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ISSN:2155-952X

Journal of Biotechnology & Biomaterials

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Special Issue Title: Biosof Materials

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Research Article Open Access

Biotechnology & BiomaterialsLópez et al. J Biotechnol Biomaterial 2011, S4

http://dx.doi.org/10.4172/2155-952X.S4-001

ISSN:2155-952X JBTBM, an open access journalResearch on Tissue Science and EngineeringJ Biotechnol Biomaterial

*Corresponding author: Tessy López, PhD, Insurgentes Sur 3877, Col La Fama,CP 14269, Tlalpan, México, DF Tel: +52 55 56063822 ext 5034; Fax: +52 55 55288036; E-mail: [email protected]

Received June 03, 2010; Accepted November 13, 2011; Published November 13, 2011

Citation: López T, Alvarez M, Arroyo S, Sánchez A, Rembao D, et al. (2011)Obtaining of SiO

2Nanostructured Materials for Local Drug Delivery of Methotrexate.

J Biotechnol Biomaterial S4:001. doi:10.4172/2155-952X.S4-001

Copyright: © 2011 López T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author andsource are credited.

López T1,2,5*, Alvarez M2, Arroyo S2,6, Sánchez A3, Rembao D5 and López R2

1Depto. de Atención a la Salud, Universidad Autónoma Metropolitana-Xochimilco, Calz. Del Hueso 1100, Col. Villa Quietud, 04960, Tlalpan, México, DF 2 Laboratorio de Nanotecnología, Instituto Nacional de Neurología y Neurocirugía “MVS”. Insurgentes Sur 3877, Col. La Fama, C. P. 14269, Tlalpan, México 3 Laboratorio de Neuropatología, Instituto Nacional de Neurología y Neurocirugía “MVS”. Insurgentes Sur 3877, Col. La Fama, C. P. 14269, Tlalpan, México 4Laboratorio de Patología Experimental, Instituto Nacional de Neurología y Neurocirugía “MVS”. Insurgentes Sur 3877, Col. La Fama, C. P. 14269, Tlalpan, México 5 Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118 USA6 Escuela Superior de Medicina. Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo Tomas, Delegación Miguel Hidalgo, C.P. 11340,

México, DF

Abstract

Local delivery of drugs has become an alternative to overcome several side effects of conventional drugadministration methods. Because of their novel properties, nanomaterials for controlled drug release have shownenormous potential in the treatment of cancer. In the present work a chemotherapeutic agent, methotrexate (MTX)

was encapsulated into a sol-gel SiO2 matrix, as a reservoir for local controlled release in the treatment of a solidbrain tumor like Glioblastoma Multiforme (GBM) in an animal model. In order to study interactions between the drugand the SiO

2, several physicochemical characterization techniques were carried out. FTIR spectroscopy showed

evidence drug incorporation into the silica matrix as the presence of bands at 1504 cm -1, 1604 cm-1 and 1637cm-1 reveals. This is supported by UV-Vis results, since silica spectrum acquired new features. The materials aremesoporous with a mean pore diameter about 78 Å. In vitro drug release was performed by UV-Vis spectroscopyfollowing the main absorption band of MTX at 348 nm. Drug release was followed and monitored by 16 days. For invivo evaluation GBM model was performed in Wistar rats once the tumor was developed a reservoir was placed bystereotactic surgery. After three months of tumor inoculation animals treated with MTX-SiO

2survived while in those

untreated survival did not exceed 30 days.

Keywords: Nanomaterials; Methotrexate; Sol-gel silica; Cancer;Controlled drug release

Abbreviations: MX: Methotrexate; GBM: GlioblastomaMultiorme; FIR: Fourier ransormed Inrared; GA:Termogravimetric Analysis; H-E: Hematoxiline Eosine

Introduction

Due to the possibility to avoid undesirable side eects, localadministration o therapeutics represents an excellent alternative totreat some reractory diseases like cancer [1]. Methotrexate (MX) is ananti-cancer drug that intereres with olic acid pathway, by competitively inhibiting dihydroolate reductase, preventing its activation to olinicacid [2]. Tis drug is administered or the treatment o neoplasticdiseases such as gestational choriocarcinoma, invasive chorioadenoma,acute lymphocytic leukemia, meningeal leukemia, breast cancer certaintypes o cancer o skin, head, neck and lung and recently in brain

tumors [3]. Its side eects involve: stomach pain, nausea, vomiting,anorexia, diarrhea and mouth ulcers.

Nanomaterials and particularly inorganic nanoparticleshaveemerged as ecient carriers or a variety o drugs [4,5] and researchin this eld has increased during the last years. Te search or ecientand non-toxic carriers to deliver drugs into cells has been a challengingand interesting area o research. In the past decades, many carriers havebeen investigated extensively. Tese carriers can be classied into ourmajor groups: viral carriers, organic cationic compounds, recombinantproteins and inorganic nanoparticles [6]. Among the advantages o inorganic nanoparticles, suitability to be used at physiological conditionsand low toxicity are the most relevant. o make possible their use inbiomedical applications, surace unctionalization is essential and –

NH2, -COOH and –SH groups play an important role or anchoring o biomolecules [7]. Regardless to the synthesis o inorganic nanoparticlesthere are several reported techniques, but one o the most versatile is

the sol-gel process, because o it is helpul or encapsulation o drugsand biological molecules [8,9]. Besides, unctionalization is easy at thebeginning o the synthesis. By controlling synthesis parameters, sol-gel

nanomaterials with desirable properties can be obtained and almost any drug can be encapsulated. With the development o nanotechnology and the subsequent advances in nanomedicine, diseases like cancer,which commonly require systemic therapeutic approach, could beclinically managed with local or regional strategies directly at thetumor site [10]. In this research, we investigated a biocompatible silica-based nanomaterial with MX encapsulated or controlled drug releaseapplications in order to use it as a reservoir or local treatment o GBM.

Group n Description

1 6Control of the procedure. Sterotactic injection of saline solutionwithout tumor.

2 6 Control of tumor growth without any treatment.

3 10 Tumor + MTX (260µg/2µl)4 5 MTX/SiO

2without tumor

5 10 Tumor + MTX/SiO2

Table 1: Animal test.

Obtaining of SiO2

Nanostructured Materials for Local Delivery of

Methotrexate

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Citation: López T, Alvarez M, Arroyo S, Sánchez A, Rembao D, et al. (2011) Obtaining of SiO2

Nanostructured Materials for Local Drug Delivery of Methotrexate. J Biotechnol Biomaterial S4:001. doi:10.4172/2155-952X.S4-001

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Materials and Methods

MX-SiO2

synthesis

10 g o Sol-gel SiO2

were prepared at pH 7, using tetraethoxisilane(EOS, SIGMA 98%) and ethanol, as previously reported [11-13]. 500mg o MX (Medsatrexate®) were added during the synthesis, in orderto encapsulate the drug. Te obtained gel was dried at room temperatureuntil solvent was completely removed. Te solid was crushed into a nepowder. A SiO

2reerence sample (without MX) was prepared under

the same conditions.

Characterization

FIR: Inrared spectra were taken in a NICOLE FIR spectrometer model MAGNA 560 with resolution o 4 cm -1, 100 scansand equipped with DGS detector. Te solid (5%wt) was milled withKBr and then 100 mg o this mixture were compressed until a thin and

transparent waer was obtained.

UV-Vis spectroscopy: Measurements were perormed in a VarianSpectrometer, Cary-1 with integration sphere. Te spectra o thepowders were taken as obtained.

GA: Te resulting gels were analyzed in a Shimadzu model D-30in conditions o static atmosphere. Te heating rate applied was 10°Cmin-1.

In vitro drug release: 50 mg waers o MX-SiO2

were made and

placed in 50 ml o deionized water at room temperature. Te sameprocedure was carried out using synthetic cerebrospinal uid. Terelease test was carried out twice. Aliquots were taken at periodical

intervals and analyzed by UV-Vis spectroscopy ollowing the band at348 nm. Calibration curve was made and concentration at time t wascalculated using the Lambert- Beer law.

In vitro cytotoxic test

Cells o the 98G cell line were cultivated in 24-well plates until getthe 90% o conuence and treated with dierent compounds (CisPlatin,SiO

2and MX/SiO

2) with dierent doses (0.15 mg, 0.25 mg, 0.5 mg

and 0.75 mg/mL) during 12 hours to reach the cytotoxic eect. Temedium was removed by suction and then washed twice with sterilePBS, methanol was added up to completely cover and lef it to stand or10 minutes, the cells were stained with crystal violet (ethanol at 10%)or 30 minutes and nally, the plates were washed extensively. Cellular

count was made to estimate cell dead percentage.Animal model: Animal experiments were made according to the

CONCENTRATION0.15 mg/ml

LIVINGCELLS

DEADCELLS

% OF LIV-ING CELLS

% OF DEADCELLS

TOTALCELL

CONTROL 135, 000 15, 000 90 10 1.5X105

SiO2 23, 400 106, 600 18 82 1.3X105

Methotrexate/SiO2

3, 200 156, 800 2 98 1.6x105

Table 2: Cytotoxic effect of Mtx/SiO2 compared to SiO2 and to a control group with-out treatment at 12 hours of exposure in a dilution of 0.15 mg/ml.

group Description Findings

1 surgical procedure Inammation associated to injection

2 Tumor 17% survival. Fast cell proliferation, inammation

and highly invasive cell behaviour

3 Tumor+MTX75% of the animals died 33 days later of inltration.

No signicant therapeutic effect was observed

4 MTX/SiO2

100% survival. Mild inammation and macrophages

were observed around particles

5 Tumor+ MTX/SiO2

80% survival. Small lesion area, moderate chronicinammation with abundant macrophages, lympho-cytes and hemosiderophages. After 6 months notumor activity was observed .

Table 3: Summarized in vivo ndings.

Figure1: Chemical structure of Methotrexate (4-ammino-10methyl pteroil-L-glutamic acid).

Figure 2: FTIR spectra of a) SiO2 and b)MTX-SiO2 samples. The inset cor-responds to the high energy region zone.

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)

Wavelenght (nm)

a)

b)

c)

a) MTXb) MTX-SiO

2

c) SiO2

Figure 3: UV-Vis absorption spectra of a) MTX powder, b) SiO2

and b)MTX-SiO

2.

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Mexican Norm (NOM-062-ZOO-1999), in the animal care acility rom the National Institute o Neurology and Neurosurgery. Te studieswere made with the ollowing model: 1 x106 C6 (ACC) cells weresuspended in 2 μL o modied Dulbecco’s medium and were inoculatedin the rontal cortex o male Wistar rats (225-250 g) through stereotacticsurgery. Te coordinates were AP=1.6 (A), L=3.0 y V=2.0.Te animalswere randomly allocated in 5 groups (able 1).

5 ±0.5 mg o nanoparticles were slightly compressed into cylinderso approximately 2x1 mm or intratumoral application in groups 4and 5. Te corresponding cylinder was placed at the 14th day afercell inoculation in the same coordinates where cells were previously injected.

In order to determine therapy eectiveness, the animals were keptunder close observation and tested with physical proves or clinical

evaluation. At the end o the experiment, the animals were sacricedand brains processed. H-E stain was perormed or the histologicalanalysis o the tumor growth and the therapy eect and GFAPImmunohistochemistry or tumor cells detection.

Results and Discussion

FIR spectra o the SiO2

reerence and the MX-SiO2

materials areshown in gure 2. In the high energy region or the SiO

2we observed

a broad band centered at 3437 cm-1 corresponding to O-H stretchingdue to the presence o water. Te band at 3643 cm-1 is attributed to O-Hbonded to Si atoms. In the MX-SiO

2sample, these bands appear at

3332 cm-1 and 3652 cm-1 respectively. Te intensity o this broad bandis due to the addition o MX, which contributes with vibrations o

its COOH and N-H groups [14]. Te main dierence between bothSiO

2and MX-SiO

2spectra is in the mid-energy region since three

bands located at 1504 cm-1, 1604 cm-1 and 1637 cm-1 assigned to C=Nand N-H stretching vibrations can be observed, these bands has beenpreviously reported or pure MX [15].

Both samples exhibited same eature bands in the low-energy region, the observed bands at 1099 cm-1 and 808 cm-1 are attributableto the asymmetric and symmetric siloxane (O-Si-O) bond vibrations,respectively.

UV-vis spectroscopy

UV-Vis spectroscopic analysis provides more inormation about

the incorporation o the drug into the silica matrix. Tis technique isthe most commonly employed or MX determinations. MX absorbsstrongly in the UV-Vis region due to the presence o heteroaromaticpterine chromophore. Te main absorption bands o pure MX aresituated at the wavelengths o 255 nm, 302 nm and 352 nm. When MXis incorporated into SiO

2, the UV-Vis spectrum o SiO

2is modied

(Figure 3) since a broad band around 302 nm is observed, but thepresence o two small shoulders at 254 and 352 nm can be detected.Tese bands can be attributable to the presence o MX and the widtho the band is due to overlapping o MX and SiO

2characteristic bands.

N2 adsorption analysis

Te adsorption-desorption isotherms o SiO2

and MX-SiO2

are

showed in gure 4 and corresponds to a type IV (IUPAQ classication).Its hysteresis loop is associated with capillary condensation taking placein mesopores, and the limiting uptake over a range o high p/p°. Teinitial part o the ype IV isotherm is attributed to monolayer-multilayeradsorption since it ollows the same path as the corresponding part o a ype II isotherm obtained with the given adsorptive on the samesurace area o the adsorbent in a non—porous orm. Te BE [ 16]surace area value was 371 m2/g, while pore size distribution curveshowed a broad peak rom 30 to 170 Å centered at 78Å. Te pores o theobtained material are in the mesopore range. Hysteresis o the isothermis type H

2, characteristic o solids consisting o particles crossed by

nearly cylindrical channels or made by aggregates o spherical particlesand pores with relative non-uniorm size or shape [17]. Te observed

hysteresis even at lower relative pressure can be associated with presenceo some micropores [18].

Termal analysis: Termograms o the samples are showed ingure 5. In MX-SiO

2a mass loss observed between 24°C to 100°C is

related with decomposition o the drug, solvent residues and removalo the surace adsorbed water o the material. Tis rst loss was about10%. Between 100°C and 800°C the sample experiments a gradual

variation in weight with a nal mass loss o 26%. Tis second step isrelated with the partial dehydroxylation o the SiO

2surace as well as the

0,0 0,2 0,4 0,6 0,8 1,0

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450

500

10 100 1000

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P o r e V o l u m e ( c m 3 / g )

Pore Diameter (Å)

V o l u m e S T P ( c c / g )

P/Po

MTX-SiO2

0,60 0,65 0,70 0,75 0,80 0,85 0,90 0,95 1,00

100

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500

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d V / d ( r p )

rp/nm

A d s o r b e d V o l u m e S T P ( c c / g )

Relative pressure (P/P0)

SiO2

Figure 4: N2

adsorption-desorption isotherm and pore size distribution curve of MTX-SiO2

and SiO2

materials.

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Citation: López T, Alvarez M, Arroyo S, Sánchez A, Rembao D, et al. (2011) Obtaining of SiO2 Nanostructured Materials for Local Drug Delivery of Methotrexate. J Biotechnol Biomaterial S4:001. doi:10.4172/2155-952X.S4-001

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Tese results suggest that the use o non-degradable materialsrepresents a good alternative to biodegradable polymers in which iscommon to observe more than two steps, not allowing an adequatekinetic study. Narayani et al. [21] reported on the synthesis o biodegradable microspheres where MX was covalently linked togelatin. Tey carried out in vitro release test and the period was about7-11 days, in their experiment release rate was dependent o surace areathey ound that the largest surace the slower release. Sol-gel materialsexhibit large surace area values, moreover porosity represent andadditional advantage, since pores with dierent sized can be obtained

leading to dierent release rates o the drug.

Since drug stability must be assed, ull spectra in the release testwere collected. In Figure 8 some spectra are showed. We observed thatdrug was release without modication and the characteristics bandswere preserved afer 12 days. In order to veriy that no other compoundcan be desorbed rom silica surace, same procedure was ollowedwith pure SiO

2. Our material was used without any treatment and the

observed spectra indicate that afer 5 days no modication that can beattributable to any other molecule present in silica surace was detected.

In vitro cytotoxic test

Cell culture: Te MX/SiO2

showed a clear eect on 98G cells,observing no cellular damage in the cells without treatment (Figure9A) whereas in the rest is notable the increase on cell death percentageas nanoparticles concentration increases (concentration o 0.15 mg/mlwith 98% o cell death, 0.25mg/ml with 98.8% o cell death, 0.50mg/mland 0.75mg/ml get 100% o cell death).

In other work, polymeric chito-PEG nanoparticles with MX wheresynthesized [22], they ound that higher eed ratios o drug leaded tolower drug loading eciency, and in vitro cytotoxicity in melanomacells showed that cell killing is directly related with MX content,more over pure MX cytotoxicity was higher than MX-incorporatedpolymeric nanoparticles.

Animal model:

a) Saline solution: We did not nd any change in the clinical behavior.In the histological study we ound the cortical puncture in the

0 200 400 600 800

0

20

40

60

80

100

W e i g h t ( % )

Temperature

a)

b)

c)

Figure 5: Thermograms of a) MTX, b) MTX-SiO2 and c) SiO2.

0 100 200 300 400

0,0

0,1

0,2

0,3

0,4

0,5

0,6

C u m

m u l a t i v e M T X r e l e a s e ( µ g )

Time (hours)

Figure 6: Release curve of MTX in deionized water.

unctionalizing agent. In pure MX, a three-step process is observed:the rst loss occurred at 85ºC and is associated with endothermicdehydration o the commercial sample associated with a mass loss o about 11% while decomposition o MX occurs at 252ºC.

MX release study

Several studies o drug release rom silica materials have been madeand have proved their suitability or controlled release applications[19,20]. In our silica sol-gel materials, we ollowed the release or amonth in deionized water and or 5 days in synthetic cerebrospinal uid.In deionized water, MX was released in two steps as can be seen in thecurve (Figure 6). Te rst step is related with the release o the druglocated on or near the surace o the nanoparticles and with the poorly entrapped drug molecules. In the rst two hours o the experiment,data showed an exponential growing. At t=72, release process becomesslower; this can be explained due to the diusion o the medium intothe SiO

2network, releasing molecules that are deeper occluded. In

synthetic cerebrospinal uid the proles showed that methotrexatereleases in controlled and sustained way (Figure 7).

Te synthesis method allows the ormation o a net o SiO2 encapsulating the drug by weak interactions to be released by diusionthrough the pores.

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Time (hr)

Concentration

Figure 7: Release prole of MTX/SiO2 in synthetic cerebrospinal uid in 120hours.

Group 1: Control – without tumor or drug

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cerebral section without inammatory response and presenting amild detachment o the leptomeninges (Figure 10a).

b) Puncture: We did not observe changes in the physical examination.Histological analysis: we ound the puncture site like a corticaldepression with the presence o a slight astrocytary reactivephenomenon, movement o the microglia (inammation at thepuncture site) and leptomeningeal swelling (Figure 10b).

Te rats showed changes in the physical exam rom day 16 post-

inltration mainly characterized by predominant right muscularweakness o the hindlimbs, adding in the next days, a general aectiono motor capacity.

Te rats died rom day 19 to 30 post-inltration, just one rat in reeevolution survived without clinical alterations, sacriced at 6 monthspost-inltration to study the tumor viability by histology analysisnding ventriculomegaly and a little area o necrosis (no viable tumor)proving a ailed tumor inoculation.

Histopathological analysis: the intracerebral inoculation o C6 linehas a similar behavior to Glioblastoma Multiorme (GBM), presenting

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( a . u )

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)

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MTX release

200 300 400 500 6000,0

0,2

0,4

0,6

0,8

A b s .

long de onda (nm)

SiO2/deionized water

Figure 8: Representative UV-Vis spectra of a) MTX-SiO2 and b) SiO2 in saline solution. The inset corresponds to MTX solution.

Figure 9: Cytotoxic effect of the MTX/SiO2 nanostructured material at different concentrations in T98G cells after 12 hours of incubation. A) Cells without treatment,B) Cells treated with a concentration of 0.15 mg/ml, C) Cells treated with a concentration of 0.25 mg/ml, D) cells treated with a concentration of 0.5 mg/ml, E) cellstreated with 0.75 mg/ml.

Group 2: Control - tumor growth without drug administration

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a) b)

Figure 10: a) Control without tumor. Saline microinjection. Cerebral cut with cortical puncture site, inammatory response and mild detachment of leptomeninges

and b) Control without tumor. Puncture only. We observe the puncture site as a cortical depression with mild localized inammation.

Figure 11: Tumor growth (C6 cell line) a) 14 days b) 18 days, c) 24 days and d)30 days cells inoculation.

the characteristic palisading necrosis with a high mitotic rate, a greatcellular pleomorphism, anaplasia, intense nuclear hyperchromasia,endothelial hyperplasia, with a distinct expansion phenomena anddierent presentation patterns. Tis tumor grows rapidly aectingalmost the entire cerebral hemisphere causing the death o the animalmodel ( Figure 11).

Physical test: the majority o the group showed a similar behaviorto the control group o tumor growing. Just tree rats had a dierent

behavior; one o them showed a delay in the presentation o the clinicalalterations; the rest, two rats, did not show changes in the physicalexamination. About survival, eight rats died rom day 21 to 33 post-

inltration. In the histopathological analysis we observed the naturalevolution o the tumor growth without changes due to the drug. Wesacriced the two survival rats 6 months later to veriy the tumor

viability, nding in the histological analysis and immunohistochemistry the presence o mild inammation reaction and suggestive results o non-viable tumor.

Group 4: Methotrexate/SiO2

without tumor

Physical exam: without clinical alterations. Survival: the ve ratssurvived without any clinical changes. Tey were sacriced 6 months

post-inltration or the histological analysis nding macrophages withparticles o nanomaterial and a mild inammatory response. Teseresults suggest the biocompatibility o the compound (Figure 12).

Group 3: Methotrexate with tumor

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Group 5: Methotrexate/SiO2

with tumor

It was applied MX/SiO2

(5.2 to 6.5 mg) to ten rats with a tumorgrowth o 14 days, leaving in ree evolution and making clinicalevaluation every day. In the physical exam ve rats showed a slightweakness o the right hindlimb at 19th day post-inltration o C6,however, two o them recovered without later clinical alterations.One rat showed muscular weakness at 25th day post-inltrationbut it recovered in a ew days without later alterations. Another o

those rats showed a ast tumor growth evolution and died at 29th day postinltration (natural tumor evolution). Te fh rat progressedslowly showing motor dysunction evident at 35th day post-inltrationand died at 38th day. Te ve missing rats maintained without clinicalalterations. Te survival eight rats were sacriced in two groups, 4 ratswere sacriced 4 months post-inltration to veriy the tumor viability,and the other 4 rats were sacriced 6 months post-inltration or thehistopathological analysis. Histological analysis: At the microscopein the slides o the survival rats sacriced 4 months post-inltrationwe ound lesion area, moderate chronic inammation with abundantmacrophages, lymphocytes and hemosiderophages. We observed thepresence o a material with calcied appearance.

We ound meningothelial hyperplasia and necrosis areas in the

lesion site. We did not nd neoplastic cells. In the slides o the 4 ratssacriced 6 months post-inltration we ound similar results with mildchronic inammation with abundant macrophages and the presence

o a calcied material (nanoparticles aggregates) without evidence o tumor cells.

In the immunohistochemical analysis we ound an intenseperilesional gliosis (healing) without tumor activity (Figure 14). Teuse o PLGA microspheres containing MX in an animal model o sarcoma 180 was reported by Singh and Udupa [23], they showedthat PLGA-MX loaded material exhibited higher antitumor activity than plain MX, however once the drug loaded has been released cell

prolieration continue and tumor grew.

Te eect o this drug against cancer is well know [24-26], but themain dierence ound in this research was the signicant eect obtainedby the controlled release o the drug (methotrexate) with a minimum

Figure 12: Cerebral sectioning of a rat administrated with MTX/SiO2 but without tumor showing the punction site and the macrophages with particles of nano-material aggregates.

Figure 13: a)Cerebral cut of post-treatment tumor with MTX/SiO2 (3 months). We observe the lesion site with an area of necrosis, mild inammatory response,

nanostructured material conglomeration (calcied aspect), without evidence of neoplastic cells; b) and c) Cerebral cut after treatment with MTX/SiO2 (6 months)

showing mild inammation with abundant macrophages and nanomaterial (calcied aspect) without tumor cells.

About the histopathological analysis o the MX/SiO2

therapy, wecan say that it is a avorable treatment with a cell response to an areao necrosis and corresponding to oreign body (mild reaction) withoutevidence o neoplasia, where it should be noted the clinical behavioro the animals, ound without systemic involvement. Te drug alonedid not aect natural evolution o the disease in the vast majority o animals and, with the immunohistochemestry, we demonstrated themeningothelial hyperplasia (a normal healing process) discardingtumor activity. Te MX/SiO

2therapy showed a signifcant result when

compared with the MX fnding that 80% o the treated rats with MX/SiO

2survive up to 6 months post-infltration o C6 cells instead with

methotrexate survived only 2% o rats.

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dose within the inorganic matrix o SiO2

and demonstrating that thesame dose o the single drug do not cause this eect, urthermore theadvantage o being a local therapy avoiding the characteristic adverseeects o the chemotherapy. In our materials, MX-SiO

2material

showed clearly higher cytotoxic eect than MX drug, both in vitroand in vivo. Tis can be explained by the enhancement due to thesynergistic eect between our sol-gel modied silica (pH and additives)and the drug. Our materials showed to be active against malignant cells,however we need to remark that in healthy animals (without tumor) nocytotoxic eect was observed.

Conclusions

A unctionalized inorganic nanostructured reservoir was obtained.In vitro experiments showed that MX was encapsulated and releasedin a controlled way rom the SiO

2structure, due to the appropriate

control o physicochemical properties o the oxide, since the beginningo the synthesis. Pore size and surace area o the material as well asthe presence o surace OH groups play an important role. Sol gelnanostructured materials have potential as drug delivery vehicles orlocal administration o chemotherapeutic agents, with the possibility toeradicate adverse side eects.

Te MX/SiO2

therapy has shown an important modication o the natural evolution o brain tumors GBM type in animal models,

increasing the survival signicantly o the aected animals with animportant reduction in tumor size. In contrast, the drug itsel to suchsmall doses showed no signicant changes in tumor behavior. Teseresults strengthened the MX/SiO

2therapy as a promising treatment

or signicant anti-carcinogenic eect avoiding the adverse eectscaused by systemic therapies.

Aknowledgements

Authors want to thank to CONACYT 99243 and FONCICYT-CONACYT 96095projects for nancial support. To ICyTDF, UAM, INNN for the facilities and to I.

Sánchez, E. Ortiz-Islas, J. Bustos and J. Manjarrez for technical assistance.

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