Journal Pre-proof

Transferrin-functionalized lipid nanoparticles for curcumin brain delivery

A.R. Neves, Putten L. van der, J.F. Queiroz, M. Pinheiro, S. Reis

PII: S0168-1656(21)00082-1
DOI: https://doi.org/10.1016/j.jbiotec.2021.03.010
Reference: BIOTEC 8871

To appear in: Journal of Biotechnology

Received Date: 16 January 2020

Revised Date: 25 September 2020
Accepted Date: 9 March 2021

Please cite this article as: Neves AR, van der PL, Queiroz JF, Pinheiro M, Reis S,
Transferrin-functionalized lipid nanoparticles for curcumin brain delivery, Journal of
Biotechnology (2021), doi: https://doi.org/10.1016/j.jbiotec.2021.03.010

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Transferrin-functionalized lipid nanoparticles for curcumin brain delivery
Neves A.R.1,2,*, van der Putten L.1 , Queiroz J.F. 1 , Pinheiro M.1 , Reis S.1

1 LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, 4050-313
Porto, Portugal.

2
CQM - Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9020-105 Funchal, Portugal.

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* Corresponding Author:

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Postal Address: CQM, Universidade da Madeira, Campus da Penteada, 9020 -105 Funchal, Portugal.

Phone: +351-291705121 (Office) / +351-291705149/249 (Fax)

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Electronic address: ana.neves@staff.uma.pt
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Graphical abstract
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Highlights

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  Curcumin-loaded lipid nanoparticle functionalized with transferrin was developed;
 No cytotoxicity of nanoparticles was detected in hCMEC/D3 cell line;
 Transferrin-nanoparticles enhance permeability across blood-brain barrier;
 Great potential for neuroprotection.

Abstract
Curcumin is an anti-inflammatory and antioxidant compound with potent neuroprotective activity. Due to its
poor water solubility, low bioavailability, rapid elimination and the challenges for crossing and transposing
the blood-brain barrier (BBB), solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) loaded

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with curcumin were successfully produced and functionalized with transferrin, in order to mediate the

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transport of these particles through the BBB endothelium to the brain.

The nanosystems revealed Z-averages under 200 nm, polydispersity index below 0.2 and zeta potential

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around -30 mV. Curcumin encapsulation around 65% for SLNs and 80% for NLCs was accomplished, while the
functionalized nanoparticles presented a value around 70-75%. A stability study revealed these
characteristics remained unchanged for at least 3 months. hCMEC/D3 cells viability was firstly analyzed by
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MTT and LDH assays, respectively, and a concentration of 10 µM of curcumin-loaded nanoparticles were
then selected for the subsequent permeability assay. The permeability study was conducted using transwell
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devices with hCMEC/D3 cells monolayers and a 1.5-fold higher permeation of curcumin through the BBB was
verified. Both SLNs and NLCs are promising for curcumin brain delivery, protecting the incorporated
curcumin and targeting to the brain by the addition of transferrin to the nanoparticles surface.
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Keywords: blood-brain barrier, transferrin receptors, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC),
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curcumin
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 1. Introduction
Curcumin is a natural phytochemical compound derived from the dietary spice turmeric. The spice is derived
from the rhizome of the plant Curcuma longa and is commonly used in India and China for cooking,
preservation of food, as cosmetic and as a yellow dye. This ginger family plant has been used for a long time
as medicine due to its antiseptic, analgesic, anti-inflammatory and antioxidant properties [1-3]. Some studies

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show curcumin may be the compound in turmeric responsible for its wide variety of beneficial health effects

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[4]. Curcumin has been demonstrated to protect against a host of degenerative conditions, especially the
ones that affect brain functions [5]. Curcumin seems to fight leading brain disorders, but its role in protecting
brain health is still under study. Researchers are finding that curcumin can treat anxiety and depression,

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promoting neurite growth and inducing brain plasticity [6, 7]. Curcumin has been shown to promote the
activity of brain-derived neurotropic factor (BDNF), a vital growth factor responsible for the strengthening of
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nerve networks that are involved in cognitive and memory skills [8-10]. Therefore, curcumin is one of the
compounds described as a potent agent with several brain benefits for both prevention and treatment of
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age-related brain diseases, such as Alzheimer’s, Parkinson’s, stroke, and brain cancer diseases [11-17]. These
diverse and complex conditions share an important relationship, related to the oxidative stress and
inflammation process. In fact, curcumin is a well-known anti-inflammatory and antioxidant compound and,
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consequently, has been shown to have potent neuroprotective activity [18, 19].
However, this compound was shown to exhibit extremely poor bioavailability [2, 20, 21], because of its
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hydrophobic characteristics, low water solubility and chemical instability [22-24], poor absorption, rapid
metabolism and systemic elimination [25-28], losing its therapeutic activity. At the same time, the challenges
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of crossing and transposing the blood-brain barrier (BBB) are crucial for an effective and successful brain
therapy [29-31], but curcumin uptake into the brain is severely restricted [20, 32]. Indeed, the passage of
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curcumin through the BBB is extremely limited and the traditional systems fail in delivering therapeutic
concentrations into the brain [33]. Considering this, the amount of curcumin that can actually reach the
brain is considered negligible, so it cannot work in the prevention or treatment of brain disorders.
In the last 20 years, a lot of research has been focused on the effective delivery of drugs across the BBB.
Several approaches have been developed so far applying nanotechnology for brain delivery [30, 34-37].
Among the different types of nanoparticles that have been engineered so far, lipid nanoparticles are readily
taken up by brain due to their lipophilic nature and their small size that also prolongs their circulation time in
the blood and make it more prone for transportation over the BBB [38-42]. In this context, curcumin-loaded
lipid nanoparticles have already shown promising results by decreasing curcumin clearance and enhancing
its bioavailability after oral or intravenous administration in rats [43-45]. Moreover, lipid nanoparticles can
be a tool for an effective delivery of curcumin through the BBB into the brain [45, 46].
Additionally, nanoparticles can be functionalized with ligands that can be recognized by endogenous BBB
transporters. Transferrin receptor is an example of transporter that is highly expressed in the luminal side of
brain capillary endothelium and might lead to receptor-mediated transcytosis across the BBB when activated
[29]. In this context, nanoparticles can be modified with transferrin on their surface, specifically recognizing
transferrin receptors overexpressed in the BBB and, consequently, promoting an active-targeting brain
delivery [47, 48]. This approach may also be effective in the treatment of cancer, since transferrin receptor is
related to cell proliferation and is overexpressed in malignant tissues compared to normal tissues because of

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the higher iron demand of malignant cells for their fast growth and division. Thereby, some studies have
been devoted to transferrin-functionalized nanoparticles loaded with curcumin for cancer treatment by
induction of apoptosis [49, 50]. Furthermore, there are some applications of lipid nanoparticles for curcumin

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brain delivery for the treatment of depression, cerebral ischemic injury, glioblastoma, Alzheimer’s,
Parkinson’s and Huntington’s diseases, but none addressed the issue of crossing the BBB with a transferrin
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moiety conjugated to the lipid nanoparticles [45].
Therefore, the main goal of this work was the development of transferrin-functionalized nanodelivery
systems based on lipid nanoparticles (solid lipid nanoparticles - SLNs and nanostructure lipid carriers – NLCs)
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for effectively deliver curcumin into the brain, taking advantage of curcumin’s brain benefits and health
promoting capacities.
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2. Materials and Methods

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2.1. Materials
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For nanoparticles synthesis, curcumin (≥ 94% curcuminoid content, ≥ 80% pure) was purchased from Sigma-
Aldrich (St. Louis, MO, USA), solid lipid cetyl palmitate was provided by Gattefossé (Nanterre, France), liquid
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lipid miglyol-812 by Acofarma (Terrassa, Spain) and polysorbate 60 (Tween 60®) was supplied by Sigma-
Aldrich (St. Louis, MO, USA). For nanoparticles functionalization, transferrin human (more than 98% pure),
EDC hydrochloride (more than 98% pure) and Sephadex G50 were provided by Sigma-Aldrich (St. Louis, MO,
USA) and DSPE-PEG(2000)-NH2 was purchased from Avanti Polar Lipids (Alabaster, AL, USA). For cell culture
studies, hCMEC/D3 cell line was obtained from the Institut National de la Santé et de la Recherche Médicale
(INSERM, Paris, France); Endothelial Cell Growth Basal Medium (EBM-2) was supplied by Lonza (Pontevedra,
Spain); fetal bovine serum (FBS), penicillin-streptomycin, chemically defined lipid concentrate (1/100) and
trypsin were purchased from Invitrogen, Gibco (Paisley, UK); hydrocortisone, ascorbic acid, basic fibroblast
growth factor (bFGF), phosphate buffered saline (PBS), thiazolyl blue tetrazolium bromide (MTT), triton X-
100, trypan blue, lucifer yellow and dimethyl sulfoxide (DMSO) were provided by Sigma-Aldrich (St. Louis,
MO, USA); HEPES (10 mM) was obtained from PAA The Cell Culture Company (Pasching, Austria); Cultrex®
Rat Collagen type I R&D system was purchased from Trevigen (Gaithersburg, MD, USA); while lactate
dehydrogenase (LDH) detection kit was supplied by Takara Bio Inc. (Otsu, Japan). Water from arium water
purification system (resistivity > 18 MΩ cm, Sartorius, Goettingen, Germany) was used for the preparation of
all solutions.

2.2. Production of nanoparticles

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Lipid nanoparticles can be subdivided in SLNs and NLCs. SLNs are made solely by solid lipids at room and

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body temperature, while NLCs are composed of solid and liquid lipids that confer a heterogeneous and
perfect matrix for accommodating drugs. In fact, the incorporation of a liquid lipid to the solid matrix of the
nanoparticles seems to create an imperfect matrix with an increased number of cavities in NLCs, facilitating

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the accommodation of the encapsulated compound, while preventing early crystallization and premature
drug release [51]. The nanoparticles were produced using a modified hot homogenization method [52, 53].
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Table 1. Composition of SLNs and NLCs formulations with no functionalization.
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SLN 0 SLN 2 SLN 5 SLN 10 SLN 25

Curcumin (mg/mL) 0 0.4 1 2 5

Cetyl palmitate (mg/mL) 100 99.6 99 98 95

Tween 60 (mg/mL) 20 20 20 20 20
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NLC 0 NLC 2 NLC 5 NLC 10 NLC 25

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Curcumin (mg/mL) 0 0.4 1 2 5

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Cetyl palmitate (mg/mL) 70 69.6 69 68 65

Miglyol-812 (mg/mL) 30 30 30 30 30
Tween 60 (mg/mL) 20 20 20 20 20
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According to Table 1, SLNs and NLCs were made of cetyl palmitate (solid lipid) together with tween 60
(surfactant) and curcumin, while NLCs contained also miglyol-812 (liquid lipid). All components were warmed
up at 70 °C, well above the melting point of the lipids. The molten lipids were dispersed in ultra-pure water
at the same temperature. Ultra-turrax T25 (Janke and Kunkel IKA-Labortechnik, Staufen, Germany) was used
to produce particles in the micrometer range, and ultrasonicator (Sonics and Materials Vibra-Cell™ CV18,
Newtown, CT, USA) reduced the formed microparticles to the nanometer range. Some parameters of the
production process were optimized in order to establish the best conditions for nanoparticles stability. The
final parameters chosen were 30 seconds of high sheer homogenization by ultra-turrax for SLNs and 120
seconds for NLCs, both at 12,000 rpm, followed by sonication for 5 minutes for SLNs at 70% of intensity and
15 minutes for NLCs at 80% of intensity. The nanoemulsions with or without curcumin were cooled down at
room temperature, to make it possible the lipids to crystallise and the nanoparticles to be formed. The
formulations without curcumin (SLN 0 and NLC 0) appeared white, while the ones containing curcumin
appeared between yellow (lower concentration) and orange (higher concentration). All formulations had a
low viscosity and were stored in glass and protected from light.

2.3. Functionalization of nanoparticles

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Nanoparticles were functionalized by covalently attaching transferrin to their surface using 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) as a coupling agent. A schematic representation of the
nanoparticles functionalization with transferrin is shown in Figure 1.

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Figure 1. Schematic representation of the functionalization of nanoparticles with transferrin. Transferrin and EDC were dissolved in
water, mixed together and stirred for 30 minutes at room temperature. EDC could react with the carboxylic group of transferri n to
form an active O-acylisourea, thereby activating transferrin. The primary amine group o f the DSPE-PEG-NH2 in nanoparticles then
formed a peptide bond with the carboxyl group of transferrin, releasing the EDC by -product, a soluble urea derivate.
As can be seen in Table 2, the nanoparticles were produced as described in the previous section, but
replacing 5 mg of cetyl palmitate by 5 mg of DSPE-PEG-NH2 , a phospholipid 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine (DSPE) associated to polyethylene glycol (PEG) spacer and a terminal amine (NH2 )
group.

Table 2. Composition of Tf SLNs and Tf NLCs formulations (functionalized with transferrin).

Tf SLN 0 Tf SLN 10 Tf NLC 0 Tf NLC 10

Curcumin (mg/mL) 0 2 0 2
Cetyl palmitate (mg/mL) 99 97 69 67
Miglyol-812 (mg/mL) 0 0 30 30
Tween 60 (mg/mL) 20 20 20 20

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DSPE-PEG-NH2 (mg/mL) 1 1 1 1

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Transferrin (mg/mL) 1 1 1 1

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Due to its lipophilic nature, DSPE moiety was incorporated into the nanoparticles matrix, while the
hydrophilic PEG and the terminal amine group were exposed on the surface to make it possible to bind the
protein transferrin. Therefore, a transferrin solution was prepared at 5 mg per millilitre of ultrapure water
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and the carboxyl groups of transferrin were activated adding 5-fold lower amount of EDC to the transferrin
solution, by stirring during 30 minutes at room temperature. To form transferrin-conjugated nanoparticles, 1
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mL of transferrin solution (5 mg) was added to the previously formed nanoformulations with DSPE-PEG-NH2
and allowed to react for 2 hours at room temperature. Finally, transferrin-functionalized nanoparticles were
dialyzed in a 10 kDa MWCO SnakeSkin Dialysis Tubing against 750 mL ultra-pure water at 37 °C with stirring,
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overnight, to remove the excess of transferrin and by-products. The amount of transferrin conjugated on the
surface of nanoparticles was determined with the Bradford dye assay using Coomassie blue G dye [54]. The
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absorbance at 595 nm was measured and compared with a blank containing the same sample but without
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transferrin and with the same amount of dye. Transferrin conjugation efficiency was expressed as milligrams
of transferrin per molarity of lipid nanoparticles.
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2.4. Morphology determination

The morphology of nanoparticles was characterized by transmission electron microscope (TEM) and Cryo-
Scanning Electron Microscopy (Cryo-SEM). For TEM, samples were mounted on 300 mesh form var copper
grids and stained with uranyl acetate. The Jeol JEM 1400 TEM (Jeol Ltd., Tokyo, Japan) was used to visualize
the samples, followed by their digitally recording by a Gatan SC 1000 ORIUS CCD camera (Warrendale, PA,
USA). For Cryo-SEM, samples were dropped on a grid and rapidly cooled in a liquid nitrogen slush (-210 °C).
They were transferred under vacuum to the cold stage of the preparation chamber. In this chamber the
samples were fractured and sublimated for 2 minutes at -90 °C. The sublimation made it possible to reveal a
greater detail. Thereafter the samples were coated by a gold-palladium alloy. Finally, the specimens were
moved under vacuum into the SEM chamber and were observed at -150 °C using a JEOL JSM-6301F (Tokyo,
Japan), an Oxford Instruments INCA Energy 350 (Abingdon, UK), and a Gatan Alto 2500 (Pleasanton, CA,
USA).

2.5. Dynamic light scattering

Particle size and polydispersity analysis were performed by dynamic light scattering (DLS) using a particle size
analyser (Brookhaven Instruments, Holtsville, NY, USA). The fixed light incidence angle was 90°, dust cut -off
value was 30% and the acquisition took place at 25 °C. The mean hydrodynamic diameter and the mean

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polydispersity index were determined after 10 runs and the measurements were performed in triplicate over
3 months.

2.6. Electrophoretic light scattering

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Zeta potential was measured using a zeta potential analyser (Brookhaven Instruments, Holtsville, NY, USA).
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The mean zeta potential was determined after 6 runs of 10 cycles each and measurements were also
performed in triplicate over 3 months, at 25 °C.
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2.7. Curcumin entrapment efficiency

The entrapment efficiency (EE) was determined by measuring the amount of curcumin inside the
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nanoparticles and comparing this value with the total amount of curcumin added to the formulations.

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒𝑑 𝑐𝑢𝑟𝑐𝑢𝑚𝑖𝑛

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𝐸𝐸 = 𝑥 100%
𝑡𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑢𝑟𝑐𝑢𝑚𝑖𝑛 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡ℎ𝑒 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛
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The formulations were diluted 200 times in water and transferred into Amicon® Ultra-4 centrifugal filters
(Merck Millipore, Billerica, MA, USA). The samples were centrifuged at 4300 rpm for 30 minutes at room
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temperature using an Allegra®X-15R centrifuge (Beckman Coulter, Brea, CA, USA). The nanoparticles were
retained in the filter while the free curcumin went down through the filter. The nanoparticles in the filter
were re-suspended in acetonitrile (ACN), vortexed and incubated for 15 minutes, in the dark and at room
temperature, to make possible the disruption of the nanoparticles matrix by ACN, releasing their content in
curcumin. The samples were then centrifuged at 10,200 rpm for 15 minutes at room temperature, forming a
pellet composed by lipids and a supernatant containing the curcumin that was released from the
nanoparticles. The amount of curcumin that was entrapped inside lipid nanoparticles was then quantified by
measuring the absorbance of the supernatant at 418 nm in a V-660 spectrophotometer (Jasco, Easton, MD,
USA) against a curcumin calibration curve in ACN. This allowed calculating the EE of curcumin in the lipid
nanoparticles.

2.8. hCMEC/D3 cell culture

The immortalized human cerebral microvascular endothelial cells (hCMEC/D3) is a very useful model of the
human BBB, commonly used for cellular and molecular studies on drug transportation through the BBB, due
to its characteristics such as the formation of tight junctions, presence of specific transporters and the
capacity to exclude most drugs. Moreover, this cell line can be easily grown and shows a similar morphology
as primary cultures of brain endothelial cells [55-57].
Cells were grown in EBM-2 medium supplemented with 5% FBS, 1% penicillin-streptomycin, 1.4 µM

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hydrocortisone, 5 µg/mL ascorbic acid, 1% of chemically defined lipid concentrate, 10 mM of HEPES, and 1
ng/mL of bFGF added extemporaneously in the cell culture medium. All flasks, plates and transwell devices
were pre-coated with rat collagen type I and cells were incubated at 37 °C with 5% CO2 with medium
changed every 3-4 days.

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2.8.1. Cell viability and cytotoxicity studies
The LDH assay was performed to assess cytotoxicity, while the MTT assay was used to evaluate the cell
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viability after nanoparticles exposure to hCMEM/D3 cells. The cells were seeded in 96 well plates in a
concentration of 30,000 cells per well. After 20 hours of incubation (37 °C, 5% CO2 ), the nanoparticles and
free curcumin were added in concentrations of 1, 5, 10, 25, 50 and 100 µM. During all assays, 2% triton X-
100 and EBM-2 medium were used as the positive and negative controls of cytotoxicity, respectively. After 4
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hours of incubation with samples, the supernatant was transferred to new plates and used for performing
the LDH assay, while the attached cells were used for MTT assay. Cells were then incubated with MTT
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solution at 0.5 mg/mL for 4 hours. Afterwards, the MTT solution was rejected by inversion and DMSO was
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added to solubilize the formazan crystals formed inside cells. After 15 minutes at room temperature
protected from light, the absorbance was measured at 550 nm and 690 nm. Cell viability was expressed as a
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percentage compared to the maximum viability of the EBM-2 medium, as follows:

𝐴𝑏𝑠 𝑜𝑓 𝑒𝑎𝑐ℎ 𝑠𝑎𝑚𝑝𝑙𝑒 (550 𝑛𝑚 ‒ 690 𝑛𝑚)
𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝑥 100
𝐴𝑏𝑠 𝑜𝑓 𝐸𝐵𝑀‐ 2 𝑚𝑒𝑑𝑖𝑢𝑚 (550 𝑛𝑚 ‒ 690 𝑛𝑚)

For the LDH assay, the new plates with the supernatant resulting from the incubation of cells for 4 hours
with the samples were centrifuged at 250 g for 10 minutes, at room temperature, using a Sigma 3K-2 plate
centrifuge (Sigma laboratory centrifuges GmbH, Osterode am Harz, Germany). Then, the supernatant was
again separated for further LDH quantification using the LDH detection kit (Takara Bio Inc., Otsu, Japan). The
absorption was read at 490 nm and 690 nm. Cytotoxicity was expressed as a percentage compared to the
maximum cytotoxicity of triton X-100 sample, as follows:

𝐴𝑏𝑠 𝑜𝑓 𝑒𝑎𝑐ℎ 𝑠𝑎𝑚𝑝𝑙𝑒 ( 490 𝑛𝑚 ‒ 690 𝑛𝑚) ‒ 𝐴𝑏𝑠 𝑜𝑓 𝐸𝐵𝑀‐ 2 𝑚𝑒𝑑𝑖𝑢𝑚 (490 𝑛𝑚 ‒ 690 𝑛𝑚)
𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 (%) = 𝑥 100
𝐴𝑏𝑠 𝑜𝑓 𝑡𝑟𝑖𝑡𝑜𝑛 𝑋‐ 100 ( 490 𝑛𝑚 ‒ 690 𝑛𝑚) ‒ 𝐴𝑏𝑠 𝑜𝑓 𝐸𝐵𝑀‐ 2 𝑚𝑒𝑑𝑖𝑢𝑚 (490 𝑛𝑚 ‒ 690 𝑛𝑚 )

2.8.2. Transwell permeability study

hCMEC/D3 cells were seeded (200,000 cells per well) in transwell devices (6-well polyester inserts, pore size
0.4 μm and a cell growth area of 4.67 cm2 ) pre-coated with rat collagen type I. The donor and the acceptor
compartments were filled with EBM-2 medium and cells were incubated for 7 days (37 °C, 5% CO2 ). The
Trans-Endothelial Electrical Resistance (TEER) was measured every 3-4 days, using a voltohmmeter (EVOM)

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from World Precision Instruments (Sarasota, FL, USA) to quickly and easily evaluate the integrity of the BBB

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monolayers. EBM-2 medium was also changed every 3-4 days from the inserts and the acceptor
compartments. After 7 days, the cells were confluent forming reliable BBB monolayers. The barrier integrity

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of hCMEC/D3 cell monolayers was previously checked using lucifer yellow as a small reference molecule with
a well reported effective permeability coefficient of 1.33x10-3 cm/min in agreement with the literature [57].
The curcumin-loaded nanoparticles and curcumin free samples with 10 µM concentration were incubated in
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the apical donor compartment for 4 hours at 37 °C in 5% CO 2 and the total amount of curcumin in the
acceptor compartment was quantified after 0.5, 1, 2, 3 and 4 hours by HPLC with fluorescence detection
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(426/539 nm). The TEER was read again at 0, 2 and 4 hours after incubation with samples. This allowed us to
evaluate the amount of compound in the basolateral compartment that was actually crossing the
monolayer. This value represents the cumulative amount of compound appearing in the receiver
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compartment of the assay system vs. time, while if we determine the amount of compound in the apical
compartment, this would give us the disappearance of a compound from the donor side of the biological
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barrier. However, usually not all compound disappears from the apical side and appears on the basolateral
side, being converted or metabolized into other compounds inside the cell’s monolayer. Therefore, the real
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interest in the present work was to verify if the compound was really able to cross the monolayer and if the
nanosystems could increment this permeability from the apical to the basolateral chamber, representing the
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permeability through the BBB.

2.9. HPLC
High-performance liquid chromatography (HPLC) was used to quantify the curcumin concentration in the
acceptor compartment in the transwell permeability study. The method used was adapted from the
literature [58]. A reversed-phase C18 monolithic column (Chromolith RP-18e, 100 mm × 4.6 mm i.d., Merck)
was connected to a Jasco (Easton, MD, USA) HPLC system (pump PU-2089, autosampler AS-2057, and LC-Net
II/ADC controller) coupled to a fluorometric detector (Jasco FP-2020, λexcitation = 426 nm, λemission = 539 nm).
Chromatographic separation was achieved by isocratic mode consisting of 20:40:40 (v/v/v) aqueous solution
(acetate buffer pH 4.6/ACN/H2 O) at a flow rate of 2.0 mL/min. The injection volume was 50 µL. A calibration
curve was made using 0.01-10 µM curcumin in PBS with 40% ACN.

2.10. Statistical analysis

All statistical analyses were performed using SPSS software (v 23.0; IBM, Armonk, NY, USA). The
measurements were repeated at least three times and data were expressed as mean ± sd. Data was analysed
using one-way analysis of variance (one-way ANOVA), followed by Bonferroni, Tukey and Dunnett post-hoc
tests. A P value of 0.05 was considered statistically significant.

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3. Results and Discussion
3.1. Transferrin conjugation efficiency

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The quantification of the actual amount of transferrin conjugated on the surface of the lipid nanoparticles
was achieved by the Bradford assay using Coomassie blue G dye [54]. Transferrin-functionalized
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nanoparticles were compared to the respective blanks containing the same nanoparticles but without
transferrin and with the same amount of dye (Table 3). The conjugation efficiency was found to be around
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30 mg of transferrin per molarity of lipid nanoparticles, corresponding to about 25 molecules of transferrin,

which is in agreement with literature that used the same method for transferrin conjugation [47-50]. This
showed a highly efficient conjugation of transferrin on the surface of nanoparticles, translated into a
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successful functionalization of lipid nanoparticles. No interference was observed between the transferrin
and the lipids used.
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Table 3. Transferrin conjugation efficiency in Tf SLNs and Tf NLCs formulations (values

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correspond to milligrams of protein per molarity of lipid nanoparticles).

Tf SLN 0 Tf SLN 10 Tf NLC 0 Tf NLC 10

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Non-funct. NPs (Blanks) 9.9 ± 0.1 9.2 ± 0.1 11.7 ± 0.2 12.4 ± 0.1
Tf-funct. NPs 40.7 ± 0.3 41.4 ± 0.2 40.4 ± 0.3 44.8 ± 0.3
Blanks Subtraction 30.8 ± 0.2 32.2 ± 0.1 28.7 ± 0.2 32.4 ± 0.2

3.2. Morphology determination

TEM images showed nanoparticles with spherical form and almost uniform shape (Figure 2). All
formulations seemed to have a low polydispersity with sizes below 200 nm and there was no visible
aggregation of nanoparticles. No significant differences in morphology were observed between SLNs and
NLCs, neither between nanoparticles with or without curcumin. The functionalization with transferrin also
did not influence the morphology of the nanoparticles, being the shape very similar and the average
diameter in the same scale.

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Figure 2. TEM images of non-functionalized (upper side) and transferrin-functionalized (lower side) lipid nanoparticles (SLNs and
NLCs) unloaded or loaded with curcumin. 20,000× magnification.

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The Cryo-SEM images confirmed the above mentioned TEM results (Figure 3). SLNs and NLCs were almost
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spherical and uniform in shape with smooth and round surfaces regardless their loading with curcumin or
functionalization with transferrin. The mean diameter was in the range of 100–200 nm and there was no
visible aggregation of particles.
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Figure 3. Cryo-SEM images of non-functionalized (upper side) and transferrin-functionalized (lower side) lipid nanoparticles (SLNs
and NLCs) unloaded or loaded with curcumin. 20,000× magnification.

3.3. Physicochemical characterization

Nanoparticles average size, polydispersity index, zeta potential and curcumin entrapment efficiency of all
formulations were measured within one week of preparation. The results are displayed in Table 4.

Table 4. Particles size, polydispersity index, zeta potential, curcumin entrapment efficiency and amount of curcumin entrapped

Particles Size Polydispersity Zeta potential Entrapment Curcumin

(nm) Index (mV) efficiency (%) entrapped (mg)

SLN 0 172 ± 7 0.15 ± 0.03 -33 ± 7 - -

SLN 2 163 ± 8 0.14 ± 0.01 -31 ± 4 65 ± 21 * 1.3 ± 0.4 *
SLN 5 165 ± 10 0.15 ± 0.03 -34 ± 3 48 ± 3 * 2.4 ± 0.2 *
SLN 10 157 ± 14 0.14 ± 0.05 -33 ± 5 44 ± 20 * 4.4 ± 2.0 *

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SLN 25 149 ± 4 0.15 ± 0.07 -34 ± 5 39 ± 10 * 9.7 ± 4.9 *

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NLC 0 182 ± 8 0.11 ± 0.01 -34 ± 2 - -
NLC 2 175 ± 14 0.12 ± 0.04 -33 ± 4 82 ± 15 * 1.6 ± 0.3 *

NLC 5 174 ± 11 0.13 ± 0.02 -32 ± 4 70 ± 9 * 3.5 ± 0.5 *

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NLC 10 168 ± 24 0.15 ± 0.06 -32 ± 5 44 ± 18 * 4.4 ± 1.8 *
NLC 25 170 ± 9 0.14 ± 0.03 -31 ± 3 37 ± 23 * 9.3 ± 5.7 *

Tf SLN 0 174 ± 22 0.15 ± 0.03 -31 ± 3 - -

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Tf SLN 10 162 ± 6 0.16 ± 0.03 -29 ± 5 75 ± 15 * 7.5 ± 1.5 *

Tf NLC 0 177 ± 15 0.13 ± 0.01 -22 ± 3 * - -

Tf NLC 10 183 ± 12 0.13 ± 0.01 -21 ± 2 * 69 ± 5 * 6.9 ± 0.5 *
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inside nanoparticles one week after production of all formulations.

All values represent the mean ± sd (n=3). * denotes statistically significant differences between formulations (P<0.05).
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Particle size is an important characteristic of nanoparticles that determines their fate inside the organism.
Nanoparticles with a size larger than 200 nm are easily captured by Küpffer cells or other phagocytic cells
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and this limits their biodistribution. On the other hand, nanoparticles with a size smaller than 100 nm have a
high likelihood of aggregation. This can cause clusters that can occlude blood flow and origin embolism [37].
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The average particle size obtained was below 200 nm for all nanoparticles tested (Table 4), which is
important to promote a longer circulation time and to enhance the permeability through the BBB, as already
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reported in the literature [41]. At the same time, a size below 200 nm helps bypassing the liver first pass
effect, that has been reported to be the major route of curcumin degradation [44]. The polydispersity index
is a measure of the uniformity of particle size distribution. The low values obtained (around 0.15) indicate
that nanoparticles were monodisperse, with low variability and no tendency to aggregate (Table 4) [51]. This
type of distribution is usual for lipid nanoparticles made using the high shear homogenization and ultrasound
method [51].
Another important parameter which predicts the tendency for particles to aggregate is the overall surface
charge that a particle acquires in a particular medium, being expressed by the zeta potential. This gives an
indication of the repulsive forces that are present between the particles and have direct influence on the
particle stability in suspension, their tendency to aggregate, and consequently, their size distribution [37]. If
the absolute value of zeta potential is substantially high, which is the case in the developed nanoparticles
that show a negative zeta potential around -30 mV (Table 4), one can consider they are stably dispersed with
no tendency to agglomerate [51]. After the addition of transferrin to the surface of nanoparticles, it is
natural that zeta potential becomes less negative, since transferrin is a protein with a positive net charge. In
the case of SLNs there is a slight decrease in this negative charge to values that are not statistically
significant. However, for NLCs this decrease is more accentuated, probably due to the fact that more
transferrin molecules are conjugated to the surface of these nanoparticles. This probably happens because

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in the composition of NLCs there is a liquid lipid (miglyol-812) that does not exist in the SLNs and that gives a
more heterogeneous structure to the nanoparticle forming cavities inside it. When transferrin is conjugated
to the nanoparticle, a chemical rearrangement should occur, which favors the interaction between the

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positively charged protein and the negatively charged miglyol-812, thus favoring a greater number of
molecules to conjugate to the nanoparticle surface, and giving rise to a less negative zeta potential in Tf-
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NLCs. This may also justify the slightly higher permeability of Tf-NLCs compared to Tf-SLNs (please see
section 3.5.2).
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As can be seen in Table 4, no statistically significant differences were found in size, polydispersity and zeta
potential between SLNs and NLCs with or with curcumin, suggesting that curcumin incorporation do not
have a significant impact on these parameters during the first week after production. However, curcumin
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entrapment efficiency seems to be totally dependent on the initial concentration of curcumin used to
prepare nanoformulations. Hence, the EE decreases from ca. 70-80% to ca. 35% with increasing initial
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amount of curcumin, which translates into ca. 1.5 to 9 mg of curcumin entrapped inside the nanoparticles.
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On the other hand, the functionalization of nanoparticles with transferrin did not significantly change size
and polydispersity, but seemed to slightly decrease the negative charge of nanoparticles immediately after
production. Besides that, functionalized nanoparticles seem to have a higher amount of curcumin entrapped
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(ca. 70-75%) compared to the respective non-functionalized nanoparticles (ca. 40%). This may indicate a
positive effect of transferrin on the entrapment of curcumin inside nanoparticles. To study if the
nanoformulations are stable over time, the same parameters were measured after 1, 2 and 3 months.
 3.4. Stability study over time
As shown in Figure 4, all nanoparticles seem to be stable over at least 3 months according to their size and
zeta potential. Only non-functionalized SLN 10 and SLN 25 had significantly increased their size with time
compared to the initial average size. In fact, there seems to be a small tendency for size to increase in non-
functionalized formulations containing curcumin. Nevertheless, the average particle size remained always
below 200 nm within the 3 months of stability study. Moreover, the tendency for average particle size to
increase is not visible in transferrin-functionalized nanoparticles. Over 3 months, no statistically significant
differences in size are found in any of the formulations, indicating that functionalization with transferrin
does not influence the size of the lipid nanoparticles, at least in the first 3 months after production.

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Concerning zeta potential, all non-functionalized and functionalized nanoformulations show a negative

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average zeta potential of around -30 mV (Figure 4). Even though there is a slight tendency for zeta potential
to decrease, this difference is not in any case significant (P>0.05). This would suggest that over at least 3
months the nanoformulations appear to be stable.

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Nevertheless, curcumin entrapment efficiency values are much more variable, which lead us to conclude
that we have to be cautious when using nanoformulations some time after production. As we had already
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seen, the entrapment efficiency of non-functionalized nanoparticles is lower than transferrin-functionalized
ones. However, after only one month, it seems to increase up to around 80%, regardless of the initial
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amount of curcumin (Figure 4) and this value is more similar to the initial value of functionalized
nanoparticles. This increase may be caused by the time necessary to set the equilibrium for curcumin
between in- and outside the nanoparticles and may be more visible with higher concentrations. However,
the entrapment efficiency for transferrin-functionalized nanoparticles sharply decreased during storage.
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After 3 months, only 20-40% of the initial curcumin amount is still entrapped in tf SLN 10 and tf NLC 10,
which corresponds to 2-4 mg of curcumin inside nanoparticles. This decrease over time may be due to a
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competitive interaction between transferrin and curcumin. Perhaps the transferrin on the surface of the NPs
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promotes the expulsion of the compound over time. This result shows the importance of using
functionalized-nanoparticles as fresh as possible, in order to avoid the release of curcumin in storage
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conditions.
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Figure 4. Effect of time of storage on particles size, zeta potential and curcumin entrapment efficiency for non-functionalized (left
side) and transferrin-functionalized SLNs and NLCs (right side), after 1, 2 and 3 months at room temperature and protected from
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light. Note: All data represent the mean ± sd (n=3). Statistically significant differences (*) are considered when P<0.05.

3.5. Cell studies

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3.5.1. Cell viability and cytotoxicity studies

Figure 5 shows the results of cytotoxicity study using LDH assay and cell viability assessed by MTT assay,
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after exposition of hCMEC/D3 cells to nanoformulations and free curcumin samples, with increasing
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concentrations. EBM-2 medium served as a negative control, as it is non-toxic for cells and does not have any
effect on cells membrane integrity nor cells metabolic activity. Detergent triton X-100 is included as a
positive control because it destroys cells membrane, compromising their viability and giving rise to 100%
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cytotoxicity. Regarding LDH assay results, neither functionalized (right side) nor non-functionalized
nanoparticles (left side) seem to influence cells membrane integrity up to 10 µM concentration, thereby this
concentration can be used in the subsequent in vitro validation tests using hCMEC/D3 cells without
compromising cells integrity. The results also revealed that, even for the highest concentration tested (100
µM), the toxicity does not exceed 30-40%, which shows the reasonable biocompatibility of the
nanoformulations even for high doses. Moreover, curcumin-loaded nanoparticles, both functionalized and
non-functionalized, apparently revealed slightly higher cytotoxicity than unloaded ones. To evaluate the
cytotoxicity of curcumin itself, the free compound was also tested. However, the values of cytotoxicity
obtained for the free compound are lower than for curcumin-loaded nanoparticles, which may indicate that
these nanoparticles induce a greater internalization of curcumin, also enhancing their effect on cells.

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Figure 5. Cytotoxicity and cell viability of hCMEC/D3 cells after 4 hours incubation with non-functionalized (left side) and transferrin-
functionalized nanoparticles (right side) with increasing curcumin concentration. Free curcumin sample with no particles was also
considered and is represented by the yellow bar. Note: All data represent the mean ± sd (n>3). (*) denotes statistically significant
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differences compared to EBM-2 medium (P<0.05).

The MTT assay was used to determine cells viability after exposition to nanoformulations and curcumin
compound itself. The results obtained (Figure 5) are in agreement with LDH cytotoxicity study and showed
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that metabolic activity decreased with increasing curcumin concentration. Once more, the functionalization
did not seem to have an influence on cells viability up to 100 µM. Regarding the presence of curcumin in the
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formulation, this is statistically significant from concentration equal to or greater than 25 µM and the
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difference between free curcumin and curcumin-loaded nanoparticles is also significant from 25 µM, for
both SLNs and NLCs, which may also pointing out a greater uptake of curcumin into cells and consequently a
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greater effect in their metabolism. The maximum concentration used without compromising the viability of
cells was again 10 μM for all nanoformulations tested. Therefore, this was the selected concentration for
being used in the subsequent permeability study, ensuring that nanoparticles did not affect cells integrity
and metabolic activity during the in vitro validation tests.
 3.5.2. BBB permeability study
hCMEC/D3 cell monolayer is a very reliable human BBB model, commonly used for cellular and molecular
studies on drug transportation through the BBB due to the well correlated permeability values compared to
the in vivo permeation [55, 56]. The barrier integrity of hCMEC/D3 cell monolayers was previously checked
using lucifer yellow as a reference molecule with an effective permeability coefficient of 1.33x10-3 cm/min
well reported in the literature [57]. TEER values were also monitored, being around 120 and 150 Ω. cm-2
throughout all study, suggesting that the nanoparticles did not compromise cells membrane integrity. The
permeability profile after 4 hours assay through the hCMEC/D3 monolayer of curcumin-loaded
nanoparticles, both non-functionalized and transferrin-functionalized ones, and the free compound is
depicted in Figure 6. In a first glance, one can conclude NLCs had slightly higher permeability compared to
SLNs. Moreover, the permeability profile of transferrin-functionalized compared to non-functionalized

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nanoparticles was 1.5-fold higher when compared to non-functionalized nanoparticles, for both SLNs and
NLCs and slightly higher compared to the free compound. In fact, some works report that transferrin can be
used as an active-targeting ligand specifically recognized by transferrin receptors that are highly expressed in

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the BBB, enabling the crossing of this barrier [47, 48]. Therefore, one can conclude that the developed
nanosystems are promising candidates for protecting curcumin during its transit and crossing into the brain,
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enhancing its permeation through the BBB, which might lead to improving curcumin brain delivery in vivo.
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Figure 6. Permeability profile of curcumin-loaded nanoparticles (functionalized and non-

functionalized) and free curcumin sample through hCMEC/D3 cell monolayer over 4 hours of study.
All values represent the mean with n = 3.

4. Conclusions
The rising incidence of neurological disorders has triggered the research field to search for compounds that
can be effective and safely used in the prevention and treatment of brain diseases. Due to the difficulty for
compounds to cross the BBB, drug delivery systems may be a promising solution for brain targeting and
nanotechnology can play an important role. Curcumin presents lots of beneficial effects in the human body,
and appears to have favourable neuroprotective effects. However, because of its extremely low solubility,
low bioavailability, rapid elimination and poor BBB permeability, little or no compound can effectively reach
the brain.
The aim of this work was to develop an effective drug delivery system for enhancing curcumin brain delivery
using SLNs and NLCs. Nanoparticles were successfully produced and functionalized with transferrin in order
to target the brain. The optimization of nanoparticles synthesis was accomplished by selecting the best
concentration of lipids and surfactant, by adding PEG as a stabilizer agent, and by decorating their surface

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with transferrin moieties. This allowed achieving an average size of less than 200 nm that, together with
PEG, is ideal to reduce their hepatic clearance and prolong their blood circulation time after intravenous
administration, also allowing increasing curcumin uptake into the brain by receptor-mediated transcytosis

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through transferrin recognition. The characterization and evaluation of the produced non-functionalized and
functionalized nanoparticles was conducted over time in order to get insights about nanoparticles stability in
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shelf conditions of storage prior to administration. This was achieved by monitoring different parameter s,
such as morphology, zeta potential, particle size, polydispersity index and curcumin entrapment efficiency.
Both non-functionalized and transferrin-functionalized nanoparticles presented a spherical shape with
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smooth and round surface, good negative zeta potential around -25 to 30 mV, narrow distribution of sizes
with average below 200 nm and reasonable high curcumin entrapment efficiency with concentration-
dependent effect. Functionalization with transferrin did not change nanoparticles morphology neither
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significantly influenced the average size, polydispersity index and zeta potential over a time period of three
months. Concerning curcumin entrapment efficiency, the initial encapsulation rate was higher for
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transferrin-functionalized nanoparticles compared to non-functionalized ones. However, the entrapment

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efficiency of curcumin in functionalized nanoparticles seems to decrease over time, reaching no more that
20-40% after 3 months of storage. This may be caused by steric hindrance and competition between
transferrin and curcumin molecules over time and indicates the importance of using fresh functionalized
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nanoparticles in future therapeutic applications.

Afterwards, cellular studies were conducted to validate the developed nanosystems. Cell metabolic activity
and cell membrane integrity were evaluated by performing MTT and LDH assays, respectively, using human
cerebral microvascular endothelial cells, as a human BBB model. The results revealed no significant
cytotoxicity and good biocompatibility of the nanoparticles up to 10 µM curcumin concentration, after 4
hours exposition to cells. The preliminary evaluation of nanoparticles permeation across the hCMEC/D3
monolayer was performed in order to see if the transferrin-functionalized nanosystems increased curcumin
permeability compared to the non-functionalized ones. In fact, the results showed a 1.5-fold higher
permeability of curcumin through the BBB, which gave us confirmation about the effectiveness and
beneficial effects of the functionalization of lipid nanoparticles with transferrin for curcumin brain delivery.
Therefore, the developed nanosystems seem to be effective, useful and safe controlled-release models
capable of being used for brain delivery of curcumin, potentially increasing its bioavailability, protecting the
incorporated curcumin and targeting to the brain by the addition of transferrin to the nanoparticles surface.
However, the potential of these nanosystems needs to be further explored in vivo in future publications. It is
very important to demonstrate the interactions with plasma proteins and recognition by the immune system
that nanoparticles encounter in vivo, as well as to study their biodistribution through body organs and
tissues. Additionally, since curcumin has poor water solubility and thus has to be bound to plasma proteins,

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such as lipoproteins, hemoglobin, and albumin, to assure its body distribution and cellular uptake through
the plasma membrane, it would be very interesting to evaluate the competition phenomenon between
these proteins and the nanoparticles for curcumin association in the plasma. For this purpose, in vivo assays

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would be of utmost importance. Besides that, these permeability studies reflect the passage of the
nanoparticles through the hCMEC/D3 cells monolayer, but the potential of functionalization can be even
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greater if we consider the entire process of biodistribution and pharmacokinetics. In fact, the present
functionalization may promote the enhancement of local concentration of the potential drug in the brain
site by avoiding and escaping the immune recognition system and reducing the premature clearance of the
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nanoparticles. This creates promising opportunities to continue curcumin research and further improve the
possibilities of using this compound as a potent therapeutic agent for the prevention and treatment of
neurological disorders.
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Authors Contribution Statements:

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Ana R. Neves: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Supervision;
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Validation; Visualization; Writing: original draft and review & editing

Lizzie van der Putten: Conceptualization; Data curation; Formal analysis; Investigation; Methodology;
Writing: review & editing
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Joana F. Queiroz: Data curation; Formal Analysis; Investigation; Validation; Methodology; Writing: review &
editing

Marina Pinheiro: Formal analysis; Investigation; Supervision; Validation; Visualization; Writing: review &
editing

Salette Reis: Funding acquisition; Investigation; Project administration; Resources; Supervision; Validation;
Writing: review & editing
Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

Acknowledgements
This work received financial support from the European Union (FEDER funds) and National Funds (FCT/MEC,
Fundação para a Ciência e a Tecnologia and Ministério da Educação e Ciência) under the Partnership
Agreement PT2020 UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265. ARN thanks her previous Post-Doc
grant under the project NORTE-01-0145-FEDER-000011. ARN also acknowledges ARDITI for her current Post-
Doc grant (ARDITI-CQM_2017_011-PDG) under the project M1420-01-0145-FEDER-000005-CQM+ and the
CQM strategic program PEst-OE/QUI/UI0674/2019. MP thanks FCT and POPH (Programa Operacional

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Potencial Humano) for her Post-Doc grant SFRH/BPD/99124/2013. The authors thank Dr. Daniela Silva
(CEMUP, UP) for technical assistance with Cryo-SEM and Dr. Rui Fernandes (i3S, UP) for expert help with
TEM. We are also thankful to Dr. Babette Weksler from Weill Cornell Medical College (New York, USA), Dr.

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Ignacio A. Romero from The Open University (Milton Keynes, UK) and Dr. Pierre-Olivier Couraud from
INSERM (Paris, France) for their support with the hCMEC/D3 cell culture.
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Disclosure
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The authors declare no conflicts of interest in this work.

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