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A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to treat Alzheimer's disease
Accepted Manuscript Title: A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to treat Alzheimer’s disease Author: Fanfei Meng Sajid Asghar Shiya Gao Zhigui Su Jue Song Meirong Huo Weidong Meng Qineng Ping Yanyu Xiao PII: DOI: Reference:
S0927-7765(15)00398-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.06.025 COLSUB 7155
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
5-4-2015 1-6-2015 10-6-2015
Please cite this article as: F. Meng, S. Asghar, S. Gao, Z. Su, J. Song, M. Huo, W. Meng, Q. Ping, Y. Xiao, A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to treat Alzheimer’s disease, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.06.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to
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treat Alzheimer’s disease
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Fanfei Menga, Sajid Asghara,b, Shiya Gaoa, Zhigui Sua, Jue Songa, Meirong Huoa, Weidong
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Mengc, Qineng Pinga, Yanyu Xiaoa*
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a
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Pharmaceutical University, Nanjing 210009, P.R. China
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b
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Faisalabad, Pakistan
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Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China
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c
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P.R. China
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Faculty of Pharmaceutical Sciences, Government College University Faisalabad,
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Department of Clinical Laboratory, People’s Hospital of Liaocheng, Liaocheng 252000,
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* Corresponding author at: Department of Pharmaceutics, State Key Laboratory of Natural
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Medicines, China Pharmaceutical University, No. 24 Tong Jia Xiang, Nanjing 210009, PR
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China.
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[email protected] (Y.Y. Xiao).
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Tel.:
+86-25-83271079;
fax:
+86-25-83271079.
E-mail
address:
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Abstract In this study, a novel low density lipoprotein (LDL)-mimic nanostructured lipid carrier
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(NLC) modified with lactoferrin (Lf) and loaded with curcumin (Cur) was designed for
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brain-targeted delivery, and its effect on controlling the progression of Alzheimer’s disease
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(AD) in rats was evaluated. NLC with the composition resembling the lipid portion of LDL
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was prepared by using solvent evaporation method. Lf was adsorbed onto the surface of NLC
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via electrostatic interaction to yield Lf modified-NLC (Lf-mNLC) as the LDL-mimic
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nanocarrier. In order to make sure more Lf was adsorbed on the surface of NLC, negatively
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charged carboxylated polyethylene glycol (100) monostearate (S100-COOH) was synthesized
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and anchored into NLC. Different levels of S100-COOH (0-0.02mmoL) and Lf modified
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NLC (0.5-2.5 mg/mL of Lf solution) were prepared and characterized. The uptake and
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potential cytotoxicities of different preparations were investigated in the brain capillary
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endothelial cells (BCECs). An AD model of rats was employed to evaluate the therapeutic
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effects of Lf-mNLC. The results indicate that Lf-mNLC with a high level of Lf showed the
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maximum uptake in BCECs (1.39 folds greater than NLC) as cellular uptake of Lf-mNLC by
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BCECs was found to be mediated by the Lf receptor. FRET studies showed Cur still wrapped
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inside NLC after uptake by BCECs, demonstrating stability of the carrier as it moved across
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the BBB. Ex vivo imaging studies exposed Lf-mNLC could effectively permeate BBB and
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preferentially accumulate in the brain (2.78 times greater than NLC). Histopathological
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evaluation confirmed superior efficacy of Lf-mNLC in controlling the damage associated
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with AD. In conclusion, Lf-mNLC is a promising drug delivery system for targeting therapy
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of brain disease.
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Keywords: Lactoferrin; Carboxylated polyethylene glycol (100) monostearate; LDL-mimic
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nanostructured lipid carrier system; Brain targeting; Alzheimer’s disease.
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1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder with no means yet known for cure or prevention. A potential difficulty in curing irreversible brain degeneration
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in AD is the existence of blood brain barrier (BBB). BBB, a structure that impedes the
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transportation of the drugs and toxic substances into the brain for the sake of its protection, is
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a challenge for the success of therapies against central nervous system (CNS) maladies [1].
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Increasing number of brain ailments in the modern world needs solution to the
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sub-therapeutic brain delivery of pharmacologically active molecules [2]. Various tactics are
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being employed for the delivery of neurotherapeutics across the BBB, but this research
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domain still needs vigorous efforts to achieve the goal [3, 4] .
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Lipid based nanocarriers have been widely reported for the treatment of brain diseases
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[5]. Recently, novel nanocarriers based on low density lipoprotein (LDL) are getting attention
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for their distinctive characteristics [6-8]. LDL is a 22-27 nm particle composed of a core of
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hydrophobic lipids, primarily cholesteryl esters with a small amount of triacylglycerides, and
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has a surface coat of phospholipids, unesterified cholesterol, and a single molecule of
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apolipoprotein B 100 (ApoB-100) [9, 10]. As a kind of naturally originated particle, LDL is
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non-immunogenic and long circulating, and possesses the innate ability to bind both
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hydrophobic and hydrophilic molecules. In addition, the overexpression of the low density
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lipoprotein receptors (LDLR) in brain capillary endothelial cells (BCECs) offers the
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advantage of brain targeting for LDL-based nanoparticles [11, 12]. However, the quantities of
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natural LDL extracted from human serum are extremely low, and it is difficult to isolate LDL
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in large quantities due to the variable composition and size. Another approach uses
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reconstituted LDL consisting of a lipid emulsion [13, 14], and ApoB-100 has been used for
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stabilizing such emulsions. It is also difficult to isolate ApoB-100 from human serum due to
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its large size and propensity to aggregate. Lactoferrin (Lf), an 80 kDa naturally occurring iron binding cationic glycoprotein, is
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abundantly found in milk. It has been reported to possess anti-inflammatory, antibacterial,
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antifungal, antiviral, and anti-cancer activities. It can also boost natural immunity, bone
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growth, and wound healing [15, 16]. Lf can be transported across BBB by the mediation of
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the lactoferrin receptors (LfR) present at the endothelial cells of the BBB [17-19]. Some
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studies showed the expression of LfR in the state of AD and Parkinson’s disease at the
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endothelial cells of the BBB would be increased [20].
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Curcumin
(Cur),
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molecular
weight
phytochemical,
possesses
diverse
pharmacological activities, including anti-oxidant, anti-inflammatory, anti-cancer, and
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antidepressant properties with low intrinsic toxicity [21, 22]. It is widely acknowledged that
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the progressive accumulation of Aβ aggregates initiates the initial development of
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neurodegenerative pathology and trigger a cascade of events such as neurotoxicity, oxidative
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damage, and inflammation that contribute to the progression of AD [23]. Cur has been shown
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to effectively reduce neurotoxic Aβ aggregates and ameliorate cognitive deficits in vivo and
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in vitro [24, 25].
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The objective of this research work was to develop a novel LDL-mimic nanocarrier
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(nanostructured lipid carrier (NLC) modified with Lf, Lf-mNLC) encapsulating Cur for
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brain-targeted delivery, and evaluate its effect on controlling the progression of AD in rats.
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Cur-loaded NLC with the composition resembling the lipid portion of LDL (PC, cholesterol 5
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oleate and glycerol trioleate) was prepared by using solvent evaporation method. Lf,
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positively charged at physiological pH, instead of ApoB-100, was used as a brain targeting
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molecule and was adsorbed onto the surface of NLC via electrostatic interaction to yield
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Lf-mNLC as the LDL-mimic nanocarrier. Carboxylated polyethylene glycol (100)
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monostearate (S100-COOH) was synthesized and its free surface carboxyl could enhance the
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negative charge of NLC, which facilitated more Lf was absorbed on the surface of NLC
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through electrostatic attraction.
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2. Experimental
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2.1 Materials
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Cur (purity, 98%) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
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Glycerol trioleate (purity, >98%) and phosphatidyl choline (PC, purity, 90%) were kindly
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donated by Evonik Degussa China Co., Ltd. (Shanghai, China). Cholesterol oleate
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(purity, >85%) and coumarin-6 (C6) were purchased from Tokyo Chemical Industry (Tokyo,
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Japan). Polyethylene glycol (100) monostearate (S100, the polymerization degree of ethylene
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glycol
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
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Sigma-Aldrich (Milwaukee, WI, USA). Aβ1-42 (purity, 95.34%) was purchased from GL
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Biochem Ltd (Shanghai, China). D-galactose (D-gal) was purchased from Sigma-Aldrich
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(Milwaukee, WI, USA). All other chemicals and reagents were of analytical grade and used
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without further purification.
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2.2 Animals
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Lf
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≥90%,
SDS-PAGE)
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Sprague-Dawley (SD) rats (180-220 g) and ICR mice (18-22 g) were obtained from 6
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Experiment Animal Center of Nantong University (Nantong, China). All animal procedures
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for this study were approved by the ethical committee of China Pharmaceutical University,
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and they were conducted in accordance with approved standards for laboratory animal care.
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2.3 Synthesis of S100-COOH
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Carboxylated polyethylene glycol (100) monostearate (S100-COOH) was synthesized
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using a one-step procedure. Briefly, 11.71 g of S100 and 0.5 g of succinic anhydride were
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mixed and activated with the catalysis of pyridine (7.5 mL) at 65 oC for 48 h. Subsequently,
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the pyridine was removed using a rotary evaporator and the residues were hydrated with 200
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mL of distilled water. The resultant solution was dialyzed with a Spectrum dialysis bag
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(MWCO of 3.5 kDa, Sigma, USA) at 4 oC for 48 h to remove the unreacted succinic
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anhydride and lyophilized to obtain S100-COOH. The structure of S100-COOH was
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characterized by 1H-NMR and 13C-NMR spectra (AVANCE AV-300, Bruker Instrument Inc.,
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Switzerland) using D2O as a solvent at 25 °C, respectively.
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2.4 Preparation of Cur-loaded Lf-mNLC
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Firstly, Cur-loaded NLC was prepared by a solvent evaporation method [26]. Briefly, PC
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(330 mg), cholesterol oleate (20 mg), glycerol trioleate (70 mg), Cur (16 mg) and different
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amounts of S100-COOH (0-0.02 mmoL) were dissolved in 10 mL of ethanol/chloroform
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mixture (1:1, v/v) and dried in a rotary evaporator under vacuum at 40 °C. The dried lipid
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film was hydrated with 10% (v/v) glycerol solution at 37 °C and ultrasonicated at 300 W for
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5 min at 4 °C, followed by extrusion through 0.22 μm cellulose nitrate membrane to remove
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the unentrapped drug and obtain Cur-loaded NLC. Afterwards, Cur-loaded Lf-mNLC was
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prepared by incubating Cur-loaded NLC with Lf solution. Briefly, 0.5, 1.5, or 2.5 mg/mL of 7
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Lf solution (dissolved in Tris-EDTA buffer (pH7.4)) was gently mixed with an equal volume
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of Cur-loaded NLC at 4 °C for 24 h. The three levels of Lf modified NLC were named as
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LfL-mNLC, LfM-mNLC and LfH-mNLC.
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2.5. Characterization of Cur-loaded NLC and Cur-loaded Lf-mNLC
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2.5.1. Morphology, particle size, zeta potential, EE and DL
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The particle size, polydispersity index (PI) and zeta potential of Cur-loaded NLC and
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Lf-mNLC were detected by Zetasizer 3000HS (Malvern, U.K.). Their morphological
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observations were performed by transmission electron microscopy (TEM, H-7000, Hitachi,
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Japan). EE of NLC and DL of NLC/Lf-mNLC were determined as previously reported [27].
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2.5.2. Fluorescence and UV-Visible absorbance spectra
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The interaction of Cur with the hydrophobic lipid core of NLC or Lf-mNLC was
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investigated by fluorescence and UV-Visible absorption spectra [28, 29]. The fluorescence
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and UV-Visible absorption spectra of Cur (dissolved in methanol), Cur-loaded NLC and
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Lf-mNLC (in aqueous solution) with the Cur concentration of 1.5 μg/mL were compared.
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The fluorescence emission spectrum was recorded from 450 to 700 nm with an excitation
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wavelength of 420 nm. Excitation and emission slit widths (modulation of magnitude and
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resolution of transmitted light) were standardized at 5 nm each. The UV-Visible absorption
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spectrum was taken from 300 to 700 nm.
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2.5.3. Plasma stability of NLC and LfH-mNLC
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NLC and LfH-mNLC were mixed with the equal volume of the rat plasma and incubated
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for prearranged time intervals (15, 30, 60, 120, 240, 360 min) in a 37 °C water bath. After
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incubation, 100 μL of the sample was diluted in 4 mL of distilled water and the particle size 8
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and zeta potential were measured by the Dynamic Light Scattering Analyzer.
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2.5.4 In vitro drug release study The in vitro release of Cur from NLC or Lf-mNLC was carried out in physiological saline
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containing 1% (w/v) tween 80 using a membrane dialysis method. 1 mL of different Cur
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preparations (containing 400 μg of Cur) was placed in a pre-swelled dialysis bag (8~10 kDa
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MW cutoff, Sigma, USA). Cur solution (containing 30%（w/w）polyethylene glycol 400) was
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taken as a control. The bag was tightened and soaked in 50 mL of release medium at 37 °C
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for 48 h (n=3). At a given time interval, 1 mL of the medium was withdrawn and replaced
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with the same amount of pre-warmed fresh medium. The withdrawn sample was then
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centrifuged at 12,000 rpm for 10 min and the supernatant was analyzed by HPLC method
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[27]. Care was taken to protect the sample from light throughout the experimental procedure.
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The release kinetic models were studied using DDsolver software.
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2.6. Cytotoxicity and cellular uptake studies
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2.6.1. Cell culture
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BCECs were cultured in the endothelial cell culture medium that contained DMEM/F12,
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20% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL
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streptomycin. BCECs were maintained in an incubator (Thermo Scientific, USA) at 37 °C
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under an atmosphere of 5% CO2.
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2.6.2. Cytotoxicity evaluation
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In order to justify the nontoxicity of different empty preparations, their cells viabilities
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were determined by MTT assay. Briefly, 5 × 103 cells/well were seeded in 96-well culture
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plates for 24 h and then incubated with empty NLC, empty LfL-mNLC, empty LfM-mNLC, 9
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and empty LfH-mNLC. The concentrations of empty carriers were in the range of 100−1600
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μg/mL. After 24 h incubation, 10 μL of 5 mg/mL MTT was added into each well for 4 h in
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dark, and then the medium was replaced with 150 μL of DMSO. The absorbance value at 570
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nm (A570nm) was read using the microplate reader (Thermo Electron Corporation. USA). Cell
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viability was expressed as a percentage of A570nm of the study group relative to that of control
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group.
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2.6.3. Cellular uptake
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C6-loaded NLC and Lf-mNLC were prepared similar to the preparation of Cur-loaded
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NLC/ Lf-mNLC, but Cur was replaced with C6. BCECs were seeded into twelve-well plates
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at a density of 2×105 cells/well and cultured in complete cell-culture medium for 24 h at
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37 °C. Then, the medium was replaced with 1 mL of different C6-loaded preparations (C6=
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300 ng/mL) and cells were further incubated for another 2 h. Cells were trypsinized and
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harvested. The fluorescence intensities of C6 in cells were determined by a BD FACS Calibur
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flow cytometer (BD Bioscience, Bedford, MA). The excitation wavelength of C6 was 465 nm,
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and FL1-H filter was used for the collection of fluorescence intensity. The data were analyzed
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through Flow Jo 7.6 software. The influences of incubation concentration, temperature and
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time on cellular uptake of the formulations were also monitored. Furthermore, uptake
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inhibition experiment was also performed by pre-incubating BCECs with excess of Lf (10
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mg/mL) for 30 min, and then the cells were incubated for 2 h at 37 °C with NLC or
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LfH-mNLC (C6=100 ng/mL).
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2.7. Intracellular stability of NLC and LfH-mNLC
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FRET analysis was conducted to evaluate the stability of NLC or Lf-mNLC in cells after 10
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uptake by BCECs in order to make sure the passage of drug across the BBB in the form of
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being wrapped in NLC [30]. A FRET pair of hydrophobic dyes, DiO as donor and DiI as
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acceptor, was loaded into the core of NLC or LfH-mNLC, with the method similar to the
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preparation of Cur-loaded NLC/ LfH-mNLC. In order to verify the occurrence of FRET, the
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fluorescence spectra of NLC or LfH-mNLC loaded with DiI and DiO were measured in the
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blank cell culture medium-DMEM/F12 or trichloromethane-methanol (1:1) with the
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excitation at 488 nm. To monitor the integrity of carriers in BCECs, NLC or LfH-mNLC were
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incubated with BCECs at 37°C for 1 h. Subsequently, NLC or LfH-mNLC was removed, and
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the cells were washed thrice with ice-cold PBS. At last, the cells were incubated with the cell
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culture medium for predetermined time periods. Fluorescence images were acquired with the
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excitation at 488 nm, and the emission between 555 nm and 655 nm for DiI detection, as well
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as the emission between 500 nm and 530 nm for DiO detection by CLSM (Leica TCS SP5).
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2.8. Pharmacokinetics
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intravenous administration of Cur solution, Cur-loaded NLC and LfH-mNLC at a dose of 10
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mg/kg of Cur (n=5). After being collected at predetermined intervals, the blood was
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centrifuged at 4000 rpm for 10 min at 4 °C and then the plasma were frozen at -20°C until
239
assay. 100 μL of acetonitrile was added into 100 μL of plasma, vortexed for 2 min and
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centrifuged at 12000 rpm for 10 min. 20 μL of supernatant was injected into the HPLC
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system equipped with a fluorescence detector (Shimadzu, Japan) to determine the
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concentration of Cur, and the excitation and emission wavelength were 436 and 518 nm,
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respectively. The plasma concentration versus time data were analyzed by Kinetica 4.4 11
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(Thermo Electron Corporation, Waltham, MA, USA) and various parameters were studied.
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2.9. Biodistribution study In order to verify the brain-targeting of Lf-mNLC, ex vivo imaging experiments were
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performed in rats. DiR, a hydrophobic near infrared dye, was loaded into NLC or LfH-mNLC.
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SD rats were intravenously administered with DiR-loaded NLC or LfH-mNLC (0.5 mg/kg)
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and anesthetized. A Kodak multimodal-imaging system IS2000MM (Kodak, USA) was used
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to perform ex vivo imaging experiments at 1, 2, 3 and 4 h post injection with the wavelength
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fixed at 720 nm for excitation and 790 nm for emission. To probe the biodistribution of NLC
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or LfH-mNLC, rats were sacrificed at 24 h post-injection, and different organs were separated
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and washed by saline, assembled for fluorescence imaging and analyzed by IS2000MM
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software (Kodak ID Image Analysis Software, Kodak).
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In addition, brain sections were used to further understand the distribution of NLC or
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LfH-mNLC in the CNS. Briefly, C6-loaded NLC and LfH-mNLC (6 mg/kg) were injected into
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the tail veins of mice, respectively. After 1 h, the mice were anaesthetized and then the brains
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were removed, fixed in 4% paraformaldehyde for 24 h, and dehydrated with 15% sucrose
259
solution for 12 h and then 30% sucrose solution for 24 h. Thereafter, the brain samples were
260
frozen in OCT (Sakura, Torrance, CA, USA) at -80 °C and cut into 20 μm thickness with a
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cryotome Cryostat (Leica, CM 1900, Germany). After staining with 5 μg/mL DAPI for 20
262
min at room temperature, slides were observed using a fluorescence microscope (Olympus,
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Japan). Fluorescence intensity of C6 in the third ventricle and cortex was measured and
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analysed using Image J software.
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2.10 Neuroprotective effects of different Cur preparations on AD model rats
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2.10.1 In vivo model of AD The in vivo model of AD was established in rats by administering Aβ1-42 and D-gal. The
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SD rats were treated with intraperitoneal injection 4% D-gal (0.3 mL/100g/d) by
269
intraperitoneal injection for six weeks. Aβ1-42 was dissolved in saline (1 mg/mL) and then
270
incubated at 37 °C for 7 days to form the aggregation. The rats were anaesthetized and fixed
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in a stereotaxic apparatus. The animals were bilaterally injected in the dorsal hippocampus
272
(2.3 mm posterior to the bregma, ±1.8 mm lateral to the Midline and 2.0 mm ventral to the
273
skull surface) with 5 μL of Aβ1-42 within 5 min. The needle was then slowly withdrawn after
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being kept for another 3 min. Rats in the sham group were injected with the same volume of
275
saline. After surgery, the rats were kept in the cages for recovery.
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2.10.2 Drug administration
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AD model rats were divided into five groups: the AD control group, sham group, and
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three test groups (n=10 for each group). One week after the surgery, Cur solution (30% (w/v)
279
PEG-400 in a 5% (v/v) glucose solution), NLC and LfH-mNLC were intravenously
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administered to the rats at 2 mg/kg/d equivalent dose of Cur for three weeks, respectively.
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2.10.3 Determination of malondialdehyde (MDA)
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The rats were anaesthetized with ether, and blood samples were taken from the
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eyeground veins. The plasma was obtained after centrifugation (5 min, 4000 rpm, 4 °C) and
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assessed for the concentration of MDA using a MDA assay kit according to the
285
manufacturer’s protocols.
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2.11 Histopathology
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Rats in each group (n=5) were anaesthetized and perfused through the heart with cold 13
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saline followed by 4% paraformaldehyde. Then the whole brains were harvested and
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immersed in 4% paraformaldehyde. Afterwards, the brains were embedded in paraffin and cut
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into 5-μm sectioned. Sections were stained with hematoxylin-eosin (HE) staining,
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respectively. The hippocampal areas were examined under a Leica optical microscope (Leica,
292
Germany).
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2.12. Statistical data analysis
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The data are given as mean ± SD. Statistical significance was tested by two-tailed Student’s t test or one-way ANOVA. Statistical significance was set at P < 0.05.
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3. Results and discussion
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3.1 Preparation and characterization of NLC and Lf-mNLC
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3.1.1. Morphology, particle size, zeta potential, EE and DL
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Based on particle size, zeta potential and EE, Cur-loaded NLC was prepared with PC,
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cholesterol oleate, glycerol trioleate and Cur according to an optimized ratio of
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1:0.06:0.21:0.05 (w/w/w/w). Effects of different amounts of S100-COOH on particle size, PI,
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zeta potential and EE of Cur-loaded NLC are listed in Table 1A. The particle size of NLC
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decreased after modification with S100-COOH and was found inversely proportional to the
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amount of S100-COOH, which might be due to its surface-active effects [31]. In addition, the
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PEG chains of S100-COOH could act as a tighter net on the outside of vesicles, thus limiting
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the increase of vesicle size [26]. The inclusion of S100-COOH in NLC also led to the
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increase in negative charge due to the ionized –COOH on the surface of NLC and the
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absolute value of zeta potential increased gradually with the increase of S100-COOH. The
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substantial increase in PI and decrease in EE as the added amount of S100-COOH increased
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to 0.02 mmoL could be due to two reasons. Firstly, the added amount of S100-COOH might
311
not totally arrange themselves on the surface of NLC and they could have spontaneously
312
formed the small sized unstable micelles [32]. The drug could be easily released from the
313
micelles and precipitate in the medium. Secondly, the decreased drug loading space owing to
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the smaller particle size might also contribute to the partial drug encapsulation. As the
315
isoelectric point of Lf is about 8.65, the protein would be positively charged in the suspension
316
of NLC (pH 6.0-6.5). The more the negative charge on the surface of NLC, the more
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favorable it is for Lf to be adsorbed on the surface of NLC. Therefore, NLC coated with
318
0.012 mmoL of S100-COOH was chosen for further modification.
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Table 1B shows the effect of different concentrations of Lf on particle size, PI, zeta
320
potential, EE, and DL of Cur-loaded NLC coated with 0.012 mmoL of S100-COOH. The
321
adsorption of Lf on the surface of NLC led to the increase of particle size and zeta potential
322
of NLC. However, Lf coating had not interfered with DL and EE of NLC. The particle size
323
and shape of NLC and Lf-mNLC were further confirmed by TEM (Fig. 1). Both NLC and
324
Lf-mNLC were quasi-spherical in shape. However, Lf-mNLC appeared a core-shell structure
325
and the thickness of the covering layer increased with the increasing amount of Lf.
326
Furthermore, a dense core was observed for nanocarrier with high concentration of Lf which
327
is not evident in NLC or Lf-mNLCs with low concentration of Lf. This could be due to
328
formation of a more compact nanocarrier owing to the greater electrostatic interactions with
329
higher concentration of Lf. TEM also showed that both NLC and Lf-mNLC were uniformly
330
dispersed without any visible sign of aggregation, indicating the stability of the formulations.
331
3.1.2. In vitro drug release study
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15
Page 15 of 38
In vitro drug release profiles of Cur from different preparations are studied (see Fig.2 of
333
the Appendix). Except for Cur solution, all preparations exhibited sustained release of Cur
334
without any burst release, which could be attributed to the encapsulation of Cur into NLC.
335
Compared with NLC, the release of Cur from Lf-mNLC decreased significantly (P < 0.05)
336
and increasing the amount of Lf further slowed the drug release, which suggests that Lf
337
adsorption further limited the diffusion of the drug from NLC [33].
us
cr
ip t
332
In order to explore the release mechanisms of Cur from Cur-loaded NLC and Lf-mNLC,
339
various models (zero-order, first-order, Higuchi, and Weibull models) were utilized to
340
simulate the release profiles (see Table 1 of the Appendix). Weibull model was found to be
341
the best-fit model to explain the release process. According to the exponent parameter (β) of
342
Weibull model, the Cur release mechanism from NLC and Lf-mNLC was found to be the
343
combined mechanism of Fickian diffusion and Case II transpobrt [34].
344
3.1.3. Fluorescence and UV-Visible absorbance spectra
M
d
te
Fluorescence and UV-Visible absorbance spectra of Cur, Cur-loaded NLC and
Ac ce p
345
an
338
346
LfH-mNLC are presented in Fig. 2. As depicted in the UV-Visible absorbance spectrum, there
347
is an intense absorption band at around 425 nm for Cur and the presence of a shoulder peak at
348
350 nm indicates the location of Cur in polar environment [29]. However, the appearance of a
349
shoulder peak at around 450 nm for the Cur-loaded NLC and LfH-mNLC could be ascribed to
350
the presence of Cur in the apolar core of NLC and LfH-mNLC [35]. For the fluorescence
351
spectra, Cur in methanol showed a sharp fluorescence peak at around 540 nm, but there was a
352
blue shift in the fluorescence spectra of Cur-loaded NLC and LfH-mNLC and Cur showed a
353
well-defined peak at around 520 nm. Sahu et al. [36] reported that when encapsulated in 16
Page 16 of 38
bovine casein micelle, Cur showed a shift in fluorescence spectra from 540 nm to 500 nm
355
because of the binding of Cur in the hydrophobic domain of the protein molecule in micelle.
356
Therefore, we inferred that when compared with that of native Cur, the blue shift in
357
fluorescence spectrum of Cur was induced by the encapsulation of Cur in the hydrophobic
358
domain of the NLC and LfH-mNLC through hydrophobic interactions.
359
3.1.4 Plasma stability
us
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In order to study the plasma stability of NLC and LfH-mNLC, variations in the particle
361
size and zeta potential of formulations were assayed in the rat plasma. The results showed
362
that NLC and LfH-mNLC had slightly smaller diameters in plasma than in distilled water, but
363
remained stable (see Fig.3A of the Appendix). The insignificant changes in the size of the
364
nanocarriers imply that the coating of Lf did not affect the stability of NLC in the plasma and
365
LfH-mNLC was stable in the plasma. The zeta potential of NLC and LfH-mNLC both dropped
366
to nearly -25 mv after 15 min incubation with the plasma and then remained steady for the
367
rest of the study (see Fig.3B of the Appendix). This could be attributed to the presence of
368
negatively charged proteins in the serum.
369
3.2. In vitro cytotoxicity assay against BCECs
M
d
te
Ac ce p
370
an
360
No significant toxicity on BCECs was found for empty NLC and Lf-mNLC as more
371
than 80% cell viability was observed even at highest concentration (1600 μg/mL) (see Fig.4
372
of the Appendix). Formulations that show greater than 80% cell viability have been regarded
373
as safe [37]. There were no significant differences in cell viability of NLC and Lf-mNLC
374
group, which shows both NLC and Lf-mNLC might be good biocompatible carriers.
375
3.3. Cellular uptake study 17
Page 17 of 38
The uptake of LfL-mNLC, LfM-mNLC and LfH-mNLC in BCECs was 1.06, 1.16 and
377
1.39 times higher, respectively, compared to that of NLC (Fig. 3A). As the cellular uptake of
378
LfH-mNLC was higher than those of LfL-mNLC and LfM-mNLC, LfH-mNLC was chosen for
379
subsequent studies.
ip t
376
As shown in Fig. 3B, the uptake amounts of both LfH–mNLC and NLC at 37 °C were
381
much higher than those at 4 °C, suggesting that their uptakes were energy-dependent. The
382
uptake increased with the increase in the concentration of C6 from 10-100 ng/mL, which
383
indicates that the uptakes of both NLC and LfH-mNLC were also concentration-dependent.
384
The uptakes of NLC and LfH–mNLC by BCECs were also dependent on the incubation time
385
(Fig. 3C). As shown in Fig. 3D, the uptake of LfH–mNLC was significantly suppressed in the
386
presence of excess free Lf, while the uptake of NLC was scarcely influenced, which confirms
387
that Lf mediated endocytosis mechanism was responsible for the enhanced uptake of
388
LfH–mNLC. BCECs have been reported to possess LfR and an increase of these receptors in
389
BCECs has also been found in Parkinson's and Alzheimer's disease [38, 39], which could
390
extend the applications of Lf-mNLC for the treatment of various brain malignancies.
391
3.4. Intracellular integrity studies of NLC and LfH-mNLC
us
an
M
d
te
Ac ce p
392
cr
380
FRET occurs through the energy transfer from the donor (DiO) to the acceptor (DiI)
393
only when both fluorescent molecules are in a range of 1-10 nm. Implicitly, with the increase
394
in distance between the donor and acceptor, the FRET ratio IR/(IG+IR) decays very rapidly,
395
where IR and IG are fluorescence intensities at 576 nm and 508 nm, respectively. When the
396
carriers disintegrate in the cells, the fluorescent molecules could not be closely retained
397
anymore, thus resulting in a lower FRET ratio [40]. Therefore, we used FRET experiment to 18
Page 18 of 38
398
monitor the stability of the carriers in cells after uptake by BECEs. LfH-mNLC in DMEM/F12 showed a strong DiI signal (see red curve in Fig. 5 of the
400
Appendix) with a FRET ratio of 0.80, which shows LfH-mNLC were stable in DMEM/F12.
401
While after using trichloromethane-methanol (1:1) to destroy the carriers, FRET phenomenon
402
almost disappeared (see green curve in Fig. 5 of the Appendix) with a FRET ratio below 0.30.
403
Same behavior was expressed by mNLC (data is not shown). The confocal images showed
404
that both NLC and LfH-mNLC in BCECs had a strong FRET phenomenon at 1 h post
405
incubation (Fig. 4), which indicates that the carriers remained intact in the cells. With the
406
passage of time (1-4 h), a decrease in red fluorescence was seen, however intracellular FRET
407
phenomenon was still existing, and implying most of the carriers didn’t disintegrate in the
408
BCECs and kept their integrity. At 8 h, the green fluorescence of DiO became much stronger
409
and FRET phenomenon disappeared, suggesting the disintegration of NLC and LfH-mNLC.
410
BBB is the unavoidable doorway for the brain targeted delivery systems, but it is not the
411
destination. The carriers intended for CNS delivery should remain intact while moving across
412
BCECs and avoid enzymatic degradation in the BCECs, which guarantees the efficient drug
413
delivery to the diseased regions of brain. Previously, Lf functionalized nanoparticles have
414
been reported to attain maximum concentration in the brain after 1 h following intravenous
415
administration [37, 41, 42]. FRET experiments revealed that NLC or LfH-mNLC were stable
416
in BCECs for at least 4 h, which might be long enough to permit safe passage of drug across
417
the BBB.
418
3.5. Pharmacokinetic studies
419
Ac ce p
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d
M
an
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399
The plasma concentration-time curves of Cur after intravenous injection of different 19
Page 19 of 38
preparations in rats are shown in Fig. 5. The blood concentration of Cur decreased rapidly
421
within the initial 2 h and could not be detected at 4 h after intravenous administration of Cur
422
solution. However, the concentration of Cur after intravenous administration of Cur-loaded
423
NLC and Lf-mNLC at 4 h were 13.03 and 5.49 ng/mL, respectively.
ip t
420
The pharmacokinetic data of Cur were simulated by non-linear least squares. The results
425
showed that the kinetics of a two-compartment open model was best fitted to Cur plasma
426
concentration-time curves in rats. The area under the curve (AUC) and MRT dramatically
427
increased and the clearance (CL) decreased (P<0.05) for Cur encapsulated in NLC when
428
compared with Cur solution (Table 1C). Compared to the drug solution, NLC and LfH-mNLC
429
showed 2.53 and 1.55 folds higher AUC, respectively and prolonged residence in body with
430
4.49 and 3.02 times greater MRT, respectively. While, 1.81 and 1.05 times lesser clearance
431
from the body than the drug solution was observed for NLC and LfH-mNLC, respectively.
te
d
M
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us
cr
424
Comparing the pharmacokinetic parameters of NLC and LfH-mNLC, an even lower
433
AUC and higher CL were observed for the latter. The coupling of NLC’s surface with Lf
434
affected the pharmacokinetics of NLC. These results are in good agreement with a previously
435
published study [43]. The main reason may be the specific targeting capability of Lf which
436
could have accelerated the uptake of Lf-mNLC by the target tissues or organs [44].
437
3.6. Biodistribution studies
Ac ce p
432
438
To investigate the brain-targeting capability of Lf-mNLC, ex vivo fluorescence images
439
of the rats were acquired after intravenous administration of DiR-loaded NLC and
440
LfH-mNLC. As displayed in Fig. 6A, the LfH-mNLC group showed significantly higher
441
fluorescence intensity in the brain than the NLC group at any time point post-administration. 20
Page 20 of 38
Additionally, the brain and major organs were harvested and imaged at 24 h
443
post-administration (Fig. 6B). As shown in Fig. 6C, The fluorescence intensity in brain tissue
444
for the LfH-mNLC group was 2.78 times high as that for the NLC group according to the
445
quantitative DiR fluorescence intensity, indicating that LfH-mNLC accumulated more than
446
NLC in brain. The presence of NLC in the brain could be attributed to the presence of PEG. It
447
has been previously demonstrated that PEG coated nanoparticles might adsorb the
448
apolipoprotein E (ApoE), and utilize the LDLR mediated transport to cross the BBB [45].
449
Our results suggested that LfH-mNLC could cross the BBB and penetrate the brain more
450
efficiently than NLC that might be attributed to the interaction between Lf and Lf receptors
451
over-expressed at the BBB.
M
an
us
cr
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442
Fig. 7A shows coronal slices of the mouse brain. Periventricular region of the third
453
ventricle and cortex showed the green fluorescence of C6. The stronger and widely
454
distributed fluorescence intensity of LfH-mNLC than that of NLC quantified with help of
455
Image J software (Fig. 7B), in these regions, indicates that Lf-receptor mediated transcytosis
456
process might have allowed LfH–mNLC to penetrate the BBB and access the brain
457
parenchyma. The therapeutic targets for the treatment of various disorders (insomnia,
458
neurodegenerative disorders, obesity) are hypothalamus and thalamus which are present at
459
the sides of the third ventricle [46, 47]. The outreach of this novel Lf-mNLC to these areas of
460
brain makes it as a suitable candidate for the delivery of therapeutic moieties.
461
3.7. Pharmacodynamic study
462
3.7.1. In vivo model of AD
463
Ac ce p
te
d
452
There
are
many
reports
about
establishing
the
model
of
AD
through
21
Page 21 of 38
intracerebroventricular injection of Aβ1-42 [48]. D-gal is a normal substance in the body and
465
can be metabolized at normal concentrations. However, at high levels it can be oxidized into
466
aldose and hydroperoxide under the catalysis of galactose oxidase, resulting in the formation
467
of a superoxide anion and oxygen-derived free radicals. These free radicals can induce
468
neuronal degeneration and death [49]. Some studies have demonstrated that long-term
469
subcutaneous injection of D-gal lead to the oxidative modifications in cerebral tissue, and
470
further contributes to lipofuscin deposition, slight neuronal damage and memory decay which
471
are similar to the pathological characters of natural aging model [50]. The animal model of
472
AD with combined administration D-gal and Aβ1-42 was better to reflect the complexity of the
473
disease and was more similar to the symptoms and pathological alteration of AD.
474
3.7.2. Determination of MDA level in blood
M
an
us
cr
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464
Oxidative stress can cause damage to membrane lipids, proteins and antioxidative
476
enzyme defense system, thus resulting in the oxidative modifications in cerebral tissue. This
477
further leads to lipofuscin deposition, neuronal damage and memory decay which are all
478
prominent and early changes in AD [50]. MDA increase is an important indicator of lipid
479
peroxidation, which is a well-known paradigm of damage to membranes under conditions of
480
oxidative stress [51]. Furthermore, MDA plasma levels have often been used as the oxidative
481
biomarkers for D-gal-induced aging models [50].
Ac ce p
te
d
475
482
The decrease in MDA levels for Cur solution group, NLC and LfH-mNLC group were
483
found to be 8.29%, 14.09% and 21.2%, respectively (see Fig. 6 of the Appendix). The content
484
of MDA in the blood of the AD model rats increased almost by 50% compared with the sham
485
control group, while the MDA levels significantly (P<0.05) decreased in the NLC and 22
Page 22 of 38
LfH-mNLC compared with Cur solution treated group. For LfH-mNLC group, the decrease of
487
MDA was 1.50 and 2.68 folds of that in NLC and Cur solution group, respectively. This
488
suggests that LfH-mNLC group could successfully cross the BBB and effectively reduce the
489
damage associated with oxidative stress.
490
3.7.3. Histopathology
cr
ip t
486
Neuronal loss in the hippocampus is one of the key hallmarks of AD [52]. Histological
492
observations were conducted to examine the state of the nerve cells in hippocampus region
493
and to evaluate the treatment effectiveness of Cur loaded formulations. No neuronal damage
494
was discovered in the rats of sham group while obvious neuronal loss, karyopyknosis and
495
perikaryon shrinkage were observed in hippocampus of AD control group (see Fig. 7 of the
496
Appendix). After treatment with Cur loaded formulations, the pathological damages were
497
ameliorated to varying extents. The ameliorative effects were more significant for LfH-mNLC
498
as compared to the NLC or Cur solution, which indicates that LfH-mNLC could effectively
499
bypass the BBB and deliver the drug to the brain, making it be a capable carrier for further
500
investigations in the treatment of brain malignancies.
an
M
d
te
Ac ce p
501
us
491
To construct an animal model of AD, primates are the ideal animals owing to their
502
structural and behavioral similarities to humans. But, their use in the laboratory research is
503
limited due to the cost and availability constraints. Presently, rodents (rats and mice) are
504
preferred for the construction of animal AD models [42, 48, 53]. The models of rodents are
505
developed on the basis of pathogenesis of AD reflecting the pathological characters of disease.
506
During current research, the positive results of LfH-mNLC traversing the BBB and
507
ameliorating the pathological characters in the models of rodents indicate its potential 23
Page 23 of 38
508
effectiveness in primates. Nevertheless, future studies should be conducted on primates to
509
evaluate the immunogenicity and safety of LfH-mNLC over a long period of time.
510
4. Conclusion In this study, a novel LDL-mimic nanostructured lipid carrier system (Lf-mNLC) was
512
developed for brain-targeted delivery. Lf-mNLC showed sustained release of active moiety
513
with no significant difference for formulations with different levels of Lf. The significantly
514
increased uptake of Lf–mNLC by BCECs compared with that of NLC diminished in the
515
presence of free Lf, which exposes the Lf receptor mediated endocytosis process. The brain
516
coronal sections displayed a higher accumulation of Lf–mNLC in the cortex and the third
517
ventricle than that of NLC. The pharmacodynamic studies revealed that Lf-mNLC could
518
efficiently control the progression of the AD. In conclusion, Lf–mNLC may provide a
519
flexible drug delivery platform for brain targeting.
520
522
Acknowledgments
Ac ce p
521
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d
M
an
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cr
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511
This work was supported by the Natural Science Foundation of Jiangsu Province
523
(Program No. BK20130655, No. BK2012761), the Open Project Program of State Key
524
Laboratory of Natural Medicines, China Pharmaceutical University (Program No.
525
SKLNMKF201204) and College Students Innovation Project for the R&D of Novel Drugs
526
(Program
527
entrepreneurial training program (Program No. G13076).
No.
J1030830)
and
the
National College
Students'
innovation
528 529 24
Page 24 of 38
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[52] Q.X. Song, M. Huang, L. Yao, X.L. Wang, X. Gu, J. Chen, J. Chen, J.L. Huang, Q.Y. Hu, T. Kang, Z.X. Rong, H. Qi,
596
G. Zheng, H.Z. Chen, and X.L. Gao, ACS Nano, 8 (2014) 2345. 27
Page 27 of 38
597 598
[53] Y. Luo, F.N. Niu, Z.Z. Sun, W.S. Cao, X. Zhang, D.N. Guan, Z.M. Lv, B. zhang, and Y. Xu, Mech. Ageing. Dev., 130 (2009) 248.
Ac ce p
te
d
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28
Page 28 of 38
ip t cr us
Fig. 1. TEM images of NLC (A), LfL-mNLC (B), LfM-mNLC (C), and LfH-mNLC (D). Bar is 100 nm.
an
599 600 601
M
602 603
Ac ce p
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d
604
29
Page 29 of 38
ip t cr us
604
Fig. 2. UV-Vis absorption spectra (Left) and fluorescence emission spectra (Right) of Cur solution (a),
606
Cur- loaded NLC (b) and LfH-mNLC (c).
Ac ce p
te
d
M
607 608
an
605
30
Page 30 of 38
ip t cr us an
608
Fig. 3. (A) Uptake of different formulations in BCECs at a dose of 300 ng/mL of C6 at 37 °C (n = 3); Uptake
610
of NLC and LfH-mNLC in BCECs (B) for different concentrations of C6 at 37°C or 4°C (n = 3), (C) at a dose of
611
100 ng/mL of C6 at different time at 37 °C (n =3), and (D) at a dose of 100 ng/mL of C6 in the presence of
612
excess free Lf (10 mg/mL) at 37 °C (n = 3). Data are expressed as mean ± SD. # P < 0.05 compared with NLC.
613
◆
615
d
te
P < 0.05 compared with LfM-mNLC.
Ac ce p
614
M
609
31
Page 31 of 38
ip t cr us an M
615
Fig. 4. Intracellular integrity assessment of Dil and Dio loaded NLC and LfH-NLC after incubation with
617
BCECs at 37 °C between 1 h and 8 h by monitoring the FRET phenomenon using CLSM.
te Ac ce p
618 619
d
616
32
Page 32 of 38
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Fig. 5. Plasma concentration–time profiles of Cur in rats after intravenous administration of Cur solution, Cur-loaded NLC and LfH–mNLC at a dose of 10 mg/kg of Cur (n=5).
Ac ce p
619 620 621 622
33
Page 33 of 38
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622
Fig. 6. (A) Whole body fluorescence images of SD mouse after intravenous injection of DiR-loaded NLC
624
and LfH–mNLC (0.5 mg/kg). (B) Fluorescence images of excised brain and other organs at 24 h
625
post-injection of DiR-loaded NLC and LfH–mNLC. (C) The fluorescence intensity of DiR in brain and other
626
organs at 24 h post-injection of DiR-loaded NLC and LfH–mNLC. Data are shown as mean ± SD (n = 3).
627
P<0.05 versus NLC .
629 630
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◆
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628
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623
34
Page 34 of 38
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630
Fig.7.
The coronal slices of the mouse brain. (A) Distribution of NLC and LfH–mNLC in the third ventricle and
632
cortex. The green fluorescence signals of C6 were visualized using the FITC filter of brain sections 1 h
633
after i.v. injection. The cell nuclei were stained with 5 μg/mL DAPI for 20 min and visualized using the
634
UV filter (100×). (B) Fluorescence intensity of C6 in the third ventricle and cortex was measured and
635
analysed using Image J software. Data are shown as mean±SD (n = 3).
M
d
P<0.05 versus NLC.
te
638
◆
Ac ce p
636 637
an
631
35
Page 35 of 38
638
Table 1A
639
Effects of different amounts of S100-COOH on particle size, PI, zeta potential, and EE of Cur-loaded NLC.
640
Data were presented as the mean±SD (n=3). Particle size
S100-COOH
Zeta potential PI
(nm)
EE(%)
(mv)
(mmoL)
ip t
Added amounts of
163.4±2.8
0.17±0.017
-3.56±1.05
91.57±0.87
0.002
119.4±1.1
0.12±0.010
-6.95±0.72
0.004
113.7±2.7
0.15±0.012
0.008
105.7±1.8
0.16±0.037
0.012
92.4±0.9
0.17±0.012
-16.87±1.91
96.74±0.95
0.020
74.7±0.7
0.26±0.023
-16.94±1.82
87.65±1.30
cr
0
us
90.39±2.51
92.09±0.39
-13.90±0.43
94.05±1.50
M
an
-8.90±1.32
641 Table 1B
643
Effects of different amounts of Lf on particle size, PI, zeta potential, EE, and DL of Cur-loaded NLC
644
coated with 0.012 mmoL of S100-COOH. Data were presented as the mean±SD (n=3). Preparations
Lf (mg/mL)
te
d
642
Particle size
Zeta potential
PI
Ac ce p
(nm)
NLC
LfL–mNLC
LfM–mNLC LfH–mNLC 645
0
0.5
1.5
2.5
EE(%)
DL(%)
(mv) 96.74±0.95
92.4±0.9
0.17±0.012
-16.87±1.91
2.65±0.23
99.7±1.6
0.20±0.014
-17.90±0.64
-
-
100.9±1.4
0.17±0.020
-12.94±0.62
-
-
103.8±0.6
0.15±0.022
-5.80±0.73
96.51±1.87
2.60±0.17
-: no detection
646 647
Table 1C
648
Pharmacokinetics parameters of Cur after intravenous administration of Cur solution, Cur-loaded NLC and
649
LfH-mNLC at a dose of 10 mg/kg of Cur to rats. Data are presented as the mean±SD (n=5). Preparations
Parameters 36
Page 36 of 38
t1/2 (h)
MRT(h)
AUC0-1
Clearance (L/h)
(ng×h/mL) Cur solution NLC LfH-mNLC
117±10.2
1.31±0.06
*
296±11.6
*
1.16±0.14
*
181±14.6
*
0.41±0.04
6.45±2.63
*
11.08±0.89*
1. 84±0.03 1.24±0.06
17.7±2.00
*
*P<0.05 versus Cur Solution
ip t
650
0.07±0.04
651
Ac ce p
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cr
652
37
Page 37 of 38
ip t
652
cr
653
us
654
Ac ce p
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655
38
Page 38 of 38
S0927-7765(15)00398-7 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.06.025 COLSUB 7155
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
5-4-2015 1-6-2015 10-6-2015
Please cite this article as: F. Meng, S. Asghar, S. Gao, Z. Su, J. Song, M. Huo, W. Meng, Q. Ping, Y. Xiao, A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to treat Alzheimer’s disease, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.06.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel LDL-mimic nanocarrier for the targeted delivery of curcumin into the brain to
2
treat Alzheimer’s disease
3
Fanfei Menga, Sajid Asghara,b, Shiya Gaoa, Zhigui Sua, Jue Songa, Meirong Huoa, Weidong
4
Mengc, Qineng Pinga, Yanyu Xiaoa*
ip t
1
5 6
a
7
Pharmaceutical University, Nanjing 210009, P.R. China
8
b
9
Faisalabad, Pakistan
us
cr
Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China
10
c
11
P.R. China
an
Faculty of Pharmaceutical Sciences, Government College University Faisalabad,
M
Department of Clinical Laboratory, People’s Hospital of Liaocheng, Liaocheng 252000,
12
* Corresponding author at: Department of Pharmaceutics, State Key Laboratory of Natural
14
Medicines, China Pharmaceutical University, No. 24 Tong Jia Xiang, Nanjing 210009, PR
15
China.
16
[email protected] (Y.Y. Xiao).
18 19
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Tel.:
+86-25-83271079;
fax:
+86-25-83271079.
address:
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Abstract In this study, a novel low density lipoprotein (LDL)-mimic nanostructured lipid carrier
26
(NLC) modified with lactoferrin (Lf) and loaded with curcumin (Cur) was designed for
27
brain-targeted delivery, and its effect on controlling the progression of Alzheimer’s disease
28
(AD) in rats was evaluated. NLC with the composition resembling the lipid portion of LDL
29
was prepared by using solvent evaporation method. Lf was adsorbed onto the surface of NLC
30
via electrostatic interaction to yield Lf modified-NLC (Lf-mNLC) as the LDL-mimic
31
nanocarrier. In order to make sure more Lf was adsorbed on the surface of NLC, negatively
32
charged carboxylated polyethylene glycol (100) monostearate (S100-COOH) was synthesized
33
and anchored into NLC. Different levels of S100-COOH (0-0.02mmoL) and Lf modified
34
NLC (0.5-2.5 mg/mL of Lf solution) were prepared and characterized. The uptake and
35
potential cytotoxicities of different preparations were investigated in the brain capillary
36
endothelial cells (BCECs). An AD model of rats was employed to evaluate the therapeutic
37
effects of Lf-mNLC. The results indicate that Lf-mNLC with a high level of Lf showed the
38
maximum uptake in BCECs (1.39 folds greater than NLC) as cellular uptake of Lf-mNLC by
39
BCECs was found to be mediated by the Lf receptor. FRET studies showed Cur still wrapped
40
inside NLC after uptake by BCECs, demonstrating stability of the carrier as it moved across
41
the BBB. Ex vivo imaging studies exposed Lf-mNLC could effectively permeate BBB and
42
preferentially accumulate in the brain (2.78 times greater than NLC). Histopathological
43
evaluation confirmed superior efficacy of Lf-mNLC in controlling the damage associated
44
with AD. In conclusion, Lf-mNLC is a promising drug delivery system for targeting therapy
45
of brain disease.
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Page 2 of 38
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Keywords: Lactoferrin; Carboxylated polyethylene glycol (100) monostearate; LDL-mimic
47
nanostructured lipid carrier system; Brain targeting; Alzheimer’s disease.
48
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68 69
1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder with no means yet known for cure or prevention. A potential difficulty in curing irreversible brain degeneration
71
in AD is the existence of blood brain barrier (BBB). BBB, a structure that impedes the
72
transportation of the drugs and toxic substances into the brain for the sake of its protection, is
73
a challenge for the success of therapies against central nervous system (CNS) maladies [1].
74
Increasing number of brain ailments in the modern world needs solution to the
75
sub-therapeutic brain delivery of pharmacologically active molecules [2]. Various tactics are
76
being employed for the delivery of neurotherapeutics across the BBB, but this research
77
domain still needs vigorous efforts to achieve the goal [3, 4] .
M
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Lipid based nanocarriers have been widely reported for the treatment of brain diseases
79
[5]. Recently, novel nanocarriers based on low density lipoprotein (LDL) are getting attention
80
for their distinctive characteristics [6-8]. LDL is a 22-27 nm particle composed of a core of
81
hydrophobic lipids, primarily cholesteryl esters with a small amount of triacylglycerides, and
82
has a surface coat of phospholipids, unesterified cholesterol, and a single molecule of
83
apolipoprotein B 100 (ApoB-100) [9, 10]. As a kind of naturally originated particle, LDL is
84
non-immunogenic and long circulating, and possesses the innate ability to bind both
85
hydrophobic and hydrophilic molecules. In addition, the overexpression of the low density
86
lipoprotein receptors (LDLR) in brain capillary endothelial cells (BCECs) offers the
87
advantage of brain targeting for LDL-based nanoparticles [11, 12]. However, the quantities of
88
natural LDL extracted from human serum are extremely low, and it is difficult to isolate LDL
89
in large quantities due to the variable composition and size. Another approach uses
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Page 4 of 38
90
reconstituted LDL consisting of a lipid emulsion [13, 14], and ApoB-100 has been used for
91
stabilizing such emulsions. It is also difficult to isolate ApoB-100 from human serum due to
92
its large size and propensity to aggregate. Lactoferrin (Lf), an 80 kDa naturally occurring iron binding cationic glycoprotein, is
94
abundantly found in milk. It has been reported to possess anti-inflammatory, antibacterial,
95
antifungal, antiviral, and anti-cancer activities. It can also boost natural immunity, bone
96
growth, and wound healing [15, 16]. Lf can be transported across BBB by the mediation of
97
the lactoferrin receptors (LfR) present at the endothelial cells of the BBB [17-19]. Some
98
studies showed the expression of LfR in the state of AD and Parkinson’s disease at the
99
endothelial cells of the BBB would be increased [20].
cr
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Curcumin
(Cur),
a
low
M
100
ip t
93
molecular
weight
phytochemical,
possesses
diverse
pharmacological activities, including anti-oxidant, anti-inflammatory, anti-cancer, and
102
antidepressant properties with low intrinsic toxicity [21, 22]. It is widely acknowledged that
103
the progressive accumulation of Aβ aggregates initiates the initial development of
104
neurodegenerative pathology and trigger a cascade of events such as neurotoxicity, oxidative
105
damage, and inflammation that contribute to the progression of AD [23]. Cur has been shown
106
to effectively reduce neurotoxic Aβ aggregates and ameliorate cognitive deficits in vivo and
107
in vitro [24, 25].
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108
The objective of this research work was to develop a novel LDL-mimic nanocarrier
109
(nanostructured lipid carrier (NLC) modified with Lf, Lf-mNLC) encapsulating Cur for
110
brain-targeted delivery, and evaluate its effect on controlling the progression of AD in rats.
111
Cur-loaded NLC with the composition resembling the lipid portion of LDL (PC, cholesterol 5
Page 5 of 38
oleate and glycerol trioleate) was prepared by using solvent evaporation method. Lf,
113
positively charged at physiological pH, instead of ApoB-100, was used as a brain targeting
114
molecule and was adsorbed onto the surface of NLC via electrostatic interaction to yield
115
Lf-mNLC as the LDL-mimic nanocarrier. Carboxylated polyethylene glycol (100)
116
monostearate (S100-COOH) was synthesized and its free surface carboxyl could enhance the
117
negative charge of NLC, which facilitated more Lf was absorbed on the surface of NLC
118
through electrostatic attraction.
119
2. Experimental
120
2.1 Materials
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Cur (purity, 98%) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
122
Glycerol trioleate (purity, >98%) and phosphatidyl choline (PC, purity, 90%) were kindly
123
donated by Evonik Degussa China Co., Ltd. (Shanghai, China). Cholesterol oleate
124
(purity, >85%) and coumarin-6 (C6) were purchased from Tokyo Chemical Industry (Tokyo,
125
Japan). Polyethylene glycol (100) monostearate (S100, the polymerization degree of ethylene
126
glycol
127
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
128
Sigma-Aldrich (Milwaukee, WI, USA). Aβ1-42 (purity, 95.34%) was purchased from GL
129
Biochem Ltd (Shanghai, China). D-galactose (D-gal) was purchased from Sigma-Aldrich
130
(Milwaukee, WI, USA). All other chemicals and reagents were of analytical grade and used
131
without further purification.
132
2.2 Animals
133
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is
100),
Lf
(human,
≥90%,
SDS-PAGE)
and
Sprague-Dawley (SD) rats (180-220 g) and ICR mice (18-22 g) were obtained from 6
Page 6 of 38
Experiment Animal Center of Nantong University (Nantong, China). All animal procedures
135
for this study were approved by the ethical committee of China Pharmaceutical University,
136
and they were conducted in accordance with approved standards for laboratory animal care.
137
2.3 Synthesis of S100-COOH
ip t
134
Carboxylated polyethylene glycol (100) monostearate (S100-COOH) was synthesized
139
using a one-step procedure. Briefly, 11.71 g of S100 and 0.5 g of succinic anhydride were
140
mixed and activated with the catalysis of pyridine (7.5 mL) at 65 oC for 48 h. Subsequently,
141
the pyridine was removed using a rotary evaporator and the residues were hydrated with 200
142
mL of distilled water. The resultant solution was dialyzed with a Spectrum dialysis bag
143
(MWCO of 3.5 kDa, Sigma, USA) at 4 oC for 48 h to remove the unreacted succinic
144
anhydride and lyophilized to obtain S100-COOH. The structure of S100-COOH was
145
characterized by 1H-NMR and 13C-NMR spectra (AVANCE AV-300, Bruker Instrument Inc.,
146
Switzerland) using D2O as a solvent at 25 °C, respectively.
147
2.4 Preparation of Cur-loaded Lf-mNLC
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cr
138
Firstly, Cur-loaded NLC was prepared by a solvent evaporation method [26]. Briefly, PC
149
(330 mg), cholesterol oleate (20 mg), glycerol trioleate (70 mg), Cur (16 mg) and different
150
amounts of S100-COOH (0-0.02 mmoL) were dissolved in 10 mL of ethanol/chloroform
151
mixture (1:1, v/v) and dried in a rotary evaporator under vacuum at 40 °C. The dried lipid
152
film was hydrated with 10% (v/v) glycerol solution at 37 °C and ultrasonicated at 300 W for
153
5 min at 4 °C, followed by extrusion through 0.22 μm cellulose nitrate membrane to remove
154
the unentrapped drug and obtain Cur-loaded NLC. Afterwards, Cur-loaded Lf-mNLC was
155
prepared by incubating Cur-loaded NLC with Lf solution. Briefly, 0.5, 1.5, or 2.5 mg/mL of 7
Page 7 of 38
Lf solution (dissolved in Tris-EDTA buffer (pH7.4)) was gently mixed with an equal volume
157
of Cur-loaded NLC at 4 °C for 24 h. The three levels of Lf modified NLC were named as
158
LfL-mNLC, LfM-mNLC and LfH-mNLC.
159
2.5. Characterization of Cur-loaded NLC and Cur-loaded Lf-mNLC
160
2.5.1. Morphology, particle size, zeta potential, EE and DL
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156
The particle size, polydispersity index (PI) and zeta potential of Cur-loaded NLC and
162
Lf-mNLC were detected by Zetasizer 3000HS (Malvern, U.K.). Their morphological
163
observations were performed by transmission electron microscopy (TEM, H-7000, Hitachi,
164
Japan). EE of NLC and DL of NLC/Lf-mNLC were determined as previously reported [27].
165
2.5.2. Fluorescence and UV-Visible absorbance spectra
M
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The interaction of Cur with the hydrophobic lipid core of NLC or Lf-mNLC was
167
investigated by fluorescence and UV-Visible absorption spectra [28, 29]. The fluorescence
168
and UV-Visible absorption spectra of Cur (dissolved in methanol), Cur-loaded NLC and
169
Lf-mNLC (in aqueous solution) with the Cur concentration of 1.5 μg/mL were compared.
170
The fluorescence emission spectrum was recorded from 450 to 700 nm with an excitation
171
wavelength of 420 nm. Excitation and emission slit widths (modulation of magnitude and
172
resolution of transmitted light) were standardized at 5 nm each. The UV-Visible absorption
173
spectrum was taken from 300 to 700 nm.
174
2.5.3. Plasma stability of NLC and LfH-mNLC
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175
NLC and LfH-mNLC were mixed with the equal volume of the rat plasma and incubated
176
for prearranged time intervals (15, 30, 60, 120, 240, 360 min) in a 37 °C water bath. After
177
incubation, 100 μL of the sample was diluted in 4 mL of distilled water and the particle size 8
Page 8 of 38
178
and zeta potential were measured by the Dynamic Light Scattering Analyzer.
179
2.5.4 In vitro drug release study The in vitro release of Cur from NLC or Lf-mNLC was carried out in physiological saline
181
containing 1% (w/v) tween 80 using a membrane dialysis method. 1 mL of different Cur
182
preparations (containing 400 μg of Cur) was placed in a pre-swelled dialysis bag (8~10 kDa
183
MW cutoff, Sigma, USA). Cur solution (containing 30%（w/w）polyethylene glycol 400) was
184
taken as a control. The bag was tightened and soaked in 50 mL of release medium at 37 °C
185
for 48 h (n=3). At a given time interval, 1 mL of the medium was withdrawn and replaced
186
with the same amount of pre-warmed fresh medium. The withdrawn sample was then
187
centrifuged at 12,000 rpm for 10 min and the supernatant was analyzed by HPLC method
188
[27]. Care was taken to protect the sample from light throughout the experimental procedure.
189
The release kinetic models were studied using DDsolver software.
190
2.6. Cytotoxicity and cellular uptake studies
191
2.6.1. Cell culture
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180
BCECs were cultured in the endothelial cell culture medium that contained DMEM/F12,
193
20% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL
194
streptomycin. BCECs were maintained in an incubator (Thermo Scientific, USA) at 37 °C
195
under an atmosphere of 5% CO2.
196
2.6.2. Cytotoxicity evaluation
197
In order to justify the nontoxicity of different empty preparations, their cells viabilities
198
were determined by MTT assay. Briefly, 5 × 103 cells/well were seeded in 96-well culture
199
plates for 24 h and then incubated with empty NLC, empty LfL-mNLC, empty LfM-mNLC, 9
Page 9 of 38
and empty LfH-mNLC. The concentrations of empty carriers were in the range of 100−1600
201
μg/mL. After 24 h incubation, 10 μL of 5 mg/mL MTT was added into each well for 4 h in
202
dark, and then the medium was replaced with 150 μL of DMSO. The absorbance value at 570
203
nm (A570nm) was read using the microplate reader (Thermo Electron Corporation. USA). Cell
204
viability was expressed as a percentage of A570nm of the study group relative to that of control
205
group.
206
2.6.3. Cellular uptake
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200
C6-loaded NLC and Lf-mNLC were prepared similar to the preparation of Cur-loaded
208
NLC/ Lf-mNLC, but Cur was replaced with C6. BCECs were seeded into twelve-well plates
209
at a density of 2×105 cells/well and cultured in complete cell-culture medium for 24 h at
210
37 °C. Then, the medium was replaced with 1 mL of different C6-loaded preparations (C6=
211
300 ng/mL) and cells were further incubated for another 2 h. Cells were trypsinized and
212
harvested. The fluorescence intensities of C6 in cells were determined by a BD FACS Calibur
213
flow cytometer (BD Bioscience, Bedford, MA). The excitation wavelength of C6 was 465 nm,
214
and FL1-H filter was used for the collection of fluorescence intensity. The data were analyzed
215
through Flow Jo 7.6 software. The influences of incubation concentration, temperature and
216
time on cellular uptake of the formulations were also monitored. Furthermore, uptake
217
inhibition experiment was also performed by pre-incubating BCECs with excess of Lf (10
218
mg/mL) for 30 min, and then the cells were incubated for 2 h at 37 °C with NLC or
219
LfH-mNLC (C6=100 ng/mL).
220
2.7. Intracellular stability of NLC and LfH-mNLC
221
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FRET analysis was conducted to evaluate the stability of NLC or Lf-mNLC in cells after 10
Page 10 of 38
uptake by BCECs in order to make sure the passage of drug across the BBB in the form of
223
being wrapped in NLC [30]. A FRET pair of hydrophobic dyes, DiO as donor and DiI as
224
acceptor, was loaded into the core of NLC or LfH-mNLC, with the method similar to the
225
preparation of Cur-loaded NLC/ LfH-mNLC. In order to verify the occurrence of FRET, the
226
fluorescence spectra of NLC or LfH-mNLC loaded with DiI and DiO were measured in the
227
blank cell culture medium-DMEM/F12 or trichloromethane-methanol (1:1) with the
228
excitation at 488 nm. To monitor the integrity of carriers in BCECs, NLC or LfH-mNLC were
229
incubated with BCECs at 37°C for 1 h. Subsequently, NLC or LfH-mNLC was removed, and
230
the cells were washed thrice with ice-cold PBS. At last, the cells were incubated with the cell
231
culture medium for predetermined time periods. Fluorescence images were acquired with the
232
excitation at 488 nm, and the emission between 555 nm and 655 nm for DiI detection, as well
233
as the emission between 500 nm and 530 nm for DiO detection by CLSM (Leica TCS SP5).
234
2.8. Pharmacokinetics
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Healthy male SD rats were used to compare the pharmacokinetics of Cur after
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236
intravenous administration of Cur solution, Cur-loaded NLC and LfH-mNLC at a dose of 10
237
mg/kg of Cur (n=5). After being collected at predetermined intervals, the blood was
238
centrifuged at 4000 rpm for 10 min at 4 °C and then the plasma were frozen at -20°C until
239
assay. 100 μL of acetonitrile was added into 100 μL of plasma, vortexed for 2 min and
240
centrifuged at 12000 rpm for 10 min. 20 μL of supernatant was injected into the HPLC
241
system equipped with a fluorescence detector (Shimadzu, Japan) to determine the
242
concentration of Cur, and the excitation and emission wavelength were 436 and 518 nm,
243
respectively. The plasma concentration versus time data were analyzed by Kinetica 4.4 11
Page 11 of 38
244
(Thermo Electron Corporation, Waltham, MA, USA) and various parameters were studied.
245
2.9. Biodistribution study In order to verify the brain-targeting of Lf-mNLC, ex vivo imaging experiments were
247
performed in rats. DiR, a hydrophobic near infrared dye, was loaded into NLC or LfH-mNLC.
248
SD rats were intravenously administered with DiR-loaded NLC or LfH-mNLC (0.5 mg/kg)
249
and anesthetized. A Kodak multimodal-imaging system IS2000MM (Kodak, USA) was used
250
to perform ex vivo imaging experiments at 1, 2, 3 and 4 h post injection with the wavelength
251
fixed at 720 nm for excitation and 790 nm for emission. To probe the biodistribution of NLC
252
or LfH-mNLC, rats were sacrificed at 24 h post-injection, and different organs were separated
253
and washed by saline, assembled for fluorescence imaging and analyzed by IS2000MM
254
software (Kodak ID Image Analysis Software, Kodak).
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In addition, brain sections were used to further understand the distribution of NLC or
256
LfH-mNLC in the CNS. Briefly, C6-loaded NLC and LfH-mNLC (6 mg/kg) were injected into
257
the tail veins of mice, respectively. After 1 h, the mice were anaesthetized and then the brains
258
were removed, fixed in 4% paraformaldehyde for 24 h, and dehydrated with 15% sucrose
259
solution for 12 h and then 30% sucrose solution for 24 h. Thereafter, the brain samples were
260
frozen in OCT (Sakura, Torrance, CA, USA) at -80 °C and cut into 20 μm thickness with a
261
cryotome Cryostat (Leica, CM 1900, Germany). After staining with 5 μg/mL DAPI for 20
262
min at room temperature, slides were observed using a fluorescence microscope (Olympus,
263
Japan). Fluorescence intensity of C6 in the third ventricle and cortex was measured and
264
analysed using Image J software.
265
2.10 Neuroprotective effects of different Cur preparations on AD model rats
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266
2.10.1 In vivo model of AD The in vivo model of AD was established in rats by administering Aβ1-42 and D-gal. The
268
SD rats were treated with intraperitoneal injection 4% D-gal (0.3 mL/100g/d) by
269
intraperitoneal injection for six weeks. Aβ1-42 was dissolved in saline (1 mg/mL) and then
270
incubated at 37 °C for 7 days to form the aggregation. The rats were anaesthetized and fixed
271
in a stereotaxic apparatus. The animals were bilaterally injected in the dorsal hippocampus
272
(2.3 mm posterior to the bregma, ±1.8 mm lateral to the Midline and 2.0 mm ventral to the
273
skull surface) with 5 μL of Aβ1-42 within 5 min. The needle was then slowly withdrawn after
274
being kept for another 3 min. Rats in the sham group were injected with the same volume of
275
saline. After surgery, the rats were kept in the cages for recovery.
276
2.10.2 Drug administration
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AD model rats were divided into five groups: the AD control group, sham group, and
278
three test groups (n=10 for each group). One week after the surgery, Cur solution (30% (w/v)
279
PEG-400 in a 5% (v/v) glucose solution), NLC and LfH-mNLC were intravenously
280
administered to the rats at 2 mg/kg/d equivalent dose of Cur for three weeks, respectively.
281
2.10.3 Determination of malondialdehyde (MDA)
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The rats were anaesthetized with ether, and blood samples were taken from the
283
eyeground veins. The plasma was obtained after centrifugation (5 min, 4000 rpm, 4 °C) and
284
assessed for the concentration of MDA using a MDA assay kit according to the
285
manufacturer’s protocols.
286
2.11 Histopathology
287
Rats in each group (n=5) were anaesthetized and perfused through the heart with cold 13
Page 13 of 38
saline followed by 4% paraformaldehyde. Then the whole brains were harvested and
289
immersed in 4% paraformaldehyde. Afterwards, the brains were embedded in paraffin and cut
290
into 5-μm sectioned. Sections were stained with hematoxylin-eosin (HE) staining,
291
respectively. The hippocampal areas were examined under a Leica optical microscope (Leica,
292
Germany).
293
2.12. Statistical data analysis
cr us
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The data are given as mean ± SD. Statistical significance was tested by two-tailed Student’s t test or one-way ANOVA. Statistical significance was set at P < 0.05.
296
3. Results and discussion
297
3.1 Preparation and characterization of NLC and Lf-mNLC
298
3.1.1. Morphology, particle size, zeta potential, EE and DL
M
an
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Based on particle size, zeta potential and EE, Cur-loaded NLC was prepared with PC,
300
cholesterol oleate, glycerol trioleate and Cur according to an optimized ratio of
301
1:0.06:0.21:0.05 (w/w/w/w). Effects of different amounts of S100-COOH on particle size, PI,
302
zeta potential and EE of Cur-loaded NLC are listed in Table 1A. The particle size of NLC
303
decreased after modification with S100-COOH and was found inversely proportional to the
304
amount of S100-COOH, which might be due to its surface-active effects [31]. In addition, the
305
PEG chains of S100-COOH could act as a tighter net on the outside of vesicles, thus limiting
306
the increase of vesicle size [26]. The inclusion of S100-COOH in NLC also led to the
307
increase in negative charge due to the ionized –COOH on the surface of NLC and the
308
absolute value of zeta potential increased gradually with the increase of S100-COOH. The
309
substantial increase in PI and decrease in EE as the added amount of S100-COOH increased
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14
Page 14 of 38
to 0.02 mmoL could be due to two reasons. Firstly, the added amount of S100-COOH might
311
not totally arrange themselves on the surface of NLC and they could have spontaneously
312
formed the small sized unstable micelles [32]. The drug could be easily released from the
313
micelles and precipitate in the medium. Secondly, the decreased drug loading space owing to
314
the smaller particle size might also contribute to the partial drug encapsulation. As the
315
isoelectric point of Lf is about 8.65, the protein would be positively charged in the suspension
316
of NLC (pH 6.0-6.5). The more the negative charge on the surface of NLC, the more
317
favorable it is for Lf to be adsorbed on the surface of NLC. Therefore, NLC coated with
318
0.012 mmoL of S100-COOH was chosen for further modification.
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Table 1B shows the effect of different concentrations of Lf on particle size, PI, zeta
320
potential, EE, and DL of Cur-loaded NLC coated with 0.012 mmoL of S100-COOH. The
321
adsorption of Lf on the surface of NLC led to the increase of particle size and zeta potential
322
of NLC. However, Lf coating had not interfered with DL and EE of NLC. The particle size
323
and shape of NLC and Lf-mNLC were further confirmed by TEM (Fig. 1). Both NLC and
324
Lf-mNLC were quasi-spherical in shape. However, Lf-mNLC appeared a core-shell structure
325
and the thickness of the covering layer increased with the increasing amount of Lf.
326
Furthermore, a dense core was observed for nanocarrier with high concentration of Lf which
327
is not evident in NLC or Lf-mNLCs with low concentration of Lf. This could be due to
328
formation of a more compact nanocarrier owing to the greater electrostatic interactions with
329
higher concentration of Lf. TEM also showed that both NLC and Lf-mNLC were uniformly
330
dispersed without any visible sign of aggregation, indicating the stability of the formulations.
331
3.1.2. In vitro drug release study
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Page 15 of 38
In vitro drug release profiles of Cur from different preparations are studied (see Fig.2 of
333
the Appendix). Except for Cur solution, all preparations exhibited sustained release of Cur
334
without any burst release, which could be attributed to the encapsulation of Cur into NLC.
335
Compared with NLC, the release of Cur from Lf-mNLC decreased significantly (P < 0.05)
336
and increasing the amount of Lf further slowed the drug release, which suggests that Lf
337
adsorption further limited the diffusion of the drug from NLC [33].
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In order to explore the release mechanisms of Cur from Cur-loaded NLC and Lf-mNLC,
339
various models (zero-order, first-order, Higuchi, and Weibull models) were utilized to
340
simulate the release profiles (see Table 1 of the Appendix). Weibull model was found to be
341
the best-fit model to explain the release process. According to the exponent parameter (β) of
342
Weibull model, the Cur release mechanism from NLC and Lf-mNLC was found to be the
343
combined mechanism of Fickian diffusion and Case II transpobrt [34].
344
3.1.3. Fluorescence and UV-Visible absorbance spectra
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Fluorescence and UV-Visible absorbance spectra of Cur, Cur-loaded NLC and
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an
338
346
LfH-mNLC are presented in Fig. 2. As depicted in the UV-Visible absorbance spectrum, there
347
is an intense absorption band at around 425 nm for Cur and the presence of a shoulder peak at
348
350 nm indicates the location of Cur in polar environment [29]. However, the appearance of a
349
shoulder peak at around 450 nm for the Cur-loaded NLC and LfH-mNLC could be ascribed to
350
the presence of Cur in the apolar core of NLC and LfH-mNLC [35]. For the fluorescence
351
spectra, Cur in methanol showed a sharp fluorescence peak at around 540 nm, but there was a
352
blue shift in the fluorescence spectra of Cur-loaded NLC and LfH-mNLC and Cur showed a
353
well-defined peak at around 520 nm. Sahu et al. [36] reported that when encapsulated in 16
Page 16 of 38
bovine casein micelle, Cur showed a shift in fluorescence spectra from 540 nm to 500 nm
355
because of the binding of Cur in the hydrophobic domain of the protein molecule in micelle.
356
Therefore, we inferred that when compared with that of native Cur, the blue shift in
357
fluorescence spectrum of Cur was induced by the encapsulation of Cur in the hydrophobic
358
domain of the NLC and LfH-mNLC through hydrophobic interactions.
359
3.1.4 Plasma stability
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In order to study the plasma stability of NLC and LfH-mNLC, variations in the particle
361
size and zeta potential of formulations were assayed in the rat plasma. The results showed
362
that NLC and LfH-mNLC had slightly smaller diameters in plasma than in distilled water, but
363
remained stable (see Fig.3A of the Appendix). The insignificant changes in the size of the
364
nanocarriers imply that the coating of Lf did not affect the stability of NLC in the plasma and
365
LfH-mNLC was stable in the plasma. The zeta potential of NLC and LfH-mNLC both dropped
366
to nearly -25 mv after 15 min incubation with the plasma and then remained steady for the
367
rest of the study (see Fig.3B of the Appendix). This could be attributed to the presence of
368
negatively charged proteins in the serum.
369
3.2. In vitro cytotoxicity assay against BCECs
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an
360
No significant toxicity on BCECs was found for empty NLC and Lf-mNLC as more
371
than 80% cell viability was observed even at highest concentration (1600 μg/mL) (see Fig.4
372
of the Appendix). Formulations that show greater than 80% cell viability have been regarded
373
as safe [37]. There were no significant differences in cell viability of NLC and Lf-mNLC
374
group, which shows both NLC and Lf-mNLC might be good biocompatible carriers.
375
3.3. Cellular uptake study 17
Page 17 of 38
The uptake of LfL-mNLC, LfM-mNLC and LfH-mNLC in BCECs was 1.06, 1.16 and
377
1.39 times higher, respectively, compared to that of NLC (Fig. 3A). As the cellular uptake of
378
LfH-mNLC was higher than those of LfL-mNLC and LfM-mNLC, LfH-mNLC was chosen for
379
subsequent studies.
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As shown in Fig. 3B, the uptake amounts of both LfH–mNLC and NLC at 37 °C were
381
much higher than those at 4 °C, suggesting that their uptakes were energy-dependent. The
382
uptake increased with the increase in the concentration of C6 from 10-100 ng/mL, which
383
indicates that the uptakes of both NLC and LfH-mNLC were also concentration-dependent.
384
The uptakes of NLC and LfH–mNLC by BCECs were also dependent on the incubation time
385
(Fig. 3C). As shown in Fig. 3D, the uptake of LfH–mNLC was significantly suppressed in the
386
presence of excess free Lf, while the uptake of NLC was scarcely influenced, which confirms
387
that Lf mediated endocytosis mechanism was responsible for the enhanced uptake of
388
LfH–mNLC. BCECs have been reported to possess LfR and an increase of these receptors in
389
BCECs has also been found in Parkinson's and Alzheimer's disease [38, 39], which could
390
extend the applications of Lf-mNLC for the treatment of various brain malignancies.
391
3.4. Intracellular integrity studies of NLC and LfH-mNLC
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cr
380
FRET occurs through the energy transfer from the donor (DiO) to the acceptor (DiI)
393
only when both fluorescent molecules are in a range of 1-10 nm. Implicitly, with the increase
394
in distance between the donor and acceptor, the FRET ratio IR/(IG+IR) decays very rapidly,
395
where IR and IG are fluorescence intensities at 576 nm and 508 nm, respectively. When the
396
carriers disintegrate in the cells, the fluorescent molecules could not be closely retained
397
anymore, thus resulting in a lower FRET ratio [40]. Therefore, we used FRET experiment to 18
Page 18 of 38
398
monitor the stability of the carriers in cells after uptake by BECEs. LfH-mNLC in DMEM/F12 showed a strong DiI signal (see red curve in Fig. 5 of the
400
Appendix) with a FRET ratio of 0.80, which shows LfH-mNLC were stable in DMEM/F12.
401
While after using trichloromethane-methanol (1:1) to destroy the carriers, FRET phenomenon
402
almost disappeared (see green curve in Fig. 5 of the Appendix) with a FRET ratio below 0.30.
403
Same behavior was expressed by mNLC (data is not shown). The confocal images showed
404
that both NLC and LfH-mNLC in BCECs had a strong FRET phenomenon at 1 h post
405
incubation (Fig. 4), which indicates that the carriers remained intact in the cells. With the
406
passage of time (1-4 h), a decrease in red fluorescence was seen, however intracellular FRET
407
phenomenon was still existing, and implying most of the carriers didn’t disintegrate in the
408
BCECs and kept their integrity. At 8 h, the green fluorescence of DiO became much stronger
409
and FRET phenomenon disappeared, suggesting the disintegration of NLC and LfH-mNLC.
410
BBB is the unavoidable doorway for the brain targeted delivery systems, but it is not the
411
destination. The carriers intended for CNS delivery should remain intact while moving across
412
BCECs and avoid enzymatic degradation in the BCECs, which guarantees the efficient drug
413
delivery to the diseased regions of brain. Previously, Lf functionalized nanoparticles have
414
been reported to attain maximum concentration in the brain after 1 h following intravenous
415
administration [37, 41, 42]. FRET experiments revealed that NLC or LfH-mNLC were stable
416
in BCECs for at least 4 h, which might be long enough to permit safe passage of drug across
417
the BBB.
418
3.5. Pharmacokinetic studies
419
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The plasma concentration-time curves of Cur after intravenous injection of different 19
Page 19 of 38
preparations in rats are shown in Fig. 5. The blood concentration of Cur decreased rapidly
421
within the initial 2 h and could not be detected at 4 h after intravenous administration of Cur
422
solution. However, the concentration of Cur after intravenous administration of Cur-loaded
423
NLC and Lf-mNLC at 4 h were 13.03 and 5.49 ng/mL, respectively.
ip t
420
The pharmacokinetic data of Cur were simulated by non-linear least squares. The results
425
showed that the kinetics of a two-compartment open model was best fitted to Cur plasma
426
concentration-time curves in rats. The area under the curve (AUC) and MRT dramatically
427
increased and the clearance (CL) decreased (P<0.05) for Cur encapsulated in NLC when
428
compared with Cur solution (Table 1C). Compared to the drug solution, NLC and LfH-mNLC
429
showed 2.53 and 1.55 folds higher AUC, respectively and prolonged residence in body with
430
4.49 and 3.02 times greater MRT, respectively. While, 1.81 and 1.05 times lesser clearance
431
from the body than the drug solution was observed for NLC and LfH-mNLC, respectively.
te
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424
Comparing the pharmacokinetic parameters of NLC and LfH-mNLC, an even lower
433
AUC and higher CL were observed for the latter. The coupling of NLC’s surface with Lf
434
affected the pharmacokinetics of NLC. These results are in good agreement with a previously
435
published study [43]. The main reason may be the specific targeting capability of Lf which
436
could have accelerated the uptake of Lf-mNLC by the target tissues or organs [44].
437
3.6. Biodistribution studies
Ac ce p
432
438
To investigate the brain-targeting capability of Lf-mNLC, ex vivo fluorescence images
439
of the rats were acquired after intravenous administration of DiR-loaded NLC and
440
LfH-mNLC. As displayed in Fig. 6A, the LfH-mNLC group showed significantly higher
441
fluorescence intensity in the brain than the NLC group at any time point post-administration. 20
Page 20 of 38
Additionally, the brain and major organs were harvested and imaged at 24 h
443
post-administration (Fig. 6B). As shown in Fig. 6C, The fluorescence intensity in brain tissue
444
for the LfH-mNLC group was 2.78 times high as that for the NLC group according to the
445
quantitative DiR fluorescence intensity, indicating that LfH-mNLC accumulated more than
446
NLC in brain. The presence of NLC in the brain could be attributed to the presence of PEG. It
447
has been previously demonstrated that PEG coated nanoparticles might adsorb the
448
apolipoprotein E (ApoE), and utilize the LDLR mediated transport to cross the BBB [45].
449
Our results suggested that LfH-mNLC could cross the BBB and penetrate the brain more
450
efficiently than NLC that might be attributed to the interaction between Lf and Lf receptors
451
over-expressed at the BBB.
M
an
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Fig. 7A shows coronal slices of the mouse brain. Periventricular region of the third
453
ventricle and cortex showed the green fluorescence of C6. The stronger and widely
454
distributed fluorescence intensity of LfH-mNLC than that of NLC quantified with help of
455
Image J software (Fig. 7B), in these regions, indicates that Lf-receptor mediated transcytosis
456
process might have allowed LfH–mNLC to penetrate the BBB and access the brain
457
parenchyma. The therapeutic targets for the treatment of various disorders (insomnia,
458
neurodegenerative disorders, obesity) are hypothalamus and thalamus which are present at
459
the sides of the third ventricle [46, 47]. The outreach of this novel Lf-mNLC to these areas of
460
brain makes it as a suitable candidate for the delivery of therapeutic moieties.
461
3.7. Pharmacodynamic study
462
3.7.1. In vivo model of AD
463
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452
There
are
many
reports
about
establishing
the
model
of
AD
through
21
Page 21 of 38
intracerebroventricular injection of Aβ1-42 [48]. D-gal is a normal substance in the body and
465
can be metabolized at normal concentrations. However, at high levels it can be oxidized into
466
aldose and hydroperoxide under the catalysis of galactose oxidase, resulting in the formation
467
of a superoxide anion and oxygen-derived free radicals. These free radicals can induce
468
neuronal degeneration and death [49]. Some studies have demonstrated that long-term
469
subcutaneous injection of D-gal lead to the oxidative modifications in cerebral tissue, and
470
further contributes to lipofuscin deposition, slight neuronal damage and memory decay which
471
are similar to the pathological characters of natural aging model [50]. The animal model of
472
AD with combined administration D-gal and Aβ1-42 was better to reflect the complexity of the
473
disease and was more similar to the symptoms and pathological alteration of AD.
474
3.7.2. Determination of MDA level in blood
M
an
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464
Oxidative stress can cause damage to membrane lipids, proteins and antioxidative
476
enzyme defense system, thus resulting in the oxidative modifications in cerebral tissue. This
477
further leads to lipofuscin deposition, neuronal damage and memory decay which are all
478
prominent and early changes in AD [50]. MDA increase is an important indicator of lipid
479
peroxidation, which is a well-known paradigm of damage to membranes under conditions of
480
oxidative stress [51]. Furthermore, MDA plasma levels have often been used as the oxidative
481
biomarkers for D-gal-induced aging models [50].
Ac ce p
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d
475
482
The decrease in MDA levels for Cur solution group, NLC and LfH-mNLC group were
483
found to be 8.29%, 14.09% and 21.2%, respectively (see Fig. 6 of the Appendix). The content
484
of MDA in the blood of the AD model rats increased almost by 50% compared with the sham
485
control group, while the MDA levels significantly (P<0.05) decreased in the NLC and 22
Page 22 of 38
LfH-mNLC compared with Cur solution treated group. For LfH-mNLC group, the decrease of
487
MDA was 1.50 and 2.68 folds of that in NLC and Cur solution group, respectively. This
488
suggests that LfH-mNLC group could successfully cross the BBB and effectively reduce the
489
damage associated with oxidative stress.
490
3.7.3. Histopathology
cr
ip t
486
Neuronal loss in the hippocampus is one of the key hallmarks of AD [52]. Histological
492
observations were conducted to examine the state of the nerve cells in hippocampus region
493
and to evaluate the treatment effectiveness of Cur loaded formulations. No neuronal damage
494
was discovered in the rats of sham group while obvious neuronal loss, karyopyknosis and
495
perikaryon shrinkage were observed in hippocampus of AD control group (see Fig. 7 of the
496
Appendix). After treatment with Cur loaded formulations, the pathological damages were
497
ameliorated to varying extents. The ameliorative effects were more significant for LfH-mNLC
498
as compared to the NLC or Cur solution, which indicates that LfH-mNLC could effectively
499
bypass the BBB and deliver the drug to the brain, making it be a capable carrier for further
500
investigations in the treatment of brain malignancies.
an
M
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Ac ce p
501
us
491
To construct an animal model of AD, primates are the ideal animals owing to their
502
structural and behavioral similarities to humans. But, their use in the laboratory research is
503
limited due to the cost and availability constraints. Presently, rodents (rats and mice) are
504
preferred for the construction of animal AD models [42, 48, 53]. The models of rodents are
505
developed on the basis of pathogenesis of AD reflecting the pathological characters of disease.
506
During current research, the positive results of LfH-mNLC traversing the BBB and
507
ameliorating the pathological characters in the models of rodents indicate its potential 23
Page 23 of 38
508
effectiveness in primates. Nevertheless, future studies should be conducted on primates to
509
evaluate the immunogenicity and safety of LfH-mNLC over a long period of time.
510
4. Conclusion In this study, a novel LDL-mimic nanostructured lipid carrier system (Lf-mNLC) was
512
developed for brain-targeted delivery. Lf-mNLC showed sustained release of active moiety
513
with no significant difference for formulations with different levels of Lf. The significantly
514
increased uptake of Lf–mNLC by BCECs compared with that of NLC diminished in the
515
presence of free Lf, which exposes the Lf receptor mediated endocytosis process. The brain
516
coronal sections displayed a higher accumulation of Lf–mNLC in the cortex and the third
517
ventricle than that of NLC. The pharmacodynamic studies revealed that Lf-mNLC could
518
efficiently control the progression of the AD. In conclusion, Lf–mNLC may provide a
519
flexible drug delivery platform for brain targeting.
520
522
Acknowledgments
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521
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d
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This work was supported by the Natural Science Foundation of Jiangsu Province
523
(Program No. BK20130655, No. BK2012761), the Open Project Program of State Key
524
Laboratory of Natural Medicines, China Pharmaceutical University (Program No.
525
SKLNMKF201204) and College Students Innovation Project for the R&D of Novel Drugs
526
(Program
527
entrepreneurial training program (Program No. G13076).
No.
J1030830)
and
the
National College
Students'
innovation
528 529 24
Page 24 of 38
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Ac ce p
te
d
M
an
us
cr
ip t
599
28
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ip t cr us
Fig. 1. TEM images of NLC (A), LfL-mNLC (B), LfM-mNLC (C), and LfH-mNLC (D). Bar is 100 nm.
an
599 600 601
M
602 603
Ac ce p
te
d
604
29
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ip t cr us
604
Fig. 2. UV-Vis absorption spectra (Left) and fluorescence emission spectra (Right) of Cur solution (a),
606
Cur- loaded NLC (b) and LfH-mNLC (c).
Ac ce p
te
d
M
607 608
an
605
30
Page 30 of 38
ip t cr us an
608
Fig. 3. (A) Uptake of different formulations in BCECs at a dose of 300 ng/mL of C6 at 37 °C (n = 3); Uptake
610
of NLC and LfH-mNLC in BCECs (B) for different concentrations of C6 at 37°C or 4°C (n = 3), (C) at a dose of
611
100 ng/mL of C6 at different time at 37 °C (n =3), and (D) at a dose of 100 ng/mL of C6 in the presence of
612
excess free Lf (10 mg/mL) at 37 °C (n = 3). Data are expressed as mean ± SD. # P < 0.05 compared with NLC.
613
◆
615
d
te
P < 0.05 compared with LfM-mNLC.
Ac ce p
614
M
609
31
Page 31 of 38
ip t cr us an M
615
Fig. 4. Intracellular integrity assessment of Dil and Dio loaded NLC and LfH-NLC after incubation with
617
BCECs at 37 °C between 1 h and 8 h by monitoring the FRET phenomenon using CLSM.
te Ac ce p
618 619
d
616
32
Page 32 of 38
ip t cr
te
d
M
an
us
Fig. 5. Plasma concentration–time profiles of Cur in rats after intravenous administration of Cur solution, Cur-loaded NLC and LfH–mNLC at a dose of 10 mg/kg of Cur (n=5).
Ac ce p
619 620 621 622
33
Page 33 of 38
ip t cr us an
622
Fig. 6. (A) Whole body fluorescence images of SD mouse after intravenous injection of DiR-loaded NLC
624
and LfH–mNLC (0.5 mg/kg). (B) Fluorescence images of excised brain and other organs at 24 h
625
post-injection of DiR-loaded NLC and LfH–mNLC. (C) The fluorescence intensity of DiR in brain and other
626
organs at 24 h post-injection of DiR-loaded NLC and LfH–mNLC. Data are shown as mean ± SD (n = 3).
627
P<0.05 versus NLC .
629 630
d
te
◆
Ac ce p
628
M
623
34
Page 34 of 38
ip t cr us
630
Fig.7.
The coronal slices of the mouse brain. (A) Distribution of NLC and LfH–mNLC in the third ventricle and
632
cortex. The green fluorescence signals of C6 were visualized using the FITC filter of brain sections 1 h
633
after i.v. injection. The cell nuclei were stained with 5 μg/mL DAPI for 20 min and visualized using the
634
UV filter (100×). (B) Fluorescence intensity of C6 in the third ventricle and cortex was measured and
635
analysed using Image J software. Data are shown as mean±SD (n = 3).
M
d
P<0.05 versus NLC.
te
638
◆
Ac ce p
636 637
an
631
35
Page 35 of 38
638
Table 1A
639
Effects of different amounts of S100-COOH on particle size, PI, zeta potential, and EE of Cur-loaded NLC.
640
Data were presented as the mean±SD (n=3). Particle size
S100-COOH
Zeta potential PI
(nm)
EE(%)
(mv)
(mmoL)
ip t
Added amounts of
163.4±2.8
0.17±0.017
-3.56±1.05
91.57±0.87
0.002
119.4±1.1
0.12±0.010
-6.95±0.72
0.004
113.7±2.7
0.15±0.012
0.008
105.7±1.8
0.16±0.037
0.012
92.4±0.9
0.17±0.012
-16.87±1.91
96.74±0.95
0.020
74.7±0.7
0.26±0.023
-16.94±1.82
87.65±1.30
cr
0
us
90.39±2.51
92.09±0.39
-13.90±0.43
94.05±1.50
M
an
-8.90±1.32
641 Table 1B
643
Effects of different amounts of Lf on particle size, PI, zeta potential, EE, and DL of Cur-loaded NLC
644
coated with 0.012 mmoL of S100-COOH. Data were presented as the mean±SD (n=3). Preparations
Lf (mg/mL)
te
d
642
Particle size
Zeta potential
PI
Ac ce p
(nm)
NLC
LfL–mNLC
LfM–mNLC LfH–mNLC 645
0
0.5
1.5
2.5
EE(%)
DL(%)
(mv) 96.74±0.95
92.4±0.9
0.17±0.012
-16.87±1.91
2.65±0.23
99.7±1.6
0.20±0.014
-17.90±0.64
-
-
100.9±1.4
0.17±0.020
-12.94±0.62
-
-
103.8±0.6
0.15±0.022
-5.80±0.73
96.51±1.87
2.60±0.17
-: no detection
646 647
Table 1C
648
Pharmacokinetics parameters of Cur after intravenous administration of Cur solution, Cur-loaded NLC and
649
LfH-mNLC at a dose of 10 mg/kg of Cur to rats. Data are presented as the mean±SD (n=5). Preparations
Parameters 36
Page 36 of 38
t1/2 (h)
MRT(h)
AUC0-1
Clearance (L/h)
(ng×h/mL) Cur solution NLC LfH-mNLC
117±10.2
1.31±0.06
*
296±11.6
*
1.16±0.14
*
181±14.6
*
0.41±0.04
6.45±2.63
*
11.08±0.89*
1. 84±0.03 1.24±0.06
17.7±2.00
*
*P<0.05 versus Cur Solution
ip t
650
0.07±0.04
651
Ac ce p
te
d
M
an
us
cr
652
37
Page 37 of 38
ip t
652
cr
653
us
654
Ac ce p
te
d
M
an
655
38
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