**1. Introduction**

When the nervous system is damaged or infected, microglia cells are activated and transformed to be branched, resulting in excessive expression of a large amount of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin 6 (IL-6), and inflammatory mediators such as nitric oxide (NO) and reactive oxygen species (ROS). Finally, nerve cells are damaged, degenerated, or die from these

**Citation:** Cheng, C.-Y.; Barro, L.; Tsai, S.-T.; Feng, T.-W.; Wu, X.-Y.; Chao, C.-W.; Yu, R.-S.; Chin, T.-Y.; Hsieh, M.F. Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection. *Int. J. Mol. Sci.* **2021**, *22*, 3037. https://doi.org/10.3390/ ijms22063037

Academic Editor: Anne Vejux

Received: 8 February 2021 Accepted: 12 March 2021 Published: 16 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

inflammatory mediators. It is recently found that in the brain of patients with neurodegenerative diseases such as Parkinson s disease (PD), Alzheimer s disease, Huntington s disease, and Creutzfeldt-Jakob disease, large amounts of microglia cells are activated and over-expressed [1–3]. Epidemiologically, PD's cause is mostly linked to the neuroinflammatory reaction. The inflammatory mediators resulting, such as TNF-α, IL-1β, IL-6, NO, and ROS, are found in the striatum of the brain [1,4–7]. The degradation of dopaminergic neurons can be regulated by microglia cells [8].

The neuroinflammation process that causes PD is preceded by primary damage of neurons caused by environmental toxins, including rotenone [9], lipopolysaccharide (LPS) [5,7], and the effects of abnormal protein accumulation [10]. The damage will cause lesions and even apoptosis of dopaminergic neurons. Then microglia cells are activated to release cytokines, resulting in inflammation and death of the neurons and finally leading to PD.

When the microglia cells are stimulated by LPS, LPS binds to the surface receptor CD14 binding site of microglia cells. The LPS-CD14 complex is linked with MD2 linker through toll-like receptor-4 (TLR4) transmembrane proteins and then involved in multiple message transmission pathways generated by mitogen-activated protein kinases (MAPK) and activating transcription factors (nuclear factor-kappa B, NF-κB). After gene transcription [5,11,12], microglial cells release cytokines such as TNF-α and IL-1β, or express genes of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2), resulting in the release of prostaglandins or NO. In addition, destructive ONOO free radicals are generated by combining superoxide anions produced from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with NO produced from iNOS, leading to the death of dopaminergic neurons [13]. Therefore, in this study, LPS was used to induce neuroinflammation of microglia cells as an in vitro model of PD.

Catechins are natural antioxidants that can prevent cell damage and provide many pharmacological benefits such as anti-tumor, anti-cancer, anti-aging, anti-pharmacological radiation, and free radical scavenging [14]. Green tea contains about 10% polyphenols by weight, including large amounts of a catechin called epigallocatechin gallate (EGCG). EGCG has the highest antioxidant activity and free radical scavenging capacity in all green tea catechins and can capture ROS to protect cells from the damage of oxidative stress [15]. EGCG also has high anti-inflammatory efficacy, which can effectively inhibit secretion of cytokines (TNF-α, IL-2, and IL-8) by macrophages [16], phosphorylation of Akt signaling proteins and IκB proteins in inflammatory pathways to reduce NF-κB expression, or AP-1 transcription by inhibiting phosphorylation of upstream MAPK proteins to balance COX-2 expression and reduce the production of pro-inflammatory cytokines [17].

Recently, EGCG was reported to be potentially therapeutic or prophylactic for PD due to suppressing active oligomers of α-synuclein (αS) [18]. EGCG also prevents αS aggregation in vitro [19–21], and cytoplasmic αS aggregation in dopaminergic neurons is one possible pathogenesis of PD leading to damage of dopaminergic neurons in substantia nigra [22]. Furthermore, EGCG can recover 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurochemical or functional damage and regulate ferroportin in substantia nigra and reduce oxidative stress [23]. EGCG also has neuroprotective and immuneprotective effects in MPTP-treated mice and can modulate neuroinflammation and protect dopaminergic neuron loss in MPTP-induced PD [24].

The anti-inflammation effects of EGCG were investigated. EGCG suppressed LPSinduced NO production and expression of iNOS in BV-2 microglial cells. EGCG can effectively inhibit the expressions of pro-inflammatory cytokines such as TNF-α and IL-1β in BV-2 cells [25]. EGCG pretreatment of human macrophages significantly inhibited LPSinduced expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [26]. In addition, post-treatment of EGCG on LPS-injured mice decreased production of a proinflammatory cytokine through modulating the TLR4-NF-κB pathway [27]. Moreover, poly(lactide-co-glycolide) (PLGA) microspheres loaded with EGCG and optimized by the addition of β-cyclodextrin (β-CD) could effectively suppress NO production from BV-2

cells in the in vitro model of murine BV-2 microglial cells stimulated by LPS, indicating the microspheres can suppress inflammation of activated microglial cells [28].

Although green tea is a daily drink, the efficacy of catechins is ineffective due to low oral bioavailability; thus, effective pharmaceutical dosage forms are required. Nano drug carrier has the benefits of avoiding premature metabolism, extending drug action time, and targeting drug delivery. Therefore, this study intends to develop liposomes containing phosphatidylcholine (PC) and phosphatidylserine (PS), similar components to the cell membrane, as anti-inflammatory dosage forms. EGCG extracted from green tea leaves was loaded in liposomes to slow down inflammatory reaction in microglia cells induced by LPS. The therapeutic effect of EGCG-loaded liposomes on the in vivo model of PD for neuroprotection was also evaluated.
