Journal of Inorganic Biochemistry 180 (2018) 171–178

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Incorporation of the chemotherapy medication cisplatin into polyamide T

membrane
⁎
Érica A. de Souzaa, Lucas A. Rochaa, Emerson H. de Fariaa, Katia J. Ciuffia, Eduardo J. Nassara, ,
Jorge V.L. Silvab, Marcelo F. Oliveirab, Izaque A. Maiab
a
Universidade de Franca, Av. Dr. Armando Salles Oliveira, 201 Pq. Universitário, 14046-000 Franca, SP, Brazil
b
Centro da Tecnologia da Informação Renato Archer (CTI), Rod. Dom Pedro I, Km 143,6, 13069-901 Campinas, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The search for more efficient and less aggressive cancer treatment methods has intensified over the last decades
Sol–gel and has involved many scientific areas. To provide skin cancer patients with better quality of life, this work aims
Additive manufacturing to incorporate chemotherapy into polyamide membranes, functionalized by the sol–gel methodology, for con-
Cytotoxicity trolled drug release at the target tissue. A 200-micrometer-thick flexible polyamide membrane prepared by
Functionalization
Additive Manufacture was activated and functionalized with the alkoxide 3-chloropropyltriethoxysilane, which
was followed by incorporation of the antitumor agent cisplatin. Membrane functionalization with the alkoxide
was attested by infrared absorption spectroscopy, which evidenced a band at 1100 cm− 1, due to Si-O-Si vi-
bration, and typical cisplatin bands at 3200 and 1600 cm− 1. The thermogravimetric curve revealed increased
silicon oxide and platinum residues. Drug release was tested in simulated body fluid. Cytotoxicity was evaluated
by the Cell Proliferation Kit, which gave IC50 of 23.95 μgM.

1. Introduction PA membrane obtained by AM can be functionalized by the sol–gel

Additive Manufacturing (AM) has improved. It has attracted the
attention of numerous researchers especially because it affords pieces of
various shapes, sizes, and complexities, with several applications [1–7].
Within AM, the Laser Sintering technique (SLS) stands out because it
uses polyamide (PA) as raw material, which is excellent for bioappli-
cations due to its biocompatibility, good mechanical properties, low
cost, and easy handling [8–10] PA consists of polyethylene (CH2)n
segments separated by amide monomers (NH-CO). These peptide units
allow the polymeric chains to form hydrogen bonds with other com-
pounds.
The sol–gel process is used to prepare highly pure, homogeneous
materials at mild temperatures [11–14]. The process encompasses hy-
drolysis and condensation reactions of the precursor alkoxide groups, to
form silanol groups (eSieOH) that can interact with other materials
and substances by hydrogen bonding. In studies involving calcium salts,
polymer surface modification with silanol groups has provided the
polymer with bioactivity and flexibility [15–17]. Surface modification
of PA pieces obtained by AM alters the physical and chemical properties
of PA, leading to applications in which the modified material functions
as an anticorrosive layer or as a biomaterial. Modified PA can also be
endowed with temperature resistance [1,8,18,19].
methodology and incorporated with light-emitting compounds and may
have potential application in skin cancer treatment [20]. Here, we have
incorporated a PA membrane functionalized via the sol–gel metho-
dology with the chemotherapy medication cisplatin for use in con-
trolled drug release during skin cancer treatment.
2. Experimental
2.1. Polyamide (PA) membrane preparation, washing, and activation
PA membrane obtained by AM at the Tridimensional Technology
Division of the Information Technology Center Renato Archer was used.
First, the PA membrane was washed with 30.0 mL of distilled water,
under stirring, to remove the PA powder that had not been sintered.
Next, the PA membrane, which measured 8.0 × 5.0 cm and had
thickness of 200 μm, was cut into 1.0-cm2 units with approximate mass
of 25.30 mg and dried. Then, the PA membrane was pretreated as de-
scribed in [21]. Briefly, the PA membrane was immersed in
1.0 mol L− 1 acetic acid under stirring for 24 h, washed with distilled
water in ultrasound bath, and dried at ambient temperature for 24 h.
This treatment activated the membrane surface by protonating the PA
amide groups.

⁎
Corresponding author at: Universidade de Franca, Av. Dr. Armando Salles Oliveira, 201, CP 82 - CEP: 14404-600 Franca, SP, Brazil.
E-mail address: eduardo.nassar@unifran.edu.br (E.J. Nassar).

https://doi.org/10.1016/j.jinorgbio.2017.12.013
Received 2 October 2017; Received in revised form 20 November 2017; Accepted 19 December 2017
Available online 24 December 2017
0162-0134/ © 2017 Elsevier Inc. All rights reserved.
É.A. de Souza et al.
2.2. PA membrane functionalization with the alkoxides tetraethyl
orthosilicate (TEOS) and 3-chloropropyltriethoxysilane (CPTES)
The 1.0-cm2 PA membrane was placed in a two-neck round-bottom
flask containing 15.0 mL of ethanol (EtOH), 3.7 mL of TEOS, and
5.0 mL of distilled water. The mixture was kept under reflux and con-
stant stirring at 80 °C for 24 h. After this process, the membrane was
washed with distilled water in an ultrasound bath and dried at 60 °C for
2 h. The resulting sample, designated PA TEOS, was functionalized with
3.4 mL of the alkoxide CPTES in the same conditions described above,
172
Journal of Inorganic Biochemistry 180 (2018) 171–178
Fig. 1. (a) Functionalized polyamide membrane (PA
TEOS CPTES) obtained via the sol–gel methodology;
(b) PA TEOS CPTES after cisplatin (cis-DDP) in-
corporation.
Flowchart 1. Representation of functionalized polyamide
membrane (PA TEOS CPTES) preparation and incorpora-
tion with the chemotherapy medication cisplatin (cis-DDP).
to give the sample labeled as PA TEOS CPTES [20].
2.3. Cisplatin (cis-DDP) incorporation into PA TEOS CPTES
The cis-DDP solution was prepared by dissolving 5.0 mg
(1.66.10− 2 mols) of cis-DDP in 10.0 mL of distilled water under con-
stant stirring for 20 min.
Drug incorporation into PA TEOS CPTES was accomplished by the
casting method. PA TEOS CPTES was placed in a cis-DDP solution at
40 °C until the solvent evaporated completely. PA TEOS CPTES became
É.A. de Souza et al. Journal of Inorganic Biochemistry 180 (2018) 171–178

Fig. 2. cis-DDP UV–Vis absorption spectrum in aqueous solution.

203

275
306

Absorbance
Absorbance

400

Wavelength (nm)

200 400 600 800

Wavelength (nm)

20.9 a bath at 37 °C for 24 h to simulate the human body dynamics. Drug

release was monitored by UV–Vis spectroscopy.
13.9 16.5
PA TEOS CPTES Cis-DDP
2.6. Cell viability
Cis-DDP
Cytotoxicity was evaluated by using the Cell Proliferation Kit (an
XTT-based colorimetric assay, Roche, Mannheim, Germany), according
to the manufacturer's instructions. The cytotoxicity assay requires a
CPS

24.9
10 20 30 40 50 60
2 θ (degree)
Fig. 3. X-ray diffractogram of pure cis-DDP and of PA TEOS CPTES incorporated with cis-
DDP.
yellowish, the typical color of cis-DDP. Spectrophotometry revealed
that 3.52 mg of the drug was incorporated into PA TEOS CPTES. Fig. 1
shows PA TEOS CPTES photos before and after cis-DDP incorporation.
Flowchart 1 illustrates PA membrane treatment, functionalization,
and incorporation with the drug:
2.4. SBF (simulated body fluid) solution preparation
SBF solution was prepared according to references [22,23]. Briefly,
1 L of solution containing 8.17 g of NaOH, 0.35 g of NaHCO3, 0.224 g of
KCl, 0.228 g of Na2HPO4, 0.305 g of MgCl2, and 0.071 g of NaSO4 in
distilled water was stirred for 24 h, until complete dissolution; pH was
adjusted to 7.4 with HCl. The solution was kept in the refrigerator.
2.5. Controlled drug release simulation
A 10.0-mL aliquot of SBF solution was placed in a beaker containing
PA TEOS CPTES incorporated with cis-DDP. The mixture was stirred in
selection based on the suitability of the cell employed in the test
method. The present study used Lung human fibroblasts (GM07492A
cells) according to protocol ISO 0993-12:2007, which is the goal of in
vitro cytotoxicity tests of biomedical devices.
The cell line was maintained as monolayers in plastic culture flasks
(25 cm2) containing HAM-F10 plus DMEM (1:1; Sigma-Aldrich) sup-
plemented with 10% fetal bovine serum (Nutricell) and 2.38 mg/mL
Hepes (Sigma-Aldrich) at 37 °C, in humidified 5% CO2 atmosphere.
70 Antibiotics (0.01 mg/mL streptomycin and 0.005 mg/mL penicillin;
Sigma-Aldrich) were added to the medium to prevent bacterial growth.
The cis-DDP-incorporated PA TEOS CPTES (1 cm2) was cut into pieces
measuring 0.5 × 0.2 mm2 and maintained in 1 mL of culture medium
at 37 °C for 24 h. Immediately before cell treatment, the sample was
vortexed for 2 min. According to the release analysis performed by
UV–Vis spectroscopy, the solution contained 2.3 mg/mL cis-DDP.
2.7. XTT assay
For the experiments, 104 cells were plated onto 96-well microplates.
Each well received 100 μL of culture medium containing cis-DDP at
concentrations ranging from 0.37 to 766.64 μM. The cells were cultured
in 5% CO2 atmosphere at 37 °C for 24 h. After incubation, the culture
medium was removed, and the cells were washed with 100 μL of
phosphate-buffered saline (PBS) and exposed to 100 μL of HAM-F10
culture medium without phenol red. Next, 25 μL of XTT (Roche
Diagnostics) was added to each well, and the microplates were in-
cubated at 37 °C for 17 h. Sample absorbance was read in a microplate
reader (ELISA, Asys UVM 340/Microwin 2000) at a wavelength of
450 nm and at a reference length of 620 nm. The amount of soluble
product (formazan) was proportional to the number of viable cells. IC50
(50% cell growth inhibition), calculated with the GraphPad Prism
program, was considered as response parameter to assess cytotoxicity.
The experiments were performed in triplicate.

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É.A. de Souza et al. Journal of Inorganic Biochemistry 180 (2018) 171–178

100 2.8. Characterizations

2.8.1. UV–vis absorption spectroscopy

Measurements were conducted at ambient temperature on the UV/
80
Vis Hewlett-Packard 8453 Diode Array spectrophotometer.
64.3
Mass (%)

2.8.2. X-ray diffractometry (XRD)

60
X-ray diffractograms were obtained at ambient temperature on a
Rigaku Geigerflex D/Max-c diffractometer equipped with CuKα radia-
tion (λ = 1.5405 Å) monochromator. Diffractograms were recorded for
40 2θ values ranging from 5 to 70°, at 0.02°/10 s.
PA TEOS CPTES Cis-DDP
Cis-DDP
21.0% 2.8.3. Thermal analyses (TGA)
20 TGA thermal analyses were accomplished on a TA Instruments-SDT
Q600-Simultaneous DTA-TGA analyzer from ambient temperature
0 100 200 300 400 500 600 700 (~ 25 °C) to 700 °C, at 20 °C/min, with nitrogen flow of 100 mL/min.
o
Temperature ( C)
2.8.4. Infrared absorption (IR) spectroscopy
Fig. 4. Thermogravimetric curves recorded for cis-DDP and for PA TEOS CPTES in- IR analyses were performed on a Perkin Elmer Frontier spectro-
corporated with cis-DDP. photometer operating from 400 to 4000 cm− 1. The IR spectrum of the
drug in KBr pellet was analyzed by diffuse reflectance spectroscopy, and
o the membrane was analyzed by ATR (IR absorption spectroscopy by
479 attenuated total reflectance). Sixteen scans were recorded.

2.8.5. Scanning electron microscopy (SEM) and energy dispersive

spectroscopy (EDS)
Samples measuring approximately 0.5 cm2 were fixed on an alu-
300 400 minum stub with a carbon tape and coated with a conducting gold layer
Derivative

o
Temperature ( C)
in a QUORUM mod. SC7620 Sputter Coater for 120 min. The mor-
phology of the membrane was investigated under an optical microscope
TESCAN (model VEGA 3 SBH). EDS results were obtained by using a
detector with 130-eV resolution, model X-ACT IE 150, coupled to the
PA TEOS CPTES Cis-DDP SEM apparatus.
Cis-DDP

3. Results and discussion

3.1. UV–vis absorption spectroscopy

0 100 200 300 400 500 600 700
o
Fig. 2 depicts the cis-DDP UV–Vis absorption spectrum with am-
Temperature ( C) plification of the region between 275 and 306 nm. The most intense
Fig. 5. Derivative thermogravimetric curves recorded for cis-DDP and for PA TEOS CPTES band emerges at 203 nm, which is typical of cis-DDP in aqueous solu-
incorporated with cis-DDP. tion [24].

3.2. X-ray diffractometry (XRD)

ν CΗ
Fig. 3 shows the X-ray diffractograms of pure cis-DDP and of PA
TEOS CPTES incorporated with cis-DDP.
The pure cis-DDP X-ray diffractogram displays narrow peaks at
2θ = 13.9, 15.0, and 16.4°, which are characteristic of the crystalline
Transmittance (%)

pattern described in the literature [25]. PA TEOS CPTES incorporated

with cis-DDP presents peaks at 2θ = 20.9 and 24.9°, due to crystalline
phase α, as described in the literature [26,27]. PA TEOS CPTES in-
corporated with cis-DDP also displays the typical peaks of cis-DDP at
2θ = 13.9 and 16.5° [25]. Ajima et al. also detected these peaks when
they incorporated cis-DDP into carbon nanotubes [28]. These peaks are
δ ΝΗ the first evidence that cis-DDP was incorporated into PA TEOS CPTES.

cis-DDP
δ ΝΗ
ν ΝΗ ν Si-O-Si 3.3. Thermal analyses (TG/DTG)
PA TEOS CPTES cis-DDP

4000 3500 3000 2500 2000 1500 1000 500 Figs. 4 and 5 illustrate the thermogravimetric (TGA) curves and the
-1 respective derivative (DTG) curves, recorded for cis-DDP and for PA
Wave number (cm )
TEOS CPTES incorporated with cis-DDP, respectively.
Fig. 6. IR spectra of cis-DDP and of PA TEOS CPTES incorporated with cis-DDP. PA TEOS CPTES presents a 19.0% residue that can be attributed to
silica (SiO2), obtained after the polyamide, TEOS ethoxide, and CPTES
chloropropyl organic groups were burned [20]. The TGA curve of PA
TEOS CPTES incorporated with cis-DDP in Fig. 3 reveals a 21.0%

174
É.A. de Souza et al.
residue, which indicates a 3% increase in mass corresponding to cis-
DDP.
cis-DPP decomposes between 250 and 370 °C, as seen in the TGA
and DTG curves. The DTG curve (Fig. 4) reveals that this decomposition
involves two steps. PA TEOS CPTES incorporated with cis-DDP de-
composes from 250 to 550 °C, with maximum decomposition at 479 °C,
which is 30 °C higher than the temperature at which untreated PA de-
composes [20]. Amplification of Fig. 4 shows the cis-DPP decomposi-
tion region and attests that the drug was incorporated into PA TEOS
CPTES.
3.4. Infrared absorption (IR) spectroscopy
The IR spectra were recorded in the ATR mode, to identify the ty-
pical PA vibrations after functionalization with TEOS and CPTES and
175
Journal of Inorganic Biochemistry 180 (2018) 171–178
Fig. 7. (A) PA membrane SEM analysis; (B) EDS analysis of
atom distribution on the membrane surface.
cis-DDP incorporation qualitatively. Fig. 6 contains the IR spectrum of
cis-DDP and of PA TEOS CPTES incorporated with cis-DDP.
The band at 1100 cm− 1, typical of silica (SieOeSi), confirms PA
membrane functionalization with TEOS and CPTES [20,29]. Vibrations
between 698 and 695 cm− 1 evidence the presence of CeCl bonds
[30,31]. The band at 1277 cm− 1 is associated with deformation of the
SieC bond, resulting from PA membrane functionalization with the
alkoxide CPTES [29].
The characteristic bands of cis-DPP in Fig. 5 confirm cis-DPP in-
corporation into PA TEOS CPTES. The bands at 3298, 3203, and
1653 cm− 1 are due to primary amine (NH3) vibrations. The band at
1535 cm− 1 corresponds to primary amine (NH3) symmetric and
asymmetric angular deformation. The bands at 1312 and 1295 cm− 1
refer to NH3 symmetric angular deformation, and the intense band at
808 cm− 1 is also typical of the angular deformation of these groups.
É.A. de Souza et al.
Bands below 600 cm− 1 are typical of bonds involving Pt. The band at
509 cm− 1 can be attributed to asymmetric vibration of the PteN bond
[32–35].
3.5. Scanning electron microscopy (SEM) and energy dispersive
spectroscopy (EDS)
Fig. 7 illustrates the PA membrane structure and morphology ob-
tained by SEM and EDS as well as the mapping of the elements carbon,
oxygen, and nitrogen.
The SEM micrograph in Fig. 7A shows the typical topography of PA
pieces prepared by Additive Manufacturing (AM) [1,8]. The PA
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Journal of Inorganic Biochemistry 180 (2018) 171–178
Fig. 8. (A) PA TEOS CPTES SEM and EDS analyses; (B) EDS
analysis of atom distribution on the membrane surface.
membrane surface is rough and irregular due to the AM process. The PA
particles were sintered with CO2 laser, and the particles combined to
form the pieces. Fig. 7B shows the atom distribution of the main PA
constituents.
Fig. 8 contains the SEM and EDS analyses as well as the atom dis-
tribution for PA TEOS CPTES.
The SEM and EDS analyses in Fig. 8 help to confirm the presence of
the functionalizing agents in PA TEOS CPTES. EDS analysis attests to
the presence of C, O, Cl, and Si in the PA membrane, confirming PA
membrane surface coating with TEOS and CPTES. The presence of C is
mainly related to the polymer PA. Au remains in the sample after
sample treatment for the analyses. Cl and Si are exclusively due to the
É.A. de Souza et al.
Fig. 9. (A) SEM analysis of PA TEOS CPTES incorporated with cis-DPP; (B) EDS analysis
of atom distribution on the PA TEOS CPTES surface incorporated with cis-DPP.
alkoxides TEOS and CPTES. This technique demonstrates that the ele-
ments are homogeneously distributed all over the PA TEOS CPTES
surface after functionalization.
Fig. 9 brings the SEM and EDS analyses conducted after in-
corporation of cis-DPP into PA TEOS CPTES.
The SEM micrographs in Fig. 9A clearly show the presence of
crystals due to cis-DDP on the PA TEOS CPTES surface, further con-
firmed by detection of Pt during EDS analysis. Zhu, W. et al. [36] in-
corporated cis-DDP into micelles intended for application as drug car-
riers. The morphology data of these micelles resembled our findings
with respect to cis-DDP crystallinity.
3.6. cis-DDP release into SBF solution
We investigated controlled cis-DDP release from PA TEOS CPTES
into a solution that mimicked body fluid; i.e., SBF solution, at 37 °C, for
177
Journal of Inorganic Biochemistry 180 (2018) 171–178
16
14
12
10
-1
C (mg/mL) x 10
8
-2
0 5 10 15 20 25
Time (h)
Fig. 10. Controlled cis-DPP release from PA TEOS CPTES into SBF solution as a function
of time, as monitored at 208 nm.
24 h. cis-DPP UV–Vis absorption in the SBF solution was measured after
1, 2, 3, 20, and 24 h PA TEOS CPTES incorporated with cis-DPP had
remained in contact with the SBF solution. Fig. 10 presents the con-
trolled cis-DPP release monitored at 208 nm.
PA TEOS CPTES incorporated with cis-DPP became less yellow after
immersion in SBF solution at 37 °C as a function of time, which in-
dicated cis-DPP release. After 2 h, a large percentage of cis-DPP had
been released into the solution. Spectrophotometric analysis revealed
42.6% cis-DPP release into SBF, pH = 7.4, after 24 h.
3.7. Cytotoxic activity
Fig. 11 displays the XTT assays after treatment with GM07492A
cells.
The results of cell viability of culture medium containing cisplatin
released from the membrane showed IC50 of 23.95 μgM.
4. Conclusion
During PA membrane treatment for sample preparation, HCl played
a fundamental role: it promoted a site for interaction between the PA
membrane and the alkoxides, which was further confirmed by XRD, IR,
SEM, and EDS analyses. PA membrane functionalization by the sol–gel
methodology reached a functionalization percentage of 19%, which
increased to 22% after addition of cis-DDP. cis-DDP release within the
first hours was high, which indicated that cis-DDP interacted weakly
with PA TEOS CPTES.
In vitro cytotoxicity studies showed that incorporated cis-DDP effi-
ciency remained unaltered after contact with culture medium for 24 h
and cis-DPP release therein.
New studies on drug release from functionalized membranes must
be accomplished, so that drug release is made slower. Such systems
could have potential application in skin cancer treatment without
producing undesirable side effects.
Our results demonstrated that it is possible to incorporate cis-DDP
and/or other drugs into functionalized polyamide membrane obtained
by Additive Manufacturing and sol–gel methodologies. These meth-
odologies are satisfactory and open good perspectives for skin disease
treatment.
É.A. de Souza et al.
Acknowledgments
The authors acknowledge CNPq 304348/2013-9 (E.J.N.) and CAPES
(Brazilian research funding agencies), FAPESP (São Paulo Research
Foundation) processes 2015/20298-0 (L.A.R.) and 2013/20443-4
(E.A.S.) for financial support, and UNIFRAN Mutagenese Laboratory
(Profa. Denise C. Tavares and Prof. Ricardo A. Furtado).
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