Nanoparticles effects on psychosine-induced changes in astrocytes morphology

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distribution and zeta potential of lecithin/chitosan nanoparticles dispersed at increasing concentrations in cell culture medium with 20µM of psychosine. psychosine. When psychosine alone at 20µM was dispersed in DEM-F/12 medium, particles with an average diameter around 300 nm where found, with a relatively large particle size distribution (PDI 0.292).

Following, nanoparticles at 0.02 µl, 0.2 µl and 2 µl were dispersed in DME-F/12 serum free containing 20 µM of psychosine, to mimic conditions close to those occurring in the experiments with astrocytes. As shown in Table 1, there is a substantial increase of nanoparticles diameter in the presence of psychosine. Moreover, the increase in particles size is dependent on the number of particles available to interact: while in the smallest nanoparticles concentration the diameter is around 700 nm, in the highest amount the it increased to more than 1000 nm. Also, PDI values variate according to the ratio between nanoparticles and psychosine: increasing the number of particles available for interaction with psychosine, the homogeneity of the dispersion increases (PDI 0.673 with LCN at 0.02 µl; PDI 0.321 with LCN at 0.2 µl; PDI 0.218 with LCN at 2 µl).

Finally, psychosine caused also a reduction of nanoparticles zeta potential reducing the surface charge close to neutral values (+0.3 mV, Table 1). These data suggested that nanoparticles structural interactions with psychosine may represent the key factor for the inhibition of the in vitro effects of psychosine on astrocytes.

4.6 Nanoparticles Effects on Psychosine-Induced Changes in

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filament marker was particularly relevant when observing astrocytes processes (15 µM: 0.77

± 0.06; 20 µM: 0.24 ± 0.08), suggesting an alteration in the cellular cytoskeleton. Once again, SVT/CoQ10-LCN nanoparticles (10 µM) significantly preserve astrocytes from psychosine damages, maintaining the basal levels of the filament marker (fluorescence around 100%) and the number of cellular processes per cell (Psy 20µM: 2.07 ± 0.10).

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Figure 5: Psychosine-induced changes in vimentin from human astrocytes. Human astrocytes were seeded at 3.9x10⁵ density per well and grown for 36h. Serum preserved cells were treated for 4h with 10 µM, 15 µM and 20 µM of psychosine ± SVT/CoQ10-LCN nanoparticles at 10 µM. Representative fluorescent imagines with a 20x magnification are displaying DAPI (blue) and vimentin (green) under treatment conditions as indicated on the figure. A 50 µM concentric circle from cell nucleus was drawn and the number of astrocytes extensions beyond was counted. 30 cells were counted to each condition. Fluorescence analysis was recorded using ImageJ software and expressed as percentage in a direct comparison to untreated cells control.

0 50 100 150

Vimentin fluorescence, %

Ctr 10 15 20 10 15 20

[Psychosine, µM]

[SVT/CoQ10-LCN, 10µM]

0 1 2 3

N° atrocytes extensions

Ctr 10 15 20 10 15 20

[Psychosine, µM]

[SVT/CoQ10-LCN, 10µM]

Control

Psy 10µM Psy 15µM Psy 20µM

S VT/CoQ10-LCN 10 µM Control

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To compare free drugs with drug-loaded nanoparticles, cells were incubated for 4h with psychosine (20µM) alone or supplemented with 10µM of SVT/CoQ10 raw material or SVT/CoQ10-nanoparticles (Figure 6). Again, fluorescence levels of vimentin marker decreased significantly in the presence of psychosine (reduced by 62%). While simvastatin co-treatment as free drug was unable to counteract psychosine toxic effect (data not shown), the combination with CoQ10 provided a partial but significant attenuation (vimentin fluorescence: 56% vs 38%; astrocytes processes: 0.60 ± 0.04 vs 1.46 ± 0.08) of the toxic-induced morphology changes. Nevertheless, SVT/CoQ10-loaded nanoparticles provided the best performance in preventing the loss of vimentin fluorescence (102.8 ± 3.1%) and astrocytes processes (Crt: 1.95 ± 0.09 vs 1.96 ± 0.13).

Vimentin staining of astrocytes incubated with the corresponding volume of blank nanoparticles showed results similar to those obtained with drugs-loaded particles (Figure 6).

In fact, the fluorescence levels of vimentin were found to be around 98.3 ± 1.8 % and cells presented an average of 2.05 (± 0.11) of astrocytes extensions.

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Figure 6: Psychosine-induced decrease in vimentin marker in human astrocytes is prevented by SVT/CoQ10 nanoparticles. Human astrocytes were seeded at 3.9x10⁵/mldensity and grown for 36h. Serum preserved cells were treated for 4h with 20µM of psychosine ± 10µM SVT/CoQ10 free drugs, SVT/CoQ10-LCN or empty nanoparticles. Representative fluorescent imagines with a 20x magnification are displaying DAPI (blue) and vimentin (VMT, green) under the treatment conditions indicated on the figure. A 50µM concentric circle from cell’s nucleus was drawn and the number of astrocytes extensions beyond was counted. 30 cells were counted to each condition. Fluorescence analysis was recorded using ImageJ software and expressed as percentage in a direct comparison to untreated cells control.

Control Psy 20

µM SVT/CoQ10

SVT/CoQ10-LCN B-LCN

VMT

DAPI

Merge

Ctr Psy20µM

SVT /CoQ10

SVT /CoQ10-LCN

B-LCN 0

50 100 150

Vimentin fluorescence, %

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0.5 1.0 1.5 2.0 2.5

N° atrocytes extensions

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These results corroborate with previous finds that pyschosine-induced vimentin loss in astrocytes extensions. Moreover, the protective effect in vimentin fluorescence intensity by loaded and unloaded nanoparticles supports a role of nanoparticles in the prevention of psychosine cytotoxicity.

4.7 Simvastatin and Coenzyme Q10 Attenuate TNF-a/IL-17A-Induced Release of the Pro-Inflammatory Cytokine IL-6 from Astrocytes

To investigate the effect of SVT/CoQ10 free drugs and nanoparticles-loaded in the management of the CNS inflammation, their effect on IL-6 release from astrocytes was investigated. In a previous study, Elain and co-workers demonstrated that the co-stimulation of human astrocytes with TNF-a and IL-17 A cytokines increase the release of IL-6 within 24 hours ²⁷. The authors showed that human astrocytes express IL17A receptors and consequently, an in vitro supplement of IL-17A increases the protein levels of IL-6 in the culture medium of those cells. Moreover, this effect was enhanced in the presence of TNF-a by its induction of IL-8 mRNA over-expression.

Here, we investigated whether the TNF-a/IL-17A-mediate release of IL-6 from astrocytes occurs also in a shorter time of stimulus and if the treatment with drugs nanoformulation affects the IL-6 release. Hence, human astrocytes were treated with 10 ng/ml of TNF-a and 50 ng/ml of Il-17 A, for increasing periods of time, i.e. 2, 4 and 6 hours.

As highlighted on Figure 7, stimulation of astrocytes with TNF-a and IL-17 A increased IL-6 release in a time-dependent manner. While at 2h of incubation TNF-a/IL-17A did not increase the levels of IL-6 protein released in the cell culture medium, at 4 hours and 6 hours increasing and significant levels of the cytokine were detected.

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Figure 7: TNF-a and IL-17 A increases the level of IL-6 secretion in human astrocytes.

Human astrocytes were seeded at 3.9x10⁵ cells/ml and grown for 48h, until confluent. Serum preserved cells were treated with 10 ng/ml of TNF-a and 50 ng/ml of IL-17 A for 2h, 4h and 6h. Graph shows the secretion of IL-6 over time under cytokines stimulus. Control bar represent values of untreated cells incubation. *** p < 0.001 compared to control.

To investigate the drugs and nanoparticles effect on the TNF-a/IL-17A-mediate increase in the IL-6 protein levels, human astrocytes were pre-treated for 1 hour simvastatin alone or in combination with CoQ10 or the two compounds co-encapsulated into nanoparticles. Afterwards, cells were co-incubated with the pro-inflammatory cytokines for further 6 hours. Controls incubation, as well as blank nanoparticles, were maintained in the same incubation conditions for all treatments.

Confirming previous findings, treatment of human astrocytes with TNF-a/IL-17A caused an increase in the levels of IL-6 within 6 hours (Figure 8). Importantly, all the treatment conditions significantly suppressed the secretion of IL-6 from astrocytes.

Simvastatin drug solution reduced IL-6 secretion by 24% at the smallest drug concentration and by about 45% at 1 µM and 10 µM. In this case, empty nanoparticles did not show any effect and did not affect the release of IL-6 from astrocytes. SVT/CoQ10-LCN nanoparticles reduced TNF-a/IL-17A-induced levels of IL-6 in a concentration-dependent manner, achieving 47% of inhibition. Interesting, the best performance in the inhibition of the TNF-a/IL-17A-induced increase in the protein levels of IL-6 was presented from the association of simvastatin and CoQ10 as drugs solution. In fact, the addition of coenzyme Q10 potentiate

Ctr 2h 4h 6h

0 200 400 600 800

[10ng/ml TNF-a / 50ng/ml IL-17A

]

IL-6 (pg/ml)

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the effect observed for simvastatin alone (2-fold), suppressing almost 60% of IL-6 the release from TNF-a/IL-17A-stimulated astrocytes compared to positive control (Figure 8). These results confirm that both, simvastatin and coenzyme Q10, attenuate the pro-inflammatory signals of TNF-a/IL-17A in human astrocytes, decreasing IL-6 secretion.

Figure 8: TNF-a/IL-17-induced IL-6 secretion in human astrocytes is attenuated by unloaded and nanoparticles-loaded compounds. (A) Experimental treatment diagram (B) Human astrocytes were seeded at 3.9x10⁵ cells/ml and grown for 48h, until confluent. Serum preserved cells were pre-treated with 0.1µM, 1µM and 10µM of SVT, SVT/CoQ10 and SVT/CoQ10-LCN for 1h before treatment with 10 ng/ml of TNF-a and 50 ng/ml of IL-17 for 6h. Treatment of human astrocytes with TNF-a/IL-17A showed an increase of IL-6, that was attenuated by drugs and nanoparticles treatments. **p < 0.01 and ***p < 0.001 compared to control.

4.8 SVT/CoQ10-LCN Nanoparticles Effect on Psychosine-Induced Demyelination in Cerebellar Slices Culture: Preliminary Results

Increasing evidences have suggested the involvement of early demyelination in the establishment of a number of neurodegenerative pathogenesis, such as multiple sclerosis

ON 6h

Serum Free

Compounds 1h

TNF-a/IL-17A

Elisa Kit

A B

0.1 1 10 0.1 1 10 0.1 1 10 0.1

0 250 500 750

IL-6 (pg/ml)

SVT B-LCN

SQ10-LCN

**

***

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[TNF-α/ IL-17A]

SVT/CoQ10-LCN

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(MS) ^18,21,49, Krabbe’s diseases (KD) ^19,50 and more recently Alzheimer diseases (AD) ^22,51. Moreover, enhanced brain accumulation of psychosine has been correlated with the dysfunction on oligodendrocytes, the cells responsible for myelin formation, and mediation of demyelination process. To determine whether SVT/CoQ10-loaded nanoparticles are able to protect myelin in a condition of psychosine accumulation, cerebellar slices were exposed to psychosine (1µM) in the presence or absence of nanoparticles (1µM) for 18 hours. Slices were further treated for 30 hours with SVT/CoQ10-LCN nanoparticles or fresh medium as control.

In agreement with previous studies ^28,30,48, the exposure of cerebellar slices culture to psychosine induced demyelination, as observed by the reduced expression of myelin basic protein (MBP) (Figure 9). Preliminary results showed that after 18 hours of treatment with psychosine, the fluorescence levels of MBP was significantly decreased in psychosine treated cerebellar slices (39%). It is also possible to note an increase in the protein expression of astrocytes marker glial fibrillary acid protein GFAP, expressed by the increase in fluorescence levels compared to control (155%, Figure 9 C).

Importantly, SVT/CoQ10-LCN nanoparticles (1µM) prevented the psychosine-induced decrease in MPB expression (92% vs 39%, Figure 9) and reduced psychosine activation of GFAP expression (102% vs 155%) on cerebellar slices. As already pointed out, SVT/CoQ10-LCN nanoparticles plays an important role in the inhibition of psychosine effects. These results demonstrate that nanoparticulate systems may have a protective effect on the demyelination processes of cerebellar slices culture.

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Figure 9: SVT/CoQ10-LCN nanoparticles inhibits psychosine-induced demyelination on cerebellar slices. (A) Experimental diagram. (B) Cerebellar cultures immunofluorescently labelled with MBP and GFAP under treatment conditions indicated. Fluorescence images captured at 10x magnification. Bar graph illustrates changes in (C) MBP and (D) GFAP staining after psychosine (1µM) and SVT/CoQ10-LCN (1µM) treatments. Fluorescence analysis was recorded using ImageJ software and expressed as percentage in a direct comparison to untreated cells control.

Ctrl Psy NP

0 50 100 150 200

GFAP fluorescence (% of control)

Psy 1µM 18h

12 days cultured

30h

Immuno-histochemical analysis Change media

Psy SVT/CoQ10 -LCN

Ctrl Psy NP

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MBP fluorescence (% of control)

Psy 1µM

GFAP MPB MERGE

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Psy + NP

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