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

Turjeman K, Bavli Y, Kizelsztein P, Schilt Y, Allon N, Katzir TB, Sasson E, Raviv U, Ovadia H, Barenholz Y - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0130442.g008: 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.

Mentions: The therapeutic effect was accompanied with changes in the brain tissue characteristics compared to control group observed by MRI (Fig 8A–8G). A short course of high-dose intravenous methylprednisolone (IVMP) is widely used to treat clinical relapses in MS [61–64]. The mechanism of action of IVMP, as of other forms of steroids, is uncertain. Suggestions have included the reduction of edema [65], immunosuppression [15], direct inhibition of demyelination [66], direct effect on axonal conduction, and reversal of blood-brain barrier (BBB) abnormalities [67]. MRI is increasingly used for diagnosing multiple sclerosis and assessing disease prognosis [68]. It has provided evidence regarding the therapeutic efficacy of steroids in MS relapses. In this study, mice were treated with 10mg/kg NSSL-MPS or saline (control) on days 8, 10, and 12 after T-cell transfer and were scanned in a 7T MRI system on day 16 after T-cell transfer. Fig 8B and 8C represents the average ventricles volume normalized to total brain volume in each group. T2 relaxation maps are suited to image edema and can also be used for measurement of brain ventricles volume. The ventricles measurements showed significantly larger ventricles in the EAE group compared to the naïve and treated mice. This pathology was previously indicated in the EAE model [69]. In diffusion tensor imaging (DTI) we can estimate the trace of the diffusion tensor or average diffusivity (ADC), a putative measure of edema or reduced tissue integrity. ADC maps showed a higher ADC parameter in the EAE group compared to the naïve and the treated groups (Fig 8D). This indicates that the diffusion of water molecules in the EAE brain tissue is higher and might result in changes in cellular/axonal damage and reduced tissue integrity. One-way ANOVA analysis shows several regions with significantly higher ADC in the EAE group compared to the naïve and treated groups (Fig 8E). This shows that in specific regions the differences in gray and white matter integrity were observed in the EAE model and following treatment. Extraction of ADC parameters in the significant clusters demonstrated higher ADC value in the EAE group compared to the naïve and treated groups. Such changes may occur due to increased tissue repair, including increased myelin density (as we have seen in LFB staining, Fig 7B), as well as increased activation and gliosis (microglia/astrocyte) in the EAE treated group (lower ADC parameters). T1-weighted images were performed before and after injection of gadolinium (Gd). Areas of Gd enhancement demonstrated on T1-weighted MRI are believed to reflect underlying blood-brain barrier disruption at active perivascular infiltrate and inflammation. Fig 8F shows a representative mouse brain from each group (G0-488 naïve; G1-492 EAE untreated; G2-482 EAE treated with NSSL-MPS) with the percentage intensity of change after Gd injection in the spinal cord, cerebellum, and the brain stem. There were higher enhancements of the signal (30–40% intensity) in both represented brains from treated and untreated groups (G1-492 and G2-482); in the mouse from the naïve group (G0-488) there was a slighter enhancement (10–20% intensity). This Gd enhancement may indicate a BBB penetration/breakdown/ [70]. There are conflicting reports regarding BBB disruption as a consistent component in the development of EAE and in the sequence of events leading to the development of signs of disease. Some studies have indicated that vascular permeability is a distinct event that precedes cellular infiltration [6, 71, 72], while other studies have found BBB permeability to be present only during inflammation [73–75]. Though these observations may be dependent on the specific model of EAE, the role of BBB permeability in the initial development and during the progression of the disease is unclear. Schmidt et al. [20] studied the effect of pegylated liposomes encapsulating prednisolone (PL) in the adoptive transfer EAE rat model. BBB integrity was determined by immunohistochemical staining for albumin. BBB disruption was greatly reduced by 10 mg/kg PL, which was superior to a 5-fold higher dose of free methylprednisolone (MP) on day 5 after cell transfer. Previously, serial iodine-enhanced CT scanning had shown that IVMP (high dose intravenous methylprednisolone) may rapidly reverse BBB abnormalities in MS lesions [76]. Treatment with IVMP showed a rapid reduction of BBB abnormalities within hours of administering methylprednisolone [76, 77], however the putative BBB abnormality may later reappear and many lesions re-enhanced within a few days of stopping IVMP. Miller et al. [77] reported that on day 7 after IVMP treatment, many lesions displayed increased enhancement in comparison with day 3, while at the same time all patients were clinically stable or improving. Therefore, given the therapeutic efficacy of 10mg/kg NSSL-MPS under the conditions employed (starting treatment at the onset of clinical signs and not shortly after T-cell transfer), it is reasonable to assume that the BBB was restored as a consequence of the treatment. Indeed, H&E staining demonstrated fewer infiltrating cells in the brains and spinal cords of EAE mice that were treated with NSSL-MPS compared to the untreated group, as well as to the free MPS-treated group (Fig 7B). However, structure abnormalities were visualized, an effect that can be explained by the re-appearance of BBB leakage due to the 4-day lag time until the scanning process. Even so, it is important to remember that there are pathophysiological mechanisms in MS relapses that are independent of the BBB, and that may or may not be modified by steroids, for example, immunological changes. Fig 8G demonstrates a cerebellum slice before Gd injection (left) and after (right). The marked regions indicate reduced signal in the suspected lesions. After Gd injection, one animal from the EAE untreated group (G1-492) seemed to show appearance of lesions. The appearance of lesions in the EAE untreated group may indicate the pathology of the disease [78]. Clinically, lesions that show such contrast enhancement on MRI are often identified as active lesions, and in many cases these lesions are found to correlate with clinical symptoms [79, 80].


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

Turjeman K, Bavli Y, Kizelsztein P, Schilt Y, Allon N, Katzir TB, Sasson E, Raviv U, Ovadia H, Barenholz Y - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492950&req=5

pone.0130442.g008: 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.
Mentions: The therapeutic effect was accompanied with changes in the brain tissue characteristics compared to control group observed by MRI (Fig 8A–8G). A short course of high-dose intravenous methylprednisolone (IVMP) is widely used to treat clinical relapses in MS [61–64]. The mechanism of action of IVMP, as of other forms of steroids, is uncertain. Suggestions have included the reduction of edema [65], immunosuppression [15], direct inhibition of demyelination [66], direct effect on axonal conduction, and reversal of blood-brain barrier (BBB) abnormalities [67]. MRI is increasingly used for diagnosing multiple sclerosis and assessing disease prognosis [68]. It has provided evidence regarding the therapeutic efficacy of steroids in MS relapses. In this study, mice were treated with 10mg/kg NSSL-MPS or saline (control) on days 8, 10, and 12 after T-cell transfer and were scanned in a 7T MRI system on day 16 after T-cell transfer. Fig 8B and 8C represents the average ventricles volume normalized to total brain volume in each group. T2 relaxation maps are suited to image edema and can also be used for measurement of brain ventricles volume. The ventricles measurements showed significantly larger ventricles in the EAE group compared to the naïve and treated mice. This pathology was previously indicated in the EAE model [69]. In diffusion tensor imaging (DTI) we can estimate the trace of the diffusion tensor or average diffusivity (ADC), a putative measure of edema or reduced tissue integrity. ADC maps showed a higher ADC parameter in the EAE group compared to the naïve and the treated groups (Fig 8D). This indicates that the diffusion of water molecules in the EAE brain tissue is higher and might result in changes in cellular/axonal damage and reduced tissue integrity. One-way ANOVA analysis shows several regions with significantly higher ADC in the EAE group compared to the naïve and treated groups (Fig 8E). This shows that in specific regions the differences in gray and white matter integrity were observed in the EAE model and following treatment. Extraction of ADC parameters in the significant clusters demonstrated higher ADC value in the EAE group compared to the naïve and treated groups. Such changes may occur due to increased tissue repair, including increased myelin density (as we have seen in LFB staining, Fig 7B), as well as increased activation and gliosis (microglia/astrocyte) in the EAE treated group (lower ADC parameters). T1-weighted images were performed before and after injection of gadolinium (Gd). Areas of Gd enhancement demonstrated on T1-weighted MRI are believed to reflect underlying blood-brain barrier disruption at active perivascular infiltrate and inflammation. Fig 8F shows a representative mouse brain from each group (G0-488 naïve; G1-492 EAE untreated; G2-482 EAE treated with NSSL-MPS) with the percentage intensity of change after Gd injection in the spinal cord, cerebellum, and the brain stem. There were higher enhancements of the signal (30–40% intensity) in both represented brains from treated and untreated groups (G1-492 and G2-482); in the mouse from the naïve group (G0-488) there was a slighter enhancement (10–20% intensity). This Gd enhancement may indicate a BBB penetration/breakdown/ [70]. There are conflicting reports regarding BBB disruption as a consistent component in the development of EAE and in the sequence of events leading to the development of signs of disease. Some studies have indicated that vascular permeability is a distinct event that precedes cellular infiltration [6, 71, 72], while other studies have found BBB permeability to be present only during inflammation [73–75]. Though these observations may be dependent on the specific model of EAE, the role of BBB permeability in the initial development and during the progression of the disease is unclear. Schmidt et al. [20] studied the effect of pegylated liposomes encapsulating prednisolone (PL) in the adoptive transfer EAE rat model. BBB integrity was determined by immunohistochemical staining for albumin. BBB disruption was greatly reduced by 10 mg/kg PL, which was superior to a 5-fold higher dose of free methylprednisolone (MP) on day 5 after cell transfer. Previously, serial iodine-enhanced CT scanning had shown that IVMP (high dose intravenous methylprednisolone) may rapidly reverse BBB abnormalities in MS lesions [76]. Treatment with IVMP showed a rapid reduction of BBB abnormalities within hours of administering methylprednisolone [76, 77], however the putative BBB abnormality may later reappear and many lesions re-enhanced within a few days of stopping IVMP. Miller et al. [77] reported that on day 7 after IVMP treatment, many lesions displayed increased enhancement in comparison with day 3, while at the same time all patients were clinically stable or improving. Therefore, given the therapeutic efficacy of 10mg/kg NSSL-MPS under the conditions employed (starting treatment at the onset of clinical signs and not shortly after T-cell transfer), it is reasonable to assume that the BBB was restored as a consequence of the treatment. Indeed, H&E staining demonstrated fewer infiltrating cells in the brains and spinal cords of EAE mice that were treated with NSSL-MPS compared to the untreated group, as well as to the free MPS-treated group (Fig 7B). However, structure abnormalities were visualized, an effect that can be explained by the re-appearance of BBB leakage due to the 4-day lag time until the scanning process. Even so, it is important to remember that there are pathophysiological mechanisms in MS relapses that are independent of the BBB, and that may or may not be modified by steroids, for example, immunological changes. Fig 8G demonstrates a cerebellum slice before Gd injection (left) and after (right). The marked regions indicate reduced signal in the suspected lesions. After Gd injection, one animal from the EAE untreated group (G1-492) seemed to show appearance of lesions. The appearance of lesions in the EAE untreated group may indicate the pathology of the disease [78]. Clinically, lesions that show such contrast enhancement on MRI are often identified as active lesions, and in many cases these lesions are found to correlate with clinical symptoms [79, 80].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus