Multifunctional Thin Films and Coatings from Ca
ffeic Acid and a
Cross-Linking Diamine
Mariagrazia Iacomino,
†Julieta I. Paez,
‡Roberto Avolio,
§Andrea Carpentieri,
†Lucia Panzella,
†Geppino Falco,
∥Elio Pizzo,
∥Maria E. Errico,
§Alessandra Napolitano,
†Aranzazu del Campo,
‡,⊥and Marco d’Ischia*
,††
Department of Chemical Sciences, University of Naples
“Federico II”, Via Cintia 4, I-80126 Naples, Italy
‡INM
− Leibniz Institute for New Materials, Campus D2 2, 66123 Saarbrücken, Germany
§
Institute of Chemistry and Technology of Polymers, National Council of Research (CNR), Via Campi Flegrei 34, Pozzuoli I-80078,
Italy
∥
Department of Biology, University of Naples
“Federico II”, Via Cintia 4, I-80126 Naples, Italy
⊥Chemistry Department, Saarland University, 66123 Saarbru
̈cken, Germany
*
S Supporting InformationABSTRACT:
The exploitation of easily accessible and nontoxic natural catechol compounds for surface functionalization and
coating is attracting growing interest for biomedical applications. We report herein the deposition on di
fferent substrates of
chemically stable thin
films by autoxidation of 1 mM caffeic acid (CA) solutions at pH 9 in the presence of equimolar amounts of
hexamethylenediamine (HMDA). UV
−visible, mass spectrometric, and solid state
13C and
15N NMR analysis indicated covalent
incorporation of the amine during CA polymerization to produce insoluble trioxybenzacridinium sca
ffolds decorated with
carboxyl and amine functionalities. Similar coatings are obtained by replacing CA with 4-methylcatechol (MC) in the presence of
HMDA. No signi
ficant film deposition was detected in the absence of HMDA nor by replacing it with shorter chain
ethylenediamine, or with monoamines. The CA/HMDA-based
films resisted oxidative and reductive treatments, displayed
e
fficient Fe(II) and Cu(II) binding capacity and organic dyes adsorption, and provided an excellent cytocompatible platform for
growing embryonic stem cells. These results pointed to HMDA as an e
fficient cross-linking mediator of film deposition from
natural catechols for surface functionalization and coatings.
■
INTRODUCTION
Deposition of thin
films from organic molecules or polymers is
an increasingly used strategy to confer speci
fic physical or
chemical properties to materials and devices for a variety of
biomedical and technological applications.
1,2A paradigm
methodology in this context relies on polydopamine
(PDA),
3−6a eumelanin-like material with universal adhesion
and coating properties inspired to catechol- and amine-rich
mussel byssus proteins.
7By dip-coating into a dopamine
solution in Tris buffer at pH 8.5
4,8it is possible to functionalize
and modify virtually any surface, enabling conjugation with
biomolecules
9to obtain biomaterials for tissue engineering
10and surface tailoring for energetic,
11environmental,
12and
analytical
13purposes.
After a decade of dopamine-based coating chemistry, PDA
remains the reference material for surface functionalization.
However, dopamine is highly toxic and its use as monomer
might be restricted in biomedical applications. Alternative
catechols free from toxicity issues are attracting interest.
14In
this context, the
film-forming ability of catechol polymers or
natural extracts of plant origin such as ellagic acid, tannic acid,
catechin, quercetin and many others has been reported.
15,16For speci
fic applications, the introduction of functional
groups into the polycatechol layer is required. A series of
reports have demonstrated that amines can be easily
Received: November 10, 2016
Revised: January 30, 2017
Published: February 13, 2017
pubs.acs.org/Langmuir
Downloaded via UNIV OF NAPLES FEDERICO II on March 4, 2020 at 12:26:23 (UTC).
incorporated into the polycatechol layers as shown for PDA
films formed in Tris buffer
8or in the presence of
hexamethylenediamine (HMDA).
17Other small molecules
and polymers have also been incorporated into PDA coatings.
18Amine functionalization has also been introduced in
4-heptadecylcatechol layers prepared in the presence of
ammonia.
19Oxidative polymerization of gallic acid in the
presence of excess HMDA also yielded
films with a high density
of carboxyl, amine, and quinone groups, which supported
immobilization of di
fferent biomolecules.
20,21These and other
studies suggest that oxidation of catechols in the presence of
amines is a rather
flexible and general method for the
deposition of reactive polycatechol coatings of di
fferent
precursors.
Herein, we report that chemically stable thin
films can be
deposited on various materials by dip-coating at pH 9 using a
nontoxic and easily accessible natural catechol, ca
ffeic acid
(CA), in the presence of equimolar amounts of HMDA. The
chemistry of the CA-HMDA reaction is investigated and the
properties of the formed layers are characterized, demonstrating
their interest as coatings supporting growth of embryonic stem
cells, as well as in metal binding and organic dyes adsorption.
■
MATERIALS AND METHODS
All reagents are purchased from commercial sources and used without further purification. Substrates (quartzes, silicon wafers and borosilicate coverslips) are cleaned by soaking in piranha solution (H2SO4/30% H2O25:1) overnight and then rinsed with distilled water
and dried under vacuum.
General Procedure for Substrate Coating. To a 1, 10, or 50 mM solution of the appropiate amine (HMDA, ethylenediamine, butylamine or dodecylamine) in 0.05 M sodium carbonate buffer pH 9, CA, 4-methylcatechol (MC) or dopamine are added under vigorous stirring in a 1:1 molar ratio. For PDA coatings, a previously reported procedure is followed involving oxidation of dopamine at 1 mM concentration in 0.05 M TRIS buffer pH 8.5. Under these conditions, no coating is obtained using CA or MC. In other cases, the oxidation is run dissolving CA or MC in 1% NH3 at 1 mM concentration.
Substrates are dipped into the reaction mixture after complete dissolution of the catechol and left under stirring for 3−6 h, then rinsed with distilled water, sonicated and dried under vacuum. For preparation of the coated cell culture plates, a solution of 1 mM HMDA and 1 mM CA in 0.05 M Na2CO3buffer (pH 9) or 1 mM
PDA in 0.05 M Tris buffer (pH 8.5, 15 mL) is poured into the plate and left overnight in air. After 24 h, the coated plates are extensively washed with water, sonicated and dried under vacuum. C18 silica gel
resin (55−105 μm; Millipore) is used as purchased. An amount of 650 mg of resin is suspended in a 1:1 solution of 25 mM HMDA and CA or 25 mM dopamine hydrochloride in 50 mM carbonate buffer (pH 9, 45 mL) under vigorous stirring. After 15 h, the coated resins are filtered under vacuum and washed with water (4 × 10 mL) and methanol (1× 5 mL). Washing solutions are monitored by UV−vis spectrometry until no absorption is found in the eluates.
Sample Preparation for MALDI-LDI-MS, FT-IR, and CP-MAS NMR Analysis. CA (540 mg, 3 mmol) is added to a solution of HMDA (348 mg, 3 mmol) in 0.05 M sodium carbonate buffer (pH 9, 300 mL) and the reaction mixture is left under stirring overnight. The deep green precipitate that separates is collected by centrifugation (5000 rpm, 4°C, 15 min) and washed with distilled water (3 × 20 mL) (124 mg). MC (366 mg, 3 mmol) is dissolved in a 1% KCl solution (150 mL) and then added to a solution of HMDA (348 mg, 3 mmol) in 0.05 M Na2CO3buffer pH 9 (150 mL), and the reaction
mixture is left under stirring for 6 h. The orange precipitate that separates is collected by centrifugation (5000 rpm, 4°C, 15 min) and washed with distilled water (3× 20 mL) (225 mg). For LDI-MS analysis, a suspension of the solid in water (1 mg/mL), is loaded onto the target plate and allowed to dry before analysis. In the case of
MALDI analysis, 10 mg/mL 2,5-dihydroxybenzoic in water is added to the suspension at a 1:1 ratio v/v. FT-IR and CP-MAS analysis are run on the solid samples after lyophilization.
Ellipsometry. Ellipsometric thickness of thefilms on silicon wafers is measured in air using a EL X-02C ellipsometer at a wavelength ofλ = 632.8 nm and at an angle of incidence of 70°. Dry thickness values are estimated by numericalfitting of the ellipsometric data (Δ and ψ) to a four layer model (silicon/silica/film/air) taking 3.912, 1.4571, and 1 as refractive index (n) values for silicon, silica, and air, respectively. Thefilms are assumed to be transparent and homogeneous with a negligible extinction coefficient (kfilm= 0) and with an n value of 1.63.
The parameters for the ellipsometry experiments are taken from our previous extensive ellipsometry studies on catechol layers. This model gave the bestfit.22
Water Contact Angle. The water contact angles are measured on a KRÜSS DSA 10 Mk2 drop shape analysis system with 3 μL of deionized water as the probefluid. The average value of the water contact angle is obtained by measuring the same sample at three tofive different locations on the modified silicon wafer.
Atomic Force Measurements. Measurements are conducted over modified silicon wafers using a Bruker Dimension Icon with ScanAsyst in the tapping mode in air. An Olympus OMCL-AC160TS cantilever is used in noncontact mode (K = 42 N/m, range: 33.5−94.1 N/m, F0= 300 kHz, range: 278−389 kHz). The root mean square
(RMS) roughness values are determined using the following formula:
= ∑= Z −Z N RMS i ( ) N i 1 ave 2
Zaveis the average Z value within the given area, Ziis the current Z
value, and N is the number of points within a given area. Zlimit: 3μm.
X-ray Photoelectron Spectroscopy. Measurements are per-formed on a Kratos AXIS Ultra DLD instrument using a monochromatic Al Kα X-ray source. Atomic composition (%) is measured for C, N, and O.
MALDI and LDI-MS Analysis. Positive Reflectron MALDI and LDI spectra are recorded on a 4800 MALDI ToF/ToF instrument (Sciex). The spectrum represents the sum of 15 000 laser pulses from randomly chosen spots per sample position. Raw data are analyzed using the computer software provided by the manufacturers and are reported as monoisotopic masses.
Solid State CP-MAS NMR. NMR spectra are acquired on a Bruker Avance II 400 spectrometer equipped with a 4 mm Bruker MAS probe for solid samples. All spectra are recorded in cross-polarization mode with a1Hπ/2 pulse width of 3.8 s and a relaxation delay of 5 s. For 13C experiments, a contact time of 2 ms and a spinning rate of 9 kHz
are selected, for15N the contact time is set to 2.5 ms and the spinning
rate is 6 kHz.
ATR/FT-IR Analysis. Infrared spectra are acquired using a Thermo Fisher Nicolet 5700 spectrophotometer equipped with a Smart Performer accessory mounting a ZnSe crystal for the analysis of solid samples.
Metal Binding.23−25An amount of 0.4−1.8 mg of CA/HMDA or PDA in water (5 mg/mL) isfinely suspended using a glass/glass potter and then added to 3 mL of a 100μM solution of FeCl2·4H2O in water,
under stirring. Control experiments are performed in the absence of material. After 5 min, the samples are centrifuged (3 min, 7500 rpm) and 100μL of a 5 mM ferrozine aqueous solution is added to the supernatants. The absorbance at 562 nm is measured after 5 min. Experiments are run in triplicate. For the copper binding assay, 0.04− 0.18 mg of CA/HMDA or PDA isfinely suspended in water (3 mg/ mL) using a glass/glass potter and then added to 2 mL of a 66.7μM solution of copper(II) acetate in 50 mM phosphate buffer (pH 6.2), under stirring. After 5 min, the samples are centrifuged (3 min, 7500 rpm) and 75μL of an aqueous solution of 4 mM pyrocatechol violet are added to the supernatants. Control experiments are performed in the absence of material. The absorbance at 616 nm is measured after 5 min. Experiments are run in triplicate. In other experiments, 3 mL of a 100μM solution of Fe(II) in water or 2 mL of 66.7 μM solution of Cu(II) in 50 mM phosphate buffer (pH 6.2) are filtered through
coated C18 silica (50−100 mg for iron and 2−50 mg for copper
binding) using a 5 mL syringe equipped with a filter at a flux of approximately 1 mL/min. Metal concentration in eluted fractions is determined as described above. To determine the binding ability of the coated silica, 5 mg of CA/HMDA C18 or PDA C18 is incubated in
solutions of Fe2+ in water or Cu2+ solutions in 50 mM phosphate
buffer (pH 6.2) at concentrations of 10, 25, 50, and 100 μM (final volume of 20 mL). Suspensions are incubated for 2 h at 37°C under stirring, then filtered through a nylon 0.45 μm membrane, and analyzed for metal concentration as described above. Metal chelation ability of the uncoated resin and of the filtering device are tested comparatively.
Organic Dyes Adsorption. PDA/Tris and CA/HMDA coated glasses are dipped in 0.4 mg/mL solutions of Congo Red or Methylene Blue in phosphate buffer (0.1 M, 20 mL, pH 7.0). After 15 min, the glasses are washed with distilled water and dried under vacuum, UV−vis spectra are recorded.
Cell Cultures. The HaCat cell line is maintained in our laboratory. Cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Euroclone) and penicillin (100 U/mL)/streptomycin (100 mg/mL). Cells are maintained at 37°C under a 5% CO2/95% air humidified atmosphere.
Cell growth rate experiments are performed using untreated coverslips (control), or CA/HMDA or PDA/Tris coated coverslips. All coverslips are placed in a 24-well plate and resterilized by UV before use. Cells are then seeded onto coverslips (30 000 cells/1 mL DMEM for each coverslip) and grown for 24, 48, and 72 h in a 5% humidified CO2incubator at 37 °C. Cell counts are performed in triplicate on
days 1, 2, and 3. Cell morphology is observed by inverted microscope. The murine embryonic stem cell (ESCs) line E14Tg2a.4 derived from strain 129P2/OlaHsd is cultured for two passages on feeder-free low adhesion plates coated with either gelatin, CA/HMDA, or PDA/Tris. The cultures are subsequently maintained in complete Embryonic Stem (ES) medium: Glasgow Minimum Essential Medium (GMEM, Gibco), 15% fetal bovine serum (FBS, EuroClone), 1000 U/mL leukemia inhibitory factor (LIF) (EuroClone), 1.0 mM sodium pyruvate (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 2.0 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol, and
500 U/mL penicillin/streptomycin (Invitrogen). ESCs are incubated at 37°C in 6% CO2; medium is changed daily, and cells are split every
2−3 days routinely.
■
RESULTS AND DISCUSSION
Autoxidation of ca
ffeic acid (CA) (1 mM) at pH 8.5−9, in
phosphate or bicarbonate bu
ffer does not result in detectable
coatings on glass or quartz, despite apparent polymerization
and darkening of the mixture.
26Addition of ammonia, Tris
bu
ffer, butylamine, dodecylamine or ethylenediamine to the
catechol solution up to 50 mM concentration does not lead to
stable coatings either. However, addition of HMDA (1 mM)
leads to the deposition of greenish-blue
films (λ
max660 nm) on
a glass surface within a few hours which can be detected by
UV
−vis spectrophotometry (
Figure 1
). E
fficient
HMDA-mediated deposition of CA
films is observed also on quartz
and other materials (
Figure S1
).
CA/HMDA thin
films are characterized by atomic force
microscopy (AFM,
Figure S2
), water contact angle (WCA)
measurements, ellipsometry, and X-ray photoemission
spec-troscopy (XPS), and the results are compared to those of PDA
films produced in the presence of Tris and HMDA. CA/
HMDA coatings are ca. 10 nm thick, smooth, and
homogeneous and exhibit lower WCA than PDA/Tris
films.
PDA/HMDA
films show intermediate properties in terms of
thickness and hydrophobicity, but a signi
ficantly higher
roughness. XPS data show a higher N content in the PDA
films prepared in the presence of HMDA vs those obtained in
Tris bu
ffer (
Table 1
). Spectrophotometric monitoring of
film
deposition kinetics showed a steep increase of the absorbance
at 660 nm over 6 h (
Figure S3
).
Based on these
findings, the CA/HMDA film is next
subjected to structural analysis to elucidate the chemical nature
of the main species. To this aim, the greenish-blue precipitate
obtained by autoxidation of CA in the presence of HMDA in
bicarbonate bu
ffer at pH 9 is collected, extensively washed and
analyzed by
13C and
15N CP-MAS NMR combined with ATR/
FT-IR and LDI/MALDI-MS (
Figure S4
−S6
). The
13C NMR
spectrum (
Figure 2
a) displays clear aliphatic chain signals of
HMDA denoting extensive incorporation of the diamine. Major
bands at
δ 95−105 indicate the presence of shielded aromatic
carbons adjacent to amine groups. Signals at
δ 125, 138, and
148 correspond to aromatic ring signals. Intense sp
2carbon
bands at
δ 168 and 175 can be attributed to conjugated
carbonyl and carboxyl groups. The
15N NMR spectrum (
Figure
2
b) reveals an intense amine resonance and two broad bands
around
δ 90 and 120, in accord with shielded imine and
heteroaromatic structures.
27Overall, these data, integrated by LDI-MS analysis and
supported by previous literature on the reactions of CA esters
with amines,
28,29describe the CA/HMDA coating as consisting
of a mixture of trioxygenated carboxylated benzacridinium
derivatives including tethered dimeric species (
Figure 3
).
Several trihydroxybenzacridine derivatives green in color
have also been identi
fied in the reaction of caffeic acid esters
(mostly chlorogenic acids) with di
fferent amino acids, a process
that was aimed at modeling the generation of green colored
species associated with the alkaline extraction of the protein
components of sun
flower meals and coffee beans.
30−32However, at variance with previous studies, the chemistry
reported herein highlights the e
ffect of the carboxyl group, due
to use of CA as free acid, on product properties. A mechanistic
path to the proposed structures including the decarboxylation
step is shown in
Figure S7
.
Interestingly, electron paramagnetic resonance (EPR) spectra
show distinct paramagnetic properties, consistent with the
occurrence of the polycyclic systems in di
fferent redox states,
e.g. the catechol and quinone forms represented in
Figure 3
.
Inspection of the EPR spectrum suggests mainly
carbon-centered radicals (g = 2.0037) with negligible involvement of
O-centered semiquinone radicals or of nitrogen centered
radicals. Both the power saturation pro
file and the broad signal
Figure 1.UV−vis spectra of cover glasses dipped into 1 mM solution of CA in 50 mM Na2CO3buffer in the presence of equimolar amounts
of various amines over 3 h and picture of a CA/HMDA coated coverslip.
amplitude (
ΔB = 6.47 G) were consistent with a high degree of
heterogeneity (
Table S1 and Figure S8
).
33The chemical stability of the CA/HMDA
films is tested
under di
fferent conditions. Exposure of as prepared films to
aqueous solutions containing hydrogen peroxide or reducing
agents, such as glutathione, dithiothreitol or sodium
borohy-dride, does not result in detectable modi
fication of the UV−
visible absorption spectra (data not shown). Interestingly, acid
vapors cause visible color change from green to greyish, which
can be reverted by ammonia vapors, denoting pH-dependent
chromophores (
Figure S9
). No apparent
film detachment is
induced by oxidative, reductive or acidic treatments.
To better clarify the role of HMDA in
film deposition, we
have replaced CA by 4-methylcatechol (MC) in the mixture. A
deep-yellow coating (
λ
max405 nm) is obtained (
Figure 4
)
which on ellipsometric (
Table S2
) and spectral characterization
(
Figure S10
−S13
) proves to consist of variously substituted
quinine imine species deriving from the nucleophilic addition of
HMDA to O-quinone moieties (
Figure 5
). Also in this case, no
UV-detectable surface coating is observed in the absence of
HMDA.
These results unambiguously demonstrate the covalent
coupling of HMDA with autoxidatively generated quinone
intermediates as a general cross-linking mechanism underlying
film formation. It is concluded that the combination of a long
aliphatic chain and two amine groups in HMDA is a crucial
requisite to confer to catechol polymers a suitable balance of
hydrophobicity, aggregation and cross-linking, since neither
simple monoamines (propylamine or the long chain
dodecyl-amine) nor short chain diamines (ethylenedidodecyl-amine) are able to
lead to
film deposition from autoxidizing catechols. The role of
HMDA would thus be to ensure cross-linking via tethering of
quinonoid structures, and to provide long-chain
−NH
2hooks
reinforcing sticking to the substrate by oxygenated functions.
In further experiments the metal binding ability of CA/
HMDA is assayed in comparison to PDA by use of colorimetric
Table 1. Characterization of Thin Films from CA/HMDA and PDA on Silicon wafers
film thickness (nm) roughness (nm) WCA (deg) comp. (atom %)
CA/HMDA 9.4± 0.4 1.0 69.4± 1.7 C 1s 54.5± 0.4 N 1s 3.8± 0.1 O 1s 27.9± 0.1 PDA/HMDA 16.9± 1.7 5.1 76.0± 2.0 C 1s 48.5± 0.9 N 1s 3.3± 0.1 O 1s 29.9± 0.5 PDA/Tris 23.7± 0.4 0.4 86.2± 1.2 C 1s 23.1± 0.9 N 1s 0.8± 0.2 O 1s 48.2± 0.5
Figure 2.(a) 13C and (b) 15N CP-MAS NMR spectra of the CA/
HMDA polymer. Spinning sidebands are marked by a dot.
Figure 3.Proposed structures for representative components of CA/ HMDAfilms as inferred from LDI-MS. Shown are the m/z values for the pseudomolecular ions.13C NMR and15N (red) NMR resonance
assignments are highlighted.
Figure 4. UV−vis spectrum of a glass coverslip dipped into 1:1 solution of MC and HMDA (1 mM) in 50 mM Na2CO3buffer pH 9
over 3 h and picture of the MC/HMDA coated coverslip.
assays.
23−25CA/HMDA proves to be much more e
fficient than
PDA in binding iron and comparable to PDA for copper
binding (
Figure S14
). On this basis the metal-binding ability of
octadecylsilane silica (55
−105 μm particle size), as a high
surface area support, following functionalization by the CA/
HMDA or PDA coating (
Figure 6
A) is then comparatively
evaluated. Almost complete (97%) removal of iron from the
solution is obtained by passing it through 100 mg of CA/
HMDA-coated C
18while the same amount of
PDA-function-alized C
18gives 78% of Fe
2+removal. Lower amounts of
functionalized resin (50 mg) lead to the almost complete
removal of copper (97% for CA/HMDA C
18and 93% for PDA
C
18). Interestingly, the e
fficiency of the CA/HMDA C
18remains almost unaltered after 4 passages of the fresh metal
containing solution through the resin (Fe
2+chelation = 97%,
Cu
2+chelation = 77%) and still e
ffective after up to 6 passages
(Fe
2+chelation = 47%, Cu
2+chelation = 47%) (
Figure S15
).
Figure 6
C reports the metal binding capacity q
e(mg of
metal/g of C
18) for the CA/HMDA C
18and PDA C
18calculated using the equation q
e= (C
0− C
eq)(V/W), where
C
0(mg/L) is the actual starting metal concentration as
analytically determined from blank experiments, C
eq(mg/L) is
the metal concentration remaining in solution after incubation
with the resins, V (L) is the volume of the incubation mixture,
and W (g) is the resin dose. In all cases, the curves are
characterized by an increased uptake following a rise in metal
concentration. With an increase of the starting metal
concentration from 10 to 100
μM, the amount of Fe
2+binding
rises up to 4.7 mg/g for CA/HMDA C
18vs 4.0 mg/g for PDA
C
18and the amount of Cu
2+binding up to 10.1 mg/g for CA/
HMDA C
18vs 8.3 mg/g for PDA C
18.
In subsequent experiments, the ability of CA/HMDA coating
to interact with or retain other molecules is tested by
immersing coated glasses into 0.4 mg/mL solutions of
commercial dyes, such as Congo Red and Methylene Blue in
0.1 M phosphate bu
ffer at pH 7. After extensive rinsing with
water, detectable adsorption of the dye is evidenced in the
UV
−visible spectra (
Figure 7
) as well as by visual inspection.
For comparison the same experiments are run on PDA
films
(
Figure S16
) showing some binding ability only in the case of
Methylene Blue.
In a
final set of experiments, the biocompatibility of the CA/
HMDA coating and the ability to support cell growth is
assessed.
HaCaT cells adhere to CA/HMDA pretreated dishes, remain
viable and show comparable morphology as on tissue culture
plates. The CA/HMDA coating e
fficiently supports the growth
of HaCat cells (
Figure S17
), with a statistically signi
ficant
increase in the proliferation rate at 72 h compared to
PDA-coated plates (p < 0.01). These results show good
biocompatibility of CA/HMDA coatings and a positive e
ffect
as cell cultures surfaces.
We have then investigated whether CA/HMDA could favor
clonogenic cell growth in an Embryonic Stem Cell (ESC)
colony assay,
34as previously reported for poly(DOPA)
35and
PDA coatings.
36ESCs are of particular interest for their
Figure 5.Proposed structures for representative components of MC/ HMDA polymer as inferred from LDI-MS. Shown are the m/z values for the pseudomolecular ions. 13C NMR and 15N (red) NMR
resonance assignments are highlighted.
Figure 6.(A) From left to right: Uncoated C18resin, CA/HMDA C18
and PDA C18. (B) Binding of iron (left) and copper (right) on
different amounts of C18resins coated with CA/HMDA (open circles)
and PDA (full circles). (C) Binding isotherms of iron (left) and copper (right) by C18resins coated with CA/HMDA (open circles)
and PDA (full circles). Initial concentration range, 10−100 μM for iron and copper; amount of functionalized resin, 0.25 mg/mL; temperature, 37°C; contact time, 2 h. Shown are mean values of three separate experiments± SD.
Figure 7.UV−visible spectra of CA/HMDA coated glass after dipping into a 0.4 mg/mL solution of Congo Red or Methylene Blue in phosphate buffer pH 7.0.
sensitivity to substrate conditions that can modulate ESC
colony morphology. ESCs are cultured for 3 days in regular
culture medium on cell culture plates coated with either CA/
HMDA, PDA, or gelatin. The coating treatments show di
fferent
degrees of cell attachment (
Figure 8
). ESCs cultured on CA/
HMDA coated plates form round-shaped colonies (
Figure 8
A-a), while ESCs cultured on PDA-coated plates form
flat-shaped
colonies (
Figure 8
A-b) which is consistent with high
self-renewal, and low-self-self-renewal, respectively. As expected ESCs
cultured on gelatin aggregate in spheroid cell structures
floating
in the medium (
Figure 8
A-c). Altogether, the data show that
CA/HMDA coated plates improve both clonogenic and
attachment capability of ESCs. Remarkably, ESCs cultured on
CA/HMDA show signi
ficantly higher number of colonies than
ESCs cultured either on PDA coated plates or on gelatin coated
plates (
Figure 8
B).
■
CONCLUSIONS
The autoxidative coupling of a natural catechol (CA) with a
long-chain diamine (HMDA) at pH 9 leading to deposition of
thin
films is disclosed herein as an expedient strategy for surface
functionalization and coating. The speci
fic film-forming
proper-ties imparted by HMDA to autoxidizing catechols can be
attributed to the e
fficient cross-linking properties ensuring
flexibility and an optimal balance of hydrophobicity and ionic
character. The two-component coating procedure reported in
this study does not require organic solvents, oxidants, or other
additives, or long coating deposition times, is versatile (can be
extended to a range of catechols and substrates, as apparent
from preliminary observations), and is based on a cheap,
nontoxic, and easily accessible natural compound like CA.
Considering the previous studies on gallic acid and DA,
17,21the
results reported in this paper expand the current repertoire of
catechol-based coatings with the additional advantage that
HMDA is used herein in equimolar amounts with the catechol
and at 1 mM concentrations, much lower than those in
previous reports. CA/HMDA
films are cytocompatible and
appear to support more e
fficiently stem cell growth than films
obtained from PDA/Tris. Of interest in the perspective of
exploitation of these materials is the ability of CA/HMDA to
e
fficiently bind metal ions and organic dyes.
■
ASSOCIATED CONTENT
*
S Supporting InformationThe Supporting Information is available free of charge on the
ACS Publications website
at DOI:
10.1021/acs.lang-muir.6b04079
.
Coatings of di
fferent materials, AFM and spectral analysis
for ca
ffeic acid and methylcatechol/HMDA, EPR analysis
on CA/HMDA and experimental procedure, response to
acid of the CA/HMDA coating, metal-binding ability of
functionalized C
18through multiple elution, and HaCaT
cells growth curve (
)
■
AUTHOR INFORMATION
Corresponding Author*E-mail:
dischia@unina.it
.
ORCIDJulieta I. Paez:
0000-0001-9510-7254Marco d’Ischia:
0000-0002-7184-0029 NotesThe authors declare no competing
financial interest.
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ACKNOWLEDGMENTS
This work was supported by grants from Italian MIUR (PRIN
project PROxi, 2010
−11). The authors thank Dr. Longjian Xue
(ellipsometry), Mrs. Helma Burg (AFM), and Dr. Tobias
Weidner (XPS) from Mpip-Mainz for their help with
measurements. We thank Prof. Gerardino D
’ Errico and Dr.
Marco Perfetti for EPR experiments and helpful discussions.
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