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Jun 6, 2018 - carrier for Chagas diseases treatment, Microporous and Mesoporous Materials ... benznidazole delivery into T. cruzi parasites was assessed.
Accepted Manuscript Chitosan grafted into mesoporous silica nanoparticles as benznidazol carrier for Chagas diseases treatment Egídio Paulo Francisco Nhavene, Wellington Marcos da Silva, Roberto Reis Trivelato Junior, Pedro Lana Gastelois, Tiago Venâncio, Regiane Nascimento, Ronaldo J.C. Baptista, Carlos Renato Machado, Waldemar Augusto de Almeida Macedo, Edésia Martins Barros de Sousa PII:

S1387-1811(18)30345-7

DOI:

10.1016/j.micromeso.2018.06.035

Reference:

MICMAT 8988

To appear in:

Microporous and Mesoporous Materials

Received Date: 13 April 2018 Revised Date:

6 June 2018

Accepted Date: 22 June 2018

Please cite this article as: Egí.Paulo.Francisco. Nhavene, W.M. da Silva, R.R. Trivelato Junior, P.L. Gastelois, T. Venâncio, R. Nascimento, R.J.C. Baptista, C.R. Machado, W.A. de Almeida Macedo, Edé.Martins. Barros de Sousa, Chitosan grafted into mesoporous silica nanoparticles as benznidazol carrier for Chagas diseases treatment, Microporous and Mesoporous Materials (2018), doi: 10.1016/ j.micromeso.2018.06.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Benznidazol

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Chitosan grafted into Mesoporous Silica Nanoparticles as benznidazol carrier for Chagas diseases treatment Egídio Paulo Francisco Nhavene1, Wellington Marcos da Silva1, Roberto Reis Trivelato Junior3,

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Pedro Lana Gastelois1, Tiago Venâncio2, Regiane Nascimento4, Ronaldo J. C. Baptista4, Carlos Renato Machado3, Waldemar Augusto de Almeida Macedo1 and Edésia Martins Barros de Sousa1*

Centro de Desenvolvimento da Tecnologia Nuclear - CDTN/CNEN. Av. Presidente Antônio

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1

Carlos, 6627 – Campus da UFMG, Belo Horizonte, MG, Brazil, Zip code: 30270-901. Departamento de Química, Laboratório de Ressonância Magnética Nuclear, UFSCar, São Paulo,

SP, Brazil, Zip code: 13565-905. 3

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Departamento de Imunologia e Bioquímica, Laboratório de Genética Bioquímica, ICB, UFMG,

Belo Horizonte, MG, Brazil, Zip code: 31270-901l. 4

Departamento de Física, UFOP, Ouro Preto, MG, Brazil, Zip code : 35400-000.

Abstract

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*Corresponding author: [email protected]

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The use of chitosan functionalized silica for benznidazole delivery in the treatment of neglected disease such as Chagas disease is one of the forms not yet explored, but with great potential for this therapy, as little is known about nanoformulations for the treatment of Chagas disease. In

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this work, we used chitosan-succinate covalently attached to the surface pore of MSNs to act as anchor for benznidazole as a delivery system. The samples were characterized structurally and chemically with multiple techniques. The applicability of functionalized MSNs as platforms for benznidazole delivery into T. cruzi parasites was assessed. The results demonstrate that the proposed system is a potential promising nanoplatform for drug and gene delivery targeting neglected diseases such as Chagas disease.

Key words: silica nanoparticles; surface functionalization; benznidazol delivery 1

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Introduction Mesoporous silica nanoparticles (MSNs) have revolutionized nanobiotechnology due to their low toxicity and high drug loading capacity, thus they are used in controlled and target drug

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delivery systems [1]. The MSNs-like MCM-41 (Mobil Composition of Matter No. 41) samples are solid inorganic materials which possess unique physicochemical properties such as ordered pore network structure with hundreds of empty channels, tunable pore size, high pore volume, and high surface area [2]. Several studies have reported the use of MSNs to absorb or

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encapsulate relatively large amounts of bioactive molecules for drug delivery, which renders them ideal for hosting guest molecules of various sizes, shapes and functionalities [3,4].

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Over the past few years, the MSNs have transformed the field of gene delivery [1]. Among the numerous nanofunctional formulations designed to improve DNA delivery system efficiency, those based on MCM-41 have been extensively investigated, without showing undesirable immune response or off-target effects [5].

The MCM-41 are used as an alternative to viral vectors, as nonviral vectors for safe and efficient gene delivery, because they have a small average size compared to the viruses (20 to

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450 nm), proteins (5 to 50 nm) and genes (2 to 100 nm) [6]. Viral vectors are the most efficient, but still remain less safe than synthetic ones, they are immunogenic and expensive to produce. Thus, the development of alternative methods for nonviral vectors allowing an effective DNA delivery and active molecules into the cells is strongly desirable [7].

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Several studies have shown a great potential for gene delivery with chitosan (CS), bearing advantageous characteristics such as biodegradability, biocompatibility, lipophilicity,

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hydrophobicity and DNA anchoring capacity [8]. Therefore, the MSNs functionalization with chitosan, one of the biopolymers most widely used as drug and gene delivery systems, results in an increase in its potential as drug and gene carrier that can be internalized in cells. Recent studies have shown CS derivatives to have a great potential for drug and gene delivery, with the benefit of the presence of highly reactive groups, mainly hydroxyl and amino ones, which facilitate the packaging of drugs, lacking selectivity for target cells [9]. Classified by the World Health Organization (WHO) as a neglected disease, Chagas disease, also known as American trypanosomiasis, has few alternatives of available treatment 2

ACCEPTED MANUSCRIPT [10,11]. Chagas disease is caused by the flagellate Trypanosoma cruzi, and affects about 6 to 7 million people worldwide [12,13]. Only two drugs discovered in the 60s, 4-[(5nitrofurfurylidene)amino-3-methylthiomorpholine-1,1-dioxide] (Nifurtimox; NFX) and (Nbenzl-2-nitro-1-imidazole acetamide) (Benznidazol; BZ) are available for specific antiparasitic

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treatment [14,15]. These two drugs are effective against the circulating form of the parasite (trypomastigotes) during the acute phase of the disease, but not during the chronic stage [16]. The BZ is the main drug commercially available for the treatment of patients with Chagas disease. Despite its widespread use, it presents

high toxicity, low efficiency, difficulty in

crossing biological barriers and low solubility in aqueous media [15]. To address these problems,

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it is important to develop a functional nanoplatform for carrying bioactive agents like Benznidazol. Nanotechnology is a convenient tool to improve the aqueous solubility and further

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bioavailability of hydrophobic drugs.

Recently, various devices have been proposed for adsorption of a variety of bioactive agents to treat the circulating forms of the parasite T. cruzi. Sequen and collaborators [17] used nanoporous hydrogel particles produced with poly (N-isopropylacrylamide) (poly(NIPAm)) and N,N9-methylenebisacrylamide (BAAm) coupled with chemical baits via amidation reaction. This system was able to sieve the nanoporous structure, allowing proteins to penetrate inside the

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particles depending on their molecular weight and shape for diagnosis of congenital Chagas disease. Chaves and collaborators [18] tested semiconductor PEGylated CdS/Cd(OH)2 quantum dots in the labeling of T.cruzi and the images obtained by confocal fluorescence microscopy and transmission electron microscopy indicated that only the endocytic paths of parasites were the

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best ones to understand the endocytosis process and the cellular differentiation. However, there is a lack of studies on the use of MSNs for gene delivery into the T. cruzi. Therefore, the

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evaluation of BZ in a new nanoplatform such as mesoporous silica, which is able to improve its effectiveness in antiparasitic treatment, could turn it into a more promising alternative for the therapy of Chagas disease.

Taken all these considerations into account and due to the lack of reports on the

functionalized MSNs as nanoplatforms to delivery benznidazole into T. cruzi parasites, in this work, we used chitosan-succinate covalently attached to the surface of mesoporous silica nanoparticles to anchor BZ molecules for delivery system. The preparation method and main characterizations of the obtained MCM-41 are investigated. In vitro cytotoxicity tests suggest 3

ACCEPTED MANUSCRIPT that the viability of T. cruzi grows extensively in presence of MCM-41; on the other hand, MSNs functionalized with CS loaded with BZ molecules present a remarkable effect on T. cruzi growth inhibition, indicating the application of this system as potential nanocarrier to treat T. cruzi, thus showing one of the several solutions offered by the nanobiotechnology in the treatment of

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neglected diseases such as Chagas disease.

Materials and Methods Materials

Benznidazole (BZ) commercial tablets (Lafepe), chitosan (CS), pure ethyl alcohol (EtOH), (3-

(HCl),

succinic

anhydride

(Suc)

(C4H4O3),

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glycidoxypropy) ltrimethoxysilane (GPTMS), sodium hydroxide (NaOH), hydrochloric acid tetraethylorthosilicate

(TEOS),

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hexadecyltrimethylammonium bromide (CTAB), were purchased from Sigma-Aldrich (São Paulo, Brazil). The epimastigote form of T. cruzi CL Brener strain were cultivated at 28°C in LIT (Liver Infusion Tryptose) medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cultilab) and 100 µg ml-1 of penicillin and 100 µg ml-1 of streptomycin

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(Gibco – Life Technologies, Carlsbad, CA, USA) according [19].

Synthesis of Mesoporous Silica Nanoparticles - MCM-41 MCM-41 silica was prepared in accordance to a published procedure [20] using commercial CTAB as a template agent in basic conditions. CTAB (2.74 mmol) and NaOH (7.00 mmol) were

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dissolved in 480 mL of Milli-Q® water. The temperature of the mixture was adjusted to 75°C. Subsequently, 22.4 mmol of TEOS were added dropwise to the surfactant solution under

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vigorous stirring. The mixture was reacted for 2 h to give rise to a white precipitate, which was filtered, washed with Milli-Q® water and methanol, and dried at 37°C for 24 h. The surfactant was removed by calcination, which was carried out by increasing the temperature to 550°C under nitrogen flow for 2 h followed by 3 h in air.

Functionalization of Mesoporous silica MCM-41 with GPTMS GPTMS act as covalent crosslinker between the MCM-41 and chitosan process. 300 mg of MCM-41 were dispersed in 20 mL of EtOH by ultrasonication during 30 min and the pH value 4

ACCEPTED MANUSCRIPT was adjusted to 6-7, under magnetic stirring. Subsequently, 1 mL of GPTMS was added dropwise into MCM-41 in EtOH dispersion with magnetic stirring. The mixture was kept under constant magnetic stirring at 50°C for 5 h. At the end, the suspension was filtered and washed thoroughly with Milli-Q® water and acetone followed by drying at 37°C for 2 days. This sample

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was named MCM-41+GPTMS, and it will be used as a bridge to link MCM-41 and chitosan succinate.

Functionalization of Mesoporous silica MCM-41 with chitosan succinate

In the next functionalization step, 250 mg of MCM-41+GPTMS were dispersed in 20 mL of

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EtOH and kept under constant magnetic stirring at 50°C for 2 h. Meanwhile, a solution of Succinic anhydride (Suc) in Pyridine (126mg·mL-1) was added dropwise to a Chitosan (CS)

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solution in hydrochloric acid (HCl) (25mg·mL-1) and was kept under stirring to ensure total solubility with strong agitation at room temperature, and then the reaction pH was adjusted to 7 using sodium hydroxide (NaOH) (3.0M) aqueous solution. After that, this new solution was added dropwise to the solution of 12.5 mg·mL-1 MCM-41+GPTMS in EtOH. This mixture was kept under constant magnetic stirring at 50°C for 5 h. The final materials, named MCM41+GPTMS+CS, were collected by centrifugation (7000rpm) and washed thoroughly with

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acetone followed by vacuum drying at 37°C for 2 days.

Benznidazol drug carrier with MSNs-MCM-41+GPTMS+CS The Benznidazol (BZ) was dispersed in 20 mL of Milli-Q® water and kept stirring to ensure total

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solubility with strong agitation at room temperature. Then 100 mg of the MCM-41+GPTMS+CS sample was mixed with 20mL of BZ aqueous solution and kept under magnetic stirring at room

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temperature for 24h. The mixture obtained, named MCM-41+GPTMS+CS+BZ, was filtered, and the resulting solid was dried in vacuum at 37°C for 3 days.

Trypanocidal assays

The epimastigote form of T. cruzi CL Brener strain were cultivated at 28°C in LIT, with pH 7.4, supplemented with 10% heat-inactivated FBS, plus 100 µg ml-1 of penicillin and 100 µg ml-1 of streptomycin. The culture was maintained in appropriate vials and in its exponential growth, through weekly peaks. To count the number of parasites, these were diluted in PBS (0.15 M pH 5

ACCEPTED MANUSCRIPT 7.2) and erythrosine (4% in PBS) in ratio 1:20. The Epimastigote forms of T.cruzi. CL Brener strain were cultured in 6-well plates with different concentrations of MSNs incubated at 37°C for 24 h and the number of viable cells determined after different time intervals by counting the motile parasites in a Neubauer chamber. The mean values obtained were plotted on a growth

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curve with standard deviation. In addition, to detect any potential anti-trypanosomal activity of the nanoparticles compound, this study was performed with two control groups; in the first control group the epimastigote forms of T. cruzi CL Brener strain were cultivated at 28°C in LIT. In the second control group the epimastigote forms of T. cruzi CL Brener strain were cultivated at 28°C in LIT,

and

Morphological characterization of

the

MCM-41

and the

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Physicochemical

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with the addition of PBS.

functionalized systems

The samples were characterized by Zeta potential (P.ζ), transmission electron microscopy (TEM) and energy-filtered transmission electron microscopy (EFTEM), elemental analysis (CHN), X-ray photoelectron spectroscopy (XPS), solid state nuclear magnetic resonance (SSNMR), and density functional theory (DFT) techniques.

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Electron Microscope images were obtained using a Bunker Nano GmBH, model XFlash detector 410-M and Tecnai G2 - 12 – SpiritBiotwin FEI −120 kV (Hillsboro, OR, USA) at the Microscopy Center of UFMG, Belo Horizonte, Brazil, and EFTEM was performed to increase contrast and retrieve a unique effect that later can be worked upon with the software ImageJ to

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assign different colors to chemical elements and to allow the merging of two or more images. Zeta potential was measured using Zetasizer Nano ZS (Malvern Instruments, Westborough, MA,

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USA). For the analysis, an aqueous dispersion of nanoparticles (0.05mg·mL-1) was prepared. After sonication of the suspension for 5 min, the measurements were made in triplicate. The functionalization rate and drug-loading rate were determined by elemental analysis, which were performed in a Perkin-Elmer CHNS model 2400 (Waltham, MA, USA). In order to elucidate the mechanism of the functionalization, the chemical bonding states of its surface elements on the MCM-41 were characterized using X-ray photoelectron spectroscopy analysis (XPS), before and after the different steps of the functionalization process. High-intensity XPS spectra were obtained using a monochromatized Al Kα X-ray (1486.6 eV) source and an energy analyzer 6

ACCEPTED MANUSCRIPT (Specs, Phoibos-150 Surface Nano Analysis GmBH, Berlin, Germany) that enables high energy resolution and excellent signal to-noise ratio. The signal of adventitious C 1s (284.6 eV) was used as reference for the energy calibration of the survey and the high-resolution spectra. The detailed oxidation states of the elements were obtained from fitting the XPS peaks assuming their

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shape as a convolution of Lorentzian and Gaussian of different components and a Shirley background. In the NMR measurements, Bruker Avance III-400 (Billerica, MA, USA) was used in the preparation of the sample, operating in a magnetic field of 9.4 T. The samples were packaged in 4 mm outer diameter zirconia rotors and capped with a KEL-F cap. For analyzes, the observation frequency

13

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Carbon

C: 100.57 MHz. Probe: MAS - for 4mm rotors. Pulse

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sequence: cross polarization on magic angle spinning with total sideband suppression (CPTOSS). Sample Rotation: 5 kHz. Observation window: 300 ppm (-50 to 250 ppm). Number of scans: 500

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to 30000. Recycle time: 3 seconds. Contact Time: 1 ms. Acquisition time: 34 ms Chemical shift reference: Adamantane (38.5 ppm for CH2 signal). For qualitative observation frequency of

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Silicon analyzes, the

Si: 79.45 MHz. Probe: MAS - for 4mm rotors. Sequence of pulses:

cross polarization on magic angle spinning (CP MAS). Sample Rotation: 5 kHz. Observation window: 500 ppm (-250 up to 150 ppm). Number of scans: 5000 to 15000. Recycle time: 5 seconds. Contact Time: 5 ms. Acquisition time: 34 ms. Chemical Offset Reference: 4,4-

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dimethyl-4-silapentane-1-sulfonic acid (DSS) (0.0 ppm)

Chitosan reactions - a DFT investigation

The first-principles calculations based on density functional theory (DFT) was used to

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investigate the structural and energetic properties of the reactions between Chitosan (C12H24N2O9), GPTMS (SiC9H20O5), Succinic anhydride (C4H4O3) and Benznidazole

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(C12H12N4O3) molecules. The structural configuration of all molecules used in this studied are depicted in the DFT investigation results section. We have calculated the reaction enthalpy (∆H) and binding energy (Eb) through supercell calculations carried out by the Quantum-ESPRESSO software package ) [21]. All structures were considered as fully optimized when all components of forces on atoms were smaller than 0.025 eV/Ang. In those calculations we have used the generalized gradient approximation (GGA), according to Perdew-Burke-Ernzerhof (PBE) [22], as exchange-correlation functional. A 30 Ry energy cutoff was used to truncate the plane-wave basis set, and 300 Ry for the electronic density. The k-grid for Brillouin zone integrations was 7

ACCEPTED MANUSCRIPT 1×1×1 k-points. Also, the total energy and electron charge density were calculated by the FermiDirac smearing method and the ultrasoft pseudo potentials were considered to represent the core electrons. To avoid interactions between periodic images we employed a cubic supercell whose

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edge is 15 Å long.

Results and Discussion Transmission Electron Microscopy - TEM

TEM images of MCM-41, MCM-41+GPTMS+CS and MCM-41+GPTMS+CS+BZ (Figure 1) clearly show the spherical shape, highly ordered mesoporous structures with well-defined

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hexagonal arrays of uniform pores, observed when the electron beam was parallel to the main axis of the mesopores and in the [100] direction when the electron beam was perpendicular to the

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main axis. This is a typical characteristic of MCM-41 mesopores) [20] with the pore diameter measured from the image of about 3.3 nm. Thus, the TEM investigation offers consistent evidence that the ordered structure is preserved in the approach proposed in this work to obtain functionalized nanostructured systems.

a

c

Figure

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b

1.

TEM

image

of

(a)

MCM-41,

(b)

MCM-41+GPTMS+CS,

(c)

MCM-

41+GPTMS+CS+BZ

Energy-filtered transmission electron microscopy - EFTEM The EFTEM, obtained from the MCM-41+GPTMS+CS nanoparticle, combines the chemical information with spatial information, to obtain so called elemental maps showing in a qualitative way the location of specific elements in the sample. As shown in Figure 2, carbon, oxygen and 8

ACCEPTED MANUSCRIPT nitrogen are homogeneously distributed in the MCM-41+GPTMS+CS nanoparticle, suggesting that MSNs were successfully functionalized with the organic moieties chitosan succinate.

b

c

d

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a

Figure 2. EFTEM are indicating the elemental composition of (a) Carbon, (b) Oxygen, (c)

Zeta Potential - Pζ

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Nitrogen and (d) and the overall map of MCM-41+GPTMS+CS nanoparticle.

Zeta potential measurements are used to characterize the residual surface charges of the

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nanoparticles (Table 1). The zeta potential value for MCM-41 is negative (-20.8 mV), due to the high number of silanol groups on the silica surface which is a typical of MCM-41 and this result

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is similar to the values reported in other publications [23]. After the first step of functionalization with GPTMS, the values change to -23.7 mV, probably due a number of glycidoxypropyl groups and epoxy groups present in the end of side, which can contribute to the increase of the surface negative charge. After the second step of functionalization with CS, the potential drastically changes, increasing to + 45.5 mV, due to the presence of a large numbers of free amino groups on the polymeric protonated chains [24]. The addition of BZ reduced significantly the zeta potential to -11.5 mV; this change could be probably attributed to the presence of amides in the aromatic rings in the BZ structure [25]. In short, the change in zeta potentials also indicated the 9

ACCEPTED MANUSCRIPT successful modification of MSN nanoparticles surface. Moreover, the Zeta potential can change the behavior of nanoparticles within in vivo environments, given that they have profound effect on how a nanoparticle acts with and behave in biological environment.

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Table 1. The zeta potential values of MCM-41 and its modified surface samples. Samples

ζ Potential ± SD (mV)

MCM-41

-20.8 ± 0.9

MCM-41+GPTMS

-23.7 ± 1.0 45.5 ± 0.5

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MCM-41+GPTMS+CS BZ

-13.9 ± 0.5

-11.5 ± 0.5

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MCM-41+GPTMS+CS +BZ

Elemental Analysis - CHN

The CHN analysis of MCM-41 matrices and the functionalized ones was used to quantify the organic molecules anchored to its surface and the amount of BZ incorporated into the MSNs materials. The results are shown in Table 2. The MCM-41 showed only traces of C and H, likely due to remnants of CTAB leftover surfactant after calcination. After functionalization, the

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carbon, hydrogen and nitrogen concentrations of MCM-41+GPTMS, MCM-41+GPTMS+CS and MCM-41+GPTMS+CS+BZ increased significantly in relation to the MCM-41, indicating that the matrices were successfully functionalized with CS and the BZ incorporation was evidenced.

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Table 2. Elemental analysis of MCM-41 and its modified surface samples. C(%)

∆C (%)

H(%)

∆H (%)

N(%)

∆N (%)

0.59

-

1.8

-

-

-

2

1.4

2.8

0.9

-

-

MCM-41+GPTMS+CS

18.8

16.7

2.8

-

3.2

3.2

CS-Cs

28.1

Samples

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MCM-41

MCM-41+GPTMS

MCM-41+GPTMS+CS +BZ

28.9

BZ

55.4

5.6 10.1

3.2 6.7

5.1 0.4

5.7

2.5

10.6

10

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X-Ray Photoelectron Spectroscopy – XPS X-Ray photoelectron spectroscopy (XPS) was used to characterize the chemical composition and bonding state of each element present at the surface of the nanoparticles before

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and after the different steps of the functionalization process. The survey and high-resolution spectra were obtained using a Specs Phoibos-150 electron energy analyzer and monochromatized Al Kα X-ray source (1486.6 eV). The spectra were taken in constant analyzer energy (CAE) mode using 100 eV pass energy for the survey and 30 eV pass energy for the high-resolution acquisitions. The C 1s signal of adventitious carbon was used as reference for the binding energy

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calibration. The survey spectra of the different samples are shown in Figure 3.

The survey scan of MCM-41 is shown in Fig. 3(a), where, besides Si and O in the expected

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elemental ratio for the silica, adventitious C 1s peak, at 284.6 eV, is also observed [26]. The survey scan of MCM-41+GPTMS is shown in Fig. 3(b), where the increase of C and O, when compared to the bare nanoparticles spectrum (Fig. 3(a)), reflects the presence of glycidoxypropyl and epoxy groups, indicating that GPTMS has been successfully grafted onto the surface of MCM-41 (Table 2), in agreement with the NMR analysis showed below. A similar response was observed in the MCM-41+GPTMS+CS-Suc spectrum, shown in Fig. 3(c), where

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the additional increase in the C and O peak intensities and the presence of N indicate that the CSsuccinate has successfully functionalized the MCM-41 nanoparticles as previously reported [27]. Moreover, this functionalization has also been suggested by the presence of nitrogen as seen in elemental analysis and confirmed by NMR analysis, discussed below. Though the MCM-

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41+GPTMS+CS-Suc+BZ spectrum, shown in Figure 3-d, also presents a nitrogen peak, an unambiguous confirmation of the functionalization with benznidazol could only be obtained

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from the analysis of the high resolution spectra, as presented below. The surface chemical composition of the different samples, as determined by XPS, is shown in Table 3. The high-resolution spectra of Si 2p for the nanoparticles, before (MCM-41) and after the

first functionalization step (MCM-41+GPTMS), are shown in Figure 4 (a) and (b), respectively. The peak deconvolutions were performed using one main doublet, assigned to SiO2 (103.3 eV and 103.9 eV) and an additional doublet assigned to Si-C (100.9 eV and 102.1 eV) which is present in the GPTMS. This confirms that the intermediate GPTMS molecule is successfully grafted onto the silica surface, admitting further use of the composite nanoparticles to 11

ACCEPTED MANUSCRIPT accommodate guest molecules. The additional small spectral contributions considered in the fittings (around 99 and 97 eV in Fig. 4(a) and (b), respectively), could be due to charge effects.

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O 2s

375 390 405 420

MCM-41+GPTMS+CS-Suc

b)

d)

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MCM-41

Si 2s Si 2p O 2s

N 1s

x60

C 1s

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Si 2s Si 2p

C 1s

OKKL

Intensity

N 1s

O 1s

c)

O 1s

a)

Intensity

N 1s

x75

MCM-41+GPTMS

1200

1000

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375 390 405 420

800 600 400 Binding Energy (eV)

200

MCM41+GPTMS+CS+Suc+BZ

1200 1000

800 600 400 Binding Energy (eV)

200

0

Figure 3. XPS survey spectra of (a) MCM-41, (b) MCM-41+GPTMS, (c) MCM-

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41+GPTMS+CS-Suc, and (d) MCM-41+GPTMS+CS-Suc+BZ samples, indicating the elemental composition of the surface of the nanoparticles. The arrows denote the energy regions

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correspondent to the N 1s peak that are amplified and shown in the insets.

Table 3. Surface chemical composition of the samples, as determined from the XPS survey spectra.

Element

MCM-41

MCM-41 +GPTMS

MCM-41 +GPTMS+C S-Suc

MCM-41 +GPTMS+CSSuc+BZ

Si

33.6%

31.1%

27.2%

28.0% 12

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60.9%

58.6%

48.0%

53.3%

C

5.5%

10.3%

25.4%

18.2%

1.4%

0.5%

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N

Si 2p

a)

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Intensity

SiO2

SiO2*

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b)

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Intensity

SiO2

108

106

Si-C SiO2*

104 102 100 98 Binding Energy (eV)

96

94

Figure 4. XPS Si 2p high-resolution spectra of (a) pure MCM-41 nanoparticles and (b) after the first functionalization step, with GPTMS (sample MCM-41+GPTMS).

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ACCEPTED MANUSCRIPT The functionalization step with benznidazol was conclusively characterized through the XPS N 1s high resolution spectra of the MCM-41+GPTMS+CS and the MCM-41+GPTMS+CS+BZ samples, shown in Figure 5 (a) and (b), respectively. The spectrum in Figure 5 (a) presents two main peaks corresponding to primary amine NH2 of the chitosan and the amide of chitosan

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succinate molecule (–NHCO–) (at 399.5 eV and 401.8 eV, respectively) [28]. This indicates that the chitosan has not been completely succinized, with both structures remaining on the surface of the samples. The minor contribution around 397.0 eV may be assigned to Si-N bounds or to some charging effect. Charging effect has been previously observed in XPS spectra of silica nanoparticles and other materials which have poor electrical conductivity [20].

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The N 1s high resolution spectrum acquired after the incorporation of the benznidazol molecule is shown in Figure 5 (b), where five contributions can be identified: amine NH2 (399.1

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eV) and the amide –NHCO– (401.8 eV) groups of the chitosan succinate, and also C=N-C (399.6 eV), -C-NH-C (400.2 eV) [29] and NO2 (402.9 eV) [30] present in the imidazol group. Another additional peak may be assigned to N 1s of nitride (SiNx) (397.2 eV) or to some charging effect. The deconvolution of N 1s is characteristic of nitrogen from the imidazol group indicating the

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successful incorporation of the benznidazol molecule onto the nanoparticles.

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N 1s

a)

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-NH2

Intensity

-NH

b)

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SiNx

C-NH-C

Intensity

-NH NO2

C=N-C -NH2

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SiNx

404

400

396

392

Binding Energy (eV)

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Figure. 5 - XPS N 1s high-resolution spectra of (a) MCM-41+GPTMS+CS-Suc a) and (b) MCM-

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41+GPTMS+CS-Suc+BZ b) samples.

The C 1s high-resolution spectra for the MCM-41+GPTMS, MCM-41+GPTMS+CS-Suc and MCM-41+GPTMS+CS-Suc+BZ samples are shown in Figure 6 (a-c).

15

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C 1s

a)

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epoxy ring C-O

C-Si ; C*

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Intensity

C-C; C-H

b) C-O; N-C

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Intensity

C-C; C-H

N-C=O

C-Si; C*

c)

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C-C ; C-H; benzene

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Intensity

C-O; N-C

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292

Figure 6.

N-C=O

288

C-Si; C*

284

280

Binding Energy (eV)

XPS C 1s high-resolution spectra of the (a) MCM-41+GPTMS, (b) MCM-

41+GPTMS+CS-Suc and (c) MCM-41+GPTMS+CS-Suc+BZ samples.

For the MCM-41 modified with GPTMS (Fig. 6 (a)), the spectrum was fitted with three main components: C-C and C-H features (overlapping at 285.30 eV), C–O bonds (at 287.5 eV) which 16

ACCEPTED MANUSCRIPT corresponds to the epoxy ring and other moieties of the GPTMS molecule, respectively [31,32], and an additional peak assigned to C-Si bindings or charging effects. For MCM-41+GPTMS modified with CS-Suc nanoparticles (Figure 6 (b)), the appearance of a spectral component at 287.7 eV, corresponding to N-C=O, confirms the bonding of chitosan and Succinic anhydride

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[33-35] For the MCM-41+GPTMS+CS-Suc nanoparticles modified with BZ (Fig. 6(c)) the C 1s component at 286.6 eV was assigned to C-O, C-N from the amino groups –C=N-C(imidazol), and the peak at 287.7 eV to N-C=O, results from the binding of BZ with the chitosan succinate

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and its incorporation onto the nanoparticles.

Solid-State Nuclear Magnetic Resonance – CP MAS NMR

CP MAS NMR spectroscopy is very well suited to the study of mesoporous materials. The

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sensitivity of the NMR is essential to characterize surfaces containing gyromagnetic ratios and/or low natural abundance, such as 29Si and 13C.

The SiO4 units are referred as Qn with formula Si(OSi)n(OH)4-n. 29Si CP MAS NMR spectra for all Si samples are shown in Figure 7. All samples (Fig. 7a, 7b, 7c and 7d) exhibit peaks with chemical shift (δ) at about -93, -102 and -111 ppm (Q2, Q3 and Q4). The first peak (Q2) is attributed to silicon atoms with geminal silanol groups (Si-O)2Si(OH)2, the second (Q3) indicates

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the presence of silicon atoms with isolated OH groups (Si-O)3 Si-OH and the third and last peak (Q4) is attributed to silicon atoms which do not have OH groups (Si(SiO)4) in its structure [36]. Initially, the contribution of these groups (Figure 7a) in the composition of the sample is 11.54, 79.60 and 8.86%. After grafted the GPTMS into the MCM-41 (Figure. 5b) two new chemical

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shifts with resonance signals at -39.52 (T1) and -56.80 (T2) ppm were identified. Both are attributed to the presence of alkyl groups (R) in the structure of the compound (RSi(OSi)(OH)2)

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for T1 and RSi(OSi)2OH) for T2 [37]. After the reactions with chitosan succinate (Figure 7c) and BZ (Figure 7d), it is clearly noticeable that there is a decrease in the composition of the geminal silanol groups (Q2) of 11.54 to 6.00% and new contributions are observed at -58.6 and -67.0 ppm. These two contributions are associated with the condensation reaction of the silanol groups that occurs due to the opening of the epoxy groups present in the GPTMS. 17

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29

Si CP MAS NMR spectra for (a) MCM-41, (b) MCM-41+GPTMS, (c) MCM-

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Figure 7.

41+GPTMS+CS and (d) MCM-41+GPTMS+CS +BZ.

Figure 8 shows the spectra of CP MAS

13

C NMR for MCM-41+GPTMS (Figure 8a), MCM-

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41+GPTMS+CS (Figure 8b) and GPTMS+CS +BZ (Figure 8c). The resonance peaks observed in Figure 10a correspond to carbon atoms of the chemically displaced epoxy ring at 45.6 and

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52.1 ppm (C3 and C4), indicating that most of the epoxy groups are preserved during the reaction conditions. Another remarkable feature was the appearance of a peak at 64.2 ppm (C5), and a doublet at 72.1 and at 73.8 ppm (C6 and C7), which can be assigned to either carbons of methylether and oligo or poly (ethylene oxide) originated from the side reactions of the epoxy ring of GPTMS. The resonance peaks observed at 8.6 (C1) and 22.6 (C2) ppm are attributed to the carbons of the methylene group (-CH2-) in the alpha (α) position on the silicon atom and to the methyl (-CH3-) groups [38]. The chemical reaction of MCM41+GPTMS with chitosan succinate molecules occurs through of the nucleophilic addition of the nitrogen atoms present in 18

ACCEPTED MANUSCRIPT the chitosan succinate structure to the carbon atom with the lowest steric hindrance present in the epoxy ring. The result is the formation of a covalent bond between the two structures. This binging will be discussed later by DFT investigation. The MCM-41+GPTMS+CS (Figure 8b) exhibits five new contributions with characteristic shifts of the peak signals at 78.3, 99.1, 128.0, 141.5 and 147.5 ppm, referring to the carbons C8, C9, C10, C11 and C12, respectively, according to

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the numbering of the structure, as well as the peak signals between C1 and C7 previously discussed. The signal at 78.3 confirms -C-O-C- linkage of chitosan chain adjacent to CH2OH, whereas the signal appearing at 99.1 is due to -C-O-C- linkage adjacent to oxygen present in the glycosidic ring. The peaks at 128.0, 141.5 and 147.5 are ascribed to vinyl group present in the

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hybrid. The succinate molecule acts as an anchor for the incorporation of the drug (BZ) into the

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system through an interaction of the type of hydrogen bond.

Figure 8. CP MAS NMR spectra for

13

C in (a) MCM-41+GPTMS, (b) MCM-41+GPTMS+CS

and (c) MCM-41+GPTMS+CS +BZ. 19

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Figure 8c shows the resonance peaks after incorporation of the BZ drug into the system. It is important to note that there may be overlapping of the carbon atom signals present in the other molecules with the BZ molecule. However, a new resonance signal appears at 39.1 ppm (C13). 13

C dipole coupling with

14

N in a quadrupole

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This resonance signals can occur due to the

nucleus. [39]. Solid-state NMR results indicate that the BZ was successfully incorporated into the system. It is worth noting that the results obtained from RMN characterization are in

Chitosan reactions - a DFT investigation

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accordance with the discussed in XPS analysis.

Following the experimental results, we have first simulated the reaction between GPTMS

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(Figure 9) and MSNs-MCM-41, whose products are modified silica (Figure 10) and modified GPTMS, that is,

SiC9H20O5 + SiO4H4 →∆H SiC6H14O5 +SiO4C3H10 . The ∆H for this reaction is 0.03 eV, which was computed according to Eq. 1.

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∆H = Eproducts − Ereagents .

(1)

Where, Eproducts and Ereagents are the sum of the total energies of the products and reagents, respectively. The binding energies are calculated as follows: (2)

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Eb = Ecombined system − ∑i Ei

where Ecombined system represents calculated total energy of the combined system (for instance, a

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Chitosan molecule bond to modified GPTMS), and Ei is the total energy of element of the combined system, that is, in this work we can stand for chitosan, modified GPTMS, modified silica, GPTMS, or MCM-41

20

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Figure 9. Structural configuration of the molecules considered in the studied reactions. (a)

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MCM-41, (b) GPTMS, (c) Chitosan, (d) Benznidazol and (e) Succinic-anhydride.

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GPTMS modified.

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Figure 10. Products of reaction between MCM-41 and GPTMS; (a) MCM-41 modified and (b)

Two different reaction sites were considered for the reaction between chitosan and modified GPTMS: one where an N-C bond is formed, (Figure 11); and another where an O-C bond is formed (Fig. 3-b). The values of Eb are -0.92 and -0.72 eV for the sites where O-C and N-C bonds are formed, respectively, which shows that the reaction sites where C-N bond is formed is more energetically favorable than that where C-B bond is formed.

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Figure 11. Products of reaction between Chitosan and GPTMS modified: (a) modified GPTMS

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binding Chitosan at N site (Eb = −0.92 eV ); and (b) the bond occurs at O site (Eb = −0.72 eV). We have also investigated possible reactions between chitosan, succinic anhydride, benznidazol

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and H2 (Eq. 3 and 4). Besides water, the products of those reactions are molecules in which the succinic anhydride binds chitosan to benznidazol through O-C and N-C covalent bonds or through an N-C covalent bond and a O-H hydrogen bond, see Figure 12. The results for ∆H (0.36 eV and -1.56 eV for benznidazol forming O-C and hydrogen bonds with the succinic anhydride, respectively) indicate that the most stable configuration is the one with hydrogen

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bond.

C12H24N2O9 + C4H4O3 + C12H12N4O3 + H2 →∆H C28H40N6O14 + H2O.

(4)

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C12H24N2O9 + C4H4O3 + C12H12N4O3 + H2 →∆H C16H28N2O12 + C12H12N4O2 + H2O.

(3)

22

ACCEPTED MANUSCRIPT Figure 12. Figure Products of reaction between Chitosan, Succinic anhydride and Benznidazol. (a) The Benznidazol binding the succinic anhydride modified in the O site and in (b). They bond through hydrogen bond.

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Biological assays - Trypanocidal assays In this study we used the non-infective epimastigote form, as has been done in several other studies [14], which is a suitable model for studying the mode of action of the compounds employed as potential trypanocidal agents. The efficacy of the MCM-41+GPTMS+CS+BZ sample as a relevant agent to the epimastigote forms of T. cruzi CL Brener strain was evaluated

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from its capacity to promote parasite death through this treatment. This assay was performed with BZ-free samples (MCM-41 and MCM-41+GPTMS+CS) to compare the final effects and to

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discriminate whether possible therapeutic effects would be the result of BZ or others materials. Representative curves of normal growth of epimastigote forms of T. cruzi CL Brener treated with samples are presented in Figures 13. The results from BZ-free samples and control groups (control group in LIT and control group with the addition of PBS), Figure 13, exhibit high percentage of live parasites, showing a normal growth of the epimastigote forms of T. cruzi CL Brenner, which increased until a plateau growth phase. Thus, the samples MCM-41 (Figure 13a)

any damage to them.

(b)

80 60

10 6 cells

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100

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10 6 cells

(a)

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and MCM-41+GPTMS+CS (Figure 13b) are non-toxic for the parasites, and it does not induce

40 20

24

48

72

100 80 60 40 20 0

0

0

120

96

120

144

0

24

48

72

96

120

144

Time(hours) Control Control+PBS

Time(hours) Control Control+PBS

MCM-41 [150 µg.mL-1 ]

MCM-41+GPTMS+CS [120 µg.mL-1 ]

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ACCEPTED MANUSCRIPT Figure 13. Normal growth of epimastigote forms of T. cruzi CL Brener treated with 150 µg mL-1 of MCM-41 (a) and 120 µg mL-1 of MCM-41+GPTMS+CS (b).

Next, an assay involving samples containing BZ was carried out. The BZ molecules anchored

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onto the surface of MCM-41+GPTMS+CS dissolved in PBS were added to the culture media to give 0.002 and 0.004 mM final concentrations as shown in Table 4. As observed in Figure 14 (a and b), both pure BZ and BZ encapsulated into nanoparticles decreased the survival rate of the parasites, producing an inhibitory effect against epimastigote forms of T. cruzi CL Brener strain. Furthermore, the amount of free BZ is approximately thirty times greater than the amount of BZ

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contained in the functionalized silica. It means that the BZ anchored on to the surface of MCM41+GPTMS+CS exhibited a remarkable effect on T. cruzi in comparison to pure BZ, since the

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lower concentration of BZ in MSN reduced the number of parasites significantly with respect to free-BNZ content in this assay (high ratio 1:30). These results confirm the success of the proposed system (MCM-41+GPTMS+CS) since the BZ drug was efficaciously released from these nanoparticles into the parasites provoking its death. Thus, functionalized MSNs showed efficiency in delivering BZ, indicating the suitability of this sample to treat the chronic phase infections.

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CS is a biodegradable cationic polymer, which is easily anchored onto surfaces of MCM-41, enhancing the biocompatibility and its presence on the structure of MCM-41 did not affect the cellular viability (results not shown). Thus, the concentrations of nanoparticles used in this work are considered ideal for biological applications. On the other hand, the MCM-41+GPTMS+CS

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has the presence of highly reactive groups, mainly hydroxyl and amino ones which facilitate the packaging of BZ drugs which targets the DNA of the T.cruzi.

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According with Rajão et al. [16], BZ acts through the formation of free radicals and electrophilic metabolites that are generated when its nitro group is reduced to an amino group by the action of nitroreductases. The electrophilic metabolites can react with DNA, which can lead to the generation of DNA lesions, if the DNA lesions are not repaired, they can lead to the death of the T.cruzi. Results from Vieira et al. studies [10], testing the toxicity of Cadmium Telluride Quantum Dots in T. cruzi, showed that the cell damage occurred only with the use of higher concentrations of Quantum Dots. Others studies using Quantum Dots capped with

24

ACCEPTED MANUSCRIPT mercaptoacetic acid shows that the high Quantum Dots concentrations are toxic to T. cruzi, inducing cell death by autophagy [11,40].

Table 4. Amount of BZ molecules anchored on the surface of MCM-41+GPTMS+CS calculated

Samples

Concentration (mM)

BZ pure

0.06

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for each sample and correlated with BZ pure. Concentration (µg/mL) 15.61

0.12 0.002

0.52

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MCM-41+GPTMS+CS +BZ

30.82

(a)

120

120

(b)

100

100

106 cells

80 60 40 20 0 0

24

48

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106 cells

1.04

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0.004

72

96

120

144

Time(hours)

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Control MCM-41+GPTMS+CS+BZ [0.002 mM] BZ [0.06 mM]

80 60 40 20

0 0

24

48

72

96

120

144

Time(hours) Control MCM-41+GPTMS+CS+BZ [0.004 mM] BZ [0.12 mM]

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Figure 14. The MCM-41+GPTMS+CS +BZ are cytotoxic to the epimastigote forms of T. cruzi CL Brener parasites.

Our results show that BZ loaded in MCM-41 clearly display a higher trypanocide activity

on epimastigote forms of T. cruzi CL Brener strain, observed for free-BZ as well. This fact is highly relevant as MCM-41 with lower doses of BZ can be used with the same efficacy.

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ACCEPTED MANUSCRIPT Conclusions The mesoporous silica nanoparticles were obtained with an ordered pore network, uniform and tunable size and shape of the particles. Its modifications with GPTMS and chitosan-succinate were successful. The characterization showed that BZ molecules are effectively anchored to the

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chitosan succinate on the surface of the mesoporous silica nanoparticles. For the first time it was shown that functionalized MCM-41 nanoparticles content BZ exhibited a remarkable effect on T. cruzi growth inhibition. It was observed that a smaller amount of BZ molecules anchored on the surface of MCM-41+GPTMS+CS were successfully delivered with an effect similar to the high number of pure BZ molecules to epimastiogotes T.cruzi CL Brenner parasites. Therefore, this

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work revealed that functionalized mesoporous silica nanoparticles with chitosan are potential nanoplatforms for drug and gene delivery targeting neglected diseases such as Chagas disease or

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American trypanosomiasis.

Acknowledgments

This work has been supported by CAPES, CNPq and FAPEMIG.

Conflict of interest

publication of this article.

[1]

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References

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The authors declare that they have no conflict of interests, financial, scientific or otherwise in the

I. I. Slowing, J. L. Vivero-Escoto, C.-W. Wu, and V. S.-Y. Lin, “Mesoporous silica

AC C

nanoparticles as controlled release drug delivery and gene transfection carriers.,” Adv. Drug Deliv. Rev., vol. 60, no. 11, pp. 1278–88, Aug. 2008.

[2]

J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T.W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins, and J. L.

Schlenkert, “A New Family of Mesoporous Molecular Sieves Prepared with Liquid Crystal Templates,” J. Am. Chem. Soc., no. 114, pp. 10834–10843, 1992.

[3]

F. Liebau, “Ordered microporous and mesoporous materials with inorganic hosts: Definitions of terms, formula notation, and systematic classification,” Microporous 26

ACCEPTED MANUSCRIPT Mesoporous Mater., vol. 58, no. 1, pp. 15–72, 2003. [4]

M. Grün, K. K. Unger, A. Matsumoto, and K. Tsutsumi, “Novel pathways for the preparation of mesoporous MCM-41 materials: control of porosity and morphology,” Microporous Mesoporous Mater., vol. 27, no. 2–3, pp. 207–216, 1999. M. Varache, I. Bezverkhyy, L. Saviot, F. Bouyer, F. Baras, and F. Bouyer, “Optimization

RI PT

[5]

of MCM-41 type silica nanoparticles for biological applications: Control of size and absence of aggregation and cell cytotoxicity,” J. Non. Cryst. Solids, vol. 408, pp. 87–97, Jan. 2015. [6]

F. Tamanoi and R. Yanes, “Development of mesoporous silica nanomaterials as a vehicle

[7]

SC

fo anticancer drug delivery,” Ther Deliv., vol. 3, no. 3, pp. 389–404, 2013.

A. Suwalski, H. Dabboue, A. Delalande, S. F. Bensamoun, F. Canon, P. Midoux, G.

M AN U

Saillant, D. Klatzmann, J.-P. Salvetat, and C. Pichon, “Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles.,” Biomaterials, vol. 31, no. 19, pp. 5237–45, Jul. 2010. [8]

M. D. Brown, A. G. Scha, and I. F. Uchegbu, “Gene delivery with synthetic ( non viral ) carriers,” vol. 229, pp. 1–21, 2001.

[9]

W. Guang Liu and K. De Yao, “Chitosan and its derivatives--a promising non-viral vector

[10]

TE D

for gene transfection.,” J. Control. Release, vol. 83, no. 1, pp. 1–11, 2002.

C. S. Vieira, D. B. Almeida, A. A. de Thomaz, R. F. S. Menna-Barreto, J. R. dos SantosMallet, C. L. Cesar, S. A. O. Gomes, and D. Feder, “Studying nanotoxic effects of CDTE

165, 2011.

W. A. Marques, S. A. O. Gomes, D. B. Almeida, J. P. F. Pacheco, R. F. S. Menna-Barreto,

AC C

[11]

EP

quantum dots in Trypanosoma cruzi,” Mem. Inst. Oswaldo Cruz, vol. 106, no. 2, pp. 158–

J. R. Santos-Mallet, C. L. Cesar, and D. Feder, “Evidence of Autophagy in Trypanosoma Cruzi Cells By Quantum Dots,” Acta Microsc., vol. 23, no. 1, pp. 1–10, 2014.

[12]

G. A. Schmunis, “Prevention of Transfusional Trypanosoma cruzi Infection in Latin America,” Mem. Inst. Oswaldo Cruz, vol. 94, no. SUPPL. 1, pp. 93–101, 1999.

[13]

M. Steindel, L. Kramer Pacheco, D. Scholl, M. Soares, M. H. de Moraes, I. Eger, C. Kosmann, T. C. M. Sincero, P. H. Stoco, S. M. F. Murta, C. J. de Carvalho-Pinto, and E. C. Grisard, “Characterization of Trypanosoma cruzi isolated from humans, vectors, and 27

ACCEPTED MANUSCRIPT animal reservoirs following an outbreak of acute human Chagas disease in Santa Catarina State, Brazil,” Diagn. Microbiol. Infect. Dis., vol. 60, no. 1, pp. 25–32, 2008. [14]

J. D. Maya, S. Bollo, L. J. Nuñez-Vergara, J. A. Squella, Y. Repetto, A. Morello, J. Périé, and G. Chauvière, “Trypanosoma cruzi: Effect and mode of action of nitroimidazole and

[15]

RI PT

nitrofuran derivatives,” Biochem. Pharmacol., vol. 65, no. 6, pp. 999–1006, 2003. J. D. Maya, B. K. Cassels, P. Iturriaga-Vásquez, J. Ferreira, M. Faúndez, N. Galanti, A. Ferreira, and A. Morello, “Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host,” Comp. Biochem. Physiol. - A Mol. Integr. Physiol., vol. 146, no. 4, pp. 601–620, 2007.

M. A. Rajão, C. Furtado, C. L. Alves, D. G. P. Silva, M. B. de Moura, B. L. S. Reis, M. K.

SC

[16]

Lima, A. A. Zuma, J. P. V. da Rocha, J. B. F. Garcia, I. C. Mendes, S. D. J. Pena, A. M.

M AN U

Macedo, G. R. Franco, N. C. de Souza-Pinto, M. H. G. de Medeiros, A. K. Cruz, M. C. M. Motta, S. M. R. Teixeira and C. R. Machado, “Unveiling Benznidazole’s Mechanismof Action Through Overexpression of

DNA Repair Proteins in Trypanosoma cruzi,”

Environ. Mol. Mutagen., vol. 55, no. 1, pp. 309–321, 2014. [17]

Y. E. Castro-Sesquen, R. H. Gilman, G. Galdos-Cardenas, L. Ferrufino, G. S??nchez, E. Valencia Ayala, L. Liotta, C. Bern, and A. Luchini, “Use of a Novel Chagas Urine

TE D

Nanoparticle Test (Chunap) for Diagnosis of Congenital Chagas Disease,” PLoS Negl. Trop. Dis., vol. 8, no. 10, pp. 4–11, 2014. [18]

C. R. Chaves, A. Fontes, P. M. A. Farias, B. S. Santos, F. D. de Menezes, R. C. Ferreira, C. L. Cesar, A. Galembeck, and R. C. B. Q. Figueiredo, “Application of core-shell

EP

PEGylated CdS/Cd(OH)2 quantum dots as biolabels of Trypanosoma cruzi parasites,” Appl. Surf. Sci., vol. 255, no. 3, pp. 728–730, 2008. C. R. Machado, J. P. Vieira-da-Rocha, I. C. Mendes, M. A. Rajão, L. Marcello, M. Bitar,

AC C

[19]

M.G. Drummond, P. Grynberg, D. A. Oliveira, C. Marques, B. Van Houten, and R. McCulloch, “Nucleotide excision repair in Trypanosoma brucei : specialization of transcription-coupled repair due to multigenic transcription,” vol. 92, no. April, pp. 756– 776, 2014.

[20]

L. B. de O. Freitas, I. J. G. Bravo, W. A. de A. Macedo, and E. M. B. de Sousa, “Mesoporous silica materials functionalized with folic acid : preparation , characterization and release profile study with methotrexate,” J Sol-Gel Sci Technol, vol. 77, no. 1, pp. 28

ACCEPTED MANUSCRIPT 186–204, 2016. [21]

P. Giannozzi et al., “Q UANTUM ESPRESSO : a modular and open-source software project for quantum simulations of materials.”

[22]

J. P. Perdew, K. Burke, and M. Ernzerhof, “Generalized Gradient Approximation Made

[23]

RI PT

Simple,” no. 3, pp. 3865–3869, 1996. D. Lee, Z. Gemici, M. F. Rubner, and R. E. Cohen, “Multilayers of oppositely charged SiO2 nanoparticles: effect of surface charge on multilayer assembly.,” Langmuir, vol. 23, no. 17, pp. 8833–8837, 2007. [24]

S. H. Chang, H. T. V. Lin, G. J. Wu, and G. J. Tsai, “pH Effects on solubility, zeta

Carbohydr. Polym., vol. 134, pp. 74–81, 2015.

M. L. Scalise, E. C. Arrúa, M. S. Rial, M. I. Esteva, C. J. Salomon, and L. E. Fichera,

M AN U

[25]

SC

potential, and correlation between antibacterial activity and molecular weight of chitosan,”

“Promising efficacy of benznidazole nanoparticles in acute trypanosoma cruzi murine model: In-vitro and in-vivo studies,” Am. J. Trop. Med. Hyg., vol. 95, no. 2, pp. 388–393, 2016. [26]

Y. Zhao, J. Zou, W. Shi, and L. Tang, “Preparation and characterization of mesoporous silica spheres with bimodal pore structure from silica/hyperbranched polyester

[27]

TE D

nanocomposites,” Microporous Mesoporous Mater., vol. 92, no. 1–3, pp. 251–258, 2006. Z. Lei, S. Bi, and H. Yang, “Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization,” Food Chem., vol. 104, no. 2, pp. 577–584, 2007. [28]

U. Janciauskaite and R. Makuska, “Cationic polyelectrolytes from natural building blocks

[29]

EP

of chitosan and inulin,” React. Funct. Polym., vol. 69, no. 5, pp. 300–305, 2009. O. Olivares-Xometl, N. V. Likhanova, R. Martínez-Palou, and M. A. Domínguez-Aguilar,

AC C

“Electrochemistry and XPS study of an imidazoline as corrosion inhibitor of mild steel in an acidic environment,” Mater. Corros., vol. 60, no. 1, pp. 14–21, 2009.

[30]

J. Baltrusaitis, P. M. Jayaweera, and V. H. Grassian, “XPS study of nitrogen dioxide

adsorption on metal oxide particle surfaces under different environmental conditions,” Phys. Chem. Chem. Phys., vol. 11, no. 37, p. 8295, 2009.

[31]

C. Funk, P. M. Dietrich, T. Gross, H. Min, W. E. S. Unger, and W. Weigel, “Epoxyfunctionalized surfaces for microarray applications: Surface chemical analysis and fluorescence labeling of surface species,” Surf. Interface Anal., vol. 44, no. 8, pp. 890– 29

ACCEPTED MANUSCRIPT 894, 2012. [32]

T. Kamra, S. Chaudhary, C. Xu, N. Johansson, L. Montelius, J. Schnadt, and L. Ye, “Covalent immobilization of molecularly imprinted polymer nanoparticles using an epoxy silane,” J. Colloid Interface Sci., vol. 445, pp. 277–284, 2015. C. Zhang, H. Zhang, R. Li, and Y. Xing, “Morphology and adsorption properties of

RI PT

[33]

chitosan sulfate salt microspheres prepared by a microwave-assisted method,” RSC Adv., vol. 7, no. 76, pp. 48189–48198, 2017. [34]

I. F. Amaral, P. L. Granja, and M. a Barbosa, “Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study.,” J. Biomater. Sci. Polym. Ed., vol. 16,

[35]

SC

no. 12, pp. 1575–1593, 2005.

W. F. Yap, W. M. M. Yunus, Z. A. Talib, and N. A. Yusof, “X-ray photoelectron

M AN U

spectroscopy and atomic force microscopy studies on crosslinked chitosan thin film,” Int. J. Phys. Sci., vol. 6, no. 11, pp. 2744–2749, 2011. [36]

S. O. Akpotu and B. Moodley, “Journal of Environmental Chemical Engineering Synthesis and characterization of citric acid grafted MCM-41 and its adsorption of cationic dyes,” Biochem. Pharmacol., vol. 4, no. 4, pp. 4503–4513, 2016.

[37]

Z. A. Alothman and A. W. Apblett, “Preparation of mesoporous silica with grafted

[38]

TE D

chelating agents for uptake of metal ions,” vol. 155, pp. 916–924, 2009. L. S. Connell, F. Romer, M. Suárez, E. M. Valliant, Z. Zhang, P. D. Lee, M. E. Smith, J. V. Hanna, and J. R. Jones, “Chemical characterisation and fabrication of chitosan–silica hybrid scaffolds with 3-glycidoxypropyl trimethoxysilane,” J. Mater. Chem. B, vol. 2, no.

[39]

EP

6, pp. 668–680, 2014.

J. Priotti, M. J. G. Ferreira, M. C. Lamas, D. Leonardi, C. J. Salomon, and T. G. Nunes,

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“First solid-state NMR spectroscopy evaluation of complexes of benznidazole with cyclodextrin derivatives,” vol. 131, pp. 90–97, 2015.

[40]

S. A. O. Gomes, C. S. Vieira, D. B. Almeida, J. R. Santos-Mallet, R. F. S. Menna-Barreto,

C. L. Cesar, and D. Feder, “CdTe and CdSe quantum dots cytotoxicity: A comparative study on microorganisms,” Sensors, vol. 11, no. 12, pp. 11664–11678, 2011.

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ACCEPTED MANUSCRIPT Highlights

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• MCM-41 were successfully functionalized with chitosan-succinate. • The functionalized material was efficiently loaded with benznidazole. • Functionalized MCM-41- benznidazole was assessed into T. cruzi parasites. • Materials loaded with BZ present a remarkable effect on T. cruzi growth inhibition.