Brain-Targeted Polysorbate‑80‑Emulsified Donepezil Nanoparticles: A Novel Neuroprotective Strategy

Abstract

Alzheimer’s disease (AD) treatments often fail to reach therapeutic concentrations in the brain because of the blood–brain barrier (BBB). We engineered a cholesterol‑modified pullulan (CHP) nanoparticle loaded with the acetylcholinesterase inhibitor donepezil (DZP) and coated its surface with polysorbate 80 (PS) to achieve active BBB transcytosis. Using dynamic light scattering and isothermal titration calorimetry, we identified a 1:5 (w/w) DZP:CHP ratio as optimal, yielding high drug entrapment (86.5 %) and loading (13.4 %). In vitro release studies demonstrated sustained DZP release over 72 h. In vivo fluorescence imaging in mice confirmed PS‑coated nanoparticles accumulate preferentially in brain tissue, with negligible off‑target organ deposition. Functional assays in PC12 and SH‑SY5Y cells pre‑treated with Aβ25–35 revealed that DZP‑CHP nanoparticles restored cell viability, reduced lactate dehydrogenase (LDH) leakage, preserved mitochondrial membrane potential (MMP), and inhibited apoptosis more effectively than free DZP. Micro‑thermophoresis showed strong ApoE binding (K_D = 3.6 µM), indicating receptor‑mediated BBB transport. These results support the clinical potential of PS‑coated DZP‑CHP nanoparticles for targeted neuroprotection in AD.

Introduction

AD is characterized by progressive cognitive decline and amyloid‑β (Aβ) deposition. Conventional oral donepezil suffers from poor aqueous solubility, low oral bioavailability, and limited BBB penetration, necessitating daily dosing and compromising patient adherence. Nanoparticle drug delivery systems (nano‑DDS) offer a promising platform to overcome these hurdles by facilitating passive diffusion and receptor‑mediated transport across the BBB. Polysorbate 80, a non‑ionic surfactant, has been demonstrated to adsorb serum apolipoproteins (ApoE, ApoB), thereby mimicking low‑density lipoprotein particles that can hijack LDL receptors on endothelial cells and traverse the BBB. Cholesterol‑modified pullulan (CHP) self‑assembles into amphiphilic nanoparticles with a hydrophobic core suitable for encapsulating lipophilic drugs and a hydrophilic shell that reduces opsonization.

In this study, we combined CHP and PS to create a dual‑functional nanoparticle that encapsulates DZP, promotes BBB crossing, and delivers sustained therapeutic levels to the brain while mitigating Aβ toxicity in vitro.

Materials and Methods

Materials

CHP (home‑prepared), donepezil (Shanghai Ziqi Biotechnology Co., Ltd.), polysorbate 80 (Tianjin Fuchen Reagent Institute), indocyanine green (ICG) dye (Tianjin Baiying Biological Technology Co., Ltd.), black C57BL/6 mice, Aβ25–35 (US Sigma), MTT reagent (US Sigma), lactate dehydrogenase (LDH) kit (Nanjing Jiancheng Biological Co., Ltd.), AO/EB staining kit (Sino Pharmaceutical Group Chemical Reagent Co., Ltd.), PC12 (rat adrenal pheochromocytoma) and SH‑S5Y (human neuroblastoma) cell lines (ATCC).

Nanoparticle Preparation

Three DZP:CHP ratios (10:20, 4:20, 2:20 w/w) were dissolved in aqueous media and dialyzed (MWCO 1 kDa) to form nanoparticles. PS (0.7 mM) was added to 10 mL of each nanoparticle suspension, stirred 1 h, and sonicated (100 W, 2 s pulses) until homogeneous. The PS‑coated DZP‑CHP (PS‑DZP‑CHP) nanoparticles were filtered to remove impurities.

Characterization

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) confirmed spherical morphology. Dynamic light scattering (DLS) measured size and zeta potential. ISothermal titration calorimetry (ITC) assessed PS binding affinity and coverage. In vitro release was evaluated via dialysis against PBS pH 7.4 at 37 °C, sampling up to 72 h and quantifying DZP by UV‑vis (312 nm) against a standard curve.

In Vivo Brain Targeting

ICG‑labeled DZP‑CHP nanoparticles were prepared by dialysis of 400 mg CHP‑DZP with 20 mg ICG, followed by sonication. PS‑ICG‑DZP‑CHP nanoparticles were then formulated as above. Ten black mice received tail‑vein injections of 200 µL (200 µg/mL) of PS‑ICG‑DZP‑CHP or ICG‑DZP‑CHP. Fluorescence imaging (excitation 765–815 nm, emission 815–845 nm) captured whole‑body and organ distribution 30 min post‑injection.

Tissue Distribution

Forty‑five C57BL/6 mice were allocated to free DZP, DZP‑CHP, and PS‑DZP‑CHP groups (0.25 mg/kg, IV). Blood and organs (brain, heart, liver, kidney) were collected at 1, 3, 6 h. Homogenates were extracted with methanol, centrifuged, and analyzed by LC–MS/MS (mobile phase: 0.1% formic acid water / methanol 70:30, flow 0.3 mL/min). Quantification used multiple reaction monitoring (MRM) with positive electrospray ionization.

Cell Models and Neuroprotection Assays

PC12 and SH‑S5Y cells were cultured in DMEM + 10 % FBS, seeded at 1×10⁴ cells/mL (96‑well plates) or 2–3×10⁵ cells/mL (other assays). Aβ25–35 (20 µM) induced neurotoxicity. Cell viability was measured by MTT (490 nm). LDH release (450 nm) assessed membrane integrity. MMP was evaluated with Rhodamine 123 fluorescence. Apoptosis morphology was visualized via AO/EB staining.

Results

Nanoparticle Characterization

CHP alone formed ~257 nm spheres (zeta –2.8 mV). DZP loading increased size slightly to 260–274 nm and shifted zeta to –5 to –9 mV, depending on drug ratio. The 1:5 DZP:CHP formulation (DCN2) achieved the highest drug entrapment (86.5 %) and loading (13.4 %). PS coating increased size to 335 nm and zeta to –2.2 mV, confirming successful surface modification.

PS Binding Affinity

ITC revealed PS binding affinities of 1.47×10⁵, 2.98×10⁵, and 3.67×10⁵ M⁻¹ for DCN1, DCN2, DCN3, respectively, with coverage ratios of 2.65, 2.70, and 1.49 mol PS per mole nanoparticle. Endothermic, entropy‑driven interactions suggest hydrophobic association.

In Vitro Release

Free DZP dissolved rapidly. PS‑DZP‑CHP released 35 % in 6 h, 70 % by 24 h, and 90 % at 72 h, indicating sustained release. Non‑coated DZP‑CHP released faster, likely due to weaker PS shielding.

Brain Targeting

Fluorescence imaging showed negligible signal in free ICG controls. PS‑ICG‑DZP‑CHP produced bright brain fluorescence 30 min post‑injection, with minimal signal in peripheral organs. Tissue quantification confirmed a 3–4‑fold higher brain DZP concentration for PS‑DZP‑CHP versus free DZP, and a delayed peak (6 h vs 1 h) indicative of sustained release.

ApoE Binding

MST yielded a K_D of 3.63 µM for PS‑ICG‑CHP–ApoE interaction, supporting receptor‑mediated BBB transcytosis.

Neuroprotection in Cell Models

Aβ25–35 at 20 µM reduced PC12 viability to 49.5 % and SH‑S5Y to 49.7 %. Pretreatment with DZP‑CHP (5 µM, 10 µM) restored viability to 68–75 % (vs 48 % with free DZP). LDH release decreased by 30 % with DZP‑CHP, and MMP remained >80 % of control, whereas free DZP only achieved ~70 %. AO/EB staining revealed reduced apoptotic morphology in DZP‑CHP groups.

Discussion

Our PS‑coated DZP‑CHP nanoparticles achieved targeted brain delivery, sustained drug release, and superior neuroprotective efficacy in vitro. The high PS affinity and ApoE adsorption facilitate LDLR‑mediated BBB transcytosis, while the CHP core provides a lipophilic reservoir that reduces Aβ aggregation. Compared to conventional free DZP, the nanoparticle platform extends dosing intervals, potentially improving patient adherence and reducing side effects. Future in vivo studies will focus on pharmacokinetics, toxicity, and therapeutic efficacy in AD animal models.

Conclusion

Cholesterol‑modified pullulan nanoparticles loaded with donepezil and surface‑coated with polysorbate 80 demonstrate optimal drug loading (1:5 DZP:CHP), sustained release, efficient BBB crossing, and enhanced neuroprotection against Aβ‑induced toxicity. These findings endorse PS‑DZP‑CHP as a promising nanomedicine for Alzheimer’s disease treatment.