Materials Chemistry and Physics 120 (2010) 518–525

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Materials Chemistry and Physics

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

Catalytic nanomedicine: Functionalisation of nanostructured cryptomelane

Tessy López a,b,∗ , Emma Ortiz b , Mayra Alvarez b , Joaquin Manjarrez b , Mario Montes c ,
Paloma Navarroc, José Antonio Odriozola d
a
Dept. Atención a la Salud, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100, C.P. 04960, Tlalpan, México, D.F., Mexico
b
Lab. Nanotecnología, Instituto Nacional de Neurología y Neurocirugía “MVS”, Insurgentes Sur 3877, col. La Fama, C.P. 14269, Tlalpan, México, D.F., Mexico
c
Departamento de Química Aplicada, Universidad del País Vasco, P-◦ Manuel de Lardizábal 3, E-20018 San Sebastián, Spain
d
Instituto de Ciencia de Materiales US-CSIC, Av. Américo Vespusio 49, Sevilla, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Nanostructured cryptomelanes (KMn 8O16) were synthesized from manganese sulphate and manganese
Received 6 May 2009 acetate precursors by the reflux method. The respectively obtained samples, CRYSO 4 and CRYAc, were
Received in revised form functionalised with hydroxyl, ammonium, sulphate and phosphate groups in order to modify the bio-
24 November 2009
compatibility and surface properties of cryptomelane. Characterization by FTIR and XRD confirmed bond
Accepted 28 November 2009
formation of CRY–NH, CRY S O, CRY–NH, and CRY PO4. In both functionalise samples (CRYSO4-F and
CRYAc-F), IR bands occurred at 1399 cm−1, corresponding to the sulphate species, and 1106 cm−1, related
Keywords:
to phosphate vibrations; along with the OH and NH characteristic vibration bands in the high energy
Catalytic nanomedicine
Cryptomelane
region. Biocompatibility of functionalised samples was tested by implantation of cryptomelane reservoir
Biocompatibility in the basolateral amygdala and caudal nucleus of Wistar rats using stereotactic surgery. The brains of
Molecular photodynamic the rats were processed in order to evaluate any damage associated with the implant. The results showed
that functionalised cryptomelanes did not cause tissue damage or inflammation while not functionalised
cryptomelanes caused cell death.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction it has photosensitivity properties [15–18]. Several studies with sil-

Materials reduced to the nanometric scale normally exhibit
new and improved magnetic, catalytic, optical and biological
properties compared to when they are at larger scales [1–5]. Appli-
cation of nanotechnology in treatment of human disease holds
great promise, and technological advances in nanometric medicine
include pharmaceutical release systems, disease diagnosis and can-
cer treatment [6,7]. Because they are considered safe for the human
organism, organic polymeric bio-nanomaterials such as poly(lactic-
co-glycolic acid) (PLGA), liposomes and intelligent biodegradable
polymers have played an important role in most of these appli-
cations [8–14]. However, one of the disadvantages is related to
the low stability of these materials in the blood stream and their
inability to cross the blood–brain barrier. For this reason, we use
inorganic nanostructured materials. They are easy to manipulate
at the molecular level, can be functionalised with any chemical
species, are porous and have a high capacity for housing drugs
within their structure for direct release at the damaged site; as well
ica and titania functionalised materials were made since 2004 in C6
model over Wistar rats by López et al. [19–22]. Also, nanostructured
titania with zinc phtalocianine was probed in different cellular
cancer lines by photodynamic therapy (PDT). The photocatalytic
action in PDT is cell damage caused by a phototsensitizer (PS) in
the presence of visible light and molecular oxygen, which generates
reactive and cytotoxic oxygen species that cause tumour destruc-
tion [23]. Research is in progress on new PS’s with better properties
like functionalised cryptomelanes to be used in treatment of skin
cancer [24]. In the present study, Octahedral Molecular Sieve (OMS-
2) cryptomelane type manganese oxide (KMn8O16) was prepared
from different precursors, functionalised to make it biocompati-
ble with living tissue, and then implanted in rats for 6 months.
Samples were characterized by FTIR, XRD, BET, pyridine adsorption
and SEM. Also, histopathology analysis of cryptomelane-containing
tissue was performed.
2. Experimental

2.1. Sample preparation

∗ Corresponding author at: Dept. Atención a la Salud, Universidad Autónoma
Metropolitana-Xochimilco, Calzada del Hueso 1100, C.P. 04960, Tlalpan, México,
D.F., Mexico. Fax: +01 52 55 56 71 68 18.
E-mail address: tessy3@prodigy.net.mx (T. López).
CRYAc. Eleven grams Mn(CH3 –COO)2 ·4H2 O (Aldrich) was dissolved in 40 mL dis-
tilled water, and a buffer solution (5 mL glacial acetic acid with 5 g of potassium
acetate (KAc) and 40 mL distilled water) was added. This solution was heated for
30 min at boiling temperature under reflux. A solution of 6.5 g KMnO4 (Aldrich) in
150 mL distilled water was added to the mixture and kept under reflux and vigorous

0254-0584/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.matchemphys.2009.11.049
 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525 519

stirring for 24 h. The resulting solid was filtered, washed with distilled water and
dried at 120 ◦C for 12 h.
CRYSO4 . A solution of 19.8 g MnSO4 ·H2 O (Aldrich) in 67.5 mL distilled water was
prepared and mixed with 6.8 mL concentrated HNO3, then heated under reflux for
30 min. Then, 13.3 g KMnO4 (Aldrich) dissolved in 225 mL distilled water was slowly
added, and the overall mixture kept under reflux with vigorous agitation for 24 h.
The resulting solid was filtered, washed with distilled water and dried at 120 ◦ C for
12 h.
CRYSO4-F and CRYAc-F were functionalised, by adding a solution of ammonium
phosphate 3.35 × 10−4 M to 10 g of each sample and leaving the resulting suspen-
sion in reflux for 72 h. The samples were dried at room temperature for 72 h in an
inert atmosphere. The suspention was then mixed with a 3.78 × 10−4 M ammonium
sulphate solution, kept in reflux for 72 h and then dried at room temperature until
completely dry. The functionalised samples are referred to CRYSO4-F and CRYAc-F.

3. Characterization

3.1. X-ray diffraction

X-ray diffraction patterns were registered with a Phillips PW

1729–1820 using Cu Ka radiation and a 1.5418 Å wavelength. The
applied tube energy and current was 45 kV and 40 mA, respectively. Fig. 1. X-ray diffraction patterns of cryptomelane samples.
The crystalline phase was identified with the ICDD PDF2 file.

implantation, tests were done to determine the presence of deficits

3.2. Nitrogen adsorption that might indicate neurotoxic damage.
The first test was to observe the conduct of the rats and eval-
Nitrogen adsorption–desorption isotherms were determined in uate its ability to hold on; another test was to place the animal in
a Micrometrics ASAP 2020. Specific areas were calculated with a supine position and evaluate the time that the rat return to an
the BET [25] equation and pore size distribution with the BJH
[26] method. Adsorption–desorption branch and total pore volume
were calculated by Gurvistch’s method.

3.3. Fourier transformed infrared (FTIR) spectroscopy

Samples were analyzed with FTIR spectroscopy using Perkin-

Elmer Spectrum One equipment. Cryptomelanes were mixed with
KBr in a 5% sample to KBr proportion, and moulding the powder into
disks by applying pressure. Sixteen scans were done per spectrum.

3.4. Pyridine adsorption – FTIR

The types of acid sites in the sample were determined by pyri-

dine adsorption followed by FTIR spectroscopy using a Nicolet
710SX with an attached pyridine adsorption cell. The equipment
was heated in situ in a cell with interchangeable windows. Sam-
ple tablets previously prepared in a hydraulic press were placed
in the cell, which was evacuated at 1 × 10.6 Torr in situ pressure
and 500 ◦C for 30 min. It was then cooled to room temperature and
the pyridine adsorbed. Excess pyridine was evacuated by heated
desorption.

3.5. Scanning electron microscopy (SEM)

Micrographs of the samples were taken using a Hitachi S-2700

(15 kV).

3.6. Biocompatibility test

Two groups of male Wistar rats (250–270 g) were anaesthetized

with ketamine–xilazine (80 and 12 mg kg−1, i.p., respectively). A
cylindrical device (1 mm diam×1.2 mm height) of functionalised
cryptomelane was then placed in the basolateral amygdala and
the caudal nucleus of the Wistar rats in the first group by means
of stereotactic surgery, using Paxinos and Watson atlas reference
[27]. In the second group, the cannula was inserted without the
device into the same cerebral structure. The rats were left to recover
Fig. 2. (a and b) BET isotherms and pore distribution of cryptomelanes: (a) CRYSO 4
in individual acrylic cages with water and food ad libitum. After and CRYSO4-F; (b) CRYAc and CRYAc-F.
522 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525

Fig. 3. Scanning electron microscopy images of nanostructured bio-photocatalyzers.

original position. After 6 months the rats were sacrificed with an
anaesthetic overdose (sodium phenobarbitol) and perfused with
saline solution.
3.7. Histological study
3.7.1. Hematoxiline and eosine stain
To evaluate the cell and tissue integrity we used a classical
hematoxiline and eosine stain, following the traditional proto-
cols. First we dehydrated slices and deparafinized them with a
decrease gradient concentrations of xilol and EtOH. The slices were
incubated for 20 min with hematoxiline rinsed with water and
incubated for 10 min with eosine, and finally rinsed with EtOH 100%
3.7.2. Cell viability test: trypan blue
Trypan blue is used as a viability cell marker. Live cells without
color and dead cells stained in blue. Trypan blue was prepared with
the following sterilized solutions 10 ×PBS, 1% tirpan blue (Sigma)
in PBS, Tripsine-EDTA. The brain tissue slices were obtained from
frozen sections made in a criostate at −17 ◦C of 10 micras, followed
by a post-fix with PFA 4% at −4 ◦C. Then was incubated with trypan
blue during 10 min and then were rinsed with decrease gradient
concentrations of EtOH.
4. Results and discussion
X-ray diffraction (XRD) of CRYSO4 and CRYAc samples showed
the characteristic peaks of cryptomelane (Fig. 1), localized at

Fig. 4. High resolution transmission electronic micropgraphs images of cryptomelane materials.

 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525 521

Fig. 5. FTIR spectra for pure cryptomelanes (below lines) and functionalised bio-
nanocatalyzers (above lines). (a) CRYAc and CRYAc-F and (b) CRYSO4 and CRYSO4-F.

2& = 12.8◦, 18.5◦, 28.9◦, 37.5◦, 42◦ and 50◦ [28]. The diffractograms
of CRYSO4-F and CRYAc-F samples are similar, indicating that the
crystalline structure was not modified.
Fig. 2 shows the nitrogen adsorption–desorption isotherms and
the corresponding pore size distributions of the conventional and
functionalised cryptomelane materials. The isotherms for all sam-
ples are similar and correspond to Type IV adsorption isotherms
and at low p/p0 values micropore filling of Type I adsorption occurs,
confirming the presence of both micro and macropores. A hystere-
sis loop of H1-type for all nanostructured materials, according to
IUPAC classification, is observed at high relative pressure near to
saturation, suggesting the existence of slit-shaped capillaries with Fig. 6. (a) Sulphate and phosphate ions linked on the surface of cryptomelane.
parallel plates [29]. The pore size distribution of CRYSO4 shows (b) Phospholipid bilayer of cellular membrane and chemical composition of phos-
pholipids. (c) Interactions between phosphate and sulphate ions on cryptomelane
maximum peak at 4.47 nm, which demonstrates the presence of
surface and polar head of phospholipids in celluar membrane.
an interlayer fibers structure. However, CRYSO4-F exhibits a pore
size of 6.46 nm. The CRYAc had almost twice the specific area
(94 m2 g−1) of the CRYSO4 (50 m2 g−1). It has been reported the CRYAc-F sample. This means that biocompatibility was attained
influence of the preparation procedure on the physical properties of without altering the physicochemical, structural, morphological
cryptomelane, notably on the textural properties, mainly due to the and surface properties of the materials. Fig. 4 shows the morpholo-
different sizes and forms of cryptomelane materials obtained [30]. gies obtained by high resolution transmission electron microscopy
Textural changes in the functionalised samples were small, with of cryptomelanes. All materials have nanofibers shapes with diam-
specific area decreasing slightly in the CRYSO4-F, since a stronger eters of 5–10 nm, and the length of these nanofibers vary from a few
sinterization effect in CRYAc-F was observed. hundred nanometers to micrometers. Similar to the SEM observa-
Fig. 3 illustrates the results of the scanning electron microscopy tions, also these HRTEM images suggest the (1, 1, 0) orientation of
studies of the conventional and functionalised manganese oxides the fibers. At the end of one fiber, it can be see the × 2 2 tunnel
materials. All samples contain regular fibrous morphologies structure of cryptomelane.
characteristic of cryptomelane with length less to 200 µm. No mor- The FTIR spectra of the cryptomelanes showed a band at
phological changes were observed in the SEM images, since the 3419 cm−1 corresponding to stretching vibrations of the O–H in
fibres were unchanged, with a slightly larger separation in the H2O and Mn–OH, followed by one at 1525 cm−1 generated by the
522 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525

Fig. 7. FTIR spectra of pyridine adsorption in functionalised cryptomelane samples: (a) CRYAc-F and (b) CRYSO4-F.

symmetrical vibrations of OH flexion (Fig. 5). Bands at 708, 521
and 460 cm−1, corresponded to vibrations of the Mn–O bond in
CRYSO4 and CRYAc. Additional bands occurred after functionalisa-
tion of the samples: A peak at 3123 cm −1 was caused by vibrations
of the amino group (N–H), a band at 1397 cm−1 corresponded to sul-
phate ions and another at 1094 cm−1 to phosphate ions. The SO 4,
PO4 and OH functional groups are linked to the sample surfaces
(Fig. 6(a)). These results indicate that the cryptomelane surface was
successfully functionalised.
On the other hand, the cell membrane is a fluid mosaic of lipids,
proteins, and carbohydrates. Its framework consists of a double
layer of phospholipids. Two layers of phospholipids molecules self
assemble so that their water soluble (hydrophilic) heads form the
surface and the interior of the membrane and the water insolu-
ble (hydrophobic) tails face each other (Fig. 6(b)). The phosphate
and sulphate linked groups in our functionalised cryptomelane
interacts with the extracellular polar head of phospholipids in
membrane as show in Fig. 6(c). The amine groups are posi-
tively charged therefore, attract negative charges on cryptomelane.
They come from the sulphate and phosphate ions. These inter-
actions are stable and generate high biocompatibility with the
cells.
Materials acidity was also characterized by FTIR, and the
pyridine adsorption analysis showed both functionalised cryp-
tomelanes to have Lewis-type acid sites but not Brönsted-type
sites (Fig. 7). This can be inferred by the intense bands observed
at 1444 and 1587 cm−1, which correspond to the vibration of pyri-
dine adsorbed over strong Lewis acid sites. The band at 1573 cm−1
resulted from the vibration of pyridine adsorbed over weak Lewis
acid sites.
The stereotactic surgery used to implant the nanoreservoir
device in the amygdala is minimally invasive and relatively easy
to perform by specialized neurosurgeons, both in small species and
humans (Fig. 8(a)). Six months after implantation, the rats were
euthanized and the brains extracted for further analysis, exposing
the hole where the cannula entered to implant the device (Fig. 8(b)).
In the histopathological images showed in Fig. 9(a)–(c), dark
brown fragments that correspond to functionalised cryptomelane
implant can be observed. These fragments are distinguished from
cerebral tissue colored in a rosy color. In appearance, brain tissue
was altered, in comparison to the control (Fig. 9(d)–(f)). Regarding
to cellular distribution, surrounding cells around the material, seem
to be adapted all over the implant outline. However, cellular mor-
phology is apparently normal, since the nucleus and membrane are

Fig. 8. Stereotaxic surgery (a). Rat brain (b) with arrow indicating cannula entrance point. Histological sections showing necrosis (c) and haemorrhaging (d) in tissue around
pure cryptomelane implants.
 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525 523

Fig. 9. (a–c) Micrographics of cerebral court treated with hematoxiline–eosine stain of medial amygdale rat at 5×, 10× and 40×, respectively of functionalised cryptomelane
implant. (d–f) Treatment with trypan blue at 5×, 10× and 40×, respectively.

clearly defined. Minimal scar tissue was observed. The results of try-
pan blue staining for the same material are shown in Fig. 9(d)–(f).
In the photographs the implants can be clearly observed in dark
brown color. In this tint type the integrity of the cellular mem-
brane is evaluated, and indicates if the cell is alive or dead. In these
cuts can be observed that membrane integrity of the cell remains
intact, because of the clear blue color of the tint. Joining the results
observed with the hematoxiline–eosine together with those of try-
pan blue then we can say that the functionalised cryptomelane
material did not altered the cell survival and no severe collateral
damages were caused, due to the absence of inflammation and
minimal scar tissue.
Fig. 10 shows the histological studies of the tissue around
of conventional cryptomelane implant. As can be observed in
Fig. 10(a)–(c), the scaring caused by the material was severe, indi-
cating that the quality of the tissue is bad. In the scar surrounding
area of we observed healthy cells, since they show well-defined
nuclei and membranes. However, the same as with the material
functionalised an alteration is observed in the distribution pattern
and cellular orientation when we compare these with intact tis-
sue. It is necessary to point out that when the histological cut was
made the tissue was damaged since it broke before any external
manipulation. From Fig. 10(d)–(f) we can observe damaged cellu-
lar tissue, the tint is dark blue which indicates that conventional
cryptomelane material, besides altering the orientation and cellu-
lar distribution of the nervous tissue affects in a negative way cell
survival, for that reason we conclude that implants of conventional
cryptomelane are not biologically compatible with the brain tissue.

Fig. 10. (a–c) Micrographics of cerebral court treated with hematoxiline–eosine stain of medial amygdale rat at 5×, 10× and 40×, respectively of conventional cryptomelane
implant. (d–f) Treatment with trypan blue at 5×, 10× and 40×, respectively.
524 T. López et al. / Materials Chemistry and Physics 120 (2010) 518–525
Fig. 11. Action of conventional cryptomelane over cerebral tissue. (a–c) Hematoxiline–eosine histological studies of conventional cryptomelane action over cerebral tissue.
(d–f) Intact tissue at 5×, 10× and 40×, respectively. (g–h) Damaged tissue by conventional cryptomelane. (i–k) Hematoxiline-eosine studies of functionalised cryptomelane
at 5×, 10× and 40×, respectively (Intact tissue is observed).
Fig. 11 shown cellular tissues with conventional cryptomelane
implant. In Fig. 11(a)–(c) we can observe in the border of the
scar how both, tissue and implant are strongly linked and through
invagination process tissue and implant are permanently joined.
This leads to scaring process, leaving the tissue seriously damaged.
When the same area was stained with trypan blue, a dark col-
oration was obtained reinforcing the H–E observations related to
cell membrane damage. These biological results prove the evidence
of biocompatibility of the functionalised cryptomelane. Cryptome-
lane materials have been demonstrated to be photoactive materials
with potential applications in light induced processes. These prop-
erties make functionalised cryptomelane a promising material for
further applications in PDT as an alternative in the treatment of
certain types of cancer.
5. Conclusions
New functionalised, nanostructured cryptomelane compounds
were synthesized. These materials exhibited surface hydroxyl,
ammonium, sulphate and phosphate groups. The obtained solids
have a high concentration of Lewis acid sites. When implanted in
the brains of Wistar rats, no changes in cellular morphology as well
as effectively scaring were observed, which is indicative of their
biocompatibility with brain tissue. These results suggest that func-
tionalised nanostructured cryptomelanes are promising materials
to be used in cancer treatment.
Acknowledgements
The authors thank the Universidad Autónoma Metropolitana,
CONACYT-FONCICYT Project 96095, the Instituto Nacional de Neu-
rología y Neurocirugía in México and the CYTED program (PI0269)
for financially and technically supporting this research, also D.H.
Aguilar for technical support.
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