Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases

<div><p>The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs’ unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a “water-soluble” amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug’s therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.</p></div

Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.

<p>^a Significant difference from the control group P<0.000001</p><p>^b Significant difference from the NSSL-MPS group P<0.0005</p><p>^c Significant difference from the control group P<0.00005</p><p>^d Significant difference from the NSSL-MPS group P<0.005.</p><p>Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.</p

Comparison of passively targeted NSSL and actively targeted peptide-conjugated NSSL.

<p><b>(A)</b> 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>(B)</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 (<b>■</b>). * p-value < 0.0001.</p

Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide conjugated NSSL-MPS in the acute EAE mice model.

<p>^a Significant difference from the control group P<0.00001</p><p>^b Significant difference from the control group P<0.001</p><p>^c Significant difference from the β-amyloid NSSL-MPS group P<0.0005</p><p>^d Significant difference from the β-amyloid NSSL-MPS group P<0.0001</p><p>^e Significant difference from the control group P<0.005</p><p>^f Significant difference from the control group P<0.0001.</p><p>Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide conjugated NSSL-MPS in the acute EAE mice model.</p

Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.

<p>^a Significant difference from the control group P<0.0001</p><p>^b Significant difference from the EPC NSSL-TMN treated group P<0.001.</p><p>Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.</p

Thermotropic characterization of SUV aqueous dispersions assessed from the DSC scans.

<p>Thermotropic characterization of SUV aqueous dispersions assessed from the DSC scans.</p

Comparison of the therapeutic efficacy of 50 and 10mg/kg NSSL-MPS in the acute EAE mice model.

<p>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 (■).</p

Effect of membrane lipid composition (EPC:Chol:PEG-DSPE or DMPC:DPPC:Chol:PEG-DSPE) on TMN retention in NSSL.

<p>% Free TMN was determined by ESR for both lipid compositions, EPC:Chol:PEG-DSPE (■) and DMPC:DPPC:Chol:PEG-DSPE (</p><p></p><p><mi>■</mi></p><p></p>) in vitro at 5°C <b>(A)</b>, 25°C <b>(B)</b>, and 37°C <b>(C)</b> during 4.5 months.<p></p

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.

<p>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) (●).</p

Small angle X-ray scattering (SAXS) measurements of NSSL-TMN.

<p><b>(A)</b> 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>(B)</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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130442#pone.0130442.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130442#pone.0130442.ref041" target="_blank">41</a>]. 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. <b>(C)</b> 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. <b>(D)</b> 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.</p