"Cell-addictive" dual-target traceable nanodrug for Parkinson's disease treatment via flotillins pathway

Abstract

α-synclein (αS) aggregation is a representative molecular feature of the pathogenesis of Parkinson's disease (PD). Epigallocatechin gallate (EGCG) can prevent αS aggregation in vitro. However, the in vivo effects of PD treatment are poor due to the obstacles of EGCG accumulation in dopaminergic neurons, such as the blood brain barrier and high binding affinities between EGCG and membrane proteins. Therefore, the key to PD treatment lies in visual examination of EGCG accumulation in dopaminergic neurons. Methods: DSPE-PEG-B6, DSPE-PEG-MA, DSPE-PEG-phenylboronic acid, and superparamagnetic iron oxide nanocubes were self-assembled into tracing nanoparticles (NPs). EGCG was then conjugated on the surface of the NPs through the formation of boronate ester bonds to form a "cell-addictive" dual-target traceable nanodrug (B6ME-NPs). B6ME-NPs were then used for PD treatment via intravenous injection. Results: After treatment with B6ME-NPs, the PD-like characteristics was alleviated significantly. First, the amount of EGCG accumulation in PD lesions was markedly enhanced and traced via magnetic resonance imaging. Further, αS aggregation was greatly inhibited. Finally, the dopaminergic neurons were considerably increased. Conclusion: Due to their low price, simple preparation, safety, and excellent therapeutic effect on PD, B6ME-NPs are expected to have potential application in PD treatment.

Keywords: EGCG; Parkinson's disease; dopaminergic neurons; nanoparticles; α-synclein aggregation.

Scheme 1

(A) The structural composition and preparation of B6ME-NPs. DSPE-PEG-B6, DSPE-PEG-MA and DSPE-PEG-phenylboronic acid were used to improve the biocompatibility of magnetic nanoparticles through a micelle formation procedure. EGCG was then grafted onto the surface of the nanoparticles through the formation of a boronate ester bond. (B) The schematic diagram of B6ME-NPs in vivo: (1) B6ME-NPs target TfR on BBB and cross the BBB. (2) The NPs can target DAT and accumulate in the lesions area. (3) The NPs are internalized by dopaminergic neurons via DAT. (4) EGCG is released from the NPs due to high ROS response. (5) Free EGCG inhibits αS aggregation. (6) The high concentration of EGCG induces flotillins, the formation of membrane curvature and vesicle budding.

Figure 1

Synthesis and characterization of B6ME-NPs. (A) Average sizes of different nanoparticles. The polydispersity indices of B6ME-NPs, B6ME-NPs, B6ME-NPs, and B6ME-NPs were 0.195, 0.224, 0.106, and 0.249, respectively. (B) Zeta potentials of different nanoparticle dispersions in 0.01 × 10^-3 M PBS at 25 ºC. (C) TEM image of B6ME-NPs. Scale bar: 50 nm. Inset: TEM image with negative staining. Scale bar: 50 nm. (D) r₂ as a function of Fe concentration from B6ME-NPs. The slope of Fe concentration-R₂ regression curve is r₂ relaxivity. (E) Serum stability of B6ME-NPs after incubation in culture medium supplemented with 10% FBS. (F) EGCG released from B6ME-NPs triggered by different concentrations of H₂O₂. Error bars indicate mean ± SD.

Figure 2

Transport of EGCG-loaded NPs across an in vitro BBB model. (A) The structure of the trans-well chambers. SH-SY5Y and bEND.3 cells were co-cultured in trans-well chambers to imitate the BBB. Cellular uptake of different NPs in (B) bEND.3 and (C) SH-SY5Y cells was observed by flow cytometry. (D) B6ME-NPs and ME-NPs in SH-SY5Y cells was detected via CLSM. Nuclei: DAPI, blue; NPs: Cy5, red; lysosome: LysoTracker, green. Scale bar: 100 μm. (E) Quantitation of (D). Error bars indicate mean ± SD (n= 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Dopaminergic neurons-specific drug delivery analysis. (A) Cell uptake of B6ME-NPs and B6E-NPs in SH-SY5Y cells observed by CLSM. Nuclei: DAPI, blue; NPs: Cy5, red; lysosome: LysoTracker, green. Scale bar: 50 μm. (B) Quantitation of (A). (C) Cellular uptake of B6ME-NPs, B6E-NPs and MA in SH-SY5Y cells (2 h incubation) observed via flow cytometry. (D) Ultrathin sections of SH-SY5Y cells incubated with B6ME-NPs and B6E-NPs for 8 h were observed via TEM. Scale bar: 2 μm. (E) Quantitation of (D). Error bars indicate mean ± SD (n= 3). *P < 0.05, **P < 0.01, ***P < 0.001; (Tg + PBS), (Tg + B6ME-NPs) vs. WT. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; (Tg + EGCG) vs. (Tg + B6ME-NPs).

Figure 4

The role of B6ME-NPs in vitro. (A) αS-overexpression in SH-SY5Y cells as a PD cell model was detected via CLSM. Nuclei: DAPI, blue; control cells: mCherry, red; αS-SH: mCherry-SNAC, red. Scale bar: 20 μm. (B) The TEM images showing the differences of of vesicles and cell membrane curvature induced by B6M-NPs or B6MA-NPs in SH-SY5Y cells (48 h). Red triangles mark curvature, blue triangles mark vesicles. Scale bar: 2 μm. (C) Quantitation of (A). (D) Quantitation of vesicular NPs in (B). (E) Expressions of lipid raft-related proteins (EGFR, Contactin1, flotillin1, flotillin2 and neurotrimin) detected by Western Blot. (F-J) Quantitation of each protein in (E). Error bars represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; (Tg + PBS), (Tg + B6ME-NPs) vs. WT. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; (Tg + EGCG) vs. (Tg + B6ME-NPs).

Figure 5

Pharmacokinetics and accumulation of the traceable NPs in the SN region. (A) Representative fluorescence images of mice 24 h after i.v. treatment with different nanoparticles (PBS, Cy7-labeled ME-NPs and Cy7-labeled B6ME-NPs). (B) T2* MR images of mice brains 24 h after i.v. treatment with different nanoparticles (PBS, SPIONs-labeled ME-NPs and SPIONs-labeled B6ME-NPs). (C) SN concentration of EGCG from different nanoparticles at different time points post i.v. injection detected with liquid MS. (D) Plasma concentration of EGCG from different nanoparticles at different time points post i.v. injection detected with HPLC. Error bars represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 6

In vivo PD therapy studies. The WT mice as control. The representative after different samples (PBS, bulk EGCG and B6ME-NPs) treating for 1.5 months. αS overexpression transgene mice as PD model. Both EGCG and the NPs via intravenous injection. (A) Rota-rod test is for the coordinated motion detection. (B) Pole test is used to assess motor function. (C) Immunofluorescence staining of the SN region of treated mouse brain. Nuclei: DAPI, blue; DAergic neuronal: TH, red; aggregation: ThT, green. Scale bar: 20 μm. (D) Quantitation of (C). (E) TH immunohistochemical slices of the SN region of treated mouse brain. Scale bar: 100 μm. (F) Quantitation of (E). Error bars represent mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001; (Tg + PBS), (Tg + B6ME-NPs) vs. WT. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; (Tg + EGCG) vs. (Tg + B6ME-NPs).