TPGS-coated zein nanoparticles encapsulating Haematococcus pluvialis extract for Alzheimer’s disease: An in vitro evaluation towards brain-targeted delivery

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by β-amyloid (Aβ) aggregation and limited treatment efficacy due to the restrictive nature of the blood-brain barrier (BBB). To address this, we developed d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-coated Zein nanoparticles (Hp-Zein-TPGS NPs) encapsulating Haematococcus pluvialis (Hp) extract as a brain-targeted drug delivery system. Hp-Zein-TPGS NPs exhibited high physical stability over 14 days, sustained astaxanthin release, and potent antioxidant activity. The NPs also demonstrated excellent biocompatibility, showing minimal cytotoxicity in both bEnd.3 and SH-SY5Y cells, along with enhanced cellular uptake in vitro. Based on the reported effects of TPGS on P-glycoprotein (P-gp) inhibition and membrane fluidity, a delivery strategy was designed to facilitate BBB transport. In an in vitro BBB model, Hp-Zein-TPGS NPs exhibited increased transport, and Rhodamine 123 (Rh123) accumulation analysis indicated properties associated with the regulation of P-gp mediated efflux. In addition, Thioflavin T (ThT) fluorescence and morphological analyses confirmed that Hp-Zein-TPGS NPs effectively inhibited Aβ_1–42 aggregation and fibril formation, while WST and Annexin V-FITC/PI assays demonstrated that Hp-Zein-TPGS NPs significantly attenuated Aβ_1–42-induced neuronal toxicity, indicating their neuroprotective effects. Taken together, these findings suggest that Hp-Zein-TPGS NPs possess favorable stability, biocompatibility, BBB transport potential, and neuroprotective effects, highlighting their promise as a nanocarrier system for brain-targeted therapeutic delivery in AD.

Introduction

AD is a progressive neurodegenerative disease characterized by memory loss, cognitive decline, and neuronal damage, making it one of the most significant global medical challenges [1]. The primary pathological hallmark of AD is the aggregation of Aβ peptides, which induce neuronal toxicity and ultimately lead to neuronal cell death, exacerbating disease progression [2]. Extensive research has been conducted to elucidate the pathological mechanisms of AD and develop effective therapeutic strategies. However, one of the biggest challenges in AD treatment is the presence of the BBB, which severely limits drug delivery to the brain. The BBB is a highly selective biological barrier that protect the central nervous system (CNS) by restricting the entry of most macromolecules and small organic compounds, thereby making the delivery of therapeutic agents for brain disorders extremely difficult [3]. To overcome this challenge, the development of nanocarriers capable of crossing the BBB has become a crucial focus in drug delivery research.

NPs have been widely investigated as potential carriers for transporting drugs across the BBB [4]. Among them, protein-based NPs have attracted considerable attention due to their biocompatibility, biodegradability, and relatively low toxicity. Zein, a hydrophobic storage protein derived from corn, has been widely used in drug encapsulation systems due to its excellent biodegradability and biocompatibility, and its zein scaffold provides various interaction sites, enabling high drug loading efficiency [5]. However, Zein NPs have poor physical stability, susceptibility to aggregation, and instability under specific pH conditions, high temperatures, and high salinity, particularly after freeze-drying [6].

To improve the stability and functionality of Zein NPs, TPGS was employed as a coating agent. TPGS is a water-soluble derivative of vitamin E, composed of vitamin E succinate and polyethylene glycol (PEG), and has been widely used in nanomedicine as a stabilizer, emulsifier, and solubilizer due to its amphiphilic nature [7]. Importantly, inhibition of permeability P-gp has been suggested as a mechanism that may enhance drug transport across the BBB [8]. In this context, TPGS, which has been investigated for its potential P-gp inhibitory activity, could contribute to facilitating the delivery of brain-targeted therapeutics. In addition, TPGS enhances membrane fluidity and promotes endocytosis-mediated uptake in brain endothelial cells, contributing to its unique role in enabling NPs to overcome the BBB and achieve efficient brain-targeted delivery. To further enhance the therapeutic efficacy, a bioactive compound capable of exerting neuroprotective effects within the brain was incorporated into the system.

Hp, a unicellular green microalga, is the richest natural source of astaxanthin and is found in diverse aquatic environments worldwide [9]. Astaxanthin, a natural carotenoid with potent antioxidant, anti-inflammatory, and neuroprotective properties, has shown promise in AD treatment [10]. Notably, astaxanthin can cross the BBB and has been reported to significantly improve BBB integrity [11]. In addition, previous studies have shown that astaxanthin reduces Aβ accumulation, protects neurons from oxidative stress, and improves cognitive function, supporting the therapeutic potential of Hp extract in the treatment of AD [12]. Moreover, clinical trials have demonstrated that daily supplementation with astaxanthin-rich Hp extract is safe and may improve cognitive function in healthy older adults [13]. However, the thick cell walls of Hp pose a challenge to the efficient extraction of astaxanthin [14]. To address this issue, HME technology was introduced to enhance the extraction efficiency and bioavailability of astaxanthin and other bioactive compounds from Hp. HME technology facilitates the solubilization of poorly water-soluble compounds through the incorporation of suitable excipients [15]. Additionally, HME enables sustained and targeted drug release, making it a promising approach for controlled drug delivery applications.

In this study, we developed a brain-targeted drug delivery system encapsulating astaxanthin-rich Hp extract within TPGS-coated Zein NPs (Hp-Zein-TPGS NPs) to mitigate neuronal damage caused by Aβ aggregation. Hp-Zein-TPGS NPs were fabricated under various conditions and thoroughly characterized. The sustained release profile of Hp was assessed, along with its antioxidant activity. Furthermore, the biocompatibility of the NPs was evaluated through hemolysis analysis and cell viability assays. To confirm cellular uptake and BBB penetration, in vitro BBB models were constructed, and permeability experiments were conducted. Additionally, an AD model was established by treating SH-SY5Y cells with Aβ1–42, and the neuroprotective effects of Hp-Zein-TPGS NPs were investigated.

Unlike previous studies that primarily focused on either astaxanthin delivery or the stabilization of zein NPs, this study integrates HME-assisted extraction of Hp with TPGS-coated zein nanocarriers to improve extractability, enhance physicochemical stability, and suggest potential for brain-targeted delivery. To our knowledge, this is the first report to combine HME processing with TPGS encapsulated protein nanocarriers for astaxanthin-rich Hp extract, thereby addressing the dual challenges of poor extractability and limited BBB permeability at a preliminary stage. Compared to conventional delivery systems, this novel formulation provides enhanced stability, favorable biocompatibility, and improved in vitro BBB permeability, suggesting its potential value as a promising strategy for developing advanced therapeutic approaches for AD. Nevertheless, as these findings are limited to in vitro models, further in vivo investigations are warranted to validate BBB penetration and therapeutic efficacy under physiologically relevant conditions.

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Materials

Zein, TPGS, Coumarin-6 (C6), 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and ThT were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modification of Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and phosphate-buffered saline (PBS) were obtained from Thermo Fisher Scientific (Massachusetts, USA). Sheep blood defibrinated was obtained from Kisanbio (Seoul, Republic of Korea).

Yoo-Na Jeon, Jong-Suep Baek, TPGS-coated zein nanoparticles encapsulating Haematococcus pluvialis extract for Alzheimer’s disease: An in vitro evaluation towards brain-targeted delivery, International Journal of Biological Macromolecules, 2026, 150378, ISSN 0141-8130, https://doi.org/10.1016/j.ijbiomac.2026.150378.

See also the interesting video on Vitamin E TPGS below and read more: here