMS; p<0.0001, non-treated vs. 10 mg/kg nSSL-BMS, non-treated vs. 20 mg/kg nSSL-BMS; p = 0.3, 10 mg/kg vs. 20 mg/kg nSSL-BMS. Arrows represent injections.</p

Reduction in (a) microglial and (b) astrocyte activation in PbAus-infected C57Bl/6 mice treated with nSSL-BMS.

<p>Infected mice were administered placebo (5% dextrose, n = 22), 20 mg/kg free BMS (n = 17), or nSSL-BMS (n = 18) on days 3, 5, 7, and 9 p.i. Infection with PbAus induced significant activation of microglia (7a) and astrocytes (7b). Administration of nSSL-BMS resulted in reduced microglial and astrocyte activation and cell numbers; a lesser effect was observed after treatment with free BMS. 3–5 mice were examined at each time-point. Scale bar 100 µm.</p