Gallate-induced nanoparticle uptake by tumor cells: Structure-activity relationships

Article abstract of DOI:10.1016/j.colsurfb.2019.03.048

How nanoparticles interact with biological systems determines whether they can be used in theranostic applications. It has been demonstrated that tea catechins, may enhance interactions of magnetic nanoparticles (MNPs) with tumor cells and the subsequent cellular internalization of MNPs. As part of the chemical structure of the major tea catechins, gallates are found in a variety of plants and thus food components. We asked whether the structure of gallate might act as a pharmacophore in the enhancement of the effects of MNP-cell interactions. Uptake of dextran-coated MNPs by glioma cells and cell-associated MNPs (MNP_cell) were respectively analyzed by confocal microscopy and a colorimetric iron assay. Co-incubation of MNPs and gallates, such as gallic acid and methyl gallate, induced a concentration-dependent increase in MNP_cell, which was associated with co-localization of internalized MNPs and lysosomes. An analysis of the structure-activity relationship (SAR) revealed that the galloyl moiety exerted the most prominent enhancement effects on MNP_cell which was further potentiated by the application of magnetic force; catechol coupled with a conjugated carboxylic acid side chain displayed comparable effects to gallate. Blockade or reduction in the number of hydroxyl groups rendered these compounds less effective, but without inducing cytotoxicity. The SAR results suggest that neighboring hydroxyl groups on the aromatic ring form an essential scaffold for the uptake effects; a similar SAR on antioxidant activities was also observed using a free radical-scavenging method. The results provide pivotal information for theranostic applications of gallates by facilitating nanoparticle-cell interactions and nanoparticle internalization by tumor cells.

Full text of DOI:10.1016/j.colsurfb.2019.03.048

Accepted Manuscript
Title: Gallate-Induced Nanoparticle Uptake by Tumor Cells:
Structure-Activity Relationships
Authors: Min-Chi Cheng, Yi-Ching Lu, Jender Wu,
Yunn-Hwa Ma
PII:
S0927-7765(19)30197-3
DOI:
Reference:
https://doi.org/10.1016/j.colsurfb.2019.03.048
COLSUB 10098
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date:
Revised date:
Accepted date:
18 September 2018
15 March 2019
21 March 2019
Please cite this article as: Cheng M-Chi, Lu Y-Ching, Wu J, Ma Y-Hwa, Gallate-Induced
Nanoparticle Uptake by Tumor Cells: Structure-Activity Relationships, Colloids and
Surfaces B: Biointerfaces (2019), https://doi.org/10.1016/j.colsurfb.2019.03.048
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Gallate-Induced Nanoparticle Uptake by Tumor Cells:
Structure-Activity Relationships
Min-Chi Cheng,1* Yi-Ching Lu,2* Jender Wu,1# Yunn-Hwa Ma2,3#
1School of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031,
Taiwan, ROC
2Department of Physiology and Pharmacology & Healthy Aging Research Center, Chang
Gung University, Guishan, Taoyuan 33302, Taiwan, ROC
3Department of Neurology, Chang Gung Memorial Hospital, Guishan, Taoyuan 33305,
Taiwan, ROC
Running title: Gallates enhance MNP-cell interactions
* Both authors contributed equally to this work.
# Correspondence and requests for reprints should be addressed to:
Yunn-Hwa Ma yhma@mail.cgu.edu.tw or
Jender Wu jd0332@tmu.edu.tw

Graphical abstract
Highlights


Co-incubation of gallates and MNPs enhanced MNP internalization by tumor cells.
The galloyl moiety may be the pharmacophore of MNP-cell interaction enhancement.
1




Two adjacent hydroxyl groups on the benzene ring are crucial for the enhancement.
Methyl gallate and gallamide exerted most potent effects among derivatives studied.
Magnetic force and gallates exerted a synergetic effect on cellular uptake of MNPs.
Abstract
How nanoparticles interact with biological systems determines whether they can be used in
theranostic applications. It has been demonstrated that tea catechins, may enhance interactions
of magnetic nanoparticles (MNPs) with tumor cells and the subsequent cellular internalization
of MNPs. As part of the chemical structure of the major tea catechins, gallates are found in a
variety of plants and thus food components. We asked whether the structure of gallate might
act as a pharmacophore in the enhancement of the effects of MNP-cell interactions. Uptake of
dextran-coated MNPs by glioma cells and cell-associated MNPs (MNPcell) were respectively
analyzed by confocal microscopy and a colorimetric iron assay. Co-incubation of MNPs and
gallates, such as gallic acid and methyl gallate, induced a concentration-dependent increase in
MNPcell, which was associated with co-localization of internalized MNPs and lysosomes. An
analysis of the structure-activity relationship (SAR) revealed that the galloyl moiety exerted
the most prominent enhancement effects on MNPcell which was further potentiated by the
application of magnetic force; catechol coupled with a conjugated carboxylic acid side chain
displayed comparable effects to gallate. Blockade or reduction in the number of hydroxyl
groups rendered these compounds less effective, but without inducing cytotoxicity. The SAR
results suggest that neighboring hydroxyl groups on the aromatic ring form an essential
scaffold for the uptake effects; a similar SAR on antioxidant activities was also observed using
a free radical-scavenging method. The results provide pivotal information for theranostic
2

applications of gallates by facilitating nanoparticle-cell interactions and nanoparticle
internalization by tumor cells.
Key words: Gallic acid; Magnetic nanoparticle; Structure-activity relationship; Polyphenols
3

1. Introduction
Over the past few decades, intensive investigations on nanoparticles have brought about
great potential applications ranging from chemistry, physics, and biotechnology to medicine.
Biomedical applications of nanoparticles, particularly in the field of drug delivery, have
garnered much attention and have been extensively studied [1-4]. Among various nanoparticles,
magnetic nanoparticles (MNPs) are one of the most established drug delivery carriers. MNPs
have a myriad of potential applications, including drug delivery for targeted therapy [4,5], gene
delivery [2], contrast agents in magnetic resonance imaging [4-6], magnetic hyperthermia
[3,5,7], biosensors[8], etc. MNPs are composed of a magnetite (Fe3O4) or maghemite (γ-Fe2O3)
core and a polymeric coating, which may be designed to increase the particle stability/water
dispersibility in physiological conditions and targeting effects in vivo [3,9].
Superparamagnetism, a unique characteristic of MNPs, allows MNPs to rapidly acquire or lose
magnetic responsiveness in response to the presence or absence of an external magnetic field,
respectively, thus achieving magneto-guidance.
In order to exert the intended effects of the active components, carriers have to get close
to the target site and/or interact with the target on the cell membrane or inside the cell.
Therefore, the efficiency of cellular uptake and membrane adsorption is critical and may affect
the outcome of the therapy or diagnosis. In general, cellular uptake of nanoparticles is
dependent on certain characteristics of the particles, including the size, shape, functional
groups on the surface, etc. [9-11]. An external magnetic force may guide MNPs to a desired
target site, improve accumulation in target tissues, and enhance their cellular uptake [12,13].
Magnetic targeting techniques were demonstrated to be feasible in preclinical studies for
treating gliomas and breast cancer [5,14]. The surface charge is another factor that may
modulate particle-cell interactions. It is generally considered that positively charged
nanoparticles tend to interact with negatively charged components on cell membranes; such
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electrostatic attraction facilitates more efficient internalization of cationic nanoparticles than
neutral or negatively charged nanoparticles [10,15]. Many synthetic and natural polymers, such
as dextran, have also been employed to modify the surface of MNPs to improve the diagnostic
and therapeutic efficacies [3,6].
Phenolic acids are naturally occurring compounds in plants and are widely found in fruits,
vegetables, and beverages. Phenolic acids are non-flavonoid polyphenolic compounds that can
be further divided into two major groups, hydroxybenzoic acids and hydroxycinnamic acids
[16]. Chemically, they are composed of at least one aromatic ring with one or more hydroxyl
groups [17]. Gallic acid, which belongs to the hydroxybenzoic acid type, is one of the most
important phenolic compounds. Gallic acid was proven to be effective against oxidative
damage induced by superoxide, hydrogen peroxide, peroxyl, and non-radicals, which are often
encountered in biological systems [18]. Due to their antioxidative properties, gallic acid and
its ester derivatives have been used as additives or excipients in the pharmaceutical, cosmetic,
and food industries [18,19]. Moreover, gallic acid and related derivatives were reported to
exhibit a diverse range of bioactivities, such as antineoplastic, antimicrobial, anti-inflammatory
[20], neuroprotective [18], chemopreventive, and lipid-regulatory effects [18,20]. Due to their
inherent antioxidative ability, phenolic compounds are adopted as eco-friendly reducing and
stabilizing agents in nanoparticle preparations. Gallic acid had been used to prepare silver and
selenium nanoparticles [21-23]. Furthermore, gallic acid‒nanoparticle conjugates showed
diverse bioactivities. For example, gallic acid grafted onto chitosan inhibited linoleic acid
autoxidation better than did free gallic acid [24]. Silica nanoparticles with gallic acid covalently
bonded to the surface exerted antioxidant and free radical-scavenging effects [25]; MNPs
functionalized with gallic acid also exhibit antioxidant and antimicrobial activities with good
biocompatibility [26,27].
In our previous studies, we found that epigallocatechin-3-gallate (EGCG), the major
catechin in green tea, can be adsorbed onto MNPs, thereby modifying the surface properties of
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MNPs, enhancing MNP-cell interactions, and subsequently enhancing MNP uptake by glioma
cells [28,29]. Other catechins, such as epicatechin and epigallocatecin, were also demonstrated
to exert similar enhancing effects; it was therefore deduced that the catechin backbone structure
may participate in facilitating MNP-cell interactions [28]. In many studies, the trihydroxyl
structure on the B and D rings of EGCG were thought to contribute to the antioxidative activity
and also many other biochemical activities [30]. A molecular dynamic study indicated that the
gallate ester moiety in EGCG critically affected the affinity toward lipid bilayers [31].
Moreover, the galloyl moiety was previously found to be an anchor group that showed high
binding affinity and strong absorption onto inorganic materials [32]. Hence, galloyl-containing
compounds, such as gallic acid, are commonly employed as linker molecules to crosslink
functional groups and nanoparticles, and those nanomaterials showed improved uptake
efficiency compared to unmodified materials [33]; multivalent polygalloyl constructs were also
reported to bind to the surface of intestinal cells [34,35]. It is anticipated that the galloyl moiety
may play an important role in many biological interactions. Gallic acid and its derivatives have
also been tested as chemotherapeutic agents and used in the construction of nanocomposites
for targeted delivery and such nanocomposites were demonstrated high cellular uptake [36-
38]; however, whether gallates facilitate the cellular uptake of nanoparticles has not been
studied. Therefore, in this study, we hypothesized that the trihydroxy benzene structure may
act as a pharmacophore in enhancing the cellular uptake of MNPs. Effects of different phenolic
acid derivatives on MNP-cell interactions were studied.
2. Experimental Section
2.1. Materials Dextran-coated MNPs (nanomag®-D COOH; 250 nm) were purchased from
micromodPartikel-technologie (Rostock, Germany); green fluorescent carboxymethyl-
dextran-coated MNPs (nano-screen MAG-CMX; 200 nm) was purchased from Chemicell
6

(Berlin, Germany). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential media
(MEM), and trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY).
Penicillin/streptomycin/amphotericin B was purchased from Upstate (Lake Placid, NY). Fetal
bovine serum (FBS), ammonium persulfate, potassium thiocyanate, caffeic acid, ferulic acid,
hydrochloric acid, paraformaldehyde, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Cell
Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich (St. Louis, MO). Lysotracker®
and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA).
Trimethoxybenzoic acid, protocatechuic acid, and α-resorcylic acid were purchased from TCI
(Tokyo, Japan). Gallic acid monohydrate was from Janssen Chimica (Beerse, Belgium);
gallamide (3,4,5-trihydroxybenzamide) was from Alfa Aesar (Heysham, Lancashire, UK); p-
hydroxybenzoic acid was purchased from Merck (Darmstadt, Germany).
2.2. Synthesis Unless otherwise specified, all chemicals were of reagent grade and were
used as received without further purification. The following compounds were synthesized and
analyzed using a Bruker Fourier 300 NMR spectrometer or Bruker Avance DRX 500 NMR
spectrometer (Bruker, Billerica, MA).
2.2.1 Methyl gallate (2) To a stirred suspension of gallic acid monohydrate (4.09 g, 21.7 mmol)
in methanol (16 mL) at 0°C, thionyl chloride (2 mL, 27.6 mmol) was added dropwise. The
mixture was heated under reflux for 3.5 h. The reaction mixture was then poured into a crushed
ice bath. The resulting precipitate was filtered and washed thoroughly with water. The crude
product was recrystallized from n-hexane-ethyl acetate to provide methyl gallate as a white
crystal, mp 199~201°C [39]. 1H NMR (300 MHz, CD3OD) δ (ppm) 3.82 (s, 3H), 7.04 (s, 2H);
13C NMR (125 MHz, CD3OD) δ(ppm) 52.4, 110.2, 121.6, 139.9, 146.7, 169.2 (Figs. S1, S2).
2.2.2. Propyl gallate (3). To a stirred suspension of gallic acid monohydrate (0.30 g, 1.6 mmol)
in 1-propanol (5 mL), thionyl chloride (0.8 mL, 11 mmol) was added dropwise. The mixture
was heated under reflux for 5.5 h. The reaction mixture was poured into a crushed ice bath and
7

extracted three times with 70 mL of ethyl acetate. The combined organic layer was washed
with water several times, dried over MgSO4, and evaporated under reduced pressure. The crude
product was recrystallized from aqueous ethanol to provide propyl gallate as a white crystal,
mp 145~148°C. 1H NMR (300 MHz, CD3OD) δ(ppm) 1.03 (t, J = 7.5 Hz, 3H), 1.76 (m, 2H),
4.18 (t, J = 6.5 Hz, 2H), 7.05 (s, 2H)[40]; 13C NMR (125 MHz, CD3OD) δ(ppm) 11.0, 23.4,
67.4, 110.2, 121.9, 139.9, 146.6, 168.8 (Figs. S3, S4).
2.2.3. Dihydrocaffeic acid (10). In a hydrogenation bottle was placed 51.5 mg of 5% Pd/C
and a solution of caffeic acid (0.5047 g, 2.8 mmol) in methanol (16 mL). Hydrogenation was
performed in a Parr hydrogenation apparatus at a pressure of 26~35 psi for 3.5 h. The Pd/C
was filtered off through a pad of celite, and the filtrate was evaporated under reduced pressure.
The crude product was recrystallized from water to provide dihydrocaffeic acid as an off-white
crystal, mp 138.5~140°C[41]. 1H NMR (300 MHz, CD3OD) δ(ppm) 2.51 (t, J = 7.8 Hz, 2H),
2.76 (t, J = 7.8 Hz, 2H), 6.52 (dd, J = 7.8, 2.2 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 6.66 (d, J =
13
7.8 Hz, 1H); C NMR (125 MHz, CD3OD) δ(ppm) 31.7, 37.4, 116.5, 116.6, 120.6, 133.9,
144.7, 146.3, 177.2 (Figs. S5, S6).
2.3. Cell culture LN-229 cells, human glioma cells, were cultured in DMEM; whereas
HeLa cells, human cervical cancer cells, were cultured in MEM. Both media were
supplemented with 10% FBS and a 1% penicillin/streptomycin/amphotericin B solution. Cells
were maintained in a 37°C incubator supplied with 5% CO2 and subcultured every 3~4 days.
2.4. Determination of cell-associated MNPs (MNPscell) LN-229 or HeLa cells were
seeded and grown in 24-well plates. After attaining 80%~90% confluence, cells were exposed
to MNPs (100 μg/mL; 26.32 μg/cm2) and phenolic compounds (10 or 30 μM) in the presence
or absence of an NdFeB magnet underneath the plate for 24 h. In some experiments, cells were
equilibrated at 4°C for 20 min, followed by an additional 2 h incubation with MNPs at 4°C in
the absence and presence of the magnet. A home-made magnetic plate with 24 pieces of a
cylindrical NdFeB magnet were used to provide a magnetic field of 3.4 kG at the center of
8

each well [12]. For the group without a magnet during incubation with MNPs, the magnetic
plate was placed underneath the culture plates for 5 min after MNP administration to ensure
MNP sedimentation at the beginning of incubation. Cells were then washed twice with
phosphate-buffered saline (PBS) and trypsinized prior to colorimetric quantification. The cell
pellet containing MNPcell was treated with 10% hydrochloric acid at 55°C for 4 h, followed by
the addition of an ammonium persulfate (1 mg/mL) and potassium thiocyanate solution (1 M).
The amount of cell-associated iron was determined with a VICTOR3 Multilabel Plate Reader
(PerkinElmer, Waltham, MA) at OD490. A calibration curve was prepared under identical
conditions.
2.5. Confocal microscopy LN-229 cells were seeded onto poly-lysine-coated coverslips 18
h before the experiments. Cells were incubated with green fluorescent MNPs with a
carboxylmethyl-dextran coating (nano-screenMAG-CMX; 100 µg/mL; 26.32 μg/cm2) and
methyl gallate (30 µM) at 37 °C for 2 h in the presence of an NdFeB magnet underneath the
culture plate. Cells were then washed twice with PBS and then incubated with Lysotracker®
(0.125 µM) for 30 min to stain lysosomes. Cells were than washed with PBS and fixed with
4% paraformaldehyde for 20 min at room temperature. Cells were counterstained with DAPI
and imaged with a Zeiss LSM 510 Meta laser confocal microscope system (Carl Zeiss, Jena,
Germany) equipped with a 100×/1.4 oil immersion objective lens. Image processing and
analysis of fluorescent MNPs colocalized with cells were performed using Fiji Image J.
2.6. Antioxidant activity assay A stable and commercially available organic nitrogen
radical reagent, DPPH, was used to determine the scavenging activity of gallate derivatives.
Briefly, 6 µL of 2% DMSO containing 0.1, 1, or 5 mM of gallate derivatives was mixed with
194 µL of DPPH in ethanol (0.1 mM) to a final gallate derivative concentration of 3, 30, or
150 µM, respectively. Samples were reacted at room temperature for 20 min, followed by
9

determination of OD517.The free radical-scavenging activity was calculated by subtracting the
decrease of DPPH absorption induced by gallate compounds to that of the vehicle control.
2.7. Cellular toxicity assay The cytotoxicities of gallate compounds toward LN-229 cells
were measured using a CCK-8 kit according to the manufacturer’s instructions. Briefly, LN-
229 cells were cultured in a 24-well plate to 80%~90% confluence before incubation with
gallate compounds (10 or 30 µM) or in combination with MNPs (100 µg/mL; 26.32 μg/cm2).
After administration of MNPs, a magnet was placed underneath for 5 min or 2 h. Then, cells
were washed with PBS and incubated with medium containing a 10% CCK-8 solution for
additional 1 h. The absorbance of each sample at 450 nm (OD450) was determined with a
microplate reader (VICTOR3 Multilabel Plate Reader, PerkinElmer, Waltham, MA). The
percentage of viability was calculated as follows: (viable cells) % = (OD of the drug-treated
sample / OD of the untreated sample) × 100.
2.8. Statistical analysis Results are expressed as the mean ± standard error (SE). Statistical
evaluation of the data was performed with Student's t-test for simple comparisons between two
values when appropriate. For multiple comparisons, results were analyzed by a 2-way analysis
of variance (ANOVA) followed by Duncan's post-hoc test. A value of p<0.05 was considered
statistically significant.
3. Results
3.1. Gallates enhanced tumor cell-MNP interactions
Based on our previous study that EGCG exerted an enhancing effect on MNP uptake,
effects of gallic acid and methyl gallate on MNPcell were measured to determine whether the
gallate moiety of EGCG may be the key structure that enhances MNP internalization. Figure 1
depicts that both gallic acid (Fig. 1A, B) and methyl gallate (Fig. 1C, D) induced concentration-
dependent increments in cell-associated MNPs (MNPcell) with or without a magnet underneath
the cells, whereas the magnet significantly enhanced MNPcell in all conditions studied.
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Compared to the vehicle group, gallic acid at a concentration of as low as 6 μM induced a
significant increase in LN-229 cell-associated MNPs both with and without the magnet; at 50
μM, gallic acid increased MNPcell by 5.4- and 3.9-fold in the absence and presence of the
magnet, respectively (Fig. 1A). Similar effects were observed in HeLa cells, where gallic acid
at 10 μM increased MNPcell by 1.8- and 1.9-fold in the absence and presence of the magnet,
respectively (Fig. 1B).
Methyl gallate, a methyl ester of gallic acid, also enhanced MNPcell in a concentration-
dependent manner both with and without the magnet (Fig. 1C). In LN-229 cells, methyl gallate
increased MNPcell by 1.4-fold at 10 μM and further increased MNPcell by 4.5-fold at 50 μM in
the absence of the magnet (Fig. 1C). With the magnet underneath LN-229 cells, methyl gallate
increased MNPcell by 2.1-fold at 10 μM and 2.9-fold at 50 μM (Fig. 1C). Similar synergetic
effects were observed in HeLa cells, where methyl gallate at 10 μM induced a 0.6- vs. 1.0-fold
increase of MNPcell in the absence and presence of the magnet (Fig. 1D), respectively.
3.2. Methyl gallate enhanced MNP internalization
To determine whether gallate compound-induced increases in MNPcell were associated
with increased MNP internalization, green fluorescent CMX-MNPs were incubated with LN-
229 cells in the presence of methyl gallate. Figure 2 depicts the representative distribution of
MNPcell relative to those of lysosomes and nuclei. Compared to the vehicle (Fig. 2A), methyl
gallate (30 µM; 2 h) greatly increased the internalization of MNPs (Fig. 2B). Green fluorescent
signals were distributed outside nuclei in blue, suggesting that MNPs were internalized in the
cytoplasm but not in nuclei. In addition, co-localization of internalized MNPs with lysosomes
was observed in the cytoplasm (Fig. 2B), suggesting that MNP internalization was
predominantly mediated by endocytosis. The overlap coefficient, Mander’s coefficients, of
MNPs relative to lysosomes were 0.51 ± 0.08 (n = 6) and 0.65 ± 0.03 (n = 8) in the absence
and presence of methyl gallate, respectively, suggesting an increase in MNPs transported into
lysosomes in the cells.
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Furthermore, incubation of gallates and MNPs with LN-229 cells at 4°C significantly
reduced MNPcell in all groups studied, suggesting that endocytosis was inhibited by a low
temperature (Fig. 2C). Compared to 37°C, MNP uptake at 4°C reduced to 42% and 43% in the
absence and presence of the magnet, respectively. At 4°C, gallic acid or methyl gallate
enhanced MNPcell by 1.9- or 1.4-fold without the magnet and 0.6- or 0.4-fold with the magnet,
respectively. These results suggested that the magnetic force- and gallate compound-induced
increases in MNPcell might not only occur by enhancing MNP internalization but also by MNP-
cell interactions.
3.3. Structural features of the galloyl moiety in relation to uptake enhancement
We surmised that gallic acid (1) may be a pharmacophore in the enhancement effect of
nanoparticle-cell interactions, and related gallate derivatives (2~4, 8) and congeners (5~7, 9~11)
were selected to evaluate the effects on MNPcell (Fig. 3). LN-229 cells were incubated with
compounds 1~11 at 10 or 30 μM with MNPs for 24 h followed by a quantitative analysis of
MNPcell (Fig. 4). Compared to the corresponding vehicle group, gallic acid (1) and derivatives
with a modified side chain (2~4) at 10 μM enhanced MNPcell by 1.9~4.0-fold in Mag (-) and
2.5~3.9-fold in Mag (+), respectively (Fig. 4A). At 30 μM, gallic acid and its derivatives (1~4)
increased MNPcell by 2.8~9.5-fold in Mag (-) and 2.9~5.0-fold in Mag (+), respectively (Fig.
4C).
To determine whether the locations and numbers of hydroxyl groups on the benzene ring
of gallates may be pivotal in the enhancing effects on MNPcell, a gallate derivative (8) and three
congeners (5~7) were tested. Compared to gallic acid, protocatechuic acid (5) reduced MNPcell
to 35%~36% and 55%~56%, respectively, in the absence and presence of the magnet at the
concentrations studied (Fig. 4A, C). Furthermore, p-hydroxybenzoic acid (6), α-resorcylic acid
(7), and trimethoxybenzoic acid (8) exerted no effects on MNPcell regardless of the absence or
presence of the magnet.
12

In addition to benzoic acid-based phenols, we also examined cinnamic acid-based phenol
compounds (9~11) to gain insights into molecular features associated with the enhancing effect.
At 10 µM, caffeic acid (9) enhanced MNPcell by 1.5- and 2.3-fold without and with the magnet
(Fig. 4B), respectively. At 30 µM, caffeic acid produced an approximately 4-fold MNPcell
enhancement either with or without the magnet (Fig. 4D), suggesting that caffeic acid (9)
exerted a concentration-dependent increase in MNPcell with or without the magnet. Compared
to caffeic acid (9), dihydrocaffeic acid (10) reduced MNPcell to 53%~72% and 70%~71% of
that of caffeic acid in the absence and presence of the magnet (Fig. 4B, D), respectively. In
addition, ferulic acid (11) exerted no effect on MNPcell in all condition studied compared to the
vehicle (v) groups (Fig. 4B, D).
3.4 Antioxidant activity
Gallic acid and its derivatives are often employed as antioxidants in food [42,43], therefore,
we asked whether the pattern of the enhancement effect of these compounds on MNPcell might
be associated with their antioxidant activities by determining their free radical-scavenging
activity. Figure 5 demonstrates antioxidant activities of compounds 1~11 according to a DPPH-
scavenging assay. Gallic acid (1), methyl gallate (2), propyl gallate (3), and gallamide (4)
exerted similar concentration-dependent antioxidant activities by scavenging 19%~26% of free
radicals at 0.1 mM and 74%~80% of free radicals at 5 mM. Compared to gallic acid and related
derivatives with similar structures (1~4), activities of protocatechuic acid (5) were reduced to
0.4~0.6-fold at the concentrations studied. In addition, p-hydroxybenzoic acid (6), α-resorcylic
acid (7), and trimethoxybenzoic acid (8) exhibited no scavenging efficiency against DPPH
(3%~7%) at all concentrations studied (Fig. 5A). Scavenging efficiencies of cinnamic acid-
based phenols, such as caffeic acid (9) and dihydrocaffeic acid (10), were less than those of the
gallic derivatives (1~4) described above. The scavenging efficiencies against DPPH of caffeic
acid (9) and dihydrocaffeic acid (10) were 10.9% ± 0.88% and 17.3% ± 0.46% at 0.1 mM,
13

whereas the efficiencies rose to 60.4% ± 3.52% and 73.1% ± 2.64% at 5 mM, respectively.
Ferulic acid (11) exerted lower antioxidant activity at most concentrations studied (Fig. 5B).
3.5. Cell Viability
In order to determine whether variations in MNPcell were due to cellular toxicity induced
by some gallate derivatives, a cell viability assay was conducted on LN-229 cells exposed to
these compounds. After a 2-h incubation with gallate derivatives, no changes in cell viability
were observed even at 30 µM of gallates in the absence (Fig. 6A) or presence (Fig. 6B) of
MNPs. These results implied that the concentrations of these compounds used in this study
may have been below the threshold for inducing cytotoxicity.
4. Discussion
Our previous study demonstrated equivalent enhancement of tea catechins on MNP
uptake by glioma cells, suggesting that the galloyl or catechol moiety may play a critical role
in the enhancement of MNP-cell interactions [28]. In this study, eleven phenolic acids with
varying number and position of phenol groups were prepared and tested for their MNP uptake
enhancement activities by a colorimetric iron assay. The results demonstrated that galloyl and
catechol functionality are vital for the enhancement of cellular uptake of MNPs, and
methylation of galloyl moiety to produce trimethoxybenzoic acid, aiming at the blockade of
hydrogen bonds, greatly reduced MNP-cell interactions, suggesting the importance of the free
phenolic hydroxyl components in the structure. This finding is consistent with a previous report
that the galloyl moiety may play an important role in many of the biological activities of
catechins and other polyphenol compounds, including antiproliferative effects and radical-
scavenging activities [42].
Previous studies also demonstrated that the catecholic functionality of 3,4-
dihydroxyphenylalanine (DOPA) or dopamine in marine mussels may afford adhesive and
14

anchoring properties, which can bind strongly to various inorganic and organic surfaces or
increase absorption, binding, and adhesion capabilities to different surfaces and tissues [44,45].
Since gallic acid and dopamine share a similar hydroxyl groups substituted benzene structure,
it is anticipated that both galloyl and catechol may possess similar properties and mechanisms
toward the cell membrane adhesion by formation of non-specific hydrogen bonds with polar
groups present on cell membranes. Furthermore, galloyl and catechol may be oxidized to form
o-quinones, which can undergo Michael addition or Schiff base reaction with nucleophilic
groups, and subsequently react with amino and thiol groups of polysaccharides and proteins
on cell membranes [46]. In addition, high affinity between the galloyl moiety of catechins and
phosphatidylcholine on cell membranes by cation-π interactions was reported [47]. These
combined interactions may give rise to the enhancing effects on MNP-cell interactions of
gallate derivatives.
The uptake of MNPs was greatly attenuated at 4°C (Fig. 2), suggesting involvement of
endocytosis in the enhancement effects of gallates at 37°C. In addition, the synergetic effects
of gallates and the magnet observed in the current study are consistent with previous findings
with EGCG [28]. Our results support a role of energy-dependent endocytosis in the synergetic
effects. The enhanced MNPcell observed in LN-229 and HeLa cells suggest that such effects of
gallates may occur in various cancer cells.
According to the SAR results, ester and amide derivatives of gallic acid exerted notable
enhancement effects among the tested compounds. Gallic acid (1), methyl gallate (2), propyl
gallate (3), and gallamide (4) exerted similar concentration-dependent effects on MNP
internalization and antioxidant activity. However, replacing the trihydroxyl groups with
methoxy groups, i.e., trimethoxybenzoic acid (8), rendered the compound nearly inactive,
suggesting that the galloyl moiety is crucial. In addition, protocatechuic acid (5), with one
hydroxyl group at the meta-position removed from the benzene ring of gallic acid, exhibited
significantly attenuated effects on MNPcell and also the antioxidant activity compared to gallic
15

acid and related derivatives (1~4). Mono-hydroxyl substitution at the para-position (6) or di-
hydroxyl substitution at the meta-position (7) of the carboxylic group also resulted in complete
loss of both activities studied under all conditions (Figs. 4, 5). These results suggest that
radical-scavenging activities and enhancement effects depend on the number and also the
distribution of hydroxyl groups on the aromatic rings [42]. Antioxidant activities of phenolics
are often estimated by bond dissociation enthalpy (BDE). Lower BDE value indicates a higher
phenolic hydrogen atom donating ability by cleaving the O–H bond, and might associated with
the easier formation of quinone [48]. The formed quinone may react with the nucleophilic
groups on cell membrane and thus increase the interaction [46]. More hydroxyl groups
substituted on the aromatic ring tend to decrease the O–H BDEs. The BDE values of tested
phenolics in ascending order are propyl gallate (69.6 kcal/mol), gallic acid (70.2), protochuic
acid (75.5), and p-hydroxybenzoic acid (84.7) [49]. This tendency is correlated to the uptake
enhancement effects. In contrast, N-acetylcysteine, a commonly used antioxidant, did not show
the uptake enhancement effect [28] due to a distinct antioxidant mechanism.
It was noted that protocatechuic acid (5) still increased MNPcell with the magnet,
suggesting that replacement of the galloyl moiety with the catechol structure on benzoic acid
may still exert a partial enhancement effect on MNPcell in the presence of the magnet. Therefore,
phenolic acids with a catechol structure are worthy of further evaluation. The cinnamic acid-
based phenol compounds, caffeic acid (9) and dihydrocaffeic acid (10), which have a
conjugated carboxylic acid and catechol moiety, also exhibited concentration-dependent
enhancing effects (Fig. 4B, D). At 10 µM, caffeic acid (9) enhanced MNPcell in the presence
but not in the absence of the magnet, suggesting that caffeic acid (9) may exert a synergetic
effect similar to protocatechuic acid (5) due to the catechol structure. Dihydrocaffeic acid (10),
in which the conjugated double bond at the side chain of caffeic acid is replaced with a single
bond, hindered the interaction of MNPs with cells compared to caffeic acid (9). Ferulic acid
(11), with methylation of the meta-hydroxyl group on caffeic acid, showed no uptake
16

enhancement effect at both concentrations studied compared to the vehicle (v) groups (Fig. 4B,
D). These results suggested that at least two adjacent hydroxyl groups on the benzene ring,
such as the catechol structure, are essential for the enhancement of MNPcell. Additionally, the
conjugated double bond on the side chain may facilitate such an effect.
In the current study, gallate derivatives (2~4) showed significantly higher internalization
effects than did the parent compound (1), suggesting that the side chain of gallate may modulate
the enhancing effect of gallates on MNPcell. In a physiological solution, gallic acid ionizes to
become a carboxylate anion, which has strong ionic interactions and serves as a strong
hydrogen bond acceptor and donor. The ester group can only act as a hydrogen bond acceptor
but cannot form an anion. Furthermore, the amide side chain does not form an anion in
physiological conditions, and the hydrogen bond strength is weaker than that of carboxylic
acid. Such a chemical transformation implies that the formation of hydrogen bonds and ionic
bonds of the side chains may modulate the interaction among gallates, nanoparticles, and
plasma membranes; however, the detailed surface interaction remains to be defined.
Bai et al. reported that polymeric organic nanoparticles with more lipophilic surface
modifications entered cells more easily [50]. Replacement of the carboxylic acid group with
an ester group increased the hydrophobicity of the molecules, which was associated with a
higher logarithm of the octanol/water partition coefficient, log P [51]. Methyl gallate (2; log P
= 0.9) and propyl gallate (3; log P = 1.8) exhibited higher log P values than gallic acid (1; log
P = 0.7), which was also in line with the current results that higher hydrophobicity may increase
the interaction of MNP-gallate conjugates with membranes. However, gallamide (4) is more
hydrophilic with a much lower log P value (0), but a greater enhancing effect than gallic acid
(1), indicating lipophilicity might not be a dominant parameter. These results suggested that
both ester and amide modifications of the galloyl structure may facilitate MNP-cell interactions.
Our results demonstrated that the enhancing effects on MNPcell are highly dependent on
the structure of gallate derivatives. The number and arrangement of phenolic hydroxyl groups
17

seems to be critical structural features. Other than the versatile adhesive/anchoring properties
of the galloyl or catechol structure, it is likely that a specific binding site, or a receptor, on the
plasma membrane may mediate the enhancing effects of gallate derivatives to enhance
nanoparticle-cell interactions. Previous studies reported that gallate may bind to G protein-
coupled receptor 35 (GPR35) to induce anti-inflammatory effects [52]. Although there is no
direct evidence suggesting the participation of GPR35 in endocytosis, we cannot rule out the
possibility that the effects of gallate on MNPcell might be mediated by receptor activation-
induced signaling.
5. Conclusions
In the present study, we demonstrated a feasible application of combining gallate
derivatives and MNPs for a targeted delivery system. A simple mix of gallic acid or methyl
gallate with MNPs provided higher internalization compared to vehicle groups in LN-229 and
HeLa cells. Further study of structure-activity relationships showed that the galloyl moiety
enhanced MNP-cell interactions most prominently, while the catechol structure along with a
conjugate carboxylic acid also showed comparable effects. Use of a magnetic field further
facilitated particle internalization, suggesting a synergetic effect of phenolic acids and
magnetic forces. An antioxidant assay displayed a similar pattern of activities to the enhancing
effects, implying that enhancement of the uptake of MNPs and antioxidation may arise from
common structural features. Such structure-activity relationships cannot be interpreted by
cytotoxicity of the tested compounds with or without MNP conjugates at the concentrations
tested. Co-administration of gallates and nanocarriers may be a potential strategy to enhance
tumor cell-nanoparticle interactions, and thus improve cancer diagnosis and targeted therapy.
Whether gallates can enhance nanoparticles with different coatings remains to be determined;
18

nevertheless, gallates may serve as a platform for further modifications for versatile
applications.
Acknowledgements
We thank the Ministry of Science and Technology of the Republic of China (NSC 101-2320-
B-182-038- and MOST 106-2320-B-182-002-), Chang Gung Memorial Hospital
(CMRPD1D0231 and BMRP432), and the Ministry of Education of Taiwan
(EMRPD1F0261) for funding support.
19

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Figure Legends
Figure 1. Synergistic effects of gallates and magnetic force on cell-associated magnetic
nanoparticles (MNPs). Gallic acid (A, B) and methyl gallate (C, D) induced a concentration-
dependent increase in cell-associated MNPs (MNPcell) in LN-229 (A, C) and HeLa (B, D) cells
*
with (+) or without (-) the magnet (Mag). Values are the mean ± SE (n = 4). p<0.05 compared
to the corresponding control group. # p<0.05 compared to corresponding values in Mag (-).
Figure 2. Gallates enhanced magnetic nanoparticle (MNP)-cell interactions and
internalization. LN-229 cells were incubated with CMX-MNP (100 µg/mL; 26.32 μg/cm2) in
the presence of the vehicle (PBS; A, C), methyl gallate (30 µM; B, C), or gallic acid (30 µM;
C) at 37°C or 4°C (C only) with (C only) or without the magnet for 2 h. After incubation, cells
were counterstained with LysoTracker (red) for lysosomes and DAPI (blue) for nuclei (A, B).
*,†
§
p<0.05 compared to the corresponding groups of vehicle and gallic acid, respectively.
#
p<0.05 compared to the corresponding groups at 4°C. p<0.05 compared to corresponding
values in Mag (-).
Figure 3. Chemical structure of gallate derivatives used in this study. The structures of
gallic acid (1), methyl gallate (2), propyl gallate (3), gallamide (4), protocatechuic acid (5), p-
hydroxybenzoic acid (6), α-resorcylic acid (7), trimethoxybenzoic acid (8), caffeic acid (9),
dihydrocaffeic acid (10), and ferulic acid (11) are illustrated to determine the relative
importance of the carboxyl or phenolic groups of gallates on cell-associated magnetic
nanoparticles (MNPcell). The dashed square indicates the gallic acid moiety of
epigallocatechin-3-gallate (EGCG), which is the proposed pharmacophore of the effects
induced by EGCG.
Figure 4. Structure-activity relationship of gallate derivatives on cell-associated magnetic
nanoparticles (MNPcell). LN-229 cells were incubated with MNPs (100 µg/mL; 26.32 μg/cm2)
and various gallate derivatives at 10 (A, B) or 30 µM (C, D) for 24 h in the presence (+) or
absence (-) of the magnet (Mag). Values are the mean ± SE (n = 4). *,†,§,# p<0.05 compared to
26

the corresponding vehicle (v), gallic acid (1), methyl gallate (2), or caffeic acid (9) groups,
respectively.
Figure 5. DPPH radical-scavenging activity of gallate derivatives. The antioxidant
activities of gallate derivatives (3~150 µM) were determined by the DPPH method at different
*
concentrations. Values are the mean ± SE (n = 3). p<0.05 compared to the corresponding 3
µM groups of each gallate derivative. †,§,# p<0.05 compared to gallic acid (1), methyl gallate
(2), and caffeic acid (9) groups at the same concentration, respectively.
Figure 6. Gallate derivatives exerted no cytotoxicity. LN-229 cells were incubated with the
vehicle (v) or gallates (10 or 30 µM) in the absence (A) or presence (B) of magnetic
nanoparticles (MNPs) (100 µg/mL; 26.32 μg/cm2) for 2 h. In the presence of both gallate
derivatives (30 µM) and MNPs, the effects of the magnetic field (Mag) were also evaluated
(B). Values are the mean ± SE (n = 4).
27

Figure 1
28