Anthocyanins encapsulated by PLGA@PEG nanoparticles potentially improved its free radical scavenging capabilities via p38/JNK pathway against Aβ1-42-induced oxidative stress

Abstract

Background: In order to increase the bioavailability of hydrophilic unstable drugs like anthocyanins, we employed a polymer-based nanoparticles approach due to its unique properties such as high stability, improved bioavailability and high water-soluble drug loading efficiency. Anthocyanins constitute a subfamily of flavonoids that possess anti-oxidative, anti-inflammatory and neuroprotective properties. However, anthocyanins are unstable because their phenolic hydroxyl groups are easily oxidized into quinones, causing a reduced biological activity. To overcome this drawback and improve the free radical scavenging capabilities of anthocyanins, in the current study we for the first time encapsulated the anthocyanins in biodegradable nanoparticle formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-2000. The biological activity and neuroprotective effect of anthocyanin loaded nanoparticles (An-NPs) were investigated in SH-SY5Y cell lines.

Results: Morphological examination under transmission electron microscopy (TEM) showed the formation of smooth spherically shaped nanoparticles. The average particle size and zeta potential of An-NPs were in the range of 120-165 nm and -12 mV respectively, with a low polydispersity index (0.4) and displayed a biphasic release profile in vitro. Anthocyanins encapsulation in PLGA@PEG nanoparticles (NPs) did not destroy its inherent properties and exhibit more potent neuroprotective properties. An-NPs were nontoxic to SH-SY5Y cells and increased their cell viability against Aβ_1-42 by its free radical scavenging characteristics and abrogated ROS generation via the p38-MAPK/JNK pathways accompanied by induction of endogenous nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Comparative to native bulk anthocyanins, An-NPs effectively attenuated Alzheimer's markers like APP (amyloid precursor protein), BACE-1 (beta-site amyloid precursor protein cleaving enzyme 1), neuroinflammatory markers such as p-NF-kB (phospho-nuclear factor kappa B), TNF-α (tumor necrosis factor) and iNOS (inducible nitric oxide synthase) and neuroapoptotic markers including Bax, Bcl₂, and Caspase-3 protein expressions accompanied by neurodegeneration against Aβ_1-42 in SH-SY5Y cell lines.

Conclusions: Overall, this data not only confirmed the therapeutic potential of anthocyanins in reducing AD pathology but also offer an effective way to improve the efficiency of anthocyanins through the use of nanodrug delivery systems.

Keywords: Alzheimer’s disease; Anthocyanins; Neuroprotection; Oxidative stress; PLGA@PEG-nanoparticles.

Fig. 1

Transmission electron microscopy and DLS observations of An-NPs and its beneficial effects against Aβ_1–42-induced neurotoxicity; a TEM micrograph of An-NP (scale bar 0.5 µm). b DLS analysis for the particle size of An-NPs. c In vitro cytotoxicity of PLGA@PEG NPs, native anthocyanin and An-NPs incubated with normal SH-SY5Y cells. Cell viability was measured by MTT assay. Four different concentrations of the test samples were added to the cells and incubated for 24 h before adding the respective assay reagents. We have observed that the nanoparticles were highly biocompatible. d Shown is the cell viability (MTT assay) histogram. Aβ_1–42 (5 µM) reduced cell viability while anthocyanins and An-NPs at three different concentrations (50, 100 and 200 µg/ml) increased the cell viability of SH-SY5Y cell lines. e Representative ROS assay histogram. Anthocyanins and An-NPs in all three different concentrations (50, 100 and 200 µg/ml) significantly reduced Aβ_1–42-induced (5 μM) ROS production. e The ApoTox-Glo Triplex Assay was performed (Promega, Promega BioSciences, LLC., San Luis Obispo, CA, USA). Histogram showing, cell viability. f Cytotoxicity and (g) Caspase-3/7 assays. All the related experimental details are provided in the “Methods” section. All these assays were performed in triplicate (±SEM). *Significantly different from the control; ^#significantly different from Aβ_1–42-treated group. Significance = **p < 0.01, ^#p < 0.05, ^##p < 0.01

Fig. 2

Microscopic study showing the cellular internalization of the rhodamin123-loaded PLGA@PEG NPs; Confocal laser scanning microscopy (CLSM) images of the SH-SY5Y cells treated with rhodamin123-loaded PLGA@PEG NPs for 12 h (scale bar 50 and 10 µm)

Fig. 3

An-NPs attenuated Aβ pathology and prevents Aβ_1–42-induced activation of P38/JNK Pathways. a Effect of An-NPs treatment on the expression of Alzheimer markers analyzed by Western Blot in Aβ_1–42 treated SHSY-5Y cell line. Shown are representative Western Blots probed with antibodies of APP and BACE-1 in the Aβ_1–42 treated SHSY-5Y cells. Data are the representative of three individual experiments (n = 3). The protein bands were quantified using sigma gel software. β-Actin was used to show equivalent amounts of protein loading. b Representative Western Blots of p-P38, p-JNK, p-NF-kB, TNF-α and iNOS in Aβ_1–42-treated SH-SY5Y cells. Data are the representative of three individual experiments (n = 3). The protein bands were quantified using sigma gel software. β-Actin was used to show equivalent amounts of protein loading. c The double immunofluorescence images of SH-SY5Y cells after Aβ_1–42 and An-NPs treatment, showing p-JNK (green) and BACE-1 (red), proteins and their respective relative density histograms. The DAPI (blue) was used to stain the nucleus. All the experiments were performed in triplicate. The details are given in the "Methods" section. *Significantly different from the control; ^#significantly different from Aβ_1–42-treated group. Significance = **p < 0.01, ^#p < 0.05, ^##p < 0.01

Fig. 4

An-NPs treatment alleviated oxidative stress and upregulated Nrf-2 and its downstream targets genes HO-1 expressions. a Shown are representative Western Blots probed with antibodies of Nrf2, and HO-1 in the Aβ_1–42 treated SHSY-5Y cells. Data are the representative of three individual experiments (n = 3). The protein bands were quantified using sigma gel software. β-Actin was used to show equivalent amounts of protein loading. b The double immunofluorescence images of SH-SY5Y cells after Aβ_1-42 and An-NPs treatment, showing 8-Oxo-G (green) and Nrf2 (red), proteins and their respective relative density histograms. The DAPI (blue) was used to stain the nucleus. All the experiments were performed in triplicate. The details are given in the “Methods” section. *Significantly different from the control; ^#significantly different from Aβ_1–42-treated group. Significance = **p < 0.01, ^#p < 0.05, ^##p < 0.01

Fig. 5

The beneficial effects of An-NPs against Aβ_1–42-induced apoptosis and neurodegeneration; a effect of An-NPs treatment on the activation of apoptotic induced proteins analyzed by Western Blot in Aβ_1–42 treated SH-SY5Y cell line. Induction of Bax, Bcl-2 and caspase-3 in Aβ_1–42-treated SH-SY5Y cells were analyzed by Western Blot. Data are the representative of three individual experiments (n = 3). The protein bands were quantified using sigma gel software. β-Actin was used to show equivalent amounts of protein loading. b Representative photomicrographs of TUNEL and DAPI stained cells (magnification ×10 objective field, scale bar 50 µm) showing a high number of apoptotic neurons in Aβ_1–42-treated group and comparatively low number of apoptotic neurons in Aβ plus An-NPs treated group. Data are the mean value (±SEM) for three independent experiments (n  =  3). *Significantly different from the control; ^#significantly different from Aβ_1–42-treated group. Significance = **p < 0.01, ***P < 0.001, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001

Fig. 6

Schematic representation of intracellular uptake of anthocyanin loaded PEGylated NPs by SH-SY5Y cell via endocytosis; inside the cellular cytoplasm, the endosome is broken down by lysosomal enzymes and the drug is released in the cytoplasm and reverts the Aβ_1–42-induced Aβ pathology by abrogating ROS generation via the P38-MAPK/JNK pathways accompanied by induction of endogenous nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1)