Nose-to-brain delivery of self-assembled curcumin-lactoferrin nanoparticles: Characterization, neuroprotective effect and in vivo pharmacokinetic study

Abstract

Curcumin (CUR) is a natural polyphenol extract with significant antioxidant and anti-inflammatory effects, which indicates its great potential for neuroprotection. Lactoferrin (LF), a commonly used oral carrier and targeting ligand, has not been reported as a multifunctional nanocarrier for nose-to-brain delivery. This study aims to develop a nose-to-brain delivery system of curcumin-lactoferrin nanoparticles (CUR-LF NPs) and to further evaluate the neuroprotective effects in vitro and brain accumulation in vivo. Herein, CUR-LF NPs were prepared by the desolvation method with a particle size of 84.8 ± 6.5 nm and a zeta potential of +22.8 ± 4.3 mV. The permeability coefficient of CUR-LF NPs (4.36 ± 0.79 × 10^-6 cm/s) was 50 times higher than that of CUR suspension (0.09 ± 0.04 × 10^-6 cm/s) on MDCK monolayer, indicating that the nanoparticles could improve the absorption efficiency of CUR in the nasal cavity. Moreover, CUR-LF NPs showed excellent protection against Aβ_25-35-induced nerve damage in PC12 cells. In vivo pharmacokinetic studies showed that the brain-targeting efficiency of CUR-LF NPs via IN administration was 248.1%, and the nose-to-brain direct transport percentage was 59.7%. Collectively, nose-to-brain delivery of CUR-LF NPs is capable of achieving superior brain enrichment and potential neuroprotective effects.

Keywords: Aβ25-35; curcumin-lactoferrin nanoparticles; neuroprotective effect; nose-to-brain; pharmacokinetic.

FIGURE 1

Schematic illustration of the preparation of curcumin-lactoferrin nanoparticles (CUR-LF NPs) and to evaluate the prospective and feasibility for neuroprotection via intranasal administration.

FIGURE 2

Characterization of CUR-LF NPs (A) Particle size distribution and appearance of CUR-LF NPs; (B) Zeta potential fitting diagram of CUR-LF NPs; (C) Transmission electron microscopy (× 100 000) of CUR-LF NPs; (D) Determination of secondary structure of native lactoferrin and CUR-LF NPs by circular dichroism; (E) X-ray diffractogram of curcumin, lactoferrin, curcumin/lactoferrin physical mixture, and CUR-LF NPs; (F) Differential scanning calorimetry of curcumin, lactoferrin, curcumin/lactoferrin physical mixture, and CUR-LF NPs; (G) Evaluation of the stabilization ratio of CUR-LF NPs incubated in PBS (pH 7.4), Dulbecco’s modified Eagle medium (DMEM), simulated nasal fluid (SNF), and artificial cerebrospinal fluid (ACSF) for 24 h; (H) Evaluation of the mean particle size and polydispersity index of CUR-LF NPs incubated in PBS (pH 7.4), DMEM, SNF, and ACSF for 4 h. Data were presented as mean ± SD (n = 3).

FIGURE 3

In vitro delivery evaluation (A) In vitro release of curcumin solution, 0.5% carboxymethyl cellulose sodium (CMC-Na) curcumin suspension, and CUR-LF NPs (B) The apparent permeability coefficient of curcumin solution, 0.5% CMC-Na curcumin suspension, and CUR-LF NPs on MDCK monolayer. Data were presented as mean ± SD (n = 3).

FIGURE 4

Cellular uptake of different curcumin formulations in PC12 cells (A) Fluorescence microscopy qualitative analysis of cellular uptake after incubation for 60 min with free curcumin and CUR-LF NPs with or without lactoferrin pretreatment at the concentration of 20 μM; (B) HPLC quantitative analysis of cellular uptake after incubation for 5, 30, 60, 120, and 240 min with the concentration of 20 μM. Data were presented as mean ± SD (n = 3).

FIGURE 5

Cell viability assay (A) PC12 cells treated with free curcumin and CUR-LF NPs (1, 10, 20, 30, and 50 μM) for 24 h ^* p < 0.05, ^** p < 0.01 free curcumin group versus solvent control group; ^# p < 0.05, ^## p < 0.01 CUR-LF NPs group versus solvent control group; ^& p < 0.05, ^&& p < 0.01 free curcumin group versus CUR-LF NPs group (B) Investigation of induction concentration (1, 10, 25, and 50 μM) and time (24 and 48 h) of Aβ_25-35-induced PC12 cell damage. ^* p < 0.05, ^** p < 0.01 Aβ_25-35 group versus control group at 24 h induction time; ^# p < 0.05, ^## p < 0.01 Aβ_25-35 group versus control group at 48 h induction time; ^& p < 0.05, ^&& p < 0.01 24 h versus 48 h induction time; ^$ p < 0.01, ^$$ p < 0.01 indicating the induction concentration and time was significantly different from other groups (C) Protective effect of lactoferrin nanoparticles, free curcumin, lactoferrin nanoparticles + free curcumin, and CUR-LF NPs on Aβ_25-35-induced damage in PC12 cells. ^* p < 0.05, ^** p < 0.01 indicating the treatment group was significantly different from other treatment groups. Data were presented as mean ± SD (n = 3).

FIGURE 6

Protective effect of CUR-LF NPs on Aβ_25-35-induced oxidative stress and apoptosis in PC12 cells (A) DCFH-DA fluorescence probe was used to detect intracellular ROS generation in PC12 cells (B) The intracellular SOD activity was measured using an SOD assay kit (C) The intracellular MDA level was evaluated with an MDA assay kit (D) Apoptosis of the treated PC12 cells was analyzed by flow cytometry using Annexin V-FITC/PI kit (E) Early and late apoptosis percent of PC12 cells (F) Representative western blotting images of Bax, Bcl-2, Caspase 3, cleaved-Caspase 3, and β-tubulin; Quantitation of Bcl-2/Bax ratio (G) and cleaved-Caspase 3/Caspase 3 ratio (H). ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus Aβ_25–35 group. ^& p < 0.05, ^&& p < 0.01 low dose treatment group (1 μM) versus high dose treatment group (10 μM). Data were presented as mean ± SD (n = 3).

FIGURE 7

Concentration-time profiles of curcumin up to 8 h post-dosing in (A) plasma, (B) brain following intranasal administration of curcumin suspension and CUR-LF NPs, and intravenous injection administration of CUR-LF NPs at a dose of 10 mg/kg. Data were presented as mean ± SD (n = 3).