In this study, 5 types of nanoliposomes were synthesized and characterized, including:

• 1.

  PEGlated nanoliposomes 1 (PNL1) containing sodium percarbonate (SPC) (Merk, Germany), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) (Merk, Germany), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine-polyethylene glycol-2000 (DSPE-PEG-2000) (Merk, Germany), carboxy-PEG (Merk, Germany), omega-3 (Merk, Germany), mannitol (Merk, Germany), 7-hydroxyflavone (Merk), and antisense oligonucleotides (Bioneer, South Korea).

• 2.

  PEGlated nanoliposomes 2 (PNL2) containing SPC, DOTAP, DSPE-PEG-2000, carboxy-PEG, omega-3, mannitol, and 7-hydroxyflavone.

• 3.

  PEGlated nanoliposomes 3 (PNL3) containing SPC, DOTAP, DSPE-PEG-2000, carboxy-PEG, omega-3, and mannitol.

• 4.

  PEGlated nanoliposomes 4 (PNL4) containing SPC, DOTAP, DSPE-PEG-2000, carboxy-PEG, and omega-3.

• 5.

  PEGlated nanoliposomes 5 (PNL5) containing SPC, DOTAP, DSPE-PEG-2000, and carboxy-PEG.

To synthesize PNLs, reverse-phase evaporation method was used.16 Briefly, SPC, DOTAP, DSPE-PEG-2000, and carboxy-PEG were dissolved in chloroform, and then omega-3, mannitol, 7-hydroxyflavone, and antisense oligonucleotides (5′-CTACATAGAGAACAC-3′) were separately added based on each formulation. The mixture was hardly mixed and homogenized using a probe ultrasound. The pulse was 5s, 2s off, 100% amplitude, and 36joles per pulse. Subsequently, the chloroform was evaporated by a rotary vacuum pump. To hydrate PNLs, PBS was used and their concentrations were adjusted to 1%, 2%, and 4%. Finally, their size distribution was measured by dynamic light scattering (DLS) and their stability was checked until one week at 25°C.

First, 100μL of PNLs at concentration of 1%, 2%, and 4% was separately incubated with 100μL of 100nM recombinant monomeric α-syn (Sigma-Aldrich) in presence of Thioflavin T (ThT) (Acros Organics) for 60min at 37°C, as previously described.17 The α-syn fibrillization was assessed each 20min at the emission wavelength of 490nm and the excitation wavelength of 450nm

Also, after incubation of PNLs with recombinant monomeric α-syn for 60min, 50μL of each mixture was separately placed on a Butvar-coated copper grid, dried, and then negatively stained by uranyl acetate (1.0%, w/v). Here, a transmission electron microscopy (TEM) (Zeiss EM10, Germany) with an excitation voltage of 100kV was applied to observe and compared the size and morphology of α-syn aggregates.

BV2 microglia were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Atlanta Biologics) and 1% Penicillin-Streptomycin (Merck, Germany). Then, they were plated at 50,000cells/well in a 96-well plate to adhere overnight. Cells were first treated with 100nM A53T α-syn and then with PNLs at a final concentration of 1%, 2%, and 4%. After 24h, supernatants were collected and assayed for TNF-a and IL-6 by ELISA kit (R&D systems) as previously described.11 Briefly, the high binding ELISA plates (Biomat, Italy) were separately coated with anti-TNF-a IgG and anti-IL-6 IgG (R&D systems) at 1μgmL−1. After washing, 50μL of supernatants was added and incubated for 1h at 37°C. Then, plates were washed and 100μL of secondary antibody (anti-TNF-a IgG and anti-IL-6 IgG (R&D systems)) was added. After incubation and washes, 50μL of 3,3′, 5,5′-tetramethylbenzidine was added and then stopped by a stopping solution. Finally, the absorbance of each well was read by a Spectrophotometer at 450nm (BioTek Industries) and the concentration of TNF-a and IL-6 was calculated using standard curve.

To quantify the expression of SNCA at mRNA level, BV2 microglia cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin, plated, and treated with PNLs at a final concentration of 1%, 2%, and 4% for 24h. Then, total RNA was extracted by extraction buffer (QIAGEN) and then cDNA was synthesized by cDNA mastermix (Life Technologies). Finally, quantitative real-time PCR was performed on an ABI real-time PCR system using qPCR Master Mix (Life Technologies). The GAPDH was used as a reference gene and the delta-delta CT formula was applied to evaluate the relative expression. The primers used in this study are listed in Table 1.

To quantify the expression of SNCA at the protein level, BV2 microglia cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin, plated, and treated with PNLs at a final concentration of 1%, 2%, and 4% for 24h. Cell lysates were mixed with Laemlli sample buffer, boiled, and separated on polyacrylamide gel (Thermo Fisher Scientific). After protein transfer to polyvinylidene fluoride membranes (Thermo Fisher Scientific), the blots were probed with the primary antibodies (α-syn (BD Transduction Laboratories) and beta-actin (Millipore)), visualized with secondary antibodies, HRP-conjugated anti-mouse IgG (GE Healthcare), and developed using ECL Prime Western Blotting Detection Reagent. The signals of immunoblots were recorded with a Chemidoc Touch Imaging System. Images of blots were cropped from different parts of the same gel for densitometry analysis.

For this part of the study, we used α-syn transgenic mouse model (https://qpsneuro.com/in vivo-services/animal-models/alpha-synuclein-transgenic-mouse-models) to check the efficacy of PNLs. Briefly, transgenic mice were separately treated with 100μL of PNLs 1–5 at a concentration of 4% which taken orally for 5 days. After treatment period, all mice were killed under high dose of anesthesia and then their brains were removed. Then, tissue sections were prepared and fixed. After passing from the serial dilutions of alcohols, the slides were immersed in distilled water and then antigen retrieval was carried out by microwaving in the citric acid buffer. In the next step, 50μL of primary antibody (anti-α-syn IgG (BD Transduction Laboratories)) were added and incubated for 12h. After washing, 50μL of the secondary antibody (FITC-conjugated anti-mouse IgG (GE Healthcare)) were added and incubated for 3h. Finally, the slides were examined by a fluorescent microscopy. The amount of α-syn accumulation was scored and finally normalized to control. To evaluate the neuroinflammation, cerebrospinal fluid (CSF) of mice was used. After treatment of mice, the concentration of TNF-a and IL-6 in CSF was evaluated by ELISA kit (R&D systems) as previously described.11 To evaluate the expression of SNCA, several pieces of brain were cut and hardly crushed by a mechanical pressure. In the next step, the mixture was centrifuged at 5000RPM for 5min and the supernatant is separated for the evaluation of SNCA expression at both mRNA and protein by real-time PCR and Western blot with above mentioned details.

Data are presented as mean±SD with 3 independent repeats (n=3) for in vitro experiments and with 5 independent biological repeats for in vivo experiments. Analysis of different study groups was performed using one-way ANOVA with Tukey's post hoc test with p<0.05.

The size distribution of all synthesized PNLs was approximately between 120 and 130nm and all of them were stable during one week at 25°C.

First we explored the effect of PNLs on α-syn fibrillization using a standard ThT kinetic fibrillization assay. We found that all PNLs shortened the α-syn growth phase, suggesting inhibition of α-syn fibrillization (Fig. 1a–e). Importantly, the endpoint ThT assay showed both PNL1 and PNL2 had significantly less fibrillization when compared with other PNLs (Fig. 1f).

Figure 1.

The effect of PNL1 (a), PNL2 (b), PNL3 (c), PNL4 (d), and PNL5 (e) on α-syn fibrillization by ThT kinetic fibrillization assay. Endpoint ThT results for different concentrations of PNLs (f). TEM images showed the inhibition of fibrillization by PNL1 and PNL2 (g). As seen, very low fibrils were seen when α-syn treated with PNL1 and PNL2 at a concentration of 1%, 2%, and 4% for 60h. The scale bar is 100nm Data are presented as mean±SD; n=3; *p<0.05 when compared with PNL3, PNL4, and PNL5 by one-way ANOVA. Control was α-syn alone without PNLs.

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After incubation of PNLs with recombinant monomeric α-syn, they were negatively stained by uranyl acetate and observed by TEM at 100kV. The inhibition of α-syn fibrillization was also seen when monomeric α-syn incubated with PNL1 or PNL2 at concentration of 1%, 2%, and 4%. In other treated groups, we saw high degrees of fibrillization (Fig. 1g).

Activation of microglia leads to the release of inflammatory agents which particularly are harmful for neurons.18 Here, we investigated the effect of PNLs on the production of TNF-a and IL-6 when microglial cells exposed to 100nM α-syn. It was found that the production of TNF-a (Fig. 2a), and IL-6 (Fig. 2b) was significantly decreased when microglial cells were treated with PNL1 or PNL2 at concentrations of 1%, 2%, and 4%.

Figure 2.

Production of TNF-a (a), and IL-6 (b) when microglial cells were first exposed to 100nM α-syn and then treated with PNLs. The expression of SNCA mRNA (c) and SNCA protein (d) when microglial cells were treated with PNLs for 24h. Data are presented as mean±SD; n=3; *p<0.05 when compared with PNL3, PNL4, and PNL5 by one-way ANOVA. Control was microglial cells were exposed to 100nM α-syn and did not treat with PNLs.

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The expression of SNCA at the mRNA level was significantly decreased when BV2 microglia cells were treated with PNL1 or PNL2 (Fig. 2c). Also, the expression of SNCA at the protein level was significantly decreased when BV2 microglia cells were treated with PNL1 or PNL2 (Fig. 2d).

Based on in vivo experiments, we confirmed the inhibition of α-syn fibrillization, attenuation of microglial activation, and silencing of SNCA in α-syn transgenic mice when treated with PNL1 (Fig. 3a) or PNL2 (Fig. 3b) at concentration of 4%. Importantly, we could not confirm the efficacy of PNL3 (Fig. 3c), PNL4 (Fig. 3d), and PNL5 (Fig. 3e), suggesting these are not good candidates.

Figure 3.

Levels of α-syn fibrillization, TNF-a, IL-6, SNCA mRNA, and SNCA protein in α-syn transgenic mouse model when treated with PNL1 (a), PNL2 (b), PNL3 (c), PNL4 (d), and PNL5 (e) for 5 days. Data are presented as mean±SD; n=5; *p<0.05 when compared with PNL3, PNL4, and PNL5 by one-way ANOVA. Control was α-syn transgenic mouse model which was not treated with PNLs.

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