Table 1.
Physicochemical properties of NSSL-MPS and EPC-based NSSL-TMN.
Fig 1.
(A) DSC measurements. Samples of SUVs (DMPC:DPPC:Chol:PEG-DSPE, DMPC:DPPC:PEG-DSPE, DMPC:DPPC, DMPC:PEG-DSPE, DPPC:PEG-DSPE, DMPC, DPPC) in saline, and saline in the reference cell, were scanned in the range 10°-80°C, at the heating rate of 1°C/min. (B) Zooming in: Samples of SUVs DMPC:DPPC:Chol:PEG-DSPE, DMPC:DPPC:PEG-DSPE.
Table 2.
Thermotropic characterization of SUV aqueous dispersions assessed from the DSC scans.
Fig 2.
Effect of membrane lipid composition (EPC:Chol:PEG-DSPE or DMPC:DPPC:Chol:PEG-DSPE) on TMN retention in NSSL.
% Free TMN was determined by ESR for both lipid compositions, EPC:Chol:PEG-DSPE (■) and DMPC:DPPC:Chol:PEG-DSPE () in vitro at 5°C (A), 25°C (B), and 37°C (C) during 4.5 months.
Fig 3.
Small angle X-ray scattering (SAXS) measurements of NSSL-TMN.
(A) Radially integrated background-subtracted scattering data (symbols) of DMPC:DPPC NSSL with and without drug, at 4 and 37°C, as indicated in the figure. Note that the curves are shifted in the intensity axis only for clarity of presentation. The solid curves are the corresponding form-factor models of a stack of infinite slabs with a Gaussian electron density profile along the vertical direction. (B) The electron density profiles of the DMPC:DPPC NSSL bilayers (with and without drug at 4 and 37°C) along the normal direction. The density profiles are obtained by fitting the scattering data to the models (see A) with the software X+, choosing a Gaussian electron density profile for the liposome membrane [40, 41]. The profile is almost symmetric and very slightly affected by the temperature or the presence of the drug. The arrows point to the profile of the inner and outer PEG layers. (C) The integrated scattering patterns as a function of the magnitude of the scattering vector, q, for EPC liposomes. Note that the curves are shifted in the intensity axis for clarity of presentation. The scattering curves of the EPC NSSL with and without drug, at 4 and 37°C are very similar. These curves are analyzed using the software X+, as in (A). The liposome bilayer is described by a Gaussian electron density profile. (D) The electron density profile in the direction normal to the membrane, calculated using the software X+, is presented for EPC NSSL, with and without drug at 4 and 37°C. The density profile of the membrane is almost unaffected by the temperature or the presence of the drug. Notice that this profile is asymmetric, suggesting that the inner and the outer PEG layers (pointed by an arrow) of the liposome are different.
Table 3.
Membrane width (head-to-head distance) of the NSSL at 4°C and 37°C, with and without TMN using small angle X-ray scattering measurements.
Fig 4.
Comparison of the therapeutic efficacy of EPC:Chol:PEG-DSPE NSSL-TMN and DMPC:DPPC:Chol:PEG-DSPE NSSL-TMN in acute EAE mice model.
SJL/J mice (n = 10) were treated by IV injections every other day starting on day 8 with: EPC:Chol:PEG-DSPE NSSL-TMN 8.5 mg/kg BW (■), DMPC:DPPC:Chol:PEG-DSPE NSSL-TMN 8.5mg/kg BW (▲), and dextrose 5% (control) (●).
Table 4.
Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.
Fig 5.
Comparison of the therapeutic efficacy of 50 and 10mg/kg NSSL-MPS in the acute EAE mice model.
SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), 10mg/kg NSSL-MPS (▲) or 50mg/kg NSSL-MPS (■).
Table 5.
Comparison of the therapeutic efficacy of 50 and 10mg/kg NSSL-MPS in the acute EAE mice model.
Fig 6.
Comparison of passively targeted NSSL and actively targeted peptide-conjugated NSSL.
(A) Representative fluorescent microscopy images comparing brain accumulation of NSSL and their payload as is (A, A1), β-amyloid NSSL(B,B1), and ApoE NSSL (C,C1) in healthy mice brain showing an increase in the amount of actively targeted NSSL and their payload accumulating, compared to passively targeted NSSL. (B) Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide-conjugated NSSL-MPS in the acute EAE mice model. SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), NSSL-MPS (●), Apo-E NSSL-MPS (▲) or β-amyloid NSSL-MPS (■). * p-value < 0.0001.
Table 6.
Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide conjugated NSSL-MPS in the acute EAE mice model.
Fig 7.
Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.
(A) SJL mice were treated by IV injections on days 8, 10, 12 post-T cell transfer with saline (control) (▲), free MPS (50mg/kg) (■) or NSSL-MPS (50mg/kg) (◆). (B) Treatment with NSSL-MPS reduced inflammation and demyelination in brains and spinal cords of mice with AT-EAE compared with free MPS treated mice and control mice. Brains and spinal cords were obtained on day 13 post T- cell transfer. Black arrows indicate infiltrating inflammatory cells; white arrow indicates demyelination. Representative H&E-stained brains (A,B,D,E,G) and spinal cord (C,F,H) sections from control (A-C), as well as NSSL-MPS (D-F) and free MPS treated EAE mice (G-H) show extensive inflammation involving perivascular infiltrates of mononuclear leukocytes (arrows) within the cerebral parenchyma and spinal cords of CTRL mice and free MPS treated mice (arrows). In the NSSL-MPS treated group, much fewer infiltrating cells were observed. LFB staining demonstrates extensive demyelination in the control spinal cord (J) and a decrease in myelin density in the free MPS treated group (I,L) around blood vessels compared with NSSL-MPS treated mice (K), demonstrating densely organized myelin sheaths. Original magnification of x 40.
Table 7.
Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.
Fig 8.
Brain tissue characteristics of acute EAE mice using magnetic resonance imaging.
A: The therapeutic efficacy of NSSL-MPS (10mg/kg) in the adoptive transfer EAE mice model. SJL mice were treated by IV injections on days 8, 10, 12 post-T cell transfer with saline (control) (■), or NSSL-MPS (10mg/kg) (◆) and were scanned in a 7T MRI system on day 16 after T-cell transfer. B: Ventricles volume measurement normalized to total brain volume. The ventricles volume in the EAE group (G1) was bigger than the volume in the naïve and NSSL-MPS (10mg/kg) treated groups (G0 and G2). C: T2 images of representative mice brains. (A) G0 –naïve group, (B) G1 –EAE group, (C) G2 –EAE treated with NSSL-MPS (10mg/kg) group. D: Voxel-based one-way ANOVA between naïve, treated with NSSL-MPS, and untreated group. The significant clusters are presented on a brain atlas. E: Graphs of the ADC value in each significant cluster in each group. In all regions, ADC value is higher in the EAE group (G1) compared to the treated (G2) and naive group (G0). F: Representative mouse brain from each group (G0-488; G1-492; G2-482) with the % intensity of change after Gd injection in the spinal cord, cerebellum, and the brain stem. G: Cerebellum slices of G1-492 mouse before and after Gd injection. Lesions appearance after Gd injection, indicate by reduced of signal marked in red in the image.